Osteonecrosis modulates extracellular matrix deposition through collagen I deposition in obese rats via the TGF-β protein

Osteonecrosis modulates extracellular matrix deposition through collagen I deposition in obese rats via the TGF-β protein | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Osteonecrosis modulates extracellular matrix deposition through collagen I deposition in obese rats via the TGF-β protein Wilson José de Miranda Lima, Jannerson Cesar Xavier Pontes, Rubens Silva Araújo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5361050/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Osteonecrosis, characterized by the death of bone tissue in the jaws, is termed bisphosphonate-related osteonecrosis of the jaws (BRONJ) when caused by bisphosphonate use. Obesity, a significant public health issue, has been associated with both BRONJ and other oral conditions, such as caries and periodontitis, highlighting the relationship between systemic factors and oral health. This study investigated the influence of TGF-ß, TNF-α, and collagen I on bone tissue and their correlation with mandibular osteonecrosis in obese rats. Twenty-four male Wistar rats ( Rattus norvegicus ) were divided into four groups: healthy, osteonecrotic, obese, and obese with osteonecrosis. Osteonecrosis was induced with zoledronic acid (250 µg/kg), which was administered weekly for eight weeks, combined with tooth extraction, while obesity was induced by a high-glycemic diet. The analyses revealed that, compared with the patients in the osteonecrosis group, the obese group with osteonecrosis had a 67.99% increase in the necrotic area, whereas the obese group had a 43.85% reduction. The healthy group had the largest reduction (97.11%). For TNF-α, there was intense staining in the osteonecrosis (27.59 ± 7.65 µm²) and obese (25.52 ± 8.31 µm²) groups, whereas the level of TGF-β was greater in the obese with osteonecrosis group (44.98 ± 3.93 µm²). Collagen I staining was more intense in healthy animals. The potential interaction between TGF-ß, TNF-α, and collagen I in bone tissue may be essential for understanding bone remodeling; however, further studies are needed to explore these mechanisms. Bone remodeling Metabolic Diseases Bisphosphonates Mandible Immunohistochemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Obesity is widely recognized as a major public health condition associated with a range of comorbidities that affect various body systems. Among the pathophysiological characteristics of obesity, a chronic state of systemic inflammation stands out because of the excessive accumulation of adipose tissue. This increase in adipocytes results in the exacerbated secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), as well as acute-phase proteins such as C-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP), which also contribute to endothelial dysfunction (Singh et al. 2017 ). This inflammatory state is associated with the infiltration of macrophages into adipose tissue, which shifts from an anti-inflammatory M2 phenotype to a proinflammatory M1 phenotype, further aggravating inflammation and leading to insulin resistance (Junqueira and Carneiro 2017 ). Thus, obesity affects not only energy metabolism but also the endocrine, cardiovascular, pulmonary, and musculoskeletal systems, contributing to the development of diseases such as dyslipidemia, hypertension, and even cancer (Kypridemos et al. 2014 ; Lopategi et al. 2016 ; Kovesdy et al. 2017 ; Gasparin et al. 2018 ; Lakkis and Weir 2018 ; Vekic et al. 2019 ). Among the various systems impacted by obesity, the skeletal system stands out as a vital reservoir of calcium and phosphate and serves as support for soft tissues and vital organs. Bone tissue undergoes constant remodeling through complex cellular processes involving osteoblasts, osteoclasts, and osteocytes. These processes are mediated by bioactive molecules, such as transforming growth factor-beta (TGF-β) and type I collagen, which play crucial roles in maintaining bone homeostasis. During remodeling, osteoclasts secrete cytokines, such as TGF-β, which regulate both bone resorption and osteoblast differentiation (HP Rang et al. 2007 ; Van Schepdael et al. 2013 ). This balance is essential for bone tissue renewal and repair capacity in response to injuries; however, when this equilibrium is disrupted, as in the case of jaw osteonecrosis, the inflammatory process plays a central role. Medication-related osteonecrosis of the jaw (MRONJ) has been associated with the use of bisphosphonates, substances frequently used to treat bone diseases, such as osteoporosis. Bisphosphonates inhibit bone resorption by interfering with osteoclast activity, which, while effective in preventing fractures, can lead to osteonecrosis under adverse conditions (Ruggiero et al. 2014 ; O’Carrigan et al. 2017 ; Farrell et al. 2018 ; Borgo et al. 2019 ). These medications, by accumulating in the bone matrix, negatively affect local vascularization and bone repair capacity, resulting in bone tissue exposure and necrosis (Fernandes et al. 2005 ; Edwards et al. 2008 ; Santos 2010 ; Rasmusson and Abtahi 2014 ; Tardast et al. 2015 ; Zandi et al. 2016 ). Owing to its lower vascularization than the maxilla, the mandible is particularly affected by this condition (Andrade 2015 ; Kim et al. 2016 ; Gong et al. 2017 ). The relationship between obesity and jaw osteonecrosis is known but not well understood, and the impact of chronic inflammation and oxidative stress—both exacerbated by obesity—on bone tissue becomes evident. The continuous systemic inflammation in these individuals can worsen local inflammatory processes in bone tissue, especially in the presence of bisphosphonates, contributing to the development of osteonecrosis (Lima et al. 2023 ). Thus, the interaction between proinflammatory factors, such as TNF-α and IL-6, and the bone remodeling mechanisms mediated by TGF-β play crucial roles in both obesity and osteonecrosis (Van Schepdael et al. 2013 ). This study aimed to explore the influence of TGF-β, TNF-α, and type I collagen on bone tissue, as well as their correlation with bisphosphonate-induced jaw osteonecrosis and obesity. Materials and methods Sample The experiment consisted of 24 male Wistar rats ( Rattus norvegicus ) aged approximately 30 days and weighing 180 g, which were divided into four groups: healthy, osteonecrotic, obese, and obese with osteonecrosis (n = 6 per group). The animals were housed in the animal facility of the Institute for Drug and Medication Research (IPeFarM) at the Federal University of Paraíba. The research protocol was approved by the Animal Ethics Committee (CEUA) under number 8738120220 (ID 000661) and complies with all the guidelines of Law 11,794 of October 8, 2008, and Decree 6,899 of July 15, 2009, as well as the regulations set forth by the National Council for Animal Experimentation Control (CONCEA). Osteonecrosis induction - Healthy and Osteonecrosis Groups Osteonecrosis was induced in the designated groups through the administration of zoledronic acid, following an adaptation of the methodology of Biguetti et al. ( 2019 ). This induction was combined with the extraction of the left lower first molar. The animals in the healthy group received saline solution intraperitoneally, whereas those in the osteonecrosis group received 250 µg/kg of zoledronic acid (Blau Farmacêutica) intraperitoneally once per week for eight weeks (Fig. 1 a and 1 b). The animals underwent tooth extraction between the fourth and fifth doses of the medication. For this procedure, they were sedated with a combination of ketamine and xylazine administered intraperitoneally. A number five exploratory probe was used to dislocate the tooth. - Obese and obese with osteonecrosis groups Osteonecrosis induction in these groups was performed as described in section 2.2.1 but at a different time, as the animals had to undergo the obesity induction protocol in parallel (described in section 2.3), which lasted 16 weeks (Fig. 1 c) (Biguetti et al. 2019 ). Obesity Induction The obese and obese with osteonecrosis groups were provided with an obesogenic diet composed of pellet-type chow (Nuvilab®), refined sugar (Alegre®), and whole condensed milk (Camponesa®). This high-glycemic index diet had a value of 77.6 and a high glycemic load of 38.8. To prepare 100 g of feed, 45.2 g of chow was ground and mixed with 9.6 g of sugar, followed by the addition of 45.2 mL of condensed milk. This mixture was homogenized, shaped to resemble conventional chow, dried in an oven (55°C) for 24 hours, and stored under refrigeration. The animals were provided 800 g of pellets per week throughout the experiment (16 weeks). To confirm obesity, the Lee index (defined as the ratio between the cube root of the animal's body mass and the nasoanal length) and the adiposity index (defined as the sum of the individual masses of the epididymal, inguinal, and retroperitoneal fat layers multiplied by 100 and divided by the final body weight) were used (Lima et al. 2023 ). Histological processing After euthanasia, each specimen (comprising the mandible) was fixed for 48 hours and then decalcified in a 5% nitric acid solution. Following demineralization, the material was sectioned and subjected to processing steps, including dehydration in increasing concentrations of ethanol, clearing in xylene, impregnation in paraffin baths at a maximum temperature of 60°C, embedding in paraffin molds, and microtomy, with the resulting sections cut to a thickness of 4 µm. The samples were stained with hematoxylin and eosin and Masson’s trichrome. Immunohistochemical and histomorphometric analyses For immunohistochemical analysis, the samples were reactivated with a pH 6.0 citrate retrieval solution (EasyPath Diagnósticos) in a Pascal pressurized chamber (Dako Cytomation, Denmark), and endogenous peroxidase was blocked for 30 minutes (EasyPath Diagnósticos). Following this step, nonspecific protein blocking was performed, and the sections were incubated with the following primary antibodies: anti-TNF-α (Cloud-Clone Corp/PAA133Hu01/Polyclonal Antibody; 1:100), anti-TGF-β (Cloud-Clone Corp/PAA124Ra01/Polyclonal Antibody; 1:100), and anti-collagen I (Boster/PA2140-2; 1:100). A biotinylated secondary antibody (goat anti-rabbit or anti-mouse biotinylated antibody at a dilution of 1:100, Link Sistem - HRP - Dako 