Effects of boric acid on alveolar sockets filling after dental extraction in rats

doi:10.21203/rs.3.rs-2062805/v1

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Effects of boric acid on alveolar sockets filling after dental extraction in rats

https://doi.org/10.21203/rs.3.rs-2062805/v1

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published 05 Apr, 2023

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Purpose: After extraction, dental alveolus filling aims to reduce bone loss and maintain the alveolus volume to patient rehabilitation. Boric acid (BA) is a boron-derived compound that displays osteogenic properties, being an interesting candidate for alveoli filling. This study aims to investigate the osteogenic capacity of the local application of BA in dental socket preservation.

Methods:32 male Wistar rats were submitted to right upper incisor extraction and randomly divided in 4 groups (n=8): control group (no intervention), BA (8 mg/kg) socket filling, bone graft (Cerabone®, Botiss, Germany), and BA + bone graft in equal parts. Animals were euthanized 28 days after dental extraction. MicroCT and histological analysis were performed to evaluate the newly formed bone on the dental alveolus.

Results: MicroCT analysis demonstrated that bone volume fraction (BV/TV), bone surface (BS), bone surface/bone volume ratio (BS/BV), bone surface density (BS/TV), trabecular thickness (Tb.Th), total bone porosity (Po-tot), and total volume of pore space (Po.V(tot)) from BA and BA + bone graft rats were significantly different from the control group. Histological evaluation displayed a delayed bone repair in BA rats, with the presence of connective tissue and inflammatory infiltrate. The BA + bone graft group demonstrated histological aspects like the bone graft animals, however, with less organized osteoblasts, suggesting inferior bone repair.

Conclusion:Osteogenic capacity did not depend on the BA local application after 28 days of dental extraction. The presence of inflammation in the BA group can represent toxicity induced by the substance dosage used.

Boric acids

tooth extraction

osteogenic

biomaterial

animal experiments

rats.

The alveolar ridge is remodeled after dental loss [1–3]. Maintaining the alveolar bone is important for the rehabilitation of the edentulous area. After dental extraction, the alveolus is filled with a blood clot, and the bone repair process commences. An osteoblast-induced bone matrix replaces the blood clot during the first stage, which originates the trabecular bone. Afterward, the trabeculae thicken, the intertrabecular spaces become smaller, and the bone denser[4]. Nevertheless, despite the alveolus repair, the alveolus walls suffer a remodeling process, diminishing the ridge amplitude [2]. This decrease in bone thickness leads to periodontal defects and impairs prosthetics rehabilitation.

The boric acid (BA) is a boron-derived compound and presents osteogenic properties [5–9]). Dietary supplementation with BA demonstrated beneficial effects on bone structure and resistance in rats [10]. In stromal cell culture, BA treatment induced the mRNA expression of alkaline phosphatase, osteocalcin, type I collagen, bone morphogenetic protein (BMP) 7, and calcium deposition [11]. Additionally, in pre-osteoblasts cells, BA increased BMP-4, -6, and − 7 protein levels [12].

The local BA application demonstrated a positive effect in the repair process in bone fractures [5]. Hadidi et al [13] used BA treatment in bone defects in rats’ femurs and observed an increase of osteoblast activity in the initial phases of repair dependent on β-catenin augmentation [13]. Furthermore, in rabbits’ maxillary sinus elevation, the use of BA, when combined with bone graft, evoked an increase of the initial bone repair process [6].

Considering the existent literature evidence, this study aims to investigate the role of BA, when combined or not with bone graft, in the bone repair process in dental alveoli after dental extraction.

Animal care

This study followed the Brazilian guidelines for the care and use of animals for scientific and didactic procedures from the National Council for the Control of Animal Experimentation (CONCEA/Brazil). Also, the experimental protocol was approved by the local Animal Ethics Committee (CEUA/PUCRS 9820/2020) and complied with the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines [14].

Male Wistar-specific pathogen-free rats (250 g-320 g; 8 weeks-old; n = 32) were obtained from the Center for Experimental Biological Models (CeMBE/PUCRS). There was no reduction or loss of the sample during the course of the experiments.

Rats were housed under standard conditions of temperature (22 ± 2ºC), light (12-h light-dark cycle), and humidity (50–70%), in ventilated cages, with autoclaved wood chips bedding. They received a standard rat chow diet (Nuvilab®), with free access to filtered water and food. Before dental extraction, rats were offered chow 3 h after the dental procedure.

