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     

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.     

Effects of enamel matrix derivative on proliferation and differentiation of primary osteoblasts

OBJECTIVE: The aim of this study was to investigate the effects of enamel matrix derivative (EMD) on proliferation, protein synthesis, and mineralization in primary mouse osteoblasts. STUDY DESIGN: Osteoblasts were obtained from mouse calvaria by enzymatic digestion and grown in monolayer together with EMD (2-100 microg/ml). Metabolic activity and cell proliferation were determined by tetrazolium salt assay (MTT) and by 5-bromo-2'-deoxyuridine (BrdU) incorporation. For differentiation studies, a 3-dimensional organoid culture system was used. Osteoblastic differentiation was estimated by alkaline phosphatase (ALP) activity and calcium content. Collagen synthesis was assessed by [(3)H]-proline incorporation. Morphologic observations were made by electron microscopy. RESULTS: EMD treatments increased metabolic cell activity and BrdU incorporation. In the organoid cultures, ALP activity and calcium accumulation were enhanced by EMD treatment, but [(3)H]-proline incorporation was not. Morphologically, an increased deposition of mineralized nodules was found. CONCLUSIONS: EMD treatment enhanced cellular activities of primary osteoblasts and might support the regeneration of periodontal bony defects.     

釉基质衍生物对人牙周膜干细胞成骨分化的影响

目的研究釉基质衍生物对人牙周膜干细胞增殖和成骨分化的影响并探究其可能的机制。方法原代培养人牙周膜干细胞,经过流式鉴定后选取第3代细胞进行实验。采用CCK-8试剂盒检测不同浓度(0、20、50、100 mg·L^-1)的釉基质衍生物对人牙周膜干细胞增殖的影响;实时荧光定量聚合酶链式反应(qRT-PCR)检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞成骨分化的影响;通过Trichrome染色和Von Kosa’s染色检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞胶原合成和矿化结节形成的影响;不同浓度釉基质衍生物和DDK1作用人牙周膜干细胞之后,通过Western blot和qRT-PCR检测β-连环蛋白、RunX2、CaMKⅡ及NLK表达情况。结果釉基质衍生物对人牙周膜干细胞的增殖具有明显的促进作用,并呈现剂量和时间依赖性;釉基质衍生物处理人牙周膜干细胞之后,矿化结节形成和胶原合成显著增多,骨钙素、Ⅰ型胶原、RunX2的表达明显增多;另外,釉基质衍生物处理能显著增加β-连环蛋白、RunX2、CaMKⅡ和NLK的表达,且该作用可被DDK1抑制。结论釉基质衍生物对体外培养的人牙周膜干细胞有促进增殖和成骨分化的作用,其作用可能是通过Wnt/β-连环蛋白实现的。     

In vitro studies on periodontal ligament cells and enamel matrix derivative

Abstract The recognition that periodontal regeneration can be achieved has resulted in increased efforts focused on understanding the mechanisms and factors required for restoring periodontal tissues so that clinical outcomes of such therapies are more predictable than those currently being used. In vitro models provide an excellent procedure for providing clues as to the mechanisms that may be required for regeneration of tissues. The investigations here were targeted at determining the ability of enamel matrix derivative (EMD) to influence specific properties of periodontal ligament cells in vitro. Properties of cells examined included migration, attachment, proliferation, biosynthetic activity and mineral nodule formation. Immunoassays were done to determine whether or not EMD retained known polypeptide factors. Results demonstrated that EMD under in vitro conditions formed protein aggregates, thereby providing a unique environment for cell-matrix interaction. Under these conditions, EMD: (a) enhanced proliferation of PDL cells, but not of epithelial cells; (b) increased total protein production by PDL cells; (c) promoted mineral nodule formation of PDL cells, as assayed by von Kossa staining; (d) had no significant effect on migration or attachment and spreading of cells within the limits of the assay systems used here. Next, EMD was screened for possible presence of specific molecules including: GM-CSF, calbindin D, EOF, fibronectin, bFGF, γ-interferon. IL-1β, 2, 3, 6; IGF-1,2; NGF, PDGF, TNF, TGFβ. With immunoassays used, none of these molecules were identified in EMD. These in vitro studies support the concept that EMD can act as a positive matrix for cells at a regenerative site.     

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.     

