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Antibacterial and Odontogenesis Efficacy of Mineral Trioxide Aggregate Combined with CO2 Laser Treatment
Basic Research—Biology
Antibacterial and Odontogenesis Efficacy of Mineral Trioxide Aggregate Combined with CO2 Laser Treatment Tuan-Ti Hsu, MS,* Chia-Hung Yeh, PhD,† Chia-Tze Kao, DDS, PhD,‡§ Yi-Wen Chen, PhD,† Tsui-Hsien Huang, DDS, PhD,‡§ Jaw-Ji Yang, PhD,* and Ming-You Shie, PhD† Abstract Introduction: Mineral trioxide aggregate (MTA) has been successfully used in clinical applications in endodontics. Studies show that the antibacterial effects of CO2 laser irradiation are highly efficient when bacteria are embedded in biofilm because of a photothermal mechanism. The aim of this study was to confirm the effects of CO2 laser irradiation on MTA with regard to both material characterization and cell viability. Methods: MTA was irradiated with a dental CO2 laser using directly mounted fiber optics in the wound healing mode with a spot area of 0.25 cm2 and then stored in an incubator at 100% relative humidity and 37 C for 1 day to set. The human dental pulp cells cultured on MTA were analyzed along with their proliferation and odontogenic differentiation behaviors. Results: The results indicate that the setting time of MTA after irradiation by the CO2 laser was significantly reduced to 118 minutes rather than the usual 143 minutes. The maximum diametral tensile strength and x-ray diffraction patterns were similar to those obtained without CO2 laser irradiation. However, the CO2 laser irradiation increased the amount of Ca and Si ions released from the MTA and regulated cell behavior. CO2 laser–irradiated MTA promoted odontogenic differentiation of hDPCs, with the increased formation of mineralized nodules on the substrate’s surface. It also up-regulated the protein expression of multiple markers of odontogenic and the expression of dentin sialophosphoprotein protein. Conclusions: The current study provides new and important data about the effects of CO2 laser irradiation on MTA with regard to the decreased setting time and increased ion release. Taking cell functions into account, the Si concentration released from MTA with laser irradiation may be lower than a critical value, and this information could lead to the development of new regenerative therapies for dentin and periodontal tissue. (J Endod 2015;-:1–8)
Key Words Antibacterial, CO2 laser, human dental pulp cell, mineral trioxide aggregate, odontogenic
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eriradicular surgery may be performed in the presence of infection or when orthograde endodontic treatment is contraindicated or considered unfeasible (1). Despite the high antimicrobial efficacy of traditional disinfectants in vitro, clinical research has shown bacterial persistence within the root canal after cleaning and shaping procedures (2–4). Therefore, it is very important to investigate advanced endodontic disinfection strategies that are effective in eliminating biofilm bacteria in the root canals. Several root filling materials play an essential role in the control of reinfection by entombing residual organisms that may remain after instrumentation, irrigation, and intracanal medication. The ideal root-end filling material should have antibacterial activity to prohibit bacterial growth, in addition to possessing good cellular properties to enhance tissue formation (5), even though the antibacterial ability of these materials is well-known (6). Mineral trioxide aggregate (MTA) has been successfully used in clinical applications in endodontics (7). Not only does MTA have good biocompatibility (8), but it also has been verified to promote hard tissue formation (9, 10). Calcium silicate–based materials have been used in dentin replacement restorative materials in dentistry (11–13). We recently showed that calcium silicate–based materials stimulate the proliferation and differentiation of primary human dental pulp cells (hDPCs) (14– 16) and human periodontal ligament cells (17) in vitro. The inorganic ions released from calcium silicate–based materials have been found to affect cell behavior and osteogenic and angiogenic marker protein secretion (15–19). In addition, although many studies have been published on the antibacterial properties of MTA, there remains some controversy regarding the underlying mechanisms (5). Although MTA releases Ca ions and leads to an alkaline microenvironment with a higher pH value, which may suppress bacterial growth (5), it has also been shown that the calcium silicate particles themselves may permeate the bacteria (20) with possible antibacterial consequences (21). Laser light can be applied to stimulate bacteria capable of bioremediation (22), and lasers have been successfully used in many fields, including medicine, industry, and the military. With regard to clinical use, there are several commercially available lasers, including CO2, diode, and erbium:yttrium aluminum garnet lasers (17). The advantage of laser light over conventional treatments with chemical agents is its ability to partially travel through the biofilm (23). Research shows that CO2 laser irradiation has highly efficient antibacterial effects when bacteria are embedded in biofilm because of a photothermal mechanism (24). However, another study reported that laser light can increase fibroblast proliferation and collagen synthesis and reduce inflammation
From the *Institute of Oral Science, ‡School of Dentistry, and §Department of Stomatology, Chung Shan Medical University Hospital, Taichung City, Taiwan; and 3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan. Address requests for reprints to Dr Ming-You Shie, 3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan or Prof Jaw-Ji Yang, Institute of Oral Science, Chung Shan Medical University Hospital, Taichung City, Taiwan. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.02.031 †
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Basic Research—Biology (25, 26). In addition, laser light can promote periodontal cell differentiation and, thus, has the potential to enhance periodontal tissue regeneration (17). The aim of this study was to assess the effects of CO2 laser irradiation on MTA with regard to material characterization and cell viability. We hypothesized that CO2 laser irradiation destroyed the surface structure of MTA and increased the release of ions. After CO2 laser irradiation, the antibacterial ability of MTA was investigated and compared with that of calcium hydroxide. This study also investigated the odontogenic protein expression for regenerative endodontics.
Materials and Methods Preparation of MTA Specimens The MTA used in this study was prepared according to the manufacturer’s instructions. MTA powder (0.2 g) was mixed with distilled H2O in a liquid/powder ratio of 0.3 mL/g to make the cement. The resulting cement was then used to fully cover each well of a 96-well plate (GeneDireX, Las Vegas, NV) to a thickness of 2 mm. The specimens were then irradiated with a dental CO2 laser with an output of 10,600 nm (Opelaser Pro; Yoshida Co Ltd, Tokyo, Japan) using directly mounted fiber optics in the wound healing mode with a spot area of 0.25 cm2 and stored in an incubator at 100% relative humidity and 37 C for 1 day to set. Before cell experiments, all specimens were sterilized by immersion in 75% ethanol followed by exposure to ultraviolet light for 1 hour. Setting Time, pH Variation, and Strength After the powder was mixed with distilled H2O, the cements were placed into a cylindric mold and treated with the CO2 laser before being stored in an incubator at 37 C and 100% relative humidity for hydration. The setting time of the cements was tested according to standards set by ISO 9917-1. To evaluate the setting time, each material was analyzed using Gilmore needles (456.5 g), and setting was completed when the needle failed to create a 1-mm-deep indentation in 3 separate areas. The variation in the pH value of the MTA during the setting process was measured with a pH meter (IQ120 miniLab pH meter; IQ Scientific Instruments, San Diego, CA). The diametral tensile strength (DTS) testing was conducted on an EZ Test machine (Shimadzu, Kyoto, Japan) at a loading rate of 1 mm/min. The maximal compression load at failure was obtained from the recorded load-deflection curves. At least 10 specimens from each group were tested. Phase Composition and Morphology The crystalline phases of MTA treated with the CO2 laser were examined using x-ray diffraction (XRD; Shimadzu XD-D1, Kyoto, Japan) over the 2-theta range from 20 –50 , with a scanning speed of 1 /min. The morphologies of the MTA specimens before and after immersion in simulated body fluid (SBF) solution were examined under a scanning electron microscope (JSM-6700F, JEOL, Tokyo, Japan) operated in the lower secondary electron image mode at 3-kV accelerating voltage.
