Proliferation Of Cementoblasts Research Articles

Differential effects of resolvin D1 and resolvin E1 on cementoblast function.

Resolvins are endogenous mediators of the resolution of inflammation. They are derived from omega-3 polyunsaturated fatty acid precursors. Resolvin D1 (RvD1) and Resolvin E1 (RvE1) are the best-characterized members for actively promoting periodontal regeneration in experimental animal models. Here, we evaluated the efficacy of RvD1 and RvE1 on cementoblasts, the key cells involved in dental cementum regeneration and the attachment of the tooth to the alveolar bone. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1-1000ng/mL) of RvD1 and RvE1. Cell proliferation was measured using an electrical impedance-based real-time cell analyzer. Mineralization was evaluated with von Kossa staining. The mRNA expression of mineralized tissue-associated markers of bone sialoprotein (BSP), Type I collagen (COL I), osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RunX2), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (NF-κB) (RANK), receptor activator of NF-κB ligand (RANKL), and extracellular matrix-degrading enzymes [matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and their tissue inhibitors (TIMP-1, TIMP-2)], RvE1 receptor (ChemR23) and RvD1 receptor (ALX/PFR2), cytokines (tumor necrosis factor-alpha {TNF-α}, interleukin {IL}-1β, IL-6, IL-8, IL-10, IL-17), oxidative stress enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPX), and cyclooxygenase-2 (Cox-2)] were analyzed using quantitative polymerase chain reaction (qPCR). Both RvD1 and RvE1 (10-100ng/mL) significantly increased the proliferation of cementoblasts and mineralized nodules at all concentrations (p<0.05). RvE1 increased BSP, RunX2, and ALP compared with the RvD1 dose and time-dependently, while RvD1 and RvE1 differentially regulated COL-I. RvE1 increased OPG mRNA expression, whereas RANK-RANKL mRNA expression decreased by RvE1. MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 expressions were reduced by RvE1 compared with RvD1. Treatment of cementoblasts with RvD1 and RvE1 differentially affected cytokine and oxidative stress enzymes while significantly increasing their receptor expressions (ChemR23 and ALX/PFR2). RvD1 and RvE1 regulate proliferation, mineralization, and gene expression in cementoblasts using similar pathways while differentially affecting tissue degradation, suggesting a targeted therapeutic approach for cementum turnover during periodontal regeneration.

Nanomaterials for Periodontal Tissue Engineering: Chitosan-Based Scaffolds. A Systematic Review.

Introduction. Several biomaterials are used in periodontal tissue engineering in order to obtain a three-dimensional scaffold, which could enhance the oral bone regeneration. These novel biomaterials, when placed in the affected area, activate a cascade of events, inducing regenerative cellular responses, and replacing the missing tissue. Natural and synthetic polymers can be used alone or in combination with other biomaterials, growth factors, and stem cells. Natural-based polymer chitosan is widely used in periodontal tissue engineering. It presents biodegradability, biocompatibility, and biological renewability properties. It is bacteriostatic and nontoxic and has hemostatic and mucoadhesive capacity. The aim of this systematic review is to obtain an updated overview of the utilization and effectiveness of chitosan-based scaffold (CS-bs) in the alveolar bone regeneration process. Materials and Methods. During database searching (using PubMed, Cochrane Library, and CINAHL), 72 items were found. The title, abstract, and full text of each study were carefully analyzed and only 22 articles were selected. Thirteen articles were excluded based on their title, five after reading the abstract, twenty-six after reading the full text, and six were not considered because of their publication date (prior to 2010). Quality assessment and data extraction were performed in the twelve included randomized controlled trials. Data concerning cell proliferation and viability (CPV), mineralization level (M), and alkaline phosphatase activity (ALPA) were recorded from each article Results. All the included trials tested CS-bs that were combined with other biomaterials (such as hydroxyapatite, alginate, polylactic-co-glycolic acid, polycaprolactone), growth factors (basic fibroblast growth factor, bone morphogenetic protein) and/or stem cells (periodontal ligament stem cells, human jaw bone marrow-derived mesenchymal stem cells). Values about the proliferation of cementoblasts (CB) and periodontal ligament cells (PDLCs), the activity of alkaline phosphatase, and the mineralization level determined by pure chitosan scaffolds resulted in lower than those caused by chitosan-based scaffolds combined with other molecules and biomaterials. Conclusions. A higher periodontal regenerative potential was recorded in the case of CS-based scaffolds combined with other polymeric biomaterials and bioceramics (bio compared to those provided by CS alone. Furthermore, literature demonstrated that the addition of growth factors and stem cells to CS-based scaffolds might improve the biological properties of chitosan.

