Mechanisms involved in the enhancement of osteoclast formation by enamel matrix derivative

BACKGROUND AND OBJECTIVE: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, and it has been reported that EMD can induce the formation of osteoclasts in mouse marrow cultures. In the present study, we investigated the mechanisms of EMD-induced osteoclast formation using a mouse monocytic cell line, RAW 264.7. MATERIAL AND METHODS: Bioactive fractions were purified from EMD by reverse-phase HPLC using a C18 hydrophobic support, following which RAW 264.7 cells were cultured with EMD or its purified fractions in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) for 8 d. Following staining with tartrate-resistant acid phosphatase (TRAP), TRAP-positive multinucleated cells were counted. The expression of receptor activator of nuclear factor-kappaB (RANK), as well as phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, in RAW 264.7 cells were detected using immunoblotting. To determine whether EMD has an effect on osteoclast function, differentiated RAW 264.7 cells were cultured on Osteologic Multitest slides with RANKL in the presence of EMD. RESULTS: Purified EMD fractions (fraction numbers 21-25; EMD peak 2) were found to enhance the formation and function of RAW 264.7 cells induced by RANKL. Moreover, EMD peak 2 enhanced the levels of phosphorylation of ERK p38 and RANK in RAW 264.7 cells stimulated with RANKL. CONCLUSION: Our results indicate that EMD induces the formation of osteoclasts through interaction with RANKL, while ERK and p38 MAPK may play a critical role in the enhancement of osteoclast formation in RAW 264.7 cells.     

Inhibitory effect of titanium particles on osteoclast formation generated by treatment of mouse bone marrow cells with PGE2

OBJECTIVE: The present study was designed to evaluate the effect of titanium (Ti) particles with no endotoxin on osteoclast differentiation and osteoclast activity in in vitro experiments. METHODS: Osteoclast formation as well as osteoclastic bone resorbing activity were examined using the mouse bone marrow culture system and purified rabbit osteoclasts treated with Ti particles (2.5-20 microgram cm-2). RESULTS: Ti particles, with no adherent endotoxin, inhibited osteoclastogenesis and receptor activator of NF-kappaB ligand (RANKL) expression in bone marrow cells treated with prostaglandin E2 (PGE2) (100 nM). The inhibitory effect of Ti particles was concentration-dependent (5-20 microgram cm-2), and was observed only on the generation of osteoclasts by PGE2, but not by 1,25-dihydroxyvitamin D3 or soluble RANKL. This suggests that Ti particles did not act uniformly on a common process in the generation of osteoclasts, but specifically on signal transduction for PGE2 in generating osteoclasts. In highly purified osteoclasts, Ti particles showed no effect on survival and bone resorbing activity. CONCLUSION: Ti particles inhibited osteoclast differentiation and RANKL expression in mouse bone marrow cells treated with PGE2, without affecting mature osteoclast survival or activity. Thus, Ti particles may alter the osteoclastogenetic action of PGE2, which is one of the regulatory factors of bone remodeling.     

The effects of enamel matrix derivative (EMD) on osteoblastic cells

Enamel matrix derivative (EMDOGAIN, EMD) has been clinically used to promote regeneration of periodontal tissue, including cementum, periodontal ligament (PDL), and alveolar bone. However, it has not been clear whether EMD directly affects osteoblastic cells. To answer this question, we examined EMD effects on bovine PDL cells, rat and mouse bone marrow cells (RBM cells and MBM cells, respectively), and mouse osteoblastic cells (Kusa/A 1 cells). EMD was dissolved in 10 mM acetic acid and added to the culture medium at a final concentration of 50 micrograms/ml. EMD stimulated mineralized-nodule formation of PDL cells, RBM cells, and Kusa/A 1 cells. In Kusa/A 1 cells, EMD enhanced ALP activity, together with DNA content. Northern blotting analysis on Kusa/A 1 cells demonstrated stimulatory effects of EMD on the gene expression of type I collagen and osteopontin. Further, application of EMD on MBM cell culture, under 1,25(OH)2 vitamin D3 supplementation, stimulated osteoclast-like cell formation. These results indicate that osteoblastic cells respond to EMD, and that EMD would be potentially useful for bone regeneration.     

