2.1.

Irradiation Protocol Settings

The LLLI experiments were performed by using a semiconductor laser-diode, which emits a maximum output power of 130 mW at 659 nm (Table 1). The particular laser diode used for these experiments is based on an AlGaInP semiconductor (model ML101J27-01, produced by Mitsubishi Electric Corp., Tokyo, Japan, Fig. 1), which emits a single transverse-mode laser beam with a typical divergence of 10 and 17 deg in the two directions transverse to the beam propagation axis. After free propagation in air, the central portion of the beam was selected through an iris diaphragm in order to guarantee a constant beam intensity over the entire irradiated field (a circular well with radius $r=8\mathrm{mm}$). The relative positions of the laser source, the mask, and the samples were appropriately chosen to obtain complete illumination of the well containing the test sample, while simultaneously avoiding illumination of adjacent wells. Both the operating temperature and current of the laser source were monitored by proper drivers (by Thorlabs GmbH, Germany). The value of the optical power incident on the samples, taking into account losses due to Fresnel reflection occurring on the cover, was set at 10 mW, corresponding to an intensity of $5\mathrm{mW}/{\mathrm{cm}}^2$. The optical power was periodically monitored to check for any laser performance degradation.

Table 1

Laser irradiation parameters (Mitsubishi laser, model ML101J27-01, Tokyo, Japan).

Fig. 1

Setup used for cell irradiation. Aluminum-gallium-indium-phosphide semiconductor diode laser emitting at 659 nm was perpendicularly positioned above the tissue culture plate containing the SAOS-2 cell monolayer.

2.2.

Temperature Measurement

In order to evaluate the possible thermal effects induced by the laser irradiation on cell cultures, a commercial temperature probe was used to measure the temperature in the culture dish before turning on the laser source, and also during laser irradiation. A T-type thermocouple (RS Components, code 621-2209, Mi, Italia) was used as the sensor; this has a good sensitivity (0.1°C) and a very broad operating range (from $-200$ to $+350°\mathrm{C}$). The temperature in the culture dish before laser treatment was $22\pm 0.1°\mathrm{C}$. The samples were then irradiated for 600 s in a single exposition at dose of $3\mathrm{J}/{\mathrm{cm}}^2$. The final temperature was of $21.8\pm 0.1°\mathrm{C}$, indicating that the temperature remained unchanged during irradiation.

2.3.

Reagents

The human osteosarcoma cell line Saos-2 was obtained from the American Type Culture Collection (HTB85, ATCC, Manassas, Virginia).^26,27 Unless otherwise specified, all reagents were from Sigma Aldrich (St. Louis, Missouri). Dr. Larry W. Fisher (National Institutes of Health, Bethesda, Maryland) provided us with the rabbit polyclonal anti type-I and III collagen, anti-decorin, anti-osteopontin, anti-osteocalcin, anti-osteonectin, and anti-alkaline phosphatase. Polyclonal antibody against human fibronectin (FN) was produced as previously described.²⁸ For the Western blot analysis, we used the anti-actin antibody and the phospho site-specific antibody against Akt-P on Ser 473 and Akt from Cell Signaling Technologies^®, Danvers, Massachusetts. Horseradish peroxidase (HRP)-conjugated secondary antibodies (HRP-conjugated from Dako) were used, and detection was performed by enhanced chemiluminescent substrate (ECL) solutions (Pierce Thermo Fisher Scientific, Rockford, Illinois). The TRAPeze™ kit used to detect telomerase activity was purchased from CHEMICON International (Millipore, Billerica, Massachusetts).

2.4.

