Effect of a Connective Tissue Graft in Combination With a Single Flap Approach in the Regenerative Treatment of Intraosseous Defects.

BACKGROUND: In the attempt to limit the post-surgery increase in buccal gingival recession (bREC), effect of a connective tissue graft (CTG) when combined with a buccal single flap approach (SFA) in the regenerative treatment of intraosseous defects is evaluated. METHODS: Data related to 30 patients with an intraosseous defect treated with a buccal SFA with (SFA+CTG group; n = 15) or without (SFA group; n = 15) placement of a CTG and regenerative treatment were retrospectively derived at three clinical centers. bREC and probing parameters were assessed at presurgery and 6 months post-surgery. RESULTS: In addition to a significant attachment gain and probing depth reduction, adjunctive use of a CTG to a buccal SFA in the regenerative treatment of periodontal intraosseous defects associated with a buccal bone dehiscence resulted in a limited post-surgery bREC, a lower prevalence of defects with a clinically detectable apical displacement of the gingival margin, and an increase in gingival width and thickness. CONCLUSION: Adjunctive use of a CTG in the regenerative treatment of intraosseous defects associated with buccal bone dehiscence accessed by buccal SFA may support the stability of the gingival profile.

Treatment of Non-Contained Infrabony Defects With Enamel Matrix Derivative Alone or in Combination With Biphasic Calcium Phosphate Bone Graft: A 12-Month Randomized Controlled Clinical Trial.

BACKGROUND: Use of enamel matrix derivative (EMD) when dealing with non-contained defects may be limited because EMD does not maintain a space itself. Use of combined therapy has been proposed, using a bone graft in combination with EMD to avoid collapse of the flap into the bony defect during healing time. Therefore, the aim of this study is to evaluate the clinical and radiologic healing response of non-contained infrabony defects after treatment with a combination of EMD and biphasic calcium phosphate (BC) or EMD alone. METHODS: Fifty-two patients with at least one infrabony defect ≥3 mm in depth with a probing depth (PD) ≥6 mm were randomly treated with EMD/BC or EMD alone. Clinical and radiographic parameters were evaluated at baseline, 6, and 12 months after surgery. To standardize the procedure, an acrylic stent and millimeter radiographic grid were used. The primary outcome was the change in clinical attachment level (CAL). RESULTS: Analysis of the data demonstrated a statistically significant difference from baseline within each group (P <0.05), with a difference in clinical and radiographic parameters at 6 and 12 months. After 1 year, mean PD reductions of 3.14 ± 1.95 mm (39.6%) in the EMD/BC group and 3.30 ± 1.89 mm (48.7%) in the EMD group were achieved. A mean CAL gain of 2.38 ± 2.17 mm (24.9%) in the EMD/BC group and 2.65 ± 2.18 mm (36.2%) in the EMD group were obtained. Reduction in the infrabony component was 2.71 ± 1.79 mm (57.9%) in the test group and 2.60 ± 2.03 mm (28.5%) in the control group. There were no statistically significant differences between treatment groups. CONCLUSIONS: It was concluded that treatment of non-contained infrabony defects with EMD, with or without BC, resulted in statistically significantly better results after 12 months compared with baseline measurements. In contrast, the combined approach did not result in a statistically significant improvement.

Evaluation of gingival crevicular fluid transforming growth factor-ß1 level after treatment of intrabony periodontal defects with enamel matrix derivatives and autogenous bone graft: A randomized controlled clinical trial.

AIM: The present study aimed to evaluate the effects of enamel matrix derivatives (EMD) either alone or combined with autogenous bone graft (ABG) applied to intrabony defects in chronic periodontitis patients on clinical/radiographic parameters and gingival crevicular fluid (GCF) transforming growth factor-ß1 (TGF-ß1) level and to compare with open flap debridement (OFD). MATERIALS AND METHODS: A total of 30 deep intrabony defects in 12 patients were randomly treated with EMD + ABG (combination group), EMD alone (EMD group), or OFD (control group). Clinical parameters, including plaque index, gingival index, bleeding on probing, probing depth, relative attachment level, and recession were recorded at baseline and 6 months postsurgery. Intrabony defect fill percentage was calculated on the standardized radiographs. TGF-ß1 level was evaluated in GCF just before surgery and 7, 14, 30, 90, 180 days after surgery using enzyme-linked immunosorbent assay. RESULTS: All treatment procedures led to significant improvements at 6 months (P < 0.01). Gain in attachment level (P < 0.01) and radiographic defect fill (P < 0.05) of the combination and EMD groups were found to be significantly higher than those of the control group, while the use of EMD either with ABG or alone was observed to produce significantly less recession than the OFD (P < 0.05). CONCLUSION: The findings suggest no clinical and radiographic differences between the combination and EMD groups whereas GCF TGF-ß1 level demonstrates an increase during the healing phase and is positively affected from EMD.