4061) was subsequently added. The reaction was visualized via 0.024% diaminobenzidine (DAB - HRP) solution (EasyPath Diagnosticos) and contrasted with Harris hematoxylin (Pontes et al. 2024 ). To calculate the area of extracellular matrix deposition, as well as the positive areas for all immunohistochemical reactions, all pixels with shades of blue (Masson's trichrome) or brown (positive immunohistochemical staining) were selected to create a binary image. Digital processing and area calculation in µm² were conducted via a 40X objective and quantified interactively with Image-Pro Plus software (Pontes et al. 2024 ). Statistical analysis The results were initially analyzed via descriptive statistics, and the maximum and minimum values, ranges, means, and standard deviations were calculated. The data from the macroscopic and histomorphometric analyses were tabulated and analyzed via one-way analysis of variance (ANOVA) followed by Tukey's post hoc test, with a statistical significance level of 5% (p < 0.05) to establish differences among the experimental groups. All the data were analyzed via GraphPad Prism® version 5.04. Results Obesity Induction There was a change in the Lee index when the healthy group (0.26 ± 0.01 g/cm) and the osteonecrosis group (0.25 ± 0.00 g/cm) were compared with the obese and obese with osteonecrosis groups (0.31 ± 0.00 g/cm), with the latter having the highest index value. The Lee index of the obese group was 19.23% greater than that of the healthy group and 24% greater than that of the osteonecrosis group (Fig. 2 a). The adiposity indices of the animals that received the high-glycemic index diet, both in the obese group (10.0 ± 1.03 g) and in the obese with osteonecrosis group (10.4 ± 0.86 g), were greater than those of the healthy (6.4 ± 1.00 g) and osteonecrosis (5.8 ± 0.68 g) groups. The adiposity index of the obese group was 56.25% greater than that of the healthy group and 72.41% greater than that of the osteonecrosis group, whereas the adiposity index of the obese with osteonecrosis group was 62.5% greater than that of the healthy group and 79.31% greater than that of the osteonecrosis group (Fig. 2 b). Anatomopathological and morphometric analysis Microscopically, the healthy group (Fig. 3 a) presented bone tissue with nucleated osteocytes showing no morphological alterations. In contrast, the groups with osteonecrosis (Fig. 3 b, black arrows), obesity (Fig. 3 c, black arrows), and obesity with osteonecrosis (Fig. 3 d, black arrows) presented anucleated osteocytes, characteristic of osteonecrosis. The results of the morphometric analysis revealed significant variations in the area of necrosis among the different groups (Fig. 4 ). Compared with the second group, the obese with osteonecrosis group presented the greatest average necrotic area (290.33 ± 15.77 µm²), followed by the osteonecrosis group (172.83 ± 18.19 µm²), which presented a 67.99% increase. The obese group, in turn, presented an average size of 97.00 ± 2.13 µm², corresponding to a 43.85% reduction compared with the osteonecrosis group. Finally, the healthy group had the smallest necrotic area, with an average of 5.00 ± 3.21 µm², indicating a 97.11% reduction compared with that of the osteonecrosis group. The differences observed between the groups were significant (p < 0.0001), highlighting a strong correlation between obesity, the presence of osteonecrosis, and the increase in the area of necrosis. Histological and histomorphometric evaluation of the extracellular matrix The histological sections of the mandible stained with Masson's trichrome, which represent the evaluated experimental conditions, revealed the following characteristics: in the healthy (a) and obese (c) groups, there was intense deposition of extracellular matrix (visualized in blue) as well as the formation of new blood vessels at the site of tooth extraction, indicating the formation of granulation tissue. However, under the other experimental conditions, namely, osteonecrosis (b) and obesity with osteonecrosis (d), there was a reduction in the formation of granulation tissue. These results were confirmed by morphometric analysis, where the mean areas marked with Masson's Trichrome were greater in the healthy group (54.66 ± 16.31 µm²) and the obese group (50.93 ± 14.19 µm²), followed by the obese with osteonecrosis (5.81 ± 2.40 µm²) and osteonecrosis groups (4.12 ± 2.43 µm²) (p < 0.0001) (Fig. 5 and Fig. 6 ). Immunohistochemical and histomorphometric evaluation of TNF-α The histological sections of the mandible from the immunohistochemical reaction against TNF-α were counterstained with hematoxylin, which represented the evaluated experimental conditions, and the following characteristics were observed. In the healthy group of animals (a), discrete and focal labeling was observed in the osteocytes (arrow). However, under the other experimental conditions, namely, osteonecrosis (b), obesity (c), and obesity with osteonecrosis (d), intense and multifocal labeling is observed in the osteocytes, which are enlarged and have an irregular morphological appearance, in addition to the marked mineral matrix. These results were confirmed by morphometric analysis, where the average areas marked in brown (positive reactions) were greater in the obese with osteonecrosis group (27.59 ± 7.65 µm²) and the obese group (25.52 ± 8.31 µm²), followed by the osteonecrosis group (17.38 ± 5.69 µm²) and the healthy group (4.34 ± 2.51 µm²) (p < 0.0001) (Fig. 7 and Fig. 8 ). Immunohistochemical and histomorphometric evaluation of TGF-β The histological sections of the mandible subjected to immunohistochemical staining against TGF-β and counterstained with hematoxylin, representing the evaluated experimental conditions, revealed the following characteristics: In the healthy group (a) and osteonecrosis group (b), discrete and focal staining was observed in the osteocytes (arrow). However, in the other experimental conditions, obese (c) and obese with osteonecrosis (d), intense and multifocal staining was noted. In particular, in Group D, enlarged osteocytes with irregular morphological features were also observed. These results were confirmed through morphometric analysis, where the mean areas stained brown (reaction positivity) were greater in the obese with osteonecrosis group (44.98 ± 3.93 µm²) and the osteonecrosis group (25.09 ± 4.65 µm²), followed by the obese group (19.57 ± 4.37 µm²) and the healthy group (4.27 ± 0.64 µm²) (p < 0.0001) (Fig. 9 and Fig. 10 ). Immunohistochemical and histomorphometric evaluation of the presence of collagen I The histological sections of the mandible subjected to immunohistochemical staining against Collagen I and stained with hematoxylin, which represent the evaluated experimental conditions, revealed the following characteristics: in the healthy group (a), intense and multifocal staining of Collagen I in the osteocytes and matrix (arrow) was observed. However, under the other experimental conditions, the osteocytes (b), Obese (c), and Obese with Osteonecrosis (d) exhibited discrete and focal staining (arrow). These results were confirmed through morphometric analysis, where the mean areas marked in brown (reaction positivity) were greater in the healthy group (26.73 ± 8.29 µm²), followed by the obese groups (4.05 ± 2.37 µm²), the osteonecrosis group (2.94 ± 2.11 µm²), and the obese with osteonecrosis group (1.56 ± 0.61 µm²) (p < 0.0001) (Fig. 11 and Fig. 12 ). Discussion This study experimentally evaluated the conditions associated with bisphosphonate-induced osteonecrosis of the jaw and obesity. To achieve this goal, four experimental groups were assessed (healthy, osteonecrotic, obese, and obese with osteonecrosis) to understand the mechanisms of tissue remodeling, highlighting the importance of the cytokines TGF-β and TNF-α, as well as the deposition of type I collagen in bone tissue. To confirm the experimental conditions (osteonecrosis and obesity), two protocols were employed: the first related to the induction of osteonecrosis (Biguetti et al. 2019 ) and the second related to obesity (Luz et al. 2018 ). Obesity was induced by a high glycemic index (HGID) diet, whereas osteonecrosis was induced via bisphosphonate (zoledronic acid). Changes that include increases in the Lee index and adiposity index confirm the induction of the obesity process. Studies have shown that such changes are important in association with the use of diets rich in high glycemic indices and fat to confirm this process of experimentally induced obesity (Bernardis and Patterson; Sadek et al. 2016 ; Bakhtiarzadeh et al. 2018 ; Lima et al. 2023 ). Microscopic changes, such as the presence of empty osteoclasts (confirmed through microscopy and morphometry), characterize the onset of osteonecrosis (Lima et al. 2023 ; Pontes et al. 2024 ). According to Alsalih et al. ( 2021 ), the use of zoledronic acid is associated with a reduction in the migration and proliferation of epithelial, endothelial, and fibroblasts. Additionally, it compromises the expression of vascular endothelial growth factor (VEGF), leading to decreased vascularization (Alsalih et al. 2021 ). Similar results were observed in the present study, with a reduction in granulation tissue in the osteonecrosis (4.12 ± 2.43 µm²) and obese with osteonecrosis (5.81 ± 2.40 µm²) groups, demonstrating that the remodeling process of the damaged tissues was compromised and that repair was hindered, favoring the development of the disease. In contrast, the healthy (54.66 ± 16.31 µm²) and obese (50.93 ± 14.19 µm²) groups presented intense deposition in the extracellular matrix and angiogenesis. Obesity has also been linked to changes in bone tissue homeostasis through mechanisms such as hormonal alterations in bone metabolism regulators, increased oxidative stress, and inflammation (Shapses et al. 2017 ). Adipose tissue produces several proinflammatory cytokines (such as TNF-α) and adipokines that can negatively influence bone tissue health (Kirk et al. 2020 ; Aaron et al. 2022 ). The deposition of the extracellular matrix is a fundamental mechanism for the formation and maintenance of bone tissue structure. However, in obese individuals, the changes present in adipocytes lead to complex and detrimental interactions resulting from the release of cytokines by cells, which can modulate osteoblasts and osteoclasts, causing damage to