BACKGROUND AND OBJECTIVES: Adenosine diphosphate (ADP), which is usually secreted from activated platelets, may activate integrins on vascular smooth muscle cells, resulting in adhesion and proliferation. Integrins, mediating the ADP-stimulated adhesion and proliferation of vascular smooth muscle cells, was investigated in this study. MATERIALS AND METHODS: Prothrombin (PT) and bone sialoprotein (BSP) were used as activation-dependent ligands in an adhesion assay. The adhesion of human aortic smooth muscle cells (HASMC) were measured after ADP stimulation, using ligand-coated 24-well plates. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the ADP-stimulated proliferation of the HASMC.RESULTS: ADP activated the HASMC to increase their adhesion to the PT or BSP, and their proliferation in a dose-dependent manner. The adhesion of the ADP-stimulated HASMC to the PT was completely blocked by P5H9, a blocking monoclonal Ab (mAb) to integrin alphavbeta5 (92% inhibition), but was only slightly inhibited by LM609, a blocking mAb to integrin alphavbeta3 (30% inhibition). The adhesion of the ADP-stimulated HASMC to the BSP was partially inhibited by both P5H9 (46% inhibition) and JBS5, a blocking mAb to integrin alpha5beta1 (75% inhibition), but was not affected by c7E3, a blocking mAb to integrin beta3. The ADP-stimulated proliferation of the HASMC was inhibited by both c7E3 and LM609 (98% and 93% inhibition, respectively), but not by either P1F5, a blocking mAb to integrin alphavbeta5 or JBS5. CONCLUSION: These results indicate the different roles of integrins on vascular smooth muscle cells after ADP stimulation; the integrins alphavbeta5 and alpha5beta1 for adhesion, and the integrin alphavbeta3 for proliferation.
Adenosine Diphosphate ; Humans ; Integrin alpha5beta1 ; Integrin alphaVbeta3 ; Integrin beta3 ; Integrin-Binding Sialoprotein ; Integrins* ; Ligands ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Negotiating* ; Prothrombin

PURPOSE: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-E1 osteoblastic cell line, especially on the osteonectin and bone sialoprotein. MATERIALS AND METHODS: Cells were irradiated with a single dose of 0.5, 1, 4 and 8 Gy at a dose rate of 5.38 Gy/min using Cs-137 irradiator. After specimens were harvested, total RNA was extracted on the 3rd, 7th, 14th, 21st day after irradiation. The total RNA was reverse-transcribed and the resulting cDNAs were subjected to amplification by PCR with a pair of primers. RESULTS: The irradiated cells showed a dose-dependent increase in osteonectin mRNA expression when compared with the unirradiated control group. The irradiated cells showed no difference in bone sialoprotein mRNA expression when compared with the unirradiated control group. In accordance with the duration of culture period after irradiation, the level of osteonectin mRNA expression showed no difference, but it increased a little at the 21st day in the 4 and 8 Gy exposure groups. In the case of bone sialoprotein, however, the level of mRNA expression increased significantly at the 3rd and 7th day after irradiation, but it showed no difference at the 14th and 21st day when compared with the control group. CONCLUSION: These results showed that each single dose of 0.5, 1, 4 and 8 Gy influenced the mRNA expression of osteonectin and bone sialoprotein at the calcification stage of osteoblastic cells, suggesting that single dose of irradiation affected the osteoblastic bone formation at the cell level.
Cell Line* ; DNA, Complementary ; Integrin-Binding Sialoprotein* ; Osteoblasts* ; Osteogenesis ; Osteonectin* ; Polymerase Chain Reaction ; RNA ; RNA, Messenger*

OBJECTIVETo investigate the timing and location of the expression of bone sialoprotein (BSP) and osteopontin (OPN) in developing dental tissues of rats.

METHODSEvery three neonatal rats were sacrificed at day 1, weeks 1, 2, 3, 5 and 8. The mandibles were dissected and the first molar and the surrounding tissue were fixed, then demineralized with 15% EDTA. Immunohistochemical technique was used to determine the expression of BSP and OPN in dental tissues and the surrounding bone at different time points.

