Influence of osteoporosis on tissue repair promoted by different dental materials: an in vivo study in a rat model

Article information

Restor Dent Endod. 2026;.e44

Publication date (electronic) : 2026 August 12

doi : https://doi.org/10.5395/rde.2026.51.e44

Guilherme Ferreira da Silva¹, Antônio Carlos Medina Junior², Mariza Akemi Matsumoto³, Roberta Okamoto³, João Pedro Gasparin Tadano¹, João Vitor Oliveira de Amorim^1,, Marco Antônio Húngaro Duarte¹, Murilo Priori Alcalde¹, Rodrigo Ricci Vivan¹

¹Departamento de Dentística, Endodontia e Materiais Dentários, Faculdade de Odontologia de Bauru, Universidade de São Paulo, Bauru, Brazil

²Departamento de Odontologia, Centro Universitário Sagrado Coração (UNISAGRADO), Bauru, Brazil

³Departamento de Ciências Básicas, Faculdade de Odontologia, Universidade Estadual Paulista (UNESP), Araçatuba, Brazil

*Correspondence to João Vitor Oliveira de Amorim, MSc Departamento de Dentística, Endodontia e Materiais Dentários, Faculdade de Odontologia de Bauru, Universidade de São Paulo, Alameda Doutor Octávio Pinheiro Brisolla 9-75, Bauru, SP 17012-901, Brazil Email: joaovitoramorim91@gmail.com

Citation: da Silva GF, Medina Junior AC, Matsumoto MA, Okamoto R, Tadano JPG, de Amorim JVO, Duarte MAH, Alcalde MP, Vivan RR. Influence of osteoporosis on tissue repair promoted by different dental materials: an in vivo study in a rat model. Restor Dent Endod 2026;51(4):e43.

Received 2026 February 10; Revised 2026 April 18; Accepted 2026 May 21.

Abstract

INTRODUCTION

The biological mechanisms involved in tissue repair are substantially influenced by genetic factors [1] and the systemic health status of the patient [2]. Therefore, systemic diseases can interfere with tissue repair following endodontic procedures [3]. As a result, these systemic conditions can decrease the treatment success rate and provoke incomplete wound healing [4].

Osteoporosis is one of the most common metabolic disorders in the elderly [5] and can affect tissue healing after endodontic procedures. López-López et al. [6] investigated the relationship between chronic apical periodontitis (AP) and bone mineral density in postmenopausal women, finding that low bone mineral density was associated with a higher frequency of AP. Another study investigated the prevalence of periapical lesions in patients with osteoporosis, compared with the general population, finding that the prevalence of periapical lesions was significantly higher in osteoporotic patients [7].

MTA-Angelus (Angelus, Londrina, Brazil) is one of the most widely used repair cements worldwide as it promotes a biological response that favors cell survival, proliferation, and differentiation, promoting tissue repair [8,9]. However, there are some concerns about its solubility, which can cause degradation and failure over time [10]. Epoxy resin-based sealers with calcium hydroxide are known for their excellent sealing properties [11] and low solubility [12]. The literature indicates that increasing the powder-to-resin ratio to achieve a thicker consistency allows these sealers to be used as repair materials. In this consistency, certain epoxy resin-based sealers have demonstrated high sealing ability [13,14] and biocompatibility comparable to that of MTA [15].

The tissue response to biomaterials depends on the immune response [16], so systemic disorders can alter immune function and affect the healing process [4]. However, there is limited information concerning the links between osteoporosis and repair materials, especially when comparing a calcium silicate and an epoxy resin-based product. Therefore, this study aimed to evaluate the influence of osteoporosis on the tissue repair promoted by two different repair materials, MTA-Angelus (MTA) and an epoxy resin-based sealer containing calcium hydroxide (ER-Ca), after implantation in a rat dental socket. The null hypothesis was that osteoporosis would not interfere with the intensity of the tissue reaction induced by both materials.

