A Comparative Study On Preparation Of Decellularized Articular Cartilage Matrix Scaffold With Different Parameters

Objective:By comparing different methods for preparing cartilage extracellular matrix(ECM),the effects of different preparation parameters(such as the decellularization reagent,cartilage particles size,cross-linking methods,etc.)on the scaffold were explored to construct a cartilage extracellular matrix scaffold with optimized performance and provide new clues for improving the method of cartilage tissue engineering preparation in the future.Methods:We selected two decellularization parameters for comparative studies,cartilage particle size and decellularization reagent,which are highly correlated with scaffold performance and had not been systematically tested.Cartilage microparticles with diameters of 0-100 ?m and 0-300 ?m were decellularized with 0.5% SDS,1% SDS,and 1% TritonX-100 reagents respectively.After lyophilization,cross-linking with dehydration heat treatment or genipin were performed respectively to construct decellularized scaffolds.Biochemical analysis and physical performance testing were performed on the decellularized scaffolds : Picogreen was used to quantify the scaffold nucleic acid content to evaluate the effect of decellularization;DMMB was used to quantify the level of scaffold glycosaminoglycans(GAG);OHP assay was used to measure the total collagen content;Mechanical properties were quantitatively measured;micro-CT scan was used to evaluate sample porosity.Results:The freeze-dried cartilage matrix particles can be used to prepare the scaffolds with three-dimensional morphology and interconnected porous microstructure.The scaffold has a certain degree of elasticity and can rebound after being compressed,showing the similar characteristics to the sponge.After decellularization,the cartilage particles(?0~100 ?m)can obtain a satisfactory residual DNA content.0.5% SDS,1% SDS,and 1% TritonX-100 all reduced DNA by 90%,which means the decellularization effects were good.Extending treatment time of the decellularization tend to reduce the DNA content of the scaffold,and this trend is statistically significant in the larger diameter cartilage particles.0.5% SDS,1% SDS,and 1% TritonX-100 have obvious differences in the reduction of proteoglycans.1% TritonX-100 can maintain relatively high GAG levels,significantly higher than 1% SDS and 0.5% SDS.The remaining amount of GAG is also related to the size of cartilage particles,and larger diameter cartilage particles can retain more GAG components.Extending treatment time has a tendency to reduce GAG content.Decellularization has little effect on the amount of collagen in the matrix scaffold.The scaffold with dehydration heat treatment has better compression resistance than that of the scaffold with genipin cross-linking treatment.The acellular matrix scaffold composed of 0~100 ?m cartilage microparticles has a higher Young's modulus,and the effect of different decellularization reagents is not obvious;The scaffolds with particle diameters of less than 300 ?m exhibited poorer compressive properties,and the type of detergents had a more obvious effect.Among them,the 1% SDS group had the lowest Young's modulus.1% TritonX-100 has a tendency to maintain higher compressive performance,but the difference is not significant.The cross-linked scaffold has a large porosity,and the difference diameter of the cartilage particles has a significant effect on the porosity.The porosity of the <100?m group is higher than <300?m group.In the <100?m group,the porosity of the scaffold treated with 1% TritonX-100 was higher,and the effect of the type of detergents in the <300?m group on the porosity was not significant.Compared to genipin,the scaffold with dehydration heat treatment tend to maintain relatively larger porosity.Conclusion:TritonX-100,as a milder detergent,can maintain a satisfactory DNA clearance and a relatively higher level of GAG.The scaffold composed of <100 ?m of cartilage particles exhibited better performance in terms of porosity and mechanical properties than the <300 ?m group.Dehydration heat treatment can obtain good mechanical properties,and the cross-linked scaffold has better compressive strength than genipin.