Research On Differentiation Of Bmscs Into Hypertrophic Chondrocytes And Osteochondral Regeneration

The repair of articular cartilage defects has always been a major challenge in clinical sports medicine.The current clinical methods mostly focus on relieving pain rather than curing,while tissue engineering is expected to achieve regeneration of osteochondral defects.Materials for bone and cartilage regeneration and transitional intermediate materials which can prevent the bone and cartilage from invading each other have been designed.As a result,various two-phase,three-phase and even multi-phase scaffolds have been developed.In natural articular cartilage,the calcified cartilage intermediate layer(tide line structure),which between the bone and the cartilage plays an important physiological role.However,most of the existing osteochondral tissue engineering scaffolds are not specifically designed for the formation of the calcified cartilage intermediate layer,which will undoubtedly affect the integration of new cartilage tissue and subchondral bone tissue.Therefore,exploring the mechanism that promotes the formation of the calcified cartilage intermediate layer and designing an implant with bionic natural bone-cartilage gradient characteristics for the regeneration of the osteochondral interface is still one of the challenges in this field.In this study,we first combined electrospinning and cell layer technology to prepare a multi-layer cell/mesh implant that mimics the transition characteristic of cartilage-calcified cartilage-subchondral bone through layer-by-layer stacking for full-thickness regeneration of osteochondral defects.According to the characteristics of the extracellular matrix composition of natural cartilage and bone tissue,the cartilage layer of the implant is composed by polylactic acid/gelatin/hyaluronic acid/chondroitin sulfate(PLLA/Gel/HA/CS)fibrous mesh loaded with a layer of bone marrow mesenchymal stem cells(BMSCs),which is pre-differentiated with chondrogenic medium;The calcified cartilage intermediate layer is composed by polylactic acid/gelatin(PLLA/Gel)fibrous membrane loaded with a layer of BMSCs,which is pre-differentiated with a 1:1 mixture of cartilage and osteogenesis induction medium;The bone layer of the scaffold is composed by polylactic acid/gelatin(PLLA/Gel)fibrous membrane loaded with a layer of BMSCs,which is pre-differentiated with osteogenic medium.The above-mentioned gradient implant was implanted under the skin of New Zealand white rabbits.Histological staining results showed that the introduction of the intermediate layer not only effectively prevented the mutual invasion of the bone and the cartilage,but also improved the continuity of the interface between the two phases.Then,the above-mentioned gradient implant was implanted into New Zealand rabbit knee joint osteochondral defects at 4 W and 12 W.Compared with the blank control group,the regeneration rate of hyaline cartilage and subchondral bone in the experimental group was significantly accelerated.At 12 W,in the implanted group,the cartilage regenerated on the surface of the defect integrated well with the surrounding natural hyaline cartilage,abundant Col-? was distributed in the new cartilage and a clear tide line structure can be observed between the surface cartilage and the subchondral bone.The above results proved that the introduction of the transitional intermediate layer played an important role in the regeneration of full-thickness osteochondral defects.Subsequently,this study selected several cells that are closely related to the process of osteochondral tissue regeneration,including BMSC,osteogenic precursor cells(MC3T3),chondrocyte(Cho),and hypertrophic chondrocyte(HCho)derived from induced differentiation.Incubating the cell layers with electrospinning PLLA/Gel fiber membranes as supports,and then decellularized the cell layers to obtain four decellularized extracellular matrix(D-ECM)materials.The effects of D-ECMs on the differentiation of re-inoculated BMSCs into hypertrophic chondrocytes was explored to provide a reference for the design of the transitional intermediate layer of the osteochondral scaffold.Studies found that all D-ECMs are beneficial to the attachment and proliferation of BMSCs,and D-ECM derived from BMSCs has the the strongest ability to promote the proliferation of re-inoculated BMSCs.In the study of re-inoculation of BMSCs for hypertrophy,it was found that D-ECMs derived from HCho and BMSCs exhibited the ability to promote the hypertrophy of re-inoculation of BMSCs to varying degrees.Due to the richer Col-X and alkaline phosphatase(ALP)deposition,D-ECM derived from HCho has a more obvious hypertrophy-promoting ability.Further analysis of related gene expression by RT-PCR,it was believed that D-ECM derived from HCho promoted the expression of COL-X,MMP-13,ALP and RunX-2 genes in the re-inoculated cells,which in turn made the cells exhibite a phenotype similar to hypertrophic chondrocytes.Four kinds of D-ECM/mesh composites were implanted into the back of rats and sampled at 2 W and 4 W.The histological staining results showed that,compared with PLLA/Gel mesh,the introduction of D-ECMs significantly reduced the inflammatory response caused by fiber membranes,and D-ECM derived from HCho has a more significant ability to induce cell hypertrophy.In summary,the calcified cartilage intermediate layer plays an important role in the regeneration of osteochondral.The biomimetic gradient osteochondral implants constructed by layer-by-layer technology,in which the intermediate transition layer was based on cell layer/mesh technology combined with in vitro pre-induction differentiation treatment or constructed by D-ECM derived from HCho,have a good application prospect in the field of osteochondral tissue engineering.