In Vitro Study On The Construction Of Tissue Engineered Nucleus Pulposus By Type Ⅰ/Ⅱ Collagen Composite Scaffold Combined With Human Nucleus Pulposus Mesenchymal Stem Cells

Purpose: The purpose of this study was to investigate the cell viability,proliferation of nucleus pulposus mesenchymal stem cells(NPMSCs)in collagenⅠscaffold and collagenⅡscaffold.In addition,a composite collagen scaffold was constructed to explore the anti-degradation,compressive strength,biocompatibility and other properties of the composite scaffold in order to provide a new choice for tissue engineering nucleus pulposus scaffold materials.Methods: Type I collagen was extracted from porcine tendon.Type II collagen was extracted from porcine articular cartilage.Two kinds of collagen scaffolds were prepared by applying uniform pressure on collagen with cover glass and vacuum lyophilization.The structures of the collagen scaffolds in the two groups were observed by scanning electron microscopy,and the effects of the two scaffolds on the proliferation of NPMSCs were evaluated by CCK-8 test.Then,double-layer collagen composite scaffolds with typeⅡcollagen on the upper layer and typeⅠcollagen on the lower layer were prepared by the above method.Crosslinking was performed by EDC/NHS to stabilize the structure.The porosity,biodegradation rate,and compressive strength of the crosslinked composite scaffolds were measured and compared with those of the non-cross-linked group.The effects of composite scaffolds on cell proliferation were evaluated by CCK-8 assay.The effect of composite scaffolds on the expression of genes related to nucleus pulpoblastic differentiation of NPMSCs was evaluated by RT-PCR.The three-dimensional structure of the composite scaffold was observed by scanning electron microscopy,and the adhesion of mesenchymal stem cells on the composite collagen scaffold and cell growth were also observed by scanning electron microscopy.Results: The results of electron microscope showed that typeⅠcollagen had dense structure,typeⅡcollagen had loose and porous structure.OD value of typeⅡcollagen group was higher than that of typeⅠcollagen group(p<0.05).Based on the above results,a bilayer collagen composite scaffold was constructed.The scaffold porosity was(88.15±2.65)%,and the degradation rate after crosslinking was significantly lower than that in the non-crosslinking group(p<0.05).The compressive strength of the cross-linked group was 14.86±0.41 Kpa,which was significantly higher than that of the non-cross-linked group(p<0.01).The results of CCK-8 test showed that the OD value of the composite collagen scaffold group was significantly higher than that of the flat culture group(p<0.05).After 21 d culture,the m RNA expressions of SOX9,typeⅠcollagen,typeⅡcollagen and aggrecan were increased significantly in collagen scaffold groups compared to control group(p<0.05).Scanning electron microscopy showed the scaffold was a good adhesive and growing material for NPMSCs.Conclusion: Compared with typeⅠcollagen,typeⅡcollagen is more suitable as a scaffold material for tissue engineering nucleus pulposus in promoting the proliferation of mesenchymal stem cells.After crosslinking with EDC and NHS,the anti-collagen properties and compressive strength of the composite collagen scaffold were improved.The typeⅠ/Ⅱcollagen composite scaffold promotes the proliferation of nucleus pulposus mesenchymal stem cells and the expression of genes related to nucleus pulposus differentiation.The cross-linked typeⅠ/Ⅱcollagen composite scaffolds can be used as a good choice for cell scaffolds in nucleus pulposus tissue engineering.