Nicotine suppresses proliferation and mineralized tissue-associated gene expressions of cementoblasts.

Previous studies reported that nicotine, which is the prominent constituent of tobacco, has negative effects on periodontium cells. However, the precise role of nicotine in cementoblast functions remains unclear. In the present study, we investigated the effects of nicotine on the functions of cementoblasts (OCCM-30) in terms of proliferation, migration, and mineralized tissue-associated gene expression. Immortalized murine cementoblasts were exposed to various concentrations (0, 10-6 , 10-5 , 10-4 , 10-3 , 10-2 , 10-1 , 1, 2.5, 5, and 10mM) of nicotine, and cementoblast proliferation was then evaluated using a real-time cell analyzer for 142 hours. Using an in vitro wound healing assay, cell migration was evaluated 2, 4, 6, and 24 hours after exposure to different concentrations of nicotine (1, 2.5, 5, and 10mM). The mRNA expressions of bone sialoprotein (BSP), collagen type I (COL-I), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), and alkaline phosphatase (ALP) were assessed in the nicotine-treated (0, 10-3 , 10-2 , 10-1 , 1, 2.5, 5, and 10mM) OCCM-30 cells by reverse transcription quantitative polymerase chain reaction at 8 and 24 hours exposure. At concentrations of 1 to 10mM, nicotine significantly reduced cementoblast proliferation (P<0.01). Exposure to nicotine at other concentrations (1, 2.5, and 5mM) significantly reduced wound healing rates, whereas nicotine at a concentration of 10mM immediately decreased the viability of OCCM-30 cells. Similar results were observed in inverted microscopy images at the highest nicotine concentrations. All concentrations of nicotine decreased the transcripts of BSP and COL-I in a dose- and time-dependent manner (P<0.001). Nicotine concentrations higher than 1mM reduced the expression of OCN, RunX2, and ALP in a time-dependent manner (P<0.001). This study indicated that nicotine inhibited the proliferation, migration, and mineralized tissue-associated gene expression of OCCM-30 cells. These findings suggest that nicotine negatively affects cementoblast function and the formation of new cementum, which is critical for new attachment.

MiR-361-3p/Nfat5 Signaling Axis Controls Cementoblast Differentiation

The development of periodontal tissue is a complex process, including cementoblast proliferation and differentiation. Emerging reports suggest that microRNAs (miRNAs) play crucial roles in gene regulatory networks governing numerous biological processes. However, how miRNAs modulate cementoblast proliferation and differentiation remains largely unknown. In a previous study, we performed miRNA microarray profiling to fully reveal the expression patterns of miRNAs involved in cementoblast differentiation. We focused on miR-361-3p, which decreased during cementoblast differentiation. Overexpression of miR-361-3p resulted in decreased cementoblast differentiation, whereas the functional inhibition of miR-361-3p yielded the opposite effect. The bioinformatics approach identified nuclear factor of activated T-cell 5 (Nfat5) as a potential target of miR-361-3p, which was further verified by dual luciferase assay. Meanwhile, the expression pattern of Nfat5 was verified both in vitro and in vivo. Furthermore, knockdown of Nfat5 mimicked the inhibitory effect of overexpressing miR-361-3p in cementoblasts. Moreover, multiple signaling pathways, including the Erk1/2, JNK, p38, PI3K-Akt, and NF-κB pathways, were notably activated, and the Wnt/ß-catenin pathway was blocked by downregulation of Nfat5 or forced expression of miR-361-3p in cementoblast differentiation. Finally, the complementary approach demonstrated that miR-361-3p regulated cementoblast differentiation via or partially via Erk1/2 and PI3K-Akt. Overall, our study elucidated that the JNK, p38, NF-κB, and Wnt/ß-catenin pathways act as balancing players in the miR-361-3p/Nfat5 signaling axis during cementoblast differentiation.

