Methods of using the brain-derived osteogenic factor ccn3 for treating bone and cartilage degeneration

Abstract

Methods of using cell communication network factor 3 (CCN3) for the treatment of bone or cartilage disorders are provided. Also provided are methods of gene therapy, including methods of expressing CCN3 in vivo in an amount effective to promote new bone or cartilage growth. In addition, methods of screening for agonists, mimetics, and analogs of CCN3 are provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of provisional application 63 / 578,795, filed August 25, 2023, pursuant to 35 USC § 119(e), which is incorporated herein by reference in its entirety.

[0003] Statement regarding federally funded research or development

[0004] This invention was made with government support under contract number R01 AG062331 granted by the National Institutes of Health. The government has certain rights in this invention.

[0005] By referencing and incorporating into the sequence list

[0006] The sequence list is provided as a sequence list XML file "UCSF-749WO" created on August 15, 2024, with a size of 24,285 bytes. The contents of the sequence list XML file are incorporated herein by reference in their entirety.

[0007] introduction

[0008] Musculoskeletal disorders constitute a significant global health burden. Osteoporosis significantly impacts healthy aging and is typically experienced more frequently by women than men. Women utilize estradiol E2 as an anabolic hormone to increase energy expenditure through the regulation of bone remodeling via osteocytes (Doolittle et al., *J Bone Miner Res*, 37, 1750-1760 (2022)), osteoblasts (Almeida et al., *J Clin Invest*, 123, 394-404 (2013)), and osteochondral skeletal stem cells (ocSSCs) (Khosla et al., *Trends Endocrinol Metab*, 23, 576-581 (2012)) (Ingraham et al., *Annu Rev Physiol*, 84). These cells (59-85 (2022)) and maintain bone mass, playing a crucial role in both bone and cartilage (Chan et al., Cell, 160, 285-298 (2015); Chan et al., Cell, 175, 43-56 e21 (2018)). In women, naturally or drug-induced estrogen depletion during menopause or anti-hormonal therapy leads to a slow decline in bone mass, highlighting the anabolic role of estrogen in bone metabolism. However, during lactation, when ovarian estrogen shuts down and bone formation and resorption surge to meet the high calcium demands of offspring, the close link between estrogen and bone is strangely broken (reviewed in Kovacs, Journal of Bone and Mineral Research, 32, 676-680 (2017)). Parathyroid hormone-related protein (PTHrP), a close ortholog of parathyroid hormone (PTH) from the mammary gland, is the main driving force for the stripping of calcium from maternal bones to obtain milk (Ardawi et al., Eur J Endocrinol 137, 402-409 (1997); Ardeshirpour et al., Bone 38, 787-793 (2006)).The relentless calcium requirements of newborns ultimately lead to significant bone loss in mothers, decreasing by nearly 30% in rodents due to large litters (VanHouten & Wysolmerski, Endocrinology 144, 5521-5529 (2003)) and by 10% in humans (Kalkwarf & Specker, Obstet Gynecol 86, 26-32 (1995); Bjornerem et al., Journal of Bone and Mineral Research 32, 681-687 (2017)); most of these losses normalize after lactation (VanHouten & Wysolmerski, ibid.; Bjornerem et al., ibid.). It is hypothesized that without this lactation-anabolism phase, maternal skeletal (and infant) bone would be severely damaged, as inferred from increased bone mass in lactating mothers after conditional PTHrP knockout (VanHouten et al., *Journal of Clinical Investigation* 112, 1429-1436 (2003); Karaplis et al., *Genes & Development* 8, 277-289 (1994)). The underlying mechanisms driving new bone formation during lactation are lacking; this idea remains to be proven.

[0009] Currently, 52.2 million Americans are diagnosed with arthritis, and this number is projected to rise to 78.4 million by 2040. Of these conditions, the most common is osteoarthritis (OA), which carries a 40% lifetime risk. Since no effective treatment has been approved to prevent the progression of OA, symptom relief and eventual joint replacement are the standard of care. In an attempt to regenerate cartilage in OA, surgeons perform microfracture (MF) surgery, a technique developed in the 1950s and widely used today. During MF surgery, surgeons drill holes in the debridement of the cartilage until they reach the medullary cavity. A hematoma forms at the MF site, which is absorbed and replaced by fibrous tissue. The resulting “fibrocartilage” provides some symptom relief but has significantly reduced mechanical properties compared to normal articular cartilage.6 Little is known about the mechanisms by which MF leads to fibrocartilage formation, the effects of this technique on resident stem cell populations, or how this technique can be used for tissue regeneration.

[0010] The limitations of current methods for addressing osteoarthritis (OA), including myeloblastic stromal transplantation (MF), have led to widespread interest in the potential of stem cell therapies for regenerating cartilage. Numerous clinical trials have explored the use of autologous stem cell transplantation as an alternative therapy for OA of the hip, knee, and thumb. Most stem cell-specific trials for treating hip and knee OA are pilot or feasibility studies investigating the application of plastic-adherent, cultured, expanded mesenchymal stem cells (MSCs). These MSCs are derived from bone marrow or adipose tissue. However, these MSCs do not constitute a validated stem cell population, and it is difficult to determine the extent of cell engraftment and its contribution to changes in functional outcomes. These trials have also failed to elucidate the specific mechanisms of action behind cases of significant symptom improvement.

[0011] Recently, several groups have made progress in identifying skeletal stem cell (SSC) in mice (mSSC) and humans (hSSC). Purified SSCs are defined by their self-renewal capacity and their multi-lineage contribution to bone, cartilage, and matrix, but not adipocytes. However, it is important to consider that SSCs sorted using different strategies may be similar, but not necessarily identical, for example, in their ability to produce adipocytes. Furthermore, SSCs in the skeleton of mice and humans are stimulated to proliferate after injury, and injury-activated progenitor cell populations also show enhanced osteogenic potential relative to the homeostatic population. However, whether the activation of resident SSCs can be used to regenerate cartilage as a means of treating osteoarthritis (OA) remains undetermined.

[0012] Better methods for treating and preventing bone and cartilage degeneration are still needed. Summary of the Invention

[0013] Methods for using cell communication network factor 3 (CCN3) to treat bone or cartilage disorders are provided. Gene therapy methods are also provided, including methods for expressing CCN3 in vivo at an effective amount sufficient to promote new bone or cartilage growth. Furthermore, methods for screening agonists, mimics, and analogs of CCN3 are provided.

[0014] In one aspect, a method is provided for treating a bone condition or symptom associated with bone degeneration in a subject, the method comprising administering a therapeutically effective amount of CCN3 to the subject.

[0015] The "therapeutic effective dose or amount" of CCN3 protein is intended to refer to the amount that, when administered as described herein, produces a positive therapeutic response, such as improved recovery from bone conditions or symptoms associated with bone degeneration. Improved recovery may include increased bone mineral density, increased bone mass, increased bone strength, increased bone mineral density, and / or reduced adipose bone marrow. Furthermore, the therapeutic effective dose or amount may stimulate osteochondral skeletal stem cells to generate bone.

[0016] Bone conditions and symptoms associated with bone degeneration include any disease or condition that leads to decreased bone mineral density, reduced bone mass, bone fragility, low bone mineral density, and / or increased fatty bone marrow, such as, but not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

[0017] In some embodiments, the CCN3 protein comprises an amino acid sequence having at least about 80% to 100% sequence identity with the sequence of SEQ ID NO:1, including any percentage identity within that range, such as having 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0018] In some implementations, the treatment increases bone mass compared to the subject's bone mass before treatment.

[0019] In some implementations, CCN3 is administered intravenously or intraperitoneally.

[0020] In some implementations, CCN3 is applied locally to the bone of the subject.

[0021] In some implementations, multiple therapeutically effective doses of CCN3 are administered to the subject. In some implementations, CCN3 is administered according to a daily dosing regimen or intermittently.

[0022] In some embodiments, the method further includes administering a bone anabolic agent or an anti-resorbent agent to the subject. Exemplary bone anabolic agents include, but are not limited to, parathyroid hormone, teriparatide, abaloparatide, and romosozumab. Exemplary anti-resorbent agents include, but are not limited to, estrogens, estrogen agonists, bisphosphonates, and denosumab.

[0023] In some implementations, the subjects are humans.

[0024] In another embodiment, a method for reducing or preventing bone degeneration in a female subject during lactation is provided, the method comprising administering a therapeutically effective amount of CCN3 to the subject.

[0025] In another aspect, a composition comprising CCN3 is provided for use in methods of treating bone conditions or symptoms associated with bone degeneration. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of creams, emulsions, gels, liposomes, nanoparticles, or ointments.

[0026] In another aspect, a method is provided for treating bone disorders or symptoms associated with bone degeneration in subjects, the method comprising administering to the subject a vector containing an expression cassette containing a coding sequence encoding CCN3.

[0027] In some implementations, the expression box contains a promoter operatively linked to the coding sequence encoding CCN3.

[0028] In some implementations, the coding sequence for CCN3 is integrated into a chromosomal locus in the subject's genome. In some implementations, an endogenous promoter is operatively linked to the integrated coding sequence for CCN3 at the chromosomal locus.

[0029] In some implementations, the vector is a plasmid or a viral vector. Exemplary viral vectors include, but are not limited to, adeno-associated virus vectors, adenovirus vectors, lentiviral vectors, and retroviral vectors.

[0030] In some implementations, the carrier is administered intravenously or intraperitoneally.

[0031] In some implementations, the carrier is administered via the portal vein.

[0032] In some implementations, the carrier is applied locally to the bone.

[0033] In another aspect, a method is provided for providing a subject with CCN3 to promote bone growth in the subject, the method comprising introducing a vector containing a promoter operatively linked to a coding sequence encoding CCN3 into a cell, wherein the cell expresses CCN3 in vivo in the subject in an effective amount sufficient to promote bone growth in the subject.

[0034] In some implementations, the vector is introduced into cells either ex vivo or in vivo.

[0035] In some implementations, the cell is a hepatocyte or osteochondral skeletal stem cell.

[0036] In some implementations, the subject has a bone condition or symptom associated with bone degeneration, such as, but not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disease, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

[0037] In another aspect, a method is provided for stimulating osteochondral skeletal stem cells to generate bone, the method comprising contacting the osteochondral skeletal stem cells with an effective amount of CCN3, wherein bone is generated by the osteochondral skeletal stem cells.

[0038] In another aspect, a method is provided for screening CCN3 agonists that increase bone growth, the method comprising: contacting cells with CCN3 and a candidate agent, wherein the cells are osteochondral skeletal stem cells, preosteoblast cells, osteoblasts, osteoprogenitor cells, or osteosarcoma cells; and measuring bone production through the cells, wherein an increase in bone production in the presence of the candidate agent, compared to a reference range of bone production in control cells in which the candidate agent is absent, indicates that the candidate agent is a CCN3 agonist.

[0039] In another aspect, a method is provided for screening mimics or analogs of CCN3 that stimulate bone growth, the method comprising: contacting cells with a candidate agent, wherein the cells are osteochondral skeletal stem cells, pre-osteoblasts, osteoblasts, osteoprogenitor cells, or osteosarcoma cells; and measuring bone mineralization through the cells, wherein an increase in bone mineralization in the presence of the candidate agent, compared to a reference range of bone mineralization in control cells in which the candidate agent is absent, indicates that the candidate agent is a mimic or analog of CCN3.

[0040] In some embodiments, the cells used in the screening method are derived from stem cells. In some embodiments, the stem cells are adult stem cells, embryonic stem cells, or induced pluripotent stem cells. In some embodiments, the adult stem cells are mesenchymal stem cells. In some embodiments, the stem cells are derived from patients suffering from bone diseases or conditions associated with bone degeneration. In some embodiments, the cells are immortalized. In some embodiments, the cells are derived from cell lines. Exemplary cell lines include, but are not limited to, the MC3T3-E1 pre-osteoblast cell line, the SaOs2 osteosarcoma cell line, the MG-63 osteosarcoma cell line, the hFOB osteoblast cell line, the ASC52telo immortalized adipose-derived mesenchymal stem cell line, the human telomerase reverse transcriptase-immortalized bone marrow mesenchymal stromal cell (hTERT-BMSC) line, and the ATDC5 chondrogenic mouse teratogenic carcinoma cell line.

[0041] In another aspect, a method for producing bone grafts is provided, the method comprising culturing osteochondral skeletal stem cells under suitable conditions in the presence of CCN3, wherein the osteochondral skeletal stem cells produce bone for the bone graft.

[0042] In another aspect, a bone graft is provided, wherein the bone graft is generated by culturing osteochondral skeletal stem cells in the presence of CCN3 using the methods described herein.

[0043] In another aspect, a method for transplanting a bone graft into a subject is provided, the method comprising transplanting a bone graft, as described herein, into the subject's transplantation site.

[0044] In some implementations, the osteochondral stem cells used to generate bone grafts are autologous, allogeneic, or xenogeneic.

[0045] In some implementations, bone grafts replace missing bone to repair fractures at the graft site. In some implementations, the fracture is a complex fracture.

[0046] In some implementations, bone grafts provide new bone to repair congenital bone defects at the transplant site.

[0047] In some implementations, the method further includes the local application of CCN3 at the transplant site to stimulate new bone growth at the transplant site.

[0048] In another aspect, a method is provided for monitoring lactating female subjects to determine the risk of bone degeneration from breastfeeding, the method comprising: obtaining a biological sample from the lactating female subject; measuring the level of CCN3 in the biological sample; comparing the level of CCN3 in the biological sample with a reference range of CCN3 from control subjects, wherein a level of CCN3 below a threshold indicates that the subject is at risk of bone degeneration from breastfeeding, and wherein a level of CCN3 equal to or greater than the threshold indicates that the female subject can continue breastfeeding without substantial risk of bone degeneration.

[0049] In some implementations, if the level of CCN3 indicates that a female subject is at risk of bone degeneration from breastfeeding, the female subject stops breastfeeding.

[0050] In some implementations, the method further includes administering a therapeutically effective amount of CCN3 to female subjects to reduce the risk of bone degeneration from breastfeeding.

[0051] In some implementations, the biological sample is blood or plasma.

[0052] In some implementations, measuring CCN3 levels includes performing enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, immunohistochemistry, fluorescence-activated cell sorting (FACS), Western blotting, mass spectrometry, tandem mass spectrometry, biochemical assay, liquid chromatography, or NMR.

[0053] In another aspect, CCN3 was provided as a biomarker for monitoring lactating female subjects to determine the risk of bone loss from breastfeeding.

[0054] In another aspect, a method for regenerating mammalian cartilage in a subject is provided, comprising: activating skeletal stem cells with mechanical stimulation; and administering to the subject a combination of a therapeutically effective amount of cell communication network factor 3 (CCN3) and a therapeutically effective amount of a vascular endothelial growth factor (VEGF) inhibitor.

[0055] In some embodiments, the CCN3 protein comprises an amino acid sequence having at least about 80% to 100% sequence identity with the sequence of SEQ ID NO:1, including any percentage identity within that range, such as having 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0056] In some implementations, the VEGF inhibitor is cabozantinib.

[0057] In some implementations, the cartilage is articular cartilage.

[0058] In some implementations, mechanical stimulation is an acute local injury.

[0059] In some implementations, acute local injury is a surgical microfracture of bone tissue performed at the desired site for cartilage regeneration.

[0060] In some implementations, VEGF inhibitors and CCN3 are applied topically to acute local injuries.

[0061] In some implementations, VEGF inhibitors and CCN3 are administered to the subject immediately after the mechanical stimulation of skeletal stem cells.

[0062] In some implementations, VEGF inhibitors and CCN3 are administered to subjects within three days following the activation of skeletal stem cells by mechanical stimulation.

[0063] In some implementations, the VEGF inhibitor and CCN3 are encapsulated in a hydrogel.

[0064] In some embodiments, the hydrogel comprises alginate. In some embodiments, the alginate is ionicly crosslinked. In some embodiments, the alginate is ionicly crosslinked by divalent cations, including but not limited to divalent calcium cations. In some embodiments, the concentration of alginate in the hydrogel is in the range of 2 to 10 weight percent (wt%).

[0065] In some embodiments, VEGF inhibitors and CCN3 are administered using a drug delivery device. In some embodiments, the drug delivery device provides continuous delivery of VEGF inhibitors and CCN3. In some embodiments, the drug delivery device is a reservoir implant or a monolithic implant. In some embodiments, the drug delivery device is a non-biodegradable implant. In some embodiments, the drug delivery device is a biodegradable implant. In some embodiments, the drug delivery device is implanted at the site of a local acute injury.

[0066] In some implementations, the subject suffers from a cartilaginous disorder. Exemplary cartilaginous disorders include, but are not limited to, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, and malignancy.

[0067] In some implementations, the method further includes administering a therapeutically effective amount of bone stem cells to the subject.

[0068] In another aspect, a composition comprising CCN3 and a VEGF inhibitor is provided for use in a method for regenerating mammalian cartilage.

[0069] In some embodiments, the VEGF inhibitor in the composition is cabozantinib.

[0070] In some embodiments, CCN3 and VEGF inhibitors are encapsulated in a hydrogel. In some embodiments, the hydrogel contains alginate. In some embodiments, the alginate is ionicly crosslinked. In some embodiments, the alginate is ionicly crosslinked via divalent cations, including but not limited to divalent calcium cations. In some embodiments, the concentration of alginate in the hydrogel is from 2% to 10% by weight.

[0071] In another aspect, a method is provided for screening CCN3 agonists that increase cartilage production, the method comprising: activating skeletal stem cells with mechanical stimulation; contacting the skeletal stem cells with CCN3, a VEGF inhibitor, and a candidate drug; and measuring cartilage production through the skeletal stem cells, wherein the amount of increase in cartilage production in the presence of the candidate drug, compared to a reference range of cartilage production in control skeletal stem cells in which the candidate drug is absent, indicates that the candidate drug is a CCN3 agonist.

[0072] In another aspect, a method is provided for screening CCN3 mimics or analogs that stimulate cartilage production, the method comprising: activating skeletal stem cells with mechanical stimulation; contacting the skeletal stem cells with a VEGF inhibitor and a candidate agent; and measuring cartilage production through the skeletal stem cells, wherein an increase in cartilage production in the presence of the candidate agent, compared to a reference range of cartilage production in control skeletal stem cells in which the candidate agent is absent, indicates that the candidate agent is a CCN3 mimic or analog. Attached Figure Description

[0073] Figures 1A to 1I Brain-dependent circulating factors function as hormones involved in bone synthesis. Figure 1AThis demonstrates stereotactic deletion of ERα in ARC using the AAV2-Cre vector and the ERα-control virus injected with AAV2. ARC Females and ERα-KO ARC A schematic diagram of a representative µCT scan of the distal femur in a female, as previously reported. 1 . Figure 1B From Esr1 Nkx2.1-Cre Esr1 Kiss-Cre and Esr1 Pdyn-Cre µCT images of mice aged 4.5–5 weeks, with BV / TV indicated in the lower right corner. Figure 1C Esr1 fl / fl (WT) and Esr1 Nkx2.1-Cre Timeline of in vivo µCT imaging of (mutant) female mice after postoperative pairing. Figure 1D Representative in vivo µCT images of the distal femur at baseline (week 0) and 3 weeks later (week 3), showing BV / TV. Figure 1E The bar chart shows the percentage change in BV / TV% in weeks 3, 6, and 17 compared to week 0. Figure 1F Esr1 in WT:WT (black) and WT:mutant pairs (red) fl / fl Absolute BV / TV% plotted for females shows the values ​​for each animal at baseline (0) and 17 weeks later (17), (N = 5WT:5WT, 5WT:5Mut). Figure 1G Wild-type female and male bone grafts to Esr1 fl / fl (WT) and Esr1 Nkx2.1-Cre A schematic diagram of a 6-week-old female mutant mouse. Figure 1H Representative images of µCT scans of control femurs transplanted into WT (WT:WT) or mutant females (WT:mutant). Figure 1I Ported to Esr1 fl / fl Removal of bone from females (black pillars), or transplantation into Esr1 Nkx2.1-Cre Bone volume fraction of female bone (red column) or male bone (blue column) in females (N = 4–6). Two-way ANOVA with repeated measures in Figure e (left) and one-way ANOVA (Šidák multiple comparison test) in Figure f (right). Unpaired Student's t-test, two-tailed in Figure i, *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Error bars ± SEM.

[0074] Figures 2A to 2K Brain-dependent bone factor increases the osteogenic capacity of ocSSCs. Figure 2AA schematic diagram showing the FACS-separation and fate of ocSSC (left) and pvSSC (right) using the listed cell surface markers. Figure 2B Ported to Esr1 fl / fl and Esr1 Nkx2.1-Cre A schematic diagram of wild-type female ocSSCs (approximately 15,000 live cells) in the renal capsule of a female mouse. Figure 2C Image of the outline of the graft region with host-derived hematopoietic characteristics (top image, white arrow). Volumetric bone density image (left image) and slices of graphic regions stained with mineralized bone (yellow), cartilage (blue), and bone marrow (red) in the right image. Figure 2D Fractional areas of bone marrow, cartilage, and bone quantified from stained sections of individual kidney grafts (N = 6, 5), and Figure 2E Bone density of individual cohorts (N = 4, 4). Figure 2F FACS-purified control ocSSCs (approximately 550 live cells) were obtained from Esr1. fl / fl-CAG-Luc,-GFP Stereoscopic positioning and bilateral delivery to Esr1 fl / fl and Esr1 Nkx2.1-Cre A schematic diagram of the MBH in a female mouse. Figure 2G Six weeks after injection, Pentachrome (top) with representative images of anti-GFP ossicles from imaging brain (bottom left) and stained brain slices (bottom right). Figure 2H Esr1 fl / fl (Black) and Esr1 Nkx2.1-Cre (Red) Measured volumetric bone mineral density of small bones in the MBH of female mice. Figure 2I Esr1 from 3 weeks old (N = 3, 3) and 10 weeks old females (N = 5, 5) and males (5, 3) fl / fl and Esr1 Nkx2.1-Cre The percentages of FACS-purified ocSSC, pvSSC, and APC described in the separation method are shown in the legend of the bar chart above. Figure 2J ocSSCs isolated from aggregates of 3-week-old and 10-week-old females differentiated in defined media and stained with Alizarin Red (left panel) or Alcian Blue (right panel), with bright-field images from representative wells. Figure 2K ), including staining with Oil Red O (n = 3, 3 in each group). Figure 2D , Figure 2I and Figure 2J One-way ANOVA (Šidák's multiple comparison test) in [the context of the test]. Unpaired Student's t-test. Figure 2E , Figure 2H and Figure 2ITwo-tailed (3 weeks). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error bars ± SEM.

[0075] Figures 3A to 3G Identification of CCN3 as a candidate brain-derived bone anabolic factor. Figure 3A Feeding Esr1 to SD or HFD for 17 weeks fl / fl and Esr1 Nkx2.1-Cre Bone volume fraction, mechanical strength, and BMAT level in long bones of females (N = 4-6 per group). Figure 3B Representative images of the tibia stained with TRAP (top row), calcein / alizarin red (middle row), and osmium (bottom row); lipid droplets (yellow arrows). Figure 3C Normalized readings of candidate genes; Penk in the pituitary gland (N = 2-4). Figure 3D 12-week-old (adapted from RE) and 27-week-old Esr1 fed SD or HFD. Nkx2.1-Cre Heatmap of the highest DEG change in females; scale is a multiple of Log2. Figure 3E The transcriptional levels of Ccn3 and Penk in mutant female ARC were determined by qPCR. Figure 3F From Esr1 fl / fl Female (10 weeks) and Esr1 Nkx2.1-Cre Staining of ERα (pink) and CCN3 (green) in brain slices of the post-ARC and SCN regions of females and males (12 weeks), scale bar = 100 µm and 200 µm. Figure 3G Esr1 Nkx2.1-Cre The merging of CCN3 and KISS1 in female ARC. Figure 3A and Figure 3C One-way ANOVA (Šidák's multiple comparison test) in [the context of the test]. Unpaired Student's t-test. Figure 3E Two-tailed. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error bar ± SEM. Abbreviations: ME (Median Eminence), ARC (Arcuate Nucleus), 3V (Third Ventricle), SCN (Suprachiasmatic Nucleus), oc (Optic Chiasm).

[0076] Figures 4A to 4J Independent of sex and age, CCN3 promotes new bone formation through enhanced osteogenic activity of ocSSC in mice and humans. Figure 4ACompared with Esr1 fl / fl Bone volume fraction of female (red circle) and male (blue circle) femurs, said femurs cultured in vitro and used from Esr1 Nkx2.1-Cre Plasma isolated from mutant females (6–12 weeks) was treated daily for 5 days; the contralateral femur was treated with control plasma (white circle) (N = 19, 10). Figure 4B The data from Figure a show the percentage change in bone volume of the contralateral female (red column) or male (blue column) femur after the addition of the mutant, relative to control plasma. Figure 4C Representative µCT images of female and male femur scans with corresponding BV / TV% processed with plasma. Figure 4D Bone volume fraction of female (N=11) and male (N=8) control femurs treated daily with recombinant mouse CCN3 (mCCN3, 3.0 nM) compared to untreated baseline controls (baseline). Figure 4E The percentage change in bone volume of female (red column) or male (blue column) femurs treated with CCN3 or saline was recorded and then compared with the baseline value of freshly isolated and stored contralateral femurs (N = 5-10). Figure 4F Representative images of the femur at baseline and after H&E staining with CCN3. Figure 4G After daily injections of CCN3 (7.5 µg / kg intraperitoneally) or saline for 21 days, Esr1 fl / fl The percentage change in BV / TV% and trabecular thickness in females and males. All data were normalized relative to the mean of control females injected with saline only (N = 6, 8 for females and N = 7, 6 for males). Figure 4H Representative µCT images of the femur from female and male patients with corresponding BV / TV% treatments. Figure 4I Osteogenic differentiation assays were performed on purified mouse ocSSCs treated with recombinant mouse (m)CCN3 protein or Penk-encoded met-ENK and Bam22P peptides. Plotted values ​​were normalized relative to alizarin staining of control wells in defined media only, set to 100%, with bright-field images of the wells in the absence or presence of mCCN3 shown below (n = 3 for each condition). Figure 4J Osteogenic differentiation assay of purified human ocSSCs treated with recombinant human (h)CCN3 protein. Plotted values ​​were normalized relative to alizarin staining of control wells in defined media only, set to 100%, and representative images of wells treated with or without mCCN3 are shown below (n = 3 for each condition). One-way ANOVA (Šidák multiple comparison test) in Figures e, k, and i. Paired-student t-test. Figure 4A and Figure 4DThe double tail and Figure 4B and Figure 4G Unpaired Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error bars ± SEM.

[0077] Figures 5A to 5I Brain-derived CCN3 drives higher bone mass and is activated in lactating females during a sudden drop in estrogen. Figure 5A , with from Esr1 fl / fl Bone volume and dynamic histomorphometry of female mice transduced with the lowest dose of AAVdj-CAG-Ccn3 viral vector after double labeling with calcein and alizarin red (5 days apart) compared to the control vector (black) obtained from the femur of control females. Figure 5B Bone formation rate and mineral deposition rate (MAR) from dynamic histomorphometric measurements, with representative images of the femoral sections described above from elderly Esr1 patients injected with AAVdj-CAG-CCN3. fl / fl Female (20-23 months old). Figure 5C BV / TV (%) from plotted µCT and bone formation rate (BFR), as determined by dynamic histomorphometry and assessed by calcein (green) and alizarin red (red). Low dose (N = 4, 6, high dose N = 7, 8). Figure 5D siRNA targeting mCcn3 was injected into mutant Esr1. Nkx2.1-Cre In the ARC of mutant females. Figure 5E Ccn3 expression in post-ARC was shown for disordered control oligonucleotides, unilateral hits, and optimal knockdown levels. Scale bar = 100 µm. The figure below shows the corresponding µCT scans of the sagittal and longitudinal sections of the distal femur; complete deletions were added to the control group (N = 4, 4). Figure 5F XY plot of the number of CCN3 positive neurons and bone volume fraction in ARC. Figure 5G Representative images of coronal brain slices stained for ERα (pink) and CCN3 (green) in the post-ARC region of females during pregnancy or at different postpartum stages as indicated; higher resolution images are shown in the lower row (N ≥ 2). Scale bar = 100 µm for the top row and 50 µm for the bottom row. Figure 5H From Esr1 fl / fl Non-reproductive females, Esr1 Nkx2.1-Cre Non-fertile females and Esr1 fl / flQuantification of Ccn3 transcripts in microdissected ARC tissues obtained from lactating (7DPP) females (N = 5, 3, 3). Figure 5I A schematic diagram of brain-derived CCN3 as a bone anabolic hormone, which counteracts the catabolic effect of mammary PTHrP to maintain adequate calcium and protect maternal bones during lactation when E2 has disappeared. Figure g shows a one-way ANOVA (Šidák multiple comparison test). Figure 5B Unpaired Student's t-test. *p < 0.05, **p < 0.01, ****p < 0.0001, ns = not significant. Error bars ± SEM. Abbreviations: ME (median eminence), ARC (arctic nucleus), 3V (third ventricle).

[0078] Figures 6A to 6E In a genetic model of KNDy ARC neurons, sex-dependent high bone mass occurs before puberty. Figure 6A Representative images of the distal femur in females and males at weeks 4.5 and 6.5 using μCT imaging. Figure 6B Compared with Esr1 fl / fl and mutant Esr1 Pdyn-Cre Box-and-whisker diagrams of structural skeletal parameters for (mutant) females (red) and males (blue); legend at top. Figure 6C Esr1 starting from 1 week old fl / fl (Black) or Esr1 Nkx2.1-Cre (Red) Time progression of high bone mass in females, data at the 4.5-week time point were taken from (RE) and replotted (N = 4 for all groups except for the 4-week-old mutant N = 2), legend at top. Figure 6D µCT imaging of 3- and 4-week-old females. Figure 6E Modified Pentachrome staining of sections from the distal femur of 4-week-old controls and mutants, with modified Movat staining showing enhanced mineralized bone (red) in the mutant femur. Figure g shows one-way ANOVA (Šidák's multiple comparison test). Figure 6B Unpaired Student's t-test. ****p < 0.0001, ns = not significant. Error bar SEM.

[0079] Figures 7A to 7E In parabiosis of WT:MUT and MUT:WT, there is an increase in trabecular bone, but no change in the weight of the whole body or other tissues. Figure 7A The bar chart shows the concentration of Evans blue in the blood of paired mice, injected into control or mutant female mice 14 days after surgery. Figure 7B Following WT:MUT pairing, Esr1 within the indicated timeframes as determined by in vivo µCT imaging.fl / fl Box plot showing the percentage change in structural bone parameters of the distal femur. Figure 7C Weight and other tissue measurements obtained 17 weeks after euthanasia (N = 6, 4). Figure 7D For example, Esr1 in MUT:WT parabionts identified by in vivo µCT scans. Nkx2.1Cre Percentage change in femur (N=5) (left figure) and bone volume fraction (BV / TV%, right figure). Figure 7E Esr1 from MUT:WT pairing Nkx2.1Cre Representative µCT images of the distal femur. Legend shown at the top. Unpaired Student's t-test for Figure a. Two-way and one-way ANOVA (Šidák multiple comparison test) for repeated measures in Figures b and d, respectively. Paired Student's t-test for ratios in Figure d (right). *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Error bars ± SEM.

