Next Article in Journal
Concrete Gas Permeability: Implications for Hydrogen Storage Applications
Previous Article in Journal
A Technological Framework to Support Asthma Patient Adherence Using Pictograms
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Applied Sciences
Volume 14
Issue 15
10.3390/app14156411
applsci-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Evaluation of the Effects of Locally Applied Resveratrol and Cigarette Smoking on Bone Healing

by
Muhsin Fırat İskender
1,
Müge Çına
2,*,
Şevket Tolga Çamlı
3,
İbrahim Metin Çiriş
4 and
Ramazan Oğuz Yüceer
5
1
Private Practice, 07000 Antalya, Turkey
2
Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Süleyman Demirel University, 32260 Isparta, Turkey
3
Silika Chemistry Co., Ltd., 42050 Konya, Turkey
4
Department of Medical Pathology, Faculty of Medicine, Süleyman Demirel University, 32260 Isparta, Turkey
5
Department of Medical Pathology, Faculty of Medicine, Sivas Cumhuriyet University, 58140 Sivas, Turkey
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(15), 6411; https://doi.org/10.3390/app14156411
Submission received: 5 June 2024 / Revised: 17 July 2024 / Accepted: 20 July 2024 / Published: 23 July 2024
(This article belongs to the Section Applied Dentistry and Oral Sciences)
Browse Figures
Versions Notes

Abstract

:
Background: Bone healing is a complex process controlled by various mechanisms. It is known that cigarette smoking (CS) negatively affects bone healing by disrupting many of these mechanisms. In an effort to find ways to eliminate these negative effects caused by CS, studies have been conducted on various vitamins, antioxidants, and medications. Since high doses and repeated injections are required to increase the therapeutic effect of conventional drug applications, controlled drug delivery systems have been developed to avoid such problems. This study aimed to investigate the effects of resveratrol (RES), which has been made into a controlled drug delivery system, on bone healing in rats that were experimentally exposed to cigarette smoke to create a chronic smoking model. Methods: After establishing a chronic CS model by exposing the subjects to cigarette smoke of six cigarettes/day for four weeks, monocortical critical size defects of 3 mm (SD ± 0.02 mm) in diameter were created in the femur using a trephine bur. During the operation, the defects in RES groups were filled locally with a gel-formed solution of RES (50 µM) and Pluronic F-127 (14 µL). CS exposure was continued during the bone healing period after surgery. All groups were sacrificed one month after the operation, and femur samples were taken. Results: The obtained samples were examined by histomorphometric and immunohistochemical techniques; osteoblast count, new bone area, macroscopic filling score, vascularization, and proliferation were evaluated. Conclusion: The results of this study indicate that CS negatively affects bone healing and that local application of RES reduces this effect.

1. Introduction

Osseous healing is a multifaceted process influenced by both local and systemic factors. The success of oral surgical procedures is compromised by delays in bone healing. Studies have reported that cigarette smoking delays bone healing and, thus, disrupts surgical planning and success. In fact, it has been found to adversely affect osteogenesis by causing changes in testosterone hormones, calciotropic hormone metabolism, adrenal cortical hormone metabolism, the receptor activator of nuclear factor (NF)-kB-ligand (RANKL), the receptor activator of NF-kB (RANK), the soluble decoy receptor osteoprotegerin (OPG) system, collagen metabolism, and bone angiogenesis [1,2,3,4].
Cigarette smoke (CS) is an aerosol with about 1010 particles per ml of carbonaceous polymeric material containing heavy metals, polycyclic aromatic hydrocarbons, N-nitrosamines, and various other organic chemicals. Tobacco smoke contains approximately 3500 chemical compounds, many of which are toxic, carcinogenic, or mutagenic (e.g., benzene, cadmium, benzopyrene, etc.) [5]. Thus, it can cause high levels of free radical formation, which can then increase bone resorption and reduce bone mass [5], potentially leading to osteoporosis.
Many studies have been conducted on various vitamins, hormones, and antioxidant substances, such as vitamins C, E, and D, to reduce the effects of CS on bone turnover [6,7,8].
Studies have shown that resveratrol (RES), an antioxidant substance, inhibits osteoclast formation while increasing osteoblast differentiation [9,10]. RES (trans-3,4,5-trihydroxystilbene), a natural polyphenol, is found in the seeds of red grapes. It has been found to have osteogenic, antioxidant, anti-inflammatory, analgesic, and anti-aging effects and has also been shown to have regenerative potential [11,12,13,14]. Studies in the literature have reported that RES inhibits osteoclastogenesis and bone resorption, increases osteoblastogenesis, and stimulates vitamin D receptor expression in osteoblast precursor cells in bone marrow [15,16,17]. Due to its anti-inflammatory properties, it has been reported that RES prevents bone resorption due to increased osteoclast activity as a result of the release of inflammatory mediators and that it increases bone mineral density, ossification amount, bone morphogenetic proteins (BMPs), and osteopontin levels [18,19,20,21]. Song et al. and Dai et al. reported that RES increased osteoblast formation by directly affecting the NO/cGMP signaling pathway of estrogen receptors. It was also found to increase osteoblastic differentiation and inhibit RANKL-induced osteoclast differentiation and bone resorption [17,22].
In conventional drug applications, it is necessary to use high and repeated doses to increase the therapeutic effects. To eliminate these problems and negative effects in conventional drug applications, serious studies have been carried out in recent years on the use of controlled drug delivery systems. In these controlled delivery systems, the drug reaches the cells and tissues in the required doses over a long period [23]. Another advantage is that it can be safely and naturally eliminated from the body, so there is no need to remove the drug carrier system after drug release [24,25]. Pluronic F-127 was chosen as a release matrix because of its unique properties as well as its ease of application to drug–polymer complexes in vivo [26,27].
The present study aimed to investigate the effects of CS and locally applied RES on bone healing histomorphometrically and to evaluate the effects of RES applied locally with a controlled delivery system on the negative effects of CS.

