Study of mandibular buccal gingival mucosa pain sensitivity / 口腔疾病防治

Objective@# Through a pain study of buccal gingival mucosa sensitivity of the mandible, the corresponding sensitive area of pain was determined, which provided the basis for reducing the pain and discomfort of oral diagnosis and treatment.@*Methods@#400 patients with mandibular tooth extraction in the outpatient department of stomatology were selected. During tooth extraction, articaine epinephrine injection was used for infiltration anesthesia. The injection needle size was 0.3 mm × 21 mm, and the injection site was about 5 mm away from the buccal gingival margin. The pain degree of the patients was recorded. The data were statistically analyzed using the modified International pain classification method.@*Results @#Among the 400 patients who underwent mandible extraction, 75% (300 patients) graded their pain from painless to moderate, and 25% (100 patients) reported moderate to severe and severe pain. Of those in the moderate to severe and severe groups, 50% and 42% reported pain in the central and lateral incisors, respectively, and 38% were in the canine group. When comparing the moderate to severe and the severe groups, 16% and 10% were in the bicuspid group, 16% and 12% and 16% were in the molar group, respectively . There were significant differences in the pain sensitivity of different teeth positions (χ2=54.203, P < 0.001). The proportion of moderate to severe and severe pain in the anterior teeth group was higher than it was in the posterior teeth group (χ2=55.555, P < 0.001). There were significant differences in the pain sensitivity of different ages (χ2=96.501, P=0.000), and there was a positive correlation between pain and age (r=0.465, P < 0.001). The proportion of women with at least a moderate degree of pain was higher than that of men (χ2=12.298, P=0.031). @*Conclusion@# The sensitivity of the buccal gingival mucosa to pain is different in different positions of the mandible. The sensitivity of the anterior gingiva is higher than that of the posterior gingiva. Age is positively correlated with the degree of pain. Further, compared with men, women are more sensitive to pain.

ANTI-CANCER ACTIVITY OF ASTER TATARICUS ON SCC-9 HUMAN ORAL SQUAMOUS CARCINOMA.

BACKGROUND: Oral squamous carcinoma is a head and neck cancer, which is one of the types of malignant cancers. Present study evaluates the anticancer activity of Aster tataricus (AT) on SCC-9 human oral squamous carcinoma. MATERIALS AND METHODS: Ethanol extract of AT was prepared by a standard procedure of maceration. AT extract was used in different concentrations like 10, 20, 40, 80, 160, 320 and 640 µg/ml for the evaluation of its anticancer activity. Effect of AT extract on SCC9 cells were observed by microscope and cytotoxicity by 3-(4, 5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Moreover, clonogenic assay was used for the estimation of effect of AT extract on colony forming ability of SCC9 cells. RESULT: Result of the study suggested that treatment with AT extract causes cytotoxicity to SCC9 cancerous cells. In addition, AT extract treatment reduces clonogenic potential of SCC9 cell and it also inhibits the proliferation of cell significantly (p<0.001) in G2/M phase. CONCLUSION: Thus, given study concludes that AT extract effectively attenuates the growth of SCC-9 cancerous cells by the virtue of its cytotoxic and anti clonogenic activity.

Nuclear Factor I-C promotes proliferation and differentiation of apical papilla-derived human stem cells in vitro.

The transcription factor Nuclear Factor I-C (NFIC) has been implicated in the regulation of tooth root development, where it may be anticipated to impact on the behavior of stem cells from the apical papilla (SCAPs) and root odontoblast activity. We hypothesized that NFIC may provide an important target for promoting dentin/root regeneration. In the present study, the effects of NFIC on the proliferation and differentiation of SCAPs were investigated. Over-expression of NFIC increased cell proliferation, mineralization nodule formation and alkaline phosphatase (ALP) activity in SCAPs. Furthermore, NFIC up-regulated the mRNA levels of odontogenic-related markers, ALP, osteocalcin and collagen type I as well as dentin sialoprotein protein levels. In contrast, knockdown of NFIC by si-RNA inhibited the mineralization capacity of SCAPs and down-regulated the expression of odontogenic-related markers. In conclusion, the results indicated that upregulation of NFIC activity in SCAPs may promote osteo/odontoblastic differentiation of SCAPs.

