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Effects of Systemic Propranolol Application on the New Bone Formation in Periimplant Guided Bone Regeneration

ABSTRACT Objectives The aim of this experimental animal study was to evaluate the effects of systemic propranolol on new bone formation in peri-implant bone defects. Material and Methods Implant slots were created 4mm long and 2.5 mm wide. After the titanium implants were placed in the sockets, 2 mm defects were created in the neck of the implants. Bone grafts were placed in these defects. Then the rats were randomly divided into three equal groups: control (n = 8), propranolol dose-1 (PRP-1) (n = 8), and propranolol dose-2 (PRP-2) (n = 8) groups. In the control group, the rats received no further treatment during the eight-week experimental period after the surgery. The rats in the PRP-1 and PRP-2 groups were given 5 mg/kg and 10 mg/kg propranolol, respectively, every three days for the eight-week experimental period after the surgery. At the end of the experimental period, the rats were euthanized. Blood serum was collected for biochemical analysis, and the implants and surrounding bone tissues were used for the histological analysis. Results There were no significant differences in the histological analysis results and the biochemical parameters (alkaline phosphatase, calcium, creatinine and phosphorus) of the groups (P > 0.05). Also, in the test groups, there was numerically but not statistically more new bone formation detected compared with the controls. Conclusions Within the limitations of this study, propranolol did not affect the new bone formation in peri-implant defects.

INTRODUCTION β-blockers (or beta blockers) are a group of drugs that have a well-known antihypertensive effect by decreasing cardiac output, releasing renin from the kidneys and inhibiting the effect of endogenous catecholamines on β-adrenoceptors [1]. They act by antagonizing β-adrenergic receptors and are among the most frequently prescribed cardiovascular drugs, their safe use having long been registered [2,3]. Systemic-acting β-blockers are broad-spectrum drugs used as antianginal and antihypertensive treatments, for heart failure and arrhythmia and for the symptoms of migraine, anxiety and thyrotoxicosis [1]. Propranolol is reported to inhibit all beta receptors and was the first certified β-blocker. Although these receptors are secreted primarily from cardiovascular and respiratory cells, they are also secreted from bone cells such as osteoblasts and osteoclasts [4][5][6]. Studies have reported that antihypertensive drugs are also associated with the bone regeneration process [7][8][9].
In spontaneously hypertensive rats with ligatureinduced experimental periodontitis, it was observed that the expression of the receptor activator of NK-kB ligand (RANKL) and the RANKL/osteoprotegerin (OPG) ratio were higher in the alveolar bone around the non-ligature teeth, the number of osteoclast cells increased, bone loss increased and bone density decreased. This result is thought to be directly affected by the alveolar bone, regardless of the ligature state [10]. In epidemiological studies, it has been reported that there is a relationship between increased bone loss and high blood pressure. It has also been suggested that β-blockers are a useful therapeutic in fracture healing and osteoporosis [11][12][13]. In an implant study, it was reported that the patients were generally healthy, but 38.2% of the patients had drugcontrolled hypertension and 10% had controlled type 2 diabetes [14]. In addition, one study suggested that the use of β-blockers had no effect on bone mineral density [15]. Other research reported that low-dose propranolol use was associated with osteoclastogenesis and inflammatory markers, without affecting haemodynamic parameters. It has been reported that it may suppress periodontal bone resorption [16]. Bone-grafting techniques entail different strategies, such as alveolar distraction and guided bone regeneration (GBR). When the implants are functionally loaded, bone grafting may be undertaken to increase the bone around the implant to ensure osteointegration and maintain implant survival. GBR, which involves reconstruction of the alveolar bone and the treatment of peri-implant bone defects, is one of the commonest methods of bone grafting [17,18]. Implanted bone regeneration is required in 40% of patients who have undergone an implant to complete the healthy osteointegration process [19]. It has been shown that the survival rates of implants placed in areas where GBR is applied are similar to those of implants in areas where it has not been applied [20][21][22]. The survival rate of implants in the GBR-treated areas varies between 79 and 100%, and the majority of the implants show a more than 90% survival rate after at least one year of function [22]. Similarly, the implant survival rate has been reported as 95% after the horizontal or vertical GBR procedure. Thus, GBR is a successful treatment option that is widely used in alveolar bone defects and implant applications [17,23,24]. In this study, we postulate that a β-blocker (propranolol) can be used together with a bone graft to reduce defects and bone loss around dental implants. As far as we know, there is no specific information about the low-, medium-and high-dose effects of propranolol on tissues surrounding peri-implants or on the mechanism of action. Accordingly, the effects of different doses of propranolol and graft material on bone defects around experimental implants were evaluated. The aim of this experimental animal study was to evaluate the effect of propranolol on graft application in periimplant bone defects in rats treated with systemic propranolol.

