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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2017 May 29;98(2):75–85. doi: 10.1111/iep.12226

Low‐level laser therapy (780 nm) combined with collagen sponge scaffold promotes repair of rat cranial critical‐size defects and increases TGF‐β, FGF‐2, OPG/RANK and osteocalcin expression

Lana Sarita de Souza de Oliveira 1, Aurigena Antunes de Araújo 2,✉, Raimundo Fernandes de Araújo Júnior 3, Carlos Augusto Galvão Barboza 4, Boniek Castillo Dutra Borges 1, José Sandro Pereira da Silva 1
PMCID: PMC5485360  PMID: 28556971

Summary

The aim of this study was to evaluate the effect of collagen sponge scaffold (CSS) implantation associated with low‐level laser therapy (LLLT) on repairing bone defects. A single 5‐mm cranial defect was surgically created in forty Wistar rats, which then received one of the following four interventions (n = 10 per group): no treatment (G0); bone defect implanted with collagen sponge scaffold (CSS) alone (G1); defect treated with low‐level laser therapy (LLLT) (wavelength 780 nm; total energy density 120 J/cm2; power 50 mW) alone (G2); and CSS associated with LLLT treatment (G3). After surgery, animals in each group were euthanized at 21 days and 30 days (n = 5 per euthanasia time group). Bone formation was monitored by X‐ray imaging analysis. Biopsies were collected and processed for histological analysis and immunohistochemical evaluation of transforming growth factor‐beta (TGF‐β), fibroblast growth factor‐2 (FGF‐2), osteoprotegerin (OPG) and receptor activator of nuclear factor ƙ (RANK). Osteocalcin (OCN) was detected by immunofluorescence analysis. Compared to the G0 group, defects in the 30‐day G3 group exhibited increased bone formation, both by increase in radiopaque areas (P < 0.01) and by histomorphometric analysis (P < 0.001). The histopathological analysis showed a decreased number of inflammatory cells (P < 0.001). The combined CCS + LLLT (G3) treatment also resulted in the most intense immunostaining for OPG, RANK, FGF‐2 and TGF‐β, and the most intense and diffuse OCN immunofluorescent labelling at 30 days postsurgery (G3 vs. G0 group, P < 0.05). Therefore, the use of CCS associated with LLLT could offer a synergistic advantage in improving the healing of bone fractures.

Keywords: bone defect, collagen sponge scaffold, FGF‐2, low‐level laser therapy, osteocalcin, TGF‐β

The ability to heal and regenerate is an important feature of bone tissue. Bone healing and regeneration involve the differentiation of pluripotent cells into specialized tissues (e.g. cartilage and bone) by processes that are directly influenced by the mechanical environment. Not all bone fractures are completely repaired. No unions or delayed fractures occur depending on specific geometric, mechanical and biological factors. Such abnormalities justify the use of many different kinds of fixation or osteoinduction devices to improve fracture stabilization or accelerate the repair process. Studies have shown that successful bone reconstruction requires osteoproduction, osteoinduction, osteoconduction, mechanical stimulation and vascularization (Santoni et al. 2007; Matassi et al. 2011; Nagaraja & Jo 2014; Garcia et al. 2016; Meagher et al. 2016; Miron et al. 2016).

In humans, bone is composed of calcium phosphate (69–80 wt.%, mainly hydroxyapatite), collagen (17–20 wt.%), water, proteins and other components (Suchanek & Yoshimura 1998). Two types of cells play important roles in the formation of bone, namely osteoblasts and osteoclasts. During intramembranous ossification in the skull, neural crest‐derived mesenchymal cells proliferate and condense into compact nodules. Some of these cells develop into capillaries, whereas others develop into committed bone precursor cells called osteoblasts. These osteoblasts secrete a collagen–proteoglycan extracellular matrix (ECM) that is able to bind calcium salts (Gilbert & Singer 2000). Osteoblasts also produce osteocalcin (OCN). OCN is a small protein synthesized by a mature osteoblast which is primarily deposited in the ECM, but a small amount of it enters the blood. Hence, OCN represents a sensitive and specific serum marker of osteoblast activity (Lumachi et al. 2012). Studies have shown that higher serum osteocalcin levels are relatively well correlated with increases in bone mineral density (Bharadwaj et al. 2009; Lumachi et al. 2012; Karsenty & Oury 2014; Lambert et al. 2016; Zhong et al. 2016).

