To assess in vitro the effect of experimental mesoporous BAG, on human dental pulp cells (hDPCs) behavior in terms of cytocompatiblity and bioactivity via mineralization potential.
Fine (FP) and large particles (LP) of a fixed BAG composition named 0NaMBG have been elaborated by a sol-gel process. In vitro assessment was achieved on cultured primary hDPCs using indirect contact. The effect of the concentration of 800 μg/mL on cell metabolic activity and cytotoxicity were examined by performing Alamar blue and crystal violet assays. Alizarin Red staining was used to detect and quantify the formation of mineralized nodules and ALP activity was colourimetrically quantified. The expression of Odontogenic markers: DMP-1 and osteopontin (OPN) expression and cell morphology was evaluated by confocal microscopy.
According to the Alamar blue and crystal violet assay, 0NaMBG samples were non-cytotoxic. Cells treated with 0NaMBG particles expressed higher metabolic activity than control cells, especially for LP. Both FP and LP significantly increased both extra and intra cellular ALP activity. hDPCs exhibited good cell spreading and adhesion in the presence of FP and LP extracts by confocal imaging. Further, Alizarin red S assay demonstrated more mineralization nodules and significant enhancement of the extracellular calcium deposition when cells were interfaced with both FP and LP compared to the control cells. Moreover, LP extracts enhanced the production and secretion of odontogenic markers: dentin matrix protein 1 (DMP-1) and osteopontin.
LP have a higher surface area and pore volume, which could explain their greater bioactivity in contact with pulp cells. The clinical relevance of these findings implicate that 0NaMBG could be used as fillers in dental therapeutic materials suitable for dentin and/or pulp tissues preservation.
Carious disease is still a neglected topic despite the acknowledgment of the World Health Organization (WHO) that it is still a major health problem in most industrialized countries. Report highlights that 60–90 percent of children and the vast majority of adults are affected by dental caries [ , ]. Erosion (a non-bacteria-mediated process) and carious lesions are the two main consequences of tooth demineralization resulting in the mineral structures loss of the enamel and dentin tissues [ ]. Moreover, dental erosion prevalence is increasing steadily [ , ]. The approach of restorative dentistry has significantly changed recently: surgical strategies are decreasing in favor of biological approaches based on preventive dentistry and minimal or non-invasive options [ , ]. Mineral ions from hydroxyapatite crystals (HA) present in enamel and dentin are removed due to acidic attack (erosive and/or carious lesions), resulting in the process of demineralization. Remineralization is a non-invasive treatment used to regenerate mineralized tissues after demineralization [ ]. Remineralization may occur with a dissolution / re-precipitation process in enamel or dentin although stimulation of pulp cells could also evoke tertiary dentin deposition along the walls of the pulpal chamber [ ].
Bioactive glasses were initially used for medical application due to their ability to form a bond with bone tissue [ ]. Some products containing BAG particles are already available in the market such as bone filling materials (Biogran® – Perioglas®). Besides bone regeneration, bioactive glass has found its niche in dentistry. Novamin® and BiominF® are two examples of recently developed toothpastes containing BAG (with or without Fluor) for enamel remineralization and dental hypersensitivity. Finally, BAG particles are used for air-abrasion (ProSylc (Novamin®) [ ]. Research is also focused on using bioactive glasses in air polishing – techniques to stimulate mineralization of tooth tissue through apatite formation [ ]. The first BAG was made in 1969 by professor Larry Hench (BAG 45S5). The composition was: 45% Silicon Oxide (SiO 2 ) – 24.5% Calcium Oxide (CaO) – 24.5% Sodium Oxide (Na 2 O) – 6% Phosphorus Oxide (P 2 O 5 ). The original composition (45S5) and manufacturing methodology for BAG are modified to create mesoporous BAG, to change particle size (nano or micro-scale) or to introduce additives. In a previous study, experimental BAG have been developed (0NaMBG) by our group. The typical composition of these particles is without sodium. The enhanced fluxing in compositions with high sodium affects the textural features by reducing the porosity due to fusion of pores. Therefore, the porosity increases with increasing calcium oxide as opposed to sodium oxide [ ]. The porosity is an interesting property to enhance surface area and therefore the bioactivity of particles [ ].
