Bacitracin upregulates mbrAB transcription via mbrCD to confer bacitracin resistance in Streptococcus mutans.

Streptococcus mutans is a bacterial cause of dental caries that is resistant to bacitracin. The aim of this study was to elucidate the mbrABCD-related bacitracin resistance mechanism of S. mutans. Transcriptome data demonstrated that the expression levels of 33 genes were induced more than twofold by bacitracin. Fourteen genes were selected from the upregulated genes, and defective mutants of these genes were constructed for measurement of their sensitivity to bacitracin. Among the mutants, only the mbrA- or mbrB-deficient mutants exhibited 100- to 121-fold greater sensitivity to bacitracin when compared with the wild-type strain. Moreover, knockout of the mbrC and mbrD genes abolished the bacitracin-induced mbrAB upregulation. These results suggest that both mbrC and mbrD are required for mbrAB upregulation that confers the bacitracin-resistant phenotype on S. mutans.

bFGF rescues imatinib/STI571-induced apoptosis of sis-NIH3T3 fibroblasts.

PDGF-B-transfected, sis-NIH3T3 fibroblasts serve as a model system for examining the role of PDGF signaling in tumors. We have found that imatinib/STI571, a tyrosine kinase inhibitor targeting PDGF receptors, induces apoptosis of sis-NIH3T3 fibroblasts cultured under serum free conditions, which was rescued by the addition of 10% newborn calf serum (NCS). Therefore, growth factors included in serum were tested with regard to their ability to rescue imatinib-induced apoptosis. While PDGF-AB, EGF, and IGF-I failed to protect imatinib-induced sis-NIH3T3 cell apoptosis, bFGF rescued it. The effects of bFGF were confirmed by both cell viability assays and Bax/Bcl-2 gene expression ratio. An FGF receptor inhibitor, PD166866, invalidated the protective effect of bFGF. However, combination of imatinib and PD166866 failed to induce cell death of sis-NIH3T3 cells when cultured in 10% NCS. These results indicate that synergistic administration of some types of tyrosine kinase inhibitors need to be tested under in vivo-like conditions to establish novel strategies in anti-cancer therapy.

Effects of lactoferrin on the differentiation of pluripotent mesenchymal cells.

Lactoferrin accelerates bone formation, but the precise cellular mechanism behind this is still unclear. We examined the effect of lactoferrin on the differentiation of pluripotent mesenchymal cells using a typical pluripotent mesenchymal cell line, C2C12. Cells were cultured in low-mitogen differentiation medium to induce cell differentiation, with or without the addition of lactoferrin. The cell lineage was determined by alkaline phosphatase (ALPase) activity, mRNA expression of cellular phenotype-specific markers using real-time polymerase chain reaction (PCR), and protein synthesis using Western blotting. The expression of low-density lipoprotein lipase receptor-related proteins (LRPs) 1 and 2, both lactoferrin receptors, was determined by reverse transcription-PCR. ALPase activity increased after the addition of lactoferrin. The mRNA expression of Runx2, osteocalcin, and Sox9 increased markedly as a result of lactoferrin treatment, whereas the expression of MyoD, desmin, and PPARgamma decreased significantly. Western blots showed that lactoferrin stimulation increased Runx2 and Sox9 proteins, whereas it decreased MyoD and PPARgamma synthesis. C2C12 cells expressed the LRP1 lactoferrin receptor. These results indicate that lactoferrin treatment converts the differentiation pathway of C2C12 cells into the osteoblastic and chondroblastic lineage.

The oral health of children in a rural area of the Lao People's Democratic Republic.