RESULTSImmunoreactivitis of BSP and OPN were present in matured ameloblasts, dentinoblasts, cementoblasts, osteoblasts, and the matrix. The expression of BSP and OPN in cementum and alveolar bone was stronger than that in enamel and dentine. In cementum and alveolar bone, BSP appeared to be concentrated in unmineralized and mineralized tissues, but OPN was concentrated in the mineralizing frontier and reversal line.

CONCLUSIONSBSP and OPN play an important role in the development and mineralization of rat mineralized tissues. The expression of BSP was different from OPN, indicating their different functions.

Animals ; Immunohistochemistry ; Integrin-Binding Sialoprotein ; Male ; Osteopontin ; Rats ; Rats, Sprague-Dawley ; Sialoglycoproteins ; analysis ; Tooth ; chemistry

PURPOSE: To investigate the effects of 2-deoxy-D-glucose (2-DG) and quercetin (QCT) on gene expression of bone sialoprotein (BSP) and osteocalcin (OC) during the differentiation in irradiated MC3T3-E1 osteoblastic cells. MATERIALS AND METHODS: When MC3T3-E1 osteoblastic cells had reached 70-80% confluence, cultures were transferred to a differentiating medium supplemented with 5 mM 2-DG or 10 micrometer QCT, and then irradiated with 2, 4, 6, and 8 Gy. At various times after irradiation, the cells were analyzed for the synthesis of type I collagen, and expression of BSP and OC. RESULTS: The synthesis of type I collagen in cells exposed to 2 Gy of radiation in the presence of 2-DG or QCT showed no significant difference compared with the control group within 15 days post-irradiation. When the cells were irradiated with 8 Gy, 2-DG facilitated the irradiation mediated decrease of type I collagen synthesis, whereas such decrease was inhibited by treating with QCT. During MC3T3-E1 osteoblastic cell differentiation, the mRNA expression of BSP and OC showed the peak value at 14 days and 21 days, respectively. 2-DG or QCT treatment alone decreased the level of BSP mRNA, but increased the OC mRNA level only at early time of differentiation (day 7). In the cells irradiated with 2, 4, 8 Gy, the mRNA expression of BSP and OC decreased at 7 days after the irradiation. The cells were treated with various dose of radiation in the presence of 2-DG or QCT, the mRNA level of both BSP and OC increased although this increase was observed at low dose of radiation (2 Gy) and at the early stage of differentiation. However, when the cells were exposed to 4, 6, or 8 Gy, the increase of BSP and OC mRNAs was detected only in cells co-incubated with QCT. CONCLUSION: This study demonstrates that 2-DG and QCT affect differently the expression of bone formation related factors, type I collagen, BSP, and OC in the irradiated MC3T3-E1 osteoblasic cells, according to the dose of radiation and the times of differentiation. Overall, the present findings suggest that 2-DG and QCT could have the regulatory roles as radiation-sensitizer and -protector, respectively.
Cell Differentiation ; Collagen Type I ; Deoxyglucose ; Gene Expression ; Integrin-Binding Sialoprotein ; Osteoblasts ; Osteocalcin ; Osteogenesis ; Quercetin ; RNA, Messenger

OBJECTIVETo study the influence of transfection with human transforming growth factor-beta1 (hTGF-beta1) gene on the osteogenic potential and differentiation of the cultured human gingival fibroblast (GF).

METHODSEnzyme kinetics method was used to measure the effects of the transfection on the alkaline phosphatase (ALP) activity. Immunohistochemistry stain and image analysis were applied to evaluate the alteration of the content of osteopontin (OPN), bone sialoprotein (BSP), osteonectin (ON), osteocalcin (OC) in the GF with transfection. Mineralization nodules formation in vitro was also used.

RESULTSThe ALP activity of the GF after transfection was higher than the GF without transfection significantly (P<0.05), and was close to that of the PDLCs (P>0.05). The content of OC in GF was not improved after transfection, was similar with that of PDLCs (P>0.05). Under immunohistochemistry stain, the contents of OPN, ON, BSP expressed in GF with transfection were higher than those of GF without transfection (P<0.05), but similar to those of PDLCs (P>0.05). In the mineralized cultured medium, the nodules were observed in the GF with transfection and PDLCs after 21 days and 24 days alternatively. After von Kossa stain, purple mineralization nodules were observed.

