Evaluation of Bone Repair in the Mandible of Rabbits Using Biphasic Calcium Phosphate Micro-Macroporous Hydroxyapatite Bioceramics and Beta-Tricalcium Phosphate

Objective: To perform a clinical and histological evaluation, characterizing and proving the feasibility of the use of beta tricalcium phosphate (HA/βTCP) bioceramics as a bone defect repair material, comparing it with autogenous bone and blood clot in terms of osteoinductive, conductive, and genic capacities. Material and Methods: The experiment was based on 3 critical defects in the mandible of 11 New Zealand rabbits. The defects were filled with HA/βTCP bioceramics and autogenous bone, respectively, collected and ground during the development of defects and blood clots. The animals were euthanized after the 90-day experiment and samples were collected for histomorphological examination. To evaluate differences between the groups, a one-way analysis of variance (ANOVA) was performed with Tukey’s post hoc test. An α value lower than 0.05 was considered statistically significant. Results: Microscopy revealed the presence of osteoblasts, osteoclasts, and osteocytes associated or not associated with the presence of mature or immature bone. All the studied materials presented bone neoformation in all cases, with the presence of mature and immature bone. Regarding the presence of HA/βTCP bioceramic residual material, the same was found in 7 of 11 slides. Conclusion: HA/βTCP bioceramics were shown to be a biocompatible bone substitute, with osteoinductive and osteoconductive characteristics, accelerating the process of new bone formation when compared with autogenous and blood clotted bone, thereby showing promise for bone defect repair with safety and efficacy.


