A study on repairing mandibular defect by means of tissue-engineering and human bone morphogenetic protein-2 gene transfection in osteoporotic rats / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of repairing bone defect with methods of tissue-engineering and human bone morphogenetic protein-2 (hBMP-2) gene transfection in osteoporotic rats.</p><p><b>METHODS</b>Twenty-four 6-month-old female Sprague-Dawley rats underwent ovariectomy, while 8 rats received sham-operations. Three months later, bone mesenchymal stem cells (BMSC) harvested from osteoporotic rats were divided into two groups randomly. Experimental group were transfected by recombinant plasmid carrying hBMP-2 gene, and control group left untreated. All BMSC were seeded into coralhydroxyapatite scaffolds. Then the cell/scaffold constructs were implanted into the defect site created in the ramus of mandible of osteoporotic rats respectively.</p><p><b>RESULTS</b>Positive results were confirmed by immunohistochemistry and in situ hybridization in experimental group. New bone formation was found at the margin of the defect treated with the BMSC modified by hBMP-2 gene transfer at 4 weeks after implantation and appeared mature 8 weeks after the treatment. However, the amount of newly formed bone was much less and there was some adipose tissue at defect margins 8 weeks after implantation in control group.</p><p><b>CONCLUSIONS</b>The results of this experiment indicate that BMSC-mediated rhBMP-2 gene therapy in conjunction with bone tissue engineering may allow for successful treatment of large bone defects in osteoporosis rats.</p>

Cultivation and induced differentiation of bone marrow stromal cells of SD rats with type I osteoporosis in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The model of Type I osteoporosis has been established successfully. BMSCs from SD rats with osteoporosis maintain their differentiation potential.</p>

Study on multi-lineage potential of bone marrow mesenchymal stem cells derived from green fluorescent protein transgenic mice / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the multi-lineage potential of bone marrow mesenchymal stem cells (MSCs) derived from transgenic mice with green fluorescent protein (GFP) gene in vitro.</p><p><b>METHODS</b>A 6-week-old GFP transgenic mouse was executed by dislocation of cervical vertebra, and the marrow in tibia and thighbone was washed out with asepsis. The limited cell strains of MSCs derived from GFP transgenic mice (GFP-MSCs) were obtained with density gradient centrifugation. The passage 3 GFP-MSCs were induced to differentiate into osteoblast, adippcyte, neuron with solution of calcium induction medium, adipogenic medium and neural induction medium respectively. After being calcium-induced, the activity of alkaline phosphatase on GFP-MSCs was determined by micro-plate reader, and alizarin red staining was performed to test the formation of calcium concentration. The adipo-induced MSCs were detected with oil red O staining. Immunocytochemical staining was performed to detect the expression of NSE on neuron-induced MSCs.</p><p><b>RESULTS</b>The ALP activity of GFP-MSCs heightened gradually along with being calcium-induced, and alizarin red staining showed positive. Oil red O staining of adipo-induced cells and NSE immunocytochemical staining of neuron-induced cells demonstrated positive.</p><p><b>CONCLUSION</b>The limited cell strain of GFP-MSCs possesses multi-lineage potential, which can be used as an efficient tracking facility for studying the mechanism of multi-lineage potential on the MSCs.</p>

Culture and characteristics of human dental papilla cells in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To culture human dental papilla cells (HDPCs)and to study its cytobiological characters in vitro.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

A study on the chondrogenesis of the compound of alginate gelatin and bone marrow stromal cells in vivo / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the chondrogenisis by alginate gelatin and rats' bone marrow stromal cells (BMSCs) chondrogenicly induced in vitro.</p><p><b>METHODS</b>Thirty-two male adult SD rats were assigned randomly to experimental and control groups. In experimental group, bone marrow was obtained from the right tibias of all the rats. After expanding and culturing 3 passages, induced BMSCs by chondrogenic culture medium for 10 days. Suspended induced cells in alginate gelatin, and injected the complex into the hypodermic tissue of the backs of rats autogenously. In control group only alginate gelatins were injected. The grafts were taken out for examinations 4 and 8 weeks after the operations.</p><p><b>RESULTS</b>Considerable cartilage appeared in experimental group 8 weeks after operations. Regular HE staining and alcian blue staining showed a great deal of cartilage holding chondrocyte masses surrounded by abundant matrix. Alginate gelatin decompounded obviously, and the rest distributed among newly formed cartilage. No cartilage appeared in control group all through.</p><p><b>CONCLUSION</b>BMSCs and alginate gelatin have a beautiful future in cartilage tissue engineering.</p>

