668 LL 0 SGZL 8 mm 3333333 m[mywumuwww THESIS 9 W 95>“ LIBRARY ll Michigan State .9- University This is to certify that the thesis entitled VIRUCIDAL ACTIVITY AND BONE INCORPORATION EFFECTS OF 98% GLYCEROL TREATMENT OR ETHYLENE OXIDE STERILIZATION OF BONE ALLOGRAFTS: IN VIVO AND IN VITRO STUDIES presented by George Steven Coronado, Jr. has been accepted towards fulfillment of the requirements for Small Animal Clinical Sciences Major professor Masters degree in Date jig-21L 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c:/C|RC/DatoDue.p65-p. 15 VIRUCIDAL ACTIVITY AND BONE INCORPORATION EFFECTS OF 98% GLYCEROL TREATMENT OR ETHYLENE OXIDE STERILIZATION OF BONE ALLOGRAFTS: IN VIVO AND IN VITRO STUDIES BY George Steven Coronado, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Small Animal Clinical Sciences 2000 ABSTRACT VIRUCIDAL ACTIVITY AND BONE INCORPORATION EFFECTS OF A 98% SOLUTION OF GLYCEROL OR ETHYLENE OXIDE STERILIZATION ON BONE ALLOGRAFTS: IN VIVO AND IN VITRO STUDIES BY George Steven Coronado, Jr. The feline leukemia virus (FeEV) was used to measure the affect of sterilization with ethylene oxide (ETO) or a 98% glycerol solution on antiviral activity and bone incorporation. FeLV-infected bone grafts were treated with ETO or glycerol and transplanted into 8-week-old specific pathogen-free (SPF) cats and introduced into cell cultures. Blood samples were obtained to monitor FeLV p27 antigen and antibody titers. Quantification of FeLV provirus was performed on blood and bone graft samples by the polymerase chain reaction (PCR). Cell culture and media samples were collected to monitor FeLV p27 antigen and FeLV provirus. There was no evidence of transmission of the virus to cats or cell culture samples in the ETO groups. Transmission of virus to a cat in the glycerol group was evident, and. glycerol-preserved. bone samples contained. a large amount of amplifiable provirus. Incorporation of bone grafts was similar among all groups. This work is dedicated to those cats that gave their lives for the benefit of this research .Hand to my cat and friend, Sid. iii q- ACKNOWLEDGMENTS The author wishes to extend his sincere gratitude for the help and support provided by the members of the graduate committee and those that assisted in carrying out this work: To Dr. Charles T. Lowrie, for his patience in helping me succeed through this program and for his great teaching in the subject of neurology and neurosurgery. To Dr. Steven P. Arnoczky, for his support and ability to always be a good sounding board. To Dr. Cheryl L. Swenson, for her patience and guidance in the lab and with her help in evaluating the data. To Dr. Charles E. DeCamp, for his support with this masters program and for being a role model for a hard- working and dedicated surgeon. To Marlee Richter, for her guidance in teaching all of the procedures performed in the lab and for her hard work. To Dr. Kim S. Burkhardt, for her enthusiasm and hard work in the lab and in handling and working with the cats. This work was supported by funds from the Companion Animal Fund, Michigan State University, the Markum Foundation, the Department of Small Animal Clinical Sciences, and the College of Veterinary Medicine at Michigan State University. v0. x TABLE OF CONTENTS Page LIST OF TABLES .......................................... vii LIST OF FIGURES ........................................ viii KEYS TO SYMBOLS OR ABBREVIATIONS ......................... ix INTRODUCTION .............................................. 1 LITERATURE REVIEW ......................................... 4 The use of cortical bone allografts in human and veterinary medicine .................................. 4 Cortical bone allograft incorporation ........... 5 Osteoinductive and osteoconductive properties ...7 Immunologic response to bone allografts ......... 9 Concerns of retroviral transmission in humans through allografts .................................. 13 Reports of retroviral transmission through soft tissue and bone allografts .......................... l6 Bone allograft harvesting and sterilization procedures .......................................... 20 Ethylene oxide ................................. 21 Glycerol solutions ............................. 25 Gamma irradiation .............................. 29 FeLV as a model for other retroviruses .............. 33 Infectivity of FeLV ................................. 37 Testing methods for FeLV ............................ 44 BONE INCORPORATION AND VIRUCIDAL EFFECTS OF A 98% SOLUTION OF GLYCEROL OR ETHYLENE OXIDE STERILIZATION ON BONE ALLOGRAFTS IN CATS ............................... 50 Materials and methods ............................... 50 Results ............................................. 63 VIABILITY OF RETROVIRUS (FELINE LEUKEMIA VIRUS) IN CORTICAL BONE GRAFTS AFTER ETHYLENE OXIDE STERILIZATION OR 98% GLYCEROL PRESERVATION ............... 77 Materials and methods ............................... 77 Results ............................................. 81 DISCUSSION ............................................... 86 Page CONCLUSION ............................................... 97 RECOMMENDATIONS ......................................... 100 REFERENCES .............................................. 101 W .3 c _: :~ .»~ LIST OF TABLES Table 1: Positive FeLV p27 antigen results in blood samples from cats after implantation with a cortical bone allograft ......................................... Table 2: Antibody titers to feline oncornavirus cell membrane-associated antigen (FOCMA) 2 1:16 in blood samples from cats following implantation with a cortical bone allograft ................................ Table 3: Quantitative polymerase chain reaction test results in blood and bone graft samples from cats that received a cortical bone allograft .................... Table 4: Positive FeLV p27 antigen results for cell culture media from negative control, ETC-sterilized, glycerol-treated, and positive bone and virus control groups ................................................ Table 5: Results of a quantitative polymerase chain reaction test to detect the number of copies of FeLV provirus in negative control, ETC-sterilized, glycerol-treated, and positive bone and virus control group cell cultures ........................... vfi Page ..68 ..69 ...70 ...84 ...85 A». v- a.. A.» :~ : . . ._. n\~ v. F~a w.. .r. ~ ~ .5. :. _.. . .r‘ v; t» A v LIST OF FIGURES Figure 1: Photograph of a harvested metatarsal cortical bone allograft. The metaphyseal segments of the bones were removed before implantation and saved for quantification of FeLV provirus ............. Figure 2: Photograph of a metatarsal cortical bone allograft ready for implantation. The metaphyseal segments were removed with a No. 10 scalpel blade, producing a 1 cm segment of bone ...................... Figure 3: Intraoperative photograph of a cortical bone allograft implanted into the middiaphyseal segmental ulnar ostectomy site ........................ Figure 4: Postoperative lateral radiograph of a radius and ulna. The cortical bone allograft was stabilized into the ulnar ostectomy site using a 0.028 inch Kirschner wire ............................. Figure 5: Photograph of a harvested implanted ulna after euthanasia 8 weeks after implantation. All grafts had completely healed in all of the groups .... Figure 6: High-resolution radiographs of a harvested radius and ulna 8 weeks after implantation with a cortical bone allograft. Moderate callus formation was present around the graft site in all cats. a) Negative control, b) Positive control, c) Glycerol group, d) ETO group ....................... Figure 7: Photomicrograph of the host-graft interface in the ulna of a cat that received a cortical bone allograft. Dead bone graft can be visualized as empty lacunae surrounded by new woven host one.