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. 2022 Jun 27;56(8):1424–1430. doi: 10.1007/s43465-022-00673-w

Effect of Locally Administered Denosumab on Bone Graft Healing in Rabbit Critical-Size Calvarial Defects.

Taha Özer 1,✉, Özgür Başlarlı 1, Alper Aktaş 1, Emre Barış 2, Mert Ocak 3
PMCID: PMC9283553  PMID: 35928658

Abstract

Background/Aim

Denosumab is a human monoclonal immunoglobulin G2 antibody developed from the ovarian cells of Chinese hamsters. We aimed to histomorphometrically and radiologically evaluate the effects of xenografts used with local denosumab on the healing of defect sites using rabbit skulls.

Materials and Methods

Two 10-mm diameter critical-size defects were created in 16 rabbits. The defect areas were filled with xenografts and xenograft + 3 mg denosumab in the control and denosumab groups (DEN), respectively. We evaluated new bone, residual graft, soft tissue areas, and bone volume in 4- and 8-week study groups.

Results

Histomorphometrically, there were no statistically significant differences between groups at both 4 and 8 weeks regarding residual graft, new bone, and soft tissue area (p > 0.05). The 4-week residual graft control group values were significantly higher than the 8-week values (p < 0.05). The soft tissue area was significantly greater in the 4-week compared with the 8-week DEN group (p < 0.05). The radiologically measured total bone volume was significantly greater in the 8-week specimens than in the 4-week specimens (p < 0.05).

Conclusion

In this study, denosumab used locally with bone grafts, showed no direct effect on new and total bone volume.

Keywords: Models, Animal, Bone transplantation, Bone histomorphometry, Denosumab, x-ray microtomography

Introduction

Orthopedic bone defects may be caused by bone disease, trauma, severe infection, congenital malformations, and tumor resections, and often require bone tissue reconstruction for medical or esthetic reasons [1]. Bone grafting is a common surgical method for defect repair, and approximately 2.2 million bone grafting operations are performed annually worldwide [2]. Autogenous grafts are the most successful and are considered the gold standard owing to their osteoinductive, osteoconductive, and osteogenic properties. However, they also have several disadvantages, including limited availability, donor site morbidity, long-term postoperative pain, and loss of function [3]. Therefore, the use of other graft materials alone or in combination with different chemical agents has become widespread recently [4].

Xenografts are defined as tissues transplanted between animals of different species and are usually of equine or bovine origin. Since xenografts are obtained from different species, the sterilization process is more sensitive than that used for allogeneic grafts and results in reduced osteoinductive properties [5, 6]. A distinctive advantage of this bone graft is that it has abundant availability, and the material cost is significantly lower than that of allogeneic bone. However, because of the reduced osteoinductive features, its use is recommended in combination with additional biological or chemical molecules [7].

Denosumab is a human monoclonal immunoglobulin G2 antibody developed by engineers from the ovarian cells of Chinese hamsters. It consists of two heavy chains and two light chains. Each light chain consists of 215 amino acids, and each heavy chain consists of 448 amino acids containing four intramolecular disulfide bonds. The mechanism of action is described as the disruption of the receptor activator of nuclear factor kappa-B (RANK)-RANK ligand (RANKL) interaction through RANKL. This prevents the formation of osteoclasts from osteoclast precursor cells. As a result, bone resorption and bone turnover decrease, whereas the mass of the bone increases in cortical and trabecular layers [8]. The pharmacokinetics of denosumab is dose-dependent, as denosumab reaches the maximum serum concentration within 10 days. The half-life of denosumab is 25–26 days, and after reaching the maximum concentration, its concentration in serum gradually decreases over 3–5 months. Six months after application, the concentration significantly decreases and cannot be detected in the serum. Therefore, the recommended dose of subcutaneously administered denosumab is 60 mg every 6 months [9]. Since denosumab is expected to be metabolized into peptides and amino acids independent of hepatic metabolism, it is not expected to be affected by systemic conditions such as liver failure. However, no study has been conducted on this subject [10]. Denosumab is used to prevent osteoporosis, particularly in the postmenopausal period in women, and to prevent metastasis in the treatment of tumors that are likely to metastasize to bone. Considering the potential systemic side effects of denosumab, local administration is often considered.

