Effects of rhBMP-2 on Bone Formation Capacity of Rat Dental Stem/Progenitor Cells from Dental Follicle and Alveolar Bone Marrow.

Dental stem/progenitor cells are a promising cell sources for alveolar bone (AB) regeneration because of their same embryonic origin and superior osteogenic potential. However, their molecular processes during osteogenic differentiation remain unclear. The objective of this study was to identify the responsiveness of dental follicle cells (DFCs) and AB marrow-derived mesenchymal stem cells (ABM-MSCs) to recombinant human bone morphogenetic protein-2 (rhBMP-2). These cells expressed vimentin and MSC markers and did not express cytokeratin and hematopoietic stem cell markers and showed multilineage differentiation potential under specific culture conditions. DFCs exhibited higher proliferation and colony-forming unit-fibroblast efficiency than ABM-MSCs; rhBMP-2 induced DFCs to differentiate toward a cementoblast/osteoblast phenotype and ABM-MSCs to differentiate only toward a osteoblast phenotype; and rhBMP-2-induced DFCs exhibited higher osteogenic differentiation potential than ABM-MSCs. These cells adhered, grew, and produced extracellular matrix on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA). During a 14-day culture on nHAC/PLA, the extracellular alkaline phosphatase (ALP) activity of DFCs decreased gradually and that of ABM-MSCs increased gradually; rhBMP-2 enhanced their extracellular ALP activity, intracellular osteocalcin (OCN), and osteopontin (OPN) protein expression; and DFCs exhibited higher extracellular ALP activity and intracellular OCN protein expression than ABM-MSCs. When implanted subcutaneously in severe combined immunodeficient mice for 3 months, DFCs+nHAC/PLA+rhBMP-2 obtained higher percentage of bone formation area, OCN, and cementum attachment protein expression and lower OPN expression than ABM-MSCs+nHAC/PLA+rhBMP-2. These results showed that DFCs possessed superior proliferation and osteogenic differentiation potential in vitro, and formed higher quantity and quality bones in vivo. It suggested that DFCs might exhibit a more sensitive responsiveness to rhBMP-2, so that DFCs enter a relatively mature stage of osteogenic differentiation earlier than ABM-MSCs after rhBMP-2 induction. The findings imply that these dental stem/progenitor cells are alternative sources for AB engineering in regenerative medicine, and developing dental tissue may provide better source for stem/progenitor cells.

Combined Use of Recombinant Human BMP-7 and Osteogenic Media May Have No Ideal Synergistic Effect on Leporine Bone Regeneration of Human Umbilical Cord Mesenchymal Stem Cells Seeded on Nanohydroxyapatite/Collagen/Poly (l-Lactide).

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a promising alternative source of mesenchymal stem cells (MSCs) that are enormously attractive for clinical use. This study was designed to investigate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) and/or osteogenic media (OMD) on bone regeneration of hUC-MSCs seeded on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA) in a rabbit model. The characteristics of stem cells were analyzed by plastic adherence, cell phenotype, and multilineage differentiation potential. Cell proliferation was examined using cell counting kit-8 assay. Osteogenic differentiation was evaluated by quantitative Ca2+ concentration, PO43- concentration, alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralized matrix formation. Bone regeneration was investigated in jaw bone defect repair in rabbit by microcomputed tomography, fluorescent labeling, and hematoxylin and eosin staining. Except for initial stress response, OMD and OMD + rhBMP-7 inhibited the proliferation of hUC-MSCs seeded on nHAC/PLA; rhBMP-7 inhibited cell proliferation in the nonlogarithmic phase and attenuated the inhibitory effect of OMD on cell proliferation. The inhibitory effects of OMD, rhBMP-7, and OMD + rhBMP-7 on cell proliferation were ranked as OMD > OMD + rhBMP-7 > rhBMP-7. OMD, rhBMP-7, and OMD + rhBMP-7 promoted Ca2+ concentration, PO43- concentration, ALP activity, OCN secretion, and mineralized matrix formation of hUC-MSCs seeded on nHAC/PLA. The promoting effects of OMD, rhBMP-7, and OMD+rhBMP-7 on Ca2+ concentration, PO43- concentration, ALP activity, OCN secretion, and mineralized matrix formation were ranked as rhBMP-7 > OMD > OMD + rhBMP-7, OMD > OMD + rhBMP-7 > rhBMP-7, OMD > rhBMP-7 > OMD + rhBMP-7, rhBMP-7 > OMD + rhBMP-7 > OMD, and OMD > rhBMP-7 > OMD + rhBMP-7, respectively. In rabbit jaw bone defect repair, OMD, rhBMP-7, and OMD + rhBMP-7 enhanced bone regeneration of hUC-MSCs seeded on nHAC/PLA, but the largest bone mineral apposition rate and bone formation were presented in cultures with rhBMP-7. These findings suggested that the combined use of rhBMP-7 and OMD may have no ideal synergistic effect on bone regeneration of hUC-MSCs seeded on nHAC/PLA in rabbit jaw bone defect.