The lack of information on oral health in Laos makes it difficult to estimate the need and methods for preventing oral disease. This study identified problems concerning the oral health of Lao children. The study subjects were 59 school children who lived in Pakkading District. Dental caries, gingivitis malocclusions, temporomandibular joint (TMJ) disorders, dental plaque, and calculus were examined. We observed an average of 1.6 decayed, missing, and filled teeth (DMFT) and 4.1 decayed and filled deciduous teeth (dft) per child. 25.4% had gingivitis scores from 16 to 20 on the papillary, marginal, and attached (PMA) index; 29.6% had one or more occlusal abnormality; and 0% had signs of TMJ disorders. 93.5% of the children had at least one buccal or lingual tooth surface with plaque covering more than two thirds of the surface; 32.6% had dental calculus. Oral health promotion programs for children should prioritise prevention and treatment of caries. It is likely that the high rate of gingivitis in Lao children is due mainly to unsuccessful plaque control in daily life. In addition to descriptive epidemiological studies of dental diseases in other areas, the influence of sociological and behavioural factors on oral health should be analyzed epidemiologically to promote child health.

In vitro characterization of the cytokine profile of the epithelial cell rests of Malassez.

BACKGROUND: The epithelial cell rests of Malassez (ERM) are an integral part of the periodontal ligament and are considered to play an important role in dental pathology. Surprisingly, this cell type is poorly described and is often disregarded in the context of periodontal research. The aim of this study was to establish primary cell cultures of human ERM, characterize the cytokine profile, and compare it to other periodontal cell entities. METHODS: ERM-derived epithelial cells were isolated from the periodontal ligament of three subjects. A cytokine antibody array, including 120 cytokines in two membranes, was used to determine the cytokine profile of conditioned medium from the ERM-derived epithelial cells. The results were compared to those of gingival epithelial cells and periodontal ligament fibroblasts. RESULTS: ERM-derived epithelial cells expressed 29 of 120 cytokines in significant amounts, including cytokines, chemokines, growth factors, and related proteins, such as interleukin (IL)-1, -6, -8, and -10; granulocyte macrophage-colony stimulating factor; monocyte chemoattractant protein (MCP)-1, -2, and -3; amphiregulin; glial-derived neurotrophic factor; vascular endothelial growth factor; and insulin-like growth factor binding protein-2. The cytokine profile of ERM cells was similar to that of gingival epithelial cells but strikingly different from the profile of periodontal ligament fibroblasts. CONCLUSIONS: The results indicated that, via paracrine secretion of a variety of soluble factors, the ERM cells actively take part in the homeostasis of the periodontium. Therefore, future research on the pathophysiology of periodontal tissue should include this often overlooked cell type.

Profiling of radicular cyst and odontogenic keratocyst cytokine production suggests common growth mechanisms.

The aim of this study was to compare the cytokine expression profiles of cyst fluids (CFs) and tissue culture supernatants (SUPs) from 7 radicular cysts (RCs) and 7 odontogenic keratocysts (OKCs) by using Human Cytokine Antibody Array to identify the specific cytokines involved in formation and expansion of RCs and OKCs, respectively. There were significant differences in relative expression levels of IL-1 beta, MCP1, MIP1 beta, FGF-9, GDNF, HGF, IGFBP-3, Ang, IP-10, MIF, OPG, and TGF-beta2 between RC-CF and OKC-CF (P < .05). On the other hand, the cytokine expression patterns of RC-SUP (HGF, IL-8, NAP-2, IL-6, TIMP-1 and 2, GRO, IP-10, and Ang) were similar to those of OKC-SUP. Only the relative expression level of GRO differed between RC-SUP and OKC-SUP (P < .05). The similarities of cytokine production by tissue cultures derived from RC and OKC indicate that the expansion mechanisms of RC and OKC might involve similar biologic mechanisms other than infection.

Compressive force stimulates the expression of osteogenesis-related transcription factors in ROS 17/2.8 cells.