METHODS

This research was approved by the Ethical Committee for Animal Research of Sagrado Coração University (Research Protocol 22/13) in compliance with Brazilian national law on animal use. The study was carried out in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, 1996). The experiment and analysis methods were conducted in accordance with ARRIVE guidelines 2.0 (Animal Research: Reporting of In Vivo Experiments). Animal use adhered to the principles of Replacement, Reduction, and Refinement (3Rs). Any anesthesia and euthanasia methods employed conformed to the American Veterinary Medical Association Guidelines for the Euthanasia of Animals (2020).

The experimental groups (n = 6/group) were described in Table 1. The sample calculation was performed using G*Power ver. 3.1 for Mac by selecting fixed-effects analysis of variance. Data were obtained from a previous study [17], in which a biocompatibility test was used to evaluate endodontic sealers. The effect size utilized in the present study was set at 0.26. The alpha error level was set at 0.05, and statistical power was set at 0.90. Based on the study design, the animals were allocated according to two systemic conditions (ovariectomized [OVX] and sham-operated [SHAM]), three material conditions (MTA-Angelus, ER-Ca, and empty tube as control), and two experimental periods (30 and 60 days). Thus, 12 experimental groups were established, with six animals per group, resulting in a total sample of 72 rats.

Table 1.

Experimental groups

Seventy-two female Holtzman rats (Rattus norvegicus albinus) weighing ±250–300 g were anesthetized via an intraperitoneal injection with ketamine hydrochloride (80 mg/kg of body weight; Virbac do Brasil Indústria e Comércio Ltda., São Paulo, Brazil) and xylazine hydrochloride (8 mg/kg of body weight; União Química Farmacêutica Nacional S/A, São Paulo, Brazil). The animals were randomly divided into two groups: an ovariectomized group (OVX) and a sham-operated group (SHAM). The ovariectomy surgery was performed in animals assigned to the OVX group, while animals in the SHAM group underwent a similar surgical procedure up to the stage of ovary exposure, at which point the ovaries were replaced intact into the abdominal cavity [18]. To evaluate the effectiveness of the ovariectomy surgery, the animals were weighed weekly on a digital scale with a precision of 1 g. The weights of the animals were compared at each milestone of the experiment. Additionally, after euthanasia, the uteri of the animals were dissected, washed with saline solution, and weighed on a digital scale with a precision of 0.001 g [18].

The animals were kept in individual cages, in an environment with a temperature between 22°C and 24°C, with a controlled light cycle (12 hours light and 12 hours dark) and fed a standard solid diet with water ad libitum during the experiment. A senior veterinarian conducted all the nutritional recommendations and was responsible for the care, pre- and postoperative fasting of the animals.

To confirm the establishment of osteoporosis, indirect parameters associated with estrogen deficiency were assessed. Body weight was monitored weekly throughout the experimental period, as OVX animals typically exhibit a significant increase in body mass compared with SHAM controls [18]. Additionally, after euthanasia, the uteri were collected and weighed, since uterine atrophy is an indicator of successful ovariectomy and consequent estrogen depletion [18]. Animals were anesthetized using the same protocol as previously described, and the maxillae were prepared aseptically using a 10% polyvinylpyrrolidone-iodine solution. The upper right incisor was carefully luxated, displaced, and then extracted. Surgical procedures were conducted by a single surgeon. The materials (Table 1) were inserted into polyethylene tubes (3.0 mm in length and 1.0 mm in diameter) and subsequently implanted in the alveolar sockets of the animals. MTA-Angelus was manipulated according to the manufacturer’s instructions, and the epoxy resin sealer was mixed in the proportion of 5:1 powder:resin by weight (thick consistency) as a repair material [15]; empty tubes were used as a control. The polyethylene tubes were placed in the alveolar socket, and immediately, the gingival margins of the surgical wounds were sutured with sterile Vicryl 4-0 (Ethicon; Johnson & Johnson Brazil Ltda, São Jose dos Campos, Brazil). After 30 and 60 days, the animals were deeply anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). After confirmation of a surgical plane of anesthesia (absence of pedal and corneal reflexes), an additional dose of ketamine (300 mg/kg, intraperitoneally) was administered to induce respiratory and cardiac arrest. Death was confirmed by cessation of heartbeat and respiration, followed by cervical dislocation to ensure complete euthanasia.

