Local Single-Dose Teriparatide Administration for BRONJ Prevention: Insights from a Rat Model Study

Abstract

Background

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) presents significant clinical challenges with uncertain treatment outcomes. Teriparatide, a fragment of human parathyroid hormone, shows potential in prevention strategies for BRONJ.

Objective

This study investigates the impact of a single local dose of teriparatide on BRONJ prevention in an animal model.

Methods

BRONJ was induced on 26 male Wistar rats via intraperitoneal injections of 0.06 mg/kg Zoledronic acid weekly for four weeks. Following tooth extraction under general anesthesia, rats were divided into two groups: one received teriparatide via Gelatamp in the tooth socket and the other as a local injection in the vestibule. Control groups received the treatment without teriparatide. Histopathological assessments for bone remodeling, osteoclast number, inflammation, angiogenesis, and necrosis were performed after four weeks.

Results

Histopathological analysis indicated a significant improvement in bone remodeling and new bone formation in both treatment groups compared to controls, with notably better outcomes in the injection group (p = 0.01). Necrosis in the Gelatamp group was significantly higher in the control group than in the treatment group (p = 0.01), and bone formation was significantly higher in the injection group compared to the Gelatamp group (p = 0.03).

Conclusion

Local administration of teriparatide significantly enhances bone remodeling and reduces necrosis, suggesting a potential role in the prevention of BRONJ. These findings support further clinical investigation into teriparatide as a preventive strategy against BRONJ.

Introduction

Teriparatide, a recombinant human parathyroid hormone fragment, has gained approval from the FDA for treating osteoporosis [1]. This drug, known for stimulating osteoblasts, plays a pivotal role in bone formation and further enhances bone remodeling by increasing both the number and activity of osteoclasts, leading to a balanced cycle of bone resorption and formation [2]. Additionally, teriparatide has been shown to augment blood circulation in bone tissues, [3] thereby promoting bone formation and accelerating healing post-fracture [4].

Medication-related osteonecrosis of the jaw (MRONJ) is a condition marked by the exposure of necrotic bone in the maxillofacial region, persisting for over eight weeks in patients on anti-angiogenic or anti-osteoporosis medication, but with no history of maxillofacial radiotherapy [5]. The pathogenesis of MRONJ is complex and not fully understood, possibly involving inhibited osteoclast function, suppressed bone metabolism, and reduced bone vascular supply [5, 6]. While treatments for MRONJ, including surgical debridement, systemic antibiotics, and antimicrobial mouthwashes, exist, their effectiveness is limited, necessitating the exploration of less invasive preventive measures for high-risk patients, such as the application of teriparatide [5, 7].

In recent studies, teriparatide has shown promise in the treatment and prevention of MRONJ, with several case studies [8–17] and a randomized clinical trial underscoring its efficacy and safety [18]. It has been particularly effective when used in combination with antibiotic therapy [19, 20]. However, these studies have predominantly focused on the systemic effects of teriparatide administered daily or weekly, leaving a gap in our understanding of its local, single-dose effects. Preliminary animal studies have indicated the potential of locally administered single-dose teriparatide in repairing significant calvarial defects [21, 22], suggesting a novel approach to MRONJ management.

This study endeavors to explore the efficacy of local, single-dose teriparatide administration in preventing MRONJ using a rat model. This approach not only provides an opportunity to deepen our understanding of teriparatide’s local effects but also offers a relevant physiological framework for extrapolating these findings to potential human applications.

Methods

Animal Model and Study Design

We utilized 30 male Wistar rats, aged 12 weeks and weighing between 250 and 350 g. The rats were acclimatized for two weeks before the study, provided with standard laboratory nutrition, and maintained in a controlled environment with a temperature of 22 ± 2 °C, humidity of 40–60%, and a 12-h light–dark cycle.

Induction of MRONJ

Following the protocol by Zandi et al. [23], MRONJ was induced by administering intraperitoneal injections of 0.06 mg/kg Zoledronic acid weekly for four weeks. Subsequently, under general anesthesia (75 mg/kg ketamine hydrochloride and 7.5 mg/kg midazolam), the first maxillary molars on both sides were extracted. Post-extraction, buprenorphine (0.15 mg/kg) was administered for two days for analgesia.

More than 50% of human cases develop MRONJ after tooth extraction [24]. Thereby, molar tooth extraction has been selected as the study intervention. After placing the rat on its back, the gingiva around the first molar was removed, and the teeth were extracted with a narrow head probe. After tooth extraction, buprenorphine (0.15 mg/kg) was provided to the animals for two days as an analgesic.