Soft tissue image reconstruction using cone-beam computed tomography combined with laser scanning: a novel method to evaluate the masticatory mucosa.

OBJECTIVE: To construct a 3-D model of the masticatory mucosa to measure the thickness of the facial/lingual gingiva and palatal mucosa. STUDY DESIGN: Maxillofacial regions of 8 volunteers were scanned using cone-beam computed tomography to generate 3-D maxillary and mandibular models. Digital models were obtained by laser scanning of the impressions. Models were constructed using global data registration and Boolean subtraction. Accuracy was assessed by comparison against control patients with a periodontal pack around their gingival boundaries. Inter- and intra-observer variability were determined. RESULTS: Masticatory mucosa models (in stereolithography format) showed the gingival and mucosal contours. The gingival thickness of the 3-D models and controls were not significantly different (P > .05). The interclass correlation coefficient and Kappa values indicated good intra-observer and inter-observer agreement, respectively. CONCLUSIONS: Cone-beam computed tomography combined with laser scanning can be reliable for visualizing and measuring the thickness of the masticatory mucosa.

[Effects of hypergravity exposure after 30-days simulated weightlessness on chemokine CCL20 and its receptor CCR6 in lingual mucosa of rhesus macaque].

OBJECTIVE: To study the influence of hypergravity exposure after 30-days simulated weightlessness on the expression of chemokine CCL20 and its receptor CCR6 in lingual mucosa of rhesus macaque. METHODS: Twenty-three male rhesus monkeys were divided according to the random number method into one control group (A, n = 3) and three experimental groups, including the weightlessness group (B, n = 3), the hypergravity group (C, n = 3) and hypergravity exposure after 30-days simulated weightlessness group (D, n = 14), which was further divided into four subgroups according to the values of overload as: +11 Gx/270s group (D1, n = 3), +13 Gx/230s group (D2, n = 4) and +15 Gx/200s group (D3, n = 4) and +13 Gx/230s with 9-days recovery group (D4, n = 3). Histopathological changes of lingual mucosa were observed by hematoxylin-eosin staining and the expressions of CCL20 and CCR6 were detected by immunohistochemistry (IHC) and quantitative real-time PCR (Q-PCR). RESULTS: Histological observation showed that no significant histopathological changes were found in the lingual mucosa in all experimental groups of the animals, however, there were more infiltrated lymphocyte and neutrophils in the experimental groups. The immunohistochemical scores of CCL20 in group C, group D2 and group D3 were 1.30 ± 0.11, 1.68 ± 0.62 and 2.26 ± 1.00, respectively, significantly higher than that in A group (0.47 ± 0.12, P < 0.05). The immunohistochemical scores of CCR6 expression in those three groups were 4.40 ± 1.48, 6.67 ± 2.04 and 7.02 ± 2.11, respectively, also remarkably higher than that in A group (1.33 ± 0.66, P < 0.05). As far as the mRNA expression was concerned, the expressions of CCL20 and CCR6 had similar change trend with their protein expressions. CONCLUSION: Hypergravity exposure after 30-days simulated weightlessness will not lead to significant pathological changes in lingual mucosa, but can induce the expression of chemokine CCL20 and its receptor CCR6.

[Effects of hypergravity exposure after 30 days of simulated weightlessness on chemokine CCL20 and its receptor CCR6 in gingival tissue of rhesus macaque].

