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.     

In vitro biologic response of human bone marrow stromal cells to enamel matrix derivative

BACKGROUND: In vitro investigations suggest that enamel matrix derivative (EMD) may affect the biologic response of periodontal-related cells, including osteoblasts and their precursors, the bone marrow stromal cells (BMSCs), which could play a crucial role in the regenerative process. In this study, we investigated the effects of EMD on human BMSCs. METHODS: Primary cultures of BMSCs were obtained from bone marrow samples of healthy donors. Cell proliferation and osteogenic marker expression in response to serial dilutions of EMD (12.5, 25, and 50 microg/ml) were assessed. Cell growth was measured by 3H-thymidine incorporation and type I collagen synthesis by immunoblotting. Alkaline phosphatase (AP)-specific activity in the early phase (7 days), in vitro mineralization by von Kossa staining and calcium quantification, and osteocalcin levels at prolonged times (3 weeks) also were evaluated. RESULTS: EMD stimulated BMSC growth in a dose-dependent manner. When EMD 50 microg/ml was followed over time, the highest proliferative effect was evident at 24 hours (3.4-fold of the control). Type I collagen level was significantly lower than the control after a 7-day incubation with EMD 50 microg/ml. AP activity was reduced in a dose-dependent manner down to 55% of the control. Also, the extracellular matrix mineralization decreased in EMD-treated cells with respect to the control, whereas only a slight, not significant, decrease in osteocalcin levels was found. CONCLUSIONS: EMD significantly increased BMSC growth and simultaneously decreased their osteogenic differentiation. The clinical efficacy of EMD in regenerating periodontal tissues can be attributed, in part, to the biologic effects exerted on the bone marrow stromal component of resident cells.     

Immunohistochemical evaluation of matrix molecules associated with wound healing following treatment with an enamel matrix protein derivative in humans

Application of enamel matrix protein derivative (EMD) onto a debrided and conditioned root surface has been shown to promote periodontal regeneration in animals and humans. However, until now there is virtually no information from humans describing the expression of different matrix molecules in the newly formed periodontal tissues following treatment with EMD. This study investigated immunohistochemically in humans the expression of matrix molecules associated with periodontal tissues reformed after treatment with EMD. Eight patients with intrabony defects were treated with EMD. Six months after surgery teeth together with some of their surrounding soft and hard tissues were removed, fixed in buffered formalin, decalcified in EDTA, and embedded in paraffin. Serial sections of 6 µm were cut in mesiodistal direction. Sections were evaluated immunohistochemically by means of polyclonal antibodies against osteopontin, collagen I and collagen III. The original (non-treated) parts of the periodontium served as controls. In all specimens the healing resulted to a varying extent in formation of cementum, periodontal ligament and alveolar bone. In all specimens the expression of the investigated matrix molecules was stronger at the reformed than at the original sites. Osteopontin expression was most intense at the border near the newly formed cementum and bone. In the regenerated periodontal ligament, collagen I and III were localized throughout the entire periodontal ligament connective tissue. Within the newly formed PDL connective tissue the immunohistochemical staining appeared stronger for collagen III than for collagen I. The present findings suggest that (a) treatment of human intrabony defects with EMD creates an environment favourable for periodontal regeneration and, (b) in humans the healing and/or remodelling process of the reformed tissues may be followed immunohistochemically for a period of 6 months.     

釉基质蛋白对牙周细胞覆盖体外创面的影响

目的：评价体外创面模型环境中,釉基质蛋白对牙周膜细胞、牙龈成纤维细胞覆盖创面能力的影响。方法：利用在盖玻片上形成的人牙周膜和牙龈成纤维细胞的融合培养物,以机械方法去除7mm宽的细胞层条带,形成体外创面模型。受损培养物在含有10％胎牛血清的培养液中继续培养2、6、9d,同时以100μg／ml的釉基质蛋白分别刺激牙周膜、牙龈成纤维细胞,并与不加釉基质蛋白者作对照。玻片经同定后作甲紫染色。以计算机辅助图像分析系统对细胞覆盖体外创面的面积进行百分比定量,采用SAS6、12软件包进行样本均数的t检验。结果：对照组组内牙周膜、牙龈成纤维细胞覆盖创面的面积存在差异,牙龈成纤维细胞覆盖创面面积在创面形成后第9天显著高于牙周膜细胞,差异有显著性（P〈0.05）。在加用100μg／ml釉基质蛋白的条件下,牙周膜细胞覆盖体外创面的面积较对照组明显增加．创面形成后第6、9天,牙周膜、牙龈成纤维细胞覆盖体外创面面积均无显著差异（P〉0．05）。结论：牙龈成纤维细胞覆盖创面的能力高于牙周膜细胞。釉基质蛋白有增进创面愈合,特别是牙周膜细胞创面覆盖的作用。     

