Effect of functionalized titanium particles with dexamethasone-loaded nanospheres on macrophage polarization and activity.

OBJECTIVE: The aim of this study was to determine the effect of titanium micro particles (TiP) previously functionalized with nanoparticles doped with dexamethasone (Dex) and doxycycline (Dox), on macrophage polarization and activity. METHODS: Macrophages RAW264.7 were cultured in the presence TiP loaded with dexamethasone -NPs (Dex)- and doxycycline -NPs (Dox)-, and as control, TiP with or without doped NPs. Cells were tested with and without previous bacterial lipopolysaccharide endotoxin (LPS) stimulation. Their morphology, proliferation, cytotoxicity, phenotypic change, and cytokines release were assessed by LIVE/DEAD, DNA release, metabolic activity, brightfield and scanning electron microscopy. The test Kruskall-Wallis was used for comparisons, while the cytokine expression profiles were examined by hierarchical clustering (p < 0.05). RESULTS: Upon exposure with TiP macrophages were activated and polarized to M1, but without depicting cytotoxic effects. The particles were phagocytised, and vacuolized. When exposed to functionalised TiP with NPs(Dex) and NPs(Dox), the ratio M1/M2 was up to forty times lower compared to TiP alone. When exposed to LPS, TiP reduced cell viability in half. Functionalised TiP with NPs(Dex) inhibited the cytokine release exerted by TiP on macrophages. When macrophages were exposed to functionalised TiPs with NPs(Dex) with and without LPS, the effect of TiP on cytokine secretion was inhibited. SIGNIFICANCE: Functionalised TiPs with NPs(Dex) and NPs(Dox) may potentially have beneficial effects on modulating titanium and LPS-related inflammatory reactions.

Effect of doxycycline doped nanoparticles on osteogenic/cementogenic and anti-inflammatory responses of human cells derived from the periodontal ligament.

OBJECTIVES: This work aimed to evaluate if doxycycline-doped polymeric nanoparticles possessed any anti-inflammatory effect and promote osteogenic/cementogenic differentiation of stem cells from human periodontal ligament (PDLSCs). METHODS: The polymeric nanoparticles (NPs) were produced by a polymerization/precipitation process and doped with doxycycline (Dox-NPs). PDLSCs were cultured in the presence or absence of the NPs under osteogenic medium or IL-1ß treatment. Cells' differentiation was assessed by gene expression analysis of osteogenic/cementogenic markers alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2). An anti-inflammatory effect was also ascertained by analyzing IL-1ß gene expression. Adipogenic and chondrogenic differentiation was used to confirm the multipotency of PDLSCs. RESULTS: Gene expression of ALP and RUNX2 in PDLSCs was significantly upregulated by the osteogenic medium (ALP: p<0.001; RUNX2: p = 0.005) while Dox-NPs further enhanced ALP gene expression of PDLSCs treated with the osteogenic medium. Furthermore, Dox-NPs suppressed the up-regulation of IL-1ß when cells were subjected to an inflammatory challenge. CONCLUSIONS: Dox-NPs enhanced PDLSCs differentiation into osteoblasts/cementoblasts lineages while providing an anti-inflammatory effect. CLINICAL SIGNIFICANCE: Due to their biocompatibility as well as anti-inflammatory and osteogenic/cementogenic effects, Dox-NPs are potential candidates for being used in periodontal regeneration.

Deciphering the Cell-Specific Effect of Osteoblast-Macrophage Crosstalk in Periodontitis.

