Heterotopic Ossification Model Research Articles

Elucidating the Mechanism of Radiation Therapy on Mesenchymal Cell Fate in Preventing Heterotopic Ossification.

Radiation therapy is a clinically proven, localized preventive measure for heterotopic ossification (HO). Despite its efficacy, there is a lack of standardization of radiation prescription dosing and fractionation, and the mechanism of the impact of radiation in HO prevention remains unknown. Here, using an established mouse model of traumatic HO induced by burn and tenotomy, we demonstrate that 7Gy in one fraction delivered to the injury site within 72 hours postoperatively significantly decreases HO formation and improves hindlimb range of motion. In-depth single-cell transcriptomic analyses, in combination with immunofluorescent staining, demonstrate decreased cellular numbers as well as aberrant endochondral differentiation and downregulation of associated upstream signaling pathways in irradiated mesenchymal progenitor cells. Our study provides the framework for future mechanistic and clinically relevant studies exploring radiation efficacy in preventing HO formation.

Exploring the Link Between Autophagy-Lysosomal Dysfunction and Early Heterotopic Ossification in Tendons.

Heterotopic ossification (HO), the pathological formation of bone within soft tissues such as tendon and muscle, is a notable complication resulting from severe injury. While soft tissue injury is necessary for HO development, the specific molecular pathology responsible for trauma-induced HO remains a mystery. The previous study detected abnormal autophagy function in the early stages of tendon HO. Nevertheless, it remains to be determined whether autophagy governs the process of HO generation. Here, trauma-induced tendon HO model is used to investigate the relationship between autophagy and tendon calcification. In the early stages of tenotomy, it is observed that autophagic flux is significantly impaired and that blocking autophagic flux promoted the development of more rampant calcification. Moreover, Gt(ROSA)26sor transgenic mouse model experiments disclosed lysosomal acid dysfunction as chief reason behind impaired autophagic flux. Stimulating V-ATPase activity reinstated both lysosomal acid functioning and autophagic flux, thereby reversing tendon HO. This present study demonstrates that autophagy-lysosomal dysfunction triggers HO in the stages of tendon injury, with potential therapeutic targeting implications for HO.

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.

Periodic static compression of micro-strain pattern regulates endochondral bone formation.

Introduction: Developmental engineering based on endochondral ossification has been proposed as a potential strategy for repairing of critical bone defects. Bone development is driven by growth plate-mediated endochondral ossification. Under physiological conditions, growth plate chondrocytes undergo compressive forces characterized by micro-mechanics, but the regulatory effect of micro-mechanical loading on endochondral bone formation has not been investigated. Methods: In this study, a periodic static compression (PSC) model characterized by micro-strain (with 0.5% strain) was designed to clarify the effects of biochemical/mechanical cues on endochondral bone formation. Hydrogel scaffolds loaded with bone marrow mesenchymal stem cells (BMSCs) were incubated in proliferation medium or chondrogenic medium, and PSC was performed continuously for 14 or 28days. Subsequently, the scaffold pretreated for 28days was implanted into rat femoral muscle pouches and femoral condylar defect sites. The chondrogenesis and bone defect repair were evaluated 4 or 10weeks post-operation. Results: The results showed that PSC stimulation for 14days significantly increased the number of COL II positive cells in proliferation medium. However, the chondrogenic efficiency of BMSCs was significantly improved in chondrogenic medium, with or without PSC application. The induced chondrocytes (ichondrocytes) spontaneously underwent hypertrophy and maturation, but long-term mechanical stimulation (loading for 28days) significantly inhibited hypertrophy and mineralization in ichondrocytes. In the heterotopic ossification model, no chondrocytes were found and no significant difference in terms of mineral deposition in each group; However, 4weeks after implantation into the femoral defect site, all scaffolds that were subjected to biochemical/mechanical cues, either solely or synergistically, showed typical chondrocytes and endochondral bone formation. In addition, simultaneous biochemical induction/mechanical loading significantly accelerated the bone regeneration. Discussion: Our findings suggest that microstrain mechanics, biochemical cues, and in vivo microenvironment synergistically regulate the differentiation fate of BMSCs. Meanwhile, this study shows the potential of micro-strain mechanics in the treatment of critical bone defects.

