Presence Of Enamel Matrix Derivative Research Articles

Effects of conservatively treated diseased cementum with or without EMD on in vitro cementoblast differentiation and in vivo cementum-like tissue formation of human periodontal ligament cells.

The present study aimed to evaluate the effects of conservatively treated diseased cementum on in vitro cementoblast differentiation and in vivo cementum-like tissue formation of human periodontal ligament cells (hPDLCs), and observe differential effects of enamel matrix derivative (EMD) on in vivo cementum formation by hPDLCs. Forty-eight cementum slices and 48 dentin slices were prepared from periodontitis compromised teeth, and hPDLCs were inoculated on to all root slices. Twenty-four co-cultured root slices of each group were used for mRNA expression of cementum attachment protein and CEMP1. With application of EMD, 24 co-cultured root slices (divided into groups C, D, C+E, D+E) were transplanted subcutaneously into nude mice. All root fragments were reviewed by histological analysis and immunohistochemical staining for bone sialoprotein. mRNA expressions of cementum attachment protein and cementum protein - 1 from hPDLCs on cementum slices were statistically higher than those of dentin slices. Seven specimens of group C and 10 specimens of group C+E revealed a layer of cementum-like tissue (NFC) on surfaces of pre-existing cementum. NFC was thicker in group C+E than in group C. All NFCs were positively stained for bone sialoprotein, however, there was no NFC formation on dentin slices. Conservatively treated diseased cementum promoted in vitro cementoblast differentiation and in vivo cementum-like tissue formation by hPDLCs, and the in vivo effect was enhanced by the presence of EMD.

Glycol Chitin–based Thermoresponsive Hydrogel Scaffold Supplemented with Enamel Matrix Derivative Promotes Odontogenic Differentiation of Human Dental Pulp Cells

IntroductionHydrogels have been widely studied as tissue engineering scaffolds over the past 2 decades because of their favorable biological properties. Recently, a new biodegradable glycol chitin–based thermoresponsive hydrogel scaffold (GC-TRS) was developed that can be easily applied as a mild viscous solution at room temperature but quickly transforms into a durable hydrogel under physiological conditions. The aim of this study was to investigate the effects of GC-TRS on the proliferation and odontogenic differentiation of colony-forming human dental pulp cells (hDPCs) in the presence of enamel matrix derivative. MethodsGlycol chitin was synthesized by N-acetylation of glycol chitosan. The morphology of the thermoresponsive hydrogel scaffold was observed by using scanning electron microscopy. The sol gel phase transition of the aqueous solution of glycol chitin was investigated by using the tilting method and rheometer studies. hDPCs were isolated based on their ability to generate clonogenic adherent cell clusters. The effect of GC-TRS and collagen on cell viability was examined by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of markers for odontogenic/osteogenic differentiation (ie, dentin sialophosphoprotein, dentin matrix protein-1, osteonectin, and osteopontin) was analyzed by performing real-time polymerase chain reaction. ResultsGC-TRS exhibited a highly macroporous and well-interconnected porous structure. The polymer solution existed in a mildly viscous sol state, but it transitioned to a gel state and did not flow above approximately 37°C. Rheometer studies showed that the glycol chitin solution exhibited a fast sol gel transition approximately at body temperature. GC-TRS and collagen did not inhibit cell viability until 7 days. Dentin sialophosphoprotein and dentin matrix protein-1 were expressed by cells cultured in GC-TRS at a higher level than that in cells cultured in collagen (P < .05). In both the scaffold groups, dentin sialophosphoprotein, dentin matrix protein-1, and osteopontin messenger RNA was up-regulated significantly in EMD-treated hDPCs when compared with the nontreated cells (P < .05). ConclusionsGC-TRS allowed the proliferation and odontogenic differentiation of hDPCs. Furthermore, the differentiation was facilitated by EMD. These results suggest that GC-TRS has the potential to be used in tissue engineering techniques for dentin regeneration.

Regulation of regenerative periodontal healing by NAMPT.

Periodontitis is an inflammatory disease characterized by destruction of the tooth-supporting tissues. Obese individuals have an increased risk of periodontitis, and elevated circulating levels of nicotinamide phosphoribosyltransferase (NAMPT) may be a pathomechanistic link between both diseases. Recently, increased levels of NAMPT have also been found in patients with periodontitis, irrespective of the presence of obesity. This in vitro study sought to examine the effects of NAMPT on the regenerative capacity of human periodontal ligament (PDL) cells and, thereby, periodontal healing. PDL cells treated with enamel matrix derivative (EMD), which was used to mimic regenerative healing conditions in vitro, were grown in the presence and absence of NAMPT for up to 14 d. EMD stimulated significantly (P < 0.05) the expression of growth factors and their receptors, matrix molecules, osteogenesis-associated factors, and wound closure and calcium accumulation. In the presence of NAMPT, all these stimulatory effects were significantly (P < 0.05) reduced. In conclusion, the beneficial effects of EMD on a number of PDL cell functions critical for periodontal regeneration are counteracted by NAMPT. Enhanced levels of NAMPT, as found in obesity and periodontal inflammation, may compromise the regenerative capacity of PDL cells and, thereby, periodontal healing in the presence of EMD.