Clinical evaluation of wound healing following multiple exposures to enamel matrix protein derivative in the treatment of intrabony periodontal defects

BACKGROUND: Multiple exposures to enamel matrix protein derivative (EMD) during periodontal therapy have been shown to be safe for the patient. The purpose of this study was to clinically determine if an altered course of wound healing would occur after multiple exposures to EMD in the treatment of intrabony defects. A secondary aim was to assess the efficacy of EMD in probing depth reduction and clinical attachment level gain. METHODS: Thirty-two systemically healthy patients (18 females, 14 males, 33 to 69 years old) who were being treated for moderate to advanced periodontal disease were selected for the study. Surgical procedures involving 2 sites were separated by at least 8 weeks, and wound healing comparisons were made between the first and second procedure. Patients were given a diary card the day of surgery, which consisted of questions concerning the presence and severity of headaches, root hypersensitivity, tooth pain, swelling, and itching. Patients were also examined at postoperative visits to clinically assess wound healing and discuss responses to the questionnaire. Soft tissue measurements were taken the day of surgery and 6 months postoperatively to ascertain probing depth reduction (PD) and gains in clinical attachment levels (CAL). RESULTS: The results revealed no clinically detectable reaction that could not be attributed to normal postoperative sequelae. There were no differences in reported symptoms between patient gender, first and second procedures, or intrabony and non-intrabony defects. Smokers were found to have a statistically significantly higher incidence of severe symptoms in root hypersensitivity, tooth pain, and swelling compared to non-smokers (n = 21). The mean probing depth reduction was 3.8 +/- 1.5 mm (2 to 9 mm), while the mean clinical attachment level gain was 2.8 +/- 1.7 mm (0 to 8 mm). CONCLUSIONS: The findings of this study demonstrate that EMD is a clinically safe product to use in the treatment of periodontal defects and that multiple uses do not have a negative impact on periodontal wound healing. In addition, a statistically significant gain in clinical attachment and reduction in probing depth were demonstrated.     

Enamel matrix derivative,inflammation and soft tissue wound healing

Over 15 years have now passed since enamel matrix derivative (EMD) emerged as an agent capable of periodontal regeneration. Following thorough investigation, evidenced‐based clinical application is now established for a multitude of clinical settings to promote regeneration of periodontal hard tissues. Despite the large number of studies and review articles written on this topic, no single review has compiled the influence of EMD on tissue inflammation, an area of research that merits substantial attention in periodontology. The aim of the present review was to gather all studies that deal with the effects of EMD on tissue inflammation with particular interest in the cellular mechanisms involved in inflammation and soft tissue wound healing/resolution. The effects of EMD on monocytes, macrophages, lymphocytes, neutrophils, fibroblasts and endothelial cells were investigated for changes in cell behavior as well as release of inflammatory markers, including interleukins, prostaglandins, tumor necrosis factor‐α, matrix metalloproteinases and members of the OPG‐RANKL pathway. In summary, studies listed in this review have reported that EMD is able to significantly decrease interleukin‐1b and RANKL expression, increase prostaglandin E₂ and OPG expression, increase proliferation and migration of T lymphocytes, induce monocyte differentiation, increase bacterial and tissue debris clearance, as well as increase fibroplasias and angiogenesis by inducing endothelial cell proliferation, migration and capillary‐like sprout formation. The outcomes from the present review article indicate that EMD is able to affect substantially the inflammatory and healing responses and lay the groundwork for future investigation in the field.     

