Enamel matrix derivative stimulates human gingival fibroblast proliferation via ERK

Emdogain, a formulation of Enamel Matrix Proteins, is used clinically for periodontal regeneration to stimulate PDL (periodontal ligament), cementum, and bone formation. Its effects on gingival fibroblasts and tissue have not been thoroughly studied. Therefore, we investigated the mechanisms by which Emdogain affects the cell cycle of human gingival fibroblasts. Without serum, Emdogain (50 microg/mL) induced human gingival fibroblast entry into the S phase and DNA synthesis, but not completion of the cell cycle. With low serum concentrations (0.2-0.5%), Emdogain synergistically induced completion of the cell cycle, resulting in increased cell numbers. The mitogenic response to Emdogain depended on Extracellular Regulated Kinase (ERK) activation, which occurred in two waves, peaking after 15 min and 4 to 6 hrs, since it was abolished by U0126, a specific MAPK inhibitor. Inhibition of the second wave was sufficient to abrogate mitogenesis. This study characterized the mitogenic effect of Emdogain on primary human gingival fibroblasts, its cooperation with serum growth factors, and the key mediatory role of the ERK cascade.     

Enamel matrix derivative improves gingival fibroblast cell behavior cultured on titanium surfaces

Objective

Although an extensive amount of research has demonstrated the positive effects of an enamel matrix derivative (EMD) on soft tissue wound healing around intrabony defects, little information is available describing its effect on peri-implant soft tissues, an area that has recently gained tremendous awareness due to the increasing prevalence of peri-implantitis. The aim of the present study was to assess the role of EMD when gingival fibroblasts were cultured on titanium surface with different surface topographies.

Methods

Human primary gingival fibroblasts were cultured on pickled (PT) and sand-blasted with large grit followed by acid etching (SLA) surfaces and assessed for cell adhesion at 2, 4, and 8 h, cell morphology at 2, 4, 8, and 24 h as well as cell proliferation at 1, 3, and 5 days post-seeding. Furthermore, genes encoding collagen 1a1, vascular endothelial growth factor-A (VEGF-A), and fibronectin were assessed by real-time PCR. Human gingival fibroblasts were also quantified for their ability to synthesize a collagen matrix on the various titanium surfaces with and without EMD by immunofluorescence staining.

Results

The results from the present study demonstrate that EMD significantly increased cell spreading at 2, 4, 8, and 24 h on PT surfaces and 4, 8, and 24 h on SLA surfaces. Furthermore, proliferation at 5 days on PT surfaces and 3 and 5 days on SLA surfaces was also increased for groups containing EMD. Real-time PCR results demonstrated that the culture of gingival fibroblasts with EMD significantly increased extracellular matrix synthesis of collagen 1 as well as improved mRNA levels of VEGF-A and fibronectin. Collagen1 immuno-fluorescent staining revealed a significantly higher area of staining for cells seeded on PT + EMD at 7 and 14 days and 14 days for SLA + EMD when compared to control samples.

Conclusion

The results from the present study favor the use of EMD for colonization of gingival fibroblasts on titanium surfaces by increasing cell growth, spreading, and synthesis of an extracellular matrix. The improvements were primarily irrespective of surface topography. Future animal and human studies are necessary to fully characterize the beneficial effects of incorporating EMD during soft tissue regeneration of implant protocols.

Clinical relevance

The use of EMD may speed up the quality of soft tissue integration around dental implants by facilitating gingival cell attachment, proliferation, and matrix synthesis of collagen 1.

     

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.     

Enamel matrix derivative promotes human periodontal ligament cell differentiation and osteoprotegerin production in vitro

Enamel matrix derivative (EMD) has been used successfully to aid periodontal repair. We sought to elucidate the mechanism of action of EMD and hypothesized that combined exposure to EMD and parathyroid hormone (PTH), which acts anabolicly when administered intermittently, would enhance periodontal ligament cell proliferation, differentiation, and local factor production. Confluent human periodontal ligament cells were exposed to EMD continuously or to PTH(1-34) intermittently, or a combination of both. Cell number, alkaline phosphatase activity, osteocalcin, and osteoprotegerin production were determined. Continuous challenge with EMD resulted in an increase of the differentiation parameters and osteoprotegerin production, while simultaneously inhibiting proliferation. Intermittent PTH(1-34) administration exerted opposite effects. Combined administration of EMD and PTH(1-34) weakened or even nullified the effects seen for the agents alone. These results suggest that EMD promotes periodontal ligament cell differentiation and osteoprotegerin production, potentially resulting in a microenvironment supporting periodontal repair, whereas combining EMD and PTH(1-34) failed to prove beneficial in this respect.     