PURPOSE: To study the effect of hypergravity exposure after 30 days of simulated weightlessness on the expression of chemokine CCL20 and its receptor CCR6 in gingival tissue of rhesus macaque. METHODS: Twenty-three male rhesus monkeys were randomly divided into 4 groups, namely control group (A,n=3), weightlessness group (B,n=3), hypergravity group (C,n=3) and hypergravity exposure after 30 days of simulated weightlessness group (D, n=14). Group D was further divided into 4 subgroups according to the values of overload as: +11 Gx /270 s group (D1, n=3), +13 Gx /230 s group (D2,n=4), +15 Gx/200 s group (D3,n=4) and +13 Gx /230 s with 9 days of recovery group (D4, n=3). Histopathological changes of gingival tissues were observed by hematoxylin-eosin staining and the expressions of CCL20 and CCR6 were detected by immunohistochemistry (IHC) and quantitative real-time PCR (Q-PCR). SPSS 17.0 software package was used for statistical analysis. RESULTS: Histological observation showed that no significant histopathological change was found in the gingival tissues in all experimental groups. However, there were more infiltrated lymphocytes and neutrophils in the experimental groups. Normal gingival epithelial cells were hardly stained by anti-CCL20 but weakly stained by anti-CCR6. In the experimental groups, CCL20 could be detected in the basal layer of the gingival epithelial tissue, and CCR6 could be detected in the spinous layer and the basal layer of the gingival epithelium. The CCL20 and CCR6 expression in the gingival tissues of each experimental group were significantly higher than those of the control group, not only at the protein level but at the mRNA level (P<0.05) except the CCL20 expression in the weightlessness group. CONCLUSIONS: Hypergravity exposure after 30 days of simulated weightlessness will not lead to significant pathological changes in gingival tissues, but can induce the expression of chemokine CCL20 and its receptor CCR6 in gingival tissue.

Prospects application of polypyrrole-based immunosensor to Porphyromonas gingivalis quantification in subgingival plaque samples.

BACKGROUND: Periodontitis still poses a serious threat to oral and systemic health condition of humans. The proportion of the main pathogenic bacteria change in localized sites was associated with the initiation of the disease process. However, the limitations of microbiological diagnostic aids rendered the diagnosis of active periodontitis status point-of-care or chair-side based on microbiological data difficult. METHODS: Porphyromonas gingivalis, a major putative etiological agent in the initiation and progression of chronic periodontitis, was used as the experimental subject. An immunosensor based on polypyrrole-coated interdigitated array microelectrodes was developed to quantify Porphyromonas gingivalis in pure culture, gingival crevicular fluid and saliva samples. The regression equation for the normalized impedance change (NIC) versus Porphyromonas gingivalis concentration (C) was measured. The correlation between results of the immunosensor and quantitative real-time PCR method in quantifying Porphyromonas gingivalis in subgingival plaque samples was evaluated. RESULTS: Results of the study revealed that the lowest detection limits of the immunosensor was 1.9 x 10(4), 2.7 x 10(5), and 2.7 x 10(6) cells/mL in pure culture, gingival crevicular fluid, and saliva samples respectively. The values determined using the immunosensor strongly correlated with those obtained using quantitative real-time PCR method (R2 = 0.91, p < 0.05). The immunosensor did not require any labels and amplification steps, and the total detection time from sampling to measurement was less than one hour. CONCLUSIONS: The immunosensor developed in the present study offered some insight into monitoring the change in the number of periodontal bacteria chair-side during routine clinical practice.

Response of human periodontal ligament cells to baicalin.

BACKGROUND: Periodontitis is the most common cause of tooth loss in adults. Periodontal ligament cell (PLC)-based therapy is considered one of the most promising methods in periodontal tissue regeneration. The traditional Chinese medicine baicalin has been shown to possess antimicrobial and anti-inflammatory activities and enhance cell proliferation and alkaline phosphatase activity. The aim of this study is to investigate the response of human PLCs (HPLCs) to baicalin. METHODS: The effect of baicalin on cultured HPLC proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of baicalin on the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), core binding factor α1 (Cbfα1), and osteocalcin (OC) was determined by quantitative real-time polymerase chain reaction and immunodetection. RESULTS: Baicalin at a concentration of 0.01 µg/mL promoted HPLC proliferation, upregulated OPG messenger RNA (mRNA) and protein expression, and downregulated RANKL mRNA and protein expression. In addition to reducing the RANKL/OPG expression ratio significantly, it also increased Cbfα1 and OC mRNA and protein expression. CONCLUSION: Baicalin showed multifaceted regulation of genes with important roles in tissue growth and differentiation, and thus it has the potential to be a promising candidate for HPLC-based periodontal regeneration therapy.

Temporal expression of Pelp1 during proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells.