2. Materials and Methods

2.1. Study Design

This study was a longitudinal, in vivo animal study that used a CS model via a sample of 44 male 6- to 8-week-old Wistar rats that each weighed 250–300 g. The rats were divided randomly into four groups. The histomorphometric measurements for the number of osteoblast values were considered for power analysis in the reference study [28]. The sample size was calculated as 9 rats in each group for 5% error, 95% power, and a 0.754 effect size; but as the mortality effect was projected as 10%, 11 rats were finally included in each group. All of the rats were kept in a room with a 12:12 h light:dark-cycle temperature of 22 °C and humidity control at 40–60% and were given a standard pellet diet and tap water supplies ad libitum. In the first four weeks of the study, the CS-exposed groups (i.e., the CS + RES and CS groups) were exposed to CS (Tekel 2000; İstanbul, Turkey) that contained tar (10 mg), nicotine (1 mg), and carbon monoxide (10 mg) in 75 × 75 × 50 cm glass cages, as Kolkesen Şahin et al. described [4]. To ensure that the non-CS rats experienced the same level of stress, they were exposed to room air in glass cages. The CS was produced in a glass smoke generator—a 25 × 15 × 15 cm reservoir with a 2 cm2 opening at the top and an open bottom fitted to a cap that covered the chamber—by burning one cigarette every 10 min in the open-base chamber, albeit in a filter to prevent the spilling of the ash into the cage. After each cigarette was burned, the glass cage was ventilated for 5 min. Two sessions of this procedure were performed every day, and the rats were exposed to a total of six cigarettes.
This study was approved by the Ethics Committee of Suleyman Demirel University (approval date and number: 21 October 2021-01). The study and details relating to the reporting of animal experiments were carried out in accordance with the ARRIVE guidelines. Power analysis was carried out with the G Power 9.1.2 program (Universitaet Kiel, Germany).

2.2. Surgical Procedures

An intraperitoneal injection of 10 mg/kg xylazine HCl (Alfazyne; Ege-Vet, İzmir, Turkey) and 80 mg/kg ketamine hydrochloride (Alphamine; Ege-Vet, İzmir, Turkey) was used to induce general anesthesia. Under sterile conditions, an approximately 1 cm-long skin incision was created via blunt dissection. The periosteum was elevated, A monocortical defect of critical size (diameter: 3 ± 0.02 mm) was osteotomized from the femur with a trephine under irrigation with saline.

2.3. Preparation and Administration of RES

A fresh mixture of RES (Sigma-Aldrich, St. Louis, MO, USA) in water with Pluronic F-127 (Sigma-Aldrich, St. Louis, MO, USA) was prepared on the day of surgery. The RES concentration in this mixture was 50 µM. Following injection, the polymer structure turned into a gel format at 37 °C, which was body temperature. The encapsulated RES was homogeneously dispersed and released through the degradation of the Pluronic F-127. The defects in the RES and CS + RES groups were filled locally with RES (50 µM), Pluronic F-127 (14 µL), and a water solution in gel form. Defects were left blank in the control and CS groups.
The muscles dissected from the bone surface were sutured primarily with 3/0 catgut suture (Doğsan, Trabzon, Turkey), and the skin was sutured primarily using 3/0 silk suture (Doğsan, Trabzon, Turkey). Ketoprofen (3 mg/kg body weight daily, i.m.; Profenid® bulb; Sanofi-Aventis, İstanbul, Turkey) was administered for controlling pain, and oxytetracycline hydrochloride (Neo Spray CAF®; MSD Animal Health, Aprilia, Italy) was administered topically for infection control for five days after surgery. CS exposure continued for four weeks postoperatively. The rats were sacrificed at week eight. The right femurs were dissected and preserved in 10% formalin.

2.4. Histomorphometric and Immunohistochemical Evaluations

The samples were decalcified for two weeks using Osteosoft (Merck, Rahway, NJ, USA). The tissues were then routinely processed by an automatic tissue processor (Leica ASP300S; Leica Microsystems, Wetzlar, Germany) and embedded in paraffin. Selected samples were cut into 5 µm thick sections with a rotary microtome (Leica RM2125RTS; Leica Microsystems, Wetzlar, Germany). All sections were stained with hematoxylin and eosin (H&E). The samples were visualized and examined under a light microscope (Eclipse E400; Nikon, Tokyo, Japan).
Osteoblasts and osteoclasts were counted at 400× magnification. Osteoblastic activity was assessed by counting the cuboid osteoblast cells bordering the osteoid. Osteoclasts, identified as large multinucleated cells near the border of the resorption surface, were counted. For immunohistochemical examination, sections 4 μm thick were taken from paraffin blocks onto positively charged slides, with one section from each of two different tissues per slide. Tissue samples on lysine-coated slides were incubated at 60 °C for 8 h for deparaffinization. Staining was performed using the antibodies SATB2 (clone MD120R, 1/150 dilution; Medaysis, Reno, Nevada, USA), PSMA (clone 3E6, 1/200 dilution; Agilent, Santa Clara, CA, USA), and Ki-67 (clone MIB-1, 1/100 dilution; Agilent). The stains were applied using a fully automated Dako Omnis (Agilent) staining device with Agilent secondary kits. Bone tissue for SATB2, prostate tissue for PSMA, and tonsil tissue for Ki-67 were used as positive external controls.
Following a traditional microscopic examination, computer-assisted histomorphometric measurements were performed. All slide images were digitally evaluated by scanning SATB2-, PSMA-, and Ki-67-stained slides, which were applied as immunohistochemical tests under a Nikon Eclipse Ni-U light microscope with 10× and 20× objectives in the hotspot areas. This was carried out using the Microvisioneer manual whole slide scanner equipment and its associated Microvisioneer MANUALWSI 2020A-05 software. The slides of the new bone area were scanned using QuPath v 0.5.0 open-source digital pathology software, which allowed for annotations (drawings on the slide) and the creation of 3D digitalized graphic data on these digitized slides. Using the image analysis program, the total number and percentage of cells expressing SATB2, PSMA, and Ki-67 markers immunohistochemically were evaluated in normal and neoplastic bladder tissues.

2.5. Statistical Analyses

All data were analyzed with the IBM SPSS Statistics 22 software package (IBM, Armonk, NY, USA). The normality of the distribution was determined using the Shapiro–Wilk test. The Kruskal–Wallis test was used to compare more than two independent groups, and the Bonferroni test was used as a post hoc test to determine the source of the difference. The relationship between categorical variables was examined with the Fisher–Freeman–Halton Exact Test. In this study, p-values less than 0.05 were considered significant.

