Effects of azelnidipine, a calcium channel blocker, in a rat femur fracture model

Abstract

Background

Fractures are an important public health problem as they reduce quality of life due to the occurrence of pain and complications. Osteoblasts originate from mesenchymal stem cells and play a vital role in bone formation. They also contain voltage-gated calcium channels. Azelnidipine is a dihydropyridine-derivative blocker of L- and T-type calcium channels. The signalling process involving the L-type calcium channel is important for the function of osteoblasts. Nevertheless, the impact of dihydropyridine chemicals on bone metabolism remains largely unexplored. To the best of our knowledge, no study has yet evaluated the specific effects of azelnidipine on bone healing. The aim of our study was to examine, for the first time, the impact of azelnidipine on the pathophysiology of bone fractures by radiographic and biochemical techniques.

Methods

The study included 40 female rats in 5 groups (n = 8 in each group): a healthy sham group, femoral fracture control group, femoral fracture + 4 mg/kg azelnidipine group, femoral fracture + 8 mg/kg azelnidipine group, and femoral fracture + 16 mg/kg azelnidipine group.

Results

Serum tumour necrosis factor-α, interleukin-1β, interleukin-17, osteocalcin, osteopontin, total oxidant capacity, and bone-specific alkaline phosphatase levels significantly increased in the fracture control group compared to the sham group (p < 0.05). Azelnidipine treatment significantly reversed these elevations, particularly at the 16 mg/kg dose, restoring TNF-α, IL-1β, IL-17 levels back toward baseline (p < 0.05). Total antioxidant capacity measured in serum decreased in the fracture group 6.47 ± 1.46 U/mL, whereas azelnidipine significantly increased this parameter to 10.01 ± 1.85 U/mL in a dose-dependent manner (p < 0.05). In radiological findings, azelnidipine contributed positively to fracture healing.

Conclusion

These results showed that azelnidipine, a calcium channel blocker, exhibits a potential healing effect in this rat femur fracture model, providing the first preclinical evidence of its potential therapeutic utility in bone recovery.

Introduction

Fracture is a condition in which the anatomical integrity of the bone structure is partially or completely disrupted and the soft tissues around the fracture are also affected [1]. After a fracture occurs, attempts are made to restore bone integrity by various physiological methods [2]. Fracture healing is a three-stage process defined by the phases of inflammation, repair, and remodelling [3]. Shortly after the fracture, the inflammation phase begins and persists for approximately one week. It is characterised by the injured area becoming inflamed and granulation tissue forming. The repair phase begins immediately after the inflammation phase and is associated with the formation of the fracture callus. Subsequently, the process culminates in the remodelling phase, which is characterized by the gradual replacement of woven bone with lamellar bone. In rat models, while hard callus formation peaks around the fourth week, this active remodelling phase extensively continues for at least two months post-fracture [4].

Many different mechanisms work together during healing because bone tissue is a tissue comprising many different components. The basic cells that manage these different mechanisms are osteoblasts. Osteoblasts have a variety of tasks. Osteoblastic activity contributes to the storage of inorganic constituents in bone. Osteoblasts also increase local phosphorus ion concentrations by secreting membrane-bound matrix vesicles, which are especially rich in alkaline phosphatase (ALP) and other enzymes. Hydroxyapatite crystals [Ca₁₀(PO₄)₆(OH)₂] are formed by the combination of calcium and phosphorus ions. These molecules are accepted as constituting the first visible stage of calcification. They grow rapidly as a result of mineral deposition and turn into a calcified mass containing collagen fibres and proteoglycans [5]. While performing these tasks, it is necessary to interact with many different cells and endogenous substances. Therefore, osteoblasts have many different receptors and ion channels, each of which has different functions. In recent studies, it has been determined that different calcium channels are also present in osteoblasts. The initial identification of voltage-gated calcium channels in bone tissue occurred in primary murine osteoblasts. These channels are essential for skeletal development, the regulation of bone remodeling, and the conversion of mechanical inputs into biochemical signals [6]. Crucially, these calcium-mediated signaling cascades do not operate in isolation; they are deeply integrated into the broader molecular networks that dictate how biological systems adapt to physiological and desynchronization stress [7]. Furthermore, such adaptive cellular responses and downstream bone remodeling processes are fundamentally driven by metabolic flexibility, which is heavily regulated by epigenetic mechanisms including acetylation and sirtuin pathways during cellular challenges [8].

