[Frontiers in Bioscience E4, 2647-2653, June 1, 2012]

Use of BMPs and bisphosphonates to improve bone fracture healing

Nicole Y.C Yu1,3, Aaron Schindeler1,2, Magnus Tagil4, Andrew J. Ruys3, David G. Little1,2

1Department of Orthopaedic Research and Biotechnology, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW 2145, Australia, 2Discipline of Paediatrics and Child Health, Faculty of Medicine, A27 University of Sydney, NSW 2006, Australia, 3School of Aerospace, Mechanical and Mechatronic Engineering, J07 University of Sydney, NSW 2006, Australia, 4Department of Orthopedics, Lund University Hospital, SE-221 85 Lund, Sweden

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Bisphosphonates and bone repair
4. Local BMP and systemic bisphosphonate treatment
5. Local BMP and bisphosphonate co-treatment
6. Conclusions

1. ABSTRACT

In orthopaedics, focus is often placed on increasing bone formation by an anabolic drug like the recombinant human bone morphogenetic protein (rhBMP). However, premature or excessive bone resorption, due to stress-shielding, instability or infection/inflammation can lead to poor, delayed, or absent bone union. Anti-catabolic drugs such as bisphosphonates have therefore been explored to improve bone repair. This short review discusses the current literature underlying the anabolic-catabolic paradigm for bone repair with a focus on BMP and bisphosphonate combination approaches.

2. INTRODUCTION

Recombinant human bone morphogenetic proteins (rhBMPs) are potent stimulators of bone anabolism that are in clinical use for the treatment of non-union and critical sized bone defects. Although BMPs stimulate bone anabolism, they also increase osteoclast differentiation, mature osteoclast survival, and osteoclastic bone resorption (1-3). Furthermore, BMPs can stimulate osteoclastogenesis indirectly through osteoblasts via the RANK/RANKL pathway (4). In a clinical setting, this can lead to the premature resorption of BMP-induced bone if early resorptive stimuli predominate. Several clinical reports of failure exist because of premature catabolism associated with BMP use. These include loss of fixation in vertebral fractures, premature allograft resorption in the spine (5), and some cases of failure and loosening in hip revision arthroplasty (6).

One approach to prevent premature or excessive bone catabolism associated with BMP application is by pharmaceutical manipulation of the osteoclasts using an anti-resorptive agent (7). Bisphosphonates (BPs) are a class of drugs that bind to bone and inhibit osteoclast-mediated resorption. BPs have been extensively studied experimentally (8-15) and are in clinical use for the treatment of osteoporosis and other metabolic bone disorders. The present review will describe examples to maximise net bone production in an orthopaedic setting by combining BMPs and BPs, aiming for maximal formation and minimal resorption. First we will outline the experimental findings with bisphosphonates and fracture repair that led to such a therapeutic approach and describe a number of instances where systemic BPs have been successfully used in combination with BMPs. Finally, we will summarise the findings of several recent studies focused on combining BMPs with local bisphosphonates.

3. BISPHOSPHONATES AND BONE REPAIR

Bisphosphonates (BPs) are a class of drug that have clinical efficacy for the treatment of osteoporosis and other metabolic bone disorders, and are increasingly being explored for orthopaedic indications. Newer bisphosphonates have increased potency due to the presence a nitrogen-containing side chain (15). Nitrogen-containing bisphosphonates (pamidronate, alendronate, risedronate, ibandronate, zoledronic acid) inhibit an enzyme called farnesyl diphosphate (FFP) synthase which leads to cellular dysfunction and apoptosis, while the older non-N-BPs (clodronate) produce toxic metabolites in the mitochondria (14).

Bisphosphonates accumulate at sites of bone mineral deposition, in particular in regions of high bone turnover such as a fracture site (12). The drug is released during subsequent bone resorption and internalised by osteoclasts leading to inhibition of osteoclast activity and/or osteoclast apoptosis. Consequently, bisphosphonates are potent inhibitors of osteoclast mediated bone resorption (14).