Group Allocation

The rats were randomly assigned to two primary groups u- the Gelatamp group and the Injection group—each with a treatment and control subgroup (Teriparatide Gelatamp: TG, Gelatamp Control: GC, Teriparatide Injection: TI, Injection Control: IC). Figure 1 graphically illustrates the experimental groups allocations and study design.

Fig. 1.

Experimental groups allocations and study design

In the Gelatamp group, after molar extraction, 4 µg/kg teriparatide (Teriparatide, Sinagen, Iran) was applied to Gelatamp (Gelita-Spon, Eberbach, Germany) and placed in the tooth socket on one side, and sutured (TG). The opposite side received Gelatamp without teriparatide (GC).

In the Injection group, 4 µg/kg teriparatide was injected locally into the vestibule next to the extracted socket on one side and sutured (TI). The opposite side was sutured without teriparatide (IC).

Outcome Measures

After four weeks, the rats were euthanized. Histological evaluations included assessing bone remodeling, osteoclast number, inflammation, angiogenesis, and necrosis in the socket area. Clinical assessments for fistulas, abscesses, exposed bone necrosis, and mucosal ulcers were performed on days 7, 14, 21, and 28 post-extraction. Blinded evaluations were conducted by a pathologist and a maxillofacial surgeon. Rats were randomly numbered, and evaluators were blind to the type of intervention. Also, the frequency of examination for each rat was the same. Due to the objectivity of the outcomes, the possibility of information bios was minimized.

Histomorphometry

Tissue specimens were fixed in 10% formalin, decalcified in 10% formic acid, dehydrated, and embedded in paraffin. Sections (6 microns thick) were stained with hematoxylin and eosin and examined under a light microscope (OLYMPUS CXZIFS, Tokyo, Japan). The percentage of connective tissue and cell types was quantified using a graded lens. Digital image analysis was performed using Photoshop software to calculate the percentage of newly formed bone based on pixel ratios [25].

For bone remodeling rate, we employed a two-step process: firstly, calculating the remodeling rate as a percentage using appropriate software, and secondly, converting this percentage into a remodeling score, graded from 1 to 5 (1: no bone resorption, 2: 25% bone resorption, 3: 50% bone resorption, 4: 75% bone resorption, 5: 100% bone resorption and replacement with fibrotic tissue and new trabeculae). Blood vessel count and inflammation intensity were evaluated in three distinct microscopic fields at 40× magnification, with the former graded from 1 to 4 (grade 1: 0–2 blood vessels, grade 2: 3–4 blood vessels, grade 3: 5–7 blood vessels, grade 4: 8 blood vessels or more) and the latter from 0 to 3 (grade 0: 0 lymphocyte, grade 1: 1 to 29 lymphocytes, grade 2: 30 to 59 lymphocytes, grade 3: more than 60 lymphocytes) [26, 27].

Statistical Analysis

Data were presented using descriptive statistics (mean, standard deviation). Data were analyzed using SPSS software (Version 26 IBM, SPSS, Statistics, Chicago, IL, USA). Data distribution was measured using the Kolmogorov–Smirnov test. In the case of normal distribution, parametric tests and otherwise non-parametric equivalents were used. Mann–Whitney and Willcoxon tests were used to analyze the data. Comparisons between 4 groups were used by ANOVA test in quantitative data with normal distribution and Kruskal–Wallis test in non-parametric data.

Results

During the study, four rats expired, two from the Gelatamp group and two from the injection group. The cause of death was not related to the treatments administered. Clinical evaluations of the surviving rats did not indicate the presence of fistulas, exposed bones, fissures, or wounds. Notably, necrosis was confined to the alveolar bone and did not extend beyond this region. Bone necrosis was observed more prevalent in the control subgroups of both Gelatamp and injection groups, compared to their respective treatment subgroups. Significantly, the injection group exhibited a lower incidence of necrosis compared to the Gelatamp group.

The histopathological examination focused on several therapeutic indicators, including bone remodeling, fibrosis, ossification, and necrosis (Fig. 2). In the Gelatamp group, no significant differences were observed between the control and treatment subgroups in terms of bone remodeling and new bone formation. Conversely, the injection group demonstrated a significant increase in these parameters within the treatment subgroup compared to its control subgroup. Additionally, bone formation was markedly higher in the injection group than in the Gelatamp group. A significant reduction in the rate of necrosis was noted in the treatment subgroup of the Gelatamp group in comparison to its control subgroup. This pattern was not replicated in the injection group. However, a comparison between control subgroups of both Gelatamp and injection groups revealed significantly lower necrosis in the injection control group.