The Association of Guided Bone Regeneration and Enamel Matrix Derivative for Suprabony Reconstruction in the Esthetic Area: A Case Report.

This case report presents the correction of severe alveolar ridge atrophy due to congenital and iatrogenic factors. Implants that compromised the natural adjacent teeth and overall esthetics for this young patient were removed and replaced after significant vertical bone and soft tissue regenerative procedures. A treatment combination of bone graft particles, a nonresorbable membrane, and enamel matrix derivatives was used. Significant and stable improvement in esthetics was achieved 12 months after final prosthetic restoration, demonstrating the ability of such a combined treatment to correct the esthetic deformity, improve the health of the adjacent natural teeth, and allow for successful implant treatment.

Histological Effects of Enamel Matrix Derivative on Exposed Dental Pulp.

INTRODUCTION: Direct pulp capping procedure is a therapeutic application of a drug on exposed tooth pulp in order to ensure the closure of the pulp chamber and to allow the healing process to take place. OBJECTIVE: The aim of this study was to examine the histological effects of Emdogain® on exposed tooth pulp of a Vietnamese pig (Sus scrofa verus). METHODS: The study comprised 20 teeth of a Vietnamese pig. After class V preparation on the buccal surfaces of incisors, canines and first premolars, pulp was exposed. In the experimental group, the perforations were capped with Emdogain® (Straumann, Basel, Switzerland), while in the control group pulp capping was performed with MTA® (Dentsply Tulsa Dental, Johnson City, TN, USA). All cavities were restored with glass-ionomer cement (GC Fuji VIII, GC Corporation, Tokyo, Japan). The observational period was 28 days, after which the animal was sacrificed and histological preparations were made. A light microscope was used to analyze dentin bridge formation, tissue reorganization and inflammation, and the presence of bacteria in the pulp. RESULTS: The formation of dentin bridge was observed in the experimental and control groups. Inflammation of the pulp was mild to moderate in both groups. Angiogenesis and many odontoblast-like cells, responsible for dentin bridge formation, were observed. Necrosis was not observed in any case, nor were bacteria present in the pulp. CONCLUSION: Histological analysis indicated a favorable therapeutic effect of Emdogain® Gel in direct pulp capping of Vietnamese pigs. Pulp reaction was similar to that of MTA®.

Study on the influence of leucine-rich amelogenin peptide (LRAP) on the remineralization of enamel defects via micro-focus x-ray computed tomography and nanoindentation.

Regeneration of severely damaged enamel (e.g. deep demineralized lesions) is currently not possible, because the structural units of enamel crystal construction are removed after its maturation. The aim of this in vitro study was to evaluate the effect of surface impregnation by leucine-rich amelogenin peptide (LRAP) on the remineralization of eroded enamel using micro-focus x-ray computed tomography (µCT). Fifteen bovine enamel blocks were embedded in resin and three zones (sound, demineralization, and remineralization) were defined on each specimen. Lesions were prepared by immersing the samples in demineralization solution for 7 d. The samples were soaked in distilled water or 60 or 120 µg mL(-1) solution of LRAP in water for 30 min. After the surface treatment, specimens were incubated in artificial saliva for either 5 or 10 d at 37 °C. The amount of mineral gain (dΔZ%) and the relative changes in the lesion depth (dLD%), obtained from µCT, were used to evaluate the effect of LRAP on the remineralization of lesions. The effects of LRAP on cross-sectional integrated hardness ΔINH were studied after 10 d using nanoindentation. ANOVA test was used to determine the effect of time and/or LRAP concentration on dΔZ%, dLD% and ΔINH mean values. Tukey's analysis was used for multiple comparison testing (α = 0.05). Analysis of µCT data showed significant effect of time and LRAP concentration on the dΔZ% (p = 0.013, p = 0.003) and the dLD% (p < 0.001, p = 0.002) mean values. The nanoindentation hardness was significantly improved by 120 µg mL(-1) LRAP (p = 0.02). Also, the peptide treatment affected the mineral distribution throughout the lesion by inhibiting of superficial deposition. This study showed that the treatment of eroded lesions in enamel by LRAP can improve and regulate the pattern of remineralization in vitro.

