Dental implant is an accepted technique for the treatment of patients with partial or complete edentulism. ¹ Osseointegration of the implant is known as the final goal of implant surgery which is considered one of the long-term implant success factors. ² When an implant is inserted into bone cavity, the recovery process will continue to osseointegration point. ³ , ⁴

Several factors can either enhance or disrupt the osseointegration process. ⁵ Factors that enhance the osseointegration process include implant design, chemical composition, implant surface topography, material, shape, and diameter of the implant, ⁶ the status of host bone and its intrinsic healing potential, ⁷ the mechanical stability and loading conditions, ⁸ and pharmacological agents such as simvastatin and bisphosphonate. ⁹ , ¹⁰

Nonspecific β-blockers are a type of systemic drugs with the ability to induce the osseointegration process. Propranolol is a commonly used nonselective β-adrenergic receptor antagonist used to treat hypertension, angina, anxiety, cardiac arrhythmia, hyperthyroidism, essential tremor, and as prophylaxis against migraine, variceal bleeding, and myocardial infarction. ¹¹

Intravenous propranolol resulted in significant decreases in cardiac output (‒25%) and heart rate (‒14%) and increases in total peripheral resistance (28%) and renal vascular resistance (37%). ¹²

Propranolol suppresses bone resorption by inhibiting RANKL-mediated osteoclastogenesis, ¹³ resulting in increased bone mass accrual and bone mineral density and reduced risk of osteoporotic fractures. ¹⁴

Propranolol appears to have significant effects on normal bone and endochondral bone formation in the defect model. Biomechanical studies on nonsurgical animals showed increased strength in propranolol compared to saline-treated rats. Propranolol may systemically stimulate bone metabolism or directly affect osteoblasts through β-adrenergic or membrane-stabilizing mechanisms. This may result from the disinhibition of an osseous metabolic pathway. ¹⁵

The suppression of β-receptors by β-blockers improves bone accrual by downregulating osteoclastic proliferation while enhancing collagen production and mineralization. ¹³ Moreover, while adrenergic stimulation through β-receptors enhances RANKL and suppresses OPG expression, ¹⁶ β-blockers (i.e., propranolol) have the opposite effect, thus dampening osteoclast differentiation. ¹³

Propranolol enhanced collagen synthesis in bone defects. It could be other reason behind the accelerated bone healing in propranolol-treated rats since collagen production is crucial for bone healing, ¹⁷ and it can be related to the fact that β-antagonists upregulate collagen synthesis through the cAMP-dependant pathway. ¹⁸

Propranolol increases intestinal absorption and decreases urinary secretion of calcium, phosphorous, and magnesium, ¹⁹ resulting in increased calcium serum levels. ¹³

It was observed that propranolol-treated rats had fewer osteoclasts in their bone defects than saline-treated ones. Propranolol enhanced collagen synthesis in bone defects; meanwhile, it increased mineralization in those defects. ²⁰

In an animal study conducted on white albino rats, it was concluded propranolol administration seems to act positively on the osseointegration procedure of stainless-steel bone implants, albeit in a nonstatistically significant manner. ²¹

Other study carried out on rats showed though there were no significant differences in biochemical parameters (alkaline phosphatase, calcium, and phosphor) of groups (P > 0.05), yet bone implant connection (BIC) ratios were detected higher in propranolol-treated animals compared to the controls (P < 0.05). ²²

Considering the lack of detailed studies on β-blockers' systemic effect on the osseointegration of titanium implants in the canine jaw, we were inspired to study propranolol's effect on osseointegration around implants in the canine jaw by histomorphometric measures.

The present study was an experimental animal study performed on dogs. Four native nondomestic street male dogs with 11–13 kg of weight and 16–20 months of age were selected for the study. The dogs were generally healthy with no preexisting systemic conditions. The inclusion criteria contain systemic health and the absence of any systemic disease. The exclusion criteria included the risk of the dog's life being at stake during the study. This animal study was approved by research and ethics committee of Isfahan university of medical sciences code: (396884). First, the necessary experiments were performed to confirm the dogs' health make sure that the dogs do not suffer from diseases such as rabies. Due to the standard protocol of the veterinarian of Research Center, dogs were vaccinated 2 weeks before the onset of the experiments. The animals were kept in separate cages for 10 days to help them get used to life in confinement. The shed at the dentistry faculty of Isfahan University of Medical Sciences had been washed and disinfected regularly and was equipped with ventilation and sewage facilities. A veterinarian measured the animals' health daily, and in case, the animals' life was at risk, they were excluded from the study for treatment.