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.

Suppression of Overactive Insulin-Like Growth Factor 1 Attenuates Trauma-Induced Heterotopic Ossification in Mice

Heterotopic ossification (HO) is the ectopic bone formation in soft tissues. Aside from hereditary HO, traumatic HO is common after orthopedic surgery, combat-related injuries, severe burns, or neurologic injuries. Recently, mTOR has been demonstrated to be involved in the chondrogenic and osteogenic processes of HO formation. However, its upstream signaling mechanism remains unknown. In this study, using an Achilles tendon puncture-induced HO model, we revealed that overactive insulin-like growth factor 1 (IGF-1) was involved in the progression of HO in mice. Micro-CT imaging showed that IGF-1 not only accelerated the rate of osteogenesis and increased ectopic bone volume but also induced spontaneous ectopic bone formation in undamaged Achilles tendons. Blocking IGF-1 activity with IGF-1 antibody or IGF-1 receptor (IGF-1R) inhibitor Picropodophyllin significantly inhibited HO formation. Mechanistically, IGF-1/IGF-1R activates PI3K/Akt signaling to promote the phosphorylation of mTOR, resulting in the chondrogenic and osteogenic differentiation of tendon-derived stem cells (TDSCs) into chondrocytes and osteoblasts in vitro and in vivo. Inhibitors of PI3K (LY294002) and mTOR (Rapamycin) both suppressed the IGF-1-stimulated mTOR signal and mitigated the formation of ectopic bones significantly. In conclusion, our results indicated that IGF-1 mediated the progression of traumatic HO through PI3K/Akt/mTOR signaling, and suppressing IGF-1 signaling cascades attenuated HO formation, providing a promising therapeutic strategy targeting HO.

NIR-triggered photodynamic therapy of traumatic heterotopic ossification with a type II collagen-targeted photosensitizer

Traumatic heterotopic ossification (HO) represents an intractable sequela following trauma with no currently effective prophylaxis or treatment. Photodynamic therapy (PDT) is a non-invasive treatment for various proliferative diseases. However, the specific effects of PDT on HO development remain unclear. In this study, the therapeutic potential of a near-infrared (NIR) probe-WL-808, composed of type II collagen-binding peptide (WYRGRL) and a PDT photosensitizer (IR-808), was evaluated for the innovative HO-targeted PDT approach. In vitro studies indicated that WL-808 could induce chondrocyte apoptosis and inhibit cell viability through ROS generation under NIR excitation. In vivo, the efficacy of WL-808-mediated PDT was tested on the tenotomy HO model mice. WL-808 specifically targeted the type II collagen cartilaginous template of HO, promoting cell apoptosis and enhancing extracellular matrix (ECM) degradation under 808 nm NIR excitation, which inhibited the final ectopic bone formation. Moreover, no obvious toxicity or side effects were detected after treatment with WL-808. Taken together, WL-808-mediated PDT significantly diminished ectopic cartilage and subsequent bone formation, providing a new perspective for HO prophylaxis and treatment.

Macrophage-Derived Extracellular DNA Initiates Heterotopic Ossification.

Heterotopic ossification (HO) severely affects people's lives; however, its pathological mechanism remains poorly understood. Although extracellular DNA (ecDNA) has been shown to play important roles in pathological calcification, its effects in HO development and progression remain unknown. The in vivo rat Achilles tendon injury model and in vitro collagen I calcification model were used to evaluate the effects of ecDNA in the ectopic calcifications and the main cell types involved in those pathological process. Histology, immunofluorescent staining, reverse transcriptase-polymerase chain reaction analysis and micro-computed tomography were used to identify the distribution of macrophage-derived ecDNA and elucidate their roles in HO. The results showed that the amount of ecDNA and ectopic calcification increased significantly and exhibited a strong correlation in the injured tendons of HO model compared with those of the controls, which was accompanied by a significantly increased number of M2 macrophages in the injured tendon. During in vitro co-culture experiments, M2 macrophages calcified the reconstituted type I collagen and ectopic bone collected from the injured tendons of HO rats, while those effects were inhibited by deoxyribonuclease. More importantly, deoxyribonuclease reversed the pathological calcification in the injured rat tendon HO model. The present study showed that ecDNA from M2 macrophages initiates pathological calcification in HO, and the elimination of ecDNA might be developed into a clinical strategy to prevent ectopic mineralization diseases. The use of deoxyribonuclease for the targeted degradation of ecDNA at affected tissue sites provides a potential solution to treat diseases associated with ectopic mineralization.