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor‐β1 (TGF‐β1), or fibroblast growth factor 2 (FGF‐2), with or without antibodies to TGF‐β1 or FGF‐2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen‐activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal‐regulated kinase (ERK) inhibitor], SP600125 [a c‐Jun N‐terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3‐kinase (PI3K)/Akt inhibitor] on EMD‐induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose‐ and time‐dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF‐β1 or FGF‐2 significantly decreased EMD‐induced VEGF production, whereas the addition of exogenous TGF‐β1 and FGF‐2 stimulated VEGF production. Enamel matrix derivative‐induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF‐β1 and FGF‐2 in human gingival fibroblasts and that EMD‐induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative‐induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.     

Enamel matrix derivative and titanium implants

AIM: The aim of present study was to evaluate if an enamel matrix derivative (Emdogain) may enhance bone formation and osseointegration of titanium implants, using a well-documented rabbit model. MATERIAL AND METHODS: Thirty-six threaded commercially pure titanium (cp.ti.) implants were inserted in six New Zealand white rabbits. One implant was placed in each femur and two in each tibia. Prior to implant insertion approximately 0.5 mL of Emdogain (EMD) (test) or the vehicle gel (PGA: propylene glycol alginate) (control) was injected into the surgically prepared implant site. The follow-up time was 6 weeks. Biomechanical evaluations by resonance frequency analysis (RFA) and removal torque measurements (RTQ) were performed. Histomorphometrical quantifications were made on ground sections by measurements of the percentage of bone-to-metal contact, bone area inside the threads as well as outside the threads (mirror image). Bone lengths along the implant surface were also measured and used for shear strength calculations. RESULTS: The results demonstrated no beneficial effects from the EMD treatment on bone formation around titanium implants in any of the tested parameters. Significant differences were demonstrated with removal torque test and shear force calculations for the control implants. No other parameter demonstrated a statistically significant difference. CONCLUSION: The results of the present study may indicate that EMD does not contribute to bone formation around titanium implants. This observation may indicate that the bone formation that occurs after EMD treatment in periodontal defects is the result of functional adaptation. However, further research is required to evaluate the effect of EMD treatment on bone formation.     

Enamel matrix derivative (Emdogain®) for periodontal tissue regeneration in intrabony defects

