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 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.

[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.

[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.

[Transcriptional regulation of dentin sialophosphoprotein by c-Jun/c-Fos].

OBJECTIVE: To investigate the role of c-Jun and c-Fos as transcriptional factors in regulation of dentin sialophosphoprotein (DSPP) gene by a promoter-luciferase reporter gene construct in odontoblast cell line MDPC-23. METHODS: Endogenous c-Jun or c-Fos protein was determined by immunocytochemistry. The role of c-Jun or c-Fos in transcription of DSPP was investigated in co-transfection experiments using promoter-luciferase reporter gene construct containing the sequence between -791 bp and +54 bp of mouse DSPP gene. RESULTS: c-Jun and c-Fos was expressed by MDPC-23 cells, and located in the nucleus of MDPC-23 cells. Overexpression of c-Jun or c-Fos significantly inhibited luciferase activity of DSPP promoter. CONCLUSION: These findings suggest c-Jun and c-Fos down-regulated the transcription of DSPP gene as a transcriptional factor in odontoblast.

[Tissue engineering of dentin-pulp complex-like structures by human dental mesenchymal cells].

OBJECTIVE: To establish three-dimensional culture model of human dental mesenchymal cells and bioengineer in vivo with ceramic bovine bone (CBB) and Collagraft as scaffolds. METHODS: Human dental mesenchymal cells induced upon stimulation of bFGF and IGF-1 or TGF-beta(1) were implanted onto CBB and Collagraft containing the same kinds of growth factors respectively. Then cell/scaffold constructs were transplanted into nude mice to establish in vivo culture model of dental mesenchymal cells. Control groups were set up at the same time. After 4 weeks or 10 weeks, the implants were taken out for histological and immunohistochemical analysis. RESULTS: Within 10-week implant tissues, typical dentin-pulp complex-like structures were generated in scaffolds containing growth factors. Human dentin sialoprotein (DSP) was expressed in the newly formed dentin. This phenomenon wasn't observed in control groups and 4-week implants. CONCLUSIONS: Dentin-pulp complex-like structures could be bioengineered successfully with human dental mesenchymal cells and CBB or Collagrafts containing growth factors in nude mice.

Smad protein mediated transforming growth factor beta1 induction of apoptosis in the MDPC-23 odontoblast-like cell line.

OBJECTIVE: The function of apoptosis and its regulation in odontoblasts remain unclear. In this study, we characterize the possible role of transforming growth factor (TGF)-beta 1 in the induction of apoptosis and the molecular mechanisms that mediate TGF-beta1-induced apoptosis in odontoblasts. METHODS: Annexin V/propidium iodide staining, cell Death Detection ELISA and DNA ladder were used to examine the effect of TGF-beta1 on apoptosis in a mouse odontoblast-like cell line, MDPC-23. Stable cell clones expressing Smad2 or Smad3 dominant negative mutants, or wild-type Smad7 were constructed to investigate the role of Smad proteins in the mediation of apoptosis by TGF-beta1 in MDPC-23 cells. The TGF-beta1-induced transcriptional activity in stable cell clones expressing Smad proteins was analyzed by a transient transfected TGF-beta-responsive reporter gene, p3TP-Lux. RESULTS: TGF-beta1 can induce apoptotic cell death in MDPC-23 cells in a dose-dependent manner. Transfection of dominant negative mutant forms of Smad2 or Smad3 blocked TGF-beta1-induced apoptosis; moreover, the Smad3 mutant was more efficient than the Smad2 mutant. Transfection of Smad7, an inhibitory Smad, also significantly inhibited TGF-beta1-induced apoptosis of these cells. Over-expression of Smad3 dominant negative mutant or Smad7 significantly inhibited TGF-beta1-induced transcriptional activity. CONCLUSION: These results suggest that Smad proteins are involved in TGF-beta1-induced apoptosis of odontoblast cells.

