Studies On Direct Construction Of Tissue-engineered Cartilage With Three-dimensional Cell-assembly Technique

Cartilage defect is very common in clinical practice. Until now , many methods have been developed to repair cartilage defects, but no one is perfect. Tissue engineering brings new hopes for the functional and biological replacement of cartilage defects. The traditional tissue engineering approach involves transplantation of cells onto scaffolds. In this study, we fabricate the engineering cartilage through 3D cell-assembly technique, which combined the principles of tissue engineering and bio-manufacturing. The formed 3D structure possesses the microenvironment suitable for cell growth from the viewpoint of microstructure ,and possesses the pre-designed structure and shape from the viewpoint of macrostructure.We firstly fabricated the 3D structure with the mixture of alginate and gelatin,then transplanted it into the subcutaneous pocket of nude mice.After 8 weeks of transplantation,the result shows that the engineering cartilage was successfully formed.The contents are divided into three parts as follow.1. Preparation and evaluation of the mixture of alginate and gelatin.Aim: Through in vitro culture the microballoons made from chondrocytes and the mixed hydrogel, evaluate whether the mixed hydrogel is feasible to chondrocytes. Method:alginate and gelatin were selected to form mixed hydrogel S20G0,S10G10,S5G15,S3G17,S1G19,which was used to fabricate microballoons with chondrocytes.After in vitro culturing 4 weeks,MTT assay was used to analyse proliferation of chondrocytes in gel. Results:MTT assay indicated that the number of chondrocytes in the S3G17 increased with time. Conclusion:The chondrocytes can normally grow in the S3G17 ,so it was selected to fabricate the 3D structure.2. Fabrication and analysis of engineered cartilage in vitro.Aim: Combining the principles of tissue engineering and bio-manufacturing ,this experiment is designed to fabricate tissue-engineered cartilage constructs by three-dimensional(3D) cell-assembly technique. Methods:Alginate and gelatin were selected to form mixed hydrogel S3G17,which was used as biomaterial of scaffold.Chondrocyte suspension was added into the mixed hydrogel. This mixture of hydrogel and chondrocytes was used to fabricate tissue-engineered cartilage constructs by 3D cell-assembly technique.Then the 3D constructs were cultured in vitro.After culturing for 1-4 weeks, the samples were observed by histological analysis. Results: MTT assay indicated that the number of chondrocytes in the 3D constructs increased with time.The histological analysis revealed that the chondrocytes in gel had the normal morphology of functional chondrocytes. Conclusion: The chondrocytes in the 3D cartilage constructs can grow, proliferate and maintain the chondrocytic phenotype and activity, which demonstrate the 3D cell-assembly technique could fabricate tissue engineering cartilage in vivo.3. Fabrication and analysis of engineered cartilage in vivo.Aim:To construct the engineered cartilage in nude mice model. Methods: the 3D structures were fabricated with the mixture of alginate and gelatin,then transplanted it into the subcutaneous pockets of nude mice.After 8 weeks of transplantation,the nude mice were sacrificed and specimens were dissected free of surrounding soft tissue for gross and histological analysis. Results: The gross and histological analysis showed that the 3D constructs formed cartilage in vivo very well. Conclusion:the 3D cell-assembly technique is a very potential technique to fabricate tissue engineering cartilage.