Experimental protocol

After a 40-min of habituation, the animals were slightly anesthetized with isoflurane (Isoflurane®, Cristália, Brasil 100% − 1 mL/L) and 5% oxygen for 2.4 mL/min. This was performed to reduce stress before the ketamine (Ketalar®, Pfizer, USA, 50 mg/mL) and xylazine (Rompun®, Bayer, Canada, 20 mg/mL) injection, administered intraperitoneally. During surgery, rats were placed over a heated table to maintain core temperature.

Using a 15C blade, an incision was made between the interdental and intrasulcular papillae of the upper right incisor. The rupture of the periodontal fibers was made by a Hollemback 3S spatula, followed by dental dislocation movements. Using a pediatric forceps, no 21, the upper right incisor was extracted after detecting dental mobility and diastema formation between upper teeth.

The rats were randomly assigned into four experimental groups: tooth extraction (control group), tooth extraction + BA local application, tooth extraction + bone graft, and tooth extraction + bone graft + BA local application. BA (8 mg/kg) with a 0.9% saline solution was applied directly to the dental alveolus, followed by suturing of the dental cavity. The application of bone graft was carried out with small particles (0.5-1.0mm) of bone graft (Cerabone®, Botiss Dental, Germany), filling the dental cavity. When both interventions were combined, the dental alveolus was filled with bone graft + BA (8 mg/kg) in equal parts. After 28 days, euthanasia by isoflurane inhalation was performed, and the maxillae were collected for microCT and histological analysis.

MicroCT Analysis

The samples were submitted to a microCT analysis to investigate the bone microstructures from rats, as previously described [15]. A desktop microCT system (SkyScan 1174v2; Bruker Micro-CT, Kontich, Belgium) was used to evaluate the bone mineral density (gr/cm³) and microarchitecture parameters of the rat socket. Scanning of the specimens was placed in a 15 mm diameter tube filled with 2.5 mL of a solution containing 4% paraformaldehyde and 0.1% glutaraldehyde and was carried out at 50 kV and 800 mA. Images were captured and reconstituted by the CTan program (version 1.16.4.1+, Bruker MicroCT), and used to determine region of interest (ROI). Alveolar socket images were used to standardize the ROI, using a cylinder measuring 1 mm of diameter, covering the beginning of the palatine bone (anterior limit) and the beginning of the contralateral incisor dental root (posterior limit) (Fig. 1A-C). The analysis of bone volume (in mm³), bone surface (BS in mm²), bone volume fraction (BV/TV in percentage), trabecular thickness (Tb.Th in mm), trabecular separation (Tb.Sp in mm), number of trabeculae (Tb.N in mm^− 1), bone surface / volume ratio (BS/BV in mm^− 1), bone surface density (BS/TV in mm^− 1), total volume of pore space (Po.V(tot) in mm³) and total bone porosity (Po-tot in percentage), were performed using the same software.

Histological analysis

The maxillae were decalcified with a 10% ethylenediaminetetraacetic acid (EDTA) solution for 15 days and embedded in paraffin. Slices of five-µm thick were made in longitudinal sections of the dental alveolus and submitted to hematoxylin-eosin (H&E) staining. The histological images were obtained using a microscope (Nikon E200) in 10x magnification, and qualitative morphological analysis was performed.

Statistical analysis

Data were submitted to Shapiro-Wilk tests to confirm normal distribution among the final four experimental groups, resulting in a normal distribution in the microCT samples. Afterward, a one-way analysis of variance (One-way ANOVA) was performed first to evaluate significance among groups. When group interactions were statistically significant (P < 0.05), Sidak’s post hoc test was utilized. Statistical tests and graphs designs were held using GraphPad Prism Software 9.3.1. (GraphPad Software Inc. San Diego, CA).

MicroCT analysis

The BV, BV/TV, BS, BS/TV, Tb.Th, Po-tot, and Po(t) parameters were significantly decreased in rats from the bone graft groups, regardless of BA application, when compared to the control group (Fig. 2A-D, I, and J). Regarding BV/TV, the bone graft application reduced this parameter in relation to the BA treatment (p = 0.0509) (Fig. 2B).

Additionally, BS, BS/BV, BS/TV, Tb.Th, and Po-tot parameters from bone graft groups, regardless of the combination with BA, display significant alteration independent of its association with BA (Fig. 2C, D, E, F, and I). As for the Tb.N, BA + bone graft treated rats present at significant increase compared to the BA + extraction group (Fig. 2H). Furthermore, the Tb.Sp did not alter among the experimental groups (Fig. 2S).