Effects of fibroblast growth factor-2 on cell proliferation of cementoblasts

Background/purposeFibroblast growth factor (FGF)-2 is known as a signaling molecule that induces tissue regeneration. Little is known about the effect of FGF-2 on cementoblasts for periodontal and periapical regeneration. The aim of this study was to investigate the effects of FGF-2 on murine immortalized cementoblast cell line (OCCM.30). Materials and methodsCell growth and proliferation was judged by using alamar blue reduction assay. Flow cytometry analysis was used to evaluate Stro-1 positive cells expression with or without FGF-2. Western blot was used to evaluate the expression of phosphorylated serine–threonine kinase Akt (p-Akt) and extracellular signal-regulated protein kinase (p-ERK) in cementoblasts. ResultsFGF-2 was found to increase cell growth in a dose-dependent manner (P<0.05). The concentration of 10 ng/mL FGF-2 enhanced cell proliferation in a time-dependent manner (P<0.05). In addition, 10 ng/mL FGF-2 significantly increased the number of Stro-1 positive cells in the first 24 hours (P<0.05). Moreover, 10 ng/mL FGF-2 was found to upregulate p-Akt and p-ERK in a time-dependent manner (P<0.05). ConclusionTaken together, FGF-2 could increase cementoblast growth, proliferation, and Stro-1 positive cells. These enhancements are associated with the upregulation of p-Akt and p-ERK expression. The application of FGF-2 may provide benefit for periodontal and periapical regeneration during the early phase of wound healing.

Effects of C-Terminal Amelogenin Peptide on Proliferation of Human Cementoblast Lineage Cells.

Extracts of enamel matrix proteins are used to regenerate periodontal tissue; amelogenin, the most abundant enamel protein, plays an important role in this regeneration. Studies have demonstrated that amelogenin fragments promote tissue regeneration, but the bioactive site of amelogenin remains unclear. This study explores the functional domain of amelogenin by investigating effects of four amelogenin species on cementoblast proliferation. Four amelogenin species based on amelogenin cleavage products were investigated: 1) recombinant human full-length amelogenin (rh174); 2) amelogenin cleavage product lacking the C-terminal (rh163); 3) amelogenin cleavage product lacking the N-terminal (rh128); and 4) the C-terminal region of rh174 (C11 peptide), which was synthesized and purified. Human cementoblast-like cell line (HCEM) cells were cultured and treated with rh174, rh163, rh128, or C11 peptide. Cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium assay and cell proliferation enzyme-linked immunosorbent assay. Mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) (MAPK-ERK) pathway was examined by Western blot analysis. Proliferation of HCEM cells was significantly enhanced on treatment with rh174, rh128, or C11 peptide. However, rh163 had no effect compared with the untreated control group. Western blot analysis revealed enhanced phosphorylated ERK1/2 signaling after addition of rh128 or C11 peptide and reduced phosphorylated ERK1/2 signaling after blocking with a specific MAPK inhibitor (U0126). C-terminal amelogenin cleavage product increased proliferation of HCEM through MAPK-ERK signaling pathway, indicating possible application of C11 peptide for periodontal tissue regeneration.

Osterix controls cementoblast differentiation through downregulation of Wnt-signaling via enhancing DKK1 expression.

Osterix (Osx), a transcriptional factor essential for osteogenesis, is also critical for in vivo cellular cementum formation. However, the molecular mechanism by which Osx regulates cementoblasts is largely unknown. In this study, we initially demonstrated that overexpression of Osx in a cementoblast cell line upregulated the expression of markers vital to cementogenesis such as osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP) at both mRNA and protein levels, and enhanced alkaline phosphatase (ALP) activity. Unexpectedly, we demonstrated a sharp increase in the expression of DKK1 (a potent canonical Wnt antagonist), and a great reduction in protein levels of β-catenin and its nuclear translocation by overexpression of Osx. Further, transient transfection of Osx reduced protein levels of TCF1 (a target transcription factor of β-catenin), which were partially reversed by an addition of DKK1. We also demonstrated that activation of canonical Wnt signaling by LiCl or Wnt3a significantly enhanced levels of TCF1 and suppressed the expression of OPN, OCN, and BSP, as well as ALP activity and formation of extracellular mineralized nodules. Importantly, we confirmed that there were a sharp reduction in DKK1 and a concurrent increase in β-catenin in Osx cKO mice (crossing between the Osx loxP and 2.3 Col 1-Cre lines), in agreement with the in vitro data. Thus, we conclude that the key role of Osx in control of cementoblast proliferation and differentiation is to maintain a low level of Wnt-β-catenin via direct up-regulation of DKK1.