Neutrophil and NETosis Modulation in Traumatic Heterotopic Ossification.

To characterize the role of neutrophil extracellular traps (NETs) in heterotopic ossification (HO) formation and progression and to use mechanical and pharmacological methods to decrease NETosis and mitigate HO formation. Traumatic HO is the aberrant osteochondral differentiation of mesenchymal progenitor cells after traumatic injury, burns, or surgery. While the innate immune response has been shown to be necessary for HO formation, the specific immune cell phenotype and function remain unknown. Neutrophils, one of the earliest immune cells to respond after HO-inducing injuries, can extrude DNA, forming highly inflammatory NETs. We hypothesized that neutrophils and NETs would be diagnostic biomarkers and therapeutic targets for the detection and mitigation of HO. C57BL6J mice underwent burn/tenotomy (a well-established mouse model of HO) or a non-HO-forming sham injury. These mice were either (1) ambulated ad libitum, (2) ambulated ad libitum with daily intraperitoneal hydroxychloroquine, ODN-2088 (both known to affect NETosis pathways), or control injections, or (3) had the injured hind limb immobilized. Single-cell analysis was performed to analyze neutrophils, NETosis, and downstream signaling after the HO-forming injury. Immunofluorescence microscopy was used to visualize NETosis at the HO site and neutrophils were identified using flow cytometry. Serum and cell lysates from HO sites were analyzed using enzyme-linked immunosorbent assay for myeloperoxidase-DNA and ELA2-DNA complexes to identify NETosis. Micro-computerized tomography was performed on all groups to analyze the HO volume. Molecular and transcriptional analyses revealed the presence of NETs within the HO injury site, which peaked in the early phases after injury. These NETs were highly restricted to the HO site, with gene signatures derived from both in vitro NET induction and clinical neutrophil characterizations showing a high degree of NET "priming" at the site of injury, but not in neutrophils in the blood or bone marrow. Cell-cell communication analyses revealed that this localized NET formation coincided with high levels of toll-like receptor signaling specific to neutrophils at the injury site. Reducing the overall neutrophil abundance within the injury site, either pharmacologically through treatment with hydroxychloroquine, the toll-like receptor 9 inhibitor OPN-2088, or mechanical treatment with limb offloading, results in the mitigation of HO formation. These data provide a further understanding of the ability of neutrophils to form NETs at the injury site, clarify the role of neutrophils in HO, and identify potential diagnostic and therapeutic targets for HO mitigation.

Ethyl caffeate inhibits macrophage polarization via SIRT1/NF-κB to attenuate traumatic heterotopic ossification in mice

Heterotopic ossification (HO) denotes the presence of mature bone tissue in soft tissues or around joints. Inflammation is a key driver of traumatic HO, and macrophages play an important role in this process. Ethyl caffeate (ECF), a critical active compound found in Petunia, exerts significant anti-inflammatory effects. Herein, we established a mouse model of HO by transection of the Achilles tendon and back burn and found abundant macrophage infiltration in the early stage of HO, which decreased with time. In vitro and in vivo experiments indicated that ECF inhibited macrophage polarization, and mechanistic studies showed that it inhibited the SIRT1/NF-κB signalling pathway, thereby suppressing the release of downstream inflammatory cytokines. ECF reduced HO in mice, and its effect was comparable to indomethacin (INDO). In vitro studies revealed that ECF did not directly affect the mineralization of mesenchymal stem cells (MSCs) or osteogenic differentiation but inhibited these processes by reducing the level of inflammatory cytokines in the conditioned medium (CM). Thus, M1 macrophages may play a crucial role in the pathogenesis of HO, and ECF is a prospective candidate for the prevention of trauma-induced HO. Data availabilityData will be made available on request

MTORC1 coordinates NF-κB signaling pathway to promote chondrogenic differentiation of tendon cells in heterotopic ossification.