This study aimed to investigate the effects of xenografts applied in combination with local denosumab on defect sites in the rabbit skull on healing through histological, histomorphometric, and radiological methods.

Patients (Subjects) and Methods

This study protocol was performed in accordance with National Institutes of Health ARRIVE guidelines for the care and use of laboratory animals, and independently reviewed and approved by the Animal Experiments Local Ethics Committee of the participating university on 29 May 2018 under the identification number, 2017/47-03. The study included 16 male/female New Zealand rabbits weighing an average of 3500 g.

Two 10-mm diameter critical-size defects were created in each 16 rabbit calvarias. The defect areas were filled with xenografts and xenograft + 3 mg denosumab in the control and denosumab groups, respectively. The durations of the study were 4 and 8 weeks. The defect was on the right side in the calvaria of each animal in the DEN group, and on the left side in the calvaria of each animal in the C group.

Prolia (Amgen Inc., Thousand Oaks, CA, USA) was used for local denosumab administration in the experimental animals. A bovine-derived bone xenograft (BEGO OSS, BEGO GmbH & Co. KG, Bremen, Germany) and a late-resorbing collagen membrane (Collagene AT, Italy) were applied to all defects to ensure guided bone regeneration. The animals were categorized into two groups such that each experimental animal had one defect each on the left and right calvaria: denosumab + xenograft group (DEN) and xenograft/control group (C).

Animals were intramuscularly anesthetized with 35 mg/kg ketamine hydrochloride (Alfamine, Alfasan International, Woerden, The Netherlands) and 2.5 mg/kg xylazine hydrochloride (Alfazyne, Alfasan International). After anesthetization, the hair in the calvarial area was shaved. The operation site was cleaned using povidone-iodine (Batticon, Adeka, Istanbul, Turkey) according to the rules of asepsis and antisepsis. Surgical drapes were placed in the surgical field around the incision site. To control bleeding, 1 mL of local anesthetic solution (Ultracain D-S Forte, Sanofi Aventis, Istanbul, Turkey) was infiltrated in the relevant site. The bone surface was exposed through a full-thickness 4-cm long incision on the midline of the calvarium over the linea media using surgical scalpel blade no. 15 to reach the periosteum. Under sterile saline cooling, two bone osteotomies were performed on the right and left sides of the linea media on the parietal bone without damaging the dura using a trephine drill with an outside diameter of 10 mm and an inner diameter of 9 mm. Disc-shaped cortical bones between the osteotomies were removed using an elevator, and the bone defects were created (Fig. 1).

Fig. 1.

Fig. 1

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Protocol followed for bone grafting of calvarial bone defects in a rabbit model

In the DEN group, each defect was grafted with a 0.2 cc bovine-derived bone xenograft with a 0.5–1-mm particle size (BEGO OSS, BEGO GmbH & Co. KG). Subsequently, 3 mg of denosumab (60 mg/mL denosumab, Prolia, Amgen Inc., Thousand Oaks, CA, USA) was injected into the grafted area, which was then covered with a late-resorbing collagen membrane (Collagene AT; Sistema AT, Padova, Italy).

In the C group, each defect was grafted with a 0.2 cc bovine-derived bone xenograft with a 0.5–1-mm particle size (BEGO OSS, BEGO GmbH & Co. KG) and covered with a late-resorbing collagen membrane (Collagene AT; Sistema AT).

Finally, the skin and subcutaneous tissues were sutured primarily via 16-mm, 3/8, sharp, 4-0 polyglactin sutures (Coated Vicryl, Ethicon, Johnson & Johnson, Machelen, Belgium) following the revision of the muscle tissue. A wound dressing spray (Opsite, Smith & Nephew, Mississauga, ON, Canada) was applied to the sutured areas to prevent postoperative infection.