Cell Culture

Saos-2 cells were cultured in McCoy’s 5A modified medium with L-glutamine and HEPES (Cambrex Bio Science, Baltimore, Maryland), supplemented with 15% fetal bovine serum, 1% L-glutamine, 0.4% antibiotics, 2% sodium pyruvate, and 0.2% fungizone. For osteogenic differentiation analysis, dexamethasone and $\beta$-glycerophosphate (both osteogenic factors) were added to the previously indicated medium at a concentration of ${10}^{-8}\mathrm{M}$ and 10 mM, respectively. Ascorbic acid, another osteogenic supplement, is a component of McCoy’s 5A modified medium. The cells were cultured at 37°C, 5% ${\mathrm{CO}}_2$, routinely trypsinized after confluence, counted, and seeded at a density of $3\times {10}^4\text{cells}/\text{well}$ in 24-well plates.

2.5.

Cell Viability

2.6.

Western Blot Analysis

Saos-2 cells treated with a laser dose of $3\mathrm{J}/{\mathrm{cm}}^2$ were analyzed for Akt and Akt phosphorylation on Ser 473 by Western blot after 10, 20, and 30 min irradiation. Briefly, cells were scraped from the dish and lysed with ice-cold lysis buffer (50 mM Tris pH 7.5, 50 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 0.1% Triton, and 1 mM sodium sodium orthovanadate) for 30 min on ice. The lysates were centrifuged at 12,000 rpm for 5 min at 4°C, and supernatant protein concentrations were determined. Equivalent samples were subjected to SDS-PAGE on 8% gel. The proteins were then transferred onto nitrocellulose membranes and probed with primary antibodies anti-phospho-Akt on Ser 473 and anti-Akt diluted $1:2000$, followed by secondary antibodies conjugated to HRP ($1:1000$). Detection was performed with ECL solution and revealed by autoradiography. Densitometry analysis of the band was performed using Image TM Software. Bands were then quantified by densitometric analysis.

2.7.

Telomerase Assay

The Trapeze^® gel-based telomerase detection kit was used to detect and evaluate telomerase activity in cells irradiated with multiple doses of $3\mathrm{J}/{\mathrm{cm}}^2$ and in control cells. This assay is a highly sensitive in vitro assay system for detecting telomerase activity and is based on an improved version of the original method described by Kim et al.²⁸ The assay is a one-buffer, two-enzyme system that uses a polymerase chain reaction (PCR) to enhance the sensitivity of telomerase detection in small samples. For visualization of the reaction products, we used a nonradioactive method: running 25 μl of the products on a 12.5% nondenaturing-page gel in 0.5x Tris/Borate/EDTA buffer. After electrophoresis, the gel was stained with SYBR^® green according to the manufacturer’s instructions (Life Technologies, ex Invitrogen, Carlsbad, California). The relative telomerase activity level is expressed as the total product generated (TPG), calculated by the following formula: $\mathrm{TPG}(\text{units})=(x-x_o)/c/(r-r_o)/c_R\times 100$; $x$ is the signal intensity of 6-bp ladders in the sample; $x_o$ is the signal intensity in heat-treated sample; $r$ is the signal intensity in TSR8 control; $r_o$ is the signal intensity in lysis buffer; $c$ is the signal intensity in 36-bp internal control in the sample; and $c_R$ is the signal intensity in TSR8 control. The intensity of the TRAP product band and standard internal control bands were determined using Image™ Software.

2.8.

Confocal Laser Scanning Microscope Analysis

Saos-2 cells ($3\times {10}^4$) were seeded on glass coverslips in growth medium and then processed for confocal laser scanning microscope (CLSM) analysis. For morphological observation, cells were stimulated with a single or multiple laser doses (1 or $3\mathrm{J}/{\mathrm{cm}}^2$). On day 7 of culture, cells were washed with PBS, fixed with 4% (w/v) paraformaldehyde solution for 30 min at 4°C, permeabilized with 0.1% Triton X-100, and finally incubated with the primary antibody anti-$\alpha$ tubulin overnight at 4°C.