核因子-κB受体活化因子配体在大鼠正畸牙压力侧骨改建中的作用

目的：初步探讨在正畸牙移动压力侧核因子-κB受体活化因子配基(receptor activator of nuclearfactor-κB ligand，RANKL)的表达在破骨细胞诱导和骨改建中的调节作用。方法：建立大鼠正畸牙移动模型，利用免疫组化的方法对压力侧RANKL的表达及其变化进行检测；并进一步观察了RANKL对大鼠骨髓破骨细胞形成的影响。结果：正畸牙移动压力侧组化结果显示，RANKL阳性表达位于牙周膜细胞和位于骨陷窝内的破骨细胞中，在正畸牙移动第3、5和7天时呈强阳性表达。体外破骨细胞诱导实验结果显示，在巨噬细胞集落刺激因子(macrophage clone stimulating factor，M-CSF)协同作用下，RANKL呈剂量依赖型诱导TRAP阳性细胞生成。结论：大鼠正畸牙移动中，RANKL在压力侧的表达上调有诱导破骨细胞形成的作用，并导致牙槽骨吸收。     

Lipopolysaccharide from Prevotella nigrescens stimulates osteoclastogenesis in cocultures of bone marrow mononuclear cells and primary osteoblasts

BACKGROUND AND OBJECTIVE: Lipopolysaccharide is thought to be a major virulence factor of pathogens associated with periodontal diseases and is believed to stimulate bone resorption in vivo. Although Prevotella nigrescens has been implicated in periodontitis, its role in osteoclastogenesis has not been reported. In this study, we investigated the effects of lipopolysaccharide from P. nigrescens on the formation of osteoclasts and the production of cytokines related to osteoclast differentiation. MATERIAL AND METHODS: Mouse bone marrow mononuclear cells were cultured in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL), with or without lipopolysaccharide. Bone marrow mononuclear cells were also cocultured with calvarial osteoblastic cells in the presence or absence of lipopolysaccharide. Osteoclast formation was determined by tartrate-resistant acid phosphatase cytochemistry. The production of osteoprotegerin (OPG), M-CSF, tumor necrosis factor alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and prostaglandin E2 (PGE2) was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: P. nigrescens lipopolysaccharide inhibited osteoclast differentiation from bone marrow mononuclear cells cultured in the presence of M-CSF and RANKL. However, in the coculture system, P. nigrescens lipopolysaccharide stimulated osteoclastogenesis. Notably, P. nigrescens lipopolysaccharide decreased OPG production but increased TGF-beta secretion. In addition, treatment with P. nigrescens lipopolysaccharide increased PGE2 production during the late stage of the culture period. There was no difference in M-CSF and TNF-alpha production. CONCLUSION: These results demonstrate that P. nigrescens lipopolysaccharide stimulates osteoclastogenesis in the coculture system by decreasing the production of OPG and increasing the production of TGF-beta and PGE2. Through the mechanisms involving these factors, P. nigrescens lipopolysaccharide may cause alveolar bone resorption in periodontal diseases.     

Role of prostaglandin in the formation of osteoclasts induced by capsular-like polysaccharide antigen of Actinobacillus actinomycetemcomitans strain Y4

We found no reports that capsular-like polysaccharide antigen purified from Actinobacillus actinomycetemcomitans either induces osteoclastic bone resorp-tion in mouse organ cultures or promotes osteoclast formation in mouse marrow cultures. In contrast, capsular-like polysaccharide antigen purified from A. actinomycetemcomitans strain Y4 induced bone resorption in mouse organ culture. To examine the mechanism of bone resorption induced by A. actinomycetemcomitans , mouse bone marrow cells were cultured with A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen. A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen stimulated osteoclast-like cell formation in mouse bone marrow cultures. However, the polysaccharide of A. actinomycetemcomitans lipopolysaccharide did not induce the formation of osteoclast-like cells. Indomethacin inhibited osteoclast-like cell formation mediated by A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen in a dose-dependent manner. There was a good correlation between the number of osteoclast-like cells formed in the marrow culture and the amount of prostaglandin E2 released into the culture media. When mouse bone marrow cells were cultured with prostaglandin E2 during the culture periods, many osteoclast-like cells were formed. These results indicate that prostaglandin E2 is involved in the mechanism of the formation of osteoclast-like cells mediated by A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen. A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen may play an important role in inflammatory bone resorption by promoting osteoclast formation in periodontal disease.     

