Dental pulp stem cells in chitosan/gelatin scaffolds for enhanced orofacial bone regeneration

Objective

Biomimetic chitosan/gelatin (CS/Gel) scaffolds have attracted great interest in tissue engineering of several tissues. However, limited information exists regarding the potential of combining CS/Gel scaffolds with oral cells, such as dental pulp stem cells (DPSCs), to produce customized constructs targeting alveolar/orofacial bone reconstruction, which has been the aim of the present study.

Dental pulp stem cells’ secretome enhances pulp repair processes and compensates TEGDMA-induced cytotoxicity

ObjectivesAim of this study was to investigate the effects of dental pulp stem cells’ (DPSCs) secretome, expressed through their culture conditioned medium (CM), on biological endpoints related to pulp repair and on TEGDMA-induced cytotoxicity.MethodsDPSCs cultures were established and characterized for stem cell markers with flow cytometry. CM was collected from DPSCs under serum deprivation conditions (SDC) and normal serum conditions (NSC) at various time-points. CM effects on DPSCs viability, migration and mineralization potential were evaluated by MTT assay, transwell insert and in vitro scratch assay and Alizarin Red staining/quantification respectively. TEGDMA (0.25–2.0 mM) cytotoxicity regarding the same biological endpoints was tested in the presence/absence of CM. TGF-β1 and FGF-2 secretion in CM was measured by ELISA.ResultsCM collected under SDC (4 d) was able to increase cell viability by 20–25% and to reduce TEGDMA cytotoxicity by 20% (p < 0.05). CM positive effects were not obvious when collected under NSC. Transwell assay showed significant increase (26%, p < 0.05) of DPSCs’ migration after CM exposure, whereas both migration assays could not support a migration rate improvement in TEGDMA-treated cultures exposed to CM compared to TEGDMA alone. CM significantly (p < 0.01) increased DPSCs mineralization potential and completely counteracted TEGDMA cytotoxicity on this process. ELISA analysis showed a time-dependent increase of TGF-β1 and a TEGDMA concentration-dependent increase of both TGF-β1 and FGF-2 in CM.SignificanceThese findings suggest that DPSCs secretome increases their viability, migration and mineralization potential and counteracts TEGDMA-induced cytotoxicy, revealing a novel mechanism of DPSCs autocrine signaling on pulp repair processes.     

Enhancing Effects of Immobilized Chondroitin Sulfate on Odontogenic Differentiation of Dental Pulp Stem Cells and Reparative Dentin Formation

IntroductionChondroitin sulfate (CS) is a major proteoglycan involved in the mineralization of the organic matrix of dentin. In this study, the roles of CS immobilized in cross-linked collagen I (Col I) hydrogels on odontogenic differentiation of dental pulp stem cells (DPSCs) and reparative dentin formation were investigated.MethodsDifferent concentrations of CS were incorporated into the genipin–cross-linked Col I hydrogels (CS-0.05, CS-0.1, and CS-0.2, respectively). The influences of CS on the proliferation and odontogenic differentiation of DPSCs were investigated. Finally, the effect of the functionalized hydrogel on the formation of reparative dentin was analyzed in a rat pulp capping model in vivo.ResultsCS improved the proliferation of DPSCs seeded on the hydrogels (P < .05). CS also enhanced the mineralization activities and increased the expression levels of the odontogenic-related proteins of DPSCs on days 7 and 14 (P < .05). In vivo, CS-0.1 hydrogel induced reparative dentin formation with higher quality compared with mineral trioxide aggregate.ConclusionsCS immobilized in Col I hydrogels could induce odontogenic differentiation of DPSCs in vitro and promote homogeneous mineralized barrier formation in vivo. CS–Col I hydrogel has the potential for reparative dentin formation of high quality in direct pulp capping.     