Identification, characterization and microRNA expression profiling of side population cells in human oral squamous cell carcinoma Tca8113 cell lines.

The present study aimed to evaluate the stem cell markers, characteristics and biological functions of cancer stem­like side population (SP) cells in human oral cancer. SP cells were isolated from the human oral squamous cell carcinoma Tca8113 cell line by Hoechst 33342 fluorescence dye and flow cytometry. The colony forming and proliferative capability of SP and non­SP cells were detected using a live­cell analysis system in vitro. The number of cells expressing stem cell markers was compared between SP cells and non­SP cells by flow cytometry. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels of stem cell genes, respectively. Differential expression of microRNAs (miRNAs) in SP and non­SP cells was determined by microarray hybridization and an miRNA regulation network was produced. With regard to the proliferation capability, SP cells reached 60.0% confluence after 40 h of growth compared with 35.1% confluence for non­SP cells (P<0.05). The number of colonies in SP cells was 43.1±9.2 compared with 33.0±8.2 of non­SP cells (P<0.05). The aldehyde dehydrogenase­1 (ALDH1)­positive cell number in the SP cells was increased by 10 times compared with the non­SP cells (P<0.01). The mRNA and protein expression levels of ALDH1, SRY­box 2, POU class 5 homeobox 1 and Nanog homeobox in SP cells were significantly higher compared with non­SP cells (P<0.05). Microarray hybridization demonstrated that 21 miRNAs were upregulated and 13 miRNAs were downregulated in SP cells compared with non­SP cells. SP cells in Tca8113 demonstrated greater capability of proliferation and colony formation compared with non­SP cells in vitro. Stem cell markers were overexpressed in SP cells compared with non­SP cells.

Tantalum-incorporated hydroxyapatite coating on titanium implants: its mechanical and in vitro osteogenic properties.

OBJECTIVE: The fabrication of bioactive coatings on metallic implants to enhance osseointegration has become a topic of general interest in orthopedics and dentistry. Hydroxyapatite (HA) coating has been shown to induce bone formation and promote bone-implant integration. Unfortunately, poor mechanical performance has hindered this from becoming a favorable coating material. The majority of present studies have focused in incorporating different elements into HA coatings to improve mechanical properties. In recent years, tantalum (Ta) has received increasing attention due to its excellent biocompatibility and corrosion resistance. The aim of on the present study was to investigate the fabrication and biological performance of Ta-incorporated HA coatings. METHODS: Ta-incorporated HA coatings were fabricated using the plasma spray technique on a titanium substrate, and the surface characteristics and mechanical properties were examined. In addition, the effects of Ta-incorporated HA coatings on the biological behavior of mesenchymal stem cells (BMSCs) were investigated. RESULTS: Ta-incorporated HA coatings with microporous structure had higher roughness and wettability. In addition, the bonding strength of Ta/HA coatings with the substrate was substantially superior to HA coatings. Furthermore, Ta-incorporated HA coatings not only facilitated initial cell adhesion and faster proliferation, but also promoted the osteogenic differentiation of BMSCs. CONCLUSION: These results indicate that the incorporation of Ta could improve mechanical performance and increase the osteogenic activity of HA coatings. The Ta-incorporated HA coating fabricated by plasma spraying is expected to be a promising bio-coating material for metallic implants.

Estrogen enhances the bone regeneration potential of periodontal ligament stem cells derived from osteoporotic rats and seeded on nano-hydroxyapatite/collagen/poly(L-lactide).