[0080] Figures 8A to 8C Transplanted into mutant Esr1 Nkx2.1-Cre Wild-type femurs in females have higher bone mass. Figure 8A Images of wild-type female bones implanted into control or mutant females 6 weeks later. Figure 8B Six weeks after implantation, wild-type female femurs entered Esr1. fl / fl (Black) or Esr1 Nkx2.1-Cre (Red, N = 5, 6) Box-and-whisker plot of µCT structural parameters in females. Figure 8C Six weeks after implantation, wild-type male bone enters Esr1. fl / fl (Black) or Esr1 Nkx2.1-Cre (Blue, N = 4, 4) Box-and-whisker plots of µCT structural parameters in females. Unpaired Student's t-tests for all plots in b and c, *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Legend and plots at the top.

[0081] Figures 9A to 9E Compared with the mutant Esr1, which exhibits youthful ocSSC and increased bone mass... Nkx2.1-Cre After females share the cycle, there is an increase in SSC from control bone. Figure 9A A box diagram of live cells obtained after FACS purification, the FACS purification being as described in the method for isolating control femurs obtained from WT:WT or WT:MUT conjoined twins (N = 6, 4) or transplanted into Esr1. Nkx2.1-Cre Femur of control females (red, N = 6, 7) or males (blue, N = 4, 4), as illustrated by the top illustration. Figure 9B The bar chart shows the CFU-F from ocSSC purified from control or mutant female long bones at the indicated ages (N = 3–6). Figure 9C Differentiation of ocSSCs in defined culture media and stained with dyes, as shown at the top of representative images of culture wells (n = 3-4 replicates); AR (Alizarin Red) and AB (Alcian Blue). Figure 9D The bone volume fraction of trabecular and cortical bone, as well as other parameters, were obtained from µCT scans. Figure 9E Representative images from µCT scans of the distal femur and midshaft region of older females (≥ 52 weeks of age) (N = 3, 3). Wet Student's t-test for all images in b and c, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error bars ± SEM.

[0082] Figures 10A to 10G The scRNA sequencing of the isolated mouse OcSSCs was expected to favor osteogenic fate. Figure 10A The unbiased Leiden clustering of 264 high-quality filtered single ocSSCs (122 control and 142 mutant cells) obtained from 7-week-old female mice was analyzed by UMAP plot SmartSeq2 scRNA sequencing. Figure 10B Dot plot of cluster-specific markers. Figure 10C Heatmap of the top 50 upregulated genes in each cluster. Figure 10D With control Esr1 fl / fl (122 cells) and mutant Esr1 Nkx2.1Cre UMAP of cell annotations (142 cells). Figure 10E The distribution of genotypes within a cluster population. Figure 10F The results showed that, compared with the control ocSSC, the mutant Esr1 Nkx2.1Cre Dot plot of higher expression of anti-inflammatory and osteogenic markers in ocSSC. Figure 10G BioPlanet 2019 pathway enrichment (top three columns) and GO Biological Process 2023 ontology (bottom three columns) show overexpression of mutants relative to wild type based on the top 200 DEGs.

[0083] Figures 11A to 11G Dietary challenges, rather than high glucose, only degraded bone in mutant females without altering other metabolic parameters. Figure 11A Esr1 fl / fl and Esr1 Nkx2.1-CreXY plots of body weight versus age for age-matched female littermates fed either a Standard Breeder Chow SD diet or a High-fat Diet HFD diet for 17 weeks from 10 weeks of age (N ≥ 4 per group). Blood glucose levels, area under the curve (AUC), fat mass, and serum triglycerides after a GTT (intraperitoneal test) were plotted for control and mutant females fed HFD for 15 weeks; legend at the top. Figure 11B Trabecular and cortical bone parameters were obtained after µCT scans of four experimental female cohorts. Figure 11C For Esr1 fl / fl and Esr1 Nkx2.1-Cre Representative images of sections of the femur, stained with TRAP and double-labeled with calcein green (green) and alizarin red (red); in the main Figure 3B The yellow square area is shown in the image. Figure 11D Dynamic histomorphometrics of tibia from four different experimental cohorts: osteocytes per bone surface (Oc / BS), bone formation rate per bone surface (BFR / BS), and mineralized surface per bone surface (MS / BS), with N = 4 per cohort. Figure 11E Esr1 fl / fl and Esr1 Nkx2.1-Cre XY plot of body weight versus age for an age-matched male experimental cohort (N = 4 per group). Figure 11F Trabecular and cortical bone parameters obtained from four male experimental cohorts via µCT imaging. Legend at the top. Figure 11G Esr1 cells obtained by µCT imaging and treated with media (N = 3, 6) or S961 (N = 5, 5) fl / fl and Esr1 Nkx2.1 -Cre Blood glucose and structural bone parameters in the female cohort were measured, with the medium or S961 delivered via an implanted osmotic pump filled with 40 nM naloxone over an 8-week timeframe (N = 5–6 per cohort). Two-way ANOVA (Šidák multiple comparison test) with repeated measures was performed on the plots (BW curves and GTT) in a and e, respectively. Figure 11B , Figure 11D , Figure 11F and Figure 11GOne-way ANOVA (Šidák multiple comparison test), unpaired Student's t-test in Figure a. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001, ns = not significant. Error bars ± SEM.

[0084] Figures 12A to 12C A cluster of ARC genes and female Esr1 Nkx2.1-Cre The mutation is associated with changes in bone mass. Figure 12A The relative expression of transcripts listed in the female hypothalamus at 2.5 weeks of age is shown in a bar chart of individual points (N = 4-8). Figure 12B From the control Esr1 fl / fl and mutant Esr1 Nkx2.1-Cre Relative expression of transcripts in microcut ARC harvested from age-matched females (red) or males (blue) (N=2–5). Figure 12C In the case of Esr1 fl / fl and Esr1 Nkx2.1-Cre Analysis of a batch RNA-Seq dataset of age-matched female littermates yielded heatmaps of the top 50 DEGs listed on the right. These littermates were fed a standard breeding diet (SD) or a high-fat diet (HFD) for 17 weeks starting at 10 weeks of age. Samples included microdissected ARC (left panel), the entire pituitary gland (middle panel), or liver tissue (right panel). Secretory protein / peptide clusters of ARC attenuated via HFD are highlighted in red text. The legend for each heatmap shows the relative Z-score. Figure 12A and 12B Unpaired Student's t-test. ns = not significant. Error bar ± SEM.

[0085] Figures 13A to 13E Low-dose CCN3 increased bone mass in control bones cultured in petri dishes and in mice after daily injections. Figure 13A A bar chart of changes in bone volume fraction from the entire femur, harvested from control females and then compared with that from Esr1. fl / fl and Esr1 Nkx2.1-Cre Age-matched females were cultured together with plasma isolated from their litters. Plasma (15 μl) was added daily for 1–7 days as described in the method. Figure 13B The bone volume fraction was determined after culturing the right tibia and right femur in a medium treated with 0.9% NS (saline). Baseline values ​​were obtained from freshly isolated left tibia or left femur from the same mouse that was immediately fixed in 4% PFA for analysis without culture (baseline). Figure 13C H&E staining of contralateral left and right femurs from sections of the same female and male mice in baseline or saline. Figure 13DBox-and-whisker diagrams of bone parameters in females after daily treatment with saline (black) or CCN3 (red). Figure 13E The efficacy of naloxone chronic infusion over 28 days, in control Esr1 infants aged 10–12 weeks at the start of treatment with either the medium delivered via an implanted microosmotic pump (0.5 mg / 24 h) or naloxone over 28 days. fl / fl and mutant Esr1 Nkx2.1-Cre Bone volume fractions were plotted for age-matched females. Legend is above the bar chart (N = 4 per group). Figure 13B Paired student t-test and Figure 13D The unpaired student t-test. Figure 13E One-way ANOVA (Šidák multiple comparison test), *p < 0.05, **p < 0.01, ns = not significant, ns = not significant. Error bars ± SEM.

[0086] Figures 14A to 14D Low-dose CCN3 increases bone formation in ocSSCs harvested from both young and older humans, whether male or female. Figure 14A Normalized alizarin staining of cultured human ocSSCs harvested from growth plates of 14-year-old males and treated with recombinant protein or purified peptides, with dosages provided and differentiation assays described in the methods section. Data for CCN3 were redrawn from the left plot of main plot 4j for comparison. Dashed lines represent 100% values ​​of cells cultured alone in osteogenic medium. Figure 14B ,and Figure 14A The difference is that the human ocSSC was harvested from a fracture site in a 15-year-old male. Figure 14C Representative images of two wells stained with alizarin and different doses of human CCN3 in control culture medium and osteogenic culture medium, with one well magnified in the rightmost image of each figure. Figure 14D Representative images of two wells stained with alizarin in a culture medium containing osteogenic defined medium minus or with human CCN3. Figure 14A , Figure 14B One-way ANOVA (Šidák multiple comparison test for control wells, black), *p < 0.05, ***p < 0.001, ****p < 0.0001, ns = not significant. Error bars ±SEM.

[0087] Figures 15A to 15C Compare with Esr1 fl / fl Ectopic hepatic mCCN3 expression in females increases bone formation. Figure 15AExpression of mCCN3 protein in female liver transduced with a low dose of AAVdj-CAG-Ccn3 two weeks after injection; the right-hand image represents a digitally magnified image of a single positive cell. Scale bar = 100 µm. Figure 15B In the low dose (5*10 9 ) or high dose (3*10) 10 AAVdj-CAG-Ccn3 viral vector (green column) and control AAVdj-empty vector (black column) from GC / mouse mice were transduced into Esr1. fl / fl The relative levels of Ccn3 transcripts in liver tissue of female littermates 5 weeks after injection (for lower titers, N = 4, 6, 6–8 months of age, and for higher titers, N = 7, 8, 3 months of age). Figure 15C In Esr1 infants aged 6-8 months fl / fl Dynamic histomorphometry of bone parameters in the femur of control females using double-labeled calcein and alizarin red. Unpaired Student's t-tests for low-dose and high-dose groups in Figures b and c. *p < 0.05, **p < 0.01, ****p < 0.0001. Error bars ± SEM.

[0088] Figure 16 Bone regeneration in mice following fracture. CCN3 was administered as a slow-release gel to the fracture site in 24-month-old male mice (NIA C57BL / 6). Images obtained 21 days post-fracture showed an approximately 400% increase in bone at the fracture site in 24-month-old mice treated with 2 µg of CCN3.

[0089] Figures 17A to 17B Cartilage regeneration via growth factor-enhanced microfractures (GEMs). Figure 17A Safranin O staining of distal femoral sections from undamaged and GEM-treated mice 4 weeks post-surgery. Mice were treated with cabozantinib, CCN3, or a combination of cabozantinib and CCN3, and compared with mice receiving PBS as a control. Injury sites are marked with black dashed lines. Figure 17B The percentage of regenerated area stained for cartilage is used to quantify the area. Data are presented as mean + / - SEM. N=3 per group. One-way ANOVA with Fisher LSD post-hoc test was used. Detailed Implementation

[0090] Methods for treating bone or cartilage disorders using CCN3 are provided. Gene therapy methods are also provided, including methods for expressing CCN3 in vivo at an effective amount sufficient to promote new bone or cartilage growth. Furthermore, methods for screening agonists, mimics, and analogs of CCN3 are provided.

[0091] Before describing exemplary embodiments of the invention, it should be understood that the invention is not limited to the specific embodiments described, and therefore variations are possible. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0092] Where a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value between the upper and lower limits of the range, up to one-tenth of the lower limit unit, is also specifically disclosed. Every smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within the stated range is encompassed within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and every range in which any, but not one or both, limits are included is also encompassed within this invention, subject to any specific exclusion of limits within the stated range. Where the stated range includes one or two limits, ranges excluding one or both of these included limits are also included in this invention.

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described and used herein may be used in the practice or testing of this invention, some potential and exemplary methods and materials are described hereafter. Any and all publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials relating to the cited publications. It should be understood that, to the extent that there is a conflict, this disclosure supersedes any disclosure in the incorporated publications.

[0094] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “cell” includes a plurality of such cells, and reference to “peptide” includes reference to one or more peptides and their equivalents, such as polypeptides and peptides known to those skilled in the art, etc.

[0095] It should also be noted that claims can be drafted to exclude any elements that may be optional. Therefore, this statement is intended to serve as a precondition for using exclusive terms such as "uniquely" or "only" in relation to the description of the elements of the claim, or for using the "negative" limitation.

[0096] The publications discussed herein are provided only for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not qualified to precede such publications by virtue of a prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification. To the extent that such publications may set forth definitions of terms that conflict with the express or implied definitions of this disclosure, the definitions of this disclosure shall prevail.

[0097] As will be readily apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any described method may be performed in the order of the described events or in any other logically possible order.

[0098] definition

[0099] The term "bone condition or symptom associated with bone degeneration" is used herein to refer to any disease or symptom that causes decreased bone mineral density, reduced bone mass, bone fragility, low bone mineral density, and / or increased fatty bone marrow. Bone conditions and symptoms associated with bone degeneration include, but are not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

[0100] The term "cartilage disorder" is used in this document to refer to any disease or condition that causes damage or loss of cartilage. Cartilage disorders include, but are not limited to, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, and malignancy.

[0101] The terms “treatment,” “treating,” “treat,” etc., are generally used herein to refer to the attainment of a desired pharmacological and / or physiological effect. This effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and / or therapeutic in relation to the partial or complete stabilization or cure of a disease and / or adverse effects attributable to the disease. The term “treatment” encompasses any treatment of a disease in mammals, particularly humans, including: (a) preventing the development of the disease and / or symptoms in a subject who may be susceptible to the disease or symptoms but has not yet been diagnosed with the disease or symptoms; (b) suppressing the disease and / or symptoms, i.e., preventing their development; and / or (c) alleviating the symptoms of the disease, i.e., causing the remission of the disease and / or symptoms. Those who require treatment include those who already have the disease (e.g., those with a bone or cartilage condition or symptom associated with bone or cartilage degeneration) and those who require prevention (e.g., those with an increased susceptibility or genetic predisposition to the development of a bone or cartilage condition or symptom associated with bone or cartilage degeneration).

[0102] Therapeutic treatment is treatment in which the subject is ill prior to administration, while prophylactic treatment is treatment in which the subject is not ill prior to administration. In some embodiments, the subject is more likely to become ill or be suspected of having the disease before treatment. In some embodiments, the subject is suspected of having an increased likelihood of becoming ill.

[0103] The terms “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to vertebrates, including but not limited to mammals, including human and non-human mammals such as non-human primates, including chimpanzees and other ape and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domesticated animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses, and cattle; amphibians such as frogs, salamanders, and caecilians; bony fish such as ray-finned fish and lobe-finned fish; reptiles such as turtles, crocodiles, snakes, lizards, and beaked lizards; and birds such as poultry, wild birds, and game birds, including chickens, turkeys, and other quail birds, ducks, and geese. In some cases, the subject approach is used for laboratory animals, veterinary applications, and the development of animal models for disease, including but not limited to rodents, including mice, rats, and hamsters, primates, and transgenic animals.

[0104] "Pharmaceutical acceptable excipients or carriers" means excipients that may be optionally included in the compositions of the present invention and will not cause significant adverse toxicological effects on patients.

[0105] "Pharmaceutically acceptable salts" include, but are not limited to, amino acid salts, salts prepared with inorganic acids such as chlorides, sulfates, phosphates, diphosphates, bromides, and nitrates, or salts prepared from any of the aforementioned corresponding inorganic acid forms, such as hydrochlorides, or salts prepared with organic acids such as malates, maleates, fumarates, tartrates, succinates, ethylsuccinates, citrates, acetates, lactates, methanesulfonates, benzoates, ascorbic acid salts, p-toluenesulfonates, palmitates, salicylates, and stearates, as well as etolates, glucono-p-ethylates, and lacturonates. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0106] The term "therapeutic effective dose or amount" of the CCN3 protein or a recombinant polynucleotide containing a coding sequence encoding the CCN3 protein is intended to refer to the amount that, when administered as described herein, produces a positive therapeutic response, such as recovery from improvement in bone or cartilage conditions or lesions associated with bone or cartilage degeneration. Recovery from improvement in bone conditions may include increased bone mineral density, increased bone mass, increased bone strength, increased bone mineral density, and / or reduced adipose bone marrow. Further, the therapeutic effective dose or amount may stimulate osteochondral skeletal stem cells to generate bone.

[0107] The term "therapeutic effective dose or amount" for CCN3 and VEGF inhibitors is intended to refer to the amount that produces a positive therapeutic response, such as improvement in cartilage damage, when administered in combination with mechanical stimulation as described herein. Improvement in cartilage damage may include the generation of new cartilage at the site of treatment (e.g., the affected joint). For example, a therapeutic effective dose or amount may be used to treat cartilage damage or loss caused by traumatic injury or degenerative diseases such as arthritis or other diseases involving cartilage degeneration. Preferably, the therapeutic effective amount restores function and / or relieves pain and inflammation associated with cartilage damage or loss.

[0108] In some embodiments, an effective amount of CCN3 protein or a recombinant polynucleotide containing a coding sequence encoding CCN3 protein, when administered alone or in combination with a VEGF inhibitor and optionally other factors, will increase bone or cartilage mass by at least about 5%, at least about 10%, at least about 20%, preferably about 20% to about 50%, and even more preferably greater than 50% (e.g., about 50% to about 100%), compared to an appropriate control, wherein the control is typically a subject not treated with the composition.

[0109] The term "microfracture" refers to a surgical technique that creates small fracture holes in the bone using tools such as picks, awls, or drills. Microfracture surgery was developed to treat cartilage defects, which are damaged areas of articular cartilage in the knee. It is typically used to treat patients with damage to the full thickness of the articular cartilage, extending into the bone. Microfractures have been used on joints, including but not limited to the shoulder, hip, ankle, and knee joints.

[0110] Microfractures are typically performed as part of arthroscopic surgery. The area undergoing microfracture is usually prepared by removing any loose or damaged cartilage. Ideally, the area undergoing microfracture will be less than about 2 cm in diameter and have good, healthy surrounding cartilage. Small picks (cones) or drills are used to create small microfracture holes in the bone. The number of microfractures created depends on the size of the joint being treated. Most patients with a 1 to 2 cm damaged area require 5 to 15 small microfracture holes in the bone. The permeability of the outer layer of bone allows blood and stem cells to form clots in the area of ​​cartilage loss.

[0111] Articular cartilage is highly specialized connective tissue in moving joints. Its main function is to provide a smooth, lubricated surface for the joint and facilitate load transmission with a low coefficient of friction. Articular cartilage lacks blood vessels, lymphatic vessels, and nerves and is subjected to harsh biomechanical environments. It possesses a limited capacity for intrinsic healing and repair. In this respect, the protection and health of articular cartilage are crucial for joint health.

[0112] The surfaces of articular bones in mammalian joints are covered with articular cartilage. Articular cartilage prevents direct contact between opposing bone surfaces and allows for movement of the joint bones relative to each other with minimal friction. Two types of articular cartilage defects are commonly observed in mammals, including full-thickness defects and partial-thickness defects. These two types of defects differ not only in the degree of physical damage but also in the nature of the repair response elicited by each similar lesion.

[0113] Full-thickness articular cartilage defects involve damage to the articular cartilage, the underlying subchondral bone, and the calcified layer of cartilage between the articular cartilage and the subchondral bone. Full-thickness defects typically occur during severe joint trauma or in the later stages of degenerative joint diseases, such as osteoarthritis. Damage to the subchondral bone is often painful because it is both innervated and vascularized. The repair response to subchondral bone damage usually results in the formation of fibrocartilage at the site of the full-thickness defect. However, fibrocartilage lacks the biomechanical properties of articular cartilage and cannot persist in the joint for long periods.

[0114] Partial-thickness articular cartilage defects are limited to the cartilage tissue itself. These defects typically include cracks or tears in the articular surface of the cartilage. Partial-thickness defects are caused by the mechanical layout of the joint, which in turn leads to wear and tear of the intra-articular cartilage tissue. In the absence of nerve innervation and vascular systems, partial-thickness defects do not elicit a repair response and therefore tend to be non-healing. Although painless, partial-thickness defects often degenerate into full-thickness defects.

[0115] Articular cartilage is hyaline cartilage, typically 2 to 4 mm thick. It consists of a dense extracellular matrix (ECM) and sparsely distributed chondrocytes. The ECM is primarily composed of water, collagen, and proteoglycans, with other non-collagenous proteins and glycoproteins present in lower amounts. Along with the collagen fiber ultrastructure and ECM, chondrocytes also contribute to various regions of the articular cartilage—superficial, intermediate, deep, and calcified zones. Within each region, three areas can be identified—the pericellular region, the territorial region, and the interterritorial region.

[0116] A thin, superficial (tangential) zone protects the deeper layers from shear stress and constitutes approximately 10% to 20% of the articular cartilage thickness. Collagen fibers (primarily type II and type IX collagen) in this zone are tightly packed and aligned parallel to the articular surfaces. The superficial layer contains a relatively high number of flattened chondrocytes, and its integrity is essential for the protection and maintenance of the deeper layers. This zone is in contact with synovial fluid and is responsible for most of the tensile properties of the cartilage, enabling it to resist shear, tensile, and compressive forces exerted by the joint.

[0117] Adjacent to the superficial zone lies the intermediate (transitional) zone, which provides an anatomical and functional bridge between the superficial and deep zones. The intermediate zone represents 40% to 60% of the total cartilage volume and contains proteoglycans and thicker collagen fibrils. In this layer, collagen is organized at an angle, and chondrocytes are spherical and at low density. Functionally, the intermediate zone is the first line of defense against compressive forces.

[0118] The deep zone is responsible for providing the greatest resistance to compressive forces because collagen fibrils are arranged perpendicular to the joint surface. The deep zone contains the largest diameter radially arranged collagen fibrils, the highest proteoglycan content, and the lowest water concentration. Chondrocytes are typically arranged in a columnar orientation, parallel to the collagen fibers and perpendicular to the joint line. The deep zone represents approximately 30% of the articular cartilage volume.

[0119] Tide marks separate the deep zone from the calcified cartilage. Given its high proteoglycan content, the deep zone is responsible for providing the maximum resistance to compressive forces. Notably, collagen fibrils are arranged perpendicular to the articular cartilage. The calcified layer plays an indispensable role in fixing the cartilage to the bone by anchoring the collagen fibrils in the deep zone to the subchondral bone. In this zone, cell populations are sparse, and chondrocytes are hypertrophic.

[0120] Collagen is the most abundant structural macromolecule in the ECM, constituting approximately 60% of the dry weight of cartilage. Type II collagen accounts for 90% to 95% of collagen in the ECM and forms fibrils and fibrous strands interwoven with proteoglycan aggregates. Types I, IV, V, VI, IX, and XI collagen also exist, but contribute only small proportions. Minor collagens contribute to the formation and stabilization of the type II collagen fibril network.

[0121] Proteoglycans comprise 10% to 15% of the wet weight of cartilage. Articular cartilage contains a variety of proteoglycans crucial for normal function, including aggregate proteoglycans, core proteoglycans, disaccharide proteoglycans, and fibrinolytic proteins. Aggregate proteoglycans are the largest in size and the most abundant by weight.

[0122] Chondrocytes are the resident cell type in articular cartilage. They are highly specialized, metabolically active cells that play a unique role in the development, maintenance, and repair of the articular cartilage matrix (ECM). Chondrocytes have a limited replication potential, which is a factor contributing to the limited intrinsic healing capacity of cartilage in response to injury. Chondrocyte survival depends on optimal chemical and mechanical environments. Biochemical markers of chondrocytes include, but are not limited to, type II collagen, chondroitin sulfate, keratin sulfate, and characteristic morphological markers of smooth muscle, including but not limited to a round morphology observed in culture, and the ability to secrete type II collagen, including but not limited to the production of tissues or matrices with hemodynamic properties of cartilage in vitro.

[0123] Fibrocartilage is formed from acute local injury at the bone site in the absence of biochemical factors that guide cartilage formation. Its mechanical properties are inferior to those of articular cartilage. For example, markers of fibrocartilage include chondrocytes and fibrotic cells that produce proteoglycans and are positive for collagen (COL) 1 and matrix metalloproteinase (MMP) 13, but negative for COL 2.

[0124] "Proliferation" refers to cell division through mitosis, that is, undergoing mitosis. "Expanded population" is a population of cells that has proliferated (i.e. undergone mitosis), resulting in an increased number of cells compared to the initial population, i.e., a greater number of cells.

[0125] The term "organism" refers to a group or layer of similarly specialized cells that work together to perform certain specific functions.

[0126] The term "organ" refers to two or more adjacent tissue layers that maintain some form of cell-cell and / or cell-matrix interaction to form a microarchitecture.

[0127] The terms “protein,” “peptide,” and “polypeptide” refer to any compound comprising a polymer or amino acid-like molecule, whether naturally occurring or synthetic, including but not limited to compounds comprising amino and / or imine molecules. The use of the terms “protein,” “peptide,” or “polypeptide” does not imply a specific size, and these terms are used interchangeably. Included in this definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), polypeptides having substituted bonds, and other modifications of both naturally occurring and non-natural (e.g., synthetic) molecules known in the art. Thus, synthetic oligopeptides, dimers, polymers (e.g., tandemly repeated, linearly linked peptides), cyclized, branched molecules, etc., are included within this definition. The term also includes molecules comprising one or more peptide-like substances (e.g., N-substituted glycine residues) and other synthetic amino acids or peptides. (For a description of peptides, see, for example, U.S. Patent Nos. 5,831,005; 5,877,278; and 5,977,301; Nguyen et al., (2000) *Chemical Biology* 7(7):463-473; and Simon et al., (1992) *Proceedings of the National Academy of Sciences* 89(20):9367-9371). Non-limiting lengths of peptides suitable for use in this invention include peptides of 3 to 5 residues, 6 to 10 residues (or any integer therebetween), 11 to 20 residues (or any integer therebetween), 21 to 75 residues (or any integer therebetween), 75 to 100 residues (or any integer therebetween), or polypeptides of greater than 100 residues. Typically, peptides used in this invention may have the maximum length suitable for the intended application. Preferably, the length of the polypeptide is between about 3 and 100 residues. Generally, given the teachings herein, those skilled in the art can readily choose the maximum length. Furthermore, proteins, peptides, and polypeptides as described herein, such as synthetic peptides, may include additional molecules, such as markers or other chemical motifs.

[0128] Therefore, references to proteins, polypeptides, or peptides also include derivatives of the amino acid sequences of the present invention, including one or more non-naturally occurring amino acids. A first polypeptide or peptide is “derived from” a second polypeptide or peptide if (i) it is encoded by a first polynucleotide derived from a second polynucleotide encoding a second polypeptide or peptide, or (ii) it has sequence identity with the second polypeptide or peptide described herein. Sequence (or percentage) identity can be determined as described below. Preferably, the derivatives exhibit at least about 50% percentage identity with the sequence from which they are derived, more preferably at least about 80%, and even more preferably between about 85% and 99% (or any value between them). Such derivatives may include post-expression modifications of the polypeptide or peptide, such as glycosylation, acetylation, phosphorylation, etc.

[0129] Amino acid derivatives may also include modifications to the native sequence, such as deletions, additions, and substitutions (generally conserved in nature), as long as the protein (or fragments thereof) retains the desired activity (e.g., the CCN3 bioactivity, the ability to increase bone density, bone mass, bone strength, and / or the rate of bone formation, and / or the ability to increase cartilage growth, the rate of cartilage formation, and / or the ability to regenerate cartilage). These modifications can be intentional, such as through site-directed mutagenesis, or can be accidental, such as through mutations in the host producing the protein or due to errors in PCR amplification. Furthermore, modifications can be made that have one or more of the following effects: increasing the rate of bone density, bone mass, bone strength, and / or bone formation, and / or increasing the rate of cartilage growth, the rate of cartilage formation, and / or the ability to promote purification, delivery, or cellular processing. The protein or its bioactive fragments may be recombinantly produced, synthetically produced, or generated in tissue culture.

[0130] As used herein, the term “cell communication network factor 3” or “CCN3” encompasses all forms of CCN3 and also includes bioactive fragments, variants, analogs, and derivatives that retain biological activity (e.g., increasing bone density, bone mass, bone strength, and / or the rate of bone formation, and / or increasing the rate of cartilage growth, cartilage formation, and / or cartilage regeneration).

[0131] CCN3 polynucleotides, nucleic acids, oligonucleotides, proteins, polypeptides, or peptides refer to molecules derived from any source. The molecule does not need to be physically derived from an organism, but can be produced synthetically or recombinantly. Many CCN3 nucleic acid and protein sequences are known. Representative sequences of the human CCN3 protein (SEQ ID NO: 1), the genomic sequence of the CCN3 gene (SEQ ID NO: 2), and the CCN3 mRNA (SEQ ID NO: 3) are presented in the sequence listing. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession numbers NM_002514, NG_009779, NM_010930.5, NM_002514, NM_030868, NM_205268, XM_058698906, XM_004580701, XM_046465005, XM_038555394, XM_057736139, XM_057791994, NP_002505, NP_035060, NP_110495, NP_990599, XP_03737995, X P_026919801, XP_032981561, XP_025306624, NP_001253825, XP_036292740, XP_010970891, XP_005564036, XP_004743541, XP_004000129, XP_037748011, XP_043457574, XP_038246527, XP_038540833 and XP_532317; all these sequences (as entered prior to the filing date of this application) are incorporated herein by reference. Any of these sequences or variants thereof containing a sequence having at least about 80% to 100% sequence identity with it, including any percentage identity within that range, such as having 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, wherein the variant CCN3 protein retains the biological activity of CCN3 (i.e., the ability to increase bone density, bone mass, bone strength, and / or the rate of bone formation, and / or the ability to regenerate cartilage, increase cartilage mass, and / or increase the rate of cartilage formation), and may be used to generate the CCN3 protein or a recombinant polynucleotide containing a coding sequence encoding the CCN3 protein, for use in the methods described herein.

[0132] "Fragment" is intended to refer to a molecule consisting only of a portion of the complete full-length sequence and structure. Fragments may include C-terminal deletions, N-terminal deletions, and / or internal deletions of peptides. An active fragment of a particular protein or peptide will typically consist of at least about 5-14 consecutive amino acid residues of the full-length molecule, but may include at least about 15-25 consecutive amino acid residues of the full-length molecule, and may include at least about 20-50 or more consecutive amino acid residues of the full-length molecule, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains biological activity (e.g., CCN3 biological activity, the ability to increase bone mineral density, bone mass, bone strength, and / or the rate of bone formation, and / or the ability to regenerate cartilage, increase cartilage mass, and / or increase the rate of cartilage formation).

[0133] "Substantially purified" typically refers to the separation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance constitutes the majority of the sample in which it resides. Typically, the substantially purified component constitutes 50% of the sample, preferably 80%-85%, more preferably 90%-95%. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and density-based sedimentation.

