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30 May 2024: Animal Study  

Enhanced Bone Healing Through Systemic Capsaicin Administration: An Experimental Study on Wistar Rats

Muhammet Bahattin Bingül ORCID logo1ABCDEFG*, Mehmet Gül ORCID logo2ABCDEFG, Serkan Dundar ORCID logo3CDF, Abdulsamet Tanık ORCID logo4BCDF, Gokhan Artas5CD, Mehmet Emrah Polat ORCID logo1CDEF

DOI: 10.12659/MSM.942485

Med Sci Monit 2024; 30:e942485

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Abstract

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BACKGROUND: The healing of bone defects is a serious challenge worldwide. One branch of dentistry deals with bone defects. Capsaicin has anti-inflammatory, anti-oxidative, and cholesterol-reducing effects. The aim of this study was to evaluate the effects of systemic capsaicin administered at different doses on bone healing.

MATERIAL AND METHODS: A total of 32 male wistar rats was used, their weight varying between 250 and 300 g. The rats were randomly divided into 4 groups of 8 rats each. The analyses served to evaluate the effect on healing of different doses of capsaicin and grafts. A significant increase was observed in the number of osteoblasts in the capsaicin-applied groups, compared with the control group.

RESULTS: The analyses served to evaluate the effect on healing of different doses of capsaicin and grafts. A significant increase was observed in the number of osteoblasts in the capsaicin-applied groups, compared with that of the control group. The inflammation scores showed a significant difference only in the control group and in the group administered with 50 mg/kg capsaicin (P=0.010). The osteoclast counts were significantly different between all groups.

CONCLUSIONS: As a result of the analyses, positive effects on bone healing were observed when capsaicin 0.25 mg/kg and 0.50 mg/kg was administered intraperitoneally. However, more studies are needed for more accurate information.

Keywords: Capsaicin, alveolar bone grafting, Bone Matrix

Introduction

The healing of bone defects is a serious challenge worldwide. One branch of dentistry deals with bone defects, which can occur for pathological or physiological reasons. These defects can be simple or complex. In a complex bone defect, epithelial integrity is mostly impaired [1,2]. Bone defect healing involves complex mechanisms because it is related to the interaction between the immune and biological systems [2,3]. After bone damage has occurred, restoration progresses through different stages. These stages are (i) coagulation and homeostasis, (ii) inflammation, (iii) proliferation, and (iv) bone defect remodelling [2–4].

Capsaicin (trans-8-methyl-N-vanilyl-6-nonenamide) is the main active ingredient of hot pepper, which is found in 0.1% to 1% in green and red peppers. It is known that capsaicin has anti-inflammatory, anti-oxidative, and cholesterol-reducing effects [5]. Capsaicin has different effects on the cell viability mechanism in different cell types. Studies have reported that cell cycle arrests and apoptosis increase in cells exposed to capsaicin [6]. The number of hepatic stellate cells responsible for the scar tissue formed in liver damage decreased with capsaicin treatment [7]. Capsaicin has a minimum level of inhibition of bone healing when applied at certain doses, and it has been suggested that it does this by activating transient receptor potential vanilloid 1(TRPV-1), tumor necrosis factor-alpha, and interleukin-6 [2,8,9].

Sympathetic and sensory nerves play an important role in bone metabolism. Recent studies have reported that the sympathetic nervous system increases bone resorption and decreases bone formation. In sensory nerve regulation, a physiological interaction between sympathetic and sensory nerves and osteoclastogenesis, based on the effects of the sensory neuropeptide calcitonin gene-related peptide (CGRP), takes place [10,11]. Capsaicin inhibits apoptosis by stimulating TRPV-1 activity in respiration and increases the proliferation of muscle cells. Increased proliferation has been reported in the gingival epithelium treated with capsaicin [12,13].

In one study, capsaicin was administered to newborn rats. When the rats became adults, tooth extraction was performed, and a 21% decrease in alveolar bone resorption was observed in the capsaicin-treated group, compared with the capsaicin-free group [14]. In another rat study, capsaicin was administered, and as a result, a 40% decrease in bone resorption was detected in adult rats. In the analysis, a decrease in the resorption surface and the number of osteoclasts was determined [15]. Contrary to these studies, opposite situations were found in some studies. For example, Offley et al reported that capsaicin decreased tarbecular integrity and increased osteoclase count in bone metabolism [16]. The reported reason for the differences is that experiments were conducted on different bones in rats, and different doses were used [17].

Bone fragments required for bone augmentation can be supplied intraorally or extraorally if autogenous use is desired [18]. The guided bone regeneration technique has emerged in response to the inability to obtain sufficient bone from the intraoral region and high risks of obtaining bone in the extraoral region. What is important in this technique is the cell aggregation potential. In addition, the suitability of the biomaterial and the selection of the correct surgical technique are important [19–21]. The aim of this study was to evaluate the effects of systemic capsaicin administered at different doses on bone healing.