Strontium promotes cementoblasts differentiation through inhibiting sclerostin expression in vitro.

Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose.

Acemannan induces cementoblast proliferation, differentiation, extracellular matrix secretion, and mineral deposition

Acemannan, a polysaccharide extract from Aloe vera gel, has been reported to induce oral wound healing and dentin regeneration in vitro and in vivo. However, the regenerative effect of acemannan on cementoblasts, cementum forming cells, has not been defined. In this study, we hypothesized acemannan would induce cementoblast proliferation, differentiation, extracellular matrix and growth factor secretion, and mineral deposition. An immortalized human cementoblast cell line (HCEM-2) was treated with acemannan in vitro. The de novo DNA synthesis, alkaline phosphatase activity, collagen type I and vascular endothelial growth factor, and osteopontin expression were determined by [3H] thymidine incorporation, biochemical assay, enzyme linked immunosorbent assay, and western blotting, respectively. In vitro mineral nodule formation was determined on day 21 of incubation using alizarin red staining. Our study revealed acemannan at concentrations of 2, 4, and 8 mg/ml significantly stimulated cementoblast proliferation approximately 2.5 fold. Acemannan also significantly enhanced alkaline phosphatase activity at day 3 and 6 of incubation. Expression of type I collagen, vascular endothelial growth factor, and osteopontin were significantly increased compared with the untreated group. At day 21, acemannan treatment markedly induced mineral deposition compared with the untreated group. These results suggest acemannan may be a possible therapeutic agent for cementum regeneration. Key words: Acemannan, cementoblast, differentiation, proliferation, mineralization.

Characterization and induction of cementoblast cell proliferation by bioactive glass nanoparticles

Cementum is a mineralized tissue that lines the surface of the tooth root enabling attachment of the periodontal ligament to the root and surrounding alveolar bone. Studies examining the mechanisms involved in the formation of root cementum have been hindered by an inability to isolate and culture the cells required for cementum production (cementoblasts). This study isolated and characterized cementoblast cells derived from rat molar periodontal ligament. It was observed that the isolated cells expressed F-Spondin, a cementoblast marker, while F-Spondin expression was not observed in the cells of other tissues such as gingival fibroblasts and osteoblasts. As expected, the isolated cementoblast cells also expressed osteocalcin (OC), bone sialoprotein (BSP), alkaline phosphatase (ALP), and type I collagen, demonstrating the presence of mineralized tissues genes in cementoblast cells. These cells showed high ALP activity and calcified nodule formation in vitro. Since cementogenesis could be a critical event for regeneration of periodontal tissues, this study investigated whether bioactive glass particles could affect the proliferation of cementoblasts since they are known to enhance osteoblast proliferation. It was found that the ionic products from bioactive glass nanoparticles increased cementoblast viability, mitochondrial activity, and induced cell proliferation. Together, these results show the characterization of cementoblast cells from rat molar periodontal ligament. Additionally, it was shown that bioactive glass nanoparticles induced cementoblast to proliferate, indicating that they could be a potential material for use in cement regeneration through tissue engineering.