BACKGROUND: Osteogenic induction and bone formation are heavily affected by environmental factors, including estrogen, estrogen receptors, and coregulatory proteins, such as the recently reported proline-, glutamic acid-, and leucine-rich protein 1(Pelp1). OBJECTIVE: To investigate Pelp1 expression in rat bone mesenchymal stem cells (rBMSCs) during cell proliferation and osteogenic differentiation. METHODS: rBMSCs were cultured in routine and osteogenic differentiation media. Cell proliferation was assessed at days 1, 3, 5, 7, 9, 11, 14, and 21. Pelp1 protein expression in the nucleus and cytoplasm were detected by immunocytochemical analysis. Real-time RT-PCR and western blot were used to detect mRNA and protein expressions of Pelp1, osteocalcin (OCN), and alkaline phosphatase (ALP). RESULTS: Over 21 days, rBMSCs in routine culture exhibited a 1-2 day lag phase and exponential growth from day 3 to 9, plateauing at day 9, and correlated with temporal mRNA expression of Pelp1, which almost reached baseline levels at day 21. In osteogenic induction cultures, Pelp1 mRNA levels rose at day 9 and steadily increased until day 21, reaching 6.8-fold greater value compared with day 1. Interestingly, Pelp1 mRNA expression in osteogenic cultures exhibited a trend similar to that of OCN expression. Pelp1 knockdown by siRNA transfection inhibited undifferentiated rBMSC proliferation, and bone markers OCN and ALP expressions in rBMSCs cultured in routine and osteogenic differentiation media. CONCLUSIONS: Pelp1 may be a key player in BMSCs proliferation and osteogenic differentiation, meriting further consideration as a target for development of therapies for pathological bone loss conditions, such as menopausal bone loss.

Human stem cells from the apical papilla response to bacterial lipopolysaccharide exposure and anti-inflammatory effects of nuclear factor I C.

INTRODUCTION: Stem cells from the apical papilla (SCAPs) are important for tooth root development and may be candidates for regenerative endodontic procedures involving immature teeth. The potential use of SCAPs for clinical applications requires a better understanding of their responses to bacterial challenge. We have investigated the effects of exposure of these cells to lipopolysaccharide (LPS). Inflammatory responses arising from bacterial challenges can constrain postinjury tissue regeneration and the effects of nuclear factor I C (NFIC), which plays a critical role in tooth root development. NFIC has been explored for its anti-inflammatory action in the context of endodontic treatment of immature teeth where continued root development is an important outcome. METHODS: SCAPs were exposed to LPS, and the expression of Toll-like receptor-4 (TLR4), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF-α) were assessed by real-time polymerase chain reaction (RT-PCR). The pLenti6.3/v5-NFIC plasmid encoding the full-length NFIC or NFIC silencing by si-RNA (small interfering RNA) in SCAPs were measured by Western blotting or RT-PCR; the effects of NFIC on IL-6, IL-8, and TNF-α were analyzed by RT-PCR. The protein levels were subsequently measured by enzyme-linked immunoassay. RESULTS: LPS induced the synthesis of IL-6, IL-8, and TNF-α in SCAPs in a time-dependent manner. Pretreatment with a TLR4 inhibitor significantly inhibited LPS-induced IL-6, IL-8, and TNF-α expression. Knockdown of NFIC increased the expression of IL-6, IL-8, and TNF-α, whereas the overexpression of NFIC resulted in the suppression of the inflammatory response stimulated by 1 µg/mL LPS, especially for IL-8. Together, these data show that LPS is recognized by the transmembranous receptor TLR4 to mediate inflammatory responses in SCAPs and NFIC overexpression can suppress LPS-initiated innate immune responses. CONCLUSIONS: The anti-inflammatory effects of NFIC overexpression provide a valuable target to dampen inflammatory responses in the infected pulp to allow tissue regeneration to occur.

Effect of icariin on cell proliferation and the expression of bone resorption/formation-related markers in human periodontal ligament cells.

