Thrombomodulin domains for promoting osteoblast function and bone healing

Abstract

The present invention relates to thrombomodulin functional domains for promoting osteoblast function and bone healing. The present invention discloses methods for stimulating bone regeneration, bone enhancement, treating bone loss, bone disorders and / or treating bone-related disorders. The method comprises administering to a subject in need thereof a therapeutically effective amount of (I): a biologically active recombinant polypeptide comprising an amino acid sequence having at least 80% identity with human thrombomodulin domains 2 and 3 (TMD23); (II): an isolated polypeptide comprising recombinant TMD23 (rTMD23) or a biologically active recombinant variant thereof; or (III): recombinant TMD23 (rTMD23) or a biologically active recombinant variant thereof, wherein the polypeptide does not contain or lacks amino acid residues from human thrombomodulin domains 4 and 5, and further, wherein the biologically active variant thereof comprises an amino acid sequence having at least 80% identity with TMD23.

Description

Technical Field

[0001] The present invention relates generally to methods for treating bone loss, promoting bone formation and fracture repair (osteoinduction, bone enhancement), and more particularly to methods for stimulating osteoblastogenesis and inhibiting osteoclastogenesis. Background Art

[0002] The bone remodeling process involves the absorption of mineralized bone by osteoclasts, followed by the formation of bone matrix and mineralization by osteoblasts. Under the stimulation of growth factors and vitamin D3, mesenchymal stem cells transform into osteoprogenitor cells and osteoblasts to initiate osteogenesis. Osteoblasts secrete type I collagen and calcium deposition to form osteoid, which is further mineralized to form bone tissue. On the other hand, under the stimulation of macrophage colony-stimulating factor 1 (M-CSF) and receptor activator of nuclear factor κB (NFκB) ligand (RANKL), macrophages transform into osteoclasts, which are responsible for the bone resorption process, called osteoclastogenesis. The balance between bone resorption and formation processes is essential to maintain bone homeostasis.

[0003] Osteoporosis is a disease that occurs when bone mass or density decreases due to an imbalance between the remodeling activities of osteoclasts and osteoblasts. Drugs that can be used to prevent and / or treat osteoporosis include bisphosphonates, calcitonin, estrogen (hormone therapy), estrogen agonists, parathyroid hormone (PTH) analogs, RANK ligand (RANKL) inhibitors, and tissue selective estrogen complex (TSEC). These drugs have side effects and cannot stimulate osteoblastogenesis and inhibit osteoclastogenesis at the same time.

[0004] Human thrombomodulin (TM) is a transmembrane protein whose structure consists of five domains, including an NH2-terminal lectin-like domain (D1), a domain with six epidermal growth factor (EGF)-like structures (D2), a domain rich in O-glycosylation sites (D3), a transmembrane domain (D4), and a cytoplasmic tail domain (D5). Cheng et al. reported that TMD1 can inhibit osteoclastogenesis by reducing pro-inflammatory high-mobility group box 1 (HMGB1) signaling (“Myeloid thrombomodulinlectin-like domain inhibits osteoclastogenesis and inflammatory bone loss” SciRep. 2016;6:28340).

[0005] US Patent No. 9,968,660 discloses a method of bone regeneration or bone enhancement. Summary of the invention

[0006] On the one hand, the present invention relates to the use of a biologically active recombinant polypeptide comprising an amino acid sequence having at least 80% identity with human thrombomodulin domains 2 and 3 (TMD23) in the preparation of a medicament for stimulating bone regeneration, bone enhancement, treating bone loss, bone disorders and / or bone-related disorders in a subject in need thereof, wherein the polypeptide does not contain or lacks amino acid residues from human thrombomodulin domains 4 and 5.

[0007] On the other hand, the present invention relates to the use of an isolated polypeptide comprising recombinant human thrombomodulin domains 2 and 3 (rTMD23) or a biologically active recombinant variant thereof in the preparation of a medicament for stimulating bone regeneration, bone enhancement, treating bone loss, bone disorders and / or bone-related disorders in a subject in need thereof, wherein the biologically active variant thereof comprises an amino acid sequence that is at least 80% identical to TMD23, ​​and further, wherein the polypeptide does not contain or lacks amino acid residues from human thrombomodulin domains 4 and 5.

[0008] In one embodiment, the polypeptide comprises amino acid residues of TMD23.

[0009] In another embodiment, the polypeptide comprises amino acid residues 224Ala to 497Ser, or 1Ala to 497Ser of SEQ ID NO:1.

[0010] In another embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:3.

[0011] In another embodiment, the polypeptide is a biologically active recombinant TMD23 variant and comprises the amino acid sequence of SEQ ID NO:3.

[0012] In another embodiment, the polypeptide further comprises amino acid residues from human thrombomodulin domain 1.

[0013] In one embodiment, the polypeptide consists essentially of domains 2 and 3, or domains 1, 2, and 3 of human thrombomodulin.

[0014] In another embodiment, the recombinant TMD23 variant consists of the amino acid sequence of SEQ ID NO:3.

