The energy density of laser light differentially modulates the skin morphological reorganization in a murine model of healing by secondary intention

Abstract

This study investigates the influence of gallium–arsenide (GaAs) laser photobiostimulation applied with different energy densities on skin wound healing by secondary intention in rats. Three circular wounds, 10 mm in diameter, were made on the dorsolateral region of 21 Wistar rats weighting 282.12 ± 36.08 g. The animals were equally randomized into three groups: Group SAL, saline solution 0.9%; Group L3, laser GaAs 3 J/cm²; Group L30, laser GaAs 30 J/cm². Analyses of cells, blood vessels, collagen and elastic fibres, glycosaminoglycans and wound contraction were performed on the scar tissue from different wounds every 7 days for 21 days. On day 7, 14 and 21, L3 and L30 showed higher collagen and glycosaminoglycan levels compared to SAL (P < 0.05). At day 21, elastic fibres were predominant in L3 and L30 compared to SAL (P < 0.05). Type-III collagen fibres were predominant at day 7 in both groups. There was gradual reduction in these fibres and accumulation of type-I collagen over time, especially in L3 and L30 compared with SAL. Elevated density of blood vessels was seen in L30 on days 7 and 14 compared to the other groups (P < 0.05). On these same days, there was higher tissue cellularity in L3 compared with SAL (P < 0.05). The progression of wound closure during all time points investigated was higher in the L30 group (P < 0.05). Both energy densities investigated increased the tissue cellularity, vascular density, collagen and elastic fibres, and glycosaminoglycan synthesis, with the greater benefits for wound closure being found at the density of 30 J/cm².

Laser photobiostimulation has been used as a non-invasive alternative to treat muscle injuries and skin wounds, and to control inflammatory processes and pain (Enwemeka et al. 2004; Reddy 2004). Although the use of laser light to accelerate the healing process was documented in the literature for the first time in 1971 (Mester et al. 1971; Shields & O'Kane 1994), and the efficacy of this therapeutic modality is proven, parameters about how it is used are still controversial (Tuner & Hode 1998; Moore et al. 2005). Parameters such as the type and source of laser light emission, number of applications, duration of treatment and mechanisms of action through which the laser light exerts its effects remain the focus of investigation in the ongoing search for efficient methodologies that justify and encourage the use of laser light in clinical practice. Several mechanisms have been proposed to explain the effects of laser light on biological tissues, including the absorption of light by the enzymes of the electron transport chain in the inner mitochondrial membrane, stimulation of the production of oxygen, and cell proliferation induced by photoactivation of the calcium channels (Shields & O'Kane 1994; Breitbart et al. 1996). Recent studies show that the main cells stimulated by laser light are macrophages and fibroblasts (Gonçalves et al. 2010a; Xavier et al. 2010). Macrophages are important cells responsible for releasing growth factors that stimulate proliferation, differentiation and synthesis of extracellular matrix components (Shields & O'Kane 1994; Reddy 2004; Gonçalves et al. 2010b). In in vitro experimental models examination of a wide range of wavelengths showed that wavelengths between 524 nm and 904 nm were related to decreased time of wound healing by stimulating fibroblast and keratinocyte differentiation, collagen production and skin neovascularization (Pogrel et al. 1997; Demidova-Rice et al. 2007).

Previous studies have shown that the gallium–arsenide laser (GaAs λ 660 nm) is able to stimulate skin wound healing in humans and laboratory animals with energy densities between 1 and 4 J/cm² (Medrado et al. 2003; Pugliese et al. 2003; Reddy 2004). However, most of the work is restricted to investigating the effect of energy densities below 4 J/cm², and reports on the effects of high energy densities in tissue repair are scarce and inconclusive. Thus, this study was designed to investigate the influence of laser photobiostimulation applied with different energy densities in a rat model of skin wound healing by secondary intention.

Materials and methods

Animals

Twenty-one male Wistar rats (Rattus norvegicus), 10 week old and weighing 282.12 ± 36.08 g, obtained from the Biological Sciences Center, Federal University of Viçosa, Minas Gerais, Brazil, were used in this study. During the experiment, the animals were allocated to individual cages that were cleaned daily and maintained in an environment with controlled temperature (22 ± 2 °C), light (12 h light/dark cycles) and humidity (60–70%).

