Body bones play a pivotal role, protecting the vital organs, articulating body movements, producing blood cells, and contributing to calcium homeostasis. A characteristic of this tissue is that when injured it heals without scarring and, despite exhibiting cellular machinery and all components to initiate a necessary regenerative process, there are some circumstances when this does not occur (Tarride et al., 2016; Wijbenga et al., 2016; Wang et al., 2018). For instance, large bone defects, such as trauma, tumor resection, and skeletal abnormalities can compromise integrative brain areas (Dimitriou et al., 2012; Wang et al., 2018), an issue with which we acquired experience and have contributed to a better understanding of the integrative brain function in health and disease (Vasquez et al., 1992, 1997, 1998; Sert Kuniyoshi et al., 2011; Viña et al., 2021).

Regarding advances in the filling of large bone defects, inorganic bovine bone (IBB), which promotes deposition and mineralization of the bone matrix, is an attractive research field in translational research (Aludden et al., 2018; Kasuya et al., 2018). Moreover, this osteoconductive material with particles of varying size preserves the space within the tissue and acts as a scaffold for deposition and mineralization of the bone matrix (Kasuya et al., 2018). It has been recommended mainly because its unlimited availability simplifies the surgical procedure (Aludden et al., 2018). But, to date and despite the scientific and biotechnological advances, there are many open questions, including its potential to interact with bone defects critical enough to preclude spontaneous and complete bone healing, including critical-sized defects.

More recently, experimental and clinical studies have focused on infrared wavelength, which seems to be more effective than the red wavelength and has also been a promisor complement for the process of critical defects (Brassolatti et al., 2018). However, irradiation of large bone defects with laser devices still needs more investigation to clear some important questions, such as the number of sessions required to achieve improvements in new therapies (Vajgel et al., 2014; Santinoni et al., 2017; Brassolatti et al., 2018). The use of low level laser therapy (LT) is considered a promising approach to improve the osteoconductive and reparative potential when combined with the biomaterial IBB (Barbosa et al., 2013; Marques et al., 2015; Acar et al., 2016; Bosco et al., 2016; de Oliveira et al., 2018). At the same time, the low-level laser therapy (LT) figures as an innovative invasive treatment of bone lesions that stimulates osteogenesis and accelerates tissue healing (Bai et al., 2021). Although the mechanisms regarding regenerative bone tissue are unclear, recent findings indicate that the light absorbed by the plasma membrane triggers a variety of biochemical reactions that activate transcription factors and modulate cell migration, mitosis, and tissue repair (Kunimatsu et al., 2018).

Considering that low-level laser therapy may improve the osteoconductive and reparative potential of the biomaterial IBB (de Oliveira et al., 2018), this study was designed to evaluate how many sessions of LT are required to improve the effects of IBB filling of large bone injuries in the head.

The present study was motivated by the challenge to contribute to the knowledge about the reparative process of large calvarial defects, and then, to create novel approaches to obtain a fast, non-invasive, not painful, not expensive, and an efficient approach to repair critical bone defects. The quantitative data generated can be expressed as summarized here. IBB-filled large defects provided a faster and better recovery of bone function than the blood clot-filled alternative. The formation of new bone into the cavities gradually increased when the bone injury was filled with IBB, and it was greatly improved when the grafting was combined with sessions of LT. The new bone formed into the cavity increased approximately 25%, 50%, and 55%, respectively, with 4, 7, and 11 sessions of LT and compared to referential Control values obtained in IBB-filled but not irradiated with low-level laser, i.e., combining the use of IBB with seven sessions of LT seems to be an optimal approach to greatly improve the recovery of calvarial defects. It would be important to verify if the optimal effects of seven sessions of LT on bone mineralization are also observed in cavities filled with other biomaterials such as the bioactive glass particles which have been used in in vitro studies from other investigators (Alves et al., 2017).