Collagen is the most abundant macromolecule in the body and exists as an extracellular matrix (ECM) of various tissues (Ueda et al. 2002). The extracellular matrix (ECM) provides physical support to tissues by occupying the intercellular space, acting as a dynamic, mobile and flexible substance defining cellular behaviours and tissue function. For most soft and hard connective tissues (bone, cartilage, tendon, cornea, blood vessels and skin), the collagen fibril network plays a dominant role in maintaining the biological and structural integrity of the ECM, and it is highly dynamic, undergoing constant remodelling for maintenance of normal physiological functions (Aszodi et al. 2006; Alford et al. 2015; Xue & Jackson 2015; Mongiat et al. 2016; Quigley et al. 2016).

Collagen fibres, while providing the framework for mineral deposits, are the principal source of bone tissue tensile strength. The mechanical properties of bone can be modified by varying fibril orientation, which may result from the degree of collagen cross‐linking and the mineral–organic composite remodelling process (Taylor 2007).

When implanted in a bone defect, biomaterial has been shown to facilitate the repair, but if the defect is large, this biomaterial alone will not perform as expected. Thus, there is a need to investigate the effectiveness of other techniques, such as the use of bone morphogenetic proteins (BMPs) (Kim et al. 2014), growth factors (Ueda et al. 2002) and low‐intensity lasers (Kazem shakouri et al. 2010), in facilitating the bone repair process.

Studies have demonstrated success in repairing bone defects with the application of chemical stimuli (e.g. BMPs) (Yang et al. 2013; Bosemark et al. 2015; Ribeiro et al. 2015), as well as physical stimuli such as ultrasound, electromagnetic fields and low‐level laser therapy (LLLT) (Maman Fracher Abramoff et al. 2014; Tim et al. 2014; Akyol et al. 2015; Acar et al. 2016). A previous study showed that infrared LLLT applied to surgically created bone defects in rats shortened the bone healing process by stimulating the modulation of the initial inflammatory response, resulting in quicker restoration of normal status (Pretel et al. 2007). Therefore, the aim of this study was to evaluate the effectiveness of a combined treatment using collagen sponge scaffold (CCS) associated with low‐level laser therapy (LLLT) on the repair of critical‐size bone defects in rats.

Materials and Methods

Animals

Male adult Wistar rats weighing 250–300 g were obtained from the vivarium of the Department of Biophysics and Pharmacology of UFRN, Brazil. All animals were housed in an animal room under standard laboratory conditions at 22 ± 2°C with a 12‐h/12‐h light/dark cycle. Animals were fed pelleted food and water ad libitum. They were acclimatized for 7 days and fasted for 12 h before the experiments. All efforts were made to minimize the number of animals used and their suffering.

Ethical approval statement

The controlled experimental study was approved by the Animal Ethics Committee (CEUA) of the Federal University of Rio Grande do Norte/UFRN/Brazil (number 067/2014).

Surgery cranial critical bone defect

Rats were anaesthetized with an intraperitoneal injection of ketamine hydrochloride 10% (80 mg/kg; Vetnil, São Paulo, Brazil) and xylazine 2% (10 mg/kg, Sytec, São Paulo, Brazil). A skin incision from the front nose area to the external occipital protuberance was made in each rat, and the entire surface of the calvaria was exposed. A single 5‐mm cranial defect was made in the parietal bone using a trephine drill driven by a 30,000‐rpm electric micromotor (Surgic XT Plus; NSK, Tochigi, Japan) under constant irrigation of sterile saline solution to avoid bone overheating (Paraguassu et al. 2012). The analgesic dipyrone (120 mg/kg/day; Neoquimica, São Paulo, Brazil) was administered orally by gavage for 5 days.