Osteoprotegerin and receptor activator of nuclear factor-kappa B ligand modulation by enamel matrix derivative in human alveolar osteoblasts

BACKGROUND: Bone regeneration techniques increasingly rely on the use of exogenous molecules able to enhance tissue formation in pathologic and traumatic defects. An enamel matrix derivative (EMD) has been largely used to promote tooth ligament regeneration within periodontal pockets. Recent evidence suggests that EMD may contribute to inducing osteoblast growth and differentiation. We investigated the effects of EMD on growth and osteogenic marker modulation in human mandibular osteoblasts. METHODS: We focused our attention on cell growth by 3-(4,5-dimethyl[thiazol-2-yl]-3,5-diphery)tetradium bromide (MTT) assay, cell differentiation, mineralized nodule formation, and, in particular, the expression of receptor activator of nuclear factor-kappa B ligand (RANKL), the main osteoclast differentiation factor, and its decoy receptor, osteoprotegerin (OPG), by enzyme-linked immunosorbent assay. RESULTS: Cell growth was significantly increased by EMD. Similarly, a significantly higher quantity of OPG and a lower amount of RANKL were detectable in groups treated with 50 and 100 microg/ml at weeks 1, 2, and 3, and alkaline phosphatase activity and osteocalcin production were enhanced in cultures treated with 50 and 100 microg/ml at weeks 2 and 3. Mineralized nodules appeared bigger and more numerous in cultures treated with 50 and 100 microg/ml EMD. CONCLUSIONS: EMD was able to enhance osteoblast cell growth and the expression of markers of osteoblastic phenotype and differentiation. EMD also seemed able to create a favorable osteogenic microenvironment by reducing RANKL release and enhancing osteoblastic OPG production.     

Cholera toxin and forskolin stimulate formation of osteoclast-like cells in mouse marrow cultures and cultured mouse calvarial bones

Osteoclasts are hematopoietic in origin and formed by proliferation, differentiation and fusion of osteoclast progenitor cells. However, the signal transducing mechanisms involved in generation of osteoclasts are not clear. We have used two well-known adenylate cyclase stimulators to examine the effect of cyclic AMP (cAMP) on the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in cultured mouse calvarial bones and in mouse bone marrow cultures. The effects of forskolin and cholera toxin were compared with those of parathyroid hormone (PTH) and 1,25(OH)2vitaminD3 (1,25(OH)2D3). PTH, as well as forskolin and cholera toxin, increased the number of osteoclast profiles/mm bone in 24-h and 120-h cultures of mouse calvarial bones. In mouse bone marrow cultures, 1,25(OH)2D3 or PTH stimulated formation of TRAP-positive multinucleated cells. Moreover, forskolin or cholera toxin produced dose-dependent stimulation of these cells at a range of concentrations correlating with their effect on cAMP production. The osteoclastic phenotype of the TRAP-positive cells was demonstrated by autoradiography of 125I-labelled calcitonin binding and by the bone-resorbing activity of the cells. The sustained presence (0-9 d) of forskolin or PTH was required to obtain maximal formation of osteoclasts. However, the presence of 1,25(OH)2D3 was required only for the last 3 d of culture for maximal osteoclast formation. We conclude that PTH may stimulate osteoclast generation using the adenylate cyclase cAMP system as a signal transduction mechanism.     

Effects of whole cell sonicates of Treponema lecithinolyticum on osteoclast differentiation

BACKGROUND: Alveolar bone destruction is a characteristic feature of periodontal diseases and multinucleated osteoclast cells derived from hemopoietic cells are responsible for bone resorption. Treponema lecithinolyticum is a novel oral spirochete isolated from the periodontal lesions. METHODS: The effect of whole cell sonicates on the osteoclast differentiation was examined in a co-culture system of hemopoietic mouse bone marrow cells and calvaria derived-osteoblastic cells to clarify the role of T. lecithinolyticum in the alveolar bone destruction associated with periodontal diseases. The differentiated osteoclasts were confirmed by tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: Sonicates of this bacterium stimulated the osteoclast formation in the co-culture system in a dose-dependent manner. The sonicates-induced osteoclast formation was partially inhibited by the heat treatment of sonicates. Indomethacin, which is a prostaglandin inhibitor, decreased the osteoclast formation induced by the bacterial sonicates. CONCLUSIONS: These findings suggest that T. lecithinolyticum induces osteoclast differentiation by a prostaglandin E2-dependent mechanism and that heat-labile components may be involved in this process.     

