Osteoblastic Differentiation Of Periodontal Ligament Cells Research Articles

Enhancement of Osteoblast Differentiation Using No-Ozone Cold Plasma on Human Periodontal Ligament Cells.

Periodontitis is an inflammatory disease that leads to periodontal tissue destruction and bone resorption. Proliferation and differentiation of cells capable of differentiating into osteoblasts is important for reconstructing periodontal tissues destroyed by periodontitis. In this study, the effects of the nozone (no-ozone) cold plasma (NCP) treatment on osteoblastic differentiation in periodontal ligament (PDL) cells were investigated. To test the toxicity of NCP on PDL cells, various NCP treatment methods and durations were tested, and time-dependent cell proliferation was analyzed using a water-soluble tetrazolium salts-1 assay. To determine the effect of NCP on PDL cell differentiation, the cells were provided with osteogenic media immediately after an NCP treatment to induce differentiation; the cells were then analyzed using alkaline phosphatase (ALP) staining, an ALP activity assay, real time PCR, and Alizarin Red S staining. The NCP treatment without toxicity on PDL cells was the condition of 1-min NCP treatment immediately followed by the replacement with fresh media. NCP increased ALP, osteocalcin, osteonectin, and osteopontin expression, as well as mineralization nodule formation. NCP treatment promotes osteoblastic differentiation of PDL cells; therefore, it may be beneficial for treating periodontitis.

Autophagy facilitates type I collagen synthesis in periodontal ligament cells

Autophagy is a lysosomal protein degradation system in which the cell self-digests its intracellular protein components and organelles. Defects in autophagy contribute to the pathogenesis of age-related chronic diseases, such as myocardial infarction and rheumatoid arthritis, through defects in the extracellular matrix (ECM). However, little is known about autophagy in periodontal diseases characterised by the breakdown of periodontal tissue. Tooth-supportive periodontal ligament (PDL) tissue contains PDL cells that produce various ECM proteins such as collagen to maintain homeostasis in periodontal tissue. In this study, we aimed to clarify the physiological role of autophagy in periodontal tissue. We found that autophagy regulated type I collagen synthesis by elimination of misfolded proteins in human PDL (HPDL) cells. Inhibition of autophagy by E-64d and pepstatin A (PSA) or siATG5 treatment suppressed collagen production in HPDL cells at mRNA and protein levels. Immunoelectron microscopy revealed collagen fragments in autolysosomes. Accumulation of misfolded collagen in HPDL cells was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. E-64d and PSA treatment suppressed and rapamycin treatment accelerated the hard tissue-forming ability of HPDL cells. Our findings suggest that autophagy is a crucial regulatory process that facilitates type I collagen synthesis and partly regulates osteoblastic differentiation of PDL cells.

Effects of paraoxonase 1 on the cytodifferentiation and mineralization of periodontal ligament cells.

Single nucleotide polymorphisms (SNPs) of paraoxonase 1 (PON1) are known to be associated with the pathogenesis of osteoporosis and periodontitis. However, the effects of PON1 on the osteoblastic differentiation of periodontal ligament (PDL) cells are unclear. In this study, we examined the effects of PON1 on the osteoblastic differentiation of PDL cells, and analysed the role of PON1 SNPs on the pathogenesis of aggressive periodontitis (AgP) in the Japanese population. Human PDL (HPDL) cells were exposed to the PON1 plasmid and PON1 inhibitor, 2-hydroxyquinoline, and cultured in mineralization medium. Expression of calcification-related genes and calcified nodule formation were assessed by real-time PCR, an alkaline phosphatase (ALPase) activity assay and Alizarin red staining. Sanger sequencing was performed to evaluate whether PON1 SNPs are associated with the pathogenesis of AgP in Japanese people. During osteoblastic differentiation of HPDL cells, expression of PON1 mRNA increased in a time-dependent manner. PON1 stimulated an increase in expression of mRNA for calcification-related genes, as well as ALPase activity. In contrast, 2-hydroxyquinoline clearly inhibited the expression of calcification-related genes, ALPase activity and calcified nodule formation in HPDL cells. Moreover, there was a statistically significant difference in the minor allele frequency of PON1 SNP rs854560 between the Japanese control database and patients with AgP in the Japanese population (P=.0190). PON1 induced cytodifferentiation and mineralization of HPDL cells, and PON1 SNP rs854560 may be associated with the pathogenesis of AgP in the Japanese population.

