2.1.

Surgical Procedure

Twenty male rats (Rattus norvegicus, Wistar) ($350\pm 10\mathrm{g}$; 12 weeks) were used in this study. After intraperitoneal anesthesia with ketamine/xylazine ($80/10\mathrm{mg}/\mathrm{Kg}$), a 1-cm incision was made in the skin to expose the tibia and after that, a standardized 3.0-mm-diameter bone defect was created bilaterally by using a motorized round drill (BELTEC^®, Araraquara-SP, Brazil), under constant physiologic saline solution irrigation. Thereafter, the cutaneous flap was joined and sutured with resorbable Vicryl^® 5-0 (Johnson & Johnson, St. Stevens-Woluwe, Belgium). In order to minimize postoperative discomfort, the animals received analgesia (i.m., $0.05\mathrm{mg}/\mathrm{kg}$ buprenorphine Temgesic; Reckitt Benckiser Health Care Limited, Schering Plough, Hoddesdon, UK) and were returned to their cages. The health status of the rats was monitored daily.

2.2.

Experimental Groups

Animals were randomly divided into two groups of 10 animals each:

2.3.

Low Level Laser Therapy

A laser (Thera laser, DMC^® São Carlos, Brazil) of 0.6 mm beam diameter with a continuous wavelength ($\lambda$) of 830 nm, $0.028{\mathrm{cm}}^2$ spot area, 30 mW, 94 s, 2.8 J, $1.071\mathrm{W}/{\mathrm{cm}}^2$, and $100\mathrm{J}/{\mathrm{cm}}^2$ was used. Laser irradiation started immediately after the surgery at one transcutaneous point, above the site of the injury, and it was performed with an interval of 24 h between each session, totaling five sessions (Table 1). Laser irradiation was performed, at one point per tibia, above the site of the injury (using the punctual contact technique) and was applied bilaterally.

Table 1

LLLT parameters.

2.4.

Retrieval of Specimens

Five days postsurgery (24 h after the last laser application), all animals were euthanized individually by carbon dioxide asphyxia. Then, both tibias were removed and rapidly dissected. The left tibias were immediately fixed in 10% formaldehyde (Merck, Darmstadt, Germany) for 24 h. The right tibias were frozen in liquid nitrogen and stored in freezer at $-80°\mathrm{C}$ until microarray analysis was carried out.

2.5.

Histological Analysis

After fixed, the left tibias were decalcified in 4% diamine tetra-acetic acid (Merck, Darmstadt, Germany) for 40 days. Then, the sample was dehydrated in a graded series of ethanol and embedded in paraffin. Therefore, thin sections ($5\mu \mathrm{m}$) were prepared in the longitudinal plane, using a micrometer (Leica RM—2145, Germany).

2.6.

Qualitative Analysis

For the qualitative histopathological analysis, the laminas were stained with hematoxylin and eosin (H.E stain, Merck, Darmstadt, Germany). Histopathological evaluation was performed in a blinded manner, under a light microscope (Olympus, Optical Co. Ltd, Tokyo, Japan). Any changes in the bone defect, such as presence of blood clots, fibrin, inflammatory processes, granulation tissue, woven bone, or even tissues undergoing hyperplastic, metaplastic, and/or dysplastic transformation were investigated in each animal. In addition, histological sections stained by the picrosirius-polarization method were viewed under polarized light to assess the structural changes in the neoforming trabecular matrix. This method allows a qualitative and quantitative evaluation of the stage of bone matrix organization based on the birefringence of the collagen fiber bundles after staining with picrosirius-red and hematoxylin. To perform the analysis, the software ImageJ (version 1.36) was used, and it is possible to identify the brightness of birefringence by calculating the intensity in “pixels” of color given picrosirius-red under polarized light. The intensity of pixels is proportional to the organization of collagen fibers. The collagen fibers are thicker and strongly birefringent presents stained in shades of orange to red, which is the most anisotropic collagen.⁸ Ten fields were evaluated in the defect region, extension of $100\times$, allowing the analysis of the entire focus of the lesion. The values corresponding to each field were added, resulting in the birefringence of collagen fibers of the bone defect per animal.

2.7.