the tissue (Forte et al. 2023 ). TNF-α has various biological functions, such as the activation of T cells and macrophages, positive feedback of proinflammatory cytokines, and promotion of neovascularization in tissue healing processes (Prado et al. 2009 ). It is also associated with cell death via apoptosis (Lee et al. 2003 ). In addition, in bone tissue, it promotes bone erosion by interfering with the differentiation and maturation process of osteoclasts or by exposing the bone matrix (Yellon et al. 1991 ). For this process to occur in the area of the bone lesion, IL-1 and IL-6 (Goldring 2002 ), along with the ligand for receptor activation of NF-kB (RANKL), are also present and linked to TNF-α, promoting an increase in the recruitment, differentiation, and activation of osteoclasts (Bingham 2002 ). Cheung et al. ( 2011 ) conducted a study that demonstrated that chronic administration of zoledronic acid was associated with increased levels of TNF-α (Cheung et al. 2011 ). This marker is expressed in the dental pulp and can activate cell death domains, stimulating the infiltration and activation of macrophages (Paula-Silva et al. 2009 ). CVIKL et al. ( 2011 ) conducted an in vitro study on the apoptosis rates of pulp cells incubated with zoledronic acid, which revealed a reduction in cell survival due to the direct toxic effect of zoledronic acid (Cvikl et al. 2011 ). In a clinical study in which TNF-α inhibitors were used to treat systemic diseases, their efficacy in inhibiting the process of bone resorption was demonstrated (Elliott et al. 1994a ). In periodontitis, TNF-α is one of the main factors contributing to the worsening of the disease (Lappin et al. 2001 ). The injection of a solution containing TNF-α antagonists can inhibit the formation of osteoclasts, decrease the recruitment of inflammatory cells, and reduce bone loss (Assuma et al. 1998 ). It has also been demonstrated that the use of TNF-α antagonists reduces bone damage in rheumatoid arthritis (Elliott et al. 1994b ). Thus, its reduction presents a potential alternative for mitigating tissue damage. In the present study, in the healthy group, there was discrete and focal labeling for TNF-α in the osteocytes, with a smaller average marked area. In contrast, in the osteonecrosis, obese, and obese with osteonecrosis groups, the labeling was intense and multifocal, and the osteocytes exhibited irregular and enlarged appearances, along with larger averages of marked areas. TNF-α is important for bone resorption and contributes to tissue damage in diseases related to inflammatory processes (Bingham 2002 ; Goldring 2002 ); possibly, this finding is related to the results observed in the groups affected by the disease. Furthermore, adipose tissue produces a relatively large amount of TNF-α (Weisberg et al. 2003 ), which supports the results of the pronounced labeling in the groups affected by obesity. Relationships between circulating TNF-α and obesity indices have been studied. Jellema et al. ( 2004 ) reported that weight loss decreases the circulating concentration of this adipokine (Jellema et al. 2004 ). Yudkin et al. ( 2005 ) suggested that TNF-α production by fat around the arterioles inhibits insulin stimulus for nitric oxide synthesis, resulting in vasoconstriction (Yudkin et al. 2005 ). Effects such as the inhibition of the insulin receptor signaling pathway have been described, including some studies involving obese rats (Hotamisligil et al. 1993; Coppack 2001 ). Another important cytokine for the process of bone remodeling is TGF-β, which is involved in various biological processes, including the regulation of cell proliferation and differentiation, apoptosis, the synthesis of extracellular matrix components, and immune responses. Dysregulation of this cytokine is associated with the onset of certain diseases, such as cancer and fibrosis (Zhao and Chen 2014 ). In the present study, discrete and focal staining for TGF-β was detected in the osteocytes of the healthy and osteonecrotic groups, whereas in the obese and obese with osteonecrosis groups, intense and multifocal staining was detected. Since TGF-β is an important cytokine for the healing process, it is believed that this cytokine may have bound to its receptors, thereby increasing the collagen concentration. TGF-β is an anti-inflammatory cytokine that plays a significant role in maintaining tissue homeostasis, thus facilitating resolution and inducing the repair process. In contrast, it negatively regulates the recruitment and activation of leukocytes while recruiting fibroblasts, thereby stimulating the synthesis of extracellular matrix components (Wahl 1994 ). This anti-inflammatory property may have contributed to the findings in the obese groups, where the staining for TGF-β was more intense. Notably, the staining for TNF-α in these groups was also intense and multifocal, supporting the hypothesis of the influence of the inflammatory process. Concomitantly, intense and multifocal staining for collagen I was identified in the osteocytes and matrix of the healthy group, indicating a possible healing process. In contrast, in the osteonecrosis, obese, and obese with osteonecrosis groups, this staining was discrete and focal, indicating the opposite of the healing process. The inflammatory process likely also influenced these findings, as it is present in both conditions. studies have shown that zoledronic acid interferes with the collagen synthesis process in cultures of periodontal fibroblasts, and one of the causes of these findings is that this drug acts by increasing the rate of apoptosis (Scheper et al. 2009 ; De Colli et al. 2015 ; Komatsu et al. 2016 ). One of the potential causes of this toxic effect is the oxidative stress generated at the target area of the drug. According to Colli et al. ( 2015 ), with the interaction of zoledronic acid, fibroblasts present in the periodontal ligament undergo a process of increased synthesis of reactive oxygen species through a pathway dependent on the constitutive synthesis of nitric oxide, thereby causing inflammatory stress (De Colli et al. 2015 ); moreover, osteocytes are sensitive to this inflammatory stress (Wimalawansa 2010 ). In the present study, the induction of osteonecrosis was associated with tooth extraction between the fourth and fifth weeks of administration of zoledronic acid. This drug irreversibly binds to the hydroxyapatite of the bone tissue (Endele et al. 2005 ); thus, when trauma occurs, the tissues are already in an inflammatory process, and this event possibly stimulates an increase in the release of proinflammatory cytokines. Studies in experimental and clinical models suggest an association between the inflammatory process and bisphosphonate-induced osteonecrosis of the jaws (Maahs et al. 2011 ; Ali-Erdem et al. 2011 ; Marino et al. 2012 ). Therefore, one of the key factors for a better understanding of these aspects is the study of the immunological profile of this disease. Zoledronic acid can have distinct effects depending on its concentration. At high concentrations, the synthesis of alkaline phosphatase and the morphology of odontoblasts may be compromised; conversely, at lower concentrations, the drug may increase the expression of collagen I (Basso et al. 2013 ). In the present study, lower expression of collagen I was observed in the osteonecrosis and obese with osteonecrosis groups, findings that corroborate those of Basso et al. ( 2013 ). Furthermore, according to Scheper et al. ( 2009 ) and Naidu et al. ( 2008 ), this drug is toxic to various cell types, such as fibroblasts and osteoblasts; thus, a reduction in collagen I synthesis is expected (Naidu et al. 2008 ; Scheper et al. 2009 ). Obesity is a disease that negatively impacts bone metabolism, primarily through alterations in the inflammatory process (Shapses et al. 2017 ). Bisphosphonates are responsible for increasing the production of acute inflammatory mediators and altering the immune and cellular profiles of patients (Norton et al. 2012 ; Rossini et al. 2012 ; Welton et al. 2013 ; Muratsu et al. 2013 ). The association of these two conditions, which are linked to alterations primarily generated in the inflammatory process, may have contributed to the low deposition of collagen I in the osteonecrosis, obese, and obese with osteonecrosis groups. Associated with the reduction in the deposition of collagen type I in the osteonecrosis, obese, and obese with osteonecrosis groups, an increase in the level of TGF-β was found in the same groups. This finding may demonstrate that in the healthy group, TGF-β was synthesized and used to activate bone tissue cells to produce collagen, thus facilitating the healing process, unlike in the other groups, where this mechanism was possibly not achieved, and the healing process, compared with that in the healthy group, was not the same. The association between TGF-β and collagen I production suggests that osteonecrosis possibly modulates the deposition of collagen I in obese rats via TGF-β. Another important factor is the inflammatory process present in both conditions, where it is believed that both conditions negatively influence the modulation of collagen I deposition. The associations among obesity, osteonecrosis, and the parameters investigated in this study provide a relevant foundation for understanding the complexities of this condition. Although research on the specific impact of zoledronic acid on the expression of inflammatory markers in patients with osteonecrosis associated with obesity is scarce, the results of the present study suggest a promising relationship that warrants further investigation. Future research on these factors is essential to confirm these hypotheses and enhance the clinical and therapeutic understanding of this condition. Conclusion Through microscopy and morphometry, this study revealed that TGF-β, TNF-α, and collagen I play important roles in bone tissue in the context of obesity and osteonecrosis, where osteonecrosis modulates the deposition of collagen I in obese rats via TGF-β. Osteonecrosis induced by the use of bisphosphonates in obese rats may interfere with the process of bone homeostasis, potentially influenced by the inflammatory process present in both conditions, contributing to the abnormal deposition of the extracellular matrix in bone tissue. This abnormal deposition may increase the risk of developing and worsening disease. Research relating obesity and medication-induced osteonecrosis of the jaws is important for a better understanding of their respective relationships, especially with respect to the regulation of bone metabolism, to develop proposals for prevention and treatment. Declarations In the context of this study, we declare that funding was provided by the Paraíba State Research Support Foundation (FAPESQ/PB) through grant number 010/2021 FAPESQ/PB - MCTIC/CNPQ under the Infrastructure Program for Young Researchers/First Projects Program (PPP). This support was formalized through Grant Agreement number 3224/2021. We emphasize that the funding agency was not involved in the study design, data collection, analysis, interpretation, or writing of this manuscript. The conclusions and interpretations expressed in this work are solely the responsibility of the authors, with no influence from the funders. Author Contribution W.J.M.L. (corresponding author) was the primary author and contributed to all stages of the study, including conceptualization, experimental planning, data collection, data analysis, interpretation of results, and manuscript drafting.