Voltage-gated calcium channels are protein complexes that control various metabolic events by converting electrical potential into intracellular signals. Intracellular calcium (Ca2+), functioning as a second messenger, influences physiological processes including muscular contraction, transcriptional control, neurotransmission, and hormone production [9]. Voltage-sensitive calcium channels belong to a superfamily of transmembrane cation channel complexes, which also encompasses potassium and voltage-gated sodium channels [10]. They are categorized into three types: L-type calcium channels, N/P/Q/R-type calcium channels, and T-type channels [11]. While L-type and T-type calcium channels are expressed across a variety of tissues, N-type, P/Q-type, and R-type channels are predominantly localized in neuronal cells. L-type calcium channels are distinguished by their elevated voltage activation thresholds and significant single-channel conductance, making them the most prevalent subtype of calcium channels. Because of these properties, L-type calcium channels remain open longer. They are called ‘L’ type due to their ‘long-lasting’ properties [12].

Regulatory factors involved in osteogenesis influence the expression of L-type calcium channels, suggesting a functional contribution of these channels to the process of bone remodelling [13]. Voltage-gated calcium channels are proposed to regulate osteogenic differentiation in human bone marrow-derived mesenchymal stem cells, indicating a functional involvement in this process. Thus, they may be a target for the pharmacological modulation of this process [14]. Current knowledge suggests that calcium channels play an important role in the regulation of osteoblast function and bone metabolism.

Differentiation of osteoblasts into osteocytes is marked by reduced L-type channel expression, permitting T-type calcium channels to mediate essential cellular functions. Throughout murine skeletal development, osteoblasts predominantly express T-type and L-type calcium channels at loci of active bone formation during the osteogenic process [15]. Although the majority of studies have concentrated on the functional roles of L-type voltage-gated calcium channels in osteoblasts, recent studies have shown that T-type voltage-gated calcium channels are essential for the functional regulation of osteocytes [16]. Since azelnidipine, the compound investigated in the present study, is an L- and T-type calcium channel blocker, T-type calcium channels are important in its administration.

Amlodipine, lacidipine, and nifedipine are dihydropyridine-derivative calcium channel blockers with high affinity for L-type calcium channels [17]. Studies have shown that amlodipine increases phosphate and calcium amounts in the femurs of ovariectomised rats [18] and preserves bone density in rats [19]. Evidence also indicates that amlodipine aids fracture healing by promoting bone growth and improving callus remodeling [20]. Amlodipine and nifedipine exhibit weak inhibitory effects on calcium channels in osteoblasts; however, at elevated concentrations, both amlodipine and lacidipine partially enhance alkaline phosphatase (ALP) activity [21].

It blocks L-type calcium channels as well as T-type calcium channels in both afferent and efferent arterioles [22]. Azelnidipine exhibits greater lipid solubility compared to earlier-generation calcium channel blockers such as amlodipine and it exhibits prolonged retention within the vascular wall [23]. Azelnidipine has anti-inflammatory activity [24]. Antioxidant benefits of this compound have been documented in recent clinical studies [25]. Given that L-type and T-type calcium channels regulate intracellular calcium homeostasis in osteoblasts and osteocytes, modulating these channels with a dual blocker like azelnidipine may alleviate fracture-induced oxidative stress and inflammation, thereby promoting a more favorable microenvironment for bone regeneration.

Methods and materials

Animals

This research involved 40 female albino Wistar rats acquired from the Medical Experimental Research Centre of Atatürk University (ATADEM). Prior to the experiments, the animals, weighing between 200 and 250 g, were housed in separate groups and maintained under standard environmental conditions at a temperature of 22 °C. The study’s animal care and experimental protocols were approved by the Experimental Animal Ethics Committee of Atatürk University (Erzurum, Turkey; 20702.2020/26). The rats were accommodated in conventional plastic enclosures with sawdust substrate. The cages were situated in a climate-controlled room maintained at 22 °C, with regulated lighting settings (14-hour light and 10-hour dark cycles). Animals were provided with regular rat chow and tap water ad libitum.