This study investigated the effects of estrogen on the bone regeneration potential of periodontal ligament stem cells (PDLSCs) derived from osteoporotic rats and seeded on a collagen-based composite scaffold [nano-hydroxyapatite/collagen/poly(L-lactide) (nHAC/PLA)]. For this purpose, 48 healthy 3­month-old Sprague-Dawley female rats were divided into 2 groups as follows: the bilaterally ovariectomized (OVX) rats and sham­operated rats. The PDLSCs were isolated at 3 months after surgery (by which time postmenopausal osteoporosis had developed). The effects of estrogen on the characteristics of these cells seeded in a culture plate and of the cells seeded on nHAC/PLA were then investigated. The PDLSC + nHAC/PLA constructs were implanted subcutaneously into the backs of severe combined immunodeficient (SCID) mice for 12 weeks in order to examine the role of estrogen in the bone formation ability of PDLSCs derived from osteoporotic rats. The results from methyl thiazolyl tetrazolium (MTT) assay revealed that the proliferation of the cells derived from the rats in the OVX group was significantly higher than that of the cells derived from the rats in the sham-operated group at the stage of logarithmic growth. The staining intensity of alkaline phosphatase (ALP) and the mineralization of the cells derived from the rats in the OVX group was significantly weaker than that of the cells from the rats in the sham-operated group. When the PDLSCs were seeded on nHAC/PLA, ALP activity, osteocalcin (OCN) secretion, mineral formation and the mRNA expression levels of ALP, OCN, estrogen receptor (ER)α and ERß in the cells derived from the rats in the OVX group were markedly decreased. Treatment with 17ß-estradiol (E2) significantly weakened the proliferative ability of the cells derived from the OVX group rats, and enhanced their osteogenic differentiation ability and the mRNA expression levels of ALP, OCN, ERα and ERß. When the constructs were implanted into the backs of SCID mice for 12 weeks, the results of histological analysis indicated that the constructs derived from the OVX group rats had a few newly formed bones and osteoids; however, a great number of newly formed bones and osteoids were present in the ones from the sham-operated group and the OVX + E2 group rats. Our findings further indicate that estrogen deficiency impairs the osteogenic differentiation potential of PDLSCs, and that ER plays an important role in the bone regeneration ability of PDLSCs. Estrogen enhances the bone regeneration potential of PDLSCs derived from osteoporotic rats and seeded on nHAC/PLA. This study may provide insight into the clinical management of periodontal bone tissue repair in postmenopausal women with the use of estrogen-mediated PDLSCs seeded on nHAC/PLA.

Enhancement of periodontal tissue regeneration by transplantation of osteoprotegerin-engineered periodontal ligament stem cells.

INTRODUCTION: The objective of the present study was to evaluate the capacity of a tissue-engineered complex of human osteoprotegerin (hOPG)-transfected periodontal ligament stem cells (PDLSCs) seeding on beta-tricalcium phosphate (ß-TCP) to regenerate alveolar bone defects in New Zealand rabbits. METHODS: PDLSCs were isolated from rabbit periodontal ligament tissues and expanded in vitro to enrich PDLSC numbers, and their proliferative activities and differentiation capability were evaluated under specific induction conditions. Lentiviral vector containing hOPG and enhanced green fluorescent protein (EGFP) was constructed by using Gateway technology and transfected into rabbit PDLSCs. The expression of hOPG was determined with quantitative real-time reverse transcription-polymerase chain reaction and Western blot. The PDLSCs with or without engineered hOPG were seeded on ß-TCP scaffolds prior to transplantation. Morphological characterization of cells and materials was done by scanning electron microscope. Twenty rabbits with alveolar bone defects were randomly allocated into four groups and transplanted with ß-TCP, PDLSCs/ß-TCP, and hOPG-transfected PDLSCs/ß-TCP or were left untreated as a control. Animals were sacrificed 12 weeks after operation for histological observation and histomorphometric analysis. RESULTS: PDLSCs expressed STRO-1 and vementin and favored osteogenesis and adipogenesis in conditioned media. Expressions of hOPG were significantly upregulated after transfection of the lentiviral vector into PDLSCs. PDLSCs attached and spread well on ß-TCP, and there was no significant difference in growth of PDLSCs on ß-TCP between the hOPG transfection group and the non-transfection group. The histological observation and histomorphometric analysis showed that the hOPG-transfected PDLSCs/ß-TCP complex exhibited an earlier mineralization and more bone formation inside the scaffold than control, ß-TCP, and PDLSCs/ß-TCP complexes. Implantation of hOPG-transfected PDLSCs contributed to new bone formation as determined by EGFP gene expression under circularly polarized light microscopy. CONCLUSIONS: The present study demonstrated the feasibility of ß-TCP scaffolds for primary PDLSC culture and expression of hOPG gene in vitro and in vivo, and hOPG-transfected PDLSCs could serve as a potential cell source for periodontal bone regeneration, which may shed light on the potential of systemic hOPG gene therapy in combination with PDLSC tissue engineering as a good candidate in periodontal tissue engineering for alveolar bone regeneration.

Lipofuscin in saliva and plasma and its association with age in healthy adults.