• A.F.A. supervised and guided the research, providing technical and intellectual support throughout all stages and assisting in the final review of the manuscript.• W.F.B.P. contributed to the critical review of the manuscript, enhancing its scientific content and clarity.• J.C.X.P., R.S.A., and M.C.P.S. provided specific technical contributions during the study, including assistance with sample processing and data analysis.All authors reviewed and approved the final version of the manuscript for submission, taking responsibility for the accuracy and integrity of the work in all its aspects. References Aaron N, Costa S, Rosen CJ, Qiang L (2022) The Implications of Bone Marrow Adipose Tissue on Inflammaging. 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Laryngoscope 101:165???169. https://doi.org/10.1288/00005537-199102000-00011 Yudkin JS, Eringa E, Stehouwer C DA (2005) “Vasocrine” signaling from perivascular fat: a mechanism linking insulin resistance to vascular disease. The Lancet 365:1817–1820. https://doi.org/10.1016/S0140-6736(05)66585-3 Zandi M, Dehghan A, Malekzadeh H, et al (2016) Introducing a protocol to create bisphosphonate-related osteonecrosis of the jaw in rat animal model. Journal of Cranio-Maxillofacial Surgery 44:271–278. https://doi.org/10.1016/j.jcms.2015.12.010 Zhao B, Chen Y-G (2014) Regulation of TGF-β Signal Transduction. Scientifica (Cairo) 2014:874065. https://doi.org/10.1155/2014/874065 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5361050","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376577139,"identity":"f08b1672-3792-4a84-baf7-7fb57b07164e","order_by":0,"name":"Wilson José de Miranda 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\u003cem\u003eb\u003c/em\u003e Obesity and obesity with osteonecrosis \u003cem\u003ec\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/7020dfdbf086aaeedf4d3e24.png"},{"id":69445818,"identity":"d8ae0e60-f66e-42a4-ac39-5bbe3887ced9","added_by":"auto","created_at":"2024-11-20 11:59:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ea\u003c/em\u003e Means of the Lee index for animals in each study group The symbols and vertical bars represent the means (n = 6) One-way ANOVA followed by Tukey's post hoc test was used □p\u0026lt;0.05 (obese vs. healthy and obese vs. osteonecrosis) ○p\u0026lt;0.05 (obese with osteonecrosis vs. healthy and obese with osteonecrosis vs. osteonecrosis) \u003cem\u003eb\u003c/em\u003e Means of the adiposity indices for animals in each study group The symbols and vertical bars represent the means (n = 6) \u0026nbsp;One-way ANOVA followed by Tukey's post hoc test. □p\u0026lt;0.05 (obese vs. healthy and obese vs. osteonecrotic) ○p\u0026lt;0.05 (obese with osteonecrosis vs. healthy and obese with osteonecrosis vs. osteonecrotic)\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/02d413da5f8f260eb01aab0f.png"},{"id":69444993,"identity":"4d412c23-c350-4f70-9e33-d492c85cbc8f","added_by":"auto","created_at":"2024-11-20 11:51:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1138344,"visible":true,"origin":"","legend":"\u003cp\u003eHistological sections of mandibles stained with hematoxylin and eosin \u003cem\u003ea\u003c/em\u003e Healthy \u003cem\u003eb\u003c/em\u003e Osteonecrosis \u003cem\u003ec\u003c/em\u003e Obesity and \u003cem\u003ed\u003c/em\u003e Obesity with osteonecrosis Magnification 400x\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/b5b20fbc3c68a4b39440550f.png"},{"id":69444990,"identity":"26535812-d04f-44d8-87bf-531abe8afce1","added_by":"auto","created_at":"2024-11-20 11:51:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":23321,"visible":true,"origin":"","legend":"\u003cp\u003eAverages of the necrotic areas marked with hematoxylin and eosin. The symbols and vertical bars represent the means (n = 6) of one-way ANOVA followed by Tukey's post hoc test. ***p \u0026lt; 0.05 (obese vs. healthy) **p \u0026lt; 0.05 (osteonecrosis vs. healthy) and *p \u0026lt; 0.05 (obese vs. healthy)\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/715eddd5f15d334a984cfbfa.png"},{"id":69445819,"identity":"debbb20b-7e26-4d57-ac44-883e821e4cf8","added_by":"auto","created_at":"2024-11-20 11:59:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1250047,"visible":true,"origin":"","legend":"\u003cp\u003eHistological section of mandibles stained with Masson's Trichrome, where the blue color indicates the presence of an extracellular matrix. \u003cem\u003ea\u003c/em\u003e Healthy \u003cem\u003eb\u003c/em\u003eOsteonecrosis \u003cem\u003ec\u003c/em\u003e Obese and \u003cem\u003ed\u003c/em\u003e Obese with osteonecrosis Magnification 400x (*) Positive staining for Masson's Trichrome\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/b8964c0cb63ef8702298b180.png"},{"id":69444657,"identity":"e6ad3fda-27bc-44db-a25b-7b32c9b4d2fe","added_by":"auto","created_at":"2024-11-20 11:43:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35071,"visible":true,"origin":"","legend":"\u003cp\u003eMeans of the areas of the extracellular matrix marked with Masson's Trichrome. The symbols and vertical bars represent the means (n = 20) of one-way ANOVA followed by Tukey's post hoc test. *p\u0026lt;0.05 (healthy vs. osteonecrotic and healthy vs. obese with osteonecrosis) #p\u0026lt;0.05 (obese vs. obese with osteonecrosis)\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/c651b5bf4274d3cb4bab4816.png"},{"id":69444653,"identity":"44ddf23e-15ea-48d4-ab8d-163db2b0ea3c","added_by":"auto","created_at":"2024-11-20 11:43:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1015345,"visible":true,"origin":"","legend":"\u003cp\u003eHistological sections of mandibles subjected toimmunohistochemistry for TNF-α. \u003cem\u003ea\u003c/em\u003e Healthy \u003cem\u003eb\u003c/em\u003e Osteonecrosis \u003cem\u003ec\u003c/em\u003e Obesity and \u003cem\u003ed\u003c/em\u003e Obesity with osteonecrosis Magnification 400x (*) Positive labeling for TNF-α\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/b72e234ba551d791a92e4d8d.png"},{"id":69444656,"identity":"a08c2713-a99e-4c56-941d-f8478dd4fa6b","added_by":"auto","created_at":"2024-11-20 11:43:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":34198,"visible":true,"origin":"","legend":"\u003cp\u003eMeans of the areas affected by TNF-α The symbols and vertical bars represent the means (n = 20) One-way ANOVA followed by Tukey's post hoc test was used #p\u0026lt;0.05 (obese with osteonecrosis vs. osteonecrosis and obese with osteonecrosis vs. healthy) *p\u0026lt;0.05 (obese vs. healthy and obese vs. osteonecrosis) +p\u0026lt;0.05 (osteonecrosis vs. healthy)\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/2fd1200c15778ae1b5c91e71.png"},{"id":69444650,"identity":"b6c450a1-e9e6-4125-a0b5-bbd691703d59","added_by":"auto","created_at":"2024-11-20 11:43:59","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":888402,"visible":true,"origin":"","legend":"\u003cp\u003eHistological section of mandibles stained through immunohistochemistry for TGF-β \u003cem\u003ea\u003c/em\u003e Healthy \u003cem\u003eb\u003c/em\u003eOsteonecrosis \u003cem\u003ec\u003c/em\u003e Obesity and \u003cem\u003ed\u003c/em\u003e Obesity with osteonecrosis Magnification: 400x (*) Positive staining for TGF-β\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/6defbadf760e1b56a3d77602.png"},{"id":69444991,"identity":"6a079fe4-a9ca-4a48-8944-1a7ba55c995d","added_by":"auto","created_at":"2024-11-20 11:51:59","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":32063,"visible":true,"origin":"","legend":"\u003cp\u003eMeans of areas for TGF-β The symbols and vertical bars represent the means (n = 20) One-way ANOVA followed by Tukey's posthoc test was used *p\u0026lt;0.05 (obese with osteonecrosis vs. healthy, obese with osteonecrosis vs. osteonecrosis, and obese with osteonecrosis vs. obese) #p\u0026lt;0.05 (osteonecrosis vs. healthy and osteonecrosis vs. obese) +p\u0026lt;0.05 (obese vs. healthy)\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/8765d2113efa9026ffdb9191.png"},{"id":69444659,"identity":"4cd83d27-8f5b-4c68-97b6-f684ffb2733b","added_by":"auto","created_at":"2024-11-20 11:43:59","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":953946,"visible":true,"origin":"","legend":"\u003cp\u003eHistological section of mandibles marked through immunohistochemistry for collagen I \u003cem\u003ea\u003c/em\u003e Healthy \u003cem\u003eb\u003c/em\u003e Osteonecrosis \u003cem\u003ec\u003c/em\u003e Obesity and \u003cem\u003ed \u003c/em\u003eObesity with osteonecrosis Magnification: 400x (*) Positive staining for collagen I\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/cb1e4ae4a0cf069d8fa86385.png"},{"id":69444658,"identity":"a7141326-e5ca-46a2-8cbc-5154316a3e62","added_by":"auto","created_at":"2024-11-20 11:43:59","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":34185,"visible":true,"origin":"","legend":"\u003cp\u003eMeans of areas for Collagen I The symbols and vertical bars represent the means (n = 20) One-way ANOVA followed by Tukey's posthoc test *p\u0026lt;0.05 (healthy vs. osteonecrotic, healthy vs. obese, and healthy vs. obese with