Chronic psychosocial stress compromises the immune response and endochondral ossification during bone fracture healing via ß-AR signaling.

Chronic psychosocial stress/trauma represents an increasing burden in our modern society and a risk factor for the development of mental disorders, including posttraumatic stress disorder (PTSD). PTSD, in turn, is highly comorbid with a plethora of inflammatory disorders and has been associated with increased bone fracture risk. Since a balanced inflammatory response after fracture is crucial for successful bone healing, we hypothesize that stress/trauma alters the inflammatory response after fracture and, consequently, compromises fracture healing. Here we show, employing the chronic subordinate colony housing (CSC) paradigm as a clinically relevant mouse model for PTSD, that mice subjected to CSC displayed increased numbers of neutrophils in the early fracture hematoma, whereas T lymphocytes and markers for cartilage-to-bone transition and angiogenesis were reduced. At late stages of fracture healing, CSC mice were characterized by decreased bending stiffness and bony bridging of the fracture callus. Strikingly, a single systemic administration of the ß-adrenoreceptor (AR) blocker propranolol before femur osteotomy prevented bone marrow mobilization of neutrophils and invasion of neutrophils into the fracture hematoma, both seen in the early phase after fracture, as well as a compromised fracture healing in CSC mice. We conclude that chronic psychosocial stress leads to an imbalanced immune response after fracture via ß-AR signaling, accompanied by disturbed fracture healing. These findings offer possibilities for clinical translation in patients suffering from PTSD and fracture.

Wireless Measurements Using Electrical Impedance Spectroscopy to Monitor Fracture Healing.

There is an unmet need for improved, clinically relevant methods to longitudinally quantify bone healing during fracture care. Here we develop a smart bone plate to wirelessly monitor healing utilizing electrical impedance spectroscopy (EIS) to provide real-time data on tissue composition within the fracture callus. To validate our technology, we created a 1-mm rabbit tibial defect and fixed the bone with a standard veterinary plate modified with a custom-designed housing that included two impedance sensors capable of wireless transmission. Impedance magnitude and phase measurements were transmitted every 48 h for up to 10 weeks. Bone healing was assessed by X-ray, µCT, and histology. Our results indicated the sensors successfully incorporated into the fracture callus and did not impede repair. Electrical impedance, resistance, and reactance increased steadily from weeks 3 to 7-corresponding to the transition from hematoma to cartilage to bone within the fracture gap-then plateaued as the bone began to consolidate. These three electrical readings significantly correlated with traditional measurements of bone healing and successfully distinguished between union and not-healed fractures, with the strongest relationship found with impedance magnitude. These results suggest that our EIS smart bone plate can provide continuous and highly sensitive quantitative tissue measurements throughout the course of fracture healing to better guide personalized clinical care.

Cartilage to bone transformation during fracture healing is coordinated by the invading vasculature and induction of the core pluripotency genes.

Fractures heal predominantly through the process of endochondral ossification. The classic model of endochondral ossification holds that chondrocytes mature to hypertrophy, undergo apoptosis and new bone forms by invading osteoprogenitors. However, recent data demonstrate that chondrocytes transdifferentiate to osteoblasts in the growth plate and during regeneration, yet the mechanism(s) regulating this process remain unknown. Here, we show a spatially-dependent phenotypic overlap between hypertrophic chondrocytes and osteoblasts at the chondro-osseous border in the fracture callus, in a region we define as the transition zone (TZ). Hypertrophic chondrocytes in the TZ activate expression of the pluripotency factors [Sox2, Oct4 (Pou5f1), Nanog], and conditional knock-out of Sox2 during fracture healing results in reduction of the fracture callus and a delay in conversion of cartilage to bone. The signal(s) triggering expression of the pluripotency genes are unknown, but we demonstrate that endothelial cell conditioned medium upregulates these genes in ex vivo fracture cultures, supporting histological evidence that transdifferentiation occurs adjacent to the vasculature. Elucidating the cellular and molecular mechanisms underlying fracture repair is important for understanding why some fractures fail to heal and for developing novel therapeutic interventions.

Origin of Reparative Stem Cells in Fracture Healing.

