IL-1Ra gene transfer potentiates BMP2-mediated bone healing by redirecting osteogenesis toward endochondral ossification.

An estimated 100,000 patients each year in the United States suffer severe disability from bone defects that fail to heal, a condition where bone-regenerative therapies could provide substantial clinical benefits. Although recombinant human bone morphogenetic protein-2 (rhBMP2) is an osteogenic growth factor that is clinically approved for this purpose, it is only effective when used at exceedingly high doses that incur substantial costs, induce severe inflammation, produce adverse side effects, and form morphologically abnormal bone. Using a validated rat femoral segmental defect model, we show that bone formed in response to clinically relevant doses of rhBMP2 is accompanied by elevated expression of interleukin-1 (IL-1). Local delivery of cDNA encoding the IL-1 receptor antagonist (IL-1Ra) achieved bridging of segmental, critical size defects in bone with a 90% lower dose of rhBMP2. Unlike use of high-dose rhBMP2, bone formation in the presence of IL-1Ra occurred via the native process of endochondral ossification, resulting in improved quality without sacrificing the mechanical properties of the regenerated bone. Our results demonstrate that local immunomodulation may permit effective use of growth factors at lower doses to recapitulate more precisely the native biology of healing, leading to higher-quality tissue regeneration.

Investigation Into the Effects of Intra-Articular Steroid on Post-Traumatic Osteoarthritis in Distal Radius Fractures: A Randomized Controlled Pilot Study.

PURPOSE: The aim of this prospective, randomized, controlled, double-blinded pilot study was to determine the rates of post-traumatic osteoarthritis and assess joint space width in the presence or absence of a single intra-articular injection of corticosteroid after an acute, intra-articular distal radius fracture (DRF). METHODS: Forty patients received a single, intra-articular, radiocarpal joint injection of 4 mg of dexamethasone (DEX) (n = 19) or normal saline placebo (n = 21) within 2 weeks of a surgically or nonsurgically treated intra-articular DRF. The primary outcome measure was minimum radiocarpal joint space width (mJSW) on noncontrast computed tomography scans at 2 years postinjection. Secondary outcomes were obtained at 3 months, 6 months, 1 year, and 2 years postinjection and included Disabilities of the Arm, Shoulder, and Hand; Michigan Hand Questionnaire; Patient-Rated Wrist Evaluation; wrist range of motion; and grip strength. RESULTS: At 2-year follow-up, there was no difference in mean mJSW between the DEX group (2.2 mm; standard deviation, 0.6; range, 1.4-3.2) and the placebo group (2.3 mm; standard deviation, 0.7; range, 0.9-3.9). Further, there were no differences in any secondary outcome measures at any postinjection follow-up interval. CONCLUSIONS: Radiocarpal joint injection of corticosteroid within 2 weeks of an intra-articular DRF does not appear to affect the development of post-traumatic osteoarthritis within 2 years follow-up in a small pilot cohort. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic II.

Prevalence of AAV2.5 neutralizing antibodies in synovial fluid and serum of patients with osteoarthritis.

Osteoarthritis (OA) is a leading cause of disability with no cure and only supportive therapy. Adeno-associated virus (AAV) serotype 2.5 is being used in a Phase I clinical trial to deliver the interleukin-1 receptor antagonist into knee joints with OA. Neutralizing antibodies (Nab) directed against AAV2.5, if present, could inhibit gene transfer. Here, we report the prevalence of AAV2.5 Nab in the sera and synovial fluids of patients with OA. Nab titers were measured by their ability to inhibit in vitro transduction by AAV2.5 encoding GFP. Of 44 synovial fluids from patients with mid-stage and advanced OA, 43% had undetectable Nab; 25% had low titers (<1:100), 16% had medium titers (1:100-1:1000) and 16% had high titers (>1:1000) of Nab. Titers of AAV2.5 Nabs correlated with those of AAV2, but not with those of AAV5. Serum titers of AAV2.5 Nab correlated positively with titers in synovial fluid, and were never less than the matched synovial fluid titers. These findings suggest that high titers of Nab against AAV2.5 are uncommon in the synovial fluids of patients with OA, and individuals with high synovial fluid Nab titers can be identified by measuring titers in the serum.