osteonecrosis)\u003c/p\u003e","description":"","filename":"image13.png","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/ee3057eec11fa5a8bced78ff.png"},{"id":70476314,"identity":"7c28a632-8a6d-46f6-8ffe-32c2eca3c5ae","added_by":"auto","created_at":"2024-12-03 14:17:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9668023,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5361050/v1/389c4710-e8a4-4314-8f6a-00701e2dfd9e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Osteonecrosis modulates extracellular matrix deposition through collagen I deposition in obese rats via the TGF-β protein","fulltext":[{"header":"Introduction","content":"\u003cp\u003eObesity is widely recognized as a major public health condition associated with a range of comorbidities that affect various body systems. Among the pathophysiological characteristics of obesity, a chronic state of systemic inflammation stands out because of the excessive accumulation of adipose tissue. This increase in adipocytes results in the exacerbated secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), as well as acute-phase proteins such as C-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP), which also contribute to endothelial dysfunction (Singh et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This inflammatory state is associated with the infiltration of macrophages into adipose tissue, which shifts from an anti-inflammatory M2 phenotype to a proinflammatory M1 phenotype, further aggravating inflammation and leading to insulin resistance (Junqueira and Carneiro \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, obesity affects not only energy metabolism but also the endocrine, cardiovascular, pulmonary, and musculoskeletal systems, contributing to the development of diseases such as dyslipidemia, hypertension, and even cancer (Kypridemos et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lopategi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kovesdy et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gasparin et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lakkis and Weir \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Vekic et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the various systems impacted by obesity, the skeletal system stands out as a vital reservoir of calcium and phosphate and serves as support for soft tissues and vital organs. Bone tissue undergoes constant remodeling through complex cellular processes involving osteoblasts, osteoclasts, and osteocytes. These processes are mediated by bioactive molecules, such as transforming growth factor-beta (TGF-β) and type I collagen, which play crucial roles in maintaining bone homeostasis. During remodeling, osteoclasts secrete cytokines, such as TGF-β, which regulate both bone resorption and osteoblast differentiation (HP Rang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Van Schepdael et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis balance is essential for bone tissue renewal and repair capacity in response to injuries; however, when this equilibrium is disrupted, as in the case of jaw osteonecrosis, the inflammatory process plays a central role. Medication-related osteonecrosis of the jaw (MRONJ) has been associated with the use of bisphosphonates, substances frequently used to treat bone diseases, such as osteoporosis. Bisphosphonates inhibit bone resorption by interfering with osteoclast activity, which, while effective in preventing fractures, can lead to osteonecrosis under adverse conditions (Ruggiero et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; O\u0026rsquo;Carrigan et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Farrell et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Borgo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These medications, by accumulating in the bone matrix, negatively affect local vascularization and bone repair capacity, resulting in bone tissue exposure and necrosis (Fernandes et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Edwards et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Santos \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Rasmusson and Abtahi \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tardast et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zandi et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Owing to its lower vascularization than the maxilla, the mandible is particularly affected by this condition (Andrade \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gong et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe relationship between obesity and jaw osteonecrosis is known but not well understood, and the impact of chronic inflammation and oxidative stress\u0026mdash;both exacerbated by obesity\u0026mdash;on bone tissue becomes evident. The continuous systemic inflammation in these individuals can worsen local inflammatory processes in bone tissue, especially in the presence of bisphosphonates, contributing to the development of osteonecrosis (Lima et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, the interaction between proinflammatory factors, such as TNF-α and IL-6, and the bone remodeling mechanisms mediated by TGF-β play crucial roles in both obesity and osteonecrosis (Van Schepdael et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This study aimed to explore the influence of TGF-β, TNF-α, and type I collagen on bone tissue, as well as their correlation with bisphosphonate-induced jaw osteonecrosis and obesity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample\u003c/h2\u003e \u003cp\u003eThe experiment consisted of 24 male Wistar rats (\u003cem\u003eRattus norvegicus\u003c/em\u003e) aged approximately 30 days and weighing 180 g, which were divided into four groups: healthy, osteonecrotic, obese, and obese with osteonecrosis (n\u0026thinsp;=\u0026thinsp;6 per group). The animals were housed in the animal facility of the Institute for Drug and Medication Research (IPeFarM) at the Federal University of Para\u0026iacute;ba. The research protocol was approved by the Animal Ethics Committee (CEUA) under number 8738120220 (ID 000661) and complies with all the guidelines of Law 11,794 of October 8, 2008, and Decree 6,899 of July 15, 2009, as well as the regulations set forth by the National Council for Animal Experimentation Control (CONCEA).\u003c/p\u003e \u003cp\u003e \u003cem\u003eOsteonecrosis induction\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e- Healthy and Osteonecrosis Groups\u003c/h3\u003e\n\u003cp\u003eOsteonecrosis was induced in the designated groups through the administration of zoledronic acid, following an adaptation of the methodology of Biguetti et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This induction was combined with the extraction of the left lower first molar. The animals in the healthy group received saline solution intraperitoneally, whereas those in the osteonecrosis group received 250 \u0026micro;g/kg of zoledronic acid (Blau Farmac\u0026ecirc;utica) intraperitoneally once per week for eight weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The animals underwent tooth extraction between the fourth and fifth doses of the medication. For this procedure, they were sedated with a combination of ketamine and xylazine administered intraperitoneally. A number five exploratory probe was used to dislocate the tooth.\u003c/p\u003e \u003cp\u003e- \u003cem\u003eObese and obese with osteonecrosis groups\u003c/em\u003e\u003c/p\u003e \u003cp\u003eOsteonecrosis induction in these groups was performed as described in section 2.2.1 but at a different time, as the animals had to undergo the obesity induction protocol in parallel (described in section 2.3), which lasted 16 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) (Biguetti et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eObesity Induction\u003c/h3\u003e\n\u003cp\u003eThe obese and obese with osteonecrosis groups were provided with an obesogenic diet composed of pellet-type chow (Nuvilab\u0026reg;), refined sugar (Alegre\u0026reg;), and whole condensed milk (Camponesa\u0026reg;). This high-glycemic index diet had a value of 77.6 and a high glycemic load of 38.8. To prepare 100 g of feed, 45.2 g of chow was ground and mixed with 9.6 g of sugar, followed by the addition of 45.2 mL of condensed milk. This mixture was homogenized, shaped to resemble conventional chow, dried in an oven (55\u0026deg;C) for 24 hours, and stored under refrigeration. The animals were provided 800 g of pellets per week throughout the experiment (16 weeks). To confirm obesity, the Lee index (defined as the ratio between the cube root of the animal's body mass and the nasoanal length) and the adiposity index (defined as the sum of the individual masses of the epididymal, inguinal, and retroperitoneal fat layers multiplied by 100 and divided by the final body weight) were used (Lima et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eHistological processing\u003c/h3\u003e\n\u003cp\u003eAfter euthanasia, each specimen (comprising the mandible) was fixed for 48 hours and then decalcified in a 5% nitric acid solution. Following demineralization, the material was sectioned and subjected to processing steps, including dehydration in increasing concentrations of ethanol, clearing in xylene, impregnation in paraffin baths at a maximum temperature of 60\u0026deg;C, embedding in paraffin molds, and microtomy, with the resulting sections cut to a thickness of 4 \u0026micro;m. The samples were stained with hematoxylin and eosin and Masson\u0026rsquo;s trichrome.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemical and histomorphometric analyses\u003c/h3\u003e\n\u003cp\u003eFor immunohistochemical analysis, the samples were reactivated with a pH 6.0 citrate retrieval solution (EasyPath Diagn\u0026oacute;sticos) in a Pascal pressurized chamber (Dako Cytomation, Denmark), and endogenous peroxidase was blocked for 30 minutes (EasyPath Diagn\u0026oacute;sticos). Following this step, nonspecific protein blocking was performed, and the sections were incubated with the following primary antibodies: anti-TNF-α (Cloud-Clone Corp/PAA133Hu01/Polyclonal Antibody; 1:100), anti-TGF-β (Cloud-Clone Corp/PAA124Ra01/Polyclonal Antibody; 1:100), and anti-collagen I (Boster/PA2140-2; 1:100).