Bone repair is a dynamic and complex process that features an early inflammatory response, followed by formation of a soft callus, its replacement with a hard mineralized callus, and finally remodeling (16). Premature and excessive remodelling of the hard callus might occur also during fracture healing, due to stress-shielding or instability, and might result in a weaker initial union, or even a delayed or non-union. The capacity of bisphosphonates to improve repair by preventing early remodelling have been examined in a number of models.

In a rat open osteotomy model, significant increases in callus volume and bone mineral content were seen with pamidronate (PAM) treatment. This led to a 60% increase in strength compared to controls at 6 weeks post-operatively (11). In a closed fracture model, rats treated with the more potent bisphosphonate zoledronic acid (ZA), again showed increases in callus size and strength (12). Subsequent studies investigating various dosing regimens indicated that a single bolus systemic dose could provide equivalent benefits to bone strength compared to multiple smaller intermittent doses. The single dose, however featured superior bone remodelling following union (17). Benefits were also seen in rabbit models of distraction osteogenesis, an orthopaedic process that is also depending on an adequate anabolic response (8, 10). In a randomised study of patients operated with a tibial osteotomy for gonarthrosis, the time to clinical healing was evaluated (18). The patients were given either ZA 5 mg i.v. or saline four weeks postoperatively as a single infusion. The extraction torque of the screws for the external fixator used to secure the osteotomy during healing was higher in the treated group, however, no effect could be seen by the ZA infusion regarding time to healing.

However, bone repair is primarily an anabolic process and bisphosphonate treatment only has the potential to maximise the effects of a system's intrinsic bone forming potential (6). Thus it was speculated that bisphosphonates could be effectively combined with bone forming agents such as BMPs, which themselves can also stimulate bone resorption (1-4).

4. LOCAL BMP AND SYSTEMIC BISPHOSPHONATE TREATMENT

A simple model to demonstrate synergy between BMPs and BPs is the rat tibial bone chamber model (Figure 1) (19). An allograft is inserted into the chamber and ingrowth can occur from one end of the graft. Using the ingrowth distance into the graft at 6 weeks as anabolic equivalent, an increase was caused by rhBMP-7 compared to saline or ZA alone. Similarly, using the BV/TV as anti-catabolic outcome, a more dense bone was found in the remodelled part of the allograft in the group receiving a single systemic dose of 0.1 mg/kg ZA at 2 weeks, compared to BMP or saline. Combining the two, rhBMP-7 + ZA co-treatment increased both parameters resulting in both increased ingrowth distance and increased density and with an increased net bone formation, compared to all other groups (bone graft only, rhBMP-7, and ZA only) (Figure 2). It was concluded that the unloaded bone chamber constitutes an extremely pro-catabolic environment and that systemic ZA was effective in preventing resorption and optimising the net bone. However, one study has shown less positive effects with local BMP and bisphosphonate icadronate co-delivered via type I collagen carrier in a rat ectopic bone formation model. Gong et al. demonstrated adjunct treatment with 5 microg rhBMP-7 and systemic incadronate (1 microg/kg thrice weekly, 3rd-7th weeks post-implantation) to block bone maturation and formation, as well as significantly reducen tartrate resistant acid phosphatase-positive cells numbers (20).

Further convincing evidence for synergy between local BMP and systemic BP reported also in a more clinically relevant model was shown in a rat critical sized defect model (9). This model features a deficient anabolic response that leads to a 100% healing failure. Addition of 50 microg rhBMP-7 (OP-1) to the fracture gap was sufficient to lead to a tenuous union in the majority of animals at 8 weeks. The addition of a single systemic bolus dose of ZA further dramatically increased callus formation leading to an 87% increase in callus volume and an 107% increase in callus strength compared to rhBMP-7 alone. Notably, there was a significant benefit delaying the dosing of systemic ZA to 2 weeks after surgery rather than administer the drug at the time of surgery (Figure 3). By speculation, the early bony callus at 2 weeks may be able to capture and retain additional bisphosphonate compared to a newly fractured bone.

