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.     

Direct contact between enamel matrix derivative (EMD) and osteoblasts is not required for EMD-induced cell proliferation

OBJECTIVE: The purpose of this study was to determine whether the effect of enamel matrix derivative (EMD) on osteoblast proliferation is dependent on direct contact between EMD and the cells. STUDY DESIGN: MC3T3-E1 cells were seeded onto 6-well culture plates at an initial density of 5000/cm(2) in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS). Serum was removed from the culture medium after 24 hours with or without the addition of EMD. Four groups were evaluated: group 1, DMEM only; group 2, DMEM with 100 microg/mL EMD directly added to the culture medium; group 3, DMEM with a culture plate insert (30-mm diameter; 0.4-microm pore size) only; group 4, DMEM with 100 microg/mL EMD added onto a culture plate insert. The porous membrane of the insert prevented direct contact between EMD and the cells. After 3-day incubation, cell morphology was examined and the total cell number per well was counted and analyzed using 1-way ANOVA. RESULTS: EMD formed precipitated aggregates on the membrane of the culture insert with the same appearance as when it was added directly to the medium. The culture plate insert alone did not cause any changes in cell morphology or proliferation. The addition of EMD significantly increased cell number regardless the presence of the culture plate insert. CONCLUSION: This study suggests that direct contact between EMD and osteoblasts is not required to induce cell proliferation. Soluble peptides released from EMD may contribute to the stimulating effects of EMD on cell proliferation.     

Enamel matrix derivative prolongs primary osteoblast growth

Enamel matrix derivative (EMD) secreted by cells of the epithelial root sheath plays an important role in cementogenesis and periodontal tissue formation. The mechanisms by which EMD influences cell function are not known. The purpose of this study was to determine the effect of EMD on cell growth of primary mouse osteoblasts. Osteoblasts were digested from 6- to 8-day-old mouse calvaria and plated into 6-well cell culture plates at an initial density of 5000 cells/cm2. After 24-h incubation with Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, cells were incubated in three different groups of media: DMEM only as control, DMEM with 25 microg/ml EMD, and DMEM with 100 microg/ml EMD. At days 3, 7, 10, and 14, the total cell number per well was calculated, and cell morphology was examined. At each observation period the number of cells in the EMD groups was significantly greater (ANOVA, p < 0.01) than that in the control group. EMD had a greater effect on osteoblast survivor in the higher concentration than in the lower concentration. Furthermore normal morphology of the primary osteoblasts was maintained in the EMD groups. These results suggest that EMD prolongs primary osteoblast growth and may have an effect on osteoblasts during periodontal regeneration.     

Enamel matrix derivative stimulates expression and secretion of resistin in mesenchymal cells

In this study we wanted to identify the effect of enamel matrix derivative (EMD) on adipocytokines, so-called adipokines. Primary human cells of mesenchymal origin (osteoblasts, periodontal ligament cells, mesenchymal stem cells, and pulp cells) and hematopoietic origin (monocytes) were incubated with EMD. The levels of adipokines in cell culture medium were quantified using the Lincoplex human adipocyte panel (Luminex) and by real-time PCR of mRNA isolated from cell lysates. Rats were injected with 2 mg of EMD or saline intramuscularly every third day for 14 d. Blood samples were taken before and after injections, and the level of resistin in rat plasma was measured by ELISA. We found a dramatic increase in the secretion of resistin from mesenchymal stem cells, and verified this result in all the cells of mesenchymal origin tested. However, we observed no significant changes in the amount of resistin secreted from monocytes exposed to EMD compared with the control. Injections of EMD significantly enhanced the circulating levels of resistin in rats, and EMD also significantly enhanced the activity of the resistin promoter in transfected mesenchymal stem cells, indicating a direct effect on resistin expression. Our results indicate that resistin may play a role in mediating the biological effect of EMD in mesenchymal tissues.     

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.     

Equibiaxial tensile strain affects calvarial osteoblast biology

Mechanical tensile strain is believed to play an important role in regulating calvarial morphogenesis. To better understand the effects of mechanical strain on pathologic calvarial growth, we applied 10% constant equibiaxial tensile strain to neonatal rat calvarial osteoblast cultures and examined cellular proliferation, cytokine production, and extracellular matrix molecule expression. Mechanical strain markedly increased osteoblast proliferation as demonstrated by increased proliferating cell nuclear antigen (PCNA) protein. In addition, both transforming growth factor-beta1 (TGF-beta1) mRNA expression and fibroblast growth factor-2 (FGF-2) protein production were increased with exposure to strain. Moreover, mechanical strain induced expression of the extracellular matrix molecule collagen IalphaI. To further explore the relationship between mechanotransduction, osteogenesis, and angiogenesis, we examined the effect of mechanical strain on calvarial osteoblast expression of vascular endothelial growth factor (VEGF). Interestingly, we found that mechanical strain induced a rapid (within 3 hrs) increase in osteoblast VEGF expression. These data suggest that constant equibiaxial tensile strain-induced mechanotransduction can influence osteoblasts to assume an "osteogenic" and "angiogenic" phenotype, and these findings may have important implications for understanding the mechanisms of pathologic strain-induced calvarial growth.     

