Experimental Study On Transgenetic Bone Marrow Mesenchymal Stem Cells And Decellularized Scaffolds To Develop Tissue-engineered Ligaments

Objective: To investigate feasible method of constructing tissue-engineered ligament (TEL). Methods: (1) Construction of recombinant adenovirus vectors Ad.bFGF-eGFP coding for basic fibroblast growth factor (bFGF) gene and enhanced green fluorescent protein (eGFP) report gene, identification and amplification. (2)Isolation, culture of bone marrow mesenchymal stem cells (BMSCs) , and to investigate expression, secretion of bFGF after transfection, and the effect of bFGF on proliferation , differentiation, and production of specific extracellular matrix proteins of BMSCs. (3)Rabbit patellar tendons were decellularized with two methods including 0.01% trypsin + 1% DCA + nuclease and TBP. After treatment, measure the tissue structures and mechanical properties of the decellularized ligament scaffolds. Select the best method to construct the TEL scaffolds with internal cells removed completely, integrity of collagen structures, and good biomechanical properties. (4)Tissue-engineered ligaments were fabricated in vitro with transgenetic BMSCs seeding onto the decelluarized ligament scaffolds. Examin the growth and proliferation of BMSCs on the scaffolds.Results: (1) Ad.bFGF-eGFP was constructed successfully; (2) BMSCs can express and secrete bFGF efficiently after transfected. The bFGF secreted increase BMSCs proliferation, induces BMSCs to differentiate to fibroblasts, and stimulating expression of specific extracellular matrix proteins. (3) Acellular matrices with excellent biomechanical properties were developed with the method of 0.01% trypsin + 1% DCA + nuclease, in which native cells were completely eliminated and collagen structures preserved undamaged. (4) Compared to control groups, transgenetic BMSCs show better growth and greater proliferation on acellular matrices. Conclusions: (1) The ability of proliferation, differentiation to fibroblasts and expression of specific extracellular matrix proteins of BMSCs was enhanced greatly by Ad.bFGF-eGFP transfected. (2) After decellularized through the method of 0.01% trypsin + 1% DCA + nuclease, the ligaments were developed to TEL scaffolds with internal cells removed completely, integrity of collagen structures, and excellent biomechanical properties. (3) Creating TEL by seeding transgenetic BMSCs onto acellular rabbit ligament matrices was efficient.