Volumetric muscle loss (VML)—defined as the irrecoverable loss of skeletal muscle tissue with associated persistent functional deficits—is among the most common and highly debilitating combat-related extremity injuries. This is particularly true in cases of severe polytrauma wherein multiple extremities may be involved as a result of high energy wounding mechanisms. As such, significant investment and effort has been made toward developing a clinically viable intervention capable of restoring the form and function of the affected musculature. While these investigations conducted to date have varied with respect to the species, breed, and sex of the chosen pre-clinical in-vivo model system, the majority of these studies have been performed in unilateral injury models, an aspect which may not fully exemplify the clinical representation of the multiply injured patient. Furthermore, while various components of the basal pathophysiology of VML (e.g., fibrosis and inflammation) have been investigated, relatively little effort has focused on how the pathophysiology and efficacy of pro-regenerative technologies is altered when there are multiple VML injuries. Thus, the purpose of this study was two-fold: (1) to investigate if/how the pathophysiology of unilateral VML injuries differs from bilateral VML injuries and (2) to interrogate the effect of bilateral VML injuries on the efficacy of a well-characterized regenerative therapy, minced muscle autograft (MMG). In contrast to our hypothesis, we show that bilateral VML injuries exhibit a similar systemic inflammatory response and improved muscle functional recovery, compared to unilateral injured animals. Furthermore, MMG treatment was found to only be effective at promoting an increase in functional outcomes in unilateral VML injuries. The findings presented herein add to the growing knowledge base of the pathophysiology of VML, and, importantly, reiterate the importance of comprehensively characterizing preclinical models which are utilized for early-stage screening of putative therapies as they can directly influence the translational research pipeline.

Intra-articular fractures (IAF) result in significant and prolonged inflammation, increasing the chances of developing post-traumatic osteoarthritis (PTOA). Interleukin-one beta (IL-1β) and Tumor Necrosis Factor-alpha (TNF-α) are key inflammatory factors shown to be involved in osteochondral degradation following IAF. As such, use of targeted biologics such as Infliximab (INX), a TNF-α inhibitor, and Anakinra (ANR), an interleukin-one (IL-1) receptor antagonist (IL1RA), may protect against PTOA by damping the inflammatory response to IAF and reducing osteochondral degradation. To test this hypothesis, IAFs were induced in the hindlimb knee joints of rats treated with INX at 10 mg/kg/day, ANR at 100 g/kg/day, or saline (vehicle control) by subcutaneous infusion for a period of two weeks and healing was evaluated at 8-weeks post injury. Serum and synovial fluid (SF) were analyzed for soluble factors. In-vivo microcomputed tomography (µCT) scans assessed bone mineral density and bone morphometry measurements. Cationic CA4⁺ agent assessed articular cartilage composition via ex vivo µCT. Scoring according to the Osteoarthritis Research Society International (OARSI) guidelines was performed on stained histologic tibia sections at the 56-day endpoint on a 0–6 scale. Systemically, ANR reduced many pro-inflammatory cytokines and reduced osteochondral degradation markers Cross Linked C-Telopeptide Of Type II (CTXII, p < 0.05) and tartrate-resistant acid phosphatase (TRAP, p < 0.05). ANR treatment resulted in increased chemokines; macrophage-chemotractant protein-1 (MCP-1), MPC-3, macrophage inhibitory protein 2 (MIP2) with a concomitant decrease in proinflammatory interleukin-17A (IL17A) at 14 days post-injury within the SF. Microcomputed tomography (µCT) at 56 days post-injury revealed ANR Treatment decreased epiphyseal degree of anisotropy (DA) (p < 0.05) relative to saline. No differences were found with OARSI scoring but contrast-enhanced µCT revealed a reduction in glycosaminoglycan content with ANR treatment. These findings suggest targeted cytokine inhibition, specifically IL-1 signaling, as a monotherapy has minimal utility for improving IAF healing outcomes but may have utility for promoting a more permissive inflammatory environment that would allow more potent disease modifying osteoarthritis drugs to mitigate the progression of PTOA after IAF.

The use of a rehabilitation approach that promotes regeneration has the potential to improve the efficacy of pro-regenerative therapies and maximize functional outcomes in the treatment of volumetric muscle loss (VML). An adjunct antifibrotic treatment could further enhance functional gains by reducing fibrotic scarring. This study aimed to evaluate the potential synergistic effects of losartan, an antifibrotic pharmaceutical, paired with a voluntary wheel running rehabilitation strategy to enhance a minced muscle graft (MMG) pro-regenerative therapy in a rodent model of VML. The animals were randomly assigned into four groups: (1) antifibrotic with rehabilitation, (2) antifibrotic without rehabilitation, (3) vehicle treatment with rehabilitation, and (4) vehicle treatment without rehabilitation. At 56 days, the neuromuscular function was assessed, and muscles were collected for histological and molecular analysis. Surprisingly, we found that the losartan treatment decreased muscle function in MMG-treated VML injuries by 56 days, while the voluntary wheel running elicited no effect. Histologic and molecular analysis revealed that losartan treatment did not reduce fibrosis. These findings suggest that losartan treatment as an adjunct therapy to a regenerative rehabilitation strategy negatively impacts muscular function and fails to promote myogenesis following VML injury. There still remains a clinical need to develop a regenerative rehabilitation treatment strategy for traumatic skeletal muscle injuries. Future studies should consider optimizing the timing and duration of adjunct antifibrotic treatments to maximize functional outcomes in VML injuries.