PURPOSE OF REVIEW: The identity and functional roles of stem cell population(s) that contribute to fracture repair remains unclear. This review provides a brief history of mesenchymal stem cell (MSCs) and provides an updated view of the many stem/progenitor cell populations contributing to fracture repair. RECENT FINDINGS: Functional studies show MSCs are not the multipotential stem cell population that form cartilage and bone during fracture repair. Rather, multiple studies have confirmed the periosteum is the primary source of stem/progenitor cells for fracture repair. Newer work is also identifying other stem/progenitor cells that may also contribute to healing. Although the heterogenous periosteal cells migrate to the fracture site and contribute directly to callus formation, other cell populations are involved. Pericytes and bone marrow stromal cells are now thought of as key secretory centers that mostly coordinate the repair process. Other populations of stem/progenitor cells from the muscle and transdifferentiated chondroctyes may also contribute to repair, and their functional role is an area of active research.

Preclinical models of orthopaedic trauma: Orthopaedic Research Society (ORS) and Orthopaedic Trauma Association (OTA) symposium 2022.

Orthopaedic trauma remains a leading cause of patient morbidity, mortality, and global health care burden. Although significant advances have been made in the diagnosis, treatment, and rehabilitation of these injuries, complications such as malunion, nonunion, infection, disuse muscle atrophy and osteopenia, and incomplete return to baseline function still occur. The significant inherent clinical variability in fracture care such as differing patient demographics, injury patterns, and treatment protocols make standardized and replicable study, especially of cellular and molecular based mechanisms, nearly impossible. Hence, the scientists dedicated to improving therapy and treatments for patients with orthopaedic trauma rely on preclinical models. Preclinical models have proven to be invaluable in understanding the timing between implant insertion and bacterial inoculation on the bioburden of infection. Posttraumatic arthritis (PTOA) can take years to develop clinically, but with a porcine pilon fracture model, posttraumatic arthritis can be reliably induced, so different surgical and therapeutic strategies can be tested in prevention. Conversely, the racehorse presents a well-accepted model of naturally occurring PTOA. With preclinical polytrauma models focusing on chest injury, abdominal injury, multiple fractures, and/or head injury, one can study how various injury patterns affect fracture healing can be systemically studied. Finally, these preclinical models serve as a translational bridge to for clinical application in human patients. With selection of the right preclinical model, studies can build a platform to decrease the risk of emerging technologies and provide foundational support for therapeutic clinical trials. In summary, orthopaedic trauma preclinical models allow scientists to simplify a complex clinical challenge, to understand the basic pathways starting with lower vertebrate models. Then, R&D efforts progress to higher vertebrate models to build in more complexity for translation of findings to the clinical practice.

Evaluating the Morphology of Unique Superficial Fissured Cartilage Lesions at the Femoral Head-Neck Junction in Patients with Femoroacetabular Impingement Syndrome.

Background: While an association between femoroacetabular impingement (FAI) and osteoarthritis (OA) has been reported, the mechanistic differences and transition between the 2 conditions is not fully understood. In FAI, cartilage lesions at the femoral head-neck junction can sometimes be visualized during hip arthroscopy. Purpose/Hypothesis: The purpose of this study was to describe a unique dimpled pattern of superficial fissured cartilage lesions on the femoral head-neck junction at impingement site in patients with FAI syndrome (FAIS) and to evaluate the clinical, histological, and genetic phenotype of this cartilage. We hypothesized that the cartilage lesions may indicate risk for, or predict occurrence of, OA. Study Design: Controlled laboratory study. Methods: Six hips (6 patients; mean age, 34.2 ± 12.9 years; range, 19-54 years) with dimpled or fissured cartilage were included among patients who underwent hip arthroscopy for treatment of FAIS from October 2020 through December 2021. This affected cartilage (dimple-pattern group) and normal cartilage (control group) on the femoral head-neck junction were collected from the same patients and evaluated for histological quantification by Mankin scores and expression of proteins related to cartilage degeneration (eg, matrix metalloproteinase [MMP]-1, MMP-2, MMP-3, MMP-10, and MMP-12, tissue inhibitor of metalloproteinase [TIMP]-1 and TMP-2, aggrecan neopepitope CS846, and hyaluronic acid [HA]) with the use of Milliplex Multiplex Assays. Results: All 6 hips were of the mixed FAI subtype. Preoperatively, 4 of 6 hips had Tönnis grade 1 radiographic changes, which was associated with greater femoral head chondral damage visualized intraoperatively. Mankin scores for the normal cartilage group and the dimple-pattern group were 0.67 ± 0.82 and 3.3 ± 0.82, respectively. Dimple pattern fissured cartilage showed a significant increase in Mankin score (P = .031) and a significant increase in protein expression of CS846 (P = .031) compared with normal cartilage. There were no significant differences in MMPs, TIMPs, or HA levels between the 2 groups. Conclusion: The dimple pattern fissured cartilage, compared to normal cartilage, showed histologically significant cartilage degeneration and a significant increase in protein expression of CS846, a biomarker for early OA. Clinical Relevance: This lesion serves as helpful visual indicator of early degeneration of the cartilage of femoral head-neck junction caused by FAIS.

