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. 2021 Oct 1;11(1):19535.
doi: 10.1038/s41598-021-98713-2.

Alveolar socket healing in 5-lipoxygenase knockout aged female mice treated or not with high dose of zoledronic acid

Ramez H Mahmoud #  1 , Claudia C Biguetti #  2 , Gustavo B Simionato  1 , Isabela C Custódio  3 , Raquel B P Silva  1 , Marco A H Duarte  4 , Leonardo P Faverani  5 , Edilson Ervolino  1 , Walid D Fakhouri  6 , Mariza A Matsumoto  7
Affiliations

Alveolar socket healing in 5-lipoxygenase knockout aged female mice treated or not with high dose of zoledronic acid

Ramez H Mahmoud et al. Sci Rep. .

Abstract

This study investigated the role 5-lypoxigenase (5-LO) on alveolar socket healing in aged female mice treated with zoledronic acid (ZL). Forty 129/Sv female mice (64-68 weeks old), 20 wild type (WT) and 20 5-LO knockout (5LOKO) were equally distributed according to ZL treatment: WT Control, WT ZL, 5LOKO Control, and 5LOKO ZL. ZL groups were treated with an intraperitoneal injection of 250 µg/Kg of ZL, while controls were treated with saline. Treatments were administered once a week, starting four weeks before surgery for tooth extraction and until 7 and 21 days post-surgery. Mice were euthanized for a comprehensive microscopic analysis (microCT, histomorphometry and immunohistochemistry). WT ZL mice presented intense inflammatory infiltrate (7 days), delayed bone formation (21 days), reduced collagenous matrix quality, and a deficiency in Runx-2 + , TRAP + , and macrophages as compared to controls. 5LOKO ZL animals presented decreased number of Runx-2 + cells in comparison to 5LOKO Control at 7 days, but no major changes in bone healing as compared to WT or 5LOKO mice at 21 days. The knockout of 5LO favored intramembranous bone healing in aged female mice, with a direct impact on inflammatory response and bone metabolism on the development of ONJ-like lesions.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Qualitative results of microCT and histological analysis for WT vs. 5LOKO mice treated or not with ZL on skeletal phenotyping WT and 5LOKO aged female mice. Aged 129 Sv WT and 5LOKO female mice were treated with 0.9% saline solution (Vehice) or ZL (250ug/Kg), once a week, for 7 weeks. Mice were euthanized and femur and L5 vertebrae were collected for analysis by microCT (A), H&E (B) and Picro-thionin (Schmorl) staining for osteocytes lacunae (C). (C) Scale bar 100 µm; (D) scale bar 50 µm. (A) Three-dimensional representative images obtained with the CT-Vox software (Skyscan, Kontich, Belgium).
Figure 2
Figure 2
MicroCT analysis of alveolar sockets post tooth extraction from WT vs. 5LOKO mice treated or not with ZL. Aged 129 Sv WT and 5LOKO female mice received IP injections of 0.9% saline solution (C) or 250 μg/Kg (ZL groups) once a week, and upper right incisor were extracted at 4 week of each treatment. Mice were euthanized for maxillary bones removal after 7 days and 21 days post tooth extraction. (A) Representative transaxial images of bone hyperdensity. Scale bar 1.5 mm. (B) Bone volume/Tissue Volume (BV/TV, %), (C) Trabecular Separation (Tb.Sp), (D) Trabecular Thickness (Tb.Th), and (E) Trabecular Number (Tb.N) for new bone in alveolar sockets and 7 and 21 days post tooth extraction. Quantitative results are presented with Box and whiskers (Min to Max). Symbol * indicates a statistically significant differences between different treatments and/or genotypes (p < 0.05).
Figure 3
Figure 3
Histopathological analysis of alveolar sockets post tooth extraction from WT vs. 5LOKO mice treated or not with ZL. Aged 129 Sv WT and 5LOKO female mice received IP injections of 0.9% saline solution (C) or 250 μg/Kg (ZL groups) once a week, and upper right incisor were extracted at 4 weeks of each treatment. Mice were euthanized for maxillary bones removal after 7 days and 21 days post tooth extraction. (A) Representative transversal sections are observed throughout days 7 and 21, from the central area of alveolar sockets. Histological slides were stained with HE (A) and modified Goldner trichrome + Alcian blue (B). Images were captured at 10 × and 100 × magnification (panels). Black scale bar = 100 µm, Blue scale bar 50 µm. NB New bone formation, BC Blood clot, MB mature remodeling trabeculae, blue arrowheads = osteoclasts; red arrows = non-viable old bone.