Effect of Intermittent PTH(1–34) on Human Periodontal Ligament Cells Transplanted into Immunocompromised Mice

Residual periodontal ligament (PDL) cells in the damaged tissue are considered a prerequisite for a successful regeneration of the periodontal architecture with all its components, including gingiva, PDL, cementum, and bone. Among other approaches, current concepts in tissue engineering aim at a hormonal support of the regenerative capacity of PDL cells as well as at a supplementation of lost cells for regeneration. Here, we investigated how far an anabolic, intermittent parathyroid hormone (iPTH) administration would enhance the osteoblastic differentiation of PDL cells and the cellular ability to mineralize the extracellular matrix in an in vivo transplantation model. PDL cells were predifferentiated in a standard osteogenic medium for 3 weeks before subcutaneous transplantation into CD-1 nude mice using gelatin sponges as carrier. Daily injections of 40 μg/kg body weight PTH(1-34) or an equivalent dose of vehicle for 4 weeks were followed by explantation of the specimens and an immunohistochemical analysis of the osteoblastic marker proteins alkaline phosphatase (ALP), osteopontin, and osteocalcin. Signs of biomineralization were visualized by means of alizarin red staining. For verification of the systemic effect of iPTH application, blood serum levels of osteocalcin were determined. The osteogenic medium stimulated the expression of ALP and PTH1-receptor mRNA in the cultures. After transplantation, iPTH resulted in an increased cytoplasmic and extracellular immunoreactivity for all markers investigated. In contrast to only sporadic areas of mineralization under control conditions, several foci of mineralization were observed in the iPTH group. Blood serum levels of osteocalcin were elevated significantly with iPTH. These data indicate that the osteoblastic differentiation of human PDL cells and their ability for biomineralization can be positively influenced by iPTH in vivo. These findings hold out a promising prospect for the support of periodontal regeneration.

Disease-Associated Extracellular Matrix Suppresses Osteoblastic Differentiation of Human Periodontal Ligament Cells Via MMP-1

Fibronectin (FN) fragments found in chronic inflammatory diseases, including periodontal disease and arthritis, may contribute to tissue destruction in part via induction of matrix metalloproteinases (MMPs). We previously showed that the 120-kDa FN fragment containing the central cell binding domain (120FN) dose dependently induces MMP-1 (collagenase-1) in human periodontal ligament (PDL) cells, whereas intact FN did not elicit this response. Recently, we found that an increase in MMP-1 expression is accompanied by a decreased osteoblastic phenotype in PDL cells. We hypothesized that 120FN inhibits osteoblastic differentiation of PDL cells by inducing MMP-1. Effects of increasing concentrations of 120FN on MMP-1 expression and on osteoblastic markers were assessed in cultured PDL cells using Western blotting, qRT-PCR, and collagen degradation and alkaline phosphatase (AP) activity assays. The 120FN dose dependently increased MMP-1 expression and activity, concomitant with a decrease in AP activity. The increase in collagenase activity was largely attributed to increased MMP-1 expression. Concurrent with the decrease in AP activity, the 120FN reduced baseline and dexamethasone-induced gene expression of specific osteoblastic markers, Runx2 and osteonectin, and diminished mineralized nodule formation. Finally, siRNA inhibition of 120FN-induced MMP-1 reduced collagenase expression and rescued the AP phenotype to baseline levels. These findings suggest that disease-associated 120FN, in addition to having direct effects on tissue destruction by upregulating MMPs, could contribute to disease progression by impeding osteoblastic differentiation of osteogenic PDL cells and, consequently, diminish bone regeneration.

Endoglin is involved in BMP‐2‐induced osteogenic differentiation of periodontal ligament cells through a pathway independent of Smad‐1/5/8 phosphorylation

