Phosphorylation Modulates Ameloblastin Self-assembly and Ca 2+ Binding.

Ameloblastin (AMBN), an important component of the self-assembled enamel extra cellular matrix, contains several in silico predicted phosphorylation sites. However, to what extent these sites actually are phosphorylated and the possible effects of such post-translational modifications are still largely unknown. Here we report on in vitro experiments aimed at investigating what sites in AMBN are phosphorylated by casein kinase 2 (CK2) and protein kinase A (PKA) and the impact such phosphorylation has on self-assembly and calcium binding. All predicted sites in AMBN can be phosphorylated by CK2 and/or PKA. The experiments show that phosphorylation, especially in the exon 5 derived part of the molecule, is inversely correlated with AMBN self-assembly. These results support earlier findings suggesting that AMBN self-assembly is mostly dependent on the exon 5 encoded region of the AMBN gene. Phosphorylation was significantly more efficient when the AMBN molecules were in solution and not present as supramolecular assemblies, suggesting that post-translational modification of AMBN must take place before the enamel matrix molecules self-assemble inside the ameloblast cell. Moreover, phosphorylation of exon 5, and the consequent reduction in self-assembly, seem to reduce the calcium binding capacity of AMBN suggesting that post-translational modification of AMBN also can be involved in control of free Ca2+ during enamel extra cellular matrix biomineralization. Finally, it is speculated that phosphorylation can provide a functional crossroad for AMBN either to be phosphorylated and act as monomeric signal molecule during early odontogenesis and bone formation, or escape phosphorylation to be subsequently secreted as supramolecular assemblies that partake in enamel matrix structure and mineralization.

Ameloblastin Peptides Modulates the Osteogenic Capacity of Human Mesenchymal Stem Cells.

During amelogenesis the extracellular enamel matrix protein AMBN is quickly processed into 17 kDa (N-terminus) and 23 kDa (C-terminus) fragments. In particular, alternatively spliced regions derived by exon 5/6 within the N-terminus region are known to be critical in biomineralization. Human mesenchymal stem cells (hMSC) also express and secrete AMBN, but it is unclear if this expression has effects on the hMSC themselves. If, as suggested from previous findings, AMBN act as a signaling molecule, such effects could influence hMSC growth and differentiation, as well as promoting the secretion of other signaling proteins like cytokines and chemokines. If AMBN is found to modulate stem cell behavior and fate, it will impact our understanding on how extracellular matrix molecules can have multiple roles during development ontogenesis, mineralization and healing of mesenchymal tissues. Here we show that synthetic peptides representing exon 5 promote hMSC proliferation. Interestingly, this effect is inhibited by the application of a 15 aa peptide representing the alternatively spliced start of exon 6. Both peptides also influence gene expression of RUNX2 and osteocalcin, and promote calcium deposition in cultures, indicating a positive influence on the osteogenic capacity of hMSC. We also show that the full-length AMBN-WT and N-terminus region enhance the secretion of RANTES, IP-10, and IL-8. In contrast, the AMBN C-terminus fragment and the exon 5 deleted AMBN (DelEx5) have no detectable effects on any of the parameters investigated. These findings suggest the signaling effect of AMBN is conveyed by processed products, whereas the effect on proliferation is differentially modulated through alternative splicing during gene expression.

Antibacterial effect of doxycycline-coated dental abutment surfaces.

Biofilm formation on dental abutment may lead to peri-implant mucositis and subsequent peri-implantitis. These cases are clinically treated with antibiotics such as doxycycline (Doxy). Here we used an electrochemical method of cathodic polarization to coat Doxy onto the outer surface of a dental abutment material. The Doxy-coated surface showed a burst release in phosphate-buffered saline during the first 24 h. However, a significant amount of Doxy remained on the surface for at least 2 weeks especially on a 5 mA-3 h sample with a higher Doxy amount, suggesting both an initial and a long-term bacteriostatic potential of the coated surface. Surface chemistry was analyzed by x-ray photoelectron spectroscopy and secondary ion mass spectrometry. Surface topography was evaluated by field emission scanning electron microscopy and blue-light profilometry. Longer polarization time from 1 h to 5 h and higher current density from 1 to 15 mA cm(-2) resulted in a higher amount of Doxy on the surface. The surface was covered by a layer of Doxy less than 100 nm without significant changes in surface topography. The antibacterial property of the Doxy-coated surface was analyzed by biofilm and planktonic growth assays using Staphylococcus epidermidis. Doxy-coated samples reduced both biofilm accumulation and planktonic growth in broth culture, and also inhibited bacterial growth on agar plates. The antibacterial effect was stronger for samples of 5 mA-3 h coated with a higher amount of Doxy compared to that of 1 mA-1 h. Accordingly, an abutment surface coated with Doxy has potential for preventing bacterial colonization when exposed to the oral cavity. Doxy-coating could be a viable way to control peri-implant mucositis and prevent its progression into peri-implantitis.