(lOX) .. Figure 8: A magnified photomicrograph of the host-graft interface as seen in Figure 7.(20X) ........ vm Page ...51 ...55 ...56 ...58 62 ...74 ...75 ...76 —‘ Anv- » . ‘u- h- v d C KEYS TO SYMBOLS OR ABBREVIATIONS AIDS ..................... Acquired Immunodeficiency Syndrome ANOVA .................................. Analysis of Variance AZT .............................. 3'-Azido-3’-Deoxythymidine BMP ................................ Bone Morphogenic Protein C ................................................... Celsius cm ............................................... Centimeter DMSO ..................................... Dimethyl Sulfoxide DNA ................................... Deoxyribonucleic Acid ELISA ..................... Enzyme—Linked Immunosorbent Assay ETO .......................................... Ethylene Oxide FEA ............................. Feline Fibroblast Cell Line FeLV .................................. Feline Leukemia Virus FIV ........................... Feline Immunodeficiency Virus FL74 ........................ Feline Lymphoblastoid Cell Line FOCMA ..Feline Oncornavirus Cell Membrane-Associated Antigen gp7O ...... Envelope Glycoprotein (for Feline Leukemia Virus) gsa .................................. Group-Specific Antigen H&E ................................... Hematoxylin and Eosin HIV ............................ Human Immunodeficeincy Virus HSV-l ........................... Herpes Simplex Type 1 Virus IFA .............................. Indirect Fluorescent Assay IM ............................................ Intramuscular IV .............................................. Intravenous m -y \s ‘I LSA ........................................... Lymphosarcoma mrads .............................................. Megarads pg/ml .............................. Microgram per Milliliter mg/kg ................................ Milligram per Kilogram mg/kg/h ..................... Milligram per Kilogram per Hour ml ............................................... Milliliter n ........................................... Number (amount) NaCl ........................................ Sodium Chloride NaHCO3 .................................... Sodium Bicarbonate OD .......................................... Optical Density plSE ...... Envelope Glycoprotein (for Feline Leukemia Virus) p27 .......... Major Core Protein (for Feline Leukemia Virus) PBMC ...................... Peripheral Blood Mononuclear Cell PCR ............................... Polymerase Chain Reaction QPCR ................. Quantitative Polymerase Chain Reaction S/P ................................ Sample to Positive Ratio SE ........................................... Standard Error SIV ........................... Simian Immunodeficiency Virus SPF .................................. Specific Pathogen Free VN ....................................... Virus Neutralizing INTRODUCTION The most common indication for cortical bone allografts in veterinary and human orthopedics is for repair of large diaphyseal long bone defects. The inconvenience of harvesting, processing, storing, and assuring quality has restricted use of allografts in most veterinary practices. Preservation of bone allografts has been limited to ultra- low freezing or treatment with ethylene oxide (ETO). Gamma irradiation also has been used to sterilize and reduce immunogenicity of allografts.33 However, problems have been encountered with use of these sterilization procedures. One major disadvantage is that ETO may reduce bone induction and incorporation of host bone at the graft site.3'142 Ethylene oxide also may negatively affect mechanical strength of the bone graft and have a toxic affect on fibroblast activityf””u$1”””2 As an alternative to sterilization with ETO, canine Cortical bone allografts stored in a 98% solution of glycerol appeared to have good incorporation into host bone, although quantification of this assessment was lacking?7 Viral and bacterial transmission are possible adverse outcomes when stored tissues are used for transplantation. Concerns that current freezing and storage practices may not be adequate to inactivate retroviruses have been L» p Y. .~ -¢. Au‘ \— . r. a.. p . , 2. u. .2 ~.. . .3 .. .3 . ‘.. substantiated by recent studies in animals that investigated viral transmission from retrovirus—infected transplanted allogenic cortical and cancellous bone and connective tissue grafts to recipient animals.10“'105 Specific pathogen—free (SPF) cats had evidence of exposure to (positive results for antibody), or infection with (positive results for antigen), the retrovirus feline leukemia virus (FeLV) by 2 to 6 weeks after implantation of infected allogenic donor tissues that had undergone l or 2 freeze-thaw cycles before implantation. These studies were important, because they documented that retroviruses may be transmitted through transplantation of infected bone and connective tissues and that freeze-thaw cycles were inadequate to prevent transmission.1°4'105 Although some biological effects have been d,3'73'142 we are not aware of studies that have investigate examined virucidal properties of ETO on cortical bone allografts. Reports of studies from Europe”, and South America” advocated use of a 98% solution of glycerol for storage of bone and skin allografts. It is stated in other reports“97 that a 98% solution of glycerol is bactericidal and virucidal against enveloped and non-enveloped viruses, suggesting that efficacy against human immunodeficiency virus also might be possible. This latter claim was extrapolated from the observation that a solution of 98% glycerol appears to have in vitro virucidal effects on herpes simplex ‘virus, type it (HSV>1) and. poliovirus. If proven effective against pathogens, a solution of 98% glycerol would be a simpler, less toxic, and more cost effective alternative to sterilization with ETO. The purpose of the studies reported here was to compare the antiviral properties and effects on bone incorporation of allografts obtained from FeLV-infected cats, then treated with a 98% solution of glycerol or ETO sterilization. we hypothesized that use of the 98% solution of glycerol would have similar virucidal effects as ETO, but would not be detrimental to incorporation of bone allografts in SPF cats. LITERATURE REVIEW Use of cortical bone allografts in human and veterinary medicine It is estimated that tissues from approximately 5000 cadaver donors are transplanted into more than 220,000 bone or soft—tissue human recipient patients annually in the United States.11 Many advantages have been found with the use of bone allografts in both human and veterinary medicine. The use of allografts eliminates donor site morbidity, operative time, and length of hospital stay; there is no limit on the size, shape, or quantity of the graft; and most grafts are amenable to long-term storage.76'95 Cortical bone allografts have been used to replace bone segments affected by tumors, to replace bone lost to trauma or autolysis of bone from cemented prostheses, and for oral reconstruction.“2'91'92'99'114 In addition, cortical bone allografts have been used in spinal surgeries either to assist in fusions or to act as a structural element.17 Twenty-five to 35% of patients who receive allograft transplants for limb salvage have a complication within the --- (I) -..— . o..