3. Results

3.1. Study Groups

One of the 11 rats in the Control group died one day before sacrification, and one femur each in the CS + RES and CS groups was fractured. Thus, this study was completed with a total of 41 rats—10 rats each in the Control, CS + RES, and CS groups, and 11 rats in the RES group.

3.2. New Bone Area

H&E-stained preparations were examined microscopically and scored according to the amount of new bone formation. The difference between the groups in new bone formation levels was found to be statistically significant (p < 0.05). The difference between the groups was examined by pairwise comparison tests with Bonferroni correction. It was determined that the level at which new bone formation was not observed (Score 0) was significantly higher in the control and CS groups (60%) than in the RES group (0%). Low (Score 1) bone formation was found to be significantly higher in the CS + RES group (50%) than in the RES group (0%). Although it was not statistically significant, it was observed that the level of new bone formation was highest in the RES group at medium, medium-high, and high levels. While new bone formation was not observed at medium, medium-high, and high levels in the CS group, new bone formation was observed at medium and medium-high levels in the CS + RES group. In the control group, medium-high and high levels of new bone formation were not observed, while in the RES group, medium-high and high levels of bone formation were observed (Figure 1, Table 1).

3.3. Vascularization

Preparations stained with PSMA were evaluated for vascularization and scored according to the degree of vascularization as follows: 0, no neovascularization was shown; 1, a slight amount of vascularization was shown; and 2, a moderate level of vascularization was shown. The difference between the groups in terms of vascularization levels was found to be statistically significant (p < 0.05). When the difference between the groups was examined by pairwise comparison tests with Bonferroni correction, it was determined that the level of no neovascularization (Score 0) was significantly lower in the RES group (36.4%) than in the control group (100%). Although it was not statistically significant, moderate vascularization was not seen in any group, but it was seen in the RES group (Figure 2, Table 2).

3.4. Macroscopic Bone-Filling Levels

The prepared preparations were evaluated macroscopically in terms of bone filling levels and scored as 0–25%, 25–50%, 50–75%, 75% and above. The difference between the groups in terms of macroscopic bone filling levels was found to be statistically significant (p < 0.05). The difference between the groups was evaluated by pairwise comparison tests with Bonferroni correction.
The macroscopic filling rate was observed at the 50–75% level in the CS + RES group. The macroscopic filling rate in the RES group was found to be statistically significantly higher than in the other groups at the 75% and above level. It was determined that macroscopic bone filling in the CS group was mostly seen at the 0–25% level. The group in which macroscopic bone filling was most observed was the RES group, while the group in which it was least observed was the CS group (Table 3).

3.5. Osteoblast Levels

The ratio of osteoblasts to other cells was evaluated in preparations stained with SATB2. A statistically significant difference was found between the groups (X2: 29.49; p ˂ 0.05). When the difference between the groups was examined using paired comparison tests with Bonferroni correction, it was determined that the values of the CS group were significantly lower than those of the RES and CS + RES groups, and the values of the CS group were significantly lower than those of the control group. It was observed that the group with the highest ratio of osteoblasts to other cells in the environment was the RES group, and the group with the lowest ratio was the CS group (Figure 2, Table 4).

3.6. Proliferation

Preparations stained with Ki67 were evaluated for proliferation and scored according to proliferation levels: 0, no proliferation; 1, 0–1% proliferation; 2, 1–3% proliferation; and 3, 3% and above proliferation. No statistically significant difference was detected between the groups in terms of proliferation levels (p > 0.05).
Although there was no statistically significant difference between the groups, the highest proliferation scores (3% and above) were found in the RES group (Figure 2, Table 5).