Murine Progeria Model Exhibits Delayed Fracture Healing with Dysregulated Local Immune Response.

Background: Bone fracture is one of the most globally prevalent injuries, with an estimated 189 million bone fractures occurring annually. Delayed union or nonunion occurs in up to 15% of fractures and involves the interruption or complete failure of bone continuity following fracture. Preclinical testing is essential to support the translation of novel strategies to promote improved fracture repair treatment, but there is a paucity of small animal models that recapitulate clinical attributes associated with delayed fracture healing. This study explores whether the Zmpste24 -/- (Z24 -/- ) knockout mouse model of Hutchinson-Gilford progeria syndrome presents with delayed fracture healing. Leveraging the previously characterized Z24 -/- phenotype of genomic instability, epigenetic changes, and fragility, we hypothesize that these underlying alterations will lead to significantly delayed fracture healing relative to age-matched wild type (WT) controls. Methods: WT and Z24 -/- mice received intramedullary fixed tibia fractures at ∼12 weeks of age. Mice were sacrificed throughout the time course of repair for the collection of organs that would provide information regarding the local (fracture callus, bone marrow, inguinal lymph nodes) versus peripheral (peripheral blood, contralateral tibia, abdominal organs) tissue microenvironments. Analyses of these specimens include histomorphometry, µCT, mechanical strength testing, protein quantification, gene expression analysis, flow cytometry for cellular senescence, and immunophenotyping. Results: Z24 -/- mice demonstrated a significantly delayed rate of healing compared to WT mice with consistently smaller fracture calli containing higher proportion of cartilage and less bone after injury. Cellular senescence and pro-inflammatory cytokines were elevated in the Z24 -/- mice before and after fracture. These mice further presented with a dysregulated immune system, exhibiting generally decreased lymphopoiesis and increased myelopoiesis locally in the bone marrow, with more naïve and less memory T cell but greater myeloid activation systemically in the peripheral blood. Surprisingly, the ipsilateral lymph nodes had increased T cell activation and other pro-inflammatory NK and myeloid cells, suggesting that elevated myeloid abundance and activation contributes to an injury-specific hyperactivation of T cells. Conclusion: Taken together, these data establish the Z24 -/- progeria mouse as a model of delayed fracture healing that exhibits decreased bone in the fracture callus, with weaker overall bone quality, immune dysregulation, and increased cellular senescence. Based on this mechanism for delayed healing, we propose this Z24 -/- progeria mouse model could be useful in testing novel therapeutics that could address delayed healing. The Translational Potential of this Article: This study employs a novel animal model for delayed fracture healing that researchers can use to screen fracture healing therapeutics to address the globally prevalent issue of aberrant fracture healing.

Clinical validation of C12FDG as a marker associated with senescence and osteoarthritic phenotypes.