- y ‘ Hi m Cort a -—, 1 I in {U f) i" _l lfl'n6 Problems associated with first 3 years after surgery. allografts include nonunion of the graft-recipient bone interface, resorption and/or fracture of the graft, and infection. 'Nonunion is aa relatively common event, and it has been proposed that nonunion may represent a subtle form of rejection.20 Cortical bone allograft incorporation Incorporation of cortical bone allografts following implantation is a long process that often is not ever completed. The term “creeping substitution" has been used to describe the process whereby transplanted bone graft is invaded by osteoclasts. Initially, the graft is the focus of an inflammatory response characterized by vascular buds infiltrating the grafted bed. By the second week, fibrous granulation tissue becomes increasingly dominant in the graft bed, the number of inflammatory cells decreases, and osteoclastic activity increases. These osteoclasts channel into the bone graft and create a tunnel known as Howships lacunae. These tunnels become vascularized by capillaries that bring osteoblasts to the area. Osteoblasts are the cells responsible for laying down new bone in concentric layers until the tunnel is filled with new bone. The final 'I' WV up- -, a. ‘7' we (I) I“ or, structure formed, containing a central arteriole canal, is known as a Haversian system or osteon.15"M This process of incorporation continues until the bone graft has been replaced with Haversian systems belonging to the recipient. After a full year of bone incorporation, the graft may appear to approach its preoperative mechanical strength. Even then, only approximately 60% of the structure is composed of new bone.4 Cortical grafts tend to remain a combination of necrotic and viable bone. Incomplete incorporation of bone grafts has been demonstrated by scintigraphy with a decline in scintigraphic activity with increasing distance from the host-graft interface 9 to 367 weeks after cortical bone allograft transplantation . 132 Until the transplanted graft becomes vascularized, it does not have the potential to respond to loads physiologically by remodeling or by repairing subfailure damage.110 Initially, as there is an increase in osteoclastic activity and a decrease in osteoblastic activity, the porosity of the bone increases, and the mechanical properties are impaired.15 Microfractures may occur which are thought to be associated with rapid _n.- r;"“ “ I‘uw .v I.--‘. 'w .- .:‘u--‘-‘ -.yara you»- .5. “AAA ‘p i.“ b»~~-~- A -C \«s "v-Ao-n.' .. ~~v-\.-. u...‘ by \“ F“ a ‘VL. ”) h c. S‘r‘l-JC revascularization of tflma graft with resultant resorption leading tx> weakness. These ndcrofractures ck) not necessarily cause harm as they can be filled with mineralized non-lamellar woven bone, and thus may' be a possible route for new bone apposition. Osteoinductive factors, suCh as bone morphogenic protein (BMP) contained in the cortical bone matrix, also are thought to be released by microfractures and may assist with healing.41 The released BMP is believed to induce mesenchymal cells to differentiate into cartilage and bone and to stimulate DNA synthesis and cell replication.72 Osteoinductive and osteoconductive properties In general, bone grafts may provide several different functions: osteogenesis, osteoinduction, and osteoconduction. .Although cortical bone allografts cannot contribute living cells for osteogenesis, they are capable of osteoinduction in the recipient and of providing structural support for osteoconduction. Osteogenesis is the pmocess whereby cellular elements within a graft survive the transplantation process and produce new bone in the recipient site. In cortical bone ,r-r :V‘ .-‘ahv wv... ".qu “'~m:‘ allografts, few, if any, cells will survive the transplantation process, and.iJ:.is generally accepted that cortical bone allografts do not contain osteogenic capabilities. Osteoinduction is the mechanism whereby nonosseous tissue is influenced to change its cellular function and become osteogenic. For this to occur, there must be an inducing stimulus, a potentially osteogenic cell, and a favorable tissue environment. Although there is some controversy as to how this mechanism is initiated, it is generally thought that the cell responsible for bone formation is the osteoblast. It remains to be established where the progenitors of osteoblasts originate. It has been suggested that the endothelial cells in blood vessels may become osteogenic, however, there also is evidence that cells resembling fibroblasts in the limiting membrane of bone and. in surrounding soft tissues may' be induced to differentiate into osteoblasts. Another theory is that all somatic cells have the genetic potential for osteoblastic transformation.4 The collagen matrix of type I collagen in the bone matrix also may play a significant role in bone ‘ o “..A ...‘v-r ' arc~_~‘ V-UV-" {or A L»- V pa-- “$ 't Ar~;‘ -A uv ‘v- I.‘.. ~Av‘v..v. -r‘-,A . v Q C) Oh induction.117 The geometry of the bone matrix may play a crucial role in determining its suitability as a foundation for osteoinduction. The implantation of a bone matrix powder with particle size 74 to 420 um resulted in bone formation in one study, whereas matrix with particle size 44 to 74 um did not induce bone formation. The inability of a fine matrix to induce bone formation was thought to be due to the role of the matrix geometry in triggering the biochemical cascade of endochondral bone differentiation in Vivo. “7 Osteoconduction, or “trellis” function, is the process of ingrowth of capillaries, perivascular tissue, and osteoprogenitor cells from the recipient bed into the graft. The bone graft in this case serves as a passive support for ingrowth of blood vessels and subsequent deposition of new bone from the recipient bed.15 Immunologic response to bone allografts Acceptance or rejection of a graft is determined by the presence or absence of alien, genetically determined antigens in the grafted cells, known as transplantation antigens. The genes responsible for formation of these cellular antigens are known as histocompatibility genes, Li—i. c- yo.‘v 1.; . * rhn. --Hh‘- () Ff .1. an.“ ‘ a 'V'.A.~ ‘ A ~— () ‘n'.“"-‘ “'.‘Ld~ ,. l- . -~,_“ ‘Llw‘... “r fi‘.‘ 1: - ra..Q: t and the chromosomal locations of these genes are histocompatibility loci. Class I inajor histocompatibility antigens are found on virtually all nucleated cells, while Class II antigens are present on the surface of B lymphocytes and other antigen presenting cells. Class I and II antigens have been identified on osteocytes, although it is difficult to correlate the presence of circulating antibodies to these antigens with an adverse clinical or functional outcome. In one study, the highest titers of antibody were found in major histocompatibility complex Class I mismatched animals, and antidonor antibodies were identified 3 weeks after transplantation of a bone allograft. The strongest response was when both Class I and Class II antigens were present.1'36 Recipients of bone autografts have a minimal immunologic response to the graft compared with transplanted bone allografts. This immunologic response to an allograft may result in a reduced osteoinductive process and a slower increase of union strength.151 The resulting inflammatory environment could potentially interfere with osteogenic cells that eventually will bind the graft in place. 10 f ." ~ .«r ~4\ w-f ‘4 "\ A at 18 “V Up. -..u“ k . p. t'l a . rho if.“ t T «n v..