Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts.

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting the musculoskeletal system. These tissues have a strictly arranged collagen type I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGFß is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Sclerostin modulates mineralization degree and stiffness profile in the fibrocartilaginous enthesis for mechanical tissue integrity.

Fibrocartilaginous entheses consist of tendons, unmineralized and mineralized fibrocartilage, and subchondral bone, each exhibiting varying stiffness. Here we examined the functional role of sclerostin, expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of unmineralized fibrocartilage and concurrent replacement of epiphyseal hyaline cartilage by bone, unmineralized fibrocartilage reexpanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin at the base of mineralized fibrocartilage adjacent to subchondral bone. In Scx-deficient mice with less mechanical loading due to defects of the Achilles tendon, sclerostin+ fibrochondrocyte count significantly decreased in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, encoding sclerostin, elevated mineral density in mineralized zones of fibrocartilaginous entheses. Atomic force microscopy analysis revealed increased fibrocartilage stiffness. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of fibrocartilaginous entheses.

A Narrative Review of the Roles of Chondromodulin-I (Cnmd) in Adult Cartilage Tissue.

Articular cartilage is crucial for joint function but its avascularity limits intrinsic repair, leading to conditions like osteoarthritis (OA). Chondromodulin-I (Cnmd) has emerged as a key molecule in cartilage biology, with potential implications for OA therapy. Cnmd is primarily expressed in cartilage and plays an important role in chondrocyte proliferation, cartilage homeostasis, and the blocking of angiogenesis. In vivo and in vitro studies on Cnmd, also suggest an involvement in bone repair and in delaying OA progression. Its downregulation correlates with OA severity, indicating its potential as a therapeutic target. Further research is needed to fully understand the mode of action of Cnmd and its beneficial implications for managing OA. This comprehensive review aims to elucidate the molecular characteristics of Cnmd, from its expression pattern, role in cartilage maintenance, callus formation during bone repair and association with OA.

Corrigendum to "Development of a highly concentrated collagen ink for the creation of a 3D printed meniscus".

[This corrects the article DOI: 10.1016/j.heliyon.2023.e23107.].

Modeling Myeloma Dissemination In Vitro with hMSC-interacting Subpopulations of INA-6 Cells and Their Aggregation/Detachment Dynamics.

Multiple myeloma involves early dissemination of malignant plasma cells across the bone marrow; however, the initial steps of dissemination remain unclear. Human bone marrow-derived mesenchymal stromal cells (hMSC) stimulate myeloma cell expansion (e.g., IL6) and simultaneously retain myeloma cells via chemokines (e.g., CXCL12) and adhesion factors. Hence, we hypothesized that the imbalance between cell division and retention drives dissemination. We present an in vitro model using primary hMSCs cocultured with INA-6 myeloma cells. Time-lapse microscopy revealed proliferation and attachment/detachment dynamics. Separation techniques (V-well adhesion assay and well plate sandwich centrifugation) were established to isolate MSC-interacting myeloma subpopulations that were characterized by RNA sequencing, cell viability, and apoptosis. Results were correlated with gene expression data (n = 837) and survival of patients with myeloma (n = 536). On dispersed hMSCs, INA-6 saturate hMSC surface before proliferating into large homotypic aggregates, from which single cells detached completely. On confluent hMSCs, aggregates were replaced by strong heterotypic hMSC-INA-6 interactions, which modulated apoptosis time dependently. Only INA-6 daughter cells (nMA-INA6) detached from hMSCs by cell division but sustained adherence to hMSC-adhering mother cells (MA-INA6). Isolated nMA-INA6 indicated hMSC autonomy through superior viability after IL6 withdrawal and upregulation of proliferation-related genes. MA-INA6 upregulated adhesion and retention factors (CXCL12), that, intriguingly, were highly expressed in myeloma samples from patients with longer overall and progression-free survival, but their expression decreased in relapsed myeloma samples. Altogether, in vitro dissemination of INA-6 is driven by detaching daughter cells after a cycle of hMSC-(re)attachment and proliferation, involving adhesion factors that represent a bone marrow-retentive phenotype with potential clinical relevance. SIGNIFICANCE: Novel methods describe in vitro dissemination of myeloma cells as detachment of daughter cells after cell division. Myeloma adhesion genes were identified that counteract in vitro detachment with potential clinical relevance.