This combination of an anabolic and an anti-catabolic drug may have additional benefit in the context of genetic disease, such as Neurofibromatosis type 1 (NF1) - a condition featuring decreased bone anabolism and an increased potential for catabolism (21). In a study with NF1-deficient mice that showed a reduced response to intramuscular implantation of 20 �g rhBMP-2, co-treatment with systemic ZA (5 doses of 0.02 mg/kg over 3 weeks) was able to maximize the amount of bone produced (22). This approach has been found to be effective in a clinical case series for NF1 tibial pseudarthrosis patients treated with BMP and BP (23), although adjunctive pharmaceutical treatment was also found to depend on effective surgical fixation. Further studies will be needed to demonstrate the value of this strategy for intervening in other genetic bone diseases.

5. LOCAL BMP AND BISPHOSPHONATE CO-TREATMENT

The first report of a combined local application of BMP and BP to a bone graft was in the rat tibial bone chamber (24). Clodronate (CLOD) a first generation BP was administered locally to an impacted bone graft model with non-impacted grafts as controls with or without addition of BMP-7. CLOD was found to increase bone graft density and eliminate the transient bone resorptive effect of BMP-7. However, local CLOD did also reduce the ingrowth distance of new bone into the graft. Adding BMP, the negative effect of both graft impaction as well as CLOD administration could be at least partly reversed (24). Interestingly, this study implied that the anti-anabolic effects of CLOD may be more significant when applied locally. Recently, the same rat bone chamber model was used to explore the combination of locally applied BMP-7 + ZA, added to non-impacted allografts (25). An almost four-fold increase in net formation of bone was found by the combination of BMP and ZA compared to saline. The ingrowth distance of both ZA alone as well as the combination BMP-7 and ZA, however appeared to be less than in the previous series using systemic bisphosphonates, implying an anti-anabolic effect of the bisphosphonate.

In another study, rhBMP-2 and the bisphosphonate minodronate were locally co-delivered in a rat intramuscular ectopic bone formation model. Addition of the bisphosphonate prevented bone loss over 4 weeks and led to a net increase in bone density and mechanical strength over rhBMP-2 alone (26). In a follow-up study, the ectopic bone was successfully used as a graft material in a femoral muscle pedical flap model (27).

Lastly the combination of BMP-2 and a potent bisphosphonate, ibandronate, has been trialled in the piglet model of ischemic necrosis, where collapse of the femoral head occurs routinely (28). In comparison to the control group, the combined therapy group had a significant decrease in femoral head deformity with a significant increase in the trabecular bone volume and osteoblast surface. One cautionary finding was the presence of heterotopic ossification in the hip joint capsule, requiring further refinement of delivery.

Not all local bisphosphonate studies have yielded favourable outcomes. Local delivery of high doses of PAM (2 mg and 3 mg) in a rat calvarial defect model was found to impair bone repair (29). While this model did not feature rhBMP treatment, similar impairment was found in a canine titanium implant fixation model with morselised bone graft soaked in high dose Alendronate (ALN). Furthermore, local ALN-treated grafts showed inhibited new bone formation and reduced resorption of the graft material (30). Later, another canine implant fixation model by the same group also found similar impaired implant fixation with 450 microg rhBMP-2 and bone allograft were treated with 9 mg/ml PAM (31). Notably, all of these studies featured a high local concentration of bisphosphonate, and as reviewed by Schindeler and Little, such doses have been reported to be cytotoxic based on in vitro studies (13).