- be AC There are primarily two possible sources of antigen in a cortical bone graft - its cells and its intercellular substance. Since the mineral content of intercellular substances should not differ quantitatively from one animal to another, it has been suggested that the matrix of the transplant, which is composed. of glycoprotein, collagen, and mucopolysaccharide, would not be sufficiently antigenic to incite a response.15 The most immunogenic element of bone is thought to be the bone marrow. The bone marrow is contained within a righd cancellous meshwork surrounded km! thick dense cortical bone and therefore is effectively sequestrated. The bone marrow of a bone graft is resorbed very slowly by the host, so the exposure of marrow (donor antigen) to host immunocompetent cells occurs over an extended period. This may allow a balance to be established between release of antigen and formation of antibody.85 It also has been found that marrow-free bone can produce an antigenic response. This would suggest that a major histocompatibility antigen might be present in the bone itself.4 One study concluded that since no immunologic response was detected from. implanted frozen allografts, the H technique of freezing bone grafts was assumed to kill all live cells. The inmmnogenb: cell in bone was, therefore, thought to be alive to cause an optimal immunologic response. In addition, frozen grafts in a rat model elicited a weaker response and a response in fewer animals 6 It also has been found that even with than fresh grafts.13 reaming or further irrigation, bone marrow remnants were never completely removed“ Therefore, these residual bone marrow cells may incite the immune response.43 In} a study that neasured the inmmnologic response to bone allografts, humoral cytotoxic antibodies in recipients of fresh allografts couLd be detected beginning the first week after transplantation. The maximum levels detected were at one and two weeks, the same times when the maximum degree of cellular immunity was observed. Humoral cytotoxic antibodies where shown to persist up to four weeks after transplantation. During this period of observation, an inflammatory reaction was found to be confined first to the periosteum, and later the outer cortex and metaphyseal portion of the medulla also were affected.85 One report suggested that cellular immunity is more important than humoral immunity in the destruction of 12 allografts. Humoral immune responses were judged tx> be generally insignificant in primary allograft rejection. A possible explanation for tflUJ; is that although antibodies are formed to bone allografts, they may not be directly involved in the rejection episode. Other rejection factors may include chemotactic and osteoclastic activating factors, release of anaphylactic toxins, and features associated with inflammation, such as vasoconstriction, vasodilation, platelet activation, and thrombus formation.15 The incidence, strength, and duration of the recipient antidonor antibody response have been sflxnni to be affected by both graft treatment and size. Massive bone allografts have been shown to elicit a sustained response compared with small cortical grafts. Concerns of retroviral transmission via allografts in people In 1989, the reported risk of obtaining an allograft from an unrecognized human immunodeficiency virus (HIV) infected donor was approximately one in 1.6 million. Since then, the epidemic has grown, but intense screening of donors and serologic testing for HIV antibodies, HIV antigen, and the polymerase chain reaction (PCR) for HIV l3 J- .o C. A» ..‘I- » afla .F.. a .c I I c C a . a; «G ‘» Vs Q» .~. v1 hats . .i P. lnfe have prevented this risk from increasing. Currently, the risk is not believed to be greater than it was in 1989.11 Human immunodeficiency virus antibody testing was first implemented for organ and tissue transplantation in 1985.4 The rapid enzyme-linked immunosorbent assay (ELISA) is the currently used screening test. It is believed that persons who have HIV antibody in their serum are infected with HIV. Following exposure to and infection with HIV, 95% of individuals will have HIV antibody at detectable levels by 6 months. The HIV antigen is thought to be circulating at detectable levels within 1 to 2 weeks of exposure. This viral antigen is seen to decline with the production of HIV antibodies.4 The period of time associated with increased antigen levels is believed to correlate with early HIV infection, before antibody production, and may be the most infectious period.”137 Various studies have been performed screening blood donors for HIV p24 antigen, but the test failed to significantly demonstrate improved detection of HIV infected persons over the antibody test.2""16'18'130 l4 The PCR on blood can detect HIV infection before seroconversion, but false negative results were obtained by PCR in about 50% of blood samples in one study.36 Plasma PCR also has been shown to be less sensitive than leukocyte PCR.127 Several factors including PCR inhibitors, variability relating to the degree of cellular lysis and site of blood collection might interfere with PCR results. Nested PCR on postmortem skin samples has been shown to detect HIV more reliably than PCR performed on blood. The rationale for performing PCR on skin is the presence of HIV proviral DNA and RNA in epidermal Langerhans’ cells isolated from EUR! infected individuals}36 In general, the technical difficulty of performing a PCR has somewhat limited its use. Coculture of patients’ peripheral blood mononuclear cells (PBMCs) and detection of amplified provirus are sensitive methods that identify more than 90% of seropositive subjects. The limitation is that they do not distinguiSh between integrated nonreplicating provirus (latent infection) and actively replicating virus (persistent infection). In contrast, the presence of circulating virions capable of infecting normal PBMCs, and HIV antigen, does correspond tx> active (persistent) viral 15 replication. However, detection of HIV antigen is not as sensitive as HIV culture.137 Reports of retroviral transmission through soft tissue and bone allografts Since 1985, there have been many reports of transplantation associated HIV transmission from seropositive donors to organ recipients.4 Other reported sources of HIV transmission are from artificial insemination22 and skin graft transplantationx”fl. The first reported transplantation-associated transmission of HIV occurred following transplantation of 1 Since then there have an HIV infected kidney and liver.2 been multiple reports of transmission of HIV through kidney transplantation, some with subsequent development of AIDS.79'88'113'116’128'155 Two possible mechanisms for transmisshmn of the virus were proposed” Either infected blood in the donor kidney transmitted the virus, or the donor kidney itself was infected with HIV.88 Infection with HIV also may negatively influence survival of renal allograft recipients. Moreover, these individuals are at riSk for HIV-associated nephropathy, with anr incidence of 6-10%.