Effects of enamel matrix derivative and transforming growth factor-ß1 on connective tissue growth factor in human periodontal ligament fibroblasts.

BACKGROUND: Enamel matrix derivative (EMD) is suggested to stimulate transforming growth factor-ß (TGF-ß) production. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-ß. This study explores the effects of EMD and TGF-ß1 on CTGF in periodontal ligament (PDL) fibroblasts and their interactions in PDL proliferation and development. METHODS: Human PDL cells were stimulated with EMD. To explore the effects of EMD and TGF-ß1 on CTGF expression, cells were treated with and without TGF-ß inhibitor, and CTGF protein levels were assayed by Western blot analysis. To study the role of CTGF in PDL development, cells were treated with CTGF inhibitor. DNA synthesis was analyzed by bromodeoxyuridine enzyme-linked immunosorbent assay. Reverse-transcription polymerase chain reaction was performed to examine messenger RNA expression of PDL osteoblastic differentiation markers: type I collagen, alkaline phosphatase, and osteocalcin. RESULTS: EMD induced a concentration-dependent increase of CTGF protein expression in PDL cells. EMD- and TGF-ß1-stimulated CTGF expression was significantly reduced in the presence of TGF-ß inhibitor. CTGF inhibition downregulated both EMD- and TGF-ß1-induced DNA synthesis. The effect of CTGF and EMD on osteoblastic mRNA expression in PDL cells is not obvious. CONCLUSIONS: EMD stimulates CTGF expression in human PDL cells, a process modulated by the TGF-ß pathway. CTGF can affect EMD- and TGF-ß1-induced PDL cell proliferation, but its effects on PDL with regard to osteoblastic differentiation remain inconclusive. The results provide novel insights into EMD-CTGF interaction in PDL cells.

Enamel matrix derivative in propylene glycol alginate for treatment of infrabony defects with or without systemic doxycycline: 12- and 24-month results.

BACKGROUND: This aim of this study is to compare regenerative therapy of infrabony defects with and without administration of post-surgical systemic doxycycline (DOXY) 12 and 24 months after therapy. METHODS: In each of 57 patients, one infrabony defect (depth ≥ 4 mm) was treated regeneratively using enamel matrix derivative at two centers (Frankfurt am Main and Heidelberg). By random assignment, patients received either 200 mg DOXY per day or placebo (PLAC) for 7 days after surgery. Twelve and 24 months after surgery, clinical parameters (probing depths [PDs] and vertical clinical attachment level [CAL-V]) and standardized radiographs were obtained. Missing data were managed according to the last observation carried forward. RESULTS: Data of 57 patients (DOXY: 28; PLAC: 29) were analyzed (26 males and 31 females; mean age: 52 ± 10.2 years; 13 smokers). In both groups, significant (P <0.01) PD reduction (DOXY: 3.7 ± 2.2 mm; PLAC: 3.4 ± 1.7 mm), CAL-V gain (DOXY: 2.7 ± 1.9 mm; PLAC: 3.0 ± 1.9 mm), and bone fill (DOXY: 1.6 ± 2.7 mm; PLAC: 1.8 ± 3.0 mm) were observed 24 months after surgery. However, the differences between both groups failed to be statistically significant (PD: P = 0.574; CAL-V: P = 0.696; bone fill: P = 0.318). CONCLUSIONS: Systemic DOXY, 200 mg/day for 7 days, after regenerative therapy of infrabony defects did not result in better PD reduction, CAL-V gain, or radiographic bone fill compared with PLAC 12 and 24 months after surgery, which may be attributable to low power and, thus, random chance.

In vitro inhibition of bovine enamel demineralization by enamel matrix derivative.

