| ObjectiveThe self-healing of articular cartilage defects under normal physiological circumstances is difficult due to the histological characteristics of cartilage and the aneural and avascular tissue. One of the treatments of cartilage defects involves the use of engineered functional cartilage produced using living cells, which requires the use of a biocompatible polymer and stimulation to induce chondrogenesis. Although autologous chondrocytes are useful in cartilage regeneration, their wider application is limited due to the proliferative capacity of terminally differentiated chondrocytes, the formation of fibrous cartilage, and the morbidity of donor sites. Thus, there have been many studies conducted to evaluate the use of adult stem cells in cartilage tissue engineering. Recently, adipose tissue-derived stem cells (ADSCs) have been regarded as good candidates for the repair and regeneration of articular hyaline cartilage due to their self renewal, long term cell viability and multilineage differentiation potential. In addition, ADSCs have advantages such as the ability to be obtained in large quantities from various body parts under local anesthesia with minimal discomfort. The goal of our study is to induce adipose tissue-derived stem cells chondrogenesis by means of an innovative co-culture way, to inspect the feasibilty in practice through detecting some targets,and to evaluate the results.Materials and Methods1.Using healthy New Zealand white rabbits approximately 3-4 months of age, adipose tissue was obtained from regions surrounding kidney after anesthesis by 3% pentobarbital and adipose tissue-derived stem cells (ADSCs) could be harvested through type I collagenase digestion.ADSCs were cultured primarily and incubated in vitro and purified by differential adhesion. CD49d, CD105, CD34 and CD106 were identified by flow cytometry analysis.2.Articular cartilage and intra-articular synovium were obtained from the non-weight regions of healthy New Zealand white rabbits,approximately 3-4 months of age with aseptic technique. The articular cartilage mass was cut into 1-3mm3 thin pieces,digested for 30 to 35 minutes by 0.25% trypsase,and then digested for 6-8 hours by 0.1% typeⅡcollagenase.ADSCs were filtered,centrifuged,collected and counted,then,inoculated into culture bottles by 2×105/ml cell density.Intra-articular synovium was cut into chips, digested by 4mg/ml type I collagenase.Synovialcells were filtered,centrifuged,collected and counted,then,inoculated into culture bottles by 1 X 105ml cell density. Chondrocytes and synovialcells were cultured and amplificated in vitro conventionally and the third generation cells were used in experiment.3.The third generation ADSCs were classified into three groups according to different inducement methods.The first group was blank control,which without inducement.The second group was induced by chondrocyte induction medium constituted of TGF-β1 lOn g/ml, bFGF-2 5ng/ml,Vc 50μg/ml and Dexamethasone 10-7M/L.The last group was coincubated with the third generation ADSCs, the same generation chondrocytes and synovialcells in 35mm X 10mm culture dishes.Three dishes were put into the same 200mm X 20mm culture dish in the shape of "three mouths stacking".Culture medium(added 10% fetal calf serum)was added into the large dish. ADSCs were collected from the three groups,which mRNA were extracted to detect the expression of SOX-9,Aggrecan and Type II collagen by electrophoresis of the products of reverse transcription-polymerase chain reaction (RT-PCR) detection and real time PCR.Results1.Changes of cell morphology The growth of the ADSCs was observed with the phase contrast microscope. During the beginning of 24 hours, sparse primary cells fastened wall, the shape of the primary ADSCs was short spear-like.Most cells had fastened wall and began to extent and split up,morover, clusters which formed of single cell division appeared after 48 hours. After changing medium of 2-3 times, most of the floating cells were cleared.5-7 days later, the clone cluster of the cells showed up along with gradual division and proliferation of cells. The subcultured cells grew faster. After 7-8 days, monolayer cells, with high nuclears and many grains in cytoplasm, were observed in the shape of parallel or swirl.2. Flow cytometry analysis of ADSCs immune phenotype Flow cytometry analysis showed that the cell surface maker CD49d and CD 105 were positive, but CD 106 and CD34 were negative.3. RT-PCR and real time PCR detection In three target genes, only SOX-9 and aggrecan expressed in the 7th and 14th day after grouping.But three genes all expressed in the 21st day.The levels of SOX-9 and type II collagen genes expression in co-culture group were higher than that in induction group,which shew statistical difference.While the level of aggrecan gene expression in above two groups had no statistical difference.Conclusion1. ADSCs can be easily obtained,generated and amplificated, a mass of which can be harvested through type I collagenase digestion and which characteristics were stable in vitro. After proliferation multi-generation in vitro, ADSCs can have specific cell makers in good condition and continuously keep the abilty of differentiation. Therefore, ADSCs are the suitable seed cells for cartilage tissue engineering.2. Chondrocytes and synovialcells can secrete multiple growth factors,such as TGF- βand IGF.These factors are accepted as induction factors that can promote MSCs to induce chondrogenesis,which play a important role in the process of ADSCs chondrogenesis possibly.3.ADSCs can be induced differentiation to chondrocytes with latter phenotypes by multiple growth factors secreted by chondrocyte and synovialcells through the innovative coincubation method in our study,which effect of induction is better than that of exogenous transforming growth factor (TGF-β) induction. |
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