The present study showed that a single dose if irradiation (15 Gy) impairs bone healing characterized by alterations in the woven bone microstructure. This effect was observed in both grafted and non-grafted defects. Bone repair process in irradiated areas has been extensively evaluated in the literature. Despite that, few studies have focused on the effect of radiotherapy in ongoing healing surgical sites [17, 24, 25]. It is assumed to posse great relevance considering the possibility of defect reconstruction and dental implant osseointegration before the onset of radiotherapy sessions.
The literature shows a tendency towards implant placement in the same intervention of the ablative surgery in order to prevent surgical procedure in irradiated tissues and to shorten the time for prosthetic rehabilitation [13]. This approach usually involves free-flap reconstruction of the jaws and has shown to be a feasible option for prosthetic rehabilitation of oral cancer [26, 27]. Data from a retrospective study involving 210 patients revealed a higher success rate of implant osseointegration in irradiated patients when implants were installed immediately at the time of flap transfer than those where a delayed setting was adopted (86 versus 64%) [12]. Evidence of increased damage when radiotherapy had been delivered preoperatively and minimal effects on bone healing when it had been applied after the surgery, can sustain this clinical finding [25, 28]. It seems that planning the rehabilitation treatment before radiotherapy sessions provides more time for integration of the graft and the implant [29]. Therefore, the rationale to investigate the effect of radiation therapyon bone repair of defects filled with particulate bone graft has been based on these findings.
In a previous study from our group [17], 30 Gy delivered 2 weeks post surgically did not affect the bone volume fraction of defects in rabbits. Interestingly, a decreased amount of bone volume fraction was evident in this experiment after 7 days of the irradiation procedure. To explain these findings, the mechanisms involved in the repair process and radiation delivery timing have to be considered. Bone healing of burr “hole” defects in long bones comprises, in summary, two healing stages. First, a phase of predominant woven bone formation along the lateral periosteal bridging the cortical bone, and then a second phase characterized by woven bone replacement by lamellar bone. In rats, the first phase was observed until day 7. Then, the woven bone is remodeled to lamellar bone to reach the defect closure and resorption of the woven bone inside the medullary cavity is observed [30]. Associating the healing phase with the irradiation timing is determinant for a proper interpretation of the results. It has been demonstrated that the effect of the radiotherapy on bone healing diverges as the degree of maturation of the tissue changes. The interval of 4 days or more between the surgery and the postoperative irradiation dramatically decreased the deleterious effect of radiation therapy on bone healing in rats. At the same time, when radiotherapy had been delivered before the surgical intervention or in an early postoperative period (within 3 days) a decrease in bone formation was observed [25]. Thus, it seems that the divergent results among the studies might be due to the distinct intervals between the surgery and the postoperative irradiation and because of the different animal models used.
The effect observed on bone microstructure could probably be observed in a short period after irradiation because radiotherapy affects mainly the proliferative capacity of osteogenic precursor cells [31]. The lesion is inducted as a challenge for the physiologic environment that requires the recruitment and differentiation of the mesenchymal cells [32], which were affected by high dose of ionizing radiation amid this process. This mechanism involves metabolism acceleration in both hard and connective tissues in order to potentiate tissue healing and local tissue defensive reactions [30]. Without this trigger, the effect of radiation on the mineralized portion of the bone probably would take longer to appear as reported previously [33]. The ionizing radiation was delivered in a single dose of 15 Gy. It promotes a rapid death of the most cells sensitive to ionizing radiation, whereas the fractionated applications induce a dose- and delay dependent response [34, 35]. The most commonly used protocol for head and neck cancer, when radiotherapy is given alone, is 2 Gy in a single fraction per day, five days a week, for six to seven weeks [36]. However, fractionated dose delivery in animal experiment was not suitable due to the tight schedule of the linear accelerator device used for the treatment and anesthetics difficulties related to animal experimentation. For this reason, the application of 15 Gy in a single dose was performed in order to simulate the effect of 60 Gy delivered in 2 Gy fractions. The more fractioned the dose, the higher the total final dose must be to achieve the same biologic effect.
Regarding the grafted defects, the radiation did not interrupt the bone integration with the grafted particles. In the histomorphometric analysis only the area of actual bone was measured precluding the area of graft particles. Deproteinized bovine bone mineral particles (DBBM) degrade very slowly. These bone graft particles have been found after 4 to 10 years after implantation [37]. This characteristic allows the volume maintenance of the grafted areas to persist over time, being advantageous especially in areas where bone dehiscences affect function and esthetics [16]. Thus, it was expected to find a lower percentage of newly formed bone in grafted groups in this initial healing period. Bone substitute material that take long periods for complete resorption result in a reduction of newly formed bone because of the continued space occupied by the particle [38]. An ideal experimental model to assess the bone formation in grafted areas for longer periods is to create critical defects, in which spontaneous bone healing does not occur during the lifetime of the animal. Larger defects in rats’ tibiae might lead to fractures, thus calvarial critical defects are the most frequently used. However, radiation delivery of 15 Gy in a single dose is not possible in this area making this model unfeasible for the present study. In the present study, the defect was analyzed after a 14-day period, during which spontaneous healing of tibia defects did not occur. Consequently, there was no need to employ a larger defect size to evaluate the effects of radiotherapy on defect healing.
