Experiment Of Repair Of Ostechondral Defects With Adipose-derived Stem Cells/fibrin Gel Complex In Skeletally Mature Rabbit

PartⅠ. The isolation, cultivation and Chondrogenic differentiationof rabbit adipose- derived stem cells in vitroObjectives: To explore the method of isolating and cultivatingrabbit adipose-derived stem cells, and study their capacity of proliferationand potential of chondrogenic differentiation in vitro.Methods: Adipose tissue from skeletally mature rabbit was digestedusing typeⅠcollagenase for obtaining cells. And the cells was culturedwith DMEM contains 10% new-born calf serum. Morphology of cellswas observed with inverted microscopy. MTT assay was used to examinegrowth kinetics of the 4th and 8th passage. The cells induced Chondro-genesis in high density with chondrogenic media. Seven days afterinduced with chondrogenic media, cells were stained by Safranin O andanti-type-Ⅱcollagen antibody. The expression of typeⅡcollagen mRNAof induced cells and uninduced cells were detected using RT-PCR.Results: Primary cells began to adhere after 2～3 days being culturedwith DMEM contains 10% new-born calf serum, and then becomespindle-shape or polygon-shape. After 7～9 days being cultured, primarycells reached almost confluence and presented colony whirlpool.Morphological change was not found within 8 passages. The doublingtime of the 4th and 8th passage were 31.5±2.7h and 33.9±6.0h, there wereno significant difference between them (p＞0.05). Induced cells began tocondense to form spheroids after 2～3 days being induced withchondrogenic media. After 1week being induced, positive staining withSafranin O and anti-type-Ⅱcollagen antibody were observe in thespheroids, weakly positive or negative stain were observe in the cells didnot condense. The expression of typeⅡcollagen mRNA was observed ininduced cells but not in uninduced cells.Conclusion: Adipose-derived stem cells from skeletally maturerabbit have great capacity of proliferation and potential of chondrogenicdifferentiation in vitro. Adipose-derived stem cells are an alternativesource for seed cells of tissue engineering of cartilage. PartⅡ. Biocompatibility of fibrin gel with rabbit adipose- derivedstem cellsObjective: To study biocompatibility of fibrin gel with rabbitadipose-derived stem cells.Methods: Fourth passage rabbit adipose-derived stem cells wereseeded in fibrin gel (group A), on the surface of fibrin gel (group B) andsurface of tissue culture plate (group C). Each group for 5 wells every96-well plate. Totally 14 96-well plates were used. The cells were seededat 5×10~3/well. Growth kinetics of each group was examined using MTTassay. And morphology of cells was observed with inverted microscopy.Results: The cells were cultured on the surface of fibrin gel and onthe surface of tissue culture plates exhibited an almost spindle-shapemorphology, and reached confluence 7～8 days after being seeded. Thedoubling time of group B and group C were 33.3±6.1 and 31.5±2.7h,there was no significant difference between them (p=0.419＞0.05). Thecells, which were cultured in fibrin gel, have a low proliferation rate, andremained almost round or star-shape. The doubling time of group A was39.7±10.6h. There was significant difference between the doubling timeof group A and group C (p=0.03＜0.05).Conclusions: Fibrin gel has great biocompatibility with rabbitadipose-derived stem cells. Rabbit adipose-derived stem cells have verylow proliferation rate when cultured in three-dimensional environmentwithin fibrin gel.PartⅢ. Repair of ostechondral defects with adipose-derived stemcells/fibrin gel complex in skeletally mature rabbitObjectives: To investigate whether ostechondral defects inskeletally mature rabbit can be successfully repaired using adipose-derived stem cells/fibrin gel complex without using additionalgrowth factors before or after the implantation.Methods: An ostechondral defect, which was 4mm in diameter and4～5mm deep, was made on the femur patellar groove in skeletally matureChinese white rabbit. The defects were respectively implanted withadipose-derived stem cells/fibrin gel complex (adipose-derived stemcells/fibrin gel complex group), cell-free fibrin gel (ell-free fibrin gelgroup), or left with no treat (spontaneous repair group). At 6and 12 weeksafter the operation, animals were killed, and macroscopic and histologicalevaluations of the repair tissue were made.Results: At 6 weeks after the operation, in adipose-derived stemcells/fibrin gel complex group, the defects were repaired with hyalinecartilage-like tissue, macroscopic and histological scales were 9.6±0.8and 1.4±0.5; in cell-free fibrin gel group, the defects were still defectswith fibrous tissue at the bottom, macroscopic and histological scaleswere 4.7±0.5 and 14.0±0.0; in spontaneous repair group, the defects wererepaired mostly with fibrous tissue, macroscopic and histological scaleswere 7.4±1.3 and 98.4±2.0. At 12 weeks after the operation, inadipose-derived stem cells/fibrin gel complex group, the defects wererepaired with more mature hyaline cartilage-like tissue, macroscopic andhistological scales were 9.7±0.5 and 0.7±0.8; in cell-free fibrin gel group,the repair fibrous tissue still can not fill the defects, macroscopic andhistological scales were 4.0±0.0 and 14.0±0.0; in spontaneous repairgroup, the defects were repaired mostly with fibrous tissue, macroscopicand histological scales were 8.5±0.8 and 8.8±0.9. At 6 and 12 weeks afterthe operation, macroscopic and histological scales were significantlyhigher in adipose-derived stem cells/fibrin gel complex group, compare toother groups (P＜0.05).Conclusions: Ostechondral defects as large as 4mm in diameter inskeletally mature rabbit can be successfully repaired using adipose-derived stem cells/fibrin gel complex without using additional growthfactors.