Participation of endogenous IGF-I and TGF-beta 1 with enamel matrix derivative-stimulated cell growth in human periodontal ligament cells

Previous studies have provided the biological basis for the therapeutic use of enamel matrix derivative (EMD) at sites of periodontal regeneration. A purpose of this study is to determine effects of EMD on cell growth, osteoblastic differentiation and insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 (TGF-beta 1) production in human periodontal ligament cells (HPLC). We also examined participation of endogenous IGF-I and TGF-beta 1 with EMD-stimulated cell growth in these cells. HPLCs used in this study were treated with EMD alone or in combination with antihuman IGF-I antibody (anti-hIGF-I) or anti-hTGF-beta 1, recombinant human bone morphogenetic protein-2 (rhBMP-2), 1,25-dihydroxyvitamin D3[1,25(OH)2D3], rhTGF-beta 1 or rhIGF-I. After each treatment, cell growth, the production of IGF-I and TGF-beta 1 and the expression of osteoblastic phenotypes were evaluated. EMD stimulated cell growth in dose-dependent and time-dependent manners. EMD was also stimulated to express IGF-I and TGF-beta 1 at protein and mRNA levels. The EMD-stimulated cell growth was partially suppressed by cotreatment with anti-hIGF-I or anti-hTGF-beta 1, and cell growth was also stimulated by treatment with rhIGF-I or rhTGF-beta 1. rhBMP-2 stimulated alkaline phosphatase (ALPase) activity and ALPase mRNA expression, and 1,25(OH)2D3 stimulated ALPase and osteocalcin mRNA expression. However, EMD showed no effect on the osteoblastic phenotypes expression. These results demonstrated that EMD has no appreciable effect on osteoblastic differentiation, however it stimulates cell growth and IGF-I and TGF-beta 1 production in HPLC, and that these endogenous growth factors partially relate to the EMD-stimulated cell growth in HPLC.     

The effects of enamel matrix derivative (EMD) on osteoblastic cells in culture and bone regeneration in a rat skull defect

OBJECTIVE: Enamel matrix derivative (EMD) has been clinically used to promote periodontal tissue regeneration. The purpose of the present study is to clarify EMD affects on osteoblastic cells and bone regeneration. MATERIALS AND METHODS: Mouse osteoblastic cells (ST2 cells and KUSA/A1 cells) are used in culture experiments. After cells were treated with EMD, cell growth was evaluated with DNA measurement, 5-bromo-2'-deoxyurydine (BrdU) incorporation assay. Measurement of alkaline phosphatase (ALP) activity and mineralized-nodule (MN) formation, Northern blotting analysis and zymography are also performed. In addition, EMD was applied to a rat skull defect and the defect was radiographically and histologically evaluated 2 weeks after the application. RESULTS: EMD did not stimulate ST2 cell growth; however, it enhanced KUSA/A1 cell proliferation. Although EMD stimulated ALP activity in both the cells, ALP activity in KUSA/A1 cells was affected to a much greater degree. Corresponding to the increase in ALP activity, MN formation in KUSA/A1 cells was enhanced by EMD. EMD stimulated osteoblastic phenotype expression of KUSA/A1 cells such as type I collagen, osteopontin, transforming growth factor beta 1 and osteocalcin. EMD treatment also stimulated matrix metalloproteinase production in KUSA/A1 cells. Although the effects of EMD on osteoblastic cells depend on cell type, the overall effect of EMD on osteoblastic cells is stimulatory rather than inhibitory. Finally, EMD application to a rat skull defect accelerated new bone formation. CONCLUSION: These results indicate that EMD affects osteoblastic cells and has potential as a therapeutic material for bone healing.     