Involvement of prostaglandin E2 and interleukin-1α in the differentiation and survival of osteoclasts induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions. LPS from various periodontal pathogens is supposed to be a major virulence factor of periodontal diseases. In the present study, we demonstrated that LPS from periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (Y4 LPS) stimulated osteoclast formation in mouse bone marrow culture systems. Addition of anti-interleukin-lα (IL-lα) antibody or indomethacin in the marrow cultures resulted in the suppression of osteoclast differentiation. Quantitative analyses revealed that Y4 LPS stimulated the production of IL-lα and prostaglandin E₂ (PGE₂) by bone marrow cells. Furthermore, an immunoblot analysis showed that Y4 LPS stimulated bone marrow cells to upregulate the expression of cyclooxygenase-2, a rate-limiting enzyme for the conversion of arachidonic acid to prostanoids. These findings suggest that both IL-lα and PGE₂ are involved in the LPS-mediated osteoclast differentiation. In addition, we found that Y4 LPS supported the survival of osteoclasts. Addition of anti-IL-lα antibody in the osteoclast culture resulted in a reduction of osteoclast survival. Indomethacin, however, showed no effect on osteoclast survival. These findings suggest that the increased PGE₂ and IL-lα synthesis by bone marrow cells may play an important role in the differentiation and survival of osteoclasts induced by A. actinomycetemcomitans LPS.     

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 micro g/mL) increased the osteogenic capacity of bone marrow, as evidenced by approximately three-fold increase in BMSC cell number and approximately 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 enamel matrix derivative (EMD) on osteoblastic cells in culture and bone regeneration in a rat skull defect

OBJECTIVE: Enamel matrix derivative (EMD) has been clinically used to promote periodontal tissue regeneration. The purpose of the present study is to clarify EMD affects on osteoblastic cells and bone regeneration. MATERIALS AND METHODS: Mouse osteoblastic cells (ST2 cells and KUSA/A1 cells) are used in culture experiments. After cells were treated with EMD, cell growth was evaluated with DNA measurement, 5-bromo-2'-deoxyurydine (BrdU) incorporation assay. Measurement of alkaline phosphatase (ALP) activity and mineralized-nodule (MN) formation, Northern blotting analysis and zymography are also performed. In addition, EMD was applied to a rat skull defect and the defect was radiographically and histologically evaluated 2 weeks after the application. RESULTS: EMD did not stimulate ST2 cell growth; however, it enhanced KUSA/A1 cell proliferation. Although EMD stimulated ALP activity in both the cells, ALP activity in KUSA/A1 cells was affected to a much greater degree. Corresponding to the increase in ALP activity, MN formation in KUSA/A1 cells was enhanced by EMD. EMD stimulated osteoblastic phenotype expression of KUSA/A1 cells such as type I collagen, osteopontin, transforming growth factor beta 1 and osteocalcin. EMD treatment also stimulated matrix metalloproteinase production in KUSA/A1 cells. Although the effects of EMD on osteoblastic cells depend on cell type, the overall effect of EMD on osteoblastic cells is stimulatory rather than inhibitory. Finally, EMD application to a rat skull defect accelerated new bone formation. CONCLUSION: These results indicate that EMD affects osteoblastic cells and has potential as a therapeutic material for bone healing.     

The W9 peptide directly stimulates osteoblast differentiation via RANKL signaling

Objective

A RANKL-binding peptide, WP9QY (W9), is known to inhibit mouse osteoclastogenesis by stimulating the production of autocrine factors such as bone morphogenetic proteins (BMPs) to induce osteoblast differentiation. In the present study, we investigated whether osteoblastic differentiation is mediated by RANKL signaling.

