The HIF-1α/PLOD2 axis integrates extracellular matrix organization and cell metabolism leading to aberrant musculoskeletal repair.

While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism-linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here, we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses of the HO site, we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α (Hoxa11-CreERT2; Hif1afl/fl) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreERT2; Hif1afl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion. From a translational aspect, a pan-LOX inhibitor significantly decreased HO. A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism. This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate. These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.

Technical Precision with Autologous Fat Grafting for Facial Rejuvenation: A Review of the Evolving Science.

SUMMARY: The scientific study of facial aging has transformed modern facial rejuvenation. As people age, fat loss in specific fat compartments is a major contributor to structural aging of the face. Autologous fat grafting is safe, abundant, readily available, and completely biocompatible, which makes it the preferred soft-tissue filler in the correction of facial atrophy. The addition of volume through fat grafting gives an aging face a more youthful, healthy, and aesthetic appearance. Harvesting and preparation with different cannula sizes and filter-cartridge techniques have allowed for fat grafts to be divided based on parcel size and cell type into three major subtypes: macrofat, microfat, and nanofat. Macrofat and microfat have the benefit of providing volume to restore areas of facial deflation and atrophy in addition to improving skin quality; nanofat has been shown to improve skin texture and pigmentation. In this article, the authors discuss the current opinions regarding fat grafting and how the evolving science of fat grafting has led to the clinical utility of each type of fat to optimize facial rejuvenation. The opportunity exists to individualize the use of autologous fat grafting with the various subtypes of fat for the targeted correction of aging in different anatomic areas of the face. Fat grafting has become a powerful tool that has revolutionized facial rejuvenation, and developing precise, individualized plans for autologous fat grafting for each patient is an important advancement in the evolution of facial rejuvenation.

Oncological Safety of Autologous Fat Grafting for Breast Reconstruction.

BACKGROUND: Autologous fat grafting has become a vital component of breast reconstruction. However, concerns remain regarding the safety of fat grafting after oncological resection and breast reconstruction. The purpose of the study was to evaluate the association of fat grafting after breast reconstruction with metastasis and death in breast cancer patients. METHODS: A retrospective, population-based cohort study was conducted using deidentified claims data from 2001 to 2018 and included privately insured patients with breast cancer who underwent breast reconstruction after surgical resection. Breast reconstruction patients who underwent fat grafting were compared with those not undergoing fat grafting, evaluating metastasis and death up to 15 years after reconstruction. One-to-one propensity score matching was used to account for selection bias on patient risk factors comparing those with and without fat grafting. RESULTS: A total of 4709 patients were identified who underwent breast reconstruction after lumpectomy or mastectomy, of which 368 subsequently underwent fat grafting. In the propensity score-matched patients, fat grafting was not associated with an increased risk of lymph node metastasis (9.7% fat-grafted vs 11.4% in non-fat-grafted, P = 0.47) or distant metastasis (9.1% fat-grafted vs 10.5% in non-fat-grafted, P = 0.53). There was no increased risk of all-cause mortality after fat grafting for breast reconstruction (3.9% fat-grafted vs 6.6% non-fat-grafted, P = 0.10). CONCLUSIONS: Among breast cancer patients who subsequently underwent fat grafting, compared with no fat grafting, no significant increase was observed in distant metastasis or all-cause mortality. These findings suggest that autologous fat grafting after oncologic resection and reconstruction was not associated with an increased risk of future metastasis or death.

Attributions and perpetrators of incivility in academic surgery.