Morphometry Analysis

Sections stained with H.E were used to perform the morphometry of the area of newly formed bone. The analysis was measured in a blind fashion by two observers, using the image analysis system Motican 5.0. Five areas of bone defect region were selected and named C1 (upper region of the border), C2 (right border), C3 (bottom region of the border), C4 (left border), and C5 (central region of the bone defect). The amount of newly formed bone was determined in $\mu {\mathrm{m}}^2$ in each region, and the total newly formed bone was represented as $\mathrm{C}1+\mathrm{C}2+\mathrm{C}3+\mathrm{C}4+\mathrm{C}5$.

2.8.

Immunohistochemistry

After deparaffinization and rehydration in graded ethanol, each specimen was pretreated in a Steamer with buffer Diva Decloaker (Biocare Medical, California) for 5 min for antigen retrieval. The material was preincubated with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) solution for 30 min in order to inactivate endogenous peroxidase and then blocked with 5% normal goat serum in PBS solution for 20 min. Three sections of each specimen were incubated with anticollagen (COL-I, Cat. no. sc-59772) polyclonal primary antibody at a concentration of $1:1200$ (Santa Cruz Biotechnology, Santa Cruz, California). Incubation was performed overnight at 4°C in refrigerated environment. Afterward, two washes were done in PBS for 10 min. Then, incubation of the sections was performed making use of biotin-conjugated secondary antibody anti-rabbit immunoglobulin G (IgG) (Vector Laboratories, Burlingame, California) at a concentration of $1:200$ in PBS for 30 min. The sections were washed twice with PBS followed by the application of preformed avidin/biotin complex conjugated to peroxidase (Vector Laboratories, Burlingame, California) for 30 min. The bound complexes were visible by the application of a 0.05% solution of 3-3′-diaminobenzidine solution and counterstained with Harris hematoxylin. In order to carry out control studies of the antibodies, the serial sections were treated with rabbit IgG (Vector Laboratories, Burlingame, California) at a concentration of $1:200$ instead of the primary antibody. Furthermore, internal positive controls were performed with each staining bath.

Collagen immunoexpression was assessed both qualitatively (presence of the immunomarkers) and semiquantitatively in five predetermined fields using a light microscopy (Leica Microsystems AG, Wetzlar, Germany) according to a previously described scoring scale ranging from 1 to 4 (1 = absent, 2 = slight, 3 = moderate, and 4 = intense) for immunohistochemical analysis.²⁰ The analysis was performed by two observers (PB and CT), in a blinded manner.

2.9.

RNA Sample Preparation

Total RNA was isolated from the right tibias using the TRIzol^® reagent (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions. After the RNA isolation, the samples were purified using the illustra RNAspin Mini RNA Kit (GE Healthcare Life Sciences) according to the manufacturer’s instructions, quantified by NanoVue spectrophotometer (GE Healthcare Life Sciences). The quality and integrity of the total RNA were confirmed with an Agilent 2100 Bioanalyzer (GE Healthcare Life Sciences) and samples presenting RNA integrity numbered $\ge 8$ were used for cRNA synthesis.

2.10.

Microarray Hybridizations

Agilent whole rat genome microarray $4\times 44\mathrm{K}$ was used to perform microarray hybridizations. The labeling and microarray hybridizations were performed by Agilent using a two-color microarray-based gene expression analysis (Agilent Technologies). Briefly, for cDNA synthesis and labeling, 200 ng of total RNA was used. After, cDNA was transcribed into cRNA, and it was labeled using Agilent low RNA input fluorescent linear amplification kit (Agilent Technologies, Santa Clara, California). Then, cRNA labels were purified, mixed with hybridization buffer, and hybridized to an Agilent whole rat genome microarray $4\times 44\mathrm{K}$ for 17 h at 65°C, according to the manufacturer’s instructions. The hybridized microarrays were washed as the manufacturer’s washing protocol (Agilent Technologies, Santa Clara, California). After hybridization, microarrays were sequentially washed: 1 min at room temperature in GE wash buffer 1 (Agilent Technologies), then 1 min at 37°C in GE wash buffer 2 (Agilent Technologies), followed by 10 s in acetonitrile wash (Agilent Technologies), and finally 30 s in stabilization and drying solution wash (Agilent Technologies). Afterward, microarray slides were scanned using GenePix^® 4000B microarray scanner (Molecular Devices) while simultaneously scanning the Cy3 and Cy5 channels at a resolution of $5\mu \mathrm{m}$. Laser was set at 100% and PMT gain was automatically adjusted for each slide using the program GenePix 4000B according to the intensity of the signal in each array.