The use of BAG as pulp capping materials has been applied over many years [ ]. Currently biomaterials with BAG are not yet commercially available for this application. Therefore, development of in vitro studies to assess the effects of BAG on human dental pulp cells (hDPCs) is fundamental. In this context, the development of bioactive materials, as BAG, for pulp regeneration is an interesting perspective [ , ]. Human dental pulp cells may represent a different population of differentiated and undifferentiated cells with great potential for pulp tissue and dentin regeneration. They can be successfully used to evaluate new biomaterials for different dental applications [ ]. The primary pulp function is dentin formation, which begins at the moment that the peripheral mesenchymal cells differentiate into odontoblasts and start the deposition of the collagen matrix, in a mineralization process resulting in the complete tooth formation [ ].
In this study, we aimed to assess the effects of fine and large 0NaMBG particles on biocompatibility and mineralization ability of human dental pulp cells. The investigated 0NaMBG particles composition has been defined as 75SiO 2 :15CaO:0Na 2 O:10P 2 O 5 and it has been synthesized using an acid catalysed sol–gel assisted method [ ]. Therefore, we evaluated the influence of particles size and surface area for a given composition of sol-gel bioactive glasses on pulp cells behavior (cell metabolic activity, cell morphology, viability, ALP activity, and mineralization ability). Two hypotheses were suggested (1) both large and fine 0NaMBG particles would enhance pulp cells behavior; (2) a very high surface area of the investigated large particles would better improve the dental pulp cells behavior than the fine particles.
Materials and methods
Bioactive glass synthesis and characterization
0NaMBG particles with 75%SiO 2 , 15%CaO, 0%Na 2 O and 10%P 2 O 5 composition were synthesized by an acid catalysed sol-gel method assisted by an evaporation-induced self-assembly (EISA) process [ ]. Tetraethyl orthosilicate (TEOS), Triethyl phosphate (TEP) and calcium acetate monohydrate were used as precursors. The temperature of drying and calcination was 60 °C and 310 °C, respectively. The particles were characterized by X-ray fluorescence (XRF), X-ray diffractometer (XRD), Brunauer-Emmett-Teller (BET/BJH), Fourier Transform Infra-Red Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Inductive Coupled Plasma Spectrometry (ICP) [ ]. Two sizes of 0NaMBG were obtained: large (LP) and fine (FP). 90% of large particles were less than 623 μm (D90), 50% were less than 177 μm (D50) and 10% were less than 18 μm (D10) ( Table 1 ). 90% of fine particles were less than 2.97 μm (D90), 50% were is less than 0.157 μm (D50) and 10% were less than 0.003 μm (D10) ( Table 1 ). As shown in Table 1 , large particles have a higher surface area and pore volume and lower pore size than fine particles. According to the results of our previous study [ ], large particles released Ca 2+ (day 1: 50 ppm – day 3: 70 ppm – Day 7: 80 ppm) as revealed by inductive coupled plasma spectrometry after immersion in deionised water. Before cell contact, the samples were sterilized under UV for 30 min after which they were suspended in the culture media at a concentration of 800 μg/mL. Preliminary assays have been carried out to determine the most efficient concentration in the current experimental conditions (data not shown).
|Samples||SURFACE AREA (m 2 g −1 )||PORE VOLUME (cm 3 g −1 )||PORE SIZE (nm)|
Dental Pulp cells (hDPCs cells) were isolated and harvested from the pulp of sound human third molar germ (14–16 years old) extracted for orthodontic reasons. Informed consent was obtained from the patients at the University of Lyon 1 – Hospices civils of Lyon (HCL), France. The culture protocol was carried out using a modification method of Couble et al. 2000 [ ]. Whole pulps (15 specimens) were separated from the crowns and roots and removed through the developing apical end, except the apical part of the pulps to prevent periodontal fibroblast contamination. Selected specimens were minced into small explants and were grown on 35 mm diameter EASY GRIP culture dishes (Thermo Scientific France). They were cultured in Basel Medium Eagle (BME) with 10% fetal bovine serum, 1% of vitamin C, 2% penicillin/streptomycin, and 1% amphotericin B. Cultures were maintained at 37◦C under a humidified atmosphere of 5% CO 2 in air for 4–7 weeks. The medium associated with the generated cells was changed every 2 days, and cells were passaged after 5 days of culture. After reaching confluence, cells were trypsinized and resuspended in the culture medium . Cell cultures were examined routinely under an inverted microscope.
