Laser-Modified Ti Surface Improves Paracrine Osteogenesis by Modulating the Expression of DKK1 in Osteoblasts.

Titanium surface modifications are widely used to modulate cellular behavior by recognition of topographical cues. However, how those modifications affect the expression of mediators that will influence neighboring cells is still elusive. This study aimed to evaluate the effects of conditioned media from osteoblasts cultured on laser-modified titanium surfaces on the differentiation of bone marrow cells in a paracrine manner and to analyze the expression of Wnt pathway inhibitors. Mice calvarial osteoblasts were seeded on polished (P) and Yb:YAG laser-irradiated (L) Ti surfaces. Osteoblast culture media were collected and filtered on alternate days to stimulate mice BMCs. Resazurin assay was performed every other day for 20 days to check BMC viability and proliferation. After 7 and 14 days of BMCs maintained with osteoblasts P and L-conditioned media, alkaline phosphatase activity, Alizarin Red staining, and RT-qPCR were performed. ELISA of conditioned media was conducted to investigate the expression of Wnt inhibitors Dickkopf-1 (DKK1) and Sclerostin (SOST). BMCs showed increased mineralized nodule formation and alkaline phosphatase activity. The L-conditioned media enhanced the BMC mRNA expression of bone-related markers Bglap, Alpl, and Sp7. L-conditioned media decreased the expression of DKK1 compared with P-conditioned media. The contact of osteoblasts with Yb:YAG laser-modified Ti surfaces induces the regulation of the expression of mediators that affect the osteoblastic differentiation of neighboring cells. DKK1 is among these regulated mediators.

Biocompatibility of new calcium aluminate cement: tissue reaction and expression of inflammatory mediators and cytokines.

INTRODUCTION: The aim of this study was to evaluate the biocompatibility of a new calcium aluminate cement (EndoBinder) in subcutaneous tissue of rats in comparison with mineral trioxide aggregate and calcium hydroxide hard-setting cement. METHODS: Polyethylene tubes (1.5 × 10 mm) containing the dental cements were implanted into dorsal subcutaneous tissue of 30 rats. After experimental periods of 7, 30, and 90 days, biopsies were performed for tissue response analysis under optical light microscope. The mRNA extraction was performed for molecular evaluation of the inflammatory process in the peri-implant tissue, which was submitted to quantitative real-time polymerase chain reaction analysis for inflammatory mediators and cytokines TNF-α, Ptges2, Il-1ß, Il-4, and Il-10. RESULTS: On the basis of the score used to grade the tissue reaction (0-3), EndoBinder (0) presented no inflammatory reaction after the 90-day period, a similar result to mineral trioxide aggregate and calcium hydroxide. The thickness of inflammatory capsules (µm) also presented significant decrease during the course of periods (P < .05). As regards expression of inflammatory mediators, Ptges2 and Il-10 were detected only at 7 and 30 days, with no statistically significant difference among the experimental groups (P > .05). CONCLUSIONS: EndoBinder induced limited inflammatory reaction. It was considered biocompatible when tested in subcutaneous tissue of rats.

Osteogenesis-inducing calcium phosphate nanoparticle precursors applied to titanium surfaces.

This study investigated the effects of the morphology and physicochemical properties of calcium phosphate (CaP) nanoparticles on osteogenesis. Two types of CaP nanoparticles were compared, namely amorphous calcium phosphate (ACP) nano-spheres (diameter: 9-13 nm) and poorly crystalline apatite (PCA) nano-needles (30-50 nm × 2-4 nm) that closely resemble bone apatite. CaP particles were spin-coated onto titanium discs and implants; they were evaluated in cultured mouse calvarial osteoblasts, as well as after implantation in rabbit femurs. A significant dependence of CaP coatings was observed in osteoblast-related gene expression (Runx2, Col1a1 and Spp1). Specifically, the PCA group presented an up-regulation of the osteospecific genes, while the ACP group suppressed the Runx2 and Col1a1 expression when compared to blank titanium substrates. Both the ACP and PCA groups presented a more than three-fold increase of calcium deposition, as suggested by Alizarin red staining. The removal torque results implied a slight tendency in favour of the PCA group. Different forms of CaP nanostructures presented different biologic differences; the obtained information can be used to optimize surface coatings on biomaterials.

The expression of chemokines CCL19, CCL21 and their receptor CCR7 in oral squamous cell carcinoma and its relevance to cervical lymph node metastasis.

The purpose of this study is to determine the expression of CCL19, CCL21, and CCR7 in samples of oral squamous cell carcinoma (OSCC) and their relationship with clinical and microscopic parameters. A comparative analysis was made of the mRNA expression of these chemokines and receptor in OSCC and normal oral mucosa. The immunoexpression of CCR7, CCL19, and CCL21 was also verified in OSCC and lymph nodes. Statistical significance was accepted at P < 0.05. Similar levels of CCR7, CCL19, and CCL21 mRNA in OSCC and normal oral mucosa were seen. A low expression of CCL19 and CCL21 in the intra- and peritumoral regions was observed. Scarce CCL19(+) and CCL21(+) cells were also noted in metastatic and non-metastatic lymph nodes. No association was found between the expression of these chemokines and clinical and microscopic parameters. Our findings would suggest that CCL19 and CCL21 may not be associated with cervical lymph node metastasis or other clinical and microscopic factors in OSCC.

