In this study, we enhanced the osteoconductive properties of autogenous bone grafts and created better quality bones rapidly using locally applied risedronate.
Recently, the rapid ossification of grafts in bone surgery has become a popular subject of study. To this end, researchers have investigated the use of various chemical and biological molecules such as statins, stem cells, and bisphosphonates under different methods. Bisphosphonates are typically used orally in low doses for the treatment of osteoporosis; however, they are also used parenterally and in high doses in some types of cancer to prevent bone metastasis. However, this use causes a series of complications owing to a high level of suppression of osteoclastic activity resulting in deterioration of bone turnover [9]. While their current use has been successful in conditions ranging from postmenopausal osteoporosis to Paget’s disease and malignant hypercalcemia, bisphosphonates used in the long term have recently been associated with systemic side effects including renal toxicity, osteonecrosis of the jawbone, atypical femur fractures, and hypocalcemia [10].
Numerous efforts to take advantage of the local effects of bisphosphonates while preventing side effects have been reported, and evidence shows that the concentration level can be maintained at the targeted site [11, 12]. For example, Toker et al. reported no significant difference between local and systemic applications of bisphosphonate in defect sites in rat skulls [13]. Similarly, Küçük et al. found no significant difference between local and systemic applications of bisphosphonates in a rabbit distraction model [14]. Numerous researchers have also shown the positive effects of drugs belonging to the bisphosphonate group in cases where bone healing is prominent given the local suppression of osteoclastic activity. Hence, the administration of bisphosphonates by this local method with minimal systemic side effects has become much more popular. The current study supports the relevant literature in this regard by concluding that the local use of risedronate, a potent bisphosphonate, increases both the total bone and newly formed bone volume.
Risedronate has a strong anti-resorptive effect without strong inhibition of mineralization [15]. Çetinkaya et al. investigated the daily use of low- (0.1 mg/kg) and high-dose (1 mg/kg) oral risedronate on alveolar bone loss in mice with periodontitis, concluding that short-term low-dose risedronate use inhibited bone resorption, whereas high-dose long-term use led to deteriorated bone formation and angiogenesis [16]. Aghayan et al. used risedronate in a 2% gel form (2 g) in rabbits and evaluated its impact on bone healing using histomorphometric analysis, and found that the experimental group displayed more ossification and more osteoblast cells at the end of two months compared to the control group [17].
Unlike Aghayan et al., we administered risedronate in its pure form and at a dose of 5 mg/ml. This has some significance for demonstrating the impact of risedronate and isolating those of additional biological materials. Khajuria et al. used biodegradable chitosan as a carrier for risedronate in the treatment of periodontitis in a rat model. Despite their positive findings, the potential share of chitosan in this study remains controversial [18]. In the current study, we used collagen membranes and autogenous graft materials as carriers of risedronate. Using this method, we maintained autogenous grafts and collagen membranes in a solution prepared with pure risedronate for 5 min, thus excluding the biological effects of other materials used as carriers.
Guided bone regeneration (GBR) was first described in 1959 to prevent the invasion of rapidly growing tissues into the bone regeneration zone, thereby optimizing bone healing and ensuring that relevant growth factors remain in the regeneration zone. This technique has been used safely by numerous surgeons in vertical and horizontal bone augmentation and peripheral nerve surgery for many years. Today, surgeons continue to perform successful bone reconstructions using various GBR methods and materials [19, 20].
With successful clinical use for over 100 years, autogenous bone grafts remain the gold standard and first choice in bone graft selection because of the osteogenic capacity of living cells. While some disadvantages such as donor site morbidity and limited availability exist, they are gradually being eliminated owing to recently defined autogenous bone harvesting methods [21]. We selected autogenous bone grafts for this study because of the osteogenic, osteoinductive, and osteoconductive properties provided by their osteoprogenitor cells, growth factors, and connective tissue proper matrices, respectively.