Histological analysis

Dental alveolus without filling (control group) demonstrated newly formed bone with thick trabeculae, signaling the main presence of lamellar bone. Small portions of less organized trabecular bone were observed, as well as small areas of conjunctive tissue in the central area of the alveoli. Furthermore, sometimes it was observed the presence of remaining blood clots and the absence of inflammatory exudate (Fig. 3A).

Rats that received BA socket filling demonstrated a majority of newly formed bone in the interior of the dental alveoli. The trabeculae vary its thickness, suggesting a delay in the bone repair process and tissue frailty. Additionally, the presence of inflammatory infiltrate with high cellularity and connective tissue confirms this delayed bone repair compared to the control group (Fig. 3B).

The dental alveoli submitted to biomaterial filling (bone graft group) displayed trabeculae being organized, demonstrating a recent synthetized bone adjacent to granule, indicating its integration. Furthermore, it was observed connective tissue portions and distinct blood clots. It is possible to see bone graft particles in different sizes being involved by newly formed bone and followed by osteoblasts throughout the vicinity (Fig. 3C).

The association of bone graft with BA evoked aspects similar to the group that was exposed only to the biomaterial. It can be observed the wide presence of newly formed bone with varying trabeculae thickness, with residual graft particles. Distinct portions of connective tissue indicate small areas of bone formation. However, the osteoblast organization surrounding the bone graft particles seems smaller (Fig. 3D).

Dental filling and alveoli repair studies have been widely reported. The alveoli bone repair and loss after dental extraction stimulate the search for substances that impair these processes. Despite the fact that the first studies using microCT in rodent maxillae are from 2010, this analysis represents the most efficient method to investigate the bone repair process [16]. Additionally, the correlation of microCT data with histological evaluations can provide precise information regarding bone remodeling. Herein, this study investigated the effect of BA in alveolus repair after dental extraction, with or without the association with bone graft, and demonstrated that the application of this boron-derived compound did not affect bone remodeling, regardless of its association with the bone graft application.

The use of BA demonstrates an osteogenic potential by stimulating human bone marrow-derived stromal cells gene markers related to cell proliferation [11]. Additionally, in MC3T3-E1 cells, which is a mouse osteoblast cell line, BA acted on cell proliferation and induced the mRNA expression of mineralized tissue-associated proteins [12] Furthermore, it was recently observed that BA regulated the tuftelin1 gene in osteoblasts, demonstrating its role in the alveolus bone and dental formation [8]. Due to this, it could be suggested that the association between BA and a biomaterial could be an alternative management to preserve the alveolus ridge; nevertheless, our data failed to confirm this hypothesis.

The dietary BA supplementation [9, 10, 17, 18] and local BA application [5–7] to promote the osteogenic process have been widely investigated. However, the local BA filling of the dental alveolus is an alternative therapeutic route that has not been investigated. Uysal et al [18] claim that local BA application minimizes the dietary BA supplementation-evoked adverse effects and delimitates the exposed area to the substance.

Gölge et al [5] observed that the local BA application in fractured femur rats displayed enhanced bone repair. Also, in rabbits, the association between BA and an autogenous bone/xenograft mixture in maxillary sinus augmentation demonstrated an improved bone repair in a 4-week period of observation, with higher expression of alkaline phosphatase (ALP), osteocalcin, proliferating cell nuclear antigen (PCNA), and vascular endothelial growth factor (VEGF), suggesting that the osteoblasts differentiation and bone synthesis are promoted by the presence of BA [6]. Thus, the improved bone repair might be related to the higher activity of osteoclast and osteoblasts throughout the four weeks of the experiment [6, 17]. In this study, the initial bone repair process was investigated using microCT and histological analysis in a 4-week period (28 days), but with no demonstration of BA impact in bone remodeling.

Hassumi et al.[19] characterized the alveolar repair at 7, 14, and 28 days by immunohistochemical, molecular and microCT analyses. The authors described highest values BV, BV/TV, Tb.Th, Tb.N and lowest values in Po-tot and Tb.Sp at 28 days post-extraction. In our study, the data compared to the control group with the tooth extraction + BA group reflect the inverse situation, suggesting that there is a delay in repair from bone microarchitecture analysis in the presence of the BA.