Experimental groups

Forty Wistar rats were randomly divided into the following four surgical intervention groups (n = 10 per group): G0, no treatment; G1, bone defect implanted with collagen sponge scaffold (CSS) alone; G2, defect treated with low‐level laser therapy (LLLT) (wavelength 780 nm; total energy density 120 J/cm2; power 50 mW) alone; and G3, combined CCS associated with LLLT treatment. Half of the rats in each group were designated to be euthanized at 21 days postsurgery, while the other half in each group were designated to be euthanized at 30 days postsurgery (n = 5 per euthanasia time group). Selection of the 21‐and 30‐day observation periods before euthanasia is based on the previously established characteristics of cranial bone wound healing. The 21‐day period represents the bone repair phase, and the 21‐to 30‐day period represents the remodelling phase, both of which are mediated by growth factors (Stroncek & Reichert 2008).

Experimental protocols

In the G1 (CSS treatment) and G3 (CSS + LLLT treatment) groups, the surgical defects were immediately filled with a collagen sponge (Hemospon®; Technew, São Paulo, Brazil). In the G2 (LLLT) and G3 (CSS + LLLT treatment) groups, the surgical areas were irradiated with a GaAlAs diode laser (MMOptics, Brazil) in continuous mode using a power of 50 mW, wavelength of 780 nm and total energy density of 120J/cm2. Applications were made at four equidistant points 1 mm distant from the edge of the defect, immediately after surgery and at intervals of 48 and 96 h (Marques et al. 2015). The surgical defects in the G3 group were first filled with collagen sponge and then irradiated.

Radiographic (X‐rays) analysis

The animals were submitted to euthanasia after the designated postsurgery observation period by intraperitoneal administration of thiopental 100 mg/kg (Thiopentax; Cristália, São Paulo, Brazil). The skull cap was removed and preserved in 10% formaldehyde for 24 h. On the following day, the skull cap was placed in ethanol solution and subjected to radiographic evaluation performed with dental X‐ray equipment (Timex 70c, 60 Hz; Gnatus, Brazil). The specimens were fixed at 40 cm distance from the equipment, and the X‐ray exposure time was 0.32 s. Radiographs were scanned and analysed with imagetool software (version 3.0; University of Texas Health Science Center, San Antonio, TX, USA). As the rat calvarial defect is relatively two‐dimensional, planar radiography can assess the bridging of the defect by mineralized tissue (Spicer et al. 2012). Skull images were scored based on the following percentages of total area with visible radiopacity consistent with mineralized bone tissue (Pryor et al. 2005): 0 = 0–25%; 1: 25.1–50%; 2: 50.1–75%; and 3: 75.1–100%.

Histological analysis

Bone tissue was paraffin‐fixed and sectioned in the laboratory and subsequently analysed by light microscopy in the Department of Morphology. Briefly, 10 cranial specimens per surgical intervention group were fixed in 10% neutral buffered formalin and demineralized in 5% nitric acid. The specimens were then dehydrated and embedded in paraffin, the calvarial defects were cut into 5‐μm transverse sections and the sections were stained with haematoxylin and eosin. The slides were examined under light microscopy by two pathologists blinded to the treatment of each group. The histological score was based on the following criteria (Pretel et al. 2007): (i) the degree of inflammation: 1 =  absence of inflammatory cells, 2 =  moderate presence of inflammatory cells and 3 =  intense presence of inflammatory cells; (ii) formation and quality of bone tissue: 1 =  new tissue formation (filling of the defect with connective tissue containing blood capillaries, fibroblasts, macrophages and newly formed collagen fibres), 2 =  dense connective tissue suggesting bone tissue differentiation with presence of a large number of osteogenic and osteoprogenitor cells and organizing fibres, 3 =  new bone formation in which the connective tissue is differentiating to form a bone matrix or osteon and 4 =  presence of mature bone tissue; and (iii) collagen maturation: 1 =  surgical wound filled with connective tissue, but no evidence of bone union – isotropy (absence of birefringence); 2 =  osteon (formation of connective tissue in bone with osteoprogenitor and osteogenic cells) – low anisotropy; 3 =  isolate immature bone spicules – moderate anisotropy; and 4 =  compact bone formation – intense anisotropy (total polarization). The scores for each of the criteria (i), (ii) and (iii) were added, and the mean and standard error (SE) were calculated for each group.