OBJECTIVE: To determine how compressive force affects the expression of osteogenesis-related transcription factors in osteoblasts. DESIGN: Cells of ROS 17/2.8, a typical osteoblastic cell line, were cultured with or without continuous compressive force (0.5-2.0 g/cm(2)). Expression of mRNA encoding the osteogenesis-related transcription factors Runx2, Osterix, Msx2, Dlx5 and AJ18 was measured using real-time polymerase chain reaction. Protein expression of these transcription factors was determined by Western blotting. RESULTS: A compressive force of 1.0 g/cm(2) significantly increased mRNA and protein expression of Runx2, Osterix, Msx2 and Dlx5, which are critical for osteoblast differentiation. In contrast, mRNA and protein expression of AJ18, which downregulates osteoblast differentiation, were decreased with 1.0 g/cm(2) of compressive force. CONCLUSIONS: A compressive force of 1.0 g/cm(2), which was considered optimal for bone formation under the present experimental conditions, stimulates osteoblastic differentiation via the modulation of osteogenesis-related transcription factors.

Lactoferrin suppress the adipogenic differentiation of MC3T3-G2/PA6 cells.

Lactoferrin accelerates the differentiation of osteogenic and chondrogenic lineage cells, whereas it inhibits the myogenic and adipogenic differentiation of pluripotent mesenchymal cells; however, the effect of lactoferrin on the differentiation of preadipocytes is unknown. In this study, we examined the effect of lactoferrin on adipogenic differentiation using a mouse preadipocyte cell line, MC3T3-G2/PA6. The cells were cultured in differentiation medium with or without lactoferrin to induce cellular differentiation. The cell lineage was then determined by Oil Red O staining, real-time PCR screening for the mRNA expression of phenotype-specific markers, and Western blot analysis. The number of Oil Red O-positive lipid droplets decreased following treatment with lactoferrin, as did the mRNA expression of C/EBPalpha, PPARgamma, aP2, and adiponectin. Furthermore, our Western blot data revealed a decrease in PPARgamma expression attributable to lactoferrin exposure. These results suggest that lactoferrin suppresses the adipogenic differentiation of MC3T3-G2/PA6 cells.

Effects of compressive force on the differentiation of pluripotent mesenchymal cells.

The purpose of this study was to determine the effect of mechanical stress on the differentiation of the pluripotent mesenchymal cell line C2C12. C2C12 cells were cultured continuously under compressive force (0.25-2.0 g/cm(2)). After mechanical stress loading, the levels of expression of mRNAs and proteins for phenotype-specific markers of osteoblasts (Runx2, Msx2, Dlx5, Osterix, AJ18), chondroblasts (Sox5, Sox9), myoblasts (MyoD), and adipocytes (PPAR gamma) were measured by real-time polymerase chain reaction analysis and Western blot analysis, respectively. The expression of activated p38 mitogen-activated protein kinase (p38 MAPK) was measured by Western blotting and/or ELISA. Loading 0.5 g/cm(2) of compressive force significantly increased the expression levels of Runx2, Msx2, Dlx5, Osterix, Sox5, and Sox9. In contrast, the expression levels of AJ18, MyoD, and PPAR gamma were decreased by exposure to 0.5 g/cm(2) of compressive force. Loading 0.5 g/cm(2) of compressive force also induced the phosphorylation of p38 MAPK. SB203580, which is a specific inhibitor of p38 MAPK, inhibited the compressive force-induced phosphorylation of p38 MAPK and partially blocked compressive force-induced Runx2 mRNA expression. These results demonstrate that compressive force stimulation directs the differentiation pathway of C2C12 cells into the osteoblast and chondroblast lineage via activated phosphorylation of p38 MAPK.

Low-intensity pulsed ultrasound stimulates osteogenic differentiation in ROS 17/2.8 cells.

