Single cell analysis reveals inhibition of angiogenesis attenuates the progression of heterotopic ossification in Mkx−/− 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.

Role of the P2X7 Receptor in the Osteogenesis of Heterotopic Ossification of the Achilles Tendon

Background Heterotopic ossification (HO) refers to a painful and complex disease. HO occurs in the setting of persistent systemic inflammation and appears in flare-ups during inflammation, following injury. In the recent research, the P2X7 receptor (P2X7R) is tightly involved in the osteogenesis of periodontal ligament stem cells under the inflammatory conditions. The ionotropic P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed in the majority of stem cells. However, the function of P2X7R in the pathological formation of HO is unclear. Here, this paper hypothesizes that in the model of Achilles tendon ectopic ossification, P2X7R is overexpressed in tendon-derived stem cells and promote osteogenesis of tendon-derived stem cells under inflammatory conditions. Methods The tenotomy puncture and burn injury-induced HO model was constructed. The qPCR and immunofluorescence were used to detect the expression of P2X7R at the site of injured Achilles tendon where HO occurs. Achilles tendon stem cells (SCs) from control group and experimental group sources were cultivated separately under inflammatory conditions. The cells from the two groups were cultured for osteogenic analysis. In addition, a specific antagonist of P2X7R, BBG was used to detect whether reversed the above process. At last, BBG was used to intervene in animal models of heterotopic ossification. Results Under inflammatory conditions, P2X7R expression of the Achilles tendon and osteogenic capability of SCs is higher in heterotopic ossification group (HOG) than in other two groups. The P2X7R expression was positive correlated with the capacity of osteogenesis of SCs. BBG can inhibit osteogenic differentiation and subsequent bone formation in the P2X7R overexpress of SCs. BBG impeded the heterotopic bone formation in animal model. Conclusions P2X7R is one of the crucial mediators in the formation of the HO, blocking which may represent a potential therapeutic target for HO.

Identification of the occurrence and potential mechanisms of heterotopic ossification associated with 17-beta-estradiol targeting MKX by bioinformatics analysis and cellular experiments

Background Tendon heterotopic ossification (HO) is a common condition occurring secondary to tendon injury or surgical trauma that significantly affects the patient’s quality of life. The treatment of tendon HO remains challenging due to a lack of clarity regarding the pathological mechanism. Mohawk (MKX) is a key factor in preventing tendon HO; however, its upstream regulatory mechanism remains to be understood. This study aimed to identify potential compounds that target and regulate MKX and explore their functional mechanisms. Methods Bioinformatics analysis of MKX-related compounds and proteins was performed based on data from the STITCH and OncoBinder databases. Subsequently, the SymMap database was used to study MKX-related traditional Chinese medicine drugs and symptoms. Next, the OncoBinder genomic and proteomic discovery model was applied to identify potential regulators of MKX. The analytical tool Expert Protein Analysis System for proteomics was used to predict the three-dimensional structure of MKX, and the AutoDockTools software was used to identify pockets of activity at potential sites for molecular docking. Furthermore, we evaluated the effect of different doses of 17-beta-estradiol on bone marrow-derived mesenchymal stem cells (BM-MSCs). Results By predicting the three-dimensional structure of MKX and simulating molecular docking, Pro-Tyr and 17-beta-Estradiol were found to target and bind to MKX. Analysis of the STITCH and OncoBinder databases showed that MKX had a significant regulatory correlation with suppressor interacting 3 A/histone deacetylase 1 (SIN3A/HDAC1). The GO and KEGG pathway enrichment analysis revealed that the functions of MKX and its associated proteins were mainly enriched in osteogenic-related pathways. Assessment of the proliferation of BM-MSCs revealed that 17-beta-estradiol possibly upregulated the mRNA expression of the HDAC1-SIN3A/BMP pathway-related RUNX2, thereby promoting the proliferation of BM-MSCs. Conclusions The compounds Pro-Tyr and 17-beta-Estradiol may bind to MKX and thus affect the interaction of MKX with SIN3A/HDAC1.

