Introduction

Bone implants are widely used in both elective maxillofacial and orthopaedic surgery and trauma. One of the main issues when dealing with operations involving the use of bone implants, is the primary fixation of the implant to the bone, which probably secures long-lasting survivorship of the implant itself2, 1 . This fixation procedure partly depends on the growth of bone at the implant-bone interface and may be the end result of a biological process, which is similar in many ways to the fracture healing response4, 3 . This procedure is elicited by implantation 6, 5 .

Beta-adrenergic receptor antagonists (beta-blockers) are important pharmacological agents in the treatment of angina pectoris, hypertension and arrhythmias 7. They also have a significant effect on bone metabolism, and therefore may play a potentially important role in reducing fracture occurrence and promoting fracture healing 8, 7. Fracture healing and implants’ osseointegration procedures share common pathways (such as osteoinduction and osteoconduction)1 , hence beta-blocker administration may enhance the latter as well.

This experimental animal study in rats aimed to assess the potentially enhancing action of beta-blocker propranolol on the osseointegration procedure of a stainless-steel bone implant following its implantation.

Methods

This blinded, in vivo animal study, was approved by the Department of Animal Health and Welfare, Veterinarian Drugs and Applications (Ref. 5741/2014; December 15th, 2014) in accordance with the Presidential Decree 56/2013 and the European Union’s Directive 63/2010. It was performed in propranolol treated and control groups, each consisting of 15 young adult 12-week-old male Wistar albino rats. The number of animals that were included in each group was pre-determined based on the available related literature 9, 4, 3 . This study was performed on male rats only, in order to avoid unnecessary discrepancies between the two groups. Animals were group-housed (two per cage) in a temperature (22°C)- and humidity (50%) -controlled vivarium, they were maintained on a 12-hour light-dark cycle (lights off at 8 AM) and had ad libitum access to commercial food and fresh water. They all followed the same pharmaceutical perioperative protocol 9 . On day 0, a custom-designed stainless steel 316 L screw (Sanmac®, Sandviken-Sweden) was implanted in the proximal metaphysis of the tibias Figure 1 , with the rats under a standard general anesthesia protocol Figure 2 and following the pre-operative administration of antibiotic prophylaxis 9 .

Figure 1 The characteristics of the custom-designed stainless steel 316 L screw (Sanmac®, Sandviken-Sweden). Hole “A” facilitated the measurement of the pullout-strength and the removal-torque. Indentation “B”served as a guide for the screwdriver.

Figure 2 The implantation procedure of the custom-designedstainless-steel screw in the proximal metaphysis of the tibia of a rat undergeneral anesthesia.

Starting on day 1, animals were randomly assigned to two groups; either receive 2.5mg/kgr of propranolol daily intraperitoneally (Dociton Injektionslösung® 1mg/ml, MIBE GmbH Arzneimittel, Brehna-Germany) forming the study group, or the same volume of saline, forming the control group. The injections (propranolol and saline) were prepared and administered daily at 09.00 A.M, in a blinded manner by a non-author caretaker. All animals were also labeled in a blinded manner, to ensure that the authors were not aware of which animal belonged to which group. Each animal was weighted at the end of each week during the experiment and the dosage of propranolol or saline were adjusted accordingly. On day 29, all animals were euthanized by exsanguination (cardiac puncture) under general anesthesia, induced by the same procedure as preoperatively 9 .

Following euthanasia, both tibiae were harvested and stripped clear from surrounding soft-tissues. They were then placed in sterile canisters, where they were covered with ambient temperature salineFigure 3 .

Figure 3 Following euthanasia, both tibiae of each animal wereharvested, stripped clear from soft-tissues and placed in a sterile canister.

The implants’ pullout strength and torque were assessed strictly within the first 2 hours following the sacrifice of the animals, with the use of a specially designed and manufactured experimental device consisting of two basic mechanisms. The first mechanism holds stable the position of the bone. The second mechanism consists of a cylindrical shaped piston that uses air pressure in order to pull the screw out of the specimen. The device also runs torque tests after proper modificationFigure 4 .