Volumetric muscle loss (VML) is a severe injury resulting in substantial skeletal muscle loss, leading to a complex pathology that culminates in suboptimal tissue repair and significant long-term functional deficits. This study employs a rodent model of VML to investigate the impact of biological sex on the injury pathobiology and its potential influence on the response to autologous minced muscle grafting (MMG) as a regenerative therapy. While no significant differences were observed between the end-stage male and female responses to VML, both male and female subjects appeared to benefit from MMG treatment. Females demonstrated improved neuromuscular function, while males exhibited reduced fibrosis at the site of injury. Additional differences in the wound healing response included distinct variations in myofiber characteristics, with females exhibiting a lower proportion of Type 2a fibers and elevated levels of myogenin. These findings suggest that regenerative therapies, such as MMG, may exhibit sex-specific benefits. Future studies will further explore sexual dimorphism in the acute response to injury to identify potential therapeutic targets that may yield greater therapeutic efficacy for each sex.

Aims: This study aimed to assess the effects of chondrogenic agents kartogenin (KGN) and KA9 in combination with anakinra (ANR), an interleukin (IL) 1 receptor antagonist (IL1RA) on post-traumatic osteoarthritis (PTOA) progression following intra-articular fractures (IAF). The hypothesis was that this immunoregenerative approach would promote osteochondral healing and alleviate PTOA more effectively than individual treatments or controls. Methods: Male Lewis rats (n = 78) underwent IAF, followed by weekly intra-articular injection of saline (control), KGN, or KA9 in conjunction with the systemic administration of saline (control) or ANR via osmotic pumps. Inflammatory and osteochondral markers were assessed through serum and synovial fluid analysis, micro-CT (µCT) for bone parameters, contrast-enhanced µCT for cartilage evaluation, histopathological analysis, and immunohistochemistry (IHC). Results: Immunoregenerative treatments did not adversely affect animal wellbeing. While local inflammation markers were minimally affected, some osteochondral remodelling markers indicated that KGN, KA9, and their combination with ANR reduced markers of osteochondral degradation. Bone analysis showed improved trabecular morphometry parameters and reduced bone surface with some treatments, and cartilage composition and thickness improved with treatments, although the effects were inconsistent across groups. Osteoarthritis Research Society International scores revealed location-specific variations in pathology, and IHC staining demonstrated intergroup differences in protein expression at different timepoints. Conclusion: This study suggests that KGN and KA9 may offer some protection against PTOA by influencing cartilage and subchondral bone health, and that the combination with ANR shows potential for enhanced effects. However, the results were mixed, with no treatment group consistently demonstrating positive improvements across all outcomes. The complexity of developing a novel immunoregenerative therapy for PTOA is highlighted by the variability in treatment response. Cite this article: Bone Joint Res 2026;15(3):264–277.

Repurposing clinically approved antifibrotic agents presents a potential near‐term therapeutic strategy to mitigate the extensive fibrotic scarring that follows severe volumetric muscle loss (VML) injury and inhibits functional recovery. We hypothesized that daily oral administration of pirfenidone, an FDA‐approved antifibrotic, would mitigate the overwhelming fibrotic response and facilitate functional recovery in a clinically relevant porcine VML model. Yorkshire‐cross pigs ( n = 12) underwent VML or sham surgery in the peroneus tertius muscle and were subsequently allocated to pirfenidone treatment or untreated controls. At the 4‐week study endpoint, the affected muscles underwent comprehensive histological, biochemical, transcriptional and neuromuscular functional assessments. Transcription profiling demonstrated that pirfenidone downregulated key pro‐inflammatory cytokine and chemokine regulators involved in excessive fibrosis, including TNF, CCL3L1 and CCL2. While pirfenidone treatment failed to reduce total collagen deposition, evidenced by picrosirius red quantification and hydroxyproline content, it did result in a less dense collagen fibre architecture, suggestive of altered tissue remodelling. However, pirfenidone treatment impaired functional recovery through reduced contractile output and resulted in stagnant weight gain compared to the untreated cohort. This work demonstrates that systemic pirfenidone administration failed to meaningfully reduce fibrosis and subsequently worsened functional capacity following VML, similar to previous antifibrotic studies. The functional deficits and stagnant weight gains observed here underscore a critical knowledge gap regarding the impact of systemic antifibrotics on contractile recovery, necessitating a rigorous re‐evaluation of delivery routes, dosages and administration timelines to ensure that future therapies for fibrotic scarring do not inadvertently hinder the physiological recovery of VML‐injured muscle.