The stained sections were digitally photographed under a microscope (Digital Camera DXM200F; Nikon, Japan). Image analysis was used to estimate the newly forming bone at 21 days and 30 days to calculate the percentage (quantitative analysis) of newly formed bone within the defect using computerized image analysis software ImageJ 6.0. Briefly, the image was corrected for optical density before the region of interest (ROI) in mm was selected in accordance with the specific colour for the region of new bone formation in the histological slide. Then, ROI parameters such as area were calculated. Automation of these steps is included in an algorithm referred to as a macro. The macro was used to normalize the selection of other ROIs. The percentage of newly formed bone was determined by ROI area/total area of images. Five sections from each sample were used for semiquantitative image analysis. Five regions in each section were photographed at 200× magnification. A total of 25 images for each sample were digitalized (Zong et al. 2010). Furthermore, images of tissue fragments stained by H&E were captured as described by the inflammation quantification. The number of inflammatory cells (μm2) was determined using ImageJ software (Dantas et al. 2014).

Immunohistochemical analysis

Each paraffin tissue section (three for each calvarial defect) was deparaffinized, rehydrated and washed with 0.3% Triton X‐100 in phosphate buffer, and the endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Tissue sections were incubated overnight at 4°C with primary antibodies (Santa Cruz Biotechnology, INTERPRISE, Brazil) against receptor activator of nuclear factor κB (RANK), osteoprotegerin (OPG), fibroblast growth factor‐2 (FGF‐2) and transforming growth factor‐beta (TGF‐β) (all at 1:400 dilution). Sections were then washed with phosphate buffer and incubated with streptavidin–horseradish peroxidase (HRP)‐conjugated secondary antibody (Biocare Medical, Concord, CA, USA) for 30 min. Immunoreactivity to RANK, OPG, FGF‐2 and TGF‐β was visualized with the colorimetric‐based TrekAvidin‐HRP Label + Kit (Dako‐Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's protocol. Immunohistochemical analysis of all four aforementioned markers was performed on the best specimens of the 30‐day groups (G0 and G3). Tissue was processed as previously described (Medeiros et al. 2011). Six 4‐μm‐thick sections of the surgical area were obtained from each group using a microtome. Samples were transferred to gelatine‐coated slides.

Immunofluorescence

Three tissue sections from each animal (three animals per group) were deparaffinized in xylene and washed in a series of ethanol and PBS concentrations. Antigen retrieval was performed by placing the sections in 10 mm sodium citrate with 0.05% Tween‐20 for 40 min at 95°C. Autofluorescent background noise was reduced by incubating the sections in 0.1% Sudan black in 70% ethanol for 20 min at room temperature (RT). The sections were incubated overnight with rabbit anti‐osteocalcin primary antibody (1:100, respectively, in 1% normal goat serum; Santa Cruz Biotechnology, USA) at 4°C, washed three times in 0.2% Triton X‐100 for 5 min and incubated with Alexa Fluor 488‐conjugated goat anti‐rabbit secondary antibody (1:500 in BSA 1%) and DAPI nuclear counterstain (SIGMA, USA). Finally, the sections were mounted with Vectashield medium.

Fluorescent images were obtained on a Carl Zeiss Laser Scanning Microscope (LSM 710, 20 ×  objective, Carl Zeiss, Jena, Germany, Brazil). Known positive controls were included in each batch of samples. Tissue reactivity in all groups (G0‐30d, G1‐30d, G2‐30d and G3‐30d) was assessed by computerized densitometry analysis of digital images captured with the aforementioned immunofluorescence microscope. Average densitometric values were calculated in imagej software (http://rsb.info.nih.gov/ij/). Contrast index measurements were obtained from the formula (selected area × 100)/total area after removal of background in regions of interest (three samples per animal).

Statistical analysis

Data are presented as the mean ± standard error of the mean or as the median (range), when appropriate. Analysis of variance (anova) followed by Bonferroni's test was used to calculate the means. The Kruskal–Wallis test followed by Dunn's test was used to compare medians/scores (graphpad prism 5.0 Software, La Jolla, CA, USA). A P‐value <0.05 indicated a statistically significant difference.