Inhibitory effect of enamel matrix derivative on osteoblastic differentiation of rat calvaria cells in culture

Background and Objective:  The effect of enamel matrix derivative (EMD) on bone differentiation remains unclear. Transforming growth factor β1 (TGF - β1) is reported to be contained in EMD. The aim of this study was to clarify the effect of EMD on osteoblastic cell differentiation and the possible role of TGF - β1.
Material and Methods:  Fetal rat carvarial cells were treated with 10, 50 or 100 µg/ml EMD for 5–17 days. Alkaline phosphatase (ALP) activity and bone nodule formation were measured, and mRNA expressions of bone matrix proteins and core binding factor were analysed.
Results:  Enamel matrix derivative inhibited ALP activity from the early stage of culture (29–44% inhibition) on days 5 and 10 and decreased bone nodule formation by 37–67% on day 17. These effects of EMD were concentration dependent. Enamel matrix derivative inhibited mRNA expression of osteocalcin and core binding factor. A high level of the active form of TGF - β1 protein was detected in the conditioned medium treated with 100 µg/ml EMD. Treatment with TGF - β1 antibody partly restored the inhibitory effect of EMD on ALP activity.
Conclusion:  Enamel matrix derivative inhibited the osteoblastic differentiation of rat carvarial cells and this was partly mediated by an increase in the activated form of TGF - β1, suggesting that EMD may function initially to inhibit osteoblastic differentiation to allow a predominant formation of other periodontal tissues.     

Novel isolation of alkaline phosphatase-positive subpopulation from periodontal ligament fibroblasts

BACKGROUND: Periodontal ligament fibroblasts (PDLFs) are the cells essential for periodontal regeneration. PDLFs comprise a heterogeneous cell population and consist of several cell subsets that differ in their function. It is known that PDLFs produce osteoblast-related extracellular matrix proteins and show higher alkaline phosphatase (ALP) activity compared with gingival fibroblasts (GFs), implying that PDLFs have osteogenic characterisitics. The aim of the present study was to isolate the osteogenic population of PDLFs according to their expression of ALP. METHODS: PDLFs and gingival fibroblasts were separated into two populations, ALP-positive and ALP-negative, with an immunomagnetic method using a monoclonal antibody against human bone type ALP and magnetic beads conjugated with a secondary antibody. Expression of basic fibroblast growth factor (bFGF) receptor and transforming growth factor (TGF)-beta receptor was investigated in these two populations. Osteoblast-related molecules, osteocalcin, and bone sialoprotein; ALP activity; and effect of bFGF on proliferation were also compared. RESULTS: Effective separation was confirmed in both PDLFs and GFs by flow cytometry. The expression of FGF receptor (FGFR) and TGF-beta receptor was significantly higher in ALP-positive PDLFs than in ALP-negative PDLFs. ALP-positive PDLFs also expressed higher mRNA levels of osteocalcin and bone sialoprotein compared with ALP-negative PDLFs. The mitogenic effect of bFGF on ALP-positive PDLFs was greater than that of ALP-negative PDLFs. CONCLUSIONS: These results indicate that osteoblastic and/or cementoblastic PDLF subsets could be isolated from the PDLF populations using an immunomagnetic method. Magnetic isolation of PDLFs may be a useful tool to obtain the cells which will potentially induce mineralization on the root surface.     

Enamel matrix derivative promotes osteoclast cell formation by RANKL production in mouse marrow cultures

OBJECTIVES: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, however, there are few reports regarding effects of EMD on bone metabolism. We evaluated the influence of EMD on osteoclast formation using in vitro bone marrow culture. METHODS: Bioactive fractions were purified from EMD by reverse-phase HPLC on a C18 hydrophobic support, then mouse bone marrow cells were cultured with EMD or its purified fractions for 8 days. Following tartrate resistant acid phosphatase (TRAP) staining, TRAP-positive multinucleated cells were counted. The expression of receptor activator of NF-kappaB ligand (RANKL) in osteoblastic cells was detected using immunoblotting. RESULTS: EMD was dissolved in 0.1% (vol/vol) trifluoroacetic acid and applied to a C18 column for HPLC. Two major peaks were obtained of which the second (fraction numbers 21-25) was found to induce the formation of osteoclasts in mouse marrow cultures. Further, osteoprotegerin completely inhibited osteoclast formation in mouse marrow cultures with or without osteoblastic stromal cells, when being cultured with EMD or its purified fractions. In addition, Western blot analysis revealed the presence of RANKL in mouse osteoblastic cells stimulated with EMD or its purified fractions. CONCLUSION: Our results indicate that EMD induces the formation of osteoclasts through RANKL expressed by osteoblastic cells, and suggest that EMD may regulate both bone formation and bone resorption during periodontal tissue regeneration.     