Introduction
Recently, much effort has been made to find solutions to correct bone defects for placing osteointegration implants. Biomaterials are remarkable in this regard, as they eliminate the need to remove autogenous bone. One of the referred biomaterials, highlighted in the present study, is beta tricalcium phosphate (βTCP) bioceramics associated with hydroxyapatite.
It has been demonstrated that βTCP bioceramics is a material with properties that can promote osteoinduction, i.e., the capacity to store proteins and cells that are viable, specific, and particular to the individual receiving the implant; thus, allowing natural bone neo-formation and biocompatibility, with problems restricted to its accelerated absorption [1,2].
Synthetic hydroxyapatites (HA), despite having a different composition and morphology, resemble natural bone the most, and are, therefore, an option for cases in which it is necessary to recover lost bone structure and fill in defects, because of its high osteogenic potential and structural maintenance with slow resorption [2,3].
To analyze alveolar repair using biomaterials, a study filled in the alveoli using an average 0.78 g of βTCP bioceramics without membranes. One first subjective visual clinical analysis rated it as very good to good for 87% of the cases post grafting. Radiographic analysis after 12 months revealed the presence of βTCP bioceramics granules in 30% of the cases, as well as reabsorption in height and width in 10% of the cases. The effectiveness of the treatment was rated as good and very good in 89% of the cases. For 100% of the cases there was no reactions or complications regarding the material. Clinically, it was observed that cases that used membranes were superior. It was concluded that the technique using membranes and βTCP bioceramics is recommended for the maximum preservation of alveoli for further implant prosthetic rehabilitation [4].
Another study performed a histological and histomorphometric analysis of autogenous bone versus βTCP bioceramics alone in 20 cases of bilateral sinus lift. On one side, βTCP bioceramics alone were placed, and on the other autogenous bone, in each patient. It was found no significant difference in the histologic and histomorphometric analyses in the experimental and control groups after 6 months; thus, being considered a good material for this type of treatment [5].
Studies have demonstrated mandibular reconstructions with βTCP bioceramics associated with autogenous bone in a proportion of 30% to 50% depending on the case. A total of 152 patients with various defects such as: bone repair after cystic exeresis, alveolar reconstruction, maxillary sinus lift, fractures of alveolar fractures, periodontal regeneration, reconstruction after removal of tumors and apicectomies, all defects being > 2 cm, were analyzed. The βTCP bioceramics in particles of 500-2,000 µm and post-operative control of 4, 12, and 52 weeks were used. The authors reported easy application of the material; in 9.2% of the cases there was local irritation with granule loss; in 2% of the cases there was total loss of the material; and in 88.8% of the cases there was complete reabsorption of the material with simultaneous bone replacement, observed radiographically and histomorphometrically. It was also reported that the insertion of dental implants in the grafted region could be performed 5-6 months after the graft [6,7].
Another study reported the use of composites of HA + carbon and HA + carbon + sodium bicarbonate, in bone defects in rabbit ulnas. The evidence of early bone regeneration, absence of infection, rejection, efficiency, and high reliability of the materials could be seen [8].
Researchers have reported studies of maxillary sinus lift with βTCP. For this purpose, 17 bilateral edentulous patients had βTCP bioceramics grafted on one side and autogenous bone was used on the other. After 6 months, 68 implants were placed, and bone removed from the site was histomorphometrically analyzed. The bone density showed no significant difference between the sides, but the biodegradation was significantly slower on the βTCP bioceramic side, whereas the bone trabeculation pattern on the β-TCCP side was lower than that of the autogenous bone, but without significant differences. It was concluded that after 6 months, implants could be anchored without major problems in grafting with βTCP bioceramics [9,10].
The performance of the resorbable bioceramics was evaluated using tricalcium phosphate silicon as a stabilizer in the repair of extensive defects in the long bones of sheep. The evaluation was performed through sequential radiographs, tomography, histology, immunohistology, and microradiography to analyze the density and percentage of bone growth. They concluded that there was excellent integration and significant bone regeneration besides evident osteoclastic biomaterial reabsorption. At the end of the first year the remainder of the biomaterial was 10-20% and after the second year it was completely reabsorbed, with the defect completely filled by new, highly mineralized, lamellar bone [11].
In an animal study carried out with 8 rabbits, βTCP bioceramic blocks and βTCP bioceramic blocks treated with PRP for onlay growth were compared. It was concluded after 3 months that there was no inflammatory process in any of the blocks, and the association with PRP did not result in any significant improvement in bone growth [12,13].
Therefore, animal studies are of paramount importance for the timely and safe comparison of biomaterials of varied characteristics, with control groups, such as autogenous bone and blood clot, and for treatments, if they were tested in human patients, avoiding, in this way, suffering and damage to humans [14].
The objective of this study was to perform a clinical and histological evaluation, characterizing and proving the feasibility of the use of HA/βTCP bioceramics as a bone defect repair material, comparing it with autogenous bone and blood clot in terms of osteoinductive, conductive, and genic capacities.