Results

Radiographic (X‐rays) analysis

The healing process was radiologically monitored for 21 days and 30 days. The results showed no significant differences in scores for X‐ray analysis in the groups [G1 – 0 (0–1); G2 – 0 (0–0.5); G3 – 0 (0–1)] treated for 21 days when compared to the G0 – 0 (0‐0) (P > 0.05; Figure 1). It was possible to visualize increased radiopaque area percentage in groups with treatment at 30 days when compared to the G0 – 0 (0–0): G1 – 1 (0–2); G2 – 1 (0.5–1.5) (P < 0.05); and G3 – 2 (0.75–2) (P < 0.01) respectively (Figure 1). It was possible to verify between 50 and 75% of new bone formation in the cranial defect area of group G3, 30 days (Figure 1). Quantification of the X‐ray films showed a significantly homogenous callus in the 30‐day G3 group.

Figure 1.

Figure 1

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Radiographic images of each group. D: interval of experiment (days). Key: G0 21d 0 (0–0); G0 30d 0 (0–0), G1 21d 0 (0–1); G1 30d 1 (0–2); G2 21d 0 (0–0.5); G2 30d 1 (0.5–1.5); G3 21d 0 (0–1); G3 30d 2 (0.75–2). Results of X‐ray analysis of scores between the groups. 21D: interval of 21 days; 30D: interval of 30 days. *P < 0.05; **P < 0.01. Images are shown at 40× magnification.

Histological analysis

At 21 days, the G0 group showed intense infiltration of inflammatory cells, new tissue formation and no evidence of union bone. At 30 days, this group exhibited the same characteristics present at 21 days, except for a reduction in inflammatory cells (Figure 2a,e), and a histological score of 5 (5–5). In the 21‐day G1 group, moderate inflammatory infiltrate, dense connective tissue and new bone tissue were found, a score of 6 (6–6) (P < 0.05) when compared to the G0, whereas at 30 days the G1 group exhibited new compact bone formation (Figure 2b,f), a score of 7 (7–7) (P < 0.001). The 21‐day G2 group showed intense inflammatory infiltrate and dense connective tissue, a score of 6.75 (6.25–7) (P < 0.001), while at 30 days the G2 group exhibited formation of new compact bone (Figure 2c,g), a score of 7 (7–7) (P < 0.001). The ImageJ 6.0 software image analysis showed qualitative and quantitative newly formed bone and inflammatory cells (Figure 3), as observed in the respective G1 group vs G3 group. The 21‐day G3 group showed significantly more optical density in ROI when compared to the G0 (P < 0.01) and exhibited moderate inflammatory infiltrate (Figure 3, d1 and a1, respectively). The 30‐day G3 group also exhibited the greatest extent of new bone formation, 8.45 (8–8.75), compared to that of the G0 group (P < 0.001; Figure 3). The number of inflammatory cells (μm2) significantly decreased among groups: 30‐day G3 group and G0 (P < 0.001), and G3 group and G1 (P < 0.01) (Figure 3h,f respectively).

Figure 2.

Figure 2

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Histopathological analysis. Capital letters indicate groups and intervals: (a) G0 21 days; (b) G1 21 days; (c) G2 21 days; (d) G3 21 days; (e) G0 30 days; (f) G1 30 days; (g) G2 30 days; (h) G3 30 days. Small letters refer to (a) secondary bone; (b) immature bone (osteoid); and (c) newly formed connective tissue (periosteum). Histological scores * p< 0.05, *** p< 0.001.

Figure 3.

Figure 3

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Histomorphometric analysis. The bone formation (blue arrow) was calculated by the ImageJ program. Number of inflammatory cells (in red) (a). Key: a, a.1: G0 21d; e, e.1: G0 30d; b, b.1: G1 21d; f, f.1: G1 30d; c, c.1: G2 21d; g, g.1: G2 30d; d, d.1: G3 21d 0 (0–1); h, h.1: G3 30d. The 30‐day G3 group had a higher area of repair (h) than the 30‐day G0 group (e). Besides, inflammatory cells (in red) were quantified where the number decreased in all the groups (b.1–d.1 and f.1–h.1) when compared to G0 21 days (a.1) and GO 30 days (e.1) when compared to G0 21 days (a.1) and GO 30 days (e.1). Percentage of optical density ROI (area/total area of images in mm). Number of inflammatory cells per μm² of the groups at 21 and 30 days. *P < 0.05, **P < 0.01, ***P < 0.001. Images are shown at 40× magnification. Scale bar = 100 μm

Immunohistochemistry analysis

Quantitative analysis of cranial bone expression levels of RANK, OPG, FGF‐2 and TGF‐β in the 30‐day G0 and G3 groups revealed notable differences. Compared to G0, the G3 group exhibited increased immunoreactivity of all four markers (Figure 3). Particularly strong OPG immunostaining was detected on the immature bone surface. Similarly, strong FGF‐2 and TGF‐β immunoreactivity was detected in neoformed connective tissue (Figure 4).