These observations led us to hypothesise that the capacity of local bisphosphonates to positively modulate the formation and retention of rhBMP-induced bone would be dose dependent. This concept was confirmed in recent study in a mouse intramuscular ectopic bone formation model (32). Variable doses of the bisphosphonate PAM were combined with a constant 25 microg dose of rhBMP-7 in a solvent-cast poly-D,L-lactic acid (PDLLA) polymer delivery system. It was found that high 2 mg local dose of PAM dramatically impaired the formation of rhBMP-7 induced bone. In contrast, lower dose of 0.02 mg PAM was able to yield an overall increase in net bone. Similarly, in a follow up study to their bone grafting work, Jakobsen et al. also showed that the effects of local zoledronic acid differed between a low dose where a favourable increase of bone in the gap and on the implant surface was seen, whereas higher doses were inhibitory. These data illustrate the importance of appropriate bisphosphonate dose selection for pre-clinical and clinical applications (30).

6. CONCLUSIONS

There is a growing body of pre-clinical evidence, particularly in rodent models, to support the biology behind BMP and bisphosphonate as dual interventions. In this review we have discussed the progression from use of bisphosphonates alone in systems featuring sufficient anabolism to facilitate bone healing to combinations of BMPs with systemic and local bisphosphonates. One continually emerging theme is that bisphosphonate dosage, particularly when applied locally, is critically important for the eventual outcome.

The number of studies specifically examining synergy between BMPs and bisphosphonates are limited. More research, particularly focused on timing of systemic doses, dosing rates, as well as local co-delivery systems for both agents will be required for translation to clinical practice. The combination of other anabolic and anti-catabolic agents is also being actively explored.

7. ACKNOWLEDGEMENTS

Nicole Yu receives a Biomedical Postgraduate Scholarship from the National Health & Medical Research Council. Dr. Aaron Schindeler's salary is supported by a Children's Tumor Foundation Young Investigator Award.

8. REFERENCES

1. Itoh K, Udagawa N, Katagiri T, Iemura S, Ueno N, Yasuda H, Higashio K, Quinn MW, Julian MW, Gillespie MT, Martin TJ, Suda T, Takahashi N. Bone morphogenetic protein 2 stimulates osteoclast differentiation and survival supported by receptor activator of nuclear factor-kB Ligand. Endocrinology 142: 3656-3662 (2001)
http://dx.doi.org/10.1210/en.142.8.3656

2. Kaneko H, Arakawa T, Mano H, Kaneda T, Ogasawara A, Nakagawa M, Toyama Y, Yabe Y, Kumegawa M, Hakeda Y. Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts. Bone 27: 479 (2000)
http://dx.doi.org/10.1016/S8756-3282(00)00358-6

3. Paul S, Lee J, Yeh L. A comparative study on BMP-induced osteoclastogenesis and osteoblastogenesis in primary cultures of adult rat bone marrow cells. Growth Factors 27 (2009)

4. Katagiri T, Takahashi N. Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Diseases 8: 147-159 (2002)
http://dx.doi.org/10.1034/j.1601-0825.2002.01829.x

5. Pradhan B, Bae H, Dawson E, Patel V, Delamarter R. Graft resorption with the use of bone morphogenetic protein: lessons from anterior lumbar interbody fusion using femoral ring allografts and recombinant human bone morphogenetic protein-2. Spine (Phila Pa 1976) 31: E377-384 (2006)

6. Kärrholm J, Hourigan P, Timperley J, Razaznejad R. Mixing bone graft with OP-1 does not improve cup or stem fixation in revision surgery of the hip: 5-year follow-up of 10 acetabular and 11 femoral study cases and 40 control cases. Acta Orthop 77: 39048 (2006)

7. Little DG, Ramachandran M, Schindeler A. The anabolic and catabolic responses in bone repair. J Bone Joint Surg Br 89-B: 425-433. (2007)
http://dx.doi.org/10.1302/0301-620X.89B4.18301
PMid:17463107

8. Little D, Cornell M, Hile M, Briody J, Cowell C, Bilston L. Effect of pamidronate on distraction osteogenesis and fixator-related osteoporosis. Injury 32: SD14-20 (2001)
http://dx.doi.org/10.1016/S0020-1383(01)00161-9