MATERIAL AND METHODS Study design
This study was conducted at Fırat University Experimental Research Center between December 16, 2019 and February 17, 2020, after obtaining approval from Fırat University Animal Experiments Local Ethics Committee (Protocol No. 2019/143). The subjects were provided by Fırat University Experimental Research Center. Twenty-four Sprague-Dawley rats were used in the experiment. The rats were randomly divided into three groups (n = 8 in each group). Rats were kept on a light/dark cycle for an equal period of time. In addition, humidity (55%) and temperature (22 [SD 2] °C) were continuously controlled. The subjects were kept in pairs in cages. Normal diet, water and ad libitum nutrition were provided. The implants which we used in this study have resorbable blast material (RBM) surfaced and threat depth 0.3 mm, threat pitch 0.4 mm, 2.5 mm diameter and 4 mm long with 8 threads (AGS Medical Corporation; Istanbul, Turkey).

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In order to standardize the defects, a 3-walled standard defect with 2.5 mm width and 2 mm length was opened on one side of the implants with the drill in which the implant slots were opened. After the grafts were placed, the defect area was closed with resorbable collagen membrane (Jason membrane ® -Botiss Biomaterials GmbH; Zossen, Germany).

Surgical procedures
General anaesthesia was created by intraperitoneal injection of ketamine hydrochloride (50 mg/kg, Ketasol ® -Richter Pharma AG; Wels, Austria) and xylazine (5 mg/kg, Rompun ® -Bayer HealthCare AG; Leverkusen, Germany) to rats. All surgical procedures were performed under sterile conditions. After anaesthesia, the operation area was cleaned with povidone-iodine and shaved. RBM titanium implants were placed in the metaphyseal part of the tibial bone.
After placing the titanium implants, the flap was returned to its original position and sutured with 4-0 polyglactin sutures. Antibiotics (50 mg/kg penicillin) and analgesic (0.1 mg/kg tramadol hydrochloride) were administered intramuscularly daily to rats for three days after surgical procedures. Group 1 (control group): implant sockets were created in the corticocancellous bone in the metaphyseal parts of the right tibial bones of the rats. These sockets were fitted with 2.5 mm-diameter, 4 mm-long titanium screws, and the hydroxyapatite bone graft of bovine origin (Cerabone ® -Botiss Biomaterials GmbH; Zossen, Germany) was placed in the bone defect that would occur in the neck region, corresponding to 2 mm of the implant length. No additional treatment was performed during the eight-week experimental setup. Group 2 (propranolol-graft dose-1 [PRP-1] group): implant sockets were created, the sockets fitted with titanium screws and the bone graft placed in the bone defect, as in Group 1. In this group, 5 mg/kg propranolol was administered orally three days a week during the eight-week experiment [25].
implant sockets were created, the sockets fitted with titanium screws and the bone graft placed in the bone defect, as in Group 1. The subjects in this group were administered 10 mg/kg propranolol oral gavage three days a week during the eight-week experiment [25].
At the end of the eight-week experimental setup, all rats were sacrificed.

Histological analysis
The implants and bone tissue around them were removed and decalcified before being subjected to histological analysis at the Fırat University Faculty of Medicine Pathology Laboratory. The histological analysis was performed on the original graft bone tissue. The samples were stored in 10% formaldehyde solution for 72 hours. Minerals were eliminated in 10% formic acid solution. The implants were removed from the samples without damaging the bone tissue. Dehydration of the bone tissue samples was performed for analysis. The samples were embedded in a paraffin wax block. Microscopic haematoxylin and eosin analysis was performed.
Light microscopy was used to examine 6 µmthick sections of bone graft samples. An image analysis program was used to evaluate the new bone formation. The newly formed bone area was calculated as a percentage of the total area of new bone area formed in peri-implant bone tissues. All histological samples were examined by means of images taken with a digital camera connected to a light microscope and transferred to a computer with original magnification [26]. Histomorphometric analyses were performed with a device fitted with an Olympus DP71 (Olympus Corp.; Tokyo, Japan) software imaging system. The new boneformation rate for each implant was calculated by the ratio of the newly formed bone surface to the grafted surface. The data were recorded as percentages (%).

Biochemical analysis
While the rats were in deep anaesthesia, blood samples were collected through cardiac puncture without anticoagulant to measure the serum alkaline phosphatase (ALP), calcium (Ca), creatinin (Cr) and phosphorus (P). Biochemical data were measured for each rat at the central biochemistry laboratory of the Faculty of Medicine Fırat University.

Statistical analysis
Statistical analyses were performed using IBM SPSS version 23.0 (IBM Inc.; Chicago, Illinois, USA). The data in each group were given as mean and standard deviation (M [SD]). One-way ANOVA was used to determine the differences between groups. Tukey's honest significant difference test was used to determine the group that caused the differences (P < 0.05). The student's t-test was used to determine the differences between the test groups (PRP-1 and PRP-2) and the control group (P < 0.05).