Material and Methods
The rabbits used in this study were obtained from the Santo Amaro University (UNISA) Animal Facility, and were monitored and medicated by the veterinarian team of the facility. All surgical procedures were performed at the UNISA Multidisciplinary Laboratory of Veterinary Surgery Techniques, and the rabbits had undergone 12 hours absolute fasting as per veterinary instruction and supervision. Eleven female New Zealand rabbits were selected for the experimental surgical procedures, with ages ranging between 4 and 6 months, and weights between 3.0 and 3.5 kg. The animals were weighed and on the day of the procedure the selected animals received pre-anesthetic medication (intramuscular), consisting of 0.2 ml Acepran 1%. In every procedure, the animals were anesthetized using an association (intramuscular) of Cetamin (40 mg/kg), Xylazin (5 mg/kg), and Meperidine (10 mg/kg), and were monitored by the responsible veterinary team, receiving further half-dose applications according to their need during the procedure [11].
The area was shaved and antisepsis was performed with povidone-iodine followed by infiltrative local anesthesia with Mepivacaine 2% without vasoconstrictor. Two separate bilateral rectilinear incisions were made in the antero-posterior mandible of the rabbits, according to the anatomy verified through palpation, using scalpel number 15. A patch was then removed to expose the bone tissue of the mandible, using periosteum lifters. In every procedure, a veterinarian of the same team monitored the heart rate, respiration, and palpebral reflexes of the animals.
Next, three inlay bony defects were made mechanically on the mandible of the rabbits, using a straight surgical instrument, electric motor, and trephines, under constant irrigation using physiological saline (0.9%). The defects were 7 mm in diameter and 3 mm deep; two were made on the left side of the mandible and one on the right (Figure 1). To facilitate posterior identification and standardization of the defects, the animal's teeth were used as reference. On the left side, the anterior defect was made in the direction of the second posterior tooth, and the posterior defect was made after the last posterior tooth. On the right side, the defect was made in the direction between the third and fourth posterior teeth. All defects were made 4 mm above the mandibular ridge. After making the defects, we carefully marked the external cortical of the mandible in the region of the defects and stored this cortical bone with physiological saline at 0.9% for further use [12].
The defects were filled in as follows: the first, on the right mandible side with a blood clot (control group); the second, on the anterior left side with autogenous bone removed when the defects were trephined and crushed using a bone grinder; and the third, on the left posterior side with HA/βTCP bioceramics, always respecting a distance of at least 3 mm between the 2 defects. After filling in the defects, the full patch was repositioned and the tissues were sutured with simple stitches by using nylon 3.0.
The rabbits were kept for 7 days in individual cages, which were cleaned daily, and then moved to cells that housed three animals, thus allowing minor motility and reducing stress during the observation period. The environment was maintained under controlled temperature and luminosity, and the animals were fed 3 times a day with Nutriara ® ration (Nutriara Alimentos Ltd., Cuiabá, MT, Brazil) and had water ad libitum. The animals received subcutaneous postoperative medication for 7 days: Enrofloxacin (5 mg/kg) and Flunixin Meglumine (1.1 mg/kg) [11].
The rabbits were sacrificed after 12 weeks, by using a technique recommended by Brazilian College of Animal Experimentation, consisting of an overdose of general anesthetic (Thiopentax ® ) associated with an intravenous injection of potassium chloride (19.1%). The assessment time was standardized as 3 months, as this period corresponded to approximately 9 months in humans. This time interval used for the assessment was justified by previous studies that showed the bone metabolism in rabbits is approximately 3 times faster than that in humans, and could be used as the foundation for research with an allometric scale [11,13,14].
Next, material was removed from the respective location with 7-mm diameter trephines. A clinical analysis was performed and any changes in the defects were registered. The specimens were stored in formaldehyde at 10%, stained with hematoxylin and eosin (HE) by histology technicians, and forwarded to the pathologic anatomy laboratory to prepare the histologic blades.
The blades were photographed and digitalized (micrography) and then analyzed (Plínio S

Results
The results were analyzed considering the clinical evolution of the animals, as well as the healing process of the bone neo-formation of the defects made on the animal's mandibles. Regarding the immediate postoperative clinical aspects, after post-anesthetic recovery, all animals were healthy and all local masticatory and sensorial functions were preserved (Table 1). The animals were followed and monitored daily, and no adverse events were observed from  It was observed that there were a greater number of osteoblasts and osteoclasts in addition to a greater presence of immature bone on the autogenous bone blades than that of the the HA/βTCP bioceramics blades. This shows an acceleration of the bone maturation process when HA/βTCP bioceramics were present (Table 2). However, when compared with that of the blood clot, the presence of osteoblasts and osteoclasts was greater in the HA/βTCP bioceramics blades, with a smaller presence of mature bone, thus evincing faster bone neoformation in the defects filled in with blood clots, with a clinical loss of the filling structure (Table 3). The presence of mature bone and osteocytes were observed in every HA/βTCP bioceramics blade; however, immature bone was observed with great significance only in one blade, which also had a greater number of osteoblasts and osteoclasts (Table 4). It was also observed that in all HA/βTCP biomaterial blades, there was a greater presence of mature bone when the material was directly in contact with the defect walls. Residual HA/βTCP material was observed in 7 of the 11 blades, but at low amounts and always located more towards the center of the defect, which showed that growth is centripetal, starting by the walls directly in contact with the material gradually towards the center of the defect. The presence of residual material on these blades also shows individual factors in relation to absorption and the metabolism time in animals also evincing the biphasic feature of the material (Figure 4).