Half of the experimental animals were sacrificed at the end of the fourth week to evaluate early ossification, whereas the other half were sacrificed at the end of the eighth week to evaluate late ossification. In both groups, intramuscular xylazine HCl (30 mg/kg, Alfazyne, Alfasan International) and ketamine HCl (70 mg/kg, Alfamine, Alfasan International) were administered at lethal doses. After the elevation of the periosteum, regions where the defects were formed were identified and removed with intact bone tissue around them. Samples were separated for each rabbit and fixed in 10% buffered formaldehyde for 48 h. After the completion of micro-computed tomography (CT) screening (for evaluation of total bone volume), the samples were assessed and decalcified in a modified 10% acetic acid solution, which was changed every 3 days, for 21 days. Following the decalcification process, the tissues were washed with distilled water and dehydrated by passing through a series of alcohol solutions. Subsequently, a series of xylene solutions was used for tissue processing; the samples were embedded into paraffin blocks and stained separately with hematoxylin and eosin. Five different photographs were taken from each section of the samples at × 100 magnification. New bone trabeculae, soft tissue, and graft areas filling the defect area in each photograph were calculated in µm2.

Statistical analysis was performed using SPSS version 24.0 software (IBM Corp., Armonk, NY, USA). Descriptive data are expressed as mean, median, and standard deviation, and the Mann–Whitney U test was used for comparisons between different groups. The Wilcoxon test was used to compare the fourth- and eighth-week data for each group. A p value of < 0.05 was considered statistically significant.

Results

Histological examination of the animals in the 4-week groups confirmed that the defect area was composed of new bone trabeculae and loose collagenous connective tissue. The connective tissue increasingly comprised cellular collagen as it approached the center of the defect. Centripetal new bone growth was observed by activation of the injured periosteum, implying that it was induced from the edges of the defect toward the center. This centripetal growth was more significant in the DEN group.

Considering the histological preparations of samples in the 8-week groups, more bone trabeculae were found in this group than in the 4-week samples of the same group. Graft particles were observed between these trabeculae; however, there were fewer graft particles in the 8-week samples than in the 4-week samples. Loose collagenous connective tissue, which exhibited cellular collagenization as it approached the defect center, was observed between the bone trabeculae. Although bone formation was slightly greater toward the center, bone formation in the entire defect site was not observed in any of the groups (Fig. 2).

Fig. 2.

Fig. 2

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Histological findings at 4 and 8 weeks after surgery for calvarial bone defects in a rabbit model (hematoxylin and eosin, ×40) C group, no treatment; DEN group, denosumab irrigation with xenogeneic bone grafting; nb new bone trabeculae, g graft material, st soft tissue, hb host bone

Radiological comparison of the groups revealed no significant difference between the groups at two different times in terms of total bone area values (p > 0.05). In both groups, the total bone area value at the eighth week was significantly higher than that at the fourth week (p < 0.05) (Figs. 3 and 4).

Fig. 3.

Fig. 3

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Micro-computed tomography images of all groups at 4 weeks and 8 weeks

Fig. 4.

Fig. 4

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Comparison of the percentages of total bone derived from radiographs obtained at 4 and 8 weeks after surgery (C group, no treatment; DEN group, denosumab irrigation with xenogeneic bone grafting; a significantly different from values obtained at 8 weeks after surgery)

Histomorphometric comparison of the groups revealed that there was no statistically significant difference between the groups in terms of residual graft, new bone formation, and soft tissue measurement averages at the fourth and eighth weeks (p > 0.05). In the control group, the residual graft value at the fourth week was significantly higher than that at the eighth week (p < 0.05). In both groups, the mean soft tissue measurements at the eighth week were significantly lower than those at the fourth week (p < 0.05) (Fig. 5).

Fig. 5.