The effects of enamel matrix derivative (EMD) on osteoblastic cells

Enamel matrix derivative (EMDOGAIN, EMD) has been clinically used to promote regeneration of periodontal tissue, including cementum, periodontal ligament (PDL), and alveolar bone. However, it has not been clear whether EMD directly affects osteoblastic cells. To answer this question, we examined EMD effects on bovine PDL cells, rat and mouse bone marrow cells (RBM cells and MBM cells, respectively), and mouse osteoblastic cells (Kusa/A 1 cells). EMD was dissolved in 10 mM acetic acid and added to the culture medium at a final concentration of 50 micrograms/ml. EMD stimulated mineralized-nodule formation of PDL cells, RBM cells, and Kusa/A 1 cells. In Kusa/A 1 cells, EMD enhanced ALP activity, together with DNA content. Northern blotting analysis on Kusa/A 1 cells demonstrated stimulatory effects of EMD on the gene expression of type I collagen and osteopontin. Further, application of EMD on MBM cell culture, under 1,25(OH)2 vitamin D3 supplementation, stimulated osteoclast-like cell formation. These results indicate that osteoblastic cells respond to EMD, and that EMD would be potentially useful for bone regeneration.     

Osteoprotegerin and receptor activator of nuclear factor-kappa B ligand modulation by enamel matrix derivative in human alveolar osteoblasts

BACKGROUND: Bone regeneration techniques increasingly rely on the use of exogenous molecules able to enhance tissue formation in pathologic and traumatic defects. An enamel matrix derivative (EMD) has been largely used to promote tooth ligament regeneration within periodontal pockets. Recent evidence suggests that EMD may contribute to inducing osteoblast growth and differentiation. We investigated the effects of EMD on growth and osteogenic marker modulation in human mandibular osteoblasts. METHODS: We focused our attention on cell growth by 3-(4,5-dimethyl[thiazol-2-yl]-3,5-diphery)tetradium bromide (MTT) assay, cell differentiation, mineralized nodule formation, and, in particular, the expression of receptor activator of nuclear factor-kappa B ligand (RANKL), the main osteoclast differentiation factor, and its decoy receptor, osteoprotegerin (OPG), by enzyme-linked immunosorbent assay. RESULTS: Cell growth was significantly increased by EMD. Similarly, a significantly higher quantity of OPG and a lower amount of RANKL were detectable in groups treated with 50 and 100 microg/ml at weeks 1, 2, and 3, and alkaline phosphatase activity and osteocalcin production were enhanced in cultures treated with 50 and 100 microg/ml at weeks 2 and 3. Mineralized nodules appeared bigger and more numerous in cultures treated with 50 and 100 microg/ml EMD. CONCLUSIONS: EMD was able to enhance osteoblast cell growth and the expression of markers of osteoblastic phenotype and differentiation. EMD also seemed able to create a favorable osteogenic microenvironment by reducing RANKL release and enhancing osteoblastic OPG production.     

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

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: EMD affects early states of osteoblastic maturation by stimulating proliferation, but as cells mature in the lineage, EMD enhances differentiation.     

Effect of estrogen receptor beta on the osteoblastic differentiation function of human periodontal ligament cells

OBJECTIVE: To investigate the effect of estrogen receptor beta (ERbeta) on osteoblastic differentiation function of human periodontal ligament (hPDL) cells by measuring the alkaline phosphatase (ALP) activity and the production of osteocalcin (OCN) in vitro. DESIGN: We employed a short interfering RNA (siRNA) technique to inhibit ERbeta expression in hPDL cells; the cells were cultured with a saturating concentration of 17beta-estradiol (10(-7)M). ALP activity was analysed by colorimetric assay using ALP kit and the amount of OCN was assessed by osteocalcin ELISA kit. RESULTS: It was shown that estradiol significantly enhanced the ALP activity and the production of OCN in hPDL cells. However, the ALP activity and the production of OCN in hPDL-siERbeta cells were not significantly changed after estradiol treatment. CONCLUSIONS: These results indicate that ERbeta may play important roles in estrogen-induced effects on osteoblastic differentiation function of PDL cells and estrogen influences the bone formation capacity of PDL cells mainly via ERbeta.     

