Carlos E. Nemcovsky

In vitro Effects of Enamel Matrix Proteins on Rat Bone Marrow Cells and Gingival Fibroblasts

Emdogain® (EMD), a formulation of Enamel Matrix Proteins (EMP), is used clinically for periodontal regeneration, where it stimulates cementum formation and promotes gingival healing. In this study, we investigated the in vitro effects of EMD on rat bone marrow stromal cells (BMSC) and gingival fibroblasts (GF). EMD (at 25 μg/mL) increased the osteogenic capacity of bone marrow, as evidenced by ~ three-fold increase in BMSC cell number and ~ two-fold increase in alkaline phosphatase (ALP) activity and mineralized nodule formation. The presence of EMD in the initial stages (first 48 hrs) of the culture was crucial for this effect. In contrast, EMD did not induce osteoblastic differentiation of GF (evidenced by lack of mineralization or ALP activity) but increased up to two-fold both their number and the amount of matrix produced. These in vitro data on BMSC and GF could explain the promotive effect of EMD on bone formation and connective tissue regeneration, respectively.

Histomorphometric assessment of bone formation in sinus augmentation utilizing a combination of autogenous and hydroxyapatite/biphasic tricalcium phosphate graft materials: at 6 and 9 months in humans

OBJECTIVE The aim of this study was to examine the efficacy of a new biphasic hydroxyapatite/tricalcium phosphate (HA/TCP) bone substitute in combination with particulate autogenous bone in sinus floor augmentation procedures. MATERIAL AND METHODS A simultaneous or a two-stage sinus augmentation and implant placement were conducted in 28 patients. A mixture of HA/TCP and autogenous bone chips in a 1 : 1 ratio was used as the grafting biomaterial. Cylindrical specimen bone retrieval was performed in all patients except one. Specimens were harvested either at 6 (n=14) or 9 (n=13) months post-augmentation. For histologic and histomorphometric evaluations, the non-decalcified tissue processing (Donaths technique) was performed. RESULTS Newly formed bone around the grafted particles was found in all samples. The encircling, highly cellular bone followed the outline of the grafted particles in direct contact. Both woven and lamellar types of bone were observed. Morphometrically, the total mean bone area fraction of all sections was 34.8+/-10.3%, increasing from 28.6+/-7.8% at 6 months to 41.6+/-8.3% at 9 months (P<0.001). Mean particle area fraction average was 25.5+/-11.6% and 23.5+/-9.3% at 6 and 9 months, respectively, with a total mean of 24.5+/-10.4%. The increase in bone area fraction was not significantly correlated to the decrease of the grafted particles area fraction. CONCLUSIONS The biphasic HA/TCP showed biocompatible and osteoconductive properties. This alloplast as a composite with autogenous bone chips promotes newly formed bone, which increases in its fraction along an extended healing period.

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 μg/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.

Long‐term bio‐degradation of cross‐linked and non‐cross‐linked collagen barriers in human guided bone regeneration

OBJECTIVE This double-blind study clinically and histologically evaluated long-term barrier bio-durability of cross-linked and non-cross-linked collagen membranes (CLM and NCLM) in sites treated by guided bone regeneration procedures. MATERIALS AND METHODS In 52 patients, 52 bony defects were randomly assigned to treatment with either a CLM or a NCLM. Post-surgical spontaneous membrane exposures were recorded. Before implant placement, full-thickness standard soft tissue discs were retrieved wherever suitable for histologic examination. RESULTS Spontaneous membrane exposure was observed in 13 (50%) CLM sites and in six (23.1%) NCLM sites (P<0.05). Clinical healing at exposed sites lasted 2-4 weeks. CLM were histologically intact in all non-perforated sites, were interrupted in five perforated sites, and undetected in four. NCLMs were undetected in all 18 specimens examined. In three non-perforated CLM sites, bone apposition and ossification at or within the membrane was observed. CONCLUSIONS CLMs were more resistant to tissue degradation than NCLMs, and maintained integrity during the study. Neither membrane was resistant to degradation when exposed to the oral environment. CLMs were associated with a higher incidence of tissue perforations. In non-perforated sites, CLM ossification at or within the membrane was occasionally observed.