Heterotopic ossification (HO) is a pathological bone formation based on endochondral ossification distinguished by ossification within muscles, tendons, or other soft tissues. There has been growing studies focusing on the treatment with rapamycin to inhibit HO, but the mechanism of mTORC1 on HO remains unclear. Tendon cells (TDs) are the first cells to form during tendon heterotopic ossification. Here, we used an in vivo model of HO and an in vitro model of chondrogenesis induction to elucidate the effect and underlying mechanism of mTORC1 in HO. The current study highlights the effect of rapamycin on murine Achilles tenotomy-induced HO and the role of mTORC1 signaling pathway on TDs. Our result showed that mTORC1 was activation in the early stage of HO, whereas the mTORC1 maintained low expression in the mature ectopic cartilage tissue and the ectopic bone formation sites. The use of mTORC1-specific inhibitor (rapamycin) immediately after Achilles tendon injury could suppress the formation of HO; once ectopic cartilage and bone had formed, treatment with rapamycin could not significantly inhibit the progression of HO. Mechanistically, mTORC1 stimulation by silencing of TSC1 promoted the expression of the chondrogenic markers in TDs. In TDs, treated with mTORC1 stimulation by silencing of TSC1, mTORC1 increased the activation of the NF-κB signaling pathway. NF-κB selective inhibitor BAY11-7082 significantly suppressed the chondrogenesis of TDs that treated with mTORC1 stimulation by silencing of TSC1. Together, our findings demonstrated that mTORC1 promoted HO by regulating TDs chondrogenesis partly through the NF-κB signaling pathway; and rapamycin could be a viable HO therapeutic regimen.

9. Novel murine model of heterotopic ossification induced by spinal cord injury

<h3>BACKGROUND CONTEXT</h3> Heterotopic ossification (HO) is a painful and debilitating condition causing abnormal bone formation in the soft tissues reported in up to 50% of polytrauma patients with spinal cord injury (SCI) and limb damage. In the absence of knowledge regarding the pathogenesis of HO, prophylactic treatment with NSAIDs, bisphosphonates, and radiation therapy do little to relieve the chronic pain and joint contractures resulting from HO or prevent its recurrence after surgical intervention to remove ectopic bone. A lack of clinically relevant animal models has been a major obstacle to understanding the pathogenesis of SCI-induced HO. <h3>PURPOSE</h3> The goal of this research is to characterize a clinically relevant mouse model of SCI-induced HO based on the hypothesis that simultaneous SCI and musculotendinous injury (MTI) can cause HO. <h3>METHODS</h3> Four-week-old C57BL/6J wild type mice were subjected to spinal cord transection between T10 and T11 using a pair of spring scissors via posterior open incision. The quadriceps were then crushed using a surgical clamp and a No. 15 surgical blade used to induce a 2mm MTI at the quadriceps insertion into patella in the LEFT hindlimb. The RIGHT hindlimb remained intact as internal procedural control. During the postoperative course, urine was expelled from the paraplegic using twice daily bladder massage. Soft food and water were placed in petri dishes on the bottom of the cages. Buprenorphine SR at 1mg/kg was administered subcutaneously on Day 0 and Day 4 to provide postoperative analgesia. All mice were euthanized at postoperative Day 21 when both hindlegs were dis-articulated at hip and ankle and fixed in formaldehyde overnight. High resolution micro-CT scans of the specimens were captured at the spatial resolution of 8µm. The volume of ectopic bone around the knee joint was quantified in a 3D reconstructed model. Undecalcified samples were then dehydrated, infiltrated, and embedded in low temperature MMA, and sectioned at 5µm. Consecutive sections were assessed by Von Kossa and Toluidine Blue staining to show ossification of peri-articular soft tissues, alkaline phosphatase (ALP) staining for osteoblasts, and tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts. Mann-Whitney U test was used for statistical analysis of ectopic bone volumes. <h3>RESULTS</h3> Of the seven mice euthanized at three weeks post SCI/MTI, two sustained patellar dislocation in the LEFT knee and were excluded from analyses. Quantitative microCT analysis of the remaining five revealed ectopic bone deposits in the peri-articular region of all LEFT knees but none in the RIGHT knees. The HO volume of the LEFT hindlimb was consistently higher than that of RIGHT hindlimb of each mouse (25th - 75th percentile 0.02 - 0.34 vs 0.00 - 0.00, P=0.09). Histological evaluation confirmed ectopic bone deposited in the peri-articular soft tissues of the LEFT knees while the musculotendinous tissue was intact on the RIGHT side. <h3>CONCLUSIONS</h3> This mouse model represents a novel, reproducible and clinically relevant preclinical model of SCI-induced HO that will facilitate investigation of pathogenic mechanisms and assessment of novel therapeutics for this condition. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