IL-6 induces osteoblastic differentiation of periodontal ligament cells

Interleukin (IL)-6 has been considered as an osteolytic factor involved in periodontal disease. However, the function of IL-6 in osteoblastic differentiation of periodontal ligament cells is not clear. We examined the effects of IL-6 and its soluble receptor (sIL-6R) on osteoblastic differentiation of periodontal ligament cells. Osteoblastic differentiation was induced by ascorbic acid. Osteoblast markers, including alkaline phosphatase activity and Runx2 gene expression, were examined. The mechanism of action of IL-6 on osteoblastic differentiation was evaluated by insulin-like growth factor (IGF)-I production and specific inhibitors for the IL-6-signaling molecule. IL-6/sIL-6R enhanced alkaline phosphatase activity and Runx2. Alkaline phosphatase activity was reduced by anti-IGF-I antibody. Mitogen-activated protein kinase and Janus protein tyrosine kinase inhibitors diminished alkaline phosphatase induced by IL-6/sIL-6R. We conclude that IL-6/sIL-6R increases ascorbic-acid-induced alkaline phosphatase activity through IGF-I production, implying that IL-6 acts not only as an osteolytic factor, but also as a mediator of osteoblastic differentiation in periodontal ligament cells.     

The effect of enamel matrix derivative on gene expression in osteoblasts

Observations that amelogenins, in the form of enamel matrix derivative (EMD), have a stimulatory effect on mesenchymal cells and tissues, and on the regeneration of alveolar bone, justified investigations into the effect of EMD on bone-forming cells. The binding and uptake of EMD in primary osteoblastic cells was characterized, and the effect of EMD on osteoblast gene expression, protein secretion, and mineralization was compared with the effect of parathyroid hormone (PTH). Although no specific receptor(s) has yet been identified, EMD appeared to be taken up by osteoblasts through clathrin-coated pits via the interaction with clathrin adaptor protein complex AP-2, the major mechanism of cargo sorting into coated pits in mammalian cells. EMD had a positive effect on factors involved in mineralization in vitro , causing an increased alkaline phosphatase (ALP) activity in the medium as well an as increased expression of osteocalcin and collagen type 1. Several hundred genes are regulated by EMD in primary human osteoblasts. There appear to be similarities between the effects of EMD and PTH on human osteoblasts. The expression pattern of several mRNAs and proteins upon EMD stimulation also indicates a secondary osteoclast stimulatory effect, suggesting that the osteogenic effect of EMD in vivo , at least partly, involves stimulation of bone remodelling.     

牙囊细胞条件培养液在大鼠脂肪间充质干细胞向成牙骨质分化中的作用

目的：探讨牙囊细胞条件培养液（DFCCM）在诱导大鼠脂肪间充质干细胞（ADSCs）向成牙骨质样细胞分化中的作用。方法：分离培养大鼠脂肪间充质干细胞、牙囊细胞,制备DFCCM。用DFCCM诱导ADSCs,通过MTT检测ADSCs增殖能力的变化;免疫荧光及实时定量PCR方法检测成骨关键蛋白一骨钙素（OCN）、碱性磷酸酶（ALP）及成牙骨质关键蛋白一牙骨质附着蛋白（CAP）,牙骨质蛋白（CP23）的情况。结果：牙囊细胞条件培养液诱导ADSCs后可抑制ADSCs的增殖能力。DFCCM诱导7d后,ADSCs细胞浆内表达OCN及CAP。同时DFC—CM诱导ADSCs后CAP,CP23,ALP及OCNmRNA表达水平显著高于对照组。结论：牙囊细胞条件培养液构建的微环境可使ADSCs向成牙骨质样细胞分化,为于细胞介导的牙骨质再生提供新的思路。     

Simvastatin promotes cell metabolism, proliferation, and osteoblastic differentiation in human periodontal ligament cells

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.