Fig. 5

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Comparison of values for the new bone, soft tissue, and residual graft area derived by histomorphometry obtained at 4 and 8 weeks after surgery (C group, no treatment; DEN group, denosumab irrigation with xenogeneic bone grafting; a significantly different from values obtained at 8 weeks after surgery)

Discussion

Bone healing is an extensive physiological process. Osteoblasts and osteoclasts are the two main cell types responsible for this mechanism. For successful bone grafting, the relationship between these cells must be balanced. A disturbance in the balance, which can be caused by bone diseases (e.g., osteomalacia and Paget’s disease), medications used (e.g., bisphosphonates, denosumab, and vitamin D), and systemic diseases (e.g., hyperparathyroidism and hypercalcemia), is bidirectional and can result in deterioration or delay in the healing process. If the balance is disrupted in favor of osteoclasts, conditions such as osteoporosis may occur, whereas osteopetrosis may develop if the balance is disrupted in favor of osteoblasts [11]. Currently, medicaments that have been recommended for use in various systemic diseases and are known to have serious side effects on bone metabolism are frequently used. These include denosumab, which is used as an alternative to bisphosphonates in the treatment of osteoporosis and hypercalcemia of malignancies. This study aimed to evaluate the effectiveness of local denosumab in bone defect healing in a rabbit calvarial defect model.

Gerstenfeld et al. systemically administered 10 mg/kg denosumab and 0.1 mg/kg alendronate twice weekly to rats and compared the effects of both on created femoral fractures. The authors observed a greater amount of unresorbed cartilage tissue in both antiresorptive agent groups than in the control group, but there was no statistically significant difference between the two antiresorptive agents [12].

The most important side effects of orally administered bisphosphonates are gastrointestinal inflammation and ulcers. Intravenous administration may result in nephrological damage and eye infections. Furthermore, ulceration occurs when the drug remains in the oral cavity for a long time and osteonecrosis develops in the jawbone as a result of chronic bisphosphonate treatment [13]. Previous studies showed that denosumab also has several effects, including severe hypocalcemia, jaw osteonecrosis, and atypical stress fractures [14–16]. Teng et al. showed that denosumab can lead to hypocalcemia, especially in patients with vitamin D deficiency and severe renal impairment (creatinine clearance < 30 mL/min) or those receiving hemodialysis [17]. Moreover, denosumab has been associated with osteonecrosis of the jaw (1.8%), similar to that observed with intravenous bisphosphonates (1.3%) [16]. Considering these systemic side effects, this study investigated the effectiveness of its local use.

There are many studies on locally applied bisphosphonates in the literature, and their antiresorptive effects have been shown [18–22]. However, according to the results of the present study, no statistically significant difference was found between the locally applied denosumab group and the control group. These results distinguish the local efficacy of denosumab from that of bisphosphonates. The efficacy of locally applied denosumab was not demonstrated in this study; therefore, there is a need for further studies comparing the systemic and local efficacies of denosumab.

Our literature review revealed that there were no reported studies about locally administered denosumab. Hence, to the best of our knowledge, this is the first study completed on this subject. In this study, 3 mg of denosumab dosage is used while the doses from the bisphosphonate studies are referred and we believe that it will lead to future dose studies.

Reported experimental defect areas in rabbits include the mandible, calvaria, femur, tibia, fibula, and radius. In this study, the calvaria bone was selected as the operative site because its embryological developmental characteristics resemble those of the human maxillofacial bone [23]. The skeletal system contains bones that are inherently loaded by distinct mechanical force patterns. Long bones are loaded predominantly along the longitudinal direction with significantly higher amplitude than flat bones, such as calvaria, which are loaded radially and tangentially due to intracranial pressure and mastication. For example, the human fibula is estimated to have a load that is nearly twice that of the skull bone. Extracellular mechanical forces are coupled with the intracellular organization of the cytoskeleton, thus affecting cell shape and functional outputs. Therefore, the morphology of osteocytes, the pattern of the osteocyte network, and their function are determined by external loading, the directional patterns and amplitudes of which are physiologically different in each bone [24]. This study is valuable for showing the effect of denosumab on healing in flat bones. However, further studies are needed to compare this effect with the effects of denosumab on long bones.

Previous studies on bone formation in rabbit calvarial defects used 4 weeks and 8 weeks as the healing periods. Spontaneous healing potential of critical-size defects in rabbit calvaria were examined in previous studies and stated that 2 or 4 weeks would be appropriate for observing the early effects of the healing period in this defect model, and 8 or 12 weeks to observe the late effects. The present study used 4 and 8 weeks as the healing periods to evaluate both the early and late phases of bone formation, along with the resorption of the materials used.

