In vivo screening identifies SPP2, a secreted factor that negatively regulates liver regeneration.

BACKGROUND AND AIMS: The liver is remarkably regenerative and can completely recover even when 80% of its mass is surgically removed. Identification of secreted factors that regulate liver growth would help us understand how organ size and regeneration are controlled but also provide candidate targets to promote regeneration or impair cancer growth. APPROACH AND RESULTS: To enrich for secreted factors that regulate growth control, we induced massive liver overgrowth with either YAP or MYC . Differentially expressed secreted factors were identified in these livers using transcriptomic analysis. To rank candidates by functionality, we performed in vivo CRISPR screening using the Fah knockout model of tyrosinemia. We identified secreted phosphoprotein-2 (SPP2) as a secreted factor that negatively regulates regeneration. Spp2 -deficient mice showed increased survival after acetaminophen poisoning and reduced fibrosis after repeated carbon tetrachloride injections. We examined the impact of SPP2 on bone morphogenetic protein signaling in liver cells and found that SPP2 antagonized bone morphogenetic protein signaling in vitro and in vivo. We also identified cell-surface receptors that interact with SPP2 using a proximity biotinylation assay coupled with mass spectrometry. We showed that SPP2's interactions with integrin family members are in part responsible for some of the regeneration phenotypes. CONCLUSIONS: Using an in vivo CRISPR screening system, we identified SPP2 as a secreted factor that negatively regulates liver regeneration. This study provides ways to identify, validate, and characterize secreted factors in vivo.

Macrophage-mediated PDGF Activation Correlates With Regenerative Outcomes Following Musculoskeletal Trauma.

OBJECTIVE: Our objective was to identify macrophage subpopulations and gene signatures associated with regenerative or fibrotic healing across different musculoskeletal injury types. BACKGROUND: Subpopulations of macrophages are hypothesized to fine tune the immune response after damage, promoting either normal regenerative, or aberrant fibrotic healing. METHODS: Mouse single-cell RNA sequencing data before and after injury were assembled from models of musculoskeletal injury, including regenerative and fibrotic mouse volumetric muscle loss (VML), regenerative digit tip amputation, and fibrotic heterotopic ossification. R packages Harmony , MacSpectrum , and Seurat were used for data integration, analysis, and visualizations. RESULTS: There was a substantial overlap between macrophages from the regenerative VML (2 mm injury) and regenerative bone models, as well as a separate overlap between the fibrotic VML (3 mm injury) and fibrotic bone (heterotopic ossification) models. We identified 2 fibrotic-like (FL 1 and FL 2) along with 3 regenerative-like (RL 1, RL 2, and RL 3) subpopulations of macrophages, each of which was transcriptionally distinct. We found that regenerative and fibrotic conditions had similar compositions of proinflammatory and anti-inflammatory macrophages, suggesting that macrophage polarization state did not correlate with healing outcomes. Receptor/ligand analysis of macrophage-to-mesenchymal progenitor cell crosstalk showed enhanced transforming growth factor ß in fibrotic conditions and enhanced platelet-derived growth factor signaling in regenerative conditions. CONCLUSION: Characterization of macrophage subtypes could be used to predict fibrotic responses following injury and provide a therapeutic target to tune the healing microenvironment towards more regenerative conditions.

Tuning Macrophage Phenotype to Mitigate Skeletal Muscle Fibrosis.

Myeloid cells are critical to the development of fibrosis following muscle injury; however, the mechanism of their role in fibrosis formation remains unclear. In this study, we demonstrate that myeloid cell-derived TGF-ß1 signaling is increased in a profibrotic ischemia reperfusion and cardiotoxin muscle injury model. We found that myeloid-specific deletion of Tgfb1 abrogates the fibrotic response in this injury model and reduces fibro/adipogenic progenitor cell proliferation while simultaneously enhancing muscle regeneration, which is abrogated by adaptive transfer of normal macrophages. Similarly, a murine TGFBRII-Fc ligand trap administered after injury significantly reduced muscle fibrosis and improved muscle regeneration. This study ultimately demonstrates that infiltrating myeloid cell TGF-ß1 is responsible for the development of traumatic muscle fibrosis, and its blockade offers a promising therapeutic target for preventing muscle fibrosis after ischemic injury.

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.

