The synergistic effects of CO2 laser treatment with calcium silicate cement of antibacterial, osteogenesis and cementogenesis efficacy

The Synergistic Effects of CO

Laser Treatment with Calcium Silicate Cement of Antibacterial, Osteogenesis, and Cementogenesis Efficacy

Tuan-Ti Hsu

, Chia-Tze Kao

, Yi-Wen Chen

, Tsui-Hsien Huang

, Jaw-Ji Yang

, Ming-You Shie

1

Institute of Oral Science, Chung Shan Medical University, Taichung City, Taiwan

2

School of Dentistry, Chung Shan Medical University, Taichung City, Taiwan

3

Department of Stomatology, Chung Shan Medical University Hospital, Taichung City, Taiwan

4

3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan

5

Both authors contributed equally to this work

6

Author to whom any correspondence should be addressed

Short title: The synergistic of antibacterial and osteogenesis on CS by CO

laser Classification numbers: 87

Correspondence:

Ming-You Shie, 3D Printing Medical Research Center, China Medical University

Hospital, Taichung City, Taiwan (E-mail: [email protected]; tel: +886-4-

22052121; fax: +886-4-24759065)

Abstract

Calcium silicate-based material (CS) has been successfully used in dental clinical applications. Some researches show that the antibacterial effects of CO

laser irradiation are highly efficient when bacteria are embedded in biofilm, due to a photo- thermal mechanism. The purpose of this study was to confirm the effects of CO

laser irradiation on CS, with regard to both material characterization and human periodontal ligament cell (hPDLs) viability. CS was irradiated with a dental CO

laser using directly mounted fiber optics in wound healing mode with a spot area of 0.25 cm

, and then stored in an incubator at 100% relative humidity and 37 °C for 1 day to set. The hPDLs cultured on CS were analyzed, along with their proliferation and odontogenic differentiation behaviors. The results indicate that the CO

laser irradiation increased the amount of Ca and Si ions released from the CS, and regulated cell behavior. CO

laser-irradiated CS promoted cementogenic differentiation of hPDLs, with the increased formation of mineralized nodules on the substrate’s surface. It also up-regulated the protein expression of multiple markers of cementogenic and the expression of cementum attachment protein. The current study provides new and important data about the effects of CO

laser irradiation on CS.

Taking cell functions into account, the Si concentration released from CS with laser irradiated may be lower than a critical value, and this information could lead to the development of new regenerative therapies for dentin and periodontal tissue.

Keywords: Calcium silicate cement, CO

laser, human periodontal ligament cell, anti-

bacterial, cementogenesis.

Introduction

Laser is classified as light amplification by stimulated involves the modification of the environment to affect existing bacteria capable of bioremediation [1,2]. The application of low level laser light was in many fields including medical, industrial, and the military [3,4]. There are several commercial product lasers, including CO

, diode, and erbium (Er):yttrium aluminum garnet (YAG) lasers in the clinical [5]. The potential safety of laser regeneration is connected with the nondestructive character of laser effect on tissue matrix and with arrangement of favorable conditions for cell proliferation and functioning [6,7]. In clinical used, periradicular surgery may be carried out in the presence of infection, or when orthograde endodontic treatment is contraindicated or considered unfeasible [8]. In spite of the high antimicrobial efficacy of traditional disinfectants in vitro, clinical research has shown bacterial persistence within the root canal after cleaning and shaping procedures [9-11]. In addition, the laser light can promote periodontal cell differentiation and it has potentially be used to enhance periodontal tissue regeneration [5,12,13]. In addition, the CO

laser is able to modulate cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, such as the function of osteoclasts [14]. Therefore, it is very important to investigate advanced endodontic disinfection strategies that are effective in eliminating biofilm bacteria in the root canals. Regrettably, even though the antibacterial ability of these materials are well known [15].

In the recent years, calcium silicate-based (CS) cements have been developed

as potential bioactive materials for bone substitute materials, in which some of them

demonstrate excellent promoted hard tissue regeneration [16-19]. In addition, CS-

based materials had been successfully used in clinical applications in endodontics

[20]. In our previous study, we produced a fast setting CS cement that contains CaO, SiO

, and Al

O

, which were demonstrated to have a reduced setting time [21]. Not only does CS have good biocompatibility [22], it has also been verified to promote hard-tissue formation [23,24]. CS-based materials have been used in dentin replacement restorative materials in dentistry [16,25,26]. We recently showed that CS-based materials stimulate the proliferation and differentiation of human dental pulp cells (hDPCs) [19,27,28], and human periodontal ligament cells (hPDLs) [5].