In periodontitis, the bone remodeling process is disrupted by the prevalent involvement of bacteria-induced proinflammatory macrophage cells and their interaction with osteoblast cells residing within the infected bone tissue. The complex interaction between the cells needs to be deciphered to understand the dominant player in tipping the balance from osteogenesis to osteoclastogenesis. Yet, only a few studies have examined the crosstalk interaction between osteoblasts and macrophages using biomimetic three-dimensional (3D) tissue-like matrices. In this study, we created a cell-laden 3D tissue analog to study indirect crosstalk between these two cell types and their direct synergistic effect when cultured on a 3D scaffold. The cell-specific role of osteoclast differentiation was investigated through osteoblast- and proinflammatory macrophage-specific feedback studies. The results suggested that when macrophages were exposed to osteoblasts-derived conditioned media from the mineralized matrix, the M1 macrophages tended to maintain their proinflammatory phenotype. Further, when osteoblasts were exposed to secretions from proinflammatory macrophages, they demonstrated elevated receptor activator of nuclear factor-κB ligand (RANKL) expression and decreased alkaline phosphate (ALP) activities compared to osteoblasts exposed to only osteogenic media. In addition, the upregulation of tumor necrosis factor-alpha (TNF-α) and c-Fos in proinflammatory macrophages within the 3D matrix indirectly increased the RANKL expression and reduced the ALP activity of osteoblasts, promoting osteoclastogenesis. The contact coculturing with osteoblast and proinflammatory macrophages within the 3D matrix demonstrated that the proinflammatory markers (TNF-α and interleukin-1ß) expressions were upregulated. In contrast, anti-inflammatory markers (c-c motif chemokine ligand 18 [CCL18]) were downregulated, and osteoclastogenic markers (TNF receptor associated factor 6 [TRAF6] and acid phosphatase 5, tartrate resistant [ACP5]) were unchanged. The data suggested that the osteoblasts curbed the osteoclastogenic differentiation of macrophages while macrophages still preserved their proinflammatory lineages. The osteoblast within the 3D coculture demonstrated increased ALP activity and did not express RANKL significantly different than the osteoblast cultured within a 3D collagen matrix without macrophages. Contact coculturing has an anabolic effect on bone tissue in a bacteria-derived inflammatory environment.

Doxycycline-doped collagen membranes accelerate in vitro osteoblast proliferation and differentiation.

OBJECTIVE: The aim of the study was to evaluate the effect of doxycycline- and dexamethasone-doped collagen membranes on the proliferation and differentiation of osteoblasts. BACKGROUND: Collagen barrier membranes are frequently used to promote bone regeneration and to boost this biological activity their functionalization with antibacterial and immunomodulatory substances has been suggested. METHODS: The design included commercially available collagen membranes doped with doxycycline (Dox-Col-M) or dexamethasone (Dex-Col-M), as well as undoped membranes (Col-M) as controls, which were placed in contact with cultured MG63 osteoblast-like cells (ATCC). Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and differentiation by measuring the alkaline phosphatase (ALP) activity using spectrophotometry. Real-time quantitative polymerase chain reaction was used to study the expression of the genes: Runx-2, OSX, ALP, OSC, OPG, RANKL, Col-I, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3. Scanning electron microscopy was used to study osteoblast morphology. Data were assessed using one-way analysis of variance or Kruskal-Wallis tests, once their distribution normality was assessed by Kolmogorov-Smirnov tests (p > .05). Bonferroni for multiple comparisons were carried out (p < .05). RESULTS: Osteoblast proliferation was significantly enhanced in the functionalized membranes as follows: (Col-M < Dex-Col-M < Dox-Col-M). ALP activity was significantly higher on cultured osteoblasts on Dox-Col-M. Runx-2, OSX, ALP, OSC, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3 were overexpressed, and RANKL was down-regulated in osteoblasts cultured on Dox-Col-M. The osteoblasts cultured in contact with the functionalized membranes demonstrated an elongated spindle-shaped morphology. CONCLUSION: The functionalization of collagen membranes with Dox promoted an increase in the proliferation and differentiation of osteoblasts.

Biomimetic Collagen Membranes as Drug Carriers of Geranylgeraniol to Counteract the Effect of Zoledronate.

To counteract the effect of zoledronate and decrease the risk of osteonecrosis of the jaw (BRONJ) development in patients undergoing guided bone regeneration surgery, the use of geranylgeraniol (GGOH) has been proposed. Collagen membranes may act as biomimetical drug carriers. The objective of this study was to determine the capacity of collagen-based membranes doped with GGOH to revert the negative impact of zoledronate on the growth and differentiation of human osteoblasts. MG-63 cells were cultured on collagen membranes. Two groups were established: (1) undoped membranes and (2) membranes doped with geranylgeraniol. Osteoblasts were cultured with or without zoledronate (50 µM). Cell proliferation was evaluated at 48 h using the MTT colorimetric method. Differentiation was tested by staining mineralization nodules with alizarin red and by gene expression analysis of bone morphogenetic proteins 2 and 7, alkaline phosphatase (ALP), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), type I collagen (Col-I), osterix (OSX), osteocalcin (OSC), osteoprotegerin (OPG), receptor for RANK (RANKL), runt-related transcription factor 2 (Runx-2), TGF-ß1 and TGF-ß receptors (TGF-ßR1, TGF-ßR2, and TGF-ßR3), and vascular endothelial growth factor (VEGF) with real-time PCR. One-way ANOVA or Kruskal-Wallis and post hoc Bonferroni tests were applied (p < 0.05). Scanning electron microscopy (SEM) observations were also performed. Treatment of osteoblasts with 50 µM zoledronate produced a significant decrease in cell proliferation, mineralization capacity, and gene expression of several differentiation markers if compared to the control (p < 0.001). When osteoblasts were treated with zoledronate and cultured on GGOH-doped membranes, these variables were, in general, similar to the control group (p > 0.05). GGOH applied on collagen membranes is able to reverse the negative impact of zoledronate on the proliferation, differentiation, and gene expression of different osteoblasts' markers.