Development of a highly concentrated collagen ink for the creation of a 3D printed meniscus.

The most prevalent extracellular matrix (ECM) protein in the meniscus is collagen, which controls cell activity and aids in preserving the biological and structural integrity of the ECM. To create stable and high-precision 3D printed collagen scaffolds, ink formulations must possess good printability and cytocompatibility. This study aims to overlap the limitation in the 3D printing of pure collagen, and to develop a highly concentrated collagen ink for meniscus fabrication. The extrusion test revealed that 12.5 % collagen ink had the best combination of high collagen concentration and printability. The ink was specifically designed to have load-bearing capacity upon printing and characterized with respect to rheological and extrusion properties. Following printing of structures with different infill, a series of post-processing steps, including salt stabilization, pH shifting, washing, freeze-drying, crosslinking and sterilization were performed, and optimised to maintain the stability of the engineered construct. Mechanical testing highlighted a storage modulus of 70 kPa for the lower porous structure while swelling properties showed swelling ratio between 9 and 11 after 15 min of soaking. Moreover, human avascular and vascular meniscus cells cultured on the scaffolds deposited a meniscus-like matrix containing collagen I, II and glycosaminoglycans after 28 days of culture. Finally, as proof-of-concept, human size 3D printed meniscus scaffold were created.

Dynamics of single cell femtosecond laser printing.

In the present study, we investigated the dynamics of a femtosecond (fs) laser induced bio-printing with cell-free and cell-laden jets under the variation of laser pulse energy and focus depth, by using time-resolved imaging. By increasing the laser pulse energy or decreasing the focus depth thresholds for a first and second jet are exceeded and more laser pulse energy is converted to kinetic jet energy. With increasing jet velocity, the jet behavior changes from a well-defined laminar jet, to a curved jet and further to an undesired splashing jet. We quantified the observed jet forms with the dimensionless hydrodynamic Weber and Rayleigh numbers and identified the Rayleigh breakup regime as the preferred process window for single cell bioprinting. Herein, the best spatial printing resolution of 42 ± 3 µm and single cell positioning precision of 12.4 µm are reached, which is less than one single cell diameter about 15 µm.

Tendon Aging.

Tendons are mechanosensitive connective tissues responsible for the connection between muscles and bones by transmitting forces that allow the movement of the body, yet, with advancing age, tendons become more prone to degeneration followed by injuries. Tendon diseases are one of the main causes of incapacity worldwide, leading to changes in tendon composition, structure, and biomechanical properties, as well as a decline in regenerative potential. There is still a great lack of knowledge regarding tendon cellular and molecular biology, interplay between biochemistry and biomechanics, and the complex pathomechanisms involved in tendon diseases. Consequently, this reflects a huge need for basic and clinical research to better elucidate the nature of healthy tendon tissue and also tendon aging process and associated diseases. This chapter concisely describes the effects that the aging process has on tendons at the tissue, cellular, and molecular levels and briefly reviews potential biological predictors of tendon aging. Recent research findings that are herein reviewed and discussed might contribute to the development of precision tendon therapies targeting the elderly population.

Dexamethasone Is Not Sufficient to Facilitate Tenogenic Differentiation of Dermal Fibroblasts in a 3D Organoid Model.