4. Discussion

In this study, it was hypothesized that local application of low doses of RES in hydrogel would increase its efficacy on bone healing in CS and the damage caused by CS in bone healing could be prevented. For this purpose, a critical size defect model was used in the rat femur. The results of the study revealed that local application of low-dose RES with controlled drug release systems increased new bone area, vascularization, osteoblast numbers, and microscopic bone filling rates, similar to repeated applications of systemic high doses.
CS affects bone metabolism due to its inhibitory effects on osteoblast activity, osteogenesis, and angiogenesis [29]. CS inhibits osteoblastic cells and causes changes in bone metabolism by changing parathyroid hormone levels, disrupting the absorption of vitamin D and calcium, and causing a decrease in bone mass by increasing cortisol levels [30]. It has been observed that CS impairs healing in bone fractures and increases the risk of complications that may occur after the operation [31]. It has also been reported to reduce the tensile strength of healing bone [32,33].
Studies have reported that RES prevents bone resorption that occurs as a result of increased osteoclast activity due to inflammatory mediator release [18]. RES has been reported to increase bone mineral density, ossification amount, BMP proteins, and osteopontin levels [19,20,21]. Bhattarai et al. [34], in a study conducted with a periodontitis model they created in rats, injected 5 mg/kg daily subcutaneous RES into rats and reported that inflammatory mediators and osteoclast formation decreased and bone mineral density increased by the end of the experiment.
Polymeric controlled delivery systems ensure that the drug remains within the therapeutic concentration range in a single dose while releasing it in a local area of the body, keeping the systemic drug level at a minimum and increasing patient comfort by facilitating patient monitoring [23,35].
In this study, based on the knowledge that the negative effects of smoking on bone healing can be improved by systemically administered RES, it was thought that the same effect could be achieved by local application of lower doses of RES. For this purpose, Wistar rats were exposed to CS for one month. At the end of one month, a critical size defect was created in the femur. In the RES groups, RES was applied locally to the defect, and the CS groups continued to be exposed to CS for one month after the operation until the day the experiment was terminated.
Özkan et al. evaluated the healing of tooth extraction sockets in rats that were exposed to CS twice a day for two months before the surgical operation, and they found that smoking negatively affected the early and late healing components of the extraction socket, especially the formation of granulation tissue and new bone trabeculae [36]. In a clinical study on smoking patients, Saldanha et al. radiographically examined changes in the extraction socket and alveolar ridge dimensions after tooth extraction [37]. They reported that only in the smoking group was there a significant decrease in alveolar bone width, bone density at the center of the extraction socket, and bone density at the apical part of the socket before extraction. Bergstrom [38] investigated the long-term effect of chronic CS on periodontal bone height and reported that bone height decreased 2.7 times more in smokers than in non-smokers. In a meta-analysis evaluating the relationship between smoking and bone mass, Ward and Klesges [39] reported that smokers experienced significantly reduced bone mass in all regions compared with non-smokers. Consistent with the literature, the results of our study show that new bone area, macroscopic bone filling rate, and osteoblast number in rats exposed to cigarettes were significantly lower than those of the control group.
Frank et al. [40] evaluated bone healing in calvarial defects created in rats exposed to CS, administered 10 mg/kg oral RES to rats for 30 days, and found a decrease in the expression of RANKL/OPG, Dkk1, which plays a role in osteoblast differentiation, and an increase in the expression of RUNX2 gene, which plays important functions in osteoblast differentiation and bone mineralization. The effects of RES on bone healing and the effect of osteogenic markers on gene expression were also researched by Casarin et al. [21] In the study, two defects were created in the tibia of rats to which 10 mg/kg/RES was administered via gavage for 30 days, and a screw-shaped titanium implant was placed. As a result of the histomorphometric analysis, it was seen that the number of defects healed was the highest in the RES group. Additionally, immunohistochemical results showed that the expression of bone morphogenetic proteins (BMP)-2, BMP-7, and osteopontin (OPN) increased in the RES group [21]. Kolkesen Şahin et al. [4] evaluated bone healing in critical size defects in the femur of CS-exposed rats preoperatively for 28 days. After the surgical operation, CS exposure was continued postoperatively for 28 days, after which the rats were sacrificed and bone healing in the defects was evaluated. They reported that the number of osteoblasts in the defect area was the fewest in the smoking group and that the new bone area formed was less in the smoking group. They reported that the number of osteoblasts and new bone area increased in the groups that had been administered 20 mg/kg RES via oral gavage compared with the control and smoking groups [4]. Uysal et al. injected a single dose of 10 μmol/kg resveratrol 24 h after the orthodontic device was placed on the interpremaxillary suture line of rats in which widening was performed. They evaluated the bone healing in the suture histomorphometrically and reported a significant increase in the new bone area and the osteoblast numbers in the resveratrol group [28]. Özcan-Küçük et al. evaluated the extraction socket and administered intraperitoneal RES injection to rats at 10 µmol/kg per day, and at the end of the study, they observed an increase in osteopontin, new bone formation, and epithelialization and a decrease in inflammation [41]. In our study, similar to these studies, the highest osteoblast numbers and the most new bone areas were found in the RES group than in the RES + CS and control groups, respectively, and the lowest osteoblast numbers were found in the CS group. Consistent with studies in the literature showing that CS inhibits osteogenesis and disrupts bone turnover by changing the balance between bone mineral content and osteoblast–osteoclast numbers [42], our study showed that CS reduces the number of osteoblasts. We found that RES given with CS increased the number of osteoblasts compared with the control group. At the same time, it is noteworthy that the study showed similar results to those of studies in the literature showing the effect of RES on osteoblasts at high and repeated doses (10 mg/kg–20 mg/kg) or low but repeated doses (10 µmol/kg) with low- (50 µmol) and single-dose local application.
RES, acting as an aryl-hydrocarbon receptor antagonist, has been demonstrated in the literature to possibly have a positive effect on periodontal tissue regeneration and significantly reduce bone tissue loss [18]. However, due to its poor aqueous solubility, the use of RES for effective treatment is limited by its pharmacokinetic properties. In addition, RES has a short plasma half-life due to its extremely rapid and extensive metabolism [13,43]. Therefore, it is important to develop a delivery system that can overcome biopharmaceutical barriers and fully realize the therapeutic potential of RES [44].
Vascularization provides bone tissue with the necessary nutrients, oxygen, growth factors, and hormones and plays an essential role in regulating bone formation [45]. In an animal study investigating early bone healing, Chang et al. [46] examined angiogenesis and vascularization in rats exposed to CS for two hours per day. They showed a lower expression of VEGF in the smoking group at two and four weeks after surgery with Western blot analyses. They also reported that CS reduces the expression of the angiogenic factors in the fracture callus, and after the fourth week of healing, in the control group, vWF expression was mainly observed around the cartilage area of the soft calluses, and in the smoking group, vWF expression and cartilage formation were low. There are many in vivo and in vitro studies in the literature that have evaluated the effects of RES on bone healing, but there is no study that has evaluated vascularization on bone defects. In a study conducted on rats, myocardial infarction occurred after pre-treatment with RES (1 mg/kg/day) for two weeks. After 24 h, it was observed that the size of the infarct decreased and the capillary density increased. Moreover, myocardium treated with RES after myocardial infarction showed that Trx-1, HO-1, and VEGF expression were observed to be significantly induced. It has been found that RES increases myocardial angiogenesis both in vitro and in vivo by accelerating the tubular morphogenesis of human coronary arteriolar endothelial cells and increasing capillary density in the peri-infarct myocardium [47]. Similar to studies in the literature, in this study, vascularization was observed to be higher in the local RES-applied group than in the control and smoking groups. The fact that vascularization was seen more in the CS + RES group than in the group in which only smoking was administered shows that RES application is effective in eliminating these negative effects of smoking.
In the available literature, there are few articles investigating the effect of resveratrol and smoking on proliferation in bone healing. Heikkinen et al. [48] reported that mesenchymal stromal cells exposed to CS standardized for nicotine concentration exhibited impaired wound healing ability, and at high concentrations, increased cell death in an in vitro study. At lower concentrations, CS dose-dependently impaired proliferation and osteogenic differentiation. Min et al. [49] formed the healthy tooth extraction socket and investigated the effects of resveratrol for bone formation in vitro and suggested that resveratrol may improve the cellular physiology of periodontal ligament and osteoblasts, including proliferation and differentiation during wound healing after tooth extraction. In the presented study, however, there was no statistically significant difference between the groups in terms of proliferation; the group with the highest proliferation was the resveratrol group. This finding suggests that because the study was conducted in vivo, there may be differences even though similar doses of resveratrol were administered.

5. Conclusions

To the best of our knowledge, this study is the first in vivo study in the literature that examined the effects of CS and RES application that has been converted into a local delivery system. In the RES group, it was observed that due to the anti-inflammatory and osteogenic effects of RES, the negative effects of CS on bone healing were reduced by local application in low doses, similar to systemic application. We recommend that further studies be conducted on local application of low doses of RES that are comparable with its systemic applications.