For osteogenic protein labeling, cells were irradiated with multiple doses at $3\mathrm{J}/{\mathrm{cm}}^2$ and stained on day 14 of culture both in proliferative medium (PM, without osteogenic factors) and in osteogenic medium (OM, with osteogenic factors). Paraformaldehyde-fixed samples were blocked with PAT [PBS containing 1% (w/v) bovine serum albumin and 0.02% (v/v) Tween 20] for 1 h at RT. Cells were then incubated with specific primary antibodies (anti-type-I collagen, anti-alkaline phosphatase, and anti-osteocalcin rabbit polyclonal antisera) diluted $1:1000$ in PAT overnight at 4°C. Following incubation with the primary antibody, cells were washed once with PBS and incubated with Alexa-Fluor-488 conjugated secondary antibody (diluted $1:500$ in PAT, Invitrogen, Carlsbad, California) for 1 h at RT. After extensive washes in PBS, nuclei were counterstained with propidium iodide ($2\mu \text{g}/\mathrm{mL}$) for morphological observation and Hoechst 33342 ($2\mu \text{g}/\mathrm{mL}$) for osteogenic protein labeling. Finally, samples were observed with a confocal fluorescence microscope (Leica TCS SPII Microsystems, Wetzlar, Germany).

2.9.

Osteogenic Differentiation

To investigate the effect of LLLI on Saos-2 osteogenic differentiation, cells treated with three consecutive doses at $3\mathrm{J}/{\mathrm{cm}}^2$ and cultured in PM or OM were analyzed on day 14 of culture (Table 2). Control cell groups were unexposed and cultured in PM or OM, respectively.

2.10.

Alkaline Phosphatase Activity

On day 14 of culture, the alkaline phosphatase (ALP) activity from LLLI stimulated and unstimulated samples cultured either in PM or OM was evaluated by a colorimetric end point assay as previously described.² The assay measures the conversion of the colorless substrate $p$-nitrophenol phosphate (PNPP) by the enzyme ALP to the yellow product p-nitrophenol, where the rate of the color change corresponds to the amount of enzyme present in the solution. Briefly, an aliquot (1 mL) of 0.3 M PNPP (dissolved in glycine buffer, pH 10.5) was added to each sample at 37°C. After incubation, the reaction was stopped by the addition of 100 mL 5 M NaOH. Standards of PNPP in concentrations ranging from 0 to 50 mM were freshly prepared from dilutions of a 500 mM stock solution and incubated for 10 min with 7U of ALP (Sigma-Aldrich) previously dissolved in 500 mL of ddH2O. The absorbance reading was performed at 405 nm with a microplate reader (BioRad Laboratories, Hercules, California) using 100 mL of standard or sample placed into individual wells of a 96-well plate. Samples were run in triplicate and compared against a calibration curve of p-nitrophenol standards. The enzyme activity was expressed as micromoles of p-nitrophenol produced per minute per milligram of enzyme.

2.11.

Calcium Quantification

On day 14 of culture, the calcium deposition from LLLI stimulated and unstimulated samples cultured either in PM or OM was determined by calcein detection and calcium cresolphthalein complexone methods as previously described.²⁹

2.12.

Assay for Gene Expression

Total RNA from LLLI stimulated and unstimulated samples cultured in PM or OM was extracted on day 14 of culture using the Trizol reagent, according to the manufacturer’s protocol (Invitrogen). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed in order to evaluate the gene expression for bone sialoprotein (BOSP), decorin (DEC), fibronectin (FN), osteocalcin (OC), osteopontin (OP), type-I collagen (COL-I), type-III collagen (COL-III), alkaline phosphatase (ALP), osteonectin (OSN), and the housekeeping gene expression for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The reverse transcriptase reaction was performed with 300 ng total RNA using the iScript™ cDNA Synthesis kit from Bio-Rad. The primers (Primm s.r.l., Milan, Italy) were designed according to the published gene sequences, and the PCRs were performed with the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, California) as previously reported.²⁹ The primers used are indicated in Table 3.

Table 3

Primers used for qRT-PCR.

2.13.