In this study, cells were placed in indirect contact with 0NaMBG. In the indirect contact the samples (concentration 800 μg/mL) are immersed in culture media for 24 h and the ion extracts are collected and contacted with cells for the evaluations [ , ].
Alamar blue – metabolic activity
Cell metabolic activity was quantified using Alamar Blue assay. Alamar blue® solution (DAL1025, Thermo Scientific France) was used in this study. The assay was carried out using a modification method of McNicholl et al. [ ]. Briefly, a 24-microwell plate was used as a ‘feeder tray’ in which 1 mL of cell suspension 10 4 cells/mL was seeded overnight, then exposed to the extract of bioactive glass particles (indirect contact, as described above) for 1, 2 and 3 days. Unexposed control cultures were maintained in the same conditions. Alamar Blue solution was added directly into wells at the final concentration of 10% (v/v) and plates were incubated at 37 °C for 4 h without shaking. The amount of Resorufin formed was determined by measuring the absorbance at 570 and 600 nm using a micro-plate reader (Infinite® M 200 PRO, NanoQuant plate, Tecan, France). For each treatment, four wells were analyzed with three independent experiments carried out. The results were expressed as percentage of viability, of the untreated control (100%).
Quantitative assay of ALP activity
The kit K412-500 was used according to the manufacturer’s instructions (BioVision Incorporated, USA). The hDPCs were seeded at a density of 10 4 cells/mL into 24-well plates and were cultured for 7 days in indirect contact with (i) FP, LP 0NaMBG at a concentration of 800 μg/mL and (ii) a control group (cells with culture media and without any particles extracts). Alkaline phosphatase (ALP) activity was measured by adding 50 μL (at the concentration of 5 mM) of ALP reaction solution (2 4-nitropheyl-phosphate disodium salt hexahydrate tablets dissolved in 5,4 mL ALP Assay Buffer) to cell lysate, incubated in the dark at 37 °C for 1 h. Afterwards, the reaction was stopped by adding 20 μL of Stop solution. Then, the absorbance of the resultant colored reaction product pNPP, within the supernatant was measured at 405 nm using a micro-plate reader (Infinite® M 200 PRO, NanoQuant plate, Tecan, France). Finally, ALP activity was calculated according to the following equation:
ALP activity = ([pNPP] (μmol)/ΔT (min) x (ml) x D (dilution factor)
ΔT = reaction time and V (initial voulme added to each well)
Crystal violet (CV) staining
Cell cytotoxicity was also measured (after 72 h of particles extracts contact) using crystal violet staining and colorimetric assay (K329-1000, Biovision, France). After 72 h in contact with 0,5% DMSO and with 3 μL of 20 mM doxorubicin for inhibitor controls, fixed cells were rinsed with wash buffer 1 X and stained with 200 μL crystal violet with 20% methanol per well for 20 min at room temperature (RT). Before visualization, the unbound stains were removed by five washes with buffer. Cells were solubilized by adding 300 μL solubilisation solution per well and leaving for 20 min at room temperature on a shaker. The absorbance was measured at 595 nm. Cell viability was calculated as percentage of cytotoxicity using the following formula:
% cytotoxicity: [(OD DMSO – OD sample)/OD DMSO] x100.