Clinical evaluation of particulate allogeneic with and without autogenous bone grafts and resorbable collagen membranes for bone augmentation of atrophic alveolar ridges

OBJECTIVES The evaluate the clinical outcome of bone augmentation with the use of particulate mineralized freeze-dried bone allograft (FDBA) with or without the addition of autogeneous bone chips, applied in a bi-layered (BL) technique, covered by a resorbable cross-linked collagen membrane. MATERIAL AND METHODS Fifty patients presenting with a vertical and/or lateral ridge deficiency of at least 3 mm were included: Group FDBA, N=27 patients, particulate FDBA was the only graft; and Group BL, N=23 patients, a BL bone grafting procedure where autogenous bone chips were the inner layer and FDBA the outer. Bone graft was covered with a ribose cross-linked collagen barrier membrane. Ridge dimensions were clinically or radiographically (computerized tomography scan) measured at the time of the bone augmentation procedure and at implant placement or uncovering and the maximum linear vertical or horizontal calcified tissue gain was calculated. Statistical analysis consisted of linear regression analysis, with maximum bone gain being the dependent variable. RESULTS In the FDBA group, mean vertical bone gain was 3.47 mm (SD 1.25) and the horizontal, 5 mm (SD 1.28), while in the BL values were 3.5 mm (SD 1.2) and 3.6 mm (SD 1.72), respectively. Addition of autogenous bone does not appear to statistically significantly enhance the results. Spontaneous membrane exposure occurred in 24% of the cases and was the only variant that significantly influenced results (P<0.001). CONCLUSIONS Large vertical and/or horizontal ridge deficiencies may be treated with FDBA and ribose cross-linked collagen barrier membranes with good clinical outcome. No added effect of the application of a layer of autogenous bone in these bone augmentation procedures could be demonstrated. Spontaneous membrane exposure was the only parameter to affect the degree of new calcified tissue formation.

Bio-degradation of a resorbable collagen membrane (Bio-Gide) applied in a double-layer technique in rats.

OBJECTIVE The aim of this study was to evaluate histologically the bio-degradation of two layers of Bio-Gide((R)) (BG) membrane, as compared with that of a single layer. MATERIAL AND METHODS Two circular calvarial bony defects, 5 mm in diameter, were made in 24 Wistar rats. BG membrane, labeled with biotin, was cut into 5-mm-diameter disks, and placed in defects either as a mono-layer membrane (MLM) or as a double-layer membrane (DLM). Rats were sacrificed after 4 or 9 weeks and histology was performed. Membranes were stained with horseradish peroxidase-conjugated streptavidin and aminoethyl carbazole as a substrate for detection of biotinylated collagen. The area of collagen and thickness of the residual membranes were measured by image analysis software. Statistical analysis was performed using the non-parametric Wilcoxons signed-ranks test. RESULTS At 4-week collagen area per measurement window within the DLM sites (0.09+/-0.05 mm(2)) was significantly greater (P<0.01) than that in the MLM sites (0.047+/-0.034 mm(2)). At 9 weeks, the collagen area was also greater in the DLM sites (0.037+/-0.026 mm(2)) compared with that of the MLM sites (0.025+/-0.016 mm(2)); however, this difference did not reach statistical significance. The rate of membrane degradation, calculated as percent membrane lost compared with baseline, was similar for the DLM and MLM at both time points ( approximately 60% at 4 weeks and approximately 80% at 9 weeks). In addition, the residual DLM thickness at 4 weeks (475.5+/-73.77 mum) was significantly (P<0.01) greater than that of MLM (262.38+/-48.01 mum). At 9 weeks, membrane thickness was also greater in the DLM sites (318.22+/-70.45 mum) compared with that of the MLM sites (183.32+/-26.72 mum); however, this difference did not reach statistical significance. The reduction in thickness between 4 and 9 weeks was 30% for MLM and 33% for DLM. DISCUSSION The use of a double layer of BG membrane results in a barrier of increased collagen area and thickness, compared with application of a single layer.

