Trauma-induced heterotopic ossification (tHO) is a condition of pathologic wound healing, defined by the progressive formation of ectopic bone in soft tissue following severe burns or trauma. Because previous studies have shown that genetic variants of HO, such as fibrodysplasia ossificans progressiva (FOP), are caused by hyperactivating mutations of the type I bone morphogenetic protein receptor (T1-BMPR) ACVR1/ALK2, studies evaluating therapies for HO have been directed primarily toward drugs for this specific receptor. However, patients with tHO do not carry known T1-BMPR mutations. Here we show that, although BMP signaling is required for tHO, no single T1-BMPR (ACVR1/ALK2, BMPR1a/ALK3, or BMPR1b/ALK6) alone is necessary for this disease, suggesting that these receptors have functional redundancy in the setting of tHO. By utilizing two different classes of BMP signaling inhibitors, we developed a translational approach to treatment, integrating treatment choice with existing diagnostic options. Our treatment paradigm balances either immediate therapy with reduced risk for adverse effects (Alk3-Fc) or delayed therapy with improved patient selection but greater risk for adverse effects (LDN-212854).
Significance Heterotopic ossification (HO) is a debilitating condition in which bone forms inappropriately within soft tissues. Two vastly different patient populations are at risk for developing HO: those with musculoskeletal trauma or severe burns and those with a genetic mutation in the bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). In this study, we demonstrate that both forms of HO share a common signaling pathway through hypoxia inducible factor-1α, and that pharmacologic inhibition or genetic knockout of this signaling pathway can mitigate and even abolish HO formation. These findings pave the way for pharmacologic inhibitors of hypoxia inducible factor-1α as therapeutic options for heterotopic ossification.
Abstract 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-CreER T2 ; Hif1a fl/fl ) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreER T2 ; Hif1a fl/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.
Purpose: Muscle has the remarkable ability to regenerate small defects, however, when the defect is large, the injured tissue results in fibrosis. Fibrotic volumetric muscle loss (VML) injuries are painful and result in dysfunctional muscle tissue characterized by dense collagen deposition. We discovered that mesenchymal progenitor cells (MPCs) regulate collagen extracellular matrix (ECM) organization after tendon injury using discoidin domain receptor 2 (DDR2). The current study aims to identify the role of DDR2 expression by fibroadipogenic progenitor cells (FAPs) after VML injury. Methods: A previously published single-cell RNA sequencing (scRNA-seq) of uninjured, regenerative 2mm, and fibrotic 3mm quadricep VML defects were analyzed (GSE163376). Analyses were performed using Seurat v4 (Hao et al., 2022). Uninjured and fibrotic injury samples were isolated for analysis. Pdgfra+ clusters were isolated and clustered for further analysis. Differential gene expression analyses were performed utilizing negative binomials. Clusters were identified by marker gene expression. Published Visium spatial transcriptomics (GSE205707) analyses of fibrotic 2mm tibialis anterior VML defects and uninjured muscle were used. VML defect zones were previously labeled (Larouche et al. 2023). Novel spatial distance analyses were performed by marking the defect region in ImageJ, matching pixel regions to spots on Visium sequencing spots, performing distance analysis from the defect to each Visium spot, and scaling the distance value to a score of 0 to 1. Monocle2 (Trapnell et al., 2014) was used to determine genes changing over distance by replacing the pseudo-time values with distance score per spot. Results: ScRNA-seq analyses of previously labeled Pdgfra+ FAPs isolated from a dataset of uninjured and fibrotic VML defect tissue showed 6 mesenchymal lineage clusters, defined by marker gene expression (A-B). All clusters were Ddr2+ (C). Visium spatial transcriptomics of VML injuries 7 and 14 days after injury showed high transcription of type 1 collagen (Col1a1) within the defect region (D). Interestingly, Pdgfra and Ddr2 expression was upregulated in the defect region compared to transition and intact muscle zones (E). Pdgfra/Ddr2 spots were found at higher proportions within the defect zone compared to transition and intact muscle zones (F). This suggests that Pdgfra+/Ddr2+ FAPs populate in the injury site and may be contributing to fibrotic collagen deposition following injury. Distance analysis was used to score spots based on distance from the defect region and determine trends of gene expression over distance (G). Col1a1 was found to decrease as distance score increased (H) compared to Desmin, which increased with increasing score (I), as expected. To determine the spatial trends in FAPs, Pdgfra and Ddr2 expression was visualized with respect to distance, which were both found to decrease as distance increased (J-K). Conclusion: These findings suggest that Ddr2-expressing FAPs are within VML defects compared to intact muscle and may be contributing to the fibrotic tissue after injury. DDR2 may serve as a therapeutic target in the prevention of post-traumatic muscle fibrosis.