Chronic conditions associated with aging have proven difficult to prevent or treat. Senescence is a cell fate defined by loss of proliferative capacity and the development of a pro-inflammatory senescence-associated secretory phenotype comprised of cytokines/chemokines, proteases, and other factors that promotes age-related diseases. Specifically, an increase in senescent peripheral blood mononuclear cells (PBMCs), including T cells, is associated with conditions like frailty, rheumatoid arthritis, and bone loss. However, it is unknown if the percentage of senescent PBMCs associated with age-associated orthopedic decline could be used for potential diagnostic or prognostic use in orthopedics. Here, we report senescent cell detection using the fluorescent compound C12FDG to quantify PBMCs senescence across a large cohort of healthy and osteoarthritic patients. There is an increase in the percent of circulating C12FDG+ PBMCs that is commensurate with increases in age and senescence-related serum biomarkers. Interestingly, C12FDG+ PBMCs and T cells also were found to be elevated in patients with mild to moderate osteoarthritis, a progressive joint disease that is strongly associated with inflammation. The percent of C12FDG+ PBMCs and age-related serum biomarkers were decreased in a small subgroup of study participants taking the senolytic drug fisetin. These results demonstrate quantifiable measurements in a large group of participants that could create a composite score of healthy aging sensitive enough to detect changes following senolytic therapy and may predict age-related orthopedic decline. Detection of peripheral senescence in PBMCs and subsets using C12FDG may be clinically useful for quantifying cellular senescence and determining how and if it plays a pathological role in osteoarthritic progression.

Cellular expansion of MSCs: Shifting the regenerative potential.

Mesenchymal-derived stromal or progenitor cells, commonly called "MSCs," have attracted significant clinical interest for their remarkable abilities to promote tissue regeneration and reduce inflammation. Recent studies have shown that MSCs' therapeutic effects, originally attributed to the cells' direct differentiation capacity into the tissue of interest, are largely driven by the biomolecules the cells secrete, including cytokines, chemokines, growth factors, and extracellular vesicles containing miRNA. This secretome coordinates upregulation of endogenous repair and immunomodulation in the local microenvironment through crosstalk of MSCs with host tissue cells. Therapeutic applications for MSCs and their secretome-derived products often involve in vitro monolayer expansion. However, consecutive passaging of MSCs significantly alters their therapeutic potential, inducing a broad shift from a pro-regenerative to a pro-inflammatory phenotype. A consistent by-product of in vitro expansion of MSCs is the onset of replicative senescence, a state of cell arrest characterized by an increased release of proinflammatory cytokines and growth factors. However, little is known about changes in the secretome profile at different stages of in vitro expansion. Some culture conditions and bioprocessing techniques have shown promise in more effectively retaining the pro-regenerative and anti-inflammatory MSC phenotype throughout expansion. Understanding how in vitro expansion conditions influence the nature and function of MSCs, and their associated secretome, may provide key insights into the underlying mechanisms driving these alterations. Elucidating the dynamic and diverse changes in the MSC secretome at each stage of in vitro expansion is a critical next step in the development of standardized, safe, and effective MSC-based therapies.

Mechanical strain drives exosome production, function, and miRNA cargo in C2C12 muscle progenitor cells.

Mesenchymal stem cells (MSCs) have been proven to promote tissue repair. However, concerns related to their clinical application and regulatory hurdles remain. Recent data has demonstrated the proregenerative secretome of MSCs can result in similar effects in the absence of the cells themselves. Within the secretome, exosomes have emerged as a promising regenerative component. Exosomes, which are nanosized lipid vesicles secreted by cells, encapsulate micro-RNA (miRNA), RNA, and proteins that drive MSCs regenerative potential with cell specific content. As such, there is an opportunity to optimize the regenerative potential of MSCs, and thus their secreted exosome fraction, to improve clinical efficacy. Exercise is one factor that has been shown to improve muscle progenitor cell function and regenerative potential. However, the effect of exercise on MSC exosome content and function is still unclear. To address this, we used an in vitro culture system to evaluate the effects of mechanical strain, an exercise mimetic, on C2C12 (muscle progenitor cell) exosome production and proregenerative function. Our results indicate that the total exosome production is increased by mechanical strain and can be regulated with different tensile loading regimens. Furthermore, we found that exosomes from mechanically stimulated cells increase proliferation and myogenic differentiation of naïve C2C12 cells. Lastly, we show that exosomal miRNA cargo is differentially expressed following strain. Gene ontology mapping suggests positive regulation of bone morphogenetic protein signaling, regulation of actin-filament-based processes, and muscle cell apoptosis may be at least partially responsible for the proregenerative effects of exosomes from mechanically stimulated C2C12 muscle progenitor cells.