In Vitro Soaking It is believed that the prerequisite for materials to bond to natural bone is the formation of a ‘‘bonelike’’ apatite layer, an indicator of bioactivity (the ability to form a chemical bond with living tissue) (21). To evaluate the in vitro bioactivity, the cements were immersed in a 10-mL SBF solution at 37 C. The SBF solution, of which the ionic composition is similar to that of human blood plasma, consisted of 7.9949 g NaCl, 0.3528 g NaHCO3, 0.2235 g KCl, 0.147 g K2HPO4, 0.305 g MgCl2 6H2O, 0.2775 g CaCl2, and 0.071 g Na2SO4 in 2
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1000 mL distilled H2O and was buffered to a pH of 7.4 with hydrochloric acid and trishydroxymethyl aminomethane ([CH2OH]3CNH2) (7, 11, 13, 21, 27). All chemicals used were of reagent grade. The solution in the shaker water bath exhibited no change under static conditions. After soaking for 1 day, the specimens were removed from the tube, and their surface microstructures were examined.
Antibacterial Properties To investigate the antibacterial effects of the MTA irradiated with CO2 laser treatment, Staphylococcus aureus in Luria Broth (LB) culture media (4.0 104 bacteria per mL) was cultured with MTA materials for 24 hours. Aliquots of 0.1 mL from each group were then mixed with 0.9 mL PrestoBlue (Invitrogen, Grand Island, NY) for 10 minutes; after which, the solution in each well was transferred to a new 96-well plate. Plates were then read in a multiwell spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. Cells cultured on the tissue culture plate without the cement were used as the control (Ctl). The results were obtained in triplicate from 3 separate experiments in terms of optical density. The agar diffusion test is considered the standard method to test for antibiotic susceptibility (8, 28, 29); thus, to further confirm the inhibitory effects of MTA treated with the CO2 laser on the growth of bacterial strains, we performed an agar diffusion assay. Four sterile 6-mm disk-shaped samples from each of the 4 experimental groups were placed on cultured agar plates containing bacterial lawns of S. aureus. The inhibition zones (in mm) were determined after 1 day of incubation. Ion Concentration The Ca and Si ion concentrations on Dulbecco modified Eagle medium (DMEM) were analyzed using an inductively coupled plasma atomic emission spectrometer (OPT 1 MA 3000DV; Perkin-Elmer, Shelton, CT) after culturing for different time points. Six samples were immersed in 10 mL DMEM and measured for each data point. The results were obtained in triplicate from 3 separate samples for each test. hDPC Isolation and Culture hDPCs were freshly derived from caries-free, intact premolars that were extracted for orthodontic treatment purposes as described previously (13, 14). The patient gave informed consent, and approval from the Ethics Committee of the Chung Shan Medicine University Hospital was obtained (CSMUH No. CS14117). The tooth was split sagittally with a chisel. The pulp tissue was then immersed in phosphatebuffered saline (PBS; Caisson, North Logan, UT) solution and digested in 0.1% collagenase type I (Sigma-Aldrich, St Louis, MO) for 30 minutes. After being transferred to a new plate, the cell suspension was cultured in DMEM (Caisson) supplemented with 20% fetal bovine serum (GeneDireX) and 1% penicillin (10,000 U/mL)/streptomycin (10,000 mg/mL) (Caisson) in a humidified atmosphere with 5% CO2 at 37 C, and the medium was changed every 3 days. The cells were subcultured through successive passaging at a 1:3 ratio until they were used for experiments (passages 3–7). The odontogenic differentiation medium was DMEM supplemented with 10 8 mol/L dexamethasone (Sigma-Aldrich), 0.05 g/L L-Ascorbic acid (Sigma-Aldrich), and 2.16 g/L glycerol 2-phosphate disodium salt hydrate (Sigma-Aldrich). Cell Viability Before the in vitro cell experiments, MTA specimens were sterilized by soaking in 75% ethanol followed by exposure to ultraviolet light for 1 hour. After direct culture for various time periods, cell viability was evaluated using the PrestoBlue assay, which is based on the detection of mitochondrial activity. Thirty microliters of PrestoBlue solution and JOE — Volume -, Number -, - 2015
Basic Research—Biology 300 mL DMEM were added to each well followed by 30 minutes of incubation. After incubation, 100 mL of the solution in each well was transferred to a 96-well enzyme-linked immunosorbent assay plate. The plates were read in a Sunrise microtiter plate reader (Hitachi) at 570 nm with a reference wavelength of 600 nm. The results were obtained in triplicate from 3 separate experiments in terms of optical density. Cells cultured on the tissue culture plate without the cement were used as the Ctl.
Fluorescent Staining We observed the cell morphology of hDPCs after culturing MTA treatment with CO2 laser using F-actin cytoskeleton stains. After incubation for 1 day, the cells were washed with PBS, fixed in 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 20 minutes, and then permeabilized with PBS containing 0.1% Triton X-100 (Sigma-Aldrich). The F-actin filaments were stained with phalloidin conjugated to Alexa Fluor 594 (Invitrogen) for 1 hour. The nuclei were stained with 300 nmol/L 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen) for 30 minutes. After washing, morphology was obtained using a Zeiss Axioskop2 microscope (Carl Zeiss, Thornwood, NY). Western Blot Western blot analysis was performed using cell lysates and a culture medium prepared using hDPCs that had been cultured for 7 days. Cells were lysed in NP-40 lysis buffer (Invitrogen) at 4 C for 30 minutes, and the lysates were centrifuged at 13,000g.
The cell lysates (40 mg protein) were separated using sodium dodecyl sulfate-polyacrylamide (SDS)-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membrane was blocked in 5% bovine serum albumin (Gibco) for 1 hour; immunoblotted with the primary antibodies anti– DMP-1 (Santa Cruz Biotechnology, Santa Cruz, CA) and b-actin (GeneTex, San Antonio, TX) for 2 hours; and then washed 3 times in a trishydroxymethyl aminomethane–buffer saline containing 0.05% Tween-20 (Sigma-Aldrich). A horseradish peroxidase–conjugated secondary antibody was subsequently added, and the proteins were visualized using enhanced chemiluminescent detection kits (Invitrogen). The stained bands were scanned and quantified using a densitometer (Syngene Bioimaging System; Syngene, Frederick, MD) and ImageJ software (National Institutes of Health, Bethesda, MD). Protein expression levels were normalized to the b-actin band for each sample. The results were obtained in triplicate from 3 separate samples for each test.
Osteogenesis Assay The level of alkaline phosphatase (ALP) activity was determined after cell seeding for 7 days. The process was as follows: the cells were lysed from discs using 0.2% NP-40 and centrifuged for 10 minutes at 2000 rpm after washing with PBS. ALP activity was determined using p-nitrophenyl phosphate (Sigma-Aldrich) as the substrate. Each sample was mixed with p-nitrophenyl phosphate in 1 mol/L diethanolamine buffer for 15 minutes; after which, the reaction was stopped by the
Figure 1. The effects of CO2 laser treatment on (A) setting time and (B) DTS of MTA cements. Statistical data are presented as means standard deviations for n = 6. *Statistically significant differences (P < .05) from those obtained from MTA without laser irradiation. (C) XRD patterns of MTA with and without CO2 laser irradiation after hydration at 37 C for 1 day. (D) Scanning electron microscopic micrographs of MTA surfaces before and after immersion in SBF for 1 day.