Chemicals

Azelnidipine was acquired from Acros, and thiopental sodium was obtained from İE Ulagay A.Ş. (Istanbul, Turkey). Metamizole sodium (Novalgin, 1 g/2 mL IM/IV ampoule) was purchased from Sanofi-Aventis (Ankara, Turkey), xylazine from İntermed Ecza Deposu Ltd. Şti. (Ankara, Turkey), ketamine (Ketalar, 50 mg/mL vial) from Pfizer A.Ş. (Istanbul, Turkey), cefazolin sodium (İESPOR, 1 g IM vial) from İE Ulagay A.Ş. (Istanbul, Turkey), and paracetamol from Doğa İlaç Hammaddeleri Tic. Ltd. Şti. (Istanbul, Turkey).

Rat femur bone fracture model

All surgical procedures in this study were performed by a specialized orthopedic surgeon under strictly sterile conditions. In the following manner, the rats were allocated randomly to five different experimental groups, with eight animals in each of the groups:

• Group 1 (SHAM): Sham group.

• Group 2 (FRACTURE): Femoral fracture control group.

• Group 3 (FR + AZE4): Femoral fracture + 4 mg/kg azelnidipine (dissolved in % 0.5 carboximetilcelulose (CMC)).

• Group 4 (FR + AZE8): Femoral fracture + 8 mg/kg azelnidipine (dissolved in % 0.5 CMC).

• Group 5 (FR + AZE16): Femoral fracture + 16 mg/kg azelnidipine (dissolved in % 0.5 CMC).

The dose regimen of azelnidipine (4, 8, and 16 mg/kg/day administered via oral gavage for 21 days) was selected based on previous pharmacokinetic and safety profiles in rodent models. In literature investigating the systemic and tissue-specific effects of azelnidipine in rats, a dose range of 3 to 10 mg/kg/day is commonly utilized to achieve effective plasma concentrations and stable calcium channel-mediated physiological responses [26]. Building upon these pharmacokinetic baselines, we kurguladık low (4 mg/kg), medium (8 mg/kg), and high (16 mg/kg) doses to evaluate a potential dose-dependent therapeutic curve on bone healing. Regarding safety, azelnidipine exhibits a wide therapeutic index in rodents; preclinical toxicity studies have demonstrated that oral doses up to 20 mg/kg/day do not induce significant subacute or chronic systemic toxicity, adverse events, or mortality [27]. In the present study, the highest dose (16 mg/kg/day) remained well within the safe threshold, and no behavioral anomalies, abnormal weight loss, or signs of toxicity were observed in any animal throughout the 21-day treatment period.

Surgical procedures were performed under aseptic conditions by a board-certified orthopedic surgeon. Xylazine (5 mg/kg) and ketamine (50 mg/kg) were administered intraperitoneally to all of the rats in order to establish anaesthesia at the beginning of the experiment, followed by maintenance with sevoflurane at a constant concentration of 2% delivered via a nose cone mask throughout the surgical procedure. Postoperative analgesia was provided by intraperitoneal administration of metamizole sodium (150 mg/kg). Following the shaving of the right hind limb of each rat, a lateral parapatellar incision measuring 2 cm was carried out in preparation for the surgical procedure, followed by lateral displacement of the patella to expose the distal femoral condyle. A mid-diaphyseal femoral fracture was then created by transversely sectioning the femur. Following manual reduction of the fracture, stabilization was achieved using intramedullary Kirschner wires. The surgical site was irrigated, and closure was performed in layers: the muscle and subcutaneous tissues were closed using 4 − 0 Vicryl sutures, and 4 − 0 nylon sutures were used to seal the wound on the skin. A similar surgical procedure was performed on the animals that were part of the sham group, with the exception that no femoral osteotomy was performed. After the operation, the animals were given the opportunity to consume anything they pleased. The rats in the experimental groups were given three different dosages of azelnidipine, whereas the rats in the control and sham groups were given the same volume of motor vehicle. Members of the experimental groupings of animals received 4, 8 or 16 mg/kg of azelnidipine orally (by gavage) everyday for a period of 21 days, beginning on the first postoperative day. Twenty-one days after the procedures, all rats were deeply anesthetized with an intraperitoneal injection of 20 mg/kg sodium thiopental. After confirming complete unconsciousness and the absence of the pedal withdrawal reflex, large-volume terminal blood samples were collected via cardiac puncture. The animals were humanely euthanized by exsanguination under continued deep anesthesia. Immediately after blood collection X-ray imaging was performed.