There have been no studies investigating the effects of the mechanical stimulation provided by Low-intensity pulsed ultrasound (LIPUS) treatment on periodontal disease accompanying bone loss. LIPUS is known to accelerate mineralization and bone regeneration, but the precise cellular mechanism is unclear. Here, we investigated the effect of LIPUS on osteogenesis by examining the effect of LIPUS stimulation on cell proliferation, alkaline phosphatase (ALPase) activity, osteogenesis-related gene expression, and mineralized nodule formation in a rat osteosarcoma cell line. The cells were cultured in medium with or without the addition of LIPUS stimulation. The ultrasound signal consisted of 1.5 MHz at an intensity of 30 mW/cm(2) for 20 min for all cultures. LIPUS stimulation did not affect the rate of cell proliferation. ALPase activity was increased at day 7 of culture after LIPUS stimulation. Real-time PCR analysis indicated that LIPUS significantly increased the expression of mRNA for the transcription factors Runx2, Msx2, Dlx5, and Osterix and for bone sialoprotein, whereas the mRNA expression of AJ18 was significantly reduced. The mineralized nodule formation and the calcium content in mineralized nodules were markedly increased on day 14 of culture after LIPUS stimulation. Our study demonstrates that LIPUS stimulation directly affects osteogenic cells, leading to mineralized nodule formation. In view of the widespread use of LIPUS for the clinical therapy of periodontal disease, it is likely that LIPUS has an important influence on key functional activities of osteoblasts in alveolar bone.

Effects of low-intensity pulsed ultrasound on the differentiation of C2C12 cells.

Low-intensity pulsed ultrasound (LIPUS) is known to accelerate bone regeneration, but the precise cellular mechanism is still unclear. The purpose of this study was to determine the effect of LIPUS on the differentiation of pluripotent mesenchymal cell line C2C12. The cells were cultured in differentiation medium with or without the addition of LIPUS stimulation. The ultrasound signal consisted of 1.5 MHz at an intensity of 70 mW/cm2 for 20 min for all cultures. To verify the cell lineage after LIPUS stimulation, mRNA expression of cellular phenotype-specific markers characterizing osteoblasts (Runx2, Msx2, Dlx5, AJ18), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (C/EBP, PPARgamma) was studied using real-time polymerase chain reaction analysis. The protein expression of Runx2 and activated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) were performed using Western blotting. The mRNA expression of Runx2, Msx2, Dlx5, AJ18, and Sox9 was increased markedly by the LIPUS stimulation, whereas the expression of MyoD, C/EBP, and PPARgamma was drastically decreased. In the Western blot analysis, LIPUS stimulation increased Runx2 protein expression and phosphorylation of ERK1/2 and p38 MAPK. Our study demonstrated that LIPUS stimulation converts the differentiation pathway of C2C12 cells into the osteoblast and/or chondroblast lineage via activated phosphorylation of ERK1/2 and p38 MAPK.

Optimal compressive force induces bone formation via increasing bone morphogenetic proteins production and decreasing their antagonists production by Saos-2 cells.

Orthodontic tooth movement induced alveolar bone resorption and formation around the teeth applied mechanical force. Although mechanical force can promote bone formation, the molecular mechanism that underlies this phenomenon is not fully understood. The purposes of this study were to determine how mechanical stress affects the osteogenic response of human osteoblastic cells (Saos-2), and also to examine the optimal compressive force for osteogenesis in vitro. Saos-2 cells were cultured with or without continuously compressive force (0.5-3.0 g/cm2). The expression of bone morphogenetic proteins (BMPs), their antagonists, and transcription factors which involved in osteogenesis were measured using real-time PCR and/or Western blot analysis. Phosphorylation of Smad1 was determined by Western blot. Loading with 1.0 g/cm2 of compressive force significantly increased the expression of BMPs, Runx2 and osterix. In contrast, the expression of BMP antagonists and AJ18 was decreased with 1.0 g/cm2 of compressive force. Loading with 1.0 g/cm2 of compressive force also induced phosphorylation of Smad1. Noggin inhibited the compressive force-induced phosphorylation of Smad1 markedly, and also partially blocked compressive force-induced Runx2 mRNA expression. Moreover, the conditioned medium from 1.0 g/cm2 of compressive force applied cells apparently increased calcium content in mineralized nodules of Saos-2 culture. This study demonstrates that an optimal compressive force stimulates in vitro mineralization via increasing BMPs production and decreasing their antagonists production.