Intersections of Fibrodysplasia Ossificans Progressiva and Traumatic Heterotopic Ossification.

Heterotopic ossification (HO) is a debilitating pathology where ectopic bone develops in areas of soft tissue. HO can develop as a consequence of traumatic insult or as a result of dysregulated osteogenic signaling, as in the case of the orphan disease fibrodysplasia ossificans progressiva (FOP). Traumatic HO (tHO) formation is mediated by the complex interplay of signaling between progenitor, inflammatory, and nerve cells, among others, making it a challenging process to understand. Research into the pathogenesis of genetically mediated HO (gHO) in FOP has established a pathway involving uninhibited activin-like kinase 2 receptor (ALK2) signaling that leads to downstream osteogenesis. Current methods of diagnosis and treatment lag behind pre-mature HO detection and progressive HO accumulation, resulting in irreversible decreases in range of motion and chronic pain for patients. As such, it is necessary to draw on advancements made in the study of tHO and gHO to better diagnose, comprehend, prevent, and treat both.

Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.

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.

IGFBP2 expressing midlobular hepatocytes preferentially contribute to liver homeostasis and regeneration.

Although midlobular hepatocytes in zone 2 are a recently identified cellular source for liver homeostasis and regeneration, these cells have not been exclusively fate mapped. We generated an Igfbp2-CreER knockin strain that specifically labels midlobular hepatocytes. During homeostasis over 1 year, zone 2 hepatocytes increased in abundance from occupying 21%-41% of the lobular area. After either pericentral injury with carbon tetrachloride or periportal injury with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), IGFBP2+ cells replenished lost hepatocytes in zones 3 and 1, respectively. IGFBP2+ cells also preferentially contributed to regeneration after 70% partial hepatectomy, as well as liver growth during pregnancy. Because IGFBP2 labeling increased substantially with fasting, we used single nuclear transcriptomics to explore zonation as a function of nutrition, revealing that the zonal division of labor shifts dramatically with fasting. These studies demonstrate the contribution of IGFBP2-labeled zone 2 hepatocytes to liver homeostasis and regeneration.

TrkA+ Neurons Induce Pathologic Regeneration After Soft Tissue Trauma.

Heterotopic ossification (HO) is a dynamic, complex pathologic process that often occurs after severe polytrauma trauma, resulting in an abnormal mesenchymal stem cell differentiation leading to ectopic bone growth in soft-tissues including tendons, ligaments, and muscles. The abnormal bone structure and location induce pain and loss of mobility. Recently, we observed that NGF (Nerve growth factor)-responsive TrkA (Tropomyosin receptor kinase A)-expressing nerves invade sites of soft-tissue trauma, and this is a necessary feature for heterotopic bone formation at sites of injury. Here, we assayed the effects of the partial TrkA agonist Gambogic amide (GA) in peritendinous heterotopic bone after extremity trauma. Mice underwent HO induction using the burn/tenotomy model with or without systemic treatment with GA, followed by an examination of the injury site via radiographic imaging, histology, and immunohistochemistry. Single-cell RNA Sequencing confirmed an increase in neurotrophin signaling activity after HO-inducing extremity trauma. Next, TrkA agonism led to injury site hyper-innervation, more brisk expression of cartilage antigens within the injured tendon, and a shift from FGF to TGFß signaling activity among injury site cells. Nine weeks after injury, this culminated in higher overall levels of heterotopic bone among GA-treated animals. In summary, these studies further link injury site hyper-innervation with increased vascular ingrowth and ultimately heterotopic bone after trauma. In the future, modulation of TrkA signaling may represent a potent means to prevent the trauma-induced heterotopic bone formation and improve tissue regeneration.

Single-cell mapping of regenerative and fibrotic healing responses after musculoskeletal injury.

After injury, a cascade of events repairs the damaged tissue, including expansion and differentiation of the progenitor pool and redeposition of matrix. To guide future wound regeneration strategies, we compared single-cell sequencing of regenerative (third phalangeal element [P3]) and fibrotic (second phalangeal element [P2]) digit tip amputation (DTA) models as well as traumatic heterotopic ossification (HO; aberrant). Analyses point to a common initial response to injury, including expansion of progenitors, redeposition of matrix, and activation of transforming growth factor ß (TGF-ß) and WNT pathways. Surprisingly, fibrotic P2 DTA showed greater transcriptional similarity to HO than to regenerative P3 DTA, suggesting that gene expression more strongly correlates with healing outcome than with injury type or cell origin. Differential analysis and immunostaining revealed altered activation of inflammatory pathways, such as the complement pathway, in the progenitor cells. These data suggests that common pathways are activated in response to damage but are fine tuned within each injury. Modulating these pathways may shift the balance toward regenerative outcomes.