[0015] In another embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

[0016] In another embodiment, the polypeptide or the biologically active variant comprises amino acid residues that are at least 85%, or at least 90%, or at least 95% identical to rTMD23.

[0017] In another aspect, the present invention relates to the use of recombinant human thrombomodulin domains 2 and 3 (rTMD23) or a biologically active recombinant variant thereof in the preparation of a medicament for stimulating bone regeneration, bone enhancement, treating bone loss, bone disorders and / or bone-related disorders in a subject in need thereof, wherein the biologically active variant thereof comprises an amino acid sequence that is at least 80% identical to human TMD23.

[0018] In another embodiment, the recombinant variant of rTMD23 comprises the amino acid sequence of SEQ ID NO:3.

[0019] In another embodiment, the recombinant TMD23 variant consists of the amino acid sequence of SEQ ID NO:3.

[0020] In another embodiment, the bone disorder is a disease or disorder selected from osteoporosis, bone loss, failure to achieve optimal bone formation, failure to achieve optimal fracture healing, achieving low peak bone mass during bone growth, impaired new bone formation, and any combination thereof.

[0021] In another embodiment, the bone-related disorder is selected from at least one of hyperparathyroidism, hyperparathyroidism-related osteopenia, hyperparathyroidism-related decreased bone density, osteopenia, bone loss, inflammatory bone loss, osteoporosis, senile osteoporosis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, osteonecrosis of the jaw, Paget's disease, and hyperphosphatemia.

[0022] Furthermore, in another embodiment, the above use is combined with the use of bone graft material for the preparation of a medicament for stimulating new bone formation. Bone graft material provides structural stability and connection and stimulates osteogenesis and bone healing in fractures.

[0023] The polypeptides or biologically active recombinant variants of rTMD23 as described above can be used to prepare a medicament for treating bone-related disorders, stimulating bone repair, promoting implantation of bone implants or stimulating osteoblast proliferation.

[0024] In one embodiment, the medicament is in a dosage form suitable for application to a part of the body in need of bone enhancement.

[0025] The body part requiring bone augmentation is selected from one of a sinus lift, a bone graft, and a ridge augmentation.

[0026] The medicament may be in the form of a gel, cream, paste, lotion, spray, suspension, solution, dispersible salves, hydrogel or ointment.

[0027] In another embodiment, the body region in need of bone augmentation is selected from a region in need of bone repair, fracture healing, or spinal fusion.

[0028] In one embodiment, the body site in need of bone augmentation is a dental implant placement site.

[0029] The drug may be a dosage form containing a daily human dose of the above polypeptide, with a unit dose ranging from 100 ng / kg to 100 mg / kg×(0.025 kg / human body weight kg) 0.33 .

[0030] The present invention also relates to a method for stimulating bone regeneration, bone enhancement, treating bone loss, bone disorders and / or bone-related disorders. The method comprises administering to a subject in need thereof a therapeutically effective amount of (I): a biologically active recombinant polypeptide comprising an amino acid sequence having at least 80% identity with TMD23; (II): an isolated polypeptide comprising recombinant TMD23 (rTMD23) or a biologically active recombinant variant thereof; or (III): recombinant TMD23 (rTMD23) or a biologically active recombinant variant thereof, wherein the polypeptide does not contain or lacks amino acid residues from human thrombomodulin domains 4 and 5, and the biologically active variant thereof comprises an amino acid sequence having at least 80% identity with TMD23.

[0031] Alternatively, the present invention relates to (I): a biologically active recombinant polypeptide comprising an amino acid sequence that is at least 80% identical to human TMD23; (II): an isolated polypeptide comprising recombinant TMD23 (rTMD23) or a biologically active recombinant variant thereof; or (III): recombinant TMD23 (rTMD23) or a biologically active recombinant variant thereof, for stimulating bone regeneration, bone enhancement, treating bone loss, bone disorders and / or bone-related disorders in subjects in need thereof, wherein the polypeptide does not contain or lacks amino acid residues from human thrombomodulin domains 4 and 5, and further, wherein its biologically active variant comprises an amino acid sequence that is at least 80% identical to TMD23.

[0032] These and other aspects will become apparent from the following description of the preferred embodiments taken in conjunction with the following drawings, although changes and modifications therein may be effected without departing from the spirit and scope of the novel concepts of the present invention.

[0033] The accompanying drawings illustrate one or more embodiments of the present invention and together with the written description serve to explain the principles of the present invention. Wherever possible, the same reference numerals are used throughout the drawings to refer to the same or similar elements of the embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1A-B shows that recombinant TMD23 (rTMD23) and TMD123 promote cell migration of MG63 cells. MG63 cells were loaded into the upper chamber, and various concentrations of rTMD23 or TMD123 as shown were loaded into the lower chamber. After incubation for 4 hours, the membrane was stained with Liu staining. The migrated cells were counted after taking pictures. **P<0.01.