17β‐Estradiol Promotes Cementoblast Proliferation and Cementum Formation in Experimental Periodontitis

Periodontitis is a chronic inflammatory condition that leads to the destruction of the tooth-supporting tissues. Its treatment includes the arrest of the inflammatory process and, in some circumstances, the restoration of the lost anatomy and function, including the formation of new cementum, periodontal ligament (PDL), and bone. With this goal, we investigated the effects of low concentrations of 17beta-estradiol on human cementoblast proliferation and its possible regenerative potential in vivo. Human cementum-derived cells obtained from a healthy human premolar were isolated and characterized by immunocytochemistry. Cell proliferation assays were performed to test the effects of 100 nM 17beta-estradiol and enamel matrix derivative (EMD). Three-wall intrabony periodontal defects were created in beagle dogs. After 1 month of plaque accumulation, 0.225 mg 17beta-estradiol impregnated in a collagen sponge was applied to randomly selected defects (test group), whereas a collagen sponge impregnated in a culture medium was applied to the control group. After 3 months, specimens were obtained, and tissue regeneration was assessed by histometric analysis. Cells spreading out from human tooth-layer explants were able to form cell colonies, produce a mineral matrix, and express osteocalcin, indicating they were cementoblast-like cells. In contrast, PDL fibroblasts did not express osteocalcin. 17beta-estradiol, but not EMD, increased the rate of human cementoblast cell proliferation in vitro by 2.5-fold. Histometric results from the treated periodontal defects revealed that 17beta-estradiol promoted the formation of 2.94 mm of new cementum, (45% of the defects) compared to 1.54 mm of new cementum in the control group (28% of the defects). Furthermore, the test group showed an inhibition of epithelial downgrowth and a gain of new connective tissue attachment. 17beta-estradiol promoted human cementoblast cell proliferation in vitro and periodontal regeneration in an experimental periodontitis model.

Role of the Wnt signaling pathway in bone and tooth

Signaling by the Wnt plays a central role in many processes during embryonic development and adult homeostasis. At least 19 types of Wnts, several families of secreted antagonists and multiple receptors have been identified. Two distinct Wnt signaling pathways, the canonical pathway and the noncanonical pathway have been described. Functional studies and experimental analysis of relevant animal models confirmed the effects of Wnt on regulation of developing mineralized tissue formation and adult homeostasis. In osteoblasts, the canonical Wnt pathway modulates differentiation, proliferation and mineralization, while it blocks apoptosis and osteoclastogenesis by increasing osteoprotegerin. Functional crosstalk between Wnt and bone morphogenetic protein signaling during osteoblastic differentiation has been reported. Recently, non-canonical Wnt signaling was shown to play a role in bone formation. The Wnt signaling pathway also plays an important role not only in tooth formation but also in differentiation and proliferation of cementoblasts and odontoblasts in the tooth. This present review provides an overview of progress in elucidating the role of Wnt signaling pathways in bone and tooth and the resulting possibilities for therapeutic potential.

Effect of the degree of clay delamination on the phase morphology, surface chemical aspects, and properties of hydrolyzable polyurethanes for periodontal regeneration

AbstractIn recent years, there has been increased interest in biodegradable polyurethanes for use in regenerative medicine because of their versatility and biocompatibility. Nevertheless, these polymers are usually produced using organic solvents that can lead to the release of toxic components. In this work, polyurethane/montmorillonite nanocomposites were designed to work as guided tissue regeneration membranes to treat periodontal diseases. Polyurethanes were synthesized in an aqueous environment. The composition, morphology, and mechanical properties of the biomaterials were evaluated. The cellular viability, proliferation, and morphology changes of rat culture cementoblasts were also investigated using a montmorillonite assay. Small‐angle X‐ray scattering, X‐ray diffraction, and infrared spectroscopy results showed that the degree of clay delamination within the polymer was able to tailor the phase morphology of the polymer, the chemical aspects of the surface, the mechanical properties, and the kinetics of hydrolysis of the materials. The produced scaffolds provided a good environment for the adhesion and proliferation of cementoblasts and thus can be considered suitable biomaterials for participating in procedures associated with periodontal regeneration. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Comparative Effect of the Ionic Products from Bioactive Glass Dissolution on the Behavior of Cementoblasts, Osteoblasts, and Fibroblasts