[0134] When referring to proteins, polypeptides, or peptides, "isolated" means that the molecule in question is separate and discrete from the entire organism in which it is found in nature, or exists in the absence of other biological macromolecules of the same type. The term "isolated" for polynucleotides refers to nucleic acid molecules that are wholly or partially lacking the sequence normally associated with them in nature; or those with the sequence they are found in nature but with a heterologous sequence associated with them; or molecules dissociated from chromosomes.

[0135] The term “derived from” is used in this article to identify the original source of a molecule, but does not imply limitation on methods that may, for example, be used to prepare the molecule by chemical synthesis or recombination.

[0136] The terms “variant,” “analyte,” and “mutant protein” refer to a biologically active derivative of a reference molecule that retains the desired activity, such as CCN3 biological activity (e.g., the ability to increase bone mineral density, bone mass, bone strength, and / or the rate of bone formation, and / or the ability to regenerate cartilage, increase cartilage mass, and / or increase the rate of cartilage formation), as described herein. Generally, the terms “variant” and “analyte” refer to a compound having the native polypeptide sequence and structure, having one or more amino acid additions, substitutions (generally conserved in nature), and / or deletions relative to the native molecule, provided that such modification does not impair biological activity and that it is “substantially homologous” to the reference molecule as defined below. Generally, the amino acid sequence of such analogs will have a high degree of sequence homology with the reference sequence, for example, more than 50%, typically more than 60%–70%, and even more specifically 80%–85% or more, such as at least 90%–95% or more amino acid sequence homology when the two sequences are aligned. Typically, analogs will comprise the same number of amino acids but will include substitutions, as explained herein. The term "mutant protein" further includes polypeptides having one or more amino acid-like molecules, including but not limited to compounds containing only amino and / or imino molecules, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids), polypeptides having substituted bonds, and other modified, cyclized, branched molecules, etc., both naturally occurring and non-natural (e.g., synthetic) known in the art. The term also includes molecules containing one or more N-substituted glycine residues ("peptides") and other synthetic amino acids or peptides. (For a description of peptides, see, for example, U.S. Patent Nos. 5,831,005; 5,877,278; and 5,977,301; Nguyen et al., Chemical Biology (2000) 7:463-473; and Simon et al., Proceedings of the National Academy of Sciences (1992) 89:9367-9371). Preferably, the analog or mutant protein has at least the same biological activity as the natural molecule. Methods for preparing polypeptide analogs and mutant proteins are known in the art and are further described below.

[0137] As explained above, analogues typically include inherently conserved substitutions, i.e., those occurring within the amino acid family associated with their side chains. Specifically, amino acids are generally classified into four families: (1) acidic – aspartic acid and glutamic acid; (2) basic – lysine, arginine, histidine; (3) nonpolar – alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar – glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonable to predict that a segregated substitution of leucine with isoleucine or valine, a substitution of aspartic acid with glutamic acid, a substitution of threonine with serine, or similar conserved substitutions of amino acids with structurally related amino acids will not have a significant impact on biological activity. For example, a polypeptide of interest may include up to about 5-10 conserved or non-conserved amino acid substitutions, or even up to about 15-25 conserved or non-conserved amino acid substitutions, or any integer between 5 and 25, as long as the desired function of the molecule remains intact. Those skilled in the art can readily identify regions of the molecule of interest that can tolerate variations by referring to the well-known Hopp / Woods and Kyte-Doolittle diagrams.

[0138] "Derivative" is intended to refer to any suitable modification of a natural polypeptide, a fragment of a natural polypeptide, or a corresponding analogue of interest, such as glycosylation, phosphorylation, polymer conjugation (such as conjugation with polyethylene glycol), or other addition of a foreign moiety, as long as the desired biological activity of the natural polypeptide is preserved. Methods for preparing polypeptide fragments, analogues, and derivatives are generally available in the art.

[0139] "Homology" refers to the percentage of identity between two polynucleotide molecules or two polypeptide molecules. Two nucleic acid sequences or two polypeptide sequences are "substantially homologous" to each other when they exhibit at least about 50% sequence identity over the defined length of the molecule, preferably at least about 75% sequence identity, more preferably at least about 80% or 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% or 98% sequence identity. As used herein, substantially homologous also means a sequence that shows complete identity with a specified sequence.

[0140] Generally, "identity" refers to the exact correspondence between nucleotides or amino acids of two polynucleotide sequences or two polypeptide sequences. Percentage identity can be determined by directly comparing the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Easily available computer programs can be used to aid analysis, such as ALIGN, Dayhoff, MO, "Atlas of Protein Sequence and Structure," MO Dayhoff (ed.), Supplement 5, 3:353-358, National Biomedical Research Foundation, Washington, DC, which uses the local homology algorithm for peptide analysis developed by Smith and Waterman in Advances in Appl. Math., 2:482-489, 1981. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI), such as the BESTFIT, FASTA, and GAP programs, which also rely on the Smith and Waterman algorithms. These programs are easily used with the manufacturer-recommended default parameters and are described in the Wisconsin Sequence Analysis Package mentioned above. For example, using the Smith and Waterman homology algorithm, the percentage identity of a specific nucleotide sequence to a reference sequence can be determined using a default scoring table and a gap penalty at six nucleotide positions.

[0141] Another method for establishing percentage identity in the context of this invention is to use the MPSRCH package, copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and published by IntelliGenetics (Mountain View, CA). From this package, the Smith Waterman algorithm can be employed, with default parameters used for the scoring table (e.g., a gap opening penalty of 12, a gap extension penalty of one, and a gap of six). From the generated data, the "match" value reflects "sequence identity." Other suitable procedures for calculating percentage identity or similarity between sequences are generally known in the art; for example, another alignment procedure is BLAST, which uses default parameters. For example, the following default parameters can be used to use BLASTN and BLASTP: Genetic Code = Standard; Filter = None; Chain = Both; Cutoff = 60; Expected Value = 10; Matrix = BLOSUM62; Description = 50 sequences; Sort By = High Score; Database = Non-Redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS Translation + Swiss Protein + Spupdate + PIR. Details of these procedures are readily available.

[0142] Alternatively, homology can be determined by hybridizing polynucleotides under conditions that form stable double strands between homologous regions, followed by digestion with a single-strand-specific nuclease, and determination of the size of the digested fragments. Essentially homologous DNA sequences can be identified in Southern hybridization experiments, for example, under stringent conditions defined for a particular system. Defining suitable hybridization conditions is within the scope of the art. See, for example, Sambrook et al., (2001), *Molecular Cloning, a Laboratory Manual* (3rd ed., Cold Spring Harbor Laboratories, NY); *DNA Cloning*, ibid.; *Nucleic Acid Hybridization*, ibid.

[0143] As used herein to describe nucleic acid molecules, "recombinant" refers to genomic, cDNA, viral, semi-synthetic, or synthetically derived polynucleotides that, due to their origin or manipulation, are not associated, in whole or in part, with all or part of the polynucleotides that are naturally associated with them. As used with respect to proteins or polypeptides, "recombinant" refers to polypeptides produced through the expression of recombinant polynucleotides. Generally, the gene of interest is cloned and then expressed in a transformed organism, as further described below. The host organism expresses the foreign gene under expression conditions to produce a protein.

[0144] The term "transformation" refers to the insertion of exogenous polynucleotides into host cells, regardless of the method used for insertion. Examples include direct uptake, transduction, or f-crossing. Exogenous polynucleotides may remain as non-integrating vectors, such as plasmids, or alternatively, may be integrated into the host genome.

[0145] "Recombinant host cell," "host cell," "cell," "cell line," "cell culture," and other such terms referring to microorganisms or higher eukaryotic cell lines cultured as single-celled entities refer to cells that can or have been used as recipients of recombinant vectors or other transferred DNA, and include the original progeny of the original transfected cells.

[0146] A “coding sequence” or “encoding” a selected polypeptide is a nucleic acid molecule that, when placed under the control of an appropriate regulatory sequence (or “control element”), is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo. The boundaries of a coding sequence can be determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Coding sequences can include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence can be located at the 3' end of the coding sequence.

[0147] Typical "control elements" include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located at the 3' of the translation termination codon), sequences for optimizing translation initiation (located at the 5' of the coding sequence), and translation termination sequences.

[0148] "Operationally linked" refers to the arrangement of elements in which the components described in this way are configured to perform their normal functions. Thus, a given promoter operably linked to a coding sequence can influence the expression of that sequence when a suitable enzyme is present. The promoter does not need to be adjacent to the coding sequence, as long as it serves to guide its expression. Therefore, for example, an untranslated but transcribed sequence can exist between the promoter and coding sequences, and the promoter sequence can still be considered "operationally linked" to the coding sequence.

[0149] "Encoded by" means a nucleic acid sequence that encodes a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids, derived from a polypeptide encoded by a nucleic acid sequence.

[0150] An "expression cassette" or "expression construct" refers to a component capable of directing the expression of a sequence or gene of interest. Expression cassettes typically include control elements as described above, such as promoters operatively linked to the sequence or gene of interest (to direct its transcription), and often also include polyadenylated sequences. In some embodiments, the expression cassettes described herein may be contained within a vector construct (e.g., a plasmid or viral vector). In addition to the components of the expression cassette, the vector may also include one or more selectable markers, signals allowing the vector to exist as single-stranded DNA (e.g., an M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., SV40 or adenovirus origin of replication).

[0151] "Purified polynucleotide" refers to a polynucleotide of interest or a fragment thereof that is substantially free of the protein to which the polynucleotide is naturally associated, for example, containing less than about 50%, preferably less than about 70%, more preferably less than about 90% of the protein. Techniques for purifying the polynucleotide of interest are well known in the art and include, for example, lysing cells containing the polynucleotide with a dissociative agent, and separating the polynucleotide and protein by ion exchange chromatography, affinity chromatography, and sedimentation based on density.

[0152] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell is "transfected" when foreign DNA has been introduced into the cell membrane. Many transfection techniques are generally known in the art. See, for example, Graham et al., (1973) *Virology*, 52:456; Sambrook et al., ibid.; Davis et al., (1995) *Basic Methods in Molecular Biology*, 2nd ed.; McGraw-Hill and Chu et al., (1981) *Gene*, 13:197. Such techniques can be used to introduce one or more portions of foreign DNA into a suitable host cell. The term refers to both stable and transient uptake of genetic material and includes the uptake of peptide-linked or antibody-linked DNA.

[0153] A "vector" is a device that can transfer nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate vectors, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct that can direct the expression of nucleic acids of interest and can transfer nucleic acid sequences to target cells. Therefore, the term includes cloning and expression vectors as well as viral vectors.

[0154] "Gene transfer" or "gene delivery" refers to methods or systems used to reliably insert DNA or RNA of interest into host cells. Such methods can result in transient expression of non-integrating transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of the host cell. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmids, viral vectors, non-viral vectors, adenoviruses, lentiviruses, alphaviruses, poxviruses, and vaccinia viruses.

[0155] A polynucleotide “derived from” a specified sequence refers to a continuous sequence of polynucleotides comprising at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides, wherein the continuous sequence corresponds to a region of the specified nucleotide sequence, i.e., is identical to or complementary to a region of the specified nucleotide sequence. The derived polynucleotide will not necessarily be physically derived from the nucleotide sequence of interest, but can be produced in any manner, including but not limited to chemical synthesis, replication, reverse transcription, or transcription, based on information provided by the sequence of bases in the region from which the polynucleotide is derived. Therefore, it can represent the sense or antisense orientation of the original polynucleotide.

[0156] As used herein, the term “determine” refers to both quantitative and qualitative determination, and therefore, the term “determine” may be used interchangeably with “measurement”, “assay”, etc.

[0157] The “effective amount” of a CCN3 agonist (e.g., small molecule, drug, receptor ligand, protein, polypeptide, peptide, peptide-like substance, aptamer) is an amount sufficient to increase the biological activity of CCN3 or to increase bone or cartilage growth. The effective amount may be administered in a single or multiple application, dose, or series of doses.

[0158] As used herein, the term "sample" refers to a material or mixture of materials containing one or more analytes of interest, typically but not necessarily in liquid form.

[0159] As used herein, “biological sample” means a sample of tissue or fluid isolated from a subject, including but not limited to, for example, blood, plasma, serum, feces, urine, bone marrow, bile, cerebrospinal fluid, lymph, skin samples, secretions from the skin, respiratory tract, intestine and genitourinary tract, tears, saliva, breast milk, blood cells, organs, biopsies and samples of in vitro cell culture components, including but not limited to conditioned media produced by the growth of cells and tissues, such as recombinant cells and cell components, in a culture medium.

[0160] The term “CCN3 sample” for an individual covers biological samples (e.g., blood or plasma samples) containing CCN3 obtained from that individual. CCN3 samples can be obtained by any suitable method, such as by venipuncture or biopsy. The definition also includes samples that have been manipulated in any way after they have been obtained, such as by treating with reagents, washing, centrifuging, or enriching a particular type of molecule (e.g., CCN3).

[0161] The terms “quantity,” “amount,” and “level” are used interchangeably herein and can refer to either an absolute quantification of a molecule or analyte in a sample or a relative quantification of a molecule or analyte in a sample, i.e., relative to another value, such as relative to a reference value taught herein, or relative to a range of values ​​for a biomarker. These values ​​or ranges can be obtained from a single patient or from a group of patients.

[0162] The term "determination" is used herein to refer to physical steps involving the manipulation of a sample to generate data related to that sample. As will be readily understood by those skilled in the art, a sample must be "obtained" before it can be determined. Therefore, the term "determination" implies that the sample has been obtained. As used herein, the terms "obtained" or "obtaining" cover the act of receiving an extracted or separated sample. For example, a testing organization may "obtain" a sample by mail (or via delivery, etc.) before determining it. In some such cases, the sample is "extracted" or "separated" from an individual by another party before being mailed (i.e., delivered, transferred, etc.) and then "obtained" by the testing organization upon the sample's arrival. Thus, the testing organization can obtain the sample and then determine it, thereby generating data related to the sample.

[0163] As used herein, the term "obtain" or "acquire" may also include the physical extraction or separation of a sample from a subject. Thus, a sample may be separated from a subject (and therefore "obtained") by the same person or entity that subsequently measures the sample. When a sample is "extracted" or "separated" from a first party or entity and then transferred (e.g., delivered, mailed, etc.) to a second party, the sample is "obtained" by the first party (and also "separated" by the first party), and then subsequently "obtained" by the second party (but not "separated" by it). Therefore, in some embodiments, the obtaining step does not include the step of separating the sample.

[0164] In some embodiments, the obtaining step includes the step of separating the sample. Methods and procedures for separating various samples (e.g., blood samples, serum samples, plasma samples, biopsy samples, aspirates, etc.) will be known to those skilled in the art, and any convenient method can be used to separate the samples.

[0165] Those skilled in the art will understand that in some cases, it is convenient to wait until multiple samples have been obtained before measuring them. Therefore, in some cases, samples are stored separately until all suitable samples have been obtained. Those skilled in the art will understand how to properly store various types of samples and that any convenient storage method suitable for a particular sample (e.g., refrigeration) can be used. In some embodiments, the untreated sample is measured before the treated sample is obtained. In some cases, the untreated sample and the treated sample are measured in parallel. In some cases, multiple different treated samples and / or one untreated sample are measured in parallel. In some cases, the sample is processed immediately or as soon as possible after acquisition.

[0166] A “reference level” or “reference value” for a biomarker (e.g., CCN3) refers to the level of a biomarker that indicates susceptibility to developing bone conditions or lesions associated with bone degeneration (e.g., bone mineral density or bone loss, bone fragility, osteoporosis, or osteopenia). A “reference level” for a biomarker can be the absolute or relative amount or concentration of the biomarker, the presence or absence of the biomarker, a range of the amount or concentration of the biomarker, the minimum and / or maximum amount or concentration of the biomarker, the average amount or concentration of the biomarker, and / or the median amount or concentration of the biomarker. Reference levels can be tailored to the specific technique used to measure the level of the biomarker in a sample (e.g., ELISA, mass spectrometry, Western blotting), where the level of the biomarker can vary based on the specific technique used.

[0167] The terms “determine,” “measure,” “evaluate,” “assess,” “determine,” and “analyze” are used interchangeably herein to refer to any form of measurement and include determining the presence of an element. These terms include both quantitative and / or qualitative determinations. A determination can be relative or absolute. For example, a “determination” can be determining whether a level is less than or “greater than or equal to” a specific threshold (which may be predetermined or can be determined by measuring a control sample). On the other hand, “determination to determine a level” can mean determining a quantitative value (using any convenient metric) representing that level. The level can be expressed in any unit associated with a particular determination (e.g., fluorescence units, such as Mean Fluorescence Intensity (MFI)) or as an absolute value with defined units (e.g., number of molecules or concentration). Furthermore, the level can be compared to the level of another biomarker or standard to derive a normalized value representing the normalized level. When evaluating multiple samples from the same individual (e.g., samples collected from the same individual at different time points), the specific metric (or unit) chosen is not critical, as long as the same units are used (or a conversion to the same units is performed). This is because units cancel each other out when calculating the multiple change in level from one sample to the next (e.g., samples collected from the same individual at different time points).

[0168] To measure protein levels, the amount or level of protein in a sample is determined. In some cases, the protein contains post-translational modifications (e.g., phosphorylation, glycosylation) associated with protein activity, such as regulation through signal transduction cascades, where the modified protein is a biomarker, and thus the amount of the modified protein is measured. In some embodiments, extracellular protein levels are measured. For example, in some cases, the protein being measured (i.e., a peptide) is a secreted protein, and therefore its concentration can be measured in blood or plasma. In some embodiments, concentration is a relative value measured by comparing the level of one protein with that of another. In other embodiments, concentration is an absolute measurement of weight / volume or weight / weight.

[0169] In some cases, the concentration of one or more additional proteins may also be measured, and the biomarker concentration may be compared to the level of one or more additional proteins to provide a normalized value for the biomarker concentration. Any convenient protocol for assessing protein levels may be used, in which the level of one or more proteins in the sample being measured is determined.

[0170] While many different methods for determining protein levels are known to those skilled in the art, and any convenient method can be used, a representative and convenient type of protocol for determining protein levels is ELISA, an antibody-based method. In ELISA and ELISA-based assays, one or more antibodies specific to the protein of interest can be immobilized onto a selected solid surface, preferably a surface exhibiting protein affinity, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, the wells of the assay plate are coated with a nonspecific “blocking” protein known to be antigenically neutral to the test sample, such as bovine serum albumin (BSA), casein, or a milk powder solution. This allows the nonspecific adsorption sites on the immobilized surface to be blocked, thereby reducing background caused by nonspecific binding of antigens to the surface. After washing to remove unbound blocking protein, the immobilized surface is brought into contact with the sample to be tested under conditions conducive to the formation of immune complexes (antigen / antibody). After incubation, the surface in contact with the antiserum is washed to remove the non-immunogenic composite material. The occurrence and amount of immune complex formation can then be determined by subjecting the bound immune complex to a second antibody that is specific to the target and different from the first antibody, and by detecting the binding of the second antibody. In some embodiments, the second antibody will have an associated enzyme, such as urease, peroxidase, or alkaline phosphatase, which will produce a colored precipitate after incubation with a suitable chromogenic substrate. After such incubation with the second antibody and washing to remove unbound material, the amount of the label is quantified, for example, by incubation with a chromogenic substrate, such as urea and bromocresol purple in the case of urease labeling, or 2,2'-azino-di-(3-ethyl-benzothiazoline)-6-sulfonic acid (ABTS) and H₂O₂ in the case of peroxidase labeling. Quantification is then achieved by measuring the degree of color production, for example, using a visible spectrophotometer.

[0171] The aforementioned approach can be modified by first binding the sample to the assay plate. The primary antibody is then incubated with the assay plate, followed by detection of the bound primary antibody using a second antibody specifically labeled to the primary antibody. The solid substrate on which one or more antibodies are immobilized can be made of a wide variety of materials and can take on a variety of shapes, such as microtiter plates, microbeads, test strips, resin particles, etc. The substrate can be selected to maximize the signal-to-noise ratio, minimize background binding, and facilitate separation and cost. Washing can be performed in a manner most suitable to the substrate used, for example, by removing beads or test strips from the reservoir, emptying or diluting the reservoir such as the wells of a microtiter plate, or rinsing the beads, particles, column, or filter with a washing solution or solvent.

[0172] Alternatively, non-ELISA-based methods can be used to measure the levels of one or more proteins in a sample. Representative exemplary methods include, but are not limited to, antibody-based methods (e.g., immunofluorescence assay, radioimmunoassay, immunoprecipitation, Western blotting, proteomics arrays, xMAP microsphere technology (e.g., Luminex technology), immunohistochemistry, flow cytometry, etc.) and non-antibody-based methods (e.g., mass spectrometry or tandem mass spectrometry).

[0173] "Providing analysis" in this document refers to the delivery of oral or written analysis (i.e., documents, reports, etc.). Written analysis can be a printed document or an electronic document. A suitable analysis (e.g., an oral or written report) provides any or all of the following information: the subject's identification information (name, age, etc.), a description of the type of sample used and / or how the sample was used, the technique used to measure the sample, the results of the measurement (e.g., the level of CCN3 measured and / or the fold change of CCN3 levels over time), an assessment of whether the individual is at risk of bone loss from continued breastfeeding, and a recommendation to discontinue breastfeeding if the CCN3 levels indicate that the subject is at risk of bone loss if breastfeeding continues, etc. The report can be in any format, including but not limited to printed information on a suitable medium or substrate (e.g., paper); or in electronic format. If in electronic format, the report can be on any computer-readable medium, such as a disk, optical disc (CompactDisk CD), flash drive, etc., on which the information has been recorded. Furthermore, the report can be presented as a website address that can be used via the Internet to access the information at a remote site.

[0174] A hydrogel is a substance formed when organic polymers (natural or synthetic) are cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open lattice structure that traps water molecules to form a gel. Biocompatible hydrogels are those formed by polymers that are non-toxic to living cells and allow sufficient diffusion of oxygen and nutrients into the trapped cells to maintain their viability.

[0175] The term "stem cell" refers to cells that maintain their self-renewal through mitotic cell division and can differentiate into various specialized cell types. Mammalian stem cells can be divided into three main categories: embryonic stem cells derived from the blastocyst, adult stem cells found in adult tissues, and umbilical cord blood stem cells found in the umbilical cord. In a developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as the body's repair system by supplementing specialized cells. Adult stem cells include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells, and neural stem cells. Totipotent stem cells are produced by the fusion of an egg cell and a sperm cell. Cells produced from the first few divisions of a fertilized egg are also totipotent. These cells can differentiate into embryonic cell types and extraembryonic cell types. Pluripotent stem cells are the offspring of totipotent cells and can differentiate into cells derived from any of the three germ layers. Pluripotent stem cells can only produce cells from closely related cell families (e.g., hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can only produce one cell type but possess the property of self-renewal, which distinguishes them from non-stem cells. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell derived from adult cells that have been reprogrammed into an embryonic-like pluripotent state. IPSCs can be derived from, for example, adult somatic cells such as peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose-derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes.

[0176] As used herein, a “reprogramming factor” refers to one or more bioactive factors, or a mixture of bioactive factors, that act on cells to alter transcription, thereby reprogramming the cells to pluripotency. The reprogramming factor may be provided to cells, for example, from somatic cells of an individual with a family history or genetic makeup of interest, such as a patient with a neurological disorder or neurodegenerative disease, either alone or as a single composition of reprogramming factors, i.e., as a premixed composition. The factor may be provided in the same molar ratio or in different molar ratios. The factor may be provided once or multiple times during the culturing of cells of the subject matter invention. In some embodiments, the reprogramming factor is a transcription factor, including but not limited to Oct3 / 4; Sox2; Klf4; c-Myc; Nanog; and Lin-28.

[0177] Somatic cells may include, but are not limited to, peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose-derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes, which are contacted with reprogramming factors as defined above in a combination and amount sufficient to reprogram the cells to pluripotency. The reprogramming factors may be provided to somatic cells alone or as a single composition of reprogramming factors, i.e., a premixed composition. In some embodiments, the reprogramming factors are provided as multiple coding sequences on a vector.

[0178] The term "skeletal stem cells" refers to pluripotent and self-renewing cells capable of producing bone marrow stromal cells, bone cells, and chondrogenic cells. Self-renewal means that when a skeletal stem cell undergoes mitosis, it produces at least one daughter cell, which is also a skeletal stem cell. Pluripotent means that skeletal stem cells are capable of producing progenitor cells (skeletal progenitor cells), which generate all cell types of the skeletal system but cannot generate cells from other organs in the body.

[0179] Bone stem cells can be derived from non-bone cells, such as embryonic stem cells, adult stem cells, and induced pluripotent stem cells. For example, bone stem cells can be generated by reprogramming mesenchymal stem cells and adipose tissue containing such cells, such as human adipose stem cells (hAASC). Induced bone cells possess characteristics derived from naturally occurring functional SSCs; that is, they can generate the same lineage.

[0180] Human SSC cell populations can be characterized by their cell surface markers, although those skilled in the art will understand that the endogenous population of SSCs does not need to be characterized for effective stimulation. Human SSCs are negative for CD45, CD235, Tie2, and CD31; and positive for podoplanin (PDPN). Cell populations with this combination of markers, such as cells isolated from bone tissue, can be referred to as [PDPN]. + / 146] cells. [PDPN + / 146] The population can be further subdivided into three groups: the monopotent subgroup capable of cartilage production [PDPN] + CD146 CD73 CD164], a subset of unipotent cells capable of osteogenic formation [PDPN] + CD146 CD73 CD164 + ] and pluripotent [PDPN] capable of intrachondral (bone and cartilage) ossification + CD146CD73 + CD164 +Cells. Regarding CD45, CD235, Tie2, and CD31, as well as PDPN, cell populations of interest for the methods of this invention can be isolated from bone. Other cell populations of interest include [PDPN]. + [CD146 CD73 CD164] cells; [PDPN] + CD146 CD73 CD164 + [cells; and [PDPN] + CD146 CD73 + CD164 + Cells. Mouse skeletal lineages were characterized as CD45-, Ter119-, Tie2-, and ocv integrin+. SSCs were further characterized as Thy1-, 6C3-, CD105-, and CD200+.

[0181] "Adipose-derived stem cells" or "adipose-derived stromal cells" refer to cells derived from adipose tissue. "Adipose" means any adipose tissue. Adipose tissue can be brown or white and is derived from the subcutaneous, omentum / visceral, mammary, gonadal, or other adipose tissue sites. Preferably, the fat is subcutaneous white adipose tissue. Such cells can be provided as primary cell cultures or immortalized cell lines. Adipose tissue can be derived from any organism possessing adipose tissue. Preferably, the adipose tissue is from mammals, and most preferably, from humans. A convenient source of adipose tissue is liposuction; however, the source or method of adipose tissue isolation is not critical to this invention.

[0182] Adipose tissue is abundant, and harvesting methods with minimal risk to patients can be used. It is estimated that each gram of adipose tissue contains more than 10... 4 These stem cells (Sen et al., 2001, Journal of Cellular Biochemistry 81:312-319) can be used immediately or cryopreserved for future autologous or allogeneic applications.

[0183] Methods for isolating, expanding, and differentiating human adipose tissue-derived cells have been reported. See, for example, Burris et al., 1999, *Molecular Endocrinology* 13:410-7; Erickson et al., 2002, *Biochem Biophys Res Commun.*, January 18, 2002; 290(2):763-9; Gronthos et al., 2001, *Journal of Cellular Physiology*, 189:54-63; Halvorsen et al., 2001, *Metabolism* 50:407-413; Halvorsen et al., 2001, *Tissue Engineering* 7(6):729-41; Harp et al., 2001, *Biochem Biophys Res Commun.* 281:907-912; Saladin et al., 1999, *Cell Growth &... Diff) 10:43-48; Sen et al., 2001, Journal of Cellular Biochemistry 81:312-319; Zhou et al., 1999, Biotechnol. Techniques 13:513-517. Adipose tissue-derived stromal cells can be obtained from thinned human adipose tissue by collagenase digestion and differential centrifugation [Halvorsen et al., 2001, Metabolism 50:407-413; Hauner et al., 1989, Journal of Clinical Research 84:1663-1670; Rodbell et al., 1966, J Biol Chem 241:130-139].

[0184] Biomarkers expressed by adipose tissue-derived stem cells have been reported, including CD13, CD29, CD44, CD63, CD73, CD90, CD166, aldehyde dehydrogenase (ALDH), and ABCG2. Adipose tissue-derived stem cells can be purified mononuclear cell populations extracted from adipose tissue that can proliferate in culture for more than one month.

[0185] To isolate cells from tissues, suitable solutions can be used for dispersion or suspension. Such solutions are typically balanced salt solutions, such as physiological saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal bovine serum or other naturally occurring factors, combined with an acceptable low concentration of buffer, usually 5-25 mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc.

[0186] Cell populations can be used immediately. Alternatively, cell populations can be frozen and stored at liquid nitrogen temperatures for extended periods, thawed, and reused. In such cases, cells are typically frozen in 10% DMSO, 50% serum, 40% buffer medium, or some other such solutions commonly used in the art for preserving cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells.

[0187] Adipocytes can be cultured in vitro under various culture conditions. The culture medium can be liquid or semi-solid, such as containing agar, methylcellulose, etc. Cell populations can be conveniently suspended in suitable nutrient media, such as Iscove's modified DMEM or RPMI-1640, which are typically supplemented with fetal bovine serum (approximately 5%-10%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics such as penicillin and streptomycin. In one embodiment of the invention, adipocytes are maintained in the culture in the absence of feeder cells, i.e., in the absence of serum, etc. The culture may contain growth factors to which cells respond. As defined herein, growth factors are molecules capable of promoting cell survival, growth, and / or differentiation in culture or in intact tissues through specific action on transmembrane receptors. Growth factors include peptide and non-peptide factors.

[0188] The terms “reprogramming efficiency,” “reprogramming efficiency,” “conversion efficiency,” or “conversion efficiency” are used interchangeably herein to refer to the ability of cells of one cell lineage to produce induced cells of another cell lineage when exposed to a suitable reprogramming system, for example, the ability of adipose tissue cells to produce iSSCs when exposed to a high dose of BMP2. In other words, the number of induced cells (e.g., iSSCs) produced by the cell is approximately 1.5 times, approximately 2 times, approximately 3 times, approximately 4 times, approximately 6 times, approximately 8 times, approximately 10 times, approximately 20 times, approximately 30 times, approximately 50 times, approximately 100 times, approximately 200 times, or more than that of the unexposed population.