Simplified conditions for storing and cryopreservation of dental pulp stem cells

ObjectivesThis study aimed to simplify the collection, isolation and cryopreservation procedure of human dental pulp stem cells (DPSCs) to ease the establishment of dental stem cell banking.DesignExtracted third molars were collected and stored either in growth medium or in gentamicin-saline (480 μg/ml) for 6, 9 or 12 h. DPSCs were isolated and subjected to cryopreservation by a controlled-rate or rapid freezing method in 5 or 10% DMSO. Flow cytometry and growth pattern of DPSCs before and after cryopreservation were conducted.ResultsRate of contamination by which the extracted teeth were stored in control and gentamicin-saline were 9.1% (N = 33) and 2.3% (N = 43), respectively. Successful cell isolation rate of teeth preserved in gentamicin-saline at 6 h (92.9%) was comparable to those of growth media group (90.3%). At 9 and 12 h, the rates dropped significantly to 75% and 54%, respectively. Cryopreservation by controlled-rate freezing either in 5 or 10% DMSO resulted in a significantly higher percentage of viable cells than by rapid freezing. Cells conserved by controlled-rate freezing in 5% DMSO showed a pattern of growth similar to control unfrozen cells; 10% DMSO significantly deteriorated the growth pattern of the cells. After thawing, DPSCs conserved by controlled-rate freezing still expressed stemness characteristics, although hematopoietic stem cell markers were slightly increased.ConclusionGentamicin-saline was effective in preserving human teeth for DPSC isolation. Controlled-rate freezing in 5% DMSO gave the highest rate of cell viability. This study simplifies the storage conditions and proposes a simple method for cryopreservation of DPSCs.     

Micropatterned hydrogels and cell alignment enhance the odontogenic potential of stem cells from apical papilla in-vitro

IntroductionAn understanding of the extracellular matrix characteristics which stimulate and guide stem cell differentiation in the dental pulp is fundamental for the development of enhanced dental regenerative therapies. Our objectives, in this study, were to determine whether stem cells from the apical papilla (SCAP) responded to substrate stiffness, whether hydrogels providing micropatterned topographical cues stimulate SCAP self-alignment, and whether the resulting alignment could influence their differentiation towards an odontogenic lineage in-vitro.MethodsExperiments utilized gelatin methacryloyl (GelMA) hydrogels of increasing concentrations (5, 10 and 15%). We determined their compressive modulus via unconfined compression and analyzed cell spreading via F-actin/DAPI immunostaining. GelMA hydrogels were micropatterned using photolithography, in order to generate microgrooves and ridges of 60 and 120 μm, onto which SCAP were seeded and analyzed for self-alignment via fluorescence microscopy. Lastly, we analyzed the odontogenic differentiation of SCAP using alkaline phosphatase protein expression (ANOVA/Tukey α = 0.05).ResultsSCAP appeared to proliferate better on stiffer hydrogels. Both 60 and 120 μm micropatterned hydrogels guided the self-alignment of SCAP with no significant difference between them. Similarly, both 60 and 120 μm micropattern aligned cells promoted higher odontogenic differentiation than non-patterned controls.SignificanceIn summary, both substrate mechanics and geometry have a statistically significant influence on SCAP response, and may assist in the odontogenic differentiation of dental stem cells. These results may point toward the fabrication of cell-guiding scaffolds for regenerative endodontics, and may provide cues regarding the development of the pulp-dentin interface during tooth formation.     

Pulp ECM-derived macroporous scaffolds for stimulation of dental-pulp regeneration process

ObjectiveRecent studies suggest xenogeneic extracellular matrices as potential regenerative tools in dental pulp regeneration. This study aimed to fabricate and characterize a novel three-dimensional macroporous pulp-derived scaffold that enables the attachment, penetration, proliferation and differentiation of mesenchymal stem cells.MethodBovine pulp was decellularized and characterized with histological and DNA content methods. This scaffold was prepared using finely milled lyophilized decellularized pulp extracellular matrix (ECM) digested with pepsin. Three different concentrations of ECM (1.50, 2.25 and 3.00 mg/ml) were freeze-dried and were tested with/without chemical crosslinking. The specimens were subjected to physicochemical characterization, cell viability and quantitative real time polymerase chain reaction assessments with human bone marrow mesenchymal stem cells (hBMMSCs). All scaffolds were subcutaneously implanted in rats for two weeks and histological and immunostaining analyses were performed.ResultsHistological and DNA analysis confirmed complete decellularization. All samples demonstrated more than 97% porosity and 1.50 mg/ml scaffold demonstrated highest water absorption. The highest cell viability and proliferation of hBMMSCs was observed on the 3.00 mg/ml crosslinked scaffolds. The gene expression analysis showed a significant increase of dmp-1 and collagen-I on 3.00 mg/ml crosslinked scaffolds compared to the other scaffolds. Histological examination of subcutaneous implanted scaffolds revealed low immunological response, and enhanced angiogenesis in cross-linked samples compared to non-crosslinked samples.SignificanceThe three-dimensional macroporous pulp-derived injectable scaffold developed and characterized in this study displayed potential for regenerative therapy. While the scaffold biodegradability was decreased by crosslinking, the biocompatibility of post-crosslinked scaffold was significantly improved.     

