Development of a High-Color Flow Cytometry Panel for Immunologic Analysis of Tissue Injury and Reconstruction in a Rat Model.

The rat model is an important resource in biomedical research due to its similarities to the human immune system and its use for functional studies. However, because of the preponderance of mouse models in foundational and mechanistic immunological studies, there is a relative lack of diverse, commercially available flow cytometry antibodies for immunological profiling in the rat model. Available antibodies are often conjugated to common fluorophores with similar peak emission wavelengths, making them hard to distinguish on conventional flow cytometers and restricting more comprehensive immune analysis. This can become a limitation when designing immunological studies in rat injury models to investigate the immune response to tissue injury. In addition, this lack of available antibodies limits the number of studies that can be done on the immune populations in lymphoid organs in other research areas. To address this critical unmet need, we designed a spectral flow cytometry panel for rat models. Spectral cytometry distinguishes between different fluorophores by capturing their full emission spectra instead of their peak emission wavelengths. This flow cytometry panel includes 24 distinct immune cell markers to analyze the innate and adaptive immune response. Importantly, this panel identifies different immune phenotypes, including tolerogenic, Type 1, and Type 2 immune responses. We show that this panel can identify unique immune populations and phenotypes in a rat muscle trauma model. We further validated that the panel can identify distinct adaptive and innate immune populations and their unique phenotypes in lymphoid organs. This panel expands the scope of previous rat panels providing a tool for scientists to examine the immune system in homeostasis and injury while pairing mechanistic immunological studies with functional studies.

Effect of Targeted Cytokine Inhibition on Progression of Post-Traumatic Osteoarthritis Following Intra-Articular Fracture.

Intra-articular fractures (IAF) result in significant and prolonged inflammation, increasing the chances of developing post-traumatic osteoarthritis (PTOA). Interleukin-one beta (IL-1ß) 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.

Effects of Adjunct Antifibrotic Treatment within a Regenerative Rehabilitation Paradigm for Volumetric Muscle Loss.

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.

Retrospective characterization of a rat model of volumetric muscle loss.

Volumetric muscle loss (VML) is a pervasive injury within contemporary combat and a primary driver of disability among injured Service members. As such, VML has been a topic of investigation over the past decade as the field has sought to understand the pathology of these injuries and to develop treatment strategies which restore the form and function of the involved musculature. To date, much of this work has been performed in disparate animal models that vary significantly in terms of the species utilized, the muscle (or muscle group) affected, and the volume of muscle lost. Moreover, variation exists in the reporting of anatomical and functional outcomes within these models. When taken together, the ability to successfully assess comparative efficacy of promising therapies is currently limited. As such, greater scrutiny on the characterization of these VML models is needed to better assess the quality of evidence supporting further translation of putative therapies. Thus, the objective of this study was to retrospectively characterize anatomical and functional outcomes associated with one such VML model - the 6 mm biopsy punch model of the rat tibialis anterior muscle. Through these efforts, it was shown that this model is highly reproducible and consistent across a large number of experiments. As such, the data presented herein represent a reasonable benchmark for the expected performance of this model with utility for drawing inferences across studies and identifying therapies which have shown promise within the preclinical domain, and thus are ready for further translation towards the clinic.

Evaluation of licofelone as an adjunct anti-inflammatory therapy to biologic scaffolds in the treatment of volumetric muscle loss.

Biologic scaffolds (BS) are the most widely studied therapeutics for the treatment of volumetric muscle loss (VML) owing to their purported effects on cell proliferation, chemotaxis, migration, and differentiation. Despite these claims, variability in reports on the nature of the immune response to their implantation suggests that BS-associated inflammation may be limiting their regenerative efficacy. To address this shortcoming, this study sought to evaluate licofelone (ML3000), a dual 5-LOX/COX inhibitor, as an anti-inflammatory adjunct therapy to a BS in the treatment of VML. Utilizing a well-established rat VML model, a micronized BS was used to treat the VML injury, with or without administration of licofelone. Functional, molecular, and histological outcomes were assessed at both 7- and 28-day post-injury time points. While the BS + licofelone group exhibited decreased transcription of pro-inflammatory markers (Tnf, Ccl5, Nos2) relative to the BS only control group, no differences in expression profile of a panel of inflammatory-related soluble factors were observed between groups. A modest reduction in type I collagen was observed in the licofelone-treated group, but no meaningful differences in histologic presentation of repaired tissue were observed between groups. Furthermore, no differences in end organ functional capacity were observed between groups. Moving forward, efforts related to modulating the wound healing environment of VML should focus on polypharmaceutical strategies that target multiple aspects of the early pathophysiology of VML so as to provide an environment that is sufficiently permissive for local regenerative therapies to promote restoration of myofiber number.