Purpose: Although incivility has been described in other specialties, little is known about the attributes and perpetrators of it in academic surgery. The goal of this study was to identify attributes and commonly associated perpetrators of incivility experienced by trainees and faculty at academic surgery programs in the U.S. Methods: A web-based survey including the Workplace Incivility Scale (WIS) and questions regarding attributions and perpetrators of incivility was sent to trainees and faculty at academic institutions across the U.S. In addition to descriptive statistics, multivariable regression models were built to determine the impact of perpetrator type and number on overall incivility scores. Results: We received 367 of 2,661 (13.8%) responses. Top three reasons for incivility were surgery hierarchy (50.1%), respondent's gender (33.8%) and intergenerational differences (28.1%). Faculty (58.6%), patients (36.8%), and nursing staff (31.9%) were the most reported parties responsible for incivility. Female surgeons reported experiencing incivility more frequently from all three top responsible parties (i.e., faculty, patients, and nurses) when compared to other gender identities. Additionally, those who reported faculty (ß = 0.61, 95%CI 0.39-0.82) or nurses (ß = 0.23, 95%CI 0.009-0.45) as perpetrators of incivility reported an increase in overall incivility scores. Conclusions: Incivility in surgery is frequently attributed to surgery hierarchy, gender, and intergenerational differences. Surgical trainees and faculty reported that faculty, patients, and nurses were the most commonly identified as responsible for uncivil events in the surgical workforce. Exposure to a greater variety of perpetrators of incivility increases overall levels of incivility, emphasizing the importance of eliminating incivility from all sources. Supplementary Information: The online version contains supplementary material available at 10.1007/s44186-023-00129-1.

Flexor Tendon Rupture Secondary to Gout.

Extra-articular deposition of monosodium urate crystals is a widely recognized manifestation of gout. However, gouty infiltration of flexor tendons in the hand resulting in tendon rupture is exceedingly rare. This case report highlights a patient with gouty infiltration of flexor tendons in the right middle finger resulting in rupture of both the flexor digitorum profundus and flexor digitorum superficialis. Given the extent of gouty infiltration and need for pulley reconstruction, the patient was treated with two-stage flexor tendon reconstruction. Febuxostat was prescribed preoperatively to limit further deposition of monosodium urate crystals and continued postoperatively to maximize the potential for long-lasting results. Prednisone was prescribed between the first- and second-stage operations to prevent a gout flare while the silicone rod was in place. In summary, tendon rupture secondary to gouty infiltration is the most likely diagnosis in patients with a history of gout presenting with tendon insufficiency.

The Current State of Fat Grafting in the Hand: A Systematic Review for Hand Diseases.

Autologous fat grafting (AFG) has traditionally been used for facial rejuvenation and soft tissue augmentation, but in recent years, its use has expanded to treat diseases of the hand. Autologous fat grafting is ideal for use in the hand because it is minimally invasive, can restore volume, and has regenerative capabilities. This review summarizes the emerging evidence regarding the safety and efficacy of AFG to the hand in several conditions, including systemic sclerosis, Dupuytren disease, osteoarthritis, burns, and traumatic fingertip injuries. A Preferred Reporting Items for Systematic Reviews and Meta-Analyses-compliant literature search on the use of AFG in hand pathologies was performed on October 8, 2020, in Ovid MEDLINE, Elsevier Embase, Clarivate Web of Science, and Wiley Cochrane Central Register of Controlled Trials. The retrieved hits were screened and reviewed by 2 independent reviewers and a third reviewer adjudicated when required. Reviewers identified 919 unique hits. Screening of the abstracts identified 22 manuscripts which described the use of AFG to treat an identified hand condition. Studies suggest AFG in the hands is a safe, noninvasive option for the management of systemic sclerosis, Dupuytren contracture, osteoarthritis, burns, and traumatic fingertip injuries. While AFG is a promising therapeutic option for autoimmune, inflammatory, and fibrotic disease manifestations in the hand, further studies are warranted to understand its efficacy and to establish more robust clinical guidelines. Studies to date show the regenerative, immunomodulatory, and volume-filling properties of AFG that facilitate wound healing and restoration of hand function with limited complications.

A Review of Laser Therapies for the Treatment of Scarring and Vascular Anomalies.

