Toll-like receptor-2 induced inflammation causes local bone formation and activates canonical Wnt signaling.

It is well established that inflammatory processes in the vicinity of bone often induce osteoclast formation and bone resorption. Effects of inflammatory processes on bone formation are less studied. Therefore, we investigated the effect of locally induced inflammation on bone formation. Toll-like receptor (TLR) 2 agonists LPS from Porphyromonas gingivalis and PAM2 were injected once subcutaneously above mouse calvarial bones. After five days, both agonists induced bone formation mainly at endocranial surfaces. The injection resulted in progressively increased calvarial thickness during 21 days. Excessive new bone formation was mainly observed separated from bone resorption cavities. Anti-RANKL did not affect the increase of bone formation. Inflammation caused increased bone formation rate due to increased mineralizing surfaces as assessed by dynamic histomorphometry. In areas close to new bone formation, an abundance of proliferating cells was observed as well as cells robustly stained for Runx2 and alkaline phosphatase. PAM2 increased the mRNA expression of Lrp5, Lrp6 and Wnt7b, and decreased the expression of Sost and Dkk1. In situ hybridization demonstrated decreased Sost mRNA expression in osteocytes present in old bone. An abundance of cells expressed Wnt7b in Runx2-positive osteoblasts and ß-catenin in areas with new bone formation. These data demonstrate that inflammation, not only induces osteoclastogenesis, but also locally activates canonical WNT signaling and stimulates new bone formation independent on bone resorption.

Stimulation of Osteoclast Formation by Oncostatin M and the Role of WNT16 as a Negative Feedback Regulator.

Oncostatin M (OSM), which belongs to the IL-6 family of cytokines, is the most potent and effective stimulator of osteoclast formation in this family, as assessed by different in vitro assays. Osteoclastogenesis induced by the IL-6 type of cytokines is mediated by the induction and paracrine stimulation of the osteoclastogenic cytokine receptor activator of nuclear factor κ-B ligand (RANKL), expressed on osteoblast cell membranes and targeting the receptor activator of nuclear factor κ-B (RANK) on osteoclast progenitor cells. The potent effect of OSM on osteoclastogenesis is due to an unusually robust induction of RANKL in osteoblasts through the OSM receptor (OSMR), mediated by a JAK-STAT/MAPK signaling pathway and by unique recruitment of the adapter protein Shc1 to the OSMR. Gene deletion of Osmr in mice results in decreased numbers of osteoclasts and enhanced trabecular bone caused by increased trabecular thickness, indicating that OSM may play a role in physiological regulation of bone remodeling. However, increased amounts of OSM, either through administration of recombinant protein or of adenoviral vectors expressing Osm, results in enhanced bone mass due to increased bone formation without any clear sign of increased osteoclast numbers, a finding which can be reconciled by cell culture experiments demonstrating that OSM can induce osteoblast differentiation and stimulate mineralization of bone nodules in such cultures. Thus, in vitro studies and gene deletion experiments show that OSM is a stimulator of osteoclast formation, whereas administration of OSM to mice shows that OSM is not a strong stimulator of osteoclastogenesis in vivo when administered to adult animals. These observations could be explained by our recent finding showing that OSM is a potent stimulator of the osteoclastogenesis inhibitor WNT16, acting in a negative feedback loop to reduce OSM-induced osteoclast formation.

RSPO3 is important for trabecular bone and fracture risk in mice and humans.

With increasing age of the population, countries across the globe are facing a substantial increase in osteoporotic fractures. Genetic association signals for fractures have been reported at the RSPO3 locus, but the causal gene and the underlying mechanism are unknown. Here we show that the fracture reducing allele at the RSPO3 locus associate with increased RSPO3 expression both at the mRNA and protein levels, increased trabecular bone mineral density and reduced risk mainly of distal forearm fractures in humans. We also demonstrate that RSPO3 is expressed in osteoprogenitor cells and osteoblasts and that osteoblast-derived RSPO3 is the principal source of RSPO3 in bone and an important regulator of vertebral trabecular bone mass and bone strength in adult mice. Mechanistic studies revealed that RSPO3 in a cell-autonomous manner increases osteoblast proliferation and differentiation. In conclusion, RSPO3 regulates vertebral trabecular bone mass and bone strength in mice and fracture risk in humans.

Post-treatments of polydopamine coatings influence cellular response.