The upper jaw was dissected, removed, and fixed in 10% neutral-buffered formalin for 48 hours. Subsequently, the jaw was demineralized in a 4.13% buffered ethylenedinitri-lotetraacetic acid (Titriplex III; Merck KGaA, Darmstadt, Germany) for 3 weeks. They were subjected to standard histological techniques. The samples were embedded in paraffin to obtain longitudinal sections (6 μm thick), which were obtained using a rotary microtome (Leica RM2125 RTS; Leica Biosystems, Wetzlar, Germany) and disposable stainless-steel knives (Leica 818; Leica Biosystems). Non-serial sections were stained using hematoxylin and eosin for morphological and morphometrical analyses.

The number of inflammatory cells (ICs) and fibroblasts (FBs) in the tissue adjacent to the implants was measured. Three sections from each animal (×40 magnification) were quantified, maintaining a minimum interval of 100 μm between the sections. Afterward, an image analysis program (Image-Pro Express ver. 6.0; Olympus, Tokyo, Japan) was used to quantify ICs and FBs in each section. A mean value was established for each experimental group according to the experimental time interval and material studied. The presence or absence of bone formation was also analyzed in these sections. Furthermore, in three sections, in images captured with a ×20 objective, the thickness of the connective tissue capsule and bone neoformation was measured. For these parameters, the ISO Standard 7405 [19] and the criteria of other studies [20] scores were established for histopathological analysis (Table 2). Two calibrated operators analyzed the sections of each specimen in a blinded manner under light microscopy [21].

Table 2.

Histopathological parameters and scores

RESULTS

No animals died during the experiment, and no adverse effects were observed from endodontic repair materials.

The tissue reaction induced by the different groups may be observed in Figure 1, and the comparisons among the histological parameters are shown in Table 3. At 30 days, many ICs were observed in the connective tissue in contact with all groups, particularly plasmocytes and lymphocytes. After 60 days, a gradual and significant reduction in the inflammatory response was seen, accompanied by an increase in the number of FBs and a reduction in the thickness of the connective tissue (p ≤ 0.05).

Figure 1.

Photomicrographs of sections of the portions adjacent to the implants performed after 30 days and 60 days (hematoxylin and eosin stain, ×600). Inflammatory cells (arrows), especially plasmocytes and lymphocytes, were present in the tissue (T) adjacent to the implants, mainly in the ovariectomy (OVX) group (A, B, C, G, H and I). D, E, G, H, J and K panels showed bone neoformation next to the defects of sealers (asterisks). Control, empty tubes; MTA, MTA-Angelus (Angelus, Londrina, Brazil); S26, Sealer 26 (Dentsply Indústria e Comércio Ltda., Petrópolis, Brazil); SHAM, sham surgery.

Table 3.

Medians of scores for different parameters

Comparison among groups showed that OVX induced a more intense inflammatory response than SHAM (p < 0.05). After 30 days, a significantly lower number of ICs and greater quantities of FBs were shown in the tissue adjacent to the control groups (p < 0.001). However, at 60 days, no statistically significant differences were observed between the SHAM and control groups. The capsule thickness was thicker at 30 days, while it was classified as thin at 60 days in MTA and ER-Ca (p ≤ 0.05); for this parameter, the control group exhibited thin capsules in both periods.

After 30 days, bone neoformation was close to the material, mainly in the MTA and ER-Ca SHAM groups. In these groups, 62.3 µm and 63.4 µm of the thickness of neoformed bone were observed, respectively. At 60 days, mineralized tissue formation was seen in all specimens except in the control groups. However, statistically significant differences were seen between the values of the OVX and SHAM groups for both materials regarding the thickness of neoformed bone (Table 3).

DISCUSSION

The mechanisms involved in tissue repair may be influenced by systemic conditions [18]. These systemic diseases can alter bone turnover and FB function, impairing or delaying wound healing [22]. Animal and epidemiological studies have suggested that several systemic conditions can influence the prognosis of endodontic treatments [3,23,24].