The ions released from CS-based materials have been found to influence cell behavior and osteogenic and angiogenic maker protein secretion [5,19,28-30]. In addition, while several researches have been published on the antibacterial properties of CS, there remains some controversy regarding the underlying mechanisms [31]. While CS releases Ca ions and leads to an alkaline microenvironment with a higher pH value, which may suppress bacterial growth [31], it has also been shown that the calcium silicate particles themselves may permeate the bacteria [32] with possible antibacterial consequences [33]. In addition, the amount of Si ions in CS-based materials can affect the adsorption of various extracellular matrix (ECM) such as collagen I, fibronectin, and vitronectin and promote the up-regulation of MAPK/ERK and MAPK/p38, signaling the pathway more effectively than Ca components [16].

The aim of this purpose was to estimate the effects of CO

laser irradiation on

CS with regard to material characterization and cell viability. We hypothesize that

CO

laser irradiation destroyed the surface structure of CS and increased the release of

ions. After CO

laser irradiation, the antibacterial ability of CS was investigated and

compared to that of calcium hydroxide. This study also investigated the cementogenic

protein expression for regenerative endodontics.

Materials and methods Preparation of specimens

The method used here for the preparation of CS powder has been described elsewhere [19]. In brief, reagent grade SiO

(High Pure Chemicals, Saitama, Japan), CaO (Sigma-Aldrich, St. Louis, MO), and MgO (Sigma-Aldrich) powders were used as matrix materials (composition: 75% CaO + MgO, and 25% SiO

) and the nominal weight ratios of CaO-SiO

–MgO were listed in Table 1. The oxide mixtures were then sintered at 1,400°C for 2 h using a high-temperature furnace and then ball-milled in ethyl alcohol using a centrifugal ball mill (S 100, Retsch, Hann, Germany) for 6 h.

The sintered powder (0.1 g) was mixed using a liquid/powder ratio of 0.35 mL/g. 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 CO

laser with an output of 10600 nm (Opelaser Pro, Yoshida Co. Ltd, Tokyo, Japan) using directly mounted fiber optics in would healing mode with a spot area of 0.25 cm

, 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 (UV) light for 1 h.

In vitro soaking

It is believed that the prerequisite for materials to bond to natural bone is the

formation of a ‘‘bone-like’’ apatite layer, an indicator of bioactivity (the ability to

form a chemical bond with living tissue) [33]. 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 of NaCl, 0.3528 g of NaHCO

, 0.2235 g of KCl, 0.147 g of K

HPO

, 0.305 g

of MgCl

• 6H

O, 0.2775 g of CaCl

, and 0.071 of g Na

SO

in 1000 mL of distilled H

O, and was buffered to a pH of 7.4 with hydrochloric acid (HCl) and trishydroxymethyl aminomethane (Tris, CH

OH)

CNH

) [16,20,25,33,34]. All chemicals used were of reagent grade. The solution in the shaker water bath exhibited no change under static conditions. After soaking for one day, the specimens were removed from the tube and their surface microstructures were examined.

Ion concentration

The Ca and Si ion concentrations on SBF were analyzed using an inductively coupled plasma-atomic emission spectrometer (ICP-AES; Perkin-Elmer OPT 1MA 3000DV, Shelton, CT, USA) after culturing for different time-points. Six samples were immersed in 10 mL SBF and measured for each data point. The results were obtained in triplicate from three separate samples for each test.

Antibacterial properties

To investigate the antibacterial effects of the CS irradiated with CO

laser

treatment, the Staphylococcus aureus in LB culture media (4.0 x 10

bacteria per mL)

were cultured with CS materials for 24 h. Aliquots of 0.1 mL from each group were

then mixed with 0.9 mL PrestoBlue

for 10 min 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 a

control (Ctl). The results were obtained in triplicate from three separate experiments

in terms of optical density (OD). In addition, the agar diffusion test is considered the

standard method to test for antibiotic susceptibility [22,35,36], and thus to further

confirm the inhibitory effects of the CS treated with CO

laser on the growth of bacterial strains, we carried out an agar diffusion assay. Four sterile 6 mm disk- shaped samples from each of the four experimental groups were placed on cultured agar plates containing bacterial lawns of Staphylococcus aureus. The inhibition zones (in mm) were determined after 1-day of incubation.