Polydopamine (PDA) is the final oxidation product of dopamine or other catecholamines. Since the first reports of PDA coatings starting around 2007, these coatings have been widely studied as a versatile and inexpensive one-step coating option for biomaterial functionalization. The coating attach to a wide range of materials and can subsequently be modified with biomolecules or nanoparticles. However, as a strong candidate for biomaterial research and even clinical use, it is important to unravel the changes in physico-chemical properties and the cell-PDA interaction as a function of heat sterilization procedures and shelf storage periods. Four groups were examined in this study: titanium (Ti), PDA-coated Ti samples and PDA-coated Ti samples either stored for up to two weeks at room temperature or heated at 121 °C for 24 h, respectively. We used X-ray Photoelectron Spectroscopy (XPS), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Water contact angle (WCA) to characterize chemical composition and surface properties of the groups. Cell adhesion and proliferation was examined by three different cell types: human primary dermal fibroblasts (hDF), human epidermal keratinocytes (HaCaTs) and a murine preosteoblastic cell line (MC3T3-E1), respectively. Cells were cultured on PDA coated samples for 4 h, 3 days and 5 days. Both thermal treatment of PDA at 121℃ for 24 h and storage of the samples for 2 weeks increased the amount of quinone groups at the surface and decreased the amount of primary amine groups as detected by XPS and ToF-SIMS. Even though these surface reactions increased the WCA of the PDA coating, we found that the post-treatments increased cell proliferation for both hDFs, HaCaTs and MC3T3-E1 s as compared to pristine PDA. This emphasizes the importance of post-treatment and shelf-time for PDA coatings.

Finding a Toll on the Route: The Fate of Osteoclast Progenitors After Toll-Like Receptor Activation.

M-CSF and RANKL are two crucial cytokines stimulating differentiation of mature, bone resorbing, multinucleated osteoclasts from mononucleated progenitor cells in the monocyte/macrophage lineage. In addition to the receptors for M-CSF and RANKL, osteoclast progenitor cells express receptors for several other pro- and anti-osteoclastogenic cytokines, which also regulate osteoclast formation by affecting signaling downstream M-CSF and RANKL receptors. Similar to many other cells originating from myeloid hematopoetic stem cells, also osteoclast progenitors express toll-like receptors (TLRs). Nine murine TLRs are expressed in the progenitors and all, with the exception of TLR2 and TLR4, are downregulated during osteoclastogenesis. Activation of TLR2, TLR4, and TLR9, but not TLR5, in osteoclast progenitors stimulated with M-CSF and RANKL arrests differentiation along the osteoclastic lineage and keeps the cells at a macrophage stage. When the progenitors are primed with M-CSF/RANKL and then stimulated with agonists for TLR2, TLR4, or TLR9 in the presence of M-CSF, but in the absence of RANKL, the cells differentiate to mature, bone resorbing osteoclasts. TLR 2, 4, 5, and 9 are also expressed on osteoblasts and their activation increases osteoclast differentiation by an indirect mechanism through stimulation of RANKL. In mice, treatment with agonists for TLR2, 4, and 5 results in osteoclast formation and extensive bone loss. It remains to be shown the relative importance of inhibitory and stimulatory effects by TLRs on osteoclast progenitors and the role of RANKL produced by TLR stimulated osteoblasts, for the bone resorbing effects in vivo.

Activation of Shc1 Allows Oncostatin M to Induce RANKL and Osteoclast Formation More Effectively Than Leukemia Inhibitory Factor.

Background and Purpose: The gp130 family of cytokines signals through receptors dimerizing with the gp130 subunit. Downstream signaling typically activates STAT3 but also SHP2/Ras/MAPK pathways. Oncostatin M (OSM) is a unique cytokine in this family since the receptor (OSMR) activates a non-redundant signaling pathway by recruitment of the adapter Shc1. We have studied the functional relevance of Shc1 for OSM-induced bone resorption. Experimental Approach: Osteoblasts were stimulated with OSM and STAT3 and Shc1 activations were studied using real-time PCR and Western blots. The role of STAT3 and Shc1 for OSM-induced RANKL expression and osteoclast formation was studied by silencing their mRNA expressions. Effects of OSM were compared to those of the closely related cytokine leukemia inhibitory factor (LIF). Key Results: OSM, but not LIF, induced the mRNA and protein expression of Shc1 and activated phosphorylation of Shc1 in the osteoblasts. Silencing of Shc1 decreased OSM-induced activation of STAT3 and RANKL expression. Silencing of STAT3 had no effect on activation of Shc1, but prevented the OSM-mediated increase of RANKL expression. Silencing of either Shc1 or STAT3 in osteoblasts decreased formation of osteoclasts in OSM-stimulated co-cultures of osteoblasts and macrophages. In agreement with these observations, OSM was a more potent and robust stimulator than LIF of RANKL formation and bone resorption in mouse calvariae and osteoclast formation in bone marrow cultures. Conclusions and Implications: Activation of the Shc1-dependent STAT3 signaling is crucial for OSM-induced osteoclast formation. Inhibition of Shc1 is a potential mechanism to specifically inhibit OSM-induced bone resorption.