Significance: Laser use has become part of the gold standard of treatment as an effective adjuvant in multimodal therapy for pathologic scarring caused by burns, trauma, acne, and surgery, as well as vascular anomalies. Understanding indications and applications for laser therapy is essential for physicians to improve patient outcomes. Recent Advances: Since the 1980s, the medical use of lasers has continuously evolved with improvements in technology. Novel lasers and fractionated technologies are currently being studied in the hopes to improve treatment efficacy, while reducing complications. Recent advancements include acne treatment with novel picosecond lasers, new hypertrophic scar therapies with simultaneous laser and intense pulsed light use, and novel systems such as lasers with intralesional optical fiber delivery devices. In addition, optimizing the timing of laser therapy and its use in multimodal treatments continue to advance the field of photothermolysis. Critical Issues: Selecting the correct laser for a given indication is the fundamental decision when choosing a laser balancing effective treatment with minimal complications. This article covers the principles of laser therapy, the preferred lasers used for the treatment of scarring and vascular anomalies, and discusses the current evidence behind these laser choices. Future Directions: To optimize laser therapy, larger randomized control trials and split scar studies are needed. Continued advancement through better randomized controlled studies will help to improve patient outcomes on a broader scale.

Discoidin domain receptor 2 regulates aberrant mesenchymal lineage cell fate and matrix organization.

Extracellular matrix (ECM) interactions regulate both the cell transcriptome and proteome, thereby determining cell fate. Traumatic heterotopic ossification (HO) is a disorder characterized by aberrant mesenchymal lineage (MLin) cell differentiation, forming bone within soft tissues of the musculoskeletal system following traumatic injury. Recent work has shown that HO is influenced by ECM-MLin cell receptor signaling, but how ECM binding affects cellular outcomes remains unclear. Using time course transcriptomic and proteomic analyses, we identified discoidin domain receptor 2 (DDR2), a cell surface receptor for fibrillar collagen, as a key MLin cell regulator in HO formation. Inhibition of DDR2 signaling, through either constitutive or conditional Ddr2 deletion or pharmaceutical inhibition, reduced HO formation in mice. Mechanistically, DDR2 perturbation alters focal adhesion orientation and subsequent matrix organization, modulating Focal Adhesion Kinase (FAK) and Yes1 Associated Transcriptional Regulator and WW Domain Containing Transcription Regulator 1 (YAP/TAZ)-mediated MLin cell signaling. Hence, ECM-DDR2 interactions are critical in driving HO and could serve as a previously unknown therapeutic target for treating this disease process.

Macrophage TGF-ß signaling is critical for wound healing with heterotopic ossification after trauma.

Transforming growth factor-ß1 (TGF-ß1) plays a central role in normal and aberrant wound healing, but the precise mechanism in the local environment remains elusive. Here, using a mouse model of aberrant wound healing resulting in heterotopic ossification (HO) after traumatic injury, we find autocrine TGF-ß1 signaling in macrophages, and not mesenchymal stem/progenitor cells, is critical in HO formation. In-depth single-cell transcriptomic and epigenomic analyses in combination with immunostaining of cells from the injury site demonstrated increased TGF-ß1 signaling in early infiltrating macrophages, with open chromatin regions in TGF-ß1-stimulated genes at binding sites specific for transcription factors of activated TGF-ß1 (SMAD2/3). Genetic deletion of TGF-ß1 receptor type 1 (Tgfbr1; Alk5), in macrophages, resulted in increased HO, with a trend toward decreased tendinous HO. To bypass the effect seen by altering the receptor, we administered a systemic treatment with TGF-ß1/3 ligand trap TGF-ßRII-Fc, which resulted in decreased HO formation and a delay in macrophage infiltration to the injury site. Overall, our data support the role of the TGF-ß1/ALK5 signaling pathway in HO.

Tourniquet use following blast-associated complex lower limb injury and traumatic amputation promotes end organ dysfunction and amplified heterotopic ossification formation.

