Construct Of Microporous Osteochondral Decellularized Matrix And ASCs Sheet As A Promising Transplant Promoting Cartilage Defect Repair And Interface Integration

Background Due to the inherent structural features and biological properties that make it difficult to self-renewal,the difficulties of cartilage regeneration and repair have occupied a lot attention.Defects in tissues or organs are the ultimate clinical manifestations of certain refractory diseases,such as trauma or osteoarthritis osteoarthritis(OA)induced by cartilage defects and defects by bone necrosis.With the creation of stem cell technology,cell therapy has been applied to repair cartilage defects,and a number of studies have shown inherent potential of adipose-derived stem cells for cartilage differentiation.Cell sheet is a complete sheet-like structure containing extracellular matrix.It contains important cell surface proteins such as ion channels,growth factor receptors,and connexins,and it has advantages comparable to traditional tissue engineering techniques.The sheet can be completely obtained through a specific culture technique without significantly destroying structures and components.The ideal regeneration of articular cartilage not only refers to the consistency and integrity of the appearance,but also includes that the structure of the repaired area be as similar to the in situ structure as possible,and the new cartilage is metabolized over time.The localized neonatal cartilage morphology produced by the defect marginal tissue suggests that it is vital that cells migrate well from the edge of the native cartilage.Therefore,scaffolds that promote cell migration,maintain structure,and provide a good microenvironment are particularly important during repair.Appropriate decellularization process can effectively remove cell components from cartilage as well as avoiding the structural damage and extracellular matrix(ECM)loss result from the graft-host immune response,and consequently maintaining similar mechanical and biological properties to the natural structure.Microporous structure contributes to the removal of deep cartilage cells during decellularized processes and to cell migration and ECM deposition.This study explored the fabrication and characteristics of microporous osteochondral decellularized matrix and the acquisition of adipose-derived mesenchymal stem cells(ASCs)sheet.The construct of microporous osteochondral decellularized matrix and ASCs sheet was transplanted to the defect of cartilage to promote the cartilage regeneration and interfacial integration in a rabbit model.Objective The purpose of this project is to explore the method of constructing ASCs sheet and to analyze the morphological characteristics,histological characteristics,potential chondrogenic differentiation ability of ASC cell sheets at different time points,and preparation and characteristics of microporous osteochondral decellularized matrix.In addition,this study testified microporous osteochondral decellularized matrix combined with ASCs sheet in promoting the repair and interfacial integration of rabbit cartilage defects.Methods The full-thickness cylindrical defect model was established in the patella trochlea groove of New Zealand rabbits.The rabbits were divided into osteochondral decellularized matrix group,the microporous osteochondral decellularized matrix group,and the microporous osteochondral decellularized matrix combined with ASCs sheet group respectively.At 6 and 12 weeks after surgery,the samples were assessed by morphological,histological,and molecular biology.Results In vitro,microporous osteochondral decellularized matrix combined with ASCs sheet were transplanted and cultured with the surrounding histological tissue closely after 4 weeks,and no obvious gap was found at the edge of the graft.Histologically,the ASCs sheet was observed filling in the gap and maintaining structural continuity.The expression of Collagen ?,Agg and Sox9 in complex construct group was higher than that in control group,Collagen X was lower than that in control group,and there was significance.In vivo,the microporous osteochondral decellularized matrix group combined with ASCs sheet had no obvious defect boundary after 6 weeks,and the repaired surface was smooth.At the 12th week,it was difficult to recognize the boundary of repaired area and the surface was smooth and continuous.Histological observation revealed that at the 6th week,more cartilage-like tissue was formed in the complex construct group,and the subchondral bone was remodeled.After 12 weeks,in the complex construct group,the upper layer of the defect is filled with cartilage-like tissue,the surface is smooth,and the new cartilage integrates well with the native cartilage and subchondral bone maintained well.At 6th and 12th week,Collagen ?,Agg and Sox9 expression was higher in microporous osteochondral decellularized matrix combined with ASCs sheet group than that in others,but Collagen X was lower than the other two groups,and there was significance.The polarized microscope observed that in the complex construct group,the collagen fibers in the repair tissue are similar to that in the normal articular cartilage tissue.The birefringence(index of collagen content)in the repair tissue is slightly weaker than the surrounding cartilage.Conclusion The new method of combining microporous osteochondral decellularized matrix scaffold with ASCs sheet can enhance cartilage regeneration and promote the integration of neo-cartilage with surrounding native cartilage.