[Construction of tissue culture model in vitro and investigation of transforming growth factor promoting pulp fibroblast to differentiate into the odontoblast].

OBJECTIVE: To construct the tissue culture model in vitro, and investigate the potential of dental pulp fibroblast differentiating into the odontoblast and the promoting role of transforming growth factor beta1 (TGF-beta1) on it. METHODS: Human pulps were cultured for 3, 7, 14 and 21 days on bone matrix gelatin (BMG) in DMEM cultural medium supplemented with TGF-beta1. The characteristics of matrix were studied through toluidine blue and Mallory stain. Meanwhile, the expression of dentin salivary protein (DSP) on the pulp cells was investigated with immunohistochemical staining. RESULTS: This experiment found that the pulp tissue in vitro were able to develop into more progressive stage, and some pulp fibroblast cells to differentiate into odontoblast-like cells. Toluidine blue and Mallory staining analysis revealed the localized deposition of mineralized bone-dentin matrix that was detected at the site of dental pulp cells. Immunohistochemical analysis proved that the DSP synthesized in these cells with the presence of TGF-beta1. CONCLUSION: The results demonstrate that this culture condition can maintain the phenotype of human pulp tissue. The model of organ culture is suitable to study the development of pulp tissue in vitro. TGF-beta1 can promote the potential of pulp cell into odontoblast, which provides an academic basis for tooth repair and regeneration.

[Spontaneous odontogenic differentiation and critical gene expression of mouse dental papilla mesenchymal cell in vitro].

OBJECTIVE: To observe the spontaneous odontogenic differentiation of mouse dental papilla mesenchymal cells in blood serum medium. And to detect the critical gene expression of correlated transcription factors what are specific to odontogenic and osteogenic differentiation. METHODS: The primary dental papilla mesenchymal cells what had been obtained from E16.5 d murine embryo were serially subcultivated in the simple serum medium and the serum medium supplemented with LIF (leukocyte inhibitory factor) respectively. It was observed whether the dental papilla mesenchymal cells differentiated into odontoblast phenotype or kept the undifferentiation phenotype. The mRNA expression of specific transcription factors were detected in cells with or without odontogenic differentiation. RESULTS: The fourth generation and behind of mouse dental papilla mesenchymal cells what were cultured in simple serum medium could spontaneously differentiate to odontoblast, while the undifferentiation phenotype of dental papilla mesenchymal cells could be lasting to ninth generation when they cultured in medium supplemented with 10(6) U/L LIF. Whether the dental papilla cells differentiate to odontogenic phenotype or not, the members of HOX gene family such as Msx1/Msx2, Pax9 and Lhx6/Lhx7 got completely expression. These transcription factors were specific to odontogenic mesenchymal cells. Also the specific gene of mineralized tissue cells such as DSPP, Sox9, Cbfa1 and Osx initiated to express after the odontoblast differentiation. CONCLUSION: Not only this spontaneous odontogenic differentiation model of mouse dental papilla mesenchymal cells can be the positive control, but also the mode of gene expression can provide an evidence for studying how gene changes when adult stem cells are induced to odontogenic differentiation.

[Culture and characteristics of human dental papilla cells in vitro].

OBJECTIVE: To culture human dental papilla cells (HDPCs)and to study its cytobiological characters in vitro. METHODS: HDPCs were isolated and cultured with explant culture technique in vitro; Type I collagen, fibronection and laminin were detected in HDPCs and its secreted matrix with the immunocyto-chemical stain; HDPCs were incubated in mineralized promoting solution containing 10 mmol/L beta-glycerophosphate, 100 mg/L of ascorbic acid and 10 nmol/L dexamethasone supplemented with 10% FBS and the form of mineralized nodules was tested with Alizarin Red S stainning. RESULTS: Cultured HDPCs in vitro were well growing in DMEM/F12. Type I collagen, fibronection and laminin staining were all positive in both HDPCs and its secreted matrix, and laminin was stained with bunchiness in matrix. Mineralized nodules formed after cultured 27 days by Alizarin Red S stainning. CONCLUSION: HDPCs isolated and cultured are well growing in vitro, have a capability of synthesizing and secreting matrix and in mineralized promoting solution, are able to form mineralizer, so, HDPCs have a capacity of seed cell of tissue engineering regeneration tooth.

