| BackgroundTendon-bone injury refers to an injury involving tendons or ligaments attached to bone tissue,including rotator cuff injury,Achilles tendon injury and anterior cruciate ligament injury,is common in daily life and exercise.The incidence of tendinous injury is increasing year by year.Further more,it is difficult to restore normal physiological function.The process of tendon-bone healing is a tissue repair process from soft tissue to hard tissue.Because of its complex anatomical structure and non-generation of the injured tissue,tendon-bone healing has become the focus of current research in sports medicine and orthopaedics.Studies over the years have revealed that bone marrow-derived mesenchymal stem cells(BMMSCs)can differentiate into osteoblasts,chondrocytes,adipocytes,and even myogenic cells,and are therefore considered multipotent cells.In fact,due to their multidirectional differentiation,self-renewal,and immunomodulatory effects,BMMSCs have become an important source of seed cells in gene therapy,tissue engineering,cell replacement therapy,and regenerative medicine.Peritunnel osteolysis can be immediately observed in patients with anterior cruciate ligament(ACL)reconstruction and in animal models of tendon-bone injury.Prevention of peritunnel osteolysis can help to avoid postoperative tendinous union failure.It is now generally accepted that increased osteoclast activity leads to local bone loss at the tendon-bone interface,and histologically,bone resorption or osteolysis affects bone-tendon ingrowth,which in turn affects the strength of tendon-bone healing,in clinical observation,the enlargement of bone tunnel can occur 3 months after operation,the extent of enlargement seriously affects the choice of operation,and the abnormal activation of osteoclasts is the cause of the enlargement of bone tunnel.Inhibition of osteoclast formation is a promising target for treatment interface osteolysis,which is associated with improved tendinous strength and better clinical outcomes.Unfortunately,anti-osteoporosis drugs showed poor therapeutic effects on osteolysis during tendon-bone healing.It was reported that systemic usage of bisphosphonate zoledronic acid(ZA)did not affect osteoclastogenesis in a rat model of tendon-bone healing.The bone mineral density and bone mineral content at the peritunnel were not improved.In contrast,local injection of osteoprotegerin(OPG)improved bone volume at the tendon-bone interface in a rabbit model.This discrepancy may be due to distinct modes of drug administration and different therapeutic targets.Together,the effects of traditional anti-osteoporosis drugs on improving tendon-bone healing may not be reliable.In the preliminary experiment,BMMSCScx was found to inhibit osteoclast formation and enhance tendon-bone healing.Therefore,how BMMSCScx inhibit osteoclast formation has been intensively investigated in this paper.Mesenchymal stem cell Exosomes(exo)have been used as potent and feasible natural nano-drug carriers and have been shown to enhance tendon-bone healing,this suggests that Mesenchymal stem cell exosomes may be a promising therapeutic strategy.Scleraxis(Scx)was found to be a transcription factor containing basicHelix-Loop-Helix(bHLH)when it was screened for tissue-specific proteins using yeast-two-hybrid system in 1995 by Cserjesi et al.One of the cDNA protein products has a BHLH structure and can form heterodimer with E12.It was first expressed in the osteogenic segment of somites and thus named.Scx is highly homologous to other bHLH family proteins,such as twist,with 61%homology.Human Scx contains 201 amino acids and the encoding gene is located on chromosome 8.While mouse Scx contains 207 amino acids with its mRNA 1140bp long and the encoding gene is located on chromosome 15.There is a proline-rich region at the carboxyl end,which is the phosphorylation site of protein kinase A,protein kinase C and tyrosine kinase,and so it is similar to the Scx gene in mice.Scx expression in the tendinocyte lineage was first identified by Schweitzer et al.in 2001.Scx is highly expressed as a specific marker in tendons,ligaments,and other connective tissues,and persists throughout tendon development.Cserjesi et al.found that during embryogenesis,the transcription of Scx could