In situ expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts of rat periodontal tissue

OBJECTIVES: This study examined the in situ expression of receptor activator of nuclear factor-kappaB ligand (RANKL), receptor activator of nuclear factor-kappaB (RANK), osteoprotegerin, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in the osteoclasts of rat periodontal tissue. BACKGROUND: In periodontal disease, osteoclasts cause resorption of the alveolar bone. The function of osteoclasts is regulated by interaction with periodontal ligament cells (PDLs). Furthermore, various kinds of molecules such as RANKL, RANK, osteoprotegerin, IL-1beta and TNFalpha are known to be related to the osteoclasts differentiation and function. It is therefore important to observe the expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts and PDLs. METHODS: Four-week-old Wistar rats were used. Tooth movement was performed by the Waldo method, and the pathological bone resorption was induced. The demineralized maxillae and mandiblae were embedded with paraffin. In situ hybridization was performed to detect RANKL, RANK, osteoprotegerin, IL-1beta, and TNFalpha mRNAs in osteoclasts and other cells using the specific RNA probes, respectively. RESULTS: Both RANKL and RANK were concomitantly expressed in some osteoclasts. RANKL was also positive in osteoblasts and PDLs. No IL-1beta- and TNFalpha-positive osteoclast was noted. The positive signals of osteoprotegerin were detected in almost all osteoblasts, PDLs and odontoblasts. No osteoprotegerin-positive osteoclasts were observed. The number and the distribution pattern of RANKL- and RANK-expressing osteoclasts changed when orthodontic excessive force was applied to periodontal tissue. In addition, IL-1beta and TNFalpha were shown to be expressed in osteoclasts under pathological status. CONCLUSION: These findings suggest that an autocrine mechanism of RANKL-RANK exists in osteoclast, which is heightened in the pathological conditions. Furthermore, the autocrine mechanism of IL-1beta and TNFalpha is also provided in osteoclast under pathological condition. These autocrine mechanisms therefore seem to regulate the osteoclast function in both physiological and pathological conditions.     

The effects of varying degrees of allograft decalcification on cultured porcine osteoclast cells

BACKGROUND: Demineralized freeze-dried bone allograft (DFDBA) is widely used in periodontal therapy as a scaffold for new bone formation in periodontal defects. It is demineralized, theoretically, to expose osteoinductive or osteoconductive bone matrix proteins that should facilitate osteogenesis. The degree of DFDBA demineralization varies between tissue banks and may affect clinical regeneration. A 2% residual calcium level in DFDBA has been shown to result in the highest alkaline phosphatase activity levels in cultured human periosteal cells and is optimally osteoinductive or osteoconductive for new bone formation. The purpose of this study was to evaluate the effect of 4 different residual calcium levels in commercially available DFDBA samples on porcine osteoclast activity as measured by resorption on calcium phosphate-coated disks. METHODS: Bone marrow was harvested from the femurs of 3-week-old farm pigs and cultured for 3 weeks. Hematopoietic stem cells were allowed to differentiate into mature active polykaryons displaying genuine osteoclast characteristics. The osteoclast cells displayed a dense actin band inside the margins of the cytoplasm under light microscopy. Culture media was decanted and collagenase added to free the attached cells. Equal cell samples were pipetted onto calcium phosphate-coated disks in 24-well plates. DFDBA samples with 1.44%, 2.41%, and 5.29% residual calcium; FDBA (30% residual calcium); and control cultures without allograft samples were prepared and all samples incubated for 1 week. Cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), Oregon Green 488-phalloidin, a stain for cytoskeletal proteins, and counterstained with propidium iodide. Specimens were examined by light and fluorescence microscopy using epi-illumination. Calcium phosphate disks were then rinsed in 5% sodium hypochlorite to remove adherent osteoclasts, and substrate surface changes were measured by white light interferometry and image analysis. RESULTS: A higher yield of TRAP-positive cells was produced without DFDBA; however, resorptive activity appears to be significantly increased in the presence of 2.41% residual calcium as compared to all other experimental groups (P<0.0065). CONCLUSION: In this in vitro model, porcine osteoclasts show significantly more resorptive activity as measured on calcium phosphate-coated disks in the presence of 2.41% residual calcium in DFDBA than in other DFDBA residual calcium levels.     