The cementum, a mineralized tissue lining the tooth root surface, is required for development of a functional periodontal ligament. The presence of healthy cementum is considered to be an important criterion for predictable restoration of periodontal tissues lost as a consequence of disease. Despite the importance of cementum to general oral health, very little is known about the cells responsible for the formation of cementum, cementoblasts. The aim of this study was to examine the effect of the ionic products from the dissolution of bioactive glass with 60% of silica ( BG60S ) on the behavior of cementoblasts, osteoblasts and fibroblasts. The cell viability was tested by MTT assay based on mitochondria activity of the cell and Trypan Blue assay based on membrane cell viability. The membrane cell viability measured by Trypan Blue assay showed the beneficial effect on all the cell types tested. It was observed a higher proliferation in the presence of ionic products from dissolution of BG60S when compared to control. In the MTT assay we also observed increased cell viability on all the cell types, but proliferation of cementoblasts was higher (107%) than observed for the other cells (104%) compared to control. The results from this study suggest that Cementoblasts, osteoblasts and fibroblasts are important cells on events that control the development of mineralizing and not mineralizing tissues and the investigation of the comparative behavior of these cells can be a useful experimental model. The observed effect of the bioactive glass particles on cementoblasts shows that this material is an interesting alternative to be used in composite membranes for cementum tissue engineering.

Effects of Endogenous and Exogenous Prostaglandin E2 on the Proliferation and Differentiation of a Mouse Cementoblast Cell Line (OCCM‐30)

Cementum formation is considered to be a critical event for successful regeneration of periodontal tissues. Cementoblasts share many characteristics with osteoblasts. Prostaglandin E(2) (PGE(2)) is an important local factor in bone metabolism. Although the effects of PGE(2) on osteoblasts are well known, its effects on cementoblasts have not yet been established. We examined the effects of PGE(2) on proliferation and differentiation in a mouse cementoblast cell line, OCCM-30 cells. OCCM-30 cells were treated with three concentrations of PGE(2) (10, 100, and 1,000 ng/ml). Cell number, alkaline phosphatase (ALP) activity, and expression for mineralization-related genes were determined. Osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) expression were also examined by real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The addition of PGE(2) at the highest dose used in this study suppressed cell proliferation of OCCM-30 cells. The expression of mineralization-related marker mRNA, such as type 1 collagen, ALP, bone sialoprotein (BSP), and osteocalcin (OCN), was constitutively detected in OCCM-30 cells. PGE(2) dose dependently stimulated ALP activity and BSP-mRNA expression in OCCM-30 cells at day 3. Transcripts for OPG and RANKL and the protein level of OPG in culture media were upregulated with PGE(2) stimulation. These results demonstrate that PGE(2) suppressed cementoblast proliferation but stimulated ALP activity and the BSP-mRNA level, suggesting a role of PGE(2) in controlling cementoblast differentiation, and further indicate that PGE(2) modulates RANKL and OPG expression in cementoblasts; the increase of OPG secreted from cementoblasts with PGE(2) stimulation may be essential to protect the root surface from resorption.

Establishment of cementoblast cell lines from rat cementum lining cells by transfection with temperature-sensitive simian virus-40 T-antigen gene

Defining the regulatory mechanisms promoting differentiation and proliferation of cementoblasts has not been well understood, because of the lack of cell models in vitro. To establish an in vitro cell model for the cementoblasts, extracted rat molars obtained from 8-week-old rats were used. Cells lining the root surface (cemetoblasts) were obtained by an enzymatic digestion method, and immediately immortalized by transfection of thermolabile SV40 T-antigen gene. The transfected cementum lining cell clones, RCM-C3 and -C4, were maintained for more than 200 population doublings (PD), while the original cells stopped their growth at 60 PD. Thus, immortalized cell lines decreased expression of SV40 T-antigen and subsequently cell proliferation at non-permissive temperature (39°C). Reverse-transcribed-polymerase chain reaction indicated expression of gene for type I collagen, alkaline phosphatase (ALP), osteopontin, and osteocalcin mRNA at both permissive (33°C) and non-permissive (39°C) temperatures. RCM-C4 expressed higher bone siaploprotein (BSP) mRNA than RCM-C3, and further RCM-C4 showed higher BSP mRNA at 39°C than 33°C. High ALP activity and mineralized nodule formation were observed at 39°C in both cell lines. These findings suggested that the cell lines, RCM-C3 and -C4, are useful model for studying the regulatory mechanisms of differentiation and proliferation of cementoblasts.