Ethical approval

The experiment was conducted in accordance with International Ethical Standards for the Care and Use of Laboratory Animals and approved by the Ethics Committee for the Care and Use of Laboratory Animals of the Federal University of Viçosa (UFV; registration 005/2008).

Experimental protocol

Before the surgical wounds were made, the animals were anaesthetized using intramuscular ketamine (50 mg/kg) and xylazine (20 mg/kg). Then, trichotomy was performed on the dorsolateral region of the animals, and the area was defatted using ethyl ether (Merck®, Rio de Janeiro, Brazil) followed by the use of 70% ethanol and 10% povidone–iodine for anti-sepsis (Johnson Diversey®, Rio de Janeiro, Brazil). Three circular secondary intention wounds 10 mm in diameter were made in the dorsolateral region of the animals by removing the skin with a scalpel until the exposure of the muscle fascia. The standardized wound area was marked with a dermographic pencil and checked using an analogical pachymeter (Kingtools®, São Paulo, Brazil) (Gonçalves et al. 2013). After completion of the wounds, the animals were randomly divided into three groups with seven animals in each. Group saline (SAL, control): saline solution 0.9%; Group L3: GaAs laser (λ 660 nm, 3 J/cm²); Group L30: GaAs laser (λ 660 nm, 30 J/cm²). The laser device (Endophoton^®, KLD, São Paulo, Brazil), which was previously calibrated by the manufacturer, presented an output of 20 mW, power density of 25.47 mW/cm², visible radiation and a 0.79 cm² circular beam. Laser light was applied transcutaneously at six equidistant points around the wound margin. The wounds were irradiated for 118.5 s in L3 to release 3 J/cm² and 1185 s in L30 to release 30 J/cm². The wounds were cleaned daily with 0.9% saline solution immediately before the laser application. The treatments were started immediately after the wound was made once a day for 21 days corresponding to the experiment duration.

Analysis of wound contraction

The progress of wound closure was evaluated by measuring the wound area every 7 days in digitized images with the dimensions of 320 × 240 pixels (24 bits/pixel) obtained using a digital video camera (W320, Sony, Tokyo, Japan). The wound areas were calculated by computerized planimetry using the Image Pro-Plus image analysis software program, version 4.5, (Media Cybernetics®, Silver Spring, MA, USA), previously calibrated. Wound contraction index (WCI) was calculated using the following ratio: initial area of the wound (Ao) − area on the day of measurement (Ai)/initial area of the wound (Ao) × 100 (Gonçalves et al. 2013). The third wound was selected for this analysis because the tissue from this wound was collected on the final day of the experiment (21st).

Analysis of total collagen and glycosaminoglycans

For each group, 35 histological sections 8 μm thick stained with Fast green and Sirius red were used to quantify the levels of collagen and total protein in scar tissue using a previously described spectrophotometric method (López-De León & Rojkind 1985). In this method, the maximal absorbance to the Sirius red (540 nm) and Fast green (605 nm) dyes, correspond to the amount of collagen and non-collagen proteins respectively. For each section used in the collagen analysis, a corresponding serial section was obtained, which was used in the analysis of glycosaminoglycans. The tissue content of glycosaminoglycans was determined according to a modified procedure described by Corne et al. (1974). Sections were transferred immediately to 10 ml of 0.1% (w/v) Alcian blue 8GX solution (0.16 M sucrose solution buffered with 0.05 ml sodium acetate at pH 5). After successive rinses in 10 ml of 0.25 M sucrose solution, dye adhered to the tissue was extracted with 10 ml of 0.5 M magnesium chloride, and the absorbance of the resultant solution was analysed in a spectrophotometer at 580 nm.