Figure 4.

Figure 4

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Immunostaining of RANK, OPG, FGF‐2 and TGF‐β in calvarial bone. Representative photomicrographs of critical defect in calvarial bone of the 30‐day G0 and G3 rats. (a) G0: 30 days, RANK (b) G3: 30 days, RANK (c) G0: 30 days, OPG (d) G3: 30 days, OPG (e) G0: 30 days, FGF (f) G3: 30 days, FGF (g) G0: 30 days, TGF‐β (h) G3: 30 days, TGF‐β. Stars indicate the strongest immunoreactivity. Images are shown at ×40 magnification. Scale bar = 100 μm.

Immunofluorescence

Cellular osteocalcin labelling (green) was strong and diffuse in the G3 group at 30 days (Figure 5d), weak in the G1 (Figure 5b) and moderate in the G2 (Figure 5c). Densitometric analysis confirmed that there was significantly increased osteocalcin immunoreactivity in the G3 group at 30 days (Figure 5d), compared to the G0 group (Figure 5a). Densitometric values were significantly different (P < 0.01) between the G3 and G0 groups (Figure 4e).

Figure 5.

Figure 5

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Representative confocal photomicrographs of osteocalcin immunofluorescence (green) in rat cranial critical‐size defect bone specimens with DAPI nuclear counterstained (blue) (a–d). G0 group – weak osteocalcin labelling (a). G1 and G2 groups – moderate osteocalcin labelling (b and c respectively); and G3 group – strong osteocalcin labelling (d). Scale bar, 100 mm; magnification, ×10. (e) Densitometric analysis showing increased osteocalcin labelling in the G3. Each column represents the mean + SE of the five sections from each of three animals (n = 15 sections) **P < 0.01 per group.

Discussion

In the present study, we evaluated the effectiveness of surgical CSS implantation with LLLT in repairing rat cranial critical‐size defects. Unlike the G0 (untreated) group, radiopaque areas indicating mineralized bone tissue could be detected in the 30‐day G1 group, the defect of which was surgically implanted with the CSS. In a previous study that evaluated the effectiveness of CSS implantation in facilitating bone formation in rat calvarial defects, the morphometric findings indicated more bone formation in CSS‐treated defects compared to the non‐treated ones (Santos Tde et al. 2015). Bone perforation may have created an environment which caused recruitment of the bone marrow cell into the sponge so that proliferation and differentiation could occur (Shimoji et al. 2009). Histological analysis revealed the occurrence of new compact bone formation in the G1 calvarial defects.

In the G2 group, LLLT treatment also resulted in the appearance of radiopaque areas at 30 days postsurgery. It is known that LLLT can stimulate pluripotent mesenchymal cells to differentiate into bone matrix‐producing osteoblasts, which rapidly become osteocytes. Laser energy can also excite intracellular chromophores (e.g. porphyrins and cytochromes) and thus induce an increase in cell activity and consequently an increase in ATP concentration, resulting in calcium release (Cunningham 1970). More recently, another study has shown that LLLT can modulate the metabolism of human osteoblasts, thereby increasing their proliferation and expression of osteogenic differentiation markers (Oliveira et al. 2016). Thus, it appears that LLLT enhances bone tissue repair by changing bone dynamics, consequently shortening the time period involved in repair (De Souza Merli et al. 2012).

The 30‐day G3 group exhibited a remodelled bone for X‐ray analysis and a high percentage of new bone compared with the G0 group. The radiographic results were further supported by histomorphometric observation of a high percentage of new lamellar bone.

LLLT has been shown to have a positive effect on fracture healing and bone tissue metabolism (Kazem shakouri et al. 2010), and in vitro studies have demonstrated that laser‐treated osteoblasts exhibit increased osteopontin and osteocalcin gene expression, alkaline phosphatase (ALP) activity and proliferation (Pires Oliveira et al. 2008; Renno et al. 2010).