BACKGROUND: Traumatic heterotopic ossification (tHO) is characterized by ectopic bone formation in extra-skeletal sites leading to impaired wound healing, entrapment of neurovascular structures, pain, and reduced range of motion. HO has become a signature pathology affecting wounded military personnel who have sustained blast-associated traumatic amputations during the recent conflicts in Iraq and Afghanistan and can compound recovery by causing difficulty with prosthesis limb wearing. Tourniquet use to control catastrophic limb hemorrhage prior to surgery has become almost ubiquitous during this time, with the recognition the prolonged use may risk an ischemia reperfusion injury and associated complications. While many factors influence the formation of tHO, the extended use of tourniquets to limit catastrophic hemorrhage during prolonged field care has not been explored. METHODS: Utilizing an established pre-clinical model of blast-associated complex lower limb injury and traumatic amputation, we evaluated the effects of tourniquet use on tHO formation. Adult male rats were subjected to blast overpressure exposure, femur fracture, and soft tissue crush injury. Pneumatic tourniquet (250-300 mmHg) applied proximal to the injured limb for 150-min was compared to a control group without tourniquet, before a trans-femoral amputation was performed. Outcome measures were volume to tHO formation at 12 weeks and changes in proteomic and genomic markers of early tHO formation between groups. RESULTS: At 12 weeks, volumetric analysis with microCT imaging revealed a 70% increase in total bone formation (p = 0.007) near the site of injury compared to rats with no tourniquet time in the setting of blast-injuries. Rats subjected to tourniquet usage had increased expression of danger-associated molecular patterns (DAMPs) and end organ damage as early as 6 h and as late as 7 days post injury. The expressions of pro-inflammatory cytokines and chemokines and osteochondrogenic genes using quantitative RT-PCR similarly revealed increased expression as early as 6 h post injury, and these genes along with hypoxia associated genes remained elevated for 7 days compared to no tourniquet use. CONCLUSION: These findings suggest that tourniquet induced ischemia leads to significant increases in key transcription factors associated with early endochondral bone formation, systemic inflammatory and hypoxia, resulting in increased HO formation.

Congenital adrenal calcifications as the first clinical indication of sphingosine lyase insufficiency syndrome: A case report and review of the literature.

Sphingosine Lyase Insufficiency Syndrome (SPLIS) or SGPL1 Deficiency is a newly described entity that is characterized by steroid-resistant nephrotic syndrome, primary adrenal insufficiency, lymphopenia, ichthyosis, and/or endocrine and neurologic abnormalities. The earliest identification of SGPL1 pathogenic variants in association with this syndrome was reported in 2017. Since then, at least 36 patients have been reported with this pediatric syndrome. Here, we report a new patient with SPLIS who had a prenatal finding of adrenal calcifications, congenital nephrotic syndrome, and abnormal newborn screening concerning for Severe Combined Immunodeficiency. We conclude that SPLIS is a clinically recognizable condition with prenatal onset. This case should increase awareness of SPLIS in the differential diagnosis for adrenal calcifications. We present a case on the severe end of the clinical spectrum of SPLIS, and a review of the literature.

Large intraperitoneal lipoleiomyoma in a pre-menopausal woman: a case report.

BACKGROUND: Lipoleiomyoma is a rare, benign variant of the commonplace uterine leiomyoma. Unlike leiomyoma, these tumors are composed of smooth muscle cells admixed with mature adipose tissue. While rare, they are most frequently identified in the uterus, but even more infrequently have been described in extrauterine locations. CASE PRESENTATION: We describe a case report of a 45-year-old woman with a history of in vitro fertilization pregnancy presenting 6 years later with abdominal distention and weight loss found to have a 30-cm intra-abdominal lipoleiomyoma. While cross-sectional imaging can narrow the differential diagnosis, histopathological analysis with stains positive for smooth muscle actin, desmin, and estrogen receptor, but negative for HMB-45 confirms the diagnosis of lipoleiomyoma. The large encapsulated tumor was resected en bloc. The patients post-operative course was uneventful and her symptoms resolved. CONCLUSIONS: Lipoleiomyoma should be considered on the differential diagnosis in a woman with a large intra-abdominal mass. While considered benign, resection should be considered if the mass is symptomatic, and the diagnosis is unclear or there is a concern for malignancy.

High Frequency Spectral Ultrasound Imaging Detects Early Heterotopic Ossification in Rodents.