Volumetric muscle loss disrupts length-dependent architectural and functional characteristics of skeletal muscle.

Purpose/Aim: Skeletal muscle architecture is a primary determinant of function. Volumetric muscle loss (VML) injury is destructive; however, the impact on muscle architecture is uncharacterized. Methods: Architectural and functional effects of VML were assessed in rat tibialis anterior (TA) muscle model 4 weeks post-injury. Results: VML caused a 31% and 33% reduction in muscle weight (p < 0.001) and fiber length (p = 0.002), respectively, culminating a 34% reduction of fiber to muscle length ratio (FL:ML; p < 0.001). Fiber pennation angle (+14%; p = 0.150) and physiological cross-sectional area (PCSA; -12%; p = 0.220) were unchanged. VML injury reduced peak isometric force (Po) by 36% (p < 0.001), specific force (sPo = Po/PCSA) by 41% (vs. Po, p > 0.999), and force per gram muscle weight (Po/mw) by 18% (vs. Po, p < 0.001). VML injury increased the length at which Po was produced (Lo) by 8% (p = 0.009), and reduced functional excursion by 35% (p = 0.035). Conclusion: The architectural changes after VML injury preserved PCSA, and therefore preserved "potential" maximal force-producing capacity. At most, only half the Po deficit was due directly to the cumulative effect of horizontal and longitudinal tissue loss. Highlighting the impact of longitudinal muscle loss, VML injury reduced fiber length, and FL:ML and grossly disrupted length-dependent functional properties. These findings raise the importance of augmenting length-dependent muscle properties to optimize functional recovery after VML injury.

The Role of the Inflammatory Response in Mediating Functional Recovery Following Composite Tissue Injuries.

Composite tissue injuries (CTI) are common among US Military Service members during combat operations, and carry a high potential of morbidity. Furthermore, CTI are often complicated due to an altered wound healing response, resulting in part from a dysregulation of the innate and adaptive immune responses. Unlike normal wound healing, in CTI, disruptions occur in innate immune responses, altering neutrophil functions, macrophage activation and polarization, further impacting the functions of T regulatory cells. Additionally, the biological underpinnings of these unfavorable wound healing conditions are multifactorial, including various processes, such as: ischemia, hypoxia, low nutrient levels, and altered cell metabolic pathways, among others, all of which are thought to trigger anergy in immune cells and destabilize adaptive immune responses. As a result, impaired wound healing is common in CTI. Herein, we review the altered innate and adaptive immune cells and their metabolic status and responses following CTI, and discuss the role a multi-pronged immunomodulatory approach may play in facilitating improved outcomes for afflicted patients.

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.

Impact of volumetric muscle loss injury on persistent motoneuron axotomy.

INTRODUCTION: Volumetric muscle loss (VML) occurs following significant traumatic injury or surgical removal of skeletal muscle, resulting in nonrecoverable loss of tissue and long-term dysfunction. Perhaps less recognized is that VML injuries inherently disrupt the neuromuscular unit, resulting in fiber denervation and presumptive motor unit rearrangement, expansion, and/or loss. To characterize neural dysfunction we quantified motoneuron axotomy, in efforts to understand how this relates to the temporal coordination of neuromuscular and morphological alterations due to injury. METHODS: In an established rat tibialis anterior (TA) VML injury model, we examined the motoneuron, skeletal muscle, and maximal isometric torque at 3, 7, 14, and 21 days postinjury. RESULTS: Significant axotomy of 57-79% of all TA muscle motoneurons was observed through 21 days postinjury, which was coupled with a 45-90% TA maximal torque deficit. DISCUSSION: A ∼20% partial ablation of the TA muscle causes disproportionate damage across the motor unit acutely postinjury. Muscle Nerve 57: 799-807, 2018.