Some effects of enamel matrix proteins on wound healing in the dento-gingival region

OBJECTIVE: The aim of the present study was to evaluate by clinical means the effect of enamel matrix proteins on the healing of a soft tissue wound produced by periodontal pocket instrumentation. MATERIAL AND METHODS: The study was performed as an intra-individual, longitudinal trial of 3 weeks duration with a double-masked, split-mouth, placebo-controlled and randomized design. The patient material was comprised of 28 subjects with moderately advanced, chronic periodontitis. Each patient presented with 3 sites in each of 2 jaw quadrants with a probing pocket depth (PPD) of >or=5 mm and bleeding following pocket probing (BoP). Baseline examination, including assessments of plaque, gingival inflammation, PPD, BoP and root dentin sensitivity, was carried out one week after oral hygiene instruction and careful self-performed plaque control. All experimental sites were scaled and root planed, and the soft tissue wall of the pocket was curetted to remove the pocket epithelium and adjacent granulation tissue. The site was carefully irrigated with saline. When the bleeding from the pocket had ceased, a 24% EDTA gel was applied in the site and retained for 2 min. This was followed by careful irrigation with saline. Left and right jaw quadrants were then randomized to subgingival application of enamel matrix derivative (Emdogain) or vehicle-control. All sites were re-examined after 1, 2 and 3 weeks. In addition, a visual analogue scale (VAS) was used to score the degree of post-treatment discomfort. The primary endpoints of treatment success were defined as (i) pocket closure (PPD 相似文献   

Assessment of early discomfort and wound healing outcomes after periodontal surgery with and without enamel matrix derivative: an observational retrospective case-control study

Clinical Oral Investigations - The aim of this study was to evaluate and compare early postoperative discomfort and wound healing outcomes between patients who underwent periodontal surgery with...     

A comparison between enamel matrix derivative and a bioabsorbable membrane to enhance healing around transmucosal immediate post-extraction implants

BACKGROUND: This clinical report compares the use of an enamel matrix derivative (EMD) and bioabsorbable barrier membrane to enhance healing following the immediate placement of transmucosal implants into extraction sockets. METHODS: Thirty-two adult patients scheduled for tooth replacement with dental implants agreed to participate. Following the insertion of a transmucosal implant into the extraction site, the subjects were assigned to one of two treatment alternatives of the remaining bone defects around the implants: 1) the residual bone defects were filled with EMD (EMD group) or 2) the residual bone defects were covered with a bioabsorbable membrane (membrane group). Flaps were then coronally positioned around implant cover screws. Patients followed weekly maintenance recalls for the first 6 weeks and then monthly recalls until the final prosthetic restoration was completed (after 6 months). The treatment outcome was evaluated after 12 months by the use of clinical variables. The null hypothesis of no treatment group differences was tested by the use of analysis of variance (ANOVA). RESULTS: At a 12-month follow-up, all of the implants were completely osseointegrated and successfully functioning, showing a success rate of 100%. The membrane group showed a significantly lower mean probing attachment level than the EMD group at proximal (0.60 mm, standard deviation (SD) 0.37 versus 1.19 mm, SD 1.10), buccal (0.80 mm, SD 0.79 versus 1.77 mm, SD 1.16), and lingual sites (0.44 mm, SD 0.52 versus 1.48 mm, SD 1.46). The difference was statistically significant at all sites (P < 0.05). With respect to the position of the soft tissue margin around the implant shoulder, the membrane group showed a consistently higher value than the EMD group at, respectively, proximal (1.30 mm, SD 2.37 versus 1.16 mm, SD 1.0), buccal (0.90 mm, SD 1.29 versus 0.22 mm, SD 1.47), and lingual sites (1.12 mm, SD 1.10 versus 0.55 mm, SD 1.42). CONCLUSIONS: The membrane group obtained more favorable results in terms of both the probing attachment level and peri-implant position of soft tissues compared to the EMD group. The use of a bioabsorbable membrane around immediately placed transmucosal implants enhanced soft and hard tissue healing and might be an advisable treatment choice particularly in areas with high esthetic demands.     