[0035] Figure 2A It shows that recombinant TMD23 (rTMD23) enhances cell proliferation of MG-63 cells. **P<0.01; ***P<0.001.

[0036] Figure 2B Shows that recombinant TMD23NA, a variant of rTMD23, ​​enhances cell proliferation of MC3T3-E1 cells. *P<0.05 compared to the absorbance of 0 ng / ml rTMD23NA.

[0037] Figure 3 Recombinant TMD23 (rTMD23) was shown to enhance cellular mineralization of MG-63 cells. Cells were incubated with different concentrations of rTMD23 (0, 10, 25, 50 ng / mL) for 28 days. Cells were fixed with 10% formaldehyde for 20 minutes and then washed twice with PBS before staining. Alizarin Red S (40%) was added to each well at 1000 μL / well and allowed to stand for 20 minutes, then rinsed at least five times with ddH2O. Photos were taken with a 10x inverted fluorescence microscope and relative intensity was measured with ImageJ software. ***P<0.001.

[0038] Figure 4A -B shows that rTMD123 inhibits RANKL-induced osteoclast formation on RAW264.7 cells. (4A) RAW264.7 cells were cultured with the indicated concentrations of rTMD123 in the presence of RANKL (30 ng / ml) and M-CSF (20 ng / ml) for 4 days. Multinucleated osteoclasts were observed by TRAP staining. (4B) TRAP + OC counts. ***P<0.001 (compared with MCSF / RANKL-treated group). Data are presented as mean ± SD and are representative of at least three experiments.

[0039] Figure 5A -D shows that rTMD23 (5A-B) and its variants (5C-D) inhibit RANKL-induced osteoclast formation in RAW264.7 cells, respectively. RAW264.7 cells were cultured with the indicated concentrations of rTMD23 or variant rTMD23NA in the presence of RANKL and M-CSF for 4 days. Multinucleated osteoclasts were observed by TRAP staining. TRAP in each well was +OC counts. Data are presented as mean ± SD and are representative of at least three experiments. ***P < 0.001 (compared with MCSF / RANKL-treated group).

[0040] Fig. 6A -B shows the effect of rTMD123 on absorption pit formation. (6A) RAW264.7 cells were cultured on Corning Osteoassay 96-well plates containing different concentrations of rTMD123 in the presence of MCSF (20 ng / ml) and RANKL (30 ng / ml) for 4 days. After incubation, the absorption pits on the plate were observed using an optical microscope. (6B) The percentage of reabsorption area relative to the control group was calculated using Image J software. ***P<0.001 (compared with the MCSF / RANKL treatment group).

[0041] Fig. 7A -B shows that recombinant TMD23 (rTMD23) enhances skull healing in STZ mice. Mice were injected with 50 mg / kg streptozotocin (STZ) daily for at least five days. Blood glucose was measured 3 days after the last injection. Skull surgery was then performed to create a hole with a diameter of 4 mm. (7A) rTMD23 (0, 10, 100 μg / kg) was injected subcutaneously once on the 7th day after surgery (week 1), and microCT (μCT) images were taken at weeks 0 and 8. (7B) For data quantification, the term "TV" represents the total volume of the hole, and the term "BV" represents the regenerated bone volume at week 8. **P<0.01.

[0042] Fig. 8A -F shows the effects of TMD1 and TMD23 on skull healing. (8A) Cell migration, (8B) proliferation, and (8C) mineralization of MG63 cells treated with the indicated doses of rTMD1. (8D) Wild-type TM wt / wt and TM domain 1 (TMD1)-deficient TM LeD / LeD Skull healing in mice. (8E) μCT scans and (8F) HE staining were used to evaluate the effects of vehicle, dichloroisocoumarin (DCI), and DCI plus rTMD23 or rTMD1 (100 μg / kg) on ​​bone healing after skull defect in mice. N = 5 / group. Black bar = 100 μm, white bar = 1 mm. Dashed lines indicate cross-sectional areas. ***P < 0.001. DETAILED DESCRIPTION

[0043] The present invention is described in more detail in the following examples, which are intended for illustration only, as it will be apparent to those skilled in the art that many modifications and variations therein will be obtained. Various embodiments of the present invention are now described in detail. With reference to the accompanying drawings, similar reference numerals represent similar components throughout the entire view. As used in the description herein and in the claims that follow, the meanings of "one", "a kind of" and "the" include plural references, except where the context clearly states otherwise. In addition, as used in the description herein and in all subsequent claims, the meaning of "in..." includes "in..." and "on...", except where the context clearly states otherwise. In addition, for the convenience of the reader, titles or subtitles may be used in the specification, which should not affect the scope of the present invention. In addition, some of the terms used in this specification are more specifically defined below.

