The Construction Of Collagen Type Ⅱ-hyaluronic Acid Composite Biomaterial And Preliminary Application In The Tissue-engineering Cartilage

ObjectionArticular cartilage defects repair is a difficult medical problem in orthopeadic area due to the limited capability for repair and self-regeneration. A minor injury of particular cartilage may lead to progressive damage and degeneration. Tissue engineering can potentially be used for cartilage repair. Establishment of a 3D system consisting of biomaterials and cells plays a vital role in tissue engineering. In this study, we prepared a collagen typeⅡ-hyaluronic acid copolymer porous scaffold(CⅡ-HA) which imitated the ingredients and ratios of natural cartilage matrix. Bone marrow mesenchymal stem cells(BMSCs) were obtained and cultured in vitro, and then seeded in the CⅡ-HA matrices. In order to investigate the feasibility of the BMSC/CⅡ-HA as tissue engineering cartilage graft, the viability and differentiation of BMSCs were detected. The expression of chondrocyte specific collagen type X in the cells were also detected.MethodType II collagen solutions were obtained by the method of our previously shown, and identified by SDS-PAGE electrophoresis and the maximum absorption wavelength. To control the pore size of materials, the initial collagen solutions were condensed with polyethylene glycol (PEG) at 4℃. The collagen typeⅡ-hyaluronic acid compound were prepared by partial homogenaization and then freeze-dried. CⅡ-HA compound were crosslinked with EDC/NHS. Collagen type I-hyaluronic acid matrics were prepared routinely as control group. The characteristics of the prepared scaffolds were analyzed by laser scanning confocal microscopy (LSCM), scanning electron microscopy (SEM), FT-IR analyzer, tensile tests and so on.Bone marrow stem cells were obtained and purified by adhesion culture in vitro. The morphology of BMSCs was observed under the phase contrast microscope Some of BMSCs were induced into adipotic cells or osteoblast cells in special supplement medium. Afterwards, specific markers of differentiation of both adipocytic and osteogenic cells were detected. BMSCs were seeded into the CⅡ-HA matrices, the proliferation of BMSCs was monitored via XTT test, and the expression of collagen type X was detected by immunofluorescence. The GFP labeled BMSCs were planted into crosslinked CⅡ-HA, the cells viability was observed by LSCM.ResultThe result of SDS-PAGE electrophoresis shows typical a andβbands of the type II collagen, which indicated that the preparation collagen is pure and free of contaminating proteins. The peak of absorption maximum wavelength was 233.8nm as the same as the collagen should be. Collagen scaffolds had the interconnected porous structure, and the pore were well-distributed. The pore size of CⅡ-HA was 92.63±32.51μm which was more suitable for the growth of cartilage tissue, compared with the control group(CⅠ-HA) that was 202.16±76.75μm, and there was significant difference between two groups. Average porosity and water capacity of CⅡ-HA scaffolds reached to 93.39% and 97.78%, respectively. The mechanical properties of cross-linked CⅡ-HA increased significantly, reached to 79.67±11 .7KPa, While CⅠ-HA was 17.38±1.8KPa. Moreover, the infrared spectrum showed collagen type II and hyaluronic acid formed a new small peak (1402cm-1) after cross-linking that indicated there was a bond formation.BMSCs were isolated and cultured in vitro. BMSCs were cultured in osteogenic or adipogenesis medium have been shown that cells we isolated could be differentiated down the osteogenic or adipogenic lineage, as evidenced by calcium deposition or droplet after 21 days. BMSCs were seeded in CⅡ-HA scaffold for 5 days, the expression of collagen type X was positive. The green fluorescent protein (GFP) gene can successfully be transducted into BMSCs by lentivirus. The BMSCs showed in normal condition after the transduction. GFP labeled BMSCs were seeded into CⅡ-HA scaffold, the cells were in normal condition and the expression of collagen type X in these cells was positive.ConclusionThe CⅡ-HA has good three-dimensional pore structure. The pore size of CⅡ-HA matrices was suitable for cell growth, Besides, the pores were interconnective and well-distributed. CⅡ-HA scaffolds had suitable mechanical properties, porosity, and water capacity for cartilage tissue engineering. This type II collagen-hyaluronic acid scaffold suppose to be an ideal biomaterial for cartilage tissue engineering.Our results indicated that CⅡ-HA scaffold may create an appropriate environment for the proliferation of BMSCs to chondrocyte differentiation. This CⅡ-HA/BMSCs graft may be suitable for implanting into full-thickness articular cartilage defect in vivo study.