Impairment of early fracture healing by skeletal muscle trauma is restored by FK506.

BACKGROUND: Heightened local inflammation due to muscle trauma or disease is associated with impaired bone regeneration. METHODS: We hypothesized that FK506, an FDA approved immunomodulatory compound with neurotrophic and osteogenic effects, will rescue the early phase of fracture healing which is impaired by concomitant muscle trauma in male (~4 months old) Lewis rats. FK506 (1 mg/kg; i.p.) or saline was administered systemically for 14 days after an endogenously healing tibia osteotomy was created and fixed with an intermedullary pin, and the overlying tibialis anterior (TA) muscle was either left uninjured or incurred volumetric muscle loss injury (6 mm full thickness biopsy from middle third of the muscle). RESULTS: The salient observations of this study were that 1) concomitant TA muscle trauma impaired recovery of tibia mechanical properties 28 days post-injury, 2) FK506 administration rescued the recovery of tibia mechanical properties in the presence of concomitant TA muscle trauma but did not augment mechanical recovery of an isolated osteotomy (no muscle trauma), 3) T lymphocytes and macrophage presence within the traumatized musculature were heightened by trauma and attenuated by FK506 3 days post-injury, and 4) T lymphocyte but not macrophage presence within the fracture callus were attenuated by FK506 at 14 days post-injury. FK506 did not improve TA muscle isometric torque production CONCLUSION: Collectively, these findings support the administration of FK506 to ameliorate healing of fractures with severe muscle trauma comorbidity. The results suggest one potential mechanism of action is a reduction in local T lymphocytes within the injured musculoskeletal tissue, though other mechanisms to include direct osteogenic effects of FK506 require further investigation.

Hydrodynamic loading in concomitance with exogenous cytokine stimulation modulates differentiation of bovine mesenchymal stem cells towards osteochondral lineages.

BACKGROUND: Mesenchymal stem cells (MSCs) are viewed as a having significant potential for tissue engineering and regenerative medicine therapies. Clinical implementation of MSCs, however, demands that their preparation be stable and reproducible. Given that environmental and bioprocessing parameters such as substrate stiffness, seeding densities, culture medium composition, and mechanical loading can result in undirected differentiation of the MSC population, the objective of this study was to systematically investigate how hydrodynamic loading influences the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) towards the osteochondral lineages both in the presence and absence of exogenous, inductive factors. METHODS: Expanded bovine MSCs were suspended in 2.5 % agarose, cast in a custom mold, and placed into either static or one of two dynamic culture environments consisting of "high" and "low" magnitude shear conditions. Constructs were supplemented with varying concentrations (0, 1, 10, 100 ng/mL) of either TGF-ß3 or BMP-2 throughout cultivation with tissue samples being collected following each week of culture. RESULTS: In the absence of exogenous supplementation, hydrodynamic loading had little effect on cell phenotype at either magnitude of stimulation. When cultures were supplemented with BMP-2 and TGF-ß3, MSCs gene expression progressed towards the osteogenic and chondrogenic pathways, respectively. This progression was enhanced by the presence of hydrodynamic loading, particularly under high shear conditions, but may point the chondrogenic cultures down a hypertrophic path toward osteogenesis reminiscent of endochondral ossification if TGF-ß3 supplementation is insufficient. CONCLUSIONS: Moving forward, these results suggest bioprocessing conditions which minimize exposure of chondrogenic cultures to fluid shear stress to avoid undesirable differentiation of the MSC population.

A Two-Stage Approach Integrating Provisional Biomaterial-Mediated Stabilization Followed by a Definitive Treatment for Managing Volumetric Muscle Loss Injuries.

Treatment of volumetric muscle loss (VML) faces challenges due to its unique pathobiology and lower priority in severe musculoskeletal injury management. Consequently, a need exists for multi-stage VML treatment strategies to accommodate delayed interventions owing to comorbidity management or prolonged casualty care in combat settings. To this end, polyvinyl alcohol (PVA) was used at concentrations of 5%, 7.5%, and 10% to generate provisional muscle void fillers (MVFs) of varying stiffness values (1.125 kPa, 3.700 kPa, and 7.699 kPa) to stabilize VML injuries as part of a two-stage approach. These were implanted into a rat model for a duration of 4 weeks, then explanted and either left untreated (control) or treated through minced muscle grafting (MMG). Additional benchmarks included acute MMG and unrepaired groups. At the MVF explant, the 7.5% PVA group exhibited superior neuromuscular function compared to the 5% and 10% PVA groups, the least fibrosis, and the largest median myofiber size among all groups at the 12-week endpoint. Despite the 7.5% PVA's superiority amongst the two-stage treatment groups, neuromuscular function was neither improved nor impaired relative to acute treatment benchmarks. This suggests that the future success of a two-stage VML treatment strategy will necessitate a more effective definitive intervention.