The Kinesin Gene KIF26B Modulates the Severity of Post-Traumatic Heterotopic Ossification.

The formation of pathological bone deposits within soft tissues, termed heterotopic ossification (HO), is common after trauma. However, the severity of HO formation varies substantially between individuals, from relatively isolated small bone islands through to extensive soft tissue replacement by bone giving rise to debilitating symptoms. The aim of this study was to identify novel candidate therapeutic molecular targets for severe HO. We conducted a genome-wide scan in men and women with HO of varying severity following hip replacement for osteoarthritis. HO severity was dichotomized as mild or severe, and association analysis was performed with adjustment for age and sex. We next confirmed expression of the gene encoded by the lead signal in human bone and in primary human mesenchymal stem cells. We then examined the effect of gene knockout in a murine model of osseous trans-differentiation, and finally we explored transcription factor phosphorylation in key pathways perturbed by the gene. Ten independent signals were suggestively associated with HO severity, with KIF26B as the lead. We subsequently confirmed KIF26B expression in human bone and upregulation upon BMP2-induced osteogenic differentiation in primary human mesenchymal stem cells, and also in a rat tendo-Achilles model of post-traumatic HO. CRISPR-Cas9 mediated knockout of Kif26b inhibited BMP2-induced Runx2, Sp7/Osterix, Col1A1, Alp, and Bglap/Osteocalcin expression and mineralized nodule formation in a murine myocyte model of osteogenic trans-differentiation. Finally, KIF26B deficiency inhibited ERK MAP kinase activation during osteogenesis, whilst augmenting p38 and SMAD 1/5/8 phosphorylation. Taken together, these data suggest a role for KIF26B in modulating the severity of post-traumatic HO and provide a potential novel avenue for therapeutic translation.

QS10. Understanding the Immune Cell Niche at the Injury Site and Draining Lymph Node Following Musculoskeletal Trauma

Purpose: Following trauma, the body responds by mounting a complex immune response involving close coordination between the local wound site and draining regional lymph nodes (LN). Mesenchymal progenitor cells (MPCs) and macrophages contribute to the aberrant injury response through alterations of the microenvironment and modulation of the immune response. Crosstalk between MPCs and immune cells through receptor-ligand interactions allow for host defense and initiation of the wound healing cascade. Aberrations to this process can lead to pathologic conditions such as heterotopic ossification (HO). Previous research has demonstrated that immune cells at the injury site play an essential role in HO development. Concurrently, we recently observed swelling of regional LNs which we have shown to necessary for HO formation. However, the cellular communication patterns at the regional LN that drive aberrant cell fate remain unknown. Given these findings we looked to evaluate the roles of immune cells at the injury site and draining LN in HO using single-cell RNA sequencing (scRNAseq). Methods: We utilized the burn/tenotomy (BT) model of HO in C57B6 mice to evaluate scRNAseq at the injury site and regional LN. The popliteal LN was collected from uninjured, 3 and 21 days post-injury and underwent scRNAseq utilizing the 10x Genomics platform and Illumina HiSeq 4000. Seurat 4.0.1 was used for downstream processing. We used a previously described HO single-cell dataset for analysis (GSE150995). CellChat and iTalk were used to evaluate ligand-receptor interactions between cell populations. Flow cytometry and immunofluorescent staining was used to validate immune cell characterization at 0, 3 and 7-days from the draining LN. Results: Analysis of receptor-ligand matching at the HO injury site demonstrated an increase in Cxcl12-Cxcr4 signaling between MPCs and macrophages, respectively, between uninjured, day 7 and day 42. Evaluation of gene expression of Cxcl12 shows relatively constitutive expression in MPCs where Cxcr4 increases in macrophages following trauma. Following BT there was a substantial increase in the size of the draining popliteal LN 3 and 7 days following injury. Flow cytometry analysis evaluating immune cell composition demonstrated an initial increase in macrophages which returned to baseline at 7 days. Unsupervised clustering of our LN single-cell dataset revealed 10 unique immune cell clusters. iTalk receptor-ligand matching demonstrated and increase TGFβ1 expression in the macrophage population with increasing signaling via TGFβr2 in the B cell population. Conclusion: ScRNAseq has allowed for a more in-depth analysis of the complex interactions between immune cells following extremity trauma at both the injury site and the regional LN. We demonstrate unique signaling between MPCs and immune cells at the HO site suggesting a role the MPCs may play in recruiting immune cells following injury. Within the draining LN we demonstrate the potential role of TGFβ1-TGFβr2 signaling between macrophages and B cells, respectively. A better understanding of the immune cell niche in HO will help with future therapeutic targets.