In order to allow a direct comparison between grafted and non-grafted groups without the intervention of the volume occupied by the particles, micro-CT analysis of the bone volume fraction was also assessed by subtracting the graft volume from the total volume. The results revealed an increased percentage of bone volume observed in both RTX and nRTX groups. The morphometric results from micro-CT agreed to the histomorphometric findings. Bio-oss® particles (Geistlich Pharma AG, Wolhusen, Switzerland) were visually distinguished from newly formed bone due to its different gray levels. The threshold for the particle segmentation was obtained by selecting the graft alone and further histogram analysis of 10 samples to minimize any possible bias that may arise from micro-CT methodological analysis. Even though misleading measurements in the bone-graft interface might occur in micro-CT analysis, and histological analysis was also performed to ensure the reliability of the results. Histological analysis showed that the osteoconductive property of the xenogeneic granules was maintained with no sign of necrosis after irradiation. Besides, irradiation delivery did not affect the bone graft incorporation as demonstrated by the percentage of particle surface in contact to bone after radiotherapy.
Only few studies have evaluated the influence of postoperative irradiation on bone properties around particulate bone substitutes [34, 39–41]. Malard et al. (2005) [34] investigated the possibility of bone reconstruction using macroporous biphasic calcium phosphate (CaP) inserted 3 weeks before radiotherapy associated to autologous bone marrow (BM) graft injected after irradiation. The results showed that CaP was osseointegrated even without marrow graft association. Histological analysis revealed the absence of fibrous tissue between bone and implanted CaP similar to the findings of the present study. The authors suggest that this integration was initiated before irradiation and was preserved contributing to bone bonding and stability. The same behavior has been noted in the osseointegration of implants coated with hydroxyapatite: the more organized the peri-implant bone was at irradiation onset, the less was the radiation damage on bone formation [29]. However, as the bone remodeling of irradiated bone has shown to be altered in a long-term period [42], future studies should evaluate the behavior of the grafted tissues over time as well as the implant osseointegration in these areas. Nonetheless, the findings of the present study are promising, mainly because osteoconductive biomaterials can enhance bone deposition in unfavorable regions [43], which has been demonstrated to be preserved after irradiation.
The literature is extensive regarding the osteoconductive property of xenogeneic bone grafts [44]. From a clinical perspective, its association with vascularized free flaps might be considered as an option for larger reconstructions considering that the particles serve as a scaffold for osteogenic cells [45] supporting the consolidation of the free flap and the host before radiotherapy. The oral rehabilitation supported by implants could be improved, in some specific cases, when bone substitutes aid in minimizing bone volume loss over time, as demonstrated in non-irradiated bone [46]. Lower survival rate of implant installed in irradiated grafted bone when compared to native bone has been reported [11]. These results were expected because of the increased complexity of the anatomical sites in cases where grafting procedures are required. However, a recent systematic review has demonstrated that the survival rate is still high (90.4%) for dental implants placed in both native and grafted bone during ablative surgery [47].
Another fundamental aspect that requires more investigation to ensure the predictability of the oncologic treatment is the backscattering effect in the presence of bone substitute materials. The particulate graft used in this experiment is a deproteinized bovine bone material, which has crystallin hydroxyapatite as constituent. Hydroxyapatite showed a minute effect whereas poly-L-lactide PLA (bioabsorbable polymer) led to minimal radiation-dose inhomogeneity for photons and electrons [48]. The biological implications of the backscatter and attenuation effects caused by the presence of bone substitute materials should also be further evaluated.
Collagen cross-linking is considered a key predictor of bone fracture risks [49]. Also, the early post-radiotherapeutic increase in osteoclastic activity is followed by long-term depletion of local osteoclasts. Consequently, collagen hyper-mineralization and hyper-orientation takes place [42]. This effect has been reported in mature bone. In newly formed bone, a decrease in collagen density has been demonstrated after a single dose of 30 Gy [17]. In the present study, 15 Gy delivered post surgically did not affect collagen-packing density in defects filled with clot. However, a small percentage of densely packed collagen in irradiated grafted defects was observed. This finding can be attributed to the fact that the proportion between soft and hard tissue in grafted defects is lower than in the clot group. Quantification analysis of fibrillar collagen levels after Picrosirius Red staining involves both mineralized and non-mineralized tissue. Thus, since components of the irradiated wound have different degrees of sensitivity [50], it is not possible to assume that the collagen packing density specifically of the newly formed bone was affected. Histological sections revealed a greater periosteum collapse inside the AOI in grafted groups. It is possible that ionizing radiation has an initial major effect in the periosteal collagen organization during this healing phase, although validation of this hypothesis requires further investigation.
In conclusion, preoperative delivery of 15 Gy led to a delay in the bone repair demonstrated by a smaller amount of newly formed bone in both grafted and non-grafted defects. However, graft particle integration with the newly formed bone was preserved after 7 days of the irradiation delivery.