To avoid possible bias and inappropriate analysis and consequent conclusion, our protocols followed rigorous procedures as follows. The development of a device (Figure 1) allowed us to produce exactly the same diameter and depth of the cavities in all animals. Moreover, the effectiveness of each kind of filling was possible to be precisely evaluated in the animals of the three main groups (non-irradiated Control and irradiated and examined at 15 vs. 30 days). In addition, all animals received the same grafting material from the same manufacture (Bio-Oss®; Geistlich Pharma, Wolhusen, Switzerland). Despite the cavity requiring a very small amount of grafted material, we are confident that in each cavity of each animal, the volume of the biomaterial was exactly the same in all of them (14.7 μL), which was reliable due to the use of a device that was used in all animals. Therefore, we believe that important sources of bias commonly occurred in this type of study were prevented, and this is the reason of our interpretation that the differences in the results are due to the therapeutic characteristics of the biomaterial, accompanied by the irradiation with low-level LT.

The preparation of two critical sized defects with both experimental and control sites or blood clot-filled and IBB in the same rat calvaria (paired split design) was provided as a goal to reduce possible bias caused by the variability between different animals (Vajgel et al., 2014). Despite the lack of consensual opinion, it is assumed that a 5-mm-diameter defect seems to avoid complete closure with newly formed bone in a short period of time (de Almeida et al., 2014; Vajgel et al., 2014; Brassolatti et al., 2018).

In this study, the histological analysis of the cavities filled with a blood clot showed that bone formation was not complete after 30 d post-surgery. Regarding the fact that this is a controversial issue (Hawkins and Abrahamse, 2005; Cunha et al., 2014; Brassolatti et al., 2018; Noba et al., 2018), in the present study, we tried to use consensual dosimetric parameters (wavelength, application time, and energy density per session) that have been reported by previous prestigious investigators (Cunha et al., 2014; de Almeida et al., 2014; Moreira et al., 2018.) Considering the premise that a beneficial effect of LT on bone tissue formation in 5-mm-diameter critical sized defects has been already evidenced and that the number of LT sessions used to be directly related to the final amount of stimulation delivered to the tissue and could interfer in the final result of the process (Brassolatti et al., 2018). The present investigation specifically focused on the number of LT sessions required to significantly improve the recovery process.

The histomorphometry analysis has been considered the “gold standard” for the evaluation of bone healing and quantification of cell activity in terms of percentage of newly formed bone (Pinheiro et al., 2012; Acar et al., 2015). Furthermore, using this technique one can measure ex vivo bone volume and represent a viable alternative to qualitative and quantitative analyses in the research field of repairment of hard tissues (Schmidt et al., 2003; Schambach et al., 2010). The microtomographic analysis was able to accurately identify newly formed bone in critical sized defects at both 15- and 30-days evaluations. The use of high-resolution images and statistical procedures of normalization of reference values (Control group), it was possible to measure trabecular structures of the calvarial defects, and to detect differences in the volume of newly formed bone and in the trabecular number between, when comparing the IBB group with a bone clot or the Control groups. These findings are not a surprise because they are in agreement with the results obtained with the analysis using HE staining shown in Figure 6. Although we did not find significant differences in the average values of trabecular thickness among groups, the lack of difference does not seem to be due to technical aspects interfering in the analysis of microtomographic parameters (Schambach et al., 2010) because we acted with maximal rigor and the procedures were performed by skilled researchers in all steps (Chatterjee et al., 2017). Moreover, some authors suggest that microtomography presents limited sensitivity to identify differences in bone tissue after only 2- and 4-weeks post-surgery since the matrix is not completely mineralized at that time (Schambach et al., 2010; Ramalingam et al., 2016; de Oliveira et al., 2018).

The histomorphometric analysis of clot-filled cavities indicates that a greater number of LT sessions accelerated bone tissue formation during the initial stages of the bone repair process, which is stabilized later. Thus, LT seems more effective at the initial phase of intense cell proliferation following deposition and mineralization of bone matrix (Ozawa et al., 1998; Matsumoto et al., 2009; Marques et al., 2015). However, our results agree with previous studies showing that LT increases the formation of bone tissue in both clot- and IBB-filled cavities (Rasouli Ghahroudi et al., 2014).

