Bone repair access of BoneCeramic™ in 5-mm defects: study on rat calvaria

Abstract Objective The aim of this study was to evaluate the osteoconductive potential of BoneCeramic™ on bone healing in rat calvaria 5-mm defects. Material and Methods A 5-mm calvaria bone defect was induced in three groups and the defect was not filled with biomaterial [Clot Group (CG)], autogenous bone (AG), or Bone Ceramic Group (BCG). Animals were euthanized after 14 or 28 days and the bone tissue within the central area of the bone defect was evaluated. Results were compared using ANOVA and Tukey test (p<0.05). Immunohistochemistry was performed using primary antibodies against osteocalcin, RUNX-2, TRAP, VEGF proteins, and 3-dimensional images of the defects in μCT were obtained to calculate bone mineral density (BMD). Results In BCG, the defect was completely filled with biomaterial and new bone formation, which was statistically superior to that in the GC group, at both time-points (p<0.001 for 14 days; p=0.002 for 28 days). TRAP protein showed weak, RUNX-2 showed a greater immunolabeling when compared with other groups, VEGF showed moderate immunostaining, while osteocalcin was present at all time-points analyzed. The μCT images showed filling defect by BCG (BMD= 1337 HU at 28 days). Conclusion Therefore, the biomaterial tested was found to be favorable to fill bone defects for the reporting period analyzed.


Introduction
Functional recovery of the jaws using dental implants requires a minimum thickness and height of ridges 1,5,15 . Several factors culminate in the absence of alveolar bone, which then becomes a limiting factor for implant placement. This usually occurs in patients who have suffered dentoalveolar trauma, traumatic tooth extractions, dental congenital absence, or other diseases involving the maxilla and mandible 2,9,17 .
The current trend is to develop and use biomaterials that accelerate or at least allow normal and complete repair of bone defects, thus reducing postoperative failure rate 7,22 . The objective of tissue engineering is to generate an osteoconductive biomaterial that is conducive to bone healing or even superior to bone grafts 17 .
Synthetic bone substitutes with osteoconductive property, such as calcium phosphate-based ceramic biomaterials, have been investigated 10,11,20 . Recently, Miramond, et al. 14 (2014) showed that isolated biphasic calcium phosphate has osteoinductive potential when used within subcutaneous tissue. The physical properties of biomaterials vary as per their surface area or the format (block or particle), porosity (dense, micro-, or macroporous) and crystallinity (crystalline or amorphous). On the other hand, chemical properties are related to the chemical composition of the material such as calcium/phosphate molar ratio, the level of elemental impurities, and ionic substitution in the atomic structure 24 . Straumann © BoneCeramic TM (BC) is a 100% synthetic bone substitute with morphology to stimulate the formation of vital bone. It has 90% porosity index, with interconnected pores with 100 to 500 microns diameter and is composed of biphasic calcium phosphate, with 60% hydroxyapatite (HA) and 40% β-tricalcium phosphate. The mechanical stability of the increased volume is maintained due to the slow resorption of HA, which prevents excessive resorption, according to the manufacturer instructions. Studies by Daculsi, et al. 4 (2003), Cordaro, et al. 3

(2008),
Frenken, et al. 6 (2010), Mardas, et al. 12 (2011), and Wang, et al. 23 (2014)   Hence, the images of the groups were analyzed using the Image J program. Using the "hands free" tools, the area of newly formed bone tissue was selected from the osteotomized bone stumps, as well as the area of connective tissue, biomaterial, and blood clot, while the program estimated the size in µm 2 .

Statistical analysis
Statistical analysis was performed using Sigma Plot  Specific analysis for BCG (14,28, and 42 days) was performed using analysis of variance (ANOVA) to a criterion 25 after having performed the normality test (Kolmogorov-Smirnov) and equal variance test.
Analysis of the time periods was performed using paired t-test 8,16 and the normality test (Shapiro-Wilk) was used. For all analyses, the Tukey test was used as a post-test. We adopted a significance level of p<0.05.

Immunohistochemical analysis
The immunohistochemistry analysis included the following steps: inhibition of endogenous peroxidase activity with hydrogen peroxide, antigen retrieval with citrate buffer at 60°C for 20 minutes, and blocking of unspecific reactions with skim milk and bovine albumin during incubation of antibodies.         The AG group showed full compatibility bone formation after 28 days; however, the defect was not fully filled in the CG as expected (Figure 9). The average BMD for the CG, AG, and BCG was 498, 850, and 935 HU at 14 days and 485, 1797, and 1337 HU at 28 days, respectively. Moreover, the BMD for BCG was 1554 HU at 42 days ( Figure 10).

Discussion
We rejected the null hypothesis that the newly

Conclusion
Therefore, within the limits of this in vivo study, we may conclude that the tested biomaterial (BC) was favorable to fill bone defects until the end of the reporting period analyzed.