Discussion
In agreement with previous observations, it was found that bone regeneration was best and fastest when there was close contact of the HA/βTCP bioceramics with the neighboring bone, because of the direct interface resulting from the integration of the cortical and medullar bone with the material [15,16].
Furthermore, complete filling of the defects, without tissue infiltration, which remained stable during bone substitution was clinically evinced in all the HA/βTCP bioceramics blades, taking into consideration the delays in the bone formation, observed histologically, because of the individual reaction to the material. Furthermore, when the process was accelerated, it also affected degradation of both the most and least soluble parts [1,5,8,9,[19][20][21][22][23].
Another factor that should be considered is that, clinically, when the surgery was performed to collect the material for analysis, we noticed that the appearance of the neoformed bone, despite not using membranes, was normal and without any tissue infiltration in the region. Thus, we believed that it is not necessary to use membranes to achieve good results with the material, as its mechanical resistance and biphasic features maintain the desired structural support until bone substitution has been completed [24][25][26].
In addition, the material was very effective in filling inlay bone defects; however, further studies are needed to show the osteogenic capacity of the material [2,6,16,26]. It was shown that the material guided the neoformation of bone trabeculae, and induced neoformation when in contact with the bone, and can thus be classified as osteoconductive and osteoinductive [22,26].
Clinically, the material appeared to be more resistant to perforation, showing higher hardness than that of the other fillings. The difficult decalcification evinced this when preparing the histologic blades, in which bone calcification required twice the time during the process [27].
However, studies in consecutive years have reported that the bone density of the grafted region when perforated for implant placement did not present clinical differences than that of contiguous natural bone sites, requiring more specific analysis for these conclusions [8,12,27].
The association between the bioceramics HA/βTCP and other materials has been studied; the use of platelet rich plasma (PRP), phosphate silicone, fibrin glue, or even autogenous bone favor increased repair speed and improvement in neoformed bone structure [6,9,10,17,18,28,29]. In another study, no changes were observed to the morphology and number of viable cells present in the site with new bone formation in any of the HA/βTCP bioceramics blades, nor in the blood clot and autogenous bone blades [30].
Based on the type of material and size of its particles, there is a greater or lesser use of that material during bone matrix formation. Hence, it is possible that particle size affects the formation of the bone trabeculae, improving or worsening its absorption, since bone deposition occurs simultaneously. In the present study, the size of the HA/βTCP bioceramics particles was 100-200 µm, which facilitated its absorption and liberation of calcium ions, favoring the osteoblast mitosis process, promoting osteoinduction [22,25].
While conducting this study, we considered the concept of critical defects in the mandible of rabbits [17]; however, the size of the defect should be considered since it could need the association of HA/βTCP bioceramics with autogenous or other osteogenic materials to promote better and faster bone repair in larger defects; thus, making it feasible to create a new cellular bone matrix in that region [6,12,26]. Nevertheless, in 2016, the material was considered as ideal for using in long bones as a substitute to autogenous bone even if used alone [17]. Another positive factor of the material that should be reported its easy application, as it is known that several products are difficult to apply, and its form of application and presentation would make it easy to use in daily practice [6].
Locally, there was acceleration in the process of bone neoformation in defects filled with HA/βTCP bioceramics, compared with those filled with blood clot. However, when compared to autogenous bone, bone neoformation was similar [16,22,23,26].

Conclusion
The bioceramic HA/βTCP was shown to be a biocompatible bone substitute, with osteoinductive and osteoconductive characteristics that accelerated the process of bone neoformation relative to autogenous bone and blood clot. Thus, it is effective for the repair of bone defects.
Financial Support: None.

Conflict of Interest:
The authors declare no conflicts of interest.