Evaluation of bone regeneration by porous alpha-tricalcium phosphate/atelocollagen sponge composite in rat calvarial defects

PurposeBoth atelocollagen and alpha-tricalcium phosphate (α-TCP) particles are widely applied as bone graft materials. In this study, we combined atelocollagen and porous α-TCP particles to form an α-TCP/atelocollagen composite sponge (α-TCP/CS), and we evaluated its effects on bone regeneration in rat calvarial defects.Materials and methodsα-TCP granules were mixed with a collagen solution. The mixture was poured into plastic molds and then frozen to ?80 °C and freeze-dried for 24 h. The composites were then cross-linked in vacuo at 140 °C for 24 h. The obtained composites were characterized by XRD and SEM and observed using micro-CT and histological analysis.ResultsIn vivo micro-CT images at 2, 4, and 6 weeks after surgery showed that in the α-TCP/CS group, the bone volume, bone mineral density, and bone mineral content were higher than those in the groups with only atelocollagen and without scaffolds at 6 weeks after surgery. Histological analysis showed that α-TCP was completely absorbed, and new bone that was continuous along the original bone was observed.ConclusionThis study demonstrated that composite sponges created using porous α-TCP particles and atelocollagen were sufficiently adaptable for treating bone defects.     

Immunoexpression of BMP-2 and BMP-4 and their receptors,BMPR-IA and BMPR-II,in ameloblastomas and adenomatoid odontogenic tumors

ObjectivesThe present study evaluated the immunohistochemical expression of BMP-2 and BMP-4 and of their receptors (BMPR-IA and BMPR-II) in solid ameloblastoma (SA), unicystic ameloblastoma (UA) and adenomatoid odontogenic tumor (AOT) in order to obtain a better understanding of their role in the development and biological behavior of these tumors.DesignThis study analyzed these proteins in 30 cases of SA, 10 cases of UA, and 30 cases of AOT. Immunoexpression was evaluated in the parenchyma and stroma by attributing the following scores: 0, no stained cells; 1, ≤10%; 2, >10% and ≤25%; 3, >25% and ≤50%; 4, >50% and ≤75%.; 5, >75% stained cells.ResultsIn SAs, positive correlations were observed between the stromal and parenchymal expression of BMP-2 (p < 0.001) and between the stromal expression of BMP-2 and BMP-4 (p = 0.020), as well as between the stromal expression of BMPR-II and BMP-4 (p = 0.001) and the stromal and parenchymal expression of BMPR-II (p < 0.001). In UAs, correlations were detected between the stromal and parenchymal expression of BMP-4 (p = 0.035) and between the stromal expression of BMP-4 and BMPR-IA (p = 0.022). In AOTs, analysis of immunoexpression in the parenchyma revealed positive correlations between all proteins.ConclusionBMPs and their receptors play an important role in the differentiation and development of ameloblastomas and AOTs, but may not explain the different biological behaviors of these lesions. The positive correlation observed in AOTs might be related to the formation of mineralized material in this tumor.     

Cell-derived Extracellular Matrix Proteins in Colloidal Microgel as a Self-Assembly Hydrogel for Regenerative Endodontics

IntroductionThis study investigated a colloidal microgel for angiogenic and odontogenic differentiation of cells in the presence of cell-derived extracellular matrix (ECM) proteins using a 3-dimensional culture model.MethodsViscoelastic properties of human dental pulp were determined to understand the native ECM environment. ECM proteins were extracted from dental pulp stem cell (DPSC) cultures, and MaxGel (Millipore Sigma, Burlington, MA) was used as a commercially available ECM protein. DPSCs were incubated in colloidal microgels in the presence of ECM proteins or gelatin methacryloyl (GelMA) as a bulk hydrogel (n = 9/group). The viability and odontogenic differentiation of DPSCs within hydrogels was determined using viability assays, mineralization staining, calcium and alkaline phosphatase assays, and quantitative polymerase chain reaction for odontogenic gene expression. Angiogenic properties of endothelial cells were determined using tubule formation assays and quantitative polymerase chain reaction to detect angiogenic gene expression.ResultsDental pulp had a higher elastic modulus than the viscous modulus, showing a solidlike response similar to hydrogels. DPSC-derived ECM showed higher collagen and GAG than MaxGel (P < .05). The viability of DPSCs was similar in colloidal microgels, whereas higher cell viability, calcium deposition, and alkaline phosphatase activity were observed in GelMA (P < .05). Colloidal microgels allowed tubule-like structures by endothelial cells, whereas no tubular formation was observed in GelMA. DPSC-derived ECM in colloidal microgel up-regulated odontogenic gene expression, whereas MaxGel up-regulated angiogenic gene expression (P < .05).ConclusionsColloidal microgels allowed cellular organization that can improve penetration and nutritional supply in a full-length root canal system. The bioactivity of cell-derived ECM proteins can be modified depending on the external stimulus.     