Author Contributions

Conceptualization, M.Ç.; methodology, M.F.İ., M.Ç., Ş.T.Ç.; software, M.Ç., R.O.Y., İ.M.Ç.; validation, M.F.İ., M.Ç., Ş.T.Ç., R.O.Y., İ.M.Ç.; formal analysis, M.F.İ., M.Ç., Ş.T.Ç., R.O.Y., İ.M.Ç.; investigation, M.F.İ., M.Ç.; resources, M.Ç.; data curation, M.Ç.; writing—original draft preparation, M.F.İ., M.Ç., Ş.T.Ç., R.O.Y., İ.M.Ç.; writing—review and editing, M.Ç.; visualization, M.F.İ., M.Ç., İ.M.Ç., R.O.Y.; supervision, M.Ç., İ.M.Ç.; project administration, M.Ç.; funding acquisition, M.Ç. All authors have read and agreed to the published version of the manuscript.

Funding

The present study was funded by the Department of Scientific Research Projects, Suleyman Demirel University (Grant/Award Number: TDH-2021-8455).

Institutional Review Board Statement

The animal study protocol was approved by the Ethics Committee of Suleyman Demirel University (date and number, 21 October 2021-01).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

Dr. Şevket Tolga Çamlı was employed by the company Silika Chemistry Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Tang, T.H.; Fitzsimmons, T.R.; Bartold, P.M. Effect of smoking on concentrations of receptor activator of nuclear factor kappa B ligand and osteoprotegerin in human gingival crevicular fluid. J. Clin. Periodontol. 2009, 36, 713–718. [Google Scholar] [CrossRef]
  2. Sørensen, L.T.; Toft, B.G.; Rygaard, J.; Ladelund, S.; Paddon, M.; James, T.; Taylor, R.; Gottrup, F. Effect of smoking, smoking cessation, and nicotine patch on wound dimension, vitamin C, and systemic markers of collagen metabolism. Surgery 2010, 148, 982–990. [Google Scholar] [CrossRef] [PubMed]
  3. Ma, L.; Zheng, L.W.; Sham, M.H.; Cheung, L.K. Uncoupled angiogenesis and osteogenesis in nicotine-compromised bone healing. J. Bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 2010, 25, 1305–1313. [Google Scholar] [CrossRef]
  4. Kolkesen Şahin, Ö.; Çina Aksoy, M.; Avunduk, M.C. Effects of resveratrol and cigarette smoking onbone healing: Histomorphometric evaluation. Turk. J. Med. Sci. 2016, 46, 1203–1208. [Google Scholar] [CrossRef] [PubMed]
  5. Valavanidis, A.; Vlachogianni, T.; Fiotakis, K. Tobacco Smoke: Involvement of Reactive Oxygen Species and Stable Free Radicals in Mechanisms of Oxidative Damage, Carcinogenesis and Synergistic Effects with Other Respirable Particles. Int. J. Environ. Res. Public. Health 2009, 6, 445–462. [Google Scholar] [CrossRef] [PubMed]
  6. DePhillipo, N.N.; Aman, Z.S.; Kennedy, M.I.; Begley, J.P.; Moatshe, G.; LaPrade, R.F. Efficacy of Vitamin C Supplementation on Collagen Synthesis and Oxidative Stress After Musculoskeletal Injuries: A Systematic Review. Orthop. J. Sports Med. 2018, 6, 2325967118804544. [Google Scholar] [CrossRef] [PubMed]
  7. Wong, S.K.; Mohamad, N.V.; Ibrahim, N.I.; Chin, K.Y.; Shuid, A.N.; Ima-Nirwana, S. The Molecular Mechanism of Vitamin E as a Bone-Protecting Agent: A Review on Current Evidence. Int. J. Mol. Sci. 2019, 20, 1453. [Google Scholar] [CrossRef]
  8. Hernigou, J.; Schuind, F. Tobacco and bone fractures: A review of the facts and issues that every orthopaedic surgeon should know. Bone Jt. Res. 2019, 8, 255–265. [Google Scholar] [CrossRef] [PubMed]
  9. Wang, W.; Zhang, L.M.; Guo, C.; Han, J.F. Resveratrol promotes osteoblastic differentiation in a rat model of postmenopausal osteoporosis by regulating autophagy. Nutr. Metab. 2020, 17, 29. [Google Scholar] [CrossRef] [PubMed]
  10. Riccitiello, F.; De Luise, A.; Conte, R.; D’Aniello, S.; Vittoria, V.; Di Salle, A.; Calarco, A.; Peluso, G. Effect of resveratrol release kinetic from electrospun nanofibers on osteoblast and osteoclast differentiation. Eur. Polym. J. 2018, 99, 289–297. [Google Scholar] [CrossRef]
  11. Cottart, C.H.; Nivet-Antoine, V.; Laguillier-Morizot, C.; Beaudeux, J. Resveratrol bioavailability and toxicity in humans. Mol. Nutr. Food Res. 2010, 54, 7–16. [Google Scholar] [CrossRef] [PubMed]
  12. Sánchez-Fidalgo, S.; Cárdeno, A.; Villegas, I.; Talero, E.; de la Lastra, C.A. Dietary supplementation of resveratrol attenuates chronic colonic inflammation in mice. Eur. J. Pharmacol. 2010, 633, 78–84. [Google Scholar] [CrossRef] [PubMed]
  13. Salla, M.; Karaki, N.; El Kaderi, B.; Ayoub, A.J.; Younes, S.; Abou Chahla, M.N.; Baksh, S.; El Khatib, S. Enhancing the Bioavailability of Resveratrol: Combine It, Derivatize It, or Encapsulate It? Pharmaceutics 2024, 16, 569. [Google Scholar] [CrossRef]
  14. Zhang, W.; Meng, L.; Lv, X.; Wang, L.; Zhao, P.; Wang, J.; Zhang, X.; Chen, J.; Wu, Z. Enhancing Stability and Antioxidant Activity of Resveratrol-Loaded Emulsions by Ovalbumin–Dextran Conjugates. Foods 2024, 13, 1246. [Google Scholar] [CrossRef]
  15. Adhikari, N.; Prasad Aryal, Y.; Jung, J.K.; Ha, J.H.; Choi, S.Y.; Kim, J.Y.; Lee, T.H.; Kim, S.H.; Yamamoto, H.; Suh, J.Y.; et al. Resveratrol enhances bone formation by modulating inflammation in the mouse periodontitis model. J. Periodontal Res. 2021, 56, 735–745. [Google Scholar] [CrossRef]