be detected as early as 9.5-10.5d in the embryo.Scx was first expressed in the mesenchymal cells of sclerotome in somites,body wall and limb bud.As the embryo develops,high levels of Scx begin to be localized to the areas where cartilage and connective tissue occur,such as tendons,ligaments,diaphragm,and bronchial cartilage.It is suggested that Scx may play a regulatory role in the formation of connective tissues such as tendons in the early stage.Brown et al.constructed Scx-/-mice by gene knockout technique.During the observation of their embryonic development,we found that the embryo of homozygous mutant mice stopped growing on the 6d(E6D)and could not form the gastrula.The embryos formed at this stage lack mesoderm and have only ectoderm and endoderm.Thus,Scx affects the development of mesoderm and,furthermore,the formation of tendon tissue.Scx plays an important role in tendon differentiation.Brown et al.found that Scx was expressed in connective tissue such as the origin of tendon ligaments on embryonic 11.5d.But Scx does not have the same effect on all tendons.Murchison et al.used gene knockout technique to construct Scx-deficient mice and found that they could survive,but had significantly restricted paw movement on their back and limbs,and could not move their tails at all.This means that the functions of the tendons transmitting mechanics and the tendons between muscles are affected to varying degrees,but the tendons and ligaments which have anchor effects are not affected.Histological examination showed that the Scx-deficient mice had decreased and disordered tendon stroma.Scott et al.established the patellar tendon injury model in mice.The qPCR results showed that the expression of Scx and Tenomodulin were significantly up-regulated,while the latter lagged behind the former in time,indicating that Scx could regulate the expression of Tenomodulin.Dyment et al.found that Scx was not expressed in the paratendon tissues(mainly the tendon sheaths)under normal conditions,but increased after tendon injury.The thickening of paratendon tissues and more extracellular matrix filling in the injury confirmed that the seed cells in tendon repair were derived from the paratendon tissues but not from the fibroblasts in the tendon.Therefore,Scx begins to express in the early stage of embryonic development and plays a regulatory role in the maturation of tendon.Levay et al.showed that the valve tissue structures and collagen arrangement directions of Scx-/-mice after birth were disordered.Liu Qiong et al.found that Scx was most strongly expressed in periodontal ligament stem cells and the expression of Scx decreased with the increase of culture generation.He Huixia analyzed the phenotype of human periodontal ligament stem cells(hPDLSCs)and considered that Scx could be used as a specific molecule of hPDLSCs,which could provide a basis for isolation and identification of hPDLSCs.In addition,Scx is also expressed in Sertoli cells and can regulate the function of this cell and promote its differentiation.Therefore,Scx is widely expressed in connective tissue cells and is a marker of connective tissue.According to a recent study of tendon transcription factors by Liu et al.,during the early stages of tendon development,Scx directly activates 32 target genes including Fmod,Tnmd,Htra3,Zfp185 and Ssc5d and inhibits 17 genes.It is suggested that Scx is a more upstream gene of all transcription factors involved in tendon development,which regulates the function of other transcription factors.Scx has been shown to be present and important in the tendons of the skull,trunk,and limbs.Scx is also regulated by other signaling pathways.The expression of Scx was induced by FGF and TGFβ signaling pathway.BMP antagonized the expression of Scx in limb tendon,and SHH antagonized the expression of Scx in axial tendon.Previous studies have reported that overexpression of Scx could promote bone formation.In addition,more researches have found that Scx(+)and Sox9(+)progenitor cells positively promote the formation of tendon-bone attachment units.Insufficient expression of Scx may affect the development of bony structure of the trunk.These studies suggest that Scx is actively involved in tendon-bone healing.Scx,on the other hand,is an important