“5 16 The first reported transmission of HIV following transplantation of bone was in 1988. The recipient was transplanted with an HIV-infected femoral head and was the first person reported to the Center for Disease Control as developing transplantation-associated. AIDS.23 Another report documented two bone recipients and one bone to patellar tendon allograft recipient developing HIV infection after receiving a transplant from an HIV infected donor.11 Three recipients of unprocessed fresh- frozen bone from an HIV-infected cadaver were infected with HIV. However, another' recipient. of 51 femoral allograft from this same cadaver tested negative for HIV-1 antibody. Unlike the other bone grafts, the shaft of the femoral allograft was extensively excavated to permit introduction of’ a metal rod. It. was theorized that the excavation removed most marrow elements and decreased the viral load of this graft. Four other recipients of organs from this cadaver also were infected with HIV-1.130 The HIV has been shown to reside in bone itself as 45J25 Fresh-frozen well as in blood. or marrow elements. connective-tissue allografts also have been shown to transmit a retrovztrus in an in VlVO study.10 This is in contrast to one in tdtro study which concluded that human bone derived cells are resistant to HIV infection using a cell to cell nethod. Tflua authors of this study believed that infection arises from nonosteoblastic cells present in whole bone and suggested that methods to sterilize bones should concentrate on inactivation of the virus in blood contaminating the graft.19 One study used the PCR to compare the amount of HIV proviral DNA in bones that were processed and unprocessed. Since the amount of proviral DNA was not significantly different between groups, they concluded that the DNA present in processed bone was inactivated and not infectious. This study also concluded that the virus could be inactivated following multiple freeze-thaw cycles. This, theoretically, would cause cell disruption and lysis, and proviral forms of HIV would not survive this cell death.125 Another in vivo study disproved the efficacy of freeze/thaw cycles by showing that multiple freeze/thaw cycles with a water flush was not sufficient to prevent transmission of a retrovirus through bone allografts.105 Although freezing allografts during processing was thought 18 to reduce HIV infectivity, it also is possible that freezing may have a preserving effect on the virus in bone.142 There have been no known cases of HIV or other viruses transmitted through freeze-dried tissue grafts. It is unknown if the processing itself or the nature of the tissue may decrease the viral load to zero or a level that is subinfectious.4 Laboratory' studies ‘have be superior tx> 12% ETO. Sterilization with 12% ETO resulted in greater dehydration which can cause the bone to become brittle and susceptible to formation of cracks on the cortical surface.73 .After sterilization with 12% ETO and 1 week storage at room temperature, pullout load or screw-stripping did not change. However, bones 22 stored. for 16 auui 32 weeks withstood. significantly less compressive and pullout loads than bones stored for 1 week. Whether this decrease in load resistance is attributable to the treatment or the storage temperature was not determined.119'146 Another study suggests that ETO- sterilization does not have an adverse affect on pullout strength, but this report failed to state the concentration of ETO or storage time used.131 Toxicity of ETO and its byproducts, ethylene glycol and ethylene chlorhydrin, and their affects on tissues have been documented, and chemical sterilization with ETO has "'“2 Persistent been associated with recipient morbidity. intraarticular reactions have been reported with ethylene oxide-sterilized allografts. In one report, detectable levels (M5 the residue, ethylene chlorhydrin, was neasured tn! gas chromatography 511 a bone-patellar tendon-bone allograft as well as in the synovium."'64 Ethylene chlorhydrin itself has been demonstrated to cause toxic reactions III biologic tissues. Implanted INK) sterilized bone-patellar tendon-bone allografts also were shown to have a high rate of graft dissolution (22%) which was 8 thought to be due to ETO byproducts.11 Sensitization with resulting anaphylaxis from ETO byproducts also has been 23 described.64 Because of these complications, use of ETO- sterilized bone-patellar tendon-bone allografts has not been highly recommended.135 Decreased incorporation associated with ethylene oxide sterilized bone has been reported and is thought to possibly be due to either alkylation of amino acids by ETO in the graft—recipient environments or to its damaging affects on bone morphogenic protein.“2J52 An effect of ETO on collagen cross-linking and ground substance has been suggested.140 Reduction in Ixxme inductive properties by ethylene oxide has been shown to be dependent upon exposure time. When a short sterilization procedure (5 minutes) was used, ETO did not destroy bone induction properties, but viable bacterial spores were still present within the bone. When exposure time was increased to 240 minutes, no viable bacterial spores were present, but bone induction properties of the implant were absent.3 Ethylene oxide treated allografts are believed to yield unacceptable results when used for spinal fusion techniques due to - . 7 decreased incorporation.1’76 It also has been suggested that ETO may be carcinogenic, especially with chronic exposure. However, 24 there is In: evidence that ethylene oxide-sterilized allografts have induced cancer.6“’148 Glycerol solutions The Swedish chemist, K.W. Scheele, discovered glycerol in 1779. It is syrupy, colorless, odorless, hygroscopic, miscible with water, and has become one of the world’s most widely used chemicals. It is used extensively in pharmaceuticals as a solvent, in creams, and as a lubricant in many products such as gelatin capsules and elixirs. The use of glycerol as a preservative for soft tissue grafts was first implemented in 1983 and quickly became the technique of choice for the Dutch National Skin Bank.57 Burn centers have successfully used glycerol preserved cadaver skin as a short-term biological dressing on wounds bed..7’1°9'13“'153 Preservation with with a questionable glycerol has been shown to be an inexpensive technique which does rum: require complicated equipment. Grafts may be stored in a refrigerator at 40 C or at room temperature (200 C). No significant ultrastructural changes have been observed if) skin grafts pmeserved ix) glycerol. Glycerol- preserved skin. maintains many of the characteristics of 25 fresh skin, including the collagenous and elastic architecture.”98 One clinical study reported that the inflammatory response seen in glycerol—preserved skin allografts was less than that seen with fresh donor skin. This may be due to the fact that glycerol has been shown to decrease antigenicity of tissues used as transplantation.56 The antigenic features of glycerol are believed to be associated vnjji its effective lyophilization properties by hygroscopic action. Lyophilization, as in freeze—dried grafts, has been shown to decrease immunologic reactions to allografts. Gflycerol, therefore, has been proposed as an effective lyophilization agent that could be used more simply and less expensively than by freeze-drying.82 Greater growth of capillaries, fibroblasts and autologous epitheliunl after application of a cause significant decreases SUI the breaking strength of irradiated bone. Irradiation below 3.0 megarads has been shown to have few effects on the grafts, but above this level a significant drop in the breaking strength of bone has been observed.“'110'131 This is thought to occur by irradiation affecting the collagen intermolecular crosslinks in the bone graft and. may be mediated by free radicals generated from water molecules, therefore affecting its mechanical stability.""”'131 Radiation produces reactive free radicals by the radiolysis of water. These cleave peptide bonds and thereby damage the collagen.