Self-assembling three-dimensional organoids that do not rely on an exogenous scaffold but maintain their native cell-to-cell and cell-to-matrix interactions represent a promising model in the field of tendon tissue engineering. We have identified dermal fibroblasts (DFs) as a potential cell type for generating functional tendon-like tissue. The glucocorticoid dexamethasone (DEX) has been shown to regulate cell proliferation and facilitate differentiation towards other mesenchymal lineages. Therefore, we hypothesized that the administration of DEX could reduce excessive DF proliferation and thus, facilitate the tenogenic differentiation of DFs using a previously established 3D organoid model combined with dose-dependent application of DEX. Interestingly, the results demonstrated that DEX, in all tested concentrations, was not sufficient to notably induce the tenogenic differentiation of human DFs and DEX-treated organoids did not have clear advantages over untreated control organoids. Moreover, high concentrations of DEX exerted a negative impact on the organoid phenotype. Nevertheless, the expression profile of tendon-related genes of untreated and 10 nM DEX-treated DF organoids was largely comparable to organoids formed by tendon-derived cells, which is encouraging for further investigations on utilizing DFs for tendon tissue engineering.

Physioxic Culture of Chondrogenic Cells.

Cartilage resides under a low oxygen tension within articulating joints. The oxygen tension within cartilage of the knee joint has been measured to be between 2% and 5% oxygen. Although the literature has historically termed this level of oxygen as hypoxia, particularly when doing experiments in vitro in this range, this is actually the physiological oxygen tension experienced in vivo and is more accurately termed physioxia. In general, culture of chondrogenic cells under physioxia has demonstrated a donor-dependent beneficial effect on chondrogenesis, with an upregulation in cartilage genes (SOX9, COL2A1, ACAN) and matrix deposition (sulfated glycosaminoglycans (sGAGs), collagen II). Physioxia also reduces the expression of hypertrophic markers (COL10A1, MMP13). This chapter will outline the methods for the expansion and differentiation of chondrogenic cells under physioxia using oxygen-controlled incubators and glove box environments, with the typical assays used for qualitative and quantitative assessment of chondrogenesis.

Practical Relevance of Institutional Guidelines in Translational Large Animal Studies of Cartilage Repair-A Multidisciplinary Survey.

Background and Objective: Translational large animal models are inevitable to transfer cartilage repair methods into clinical practice. Guidelines for these trials have been published by guiding agencies (FDA, ASTM, EMEA) including recommendations for study descriptors and study outcomes. However, practical adherence to these recommendations is not achieved in all aspects. This study includes an assessment of the recommended aspects regarding practical relevance in large animal models for cartilage repair by professionals in the field. Materials and Methods: In an online based survey, 11 aspects regarding study design and 13 aspects regarding study outcome from previously published guidelines were evaluated (0-10 points, with 10 being most important) by study participants. Additionally, the survey contained questions related to professional experience (years), professional focus (preclinical, clinical, veterinarian, industry) and the preferred translational large animal model for cartilage repair. Results: The total number of survey participants was 37. Rated as most important for study design parameters was lesion size (9.54 pts., SD 0.80) followed by study duration (9.43 pts., SD 1.21); and method of scaffold fixation (9.08 pts., SD 1.30) as well as depth of the lesion (9.03 pts., SD 1.77). The most important aspects of study outcome were considered histology (9.41 pts., SD 0.86) and defect filling (8.97 pts., SD 1.21), while gene expression was judged as the least important (6.11 pts., SD 2.46) outcome. A total of 62.2% of all participants were researchers, 18.9% clinicians, 13.5% veterinarians and 5.4% industry employees. Conclusions: In translational research, recommendations published by guiding agencies receive broad theoretical consensus within the community, including both clinically and preclinically orientated scientists. However, implementation into practical research lacks in major aspects. Ongoing re-evaluation of the guidelines under involvement of all stakeholders and approaches to overcome financial and infrastructural limitations could support the acceptance of the guidance documents and contribute to standardization in the field.

Tendon healing: a concise review on cellular and molecular mechanisms with a particular focus on the Achilles tendon.

