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ABSTRACT: The purpose of this study was to investigate the osteogenic response of human adipose-derived stem cells (hASCs) under mechanical and/or chemical stimulation. hASCs were divided into three groups. In group A, the cells were cultured without any stimulation, in group B, the cells were induced with chemical stimulation, and in group C, the cells were induced with a combination of chemical stimulation and stretch loading. Stretch loading and chemical stimulation were applied using a four-point bending apparatus (0.5 Hz, 2,000 µε, 2 h/day) and osteogenic differentiation medium, respectively. At the 1st, 2nd, 3rd, 5th and 7th day following initiation of stretch loading, we detected alkaline phosphatase activity, mRNA expression (RUNX2, ALPL, osteonectin, osteopontin and type I collagen) and protein expression (RUNX2 and osteopontin) by colorimetric assay, real-time PCR and Western blot methods, respectively. Alkaline phosphatase activity, mRNA expression and protein expression all increased in groups B and C along with the culture time, but were observed to be downregulated by the 7th day in group C (p < 0.05). Compared to group A, most of the above markers were significantly higher in groups B and C (p < 0.05). All of the above markers in group C were higher than those in group B before the 5th day (p < 0.05), except at the 1st day. These results indicated that stretch loading promoted osteogenic differentiation of hASCs and that the combination of mechanical and chemical stimulation could enhance the osteogenic capability up to the 5th day relative to chemical stimulation alone.
Cells Tissues Organs 05/2012; 196(4):313-24. · 2.20 Impact Factor
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Jie Long,
Peng Li,
Hong-ming Du,
Lei Liu,
Xiao-hui Zheng, Yun-feng Lin,
Hang Wang,
Wei Jing,
Wei Tang,
Wei-hui Chen,
Wei-dong Tian
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ABSTRACT: We investigated the effect of recombinant human bone morphogenetic protein 2 (rhBMP-2) on new bone formation during rapid-rate mandibular distraction osteogenesis. We also explored the feasibility of using local BMP-2 gene therapy to compensate for bad callus formation caused by a rapid distraction rate.
Bone marrow mesenchymal stem cells (MSCs) from Japanese rabbits were transfected with adenovirus (adv)-BMP-2. The right mandibles of the rabbits were distracted after corticotomy. The distraction rate in group A was 0.8 mm/d. The distraction rate in group B was 2.4 mm/d, and the distraction gap was injected with adv-lacZ-transfected bone marrow MSCs. The distraction rate in group C was 2.4 mm/d, and the distraction gap was injected with adv-BMP-2-transfected bone marrow MSCs. New generation bone tissue in the distraction gap was analyzed by plain radiograph examinations, microfocus computerized tomography (micro-CT) examinations, and biomechanical tests at weeks 2, 4, and 8 of the consolidation period.
Radiographic and micro-CT examinations showed a better bone quality in group C compared with group A at weeks 2 and 4 of the consolidation period. There was no obvious new bone formation in group B. The trabecular parameters (trabecular thickness, trabecular number, volumetric bone mineral density at tissue, and bone volume fraction) were significantly higher in group C than in group A at weeks 2 and 4. At week 8, no significant difference were detected for all parameters except trabecular number between groups A and C. All biomechanical stress parameters were significantly higher in group C than in group A at week 4, and only peak stress was significantly different at week 8.
Gene therapy using rhBMP-2-modified MSCs promoted new bone formation during mandibular distraction osteogenesis, and effectively compensated for the detrimental effect of rapid distraction rate on new bone formation.
Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 12/2010; 112(1):50-7. · 1.50 Impact Factor
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ABSTRACT: 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.
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.
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.
As the population of GFP labeled DPSCs can be easily identified, this will be a promising method for tracking DPSCs in vivo.
International Journal of Oral Science 03/2010; 2(1):21-7. · 1.41 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
International Journal of Oral Science 06/2009; 1(2):81-9. · 1.41 Impact Factor
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ABSTRACT: To compare the growth and development of tissue engineered tooth germ implanted into different tissues, and explore a suitable growing environment for the tissue engineered teeth in vivo.
SD rat/porcine tooth germ cells from postnatal 4 days were used as seeding cells, which combined various scaffolding biomaterials to construct the compound with tissue engineered teeth. The allografts were implanted into renal subcapsule, the mesenteries and subcutaneous tissues. Then, the implants were retrieved at special time points for histological analysis.
Further developments were not observed in the graft implanted into mesenteries and subcutaneous tissues. Partial grafts were fallen off and lost from the subcutaneous tissues after implanted, and there were obvious lymphocyte infiltrations in the mesenteries. Moreover, the enamel and pulp-dentin complex were observed within the graft implanted in the subrenal capsule, which indicated there to be good condition.
The subrenal capsule can provide a promising implantation environment for the further growth of allogeneic tissue engineered tooth germ, and the subrenal capsule implantation can be used as a new alternative method for tissue-engineering tooth in vivo.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 03/2008; 39(2):279-82.