Hypoxia increases the expression of enamel genes and cytokines in an ameloblast-derived cell line.

The aim of this study was to investigate the effect of hypoxic conditions on the expression of enamel genes and on the secretion of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), cytokines, and interleukins by an ameloblast-derived cell line. Murine ameloblast-derived cells (LS-8 cells) were exposed to 1% oxygen for 24 and 48 h and harvested after 1, 2, 3, and 7 d. The effect of culture in hypoxic conditions on the expression of structural enamel matrix genes and on the secretion of cytokines and interleukins, as well as ALP and LDH, into the cell-culture medium was calculated relative to the expression and secretion of these factors by untreated cells (controls) at each time point. Hypoxia increased expression of the structural enamel matrix genes amelogenin (Amelx), ameloblastin (Ambn), and enamelin (Enam), and the enamel protease matrix metalloproteinase-20 (Mmp20). Expression of hypoxia-inducible factor 1-alpha (Hif1α), and secretion of several vascularization factors and pro-inflammatory factors, were increased after 24 and 48 h of hypoxia. The ALP activity was reduced after 24 and 48 h of hypoxia, whereas the LDH level in the cell-culture medium was higher after 24 h of hypoxic conditions compared with 48 h. In conclusion, hypoxic exposure may disrupt the controlled fine-tuned expression and processing of enamel genes, and promote the secretion of pro-inflammatory factors.

The influence of surface nanoroughness, texture and chemistry of TiZr implant abutment on oral biofilm accumulation.

OBJECTIVES: The aim of the study was to examine surface nanoroughness, texture and chemistry of dental implant abutment and to investigate how these parameters influence oral biofilm formation in healthy subjects. MATERIALS AND METHODS: Eight different nanorough TiZr surfaces were produced by polishing, machining, cathodic polarization and acid etching. Surface topography was examined using field emission scanning electron microscope and a blue light laser profilometer. Surface chemistry was analyzed by secondary ion mass spectrometry and X-ray photoelectron spectroscopy. Surface hydrophilicity was tested by measuring contact angle on the surfaces. A human in vivo study using a splint model was employed to evaluate oral biofilm accumulation on these surfaces. RESULTS: Different surface textures (flat, grooved and irregular) were created with nanoroughness from 29 to 214 nm. Some test surfaces were incorporated with hydrogen by cathodic polarization and/or acid etching with HCl/H(2)SO(4). Nanoroughness (S(a)) positively correlated with microbial adhesion. Biofilm accumulation was less pronounced on flat and grooved than on irregular surfaces. No significant association between hydrogen content or hydrophilicity of the surface and biofilm accumulation was observed. CONCLUSIONS: Nanoroughness (< 214 nm) and surface texture influence oral biofilm accumulation independent of surface chemistry and hydrophilicity. Surface hydrogen, which has previously been shown to promote fibroblast growth, does not affect biofilm formation.

Sulcus fluid bone marker levels and the outcome of surgical treatment of peri-implantitis.

AIM: To analyse change in selected bone markers in peri-implant sulcus fluid (PISF) sampled before treatment and after 12 months and test correlation with change in disease progression. MATERIALS AND METHODS: Peri-implant sulcus fluid was sampled from 32 patients in a randomized, clinical study comparing peri-implant defect re-construction with or without porous titanium granules. Matrix metalloproteinase 8 levels were measured using the Quantikine Human Total MMP-8 (DMP800) ELISA. Multianalyte profiling of the level of bone markers [interleukin-6, osteprotegerin (OPG), osteocalcin, leptin, osteopontin, parathyroid hormone, tumour necrosis factor-α, adiponectin and insulin] was performed by Luminex using Human Bone Panel IB. Changes in bone marker levels were compared and correlation with clinical findings was tested. RESULTS: No differences in clinical parameter or bone marker levels between test and control group were found. When comparing bone marker levels irrespective of treatment allocation between baseline and 12 months, a significant reduction in total protein, matrix metalloproteinase -8, interleukin-6, OPG, leptin and adiponectin were demonstrated. Positive correlations were found between the reduction in interleukin-6 (r = 0.43), insulin (r = 0.38) and matrix metalloproteinase-8 (r = 0.47) concentration, and probing pocket depth reduction. CONCLUSION: Peri-implantitis surgical treatment induced some reduction of the studied bone markers. Conclusive evidence for correlation between change in bone marker concentrations with disease resolution was not found.