[0189] Using CCN3 to promote bone and cartilage regeneration

[0190] Compositions and methods for using CCN3 for regeneration or replacement of bone or cartilage are provided. In some embodiments, CCN3 is used as an osteogenic factor to stimulate osteochondral skeletal stem cells to generate bone. CCN3 can be used to increase bone mineral density, bone mass, bone strength, and / or the rate of bone formation, reduce fatty bone marrow in bone, and / or reverse bone loss. In some embodiments, CCN3 is used to treat bone conditions or symptoms associated with bone degeneration. Bone conditions and symptoms associated with bone degeneration include, but are not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

[0191] In other implementations, CCN3 is used as a chondrogenic factor to stimulate bone cells to produce cartilage. CCN3 can be used, for example, to treat cartilage lesions or injuries in a subject. Such lesions include any condition involving cartilage tissue that is insufficient for physiological or cosmetic purposes. Such defects include those that are congenital, including developmental malformations, as well as those caused by disease, trauma, or surgery or other medical procedures. Cartilage defects include those caused by cartilage disorders, including but not limited to osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury (e.g., caused by surgery, accidental injury, sports injury), infection, or malignancy.

[0192] It can treat various sites of articular cartilage regeneration, including but not limited to the knee joint, elbow joint, joints in the phalanges and toes, shoulder joint, hip joint, wrist joint, and ankle joint. The individual can be an adult, such as someone who has passed puberty, or an elderly person, such as someone over 55, over 65, or over 70 years old.

[0193] Individuals requiring cartilage regeneration can be treated using the methods described in this article. For the treatment of cartilage disorders, a therapeutically effective dose of CCN3 is administered in combination with a therapeutically effective dose of a VEGF inhibitor and mechanical stimulation.

[0194] VEGF inhibitors can be any substance that reduces signaling through the VEGF-VEGFR pathway. To name just a few, VEGF inhibitors include small molecules, peptides, polypeptides, proteins, and more specifically, antibodies, including anti-VEGF antibodies, anti-VEGFR antibodies, intracellular antibodies, maximal antibodies, microantibodies, biantibodies, Fc fusion proteins such as peptide antibodies, receptor antibodies, soluble VEGF receptor proteins and fragments, and various others. Many VEGF inhibitors act by binding to VEGF or VEGF receptors. Other VEGF inhibitors act more indirectly by binding to factors that bind to VEGF or VEGF receptors or other components of the VEGF signaling pathway. Still others act by altering regulatory post-translational modifications that regulate VEGF pathway signaling. VEGF inhibitors can also act through more indirect mechanisms. Regardless of the mechanism involved, as used herein, a VEGF inhibitor reduces the effective activity of the VEGF signaling pathway under given conditions to a level lower than the activity under the same conditions in the absence of the inhibitor.

[0195] In some embodiments, a dose of VEGF inhibitor is provided in the implant, such as a matrix or scaffold for local delivery of the factor. The effective dose can be determined based on the specific tissue, the rate of release from the implant, the size of the implant, etc., and can be determined empirically by those skilled in the art. This dose can provide bioactivity equivalent to 1 mg of soluble VEGF receptor, 10 mg, 100 mg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 250 mg, 500 mg, 750 mg, or 1 g of soluble VEGF receptor. The dose can be administered at a single time point, e.g., as a single implant application; or it can be administered in multiple doses, e.g., via microneedle delivery. The dose can be administered once, twice, three times, four times, five times, ten times, or more as needed to achieve the desired effect, and administration can be daily, every two days, every three days, every four days, weekly, every two weeks, monthly, or for longer periods.

[0196] Many VEGF inhibitors have been described in the literature. For descriptions of VEGF inhibitors, see, for example, the following patent documents: US 2003 / 0105091, US2006 / 0241115, US 5,521,184, US 5,770,599, US 5,990,141, US 6,235,764, US 6,258,812, US 6,515.004, US 6,630,500, US 6,713,485, WO 02005 / 070891, WO 01 / 32651, WO 02 / 68406, WO 02 / 66470, WO 02 / 55501, WO 04 / 05279, WO 04 / 07481, WO 04 / 07458, WO 04 / 09784, WO WO 02 / 59110, WO 99 / 450029, WO 00 / 59509, WO 99 / 61422, WO 00 / 12089, WO 00 / 02871 and WO 01 / 37820, particularly the parts relating to VEGF inhibitors; are incorporated herein by reference.

[0197] Exemplary VEGF inhibitors include, but are not limited to, ABT-869 (Abbott), including formulations for oral administration and closely related VEGF inhibitors; AEE-788 (Novartis) (also known as AE-788 and NVP-AEE-788, etc.), including formulations for oral administration and closely related VEGF inhibitors; AG-13736 (Pfizer) (also known as AG-013736), including formulations for oral administration and closely related VEGF inhibitors; AG-028262 (Pfizer) and closely related VEGF inhibitors; and angiostatin (EntreMed) Also known as CAS Registry No. 86090-08-6, K1-4, and rhuAngiostatin, etc.) and closely related inhibitors, such as those described in US Patent Nos. 5,792,825 and 6,025,688, particularly in the sections relating to angiostatin and closely related VEGF inhibitors, their structures and properties, and their preparation and use; Avastin™ (Genentech) (also known as bevacizumab, R-435, rhuMAB-VEGF, and CAS Registry No. 216974-75-3, etc.) and closely related VEGF inhibitors; AVE-8062 (Ajinomoto Co. and Sanofi-aventis (also known as AC-7700 and Compuratin A4 analogs, etc.), and closely related VEGF inhibitors; AZD-2171 (AstraZeneca) and closely related VEGF inhibitors; Nexavar® (Bayer AG and Onyx) (also known as CAS Registry No. 284461-73-0, BAY-43-9006, raf kinase inhibitor, sorafenib, sorafenib analogs and IDDBCP150446, etc.) and closely related VEGF inhibitors; BMS-387032 (Sunesis and Bristol-Myers) Squibb (also known as SNS-032 and CAS Registry No. 345627-80-7, etc.) and closely related VEGF inhibitors; CEP-7055 (Cephalon and Sanofi-aventis) (also known as CEP-11981 and SSR-106462, etc.) and closely related VEGF inhibitors; CHIR-258 (Chiron) (also known as CAS Registry No. 405169-16-6, GFKI and GFKI-258, etc.) and closely related VEGF inhibitors; CP-547632 (OSI Pharmaceuticals and Pfizer) (also known as CAS Registry No. 252003-65-9, etc.) and closely related VEGF inhibitors, such as CP-564959, etc.E-7080 (Eisai) (also known as CAS Registry No. 417716-92-8 and ER-203492-00, etc.) and closely related VEGF inhibitors; 786034 (GlaxoSmithKline) and closely related VEGF inhibitors; GW-654652 (GlaxoSmithKline) and closely related indazole pyrimidine Kdr inhibitors; IMC-1C11 (ImClone) (also known as DC-101 and c-p1C11, etc.) and closely related VEGF inhibitors; KRN-951 (Kirin Brewery and other closely related quinoline-urea VEGF inhibitors; PKC-412 (Novartis) (also known as CAS Registry No. 120685-11-2, benzoylstralicin, CGP-41251, midostaurin, and STI-412, etc.) and closely related VEGF inhibitors; PTK-787 (Novartis and Schering) (also known as CAS Registry Nos. 212141-54-3 and 212142-18-2, PTK / ZK, PTK-787 / ZK-222584, ZK-22584, VEGF-TKI, VEGF-RKI, PTK-787A, DE-00268, CGP-79787, CGP-79787D, vatalanib, ZK-22 2584, etc.) and closely related aniline phthalazine derivative VEGF inhibitors; SU11248 (Sugen and Pfizer) (also known as SU-11248, SU-011248, SU-11248J, Sutent®, and sunitinib malate, etc.) and closely related VEGF inhibitors; SU-5416 (Sugen and Pfizer / Pharmacia) (also known as CAS Registry No. 194413-58-6, simatinib, 204005-46-9, etc.) and closely related VEGF inhibitors; SU-6668 (Sugen and Taiho) (also known as CAS Registry No. 252916-29-3, SU-006668, and TSU-68, etc.) and closely related VEGF inhibitors, such as WO-09948868, WO- As described in 09961422 and WO-00038519, particularly in the sections relating to SU-6668 and closely related VEGF inhibitors, their structures and properties, and methods for their preparation and use;VEGF traps (Regeneron and Sanofi-aventis) (also known as AVE-0005 and systemic VEGF traps, etc.) and closely related VEGF inhibitors, such as those described in WO-2004110490, etc., particularly in the sections concerning VEGF traps and closely related VEGF inhibitors, their structures and properties, and methods for their preparation and use; thalidomide (Celgene) (also known as CAS Registry No. 50-35-1, Synovir, Thalidomide) Pharmion and Thalomid, etc.) and closely related VEGF inhibitors; XL-647 (Exelixis) (also known as EXEL-7647, etc.) and closely related VEGF inhibitors; XL-999 (Exelixis) (also known as EXEL-0999, etc.) and closely related VEGF inhibitors; XL-880 (Exelixis) (also known as EXEL-2880, etc.) and closely related VEGF inhibitors; ZD-6474 (AstraZeneca) (also known as CAS Registry No. 443913-73-3, Zactima and AZD-6474, etc.) and closely related aniline quinazoline VEGF inhibitors. Inhibitors; and ZK-304709 (Schering) (also known as a CDK inhibitor (indirubin derivative), ZK-CDK, MTGI, and multi-target tumor growth inhibitor, etc.) and other closely related compounds, including indirubin derivative VEGF inhibitors described in WO-00234717, WO-02074742, WO-02100401, WO-00244148, WO-02096888, WO-03029223, WO-02092079, and WO-02094814, particularly those related to these and closely related VEGF inhibitors, their structures and properties, and methods for their preparation and use. VEGF inhibitors can be delivered in a manner suitable to the properties of the inhibitor, such as as proteins, small molecules, nucleic acids, etc., including but not limited to suitable mediators and carriers as desired.

[0198] In some implementations, the mechanical stimulation is an acute local injury. In some implementations, an individual presenting with an acute local injury, such as due to an accident or sports injury, can be treated with a combination of CCN3 and VEGF inhibitors to reduce the development of fibrocartilage and enhance the regeneration of articular cartilage.

[0199] In other implementations, acute local injury is addressed through a microfracture procedure using a cone, drill, or similar tool. Microfracture refers to a surgical technique that creates a “microfracture” in the subchondral bone perpendicular to its surface. This technique can utilize various angled cones or “picks,” or small drills. A rough, unprocessed surface can also be created to retain the clot. For example, a pick can be used to create fracture fragments that attract and retain the clot. For tissue regeneration, cells must be present. In this procedure, controlled “microfractures” through the subchondral bone allow access to bone marrow-based progenitor cells and growth factors. A bone marrow clot forms at the base of the prepared cartilage lesion, and pluripotent cells proliferate and differentiate.

[0200] General indications for microfractures include full-thickness defects, unstable cartilage covering subchondral bone, and partial-thickness lesions where, during probing, the cartilage is scraped down only to the bone. Patient age is not a specific contraindication. While patients under 35 years of age show greater improvement, older patients also demonstrate improvement. Lesion size is also not a contraindication for microfractures. Lesions may be less than 400 mm. 2 or greater than 400 mm 2 The height of the cartilage margin surrounding the lesion may be sufficient to hold the clot in place.

[0201] MRI can be used to assess cartilage thickness and identify other associated damage. MRI enables imaging of morphological changes such as cartilage fibrosis, clefts, focal defects and corresponding fragmentation, as well as more diffuse thinning and wear—all of which manifest as changes in cartilage thickness and surface area at the interface between the cartilage and the synovial fluid and membrane. Early degenerative changes in cartilage, such as softening or blistering, and later fibrotic changes, can also be seen as changes in MRI signal and heterogeneous intramaterial regions, although such assessments remain qualitative in standard clinical practice.

[0202] Thorough diagnostic arthroscopy of the joint can be performed through three ports (inflow cannula, arthroscopy, and working instruments). Special attention should be paid to anterior space scars, folds, and lateral retinaculum, as these can potentially increase compression between cartilage surfaces. Microfracture is the last intra-articular procedure performed. This allows the initial clot in the microfracture site to be preserved. It also prevents blood and fat droplets from entering the knee through the microfracture site and obscuring its visibility.

[0203] After identifying full-thickness articular cartilage lesions, all remaining unstable cartilage is removed. Loose or marginally attached cartilage can be moved back to the stable cartilage margin using a handheld curved curette and a full-radius excision tool. Preferably, the calcified cartilage layer that still acts as a cap for many lesions is removed using a curette. The integrity of the subcartilaginous plate should be maintained. It is important that the defect is cleared deep enough to remove the calcified cartilage layer, but not so deep that the subcartilaginous plate is damaged. This prepared lesion, with a stable vertical margin of well-attached, viable healthy cartilage around the defect, provides a pool that helps retain the bone marrow clot during its formation.

[0204] Arthroscopic cones are used to create multiple holes or “microfractures.” Angled cones, typically 30° or 45°, allow the tip to be perpendicular to the bone as it advances. A 90° cone is used for the patella or other cartilage; however, it can only be advanced manually, not with a mallet. Starting at the periphery, microfracture holes are created, ending with a hole towards the center of the defect. They are created so that they do not intersect each other, and the subcartilaginous plate between them is protected. When the appropriate depth (approximately 2 to 4 mm) has been reached, fat droplets from the medullary cavity can be seen. When finished, the irrigation pump pressure is reduced to observe the release of medullary fat droplets and blood from the microfracture holes. During microfracture, a rough surface is created at the defect. This surface should not be further debrided or scraped to smooth it. This rough surface allows for easier adhesion of bone marrow clots, but the integrity of the subcartilaginous plate is preserved for articular surface shaping.

[0205] In cases of microfractures, or shortly after localized acute injury, CCN3 is administered in combination with a VEGF inhibitor to treat cartilage disorders. In some cases, a drug delivery device is implanted or otherwise positioned to deliver an effective dose of both CCN3 and the VEGF inhibitor. The CCN3 and VEGF inhibitor can be provided in separate compositions or in a single composition, i.e., as a premixed composition of the factors. The CCN3 and VEGF inhibitor can be provided in the same or different molar ratios and can be provided once or multiple times during the treatment process. For example, an implant containing both CCN3 and the VEGF inhibitor can be provided to an individual, as further described below.

[0206] The generation of CCN3

[0207] CCN3 proteins, or their biologically active variants or fragments, can be prepared in any suitable manner (e.g., recombinant expression, purification from cell cultures, chemical synthesis, etc.) and in various forms (e.g., native, fusion, labeled, lipid-modified, amidated, acetylated, PEGylated, etc.). CCN3 proteins may include naturally occurring peptides, recombinant peptides, synthetic peptides, or peptides produced by a combination of these methods. The means used to prepare the protein are well understood in the art. The protein is preferably prepared in a substantially pure form (i.e., substantially free of other host cell or non-host cell proteins).

[0208] CCN3 nucleic acid and protein sequences can be derived from any source. Many CCN3 nucleic acid and protein sequences are known. Representative sequences of the human CCN3 protein (SEQ ID NO: 1), the genomic sequence of the CCN3 gene (SEQ ID NO: 2), and the CCN3 mRNA (SEQ ID NO: 3) are presented in the sequence listing. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession numbers NM_002514, NG_009779, NM_010930.5, NM_002514, NM_030868, NM_205268, XM_058698906, XM_004580701, XM_046465005, XM_038555394, XM_057736139, XM_057791994, NP_002505, NP_035060, NP_110495, NP_990599, XP_03737995, X P_026919801, XP_032981561, XP_025306624, NP_001253825, XP_036292740, XP_010970891, XP_005564036, XP_004743541, XP_004000129, XP_037748011, XP_043457574, XP_038246527, XP_038540833 and XP_532317; all these sequences (as entered prior to the filing date of this application) are incorporated herein by reference. Any of these sequences or variants thereof containing a sequence having at least about 80% to 100% sequence identity with it, including any percentage identity within that range, such as having 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, wherein the variant CCN3 protein retains the biological activity of CCN3 (i.e., the ability to increase bone density, bone mass, bone strength, and / or the rate of bone formation, and / or the ability to regenerate cartilage, increase cartilage mass, and / or increase the rate of cartilage formation), and may be used to generate the CCN3 protein or a recombinant polynucleotide containing a coding sequence encoding the CCN3 protein, for use in the methods described herein.

[0209] In one implementation, recombinant technology is used to generate the CCN3 protein. Those skilled in the art can readily determine the nucleotide sequence encoding CCN3 using standard methods and the teachings herein. Oligonucleotide probes can be designed based on known sequences and used to probe genomic or cDNA libraries. The sequence can then be further isolated using standard techniques, and the gene can be truncated at desired portions of the full-length sequence, for example, using restriction enzymes. Similarly, known techniques such as phenol extraction can be used to directly isolate the sequence of interest from cells and tissues containing the sequence of interest, and the sequence can be further manipulated to produce the desired truncated form. For a description of techniques used to obtain and isolate DNA, see, for example, Sambrook et al., ibid.

[0210] The sequence encoding CCN3 can also be synthesized, for example, based on a known sequence. The nucleotide sequence can be engineered with codons that fit a specific amino acid sequence desired. The complete sequence is typically assembled from overlapping oligonucleotides prepared using standard methods to form the complete coding sequence. See, for example, Edge (1981), *Nature*. 292 :756; Nambair et al., (1984) Science 223 :1299; Jay et al., (1984) Journal of Biochemistry 259 :6311; Stemmer et al., (1995) Gene 164:49-53.

[0211] Recombinant technology can be readily used to clone sequences encoding CCN3, which can then be mutagenized in vitro by replacing appropriate base pairs to produce codons for the desired amino acids. Such alterations can range from as few as one base pair, affecting a single amino acid, to alterations involving several base pairs. Alternatively, mutagenesis can be achieved using mismatch primers that hybridize to the parental nucleotide sequence (typically cDNA corresponding to the RNA sequence) at temperatures below the melting temperature of the mismatched double helix. Primers can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutated bases in the central position. See, for example, Innis et al., (1990) “PCR Applications: Protocols for Functional Genomics”; Zoller and Smith, Methods Enzymol. (1983). 100468. DNA polymerase is used to extend primers, clone products, and select clones containing mutated DNA obtained by separating the extended primer strands. Selection can be accomplished using mutated primers as hybridization probes. This technique is also suitable for generating multi-point mutations. See, for example, Dalbie-McFarland et al., Proceedings of the National Academy of Sciences (1982). 79 :6409.

[0212] Once the coding sequences have been isolated and / or synthesized, they can be cloned into any suitable vector or replicon for expression. (See also Examples). As will be apparent from the teachings herein, a wide variety of vectors encoding modified polypeptides can be generated by creating expression constructs that operatively link polynucleotides encoding polypeptides having deletions or mutations therein in various combinations.

[0213] Many cloning vectors are known to those skilled in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors used for cloning and the host cells they can transform include bacteriophage λ (Escherichia coli), pBR322 (Escherichia coli), pACYC177 (Escherichia coli), pKT230 (Gram-negative bacteria), pGV1106 (Gram-negative bacteria), pLAFR1 (Gram-negative bacteria), pME290 (non-Escherichia coli Gram-negative bacteria), pHV14 (Escherichia coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Yeast), YCp19 (Yeast), and bovine papillomavirus (mammalian cells). See also DNA Cloning: Volumes I & II, ibid.; Sambrook et al., ibid.; Perbal et al., A Practical Guide to Molecular Cloning (Wiley-Liss; 2nd edition, 1988).

[0214] Insect cell expression systems, such as baculovirus systems, can also be used, and are known to those skilled in the art and described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for using baculovirus / insect cell expression systems are commercially available in kit form from Invitrogen, San Diego CA (“MaxBac” kits), etc.

[0215] Plant expression systems can also be used to produce CCN3. Typically, such systems use virus-based vectors to transfect plant cells with a heterologous gene. For a description of such systems, see, for example, Porta et al., *Molecular Biotechnology* (1996). 5 :209-221; and Hackland et al., "Literature of Virology (Arch. Virol.)" (1994) 139 :1-22.

[0216] Viral systems, such as cowpox-based infection / transmission systems, as described by Tomei et al., *Journal of Virology* (1993). 67 :4017-4026 and Selby et al., *Journal of General Virology* (1993) 74 The methods described in 1103-1113 will also be used in this invention. In this system, cells are first transfected in vitro with a recombinant vaccinia virus encoding a phage T7 RNA polymerase. This polymerase exhibits high specificity because it transcribes only templates carrying a T7 promoter. After infection, cells are transfected with the DNA of interest driven by the T7 promoter. The polymerase, expressed in the cytoplasm of the vaccinia virus recombinant, transcribes the transfected DNA into RNA, which is then translated into protein by the host translational machinery. This method provides a high level of transient cytoplasmic production of large amounts of RNA and its translation products.

[0217] A gene can be placed under the control of a promoter, a ribosome-binding site (for bacterial expression), and optionally an operon (collectively referred to herein as a "control" element), such that a DNA sequence encoding a desired polypeptide is transcribed into RNA in a host cell transformed by a vector containing the expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. For the purposes of this invention, both naturally occurring signal peptides and heterologous sequences may be used. The leader sequence can be removed by the host during post-translational processing. See, for example, U.S. Patent Nos. 4,431,739; 4,425,437; and 4,338,397. Such sequences include, but are not limited to, TPA leader sequences and bee venom peptide signal sequences.

[0218] Other regulatory sequences may also be desired, allowing the expression of protein sequences that regulate growth relative to the host cell. Such regulatory sequences are known to those skilled in the art, and examples include those sequences that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Other types of regulatory elements, such as enhancer sequences, may also be present in the vector.

[0219] Before insertion into a vector, control sequences and other regulatory sequences can be ligated to the coding sequence. Alternatively, the coding sequence can be directly cloned into an expression vector that already contains control sequences and appropriate restriction sites.

[0220] In some cases, it may be necessary to modify the coding sequence so that it can be attached to the control sequence in the appropriate orientation; that is, to maintain the correct reading frame. Mutants or analogues can be prepared by deleting a portion of the sequence encoding the protein, by inserting a sequence, and / or by substituting one or more nucleotides in the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, for example, Sambrook et al., (2001), *Molecular Cloning, a Laboratory Manual* (3rd ed., Cold Spring Harbor Laboratory, New York); *DNA Cloning, Vols. I and II*, ibid.; *Nucleic Acid Hybridization*, ibid.

[0221] The expression vector is then used to transform appropriate host cells. Many mammalian cell lines are known in the art, including immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), Vero293 cells, and other cell lines. Similarly, bacterial hosts such as *Escherichia coli*, *Bacillus subtilis*, and *Streptococcus* species will be used in the expression constructs of this invention. Yeast hosts that can be used in this invention include, in particular, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guilliermondii, Pichia pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Insect cells used for baculovirus expression vectors include, in particular, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0222] Depending on the chosen expression system and host, the CCN3 protein is produced by growing host cells transformed from the expression vector described above under conditions that express the protein. The selection of appropriate growth conditions is within the scope of the art.

[0223] In one implementation, the transformed cells secrete the CCN3 protein product into the surrounding culture medium. The vector may contain certain regulatory sequences to enhance protein product secretion, such as a tissue plasminogen activator (TPA) leader sequence, interferon (… or The secreted CCN3 protein product can then be isolated using various techniques described herein, such as, but not limited to, hydroxyapatite resin, column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, HPLC, immunoadsorption, affinity chromatography, immunoprecipitation, etc.

[0224] Alternatively, the transformed cells may be destroyed using chemical, physical, or mechanical means that lyse the cells but leave the recombinant peptides or polypeptides substantially intact. Intracellular proteins can also be obtained by removing components from the cell wall or cell membrane, for example, by using detergents or organic solvents to induce peptide leakage. Such methods are known to those skilled in the art and are described, for example, in *Protein Purification Applications: A Practical Approach* (edited by Simon Roe, 2001).

[0225] For example, methods for disrupting cells used in this invention include, but are not limited to: sonication or ultrasonic treatment; agitation; liquid or solid extrusion; heat treatment; freeze-thaw; drying; explosive decompression; osmotic shock; treatment with lysins, including proteases such as trypsin, neuraminidase, and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium lauryl sulfate, Triton, NP40, and CHAPS. The specific technique used for disrupting cells is largely a matter of selection and will depend on the cell type in which the polypeptide is expressed, the culture conditions, and any pretreatment used.

[0226] After cell disruption, cell debris is typically removed by centrifugation, and the peptides or polypeptides produced within the cells are further purified using standard purification techniques, such as, but not limited to, column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, HPLC, immunosorbent assay, affinity chromatography, and immunoprecipitation.

[0227] For example, a method for obtaining intracellular peptides involves affinity purification, such as immunoaffinity chromatography using an antibody (e.g., a previously generated antibody), or lectin affinity chromatography. Particularly preferred lectin resins are those that recognize the mannose moiety, such as, but not limited to, resins derived from: snowdrop (Galanthus nivalis) agglutinin (GNA), lentil (Lens culinaris) agglutinin (LCA or lentil agglutinin), pea (Pisum sativum) agglutinin (PSA or pea agglutinin), daffodil (Narcissus pseudodonarcissus) agglutinin (NPA), and bear garlic (Allium ursinum) agglutinin (AUA). The selection of suitable affinity resins is within the scope of the art. Following affinity purification, the peptide or polypeptide may be further purified using conventional techniques well known in the art, such as any of the techniques described above.

[0228] CCN3 protein can be readily chemically synthesized, for example, by any of several techniques known to those skilled in the art of peptide synthesis. See, for example, *Fmoc Solid Phase Peptide Synthesis: A Practical Approach* (edited by WC Chan and Peter D. White, Oxford University Press, 1st edition, 2000); *Chemistry of Peptide Synthesis* (CRC Press, 1st edition, 2005); *Peptide Synthesis and Applications* ("Methods in Molecular Biology", edited by John Howl, Humana Press, 1st edition, 2005); and *Pharmaceutical Formulation Development of Peptides and Proteins* (Taylor & Francis series in Pharmaceutical Sciences, edited by Lars Hovgaard, Sven Frokjaer, and Marco van de Weert, CRC Press, 1st edition, 1999); which are incorporated herein by reference.

[0229] Generally, these methods involve sequentially adding one or more amino acids to a growing peptide chain. Typically, the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derived amino acid can then be attached to an inert solid support, or, under conditions allowing for amide bond formation, used in solution by adding the next amino acid with a complementary (amino or carboxyl) group in the sequence that has appropriate protection. The protecting group is then removed from the newly added amino acid residue, and then the next amino acid (appropriately protected) is added, and so on. After the desired amino acids have been linked in the correct sequence, any remaining protecting groups (and any solid support, if solid-phase synthesis techniques are used) are removed sequentially or simultaneously to obtain the final peptide or polypeptide. With simple modifications to this general procedure, it is possible to add more than one amino acid at a time to the growing chain, for example, by coupling a protected tripeptide with an appropriately protected dipeptide (under conditions that do not racemic the chiral center) to form a pentapeptide after deprotection. For information on solid-phase peptide synthesis techniques, see, for example, JM Stewart and JD Young, […]. Solid Phase Peptide Synthesis Synthesis (Pierce Chemical Co., Rockford, IL 1984) and G. Barany and R.B. Merrifield, […]. Peptides: Analysis, Synthesis, Biology , E. Gross and J. Meienhofer (eds.), Volume 2, (Academic Press, New York, 1980), pp. 3-254; and on classical solution synthesis, M. Bodansky, , Principles of Peptide Synthesis Synthesis , (Springer-Verlag, Berlin, 1984) and edited by E. Gross and J. Meienhofer, , peptide: Analysis, synthesis, biology Volume 1. These methods are typically used for relatively small peptides, i.e., at most about 50-100 amino acids in length, but are also applicable to larger peptides.

[0230] Typical protecting groups include tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl); biphenylisopropoxycarboxylcarbonyl, tert-pentoxycarbonyl, isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrobenzenesulfonyl, etc.

[0231] Typical solid supports are cross-linked polymer supports. These can include polymers of styrene based on divinylbenzene crosslinking, such as divinylbenzene-hydroxymethylstyrene copolymer, divinylbenzene-chloromethylstyrene copolymer, and divinylbenzene-diphenylamino polystyrene copolymer.

[0232] CCN3 protein can also be prepared chemically by other methods, such as through simultaneous peptide synthesis. See, for example, Houghten, Proceedings of the National Academy of Sciences (1985). 82 US Patent Nos. 4,631,211: 5131-5135; 4,631,211.

[0233] Nucleic acid encoding CCN3

[0234] Nucleic acids encoding CCN3 can be used in gene therapy applications to treat bone or cartilage disorders or conditions associated with bone or cartilage degeneration. The nucleic acids described herein can be inserted into expression vectors to create expression cassettes capable of producing CCN3 in suitable host cells. The ability of the construct to produce CCN3 can be determined empirically.

[0235] Expression cassettes typically include control elements operatively linked to the coding sequence that allow the gene to be expressed in vivo in the target species. Typical promoters for mammalian cell expression include the SV40 early promoter, CMV promoters such as the CMV immediate early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Adenovirus Major Late Promoter Ad MLP), and the herpes simplex virus promoter. Other non-viral promoters, such as promoters derived from the mouse metallothionein gene, are also used for mammalian expression. Typically, transcription termination and polyadenylation sequences are also present at the 3' of the translation stop codon. Preferably, a sequence at the 5' of the coding sequence for optimizing translation initiation is also present. Examples of transcription terminators / polyadenylation signals include those derived from SV40, such as those described above by Sambrook et al., and bovine growth hormone terminator sequences.

[0236] Enhancer elements can also be used in this paper to increase the expression levels of mammalian constructs. Examples include enhancers for early SV40 genes, as described by Dijkema et al., EMPO Journal (EMPO J.) (1985) 4:761; enhancers / promoters derived from long terminal repeat (LTR) sequences of Rous sarcoma virus, as described by Gorman et al., Proceedings of the National Academy of Sciences (PNAS) (1982b) 79:6777; and elements derived from human CMV, as described by Boshart et al., Cell (1985) 41:521, such as elements contained in CMV intron A sequences.

[0237] Once completed, the construct encoding CCN3 can be administered to a subject using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466. The gene can be delivered directly to the subject, or alternatively, ex vivo to cells derived from the subject and cells re-implanted into the subject.

[0238] Numerous virus-based systems have been developed for transferring genes into mammalian cells. These include adenoviruses, retroviruses (gamma-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses (see, for example, Warnock et al., (2011) Methods Mol. Biol. 737:1-25; Walther et al., (2000) Drugs 60(2):249-271; and Lundstrom (2003) Trends Biotechnol. 21(3):117-122; incorporated herein by reference).

[0239] For example, retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered in vivo or in vitro to the cells of a subject. Many retroviral systems have been described (US Patent No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proceedings of the National Academy of Sciences 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109; and Ferry et al. (2011) Curr Pharm Des. 17(24):2516-2527). Lentivirals are a class of retroviruses particularly well-suited for delivering polynucleotides to mammalian cells because they are capable of infecting both dividing and non-dividing cells (see, for example, Lois et al., (2002) Science 295:868-872; Durand et al., (2011) Viruses 3(2):132-159; incorporated herein by reference).

[0240] Many adenovirus vectors have also been described. Unlike retroviruses that integrate into the host genome, adenoviruses persist extrachromosomally, thereby minimizing the risk associated with insertional mutations (Haj-Ahmad and Graham, Journal of Virology (1986) 57:267-274; Bett et al., Journal of Virology (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., Journal of Virology (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, KL Biotechnology (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476). Furthermore, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well-known in the art. See, for example, US patents 5,173,414 and 5,139,941; international publications WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 4, 1993); Lebkowski et al., *Molecular and Cell Biology* (1988) 8:3988-3996; Vincent et al., *Vaccines* 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, BJ, *Current Opinion in Biotechnology* (1992) 3:533-539; Muzyczka, N., *Current Topics in Microbiol. and Immunol.* (1992) 158:97-129; Kotin, RM Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., Journal of Experimental Medicine (1994) 179:1867-1875.