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ABSTRACT: 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.
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.
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.
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.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 03/2008; 39(2):283-5.
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ABSTRACT: The purpose of this study was to investigate the distribution and relationship of fibronectin (FN), bone morphogenetic protein (BMP) 2, 4 during development of mouse tooth germ.
Rat embryonic first molars were collected from E14 (bud stage) and E18 (bell stage). The expressions of FN and BMP-2, 4 were analyzed with immunohistochemistry. RESLUTS: FN was located in the epithelium and dental mesenchyme on bud stage, but on bell stage, the FN was found at the region of differentiating odontoblasts and in the inner enamel epithelium, and also BMP-2, 4 were abundant mainly at the brisk region of differentiating odontoblasts.
The results demonstrate that BMP-2, 4 and FN play the important roles during bud stage and bell stage, and there may be synergy between BMP-2, 4 and FN in regulating differentiation and maturity of odontoblasts.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 10/2007; 38(5):826-8.
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ABSTRACT: This study aimed to investigate the odontogenic potential of bone marrow mesenchymal stem cells (BM-MSCs) for seeding in tooth regeneration.
In this study, BM-MSCs were co-cultured with oral epithelial cells derived from rat embryos. Expression of the odontogenic genes Pax9, DMP1, and DSPP was detected by the reverse-transcription polymerase chain reaction (RT-PCR) technique. To further characterize the odontogenic potential of BM-MSCs, the gold standard in vivo transplantation system was used.
The results revealed that Pax9, DMP1, and DSPP expression was detected by RT-PCR only after co-culture of BM-MSCs and oral epithelial cells derived from embryos age E11.5. Histological analyses of the BM-MSCs/epithelial cell mass demonstrated the presence of tooth-like structures.
The series of experiments both in vitro and in vivo demonstrated that BM-MSCs can differentiate into functional odontoblast-like cells. This implies that BM-MSCs may become a novel source of cells for seeding in tooth regeneration research.
Journal of Oral and Maxillofacial Surgery 04/2007; 65(3):494-500. · 1.64 Impact Factor
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ABSTRACT: To investigate the molecular mechanism of osteogenetic differentiation of bone-marrow mesenchymal stem cells (BMSCs) and the probability using the BMSCs to gene therapy for bone fractures.
By gradient centrifugation and adherence to the culture plastic, the MSCs were separated and purified from mouse bone marrow. The BMSCs then were cultured and sub-cultured in the osteogenetic medium (100 nmol/L Dexamethasone, 10 mmol/L beta-glycerophosphate and 50 mg/mL ascorbic acid, osteogenic supplements, OS-medium) or the recombinant human bone morphogenetic protein-2 (rhBMP-2, 500 ng/mL) for the mineralized inductions of osteogenesis, and stained by alizarin red in inducing week 1 and 2 for the identification of calcium nodule formed. The gene expressions of Runx2, Osx, OCN, and Col I were detected by RT-PCR on day 1, 2 and 3 after doing the osteogenetic inductions.
The BMSCs induced by OS-medium and rhBMP-2 were both of positive Ca nodules with alizarin red. However, the Ca nodule induced by OS-medium formed in 1 inducing week, but the one done by rhBMP-2 occurred in 2 inducing weeks, which meant it was a late for one week. In the OS-group, the mRNA of Runx2 could not be detected on inducing day 1, 2 and 3, but the Osx mRNA appeared on inducing day 2 and 3, and also the mRNAs of OCN and Col I could be detected in all the three inducing days. In rhBMP-2 group, the Runx2 gene expressed on inducing day 2, the Osx gene expressed on inducing day 2 and 3, the OCN and Col I genes expressed on inducing day 1, 2 and 3.
The BMSCs induced by OS-medium are more likely to form bone nodules than that of rhBMP-2, because of their simpler mechanisms to differentiate into osteoblasts.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 12/2006; 37(6):856-9.
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ABSTRACT: To evaluate the expression of dentin sialophosphoprotein (DSPP) in transfected rat bone marrow mesenchymal stem cells (BM-MSC) and the influence of the transfection.
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.
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.
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.
Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology 08/2006; 41(7):426-9.
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ABSTRACT: 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.
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.
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.
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.
Hua xi kou qiang yi xue za zhi = Huaxi kouqiang yixue zazhi = West China journal of stomatology 05/2005; 23(2):152-4.
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ABSTRACT: 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.
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.
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.
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.
Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology 04/2005; 40(2):150-3.
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ABSTRACT: To isolate human adipose tissue-derived stromal cells and study the potential of osteogenic differentiation after inductive culture.
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.
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.
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.
Hua xi kou qiang yi xue za zhi = Huaxi kouqiang yixue zazhi = West China journal of stomatology 01/2005; 22(6):507-9.
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ABSTRACT: 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.
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.
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.
It seems that stromal cells derived from human adipose tissue presents a potential for chondrogenic differentiation.
Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology 08/2004; 39(4):316-9.