Dexamethasone and Doxycycline Doped Nanoparticles Increase the Differentiation Potential of Human Bone Marrow Stem Cells.

Non-resorbable polymeric nanoparticles (NPs) are proposed as an adjunctive treatment for bone regenerative strategies. The present in vitro investigation aimed to evaluate the effect of the different prototypes of bioactive NPs loaded with zinc (Zn-NPs), doxycycline (Dox-NPs) or dexamethasone (Dex-NPs) on the viability, morphology, migration, adhesion, osteoblastic differentiation, and mineralization potential of human bone marrow stem cells (hBMMSCs). Cell viability, proliferation, and differentiation were assessed using a resaruzin-based assay, cell cycle analysis, cell migration evaluation, cell cytoskeleton staining analysis, Alizarin Red S staining, and expression of the osteogenic-related genes by a real-time quantitative polymerase chain reaction (RT-qPCR). One-Way ANOVA and Tukey's test were employed. The resazurin assay showed adequate cell viability considering all concentrations and types of NPs at 24, 48, and 72 h of culture. The cell cycle analysis revealed a regular cell cycle profile at 0.1, 1, and 10 µg/mL, whereas 100 µg/mL produced an arrest of cells in the S phase. Cells cultured with 0.1 and 1 µg/mL NP concentrations showed a similar migration capacity to the untreated group. After 21 days, mineralization was increased by all the NPs prototypes. Dox-NPs and Dex-NPs produced a generalized up-regulation of the osteogenic-related genes. Dex-NPs and Dox-NPs exhibited excellent osteogenic potential and promoted hBMMSC differentiation. Future investigations, both in vitro and in vivo, are required to confirm the suitability of these NPs for their clinical application.

Antibiotic-Loaded Polymeric Barrier Membranes for Guided Bone/Tissue Regeneration: A Mini-Review.

Polymeric membranes are frequently used for bone regeneration in oral and periodontal surgery. Polymers provide adequate mechanical properties (i.e., Young's modulus) to support oral function and also pose some porosity with interconnectivity to permit for cell proliferation and migration. Bacterial contamination of the membrane is an event that may lead to infection at the bone site, hindering the clinical outcomes of the regeneration procedure. Therefore, polymeric membranes have been proposed as carriers for local antibiotic therapy. A literature search was performed for papers, including peer-reviewed publications. Among the different membranes, collagen is the most employed biomaterial. Collagen membranes and expanded polytetrafluoroethylene loaded with tetracyclines, and polycaprolactone with metronidazole are the combinations that have been assayed the most. Antibiotic liberation is produced in two phases. A first burst release is sometimes followed by a sustained liberation lasting from 7 to 28 days. All tested combinations of membranes and antibiotics provoke an antibacterial effect, but most of the time, they were measured against single bacteria cultures and usually non-specific pathogenic bacteria were employed, limiting the clinical relevance of the attained results. The majority of the studies on animal models state a beneficial effect of these antibiotic functionalized membranes, but human clinical assays are scarce and controversial.

Ex vivo investigations on bioinspired electrospun membranes as potential biomaterials for bone regeneration.

