Roles And Mechanism Of Mkx In Tendon Heterotopic Ossification

Tendon heterotopic ossification is a clinically refractory disease characterized by the existence of osteoblasts and formation of bone tissue in tendon,which is often caused by tendon injury or surgical trauma.Tendon heterotopic ossification can cause varying degrees of pain,local edema,limited joint movement,reduced mechanical properties and even rupture of tendon,which seriously affects the life quality of patients.At present,local injections of non-steroidal anti-inflammatory drugs,glucocorticoids or shock waves are often used in clinical practice to relieve symptoms.There is no effective treatment to completely cure tendon heterotopic ossification.Although the research on tendon heterotopic ossification in recent years has revealed the important role of various factors such as mechanical injury and inflammation in the pathological process,which has brought new inspiration for the development of new treatment schemes for tendon heterotopic ossification,but at present,two key issues remain to be resolved:1)What are the key internal regulatory factors of tendon heterotopic ossification? 2)How does the regulatory factor affect the cell fate of different tendon cell subpopulations? Based on the research foundation of our group on the regulation of tendon cell differentiation,we aim to conduct a systematic investigation regarding of the two questions.The contents of this study are as follows:In the first part,through data mining,clinical sample examination,it was found that the expression of the transcription factor Mohawk(Mkx)was reduced in heterotopic ossified tendons.Further analyses of Mkx knockout mice revealed that Mkx knockout resulted in increased expression of cartilage and osteogenesis-related genes,as well as the heterotopic ossification of the Achilles tendon,patellar tendon,and tail tendon,indicating that Mkx can inhibit tendon heterotopic ossification.In the second part,by using the bi-molecule fluorescence complementary technology,a list of proteins capable of interacting with Mkx was identified.It was found that Mkx proteins interacted with histones and proteins related to histone binding.Chromatin immuno-coprecipitation sequencing and transcriptome sequencing analysis revealed that Mkx deficiency resulted in increased H3K27 ac modification levels and gene expression levels of angiogenesis and ossification related genes.It is suggested that Mkx is involved in the epigenetic modification of tenocytes and the maintenance of gene expression levels.In the third chapter,we used single-cell sequencing technology to perform singlecell transcriptome sequencing analysis of wild-type and Mkx knockout tendons.It was found that the cellular heterogeneity between normal and Mkx knockout tendon cell subpopulations was similar.Differential gene expression analyses showed that all subpopulations of Mkx knockout tendon cells highly express angiogenesis-related genes,while Mkx knockout tendon progenitor cells highly express cartilage-related genes,and Mkx knockout tenocytes highly express osteoblast-related genes.These findings indicated that Mkx knockout promotes angiogenesis and specifically increases the expression of cartilage and osteogenic differentiation-related genes in different cell subsets,and finally leading to heterotopic ossification.In the fourth chapter,we explored the feasibility and effectiveness of stem cell and small molecule drugs for the treatment and prevention of tendon heterotopic ossification based on the above research findings.The results showed that adipose and bone marrowderived mesenchymal stem cells may not be suitable for the treatment of tendon heterotopic ossification;however,the angiogenesis inhibitor BIBF1120 can alleviate tendon heterotopic ossification caused by Mkx knockout and injury,and is a potential therapeutic drug.In summary,our research systematically revealed the pathological mechanism of tendon heterotopic ossification from the cellular and molecular levels.Based on this,it has been found that a small-molecule drug can be used to treat tendon heterotopic ossification.Our research provided a new perspective for deep understanding of the pathological mechanism of tendon heterotopic ossification,and provided new ideas for the innovative development of clinical therapeutic drugs for tendon heterotopic ossification.