[0241] Another vector system that can be used to deliver polynucleotides encoding CCN3 is the enteric recombinant poxvirus vaccine described by Small, Jr., PA et al. (US Patent No. 5,676,950, issued October 14, 1997, which is incorporated herein by reference).

[0242] Additional viral vectors that can be used to deliver nucleic acid molecules encoding CCN3 include those derived from the vaccinia virus family, including vaccinia virus and fowlpox virus. As an example, a vaccinia virus recombinant expressing CCN3 can be constructed as follows: First, DNA encoding a specific CCN3 coding sequence is inserted into a suitable vector such that it is adjacent to the vaccinia promoter and flanking vaccinia DNA sequences, such as those encoding thymidine kinase (TK). The vector is then used to transfect cells co-infected with vaccinia virus. Homologous recombination is used to insert a gene encoding the coding sequence of interest into the viral genome along with the vaccinia promoter. The resulting TK recombinant can be selected by culturing cells in the presence of 5-bromodeoxyuridine and selecting viral plaques resistant to it.

[0243] Alternatively, fowlpox viruses, such as chickenpox virus and canarypox virus, can also be used for gene delivery. Recombinant fowlpox viruses expressing immunogens from mammalian pathogens are known to confer protective immunity when administered to non-avian species. The use of fowlpox virus vectors in humans and other mammalian species is particularly desirable because members of the fowlpox virus genus can only be productively replicated in susceptible avian species and are therefore non-infectious in mammalian cells. Methods for producing recombinant fowlpox viruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, for example, WO 91 / 12882; WO 89 / 03429; and WO 92 / 03545.

[0244] Molecular conjugate vectors, such as the adenovirus chimeric vectors described by Michael et al., Journal of Biochemistry (1993) 268:6866-6869 and Wagner et al., Proceedings of the National Academy of Sciences (1992) 89:6099-6103, can also be used for gene delivery.

[0245] Members of the genus Alphavirus, such as, but not limited to, vectors derived from Sindbis Virus (SIN), Semliki Forest Virus (SFV), and Venezuelan Equine Encephalitis Virus (VEE), will also be used as viral vectors for delivering the polynucleotides of this invention. For a description of Sindbis Virus-derived vectors that can be used to practice this method, see Dubensky et al., (1996) *Journal of Virology* 70:508-519; and International Publications WO 95 / 07995, WO 96 / 17072; and U.S. Patent No. 5,843,723 to Dubensky, Jr., TW et al., issued December 1, 1998, and U.S. Patent No. 5,789,245 to Dubensky, Jr., TW, issued August 4, 1998, both of which are incorporated herein by reference. A particularly preferred option is a chimeric alphavirus vector containing sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, for example, Perri et al., (2003) Journal of Virology 77: 10394-10403 and International Publications WO 02 / 099035, WO 02 / 080982, WO 01 / 81609 and WO 00 / 61772; which are incorporated herein by reference in their entirety.

[0246] Vaccinia-based infection / transfection systems can be conveniently used to provide inducible transient expression of coding sequences of interest (e.g., CCN3 expression cassettes) in host cells. In this system, cells are first infected in vitro with a vaccinia virus recombinant encoding a phage T7 RNA polymerase. This polymerase exhibits high specificity because it transcribes only templates carrying a T7 promoter. Post-infection, cells are transfected with polynucleotides of interest driven by the T7 promoter. The polymerase, expressed in the cytoplasm of the vaccinia virus recombinant, transcribes the transfected DNA into RNA, which is then translated into protein by the host translational machinery. This method provides high-level, transient cytoplasmic production of large amounts of RNA and its translation products. See, for example, Elroy-Stein and Moss, Proceedings of the National Academy of Sciences (1990) 87:6743-6747; Fuerst et al., Proceedings of the National Academy of Sciences (1986) 83:8122-8126.

[0247] As an alternative to infecting with vaccinia virus or fowlpox virus recombinants or delivering genes using other viral vectors, an amplification system can be used that leads to high-level expression after introduction into host cells. Specifically, the T7 RNA polymerase promoter preceding the coding region of the T7 RNA polymerase can be engineered. Translation of RNA derived from this template will produce T7 RNA polymerase, which will then transcribe more template. Simultaneously, cDNA will be present, and its expression is under the control of the T7 promoter. Therefore, some T7 RNA polymerase produced by translating the amplified template RNA will lead to the transcription of the desired gene. Because some T7 RNA polymerase is required to initiate amplification, the T7 RNA polymerase can be introduced into the cell along with the template to trigger the transcriptional response. The polymerase can be introduced as a protein or onto a plasmid encoding the RNA polymerase. For further discussion of the T7 system and its use in transforming cells, see, for example, International Publication No. WO 94 / 26911; Studier and Moffatt, Journal of Molecular Biology (1986) 189:113-130; Deng and Wolff, Genes (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nucleic Acids Res. (1993) 21:2867-2872; Chen et al., Nucleic Acids Research (1994) 22:2114-2120; and U.S. Patent No. 5,135,855.

[0248] Synthetic expression cassettes of interest can also be delivered without viral vectors. For example, synthetic expression cassettes can be packaged as DNA or RNA in liposomes before delivery to a subject or cells derived from them. Liposome encapsulation is typically accomplished using liposomes capable of stably binding or trapping and retaining nucleic acids. The ratio of concentrated DNA to lipid formulation can vary, but is typically about 1:1 (mg DNA: micromolar lipid), or more lipids. For a review of the use of liposomes as carriers for nucleic acid delivery, see Hug and Sleight, *Biochim. Biophys. Acta.* (1991), 1097:1-17; Straubinger et al., *Methods of Enzymology* (1983), Vol. 101, pp. 512-527.

[0249] The liposome formulations used in this invention include cationic (positively charged) formulations, anionic (negatively charged) formulations, and neutral formulations, with cationic liposomes being particularly preferred. Cationic liposomes have been shown to functionally mediate the following intracellular delivery: plasmid DNA (Felgner et al., Proceedings of the National Academy of Sciences (1987) 84:7413-7416); mRNA (Malone et al., Proceedings of the National Academy of Sciences (1989) 86:6077-6081); and purified transcription factors (Debs et al., Journal of Biochemistry (1990) 265:10189-10192).

[0250] Cationic liposomes are readily available. For example, N[1-2,3-dioleoyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes can be obtained from GIBCO BRL, Grand Island, NY, under the trademark Lipofectin. (See also Felgner et al., Proceedings of the National Academy of Sciences (1987) 84:7413-7416). Other commercially available lipids include (DDAB / DOPE) and DOTAP / DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. For a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonium)propane) liposomes, see, for example, Szoka et al., Proceedings of the National Academy of Sciences (1978) 75:4194-4198; PCT Publication No. WO 90 / 11092.

[0251] Similarly, anionic and neutral liposomes are readily available, for example, from Avanti PolarLipids (Birmingham, AL), or can be readily prepared using readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), etc. These materials can also be mixed with DOTMA and DOTAP starting materials in appropriate ratios. Methods for preparing liposomes using these materials are well known in the art.

[0252] Liposomes can contain multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Various liposome-nucleic acid complexes are prepared using methods known in the art. See, for example, Straubinger et al., *Methods of Immunology* (1983), Vol. 101, pp. 512-527; Szoka et al., *Proceedings of the National Academy of Sciences* (1978) 75:4194-4198; Papahadjopoulos et al., *Chinese Journal of Biochemistry and Biophysics* (1975) 394:483; Wilson et al., *Cell* (1979) 17:77; Deamer and Bangham, *Chinese Journal of Biochemistry and Biophysics* (1976) 443:629; Ostro et al., *Research Letters in Biochemistry and Biophysics* (1977) 76:836; Fraley et al., *Proceedings of the National Academy of Sciences* (1979) 76:3348; Enoch and Strittmatter, *Proceedings of the National Academy of Sciences* (1979) 76:145; Fraley et al., *Journal of Biochemistry* (1980). 255:10431; Szoka and Papahadjopoulos, Proceedings of the National Academy of Sciences (1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.

[0253] DNA and / or peptides can also be delivered in helical lipid compositions similar to those described by Papahadjopoulos et al., Acta Biochimica et Biophysica Sinica (1975) 394:483-491. See also U.S. Patent Nos. 4,663,161 and 4,871,488.

[0254] Expression cassettes of interest can also be encapsulated, adsorbed onto, or associated with particulate carriers. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactide) and poly(lactide-co-glycolic acid) called PLG. See, for example, Jeffery et al., *Pharm. Res.* (1993) 10:362-368; McGee JP et al., *J Microencapsul.* 14(2):197-210, 1997; O'Hagan DT et al., *Vaccine* 11(2):149-54, 1993.

[0255] In addition, other particulate systems and polymers can be used for in vivo or in vitro delivery of nucleic acids of interest. For example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules can be used to transfer nucleic acids of interest. Similarly, DEAE-mediated transfection, calcium phosphate precipitation, or precipitation using other insoluble inorganic salts such as strontium phosphate, aluminum silicate including bentonite and kaolin, chromium oxide, magnesium silicate, talc, etc., will be used in the methods of the present invention. For a review of delivery systems that can be used for gene transfer, see, for example, Felgner, PL, *Advanced Drug Delivery Reviews* (1990) 5:163-187. Peptides (U.S. Patent No. 5,831,005, issued November 3, 1998, by reference, to Zuckerman, RN et al., incorporated herein by reference) can also be used to deliver the constructs of the present invention.

[0256] Furthermore, biological projectile delivery systems employing particulate carriers such as gold and tungsten are particularly suitable for delivering synthetic expression cassettes encoding CCN3. The particulate is coated with the synthetic expression cassette to be delivered and, typically in a reducing atmosphere, is accelerated to high speed using gunpowder release from a “gene gun.” For a description of such techniques and therefore useful devices, see, for example, U.S. Patent Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744. Additionally, needle-free injection systems can be used (Davis, HL et al., Vaccines 12:1503-1509, 1994; Bioject Inc., Portland, Oreg.).

[0257] Recombinant vectors carrying synthetic expression cassettes encoding CCN3 are formulated into compositions for delivery to vertebrate subjects. These compositions may be prophylactic (to prevent bone loss) or therapeutic (to treat bone or cartilage conditions or symptoms associated with bone or cartilage degeneration). The composition will contain a “therapeuticly effective amount” of the nucleic acid of interest, enabling the production in vivo of a quantity of the CCN3 protein (or a bioactive fragment thereof) to stimulate bone or cartilage regeneration and repair in the individual receiving the composition (e.g., increased bone density, increased bone mass, increased bone strength and / or reduced fatty bone marrow, and / or increased cartilage growth, increased cartilage mass, regenerated cartilage). The exact amount required will vary depending on: the subject being treated; the age and general condition of the subject to be treated; the desired level of protection; the severity of the condition being treated; the specific CCN3 protein produced and its administration method, and other factors. A suitable effective amount can be readily determined by those skilled in the art. Therefore, the “therapeuticly effective amount” will fall within a relatively wide range that can be determined through routine testing.

[0258] The composition will typically include one or more pharmaceutically acceptable excipients or mediators, such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, etc. Further, auxiliary substances, such as wetting agents or emulsifiers, pH buffers, surfactants, etc., may be present in such mediators. Certain promoters of nucleic acid uptake and / or expression may also be included in or co-administered with the composition.

[0259] Once formulated, the composition can be administered directly to a subject (e.g., as described above), or alternatively, delivered ex vivo to cells derived from the subject using methods such as those described above. For example, methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and may include, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, lipofectamine and LT-1-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the cell nucleus.

[0260] Direct delivery of the in vivo synthetic expression cassette composition is typically accomplished by injection, with or without the viral vector described above, using a conventional syringe, a needle-free device such as Bioject™, or a gene gun such as the Accell™ gene delivery system (PowderMed Ltd, Oxford, England).

[0261] Pharmaceutical Composition

[0262] The CCN3 protein, or a recombinant polynucleotide containing a coding sequence encoding the CCN3 protein, can be formulated into a pharmaceutical composition optionally containing one or more pharmaceutically acceptable excipients. Further, for the treatment of chondrodysplasia, a VEGF inhibitor can be provided in the same pharmaceutical composition as the CCN3 protein or in a separate pharmaceutical composition containing one or more pharmaceutically acceptable excipients.

[0263] Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Suitable excipients for injectable compositions include water, alcohols, polyols, glycerol, vegetable oils, phospholipids, and surfactants. Carbohydrates such as sugars, derivatized sugars such as sugar alcohols, aldonic acids, esterified sugars, and / or sugar polymers can be present as excipients. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides, such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides, such as raffinose, mesotriose, maltodextrin, dextran, starch, etc.; and sugar alcohols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucosyl sorbitol), pyranosyl sorbitol, inositol, etc. Excipients may also include inorganic salts or buffer solutions, such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and combinations thereof.

[0264] The composition may also contain an antimicrobial agent for preventing or inhibiting the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for use in this invention include benzalkonium chloride, benzyl chloride, benzyl alcohol, hexadecylpyridinium chloride, chlorobutanol, phenol, phenethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

[0265] Antioxidants may also be present in the composition. Antioxidants are used to prevent oxidation, thereby preventing the degradation of CCN3, VEGF inhibitors, or other components of the formulation. Suitable antioxidants for use in this invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphite, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0266] Surfactants can be present as excipients. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80", and pluronics, such as F68 and F88 (BASF, Mount Olive, New Jersey); dehydrated sorbitol esters; lipids, such as phospholipids, such as lecithin and other phosphatidylcholine, phosphatidylethanolamine (although preferably not in liposome form), fatty acids and fatty acid esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.

[0267] Acids or bases may be present in the composition as excipients. Non-limiting examples of acids that may be used include those selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, but are not limited to, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

[0268] The amount of CCN3 and / or VEGF inhibitors in the composition (e.g., when contained in a drug delivery system) will vary depending on many factors, but will be optimally therapeutically effective when the composition is in a unit dosage form or container (e.g., a vial). The therapeutically effective dose can be experimentally determined by repeatedly administering increasing amounts of the composition to determine which amount produces the clinically desired endpoint.

[0269] The amount of any single excipient in the composition will vary depending on the nature and function of the excipient and the specific requirements of the composition. Typically, the optimal amount of any single excipient is determined through conventional experiments, i.e., by preparing compositions containing varying amounts of excipient (ranging from low to high), examining stability and other parameters, and then determining the range from which optimal performance is obtained without significant side effects. However, generally, the excipient will be present in the composition in an amount of about 1% to about 99% by weight, preferably about 5% to about 98% by weight, more preferably about 15% to about 95% by weight, with a concentration of less than 30% by weight being most preferred. These aforementioned pharmaceutical excipients, along with other excipients, are described in the following: “Remington: The Science & Practice of Pharmacy”, 19th edition, Williams & Williams, (1995); “Physician's Desk Reference”, 52nd edition, Medical Economics, Montvale, NJ (1998); and “Handbook of Pharmaceutical Excipients”, 3rd edition, American Pharmaceutical Association, Washington, D.C., 2000.

[0270] The compositions encompass all types of formulations, and particularly those suitable for injection, such as powders or lyophilized preparations that can be reconstituted with a solvent prior to use, ready-made solutions or suspensions for injection, dried insoluble compositions for combination with a carrier prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of suitable diluents for reconstituted solid compositions prior to injection include antibacterial water for injection, 5% dextran aqueous solution, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. Regarding liquid pharmaceutical compositions, solutions and suspensions are contemplated. Additional preferred compositions include those for oral, ocular, or topical delivery.

[0271] The pharmaceutical formulations described herein can also be contained in syringes, implantable devices, etc., depending on the intended delivery and usage pattern. Preferably, the compositions containing CCN3 and / or VEGF inhibitors are in unit dose form, meaning that the amount of conjugate or composition suitable for a single dose is in a pre-measured or pre-packaged form.

[0272] The compositions described herein may optionally include one or more additional pharmaceutical agents, such as other drugs for treating bone or cartilage disorders or conditions associated with bone or cartilage degeneration, or other pharmaceutical agents for treating the condition or disease of the subject. The combination formulation may include CCN3 and / or VEGF inhibitors and one or more drugs for treating bone or cartilage disorders or conditions associated with bone or cartilage degeneration, such as bone anabolic agents or bone morphogenetic agents, including but not limited to parathyroid hormone, teriparatide, abapatide, and romozide; anti-reabsorption agents, including but not limited to estrogens and estrogen agonists, bisphosphonates, and denosumab; analgesics, including but not limited to acetaminophen, and non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs, including but not limited to aspirin, ibuprofen, dextro ibuprofen, naproxen, fenprofen, ketoprofen, dextro ketoprofen, flurbiprofen, oxaprazin, loxoprofen, piroxifen, zaltoprofen, fenbufen, tiprofenicol, carprofen, indomethacin, acemetacin, tometine, sulindac, etodoxacin, ketoprofen, diclofenac, fenclofenac, aceclofenac, bromofenac, fentimicin, nabumetone, piroxicam, ampixicam, meloxicam, tenoxicam, droxicam, lornoxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamic acid, tofenamic acid, and etodoxacin; and steroids, including but not limited to hydrocortisone, triamcinolone, prednisone, prednisolone, methylprednisolone, and dexamethasone, or other pharmaceutical agents. Alternatively, such agents may be included in a separate composition from the composition containing CCN3 and / or VEGF inhibitors, and may be administered simultaneously, before, or after the composition containing CCN3 and / or VEGF inhibitors.

[0273] In some embodiments, a dose of CCN3 is provided in an implant (e.g., a matrix or scaffold for local delivery of CCN3), wherein CCN3 is provided as the CCN3 protein or a biologically active variant or fragment thereof. The effective dose can be determined based on the specific tissue, the release rate from the implant, the size of the implant, etc., and can be determined empirically by those skilled in the art. This dose can provide the biological activity equivalent to 1 mg of CCN3 protein, 10 mg, 100 mg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 250 mg, 500 mg, 750 mg, or 1 g of CCN3 protein. The dose can be administered at a single time point, e.g., as a single implant; or it can be administered in multiple doses, e.g., via a microneedle configuration. The dose can be administered once, twice, three times, four times, five times, ten times, or more as needed to achieve the desired effect, and administration can be daily, every two days, every three days, every four days, weekly, every two weeks, monthly, or for longer periods.

[0274] Drug delivery devices include structures that can be implanted and release an active agent, such as CCN3 for treating bone conditions, or a combination of CCN3 and a VEGF inhibitor for treating cartilage conditions at a target site (e.g., a site of bone or cartilage defect or injury). Implantable drug delivery devices can be broadly classified into two groups: passive implants and active implants. The first group includes two main types of implants: biodegradable implants and non-biodegradable implants. Active systems rely on energy-dependent methods that provide a driving force to control drug release. The second group includes devices such as osmotic pressure gradients and electromechanical actuations.

[0275] Passive polymer implants are typically relatively simple devices without moving parts, relying on passive diffusion for drug release. They are usually made of drugs packaged within biocompatible polymer molecules. Several parameters, such as drug type / concentration, polymer type, implant design, and surface properties, can be varied to control the release profile. Passive implants can be divided into two main categories: non-biodegradable systems and biodegradable systems.

[0276] Non-biodegradable implants are typically made using polymers such as silicones, poly(urethane), poly(acrylate), or copolymers such as poly(ethylene-vinyl acetate). Poly(ethylene-vinyl acetate) (PEVA) is a thermoplastic copolymer of ethylene and vinyl acetate. Poly(siloxanes), or silicones, are organosilicon polymer materials composed of silicon and oxygen atoms. Side groups can be methyl, vinyl, or phenyl groups. These groups affect the properties of the polymer. Poly(siloxanes) have been widely used in medicine due to their unique combination of thermal stability, biocompatibility, chemical inertness, and elastomer properties. Silicones commonly used in medical devices are vulcanized at room temperature. They are prepared using two-component poly(dimethylsiloxanes) (PDMS) in the presence of a catalyst (platinum-based compound). The final material is formed via an addition hydrosilylation reaction. An alternative method to obtain silicones for medical applications is to use linear PDMS with hydroxyl end groups. This linear polymer is crosslinked with low molecular weight tetra(alkoxysilane) using a stannous octoate catalyst.

[0277] This type of device can be either a monolithic implant or a reservoir implant. Monolithic implants are made of a polymer matrix in which the drug is uniformly dispersed. Reservoir implants, on the other hand, comprise a dense drug core covered by a permeable, non-biodegradable membrane. The membrane thickness and the permeability of the membrane will determine the release kinetics.

[0278] Biodegradable implants are made using polymers or block copolymers that can break down into smaller fragments that are subsequently excreted or absorbed by the body. Typically, they are made using polymers such as collagen, PEG, chitosan, poly(caprolactone) PCL, poly(lactic acid) PLA, or poly(lactic-co-glycolic acid) PLGA. Many other biodegradable polymers exist for drug delivery, including poly(amides), poly(anhydrides), poly(phosphazenes), and poly(dioxanone). Poly(anhydrides) have low hydrolytic stability, resulting in rapid degradation rates, making them suitable for short-term controlled delivery systems. Poly(phosphazenes) have degradation rates that can be fine-tuned by appropriate substitution with specific chemical groups, and their use in bone tissue regeneration and drug delivery has been investigated. Poly(p-dioxanone), like PCL, is a polylactone already used for purposes such as drug delivery and tissue engineering. They do not need to be extracted after implantation because they are degraded by the patient's body. They can be manufactured into monolithic implants and reservoir implants. In addition to biopolymers such as PLA mentioned above, there are also several natural polymers that represent a promising class of materials with broad applications, including for implantable devices. These natural polymers include collagen, hyaluronic acid, cellulose, chitosan, silk fibroin, and other naturally derived proteins, as well as collagen, gelatin, albumin, elastin, and milk proteins. Compared to conventional materials (metals and ceramics) or synthetic polymers, these materials exhibit certain advantages, such as biocompatibility, biodegradability, and non-cytotoxicity, making them promising candidates for implantable drug delivery devices.

[0279] Dynamic or active polymer implants have a positive driving force to control the release of drugs from the device. Most implants in this category are electronic systems made of metallic materials. Dynamic drug delivery implants are primarily pump-type implants. The main type of active polymer implant is the osmotic pump. This type of device is mainly formed by a semi-permeable membrane surrounding a drug reservoir. This membrane should have orifices that will allow drug release. The osmotic gradient will allow a steady inflow of fluid into the implant. This process will cause an increase in pressure within the implant, which will force the drug to be released through the orifices. This design allows for constant drug release (zero-order kinetics). This type of device allows for favorable release rates, but the drug loading capacity is limited.

[0280] In some embodiments, CCN3 and / or VEGF inhibitors are prepared as an injectable paste. The paste can be injected into the implantation site. In some embodiments, the paste can be prepared prior to implantation and / or stored in a syringe below ambient temperature until needed. In some embodiments, the composite material can be administered by injection, similar to bone cement, which can be used to connect and hold bone fragments in place, or to improve adhesion, for example, in hip prostheses, to replace damaged cartilage in the joint, etc. Implantation can also be performed in a non-open surgical setting.

[0281] In other embodiments, CCN3 and / or VEGF inhibitors are prepared as a moldable bone putty. Hydrated graft bone putty can be prepared and molded into a shape approximating any implant. The putty is then pressed into the appropriate location to fill voids in cartilage, bone, alveolar bone, or other sites. In some embodiments, graft bone putty can be used to repair defects in bone nonunion, or in other cases where there are large fractures, holes, or voids to be filled and a certain degree of mechanical integrity in the implant material is required to both fill the gap and maintain its shape.

[0282] In some implementations, additional factors and / or cells are provided during the treatment process. While in many cases, endogenous SSCs are sufficient for bone or cartilage regeneration, exogenous cells can be provided at the site of local acute injury. Cells can be SSCs or non-SSCs, such as stem cells, e.g., adipose-derived stem cells. Cells can be autologous or allogeneic. Cells can be provided along with the delivery of CCN3 or a combination of CCN3 and a VEGF inhibitor, e.g., simultaneously, shortly before, shortly after, etc., and can be included with CCN3 or a combination of CCN3 and a VEGF inhibitor in a single implant, as a standalone implant, or as an injection, etc.

[0283] Systems intended for pharmaceutical use, i.e., drug delivery devices having CCN3 and / or VEGF inhibitors and optionally other factors, may include pharmaceutically acceptable, non-toxic carriers as diluents, depending on the desired formulation. These diluents are defined as media commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected to not affect the bioactivity of the composition. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextran solution, and Hank's solution. Furthermore, NR pharmaceutical compositions or formulations may include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The composition may also contain additional substances that approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, humectants, and detergents.

[0284] The toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and / or laboratory animals, including, for example, determining the LD50 (lethal dose in 50% of the population) and ED50 (the therapeutically effective dose in 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. Compounds exhibiting a large therapeutic index are preferred.

[0285] Data obtained from cell culture and / or animal studies can be used to formulate dosage ranges for human use. Dosages of the active ingredient are typically within a range that includes circulating concentrations with low toxicity, including an ED50. Dosages can vary within this range depending on the dosage form and route of administration.

[0286] The components used to formulate pharmaceutical compositions preferably have high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food NF grade, typically at least analytical grade, and more typically at least pharmaceutical grade). Furthermore, compositions intended for in vivo use are generally sterile. With regard to the synthesis of a given compound prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins that may be present during the synthesis or purification process. Compositions intended for parenteral administration are also sterile, substantially isotonic, and prepared under GMP conditions.

[0287] The effective amount of a therapeutic composition to be administered to a specific patient will depend on a number of factors, some of which will vary from patient to patient. A competent clinician will be able to determine the effective amount of the therapeutic agent administered to a patient to stop or reverse disease progression as needed. Using LD50 animal data and other available information about the agent, clinicians can determine the maximum safe dose for an individual based on the route of administration. For example, considering that a therapeutic composition is administered into more body fluids, an intravenous dose may be higher than an intrathecal dose. Similarly, compositions that are rapidly cleared from the body may be administered at higher doses or in repeated doses to maintain therapeutic concentrations. Using common techniques, a competent clinician will be able to optimize the dosage of a specific therapeutic agent during routine clinical trials.

[0288] CCN3 is used to treat bone diseases.

[0289] Treatment with the CCN3 protein or a vector containing a coding sequence encoding CCN3 for at least one effective treatment cycle will be administered to the subject for the treatment of bone conditions or symptoms associated with bone degeneration. Bone conditions and symptoms associated with bone degeneration include, but are not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury (e.g., fractures caused by falls, road traffic accidents, fights, or surgery), pathological fractures, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

[0290] A “therapeutic effective dose or amount” of the CCN3 protein or a vector containing a coding sequence encoding the CCN3 protein refers to an amount that, when administered as described herein, produces a positive therapeutic response, such as improvement in a bone condition or symptom associated with bone degeneration. Improvement may include increased bone mineral density, increased bone mass, increased bone strength, increased bone mineral density, and / or reduced adipose bone marrow. Further, a therapeutic effective dose or amount may stimulate osteochondral skeletal stem cells to generate bone.

[0291] In some embodiments, a combination of therapeutically effective doses of a composition comprising the CCN3 protein, or a vector comprising a coding sequence encoding CCN3, and / or one or more other therapeutic agents, such as other drugs for treating bone conditions or symptoms associated with bone degeneration, or other pharmaceutical agents. The compositions of the present invention are typically, but not necessarily, administered orally, by injection (subcutaneous, intravenous, intramuscular, or intraperitoneal), by infusion, or topically. Additional routes of administration are also contemplated, such as intrahepatic, intraosseous, pulmonary, rectal, percutaneous, transmucosal, intrathecal, pericardial, intraarterial, etc.

[0292] This formulation is also suitable for topical treatment. In one particular embodiment, the composition is used for the topical delivery of the CCN3 protein or a vector containing a coding sequence encoding CCN3 for the treatment of bone conditions or symptoms associated with bone degeneration. For example, the composition can be applied topically near bone defects or injuries requiring repair or regeneration. Specific formulations and appropriate methods of administration are selected to target the CCN3 protein or a vector expressing the CCN3 protein to sites requiring new bone growth or bone repair.

[0293] Pharmaceutical formulations may be in the form of a liquid solution or suspension to be administered immediately before application, but may also take other forms such as syrup, cream, ointment, tablet, capsule, powder, gel, matrix, suppository, etc. Pharmaceutical compositions containing CCN3 protein and / or other agents may be administered using the same or different routes of administration according to any medically acceptable method known in the art.

[0294] In another embodiment, a pharmaceutical composition comprising the CCN3 protein or a carrier and / or other agents comprising a coding sequence encoding the CCN3 protein is administered prophylactically, for example, to prevent bone loss. Such prophylactic application would be particularly valuable for lactating female subjects or subjects with a genetic predisposition or condition that increases the risk of developing osteoporosis or osteopenia (e.g., menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy). In another embodiment, a pharmaceutical composition comprising the CCN3 protein or a carrier and / or other agents comprising a coding sequence encoding the CCN3 protein is administered therapeutically to subjects suffering from osteoporosis or osteopenia.

[0295] In another embodiment, a pharmaceutical composition comprising the CCN3 protein or a carrier and / or other agents encoding the CCN3 protein is in a sustained-release formulation or a formulation administered using a sustained-release device. Such devices are well known in the art and include, for example, transdermal patches and microimplantable pumps, which can deliver drug over time in a continuous, steady-state manner at various doses to achieve the sustained-release effect of a non-sustained-release pharmaceutical composition.

[0296] This disclosure also provides a method for administering a conjugate containing the CCN3 protein to a patient suffering from a bone condition or symptom associated with bone degeneration or a symptom that responds to treatment with the CCN3 protein contained in the conjugate or composition. The method includes administering a therapeutically effective amount of the conjugate or drug delivery system via any of the methods described herein, preferably as part of a pharmaceutical composition. The administration method can be used to treat any symptom that responds to treatment with the CCN3 protein.

[0297] Those skilled in the art will understand that specific CCN3 proteins can effectively treat certain conditions. The actual dose to be administered will vary depending on the subject's age, weight, and general condition, as well as the specific bone condition being treated, the severity of the condition being treated, the judgment of the healthcare professional, and the specific CCN3 protein or conjugate being administered. The therapeutically effective dose can be determined by those skilled in the art and will be adjusted to the specific requirements of each particular case.