Guided bone regeneration was first defined in 1959 and has continued to be used for various indications. With this method, the connective tissue elements are prevented from invading the defect before osteogenic cells fill it [25]. The most common barrier materials used for this purpose are polytetrafluoroethylene, expanded polytetrafluoroethylene, collagen, freeze-dried dura mater, polyglactin 910, polylactic acid, polyglycolic acid, polyorthoester, polyurethane, polyhydroxybutyrate, calcium sulfate, and titanium foils. In the present study, the collagen membrane was the barrier of choice owing to its high biocompatibility and low antigenicity. Further, the membrane improves cellular penetration, is hydrophilic, decomposes into tolerable physiological components, and has successful mechanical properties [26].

The osteogenic properties of autogenous grafts stimulate new bone formation. Autogenous grafts are the gold standard because of this property and are the first choice as bone grafts. However, procuring autografts increases the duration of surgery, morbidity, postoperative discomfort, recovery time, and costs because it requires a second field of operation. Furthermore, the rate of resorption of autogenous grafts remains a serious disadvantage. Hence, the use of other graft materials has become more common in clinical practice; one such product is the bovine-derived bone xenograft. Due to the low resorption rates, xenografts prevent the need for a second surgical site as required for the procurement of an autologous graft, provide a stable scaffold for bone formation, and maintain long-term graft volume stability [27, 28]. Bovine-derived bone xenografts were used in the present study because of the aforementioned features.

According to the histomorphometric analysis results, a significant difference was found in the control group in terms of the residual graft measurement averages between the 4-week and 8-week samples. However, there was no significant difference in the denosumab group. Although these data showed the antiresorptive efficacy of denosumab on the graft, no significant difference was observed in the new bone measurement averages in the fourth and eighth weeks. Furthermore, there was no significant difference in total bone measurements in radiological or histomorphometric analyses. This can be attributed to the preservation of the residual graft area through the antiresorptive activity of denosumab, and any difference was eliminated by new bone formation in the control group.

Two-dimensional histomorphometric analysis and three-dimensional micro-CT analysis are the most reliable methods for measuring the amount of newly formed bone and soft tissue. In the literature, more accurate and detailed results have been reported in studies conducted with micro-CT analysis. This difference is mainly due to the fact that two-dimensional central examination is performed on histomorphometric analysis, whereas a wider three-dimensional examination can be performed in micro-CT analysis [29]. In the present study, the histomorphometric analysis results were supported by a more detailed micro-CT analysis, and the results were found to be in line with the histomorphometric results.

In conclusion, this study showed the antiresorptive efficacy of denosumab for use in bone defects grafted with xenografts; however, the effect of denosumab on ossification could not be demonstrated. If the predicted results in this study had been achieved, denosumab would have been clinically shown to help a much faster graft recovery by being free of all systemic side effects. The administered dose or auxiliary/carrier substances in the denosumab solution can be cited as a reason for this negative results.

There is a need for further studies on the concentration of local denosumab and its use in combination with artificial bone grafts or autografts.

Author Contribution

Conceptualization: Taha Özer, Özgür Başlarlı, Alper Aktaş; Methodology: Taha Özer, Emre Barış; Formal analysis and investigation: Taha Özer, Emre Barış, Mert Ocak; Writing - original draft preparation: Taha Özer; Writing - review and editing: Alper Aktaş; Funding acquisition: Taha Özer, Özgür Başlarlı; Resources: Emre Barış, Mert Ocak; Supervision: Alper AktaşAll authors read and approved the final manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declarations

Conflict Of Interest

The authors have no competing interests to declare that are relevant to the content of this article.

Ethical Approval

This study protocol was performed in accordance with National Institutes of Health ARRIVE guidelines for the care and use of laboratory animals and independently reviewed and approved by the Animal Experiments Local Ethics Committee of Hacettepe University on 29 May 2018 under the identification number, 2017/47-03.