Fig. 2.

Necrosis absence (A, H&E staining, 40×), High remodeling (B, H&E staining, 100 ×) and High new bone formation (C, H&E staining, 100 ×) in Teriparatide Injection group and Necrosis (D, H&E staining, 100 ×), Low remodeling (E, H&E staining, 100 ×) and Low new bone formation (F, H&E staining, 100 ×) in Gelatamp Control group (NBF: New Bone Formation, Fib: Fibroblasts, Black arrow: Osteoclast, White arrows: Osteoblast, Infla: Inflammation, Lacuna: Empty Lacunae)

The treatment subgroup of the injection group showed a significant increase in osteoclast numbers compared to its control subgroup, a trend not seen in the Gelatamp group. Regarding inflammation, a reduction was observed in the treatment subgroups of both Gelatamp and injection groups compared to their respective controls, although this difference did not reach statistical significance. Similarly, no significant differences were detected in bone fibrosis and vascularity between control and treatment subgroups in either the Gelatamp or injection groups. These findings, including statistical values and significance levels, are detailed in Table 1 and Fig. 3.

Table 1.

Histopathological results in different study groups

	Teriparatide injection: TI Mean (SD)	Injection control: IC Mean (SD)	Teriparatide Gelatamp: TG Mean (SD)	Gelatamp control: GC Mean (SD)	p Value
Remodeling	2.69 (0.48)	2.33 (0.44)	2.38 (0.65)	2.38 (0.51)	0.146
Fibrosis	21.31 (5.60)	23.31 (8.20)	28 (8.47)	26.54 (10.86)	0.321
New bone formation	36.62 (4.65)	32.46 (6.13)	27.69 (8.05)	26.54 (9.21)	0.010
Inflammation	1.54 (0.78)	1.92 (0.76)	1.85 (0.80)	2.15 (0.69)	0.203
Necrosis	0.77 (2.77)	3.08 (4.80)	3.85 (4.16)	11.54 (7.74)	0.001
Vascularity	2.31 (0.48)	2.38 (0.65)	2.46 (0.52)	2.23 (0.44)	0.676
Osteoclasts	7.85 (0.99)	6.78 (1.59)	8 (1.47)	7.07 (1.85)	0.288

Fig. 3.

Comparison of bone repair indices according to injection groups (test = TI, control = IC) and Gellatamp groups (test = TG, control = GC)

Discussion

This study aimed to evaluate the effectiveness of locally administered single-dose teriparatide in preventing medication-related osteonecrosis of the jaw (MRONJ) in a rat model. Our findings reveal significant effects on bone remodeling and osteoclast activity, both of which are crucial factors in the development of MRONJ.

Bisphosphonates (BP) are known to reduce bone remodeling by inducing apoptosis and inhibiting osteoclasts. Consequently, the number and activity of osteoclasts are critical indicators in assessing the condition of MRONJ [24]. Our study indicates that single local administration of teriparatide may counteract the effects of BP, as evidenced by a higher number of osteoclasts in both TI and IC groups. Interestingly, Keskinruzgar et al. [28] observed a similar enhancement in both the count and function of osteoclasts in rats treated with Zoledronic acid (ZA) and dexamethasone, following a regimen of bi-weekly teriparatide administration over three weeks. Complementing this, Kuroshima et al. [3] documented an increase in osteoclast surface area with daily teriparatide doses in rats also treated with alendronate and dexamethasone. Contrastingly, Dayisoylu et al. [2] reported no significant change in osteoclast number or activity in rats receiving daily teriparatide for eight weeks alongside ZA treatment. These varying outcomes may be attributed to differences in the administration of bisphosphonates (BPs) and the duration of follow-up in these studies.

Furthermore, BP has been shown to reduce bone remodeling and the ability to regenerate bone tissue [29]. In our study, a single dose of teriparatide significantly enhanced bone remodeling and new bone formation, with particularly notable effects observed in the injection method. Other studies by Yu et al. [30] and Zandi et al. [31] have reported similar findings in different animal models with systemic daily and weekly administration.