Figure 4
Figure 4
Birefringence analysis of collagen fibers in alveolar sockets post tooth extraction from WT vs. 5LOKO mice treated or not with ZL. Aged 129 Sv WT and 5LOKO female mice received IP injections of 0.9% saline solution (C) or 250 μg/Kg (ZL groups) once a week, and upper right incisor were extracted at 4 weeks of each treatment. Mice were euthanized for maxillary bones removal after 7 days and 21 days post tooth extraction. (A) Representative transversal sections alveolar socket upon polarized and conventional light. Polarized light shows green birefringence color for thinner collagen fibers; yellow and red colors at birefringence analysis indicate thick collagen fibers. Original magnification was 40x, Scale bar: 100 µm. (B) Total area (pixels2) of collagen and area for each birefringence color (pixels2) (green, yellow and red). RGB values for green spectra (R:114–255, G:78–255, B:10–255), yellow (R:196–255,G:32–255,B:10–255) and red (R:162–255,G:0–175,B:0–255). Quantitative results are presented with Box and whiskers (Min to Max). Symbol * indicates a statistically significant differences between different treatments and/or genotypes (p < 0.05).
Figure 5
Figure 5
Immunolabeling and quantification of 5LO + cells at 21 days post tooth extraction in WT C vs. WT ZL. Aged 129 Sv WT female mice received IP injections of 0.9% saline solution (C) or 250 μg/Kg (ZL groups) once a week, and upper right incisor were extracted at 4 week of each treatment. (A) Representative transversal sections from the central area of alveolar sockets stained for 5LO + cells in WT C and WT ZL group. (A’) Quantitative and comparative analysis of detached 5LO + cells in WT C vs. Scale bar 30 µm. WT ZL mice at 21 days post-extraction. Results are presented as the means (± SD) of area density (%). Symbol * indicates a statistically significant difference between groups (p < 0.05). DAB chromogen and counterstained with Fast green.
Figure 6
Figure 6
Immunolabeling and quantification of COX2 and F4/80 + cells at 7 and 21 days post tooth extraction in WT and 5LOKO mice, treated or not with ZL. Aged 129 Sv WT female mice received IP injections of 0.9% saline solution (C) or 250 μg/Kg (ZL groups) once a week, and upper right incisor were extracted at 4 week of each treatment. Mice were euthanized for maxillary bones removal after 7 days and 21 days post tooth extraction. (A) Representative transversal sections from the central area of alveolar sockets stained for COX2 + cells (A) and F4/0 + cells (B). Quantitative and comparative analysis of COX2 + cells (A’) and F4/80 + cells (B’) among groups. Scale bar: 50 µm. Quantitative results are presented with Box and whiskers (Min to Max). Symbol * indicates a statistically significant difference between groups (p < 0.05). DAB chromogen and counterstaining with Harris’ Hematoxylin.
Figure 7
Figure 7
Immunolabeling and quantification for Runx2 + , OCN + and TRAP + cells at 7 and 21 days post tooth extraction in WT and 5LOKO mice, treated or not with ZL. Aged 129 Sv WT female mice received IP injections of 0.9% saline solution (C) or 250 μg/Kg (ZL groups) once a week, and upper right incisor were extracted at 4 week of each treatment. Mice were euthanized for maxillary bones removal after 7 days and 21 days post tooth extraction. (A) Representative transversal sections from the central area of alveolar sockets stained for Runx2 + cells (A), OCN + cells (B) and TRAP + cells (C), Scale bar: 50 µm. Quantitative and comparative analysis of Runx2 + cells (A’), OCN + cells (B’), and TRAP + cells (C’) among groups. Quantitative results are presented with Box and whiskers (Min to Max). (D,E) Quantitative analysis for attached and detached TRAP + cells. Results are presented as the means (± SD) of area density (%). Symbol * indicates a statistically significant difference between groups (p < 0.05). DAB chromogen and counterstaining with Harris’ Hematoxylin.
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