Construction Of Calcified Interfacial Layer And Its Application In The Integrated Repair Of Osteochondral Defects

Background: Chondropathy of knee joint is a disease that affects almost all ages and seriously affects the quality of life of all mankind.There are many causes of osteochondral damage,such as trauma,disease or aging.Anatomically,articular cartilage and hard bone are closely related,and their biological functions influence each other.The osteochondral interface is the interface between hyaline cartilage and subchondral bone.The natural interface is composed of cartilage calcification area,which is thin and dense and ACTS as a barrier between the cartilage layer and the subchondral bone layer while integrating them organically.When osteochondral defects involve both cartilage layer and subchondral bone layer,their different mechanical properties and tissue structure highlight the importance of calcified interface,which is necessary to maintain the normal function of cartilage and subchondral bone.Therefore,the ideal composite osteo-cartilage scaffold should simulate the structure of normal osteo-cartilage tissue,so the structure of calcification layer should be constructed in the preparation of composite scaffold,so as to better promote the repair of osteo-cartilage tissue defects.The regeneration of osteo-cartilage integrated scaffold will become a hot topic in scientific research and clinical practice.Therefore,this study mainly includes the following contents: 1.The construction of cartilage layer scaffold technology,and the screening of hydrogel scaffold suitable for chondrocyte phenotype and function;2.Develop an integrated scaffold with calcification layer;3.To evaluate the characteristics of integrated scaffolds and the shielding effect of calcified interface layer in vitro.Methods: In this study,sodium alginate and chitosan hydrogels with good biocompatibility were selected to construct an integrated composite scaffold with calcification layer.First cartilage process optimization,screening of two kinds of molecular weight close to but,uronic acid(G),sodium alginate with different content of material,the distribution of tag for HG and MG,was prepared with different mechanical properties of sodium alginate-collagen composite hydrogel scaffold,focuses on G content in sodium alginate on cartilage cell phenotype and function of the influence law.The adhesion and viability of chondrocytes on materials were observed by scanning electron microscopy(sem)in the original joint chondrocytes of New Zealand rabbits.Then,Cell proliferation activities of chondrocytes were observed by co-culture of the two groups of materials with chondrocytes through Cell Counting kit-8(cck-8)experiment and live/dead Cell staining experiment.Cytoskeleton morphology staining experiment was used to determine the Cell morphology changes of chondrocytes on the two groups of hydrogels.Real-time fluorescence quantitative polymerase chain reaction(qrt-pcr)was performed to determine the activity of chondrocytes and the expression of AGG,SOX9 and Collagen Collagen II(col-ii)mRNA in 7,14 days after co-culture with chondrocytes in the two groups.The expression of extracellular matrix col-ii and glycosaminoglycan(GAGs)in chondrocytes was detected by ELISA.The calcified scaffold was further constructed,and the subchondral scaffold and the cartilage scaffold of the osteochondral composite scaffold were integrated by ion crosslinking.The structure of the stent was characterized by scanning electron microscope and micro-ct.Different color fluorescent labeling was used to observe the distribution of polymer materials in the scaffold under confocal microscope.Finally,whether the calcified layer in the integrated scaffold has good cell shielding effect was verified in vitro.The necessity of calcification layer was verified by tanswell cell co-culture experiment.Further,chondrocytes,endothelial cells and osteoblasts were labeled with fluorescent probes and seeded in cartilage layer and subchondral bone layer respectively.After co-culture for 7 days,the shielding effect of calcified scaffold cells was observed under confocal microscopy.Result: SEM observation showed that chondrocytes in MG group had good adhesion and diffusion on the pore wall of the scaffold surface.Cytoskeletal morphology staining results showed that after 7 days of co-culture,chondrocytes in MG group maintained their original morphology better.After 7 days,the growth of chondrocytes in the MG group was better than that in the HG group.In addition,CCK8 assay showed that chondrocytes maintained good proliferation on the surface of the two groups of hydrogel scaffolds.The results of qrt-pcr showed that the expression of SOX9,Agg and COLII genes related to the maintenance of cartilage function on the hydrogel in MG group were significantly up-regulated(P < 0.05).ELISA results showed that the expression levels of COLII and GAGs in MG group were significantly higher than that in HG group(P < 0.05).Conclusion: we selected the interpenetrating network hydrogel prepared by sodium alginate and collagen in MG group as the cartilage layer of cartilage tissue engineering scaffold,and the interpenetrating network hydrogel prepared by sodium alginate and collagen and nanometer hydroxyapatite in HG group as the subchondral bone layer of cartilage tissue engineering scaffold.And through the positive charge of chitosan react with sodium alginate burden electricity,between the two layers via electrostatic self-assembly form polyelectrolyte complex film forming integration support,further verify the polyelectrolyte complexation with dense membranes as calcification integration support up to the cells of the interface layer shielding effect,achieve the separation of the two kinds of microenvironment.