Effect of enamel matrix derivative on periodontal ligament cells in vitro is diminished by Porphyromonas gingivalis

BACKGROUND: Enamel matrix derivative (EMD) has been shown to possess a mitogenic effect to induce effective periodontal regeneration, however, it is unclear whether periodontal pathogens can modulate the effect of EMD. The present study examined the influence of Porphyromonas gingivalis on EMD-stimulated periodontal ligament (PDL) cells. METHODS: P. gingivalis ATCC33277 and its mutants deficient in fimbriae (delta fimA) or gingipains (delta rgpA delta rgpB, delta kgp, and delta rgpA delta rgpB delta kgp) were employed. PDL cells were grown on EMD-coated dishes and infected with P. gingivalis wild strain or a mutant. Cell migration and proliferation were then evaluated with an in vitro wound healing assay. The expression of transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor I (IGF-I) mRNA by PDL cells was examined. Further, the degradation and phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) as well as paxillin in infected PDL cells were estimated using Western blot analysis. RESULTS: P. gingivalis ATCC33277 inhibited the migration and proliferation of PDL cells on EMD-coated dishes, and the mutants delta fimA, delta rgpA delta rgpB, and delta kgp showed the same effects. Further, each of these organisms diminished the expression of TGF-beta1 and IGF-I mRNA, as well as the phosphorylation of ERK1/2 from EMD-stimulated PDL cells. In addition, total paxillin protein was markedly degraded by both the wild-type strain and each of the mutants except for delta rgpA delta rgpB delta kgp, which showed a negligible effect in all of the assays with EMD-stimulated PDL cells. CONCLUSION: These results suggest that P. gingivalis diminishes the effect of EMD on PDL cells in vitro through a cooperative action of gingipains.     

釉基质蛋白对人牙髓细胞成牙本质分化的影响

目的 探讨釉基质蛋白对人牙髓细胞(human dental pulp cells,HDPC)成牙本质分化的影响,为牙本质的组织工程学研究提供有效的生物活性物质.方法 将HDPC接种于6孔板(2×105个/孔)后分为5组,分别加入含1、10、100 mg/L釉基质蛋白(enamel matrix proteins,EMP)(分别为EMP 1、10、100 mg/L组)、10-8 mol/L地塞米松及100 mg/L抗坏血酸(dexamethasone and ascorbic acid,Dex-AA组)、单纯基础培养液(对照组).于培养1、5、10 d分别用碱性磷酸酶(alkaline phosphatase,ALP)试剂盒检测ALP活性,实时荧光定量反转录聚合酶链反应技术检测成牙本质相关基因牙本质基质蛋白1(dentine matrix protein-1,DMP-1)及牙本质涎磷蛋白(dentine sialophosphoprotein,DSPP)的表达,应用2-△△CT方法得出具体数值并进行单因素方差分析与Post Hoe 检验,用茜素红染色检测并定量分析矿化情况.结果 各组ALP水平呈时间依赖性上调,培养1d后各组ALP活性与对照组相比差异均无统计学意义;5 d后EMP 10、100 mg/L组及Dex-AA组ALP活性显著增高,分别达到7.573±0.267、6.119±0.502、5.846±0.096,与对照组相比差异均有统计学意义(P＜0.05);10 d后10 mg/L EMP组及Dex-AA组ALP活性增高更显著,分别达21.035±0.149、13.223±0.797,与对照组(5.825±0.404)相比差异均有统计学意义(P＜0.01).培养1d后各组DMP-1及DSPP mRNA水平均较对照组显著升高,差异均有统计学意义(P＜0.01);培养10 d后10 mg/L EMP组DMP-1和DSPP表达水平均显著增高,分别达到14.791±0.164、12.238±0.421.培养10 d后各组均有矿化,10 mg/L EMP组钙离子浓度[(191.8±2.0) μmol/L]显著高于对照组[(81.1±8.1)μmol/L],差异有统计学意义(P＜0.01).结论 适当浓度的EMP对HDPC的成牙本质分化有一定的促进作用.     