Enamel Matrix Derivative Protects Human Gingival Fibroblasts From TNF-Induced Apoptosis by Inhibiting Caspase Activation

Emdogain®, a formulation of enamel matrix derivative (EMD), is used clinically for regeneration of the periodontium (tooth supporting tissues), but the molecular mechanisms of its action have not been elucidated. Several clinical studies suggested that EMD may also improve gingival healing after periodontal surgery and thus affect the fate of gingival fibroblasts (GFs). Since these cells are targets for local inflammatory mediators such as TNF, a pro‐apoptotic cytokine, during the course of periodontal disease, we tested whether EMD protects human GFs (hGFs) from TNF‐induced cytotoxicity. Quiescent primary hGFs were challenged with TNF (10–100 ng/ml) with or without EMD (100 µg/ml) pretreatment. Cell viability was assessed by neutral red staining, cell death by LDH release and apoptosis by caspase activity. Signaling pathways were evaluated by Western blotting and pharmacological inhibitors. TNF induced classical signs of apoptosis in hGFs, including typical cellular morphology and increased caspase activity. TNF‐induced cytotoxicity was entirely caspase‐dependent. Pretreatment (4–24 h) with EMD dramatically inhibited the activation of initiator and executioner caspases and enhanced hGF survival. Although TNF induced the activation of p38 MAPK, JNK, ERK and PI‐3K signaling, these pathways were not crucial for EMD protection of hGFs. However, EMD increased the levels of c‐FLIPL, an anti‐apoptotic protein located upstream of caspase activation. These data demonstrate, for the first time, that EMD protects hGFs from inflammatory cytokines and, together with our recent reports that EMD stimulates rat and human GF proliferation, could help explain the mechanisms whereby in vivo use of EMD promotes gingival healing. J. Cell. Physiol. 213:750–758.

Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase-3 in human gingival fibroblasts via extracellular signal-regulated kinase

BACKGROUND AND OBJECTIVE Periodontal disease is characterized by increased expression and activity of matrix metalloproteinases (MMPs) and insufficient expression/activity of their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). This altered MMP-TIMP balance results in progressive destruction of gingival and periodontal extracellular matrix. Enamel matrix derivative (EMD), clinically used for periodontal regeneration in a device called Emdogain, has been suggested to enhance gingival healing following periodontal procedures in humans. We previously showed that EMD increases the proliferation of human and rat gingival fibroblasts and protects them from tumor necrosis factor-induced apoptosis. In the present study, the modulation of MMP and TIMP expression by EMD was investigated. MATERIAL AND METHODS Primary human gingival fibroblasts were treated in vitro with tumor necrosis factor, EMD or both in serum-free conditions, and RNA was analyzed with an extracellular matrix-focused microarray and quantitative real-time polymerase chain reaction. RESULTS Microarray analysis showed detectable expression of MMP-1, MMP-2, MMP-3, MMP-7 and MMP-13, as well as TIMP-1 and TIMP-3 in untreated cells. There was no apparent regulation of the expression of MMP-2, MMP-7, MMP-13 and TIMP-1 by either tumor necrosis factor or EMD. In contrast, tumor necrosis factor significantly increased MMP-1 expression, and EMD reduced it when both agents were present. Also, EMD significantly induced TIMP-3 expression, an effect which was dependent on activation of extracellular signal-regulated kinase 1/2, since it was totally abolished by a selective extracellular signal-regulated kinase pathway inhibitor. CONCLUSION These data suggest that EMD may affect gingival health by ways other than cell proliferation/survival, i.e. by stimulation of TIMP-3 production, which could improve the MMP-TIMP balance in gingival tissue and curb extracellular matrix destruction.