The periodontal ligament (PDL), a connective tissue located between the cementum of teeth and the alveolar bone of mandibula, plays a crucial role in the maintenance and regeneration of periodontal tissues. The PDL contains fibroblastic cells of a heterogeneous cell population, from which we have established several cell lines previously. To analyze characteristics unique for PDL at a molecular level, we performed cDNA microarray analysis of the PDL cells versus MC3T3-E1 osteoblastic cells. The analysis followed by validation by reverse transcription-polymerase chain reaction and immunochemical staining revealed that endoglin, which had been shown to associate with transforming growth factor (TGF)-beta and bone morphogenetic proteins (BMPs) as signaling modulators, was abundantly expressed in PDL cells but absent in osteoblastic cells. The knockdown of endoglin greatly suppressed the BMP-2-induced osteoblastic differentiation of PDL cells and subsequent mineralization. Interestingly, the endoglin knockdown did not alter the level of Smad-1/5/8 phosphorylation induced by BMP-2, while it suppressed the BMP-2-induced expression of Id1, a representative BMP-responsive gene. Therefore, it is conceivable that endoglin regulates the expression of BMP-2-responsive genes in PDL cells at some site downstream of Smad-1/5/8 phosphorylation. Alternatively, we found that Smad-2 as well as Smad-1/5/8 was phosphorylated by BMP-2 in the PDL cells, and that the BMP-2-induced Smad-2 phosphorylation was suppressed by the endoglin knockdown. These results, taken together, raise a possibility that PDL cells respond to BMP-2 via a unique signaling pathway dependent on endoglin, which is involved in the osteoblastic differentiation and mineralization of the cells.

Extracellular Signal‐Regulated Kinase 1/2 Is Involved in Ascorbic Acid–Induced Osteoblastic Differentiation in Periodontal Ligament Cells

Periodontal ligament (PDL) cells possess osteoblast-like properties and play key roles in periodontal regeneration. Previously, we have reported that ascorbic acid promotes the osteoblastic differentiation of PDL cells by modulating the type I collagen-integrin interaction. However, the signaling pathway activated following collagen-integrin interaction is still unclear. In this study, we examined the involvement of extracellular signal-regulated kinase (ERK)1/2 in the expression of osteoblastic marker genes such as the osteoblast-specific transcriptional factor runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) in PDL cells. PDL cells were cultured on a conventional or type I collagen-coated dish in the presence or absence of ascorbic acid and examined for ALP activity and osteoblastic marker genes. For detection of ERK1/2, cells were plated on a petri (non-adhesive) dish or type I collagen-coated dish, and Western blot analysis was performed. The effect of the ERK1/2 inhibitor on osteoblastic marker gene expression was examined. Ascorbic acid increased gene expression of Runx2, ALP, and OCN. A combination of ascorbic acid and type I collagen remarkably upregulated Runx2, ALP, and OCN gene expression and ALP activity. Western blot analysis revealed an increased level of ERK1/2 phosphorylation in cells plated on type I collagen. An ERK1/2 inhibitor suppressed ascorbic acid-induced ALP and OCN gene expression, whereas Runx2 was not affected in PDL cells. These results indicate that ERK1/2 is involved in ascorbic acid-induced osteoblastic differentiation during PDL cell attachment to type I collagen.

Dexamethasone's enhancement of osteoblastic markers in human periodontal ligament cells is associated with inhibition of collagenase expression

Although dexamethasone (Dex) substantially enhances the osteoblastic phenotype in osteogenic cells, including human periodontal ligament (PDL) cells, the basis for this response remains poorly understood. Since the accretion of a collagenous matrix is important for an osteoblastic response and dexamethasone is known to decrease collagenase expression, we examined whether osteoblastic differentiation mediated by Dex is linked to a decrease in collagenase expression in PDL cells. Early passage human PDL cells were exposed to Dex, or ascorbic acid (AA) or β-glycerophosphate (βGP) alone, or in various combinations in serum-free media for 3 or 5 days. Cells exposed to Dex alone or any combinations of treatments that included Dex demonstrated increased core binding factor alpha 1 (Cbfa1), alkaline phosphatase (AP), osteonectin (ON), osteopontin (OP), bone sialoprotein (BSP) and collagen I (α1) expression when compared to control cells or those exposed to AA or βGP. The induction of these osteoblastic markers was accompanied by a decrease in collagenase-1 expression. Collagenase activity showed a statistically significant strong negative relationship to Cbfa1 (Pearson's r = − 0.97), AP ( r = − 0.87), OP ( r = − 0.95) and BSP ( r = − 0.82) in 5-day cultures, and moderately strong relationship to ON ( r = − 0.74) from 3 days culture. Dex also produced a dose-dependent increase in AP that was paralleled by a decrease in collagenase activity ( r = − 0.98). Addition of collagenase inhibitors increased AP expression while concomitantly suppressing collagenase activity. Conversely, addition of exogenous collagenase decreased the AP phenotype of the cells, which was more marked in the absence then in the presence of Dex. The findings indicate that Dex enhances specific markers of osteoblastic differentiation in PDL cells by decreasing collagenase expression, and suggest that endogenous collagenase may regulate osteoblastic differentiation of these cells.