[Role of Smad signaling in transcription of Smad7 gene mediated by TGF-beta1 in odontoblast cell line MDPC-23].

PURPOSE: To investigate the role of Smad signaling in transcription of Smad7 gene mediated by TGF-beta1 in odontoblast cell line MDPC-23, and to explore the molecular mechanism of Smad7 gene expression mediated by TGF-beta1 at the transcriptional level. METHODS: Smad function and its role in transcription of Smad7 were investigated in cotransfection experiments using Smad7 promoter-luciferase reporter construct containing the sequence between -408 bp and +112 bp of mouse Smad7 gene. The data were analysed by one-way ANOVA. RESULTS: When the Smad7 promoter-luciferase reporter gene construct was expressed in MDPC-23 cells, its transcriptional activity was significantly induced by TGF-beta1 treatment, whereas not by BMP-2 treatment. Overexpression of Smad1, 2, 4, or 5 had no effect on transcriptional activity of Smad7 promoter. Overexpression of Smad3 markedly promoted transcriptional activity of Smad7 promoter, whereas co-transfection of Smad3 and Smad4 doubled the effect of Smad3. Overexpression of Smad3 dominant negative mutant or Smad3 antisense cDNA (AS-Smad3) significantly inhibited transcriptional activity of Smad7 promoter induced by TGF-beta1. CONCLUSION: TGF-beta1 regulated transcription of Smad7 gene through association of Smad3 and Smad4 in MDPC-23 cells.

[Bone morphogenetic protein-2-induced alpha 2 (I) collagen expression in odontoblastic MDPC-23 cells mediated by Smad proteins].

OBJECTIVE: To characterize the role of Smads proteins in alpha 2 (I) collagen (COL1A2) gene expression induced by bone morphogenetic protein-2 (BMP-2) in odontoblast cell line MDPC-23. METHODS: Endogenous Smad protein expression was determined by immunocytochemistry. Smads function and their role in COL1A2 gene expression were investigated in cotransfection experiments using promoter-luciferase reporter gene construct. RESULTS: MDPC-23 cells expressed Smad1, Smad5 and Smad6. BMP-2 promoted the activation of COL1A2 promoter reporter construct. Transient overexpression of Smad1 or Smad5 was enhanced, while overexpression of Smad6 inhibited BMP-2-induced COL1A2 promoter activity. BMP-2 inducibility could be blocked by overexpression of Smad1 or Smad5 dominant negative mutant. CONCLUSIONS: Smad signaling is functioning and appears to be involved in BMP-2-induced COL1A2 collagen transcription in MDPC-23. Smad signaling may play an important role in odontoblast differentiation and dentin extracellular matrix formation mediated by BMP-2.

TGF-beta activated Smad signalling leads to a Smad3-mediated down-regulation of DSPP in an odontoblast cell line.

OBJECTIVE: Transforming growth factor-beta (TGF-beta) regulates odontoblast differentiation and stimulates dentine extracellular matrix synthesis. However, until recently, the molecular mechanisms of action of TGF-beta have been unknown. Smad proteins have recently been identified as intracellular signalling mediators of TGF-beta. In this study, we characterise the role of Smad proteins as mediators of TGF-beta in a mouse odontoblast cell line MDPC-23. METHODS: Transcription of Smads was detected by RT-PCR. The change of intracellular location of Smad proteins treated by TGF-beta1 was evaluated immunocytochemically. Smad function and its role in transcription of dentin sialophosphoprotein (DSPP) were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs. RESULTS: MDPC-23 cells expressed Smad2, Smad3 and Smad4 mRNA. Endogenous Smad2, Smad3 and Smad4 rapidly translocated from the cytoplasm into the nucleus in response to TGF-beta1. The activity of the TGF-beta-responsive p3TP-Lux reporter construct was stimulated by 12.7-fold with TGF-beta1 treatment. Over-expression of wild-type Smad3 promoted TGF-beta1-induced luciferase activity, whereas dominant negative Smad3 inhibited it. TGF-beta1 also inhibited the activity of DSPP promoter luciferase reporter construct containing the sequence between -791 bp and +54 bp of the mouse DSPP gene. Over-expression of wild-type Smad3 potentiate the inhibitory effect of TGF-beta1 on transcriptional regulation of DSPP, while dominant negative Smad3 decreased the effect. In contrast to Smad3, wild-type Smad2 or its dominant negative mutant had little effect on TGF-beta1 regulation of the promoter activity of DSPP. CONCLUSIONS: Smad2, Smad3 and Smad4 are present and activated by TGF-beta1 in MDPC-23 cells. The Smad pathway is functional in these cells and Smad3 appears to be involved in down-regulation of DSPP by TGF-beta1. These findings raise the possibility that Smad signalling plays a role in dentinogenesis.