Figure 4 The pullout-strength (A) and the removal-torque (B)were measured with the use of a specially designed and manufacture dexperimental device [9].

The pullout strength was measured in right tibiae and the removal torque in the left 9 . Only after the completion of the study, the results of each animal were unblinded and statistically evaluated. This study was performed in compliance with the ARRIVE guidelines.

Statistical analysis.

For the statistical analysis, both parametric and non-parametric statistical tests were utilized. The assumptions of normality and homogeneity of variances were tested using the Shapiro-Wilk and Levene’s test, respectively. The statistical evaluation of the removal strength and the torque was made with the use of the student’s t-test. For the statistical evaluation of animal weight in the control group for two different time points, the paired samples t test was applied, whereas the animal weight in the propranolol group was analyzed using the Wilcoxon signed rank test. Data referring to the comparison of the final animal weight between the two groups, were processed with the Mann-Whitney test. Correlations between animal weight and torque, as well as between animal weight and removal strength were tested with Spearman’s rho. The evaluation of a possible statistical correlation between torque and pullout-strength was made with the Pearson’s correlation coefficient. Statistical significance level was set at p<0.05. All experimental data were analyzed with the SPSS version 20.0 (IBM Corp., Armonk, New York-USA) and are presented as mean ± SD.

Results

All animals completed the experimental period successfully and uneventfully. There were no cases of tissue healing problems and/or fractures at the operative sites and all harvested tibiae were suitable for evaluation.

The animal weight of both groups increased in a statistically significant manner (study group: p=0.002, control group: p<0.001). Additionally, the differences in the final animal weight between the two groups was statistically non-significant (p=0.299, Mann-Whitney test), hence propranolol administration had no influence whatsoever on the weight of the animals.

The mean value of the pullout-strength for the propranolol group and its Standard Deviation (SD) was 104.2±21.3 Newtons versus 90.8±18.2 Newtons (p=0.103). The torque mean value and its SD was 5.4±0.8 N/cm versus 4.9± 0.7 N/cm (p=0.09). This suggested better osseointegration of the implants in animals in the study group that postoperatively received beta-blocker propranolol, albeit in a non-statistically significant manner. No statistically significant correlation was found between the animal weight and the pullout strength (p=0.159, rs=-0.279), the animal weight and the torque (p=0.628, rs=-0.101) and between the pullout strength and the torque (p=0.193, r=0.258).

Discussion

Bone implants are widely used in both elective maxillofacial and orthopaedic surgery and trauma. The osseointegration procedure (initiation, progress and completion) of an implant to the host-bone, is one of the main issues when dealing with operations involving the use of bone implants 2, 1 . An implant is considered to be fully osseointegrated, when no progressive relative movement exists between the implant and the bone with which it has direct contact 10 . The evaluation of osseointegration is not an easy task 12, 11 . Depending on whether the study is performed on animals or is a clinical one, several invasive and non-invasive methods have been proposed and tested 13, 10. The evaluation of the osseointegration procedure in the clinical setting, necessitates mid- to long-term, large-scale, multicenter trials, which are very difficult to organize and perform. On the other hand, invasive methods, such as Histomorphometric analysis, tensional test, push-out/pull-out test and removal torque test, involve the removal of the implant following a certain waiting period to determine the extent of osseointegration, hence are limited to animal studies. The main -if not only- disadvantage of animal studies, is the fact that their results cannot be easily extrapolated into humans, since both bone biology and clinical settings differ substantially 13, 4, 3 . However, despite all these difficulties, it is still crucial to determine whether the primary fixation of an implant to the bone is secure.