Material and Methods

SURGICAL PROCEDURE:

Before the calvarial defects were opened, an intramuscular anesthetic was administered to the rats. This consisted of 90 mg/kg ketamine hydrochloride (Ketalar, Pfizer) and 10 mg/kg xylazine (Rompun-Bayer). Afterward, asepsis was achieved with povidone iodine (Batticon-Adeka, Turkey) in the operation area. The operation area was covered with sterile drapes, and local anaesthesia was administered to the area, namely 0.5 cc of 4% articaine (Ultracain DS-Aventis, Istanbul, Turkey) containing 0.006 mg/mL epinephrine. A full-thickness flap was opened with a no. 15 scalpel. The defect was created using a 7-mm trafen bur, without damaging the brain. No material was placed in the defect in the control group. In the other groups, the defects were filled with synthetic grafts. For infection control, 50 mg/kg cefazolin sodium was administered for 3 days after the operation, and 1 mg/kg of tramadol hydrochloride was administered for pain control for 3 days after the operation. The operation area was sutured using a 3.0 silk suture.

During the experiment, 4 rats in total, 1 from each group, died. The surviving rats were observed on day 28, at the end of which they were killed by means of a lethal dose of intraperitoneal ketamine hydrochloride (60 mg/kg). A full-thickness flap was removed in the area, using a size 15 scalpel. Then, using a piezo device (NSK, Tokyo, Japan), samples were taken from the region in healthy tissues, without damaging the bone tissues. Likewise, the bone samples were disconnected from the brain under the guidance of physiological saline, without creating heat in the tissues.

Samples obtained after death were preserved in formaldehyde and referred to pathology for histological analysis.

HISTOLOGICAL PROCEDURE:

Samples kept in 10% neutral formalin were softened in EDTA solution for decalcification. The softened samples were dehydrated, cleared with xylitol, and embedded in paraffin blocks. Sections of 5 to 6 μm were obtained with a microtome. After staining with hematoxylin-eosin, bone healing was examined under a light microscope. The stained histological preparations were left for drying overnight, after which the sample surfaces were covered with a coverslip of methyl methacrylate. Digital images of all samples were taken at 4× magnification with a digital camera (Olympus DP 70, Tokyo, Japan) connected to a light microscope (Olympus BX50). Histological bone healing was assessed using the Image J Analysis Program (Image J version 1.44; National Institutes of Health, Bethesda, MD, USA).

SEMI-QUANTITATIVE SCORING OF HISTOPATHOLOGIC PARAMETERS:

A semi-quantitative scoring was determined by examining the cells in the bone tissue, such as osteoblasts, osteocytes, and osteoclasts. In the evaluation of histological sections, 15 different areas were scanned for each slide, and then the average value of 10 randomly selected cells was determined. From these averages, 10 points were obtained for each animal group, and then these values were analyzed statistically. The method we used has also been used in previous histochemical studies of bone tissue [23,24].

Inflammation was categorized as follows: 0=13–15 inflammatory cells per histological field; 1=10–13 inflammatory cells per histological field; 2=7–10 inflammatory cells per histological field; 3=4–7 inflammatory cells per histological field; and 4=1–4 inflammatory cells per histological field [25].

STATISTICAL ANALYSIS:

Histological analysis of the rats in our study was performed with the SPSS (IBM Ver 15.0 Windows, USA) program. Arithmetic mean values and standard deviation (SD) values of histological analyzes (osteoblasts, osteocytes, inflammation, and osteoclasts) were used. Normality tests of histological data were determined using the Kolmogorov-Smirnov test. Since the Kolmogorov-Smirnov test had a P value of <0.05, it was determined that our study did not show a normal distribution. Comparisons of histological data between more than 2 experimental groups (group I, group II, group III, and group IV) used the Kruskal-Wallis H test. The Mann-Whitney U test was used to compare histological data between paired groups, with the result of the Kruskal-Wallis H test being significant (P<0.05). In all statistical tests, P value <0.05 was considered significant.

Results

Histological analysis evaluated osteoblast numbers, and, while a value of 1.57±0.535 was found in the control group, a value of 2.14±0.69 was found in the graft group, and a value of 2.57±0.53 was found in the capsaicin 25 mg/kg group. Statistically significant differences were found between the control group and the groups administered a graft and 25 mg/kg. When looking at osteocyte numbers, the highest value was found in the capsaicin 50 mg/kg group (3.43±0.53), while the lowest value was found in the control group (1.86±0.69), and statistically significant values were found between all groups (P=0.003). In addition, inflammation was evaluated and a significant difference was found only in the control group (3.00±0.58) and capsaicin 50 mg/kg (2.00±0.58) group (P=0.010) (Table 1). The osteoclast counts revealed a significant difference between all groups (P=0.005) (Figures 1, 2).