[0298] In some embodiments, multiple therapeutically effective doses of CCN3 protein are administered or administered intermittently according to a daily dosing regimen. For example, the therapeutically effective dose may be administered one day, two days, three days, four days, or five days a week, etc. "Intermittent" administration means that the therapeutically effective dose may be administered, for example, every other day, every two days, every three days, once a week, every other week, etc. For example, in some embodiments, the composition containing CCN3 protein is administered once a week, twice a week, or three times a week for extended periods, such as for 1, 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks, etc. "Twice-weekly" or "two times per week" means administering the two therapeutically effective doses of the agent discussed to the subject over a 7-day period starting from day 1 of the first week of administration, wherein there are at least 72 hours between doses and at most 96 hours between doses. "Thirdly weekly" or "three times per week" refers to administering three therapeutically effective doses to a subject over a 7-day period, allowing a minimum of 48 hours and a maximum of 72 hours between doses. For the purposes of this disclosure, this type of administration is referred to as "intermittent" therapy. Subjects may receive intermittent therapy (i.e., administering a therapeutically effective dose once, twice, or three times per week) for one or more weekly cycles according to the methods described herein until the desired therapeutic response is achieved. The agent may be administered via any acceptable route of administration as described below. The amount administered will depend on the potency of the specific CCN3 protein, the specific bone or cartilage condition being treated, the magnitude of the desired effect, and the route of administration.

[0299] Purified CCN3 protein or a vector containing a coding sequence encoding CCN3 protein (again, preferably provided as part of a pharmaceutical formulation) can be administered alone or in combination with one or more other therapeutic agents, such as bone anabolic agents or bone morphogenetic agents, including but not limited to parathyroid hormone, teriparatide, abapatide, and romozolumab; anti-resorption agents, including but not limited to estrogens and estrogen agonists, bisphosphonates, and denosumab; analgesics, including but not limited to acetaminophen and nonsteroidal anti-inflammatory drugs (NSAIDs), including but not limited to aspirin, ibuprofen, dextrobuprofen, naproxen, fenprofen, ketoprofen, dextroketoprofen, flurbiprofen, and oxaliplatin. Saprozidin, loxoprofen, piroxifen, zaltoprofen, fenbufen, tiprofenic acid, carprofen, indomethacin, acimetidine, tometamine, sulindac, etodoxacin, ketoroxyfen, diclofenac, fenclofenac, aceclofenac, bromofenac, fentimicin, nabumetone, piroxicam, ampixicam, meloxicam, tenoxicam, droxicam, lornoxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamic acid, tofenamic acid, and etodoxacin; steroids, including but not limited to hydrocortisone, triamcinolone, prednisone, prednisolone, methylprednisolone, and dexamethasone, or other pharmaceutical preparations used to treat specific symptoms or diseases according to various dosing regimens based on the clinician's judgment and the patient's needs. Specific dosing regimens will be known to those skilled in the art or determined experimentally using conventional methods. Exemplary dosing regimens include, but are not limited to, administration five times daily, four times daily, three times daily, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Preferred compositions are those requiring administration no more than once daily.

[0300] The CCN3 protein or a carrier containing a coding sequence encoding the CCN3 protein may be administered before, concurrently with, or after other pharmaceutical agents. If administered concurrently with other pharmaceutical agents, the CCN3 protein or a carrier containing a coding sequence encoding the CCN3 protein may be administered in the same or different compositions. Therefore, the CCN3 protein or a carrier containing a coding sequence encoding the CCN3 protein and / or other pharmaceutical agents can be presented to an individual via concurrent therapy. "Concurrent therapy" is intended to refer to administration to a subject that produces a therapeutic effect in the subject undergoing the therapy. For example, depending on a specific dosing regimen, concurrent therapy can be achieved by administering a dose of a pharmaceutical composition containing the CCN3 protein or a carrier containing a coding sequence encoding the CCN3 protein and a dose of a pharmaceutical composition containing at least one other pharmaceutical agent, the combination of which constitutes a therapeutically effective dose, such as another drug for treating bone conditions or symptoms associated with bone degeneration. Similarly, the CCN3 protein or a carrier containing a coding sequence encoding the CCN3 protein and one or more other therapeutic agents may be administered at at least one therapeutic dose. The administration of individual drug compositions may be performed simultaneously or at different times (i.e., sequentially, in any order, on the same day or on different days), provided that the combination of these substances produces a therapeutic effect in the subject undergoing the therapy.

[0301] The methods described herein can be used to treat bone conditions or symptoms associated with bone degeneration in human subjects. The methods described herein will also be used in veterinary applications to treat such bone conditions or symptoms in, for example, domestic animals, including but not limited to, pets such as dogs and cats; and farm animals such as sheep, goats, pigs, horses, and cattle.

[0302] Bone grafts

[0303] This article also provides a method for generating bone grafts by culturing osteochondral skeletal stem cells under suitable conditions for osteochondral skeletal stem cells in the presence of CCN3 to generate bone. In some cases, the osteochondral skeletal stem cells are encapsulated within a biocompatible scaffold such that the cells remain within the scaffold when the tissue graft is transplanted at the individual's transplantation site (e.g., a site requiring bone replacement or repair). In some embodiments, the scaffold provides support for the bone generated from the osteochondral skeletal stem cells and has a defined geometry that mimics the shape of the bone or a portion of bone requiring replacement or repair. Preferably, the scaffold has mechanical properties similar to bone.

[0304] As an example, a biocompatible scaffold can comprise any material that allows CCN3 to be incorporated and is compatible with the addition of osteochondral skeletal stem cells. The carrier, matrix, or scaffold can be primarily non-immunogenic and biodegradable. Examples of biodegradable materials include, but are not limited to, alginate, polyglycolic acid (PGA), polylactic acid (PLA), hyaluronic acid, catgut suture materials, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin, cotton, or other naturally occurring biodegradable materials. In some embodiments, the matrix is ​​biodegradable for a period of less than one year, more preferably less than six months, and most preferably more than two to ten weeks. The polymer composition and manufacturing method can be used to determine the degradation rate. It may be preferred to sterilize the matrix or scaffold material prior to application or implantation, for example, by treating it with ethylene oxide or by gamma radiation or electron beam irradiation. In addition, many other materials can be used to form scaffold or framework structures, including but not limited to: hydroxyapatite, nylon (polyamide), dacron (polyester), polystyrene, polypropylene, polyacrylate, polyethylene compounds (e.g., polyvinyl chloride), polycarbonate (PVC), polytetrafluoroethylene (PTFE, Teflon), thermox (TPX), polymers of hydroxy acids such as polylactic acid (PLA), polyglycolic acid (PGA) and polylactic-glycolic acid (PLGA), polyorthoesters, polyanhydrides, polyphosphazenes and various polyhydroxyalkanoates and combinations thereof.

[0305] Suitable matrices include meshes, sponges, or polymeric hydrogels. Hydrogels are defined as substances formed when organic polymers (natural or synthetic) are crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open lattice structure that traps water molecules to form a gel. Generally, these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcoholic solutions with charged side groups, or their monovalent ionic salts. Exemplary hydrogel polymers include, but are not limited to, natural polymers such as polysaccharides, including hyaluronic acid, chitosan, heparin, alginate, cellulose, dextrose, and agarose; and proteins, including fibrin, fibrinogen, collagen, elastin, gelatin, silk fibroin, laminin, fibronectin, albumin, thrombin, and keratin; modified natural polymers, including hydroxymethyl cellulose, hydroxyethyl cellulose, gelatin methacrylate, polyanionic N-carboxymethyl chitosan, and polycationic N-trimethyl chitosan; and synthetic polymers, including polyvinyl alcohol, N-ethylene... Polypyrrolidone, polyethylene glycol, poly(ethylene glycol) diacrylate, polyacrylamide, poly(N-isopropylacrylamide), sodium polyacrylate, acrylate polymers and copolymers, such as hydroxyethyl methacrylate, ethyl methacrylate, hydroxypropyl methacrylate, ethylene glycol dimethacrylate, methyl methacrylate, glycidyl methacrylate and hydroxyethyl methacrylate, poly(N-isopropylacrylamide-co-acrylic acid), polyester, polyurethane, nylon, synthetic polyamino acids, prolysins; and combinations thereof, as well as other such molecules, including recombinant versions of such polymers. In some embodiments, the hydrogel comprises polyacrylamide.

[0306] Methods for preparing hydrogels are well known in the art. See, for example, Barbucci, “Hydrogels: Biological Properties and Applications,” Springer, 2009; “Hydrogels in Cell-Based Therapies,” edited by Connon and Hamley, Royal Society of Chemistry, 2014; and “Tunable Hydrogels,” edited by Lavrentieva, Pepelanov, and Seliktar, Springer Nature Switzerland AG, 2020; all incorporated herein by reference. In some cases, hydrogels are cross-linked through chemical cross-linking. Exemplary chemical crosslinking agents include, but are not limited to, N,N,N',N'-tetramethylethylenediamine (TEMED), ammonium persulfate, glutaraldehyde, formaldehyde, epoxy compounds, dialdehyde, N,N'-methylenebis(acrylamide) MBA, ethylene glycol diacrylate (EGDA), ethylene glycol dimethylacrylate (EGDMA), PEG diacrylate (PEGDA), glyoxal, epichlorohydrin, and sodium borate / boric acid. The crosslinking density can vary depending on the type and concentration of the chemical crosslinking agent used. Alternatively, the hydrogel can be photocrosslinked by exposure to light. UV-sensitive photoinitiators (i.e., polymerization initiated by exposure to UV light in the 200-400 nm range) or visible-light-sensitive photoinitiators (i.e., polymerization initiated by exposure to visible light in the 400-800 nm range) can be used. Photoinitiators can be used to initiate photopolymerization. Photopolymerization can involve free radical-initiated chain polymerization or bioorthogonal click reactions. Exemplary photoinitiators include, but are not limited to, (1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one), lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LAP), and eosin-γ. The crosslinking density can vary depending on the intensity and duration of the electromagnetic radiation used to promote the photopolymerization of the hydrogel. In some embodiments, the crosslinking density of the crosslinked hydrogel is in the range of 1 × 10⁻⁶. -15 moles / cm 3 Up to 1×10 -3 moles / cm3 Within the range.

[0307] The compressive modulus (i.e., material stiffness, also referred to herein as "modulus") of a hydrogel can range from 1 kPa to 70 kPa, including any stiffness within this range, such as 1 kPa, 2 kPa, 3 kPa, 4 kPa, 5 kPa, 6 kPa, 7 kPa, 8 kPa, 9 kPa, 10 kPa, 11 kPa, 12 kPa, 13 kPa, 14 kPa, 15 kPa, 16 kPa, 17 kPa, 18 kPa, 19 kPa, 20 kPa, 21 kPa, 22 kPa, 23 kPa, 24 kPa, 25 kPa, 26 kPa, 27 kPa, 28 kPa, 29 kPa, 30kPa, 31 kPa, 32 kPa, 33 kPa, 34 kPa, 35 kPa, 36 kPa, 37 kPa, 38 kPa, 39 kPa, 40 kPa, 45kPa, 50 kPa, 55 kPa, 60 kPa, 65 kPa or 70 kPa.

[0308] The stent can have any suitable lateral dimension (e.g., width and / or length, or diameter). In some cases, the polymer stent has a lateral dimension of about 1.0 cm or larger, such as about 2.0 cm or larger, about 3.0 cm or larger, about 4.0 cm or larger, including a lateral dimension of 5 cm or larger, and in some cases, it has a lateral dimension of about 10 cm or smaller, such as about 9.0 cm or smaller, about 8.0 cm or smaller, about 7.0 cm or smaller, about 6.0 cm or smaller, including a lateral dimension of about 5.0 cm or smaller. In some embodiments, the polymer stent has a lateral dimension in the range of about 1.0 cm to about 10 cm, such as about 1.0 cm to about 9.0 cm, about 2.0 cm to about 8.0 cm, including a lateral dimension of about 3.0 cm to about 7.0 cm.

[0309] In some embodiments, the composition is injected at or near the site of the fracture or defect. Any type of bone can be treated using this method, including but not limited to bones of the arm, hand, leg, foot, neck, head, or spine.

[0310] In some implementations, the treated subject suffers from a bone condition or symptom associated with bone degeneration. Bone conditions and symptoms associated with bone degeneration include, but are not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury (e.g., fractures caused by falls, road traffic accidents, fights, or surgery), pathological fractures, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy. In some implementations, bone grafts provide new bone for craniofacial surgery (e.g., for repairing craniofacial clefts, facial fractures, or congenital abnormalities), oral and maxillofacial surgery, plastic surgery, or reconstructive surgery, or for dental implants (e.g., for repairing or replacing bone in the skull or jawbone).

[0311] Bone grafts can contain any suitable amount of osteochondral bone stem cells. The number of cells can depend on a variety of factors, such as the size of the bone graft, the length of time the graft will remain in place, the condition to be treated with the bone graft, and / or the desired treatment outcome. In some cases, bone grafts include at least 10... 5 Cells, for example, at least 10 6 10 cells, at least 10 7 10 cells, at least 10 8 10 cells, at least 10 9 10 cells, at least 10 10 One or more cells.

[0312] Treatment of chondrodysplasia with CCN3 and VEGF inhibitors

[0313] A combination of at least one therapeutically effective dose of CCN3 (or a vector containing a coding sequence encoding CCN3) with a therapeutically effective dose of a VEGF inhibitor and mechanical stimulation. The “therapeuticly effective dose or amount” of CCN3 (or a vector containing a coding sequence encoding CCN3) and the VEGF inhibitor is intended to refer to the amount that produces a positive therapeutic response, such as improved recovery from cartilage damage, when administered in combination with mechanical stimulation (e.g., microfracture surgery or accidental acute local injury), as described herein. Improved recovery may include the generation of new cartilage at the treatment site (e.g., the damaged joint). For example, a therapeutically effective dose or amount may be used to treat cartilage damage or loss caused by traumatic injury or degenerative diseases such as arthritis or other diseases involving cartilage degeneration. Preferably, the therapeutically effective amount restores function and / or relieves pain and inflammation associated with cartilage damage or loss.

[0314] Cartilage conditions treatable by the methods described herein include, but are not limited to, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, and malignancy. In some embodiments, the subject method is used to treat injuries to hyaline cartilage, elastic cartilage, or fibrocartilage. In some embodiments, joints with damaged cartilage are treated by the subject method. For example, the damaged cartilage may be located in joints of the knee, hip, elbow, shoulder, hand, ankle, or spine.

[0315] In some implementations, each of the multiple therapeutically effective doses of the CCN3 and VEGF inhibitors will be administered according to a daily dosing regimen or intermittently. For example, the therapeutically effective dose may be administered one day, two days, three days, four days, or five days a week, etc. "Intermittent" administration is intended to mean that the therapeutically effective dose may be administered, for example, every other day, every two days, every three days, etc. For example, in some implementations, the CCN3 and VEGF inhibitors will be administered twice or three times a week for extended periods, such as for 1, 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks, etc. "Twice-weekly" or "two times per week" is intended to mean administering the two therapeutically effective doses of the drug in question to the subject over a 7-day period starting from day 1 of the first week of administration, wherein there are at least 72 hours and at most 96 hours between doses. "Thirdly weekly" or "three timesper week" refers to administering three therapeutically effective doses to a subject over a seven-day period, allowing a minimum of 48 hours and a maximum of 72 hours between doses. For the purposes of this invention, this type of administration is referred to as "intermittent" therapy. According to the method of this invention, a subject may receive intermittent therapy (i.e., administering a therapeutically effective dose twice or three times per week) for one or more weekly cycles until the desired therapeutic response is achieved. The medication may be administered via any acceptable route of administration as described below.

[0316] CCN3 can be administered before, concurrently with, or after a VEGF inhibitor. If administered concurrently with a VEGF inhibitor, CCN3 can be provided in the same or different compositions. Therefore, these two agents can be presented to an individual via concurrent therapy. "Concurrent therapy" refers to administration to a human subject that produces a therapeutic effect from the combination of substances in the subject undergoing the therapy. For example, concurrent therapy can be achieved, depending on a specific dosing regimen, by administering at least one therapeutically effective dose of a pharmaceutical composition containing CCN3 and at least one therapeutically effective dose of a pharmaceutical composition containing a VEGF inhibitor. The administration of the individual pharmaceutical compositions can be simultaneous (i.e., concurrently) or at different times (i.e., sequentially, in any order, on the same day, or on different days), provided that the combination of these substances produces a therapeutic effect in the subject undergoing the therapy.

[0317] In other embodiments, the pharmaceutical composition comprising agents such as CCN3 and / or VEGF inhibitors is a sustained-release formulation, or a formulation administered using a sustained-release device. Such devices are well known in the art and include, for example, transdermal patches and micro-implantable pumps, which can deliver drug over time in a continuous, steady-state manner at various doses to achieve a sustained-release effect in the absence of a sustained-release pharmaceutical composition.

[0318] Pharmaceutical compositions containing CCN3 and / or VEGF inhibitors can be administered using the same or different routes of administration according to any medically acceptable method known in the art. Suitable routes of administration include parenteral administration, such as subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravenous (IV), or infusion, oral and pulmonary, nasal, topical, percutaneous, and suppository administration. When the composition is administered via pulmonary delivery, the therapeutically effective dose is adjusted such that the solubility level of the agent, such as CCN3 and VEGF inhibitors, in the bloodstream is comparable to that obtained with a therapeutically effective dose administered parenterally, such as via SC, IP, IM, or IV. In some embodiments, the pharmaceutical composition containing CCN3 and / or VEGF inhibitors is administered via IM or SC injection. In some embodiments, the CCN3 and VEGF inhibitors are applied topically as drops, on a patch, or in a gel. In some embodiments, the CCN3 and VEGF inhibitors are applied topically to the site of cartilage loss or damage (e.g., joint).

[0319] Factors influencing the appropriate amount of various compositions to be administered include, but are not limited to, the method of administration, frequency of administration (i.e., daily or intermittent, such as twice or three times a week), the specific type of chondropathy undergoing treatment, the location of cartilage damage, the severity of the disease, medical history, whether the individual is undergoing concurrent therapy with another therapeutic agent, and the individual's age, height, weight, health status, and physical condition. Generally, higher doses of the agent are preferred as the weight of the subject undergoing treatment increases.

[0320] Reagent test kit

[0321] Kits containing any of the compositions described herein are also provided for treating patients with bone or cartilage disorders or conditions associated with bone or cartilage degeneration. The CCN3 protein or a vector containing a coding sequence encoding the CCN3 protein and optionally other therapeutic agents may be included in separate compositions or in the same composition. In some embodiments, the kit further includes a VEGF inhibitor (e.g., cabozantinib). In some embodiments, the kit further includes tools for performing microfractures, such as a cone or drill.

[0322] In some embodiments, the kit comprises a CCN3 protein comprising or consisting of the following: the amino acid sequence of SEQ ID NO: 1, or a variant comprising a sequence showing at least about 80%-100% sequence identity, including any percentage identity within that range, such as showing 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or a bioactive fragment thereof, wherein said variant or fragment retains the ability to increase bone mineral density, bone mass, bone strength, and / or the rate of bone formation, and / or the ability to regenerate cartilage, increase cartilage mass, and / or increase the rate of cartilage formation. The kit may include at least one container containing a solution of a unit dose of CCN3 and / or a VEGF inhibitor and pharmaceutically acceptable excipients; and instructions for administering the unit dose according to a desired or exemplary regimen based on the specific bone or cartilage condition being treated, age, weight, etc.

[0323] The kit may include a unit dose of a formulation containing a CCN3 and / or VEGF inhibitor suitable for the treatment methods described herein, such as in tablet or injectable doses. In addition to the container containing the unit dose, such kits may include an informational insert describing the use and associated benefits of treating bone conditions or symptoms associated with bone degeneration. The kit may include, for example, a dosing regimen for a CCN3 protein and / or VEGF inhibitor.

[0324] Formulations suitable for intravenous or intraperitoneal administration are of particular interest, and in such embodiments, the kit may further include a syringe or other device for such administration, which may be pre-filled with CCN3 protein. Instructions may be printed on a label affixed to the container or may be a packaging insert accompanying the container.

[0325] In addition to the components described above, the subject kit may further include (in some embodiments) instructions for practicing the subject method. These instructions may exist in a variety of forms within the subject kit, including one or more forms. One form of these instructions may be as printed information on a suitable medium or substrate, such as on one or more sheets of paper with the information printed thereon, in the kit packaging, in a packaging insert, etc. Another form of these instructions may be as computer-readable media on which information is already recorded, such as a disk, optical disc (CD), DVD, Blu-ray disc, flash drive, etc. Yet another possible form of these instructions is a website address that can be used via the Internet to access information on a removed site.

[0326] filter

[0327] The inventors have discovered that CCN3 is a brain-derived osteogenic factor that promotes bone and cartilage growth (see Examples). Therefore, agonists, mimics, and analogs of CCN3 can be used to treat bone or cartilage disorders and symptoms associated with bone or cartilage degeneration. Therefore, a screening method is provided for identifying candidate agents that increase or mimic CCN3 activity.

[0328] Various assays can be used for this purpose, and in many embodiments, candidate agents will be tested in different assays to confirm their activity in stimulating bone or cartilage growth and their efficacy in treating bone or cartilage disorders. For example, cell-based assays can determine the effect of an agent on the bioactivity of CCN3 (e.g., the ability to stimulate bone or cartilage production via osteochondral skeletal stem cells and / or increase the rate of bone or cartilage formation). In some embodiments, the ability of a CCN3 mimic or analogue to stimulate osteochondral skeletal stem cells to produce bone or cartilage is determined. Any convenient form can be used for the assay, such as wells, plates, flasks, etc., preferably high-throughput forms, such as multi-well plates. The test agent of interest is added to a reaction mixture containing or not containing CCN3 protein, and the effect of the agent on bone or cartilage growth is determined.

[0329] For example, a cellular assay to identify agonists that increase bone production can be performed by contacting cells with CCN3 and a candidate agent and measuring bone mineralization through the cells, wherein an increase in bone mineralization in the presence of the candidate agent, compared to a reference range of bone mineralization in control cells (i.e., cells not contacted with the candidate agent, in which the candidate agent is absent), indicates that the candidate agent is an agonist of CCN3.

[0330] In another example, a cellular assay for identifying a mimic or analogue of CCN3 that stimulates bone growth can be performed by contacting cells with a candidate agent and measuring bone mineralization through the cells, wherein the cells are osteochondral skeletal stem cells, pre-osteoblasts, osteoblasts, osteoprogenitor cells, or osteosarcoma cells, wherein an increase in bone mineralization in the presence of the candidate agent compared to a reference range for the amount of bone mineralization in control cells indicates that the candidate agent is a mimic or analogue of CCN3.

[0331] In another example, a cellular assay to identify an agonist that increases cartilage production can be performed by: activating skeletal stem cells with mechanical stimulation; contacting the skeletal stem cells with CCN3, a VEGF inhibitor, and a candidate agent; and measuring cartilage production in the skeletal stem cells, wherein the increase in cartilage production in the presence of the candidate agent, compared to a reference range for the amount of cartilage production in control skeletal stem cells, indicates that the candidate agent is an agonist of CCN3.

[0332] In another example, a cellular assay to identify a mimic or analog of CCN3 that stimulates cartilage production can be performed by: activating skeletal stem cells with mechanical stimulation; contacting the skeletal stem cells with a vascular endothelial growth factor (VEGF) inhibitor and a candidate agent; and measuring cartilage production through the skeletal stem cells, wherein an increase in cartilage production in the presence of the candidate agent, compared to a reference range for the amount of cartilage production in control skeletal stem cells, indicates that the candidate agent is a mimic or analog of CCN3.

[0333] CCN3 agonists, mimics, or analogs can be any type of molecule, including but not limited to small molecules, drugs, receptor ligands, proteins, polypeptides, peptides, fusion proteins, nucleic acids, oligonucleotides, peptide nucleic acids, and aptamers that increase bone and / or cartilage growth. In some embodiments, the agonist increases CCN3 activity or CCN3 mRNA or protein levels by 1, 2, 3, 4, 5, 10, 100, or more, or any amount in between, compared to native or control levels. In some embodiments, the agonist or mimic increases bone mineralization through cells by 1, 2, 3, 4, 5, 10, 100, 200, 300, 400, or more, or any amount in between, compared to native or control levels.

[0334] Cell assays can be performed on, for example, osteochondral skeletal stem cells, pre-osteoblasts, osteoblasts, osteoprogenitor cells, or osteosarcoma cells. In some embodiments, cell assays are performed using skeletal stem cells derived from other types of stem cells, such as adult stem cells (e.g., mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells, or neural stem cells), embryonic stem cells, or induced pluripotent stem cells. In some embodiments, the skeletal stem cells or stem cells derived from skeletal stem cells are obtained from patients suffering from bone or cartilage diseases or conditions associated with bone or cartilage degeneration.

[0335] In some implementations, cells from cell lines are used for cell assays. Exemplary cell lines that can be used in subject screening methods include, but are not limited to, the MC3T3-E1 pre-osteoblast cell line, the SaOs2 osteosarcoma cell line, the MG-63 osteosarcoma cell line, the hFOB osteoblast cell line, the ASC52telo immortalized adipose-derived mesenchymal stem cell line, the human telomerase reverse transcriptase immortalized bone marrow mesenchymal stromal cell line (hTERT-BMSC), and the ATDC5 chondrogenic mouse teratogenic carcinoma cell line.

[0336] The assay may further include a suitable control (e.g., a sample in the absence of the test reagent). Typically, multiple assay mixtures are run in parallel with different reagent concentrations to obtain different responses to various concentrations. Typically, one of these concentrations is used as a negative control, i.e., at zero concentration or below the detection level.

[0337] Various other reagents may be included in the screening assay. These include reagents such as salts, neutral proteins such as albumin, detergents, etc., including agents to promote optimal binding activity and / or reduce nonspecific or background activity. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may be used. The components of the assay mixture are added in any order to provide the necessary activity. Incubation is performed at any suitable temperature, typically between 4°C and 40°C. The incubation period is selected for optimal activity but can also be optimized to facilitate rapid, high-throughput screening. In some embodiments, a period between 0.1 hours and 1 hour, between 1 hour and 2 hours, or between 2 hours and 4 hours will be sufficient.

[0338] A wide variety of test reagents can be screened. Candidate reagents cover many chemical categories, such as small organic compounds with molecular weights greater than 50 Daltons and less than about 10,000 Daltons, less than about 5,000 Daltons, or less than about 2,500 Daltons. Test reagents may contain functional groups necessary for interaction with protein structures, such as hydrogen bonds, and may include at least one amine, carbonyl, hydroxyl, or carboxyl group, or at least two of the functional groups. Test reagents may contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the aforementioned functional groups. Test reagents have also been found in biomolecules, including peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0339] Test agents are obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous methods are available for the random and directed synthesis of a wide range of organic compounds and biomolecules, including the randomized expression of oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily generated. Furthermore, naturally or synthetically produced libraries and compounds can be readily modified using conventional chemical, physical, and biochemical methods and can be used to generate combinatorial libraries. Known drug agents can undergo directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogs. In addition, screening can target known pharmacologically active compounds and their chemical analogs, or novel agents with unknown properties, such as those generated through rational drug design.

[0340] In some implementations, the test reagent is a synthetic compound. Many techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides. See, for example, WO 94 / 24314, which is expressly incorporated herein by reference, which discusses methods for generating new compounds, including randomized chemical methods and enzymatic methods.

[0341] In another embodiment, the test agent is provided as a library of natural compounds in the form of readily available or easily produced bacterial, fungal, plant, and animal extracts. Further, the naturally or synthetically produced libraries and compounds can be readily modified by conventional chemical, physical, and biochemical means. Known pharmaceutical agents can undergo directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.

[0342] In some embodiments, the test agent is an organic moiety. In this embodiment, the test agent is synthesized from a series of chemically modifiable substrates. "Chemically modified" as used herein includes both conventional chemical reactions and enzymatic reactions. These substrates typically include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes, and heteroalkyl groups), aryl groups (including aromatics and heteroaryl groups), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepines, β-lactams, tetracyclines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecdysone, etc.), alkaloids (including ergot, periwinkle, curare, pyrrolizidine, and mitomycin), organometallic compounds, compounds with heteroatoms, amino acids, and nucleosides. The moiety can be chemically (including enzymatically) reacted to form new substrates or candidate agents, which can then be tested using this invention.

[0343] In some implementations, well-known assays, such as trypan blue exclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazole bromide) assays, are used to assess any cytotoxic activity that the test agent may exhibit against live eukaryotic cells. Agents that do not exhibit significant cytotoxic activity are considered candidate agents.

[0344] Monitoring the risk of bone loss from breastfeeding

[0345] CCN3 levels can be used to monitor breastfeeding women to determine the risk of bone loss from breastfeeding and to provide guidance on whether to continue or discontinue breastfeeding. During lactation, calcium (Ca) levels decrease due to the increased risk of calcium loss during breastfeeding. 2+ High demand for breast milk can lead to significant bone loss in mothers. Brain-derived osteogenic factor CCN3, secreted by neurons in the arcuate nucleus (ARC), counteracts calcium loss by promoting bone production in lactating women. CCN3 levels produced by ARC neurons decline over time after birth. Therefore, if breastfeeding continues after CCN3 levels have fallen below a critical threshold, the mother is at risk of bone degeneration. Thus, CCN3 levels can be monitored in lactating female subjects to determine whether CCN3 levels remain above a critical threshold to avoid bone degeneration, or whether they have fallen below a critical threshold indicating a risk of bone degeneration if breastfeeding continues.

[0346] Samples can be obtained from lactating female subjects to measure the level of CCN3 present. Samples are typically blood or plasma samples containing CCN3 taken from the subject. Blood samples can be obtained from the subject using routine techniques. For example, blood samples can be obtained via venipuncture using methods well known in the art.

[0347] CCN3 levels can be compared to a reference range to determine whether the CCN3 level indicates a risk of bone degeneration (e.g., loss of bone mineral density or bone mass, bone fragility, osteoporosis, or osteopenia). For example, the measurement can be used to determine whether the CCN3 level is less than or “greater than or equal to” a specific threshold (which can be predetermined or determined by measuring a control sample). In some embodiments, the CCN3 level in a sample obtained from a female lactating subject is compared to a reference range representing the CCN3 level in a control sample from one or more female lactating subjects who do not have a significant risk of bone degeneration (i.e., have CCN3 levels at or above the critical threshold). That is, a CCN3 level at or above the threshold indicates that the female lactating subject can continue breastfeeding without a substantial risk of bone degeneration, while a CCN3 level below the critical threshold indicates that the female lactating subject is at risk of bone degeneration if breastfeeding continues. During the period when female subjects are breastfeeding and wish to continue breastfeeding, CCN3 levels can be monitored over time to determine whether CCN3 levels remain at or above the critical threshold level, or have decreased to below the critical threshold level, in order to determine whether continuing breastfeeding is still safe without substantial risk of bone degeneration.

[0348] Determining whether a breastfeeding female subject is at risk of bone degeneration from breastfeeding is a positive clinical application of the correlation between CCN3 levels and the risk of bone degeneration. For example, “determining” requires an active step of measuring CCN3 levels, reviewing data generated during the active measurement step, and resolving whether the individual has a substantial risk of bone degeneration from continued breastfeeding. Further, in some cases, a decision is made on whether to continue breastfeeding. In some cases, the subject approach includes the following steps: a healthcare practitioner (e.g., a nurse or physician) informs the breastfeeding female subject that continuing breastfeeding carries a risk of bone degeneration, for example, if CCN3 levels have fallen below a critical threshold.