Osteoporosis accelerates bone resorption, including in alveolar bone, and reduces bone density, which influences tissue repair after endodontic procedures [6,7]. Therefore, it is important to evaluate the influence of osteoporosis on the tissue repair promoted by two repair materials after implantation in a rat dental socket. Animal models of osteoporosis are suitable tools for studying new prevention and treatment modalities. Although no animal model is perfect, the similarities in response to estrogen deficiency and therapeutic agents between the human and rat skeleton have made the ovariectomized rat model an appropriate model in osteoporosis research [18].

The chemical composition of endodontic materials may influence tissue response due to the release of bioactive or potentially cytotoxic substances [25]. In the present study, MTA-Angelus, one of the most widely used calcium silicate materials worldwide and recognized for its excellent biological properties, was evaluated. However, it is known that this type of material may exhibit relatively high solubility [10]. Therefore, for comparison, an epoxy resin material was used due to its low solubility and high adhesiveness [11-14,26], which may contribute to improved long-term treatment outcomes.

Our findings strongly suggest that osteoporosis negatively interferes with tissue repair, as evidenced by the more intense inflammatory response after 60 days in the OVX group. Therefore, the null hypothesis was rejected. These biological mechanisms associated with osteoporosis may explain the present results. Several studies have reported that bone changes in osteoporosis were associated with loss of periodontal attachment, loss of teeth, and reduction of alveolar bone [27], impairing tissue repair. Bender and Seltzer [28] suggested that osteoporosis may help to explain why healing following root canal therapy was more favorable in the younger age groups. Although there was bone formation in all groups after 60 days, the thickness was lower in the OVX group. These findings suggest that the persistence of inflammatory infiltrate in this group contributed to delayed bone formation.

The differences observed between the OVX and SHAM groups suggest that systemic alterations associated with osteoporosis directly influence tissue response to implanted materials [3,4]. In particular, the persistence of inflammatory infiltrate and the delayed bone formation observed in the OVX groups suggest that local healing may be modulated by systemic factors related to bone metabolism and immune regulation [2]. This impaired tissue repair may be explained by alterations in bone remodeling dynamics, particularly those associated with estrogen deficiency. Estrogen deficiency, as induced in the OVX model, is known to disrupt the balance between osteoclast and osteoblast activity through alterations in the receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) signaling pathway. Reduced estrogen levels promote increased expression of RANKL and decreased production of OPG, thereby enhancing osteoclastogenesis and promoting increased bone resorption and reduced bone formation [29]. Moreover, this imbalance is associated with the upregulation of pro-inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin (IL)-1β, and IL-6, which contribute to osteoclast activation and prolongation of the inflammatory response. Collectively, these mechanisms may help explain the persistence of inflammatory infiltrate and delayed tissue repair observed in osteoporotic conditions [5,16,29].

Comparison between the materials revealed no significant differences in tissue response. It is well documented that calcium silicate-based materials are biocompatible. Although these products initially caused an intense inflammatory reaction, after 30 and 60 days, there was a regression in the inflammatory process [17,30]. In the present study, especially after 60 days, a reduction in IC infiltration and an increase in FB density were seen adjacent to MTA-Angelus, which was accompanied by bone formation. The epoxy resin-based sealer used includes bismuth oxide, calcium hydroxide, and epoxy resin. A greater proportion of powder/resin is used to obtain a thicker consistency for use as a repair material. It has been previously demonstrated that certain epoxy resin-based sealers present favorable sealing properties when used for retrograde obturation [13,14]. Tanomaru-Filho et al. [15] revealed similar periapical repair for the ProRoot MTA (Dentsply Tulsa Dental, Tulsa, OK, USA), a calcium silicate material, and Sealer 26 (Dentsply Indústria e Comércio Ltda., Petrópolis, Brazil) after retrograde filling, including the deposition of mineralized tissue in contact with the materials. These results are in agreement with those found in the present study.