Volumetric Muscle Loss: A Bibliometric Analysis of a Decade of Progress.

The consequences of military conflict, accidents, and diseases have led to the definition-and subsequent study-of the pathological condition now known as volumetric muscle loss (VML). VML is a significant injury to skeletal muscle tissue on a scale that is endogenously irrecoverable and leads to chronic functional deficits and long-term disability. Currently, there lacks a definitive approach to meaningfully restore the tissue and function lost by those afflicted, ushering a need for scientific activities and associated funding to both facilitate a deeper understanding of the pathobiology of VML as well as to develop and assess clinically relevant therapeutics and treatment strategies. Thereby, evaluation of the VML field is crucial to gauging the return on resource expenditures and to understand the evolution of the field to guide future directions. This article presents a bibliometric analysis of publicly available data to explore the growth of the VML field since its genesis and to highlight its prosperity through its expanding literature, its development and evaluation of promising treatment strategies, rising financial investments, and innovation. Altogether, the bibliometric analysis reveals the field of VML as an emergent research focus that is productive and translational. Impact statement Analyses of a research topic are fundamental toward evaluating the returns on investment and appreciating the evolution of the research toward novel directions. This study aims to highlight the growing field of volumetric muscle loss (VML), defined as a significant injury to skeletal muscle tissue that leads to functional impairment and is irrecoverable through inherent regenerative mechanisms. The analysis of bibliometric and publicly available data provides evidence that the field of VML has an expanding research interest and investment, with biomaterials at the forefront of study.

Demographics and Comorbidities of United States Service Members with Combat-Related Lower Extremity Limb Salvage.

INTRODUCTION: This retrospective study describes the demographics and injury characteristics of a recently identified cohort of US Service members with combat-related lower extremity limb salvage (LS). METHODS: US Service members with combat trauma were identified from the Expeditionary Medical Encounter Database and Military Health System Data Repository and stratified into primary amputation (PA), LS, and non-threatened limb trauma (NTLT) cohorts based on ICD-9 codes. Disparities in demographic factors and injury characteristics were investigated across cohorts and within the LS cohort based on limb retention outcome. RESULTS: Cohort demographics varied by age but not by sex, branch, or rank. The mechanism of injury and injury characteristics were found to be different between the cohorts, with the LS cohort exhibiting more blast injuries and greater injury burden than their peers with NTLT. A sub-analysis of the LS population revealed more blast injuries and fewer gunshot wounds in those that underwent secondary amputation. Neither demographic factors nor total injury burden varied with limb retention outcome, despite slight disparities in AIS distribution within the LS cohort. CONCLUSIONS: In accordance with historic dogma, the LS population presents high injury severity. Demographics and injury characteristics are largely invariant with respect to limb retention outcomes, despite secondary amputation being moderately more prevalent in LS patients with blast-induced injuries. Further study of this population is necessary to better understand the factors that impact the outcomes of LS in the Military Health System.

Development and characterization of an intra-articular fracture mediated model of post-traumatic osteoarthritis.