  16. Lee, Y.S.; Kim, Y.S.; Lee, S.Y.; Kim, G.H.; Kim, B.J.; Lee, S.H.; Lee, K.U.; Kim, G.S.; Kim, S.W.; Koh, J.M. AMP kinase acts as a negative regulator of RANKL in the differentiation of osteoclasts. Bone 2010, 47, 926–937. [Google Scholar] [CrossRef] [PubMed]
  17. Dai, Z.; Li, Y.; Quarles, L.D.; Song, T.; Pan, W.; Zhou, H.; Xiao, Z. Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation. Phytomed. Int. J. Phytother. Phytopharm. 2007, 14, 806–814. [Google Scholar] [CrossRef]
  18. Casati, M.Z.; Algayer, C.; Cardoso da Cruz, G.; Ribeiro, F.V.; Casarin, R.C.; Pimentel, S.P.; Cirano, F.R. Resveratrol decreases periodontal breakdown and modulates local levels of cytokines during periodontitis in rats. J. Periodontol. 2013, 84, 58–64. [Google Scholar] [CrossRef]
  19. Liu, Z.P.; Li, W.X.; Yu, B.; Huang, J.; Sun, J.; Huo, J.S.; Liu, C.X. Effects of trans-resveratrol from Polygonum uspidatum on bone loss using the ovariectomized rat model. J. Med. Food 2005, 8, 14–19. [Google Scholar] [CrossRef]
  20. Pearson, K.J.; Baur, J.A.; Lewis, K.N.; Peshkin, L.; Price, N.L.; Labinskyy, N.; Swindell, W.R.; Kamara, D.; Minor, R.K.; Perez, E.; et al. Resveratrol delays age-related deterioration and mimics transcriptional aspects of dietary restriction without extending life span. Cell Metab. 2008, 8, 157–168. [Google Scholar] [CrossRef]
  21. Casarin, R.C.; Casati, M.Z.; Pimentel, S.P.; Cirano, F.R.; Algayer, M.; Pires, P.R.; Ghiraldini, B.; Duarte, P.M.; Ribeiro, F.V. Resveratrol improves bone repair by modulation of bone morphogenetic proteins and osteopontin gene expression in rats. Int. J. Oral Maxillofac. Surg. 2014, 43, 900–906. [Google Scholar] [CrossRef] [PubMed]
  22. Song, L.H.; Pan, W.; Yu, Y.H.; Quarles, L.D.; Zhou, H.H.; Xiao, Z.S. Resveratrol prevents CsA inhibition of proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells through an ER/NO/cGMP pathway. Toxicol. Vitro Int. J. Publ. Assoc. BIBRA 2006, 20, 915–922. [Google Scholar] [CrossRef] [PubMed]
  23. Li, J.; Mooney, D.J. Designing hydrogels for controlled drug delivery. Nat. Rev. Mater. 2016, 1, 16071. [Google Scholar] [CrossRef] [PubMed]
  24. Lin, Y.; Jiandong, D. Injectable hydrogels as unique biomedical materials. Chem. Soc. Rev. 2008, 37, 1473–1481. [Google Scholar]
  25. Almeida, H.; Amaral, M.H.; Lobão, P. Temperature and pH stimuli-responsive polymers and their applications in controlled and selfregulated drug delivery. J. Appl. Pharm. Sci. 2012, 2, 1–10. [Google Scholar]
  26. Diniz, I.M.; Chen, C.; Xu, X.; Ansari, S.; Zadeh, H.H.; Marques, M.M.; Shi, S.; Moshaverinia, A. Pluronic F-127 hydrogel as a promising scaffold for encapsulation of dental-derived mesenchymal stem cells. J. Mater. Sci. Mater. Med. 2015, 26, 153. [Google Scholar] [CrossRef] [PubMed]
  27. Dumortier, G.; Grossiord, J.L.; Agnely, F.; Chaumeil, J.C. A Review of Poloxamer 407 Pharmaceutical and Pharmacological Characteristics. Pharm. Res. 2006, 23, 12. [Google Scholar] [CrossRef] [PubMed]
  28. Uysal, T.; Gorgulu, S.; Yagci, A.; Karslioglu, Y.; Gunhan, O.; Sagdic, D. Effect of resveratrol on bone formation in the expanded inter-premaxillary suture: Early bone changes. Orthod. Craniofac. Res. 2011, 14, 80–87. [Google Scholar] [CrossRef] [PubMed]
  29. Yoon, V.; Maalouf, N.M.; Sakhaee, K. The effects of smoking on bone metabolism. Osteoporosis international: A journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. Osteoporos. Int. 2012, 23, 2081–2092. [Google Scholar] [CrossRef]
  30. Cusano, N.E. Skeletal Effects of Smoking. Curr. Osteoporos. Rep. 2015, 13, 302–309. [Google Scholar] [CrossRef]
  31. Al-Bashaireh, A.M.; Haddad, L.G.; Weaver, M.; Chengguo, X.; Kelly, D.L.; Yoon, S. The Effect of Tobacco Smoking on Bone Mass: An Overview of Pathophysiologic Mechanisms. J. Osteoporos. 2018, 2018, 1206235. [Google Scholar] [CrossRef] [PubMed]
  32. Scolaro, J.A.; Schenker, M.L.; Yannascoli, S.; Baldwin, K.; Mehta, S.; Ahn, J. Cigarette smoking increases complications following fracture: A systematic review. J. Bone Jt. Surg. Am. 2014, 96, 674–681. [Google Scholar] [CrossRef] [PubMed]
  33. Moghaddam, A.; Zimmermann, G.; Hammer, K.; Bruckner, T.; Grützner, P.A.; von Recum, J. Cigarette smoking influences the clinical and occupational outcome of patients with tibial shaft fractures. Injury 2011, 42, 1435–1442. [Google Scholar] [CrossRef] [PubMed]
  34. Bhattarai, G.; Poudel, S.B.; Kook, S.H.; Lee, J.C. Resveratrol prevents alveolar bone loss in an experimental rat model of periodontitis. Acta Biomater. 2016, 29, 398–408. [Google Scholar] [CrossRef]
  35. Kost, J.; Langer, R. Responsive polymeric delivery systems. Adv. Drug Deliv. Rev. 2001, 46, 125–148. [Google Scholar] [CrossRef] [PubMed]
