Simvastatin induces apoptosis and disruption of the actin cytoskeleton in human dental pulp cells and periodontal ligament fibroblasts.

OBJECTIVE: Simvastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, and widely used as cholesterol-lowering agent, has been suggested for its beneficial effects on alveolar bone formation, regeneration of dental pulp tissue and periodontal ligament. High doses of simvastatin appear to induce apoptosis in several cell types, but little is known about its possible effect on tooth-associated cells. Therefore, the effects of simvastatin were studied on apoptosis and cell morphology of human dental pulp cells (HDPCs) and periodontal ligament fibroblasts (HPLFs). METHODS: HDPCs/HPLFs obtained from 4 patients were cultured with or without various concentrations of simvastatin (0.1, 1, and 10µM) for 24, 48, and 72h. The 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate cell viability. The levels of apoptosis of HDPCs and HPLFs were measured by flow cytometry after Annexin V/propidium iodide double staining. Phalloidin-FITC and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining was used to examine differences in the actin cytoskeleton and nuclear morphology, respectively. RESULTS: The viability of HDPCs and HPLFs was significantly reduced after simvastatin treatment in a dose- and time-dependent manner (p<0.05). The apoptosis of HDPCs and HPLFs was significantly increased in 10µM simvastatin-treated cells (p<0.05). The effect on apoptosis was comparable for HDPCs and HPLFs. Nuclear staining showed typical apoptotic nuclear condensation and fragmentation in simvastatin-treated HDPCs/HPLFs. A dose- and time-dependent simvastatin-induced disruption of the actin cytoskeleton was observed in both cell types. CONCLUSION: Our data demonstrated that simvastatin decreases the viability of HDPCs and HPLFs, probably by inducing apoptosis.

Oestrogen inhibits osteoclast formation induced by periodontal ligament fibroblasts.

OBJECTIVE: Since tooth-associated fibroblasts are taken to participate in the formation of osteoclasts and it is unknown whether oestrogen affects this process, the effects of 17ß-estradiol (17ß-E(2)) were studied on osteoclastogenesis induced by human periodontal ligament fibroblasts (PLFs) and gingival fibroblasts (GFs). METHODS: Human peripheral blood mononuclear cells (PBMCs) were seeded on monolayers of PLFs and GFs and cocultured for 14 days in the presence or absence of various concentrations of 17ß-E(2). The number of tartrate resistant acid phosphatase (TRACP)-positive osteoclast-like cells (OCs) was assessed. In addition, we analysed the PBMC-induced withdrawal of the fibroblasts. mRNA expression was determined of oestrogen receptor (ER)-α, ER-ß, receptor activator nuclear factor kappa B ligand (RANKL), and osteoprotegerin (OPG) by PLFs and GFs. RESULTS: PBMCs induced a higher number and larger fibroblast-free areas if cocultured with PLFs than with GFs. Concomitantly, the number of TRACP-positive OCs was significantly higher in PLF cocultures. 17ß-E(2) inhibited the formation of OCs in PLF cocultures. 17ß-E(2) did not alter the expression of RANKL, OPG, and ER-α mRNAs in either fibroblast cell population. CONCLUSION: Our data indicate that PLFs may promote osteoclastogenesis more strongly than GFs. 17ß-E(2) inhibits the PLF-induced formation of osteoclast-like cells. Thus, the inhibitory effect of oestrogen on osteoclast formation appears to be cell type dependent.

Gingival fibroblasts are better at inhibiting osteoclast formation than periodontal ligament fibroblasts.

Various studies indicate that periodontal ligament fibroblasts (PLF) have some similarities to osteoblasts, for example they have the capacity to induce the formation of osteoclast-like cells. Here, we investigated whether a second population of tooth-associated fibroblasts, gingival fibroblasts (GF), has similar osteoclastogenesis properties. PLF and GF were co-cultured with peripheral blood mononuclear cells (PBMC) in the presence and absence of dexamethasone and 1alpha,25dihydroxycholecalciferol (dex + vit D(3)) on plastic and on cortical bone slices. Tartrate resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs) were more abundant in co-cultures with PLF than in GF-PBMC co-cultures, more abundant on plastic compared to bone and more abundant in the presence of dex + vit D(3). In line with these findings was an inhibition of MNC formation and not inhibition of existing osteoclasts by medium conditioned by GF. We next investigated whether expression of molecules important for osteoclastogenesis differed between the two types of fibroblasts and whether these molecules were regulated by dex + vit D(3). OPG was detected at high levels in both fibroblast cultures, whereas RANKL could not be detected. Resorption of bone did not occur by the MNCs formed in the presence of either fibroblast subpopulation, suggesting that the fibroblasts secrete inhibitors of bone resorption or that the osteoclast-like cells were not functional. The incapacity of the MNCs to resorb was abolished by culturing the fibroblast-PBMC cultures with M-CSF and RANKL. Our results suggest that tooth-associated fibroblasts may trigger the formation of osteoclast-like cells, but more importantly, they play a role in preventing bone resorption, since additional stimuli are required for the formation of active osteoclasts.