This study aimed to determine whether enamel matrix derivative (Emdogain) affects the demineralization of bovine enamel in vitro and to assess the agent's anti-caries potential. Bovine enamel blocks were prepared and randomly divided into three groups (n = 15 per group), which were treated with distilled water (negative control), NaF (positive control), or Emdogain. All three groups were pH-cycled 12 times over 6 days. The percentage of surface enamel microhardness reduction (%SMHR), calcium demineralization rate (CDR), surface roughness, lesion depth and mineral loss after demineralization were examined. Surface morphology of specimens was studied by scanning electron microscopy. The Emdogain and positive control groups showed similar surface roughness, lesion depths and mineral loss, which were significantly lower than those in the negative control group. In addition, the enamel surfaces of both the Emdogain and NaF groups showed much narrower intercrystalline spaces than the surfaces of the negative control group, which exhibited extensive microfractures along the crystal edges. %SMHR differed significantly among all three groups, with the smallest value in the Emdogain group and the greatest in the negative control group. These results indicate that enamel matrix derivative (Emdogain) can significantly inhibit demineralization of bovine enamel in vitro, suggesting that it has potential as an anti-caries agent.

Extracellular matrix administration as a potential therapeutic strategy for periodontal ligament regeneration.

INTRODUCTION: The current strategies employed for the treatment of connective tissue disease include the application of stem cells, the use of functional molecules that can reorganize tissue integrity and cellular activities to recover connective tissue function. Approaches to the regeneration of periodontal tissue, which is the tooth-supporting connective tissue, have made some progress recently and provide a useful experimental model for the evaluation of future strategies to treat connective tissue diseases such as periodontal disease. AREAS COVERED: The ultimate goal of periodontal tissue regeneration is to reconstruct the ligament structure that will sustain the required mechanical force to connect with mineralized tissues such as cementum and alveolar bone. In this review, we discuss the proposed use of extracellular matrix (ECM) administration therapy as an additional therapeutic strategy to stem cell transplantation and cytokine administration in the current field of periodontal tissue regeneration therapy. EXPERT OPINION: Although various available tissue engineering technologies can now achieve periodontal tissue regeneration, ECM administration therapy is likely to play an essential future role in the development and regeneration of periodontal tissue and attenuate the signaling events that mediate tissue degradation. Hence, ECM administration could serve as a novel technology in periodontal tissue regeneration and also as a viable approach to alleviating connective tissue disorders such as Marfan's syndrome.

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.

Effects of enamel matrix derivative on vascular endothelial growth factor expression and microvessel density in gingival tissues of periodontal pocket: a comparative study.

BACKGROUND: Vascular endothelial growth factor (VEGF) stimulates proliferation and migration of endothelial cells, and correlates with inflammatory resolution and periodontal tissue healing. Enamel matrix derivative (EMD) seems to stimulate soft tissue healing. Our aim was to assess if topical EMD application in an instrumented periodontal pocket could affect angiogenesis at the gingival level. METHODS: A total of 56 periodontal sites in 28 patients were treated with a single session of comprehensive scaling and root planing under local anesthesia after recording the clinical attachment level (CAL). EMD gel in the test site or only the vehicle propylene glycol alginate in aqueous solution in the control site of the same mouth was applied onto the root surfaces and into the pocket and left in place for 3 minutes. After 48 hours, gingival biopsies were collected for histologic and immunohistochemical analysis for VEGF and CD34 (for microvessel density [MVD] count) antibodies. Statistical comparisons were performed by analysis of variance test. RESULTS: Endothelial VEGF expression and MVD were statistically different in the test site compared to the control site. VEGF expression and MVD of the control site were not correlated with CAL, whereas the test site showed high correlations among CAL and endothelial VEGF or MVD. CONCLUSIONS: EMD induces proliferation and viability and angiogenesis of human microvascular cells. Recent clinical and histologic studies found EMD to be useful as an adjunct to scaling and root planing in single-rooted teeth. Our findings may help to understand the mechanisms involved in soft tissue healing, through the ability of EMD to increase angiogenesis at periodontal pockets.

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.

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 gamma-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.

Effects of enamel matrix derivative on the viability, cytokine secretion, and phagocytic activity of human monocytes.