\u003c/p\u003e \u003cp\u003eA biotinylated secondary antibody (goat anti-rabbit or anti-mouse biotinylated antibody at a dilution of 1:100, Link Sistem - HRP - Dako 4061) was subsequently added. The reaction was visualized via 0.024% diaminobenzidine (DAB - HRP) solution (EasyPath Diagnosticos) and contrasted with Harris hematoxylin (Pontes et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To calculate the area of extracellular matrix deposition, as well as the positive areas for all immunohistochemical reactions, all pixels with shades of blue (Masson's trichrome) or brown (positive immunohistochemical staining) were selected to create a binary image. Digital processing and area calculation in \u0026micro;m\u0026sup2; were conducted via a 40X objective and quantified interactively with Image-Pro Plus software (Pontes et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe results were initially analyzed via descriptive statistics, and the maximum and minimum values, ranges, means, and standard deviations were calculated. The data from the macroscopic and histomorphometric analyses were tabulated and analyzed via one-way analysis of variance (ANOVA) followed by Tukey's post hoc test, with a statistical significance level of 5% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to establish differences among the experimental groups. All the data were analyzed via GraphPad Prism\u0026reg; version 5.04.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eObesity Induction\u003c/h2\u003e \u003cp\u003eThere was a change in the Lee index when the healthy group (0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g/cm) and the osteonecrosis group (0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 g/cm) were compared with the obese and obese with osteonecrosis groups (0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 g/cm), with the latter having the highest index value. The Lee index of the obese group was 19.23% greater than that of the healthy group and 24% greater than that of the osteonecrosis group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The adiposity indices of the animals that received the high-glycemic index diet, both in the obese group (10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 g) and in the obese with osteonecrosis group (10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 g), were greater than those of the healthy (6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 g) and osteonecrosis (5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 g) groups. The adiposity index of the obese group was 56.25% greater than that of the healthy group and 72.41% greater than that of the osteonecrosis group, whereas the adiposity index of the obese with osteonecrosis group was 62.5% greater than that of the healthy group and 79.31% greater than that of the osteonecrosis group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnatomopathological and morphometric analysis\u003c/h2\u003e \u003cp\u003eMicroscopically, the healthy group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) presented bone tissue with nucleated osteocytes showing no morphological alterations. In contrast, the groups with osteonecrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, black arrows), obesity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, black arrows), and obesity with osteonecrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, black arrows) presented anucleated osteocytes, characteristic of osteonecrosis.\u003c/p\u003e \u003cp\u003eThe results of the morphometric analysis revealed significant variations in the area of necrosis among the different groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Compared with the second group, the obese with osteonecrosis group presented the greatest average necrotic area (290.33\u0026thinsp;\u0026plusmn;\u0026thinsp;15.77 \u0026micro;m\u0026sup2;), followed by the osteonecrosis group (172.83\u0026thinsp;\u0026plusmn;\u0026thinsp;18.19 \u0026micro;m\u0026sup2;), which presented a 67.99% increase. The obese group, in turn, presented an average size of 97.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13 \u0026micro;m\u0026sup2;, corresponding to a 43.85% reduction compared with the osteonecrosis group. Finally, the healthy group had the smallest necrotic area, with an average of 5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21 \u0026micro;m\u0026sup2;, indicating a 97.11% reduction compared with that of the osteonecrosis group. The differences observed between the groups were significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), highlighting a strong correlation between obesity, the presence of osteonecrosis, and the increase in the area of necrosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistological and histomorphometric evaluation of the extracellular matrix\u003c/h2\u003e \u003cp\u003eThe histological sections of the mandible stained with Masson's trichrome, which represent the evaluated experimental conditions, revealed the following characteristics: in the healthy (a) and obese (c) groups, there was intense deposition of extracellular matrix (visualized in blue) as well as the formation of new blood vessels at the site of tooth extraction, indicating the formation of granulation tissue. However, under the other experimental conditions, namely, osteonecrosis (b) and obesity with osteonecrosis (d), there was a reduction in the formation of granulation tissue. These results were confirmed by morphometric analysis, where the mean areas marked with Masson's Trichrome were greater in the healthy group (54.66\u0026thinsp;\u0026plusmn;\u0026thinsp;16.31 \u0026micro;m\u0026sup2;) and the obese group (50.93\u0026thinsp;\u0026plusmn;\u0026thinsp;14.19 \u0026micro;m\u0026sup2;), followed by the obese with osteonecrosis (5.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40 \u0026micro;m\u0026sup2;) and osteonecrosis groups (4.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43 \u0026micro;m\u0026sup2;) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical and histomorphometric evaluation of TNF-α\u003c/h2\u003e \u003cp\u003eThe histological sections of the mandible from the immunohistochemical reaction against TNF-α were counterstained with hematoxylin, which represented the evaluated experimental conditions, and the following characteristics were observed. In the healthy group of animals (a), discrete and focal labeling was observed in the osteocytes (arrow). However, under the other experimental conditions, namely, osteonecrosis (b), obesity (c), and obesity with osteonecrosis (d), intense and multifocal labeling is observed in the osteocytes, which are enlarged and have an irregular morphological appearance, in addition to the marked mineral matrix. These results were confirmed by morphometric analysis, where the average areas marked in brown (positive reactions) were greater in the obese with osteonecrosis group (27.59\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65 \u0026micro;m\u0026sup2;) and the obese group (25.52\u0026thinsp;\u0026plusmn;\u0026thinsp;8.31 \u0026micro;m\u0026sup2;), followed by the osteonecrosis group (17.38\u0026thinsp;\u0026plusmn;\u0026thinsp;5.69 \u0026micro;m\u0026sup2;) and the healthy group (4.34\u0026thinsp;\u0026plusmn;\u0026thinsp;2.51 \u0026micro;m\u0026sup2;) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical and histomorphometric evaluation of TGF-β\u003c/h2\u003e \u003cp\u003eThe histological sections of the mandible subjected to immunohistochemical staining against TGF-β and counterstained with hematoxylin, representing the evaluated experimental conditions, revealed the following characteristics: In the healthy group (a) and osteonecrosis group (b), discrete and focal staining was observed in the osteocytes (arrow). However, in the other experimental conditions, obese (c) and obese with osteonecrosis (d), intense and multifocal staining was noted. In particular, in Group D, enlarged osteocytes with irregular morphological features were also observed. These results were confirmed through morphometric analysis, where the mean areas stained brown (reaction positivity) were greater in the obese with osteonecrosis group (44.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93 \u0026micro;m\u0026sup2;) and the osteonecrosis group (25.09\u0026thinsp;\u0026plusmn;\u0026thinsp;4.65 \u0026micro;m\u0026sup2;), followed by the obese group (19.57\u0026thinsp;\u0026plusmn;\u0026thinsp;4.37 \u0026micro;m\u0026sup2;) and the healthy group (4.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64 \u0026micro;m\u0026sup2;) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical and histomorphometric evaluation of the presence of collagen I\u003c/h2\u003e \u003cp\u003eThe histological sections of the mandible subjected to immunohistochemical staining against Collagen I and stained with hematoxylin, which represent the evaluated experimental conditions, revealed the following characteristics: in the healthy group (a), intense and multifocal staining of Collagen I in the osteocytes and matrix (arrow) was observed. However, under the other experimental conditions, the osteocytes (b), Obese (c), and Obese with Osteonecrosis (d) exhibited discrete and focal staining (arrow). These results were confirmed through morphometric analysis, where the mean areas marked in brown (reaction positivity) were greater in the healthy group (26.73\u0026thinsp;\u0026plusmn;\u0026thinsp;8.29 \u0026micro;m\u0026sup2;), followed by the obese groups (4.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37 \u0026micro;m\u0026sup2;), the osteonecrosis group (2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11 \u0026micro;m\u0026sup2;), and the obese with osteonecrosis group (1.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 \u0026micro;m\u0026sup2;) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study experimentally evaluated the conditions associated with bisphosphonate-induced osteonecrosis of the jaw and obesity. To achieve this goal, four experimental groups were assessed (healthy, osteonecrotic, obese, and obese with osteonecrosis) to understand the mechanisms of tissue remodeling, highlighting the importance of the cytokines TGF-β and TNF-α, as well as the deposition of type I collagen in bone tissue.