Background: Periodontitis is a chronic infective disease of the gums caused by bacteria present in dental plaque. This condition induces the breakdown of the tooth supporting apparatus until teeth are lost. Surgery may be indicated to arrest disease progression and regenerate lost tissues. Several surgical techniques have been developed to regenerate periodontal tissues including guided tissue regeneration (GTR), bone grafting (BG) and the use of enamel matrix derivative (EMD). EMD is an extract of enamel matrix and contains amelogenins of various molecular weights. Amelogenins are involved in the formation of enamel and periodontal attachment formation during tooth development. Objectives: To test whether EMD is effective, and to compare EMD versus GTR, and various BG procedures for the treatment of intrabony defects. Search strategy: We searched the Cochrane Oral Health Group Trials Register, CENTRAL, MEDLINE and EMBASE. Several journals were handsearched. No language restrictions were applied. Authors of randomized controlled trials (RCTs) identified, personal contacts and the manufacturer were contacted to identify unpublished trials. Most recent search: February 2009. Selection criteria: RCTs on patients affected by periodontitis having intrabony defects of at least 3 mm treated with EMD compared with open flap debridement, GTR and various BG procedures with at least 1 year follow‐up. The outcome measures considered were: tooth loss, changes in probing attachment levels (PAL), pocket depths (PPD), gingival recessions (REC), bone levels from the bottom of the defects on intraoral radiographs, aesthetics and adverse events. The following time‐points were to be evaluated: 1, 5 and 10 years. Data collection and analysis: Screening of eligible studies, assessment of the methodological quality of the trials and data extraction were conducted in duplicate and independently by two authors. Results were expressed as random‐effects models using mean differences for continuous outcomes and risk ratios (RR) for dichotomous outcomes with 95% confidence intervals (CI). It was decided not to investigate heterogeneity, but a sensitivity analysis for the risk of bias of the trials was performed. Main results: Thirteen trials were included out of 35 potentially eligible trials. No included trial presented data after 5 years of follow‐up, therefore all data refer to the 1‐year time point. A meta‐analysis including nine trials showed that EMD treated sites displayed statistically significant PAL improvements (mean difference 1.1 mm, 95% CI 0.61 to 1.55) and PPD reduction (0.9 mm, 95% CI 0.44 to 1.31) when compared to placebo or control treated sites, though a high degree of heterogeneity was found. Significantly more sites had <2 mm PAL gain in the control group, with RR 0.53 (95% CI 0.34 to 0.82). Approximately nine patients needed to be treated (NNT) to have one patient gaining 2 mm or more PAL over the control group, based on a prevalence in the control group of 25%. No differences in tooth loss or aesthetic appearance as judged by the patients were observed. When evaluating only trials at a low risk of bias in a sensitivity analysis (four trials), the effect size for PAL was 0.62 mm (95% CI 0.28 to 0.96), which was less than 1.1 mm for the overall result. Comparing EMD with GTR (five trials), GTR showed statistically significant more postoperative complications (three trials, RR 0.12, 95% CI 0.02 to 0.85) and more REC (0.4 mm 95% CI 0.15 to 0.66). The only trial comparing EMD with a bioactive ceramic filler found statistically significant more REC (‐1.60 mm, 95% CI ?2.74 to ?0.46) at the EMG treated sites. Authors’ conclusions: One year after its application, EMD significantly improved PAL levels (1.1 mm) and PPD reduction (0.9 mm) when compared to a placebo or control, however, the high degree of heterogeneity observed among trials suggests that results have to be interpreted with great caution. In addition, a sensitivity analysis indicated that the overall treatment effect might be overestimated. The actual clinical advantages of using EMD are unknown. With the exception of significantly more postoperative complications in the GTR group, there was no evidence of clinically important differences between GTR and EMD. Bone substitutes may be associated with less REC than EMD. Plain language summary: Enamel matrix derivative (Emdogain®) for periodontal tissue regeneration in intrabony defects. Emdogain might have some advantages over other methods of regenerating the tissue supporting teeth lost by gum disease, such as less postoperative complications, but has not been shown to save more compromised teeth or that patients noticed any aesthetic improvement 1 year after its application. Bacteria in plaque can cause gum disease (periodontitis) that breaks down tissue supporting teeth. Surgical cleaning tries to stop the disease to save loose teeth. Bone grafting, guided tissue regeneration and enamel matrix derivatives (such as Emdogain) aim to regenerate support tissues. Emdogain contains proteins (derived from developing pig teeth) believed to regenerate tooth attachment. The review found that adjunctive application of Emdogain regenerates about 1 mm more tissue than surgical cleaning alone, although it is unclear to which extent such improvement is noticeable since patients did not find any difference in the aesthetic results. Emdogain showed similar clinical results to guided tissue regeneration, but is simpler to use and determines less complications. Bone substitutes may induce less gum retraction than Emdogain. No serious adverse reactions to Emdogain were reported in trials.     

Enamel matrix derivative in the treatment of human intrabony osseous defects

BACKGROUND: There is limited information available from clinical trials regarding the performance of enamel matrix derivative (EMD) in the treatment of periodontal intrabony defects. This randomized, double-blind, placebo-controlled, split-mouth study was designed to compare the clinical and radiographical effects of EMD treatment to that of placebo-controlled treatment for intrabony defects. METHODS: Sixteen patients were included, each of whom had 1 or 2 pairs of intrabony defects located contralaterally in the same arch. Thirty-six intrabony defects were randomly assigned treatment with flap surgery plus EMD or flap surgery plus placebo. At baseline and at the 12-month follow-up evaluation visit, clinical and radiographic measurements were determined. Data were statistically analyzed using the Wilcoxon-signed rank test (alpha = 0.05). RESULTS: At the 12-month visit, bleeding on probing for the EMD group was 0.11 +/- 0.32 compared to the placebo group, 0.61 +/- 0.50 (P <0.05). Probing depth reduction was greater in the EMD group (3.00 +/- 0.97 mm) compared to the placebo group (2.22 +/- 0.81 mm) (P <0.05). Mean values for clinical attachment gain in the EMD and the placebo groups were 1.72 +/- 1.07 mm and 0.83 +/- 0.86 mm, respectively (P <0.05). Vertical relative attachment gain was 38.5 +/- 22.6% in the EMD group and 21.4 +/- 25.2% in the placebo group (P<0.05). Radiographic bone density gain was greater in the EMD (20.2 +/- 16.6%) compared to the placebo group (-3.94 +/- 23.3%) (P<0.01). CONCLUSIONS: Treatment with flap surgery and EMD, compared to flap surgery with placebo, produced a significantly more favorable clinical improvement in intrabony periodontal defects.     