Effects of enamel matrix derivative on periodontal wound healing in an inflammatory environment in vitro

This in vitro study was established to investigate whether the regenerative capacity of periodontal ligament (PDL) cells in the presence of enamel matrix derivative (EMD) is modulated by inflammation. PDL cells were grown in the presence or absence of EMD under normal and inflammatory conditions for up to 14 days. In order to mimic an inflammatory environment, cells were incubated with interleukin (IL)-1β. Cells were also exposed to transforming growth factor (TGF)-β1 and insulin-like growth factor (IGF)-1 under both conditions. For analysis of wound healing, an in vitro wound fill assay was used. The synthesis of growth factors, markers of proliferation, and osteogenic differentiation, as well as collagen was studied by real-time polymerase chain reaction, enzyme-linked immunoassay, and immunoblotting. Mineralization was assessed by alizarine red S and von Kossa staining. EMD stimulated significantly the in vitro wound fill rate, cell proliferation and adhesion, synthesis of growth factors, and collagen, as well as mineralization. In the presence of IL-1β, these EMD effects were significantly reduced. IL-1β also inhibited significantly the wound fill rate induced by TGF-β1 and IGF-1. Critical PDL cell functions that are associated with periodontal regeneration are reduced in an inflammatory environment.

Enamel matrix derivative stimulates chondrogenic differentiation of ATDC5 cells

Although enamel matrix derivative can promote chondrogenic differentiation of pluripotent mesenchymal precursor cells, the molecular mechanism that underlies this phenomenon is unclear. The purpose of this study was to determine the effect of enamel matrix derivative on chondrogenic differentiation. ATDC5 cells, which undergo a reproducible multistep chondrogenic differentiation, were cultured with or without enamel matrix derivative for up to 35 d. Cell proliferation and alkaline phosphatase activity increased markedly in cells cultured in the presence of enamel matrix derivative, compared with cells cultured in its absence. Deposition of Alcian blue-positive cartilage matrix and Alizarin red-positive mineralized nodules also increased significantly upon treatment with enamel matrix derivative. Expression of mRNAs encoding cartilage extracellular matrix proteins (type II collagen, type X collagen and aggrecan) and chondrogenic-related transcription factors (Sox9, Zfp60 and AJ18) were measured using the real-time polymerase chain reaction. Type II collagen, type X collagen and aggrecan mRNA expression increased markedly with enamel matrix derivative treatment. Transcription of Sox9, an important transcription factor that mediates chondrogenic differentiation, also increased with enamel matrix derivative treatment. The KRAB/C2H2 zinc-finger transcription factors, Zfp60 and AJ18, were transiently expressed in the prehypertrophic stage, and their expression increased with enamel matrix derivative treatment. In a western blot analysis with anti-insulin-like growth factor-I and anti-bone morphogenetic protein-6 immunoglobulin, bands corresponding to approximately 14, approximately 18 and approximately 60 kDa were found in enamel matrix derivative. Our study provides clear evidence that enamel matrix derivative promotes chondrogenic differentiation of ATDC5 cells.

Mechanisms involved in the enhancement of osteoclast formation by enamel matrix derivative

Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, and it has been reported that EMD can induce the formation of osteoclasts in mouse marrow cultures. In the present study, we investigated the mechanisms of EMD-induced osteoclast formation using a mouse monocytic cell line, RAW 264.7. Bioactive fractions were purified from EMD by reverse-phase HPLC using a C18 hydrophobic support, following which RAW 264.7 cells were cultured with EMD or its purified fractions in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) for 8 d. Following staining with tartrate-resistant acid phosphatase (TRAP), TRAP-positive multinucleated cells were counted. The expression of receptor activator of nuclear factor-kappaB (RANK), as well as phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, in RAW 264.7 cells were detected using immunoblotting. To determine whether EMD has an effect on osteoclast function, differentiated RAW 264.7 cells were cultured on Osteologic Multitest slides with RANKL in the presence of EMD. Purified EMD fractions (fraction numbers 21-25; EMD peak 2) were found to enhance the formation and function of RAW 264.7 cells induced by RANKL. Moreover, EMD peak 2 enhanced the levels of phosphorylation of ERK p38 and RANK in RAW 264.7 cells stimulated with RANKL. Our results indicate that EMD induces the formation of osteoclasts through interaction with RANKL, while ERK and p38 MAPK may play a critical role in the enhancement of osteoclast formation in RAW 264.7 cells.