Real-Time PCR

Total RNA from LLLI stimulated and unstimulated samples cultured in PM or OM was extracted on day 14 of culture with the NucleoSpin^® RNA XS kit (Macherey-Nagel, Duren, Germany) and retro-transcribed to c-DNA with the iScript cDNA Synthesis Kit (BioRad Laboratories, Marnes-La-Coquette, France). A quantitative RT-PCR analysis was performed in a 48-well optical reaction plate using a MiniOpticon^® Real Time PCR System (BioRad Laboratories) as previously described.² Gene expression was analyzed in triplicate and normalized to GAPDH gene expression, using the Livak method.³⁰ Analysis was performed in a total volume of 20 μL amplification mixture containing 2x (10 μL) Brilliant^® SYBR^® Green QPCR Master Mix (Stratagene, La Jolla, California), 2 μL cDNA, 0.4 μL of each primer, and 7.2 μL ${\mathrm{H}}_2\mathrm{O}$. Thermal cycling was initiated by denaturation at 95 deg for 3 min, followed by 40 cycles at 95 deg for 5 s and 60 deg for 20 s. To perform the real-time analysis, we used Invitrogen™ (Carlsbad, California) primers for the genes listed in Table 3.

2.14.

Extraction of the Extracellular Matrix Proteins and ELISA Assay

On day 14 of culture, in order to evaluate the amount of extracellular matrix proteins produced by LLLI stimulated and unstimulated samples cultured either in PM or OM, an ELISA assay was performed as previously described.²⁹ Briefly, the samples were washed extensively with sterile PBS to remove culture medium and then incubated for 24 h at 37°C with 1 mL of sterile sample buffer [20 mM Tris-HCl, 4 M GuHCl, 10 mM EDTA, 0.066% (w/v) sodium dodecyl sulphate (SDS), pH 8.0]. At the end of the incubation period, the total protein concentration in both culture systems was evaluated with the BCA Protein Assay Kit (Pierce Biotechnology Inc., Rockford, Illinois). Calibration curves to measure COL-I, COL-III, DEC, OP, OC, OSN, FN, and ALP were performed as previously described.² We have taken into consideration that an underestimation of the absolute protein deposition is possible because the sample buffer used for matrix extraction contains SDS, which may interfere with protein adsorption during the ELISA assay. The amount of extracellular matrix constituents from samples was expressed as pg/(cells per well).

2.15.

Scanning Electron Microscopy Analysis

Saos-2 were seeded on plastic cell culture coverslip disks (Thermanox Plastic, Nalge Nunc International, Rochester, New York) and irradiated with multiple laser doses at $3\mathrm{J}/{\mathrm{cm}}^2$ in PM or OM. Control groups were treated under the same conditions. On day 14 of culture, cells were treated as previously described.² The specimens were sputter coated with gold and observed at $250\times$ and $1000\times$ magnification, respectively, with a Leica Cambridge Stereoscan 440 microscope (Leica Microsystems) at 8 kV.

2.16.

Statistical Analysis

Each experimental condition was performed in triplicate in three separate experiments. Differences between groups were tested by one-way analysis of variance. Tukey’s test was used to correct for multiple comparisons. Statistical significance was established at two-tailed $p\le 0.05$. All calculations were generated using GraphPad Prism 5.0 (GraphPad Inc., San Diego, California).

3.1.