HPDLs isolation and culture

The hPDLs were freshly derived from caries-free, intact premolars that were extracted from 3 systemically healthy adults (18-24 years of age) for orthodontic treatment purposes. The patient gave informed consent, and approval from the Ethics Committee of the Chung Shan Medicine University Hospital was obtained (CSMUH No. CS14117). The teeth were instantly immersed into a phosphate-buffered saline (PBS; Caisson Laboratories, North Logan, UT, USA) that contained 100 U/mL penicillin/streptomycin (Caisson) for transferring to the laboratory. The PDL tissues of the root surface were separated by blade, washed several times with PBS, and then cut into cubes. The tissue were digested with 1 mL of 2 mg/mL type I collagenase (Sigma-Aldrich) and 4 mg/mL dispase (Sigma-Aldrich) for 30 min in the incubator.

Then, the tissue fragments were distributed into plates and cultured in DMEM,

supplemented with 20% fetal bovine serum (FBS; Caisson), 1% penicillin (10,000

U/mL)/streptomycin (10,000 mg/mL) (PS, Caisson) and kept in a humidified

atmosphere with 5% CO

at 37°C; the medium was changed every 3 days. The

primary cells were then sub-cultured to obtain passage 0 single cell-derived clones

(P0), and the passages P4-P6 were used for the following in vitro study. The

cementogenic differentiation medium was DMEM supplemented with 10

M

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). The angiogenic differentiation medium was DMEM supplemented with 2% fetal bovine serum, 1%

penicillin/streptomycin, and 50 ng/mL vascular endothelial growth factor (Prospec, East Brunswick, NJ).

Collagen secretion

Cells were cultured on different substrates for 1, 3, and 6 h, and the cell culture media were then collected and stored at room temperature. The enzyme linked immunosorbent assay (ELISA) kits of human collagen I were obtained from Abcam (Abcam, Cambridge, MA). Following the manufacturer’s instructions we used a 3 h assay, which has a high sensitivity. The reaction was terminated by the addition of stop solution and read at 450 nm using a multiwell spectrophotometer.

Cell adhesion and proliferation

Before the in vitro cell experiments, CS materials were sterilized by soaking in

75% ethanol followed by exposure to ultraviolet (UV) light for 1 h. After direct

cultured for various time periods, cell viability was evaluated using the PrestoBlue

assay (Invitrogen), which is based on the detection of mitochondrial activity. Thirty

microliters of PrestoBlue

solution and 300 µL of DMEM were added to each well,

followed by 30 min of incubation. After incubation, 100 µL of the solution in each

well was transferred to a 96-well ELISA plate. The plates were read in a Sunrise

microtiter plate reader (Hitachi, Tokyo, Japan) at 570 nm, with a reference

wavelength of 600 nm. The results were obtained in triplicate from three separate

experiments in terms of optical density. Cells cultured on the tissue culture plate

without the cement were used as a control (Ctl)

Fluorescent staining

We observed the cell morphology of hDPCs after culturing on CS treatment with CO

laser using F-actin cytoskeleton stains. After incubation for one day, the cells were washed with PBS, fixed in 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 20 min, and then permeabilized with PBS containing 0.1% Triton X- 100 (Sigma). The F-actin filaments were stained with phalloidin conjugated to Alexa Fluor 594 (Invitrogen) for 1 h. The nuclei were stained with 300 nM DAPI (Invitrogen) for 30 min. After washing, the morphology was obtained using a Zeiss Axioskop2 microscope (Carl Zeiss, Thornwood, NY, USA).

Osteogenesis assay

The level of alkaline phosphatase (ALP) activity was determined after cell seeding for seven days. The process was as follows: the cells were lysed from discs using 0.2 % NP-40, and centrifuged for 10 min at 2000 rpm after washing with PBS.

ALP activity was determined using p-nitrophenyl phosphate (pNPP, Sigma) as the substrate. Each sample was mixed with pNPP in 1 M diethanolamine buffer for 15 min, after which the reaction was stopped by the addition of 5 N NaOH and quantified by absorbance at 405 nm. All experiments were done in triplicate.

The 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 done in triplicate.

