Jia Tang

Effects of amelogenin on proliferation, differentiation, and mineralization of rat bone marrow mesenchymal stem cells in vitro.

The aim of this study was to clarify the function of amelogenin, the major protein of enamel matrix derivative, on the proliferation, differentiation, and mineralization of cultured rat bone marrow stem cells (BMSCs), toward the establishment of future bone regenerative therapies. No differences in the morphology of BMSCs or in cell numbers were found between amelogenin addition and additive-free groups. The promotion of ALPase activity and the formation of mineralized nodules were detected at an early stage in amelogenin addition group. In quantitative real-time RT-PCR, mRNA expression of osteopontin, osteonectin, and type I collagen was promoted for 0.5 hours and 24 hours by addition of amelogenin. The mRNA expression of osteocalcin and DMP-1 was also stimulated for 24 hours and 0.5 hours, respectively, in amelogenin addition group. These findings clearly indicate that amelogenin promoted the differentiation and mineralization of rat BMSCs but did not affect cell proliferation or cell morphology.

Biocompatibility of Novel Type I Collagen Purified from Tilapia Fish Scale: An In Vitro Comparative Study.

Type I collagen (COL-1) is the prevailing component of the extracellular matrix in a number of tissues including skin, ligament, cartilage, bone, and dentin. It is the most widely used tissue-derived natural polymer. Currently, mammalian animals, including pig, cow, and rat, are the three major sources for purification of COL-1. To reduce the risk of zoonotic infectious diseases transmission, minimize the possibility of immunogenic reaction, and avoid problems related to religious issues, exploration of new sources (other than mammalian animals) for the purification of type I collagen is highly desirable. Hence, the purpose of the current study was to investigate the in vitro responses of MDPC-23 to type I collagen isolated from tilapia scale in terms of cellular proliferation, differentiation, and mineralization. The results suggested that tilapia scale collagen exhibited comparable biocompatibility to porcine skin collagen, indicating it might be a potential alternative to type I collagen from mammals in the application for tissue regeneration in oral-maxillofacial area.

Nephronectin Stimulates the Differentiation of MDPC-23 Cells into an Odontoblast-like Phenotype

Introduction: The present study investigated the in vitro effects of nephronectin (Npnt) on the proliferation, differentiation, and mineralization of a rat odontoblast‐like cell line (MDPC‐23 cells). Methods: MDPC‐23 cells were cultured on Npnt‐coated polystyrene or in the presence of soluble Npnt. Cell proliferation was analyzed using a Cell Counting Kit‐8 kit (Dojindo, Kumamoto, Japan). Alkaline phosphatase (ALP) activity was quantified using an ALP activity assay. A reverse‐transcription polymerase chain reaction was performed to evaluate the messenger RNA (mRNA) expression level of odontogenic markers and integrin(s). Alizarin red staining was conducted to quantify the calcium deposition. Results: Soluble Npnt had no adverse effect on the proliferation of MDPC‐23 cells, but it exhibited concentration‐dependent inhibitory activity toward differentiation. In contrast, coated Npnt promoted cell proliferation dramatically and significantly up‐regulated the mRNA expression of odontogenesis‐related genes; moreover, mRNA expression of integrin &agr;1, &agr;3, &agr;5, &bgr;1, and &bgr;5 was found to be augmented. MDPC‐23 cells cultured on Npnt‐coated polystyrene displayed markedly higher ALP activity as early as day 3 after inoculation. In addition, mineralization was accelerated on Npnt‐coated polystyrene. Conclusions: Npnt in its immobilized form enhanced the proliferation of MDPC‐23 cells and induced this odontoblastic precursor cell line to differentiate into a mineralizing phenotype.

Effect of type I collagen derived from tilapia scale on odontoblast-like cells

Type I collagen is the most abundant extracellular matrix in bone and dentin. It was reported to be effective in inducing the osteoblast differentiation. Majority of the type I collagen used in scientific research are from bovine skin, porcine skin and rat tail. Recently, a novel type I collagen possessing sound thermal stability, was isolated from tilapia fish scale. It was reported to be able to induce the differentiation of human mesenchymal stem cells into osteoblasts. However, no information is available regarding its effect on odontoblast-like cells. The purpose of this study was to investigate the effect of type I collagen derived from tilapia fish scale on odontoblast-like cells. Type I collagen was immobilized to tissue culture polystyrene dish. Biocompatibility of type I collagen was evaluated in terms of initial cell number, alkaline phosphatase activity (ALP activity), odontogenic gene expression and calcific deposition. The results showed that type I collagen purified from tilapia fish scale significantly enhanced the cell attachment to nearly twofold as compared to control; moreover, cells cultured in type I collagen-modified substrate was induced to differentiate toward odontoblast lineage as demonstrated by upregulation of ALP activity on day 7, enhancement of ALP, BSP mRNA expression on day 7 and 10, and accelerated mineralization on day 9. It is therefore suggested that type I collagen derived from tilapia scale, an underutilized resource, holds promise as scaffolding material in the application of tissue engineering in dental field.

