Miron Weinreb

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.

The Effects of Titanium and Hydroxyapatite on Osteoblastic Expression and Proliferation in Rat Parietal Bone Cultures

Titanium and hydroxyapatite are used for the fabrication of dental and orthopedic implants. The longevity of these implants depends on the amount and rate of bone formation that occurs around their surfaces. In the present study, the effects of titanium, hydroxyapatite, and polystyrene (control) on the proliferation of rat calvarial cells, and on their capacity to express alkaline phosphatase and respond to parathyroid hormone (PTH) stimulation, were studied. The nature of the substrate did not affect the DNA and protein contents of experimental and control cultures throughout the experimental period. Alkaline phosphatase expression and PTH response, as assessed by DNA synthesis and adenylate cyclase activity, were higher in cultures grown on hydroxyapatite and polystyrene than in those grown on titanium. These results indicate that hydroxyapatite was a more favorable substrate than titanium for the growth and differentiation of osteoblast-like cells in vitro.

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.

Expression of the prostaglandin E2 (PGE2) receptor subtype EP4 and its regulation by PGE2 in osteoblastic cell lines and adult rat bone tissue1

Abstract Prostaglandins E (especially PGE 2 ) stimulate bone formation and increase bone mass in several species including man. The mechanism for this effect, the target cells, and the receptors involved are not known. Specific cell-surface receptors for PGE 2 (EP 1–4 ) have been cloned and characterized. EP 4 was reported to be the major receptor in embryonic and neonatal bone tissue in mice, especially in preosteoblasts; however, no data are available regarding its expression in adult bone. This study examines the expression of EP 4 in bone tissue of young adult rats, in which PGE 2 is markedly anabolic, and in various osteoblastic cell lines. Using northern blot analysis, we found that osteoblastic cell lines RCT-1, RCT-3, TRAB-11, and RP-1, primary osteoblastic cells harvested from fetal rat calvaria, as well as tibiae and calvariae of 5-week-old rats express 3.8 kb EP 4 messenger RNA (mRNA). Treatment of periosteal cells (RP-1) in vitro with 10 −6 mol/L PGE 2 increased the levels of both EP 4 mRNA and EP 4 protein, peaking at 1–2 h. Similarly, systemic administration of an anabolic dose of PGE 2 (3–6 mg/kg) to young adult rats upregulated the expression of EP 4 in the tibia and calvaria, also peaking at 1–2 h. Using in situ hybridization, we found increased expression of EP 4 in bone marrow cells of the tibial metaphysis in response to systemic PGE 2 treatment. The preosteoblastic nature of these EP 4 -expressing cells was suggested by the fact that dexamethasone-treated bone marrow stromal cells in culture express EP 4 mRNA, which is upregulated by PGE 2 . Northern blot analysis failed to detect both basal and PGE 2 -induced EP 2 mRNA in the bone samples or cell lines tested. Taken together, these data implicate EP 4 as the major cyclic AMP-related PGE 2 receptor subtype expressed in bone tissue and osteoblastic cells and indicate that this receptor is upregulated by its ligand, PGE 2 .

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.

Short-term healing kinetics of cortical and cancellous bone osteopenia induced by unloading during the reloading period in young rats

Abstract We investigated the short-term recuperation of bone mass during skeletal reloading after a period of unloading in young rats. One hind limb of 4-week-old rats was either unloaded irreversibly by sciatic neurectomy, or unloaded reversibly by external fixation. Other animals were sham-operated. After 9 days, the fixation-unloaded limbs were reloaded for 1–3 weeks and were compared with the hind limbs of age-matched unloaded (neurectomized) and sham-operated controls. Cortical and cancellous bone mass was measured using ashing and histomorphometry. Cortical bone mass (expressed as femoral dry and ash weight and tibial cortical bone area) was reduced in both unloaded groups and was accompanied by production of hypomineralized bone, as shown by a reduction in the percent ash of the dry weight. Cancellous bone mass (expressed as bone area and surface at the tibial metaphysis) was also reduced in both unloaded groups. Cortical bone mass deficit was greater in the fixation group than in the neurectomy group. Thereafter it increased in the neurectomy group despite a normal longitudinal growth rate, but returned to age-matched values in the reloaded group by 3 weeks. The changes in tibial cancellous bone mass were more pronounced but followed a similar pattern and normalized by 2 weeks. These data demonstrate that total unloading produced by external fixation causes a greater degree of bone mass deficit than partial unloading (produced by neurectomy); the rate of bone loss during unloading in the rat hind limb is more rapid than its recovery during reloading; and cancellous bone recuperates during the reloading phase faster than does cortical bone.

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.

AGEs induce caspase-mediated apoptosis of rat BMSCs via TNFα production and oxidative stress.

Diabetic humans and animals exhibit lower bone mass and healing, resulting from diminished bone formation. We have recently reported that type 1 diabetic rats have fewer bone marrow osteoprogenitor cells, and since the formation of advanced glycation end products (AGEs) in bone increases in diabetes, we explored possible mechanisms involved in AGE-induced apoptosis of rat bone marrow stromal cells (BMSCs). BMSCs isolated from 4-month-old rats were exposed to 10-400 μg/ml AGE-BSA for 16 h and apoptosis was quantified with PI/annexin V staining and flow cytometry. Signaling mechanisms were evaluated by preincubating the cells with appropriate inhibitors. The formation of reactive oxygen species (ROS) was quantified by flow cytometric analysis of DCFDA fluorescence and the expression of genes by RT-PCR analysis. AGE-BSA at a concentration of 400 μg/ml increased the apoptosis of BMSCs two- to threefold, an effect completely blocked by a pan-caspase inhibitor. BSA or high concentrations of glucose had no effect. AGE-BSA-induced BMSC apoptosis was attenuated by a p38 inhibitor but not by an NF-κB inhibitor. Treatment with AGE-BSA induced the expression of several pro-apoptotic ligands and receptors, most notably tumor necrosis factor α (TNFα), TRAIL, lymphotoxin alpha, CD40, and TNFR2. Furthermore, AGE-BSA-induced apoptosis was completely blocked by pirfenidone, an inhibitor of TNFα production/secretion. Finally, AGE-BSA increased the production of ROS in BMSCs, and its apoptogenic effect was blocked by the antioxidant N-acetylcysteine (N-acetyl-L-cysteine). Thus, AGE-BSA increases the apoptosis of rat BMSCs via the activation of caspases, involving TNFα production/secretion, p38 MAPK signaling, and oxidative stress. We propose that increased protein glycation, such as that occurring under hyperglycemia, causes the apoptosis of BMSCs, which might significantly contribute to the development of osteopenia in diabetic animals.

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.