Evaluation of photobiomodulation therapy associated with guided bone regeneration in critical size defects. In vivo study

Abstract The repair of bone defects raises the interest of investigators in several health specialties. Grafting techniques with bone substitutes and laser therapies have been investigated to replace autogenous bone and accelerate the bone healing process. Objective To evaluate the effect of photobiomodulation therapy (PBMT) associated with guided bone regeneration (GBR) in critical size defects. Material and Methods The study was conducted on 80 male rats (Rattus norvegicus albinus, Wistar) submitted to surgical creation of a critical size defect on the calvaria, divided into eight study groups: group C (control - only blood clot); group M (collagen membrane); group PBMT (photobiomodulation therapy); group AB (autogenous bone); group AB+PBMT; group AB+M; group PBMT+M; group AB+PBMT+M. The animals were killed 30 days postoperatively. After tissue processing, bone regeneration was evaluated by histomorphometric analysis and statistical analyses were performed (Tukey test, p<0.05). Results All groups had greater area of newly formed bone compared to group C (9.96±4.49%). The group PBMT+M (achieved the greater quantity of new bone (64.09±7.62%), followed by groups PBMT (47.67±8.66%), M (47.43±15.73%), AB+PBMT (39.15±16.72%) and AB+PBMT+M (35.82±7.68%). After group C, the groups AB (25.10±16.59%) and AB+M (22.72±13.83%) had the smallest quantities of newly formed bone. The area of remaining particles did not have statistically significant difference between groups AB+M (14.93±8.92%) and AB+PBMT+M (14.76±6.58%). Conclusion The PBMT utilization may be effective for bone repair, when associated with bone regeneration techniques.


Introduction
The need to repair bone defects has raised the interest of investigators in several health specialties.
Grafting techniques with bone substitutes and laser therapies 2,3,6,24 have been investigated to replace autogenous bone and accelerate the bone healing process.
Autogenous bone is considered the gold standard for regeneration of bone defects 9 , because of its osteogenic, osteoinductive and osteoconductive properties 5,9 . Also, it does not have antigenicity 9 , but a natural supporting structure and type I collagen, which promotes resilience and revascularization 9 . However, its achievement is associated with postoperative complications at the donor site, such as esthetic defects, hematomas, injuries to local anatomic structures, infections, and postoperative pain 7 .
Another aspect to be considered is the unpredictable graft resorption, which may negatively influence the postoperative outcomes. with autogenous bone grafts 2,17,26 , biomaterials, as inorganic bone, lyophilized bone, hydroxyapatite, and morphogenetic bone protein, associated or not with biological membranes 6,16 .
The utilization of biological membranes in guided bone regeneration (GBR) prevents epithelial migration into the bone defect, besides maintaining space to allow the host osteogenic cells to repopulate the defect area and accelerate the bone repair 11 . The membranes should be biocompatible, able to create space, have occlusive properties, achieve tissue integration and be easy to handle.
The association of membrane with autogenous graft improves the outcomes, since its utilization stabilizes the grafting material inside the bone defect, which may enhance the outcomes achieved so far when associated with PBMT. Thus, this study evaluated the effect of     were also identified. The TA was measured in mm 2 representing 100% of the area analyzed. The ANB and ARP were also measured in mm 2 and calculated as percentages of TA 13 .

Statistical analysis
Data were submitted to normality test (Shapiro-Wilk), which showed that they had normal distribution.
All variables showed statistical differences between means according to the ANOVA parametric test Group C -In these specimens, the extent of the surgical wound was almost entirely filled with loose connective tissue and parallel collagen fibers. There  Histometric and statistical analysis Table 1 shows the descriptive analysis of ANB for all groups and comparison between them.

Discussion
The study showed that PBMT may be effective for bone repair when associated with bone regeneration techniques, rejecting the null hypothesis.
The bone tissue has wide ability to regenerate, however there are limits for spontaneous repair. In case of very wide defects, in which the vascularization capacity is impaired or there is mechanical instability, the ability to recover the structure and function is limited. Therefore, several techniques and materials      In groups treated with PBMT, bone formation was significantly higher than in group C. When PBMT was applied in isolation, ANB analysis demonstrated good outcomes compared to non-irradiated groups, which demonstrates that utilization of this protocol per se was effective to accelerate bone repair.
The results were better when therapy with PBMT was associated with utilization of collagen membrane (group PBMT+M), achieving the best ANB outcome.
This may be explained because, in guided bone regeneration, space maintenance by utilization of membranes is a prerequisite for treatment success 14 .
Also, its utilization may avoid graft resorption and assure stability of the graft material. This may explain the fact that, when group M was compared to the others, it showed statistically significant differences in relation to groups C and AB+M. We did not find any study in the literature similar to our research, which uses collagen membrane associated with autogenous graft and/or low-level laser.
Even though no significant differences were observed between groups, group AB+PBMT+M exhibited greater quantity of new bone formation than AB+M. In these groups, when the membrane was associated with autogenous graft and/or laser, there was new bone formation below, above and inside the collagen membrane; however, the ANB observed beyond that the TA was not considered in this study due to the methodology employed, which may have In our study, both the receptor site and graft were irradiated, and even though no statistically significant differences were observed between groups AB and AB+PBMT, we believe that the laser was able to accelerate the bone repair, since histological analysis showed new bone formation toward the center of the defect, as well as presence of osteoid matrix around the remaining particles in some specimens, demonstrating that laser application at the initial stages of repair may induce osteogenesis and aid the Future studies should be conducted using advanced techniques for analysis, e.g. immunohistochemistry and microtomography, to evaluate the effect of lowlevel laser on the initial stages of bone repair, to complement the histomorphometric analysis.

Conclusion
Considering our results, the PBMT utilization may be effective for bone repair when associated with bone regeneration techniques. Thus, the null hypothesis was rejected.