Twist negatively regulates osteoblastic differentiation in human periodontal ligament cells

Periodontal ligament (PDL) is a thin fibrous connective tissue located between two mineralized tissues, alveolar bone and cementum, which maintains a constant width physiologically. The mechanisms by which PDL resists mineralization are not well understood. Twist is a basic helix loop helix protein that plays a central role in regulation of early osteogenesis. We investigated the localization of Twist in PDL and compared the expression of Twist and osteoblast-related genes in PDL cells with those in osteoblast-like cells in the presence or absence of recombinant human bone morphogenetic protein (BMP)-2. Histochemical analysis showed that Twist was expressed along alveolar bone surface in PDL. PDL cells constitutively expressed Twist gene and the expression level was higher than that in osteoblast-like cells. In osteoblast-like cell culture, BMP-2 enhanced osteoblast-related gene expression, while Twist expression was slightly decreased. In contrast, BMP-2 increased runt-related transcription factor (Runx)-2, but failed to enhance alkaline phosphatase (ALP) and osteocalcin (OCN) gene expression in PDL cells. Interestingly, unlike in osteoblast-like cells, Twist expression was upregulated by BMP-2 in PDL cells. We transiently knocked down Twist gene in PDL cells using a short interference RNA expression vector (siTwist) and found that ALP, osteopontin (OPN), bone sialoprotein (BSP) genes expression and basal level of ALP activity were slightly increased, whereas Runx2 and OCN genes were not affected. Collectively, these results suggest that Twist may act as a negative regulator of osteoblastic differentiation in PDL cells.

Simvastatin Promotes Cell Metabolism, Proliferation, and Osteoblastic Differentiation in Human Periodontal Ligament Cells

Simvastatin is one of the cholesterol lowering drugs. Recent studies demonstrated that it has a bone stimulatory effect. Periodontal ligament (PDL) cells are believed to play an important role in periodontal regeneration; that is, they may differentiate into specific cells which make cementum, bone, and attachment apparatus. It would be of interest whether simvastatin has a positive effect on PDL cells. Therefore, effects of simvastatin on cell proliferation and osteoblastic differentiation in PDL cells were analyzed. Human PDL cells were cultured in monolayer with simvastatin for 24 and 72 hours and cell metabolism and proliferation were determined. To analyze osteoblastic differentiation, human PDL cells were cultured in organoid culture for 7, 14, and 21 days and alkaline phosphatase (ALP) activity, osteopontin (OPN), bone morphogenetic protein (BMP) -2, osteocalcin (OCN), and calcium contents were measured. They were co-treated by simvastatin and mevalonate. Simvastatin enhanced cell proliferation and metabolism dose-dependently after 24 hours. Simvastatin also stimulated ALP activity of human PDL cells dose-dependently, and maximum effect was obtained at the concentration of 10(8) M. In time dependent analysis, 10(8) M simvastatin stimulated ALP activity and osteopontin content after 7 days and calcium contents after 21 days. BMP-2 and OCN contents were not detected. Moreover this statin-enhanced ALP activity was abolished by mevalonate. These results suggest that at low concentration, simvastatin exhibits positive effect on proliferation and osteoblastic differentiation of human PDL cells, and these effects may be caused by the inhibition of the mevalonate pathway.