Activation of Toll-like receptor 2 induces B1 and B2 kinin receptors in human gingival fibroblasts and in mouse gingiva.

The regulation of the kallikrein-kinin system is an important mechanism controlling vasodilation and promoting inflammation. We aimed to investigate the role of Toll-like receptor 2 (TLR2) in regulating kinin B1 and B2 receptor expression in human gingival fibroblasts and in mouse gingiva. Both P. gingivalis LPS and the synthetic TLR2 agonist Pam2CSK4 increased kinin receptor transcripts. Silencing of TLR2, but not of TLR4, inhibited the induction of kinin receptor transcripts by both P. gingivalis LPS and Pam2CSK4. Human gingival fibroblasts (HGF) exposed to Pam2CSK4 increased binding sites for bradykinin (BK, B2 receptor agonist) and des-Arg10-Lys-bradykinin (DALBK, B1 receptor agonist). Pre-treatment of HGF for 24 h with Pam2CSK4 resulted in increased PGE2 release in response to BK and DALBK. The increase of B1 and B2 receptor transcripts by P. gingivalis LPS was not blocked by IL-1ß neutralizing antibody; TNF-α blocking antibody did not affect B1 receptor up-regulation, but partially blocked increase of B2 receptor mRNA. Injection of P. gingivalis LPS in mouse gingiva induced an increase of B1 and B2 receptor mRNA. These data show that activation of TLR2 in human gingival fibroblasts as well as in mouse gingival tissue leads to increase of B1 and B2 receptor mRNA and protein.

Osteoblast differentiation is enhanced by a nano-to-micro hybrid titanium surface created by Yb:YAG laser irradiation.

OBJECTIVES: The aim of this study was to analyze the capacity of a new modified laser surface to stimulate calvarial osteoblasts isolated from neonatal mouse bones to differentiate and form mineralized nodules. METHODS: Titanium discs were subjectezd or not to laser irradiation according to specific parameters and characterized. Osteoblasts isolated from neonatal mouse calvaria were cultured over the discs, and the capacity of these cells to proliferate (MTT assay), form mineralized nodules (Alizarin red assay), and enhance alkaline phosphatase activity (ALPase activity) was analyzed. Real-time PCR was used for quantification of gene expression. RESULTS: Laser-irradiated titanium discs (L) presented a rough nano-to-micrometric oxidized surface contrasting with the smooth pattern on polished discs (P). The Ra on the micrometric level increased from 0.32 ± 0.01 µm on P surfaces to 10.57 ± 0.39 µm on L surfaces. When compared with P, L promoted changes in osteoblast morphology, increased mineralized nodule formation in osteoblasts cultured on the surfaces for 14 days, and enhanced ALPase activity at days 7 and 14. Transcription factors triggering osteoblast differentiation (Runx2 and Sp7) and genes encoding the bone extracellular matrix proteins collagen type-1 (Col1a1), osteopontin (Spp1), and osteocalcin (Bglap) were upregulated in cells on L surfaces compared with those on P surfaces at days 1-14. CONCLUSION: Laser treatment of titanium surfaces created a rough surface that stimulated osteoblast differentiation. CLINICAL RELEVANCE: Laser treatment of titanium generates a reproducible and efficient surface triggering osteoblast differentiation that can be of importance for osteointegration.

Porphyromonas gingivalis Stimulates Bone Resorption by Enhancing RANKL (Receptor Activator of NF-κB Ligand) through Activation of Toll-like Receptor 2 in Osteoblasts.