Effect of Low-Level Laser Irradiation on Cell Proliferation and Morphology

To test whether LLLI can act as a proliferative factor, the experimental setup was performed as indicated in Table 2. At all levels of applied irradiation and at each time interval, cell vitality and morphology were evaluated by MTT assay, FDA, and CLSM, respectively (Fig. 2). The results of cell viability after a single dose at 1 or $3\mathrm{J}/{\mathrm{cm}}^2$ is shown in Fig. 2(Aa). Significant differences were detected in cell proliferation after a single dose at 1 or $3\mathrm{J}/{\mathrm{cm}}^2$ with respect to the dark control ($p<0.05$) on day 2. However, on days 3 and 7, these differences were not statistically significant. The results of cells treated with daily doses are also presented in Fig. 2(Ab). The viability analysis showed that repeated irradiation on three consecutive days with doses of 1 or $3\mathrm{J}/{\mathrm{cm}}^2$ resulted in a significantly higher proliferation when compared to the dark control group on days 2, 3, and 7 ($p<0.05$). It is possible to hypothesize that single dose may exert an effect on cell proliferation in the first few days after exposure, but this effect may not last, as previously reported.²³ The effect of irradiation was then qualitatively evaluated by FDA assay at day 7 of culture in untreated/laser-treated cells after single or multiple doses [Fig. 2(B)]. Results showed comparable cell viability between the samples. The Saos-2 cell morphology after laser exposure was evaluated by CLSM and no differences in the cytoskeletal organization of alpha tubulin between the experimental groups was observed [Fig. 2(C)]. To further complete the analysis on proliferative activity following laser treatment, the activation of mitogenic kinase Akt was analyzed (Fig. 3). This protein was previously reported to be involved in LLLI-induced cell proliferation.³¹

Fig. 2

Time course of cell Saos-2 proliferation following irradiation with a single and daily laser doses. (Aa) The viability of groups irradiated with a single dose at 1 or $3\mathrm{J}/{\mathrm{cm}}^2$ on day 0. (Ab) The viability of groups irradiated with a daily dose of 1 or $3\mathrm{J}/{\mathrm{cm}}^2$ on days 0, 1, and 2. The MTT test was performed to evaluate cell viability after a single dose or daily dose exposures at days 1, 2, 3, and 7 of cell culture. After seven days of culture, all experimental groups were treated with fluorescein diacetate (green cells alive) and propidium iodide (red cells dead) (B). Dark control and groups irradiated with one or three consecutive doses at $3\mathrm{J}/{\mathrm{cm}}^2$ are shown ($20\times$ magnification, the scale bar represents 50 μm). SAOS-2 cell morphological analysis after laser treatment: alfa-tubulin is shown in green and nuclei in red ($40\times$ magnification, the scale bar represents 50 μm) (C). The dark control and irradiated sample with one or three consecutive doses at $3\mathrm{J}/{\mathrm{cm}}^2$ are shown.

Fig. 3

Representative Western blot analysis for Akt phosphorylation up to 30 min after a single laser dose at $3\mathrm{J}/{\mathrm{cm}}^2$. SAOS-2 were cultured for 24 h in serum free medium, irradiated, and collected at the indicated time after laser treatment and then analyzed for Akt phosphorylation (Ser-473) by Western blot. Akt and b-actin were used as controls. Results represent one of three replicates (a). Bar graph represents the phosphorylation level of the signal protein calculated by the ratio between the phosphorylated and total protein obtained in three different experiments. *$p<0.05$ versus time 0 (b).

Western blot analysis showed that laser treatment with a dose of $3\mathrm{J}/{\mathrm{cm}}^2$ is able to induce a transient increase in Akt phosphorylation/activation, which gradually reached a maximum level of activation 20 min after stimulation [Figs. 3(a) and 3(b)]. The correlation between telomerase activity and cell proliferation after laser irradiation was also investigated (Fig. 4). Telomerase activity was measured quantitatively with the TRAPEZE Gel Based Telomerase Detection Kit. As shown in Fig. 4, both stimulated and unstimulated samples were telomerase positive, but telomerase activity was not significantly increased in stimulated cells compared to the dark control ($p>0.05$).

Fig. 4

Electrophoresis image of telomerase activity in Saos-2 after irradiation for three consecutive days at a dose of $3\mathrm{J}/{\mathrm{cm}}^2$ in proliferative medium using the TRAPeze™ kit. One representative telomerase assay out of three similar ones is presented (a). Lane 1: TRAPeze quality control cell lysate (HeLa cells) heat-treated; lane 2: non-heat-treated extract of control HeLa cells; lane 3: heat-treated dark control cells; lane 4: non-heat-treated dark control cells; lane 5: heat-treated irradiated cells; lane 6: non-heat-treated irradiated cells; lanes 7 and 8: TSR8 control and 1X CHAP lysis buffer, respectively. Bar graph represents the telomerase products quantification (b). Telomerase activity (in total product generated units) was calculated by comparing the ratio of telomerase products to an internal standard for each lysate, as described by CHEMICON International.