Effects of enamel matrix derivative on Porphyromonas gingivalis

BACKGROUND: Enamel matrix derivative (EMD) is used during periodontal surgery for the regeneration of periodontal tissue. It consists of the amelogenin fraction of porcine enamel matrix (AMEL) suspended in a vehicle of propylene glycol alginate (PGA). EMD-treated sites appear to heal with less inflammation. It has been suggested that antimicrobial properties of EMD might account for improved healing in vivo. The objectives of this study were: 1) to determine the antibacterial effects of EMD on the periodontal pathogen Porphyromonas gingivalis and 2) to establish the component(s) of EMD that are responsible for this effect. METHODS: The antimicrobial effects were determined in vitro using the broth dilution assay. P. gingivalis at a starting inoculum of 10(9) colony forming units/ml was treated with EMD, AMEL, and PGA in Hank's balanced salt solution (HBSS) for 1, 3, and 24 hours. The CFU/ml of P. gingivalis recovered on enriched Brucella blood agar was determined at 6 and 10 days. RESULTS: EMD (containing AMEL and PGA) or PGA alone eliminated recoverable CFUs of P. gingivalis. Interestingly, AMEL in HBSS increased recoverable CFUs from 8.62 log CFU/ml to 8.93 log CFU/ml. Further analysis of the dose response at concentrations of 0.3, 3, and 30 mg/ml of AMEL in HBSS revealed that only 30 mg/ml (clinical concentration) increased CFUs of P. gingivalis relative to baseline (from 8.8 log CFU/ml to 9.2 log CFU/ml in 3 hours). Additionally, AMEL was compared to a protein control bovine serum albumin (BSA) to determine whether this effect was unique to AMEL. A marked increase in recoverable CFUs occurred with the AMEL (increasing from 8.8 log CFU/ml to 9.47 log CFU/ml), but not with BSA. CONCLUSIONS: EMD possesses antimicrobial properties that can be attributed to the propylene glycol alginate vehicle. The amelogenin fraction of porcine enamel matrix in enamel matrix derivative (i.e., AMEL) is not antibacterial for P. gingivalis and was shown to increase recoverable CFUs.     

The influence of enamel matrix derivative on the angiogenic activity of primary endothelial cells

Kasaj A, Meister J, Lehmann K, Stratul SI, Schlee M, Stein JM, Willershausen B, Schmidt M. The influence of enamel matrix derivative on the angiogenic activity of primary endothelial cells. J Periodont Res 2012; 47: 479–487. © 2011 John Wiley & Sons A/S Background and Objective: Angiogenesis plays a crucial role in early wound healing and tissue regeneration. Although enamel matrix derivative (EMD) has demonstrated the potential to stimulate periodontal regeneration, the biological effects of EMD on angiogenesis and underlying mechanisms have not been fully elucidated. The aim of the present study was to examine the angiogenic effects of EMD in vitro. Material and Methods: Human umbilical vein endothelial cells (HUVECs) were used to assess the effect of EMD on proliferation, survival, adhesion and migration. The effect of EMD on HUVEC angiogenesis was assessed by a three‐dimensional sprouting assay. In order to understand the signalling mechanism of altered cell proliferation of HUVECs caused by EMD, the phosphorylation status of ERK1/2 and of the serine/threonine protein kinase Akt was analysed by western blot using phospho‐specific antibodies. Results: The proliferation of HUVECs was stimulated by 50 μg/mL EMD, whereas higher concentrations (≥ 100 μg/mL) resulted in an increased apoptotic rate. The mitogenic response to EMD was associated with the activation of ERK1/2. Enamel matrix derivative did not affect cell adhesion, but all concentrations of EMD tested (0.1–250 μg/mL) promoted migration of HUVECs. Furthermore, EMD induced capillary‐like sprout formation from HUVEC spheroids in a dose‐dependent manner. Conclusion: Our data indicate that EMD acts as a proangiogenic factor in vitro and, as such, might contribute to periodontal tissue regeneration by stimulation of vessel formation during wound healing.     

Effect of the enamel matrix derivative Emdogain on the growth of periodontal pathogens in vitro

OBJECTIVES: The aim of this study was to evaluate the effect of Emdogain (EMD), used for periodontal regeneration, on the growth of periodontal pathogens like Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia. For comparison, we studied the effect of EMD on several microbes associated with other oral diseases as well as its effect on non-pathogenic oral inhabitants. METHODS: Freshly prepared EMD or its vehicle propylene glycol alginate (PGA) alone were added to calibrated suspensions of microbes. As a control, imitating the post-surgical subgingival situation after flap closure, a serum/NaCl-solution mixture was used. Aliquots for growth assays were taken at scheduled times for calculation of colony-forming units and cell densities over an observation period of 24 h. Additionally, EMD was spotted onto selected, newly seeded microbes growing on agar plates to see if growth inhibition zones could be produced. RESULTS: The study revealed a marked inhibitory effect of EMD on the growth of the gram-negative periodontal pathogens. A. actinomycetemcomitans showed a significant decrease (p=0.012) in viable counts after 24 h when EMD was added at baseline. P. gingivalis and P. intermedia also showed a marked growth reduction in the presence of EMD and in these cultures no viable microbes could be detected anymore after 24 h. In contrast, no significant growth inhibition was observed in gram-positive bacteria. CONCLUSIONS: The results suggest that EMD has a positive effect on the composition of bacterial species in the post-surgical periodontal wound, by selectively restricting growth of periopathogens that could hamper the wound healing and reduce the outcome of regenerative procedures.     