Stereological analysis

Tissue fragments were collected from the different wounds every 7 days. Each fragment contained tissue removed from the centre of the wound and part of the uninjured adjacent tissue that had not received laser radiation. The fragments were put into Karnovsky's solution for 24 h and processed for paraffin embedding. Semiserial 4-μm-thick vertical uniform random (VUR) sections were obtained using a rotating microtome (Leica Multicut 2045®, Reichert-Jung Products, Jena, Germany). One of every 20 sections was used to avoid repeating analysis of the same histological area. Sections mounted on histology slides were stained with haematoxylin and eosin for visualization of cells and blood vessels (Karu 2003), Verhoeff's method for elastic fibres (Verhoeff 1908) and Sirius red dye (Sirius red F3B, Mobay Chemical Co., Union, NJ, USA) for marking collagen fibres observed under polarizing microscopy (Junqueira et al. 1979). Analysis of collagen was based on the birefringence properties of the collagen fibres, because under polarization, the thick collagen fibres (type I) appear in shades of bright colour ranging from red to yellow, whereas thin reticular fibres (type III) are shown in bright green (Gonçalves et al. 2010a).

The slides were visualized, and the images captured using a BX-60® light microscope (Olympus, São Paulo, Brazil) connected with a digital camera (QColor-3®, Olympus, São Paulo, Brazil). For each wound and staining method, 10 histological sections were analysed. For each section, five images were obtained randomly with a 20× objective lens, and the cells and blood vessels were quantified in the histological area. Under each image was applied an unbiased two-dimensional test area (At) of 69 × 10³ μm² at tissue level, so that the total histological area investigated was 24 × 10⁶ μm². The proportion of the histological area occupied by type-I and type-III collagen fibres was determined using the Quantum® software program (Department of Soil Science, Federal University of Viçosa, Viçosa, Brazil) (Gonçalves et al. 2010a).

The volume density of cells (Vv [cells], %), blood vessels (Vv [bvs], %) and elastic fibres (Vv [elf], %) was estimated as:

(1)

where ΣPp [cells; bvs; elf] denotes the total number of points on the cells, blood vessels or elastic fibres, and ΣPt is the total points of the test system (ΣPt = 200).

The length density of blood vessels (Lv [bvs], mm/mm³) and elastic fibres (Lv [elf], mm/mm³) was estimated as:

(2)

where ΣQ⁻[bvs] denotes the total number of blood vessel or elastic fibre profiles counted in the At, and ΣP [tissue] is the total number of points on the tissue (Brüel et al., 2005).

The surface area density of blood vessels (Sv [bvs], mm²/mm³) was estimated as:

(3)

where ΣI [bvs] denotes the total number of intersections between the cycloid arcs (here 44) and the blood vessel surface area, and l is the length of the cycloid arcs. The Image Pro-Plus 4.5® image analysis software (Media Cybernetics) was used in the stereological analysis.

Data analysis

The data were expressed as mean and standard deviation (mean ± SD). The normalcy of the data distribution was verified using the Shapiro–Wilk test. All variables investigated were subjected to the Kruskal–Wallis test for multiple comparisons. Statistical significance was established at P < 0.05. The analysis was performed using the software Sigma Stat 3.0® (Systat Software Inc., Chicago, IL, USA).

Results

There were no significant differences in total collagen and glycosaminoglycan content in the uninjured tissues from the different groups (Table 1). At all investigated time points, the groups exposed to laser photobiostimulation had higher collagen content in the scar tissue compared with SAL (P < 0.05). At day 7, the content of glycosaminoglycans was higher in both groups exposed to laser irradiation in relation to SAL group. A similar result was observed at day 14, but only the group L30 was significantly different compared with SAL. At the end of the experiment, the content of glycosaminoglycans was significantly higher in L3 compared with the other groups.

Table 1.