Osteoarthritis gene therapy in 2022.

PURPOSE OF REVIEW: To assess the present status of gene therapy for osteoarthritis (OA). RECENT FINDINGS: An expanding list of cDNAs show therapeutic activity when introduced into the joints of animals with experimental models of OA. In vivo delivery with adenovirus or adeno-associated virus is most commonly used for this purpose. The list of encoded products includes cytokines, cytokine antagonists, enzymes, enzyme inhibitors, growth factors and noncoding RNA. Elements of CRISPR-Cas have also been delivered to mouse knees to ablate key genes. Several human trials have been initiated, using transgenes encoding transforming growth factor-ß1, interleukin-1 receptor antagonist, interferon-ß, the NKX3.2 transcription factor or variant interleukin-10. The first of these, using ex vivo delivery with allogeneic chondrocytes, gained approval in Korea which was subsequently retracted. However, it is undergoing Phase III clinical trials in the United States. The other trials are in Phase I or II. No gene therapy for OA has current marketing approval in any jurisdiction. SUMMARY: Extensive preclinical data support the use of intra-articular gene therapy for treating OA. Translation is beginning to accelerate and six gene therapeutics are in clinical trials. Importantly, venture capital has begun to flow and at least seven companies are developing products. Significant progress in the future can be expected.

Gene Delivery to Chondrocytes.

Delivering genes to chondrocytes offers new possibilities both clinically, for treating conditions that affect cartilage, and in the laboratory, for studying the biology of chondrocytes. Advances in gene therapy have created a number of different viral and non-viral vectors for this purpose. These vectors may be deployed in an ex vivo fashion, where chondrocytes are genetically modified outside the body, or by in vivo delivery where the vector is introduced directly into the body; in the case of articular and meniscal cartilage in vivo delivery is typically by intra-articular injection. Ex vivo delivery is favored in strategies for enhancing cartilage repair as these can be piggy-backed on existing cell-based technologies, such as autologous chondrocyte implantation, or used in conjunction with marrow-stimulating techniques such as microfracture. In vivo delivery to articular chondrocytes has proved more difficult, because the dense, anionic, extra-cellular matrix of cartilage limits access to the chondrocytes embedded within it. As Grodzinsky and colleagues have shown, the matrix imposes strict limits on the size and charge of particles able to diffuse through the entire depth of articular cartilage. Empirical observations suggest that the larger viral vectors, such as adenovirus (~100 nm), are unable to transduce chondrocytes in situ following intra-articular injection. However, adeno-associated virus (AAV; ~25 nm) is able to do so in horse joints. AAV is presently in clinical trials for arthritis gene therapy, and it will be interesting to see whether human chondrocytes are also transduced throughout the depth of cartilage by AAV following a single intra-articular injection. Viral vectors have been used to deliver genes to the intervertebral disk but there has been little research on gene transfer to chondrocytes in other cartilaginous tissues such as nasal, auricular or tracheal cartilage.

MiRNAs as Potential Regulators of Enthesis Healing: Findings in a Rodent Injury Model.

MicroRNAs (miRNAs) are short non-coding RNA sequences with the ability to inhibit the expression of a target mRNA at the post-transcriptional level, acting as modulators of both the degenerative and regenerative processes. Therefore, these molecules constitute a potential source of novel therapeutic tools. In this study, we investigated the miRNA expression profile that presented in enthesis tissue upon injury. For this, a rodent enthesis injury model was developed by creating a defect at a rat's patellar enthesis. Following injury, explants were collected on days 1 (n = 10) and 10 (n = 10). Contra lateral samples (n = 10) were harvested to be used for normalization. The expression of miRNAs was investigated using a "Fibrosis" pathway-focused miScript qPCR array. Later, target prediction for the aberrantly expressed miRNAs was performed by means of the Ingenuity Pathway Analysis, and the expression of mRNA targets relevant for enthesis healing was confirmed using qPCRs. Additionally, the protein expression levels of collagens I, II, III, and X were investigated using Western blotting. The mRNA expression pattern of EGR1, COL2A1, RUNX2, SMAD1, and SMAD3 in the injured samples indicated their possible regulation by their respective targeting miRNA, which included miR-16, -17, -100, -124, -133a, -155 and -182. Furthermore, the protein levels of collagens I and II were reduced directly after the injury (i.e., day 1) and increased 10 days post-injury, while collagens III and X showed the opposite pattern of expression.