Gene Expression Profiles of Periodontal Ligament Cells Treated With Enamel Matrix Proteins In Vitro: Analysis Using cDNA Arrays

A number of procedures have been used to achieve periodontal regeneration. Recently, enamel matrix derivative (EMD) has been the subject of significant basic and clinical investigations. The precise molecular events involved in EMD modulation of periodontal wound healing are not completely understood; however, cDNA microarray technology may enable rapid and accurate examination of EMD-mediated changes in gene expression in periodontal ligament (PDL) cells in vitro. The present study was undertaken to explore the selective effects of EMD on the activities of 268 cytokine, growth factor, and receptor genes in PDL. PDL cells were cultured in the absence and presence of EMD at a concentration of 100 microg/ml for 4 days. RNA was extracted and used to generate labeled cDNA probes. These were hybridized to cDNA arrays comprising 268 genes and exposed to x-ray films. Autoradiographs were digitized and analyzed. Forty-six percent (125 of 268) of the tested genes were found to be expressed by the PDL cells. Of these 125 genes, 38 were differentially expressed by PDL cells which had been cultured in the presence of EMD. Among the 38, 12 were found to be downregulated, notably mostly inflammatory genes, whereas 26 genes demonstrated upregulation, many of these coding for growth factors and growth factor receptors. The present study has shown that EMD down-regulates the expression of genes involved in the early inflammatory phases of wound healing while simultaneously upregulating genes encoding growth and repair-promoting molecules. This may partly explain the apparent efficacy of EMD application in periodontal regeneration.

Effect of enamel matrix derivative on the differentiation of C2C12 cells.

Although enamel matrix derivative (EMD) can initiate de novo cementum and bone formation by stimulating and inducing differentiation of mesenchymal cells in the periodontal ligament, the molecular mechanism of this phenomenon is not fully understood. The purpose of this study was to determine the effect of EMD on the differentiation of pluripotential mesenchymal cells. A typical pluripotential mesenchymal cell line, C2C12, was used to clarify the effect of EMD on cell differentiation. The cells were cultured in 5% serum-containing medium to induce cell differentiation, either with or without the addition of EMD. Differentiation to myoblasts was analyzed by immunostaining of desmin and type II myosin heavy chains. Osteoblast differentiation was evaluated by measuring alkaline phosphatase (ALPase) activity. Furthermore, to verify the cell lineage after culture with EMD, mRNA expression of cellular phenotype-specific markers characterizing osteoblasts (ALPase and osteocalcin), chondroblasts (type X collagen), myoblasts (desmin and MyoD), and adipocytes (lipoprotein lipase) was studied using semiquantitative reverse transcription-polymerase chain reaction. C2C12 cells cultured in differentiation medium without EMD altered their phenotype to myoblasts, exhibiting positive reactions to desmin and myosin heavy chains by immunological analysis. However, the cells cultured in the presence of EMD were strongly inhibited from developing into myoblasts, and showed high ALPase activity that was approximately 2 to 4 times greater than that of the vehicle. The mRNA expression of ALPase, osteocalcin, and type X collagen was increased markedly by the EMD-stimulated medium, whereas the expression of desmin, MyoD, and lipoprotein lipase was drastically decreased. Our study provides clear evidence that EMD converts the differentiation pathway of C2C12 cells into the osteoblast and/or chondroblast lineage.

Porcine fetal enamel matrix derivative stimulates proliferation but not differentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-like MG63 cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells.

Embryonic enamel matrix proteins are hypothesized to be involved in the formation of acellular cementum during tooth development, suggesting that these proteins can be used to regenerate periodontal tissues. Enamel matrix protein derived from embryonic porcine tooth germs is used clinically, but the mechanisms by which it promotes the formation of cementum, periodontal ligament, and bone are not well understood. This study examined the response of osteoblasts at 3 stages of osteogenic maturation to porcine fetal enamel matrix derivative (EMD). Proliferation (cell number and [3H]-thymidine incorporation), differentiation (alkaline phosphatase and osteocalcin), matrix synthesis ([35S]-sulfate incorporation; percentage of collagen production), and local factor production (prostaglandin E2 [PGE2] and transforming growth factor-beta 1 [TGF-beta1]) were measured in cultures of 2T9 cells (pre-osteoblasts which exhibit osteogenesis in response to bone morphogenetic protein-2 [BMP-2]), MG63 human osteoblast-like osteosarcoma cells, and normal human osteoblasts (NHOst cells). EMD regulated osteoblast proliferation and differentiation, but the effects were cell-specific. In 2T9 cell cultures, EMD increased proliferation but had no effect on alkaline phosphatase-specific activity. EMD decreased proliferation of MG63 cells and increased cellular alkaline phosphatase and osteocalcin production. There was no effect on collagen synthesis, proteoglycan sulfation, or PGE2 production; however, TGF-beta1 content of the conditioned media was increased. There was a 60-fold increase in cell number in third passage NHOst cells cultured for 35 days in the presence of EMD. EMD also caused a biphasic increase in alkaline phosphatase that was maximal at day 14. EMD affects early states of osteoblastic maturation by stimulating proliferation, but as cells mature in the lineage, EMD enhances differentiation.