Morphometric and histological analysis of low-power laser influence on bone morphogenetic protein in bone defects repair
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- Denadai, A.S., de Carvalho, P.T.C., dos Reis, F.A. et al. Lasers Med Sci (2009) 24: 689. doi:10.1007/s10103-008-0595-6
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Bone morphogenetic proteins (BMPs) are secreted signaling molecules belonging to the transforming growth factor-β (TGF-β) superfamily. The objective of this study was to determine how gallium–aluminum–arsenium (GaAlAs) 650 nm laser influenced the action of BMPs on bone defects created in rat femurs. The sample consisted of 24 male albino Wistar rats. Group 1 was composed of rats with bone defects filled with bone-inducing substance, with the application of low-power laser. Group 2 contained rats with bone defects filled with a bone-inducing substance, without the application of low-power laser. Group 3 rats had bone defects not filled with a bone-inducing substance, with the application of low-power laser. Group 4 rats had bone defects and no treatment (control group). A bone defect was produced with drills. In groups 1 and 2 the defects were filled with a bone-inducing substance. The animals were treated with GaAlAs (50 mW) laser, energy density 4J/cm2, for 80 ss on a 1 cm2 area. Groups 2 and 4 were used as control. Bone samples were removed for histological procedures and morphometric analysis on the 7th, 14th and 21st days after surgery. Results obtained were subjected to statistical analysis. Rejection level for the null hypothesis was 0.05. Statistical differences were found in the comparison between group 1 (G1), G2, G3 and G4 [analysis of variance (ANOVA); P < 0.0134]. There was a statistically significant correlation between groups 1 and 4 (P < 0.01). The results of other correlations by Tukey’s post-hoc test were: group 1 vs group 3 (P = 0.341), group 1 vs group 2 (P = 0.862), group 2 vs group 4 (P = 0.061), group 2 vs group 3 (P = 0.744), and group 3 vs group 4 (P = 0.249). We concluded that the association of low-power laser with a bone-inducing substance produced better results than when low-power laser or BMPs were used alone.
KeywordsLow-power laserBone repairBiomaterialsBone morphogenetic proteins
The need for bone restoration, especially as a result of consolidation anomalies, often hinders the work of clinicians and surgeons responsible for patient rehabilitation . However, recent studies have produced encouraging results regarding techniques utilized in bone repair. Among them are studies in the field of molecular biology, concerning the discovery of new biomaterials and the elucidation of repair components, particularly with regard to the action of growth factors and their capacity to promote the differentiation of immature osteo-progenitor mesenchymal cells into osteo-competent cells [2, 3, 4].
Bone contains several growth factors, including bone morphogenetic proteins (BMPs), transforming growth factor beta (TGF-β), insulin-like growth factors I and II (IGF-I and IGF-II), platelet derived growth factor (PDGF) and basic and acidic fibroblast growth factor (bFGF and aFGF).[5, 6]
Bone morphogenetic proteins are differentiation factors whose main function is to induce the transformation of undifferentiated mesenchymal cells into chondroblasts and osteoblasts in a dose-dependent manner . The potential clinical uses of these proteins include bone graft replacement in the treatment of delayed joins, joint failures, poor consolidation, arthrodesis and reconstructive orthopedic procedures, among others .
The bone-inducing ability of BMPs has already been demonstrated in a large variety of species, with different carriers, including collagen and polymers, at heterotopic and orthotopic sites. Among the functions of the carrier are the delay of rapid dispersion of the BMPs from the implant site and protection against non-specific lysis .
In the wake of the evolution of the biotechnological and medical sciences, low-power lasers have become popular and are currently a complementary non-invasive alternative for bone tissue restoration. This has been proven through studies done by several researchers [6, 7].
Seen from this perspective, our study was aimed at investigating the action of low-level laser on BMPs for repair of bone defects.
Materials and methods
The sample consisted of 24 adult male albino Wistar rats (Rattus norvegicus albinus), with body weights ranging from 230 g to 350 g. They came from the central vivarium at the University for the Development of the State and Pantanal Region (UNIDERP) and were kept in four cages (six animals per cage) under controlled lighting and temperature conditions, with standard food and water available ad libitum.
All the experimental procedures were carried out in accordance with the norms of the Brazilian College for Animal Experimentation (COBEA). The animals were cared for in accordance with the national guidelines for the humane treatment of laboratory animals.
The animals were randomly distributed into four experimental groups as follows: group 1 (n = 6), was composed of rats with bone defects filled with a bone-inducing substance, with the application of low-level laser; group 2 (n = 6) contained rats with bone defects filled with a bone-inducing substance, without the application of low-level laser; group 3 rats (n = 6) had bone defects not filled with bone-inducing substance, with the application of low-level laser; group 4 (n = 6): rats had bone defects with no treatment (control group).
All groups were further subdivided into subgroups, as follows: group 1, A7, A14 and A28; group 2, B7, B14 and B28; group 3 C7, C14 and C28; group 4 D7, D14 and D28.