The effects of enamel matrix derivative and cyclic mechanical strain on human gingival fibroblasts in an in vitro defect healing model

Gingival fibroblasts (GFs) play a considerable role in the maintenance of the gingival apparatus as well as in connective tissue repair. Mobility of a periodontal wound or soft tissue graft can impair connective tissue healing from the GFs. Enamel matrix derivative (EMD) is an enamel matrix protein used clinically for periodontal regeneration of intrabony defects and furcations, as well as treatment of gingival margin recessions. The goal of this project was to compare the effects of varying concentrations of EMD, with and without cyclic mechanical strain, on cellular wound fill of human GFs using an in vitro defect healing model. GFs were seeded and cultured in six-well flexible-bottomed plates. A 3-mm wound was created in the central portion of each confluent well. Three wells were treated with each EMD concentration of 0 Μg/mL (control), 30 Μg/mL, 60 Μg/mL, or 120 Μg/mL. The plates were placed in an incubator containing a strain unit to subject test plates to cyclic strain. An identical set of control plates were not flexed. Cells were examined on days 4, 8, 12, and 16. Microphotographs were taken and wound fill measurements made using image analysis software. The percent wound fill was calculated. All nonflexed plates, regardless of EMD concentration, reached > 90% defect fill at similar rates by day 16. However, in the flexed plates, EMD had a significant negative effect on defect fill. The defect fill was 55.7% for 0 Μg/mL EMD, 48.2% for 30 Μg/mL EMD, 36.7% for 60 Μg/mL EMD, and 34.1% for 120 Μg/mL EMD on day 16 for the flexed GFs. EMD, in concentrations as high as 120 Μg/mL, did not significantly affect the amount of defect fill with nonflexed GFs. However, when the GFs were flexed, the addition of EMD had a significant negative effect on defect fill in a dose-dependent manner.     

In vitro models for evaluation of periodontal wound healing/regeneration

Periodontal wound healing and regeneration are highly complex processes, involving cells, matrices, molecules and genes that must be properly choreographed and orchestrated. As we attempt to understand and influence these clinical entities, we need experimental models to mimic the various aspects of human wound healing and regeneration. In vivo animal models that simulate clinical situations of humans can be costly and cumbersome. In vitro models have been devised to dissect wound healing/regeneration processes into discrete, analyzable steps. For soft tissue (e.g. gingival) healing, in vitro models range from simple culture of cells grown in monolayers and exposed to biological modulators or physical effectors and materials, to models in which cells are ‘injured’ by scraping and subsequently the ‘wound’ is filled with new or migrating cells, to three‐dimensional models of epithelial–mesenchymal recombination or tissue explants. The cells employed are gingival keratinocytes, fibroblasts or endothelial cells, and their proliferation, migration, attachment, differentiation, survival, gene expression, matrix production or capillary formation are measured. Studies of periodontal regeneration also include periodontal ligament fibroblasts or progenitors, osteoblasts or osteoprogenitors, and cementoblasts. Regeneration models measure cellular proliferation, attachment and migration, as well as gene expression, transfer and differentiation into a mineralizing phenotype and biomineralization. Only by integrating data from models on all levels (i.e. a single cell to the whole organism) can various critical aspects of periodontal wound healing/regeneration be fully evaluated.     

Formulation of enamel matrix derivative for surface coating

Abstract Enamel Matrix Derivative (EMD) contains a protein complex belonging to the amelogenin family. Enamel matrix as well as EMD have been found to promote periodontal regeneration when applied onto denuded root surfaces in dehiscence models. In the present studies it is shown that propylene glycol alginate (PGA) is a suitable vehicle for EMD for its local application. EMD can be dissolved in PGA at an acidic pH, resulting in a highly viscous solution. At neutral pH and body temperature the viscosity decreases and EMD precipitates. Multilayers of EMD on mineral or protein surfaces have been analysed using ellipsometry, total internal reflection fluorescence (TIRE) and biospecific interaction analysis (BIA). The studies show that EMD adsorbs both to hydroxyapatite and collagen and to denuded dental roots. It forms insoluble spherical complexes, and detectable amounts remain at the site of application on the root surface for two weeks, as shown with radiolabelled protein in rats and pigs. Scanning electron micrograph (SEM) studies on monkey teeth further indicate that EMD in PGA may promote repopulation of fibroblast-like cells during the first weeks after application.     

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.     

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.