[0349] In some implementations, the subject-matter approach includes providing an analysis indicating whether a breastfeeding female subject is at substantial risk of bone degeneration if breastfeeding continues. Appropriate analyses (e.g., oral or written reports) provide any or all of the following information: subject identification information (name, age, etc.), a description of what type of sample was used and / or how the sample was used, the technique used to measure the sample, the results of the measurement (e.g., the level of CCN3 measured and / or the fold change of CCN3 levels over time), an assessment of whether the individual is at risk of bone degeneration from continued breastfeeding, and, if the CCN3 levels indicate that the subject is at risk of bone degeneration if breastfeeding continues, a recommendation to discontinue breastfeeding, etc. This analysis may be made available to the subject, the subject's physician, the testing facility, etc. The analysis may also be accessible via the Internet as a URL. In some such cases, the analysis may be accessible to multiple different entities (e.g., the subject, the subject's physician, the testing facility, etc.).

[0350] Detecting CCN3

[0351] It should be understood that CCN3 in a sample can be measured by any suitable method known in the art. In some embodiments, antibody-based methods are used to measure CCN3 levels using antibodies that specifically bind to CCN3. The term "specifically bind" or "specifically binding" as used herein refers to preferential binding to one molecule relative to other molecules or portions in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides or epitopes). In some embodiments, the affinity of one molecule for another molecule with which it specifically binds is characterized by 10. -5 M or smaller (e.g., 10) -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 10 -13 M or smaller, 10 -14 M or smaller, 10 -15 M or smaller, or 10 -16 K (M or smaller) d (Dissociation constant). "Affinity" refers to the strength of the bond; increased bonding affinity is associated with a lower K. d Related.

[0352] While various methods for determining protein levels are known in the art, a representative and convenient type of protocol used for determining protein levels is enzyme-linked immunosorbent assay (ELISA). In ELISA and ELISA-based assays, one or more antibodies specific to the protein of interest are immobilized onto a selected solid surface, preferably a surface exhibiting protein affinity, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, the wells of the assay plate are coated with a nonspecific “blocking” protein known to be antigenically neutral to the test sample, such as bovine serum albumin (BSA), casein, or a milk powder solution. This allows the nonspecific adsorption sites on the immobilized surface to be blocked, thereby reducing background caused by nonspecific binding of antigens to the surface. After washing to remove unbound blocking protein, the immobilized surface is brought into contact with the sample to be tested under conditions conducive to the formation of immune complexes (antigen / antibody). Such conditions include diluting the sample in phosphate-buffered saline (PBS) / Tween or PBS / Triton-X 100 with a diluent such as BSA or bovine gamma globulin (BGG). This also tends to help reduce nonspecific background and allows the sample to be incubated at temperatures on the order of approximately 25°C–27°C for approximately 2–4 hours (although other temperatures may be used). After incubation, the surfaces that came into contact with the antiserum are washed to remove the non-immunocomposite material. Exemplary washing procedures include washing with solutions such as PBS / Tween, PBS / Triton-X 100, or borate buffer. The occurrence and amount of immune complex formation can then be determined by subjecting the bound immune complex to a second antibody that is specific to the target and different from the first antibody, and detecting the binding of the second antibody. In some embodiments, the second antibody will have an associated enzyme, such as urease, peroxidase, or alkaline phosphatase, which will produce a colored precipitate upon incubation with a suitable chromogenic substrate. For example, urease- or peroxidase-conjugated anti-human IgG can be used for a period of time under conditions favorable to the formation and development of immune complexes (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS / Tween). After such incubation with a secondary antibody and washing to remove unbound material, the amount of the label is quantified, for example, by incubation with a chromogenic substrate, such as urea and bromocresol purple in the case of urease labeling, or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H₂O₂ in the case of peroxidase labeling. Quantification is then achieved by measuring the degree of color production, for example, using a visible spectrophotometer. The preceding form can be modified by first binding the sample to the assay plate.The primary antibody was then incubated with the assay plate, and the bound primary antibody was detected using a second antibody specifically labeled to the primary antibody.

[0353] The solid substrate on which one or more antibodies are immobilized can be made of a wide variety of materials and come in a variety of shapes, such as microtiter plates, microbeads, test strips, resin particles, etc. The substrate can be selected to maximize the signal-to-noise ratio, minimize background binding, and balance ease of separation and cost. Washing can be performed in a manner most suitable for the substrate used, for example, by removing beads or test strips from the reservoir, emptying or diluting the reservoir such as the wells of a microtiter plate, or rinsing the beads, particles, column, or filter with a washing solution or solvent.

[0354] Alternatively, non-ELISA-based methods for measuring CCN3 levels in a sample can be used, and any convenient method can be employed. Representative examples known to those skilled in the art include, but are not limited to, other immunoassay techniques such as radioimmunoassay (RIA), sandwich immunoassay, fluorescence immunoassay, enzyme multiplied immunoassay technique (EMIT), capillary electrophoresis immunoassay (CEIA), and immunoprecipitation assay; mass spectrometry or tandem mass spectrometry, proteomic arrays, xMAP microsphere technology, Western blotting, immunohistochemistry, flow cytometry, time-of-flight cytometry (CyTOF), multiplexed ion beam imaging (MIBI), and detection in body fluids via electrochemical sensors.

[0355] As another example, an electrochemical sensor can be employed. In such methods, a capture aptamer or antibody specific to CCN3 is immobilized on an electrode. A second aptamer or antibody, also specific to CCN3, is labeled, for example, with pyrroquinoline quinone glucose dehydrogenase (PQQ)GDH. A bodily fluid sample (e.g., blood or plasma) is introduced into the sensor by immersing the electrode in bodily fluid or by adding sample fluid to the sample chamber, allowing the CCN3 analyte to interact with the labeled aptamer / antibody and the immobilized capture aptamer / antibody. Glucose is then supplied to the sample, and the current generated by (PQQ)GDH is observed, where the amount of current flowing through the electrochemical cell is directly related to the amount of analyte captured at the electrode.

[0356] To measure protein activity levels, the amount or level of CCN3 activity in a sample is determined. For example, the ability of CCN3 in a sample to stimulate bone production via osteochondral skeletal stem cells can be measured (see, for example, examples).

[0357] In other embodiments, the amount or level of CCN3 protein in the sample is determined. Any convenient method for measuring protein levels in a sample can be used, such as antibody-based methods, such as immunoassays, such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and mass spectrometry.

[0358] The resulting data provides information about the amount and / or activity of CCN3 that has been measured, wherein the information is about the presence and level of CCN3, including guidance on whether the level is greater than or equal to a critical threshold level to avoid substantial bone degeneration or below a critical threshold level, and wherein the data may be both qualitative and quantitative.

[0359] utility

[0360] The compositions and methods disclosed herein are intended for a variety of applications, including the treatment of bone and cartilage conditions and symptoms associated with bone or cartilage degeneration. Bone conditions and symptoms associated with bone degeneration include, but are not limited to, osteoporosis, osteopenia, lactation, traumatic bone injury (e.g., fractures caused by falls, road traffic accidents, fights, or surgery), pathological fractures, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy. The compositions and methods disclosed herein are also used to treat cartilage disorders, including but not limited to osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthritis, achondroplasia, recurrent polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, and malignancy.

[0361] CCN3 can be used to stimulate osteochondral skeletal stem cells to generate new bone or cartilage in vivo, in vitro, or in vitro. Treatment with CCN3 or recombinant polynucleotides containing the coding sequence for CCN3 can increase bone mineral density, bone mass, and bone strength, reduce fatty bone marrow, increase cartilage mass, increase the rate of bone or cartilage generation, or regenerate, replace, or repair bone or cartilage. Furthermore, CCN3 can be used to generate bone for bone grafts to repair fractures or bone defects, or cartilage for cartilage grafts to repair cartilage defects or damage. CCN3 can also be used as a biomarker to determine the risk of bone degeneration in breastfeeding women during breastfeeding.

[0362] Examples of non-limiting aspects of this disclosure

[0363] The aspects of the subject matter of the invention described above, including embodiments, may be advantageous individually or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of this disclosure, numbered 1 to 125, are provided below. As will be apparent to those skilled in the art upon reading this disclosure, each of the individually numbered aspects can be used or combined with any preceding or following individually numbered aspects. This is intended to support all such combinations of aspects, and is not limited to combinations of aspects explicitly provided below:

[0364] 1. A method for treating a bone condition or symptom associated with bone degeneration in a subject, the method comprising administering a therapeutically effective amount of cell communication network factor 3 (CCN3) to the subject.

[0365] 2. The method according to aspect 1, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.

[0366] 3. The method according to aspect 1 or 2, wherein the bone condition or symptom associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

[0367] 4. The method according to any one of aspects 1 to 3, wherein the treatment increases bone mass compared to the bone mass of the subject before treatment.

[0368] 5. The method according to any one of aspects 1 to 4, wherein the treatment increases bone density compared to the bone density of the subject before treatment.

[0369] 6. The method according to any one of aspects 1 to 5, wherein the treatment reduces the amount of fatty bone marrow compared to the amount of fatty bone marrow in the subject before treatment.

[0370] 7. The method according to any one of aspects 1 to 6, wherein the CCN3 is administered intravenously or intraperitoneally.

[0371] 8. The method according to any one of aspects 1 to 6, wherein the CCN3 is locally applied to the bone of the subject.

[0372] 9. The method according to any one of aspects 1 to 8, wherein a plurality of therapeutically effective doses of CCN3 are administered to the subject.

[0373] 10. The method according to aspect 9, wherein the CCN3 is administered according to a daily dosing regimen or intermittently.

[0374] 11. The method according to any one of aspects 1 to 10, the method further comprising administering a bone anabolic agent or an anti-resorption agent to the subject.

[0375] 12. The method according to aspect 11, wherein the bone anabolic agent is parathyroid hormone, teriparatide, abapatide, or romozoltab.

[0376] 13. The method according to aspect 11, wherein the anti-reabsorption agent is an estrogen, an estrogen agonist, a bisphosphonate, or denosumab.

[0377] 14. The method according to any one of aspects 1 to 13, wherein the subject is a human.

[0378] 15. The method according to any one of aspects 1 to 14, wherein the CCN3 is encapsulated in a hydrogel.

[0379] 16. The method according to aspect 15, wherein the hydrogel comprises alginate.

[0380] 17. The method according to aspect 16, wherein the alginate is ionically cross-linked.

[0381] 18. The method according to aspect 17, wherein the alginate is ionically crosslinked by divalent calcium cations.

[0382] 19. The method according to any one of aspects 16 to 18, wherein the concentration of the alginate in the hydrogel is in the range of 2 to 10 weight percent (wt%).

[0383] 20. The method according to any one of aspects 1 to 19, wherein the CCN3 is administered using a drug delivery device.

[0384] 21. The method according to aspect 20, wherein the drug delivery device provides continuous delivery of the CCN3.

[0385] 22. The method according to aspect 20, wherein the drug delivery device is a reservoir implant or an integral implant.

[0386] 23. The method according to aspect 20, wherein the drug delivery device is a non-biodegradable implant.

[0387] 24. The method according to aspect 20, wherein the drug delivery device is a biodegradable implant.

[0388] 25. The method according to any one of aspects 1 to 21, the method further comprising administering a therapeutically effective amount of osteochondral skeletal stem cells to the subject.

[0389] 26. A method for reducing or preventing bone degeneration in a female subject during lactation, the method comprising administering a therapeutically effective amount of cell communication network factor 3 (CCN3) to the subject.

[0390] 27. The method according to aspect 26, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.

[0391] 28. The method according to aspect 26 or 27, wherein the CCN3 is administered intravenously or intraperitoneally.

[0392] 29. The method according to aspect 26 or 27, wherein the CCN3 is locally applied to the bone of the subject.

[0393] 30. The method according to any one of aspects 26 to 29, wherein the CCN3 is administered according to a daily dosing regimen or intermittently.

[0394] 31. A composition comprising CCN3, used in a method of treating bone disorders or conditions associated with bone degeneration.

[0395] 32. The composition according to aspect 31, wherein the composition further comprises a pharmaceutically acceptable excipient.

[0396] 33. The composition according to aspect 31 or 32, wherein the composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of creams, emulsions, gels, liposomes, nanoparticles or ointments.

[0397] 34. The composition according to any one of aspects 31 to 33, wherein the bone condition or symptom associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disease, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

[0398] 35. A method of treating a subject for a bone condition or symptom associated with bone degeneration, the method comprising administering to the subject a vector containing an expression cassette, the expression cassette containing a coding sequence encoding cell communication network factor 3 (CCN3).

[0399] 36. The method according to aspect 35, wherein the expression box includes a promoter operatively connected to the coding sequence encoding the CCN3.

[0400] 37. The method according to aspect 35, wherein the coding sequence encoding the CCN3 is integrated into a chromosomal locus in the genome of the subject.

[0401] 38. The method according to aspect 37, wherein an endogenous promoter is operatively linked at the chromosomal locus to the integrated coding sequence encoding the CCN3.

[0402] 39. The method according to any one of aspects 35 to 38, wherein the vector is a plasmid or a viral vector.

[0403] 40. The method according to aspect 39, wherein the viral vector is an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, or a retroviral vector.

[0404] 41. The method according to any one of aspects 35 to 40, wherein the carrier is administered intravenously or intraperitoneally.

[0405] 42. The method according to aspect 41, wherein the carrier is administered via the portal vein.

[0406] 43. The method according to any one of aspects 35 to 40, wherein the carrier is applied locally to bone.

[0407] 44. The method according to any one of aspects 35 to 43, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.

[0408] 45. The method according to any one of aspects 35 to 44, wherein the bone condition or symptom associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

[0409] 46. ​​The method according to any one of aspects 35 to 45, wherein the treatment increases bone mass compared to the bone mass of the subject before treatment.

[0410] 47. The method according to any one of aspects 35 to 46, wherein the treatment increases bone density compared to the bone density of the subject before treatment.

[0411] 48. The method according to any one of aspects 35 to 47, wherein the treatment reduces the amount of fatty bone marrow compared to the amount of fatty bone marrow in the subject's bone before treatment.

[0412] 49. A method of providing a subject with cell communication network factor 3 (CCN3) to promote bone growth in the subject, the method comprising introducing a vector comprising a promoter operatively linked to a coding sequence encoding said CCN3 into cells, wherein said cells express said CCN3 in vivo in the subject in an effective amount sufficient to promote bone growth in the subject.

[0413] 50. The method according to aspect 49, wherein the carrier is introduced into the cells either in vitro or in vivo.

[0414] 51. The method according to aspect 49 or 50, wherein the cell is a hepatocyte or osteochondral skeletal stem cell.

[0415] 52. The method according to any one of aspects 49 to 51, wherein the vector is a plasmid or a viral vector.

[0416] 53. The method according to aspect 52, wherein the viral vector is an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, or a retroviral vector.

[0417] 54. The method according to any one of aspects 49 to 53, wherein the subject suffers from a bone disease or condition associated with bone degeneration.

[0418] 55. The method according to aspect 54, wherein the bone condition or symptom associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

[0419] 56. A method of stimulating osteochondral skeletal stem cells to generate bone, the method comprising contacting the osteochondral skeletal stem cells with an effective amount of cell communication network factor 3 (CCN3), wherein bone is generated by the osteochondral skeletal stem cells.

[0420] 57. The method according to aspect 56, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.

[0421] 58. A method for screening agonists of cell communication network factor 3 (CCN3) that increase bone growth, the method comprising:

[0422] Contacting cells with the CCN3 and the candidate drug agent, wherein the cells are osteochondral skeletal stem cells, pre-osteoblasts, osteoblasts, osteoprogenitor cells, or osteosarcoma cells; and

[0423] Bone production through the cells is measured, wherein the increase in bone production in the presence of the candidate drug, compared to a reference range of bone production in control cells in which the candidate drug is absent, indicates that the candidate drug is an agonist of CCN3.

[0424] 59. A method for screening mimics or analogs of cell communication network factor 3 (CCN3) that stimulates bone growth, the method comprising:

[0425] Contacting cells with a candidate drug agent, wherein the cells are osteochondral skeletal stem cells, pre-osteoblasts, osteoblasts, osteoprogenitor cells, or osteosarcoma cells; and

[0426] Bone mineralization is measured through the cells, wherein the increase in bone mineralization in the presence of the candidate drug, compared to a reference range of bone mineralization in control cells in which the candidate drug is absent, indicates that the candidate drug is a mimic or analog of the CCN3.

[0427] 60. The method according to aspect 58 or 59, wherein the cell is derived from stem cells.

[0428] 61. The method according to aspect 60, wherein the stem cell is an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell.

[0429] 62. The method according to aspect 61, wherein the adult stem cell is a mesenchymal stem cell.

[0430] 63. The method according to aspect 60, wherein the stem cells are derived from a patient suffering from a bone disease or symptom associated with bone degeneration.

[0431] 64. The method according to aspect 59, wherein the cell is immortalized.

[0432] 65. The method according to aspect 59, wherein the cells are derived from the MC3T3-E1 pre-osteoblast cell line, the SaOs2 osteosarcoma cell line, the MG-63 osteosarcoma cell line, the hFOB osteoblast cell line, the ASC52telo immortalized adipose-derived mesenchymal stem cell line, the human telomerase reverse transcriptase immortalized bone marrow mesenchymal stromal cell line (hTERT-BMSC), or the ATDC5 chondrogenic mouse teratogenic carcinoma cell line.

[0433] 66. A method for producing a bone graft, the method comprising culturing osteochondral skeletal stem cells under suitable conditions in the presence of cell communication network factor 3 (CCN3), wherein the osteochondral skeletal stem cells produce bone for the bone graft.

[0434] 67. A bone graft, said bone graft being produced by the method according to aspect 66.

[0435] 68. The bone graft according to aspect 67, wherein the osteochondral skeletal stem cells are derived from adult stem cells, embryonic stem cells or induced pluripotent stem cells.

[0436] 69. The bone graft according to aspect 68, wherein the adult stem cells are mesenchymal stem cells.

[0437] 70. A method of transplanting a bone graft into a subject, the method comprising transplanting the bone graft according to aspect 69 into the transplantation site of the subject.

[0438] 71. The method according to aspect 70, wherein the osteochondral skeletal stem cells are autologous, allogeneic, or xenogeneic.

[0439] 72. The method according to aspect 70 or 71, wherein the osteochondral skeletal stem cells are derived from adult stem cells, embryonic stem cells or induced pluripotent stem cells.

[0440] 73. The method according to aspect 72, wherein the adult stem cell is a mesenchymal stem cell.

[0441] 74. The method according to any one of aspects 70 to 73, wherein the bone graft replaces the missing bone to repair the fracture at the transplantation site.

[0442] 75. The method according to aspect 74, wherein the fracture is a complex fracture.

[0443] 76. The method according to any one of aspects 70 to 73, wherein the bone graft provides new bone to repair congenital bone defects at the transplantation site.

[0444] 77. The method according to any one of aspects 70 to 73, wherein the bone graft provides new bone for craniofacial surgery, oral and maxillofacial surgery, plastic surgery or reconstructive surgery.

[0445] 78. The method according to any one of aspects 70 to 73, wherein the bone graft provides new bone for the dental implant.

[0446] 79. The method according to any one of aspects 70 to 78, the method further comprising locally applying cell communication network factor 3 (CCN3) at the transplant site to stimulate new bone growth at the transplant site.

[0447] 80. A method for monitoring lactating female subjects to determine the risk of bone degeneration from breastfeeding, the method comprising:

[0448] Biological samples were obtained from the lactating female subjects.

[0449] The levels of Communication Network Factor 3 (CCN3) in the biological samples were measured; and

[0450] The level of CCN3 in the biological sample was compared with a reference range of CCN3 from control subjects, wherein a level of CCN3 below a threshold indicated that the subject was at risk of bone degeneration from breastfeeding, and wherein a level of CCN3 equal to or greater than the threshold indicated that the female subject could continue breastfeeding without substantial risk of bone degeneration.

[0451] 81. The method according to aspect 80, wherein if the level of CCN3 indicates that the female subject is at risk of bone degeneration from breastfeeding, the female subject stops breastfeeding.

[0452] 82. The method according to aspect 80 or 81, the method further comprising administering a therapeutically effective amount of the CCN3 to the female subject to reduce the risk of bone degeneration from breastfeeding.

[0453] 83. The method according to any one of aspects 80 to 82, wherein the biological sample is blood or plasma.

[0454] 84. The method according to any one of aspects 80 to 83, wherein the measurement of CCN3 level comprises performing enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, immunohistochemistry, fluorescence activated cell sorting (FACS), Western blotting, mass spectrometry, tandem mass spectrometry, biochemical assay, liquid chromatography, or NMR.

[0455] 85. A communication network factor 3 (CCN3) used as a biomarker to monitor lactating female subjects to determine the risk of bone loss from breastfeeding.

[0456] 86. A method for regenerating mammalian cartilage in a subject, the method comprising:

[0457] Activating bone marrow stem cells through mechanical stimulation; and

[0458] The subjects were administered a therapeutically effective dose of a combination of cell communication network factor 3 (CCN3) and a therapeutically effective dose of a vascular endothelial growth factor (VEGF) inhibitor.

[0459] 87. The method according to aspect 86, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.

[0460] 88. The method according to aspect 86 or 87, wherein the VEGF inhibitor is cabozantinib.

[0461] 89. The method according to any one of aspects 86 to 88, wherein the cartilage is articular cartilage.

[0462] 90. The method according to any one of aspects 86 to 89, wherein the mechanical stimulation is an acute local injury.

[0463] 91. The method according to aspect 90, wherein the acute local injury is a surgical microfracture of bone tissue performed at a desired site for cartilage regeneration.

[0464] 92. The method according to aspect 90 or 91, wherein the VEGF inhibitor and the CCN3 are applied locally to the acute local injury.

[0465] 93. The method according to any one of aspects 86 to 92, wherein the VEGF inhibitor and the CCN3 are administered to the subject immediately after the bone stem cells are activated by the mechanical stimulation.

[0466] 94. The method according to any one of aspects 86 to 92, wherein the VEGF inhibitor and the CCN3 are administered to the subject within three days after the bone stem cells are activated by the mechanical stimulation.

[0467] 95. The method according to any one of aspects 86 to 94, wherein the VEGF inhibitor and the CCN3 are encapsulated in a hydrogel.

[0468] 96. The method according to aspect 95, wherein the hydrogel comprises alginate.

[0469] 97. The method according to aspect 96, wherein the alginate is ionically cross-linked.

[0470] 98. The method according to aspect 97, wherein the alginate is ionically crosslinked by divalent calcium cations.

[0471] 99. The method according to any one of aspects 96 to 98, wherein the concentration of the alginate in the hydrogel is in the range of 2 to 10 weight percent (wt%).

[0472] 100. The method according to any one of aspects 86 to 99, wherein the VEGF inhibitor and the CCN3 are administered using a drug delivery device.

[0473] 101. The method according to aspect 100, wherein the drug delivery device provides continuous delivery of the VEGF inhibitor and the CCN3.

[0474] 102. The method according to aspect 100 or 101, wherein the drug delivery device is a reservoir implant or an integral implant.

[0475] 103. The method according to any one of aspects 100 to 102, wherein the drug delivery device is a non-biodegradable implant.

[0476] 104. The method according to any one of aspects 100 to 102, wherein the drug delivery device is a biodegradable implant.

[0477] 105. The method according to any one of aspects 100 to 104, wherein the drug delivery device is implanted at the site of the local acute injury.

[0478] 106. The method according to any one of aspects 86 to 105, wherein the subject suffers from rickets.

[0479] 107. The method according to aspect 106, wherein the cartilage disease is osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthropathy, achondroplasia, relapsing polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, or malignancy.

[0480] 108. The method according to any one of aspects 86 to 107, the method further comprising administering a therapeutically effective amount of bone stem cells to the subject.

[0481] 109. The method according to any one of aspects 86 to 108, wherein the CCN3 is provided by a carrier comprising an expression box, the expression box comprising an encoding sequence encoding the CCN3.

[0482] 110. The method according to aspect 109, wherein the expression box includes a promoter operatively connected to the coding sequence encoding the CCN3.

[0483] 111. The method according to aspect 109, wherein the coding sequence encoding the CCN3 is integrated into a chromosomal locus in the genome of the subject.

[0484] 112. The method according to aspect 111, wherein an endogenous promoter is operatively linked at the chromosomal locus to the integrated coding sequence encoding the CCN3.

[0485] 113. The method according to any one of aspects 109 to 112, wherein the vector is a plasmid or a viral vector.

[0486] 114. The method according to aspect 113, wherein the viral vector is an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, or a retroviral vector.

[0487] 115. A composition comprising cell communication network factor 3 (CCN3) and a vascular endothelial growth factor (VEGF) inhibitor, for use in a method of regenerating mammalian cartilage.

[0488] 116. The composition according to aspect 115, wherein the CCN3 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.

[0489] 117. The composition according to aspect 115 or 116, wherein the VEGF inhibitor is cabozantinib.

[0490] 118. The composition according to any one of aspects 115 to 117, wherein the cartilage is articular cartilage.

[0491] 119. The composition according to any one of aspects 115 to 118, wherein the CCN3 and the VEGF inhibitor are encapsulated in a hydrogel.

[0492] 120. The composition according to aspect 119, wherein the hydrogel comprises alginate.

[0493] 121. The composition according to aspect 120, wherein the alginate is ionically crosslinked.

[0494] 122. The composition according to aspect 121, wherein the alginate is ionically crosslinked by divalent calcium cations.

[0495] 123. The composition according to any one of aspects 120 to 122, wherein the concentration of the alginate in the hydrogel is in the range of 2 to 10 weight percent (wt%).

[0496] 124. A method for screening agonists of cell communication network factor 3 (CCN3) that increase chondrogenesis, the method comprising:

[0497] Mechanical stimulation is used to activate bone stem cells;

[0498] Contact the bone stem cells with CCN3, a vascular endothelial growth factor (VEGF) inhibitor, and a candidate drug; and

[0499] The amount of increased cartilage production in the presence of the candidate drug, compared to a reference range of cartilage production in control bone stem cells in which the candidate drug is absent, indicates that the candidate drug is an agonist of CCN3.

[0500] 125. A method for screening mimics or analogs of cell communication network factor 3 (CCN3) that stimulates cartilage production, the method comprising:

[0501] Mechanical stimulation is used to activate bone stem cells;

[0502] Contact the bone stem cells with a vascular endothelial growth factor (VEGF) inhibitor and a candidate drug; and

[0503] The amount of cartilage production through the bone stem cells is measured, wherein the increase in cartilage production in the presence of the candidate drug, compared to a reference range of cartilage production in control bone stem cells in which the candidate drug is absent, indicates that the candidate drug is an analogue or analogue of the CCN3.

[0504] Example

[0505] As can be understood from the disclosure provided above, this disclosure has a wide range of applications. Therefore, the following embodiments are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors consider their invention, nor to represent that the following experiments are all or only the experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, dimensions, etc.), but some experimental errors and biases should be taken into account. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially similar results.

[0506] Example 1: Brain-derived CCN3, an anabolic hormone for bone formation in lactating females

[0507] We and others have demonstrated that central estrogen signaling exerts a sex-dependent inhibition on bone formation and its role in promoting spontaneous activity and thermogenesis. 17-20 The high bone mass in females is due to the presence of neurons ERa in the arcuate nucleus of the medial basal hypothalamus (MBH). Caused by missing 1 22 Designed to eliminate ARC, especially ARC. Kiss1 Viral and genetic mouse models of ERa in neurons, independent of high E2 levels, resulted in high trabecular bone mass in the spine and long bones, confirming the central origin of this remarkable bone phenotype. 1 .

[0508] Here, using a combination of problem-driven and discovery-based approaches, after first demonstrating the circulation of osteogenic hormone-like factors in the blood, we began to identify the osteogenic hormone-like factor that leads to high bone mass in mutant females. Cell communication network factor 3 (CCN3 / Nov) emerged as the best candidate, meeting all expected criteria; it is secreted, its appearance in ARC coincides with the onset and subsequent loss of the bone phenotype following a dietary challenge, it leads to new bone formation when ectopically delivered to bone or in vivo delivered to mice, and finally, bone mass is reduced in mutant females upon knockout of the Ccn3 transcript in ARC. We propose that CCN3 functions as a brain-derived osteosynthetic hormone in the newly identified brain-bone axis, which has evolved to maintain skeletal health in mammalian mothers and offspring.

[0509] Humoral factors mediate high bone mass in mutant females

[0510] Our previous findings on the genetic and viral deletion of ERa in ARC strongly suggest that a subset of KNDy neurons in ARC regulates bone mass and bone strength in females rather than males. Figure 1A , 1B ) 1 Deleting ERa using the prodynorphin-Cre driver further supports the involvement of KNDy neurons in this brain-bone axis. Figure 1B and Figure 6A , Figure 6B To identify the molecular origin of the high bone mass phenotype, we specifically relied on Esr1. Nkx2-1Cre A female mouse model that exhibited this unusual phenotype at four weeks of age. Figure 6C-6E Given the unique position of the ARC as one of the few periventricular organs located dorsally to the median eminence, we inquired whether this high bone mass in mutant females might originate from the circulatory factor.

[0511] Using classic conjoined symbiosis combined with in vivo µCT imaging ( Figure 1C Two groups of females were surgically bonded to create a control pair (WT-WT) and a control paired with a mutant female (WT-MUT). Shortly after surgery (2 weeks), baseline bone microstructural parameters on the contralateral femur opposite the operated side were established for each animal in the pair. Females in the WT-WT pair exhibited a net reduction in bone mass, which was readily observable starting at six weeks post-surgery. Figure 1E By week 17, this decline was normalized, with an average increase of approximately 37%. In WT-MUT pairings, higher bone volume fraction (BV / TV%) was observed in control females at all time points, increasing by approximately 152% by week 17. Figure 1F and Figure 7A , Figure 7BUterine weight and other parameters did not change between WT-WT and WT-MUT pairings, consistent with the view that higher estrogen levels do not contribute to high bone mass in mutant females. Figure 7C We noted that in some MUT-WT pairings, the already high bone mass in mutant females was further increased ( Figure 7D , Figure 7E ).

[0512] Bone transplantation studies have confirmed that humoral factors lead to high bone mass in mutant females. Female and male femurs from 4-week-old control donors were subcutaneously implanted into 8-week-old wild-type or mutant females. Figure 1G and Figure 8A Six weeks after implantation into mutant males, a significant increase in bone fraction was detected in both female and male femurs. Figure 1H , Figure 1I and Figure 8B , Figure 8C (This suggests that) it is strongly believed that this brain-dependent, female-specific bone-synthesizing hormone will play a role in both sexes.

[0513] Brain-dependent factors alter skeletal stem cell dynamics and bone anabolism activity.