Informed Consent

Not applicable.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Gaihre B, Uswatta S, Jayasuriya AC. Reconstruction of craniomaxillofacial bone defects using tissue-engineering strategies with injectable and non-injectable scaffolds. Journal Of Functional Biomaterials. 2017;8(4):49. doi: 10.3390/jfb8040049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Giannoudis P, Psarakis S, Kontakis G. Can we accelerate fracture healing? A critical analysis of the literature. Injury. 2007;38(Suppl 1):S81–9. doi: 10.1016/j.injury.2007.02.013. [DOI] [PubMed] [Google Scholar]
  • 3.Lima RG, et al. Bone volume dynamics and implant placement torque in horizontal bone defects reconstructed with autologous or xenogeneic block bone: a randomized, controlled, split-mouth, prospective clinical trial. International Journal of Oral and Maxillofacial Implants. 2018;33(4):888–894. doi: 10.11607/jomi.6288. [DOI] [PubMed] [Google Scholar]
  • 4.Bezerra BT, et al. Autogenous bone graft versus bovine bone graft in association with platelet-rich plasma for the reconstruction of alveolar clefts: a pilot study. The Cleft Palate-Craniofacial Journal. 2019;56(1):134–140. doi: 10.1177/1055665618770194. [DOI] [PubMed] [Google Scholar]
  • 5.Moussa NT, Dym H. Maxillofacial bone grafting materials. Dental Clinics of North America. 2020;64(2):473–490. doi: 10.1016/j.cden.2019.12.011. [DOI] [PubMed] [Google Scholar]
  • 6.Lee YC, et al. Comparing the osteogenic potentials and bone regeneration capacities of bone marrow and dental pulp mesenchymal stem cells in a rabbit calvarial bone defect model. International Journal of Molecular Sciences. 2019;20(20):5015. doi: 10.3390/ijms20205015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Jensen J, et al. Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model. SICOT J. 2016;2:16. doi: 10.1051/sicotj/2016004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ruggiero SL, et al. American association of oral and maxillofacial surgeons position paper on medication-related osteonecrosis of the jaw–2014 update. Journal of Oral and Maxillofacial Surgery. 2014;72(10):1938–56. doi: 10.1016/j.joms.2014.04.031. [DOI] [PubMed] [Google Scholar]
  • 9.Sutjandra L, et al. Population pharmacokinetic meta-analysis of denosumab in healthy subjects and postmenopausal women with osteopenia or osteoporosis. Clinical Pharmacokinetics. 2011;50(12):793–807. doi: 10.2165/11594240-000000000-00000. [DOI] [PubMed] [Google Scholar]
  • 10.Deeks ED. Denosumab: a review in postmenopausal osteoporosis. Drugs and Aging. 2018;35(2):163–173. doi: 10.1007/s40266-018-0525-7. [DOI] [PubMed] [Google Scholar]
  • 11.Meng ZL, et al. Reconstruction of large segmental bone defects in rabbit using the Masquelet technique with alpha-calcium sulfate hemihydrate. Journal of Orthopaedic Surgery. 2019;14(1):192. doi: 10.1186/s13018-019-1235-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Gerstenfeld LC, et al. Comparison of effects of the bisphosphonate alendronate versus the RANKL inhibitor denosumab on murine fracture healing. The Journal of Bone and Mineral Research. 2009;24(2):196–208. doi: 10.1359/jbmr.081113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Cryer B, Bauer DC. Oral bisphosphonates and upper gastrointestinal tract problems: what is the evidence? Mayo Clinic Proceedings. 2002;77(10):1031–43. doi: 10.4065/77.10.1031. [DOI] [PubMed] [Google Scholar]
  • 14.Hamadeh IS, Ngwa BA, Gong Y. Drug induced osteonecrosis of the jaw. Cancer Treatment Reviews. 2015;41(5):455–64. doi: 10.1016/j.ctrv.2015.04.007. [DOI] [PubMed] [Google Scholar]
  • 15.Diz P, et al. Denosumab-related osteonecrosis of the jaw. The Journal of the American Dental Association. 2012;143(9):981–4. doi: 10.14219/jada.archive.2012.0323. [DOI] [PubMed] [Google Scholar]
