| Battle wound and various kinds of diseases such as osteoarthrosis caused cartilage defects influence patient's outward appearance and their function. It is significant to repair it stable and convenient for improve patient's quality of life. Therefore, autologous, allogeneic cartilade and some artificial materials has been used in clinical. However, the disadvantages such as supply limited, hard to match and immunological rejection had restricted the development. Tissue engineering use the principles and methods of engineering and life sciences to repair and reconstruction of tissue defects. Seed cells and the material of scaffolds are key factors in the tissue engineering. The self-histiocyte has some question about quantity of localization, function aging and so on. The embryonic progenitor cells and marrow mesenchymal stem cells also have some ethical problems. On the other hand, the commonly used scaffolds in the tissue engineering have some disadvantages such as serious immune response, high price and so on.There is a kind of cells in the adipose tissue called adipose tissue-derived stromal cells (ADSCs )have the potential ability to differentiate into bone, cartilage, muscle and so on, ADSCs could be available in large quantities with minimal morbidity and discomfort associate with their harvest. They can be used as a new kind of seed cells in the tissue engineering. The injectable and thermosensitive Hydroxypropyl methyl cellulose (HPMC) has the advantages of convenience, plasticity and mini injury; it might be used as an ideal material of scaffolds in tissue engineering. This study focus on using ADSCs combined with HPMC to fabricate cartilage with tissue engineering technology.Materials and methods1. Cell culture and Differentiation Human adipose tissues were obtained from elective liposuction procedures under local anesthesia, then digested and centrifuge the lipoaspirates to obtain the useful cells. The cellar pellet was resuspended in proper mediums. We observed the appearance of cells and drew the growth curve.2. Flow CytometryWe detected the unique CD maker antigens-CD14, CD31, CD34, CD45, CD49d, CD56, CD105 andCD106 of the 6th generation ADSCs. 3. Cell multiplication and induction.ADSCs cultured separated in different mediums were trypsinized and seeded in two 96-well plates at a density of 2×103/200μl. When the three groups of cells described above were cultured for 1day to 8days, cell proliferation was detected by MTT colorimetric method. 4. Fabrication of engineering cartilage using induced ADSCs in nude mice We injected the mixture of induced ADSCs and HPMC into the 3 mice's hypodermis. In each mouse, 3 points were experimental group, the other 1 point was control. Mice were killed at the 3w, 5w and 7w after cell injection. Specimens were fixed in buffered formalin.Results1. ADSCs cultured in the two mediums grows and proliferate in different waysADSCs arrange like a swirl, exhibited an average population doubling time of 55h using common medium. The cells could go down to the 9th passage, after that, the cells grew slowly and aging obviously. In contrast, the induction ADSCs grew faster than the former, cells were bigger, and some pseudopodium and nucleoli were observed. These cells went into index stage at the second day, exhibited population doubling time of 30h. The cells turned aging at about the15th passage.2. Phenotypic Characterization of ADSCs population: CD Marker profileCD maker profile was examined for characterize the ADSCs population, no expression of the hematopoietic lineage markers CD31, CD34, CD45 and CD106 was observed in the cells. CD14, CD56 and CD105 were expressed in low level. ADSCs expressed CD49d, whereas this antigen was not expressed in hematopoietic lineage, it can be seen as a significant marker for ADSCs.3. Immunehistochemenical and special staining. After cultured in chondrogenic medium, the cells secreted the specific cartilaginous matrices sulfated proteglycan and collagenⅡ. And we had observed the special staining of chondrocyte induced ADSCs under polarized microscope, which was characteristic cartilage matrix.4. Observation of the nude mice induced ADSCs and HPMCThe skins of all nude mice were normal, the hypodermis became bloodshot. At the 3w, the 5w and 7w, we all observed the cartilage through the microscope.Conclusion1. Adipose derived stromal cells (ADSCs) could be obtained from the adipose tissue in elective liposuction of hypersound. The cells were identificated to be the ADSCs by flow cytometry.2. The ADSCs cultured in induced medium turned to have some characters of cartilage cells, and fabricated the cartilage in nude mice, suggested that the ADSCs were fit for the seed cells in tissue engineering.3. The HPMC and ADSCs were compatible, it wasn't infected or discharged. It can be considered to be a suitable scaffold in tissue engineering.
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