[0514] Skeletal homeostasis is tightly regulated by bone formation based on skeletal stem cells (SSCs) and bone resorption based on osteoclasts. Chan, Ambrosi, and colleagues demonstrated that stem cells with different lineage levels and cellular contributions promote new bone formation. 23 Specifically, osteochondral SSCs (ocSSCs) form bone and cartilage (and fibrous / matrix lineage cell populations, but not bone marrow adipocytes) and are present in the growth plate and periosteum of bone. ( Figure 2A Perivascular SSCs (pvSSCs) produce unilaterally directed adipogenic progenitor cells (APCs), which generate all bone marrow adipose tissue (BMAT) in bone. 23,25 Therefore, we infer that, given the increased bone formation in mutant females, brain-dependent anabolic hormones may alter the activity of ocSSCs. 1 OcSSCs from female wild-type mice were isolated by flow cytometry and transplanted subcapsularly into the kidneys of control or mutant females (Fig. 2bB). As expected, transplantation into control Esr1 resulted in successful transplantation. fl / fl Wild-type ocSSCs in female littermates developed ectopic bone grafts with host-derived hematopoietic compartments within six weeks. Figure 2C , Figure 2D However, transplanting to Esr1 Nkx2-1Cre Wild-type ocSSCs in females exhibit significantly higher mineralization and less hematopoietic bone marrow. Figure 2C-2EThis indicates that the bone anabolic hormones present in mutant females alter the ocSSC lineage to promote bone formation. Figure 2E Consistent with this hypothesis, higher bone fractions observed in WT-MUT conjoined twinning or whole bone graft studies were associated with increased ocSSC frequency. Figure 9A The robustness of this circulating bone anabolic hormone was further validated by stereotactic delivery of GFP-positive wild-type ocSSCs to the vicinity of the ARC. Figure 2F It is worth noting that mutant Esr1 Nkx2-1Cre µCT imaging of the hypothalamus revealed mineralized ossicles overlapping with transplanted GFP+ cells six weeks post-injection; no ossicles were detected in the wild-type brain. Figure 2G , Figure 2H These data further confirm the presence of circulating anabolic bone factor in mutant females, which may originate from the ARC or surrounding hypothalamic regions.

[0515] Circulating factors may promote the activity of wild-type ocSSCs, prompting us to compare the differentiation capacity of mutant and control ocSSCs. Flow cytometry analysis revealed that the frequency of ocSSCs increased in a sex-dependent manner in both pre-pubertal (3 weeks) and young adult mutant (10 weeks) females. Figure 2I This change is limited to ocSSC because it is destined for BMAT. pvSSCs and their progeny adipogenic progenitors (APCs) were identical in controls and mutants, except that APCs were less frequent in younger mutants. Figure 2I Differentiation assays revealed that, although ocSSCs from the two genotypes did not show a difference in colony-forming ability (CFU-F), Figure 9B However, mutant ocSSCs exhibit a high intrinsic potential for bone and cartilage formation. Figure 2J In the older Esr1 Nkx2-1Cre Enhanced ocSSC activity was also observed in females, consistent with their reduced bone loss compared to age-matched control littermates and the known link between ocSSC dysfunction and age-related bone loss. Figure 9C-9E ) 26 .

[0516] Identification of CCN3 as a candidate brain-derived osteoblast

[0517] Despite the amplification and enhanced osteogenic capacity of mutant ocSSCs, single-cell RNA sequencing (sc-RNAseq) data indicated that while the differentiation kinetics of mutant ocSSCs favored bone formation, only modest overall transcriptional differences were detected; therefore, unfortunately, no information regarding Esr1 was provided. Nkx2-1CreFew clues exist regarding the identity of bone-anabolic hormones in females (Figure 10). However, the first useful clue in our search for this anabolic bone factor emerged after the discovery that a chronic high-fat diet (HFD) challenge significantly disrupts the sex-dependent brain-bone axis. Although body weight, fat mass, blood triglycerides, and glucose homeostasis remain unchanged under HFD ( Figure 11A ), but Esr1 Nkx2-1Cre The high bone mass phenotype in females was completely reversed under this dietary challenge. Figure 3A The reduction in trabecular and cortical bone mass is accompanied by expected structural changes. Figure 11B and reduced bone strength and secondary cancellous bone ( Figure 3A , Figure 3B Histomorphometry showed that in mutant females fed HFD, the number of osteoclasts, bone formation, and mineralized surface area remained proportional to the bone surface area. Figure 3B and Figure 11C , Figure 11D However, through osmium staining... 27 Quantitative levels of BMAT were significantly lower in mutant females of SD or HFD compared to control littermates. Figure 3A , Figure 3B However, although Esr1 Nkx2-1Cre Dense bone in females is prone to degradation with HFD, but they fail to accumulate BMAT to a similar degree as control females, thus disrupting the normal link between BMAT expansion and bone loss. 28 Esr1 Nkx2-1Cre Bone parameters in males remained unchanged compared to their control litter. Figure 11E , Figure 11F In addition, the insulin receptor antagonist S961 Induced chronic hyperglycemia failed to degrade bone mass in mutant females, demonstrating the specificity of HFD-induced bone loss in mutant females. Figure 11G ).

[0518] Then, we analyzed Esr1 from feeding SD or HFD. Nkx2-1Cre Genetic changes in microdissected ARCs in females were profiled using dynamic changes in bone after HFD. Batch RNA-seq revealed a small group of differentially expressed genes (DEGs) encoding neuropeptides or secreted proteins in ARCs, including Ccn3, Fst, Grp, and Penk—all of which significantly decreased after HFD but increased in SDs of both young and older females. Figure 3C , Figure 3D and Figure 12A Importantly, Ccn3 and Penk expression are strongly associated with the onset of high bone mass at approximately four weeks of age. Figure 3E However, at an earlier time or in Esr1Nkx2-1Cre No change was observed in males. Figure 12B , Figure 12C Apart from Penk in the mutant pituitary, few noteworthy candidates emerged after profiling the pituitary and liver, two common tissue sources of secreted proteins. Figure 3C and Figure 12A CCN3 / Ccn3 expression was almost absent in control females or males, but was readily detected in the substrate region of mutant female ARCs that co-localized with KISS1, a known marker of KNDy neurons. Figure 3F , Figure 3G The expression levels easily exceed those detected in the suprachiasmatic nucleus (SCN). 30 .

[0519] CCN3 functions as a bone anabolic hormone.

[0520] The high expression of CCN3 in KISS1-positive and ERa-negative mutant ARC neurons prompted us to test this founding member of the CCN family. 31 This secreted protein is presumed to antagonize CCN2 to inhibit bone formation. 32,33 Although one report suggests the opposite 34 However, the anabolic potential of CCN3 was evaluated in three different assays. First, in the initial discovery from Esr1... Nkx2-1Cre Plasma collected from females increased bone mass in freshly dissected control femurs after five days. Treatment of ex vivo whole long bone cultures with CCN3 resulted in an average increase of 37% and 50% in female and male femurs, respectively. Figure 4A , Figure 4B and Figure 13A Importantly, when cultured in separate media, long bones from both sexes showed substantial degradation from their baseline values. Figure 13B , Figure 13C Using this simple yet effective assay, we found that low-dose mouse (m)CCN3 (3.0 nM) induced an upward dynamic shift in bone mass (60%) compared to saline. Figure 4D-4F and Figure 13B Second, adult wild-type mice were injected daily with mCCN3 (intraperitoneally) or saline for three weeks. Within this short timeframe and at this low dose (7.5 µg / kg), a significant increase in bone mass was observed in both female and male mice treated with mCCN3. Figure 4G , Figure 4H and Figure 13D Finally, culturing primary ocSSCs isolated from neonatal wild-type mice treated daily with mCCN3 increased mineralization by approximately 200%. Figure 4IThe two major peptides encoded by Penk, Met-ENK and BAMP-22, did not cause any changes; these negative results are consistent with the fact that naloxone, a competitive antagonist of the µ-opioid receptor, cannot degrade the high bone mass in mutant female mice. Figure 14A Furthermore, low concentrations of human CCN3 increased bone formation in primary human ocSSCs from adolescents and elderly / senior patients, regardless of sex; higher levels had no beneficial effect on bone formation and were inhibitory at the highest levels. Figure 4J and Figure 14B -14E). The osteogenic effect of ocSSCs in both young and older individuals appears to be unique to CCN3. Figure 14B , Figure 14C In summary, these data indicate that CCN3 is a bone anabolic hormone in mice and humans at low doses.

[0521] Brain-derived CCN3 is used to maintain bone mass during lactation.

[0522] In order to establish CCN3 and Esr1 Nkx2-1 The association between increased bone mineral density in females and bone parameters was measured after CCN3 overexpression or knockdown in control or mutant females. We utilized the secretory capacity of hepatocytes to increase Esr1 after systemic delivery of the highly hepatic-specific AAV-dj-CCN3 vector. fl / fl Circulating CCN3 in control females 35 Ectopic CCN3 expression in hepatocytes was detected as early as two weeks after injection. Figure 5A and Figure 15A Even at very low levels of hepatic CCN3 expression, a modest increase in bone formation rate was observed (Extended Data). Figure 10A , Figure 10B At 10-fold higher liver CCN3 expression levels, bone volume fraction increased by nearly 80%. Figure 5B , Figure 5C and Figure 15B Conversely, delivery of siRNA via stereotactic targeting in Esr1... Nkx2-1 Transient knockdown of Ccn3 expression in female ARC neurons attenuated the compact bone phenotype, with Ccn3 / CCN3 levels tracking well by BV / TV%. Figure 5D-5F In summary, these data indicate that increasing or decreasing CCN3 levels in control and mutant females affects bone mass, and Esr1... Nkx2-1Cre The maintenance of high bone mass in females depends on ARC ERα-CCN3 Sufficient CCN3 in neurons.

[0523] To extend our findings beyond artificially inherited phenomena, we inquired whether CCN3 was present in control females at any stage of life, with a focus on the postpartum period, when maternal BFR increases to maintain bone calcium reserves. Facing extremely low cycle E2 9 Similar to the non-pregnant control group, ARC expressing CCN3... ERa Neurons are absent in early and late pregnancy. Figure 5G , Figure 5H However, by seven days postpartum (DPP), CCN3 was present in the ARC of lactating female mice. ERa Highly expressed in subpopulations of neurons ( Figure 5G ), reaching close to Esr1 Nkx2-1Cre The same level found in mutant females ( Figure 5H Forced weaning reduced ARC when checked 3 or 7 days after pup removal (10 or 14 DPP, respectively). ERa CCN3 in neurons, indicating that the demand for bone-promoting CCN3 decreases when calcium requirements decrease and lactation ceases. Figure 5G These data indicate that as ovarian-derived estrogen gradually disappears, ARC... ERa-KISS1-CCN3 Brain-derived CCN3 in neurons maintains sufficient bone formation to support intergenerational resource transfer. Figure 5I ).

[0524] discuss

[0525] ARC ERa-KISS1 The role of neurons as gatekeepers of female reproduction is well established—these neurons control multiple aspects of physiology, including regulating puberty onset and the hypothalamic-pituitary-gonadal axis. Here, we find the female ARC. ERa-KISS1 Another key function of neurons is controlling bone homeostasis during lactation via the brain-derived bone-synthesizing hormone CCN3. During lactation, the production of ovarian estradiol and energy expenditure cease. 37 This raises a serious question—how can the mineralized bone surface retain its calcium-depleting properties during breastfeeding? 9 Especially in the trabecular spine, this is associated with lactation-related osteoporosis. 13 and loss of ERa signal transduction in bone Particularly sensitive. Through CCN3, ARC ERa-KISS1-CCN3 Neurons solve this problem by unrestricting the energy-intensive process of bone formation, thereby increasing the amount of trabecular bone in the spine and long bones. 1 It degrades during lactation. How does CCN3 become upregulated in female ARC during this unique life stage? Although CCN3 can be synergistically upregulated along with Kiss1 due to the sharp increase in prolactin signaling...38 However, we noted that prolactin levels were only moderately elevated in mutant females. 1 In addition to bone homeostasis, it is also of interest to determine whether the medial basal hypothalamus also directly or indirectly controls other adaptive responses, including the significant increase in intestinal length and nutrient absorption in lactating mothers. 39 .

[0526] Our data are inconsistent with the proposed function of CCN3 as an inhibitor of bone formation, such as... 40 This is a review article. However, most previous studies relied on high doses and overexpression of CCN3, which suggests that compensatory cellular responses or nonspecific receptor activation in the bone niche may be the cause of adverse effects on osteogenic formation. We also observed inhibitory effects in both mouse and human ocSSC differentiation assays at CCN3 doses exceeding the sub-nanomolar range. This is based on the presence of antiparallel b-chains and C-terminal cysteine ​​knot (CTCK) domains that mediate disulfide bond dimerization. 41 We predict that CCN3, circulating as a tightly held homodimer at low doses, binds to its homologous receptor with high affinity, similar to other growth factors such as NGF. We identified molecular targets of CCN3 in ocSSCs and potentially other cell populations, including osteocytes that reversibly remodel their pericavitary / lacrimal canaliculus matrix during lactation. 42 This will help resolve these differences. We speculate that, from ARC... KISS1 Permanent deletion of ERa in neurons leads to sustained secretion of low levels of CCN3, reproducing the anabolic phase of healthy postpartum bone remodeling. CCN3, combined with E2 and lacking PTHrP, rapidly generates strong, dense bone. Whether our findings can be translated into new therapies to reduce osteoporosis and fracture risk remains an exciting direction for the future.

[0527] References

[0528] 1Herber, CB et al. Estrogen signaling in arcuate Kiss1 neuronssuppresses a sex-dependent female circuit promoting dense strong bones. NatCommun 10, 163 (2019).

[0529] 2Ingraham, H. A., Herber, C. B. & Krause, W. C. Running the FemalePower Grid Across Lifespan Through Brain Estrogen Signaling. Annu Rev Physiol84, 59-85 (2022).

[0530] 3Khosla, S., Oursler, M. J. & Monroe, D. G. Estrogen and theskeleton. ...

Claims

1. A method for treating a bone condition or symptom associated with bone degeneration in a subject, the method comprising administering a therapeutically effective amount of cell communication network factor 3 (CCN3) to the subject.

2. The method according to claim 1, wherein the CCN3 comprises or is composed of the following: The amino acid sequence of SEQ ID NO: 1, or a sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

3. The method according to claim 1 or 2, wherein the bone condition or symptom associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

4. The method according to any one of claims 1 to 3, wherein the treatment increases bone mass compared to the bone mass of the subject before treatment.

5. The method according to any one of claims 1 to 4, wherein the treatment increases bone density compared to the bone density of the subject before treatment.

6. The method according to any one of claims 1 to 5, wherein the treatment reduces the amount of fatty bone marrow in the bone of the subject before treatment.

7. The method according to any one of claims 1 to 6, wherein the CCN3 is administered intravenously or intraperitoneally.

8. The method according to any one of claims 1 to 6, wherein the CCN3 is locally applied to the bone of the subject.

9. The method according to any one of claims 1 to 8, wherein a plurality of therapeutically effective doses of CCN3 are administered to the subject.

10. The method of claim 9, wherein the CCN3 is administered according to a daily dosing regimen or intermittently.

11. The method according to any one of claims 1 to 10, the method further comprising administering a bone anabolic agent or an anti-resorption agent to the subject.

12. The method of claim 11, wherein the bone anabolic agent is parathyroid hormone, teriparatide, abapatide, or romozolol.

13. The method of claim 11, wherein the anti-reabsorption agent is an estrogen, an estrogen agonist, a bisphosphonate, or denosumab.

14. The method according to any one of claims 1 to 13, wherein the subject is a human.

15. The method according to any one of claims 1 to 14, wherein the CCN3 is encapsulated in a hydrogel.

16. The method of claim 15, wherein the hydrogel comprises alginate.

17. The method of claim 16, wherein the alginate is ionically crosslinked.

18. The method of claim 17, wherein the alginate is ionically crosslinked by divalent calcium cations.

19. The method according to any one of claims 16 to 18, wherein the concentration of the alginate in the hydrogel is in the range of 2 to 10 weight percent (wt%).

20. The method according to any one of claims 1 to 19, wherein the CCN3 is administered using a drug delivery device.

21. The method of claim 20, wherein the drug delivery device provides continuous delivery of the CCN3.

22. The method of claim 20, wherein the drug delivery device is a reservoir implant or an integral implant.

23. The method of claim 20, wherein the drug delivery device is a non-biodegradable implant.

24. The method of claim 20, wherein the drug delivery device is a biodegradable implant.

25. The method according to any one of claims 1 to 21, the method further comprising administering a therapeutically effective amount of osteochondral skeletal stem cells to the subject.

26. A method for reducing or preventing bone degeneration in a female subject during lactation, the method comprising administering a therapeutically effective amount of cell communication network factor 3 (CCN3) to the subject.

27. The method of claim 26, wherein the CCN3 comprises or is composed of the following: The amino acid sequence of SEQ ID NO: 1, or a sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

28. The method of claim 26 or 27, wherein the CCN3 is administered intravenously or intraperitoneally.

29. The method of claim 26 or 27, wherein the CCN3 is locally applied to the bone of the subject.

30. The method according to any one of claims 26 to 29, wherein the CCN3 is administered according to a daily dosing regimen or intermittently.

31. A composition comprising CCN3, used in a method of treating bone disorders or conditions associated with bone degeneration.

32. The composition of claim 31, wherein the composition further comprises a pharmaceutically acceptable excipient.

33. The composition according to claim 31 or 32, wherein the composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of creams, emulsions, gels, liposomes, nanoparticles or ointments.

34. The composition according to any one of claims 31 to 33, wherein the bone condition or symptom associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

35. A method of treating a subject for a bone condition or symptom associated with bone degeneration, the method comprising administering to the subject a vector containing an expression cassette, the expression cassette containing a coding sequence encoding cell communication network factor 3 (CCN3).

36. The method of claim 35, wherein the expression box includes a promoter operatively connected to the coding sequence encoding the CCN3.

37. The method of claim 35, wherein the coding sequence encoding the CCN3 is integrated into a chromosomal locus in the genome of the subject.

38. The method of claim 37, wherein the endogenous promoter is operatively linked at the chromosomal locus to the integrated coding sequence encoding the CCN3.

39. The method according to any one of claims 35 to 38, wherein the vector is a plasmid or a viral vector.

40. The method according to claim 39, wherein the viral vector is an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, or a retroviral vector.

41. The method according to any one of claims 35 to 40, wherein the carrier is administered intravenously or intraperitoneally.

42. The method of claim 41, wherein the carrier is administered via the portal vein.

43. The method according to any one of claims 35 to 40, wherein the carrier is applied locally to bone.

44. The method according to any one of claims 35 to 43, wherein the CCN3 comprises or is composed of the following: The amino acid sequence of SEQ ID NO: 1, or a sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

45. The method according to any one of claims 35 to 44, wherein the bone condition or symptom associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

46. ​​The method according to any one of claims 35 to 45, wherein the treatment increases bone mass compared to the bone mass of the subject before treatment.

47. The method according to any one of claims 35 to 46, wherein the treatment increases bone density compared to the bone density of the subject before treatment.

48. The method according to any one of claims 35 to 47, wherein the treatment reduces the amount of fatty bone marrow compared to the amount of fatty bone marrow in the subject's bone before treatment.

49. A method of providing a subject with cell communication network factor 3 (CCN3) to promote bone growth in the subject, the method comprising introducing a vector comprising a promoter operatively linked to a coding sequence encoding said CCN3 into cells, wherein said cells express said CCN3 in vivo in the subject in an effective amount sufficient to promote bone growth in the subject.

50. The method of claim 49, wherein the carrier is introduced into the cells either in vitro or in vivo.

51. The method according to claim 49 or 50, wherein the cell is a hepatocyte or osteochondral skeletal stem cell.

52. The method according to any one of claims 49 to 51, wherein the vector is a plasmid or a viral vector.

53. The method according to claim 52, wherein the viral vector is an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, or a retroviral vector.

54. The method according to any one of claims 49 to 53, wherein the subject suffers from a bone condition or symptom associated with bone degeneration.

55. The method of claim 54, wherein the bone condition or symptom associated with bone degeneration is osteoporosis, osteopenia, lactation, traumatic bone injury, pathological bone injury, periprosthetic bone loss, osteolysis, menopause, obesity, anorexia nervosa, type 1 diabetes, chronic kidney disease, chronic liver disease, celiac disease, inflammatory bowel disease, lupus, rheumatoid arthritis, hyperthyroidism, hyperparathyroidism, cancer, multiple myeloma, craniofacial disorders, premature ovarian failure, oral and maxillofacial surgery, plastic surgery, reconstructive surgery, or oophorectomy.

56. A method of stimulating osteochondral skeletal stem cells to generate bone, the method comprising contacting the osteochondral skeletal stem cells with an effective amount of cell communication network factor 3 (CCN3), wherein bone is generated by the osteochondral skeletal stem cells.

57. The method of claim 56, wherein the CCN3 comprises or is composed of the following: The amino acid sequence of SEQ ID NO: 1, or a sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

58. A method for screening agonists of cell communication network factor 3 (CCN3) that increase bone growth, the method comprising: The cells are brought into contact with the CCN3 and the candidate drug, wherein the cells are osteochondral skeletal stem cells, pre-osteoblasts, osteoblasts, osteoprogenitor cells, or osteosarcoma cells; as well as Bone production through the cells is measured, wherein the increase in bone production in the presence of the candidate drug, compared to a reference range of bone production in control cells in which the candidate drug is absent, indicates that the candidate drug is an agonist of CCN3.

59. A method for screening mimics or analogs of cell communication network factor 3 (CCN3) that stimulates bone growth, the method comprising: The cells are brought into contact with the candidate drug agent, wherein the cells are osteochondral skeletal stem cells, pre-osteoblasts, osteoblasts, osteoprogenitor cells, or osteosarcoma cells; as well as Bone mineralization is measured through the cells, wherein the increase in bone mineralization in the presence of the candidate drug, compared to a reference range of bone mineralization in control cells in which the candidate drug is absent, indicates that the candidate drug is a mimic or analog of the CCN3.

60. The method of claim 58 or 59, wherein the cell is derived from stem cells.

61. The method of claim 60, wherein the stem cell is an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell.

62. The method of claim 61, wherein the adult stem cells are mesenchymal stem cells.

63. The method of claim 60, wherein the stem cells are derived from a patient suffering from a bone disease or symptom associated with bone degeneration.

64. The method of claim 59, wherein the cell is immortalized.

65. The method of claim 59, wherein the cells are derived from the MC3T3-E1 pre-osteoblast cell line, the SaOs2 osteosarcoma cell line, the MG-63 osteosarcoma cell line, the hFOB osteoblast cell line, the ASC52telo immortalized adipose-derived mesenchymal stem cell line, the human telomerase reverse transcriptase immortalized bone marrow mesenchymal stromal cell line (hTERT-BMSC), or the ATDC5 chondrogenic mouse teratogenic carcinoma cell line.

66. A method for producing a bone graft, the method comprising culturing osteochondral skeletal stem cells under suitable conditions in the presence of cell communication network factor 3 (CCN3), wherein the osteochondral skeletal stem cells produce bone for the bone graft.

67. A bone graft, said bone graft being produced by the method according to claim 66.

68. The bone graft of claim 67, wherein the osteochondral skeletal stem cells are derived from adult stem cells, embryonic stem cells or induced pluripotent stem cells.

69. The bone graft according to claim 68, wherein the adult stem cells are mesenchymal stem cells.

70. A method of transplanting a bone graft into a subject, the method comprising transplanting the bone graft of claim 69 into the transplantation site of the subject.

71. The method of claim 70, wherein the osteochondral skeletal stem cells are autologous, allogeneic, or xenogeneic.

72. The method according to claim 70 or 71, wherein the osteochondral skeletal stem cells are derived from adult stem cells, embryonic stem cells or induced pluripotent stem cells.

73. The method of claim 72, wherein the adult stem cells are mesenchymal stem cells.

74. The method according to any one of claims 70 to 73, wherein the bone graft replaces the missing bone to repair the fracture at the transplantation site.

75. The method of claim 74, wherein the fracture is a complex fracture.

76. The method according to any one of claims 70 to 73, wherein the bone graft provides new bone to repair congenital bone defects at the transplantation site.

77. The method according to any one of claims 70 to 73, wherein the bone graft provides new bone for craniofacial surgery, oral and maxillofacial surgery, plastic surgery, or reconstructive surgery.

78. The method according to any one of claims 70 to 73, wherein the bone graft provides new bone for the dental implant.

79. The method according to any one of claims 70 to 78, the method further comprising locally applying cell communication network factor 3 (CCN3) at the transplant site to stimulate new bone growth at the transplant site.

80. A method for monitoring lactating female subjects to determine the risk of bone degeneration from breastfeeding, the method comprising: Biological samples were obtained from the lactating female subjects. The level of communication network factor 3 (CCN3) in the biological sample was measured; as well as The level of CCN3 in the biological sample was compared with a reference range of CCN3 from control subjects, wherein a level of CCN3 below a threshold indicated that the subject was at risk of bone degeneration from breastfeeding, and wherein a level of CCN3 equal to or greater than the threshold indicated that the female subject could continue breastfeeding without substantial risk of bone degeneration.

81. The method of claim 80, wherein if the level of CCN3 indicates that the female subject is at risk of bone degeneration from breastfeeding, the female subject stops breastfeeding.

82. The method of claim 80 or 81, the method further comprising administering a therapeutically effective amount of the CCN3 to the female subject to reduce the risk of bone degeneration from breastfeeding.

83. The method according to any one of claims 80 to 82, wherein the biological sample is blood or plasma.

84. The method according to any one of claims 80 to 83, wherein the measurement of CCN3 level comprises performing enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, immunohistochemistry, fluorescence activated cell sorting (FACS), Western blotting, mass spectrometry, tandem mass spectrometry, biochemical assay, liquid chromatography, or NMR.

85. A communication network factor 3 (CCN3) used as a biomarker to monitor lactating female subjects to determine the risk of bone loss from breastfeeding.

86. A method for regenerating mammalian cartilage in a subject, the method comprising: Mechanical stimulation is used to activate bone stem cells; as well as The subjects were administered a therapeutically effective dose of a combination of cell communication network factor 3 (CCN3) and a therapeutically effective dose of a vascular endothelial growth factor (VEGF) inhibitor.

87. The method of claim 86, wherein the CCN3 comprises or is composed of the following: The amino acid sequence of SEQ ID NO: 1, or a sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

88. The method according to claim 86 or 87, wherein the VEGF inhibitor is cabozantinib.

89. The method according to any one of claims 86 to 88, wherein the cartilage is articular cartilage.

90. The method according to any one of claims 86 to 89, wherein the mechanical stimulation is an acute local injury.

91. The method of claim 90, wherein the acute local injury is a surgical microfracture of bone tissue performed at a desired site for cartilage regeneration.

92. The method according to claim 90 or 91, wherein the VEGF inhibitor and the CCN3 are applied locally to the acute local injury.

93. The method according to any one of claims 86 to 92, wherein the VEGF inhibitor and the CCN3 are administered to the subject immediately after the bone stem cells are activated by the mechanical stimulation.

94. The method according to any one of claims 86 to 92, wherein the VEGF inhibitor and the CCN3 are administered to the subject within three days after the mechanical stimulation activates the bone stem cells.

95. The method according to any one of claims 86 to 94, wherein the VEGF inhibitor and the CCN3 are encapsulated in a hydrogel.

96. The method of claim 95, wherein the hydrogel comprises alginate.

97. The method of claim 96, wherein the alginate is ionically cross-linked.

98. The method of claim 97, wherein the alginate is ionically crosslinked by divalent calcium cations.

99. The method according to any one of claims 96 to 98, wherein the concentration of the alginate in the hydrogel is in the range of 2 to 10 weight percent (wt%).

100. The method according to any one of claims 86 to 99, wherein the VEGF inhibitor and the CCN3 are administered using a drug delivery device.

101. The method of claim 100, wherein the drug delivery device provides continuous delivery of the VEGF inhibitor and the CCN3.

102. The method according to claim 100 or 101, wherein the drug delivery device is a reservoir implant or an integral implant.

103. The method according to any one of claims 100 to 102, wherein the drug delivery device is a non-biodegradable implant.

104. The method according to any one of claims 100 to 102, wherein the drug delivery device is a biodegradable implant.

105. The method according to any one of claims 100 to 104, wherein the drug delivery device is implanted at the site of the local acute injury.

106. The method according to any one of claims 86 to 105, wherein the subject suffers from chondrodysplasia.

107. The method according to claim 106, wherein the cartilage disease is osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthritis, achondroplasia, recurrent polychondritis, chondroma, chondrosarcoma, traumatic cartilage injury, infection, or malignancy.

108. The method according to any one of claims 86 to 107, the method further comprising administering a therapeutically effective amount of bone stem cells to the subject.

109. The method according to any one of claims 86 to 108, wherein the CCN3 is provided by a carrier comprising an expression box, the expression box comprising an encoding sequence encoding the CCN3.

110. The method of claim 109, wherein the expression box includes a promoter operatively connected to the coding sequence encoding the CCN3.

111. The method of claim 109, wherein the coding sequence encoding the CCN3 is integrated into a chromosomal locus in the genome of the subject.

112. The method of claim 111, wherein the endogenous promoter is operatively linked at the chromosomal locus to the integrated coding sequence encoding the CCN3.

113. The method according to any one of claims 109 to 112, wherein the vector is a plasmid or a viral vector.

114. The method according to claim 113, wherein the viral vector is an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, or a retroviral vector.

115. A composition comprising cell communication network factor 3 (CCN3) and a vascular endothelial growth factor (VEGF) inhibitor, for use in a method of regenerating mammalian cartilage.

116. The composition of claim 115, wherein the CCN3 comprises or is composed of the following: The amino acid sequence of SEQ ID NO: 1, or a sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

117. The composition according to claim 115 or 116, wherein the VEGF inhibitor is cabozantinib.

118. The composition according to any one of claims 115 to 117, wherein the cartilage is articular cartilage.

119. The composition according to any one of claims 115 to 118, wherein the CCN3 and the VEGF inhibitor are encapsulated in a hydrogel.

120. The composition of claim 119, wherein the hydrogel comprises alginate.

121. The composition according to claim 120, wherein the alginate is ionically crosslinked.

122. The composition of claim 121, wherein the alginate is ionically crosslinked by divalent calcium cations.

123. The composition according to any one of claims 120 to 122, wherein the concentration of the alginate in the hydrogel is in the range of 2 to 10 weight percent (wt%).

124. A method for screening agonists of cell communication network factor 3 (CCN3) that increase chondrogenesis, the method comprising: Mechanical stimulation is used to activate bone stem cells; The bone stem cells are then contacted with CCN3, a vascular endothelial growth factor (VEGF) inhibitor, and a candidate drug. as well as The amount of increased cartilage production in the presence of the candidate drug, compared to a reference range of cartilage production in control bone stem cells in which the candidate drug is absent, indicates that the candidate drug is an agonist of CCN3.

125. A method for screening mimics or analogs of cell communication network factor 3 (CCN3) that stimulates cartilage production, the method comprising: Mechanical stimulation is used to activate bone stem cells; The bone stem cells are then contacted with vascular endothelial growth factor (VEGF) inhibitors and candidate agents. as well as The amount of cartilage production through the bone stem cells is measured, wherein the increase in cartilage production in the presence of the candidate drug, compared to a reference range of cartilage production in control bone stem cells in which the candidate drug is absent, indicates that the candidate drug is an analogue or analogue of the CCN3.

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