BACKGROUND AND AIM: To compare blood and salivary levels of lipofuscin in healthy adults and to analyze the relationship between the lipofuscin level and the healthy adults' age. METHODS: One hundred and twenty-two healthy volunteers were recruited and divided into three groups according to their age: young (n = 42, 20-44 years old), middle-aged (n = 51, 45-59 years old), and elderly (n = 29, 60-74 years old). One ml saliva and 5 ml whole blood were collected from each person. An ELISA kit was used to measure both the plasma and salivary lipofuscin levels. The differences between the groups were compared with independent-sample t test, and the relationship between the salivary lipofuscin level and the age was assessed with linear regression analysis. RESULTS: The mean ± SD of the lipofuscin level in the saliva and plasma of 122 subjects was 68.93 ± 1.32 and 78.05 ± 1.75 µmol/l, respectively. No gender-dependent differences were observed in either the salivary or the plasma lipofuscin level (saliva: p = 0.443, plasma: p = 0.459). The salivary and plasma lipofuscin levels of the elderly subjects were significantly higher than those of the young (saliva: 80.72 ± 13.53 mmol/l versus 59.12 ± 1.92 mmol/l, p = 0.0003; plasma: 93.31 ± 3.14 mmol/l versus 67.43 ± 2.54 mmol/l, p = 0.0002) and middle-aged (saliva: 80.72 ± 13.53 mmol/l versus 70.31 ± 11.17 mmol/l, p = 0.0004; plasma: 93.31 ± 3.14 mmol/l versus 78.12 ± 2.40 mmol/l, p = 0.0002) subjects. Similarly, the salivary and plasma lipofuscin levels of the middle-aged subjects were significantly higher than those of the young subjects (saliva: 70.31 ± 11.17 mmol/l versus 59.12 ± 1.92 mmol/l, p < 0.0001; plasma: 78.12 ± 2.40 mmol/l versus 67.43 ± 2.54 mmol/l, p = 0.0019). The lipofuscin levels in the saliva and plasma were significantly positively correlated with the subject age (r = 0.551, p = 0.0001; r = 0.528, p < 0.0001). Furthermore, the salivary lipofuscin level and plasma lipofuscin level also were found to have a positive correlation (r = 0.621, p < 0.0001). CONCLUSION: No gender-dependent differences were observed in either the salivary or plasma lipofuscin levels. The salivary and plasma lipofuscin levels were positively correlated, and the age is positively correlated with lipofuscin content in saliva.

Synergistic cytotoxicity of cisplatin and Taxol in overcoming Taxol resistance through the inhibition of LDHA in oral squamous cell carcinoma.

The development of chemoresistance in patients represents a major challenge in cancer treatment. Lactate dehydrogenase-A (LDHA) is one of the principle isoforms of LDH that is expressed in breast tissue, controlling the conversion of pyruvate to lactate and also playing a significant role in the metabolism of glucose. The aim of this study was to identify whether LDHA was involved in oral cancer cell resistance to Taxol and whether the downregulation of LDHA, as a result of cisplatin treatment, may overcome Taxol resistance in human oral squamous cells. The OECM-1 oral epidermal carcinoma cell line was used, which has been widely used as a model of oral cancer in previous studies. The role of LDHA in Taxol and cisplatin resistance were investigated and the synergistic cytotoxicity of cisplatin and/or Taxol in oral squamous cells was analyzed. Cell viability was analyzed by MTT assay, LDHA expression was analyzed by western blot analysis and siRNA tranfection was performed to knock down LDHA expression. The present study results showed that decreased levels of LDHA were responsible for the resistance of oral cancer cells to cisplatin (CDDP). CDDP treatments downregulated LDHA expression, and lower levels of LDHA were detected in the CDDP-resistant oral cancer cells compared with the CDDP-sensitive cells. By contrast, the Taxol-resistant cancer cells showed elevated LDHA expression levels. In addition, small interfering RNA-knockdown of LDHA sensitized the cells to Taxol, but desensitized them to CDDP treatment, while exogenous expression of LDHA sensitized the cells to CDDP, but desensitized them to Taxol. The present study also revealed the synergistic cytotoxicity of CDDP and Taxol for killing oral cancer cells through the inhibition of LDHA. This study highlights LDHA as a novel therapeutic target for overcoming Taxol resistance in oral cancer patients using the combined treatments of Taxol and CDDP.

Pathological changes in the maxillary sinus mucosae of patients with recurrent odontogenic maxillary sinusitis.

