[Show abstract][Hide abstract] ABSTRACT: Enamel matrix derivative (EMD) is widely used in periodontal tissue regeneration therapy. However, because the bioactivity of EMD varies from batch to batch, and the use of a synthetic peptide could avoid use from an animal source, a completely synthetic peptide (SP) containing the active component of EMD would be useful. In this study an oligopeptide synthesized derived from EMD was evaluated for whether it contributes to periodontal tissue regeneration. We investigated the effects of the SP on cell proliferation and osteoblast differentiation of human mesenchymal stem cells (MSCs), which are involved in tissue regeneration. MSCs were treated with SP (0 to 1000 ng/mL), to determine the optimal concentration. We examined the effects of SP on cell proliferation and osteoblastic differentiation indicators such as alkaline phosphatase activity, the production of procollagen type 1 C-peptide and osteocalcin, and on mineralization. Additionally, we investigated the role of extracellular signal-related kinases (ERK) in cell proliferation and osteoblastic differentiation induced by SP. Our results suggest that SP promotes these processes in human MSCs, and that ERK inhibitors suppress these effects. In conclusion, SP promotes cell proliferation and osteoblastic differentiation of human MSCs, probably through the ERK pathway.
International Journal of Molecular Sciences 08/2014; 15(8):14026-43. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: Emdogain®(EMD) is derived from the tooth germ of juvenile swine, and is a commonly used for periodontal tissue regeneration, including the formation of alveolar bone, in the treatment of periodontitis. However, because it originates from pig tissue, some patients choose not to be treated with EMD. The active component of EMD is a peptide sequence that corresponds to an amelogenin II precursor. Angiogenesis is one of the most critical events in the wound healing process and in periodontal regeneration. As such, this peptide may function as an angiogenic factor to stimulate cell differentiation and tissue regeneration.
Method: We characterized the effects of the synthetic peptide derived from EMD on the proliferation, migration, outgrowth extension and ICAM-1 expression in human microvascular endothelial cells (HMVECs).
Result: We demonstrated that the EMD-derived peptide significantly increased HMVEC proliferation and chemotaxis over unstimulated controls. The peptide also led to an increase in outgrowth of processes from HMVEC spheroids in three-dimensional collagen cultures. ICAM-1 mRNA expression was also significantly elevated in HMVECs following treatment with the EMD-derived peptide.
Conclusion: EMD-derived synthetic peptide may act as an angiogenic factor to stimulate the proliferation, chemotaxis, adhesion and migration of microvascular endothelial cells.
[Show abstract][Hide abstract] ABSTRACT: Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) induces pro-inflammatory cytokines, such as interleukin-1 β (IL-1β), IL-6, and IL-8, which induce periodontal tissue destruction. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration and are expected to have future applications in cellular therapies for periodontitis. However, no studies have examined the effects of P. gingivalis LPS on PDLSCs. The aim of this study was to investigate how P. gingivalis LPS affects the osteoblastic differentiation and pro-inflammatory cytokine production of PDLSCs.
PDLSCs were obtained from healthy adult human mandibular third molars. The identification of PDLSCs was confirmed by immunohistochemical evaluations of the mesenchymal stem cell markers STRO-1 and SSEA-4. Cell proliferation and osteoblastic differentiation were investigated by culturing the PDLSCs in a normal or osteogenic medium with P. gingivalis LPS (0, 1, or 10μg/mL) and then measuring the alkaline phosphatase (ALP) activity and the production of collagen type 1 Alpha 1 (COL1A1), osteocalcin production, and mineralisation. Additionally, we examined the production of IL-1β, IL-6, and IL-8 in the PDLSCs.
P. gingivalis LPS inhibited the ALP activity, COL1A1 and osteocalcin production, and mineralisation in the PDLSCs, which are positive for STRO-1 and SSEA-4. P. gingivalis LPS also promoted cell proliferation and produced IL-1β, IL-6, and IL-8.
This study provides the first findings that P. gingivalis LPS inhibits osteoblastic differentiation and induces pro-inflammatory cytokines in PDLSCs. These findings will help clarify the relationship between periodontitis and periodontal tissue regeneration.
Archives of oral biology 02/2014; 59(2):167-75. · 1.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Titanium surfaces play an important role in affecting osseointegration of dental implants. Previous studies have shown that the titania nanotube promotes osseointegration by enhancing osteogenic differentiation. Only relatively recently have the effects of titanium surfaces with other nanostructures on osteogenic differentiation been investigated.
In this study, we used NaOH solutions with concentrations of 2.5, 5.0, 7.5, 10.0, and 12.5 M to develop a simple and useful titanium surface modification that introduces the nanonetwork structures with titania nanosheet (TNS) nanofeatures to the surface of titanium disks. The effects of such a modified nanonetwork structure, with different alkaline concentrations on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMMSCs), were evaluated.