The use of bone substitutes in alveoli filling aims to prevent the ridge dimensions. Lyophilized bone grafts, porous hydroxyapatite, and calcium phosphate-based are among the most used biomaterials [1]. Here, a lyophilized bovine bone graft was used for its slow degradation and to guarantee alveoli volume, which is important in procedures that aim to conserve the alveolus ridge [3].

The presence of bone graft, BA, and their association failed to demonstrated differences during the bone remodeling process, and failed to induce an enhanced repair in the alveolus bone. The absence of immunostaining investigation regarding osteoclastic and osteoblastic activity limits the analyses of the whole bone repair scenario, seen that these markers could help gain further insight of the actions of BA in the alveolus bone. Concerning the histological evaluation, the descriptive analysis did not show notable differences between the experimental groups exposed to BA and between groups not. Nonetheless, histological samples from the BA rats demonstrated regions with inflammatory cells and connective tissue, denoting a delay in the repair process. These findings go against previous studies which evaluated bone repair in dental alveoli from rats, where the presence of residual inflammatory cells was undetected in this healing time [4, 19–21]. In this study, the rats that received the local application of bone graft in the dental alveolus demonstrated a delayed bone repair in the microCT and histological analyses, which was expected. The presence of connective tissue with the biomaterial and the delayed healing process is well seen in literature data [3, 22–24]. Nonetheless, in this study, the BA experimental group demonstrated less organized osteoblasts surrounding the bone graft particles, different from what occurred in the bone graft group without the association with BA. It is possible to suspect that the BA filling might evoke toxicity and postpone the repair process due to the chosen BA dosage since previous research describe that the BA can inhibit osteoblast synthesis when used in higher concentrations [12, 25]. Notably, the non-toxic dosage of BA varied from 3 mg/kg to 8.7 mg/kg in previous studies, demonstrating interesting results regarding bone repair[5–7] According to previous studies, dietary BA 18–20 mg/kg and 6 µg/ml in mesenchymal cell culture display toxicity, nonetheless, toxic dosages for local applications lacks information [25, 26]. In this study, it was applied 8 mg/kg of BA locally, into the alveolus after dental extraction, associated or not to bone graft in equal parts, based on a previous report [5]. Nonetheless, other concentrations might gain further insights since this study failed to demonstrate significant results regarding BA and its possible osteogenic properties. The association of BA with a bone graft was firstly described by Kömürcü et al [7], in a study regarding autogenous bone associated with BA in posterolateral spinal fusion. The authors observed an improved repair process histologically when compared to the autogenous bone isolated [7]. Afterward, Ulu et al[6] identified an increased initial bone repair with BA, suggesting its use to enhance the bone formation process and to reduce the bone graft absorption [6]. In this investigation, the bone remodeling did not differ when the bone graft was associated with BA, and, similar to Kömürcü et al [7], it was not possible to observe an increase in bone repair process when pure BA was applied locally.

This investigation failed to prove the hypothesis that the use of BA could benefit the repair process of the alveolus bone after 28 days of the upper incisor extraction from rats. These data suggest a broader investigation using different dosages of the substance, with the intent to observe the osteogenic activity to maintain the alveolus ridge. Also, this wider investigation is important to gain further insights regarding cell toxicity related to the local application of BA. Moreover, its association with a biomaterial can be viable with other BA concentrations since the data obtained here did not differ when BA was associated with bone graft. Lastly, this study sheds light on the possible use of BA in bone remodeling after dental extraction.

Consent for publication:

Animal Ethics Committee Approvement:

Author’s contribution:

R.R.C. and J.B.B.W. conceived and designed research;
R.R.C. and G.C. performed experiments;
R.R.C. and R.D.S.F. analyzed data, interpreted results of experiments, prepared figures and drafted manuscript;
R.R.C. and J.B.B.W. revised manuscript;
R.R.C., G.C., R.D.S.F. and J.B.B.W approved final version of the manuscript.

Funding:

This work was supported and funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) – [Finance Code 001].

Consent to participate:

Not applicable.

Availability of data and materials:

Not applicable.

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No competing interests reported.

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Journal Publication

published 05 Apr, 2023

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You are reading this latest preprint version

Effects of boric acid on alveolar sockets filling after dental extraction in rats

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Animal care

Experimental protocol

MicroCT Analysis

Histological analysis

Statistical analysis

Results

MicroCT analysis

Histological analysis

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1