positive regulator of tendon development and plays an important role in tendon repair.Recent studies have shown that overexpressed Scx genes can promote tendon differentiation,tendon-like tissue formation and ligament cell differentiation.However,the role of Scx in tendon-bone interface remodeling has been merely studied.Therefore,overexpression of Scx in BMMSCs may enhance the repair effect of BMMSCs on tendon-bone healing.Based on the above research status,this study explored the dynamic expression of Scx in PDGFR α(+)BMMSCs during the process of natural tendon-bone healing.Then,we investigated the role of PDGFR α(+)BMMSCs in tendon-bone healing after overexpression of Scx and the underlying mechanisms.Therefore,in order to clarify the repair role in the process of tendon-bone healing,through the establishment of a mouse model of tendon-bone injury,to analyze the related factors affecting the repairing process of tendon-bone healing,the biological and mechanical properties during tendon-bone healing were studied in vivo and in vitro.Next,we used gene sequencing and online bioinformatics tools to predict potential target genes and their binding sites,and analyzed whether Scx-overexpressing BMMSCs could promote tendonbone healing.1 Preparation&identification of BMMSCScx and its repair effect on tendon-bone healingIn order to observe the effect of BMMSCScx on tendon-bone healing,the expression of Scx in BMMSCs and the percentage of BMMSCs on Day 0,Day 7,Day 14 and Day 21 were analyzed.Secondly,BMMSCs were extracted from the tibia and femur of C57 mice.The noload BMMSCs and BMMSCScx were constructed and identified by lentiviral infection.1.1 Experimental methods1.1.1 To establish a model of tendon-bone injury in C57 mice,and to isolate,identify and culture BMMSCs from C57 mice.1.1.2 Stable Scx-overexpressing BMMSCs were constructed by lentiviral infection and named BMMSCScx.BMMSCs infected with empty vector were named BMMSCAd as control group.1.1.3 Cell sternness determined by flow cytometry,quaternary differentiation inducted in vitro,observation under fluorescence microscope,analysis by qRT-PCR and WB,Scx protein and mRNA levels were detected.Proliferation and apoptosis of BMMSCs,BMMSCScx,BMMSCAd were analyzed by flow cytometry.1.1.4 The histological characteristics and biomechanical properties of the damaged tendon-bone injury were observed and compared by using the methods of Safranin O/Fast Green staining,TRAP staining and biomechanical test.1.2 Experimental results1.2.1 BMMSCs and BMMSCScx were isolated,cultured and identified successfully.1.2.2 Histochemical staining showed that BMMSCScx could prevent osteolysis and significantly reduce osteoclastogenesis.Biomechanical tests showed that the intensity of tendon-bone healing was significantly enhanced after BMMSCScx treatment.1.3 Summary1.3.1 Firstly,BMMSCs were isolated,identified,cultured and BMMSCScx were constructed.1.3.2 The repair effect of BMMSCScx on tendon-bone healing was preliminarily studied.The results showed that BMMSCScx could prevent osteolysis,improve biomechanical properties and enhance the intensity of tendon-bone healing.2 Repair effect and mechanism of BMMSCScx-exo on tendon-bone healingBased on the BMMSCScx discussed in the previous chapter,exosomes were extracted and identified in order to probe the repair effect and mechanism of BMMSCs,BMMSCAd,BMMSCScx and their exosomes on tendon-bone healing.2.1 Experimental methods2.1.1 Three kinds of exosomes were identified by electron microscopy,particle microscopy and WB.The effects of exosomes on osteoclastogenesis were observed by TRAP staining and qRT-PCR.2.1.2 Three exosomes were injected and the specimens were collected at Day 14 and Day 21.The histological characteristics and biomechanical properties of the specimens with tendonbone injuries were observed by the methods of Safranin O/Fast Green staining,TRAP staining and biomechanical tests,to explore the effect and mechanism of BMMSCScx-exo on tendonbone healing.2.2 Experimental results2.2.1 Three exosomes were successfully extracted and identified by electron microscopy,particle microscopy and WB.2.2.2 By using the methods of Safranin O/Fast Green staining,TRAP staining and biomechanical test,the main component of BMMSCScx in inhibiting