“‘1 This effect has been seen in collagen in skin or tendon as breakdown of molecules into smaller subunits or by disorganization of the secondary structure of’ the triple helix. Histologically, gamma irradiation induced. crimping and separation. of collagen fascicles.L” At standard doses of irradiation, elasticity of bone is unaffected, but the capacity to absorb work and strength are decreased.45 31 Bone grafts are thought tr>lxe further compromised if tissues are freeze-dried in addition to being irradiated. Effects of the combination of gamma irradiation and freeze- drying are dependent upon the order of procedures. Initially irradiated, then freeze-dried bone-patellar tendon-bone grafts lmuiia 35% decrease in strength, whereas a freeze-dried then irradiated graft had a 75% decrease in strength.“’110 Although the combination of freezing and irradiation is detrimental to bone, the effect of freezing is thought to give partial protection against embrittlement in comparison with irradiatbmd at room temperature. It is possible that the affect of highly-reactive oxygen free radicals, produced by irradiation from the radiolysis of water, is decreased by irradiating when frozen.44 Treatment with gamma irradiation also is believed to 6%8L86 Lymphocytes reduce immunogenecity of time allograft. are very radiosensitive cells, and exposure to low—dose radiation produces almost complete destruction of lymphoid tissue, with suppression of immunological capacities?“78 It is thought that the DNA of the cell is the principal target for radiation-induced. cell lethalitju Therefore, proliferative cells would seem more sensitive to the effects of irradiation than resting cells.40 32 FeLV as a model for other retroviruses The human immunodeficiency virus (HIV) is a retrovirus belonging to the lentivirus subfamily. It is designated as human immunodeficiency virus type 1 (HIV-1) or human immunodeficiency virus type 2 (HIV-2). The only animals susceptible to experimental HIV-1 infection are the chimpanzee, gibbon sum» and rabbit, inn: AIDS-like disease has run: yet been reported 1J1 these species. The limitations of using simians to study HIV are the practicality, cost, safety, animal welfare, availability of the animals, and appropriate animal facilities.37 During the replication of a retrovirus such as the human immunodeficiency virus, the single-stranded RNA genome is copied by a preformed viral enzyme, reverse transcriptase, into a complementary DNA form, which then is converted to double-stranded DNA, called provirus. The proviral DNA then integrates within the chromosomal DNA of the infected cells of the host, resulting in viral replication whenever the host cell divides. The provirus can be detected using several molecular biological techniques.32 33 W.F.H. Jarrett first discovered FeLV in 1969 as a naturally occurring retrovirus. Since then iJ:Ihas become one of the most studied retroviruses affecting outbred species. Over 2% of the cat population in the United States is thought to be infected with FeLV, and resulting disease processes are responsible for most deaths in this species.123'141 FeLV is a retrovirus that belongs to the type C Oncornavirinae subfamily. The FeLV-associated immunodeficiency results in an acquired immunodeficiency disease (AIDS)—like syndrome similar to human AIDS and makes the feline model attractive for retroviral studies.”9 The effect of 3’-azido-3’-deoxythymidine (AZT) on the activity of retroviruses has been studied using FeLV as a model.141 The efficacy of the drug Zidovudine, the first antiviral drug to be approved for AIDS therapy, was studied using FeLV to test its antiretroviral activity.35 Although FeLV differs from HIV in terms of its host, the mechanism of tumorogenesis, and the primary route of natural infection, it serves as a well-documented comparative model for the study of HIV.48 Both viruses share a similar structure and replication cycle. Both lead 34 to an infection in which incorporated retroviral DNA results in the production of infectious virus particles by the host cell. The basic similarities and available reliable tests make FeLV an effective model to study retroviral transmission through transplantation.104 Although HIV and FeLV have different genomic structure and resultant infections represent distinct retrovirus/host relationships, there anxe many similarities 1J1 their infectious activity. Infections with both retroviruses require close contact and transfer of secretions or bdood for contagious transmission. Both infections are characterized by sequential stages in the progression of infection and disease and produce T-cell depletion in vivo. Both retroviruses manifest viral envelope glycoprotein heterogenicity, contain virus strain-related variations in tissue tropism and pathogenicity, and exhibit viral latency and activation. The pathogenic potential of the virus within an FeLV—induced AIDS strain suggests the possibility that a similar diversity may exist in other retroviruses such. as within HIV.103 Subtle changes. in the envelope glycoprotein (gp70) of FeLV can convert a minimally pathogenic virus into one that induces an acute form of immunodeficiency.58 It is thought that these changes may similarly occur in HIV—infected people.37 35 The feline immunodeficiency virus (FIV) is also a retrovirus, but belongs to the subfamily Lentivirinae. It is structurally and. biologically similar to HIV and is associated with immunosuppression in domestic cats. Besides the simian immunodeficiency virus (SIV), FIV is one of the more closely related viruses to HIV.108 Although FIV belongs to the same lentivirus subfamily as HIV, it is not antigenically related}.7 The prevalence of anemia, lymphopenia, neutropenia, and thrombocytopenia associated with FIV infection is similar to that seen in HIV- seropositive patients with AIDS. Use of this feline virus also could provide an understanding of marrow suppression from lentivirus infections and/or the hematologic effects of new therapies.129 Feline infectious viruses are good models for HIV infection in man. Cats are well-established research animal and almost all Vivaria are equipped to handle them. They are relatively inexpensive to obtain, easy to handle, and naturally infected cats with AIDS-like disease could be recruited for therapeutic trials.103'108 36 Infectivity of FeLV The pathogenesis of FeLV in experimentally and naturally infected cats has been desribed.122’141 The major vehicle for transmitting the virus in nature is probably blood and possibly saliva. Biting enables direct injection of the virus, while licking may permit infection of cats via the ocular, oral, and nasal membranes.53 Infection of local lymphoid tissue associated with the inoculation site occurs first where replication is amplified. Infected circulating' mononuclear cells spread. to 'various systemic lymphoid tissues. Virus replication can be detected in the bone marrow Iby 14-21 days after infecticnu After this point, infection is amplified by spreading circulating leukocytes and platelets and persistent (chronic) viremia becomes established. It is thought that time presence of antigen in circulation.51 A positive IFA result is highly predictive of persistent FeLV infection. The IFA test is performed on whole blood or buffy coat smears which are examined using fluorescent dye- labeled anti-p27 monoclonal antibodies. Infected cells fluoresce when stimulated by light of the appropriate wave length.5 The IFA test has been considered the reference standard FeLV test.5°'51'80 46 The assay for FOCMA antibodies is an indirect membrane immunoflourescence assay in which the test cat serum serves as the source of primary antibody, a fluorescein-labeled anti-cat antibody serves as a secondary antibody, and an FeLV—infected, feline lymphoblastoLd cell line (FL74) serves as the target cell. The test for antibody