Tendon is a bradytrophic and hypovascular tissue, hence, healing remains a major challenge. The molecular key events involved in successful repair have to be unravelled to develop novel strategies that reduce the risk of unfavourable outcomes such as non-healing, adhesion formation, and scarring. This review will consider the diverse pathophysiological features of tendon-derived cells that lead to failed healing, including misrouted differentiation (e.g. de- or transdifferentiation) and premature cell senescence, as well as the loss of functional progenitors. Many of these features can be attributed to disturbed cell-extracellular matrix (ECM) or unbalanced soluble mediators involving not only resident tendon cells, but also the cross-talk with immigrating immune cell populations. Unrestrained post-traumatic inflammation could hinder successful healing. Pro-angiogenic mediators trigger hypervascularization and lead to persistence of an immature repair tissue, which does not provide sufficient mechano-competence. Tendon repair tissue needs to achieve an ECM composition, structure, strength, and stiffness that resembles the undamaged highly hierarchically ordered tendon ECM. Adequate mechano-sensation and -transduction by tendon cells orchestrate ECM synthesis, stabilization by cross-linking, and remodelling as a prerequisite for the adaptation to the increased mechanical challenges during healing. Lastly, this review will discuss, from the cell biological point of view, possible optimization strategies for augmenting Achilles tendon (AT) healing outcomes, including adapted mechanostimulation and novel approaches by restraining neoangiogenesis, modifying stem cell niche parameters, tissue engineering, the modulation of the inflammatory cells, and the application of stimulatory factors.Cite this article: Bone Joint Res 2022;11(8):561-574.

Evolution of implants and advancements for osseointegration: A narrative review.

Since ancient times, reduction and internal fixation has been applied to restore skeletal integrity. Despite advances in the understanding of fracture healing, the risk of complication such as implant loosening or implant-related infection still depicts a challenging complication. Nowadays, a great deal of research is devoted to unreveal the impact of implant surface modifications on osteogenic processes to enhance bone consolidation and osseointegration. This narrative review is aimed to (1) show the evolution and already achieved milestones of implant optimization, and (2) to outline the key factors that contribute to an enhanced osseointegration. Different physical and chemical roughening techniques are currently applied in various studies. Surface patterning on the nanoscale has been found to be an essential factor for the biological response, achievable by e.g. anodisation or laser texturing. Besides surface roughening, also different coating methods are vastly investigated. Next to metal or inorganic compounds as coating material, a variety of biomolecules is currently studied for their osteosupportive capacities. Osseointegration can be improved by surface modification on the micro and nanoscale. Bioactive agents can further improve the osseointegration potential. Used agents at the moment are e.g. inorganic compounds, growth factors (BMPs and non-BMPs) and antiresorptive drugs. The advancement in research on new implant generations therefore aims at actively supporting osseointegration processing.

Bone regeneration after marginal bone resection in two-stage treatment of chronic long bone infection - a combined histopathological and clinical pilot study.

INTRODUCTION: In chronic bone infection, marginal bone resection avoids large and difficult to reconstruct bone defects. However, there is still a lack of knowledge on bone regeneration during chronic bone infection and bone healing capability after marginal bone resection. Therefore, the purpose of this study was to investigate the clinical and histopathological outcomes after marginal bone resection in chronic long bone infection. We hypothesized that there is a regenerative bone healing potential after marginal bone resection that results in an acceptable clinical outcome and improved pathohistological bone healing parameters during treatment. MATERIALS AND METHODS: Nine patients were treated for chronic bone infections in a two-stage manner with marginal bone resection of the infected area and the placement of an antibiotic-loaded polymethyl methacrylate (PMMA) spacer at stage one followed by bone reconstruction at stage two combined with systemic antibiotic therapy. Comparable bone samples were harvested at the border region between vital and necrotic bone area during stage one and the identical location during stage two. Control bone samples were harvested from five healthy patients without bone infection. Clinical outcome in terms of infection eradication and bone consolidation were assessed. The phenotypic changes of osteocyte and morphological changes of lacunar-canalicular network were investigated by histological and immunohistochemical staining between the two observation periods. Furthermore, expression levels of major bone formation and resorption markers were investigated by immunohistochemical and tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: The clinical results with a follow-up of 12.9 months showed that eight of nine patients (88.9%) achieved bone consolidation after a planned two-stage procedure of marginal resection of necrotic bone and consecutive reconstruction. In four of the nine patients (44.4%), additional marginal debridements after stage two had to be performed. After marginal resection at stage one, the improved bone formation ability at stage two was demonstrated by significantly lower percentage of empty lacunae, significantly more mature osteocytes and higher BMP-2 positive cell density, whereas decreased resorption was indicated by significantly lower osteoclast density and RANKL/OPG ratio. In patients requiring additional debridement compared to patients without additional debridements, a significantly higher percentage of empty lacunae was found at stage one. CONCLUSION: Marginal bone resection combined with local and systemic antibiotic therapy is a feasible treatment option to avoid large bone defects as bone from the marginal resection area seems to have good regenerative potential. Despite a high revision rate of 44.4%, this technique avoids large bone resection and revisions can be done by further marginal debridements.

