Copyright © 2019 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited. Effects of Different Levels of Restraint Stress on Bone–Implant Contact Alihan Bozoglan, DDS, PhD,  Serkan Dundar, DDS, PhD,  Tuba T. Yildirim, DDS, PhD,  Ozgur Bulmus, DVM, PhD, y Abdullah Seckin Ertugrul, DDS, PhD, z Merve Y. Bozoglan, MD, § Samet Tekin, DDS, MSC, jj and Vesile Elif Toy, DDS, PhD ô Abstract: This study examined the effects of different levels of chronic restraint stress on bone–titanium implant contact in rats. This study included 32 adult female Sprague–Dawley rats. The machined surface titanium implants were surgically placed into the metaphyseal region of the rat tibias. Next, the rats were divided randomly into 4 groups, namely, control (CNT) (n ¼ 8), low-restraint stress (LRS) (n ¼ 8), medium-restraint stress (MRS) (n ¼ 8), and high-restraint stress (HRS) (n ¼ 8) groups. The rats in the CNT group received only the titanium implants surgically but did not receive any further treatment during the experimental period of 30 days. The rats in the LRS, MRS, and HRS groups were applied restraint stress for 1, 2, and 4 hours, respectively, daily for 28 days starting from day 2 after the surgery. At the end of the study period, the rats were sacrificed and their implants and the surrounding bone tissues were harvested for performing non- decalcified histological analysis. Moreover, blood samples were col- lected from the rats and were centrifuged for analyzing serum cortisol levels. Serum cortisol levels of the rats in the LRS, MRS, and HRS groups were higher than those of the rats in the CNT group (P < 0.05). Moreover, serum cortisol levels of the rats in the HRS group were higher than those of the rats in the MRS and LRS groups (P < 0.05). The extent of bone–implant contact was lower in the rats in the HRS group than in the rats in the CNT and LRS groups (P < 0.05). These data suggest that the application of 4-hour chronic restraint stress during the 28-day experimental period impaired the bone–implant contact. Key Words: Bone–implant connection, cortisol, osseointegration, rat, restraint stress level (J Craniofac Surg 2019;30: 1294–1297) S tress affects a large part of the world population regardless of age, sex, or social class. Chronic stress can disrupt the physical and emotional health of people. Recent studies have shown that stress is one of the most important risk factors of many illnesses. 1–4 Stress activates hypothalamic–pituitary–adrenal pathway and increases the release of corticotropin-releasing hormone from the hypothalamic paraventricular nucleus. This in turn stimulates the release of adrenal corticosteroids, which is mediated by the release of adrenocorticotropin from the anterior pituitary gland. 5–7 Chronic and acute stress affects the process of wound healing. Although mechanisms underlying stress-induced disruption of wound healing are not completely understood to date, it has been suggested that stress disrupts wound healing by triggering the hypothalamic– pituitary–adrenal pathway. In addition, some experimental animal studies have reported that glucocorticoids delay soft tissue healing and impair bone–implant contact. 8–11 Dental implant-supported prostheses are a commonly used and scientifically accepted option for treating partial and complete toothlessness. In dental implant therapy, the systemic condition of the patient, quality and quantity of the bone in which the implant is to be placed, geometric and surface characteristics of the implant, and smoking habit of the patient are important factors for osseoin- tegration. 12,13 Assessment of mechanisms underlying wound heal- ing suggests that the harmful effects of chronic stress occur during the early stage of wound healing. 9 Although diverse evidence indicates that chronic stress negatively affects wound healing, the effects of stress on bone–implant contact have not been completely elucidated to date. Therefore, this study investigated the effects of different levels of restraint stress on the osseointe- gration of titanium implants in the rat tibias. METHODS Animals and Experimental Design All the experimental procedures in this study were performed at Firat University Experimental Research Center, Elazig, Turkey. This study was ethically approved by Firat University Animal Experiments Local Ethics Committee (Protocol Number: 2016/ 76). Moreover, the study procedures completely conformed to the recommendations of the World Medical Association Helsinki Declaration on the protection of animals used in laboratory inves- tigations. This study used 32 healthy adult 270to 320 g female Sprague–Dawley rats aged 1 to 1.2 years in the same estrus cycle. All the rats were kept in plastic cages, and temperatures were recorded for areas where the animals were kept. Moreover, all the rats were given free access to food and water and were maintained at a 12-/12-h light/dark cycle. All rats were weighed at the beginning of the experimental phase. Machined surfaced titanium implants (Implance, AGS Medical, Istanbul, Turkey) were surgically placed into the right tibias of the rats. Next, the rats were randomly divided into the following 4 experimental groups: control group (n ¼ 8) (rats implanted with the From the  Department of Periodontology, Faculty of Dentistry; y Department of Physiology, Faculty of Health Sciences, Firat University, Elazig; z Department of Periodontology, Faculty of Dentistry, Izmir Katip C ¸ elebi University, Izmir; § Department of Pharmacology, Faculty of Medicine; jj Department of Prosthetics, Faculty of Dentistry, Firat University, Elazig; and ô Department of Periodontology, Faculty of Dentistry, Inonu University, Malatya, Turkey. Received July 6, 2018. Accepted for publication September 10, 2018. Address correspondence and reprint requests to Serkan Dundar, DDS, PhD, Department of Periodontology, Faculty of Dentistry, Firat University, 23119, Campus, Elazig, Turkey; E-mail: sdundar@firat.edu.tr, dtserkandundar@gmail.com This study was presented as an oral presentation in 23 International Scientific Congress of Turkish Dental Association, September 21–24, 2017, Istanbul, Turkey. The authors report no conflicts of interest. Copyright # 2018 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000005104 SCIENTIFIC FOUNDATION 1294 The Journal of Craniofacial Surgery  Volume 30, Number 4, June 2019