Levels of collagen and glycosaminoglycans in scar tissue of rats receiving laser light applied with different energy densities

Groups	Day 0	Day 7	Day 14	Day 21
Collagen (μg/g protein)
SAL	511.37 ± 60.25	203.72 ± 51.03	520.47 ± 43.91	400.34 ± 32.14
L3	515.11 ± 47.15	277.63 ± 30.81*	594.58 ± 39.07*	493.09 ± 45.70*
L30	503.55 ± 41.64	299.53 ± 33.27*	581.66 ± 34.33*	421.82 ± 54.11†
Glycosaminoglycans (μg/g protein)
SAL	40.35 ± 5.12	58.11 ± 9.05	60.19 ± 7.54	56.51 ± 6.10
L3	37.28 ± 8.07	75.23 ± 8.30*	66.80 ± 10.37	42.25 ± 8.16*
L30	39.46 ± 7.33	88.52 ± 10.62*	71.23 ± 8.18*	47.36 ± 9.02†

Day 0 represents the unharmed tissue. Data are represented as mean and standard deviation (mean ± SD). SAL, 0.9% saline solution; L3, laser 3 J/cm2; L30, laser 30 J/cm2. ^*† denote statistical differences between groups (P < 0.05)

^*vs. SAL

^†vs. L3; Kruskal–Wallis test.

The analysis of collagen fibres in the uninjured tissue showed no difference in the proportion of type-I and type-III fibres between the groups. On days 14 and 21, the groups receiving laser irradiation had higher proportion of type-I collagen fibres compared with SAL, with the best results in L30 (P < 0.05). At day 21, this variable was similar in L3 and L30. Animals in L3 and L30 had a higher proportion of type-III fibres compared with SAL on days 7 and 14, with the best results in L3 (P < 0.05). At day 21, the content of type-III fibres was similar in all groups (Figure 1).

Figure 1.

Proportion of type-I and type-III collagen fibres in the scar tissue of rats receiving laser light applied with different energy densities. In B are shown representative photomicrographs of the scar tissue at the end of the experiment (day 21; Sirius red staining under polarized light, bar = 60 μm). SAL, 0.9% saline solution; L3, laser 3 J/cm²; L30, laser 30 J/cm². Day 0 represents the unharmed tissue. Data are represented as mean and standard deviation (mean ± SD). *, † denote statistical differences between groups (P < 0.05), *vs. SAL, †vs. L3; Kruskal–Wallis test.

The analysis of elastic fibres in the uninjured tissue showed no difference in the proportion of volume and length of elastic fibres between the groups. On day 21, the groups receiving laser irradiation had a higher proportion of volume (Vv) and length (Lv) of elastic fibres (elf) compared with SAL (P < 0.05) (Figure 2).

Figure 2.

Density of volume (Vv) and length (Lv) of elastic fibres (elf) in scar tissue of rats receiving laser light applied with different energy densities. The top panels are representative photomicrographs of the scar tissue at the end of the experiment (day 21) (Verhoeff staining, bar = 40 μm), arrows show the elastic fibres. SAL, 0.9% saline solution; L3, laser 3 J/cm²; L30, laser 30 J/cm². Day 0 represents the unharmed tissue. Data are represented as mean and standard deviation (mean ± SD). *, denotes statistical differences between groups (P < 0.05), *vs. SAL; Kruskal–Wallis test.

The extent of scar tissue occupied by blood vessels is shown in Table 2. There were no significant differences in volume, length or surface densities of blood vessels in the unharmed tissues (day 0). At day 7, all these parameters were significantly higher in both groups that received laser light compared with SAL, with better results in L30 (P < 0.05). On days 14 and 21, similar results were observed in L30 compared with other groups (P < 0.05).

Table 2.

Density of length (Lv) and area (Sv) of blood vessels (bvs) in scar tissue of rats receiving laser light applied with different energy densities

