Chitosan-Calcium-Simvastatin Scaffold as an Inductive Cell-Free Platform.

The development of biomaterials based on the combination of biopolymers with bioactive compounds to develop delivery systems capable of modulating dentin regeneration mediated by resident cells is the goal of current biology-based strategies for regenerative dentistry. In this article, the bioactive potential of a simvastatin (SV)-releasing chitosan-calcium-hydroxide (CH-Ca) scaffold was assessed. After the incorporation of SV into CH-Ca, characterization of the scaffold was performed. Dental pulp cells (DPCs) were seeded onto scaffolds for the assessment of cytocompatibility, and odontoblastic differentiation was evaluated in a microenvironment surrounded by dentin. Thereafter, the cell-free scaffold was adapted to dentin discs positioned in artificial pulp chambers in direct contact with a 3-dimensional (3D) culture of DPCs, and the system was sealed to simulate internal pressure at 20 cm/H2O. In vivo experiments with cell-free scaffolds were performed in rats' calvaria defects. Fourier-transform infrared spectroscopy spectra proved incorporation of Ca and SV into the scaffold structure. Ca and SV were released upon immersion in a neutral environment. Viable DPCs were able to spread and proliferate on the scaffold over 14 d. Odontoblastic differentiation occurred in the DPC/scaffold constructs in contact with dentin, in which SV supplementation promoted odontoblastic marker overexpression and enhanced mineralized matrix deposition. The chemoattractant potential of the CH-Ca scaffold was improved by SV, with numerous viable and dentin sialoprotein-positive cells from the 3D culture being observed on its surface. Cells at 3D culture featured increased gene expression of odontoblastic markers in contact with the SV-enriched CH-Ca scaffold. CH-Ca-SV led to intense mineralization in vivo, presenting mineralization foci inside its structure. In conclusion, the CH-Ca-SV scaffold induces differentiation of DPCs into a highly mineralizing phenotype in the presence of dentin, creating a microenvironment capable of attracting pulp cells to its surface and inducing the overexpression of odontoblastic markers in a cell-homing strategy.

Transdentinal photobiostimulation of stem cells from human exfoliated primary teeth.

AIM: To evaluate the effects of infrared light-emitting diode (LED) irradiation on stem cells from human exfoliated deciduous teeth (SHEDs). METHODOLOGY: Exfoliated primary teeth were obtained (n = 3), and SHEDs obtained from the teeth were seeded on the pulpal surface of 0.2-mm-thick dentine discs produced from permanent molars. The cells were incubated for 24 h by placing the discs in plain Dulbecco's modified Eagle's medium (DMEM). The DMEM was then replaced with new culture medium formulated for odontoblast differentiation. After 12 h in the second medium, SHEDs were irradiated through the dentine discs using an infrared LED (850 nm) with a power density of 80 mW cm-2 . Energy doses (EDs) delivered to the occlusal surface of the dentine discs were 0 (control), 2 and 4 J cm-2 (n = 6). Subsequent tests were performed 72 h after irradiation. These tests included cell viability (MTT), alkaline phosphatase activity (ALP), total protein production (TP), scanning electron microscopy (SEM), as well as gene expression for ALP, Col I, DSPP and DMP-1. Data were analysed using Kruskal-Wallis and Mann-Whitney t-tests (α = 0.05). RESULTS: Both EDs (2 and 4 J cm-2 ) significantly increased cell viability and ALP activity. For TP, ALP and Col I gene expression, only the 4 J cm-2 group had significantly higher values compared to the control group. Cell morphology was not affected by irradiation. CONCLUSION: Infrared LED irradiation was capable of biostimulating SHEDs through a 0.2 mm thickness of dentine, especially at the 4 J cm-2 level.

Nutritional deprivation and LPS exposure as feasible methods for induction of cellular - A methodology to validate for vitro photobiomodulation studies.

Previous studies have demonstrated that high biostimulation takes place when cells under stress are subjected to phototherapy by laser or light-emitting-diode (LED) devices. Several studies selected nutritional deprivation by reducing the concentration of fetal bovine serum (FBS) in the culture medium or the exposure of cultured cells to lipopolysaccharide (LPS) as an in vitro cellular stress condition. However, there are no data certifying that these stimuli cause stressful conditions for cultured cells. This investigation assessed the induction of cellular stress by decreasing the concentration of FBS or adding LPS to culture medium. Odontoblast-like cells (MDPC-23) were cultured in complete culture medium (DMEM) containing 10% FBS. After a 12-hour incubation period, the DMEM was replaced by fresh medium containing 10% FBS (control), low concentrations of FBS (0, 0.2, 0.5, 2, or 5%) or LPS from Escherichia coli (10µg/ml). After an additional 12-hour incubation, cell viability, total cell-counting, total protein production, and gene expression of heat shock protein 70 (HSP70) were assessed. Data were statistically analyzed by ANOVA complemented by the Tukey test, with 5% considered significant. Cell viability was negatively affected only for 0% FBS, while reduced viable cell numbers and total protein production were detected for FBS concentrations lower than 2%. Higher HSP70 gene expression was also observed for FBS concentrations lower than 2% and for cells exposed to LPS. The nutritional deprivation model with culture medium lower than 2% of FBS can be safely used to induce cellular stress for in vitro photobiomodulation studies.