OBJECTIVES: To assess the surface characteristics and composition that may enhance osteoblasts viability on novel electrospun composite membranes (organic polymer/silicon dioxide nanoparticles). METHODS: Membranes are composed by a novel polymer blend, the mixture of two hydrophilic copolymers 2-hydroxyethylmethacrylate-co-methylmethacrylate and 2-hydroxyethylacrylate-co-methylacrylate, and they are doped with silicon dioxide nanoparticles. Then the membranes were functionalized with zinc or doxycycline. The membranes were morphologically characterized by atomic force and scanning electron microscopy (FESEM), and mechanically probed using a nanoindenter. Biomimetic calcium phosphate precipitation on polymeric tissues was assessed. Cell viability tests were performed using human osteosarcoma cells. Cells morphology was also studied by FESEM. Data were analyzed by ANOVA, Student-Newman-Keuls and Student t tests (p < 0.05). RESULTS: Silica doping of membranes enhanced bioactivity and increased mechanical properties. Membranes morphology and mechanical properties were similar to those of trabecular bone. Zinc and doxycycline doping did not exert changes but it increased novel membranes bioactivity. Membranes were found to permit osteoblasts proliferation. Silica-doping favored cells proliferation and spreading. As soon as 24 h after the seeding, cells in silica-doped membranes were firmly attached to experimental tissues trough filopodia, connected to each other. The cells produced collagen and minerals onto the surfaces. CONCLUSIONS: Silica nanoparticles enhanced surface properties and osteoblasts viability on electrospun membranes. CLINICAL SIGNIFICANCE: The ability of silica-doped matrices to promote precipitation of calcium phosphate, together with their mechanical properties, observed non-toxicity, stimulating effect on osteoblasts and its surface chemistry allowing covalent binding of proteins, offer a potential strategy for bone regeneration applications.

Novel non-resorbable polymeric-nanostructured scaffolds for guided bone regeneration.

OBJECTIVE: The aim of this study was to evaluate the bone-regeneration efficiency of novel polymeric nanostructured membranes and the effect of zinc, calcium, titanium, and bone morpho-protein loading on membranes, through an in vivo rabbit model. MATERIAL AND METHODS: Nanostructured membranes of methylmethacrylate were loaded with zinc, calcium, TiO2 nanoparticles, and bone-morphogenetic protein (BMP). These membranes covered the bone defects prepared on the skulls of six rabbits. Animals were sacrificed 6 weeks after surgery. Micro computed tomography was used to evaluate bone architecture through BoneJ pluging and ImageJ script. Three histological processing of samples, including von Kossa silver nitrate, toluidine blue, and fluorescence by the deposition of calcein were utilized. RESULTS: Zn-membranes (Zn-Ms) promoted the highest amount of new bone and higher bone perimeter than both unloaded and Ti-membranes (Ti-Ms). Ca-membranes (Ca-Ms) attained higher osteoid perimeter and bone perimeter than Zn-Ms. The skeleton analysis showed that Zn-Ms produced more branches and junctions at the trabecular bone than BMP-loaded membranes (BMP-Ms). Samples treated with Ti-Ms showed less bone formation and bony bridging processes. Both Zn-Ms and Ca-Ms achieved higher number of osteoblasts than the control group. BMP-Ms and Ca-Ms originated higher number of blood vessels than Ti-Ms and control group. CONCLUSIONS: Zn incorporation in novel nanostructured membranes provided the highest regenerative efficiency for bone healing at the rabbit calvarial defects. CLINICAL RELEVANCE: Zn-Ms promoted osteogenesis and enhanced biological activity, as mineralized and osteoid new bone with multiple interconnected ossified trabeculae appeared in close contact with the membrane.

Silver improves collagen structure and stability at demineralized dentin: A dynamic-mechanical and Raman analysis.

OBJECTIVE: This study aimed to evaluate the effect of silver loaded nanoparticles (NPs) application on dentin remineralization. METHODS: Polymethylmetacrylate-based NPs and silver loaded NPs (Ag-NPs) were applied on demineralized dentin surfaces. Dentin was characterized morphologically by scanning electron microscopy, mechanically probed by a nanoindenter to test nanohardness and Young modulus, and chemically analyzed by Raman spectroscopy after 24 h and 7 d of storage. Untreated surfaces were used as control. Data were submitted to ANOVA and Student-Newman-Keuls multiple comparisons tests (P < 0.05). RESULTS: After Raman analysis, dentin treated with Ag-NPs obtained the lowest mineralization and intensity of stoichiometric hydroxyapatite when compared with specimens treated with undoped-NPs. The lowest relative mineral concentration, expressed as the ratio phosphate or carbonate/phenyl group, and crystallinity was attained by dentin treated with Ag-NPs, after 7 d. Ag-NPs application generated the highest values of collagen crosslinking (intensity at 1032 cm-1 band). The molecular conformation of the collagen's polypeptide chains, amide-I and CH2 also attained the highest peaks in dentin treated with Ag-NPs. Staggered and demineralized collagen fibrils were observed covering the dentin surfaces treated with Ag-NPs, at both 24 h and 7 d. Samples treated with Ag-NPs attained the lowest values of nanohardness and Young's modulus at 7 d of storage. CONCLUSIONS: Peritubular and intertubular dentin were remineralized when using undoped-NPs. After 7 d, collagen treated with NPs was remineralized but dentin treated with Ag-NPs attained an improved collagen matrix structure and stability but the lowest mineralization and crystallinity. CLINICAL SIGNIFICANCE: Preservation of the demineralized organic matrix is fundamental in operative dentistry. Silver contributed to improve crosslinking, nature and secondary structure of demineralized dentin collagen, for further long-term intrafibrillar mineralization.