CONCLUSIONThe GF transfected with pcDNA3-hTGF-beta1 could express some osteogenic characters, though these characters are restricted.

Alkaline Phosphatase ; Cell Differentiation ; Fibroblasts ; Gingiva ; Humans ; Integrin-Binding Sialoprotein ; Osteocalcin ; Osteonectin ; Osteopontin ; Transfection ; Transforming Growth Factor beta1

OBJECTIVETo investigate the effects of Osterix (Osx) overexpression on the osteogenic differentiation of human periodontal ligament cells in response to mechanical force.

METHODSHuman periodontal ligament cells were isolated and cultured in vitro with explant method. Cells were transfected with either an Osx expression vector pcDNA3.1 flag-Osx or the mock control vector pcDNA3.1 flag. Then, cells were centrifuged for 6 h. After transfection and centrification, the expression of Osx mRNA and protein in untransfected cells, mock-transfected cells and Osx-transfected cells were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. Furthermore, the changes of mRNA expressions of core-binding factor cal (Cbfal), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OC), bone sialoprotein(BSP) and collagen protein al (Col I ) genes were measured to assess the differentiation of human periodontal ligament cells.

RESULTSAt 24 h after transfection, Osx mRNA and protein level increased significantly in Osx-transfected cells (P < 0.01), while there were no significant difference in Osx mRNA and protein levels between mock-transfected cells and untransfected cells(P > 0.05). Simultaneously, the upregulated mRNA expressions of all the five osteogenic genes were observed (P < 0.05, P < 0.01). After 6 h of mechanical stimulation, a significant increase in Osx expression was shown in all three groups. However, compared to mock-transfected and untransfected cells, Osx-transfected cells further showed the highest Osx mRNA and protein expression level. Furthermore, the mRNA expressions of all five osteogenic markers in Osx-transfected cells also exhibited the greater increase and showed the highest levels.

CONCLUSIONThe overexpression of Osx promotes the mechanical stress-induced osteogenic differentiation of human periodontal ligament cells. Osx may be essential for mechanical stress-induced differentiation of human periodontal ligament cells to osteoblas tic-like cells and be involved in orthodontic osteogenic remodeling.

Alkaline Phosphatase ; Cell Differentiation ; Cells, Cultured ; Humans ; Integrin-Binding Sialoprotein ; Osteocalcin ; Osteogenesis ; Osteopontin ; Periodontal Ligament ; RNA, Messenger ; Stress, Mechanical ; Transfection

OBJECTIVE: This study evaluated the effects of nitric oxide (NO) on the proliferation and differentiation of human periodontal ligament cells involved in orthodontic tooth movement. METHODS: A range of concentrations of sodium nitroprusside (SNP), a NO donor, were administered to samples of human periodontal ligament cells, followed by measurement of cell viability, alkaline phosphatase (ALP) activity, and expression of osteonectin and bone sialoprotein. RESULTS: Cell viability, ALP activity and the expression of osteonectin and bone sialoprotein were increased in human periodontal ligament cells treated with SNP concentrations of < 0.2 mM compared with controls, but were decreased with SNP concentrations of > 1.0 mM. CONCLUSION: NO has a biphasic effect on proliferation and differentiation in human periodontal ligament cells, with a stimulatory effect at low concentrations and an inhibitory effect at high concentrations.
Alkaline Phosphatase ; Cell Survival ; Humans* ; Integrin-Binding Sialoprotein ; Nitric Oxide* ; Nitroprusside ; Osteonectin ; Periodontal Ligament* ; Tissue Donors ; Tooth Movement