The nanonetwork structures with TNS nanofeatures induced by alkali etching markedly enhanced BMMSC functions of cell adhesion and osteogenesis-related gene expression, and other cell behaviors such as proliferation, alkaline phosphatase activity, extracellular matrix deposition, and mineralization were also significantly increased. These effects were most pronounced when the concentration of NaOH was 10.0 M.
The results suggest that nanonetwork structures with TNS nanofeatures improved BMMSC proliferation and induced BMMSC osteogenic differentiation. In addition, the surfaces formed with 10.0 M NaOH suggest the potential to improve the clinical performance of dental implants.
International Journal of Nanomedicine 01/2014; 9:1741-55. · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: Periodontitis is an inflammatory disease that leads to the progressive degeneration of the alveolar bone that surrounds and supports the teeth. Without effective treatment, periodontitis can cause tooth loss. One method of treating periodontitis is to use Emdogain®, a material derived from the tooth germ of juvenile swine that promotes periodontal tissue regeneration, including the formation of hard tissue as cementum, alveolar bone. The use of Emdogain® is therefore established in the field of periodontal regenerative therapy. However, because of its swine origin, some patients choose not to be treated with Emdogain®. The active component of Emdogain® has been shown to be a peptide whose sequence corresponds to an amelogenin II precursor. As such, this peptide may function as a growth factor to stimulate cell differentiation and tissue regeneration.
Method: In this study, we characterized the effects of the synthetic Emdogain®-derived peptide on the proliferation, adhesion, migration and differentiation of periodontal ligament fibroblasts (HPdLFs), which display properties similar to mesenchymal stem cells. We next investigated the effects of the peptide at a concentration of 100 ng/ml on osteogenesis and cementogenesis in HPdLF cells by assaying alkaline phosphatase activity, osteocalcin production, and mineralization.
Result: Compared to cells not treated with the synthetic Emdogain®-derived peptide, treated cells showed increased proliferation, initial adhesion, and chemotactic activity. The optimum peptide concentration that stimulated these activities was determined to be 100 ng/ml.Compared with untreated cells, cells incubated with the peptide showed increased alkaline phosphatase activity after 21 days, increased osteocalcin production after 28 days, and increased calcium deposition after 28 days.
Conclusion: Taken together, our data suggest that the Emdogain®-derived peptide stimulates periodontal hard tissue regeneration by stimulating the proliferation, adhesion, and migration of mesenchymal stem cells.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Objective: Recently, researchers for finding unusual new function of material have been going on eagerly. We found that titanium oxide generates the nanosheet structure by itself in room temperature and ordinary atmospheric pressure. The application to biocompatible materials is expected because of interaction between biological tissue and titanium nanosheet structure. The aim of this study was to evaluate the influence of titanium at nanosheet surface on initial attachment of bone marrow cells for application to biocompatible materials.
Method: In the test group, Ti discs which educed the nanosheet were used as an experimental material, and unprocessed Ti discs were also used in the control group. The titanium nanostructure surface was viewed by scanning electron microscopy (S-4000; Hitachi, Tokyo, Japan). It was measured adhesion behavior of cow’s serum albumin and fibronectin by using BCA protein assay Kit. SD rat bone marrow cells (4×104 cells/well) were plated into each well, and the number of cells was counted at 1, 3, 6, 24 hours of culture by using CellTiter-BlueTM Viabillity Assay Kit.
Result: In SPM images, nano network structure was showed in the test group. The adhesion number of both proteins on Ti surface in the test group was significantly higher than that of the control group. After 1 and 3 hours of culture, the number of cells of the test group shows significantly more than those of the control group, but after 6 and 24 hours of culture, no significant difference was observed between the test and control group.
Conclusion: These data suggest surface reforming of Ti nanostructure surface changed its molded surface with nano size and improved adhesion of protein and focal adhesion of cells.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Objective: There has been considerable recent interest in finding novel applications and functions for existing dental materials. We found that, at room temperature and atmospheric pressure, titanium oxide spontaneously generates nano-structures very similar to the “nanotubes” created by TiO2 sputter. The aim of this study is to evaluate the ability of this surface to affect the cellular osteogenic differentiation response.
Method: Titanium discs without and with a ‘nanosheet’ deposited on their surface were used as the control and test groups, respectively. Cell culture experiments were performed with SD rat bone marrow cells, which were seeded into microplate wells and cultured in media designed to induce osteogenic differentiation. We measured markers such as alkaline phosphatase (ALP) activity, osteocalcin (OCN) production,calcium deposition and Runx2 gene expression to assess the levels of differentiation.
Result: After 14 and 21 days, cellular ALP activity was significantly higher in the test group than in the control group. After 28 days, cells in the test group also showed significantly more calcium deposition and OCN production than those in the control group. Finally, there was significantly different expression of Runx2 mRNA in the test group compared to the control group after 3 days of culture.
