In Vitro Comparison Of The Osteogenic Capacity Between Stem Cells From Human Exfoliated Deciduous Teeth(SHED) And Human Bone Marrow Mesenchmal Stem Cells(BMMSCs)

Background and objectiveStem cells are characteristic of self-renewal and multiplex differentiation potential. They play an important role in tissue organ development and organ damage repair process, and for regenerative medicine and tissue engineering serving as seed cells.Studies confirm that stem cells from human exfoliated deciduous teeth(SHED) have the multiplex differentiation capacity. In vitro, SHED are capable of differentiating into various cells tissues including osteoblasts, cartilage cells, fat cells, nerve cells SHED as a kind of new stem cell in tissue engineering have very bright prospec. Existing isolating methods of stem cells need a long cycle and is complicated, we want to apply immune magnetic beads technology in vitro for the separation cultivation and amplification of SHED, exploring the growth curve, cell surface antigen markers multiplex differentiation ability of SHED, and finally to establish a kind of convenient and effective separation method for SHED which can be better applied to tissue engineering.In 2003, SHED was first reported by Miura just since then SHED have become the focus as a new stem cells, it has the same sources in embryonic development with maxillofacial bone, application to repair the maxillofacial bone defect just may be appropriate. what is more in the process of get SHED nearly no pain, more accepted by people, and the success of umbilical cord blood Banks build applications, provides us with a very good example, maybe we can also build SHED Banks for get through bad times(In Japan, Hong Kong, Singapore and other countries and regions have established the Bank of SHED). In 1976 Friedenstein confirmed bone marrow contains a kind of cells that can differentiate into cartilage cells and fat cells. Bone marrow mesenchymal stem cells(BMMSCs),are derived from mesoderm have the potential of self-renewal and multiplex differentiation, in different conditions can be induced to bone, fat, neural, cartilage, myocardial, skeletal etc. This study Comparison osteogenic capacity of SHED and BMMSCs in vitro in order to choose appropriate seed cells repair bone defect provide experimental data.In vitro comparison of the osteogenic capacity between stem cells from human exfoliated deciduous teeth (SHED) and human bone marrow mesenchmal stem cells (BMMSCs)Materials and methods1 Isolate and culture BMMSCs and SHEDAccording to Miura et al experimental method, pulp tissues from children of 6-10 years old were obtained, digested by enzyme and cultured in the medium. The filtered cells suspension were collected and cultured in the container.According to kits requirement of the magnetic-beads. Use STRO-1 for surface markers isolate SHED from the third generation deciduous teeth pulp cells.BMMSCs were obtained from the posterior ilia crests of 3 healthy adult volunteers following informed consent. After being cultured and expanded, the third generation cells were identified by phenotypic analysis and induced by adipogenic, osteogenic and media. HBMMSCs in the third generation were used in the investigations.2 Cell morphology observation and cell growth curve measurementThe growth condition, proliferation and morphologic characteristics of both the primary and passage cells were observed.Use single-celled liquid of SHED and BMMSCs and deciduous teeth pulp cells, Measure each hole by MTT method for measuring values, measuring 10 days,time for the horizontal axis the value for the vertical axis, drawing the growth curve.3 Flow cytometry testing Surface markersCollect the two kinds stem cells with good condition, test expression level of Surface markers CD29,CD105,CD34,CD45.4 Multiplex differentiation in vitroThe two kinds stem cells were cultured in the adipogenic medium and mineralization medium for 3 weeks, then respectively test multiplex differentiation ability. After 4 weeks'culture in the chondrogenic medium, immunocytochemical staining test the expression of collagen type II, cell morphology observation by HE staining.5 Fluorescence quantitative PCR technology testing the osteogenic genes expression levelAt different times Real time-PCR was used to examining mRNA expression of osteogenic genes ALP. OPN and OC in SHED and BMMSCs.6 Statistical analysisAll statistical analyses were performed with the SPSS 13.0(SPSS Inc, Chicago, IL, USA). P values<0.05 were considered statistically significant. Statistical analysis methods: Repeated Measures was used compares for the whole variance analysis, Independent-Samples T Test was used for the compares of same time points but different groups, if the data does not meet the homogeneity of variance, the approximate method of analysis of Satterthwaite method for statistical analysis.Results1 Isolation and culture SHED and BMMSCsAfter 2 days of the primary generation BMMSCs culture change media still have large amount of red cells suspended in the bottom; until the seventh day cells appeared a clonally growing trend and showed a fibroblast-like morphology.2 Cell growth curve measurementAfter 10 days culture, MTT method detected the growth curve of three kinds of cells appear the "S" shape.3 Flow cytometry testing Surface markersFlow cytometry inspection showed that both the SHED and BMMSCs could highly express mesenchymal stem cells'surface marker CD29 and CD105 but fewer express hemopoietic stem cells'surface markers CD34 and CD45.4 Multiplex differentiation in vitroStem cells were cultured in the mineralization medium of 3 weeks, Alizarin red stain visible positive calcium nodules, ALP staining was positive. Oil red O staining was positive after stem cells was cultured in the adipogenic medium of 3 weeks.Immunocytochemical staining showed positive expression of collagen type II after SHED were induced for chondrogenesis.5 Expression of osteogenic genesDifferent genes at different times the two kinds of cells showed no significant difference (P>0.05).ConclusionSHED could be inducted into osteoblasts, chondrocytes and adipocytes by induction of Osteo-,chondro-and adipogenesis; At same times two kinds of cells have the same osteogenic genes expression level, and different genes in different time expression level have corresponding change.