Considering that the cavities were grafted with IBB and analyzed after 15 days, we did not expect that a higher number of LT sessions would benefit a better outcome, and in fact, we did not observe important differences in bone formation between 4LT15d and 7LT15d sub-groups. In contrast, after 30 days we found significant differences when comparing the percentage of mineralization in 7LT30d and 11LT30d sub-groups and, in comparison, with Control groups (non-irradiated). The fact that the 4LT30d sub-group was not significantly different from 0LT30d sub-group could indicate that a minimum number of LT sessions is needed to increase bone tissue formation. Conversely, the absence of a significant difference between 7LT30d and 11LT30d sub-groups suggests that the amount of mineralized tissue tends to stabilize after 7 LT sessions.

Our findings indicated that beneficial effect of LT in IBB-filled cavities evaluated 15 and 30 days post-surgery. However, one could argue that this could be a misinterpretation because in the group of 30 days one of the Control groups had levels of mineralization completely different from the other sub-groups. To avoid this confounding factor, we decided to normalize and to use the most accepted solution when one deals with comparison of groups exhibiting marked and unexpected differences in the basal or control values. Surprisingly (see Figure 6), we found remarkably interesting results that led us to propose changes in our initial hypothesis. What we found was that the differences between the experimental sub-groups (number of sessions) and the non-irradiated Control group are not influenced by the number of sessions with LT in blood clot-filled cavities. In contrast, in IBB-filled cavities groups it was observed that the percentage of mineralization is clearly associated with the increase of LT sessions, and that the magnitude of the response does not recommend using more than seven sessions of LT is an exciting result.

Other authors have been pioneers in the demonstration of the beneficial effects of combining IBB, which has similarity to human bone, with low-level LT (Cunha et al., 2014). The present data provides a contribution to this research field, by focusing on the number of LT sessions required to speed and amplify the effects of this combination. Despite our exciting results using IBB, other bioactive materials such as bioactive glass particles (Alves et al., 2014) could also be tested. We are confident about our results because we were challenged to perform a three-dimension microtomographic analysis, which has been an important approach in this research field (Alves et al., 2014). That procedure showed that the combination seven sessions of LT with IBB resulted in a significant increase in the volume of new bone formation (∼twofold) and an increase in the trabecular number (∼threefold). However, other avenues have been opened with this translational research and need to be pursued before moving up to clinical trials.

One exciting contribution to the mechanisms involved with the osteogenic and mineralization of cellular and extracellular matrix in the recovery of calvaria bone defects was recently provided by Alves et al. (2014). Through in vitro manipulation of newborn osteogenic cells those authors revealed some conditions favoring immunolabeling proteins as important contributors to the osteoblastic activities and differentiation and formation of mineralized matrix (Alves et al., 2014). Based on this scenario, we conclude that our findings show that IBB-filled cavities irradiated during seven sessions of LT could favor the regenerative process of great-sized defects and challenge other investigators to join us in the search for rational solutions in the fighting against critical calvaria defects.

The present study is related with approaches to achieve rapid and effective repair of critical bone defects by using grafting inorganic material and low-level LT. Our data demonstrated that critical calvaria defects filled with IBB produces have much better reparative results than those filled with blood clot, and that the combination of these techniques with low-level LT accelerates and increases the bone healing. We determine the number of sessions of laser therapy required to obtain minimal and maximal effects in the regenerative process of bone critical defects. The data revealed that a maximum benefit is obtained using IBB combined with seven sessions of low-level LT. Therefore, the present results greatly contribute to the expected promotion of better conditions for those suffering from bone trauma- and other cause-related critical injuries. The present study and conclusions may provide new insights and motivate clinical trials in this research field.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Ethics Committee in the Use of Animals, Federal University of Espirito Santo, Brazil.

MS, DS, RB, SM, and EV made substantial contributions to the concept and design of the study and interpretation of the data. MS conducted the experiments and provided acquisition and analysis. KR, FS, and ED performed the microtomographic analysis. LL and EV processed the spreadsheet data, constructed the graphs, and provided statistical analysis. TP contributed with the concept, analysis, and production of Figure 6 and collaboration in the illustration of microtomographic images of a new graph suggested by the reviewers. EV coordinated the critical revision of the manuscript and provided the final version. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.