The role of cyclooxygenase-2 (COX-2) in inflammatory bone resorption

Prostaglandin E2 (PGE(2)) is an important inflammatory mediator that plays an essential role in the development and progression of periradicular diseases. Cyclooxygenase-2 (COX-2) is the inducible enzyme responsible for increased PGE(2) levels during inflammation and other pathologic processes. The purpose of this study was to determine the role of COX-2-mediated PGE(2) synthesis in osteoclast formation in response to endodontic pathogens and materials. Primary osteoblast cultures and osteoclast cultures were prepared from COX-2 knockout (K/O) and wild-type (WT) littermates. These cultured cells were exposed to lipopolysaccharide (LPS) or root canal obturation materials including gutta-percha (GP), Resilon (RS), mineral trioxide aggregates (MTAs), and AH Plus (AH+). Osteoclast formation was evaluated using tartrate-resistant acid phosphatase (TRAP) staining. The expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) was determined by real-time polymerase chain reaction (PCR) analysis. It was found that in both WT and K/O cultures, treatment with LPS led to a marked increase in osteoclast formation. The number of osteoclasts formed was significantly lower in K/O cultures compared to WT cultures. Exposure to endodontic materials did not lead to any significant osteoclast formation. LPS and endodontic materials caused a decrease in both RANKL and OPG expression in WT cells. In K/O cells, the baseline levels of RANKL and OPG expression were dramatically decreased compared to the WT cells. In conclusion, COX-2-mediated PGE(2) expression is required for LPS-induced inflammatory bone resorption and maintaining the baseline level of RANKL and OPG expression. LPS-induced osteoclast formation may be independent of the RANKL pathway.     

Effects of Porphyromonas gingivalis surface-associated material on osteoclast formation

Porphyromonas gingivalis strongly correlates with periodontitis, but the underlying mechanisms causing dentoalveolar bone resorption are not fully understood. As contradictory effects of P. gingivalis on osteoclastogenesis have been reported, this study investigates the effect of P. gingivalis extract on osteoclast formation. Osteoclast formation in mouse bone marrow (MBM) cell cultures and RAW 264.7 cells was stimulated by nuclear factor-κB ligand (RANKL) or parathyroid hormone (PTH). Cells were cultured with and without P. gingivalis surface-associated material and phenotypic characteristics were examined using microscopy, flow cytometry, and RT-PCR. P. gingivalis significantly decreased osteoclast formation and the expression of osteoclast phenotypic markers in PTH-stimulated MBM cultures. Additionally, P. gingivalis inhibited expression of osteoclast differentiation factors and stimulated expression of the mouse macrophage marker F4/80. The presence of P. gingivalis in RANKL-stimulated MBM cultures and RAW 264.7 cells inhibited osteoclastogenesis. Interestingly, a transient exposure with P. gingivalis before PTH stimulation increased osteoclastogenesis in MBM cultures. Flow cytometric analyses of cells transiently exposed to P. gingivalis demonstrated an increased proportion of potential osteoclast precursor cells. We conclude that a transient exposure of MBM cultures to P. gingivalis increases the number of osteoclast precursors and osteoclast formation, whereas a prolonged exposure completely abolishes osteoclastogenesis.     

A histopathological investigation on the effect of systemic administration of the bisphosphonate alendronate on resorptive phase following mucoperiosteal flap surgery in the rat mandible

BACKGROUND: The present study was designed to assess histopathologically whether the systemic administration of aminobisphosphonate (alendronate), 0.5 mg/kg body weight, is effective in preventing alveolar bone resorption following mucoperiosteal flap surgery, and whether alendronate modulates tissue factors. METHODS: The effect of alendronate on bone resorption was evaluated in mucoperiosteal flaps used as a resorptive model. The animals were given subcutaneous injections of either saline (control group) or 0.5 mg/kg of alendronate (experimental group). The alendronate or saline was administered subcutaneously 1 week prior to surgery, immediately prior to surgery, and 1 week after surgery. The parameters determined with a semiquantitative subjective method for histopathological evaluation were as follows: inflammatory cell infiltration (ICI) of adjacent periodontal tissue, degree of fibrosis and collagen bundle formation, number and morphology of osteoclasts of the alveolar bone and interdental septum, resorption lacunae (osteoclast surfaces), and osteoblastic activity (forming surfaces). RESULTS: There were no statistically significant differences between the saline and alendronate groups with regard to inflammatory cell infiltration, number of osteoclasts, and osteoblastic activity. Fibrosis and collagen bundle formation, osteoclast morphologies, and resorption lacunae formation were significantly different between the two groups, in favor of the alendronate group. CONCLUSIONS: The systemic administration of 0.5 mg/kg alendronate was effective in preventing alveolar bone loss and in modulating tissue factors. These findings indicate that alendronate would be a valuable addition to the therapeutic armamentarium available for the treatment of periodontal diseases, either alone or in combination with regenerative components such as anti-inflammatory drugs, bone graft materials, and guided tissue regeneration techniques, and even with dental implants.