A study on transfecting green fluorescent protein gene to rat bone marrow mesenchymal stem cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study an efficient method to transfect green fluorescent protein gene (GFP) to rat bone marrow mesenchymal stem cells (MSCs) and to determine the biological properties and differentiation potency of transfected MSCs.</p><p><b>METHODS</b>SD rats' bone marrow MSCs were separated and purified in vitro. After subculture and expansion, MSCs infected with Adenoviral vector (Ad-GFP) or transfected with liposome were observed, and their transfection efficiency was assessed with flow cytometry. The MSCs expressing GFP gene were induced to differentiate to osteoblast, and non-transfected MSCs were set as control.</p><p><b>RESULTS</b>Ad-GFP delivered GFP gene with high efficiency to rat MSCs. (41.3 +/- 1.4)% of MSCs infected with Ad-GFP expressed GFP gene, which was much higher than the control (12.5%). Expression of GFP gene of infected MSCs maintained stable from 1 to 6 weeks after infection. Infected MSCs possessed the same alkaline phosphatase activation as non-infected MSCs, and formed mineralized mouldes.</p><p><b>CONCLUSIONS</b>The infected MSCs with Ad-GFP expressed GFP with much higher efficiency than liposome transfection, and maintained the same ability of proliferation and differentiation as non-infected MSCs. Transfection with Ad-GFP is a highly effective method for labeling MSCs.</p>

Establishment of a culture system of chick embryo for mouse tooth germ development / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To establish a new culture system for mouse tooth germs in chick embryo.</p><p><b>METHODS</b>The mandibular first molar germ fragments of 15 embryonic days' Kunming mouse embryo were implanted into the lateral mesenchyme of 4-5 days' chick embryo wing buds in ove. Eggs were reincubated and implanted tissues were examined by histochemistry.</p><p><b>RESULTS</b>The cultured tooth germ development continued from cap stage to latest bell stage. The ameloblast and the odontoblast all differentiated maturely and secreted matrix.</p><p><b>CONCLUSION</b>4-5 days' wing buds chick embryo could serve as developing the mouse tooth germs and demonstrate well physiological process of differentiation and morphogenesis.</p>

A study on myogenic differentiation of human adipose tissue-derived stromal cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To isolate human adipose tissue-derived stromal cells and study the potential of osteogenic differentiation after inductive culture.</p><p><b>METHODS</b>Liposuction human adipose tissues were minced and digested with collagenase type I. The obtained stromal cells were plated in BGJb medium as primary culture for ten days. The second passage cells were harvested and plated in DMEM/F12 medium supplemented with 10% FBS, 5% horse serum and 50 micromol/L hydrocortisone for myogenic induction culture. The cell-anchored slips were removed and fixed in 4% formaldehydam polymerisatum. Toluidine blue, Mallory's phosphotungstic hematoxylin staining and monoclonal antibody to human skeletal muscle myosin heavy chain immunocytochemical methods were used to assay the differentiation of cells.</p><p><b>RESULTS</b>It was observed that the size and shape of induced cells were much different from those of non-induced cells. Toluidine blue, Mallory's phosphotungstic hematoxylin staining demonstrated there were many basophilic striations within cytoplasm and multinucleated myotubes were formed. Immunocytochemical stain indicated that characterastic skeletal myosin heavy chain was positive in myogenic induced cells.</p><p><b>CONCLUSION</b>It seems that human adipose tissue represents an abundant reservoir of adult stem cells that have multi-germline potential to differentiate into myoblasts. Adipose tissue derived stromal cells will be another alternative source for cell-based tissue engineering in skeletal muscle reconstruction.</p>

Heterotopic chondrogenesis of human adipose tissue-derived stromal cells loading on alginate gel / 中华口腔医学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>It seems that stromal cells derived from human adipose tissue presents a potential for chondrogenic differentiation.</p>