In Vitro Evaluation of Demineralized Freeze‐Dried Bone Allograft in Combination With Enamel Matrix Derivative

Background: Preclinical and clinical studies suggest that a combination of enamel matrix derivative (EMD) with demineralized freeze‐dried bone allograft (DFDBA) may improve periodontal wound healing and regeneration. To date, no single study has characterized the effects of this combination on in vitro cell behavior. The aim of this study is to test the ability of EMD to adsorb to the surface of DFDBA particles and determine the effect of EMD coating on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. Methods: DFDBA particles were precoated with EMD or human blood and analyzed for protein adsorption patterns via scanning electron microscopy. Cell attachment and proliferation were quantified using a commercial assay. Cell differentiation was analyzed using real‐time polymerase chain reaction for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen 1α1, and mineralization was assessed using alizarinred staining. Results: Analysis of cell attachment revealed no significant differences among control, blood‐coated, and EMD‐coated DFDBA particles. EMD significantly increased cell proliferation at 3 and 5 days after seeding for both osteoblasts and PDL cells compared to control and blood‐coated samples. Moreover, there were significantly higher messenger ribonucleic acid levels of osteogenic differentiation markers, including collagen 1α1, alkaline phosphatase, and osteocalcin, in osteoblasts and PDL cells cultured on EMD‐coated DFDBA particles at 3, 7, and 14 days. Conclusion: The results suggest that the addition of EMD to DFDBA particles may influence periodontal regeneration by stimulating PDL cell and osteoblast proliferation and differentiation.     

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative

Objectives

Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials.

Aims

This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells.

Materials and methods

EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining.

Results

Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points.

Conclusion

The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings.

Clinical relevance

The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.     

降钙素基因相关肽对血清饥饿作用下MC3T3-E1成骨细胞凋亡和自噬的影响

目的 研究血清饥饿条件下降钙素基因相关肽（CGRP）对小鼠MC3T3-E1成骨细胞凋亡、自噬的影响以及二者之间的关系,以进一步明确CGRP对成骨细胞的保护机制。方法 体外培养小鼠MC3T3-E1成骨细胞。采用流式细胞术和蛋白质印迹检测正常血清、无血清（血清饥饿）、3-MA预处理+血清饥饿培养的成骨细胞的凋亡和微管相关蛋白1轻链3（LC3）蛋白的表达。采用蛋白质印迹检测正常血清、血清饥饿、血清饥饿+不同浓度（10^-10、10^-9、10^-8、10^-7 mol·L^-1）CGRP培养的成骨细胞的LC3和P62蛋白表达;采用蛋白质印迹检测正常血清、血清饥饿、血清饥饿+10^-8 mol·L^-1 CGRP培养不同时间（2、6、12、24、48、72 h）的成骨细胞的LC3蛋白表达,流式细胞数检测细胞凋亡,MDC染色检测细胞自噬泡。采用流式细胞术检测正常血清、血清饥饿、血清饥饿+10^-8 mol·L^-1 CGRP、3-MA预处理+血清饥饿、3-MA预处理+血清饥饿+10^-8 mol·L^-1 CGRP培养24 h的成骨细胞的凋亡。结果 血清饥饿培养时成骨细胞的LC3Ⅱ蛋白表达及细胞凋亡较正常血清时增加,3-MA预处理+血清饥饿培养时成骨细胞的凋亡较血清饥饿时增加（P<0.01）。与正常血清相比,血清饥饿、血清饥饿+CGRP培养时LC3Ⅱ蛋白表达增加,P62蛋白表达降低,以血清饥饿+10^-8 mol·L^-1 CGRP培养24 h时LC3Ⅱ/Ⅰ比值最高;血清饥饿+10^-8 mol·L^-1 CGRP培养能抑制成骨细胞的凋亡,促进自噬泡的合成。3-MA预处理后MC3T3-E1成骨细胞凋亡增加,CGRP部分逆转3-MA预处理所增加的成骨细胞凋亡。结论 CGRP能够增强血清饥饿下MC3T3-E1成骨细胞的自噬活性,并可能通过促进自噬抑制成骨细胞的凋亡。     

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.     