3.2.

Effect of Low-Level Laser Irradiation on Cell Differentiation

To investigate whether low-level laser treatment was able to influence Saos-2 osteogenic differentiation (Table 2, for experimental setup), all analyses were performed on day 14 of culture on multiple-stimulated or unstimulated cells, with/without the addition of osteogenic factors in the culture medium. ALP activity, calcium deposition, gene expression, and bone extracellular matrix protein production were evaluated. The level of ALP activity was higher in cells in osteogenic medium (both unstimulated/laser stimulated) than cells in proliferative medium (both unstimulated/laser stimulated) [Fig. 5(B)]. The ALP activity was different between cells that were laser treated or untreated cultured in PM as well as in OM. Interestingly, the ALP activity of LLLI-exposed samples in PM was considerably higher when compared to unexposed samples in PM (*$p<0.05$); on the contrary, ALP activity of laser-exposed samples in OM was slightly lower than that of unexposed samples in OM (#$p>0.05$). These data are in accordance with the immunolocalization of ALP on the cell surface as observed by CLSM. A more intense green fluorescence signal was observed on laser-stimulated samples in PM and on stimulated/unstimulated samples in OM [Figs. 5(Ab), 5(Ac), and 5(Ad)] than on unstimulated cells in PM [Fig. 5(Aa)]. Figure 6 shows the calcium deposition detected by calcein assays: a more intense green fluorescence signal was observed on laser-stimulated cells in PM and laser-stimulated/unstimulated cells in OM [Figs. 6(Ab), 6(Ac), and 6(Ad)] than on unstimulated cells maintained in PM [Fig. 6(Aa)]. A significant difference was also observed between laser-exposed/unexposed cultures in PM. The results were quantitatively confirmed with the calcium creosolphatein complexone method [Fig. 6(B)].

Fig. 5

Alkaline phosphatase (ALP) deposition and activity of Saos-2 cells exposed to three consecutive doses at $3\mathrm{J}/{\mathrm{cm}}^2$ and cultured in proliferative medium (PM) or in osteogenic medium (OM). ALP presence was determined by confocal laser microscopy ($40\times$ magnification, the scale bar represents 50 μm) (A), and ALP activity was measured colourimetrically, corrected for the protein content measured with the BCA Protein Assay Kit and expressed as millimoles of p-nitrophenol produced per minute per milligram of protein (B). Bars express the $\text{mean values}\pm \mathrm{SD}$ of results from three experiments (*$p<0.05$ versus PM).

Fig. 6

Calcium matrix produced by Saos-2 cells irradiated with three consecutive doses at $3\mathrm{J}/{\mathrm{cm}}^2$ and cultured in PM or in OM. Calcium deposition was determined with a confocal laser scanning microscope [(A), $20\times$ magnification, the scale bar represents 50 μm] and by quantification of calcium content as reported in the Materials and Methods section (B). Results are presented as an $\text{average}\pm \mathrm{SD}$ (*$p<0.05$ versus PM).