Studies have shown that biological properties are associated with the composition of the materials. The literature reports that calcium hydroxide has essential effects on tissue, promoting an alkaline pH, antimicrobial effects, and accelerating tissue repair [31]. Additionally, calcium hydroxide induces mineralization by dissociating calcium (Ca²⁺) and hydroxyl ions (OH^–), activating alkaline phosphatase, and inducing cell differentiation. The release of calcium ions also allows the activation of calcium-dependent ATPase and the formation of calcite microcrystals that will initiate the tissue mineralization process [32]. Furthermore, the alkaline pH may stimulate the recruitment of ICs and the production of cytokines that enhance leukocyte adhesion to endothelial walls, activation of neutrophils, and differentiation of plasma cells. The inflammatory process involves a complex and coordinated cascade of cellular and molecular events that may culminate in superficial necrosis, a scaffold for the healing process and mineralization [30]. The presence of calcium hydroxide in the composition of the epoxy resin-based sealer and its formation during the hydration reaction of MTA-Angelus may stimulate bone neoformation after 60 days in the SHAM groups.

During endodontic treatments, the repair materials may come into direct contact with the surrounding tissues. Biocompatibility is an essential property of these materials [33]. The healing process is dynamic and involves distinct stages, beginning with an inflammatory phase, followed by tissue remodeling and collagen formation. In cases involving bone loss, this process may culminate in new bone formation. Although the materials evaluated have different compositions, both MTA-Angelus and the epoxy resin-based sealer showed similar biological behavior and were able to modulate the tissue repair process, allowing the regression of inflammation in both groups. However, this response was more favorable in the SHAM group, reinforcing the idea that tissue repair is less favorable in cases of osteoporosis. Despite this, the results of the present study indicate that even in this systemic condition, both materials modulate the repair process, as evidenced by the regression of inflammation, accompanied by FB proliferation and bone formation, especially after 60 days.

From a clinical perspective, the present findings suggest that osteoporosis may negatively influence the healing process following endodontic procedures, potentially affecting treatment outcomes due to delayed inflammation resolution and reduced bone formation [3,7,24]. This highlights the importance of considering systemic conditions during treatment planning and follow-up [4,25]. Moreover, although the materials evaluated demonstrated favorable biological behavior even under osteoporotic conditions, the impaired healing observed in OVX animals may indicate the need for adjunctive strategies to optimize repair in such patients. Such strategies may include closer clinical monitoring, modification of treatment protocols, or the use of biomaterials and therapeutic approaches that enhance bone regeneration [3,8,20].

Despite the relevant findings, this study presents some limitations that should be considered. First, although the sample size was determined based on a priori power analysis, the relatively small number of animals per group may reduce the sensitivity to detect subtle differences between experimental conditions, particularly in histological evaluations where biological variability is inherent [21]. Therefore, the absence of statistically significant differences between materials should be interpreted with caution. Although the ovariectomized rat model is widely accepted and validated, the use of techniques such as micro-computed tomography or densitometric analysis would provide a more precise and quantitative characterization of bone alterations [18]. Finally, as this is an in vivo animal study, the results should be interpreted with caution when extrapolating to clinical conditions, given anatomical, physiological, and metabolic differences between species. Future studies incorporating advanced imaging methods and larger sample sizes are recommended to further validate and expand the present findings.

Beyond confirming the negative impact of osteoporosis on tissue repair, the present study provides important insights into the behavior of different classes of endodontic repair materials under compromised systemic conditions. Notably, both MTA-Angelus and the ER-Ca demonstrated the ability to modulate inflammation and support bone formation, even in the presence of delayed healing associated with estrogen deficiency. These findings suggest that the biological performance of these materials is maintained despite systemic impairment, reinforcing their potential applicability in patients with osteoporosis. Furthermore, the comparable response observed between the materials indicates that alternative formulations, such as epoxy resin-based sealers in modified consistency, may represent viable options for reparative procedures.

CONCLUSIONS

Osteoporosis negatively affects tissue repair by delaying inflammation resolution and bone formation. However, both MTA-Angelus and the ER-Ca maintained favorable biological performance, demonstrating their ability to modulate tissue response and promote bone neoformation even under osteoporotic conditions. These findings provide relevant insights into the selection of repair materials for patients with compromised systemic health and support their potential clinical applicability in such scenarios.

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