| Objective:The difficult to heal the tendon-bone interface after rotator cuff repair is one of the main causes of surgical failure.Previous studies demonstrated that demineralized cortical bone(DCB)could improve the healing of enthesis.However,the tendon-bone interface contains heterogeneous areas of different tissue,and the effect of biomaterials with single structure on the healing of enthesis is poor.It is necessary to construct biomimetic scaffold for the repair of tendon-bone interface.Extracellular matrix(ECM)contains a variety of biochemical components,and it could regulate the proliferation,migration and differentiation of stem cells.At present,some studies have developed scaffolds coated with stem cell-derived ECM for tissue repair.Therefore,the purpose of this study is:(1)To evaluate the effect of DCB modified with stem cell-derived extracellular matrix(DCB-ECM)on the proliferation,migration and differentiation of stem cells;(2)To evaluate the outcomes of the hierarchically demineralized cortical bone(h DCB)modified with stem cell-derived extracellular matrix(h DCB-ECM)in the repair of rotator cuff in a rabbit model.Materials and Methods:1.DCB was prepared according to our published method,and extracellular matrix(ECM)from tendon-derived stem cells(TDSCs)was used to modify DCB.First,the surface topographies,element distribution and histological structure of scaffolds were observed with scanning electron microscope(SEM),energy dispersive X-ray spectroscopy(EDX),fourier transform infrared spectroscopy(FTIR)and histological staining.Next,DNA content detection was used to determine the best re-decellularization time.Then,the contents of stromal cell-derived factor 1(SDF-1),biglycan(Bgn)and fibromodulin(Fmod)in ECM were detected by ELISA.Finally,the elastic modulus of the scaffold surface was observed by atomic force microscopy(AFM).2.Cell proliferation of TDSCs cultured on scaffolds was evaluated with cell counting kit-8(CCK-8),and the cell viability was assessed by live/dead staining.Cell morphology was detected by fluorescent staining.The ability of scaffolds to recruit stem cells was valued by transwell migration assay.The expression levels of bone-related,cartilage-related and tendon-related genes of stem cells were assessed by real-time polymerase chain reaction(PCR).3.Rabbit TDSCs were isolated,cultured and identified.The h DCB,as a biomimical scaffold,was prepared by the graded demineralization.The h DCB-ECM was fabricated through the two-week cell culture and decellularization,and DNA content of the scaffolds was detected.The morphology and mineral content of three components within h DCB-ECM were evaluated through SEM,EDX and FTIR.The representative biochemical factors of the h DCB-ECM were evaluated by ELISA.Finally,the elastic modulus of the surface in three components within h DCB-ECM was examined by AFM.4.Twenty-four rabbits(forty-eight samples)were randomly divided into four groups: control,DCB,h DCB and h DCB-ECM.All rabbits were performed bilateral detachment of the infraspinatus tendon and the tendon-bone interface was repaired with or without the scaffolds.After surgery,sixteen samples were assessed by immunofluorescence staining at 2 weeks,and sixteen samples were assessed by micro-CT examination and histological staining at12 weeks,and another 16 samples were used for mechanical testing at 12 weeks.Results:1.The result of SEM showed that there was no obvious change on the surface of DCB before and after demineralization.The result of EDX and FTIR indicated that DCB was completely demineralized.The result of DNA content detection showed that 10 min and 15 min of Triton X-100 treatment could remove TDSCs effectively.After decellularization,the surface morphology of DCB-ECM was changed before and after modification with stem cell-derived ECM.The results of ELISA analysis indicated that the contents of SDF-1,Bgn and Fmod in the DCB-ECM were significantly higher than those in the DCB.The results of AFM showed that the elastic modulus of DCB was significantly higher than that of DCB-ECM.2.The results of cell viability analysis demonstrated that DCB-ECM had good cytocompatibility and could enhance proliferation of TDSCs when compared to DCB.The results of transwell migration assay indicated that DCB-ECM also recruited more stem cells than DCB.Real-time PCR analysis showed that DCB-ECM promoted chondrogenic and tenogenic differentiation of TDSCs,while it inhibited osteogenic differentiation of cells when compared to the DCB.3.Rabbit TDSCs were identified to have universal stem cell characteristics including cell markers,clonogenicity and multilineage differentiation.The h DCB-ECM contained three components(bone,p DCB and DCB-ECM)with a gradient of mineral content.The result of DNA content detection confirmed that h DCB-ECM had been decellularized,and the scaffold had SDF-1,Bgn and Fmod.The results of AFM showed that the elastic modulus of bone was significantly higher than that of p DCB or DCB-ECM,and the elastic modulus of p DCB was significantly higher than that of DCB-ECM.4.Macroscopic observations demonstrated the absence of infection and rupture around the enthesis.The results of immunofluorescence staining showed that h DCB-ECM promoted endogenous stromal cell recruitment.Results of micro-CT analysis,histological staining and immunohistochemical staining illustrated that the h DCB-ECM enhanced bone and fibrocartilage formation at the tendon-bone interface when compared with DCB.Biomechanical analysis showed that the h DCB-ECM group had higher ultimate tensile stress and Young’s modulus than the DCB group.Conclusion:In this study,DCB modified with ECM derived from rat TDSCs was prepared,and in vitro experiments confirmed that ECM modification could enhance proliferation,migration and differentiation of stem cells.The h DCB-ECM with the changes in mineral contents,biochemical factors and elastic moduli was prepared by the graded demineralization and ECM modification,and in vivo experiments confirmed that h DCB-ECM could promote tendon-bone interface healing by recruiting endogenous stromal cells. |
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