Periodontitis has been associated with rheumatoid arthritis. In experimental arthritis, concomitant periodontitis caused by oral infection with Porphyromonas gingivalis enhances articular bone loss. The aim of this study was to investigate how lipopolysaccharide (LPS) from P. gingivalis stimulates bone resorption. The effects by LPS P. gingivalis and four other TLR2 ligands on bone resorption, osteoclast formation, and gene expression in wild type and Tlr2-deficient mice were assessed in ex vivo cultures of mouse parietal bones and in an in vivo model in which TLR2 agonists were injected subcutaneously over the skull bones. LPS P. gingivalis stimulated mineral release and matrix degradation in the parietal bone organ cultures by increasing differentiation and formation of mature osteoclasts, a response dependent on increased RANKL (receptor activator of NF-κB ligand). LPS P. gingivalis stimulated RANKL in parietal osteoblasts dependent on the presence of TLR2 and through a MyD88 and NF-κB-mediated mechanism. Similarly, the TLR2 agonists HKLM, FSL1, Pam2, and Pam3 stimulated RANKL in osteoblasts and parietal bone resorption. LPS P. gingivalis and Pam2 robustly enhanced osteoclast formation in periosteal/endosteal cell cultures by increasing RANKL. LPS P. gingivalis and Pam2 also up-regulated RANKL and osteoclastic genes in vivo, resulting in an increased number of periosteal osteoclasts and immense bone loss in wild type mice but not in Tlr2-deficient mice. These data demonstrate that LPS P. gingivalis stimulates periosteal osteoclast formation and bone resorption by stimulating RANKL in osteoblasts via TLR2. This effect might be important for periodontal bone loss and for the enhanced bone loss seen in rheumatoid arthritis patients with concomitant periodontal disease.

M-CSF priming of osteoclast precursors can cause osteoclastogenesis-insensitivity, which can be prevented and overcome on bone.

Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone.

Non-canonical signalling and roles of the vasoactive peptides angiotensins and kinins.

GPCRs (G-protein-coupled receptors) are among the most important targets for drug discovery due to their ubiquitous expression and participation in cellular events under both healthy and disease conditions. These receptors can be activated by a plethora of ligands, such as ions, odorants, small ligands and peptides, including angiotensins and kinins, which are vasoactive peptides that are classically involved in the pathophysiology of cardiovascular events. These peptides and their corresponding GPCRs have been reported to play roles in other systems and under pathophysiological conditions, such as cancer, central nervous system disorders, metabolic dysfunction and bone resorption. More recently, new mechanisms have been described for the functional regulation of GPCRs, including the transactivation of other signal transduction receptors and the activation of G-protein-independent pathways. The existence of such alternative mechanisms for signal transduction and the discovery of agonists that can preferentially trigger one signalling pathway over other pathways (called biased agonists) have opened new perspectives for the discovery and development of drugs with a higher specificity of action and, therefore, fewer side effects. The present review summarizes the current knowledge on the non-canonical signalling and roles of angiotensins and kinins.

The role of cytokines in inflammatory bone loss.

Chronic inflammatory processes close to bone often lead to loss of bone in diseases such as rheumatoid arthritis, periodontitis, loosened joint prosthesis and tooth implants. This is mainly due to local formation of bone resorbing osteoclasts which degrade bone without any subsequent coupling to new bone formation. Crucial for osteoclastogenesis is stimulation of mononuclear osteoclast progenitors by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) which induces their differentiation along the osteoclastic lineage and the fusion to mature, multinucleated osteoclasts. M-CSF and RANKL are produced by osteoblasts/osteocytes and by synovial and periodontal fibroblasts and the expression is regulated by pro- and anti-inflammatory cytokines. These cytokines also regulate osteoclastic differentiation by direct effects on the progenitor cells. In the present overview, we introduce the basic concepts of osteoclast progenitor cell differentiation and summarize the current knowledge on cytokines stimulating and inhibiting osteoclastogenesis by direct and indirect mechanisms.

Cysteine proteinase inhibitors regulate human and mouse osteoclastogenesis by interfering with RANK signaling.

The cysteine proteinase inhibitor cystatin C inhibited RANKL-stimulated osteoclast formation in mouse bone marrow macrophage cultures, an effect associated with decreased mRNA expression of Acp5, Calcr, Ctsk, Mmp9, Itgb3, and Atp6i, without effect on proliferation or apoptosis. The effects were concentration dependent with half-maximal inhibition at 0.3 µM. Cystatin C also inhibited osteoclast formation when RANKL-stimulated osteoclasts were cultured on bone, leading to decreased formation of resorption pits. RANKL-stimulated cells retained characteristics of phagocytotic macrophages when cotreated with cystatin C. Three other cysteine proteinase inhibitors, cystatin D, Z-RLVG-CHN2 (IC50 0.1 µM), and E-64 (IC50 3 µM), also inhibited osteoclast formation in RANKL-stimulated macrophages. In addition, cystatin C, Z-RLVG-CHN2, and E-64 inhibited osteoclastic differentiation of RANKL-stimulated CD14(+) human monocytes. The effect by cystatin C on differentiation of bone marrow macrophages was exerted at an early stage after RANKL stimulation and was associated with early (4 h) inhibition of c-Fos expression and decreased protein and nuclear translocation of c-Fos. Subsequently, p52, p65, IκBα, and Nfatc1 mRNA were decreased. Cystatin C was internalized in osteoclast progenitors, a process requiring RANKL stimulation. These data show that cystatin C inhibits osteoclast differentiation and formation by interfering intracellularly with signaling pathways downstream RANK.