[0044] definition

[0045] The terms used in this specification generally have their ordinary meaning in the art, in the context of the present invention, and in the specific context in which each term is used. Certain terms used to describe the present invention are discussed elsewhere below or in the specification to provide additional guidance to practitioners with respect to the description of the present invention. It will be understood that the same thing can be expressed in more than one way. Therefore, any one or more terms discussed herein can use alternative language and synonyms, and whether a term is elaborated or discussed herein does not give any special meaning. Mentioning one or more synonyms does not exclude the use of other synonyms. The use of embodiments (including examples of any terms discussed herein) anywhere in this specification is only illustrative, and does not limit the scope and meaning of the present invention or any exemplary term in any way. Likewise, the present invention is not limited to the various embodiments provided in this specification.

[0046] Unless defined otherwise, 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 belongs. In the event of a conflict, the present document, including definitions, will control.

[0047] As used herein, "around", "about" or "approximately" generally means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. The numerical values ​​given herein are approximate, which means that if not explicitly stated, the term "around", "about" or "approximately" can be inferred.

[0048] The term "rTMD23" refers to recombinant thrombomodulin domains 2 and 3. Thrombomodulin (TM) is a cell membrane-bound glycoprotein consisting of five domains, including an N-terminal lectin-like domain (D1), six epidermal growth factor (EGF) repeats (D2), a serine-threonine-rich region (D3), a transmembrane domain (D4), and a short cytoplasmic tail (D5). Mature human TM without a signal peptide has 557 amino acid residues and comprises the amino acid sequence of SEQ ID NO: 1.

[0049] At least 80% means that all integer unit amounts within the range of 80% to 100% are specifically disclosed as part of the present invention. Therefore, 80%, 81%, 82%, 83%, 84%, 85% ... 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% unit amounts are included as embodiments of the present invention.

[0050] The term "amino acid sequence having at least 80% identity to TMD23" shall include wild-type rTMD23 and biologically active recombinant variants thereof.

[0051] The terms "bioactive" and "biologically active" are used interchangeably.

[0052] As used herein, the term "biologically active recombinant variant thereof" generally refers to a biologically active recombinant variant form of thrombomodulin domains 2 and 3 (TMD23).

[0053] "Biologically active recombinant variant of rTMD23" refers to a variant having the biological activity of wild-type rTMD23, ​​wherein the biological activity is selected from at least one of promoting cell proliferation and migration in osteoblasts, enhancing cell mineralization in osteoblasts, inhibiting osteoclastogenesis, inhibiting bone resorption activity, promoting bone regeneration, promoting osteoblast function and bone healing, and any combination thereof.

[0054] The terms "biologically active variant form of recombinant TMD23", "biologically active recombinant human TMD23 variant", "biologically active recombinant human TMD23 variant polypeptide" and "human rTMD23 variant" are interchangeable.

[0055] As used herein, the terms "thrombomodulin variant", "variant of thrombomodulin" and "mutant thrombomodulin" are interchangeable.

[0056] Thrombomodulin variants generally refer to mutant thrombomodulin that differ in amino acid residues from the wild-type thrombomodulin domain but retain the biological activity of the wild-type thrombomodulin domain in promoting wound healing effects. Such thrombomodulin variants, rTMD23NA and rTMD123NA, have been disclosed in U.S. Pat. No. 9,156,904B2, all of which are incorporated herein by reference in their entirety.

[0057] The term "rTMD23NA" refers to a recombinant TMD23 variant with two substitutions, Asn364Ala and Asn391Ala. rTMD23NA is a mutant in which Asn is replaced by Ala at residues 364 and 391 of wild-type mature human thrombomodulin (SEQ ID NO: 1).

[0058] Both TMD123NA and rTMD23NA are variants with two putative N-glycosylated Asn residues, namely, N364 and N391 of TMD23 mutated to Ala by site-directed mutagenesis. The recombinant mutant rTMD23NA has little or no protein C activation activity. Preferably, the variant of thrombomodulin comprises and / or has the following characteristics: (i) an amino acid sequence having at least 80% identity with SEQ ID NO: 1 (557aa); (ii) two amino acid residues replace Asn364Ala and Asn391Ala; (iii) little or no protein C activation activity.

[0059] The terms "treating" or "treatment" refer to the administration of an effective amount of a therapeutic agent to a subject suffering from a disease or having symptoms or a tendency for such a disease, in order to cure, alleviate, relieve, remedy, ameliorate or prevent the disease, its symptoms or tendency.

[0060] The terms "bone grafting materials" and "bone graft materials" are interchangeable. It is well known that bone graft materials can provide structural stability and connection and stimulate osteogenesis and bone healing in fractures.

[0061] The term "associated with" generally means "related to" or "pertaining to." Thus, "hyperparathyroidism associated with osteopenia" should mean "hyperparathyroidism associated with osteopenia."

[0062] The U.S. Department of Health and Human Services Food and Drug Administration's "Guide for Industry and Reviewers to Evaluate Safe Starting Doses for Clinical Trials of Therapeutics in Adult Healthy Volunteers" discloses that the "human equivalent dose" can be calculated by the following formula:

[0063] HED = animal dose in mg / kg x (animal body weight in kg / human body weight in kg) 0.33.

[0064] Sequence listing: SEQ ID NO: 1, wild-type full-length TM amino acid sequence without signal peptide (557aa); Domain 1, 1Ala-223Gly; Domain 2, 224Ala-462Cys; Domain 3, 463Asp-497Ser; Domain 4, 498Gly-521Leu; Domain 5, 522Arg-557Leu; SEQ ID NO: 2, rTMD23 sequence (274aa); SEQ ID NO: 3, rTMD23NA sequence (274aa); SEQ ID NO: 4, rTMD123 sequence (497aa); SEQ ID NO: 5, TMD123NA sequence (497aa).

[0065] Abbreviations: TM, thrombomodulin; rTM, recombinant thrombomodulin; rTMD23, ​​recombinant thrombomodulin domains 2 and 3; TMD4, thrombomodulin domain 4; TMD5, thrombomodulin domain 5; Asn (N), asparagine; Ala (A), alanine.

[0066] The present invention relates to the discovery of truncated thrombomodulin (TM) proteins comprising thrombomodulin domains 2 and 3 (TMD23) or variants thereof, which have the biological activity of inhibiting the transformation of macrophages into osteoclasts and promoting the formation of osteoblasts from mesenchymal stem cells. Studies have found that truncated TM proteins TMD23, ​​TMD123 and variant TMD23NA can simultaneously increase the generation of osteoblasts and inhibit the generation of osteoclasts. These biologically active TM domains can be used to stimulate bone formation and inhibit bone loss.

[0067] The present invention also relates to recombinant proteins TMD23, ​​TMD123 or their biologically active variants, which are used to improve and treat osteoporosis. Studies have found that in recombinant TM fragments, TM domains 2 and 3 (TMD23) rather than lectin-like domains (TMD1) promote osteoblast function and skull healing. The present invention also relates to a truncated TM, which is used to stimulate osteoblasts by systemic, local administration or in combination with bone graft materials, while inhibiting osteoclasts.

[0068] Example

[0069] Without limiting the scope of the present invention, exemplary instruments, devices, methods and related results thereof according to embodiments of the present invention are provided below. Note that titles or subtitles may be used in the examples for the convenience of the reader, which should not limit the scope of the present invention. In addition, certain theories are proposed and disclosed herein; however, as long as the present invention is implemented according to the present invention without considering any particular theory or course of action, whether they are right or wrong, they should not limit the scope of the present invention.

[0070] Materials and methods

[0071] Expression and purification of recombinant TM domain and variant proteins. The preparation of rTMD23 and variant rTMD23NA has been disclosed in US9,156,904B2, which is incorporated herein by reference in its entirety. In brief, pCR3 or pPICZA vectors (Invitrogen, Oregon) are used to express and secrete recombinant TM domain proteins in HEK293 or Pichia pastoris protein expression systems. DNA fragments encoding TMD1, TMD23 and TMD123 were obtained as described previously (Shi et al. "Lectin-like domain of thrombomodulin binds to its specific ligand Lewis Yantigen and neutralizes lipopolysaccharide-induced inflammatory response" Blood. 2008 Nov 1; 112(9): 3661-70; Cheng et al. "Functions of rhomboid family protease RHBDL2and thrombomodulin in wound healing" J Invest Dermatol. 2011 Dec; 131(12): 2486-94). The expressed recombinant protein was loaded onto a nickel chelated Sepharose column and the fraction containing the recombinant TM domain was eluted.

[0072] Cell culture and treatment. The osteoblast cell line MC3T3-E1, the human osteoblast-like cell line MG63, and the mouse macrophage cell line RAW264.7 ( Rockville, MD, USA). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, MD, USA) containing 10% fetal bovine serum (FBS, Gibco).

[0073] Transwell cell migration assay. A 24-well chemotaxis chamber with an 8-μm pore size membrane was used to assess cell migration. Cell suspensions (5 × 10 for MG63 cells) were plated in a 5% flask. 4 Cells / 100 μL of serum-free DMEM medium) were added to the upper chamber, and the recombinant TM domain in serum-free medium (600 μL) was added to the lower chamber. These chambers were incubated at 37°C for 8 hours. The non-migrated cells were wiped off with a cotton pad. The filter was developed using Liu's staining kit and the number of remaining cells was counted under a microscope.

[0074] Proliferation assay. MG63 cells were treated with different concentrations of rTMD23 for 1, 4, and 7 days. The culture medium was replaced every 2-3 days. Finally, the number of viable cells in each well was determined by the crystal violet method. The cells in each well were washed twice with PBS before staining. Crystal violet (0.5%, 300 μL) was added to each well for 15 minutes and then rinsed with ddH2O at least five times. Acetic acid (10%, 300 μL) was added to dissolve the crystal violet. Then, 100 μL / well was taken and placed in a 96-well plate to obtain OD595 measurements. MC3T3-E1 cell proliferation assays under different concentrations of TMD23 variants were performed. In short, MC3T3-E1 cells in 96-well plates were treated with different concentrations of rTMD23NA for 1, 3, and 5 days. The number of viable cells in each well was measured using WST-1 reagent. The cells in each well were washed twice with DMEM medium and then stained with 100 μL 10% WST-1 (in DMEM). Measure each well at A 450nm -A 690nm The absorbance at .

[0075] Mineralization assay. MG63 cells were seeded in 6-well plates (5×10 5 Cells / well) were added and treated with recombinant TM domains for 28 days. As previously described, calcium staining of mineralized matrix was performed using Alizarin Red staining (Stanford et al. "Rapidlyforming apatitic mineral in an osteoblastic cell line (UMR 106-01BSP)" J Biol Chem. 1995 April 21; 270(16): 9420-8). Briefly, cells were washed with PBS and then fixed in ice-cold 70% ethanol for 1 hour. After washing, cells were stained with 40mM Alizarin Red (pH 4.2) for 10 minutes at room temperature. The stained cells were further treated as follows: rinsed five times with water and then rotated in PBS for 15 minutes to wash to reduce nonspecific Alizarin Red staining. Images were taken and relative intensity was measured by ImageJ software.

[0076] Experimental animals and diabetic mice.LeD / LeD Six to eight week old mice and wild type C57BL / 6 mice (Conway et al. "The lectin-like domain of thrombomodulinconfers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways" J Exp Med. 2002 Sep 2; 196(5):565-77). Diabetic mice were generated by a single intravenous injection of streptozotocin (STZ) (150 mg / kg) dissolved in 50 mM citrate (pH 4.7). Control mice were injected with citrate buffer. Glucose content was measured using a glucose kit (Randox, Crumlin, UK) according to the manufacturer's instructions. The blood glucose concentration of diabetic mice reached 413.9 mm (250 mg / dL).

[0077] Healing of skull defects. Male mice were randomly assigned to designated groups. After anesthesia, a 4-mm diameter defect was created on the skull, and the wound was sutured. At week 1 after surgery, dichloroisocoumarin (DCI) (1 mM) or a combination of rTMD23 (0, 10, 100 μg / kg) and U0126 (20 μM) was injected subcutaneously once near the skull opening. At designated time points after surgery (weeks 0 and 8), bone healing was monitored radiographically and histologically using μCT (Skyscan 1076, Antwerp, Belgium). The μCT scanner was operated at 50 kV, 220 mA, 1.2 s exposure time, with a pixel size of 2.5 mm, an aluminum filter of 0.5 mm, an isotope of 17.09 mm voxels generated by 180° rotation, and a slice thickness of 2.5 mm. To calculate bone formation, a cylindrical volume with a diameter of 4 mm and a depth of 2 mm was selected for skull defect regeneration. The cross-sectional images were converted into volume reconstructions using the NRecon program (Skyscan). The volume of bone removed and regenerated (mm) was analyzed using 3D CT Analyzer software (CTAN; Skyscan). 3 ) for quantification.

[0078] For histological analysis, specimens were decalcified in 10% EDTA for 1 week and then embedded in paraffin. Hematoxylin-eosin (H&E) staining was performed using 10 mm thick sections.

[0079] Statistical analysis. Statistical analysis was performed using Prism 7 (GraphPad software). Data are presented as mean ± SD. For comparison of multiple groups, one-way analysis of variance (ANOVA) was used. For comparison of multiple groups at different time points, two-way analysis of variance was used. Values ​​of P < 0.05 were considered statistically significant.

[0080] result

[0081] Both rTMD23 and rTMD123 can promote cell migration of osteoblasts

[0082] Cell functions of osteoblasts include migration, proliferation, and mineralization. Cells in serum-free medium were added to the upper transwell chamber, and 1% fetal bovine serum and increasing doses of rTMD23 were added to the lower transwell chamber. After incubation at 37°C for 4 hours, the transwell membrane was removed and stained. The number of cells that migrated through the membrane to the lower surface of the membrane was counted under a microscope. Cell migration assays showed that both rTMD23 and rTMD123 increased cell migration of osteoblasts ( Figure 1A -B).

[0083] Both rTMD23 and its bioactive variants promote cell proliferation of osteoblasts

[0084] Cell proliferation assays were performed using the human osteoblastic cell line MG63 and the osteoblastic cell line MC3T3-E1. Briefly, different concentrations of rTMD23 and rTMD23NA (variant forms of rTMD23) were added to MG63 or MC3T3-E1 cell cultures containing 1% FBS, respectively. The cell cultures were washed twice with PBS and stained with crystal violet. The OD595 of the cell lysates in 10% acetic acid was measured to obtain the relative cell counts in each well. The results showed that rTMD23 and variant rTMD23NA could enhance the proliferation of the human osteoblastic cell line MG63 ( Figure 2A ) and osteoblast cell line MC3T3-E1( Figure 2B ) of cell proliferation.

[0085] rTMD23 enhances cellular mineralization of osteoblasts

[0086] MG63 cells were incubated in medium containing 1% FBS in the presence of the indicated concentrations of rTMD23 for 28 days. After fixation with 10% formaldehyde, the cells were stained with 40% Alizarin Red S. The results showed that rTMD23 could promote the mineralization of MG63 cells ( Figure 3 ).

[0087] rTMD123, rTMD23 and their variants inhibit osteoclastogenesis

[0088] To induce osteoclastogenesis, RAW264.7 cells were cultured in the presence of RANKL (30 ng / ml) and M-CSF (20 ng / ml) for 4 days to induce the formation of TRAP-positive osteoclasts. Multinucleated osteoclasts in each well were shown by TRAP staining ( Figure 4A ). For the TRAP formed in each well + OC number count ( Figure 4B ). In the presence of rTMD123, the formation of TRAP-positive osteoclasts was inhibited. rTMD23( Figure 5A -B) and variant rTMD23NA ( Figure 5C -D) also obtained similar results. The results showed that both rTMD23 and rTMD123 could inhibit the formation of osteoclasts in vitro.

[0089] rTMD123 inhibits the bone resorption activity of osteoclasts

[0090] In the presence of different concentrations of rTMD123, MCSF (20 ng / ml) and RANKL (30 ng / ml) were used to induce osteoclast formation in RAW264.7 cells cultured on Corning Osteoassay 96-well plates. After 4 days of incubation, the absorption pits on each well were observed using an optical microscope ( Fig. 6A ), and the absorption area was measured using Image J software. Compared with the control group, the percentage of bone absorption area in the rTMD123 treatment group was significantly reduced ( Figure 6B ). The results showed that rTMD123 could inhibit the bone resorption activity of RAW264.7 cells.

[0091] rTMD23 enhances skull bone healing in a streptozotocin-induced diabetic model

[0092] Diabetes was induced in mice by injecting 50 mg / kg streptozotocin (STZ) for 5 days. A 4 mm diameter hole was created in the skull of each mouse. Different doses of rTMD23 were injected subcutaneously once near the skull opening at week 1 after surgery. MicroCT images were taken at week 0 and week 8 ( Fig. 7A The rTMD23-treated group had a significant increase in regenerated bone volume within 8 weeks ( Figure 7B ). The results showed that rTMD23 can promote bone regeneration. The average weight of mice was 25g.

[0093] rTMD23 but not rTMD1 promotes skull healing

[0094] We further investigated whether TM lectin-like domain (TMD1) could also enhance cellular function in osteoblasts. Functional assays showed that rTMD1 did not promote cell migration in MG63 cells ( Fig. 8A ), or proliferation ( Figure 8B ), or mineralization ( Figure 8C Using TMD1 deletion (TMD1 LeD / LeD ) transgenic mice, the effect of TMD1 on bone regeneration was further evaluated by skull bone healing assay. As shown by μCT, at week 8, TM LeD / LeD Mouse and TM wild-type TM wt / wt There was no significant difference in bone healing between control mice ( Fig.8D These findings suggest that TMD1 cannot promote osteoblast function and bone healing. The skull bone healing assay was also used to evaluate the effect of TMD23 on bone healing. μCT measurements ( Fig. 8E ) and H&E staining ( Fig.8F ) showed that treatment with DCI to inhibit RHBDL2 and TM ectodomain shedding reduced regeneration of calvarial defects. However, rTMD23, ​​but not rTMD1, could rescue this inhibitory effect. The results suggest that rTMD23, ​​but not rTMD1, has the ability to promote osteoblast function and bone healing.

[0095] In summary, rTMD23, ​​rTMD123 or its variants can promote the migration, proliferation and mineralization of osteoblasts. As shown, TMD23 can be used as a chemical attractant to attract osteoblasts and pre-osteoblasts MG63 (a human osteoblast-like cell line), thereby promoting bone formation. MG63 cells are phenotypically in a pre-osteoblast state and can be induced to mineralize. It has been shown that TMD23 can promote bone healing in STZ-induced diabetic mice. Macrophages can be induced to form multinuclear osteoclasts by stimulation with macrophage colony stimulating factor 1 (MCSF-1) and RANK ligand (RANKL) during osteoclastogenesis. TMD23, ​​TMD123 or its biologically active variants can inhibit the generation of macrophage osteoclasts induced by MCSF-1 and RANK ligand (RANKL). As shown, rTMD23 can inhibit bone resorption of osteoclasts.

[0096] In summary, proteins containing thrombomodulin domain 23, including TMD23, ​​TMD123 and their biologically active variants, can inhibit osteoclastogenesis and promote osteoblastogenesis. TMD23, ​​TMD123 or their biologically active variants can be used to prevent and treat bone loss diseases, symptoms or conditions, including osteoporosis, osteopenia, rheumatoid arthritis, arthritis and periodontitis, and to promote bone regeneration, bone enhancement, bone injury treatment and promote bone healing.

[0097] The foregoing description of exemplary embodiments of the present invention has been presented for purposes of illustration and description only and is not intended to be exhaustive or to limit the invention to the precise form disclosed.Many modifications and variations are possible in light of the above teachings.

[0098] Several references are cited and discussed in the specification of the present invention, and these references may include patents, patent applications, and various publications. These references are cited and / or discussed only to clarify the description of the present invention, and no admission is made that any such reference is "prior art" to the invention described herein. The entire contents of all references cited and discussed in this specification are incorporated herein by reference to the same extent as if each reference were individually incorporated by reference. SEQUENCE LISTING <110> Precious Blood Purification Technology Co., Ltd. <120> Thrombomodulin domains for promoting osteoblast function and bone healing <130> 10035-005PCT <150> 62951490 <151> 2019-12-20 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 557 <212> PRT <213> Homo sapiens <400> 1 Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp 1 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln 20 25 30 Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp 65 70 75 80 Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110 Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160 Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly 165 170 175 Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro Val Gly 180 185 190 Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220 Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp 260 265 270 Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg 290 295 300 Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln 305 310 315 320 Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350 Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His 370 375 380 Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp 405 410 415 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430 Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys 435 440 445 Gly Pro Asp Ser Ala Leu Ala Arg His Ile Gly Thr Asp Cys Asp Ser 450 455 460 Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser 465 470 475 480 Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His 485 490 495 Ser Gly Leu Leu Ile Gly Ile Ser Ile Ala Ser Leu Cys Leu Val Val 500 505 510 Ala Leu Leu Ala Leu Leu Cys His Leu Arg Lys Lys Gln Gly Ala Ala 515 520 525 Arg Ala Lys Met Glu Tyr Lys Cys Ala Ala Pro Ser Lys Glu Val Val 530 535 540 Leu Gln His Val Arg Thr Glu Arg Thr Pro Gln Arg Leu 545 550 555 <210> 2 <211> 274 <212> PRT <213> Homo sapiens <400> 2 Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn 1 5 10 15 Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu 20 25 30 Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn 35 40 45 Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser 50 55 60 Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His 65 70 75 80 Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro 85 90 95 Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro 100 105 110 Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys 115 120 125 Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser 130 135 140 Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro 145 150 155 160 His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp Cys 165 170 175 Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu 180 185 190 Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly 195 200 205 Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile 210 215 220 Cys Gly Pro Asp Ser Ala Leu Ala Arg His Ile Gly Thr Asp Cys Asp 225 230 235 240 Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro 245 250 255 Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val 260 265 270 His Ser <210> 3 <211> 274 <212> PRT <213> Homo sapiens <400> 3 Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn 1 5 10 15 Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu 20 25 30 Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn 35 40 45 Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser 50 55 60 Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His 65 70 75 80 Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro 85 90 95 Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro 100 105 110 Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys 115 120 125 Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Ala Gln Thr Ser 130 135 140 Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro 145 150 155 160 His Arg Cys Gln Met Phe Cys Ala Gln Thr Ala Cys Pro Ala Asp Cys 165 170 175 Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu 180 185 190 Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly 195 200 205 Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile 210 215 220 Cys Gly Pro Asp Ser Ala Leu Ala Arg His Ile Gly Thr Asp Cys Asp 225 230 235 240 Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro 245 250 255 Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val 260 265 270 His Ser <210> 4 <211> 497 <212> PRT <213> Homo sapiens <400> 4 Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp 1 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln 20 25 30 Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp 65 70 75 80 Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110 Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160 Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly 165 170 175 Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro Val Gly 180 185 190 Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220 Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp 260 265 270 Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg 290 295 300 Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln 305 310 315 320 Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350 Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His 370 375 380 Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp 405 410 415 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430 Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys 435 440 445 Gly Pro Asp Ser Ala Leu Ala Arg His Ile Gly Thr Asp Cys Asp Ser 450 455 460 Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser 465 470 475 480 Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His 485 490 495 Ser <210> 5 <211> 497 <212> PRT <213> Homo sapiens <400> 5 Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp 1 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln 20 25 30 Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp 65 70 75 80 Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110 Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160 Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly 165 170 175 Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro Val Gly 180 185 190 Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220 Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp 260 265 270 Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg 290 295 300 Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln 305 310 315 320 Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350 Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Ala Gln Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His 370 375 380 Arg Cys Gln Met Phe Cys Ala Gln Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp 405 410 415 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430 Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys 435 440 445 Gly Pro Asp Ser Ala Leu Ala Arg His Ile Gly Thr Asp Cys Asp Ser 450 455 460 Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser 465 470 475 480 Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His 485 490 495 Ser

Claims

1. Use of a recombinant polypeptide or a recombinant variant thereof in the preparation of a medicament for stimulating bone regeneration, bone enhancement, bone healing and treating bone loss in a subject in need thereof, The polypeptide or its recombinant variant does not contain or lacks the amino acid sequence from human thrombomodulin domains 4 and 5, and has the biological activity of promoting osteoblast proliferation and migration, enhancing osteoblast mineralization, bone regeneration and / or osteoblast function, in, The recombinant polypeptide or a recombinant variant thereof is an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:

5.

2. The use as claimed in claim 1, further comprising use in the preparation of a medicament for treating bone healing after bone defect.

3. The use according to claim 1, in combination with the use of a bone graft material for the manufacture of a medicament for stimulating new bone formation in a subject in need thereof.

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