The Experimental Research On The Repair Of Articular Cartilage Defects With The Biological Support Material Compounded With Mesenchymal Stem Cells

Objectives1 To investigate the more effective methods of isolation, culture of bone marrow mesenchymal stem cells (BMSC) in vitro.2 To explore the more conformable cartilage biomaterial that conforms to the natural biomaterial in total porosity, total specific surface area, average pore radius, elastic modulus and biocompatibility, such as test for in vitro cytotoxicity, test of hemolysis and biodegradation.3 To investigate the feasibility of repairing the whole layer cartilage defects of medial femoral condyles of adult rabbits with the BMSC/ACM materials.Methods1 Bone marrow stem cells of New Zealand Rabbit were obtained and purified by gradient centrifuge an adhesion culture in vitro. The morphology of BMSC were observed with phase contrast microscope; Drawing the growth curve of BMSC:The cells(P1,P3,P5)were assimilated by trypsin and cultivated in 24一well plate.Three bores were assimilated every day for 8 days, and then growth curve with average was drawn; Identifying the cel1-surface marker:The percentage of the wel1 growth P2 cells were identified by CD44 staining by flow cytometry.2①The articular cartilage of pigs were processed into powder. The components of cells were eliminated by Trypsin, TritonX-100 and distilled water. Finally the powder was freezed out by freezedryer and irradiated by ultraviolet. The microcosmic images of ACM were showed in electron microscope and the parameters of pore radius distribution were measured by sorptomtic Instrument;②Test for in vitro cytotoxicity: The BMSC were cultured in the special L-DMEM that ACM were dipped in and then observed through microscope and MTT;③Test for acute systemic toxicity: The changes of the behavior and the avoirdupois of the SD rats were evaluated after the special L-DMEM was injected into the SD rats by intraperitoneal injection;④Test of hemolysis: The grade of hemolysis was made in the macroscopical phenomenon and the measures of optical density at 492nm;⑤The growth state of the BMSC that immerged into ACM was described. Then the biodegradation of the ACM was estimated after the ACM was hypodermically embedded.3①Bone marrow stem cells of New Zealand Rabbit were obtained and purified by gradient centrifuge and adhesion culture in vitro. The passage 3 BMSC were used for the seeding cells of cartilage tissue engineering;②The acellular pig articular cartilage was prepared by lyophilization, trypsinization and chemical subtraction.③Full thickness empty defects measuring 4 mm in diameter by 3 mm deep were prepared in the medial femoral condyles of 24 3-month-old New Zealand White rabbits, which were randomized to select two groups. Each group was randomized to receive transplantations with ACM-BMSC (8 knees), ACM (8 knees), or no grafts (8 knees) into the cartilage defects.④The repairing effects of the condyles were macroscopically observed and the morphological changes of repaired defects were evaluated 6 and 12 weeks after the operation. The histological scores and the type-Ⅱcollagen immunohistochemistrical stains were carried.Results1①Observing the morphology of BMSC with inverted microscope:Cells showed fiber figure and swirl growth.Primary cultured BMSC were oval,spindle-shaped or polygonal,and adhered to plastic surface within 48h and reached 90% confluence within 14 days.After purification and proliferation,they were uniformly long spindle-shaped form;②Cells growth cure of BMSC;The BMSC were still in latent phase after being adherent to the bottom 2 days;3-4 days later, cells were in log phase;6 days later,and cells came into platform phase;③The result of the cell-surface markers:Cell membranes were colored up evidently after CD44 staining and CD44 of the passage 2 BMSC positive rate were 93%.2①The surface of the pale yellow ACM was porous and netty and the bouncy ACM was fragile. The results showed that the Average pore radius was 47.13μm and the Total porosity came to 68.54%;②Test for in vitro cytotoxicity: At every time interval, the OD scores of three groups were not significant differences (P>0.05).The grade of the cytotoxicity of the ACM was 0;③Test for acute systemic toxicity: The avoirdupois scores of both groupⅠandⅡwere not statistically significant differences (P>0.05), but the differences between groupⅠandⅢwere statistically significant(P<0.05);④Test of hemolysis: Hemolysis Percentage was 2.92%;⑤Observing the morphology of BMSC in the ACM with inverted microscope:BMSC were oval and nucleoli appeared in a few cells. And the ACM were decomposed into particulates; the histological observation after the ACM was hypodermically embedded: There were few lymphocytes and polymorphonuclear leucocyte around the ACM. And the ACM were closed in by compact fibrosis wall and were decomposed into small parts.3①In the 6th and 12th week after the operation, the morphology, distribution and arrangement of the regenerated tissues were similar to normal cartilage in the knees with ACM-BMSC transplantation, and the regenerated tissues grew to be integrated with the surrounding normal cartilage with obscure boundary between them. In the ACM group, the rough surface of regenerated tissue sunk obviously and the fibroblasts in all layer and the few chondrocytes in the deep layer were found. While the thin reddish grey layer of soft granulation tissue formed in the defect and the fibroblasts increased in non–transplantation group;②At every time interval, the histological scores of groupⅢbased on Wakitani scoring excelled both groupⅠandⅡwith statistically significant differences(P<0.05),but the differences betweenⅠandⅡwere not significant(P>0.05);③Immunohistochemistrical stains showed that cells in the zones of repaired tissues were abundant, arranged columnedly and the typeⅡcollagen staining was positive.Conclusions1 BMSC were easily isolated and cultured in vitro and proliferate prosperously, and more purified. The protocol should make it possible to undertake.2 ACM accords with the biomaterial standards of cartilage tissue engineering in Total porosity, Total specific surface area, test for in vitro cytotoxicity, test of hemolysis, tests for acute systemic toxicity and biodegradation.3 Tissue-engineering cartilage based on BMSC seeded into ACM can repair the defects of the whole layer cartilage defects of medial femoral condyles of rabbits. The repair tissue was confirmed to be hyaline cartilage.