OBJECTIVE: To study the structural and functional changes of maxillary sinus mucosae of patients with odontogenic maxillary sinusitis, and to improve the therapeutic effects. METHODS: Ten mucosal biopsy samples collected during the surgeries of patients with recurrent odontogenic maxillary sinusitis were selected as Group A. Another ten mucosal biopsy sample were collected during retention cyst-removing surgeries and referred to as Group B. The mucosae were put in 10% neutral formalin solution for 1 day and prepared into 5-7 µm thick paraffin sections which were subjected to hematoxylin-eosin staining. The reactions included: (1) Reaction with T-lymphocyte (CD-3); (2) reaction with T-helper cell (CD-4); (3) reaction with T-suppressing cell (CD-8); (4) reaction with B-lymphocyte (CD-20). Polymeric horseradish peroxidase visualized detection system was used. The contents of CD3, CD4, CD8 and CD20 in the stained cells of the maxillary sinus mucosal layer were calculated. The responses of receptors to muramidase were classified as mild, moderate and strong. All data were analyzed by Statistica 6.0 package for Windows based on Mann-Whitney non-parametric standards. RESULTS: The epithelial tissues in the maxillary sinus mucosa of Group B were covered with multiple rows of cilia. The epithelial cells of Group A suffered from degeneration, shrinkage and desquamation. Different cells were distributed in the autologous mucosal layer, of which macrophages, fibroblasts, lymphocytes and neutrophils were dominant. The average contents of macrophages and lymphocytes accounted for 42.8%. Lymphocyte subset analysis showed that the number of CD3 cells exceeded that of CD20 ones and there were more CD4+ cells than CD8+ ones. T-helper and T-suppressing cells were distributed remarkably differently. CD8+ cells were mainly located inside and under the epithelium, while CD4+ cells were scattered in the autologous matrix. CONCLUSION: For patients with recurrent odontogenic maxillary sinusitis, the maxillary sinus mucosa mainly suffered from regeneration of epithelial tissues and inhibition of cell proliferation, which were accompanied by damages to the protective and shielding effects of the mucociliary transport system. Macrophages and lymphocytes dominated in the infiltration of autologous mucosal layer, and the coexisting copious fibroblasts indicated the onset of inflammation.

[The effect of different factors on forming-dentin differentiation of rat dental mesenchymal cells].

PURPOSE: To investigate the effects of combined use of bFGF, IGF1, BMP4 and TGF-ß1 on forming-dentin differentiation of rat dental mesenchymal cells (rDMCs). METHODS: Enzyme and differential digestions were performed to isolate and culture rDECs and rDMCs, and immunofluorescence staining against cytokeratin and vimentin were carried out to identify cell sources. Then alizarin red staining and Gomori calcium-cobalt method were used to detect the mineralization ability of rDMCs after mineralized induction. Immunohistochemistry, image analysis, real-time PCR and Western blot were utilized to determine the expression differences of DSPP/CAP/OPN/OCN in rDMCs after induction by bFGF+IGF1 (group 1), TGF-ß1+BMP4 (group 2) and bFGF+IGF1+TGF-ß1+BMP4 (group 3), respectively. Statistical analysis was performed with SPSS 14.0 software package. RESULTS: The rDECs and rDMCs were isolated, cultured and identified successfully. Calcium nodus and ALP staining were positive in cytoplasms of rDMCs after being induced by mineralization liquid. In groups 1 and 2, the expression levels of DSPP/CAP/OPN/OCN mRNA and protein were notably higher than those of control group, significant differences were found between groups (P<0.01). Among them, the expression levels of CAP/OCN in group 1 and DSPP/OPN in group 2 were the highest, respectively. CONCLUSIONS: The rDMCs possess osteogenesis property after mineralization induction. bFGF+IGF1 can notably promote the expressions of CAP/OCN, and accelerate rDMCs to differentiate into cementoblast and osteoblast, and the mineralization of cementum matrix and bone matrix. TGF-ß1+BMP4 can markedly increase the expressions of DSPP/OPN, and quicken rDMCs to differentiate into odontoblast and osteoblast, and the mineralization of dentinal matrix and bone matrix which display osteogenesis trend. Combined use of four factors had no significant synergism.

[Effect of insulin on peroxiredoxin-6 in the osteogenic differentiation of rat's mandiular bone marrow stromal cells in high glucose].

PURPOSE: To explore the effect of insulin on the expression of peroxiredoxin-6 in osteogenic differentiation of rat's mandibular bone marrow stromal cells(rBMSCs) in high glucose. METHODS: Bone marrow stromal cells were obtained from the mandible of Wistar rats and stimulated in three glucose concentrations mineral medium(5.5 mmol/L, 25 mmol/L and 45 mmol/L) with or without insulin(10-5mol/L) for 1, 3, 7, 14, and 21 days. The expression of prohibitin was quantified via enzyme-linked immuno sorbent assays (ELISA). The mineralization nodules were assessed at day 21 by alizarine red staining. Statistical analysis was performed using SPSS 15.0 soft ware package. RESULTS: High glucose of 45 mmol/L inhibited mineralization of rBMSCs and insulin can improve the mineralization in high glucose. The expression of peroxiredoxin-6 in 45 mmol/L group decreased significantly compared with 5.5 mmol/L group and 25 mmol/L group. The expression of peroxiredoxin-6 in each group achieved maximum at day 21. Insulin (10-5 mol/L) increased the expression of peroxiredoxin-6 in 25 mmol/L group and 45 mmol/L group in osteogenic differentiation of rBMSCs. CONCLUSIONS: High glucose inhibits the expression of peroxiredoxin-6 in osteogenic differentiation of rBMSCs, while insulin upregulates the expression of peroxiredoxin-6 in rBMSCs. Peroxiredoxin-6 may play an important part in later stage in osteogenic differentiation of rBMSCs.