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RESULTS
Although the histological analyses showed no statistical differences between the control group (47.63% [5. . Cr (mg/ dl) levels were 0.55 (0.08) in the control group, 0.52 (0.09) in the PRP-1 group and 0.57 (0.12) in the PRP-2 group, and no statistical difference was found between them (P > 0.05). In addition, when compared with the control group and the propranolol groups, although lower Cr values were obtained in the propranolol groups, no statistically significant difference was found between them (P > 0.05; Table 2). Not statistically significant at level P < 0.05 (One-way ANOVA).

DISCUSSION
Okada et al. [27] suggested that applying β-blockers (propranolol) at low doses can prevent periodontal bone loss by reducing bone resorption. In a study by Rodrigues et al. [16], it was suggested that the effects of β-blockers on bone at low doses are partly due to the inhibition of inflammatory markers and osteoclastogenesis, without affecting heart function. Some studies have shown that periodontal tissue is controlled by the autonomic nervous system,   especially the sympathetic nervous system, and dysfunction of this system can lead to periodontal disruption [28,29]. Togari et al. [30] indicated that the downregulation of bone formation depends on the activation of β2-adrenergic receptors, which are known to be expressed only by osteoblasts. However, Rodrigues et al. [16] stated that there is less bone resorption between bones and a protective effect on the bone in ligated animals treated with low doses of propranolol (0.1 and 5 mg/kg). Pierroz et al. [31] showed that propranolol could not completely recover bone loss in adult mice undergoing ovariectomy, and pharmacological studies on adrenergic receptors gave controversial results. Minkowitz et al. [32] formed bone fractures in rats, then applied propranolol therapy (0.1 mg/kg) for 12 weeks, and found metaphysis and increased bone formation in both the periosteum and the endosteum. They investigated whether propranolol administered systemically at low doses had an effect on bone metabolism and caused an increase in orthotopic endochondral bone formation. As a result of biomechanical tests in the non-surgical group, they reported that systemically administered propranolol increased femoral torsional strength. In rats undergoing surgical procedures, the right femoral bones were fixed with a polyethylene plate and the defect was filled with demineralized bone matrix after defects were formed in the middle part of the diaphysis. Analyses performed on imperfect rats showed that trabecular femoral metaphyseal mineral apposition rates increased when treated with propranolol. Increased callus and bone fusion was reported after 12 weeks in rats treated with propranolol. The researchers reported that systemic propranolol treatment can significantly affect bone properties [32]. Bonnet et al. [33] reported that low-dose β-blockers can be useful as a therapeutic agent in the treatment of osteoporosis by showing high selectivity in bone tissues, and this supports our results regarding periodontal bone diseases.
In the present study, different doses of propranolol were applied after bone grafting around the implant to increase bone healing and implant osteointegration. Histologically, higher levels of new bone were detected in the groups that received propranolol. However, this difference was not statistically significant. In their experimental study of rat tibiae, Al-Subaie et al. [25] reported that propranolol enhanced bone healing and implant osseointegration. Propranolol-treated rats were found to present smaller cortical defects with more bone volume/tissue volume compared to controls. Propranolol also enhanced osseointegration, as propranolol-treated rats exhibited higher bone-implant contact (and peri-implant bone volume/tissue volume) than control animals. Similarly to previous studies, we found that propanol administration increased bone-implant contact at different doses. However, no statistically significant difference was found. Propranolol has been reported to reduce serum ALP and alanine aminotransferase (ALT) levels alone and in combination with ginger [34]. In a study by Abrar et al. [35], ALT, ALP and gamma-glutamyltransferase (γGT) levels were measured and found to be significantly higher in the rifampicin (RIF) (100 mg/ kg) group compared to the control group that received distilled water, and the group that received RIF (100 mg/kg) and propranolol (30 mg/kg) daily. Our study confirmed that ALP levels were lower in the groups treated with propanol.

Limitations
Molecular mechanisms related to changes in bone metabolism after systemic propranolol administration has not been fully explained. In vivo studies are important to determine the pathways underlying the anti-bone-absorbing effects of propranolol, but the results can only be used to predict the corresponding pathways in humans. In addition, implants are applied to various bones in the body (such as mandiblemaxilla). Due to the application of the experiment to the tibia, the results may be different in implants applied to different regions [36].

CONCLUSIONS
In the present study, alkaline phosphatase values were found lower in the groups treated with propranolol, although the difference was not statistically significant. Thus, in our new bone formation study in implants, different doses of propranolol were applied in grafting, but no statistically significant differences were obtained, despite evidence of positive results from the applied propranolol compared to the control group. More studies are needed to better understand the effects of propranolol.