INTRODUCTION: There is some controversy about the effect of enamel matrix derivative (EMD) on inflammation and resorption. The aim of this study was to investigate the effect of EMD on the inflammatory response of monocytes and their phagocytic activity in vitro. METHODS: Human monocytes were incubated in complete medium (CM) and exposed to 50, 100, and 200 microg/mL EMD for different time points (12, 24, 48, and 72 hours). Untreated monocytes were considered as controls. Cellular viability was evaluated through a 3-(4, 5 dimethylthiazol-2-yl) 2, 5-diphenyl-2 tetrazolium bromide assay. For cytokine measurements, the cells were treated simultaneously with 50, 100, or 200 microg/mL EMD and 10 microg/mL Escherichia coli lipopolysaccharide. Cell-free supernatants were collected after 12, 24, 48, and 72 hours of incubation. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) concentrations were measured by an enzyme-linked immunosorbent assay kit. Phagocytic activity of the cells was assayed using the PHAGOTEST kit (Glycotope Biotechnology, Heidelberg, Germany) according to the manufacturer's instructions. RESULTS: The viability of cells exposed to 50, 100, and 200 microg/mL EMD for 12, 24, 48, and 72 hours were similar to the controls. There was no significant differences in the production of TNF-alpha and IL-1beta among samples with various concentrations (50, 100, and 200 microg/mL) of EMD and control (EMD = 0) at 12, 24, 48, and 72 hours. Phagocytic activity of monocytic cells increased significantly after 72 hours compared with 12 hours. CONCLUSIONS: Based on the results of this study, EMD does not promote releasing of the two studied proinflammatory and resorbing cytokines, TNF-alpha and IL-1beta. By increasing the phagocytic activity of monocytic cells, EMD might accelerate wound healing.

The role of macrophages in the periodontal regeneration using Emdogain gel.

BACKGROUND AND OBJECTIVE: Emdogain gel is clinically used as a periodontal regenerative material. However, the mechanism of the regeneration has not been completely elucidated. Although many studies have focused on the regenerative effect of Emdogain on connective tissue attachment and alveolar bone, the role of macrophages and the expression of growth factors remains unclear in the regeneration stimulated by Emdogain gel in vivo. The aim of this study was to investigate the effect of Emdogain gel on the expression of cytokines and growth factors by macrophages in vivo using a newly devised rat experimental periodontitis model. MATERIAL AND METHODS: Rat experimental periodontitis was induced by elevating a full-thickness gingival flap and ligating silk threads around the first molars of the mandible. At 14 d after inducing experimental periodontitis, Emdogain gel or propylene glycol alginate was applied to the furcation area. The rats were killed 7 and 14 d after treatment with propylene glycol alginate or Emdogain gel. The expression of cytokines and growth factors, and the regeneration of periodontal tissue, were examined by histochemical and immunohistochemical methods. RESULTS: Fourteen days after the induction of periodontitis, the resorption of alveolar bone at furcation was observed and cytokines such as interleukin-1beta, transforming growth factor-beta1, receptor activator of nuclear factor-kappaB ligand, receptor activator of nuclear factor-kappaB and osteoprotegerin were found. In the Emdogain-treatment group, the formation of new acellular cementum and, more remarkably, recovery of the bone, were observed. The new bone formation ratio in the Emdogain treatment group was significantly higher than that of the propylene glycol alginate treatment group. Although the expression of cytokines such as interleukin-1beta, transforming growth factor-beta1, receptor activator of nuclear factor-kappaB ligand and receptor activator of nuclear factor-kappaB was very low, bone morphogenetic protein-2- and bone morphogenetic protein-4-expressing macrophages were observed close to the root, and bone morphogenetic protein-4-expressing macrophages were mainly observed close to the bone surface at the furcation in the Emdogain-treatment group. CONCLUSION: These results suggest that wound-healing macrophages may express bone morphogenetic protein and play an important role in the regeneration of periodontal tissue at the furcation following the application of Emdogain gel.

The potential use of enamel matrix derivative for in situ anterior cruciate ligament tissue engineering: a translational in vitro investigation.

