Bone tissue is physiologically dynamic. It is in continuous remodeling cycle of bone formation and resorption, maintaining a constant calcium homeostasis and bone mass during vertebrates’ lives (Kobayashi et al., 2016). The periodontal ligament and the mineralized tissues support many functional loads and transmit them to alveolar bone. Therefore, the magnitude, the direction, the frequency and the duration of these loads determine alveolar bone remodeling (Beertsen et al., 1997; Ho et al., 2010).

Dental occlusal trauma plays an important role in alveolar bone remodeling and its effects includes many cytokines and signaling pathways. However, the exact mechanism of the traumatic stimulus for alveolar bone remodeling is still unclear (Wan et al., 2012). Changes in mechanical stimulation as a result of dental extraction can lead to changes in bone tissue. Traumatic occlusal forces are responsible for altering the expression of osteoblasts and osteoclasts genes, and thus may cause bone resorption (Walker et al., 2008).

Mechanobiology is responsible for studying the action of these mechanical changes on biological processes through cellular signaling, enabling the understanding of how these tissues are produced, maintained and adapted by the cells as a reply to mechanical stimuli. The research in this field can be divided into two fields: mechanoregulation algorithms to estimate changes in bone tissue and models that analyze cellular mechanobiology (van der Meulen and Huiskes, 2002; Giorgi et al., 2016).

Understanding of cell behavior and expression of proteins related to tissue changes is possible by means of immunohistological analysis (Lin et al., 2017). One critical and most studied Wnt pathway is the canonical Wnt signaling, that works by regulating the amount of the transcriptional co-activator β-catenin, which controls the major programs of gene expression in development (MacDonald et al., 2009).

It is established that Wnt plays an important role in the regulation of bone mass in humans and rodents, positively affecting bone formation by managing osteoblasts at different degrees, and it is recognized as an essential regulator of osteoblast activity through the translation of mechanical loading into biochemical signals (Baron and Rawadi, 2007; Tyrovola, 2019).

The Wnt signaling pathway is regulated by molecular and mechanical extracellular environment signals and its biological effects well settled (Robling and Turner, 2009). Wnt/β-catenin signaling activation induces osterix expression, a transcription factor that promotes osteoblast differentiation. Besides that, osteoprotegerin (OPG) is a target gene of this signaling pathway. Therefore, the expression of OPG is induced, when signaling is activated, in order to inhibit osteoclasts differentiation, preventing bone resorption (Kobayashi et al., 2016).

The literature (Bonewald and Johnson, 2008; Xu et al., 2016) cited the osteocytes as the major bone cell type responsible for mechanical strain sensing, acting in the bone remodeling process by sensing and transmitting external mechanical loading information to effector cells. The osteocytes use the intracellular Wnt/β-catenin pathway to transmit signals of mechanical loading to cells on the bone surface (Bonewald and Johnson, 2008).

The finite element analysis is an in silico evaluation that has been applied when studying living tissues, having a wide spectrum of clinical applications (Tsouknidas et al., 2015). The effectiveness of this analysis depends on obtaining precise models (Hohmann et al., 2011). However, it is now possible to obtain proper models, since methods such as computerized tomography or microtomography or serial sections of the samples can be used (Mehari Abraha et al., 2019). Its acceptance allowed the analysis to become vastly used in biomechanics, and the development of increasingly complex and sophisticated three-dimensional models made it easier the comprehension of the in situ mechanical responses of biological systems (Mehari Abraha et al., 2019).

Regarding alveolar bone, studies using the finite element method have shown that, whether in rodents, non-human primates or humans, there is a bone adaptation coming from different Strain regimes associated with chewing (Cox et al., 2012; Prado et al., 2016). Tooth absence can lead to occlusal hypofunction, that is a reduced bite force on the antagonist tooth and adjacent alveolar bone during mastication and, then, cause a negative impact in bone homeostasis (Shimomoto et al., 2007; Xu et al., 2016).

In this study, the right upper incisor tooth extraction was performed to cause altered mechanical loading to study the tissue response of the alveolar bone from the adjacent tooth (left upper incisor tooth), considering that the mastication force was altered. Thus, our hypothesis was that the tooth extraction condition can cause β-catenin expression and altered strain pattern of the alveolar bone of the adjacent tooth (left upper incisor). The aim of this study was to analyze the relationship between alveolar bone deformation and β-catenin expression levels in response to the mechanical load changed by tooth extraction in adult rats.

This study was approved by the Ethics Committee on Animal Experimentation (CEUA) from the Biology Institute (IB) of the University of Campinas (UNICAMP) (protocol number: 4674-1/2017).

Animal models provide relevant information about a variety of disorders in dentistry. The rat model has been widely used for studies in dentistry, including those which involve periodontal diseases, alveolar bone healing, tooth movement, osteogenesis, osseointegration, use of biomaterials, and altered occlusal loadings, for example (Okamoto and de Russo, 1973; Shimomoto et al., 2007; Walker et al., 2008; Wan et al., 2012; Xu et al., 2016; Zeng et al., 2016; Koutouzis et al., 2017; Lee et al., 2017; Hassumi et al., 2018; Palin et al., 2018; Uslu et al., 2018; Wang et al., 2018). Besides that, tooth extraction, inclosing incisor extraction, has been considered as a classical model of preclinical studies and has been applied in many studies with different aims in the last years (Avivi-Arber et al., 2010; Shoji et al., 2010; Boonyagul et al., 2014; Curra et al., 2016; Hassumi et al., 2018; de Oliveira Puttini et al., 2019).