Effect of enamel matrix derivative on collagen guided tissue regeneration-based root coverage procedure

BACKGROUND: Enamel matrix derivative (EMD) has been shown to promote periodontal wound healing and/or regeneration when applied to tooth root surfaces in soft tissue dehiscence models. In addition, guided tissue regeneration (GTR)-based root coverage using collagen membrane (GTRC) has shown promising results. However, limited information is available regarding how EMD may influence GTRC outcome. METHODS: Twenty-six patients with Miller's Class I or II gingival recession defects of 2.5 mm were recruited for the study. Subjects were randomly assigned to receive either EMD + collagen (EMDC; test group) or collagen membrane (GTRC; control group). Clinical parameters, including plaque index (PI), gingival index (GI), relative clinical attachment levels (RCAL) to the stent, recession depth (RD), recession width (RW), probing depth (PD), gingival tissue thickness (GTT), and width of keratinized gingiva (KG) were assessed at baseline, and 3 and 6 months after surgery. A repeated measure of analysis of variance (ANOVA) was used to determine differences between treatment groups and time effect. RESULTS: Both treatments (GTRC and EMDC) resulted in a statistically significant decrease in RD and RW between baseline and 6 months (P <0.05). However, no difference was noted between treatment groups. The percent of root coverage after 6 months was 75% for GTRC and 63% for EMDC. Complete 100% root coverage was achieved in five patients in the GTRC group, compared to only one patient in the EMDC group. There was a statistically significant gain (P <0.05) in the clinical attachment level (CAL) between baseline and 6 months in both groups, as reflected on the RCAL data. No other significant differences were noted on other clinical parameters (PD, GTT, KG, GI, and PI). CONCLUSIONS: GTR-based root coverage utilizing collagen membrane, with or without enamel matrix derivative, can be successfully used in obtaining gingival recession coverage. The application of EMD during GTRC procedures did not add additional benefit to the final clinical outcome.     

Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative

OBJECTIVE: Enamel extracellular matrix proteins in the form of the enamel matrix derivative EMDOGAIN (EMD) have been successfully employed to mimic natural cementogenesis to restore fully functional periodontal ligament, cementum and alveolar bone in patients with severe periodontitis. When applied to denuded root surfaces EMD forms a matrix that locally facilitates regenerative responses in the adjacent periodontal tissues. The cellular mechanism(s), e.g. autocrine growth factors, extracellular matrix synthesis and cell growth, underlying PDL regeneration with EMD is however poorly investigated. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were cultured on EMD and monitored for cellular attachment rate, proliferation, DNA replication and metabolism. Furthermore, intracellular cyclic-AMP levels and autocrine production of selected growth factors were monitored by immunological assays. Controls included PDL and epithelial cells in parallel cultures. RESULTS: PDL cell attachment rate, growth and metabolism were all significantly increased when EMD was present in cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of TGF-beta1, IL-6 and PDGF AB when compared to controls. Epithelial cells increased cAMP and PDGF AB secretion when EMD was present, but proliferation and growth were inhibited. CONCLUSION: Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-beta1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development.     

Effect of dental materials calcium hydroxide-containing cement, mineral trioxide aggregate, and enamel matrix derivative on proliferation and differentiation of human tooth germ stem cells

Introduction

Biocompatibility of pulp capping materials is important for successful use in dentistry. These materials should be nontoxic and permissive for proliferation and induction of odontogenic differentiation of pulp cells. The aim of our study was to evaluate the effects of enamel matrix derivative (EMD), mineral trioxide aggregate (MTA), and calcium hydroxide-containing cement (DYCAL) on proliferation and odontogenic differentiation of human tooth germ stem cells (hTGSCs) in which cells belonging to both pulp tissue and dental follicle exist.

Methods

The 96-well plates, 24-well plates, and special chamber slides were coated with biomaterials for cell proliferation, differentiation, and scanning electron microscopy analysis. Odontogenic differentiation of hTGSCs was evaluated by analyzing mRNA expression of dentin sialophosphoprotein (DSPP) by real-time polymerase chain reaction expression analysis, measurement of alkaline phosphatase activity, and visualization of calcium depositions by von Kossa staining.

Results

Our results demonstrate that EMD is the best material in terms of inducing differentiation and proliferation of hTGSCs. DYCAL was found to be toxic to hTGSCs; however, EMD-coated DYCAL showed less toxicity. EMD-coated MTA was not efficient at inducing proliferation and differentiation.

Conclusions

Pulp capping materials come in direct contact with dental pulp cells; thus, they require comprehensive evaluation of interactions between cells and biomaterials. Therefore, we cultured hTGSCs, capable of odontogenic differentiation, on pulp capping materials directly. Our results suggest that combination of capping materials with EMD would increase the quality of capping by increasing biocompatibility of capping materials.