Interleukin-4 and interleukin-13 inhibit the expression of leukemia inhibitory factor and interleukin-11 in fibroblasts.

Cytokines produced by inflammatory or resident mesenchymal cells play important modulatory roles in the pathogenesis of inflammation induced bone loss. In the present study, the effects of IL-4 and IL-13 on the expression of three osteotropic cytokines in the IL-6 family expressed in human gingival fibroblasts were studied. IL-4Rα and IL-13Rα1 mRNA were constitutively expressed in human gingival fibroblasts. The inflammatory cytokines IL-1ß and TNF-α increased expression of IL-6, LIF, and IL-11 mRNA and protein in the gingival fibroblasts. Addition of IL-4 or IL-13 had no effect on IL-6 expression, but significantly inhibited LIF and IL-11 mRNA and protein stimulated by IL-1ß and TNF-α. No involvement of NF-κB or STAT1 was observed in the inhibition. STAT6 was phosphorylated at Y641 by treatment with IL-4 and knockdown of STAT6 with siRNA decreased the inhibition of IL-11 and LIF expression by IL-4 in IL-1ß and TNF-α stimulated cells. This study suggests that activation of STAT6 by IL-4 and IL-13, through type 2 IL-4 receptors, inhibits production of IL-11 and LIF stimulated by IL-1ß and TNF-α in human gingival fibroblasts. A negative modulatory role of IL-4 and IL-13 in osteotropic cytokine production could be a mechanism playing an important inhibitory role in inflammation induced periodontitis.

Lithium thiazolidine-4-carboxylate: synthesis, spectroscopic characterization and preliminary in vitro cytotoxic studies in human HeLa cells.

A new water-soluble lithium salt of thiazolidine-4-carboxylic acid was synthesized and characterized by chemical and spectroscopic techniques. Elemental and mass spectrometric (ESI-MS) analyses of the solid compound fit to the composition LiC(4)H(6)NSO(2). (1)H, (13)C nuclear magnetic resonance (NMR), [(1)H-(15)N] NMR and infrared (IR) analyses permitted to elucidate the structure of the compound. Biological activity was evaluated by cytotoxic analysis using HeLa cells. Determination of cell death was assessed using a tetrazolium salt colorimetric assay, which reflects the cells viability.

Participation of kallikrein-kinin system in different pathologies.

The general description of kinins refers to these peptides as molecules involved in vascular tone regulation and inflammation. Nevertheless, in the last years a series of evidences has shown that local hormonal systems, such as the kallikrein-kinin system, may be differently regulated and are of pivotal importance to pathophysiological control. The combined interpretations of many recent studies allow us to conclude that the kallikrein-kinin system plays broader and richer roles than those classically described until recently. In this review, we report findings concerning the participation of the kallikrein-kinin system in inflammation, cancer, and in pathologies related to cardiovascular, renal and central nervous systems.

Regulation of angiotensin II receptors levels during rat induced pulpitis.

A change in the microcirculatory hemodynamic is one of the most important events in inflammation. In the dental pulp, which is a connective tissue surrounded by a mineralized dentine substrate, disturbance in the blood flow as well as plasma extravasation may increase the pulp pressure and cause local ischemia. The octapeptide angiotensin II (AngII) regulates vascular tone and stimulates the release of pro-inflammatory cytokines by acting through the AT1 and AT2 receptors. The AT1 receptor is responsible for the classical effects of AngII. The AT2 receptor is involved in other effects, such as vasodilation. Therefore, we aimed to evaluate the role of AT1 and AT2 receptors on the pulpal inflammation. The pulp tissue was mechanically exposed and after different periods the teeth were extracted and submitted to histopathological and RT-PCR analyses. The histological sections showed a number of congested and dilated blood vessels associated with a notable presence of inflammatory cells. RT-PCR data revealed that the AT1 receptor was down-regulated at 24 h after the pulp exposure. The AT2 receptor expression was up-regulated by a 9-hour period, and then decreased between 12- and 24-hour periods. It was demonstrated that the renin-angiotensin system plays an important role in the pulpal inflammation, with regulation of AngII receptor levels.