Transdentinal cell photobiomodulation using different wavelengths.

OBJECTIVE: The aim of this study was to investigate the effects of transdentinal irradiation with different light-emitting diode (LED) parameters on odontoblast-like cells (MDPC-23). METHODS AND MATERIALS: Human dentin discs (0.2 mm thick) were obtained, and cells were seeded on their pulp surfaces with complete culture medium (Dulbecco modified Eagle medium). Discs were irradiated from the occlusal surfaces with LED at different wavelengths (450, 630, and 840 nm) and energy densities (0, 4, and 25 J/cm(2)). Cell viability (methyltetrazolium assay), alkaline phosphatase activity (ALP), total protein synthesis (TP), and cell morphology (scanning electron microscopy) were evaluated. Gene expression of collagen type I (Col-I) was analyzed by quantitative polymerase chain reaction (PCR). Data were analyzed by the Mann-Whitney test with a 5% significance level. RESULTS: Higher cell viability (21.8%) occurred when the cells were irradiated with 630 nm LED at 25 J/cm(2). Concerning TP, no statistically significant difference was observed between irradiated and control groups. A significant increase in ALP activity was observed for all tested LED parameters, except for 450 nm at 4 J/cm(2). Quantitative PCR showed a higher expression of Col-I by the cells subjected to infrared LED irradiation at 4 J/cm(2). More attached cells were observed on dentin discs subjected to irradiation at 25 J/cm(2) than at 4 J/cm(2). CONCLUSION: The infrared LED irradiation at an energy density of 4 J/cm(2) and red LED at an energy density of 25 J/cm(2) were the most effective parameters for transdentinal photobiomodulation of cultured odontoblast-like cells.

Effects of low-level laser therapy on the proliferation and apoptosis of gingival fibroblasts treated with zoledronic acid.

Low-level laser therapy (LLLT) has been indicated as an adjuvant therapy for bisphosphonate-induced osteonecrosis. However, the effects of LLLT on bisphosphonate-treated cells are not yet clear. This study evaluated the effects of LLLT on the proliferation and apoptosis of gingival fibroblasts treated with zoledronic acid (ZA). Cells were exposed to ZA at 5µM for 48h. Irradiation was performed using a laser diode prototype (LaserTABLE, InGaAsP; 780nm±3nm, 25mW) at 0.5 or 3J/cm(2), three times every 24h. Cell proliferation and apoptosis were evaluated by fluorescence microscopy. Data were analyzed by Mann-Whitney test at the 5% level of significance. ZA decreased cell proliferation to 47.62% (interquartile range (IQR) 23.80-57.14%; P=0.007) and increased apoptosis of gingival fibroblasts to 27.7% (IQR 20.9-33.4%; P=0.0001). LLLT increased cell proliferation compared with non-irradiated cells, at 0.5J/cm(2) (57.14%, IQR 57.14-71.43%; P=0.003) and at 3J/cm(2) (76.19%, IQR 61.90-76.19%; P=0.0001), but did not increase cell proliferation in ZA-treated cells. Irradiated fibroblasts presented lower apoptosis rates than the ZA-treated cells, but apoptosis was no different in ZA-treated cells compared to those that were ZA-treated and also irradiated.

Infrared LED irradiation photobiomodulation of oxidative stress in human dental pulp cells.

AIM: To investigate the effect of infrared light-emitting diode (LED) irradiation on the oxidative stress induced in human dental pulp cells (HDPCs) by lipopolysaccharide (LPS). METHODOLOGY: Human dental pulp cells (HDPCs) were harvested from sound primary teeth that were near exfoliation. Cells were seeded (10(5)  cells cm(-2) ) using α-MEM supplemented with 10% FBS and after 24 h, were placed in contact with LPS (10 µg mL(-1) of culture medium). Immediately afterwards, HDPCs were subjected to a single irradiation with an infrared LED (855 nm) delivering different doses of energy (0, 2, 4, 8, 15 or 30 J cm(-2) ). For each dose, there was a control group without LPS application. Twenty-four hours after irradiation, groups were tested for nitric oxide (NO) quantification, cell viability (MTT assay) and qualitative assessment of reactive oxygen species (ROS). Data were submitted to Kruskal-Wallis and Mann-Whitney tests (α = 0.05). RESULTS: Lipopolysaccharide (LPS)-induced stress resulted in significant increase in NO production by HDPC without causing damage to cell respiratory metabolism. Irrespective of energy dose delivered, NO production was significantly reduced when LPS-stressed cells were irradiated with infrared LED (2 J cm(-2) , P = 0.003; 95% CI = 5.84-27.71; 4 J cm(-2) , P = 0.001; 95% CI = 7.52-26.39; 8 J cm(-2) , P = 0.0195; 95% CI = -2.86-16.01; 15 J cm(-2) , P = 0.0001; 95% CI = 12.10-30.96; 30 J cm(-2) , P = 0.007; 95% CI = 5.84-24.71). The highest decrease in NO production was observed when 15 J cm(-2) was delivered to cells. Infrared LED irradiation resulted in a decrease in ROS production, whilst HDPC metabolism was not significantly affected. CONCLUSION: Biomodulation of oxidative stress of HPDC can be achieved by irradiation with a single dose of infrared LED. Within the range investigated, 15 J cm(-2) resulted in the least production of NO.