Research progress on the application of framework nucleic acid in bone regeneration / 华西口腔医学杂志

Framework nucleic acid (FNA) is a set of DNA nanostructures characterized by the framework morphology. It can design rational DNA sequences and follow the principle of complementary base pairing to construct FNA. The recent discovery of FNA constructed by DNA nanotechnology has great application potential in the field of bone regene-ration. It plays a positive role in the osteogenic differentiation of stem cells, bone regeneration, vascular regeneration, neuromodulation, immune regulation, and drug delivery. Here, we reviewed the current study findings on FNA in the field of bone regeneration.

Multilineage differentiation of dental pulp stem cells from green fluorescent protein transgenic mice / 国际口腔科学杂志·英文版

<p><b>AIM</b>The aim of this study was to confirm the multilineage differentiation ability of dental pulp stem cells (DPSCs) from green fluorescent protein (GFP) transgenic mice. The expression of GFP in DPSCs was also observed during differentiation.</p><p><b>METHODOLOGY</b>DPSCs were harvested from the dental pulp tissue of transgenic nude mice, and then transferred to osteogenic, adipogenic, and chondrogenic media. The morphological characterization of induced cells was observed by microscopy and histological staining. The expression of marker genes was measured by RT-PCR.</p><p><b>RESULTS</b>The endogenous GFP and multilineage potential of transgenic DPSCs had no influence on each other. Moreover, the results of fluorescence microscopic imaging suggest that there was no significant decline of GFP expression during DPSCs differentiation.</p><p><b>CONCLUSION</b>As the population of GFP labeled DPSCs can be easily identified, this will be a promising method for tracking DPSCs in vivo.</p>

DAPT enhances the apoptosis of human tongue carcinoma cells / 国际口腔科学杂志·英文版

<p><b>AIM</b>To investigate the effect of DAPT (gamma-secretase inhibitor) on the growth of human tongue carcinoma cells and to determine the molecular mechanism to enable the potential application of DAPT to the treatment of tongue carcinoma.</p><p><b>METHODOLOGY</b>Human tongue carcinoma Tca8113 cells were cultured with DAPT. Cell growth was determined using Indigotic Reduction method. The cell cycle and apoptosis were analyzed by flow cytometry. Real-time PCR and Immuno-Fluorescence (IF) were employed to determine the intracellular expression levels.</p><p><b>RESULTS</b>DAPT inhibited the growth of human tongue carcinoma Tca8113 cells by inducing G0-G1 cell cycle arrest and apoptosis. The mRNA levels of Hairy/Enhancer of Split-1 (Hes-1), a target of Notch activation, were reduced by DAPT in a dose-dependent manner. Coincident with this observation, DAPT induced a dose-dependent promotion of constitutive Caspase-3 in Tca8113 cells.</p><p><b>CONCLUSION</b>DAPT may have a therapeutic value for human tongue carcinoma. Moreover, the effects of DAPT in tumor inhibition may arise partly via the modulation of Notch-1 and Caspase-3.</p>

An experimental study on rat bone marrow mesenchymal stem cells transfected with dentin sialophosphoprotein gene in vitro / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the expression of dentin sialophosphoprotein (DSPP) in transfected rat bone marrow mesenchymal stem cells (BM-MSC) and the influence of the transfection.</p><p><b>METHODS</b>Plasmid containing mice dentin DSPP was constructed by using the cytomegalovirus (CMV) promoter and then transfected the cultured BM-MSC by lipofectamine; The expression of Pax-9 and dentin matrix protein 1 (DMP1) gene of transfected BM-MSC were detected by RT-PCR. The expression of DSPP was examined by immunocytochemical staining, and the formation ratio of mineralized nodules of transfected BM-MSC was compared with untransfected ones after mineralized induction.</p><p><b>RESULTS</b>The constructed pcDNA3.1(+)/mDSPP could produced 3.0 kb and 5.4 kb fragments, DSPP gene and Pax9 gene were expressed 24 h and 48 h respectively, after BM-MSC were transfected Pax-9 gene was exprssed, but DMP1 gene was not; Immunohistochemical staining showed that DSPP was positive in transfected BM-MSC; The formation ratio of mineralized nodules of transfected BM-MSC was higher than that of untransfected ones after mineralized induction.</p><p><b>CONCLUSIONS</b>The expression of mice DSPP in BM-MSC by gene transfection can induce the expression of tooth development-associated gene Pax9 and enhance the formation of mineralized nodules, which suggests that DSPP gene might induce odontogenic differentiation of BM-MSC.</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>

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>

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>

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>