Groups	Day 0	Day 7	Day 14	Day 21
Vv [bvs] (%)
SAL	5.15 ± 1.37	9.11 ± 1.70	7.29 ± 1.48	5.94 ± 1.49
L3	4.59 ± 1.06	14.47 ± 2.03*	9.35 ± 1.61	6.33 ± 1.65
L30	4.82 ± 1.15	15.79 ± 2.44*	13.52 ± 2.12*,†	10.46 ± 1.97*,†
Lv [bvs] (mm/mm3)
SAL	126.64 ± 13.20	230.31 ± 21.05	229.11 ± 12.11	207.41 ± 19.52
L3	125.11 ± 15.17	282.24 ± 20.02*	227.32 ± 17.13	200.88 ± 16.95
L30	126.30 ± 18.31	338.27 ± 24.98*,†	268.65 ± 14.01*,†	240.93 ± 14.7*,†
Sv [bvs] (mm2/mm3)
SAL	8.32 ± 1.57	14.20 ± 2.15	15.18 ± 2.70	11.26 ± 2. 14
L3	7.94 ± 1.45	21.93 ± 3.07*	17.55 ± 2.94	13.40 ± 2.16
L30	8.59 ± 1.28	30.81 ± 3.44*,†	24.83 ± 2.61*,†	17.94 ± 2.01*,†

Day 0 represents the unharmed tissue. Data are represented as mean and standard deviation (mean ± SD). SAL, 0.9% saline solution; L3, laser 3 J/cm²; L30, laser 30 J/cm²; Vv, volume density; Lv, length density; Sv, surface density; bvs, blood vessels. ^{*, †} denote statistical differences between groups (P < 0.05)

^*vs. SAL

^†vs. L3; Kruskal–Wallis test.

The results of tissue cellularity are shown in Table 3. The unharmed tissue presented similar cellularity in all groups. On days 7 and 21, the groups L3 and L30 had higher cellularity in the granulation tissue compared with SAL (P < 0.05). At day 14, there was a higher volume density of cells in L3 compared with the other groups (P < 0.05).

Table 3.

Volume density (Vv [cells], %) of cells in the histological area of the scar tissue of rats receiving laser light applied with different energy densities

Groups	Day 0	Day 7	Day 14	Day 21
SAL	25.37 ± 4.91	43.38 ± 6.08	32.51 ± 4.83	21.35 ± 3.60
L3	24.89 ± 4.64	59.13 ± 6.39*	43.29 ± 4.12*	30.27 ± 3.81*
L30	26.11 ± 5.10	61.75 ± 5.47*	37.71 ± 5.03*,†	29.11 ± 4.09*

SAL, 0.9% saline solution; L3, laser 3 J/cm²; L30, laser 30 J/cm². Day 0 represents the unharmed tissue. Data are represented as mean and standard deviation (mean ± SD). ^{*, †} denote statistical differences between groups (P < 0.05)

^*vs. SAL

^†vs. L3; Kruskal–Wallis test.

Figure 3 colour shows photomicrographs of skin histological sections collected in both groups investigated. The uninjured skin showed similar cellularity and blood vessel density in all groups. On days 7, 14 and 21, there was increased cell distribution in all groups, with higher cellularity in L3 and L30 compared with the SAL (Figure 3 and Table 2). On days 7 and 14, increased density of blood vessels was observed mainly in the group L30 compared with the other groups. At day 21, there was a higher density of cells and blood vessels in both groups that received laser light compared with SAL.

Figure 3.

Representative photomicrographs showing the distribution of cells and blood vessels in scar tissue of rats receiving laser light applied with different energy densities (H&E staining, bar = 30 μm). Tissue fragments were collected every 7 days during 21 days of treatment. SAL, 0.9% saline solution; L3, laser 3 J/cm²; L30, laser 30 J/cm². Day 0 represents the unharmed tissue.

At all times investigated, the group L30 showed a significant reduction in the wound area compared with other groups (P < 0.05). At day 7, the rate of wound closure was higher in the groups receiving laser irradiation compared with SAL (P < 0.05). A high rate of wound closure was identified in SAL at the end of the experiment (day 21). Total closure of the wound was achieved in L30 by day 21, a feature not found in the other groups (Table 4 and Figure 4).

Table 4.