Surface hydride on titanium by cathodic polarization promotes human gingival fibroblast growth.

Connective tissue seal to dental abutment is crucial for peri-implant health. Several efforts have been made previously to optimize abutment surfaces, but no consensus has been reached regarding the optimal surface architecture and/or composition for soft tissue seal. Here, we report on experiments using cathodic polarization in organic acids to optimize titanium (Ti) surfaces for use as abutments. The three main factors affecting surface topography and chemistry were electrolyte composition, current density, and polarization time. Under identical conditions, oxalic acid created rougher surfaces than tartaric acid and acetic acid, and acetic acid produced more surface hydride. Surface hydride amount was suggested to first increase and then decrease with current density from 1 mA/cm(2) to 15 mA/cm(2) . The complexity of the surface topography and hydride production both increased with polarization time. Proliferation rate of human gingival fibroblasts (HGFs) was positively correlated with surface hydride content, suggesting the positive effect of surface hydride on connective tissue growth around dental abutment. Changes in surface topography and hydrophilicity did not significantly influence HGF growth.

Osseointegration of dental implants in extraction sockets preserved with porous titanium granules - an experimental study.

OBJECTIVES: This study investigated osseointegration of dental implants inserted in healed extraction sockets preserved with porous titanium granules (PTG). MATERIAL AND METHODS: Three adult female minipigs (Gøttingen minipig; Ellegaard A/S, Dalmose, Denmark) had the mandibular teeth P2, P3 and P4 extracted. The extraction sockets were preserved with metallic PTG (Tigran PTG; Tigran Technologies AB, Malmö, Sweden) n = 12, heat oxidized white porous titanium granules (WPTG) (Tigran PTG White) n = 12 or left empty (sham) n = 6. All sites were covered with collagen membranes (Bio-Gide; Geistlich Pharma, Wolhausen, Switzerland) and allowed 11 weeks of healing before implants (Straumann Bone Level; Straumann, Basel, Switzerland) were inserted. The temperature was measured during preparation of the osteotomies. Resonance frequency analysis (RFA, Osstell; Osstell AB, Gothenburg, Sweden) was performed at implant insertion and at termination. After 6 weeks of submerged implant healing, the pigs were euthanized and jaw segments were excised for microCT and histological analyses. RESULTS: In the temperature and RFA analyses no significant differences were recorded between the test groups. The microCT analysis demonstrated an average bone volume of 61.7% for the PTG group compared to 50.3% for the WPTG group (P = 0.03) and 57.1% for the sham group. Histomorphometry demonstrated an average bone-to-implant contact of 68.2% for the PTG group compared to 36.6% for the WPTG group and 60.9% for the sham group (n.s). Eight out of ten implants demonstrated apical osseous defects in the WPTG group, but similar defects were observed in all groups. CONCLUSIONS: PTG preserved extraction sockets demonstrate a similar outcome as the sham control group for all analyses suggesting that this material potentially can be used for extraction socket preservation prior to implant installment. Apical osseous defects were however observed in all groups including the sham group, and a single cause could not be determined.

Enhanced in vitro osteoblast differentiation on TiO2 scaffold coated with alginate hydrogel containing simvastatin.