Contemporary perspectives on heterotopic ossification.

Heterotopic ossification (HO) is the formation of ectopic bone that is primarily genetically driven (fibrodysplasia ossificans progressiva [FOP]) or acquired in the setting of trauma (tHO). HO has undergone intense investigation, especially over the last 50 years, as awareness has increased around improving clinical technologies and incidence, such as with ongoing wartime conflicts. Current treatments for tHO and FOP remain prophylactic and include NSAIDs and glucocorticoids, respectively, whereas other proposed therapeutic modalities exhibit prohibitive risk profiles. Contemporary studies have elucidated mechanisms behind tHO and FOP and have described new distinct niches independent of inflammation that regulate ectopic bone formation. These investigations have propagated a paradigm shift in the approach to treatment and management of a historically difficult surgical problem, with ongoing clinical trials and promising new targets.

Neuron-to-vessel signaling is a required feature of aberrant stem cell commitment after soft tissue trauma.

The functional interdependence of nerves and blood vessels is a well-established concept during tissue morphogenesis, yet the role of neurovascular coupling in proper and aberrant tissue repair is an emerging field of interest. Here, we sought to define the regulatory relationship of peripheral nerves on vasculature in a severe extremity trauma model in mice, which results in aberrant cell fate and heterotopic ossification (HO). First, a high spatial degree of neurovascular congruency was observed to exist within extremity injury associated heterotopic ossification. Vascular and perivascular cells demonstrate characteristic responses to injury, as assessed by single cell RNA sequencing. This vascular response to injury was blunted in neurectomized mice, including a decrease in endothelial proliferation and type H vessel formation, and a downregulation of key transcriptional networks associated with angiogenesis. Independent mechanisms to chemically or genetically inhibit axonal ingrowth led to similar deficits in HO site angiogenesis, a reduction in type H vessels, and heterotopic bone formation. Finally, a combination of single cell transcriptomic approaches within the dorsal root ganglia identified key neural-derived angiogenic paracrine factors that may mediate neuron-to-vascular signaling in HO. These data provide further understanding of nerve-to-vessel crosstalk in traumatized soft tissues, which may reflect a key determinant of mesenchymal progenitor cell fate after injury.

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.

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.

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.

NGF-TrkA signaling dictates neural ingrowth and aberrant osteochondral differentiation after soft tissue trauma.

Pain is a central feature of soft tissue trauma, which under certain contexts, results in aberrant osteochondral differentiation of tissue-specific stem cells. Here, the role of sensory nerve fibers in this abnormal cell fate decision is investigated using a severe extremity injury model in mice. Soft tissue trauma results in NGF (Nerve growth factor) expression, particularly within perivascular cell types. Consequently, NGF-responsive axonal invasion occurs which precedes osteocartilaginous differentiation. Surgical denervation impedes axonal ingrowth, with significant delays in cartilage and bone formation. Likewise, either deletion of Ngf or two complementary methods to inhibit its receptor TrkA (Tropomyosin receptor kinase A) lead to similar delays in axonal invasion and osteochondral differentiation. Mechanistically, single-cell sequencing suggests a shift from TGFß to FGF signaling activation among pre-chondrogenic cells after denervation. Finally, analysis of human pathologic specimens and databases confirms the relevance of NGF-TrkA signaling in human disease. In sum, NGF-mediated TrkA-expressing axonal ingrowth drives abnormal osteochondral differentiation after soft tissue trauma. NGF-TrkA signaling inhibition may have dual therapeutic use in soft tissue trauma, both as an analgesic and negative regulator of aberrant stem cell differentiation.

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.

Activin A does not drive post-traumatic heterotopic ossification.