Production of bone defects
The animals were anesthetized with a mixture of 10% ketamine and 2% xylazine, in the proportions 1:2, at doses of 0.10–0.15ml per 100g of body weight, via an intraperitoneal route.
Following the anesthesia procedure, trichotomy was performed on the lateral face of the right thigh, sufficiently long enough to allow viewing of the region to be dealt with. The animal was then positioned in ventral decubitus, and the front and hind paws were pinned in an abducted position. The incision location was then prepared with antiseptic (iodide alcohol).
A direct incision to access the femur bone was made with a scalpel. Following incision of the fascia lata, the femoral diaphysis was located and a bone defect was created on the craniolateral face at approximately 50 mm from the proximal epiphysis. This was done with a no. 1016 spherical diamond-tipped drill bit (KG Sorensen) coupled to a dental handpiece (Kawo, Brazil), operated by an implant motor (Easy Implant, Easy Equipment) at 42,000 r.p.m. During the procedure, there was constant irrigation of the site with physiological serum. The procedure consisted of drilling through the bone cortex until the medullary canal was reached.
In groups 1 and 2, the defect was filled with a paste formed by Gen-Tech bone-inducing substance (Genius Baumer Biomaterial Division S/A, code 9214.1,6, batch 012991). It consisted of an agglutination of organic bone matrix; inorganic bovine bone; bone morphogenetic proteins; hydroxyapatite and collagen in the proportions 10:1; and 9% saline solution for homogenization. To avoid dispersion of biomaterial, we covered the defects with a biological membrane consisting of Gen-Derm decalcified bovine bone cortex (Baumer Biomaterial Division S/A, code 980.S, batch 013310). Following these procedures, the incision was sutured in layers with a needle with 0.30 nylon polyamide thread.
Application of low-level laser
A gallium–aluminum–arsenium (GaAlAs) laser made by Laserline® (Inova model) was utilized, with power of 50 mW, beam width of 0.1 cm2, and wavelength (λ) of 650 nm. The laser was applied by the transcutaneous point method, with a predetermined dose of 4 J/cm2 and duration of 80 s, for a 1 cm2 area.
Groups 2 and 4 were used as comparative control groups for the histological analysis. Two animals from each group were killed by ethyl ether inhalation on the 7th, 14th and 28th days after the operation. Bone samples were then taken and sent for histological procedures and morphometric analysis.
The specimens for histological evaluation were fixed in 10% formol for 24 h. Next, they were decalcified with ethylene diamine tetra-acetic acid (EDTA) and embedded in paraffin blocks. They were cut into 5 μm-thick transversal sections, and slides containing two sections each were prepared. These were stained with hematoxylin-eosin and Masson’s trichrome.
For the morphometric evaluation, images were digitized, and computer analysis was performed with a specific image processing and analysis program (Image Pro Plus 4.5).
To quantify areas that were representative of newly formed bone, we digitized five fields, using an Axiolab microscope (Carl Zeiss, ×20 objective) coupled to a Sanyo Digital Active BLC image capture camera connected to a Pentium IV 2.8 GHz microcomputer equipped with a video-board.
Prior to the quantification process, all images were digitized, and the microscope light intensity and condenser height were standardized. We separated the areas of newly formed bone on the image by using color distribution as a discriminating parameter. For each quantified image, the same color interval was utilized to separate the area to be quantified. The standardized color interval was empirically defined at the start of the experiment. Color bands were adjusted by trial and error until the representative areas of the image had been separated.
Subsequently, the same color intervals were utilized to identify the new bone in all the digitized fields. After that, the area occupied and the quantity of light absorbed by the bone in each of the fields were calculated for each of the fields.
After tabulating the data, we subjected them to the Shapiro–Wilk normality test, because the sample size was small. Thus, if samples presented normal distribution (parametric), analysis of variance (ANOVA) would be applied with the Tukey post-test, so that the groups could be compared. In the case of a non-parametric distribution, the Kruskal–Wallis test would be adopted. For these procedures, the BioEstat® 3.0 statistical software was utilized, with a significance level (P) of 5%, i.e., P ≥ 0.05.
Means from the histomorphometric analyses of new bone formation (BMPs bone morphogenetic proteins, LLLT low-level laser therapy)
Days after creation of lesion
Group 1 (BMPs + LLLT)
Group 2 (BMPs)
Group 3 (LLLT)
Group 4 (Control)
Seven days after creation of the lesions
Histological and morphometric analysis of group 1 (bone defects filled with a bone-inducing substance, with the application of low-level laser) showed the presence of fibrous conjunctive tissue with new formation of vessels and areas of newly formed bone tissue, without inflammatory reaction. There was a large concentration of osteoblasts.
Group 2 (bone defects filled with a bone-inducing substance, without the application of low-level laser) showed the presence of fibrous conjunctive tissue with new formation of vessels and some scarce areas of newly formed bone tissue, with inflammatory reaction involving the biomaterial, which was dispersed in the inflammatory infiltrate. Osteoblasts were present.
Group 3 (bone defects not filled with a bone-inducing substance, with application of low-level laser) presented a medullary area filled with fibrous material and scarce areas of newly formed bone tissue.