Surgical protocol, Stage 1

In the first surgery, the dogs were sedated and subsequently anesthetized. Acepromazine 1% (0.2 cc/kg), ketamine 10% (10 mg/kg), and atropine 0.04 mg/kg were used for anesthetization. Halothane was administered to keep the animals in an anesthetized state. At first, a full-thickness flap was elevated under the left quadrant of the mandibular-premolar region (from the first to the fourth premolar), and the second, third, and fourth premolar teeth were sectioned buccolingually. The roots were extracted individually using periotom to avoid any damage to the bone walls. Finally, the flap was repositioned and sutured with 4-0 nonabsorbable suture. The dogs were randomly divided into two groups: saline administrated group (control) and propranolol administrated group (case). Administration of propranolol oral tablet was initiated in two case dogs (0.2 mg/kg). For two control dogs, oral saline was administered. After a 2-month recovery period, ceftriaxone and oral metronidazole were systemically administered one night before surgery to provide the ground for the second stage of surgery; the placement of implants. This dose of antibiotics provides a 4-day coverage, so the next dose was administered 4 days later to maintain the coverage until the 8 ^thday.

Surgical protocol, Stage 2

In the second stage of surgery, after sedation and administration of anesthesia, a horizontal crestal incision was made at the left mandibular premolar region of each animal, and three identical bone level implants (SNUCON, Korea) with 4 mm in diameter and 10 mm in length were placed.

Twelve implants were inserted in all dogs (three implants in each one). The flaps were sutured with nonabsorbable suture, and implants were submerged. The animals were then subjected to a soft diet for 14 days, and after that period, the sutures were pulled out. The gingival healing was periodically evaluated, and the remaining teeth were cleaned with ultrasound.

Sample preparation and statistical analysis

Treatment was resumed with the administration of propranolol and saline in the case and control groups, respectively. After 4 and 9 weeks, the dogs were anesthetized, and dental implants were removed alongside the peripheral bone using a 6-mm trephine drill. Samples were immediately stored in 10% formalin solution and subsequently mounted in acrylic blocks. Hard-tissue section tools are used for longitudinal incision with a diameter of 50–200 μ. Samples are mounted and painted on a Lam. The samples were then examined at ×40 magnification by an optical microscope.

BIC of samples was histomorphometrically analyzed using Nillo pathology analyzer software. Two-way ANOVA with a 95% confidence interval was used to compare the data.

The two-way ANOVA test was used to determine the effect of treatment types and the effect of postintervention time interval on BIC. Considering the effect of group treatment, results concluded that treatment type had a significant effect on BIC (P < 0.001). As for the effect of postoperative time interval, it was observed that time had a significant effect on mean BIC (P = 0.022). Meanwhile, the bilateral effect of treatment type and postoperative elapsed time was also significant. Table 1presents the mean and standard deviation of BIC(%) at week 4 and 9 respectively.{Table 1}

Due to the significance of the bilateral effect of treatment type and postoperative elapsed time and due to nonnormal distribution of data in each group, the Mann–Whitney test was used. The results as it can be observed in Table 2, showed that there is a significant difference among four groups. In the next stage, the four groups were separately compared.{Table 2}

Due to histological evaluation, newly formed bone was mostly of woven type in all samples and groups at week 4 and mostly of lamellar type in all samples and groups at week 9. Figure 1demonstrates the comparison between microscopic images of control speciments at week 4 and 9, while Figure 2demonstrates the comparison between microscopic images of case speciments at week 4 and 9. Figure 1

Microscopic images of control specimens at week 4 (left) and week 9 (right) week.

Due to the histomorphometric assessment, the mean score of bone-implant contact (BIC) in week 4 was significantly higher in the case group compared to control one (68.33% vs. 20.22%). In week 9, the mean BIC score was higher in case group compared to control one (68.60% vs. 50.17%) though it was not statistically significant.

Due to histomorphometric evaluation, in all samples and groups, newly formed bone at week 4 was mostly containing woven bone but altered to lamellar type at week 9. This finding is consistent to Berglundh's histologic findings on dogs. ²³

β2-adrenergic has a negative effect on bone modeling; thus, inhibiting this process will have a favorable effect. Pierroz et al. ²⁴ observed mice deficient in β2-receptors present decreased bone resorption and increased bone formation. The concomitant absence of β1- and β2-adrenergic signaling leads to low bone mass and low cancellous and cortical microarchitecture.

