Cartilage Tissue Engineering By Different Culture Systems With Adipose Tissue-Derived Mesenchymal Stem Cells

ObjectiveTo evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold by comparison of the chondrogenic differentiation,cell proliferation and phenotype expression in scaffold,plates and pellet culture systems for rat adipose tissue-derived stromal cells cultured in vitro.MethodsAdipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by typeⅠcollagenase and then cultured in vitro.TypeⅠcollagen was fully mixed with chitosan with volume ratios of 7:3,then freeze-dried and then cross-linked with 2-N-morpholino ethanesulfonic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,N-hydroxysuccinimide and chondroitin sulfate,then freeze-dried again and sterilized by ethylene oxide.The pore diameter,water content,porosity of the scaffold were tested.The passage 4 adipose tissue-derived stromal cells cultured in vitro were digested and were seeded into the scaffold,plates and centrifuged into pellet,then were induced into cartilage in the induction medium(serum free DMEM,0.1%bovine serum albumin,10ng/mlTGF-β1, 50mg/Lascorbic acid,10^-7mol/L dexamethasone,ITS+,1%penicillin-streptomycin). MTT detection for the cell proliferation at the time point of 1,3,5,7,14,21d,the OD valve were detected to draw growth curve,inverted microscope and Scanning Electron Microscope were used to observe the cell morphology in the plate and pellet culture systems,cell proliferation and adhesion on the scaffold were also observed.Histology stain,typeⅡcollagen immunohistochemistry stain were used to observe the cell morphology and distribution in 3 groups.RT-PCR for the cartilage specific mRNA,typeⅡcollagen,aggrecan,SOX-9 and the dedifferention marker of type X collagen were analyzed after 3 weeks to show the state of chondrogenic differentiation.ResultsThe pore diameter,water content,porosity tested for the scaffold showed an appropriate form.Cell proliferation was observed in all three systems showed by MTT detection but faster in the scaffold and pellet culture system after the 5 day,the difference was statistically significant,there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet culture system;cells on the scaffold proliferated densely showed by the histological staining but there was scaffold structure residues in the inner layer.The finding of typeⅡimmunohistochemistry stain showed that cells express strong positive for typeⅡcollagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system;The specific mRNA for cartilage,typeⅡcollagen,aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR,but the type X collagen was expressed continuously in the plate culture system and expressed after 21 days in the pellet culture system,whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system.ConclusionThe parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study.The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and can maintain the phenotype of chondrocytes well,it is the optimal choice for cartilage tissue engineering in the future.