Adenoviral gene transfer of a single-chain IL-23 induces psoriatic arthritis-like symptoms in NOD mice.

Previously, we demonstrated that intratumoral delivery of adenoviral vector encoding single-chain (sc)IL-23 (Ad.scIL-23) was able to induce systemic antitumor immunity. Here, we examined the role of IL-23 in diabetes in nonobese diabetic mice. Intravenous delivery of Ad.scIL-23 did not accelerate the onset of hyperglycemia but instead resulted in the development of psoriatic arthritis. Ad.scIL-23-treated mice developed erythema, scales, and thickening of the skin, as well as intervertebral disc degeneration and extensive synovial hypertrophy and loss of articular cartilage in the knees. Immunological analysis revealed activation of conventional T helper type 17 cells and IL-17-producing γδ T cells along with a significant depletion and suppression of T cells in the pancreatic lymph nodes. Furthermore, treatment with anti-IL-17 antibody reduced joint and skin psoriatic arthritis pathologies. Thus, these Ad.scIL-23-treated mice represent a physiologically relevant model of psoriatic arthritis for understanding disease progression and for testing therapeutic approaches.-Flores, R. R., Carbo, L., Kim, E., Van Meter, M., De Padilla, C. M. L., Zhao, J., Colangelo, D., Yousefzadeh, M. J., Angelini, L. A., Zhang, L., Pola, E., Vo, N., Evans, C. H., Gambotto, A., Niedernhofer, L. J., Robbins, P. D. Adenoviral gene transfer of a single-chain IL-23 induces psoriatic arthritis-like symptoms in NOD mice.

Taking the Next Steps in Regenerative Rehabilitation: Establishment of a New Interdisciplinary Field.

The growing field of regenerative rehabilitation has great potential to improve clinical outcomes for individuals with disabilities. However, the science to elucidate the specific biological underpinnings of regenerative rehabilitation-based approaches is still in its infancy and critical questions regarding clinical translation and implementation still exist. In a recent roundtable discussion from International Consortium for Regenerative Rehabilitation stakeholders, key challenges to progress in the field were identified. The goal of this article is to summarize those discussions and to initiate a broader discussion among clinicians and scientists across the fields of regenerative medicine and rehabilitation science to ultimately progress regenerative rehabilitation from an emerging field to an established interdisciplinary one. Strategies and case studies from consortium institutions-including interdisciplinary research centers, formalized courses, degree programs, international symposia, and collaborative grants-are presented. We propose that these strategic directions have the potential to engage and train clinical practitioners and basic scientists, transform clinical practice, and, ultimately, optimize patient outcomes.

Contribution of Implanted, Genetically Modified Muscle Progenitor Cells Expressing BMP-2 to New Bone Formation in a Rat Osseous Defect.

Because muscle contains osteoprogenitor cells and has a propensity to form bone, we have explored its utility in healing large osseous defects. Healing is achieved by the insertion of muscle fragments transduced with adenovirus encoding BMP-2 (Ad.BMP-2). However, it is not known whether the genetically modified muscle contributes osteoprogenitor cells to healing defects or merely serves as a local source of BMP-2. This question is part of the larger debate on the fate of progenitor cells introduced into sites of tissue damage to promote regeneration. To address this issue, we harvested fragments of muscle from rats constitutively expressing GFP, transduced them with Ad.BMP-2, and implanted them into femoral defects in wild-type rats under various conditions. GFP+ cells persisted within defects for the entire 8 weeks of the experiments. In the absence of bone formation, these cells presented as fibroblasts. When bone was formed, GFP+ cells were present as osteoblasts and osteocytes and also among the lining cells of new blood vessels. The genetically modified muscle thus contributed progenitor cells as well as BMP-2 to the healing defect, a property of great significance in light of the extensive damage to soft tissue and consequent loss of endogenous progenitors in problematic fractures.