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Basic Research—Biology addition of 5 N NaOH and quantified by absorbance at 405 nm. All experiments were performed in triplicate. The osteocalcin (OC) proteins released from pulp cells were cultured on different substrates for 14 days after cell seeding. An osteocalcin enzyme-linked immunosorbent assay kit (Invitrogen) was used to determine OC protein content following the manufacturer’s instructions. The OC protein concentration was measured by correlation with a standard curve. The analyzed blank plates were treated as controls. All experiments were performed in triplicate.
Alizarin Red S Stain Accumulated calcium deposition was observed for 7 and 14 days using alizarin red S staining as described in a previous study (30). To summarize briefly, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 minutes and then incubated in 0.5% alizarin red S (Sigma-Aldrich) at a pH of 4.0 for 15 minutes at room temperature in an orbital shaker (25 rpm). After the cells were washed with PBS, photographs were taken using an optical microscope (BH2-UMA; Olympus, Tokyo, Japan) equipped with a digital camera (Nikon, Tokyo, Japan) at 200 magnification. To quantify the stained calcified nodules after staining, samples were immersed in 1.5 mL 5% SDS in 0.5 N hydrochloric acid for 30 minutes at room temperature; after which, the tubes were centrifuged at 5000 rpm for 10 minutes, and the supernatant was transferred to the new 96-well plate (GeneDireX). At this time, absorbance was measured at 405 nm (Hitachi).
Statistical Analysis One-way analysis of variance was used to evaluate the significance of the differences between the groups in each experiment. A Scheffe multiple comparison test was used to determine the significance of the deviations in the data for each specimen. In all cases, the results were considered statistically significant with P values <.05.
Results Physicochemical Properties Setting time is one of the most clinically relevant factors in experiments of this nature. MTA set within 118 9 minutes and 143 7 minutes when treated with or without CO2 laser irradiation, respectively (Fig. 1A). Figure 1B shows the DTS values for the cement specimens. The analysis of variance results for the DTS data show that the variations in strength between specimens are not significant (P > .05). MTA had strength values of 4.1 0.2 and 3.9 0.3 MPa when treated with or without CO2 laser irradiation, respectively. In addition, there was no significant difference (P > .05) in the pH value of MTA without or with CO2 laser irradiation at the same time periods. The pH value of MTA at fresh mixing was 10.1. At the 24-hour setting, it reached a value of 12.1. Figure 1C shows the XRD patterns of the MTA cement after hydration for 1 day. Analysis of the specimens containing calcium silicate revealed 2 key points: first, an obvious diffraction peak near 2q = 29.4 , which corresponds to the calcium silicate hydrate gel, and, second,
Figure 2. (A) The growth inhibition zones and (B) the antibacterial effects of MTA with and without CO2 laser irradiation after culture in E. faecalis. (C) Ca and (D) Si ion concentrations of DMEM after immersion for different times. Data represent means standard deviation (n = 6). *Statistically significant difference (P < .05) from Ca(OH)2. @Statistically significant difference (P < .05) from MTA without laser irradiation.
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Basic Research—Biology incompletely reacted inorganic component phases of the b-dicalcium silicate (b-Ca2SiO4) at 2q between 32 and 34 . The results show lower peak intensities of the calcium silicate hydrate gel and b-Ca2SiO4 phases, with the strongest peak at 2q = 27.4 being ascribed to Bi2O3. The scanning electron microscopic micrographs of the MTA treated with or without CO2 laser irradiation before and after immersion in SBF are shown in Figure 1D. It can be noted that the MTA specimens had a porous structure. After immersion in SBF for 1 day, the MTA specimens induced the precipitation of apatite spherule aggregates, which appeared on the cement surface in the case of all groups.
Antibacterial Properties Figure 2A shows that laser treatment had no effect on the growth inhibition zone of Ca(OH)2 with regard to S. aureus. Ca(OH)2 had greater growth inhibition zones with sizes of more than 4 mm. The results of the zone of inhibition test clearly show that the antibacterial behavior of MTA is rather weak compared with that of Ca(OH)2 (P < .05). Interestingly, the inhibition zones of MTA after treatment with CO2 laser irradiation were more numerous than those seen with the specimens without laser treatment. In Figure 2B, the S. aureus cultures with the Ctl show the highest absorbance. No significant difference was detected in the absorbance readings for Ca(OH)2 groups with and without laser treatment (P > .05). Similarly, the absorbance by S. aureus cultured on CO2 laser–treated MTA is significantly lower (P < .05) than on untreated MTA.
Ion Concentrations The variations of the DMEM’s Ca and Si ion concentrations, as measured at different times, are shown in Figure 2C and D. In the MTA cement, the Ca ion concentration of the medium increased to approximately 1.68 mmol/L after being immersed for 3 hours and then went to levels higher than the baseline Ca concentration of DMEM (1.45 mmol/L) (P < .05). There were significant differences in the Ca ion concentration levels found between MTA CO2 laser treatment and MTA at all of the times they were measured (Fig. 2C). The Si concentration within all groups rose in proportion with the increased incubation time (Fig. 2D). In the MTA specimens, the Si ion concentration was approximately 0.22, 0.61, and 1.03 mmol/L at 3, 12, and 24 hours, respectively. However, the CO2 laser–treated MTA released more Si ions than the untreated MTA at all time points (P < .05). Cell Viability The cell viability of hDPCs grown on MTA and the Ctl are shown in Figure 3A. PrestoBlue showed that the cells treated with the CO2 laser had a significantly higher viability rate than those on the Ctl on days 1 and 7 (P < .05). CO2 laser–treated MTA elicited significant (P < .05) increases of 55% and 60% in optical density compared with MTA on days 1 and 7 of cell seeding, respectively. The fluorescence staining showed that on day 1 the hDPCs cultured on MTA with and without CO2 laser treatment showed strong F-actin stress fiber morphologies of the cells (Fig. 3B).
Figure 3. (A) Cell proliferation assay for hDPCs cultured on MTA on days 1 and 7. *Statistically significant difference (P < .05) from the Ctl. @Statistically significant difference (P < .05) from MTA without laser irradiation. (B) Immunofluorescence images of nuclei (blue) and F-actin (red) in the hDPCs were cultured on MTA on day 1.
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Basic Research—Biology Odontogenic Differentiation in hDPCs MTA has been shown to induce odontogenic differentiation in various cell types. To investigate the potential of hDPCs for odontoblastlike differentiation after being cultured on MTA with CO2 laser treatment, the hDPCs were cultured for up to 3 days to evaluate the protein expression levels of differentiation markers (anti-dentin matrix protein-1, anti-dentin sialophosphoprotein [DSPP] and DMP-1) by Western blot analysis (Fig. 4A). CO2 laser–treated MTA markedly upregulated DSPP and DMP-1, which increased by 24% and 26% compared with MTA, respectively, and a significance of P < .05. The ALP expression of hDPCs was also examined. Figure 4B shows the results of the analysis of the quantitative examination data and the ALP activity of cells cultured on the different substrates for 7 days. Significant 47% and 83% increases (P < .05) in the ALP levels were
found for the untreated MTA and MTA with CO2 laser irradiation in comparison with the Ctl cement after 7 days. The results for OC secretion in the cells were similar to those for ALP activity, as shown in Figure 4C. In addition, to investigate the effects of different substrates on mineralization in hDPCs, we examined mineralized nodule formation in these cells using alizarin red S staining (Fig. 4D) because this can reveal details of bone nodule formation and calcium deposition. The results showed that CO2 laser–treated MTA clearly increased the area of calcified nodules by up to 1.4-fold compared with MTA after 14 days.
