Effects Of Tissue Engineering Scaffolds Based On Cartilage Decellularized Matrix On Chondrogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objective: Due to the avascular and nerveless nature,articular cartilage has very limited self-repair capability after damage.Decellularized matrix is a natural biomaterial with bioactive molecules.Decellularized matrix is obtained by removing cells and antigenic components from tissues.Decellularized matrix scaffolds are widely used in tissue engineering.The repair effect of decellularized matrix scaffolds is related to the variables such as the source species of decellularized matrix,processing method and the acellular efficiency.Meanwhile,tissue source age also plays an important factor.We compared the differences in rabbit cartilage tissue from three different age groups: newborn(3-day),juveniles(100-day)and adults(200-day)and investigated the chondrogenic induction of bone marrow mesenchymal stem cells(BMSCs)by cartilage decellularized matrix in different scaffolds.Methods: In the first part,the articular cartilage was extracted from the knee joints of 3-day,100-day,and 200-day old New Zealand Rabbits.The content of sGAG,DNA,total collagen content,mechanical properties and the content of specific growth factors in tissues of different age groups were characterized.After grinding,articular cartilage was decellularized and the decellularization efficiency were confirmed by DAPI staining and DNA content detection.Matrix retention was determined by sGAG and total collagen quantitation and ELISA.In the second part,decellularized matrix hydrogel from different aged rabbit stimulated BMSCs to explore chondrogenic differentiation.DMPs were mixed with collagen to form collagen-DMP composite hydrogels.Collagen hydrogels were used as control.The hydrogels were mixed with BMSCs and cultured in vitro and implanted into subcutaneous implantation in nude mice.The proliferation and differentiation of cells in the four groups of hydrogels were detected by DNA,sGAG and section staining.In the third part,decellularized matrix orientated scaffolds from different aged rabbit stimulated the chondrogenic differentiation of BMSCs.DMPs were mixed with collagen to form collagen-DMP composite scaffold with orientated pore architecture.Collagen scaffolds were used as control.BMSCs were seeded into scaffolds and cultured for 28 days in vitro.sGAG quantitative detection and section staining were used to observe the secretions and mineralization in the four groups.In vivo,collagen-DMP composite scaffolds were used for the repair of cartilage defects in rabbit knee joint.Results: In the first part,the results showed that with the increased age,the content of DNA,sGAG and cytokines showed a decreasing trend,while the total collagen and the mechanical properties of cartilage showed an increasing trend.After decellularization,the results showed that the DNA removal efficiency was up to 99.2%,and the important bioactive molecules were preserved.In the second part,the group of Col3d-H better promoted the chondrogenesis of BMSCs,but also lead to obvious osteogenic differentiation of BMSCs.The Col100d-H group showed good cartilage induction and anti-calcification ability.Col200d-H group had the worst chondrogenic ability.In the third part,the Col3d-S group promoted the chondrogenesis of BMSCs but led to matrix calcification.The Col100d-S group showed good chondrogenic differentiation ability.The Col200d-S group showed a tendency of fibrosis in the late stage of cell culture.In vivo,The results showed that the collagen-DMP composite scaffold had resulted in a better cartilage repair.Conclusion:1.The decellularized matrix of cartilage in all three age groups can effectively promote the proliferation of BMSCs and induce chondrogenic differentiation of BMSCs.2.The decellularized matrix of 3d may provide better cartilage induction,but it may also lead to significant matrix mineralization.3.The decellularized matrix of 100 d achieved the best overall results in promoting chondrogenesis of BMSCs and preventing matrix calcification.4.BMSCs synthesized the lowest amount of cartilaginous matrix with decellularized matrix of 200 d.5.Therefore,juvenile ECM could be a better source than both newborn and adult ECMs.This finding provides important information for the design of decellularized matrix based biomaterials towards successful repair of articular cartilage in the future.