Sustained-release losartan from peptide nanofibers promotes chondrogenesis.

Introduction: The central pathologic feature of osteoarthritis (OA) is the progressive loss of articular cartilage, which has a limited regenerative capacity. The TGF-ß1 inhibitor, losartan, can improve cartilage repair by promoting hyaline rather that fibrous cartilage tissue regeneration. However, there are concerns about side effects associated with oral administration and short retention within the joint following intra-articular injections. To facilitate local and sustained intra-articular losartan delivery we have designed an injectable peptide amphiphile (PA) nanofiber that binds losartan. The aims of this study are to characterize the release kinetics of losartan from two different PA nanofiber compositions followed by testing pro-regenerative bioactivity on chondrocytes. Methods: We tested the impact of electrostatic interactions on nanostructure morphology and release kinetics of the negatively charged losartan molecule from either a positively or negatively charged PA nanofiber. Subsequently, cytotoxicity and bioactivity were evaluated in vitro in both normal and an IL-1ß-induced OA chondrocyte model using ATDC5. Results: Both nanofiber systems promoted cell proliferation but that the positively-charged nanofibers also significantly increased glycosaminoglycans production. Furthermore, gene expression analysis suggested that losartan-encapsulated nanofibers had significant anti-inflammatory, anti-degenerative, and cartilage regenerative effects by significantly blocking TGF-ß1 in this in vitro system. Discussion: The results of this study demonstrated that positively charged losartan sustained-release nanofibers may be a novel and useful treatment for cartilage regeneration and OA by blocking TGF-ß1.

Encapsulation of ß-NGF in injectable microrods for localized delivery accelerates endochondral fracture repair.

Introduction: Currently, there are no non-surgical FDA-approved biological approaches to accelerate fracture repair. Injectable therapies designed to stimulate bone healing represent an exciting alternative to surgically implanted biologics, however, the translation of effective osteoinductive therapies remains challenging due to the need for safe and effective drug delivery. Hydrogel-based microparticle platforms may be a clinically relevant solution to create controlled and localized drug delivery to treat bone fractures. Here, we describe poly (ethylene glycol) dimethacrylate (PEGDMA)-based microparticles, in the shape of microrods, loaded with beta nerve growth factor (ß-NGF) for the purpose of promoting fracture repair. Methods: Herein, PEGDMA microrods were fabricated through photolithography. PEGDMA microrods were loaded with ß-NGF and in vitro release was examined. Subsequently, bioactivity assays were evaluated in vitro using the TF-1 tyrosine receptor kinase A (Trk-A) expressing cell line. Finally, in vivo studies using our well-established murine tibia fracture model were performed and a single injection of the ß-NGF loaded PEGDMA microrods, non-loaded PEGDMA microrods, or soluble ß-NGF was administered to assess the extent of fracture healing using Micro-computed tomography (µCT) and histomorphometry. Results: In vitro release studies showed there is significant retention of protein within the polymer matrix over 168 hours through physiochemical interactions. Bioactivity of protein post-loading was confirmed with the TF-1 cell line. In vivo studies using our murine tibia fracture model show that PEGDMA microrods injected at the site of fracture remained adjacent to the callus for over 7 days. Importantly, a single injection of ß-NGF loaded PEGDMA microrods resulted in improved fracture healing as indicated by a significant increase in the percent bone in the fracture callus, trabecular connective density, and bone mineral density relative to soluble ß-NGF control indicating improved drug retention within the tissue. The concomitant decrease in cartilage fraction supports our prior work showing that ß-NGF promotes endochondral conversion of cartilage to bone to accelerate healing. Discussion: We demonstrate a novel and translational method wherein ß-NGF can be encapsulated within PEGDMA microrods for local delivery and that ß-NGF bioactivity is maintained resulting in improved bone fracture repair.