Western blot

Western blot analysis was carried out using cell lysates and a culture medium prepared using hDPCs that had been cultured for 14 days. Cells were lysed in NP-40 lysis buffer (Invitrogen) at 4°C for 30 min and the lysates were centrifuged at 13,000 g. The cell lysates (40 μg protein) were separated using SDS-polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. The membrane was blocked in 5% bovine serum albumin (BSA, Gibco) for 1 h and then immunoblotted with the primary antibodies anti-CAP, anti-CEMP1 (Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin (GeneTex, San Antonio, TX) for 2 h, then washed three times in a tris-buffer saline containing 0.05% Tween-20 (Sigma-Aldrich). A horseradish peroxidase (HRP)-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; Frederick, MD) and the ImageJ software (National Institutes of Health, Bethesda, MD). Protein expression levels were normalized to the β-actin band for each sample. The results were obtained in triplicate from three separate samples for each test.

Alizarin red S stain

Accumulated calcium deposition was observed for seven and 14 days using

Alizarin Red S staining, as described in a previous study [37]. To summarize briefly,

the cells were fixed with 4% paraformadedyde (Sigma-Aldrich) for 15 min and then

incubated in 0.5% Alizarin Red S (Sigma-Aldrich) at pH 4.0 for 15 min 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 200x magnification.

To quantify the stained calcified nodules after staining, samples were immersed in 1.5 mL of 5% SDS in 0.5N HCl for 30 min at room temperature, after which the tubes were centrifuged at 5,000 rpm for 10 min and the supernatant was transferred to the new 96-well plate (GeneDireX). At this time, absorbance was measured at 405 nm (Hitachi).

Statistical analysis

A one-way analysis of variance (ANOVA) was used to evaluate the

significance of the differences between the groups in each experiment. A Scheffe’s

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 < 0.05.

Results

Apatite precipitate

The SEM micrographs of the CS treated with or without CO

laser irradiation before and after immersion in SBF are shown in Fig. 1. It can be seen that the CS specimens had a dense structure. After immersion in SBF for 3 h, the CS specimens induced the precipitation of apatite spherule aggregates, which appeared on the cement surface in the case of all groups.

Ion Concentrations

The variations of the SBF’s Ca and Si ion concentrations, as measured at different times, are shown in Figs. 2A and 2B. In the CS cement, the Ca ion concentration of the medium increased to approximately 1.75 mM after being immersed for 3 h, and then went to levels higher than the baseline Ca concentration of DMEM (1.58 mM) (p < 0.05). There were significant differences in the Ca ion concentration levels found between CS-CO

laser treatment and CS at all of the times they were measured (Fig. 2A). The Si concentration within all groups rose in proportion with the increased incubation time (Fig. 2B). In the CS specimens, the Si ion concentration was approximately 2.12, 2.48, and 2.66 mM at 6, 12, and 24 h, respectively. However, the CO

laser treated-MTA released more Si ions than the untreated MTA at all time points (p < 0.05).

Antibacterial Properties

Figure 3A shows that laser treatment had no effect on the growth inhibition

zone of Ca(OH)

with regard to Staphylococcus aureus. The Ca(OH)

had greater

growth inhibition zones, with sizes of more than 9 mm. The results of the zone of

inhibition test clearly show that the antibacterial behavior of CS is rather weak compared to that of Ca(OH)

(p < 0.05). Interestingly, the inhibition zones of CS after treatment with CO

laser irradiation were more numerous than those seen with the specimens without laser treatment. In Fig. 3B, the Staphylococcus aureus cultures with Ctl show the highest absorbance. No significant difference was detected in the absorbance readings for Ca(OH)

groups with and without laser treatment (p > 0.05).

Similarly, the absorbance by Staphylococcus aureus cultured on CO

laser treated-CS is significantly lower (p < 0.05) than on untreated CS.

Cell adhesion and collagen secretion

The cell viability of hPDLs adhesion on CS and Ctl are shown in Fig. 4A. There are no significant differences (p > 0.05) between Ctl and TCP. PrestoBlue

showed that the cells on CS had a significantly higher adhesion rate than those on the Ctl after 3 h (p < 0.05). The CO

laser treated-CS elicited significant (p < 0.05) increases of 16% and 15% in the optical density compared with CS after 3 and 12 h of cell seeding, respectively. Fig. 4B shows the amounts of COL protein in the culture medium secreted from hPDLs cultured with different specimens. At 1 h, the COL secretion on CS-lase is 2.26 and 1.18 times higher (p < 0.05) than on Ctl and CS, respectively. The fluorescence staining showed that at 3 and 6 h the hPDLs cultured on CS with and without CO

laser treatment showed strong F-actin stress fiber morphologies of the cells (Fig 5).