Over-expression of wild-type ACVR1 in fibrodysplasia ossificans progressiva mice rescues perinatal lethality and inhibits heterotopic ossification

Fibrodysplasia ossificans progressiva (FOP) is a devastating disease of progressive heterotopic bone formation for which effective treatments are currently unavailable. FOP is caused by dominant gain-of-function mutations in the receptor ACVR1 (also known as ALK2), which render the receptor inappropriately responsive to activin ligands. In previous studies, we developed a genetic mouse model of FOP that recapitulates most clinical aspects of the disease. In this model, genetic loss of the wild-type Acvr1 allele profoundly exacerbated heterotopic ossification, suggesting the hypothesis that the stoichiometry of wild-type and mutant receptors dictates disease severity. Here, we tested this model by producing FOP mice that conditionally over-express human wild-type ACVR1. Injury-induced heterotopic ossification (HO) was completely blocked in FOP mice when expression of both the mutant and wild-type receptor were targeted to Tie2-positive cells, which includes fibro/adipogenic progenitors (FAPs). Perinatal lethality of Acvr1R206H/+ mice was rescued by constitutive ACVR1 over-expression and these mice survived to adulthood at predicted Mendelian frequencies. Constitutive over-expression of ACVR1 also provided protection from spontaneous HO, and the incidence and severity of injury-induced HO in these mice was dramatically reduced. Analysis of pSMAD1/5/8 signaling both in cultured cells and in vivo indicates that ACVR1 over-expression functions cell-autonomously by reducing osteogenic signaling in response to activin A. Manipulating the stoichiometry of FOP-causing and wild-type ACVR1 receptors may provide the foundation for novel therapeutic strategies to treat this devastating disease.

Compressed Sensing Image Reconstruction of Ultrasound Image for Treatment of Early Traumatic Myositis Ossificans of Elbow Joint by Electroacupuncture

This article conducts a retrospective analysis of 500 patients with posttraumatic elbow dysfunction admitted to our department from March 2019 to September 2020. The average time from injury to operation is 11 months (2–20 months). We adopt a personalized treatment method to completely remove the hyperplastic adhesion tissue and heterotopic ossification around the joint, remove part of the joint capsule and ligament, and release it to achieve maximum function. After the operation, an external fixator was used to stabilize the loosened elbow joint, and the patient was guided to perform rehabilitation exercises with the aid of a hinged external fixator, and celecoxib was used to prevent heterotopic ossification. Mayo functional scoring system was used to evaluate the curative effect before and after surgery. The rapid realization of ultrasound imaging under the framework of compressed sensing is studied. Under the premise of ensuring the quality of ultrasound imaging reconstruction, the theory of ultrasound imaging is improved, and a plane wave acoustic scattering ultrasound echo model is established. On this basis, the theory of compressed sensing is introduced, the mathematical model of compressed sensing reconstruction is established, and the fast iterative shrinkage thresholding algorithm (FISTA) of compressed sensing reconstruction is improved to reduce the computational complexity and the number of iterations. This article uses FISTA directly to reconstruct medical ultrasound images, and the reconstruction results are not ideal. Therefore, a simulation model of FISTA training and testing was established using the standard image library. By adding different intensities of noise to all images in the image library, the influence of noise intensity on the quality of FISTA reconstructed images is analyzed, and it is found that the FISTA model has requirements for the quality of the images to be reconstructed and the training set images. In this paper, Rob’s blind deconvolution restoration algorithm is used to preprocess the original ultrasound image. The clarity of the texture details of the restored ultrasound image is significantly improved, and the image quality is improved, which meets the above requirements. This paper finally formed a reconstruction model suitable for ultrasound images. The reconstruction strategy verified by the ultrasound images provided by the Institute of Ultrasound Imaging of a medical university has achieved a significant improvement in the quality of ultrasound images.