The aim of this study was to develop a three-dimensional porous bone graft material as vehicle for simvastatin delivery and to investigate its effect on primary human osteoblasts from three donors. Highly porous titanium dioxide (TiO2) scaffolds were submerged into simvastatin containing alginate solution. Microstructure of scaffolds, visualized by scanning electron microscopy and micro-computed tomography, revealed an evenly distributed alginate layer covering the surface of TiO2 scaffold struts. Progressive and sustained simvastatin release was observed for up to 19 days. No cytotoxic effects on osteoblasts were observed by scaffolds with simvastatin when compared to scaffolds without simvastatin. Expression of osteoblast markers (collagen type I alpha 1, alkaline phosphatase, bone morphogenetic protein 2, osteoprotegerin, vascular endothelial growth factor A and osteocalcin) was quantified using real-time reverse transcriptase-polymerase chain reaction. Secretion of osteoprotegerin, vascular endothelial growth factor A and osteocalcin was analysed by multiplex immunoassay (Luminex). The relative expression and secretion of osteocalcin was significantly increased by cells cultured on scaffolds with 10 µM simvastatin when compared to scaffolds without simvastatin after 21 days. In addition, secretion of vascular endothelial growth factor A was significantly enhanced from cells cultured on scaffolds with both 10 nM and 10 µM simvastatin when compared to scaffolds without simvastatin at day 21. In conclusion, the results indicate that simvastatin-coated TiO2 scaffolds can support a sustained release of simvastatin and induce osteoblast differentiation. The combination of the physical properties of TiO2 scaffolds with the osteogenic effect of simvastatin may represent a new strategy for bone regeneration in defects where immediate load is wanted or unavailable.

Microcomputed tomographic and histologic analysis of animal experimental degree II furcation defects treated with porous titanium granules or deproteinized bovine bone.

BACKGROUND: Titanium is an interesting material for osseous reconstruction given its thrombogenic properties. The aim of this study is to compare the potential of porous titanium granules (PTGs) with sham and deproteinized bovine bone mineral (DBBM) in the reconstructive treatment of surgically created buccal, degree II furcation defects in mini-pigs. METHODS: Buccal degree II furcation defects were surgically created in maxillary premolar teeth in adult, female, mini-pigs and filled with PTG or DBBM or were left empty (sham). After 6 weeks of healing, pigs were euthanized. Teeth with defects were excised en bloc and analyzed by microcomputed tomography (microCT) and histology. RESULTS: The histologic analysis showed significantly more vertical bone formation in both PTG and sham groups compared to DBBM-treated defects (P <0.01). The microCT analysis showed significantly more bucco-palatal bone formation in furcations treated with PTG compared to the DBBM and sham (P <0.05). Bucco-palatal cylindrical microCT cores demonstrated a median defect fill of 96.8% for PTG-implanted defects, which was significantly greater than sham (72.2%) and DBBM (62.0%) (P <0.001) treatments. Significantly more regenerated periodontal ligament was seen for sham than DBBM-treated defects (P <0.05). Root resorption lacunae were small and infrequent and did not differ among groups. CONCLUSIONS: The results of this study in mini-pigs suggest that PTG may integrate well in alveolar bone and supports osseous regrowth in degree II furcation defects. Moreover, PTG seems safe to use in close proximity to root surfaces. Clinical studies will be necessary to further explore these experimental animal findings.

LBP and sCD14 patterns in total hip replacement surgery performed during combined spinal/epidural anaesthesia.

BACKGROUND: Danger patterns and pattern recognition receptors have been targets in the investigation and treatment of systemic inflammatory response syndrome and sepsis. Lipopolysaccharide (LPS)-binding protein (LBP) presents LPS and gram-positive bacterial cell wall products to the receptors TLR4/MD-2 and TLR2, respectively. Low concentrations of LBP stimulate responses to LPS and peptidoglycan, whereas higher concentrations inhibit these responses. Soluble CD14 (sCD14) presents the LBP-LPS complex to CD14-negative cells, and it modulates the biological activity of circulating LPS. In this study, we aimed to elucidate the physiological reactions to LBP and sCD14 after total hip replacement surgery during spinal/epidural anaesthesia. METHODS: Seven patients with coxarthrosis were operated upon with a total hip replacement, which is a defined trauma to bone and muscles in conjunction with a certain amount of blood loss. Venous blood samples were taken before the operation and at 1 h, 3 days and 6 days after surgery. LBP and sCD14 were measured by conventional ELISA. To correct for hemodilution, each parameter was adjusted for hematocrit. A panel of cytokines was measured using Luminex technology to evaluate the trauma reaction. RESULTS: IL-6 levels peaked 24 h after the operation, whereas IL-1ß and IL-10 levels remained unchanged. Systemic levels of LBP were increased 24 h after surgery, whereas sCD14 remained steady. However, the dilution-corrected sCD14 values increased significantly, and the levels of both LBP and sCD14 peaked at day 3 after surgery. CONCLUSION: Aseptic trauma primes the innate immune system for the posttraumatic release of LBP and sCD14.

Human osteoblastic cells discriminate between 20-kDa amelogenin isoforms.