  36. Ozkan, A.; Bayar, G.R.; Altug, H.A.; Sencimen, M.; Dogan, N.; Gunaydin, Y.; Erdogan, E. The effect of cigarette smoking on the healing of extraction sockets: An immunohistochemical study. J. Craniofac. Surg. 2014, 25, 397–402. [Google Scholar] [CrossRef] [PubMed]
  37. Saldanha, J.B.; Casati, M.Z.; Neto, F.H.; Sallum, E.A.; Nociti, F.H. Smoking may affect the alveolar process dimensions and radiographic bone density in maxillary extraction sites: A prospective study in humans. J. Oral Maxillofac. Surg. 2006, 64, 1359–1365. [Google Scholar] [CrossRef]
  38. Bergström, J. Influence of tobacco smoking on periodontal bone height. Longterm bservations and a hypothesis. J. Clin. Periodontol. 2004, 31, 260–266. [Google Scholar] [CrossRef] [PubMed]
  39. Ward, K.D.; Klesges, R.C. A meta-analysis of the effects of cigarette smoking on bone mineral density. Calcif. Tissue Int. 2001, 68, 259–270. [Google Scholar] [CrossRef]
  40. Franck, F.C.; Benatti, B.B.; Andia, D.C.; Cirano, F.R.; Casarin, R.C.; Corrêa, M.G.; Ribeiro, F.V. Impact of resveratrol on bone repair in rats exposed to cigarette smoke inhalation: Histomorphometric and bone-related gene expression analysis. Int. J. Oral Maxillofac. Surg. 2018, 47, 541–548. [Google Scholar] [CrossRef]
  41. Ozcan Kucuk, A.; Alan, H.; Gul, M.; Yolcu, U. Evaluating the Effect of Resveratrol on the Healing of Extraction Sockets in Cyclosporine A-Treated Rats. Int. J. Oral Maxillofac. Surg. 2018, 76, 1404–1413. [Google Scholar] [CrossRef] [PubMed]
  42. Rothem, D.E.; Rothem, L.; Soudry, M.; Dahan, A.; Eliakim, R. Nicotine modulates bone metabolism-associated gene expression in osteoblast cells. J. Bone Miner. Metab. 2009, 27, 555–561. [Google Scholar] [CrossRef] [PubMed]
  43. Arakkunakorn, W.; Pholthien, W.; Sajomsang, W.; Basit, A.; Sripetthong, S.; Nalinbenjapun, S.; Ovatlarnporn, C. Validated HPLC method for simultaneous quantitative determination of dimethylcurcumin and resveratrol in pluronic-F127 nanomicelles: Formulation with enhanced anticancer efficacy. MethodsX 2023, 11, 102457. [Google Scholar] [CrossRef] [PubMed]
  44. Amri, A.; Chaumeil, J.C.; Sfar, S.; Charrueau, C. Administration of resveratrol: What formulation solutions to bioavailability limitations? J. Control. Release 2012, 158, 182–193. [Google Scholar] [CrossRef] [PubMed]
  45. Peng, Y.; Wu, S.; Li, Y.; Crane, J.L. Type H blood vessels in bone modeling and remodeling. Theranostics 2020, 10, 426–436. [Google Scholar] [CrossRef] [PubMed]
  46. Chang, C.J.; Jou, I.M.; Wu, T.T.; Su, F.C.; Tai, T.W. Cigarette smoke inhalation impairs angiogenesis in early bone healing processes and delays fracture union. Bone Jt. Res. 2020, 9, 99–107. [Google Scholar] [CrossRef] [PubMed]
  47. Kaga, S.; Zhan, L.; Matsumoto, M.; Maulik, N. Resveratrol enhances neovascularization in the infarcted rat myocardium through the induction of thioredoxin-1, heme oxygenase-1 and vascular endothelial growth factor. J. Mol. Cell Cardiol. 2005, 39, 813–822. [Google Scholar] [CrossRef] [PubMed]
  48. Heikkinen, J.; Tanner, T.; Bergmann, U.; Palosaari, S.; Lehenkari, P. Cigarette smoke and nicotine effect on human mesenchymal stromal cell wound healing and osteogenic differentiation capacity. Tob. Induc. Dis. 2024, 22, 10. [Google Scholar] [CrossRef]
  49. Min, K.K.; Neupane, S.; Adhikari, N.; Sohn, W.J.; An, S.Y.; Kim, J.Y.; An, C.H.; Lee, Y.; Kim, Y.G.; Park, J.W.; et al. Effects of resveratrol on bone-healing capacity in the mouse tooth extraction socket. J. Periodontal Res. 2020, 55, 247–257. [Google Scholar] [CrossRef]
Applsci 14 06411 g001
Figure 1. (A) While there were no osteoblasts in the defective area in the control group, 1–2 osteoclasts (blue arrow) were observed. (B) In the smoking group, 3–4 osteoclasts (red arrow) were observed. (C) An increase in the number of osteoblasts and osteoblastic activity (green arrow) was observed in the resveratrol group. (D) A lower number of osteoblasts (yellow arrow) were observed in the CS + RES group compared to the RES group ((A–D): H&E 200×).
Figure 1. (A) While there were no osteoblasts in the defective area in the control group, 1–2 osteoclasts (blue arrow) were observed. (B) In the smoking group, 3–4 osteoclasts (red arrow) were observed. (C) An increase in the number of osteoblasts and osteoblastic activity (green arrow) was observed in the resveratrol group. (D) A lower number of osteoblasts (yellow arrow) were observed in the CS + RES group compared to the RES group ((A–D): H&E 200×).
Applsci 14 06411 g001
Applsci 14 06411 g002
Figure 2. (A) In the sample scanned at 200× with Microvisioneer manual whole slide scanner equipment, SATB2 expression was digitally evaluated from the hotspot area using QuPath open-source software. (B) A small number of new vessel formations were observed with PSMA in the resveratrol group. (C) In the resveratrol group, 2–3% proliferation was observed in the hotspot area with Ki-67. (QuPath open-source digital pathology; A: SATB2 DAB 200×, B: PSMA DAB 200×, C:K i-67 DAB 200×).