From the Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California. Received October 20, 2010. Accepted for publication November 19, 2010. Address correspondence and reprint requests to Michael T. Longaker, MD, MBA, Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, 257 Campus Dr, Stanford University, Stanford, CA 94305-5148; E-mail: [email protected] This study was supported by National Institutes of Health, National Endowment for Plastic Surgery, the Oak Foundation, and Hagey Laboratory for Pediatric Regenerative Medicine to M.T.L. B.L. was supported by the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases. The authors report no conflicts of interest.
Abstract 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 TGFβ 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.
Advance care planning (ACP) is defined by the Institute of Medicine as an iterative process that involves discussing end-of-life issues, clarifying relevant values and goals of care, and embodying preferences through written documents and medical orders. ACP is predicated on the principle of respect for autonomy, which recognizes an individual’s right to accept or decline medical therapies. With the development of medical technologies that can sustain life (including mere physiologic existence), effective ACP has become a critical yet underused process for patients, their families, and clinicians. This review discusses the emergence of ACP, promises and pitfalls of advance directives, and promising approaches, including ACP interventions and research, as well as a focus on public engagement and future directions. Figures show a timeline of important advances in ACP since 1990, key features of the comprehensive ACP process, the three core aspects or pillars for implementation of ACP, stages of change for ACP behaviors, and two commercially available end-of-life games. Tables list theoretical pros and cons of advance directives, ACP resources, examples of recent research studies on ACP interventions, types and examples of ACP resources, and public engagement campaigns. This review contains 5 figures, 12 tables, and 101 references Keywords: Advance care planning, advance directive, end-of-life
PURPOSE: The difficulty of harvest and relative scarcity of bone marrow stromal cells (BMSCs) has limited the widespread use and clinical application of this technology, thereby necessitating inquiry into other therapies including adipose-derived stromal cells (ASCs). The goal of this study was to compare the ability of ASCs and BMSCs to heal mandibular defects and understand the mechanism through which this occurs. We hypothesize that ASCs will enhance fracture healing by improving vasculogenesis, while BSMCs will directly affect osteogenesis. METHODS: Male Lewis rats were radiated (35Gy), and subsequently underwent mandibular osteotomy with external fixation with implantation of two million BMSCs (n=12) or ASCs (n=16) marked with Green fluorescent protein (GFP). After 40 days, union rates were evaluated using microCT. Confocal microscopy visualized the contribution of ASCs/BMSCs to the bone regenerate. Quantitative polymerase chain reaction of ASCs/BMSCs compared expression of osteogenic and vasculogenic genes. Coculture of ASCs (n=3) or BMSCs (n=3) with human umbilical vein endothelial cells (HUVECs) was performed in vitro in transwells to measure tubule formation as a marker of vasculogenesis. RESULTS: ASC-implantation resulted in higher union rates than BMSC-implantation (union rate: 94% vs. 66%). These cells contribute indirectly to fracture healing, as GFP was not visualized at the site. BMSCs expressed osteogenic genes including osteopontin to a significantly greater degree than did ASCs, while ASCs expressed greater levels of vascular endothelial growth factor. This translated to greater tubule formation among HUVECs co-cultured with ASCs than with BMSCs (64.3 ± 7.3 vs. 23.3 ± 2.6, p=0.0008), and increased vasculogenesis in vivo in mandibles after ASC implantation. CONCLUSIONS: ASCs heal fracture defects better than BMSCs. This effect is likely mediated by indirect modulation of vasculogenesis, rather than by a direct effect on osteogenesis. Clinicians interested in cell-based therapies for irradiated bone injury should consider ASCs as a promising option, given their abundance, ease of acquisition, and improved fracture healing.