[Primary culture of human periodontal ligament fibroblasts by explants with enzymatic digestion].

OBJECTIVE: To increase the success rate of primary culture of human periodontal ligament fibroblasts (HPLF), and to establish an experimental model for studying HPLF in vitro. METHODS: The primary cells were isolated from human periodontal ligament by explants with enzymatic digestion method. Morphological analysis and immunocytochemical staining were used to characterize the cell lineage, and growth curve assay to evaluate the biological features of HPLF. RESULTS: The success rate of primary culture of HPLF was 77.8%. Cultured cells were spindle-shaped, and had a positive reaction to antibodies against vimentin, and a negative reaction to antibodies against keratin. Their morphological and biological characteristics were similar to those of typical HPLF. Growth of HPLF obtained by this method was satisfactory. CONCLUSION: The success rate of primary culture of HPLF is significantly increased by explants combined with enzymatic digestion. The method is simple and feasible.

[Expression of matrixmetalloproteinase-8 on the bell-stage in human and rat tooth development].

OBJECTIVE: To study the expression of MMP-8 in human and rat tooth development. METHODS: Immunohistochemistry was used to detect the localization of MMP-8 protein while in situ hybridization was used to examine the expression of MMP-8 mRNA. RESULTS: The expression of MMP-8 protein was localized in odontoblast and dentin matrix at the later bell stage in human tooth germ. The dentin was denser close to the pulp cavity. The expression of MMP-8 mRNA was found in very few polarized odontoblast at the early bell stage and all polarized odontoblast at the later bell stage in rat tooth germ. CONCLUSION: The results suggested that MMP-8 involved in dentin matrix rebuilding in the process of dentin formation in human and rat dental development.

[The roles of Smad 2/3 in transforming growth factor-beta 1 signaling in human dental pulp cells].

OBJECTIVE: To explore the roles of Smad 2/3 in transforming growth factor-beta(1) (TGF-beta(1)) signaling by human dental pulp cells. METHODS: Laser scanning confocal microscope was used to observe translocation of Smad 2/3 from plasma into nucleus in cultured dental pulp cells at early stage of TGF-beta(1) treatment, and changes of Smad 2/3 protein expression at later stage were evaluated by Western blot analyses. RESULTS: The expression of Smad 2/3 (fluorescence intensity) kept decreasing in cytoplasm but increasing in nucleus within 2 h after TGF-beta(1) treatment, forming a trend that Smad 2/3 translocated into nucleus from cytoplasma. The total amount of Smad 2 protein remained unchanged before and after TGF-beta(1) treatment, but the expression level of Smad 3 decreased markedly after 24 h treatment and kept dropping by 48 h. CONCLUSIONS: The results suggest that the Smad 2/3 may be the downstream signal transducers of TGF-beta(1) in human dental pulp cells and Smad 2/3 may mediate TGF-beta(1) signaling by translocation early in TGF-beta(1) treatment, while down-regulation of Smad 3 expression by TGF-beta(1) at later stage is involved in negative modulation of TGF-beta(1) signaling.