2.11.

Microarray Data Analysis

Microarray data analysis was performed as described by Castro et al.²¹ Data files were generated using Agilent’s feature extraction software (version 11.5, Agilent) and the default parameters, which include Lowess-based signal normalization. The dye-normalyzed values generated in the feature extraction data files were used to upload the software express converter (version 2.1, TM4 available at Ref. 22), which conveniently converts the Agilent file format to MeV (multiexperiment view) file format compatible to the TM4 softwares for microarray analysis (available at Ref. 23). The MeV files were then uploaded in the MIDAS software where the resulting data were averaged from replicated genes on each array, from three biological replicates, taking a total of three intensity data points for each gene. The MeV files generated were then loaded in MeV software where differentially expressed genes were identified using one-class $t$-test ($p<0.01$). Significantly different genes were those whose mean log2 expression ratio over all included samples was statistically different from 0, which indicates the absence of gene modulation.

2.12.

Functional Analyses Using Ingenuity Pathways Analysis Software

A network analysis was performed using the ingenuity pathways analysis (IPA) (Ingenuity Systems)²⁴ algorithm. The lists of differentially expressed genes were entered into the IPA software to explore relevant biological networks and to assess interactions with other genes. A hypothetical global gene interaction network was constructed, showing the most relevant direct and indirect connections of genes found to be regulated under LLLT.

2.13.

Quantitative Real-Time Polymerase Chain Reaction

Real-time polymerase chain reaction (RT-PCR) was performed to confirm the differential expression results obtained by the microarray experiments. Total RNA was extracted and purified using the experimental protocols described above. One microgram of total RNA was reverse-transcribed into cDNA and followed by RT-PCR amplification using rats gene-specific primers (Table 2), which were designed by primerexpress software (Applied Biosystems, Foster City, California). The optimized PCR conditions were: initial denaturation at 94°C for 10 min, followed by 40 cycles consisting of denaturation at 94°C for 15 s, annealing at 60°C for 1 min, and extension at 72°C for 45 s, with a final extension step at 72°C for 2 min. Negative-control reactions with no template (deionized water) were also included in each run. All samples were amplified simultaneously in duplicate in one assay run. Analysis of relative gene expression was performed using the $2\text{-}\mathrm{\Delta }\mathrm{\Delta }\mathrm{CT}$ method. RPS18 was used as a housekeeping gene to normalize our expression data.

Table 2

Real-time PCR primers.

2.14.

Statistical Analysis

The normality of all variables distribution was verified using Shapiro–Wilk’s W test. For morphometric and immunohistochemical analysis, comparisons among groups were performed using $t$-test. STATISTICA version 7.0 (data analysis software system—StatSoft Inc.) was used to carry out the statistical analysis. Values of $p<0.05$ were considered statistically significant.

3.1.

Histological Analysis

An overview of the representative histological sections of all experimental groups is shown in the Fig. 1. For CG, 5 days after the surgery, the bone defect area was filled by inflammatory infiltrate, granulation tissue, and immature newly formed bone [Fig. 1(a)]. At the same experimental period, for the LG, inflammatory infiltrate was presented only in the central region of the defect. The rest of the defect was filled by some areas of granulation tissue and bone ingrowth [Fig. 1(b)]. In addition, picrosirius analysis showed that collagen fiber organization in the LG was enhanced compared to the CG, demonstrated by the increased thickness of the colored areas [Figs. 2(a) and 2(b)].

Fig. 1

Representative sections of hematoxylin and eosin stain. Newly formed bone (Nb), granulation tissue (G), inflammatory infiltrate (In). (a) CG = control group, (b) LG = laser group. $\text{Scale bar}=150\mu \mathrm{m}$.