Progression of the closure of skin wounds in rats receiving laser light applied with different energy densities

Day	Area/Contraction	SAL	L3	L30
0	A (mm2)	80.02 ± 1.03	79.47 ± 1.50	79.33 ± 0.84
	mm2/day	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
7	A (mm2)	75.16 ± 5.37	71.25 ± 5.53	55.59 ± 4.14*,†
	mm2/day	0.69 ± 0.18	1.17 ± 0.32*	3.39 ± 0.51*,†
14	A (mm2)	41.66 ± 4.11	34.15 ± 4.88	25.27 ± 2.09*,†
	mm2/day	4.79 ± 0.76	5.30 ± 0.82	4.33 ± 0.40*,†
21	A (mm2)	0.75 ± 0.41	0.26 ± 0.13	0.00 ± 0.00*,†
	mm2/day	5.84 ± 0.95	4.83 ± 0.62	3.61 ± 0.38*,†

SAL, 0.9% saline solution; L3, laser 3 J/cm²; L30, laser 30 J/cm². Day 0 represents the unharmed tissue. Data are represented as mean and standard deviation (mean ± SD). ^{*, †} denote statistical differences between groups (P < 0.05)

^*vs. SAL

^†vs. L3; Kruskal–Wallis test.

Figure 4.

Representative photomicrographs showing the progression of the closure of wound skin in rats receiving laser light applied with different energy densities. Tissue fragments were collected every 7 days during 21 days of treatment. SAL, 0.9% saline solution;L3, laser 3 J/cm²; L30, laser 30 J/cm².

Discussion

The present study investigated the effect of different energy densities of the GaAs laser on skin wound healing. Using design-based stereology and spectrophotometric methods, the results indicated that the laser photobiostimulation was able to modify the morphology of the scar tissue in a time-dependent way leading to more efficient healing.

It is widely recognized that for healing to occur properly, synthesis of extracellular matrix is required, especially collagen, a protein that provides structural support for cell proliferation and neoangiogenesis (Liu et al. 2008; Gonçalves et al. 2010a,b2010b). The results of this study showed that both groups that received laser irradiation had a higher total collagen content at all time points analysed. These findings corroborate the results found by Medrado et al. (2003) and Gonçalves et al. (2010a,b2010b), which observed a significant increase in the collagen content in scar tissue 7 days after laser irradiation of skin wounds in rats. Collagen synthesis is an event directly related to the biomechanical properties of the scar tissue. In this context, the greatest collagen content gives the scar tissue greater resistance to mechanical stresses, a characteristic essential to the maintenance of tissue integrity and to reduced susceptibility to further injury (Karu 2003; Gonçalves et al. 2010a,b2010b).

Considering the different collagen types, both irradiated groups had a higher proportion of type-I and type-III collagen fibres than the control group. Both energy densities investigated were effective in stimulating the maturation of collagen in scar tissue, and the best results were found in group L30. Although laser irradiation has influenced the total levels of collagen, it is essential to identify the types of collagen produced in scar tissue. Traditionally, the assessment of type-I and type-III fibrillar collagens has provided an important indicator of the progression of the healing process (Karu 2003; Gonçalves et al. 2010a,b2010b). In the earlier stages of cutaneous wound healing the synthesis of type-III collagen predominates and is then gradually replaced by type-I collagen fibres, thicker, resilient and the type of collagen that predominate in normal tissue (unharmed). Thus, determining the proportion of type-I collagen fibres in relation to type-III fibres allows us to evaluate the level of remodelling and maturation of scar tissue, which in turn indicates how much this tissue approximates to the tissue when it is unharmed (Reddy 2004; Mendez et al. 2004; Gonçalves et al. 2010b). Considering these characteristics, it is widely recognized that therapeutic approaches that stimulate the synthesis of type-I collagen, leading to increased collagen maturation, are potentially useful strategies in the treatment of skin injuries (Medrado et al. 2003; Pugliese et al. 2003; Gonçalves et al. 2010a,b2010b).