The foremost part of the osseointegration procedure of a bone implant is the primary fixation 16, 15, 14 . This complex procedure is elicited by the implantation. Primary fixation facilitates the early postoperative mobilization of the patient (in orthopaedic surgery) and eventually secures long-lasting survivorship of the implant itself 14. Primary fixation depends to some extent on the growth of bone at the implant’s-bone interface and may be the end result of biological processes, which are similar in many ways to the fracture healing response, at least in terms of initial host response 19, 18, 17, 10 . This cascade of biological events is regulated by growth and differentiation factors released by the activated blood cells at the bone-implant interface 20 , recapitulating bone development and can be considered a form of tissue regeneration 21, 6.

Indirect (secondary) fracture healing is the most common form of fracture healing and consists of both endochondral and intramembranous bone healing 22, 6 . Many factors affecting the fracture healing procedure have been identified through extensive research 28, 27, 26, 25, 24, 23, 21. Beta-adrenergic receptor antagonists have a significant effect on bone metabolism and may play a potential role in reducing fracture occurrence and promoting fracture healing 7 . The administration of β-blockers was found to promote fracture healing in several studies 34, 33, 32, 31, 30, 29, 28, 7 . Propranolol in particular, was shown to be even able to rescue the deleterious effect of fluoxetine on fracture healing 36, 35 .

The effect exerted by several pharmacological agents on the osseointegration procedure has been studied to some extent as well 41, 40, 39, 38, 37, 36, 10 . Since fracture healing and osseointegration procedures share common pathways 10 , it could be assumed that any factor acting positively or negatively on the fracture healing procedure, will affect in the same way and the osseointegration procedure. These actions, need however to be verified for the osseointegration procedure as well.

Our study evaluated the stability achieved during the osseointegration process of a stainless-steel bone implant, by implementing a previously verified biomechanic evaluation model9 . Our results (pullout-strength and removal torque) were in favor of the propranolol-treated group, albeit in a non-statistically significant manner.

As with all similar studies, this one has certain limitations as well. A limitation of the study is the fact that an animal model was used. However, given the well-established evaluation procedures implemented 9 , the fact that the accurate evaluation of the immediate postoperative osseointegration procedure of implants in clinical studies is extremely difficult and has several limitations 42 , the animal model chosen in this study, seems as a useful alternative. It is true that the rat bone model shares similarities with the human bone, nonetheless, results derived from experimental studies should be interpreted with caution and should not be extrapolated to humans without further evaluation.

Another limitation was the fact that we did not perform an ad-hoc statistical analysis to determine the samples’ sizes needed to reach statistically significant results, and this determination was made based on literature research only, which revealed very few similar papers 9, 4, 3 . However, one can only speculate whether with a larger sample, statistical significance could have been reached.

The dosage of administered β-blocker propranolol was another important issue that had to be decided before the beginning of the study. Literature research on studies evaluating the action of propranolol on fracture healing failed to lead to a standard dosage44, 43 . We decided to avoid extremities and implement the dosage of 2.5 mg/Kg BW. An extrapolation of this dosage to the human, based on body weight only, is certainly not correct. This should be based on allometric scaling, taking also into consideration a correction factor reflecting the relationship between the body weight and the body surface44 . Based also on literature research, the time period of 4 weeks was also determined to be adequate to evaluate the primary stability of an orthopaedic implant 9, 4, 3 .

To the best of our knowledge, this is the first study examining the ability of β-blocker propranolol to enhance osseointegration of stainless-steel bone implants. Beta-blocker agents are widely used pharmacological agents which are usually prescribed to older patients. Their potential beneficial effect on bone metabolism in general and the osseointegration procedure of a bone implant in particular, may add even more value to their use. By performing a blinded biomechanical study, we tried to eliminate any bias. However, our results should be interpreted with caution and extrapolation of these results to humans should be supported by more research. Further studies with different animal models and/or different implants, and studies evaluating the β-blocker propranolol dose response should be the next logical step to securely reach firm conclusions. Based on their results, large-scale clinical trials, evaluating the effect exerted by propranolol on osseointegration, may come later.