Cell proliferation

The cell proliferation of hPDLs grown on CS and Ctl are shown in Fig. 6.

PrestoBlue

showed that the cells cultured with CS had a significantly higher viability

rate than those on the Ctl at all time-points (p < 0.05). The CO

laser treated-MTA elicited significant (p < 0.05) increases of 25%, 20% and 7% in the optical density compared with CS on days 1, 3, and 7 of cell seeding, respectively.

Osteogenic and cementogenic differentiation in hPDLs

CS has been shown to induce osteogenic and cementogenic differentiation in various cell types. To investigate the potential of the hPDLs for osteoblast-like or cementblast differentiation after being cultured on CS with CO

laser treatment, the hPDLs were cultured for up to three days to evaluate the protein expression levels of differentiation markers by ELISA (Fig 7) and Western blot analysis (Fig. 8). In osteogenesis, the CO

laser treated-CS markedly up-regulated ALP and OC, which increased by 26% and 15% compared with CS at day 3, respectively, and a significance of (p < 0.05). In cementogenic protein, the CAP and CEMP1 expression of hPDLs were also examined. Figure 8 shows the results of the analysis of the quantitative examination data and the proteins of cells cultured on the different substrates for seven days. Significant 25% and 22% increases (p < 0.05) in the CAP and CEMP1 levels were found for CS with CO

laser irradiation in comparison with the CS cement after seven days. In addition, to analyze the effects of different substrates on mineralization in the hPDLs, we examined mineralized nodule formation in these cells using Alizarin Red S staining (Fig. 9), as this can reveal details of bone nodule formation and calcium deposition. The results showed that CO

laser treated-CS clearly increased the area of calcified nodules by up to 1.2-fold

compared with CS after 7 days.

Discussion

This study evaluated the influence of CS treated with CO

laser on the resulting antibacterial effects and cell behavior. Pathological changes in the oral environment mean that is susceptible to invasion by many bacteria and other antigens.

While 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 following root canal treatment remains a significant complication. In clinical, the calcium silicate-based materials are known to possess an inherent antibacterial property that is effective against Staphylococcus aureus [33,37]. In addition, CO

lasers lead to cell signal transduction and outcomes through similar mechanoreceptors in cells and tissues and it is therefore suggested that common signaling mechanisms are involved in laser transduction pathways [13]. CO

laser irradiation of tooth enamel has been suggested as a preventive treatment for caries, as it can change the composition of enamel and the enamel in the oral environment is naturally covered by biofilms [1]. Such a system could combine the benefits of bone substrate properties and CO2 laser irradiation. During immersion in medium, the Si ions were hydrolyzed from the surfaces of Si-based materials [5,19,28,29,38,39], and affected cell behaviors [27,31,37]. The currently used CS has the ability to control the release rate of soluble Ca and Si ions, which can promote cell attachment and proliferation [5,18,31]. In addition, CS 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 [31]; the other is that the calcium silicate-based materials themselves may permeate the bacteria [32] with possible antibacterial consequences.

The SEM images obtained in this work from the CO

laser-treated CS after

immersion in SBF suggest that the apatite precipitated on the surface. In previous

study, CO

laser can be applied to melt glass and thus create a bioactive glass coating on a titanium substrate [23], this then creates a uniform surface and promotes the in vitro bioactivity. In addition, the study showed that the changes in chemical

composition introduced by the CO

laser treatment of the bioactive glass were minor, and had influence the ions released from the glass [23]. The increasing of Ca ions released from substrate can enhance the formation of calcium silicate hydrates, and decrease the setting time [40]. In addition, the variations in pH value during setting, this initial increase in pH value was a result of the formation of Ca(OH)

during the setting reaction, which was released to an aqueous environment. Examinations of the bioactivity of calcium silicate substrates show that the presence of PO4

ions in the composition is not an essential requirement for the development of an apatite layer, which consumes calcium and phosphate ions [18,25]. In addition, the ions released from the CS may change the osmolality of a culture medium [27].