Polyester scaffolds have been used as an alternative to autogenous tissues for the reconstruction of the anterior cruciate ligament (ACL). They are biocompatible and encourage tissue infiltration, leading to neoligament formation. However, rupture can occur, caused by abrasion of the scaffold against the bone tunnels through which it is implanted. Good early tissue induction is therefore considered essential to protect the scaffold from this abrasion. Enamel matrix derivative (EMD) is used clinically in the treatment of periodontal disease. It is a complex mix of proteins with growth factor-like activity, which enhances periodontal ligament fibroblast attachment, proliferation, and differentiation, leading to the regeneration of periodontal bone and ligament tissues. We hypothesized that EMD might, in a similar manner, enhance tissue induction around scaffolds used in ACL reconstruction. This preliminary investigation adopted a translational approach, modelling in vitro 3 possible clinical modes of EMD administration, to ascertain the suitability of each protocol for application in an animal model or clinically. Preliminary investigations in monolayer culture indicated that EMD had a significant dose-dependent stimulatory effect (p < 0.05, n = 6) on the proliferation of bovine primary synovial cells. However, pre-treating culture plates with EMD significantly inhibited cell attachment (p < 0.01, n = 6). EMD's effects on synovial cells, seeded onto ligament scaffolds, were then investigated in several in vitro experiments modelling 3 possible modes for clinical EMD administration (pre-, intra-, and post-operative). In the pre-operative model, EMD was adsorbed onto scaffolds before the addition of cells. In the intra-operative model, EMD and cells were added simultaneously to scaffolds in the culture medium. In the post-operative model, cells were pre-seeded onto scaffolds before EMD was administered. EMD significantly inhibited cell adhesion in the pre-operative model (p < 0.05, n = 6) and had no significant benefit in the intra-operative model. In the post-operative model, the addition of EMD to previously cell-seeded scaffolds significantly increased their total deoxyribonucleic acid content (p < 0.01, n = 5). EMD's stimulative effect on cell proliferation in vitro suggests that it may accelerate scaffold colonization by cells (and in turn tissue induction) in situ. However, its inhibitory effect on synovial cell attachment in vitro implies that it may only be suited to post-operative administration.

Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts.

AIM: The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS: Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. RESULTS: Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. CONCLUSION: These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.

The effect of the amelogenin fraction of enamel matrix proteins on fibroblast-mediated collagen matrix reorganization.

Enamel matrix proteins (EMP), extracted from developing porcine teeth, promote not only periodontal regeneration but also cutaneous wound healing presumably via the amelogenin fraction. Because it is unclear whether the effect of EMP can be ascribed to amelogenins, we compared EMP with recombinant amelogenin in the relaxed dermal equivalent (DE) in vitro model for early wound contraction. EMP and recombinant porcine amelogenin (rP172) at 1 mg/ml were incorporated into DEs composed of human dermal fibroblasts and a type I collagen matrix. The area reduction, as a measure of contraction, as well as fibroblast numbers and TGF-beta1 levels, were quantified over 7 days in culture in the presence of 10% foetal bovine serum. Both EMP and recombinant amelogenin increased contraction (p < 0.005) and fibroblast numbers (p < 0.005) compared with controls (acetic acid vehicle and 1mg/ml porcine serum albumin) and the positive control TGF-beta1 added at 10 ng/ml. Increased contraction with EMP and recombinant amelogenin was most pronounced after the first day of incubation and was associated with elevated (p < 0.005) TGF-beta1 levels in conditioned medium. In conclusion, the amelogenin component of EMP augmented fibroblast-driven collagen matrix remodelling, at least partially, by increasing the endogenous production of TGF-beta1. These effects of EMP/amelogenin may be beneficial for cutaneous wound healing.

Analysis of eosinophilic round bodies formed after injection of enamel matrix derivative into the backs of rats.

BACKGROUND: Enamel matrix derivative (EMD) is used in dental clinics for the regeneration of alveolar bone. Its effects have not yet been clarified, although it induces eosinophilic round bodies (ERBs) and cartilage formation at the injection site. The objective of this experiment was to examine the histopathologic and biochemical properties of ERBs formed after EMD injection. METHODS: The backs of Sprague-Dawley rats injected with various concentrations of EMD were examined histopathologically. For biochemical examinations, ERBs were microdissected out from the sections. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), and database analysis of ERBs were carried out. RESULTS: The histopathological findings were consistent with a foreign body reaction. Numerous ERBs were observed 7 days after injection of 30.0 mg/ml EMD. Histopathologically, ERBs did not contain polysaccharide, amyloid, or hemosiderin. The cells surrounding ERBs were not macrophages or vascular endothelial cells. SDS-PAGE of the microdissected ERBs revealed an intense band at around the 40-kDa region. MALDI-TOF MS showed that the spectrum for ERBs has only a single strong ion intensity. Analysis of the amino acid sequence revealed that the ERBs were composed of various molecular fragments, which all contained an identical seven amino acid sequence. In addition, these peptides are a component of amelogenin. CONCLUSIONS: A high concentration of EMD induces ERBs that consist of a 40-kDa protein which includes a constituent part of amelogenin. The ERBs (or remaining EMD) might promote mesenchymal cell differentiation into hard tissue-forming cells around the EMD injection site.