PURPOSE: This study aimed to develop and characterize a closed intra-articular fracture (IAF) mediated post-traumatic osteoarthritis (PTOA) model in rats to serve as a testbed for putative disease modifying interventions. METHODS: Male rats were subject to a 0 Joule (J), 1 J, 3 J, or 5 J blunt-force impact to the lateral aspect of the knee and allowed to heal for 14 and 56 days. Micro-CT was performed at time of injury and at the specified endpoints to assess bone morphometry and bone mineral density measurements. Cytokines and osteochondral degradation markers were assayed from serum and synovial fluid via immunoassays. Histopathological analyses were performed on decalcified tissues and assessed for evidence of osteochondral degradation. RESULTS: High-energy (5 J) blunt impacts consistently induced IAF to the proximal tibia, distal femur, or both while lower energy (1 J and 3 J) impacts did not. CCL2 was found to be elevated in the synovial fluid of rats with IAF at both 14- and 56-days post-injury while COMP and NTX-1 were upregulated chronically relative to sham controls. Histological analysis showed increased immune cell infiltration, increased osteoclasts and osteochondral degradation with IAF relative to sham. CONCLUSION: Based on results from the current study, our data indicates that a 5 J blunt-forced impact adequately and consistently induces hallmark osteoarthritic changes to the articular surface and subchondral bone at 56 days after IAF. Marked development of PTOA pathobiology suggest this model will provide a robust testbed for screening putative disease modifying interventions that might be translated to the clinic for militarily relevant, high-energy joint injuries.

In situ forming biomaterials as muscle void fillers for the provisional treatment of volumetric muscle loss injuries.

Volumetric muscle loss (VML) represents a devastating extremity injury which leads to chronic functional deficits and disability and is unrecoverable through normal healing pathways. When left untreated, the VML pathophysiology creates many challenges towards successful treatment, such as altered residual muscle architecture, excessive fibrosis, and contracture(s). As such, innovative approaches and technologies are needed to prevent or reverse these adverse sequelae. Development of a rationally designed biomaterial technology which is intended to be acutely placed within a VML defect - i.e., to serve as a muscle void filler (MVF) by maintaining the VML defect - could address this clinical unmet need by preventing these adverse sequelae as well as enabling multi-staged treatment approaches. To that end, three biomaterials were evaluated for their ability to serve as a provisional MVF treatment intended to stabilize a VML defect in a rat model for an extended period (28 days): polyvinyl alcohol (PVA), hyaluronic acid and polyethylene glycol combination (HA + PEG), and silicone, a clinically used soft tissue void filler. HA + PEG biomaterial showed signs of deformation, while both PVA and silicone did not. There were no differences between treatment groups for their effects on adjacent muscle fiber count and size distribution. Not surprisingly, silicone elicited robust fibrotic response resulting in a fibrotic barrier with a large infiltration of macrophages, a response not seen with either the PVA or HA + PEG. Taken together, PVA was found to be the best material to be used as a provisional MVF for maintaining VML defect volume while minimizing adverse effects on the surrounding muscle.

Mobility Solutions After a Lower Extremity Fracture and Applicability to Battlefield and Wilderness Medicine.

The potential for delayed evacuation of injured Service members from austere environments highlights the need to develop solutions that can stabilize a wound and enable mobility during these prolonged casualty care (PCC) scenarios. Lower extremity fractures have traditionally been treated by immobilization (splinting) followed by air evacuation - a paradigm not practical in PCC scenarios. In the civilian sector, treatment of extremity injuries sustained during remote recreational activities have similar challenges, particularly when adverse weather or terrain precludes early ground or air rescue. This review examines currently available fracture treatment solutions to include splinting, orthotic devices, and biological interventions and evaluates their feasibility: 1) for prolonged use in austere environments and 2) to enable patient mobilization. This review returned three common types of splints to include: a simple box splint, pneumatic splints, and traction splints. None of these splinting techniques allowed for ambulation. However, fixed facility-based orthotic interventions that include weight-bearing features may be combined with common splinting techniques to improve mobility. Biologically-focused technologies to stabilize a long bone fracture are still in their infancy. Integrating design features across these technologies could generate advanced treatments which would enable mobility, thus maximizing survivability until patient evacuation is feasible.

A Data-Driven Method to Discriminate Limb Salvage from Other Combat-Related Extremity Trauma.