X-ray imaging

X-ray imaging performed after surgery was done in order to assess the level of fracture healing that had occurred in the femur. Fracture healing stages were assessed using the scoring system described by Lane and Sandhu [28].

Biochemical studies

Measurements of serum osteocalcin (OC), osteopontin (OPN), tumour necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-17 (IL-17), bone-specific alkaline phosphatase (BSALP), total oxidant capacity (TOS) and total antioxidant capacity (TAS) levels were conducted for each sample in duplicate assays with highly sensitive ELISA kits specifically designed for rats (BT-LAB Cat No: E0270Ra; BT-LAB Cat No: E 0273Ra; BT-LAB Cat No: E0764Ra; BT-LAB Cat No: E0119Ra; BT-LAB Cat No: E0115Ra; mybiosource Cat No: MBS265845, BT-LAB Cat No: E1512Ra; BT-LAB Cat No : E1710Ra ) in accordance with the instructions provided by the manufacturer.

Statistical analyses

The experimental observations are presented as the mean ± standard deviation (SD), with p-values below 0.05 considered statistically significant. After using the post hoc Duncan test, a one-way analysis of variance (ANOVA) was employed to determine the significance of the differences among the groups.

Results

X-ray imaging

The radiological images of the resected femurs, obtained in a single anteroposterior plane using a mammography device (SENOGRAPHIE DMR, General Electric, Boston, MA, USA) were evaluated based on the Lane and Sandhu scoring system; this analysis revealed a significant dose-dependent improvement across the groups, with the mean radiological score increasing from 1.13 in the FR group to 3.25 in the FR+AZE16 group (Fig. 1).

Fig. 1.

Radiological images: (A) SHAM, (B) FR, (C) FR + AZE4, (D) FR + AZE8, (E) FR + AZE16

Biochemical results

In biochemical studies, serum OC, OPN, TAS, TOS, TNF-α, IL-17 and IL-1β amounts were analysed. When comparing the fracture group to the healthy sham group, it was discovered that the concentrations of TNF-α, IL-17, and IL-1β were significantly elevated in these individuals. In the fracture groups treated with azelnidipine, these parameters decreased statistically significantly in a dose-dependent manner (Table 1; Fig. 2).

Table 1.

Serum levels of TNF-α, IL-17 and IL-1β (different letters in columns denote statistically significant differences)

	TNF-α (ng/mL)				IL-1β (pg/mL)				IL-17 (pg/mL)
SHAM	31.98	±	3.34	a	50.03	±	5.75	a	115.76	±	33.61	a
FR	52.42	±	7.60	c	84.98	±	9.84	c	230.63	±	21.62	c
FR + AZE4	44.29	±	5.46	b, c	71.17	±	7.15	b	178.15	±	31.13	b
FR + AZE8	39.13	±	9.17	a, b	65.43	±	8.91	b	168.75	±	31.52	b
FR + AZE16	40.10	±	5.18	a, b	58.19	±	8.35	a, b	138.45	±	20.45	a, b

Groups with different letters (a, b, c) within the same row are statistically significantly different (p < 0.05)

Fig. 2.

Levels of, IL-1β, IL-17 and TNF-α measured in rat serum (different letters on bars denote statistically significant differences)

Serum OPN and OC levels increased statistically in the fracture groups reletive to the healthy sham group. In the fracture groups for which azelnidipine was administered, these parameters decreased statistically significantly in a dose-dependent manner (Table 2; Fig. 3).

Table 2.