Soares et al. (2014) evaluated femur bone healing in rats treated with biomaterials and an LLLT protocol similar to ours. They found that in the group treated with only laser therapy (780 nm, 70 mW, 140 J/cm2 total treatment, distributed among 4 points), newly formed bone, collagen, inflammatory cells and osteocytes were all present at 30 days post‐treatment (Soares et al. 2014).

In addition to increased radiopaque areas shown by bone healing histology, the 30‐day G3 group exhibited stronger expression of OPG, RANK, FGF‐2 and TGF‐β compared to the 30‐day G0 group. High expression of these markers collectively indicates the presence of high osteoblastic activity, with increased production of organic (collagen) matrix and inhibition of osteoclastogenesis. Increased RANK expression in response to the combined CSS plus LLLT treatment suggests involvement of the RANK/RANKL signalling pathway in the bone tissue repair process mediated by osteoblasts and osteoclasts (Walsh & Choi 2014). However, OPG was even more strongly expressed on the immature bone surface, indicating an inhibition of osteoclastogenesis, and thus protection against excessive tissue resorption (Walsh & Choi 2014). It has been shown that the OPG/RANKL ratio is initially increased after the injury as well as during the resorption period, but it is later decreased during secondary bone formation and bone turnover (Gerstenfeld et al. 2003). This finding suggests that bone cells are inhibiting resorption and promoting bone formation during the earlier stages of the repair period.

The high level of FGF‐2 immunostaining observed in the 30‐day G3 group indicates high infiltration of fibroblasts, which in turn signifies enhanced synthesis of collagen I, the main component of organic bone matrix (osteoid). This result was confirmed by the detection of newly formed connective tissue during the histological analysis. These collagen‐producing fibroblasts play an important role in maintenance of the ECM, and the FGF signalling in these cells may regulate intramembranous bone formation (Su et al. 2014).

TGF‐β is a growth factor associated with cell development and differentiation, and its expression was also increased in the 30‐day G3 group. It can also be involved in forming a callus during repair and is capable of inducing the expression of ECM proteins (Bostrom 1998; Hyytiainen et al. 2004). Callus formation depends on the recruitment of mesenchymal stem cells from the adjacent soft tissue, cortex, marrow and periosteum. In our study, the recruited cells we found in the treated calvarial defects were undoubtedly fibroblasts derived from periosteum, as evidenced by the newly formed connective tissue. TGF‐β is one of the factors responsible for this fibroblast recruitment, after which a spike in collagen I and II production occurs to form the osteoid (Gerstenfeld et al. 2003). The greatest production of organic bone matrix was observed in the 30‐day G3 group, which confirms previous studies that showed more efficient results when collagen was combined with some other repair‐facilitating technique (Jansen et al. 2009; Maraldi et al. 2013).

In addition to increased expression of the aforementioned immunohistochemical markers, OCN expression in the 30‐day G3 group was also increased. It has been shown that laser irradiation after tooth extraction can promote osteoblast differentiation, as evidenced by increased OCN expression; thus, this finding suggests that OCN may be a molecular mechanism involved in the laser‐induced enhancement of bone healing. It is our belief that the CSS served as a support matrix for cell differentiation and ECM formation stimulated by the laser treatment, which resulted in the greatest extent of radiographic bone healing.

One of the limitations of our study was the use of X‐rays. X‐rays are normally used in medical diagnosis to obtain a visual image of the radiographed tissue. The image results from the differential attenuation of the radiation which depends on the thickness, density and configuration of the irradiated organ and on the proportion and nature of the different chemical elements present. The nature of biological material is such that the contrast differentiation between organs or parts of an organ is frequently poor, and despite methods to increase the contrast, this remains one of its principal limitations (McKenna et al. 2012).

In conclusion, the results of our study demonstrate that either CSS or LLLT alone can facilitate repairing rat cranial critical‐size defects. The effects of these techniques on bone tissue repair are mediated by increased proliferation of osteoblasts and fibroblasts and increased collagen and osteoid synthesis. The combined use of CSS plus LLLT has a greater effect on repairing these calvarial defects than either technique alone. Therefore, this combined technical approach could offer a synergistic advantage in enhancing the healing of bone fractures.

Conflict of interest

The authors declare no conflicts of interests.

Funding source

The authors declare that there is no funding source for this article.

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