Periodontitis is a common destructive inflammatory disease that leads to changes in the tooth-supporting tissues. Human periodontal ligament cells are essential in periodontal tissue regeneration. The traditional Chinese medicine icariin promoted bone formation, stimulated the osteogenic differentiation of preosteoblastic cells and inhibited osteoclast differentiation and bone resorption. Thus, in the present study, the effect of icariin on cell proliferation and the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), core binding factor α1 (Cbfa1) and osteocalcin (OC) was investigated in human periodontal ligament cells, by an MTT assay, qPCR and western blot analysis. The results demonstrated that icariin promoted cell proliferation in a dose- and time-dependent manner, upregulated OPG, Cbfa1 and OC expression, and downregulated RANKL production and the RANKL/OPG expression ratio. This suggested the potential value of icariin in treating alveolar bone resorption and promoting periodontal tissue regeneration, due to its ability to stimulate the proliferation and osteogenic differentiation of human periodontal ligament cells and inhibit osteoclast differentiation.

Conditioned medium from periapical follicle cells induces the odontogenic differentiation of stem cells from the apical papilla in vitro.

INTRODUCTION: We investigated the biological effects of conditioned medium (CM) from periapical follicle cells (PAFCs) of root-developing tooth on the proliferation and differentiation of stem cells from the apical papilla (SCAP) in vitro. METHODS: Human SCAP and PAFCs were isolated and expanded. CM from PAFCs was prepared with the primary cells. Cell cycle analysis, methyl-thiazol-diphenyltetrazolium assay, alkaline phosphatase activity, mineralization behavior, and gene expression of odontoblast phenotype SCAP cultured with or without CM from PAFCs were evaluated. RESULTS: In the CM-treated group, the cell growth, alkaline phosphatase activity, and mineralization of SCAP were up-regulated. The expression of dentin sialophosphoprotein, alkaline phosphatase, and osteocalcin mRNA progressively increased in SCAP treated with CM from PAFCs. CONCLUSIONS: Our findings suggest that CM from PAFCs is able to provide a favorable odontogenic microenvironment to induce differentiation of SCAP along the odontoblast lineage.

Vitamin C induces periodontal ligament progenitor cell differentiation via activation of ERK pathway mediated by PELP1.

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our finding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.

Phenotypic changes in nonfimbriated smooth strains of Aggregatibacter actinomycetemcomitans grown in low-humidity solid medium.

Aggregatibacter actinomycetemcomitans is the primary etiologic agent of localized aggressive periodontitis. In vitro, it can undergo fimbriated rough to nonfimbriated smooth phenotypic transition, accompanied by an increase in invasive ability and a decrease in adhesive ability. No opposite direction phenotypic transition was reported. To better understand its pathogenicity, the authors studied the morphological changes of nonfimbriated smooth strains induced by growth environmental humidity. Transmission electron microscopy was used to identify fimbriae expression change. It was found that the lower medium humidity, the more fimbriae reexpressed. In conclusion, the smooth strain of A. actinomycetemcomitans can reexpress the fimbriae in lower humidity environment.

[The effect of Smads signal pathway on the osteogenesis of human periodontal ligament stem cells in simulated microgravity].

UNLABELLED: PUPOSE: To investigate the effect of Smads signal pathway on the osteogenesis of human periodontal ligament stem cells (hPDLSCs) in simulated microgravity. METHODS: Human periodontal ligament stem cells were isolated from the ligament of surgically extracted human teeth.Through limiting dilution assay, mono-clone of the cell was obtained, hPDLSCs were isolated from MesenPRO RS medium. Rotary cell culture system (RCCS) was enrolled to set simulated microgravity (SMG). Samples were set to control group (normal gravity group, NG) and simulated microgravity group (SMG). Real-time PCR was used to detect the expression of Smads signals and the expression of markers of osteogenesis before and after SIS3. The amount of phosphated-Smad was assayed by flow cytometry. Statistical analysis of the data was done by ANOVA with SPSS 13.0 software package. RESULTS: Compared with that of the control group, the expression of Smads 2,3,4 was significantly higher in SMG group (P<0.05)in a time-dependent manner. Flow cytometry assay showed increased expression of p-Smads at 30-min, and peak expression was observed at 2h,reaching at 91.32%. The addition of SIS3 led to significant decrease of COL I, ALP, OCN and p-Smads (P<0.05). CONCLUSION: Smads signal pathway was enrolled in the osteogenesis of hPDLSCs in simulated microgravity.

Osteogenic differentiation of human periodontal ligament stem cells expressing lentiviral NEL-like protein 1.