Inflammatory cytokines induce a unique mineralizing phenotype in mesenchymal stem cells derived from human bone marrow.

Bone marrow contains mesenchymal stem cells (MSCs) that can differentiate along multiple mesenchymal lineages. In this capacity they are thought to be important in the intrinsic turnover and repair of connective tissues while also serving as a basis for tissue engineering and regenerative medicine. However, little is known of the biological responses of human MSCs to inflammatory conditions. When cultured with IL-1ß, marrow-derived MSCs from 8 of 10 human subjects deposited copious hydroxyapatite, in which authenticity was confirmed by Fourier transform infrared spectroscopy. Transmission electron microscopy revealed the production of fine needles of hydroxyapatite in conjunction with matrix vesicles. Alkaline phosphatase activity did not increase in response to inflammatory mediators, but PPi production fell, reflecting lower ectonucleotide pyrophosphatase activity in cells and matrix vesicles. Because PPi is the major physiological inhibitor of mineralization, its decline generated permissive conditions for hydroxyapatite formation. This is in contrast to MSCs treated with dexamethasone, where PPi levels did not fall and mineralization was fuelled by a large and rapid increase in alkaline phosphatase activity. Bone sialoprotein was the only osteoblast marker strongly induced by IL-1ß; thus these cells do not become osteoblasts despite depositing abundant mineral. RT-PCR did not detect transcripts indicative of alternative mesenchymal lineages, including chondrocytes, myoblasts, adipocytes, ligament, tendon, or vascular smooth muscle cells. IL-1ß phosphorylated multiple MAPKs and activated nuclear factor-κB (NF-κB). Certain inhibitors of MAPK and PI3K, but not NF-κB, prevented mineralization. The findings are of importance to soft tissue mineralization, tissue engineering, and regenerative medicine.

Preclinical toxicity evaluation of AAV for pain: evidence from human AAV studies and from the pharmacology of analgesic drugs.

Gene therapy with adeno-associated virus (AAV) has advanced in the last few years from promising results in animal models to >100 clinical trials (reported or under way). While vector availability was a substantial hurdle a decade ago, innovative new production methods now routinely match the scale of AAV doses required for clinical testing. These advances may become relevant to translational research in the chronic pain field. AAV for pain targeting the peripheral nervous system was proven to be efficacious in rodent models several years ago, but has not yet been tested in humans. The present review addresses the steps needed for translation of AAV for pain from the bench to the bedside focusing on pre-clinical toxicology. We break the potential toxicities into three conceptual categories of risk: First, risks related to the delivery procedure used to administer the vector. Second, risks related to AAV biology, i.e., effects of the vector itself that may occur independently of the transgene. Third, risks related to the effects of the therapeutic transgene. To identify potential toxicities, we consulted the existing evidence from AAV gene therapy for other nervous system disorders (animal toxicology and human studies) and from the clinical pharmacology of conventional analgesic drugs. Thereby, we identified required preclinical studies and charted a hypothetical path towards a future phase I/II clinical trial in the oncology-palliative care setting.

Progress Toward a Gene Therapy for Arthritis.