Discussion This study evaluated the influence of MTA treated with CO2 laser on the resulting antibacterial effects and cell behavior. Pulpal and periapical diseases are biofilm-mediated infections, and the elimination of
Figure 4. (A) Immunodetection of DSPP and DMP-1 protein expression in hDPCs cultured on MTA for 3 days. (B) ALP activity and (C) OC amount of hDPCs cultured on MTA for different times. (D) Alizarin red S staining and quantification of calcium mineral deposits by hDPCs cultured on MTA for 1 and 2 weeks. The scale bar was 100 mm. *Statistically significant difference (P < .05) from the Ctl. @Statistically significant difference (P < .05) from MTA without laser irradiation.
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Basic Research—Biology bacteria from the root canal system is the major goal of root canal treatment (5). Pathological changes in the dental pulp tissue mean that it is susceptible to invasion by many bacteria and other antigens. Although there are more than 300 bacterial species in the normal human oral flora, a relatively small group is present in infected root canals, and, thus, bacterial infection after root canal treatment remains a significant complication. The invasion of bacteria and microorganisms into the pulp is able to induce pulpal inflammation and lead to pulp death. The endodontic materials, such as MTA and calcium silicate–based materials, are known to possess an inherent antibacterial property that is effective against S. aureus (21, 30). Lasers have been a part of oral surgical practice since the mid-1960s, and they are becoming increasingly popular because of their various advantages, such as hemostasis, decreased scarring, and less postoperative pain (31). In addition, CO2 lasers lead to cell signal transduction and outcomes through similar mechanoreceptors in cells and tissues, and, therefore, it is suggested that common signaling mechanisms are involved in laser transduction pathways (32). CO2 laser irradiation of tooth enamel has been suggested as a preventive treatment for caries because it can change the composition of enamel and the enamel in the oral environment is naturally covered by biofilms (22). The scanning electron microscopic images obtained in this work from CO2 laser–treated MTA after immersion in SBF suggest that the apatite precipitated on the surface. In a previous study, the CO2 laser can be applied to melt glass and thus create a bioactive glass coating on a titanium substrate (9); this then creates a uniform surface and promotes in vitro bioactivity. In addition, the study showed that the changes in chemical composition introduced by the CO2 laser treatment of the bioactive glass were minor and had an influence on the ions released from the glass (9). The increase of Ca ions released from substrate can enhance the formation of calcium silicate hydrates and decrease the setting time (33). In addition, regarding the variations in the pH value during setting, this initial increase in the pH value was a result of the formation of Ca(OH)2 during the setting reaction, which was released to an aqueous environment. Calcium silicate–based materials have been found to encourage cell adhesion, growth, and differentiation and have been used as implant materials for tissue repair and formation (11, 13). This study was designed to examine the use of CO2 laser–treated MTA, a component of some new capping agents used to preserve the viability of dental pulp tissue during reparative dentin formation. In this study, PrestoBlue analysis showed a number of differences between hDPCs cultured on MTA with and without CO2 laser treatment. First, the absorbance values were higher for hDPCs cultured on MTA in comparison with the Ctl, and these differences were significant (P < .05) for all time points. Previous studies in our laboratory have shown that the viabilities of fibroblast and osteoblastlike cells on untreated MTA were higher than the Ctl at all culture times (28). Shie et al (34) found that soluble factors from calcium silicate substrates may be more important for proliferation and osteogenesis differentiation in a growth medium. During immersion in medium, the Si ions were hydrolyzed from the surfaces of Si-based materials (15–18, 35, 36) and affected cell behaviors (5, 14, 30). The currently used MTA has the ability to control the release rate of soluble Ca and Si ions, which can promote cell attachment and proliferation (5, 17, 37). In addition, MTA can release more Ca and Si ions after laser irradiation, leading to a weak alkaline microenvironment with a higher pH value, which may restrain bacterial growth (5). The other is that the calcium silicate–based materials themselves may permeate the bacteria (20) with possible antibacterial consequences. Examinations of the bioactivity of calcium silicate substrates show that the presence of PO43 ions in the composition is not an essential requirement for the development of an apatite layer, which consumes calcium and JOE — Volume -, Number -, - 2015
phosphate ions (11, 37). In addition, the ions released from MTA may change the osmolality of a culture medium (14). In summary, the current study provides new and important data regarding the mechanisms by which MTA irradiated by CO2 laser is able to increase ion release and decrease the setting time. In addition, these results suggest that CO2 laser treatment may inhibit bacterial activity and stimulate odontogenic differentiation in hDPCs on MTA. Taking cell functions into account, the Si concentration released from MTA with laser irradiation may be lower than a critical value, and this information may help in developing new regenerative therapies for dentin and periodontal tissue.
Acknowledgments Tuan-Ti Hsu and Chia-Hung Yeh contributed equally to this study. Supported by a grant from the Ministry of Science and Technology (grants MOST 102-2314-B-040-007-MY3 and MOST 1042314-B-039-004) of Taiwan. The authors deny any conflicts of interest related to this study.
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Basic Research—Biology 20. Wu C, Chang J, Fan W. Bioactive mesoporous calcium–silicate nanoparticles with excellent mineralization ability, osteostimulation, drug-delivery and antibacterial properties for filling apex roots of teeth. J Mater Chem 2012;22:16801–9. 21. Kao CT, Huang TH, Chen YJ, et al. Using calcium silicate to regulate the physicochemical and biological properties when using b-tricalcium phosphate as bone cement. Mater Sci Eng C Mater Biol Appl 2014;43:126–34. 22. Tosun E, Tasar F, Strauss R, et al. Comparative evaluation of antimicrobial effects of Er:YAG, diode, and CO2 lasers on titanium discs: an experimental study. J Oral Maxillofac Surg 2012;70:1064–9. 23. Cohen J, Featherstone JD, Le CQ, et al. Effects of CO2 laser irradiation on tooth enamel coated with biofilm. Lasers Surg Med 2014;46:216–23. 24. Dederich DN, Pickard MA, Vaughn AS, et al. Comparative bactericidal exposures for selected oral bacteria using carbon dioxide laser radiation. Lasers Surg Med 1990; 10:591–4. 25. Huang TH, Lu YC, Kao CT. Low-level diode laser therapy reduces lipopolysaccharide (LPS)-induced bone cell inflammation. Lasers Med Sci 2012;27:621–7. 26. Kuo CL, Kao CT, Fang HY, et al. Antiosteoclastogenesis activity of a CO2 laser antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine macrophages. Laser Phys Lett 2015;12:035601. 27. Su CC, Kao CT, Hung CJ, et al. Regulation of physicochemical properties, osteogenesis activity, and fibroblast growth factor-2 release ability of b-tricalcium phosphate for bone cement by calcium silicate. Mater Sci Eng C Mater Biol Appl 2014;37: 156–63. 28. Ding SJ, Kao CT, Shie MY, et al. The physical and cytological properties of white MTA mixed with Na2HPO4 as an accelerant. J Endod 2008;34:748–51.
