The Role And Mechanism Of Tendon Stem Cell Derived Exosomes In Tendon Repair

Background: Tendon injury is a common disease in the field of sports medicine,30-50% of sports injuries are associated with tendon.Due to the lack of internal blood vessel in tendon tissue,the self-healing ability is weak.In addition,the complications such as scar,re-rupture and calcification are prone to occur after healing.Therefore,it is necessary to find an ideal method for promoting tendon repair.During the past decade,the discovery of tendon-derived stem cells(TDSCs)has provided new ideas for tendon repair.Compared with the most commonly used bone marrow mesenchymal stem cells(BMSCs),TDSC has stronger colony forming ability,proliferative ability and the ability to differentiate into tendon.Moreover,TDSC can promote tendon regeneration and anti-inflammatory effects.Therefore,TDSC will play an important role in tendon repair.However,there are still some limitations in the clinical application of stem cell therapy: 1.Allogeneic cells may cause immune rejection and the spread of pathogens;2.There is a risk of tumor formation;3.The stem cells are difficult to store and transport.Several studies have confirmed that in vivo administration of MSCs ameliorates disease in preclinical models but that these cells are rapidly cleared within 48 h.Cell differentiation and direct tissue repair contribute minimally to the beneficial effects attributed to MSCs,and paracrine Extracellular vesicles,immunomodulatory pathways are the predominant mechanisms of their in vivo effect.Exosomes,as one of the important components of paracrine vesicles,may play an important role in the regulation of target cells by stem cells.Exosomes are vesicles with a bilayer membrane structure with a diameter of 30-150 nm containing numerous proteins and lipids,as well as nucleic acid material in the form of DNA,m RNA,micro RNA(miRNA),and noncoding RNA.Exosomes enter the extracellular matrix through budding,and they are endocytosed by the target cells to release internal components and complete cell-to-cell communication.Exosomes can effectively repair damaged tissues and organs such as heart,lung,kidney,and brain,indicating that transplanted exosomes can perform similar functions as transplanted stem cells.In recent years,large numbers of reports have reported that exosomes can promote the repair of tissues such as heart,bone,kidney,brain and lung.In this research,we hypothesized that TDSC Exosomes(TDSC-Exos)could exhibit similar biological functions of TDSC,promoting the repair of injured tendons.Therefore,this study aimed to explore whether TDSC-Exos could promote the repair of tendon and what the underlying mechanism.Objective: 1.Isolation,culture and identification of TDSC,extraction and identification of exosomes derived from TDSC.2.To study the effect of TDSC-Exos on the proliferation and differenation of tenocytes(TC)in vitro,and the repair of tendon injury in vivo.3.To explore the molecular mechanism of TDSC-Exos-mediated repairing tendon injury.Methods: 1.Primary cultured TDSC were identified by their surface antigen and multilineage differentiation potential;2.The P3-P6 cell culture supernatant of TDSC was collected,TDSCs-Exos was extracted by ultracentrifugation,the morphology of TDSC-Exos was observed by transmission electron microscopy(TEM),and the particle size distribution was observed by Nanosight particle size analyzer.Western Blot was used to detect the expression of membrane specific proteins on the exosomes.3.Confocal microscopy was used to observe whether TDSC-Exos could be uptaken by tenocytes,Cell Counting Kit-8(CCK-8)was used to study the effect of TDSC-Exos on proliferation,anti-starvation and anti-oxidative stress of tendon cells;Transwell assay and Wound Healing assay were used to study the effect of TDSC-Exos on migration.Picric acid Sirius red staining was performed to detect the effect of TDSC-Exos on tenocytes type I collagen synthesis.q-PCR was used to assess the expression of tendon specific m RNA.4.Creating SD rat patellar tendon window detect model,Preparation of Photopolymerizable Hyaluronic Acid-TDSC-Exos Scaffold(p HA-TDSCs-Exos);fill the control group(without any material),p HA group and p HA-TDSC-Exos group into the tendon injury site.After 2,4,and 8 weeks postoperatively,Histomorphological analysis and biomechanical analysis was performed 8 weeks after surgery.5.To identify the exosome-associated small RNA(s)that induce proliferation,migration and another promoting effects,we selected and profiled all small RNAs in the TDSC-Exos and TC-Exos by Illumina Hi Seq TM 2500 deep sequencing.Top 10 miRNAs with statistical differences were selected,predicting the target genes of miRNAs.In addition,GO functional enrichment analysis and KEGG biological pathway enrichment analysis was performed to analyse target genes and to further speculate the biological pathways in which TDSC-Exos act.Results: 1.Established stable rat primary TDSCs and TCs culture system.Flow cytometry showed that CD90 and CD44 were positively expressed in TDSCs,CD31 and CD106 were negative.Distinctive colonies formed in low density culture.The multidifferentiation potential assay showed that isolated BMSCs had osteogenesis,chondrogenesis and adipogenesis abilities.2.Isolation TDSC-Exos by ultracentrifugation,with a diameter of about 100 nm and expressed exosome-specific markers(CD63,CD81,CD9 and TSG101).3,TDSC-Exos could promote tenocytes proliferation,migration,anti-starvation,antioxidative stress.At the same time,TDSC-Exos could promote the secretion of type I collagen and the expression of tendon specific m RNA.4.p HA-TDSC-Exos biological scaffold significantly promote the healing and functional repair in a rat patellar tendon defect model.5.Extract TDSC-Exos and TC-Exos for sequencing,screen the differentially expressed miRNAs,predict the target genes of miRNAs,and then perform GO(Gene Ontology)and KEGG(Kyoto encyclopedia of genes and genomes)on target genes.TDSC-Exos is involved in the regulation of the MAPK pathway,m TOR pathway,PI3K-Akt pathway,and HIF-1 pathway.This finding is consistent with the biological pathway of mesenchymal stem cell exosomes participated in tissue repair.Conclusion: TDSC-Exos could promote tenocytes proliferation,migration,anti-starvation,antioxidative stress.In addition,TDSC-Exos could promote the secretion of type I collagen and the expression of tendon specific m RNA.p HA-TDSC-Exos biological scaffold significantly facilitate the healing and functional repair in a rat patellar tendon defect model.These effects may be achieved by the regulation of MAPK pathway,m TOR pathway,PI3K-Akt pathway and the HIF-1 pathway.