The barriers used for GBR must have certain properties including biocompatibility, cell permeability, and space protection. In addition, resorbable membranes should not prevent bone regeneration through minimal tissue reactions during resorption. Numerous barrier membranes have been reported including polytetrafluoroethylene, expanded polytetrafluoroethylene, collagen, freeze-dried dura mater, and titanium foils [22]. In this study, we chose to use collagen membranes because of their high biocompatibility and low antigenicity. These membranes also served as carriers for risedronate, eliminating the need for another biological agent. To the best of our knowledge, our study is the first where collagen membranes have been used with risedronate in the literature and are therefore highly valuable in evaluating the pure impact of local risedronate.
The suitable sites for creating experimental defects in rabbits for research purposes include the mandible, calvaria, femur, tibia, fibula, and radius. We chose the calvaria as the defect site because of its ease of transportation and application, similar ossification patterns with maxillofacial region bones, and ease of comparison with similar previous research. There have been many successful trials using rabbit skulls as surgical sites. Furthermore, by choosing this region, which offers a sufficient surgical area, we ensured the elimination of individual differences, forming both groups on the same anatomical region of the same animal.
A critical-sized bone defect is defined as the smallest bone wound in an animal that cannot spontaneously heal with bone filling throughout its life without using osteopromotive materials. This defect is determined based on the size of each bone in each animal species. For the skull bone of rabbits, the area of this type of defect has been determined as two 10 mm-diameter circles according to multiple studies. Previous studies have created 10 mm-diameter, bilateral, calvarial defects and allowed them to heal spontaneously, reporting less than 20% ossification [23–25]. In relevant research, using critical-sized defects is valuable and necessary to indicate the contribution of grafts or chemicals to bone healing in these defects, which cannot heal spontaneously. In the current study, we used the critical-sized bone defects determined for the rabbit skulls.
The rate of bone metabolism in experimental rabbits is three times faster than that in humans. Given that humans complete ossification within six months after surgery, the corresponding process in rabbits could be interpreted as a period of eight weeks [26]. Previous research has observed angiogenesis in rabbits at the end of four weeks, indicating bone healing. Miloro et al. investigated various time frames to observe early and late bone healing, specifically examining the 2nd, 4th, 8th, and 12th weeks. The authors highlighted that the 4th week was the most appropriate time for evaluating ossification in the early period and the 8th week in the late period [26, 27]. Therefore, given this information, we chose to sacrifice the experimental rabbits at the 4th and 8th weeks to examine early and late ossification, respectively.
Newly formed bone volume measurements and soft tissue evaluations were performed using two-dimensional histomorphometric and three-dimensional micro-CT analyses, as histomorphometric analyses alone have the limitation of being two-dimensional and involve time-consuming sample preparation and formation of artificial tissue [28, 29]. Thus, current approaches tend to include micro-CT.
In addition, immunohistochemical analysis is a key method for identifying important proteins in the extracellular matrix that plays a role in bone formation. In this study, we evaluated the OSP and BSP and used h-scores to evaluate the density and prevalence of proteins in the samples, which were calculated by modifying the h-score formulation in Karataş et al. and were analyzed semi-quantitatively [30]. According to our results, there were no significant differences between the groups. Nevertheless, the increases in BSP and OSP among the 8th-week samples were noteworthy compared to those in the 4th -week samples. The fact that these proteins increased over time in both groups was interpreted to indicate strong future ossification.
In our RIS group, the newly formed bone volume was significantly higher among the 8th -week samples, which is explained by the fact that risedronate induces new bone formation in the late period. The effect of risedronate during the early period was only demonstrated by the total bone volume measured radiologically. This may also stem from the early anti-resorptive effect of risedronate on autogenous bone grafts. This change, without alteration of the existing graft area, was supported by a significant reduction in soft tissue area over time. When applied locally with grafts, bisphosphonates are expected to maintain the graft at the defect site without resorption and to increase the total bone area. In this study, we observed that risedronate had an anti-resorptive effect on the graft in the early period and induced new bone formation in the late period.
This study had some limitations, including the dosage and method of administration of the drug, possible drug diffusion between defects, and the possibility of systemic effects by the locally administered drug. Future research should determine the most appropriate dose in this context.