The Experimental Study Of Inducing Adipose-derived Adult Stem Cells Into Chondrocytes

The injury and defect of articular cartilage is one of the common problems in orthopedics. Articular cartilage shows quite limited ability of regeneration and self-repair. Traditional treatments such as abrasion, subchondral drilling and microfracture have some disadvantages. In recent years, the development of tissue engineering technology has brought new hopes to solve this problem, which may provide a new treatment method for repairing cartilage defect. Seed cells of cartilage tissue engineering are inoculated into suitable biomaterial scaffolds and then implanted in vivo to repair cartilage defect after being induced in vitro. Therefore, seed cells and biomaterial scaffolds play important roles in the construction of tissue engineered cartilage.objective: To study the differentiation of adipose-derived adult stem cells(ADASCs)which were isolated from rabbit's adipose tissue into chondrocytes induced by TGF-β1 in the presence of different concentrations of serum in vitro. After being passaged stably, ADSCs were inoculated into fibrin glue/hyaluronic acid (FG/HA) composite scaffold and cultured in chondrogenic culture medium to construct the tissue engineered cartilage so as to investigate the usefulness of FG/HA as biomaterial scaffold for cartilage tissue engineering.Methods:1. Differentiation of ADASCs into chondrocytes in the presence of different concentrations of serum: ADASCs were isolated from rabbit's subcutaneous adipose tissue using enzymatic digestion. The adipose tissue was minced and digested with collagenase type I, and the released cells were collected by centrifugation and then placed in culture. After being passaged three times, ADASCs were induced to differentiate into chondrocytes in chondrogenic culture medium containing 1% or 10% of FBS for 2 weeks. MTT, toluidine blue staining and typeⅡcollagen immunohistochemistry staining were then tested in both groups. Gray value was analyzed by Leica patho-image analysis system to observe the difference in differentiative status between the two groups.2. Tissue engineered cartilage constructed with ADASCs and FG/HA composite scaffold induced by TGF-β1: After being passaged three times, ADASCs were digested and centrifugated and then the supernatant was discarded. In the experimental group, 100μL catalytic liquid of FG was added into the cells. Then 200μL HA and 100μL major colloid of FG were added respectively and mixed adequately. Finally, the gelatum was placed into 24-hole culture plank and cultured in chondrogenic culture medium. In the control group, 200μL catalytic liquid and 200μL major colloid of FG was added and mixed with the cells. The other procedure was almost the same as the experimental group except for the absence of HA. The growth status of the cells and the degradation of the biomaterial scaffolds were observed under the inverted microscope every day. After being cultured for 14 days, the specimens of both groups were fixed with 2.5% glutaral and examined by scanning electron microscope(SEM). The other specimens were fixed with formalin and embedded with paraffin and cut into sections. HE staining, toluidine blue staining and typeⅡcollagen immunohistochemistry staining were then tested.Results: MTT showed that the cells of 10% FBS group had higher proliferation activity than 1% FBS group(P<0.05). Toluidine blue staining and typeⅡcollagen immunohistochemistry staining showed positive results in both groups, but they were more significant in 10% FBS group. The analysis of gray value showed that 10% FBS group expressed more typeⅡcollagen in the matrix(P<0.05).ADASCs were in round shape in the three dimensional scaffolds and had much higher proliferation activity. The SEM examination showed that the FG/HA composite scaffold had three dimensional structure with high potosity. HE staining of the paraffin sections showed similar histological characteristics to the normal cartilage. Toluidine blue staining and typeⅡcollagen immunohistochemistry staining also showed positive results in both groups, but they were more significant in the experimental group.Conclusions: ADASCs can be induced into chondrocytes in vitro. The chondrogenic culture medium which contained 10% FBS are more useful in promoting the proliferation and differentiation of ADASCs into chondrocytes. In the in vitro culture system, the composite of fibrin glue and hyaluronic acid (FG/HA) is a ideal scaffold material for cartilage tissue engineering because of its biocompatibility and biodegradability. A three dimensional circumstance was more useful in promoting the differentiation of stem cells into chondrocytes and in the maintenance of chondrocyte phenotype.