EGR1 Effects Differentiation Of TSCs And Its Role On Bone-tendon Healing Of Rotator Cuff Insertion

BackgroundTendon-bone healing is one of common roblems encountered in orthopaedic and sports medicine.Various biological approaches have been used to augment surgical repairs to try and improve long-term outcomes of these repair surgeries.These include augmentations with cellular components,extracellular matrix,or various individual growth factors.However,sufficient improvements in repair in humans have not yet been reported with any of these approaches,and there is a need for more investigations to identify effective biological augmentation strategies.Among the various options for supplementing rotor cuff repair surgeries,augmentation with growth factors is considered to be the more attractive procedure.Previous studies have shown that growth factors,such as bone morphogenic proteins 2,and 7(BMP-2 and BMP-7,respectively),transforming growth factors ?1 and ?3(TGF-?1 and TGF-?3,respectively)and fibroblast growth factor(FGF),can increase healing rates in animal models.However,the overemphasis on passive healing at bone site resulted in that the size of the tendons tended to increase but biomechanical strength of the repaired tendons did not significantly increased.Therefore,the conclusion that the application of growth factors in tendon repair could improve the success rate in surgical was questioned.The early growth response-1(EGR1)transcription factor has recently been shown to be involved in tendon differentiation.It was associated with increased collagen formation during tendon differentiation in embryo limbs,and it induced expression of the tendon marker scleraxis(SCX)and tendon collagens.Vivo studies further demonstrated that over-expression of EGR1 in tendon differentiation of mesenchymal stem cells(MSCs)had obvious repair effect in injury tendon.Prophase research in our research group have confirmed that tendon stem cells(TSCs)is one of the most promising precursor cells in tendon injuries.Therefore,how to induce tendon differentiation of TSCs and strengthen the initiative tendon repair is the focus of our work.In the current study,we detected the level of EGR1 in tendon differentiation of rabbit TSCs.We hypothesized that EGR1 would drive the differentiation of TSCs into tendon cells,the signal pathway of this function and that EGR1-producing TSCs would enhance healing and repair of rotator cuff tears in a rabbit model of this injury.Section 1: A preliminary study about the effect of EGR1 on the muti-differentiation of TSCsIt is imperative to set up a suitable model for rotator cuff repairs.Then it evaluated two techniques to deal bone-tendon interface in biology.1.MethodsThe EGR1 coding sequence from the p CMV-EGR1 cDNA clone was cloned into the p CDNA3.1+ vector to yield pCDNA-EGR1.TSCs were transfected with p CDNA-EGR1 or the vector alone(control)using the Lipofectamine 2000 transfection reagent.G418(800 ?g/ml)-containing medium was used to select stable transfectants.To induce tenogenic differtiation,TSCs and pCDNA-EGR1-transfected TSCs(EGR1-TSCs)were cultured in 6-well plates in DMEM supplemented.The expression of differentiation related genes was determined by immunofluorescence staining and quantitative PCR.2.ResultsImmunofluorescence staining also showed that the EGR1-overexpressing cells(EGR1-TSCs)had higher levels of the tenocyte-associated protein markers SCX and TNMD compared with TSCs,indicating that the effect of EGR1 was at the transcription level of these tenocyte-associated genes.Induction of differentiation of the TSCs in the presence of EGR1 overexpression inhibited adipocyte,osteocyte,and chondrocyte differentiation(Fig.2C).In addit ion,PPAR?,RUNX2,and SOX9 transcript levels were lower in the EGR1-TSCs compared with TSC cell(Fig.2D),indicating down-regulation of these genes by EGR1.3.Conclusion1.After TSCs were transfected with EGR1,EGR1-overexpressing cells showed a sustained tenogenic differentiation2 EGR1 induced tenogenic differentiation though mRNA and protein levels3 EGR1 induced specifically tenogenic differentiation of TSCs,and inhibited adipogenic and osteogenic differentiation.Section 2: The signaling pathway of EGR1 induced tendogenic differentiation of TSCsOn the basis of previous study,we investigated the signaling pathway of EGR1 in tendogenic differentiation of TSCs.1.MethodsTo examine the production of proteins in the BMP12/Smad1/5/8 pathway during tenogenic differentiation,TSCs or EGR1-TSCs were cultured for different time table.Total protein concentrations were measured using a BCA protein assay kit,and equal amounts of extracted proteins were resolved by SDS-polyacrylamide gel electrophoresis.Proteins were then transferred on to polyvinylidene difluoride membranes,and membranes blocked.The membranes were then incubated sequentially with primary and secondary antibodies.Proteins were visualized and images captured using a LiCoR Odyssey imager.2.ResultsBMP12 mRNA and protein levels increased as well as phosphorylated Smad1/5/8 protein levels gradually in a time-dependent manner during tenogenic differentiation in EGR1-overexpressing TSCs but not in TSCs.Total Smad1 protein levels showed no increase during tenogenic differentiation.Quantification of the transcripts of the tenocyte-associated genes SCX,TNMD,TNC,and Col I also showed gradual increase in these mRNAs in a time-dependent manner in EGR1-TSCs.3.Conclusion1.BMP12 induced tenogenic differentiation of TSCs,obtain the optimal dose and duration.2.EGR1 induced tenogenic signaling in TSCs via the BMP12/Smad1/5/8 pathway.3.BMP12 and Smad1/5/8 m RNA and protein levels increased gradually in a time-dependent manner during tenogenic differentiation in EGR1-overexpressing TSCs.4.BMP12 induced Smad through BMPR Ia.Section 3: The effects of EGR1-TSCs on chronic rotator cuff injury in rabbitsAfter establised a chronic model of rabbit rotator cuff tear,Egr1-TSCs was implanted in the interface between bone and tendon.1.MethodsThe supraspinatus tendon was severed in the right shoulder,and sham operations were performed in the left shoulder in 24 mature New Zealand white male rabbits.Six weeks following the induced tear,surgical procedures to repair the injury.The following procedures were performed in three groups: Group 1(R),repair surgery;Group 2(TSCs),repair + TSCs in a fibrin glue carrier;and Group 3(EGR1-TSCs),repair +EGR1-TSCs in a fibrin glue carrier.Application of TSCs and EGR1-TSCs in a fibrin glue was as described previously.Eight weeks after the repair surgical procedure,all rabbits were sacrificed.Histological analyses and immunohistochemistry were performed to evaluate differents between there groups.2.ResultsThe TSCs group showed parallel fibrous tissues,continuously aligned along the load axis at the tendon insertion with small amounts of Sharpey fibers.In the EGR1-TSCs group,collagen fibers were packed in a parallel pattern,with slight splitting between bundles,and many Sharpey fibers were observed at the tendon insertion site.3.Conclusion1.To establish a chronic model of rabbit rotator cuff tear,it provides a reproducible animal model for tendon bone healing.2.For chronic injury model,tenogenic differentiation in EGR1-overexpressing TSCs promote tendon bone healing.