The Experimental Study Of Co-culture Of Bone Marrow Mesenchymal Stem Cells Compounded Improved Fibrin Glue Hydrogel And Chondrocytes To Construct Tissue Engineered Cartilage In Vitro

Objective: To investigate rat bone marrow mesenchymal stem cells(BMSCs),which compound improved fibrin glue hydrogel and chondrocytesco-culture six weeks, proliferate in three-dimensional fibrin glue hydrogel andgrow into cartilage in vitro and providing experimental evidence for cartilagetissue engineering to repair cartilage defects in clinical applications. Methods:1. The bone marrow was obtained from femur and tibia of3days SD rat andthe BMSCs were isolated by whole marrow adherence culture. Aftercultivation, amplification, the3rd Passaged BMSCs were divided into twogroups: the one group was induced to an osteogenic lineage; the another groupwas induced to an adipogenic lineage; The Alizarin red staining and oil red Ostaining were used to examine the effects of adipogenic differentiation andosteogenic differentiation. Meanwhile,the articular chondrocytes wereextracted from knee cartilage of3days SD rat by improved two-stepenzymatic digestion.2. The experimental groups were divided into Agroup(articular chondrocytes group, positive control group),B group(co-culturegroup),C group(BMSCs group, negative control group).The3rd PassagedBMSCs that density was2×106/ml were seeded in improved fibrin glue hydrogel to construct the compound in Transwell co-culture system. The firstPassaged articular chondrocytes that density was1×106/ml were seeded inTranswell co-culture system. The compound were placed in the bottom andarticular chondrocytes in the upper (co-culture group or B group). Articularchondrocytes and BMSCs were compounded improved fibrin glue hydrogelalone as a control group with the same density (positive control group or Agroup, negative control group or C group).After Cultured six weeks in vitro,the compounds were used to evaluate the result by gross observed, wet weightmeasurement, glycosaminoglycan (GAG) quantification, histology,immunohistochemistry and RT-PCR method.Results:1.The BMSCs whichwere isolated by whole marrow adherence culture, were uniform morphology,fusiform and all in the whirlpool shape. Throug adipogenic differentiation andosteogenic differentiation，they were strongly positive;2. In both A group andB group, the tissue engineered cartilage could keep the original size and weremorphologically thicker than C group after6weeks of in vitro culture. And theco-culture group was strongly positive for HE staining,TB staining;3.Dead&live cell staining showed that Cells had fine adhesion to the improvedfibrin glue hydrogel scaffolds;4. The wet weight and GAG quantification of Bgroup were both nearly equivalent of A group.But there was a huge gapbetween B group and C group and statistically significant (p <0.05);5. Fromthe type II collagen immunohistochemical test, the brown granules in both Agroup and B group were seen surround the cell or extracellular matrix;6. RT- PCR method statistics Showed the B groupⅡtype collagen gene expressionlittle more than A group,but there was no statistically significant(p>0.05).Conclusion:1. The BMSCs which were isolated by whole marrowadherence culture, could be induced into chondrocytes by co-culture withallograft articular cartilage cells in vitro;2. Improved fibrin glue hydrogel hadgood biocompatibility to seed cells, and can be made using the injection formresearch;3. The BMSCs in Improved fibrin glue hydrogel could be inducedinto chondrocytes by a little chondrocytes, and the formation of neocartilagetissue, so that chondrocytes, BMSCs and Improved fibrin glue hydrogel can beconstructed together injectable cartilage tissue engineering.