[Cultivation and induced differentiation of bone marrow stromal cells of SD rats with type I osteoporosis in vitro].

OBJECTIVE: To study the biological features and osteoblast/adipocyte phenotypes of bone marrow stromal cells (BMSCs) of Sprague-Dawley (SD) rats with Type I osteoporosis by induced culture. METHODS: Six-month-old SD rats were used in this study. 16 female rats were randomly divided into two groups. Eight rats were ovariectomied as experimental group to establish the modle of Type I osteoporosis, while other rats received sham-operation. Three month later BMSCs of 16 rats were isolated by discontinueous gradient centrifugation and then plated in alpha-MEM medium as primary culture. Secondary harvested cells were cultured for 14 days in alpha-MEM medium supplemented with dexamethasone, ascorbic acid, vitamin D3, beta-glycerophosphate or dexamethasone, 3-isobutyl-1-methylxanthine, insuline, and indomethine. The cells were screened by inverted microscope each day and cell growth was studied with cell counting. The osteoblast and adipocyte phenotypes were verified by cytochemistry staining, counted the percentage of positive stained cells. RESULTS: The weight and bone mineral density of rats were statistically different between experimental group and control group. Gomori and Von Kossa's staining demonstrated positive osteoblast phenotypes of alkaline phosphatase and mineralized nods by osteogenic inducer, while Oil Red O staining identified BMSCs treated with adipogenic medium resulted in adipocyte formation and there was no significant difference in the percentage of positive stained cells between two groups. CONCLUSION: The model of Type I osteoporosis has been established successfully. BMSCs from SD rats with osteoporosis maintain their differentiation potential.

[Heterotopic chondrogenesis of human adipose tissue-derived stromal cells loading on alginate gel].

OBJECTIVE: To isolate and chondro-inductive culture of human adipose tissue-derived stromal cells and to study their heterotopic chondrogenesis by loading them on alginate gel. METHODS: Liposuction human adipose tissues were minced and digested with collagenase type I. The obtained stromal cells were primarily cultured in BGJb medium for ten days. Secondary harvested cells were cultured in DMEM-F12 medium supplemented with 10%FBS, 6.25 mg/L insulin, 10 mg/L TGF-beta1, 50 mg/L of freshly prepared L-ascorbate for 14 days. After in vitro assay of chondrogenic phenotypes, the cells at density of 10(10)/L were mixed with 1.2% alginate sodium and 102 mmol/L CaCl(2). The cross-linking cell-alginate gel were injected into four BALB/C athymic mice subcutaneously (1 ml for each mouse). Meanwhile, the auto-controls were set by injecting equal dose of simple alginate gel and pure cells in two opposite buttocks of the same mouse subcutaneously. Two mice were sacrificed at fourth and eighth week postoperatively and all samples were removed, fixed, embedded in paraffin and cut into sections of 5 micro m thick. HE staining, Alcian blue and modified Masson's trichrome staining were employed to observe chondrogenesis histologically. RESULTS: Alcian blue and immunocytochemical staining revealed chondroitin sulfate and collagen II in cell matrix after having been chondro-inductive cultured for 14 days. At intervals of fourth and eighth week, heterotopic chondrogenesis is (cartilage formed) within cell-alginate injected sites were found in all mice but negatively in auto-controls. Histologically the hypertrophic chondrocytes were among cartilage matrix in different staining. All alginate gel and solitory cells absorbed within two to three weeks postoperatively in auto-controls. CONCLUSION: It seems that stromal cells derived from human adipose tissue presents a potential for chondrogenic differentiation.