Enamel matrix derivative and systemic antibiotics as adjuncts to non-surgical periodontal treatment: biologic response

BACKGROUND: Short-term clinical observations suggest an anti-inflammatory effect of enamel matrix derivative (EMD). The purpose of this study was to evaluate the anti-inflammatory capacity of EMD, used as an adjunct to non-surgical periodontal treatment of deep lesions in chronic periodontitis patients, by monitoring inflammatory markers in gingival crevicular fluid (GCF). METHODS: Sixteen subjects were randomly assigned to treatment with EMD or placebo in contralateral dentition areas. Half of the subjects received 250 mg metronidazole and 375 mg amoxicillin three times a day for 7 days; the other half received a placebo. GCF samples were collected from one interproximal lesion in each of the contralateral quadrants before treatment and after 10 days and 2, 6, and 12 months. Total protein content was determined according to the Bradford method. Myeloid-related protein (MRP) 8/14 and interleukin (IL)-1beta were analyzed quantitatively by enzyme-linked immunosorbent assay (ELISA), and elastase activity was determined using a low molecular weight fluorogenic substrate. RESULTS: No significant differences were observed between sites treated with or without EMD for any biochemical parameter. Two months after treatment, subjects treated with antibiotics exhibited less clinical signs of inflammation. Furthermore, these subjects had lower MRP 8/14 levels only at day 10 compared to those receiving the placebo. For total protein, IL-1beta, and elastase, no statistically significant differences were noted for subjects with or without antibiotic therapy at any time point. CONCLUSIONS: Improved healing of the soft tissues has been noted clinically in non-surgically treated sites in subjects treated with antibiotics. The expression of inflammatory mediators in GCF corroborated this finding only in part. EMD did not seem to further affect the expression of inflammatory mediators.     

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.     

釉基质蛋白对人牙周膜细胞形成Ⅰ型胶原、Ⅲ型胶原的影响

目的:观察釉基质蛋白对体外培养的人牙周膜细胞形成Ⅰ型胶原、Ⅲ型胶原的影响.方法:改良组织块法培养人牙周膜细胞,免疫细胞化学方法和图像分析方法观察细胞形成Ⅰ型胶原、Ⅲ型胶原的能力.结果:50、100、200 mg/L的釉基质蛋白可以促进牙周膜细胞合成Ⅰ、Ⅲ型胶原,其中,以100 mg/L釉基质蛋白的促Ⅰ型胶原合成作用最明显,50 mg/L釉基质蛋白的促Ⅲ型胶原合成作用最明显.但是,这种促进作用有一定的时间性.结论:一定浓度的釉基质蛋白可以促进牙周膜细胞形成Ⅰ型胶原、Ⅲ型胶原.     

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.     

Effects of mechanical tension on matrix degradation by human periodontal ligament cells cultured in collagen gels

BACKGROUND: Periodontal ligament (PDL) cells are thought to play a crucial role in the remodelling of periodontal tissues during orthodontic tooth movement. OBJECTIVE: The objective of this study was to analyse the effects of mechanical tension on matrix degradation by PDL cells cultured in collagen gels. METHODS: The gels were prepared free-floating or attached to the culture wells and cultured for up to 22 d. In free-floating gels very little mechanical tension is generated within the matrix, whereas in attached gels tension is highly increased. RESULTS: At d 8, free-floating gels had contracted to 2% of their original wet weight. Attached gels had contracted to only 40%, but by d 15 all gels had spontaneously detached from the wells and had contracted rapidly. The collagen content of free-floating gels had decreased to 30% of the initial value at d 22. Collagenase activity was detected in the culture media of the free-floating gels and the presence of matrix metalloproteinases (MMPs) 2 and 9 was shown by zymography. In addition, histological sections showed matrix degradation around the cells. This shows that ligament cells in free-floating gels are actively resorbing the collagen matrix. The collagen content of attached gels did not change during the first 8 d but, after detachment, it rapidly decreased to 2%. Therefore, mechanical tension seems to prevent degradation of the matrix. In contrast, relaxation of the tension enhances the resorptive activity. CONCLUSIONS: The sensitivity of PDL cells to mechanical tension may be essential for the remodelling of periodontal tissues and their adaptation to physiological and orthodontic forces.