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29. Bottino MC, Kamocki K, Yassen GH, et al. Bioactive nanofibrous scaffolds for regenerative endodontics. J Dent Res 2013;92:963–9. 30. Su YF, Lin CC, Huang TH, et al. Osteogenesis and angiogenesis properties of dental pulp cell on novel injectable tricalcium phosphate cement by silica doped. Mater Sci Eng C Mater Biol Appl 2014;42:672–80. 31. Huang TH, Chen CC, Liu SL, et al. A low-level diode laser therapy reduces the lipopolysaccharide (LPS)-induced periodontal ligament cell inflammation. Laser Phys Lett 2014;11:075602. 32. Hsieh WH, Chen YJ, Hung CJ, et al. Osteogenesis differentiation of human periodontal ligament cells by CO2 laser-treatment stimulating macrophages via BMP2 signalling pathway. Laser Phys 2014;24:115607. 33. Huang TH, Shie MY, Kao CT, Ding SJ. The effect of setting accelerator on properties of mineral trioxide aggregate. J Endod 2008;34:590–3. 34. Shie MY, Ding SJ, Chang HC. The role of silicon in osteoblast-like cell proliferation and apoptosis. Acta Biomater 2011;7:2604–14. 35. Hung CJ, Kao CT, Chen YJ, et al. Antiosteoclastogenic activity of silicate-based materials antagonizing receptor activator for nuclear factor kappaB ligand– induced osteoclast differentiation of murine marcophages. J Endod 2013;39: 1557–61. 36. Hung CJ, Hsu HI, Lin CC, et al. The role of integrin av in proliferation and differentiation of human dental pulp cell response to calcium silicate cement. J Endod 2014; 40:1802–9. 37. Liu CH, Huang TH, Hung CJ, et al. The synergistic effects of fibroblast growth factor-2 and mineral trioxide aggregate on an osteogenic accelerator in vitro. Int Endod J 2014;47:843–53.
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Antibacterial and Odontogenesis Efficacy of Mineral Trioxide Aggregate Combined with CO2 Laser Treatment Tuan-Ti Hsu, MS,* Chia-Hung Yeh, PhD,† Chia-Tze Kao, DDS, PhD,‡§ Yi-Wen Chen, PhD,† Tsui-Hsien Huang, DDS, PhD,‡§ Jaw-Ji Yang, PhD,* and Ming-You Shie, PhD† Abstract Introduction: Mineral trioxide aggregate (MTA) has been successfully used in clinical applications in endodontics. Studies show that the antibacterial effects of CO2 laser irradiation are highly efficient when bacteria are embedded in biofilm because of a photothermal mechanism. The aim of this study was to confirm the effects of CO2 laser irradiation on MTA with regard to both material characterization and cell viability. Methods: MTA was irradiated with a dental CO2 laser using directly mounted fiber optics in the wound healing mode with a spot area of 0.25 cm2 and then stored in an incubator at 100% relative humidity and 37 C for 1 day to set. The human dental pulp cells cultured on MTA were analyzed along with their proliferation and odontogenic differentiation behaviors. Results: The results indicate that the setting time of MTA after irradiation by the CO2 laser was significantly reduced to 118 minutes rather than the usual 143 minutes. The maximum diametral tensile strength and x-ray diffraction patterns were similar to those obtained without CO2 laser irradiation. However, the CO2 laser irradiation increased the amount of Ca and Si ions released from the MTA and regulated cell behavior. CO2 laser–irradiated MTA promoted odontogenic differentiation of hDPCs, with the increased formation of mineralized nodules on the substrate’s surface. It also up-regulated the protein expression of multiple markers of odontogenic and the expression of dentin sialophosphoprotein protein. Conclusions: The current study provides new and important data about the effects of CO2 laser irradiation on MTA with regard to the decreased setting time and increased ion release. Taking cell functions into account, the Si concentration released from MTA with laser irradiation may be lower than a critical value, and this information could lead to the development of new regenerative therapies for dentin and periodontal tissue. (J Endod 2015;-:1–8)
Key Words Antibacterial, CO2 laser, human dental pulp cell, mineral trioxide aggregate, odontogenic
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eriradicular surgery may be performed in the presence of infection or when orthograde endodontic treatment is contraindicated or considered unfeasible (1). Despite the high antimicrobial efficacy of traditional disinfectants in vitro, clinical research has shown bacterial persistence within the root canal after cleaning and shaping procedures (2–4). Therefore, it is very important to investigate advanced endodontic disinfection strategies that are effective in eliminating biofilm bacteria in the root canals. Several root filling materials play an essential role in the control of reinfection by entombing residual organisms that may remain after instrumentation, irrigation, and intracanal medication. The ideal root-end filling material should have antibacterial activity to prohibit bacterial growth, in addition to possessing good cellular properties to enhance tissue formation (5), even though the antibacterial ability of these materials is well-known (6). Mineral trioxide aggregate (MTA) has been successfully used in clinical applications in endodontics (7). Not only does MTA have good biocompatibility (8), but it also has been verified to promote hard tissue formation (9, 10). Calcium silicate–based materials have been used in dentin replacement restorative materials in dentistry (11–13). We recently showed that calcium silicate–based materials stimulate the proliferation and differentiation of primary human dental pulp cells (hDPCs) (14– 16) and human periodontal ligament cells (17) in vitro. The inorganic ions released from calcium silicate–based materials have been found to affect cell behavior and osteogenic and angiogenic marker protein secretion (15–19). In addition, although many studies have been published on the antibacterial properties of MTA, there remains some controversy regarding the underlying mechanisms (5). Although MTA releases Ca ions and leads to an alkaline microenvironment with a higher pH value, which may suppress bacterial growth (5), it has also been shown that the calcium silicate particles themselves may permeate the bacteria (20) with possible antibacterial consequences (21). Laser light can be applied to stimulate bacteria capable of bioremediation (22), and lasers have been successfully used in many fields, including medicine, industry, and the military. With regard to clinical use, there are several commercially available lasers, including CO2, diode, and erbium:yttrium aluminum garnet lasers (17). The advantage of laser light over conventional treatments with chemical agents is its ability to partially travel through the biofilm (23). Research shows that CO2 laser irradiation has highly efficient antibacterial effects when bacteria are embedded in biofilm because of a photothermal mechanism (24). However, another study reported that laser light can increase fibroblast proliferation and collagen synthesis and reduce inflammation
From the *Institute of Oral Science, ‡School of Dentistry, and §Department of Stomatology, Chung Shan Medical University Hospital, Taichung City, Taiwan; and 3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan. Address requests for reprints to Dr Ming-You Shie, 3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan or Prof Jaw-Ji Yang, Institute of Oral Science, Chung Shan Medical University Hospital, Taichung City, Taiwan. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.02.031 †
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Basic Research—Biology (25, 26). In addition, laser light can promote periodontal cell differentiation and, thus, has the potential to enhance periodontal tissue regeneration (17). The aim of this study was to assess the effects of CO2 laser irradiation on MTA with regard to material characterization and cell viability. We hypothesized that CO2 laser irradiation destroyed the surface structure of MTA and increased the release of ions. After CO2 laser irradiation, the antibacterial ability of MTA was investigated and compared with that of calcium hydroxide. This study also investigated the odontogenic protein expression for regenerative endodontics.