An additional result shown in the present study was the influence of the laser photobiostimulation on the glycosaminoglycan content in irradiated tissue. This finding indicates a transient modification of some structural polysaccharides of the extracellular matrix during the healing of skin wounds. It is believed that this event is possibly related to the development of a structural and functional support able to stimulate the cell migration and differentiation (Pierce et al. 1991; Hodde 2002; Lai et al. 2006). It is known that the content and distribution of polysaccharides molecules are important to the hydration (attraction of water molecules – solvation water) and nutrition of the granulation tissue during the development of a vascular network that would allow the progression of tissue repair (Pierce et al. 1991; Hodde 2002; Lai et al. 2006). Although the quantity and quality of non-protein and protein components of the stromal tissue are important in tissue repair, currently there is not sufficient evidence as to how the laser irradiation modulates the synthesis and secretion of polysaccharide molecules to stimulate the healing of skin wounds. As the analysis of these molecules performed in this study is not as sensitive and specific as some molecular biology techniques, we cannot yet establish how much the induction of synthesis of polysaccharides contributes to the mechanism through which the laser photobiostimulation improves the healing process. Thus, further studies are needed in this area.

In addition to the increased collagen and glycosaminoglycan content, the laser-treated groups also had a higher tissue area occupied by capillaries, with the best results in the group that received the highest energy density. Furthermore, this study confirmed previous findings (Moore et al. 2005; Houreld et al. 2010) that the laser radiation, in both low and high doses, stimulates the tissue cellularity and increases the synthesis of granulation tissue, which are aspects involved in tissue repair. These data are similar to those described by Corazza et al. (2007) and Gonçalves et al. (2010a). These authors showed the efficiency of high-energy dosages in the induction of fibroblast proliferation and neoangiogenesis. However, these findings are in contrast to previous studies that show better results in these variables with the use of low doses of energy, especially 1–4 J/cm² (Tuner & Hode 1998; Medrado et al. 2003; Reddy 2004). A complex mechanism has been described through which the laser light stimulates the tissue repair. Studies with models of soft-tissue injuries have provided evidence that the photobiostimulation laser induces the synthesis and secretion of mitogens (Posten et al. 2005; Houreld et al. 2010; Xavier et al. 2010) such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and tumour necrosis factor alpha (TNF-α) by macrophages, neutrophils, endothelial cells and fibroblasts, which stimulate the reorganization and repair of damaged tissue through the induction of proliferation, cell differentiation and neoangiogenesis (Posten et al. 2005; Houreld et al. 2010; Xavier et al. 2010).

There is sufficient evidence that the synthesis and differentiation of parenchymal and stromal components of the tissue determine the progression of the reorganization of injured tissue and the quality of the neoformed tissue (Karu 2003; Posten et al. 2005; Corazza et al. 2007; Liu et al. 2008). Thus, therapeutic interventions that stimulate the production of cellular and molecular components of the granulation tissue have been effective in promoting faster closure of wounds in soft tissues (Gonçalves et al. 2010a,b2010b; Xavier et al. 2010). In the present study, the group that received a higher dose of laser radiation (L30) showed more rapid progression of wound closure compared with other groups. These data are similar to those found by Enwemeka et al. (2004) and Moore et al. (2005), which showed the influence of various parameters of laser photobiostimulation on the tissue repair, including reduction in the wound area mainly with moderate energy densities between 19 and 24 J/cm². In contrast, in these same studies, densities below 8.25 J/cm² did not improve the injuries' closing time, findings that are contrary to the results of Medrado et al. (2003), Pugliese et al. (2003) and Mendez et al. (2004) that demonstrated a higher closing speed of the injured tissue at low energy densities (2–4 J/cm²), while high doses led to a delay in tissue recovery.

The findings of the present study suggest that laser photobiostimulation can modulate the process of skin wound healing in a time-dependent way. The higher energy density investigated was more effective in modifying the morphology of the parenchyma and stroma of the scar tissue and led to a faster healing. Considering the findings of this study in relation to the contradictory results of previous investigations, it is evident that additional studies are required to investigate the effects of photobiostimulation lasers with different energy densities on biological tissues, especially in relation to ultrastructural and metabolic changes of injured tissues.

Authorship and contribution

All listed authors meet ICMJE authorship criteria and that nobody who qualifies for authorship has been excluded. Authors contributed to research design, acquisition, analysis and interpretation of data, to drafting the paper or revising it critically and to the approval of the submitted and final versions.

Disclosure statement

The authors declare that there is no conflict of interests.