9. Little D, McDonald M, Bransford R, Godfrey C, Amanat N. Manipulation of the anabolic and catabolic responses with OP-1 and zoledronic acid in a rat critical defect model. J Bone Miner Res 20: 2044-2052 (2005)
http://dx.doi.org/10.1359/JBMR.050712
PMid:16234978

10. Little D, Smith N, Williams P, Briody J, Bilston L, Smith E, Gardiner E, Cowell C. 2003. Zoledronic acid prevents osteopenia and increases bone strength in a rabbit model of distraction osteogenesis. J Bone Miner Res 18: 300-307 (2003)
http://dx.doi.org/10.1359/jbmr.2003.18.7.1300
PMid:12854841

11. Amanat N, Brown R, Bilston L, Little D. A single systemic dose of pamidronate improves bone mineral content and accelerates restoration of strength in a rat model of fracture repair. J Orthop Res 23: 1029-1034. (2005)
http://dx.doi.org/10.1016/j.orthres.2005.03.004
PMid:16140188

12. Amanat N, McDonald M, Godfrey C, Bilston L, Little D. Optimal timing of a single dose of zoledronic acid to increase strength in rat fracture repair. J Bone Miner Res 22(6):867-876 (2007)
http://dx.doi.org/10.1359/jbmr.070318
PMid:17371160

13. Schindeler A, Little D. Bisphosphonate action: revelations and deceptions from in vitro studies. J Pharm Sci 96: 1872-1878 (2007)
http://dx.doi.org/10.1002/jps.20904
PMid:17518363

14. Rogers M. New insights into the molecular mechanisms of action of bisphosphonates. Curr Pharm Des 9: 2643-2658 (2003)
http://dx.doi.org/10.2174/1381612033453640
PMid:14529538

15. Russell R, Xia Z, Dunford J, Oppermann U, Kwaasi A, Hulley P, Kavanagh K, Triffitt J, Lundy M, Phipps R. Bisphosphonates: an update on mechanisms of action and how these relate to clinical efficacy. Ann N Y Acad Sci 1117: 209-257 (2007)
http://dx.doi.org/10.1196/annals.1402.089
PMid:18056045

16. Schindeler A, McDonald M, Bokko P, Little D. Bone remodelling during fracture repair: The cellular picture. Semin Cell Dev Biol 19: 459-466 (2008)
http://dx.doi.org/10.1016/j.semcdb.2008.07.004

17. McDonald M, Dulai S, Godfrey C, Sztynda T, Little D. Osteoclasts are functionally redundant in initial endochondral fracture union but not callus remodelling: Insights into optimal bisphosphonate dosing. J Bone Miner Res 21: S398 (2006)

18. Harding A, Toksvig-Larsen S, Tägil M, W-Dahl A. A single dose zoledronic acid enhances pin fixation in high tibial osteotomy using the hemicallotasis technique. A double-blind placebo controlled randomized study in 46 patients. Bone 46: 649-654 (2010)
http://dx.doi.org/10.1016/j.bone.2009.10.040
PMid:19913119

19. Harding AK, Aspenberg P, Kataoka M, Bylski D, Tägil M. Manipulating the anabolic and catabolic response in bone graft remodeling: Synergism by a combination of local BMP-7 and a single systemic dose of zoledronate. J Orthop Res 26: n/1245-1249 (2008)

20. Gong L, Hoshi K, Ejiri S, Nakajima T, Shingaki S, Ozawa H. Bisphosphonate incadronate inhibits maturation of ectopic bone induced by recombinant human bone morphogenetic protein 2. JBMM 21: 5-11 (2003)
http://dx.doi.org/10.1007/s007740300001

21. Schindeler A, Little D. Recent insights into bone development, homeostasis, and repair in type 1 neurofibromatosis (NF1). Bone 42: 616-622 (2008)
http://dx.doi.org/10.1016/j.bone.2007.11.006