CONCLUSIONS

Beta-blocker propranolol administration seems to act positively on the osseointegration procedure of stainless-steel bone implants, albeit in a non-statistically significant manner. Further studies will be necessary in order to accurately determine whether the potential beneficial effect of beta-blocker propranolol on the osseointegration procedure of bone implants really exists.

References

  1. Hydroxyapatite ceramic coating for bone implant fixation. Mechanical and histological studies in dogs Soballe K. Acta Orthop. Scand. Suppl.1993;255:1-58.
  2. Early osseointegration driven by the surface chemistry and wettability of dental implants Sartoretto S C, Alves A T, Resende R F. J. Appl. Oral Sci.2015;23:279-287.
  3. Biological Strategies for Improved Osseointegration and Osteoinduction of Porous Metal Orthopedic Implants Lewallen Eric Alexander, Riester Scott M., Bonin Carolina A., Kremers Hilal Maradit, Dudakovic Amel, Kakar Sanjeev, Cohen Robert C., Westendorf Jennifer J., Lewallen David G., van Wijnen Andre J.. Tissue Engineering Part B: Reviews.2015;21(2):218-230.
  4. Osseointegration in arthroplasty: can simvastatin promote bone response to implants? Basarir K, Erdemli B, Can A, Erdemli E, Zeyrek T. Int. Orthop.2009;33:855-859.
  5. Early Effect of Parathyroid Hormone (1???34) on Implant Fixation Skripitz Ralf, Aspenberg Per. Clinical Orthopaedics and Related Research.2001;392:427-432.
  6. Implant fixation enhanced by intermittent treatment with parathyroid hormone Skripitz R., Aspenberg P.. The Journal of Bone and Joint Surgery. British volume.2001;83-B(3):437-440.
  7. Three-dimensional Reconstruction of Fracture Callus Morphogenesis Gerstenfeld Louis C., Alkhiary Yaser M., Krall Elizabeth A., Nicholls Fred H., Stapleton Stephanie N., Fitch Jennifer L., Bauer Megan, Kayal Rayyan, Graves Dana T., Jepsen Karl J., Einhorn Thomas A.. Journal of Histochemistry & Cytochemistry.2006;54(11):1215-1228.
  8. Evaluation in vitro and in vivo of biomimetic hydroxyapatite coated on titanium dental implants Rigo E.C.S., Boschi A.O., Yoshimoto M., Allegrini S., Konig B., Carbonari M.J.. Materials Science and Engineering: C.2004;24(5):647-651.
  9. Beta-adrenergic blockers reduce the risk of fracture partly by increasing bone mineral density: Geelong Osteoporosis Study Pasco J A, Henry M J, Sanders K M. J Bone. Miner. Res.2004;19:19-24.
  10. Drugs and fracture repair Aspenberg Per. Acta Orthopaedica.2005;76(6):741-748.
  11. Molecular aspects of fracture healing:Which are the important molecules? Tsiridis Eleftherios, Upadhyay Neil, Giannoudis Peter. Injury.2007;38(1):S11-S25.
  12. Propranolol Reverses Impaired Fracture Healing Response Observed With Selective Serotonin Reuptake Inhibitor Treatment Lee Sooyeon, Remark Lindsey H, Buchalter Daniel B, Josephson Anne M, Wong Madeleine Z, Litwa Hannah P, Ihejirika Rivka, Leclerc Kevin, Markus Danielle, Yim Nury L, Tejwani Ruchi, Bradaschia‐Correa Vivian, Leucht Philipp. Journal of Bone and Mineral Research.2020.
  13. Mechanisms of endosseous integration Davies J E. Int. J. Prosthodont.1998;11:391-401.
  14. Optimal mechanical environment of the healing bone fracture/osteotomy Mavčič Blaž, Antolič Vane. International Orthopaedics.2012;36(4):689-695.