Laminin-1 acts as an adhesive for odontoblast-like cells and promotes their differentiation toward a hard tissue-forming phenotype

The present study was designed to investigate the effect of laminin-1 (LN-1 or LN-111) on an odontoblast-like cell line, MDPC-23. Wells of non-treated polystyrene plates were coated with various concentrations of LN-1 (0.1, 1, 10, and 100 µg/mL) and left to dry for 2 days. Water-coated surfaces were used as controls. MDPC-23 cell proliferation, differentiation and mineralization were evaluated in terms of the CCK-8 assay, ALP activity, real-time RT-PCR and Alizarin red staining. The data indicated that LN-1 promoted the proliferation of MDPC-23 cells in a concentration-dependent manner. Moreover, it enhanced ALP activity and expression of key odontogenic genes (DMP-1 and DSPP) upon addition of mineralization reagents, leading to significant promotion of calcification by the cells. These results demonstrate that LN-1 acts as an adhesive for odontoblast-like cells, allowing up-regulation of odontogenic genes and accelerating matrix mineralization. In the context of the present study, the optimal LN-1 coating concentration for MDPC-23 cells was suggested to be 100 µg/mL.

Human plasma fibronectin promotes proliferation and differentiation of odontoblast

Abstract Objective To assess the effect of fibronectin (Fn) and porcine type I collagen (PCOL) on odontoblast-like cells in vitro. Material and Methods Rat odontoblast-like cells (MDPC-23 cells) were inoculated and cultured on Fn-coated or type I collagen-coated substrates. Proliferation assay, alkaline phosphatase activity (ALP activity), mRNA expression of hard tissue-forming markers, and Alizarin red staining were investigated over a period of 10 days. Results Cells maintained a high proliferation activity on Fn and PCOL even at a low seeding concentration (0.5×104/mL) as demonstrated by CCK-8 assay. The proliferation activity of cells on Fn increases in a concentration-dependent manner while it reached a plateau after 10 µg/mL. Cells adopted long, thin and spindle shape on Fn(10-50) and PCOL. Parallel actin filaments were observed in MDPC-23 cells cultured on Fn and PCOL. ALP activity was markedly up-regulated on Fn and PCOL-coated surfaces. Importantly, gene expression of BSP (Fn10: 2.44±0.32; Fn20: 3.05±0.01; Fn30: 2.90±0.21; Fn40: 2.74±0.30; Fn50: 2.64±0.12; PCOL: 2.20±0.03) and OCN (Fn10: 2.52±0.23; Fn20: 2.28±0.24; Fn30: 2.34±0.21; Fn40: 2.34±0.25; Fn50: 2.20±0.22; PCOL: 1.56±0.16) was significantly enhanced on Fn and PCOL substrates as compared with control; moreover, expression of integrin beta 1 (ITGB1), an ubiquitous cell surface receptor was augmented in Fn(10-50) and PCOL groups simultaneously. In accordance with the ALP activity and gene expression data, calcific deposition in cells grown on Fn(10-50) and PCOL was observed as well. Conclusion Despite the limitation of this study, the findings indicate that a surface coating of Fn enhances the proliferation, differentiation and mineralization of odontoblast-like cells by activation of integrin beta 1 (ITG B1). The promoting effects of Fn on MDPC-23 cells were achieved at a comparatively lower coating concentration than type I collagen (300 µg/mL). Specifically, it is suggested that the optimum coating concentration of Fn to be 10 µg/mL.

Dexamethasone stimulates nephronectin expression and mediates mineralization in MDPC-23 cell via Akt/mTOR signaling pathway

Dexamethasone (Dex) is frequently used for induction of cellular mineralization in vitro. However, the underlying mechanism of its influence on mineralization remains incompletely characterized. Nephronectin (Npnt), a novel RGD-containing matrix protein, was reported to promote osteoblast differentiation and mineralization, moreover, its mRNA expression was found to be up-regulated by bone morphogenetic protein 2 (BMP2) and 1α25-dihydroxyvitamin D3 in concentrationand time-dependent manner. The present work seeks to uncover the effect of Dex in the regulation of Npnt mRNA expression and possible signaling pathways that involved in the Dex-mediated mineralization. The results conclude that Dex promotes cellular mineralization in MDPC-23 cell in a dose-dependent manner via Akt/mTOR signaling pathway, simultaneously, it up-regulated Npnt mRNA expression during the differentiation process of MDPC-23 cell. Correspondence to: Jia Tang, Division of Biochemistry, Department of Oral Biology, School of Dentistry, Health Sciences University of Hokkaido, 1757-Kanazawa, Ishikari-Tobetsu, 061-0293, Hokkaido, Japan. E-mail: tangjia@ hoku-iryo-u.ac.jp

Elucidation on Predominant Pathways Involved in the Differentiation and Mineralization of Odontoblast-Like Cells by Selective Blockade of Mitogen-Activated Protein Kinases