Serum levels of OPN and OC (different letters in columns denote statistically significant differences)

	OPN (ng/mL)				OC (ng/mL)
SHAM	10.08	±	2.25	a	21.15	±	1.92	a
FR	29.04	±	3.54	d	42.78	±	4.89	d
FR + AZE4	22.39	±	3.84	c	34.64	±	2.99	c
FR + AZE8	16.99	±	3.39	b	30.13	±	3.17	b
FR + AZE16	13.43	±	3.13	a	29.14	±	4.53	b

Groups with different letters (a, b, c, d) within the same row are statistically significantly different (p < 0.05)

Fig. 3.

OPN and OC levels measured in rat serum (different letters on bars denote statistically significant differences)

Among the final parameters to be examined, TAS and TOS levels reflect oxidative and antioxidant balance. TOS levels signify oxidative stress and thus cell damage, and TOS levels were observed to be higher in the fracture groups as relative to the sham group but decreased statistically significantly in parallel with healing in the groups for which azelnidipine was administered after fracture. TAS levels signify antioxidant status, and antioxidant levels in the fracture groups were found to have significantly decreased but that deterioration statistically significantly improved in the groups treated with azelnidipine after fracture. Finally, we analysed serum bone-specific ALP (BSALP) as an important biochemical marker. BSALP was significantly increased in the femoral fracture groups but this deterioration was statistically significantly improved in the groups treated with azelnidipine after fracture (Table 3; Fig. 4).

Table 3.

Serum levels of TOS, TAS, and BSALP (different letters in columns denote statistically significant differences)

	TOS (U/mL)				TAS (U/mL)				Bone-specific ALP (U/mL)
SHAM	5.79	±	0.88	a	11.29	±	2.01	b	14.68	±	2.32	a
FR	8.50	±	1.38	b	6.47	±	1.46	a	24.90	±	2.28	d
FR + AZE4	5.48	±	0.66	a	9.41	±	1.48	b	20.12	±	0.65	c
FR + AZE8	5.56	±	0.79	a	9.65	±	1.22	b	18.10	±	1.88	b, c
FR + AZE16	5.41	±	1.24	a	10.01	±	1.85	b	16.83	±	2.19	a, b

Groups with different letters (a, b, c, d) within the same row are statistically significantly different (p < 0.05)

Fig. 4.

TOS, TAS and bone-specific ALP levels measured in rat serum (different letters on bars denote statistically significant differences)

Discussion

The effects of azelnidipine were investigated in this study, a new-generation blocker of both T-type and L-type calcium channels with very high lipid solubility, in a rat femur fracture model. In our study, azelnidipine was administered orally once daily for 21 days after fracture induction. To evaluate its therapeutic value, healing, radiographic fractures, serum cytokines (IL-1β, IL-17 and TNF-α), OC, OP, TAS, TOS, and BSALP values were measured. The comprehensive assessment of these specific biochemical and immunological markers is vital in rodent models, as systemic profiles and hematological/leukocyte parameters are highly sensitive to physiological disruptions and experimental stressors during development and tissue injury [29]. This study is the first investigation into the effects of azelnidipine on fracture healing, as no prior research has addressed this relationship.

Dihydropyridine-derived calcium channel blockers have been demonstrated to promote osteoblast differentiation and appear to exhibit greater efficacy in this respect compared to other classes of calcium channel blockers [30]. Another study showed that L-type calcium channel blockers inhibited bone resorption and consequently increased intracellular calcium concentration [17].

For healing to occur after fracture formation, mediators such as prostaglandins and bone-stimulating factors, which are produced locally at the fracture site or migrate to the fracture site via blood circulation, are necessary for osteoblasts, osteoclasts, and other cells with important roles in the fracture healing process to assemble and fulfil their duties [31]. During the initial stage of fracture healing, genes that play a role in the progression of the cell cycle and cell–cell interactions are upregulated [32]. These genes also play crucial roles in fibroblast proliferation, angiogenesis of capillary vessels, and the infiltration of inflammatory mononuclear cells during the healing process. A strong oxidant-antioxidant balance is needed during the realisation of these events. Therefore, the beneficial effects of antioxidant therapy on fracture healing have been explored in experimental and clinical studies [33]. Altındağ et al. reported that patients with osteoporosis exhibited significantly lower TAS, elevated TOS, and increased oxidative stress index values in comparison to healthy controls. Their findings suggest that the imbalance between oxidant and antioxidant levels may contribute to heightened osteoclastic activity and diminished osteoblastic function in postmenopausal osteoporosis [33]. In the aforementioned studies, TAS levels were found to be decreased in groups in which experimental fracture models were created, whereas TAS levels increased according to the fracture healing status in line with the drug treatments applied. In addition, while TOS levels increased significantly, TOS amounts decreased according to the fracture healing status in line with the drug treatments applied.

