Effect of different low-level intensity laser therapy (LLLT) irradiation protocols on the osseointegration of implants placed in grafted areas

Abstract Objective To evaluate the effect of different protocols of low-level intensity laser therapy (LLLT) irradiation on the osseointegration of implants placed in grafted areas. Methodology 84 rats were randomly allocated into six groups: DBB: defect filled with deproteinized bovine bone; HA/TCP: defect filled with biphasic ceramic of hydroxyapatite/β-tricalcium phosphate ; DBB-LI: defect filled with DBB and treated with LLLT after implant placement; HA/TCP-LI: defect filled with HA/TCP and treated with LLLT after implant placement; DBB-LIB: defect filled with DBB and treated with LLLT after graft procedure and implant placement; and HA/TCP-LIB: defect filled HA/TCP and treated with LLLT after graft procedure and implant placement. The bone defects were made in the tibia and they were grafted. After 60 days, the implants were placed. The rats were subsequently subjected to euthanasia 15 and 45 days after implant placement. The pattern of osseointegration and bone repair in the grafted area was evaluated by biomechanical, microtomographic, and histometric analyses. Furthermore, the expression of bone biomarker proteins was assessed. Results The LLLT groups presented higher removal torque, mineralized tissue volume, and a greater degree of osseointegration, especially when LLLT was performed only after implant placement, and these findings were associated with higher expression of BMP2 and alkaline phosphatase. Conclusion LLLT performed on implants placed in grafted areas enhances the osseointegration process.


Introduction
The improvement in bone formation in grafted areas with osteoconductive bone substitutes may diminish the time for implant loading and positive long-term outcomes of the rehabilitation with implants.
Although the use of osteoconductive bone substitutes reduces bone formation in bone defects, 1 these bone substitutes have been used extensively, since the use of autogenous bone grafts is related to donor site morbidity. 2 Low-level laser therapy (LLLT) has been successfully used in several clinical conditions, such as those involving joints, 3 muscles, 4 cutaneous tissue, 5 and nerve tissue 6 lesions. It has been proposed that the activation of mitochondrial chromophores stimulates the action of the respiratory chain with subsequent increase in cellular metabolism, producing the beneficial actions of LLLT in the process of tissue regeneration. 7 The benefits of LLLT use have also been demonstrated in bone tissue by the stimulation of the differentiation and activation of osteoblastic cells. 8 Preclinical studies have shown that the use of LLLT accelerated the repair of long bone fracture models, 9 stimulated the healing of critical-sized calvarial defects, 10,11 and accelerated the osseointegration of implants placed in native [12][13][14][15] and grafted bone. [16][17][18] Previous studies using LLLT in infrared wavelength range have shown improvement in the healing of grafted areas with different types of osteoconductive biomaterials. 11,[19][20][21] A preclinical study demonstrated that the use of LLLT at an 808 nm wavelength increased bone tissue formation in grafted areas with deproteinized bovine bone (DBB) and biphasic ceramics based on hydroxyapatite and β-tricalcium phosphate (HA/TCP) and that this effect was related to the increased expression of biological mediators that stimulate the formation of bone tissue. 22 The use of LLLT aimed to accelerate osseointegration in grafted areas has not been previously explored. In an earlier study, our research group demonstrated that the use of LLLT to DBB and HA/TCP grafted areas in the tibia of rats improved the osseointegration process of implants. 18 However, LLLT protocols to accelerate the osseointegration of implants in areas of native bone use LLLT sessions after implant placement. 13,14,23,24 Moreover, the association of the use of LLLT sessions at two different times (after grafting and after implant placement) was also not described. Thus, this study compares the effect of different LLLT protocols on osseointegration in areas grafted with DBB and HA/ TCP.

Methodology
This study was submitted and approved by the Research Ethics Committee on Animal Use of our institution (08/2014) and it was conducted according to the international guiding principles for biomedical research involving animals and followed the ARRIVE guidelines. In total, 84 animals (Rattus novergicus, Hotzman strain) aged three months old and weighing 250-300 g were used. The animals were kept in an environment with controlled temperature (21±1°C), humidity (65-70%), and light-dark cycles (12 hours) and they had access to appropriate food and water ad libitum.

Groups
The animals were randomly distributed into six LLLT was performed after implant placement in the LI groups; in the LIB groups LLLT was performed after grafting procedures and implant placement. (Figure 1). the irradiation. The grafted area was delimited after the sutures of the surgical site, aided by a tissue marker pen. Four equidistant 3-mm points were marked in order to encompass the whole area to be irradiated; these points also served as a guide for laser irradiation. The laser was irradiated with the laser tip in contact with the skin tissue for 10 seconds at each point (1 J), totaling 40 seconds of irradiation per session (4 J). Seven sessions were performed -which were repeated every 48 hours for two weeks after the grafting procedure or implant placement. The energy density at each point was approximately 354 J/cm 2 22 .
The animals of the control groups were submitted to placebo LLLT interventions to handle the animals of every group with the same frequency.

Surgical procedure
The surgical protocol was the same as that used in another preclinical study 18   were separately evaluated at the first six threads of the implants manually without establishing thresholds in the software. 18 We also performed an analysis of the The micro-CT, histometry, and immunohistochemistry analyses were repeated by the raters in 10 rats, and the data correlation was higher than 0.90.

Biomechanical analysis
It was observed that the DBB-LI group (    to non-irradiated groups. Notably, the histometric data presented significant differences that were slightly different than the biomechanical and micro-CT data since this model evaluated osseointegration in a 2D view, the biomechanical analysis evaluated osseointegration in an indirect way, and micro-CT was not able to evaluate the BIC because of the artefacts induced by the implants and bone substitutes. 18,25 The use of LLLT after implant placement in areas grafted with DBB increased the removal torque  The results presented in this study raise the possibility of using LLLT in areas with poor bone quality as a way to improve osseointegration. It is necessary to compare the effects of the infrared laser with the red laser to evaluate whether there are differences in the use of these two distinct wavelengths since the results presented in the literature to this date are conflicting. 16,29,42,43 Clinical studies evaluating LLLT with infrared lasers on osseointegration are also required.
Only one clinical study evaluated the effect of infrared laser LLLT on osseointegration, and no differences were found in achieving the secondary stability of implants.
In the initial period of evaluation, the stability obtained by the implants installed in the control and laser groups reached high values of stability. 23 However, in this study the implants were installed in the posterior region of the mandible, and this area is not considered an area with poor bone quality.
This study presents some drawbacks that need to be considered in the our data interpretation. The type of defect tested in this study was a noncritical size defect because of the limitations of space to perform a critical-sized defect in the tibia that enables implant placement, so the type of defect tested may be less