osteoclastogenesis was its exosome,and BMMSCScx-exo could significantly enhance the intensity of tendon-bone healing.2.3 Summary2.3.1 Exosomes were successfully extracted and identified by electron microscopy,granuloscope and WB.2.3.2 BMMSCScx-exo were verified to be the major component of the inhibition of osteoclastogenesis,and this effect was associated with Scx overexpression.Animal experiments showed that BMMSCScx-exo treatment could significantly improve the intensity of tendon-bone healing.3 The repair effect and mechanism of miR-6924-5p on tendon-bone healingBased on the previous two chapters,whether miR-6924-5p in exosomes could inhibit the formation of osteoclast precursor cells was observed by miRNAs sequencing,so as to probe the repair effect and mechanism of tendon-bone healing.3.1 Experimental methods3.1.1 The differential expression of miRNAs in BMMSCScx-exo and BMMSCAd-exo was compared by miRNAs sequencing.3.1.2 The effect of miR-6924-5p on osteoclast precursor cells was observed by qRT-PCR and TRAP staining.3.1.3 To investigate whether Scx upregulates miR-6924-5p expression at the transcriptional or post-transcriptional level,the transcription-blocking technique of actinomycin D was used.3.1.4 Agomir.NC and agomiR-6924-5p were injected and specimens were collected on Day 14 and Day 21.By using the methods of Safranin O/Fast Green staining,TRAP staining and biomechanical test,the effect of miR-6924-5p on intensity of tendon-bone healing was analyzed.3.1.5 The potential target genes and binding sites of miR-6924-5p were predicted by bioinformatics technique and dual-luciferase reporter system.3.1.6 Expression of human osteoclast biomarkers and osteoclastogenesis were observed by qRT-PCR and TRAP staining.3.2 Experimental results3.2.1 The results of miRNAs sequencing showed that 38 miRNAs were up-regulated and 171 miRNAs were down-regulated in BMMSCScx-exo.Gene enrichment analysis showed that these miRNAs were significantly involved in the process of myeloid cell differentiation.KEGG pathway enrichment analysis showed that the osteoclast differentiation pathway was highly enriched.3.2.2 By qRT-PCR analysis,miR-6924-5p mRNA expression level was significantly increased,and the enrichment of miR-6924-5p in BMMSCScx and BMMSCScx-exo was the most significant.3.2.3 The transcription-blocking technique of actinomycin D suggests that miR-6924-5p may be affected by Scx at a post-transcriptional level.3.2.4 Biomechanical tests showed that miR-6924-5p could effectively inhibit osteoclastogenesis and promote tendon-bone healing in vivo.3.2.5 OCSTAMP,CXCL12 and PRLR genes were predicted as target gene by online bioinformatics tools.Dual-luciferase reporter system analysis showed that miR-6924-5p could bind to the 3’ UTR of OCSTAMP and 3’ UTR of CXCL12.3.2.6 The analysis of TRAP staining showed that the number of osteoclasts decreased significantly after miR-6924-5p treatment.The expression level of biomarkers of human osteoclasts also decreased significantly.These results suggested that miR-6924-5p could inhibit osteoclastogenesis in human osteoclasts.3.3 Summary3.3.1 The results of miRNAs sequencing indicated that miR-6924-5p in BMMSCScx-exo was the key regulator inhibiting osteoclastogenesis.3.3.2 MiR-6924-5p can effectively inhibit osteoclastogenesis in vivo and in vitro,thereby promoting tendon-bone healing.3.3.3 On-line bioinformatics technology were used to show that PRLR,CXCL12 and OCSTAMP were all target genes.Dual-luciferase reporter system showed that OCSTAMP and CXCL12’s 3’UTR could bind to miR-6924-5p.3.3.4 MiR-6924-5p inhibits osteoclastogenesis by targeting and regulating multiple osteoclasts.3.3.5 MiR-6924-5p can effectively prevent osteoclastogenesis in human osteoclasts.4 ConclusionThe molecular mechanism of BMMSCScx inhibiting osteoclast differentiation and promoting tendon-bone healing through exosome pathway has been fullly demonstrated in this paper;the biological function and target gene of miR-6924-5p are reported for the first time;the potential value of BMMSCScx in the treatment of tendon-bone healing has also been confirmed.These findings provide new therapeutic options and research strategies for the biotherapy of tendon-bone healing. |
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