Bioengineered 3D Living Fibers as In Vitro Human Tissue Models of Tendon Physiology and Pathology.

Clinically relevant in vitro models of human tissue's health and disease are urgently needed for a better understanding of biological mechanisms essential for the development of novel therapies. Herein, physiological (healthy) and pathological (disease) tendon states are bioengineered by coupling the biological signaling of platelet lysate components with controlled 3D architectures of electrospun microfibers to drive the fate of human tendon cells in different composite living fibers (CLFs). In the CLFs-healthy model, tendon cells adopt a high cytoskeleton alignment and elongation, express tendon-related markers (scleraxis, tenomodulin, and mohawk) and deposit a dense tenogenic matrix. In contrast, cell crowding with low preferential orientation, high matrix deposition, and phenotypic drift leading to increased expression of nontendon related and fibrotic markers, are characteristics of the CLFs-diseased model. This diseased-like profile, also reflected in the increase of COL3/COL1 ratio, is further evident by the imbalance between matrix remodeling and degradation effectors, characteristic of tendinopathy. In summary, microengineered 3D in vitro models of human tendon healthy and diseased states are successfully fabricated. Most importantly, these innovative and versatile microphysiological models offer major advantages over currently used systems, holding promise for drugs screening and development of new therapies.

Rifampicin restores extracellular organic matrix formation and mineralization of osteoblasts after intracellular Staphylococcus aureus infection.

AIMS: Bone regeneration during treatment of staphylococcal bone infection is challenging due to the ability of Staphylococcus aureus to invade and persist within osteoblasts. Here, we sought to determine whether the metabolic and extracellular organic matrix formation and mineralization ability of S. aureus-infected human osteoblasts can be restored after rifampicin (RMP) therapy. METHODS: The human osteoblast-like Saos-2 cells infected with S. aureus EDCC 5055 strain and treated with 8 µg/ml RMP underwent osteogenic stimulation for up to 21 days. Test groups were Saos-2 cells + S. aureus and Saos-2 cells + S. aureus + 8 µg/ml RMP, and control groups were uninfected untreated Saos-2 cells and uninfected Saos-2 cells + 8 µg/ml RMP. RESULTS: The S. aureus-infected osteoblasts showed a significant number of intracellular bacteria colonies and an unusual higher metabolic activity (p < 0.005) compared to uninfected osteoblasts. Treatment with 8 µg/ml RMP significantly eradicated intracellular bacteria and the metabolic activity was comparable to uninfected groups. The RMP-treated infected osteoblasts revealed a significantly reduced amount of mineralized extracellular matrix (ECM) at seven days osteogenesis relative to uninfected untreated osteoblasts (p = 0.007). Prolonged osteogenesis and RMP treatment at 21 days significantly improved the ECM mineralization level. Ultrastructural images of the mineralized RMP-treated infected osteoblasts revealed viable osteoblasts and densely distributed calcium crystal deposits within the extracellular organic matrix. The expression levels of prominent bone formation genes were comparable to the RMP-treated uninfected osteoblasts. CONCLUSION: Intracellular S. aureus infection impaired osteoblast metabolism and function. However, treatment with low dosage of RMP eradicated the intracellular S. aureus, enabling extracellular organic matrix formation and mineralization of osteoblasts at later stage. Cite this article: Bone Joint Res 2022;11(5):327-341.

Implant-bone-interface: Reviewing the impact of titanium surface modifications on osteogenic processes in vitro and in vivo.