In order to characterize bone-specific gene expression, an RT-PCR analysis was performed at 14 days culture with/without osteogenic factors on unstimulated/stimulated samples. The qualitative RT-PCR (qRT-PCR) showed differences for the transcripts specific for COL-I, BOSP, and OP (Fig. 7). To further expand these data, a qRT-PCR for the ALP, BOSP, COL-I, and OP in laser-stimulated/unstimulated cells cultured in PM or OM was performed at 14 days using the $\mathrm{\Delta }\mathrm{\Delta }\mathrm{Ct}$ method. These genes are involved in the osteogenic process, and they are the most characterized to evaluate bone differentiation of cells.^32–36 As shown in Fig. 8, the real-time PCR data revealed no significant differences between laser-treated/untreated cells cultured in PM in the expression of ALP and COL-I, while a statistically significant down regulation of BOSP and OP expressions ($p<0.05$), which are genes related to the final phases of osteogenic differentiation, was observed [Fig. 8(a)]. No evident fold-increase was detected for these genes between laser-stimulated/unstimulated cells cultured in OM [Fig. 8(b)]. These findings were confirmed by the confocal analysis of COL-1 and osteocalcin (OCN) expression in laser-treated and untreated cultures in PM [Figs. 9(A) and 9(B)]. Instead, a more intense green fluorescence signal for COL-I and OCN in laser-stimulated/unstimulated cells was observed in osteogenic culture conditions compared with PM [Figs. 9(A) and 9(B)]. In Table 4, data are reported for the extracellular matrix protein deposition on day 14 of culture. An enhancement in COL-III, ALP, and DEC deposition was observed in laser-treated cells in PM. These were, respectively, 1.8-, 2-, and 1.8-fold greater when compared with unstimulated cells in PM ($p<0.05$). On the contrary, the level of bone proteins was not statistically different when measured in cells stimulated with laser and cultured in the presence of osteogenic factors compared with osteogenic control (Table 3; $p>0.05$). Moreover, two weeks after seeding, SEM revealed that cells formed a confluent multilayer with a more evident cellular density in laser-stimulated/unstimulated samples in PM [Figs. 10(a) and 10(b)] as compared with OM cultures [Figs. 10(c) and 10(d)]. At higher magnification, the characteristic cell morphology of PM and OM cultures following laser irradiation were observed: in PM, cells exhibited a round shape, while in OM, they were flat and elongated, typical of differentiated cells. These results suggested that the laser treatment did not interfere with proliferation and differentiation processes.

Fig. 7

Assay for gene transcription of cells cultured in PM, or irradiated with three consecutive doses at $3\mathrm{J}/{\mathrm{cm}}^2$ and cultured in PM, or cultured in OM, or irradiated with three consecutive doses at $3\mathrm{J}/{\mathrm{cm}}^2$ and cultured in OM. The indicated reverse transcriptase-polymerase chain reaction (RT-PCR) products were subjected to electrophoresis on 2% agarose gel and visualized by UV exposure. The level of specific bands was normalized for glyceraldehyde-3-phosphate dehydrogenase cDNA.

Fig. 8

Expression of the indicated bone-specific genes as determined by $q$RT-PCR. Saos-2 were stimulated for three consecutive days with a dose of $3\mathrm{J}/{\mathrm{cm}}^2$ and then cultured in PM (a) or in OM (b). The graph shows the fold induction of gene expression (arbitrary units), setting the expression of the indicated genes in cells grown in the absence of laser treatment at day 14 of culture. A $p<0.05$ was considered statistically significant. Data are representative of one of the three experiments performed.

Fig. 9

Representative images of osteoblast protein markers after laser treatment by confocal laser scanning microscope. Saos-2 were treated for three consecutive days at $3\mathrm{J}/{\mathrm{cm}}^2$ and then cultured in PM [(Aa), (Ab), (Ba), and (Bb)] or in OM [(Ac), (Ad), (Bc), and (Bd)]. Immunofluorescence analysis was performed at day 14 of culture. Coll-1 (A) and osteocalcin (B) (in green), nuclei (in blue) ($40\times$ magnification, scale bar represents 50 μm).

Table 4

Normalized amount of extracellular matrix constituents secreted and deposited by Saos-2 with or without three doses laser of 3  J/cm2 in proliferative medium (PM) or osteogenic medium (OM) after two weeks of cell culture (pg/cell×well). In comparison to unstimulated samples, a p value <0.05 was considered statistically significant (*).

Fig. 10

SEM images of Saos-2 cells after laser treatment. Cells were irradiated with three consecutive doses at $3\mathrm{J}/{\mathrm{cm}}^2$ and cultured in PM [(a) and (b)] or in OM [(c) and (d)] at $250\times$ magnification, scale bar represents 10 μm (insert at $1000\times$, scale bar represents 10 μm).