In the present study, we showed that azelnidipine administration at increasing doses significantly increased TAS and decreased TOS in fracture cases. In light of our findings, we suggest that azelnidipine plays an active role in fracture healing. This may be explained by the antioxidant effects of azelnidipine. In previous studies, it was reported that azelnidipine decreased the levels of antioxidant reactive oxygen species [25]. Azelnidipine demonstrates significant anti-inflammatory activity by inhibiting TNF, interleukins, and other pro-inflammatory cytokines. Consequently, it has been shown to exhibit considerable anti-inflammatory and antioxidant properties [24]. Another research confirmed the potent antioxidant effects of azelnidipine [25]. To understand the mechanisms driving these effects, we need to examine the possible role of calcium and its channels in oxidative stress and inflammation.

Azelnidipine, a calcium channel blocker, exhibits anti-inflammatory and antioxidant properties, highlighting the significance of calcium ions in the context of inflammation. Calcium ions are essential for the synthesis and release of pro-inflammatory mediators [34]. Experimental studies have shown that pain and inflammation decrease with the decrease of calcium ions in tissues and increase with the increase of calcium ion in tissues [35]. Suleyman et al. [36] investigated the anti-inflammatory effects of the calcium channel blockers nicardipine, amlodipine and lacidipine were investigated in a carrageenan-induced acute inflammation model in rats, demonstrating that these agents significantly attenuated and prevented inflammatory responses. A study conducted by Dursun et al. [37], the protective properties of calcium channel blockers lacidipine and amlodipine against hypertension and the effects of ovariectomy in rat hepatocytes were investigated and it was proved that both drugs were protective owing to their capacity to modulate oxidative stress and inflammation.

This study has demonstrated that azelnidipine, a blocker of T-type and L-type calcium channels with high lipid solubility, may be a critical factor in the treatment of bone fracture through anti-inflammatory and antioxidant mechanisms. (Fig. 5). The positive effects of azelnidipine on the healing of bone fracture were observed both biochemically and radiologically. This study was not designed to establish a direct mechanistic link between fracture healing and L-type calcium channel blockade. Therefore, the observed findings indicate a strong therapeutic association but do not definitively confirm direct causality. Further studies using pharmacological reversal approaches, gene silencing techniques, or gene knockout models are needed to elucidate the precise molecular mechanisms underlying azelnidipine’s effects on fracture healing. Additionally, since this preliminary investigation was conducted exclusively on female rats to eliminate inter-sex hormonal variance, subsequent research encompassing both male and female cohorts is warranted to evaluate potential sex-specific therapeutic outcomes and to fully clarify how estrogenic or androgenic factors interact with calcium channel modulation during bone regeneration.

Fig. 5.

Mechanism of action of azelnidipine

Authors’ contributions

Conception: HK, AA, AA, YB. Design: HK, AA, AA, YB. Supervision: HK, AA. Materials: HK, AA. Data Collection and/or Processing: HK, AA, AA, YB. Analysis and Interpretation: HK, AA, AA, YB. Literature: HK, AA. Review: HK, AA, AA, YB. Writing: HK, AA, ZH. Critical Review: HK, AA, YB, ZH. The authors confirm that no paper mill and artificial intelligence was used.

Funding

This study was supported by the Atatürk University Scientific Research Projects (BAP) Coordination Unit (TDK-2021-9366).

Data availability

All data supporting the results of the study are provided within the article itself and there is no need for additional source data.

Declarations

Ethics approval and consent to participate

The experiments adhered to ethical guidelines approved by the Ethics Committee of the Experimental Animal Teaching and Research Centre (E.1800039863). The rats used in this study were obtained from the Medicinal and Experimental Application and Research Centre in Erzurum, Turkey (ATADEM).

Consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.