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Demineralization for Inactivation of Infectious Retrovirus in Systemically Infected Cortical Bone

In Vitro and in Vivo Experimental Studies

Investigation performed at the Department of Pathobiology and Diagnostic Investigation and the Laboratory for Comparative Orthopedic Research, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan

Cheryl L. Swenson, DVM, PhD
Steven P. Arnoczky, DVM
Department of Pathobiology and Diagnostic Investigation (C.L.S.) and Laboratory for Comparative Orthopedic Research (S.P.A.), College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824-1314. E-mail address for C.L. Swenson: swenson{at}cvm.msu.edu E-mail address for S.P. Arnoczky: arnoczky@cvm.msu.edu

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Osteotech. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

Background: Clinical and experimental studies have demonstrated viral transmission through the transplantation of fresh-frozen infected bone. While sterilization methods sufficient to inactivate the human immunodeficiency virus (HIV) have been shown to markedly alter osteoconductive and osteoinductive properties of bone allografts, the ability of a process for creating demineralized bone matrix to abrogate transmission of a retrovirus has not been investigated, to our knowledge. We hypothesized that a clinically accepted demineralization procedure would alter the nucleic acids of the feline leukemia virus (FeLV, a retrovirus with a structure and replication cycle similar to those of HIV), inactivating the virus in infected bone and rendering it noninfectious.

Methods: Bone infected with FeLV was demineralized with a method employed for creating demineralized bone matrix powder. The effects of demineralization on cellular and (pro)viral nucleic acids were examined with use of gel electrophoresis and quantitative polymerase chain reaction, respectively. To compare the infectivity of the demineralized bone matrix with that of mineralized bone particles in cell cultures and in animals in which they had been implanted, we measured FeLV p27 antigen and (pro)viral nucleic acids as well as antiviral antibodies.

Results: Demineralization of FeLV-infected bone appeared to inactivate the virus by degradation and fragmentation of the DNA, rendering it noninfectious in both in vitro and in vivo test systems. In contrast, untreated mineralized FeLV-infected bone contained intact nucleic acids and readily transmitted the virus in both test systems.

Conclusions: The demineralization process inactivated infectious retrovirus in infected cortical bone, thereby preventing disease transmission.

Clinical Relevance: Rigorous donor screening is currently believed to be the best method for ensuring the safety of allografts, but it is not 100% effective. A process for creating demineralized bone matrix powder reliably inactivated a retrovirus from systemically infected bone while maintaining the osteoinductive properties of the bone. Therefore, use of demineralized bone matrix would provide patients and surgeons with an additional margin of safety while sustaining osteoinductive efficacy.

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