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Scientific Articles   |    
rhBMP-2/Calcium Phosphate Matrix Induces Bone Formation While Limiting Transient Bone Resorption in a Nonhuman Primate Core Defect Model
Howard J. Seeherman, PhD, VMD1; X. Jian Li, MD1; Erica Smith, PhD2; John M. Wozney, PhD1
1 Inflammation and Tissue Repair, Pfizer Discovery Research, 200 Cambridge Park Drive, Cambridge, MA 02140. E-mail address for H.J. Seeherman: howard.seeherman@pfizer.com
2 Charles River Laboratories International, Inc., 251 Ballardvale Street, Wilmington, MA 01887
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  • Disclosure statement for author(s): PDF

Investigation performed at Inflammation and Remodeling, Pfizer Discovery Research, Cambridge, Massachusetts



Disclosure: One or more of the authors received payments or services, either directly or indirectly (i.e., via his or her institution), from a third party in support of an aspect of this work. In addition, one or more of the authors, or his or her institution, has had a financial relationship, in the thirty-six months prior to submission of this work, with an entity in the biomedical arena that could be perceived to influence or have the potential to influence what is written in this work. No author has had any other relationships, or has engaged in any other activities, that could be perceived to influence or have the potential to influence what is written in this work. The complete Disclosures of Potential Conflicts of Interest submitted by authors are always provided with the online version of the article.

Copyright © 2012 by The Journal of Bone and Joint Surgery, Inc.
J Bone Joint Surg Am, 2012 Oct 03;94(19):1765-1776. doi: 10.2106/JBJS.K.00523
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Abstract

Background: 

Transient bone resorption limits the use of recombinant human bone morphogenetic protein-2 (rhBMP-2)/absorbable collagen sponge in metaphyseal bone. The purpose of the present study was to evaluate the efficacy of rhBMP-2/calcium phosphate matrix (CPM) to induce bone formation while limiting transient bone resorption in nonhuman primate core defects.

Methods: 

Metaphyseal core defects were created in eighteen cynomolgus monkeys. rhBMP-2 retention was evaluated in the distal part of the radius. Bone formation was evaluated at eight weeks following treatment with 1.5 or 4.5-mg/mL rhBMP-2/CPM, CPM alone, or no treatment in the distal part of the radius, the proximal part of the tibia, and the proximal part of the femur; at twenty-four weeks following treatment with 1.5-mg/mL rhBMP-2/CPM or CPM alone in the proximal part of the tibia; and at one, two, and four weeks following treatment with 1.5-mg/mL rhBMP-2/CPM or no treatment in the distal part of the radius. Bone resorption was evaluated at four weeks following treatment with 1.5, 2.0, 3.0, and 4.5-mg/mL rhBMP-2/CPM or CPM alone in the distal part of the femur. Evaluations were performed with use of scintigraphy, radiographs, histological analysis, and computed tomography.

Results: 

Seventy-eight percent, 64%, 50%, 35%, and 12% of the rhBMP-2 was retained in the distal part of the radius at one, seven, fourteen, twenty-one, and forty-nine days after surgery. rhBMP-2/CPM increased bone formation within core defects and surrounding trabeculae compared with CPM alone or no treatment at all anatomic locations at eight weeks, and bone formation was ongoing in the rhBMP-2/CPM-treated proximal tibial sites at twenty-four weeks. Bone formation began in the trabeculae surrounding the core defects at one week and was observed adjacent to the resorbing CPM within the core defects and in the surrounding trabecular bone at two and four weeks in the rhBMP-2/CPM-treated distal radial sites. Bone formation was confined to the region immediately surrounding the core defects in the untreated distal radial sites at all time points. Transient bone resorption was only observed in the distal femoral sites treated with 4.5 mg/mL of rhBMP-2/CPM at two weeks.

Conclusions: 

Treatment of nonhuman primate metaphyseal core defects with 1.5 to 3.0-mg/mL rhBMP-2/CPM resulted in bone formation without transient bone resorption.

Clinical Relevance: 

rhBMP-2/CPM may be useful to accelerate healing of metaphyseal bone defects in humans.

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    References

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    These activities have been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Academy of Orthopaedic Surgeons and The Journal of Bone and Joint Surgery, Inc. The American Academy of Orthopaedic Surgeons is accredited by the ACCME to provide continuing medical education for physicians.
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    Howard J. Seeherman, PhD, VMD
    Posted on November 15, 2012
    Response to Dr. Karaikovic
    Pfizer Discovery Research, Cambridge, MA, USA

    The authors certainly agree with Dr. Karaikovic’s comment concerning respect for the well-being and life of humans as well as all forms of life when conducting research. While this is not the optimal forum for discussing the use of animals for drug development or research in general, the authors would like to clarify and respond to a number of issues brought up by Dr. Karaikovic in his comments about our recent publication.

    The manuscript submitted to JBJS used the word euthanasia to describe the process of humane termination of the lives of the animals in this study. The editorial board of the journal insisted on the use of the word “kill” in the publication as a policy of the journal over the objections of the authors.