The effects of enamel matrix derivative on the proliferation and differentiation of human mesenchymal stem cells

Purpose: This study was designed to investigate the effect of enamel derivative matrix (EMD) on the proliferation, mineralization, and differentiation of human mesenchymal stem cells (hMSCs). Material and methods: For the proliferation assay, water‐soluble tetrazolium salt‐8 tests were carried out after culturing for 24 and 48 h. For the evaluation of mineralization, Alizarin red S (ARS) tests were performed after 21 days of culturing in an osteogenic medium. In order to investigate some of the bone‐related proteins, namely type I collagen (Col I A2), bone sialoprotein (BSP), and bone γ‐carboxyglutamate (Gla) protein (BGLAP, osteocalcin), real‐time polymerase chain reaction (RT‐PCR) tests were carried out after 2, 3, and 4 weeks of culturing, respectively. Results: The activity of proliferation and mineralization increased significantly depending on the concentration of EMD (P<0.05). In the control group, the expression of Col I A2 decreased, but EMD enhanced its expression over time and was correlated to the concentration. The amount of expression of BSP in this group increased over time, but EMD strikingly suppressed its expression in the fourth week. As well, the amount of expression of BGLAP increased as the culture duration lengthened in the control group. However, the expression of BGLAP was suppressed in the experimental group with EMD. Conclusion: Within the limits of this study, EMD enhanced the proliferation of hMSCs. After evaluation with ARS staining, EMD seemed to enhance mineralization, and the RT‐PCR test revealed that EMD promoted early‐stage osteoblast differentiation by enhancing Col I A2 expression, but exerted an inhibitory effect on the mineralization by lowering the gene expression of BSP and BGLAP. Mineralized nodules formed with EMD may be composed of substances other than normal bone. Because most of the organic matrix of bone is type I collagen, which acts as the mineralization site, bone or bone‐like mineralized mass might have been formed in spite of the different components of the non‐collagenous proteins. To cite this article:
Jue S‐S, Lee WY, Kwon Y‐D, Kim Y‐R, Pae A, Lee B. The effects of enamel matrix derivative on the proliferation and differentiation of human mesenchymal stem cells.
Clin. Oral Impl. Res. 21 , 2010; 741–746.
doi: 10.1111/j.1600‐0501.2009.01901.x     

Parathyroid hormone induces mitogen-activated kinase phosphatase 1 in murine osteoblasts primarily through cAMP-protein kinase A signaling

BACKGROUND: Parathyroid hormone (PTH) regulates osteoblast function by binding to the PTH receptor 1 (PTHR1) to activate downstream signaling to induce expression of primary response genes (PRGs), which affect various aspects of the osteoblast phenotype. We previously identified PTH-induced PRGs in MC3T3-E1 cells, including mitogen-activated protein kinase (MAPK) phosphatase 1 (mkp1), which dephosphorylates members of the MAPK family. The aim of this study was to explore the molecular mechanisms of PTH's induction of mkp1 in primary mouse osteoblasts. METHODS: Northern and Western analyses were used to determine mkp1 mRNA and protein expression. In vivo experiments were also performed to determine PTH's effect on mkp1 in mouse calvariae and long bones. RESULTS: A total of 10 nM PTH and PTH-related protein (PTHrP) maximally induced mkp1 mRNA levels after 1 hour in osteoblasts. PTH also increased mkp1 protein expression, and induced mkp1 mRNA independent of new protein synthesis. PTHR1 triggers protein kinase A (PKA), PKC, and calcium pathways. Although PKA and PKC agonists induced mkp1 mRNA levels, only cyclic adenosine 3':5'-monophosphate (cAMP)-PKA inhibition blocked PTH-induced mkp1 mRNA levels. These data suggest that PTH-induced mkp1 mRNA levels are primarily mediated through the cAMP-PKA pathway. Further, prostaglandin E2 (PGE2), which activates cAMP-PKA and PKC, induced mkp1 mRNA to a greater extent than PGF2alpha and fluprostenol, which activate PKC signaling only. Finally, PTH maximally induced mkp1 mRNA levels in mouse calvariae and long bones in vivo at 0.5 hours. CONCLUSIONS: mkp1's in vitro and in vivo induction in PTH-target tissues suggests its involvement in some of the effects of PTH on osteoblast function. mkp1 may be an important target gene in the anabolic effect of PTH on osteoblasts.     

Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: consequences for bone repair during periodontal disease

A prime pathogenic agent associated with periodontitis is lipopolysaccharide (LPS) derived from Porphyromonas gingivalis . This study investigated the effects of P. gingivalis LPS on osteoblasts, which are responsible for alveolar bone repair. Bone cells were obtained from explants of rat alveolar bone chips and cultured with 0–200 ng ml⁻¹ of P. gingivalis LPS. Porphyromonas gingivalis LPS significantly increased cell proliferation and inhibited osteoblast differentiation, as judged by reduced alkaline phosphatase activity. Analysis of biglycan mRNA and protein levels indicated that P. gingivalis LPS significantly delayed the normally high expression of biglycan during the early stages of culture, which are associated with cell proliferation and early differentiation of progenitor cells. In the presence of P. gingivalis LPS, decorin expression by the alveolar bone cells was reduced during periods of culture relating to collagen fibrillogenesis and mineral deposition. Analysis of glycosaminoglycan chains conjugated to these proteoglycans suggested that in the presence of P. gingivalis LPS, dermatan sulfate persisted within the matrix. This study suggests that P.  gingivalis LPS influences the expression and processing of decorin and biglycan in the matrix, altering alveolar bone cell activity and osteoblast phenotype development. The consequences of this altered expression in relation to hindering bone repair as part of the cycle of events during periodontal disease are discussed.