22. Schindeler A, Ramachandran M, Godfrey C, Morse A, McDonald M, Mikulec K, Little DG. Modeling bone morphogenetic protein and bisphosphonate combination therapy in wild-type and Nf1 haploinsufficient mice J Orthop Res 26: 65-74 (2008)
http://dx.doi.org/10.1002/jor.20481

23. Birke O, Schindeler A, Ramachandran M, Cowell C, Munns C, Bellemore M, Little D. Preliminary experience with the combined use of recombinant bone morphogenetic protein and bisphosphonates in the treatment of congenital pseudarthrosis of the tibia J Child Orthop 4: 507-517 (2010)
http://dx.doi.org/10.1007/s11832-010-0293-3

24. Jeppsson C, Åstrand J, Tägil M, Aspenberg P. A combination of bisphosphonate and BMP additives in impacted bone allografts Acta Orthopaedica Scandinavica 74: 483-489 (2003)
http://dx.doi.org/10.1080/00016470310017839

25. Belfrage O, Flivik G, Sundberg M, Kesteris U, Tägil M. Local treatment of cancellous bone grafts with BMP-7 and zoledronate increases both bone formation rate and bone density. A bone chamber study in rats. Acta Orthopedica 82(2):228-233 (2011)
http://dx.doi.org/10.3109/17453674.2011.566138

26. Chen W-J, Jingushi S, Hirata G, Matsumoto Y, Iwamoto Y. Intramuscular bone inducation by the simultaneous administration of recombinant human bone morphogenetic protein 2 and bisphosphonate for autobone graft. Tissue Eng 10: 1652-1661. (2004)
http://dx.doi.org/10.1089/ten.2004.10.1652

27. Chen W-J, Jingushi S, Jingushi K, Iwamoto Y. In vivo banking for vascularized autograft bone by intramuscular inoculation of recombinant human bone morphogenetic protein-2 and β-tricalcium phosphate. J Orthop Sci 11: 283-288 (2006)
http://dx.doi.org/10.1007/s00776-006-1017-x

28 Vandermeer J, Kamiya N, Aya-ay J, Garces A, Browne R, Kim H. Local administration of ibandronate and bone morphogenetic protein-2 stimulates bone formation and decreases femoral head deformity following ischemic osteonecrosis of the immature femoral head. J Bone Joint Surg Am 93(10):905-13 (2011)
http://dx.doi.org/10.2106/JBJS.J.00716

29. Choi J-Y, Kim H-J, Lee Y-C, Cho B-O, Seong H-S, Cho M, Kim S-G. Inhibition of bone healing by pamidronate in calvarial bony defects. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 103: 321-328 (2007)
http://dx.doi.org/10.1016/j.tripleo.2006.06.057

30. Jakobsen T, Baas J, Bechtold JE, Elmengaard B, Soballe K. Soaking morselized allograft in bisphosphonate can impair implant fixation. Clin Orthop Relat Res 463: 195-201 (2007)

31. Baas J, Elmengaard B, Jensen TB, Jakobsen T, Andersen NT, Soballe K. The effect of pretreating morselized allograft bone with rhBMP-2 and/or pamidronate on the fixation of porous Ti and HA-coated implants. Biomaterials 29: 2915-2922 (2008)
http://dx.doi.org/10.1016/j.biomaterials.2008.03.010

32. Yu NYC, Schindeler A, Peacock L, Mikulec K, Baldock PA, Ruys AJ, Little DG. In vivo local co-delivery of recombinant human bone morphogenetic protein-7 and pamidronate via poly-D, L-lactic acid. Eur Cell Mater 20: 431-442 (2010)

Key Words: Anabolism, Catabolism, Bone tissue engineering, Bone morphogenetic protein (BMP), Bisphosphonate, Review

Send correspondence to: Nicole Y.C Yu, Department of Orthopaedic Research & Biotechnology, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW 2145, Australia, Tel: 612 9845 1451, Fax: 612 9845 3078, E-mail:nicole.yu@sydney.edu.au