INTRODUCTION: The aim of this study was to address and enhance our ability to study the clinical outcome of limb salvage (LS), a commonly referenced but ill-defined clinical care pathway, by developing a data-driven approach for the identification of LS cases using existing medical code data to identify characteristic diagnoses and procedures, and to use that information to describe a cohort of US Service members (SMs) for further study. METHODS: Diagnosis code families and inpatient procedure codes were compiled and analyzed to identify medical codes that are disparately associated with a LS surrogate population of SMs who underwent secondary amputation within a broader cohort of 3390 SMs with lower extremity trauma (AIS > 1). Subsequently, the identified codes were used to define a cohort of all SMs who underwent lower extremity LS which was compared with the opinion of a panel of military trauma surgeons. RESULTS: The data-driven approach identified a population of n = 2018 SMs who underwent LS, representing 59.5% of the combat-related lower extremity (LE) trauma population. Validation analysis revealed 70% agreement between the data-driven approach and gold standard SME panel for the test cases studied. The Kappa statistic (κ = 0.55) indicates a moderate agreement between the data-driven approach and the expert opinion of the SME panel. The sensitivity and specificity were identified as 55.6% (expert range of 51.8-66.7%) and 87% (expert range of 73.9-91.3%), respectively. CONCLUSIONS: This approach for identifying LS cases can be utilized to enable future high-throughput retrospective analyses for studying both short- and long-term outcomes of this underserved patient population.

Minced muscle autografting improves bone healing but not muscle function in a porcine composite injury model.

Composite tissue injuries (CTIs) in extremities include segmental bone defects (SBDs) and volumetric muscle loss. The objective of this study was to determine if skeletal muscle autografting with minced muscle grafts (MMGs) could improve healing in an SBD and improve muscle function in a porcine CTI model that includes an SBD and adjacent volumetric muscle loss injury. Adult Yucatan Minipigs were stratified into three groups including specimens with an isolated SBD, an SBD with volumetric muscle loss (CTI), and an SBD with volumetric muscle loss treated with MMG (CTI + MMG). Bone healing was quantified with serial x-rays and postmortem computed tomography scanning. Muscle function was quantified with a custom in vivo force transducer. Muscle tissue content was determined by biochemical analyses and histology. Anterior cortex-modified Radiographic Union Score for Tibia fractures (mRUSTs) decreased from 2.7 to 1.9 (p = 0.003) in CTI versus SBD animals. MMG improved anterior mRUST scores to 2.5 in CTI + MMG specimens (p = 0.030 compared to CTI specimens) and overall mRUST scores increased from 9.4 in CTI specimens to 11.1 in CTI + MMG specimens (p = 0.049). Residual strength deficits at euthanasia were 42% in SBD (p < 0.001), 44% in CTI (p < 0.001), and 48% in CTI + MMG (p < 0.001) compared to preoperative values. There were no differences in strength deficits between the three groups. Biochemical and histologic analyses demonstrated scattered differences between the three groups compared to contralateral muscle. MMG improved bone healing. However, the primary cause of muscle dysfunction and biochemical changes was the presence of an SBD. Clinical significance: Early mitigation of SBDs may be necessary to prevent muscle damage and weakness in patients sustaining composite extremity trauma.

Exploring relationships among multi-disciplinary assessments for knee joint health in service members with traumatic unilateral lower limb loss: a two-year longitudinal investigation.

Motivated by the complex and multifactorial etiologies of osteoarthritis, here we use a comprehensive approach evaluating knee joint health after unilateral lower limb loss. Thirty-eight male Service members with traumatic, unilateral lower limb loss (mean age = 38 yr) participated in a prospective, two-year longitudinal study comprehensively evaluating contralateral knee joint health (i.e., clinical imaging, gait biomechanics, physiological biomarkers, and patient-reported outcomes); seventeen subsequently returned for a two-year follow-up visit. For this subset with baseline and follow-up data, outcomes were compared between timepoints, and associations evaluated between values at baseline with two-year changes in tri-compartmental joint space. Upon follow-up, knee joint health worsened, particularly among seven Service members who presented at baseline with no joint degeneration (KL = 0) but returned with evidence of degeneration (KL ≥ 1). Joint space narrowing was associated with greater patellar tilt (r[12] = 0.71, p = 0.01), external knee adduction moment (r[13] = 0.64, p = 0.02), knee adduction moment impulse (r[13] = 0.61, p = 0.03), and CTX-1 concentration (r[11] = 0.83, p = 0.001), as well as lesser KOOSSport and VR-36General Health (r[16] = - 0.69, p = 0.01 and r[16] = - 0.69, p = 0.01, respectively). This longitudinal, multi-disciplinary investigation highlights the importance of a comprehensive approach to evaluate the fast-progressing onset of knee osteoarthritis, particularly among relatively young Service members with lower limb loss.