Discussion

In the present study, vocacapsaicin, the active ingredient of which is capsaicin, was tested on rats. Bone healing was evaluated, and no adverse events were found. An increase in bone healing was observed in rats given moderate levels of the active ingredient. Studies have been carried out on rabbits, which found no adverse effects on bone healing. Vocacapsaicin was applied before the operation in unilateral osteoma models. No adverse situation was found in its use as a local and systemic non-opioid agent in reducing pain [26]. In our study, different doses of systemic capsaicin were administered to rats with a head defect, and a significant increase in bone healing was observed.

It has been reported that after the capsaicin active ingredient in vocacapsaicin is secreted, it affects TRPV-1 receptors in conjunction with C fibers [27–29]. In one study, rats were evaluated radiographically after the administration of vocacapsaicin at a medium dose of 0.3 mg/kg. An increase in bone healing was observed, compared with the control group. In studies, TRPV-1 has been reported to be actively involved in bone healing for rats and rabbits. In one study, a unilateral femur fracture model was created, and a delay in bone healing was observed in the TRPV-1-deficient group. In addition, a negative situation was observed in the balance of osteoblasts and osteoclasts [30]. A similar study was conducted on wild rats, which found an increase in TRPV-1 immunofluorescence [31]. Other studies administered TRPV-1 antagonist and reported that osteoclast activity was inhibited [32,33]. The effect of the use of vocacapsaicin on bone healing has not been fully clarified. However, it is currently believed that the TRPV-1 receptor family is involved in the bone healing mechanism [34].

In our study, systemic capsaicin was administered at doses of 0.25 mg/kg and 0.50 mg/kg. The results were evaluated histologically. An increase in osteoblast activity was observed. We think that this is due to the effect of capsaicin on TRPV-1 receptors.

It has been reported that capsaicin causes an increase in CGRP release from cardiac C-fibre nerve endings in the heart [35]. In particular, use of calcitonin receptor-like receptor/receptor activity-modifying protein 1 receptors (CLR/RAMP1) not only resulted in vascular relaxation from CGRP signaling, but it also reportedly modulated inflammation by regulating proinflammatory cytokine production in dendritic cells [36]. Histological evaluations revealed a significant decrease in inflammation in the capsaicin-applied groups. We think that the effect of capsaicin on CGRP release regulates proinflammatory cytokine production in dendritic cells.

Bone marrow mesenchymal stem cells (BMSCs) play a role in the mechanism of bone formation and destruction and in maintaining this balance [37,38]. The differentiation of stem cells into osteoblasts is known as osteogenic differentiation [39]. Osteoporosis occurs as a result of decreased proliferative ability and abnormal differentiation in bone mesenchymal stem cells. Sirtuin 6 (SIRT-6), a member of the sirtuin deacetylase family, plays a role in osteogenic differentiation [40,41]. Overexpression of SIRT-6 in human BMSCs disrupts the TRPV-1 mechanism, resulting in inhibition of the release of CGRP. Subsequently, osteogenic differentiation is inhibited. As a result of the capsaicin treatment, TRPV-1 is activated, the expression of CGRP increases, and the genes involved in osteogenic differentiation, namely osteocalcin, bone sialoprotein, and osteopontin, increase [42]. The rise in alkaline phosphatase activity indicates that capsaicin supports osteogenic activity [43].

Similarly, in our study, a significant increase in the amount of osteocytes was observed in the capsaicin-applied groups. As emphasized ın previous studies, we think that this was due to the positive effect of capsaicin on the osteogenic activity occurring between TRPV-1, CGRP, and SIRT-6. Capsaicin can be consumed with daily foods. This consumption is excessıve in some societies. Therefore, the positive or negative effects of the effect of capsaicin consumption rate on bone methodology has been evaluated. In the present study, positive effects were observed with the doses we used, and we determined the effects of capsaicin on various factors. Due to the positive effects of capsaicin on these factors, we determined their positive effects on bone metabolism in the doses used.

Conclusions

Some of the foods we frequently use in our daily lives contain capsaicin (hot peppers and their derivatives), which is the reason we used capsaicin in the present study. In our study, we evaluated previous studies and determined that the dose to be used to affect bone healing was very important. Therefore, we used 2 different doses. The effects of the doses used on metabolism were investigated, and the effects on bone metabolism were evaluated. In our study, the systemic effect of capsaicin in rats with a head defect was evaluated. As a result of the analyses, positive effects on bone healing were observed when capsaicin 0.25 mg/kg and 0.50 mg/kg was administered intraperitoneally. Since capsaicin is found in foods and is an herbal product, we think that it can be used clinically after adjusting the doses. There are various limitations in our study, especially the number of subjects, which was limited to avoid killing many animals. Additional studies are needed to obtain more accurate information.

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