\u003c/p\u003e \u003cp\u003eTo confirm the experimental conditions (osteonecrosis and obesity), two protocols were employed: the first related to the induction of osteonecrosis (Biguetti et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and the second related to obesity (Luz et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Obesity was induced by a high glycemic index (HGID) diet, whereas osteonecrosis was induced via bisphosphonate (zoledronic acid). Changes that include increases in the Lee index and adiposity index confirm the induction of the obesity process. Studies have shown that such changes are important in association with the use of diets rich in high glycemic indices and fat to confirm this process of experimentally induced obesity (Bernardis and Patterson; Sadek et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bakhtiarzadeh et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lima et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Microscopic changes, such as the presence of empty osteoclasts (confirmed through microscopy and morphometry), characterize the onset of osteonecrosis (Lima et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pontes et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to Alsalih et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the use of zoledronic acid is associated with a reduction in the migration and proliferation of epithelial, endothelial, and fibroblasts. Additionally, it compromises the expression of vascular endothelial growth factor (VEGF), leading to decreased vascularization (Alsalih et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similar results were observed in the present study, with a reduction in granulation tissue in the osteonecrosis (4.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43 \u0026micro;m\u0026sup2;) and obese with osteonecrosis (5.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40 \u0026micro;m\u0026sup2;) groups, demonstrating that the remodeling process of the damaged tissues was compromised and that repair was hindered, favoring the development of the disease. In contrast, the healthy (54.66\u0026thinsp;\u0026plusmn;\u0026thinsp;16.31 \u0026micro;m\u0026sup2;) and obese (50.93\u0026thinsp;\u0026plusmn;\u0026thinsp;14.19 \u0026micro;m\u0026sup2;) groups presented intense deposition in the extracellular matrix and angiogenesis.\u003c/p\u003e \u003cp\u003eObesity has also been linked to changes in bone tissue homeostasis through mechanisms such as hormonal alterations in bone metabolism regulators, increased oxidative stress, and inflammation (Shapses et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Adipose tissue produces several proinflammatory cytokines (such as TNF-α) and adipokines that can negatively influence bone tissue health (Kirk et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Aaron et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe deposition of the extracellular matrix is a fundamental mechanism for the formation and maintenance of bone tissue structure. However, in obese individuals, the changes present in adipocytes lead to complex and detrimental interactions resulting from the release of cytokines by cells, which can modulate osteoblasts and osteoclasts, causing damage to the tissue (Forte et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTNF-α has various biological functions, such as the activation of T cells and macrophages, positive feedback of proinflammatory cytokines, and promotion of neovascularization in tissue healing processes (Prado et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It is also associated with cell death via apoptosis (Lee et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In addition, in bone tissue, it promotes bone erosion by interfering with the differentiation and maturation process of osteoclasts or by exposing the bone matrix (Yellon et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). For this process to occur in the area of the bone lesion, IL-1 and IL-6 (Goldring \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), along with the ligand for receptor activation of NF-kB (RANKL), are also present and linked to TNF-α, promoting an increase in the recruitment, differentiation, and activation of osteoclasts (Bingham \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCheung et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) conducted a study that demonstrated that chronic administration of zoledronic acid was associated with increased levels of TNF-α (Cheung et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This marker is expressed in the dental pulp and can activate cell death domains, stimulating the infiltration and activation of macrophages (Paula-Silva et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). CVIKL et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) conducted an in vitro study on the apoptosis rates of pulp cells incubated with zoledronic acid, which revealed a reduction in cell survival due to the direct toxic effect of zoledronic acid (Cvikl et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a clinical study in which TNF-α inhibitors were used to treat systemic diseases, their efficacy in inhibiting the process of bone resorption was demonstrated (Elliott et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1994a\u003c/span\u003e). In periodontitis, TNF-α is one of the main factors contributing to the worsening of the disease (Lappin et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The injection of a solution containing TNF-α antagonists can inhibit the formation of osteoclasts, decrease the recruitment of inflammatory cells, and reduce bone loss (Assuma et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). It has also been demonstrated that the use of TNF-α antagonists reduces bone damage in rheumatoid arthritis (Elliott et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1994b\u003c/span\u003e). Thus, its reduction presents a potential alternative for mitigating tissue damage.\u003c/p\u003e \u003cp\u003eIn the present study, in the healthy group, there was discrete and focal labeling for TNF-α in the osteocytes, with a smaller average marked area. In contrast, in the osteonecrosis, obese, and obese with osteonecrosis groups, the labeling was intense and multifocal, and the osteocytes exhibited irregular and enlarged appearances, along with larger averages of marked areas. TNF-α is important for bone resorption and contributes to tissue damage in diseases related to inflammatory processes (Bingham \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Goldring \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e); possibly, this finding is related to the results observed in the groups affected by the disease. Furthermore, adipose tissue produces a relatively large amount of TNF-α (Weisberg et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), which supports the results of the pronounced labeling in the groups affected by obesity.\u003c/p\u003e \u003cp\u003eRelationships between circulating TNF-α and obesity indices have been studied. Jellema et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) reported that weight loss decreases the circulating concentration of this adipokine (Jellema et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Yudkin et al. (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) suggested that TNF-α production by fat around the arterioles inhibits insulin stimulus for nitric oxide synthesis, resulting in vasoconstriction (Yudkin et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Effects such as the inhibition of the insulin receptor signaling pathway have been described, including some studies involving obese rats (Hotamisligil et al. 1993; Coppack \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother important cytokine for the process of bone remodeling is TGF-β, which is involved in various biological processes, including the regulation of cell proliferation and differentiation, apoptosis, the synthesis of extracellular matrix components, and immune responses. Dysregulation of this cytokine is associated with the onset of certain diseases, such as cancer and fibrosis (Zhao and Chen \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, discrete and focal staining for TGF-β was detected in the osteocytes of the healthy and osteonecrotic groups, whereas in the obese and obese with osteonecrosis groups, intense and multifocal staining was detected. Since TGF-β is an important cytokine for the healing process, it is believed that this cytokine may have bound to its receptors, thereby increasing the collagen concentration.\u003c/p\u003e \u003cp\u003eTGF-β is an anti-inflammatory cytokine that plays a significant role in maintaining tissue homeostasis, thus facilitating resolution and inducing the repair process. In contrast, it negatively regulates the recruitment and activation of leukocytes while recruiting fibroblasts, thereby stimulating the synthesis of extracellular matrix components (Wahl \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). This anti-inflammatory property may have contributed to the findings in the obese groups, where the staining for TGF-β was more intense. Notably, the staining for TNF-α in these groups was also intense and multifocal, supporting the hypothesis of the influence of the inflammatory process.\u003c/p\u003e \u003cp\u003eConcomitantly, intense and multifocal staining for collagen I was identified in the osteocytes and matrix of the healthy group, indicating a possible healing process. In contrast, in the osteonecrosis, obese, and obese with osteonecrosis groups, this staining was discrete and focal, indicating the opposite of the healing process. The inflammatory process likely also influenced these findings, as it is present in both conditions.