Potential chemotherapeutic effects of diosgenin,zoledronic acid and epigallocatechin-3-gallate on PE/CA-PJ15 oral squamous cancer cell line

ObjectiveTo study the potential chemotherapeutic effects of Diosgenin, zoledronic acid and Epigallocatechin-3-gallate on oral squamous cell cancer (OSCC).Materials and methodsCell viability, migration, apoptosis and cell cycle evaluation assays were performed in order to assess the effects of different doses of Diosgenin, zoledronic acid and Epigallocatechin-3-gallate on the PE/CA-PJ15 cell line.ResultsDoses of 100 μM of diosgenin or zoledronic acid reduced cell viability significantly after 72 h (p < 0.001), as well as increasing apoptosis (p < 0.05 and p < 0.01 respectively). All three agents reduced cell migration and altered the cell cycle, each at a different phase of the cycle.Conclusionwhile DG and ZA reduced cell viability, increased apoptosis, inhibited cell migration and modified the cell cycle in different ways, EGCG only modified the cell cycle and reduced cell migration. These agents present a potential chemotherapeutic effect on PE/CA-PJ15 OSSC cell line, which have to be further studied.     

Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ

ObjectiveThe present in situ - investigation aimed to specify the impact of pure hydroxyapatite microclusters on initial bioadhesion and bacterial colonization at the tooth surface.DesignPellicle formation was carried out in situ on bovine enamel slabs (9 subjects). After 1 min of pellicle formation rinses with 8 ml of hydroxyapatite (HA) microclusters (5%) in bidestilled water or chlorhexidine 0.2% were performed. As negative control no rinse was adopted. In situ biofilm formation was promoted by the intraoral slab exposure for 8 h overnight. Afterwards initial bacterial adhesion was quantified by DAPI staining and bacterial viability was determined in vivo/in vitro by live/dead-staining (BacLight). SEM analysis evaluated the efficacy of the mouthrinse to accumulate hydroxyapatite microclusters at the specimens’ surface and spit-out samples of the testsolution were investigated by TEM.ResultsCompared to the control (2.36 × 10⁶ ± 2.01 × 10⁶ bacteria/cm²), significantly reduced amounts of adherent bacteria were detected on specimens rinsed with chlorhexidine 0.2% (8.73 × 10⁴ ± 1.37 × 10⁵ bacteria/cm²) and likewise after rinses with the hydroxyapatite testsolution (2.08 × 10⁵ ± 2.85 × 10⁵ bacteria/cm², p < 0.001). No demonstrable effect of HA-particles on Streptococcus mutans viability could be shown. SEM analysis confirmed the temporary adsorption of hydroxyapatite microclusters at the tooth surface. Adhesive interactions of HA-particles with oral bacteria were shown by TEM.ConclusionHydroxyapatite microclusters reduced initial bacterial adhesion to enamel in situ considerably and could therefore sensibly supplement current approaches in dental prophylaxis.     

Effect of proteoglycans at interfaces as related to location,architecture, and mechanical cues