NEL-like protein 1 (NELL1) is a newly identified secreted protein involved in craniosynostosis and has been found to promote osteogenic differentiation of mesenchymal stem cells. The objective of this study was to investigate the effect of NELL1 on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and the potential underlying mechanism. hPDLSCs underwent lentivirus-mediated NELL1 transfection (Lenti-NELL1) and markers of osteogenesis were assessed [alkaline phosphate (ALP), osteocalcin (OCN) and calcium deposition] to evaluate the effect of NELL1 on the differentiation of these cells. Quantitative polymerase chain reaction (qPCR) was employed to measure the mRNA expression of Msx2 and Runx2, and Lenti-enhanced green fluorescent protein (EGFP) served as a control. Western blot analysis and qPCR analyses confirmed that Lenti-NELL1-transfected hPDLSCs could express NELL1. Compared with the Lenti-EGFP group, ALP, OCN, calcium deposition and Msx2 mRNA expression were markedly increased (P<0.01), but there was no significant difference in Runx2 mRNA expression between the two groups (P>0.01). hPDLSCs can be transfected by Lenti-NELL1 and can stably express NELL1. NELL1 is able to promote the osteogenic differentiation of hPDLSCs, which may be related to the downregulation of Msx2 expression. Lenti-NELL1 transfection can be used during in vitro gene therapy for periodontal regeneration.

Basic fibroblast growth factor enhances stemness of human stem cells from the apical papilla.

INTRODUCTION: Stem cells from the apical papilla (SCAP) are a type of mesenchymal stem cells found in the developing tissue, apical papilla, of immature permanent teeth. Studies have shown that SCAP are likely to be a source of primary odontoblasts that are responsible for the formation of root dentin. Basic fibroblast growth factor (bFGF) is a signaling molecule and pleiotropic growth factor involved in tooth root development, and it promotes proliferation of a variety of cell types. The effects of bFGF on SCAP, however, have not been examined. METHODS: We investigated the regulatory effects of bFGF on the proliferation and differentiation potential of human SCAP in vitro. Changes in the cell cycle and proliferation, colony-forming unit-fibroblastic formation, alkaline phosphatase (ALP) activity, osteogenic/dentinogenic differentiation, and stem cell gene makers of SCAP, cultured in the presence or absence of bFGF, were evaluated. RESULTS: Treatment with 5 ng/mL bFGF significantly increased SCAP proliferation and their colony-forming unit-fibroblastic formation efficiency. The growth factor also increased the expression of STRO-1 and the stem cell gene makers Nanog, Oct4, Sox2, and Rex1 in SCAP. In contrast, bFGF reduced the ALP activity, mineral nodule formation, and the expression of ALP, osteocalcin, bone sialoprotein, and dentin sialophosphoprotein. When SCAP cultures were expanded in the presence of bFGF for 1 week, subsequent stimulation of the osteogenic/dentinogenic condition resulted in enhanced differentiation. CONCLUSIONS: Under certain conditions, bFGF enhances SCAP stemness by up-regulating stem cell gene expression, increasing proliferation ability, and potentiating differentiation potency.

[The effect of insulin-like growth factor-I on the proliferation and alkaline phosphatase activity of human periodontal ligament cells under three-dimensional culture system].

OBJECTIVE: To investigate the effect of insulin-like growth factor- I (IGF- I) on the proliferation and alkaline phosphatase (ALP) activity of human periodontal ligament cells (hPDLCs) under three-dimensional (3D) culture system. METHODS: The hPDLCs were cultured from periodontium of human teeth by the outgrowth method. Rotary cell culture system (RCCS) was enrolled to set 3D culture system. Samples were set to four groups: Negative control group, positive control group (3D group, IGF-I group), and experimental group (3D with IGF- I group). Proliferation was tested with methylthiazolyl tetrazolium (MTT), and ALP activity was assayed by spectrophotometer at 1, 3, 5, 7 d respectively. RESULTS: Compared with that of negative control group, cell proliferation increased significantly in 3D with IGF-I group since 3 d (P < 0.05). Besides, the cell proliferation of 3D with IGF-I group was significantly higher than that of 3D group (P < 0.05). ALP activity of 3D with IGF- I group was significantly higher than that of negative control group, and 3D group at 3, 5, 7 d (P < 0.05). CONCLUSION: IGF-I significantly promotes the proliferation and ALP activity of hPDLCs under 3D culture system.

