Dynamics of bone healing after osteotomy with piezosurgery or conventional drilling - histomorphometrical, immunohistochemical, and molecular analysis

Jônatas Caldeira Esteves, Elcio Marcantonio Jr, Ana Paula de Souza Faloni, Fernanda Regina Godoy Rocha, Rosemary Adriana Marcantonio, Katarzyna Wilk, Giuseppe Intini, Jônatas Caldeira Esteves, Elcio Marcantonio Jr, Ana Paula de Souza Faloni, Fernanda Regina Godoy Rocha, Rosemary Adriana Marcantonio, Katarzyna Wilk, Giuseppe Intini

Abstract

Background: Piezosurgery is an osteotomy system used in medical and dental surgery. Many studies have proven clinical advantages of piezosurgery in terms of quality of cut, maneuverability, ease of use, and safety. However, few investigations have tested its superiority over the traditional osteotomy systems in terms of dynamics of bone healing. Therefore, the aim of this study was to evaluate the dynamics of bone healing after osteotomies with piezosurgery and to compare them with those associated to traditional bone drilling.

Methods: One hundred and ten rats were divided into two groups with 55 animals each. The animals were anesthetized and the tibiae were surgically exposed to create defects 2 mm in diameter by using piezosurgery (Piezo group) and conventional drilling (Drill group). Animals were sacrificed at 3, 7, 14, 30 and 60 days post-surgery. Bone samples were collected and processed for histological, histomorphometrical, immunohistochemical, and molecular analysis. The histological analysis was performed at all time points (n = 8) whereas the histomorphometrical analysis was performed at 7, 14, 30 and 60 days post-surgery (n = 8). The immunolabeling was performed to detect Vascular Endothelial Growth Factor (VEGF), Caspase-3 (CAS-3), Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor kappa-B Ligand (RANKL), and Osteocalcin (OC) at 3, 7, and 14 days (n = 3). For the molecular analysis, animals were sacrificed at 3, 7 and 14 days, total RNA was collected, and quantification of the expression of 21 genes related to BMP signaling, Wnt signaling, inflammation, osteogenenic and apoptotic pathways was performed by qRT-PCR (n = 5).

Results: Histologically and histomorphometrically, bone healing was similar in both groups with the exception of a slightly higher amount of newly formed bone observed at 30 days after piezosurgery (p < 0.05). Immunohistochemical and qRT-PCR analyses didn't detect significant differences in expression of all the proteins and most of the genes tested.

Conclusions: Based on the results of our study we conclude that in a rat tibial bone defect model the bone healing dynamics after piezosurgery are comparable to those observed with conventional drilling.

Figures

Figure 1
Figure 1
Study design. Different time points (days after surgery) are illustrated in blue and number of animals per group (n) are illustrated in green.
Figure 2
Figure 2
Histological evaluation of the healing process over time. Light micrographs obtained at 3, 7, 14, 30, and 60 days after surgery. Healing process after drilling (Drill group, left) and after piezosuregry (Piezo group, right). Hematoxylin and eosin staining.
Figure 3
Figure 3
Histomorphometrical analysis of the healing process over time.Percentage of bone neoformation measured within the bone defects generated by drilling (Drill) or piezosuregry (Piezo). * indcates statistically significant difference between the Drill and Piezo group (p < 0.05, n = 8).
Figure 4
Figure 4
Immonohistochemical analysis of early healing markers during the bone regeneration process. Percentile ranks of immunolabeling for Vascular endothelial growth factor (VEGF) and Caspase-3 (CAS-3) at 3, 7, and 14 days after surgery, in bone defects generated by drilling (Drill) or piezosuregry (Piezo). No statistically significant differences were found across all time points (n = 3).
Figure 5
Figure 5
Immunolabeling of early healing and bone remodeling markers. Left: Immunolabeling of Vascular endothelial growth factor (VEGF) and Caspase-3 (CAS-3) of tissue sections obtained at 3, 7, and 14 days after drilling (Drill) or piezosuregry (Piezo). Right: Immunolabeling of Osteoprotegerin (OPG), Receptor activator of nuclear factor kappa-B ligand (RANKL), and Osteocalcin (OC) of tissue sections obtained at 7 and 14 days after drilling (Drill) or piezosuregry (Piezo). Sections were stained with the chromogen substrate diaminobenzidine and counterstained with hematoxylin. Staining scores were categorized as negative, positive (brown-yellow color), superpositive (brown color), and hyperpositive (intense brown color) (see Methods). Left (VEGF and CAS-3): at 3 days, only a few posivite regions in brown-yellow color are observed in both groups. Hyperpositive immunolabeling (intense brown color) is visible only at 7 days. At 14 days, the expression of VEGF and CAS-3 tended to be positive (brown-yellow) and superpositive (brown color). Right (OPG, RANKL and OC): at 7 days, hyperpositive immunostaining (intense brown color) is observed for OPG and OC, whereas superpositive immunoreaction (brown color) was detected for RANKL. At 14 days postsurgery, superpositive labeling (brown color) is observed for all three markers. In the negative controls no immunopositivity was detected at all times.
Figure 6
Figure 6
Immonohistochemical analysis of bone remodeling markers during the bone regeneration process. Percentile ranks of immunolabeling for Osteoprotegerin (OPG), Receptor activator of nuclear factor kappa-B ligand (RANKL), and Osteocalcin (OC) at 7 and 14 days after surgery, in bone defects generated by drilling (Drill) or piezosuregry (Piezo). No statistically significant differences were found across all time points (n = 3).
Figure 7
Figure 7
Gene expression analysis at 3 days after surgery. Comparative gene expression analysis between drilling (Drill) and piezosurgey (Piezo) of 21 genes involved with BMP signaling, Wnt signaling, inflammation, apoptosis and osteogenenis 3 days after surgery. * indicates statistically significant difference (p < 0.05), Nd (not detectable) indicates lack of detectable expression (n = 5).
Figure 8
Figure 8
Gene expression analysis at 7 days after surgery. Comparative gene expression analysis between drilling (Drill) and piezosurgey (Piezo) of 21 genes involved with BMP signaling, Wnt signaling, inflammation, apoptosis and osteogenenis 7 days after surgery. * indicates statistically significant difference (p < 0.05), Nd (not detectable) indicates lack of detectable expression (n = 5).
Figure 9
Figure 9
Gene expression analysis at 14 days after surgery. Comparative gene expression analysis between drilling (Drill) and piezosurgey (Piezo) of 21 genes involved with BMP signaling, Wnt signaling, inflammation, apoptosis and osteogenenis 14 days after surgery. * indicates statistically significant difference (p < 0.05), Nd (not detectable) indicates lack of detectable expression (n = 5).

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