  15. Effects of propranolol on bone metabolism in the rat Minkowitz Barbara, Boskey Adele L., Lane Joseph M., Pearlman Hubert S., Vigorita Vincent J.. Journal of Orthopaedic Research.1991;9(6):869-875.
  16. Postoperative Administration of Alpha-tocopherol Enhances Osseointegration of Stainless Steel Implants Savvidis Matthaios, Papavasiliou Kyriakos, Taitzoglou Ioannis, Giannakopoulou Aggeliki, Kitridis Dimitrios, Galanis Nikiforos, Vrabas Ioannis, Tsiridis Eleftherios. Clinical Orthopaedics and Related Research.2020;478(2):406-419.
  17. Ciprofloxacin Inhibition of Experimental Fracture-Healing* HUDDLESTON P. M., STECKELBERG J. M., HANSSEN A. D., ROUSE M. S., BOLANDER M. E., PATEL R.. The Journal of Bone and Joint Surgery-American Volume.2000;82(2):161-173.
  18. Serotonin-reuptake inhibitors act centrally to cause bone loss in mice by counteracting a local anti-resorptive effect Ortuño María José, Robinson Samuel T, Subramanyam Prakash, Paone Riccardo, Huang Yung-yu, Guo X Edward, Colecraft Henry M, Mann J John, Ducy Patricia. Nature Medicine.2016;22(10):1170-1179.
  19. Effects of ?-adrenergic blockade in an osteoblast-like cell line Majeska R. J., Minkowitz B., Bastian W., Einhorn T. A.. Journal of Orthopaedic Research.1992;10(3):379-384.
  20. A Review of Osteocyte Function and the Emerging Importance of Sclerostin Compton Jocelyn T., Lee Francis Y.. The Journal of Bone and Joint Surgery.2014;96(19):1659-1668.
  21. The effect of β-blockers on bone metabolism as potential drugs under investigation for osteoporosis and fracture healing Graham Simon, Hammond-Jones Dafydd, Gamie Zakareya, Polyzois Ioannis, Tsiridis Evgenios, Tsiridis Eleftherios. Expert Opinion on Investigational Drugs.2008;17(9):1281-1299.
  22. Hydroxyapatite coating converts fibrous tissue to bone around loaded implants Soballe K, Hansen ES, Brockstedt-Rasmussen H, Bunger C. The Journal of Bone and Joint Surgery. British volume.1993;75-B(2):270-278.
  23. Effect of nebivolol on fracture healing: An experimental rat model Metineren Hasan, Dülgeroğlu Turan, Metineren Mehmet, Aydın Ekrem. Advances in Clinical and Experimental Medicine.2017;26(6):919-923.
  24. 5-year clinical and radiographic follow-up of the uncemented Symax hip stem in an international study Kruijntjens Dennis Silvester Maria Gerardus, Kjaersgaard-Andersen Per, Revald Peter, Leonhardt Jane Schwartz, Arts Jacobus Johannes Chris, ten Broeke René Hendrikus Maria. Journal of Orthopaedic Surgery and Research.2018;13(1):191-191.
  25. Regulation of Collagen Production by the β-Adrenergic System Berg Richard A., Moss Joel, Baum Bruce J., Crystal Ronald G.. Journal of Clinical Investigation.1981;67(5):1457-1462.
  26. Combined treatment with a β-blocker and intermittent PTH improves bone mass and microarchitecture in ovariectomized mice Pierroz Dominique D., Bouxsein Mary L., Rizzoli René, Ferrari Serge L.. Bone.2006;39(2):260-267.
  27. Biological options to enhance periprosthetic bone mass Tsiridis E., Gamie Z., Conaghan P.G., Giannoudis P.V.. Injury.2007;38(6):704-713.
  28. Total antioxidant capacity as a tool to assess redox status: critical view and experimental data Ghiselli Andrea, Serafini Mauro, Natella Fausta, Scaccini Cristina. Free Radical Biology and Medicine.2000;29(11):1106-1114.