A bioresponsive hydrogel tuned to chondrogenesis of human mesenchymal stem cells.

Cartilage tissue engineering aims to replace damaged or diseased tissue with a functional regenerate that restores joint function. Scaffolds are used to deliver cells and facilitate tissue development, but they can also interfere with the structural assembly of the cartilage matrix. Biodegradable scaffolds have been proposed as a means to improve matrix deposition and the biomechanical properties of neocartilage. The challenge is designing scaffolds with appropriate degradation rates, ideally such that scaffold degradation is proportional to matrix deposition. In this study, we developed a bioresponsive hydrogel with cell-mediated degradation aligned to the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). We identified matrix metalloproteinase 7 (MMP7) as an enzyme with a temporal expression pattern that corresponded with cartilage development. By embedding MMP7 peptide substrates within a poly(ethylene glycol) diacrylate backbone, we built MMP7-sensitive hydrogels with distinct degradation rates. When MMP7-sensitive scaffolds were compared with nondegradable scaffolds in vitro, photoencapsulated hMSCs produced neocartilage constructs with more extensive collagenous matrices, as demonstrated through immunohistochemistry and biochemical quantification of matrix molecules. Furthermore, these changes translated into an increased dynamic compressive modulus. This work presents a practical strategy for designing biomaterials uniquely tuned to individual biological processes.

Bone turnover markers as surrogates of fracture healing after intramedullary fixation of tibia and femur fractures.

AIMS: Bone turnover markers (BTMs) follow distinct trends after fractures and limited evidence suggests differential levels in BTMs in patients with delayed healing. The effect of vitamin D, and other factors that influence BTMs and fracture healing, is important to elucidate the use of BTMs as surrogates of fracture healing. We sought to determine whether BTMs can be used as early markers of delayed fracture healing, and the effect of vitamin D on BTM response after fracture. METHODS: A total of 102 participants aged 18 to 50 years (median 28 years (interquartile range 23 to 35)), receiving an intramedullary nail for a tibial or femoral shaft fracture, were enrolled in a randomized controlled trial comparing vitamin D3 supplementation to placebo. Serum C-terminal telopeptide of type I collagen (CTX; bone resorption marker) and N-terminal propeptide of type I procollagen (P1NP; bone formation marker) were measured at baseline, six weeks, and 12 weeks post-injury. Clinical and radiological fracture healing was assessed at three months. RESULTS: CTX and P1NP concentrations peaked at six weeks in all groups. Elevated six-week CTX and P1NP were associated with radiological healing at 12 weeks post-injury (odds ratio (OR) 10.5; 95% confidence interval 2.71 to 53.5, p = 0.002). We found no association between CTX or P1NP and functional healing. Baseline serum 25(OH)D showed a weak inverse relationship with P1NP (p = 0.036) and CTX (p = 0.221) at 12 weeks, but we observed no association between vitamin D supplementation and either BTM. CONCLUSION: Given the association between six-week BTM concentrations and three-month radiological fracture healing, CTX and P1NP appear to be potential surrogate markers of fracture healing. Cite this article: Bone Joint Res 2022;11(4):239-250.

The intersection of fracture healing and infection: Orthopaedics research society workshop 2021.

Infection is a common cause of impaired fracture healing. In the clinical setting, definitive fracture treatment and infection are often treated separately and sequentially, by different clinical specialties. The ability to treat infection while promoting fracture healing will greatly reduce the cost, number of procedures, and patient morbidity associated with infected fractures. In order to develop new therapies, scientists and engineers must understand the clinical need, current standards of care, pathologic effects of infection on fractures, available preclinical models, and novel technologies. One of the main causes of poor fracture healing is infection; unfortunately, bone regeneration and infection research are typically approached independently and viewed as two separate disciplines. Here, we aim to bring these two groups together in an educational workshop to promote research into the basic and translational science that will address the clinical challenge of delayed fracture healing due to infection. Statement of clinical significance: Infection and nonunion are each feared outcomes in fracture care, and infection is a significant driver of nonunion. The impact of nonunions on patie[Q2]nt well-being is substantial. Outcome data suggests a long bone nonunion is as impactful on health-related quality of life measures as a diagnosis of type 1 diabetes and fracture-related infection has been shown to significantly l[Q3]ower a patient's quality of life for over 4 years.  Although they frequently are associated with one another, the treatment approaches for infections and nonunions are not always complimentary and cannot be performed simultaneously without accepting tradeoffs. Furthermore, different clinical specialties are often required to address the problem, the orthopedic surgeon treating the fracture and an infectious disease specialist addressing the sources of infection. A sequential approach that optimizes treatment parameters requires more time, more surgeries, and thus confers increased morbidity to the patient. The ability to solve fracture healing and infection clearance simultaneously in a contaminated defect would benefit both the patient and the health care system.