[Effect of insulin-like growth factor on the proliferation and early stage osteogenesis of human periodontal ligament stem cells under three-dimensional culture system].

OBJECTIVE: To investigate the effect of insulin-like growth factor-I (IGF-I) on the proliferation and osteogenesis of human periodontal ligament stem cells (hPDLC) under three-dimensional (3D) culture system. METHODS: Human periodontal cells were isolated from the ligament of surgically extracted human teeth, and through the limiting dilution assay, got mono-clone of the cell, hPDLCs were isolated from MesenPRO RS medium. Rotary cell culture system (RCCS) was enrolled to set 3D environment. Control group and experiment groups were assigned according to the concentration of IGF-I. There were 5 level of experiment groups (0.1, 1, 10, 50, 100 µg/L). Proliferation was tested with methyl thiazolyl tetrazolium (MTT), and alkine phosphatase (ALP) level was assayed by spectrophotometer to analyze the osteogenesis of hPDLCs. Gene expression of ostetocalcin (OCN) and type I collagen (Col I) were assayed by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: In 3D culture system, the effect of IGF-I on cell proliferation was significantly different between control group and experiment groups (P < 0.05), and there showed significant differences between the group of 0.1 µg/L (0.219 ± 0.021) IGF-I and the groups of 50, 100 µg/L (0.287 ± 0.011, 0.293 ± 0.012). However, there showed no significant differences among other groups. Significant differences of ALP activity were observed between the control group and experiment groups, and between the groups of 1, 10 µg/L (0.304 ± 0.020, 0.310 ± 0.013) and that of 50, 100 µg/L (0.347 ± 0.011, 0.344 ± 0.010) (P < 0.05). While no significant differences were detected between the group of 1 µg/L and that of 10 µg/L, nor between the group of 50 µg/L and that of 100 µg/L. Expressions of Col I and OCN in mRNA and protein level both showed dose-dependent increase. CONCLUSIONS: In 3D culture system, in the scale of 0.1 - 100 µg/L, the effect of IGF-I on the proliferation of hPDLCs increased dose-dependently. 100 µg/L IGF-I promotes osteogenesis of the cells significantly.

[Protective effect of resveratrol on apoptosis of human periodontal ligament cells in vitro].

OBJECTIVE: To investigate the effects of resveratrol (RES) on apoptosis of human periodontal ligament cells (HPLC). METHODS: HPLC were subjected to oxidative injury induced by H2O2 for 24 h after pretreatment with different concentration of RES. HPLC were then divided into the control, model, vector, RES 1, 10, 30, 50 micromol/L treatment group. The viability of the HPLC was determined by methyl thiazolyl tetrazolium (MTT) method. Lactate dehydrogenase (LDH) rate and malondialdehyde (MDA) in the culture medium, superoxide dismutase (SOD) in the HPLC homogenate were evaluated by spectrophotometry. The apoptotic HPLC was detected by flow cytometry (FCM) and calculated by relative apoptosis rate. Bax and Bcl-2 protein levels were detected by Western blotting. RESULTS: RES increased the cell survival rate after H2O2 injury. The survival rate of RES 30 micromol/L group was (86.1 +/- 4.1)% and the model group was (54.6 +/- 4.0)%, which was significantly different between the two groups (P < 0.01). The LDH leakage rate and MDA content of the RES 30 micromol/L group were (32.6 +/- 2.0)% and (1.70 +/- 0.21) micromol/L, which were significantly different with that in the model group (P < 0.01). At the same time RES could remarkably restore the vitality of SOD in the HPLC. RES increased Bcl-2 and reduced the expression of Bax protein. The apoptosis rate of the RES 30 micromol/L group and model group was (14.84 +/- 1.36)% and (64.37 +/- 2.34)%, respectively (P < 0.01). The protective effect of RES on the cell apoptosis was in a dose-dependent manner, reaching peak at a concentration of 30 micromol/L (P < 0.01). CONCLUSIONS: RES reduced oxidative stress and apoptosis in an experimental HPLC injury model induced by H2O2. RES plays a key role in the HPLC protection against oxidative injury.