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.
Rats; Gene expression; Osteoprotegerin; Bones and bone; Bone remodeling
Micro-CT analysis using CT Analyzer software (SkyScan, Leuven, Belgium). Coronal plane. The area outlined in red indicates the region of interest (ROI), which was positioned within the alveolar socket undergoing bone healing
Region for analysis of middle third of the socket observed using the color filter in the CT Analyzer software (SkyScan, Leuven, Belgium). Sagittal plane. Black arrows delimit the contour of the alveolar socket. Red arrows delimit the area of bone formation
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
Mean values and standard deviation of BV, BV/TV, Tb.Th, Tb.Sp and Po-tot. BV, BV/TB and Tb.Th increased progressively over time. Consequently, a gradual decrease of Tb.Sp and Po-tot was observed. The different letters (a, b and c) indicate significant statistical difference (p<0.05) between the groups
Histological images of the medium third of the alveolar socket at 7, 14, and 28 days after extraction. Hematoxylin and eosin staining (63x magnification)
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)
Graph with the scores and p-value of OPG, RANK and TRAP
Graph showing the relative gene expression and standard deviation of OPG, RANKL, Runx2, OC, OPN, OST, RANK, RANKL/OPG. The * indicates significant statistical difference (p<0.05) between time periods