Collagen X Longitudinal Fracture Biomarker Suggests Staged Fixation in Tibial Plateau Fractures Delays Rate of Endochondral Repair.

OBJECTIVES: To use a novel, validated bioassay to monitor serum concentrations of a breakdown product of collagen X in a prospective longitudinal study of patients sustaining isolated tibial plateau fractures. Collagen X is the hallmark extracellular matrix protein present during conversion of soft, cartilaginous callus to bone during endochondral repair. Previous preclinical and clinical studies demonstrated a distinct peak in collagen X biomarker (CXM) bioassay levels after long bone fractures. SETTING: Level 1 academic trauma facility. PATIENTS/PARTICIPANTS: Thirty-six patients; isolated tibial plateau fractures. INTERVENTION: (3) Closed treatment, ex-fix (temporizing/definitive), and open reduction internal fixation. MAIN OUTCOME MEASUREMENTS: Collagen X serum biomarker levels (CXM bioassay). RESULTS: Twenty-two men and 14 women (average age: 46.3 y; 22.6-73.4, SD 13.3) enrolled (16 unicondylar and 20 bicondylar fractures). Twenty-five patients (72.2%) were treated operatively, including 12 (33.3%) provisionally or definitively treated by ex-fix. No difference was found in peak CXM values between sexes or age. Patients demonstrated peak expression near 1000 pg/mL (average: male-986.5 pg/mL, SD 369; female-953.2 pg/mL, SD 576). There was no difference in peak CXM by treatment protocol, external fixator use, or fracture severity (Schatzker). Patients treated with external fixation (P = 0.05) or staged open reduction internal fixation (P = 0.046) critically demonstrated delayed peaks. CONCLUSIONS: Pilot analysis demonstrates a strong CXM peak after fractures commensurate with previous preclinical and clinical studies, which was delayed with staged fixation. This may represent the consequence of delayed construct loading. Further validation requires larger cohorts and long-term follow-up. Collagen X may provide an opportunity to support prospective interventional studies testing novel orthobiologics or fixation techniques. LEVEL OF EVIDENCE: Level II, prospective clinical observational study.

Long-term functional regeneration of radiation-damaged salivary glands through delivery of a neurogenic hydrogel.

Salivary gland acinar cells are severely depleted after radiotherapy for head and neck cancer, leading to loss of saliva and extensive oro-digestive complications. With no regenerative therapies available, organ dysfunction is irreversible. Here, using the adult murine system, we demonstrate that radiation-damaged salivary glands can be functionally regenerated via sustained delivery of the neurogenic muscarinic receptor agonist cevimeline. We show that endogenous gland repair coincides with increased nerve activity and acinar cell division that is limited to the first week after radiation, with extensive acinar cell degeneration, dysfunction, and cholinergic denervation occurring thereafter. However, we found that mimicking cholinergic muscarinic input via sustained local delivery of a cevimeline-alginate hydrogel was sufficient to regenerate innervated acini and retain physiological saliva secretion at nonirradiated levels over the long term (>3 months). Thus, we reveal a previously unknown regenerative approach for restoring epithelial organ structure and function that has extensive implications for human patients.

Growth factor delivery using extracellular matrix-mimicking substrates for musculoskeletal tissue engineering and repair.