Enamel matrix derivative (EMD) is used to stimulate healing of alveolar bone after destructive marginal periodontitis; however, the roles of the different EMD constituents are unclear. The aim here was to compare the effect of two EMD fractions (A1 and A2) on primary human osteoblasts cultured in the presence of 50 µg ml(-1) of A1, A2, or EMD. SDS-PAGE showed that A1 and A2 were comprised of amelogenins migrating at around 20 kDa. Fourier transform infrared (FTIR) analysis revealed that A1 and A2 had different secondary structures, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) identified different peptide mass values. Osteoblasts responded differently to A1 and A2. Whereas A1 enhanced the proliferation [measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU)] of osteoblasts, the expression of runt-related transcription factor-2 (RUNX2) mRNA, and the secretion of interleukin 6 (IL-6) into the cell culture medium, exposure to A2 resulted in increased alkaline phosphatase (ALP) activity, increased expression of CD44 mRNA, and increased secretion of osteoprotegrin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL). The level of osteocalcin in the cell culture medium was increased after all treatments, while A2 stimulated the expression of dentin matrix protein 1 (DMP1) mRNA. The results suggest that both A1 and A2 participate in the observed effect of EMD, but have different effects on the expression of osteoblast mRNA and the secretion of osteoblast protein, and thus might facilitate the differentiation of a different phenotype.

Serotonin and fluoxetine receptors are expressed in enamel organs and LS8 cells and modulate gene expression in LS8 cells.

The selective serotonin re-uptake inhibitor (SSRI) fluoxetine is widely used in the treatment of depression in children and fertile women, but its effect on developing tissues has been sparsely investigated. The aim of this study was to investigate if enamel organs and ameloblast-derived cells express serotonin receptors that are affected by peripherally circulating serotonin or fluoxetine. Using RT-PCR and western blot analysis we found that enamel organs from 3-d-old mice and ameloblast-like cells (LS8 cells) express functional serotonin receptors, the rate-limiting enzyme in serotonin synthesis (Thp1), as well as the serotonin transporter (5HTT), indicating that enamel organs and ameloblasts are able to respond to serotonin and regulate serotonin availability. Fluoxetine and serotonin enhanced the alkaline phosphatase activity in the cell culture medium from cultured LS8 cells, whereas the expression of enamelin (Enam), amelogenin (Amel), and matrix metalloproteinase-20 (MMP-20) were all significantly down-regulated. The secretion of vascular endothelial growth factor (VEGF), monocyte chemotactic protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10) was also reduced compared with controls. In conclusion, enamel organs and ameloblast-like cells express functional serotonin receptors. Reduced transcription of enamel proteins and secretion of vascular factors may indicate possible adverse effects of fluoxetine on amelogenesis.

The effect of hydrofluoric acid treatment of titanium surface on nanostructural and chemical changes and the growth of MC3T3-E1 cells.

Fluoride-modification of dental titanium (Ti) implants is used to improve peri-implant bone growth and bone-to-implant contact and adhesion strength. In this study, the surface topography, chemistry and biocompatibility of polished Ti surfaces treated with hydrofluoric acid solution (HF) were studied. Murine osteoblasts (MC3T3-E1) were cultured on the different groups of Ti surfaces. Surfaces treated with HF had higher roughness, lower cytotoxicity level and better biocompatibility than controls. For short treatment times (40 and 90 s), fluorine was detected only within the first 5 nm of the surface layer (X-ray Photoemission Spectroscopy, XPS), whereas longer treatment time (120 and 150 s) caused fluoride ions to penetrate deeper (Secondary Ion Mass Spectrometry, SIMS). These results suggest that submerging Ti implants in a weak HF solution instigate time-dependant specific surface changes that are linked to the improved biocompatibility of these surfaces.

Titanium implant surface modification by cathodic reduction in hydrofluoric acid: surface characterization and in vivo performance.

