Effect of insulin-like growth factor on the proliferation and early stage osteogenesis of human periodontal ligament stem cells under three-dimensional culture system / 中华口腔医学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

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 / 华西口腔医学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>IGF-I significantly promotes the proliferation and ALP activity of hPDLCs under 3D culture system.</p>

Protective effect of resveratrol on apoptosis of human periodontal ligament cells in vitro / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of resveratrol (RES) on apoptosis of human periodontal ligament cells (HPLC).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>

Transcriptional regulation of dentin sialophosphoprotein by c-Jun/c-Fos / 华西口腔医学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>These findings suggest c-Jun and c-Fos down-regulated the transcription of DSPP gene as a transcriptional factor in odontoblast.</p>

Tissue engineering of dentin-pulp complex-like structures by human dental mesenchymal cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To establish three-dimensional culture model of human dental mesenchymal cells and bioengineer in vivo with ceramic bovine bone (CBB) and Collagraft as scaffolds.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>Dentin-pulp complex-like structures could be bioengineered successfully with human dental mesenchymal cells and CBB or Collagrafts containing growth factors in nude mice.</p>

Bone morphogenetic protein-2-induced alpha 2 (I) collagen expression in odontoblastic MDPC-23 cells mediated by Smad proteins / 中华口腔医学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Expression of matrixmetalloproteinase-8 on the bell-stage in human and rat tooth development / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the expression of MMP-8 in human and rat tooth development.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The results suggested that MMP-8 involved in dentin matrix rebuilding in the process of dentin formation in human and rat dental development.</p>

The roles of Smad 2/3 in transforming growth factor-beta 1 signaling in human dental pulp cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To explore the roles of Smad 2/3 in transforming growth factor-beta(1) (TGF-beta(1)) signaling by human dental pulp cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

An immunohistochemical study of the effects of excessive fluoride on type I collagen in rat developmental dentine / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the effects of excessive fluoride on type I collagen in rat developmental dentine.</p><p><b>METHODS</b>Eighty SD rats, 5 days old, were divided into experimental and control groups, 40 in each group. The experimental group received subcutaneous injection of 0.2% NaF every 4 days (the dose was 2 mg NaF per kg body wt). The same volume of 0.9% NaCl was used in the control. Twenty rats in each group were killed 4 days after the second and the seventh injection respectively. The expression of type I collagen was assayed with immunohistochemical technique.</p><p><b>RESULTS</b>4 out of 20 rats after two injections showed abnormal distribution of type I collagen (dense stain of collagen in the odontoblast, aggregation of collagen in the dentine and disordered arrangement of collagen in the predentine; All 20 rats after seven injections showed abnormal distribution of type I collagen.</p><p><b>CONCLUSION</b>Excessive fluoride may affect the metabolism of type I collagen in rat developmental dentine.</p>