In conclusion, these data suggest that modified the surface of titanium implants modified with nanostructures promotes osteogenic differentiation, and may improve the biointegration of these implants into the alveolar bone.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Background: We obtained a synthetic peptide (SP) for useful periodontal tissue regeneration in our previous study. Periodontal ligament stem cells (PDLSCs) have a multiple potentiality to contribute the tissue regeneration. Our aim of this experiment was to investigate the effect of SP on human PDLSCs. Methods: Periodontal ligament (PDL) cells were obtained from healthy adult human third molars and used to isolate single PDLSC-derived colonies. The mesenchymal stem cell nature of the PDLSCs was confirmed by the immunohistochemical evaluation of the expression of STRO-1. Proliferation and osteoblastic differentiation were investigated by culturing PDLSCs in normal or osteogenic medium with and without the SP (100 ng/ml). Osteoblastic differentiation was assessed by measuring alkaline phosphatase (ALP) activity, osteocalcin production, the mRNA expression of osteonectin, mineralization, and calcium deposition. Results: Isolated PDLSCs were immunohistochemically positive for vimentin and STRO-1, and negative for cytokeratin. A greater number of calcified nodules were observed in osteogenic medium culture with SP. In the early and later stage of PDLSCs culture with SP, osteonectin production and osteocalcin production were increased. The SP in culture with osteogenic medium significantly enhanced the proliferation of the PDLSCs, as well as ALP activity, the expression of osteonectin, osteocalcin production, the formation of calcified nodules, and mineralization. Conclusions: The calcified nodules formation and osteocalcin production in the culture of PDLSCs into osteoblast-like cells and is a useful material for periodontal tissue regeneration.
Journal of Periodontology 11/2012; · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, there has been considerable interest in finding novel applications and functions for existing dental materials. We found that, at room temperature and atmospheric pressure, titanium oxide spontaneously generates nanostructures very similar to the "nanotubes" created by TiO(2) sputtering. The aim of this study was to evaluate the ability of this surface to affect the cellular osteogenic differentiation response.
Titanium disks without and with a 'nanosheet' deposited on their surface were used as the control and test groups, respectively. Cell culture experiments were performed with SD rat bone marrow cells, which were seeded into microplate wells and cultured in media designed to induce osteogenic differentiation. We measured alkaline phosphatase (ALP) activity, osteocalcin (OCN) production, calcium deposition and Runx2 gene expression to assess the levels of differentiation.
After 14 and 21 days, cellular ALP activity was significantly higher in the test group than in the control group. After 28 days, cells in the test group also showed significantly more calcium deposition and OCN production than those in the control group. There was significantly different expression of Runx2 mRNA in the test group compared to the control group after 3 days of culture.
In conclusion, these data suggest that titanium implants modified by the application of nanostructures promote osteogenic differentiation, and may improve the biointegration of these implants into the alveolar bone.
Journal of prosthodontic research. 05/2012; 56(3):170-7.
[Show abstract][Hide abstract] ABSTRACT: Objectives: Enamel matrix derivative (EMD) extracted from the tooth germ of young pigs has been used clinically as a convenient material for periodontal tissue regeneration. However, EMD might be rejected by patients, because it is an animal-derived material and may carry unknown pathogenic organisms. Development of a newly synthesized oligopeptide with the same function as EMD is desirable. The aim of the present study was to examine the effects on human gingival epithelial cells of the newly synthesized oligopeptide by using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry analysis of eosinophilic round bodies formed after subcutaneous injection of EMD into the backs of rats. Methods: Human gingival epithelial cell lines (HGECs) known as epi4 was obtained from Prof. Murakami, Osaka University. The in vitro effects of the oligopeptides on HGECs at three concentrations (5, 100, 500 ng/mL) and control (0 ng/mL) were determined by examining cell proliferation/viability, adhesion and chemotaxis. Cell proliferation/viability, cell adhesion and cell chemotaxis were determined using MTS assay reagent, PicoGreen dsDNA Quantitation Assay Reagent Kit and BD FALCON Fluoroblok Insert System by Boyden chamber method, respectively. Results: The proliferative response was enhanced by the oligopeptides at all concentrations with time. In cell proliferation/viability and adhesion assays, the fluorescent intensities at 100 ng/mL were significantly lower than at the other concentrations and control. In cell chemotaxis assay, HGEC chemotaxis at 100 ng/mL was significantly greater than the other concentrations and for the control. Conclusions: These findings suggest that the oligopeptide may not upregulate cell proliferation/viability, adhesion, or chemotaxis in a dose-dependent manner. Therefore, further investigation is needed to determine the optimal concentration of the oligopeptide to enhance wound healing responses by HGEC throughout the process of periodontal regeneration.