Figure 2. (A) In the sample scanned at 200× with Microvisioneer manual whole slide scanner equipment, SATB2 expression was digitally evaluated from the hotspot area using QuPath open-source software. (B) A small number of new vessel formations were observed with PSMA in the resveratrol group. (C) In the resveratrol group, 2–3% proliferation was observed in the hotspot area with Ki-67. (QuPath open-source digital pathology; A: SATB2 DAB 200×, B: PSMA DAB 200×, C:K i-67 DAB 200×).
Applsci 14 06411 g002
Table 1. Comparison of new bone formation levels.
Table 1. Comparison of new bone formation levels.
New Bone Area ScoresControl
(n, %)		RES
(n, %)		CS + RES
(n, %)		CS
(n, %)		p
New Bone AreaScore 06 (60.0) a0 (0.0) b1 (10.0) a,b6 (60.0) a0.01 *
Score 13 (30.0) a,b0 (0.0) b5 (50.0) a4 (40.0) a,b
Score 21 (10.0) a4 (36.4) a2 (20.0) a0 (0.0) a
Score 30 (0.0) a3 (27.3) a2 (20.0) a0 (0.0) a
Score 40 (0.0) a4 (36.4) a0 (0.0)0 (0.0) a
Fisher–Freeman–Halton Exact Test: 30.58. RES: resveratrol; CS: cigarette smoking. Different letters on the same line indicate a significant difference. Statistical differences are indicated by letters such as a, b, and ab. There is a statistically significant difference between groups indicated with different letters (* p ˂ 0.05). There is no statistically significant difference in groups with the same letters.
Table 2. Comparison of vascularization levels by groups.
Table 2. Comparison of vascularization levels by groups.
Vascularization ScoresControl
(n, %)		RES
(n, %)		CS + RES
(n, %)		CS
(n, %)		p
VascularizationScore 010 (100.0) a4 (36.4) b6 (60.0) a,b8 (80.0) a,b0.02 *
Score 10 (0.0) a5 (45.5) a4 (40.0) a2 (20.0) a
Score 20 (0.0) a2 (18.2) a0 (0.0) a0 (0.0) a
Fisher–Freeman–Halton Exact Test: 11.84. RES: resveratrol; CS: cigarette smoking. Different letters on the same line indicate a significant difference. Statistical differences are indicated by letters such as a, b, ab. There is a statistically significant difference between groups indicated with different letters (* p ˂ 0.05). There is no statistically significant difference in groups with the same letters.
Table 3. Comparison of macroscopic bone filling.
Table 3. Comparison of macroscopic bone filling.
Macroscopic Bone Filling (%)Control
(n, %)		RES
(n, %)		CS + RES
(n, %)		CS
(n, %)		p
Macroscopic Bone0–25%4 (40.0) a,b0 (0.0) b1 (10.0) b8 (80.0) a0.01 *
25–50%6 (60.0) a0 (0.0) b2 (20.0) a,b2 (20.0) a,b
50–75%0 (0.0) a2 (18.2) a5 (50.0) a0 (0.0) a
75+%0 (0.0) a9 (81.8) b2 (20) a0 (0.0) a
Fisher–Freeman–Halton Exact Test: 40.12 RES: resveratrol; CS: cigarette smoking. Values are presented as numbers (%). Statistical differences are indicated by letters such as a, b, and ab. There is a statistically significant difference between the groups indicated with different letters (* p ˂ 0.05). There is no statistically significant difference in groups with the same letters.
Table 4. Ratio of osteoblasts to other cells.
Table 4. Ratio of osteoblasts to other cells.
Median (Min-Maks.)X2p
Control a0.23 (0.01–2.13)29.490.01 *
RES b11.00 (5.66–38.68)
CS + RES c2.71 (0.01–10.00)
CS d0.10 (0.01–0.29)
X2: Kruskal–Wallis-H test. RES: resveratrol; CS: cigarette smoking. Statistical differences are indicated by letters such as a, b, c, and d. There is a statistically significant difference between groups indicated with different letters (* p ˂ 0.05). There is no statistically significant difference in groups with the same letters.
Table 5. Comparison of proliferation.
Table 5. Comparison of proliferation.
Proliferation (%)Control
(n, %)		RES
(n, %)		CS + RS
(n, %)		CS
(n, %)		p
Proliferation LevelsNone8 (80.0)3 (27.3)3 (30.0)6 (60.0)0.06
0–1%2 (20.0)3 (27.3)5 (50.0)4 (40.0)
1–3%0 (0.0)2 (18.2)2 (20.0)0 (0.0)
3+%0 (0.0)3 (27.3)0 (0.0)0 (0.0)
Fisher–Freeman–Halton Exact Test: 13.12. RES: resveratrol; CS: cigarette smoking. Values are presented as numbers (%). There is no statistically significant difference detected between the groups (p > 0.05).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

İskender, M.F.; Çına, M.; Çamlı, Ş.T.; Çiriş, İ.M.; Yüceer, R.O. Evaluation of the Effects of Locally Applied Resveratrol and Cigarette Smoking on Bone Healing. Appl. Sci. 2024, 14, 6411. https://doi.org/10.3390/app14156411

AMA Style

İskender MF, Çına M, Çamlı ŞT, Çiriş İM, Yüceer RO. Evaluation of the Effects of Locally Applied Resveratrol and Cigarette Smoking on Bone Healing. Applied Sciences. 2024; 14(15):6411. https://doi.org/10.3390/app14156411

Chicago/Turabian Style

İskender, Muhsin Fırat, Müge Çına, Şevket Tolga Çamlı, İbrahim Metin Çiriş, and Ramazan Oğuz Yüceer. 2024. "Evaluation of the Effects of Locally Applied Resveratrol and Cigarette Smoking on Bone Healing" Applied Sciences 14, no. 15: 6411. https://doi.org/10.3390/app14156411

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop