TGF-β1 and nitric oxide delivery platform for bone wounds

Abstract

Disclosed are compositions comprising a hydrogel, a nitric oxide donor and transforming growth factor (TGF) β1. The composition useful for treating bone injuries, such as fractures, and / or bone defects. The composition may be capable of potentiating bone regeneration in a compromised wound and is bactericidal. Also disclosed is a method comprising injecting the biomaterial into a compromised wound in a subject. The method may also decrease an infection in the wound and / or enhance wound healing.

Description

SAS / amcl 8123-113405-02 02 / 06 / 26 07032TGF-β1 AND NITRIC OXIDE DELIVERY PLATFORM FOR BONE WOUNDSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This claims the benefit of U. S. Provisional Application No. 63 / 755,994, filed February 7, 2025, which is incorporated herein by reference in its entirety.FIELD

[0002] This disclosure is related to materials, devices, and methods for regenerating bone and other tissues in compromised wounds.ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under W81XWH-21-2-0008 awarded by the Defense Health Agency, Medical Research and Development Branch. The government has certain rights in the invention.BACKGROUND

[0004] Some people, such as military personnel, are substantially burdened with traumatic bone injury to the extremities, but no ideal therapy is available to regenerate large bone volumes in compromised wounds. Some people suffer chronic non-union of bone injuries such as fracture. These wounds are sub-optimal for regeneration because the impaired vascularity and immune responses provoke hypoxia and inflammation which impair bone neogenesis and drive aberrant fibrosis. Accordingly, there is a need for improved materials, devices, and methods for regenerating bone and other tissues in compromised wounds.SUMMARY

[0005] Disclosed herein are aspects of a composition comprising a hydrogel, a transforming growth factor (TGF)-β1, and a nitric oxide (NO*) donor. The hydrogel may have a density of from 0.2% to 3% w / v and / or is enzymatically degradable. In some aspects, the nitric oxide donor has a half-life of from 6 hours to 24 hours and / or is present in an amount sufficient to provide a concentration of NO* in the hydrogel of from 3 mM to 160 mM. And in some aspects, the TGF-β1 is present in an amount of from 1 ng to less than 500 pg per mL of the hydrogel.SAS / amcl 8123-113405-02 02 / 06 / 26 07032

[0006] Also disclosed herein are aspects of a method for using the disclosed composition. In some aspects, the method comprises administering the composition to a subject to repair a bone defect, such as a fracture.

[0007] Further disclosed are aspects of a use of the composition in the manufacture of a medicament for repairing bone defects.

[0008] The foregoing and other objects, features, and advantages of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1. Appearance of NO* activated donors in solution. The nitrosylated glutathione (GSNO), nitrosylated 3-mercaptopropyl trimethoxysilane sol-gel microparticles (mSNO-MPs), and nitrosylated D-penicillamine (S-nitroso-penicillamine) within sol-gel microparticles (pSNO-MPs) are ambient temperature stable and nitrosylated immediately before use with a mixture of nitrite in HC1. Their nitrosylated forms of S-nitrosothiols are temperature and light labile.

[0010] FIG.2. Collagen type I hydrogels (RTC) loaded with mSNO-MPs and pSNO-MPs. 10 mg / ml mSNO-MPs and 24.5 mg / ml pSNO-MPs were each encapsulated in 0.265% w / v RTC at an equivalent concentration 42.5 mM NO*.

[0011] FIG.3. Treatment syringes containing 300 ul of pSNO-MP in RTC (left) and mSNO-MP in RTC (right).

[0012] FIG.4. Live / Dead staining of MSCs exposed to rat tail versus human derived collagen. Human bone marrow -derived mesenchymal stem cells (MSCs) were seeded on tissue culture plastic in basal medium and cultured at 37C, 5% pCO2 and 20% pO2. After one day, the cells were exposed to either the hydrogel made of these collagens (top row) or the neutralized collagen supplemented in solution (supplemented into medium before gelation). The human collagen led to rounder cell shape and less uniform cell distribution.

[0013] FIG.5. Comparison of mSNO- versus pSNO-MPs impact on mitochondrial respiration at equimolar NO* doses. MSCs were seeded on tissue culture dishes and exposed to equimolar NO* doses (0, 2.25, 6.63, 13.29, 21.2, 26.58 pmol) from mSNO-MP (0, 1.06, 3.12, 6.25, 10, 12.5 mg / ml) and pSNO-MP (2.599, 7.65, 15.32, 24.52, 30.65 mg / ml) within 221 µl RTC (0.265% w / v) gelled in a transwell insert to assess their effects on mitochondrial respiration. Culture conditions were basal medium (a-MEM, FBS, andSAS / amcl 8123-113405-02 02 / 06 / 26 07032antibiotics) at 37 °C under 5% and 20% pO₂. Mitochondrial respiration was assayed with MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) using the CELLTITER 96® AQueous One Solution Cell Proliferation Assay reagent. After 24 hours of treatment, both mSNO- and pSNO-MP have minimal impact on metabolic activity at 20% oxygenation. However, at 5% pO2, increasing doses of MSNO result in reduction in metabolic readings. By day 7, neither pSNO- nor mSNO-MPs significantly affect cell respiration at 20% oxygenation, regardless of concentration. In contrast, mSNO-MP induces a noticeable reduction in metabolic activity at 5% pO2 (circles and arrows).

[0014] FIG.6. Dose effect of mSNO-MP and pSNO-MP on MSC proliferation over time. MSCs were seeded at 104cells / cm2in 24-well culture dishes, cultured in basal medium (α-MEM, FBS, and antibiotics) at 37 °C and 20% pO2, and exposed to NO* at the indicated doses for 24 hours. After assaying mitochondrial respiration with the MTS assay, cell proliferation was assayed by quantifying total DNA per well with the Quant-iT™ PicoGreen™ dsDNA Assay Kit. No changes in cell proliferation were detected across all donors and doses. This finding suggested that the alterations observed in MTS readings are a genuine reflection of changes in mitochondrial respiration rather than an indication of changes in cell health.

[0015] FIG.7. Dose effect of mSNO-MP on MSC expression of “sternness” genes at 7-days treatment in 2-dimensional culture. MSCs from two donors were seeded at 104cells / cm2in 24-well culture dishes and exposed to NO* at the indicated doses for 7 days. qRT-PCR was performed using TRIzol reagent lysis and solubilization of RNA, RNA extraction with the RNeasy Mini Kit, reverse transcription with the Maxima H Minus First Strand cDNA Synthesis Kit, and qPCR with the PowerUp™ SYBR™ Green Master Mix and primers designed and validated in-house. NO* decreased expression of the sternness markers SOX2 and OCT4 in a dose dependent manner evident at 7-days culture. Oxygen tension strongly biased the response, with maker expression lower at 5% pO2 than 20% pO2. These data are consistent with potentiation of differentiation in MSCs by NO*.

[0016] FIG.8. Experimental Design of Bone Regeneration and Leukocyte Profiling Assays. Animals were treated one -day after surgical creation of the 5.0 mm defect (mid-diaphyseal osteotomy of the femur held with rigid but radiolucent plate fixation) with 350 pL of RTC+mSNO-MP, RTC+TGF-β1, and RTC+mSNO-MP+TGF-pl. Controls included RTC+mS-MP (no NO* loading of microparticle), RTC hydrogel alone, and untreatedSAS / amcl 8123-113405-02 02 / 06 / 26 07032(empty) defects. Two different time-points were used for bone regeneration versus leukocyte profiling. The leukocyte milieu was profiled at 5-days post-treatment. Immediately after euthanasia, the defect tissue was biopsied and preserved in RNA*later*. After one to three days, these were mechanically disrupted to liberate the cells. The antibody panel included anti-CD45, CD3, CD4, CD8, CD161, CD11b / c, CD86, CD163, and His48. X-rays were captured every two weeks to monitor the progression of healing. Bone regeneration was assayed at 16-weeks post-treatment. Immediately after euthanasia, the limbs were excised and fixed in 10% neutral buffered formalin for 48 hours. After rinsing in phosphate buffered saline (PBS), limbs were imaged on a SCANCO pCT 50 for quantification of bone volume, quality, and defect bridging. Subsequently, the orthopedic hardware was removed and tissues decalcified, paraffin processed for serial histology, and histochemically stained and analyzed for regenerate tissue types, e.g. bone, cartilage, and fibrous tissue.

[0017] FIGs.9A-9D. Impact of NO* and TGF-β1 on the leukocyte infiltrate into the defect site at 5-days post-treatment.. The NO* and TGF-β1 components of the RTC modulated the host immune response within the bone defect. Dissimilar letters above the groups indicate significantly different at a p-value < 0.05. Treatment with TGF-β1 significantly suppressed the Ml macrophage phenotype in the defect, indicating a shift toward a regenerative rather than inflammatory state. Additionally, results suggest mSNO-MP and TGF-β1 may decrease CD4 and CD8 (cytotoxic) positive T cells and increase granulocytes pointing to a nitric oxide mechanism modulating the leukocyte milieu.

[0018] FIG. 10. Bone healing at 16-weeks post-treatment. (Top) Movat’s pentachrome staining showing collagen and fibrous tissue, bone as dense (with immature bone also shown), elastic fibers, muscle and fibrin, mucin and cartilage, and nuclei. (Bottom). pCT image of defect sliced transversely along the length of the defect. TGF-β1+MSNO+RTC markedly promoted regeneration of critically sized defects after 16-weeks post-treatment. Some evidence of cartilage formation was detected within defects (greenish stain), but most bone appeared intramembranous in origin with immature bone (red) showing woven matrix undergoing remodeling.

[0019] FIG. 11. pCT quantification of defect bridging at 16-weeks post-treatment. Limbs were imaged on a SCANCO pCT 50 at 6 pm voxel size. The distance along the longitudinal axis of contiguous bone spanning the defect from the proximal and distal osteotomy margins was calculated. The fraction of defect bridging was calculated as the ratio of this sum to the total defect length. The error bars represent standard deviation. The numberSAS / amcl 8123-113405-02 02 / 06 / 26 07032above bars indicate the number of biological replicates. The letters above the treatments indicate significantly different bone bridging at a p-value < 0.01 between dissimilar letters. Only the RTC+mSNO+TGF-β1 and RTC+TGF-β1 treatments showed a significant difference in bridging of the defect compared to untreated defects. The RTC+mSNO-MP+TGF-β1 treatment appeared to showed 30% more bridging than the RTC+TGF-β1 (p = 0.06), indicating a synergistic interaction of NO* and TGF-β1 compared to RTC with either alone.

[0020] FIG. 12. Hematoxylin and eosin stain of representative defects 16- weeks post treatment. Bone shows as dense pink, muscle darker pink / red, cartilage purple, and nuclei purple. The yellow circles indicate bone formation bringing the defect. More complete bridging is evident with the RTC+mSNO-MP+TGF-β1 than in untreated (empty defects). Untreated defects often showed muscle tissue within the defect proper. Screw holes are evident in both images as large circular voids. Bone deposition is evident within the screw holes as a darker pink stain (more cellular).

[0021] FIG. 13. Metabolic activity of human bone marrow-derived stem cells (MSCs) treated with lab-made versus commercial GSNO and inactive formulations in vitro.MSCs were cultured in vitro on tissue culture plastic dishes in basal medium (a-MEM with 10% fetal bovine serum (FBS) and antibiotics) and subject to either lab-synthesized GSNO, UV-inactivated lab synthesized GSNO, reduced glutathione prepared in the lab (lab-synthesized GS), or commercially available GSNO. Untreated cell (Live) and methanol treated cells (Dead) served as controls. The glutathione and GSNO donors were placed in a transwell above the cells. An MTS (i.e.. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was conducted 24 hours post-treatment to assess mitochondrial respiration under 20% pO₂ conditions with the CELLTITER 96® AQueous One Solution Cell Proliferation Assay reagent. Increasing concentrations of GSNO reduced the metabolic activity of human bone marrow-derived stem cells due to increased NO* release and not to increased impurity content as evidence by similar outcomes between UV-inactivated and lab-synthesized GS. Commercially available GSNO showed a greater reduction of metabolic activity than lab-synthesized, indicating impurity in the commercial formulation. The 14.3 mg / ml dose was fully cytotoxic.

[0022] FIG. 14. Dose effect of mSNO-MP on osteogenesis by MSCs in 3-dimensional culture. MSCs were encapsulated at 30×106cells / cm2in 4% w / v gelatin+poly(ethylene glycol) hydrogels cast in cylindrical molds (8 mm diameter and 1.8 mm height). AnSAS / amcl 8123-113405-02 02 / 06 / 26 07032equivalent volume of 2% w / v hydrogel was cast above containing the indicated doses of mSNO-MP and TGF-β1. Cells were cultured in osteogenic medium (a-MEM, FBS, ascorbate, P-glycerophosphate, Vitamin-D3, dexamethasone, and antibiotics) at 37 °C under 5% pO₂, which more closely simulates oxygen tension in vivo than 20% pO₂. Gene expression of osteocalcin was analyzed after 7-days of culture. NO* alone (1.06 mg / ml dose) and TGF-β1 alone induced close to a doubling of osteocalcin expression. However, the combination of NO* and TGF-β1 led to a quadrupling of osteocalcin expression at the high mSNO-MP dose, indicating an effect of NO* and TGFB1 in promoting osteogenic differentiation of MSCs. This data shows synergy of NO* and TGFB 1 and evidences an effect via promoting MSC osteogenic differentiation.DETAILED DESCRIPTION

[0023] The disclosed technology describes materials, devices, and methods for treating fracture and segmental defect of bone, among other applications.

[0024] Treatment of open bone wounds and bone non-unions is a significant challenge in civilian and military medicine. In civilian medicine, 31 out of 100,000 persons per year suffer open bone wounds (over 100,000 open fractures per year in the USA). In military medicine, roughly 2 / 3 of modem battlefield injuries are open extremity wounds, of which 1 / 3 are open bone wounds, Advanced orthopedic care is often unavailable in first care facilities. Military personnel are substantially burdened with traumatic bone injury to the extremities, but no ideal therapy is available to regenerate large bone volumes in compromised sites. Open fractures in HPSAs have greater infection and chronic non-union rates (42% in the USA and 6-12x worldwide. About a quarter of combat-related extremity wounds develop infection, of which a fifth of those with open fracture as the most severe injury developing infection. Segmental defects, on average, spend 12 days in hospital treatment yet suffer a 33%-58% 2-year post-op infection rate at a cost of over $150,000 per patient. Infection may lead to bone loss (e.g., osteomyelitis) and chronic non-union of fractures. Surgical may require bone resection. Preemptive limb shortening and amputation are used when bone fillers and autografts are contraindicated, e.g. no viable graft source, poor vascularity, excessive acute inflammation, mal-union, and osteomyelitis. The injury sites are sub-optimal for regeneration because the impaired vascularity and immune responses provoke hypoxia and inflammation which impair bone neogenesis and drive aberrant fibrosis. This leads to delayed reconstruction and healing, multiple surgeries, and non-unions. As an example, it is estimatedSAS / amcl 8123-113405-02 02 / 06 / 26 07032that more than 1500 total American soldiers had traumatic limb amputations in Afghanistan and Iraq wars. It is estimated that the lifetime costs of care for amputees is greater than $600 billion. Even in the civilian realm, traumatic injury is the 2ndleading cause for extremity amputations, of which there are an estimated 700,000 per year, leading to yearly hospital charges of more than $8 billion. Though TGFP-1 is highly sequestered in bone, TGFPs have not previously shown clinically applicable improvement in bone healing of segmental bone defects in large animal models, likely due to potency, stability, delivery, and dosing issues. Though NO* impacts fracture healing and is produced by the immune system to clear infections, it has not been translated to clinical use, likely due to similar issues. The potential interactions of TGFPs and NO* in bone healing are not understood.

[0025] The following describes certain particular aspects of the herein disclosed technology, which aspects are designed to regenerate volumes of bone in compromised wounds. The following description does not limit the full scope of this disclosure, but rather provides a detailed description of certain representative examples of the disclosed technology.Terms

[0026] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin ’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context indicates otherwise. For example, the term “a joint” includes single or plural joints and can be considered equivalent to the phrase “at least one joint.” As used herein, the term “comprises” means “includes.”

[0027] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims, are to be understood as being modified by the term “about.” Unless context indicated otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.

[0028] It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated.

[0029] Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition,SAS / amcl 8123-113405-02 02 / 06 / 26 07032the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0030] Administration: The introduction of a composition (such as a hydrogel) into a subject by a chosen route. The route can be local or systermc. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. If the chosen route is local, the composition can be administered by introducing the composition directly into a tissue of the subject, such as to a gap or void in a bone.

[0031] Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.

[0032] Arthroplasty: A surgical procedure to replace some (partial arthroplasty) or all of a joint (total arthroplasty). The worn or damaged parts of a joint are replaced with an artificial (prosthetic) joint made of, for example, metal, plastic or ceramic. A first time replacement of joint is referred to as a primary arthroplasty, whereas second and subsequent replacements are referred to as secondary arthroplasty or a revision.

[0033] Aseptic loosening: The failure of fixation of a prosthetic implant in the absence of infection due to biologic causes. Aseptic loosening occurs when particulate from the implant causes immune cell activation and chronic inflammation in the tissue around the implant which induces periprosthetic osteolysis (PPOL) and ultimately failure of the orthopedic implant.

[0034] Biocompatible: Any material, that, when implanted in a mammalian subject, does not provoke an adverse response in the subject. A biocompatible material, when introduced into an individual, is able to perform its intended function, and is not toxic or injurious to that individual, nor does it induce immunological rejection of the material in the subject.

[0035] Bone defect: Includes any disease, break, or disorder which affects bone strength, function, and / or integrity. Bone defects can result from injury, or can be brought about during the course of surgery, infection, malignancy, or developmental malformation.Examples of bone defects include, but are not limited to, fractures and gaps. A bone defect can be a critical defect (which cannot heal on its own), a non-union fracture, a void or gap in bone, or a dental or facial defect (such as cleft palate or facial, skull, or dental injuries or malformations). Other examples of bone defects include damage to bones resulting from disorders of bone fragility, such as osteoporosis, osteopenia, and malignancies and / or cancersSAS / amcl 8123-113405-02 02 / 06 / 26 07032of the bone such as a sarcoma, such as osteosarcoma. The segmental defects are a gap or void in the bone.

[0036] Bone Healing and Fracture Healing: Bone heals (fuses) in a unique way compared with other connective tissues. Rather than develop scar tissue, it has the ability to regenerate itself completely through a process of mineralized tissue deposition and remodeling. The majority of fractures heal by secondary fracture healing and that involves a combination of intramembranous and endochondral ossification. Without being bound by theory, it is generally believed that the fracture healing sequence involves five discrete stages of healing. This includes an initial stage in which a hematoma is formed and inflammation occurs; a subsequent stage in which cartilage begins to form and angiogenesis proceeds, and then three successive stages of cartilage calcification, cartilage resorption and bone deposition, and ultimately a more chronic stage of bone remodeling. Generally, committed osteoprogenitor cells and uncommitted, undifferentiated mesenchymal stem cells contribute to the process of fracture healing. Bone that forms by intramembranous ossification is found early and further from the site of the fracture, results in the formation of a hard callus, and forms bone directly without first forming cartilage. Generally, two weeks after fracture, cell proliferation declines and hypertrophic chondrocytes become the dominant cell type in the chondroid callus. The resulting endochondral bone is formed adjacent to the fracture site.

[0037] Control: A reference standard. In some aspects, the control is a negative control sample obtained from a healthy patient (such as one without a fracture of an infection), or a subject treated with a carrier, or an untreated patient. In other aspects, the control is a positive control sample obtained from a patient that has been treated with an active agent, such as an effective amount of a composition disclosed herein. In still other aspects, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with known prognosis or outcome, or group of samples that represent baseline or normal values). In other aspects, the control is treatment of cells or an animal with a composition lacking one or more components or with no composition.

[0038] A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least 5%, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at leastSAS / amcl 8123-113405-02 02 / 06 / 26 07032100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, or greater than 500%.

[0039] Effective Amount: A quantity of a specific substance, such as a disclosed composition, sufficient to achieve a desired effect in a subject being treated. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in the bone) that has been shown to achieve a desired in vitro effect.

[0040] Hydrogel density: As used herein, hydrogel density refers to the mass of hydrogel polymer per unit volume when the hydrogel is hydrated and is provided as a percentage.

[0041] Joint: An articulation (or articular surface) that is the connection made between bones, ossicles, or other hard structures in the body which link an animal's skeletal system into a functional unit. Joints are classified anatomically into the following groups: finger / hand joints, elbow joints, wrist joints, axillary joints, sternoclavicular joints, vertebral articulations, temporomandibular joints, sacroiliac joints, hip joints, knee joints, and articulations of foot. Joints are structurally classified as fibrous joints, cartilaginous joints, synovial joints, and facet joints.

[0042] Mammal: This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.

[0043] Modulate: To alter in a statistically significant manner. Modulation can be an increase or a decrease. One of skill in the art can identify an appropriate assay to determine a statistically significant increase or decrease in a parameter. These include, but are not limited to, a student’s t-test or a paired ratio t test. Exemplary methods are provided in the Examples section.

[0044] Nitric oxide (NO*): is a pleiotropic radical gaseous molecule endogenously produced in the body that regulates several body functions including cardiovascular, immune, and skeletal growth. Following bone injury, NO* is produced by the immune system to clear infections via nitrosative and oxidative damage to microorganisms. Available NO* delivery systems are unsuitable for deep wound treatment and bone repair because they either use nitrite salts and acidifying agents with poor biocompatibility or use a donor delivery system with inadequate NO* release profiles.

[0045] Osteomyelitis: An infection of the bone. Symptoms may include pain in a specific bone with overlying redness, fever, and weakness. The cause is usually a bacterial infection but can be a fungal infection. Osteomyelitis is accompanied by increased formation and activity of bone-resorbing osteoclasts and bone loss.SAS / amcl 8123-113405-02 02 / 06 / 26 07032

[0046] Osteoporosis: A systermc skeletal disorder characterized by low bone mass, micro-architectural deterioration of bone tissue leading to bone fragility, and consequent increase in fracture risk. It is the most common reason for a broken bone among the elderly. The main consequence of osteoporosis is the increased risk of bone fractures. Osteoporotic fractures occur in situations where healthy people would not normally break a bone; they are therefore regarded as fragility fractures. Typical fragility fractures occur in the vertebral column, rib, hip and wrist. The World Health Organization (WHO defines osteoporosis as bone density 2.5 standard deviations below the bone density of a reference standard (i.e., generally a healthy young adult of about 30 years old). “Osteopenia” refers to a decrease in bone mineral density that is not as severe as osteoporosis, whether or not osteoporosis is present, as detected by a suitable diagnostic procedure, such as a radiographic technique. The WHO defines osteopenia as a bone density between 1 standard deviation and 2.5 standard deviations below the bone density of a reference standard as above.

[0047] Peri-implantitis: A destructive bacteria driven inflammatory process affecting the soft and hard tissues surrounding dental implants. The soft tissues become inflamed whereas the alveolar bone (hard tissue), which surrounds the implant for the purposes of retention, is lost over time. A healthy peri-implant is characterized by absence of erythema, bleeding on probing, swelling and suppuration.

[0048] Periprosthetic infection: A destructive bacteria driven inflammatory processes affecting hard and soft tissues surrounding orthopedic implants. The resulting bone loss causes pain, fever, and implant loosening.

[0049] Periprosthetic Osteolysis (PPOL): The most common complication after primary arthroplasty whereby aseptic deterioration of bone at the site of arthroplasty (e.g., in and around the interface of the implant and the bone) occurs, which can lead to loosening and ultimately failure of the arthroplasty. Components of the implant, usually formed of plastic or metal, used in arthroplasty generate debris, or wear particles, caused by abrasion. These wear particles trigger recruitment of cells, including macrophages, fibroblasts, lymphocytes, and osteoclasts, leading to a localized inflammatory response, which causes the release of various cytokines that affect osteoclast differentiation and activity (e.g., TNF, RANKL, IL-6, IL-1, and IL-11). The prolonged duration of inflammatory activity promotes progressive osteolysis. Periprosthetic osteolysis is progressive and may be complicated by joint failure or periprosthetic fracture with the subsequent need for surgical revision. Thus, diagnostic imaging can be used for evaluating the extent and distribution of osteolysis. For example, radiographs can be used to characterize and monitor periprosthetic osteolysis. Geographic orSAS / amcl 8123-113405-02 02 / 06 / 26 07032linear zones of periprosthetic lucency greater than 2 mm that progress on serial examinations or develop after 2 years following arthroplasty are indicative of osteolysis. Computed tomograph (CT) can sometimes provide a more sensitive detection of images characteristic of osteolysis. For example, radiolucent lesions that communicate with the joint space and have well-defined sclerotic borders- are indicative of osteolysis. Magnetic resonance imaging (MRI) is also used to diagnose periprosthetic osteolysis. MRI provides higher sensitivity than CT in detection of small (less than 3 cm) periprosthetic lesions. MRI can also be used to identify extraosseous soft tissue deposits, pathology affecting neurovascular bundles, or precursors to bone resorption. Unlike infections, osteolysis presents as well-defined lesions with low signal intensity similar to skeletal muscle by MRI. (Desai, M. A. et al. Orthopedics 2008, 31(6).)

[0050] Prosthetic: An artificial (man-made) body part. Prosthetics include, but are not limited to, implants, artificial joints, pins, screws, plates, dental implants, posts, rods, plates, springs, artificial discs, cages, or screws.

[0051] Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals, such as non-human primates, rats, mice, dogs, cats, horses, cows and pigs. In an example, a subject is a human.

[0052] Transforming Growth Factor (TGF)-β1: A polypeptide member of the transforming growth factor beta superfamily of cytokines. TGF-β1 is a secreted protein that performs many cellular functions, including the control of cell growth, cell proliferation, cell differentiation, and apoptosis. In humans, TGF-β1 is encoded by the TGFB1 gene. It is a protein that is produced as a large protein precursor (containing 390 amino acids) that is proteolytically processed to produce a mature peptide of 112 amino acids. A sequence for human TGF-β1 can be found in GENBANK® Accession No. NP_000651.3, November 21, 2025, and a sequence for murine TGF-β1 can be found in GENBANK® Accession No. NP_035707.1, January 23, 2026, both incorporated herein by reference.

[0053] Treating, Treatment, and Therapy: Any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, or improving a subject’s physical or mental well-being. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations.SAS / amcl 8123-113405-02 02 / 06 / 26 07032Composition

[0054] Disclosed here are aspects of a composition comprising a hydrogel, a transforming growth factor (TGF)-β1, and a nitric oxide (NO*) donor.

[0055] Hydrogel

[0056] The hydrogel may be any hydrogel suitable for use in the composition. In some aspects, the hydrogel is an enzymatically degradable hydrogel. In some aspects the hydrogel is selected to crosslink without irradiation by light.

[0057] In some aspects, the hydrogel is or comprises a polysaccharide, a decellularized extracellular matrix (ECM), a protein, a peptide, or a combination thereof.

[0058] In some aspects, the hydrogel is a composite hydrogel comprising one or more of a polysaccharide, decellularized extracellular matrix (ECM), protein, peptide, polyethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNIPA), poloxamer, or a naturally derived polysaccharide having a branched or a linear structure.

[0059] In some aspects, the hydrogel is or comprises a polysaccharide, and the polysaccharide may be chitosan, hyaluronan, heparin, heparin sulfated proteoglycan, or a combination thereof,

[0060] In some aspects, the hydrogel is or comprises a decellularized extracellular matrix (ECM), and in certain aspects, the decellularized extracellular matrix may be matrigel.

[0061] In some aspects, the hydrogel is or comprises a protein, and the protein may be a collagen-derived protein, such as collagen or gelatin. Or the protein may be an alternative protein hydrogel, such as fibrin.

[0062] In some aspects, the hydrogel is or comprises a peptide, and in some aspects, the peptide-containing hydrogel is a supramolecular hydrogel.

[0063] In some aspects, the hydrogel is a polysaccharide, a decellularized extracellular matrix (ECM), a protein, or a peptide. In other aspects, the hydrogel is a combination of two or more, such as two, three or four, of a polysaccharide, a decellularized extracellular matrix (ECM), a protein, or a peptide. In certain aspects, the hydrogel is a combination of two of a polysaccharide, a decellularized extracellular matrix (ECM), a protein, or a peptide, for example, gelatin and heparin.

[0064] In some aspects, the hydrogel is a composite hydrogel comprising a polysaccharide, a decellularized extracellular matrix (ECM), a protein, a peptide, or a combination thereof, and one or more, such as 1, 2, 3, or 4, of a synthetic hydrophilic polymer (such as poly(ethylene glycol) (PEG)), a synthetic self-assembling hydrophilic polymer (for example, poly(N-SAS / amcl 8123-113405-02 02 / 06 / 26 07032isopropylacrylamide) (PNIPA)), a poloxamer (for example poloxamer 407, or Pluronic F86 (poloxamer 188)), a naturally derived polysaccharide having a branched structure (for example, dextran), or a naturally derived polysaccharide having a linear structure (such as alginate, pectin, or gellan gum). In some aspects, the polyethylene glycol is from 4 kDa (PEG 4,000) to 20 kDa (PEG 20,000). In some aspects, the PEG is linear, and in certain aspects, a linear PEG is from 4 kDa (PEG 4,000) to 10 kDa (PEG 10,000). In some other aspects, the PEG is non-linear, having 3 or 4 or more arms, and in some such aspects, the PEG is from 10 kDa to 20 kDa (PEG 10,000 to PEG 20,000). Example composite hydrogels suitable for use in the disclosed technology include, but are not limited to, gelatin+heparin, gelatin+PEG, gelatin+heparin+PEG, PNIPA+gelatin, PNIPA+hyaluronan, PNIPA+chitosan.

[0065] In some aspects, the hydrogel has a mesh size of from 40 nm to 10 pm. In some aspects, the hydrogel has a mesh size of 100 nm to 10 pm, from 500 nm to 10 pm, or from 1 pm to 10 pm. In other aspects, the hydrogel has a mesh size of from 40 nm to 5 pm, such as from 40 nm to 2.5 pm, from 40 nm to 1 pm, or from 40 nm to 500 nm.

[0066] In some aspects, the hydrogel has a density of from 0.2% to 3% w / v. In some aspects, the hydrogel has a density of from 0.3% to 2% w / v, but in other aspects, the hydrogel has a density of from 1% to 3% w / v, such as from 1.5% to 3% w / v or from 2% to 3% w / v.

[0067] In some aspects, the hydrogel is selected to be at least 90% degraded 8 weeks after administration, such as at least 95% degraded, at least 97% degraded, at least 98% degraded, at least 99% degraded, or substantially completely degraded such that no hydrogel remains detectable at the administration site after 8 weeks. In some aspects, the hydrogel is selected to be at least 90% degraded 2 weeks after administration, such as at least 95% degraded, at least 97% degraded, at least 98% degraded, at least 99% degraded, or substantially completely degraded such that no hydrogel remains detectable at the administration site after 2 weeks.

[0068] In some aspects, the hydrogel is selected to be at least 90% degraded 3 weeks after administration, such as at least 95% degraded, at least 97% degraded, at least 98% degraded, at least 99% degraded, or substantially completely degraded such that no hydrogel remains detectable at the administration site after 3 weeks. In some aspects, the hydrogel is selected to be at least 90% degraded 4 weeks after administration, such as at least 95% degraded, at least 97% degraded, at least 98% degraded, at least 99% degraded, or substantially completely degraded such that no hydrogel remains detectable at the administration site after 4 weeks. In some aspects, the hydrogel is selected to be at least 90% degraded 5 weeks after administration, such as at least 95% degraded, at least 97% degraded, at least 98% degraded,SAS / amcl 8123-113405-02 02 / 06 / 26 07032at least 99% degraded, or substantially completely degraded such that no hydrogel remains detectable at the administration site after 5 weeks. In some aspects, the hydrogel is selected to be at least 90% degraded 6 weeks after administration, such as at least 95% degraded, at least 97% degraded, at least 98% degraded, at least 99% degraded, or substantially completely degraded such that no hydrogel remains detectable at the administration site after 6 weeks. In some aspects, the hydrogel is selected to be at least 90% degraded 7 weeks after administration, such as at least 95% degraded, at least 97% degraded, at least 98% degraded, at least 99% degraded, or substantially completely degraded such that no hydrogel remains detectable at the administration site after 7 weeks.

[0069] Nitric oxide donor

[0070] The nitric oxide donor can be any nitric oxide donor that is suitable for use in the disclosed technology. In some aspects, the nitric oxide donor has a half-life of from 6 hours to 24 hours as measured in PBS at 37 °C, such as from 12 to 18 hours.

[0071] In some aspects, the nitric oxide donor is an S-nitrosothiol, N-diazeniumdiolate (NONOate), furoxan. molecular hybrid nitric oxide donor, or a combination thereof.

[0072] In some aspects, the nitric oxide donor is a combination of an S-nitrosothiol, N- diazeniumdiolate (NONOate), furoxan, or molecular hybrid nitric oxide donor, or a combination thereof, and nitrosoglutathione (GSNO, glutathione (i.e., an un-nitrosylated glutathione) (GH), SNAC (s-nitroso-n-acetylcysteine) (SNAC), spermine NONOate, (Z)-[3-aminopropyl(propyl)amino]-hydroxyimino-oxidoazanium (PAPA NONOate), (Z)-[bis(3-aminopropyl)amino]-hydroxyimino-oxidoazanium (DPTA NONOate),O’ 4-(phenylsulfonyl)-3- { [(2-dimethylamino)ethyl]thio } furoxan (LL4254), or 4-hydroxymethyl-furoxan-3-carboxamide (CAS-1609).

[0073] In some aspects, the S-nitrosothiol is S-nitroso-N-acetylpenicillamine (SNAP), or nitrosylated 3 -mercaptopropyl trimethoxysilane sol-gel microparticles (mSNO-MPs).

[0074] In some aspects, the N-diazeniumdiolate (NONOate) is (Z)-l-[N-(2-aminoethyl)-N- (2-amnionioethyl)amino]diazen-l-ium-l,2-diolate (DETA NONOate), or N-(6-aminohexyl) aminopropyl-trimethoxysilane (AHAP3) sol-gel microparticle.SAS / amcl 8123-113405-02 02 / 06 / 26 07032Qo=s=oO— N*N,

[0075] In some aspects, the furoxan is(3-{[2- (dimethylamino)ethyl]oxy}-4-phenylfuroxan) (LL4212), or a phenylsulfonyl furoxan derivative.

[0076] In some aspects, the molecular hybrid nitric oxide donor is a Cephalosporin-linked diazeniumdiolate, or S-nitroso diclofenac.

[0077] In some aspects, the nitric oxide donor provides two molecules of NO* per donor molecule. But in other aspects, the nitric oxide donor provides one molecule of NO* per donor molecule.

[0078] In some aspects, the amount of the nitric oxide donor per gram of hydrogel is selected to provide from 0.014 mmoles NO* / gram hydrogel to 26.6 mmoles NO* / gram hydrogel.

[0079] In some aspects, the amount of the nitric oxide donor in the hydrogel is selected to provide a concentration of NO* in the hydrogel of from 1 millimolar (mM) to 175 mM, such as from 3.25 millimolar (mM) to 157 mM, from 32.5 millimolar (mM) to 157 mM, from 3.25 millimolar (mM) to 53.1 mM, or from 32.5 millimolar (mM) to 53.1 mM.

[0080] TGF-β1

[0081] In some aspects, the composition comprises TGF-β1 in an amount of from 1 ng per mL of the hydrogel to less than 500 pg per mL of the hydrogel, such as from 1 ng per mL to 200 pg per mL of the hydrogel, from 1 ng / mL to 1 pg / mL of the hydrogel, from 1 pg / mL to 200 pg / mL of the hydrogel, or from 50 pg / mL to 200 pg / mL of the hydrogel.

[0082] Additional components

[0083] In some aspects, the composition may further comprise one or more additional components. The additional components may include, but are not limited to, analgesics, anesthetics, antifungals, antibiotics, anti-inflammatories, antidotes, antihistamines, antimicrobials, antiseptics, anti-arthritics, antivirals, or chemotherapeutic agents, a colored or fluorescent imaging agent, corticoids (such as steroids), diagnostic aids, diuretics, enzymes,SAS / amcl 8123-113405-02 02 / 06 / 26 07032hormones, minerals, nutritional supplements, a radioisotope, sedatives, sulfonamides, stimulants, tranquilizers, vitamins, and / or growth factors.

[0084] In some aspects, the additional component is or comprises an antimicrobial (such as an antibiotic, antifungal, antiviral, or antiparasitic), anti-inflammatory, analgesic, anesthetic, or a combination thereof. Exemplary additional components suitable for use with the disclosed technology include, but are not limited to:

[0085] Antibiotics, such as, but not limited to Glycopeptides (e.g. vancomycin), lipopeptides (e.g., daptomycin), aminoglycosidic (e.g. gentamicin, tobramycin), rifamycin (e.g., Rifampin), penicillins (e.g. Nafcilin, Penicillin G, Cloxacillin, Amoxicillin), cephalosporins (e.g. Cefazolin, ceftriaxone), Nitroimidazoles (e.g. metronidazole), Quinolones (e.g., ciprofloxacin);

[0086] Metal and metal ion releasing compounds, such as gold nanoparticles (AuNPs), silver nanoparticles, and other metals including nanoselenium, coper, iron, and zinc;

[0087] Cationic polymers and cationic particles;

[0088] Imidazoles, including both antifungal and antibacterial, such as voriconazole, amphotericin B, fluconazole, clotrimazole;

[0089] For analgesic in bone injuries, non-steroidal anti-inflammatories (NS AID) such as diclofenac, ibuprofen, naproxen;

[0090] Anesthetics, such as lidocaine, bupivacaine, ropivacaine; and

[0091] Opioids, such as morphine, oxycodone, fentanyl.

[0092] Applications

[0093] In some aspects, the disclosed therapeutic is an injectable hydrogel to treat bone wounds with / without bacterial contamination. The hydrogel serves as a scaffold for cellular ingrowth and releases transforming growth factor beta-1 (TGF-f> I ) and nitric oxide (NO*) to exert antimicrobial and regenerative effects. In some aspects, the disclosed formulation comprises at least one NO* donor with half-life of about a day at 37 °C within a self-gelling hydrogel (chemical and / or physical gelation) that crosslinks within approximately 5 to 10 minutes upon mixing components. Unlike antibiotics, bacteria have no known resistance to NO. The inventors have discovered that co-delivery of TGF-β1 and NO synergistically enhance bone healing beyond the additive effects of either molecule alone. Canonical signaling by TGF-β1 may occur via the SMAD (mothers against decapentaplegic homolog) protein pathway. However, the inventors have found NO decreases the transcriptional activity of the SMAD4 complex (binding of the SMAD binding element, SBE) in response to TGF-SAS / amcl 8123-113405-02 02 / 06 / 26 07032β1 using a luciferase reporter construct. The inventors have determined that, unlike current anabolic biologics for bone formation (INFUSE), the disclosed formulation can employ much lower doses of the cytokine (TGF-β1) due to co-delivery of TGF-β1 with the NO donor. Most commercially available donors have short half-lives, from minutes to 6 hours. Equivalent doses of NO via donors with short half-lives is cytotoxic due to their rapid NO delivery. The inventors have determined that incorporation of the NO donors in the hydrogel prolongs the half-life by several hours with a negligible impact on total delivery, depending on total dose.

[0094] Clinical Need: A therapeutic formulation is needed to speed bone healing, particularly by patients with needing rapid return to service and patients with non-union risks. Further, a formulation that mitigates bacterial contamination in open and deep wounds is of great interest. NO* is a potent antimicrobial. Such a formulation would also benefit any patient, civilian and military alike, suffering major orthopedic injury.

[0095] The disclosed formulation can be used for management of traumatic extremity injuries involving bone (e.g. bone fractures, blast injuries) along the continuum of care. The end users are non-clinicians and clinicians in field and clinic care, e.g. orthopedist setting a fracture in a hospital, aid worker treating patient in medical tent through humanitarian relief agency, and military personnel in echelon I care (nonmedical soldier in battlefield) and in subsequent care facilities (medics and clinicians). The formulation can be reapplied along the continuum of care, e.g. reapplied after debridement of infected tissue. It can be used in conjunction with conventional antibiotic therapy, e.g. delivery of systermc and local Vancomycin in treatment of Methicillin-resistant Staphylococcus aureus (MRSA) infection. It can be used in conjunction with other wound temporization, hemorrhage control, and limb fixation devices, e.g. Integra skin grafts, the REBOA ballon catheter, and FastCast splint. It may be used as an adjuvant for allograft and implant procedures.

[0096] Efficacy: 1) The inventors have demonstrated efficacy in vivo using a 5 mm mid-diaphyseal segmental defect in rat femurs. The formulation comprised 10 mg / ml MSNO and 100 ug / ml TGF-β1 within an injectable collagen type I (Coll) hydrogel (0.265% w / v). The device led to more than double the bone formation compared to the controls (10 mg / ml MSNO in Coll alone, Coll hydrogel alone, and untreated defects). 2) The inventors have demonstrated antimicrobial efficacy and cytocompatibility in vitro. To investigate the impact half-life of the donor, the formulations comprised either pSNO-MP (prolonged half-life of 30 days), mSNO-MP (long half-life of 18 hours) or GSNO (short half-life) in the Coll hydrogel. A minimum 6 mg / ml dose of mSNO-MP was required for antimicrobial efficacy against Pseudomonas aeruginosa and MRSA in planktonic and biofilm cultures, while a greater doseSAS / amcl 8123-113405-02 02 / 06 / 26 07032of 12 mg / ml pSNO-MP due to its lower NO* concentration and a lower dose of GSNO due to its faster release and permeation into cells. A dose up to 53 mM mSNO-MP was cytocompatible with human bone marrow stem cells (MSCs), a progenitor pool in bone healing, while doses over 4.5 mM GSNO were cytotoxic. 3) The inventors have demonstrated that the mechanism of action of the hydrogel laden with TGF-β1 and NO* donor is not via an increase in canonical TGF-β1 signaling. To compare long half-life donors, the formulations comprised either NOC-18 or mSNO-MP in the Coll hydrogel. NO suppressed rather than increased canonical signaling by TGF-β1. The mechanism may occur on a cellular level or system level, e.g. changes in other intracellular signaling pathways or alterations in the cellular milieu and interactions within in vivo (the balance of cells involved in wound healing in the bone).

[0097] No devices employ NO to potentiate the biologic action of drugs. No interaction of TGF-β1 and NO* in intracellular signaling and synergistic effect on bone healing have been demonstrated. Potentiation of TGF-β1 signaling is particularly unique in that 1) these molecules are not expected to interact and 2) the interaction decreases canonical TGF-β1 signaling. 1) NO is a known modulator of G protein coupled receptor (GPCR) signaling primarily via action on guanylate cyclase. The TGF-β1 receptors are not GPCRs, rather receptor serine / threonine kinases. NO* would be expected to show synergistic interaction with PGE2 and PTH / PTHrP receptor signaling (both GPCR). Some literature exists on PGE2 and PTH / PTHrP signaling, but is inconsistent (e.g. up / downregulation of PGE2) and not focused on the ligand signaling per se. 2) The interaction of NO and TGF-β1 leads to decreased SMAD4 transcription factor complex activity, an unexpected finding.

[0098] No commercial therapeutics exist to simultaneously mitigate microbial contamination and promote bone repair. The TGF-β1 laded hydrogel formulation is based on the inventors patent US 12,440,604 B2. Herein, the inventors have employed NO* delivery to decrease the required TGF-β1 dose. The inventors further utilize the NO* for antimicrobial utility. When using the mSNO-MP donor instead of the naked NOC-18 molecular donor, the hydrogel further serves to lock the microparticles within the injection site. The polymers of the hydrogel have different chemical modifications (from those in the patent) to make hydrogel crosslinking compatible with the NO donors and facilitate use. First, the prior hydrogel crosslinked via radical polymerization upon UV-A irradiation. However, UV-A activates release of NO from NO* donor (e.g., NOC-18 and mSNO-MP). Thus, the prior hydrogel releases all the NO load upon crosslinking. The Coll hydrogel undergoes physical instead of chemical gelation (crosslinking) and was employed for proof-of-concept inSAS / amcl 8123-113405-02 02 / 06 / 26 07032determining the interaction of TGF-β1 with NO*. The new hydrogel formulation based on the prior patent consists of a similar primary composition that crosslinks via click-chemistry. Namely the collagen and heparin polymers are azide functionalized and react via strain-promoted azi de-alkyne cycloaddition (SPAAC) with dibenzocyclooctyne (DBCO) functionalized PEG upon mixing under aqueous conditions at room and body temperature. Second, this formulation was designed to be compatible with the nitrosylation procedure for the mSNO-MPs. In order to create a shelf-stable device (room temp stable), the nitrosylation of these microparticles must be carried out immediately before implantation. This hydrogel formulation is compatible with bi-compartment separation of the microparticle reagents in frangible packs or multi -compartment syringes.Methods for Treating Bone

[0099] Methods are disclosed herein for treating a bone defect in a subject. Treatment of the bone defect does not require a process of complete healing or a treatment which is 100% effective at restoring a defect to its pre-defect state. Successful treatment of a bone defect includes partial repair or healing, for example filling of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the bone defect with new bone material.

[0100] Methods are provided to promote fracture healing. Methods are also provided for healing a gap or void in a bone. In aspects, the subject can have a fracture, such as a simple or compound fracture. There can be an infection at the fracture site. Orthopedic classification of fractures includes closed or open and simple or multi- fragmentary fractures. In closed fractures the skin remains intact, whilst in an open fracture the bone may be exposed through the wound site, which brings a higher risk of infection. Simple fractures occur along a single line, tending to divide the bone in two. Multi -fragmentary fractures spilt the bone into multiple pieces. Other fracture types include, compression fracture, compacted fracture, spiral fracture, complete and incomplete fractures, transverse, linear and oblique fractures and comminuted fractures. The disclosed methods are of use to treat any of these types of fracture. The fracture can be a skull fracture, a sternum fracture, a rib fracture, or a fracture of an extremity, such as a leg or an arm bone. In some aspects, the fracture is in a extremity. In the case of an avian subject, the fracture can be in a wing bone. In the case of a human subject, the fracture can be in an arm or leg. In the case of a veterinary subject, the fracture can be in a leg. Any of these subjects can be treated using the biomaterials and devices disclosed here.SAS / amcl 8123-113405-02 02 / 06 / 26 07032

[0101] The fracture can be in any bone, including but not limited to cranial bones such as the frontal bone, parietal bone, temporal bone, occipital bone, sphenoid bone, ethmoid bone: facial bones such as the zygomatic bone, superior and inferior maxilla, nasal bone, mandible, palatine bone, lacrimal bone, vomer bone, the inferior nasal conchae; the bones of the ear, such as the malleus, incus, stapes; the hyoid bone; the bones of the shoulder, such as the clavicle or scapula; the bones of the thorax, such as the sternum or the ribs; the bones of the spinal column including the cervical vertebrae, lumbar vertebrae, and thoracic vertebrae; the bones of the arm, including the humerus, ulna and radius; the bones of the hands, including the scaphoid, lunate, triquetrum bone, pisiform bone, trapezium bone, trapezoid bone, capitate bone, and hamate bone; the bones of the palm such as the metacarpal bones; the bones of the fingers such as the proximal, intermediate and distal phalanges the bones of the pelvis such as the ilium, sacrum and coccyx; the bones of the legs, such as the femur, tibia, patella, and fibula; the bones of the feet, such as the calcaneus, talus, navicular bone, medial cuneiform bone, intermediate cuneiform bone, lateral cuneiform bone, cuboidal bone, metatarsal bone, proximal phalanges, intermediate phalanges and the distal phalanges; and the pelvic bones.

[0102] The subject can have a congenital, genetic, and metabolic skeletal pathology, such as a disease that affects the growth plate of long bones (e.g., dyschondroplasia and chondrodystrophy), or a pathology that affects the structure and function of the joint articular cartilage (e.g., chondrosis). The subject can have osteoporosis and / or osteopenia. The disclosed methods are of use to treat fractures in any of these subjects. However, the subject can also not have these conditions. The subject can be a military personnel; the disclosed compositions are of use in the field setting.

[0103] The subject can have a bone disease. The subject can have metabolic bone disease such as hyperparathyroidism or osteogenesis imperfecta. The subject can have a cancer. The subject can have diabetes. The methods can be used to repair gaps or voids in bone, such as those from traumatic bone injury of from surgical resection of bone, segmental defects, spinal fusions or non-unions and other bone defects. The subject can have an infection at a site in a bone impacted by disease. Any of these subjects can be treated using the biomaterials and devices disclosed here.

[0104] The subject can have a congenital, genetic, and metabolic skeletal pathology, such as a disease that affects the growth plate of long bones (e.g., dyschondroplasia and chondrodystrophy), or a pathology that affects the structure and function of the joint articular cartilage (e.g., chondrosis). The subject can be have osteoporosis and / or osteopenia. TheseSAS / amcl 8123-113405-02 02 / 06 / 26 07032subject can have an infection. Any of these subjects can be treated using the biomaterials and devices disclosed here.

[0105] The disclosed methods can reduce fracture-related infection that can lead to bone infection, such as osteomyelitis and / or bacterial chondronecrosis with osteomyelitis (BCO). Thus, in some examples, the methods prevent or treat a bone infection, such as osteomyelitis or BCO. In some example, the methods prevent or treat an infection with Staphylococcus aureus, Escherichia coli, a coagulase-negative Staphylococci or an Enterococcus species. The subject can have a MRSA infection. In aspects, the infection is a Staphylococcus infection, a Streptococcus infection, an Escherichia coli infection, a Pseudomonas infection, a. Micrococcus infection, a Chryseobacterium infection or an Enterococcus species infection. The bone may be predisposed to infection due to a recent trauma that results in a blood clot or hemostasis. The osteomyelitis can be acute or chronic.

[0106] In specific non-limiting examples, the methods can be used to prevent or treat osteomyelitis in growth plates of long bones, such as the proximal growth plate of the femur and tibiotarsus. In some embodiments, use of the material and / or device results in a decrease of infection. This can be evaluated based on the lack of one or more symptoms of osteomyelitis (e.g., including but not limited to, pain in the bone, bone tenderness, and swelling or warmth) or a negative diagnosis based on one or more diagnostic tests (e.g., including but not limited to, a bone scan, blood culture, or culture of the infectious lesion). In some embodiments, the composition is delivered directly to the site of infection.

[0107] The subject can be a human. The subject can be a veterinary subject.

[0108] The subject can be an avian subject. The bird can be any type of bird, including a bird of the order Anseriformes (swans, geese and ducks), Apodiformes (swifts and hummingbirds), Caprimulgiformes (goatsuckers), Charadriiformes (shorebirds, gulls and terns), Ciconiiformes (bitterns, herons, ibis and storks), Columbiformes (pigeons and doves), Coraciiformes (kingfishers), Falconiformes (birds of prey such as falcons), Galliformes (turkey and chicken), Gaviidae (loons), Passeriformes (Passerines such as the blackbird, thrush, warbler balckbird and sparrow), Pelecaniformes (boobies, pelicans, cormorants and anhingas), Phoenicopteriformes (flamingos), Piciformes (woodpeckers and allies), Podicipediformes (grebes), Psittaciformes (parrots and parakeets), and Strigidae (owls). An exemplary list of birds that can be treated can be found on the web, see nps.gov / ever / naturescience / birdspecieslisthtm, and birds.audubon.org / species, birds-of-north-america.net / list-of-north-american-birds.html, and birdchannel.com / bird-species / all_landing.aspx, as of October 17. 2014, which are incorporated herein by reference.SAS / amcl 8123-113405-02 02 / 06 / 26 07032In specific, non-limiting examples, the bird is a chicken, turkey, duck, pigeon, parakeet, lovebird, or a canary. The bird can be a fancy bird, such as fancy pigeon, bred for various traits relating to size, shape, color and / or behavior. Fancy pigeons are disclosed, for example, in the Australian Fancy Pigeons book of standards and the European List of Breeds of Fancy Pigeons, see the world-wide web, file entente-ee.com / deutsch / sparten / tauben / Dateien / 2012 / ELRT%2011 -06-2012_pdf. In some embodiments, the bird can be a bird of prey. The bird can be a member of any of the families of birds of prey, such as Accipitridae (a hawk, an eagle, a buzzard, a kite or an Old World vulture), Pandionidae (such as an osprey), Sagittariidae (such as a secretary bird), Falconidae (such as a falcon, a caracara and a forest falcon), or Cathartidae (such as a New World Vulture). The bird can be a nocturnal bird of prey, such as a member of the family Strigidae (such as a typical owl) or Tytonidae (such as a barn owl or a bay owls). In a specific nonlimiting example, the bird is a falcon. The falcon can be a kestrel, a hobby, a peregrine falcon or a heierofalcon. Exemplary falcons are Malagasy kestrel (Falco newtoni), Seychelles kestrel (Falco araea), Mauritius kestrel (Falco punctatus), Spotted kestrel (Falco moluccensis), Nankeen kestrel or Australian kestrel (Falco cenchroides), Common kestrel (Falco tinnunculus), Rock kestrel (Falco rupicolus), Greater kestrel (Falco rupicoloides), Fox kestrel (Falco alopex), Lesser kestrel (Falco naumanni), Grey kestrel (Falco ardosiaceus), Dickinson's kestrel (Falco dickinsoni), Banded kestrel (Falco zoniventris), Rednecked falcon (Falco chicquera ), African red-necked falcon (Falco (chicquera) ruficollis), Red-footed falcon (Falco vespertinus), Amur falcon (Falco amurensis), Eleonora's falcon (Falco eleonorae), Sooty falcon (Falco concolor), American kestrel or "sparrow hawk" (Falco sparverius), Aplomado falcon (Falco femoralis), Merlin or "pigeon hawk" (Falco columbarius), Eurasian merlin (Falco (columbarius) aesalon), Bat falcon (Falco rufigularis), Orange-breasted falcon (Falco deiroleucus), Eurasian hobby (Falco subbuteo), African hobby (Falco cuvierii), Oriental hobby (Falco severus), Australian hobby or little falcon (Falco longipennis), New Zealand falcon or karearea (Falco novaeseelandiae), Brown falcon (Falco berigora), Grey falcon (Falco hypoleucos), Black falcon (Falco subniger), Lanner falcon (Falco biarmicus), Laggar falcon (Falco jugger), Saker falcon Falco cherrug, Altai falcon (Falco cherrug altaicus), Gyrfalcon (Falco rusticolus), Prairie falcon (Falco mexicanus), Peregrine falcon Falco peregrinus, Peale's falcon (Falco peregrinus pealei), Pallid falcon (Falco peregrinus cassini var. kreyenborgi), Barbary falcon (Falco (peregrinus) pelegrinoides) and a Taita falcon (Falco fasciinucha).SAS / amcl 8123-113405-02 02 / 06 / 26 07032

[0109] In additional embodiments, the bird is a hawk, such as an Accipiter or a Buteo hawk. The hawk can be a member of the Genus Accipiter, such as a Northern goshawk (A. gentilis), Eurasian sparrowhawk (A. nisus), Grey-bellied hawk (A. poliogaster), Crested goshawk (A. trivirgatus), Sulawesi goshawk (A. griseiceps), Red-chested goshawk (A. toussenelii), African goshawk (A. tachiro), Chinese sparrowhawk (A. soloensis), Frances's sparrowhawk (A. francesii ), Anjouan sparrowhawk (Accipiter francesiae pusillus), Spot-tailed sparrowhawk (A. trinotatus), Grey goshawk (A. novaehollandiae), Brown goshawk (A. fasciatus), Christmas goshawk (Accipiter fasciatus natalis), Pied goshawk (A. albogularis), Fiji goshawk (A. rufitorques), White -bellied goshawk (A. haplochrous), Moluccan goshawk (A. henicogrammus), Grey-headed goshawk (A. poliocephalus), New Britain goshawk (A. princeps), Black sparrowhawk, (A. melanoleucus), Henst's goshawk (A. henstii), Meyer's goshawk (A. meyerianus), Chestnut-flanked sparrowhawk (A. castanilius), Nicobar sparrowhawk (A. butleri), Levant sparrowhawk (A. brevipes), Slaty-mantled sparrowhawk (A. luteoschistaceus), Imitator sparrowhawk (A. imitator), Red-thighed sparrowhawk (A. erythropus), Little sparrowhawk (A. minullus), Japanese sparrowhawk (A. gularis), Dwarf sparrowhawk (A. nanus), Rufous-necked sparrowhawk (A. erythrauchen). Collared sparrowhawk (A. cirrocephalus), New Britain sparrowhawk (A. brachyurus), Vinousbreasted sparrowhawk (A. rhodogaster), Madagascar sparrowhawk (A. madagascariensis), Ovambo sparrowhawk (A. ovampensis), Rufous-chested sparrowhawk (A. rufiventris), Shikra (A. badius), Tiny hawk (A. superciliosus), Semicollared hawk (A. collaris), Sharp-shinned hawk (A. striatus), White-breasted hawk (A.,v. chionogaster), Plain-breasted hawk (A. x. ventralis), Rufous -thighed hawk (A.,y. erythronemius), Cooper's hawk (A. cooperii), Gundlach's hawk (A. gundlachi), Bicolored hawk (A. bicolor), or a Besra (A. virgatus). The hawk can be a member of the genus Melierax, such as a Gabar goshawk (M. gabar), Dark chanting goshawk (M. metabates), Eastern chanting goshawk (M. poliopterus) or a Pale chanting goshawk (M. canorus). The hawk can be a member of the genus Urotriorchis, such as a Long -tailed hawk (U. macrourus). The hawk can be a member of the genusErythrotriorchis, such as a Red goshawk (E. radiatus) or a Chestnut-shouldered goshawk (E. buergersi). The hawk can be a member of the genus Megatriorchis such as a Doria's goshawk (M. doriae).

[0110] Methods are also disclosed for treating periprosthetic osteolysis (PPOL )at the site of a prosthetic implant in bone or cartilage. The method includes administering to the subject an effective amount of a disclosed biomaterial or composition. In some aspects, the subject is a human subject. In some aspects, the subject has experienced bone loss at the site of theSAS / amcl 8123-113405-02 02 / 06 / 26 07032prosthetic and / or aseptic loosening of the prosthetic. In some aspects, the subject has a joint replacement, wherein the joint replacement has loosened. Loosening can be due to infection or aseptic.

[0111] Methods are disclosed herein for treating PPOL. 'The PPOL can be occur at any joint that has received an implant. Methods are also disclosed for treating implant loosening from periprosthetic infection. These joints include hand joints (fingers, thumb), elbow joints, wrist joints, axillary joints, sternoclavicular joints, vertebral articulations, temporomandibular joints, sacroiliac joints, hip joints, knee joints, and articulations of foot (e.g., ankles, toes). The joint can be a fibrous joint, cartilaginous joint, synovial joint, or facet joint.

[0112] The PPOL can occur at a site that has received an implant that is not a joint. For example, when an implant is positioned in bone due to a break or crush of the bone or other bone void in a location that is not at a joint, PPOL may occur at that site. For example, PPOL may occur at the site of a plate, pin, screw, or the like that has been implanted in a bone. For example, the site may be in a bone of the leg, arm, skull, spine, hip, rib, hand, foot, ankle, or wrist.

[0113] In aspects, the subject has experienced a total arthroplasty of a joint. In further aspects, the subject has a partial arthroplasty of a joint. In these aspects, the prosthetic is implanted to totally or partially replace the joint. The partial or total arthroplasty of the joint may be of a joint in the finger or hand, the elbow, the wrist, the axilla (shoulder or acromioclavicular joint), a sternoclavicular joint, a facet joint of the spine, neck joint, temporomandibular joint, a sacroiliac joint, a hip joint, a knee joint, and / or articulations of foot (ankle joint, toe joints). For example, the PPOL may be occurring in the knee at the site of total or partial arthroplasty, e.g., a “knee replacement.’’ For example, the PPOL may be occurring in the hip at the site of a total or partial arthroplasty, e.g., a “hip replacement.”

[0114] A subject can be selected for treatment with an effective amount of a disclosed biomaterial or composition that has a joint replacement. The joint can be any joint of interest, including, but not limited to, a knee, hip, shoulder, elbow, wrist, ankle, finger, toe, or temporomandibular joint. The prosthetic can be an artificial knee, artificial hip, artificial shoulder, artificial elbow, artificial wrist, artificial ankle, artificial finger joint, artificial toe joint, or artificial temporomandibular joint, respectively. In certain aspects, the prosthetic is an artificial finger joint. The finger joint can be a metacarpophalangeal joint (MCP), a proximal interphalangeal joint (PIP), a distal interphalangeal joint (DIP), or an interphalangeal joint (IP). In other aspects, the prosthetic is an artificial toe joint, and the toe joint can be a metatarsophalangeal joint (MCP), a proximal interphalangeal joint (PIP), aSAS / amcl 8123-113405-02 02 / 06 / 26 07032distal phalangeal joint (DP), or an interphalangeal joint (IP). In specific non-limiting examples, the joint is a hip or a knee, and the subject has received a total hip arthroplasty (TH A) and / or a total knee arthroplasty (TKA).

[0115] The disclosed methods can be used to treat PPOL and / or periimplantitis in a dental implant. The disclosed methods can be used to treat periimplantitis. In these methods, the subject has a dental implant. Dental implants may include implants into the mandible or maxilla to support crowns or other prosthetic dental structures or other implants that support the teeth. Pins and plates may be inserted to strengthen a bone or joint after an injury, such as a fracture. For example, a dental implant is a replacement tooth root, e.g., that connects directly to a jaw bone. In some examples, a dental implant is an endosteal (in the bone) implant or a subperiosteal (on the bone) implant. Exemplary dental implants include screws, cylinders or blades surgically placed into the jawbone. In some aspects, each implant holds at least one tooth (e.g., prosthetic tooth). Subperiosteal implants are placed on top of the jaw, and the implant framework contains posts that protrude through the gum of the subject in order to secure the implant. Osteointegrated implants, e.g. teeth, digits, facial prosthesis and implanted hearing devices can also be treated using a disclosed biomaterial or composition.

[0116] PPOL. can also be treated at the site other types of orthopedic implants. Exemplary orthopedic implants are bone screws, orthopedic pins, mechanical devices for the fixation and stabilization of an orthopedic fracture, bone cement, elbow replacements, synthetic joints, synthetic cartilage, synthetic spin discs, bone plates, orthopedic nails, orthopedic rods, orthopedic rectangles, compression plates, shoulder replacements, bone wires, and prostheses.

[0117] The disclosed biomaterials and compositions can also of use in treating PPOL, resulting from disintegration of metal, ceramic, or plastic implants. For example, the implant (e.g., orthopedic or dental) comprises a metal (e.g., tantalum, titanium, a titanium alloy such as Ti6A14V, cobalt, chromium, a cobalt-chromium alloy, zirconium, or a zirconium alloy such as oxinium oxidized zirconium), a ceramic, hydroxyapatite, and / or polyethylene. In certain aspects, the prosthetic is made of polyethylene, cobalt-chromium-molybdenum, titanium or titanium alloy, stainless steel, or ceramic. In non-limiting examples, the polyethylene is ultra-high molecular weight polyethylene or cross-linked polyethylene. In more non-limiting examples, the ceramic is zirconia or alumina. An effective amount of a disclosed biomaterial or composition can be used to treat PPOL resulting from any of these implants.SAS / amcl 8123-113405-02 02 / 06 / 26 07032

[0118] The prosthetic can be implanted into a bone in a subject that is not an articular joint. The prosthetic can be implanted into any bone, including, but not limited to, the skull, jaw, neck (cervical spine), thoracic spine, lumbar spine, or coccyx. The prosthetic can be implanted into a bone of the leg, arm, foot, hand, chest, or pelvis. The prosthetic can be, for example, a pin, post, rod, plate, spring, artificial disc, cage, or screw. These subjects can be selected for treatment, and an effective amount of a disclosed biomaterial or composition can be used to treat periprosthetic osteolysis resulting from any of these implants.

[0119] A therapeutic effect experienced by the subject may be (i) a reduction in the severity of the symptoms for the period of time as compared to the severity of the symptoms prior to the treatment course, (ii) remission of the PPOL or its symptoms for the period of time, (iii) prevention of flare up or relapse of the periprosthetic osteolysis during the period of time, (iv) reduction in the severity of symptoms experienced during a flare-up or relapse during the period of time compared to the severity of symptoms experienced prior to the treatment course, (v) reduction in the frequency of relapse or flare-up during the period of time as compared to frequency of relapse or flare up prior to the treatment course, or (vi) absence of signs of progression of the PPOL during the period of time after completing the treatment course. For any given subject, a reduction or improvement in the severity of symptoms or remission of the disorder can be measured according to relevant clinical indicia and relevant clinical objective standards, for example, a scoring system for joint mobility or immune reactivity. For example, a patient may experience a a clinical outcome during the time period indicative of improvement of the disorder as a result of the treatment as compared to the score prior to the treatment course.

[0120] Diagnostic imaging can be used for evaluating the extent and distribution of osteolysis at a site of an implant or of aseptic loosening of the implant. For example, radiographs can be used to characterize and monitor periprosthetic osteolysis. Geographic or linear zones of periprosthetic lucency greater than 2 mm that progress on serial examinations or develop after 2 years following arthroplasty are indicative of osteolysis. Computed tomograph (CT) may provide a more sensitive detection of images characteristic of osteolysis. For example, radiolucent lesions that communicate with the joint space and have well-defined sclerotic borders- are indicative of osteolysis. Magnetic resonance imaging (MRI) is also used to diagnose periprosthetic osteolysis. MRI provides higher sensitivity than CT in detection of small (less than 3 cm) periprosthetic lesions. MRI can also be used to identify extraosseous soft tissue deposits, pathology affecting neurovascular bundles, or precursors to bone resorption. Unlike infections, osteolysis presents as well-defined lesionsSAS / amcl 8123-113405-02 02 / 06 / 26 07032with low signal intensity similar to skeletal muscle by MRI. (Desai, M. A. et al. Orthopedics 2008, 31(6).) These methods can be used to assess the efficacy of treatment.

[0121] For administration to a joint, or the site of an implant, administration of the composition can be performed by procedures including but not limited to injection, endoscopic delivery, minimally invasive surgery, arthroscopy, infusion, or surgical implantation. For administration to a fracture, the administration can be performed, without limiting, by injection or infusion, or during a surgical procedure, such as when the bone is set.

[0122] The disclosed compositions can be administered with other agents. Suitable agents include, but are not limited to, antibiotics and growth factors. Suitable antibiotics are disclosed herein.OVERVIEW

[0123] Paragraph 1. A composition comprising a hydrogel, a transforming growth factor (TGF)-β1, and a nitric oxide (NO*) donor, wherein:the hydrogel has a density of from 0.2% to 3% w / v and is enzymatically degradable; the nitric oxide donor has a half-life of from 6 hours to 24 hours and is present in an amount sufficient to provide a concentration of NO* in the hydrogel of from 3 mM to 160 mM; andthe TGF-β1 is present in an amount of from 1 ng to less than 500 pg per mL of the hydrogel.

[0124] Paragraph 2. The composition of paragraph 1, wherein the hydrogel has a mesh size of 40 nm to 10 pm.

[0125] Paragraph 3. The composition of paragraph 1 or paragraph 2, wherein the hydrogel is selected to crosslink without irradiation by light.

[0126] Paragraph 4. The composition of any one of paragraphs 1-3, wherein the hydrogel is a polysaccharide, decellularized extracellular matrix (ECM), protein, peptide, or a combination thereof.

[0127] Paragraph 5. The composition of any one of paragraphs 1-3, wherein the hydrogel is a composite hydrogel comprising one or more of a polysaccharide, decellularized extracellular matrix (ECM), protein, peptide, polyethylene glycol (PEG), poly(N-isopropylacrylamide) (PNIPA), poloxamer, or a naturally derived polysaccharide having a branched or a linear structure.SAS / amcl 8123-113405-02 02 / 06 / 26 07032

[0128] Paragraph 6. The composition of paragraph 4, wherein the hydrogel is chitosan, hyaluronan, heparin, heparin sulfated proteoglycan, matrigel, collagen, gelatin, fibrin, gelatin+heparin, or a combination thereof.

[0129] Paragraph 7. The composition of paragraph 5, wherein the hydrogel comprises: i) one or more of chitosan, hyaluronan, heparin, heparin sulfated proteoglycan, matrigel, collagen, gelatin, fibrin, or gelatin+heparin; andii) one or more of PEG, PNIPA, Poloxamer 407, Pluronic F86, dextran, alginate, pectin, or gellan gum.

[0130] Paragraph 8. The composition of any one of paragraphs 1-7, wherein the nitric oxide donor has a half-life of from 12 hours to 18 hours.

[0131] Paragraph 9. The composition of any one of paragraphs 1-8, wherein an amount of the nitric oxide donor per gram of the hydrogel is selected to provide from 0.014 mmoles NO* / gram hydrogel to 26.6 mmoles NO* / gram hydrogel.

[0132] Paragraph 10. The composition of any one of paragraphs 1-8, wherein an amount of the nitric oxide donor in the hydrogel is selected to provide a concentration of NO* in the hydrogel of from 3.25 millimolar (mM) to 157 mM.

[0133] Paragraph 11. The composition of paragraph 10, wherein the amount of the nitric oxide donor is selected to provide a concentration of NO* in the hydrogel of from 32.5 millimolar (mM) to 157 mM.

[0134] Paragraph 12. The composition of paragraph 10, wherein the amount of the nitric oxide donor is selected to provide a concentration of NO* in the hydrogel of from 3.25 millimolar (mM) to 53.1 mM.

[0135] Paragraph 13. The composition of paragraph 10, wherein the amount of the nitric oxide donor is selected to provide a concentration of NO* in the hydrogel of from 32.5 millimolar (mM) to 53.1 mM.

[0136] Paragraph 14. The composition of any one of paragraphs 1-13, wherein the nitric oxide donor is:i) an S-nitrosothiol, N-diazeniumdiolate (NONOate), furoxan, or molecular hybrid nitric oxide donor, andii) nitrosoglutathione (GSNO), glutathione (GSH), s-nitroso-n-acetylcysteine (SNAC), spermine NONOate, (Z)-[3-aminopropyl(propyl)amino]-hydroxyimino-oxidoazanium (PAPA NONOate), (Z)-[bis(3-aminopropyl)amino]-hydroxyimino-oxidoazanium (DPTA NONOate),SAS / amcl 8123-113405-02 02 / 06 / 26 070324-(phenylsulfonyl)-3-{ [(2-dimethylamino)ethyl]thio }furoxan (LL4254), or 4-hydroxymethyl-furoxan-3-carboxamide (CAS-1609).

[0137] Paragraph 15. The composition of paragraph 14 wherein:the S-nitrosothiol is S-nitroso-N-acetylpenicillamine (SNAP), or nitrosylated 3-mercaptopropyl trimethoxysilane sol-gel microparticles (mSNO-MPs);the N-diazeniumdiolate (NONOate) is (Z)-l-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-l-ium-l,2-diolate (DETA NONOate), or N-(6-aminohexyl) aminopropyl-trimethoxysilane (AHAP3) sol-gel microparticle;O=S=OGVO-Ns+the furoxan is(3 - { [2-(dimethylamino)ethyl]oxy } -4-phenylfuroxan) (LL4212), or a phenylsulfonyl furoxan derivative; orthe molecular hybrid nitric oxide donor is a Cephalosporin-linked diazeniumdiolate, or S-nitroso diclofenac.

[0138] Paragraph 16. The composition of any one of paragraphs 1-15, wherein the composition comprises TGF-β1 in an amount of from 1 ng per mL of the hydrogel to 200 pg per mL of the hydrogel.

[0139] Paragraph 17. The composition of paragraph 16, wherein the amount of TGF-β1 is from 1 ng / mL to 1 μg / mL of the hydrogel.

[0140] Paragraph 18. The composition of paragraph 16, wherein the amount of TGF-β1 is from 1 μg / mL to 200 μg / mL of the hydrogel.

[0141] Paragraph 19. The composition of paragraph 18, wherein the amount of TGF-β1 is from 50 μg / mL to 200 μg / mL of the hydrogel.

[0142] Paragraph 20. The composition of any one of paragraphs 1-19, wherein the hydrogel is selected to be at least 95% degraded 8 weeks after administration.

[0143] Paragraph 21. The composition of paragraph 20, wherein the hydrogel is selected to be completely degraded 8 weeks after administration.SAS / amcl 8123-113405-02 02 / 06 / 26 07032

[0144] Paragraph 22. The composition of any one of paragraphs 1-21, wherein the composition further comprises an additional component.

[0145] Paragraph 23. The composition of paragraph 22, wherein the additional component is an antimicrobial, anti-inflammatory, analgesic, aanesthetic, or a combination thereof.

[0146] Paragraph 24. The composition of paragraph 22, wherein the additional component is a glycopeptide, lipopeptide, aminoglycosidic, rifamycin, penicillin, cephalosporin, nitroimidazole, quinolone, metal nanoparticle, cationic polymer, cationic particle, imidazole, non-steroidal anti-inflammatory, Anesthetic, opioid, or a combination thereof.

[0147] Paragraph 25. The composition of paragraph 22, wherein the additional component is vancomycin, daptomycin, gentamicin, tobramycin, rifampin, nafcilin, penicillin G, cloxacillin, amoxicillin, cefazolin, ceftriaxone, metronidazole, ciprofloxacin, gold nanoparticles, silver nanoparticles, nanoselenium, copper nanoparticles, iron nanoparticles, zinc nanoparticles, voriconazole, amphotericin B, fluconazole, clotrimazole, diclofenac, ibuprofen, naproxen, lidocaine, bupivacaine, ropivacaine, morphine, oxycodone, fentanyl, or a combination thereof.

[0148] Paragraph 26. The composition of paragraph 25, wherein the additional component is vancomycin, amoxicillin, cefazolin, lidocaine, or a combination thereof.

[0149] Paragraph 27. A method of repairing a bone defect in a subject, comprising administering locally to the bone defect a composition comprising a hydrogel, and effective amount of transforming growth factor (TGF)-β1, and an effective amount of an NO* donor, thereby repairing the bone defect.

[0150] Paragraph 28. The method of paragraph 27, wherein the composition is a composition according to any one of paragraphs 1-26.

[0151] Paragraph 29. The method of paragraph 27 or paragraph 28, wherein the bone defect is a fracture.

[0152] Paragraph 30. The method of paragraph 29, wherein the fracture is a simple or complex fracture.

[0153] Paragraph 31. The method of paragraph 27 or paragraph 28, wherein the bone defect is a gap or void in a bone.

[0154] Paragraph 32. The method of any one of paragraphs 27-31, wherein the defect is cranofacial, in a sternum, in a rib, in a vertebrae, or a in an extremity.

[0155] Paragraph 33. The method of any one of paragraphs 27-32, wherein the bone defect is infected.SAS / amcl 8123-113405-02 02 / 06 / 26 07032

[0156] Paragraph The method of paragraph 33, wherein the infection is a Staphylococcus infection, a Streptococcus infection, an Escherichia coli infection, a Pseudomonas infection, a Micrococcus infection, a Chryseobacterium infection or an Enterococcus species infection.

[0157] Paragraph 35. The method of any one of paragraphs 29-34, wherein the defect is a result of trauma.

[0158] Paragraph 36. The method of any one of paragraphs 29-35, wherein the defect is result of an underlying physiological condition.

[0159] Paragraph 37. The method of paragraph 36, wherein the underlying physiological condition is osteopenia, osteoporosis, diabetes, or a metabolic bone disease such as hyperparathyroidism or osteogenesis imperfecta.

[0160] Paragraph 38. The method of paragraph 27 or paragraph 28, wherein the subject has a prosthetic in bone or cartilage, and wherein the method treats periprosthetic osteolysis in the subject.

[0161] Paragraph 39. The method of paragraph 38, wherein the subject has experienced bone loss at the site of the prosthetic and / or aseptic loosening of the prosthetic.

[0162] Paragraph 40. The method of paragraph 38 or paragraph 39, wherein the subject has experienced a total or partial arthroplasty of a joint, and the prosthetic is implanted to totally or partially replace the joint.

[0163] Paragraph 41. The method of any one of paragraphs 38-40, wherein the joint is a knee, hip, shoulder, elbow, wrist, ankle, finger, toe, or temporomandibular joint, and wherein the prosthetic is an artificial knee, artificial hip, artificial shoulder, artificial elbow, artificial wrist, artificial ankle, artificial finger joint, artificial toe joint, or artificial temporomandibular joint, respectively.

[0164] Paragraph 42. The method of any one of paragraphs 38-40, wherein the subject has received a total hip arthroplasty and / or a total knee arthroplasty.

[0165] Paragraph 43. The method of paragraph 37, wherein the prosthetic is a dental implant into the jaw.

[0166] Paragraph 44. The method of paragraph 43, wherein the subject has periimplantitis.

[0167] Paragraph 45. The method of any one of paragraphs 37, 38, 43, or 44, wherein the prosthetic is implanted into a bone in the subject that is not at an articular joint.

[0168] Paragraph 46. The method of any one of paragraphs 37-45, wherein the prosthetic is a pin, post, rod, plate, spring, artificial disc, cage, or screw.

[0169] Paragraph 47. The method of any one of paragraphs 27-46, wherein the subject is a human subject or an avian subject.SAS / amcl 8123-113405-02 02 / 06 / 26 07032

[0170] Paragraph 48. The method of any one of paragraphs 38-47, wherein the composition is administered at an interface between the prosthetic and bond.

[0171] Paragraph 49. The method of any one of paragraphs 27-47, wherein the composition is implanted into the skull, jaw, cervical spine, thoracic spine, lumbar spine, or coccyx.

[0172] Paragraph 50. Use of the composition of any one of paragraphs 1-26 in the manufacture of a medicament for repairing a bone defect.

[0173] Paragraph 51. The composition of any one of paragraphs 1 -26 for use in a method of administration to a subject having a bone defect.

[0174] Paragraph 52. The composition of any one of paragraphs 1-26 for use in a method for repairing a bone defect.

[0175] Paragraph 53. The use of paragraph 50, or the composition for use of paragraph 51 or paragraph 52, wherein the bone defect is a fracture.

[0176] Paragraph 54. The composition of any one of paragraphs 51-53, for use in the method of any one of paragraphs 27-49.Examples

[0177] As disclosed herein, a single-dose, injectable, prolonged nitric oxide (NO*) hydrogel-delivery system was developed to treat deep wound infection and promote tissue regeneration. NO* is a natural antimicrobial produced by the human immune system. NO* has broad-spectrum antimicrobial action against numerous microbial components. NO* is also known to enhance bone regeneration. After the inflammatory phase of fracture healing, NO* concentrations drop, promoting wound contraction, osteogenesis, and angiogenesis to support bone regeneration Exogenous delivery of NO* supports bone healing, enhancing callus size and fracture stability. Though the potential regenerative benefits of NO* are clear, NO* systems have not been developed for deep tissue applications because endogenous NO* has a short physiological half-life and rapid diffusion. Attempts to exogenously deliver NO* are complicated by burst release, lack of prolonged release, and donor impurities that exert cytotoxic effects. Current low-cost NO* delivery systems rely on acid-based reduction of nitrite salts, which limits application to topical wounds and not deep-tissue wounds. Currently available donors with prolonged delivery are costly, readily diffuse from the defect size due to their small molecular size, and emit a burst release that is toxic to human cells at the required antimicrobial concentrations, e.g., S -nitrosoglutathione (GSNO) and SNAP. To address these issues with NO* delivery, a hydrogel carrier was utilized and several NO*SAS / amcl 8123-113405-02 02 / 06 / 26 07032donors were developed that are efficient carriers capable of delivering NO* over time, from brief delivery for a day to prolonged delivery for more than a week.

[0178] Experiments were performed to (1) evaluate the NO release, bactericidal efficacy, and biocompatibility of GSNO, short-term delivery microparticle (mSNO-MP), and longterm delivery microparticle (pSNO-MP) devices in vitro and (2) determine the efficacy in clearing infection (bactericidal) and in regenerating bone of hydrogels with mSNO-MP injected into infected femoral segmental bone defects in rats.EXAMPLE 1: Fabrication of device formulations

[0179] Manufacture the different NO* donors and hydrogel components. Zylo Therapeutics synthesized both mSNO-MP and pSNO-MP. The neutralization of the mSNO-MPs was optimized, eliminating the cytotoxicity on MSCs due to insufficient pH neutralization after nitrosylation. The pSNO-MP particle was rougher and larger than mSNO-MP, as the microparticle (MP) was produced from a ground stock. A protocol was developed for optimum activation of the pSNO-MPs (loading of the MP with NO*).

[0180] GSNO was synthesized, and activity was confirmed (FIG. 1). Increasing concentrations of GSNO reduced the metabolic activity of bone marrow stem cells due to increased NO* release and not to increased impurity content (FIG. 13). Both reduced glutathione (GS) and GSNO treated with UV light (which releases the nitric oxide load of GSNO reverting it to GS) minimally affected cellular metabolic activity, indicating that neither GS nor residual sodium nitrite and sodium chloride salts (used in nitrosylation of GS) were responsible for the observed effects. Rather, the decrease in metabolic activity is attributed to the delivery of NO* from GSNO.

[0181] It was verified that RTC loaded with microparticles consistently formed hydrogels across different commercial RTC batches (FIG. 2). Additionally, routine quality checks on the collagen stock were conducted, e.g., using prolong culture of antibiotic free medium inoculated with the collagen to check for potential microbial contamination. The treatment in the delivery system is shown in FIG. 3.

[0182] An alternate human derived collagen source was tested with clinical translation in mind. The stock was provided as an 8% w / v solution, which also afforded the potential to create stiffer gels with slower degradation compared to the 0.3% w / v of rat tail collagen stock. The human collagen proved inferior in gelation (inadequate mechanical consistency for use as a carrier for the NO* donors) and deleterious on cell spreading (FIG. 4). Thus, it was decided to use rat tail collagen throughout the rest of these studies.SAS / amcl 8123-113405-02 02 / 06 / 26 07032EXAMPLE 2: Evaluation of cytocompatibility on human MSCs

[0183] It was evaluated if the MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) doses are cytocompatible on human MSCs, because these are one of the cell pools that lead to regeneration in the segmental defects. It was further investigated what the effect of NO* on the “sternness” phenotype of MSCs, which would inform the mechanism of action in vivo. The MPs overcome limitations of conventional NO* donors, such as GSNO, in achieving high NO* dosing without cytotoxic effects on mammalian cells. Based on the NO* delivery assays above, it was concluded that the significant burst release from the GSNO is responsible for its cytotoxicity at required antimicrobial doses compared to mSNO-MP.

[0184] Next, it was investigated whether the dose effect of the mSNO-MPs and pSNO-MPs, with and without NO* loading, on the human MSC mitochondrial respiration via the MTS assay (FIG. 5). Results showed NO* delivery from mSNO-MP impacts respiration depending on ambient oxygen levels. Given the cytotoxic effect of GSNO at required microcidal doses, further investigation of additional GSNO concentrations was not pursued. The dose effect of mSNO-MPs on cell proliferation via DNA quantification was investigated using the PICOGREEN™ assay (FIG. 6). Results confirmed NO* changes in MTS assay were due to impact on mitochondrial respiration, not cell proliferation, consistent with viability (Live / Dead staining) results.

[0185] Quantitative PCR (qPCR) was used to measure the expression of Sox2 and Oct4, which are key markers of self-renewal and multipotency in bone marrow stem cells. Cells cultured on tissue culture plates served as untreated controls. Overall, the results showed a dose dependent decrease in the expression of these sternness markers 7 days after treatment, but a strong underlying bias to oxygen tension (pO2), with 20% pO2 showing greater expression than 5% pO2 (FIG. 7). These results suggested that NO* may potentiate the differentiation potential of MCS. qPCR was used to determine the effect of NO* on osteogenesis by MSCs in 3D cultures within hydrogels (FIG. 14). NO* and TGF-β1 each induced about a doubling in osteocalcin expression. However, the combination of NO* and TGF-β1 led to a quadrupling of osteocalcin expression at the high mSNO-MP dose, indicating a additive effect of NO* and TGFB1 in promoting osteogenic differentiation of MSCs.SAS / amcl 8123-113405-02 02 / 06 / 26 07032EXAMPLE 3: Rat model to study the immunomodulatory effects in regenerate tissue via flow cytometry

[0186] Eighty-two (82) animals were operated on, and treated with RTC, saline, or the defect empty was left empty to provide tissue samples for flow cytometry optimization. In addition, a further 36 animals were operated on, and of which biopsies were processed for the flow cytometry (accounting for attrition). The experimental design is depicted in FIG. 8.

[0187] The flow cytometry panel was optimized using control spleen tissue from the rats. A comparative assay was run at 5-days post treatment. Inclusion of TGF-β1 shifted the macrophage phenotype to a regenerative phenotype. mSNO-MP and TGF-β1 appeared to increase granulocytes, pointing to a nitric oxide mechanism modulating the leukocyte milieu. The apparent upregulation of CD4+T cells with RTC alone was consistent with collagen’s known bioactivity and potential antigenicity (FIG. 9).EXAMPLE 4: Quantification of Bone Regeneration

[0188] X-ray imaging was conducted every two weeks to monitor bone regeneration, which was supplemented by a terminal high-resolution μCT scan (FIG. 10). The RTC+mSNO-MP+TGF-β1 showed the earliest bridging of the defect at approximately 50% by 8-weeks.

[0189] A synergistic interaction of NO* and TGF-β1 was seen on bone healing in the RTC+mSNO-MP+TGF-β1 group (FIGs. 11-12). The RTC+pSNO-MP did not show significant bridging. The incomplete bone bridging is attributable to lack of micromotion from the rigid plate system. This data supports prior works showing NO* delivery accelerates osteogenesis and indicates potential mechanisms. The results suggest a direct mechanism in promoting osteogenesis by MSCs in vitro, potentially driving the earlier bony bridging compared to controls in vivo. Further, NO*’s mechanism on healing seems to involve modulation of the leukocyte milieu. NO* synergized with TGF-β1, which is released from bone and some leukocytes, yielding greater osteogenesis than either alone. In addition to promoting repair via early delivery into bone wounds, mSNO-MP+TGF-β1 hydrogel treatment can serve as a potential adjuvant with other biologies or scaffolds like demineralized bone matrix to promote bone regeneration in compromised wounds.

[0190] An injectable therapeutic was created with prolonged release of nitric oxide (NO*) and transforming growth factor-betal (TGF-β1) that successfully treated deep wound infection and promoted bone regeneration in rats. The NO* is delivered from the mSNO-MPs, which are comprised of mercaptopropyl trimethoxy silane that stabilizes and prolongSAS / amcl 8123-113405-02 02 / 06 / 26 07032NO* release over three days. The incorporated recombinant human TGF-β1 is cytokine naturally sequestered in bone that regulates bone homeostasis and promotes fracture healing by enhancing MSC differentiation into chondrocytes and osteoblasts. It has previously been shown that TGF-β1 promotes bone healing but at supraphysiologic concentrations, which limited translational potential and clinical utility. After several design development steps including dose response assays for antibacterial efficacy and biocompatibility, a therapeutic composition of 10 mg / mL of mSNO-MP and 100 pg / ml of human TGF-β1 in a 0.265% w / v rat tail collagen (RTC) hydrogel was determined for validation in vivo.

[0191] The NO* and TGF-β1 components of the therapeutic synergize to drive osteogenesis of progenitor cells and bone healing, and at 1 / 10ththe dose of TGF-β1 compared to prior work. Rats treated with the NO* and TGF-β1 releasing hydrogel showed a 2.2 to 1.63 greater degree of defect bridging at 16-weeks compared to the NO* and TGF-β1 alone in hydrogels, respectively. The mSNO-MP loaded therapeutic is an effective bactericidal for methicillin-resistant. S', aureus (MRSA) biofilms in vitro and significantly reduces the bacterial load in 5.0 mm mid-diaphyseal segmental femoral bone defects inoculated with a supraphysiologic dose of 500,000 CFU of MRSA. This is the basis for a novel therapeutic that enables early treatment of contaminated open bone wounds in low resource settings such as battlefields, rural communities and low-income countries

[0192] The therapeutic’s mechanism of action is likely multifold, involving interaction across multiple cell types including osteoprogenitors and leukocytes. This data demonstrates a synergistic effect on immunomodulation and bone healing in vivo. Regarding osteoprogenitors, human bone marrow stromal cells (MSC) were subjected to co-culture with mSNO-MP laden hydrogels in basal medium in vitro to probe inherent effects of NO* on “sternness” and osteogenesis (no other osteogenic supplements in the medium). MSCs were subjected to co-culture with mSNO-MP, TGF-β1, and mSNO-MP +TGF-P1 laden hydrogels in osteogenic medium in vitro to probe the effect on osteogenic differentiation. Regarding leukocytes, the leukocyte milieu was profiled in the defect site 5-days post therapeutic implantation. The device strongly modulated the leukocyte infiltrate, decreasing Ml -like macrophages and increasing granulocytes at 5-days post treatment. This synergistic mechanism may play a role endogenous fracture healing, given TGF-β1 is one cytokine sequestered in bone and NO* is rapidly produced during the initial inflammatory phase of healing.SAS / amcl 8123-113405-02 02 / 06 / 26 07032

[0193] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following paragraphs. We therefore paragraph as our invention all that comes within the scope and spirit of these paragraphs.

Claims

SAS / amcl 8123-113405-02 02 / 06 / 26 07032We claim:

1. A composition comprising a hydrogel, a transforming growth factor (TGF)-β1, and a nitric oxide (NO*) donor, wherein:the hydrogel has a density of from 0.2% to 3% w / v and is enzymatically degradable; the nitric oxide donor has a half-life of from 6 hours to 24 hours and is present in an amount sufficient to provide a concentration of NO* in the hydrogel of from 3 mM to 160 mM; andthe TGF-β1 is present in an amount of from 1 ng to less than 500 pg per mL of the hydrogel.

2. The composition of claim 1, wherein the hydrogel has a mesh size of 40 nm to 10 pm.

3. The composition of claim 1 or claim 2, wherein the hydrogel is selected to crosslink without irradiation by light.

4. The composition of any one of claims 1-3, wherein the hy drogel is a polysaccharide, decellularized extracellular matrix (ECM), protein, peptide, or a combination thereof.

5. The composition of any one of claims 1-3, wherein the hydrogel is a composite hydrogel comprising one or more of a polysaccharide, decellularized extracellular matrix (ECM), protein, peptide, polyethylene glycol (PEG), poly(N-isopropylacrylamide) (PNIPA), poloxamer, or a naturally derived polysaccharide having a branched or a linear structure.

6. The composition of claim 4, wherein the hydrogel is chitosan, hyaluronan, heparin, heparin sulfated proteoglycan, matrigel, collagen, gelatin, fibrin, gelatin+heparin, or a combination thereof.

7. The composition of claim 5, wherein the hydrogel comprises:i) one or more of chitosan, hyaluronan, heparin, heparin sulfated proteoglycan, matrigel, collagen, gelatin, fibrin, or gelatin+heparin; andSAS / amcl 8123-113405-02 02 / 06 / 26 07032ii) one or more of PEG, PNIPA, Poloxamer 407. Pluronic F68, dextran, alginate, pectin, or gellan gum.

8. The composition of any one of claims 1-7, wherein the nitric oxide donor has a half-life of from 12 hours to 18 hours.

9. The composition of any one of claims 1-8, wherein an amount of the nitric oxide donor per gram of the hydrogel is selected to provide from 0.014 mmoles NO* / gram hydrogel to 26.6 mmoles NO* / gram hydrogel.

10. The composition of any one of claims 1-8, wherein an amount of the nitric oxide donor in the hydrogel is selected to provide a concentration of NO* in the hydrogel of from 3.25 millimolar (mM) to 157 mM.

11. The composition of claim 10, wherein the amount of the nitric oxide donor is selected to provide a concentration of NO* in the hydrogel of from 32.5 rnillimolar (mM) to 157 mM.

12. The composition of claim 10, wherein the amount of the nitric oxide donor is selected to provide a concentration of NO* in the hydrogel of from 3.25 millimolar (mM) to 53.1 mM.

13. The composition of claim 10, wherein the amount of the nitric oxide donor is selected to provide a concentration of NO* in the hydrogel of from 32.5 rnillimolar (m ) to 53.1 mM.

14. The composition of any one of claims 1-13, wherein the nitric oxide donor is: i) an S-nitrosothiol, N-diazeniumdiolate (NONOate), furoxan, or molecular hybrid nitric oxide donor, andii) nitrosoglutathione (GSNO), glutathione (GSH), s-nitroso-n-acetylcysteine (SNAC), spermine NONOate, (Z)-[3-aminopropyl(propyl)amino]-hydroxyimino-oxidoazanium (PAPA NONOate), (Z)-[bis(3-aminopropyl)amino]-hydroxyimino-oxidoazanium (DPTA NONOate),SAS / amcl 8123-113405-02 02 / 06 / 26 07032O’ 4-(phenylsulfonyl)-3-{ [(2-dimethylamino)ethyl]thio }furoxan (LL4254), or 4-hydroxymethyl-furoxan-3-carboxamide (CAS-1609).

15. The composition of claim 14 wherein:the S -nitrosothiol is S-nitroso-N-acetylpenicillamine (SNAP), or nitrosylated 3- rnercaptopropyl trirnethoxysilane sol-gel microparticles (raSNO-MPs);the N-diazeniumdiolate (NONOate) is (Z)-l-[N-(2-aminoethyl)-N-(2-animonioethyl)aniino]diazen-l -ium- 1.2-diolate (DETA NONOate), or N-(6-aminohexyl) aminopropyl-trimethoxvsilane (AHAP3) sol-gel microparticle;9O-NX+thefuroxan isO' (3-{[2-(dimethylamino)ethyl]oxy}-4-phenylfuroxan) (LL4212), or a phenylsulfonyl furoxan derivative; orthe molecular hybrid nitric oxide donor is a Cephalosporin-linked diazeniuunidiolate, or S-nitroso diclofenac.

16. The composition of any one of claims 1-15, wherein the composition comprises TGF-pl in an amount of from 1 ng per mL of the hydrogel to 200 pg per mL of the hydrogel.

17. The composition of claim 16, wherein the amount of TGF-pl is from 1 ng / mL to 1 pg / mL of the hydrogel.

18. The composition of claim 16. wherein the amount of TGF-β1 is from 1 pg / mL to 200 pg / L of the hydrogel.SAS / amcl 8123-113405-02 02 / 06 / 26 0703219. The composition of claim 18, wherein the amount of TGF-pl is from 50 pg / mL to 200 pg / mL of the hydrogel.

20. The composition of any one of claims 1-19, wherein the hydrogel is selected to be at least 95% degraded 8 weeks after administration.

21. The composition of claim 20. wherein the hydrogel is selected to be completely degraded 8 weeks after administration.

22. The composition of any one of claims 1-21, wherein the composition further comprises an additional component.

23. The composition of claim 22. wherein the additional component is an antimicrobial, anti-inflammatory, analgesic, anesthetic, or a combination thereof.

24. The composition of claim 22, wherein the additional component is a glycopeptide, lipopeptide, aminoglycosidic, rifamycin, penicillin, cephalosporin, nitroimidazole, quinolone, metal nanoparticle, cationic polymer, cationic particle, imidazole, non-steroidal anti-inflammatory, Anesthetic, opioid, or a combination thereof.

25. The composition of claim 22, wherein the additional component is vancomycin, daptomycin, gentamicin, tobramycin, rifampin, nafcilin, penicillin G, cloxacillin, amoxicillin, cefazolin, ceftriaxone, metronidazole, ciprofloxacin, gold nanoparticles, silver nanoparticles, nanoselenium, copper nanoparticles, iron nanoparticles, zinc nanoparticles, voriconazole, amphotericin B, fluconazole, clotrimazole, diclofenac, ibuprofen, naproxen, lidocaine, bupivacaine, ropivacaine, morphine, oxycodone, fentanyl, or a combination thereof.

26. The composition of claim 25, wherein the additional component is vancomycin, amoxicillin, cefazolin, lidocaine, or a combination thereof.

27. A method of repairing a bone defect in a subject, comprising administering locally to the bone defect a composition comprising a hydrogel, and effective amount ofSAS / amcl 8123-113405-02 02 / 06 / 26 07032transforming growth factor (TGF)-pi, and an effective amount of an NO* donor, thereby repairing the bone defect.

28. The method of claim 27, wherein the composition is a composition according to any one of claims 1 -26.

29. The method of claim 27 or claim 28, wherein the bone defect is a fracture.

30. The method of claim 29, wherein the fracture is a simple or complex fracture.

31. The method of claim 27 or claim 28, wherein the bone defect is a gap or void in a bone.

32. The method of any one of claims 27-31, wherein the defect is craniofacial, in a sternum, in a rib, in a vertebra, or in an extremity.

33. The method of any one of claims 27-32, wherein the bone defect is infected.

34. The method of claim 33, wherein the infection is a Staphylococcus infection, a Streptococcus infection, an Escherichia coli infection, a Pseudomonas infection, a Micrococcus infection, a Chryseobacterium infection or an Enterococcus species infection.

35. The method of any one of claims 29-34, wherein the defect is a result of trauma.

36. The method of any one of claims 29-35, wherein the defect is result of an underlying physiological condition.

37. The method of claim 36, wherein the underlying physiological condition is osteopenia, osteoporosis, diabetes, or a metabolic bone disease such as hyperparathyroidism or osteogenesis imperfecta.

38. The method of claim 27 or claim 28, wherein the subject has a prosthetic in bone or cartilage, and wherein the method treats periprosthetic osteolysis in the subject.SAS / amcl 8123-113405-02 02 / 06 / 26 0703239. The method of claim 38, wherein the subject has experienced bone loss at the site of the prosthetic and / or aseptic loosening of the prosthetic.

40. The method of claim 38 or claim 39, wherein the subject has experienced a total or partial arthroplasty of a joint, and the prosthetic is implanted to totally or partially replace the joint.

41. The method of any one of claims 38-40, wherein the joint is a knee, hip, shoulder, elbow, wrist, ankle, finger, toe, or temporomandibular joint, and wherein the prosthetic is an artificial knee, artificial hip, artificial shoulder, artificial elbow, artificial wrist, artificial ankle, artificial finger joint, artificial toe joint, or artificial temporomandibular joint, respectively.

42. The method of any one of claims 38-40, wherein the subject has received a total hip arthroplasty and / or a total knee arthroplasty.

43. The method of claim 38 or claim 39, wherein the prosthetic is a dental implant into the jaw.

44. The method of claim 43, wherein the subject has periimplantitis.

45. The method of any one of claims 37, 38, 43, or 44, wherein the prosthetic is implanted into a bone in the subject that is not at an articular joint.

46. The method of any one of claims 37-45, wherein the prosthetic is a pin, post, rod, plate, spring, artificial disc, cage, or screw.

47. The method of any one of claims 27-46, wherein the subject is a human subject or an avian subject.

48. The method of any one of claims 38-47, wherein the composition is administered at an interface between the prosthetic and bond.SAS / amcl 8123-113405-02 02 / 06 / 26 0703249. The method of any one of claims 27-47, wherein the composition is implanted into the skull, jaw, cervical spine, thoracic spine, lumbar spine, or coccyx.

50. Use of the composition of any one of claims 1-26 in the manufacture of a medicament for repairing a bone defect.

51. The composition of any one of claims 1 -26 for use in a method of administration to a subject having a bone defect.

52. The composition of any one of claims 1-26 for use in a method for repairing a bone defect.

53. The use of claim 50, or the composition for use of claim 51 or claim 52, wherein the bone defect is a fracture.

54. The composition of any one of claims 51-53, for use in the method of any one of claims 27-49.

Images