Materials and Methods Preparation of MTA Specimens The MTA used in this study was prepared according to the manufacturer’s instructions. MTA powder (0.2 g) was mixed with distilled H2O in a liquid/powder ratio of 0.3 mL/g to make the cement. The resulting cement was then used to fully cover each well of a 96-well plate (GeneDireX, Las Vegas, NV) to a thickness of 2 mm. The specimens were then irradiated with a dental CO2 laser with an output of 10,600 nm (Opelaser Pro; Yoshida Co Ltd, Tokyo, Japan) using directly mounted fiber optics in the wound healing mode with a spot area of 0.25 cm2 and stored in an incubator at 100% relative humidity and 37 C for 1 day to set. Before cell experiments, all specimens were sterilized by immersion in 75% ethanol followed by exposure to ultraviolet light for 1 hour. Setting Time, pH Variation, and Strength After the powder was mixed with distilled H2O, the cements were placed into a cylindric mold and treated with the CO2 laser before being stored in an incubator at 37 C and 100% relative humidity for hydration. The setting time of the cements was tested according to standards set by ISO 9917-1. To evaluate the setting time, each material was analyzed using Gilmore needles (456.5 g), and setting was completed when the needle failed to create a 1-mm-deep indentation in 3 separate areas. The variation in the pH value of the MTA during the setting process was measured with a pH meter (IQ120 miniLab pH meter; IQ Scientific Instruments, San Diego, CA). The diametral tensile strength (DTS) testing was conducted on an EZ Test machine (Shimadzu, Kyoto, Japan) at a loading rate of 1 mm/min. The maximal compression load at failure was obtained from the recorded load-deflection curves. At least 10 specimens from each group were tested. Phase Composition and Morphology The crystalline phases of MTA treated with the CO2 laser were examined using x-ray diffraction (XRD; Shimadzu XD-D1, Kyoto, Japan) over the 2-theta range from 20 –50 , with a scanning speed of 1 /min. The morphologies of the MTA specimens before and after immersion in simulated body fluid (SBF) solution were examined under a scanning electron microscope (JSM-6700F, JEOL, Tokyo, Japan) operated in the lower secondary electron image mode at 3-kV accelerating voltage.
In Vitro Soaking It is believed that the prerequisite for materials to bond to natural bone is the formation of a ‘‘bonelike’’ apatite layer, an indicator of bioactivity (the ability to form a chemical bond with living tissue) (21). To evaluate the in vitro bioactivity, the cements were immersed in a 10-mL SBF solution at 37 C. The SBF solution, of which the ionic composition is similar to that of human blood plasma, consisted of 7.9949 g NaCl, 0.3528 g NaHCO3, 0.2235 g KCl, 0.147 g K2HPO4, 0.305 g MgCl2 6H2O, 0.2775 g CaCl2, and 0.071 g Na2SO4 in 2
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1000 mL distilled H2O and was buffered to a pH of 7.4 with hydrochloric acid and trishydroxymethyl aminomethane ([CH2OH]3CNH2) (7, 11, 13, 21, 27). All chemicals used were of reagent grade. The solution in the shaker water bath exhibited no change under static conditions. After soaking for 1 day, the specimens were removed from the tube, and their surface microstructures were examined.
Antibacterial Properties To investigate the antibacterial effects of the MTA irradiated with CO2 laser treatment, Staphylococcus aureus in Luria Broth (LB) culture media (4.0 104 bacteria per mL) was cultured with MTA materials for 24 hours. Aliquots of 0.1 mL from each group were then mixed with 0.9 mL PrestoBlue (Invitrogen, Grand Island, NY) for 10 minutes; after which, the solution in each well was transferred to a new 96-well plate. Plates were then read in a multiwell spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. Cells cultured on the tissue culture plate without the cement were used as the control (Ctl). The results were obtained in triplicate from 3 separate experiments in terms of optical density. The agar diffusion test is considered the standard method to test for antibiotic susceptibility (8, 28, 29); thus, to further confirm the inhibitory effects of MTA treated with the CO2 laser on the growth of bacterial strains, we performed an agar diffusion assay. Four sterile 6-mm disk-shaped samples from each of the 4 experimental groups were placed on cultured agar plates containing bacterial lawns of S. aureus. The inhibition zones (in mm) were determined after 1 day of incubation. Ion Concentration The Ca and Si ion concentrations on Dulbecco modified Eagle medium (DMEM) were analyzed using an inductively coupled plasma atomic emission spectrometer (OPT 1 MA 3000DV; Perkin-Elmer, Shelton, CT) after culturing for different time points. Six samples were immersed in 10 mL DMEM and measured for each data point. The results were obtained in triplicate from 3 separate samples for each test. hDPC Isolation and Culture hDPCs were freshly derived from caries-free, intact premolars that were extracted for orthodontic treatment purposes as described previously (13, 14). The patient gave informed consent, and approval from the Ethics Committee of the Chung Shan Medicine University Hospital was obtained (CSMUH No. CS14117). The tooth was split sagittally with a chisel. The pulp tissue was then immersed in phosphatebuffered saline (PBS; Caisson, North Logan, UT) solution and digested in 0.1% collagenase type I (Sigma-Aldrich, St Louis, MO) for 30 minutes. After being transferred to a new plate, the cell suspension was cultured in DMEM (Caisson) supplemented with 20% fetal bovine serum (GeneDireX) and 1% penicillin (10,000 U/mL)/streptomycin (10,000 mg/mL) (Caisson) in a humidified atmosphere with 5% CO2 at 37 C, and the medium was changed every 3 days. The cells were subcultured through successive passaging at a 1:3 ratio until they were used for experiments (passages 3–7). The odontogenic differentiation medium was DMEM supplemented with 10 8 mol/L dexamethasone (Sigma-Aldrich), 0.05 g/L L-Ascorbic acid (Sigma-Aldrich), and 2.16 g/L glycerol 2-phosphate disodium salt hydrate (Sigma-Aldrich). Cell Viability Before the in vitro cell experiments, MTA specimens were sterilized by soaking in 75% ethanol followed by exposure to ultraviolet light for 1 hour. After direct culture for various time periods, cell viability was evaluated using the PrestoBlue assay, which is based on the detection of mitochondrial activity. Thirty microliters of PrestoBlue solution and JOE — Volume -, Number -, - 2015
Basic Research—Biology 300 mL DMEM were added to each well followed by 30 minutes of incubation. After incubation, 100 mL of the solution in each well was transferred to a 96-well enzyme-linked immunosorbent assay plate. The plates were read in a Sunrise microtiter plate reader (Hitachi) at 570 nm with a reference wavelength of 600 nm. The results were obtained in triplicate from 3 separate experiments in terms of optical density. Cells cultured on the tissue culture plate without the cement were used as the Ctl.
Fluorescent Staining We observed the cell morphology of hDPCs after culturing MTA treatment with CO2 laser using F-actin cytoskeleton stains. After incubation for 1 day, the cells were washed with PBS, fixed in 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 20 minutes, and then permeabilized with PBS containing 0.1% Triton X-100 (Sigma-Aldrich). The F-actin filaments were stained with phalloidin conjugated to Alexa Fluor 594 (Invitrogen) for 1 hour. The nuclei were stained with 300 nmol/L 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen) for 30 minutes. After washing, morphology was obtained using a Zeiss Axioskop2 microscope (Carl Zeiss, Thornwood, NY). Western Blot Western blot analysis was performed using cell lysates and a culture medium prepared using hDPCs that had been cultured for 7 days. Cells were lysed in NP-40 lysis buffer (Invitrogen) at 4 C for 30 minutes, and the lysates were centrifuged at 13,000g.