Osteoarthritis (OA) is a highly prevalent, disabling, incurable, and expensive disease that is difficult to treat nonsurgically. The pharmacokinetics of drug delivery to joints are such that it is not possible to target antiarthritic agents, especially biologics, to individual joints with OA at sustained, therapeutic concentrations. More than 30 years ago, we proposed that local, intra-articular gene transfer can overcome this barrier to therapy by engineering articular cells to synthesize antiarthritic gene products endogenously. This article summarizes the progress toward this goal. Initially, a retroviral vector was used to deliver cDNA encoding the interleukin-1 receptor antagonist (IL-1Ra) to the joints of experimental animals. Using an ex vivo strategy, cultures of autologous synovial fibroblasts were genetically modified in cell culture and introduced into joints by means of intra-articular injection. Successful development of this technology led to the first-in-human gene therapy trial for arthritis. This Phase I study targeted metacarpophalangeal joints with rheumatoid arthritis. Although successful, for various reasons, subsequent research targeted OA and used adeno-associated virus as a vector to deliver IL-1Ra by direct in vivo injection into the joint. A Phase I human clinical trial has just been completed successfully in subjects with mid-stage OA of the knee, leading to a Phase Ib study that is in progress.

Viral Gene Delivery in Chondrocytes.

Viral gene transfer, known as transduction, is a powerful research tool for studying the biology of chondrocytes in novel ways and also a technology enabling the use of gene therapy for regenerating cartilage and treating diseases that affect cartilage, such as osteoarthritis. Adenovirus, retrovirus, lentivirus, and adeno-associated virus (AAV) are most commonly used to transduce chondrocytes. Although AAV is able to transduce chondrocytes in situ by intra-articular injection, chondrocytes are most commonly transduced in monolayer culture using the four vectors mentioned above. Protocols for achieving this are described, along with a discussion of the variables that can influence transduction efficiency.

Transcriptomic changes during the replicative senescence of human articular chondrocytes.

Osteoarthritis (OA) is a degenerative joint disease and a leading cause of disability worldwide. Aging is a major risk factor for OA, but the specific mechanisms underlying this connection remain unclear. Although chondrocytes rarely divide in adult articular cartilage, they undergo replicative senescence in vitro which provides an opportunity to study changes related to aging under controlled laboratory conditions. In this pilot study, we performed bulk RNA sequencing on early- and late-passage human articular chondrocytes to identify transcriptomic changes associated with cellular aging. Chondrocytes were isolated from the articular cartilage of three donors, two with OA (age 70-80 years) and one with healthy cartilage (age 26 years). Chondrocytes were serially passaged until replicative senescence and RNA extracted from early- and late-passage cells. Principal component analysis of all genes showed clear separation between early- and late-passage chondrocytes, indicating substantial age-related differences in gene expression. Differentially expressed genes (DEGs) analysis confirmed distinct transcriptomic profiles between early- and late-passage chondrocytes. Hierarchical clustering revealed contrasting expression patterns between the two isolates from osteoarthritic samples and the healthy sample. Focused analysis of DEGs on transcripts associated with turnover of the extra-cellular matrix and the senescence-associated secretory phenotype (SASP) showed consistent downregulation of Col2A1 and ACAN, and upregulation of MMP19, ADAMTS4, and ADAMTS8 in late passage chondrocytes across all samples. SASP components including IL-1α, IL-1ß, IL-6, IL-7, p16INK4A (CDKN2A) and CCL2 demonstrated significant upregulation in late passage chondrocytes originally isolated from OA samples. Pathway analysis between sexes with OA revealed shared pathways such as extracellular matrix (ECM) organization, collagen formation, skeletal and muscle development, and nervous system development. Sex-specific differences were observed, with males showing distinctions in ECM organization, regulation of the cell cycle process as well as neuron differentiation. In contrast, females exhibited unique variations in the regulation of the cell cycle process, DNA metabolic process, and the PID-PLK1 pathway.

Efficient autocrine and paracrine signaling explain the osteogenic superiority of transgenic BMP-2 over rhBMP-2.