against FOCMA is used to identify the presence of antibodies in plasma and is indicative of exposure to the virus. Cats that develop FOCMA antibody titer >1:8 are thought to be protected against tumor development.12 Healthy persistently viremic cats do not have detectable amount of antibody to the viral structural proteins, but (RHI develop protective antibody titers to FOCMA in some cases. The amount of humoral antibodies to FOCMA present in cats is inversely correlated with tumor progression.88 An association between cats with positive FOCMA antibody titers and a history of disease has been reported. Lower FOCMA antibody titer in young diseased cats was thought to 1x3 related to inmmnosuppression resulting from transient, latent, or low-activity FeLV infection.138 More specifically, FOCMA was reported to have specific reactivity to the envelope glycoprotein (gp70) FeLV c.133'150 The FOCMA is thought to be virus encoded and not a tumor- 47 specific antigen. It binds to nascent but not mature virus particles.150 One study reported antibodies to FOCMA in kittens that nursed from mothers who previously nursed kittens injected with FeLV. This showed that the mothers were infected with FeLV but were able to mount an immune response which resulted in the production of humoral antibody.31 The polymerase chain reaction test is a powerful technique in which as little as one copy of a gene in chromosomal DNA can be amplified to yield as many as 106 or 109 copies that can be easily detected.32 The test can be particularly useful in instances where infectious virus particles are nonreplicating or are present in very low numbers. The use of the PCR has been useful in identifying FeLV proviral DNA in many lymphosarcomas in cats that are otherwise antigen negative.65'66 A positive PCR result on a blood sample indicates the presence of FeLV proviral DNA in peripheral blood leukocytes irrespective of the transcriptional activity of the proviral DNA. Therefore a positive result by the PCR test does Inn: necessarily indicate viremia. Although the 48 PCR has been shown to correlate well with the ELISA assay, discordant results have been found.101 One study compared the results of detection of FeLV antigen by ELISA and detection of FeLV DNA by PCR in peripheral blood samples. The study did not find a significant difference in detection of infected cats. Therefore, the authors suggested that the PCR may be more suitable to explore the question of a latent or replication-defective FeLV infection in an antigen—negative cat using tissues other than peripheral blood.66 49 BONE INCORPORATION AND VIRUCIDAL EFFECTS OF A 98% SOLUTION OF GLYCEROL OR ETHYLENE OXIDE STERILIZATION ON BONE ALLOGRAFTS IN CATS MATERIALS AND METHODS Harvesting and preparation of bone allografts Four weeks before implantation, 5 metatarsal bones from an uninfected SPF cat plus 3 groups of bones (n = 5 bones/group) from 5 SPF cats (3 bones from each cat) infected with the Rickard strain of FeLV were aseptically harvested and stored fresh-frozen at -700 C. The Rickard strain was chosen to expand upon previous studies using 9 and. because most cats infected with this this strainm strain will not exhibit disease during the 8 week study period. Bones harvested from the uninfected SPF cat (negative- control allografts) were thawed, placed into separate sterile plastic conical tubes, and refrozen at —700 (I (Figure 1). The 3 metatarsal bones from each of the FeLV- infected cats were assigned to 1 of 2 FeLV—infected 50 Figure 1: Photograph (of a harvested Inetatarsal cortical bone allograft. The metaphyseal segments of the bones were removed before implantation and saved for quantification of FeLV provirus. treatment groups or to the positive-control group. After thawing, intact. bones for the IETO verify an1 FeLV' negative status. Serial blood samples (3 ml) were collected weekly from each cat starting 22 weeks after surgery. When necessary, cats were sedated with a combination of ketamine hydrochloride (10 57 Figure 4: Postoperative lateral radiograph of a radius and ulna. The cortical bone allograft was stabilized into the ulnar ostectomy site using a 0.028 inch Kirschner wire. mg/kg, IM), midazolam (0.2 mg/kg, IM), and butorphanol (0.1 mg/kg, IM). FeLV p27 antigen The enzyme-linked immunosorbent assay (ELISA) was used to test for FeLV p27 antigen in plasma samples obtained at weeks 0 and 2 through 8. An ELISA microplate reader was used to quantify antigen on the basis of optical density (OD). The sample-to-positive control ratio (S/P) was calculated, using the following formula: (OD of sample) — (OD of negative-control sample) (OD of positive-control sample) — (CD of negative-control sample) Calculated S/P values of 2 0.1 were considered positive.101 FeLV antibody Antibody titers to feline oncornavirus cell membrane- associated antigen (FOCMA) were measured in plasma samples at weeks 0 and 2 through 8, using a live—cell immunofluorescence assay, as described elsewhere.101 Antibody titers of 2 1:16 were considered positive. 59 Extraction and quantification of DNA The DNA was extracted from buffy coats of anticoagulated. blood samples collected from each cat at weeks 0, 4, and 8, using a DNA extraction kit. Remaining sections of bone graft stored at the time of surgery, as well as the bone sample stored untreated at ambient temperature, were ground to powder, using a freezer-mill. The DNA was extracted from each sample of bone powder by use of a phenol-chloroform techniqueue; extracted solutions were electrophoresed through a 1% agarose gel and stained with ethidium bromide. The DNA extracted from blood and bone samples was quantified, using a DNA flourometer. Samples of DNA were digested with the restriction endonuclease EcoRl, electrophoresed through a 3% agarose gel, and stained with ethidium bromide to confirm detection and uniformity of DNA in each sample. Digested DNA samples were stored at 4°(3 in sterile microfuge tubes. Although all bone grafts underwent the same process of DNA extraction, a low quantity of DNA was obtained from 60 ETO—sterilized bone grafts, impeding quantification of FeLV provirus. Consequently, larger bone samples were collected from the ulna of the corresponding limb of each donor cat. These samples were subjected to ETO sterilization, DNA extraction, and quantitative polymerase chain reaction (QPCR). FeLV provirus A QPCR assay was used to quantify FeLV proviral DNA in 100 ng of digested DNA extracted from blood and bone samples. Negative and positive calibration standards were assayed in parallel with test samples. Other PCR assays for detection of FeLV provirus have been reported.“'101 Radiographic and histologic evaluation of implanted limbs After recipient cats were euthanatized, each implanted ulna was harvested and placed in neutral-buffered 10% formalin (Figure 5). High-resolution radiographs were taken of each harvested ulna, using a faxitron and kodalith—ortho film. Each ulna then was decalcified, using 5% nitric acid, and sections were prepared and stained with H&E for histologic examination. Subjective histologic 61 Figure 5: Photograph of a harvested implanted ulna after euthanasia 8 weeks after implantation. All grafts had completely healed in all of the groups. 