Medical and dental science has benefited from the rat as a model through the investigation of pathological conditions affecting craniofacial morphology. As the rat has in the past, and continues to play, such a crucial role in the advancement of understanding of craniofacial development, morphology and evolution, it is surprising that very little published data exists regarding the anatomy, and in particular the craniofacial biomechanics behavior.

Mechanobiology is governed by cells response within the tissue to mechanical forces, and is essential to bone adaptation, since the skeletal tissues morphology and structural fitness are modulated by mechanical forces. Mechanically load-induced strains in the cells and stresses of fluid flow can bring on changes in gene expression in bone tissue. It is known that bone is a complex tissue made up of cells and collagenous matrix, which includes minerals, and its homeostasis is strongly conditioned on mechanosensory regulatory signals of environmental forces in biochemical responses. Bone architecture and mass modifying occurs to make possible a more efficient performance of the bone tissue. It is clear that bone cells act as mechanosensors, but the central issue in this study field is how external muscle loads are to skeletal tissues, how bone cells perceive these loads, and how these signals are translated into biochemical reactions to produce cell expression or differentiation, resulting in macroscopic changes in the bone structure (van der Meulen and Huiskes, 2002; Feller et al., 2015; Ruggiu and Cancedda, 2015).

According to the results of the present study, it is believed that the incisor tooth adjacent to the extracted tooth can present a modified masticatory loading regime. It is worth mentioning that the scarcity of information on how the rat behaves in vivo in chewing after extraction should be considered.

Little is known about the transduction of osteogenic signals to stimulate bone formation or bone resorption, because this pathway is hard to illustrate. Nevertheless, some recent molecular advances allow looking for genes and molecules to understand their role in the adaptation pathways (van der Meulen and Huiskes, 2002). Thus, the present study had the aim to evaluate Wnt/β-catenin signaling in bone, once this protein is responsible for the transmission of mechanical stimuli to biological signals for bone formation.

It is accepted that mechanical loads placed on bone generate a variety of stimuli that could be detected by the osteocyte, but the mechanism by which osteocytes sense mechanical strain has yet to be fully understood and described (Oftadeh et al., 2015). The Wnt/β-catenin pathway has been identified using in vitro and in vivo approaches to be an important pathway in osteocytes to sense and transduce the signals of mechanical stimuli to bone cells (Duan and Bonewald, 2016). Activation of Wnt/β-catenin signaling was related as a normal response to bone stimulation (Robinson et al., 2006; Javaheri et al., 2014; Lara-Castillo et al., 2015).

In the present study, when we induced the change in the normal stimulus, i.e., extracted the right upper incisor, we observed the activation of Wnt/β-catenin signaling in alveolar bone. We can suggest that the bone cells mechanosensing by an altered loading pattern microenvironment regulate the bone tissue behavior coordinating adaptive alterations in bone mass and architecture. In response to unusual signals several mechanotransduction pathways are activated (Shivashankar et al., 2015; Giorgi et al., 2016).

In the present study, the mechanical load pattern was altered by tooth extraction. It was possible to note the Wnt/β-catenin signaling in response to this altered load pattern, as it can be seen by a greater positive β-catenin staining osteocyte of the lingual bone in the 14-day group. β-catenin signaling in the alveolar bone of the adjacent tooth allows the understanding that the bone tissue had to adapt its structure to these new loads that were applied. Lara-Castillo et al. (2015) observed that β-catenin signaling activation in osteocytes occurred in regions that suffered both compression and tension after a mechanical stimulus. They suggested that the critical factor is the magnitude of the change, not necessarily the type of deformation applied.

To date, little is exposed about how β-catenin signaling is physiologically modulated by masticatory loads (Galli et al., 2010; Oftadeh et al., 2015). It is necessary developing additional studies to verify if the β-catenin activation pattern corresponds spatially and temporally with bone remodeling against mechanical stimuli. In the literature, it has been consistent that β-catenin plays an important role in bone formation and remodeling (Herencia et al., 2016).

Frost introduced the “mechanostat theory” in bone biology to understand the “minimum effective strain (MES),” which relates bone architecture changes to mechanical loads (Tyrovola, 2019). In our study, the FEA was used in order to figure out about the bone tissue threshold for strain associated with the β-catenin activation in alveolar bone, and how is the behavior of the alveolar bone after induction of changes in mechanical stimulus on adjacent tooth (upper left incisor) due to tooth extraction. We verified that the alveolar bone was deformed with changes in mechanical strain in comparison of two group.

The computational simulation by FEA in this present study has the limitation being a static analysis, presenting only the strain distribution from a bite load. In addition, we predicted that the experimental group apply the same bite force during the power stroke phase. In fact, dynamic features must be considered in a future study, as the frequency of bite loads as well as changes in the force orientation in result of a possible changes in the mandible position during incisor biting. Thus, beyond to establish the relation between the β-catenin expression to the changes in the mechanical strain distribution, this relationship could include dynamic features during feeding.

In conclusion, the findings of the present study were in accordance with our hypothesis that the condition of tooth extraction can cause the expression of β-catenin and alter the regimes of bone deformation.

All datasets generated for this study are included in the article/Supplementary Material.

The animal study was reviewed and approved by Ethics Committee on Animal Experimentation (CEUA) 4674-1/2017.

BF, AR, and AF contributed to conception of the manuscript. AR and BF contributed to the experiment. BF, AF, RA, GA-S, RO, and AR contributed to the data acquisition. BF, AR, FP, and AR contributed to the analyses and interpretation of the results. All authors contributed to design of the experiments and reviewed the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.