  29. Clinical evaluation of osseointegration using resonance frequency analysis Chowdary SheebaGlory, Satwalekar Parth, Nalla Sandeep, Reddy Ramaswamy. The Journal of Indian Prosthodontic Society.2015;15(3):192-192.
  30. Osteoinduction, osteoconduction and osseointegration T. Albrektsson, C. Johansson. European Spine Journal.2001;10(0):S96-S101.
  31. The biology of fracture healing Marsell Richard, Einhorn Thomas A.. Injury.2011;42(6):551-555.
  32. Differential inhibition of fracture healing by non-selective and cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs Gerstenfeld Louis C., Thiede Mark, Seibert Karen, Mielke Cindy, Phippard Deborah, Svagr Bohus, Cullinane Dennis, Einhorn Thomas A.. Journal of Orthopaedic Research.2003;21(4):670-675.
  33. FACTORS AFFECTING THE DURABILITY OF PRIMARY TOTAL KNEE PROSTHESES RAND JAMES A., TROUSDALE ROBERT T., ILSTRUP DUANE M., HARMSEN W. SCOTT. The Journal of Bone and Joint Surgery-American Volume.2003;85(2):259-265.
  34. Biology of implant osseointegration Mavrogenis A F, Dimitriou R, Parvizi J, Babis G C. J. Musculoskelet. Neuronal Interact.2009;9:61-71.
  35. Low dose of propranolol does not affect rat osteotomy healing and callus strength Smitham Peter, Crossfield Lawrence, Hughes Gillian, Goodship Allen, Blunn Gordon, Chenu Chantal. Journal of Orthopaedic Research.2014;32(7):887-893.
  36. Role of primary stability for successful osseointegration of dental implants: Factors of influence and evaluation Javed Fawad, Ahmed Hameeda Bashir, Crespi Roberto, Romanos Georgios E.. Interventional Medicine and Applied Science.2013;5(4):162-167.
  37. Influence of cyclosporin A on quality of bone around integrated dental implants: a radiographic study in rabbits Sakakura Celso Eduardo, Marcantonio Elcio, Wenzel Ann, Scaf Gulnara. Clinical Oral Implants Research.2007;18(1):34-39.
  38. Effects of Nonsteroidal Anti-Inflammatory Drugs on Bone Formation and Soft-Tissue Healing Dahners Laurence E., Mullis Brian H.. Journal of the American Academy of Orthopaedic Surgeons.2004;12(3):139-143.
  39. Adrenergic receptor blockade attenuates placental ischemia-induced hypertension Spradley Frank T., Ge Ying, Haynes B. Peyton, Granger Joey P., Anderson Christopher D.. Physiological Reports.2018;6(17):e13814-e13814.
  40. A simple practice guide for dose conversion between animals and human Nair AnroopB, Jacob Shery. Journal of Basic and Clinical Pharmacy.2016;7(2):27-27.
  41. The bisphosphonate ibandronate accelerates osseointegration of hydroxyapatite-coated cementless implants in an animal model Eberhardt C, Habermann B, Müller S. J. Orthop. Sci.2007;12:61-66.
  42. Effect of low molecular weight heparin on fracture healing in a stabilized rat femur fracture model Hak David J., Stewart Rena L., Hazelwood Scott J.. Journal of Orthopaedic Research.2006;24(4):645-652.
  43. Vitamin E Biochemistry and Function: A Case Study in Male Rabbit Castellini C, Mourvaki E, Dal Bosco A, Galli F. Reproduction in Domestic Animals.2007;42(3):248-256.
  44. Effects of Chemotherapy on Osseointegration of Implants: A Case Report McDonald Alexander R., Pogrel M. Anthony, Sharma Arun. Journal of Oral Implantology.1998;24(1):11-13.