[Effects of glimepiride on proliferation, differentiation and mineralization of rat mandibular osteoblasts in hyperglycemia].

PURPOSE: To evaluate the effects of hyperglycemia and glimepiride on proliferation, differentiation and mineralization of rat mandibular osteoblasts to verify the hypothesis of dental implant administration. METHODS: Primary osteoblasts were isolated and cultured. Then the cells were placed in an osteogenic medium, containing 2 different concentrations of glucose (5.5 mmol/L and 16.5 mmol/L), with or without glimepiride (10 µmol/L). Cell proliferation was evaluated through MTT assay. Alkaline phosphatase (ALP) activity was determined by biochemistric method. Col I protein levels were determined by Western blot. OCN mRNA levels were tested by RT-PCR. SPSS 13.0 software package was used for statistical analysis. RESULTS: Hyperglycemic conditions interfered with the proliferation, ALP activity and OCN mRNA expression of rat osteoblasts, but improved the expression of Col I on day 14. Glimepiride stimulated rat osteoblast proliferation, ALP activity and OCN mRNA expression. The addition of glimepiride to normoglycemic (5.5 mmol/L) cultures registered a significant increase of Col I expression at 7 d and 14 d. Glimepiride significantly increased Col I expression in cells cultured with 16.5 mmol/L glucose for 7 days, but failed to increase at 14 d. CONCLUSIONS: Hyperglycemic conditions interfered with the proliferation, differentiation and mineralization of osteoblasts in rats; however, glimepiride improved the proliferation, differentiation and mineralization of osteoblasts in rats.

[Proteomics analysis of mandibles in type 2 diabetic rat].

PURPOSE: To investigate the differentially expressed proteins in mandible between normal rats, and type 2 diabetic rats. METHODS: Wistar rats and Goto-Kakizaki (GK) rats were selected as normal controls and experimental rats respectively. Weight and blood glucose were measured before sacrificed. The mandibles of each group were removed at the same time. After protein extraction, differentially expressed proteins were separated by two-dimensional gel electrophoresis (2-DE) and identified by MALDI-TOF/TOF. Statistical analysis was performed using SPSS 15.0 software package. RESULTS: Blood glucose levels of the experimental group were significantly higher than that of the control group(P<0.05). The weights of the two groups were not significantly different. 20 proteins were differentially expressed at least 3-fold between GK rats and Wistar rats. Of the 20 proteins, 5 proteins were more than 20-fold different between the two groups. The 5 proteins fell into 3 functional categories as follows: metabolism, binding proteins, and signal transduction. CONCLUSIONS: The differentially expressed proteins of mandible between GK rats and Wistar rats may be new targets for investigating the mechanisms of the effect of type 2 diabetes on mandible.

[Effect of insulin on prohibitin in the osteogenic differentiation of rat's mandibular bone marrow stromal cells in high glucose].

PURPOSE: To explore the effect of insulin on the expression of prohibitin in osteogenic differentiation of rat's mandibular bone marrow stromal cells(BMSC) in high glucose. METHODS: Bone marrow stromal cells were obtained from the mandible of Wistar rats and stimulated in three glucose concentrations mineral medium(5.5, 25, 45 mM) with or without insulin(10-5 M) for 1, 3, 7,14 and 21 days. The expression of prohibitin was quantified via enzyme-linked immunosorbent assays (ELISA). The mineralization nodules assessment was performed at day 21 by alizarine red staining. The statistics analysis was performed using SPSS 15.0 software package. RESULTS: High glucose of 45 mM inhibited mineralization of rBMSC and insulin could improve the mineralization in high glucose. The expression of prohibitin of 45 mM group decreased significantly compared with 5.5 mM group and 25 mM group. The expression of prohibitin of each group achieved maximum at day 3. Insulin (10-5 M) increased the expression of prohibitin of 25 mM group and 45 mM group in osteogenic differentiation of rBMSC. CONCLUSIONS: High glucose inhibited the expression of prohibitin in osteogenic differentiation of rBMSC, and insulin can improve the effect. Prohibitin may play an important part in early stage in osteogenic differentiation of rBMSC.