Effect of compressive force on the expression of MMPs, PAs, and their inhibitors in osteoblastic Saos-2 cells.

Bone matrix turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), and the plasminogen activation system, including tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor type-1 (PAI-1). We previously demonstrated that 1.0g/cm(2) of compressive force was an optimal condition for inducing bone formation by osteoblastic Saos-2 cells. Here, we examined the effect of mechanical stress on the expression of MMPs, TIMPs, tPA, uPA, and PAI-1 in Saos-2 cells. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and with or without continuously compressive force (0.5-3.0g/cm(2)) for up to 24h. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 gene expression were estimated by determining the mRNA levels using real-time PCR, and the protein levels were determined using ELISA. The expression levels of MMP-1, MMP-2, MMP-14, and TIMP-1 markedly exceeded the control levels at 1.0g/cm(2) of compressive force, whereas the expression levels of MMP-3, MMP-13, TIMP-2, TIMP-3, TIMP-4, tPA, uPA, and PAI-1 markedly exceeded the control levels at 3.0g/cm(2). These results suggest that mechanical stress stimulates bone matrix turnover by increasing these proteinases and inhibitors, and that the mechanism for the proteolytic degradation of bone matrix proteins differs with the strength of the mechanical stress.

Profiling the cytokines in gingival crevicular fluid using a cytokine antibody array.

BACKGROUND: Various compounds have been detected in gingival crevicular fluid (GCF) as indicators of periodontal disease activity. Therefore, the analysis of GCF may be especially beneficial for diagnosing current periodontal status and addressing the effects of treatment. Moreover, the identification of new markers in GCF may also contribute to elucidating novel mechanisms involved in periodontal disease. This study sought novel marker proteins specific to chronic periodontitis by profiling cytokines in GCF using a cytokine antibody array system. METHODS: Human cytokine array V, which detects 79 cytokines on one membrane, was used to determine the profile of cytokines in GCF from seven subjects with chronic periodontitis and seven subjects with healthy periodontia. The profile was exposed to x-ray film and quantified using image analysis software. Healthy and diseased sites were compared statistically. RESULTS: We detected 10 cytokines in periodontally healthy sites and 36 cytokines in periodontally diseased sites. Interleukin-8 (IL-8) and transforming growth factor-beta 2 (TGF-beta2) were detected at high levels in healthy and diseased subjects. There were significant differences between healthy and diseased subjects in the levels of tissue inhibitor of metalloproteinases-2 (TIMP-2), tumor necrosis factor-beta (TNF-beta), growth-related oncogene (GRO), interferon-inducible protein-10 (IP-10), angiogenin (Ang), vascular endothelial growth factor (VEGF), insulin-like growth factor binding protein-3 (IGFBP-3), osteoprotegerin (OPG), epidermal growth factor (EGF), glial-derived neurotrophic factor (GDNF), pulmonary and activation-regulated chemokine (PARC), oncostatin M (OSM), fibroblast growth factor-4 (FGF-4), IL-16, homologous to lymphotoxins (LIGHT), and placenta growth factor (PlGF). Of these, the newly detected cytokines were GRO, Ang, IGFBP-3, GDNF, PARC, OSM, FGF-4, IL-16, LIGHT, and PlGF. CONCLUSIONS: In this study, we detected several cytokines in GCF using a cytokine antibody array system, including both inflammatory cytokines and various growth factors. Therefore, periodontal disease may participate in the wound healing process and in tissue destruction via the inflammatory process. Our results suggest that the quantification of these cytokines in GCF provides useful information for the diagnosis of periodontal disease status.

Optimal compressive force induces bone formation via increasing bone sialoprotein and prostaglandin E(2) production appropriately.