Group 4 (bone defects without any treatment—control group) presented a cavitary lesion with necrotic tissue, severe inflammatory reaction and connective tissue.
Fourteen days after creation of the lesions
Group 1 (bone defect filled with bone-inducing substance, with application of low level laser) showed newly formed bone trabeculae displaying numerous osteoblasts aligned on the periphery and intense presence of new vessel formation.
Group 2 (bone defect filled with bone-inducing substance, without application of low level laser) showed the presence of infill tissue, newly formed bone trabeculae, numerous unaligned osteoblasts, giant cells, and an area with inflammatory infiltrate.
Group 3 (bone defect not filled with bone-inducing substance, with application of low level laser) showed well-vascularized conjunctive tissue rich in fibroblasts, newly formed bone trabeculae, and numerous osteoblasts.
Group 4 (bone defect without any treatment—control group) showed an evident cavity lesion and neovascularization, inflammatory infiltrate and lower osteoblast concentration than in the other groups.
Twenty-eight days after creation of the lesions
Studies  have revealed that biomaterials (BMPs) promote slow and gradual formation of new bone and that associating these with low-level laser treatment promotes bone repair, as there is stimulation of tissue cells and microcirculation. Thus, the objective of our study was to investigate and compare new bone formation in four groups as follows: group 1 (laser with BMPs); group 2 (BMPs alone); group 3 (laser without BMPs); and group 4 (no treatment).
In this study, Wistar rats were used as the experimental model, because of the positive bone tissue responses in this mammal, which resemble those found in humans, and also because of the ease of acquiring and handling these animals [6, 7, 9–12].
When the animals in this study were examined on the seventh day after the creation of bone defects, it was observed that group 1 (treated with laser associated with BMPs) was at a more advanced stage of repair than were the other groups being studied, presenting an area of new vessel formation, and newly formed bone tissue with a large concentration of osteoblasts and the absence of inflammatory reaction. On the other hand, in the other groups, the presence of inflammatory reaction and necrotic tissue (group 4 only) was very evident.
Salgado  and Ribeiro et al.  used the tibia as the lesion site and evaluated the results after 5 days. In our study, however, the femur was used, and samples were collected after 7 days and laser was used. Comparing both studies we noticed that repair was more evident after 7 days.
Analysis of the results obtained after 14 days for group 1 (treated with laser in association with in-filling with BMPs) showed newly formed bone trabeculae with osteoblasts aligned on the periphery. In contrast, group 2 presented unaligned osteoblasts an an inflammatory infiltrate. Group 4 (control) was at an earlier stage of repair than the other groups, with a cavity lesion, lower osteoblast concentration and more inflammatory infiltrate.
Another study presented similar results on the 15th day after the lesion had been created: the group was treated with laser and biomaterials (BMPs), showed the presence of large numbers of osteoblasts, cells, and blood capillaries and a bed filled with biomaterials that were in contact with the conjunctive tissue. There was no sign of bone joining or maturation of the collagen fibers in that study’s control group. The inflammatory process still persisted, the conjunctive tissue was disorganized, and cells were undergoing a regeneration process
Group 1, whose samples were collected on the 28th day after the bone lesion had been created, showed signs of medullary canal reconstruction. Groups 2 and 3 also presented favorable results, with the presence of bone trabeculae and osteoblasts aligned within them. In addition, a poorly defined medullary canal of small dimension could be seen in group 2.
The findings in our study also corroborate those of studies that presented favorable results from grafts using BMPs, in which bone incorporation occurred more rapidly and the repair took place with intense formation of new bone [3, 14].
An investigation using GaAlAs laser (650–680 nm) with a density of 5 J/cm2 for guided bone regeneration in rabbits, showed results similar to those achieved in our study, since the histological analysis showed the early formation of immature bone, thereby accelerating cell activity .
Analyzing the morphometry results, we observed that group 1 (bone defects filled with a bone-inducing substance, with application of low level laser) presented a greater general mean for the quantity of newly formed bone than did group 4 (control), which was statistically significant,
Analyzing the morphometry results, we found that group 1 (bone defect filled with bone-inducing substance, with low-level laser) had a higher mean amount of newly formed bone than did group 4 (control); this difference was statistically significant. Analyses comparing group 1 with groups 2 and 3, revealed no significant differences, although the numerical results from groups 2 and 3 were lower than those from group 1. The same was true of the histopathological analysis, results of which corroborated those of similar studies. Another study comparing different biomaterials concluded that, although devitalized bovine bone exhibited a greater inflammatory reaction with the presence of foreign body giant cells, formation of cystic cavities containing implant residues and slower pore invasion by bone tissue, this bone material demonstrated osteo-conductive properties in bone defect repairs in comparison with other implants .
Our study results indicated that there was an inflammatory reaction of lesser intensity and duration in groups subjected to irradiation with low-power laser, which is corroborated by results achieved in other studies using laser [14, 15, 17].
The association of GaAlAs 650 nm low-power laser and a bone morphogenetic protein bone-inducing substance achieved a better result than did laser application alone or the use of BMPs alone.