Influence of phosphoproteins' biomimetic analogs on remineralization of mineral-depleted resin-dentin interfaces created with ion-releasing resin-based systems.

OBJECTIVE: The study aimed at evaluating the remineralization of acid-etched dentin pre-treated with primers containing biomimetic analogs and bonded using an ion-releasing light-curable resin-based material. METHODS: An experimental etch-and-rinse adhesive system filled with Ca(2+), PO4(3-)-releasing Ca-Silicate micro-fillers was created along with two experimental primers containing biomimetic analogs such as sodium trimetaphosphate (TMP) and/or polyaspartic acid (PLA). Dentin specimens etched with 37% H3PO4 were pre-treated with two different aqueous primers containing the polyanionic biomimetic analogs or deionized water and subsequently bonded using the experimental resin-based materials. The specimens were sectioned and analyzed by AFM/nanoindentation to evaluate changes in the modulus of elasticity (Ei) across the resin-dentin interface at different AS storage periods (up to 90 days). Raman cluster analysis was also performed to evaluate the chemical changes along the interface. The phosphate uptake by the acid-etched dentin was evaluated using the ATR-FTIR. Additional resin-dentin specimens were tested for microtensile bond strength. SEM examination was performed after de-bonding, while confocal laser microscopy was used to evaluate the interfaces ultramorphology and micropermeability. RESULTS: Both biomimetic primers induced phosphate uptake by acid-etched dentin. Specimens created with the ion-releasing resin in combination with the pre-treatment primers containing either PLA and TMA showed the greatest recovery of the Ei of the hybrid layer, with no decrease in µTBS (p>0.05) after 3-month AS storage. The ion-releasing resin applied after use of the biomimetic primers showed the greatest reduction in micropermeability due to mineral precipitation; these results were confirmed using SEM. SIGNIFICANCE: The use of the ion-releasing resin-based system applied to acid-etched dentin pre-treated with biomimetic primers containing analogs of phosphoproteins such as poly-l-aspartic acid and/or sodium trimetaphosphate provides a suitable bonding approach for biomimetic remineralization of resin-dentin interfaces.

Zinc incorporation improves biological activity of beta-tricalcium silicate resin-based cement.

INTRODUCTION: Matrix metalloproteinase (MMP) inhibition may improve endodontic treatment prognosis. The purpose of this study was to determine if zinc incorporation into experimental resin cements containing bioactive fillers may modulate MMP-mediated collagen degradation of dentin. METHODS: Human dentin samples untreated and demineralized using 10% phosphoric acid or 0.5 mol/L EDTA were infiltrated with the following experimental resins: (1) unfilled resin, (2) resin with Bioglass 45S5 particles (OSspray, London, UK), (3) resin with beta-tricalcium silicate particles (ßTCS), (4) resin with zinc-doped Bioglass 45S5, and (5) resin with zinc-doped ßTCS particles. The specimens were stored in artificial saliva (for 24 hours, 1 week, and 4 weeks) and submitted to radioimmunoassay to quantify C-terminal telopeptide. Scanning electron microscopy analysis was also undertaken on dentin samples after 4 weeks of storage. RESULTS: Collagen degradation was prominent both in phosphoric acid and EDTA-treated dentin. Resin infiltration strongly reduced MMP activity in demineralized dentin. Resin containing Bioglass 45S5 particles exerted higher and stable protection of collagen. The presence of zinc in ßTCS particles increases MMP inhibition. Different mineral precipitation was attained in dentin infiltrated with the resin cements containing bioactive fillers. CONCLUSIONS: MMP degradation of dentin collagen is strongly reduced after resin infiltration of dentin. Zinc incorporation in ßTCS particles exerted an additional protection against MMP-mediated collagen degradation. However, it did not occur in resin containing Bioglass 45S5 particles, probably because of the formation of phosphate-zinc compounds.

Digital image analysis method to assess the performance of conventional and self-limiting concepts in dentine caries removal.

OBJECTIVE: To assess dentine caries removal effectiveness (CRE) and minimal invasiveness potential (MIP) of carbide and polymer burs. METHODS: Sectioned carious molars were photographed. Digital images were taken, before and after caries removal, using a Digital Single Lens Reflex camera. The following regions of interest were measured using visual criteria: Residual Infected Dentine (RI), Residual Affected Dentine (RA), Removal Sound Dentine (RA), Prepared Cavity (PC) and Removed Sound Dentine (RS). CRE was determined on basis of: relative residual infected dentine (RI/II), relative residual carious-affected dentine (RA/IA) and total relative residual dentine (RI+RA/II-IA). MIP was determined on basis of: infected dentine cavity size (PC/II), total relative cavity size (PC/II+IA), and corrected relative cavity size (PC-RS/II+IA). RESULTS: The polymer bur showed the highest preservation of carious-affected dentine after excavation, when the RA/IA ratio was studied. Both kind of burs showed similar values after assessing the RI/II and RI+RA/II-IA ratios. The infected dentine relative cavity size (PC/II) was higher when the carbide bur was used. Both burs attained similar PC/II+IA and PC-RS/II+IA ratios. CONCLUSIONS: Polymer burs accomplished the concept of minimal-invasive dentistry, showing its self-limiting ability. The minimal-invasiveness potential showed that carbide burs resulted in the worst compromise between effective and selective infected-caries removal.

MMPs activity and bond strength in deciduous dentine-resin bonded interfaces.

OBJECTIVES: To determine effect of ageing on deciduous dentine-resin interfaces bond strength and the metalloproteinases (MMPs) activity at the hybrid layer compared to permanent dentine. METHODS: Microtensile bond strength (MTBS) tests were performed in human deciduous and permanent dentine after 24h, 3 and 6 months using an etch and rinse adhesive. C-terminal telopeptide concentrations (ICTP) were calculated, in order to determine MMPs mediated collagen degradation at the hybrid layer. RESULTS: The highest MMPs-mediated collagen degradation values occurred in phosphoric acid demineralized dentine, ICTP values were similar for deciduous and permanent dentine after 1 week. Resin infiltration decreased collagen degradation in both dentins and ICTP values were similar to those attained by for untreated dentine. In resin infiltrated and untreated dentine specimens collagen degradation was always higher for deciduous dentine. At 24h, MTBS was higher in permanent dentine. After ageing MTBS decreased and performed similarly in both dentins. CONCLUSIONS: Higher collagenollytic activity is found in deciduous than in permanent dentine. At 24h, collagen cleavage by MMPs at the hybrid layer is higher in deciduous dentine leading to a lower MTBS. CLINICAL SIGNIFICANCE: The presence of resin monomers reduced collagen degradation when applied on demineralized dentine, but exerted protection was lower in deciduous dentine.

Experimental resin cements containing bioactive fillers reduce matrix metalloproteinase-mediated dentin collagen degradation.

INTRODUCTION: Collagen dentin matrix may represent a suitable scaffold to be remineralized in the presence of bioactive materials. The purpose of this study was to determine if experimental resin cements containing bioactive fillers may modulate matrix metalloproteinase-mediated collagen degradation of etched dentin. METHODS: Human dentin beams demineralized using 10% phosphoric acid or 0.5 mol/L EDTA were infiltrated with the following experimental resins: (1) unfilled resin, (2) resin with Bioglass 45S5 particles (Sylc; OSspray Ltd, London, UK), and (3) resin with ß-tricalcium phosphate-modified calcium silicate cement (HCAT-ß) particles. The filler/resin ratio was 40/60 wt%. The specimens were stored in artificial saliva, and the determination of C-terminal telopeptide (ICTP) was performed by radioimmunoassay after 24 hours, 1 week, and 4 weeks. Scanning electron microscopic analysis of dentin surfaces after 4 weeks of storage was also executed. RESULTS: Collagen degradation was prominent both in phosphoric acid and EDTA-treated dentin. Resin infiltration strongly reduced the MMP activity in demineralized dentin. Resin-containing Bioglass 45S5 particles exerted higher and more stable protection of collagen at all tested dentin states and time points. HCAT-ß induced collagen protection from MMPs only in EDTA-treated specimens. Dentin remineralization was achieved when dentin was infiltrated with the resin cements containing bioactive fillers. CONCLUSIONS: MMP degradation of dentin collagen is strongly reduced in resin-infiltrated dentin. The inclusion of Bioglass 45S5 particles exerted an additional protection of collagen during dentin remineralization.

A ZnO-doped adhesive reduced collagen degradation favouring dentine remineralization.

OBJECTIVES: The objective of the study was to determine the efficacy of a ZnO-doped etch and rinse adhesive in decreasing MMPs-mediated collagen degradation at the resin-dentine hybrid layer, and increasing bonding stability. METHODS: C-terminal telopeptide concentrations (ICTP) were determined after 24h, 1wk and 4wk in human dentine beams. Dentine was treated: (1) 37% phosphoric acid for 15s (PA), (2) PA-etched dentine infiltrated with Single Bond (SB), (3) PA-etched dentine infiltrated with ZnO doped SB (ZnO particles--10wt%--were added to the bonding resin) (ZnO-SB), and (4) Clearfil SE Bond primed-dentine was infiltrated with Clearfil SE bonding resin (CSE). Microtensile bond strength (MTBS) was assessed for the different groups at 24h and after 3months. Debonded dentine surfaces were studied by scanning electron microscopy. RESULTS: MMPs-mediated collagen degradation occurred in demineralized dentine (PA). Resin infiltration decreased collagen degradation. The lowest collagen degradation was found for Zn-doped SB, followed by CSE. When these adhesives were applied, ICTP values did not change throughout the study period. At 24h, similar MTBS was attained for all adhesives. Only SB decreased MTBS after three months. CONCLUSIONS: Addition of ZnO particles to SB produced a reduction in dentine collagen degradation and increased resin-dentine bonds durability. In Zn-doped adhesive interfaces, a calcium phosphate layer and tubular occlusion was encountered at the debonded interface. CLINICAL SIGNIFICANCE: ZnO particles addition into the bonding resin of SB makes a breakthrough to prevent the hybrid layer degradation and to preserve its bonding efficacy overtime.

Bleaching agents increase metalloproteinases-mediated collagen degradation in dentin.

INTRODUCTION: Tooth bleaching is based on hydrogen peroxide application. The Objective of this study was to determine whether dental bleaching agents affect metalloproteinases-mediated dentin collagen degradation. METHODS: Human dentin specimens were subjected to different treatments: (1) untreated dentin; (2) demineralization by 37% phosphoric acid (PA); (3) demineralization by 37% PA, followed by application of Single Bond (SB); (4) 2 immersions of 7 days each in a nonvital bleaching agent, followed by PA; (5) 2 immersions of 7 days each in nonvital bleaching, followed by PA and SB application; (6) 3 immersions by using in-office bleaching gel for 20 minutes; (7) 3 immersions by using in-office bleaching gel for 20 minutes plus activation with a light source; and (8) immersion in home bleaching gel for 8 hours per day during 3 weeks. Specimens were stored in artificial saliva. C-terminal telopeptide determinations (radioimmunoassay) were performed after 24 hours, 1 week, and 4 weeks. RESULTS: Bleaching agents increased collagen degradation, but C-terminal telopeptide of type I collagen (ICTP) values were higher when dentin was PA-demineralized. Nonvital bleaching plus PA promoted the highest collagenolytic activity, which was reduced after SB infiltration. Halogen light application did not influence ICTP values. At 24 hours, home bleaching exhibited high collagenolytic activity, which decreased up to 4 weeks. After 4 weeks of storage, all bleaching procedures showed similar values of collagen degradation, which were not different from those of PA-demineralized and resin-infiltrated dentin. CONCLUSIONS: All tested bleaching agents increase matrix metalloproteinases-mediated collagen degradation in dentin. This effect was not completely reverted after 4 weeks. Home bleaching induced the highest collagen degradation.

Zinc-doped dentin adhesive for collagen protection at the hybrid layer.

The aim of the study was to ascertain whether the addition of zinc to adhesives may decrease metalloproteinase-mediated collagen degradation without affecting bonding efficacy. Human dentin beams were treated with phosphoric acid, with Clearfil SE Bond Primer or with Clearfil SE Bond Primer plus ZnCl(2) (2 wt%). Acid-etched dentin was infiltrated with Single Bond, Single Bond plus ZnCl(2) (2 wt%), or Single Bond plus ZnO nanoparticles (10 wt%), and Clearfil SE Bond-primed dentin was infiltrated with Clearfil SE Bonding resin, Clearfil SE-Bonding resin with ZnCl(2) (2 wt%), or Clearfil SE-Bonding resin with ZnO nanoparticles (10 wt%). The C-terminal telopeptide concentrations were determined 24 h, and 1 and 4 wk after treatment. Microtensile bond strength to dentin was determined for the tested adhesives. Matrix metalloproteinases-mediated collagen degradation occurred in acid-etched and SE-primed dentin. Resin infiltration decreased collagen degradation. Lower collagen degradation was found for SE Bond than for Single Bond. Zinc-doped Single Bond resin always reduced collagen degradation, the ZnO particles being more effective than ZnCl(2) . Zinc-doped SE Bond reduced the liberation of C-terminal telopeptide only at 24 h. Bond strength to dentin was not decreased when Zn-doped resins were employed, except when ZnCl(2) was added to SE Primer. Zinc-doped resin reduced collagen degradation in Single Bond hybrid layers, but did not affect bond strength. The addition of zinc to SE Bond had no beneficial effects.

Comparison of bond stability between dual-cure resin cements and pretreated glass-infiltrated alumina ceramics.

OBJECTIVES: To evaluate the bond stability of resin cements used for luting alumina-based all-ceramic dental restorations. BACKGROUND DATA: Although different pretreatments may be applied on alumina to improve bond strengths, any previous study investigated the bond stability of resin-based cements luted to laser-irradiated alumina. METHODS: 64 sintered, glass-infiltrated alumina blocks were sandblasted and randomly assigned to the following subgroups: 1. no additional treatment (NT); 2. Rocatec (Roc); 3. Nd:YAG laser (L); and 4. Nd:YAG laser plus Rocatec (LRoc). Composite samples were bonded to conditioned ceramics using two different resin based cements: a self-etching adhesive cement-Panavia F (PF) and a self-adhesive resin cement-RelyX Unicem (RXU). After 24 h, bonded specimens were cut into microtensile sticks (1 ± 0.1 mm(2)). One-half of the beams were loaded in tension until failure. The remaining one-half was immersed in 10%-NaOCl aqueous solution (NaOCl(aq)) for 12 h before testing. ANOVA and Student-Newman-Keuls tests were run (P < 0.05). Failure mode was recorded. Ceramic topography was SEM-analyzed. RESULTS: After 24 h, L-sticks achieved the highest MTBS despite the cement type, whereas NT-samples recorded the lowest values. After NaOCl(aq) immersion bond strengths decreased except for RXU luted to NT-alumina. PF luted to L- and LRoc-samples, and RXU luted to L-sticks attained the highest bond strength. CONCLUSIONS: Nd:YAG laser irradiation improved bond strength between alumina ceramics and resin cements (PF or RXU). Chemical challenging impaired adhesion, mainly through resin matrixes and silane coupling degradation. Laser-treated specimens remained with the highest bond strength.

Effect of dentin etching and chlorhexidine application on metalloproteinase-mediated collagen degradation.

Dentin matrix metalloproteinases (MMPs) are involved in the degradation of collagen in resin-dentin interfaces. This study evaluated whether collagen degradation can be prevented by chlorhexidine digluconate (CHX) after different dentin demineralization procedures. The demineralization of human dentin was performed with phosphoric acid (PA), EDTA or acidic monomers (Clearfil SE Bond and Xeno V). Specimens were stored (for 24 h, or for 1 or 3 wk) in the presence or absence of CHX. In half of the groups, active MMP-2 was incorporated into the storage solution. At the end of each storage period, the C-terminal telopeptide (ICTP) concentration (which indicates the amount of collagen degradation) was measured in the storage solution. Collagen degradation was higher in PA- and EDTA-demineralized dentin. Chlorhexidine digluconate reduced collagen degradation in these groups only for 24 h. When dentin was demineralized with Clearfil SE Bond or Xeno V, collagen degradation was reduced by up to 30%, but the addition of exogenous MMP-2 significantly increased collagen degradation. In self-etchant-treated dentin, the inhibitory effect of CHX on MMPs lasted for up to 3 wk. Treating dentin with EDTA, PA or self-etching agents produces enough demineralization to permit cleavage of the exposed collagen. Monomer infiltration may exert protection on demineralized collagen, probably through immobilization of MMPs. The partial inhibitory action of CHX on MMP activity produced by self-etching adhesives was prolonged compared with the short-acting PA- or EDTA-treated dentin.