\u003c/p\u003e \u003cp\u003estudies have shown that zoledronic acid interferes with the collagen synthesis process in cultures of periodontal fibroblasts, and one of the causes of these findings is that this drug acts by increasing the rate of apoptosis (Scheper et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; De Colli et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Komatsu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). One of the potential causes of this toxic effect is the oxidative stress generated at the target area of the drug. According to Colli et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), with the interaction of zoledronic acid, fibroblasts present in the periodontal ligament undergo a process of increased synthesis of reactive oxygen species through a pathway dependent on the constitutive synthesis of nitric oxide, thereby causing inflammatory stress (De Colli et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); moreover, osteocytes are sensitive to this inflammatory stress (Wimalawansa \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, the induction of osteonecrosis was associated with tooth extraction between the fourth and fifth weeks of administration of zoledronic acid. This drug irreversibly binds to the hydroxyapatite of the bone tissue (Endele et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e); thus, when trauma occurs, the tissues are already in an inflammatory process, and this event possibly stimulates an increase in the release of proinflammatory cytokines. Studies in experimental and clinical models suggest an association between the inflammatory process and bisphosphonate-induced osteonecrosis of the jaws (Maahs et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ali-Erdem et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Marino et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, one of the key factors for a better understanding of these aspects is the study of the immunological profile of this disease.\u003c/p\u003e \u003cp\u003eZoledronic acid can have distinct effects depending on its concentration. At high concentrations, the synthesis of alkaline phosphatase and the morphology of odontoblasts may be compromised; conversely, at lower concentrations, the drug may increase the expression of collagen I (Basso et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the present study, lower expression of collagen I was observed in the osteonecrosis and obese with osteonecrosis groups, findings that corroborate those of Basso et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Furthermore, according to Scheper et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and Naidu et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), this drug is toxic to various cell types, such as fibroblasts and osteoblasts; thus, a reduction in collagen I synthesis is expected (Naidu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Scheper et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eObesity is a disease that negatively impacts bone metabolism, primarily through alterations in the inflammatory process (Shapses et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Bisphosphonates are responsible for increasing the production of acute inflammatory mediators and altering the immune and cellular profiles of patients (Norton et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rossini et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Welton et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Muratsu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The association of these two conditions, which are linked to alterations primarily generated in the inflammatory process, may have contributed to the low deposition of collagen I in the osteonecrosis, obese, and obese with osteonecrosis groups.\u003c/p\u003e \u003cp\u003eAssociated with the reduction in the deposition of collagen type I in the osteonecrosis, obese, and obese with osteonecrosis groups, an increase in the level of TGF-β was found in the same groups. This finding may demonstrate that in the healthy group, TGF-β was synthesized and used to activate bone tissue cells to produce collagen, thus facilitating the healing process, unlike in the other groups, where this mechanism was possibly not achieved, and the healing process, compared with that in the healthy group, was not the same.\u003c/p\u003e \u003cp\u003eThe association between TGF-β and collagen I production suggests that osteonecrosis possibly modulates the deposition of collagen I in obese rats via TGF-β. Another important factor is the inflammatory process present in both conditions, where it is believed that both conditions negatively influence the modulation of collagen I deposition. The associations among obesity, osteonecrosis, and the parameters investigated in this study provide a relevant foundation for understanding the complexities of this condition. Although research on the specific impact of zoledronic acid on the expression of inflammatory markers in patients with osteonecrosis associated with obesity is scarce, the results of the present study suggest a promising relationship that warrants further investigation. Future research on these factors is essential to confirm these hypotheses and enhance the clinical and therapeutic understanding of this condition.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThrough microscopy and morphometry, this study revealed that TGF-β, TNF-α, and collagen I play important roles in bone tissue in the context of obesity and osteonecrosis, where osteonecrosis modulates the deposition of collagen I in obese rats via TGF-β.\u003c/p\u003e \u003cp\u003eOsteonecrosis induced by the use of bisphosphonates in obese rats may interfere with the process of bone homeostasis, potentially influenced by the inflammatory process present in both conditions, contributing to the abnormal deposition of the extracellular matrix in bone tissue. This abnormal deposition may increase the risk of developing and worsening disease.\u003c/p\u003e \u003cp\u003eResearch relating obesity and medication-induced osteonecrosis of the jaws is important for a better understanding of their respective relationships, especially with respect to the regulation of bone metabolism, to develop proposals for prevention and treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eIn the context of this study, we declare that funding was provided by the Para\u0026iacute;ba State Research Support Foundation (FAPESQ/PB) through grant number 010/2021 FAPESQ/PB - MCTIC/CNPQ under the Infrastructure Program for Young Researchers/First Projects Program (PPP). This support was formalized through Grant Agreement number 3224/2021. We emphasize that the funding agency was not involved in the study design, data collection, analysis, interpretation, or writing of this manuscript. The conclusions and interpretations expressed in this work are solely the responsibility of the authors, with no influence from the funders.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e W.J.M.L. (corresponding author) was the primary author and contributed to all stages of the study, including conceptualization, experimental planning, data collection, data analysis, interpretation of results, and manuscript drafting.\u0026bull; A.F.A. supervised and guided the research, providing technical and intellectual support throughout all stages and assisting in the final review of the manuscript.\u0026bull; W.F.B.P. contributed to the critical review of the manuscript, enhancing its scientific content and clarity.\u0026bull; J.C.X.P., R.S.A., and M.C.P.S. provided specific technical contributions during the study, including assistance with sample processing and data analysis.All authors reviewed and approved the final version of the manuscript for submission, taking responsibility for the accuracy and integrity of the work in all its aspects.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAaron N, Costa S, Rosen CJ, Qiang L (2022) The Implications of Bone Marrow Adipose Tissue on Inflammaging. 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Scientifica (Cairo) 2014:874065. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2014/874065\u003c/span\u003e\u003cspan address=\"10.1155/2014/874065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bone remodeling, Metabolic Diseases, Bisphosphonates, Mandible, Immunohistochemistry","lastPublishedDoi":"10.21203/rs.3.rs-5361050/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5361050/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteonecrosis, characterized by the death of bone tissue in the jaws, is termed bisphosphonate-related osteonecrosis of the jaws (BRONJ) when caused by bisphosphonate use. Obesity, a significant public health issue, has been associated with both BRONJ and other oral conditions, such as caries and periodontitis, highlighting the relationship between systemic factors and oral health. This study investigated the influence of TGF-\u0026szlig;, TNF-α, and collagen I on bone tissue and their correlation with mandibular osteonecrosis in obese rats. Twenty-four male Wistar rats (\u003cem\u003eRattus norvegicus\u003c/em\u003e) were divided into four groups: healthy, osteonecrotic, obese, and obese with osteonecrosis. Osteonecrosis was induced with zoledronic acid (250 \u0026micro;g/kg), which was administered weekly for eight weeks, combined with tooth extraction, while obesity was induced by a high-glycemic diet. The analyses revealed that, compared with the patients in the osteonecrosis group, the obese group with osteonecrosis had a 67.99% increase in the necrotic area, whereas the obese group had a 43.85% reduction. The healthy group had the largest reduction (97.11%). For TNF-α, there was intense staining in the osteonecrosis (27.59\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65 \u0026micro;m\u0026sup2;) and obese (25.52\u0026thinsp;\u0026plusmn;\u0026thinsp;8.31 \u0026micro;m\u0026sup2;) groups, whereas the level of TGF-β was greater in the obese with osteonecrosis group (44.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93 \u0026micro;m\u0026sup2;). Collagen I staining was more intense in healthy animals. The potential interaction between TGF-\u0026szlig;, TNF-α, and collagen I in bone tissue may be essential for understanding bone remodeling; however, further studies are needed to explore these mechanisms.\u003c/p\u003e","manuscriptTitle":"Osteonecrosis modulates extracellular matrix deposition through collagen I deposition in obese rats via the TGF-β protein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-20 11:43:54","doi":"10.21203/rs.3.rs-5361050/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"573c86bc-1f37-47bb-a853-4d1bea59318e","owner":[],"postedDate":"November 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-03T14:09:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-20 11:43:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5361050","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5361050","identity":"rs-5361050","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}