The cell lysates (40 mg protein) were separated using sodium dodecyl sulfate-polyacrylamide (SDS)-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membrane was blocked in 5% bovine serum albumin (Gibco) for 1 hour; immunoblotted with the primary antibodies anti– DMP-1 (Santa Cruz Biotechnology, Santa Cruz, CA) and b-actin (GeneTex, San Antonio, TX) for 2 hours; and then washed 3 times in a trishydroxymethyl aminomethane–buffer saline containing 0.05% Tween-20 (Sigma-Aldrich). A horseradish peroxidase–conjugated secondary antibody was subsequently added, and the proteins were visualized using enhanced chemiluminescent detection kits (Invitrogen). The stained bands were scanned and quantified using a densitometer (Syngene Bioimaging System; Syngene, Frederick, MD) and ImageJ software (National Institutes of Health, Bethesda, MD). Protein expression levels were normalized to the b-actin band for each sample. The results were obtained in triplicate from 3 separate samples for each test.
Osteogenesis Assay The level of alkaline phosphatase (ALP) activity was determined after cell seeding for 7 days. The process was as follows: the cells were lysed from discs using 0.2% NP-40 and centrifuged for 10 minutes at 2000 rpm after washing with PBS. ALP activity was determined using p-nitrophenyl phosphate (Sigma-Aldrich) as the substrate. Each sample was mixed with p-nitrophenyl phosphate in 1 mol/L diethanolamine buffer for 15 minutes; after which, the reaction was stopped by the
Figure 1. The effects of CO2 laser treatment on (A) setting time and (B) DTS of MTA cements. Statistical data are presented as means standard deviations for n = 6. *Statistically significant differences (P < .05) from those obtained from MTA without laser irradiation. (C) XRD patterns of MTA with and without CO2 laser irradiation after hydration at 37 C for 1 day. (D) Scanning electron microscopic micrographs of MTA surfaces before and after immersion in SBF for 1 day.
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Basic Research—Biology addition of 5 N NaOH and quantified by absorbance at 405 nm. All experiments were performed in triplicate. The osteocalcin (OC) proteins released from pulp cells were cultured on different substrates for 14 days after cell seeding. An osteocalcin enzyme-linked immunosorbent assay kit (Invitrogen) was used to determine OC protein content following the manufacturer’s instructions. The OC protein concentration was measured by correlation with a standard curve. The analyzed blank plates were treated as controls. All experiments were performed in triplicate.
Alizarin Red S Stain Accumulated calcium deposition was observed for 7 and 14 days using alizarin red S staining as described in a previous study (30). To summarize briefly, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 minutes and then incubated in 0.5% alizarin red S (Sigma-Aldrich) at a pH of 4.0 for 15 minutes at room temperature in an orbital shaker (25 rpm). After the cells were washed with PBS, photographs were taken using an optical microscope (BH2-UMA; Olympus, Tokyo, Japan) equipped with a digital camera (Nikon, Tokyo, Japan) at 200 magnification. To quantify the stained calcified nodules after staining, samples were immersed in 1.5 mL 5% SDS in 0.5 N hydrochloric acid for 30 minutes at room temperature; after which, the tubes were centrifuged at 5000 rpm for 10 minutes, and the supernatant was transferred to the new 96-well plate (GeneDireX). At this time, absorbance was measured at 405 nm (Hitachi).
Statistical Analysis One-way analysis of variance was used to evaluate the significance of the differences between the groups in each experiment. A Scheffe multiple comparison test was used to determine the significance of the deviations in the data for each specimen. In all cases, the results were considered statistically significant with P values <.05.
Results Physicochemical Properties Setting time is one of the most clinically relevant factors in experiments of this nature. MTA set within 118 9 minutes and 143 7 minutes when treated with or without CO2 laser irradiation, respectively (Fig. 1A). Figure 1B shows the DTS values for the cement specimens. The analysis of variance results for the DTS data show that the variations in strength between specimens are not significant (P > .05). MTA had strength values of 4.1 0.2 and 3.9 0.3 MPa when treated with or without CO2 laser irradiation, respectively. In addition, there was no significant difference (P > .05) in the pH value of MTA without or with CO2 laser irradiation at the same time periods. The pH value of MTA at fresh mixing was 10.1. At the 24-hour setting, it reached a value of 12.1. Figure 1C shows the XRD patterns of the MTA cement after hydration for 1 day. Analysis of the specimens containing calcium silicate revealed 2 key points: first, an obvious diffraction peak near 2q = 29.4 , which corresponds to the calcium silicate hydrate gel, and, second,
Figure 2. (A) The growth inhibition zones and (B) the antibacterial effects of MTA with and without CO2 laser irradiation after culture in E. faecalis. (C) Ca and (D) Si ion concentrations of DMEM after immersion for different times. Data represent means standard deviation (n = 6). *Statistically significant difference (P < .05) from Ca(OH)2. @Statistically significant difference (P < .05) from MTA without laser irradiation.
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Basic Research—Biology incompletely reacted inorganic component phases of the b-dicalcium silicate (b-Ca2SiO4) at 2q between 32 and 34 . The results show lower peak intensities of the calcium silicate hydrate gel and b-Ca2SiO4 phases, with the strongest peak at 2q = 27.4 being ascribed to Bi2O3. The scanning electron microscopic micrographs of the MTA treated with or without CO2 laser irradiation before and after immersion in SBF are shown in Figure 1D. It can be noted that the MTA specimens had a porous structure. After immersion in SBF for 1 day, the MTA specimens induced the precipitation of apatite spherule aggregates, which appeared on the cement surface in the case of all groups.
Antibacterial Properties Figure 2A shows that laser treatment had no effect on the growth inhibition zone of Ca(OH)2 with regard to S. aureus. Ca(OH)2 had greater growth inhibition zones with sizes of more than 4 mm. The results of the zone of inhibition test clearly show that the antibacterial behavior of MTA is rather weak compared with that of Ca(OH)2 (P < .05). Interestingly, the inhibition zones of MTA after treatment with CO2 laser irradiation were more numerous than those seen with the specimens without laser treatment. In Figure 2B, the S. aureus cultures with the Ctl show the highest absorbance. No significant difference was detected in the absorbance readings for Ca(OH)2 groups with and without laser treatment (P > .05). Similarly, the absorbance by S. aureus cultured on CO2 laser–treated MTA is significantly lower (P < .05) than on untreated MTA.
Ion Concentrations The variations of the DMEM’s Ca and Si ion concentrations, as measured at different times, are shown in Figure 2C and D. In the MTA cement, the Ca ion concentration of the medium increased to approximately 1.68 mmol/L after being immersed for 3 hours and then went to levels higher than the baseline Ca concentration of DMEM (1.45 mmol/L) (P < .05). There were significant differences in the Ca ion concentration levels found between MTA CO2 laser treatment and MTA at all of the times they were measured (Fig. 2C). The Si concentration within all groups rose in proportion with the increased incubation time (Fig. 2D). In the MTA specimens, the Si ion concentration was approximately 0.22, 0.61, and 1.03 mmol/L at 3, 12, and 24 hours, respectively. However, the CO2 laser–treated MTA released more Si ions than the untreated MTA at all time points (P < .05). Cell Viability The cell viability of hDPCs grown on MTA and the Ctl are shown in Figure 3A. PrestoBlue showed that the cells treated with the CO2 laser had a significantly higher viability rate than those on the Ctl on days 1 and 7 (P < .05). CO2 laser–treated MTA elicited significant (P < .05) increases of 55% and 60% in optical density compared with MTA on days 1 and 7 of cell seeding, respectively. The fluorescence staining showed that on day 1 the hDPCs cultured on MTA with and without CO2 laser treatment showed strong F-actin stress fiber morphologies of the cells (Fig. 3B).