Differential effects of prostaglandin E2 and enamel matrix derivative on the proliferation of human gingival and dermal fibroblasts and gingival keratinocytes

BACKGROUND AND OBJECTIVE Elevated levels of prostaglandins contribute to periodontal destruction but can impair gingival healing by affecting local fibroblasts. Enamel matrix derivative (EMD) has beneficial effects on supporting and gingival tissues. We showed that prostaglandin E(2) (PGE(2) ) inhibits the proliferation of human gingival fibroblasts (hGFs) and that EMD stimulates it. Prostaglandins and EMD may also affect skin healing by targeting dermal fibroblasts (DFs). Thus, we compared the effects of these two agents on the proliferation of hGFs, human gingival keratinocytes (hGKs) and hDFs. MATERIAL AND METHODS Cells from healthy human gingiva or skin were treated with PGE(2) and/or EMD, and proliferation was assessed by measuring cell number and DNA synthesis. RESULTS In hGFs, PGE(2) (1 μm) inhibited proliferation while EMD stimulated it. When present together, EMD abolished the PGE(2) -induced inhibition. Serum increased (by a factor of 10) the amount of phosphorylated extracellular signal-regulated kinase (p-ERK), PGE(2) reduced it (by 70-80%) and EMD restored it when present with PGE(2). Prostaglandin E(2) stimulated cAMP production in hGFs while serum or EMD did not. Enamel matrix derivative stimulated hDF proliferation, but the inhibitory effect of PGE(2) was milder than with hGFs. When present together, EMD abolished the PGE(2) -induced inhibition. Enamel matrix derivative inhibited the proliferation of primary hGKs, but PGE(2) had no effect. Finally, we found that hDFs contained about five times less prostaglandin EP(2) receptor mRNA than hGFs, while hGKs contained none. CONCLUSION Prostaglandin E(2) inhibits and EMD stimulates hGF proliferation via distinct pathways. The different sensitivities of hDFs and hGKs to PGE(2) can be explained by the levels of EP(2) expression.

Prostaglandin E2 inhibits the proliferation of human gingival fibroblasts via the EP2 receptor and Epac

Elevated levels of prostaglandins such as PGE2 in inflamed gingiva play a significant role in the tissue destruction caused by periodontitis, partly by targeting local fibroblasts. Only very few studies have shown that PGE2 inhibits the proliferation of a gingival fibroblast (GF) cell line, and we expanded this research by using primary human GFs (hGFs) and looking into the mechanisms of the PGE2 effect. GFs derived from healthy human gingiva were treated with PGE2 and proliferation was assessed by measuring cell number and DNA synthesis and potential signaling pathways were investigated using selective activators or inhibitors. PGE2 inhibited the proliferation of hGFs dose‐dependently. The effect was mimicked by forskolin (adenylate cyclase stimulator) and augmented by IBMX (a cAMP‐breakdown inhibitor), pointing to involvement of cAMP. Indeed, PGE2 and forskolin induced cAMP generation in these cells. Using selective EP receptor agonists we found that the anti‐proliferative effect of PGE2 is mediated via the EP2 receptor (which is coupled to adenylate cyclase activation). We also found that the effect of PGE2 involved activation of Epac (exchange protein directly activated by cAMP), an intracellular cAMP sensor, and not PKA. While serum increased the amount of phospho‐ERK in hGFs by ∼300%, PGE2 decreased it by ∼50%. Finally, the PGE2 effect does not require endogenous production of prostaglandins since it was not abrogated by two COX‐inhibitors. In conclusion, in human gingival fibroblasts PGE2 activates the EP2—cAMP—Epac pathway, reducing ERK phosphorylation and inhibiting proliferation. This effect could hamper periodontal healing and provide further insights into the pathogenesis of inflammatory periodontal disease. J. Cell. Biochem. 108: 207–215, 2009.