    The authors of the current study have previously published a study in JBJS demonstrating that recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered in an absorbable collagen sponge (ACS) induced transient bone resorption followed by bone formation in nonhuman primate metaphyseal core defects (Seeherman HJ, Li XJ, Bouxsein ML, Wozney JM. rhBMP-2 induces transient bone resorption followed by bone formation in a nonhuman primate core-defect model. J Bone Joint Surg Am. 2010;92:411-26). This initial study was intended to mandate caution in considering the use of rhBMP-2/ACS for metaphyseal fracture repair when bone resorption may lead to loss of fixation or structural support.  The current study demonstrates that the cause of this transient bone resorption is related to the rapid release of rhBMP-2 from the ACS carrier and demonstrates that alternative carriers with prolonged rhBMP-2 retention result in bone formation without transient bone resorption.   The authors will leave it to the orthopedic community and the editors of JBJS to determine whether this use of animals to identify potential adverse events, to determine the cause of the adverse event and offer potential solutions that could affect the outcome of an orthopedic procedure in people and in animals is justified.

    The authors are very much aware of the need to carefully design studies that involve the use of animals. Nonhuman primates have historically been used to determine safety, efficacy and dose ranging of BMPs intended for use in people based on the observation that studies in other animal species are not predictive of the response to BMPs in people (Seeherman H, Wozney J, Li R. Bone morphogenetic protein delivery systems. Spine. 2002 Aug 15;27(16 Suppl 1):S16-23).

    The authors agree with Dr. Karaikovic’s suggestion that animal models that do not require euthanasia should be implemented whenever possible.  The authors have published studies specifically evaluating rhBMP-2 in nonhuman primate fibula osteotomy models where the outcome measurements including histology and mechanical testing do not require euthanasia (Seeherman HJ, Bouxsein M, Kim H, Li R, Li XJ, Aiolova M, Wozney JM. Recombinant Human Bone Morphogenetic Protein-2 Delivered in an Injectable Calcium Phosphate Paste Accelerates Osteotomy Healing in Nonhuman Primate Model.  J Bone Joint Surg Am. 2004 Sep;86-A(9):1961-72.  Seeherman H, Li R, Bouxsein M, Kim H, Li XJ, Smith-Adaline EA, Aiolova M, Wozney JM. rhBMP-2/Calcium Phosphate Matrix Accelerates Osteotomy-Site Healing in a Non-Human Primate Model at Multiple Treatment Times and Concentrations.  J Bone Joint Surg Am. 2006 Jan;88(1):144-60).  The authors in collaboration with the Institutional Animal Care and Use Committee (IACUC) carefully assessed the potential for using a similar strategy for this series of studies.  The conclusion was reached that survival surgery for retrieval of core defects of any size was not possible due to the small size of the metaphyseal segments at any of the anatomic locations used in this study.   A complex study design was implemented to limit the number of animals required for the study while providing sufficient statistical power to draw definitive conclusions.

    The authors do take exception with Dr Karaikovic’s opinion that scientists in industry are biased towards unjustified use of animals for research compared to our colleagues in academia.  Each proposed use of animals is thoroughly evaluated and approved by the IACUC before any study activity is initiated.  Industry IACUCs are subject to the same guidelines outlined in the Animal Welfare Act (AWA) and National Research Council’s guide for the Care and Use of Laboratory Animals as academic IACUCs. The Pfizer IACUC includes two unaffiliated/public members on the committee responsible for reviewing each protocol. In addition, the study site is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and inspected annually by USDA, meeting the same or higher animal welfare quality standards as academic animal facilities. 


    Eldin E. Karaikovic, MD, PhD
    Posted on November 01, 2012
    Unnecessary euthanasia
    Northshore University Health System, Department of Orthopaedic Surgery, Chicago, IL

    Without judging the hard work and very intriguing findings of the study, I am outraged and deeply saddened to learn that the authors of the paper “rhBMP-2/Calcium Phosphate” had to euthanize or sacrifice (or however the authors preferred to call it “kill”) 18 healthy macaca monkeys in their study. The research they conducted is not associated with analysis of a medication or a substance with a lethal effect on humans or other species. Their research was only directed to analyze an effect of a non-lethal substance (rhBMP-2/CaP) on bony healing. With a better thought-through methodology they might have gotten the same results without sacrificing the animals. A couple of examples: using a smaller core defect then 3.5 mm and retaking it by removing a core around it 2 mm wider then the defect at the end of the study. Also only larger bones could be used since that would make less structural damage to the studied bones if a 3.5 mm core defect was used. A larger core could be removed later eliminating a possibility for a fracture through the bone defects. The process of physiologic ossification and re-ossification is the same in the radius, tibia and femur; and probably are the same as the effects of the substance used in the study (as it is proven with the study). This certainly would eliminate a need for an unfortunate euthanasia of the animals if the bone defect was the reason for it. Anyway, the authors did not explain why it was necessary to sacrifice 18 healthy monkeys to reach their conclusions.

    Although the study was approved by the “Institutional Animal Care and Use Committee” their institution is The Pfizer research plant e.g. a pharmaceutical company plant (which is obviously not an academic institution). The funding was provided by Pfizer Discovery Research. It is hard to believe that the Committee’s decision was free from any bias and therefore it would have been more appropriate to include an outside independent IRB review of the project. Besides, in the disclosure it is written that “one or more authors received the payments or services from a third party in support” and “has had a financial relationship…with the entity in the biomedical arena”.

    Since the process of legally regulating unnecessary “killings” of research animals is lengthy, and because the unfortunate 18 monkeys did not have an ability to express their dismay with somebody else’s decision to end their lives prematurely, they neither had an advocate to speak on their behalf. Therefore I kindly ask the Editorial Board of the JBJS to consider at least not publishing research in which a by-product of the research is a loss of life regarding other species in experiments in which products tested usually do not cause a lethal effect on human beings. I believe we should not only respect the well-being and life of humans when conducting research; but all forms of life.

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