To Investigate A Cartilage Extracellular Matrix-derived Scaffold

?Objective? To evaluate the advantage of the modified procedure to process cartilage extracellular matrix scaffold.?Methods?(1)Using an existing procedure as homogenate-decellularization to product cartilage extracellular matrix(ECM)powder,then to grind and filtrate ECM to prepare fine cartilage extracellular matrix(FECM)powder.(2)To freeze-dry the suspension of ECM powder and FECM powder in concentration of 10%(w/v)to preparate ECM scaffold and FECM scaffold as control group and experimental group.(3)The scaffolds were assessed via macroscopic observation,scanning electron microscope.And then the pore diameter was observed.(4)The decellularization of ECM scaffold and FECM scaffod was assessed by DAPI stain,then calculated the porosity and water absorption of scaffolds,and performed the biological safety experiments by CCK-8 test.(5)The third generation bone mesenchymal stem cells(BMSCs)were inoculated on to ECM scaffold and FECM scaffold.The seeding efficiency and adhesive rate of seed cells were calculated after seeding.(6)Both of ECM-scaffolds and FECM-scaffolds were seeded with bone marrow mesenchymal stem cells(BMSCs)as control group and experimental group.The PLGA scaffold were seeded with BMSCs which were induced to differentiate into chondrocytes for 21 days as postive control group.After culture in vitro for 12 h,the cell-scaffold constructs were implanted in the nude mice subcutaneously.(7)The cell-scaffold constructs were harvested and detected on histology and immunohistochemistry at day 28.?Results?(1)The diameter and the height of the scaffold were 6mm and 2mm.The surface of ECM scaffolds were coarse,and internal clearances were wider,however,the surface of FECM scaffolds were fine and smooth.The pore diameter of ECM scaffold was 239.082±76.703?m,and the pore diameter of FECM scaffold was 69.492±17.316?m.(2)Through DAPI staining,it was observed that the cell components in the two kinds of scaffolds disappeared and the decellularization was completely.(3)The porosity and water absorption of ECM scaffold were 92.11±0.92% and 2156.3±276.9%.The porosity and water absorption of FECM scaffold were 83.63±0.27% and 1763.2±103.1%.(4)CCK-8 test showed that the scaffolds did not induce any cytotoxicity to BMSCs.(5)The seeding efficiency and adhesive rate of ECM scaffold were 81.4% and 73.1%.The seeding efficiency and adhesive rate of FECM scaffold were 89.6% and 80.8%.(6)The cell-scaffold constructs were assessed via macroscopic observation.The specimens in experimental group were rounded and full,the color is porcelain white that matched to normal cartilage.But the cell-scaffold constructs of control group were out-of-shape with collapse.All the cell-scaffold constructs of experimental group and the positive control group have a full shape,with smooth surface and uniform color.(7)HE staining and Safranin O staining:in experimental group and the positive control group,Safranin O staining showed strong positive reaction.Chondrocytes distributed evenly with good growth and rapid proliferation,and encompassed by massive diffuse ECM deposition.But in control group,chondrocytes distributed sparse with low density,ECM were fragmented,meanwhile Safranin O staining showed very weakly positive reaction.(8)Immunohistochemical staining:in experimental group and the positive control group,immunohistochemical staining for type II collagen showed very strong positive reaction,but weakly positive reaction in control group.?Conclusion? FECM scaffold with excellent shape fabricated by a modified procedure can mimic native healthy microenvironment of articular chondrocyte more better,it is more conducive to BMSCs to adhesion,growth,proliferation and differentiation.FECM-scaffold can promote BMSCs to differentiation into chondrocytes without exogenous TGF-?1 to construct quality tissue engineered cartilage.So FECM scaffold have potential of application in repairing of cartilage defects with tissue engineering.