Figure 3. (A) Cell proliferation assay for hDPCs cultured on MTA on days 1 and 7. *Statistically significant difference (P < .05) from the Ctl. @Statistically significant difference (P < .05) from MTA without laser irradiation. (B) Immunofluorescence images of nuclei (blue) and F-actin (red) in the hDPCs were cultured on MTA on day 1.
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Basic Research—Biology Odontogenic Differentiation in hDPCs MTA has been shown to induce odontogenic differentiation in various cell types. To investigate the potential of hDPCs for odontoblastlike differentiation after being cultured on MTA with CO2 laser treatment, the hDPCs were cultured for up to 3 days to evaluate the protein expression levels of differentiation markers (anti-dentin matrix protein-1, anti-dentin sialophosphoprotein [DSPP] and DMP-1) by Western blot analysis (Fig. 4A). CO2 laser–treated MTA markedly upregulated DSPP and DMP-1, which increased by 24% and 26% compared with MTA, respectively, and a significance of P < .05. The ALP expression of hDPCs was also examined. Figure 4B shows the results of the analysis of the quantitative examination data and the ALP activity of cells cultured on the different substrates for 7 days. Significant 47% and 83% increases (P < .05) in the ALP levels were
found for the untreated MTA and MTA with CO2 laser irradiation in comparison with the Ctl cement after 7 days. The results for OC secretion in the cells were similar to those for ALP activity, as shown in Figure 4C. In addition, to investigate the effects of different substrates on mineralization in hDPCs, we examined mineralized nodule formation in these cells using alizarin red S staining (Fig. 4D) because this can reveal details of bone nodule formation and calcium deposition. The results showed that CO2 laser–treated MTA clearly increased the area of calcified nodules by up to 1.4-fold compared with MTA after 14 days.
Discussion This study evaluated the influence of MTA treated with CO2 laser on the resulting antibacterial effects and cell behavior. Pulpal and periapical diseases are biofilm-mediated infections, and the elimination of
Figure 4. (A) Immunodetection of DSPP and DMP-1 protein expression in hDPCs cultured on MTA for 3 days. (B) ALP activity and (C) OC amount of hDPCs cultured on MTA for different times. (D) Alizarin red S staining and quantification of calcium mineral deposits by hDPCs cultured on MTA for 1 and 2 weeks. The scale bar was 100 mm. *Statistically significant difference (P < .05) from the Ctl. @Statistically significant difference (P < .05) from MTA without laser irradiation.
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Basic Research—Biology bacteria from the root canal system is the major goal of root canal treatment (5). Pathological changes in the dental pulp tissue mean that it is susceptible to invasion by many bacteria and other antigens. Although there are more than 300 bacterial species in the normal human oral flora, a relatively small group is present in infected root canals, and, thus, bacterial infection after root canal treatment remains a significant complication. The invasion of bacteria and microorganisms into the pulp is able to induce pulpal inflammation and lead to pulp death. The endodontic materials, such as MTA and calcium silicate–based materials, are known to possess an inherent antibacterial property that is effective against S. aureus (21, 30). Lasers have been a part of oral surgical practice since the mid-1960s, and they are becoming increasingly popular because of their various advantages, such as hemostasis, decreased scarring, and less postoperative pain (31). In addition, CO2 lasers lead to cell signal transduction and outcomes through similar mechanoreceptors in cells and tissues, and, therefore, it is suggested that common signaling mechanisms are involved in laser transduction pathways (32). CO2 laser irradiation of tooth enamel has been suggested as a preventive treatment for caries because it can change the composition of enamel and the enamel in the oral environment is naturally covered by biofilms (22). The scanning electron microscopic images obtained in this work from CO2 laser–treated MTA after immersion in SBF suggest that the apatite precipitated on the surface. In a previous study, the CO2 laser can be applied to melt glass and thus create a bioactive glass coating on a titanium substrate (9); this then creates a uniform surface and promotes in vitro bioactivity. In addition, the study showed that the changes in chemical composition introduced by the CO2 laser treatment of the bioactive glass were minor and had an influence on the ions released from the glass (9). The increase of Ca ions released from substrate can enhance the formation of calcium silicate hydrates and decrease the setting time (33). In addition, regarding the variations in the pH value during setting, this initial increase in the pH value was a result of the formation of Ca(OH)2 during the setting reaction, which was released to an aqueous environment. Calcium silicate–based materials have been found to encourage cell adhesion, growth, and differentiation and have been used as implant materials for tissue repair and formation (11, 13). This study was designed to examine the use of CO2 laser–treated MTA, a component of some new capping agents used to preserve the viability of dental pulp tissue during reparative dentin formation. In this study, PrestoBlue analysis showed a number of differences between hDPCs cultured on MTA with and without CO2 laser treatment. First, the absorbance values were higher for hDPCs cultured on MTA in comparison with the Ctl, and these differences were significant (P < .05) for all time points. Previous studies in our laboratory have shown that the viabilities of fibroblast and osteoblastlike cells on untreated MTA were higher than the Ctl at all culture times (28). Shie et al (34) found that soluble factors from calcium silicate substrates may be more important for proliferation and osteogenesis differentiation in a growth medium. During immersion in medium, the Si ions were hydrolyzed from the surfaces of Si-based materials (15–18, 35, 36) and affected cell behaviors (5, 14, 30). The currently used MTA has the ability to control the release rate of soluble Ca and Si ions, which can promote cell attachment and proliferation (5, 17, 37). In addition, MTA can release more Ca and Si ions after laser irradiation, leading to a weak alkaline microenvironment with a higher pH value, which may restrain bacterial growth (5). The other is that the calcium silicate–based materials themselves may permeate the bacteria (20) with possible antibacterial consequences. Examinations of the bioactivity of calcium silicate substrates show that the presence of PO43 ions in the composition is not an essential requirement for the development of an apatite layer, which consumes calcium and JOE — Volume -, Number -, - 2015
phosphate ions (11, 37). In addition, the ions released from MTA may change the osmolality of a culture medium (14). In summary, the current study provides new and important data regarding the mechanisms by which MTA irradiated by CO2 laser is able to increase ion release and decrease the setting time. In addition, these results suggest that CO2 laser treatment may inhibit bacterial activity and stimulate odontogenic differentiation in hDPCs on MTA. Taking cell functions into account, the Si concentration released from MTA with laser irradiation may be lower than a critical value, and this information may help in developing new regenerative therapies for dentin and periodontal tissue.
Acknowledgments Tuan-Ti Hsu and Chia-Hung Yeh contributed equally to this study. Supported by a grant from the Ministry of Science and Technology (grants MOST 102-2314-B-040-007-MY3 and MOST 1042314-B-039-004) of Taiwan. The authors deny any conflicts of interest related to this study.
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JOE — Volume -, Number -, - 2015