Expression of suppressor of cytokine signaling 1 and 3 in ligature-induced periodontitis in rats.

OBJECTIVE: Evaluate expression of inducible negative regulators of JAK/STAT pathway and their target proteins during the course of ligature-induced experimental periodontal disease in rats. DESIGN: Rats were sacrificed 07, 15 and 30 days after disease induction for histological evaluation of periodontal inflammation and macroscopic analysis of alveolar bone loss. SOCS expression and the activation status of STAT1 and STAT3 were evaluated in gingival biopsies by real time PCR and Western blot. RESULTS: Ligature-induced model presented significant progressive bone loss from 7 to 30 days. Inflammation was evident and similar for 07 and 15 days; however, a decrease on severity at the end of the experimental period was observed. There was a significant (p<0.05) increase on SOCS1 and SOCS3 gene expression in PD compared to control group at 15 and 30days. The SOCS1 and SOCS3 protein expression and activation of STAT1 and STAT3 were increased in earlier periods in the ligature model. CONCLUSION: This study suggests that SOCS1 and SOCS3 were directly correlated with regulatory mechanism of the inflammatory process responsible for the periodontal disease destruction.

Effects of intrapulpal temperature change induced by visible light units on the metabolism of odontoblast-like cells.

PURPOSE: To investigate the effects of intrapulpal temperature changes induced by a quartz tungsten halogen (QTH) and a light emitting diode (LED) curing units on the metabolism of odontoblast-like cells. METHODS: Thirty-six 0.5 mm-thick dentin discs obtained from sound human teeth were randomly assigned into three groups: QTH, LED and no light (control). After placement of the dentin discs in pulp chamber devices, a thermistor was attached to the pulpal surface of each disc and the light sources were applied on the occlusal surface. After registering the temperature change, odontoblast-like cells MDPC-23 were seeded on the pulpal side of the discs and the curing lights were again applied. Cell metabolism was evaluated by the MTT assay and cell morphology was assessed by SEM. RESULTS: In groups QTH and LED the intrapulpal temperature increased by 6.4 degrees C and 3.4 degrees C, respectively. The difference between both groups was statistically significant (Mann-Whitney; P < 0.05). QTH and LED reduced the cell metabolism by 36.4% and 33.4%, respectively. Regarding the cell metabolism, no statistically significant difference was observed between both groups (Mann-Whitney; P > 0.05). However, when compared to the control, only QTH significantly reduced the cell metabolism (Mann-Whitney; P < 0.05). It was concluded that the irradiance of 0.5 mm-thick human dentin discs with a QTH in comparison to a LED curing unit promoted a higher temperature rise, which propagates through the dentin negatively affecting the metabolism of the underlying cultured pulp cells.

Cytotoxic effects and pulpal response caused by a mineral trioxide aggregate formulation and calcium hydroxide.

PURPOSE: To evaluate the in vivo pulpal response after pulpotomy with different capping agents. In addition, the in vitro cytotoxic effects of both materials were assessed by applying them on culture of pulp cells. METHODS: For the in vivo test, the coronal pulp of 28 teeth of dogs was mechanically removed and the root pulps were capped with the following dental materials: Group 1: Pro-Root MTA (PRMTA); and Group 2 (control): calcium hydroxide saline paste (CH). After 60 days, the animals were sacrificed and the teeth processed for histological analysis. In the in vitro test, experimental extracts obtained from both capping agents were applied on the cultured MDPC-23 odontoblast-like cells. RESULTS: In the root pulps capped with PRMTA or CH, coagulation necrosis partially replaced by dystrophic calcification as well as tubular dentin matrix laid down by elongated pulp cells was observed. None or mild inflammatory response occurred beneath the capped pulpal wound. Regarding the pulpal response, PRMTA and CH presented no statistical difference. However, the teeth capped CH presented greater healthy pulp loss which resulted in convex shape of the hard barrier than PRMTA. When applied on the cultured cells, it was demonstrated that PRMTA and CH solutions decreased the cell metabolic activity by 9.9% and 29.4%, respectively. CH caused higher cytotoxic effects to the MDPC-23 cells as well as deeper healthy pulp tissue loss than PRMTA. However, similar sequence of healing occurred after pulpotomy with both dental materials.

Cytotoxic effects of different concentrations of chlorhexidine.

PURPOSE: To evaluate the cytotoxic effects of different concentrations of Chlorhexidine (Chx) to the odontoblast cell line MDPC-23. METHODS: The odontoblast-like cells were seeded (30,000 cells/cm2) in 60 wells of 24-well dishes and then incubated in contact with the following experimental and control solutions: Group 1: 0.0024% Chx; Group 2: 0.004% Chx; Group 3: 0.02% Chx; Group 4: Phosphate buffer saline solution (PBS, negative control); and Group 5: 0.06% H2O2 (positive control). Cell metabolic activity was measured by MTT assay and the cell morphology was analyzed by SEM. RESULTS: The cytotoxic effects of Chx are dose-dependent. The reduction in the cell metabolism for Groups 1, 2, and 3 was 24.8%, 29.9% and 70.8%, respectively. No statistical difference was observed between the Groups 1 and 2 in which no significant cell morphology changes occurred. Consequently, it was concluded that 0.02% Chx solution presents high cytotoxicity to the odontoblast-like cells MDPC-23. On the other hand, 0.0024% and 0.004% Chx causes slight cytopathic effects to the cultured cells.