Alveolar bone healing in rats: micro-CT, immunohistochemical and molecular analysis

Abstract Alveolar bone healing after upper incisor extraction in rats is a classical model of preclinical studies. The underlying morphometric, cellular and molecular mechanism, however, remains imprecise in a unique study. Objectives The aim of this study was therefore to characterize the alveolar bone healing after upper incisor extraction in rats by micro computed tomographic (Micro-CT), immunohistochemical and real-time polymerase chain reaction (RT-PCR) analysis. Material and Methods Thirty animals (Rattus norvegicus, Albinus Wistar) were divided into three groups after upper incisors extraction at 7, 14, and 28 days. Micro-CT was evaluated based on the morphometric parameters. Subsequently, the histological analyses and immunostaining of osteoprotegerin (OPG), receptor activator of nuclear kappa B ligand (RANKL) and tartrate resistant acid phosphate (TRAP) was performed. In addition, RT-PCR analyses of OPG, RANKL, the runt-related transcription factor 2 (RUNX2), osteocalcin (OC), osteopontin (OPN), osterix (OST) and receptor activator of nuclear kappa B (RANK) were performed to determine the expression of these proteins in the alveolar bone healing. Results Micro-CT: The morphometric parameters of bone volume and trabecular thickness progressively increased over time. Consequently, a gradual decrease in trabecular separation, trabecular space and total bone porosity was observed. Immunohistochemical: There were no differences statistically significant between the positive labeling for OPG, RANKL and TRAP in the different periods. RT-PCR: At 28 days, there was a significant increase in OPG expression, while RANKL expression and the RANKL/OPG ratio both decreased over time. Conclusion Micro-CT showed the newly formed bone had favorable morphometric characteristics of quality and quantity. Beyond the RUNX2, OC, OPN, OST, and RANK proteins expressed in the alveolar bone healing, OPG and RANKL activity showed to be essential for activation of basic multicellular units during the alveolar bone healing.

, which also results in the morphometric parameters evaluated by micro-CT during the alveolar bone healing 30 . In addition, physiologic conditions such as osteoporosis, uncontrolled diabetes and hypertension have been associated with impaired bone metabolism 6,20  of significance of 5% and power test of 95% were adopted, and it was suggested four animals per group.
Thus, with a possible animal loss, it was used five per period of analysis.
The animals were kept in cages in an environment at a stable temperature (22±2°C) and a controlled light cycle (12 h    3 is intense immunostaining and more than 75% of the area, according to previous studies 8,18,19,25 . The representation of the immunolabeling data was performed through the most frequency score attributed to the animals of each period of evaluation. Three-dimensional surface models of the socket observed using the Mimics software (Materialise, Leuven, Belgium). Sagittal plane. White color represents the bone formation in the alveolar socket healing  There was no signal of inflammatory response that could be associated to alveolitis process.

Immunohistochemical analysis
Immunostaining of OPG and RANKL protein at 7, 14, and 28 days after extraction are shown in Figure   6. Representative immunostaining of OPG, RANKL, and TRAP at 7, 14, and 28 days after right upper incisor extraction. OPG and RANKL showed moderate staining all periods analyzed. TRAP was moderate at 14 days and mild at 7 and 28 days. Red arrows indicate intensity of protein tags (63x magnification)    Figure 8E). OST expression increased at 28 and 14 days compared with 7 days (p<0.05, Shapiro-Wilk) ( Figure 8F). RANK was not significantly increased (p >0.05, ANOVA-1 factor) ( Figure 8G). Bone turnover, calculated using the RANKL/OPG ratio, was decreased at 28 days (p<0.05, Tukey test) when compared with 7 and 14 days ( Figure 8H).

Discussion
This study characterized the alveolar bone healing after the upper right incisor extraction in rats. The alveolar bone healing showed to be complete at 28 days after surgery, supporting previous studies 15,16,22,23 . Thus, it is essential to determinate the alveolar healing nature of a model in order to compare with no physiological conditions. Therefore, the alveolar healing characterization provides knowledge to further researches for a favorable bone healing. expression was higher at 14 days. These findings are consistent with effective alveolar healing, since should have an initial organization of granulation tissue, following a gradual replacement by connective tissue and bone. This process is proportional to an increase of extracellular OPG, which blocks the bone resorption 13 .
Also RANKL that regulates osteoclast activity and bone resorption 9 had its expression increased after 14 days, succeeding the bone formation. After 28 days, RANKL expression was lower due to inhibition by OPG, which took control of bone formation. TRAP activated by RANKL, represents the osteoclast activity resorption 25 . The question arises about the correlation of mRNA and protein expression in the developing bone. A direct relationship of mRNA and protein is expected. However, in the current study, it was true only for RANKL. The OPG mRNA expression was increased after 28 days, but it did not correspond to the protein expression over the same period. It could be explained by the fact that OPG is a soluble receptor 14 . Consequently, after synthesized by osteoblasts, OPG is released into the extracellular medium, making its detection more difficult by immunohistochemical staining. Despite this, our molecular findings indicate an increase of OPG expression. These data demonstrate the significance of multiple techniques to describe more completely the behavior of a specific protein during the bone healing.
Taken together, these findings demonstrate a significant function of the OPG/RANK/RANKL system in the osteoclasts and osteoblasts response, and BMU activation. Once activated, BMUs provide valuable information of amount and quality to form the bone 17 .
It will allow the alveolar socket to fill a thicker and slightly separated trabecular bone. These findings complete previous studies that described the alveolar bone healing in rat ended at 28 days 22,23 . Another aspect evaluated was the metabolic activity inside the alveolar socket. At 14 days after extraction, immunostaining revealed high activity of proteins involved in synthesis of the mineralized matrix.
Additionally, OPG and RANKL protein showed strong activity at 14 days after extraction, which can be related to higher BMU activity during this time.
In summary, a favorable alveolar bone healing is 2) Beyond other proteins expressed, the mutual activity of OPG and RANKL is essential for BMUs activation during the alveolar bone healing.