Heterotopic ossification (HO) is a devastating condition in which ectopic bone forms inappropriately in soft tissues following traumatic injuries and orthopedic surgeries as a result of aberrant mesenchymal progenitor cell (MPC) differentiation. HO leads to chronic pain, decreased range of motion, and an overall decrease in quality of life. While several treatments have shown promise in animal models, all must be given during early stages of formation. Methods for early determination of whether and where endochondral ossification/soft tissue mineralization (HO anlagen) develop are lacking. At-risk patients are not identified sufficiently early in the process of MPC differentiation and soft tissue endochondral ossification for potential treatments to be effective. Hence, a critical need exists to develop technologies capable of detecting HO anlagen soon after trauma, when treatments are most effective. In this study, we investigate high frequency spectral ultrasound imaging (SUSI) as a noninvasive strategy to identify HO anlagen at early time points after injury. We show that by determining quantitative parameters based on tissue organization and structure, SUSI identifies HO anlagen as early as 1-week postinjury in a mouse model of burn/tenotomy and 3 days postinjury in a rat model of blast/amputation. We analyze single cell RNA sequencing profiles of the MPCs responsible for HO formation and show that the early tissue changes detected by SUSI match chondrogenic and osteogenic gene expression in this population. SUSI identifies sites of soft tissue endochondral ossification at early stages of HO formation so that effective intervention can be targeted when and where it is needed following trauma-induced injury. Furthermore, we characterize the chondrogenic to osteogenic transition that occurs in the MPCs during HO formation and correlate gene expression to SUSI detection of the HO anlagen.

Fat Grafting Subjectively Improves Facial Skin Elasticity and Hand Function of Scleroderma Patients.

Systemic scleroderma is a chronic connective tissue disease characterized by internal organ and skin fibrosis. Unfortunately, there is a lack of efficacious treatments for cutaneous manifestations, and alternative interventions should be considered. Fat grafting has gained significant attention due to its regenerative properties and success in improving skin quality and volume deficits in fibrotic diseases. While some studies have investigated the efficacy of autologous fat grafting, we utilized the Coleman method for harvesting and processing to determine the efficacy of fat grafting to improve skin fibrosis in the hands and face of scleroderma patients without excess processing of adipose tissue. Patients with a diagnosis of scleroderma who underwent fat grafting between March 2015 and March 2019 at the University of Michigan were included. Ten female patients were identified that met inclusion criteria. The mean age at the time of surgery was 48.7 (± 17.6) years. An average of 53.2 (± 15.5) ml of fat was injected into the hands and 26.1 (± 16.4) ml into the face. Patients were treated with 1-4 rounds of grafting depending on the initial severity of skin fibrosis and volume deficiency. Fat grafting subjectively and qualitatively improved perioral skin quality, facial animation, hand range of motion, and hand pain for patients with systemic scleroderma. No complications were identified. Additional studies are necessary to determine the ideal volume, timing of treatments, and type of fat to optimize the efficacy of autologous fat grafting for the treatment of systemic scleroderma.

Novel Lineage-Tracing System to Identify Site-Specific Ectopic Bone Precursor Cells.

Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; however, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and inducible lineage-tracing mouse (Hoxa11-CreERT2;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors specifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and adipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion.

Achieving the Optimal Aesthetic Benefit While Correcting Midface Deficiency: Utilizing A High Winged Le Fort I in Cleft and Craniofacial Patients.

ABSTRACT: Craniofacial anomalies are congenital disorders that affect the cranium and facial bones, with cleft lip and palate being the most common. These anomalies are often associated with abnormal development of pharyngeal arches and can result in the development of class III malocclusion and severe maxillary retrusion. Current treatment includes orthodontic decompensation and Le Fort I osteotomy to correct the maxillomandibular relationship. However, the traditional Le Fort I (LFI) advancement does not fully address the lack of skeletal volume in the midface. The high winged Le Fort I osteotomy (HWLFI) is an excellent surgical option for simultaneous correction of the midface deficiency and malocclusion while restoring optimal esthetic convexity. A retrospective chart review was conducted to include all cleft and craniofacial patients who underwent HWLFI advancement from 2002 to 2018. Patients had a minimum of 12 months of follow-up. Patient data and complications were reviewed. Standardized facial photographs were analyzed for esthetic improvement, occlusion, and beneficial salutary effects on the midface. Forty-three patients met the inclusion criteria. The mean age at surgery was 18.9 years. The mean follow-up was 32 months. Early complications included infection (9.3%) and temporary nerve paresthesia (2.3%). Late complications included infection (6.5%), wound dehiscence (4.3%), and painful hardware (2.3%). One patient (2.3 percent) had clinically significant relapse that required surgery. Postoperatively, patients demonstrated excellent midface projection and correction of the skeletal malocclusion. The HWLFI advancement significantly improves both the malocclusion and esthetic concerns of cleft and craniofacial patients by reestablishing maximal midfacial support. Important advantages of the HWLFI are avoidance of alloplastic implant use and extensive and potentially unstable surgical procedures that increase orbital volume.