  • 16.Thosani S, Hu MI. Denosumab: a new agent in the management of hypercalcemia of malignancy. Future Oncology. 2015;11(21):2865–71. doi: 10.2217/fon.15.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Teng J, et al. Protracted hypocalcaemia following a single dose of denosumab in humoral hypercalcaemia of malignancy due to PTHrP-secreting neuroendocrine tumour. Clinical Endocrinology (Oxford) 2014;81(6):940–2. doi: 10.1111/cen.12519. [DOI] [PubMed] [Google Scholar]
  • 18.Kitasato S, et al. Local application of alendronate controls bone formation and beta-tricalcium phosphate resorption induced by recombinant human bone morphogenetic protein-2. Journal of Biomedical Materials Research Part A. 2020;108(3):528–536. doi: 10.1002/jbm.a.36833. [DOI] [PubMed] [Google Scholar]
  • 19.Saulacic N, et al. Effects of local application of alendronate on early healing of extraction socket in dogs. Clinical Oral Investigations. 2020;24(4):1579–1589. doi: 10.1007/s00784-019-03031-7. [DOI] [PubMed] [Google Scholar]
  • 20.Heidari P, et al. Effect of risedronate on bone resorption during consolidation phase of distraction osteogenesis: a rabbit model. Journal of Orthopaedic Surgery (Hong Kong) 2010;18(2):228–34. doi: 10.1177/230949901001800219. [DOI] [PubMed] [Google Scholar]
  • 21.He Y, et al. Effects of systemic or local administration of zoledronate on implant osseointegration: a preclinical meta-analysis. BioMed Research International. 2019;2019:9541485. doi: 10.1155/2019/9541485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kellesarian SV, et al. Effect of local zoledronate delivery on osseointegration: a systematic review of preclinical studies. Acta Odontologica Scandinavica. 2017;75(7):530–541. doi: 10.1080/00016357.2017.1350994. [DOI] [PubMed] [Google Scholar]
  • 23.Leucht P, et al. Embryonic origin and hox status determine progenitor cell fate during adult bone regeneration. Development. 2008;135(17):2845–54. doi: 10.1242/dev.023788. [DOI] [PubMed] [Google Scholar]
  • 24.Himeno-Ando A, et al. Structural differences in the osteocyte network between the calvaria and long bone revealed by three-dimensional fluorescence morphometry, possibly reflecting distinct mechano-adaptations and sensitivities. Biochemical and Biophysical Research Communications. 2012;417(2):765–70. doi: 10.1016/j.bbrc.2011.12.031. [DOI] [PubMed] [Google Scholar]
  • 25.Zhang HY, et al. Comparing properties of variable pore-sized 3D-printed PLA membrane with conventional PLA membrane for guided bone/tissue regeneration. Materials (Basel) 2019;12(10):1718. doi: 10.3390/ma12101718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Lundgren AK, et al. Influence of decortication of the donor bone on guided bone augmentation. An experimental study in the rabbit skull bone. Clinical Oral Implants Research. 2000;11(2):99–106. doi: 10.1034/j.1600-0501.2000.00002.x. [DOI] [PubMed] [Google Scholar]
  • 27.Orsini G, et al. Histological and ultrastructural evaluation of bone around Bio-Oss particles in sinus augmentation. Oral Diseases. 2007;13(6):586–93. doi: 10.1111/j.1601-0825.2006.01343.x. [DOI] [PubMed] [Google Scholar]
  • 28.Jensen T, et al. Volumetric changes of the graft after maxillary sinus floor augmentation with Bio-Oss and autogenous bone in different ratios: a radiographic study in minipigs. Clin Oral Implants Research. 2012;23(8):902–10. doi: 10.1111/j.1600-0501.2011.02245.x. [DOI] [PubMed] [Google Scholar]
  • 29.Deluiz D, et al. Time-dependent changes in fresh-frozen bone block grafts: tomographic, histologic, and histomorphometric findings. Clinical Implant Dentistry and Related Research. 2015;17(2):296–306. doi: 10.1111/cid.12108. [DOI] [PubMed] [Google Scholar]

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