Fig. 2

Representative sections of picrosirius red stain. Photomicrographs using polarized light illustrating the collagen. Arrow indicates the yellowish and reddish birefringent zones. (a) CG = control group, (b) LG = laser group. $\text{Scale bar}=150\mu \mathrm{m}$.

3.2.

Morphometric Analysis

Figure 3 shows that the animals exposed to laser therapy presented a statistically significant increase in the newly formed bone at the defect area compared to the CG (Fig. 2).

Fig. 3

Means and SD of the newly formed bone tissue of bone area ($\mu {\mathrm{m}}^2$) of the defect after treatments. Significant differences of $p<0.001$ are represented by a single asterisk (*).

3.3.

Immunohistochemical Analysis

Qualitative immunohistochemical analysis demonstrated that COL-I expression was detected in granulation tissue, osteoblast, and bone matrix for both groups [Fig. 4(a)]. However, for the LG, the immunoreactivity of COL-I was mainly observed in osteoblast and newly formed bone, when compared to the CG. Semiquantitative analysis revealed a significant increase in COL-I immunoexpression for the LG compared to the CG, 5 days after surgery [Fig. 4(b)].

Fig. 4

(a) representative sections of COL-I immunohistochemistry. Newly formed bone (Nb), granulation tissue (G), osteoblast, and (Ob) intact bone (*). (b) means and standard error of the mean of scores immunohistochemistry of COL-I. Significant differences of $p<0.05$ are represented by a single asterisk (*).

3.4.

Microarray Analysis

In order to examine genes modulated by LLLT during bone repair on a genome-wide basis, microarray expression profiling was carried out on skeletal tissues. Thus, a total of 382 genes modulated were identified after laser irradiation. It was found that 127 genes downregulate and 255 genes upregulate.

3.5.

Functional Network Analysis

In order to analyze these expression data, a reliable new bioinformatics approach, IPA (Ingenuity, California) was applied to set up a potential network, which is based on the regulated genes in order to identify the molecular events and further unveil the molecular mechanisms regulating the processes of LLLT effects in bone. In order to start building networks, IPA queries the ingenuity pathways knowledge base for interactions between the regulated genes and the genes stored in the database. The networks were identified and ranked according to the score $p\text{-}\text{score}=-\log 10$ ($p$-value). The score takes into account the number of network eligible molecules in the network and its size, as well as the total number of network eligible molecules analyzed and the total number of molecules in the ingenuity knowledge base, which could potentially be included in networks. A $\text{score}>3$ ($p<0.001$) indicates a $>99.9\%$ confidence that a gene network was not generated by chance alone. Furthermore, the IPA score can be interpreted as the probability of getting a network containing at least the same number of network eligible molecules by chance when randomly picking genes that can be in networks from the ingenuity knowledge base. Thus, the major biological processes identified for increased gene expressions related to inflammatory responses, connective tissue development, and skeletal and muscular system activity were investigated (Table 3).

Table 3

Top genetic network.

In view of the aforementioned, several functional groupings of differentially expressed genes were identified. The up- and downregulated connective tissue development and function genes were further examined and revealed some COX-2-related genes, interleukins, growth factors, angiogenic, and osteogenic signaling and provided an initial analysis. However, this report focused on expression of bone collagen of early events of bone healing and the downregulation and upregulation connective tissue genes were examined and revealed the significantly upregulated of type I collagen gene. The altered expression of this gene suggests that LLLT acts on the stimulation of fibriblasts and osteoblast, consequently in bone forming.

3.6.

Validation of Microarray Data by Real-Time Polymerase Chain Reaction

Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis. Thus, qPCR was performed to validate our results of the microarray assays. The genes were selected according to the canonical pathway analysis and the expression profiles of the genes confirmed the microarray results. Our PCR results were consistent with the pathway prediction, showing that the relative mRNA levels of those osteogenic genes were modulated by LLLT (Fig. 5).

Fig. 5

Validation of up- and downregulated gene expression by real-time RT-PCR in irradiated animals. The results are normalized as a ratio of each specific mRNA signal to the RPS18 gene signal within the same sample and the values expressed. The significance of differences was determined using Student’s t-test. *$p<0.05$.