BMP Ligand Trap ALK3-Fc Attenuates Osteogenesis and Heterotopic Ossification in Blast-Related Lower Extremity Trauma.

Traumatic heterotopic ossification (tHO) commonly develops in wounded service members who sustain high-energy and blast-related traumatic amputations. Currently, no safe and effective preventive measures have been identified for this patient population. Bone morphogenetic protein (BMP) signaling blockade has previously been shown to reduce ectopic bone formation in genetic models of HO. In this study, we demonstrate the efficacy of small-molecule inhibition with LDN193189 (ALK2/ALK3 inhibition), LDN212854 (ALK2-biased inhibition), and BMP ligand trap ALK3-Fc at inhibiting early and late osteogenic differentiation of tissue-resident mesenchymal progenitor cells (MPCs) harvested from mice subjected to burn/tenotomy, a well-characterized trauma-induced model of HO. Using an established rat tHO model of blast-related extremity trauma and methicillin-resistant Staphylococcus aureus infection, a significant decrease in ectopic bone volume was observed by micro-computed tomography imaging following treatment with LDN193189, LDN212854, and ALK3-Fc. The efficacy of LDN193189 and LDN212854 in this model was associated with weight loss (17%-19%) within the first two postoperative weeks, and in the case of LDN193189, delayed wound healing and metastatic infection was observed, while ALK3-Fc was well tolerated. At day 14 following injury, RNA-Seq and quantitative reverse transcriptase-polymerase chain reaction analysis revealed that ALK3-Fc enhanced the expression of skeletal muscle structural genes and myogenic transcriptional factors while inhibiting the expression of inflammatory genes. Tissue-resident MPCs harvested from rats treated with ALK3-Fc exhibited reduced osteogenic differentiation, proliferation, and self-renewal capacity and diminished expression of genes associated with endochondral ossification and SMAD-dependent signaling pathways. Together, these results confirm the contribution of BMP signaling in osteogenic differentiation and ectopic bone formation and that a selective ligand-trap approach such as ALK3-Fc may be an effective and tolerable prophylactic strategy for tHO.

The role of neutrophil extracellular traps and TLR signaling in skeletal muscle ischemia reperfusion injury.

Ischemia reperfusion (IR) injury results in devastating skeletal muscle fibrosis. Here, we recapitulate this injury with a mouse model of hindlimb IR injury which leads to skeletal muscle fibrosis. Injury resulted in extensive immune infiltration with robust neutrophil extracellular trap (NET) formation in the skeletal muscle, however, direct targeting of NETs via the peptidylarginine deiminase 4 (PAD4) mechanism was insufficient to reduce muscle fibrosis. Circulating levels of IL-10 and TNFα were significantly elevated post injury, indicating toll-like receptor (TLR) signaling may be involved in muscle injury. Administration of hydroxychloroquine (HCQ), a small molecule inhibitor of TLR7/8/9, following injury reduced NET formation, IL-10, and TNFα levels and ultimately mitigated muscle fibrosis and improved myofiber regeneration following IR injury. HCQ treatment decreased fibroadipogenic progenitor cell proliferation and partially inhibited ERK1/2 phosphorylation in the injured tissue, suggesting it may act through a combination of TLR7/8/9 and ERK signaling mechanisms. We demonstrate that treatment with FDA-approved HCQ leads to decreased muscle fibrosis and increased myofiber regeneration following IR injury, suggesting short-term HCQ treatment may be a viable treatment to prevent muscle fibrosis in ischemia reperfusion and traumatic extremity injury.