Cell morphology spreading by ConfocaL Laser Scanning Microscopy (CLSM)
Fluorescence staining was performed to observe the formation and the organization of stress fibers and morphological changes. Cells were seeded on a μ_dish glass bottom chamber (Ibidi GmbH, Germany) placed in a complete medium and incubated at 37 °C at a density of 10 4 cells/mL for 24 h. The culture medium was replaced with the corresponding extract and incubated again at 37 °C for 24 h. Cells were harvested and washed three times with PBS. Then the cells were fixed for 30 min by incubating in 3.7% formaldehyde in PBS followed by further washing. The cells were permeabilized with 1% Triton X100 in PBS and then blocked with 1% bovine serum albumin in PBS. Actin microfilaments were stained by Alexa Fluor® 488 phalloidin (green fluorescence) at a 1: 100 ratio to visualize pulp cell actin-filaments. Cell nuclei were identified using Propidium Iodide (red fluorescence) at a 1:3000 ratio at room temperature. Supercontinuum white light laser was used to excite Alexa Fluor® 488 and Propidium Iodide. Acquisitions were collected sequentially (green fluorescence/red fluorescence) to avoid potential cross-talking between the two channels. The resulting stained cells on the glass bottom chamber in 1% bovine serum albumin in PBS were examined under a Confocal Lase Scanning Microscope CLSM LEICA SP5 X (Leica, Wetzlar, Germany).
Alizarin red S staining – mineralization
Alizarin red S staining was used to assess matrix mineralization. After Treatment, HDPCs were fixed using formaldehyde (3,7 %, 30 min) and washed with deionized water. 40 mM of alizarin red staining solution (pH 4.2) was then added into the 24-well plates.The cells were incubated at room temperature for 40 min, then washed with deionized water 3 times and viewed under a microscope, with images captured. For quantitative calcium analysis (semi-quantification) of mineralized matrix nodules generated from human pulp cells, the cells were treated with 10% cetylpyridinium chloride solution (Sigma-Aldrich) for 15 min at room temperature to dissolve and release the calcium-combined Alizarin Red S into solution. The OD values were read at 560 nm, which represented the relative quantity of mineralization nodules. The experiments were repeated at least 3 times.
Odontogenic markers: OPN expression and DMP-1
The evaluation of osteopontin (OPN) and Dentin Matrix Protein 1 (DMP-1) expression was performed following cell exposure to 0NaMBG particles extracts, cells fixation and permeabilization. For these endpoints, OPN staining was performed using Rabbit Anti-Osteopontin Polyclonal Antibody, ALexa Fluor® 555 Conjugated (bs-0019R-A555, CliniSciences, France), diluted 1:200 (40 min of incubation with the cells at room temperature). DMP-1 expression was analyzed using two fluorescents stains. Monoclonal Anti-DMP, antibody (SAB140275-100 G) was used as the primary antibody at the dilution ratio 1: 50 (2 h at 37 °C). This was followed by a second staining using Cyanine 3 (AS008, CliniSciences, France) at 1:100 (1 h at 37 °C). Subsequently, cell nuclei were identified using Dapi (4′,6-diamidino-2-phenylindole) at a 1:3000 ratio at room temperature. The stained cells were observed using the microscope CLSM LEICA SP5 X (Leica, Wetzlar, Germany). Tow laser sources were used to excite the indicated stains: a supercontinuum white light laser and a laser Diode-Pumped Solid-State (405 nm).
Data were analyzed using statistical software SPSS™ (V21.0, IBM, IL, USA) and fond to be normally distributed. Non-parametric analysis and multiple comparison were achieved using One-way Analysis of Variance (ANOVA) with a repetition test followed by Post Hoc tests. A comparison was made between the two tested particle groups (fine vs large particles) and exposed cells were compared to control group cells (fine or large particles vs controls cells). Results were reported as mean standard deviation (±SD) and statistical significance was accepted at p < 0.05.
Cell metabolic activity by Alamar blue assay
Cells were treated with 0NaMBG for 24, 48 and 72 h. The results of cell metabolic activity comparing the fine and the large particles are shown in Fig. 1 . At 24 h, the metabolic activity of cells treated with samples were similar to the control cells. No cytotoxic effects were observed for both conditions. Interestingly at day 3, cells treated with particles expressed higher metabolic activity (p = 0.03) than the control cells with a significant enhancement for the large particles after 72 h of incubation (p = 0.02).