Etching is used for the surface modification of titanium to improve the implant performance in bone. In this study, pure titanium implants were surface modified by a cathodic reduction process by using hydrofluoric acid (HF) at various concentrations (0.001, 0.01, and 0.1 vol %) and a constant current of 1 mA/cm(2). The resulting surface microtopographies were analyzed by atomic force microscopy, scanning electron microscopy, and profilometry, while the surface chemical contents were evaluated by time of flight secondary ion mass spectrometry. The competitive forces between ionic surface implementation induced by the current direction and the HF etching effect on titanium were highlighted. The implant performance was evaluated in an in vivo rabbit model by using a pull-out test method. The group of implants modified with 0.01% HF showed the highest retention in bone. Fluoride and hydride amounts measured in the surfaces, as well as surface skewness (S(sk)), kurtosis (S(ku)), and core fluid retention (S(ci)) were positively correlated to the implant's retention in bone in vivo. Frequently used parameters for characterizing the implant, such as oxide content and the average height deviation from the mean plane (S(a)), were not correlated to implant performance, suggesting that these parameters are not the most important in predicting the implant performance.

Systemic administration of enamel matrix derivative to lipopolysaccharide-challenged pigs: effects on the inflammatory response.

BACKGROUND: Periodontitis is the primary clinical indication for enamel matrix derivative (EMD). Recent investigations, showing that EMD inhibits the production of tumor necrosis factor-alpha (TNF-alpha) when added to human whole blood, indicate a novel role for EMD as a modulator of systemic inflammation. In the present study, we investigated the systemic effects of EMD in lipopolysaccharide (LPS)-challenged pigs. METHODS: In a preparatory study, seven pigs received a prophylactic EMD bolus injection (5 mg/kg), followed by a continuous infusion (50 mg/kg/min). Thirty minutes later, a continuous infusion of LPS (1.7 mcg/kg/h) was started. An additional 12 pigs were randomized into two groups. Six of these animals were given the same treatment, except that EMD was administered 30 min after LPS. The remainder served as controls. The groups were compared according to organ injury and function, hemodynamics, and systemic markers of inflammation. RESULTS: Prophylactic administration of EMD triggered transient hemodynamic instability in two of seven pigs. In the randomized pigs, no or only nonspecific changes were observed in biopsies from vital organs, independent of treatment. Enamel matrix derivative did not modify systemic TNF-alpha, interleukin (IL)-1 beta, or IL-6 concentrations. CONCLUSIONS: In the formulation and dosages used, EMD did not modulate the inflammatory response. No true allergic or immunotoxic reactions were seen. To be usable for systemic application, a new formulation should be developed, or the active part of the protein(s) should be identified and produced in a soluble form designed for infusion. The potential of EMD as a systemic immune modulator is still unsettled.

Liver X receptor is a key regulator of cytokine release in human monocytes.

Aberrant regulation of innate immune responses and uncontrolled cytokine bursts are hallmarks of sepsis and endotoxemia. Activation of the nuclear liver X receptor (LXR) was recently demonstrated to suppress inflammatory genes. Our aim was to investigate the expression of LXR in human monocytes under normal and endotoxemic conditions and to study the influence of LXR activation on endotoxin-induced cytokine synthesis and release. Adherent human monocytes or whole blood were pretreated with a synthetic LXR agonist (3-{3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy}-phenyl)-acetic acid) and subsequently challenged with LPS (from Escherichia coli) or peptidoglycan (from Staphylococcus aureus). Cytokine release was assessed by a Multiplex antibody bead kit, and cytokine mRNA levels were measured by real-time reverse-transcriptase-polymerase chain reaction. We found that LXRalpha mRNA was up-regulated in CD14+ monocytes in LPS-challenged blood, whereas LXRbeta mRNA was not altered. Addition of 3-{3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy}-phenyl)-acetic acid to monocytes suppressed the LPS-induced release of IL-1beta, IL-6, IL-8, IL-10, IL-12p40, TMF-alpha, macrophage inflammatory protein 1alpha, macrophage inflammatory protein 1beta, and monocyte chemoattractant protein 1 in a concentration-dependent manner. Surprisingly, an accompanying decrease in cytokine mRNA accumulation was not observed. The suppressed cytokine release could not be explained by a diminished transport of mRNA out of the nucleus or a decreased secretion of cytokines. We propose that LXR is a key regulator of cytokine release in LPS-challenged human monocytes, possibly by interfering with translational events.

Determining optimal dose of laser therapy for attachment and proliferation of human oral fibroblasts cultured on titanium implant material.