IntroductionCovalently bound functional GAGs orchestrate tissue mechanics through time-dependent characteristics.ObjectiveThe role of specific glycosaminoglycans (GAGs) at the ligament–cementum and cementum–dentin interfaces within a human periodontal complex were examined. Matrix swelling and resistance to compression under health and modeled diseased states was investigated.Materials and methodsThe presence of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs at the ligament–cementum and cementum–dentin interfaces in human molars (N = 5) was illustrated by using enzymes, atomic force microscopy (AFM), and AFM-based nanoindentation. The change in physical characteristics of modeled diseased states through sequential digestion of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs was investigated. One-way ANOVA tests with P < 0.05 were performed to determine significant differences between groups. Additionally, the presence of mineral within the seemingly hygroscopic interfaces was investigated using transmission electron microscopy.ResultsImmunohistochemistry (N = 3) indicated presence of biglycan and fibromodulin small leucine rich proteoglycans at the interfaces. Digestion of matrices with enzymes confirmed the presence of KS and CS GAGs at the interfaces by illustrating a change in tissue architecture and mechanics. A significant increase in height (nm), decrease in elastic modulus (GPa), and tissue deformation rate (nm/s) of the PDL-C attachment site (215 ± 63–424 ± 94 nm; 1.5 ± 0.7–0.4 ± 0.2 GPa; 21 ± 7–48 ± 22 nm/s), and cementum–dentin interface (122 ± 69–360 ± 159 nm; 2.9 ± 1.3–0.7 ± 0.3 GPa; 18 ± 4–30 ± 6 nm/s) was observed.ConclusionsThe sequential removal of GAGs indicated loss in intricate structural hierarchy of hygroscopic interfaces. From a mechanics perspective, GAGs provide tissue recovery/resilience. The results of this study provide insights into the role of GAGs toward conserved tooth movement in the socket in response to mechanical loads, and modulation of potentially deleterious strain at tissue interfaces.     

Role of proteoglycans on the biochemical and biomechanical properties of dentin organic matrix

ObjectiveProteoglycans (PGs) are multifunctional biomacromolecules of the extracellular matrix of collagen-based tissues. In teeth, besides a pivotal regulatory role on dentin biomineralization, PGs provide mechanical support to the mineralized tissue and compressive strength to the biosystem. This study assessed enzymatic protocols for selective PGs removal from demineralized dentin to determine the roles of these biomacromolecules in the bulk mechanical properties and biostability of type I collagen.MethodsSelective removal of glycosaminoglycans chains (GAGs) and PGs from demineralized dentin was carried out by enzymatic digestion protocols using chondroitinase ABC (c-ABC) and trypsin (Try). A comprehensive study design included assessment of dentin matrix mass loss, biodegradability of the PGs/GAGs-depleted dentin matrix, ultimate tensile strength (UTS) and energy to fracture tests. Quantitative data was statistically analyzed by two-way and one-way ANOVA followed by the appropriate post hoc tests (α = 0.05).ResultsTransmission electron microscopy images show effective GAGs removal by c-ABC and Try and both enzymatic methods released statistically similar amounts of GAGs from the demineralized dentin. Try digestion resulted in about 25% dentin matrix mass loss and increased susceptibility to collagenolytic digestion when compared to c-ABC (p = 0.0224) and control (p = 0.0901). Moreover, PGs digestion by Try decreased the tensile strengths of dentin. Statistically lower energy to fracture was observed in c-ABC-treated dentin matrix.ConclusionsGAGs plays a pivotal role on tissue mechanics and anisotropy, while the core protein of PGs have a protective role on matrix biostability.     

Effects of connective tissue growth factor on human periodontal ligament fibroblasts

ObjectiveThe aim of this study was to evaluate the effects of different concentrations of connective tissue growth factor (CTGF) on human periodontal ligament fibroblasts(HPLFs).DesignHPLFs were cultured and identified. Then, different concentrations of CTGF (1, 5, 10, 50, 100 ng/ml) were added to the HPLF culture. Next, CCK-8 assays, alkaline phosphatase (ALP) assays, hydroxyproline determination, alizarin red staining methods, Transwell chambers and real-time PCR methods were applied to observe the effects of CTGF on the proliferation, ALP activity, synthesis of collagen, formation of mineralized nodules and migration. We also studied expression of ALP, fiber link protein (FN), integrin-binding sialoprotein (IBSP), osteocalcin (OC), and integrin beta 1 (ITGB1) mRNA by HPLFs. Statistical significance was assumed if P < 0.05 or P < 0.01.ResultsThe addition of CTGF (1, 5, 10 ng/ml) remarkably promoted the proliferation and collagen synthesis of HPLFs compared with controls. CTGF (1, 5, 10, 50 ng/ml) improved ALP activity of HPLFs, and at all concentrations, CTGF (1, 5, 10, 50, 100 ng/ml) improved the expression of ALP, FN, IBSP and ITGB1 mRNA. In addition, CTGF (1, 5, 10, 50, 100 ng/ml) promoted the migration of HPLFs, which was dose-dependent, with maximal promotion in the 10 ng/ml group (P < 0.05 or P < 0.01).ConclusionsThus, in a certain range of concentrations, CTGF can promote the biological effects, including proliferation, migration and collagen synthesis of HPLFs, to promote the differentiation of HPLFs in the process of osteogenesis.     

Cell migration,viability and tissue reaction of calcium hypochlorite based-solutions irrigants: An in vitro and in vivo study

ObjectiveThis study aimed to analyze in vitro cytotoxicity to cultured 3T3 fibroblasts and in vivo inflammatory reaction in rats by calcium hypochlorite (Ca(OCl)₂) solutions compared with sodium hypochlorite (NaOCl) solutions.DesignCultured 3T3 fibroblasts were exposed to different concentrations of (Ca(OCl)₂) and NaOCl solutions, and a scratch assay was performed. The viability rate was analyzed with trypan blue assay. Both solutions of 1% and 2.5% concentrations were injected into the subcutaneous tissue of 18 male Wistar rats aged 18 weeks. The inflammatory tissue reaction was evaluated at 2 h, 24 h, and 14 days after the injections. The samples were qualitatively analyzed using a light microscope. Statistical analysis was performed with ANOVA and Tukey post hoc tests for in vitro assays and Kruskal–Wallis and Dunn post hoc tests for in vivo assays (α = 0.05).ResultsIn the scratch assay, Ca(OCl)₂ showed no significant difference compared with the control group (culture medium) at 24 h (p < 0.05). Solutions of 0.0075% and 0.005% NaOCl and Ca(OCl)₂ concentrations presented similar results compared with those in the positive control group (hydrogen peroxide) (p > 0.05) in the trypan blue assay. In the in vivo assay, 1% Ca(OCl)₂ group showed a significant decrease in neutrophils at 2 h and 24 h (p = 0.041) and 2 h and 14 days (p = 0.017). There was no statistically significant difference for lymphocyte/plasmocyte and macrophage counts among the different concentration groups.ConclusionsCa(OCl)₂ showed favorable results of viability and induced a low-level inflammatory response. Ca(OCl)₂ presented acceptable cytotoxicity and biocompatibility as an irrigant solution.     

The repair of critical-size defects with porous hydroxyapatite/polyamide nanocomposite: an experimental study in rabbit mandibles

This study was conducted to evaluate the healing of critical-size surgical defects after implantation of porous nano-hydroxyapatite/polyamide composite (nHA/PA) blocks based on a bilateral mandible model using adult New Zealand white rabbits. 15 rabbits were divided randomly into three groups according to the observation period: 4, 12 and 24 weeks. The defects on one side were implanted with nHA/PA blocks and the contralateral defects were kept empty as blank controls. A combination of macroscopic, radiographic, histological and histomorphometric studies were performed up to 24 weeks postoperatively and compared with normal healing. Large amounts of callus and active osteoblasts were found in the pore structure after 4 weeks of implantation, and the defects were completely occupied by neo-bone with density comparable with that of host bone at 24 weeks. Significant difference was found between nHA/PA groups and blank controls regarding X-ray opacity over the whole period and bone parameters at 4 weeks postoperation (P < 0.05). The porous nHA/PA composite promotes bone formation over the extension of the defect, particularly in the early stage. Porous nHA/PA offers interesting potential for maxillofacial reconstructive procedures in load-free areas.     

Cytotoxicity and potential anti-inflammatory activity of velutin on RAW 264.7 cell line differentiation: Implications in periodontal bone loss

ObjectivesHypoxia-inducible factor-1α (HIF-1α) has been implicated in periodontal tissue inflammation and possibly in osteoclast differentiation, while polyphenols are known to be anti-inflammatory natural compounds that are capable of regulating the NF-κB protein complex pathway. The objective of this study was to investigate cytotoxicity and HIF-1α expression through the NF-κB pathway by polyphenol velutin (Euterpe oleracea Mart.), found in the pulp of acai fruit, during inflammatory RAW 264.7 differentiation.DesignRAW 264.7 mouse monocyte macrophage cells were stimulated with RANKL (30 ng/mL) and Porphyromonas gingivalis lipopolysaccharide (1 μg/mL). Cells were treated with various concentrations of velutin (0.5–2 μM) to check for viability, morphology, osteoclast differentiation, and HIF-1α expression (Western blot).ResultsAlamar blue cell viability assay showed no toxicity to RAW cells with the use of velutin in all concentrations tested (p > 0.05). Velutin did not induce cell apoptosis based on caspase 3/7 assay (p > 0.05). Fluorescence images stained by DAPI showed no alteration in the morphology of RAW cell nuclei (p > 0.05) treated with velutin. TRAP assays demonstrated a dose-dependent reduction in osteoclast formation by velutin when compared with control (p < 0.05). Velutin showed a reduction in HIF-1α expression related to IκB phosphorylation when compared with control (p < 0.001).ConclusionsAt the tested concentrations, velutin was not cytotoxic to RAW 264.7 and differentiated cells. Velutin reduced osteoclast differentiation and downregulated HIF-1α through the NF-κB pathway.     

Effects of risedronate on osteoblastic cell cultures

ObjectiveBisphosphonates (BPs) have been widely used in the treatment of bone disorders due to their ability to modulate bone turnover. The biological mechanisms through BFs exert their effects on osteoclasts are well established. However, the role of BFs on the osteoblasts is controversial. The present study aimed to evaluate the effects of risedronate on osteoblastic cells.DesignMC3TE-E1 cells were exposed to risedronate at 0, 10⁻⁸, 10⁻⁶, 10⁻⁴, and 10⁻³ M. The following parameters were assayed: (1) cell proliferation by hemocytometer counting after 24, 48 and 72 h, (2) cell viability by MTT assay after 24, 48 and 72 h, (3) Type I Collagen quantification by ELISA after 24, 48 and 72 h, (3) alkaline phosphatase activity after 7 and 10 days and (4) matrix mineralization after 14 days.ResultsAfter 24 h, risedronate did not affect both cell proliferation and viability (p > 0.05). However, after 48 and 72 h, a decrease in cell proliferation and viability was detected in osteoblastic cultures exposed to risedronate at 10⁻⁴ and 10⁻³ M (p < 0.05). After 48 and 72 h, Type I Collagen synthesis was stimulated by risedronate at 10⁻⁴ M (p < 0.05). High levels of ALP activity were detected in cultures exposed to risedronate at 10⁻⁴ M after 7 and 10 days (p < 0.05). After 14 day, high calcium content was observed in cultures exposed to risedronate at 10⁻⁴ M (p > 0.05).ConclusionThese results indicated that risedronate can promote osteoblast differentiation.     

One-bottle self-etching adhesives applied to dentine air-abraded using bioactive glasses containing polyacrylic acid: An in vitro microtensile bond strength and confocal microscopy study

ObjectivesThe aim of this study was to test the microtensile bond strength (μTBS) of two “simplified” self-etching adhesives bonded to air-abraded dentine using experimental bioactive glass powders containing polyacrylic acid.MethodsSound dentine specimens were air-abraded using a pure Bioglass 45S5 (Bioglass) powder or two Bioglass powders containing different concentration of polyacrylic acid (PAA: 15 wt% or 40 wt%). The bonding procedures were accomplished by the application of two self-etching adhesives (CS3: Clearfil S3 Bond; Kuraray, Osaka, Japan or GB: G Bond; GC Ltd. Tokyo, Japan). The resin-bonded specimens were cut in beams (0.9 mm²) and the μTBS testing was performed after 24 h or 6 months of phosphate buffer solution (PBS) storage. The results were statistically analysed by three-way ANOVA and Student–Newman–Keuls test used (α = 0.05). Further bonded-dentine specimens were used for the confocal microscopy interfacial characterisation and micropermeability analysis.ResultsThe CS3 adhesive system achieved higher μTBS than those attained in the specimens bonded with GB both after 24 h and 6 months of PBS storage. The CLSM analysis performed after 6 months of PBS storage indicated severe micropermeability within the bonded-dentine interfaces created using GB applied onto dentine air-abraded with Bioglass/PAA-15 and Bioglass/PAA-40. Conversely, CS3 exhibited no dye penetration (micropermeability) at the resin–dentine interface.ConclusionIt is possible to affirm that air-abrasion procedures performed using pure Bioglass or Bioglass containing 15 wt% PAA do not interfere with the immediate bonding performance of self-etching adhesives. However, the durability of the bonded-dentine interfaces created subsequent air-abrasion procedures using bioactive glasses will depend also upon the chemical composition of the self-etch adhesive systems.