The Experimental Study Of The Repairing Of Cartilage Defects Using Chondrocyte And Osteoblast With Scaffold

ObjectiveArticular cartilage injury is a common disease in the field of orthopedics, whose incidence caused by aging, articular wear, degeneration, trauma and sports injury is increasing. It has been a very important factor leading to disability and reduced quality of life. The previous methods to repair the articular cartilage defects do not always attain satisfying results. Reconstruction of cartilage defects still remains one of the most difficult problems for joint surgeons. Significant achievements in co-culturing chondrocytes and scaffolds to repair the cartilage defects by using techniques of cartilage tissue engineering have been obtained. In the meantime, the work to seek after suitable seed cells and scaffold materials for cartilage tissue engineering is ongoing strenuously. The goal of our study is to evaluate the effect of treating the cartilage defects with cell-β-TCP composites implanted into osteochondral defects on canine models by using techniques of mosaicplasty, chondrocyte-β-TCP scaffold composites in the top of the defect and osteoblast -β-TCP scaffold composites in the bottom of the defect, osteochondral composites were constructed in vivo, looking forward to acquire desirable seed cells and scaffold materials.Materials and Methods1 Three healthy Beagles, approximately 10-12 months of age were used in this study. A 7-8ml of bone marrow was isolated from the iliacs of each canie. The MSCs suspension was aspired after bone marrow was centrifuged by percoll separating medium, cultured primarily, and then generated. The third generation MSCs were induced.Three groups were divided : Group I receiving 10ng/ml TGF-β₁/L, insulin trypsin 10μ/ml, dexamethasone 100nmol/L ; Group II receiving dexamethason 10nmol/L,β—Glycerin phosphoric acid sodium 10mmol/L , VitaminC50mg/L; GroupIII as the control group receiving no cell growth factors, and three groups were cultured conventionally in DMEM medium containing 10% fetal bovine serum. The induced MSCs were viewed at different stages with phase contrast microscope. After 15-day culture, chondrocyte cell viability was estimated by the MTT assay, and cell function was assessed by measureing sulfated glycosaminoglycan (GAG) secreted by chondrocytes. Immunohistochemistry was applied to detect the secretion of collagen type II. steoblast cell viability was estimated by aikaline phosphatase dye.2 Adjust the cell density of 14- day induced cultured MSCs to 5×10⁶ml, then co-culture chondrocyte Osteoblast andβ-TCP scaffolds for 7 days. The biocomposites were ready for animal model experiment. Theβ-TCP scaffold and chondrocyte -Osteoblast -β-TCP scaffold constructs were harvested for scanning electron microscopic examination(SEM).3 Ten Beagles, approximately 10-12months of age, were used in this animal experiment. A full- thickness cylindrical osteochondral defect of 5mm diameter and 5mm depth was created in both femoral humeris in every canie. These canies were randomized and divided into 3 groups: Group A(n=10), as experimental group, implanted with osteoblast -β-TCP scaffold composites in the base and induced chondrocyte -β-TCP scaffold composites in the top into the defects of the left knees; Group B(n=10),as negative group, implanted with chondrocyte - MSC-β-TCP scaffold composites into the defects of the left knees; Group C, as control group, implanted without any construct into the defects of the right knees. The canies (5 of each group) were sacrificed at 12,16 weeks. Each detect area was evaluated both with naked eyes and histology.Results1 Changes of cell morphology The growth of the MSCs was observed with the phase contrast microscope. During the beginning of 1-3 days, sparse primary cells fastened wall, the shape of the primary MSCs was short spear-like. After changing medium of 2-3 times, most of the floating cells were cleared. Three days later, the clone cluster of the cells showed up. After 7-8 days, the cells reached confluence. After 12-14 days, monolayer cells were established. The subcultured cells grew faster. After 7-8 days, monolayer cells, with high nuclears and many grains in cytoplasm, were observed. After adding cell growth factors, cells proliferated significantly.2 MTT OD value, GAG content in culture mediums and positive secretion of collagen type II with Immunohistochemistry in 10ng/ml TGF-β₁ experimental group was obviously higher than the control group. With Immunohistochemistry of collagen type II, cytoplasm of positive MSCs was stained yellow or brown-yellow. Electron microscope observation.β-TCP scaffold had multi-pore shape, with a good pore of 200-300um. The induced MSCs had fine adhesion progression and proliferation in theβ-TCP scaffold.2 Animal experiment resultsObservation with naked eyes At 16 weeks after transplantation the defects of Group A were covered with semi-transparent smooth white tissue and the margins between the repair tissue and the surrounding cartilage were not recognized. The defects of Group B were covered with repair tissues, partial of them were conformed with original cartilage. Only in limited area some little pores were noticeable. Compared to Experimental Group, the luster of repair tissues of Negative Group was not good. However, the defects of Control Group were repaired with soft fibrous tissues without luster, and an obvious boundary between reparative and original cartilage was seen.3 Histological observationsAt 16 weeks after operation: the repair tissues of Experimental Group maintained their thickness to the full depth of the original defects. Chondrocytes arranged regularly. Arrangement of chondrocytes on the surface tended to be parallel to the joint line, while that of the deeper layer liable to be vertical. The defects were repaired with hyaline-like cartilage. The subchondral bone and tidemark were well remodeled. There was no clear gap between reparative and surrounding cartilage. In the Negative Group chondrocytes arranged irregularly, partial fibrous repair tissue was viewed. However, the defects of Control Group were only repaired with fibrous tissue.Conclusion1 MSCs can be easily obtained, percoll separation method can get huge MSCs, their characteristics were stable in vitro, easy to generate. Under some conditions, MSCs can be induced to cartilage cells in vitro. Therefore MSCs are the suitable seed cells for constructing tissue engineered cartilage.2 It was demonstrated that combination of growth factors played an important role in chondrogenic differentiation. Proliferation and expression of chondrogenic phenotype of MSCs induced by TGF-β₁ were obviously higher than the control group.3β-TCP scaffold, with a good pore rate and biocompatibility, can be used as satisfying scaffold materials for cartilage tissue engineering. MSCs in this scaffold have a fine adhesion, progression and proliferation.4 Chondrocyte -osteoblast -β-TCP scaffold composite was the ideal construct for repairing cartilage defects. Animal experiments demonstrated that the effect of osteoblast in the bottom and chondrocyte on the top composited withβ-TCP scaffolds repairing cartilage defects was obviously better than with chondrocyte -MSCs-β-TCP compostites. The repair tissue was well-differentiated into nearly normal morphology of cartilage.