Although orthodontic tooth movement can promote bone formation, the molecular mechanism that underlies this phenomenon is not fully understood. The purposes of this study were to determine how mechanical stress affects the osteogenic response of human osteoblastic cells (Saos-2), and also examine the optimal compression for osteogenesis in vitro. Saos-2 cells cultured with or without continuously compressive force (0.5 approximately 3.0 g/cm(2)). The expression of bone sialoprotein (BSP), osteopontin, and cyclooxygenase-2 (COX-2) were measured using real-time PCR, Western blot analysis and immunoassay. The calcium content in the mineralized nodules was determined using Calcium C-Test kit. Only one loading with 1.0 g/cm(2) of compressive force significantly increased the expression of BSP mRNA and protein, COX-2 mRNA expression and PGE(2) synthesis. Indomethacin, an inhibitor of PGE(2) synthesis, inhibited the compression-induced above phenomenon. Moreover, the conditioned medium from 1.0 g/cm(2) of compressive force apparently stimulated calcium content in mineralized nodules. This study demonstrates that an optimal compressive force stimulates in vitro mineralization by BSP synthesis through the autocrin action of PGE(2) production.

Enamel matrix derivative is a potent inhibitor of breast cancer cell attachment to bone.

This study examined whether enamel matrix derivative (EMD) inhibits the adhesion of cancer cells to bone. A typical breast cancer cell line, MCF-7, was used. Conditioned human osteosarcoma cell (Saos-2) medium was used as extracellular bone matrix (ECBM) to measure cell attachment. MCF-7 cells were incubated on ECBM-coated culture plates with or without soluble EMD, Arg-Gly-Asp (RGD) sequence blocking peptides, recombinant bone sialoprotein (rBSP), or specific integrin antibodies, and the attached cells were quantified using toluidine blue staining. EMD markedly reduced the attachment of MCF-7 cells to ECBM in a dose-dependent manner. An RGD peptide (GRGDSP) and recombinant BSP inhibited cell attachment to the same degree as EMD. Similarly, anti-alphavbeta3 integrin antibody strongly reduced cell attachment, whereas anti-alphavbeta5 and anti-beta1 integrin antibodies had less marked effects on cell attachment. These results show that EMD inhibits MCF-7 cell attachment to a bone matrix and that it might be useful as an anti-adhesive agent for breast cancer cells to bone in vivo.

Enamel matrix derivative stimulates core binding factor alpha1/Runt-related transcription factor-2 expression via activation of Smad1 in C2C12 cells.

BACKGROUND: Although enamel matrix derivative (EMD) can promote osteogenic differentiation of the pluripotent mesenchymal precursor cell line, C2C12, the molecular mechanism that underlies this phenomenon is unclear. The purpose of this study was to determine which molecules in EMD stimulate osteogenic differentiation. METHODS: C2C12 cells were cultured in 5% serum-containing medium to induce differentiation, either with or without the addition of EMD. The expression of core binding factor alpha1/runtrelated transcription factor-2 (Cbfa1/Runx2) was measured using Northern blot, Western blot, and/or real-time polymerase chain reaction (R-PCR) analysis. Phosphorylation of mothers against decapentaplegic homolog 1 (Smad1) and bone morphogenetic protein (BMP)-like molecules in EMD was determined by Western blot. RESULTS: EMD increased Cbfa1/Runx2 mRNA and protein expression substantially. EMD also induced phosphorylation of Smad1. Noggin inhibited the EMD-induced phosphorylation of Smad1 markedly, and also partially blocked EMD-induced Cbfa1/ Runx2 mRNA expression. In the Western blot analysis, single bands that corresponded to approximately 15 and approximately 17.5 kDa proteins were recognized in EMD by anti-BMP-2/4 and anti-BMP-7 antibodies, respectively. CONCLUSIONS: Our study demonstrates that EMD stimulates Cbfa1/Runx2 expression and the phosphorylation of Smad1, and that both of these processes can be blocked by noggin. Therefore, the osteogenic activity of EMD may be mediated by BMPlike molecules in EMD.