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 [16,25]. This study was designed to examine the use of CO

laser-

treated CS, 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 hPDLs cultured on CS with and

without CO

laser treatment. First, the absorbance values were higher for hPDLs

cultured on CS in comparison to Ctl, and these differences were significant (p < 0.05)

for all time-points. Previous studies in our laboratory have demonstrated that the

viabilities of fibroblast and osteoblast-like cells on untreated CS were higher than Ctl

at all culture times [35]. Shie et al. found that soluble factors from calcium silicate

substrates may be more important for proliferation and osteogenesis differentiation in

a growth medium [41]. These proteins are one of the important factors in the initial

adhesion of cells when biomaterials are implanted in living tissue [42]. The

complexity of the tooth and bone supporting apparatus makes periodontal tissue

regeneration a challenging field, as it involves the regeneration of cementum, a

functionally oriented PDL and an alveolar bone in the periodontal defect. It has been

proved that hPDLCs could be differentiated into fibroblastic, osteoblastic, and

cementoblastic cell [12]. In recent years, several researches had been focused on the

interaction of biomaterials and mesenchyme-derived cell lines to accelerate the

process of periodontal tissue regeneration [43]. ALP enzyme activity is also

associated with bone formation, and it is produced in high levels during the bone

formation phase [44]. CAP has been found as a collagen-like protein and serves as a

marker for cementoblastic progenitors of the hPDLs. CEMP1 protein has been

characterized as a novel, cementum-specific protein expressed by PDL

subpopulations and cementoblasts [45]. It was found that CS materials promoted cell

attachment and proliferation of hPDLCs. Interestingly, CS-laser cement significantly

increased relative cementogenic protein (CEMP1 and CAP) and osteogenic protein

(ALP and OPN) expression of hPDLCs compared to those on the CS cement. The

results indicate that CS-laser irradiation possess excellent in vitro

cementogenic/osteogenic stimulation for hPDLCs.

5. Conclusion

In summary, the current study provides new and important data regarding the

mechanisms by which CS-irradiated by CO

laser is able to increase ion release and

decrease the setting time. In addition, these results suggest that CO

laser treatment

may inhibit bacterial activity and stimulate cementogenic/osteogenic differentiation in

hPDLs on CS. Taking cell functions into account, the Si concentration released from

CS 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.

Acknowledgements

The authors acknowledge receipt of a grant from the National Science Council

grants MOST 102-2314-B-040-007-MY3 and MOST 104-2314-B-039-004 of

Taiwan. The authors declare that they have no conflicts of interest.

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Figure Legends

Figure 1. SEM micrographs of CS surfaces before and after immersion in SBF for different time-points.

Figure 2. (A) Ca and (B) Si ion concentrations of SBF after immersion for different times. Data represent means ± SD (n = 6). “*”, statistically significant difference (p <

0.05) from CS.

Figure 3. (A) The growth inhibition zones and (B) the anti-bacterial effects of MTA with and without CO

laser irradiation after culture in E. faecalis. Data represent means ± SD (n = 6). “*”, statistically significant difference (p < 0.05) from Ca(OH)

;

“@”, statistically significant difference (p < 0.05) from CS without laser irradiation.

Figure 4. (A) Cell adhesion and (B) collagen I secretion from hPDLs cultured on CS at 3,6, and 12 h. “*”, statistically significant difference (p < 0.05) from Ctl; “@”, statistically significant difference (p < 0.05) from CS without laser irradiation.

Figure 5. Immunofluorescence images of nuclei (blue), and F-actin (red) in the hDPCs were cultured on CS on 3 and 6 h.

Figure 6. Cell proliferation assay for hDPCs cultured on CS at days 1, 3, and 7. “*”, statistically significant difference (p < 0.05) from Ctl; “@”, statistically significant difference (p < 0.05) from CS without laser irradiation.

Figure 7. (A) ALP activity and (B) OC amount of hPDLs were cultured on CS for different time-points. “*”, statistically significant difference (p < 0.05) from Ctl; “@”, statistically significant difference (p < 0.05) from CS without laser irradiation.

Figure 8. (A) Immunodetection and (B) quantification of CAP and CEMP1-1 protein expression in hPDLs were cultured on CS for 3 days. “*”, statistically significant difference (p < 0.05) from Ctl; “@”, statistically significant difference (p < 0.05) from CS without laser irradiation.

Figure 9. (A) Alizarin Red S staining and (B) quantification of calcium mineral

deposits by hPDLs cultured on CS for one and two weeks. “*”, statistically significant

difference (p < 0.05) from Ctl; “@”, statistically significant difference (p < 0.05) from

CS without laser irradiation.