Minkowitz et al. ¹⁵ showed the beneficial effect of propranolol on bone in a fractured rat model. They concluded that 9 weeks of low-dose propranolol treatment (0.1 mg/kg/day) increased bone formation parameters, and the process occurred at the periosteum and endosteum compartments.

Takeda et al. ²⁵ indicated that propranolol could prevent the loss of vertebral trabecular bone induced by central leptin infusion and ovariectomized (OVX) in young/growing mice. They observed positive expression of Adrβ2R on rat osteoblasts in Adrβ2R-deficient mice. Bonnet et al. (2008) ²⁶ compared the effects of low (0.1 mg/kg/day), medium (5 mg/kg/day), or high (20 mg/kg/day) doses of propranolol given 5 days/week for 10 weeks in OVX rats. They observed low-dose propranolol prevented OVX-induced bone loss by increasing bone formation (+30% of MAR vs. placebo, P = 0.01) and decreasing bone resorption (‒52% of osteoclast surface on the bone surface vs. placebo, P = 0.01). In contrast, medium and high doses of propranolol had a negative effect on heart functions, no significant protective effects on bone mass in OVX rats.

In a study by Al-Subaie et al., ²⁰ on 24 Sprague–Dawley rats, a unicortical defect was created in the right tibial metaphysis of each rat, and a custom-made titanium implant was placed in the left tibia. Then, animals were assigned into two groups (n = 12, each group) and treated daily with either propranolol (5 mg/kg: subcutaneous) or saline for 2 weeks. Then, after killing, the volume of cortical defects (mm ³) and the percentages of newly formed bone in the defects were assessed with microcomputed tomography; bone-implant contact percentage and peri-implant bone volume/tissue volume were assessed by histomorphometry. The results showed 2-week administration of propranolol increased osseointegration by almost 50%, from 42.5% in controls to 73.8% among propranolol-treated ones, which is over two folds higher than the improvements reported for Bone Morphogenetic Proteins (BMPs) and parathyroid therapy, two drugs that have been used to enhance osseointegration.

In an experimental study by Karanassos et al. ^,[21] aimed to assess potential positive effect of administrating propranolol on the osseointegration procedure of stainless-steel bone implants, a custom-designed screw was implanted in tibia of 30 adult male albino rats on day 0. Starting on the 1 ^stpostoperative day, case group animals (15 rats) received 2.5 mg/kg (1 mg/ml) of propranolol daily intraperitoneally. The control group (15 rats) received the same volume of saline. On day 29, all animals were euthanized, both tibias from each animal were harvested, and the implants' pullout-strength and removal torque were assessed. Propranolol administration seems to act positively on the osseointegration procedure of stainless-steel bone implants, albeit in a nonstatistically significant manner.

The present study also resulted in a significant increase in osseointegration around titanium implants in the propranolol administrated group compared to control one after 4 weeks of implant placement; though after 9 weeks, the difference between the two groups was not significant, probably indicating the decrease of propranolol's effect on osseointegration over time. Due to this result, administrating low-dose proponalol can be beneficial when the clinician plans for early loading of the dental implant.

The present study is the first study conducted to investigate the association of β-blockers with osteointegration in the canine jaw, as an animal model comparable to humans; therefore, the possibility of generalizing the results of the study to human groups was investigated. Besides, propranolol was administered due to the usual treatment protocol, and its effects were studied and compared to the effects of preimplant and postimplant administration of drug in prospective studies.

As with all similar studies, the study at hand has certain limitations as well. A limitation of the study is the fact that an animal model was used. However, regarding the evaluation of bone implant contact of implants in clinical studies is difficult and has several limitations, the animal model chosen in this study, seems as a useful alternative.

The results of this study confirmed that administration of low-dose propranolol could have a positive effect on the mean bone-implant contact, especially in earlier rather than later phases of osseointegration which can be critical in immediate loading of dental implants or in patients with suspicious bone formation.

It is true that the canine bone model shares similarities with the human bone; nonetheless, results derived from experimental studies should be interpreted with caution. It is highly recommended to conduct clinical studies to confirm this relationship and further explore the relevant mechanisms.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.