Bone morphogenetic protein-2 (BMP-2) is an osteogenic protein used clinically to enhance bone healing. However, it must be applied in very high doses, causing adverse side effects and increasing costs while providing only incremental benefit. Preclinical models of bone healing using gene transfer to deliver BMP-2 suggest that transgenic BMP-2 is much more osteogenic than rhBMP-2. Using a reporter mesenchymal cell line, we found transgenic human BMP-2 cDNA to be at least 100-fold more effective than rhBMP-2 in signaling. Moreover, a substantial portion of the BMP-2 produced by the transduced cells remained cell associated. Signaling by transgenic BMP-2 occurred via binding to the type I receptor, activating the associated kinase and generating phospho-smads. Signaling was partially resistant to noggin, an important extracellular inhibitor of BMP-2, possibly because nascent BMP-2 binds to its cell surface receptor during secretion and thus signals in a protected peri-cellular environment. Although the amounts of BMP-2 secreted by the transduced cells were too low to affect distant cells, transduced cells were able to induce signaling in a paracrine fashion that required close proximity of the cells, possibly cell-to-cell contact. The greater osteogenic potency of transgenic BMP-2 was confirmed with human bone marrow stromal cells.

Segmental defect healing in the presence or absence of recombinant human BMP2: Novel insights from a rat model.

This study defined and compared the course of native, impaired and growth factor-stimulated bone regeneration in a rat femoral defect model. A mid-diaphyseal defect with rigid internal fixation was surgically created in the right femur of male Fischer rats and serially analyzed over 36 weeks. Native bone regeneration was modeled using a sub-critical, 1 mm size defect, which healed uneventfully. Critical size defects of 5 mm were used to analyze impaired bone regeneration. In a third group, the 5 mm defects were filled with 11 µg of recombinant human bone morphogenetic protein 2 (rhBMP2) impregnated onto an absorbable collagen sponge, modeling its clinical use. Native bone regeneration was characterized by endochondral ossification with progressive remodeling to ultimately resemble intact femora. An endochondral response was also observed under conditions of impaired bone regeneration, but by week 8 medullary capping occurred with fibrofatty consolidation of the tissue within the defect, resembling an atrophic non-union. rhBMP2 treatment was associated with prolonged inflammatory cytokine expression and rapid intramembranous bone formation occurring with reduced expression of cartilage-associated collagens. Between weeks 4 and 36, rhBMP2-treated bones demonstrated decreased trabecular number and increased trabecular separation, which resulted in inferior mechanical properties compared with bones that healed naturally. Clinical Significance: Recombinant human bone morphogenetic protein 2 (rhBMP2) is used clinically to promote healing of long bones. Our data suggest that it drives intramembraneous ossification producing an inferior regenerate that deteriorates with time. Clinical outcomes would be improved by technologies favoring endochondral regenerative ossification.

Improvement of tendon repair using muscle grafts transduced with TGF-ß1 cDNA.

Tendon rupture is a common injury. Inadequate endogenous repair often leaves patients symptomatic, with tendons susceptible to re-rupture. Administration of certain growth factors improves tendon healing in animal models, but their delivery remains a challenge. Here we evaluated the delivery of TGF-ß1 to tendon defects by the implantation of genetically modified muscle grafts. Rat muscle biopsies were transduced with recombinant adenovirus encoding TGF-ß1 and grafted onto surgically transected Achilles tendons in recipient animals. Tissue regenerates were compared to those of controls by biomechanical testing as well as histochemical and immunohistochemical analyses. Healing was greatly accelerated when genetically modified grafts were implanted into tendon defects, with the resulting repair tissue gaining nearly normal histological appearance as early as 2 weeks postoperatively. This was associated with decreased deposition of type III collagen in favour of large fibre bundles indicative of type I collagen. These differences in tendon composition coincided with accelerated restoration of mechanical strength. Tendon thickness increased in gene-treated animals at weeks 1 and 2, but by week 8 became significantly lower than that of controls suggesting accelerated remodelling. Thus localised TGF-ß1 delivery via adenovirus-modified muscle grafts improved tendon healing in this rat model and holds promise for clinical application.

Effect of BMP-12, TGF-ß1 and autologous conditioned serum on growth factor expression in Achilles tendon healing.

PURPOSE: Achilles tendon ruptures are devastating and recover slowly and incompletely. There is a great demand for biomolecular therapies to improve recovery, yet little is understood about growth factors in a healing tendon. Here, the role of growth factors during tendon healing in a rat model and their reaction to single and multiple growth factor treatment are explored. METHODS: Rat tendons were transected surgically and resutured. The expression of bFGF, BMP-12, VEGF and TGF-ß1 was assessed by immunohistochemical analysis one to 8 weeks after surgery. Paracrine effects of TGF-ß1 or BMP-12 added by adenoviral transfer, as well as the effect of autologous conditioned serum (ACS) on growth factor expression, were evaluated. RESULTS: bFGF, BMP-12 and VEGF expression was highest 1 week after transection. bFGF and BMP-12 declined during the remaining period whereas VEGF expression persisted. TGF-ß1 expression dramatically increased after 8 weeks. ACS treatment increased bFGF (P = 0.007) and BMP-12 (P = 0.004) expression significantly after 8 weeks. Also overall expression of bFGF, BMP-12 and TGF-ß1 regardless of time point was significantly greater than controls with ACS treatment (P < 0.05). Both BMP-12 and TGF-ß1 treatments had no significant effect. No effect was observed in VEGF with any treatment. CONCLUSION: bFGF, BMP-12, VEGF and TGF-ß1 are differentially expressed during tendon healing. Additional BMP-12 or TGF-ß1 has no significant influence, whereas ACS generally increases expression of all factors except VEGF. Staged application of multiple growth factors may be the most promising biomolecular treatment.

Gene Therapy in Orthopaedics: Progress and Challenges in Pre-Clinical Development and Translation.

In orthopaedics, gene-based treatment approaches are being investigated for an array of common -yet medically challenging- pathologic conditions of the skeletal connective tissues and structures (bone, cartilage, ligament, tendon, joints, intervertebral discs etc.). As the skeletal system protects the vital organs and provides weight-bearing structural support, the various tissues are principally composed of dense extracellular matrix (ECM), often with minimal cellularity and vasculature. Due to their functional roles, composition, and distribution throughout the body the skeletal tissues are prone to traumatic injury, and/or structural failure from chronic inflammation and matrix degradation. Due to a mixture of environment and endogenous factors repair processes are often slow and fail to restore the native quality of the ECM and its function. In other cases, large-scale lesions from severe trauma or tumor surgery, exceed the body's healing and regenerative capacity. Although a wide range of exogenous gene products (proteins and RNAs) have the potential to enhance tissue repair/regeneration and inhibit degenerative disease their clinical use is hindered by the absence of practical methods for safe, effective delivery. Cumulatively, a large body of evidence demonstrates the capacity to transfer coding sequences for biologic agents to cells in the skeletal tissues to achieve prolonged delivery at functional levels to augment local repair or inhibit pathologic processes. With an eye toward clinical translation, we discuss the research progress in the primary injury and disease targets in orthopaedic gene therapy. Technical considerations important to the exploration and pre-clinical development are presented, with an emphasis on vector technologies and delivery strategies whose capacity to generate and sustain functional transgene expression in vivo is well-established.

Fracture hematoma micro-architecture influences transcriptional profile and plays a crucial role in determining bone healing outcomes.

The hematoma that forms between broken fragments of bone serves as a natural fibrin scaffold, and its removal from the defect site delays bone healing. The hypothesis of this study is that the microarchitectural and mechanical properties of the initially formed hematoma has a significant effect on the regulation of the biological process, which ultimately determines the outcome of bone healing. To mimic three healing conditions in the rat femur (normal, delayed, and non-healing bone defects), three different defect sizes of 0.5, 1.5, and 5.0 mm, are respectively used. The analysis of 3-day-old hematomas demonstrates clear differences in fibrin clot micro-architecture in terms of fiber diameter, fiber density, and porosity of the formed fibrin network, which result in different mechanical properties (stiffness) of the hematoma in each model. Those differences directly affect the biological processes involved. Specifically, RNA-sequencing reveals almost 700 differentially expressed genes between normally healing and non-healing defects, including significantly up-regulated essential osteogenic genes in normally healing defects, also differences in immune cell populations, activated osteogenic transcriptional regulators as well as potential novel marker genes. Most importantly, this study demonstrates that the healing outcome has already been determined during the hematoma phase of bone healing, three days post-surgery.