Assessment of quality and interpretation of panoramic radiographs obtained in the Lao People's Democratic Republic as part of a teleradiology collaboration with Japan.

As part of quality assessment of a teleradiology program we evaluated the validity of patient information received, the quality of panoramic radiography imaging in Laos, and the ability of a Laotian radiologist to detect temporomandibular joint abnormalities. The amount of patient information gathered from 2,021 scans of panoramic radiographs was evaluated by triage before image diagnosis. Among the radiographs from 2,021 patients, primary triage indicated that there was insufficient information for 794 (39.3%) patients. Secondary triage to assess imaging failure included 1,227 radiographs, four of which were excluded from imaging diagnosis because of unacceptable image flaws. In total, 2,446 joints from 1,223 radiographs were evaluated for temporomandibular joint abnormalities in order to compare the image interpretation abilities of Laotian and Japanese radiologists. The kappa coefficient was 0.836 (P < 0.01) for the agreement between the two observers in detecting temporomandibular joint abnormalities on radiographs. We conclude that additional efforts are needed in order to overcome the challenges of maintaining quality in imaging techniques and diagnoses in Laos.

Effect of beta-alanyl-L-histidinato zinc on the differentiation of C2C12 cells.

Although beta-alanyl-L-histidinato zinc (AHZ) can promote osteoblast differentiation, the molecular mechanism responsible is not fully understood. The purpose of this study was to determine the effect of AHZ on undifferentiating mesenchymal cells. C2C12, a typical pluripotential mesenchymal cell line, was used. The cells were cultured in 5% serum-containing medium to induce differentiation, either with or without the addition of AHZ. Cell lineage was determined by immunostaining of type II myosin heavy chains, alkaline phosphatase (ALPase) activity, mRNA expression of cellular phenotype-specific markers using semi-quantitative reverse transcriptase-polymerase chain reaction, and core binding factor alpha1/runt-related transcription factor-2 (Cbfa1/Runx2) protein synthesis using Western blot analysis. C2C12 cells cultured in the presence of AHZ were strongly inhibited from developing into myoblasts, and showed high ALPase activity that was approximately double that in the vehicle. The expression of mRNA for Cbfa1/Runx2, ALPase, Sox9 and type X collagen was increased markedly by the AHZ-stimulated medium, whereas that of desmin and MyoD mRNA was drastically decreased. AHZ increased Cbfa1/Runx2 protein expression substantially. These results provide clear evidence that AHZ converts the differentiation pathway of C2C12 cells to the osteoblast and/or chondroblast lineage.

Effect of beta-alanyl-L-histidinato zinc on the differentiation pathway of human periodontal ligament cells.

We examined the effect of beta-alanyl-L-histidinato zinc (AHZ) on the differentiation of human periodontal ligament (HPDL) cells. HPDL cells were cultured with alpha-minimum essential medium containing 10% fetal bovine serum with or without 10(-4) or 10(-5) M AHZ for up to 10 days. The expression of runt-related transcription factor-2/core binding factor alpha-1 (RUNX2/Cbfa1), Sox9, bone morphogenetic proteins (BMPs), and BMP receptors was measured using semiquantitative reverse transcription-polymerase chain reactions. Phosphorylation of Smad1 was determined using a Western blot analysis. RUNX2/Cbfa1 expression increased markedly in cells cultured with AHZ, while Sox9 expression increased slightly. BMP-7 expression was much higher than that of controls in cultures with AHZ, whereas BMP-2 expression was only slightly higher. The expression of BMP receptors increased markedly in cells cultured with AHZ. The phosphorylation of Smad1, a signal-transducing molecule for BMP-2 and BMP-7, was increased markedly in cultures with AHZ. The results suggest that AHZ diverts the differentiation pathway of HPDL cells to the osteoblast lineage via BMP-2 or BMP-7.