Periodontal therapy has dealt primarily with attempts at arresting progression of disease, however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. The effect of chitosan, a carbohydrate biopolymer extracted from chitin, on periodontal ligament regeneration is of particular interest. The purpose of this study was to evaluate the effect of chitosan on the expression of extracellular matrix proteins in primary rat calvarial cells in vitro. In the control group, cells was cultured with BGJb media. In the experimental groups, cells were cultured with chitosan in concentration of 0.01, 0.1, 1.0 and 2.0 mg/ml. Then each group was characterized by examining alkaline phosphatase activity at 3 and 7 days, and the ability to produce mineralized nodules of rat calvarial cells at 14 and 21 days. Synthesis of type I collagen (COL-I), osteocalcin (OCN), bone sialoprotein (BSP) was evaluated by RT-PCR at 14 days. The results were as follows: 1. Alkaline phosphatase activity was significantly higher in the concentration of chitosan 0.01mg/ml, 0.1mg/ml and 1.0mg/ml compared to control (p<0.05). 2. The percentage of mineralized bone nodule was more in the concentration of chitosan 0.1mg/ml and 1.0 mg/ml than the control. 3. At 14 day culture, the expression of OCN was increased by chitosan in concentration of 1.0 mg/ml and 2.0 mg/ml. These results suggested that chitosan in concentration of 0.1 and 1.0 mg/ml stimulate the extracellular matrix of primary rat calvarial cells and may facilitate the formation of bone.
Alkaline Phosphatase ; Animals ; Biopolymers ; Bone Matrix* ; Chitin ; Chitosan* ; Collagen Type I ; Extracellular Matrix ; Extracellular Matrix Proteins ; Integrin-Binding Sialoprotein ; Osteocalcin ; Periodontal Ligament ; Periodontium ; Rats* ; Regeneration

OBJECTIVE: To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation-mimicking agent cobalt chloride (CoCl2) on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and elucidate the underlying molecular mechanisms. STUDY DESIGN: The dose and exposure periods for CoCl2 in hMSCs were optimized by cell viability assays. After confirmation of CoCl2-induced HIF-1alpha and vascular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with CoCl2 on hMSC osteogenic differentiation were evaluated by RT-PCR analysis of osteogenic gene expression, an alkaline phosphatase (ALP) activity assay and by alizarin red S staining. RESULTS: Variable CoCl2 dosages (up to 500 microM) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After CoCl2 treatment of hMSCs at 100 microM for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocalcin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP activity was increased in these treated cells in which an accelerated osteogenic capacity was also verified by alizarin red S staining. CONCLUSIONS: The osteogenic differentiation potential of hMSCs could be preserved and even enhanced by CoCl2 treatment.
Alkaline Phosphatase ; Anoxia ; Anthraquinones ; Cell Survival ; Cobalt ; Durapatite ; Gene Expression ; Humans ; Integrin-Binding Sialoprotein ; Mesenchymal Stromal Cells ; Osteocalcin ; Osteopontin ; RNA, Messenger ; Vascular Endothelial Growth Factor A

Osteoblasts from alveolar bone may have an important role in the bone regeneration for periodontium, but their culture and characterization are not determined yet. The purpose of this study was to investigate the biological characteristics of primary explant cultured osteoblasts(PECO) from alveolar bone. Osteoblasts were isolated and cultured from alveolar socket of extracted tooth in children. To compare the characteristics, osteoblasts and gingival fibroblasts were cultured with DMEM at 37degrees C, 5% CO2, 100% humidity incubator, and human fetal osteoblasts cell line(hFOB1) were cultured with DMEM at 34degrees C, 5%, CO2, 100% humidity incubator. To characterize the isolated bone cells, morphologic change, cell proliferation and differentiation were measured. Morphology of PECO was small round body or cuboidal shape on inverted microscope and was similar with hFOB1. PECO became polygonal shape with stellate and had an amorphous shape at 9th passage in culture. PECO had significantly higher activity than that of gingival fibroblasts and hFOB1 in alkaline phosphatase activity. The expression of osteocalcin and bone sialoprotein in PECO was notably increased when compared with hFOB1 and gingival fibroblasts. These result indicated that PECO from alveolar bone in children has an obvious characteristics of osteoblast, may be applied for the regeneration of bone.
Alkaline Phosphatase ; Bone Regeneration ; Cell Proliferation ; Child ; Fibroblasts ; Humans ; Humidity ; Incubators ; Integrin-Binding Sialoprotein ; Osteoblasts* ; Osteocalcin ; Periodontium ; Population Characteristics* ; Regeneration ; Tooth