The purpose of this study was to investigate the influence of single or multiple doses of low-level laser therapy (LLLT) on attachment and proliferation of human gingival fibroblasts in a standardized, reproducible in vitro model. Titanium discs were randomly allotted to one of three groups: group I served as a control, group II was exposed to a single laser dose of 3 J/cm2, and the three subgroups in group III were exposed to laser doses of 0.75, 1.5, and 3 J/cm2. To examine the possible thermal effects of laser exposure on the cell culture, the temperature in the Petri dish was measured for every dose used, before and during irradiation. For attachment assays, groups II and III were exposed to laser irradiation and then seeded onto titanium discs. In group III, the exposures were repeated after 3 and 6 h. Cells were cultured for 6 and 24 h and stained with Hoechst and Propidium. Attached cells were counted under a light microscope. To investigate the effect of LLLT on cell proliferation after 48 h, 72 h, and 7 days, cells were cultured on titanium discs for 24 h and then exposed to laser irradiation for 1 day and 3 consecutive days, respectively. Cell proliferation was determined by counting cells under the microscope and by a cell proliferation enzyme-linked immunosorbent assay system. No increase of temperature of the cell cultures occurred before or during laser exposure at any of the doses tested. Both single and multiple doses of LLLT significantly enhanced cellular attachment (p<0.05). The proliferation assays showed higher cell proliferation (p<0.05) in group III at doses of 1.5 and 3 J/cm2 after 72 h and 7 days, with agreement between staining and enzyme-linked immunosorbent assay. It is concluded that, in this cellular model, the attachment and proliferation of human gingival fibroblasts are enhanced by LLLT in a dose-dependent manner.

Effect of laser therapy on attachment, proliferation and differentiation of human osteoblast-like cells cultured on titanium implant material.

The aim of this in vitro study was to investigate the effect of low-level laser therapy (LLLT) on the attachment, proliferation, differentiation and production of transforming growth factor-ss(1) (TGF-beta(1)) by human osteoblast-like cells (HOB). Cells derived from human mandibular bone were exposed to GaAlAs diode laser at dosages of 1.5 or 3 J/cm(2) and then seeded onto titanium discs. Non-irradiated cultures served as controls. After 1, 3 and 24h, cells were stained and the attached cells were counted under a light microscope. In order to investigate the effect of LLLT on cell proliferation after 48, 72 and 96 h, cells were cultured on titanium specimens for 24h and then exposed to laser irradiation for three consecutive days. Specific alkaline phosphatase activity and the ability of the cells to synthesize osteocalcin after 10 days were investigated using p-nitrophenylphosphate as a substrate and the ELSA-OST-NAT immunoradiometric kit, respectively. Cellular production of TGF-beta(1) was measured by an enzyme-linked immunosorbent assay (ELISA), using commercially available kits. LLLT significantly enhanced cellular attachment (P<0.05). Greater cell proliferation in the irradiated groups was observed first after 96 h. Osteocalcin synthesis and TGF-beta(1) production were significantly greater (P<0.05) on the samples exposed to 3 J/cm(2). However, alkaline phosphatase activity did not differ significantly among the three groups. These results showed that in response to LLLT, HOB cultured on titanium implant material had a tendency towards increased cellular attachment, proliferation, differentiation and production of TGF-beta(1), indicating that in vitro LLLT can modulate the activity of cells and tissues surrounding implant material.

Enhancement of bone formation in rat calvarial bone defects using low-level laser therapy.

OBJECTIVE: To evaluate the effect of low-level laser therapy (LLLT), using a GaAlAs diode laser device, on bone healing and growth in rat calvarial bone defects. STUDY DESIGN: An animal trial of 4 weeks' duration was conducted using a randomized blind, placebo-controlled design. Standardized round osseous defects of 2.7 mm diameter were made in each parietal bone of 20 rats (n=40 defects). The animals were randomly divided into an experimental and a control group of 10 animals each. In the experimental group, a GaAlAs diode laser was applied immediately after surgery and then daily for 6 consecutive days. The control group received the same handling and treatment, but with the laser turned off. Five rats from each group were killed on day 14 and the remainder on day 28 postoperatively. From each animal, tissue samples from one defect were prepared for histochemistry and samples from the contralateral defect for histology. Levels of calcium, phosphorus, and protein were determined by using atomic absorption spectrometry, colorimetry, and photometry, respectively. Student t-test and Mann-Whitney were used for statistical analyses. RESULTS: At both time points the tissue samples from the experimental animals contained significantly more calcium, phosphorus, and protein than the controls. Similarly, histological analyses disclosed more pronounced angiogenesis and connective tissue formation, and more advanced bone formation in the experimental group than in the controls. CONCLUSION: LLLT may enhance bone formation in rat calvarial bone defects.