Inhibition of eukaryotic translation initiation factor 5A (eIF5A) hypusination impairs melanoma growth.

The eukaryotic translation initiation factor 5A (eIF5A) undergoes a specific post-translational modification called hypusination. This modification is required for the functionality of this protein. The compound N1-guanyl-1,7-diaminoheptane (GC7) is a potent and selective inhibitor of deoxyhypusine synthase, which catalyses the first step of eIF5A hypusination process. In the present study, the effects of GC7 on cell death were investigated using two cell lines: melan-a murine melanocytes and Tm5 murine melanoma. In vitro treatment with GC7 increased by 3-fold the number of cells presenting DNA fragmentation in Tm5 cells. Exposure to GC7 also decreased viability to both cell lines. This study also describes, for the first time, the in vivo antitumour effect of GC7, as indicated by impaired melanoma growth in C57BL/6 mice.

Effect of curing regime on the cytotoxicity of resin-modified glass-ionomer lining cements applied to an odontoblast-cell line.

OBJECTIVE: The aim of this in vitro study was to evaluate the cytotoxicity of resin-modified glass-ionomer lining cements submitted to different curing regimes and applied to an immortalized odontoblast-cell line (MDPC-23). METHODS: Forty round-shaped specimens of each experimental material (Fuji Lining LC and Vitrebond) were prepared. They were light-cured for the manufacturers' recommended time (MRT = 30 s), under-cured (0.5 MRT = 15 s), over-cured (1.5 MRT = 45 s) or allowed to dark cure (0 MRT). Sterilized filter papers soaked with either 5 microL of PBS or HEMA were used as negative and positive control, respectively. After placing the specimens individually in wells of 24-well dishes, odontoblast-like cells MDPC-23 (30,000 cells/cm2) were plated in each well and incubated for 72 h in a humidified incubator at 37 degrees C with 5% CO2 and 95% air. The cytotoxicity was evaluated by the cell metabolism (MTT assay) and cell morphology (SEM). RESULTS: Fuji Lining LC was less cytotoxic than Vitrebond (p < 0.05) in all the experimental conditions. However, the cytotoxicity of Fuji Lining LC was noticeably increased in the absence of light-curing while the same was not observed for Vitrebond. The length of light-curing (15, 30 or 45 s) did not influence the toxicity of both lining materials when they were applied on the odontoblast-cell line MDPC-23. SIGNIFICANCE: The light-activation plays an important role in reducing the cytotoxicity of Fuji Lining LC. Following the manufacturer' recommendation regarding the light-curing regime may prevent toxic effect to the pulp cells.

In vitro cytotoxicity and in vivo biocompatibility of contemporary resin-modified glass-ionomer cements.

OBJECTIVES: To evaluate the effects of current resin-modified glass-ionomer cements (RMGICs) applied on culture of cells or implanted into subcutaneous tissue of rats. METHODS: Experiment 1 - Thirty round-shaped samples of every RMGICs: Rely X Luting Cement (RL), Vitremer (VM), and Vitrebond (VB) were placed into wells with 1.1 mL of culture medium (DMEM), and incubated for 24, 48 or 72 h. The extracts from every sample were applied on the MDPC-23 cells. Fresh DMEM was used as control group. The MTT assay was carried out for mitochondrial respiration. Experiment 2 - Fifty-four polyethylene tubes filled with the experimental materials were implanted into the dorsal subcutaneous tissue of rats. At 7, 30, and 90 days the animals were killed and the biopsies were processed for histological evaluation. RESULTS: Experiment--Both time of elution and material significantly influenced cell respiratory activity. In general, the extracts obtained at 24 h were less cytotoxic than 48 and 72 h incubation. The cytotoxic effect of VM and RL were not statistically different (p < 0.05) for the 24-hour period. VB showed the highest cytotoxic effect. Experiment 2--All RMGICs elicited at 7 days a moderate to intense inflammatory reaction which decreased over time. However, connective healing occurred for most of samples at 90-day evaluation. SIGNIFICANCE: Glass-ionomer cements may cause noticeable inflammatory response when in direct contact to connective tissue. The toxic effects of this kind of soluble material depend on the amount of components released in the aqueous environment.