Titanium is commonly and successfully used in dental and orthopedic implants. However, patients still have to face the risk of implant failure due to various reasons, such as implant loosening or infection. The risk of implant loosening can be countered by optimizing the osteointegration capacity of implant materials. Implant surface modifications for structuring, roughening and biological activation in favor for osteogenic differentiation have been vastly studied. A key factor for a successful stable long-term integration is the initial cellular response to the implant material. Hence, cell-material interactions, which are dependent on the surface parameters, need to be considered in the implant design. Therefore, this review starts with an introduction to the basics of cell-material interactions as well as common surface modification techniques. Afterwards, recent research on the impact of osteogenic processes in vitro and vivo provoked by various surface modifications is reviewed and discussed, in order to give an update on currently applied and developing implant modification techniques for enhancing osteointegration.

Cell-based treatment options facilitate regeneration of cartilage, ligaments and meniscus in demanding conditions of the knee by a whole joint approach.

PURPOSE: This article provides an update on the current therapeutic options for cell-based regenerative treatment of the knee with a critical review of the present literature including a future perspective on the use of regenerative cell-based approaches. Special emphasis has been given on the requirement of a whole joint approach with treatment of comorbidities with aim of knee cartilage restoration, particularly in demanding conditions like early osteoarthritis. METHODS: This narrative review evaluates recent clinical data and published research articles on cell-based regenerative treatment options for cartilage and other structures around the knee RESULTS: Cell-based regenerative therapies for cartilage repair have become standard practice for the treatment of focal, traumatic chondral defects of the knee. Specifically, matrix-assisted autologous chondrocyte transplantation (MACT) shows satisfactory long-term results regarding radiological, histological and clinical outcome for treatment of large cartilage defects. Data show that regenerative treatment of the knee requires a whole joint approach by addressing all comorbidities including axis deviation, instability or meniscus pathologies. Further development of novel biomaterials and the discovery of alternative cell sources may facilitate the process of cell-based regenerative therapies for all knee structures becoming the gold standard in the future. CONCLUSION: Overall, cell-based regenerative cartilage therapy of the knee has shown tremendous development over the last years and has become the standard of care for large and isolated chondral defects. It has shown success in the treatment of traumatic, osteochondral defects but also for degenerative cartilage lesions in the demanding condition of early OA. Future developments and alternative cell sources may help to facilitate cell-based regenerative treatment for all different structures around the knee by a whole joint approach. LEVEL OF EVIDENCE: IV.

Tenomodulin knockout mice exhibit worse late healing outcomes with augmented trauma-induced heterotopic ossification of Achilles tendon.

Heterotopic ossification (HO) represents a common problem after tendon injury with no effective treatment yet being developed. Tenomodulin (Tnmd), the best-known mature marker for tendon lineage cells, has important effects in tendon tissue aging and function. We have reported that loss of Tnmd leads to inferior early tendon repair characterized by fibrovascular scaring and therefore hypothesized that its lack will persistently cause deficient repair during later stages. Tnmd knockout (Tnmd-/-) and wild-type (WT) animals were subjected to complete Achilles tendon surgical transection followed by end-to-end suture. Lineage tracing revealed a reduction in tendon-lineage cells marked by ScleraxisGFP, but an increase in alpha smooth muscle actin myofibroblasts in Tnmd-/- tendon scars. At the proliferative stage, more pro-inflammatory M1 macrophages and larger collagen II cartilaginous template were detected in this group. At the remodeling stage, histological scoring revealed lower repair quality in the injured Tnmd-/- tendons, which was coupled with higher HO quantified by micro-CT. Tendon biomechanical properties were compromised in both groups upon injury, however we identified an abnormal stiffening of non-injured Tnmd-/- tendons, which possessed higher static and dynamic E-moduli. Pathologically thicker and abnormally shaped collagen fibrils were observed by TEM in Tnmd-/- tendons and this, together with augmented HO, resulted in diminished running capacity of Tnmd-/- mice. These novel findings demonstrate that Tnmd plays a protecting role against trauma-induced endochondral HO and can inspire the generation of novel therapeutics to accelerate repair.