Palovarotene inhibits the NF-κB signalling pathway to prevent heterotopic ossification.

Heterotopic ossification (HO) is a common disease characterized by pain, dysfunction and calcification. The mechanisms underlying HO have not been completely elucidated. Palovarotene, a retinoic acid receptor γ agonist, significantly inhibits the formation of HO in vivo. However, its specific mechanism of action remains unclear. Therefore, we aimed to evaluate the signalling pathways related to the formation of HO as well as the mechanism of palovarotene action. We constructed in vitro and in vivo models of HO. Osteogenic differentiation of bone mesenchymal stem cells (BMSCs) was observed by alizarin red and alkaline phosphatase staining assays in vitro. X-ray and haematoxylin-eosin staining were performed in vivo. Western blots and reverse transcription-polymerase chain reaction were performed to determine the levels of osteogenic- and inflammation-related genes. Immunofluorescence and immunocytochemistry were used to assess the levels of p65, the core molecule of the nuclear factor κ-B (NF-κB) signalling pathway. We demonstrated that, in vitro, under inflammatory stimulation, pathological calcium deposition increased in BMSCs. The levels of osteogenesis- and inflammation-related genes were also upregulated, along with an enhanced expression of p65. Immunofluorescence assays revealed that p65 entered the nucleus, thereby stimulating the downstream effectors of the NF-κB pathway. The above trends were reversed after palovarotene treatment. In conclusion, the NF-κB signalling pathway played an important role in HO, and palovarotene could alleviate HO by blocking the NF-κB cascade. Our results may provide a theoretical basis for palovarotene in the treatment of HO. Further studies on the side effects of palovarotene are warranted in the future.

Galunisertib attenuates progression of trauma-induced heterotopic ossification via blockage of Smad2/3 signaling in mice

Heterotopic ossification (HO) is the formation of bony tissues in the extraskeletal system. To date, no effective therapy has been developed for the treatment of HO, although increasing evidences have shown that inhibition of TGF-β signaling has potential as a new therapeutic approach for attenuating HO progression. Results from previous clinical trials have demonstrated that patients with malignant tumors exhibit excellent tolerability to Galunisertib, a TGF-β receptor I kinase inhibitor. However, its therapeutic potential in preventing HO and inhibitory effect on osteogenesis remain unclear. In this study, we demonstrated that intragastrical administration of Galunisertib, at a concentration as low as 10 mg/kg, was not only fairly effective in preventing HO development in a dose-dependent manner, but also generated a non-toxic response in a novel Achilles tendon puncture-induced traumatic HO model in mice. Moreover, Galunisertib treatment in the early phases of HO development, including the inflammatory and chondrogenic period, resulted in better therapeutic effects instead of eliminating already formed bony tissues. Mechanistically, Galunisertib suppressed the osteogenic differentiation capacity of tendon-derived stem cells (TDSCs) by interfering with the Smad2/3 signaling pathway, blocking the phosphorylation of Smad2/3 translocated from cytoplasm into the nucleus to regulate the expression of both osteogenesis-related transcription factors and related proteins. Results from in vivo experiments further validated Galunisertib's effect on HO attenuation, by intercepting the TGF-β/Smad2/3 signaling pathway. In conclusion, our findings demonstrated Galunisertib's potential as a prophylactic drug for the treatment of traumatic HO or other related diseases triggered by over-expressed TGF-β.