Heterotopic ossification (HO), the formation of ectopic bone in soft tissues, has been extensively studied in its two primary forms: post-traumatic HO (tHO) typically found in patients who have experienced musculoskeletal or neurogenic injury and in fibrodysplasia ossificans progressiva (FOP), where it is genetically driven. Given that in both diseases HO arises via endochondral ossification, the molecular mechanisms behind both diseases have been postulated to be manifestations of similar pathways including those activated by BMP/TGFß superfamily ligands. A significant step towards understanding the molecular mechanism by which HO arises in FOP was the discovery that FOP causing ACVR1 variants trigger HO in response to activin A, a ligand that does not activate signaling from wild type ACVR1, and that is not inherently osteogenic in wild type settings. The physiological significance of this finding was demonstrated by showing that activin A neutralizing antibodies stop HO in two different genetically accurate mouse models of FOP. In order to explore the role of activin A in tHO, we performed single cell RNA sequencing and compared the expression of activin A as well as other BMP pathway genes in tHO and FOP HO. We show that activin A is expressed in response to injury in both settings, but by different types of cells. Given that wild type ACVR1 does not transduce signal when engaged by activin A, we hypothesized that inhibition of activin A will not block tHO. Nonetheless, as activin A was expressed in tHO lesions, we tested its inhibition and compared it with inhibition of BMPs. We show here that anti-activin A does not block tHO, whereas agents such as antibodies that neutralize ACVR1 or ALK3-Fc (which blocks osteogenic BMPs) are beneficial, though not completely curative. These results demonstrate that inhibition of activin A should not be considered as a therapeutic strategy for ameliorating tHO.

Small molecule inhibition of non-canonical (TAK1-mediated) BMP signaling results in reduced chondrogenic ossification and heterotopic ossification in a rat model of blast-associated combat-related lower limb trauma.

Heterotopic ossification (HO) is defined as ectopic bone formation around joints and in soft tissues following trauma, particularly blast-related extremity injuries, thermal injuries, central nerve injuries, or orthopaedic surgeries, leading to increased pain and diminished quality of life. Current treatment options include pharmacotherapy with non-steroidal anti-inflammatory drugs, radiotherapy, and surgical excision, but these treatments have limited efficacy and have associated complication profiles. In contrast, small molecule inhibitors have been shown to have higher specificity and less systemic cytotoxicity. Previous studies have shown that bone morphogenetic protein (BMP) signaling and downstream non-canonical (SMAD-independent) BMP signaling mediated induction of TGF-ß activated kinase-1 (TAK1) contributes to HO. In the current study, small molecule inhibition of TAK1, NG-25, was evaluated for its efficacy in limiting ectopic bone formation following a rat blast-associated lower limb trauma and a murine burn tenotomy injury model. A significant decrease in total HO volume in the rat blast injury model was observed by microCT imaging with no systemic complications following NG-25 therapy. Furthermore, tissue-resident mesenchymal progenitor cells (MPCs) harvested from rats treated with NG-25 demonstrated decreased proliferation, limited osteogenic differentiation capacity, and reduced gene expression of Tac1, Col10a1, Ibsp, Smad3, and Sox2 (P < 0.05). Single cell RNA-sequencing of murine cells harvested from the injury site in a burn tenotomy injury model showed increased expression of these genes in MPCs during stages of chondrogenic differentiation. Additional in vitro cell cultures of murine tissue-resident MPCs and osteochondrogenic progenitors (OCPs) treated with NG-25 demonstrated reduced chondrogenic differentiation by 10.2-fold (P < 0.001) and 133.3-fold (P < 0.001), respectively, as well as associated reduction in chondrogenic gene expression. Induction of HO in Tak1 knockout mice demonstrated a 7.1-fold (P < 0.001) and 2.7-fold reduction (P < 0.001) in chondrogenic differentiation of murine MPCs and OCPs, respectively, with reduced chondrogenic gene expression. Together, our in vivo models and in vitro cell culture studies demonstrate the importance of TAK1 signaling in chondrogenic differentiation and HO formation and suggest that small molecule inhibition of TAK1 is a promising therapy to limit the formation and progression of HO.

Immobilization after injury alters extracellular matrix and stem cell fate.

Cells sense the extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction, which alters cell maintenance, proliferation, and differentiation. Here we use a mouse model of trauma-induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact mesenchymal progenitor cell (MPC) fate. After injury, single-cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional coactivator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, and signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, whereas in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.