Aim To analyze the effect of three mitogen-activated protein kinase (MAPK) inhibitors, namely, SB202190 (p38 inhibitor), SP600125 (JNK inhibitor), and PD98059 (ERK inhibitor) in Dex-stimulated MDPC-23 cell differentiation and mineralization. Methods Experiment was divided into five groups, control (cells without Dex and inhibitors treatment), Dex (cells with Dex treatment but without inhibitors), Dex + SB202190, Dex + SP600125, and Dex + PD98059. Cell differentiation was assessed by alkaline phosphatase (ALP) activity assay and real time RT-PCR. Cell mineralization was investigated by alizarin red staining. Results Exposure to SB202190 (20 μM) significantly decreased the mineral deposition in Dex-treated cells as demonstrated by alizarin red staining. Treatment of SP600125 (20 μM) attenuated the mineralization as well, albeit at a lower degree as compared to SB202190 (20 μM). Similarly, SB202190 (20 μM) completely abrogated the ALP activity stimulated by Dex at six days in culture, while no changes were observed with regard to ALP activity in SP600125 (20 μM) and PD98059 (20 μM) treated cells. The upregulation of bone sialoprotein (BSP), ALP, and osteopontin (OPN) in Dex challenged cells was completely inhibited by SB202190. Conclusion Blockade of p38-MAPK signaling pathway resulted in significant inhibition of ALP activity, mineralization, and downregulation of osteogenic markers. The data implicated that p38 signaling pathway plays a critical role in the regulation of MDPC-23 cells differentiation and mineralization.

iMatrix-511 Stimulates the Proliferation and Differentiation of MDPC-23 Cells into Odontoblastlike Phenotype

Introduction iMatrix‐511 is a novel integrin‐binding fragment derived from laminin‐511. Previous studies showed its superiority as a culture substrate for xeno‐free culture and maintenance of pluripotency in stem cells. However, its effects in the dental field remain largely unknown. The aim of the present study was to unravel the in vitro effects of iMatrix‐511 in comparison with vitronectin (VN). Methods Biochemical assays were performed in vitro in MDPC‐23 cells. The optimal coating density for 2 proteins was determined using the cell counting kit‐8. To evaluate cell proliferation to both proteins, MDPC‐23 cells were directly seeded onto the iMatrix‐511 or VN‐modified polystyrene and analyzed by the cell counting kit‐8. The phenotype of cells seeded on iMatrix‐511 and VN was characterized. Phenotypic characterization included real‐time reverse‐transcription polymerase chain reaction and alizarin red staining. Results The optimal coating density for iMatrix‐511 and VN was determined to be 1 &mgr;g/cm2 and 0.25 &mgr;g/cm2, respectively. Cells cultured on iMatrix‐511 showed higher cell proliferative activity than the noncoated control and VN on days 1, 2, and 4. Cell morphology observation revealed MDPC‐23 cells attach preferentially to iMatrix‐511 and start to spread as early as 1 hour after inoculation. MDPC‐23 cells exhibited more potent odontogenic differentiation on iMatrix‐511 than the control and VN as shown by the marked enhancement of dentin matrix protein 1 and dentin sialophosphoprotein messenger RNA expression. Although both proteins showed more mineralized nodule formation than the control, iMatrix‐511 remained to be the one that elicited stronger calcific deposition. Conclusions iMatrix‐511 supported the proliferation and acquisition of odontogenic cell phenotype in vitro, rendering this novel material a potential candidate for dentin regeneration.

A Novel Fragment Derived from Laminin-411 Facilitates Proliferation and Differentiation of Odontoblast-Like Cells

The aim for the present study was to evaluate the in vitro effects of iMatrix-411 in odontoblast-like cells. To that end, iMatrix-411 was coated to both nontissue culture treated- (Non-PS) and tissue culture treated-polystyrene (TCPS) multiwells. MDPC-23 cells were seeded into noncoated (control) or coated wells. Optimal coating density and cell proliferation were assessed by cell counting kit-8 (CCK-8) at day two, day three, and day five. Osteo/odontogenic differentiation was evaluated by real-time RT-PCR and alkaline phosphatase (ALP) activity at days seven and eight, respectively. Calcific deposition of cells was visualized by alizarin red staining. Data were analyzed with post hoc Tukey HSD test (p < 0.05). Optimal coating density for iMatrix-411 was 8 μg/cm2. Exposure of MDPC-23 cells to iMatrix-411 in either non-PS or TCPS significantly enhanced proliferative activity. iMatrix-411 elevated ALP activity in both types of culture plates. iMatrix-411 significantly increased the mRNA level of OCN, BSP, OPN, ALP, and DMP-1. Meanwhile, it enhanced the expression of several integrin subunits: ITGA1, ITGA5, ITGAV, ITGB1, and ITGB5. Finally, iMatrix-411 also accelerated the mineralization at day eight in Non-PS. The results indicated iMatrix-411 stimulates proliferation and favours differentiation of odontoblast-like cells.