A Network Pharmacology Study: Reveal the Mechanisms of Palovarotene Against Heterotopic Ossification.

Heterotopic ossification (HO) occurs when bone forms within non-ossifying tissues, such as in muscle. Palovarotene, an activator of retinoic acid receptor γ (RAR-γ), has been shown to inhibit the formation of ectopic bone in HO model mice, but its specific mechanism of action remains unclear. This study will explore the target and molecular mechanism of Palovarotene's action on HO by network pharmacology study. We collected the relevant targets of Palovarotene and HO from the database, obtained the potential targets of Palovarotene acting on HO through Venn analysis, and constructed the protein-protein interaction (PPI) network. Then, Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment Analysis and Module-based Network Analysis were performed for potential targets, and in addition, PPI Network Topology Analysis and Gene-Phenotype Correlation Analysis were performed. The results suggested that MAPK1, MDM2, and other targets as well as P53 signaling pathway and PI3K–Akt signaling pathway may be closely related to Palovarotene treatment of HO. We carried out verification experiments to confirm our finding, alkaline phosphatase and alizarin red staining in vitro and Micro-CT as well as hematoxylin-eosin staining in vivo were performed to verify treatment for HO of Palovarotene, reverse transcription polymerase chain reaction was also used to explore the transcription changes of MAPK1, MDM2, and osteogenic genes. This study systematically elucidated the possible mechanism of Palovarotene in the treatment of HO through network pharmacology study, revealing a new direction for the further application of Palovarotene in the treatment of HO.

Palovarotene Can Attenuate Heterotopic Ossification Induced by Tendon Stem Cells by Downregulating the Synergistic Effects of Smad and NF-κB Signaling Pathway following Stimulation of the Inflammatory Microenvironment.

Heterotopic ossification (HO) is defined as the formation of bone tissues outside the bones, such as in the muscles. Currently, the mechanism of HO is still unclear. Tendon stem cells (TSCs) play important roles in the occurrence and development of HO. The inflammatory microenvironment dominated by macrophages also plays an important role in the course of HO. The commonly used clinical treatment methods, such as nonsteroidal anti-inflammatory drugs and radiotherapy, have relatively large side effects, and more efficient treatment methods are needed in clinical practice. Under physiological conditions, retinoic acid receptor (RAR) signal transduction pathway inhibits osteogenic progenitor cell aggregation and chondrocyte differentiation. We focus on palovarotene, a retinoic acid γ-receptor activator, showing an inhibitory effect on HO mice, but the specific mechanism is still unclear. This study was aimed at exploring the specific molecular mechanism of palovarotene by blocking osteogenic differentiation and HO formation of TSCs in vitro and in vivo in an inflammatory microenvironment. We constructed a coculture model of TCSs and polarized macrophages, as well as overexpression and knockdown models of the Smad signaling pathway of TCSs. In addition, a rat model of HO, which was constructed by Achilles tendon resection, was also established. These models explored the role of inflammatory microenvironment and Smad signaling pathways in the osteogenic differentiation of TSCs which lead to HO, as well as the reversal role played by palovarotene in this process. Our results suggest that, under the stimulation of inflammatory microenvironment and trauma, the injured site was in an inflammatory state, and macrophages were highly concentrated in the injured site. The expression of osteogenic and inflammation-related proteins, as well as Smad proteins, was upregulated. Osteogenic differentiation was performed in TCSs. We also found that TCSs activated Smad and NF-κB signaling pathways, which initiated the formation of HO. Palovarotene inhibited the aggregation of osteogenic progenitor cells and macrophages and attenuated HO by blocking Smad and NF-κB signaling pathways. Therefore, palovarotene may be a novel HO inhibitor, while other drugs or antibodies targeting Smad and NF-κB signaling pathways may also prevent or treat HO. The expressions of Smad5, Id1, P65, and other proteins may predict HO formation.