Therapeutic approaches for musculoskeletal tissue regeneration commonly employ growth factors (GFs) to influence neighboring cells and promote migration, proliferation, or differentiation. Despite promising results in preclinical models, the use of inductive biomacromolecules has achieved limited success in translation to the clinic. The field has yet to sufficiently overcome substantial hurdles such as poor spatiotemporal control and supraphysiological dosages, which commonly result in detrimental side effects. Physiological presentation and retention of biomacromolecules is regulated by the extracellular matrix (ECM), which acts as a reservoir for GFs via electrostatic interactions. Advances in the manipulation of extracellular proteins, decellularized tissues, and synthetic ECM-mimetic applications across a range of biomaterials have increased the ability to direct the presentation of GFs. Successful application of biomaterial technologies utilizing ECM mimetics increases tissue regeneration without the reliance on supraphysiological doses of inductive biomacromolecules. This review describes recent strategies to manage GF presentation using ECM-mimetic substrates for the regeneration of bone, cartilage, and muscle.

A quantitative serum biomarker of circulating collagen X effectively correlates with endochondral fracture healing.

Currently, there are no standardized methods for quantitatively measuring fracture repair. Physicians rely on subjective physical examinations and qualitative evaluation of radiographs to detect mineralized tissue. Since most fractures heal indirectly through a cartilage intermediate, these tools are limited in their diagnostic utility of early repair. Prior to converting to the bone, cartilage undergoes hypertrophic maturation, characterized by the deposition of a provisional collagen X matrix. The objective of this study was to characterize the kinetics of a novel collagen X biomarker relative to other biological measurements of fracture healing using a murine model of endochondral fracture repair in which a closed, mid-shaft tibia fracture was created using the classic drop-weight technique. Serum was collected 5 to 42 days post-fracture in male and female mice and compared to uninjured controls (n = 8-12). Collagen X in the serum was quantified using a recently validated ELISA-based bioassay ("Cxm")1 and compared to genetic and histological markers of fracture healing and inflammation. We found the Cxm biomarker reliably increased from baseline to a statistically unique peak 14 days post-fracture that then resolved to pre-fracture levels by 3 weeks following injury. The shape and timing of the Cxm curve followed the genetic and histological expression of collagen X but did not show a strong correlation with local inflammatory states. Assessment of fracture healing progress is crucial to making correct and timely clinical decisions for patients. This Cxm bioassay represents a minimally invasive, inexpensive technique that could provide reliable information on the biology of the fracture to significantly improve clinical care.

Chondrocyte-to-osteoblast transformation in mandibular fracture repair.

The majority of fracture research has been conducted using long bone fracture models, with significantly less research into the mechanisms driving craniofacial repair. However, craniofacial bones differ from long bones in both their developmental mechanism and embryonic origin. Thus, it is possible that their healing mechanisms could differ. In this study we utilize stabilized and unstabilized mandible fracture models to investigate the pathways regulating repair. Whereas fully stable trephine defects in the ramus form bone directly, mechanical motion within a transverse fracture across the same anatomical location promoted robust cartilage formation before boney remodeling. Literature investigating long bone fractures show chondrocytes are a direct precursor of osteoblasts during endochondral repair. Lineage tracing with Aggrecan-CreERT2 ::Ai9 tdTomato mice demonstrated that mandibular callus chondrocytes also directly contribute to the formation of new bone. Furthermore, immunohistochemistry revealed that chondrocytes located at the chondro-osseous junction expressed Sox2, suggesting that plasticity of these chondrocytes may facilitate this chondrocyte-to-osteoblast transformation. Based on the direct role chondrocytes play in bone repair, we tested the efficacy of cartilage grafts in healing critical-sized mandibular defects. Whereas empty defects remained unbridged and filled with fibrous tissue, cartilage engraftment produced bony-bridging and robust marrow cavity formation, indicating healthy vascularization of the newly formed bone. Engrafted cartilage directly contributed to new bone formation since a significant portion of the newly formed bone was graft/donor-derived. Taken together these data demonstrate the important role of chondrocyte-to-osteoblast transformation during mandibular endochondral repair and the therapeutic promise of using cartilage as a tissue graft to heal craniofacial defects.