[Soft and hard tissue changes in Class II division 1 patients treated with Tip-Edge plus appliance].

OBJECTIVE: To investigate the soft and hard tissue changes in Class II division 1 patients treated with Tip-Edge plus technique. METHODS: Sixteen Class II division 1 patients (7 boys and 9 girls) with mandibular retrusion in permanent dentition were selected and treated with Tip-Edge plus appliance. Lateral cephalometric films were analyzed before and after treatment. The effects were evaluated with Holdaway soft tissues analysis and routine cephalometric analysis methods. The arithmetic mean and standard deviation were calculated for each variable. Paired t-test was performed. RESULTS: The average treatment time was 16 months. Normal overjet and overbite were established with retroclination of upper incisors and proclination of lower incisors. U1-NA(°) and U1-NA (mm) decreaed by (15.40 ± 5.31)° and (4.16 ± 1.82) mm (P < 0.01). NLA showed an average increase of (-16.60 ± 5.29)° (P < 0.01). Remarkable soft tissue change was noted after the treatment. CONCLUSIONS: The profile in Class II division 1 patients could be quickly and efficiently improved after treatment with Tip-Edge plus technique.

[Experimental study on co-culture of salivary adenoid cystic carcinoma cells and ganglia].

OBJECTIVE: To construct the co-culture models of salivarya denoid cystic carcinoma (SACC) cells and dorsal root ganglia (DRG) of chickens and investigate the promotive effects of SACC on neural tissue. METHODS: Glass-base culture dish was adopted to construct co-culture model of SACC-83 cells and DRG. SACC-83 cells were seeded in the medium pore with DRG around them. Outgrowth of neuronal processes was observed. Then DRG was cultured in the conditioned medium of SACC-83, with the groups of conditioned medium of MC3T3-E1 and HGF, the group of cell lysis buffer, the groups of serum-free medium and serum-plus medium as the controls. Outgrowth of neuronal processes was also recorded and compared with control groups. RESULTS: In the co-culture model of tumor and neuronal tissue, SACC-83 cells produced a suitable microenvironment in which neuronal processes remarkably grow. Neuronal processes of most DRG displayed growth tendency toward SACC. The group of conditioned medium from SACC-83 manifested obvious promotive effects on DRG. CONCLUSIONS: Co-culture model of tumor and neuronal tissue was successfully constructed, with which the promotive effects of tumor on outgrowth of neuronal processes could be observed. So hypothesized that SACC could secrete some neurotrophic factors to guide peripheral nerves gemmating and to trigger the cascade of the neural invasion in succession.

[Effect of ultrasound on osteoprotegerin and receptor activator nuclear factor kappaB ligand expression during root resorption in rats].

OBJECTIVE: To evaluate the effect of pulsed ultrasound on the expressions of osteoprotegerin (OPG) and receptor activator nuclear factor kappaB ligand (RANKL) during root resorption in a mouse model of orthodontic tooth movement. METHODS: Thirty-two male Wistar rats (6-8 weeks old) were randomly assigned into 4 equal groups, including the blank control group, two ultrasound exposure groups with daily local LIPUS stimulation (100 and 150 MW/cm(2)) for 10 days during mechanical loading, and the control group with mechanical loading but not LIPUS exposure. Nickel-titanium closed-coil springs were used to generate 100 g mesial force for 10 days to move the maxillary right first molars. The expression of OPG and RANKL proteins at the compression sites was detected by immunohistochemistry. RESULTS: Ultrasound stimulation significantly up-regulated the expression of OPG and down-regulated RANKL expression (P<0.05). The expressions of OPG and RANKL showed significant differences between the two ultrasound exposure groups (P<0.05). CONCLUSION: Ultrasound stimulation might be useful to protect against root resorption and accelerate its repair by regulating the expressions of OPG and RANKL.

Reconstruction of alveolar bone defects using bone morphogenetic protein 2 mediated rabbit dental pulp stem cells seeded on nano-hydroxyapatite/collagen/poly(L-lactide).

