The Study On United Culture Of Acellular Meniscus Framework And Bone Marrow Stromal Stem Cells

The treatment of meniscus cartilage defect is a difficult problem in medicine. Actual major treatment means are meniscectomy and incomplete resection, which often cause degeneration of knee joint. Along with the development of tissue engineering, scientist attempted to use tissue engineering meniscus to repair damage. 3D structure and organization of seed cells is an important factor in the project. On the foundations of prophase study, using meniscus cartilage through acellular treatment, we make the framework which united culture and study with bone marrow stem cells. The purpose of this study is to provide evidences for making production of tissue engineering knee joint.Materials and methods1. The culture of bone marrow stem cellsA Japanese oryctolagus cuniculus(male or female) was hocused and bored in the upside metaphysis of femur. The marrow was got and inoculated to culture flask sterilely and culture with the low glucose DMEM culture medium with fetal bovine serum and choose the third generation cells as seed cell.2. Production of acellular exo - framework of swine meniscus cartilage The meniscus and knee joint cartilage of swine (three months old) weresheared into 1x1 cm lamellar structure. We chose Tris - HCI (PH7. 4 ) as buffer solution, and use TritonX -100 doff cells. The acellular cartilage was put into Tris - HCL (PH7. 4 ) and stored at 4℃.3. The united culture of rabbit bone marrow stem cells and acellular frame-work of swine meniscus cartilage frameworkCollecting the third generation matrix stem cells sterilely, bone marrow stem cells were inoculated into meniscus cartilage by reduplicative titration, and united culture at the condition of 31°C , 5% C02 and saturated humidity. Thenceforth, changing medium every other day, and observing after 7days.4. Histological examinationThe third generation cells were washed with PBS three times, then cultured with medium without serum three days. Using medium without serum as control, the cell culture medium was analyzed by gelatin zymography analysis to check the expression level of matrix metalloproteinase - 2 ( MMP2 ). Normal and acel-lular meniscus were fixed with 4% polyoxymethylene, generally paraffine embedded, sliced up, then HE and improved MASSON stained. The acellular meniscus and united cultured meniscus were fixed with 2.5% Glutaraldehyde, and check by Scanning Electron Microscope.Results1. The medium cultured without serum of stem cell showed a piece of negative staining strip (MMP2 ). The medium without serum as control showed nothing.2. The HE staining showed acellular meniscus had not cellular component, space in matrix became larger and connected each other to be reticular structure. Improving MASSON staining showed the matrix of acellular meniscus cartilage was green, which was proved a great deal of collagen fibers here under Scanning electron microscope, we could discover that space between the acellular meniscus cartilage framework became larger, walls of the space were not slippery, their diameters were 20 -200fxm, the radio of space was more than 80% , and the space connected each other to form 3D reticular structure. And this also showed that the bone marrow matrix stem cells combine with meniscus framework well, the cells and the framework connected tightly and protruded pseudopodia, and excreted some kind of protein. The number and growth of cells were better.Conclusions1. The bone marrow matrix stem cells was really express MMP2 which proved that they had invasive ability, and could connect with framework very well.2. Acellular meniscus cartilage was a kind of ideal material for framework of cartilage tissue engineering.3. Simple acellular arthral cartilage could not be suited for the demanding of material for tissue engineered framework.New points1. It was the first time to prove MMP2 could express in the bone marrow matrix stem cell interiorly.2. Acellular meniscus cartilage framework showed unique innovation, and proved 3D reticular structure. The united culture with bone marrow matrix stem cell also proved that it was a kind of ideal framework material, which inaugurated a new way for studying of meniscus framework.3. The contrastive study illuminated that acellular meniscus cartilage framework was ideal than that of the acellular arthral cartilage framework for repairing the damage mansions.