Single cell analysis reveals inhibition of angiogenesis attenuates the progression of heterotopic ossification in Mkx\u2212/\u2212 mice

Tendon heterotopic ossification (HO) is characterized by bone formation inside tendon tissue, which severely debilitates people in their daily life. Current therapies fail to promote functional tissue repair largely due to our limited understanding of HO pathogenesis. Here, we investigate the pathological mechanism and propose a potential treatment method for HO. Immunofluorescence assays showed that the Mohawk (MKX) expression level was decreased in human tendon HO tissue, coinciding with spontaneous HO and the upregulated expression of osteochondrogenic and angiogenic genes in the tendons of Mkx−/− mice. Single-cell RNA sequencing analyses of wild-type and Mkx−/− tendons identified three cell types and revealed the excessive activation of osteochondrogenic genes during the tenogenesis of Mkx−/− tendon cells. Single-cell analysis revealed that the gene expression program of angiogenesis, which is strongly associated with bone formation, was activated in all cell types during HO. Moreover, inhibition of angiogenesis by the small-molecule inhibitor BIBF1120 attenuated bone formation and angiogenesis in the Achilles tendons of both Mkx mutant mice and a rat traumatic model of HO. These findings provide new insights into the cellular mechanisms of tendon HO and highlight the inhibition of angiogenesis with BIBF1120 as a potential treatment strategy for HO.

QS7: Digit Tip Regeneration Following Amputation Is Not True Regeneration

Purpose:Repair of distal phalanx amputation is widely studied and thought to occur through a regenerative process. Single-cell RNA sequencing (scRNA) has greatly improved the ability to characterize the cells that are involved in regeneration and abnormal healing of bone and soft tissue. Given the disorganized architecture of digit tip “regenerated” bone and its common progenitor cell to the pathologic process of heterotopic ossification, we hypothesized that digit tip repair after injury follows a program of aberrant wound repair rather than true regeneration of normal bone tissue.Methods:ScRNA data of mouse digit tip amputation (DTA) and embryonic limb development (EMB) was downloaded from NBCI GEO data base ascension number GSE135985. Additionally, scRNA data from heterotopic ossification (HO) was downloaded from GSE126060. HO, DTA, and EMB datasets were clustered individually. HO was merged and aligned with EMB and DT in separate analyses. Cells expressing the mesenchymal progenitor cell (MPC) marker Pdgfra, which we have shown to differentiate into HO and others have shown to differentiate into bone after DTA, were identified and analyzed in the HO-DTA and HO-EMB datasets. Monocle 2, Seurat 3.1.1, and Pearson correlation analysis were used for analysis and visualization. Immunostaining for markers present in DTA (ACAN, FBN-2, LTBP2) was performed on sections collected from a murine model of traumatic HO in Pdgfra-CreER;TdTomato mice induced with tamoxifen one-week prior to HO-forming injury.Results:MPC clusters from DTA and HO and from EMB and HO were aligned and projected using UMAP to compare transcription profiles. Across the timepoints, MPCs in the DTA and HO occupy similar regions of the UMAP plot, while MPCs in EMB and HO occupy largely independent regions, with only overlap at the latest time points. Using Monocle 2, cells were placed on a virtual timeline based on similarities in transcription. MPCs from DTA and HO meet at the same location on the trajectory following injury. In the EMB to HO comparison, cells only occupy similar regions of the trajectory at post-natal day 3 suggesting that bone is formed by different pathways. To understand correlations between time points, transcriptomes were also compared by Pearson analysis. MPCs following HO and DTA injuries occupy similar regions of the plot while MPCs in EMB and HO only occupy similar regions of the correlation plot at P3. Markers previously identified to be upregulated in MPCs in DTA were also upregulated in MPCs in HO by both scRNA and immunofluorescent histology.Conclusions:Cells responsible for bone repair following DTA show similar transcriptional profiles as MPCs in traumatic HO and dissimilar to MPCs in EMB. Furthermore, MPCs in both digit tip repair and HO follow trajectories that do not overlap with embryonic limb development. Thus, we demonstrate for the first time, that the process following DTA is not truly regeneration and follows programs of aberrant repair as seen in HO.