The objective of the present study was to evaluate the capacity of a tissue-engineered bone complex of recombinant human bone morphogenetic protein 2 (rhBMP-2)-mediated dental pulp stem cells (DPSCs) and nano-hydroxyapatite/collagen/poly(L-lactide) (nHAC/PLA) to reconstruct critical-size alveolar bone defects in New Zealand rabbit. Autologous DPSCs were isolated from rabbit dental pulp tissue and expanded ex vivo to enrich DPSCs numbers, and then their attachment and differentiation capability were evaluated when cultured on the culture plate or nHAC/PLA. The alveolar bone defects were treated with nHAC/PLA, nHAC/PLA+rhBMP-2, nHAC/PLA+DPSCs, nHAC/PLA+DPSCs+rhBMP-2, and autogenous bone (AB) obtained from iliac bone or were left untreated as a control. X-ray and a polychrome sequential fluorescent labeling were performed postoperatively and the animals were sacrificed 12 weeks after operation for histological observation and histomorphometric analysis. Our results showed that DPSCs expressed STRO-1 and vementin, and favored osteogenesis and adipogenesis in conditioned media. DPSCs attached and spread well, and retained their osteogenic phenotypes on nHAC/PLA. The rhBMP-2 could significantly increase protein content, alkaline phosphatase activity/protein, osteocalcin content, and mineral formation of DPSCs cultured on nHAC/PLA. The X-ray graph, the fluorescent, histological observation, and histomorphometric analysis showed that the nHAC/PLA+DPSCs+rhBMP-2 tissue-engineered bone complex had an earlier mineralization and more bone formation inside the scaffold than nHAC/PLA, nHAC/PLA+rhBMP-2, and nHAC/PLA+DPSCs, or even autologous bone. Implanted DPSCs' contribution to new bone was detected through transfected eGFP genes. Our findings indicated that stem cells existed in adult rabbit dental pulp tissue. The rhBMP-2 promoted osteogenic capability of DPSCs as a potential cell source for periodontal bone regeneration. The nHAC/PLA could serve as a good scaffold for autologous DPSC seeding, proliferation, and differentiation. The tissue-engineered bone complex with nHAC/PLA, rhBMP-2, and autologous DPSCs might be a better alternative to autologous bone for the clinical reconstruction of periodontal bone defects.

[Effects of injectable chitosan thermosensitive hydrogel on dog bone marrow stromal cells in vitro].

PURPOSE: To investigate the biological effects of injectable chitosan(CS) thermosensitive hydrogel on dog bone marrow stromal cells(BMSCs) in vitro. METHODS: Dog BMSCs were obtained from dog bone marrow aspiration by density gradient centrifugation method, cultured in DMEM medium with 10% fetal bovine serum (FBS) and identified. Chitosan thermosensitive hydrogel was prepared and the BMSCs cultured in a medium containing hydrogel were observed under light microscope. The extract liquid from hydrogel was made and its effects on cell proliferation and differentiation were evaluated. Statistical significance was assessed with SPSS 13.0 software package for one -way ANOVA. RESULTS: Dog BMSCs survived in the extract liquid and there was no significant difference. The proliferation of BMSCs in mineralization condition medium was lower than normal medium after 1,4,7 days culture and was similar to that in normal medium after 10 days. The osteogenic extract liquid improved the secretion of alkaline phosphatase (ALP) and osteocalcin (OC). CONCLUSIONS: The CS thermosensitive hydrogel possesses good biocompatibility and can be used for in vitro and in vivo experimental studies of CS hydrogel loaded with BMSCs.

[Effects of ADAM28 on biological functions of human dental pulp stem cells].

PURPOSE: To investigate the effects of a disintegrin and metalloproteinase 28 (ADAM28) on proliferation, differentiation and apoptosis of human dental pulp stem cells (HDPSCs) and the possible mechanism. METHODS: Firstly, HDPSCs were isolated and cultured in vitro and identified. ADAM28 eukaryotic expression plasmid was constructed via gene rebuilt technique and transfected into HDPSCs. Then MTT chromatometry, enzyme dynamics and flow cytometry (FCM) techniques were performed to detect the effects of ADAM28 on biological characteristics of HDPSCs. Immunocytochemical and image analysis techniques were used to determine the influence of ADAM28 on HDPSCs expressing dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP) and osteopontin (OPN). Statistical significance was assessed by the Student-Newman-Keuls (SNK) test with SPSS 13.0 software package. RESULTS: ADAM28 eukaryotic plasmid was constructed and transfected into HDPSCs for 48 hours successfully. In ADAM28 eukaryotic plasmid group, proliferation activity and index of HDPSCs were lower than those of pcDNA3.1(+) group and untransfected group significantly.Alkaline phosphatase (ALP) secretion level and percentage of apoptotic cells went up remarkly. Significant difference was detected between eukaryotic plasmid group and other groups (P<0.05). The expression level of DSPP in HDPSCs elevated significantly (P<0.05). CONCLUSIONS: ADAM28 could inhibit HDPSCs proliferation, promote ALP secretion activity and DSPP expression in HDPSCs and induce HDPSCs apoptosis significantly.