Role of Ascorbic Acid in Periodontal Ligament Cell Differentiation

Periodontal ligament (PDL) cells form mineralized nodules in vitro. Ascorbic acid is known to be required in this process, although its effect on osteoblastic differentiation of PDL cells remains unclear. The purpose of this study was to determine the role of ascorbic acid on the early osteoblastic differentiation of PDL cells, with regard to alkaline phosphatase (ALP) activity, type I collagen production and integrin expression. Cultured PDL cells were stimulated at confluence with ascorbic acid in the presence or absence of type I collagen inhibitor and blocking antibodies to integrins. After stimulation, the cells and culture supernatants were examined for ALP activity, type I collagen production, and integrin expression. The ALP activity was measured using a colorimetric assay with p-nitrophenyl phosphate and ALP staining. Enzyme-linked immunosorbent assay (ELISA) was used to determine type I collagen production, and ELISA and flow cytometric analysis were employed for assessment of integrin expression. Both ALP activity and type I collagen production were upregulated when PDL cells were cultured in the presence of ascorbic acid (200 microM). Inhibitor of the formation of collagen triple helices and blocking antibodies to alpha2beta1 integrin inhibited ALP activity by 50% in ascorbic acid-stimulated PDL cells. Furthermore, ascorbic acid increased the cell surface expression of alpha2beta1 integrin. Our findings indicated that ascorbic acid increases the ALP activity of PDL cells via type I collagen production and also enhances the expression of alpha2beta1 integrin, which is a major receptor of type I collagen. These results suggest that ascorbic acid promotes the osteoblastic differentiation of PDL cells by modulating type I collagen-alpha2beta1 integrin interaction.

Role of epidermal growth factor and its receptor in mechanical stress-induced differentiation of human periodontal ligament cells in vitro

The periodontal ligament (PDL) contains precursor cells for osteoblasts and cementoblasts. It has been shown that epidermal growth factor (EGF) inhibits dexamethasone-induced differentiation and up-regulates EGF-receptor (EGF-R) expression, whereas EGF-R is down-regulated in the course of differentiation. Thus it was suggested that EGF and its receptors act as a negative regulator of osteoblastic differentiation in PDL cells. In order to investigate further this hypothesis, human PDL cells were now used to elucidate the role of EGF and EGF-R in their proliferation and differentiation under mechanical stress-loaded conditions in vitro, as the PDL regularly receives mechanical stress from occlusal forces. As a model of mechanical stress, a cyclic stretch of 9 or 18% elongation was applied to the cells with a Flexercell cell-strain unit system. Alkaline phosphatase activity and osteocalcin mRNA expression were significantly induced by loading cyclic stretch for more than 4 days, whereas stretch slightly inhibited cell proliferation. Visualization of the actin stress fibres of the cells by rhodamine phalloidin revealed that approx. 10% of the total number of cells had become aligned perpendicularly to the direction of the stretch. The effects of stretch on alkaline phosphatase activity and cell proliferation were totally abolished by the presence of 10 ng/ml EGF. Western blotting of EGF-R protein demonstrated that stretch-induced differentiation accompanied the decreased expression of EGF-R protein in the cells. However, the amount of tyrosine-phosphorylated EGF-R upon EGF stimulation was restored to the control level in stretched cells. These results suggest that the EGF/EGF-R system acts as a negative regulator of differentiation of PDL cells regardless of the type of differentiation stimuli. Also, interaction between mechanical stress and the EGF/EGF-R system may participate in the osteoblastic differentiation of PDL cells and thereby regulate the source of cementoblasts and osteoblasts.

Proliferation and Differentiation of Human Osteoblastic Cells Associated with Differential Activation of MAP Kinases in Response to Epidermal Growth Factor, Hypoxia, and Mechanical Stressin Vitro

In an attempt to elucidate the specificity of pathways from environmental stress to cellular outcome via mitogen activated protein kinases (MAPKs) activation, we examined the responsiveness of cultured human osteoblastic periodontal ligament (PDL) cells to epidermal growth factor (EGF), hypoxia, and mechanical stress, in terms of cell proliferation, differentiation, and associated activation of three different types of MAPK. Cell proliferation was promoted in the presence of 10ng/ml of EGF or in hypoxic conditions (5% O2), whereas it was inhibited by cyclic stretch (9% strain, 6 cycles/min), which was used as a model of mechanical stress. Conversely, the alkaline phosphatase activity, a marker for osteoblastic differentiation of the cells, was increased by cyclic stretch but decreased by EGF and hypoxia. The mitogenic response of PDL cells to EGF or hypoxia was associated with the selective phosphorylation and activation of extracellular-related kinase (ERK) 1/2, while phosphorylation and activation of c-Jun N-terminal kinase (JNK) was observed in mechanical stretch loaded cells. No such changes were seen in p38 protein. These findings suggested that stress-responsive changes in proliferation and osteoblastic differentiation of PDL cells are selectively mediated by ERK 1/2 and by JNK, respectively, and that a balance between these two pathways determines the cell fate.