The effectiveness of local and single-dose teriparatide on bone formation has also been explored in the context of bone defects. Özer et al. [22] found that applying teriparatide locally, in conjunction with xenograft, markedly increased bone regeneration in rabbit calvaria defects. Similarly, Koca et al. [21] observed an enhanced bone formation in rat mandible defects when teriparatide was used alongside allograft. In research by Kajii et al., a critical-sized defect in rat calvarium was treated with a local application of a mixture comprising calcium phosphate and teriparatide. This approach proved significantly more effective in regenerating bone than using calcium phosphate alone [32]. Additionally, Auersvald et al. employed a collagen sponge soaked in teriparatide and covered with a bovine cortical membrane to treat rat calvaria defects. Histological analyses revealed a substantially higher percentage of bone reconstruction in these cases compared to the control group [33]. Despite the differences in the specifics of these studies, such as their design, settings, dosages, and administration methods, the consistency in the outcomes they present, alongside our own findings, underscores the potential of teriparatide as a bone-reconstructive agent, even when administered in a single, localized dose.

In our study, both clinical and histological evaluations indicated that the formation of necrotic foci was substantially mitigated through the administration of a single local dose of teriparatide, particularly via injection. This observation is in line with findings from Ersan et al. [34] who reported a significant decrease in necrotic bone percentage in rats treated with ZA and receiving daily teriparatide. Kuroshima et al. [3] observed similar results in rats with injuries to the jaw and tibia, and the study by Keskinruzgar revealed that systemic administration of teriparatide twice weekly also led to a reduction in necrotic areas [28]. Notably, in our study, the most pronounced anti-necrotic effects were seen with teriparatide injections directly adjacent to the extracted tooth socket. This outcome suggests the potential benefits of this protocol in future human trials particularly considering that a single, localized dose could minimize side effects and potentially lower treatment costs.

In our research, we evaluated the efficacy of combining teriparatide with Gelatamp, a gelatin sponge enhanced with colloidal silver, typically employed in the healing of tooth sockets [35]. The rationale behind this approach was that Gelatamp might facilitate a slower, more prolonged release of the active ingredient. However, our findings indicated that direct injection of teriparatide was superior to the Gelatamp method in preventing necrosis, enhancing new bone formation, and aiding bone remodeling. It was even observed that the necrosis rates in the injection control group were comparable to those in the teriparatide Gelatamp group, yet both were lower than in the Gelatamp control group. This observation might reflect the systemic protective properties that are activated by the localized injection of teriparatide. Nonetheless, the efficacy of teriparatide might vary with different delivery systems. For instance, Auersvald et al.’s [33] use of a gelatin sponge soaked in teriparatide showed improved bone formation. Additionally, Kajii et al. [32] found that a scaffold comprising octacalcium phosphate and collagen significantly enhanced bone regeneration in calvarial bone defects. These variations indicate that the delivery method, including the use of different scaffolds or potential teriparatide gel forms, could significantly impact the therapeutic outcomes of teriparatide.

While teriparatide has received FDA approval, its application in patients with malignant bone diseases presents significant challenges. The drug’s osteoanabolic action raises concerns about the potential stimulation of cellular proliferation in bone tissues, which could aggravate active malignant bone diseases or trigger a recurrence in previously treated patients [18]. Consequently, the duration of teriparatide therapy is generally capped at a cumulative period of 24 months to lower the risk of developing osteosarcoma [36]. The approach of employing a single local dose of teriparatide, as explored in our study, offers a promising alternative. This method could potentially provide an effective treatment option for patients at high risk, mitigating the concerns related to teriparatide’s possible carcinogenic effects.

This study encountered certain limitations. Primarily, due to the inherent risks associated with human research and the challenges in conducting histopathological examinations on humans, the study was firstly conducted using an animal model. While providing valuable insights, this model does not completely replicate human physiological responses. Additionally, although the split-mouth design offers the benefit of uniform conditions, it does not entirely eliminate the possibility that the local administration of the treatment could have systemic effects, potentially influencing the untreated side of the rat’s jaw.

In conclusion, this study on the effectiveness of local single-dose teriparatide administration in a rat model has demonstrated its significant potential in preventing medication-related osteonecrosis of the jaw (MRONJ). The findings reveal that teriparatide effectively enhances bone remodeling and osteoclast activity, countering the suppressive effects of bisphosphonates, and indicating its potential as a preventive agent for MRONJ. While the results are promising, particularly with local injection, the variation in outcomes with different delivery systems like Gelatamp highlights the need for further exploration. Additionally, the limitations inherent in an animal model study, underscore the necessity for more extensive human clinical trials. This research paves the way for future studies to fully understand the potential of teriparatide in MRONJ management, potentially leading to safer and more effective clinical applications.

Declarations

Conflict of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.

Ethical Approval

This study was conducted with the approval of the ethics committee of Isfahan University of Medical Sciences, under the code “IR.MUI.RESEARCH.REC.1400.151”. All procedures followed the established ethical guidelines for research on laboratory animals.