| ObjectiveAdult human stem cells that are intrinsic to various tissues have been described and characterized recently. These cells are capable of maintaining, generating , and replacing terminally differentiated cells within their own specific tissue as a consequence of physiologic cell turnover or tissue damage due to injury. Recent data suggest that adult stem cells generate differentiated cells beyond their own tissue boundaries, a process termed " developmental plasticity. " In this article we focus on in vitro models of adult stem cells derived from bone marrow and their potential therapeutic applications. Stem cells are defined as cells that have clonogenic and self - renewing capabilities and that differentiate into multiple cell lineages.Stem cells can be expanded in large numbers and retain their ability to differentiate into mature functional cells. According to different developmental stage, the stem cells can be divided into two categories: adult and embryonic stem cells, the research in embryonic stem cells is restricted by its limited source and ethical controversy, so research on adult stem cells attracts more attentions. It is demonstrated that bone marrow stromal stem cells ( BMSCs) have multiple potential to differentiate into muscle, bone, cartilage, adipose, blood vessel endo-thelial,liver and neuron cells. BMSCs can be used as cell source in tissue engi-neering, cell implant and gene therapy with the possibility to be distributed to different tissue and organs by normal circulation. They hold great promise in future therapeutic application in that they are easy to obtain,separate,expand and transfect ,the procedure of bone marrow extraction is less harmful and the replantation induces no immune rejection.The differentiation of BMSCs is closely related to the microenvironment they grow in. it is probably because certain factors in the microenvironment are necessary for BMSCs to differentiate into target cell lineages.Our purpose in this study is to investigate the differentiation of cultured bone marrow stromal stem cells of rabbit into osteoblast to provide seed cells for bone tissue engineeringMethodsUnder aseptic conditions, approximately 3 ml bone marrow was obtained by routine great trochanter aspiration from an adult male or female New Zealand white rabbit under sodium pentobarbital anesthesia (30 mg/Kg). Rabbit MSCs were isolated from the marrow aspirates using methods described previously. Marrow for 3 ml was added to 8 ml of 20% Dulbecco"s minimum essential medium (DMEM) (containing 20% calf serum from selected lots) , and centrifuged at 1 000 g for 5 minutes to pellet the cells and remove the fat layer. Cell pellets were then resuspended. The MSCs-enriched was collected, rinsed with 20% DMEM, plated at 50 ml culture flask of six, and cultured at 37℃ in a humidified atmosphere containing 5% CO2. Nonadherent cells were removed on day 1 at the time of the first medium change, and fresh medium was changed every 3 days thereafter. When adherent MSCs became nearly confluent, cells were detached with 0.25 % trypsin for 4 minutes at room temperature. After passaged, MSCs were seeded onto the sterile glass slides preset in the culture flasks, cultured under control media (20% DMEM) and osteogenic inducing conditions(OGC) media (20% DMEM containing 0. 1μmol/L dexamethasone, 50μg/ml ascorbic acid, 10mmol/L β - glycerol phosphate) and observed under phase contrast microscope every day. Furthermore,BMSCs were isolated from rabbit of two month and cultured to observe live speciability ,and the osteogenic potential of bone marrow stromal stem cells was investigated by means of alkaline phos-phatase(ALP) , alizarin red S(ARS) stainings, immunohistochemical staining , transmission electron microscope ( TEM) and scanning electron microscope (SEM).ResultsMass clones of BMSCs and lots of globular cells were observed in primary culture at the seventh day and arranged in spite shape in passage culture. BMSCs were seen to be differentiated into osteoblast after the conditional fluid was added,ALP positive dark granules and collagen I positive product were found in the cytoplasm of the cultured cells, and ARS positive calcium nodule was seen formed by several cultured cells, and a lot of matrix vesicles and collagen product was observed by TEM. The results by SEM were as follows: Numerous mi-crovilli could be detected over the cytoplasmic memberane in primary culture. In the space between the spindle - shaped cells and the polygonal cells or globular cells, as microvilli over the cytoplasmic membrane and round granules on or a-round the cells could be discerned. The cytoplasmic processes of the globular cells were shorter than that of the polygonal cells. Thus the globular cells were easily discerned with other type of cells. With lapse of time, the cytoplasmic processes became shorter or disappeared from the globular cell body. And the cells excreted numerous round granules, with diameters of 0. 3 -2. 0 |xm. The granules were distributed over the cell surface and the surrounding areas, showing tendency to agglomerate and then heaped up to form nodule - like structure with the globular cell as the center. The bony nodes showed various configura-...
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