62 analysis was performed to evaluate host—graft interfaces, healing of the bone graft, and incorporation of the bone graft by the host. Analysis was concentrated at the proximal and distal host-graph interfaces. Amounts of periosteal overgrowth, dead graft bone (identified as empty lacunae), incorporation (Hf host bone ixux> graft bone, and inflammatory cells were evaluated. RESULTS Cats At the time» of implantation, cats were 6.4 to 8.0 weeks old (mean, 7.2 weeks) and weighed from 0.45 to 0.90 kg (mean, 0.68 kg). At the time of euthanasia, cats weighed from 1.36 to 1.80 kg (mean, 1.52 kg). Surgical outcome All cats recovered from anesthesia without complications. Nine cats luui bone allografts implanted in the left ulna, and 11 cats had bone allografts implanted in the right ulna. On the basis of examination of radiographs taken immediately before and after surgery, all bone grafts 63 had good apposition within the ostectomy site, and the position of the Kirschner wire was satisfactory, except for 2 cats (1.:Ui group ETO and 1.:U1 group glycerol). The pin was placed medially to the distal portion of the ulna in the cat in group ETO. That cat recovered well and was not lame. The cat in group glycerol was reanesthetized, and the pin was repositioned because it had passed into the carpus. That cat and 2 other cats were lame in the affected limb for 1 week after surgery but subsequently used the limb appropriately. Complications with nonunion or malunion were not observed, and all grafts were incorporated into host bone. All 20 cats eventually used the grafted limb well. One cat in the positive-control group had early signs of carpal valgus at Vfififi( 2; this condition slowly progressed during the duration of the study. One cat in the glycerol group developed a seroma at the olecranon area of pin placement at week 7, which subsequently resolved over a few days. Another cat in the glycerol group developed acute lameness of the implanted limb at week 8. That cat did not exhibit signs of pain during manipulation of the limb; however, a transverse fracture of the radius and ulna at the distal graft site was discovered. when the limb was examined after the cat was euthanatized. We hypothesized that fractures of the radius and implanted ulna were 64 attributable to trauma from the cage, because adequate incorporation of the graft was evident at proximal and distal host-graft interfaces, and a bent Kirschner wire was evident on radiographs of the limb. Health status of cats Six cats developed transient vomiting or diarrhea during the study (2 cats from the negative-control group, 3 from the ETO group, and 1 from the positive-control group). Five cats developed transient sneezing or coughing (1 from the negative-control group, 1 from the ETO group, and 3 from the positive-control group). All but 1 of the cats were in good general health throughout the study. One of the cats in the positive-control group was given antibiotics because of aa respiratory tract infection at week 4. That cat initially responded to treatment, but relapsed and died at week 6. We hypothesized that the cat was immunocompromised as a consequence of FeLV infection. FeLV p27 antigen All cats had negative results when tested for FeLV p27 antigen just before surgery (week 0). Cats in the negative- 65 control and ETO groups had negative results for FeLV p27 antigen throughout the entire 8 week study. One cat from the glycerol group had positive results for FeLV p27 antigen at weeks 5 and 6, then reverted to negative results at weeks 7 and 8. All positive-control cats had positive results for FeLV p27 antigen at weeks 2 and 3. Two of these cats reverted and had negative results for the remainder of the study, whereas the other 3 positive-control cats had positive results throughout the remainder of the study, including the cat that died at week 6 (Table 1). FeLV antibody All negative-control cats were seronegative to FOCMA at week (1 and remained seronegative fer time remainder of the study (Table 2). One of the cats in group ETO had low titers (1:16) from weeks 3 through 8, but all other cats in that group were seronegative to FOCMA. One cat in the glycerol group had moderate antibody titers to FOCMA (1:32 to 1:128) at weeks 6 through 8. Four of the cats in the positive-control group had positive titers (range, 1:16 to 1:128) at week 2. All cats in the positive-control group had moderate to marked antibody titers to FOCMA from week 3 66 through the remainder of the study (range of titers at week 8, 1:128 to 1:2,096). FeLV provirus in blood samples All cats had negative results when tested for FeLV proviral DNA by QPCR before surgery (week 0). All cats in the negative-control and ETO groups had negative results for FeLV proviral DNA throughout the study. One cat in the glycerol group had positive results for FeLV proviral DNA at weeks 4 (257 copies) and 8 (8,729 copies), whereas all other cats iriiflua glycerol group had negative results for FeLV provirus. All positive-control cats had detectable FeLV proviral DNA at weeks 4 (range, 2,772 to 468,021 copies; mean, 204,428 copies) and 8 (range, 661 to 521,192 copies; mean, 243,178 copies). Three cats in the positive- control group had decreased proviral loads, and 1 cat had an increased proviral load, from weeks 4 to 8. The remaining cat in the positive-control group died at week 6 (Table 3). 67 Table 1: Positive FeLV p27 antigen results in blood samples from. cats after implantation ‘with aa cortical bone allograft. Number of Cats with Positive FeLV p27 Antigen Results Week after implantation Group 0 2 3 {4 5 6 7 8 Negative - control (n =5) 0 0 0 O 0 O O 0 ETO(n=5) O () O O O O O 0 _ 1 1 Glycerol (n— 5) O 0 0 O (0.05) (0.11) O 0 Positive-control O 5 5 3 2 3 2* 2* (n = 5) (0.27) (0.56) (0.60) (0.39) (0.55) (0.62) (0.84) * n = 4. Negative-control group = untreated allografts from uninfected SPF cats. ETO group = ethylene oxide-treated allografts from FeLV-infected cats. Glycerol group = 98% solution (n3 glycerol-treated allografts from FeLV—infected cats. Positive-control group == untreated. allografts from FeLV-infected cats . Values reported as number of blood samples within a group with positive results (and mean S/P ratio). 68 Table 2: Antibody titers to feline oncornavirus cell membrane-associated antigen (FOCMA) 2 1:16 in blood samples from cats following implantation with a cortical bone allograft. Number of Cats with Antibody Titers to FOCMA 2 1:16 Week after implantation Group 0 2 3 4 5 6 7 8 Igoengtigrfnfi) 0 0 0 0 0 0 0 0 ETO (n=5) 0 0 (1 :116) (1:16) (1:16) (1 :11 6) (1:16) (1 :116) 313;?“ O O 0 0 O (1:132) (1:128) (1:128) Positive 4 5 5 5 5 4* 4* o (1:16- (1264- (1:64- (1:64- (1:128- (1:128- (1:128- °°mr°1 1:128) 1:128) 1:128) 1:512) 1:2096) 1:1024) 1:2096) Titers 2 1:16 were considered positive. See Table 1 for key. Values reported indicate number of cats (range of titers). * n = 4. 69 Table) 13: Quantitative polymerase chain reaction test results in blood and bone graft samples from cats that received a cortical bone allograft. Copies of FeLV Provirus Blood samples G (week after Bone roup implantation) samples* 0 4 8 Negative Control 0 O O 0 (n=5) ETO Grou 3 (“25) P o o 0 (5,733 .+_ 3,400) Glycerol l l 5 Group 0 (1746 :1: (47,120 i (n=5) (511' 51) 1746) 11,690) Positive 5 4** 5 Control 0 (204,428 i (243,178 i (49,459 i (n=5) 107,716) 140,668) 15,760) Ambient temperaturet 3J50 (n=1) Values reported indicate the number of cats or bones with positive copy numbers for FeLV provirus (mean copies i SE). A value of 22 or greater copies was considered positive. *Bone samples were obtained from sections of bones implanted into recipient cats. ** n = 4. TResults (recorded as copies) for a bone sample stored at ambient temperature for 4 weeks. 70 Agarose gel electrophoresis Following agarose gel electrophoresis and ethidium bromide staining, DNA from bones of negative-control, glycerol-treated, enui positive—control groups appeared intact and had distinct bands. In contrast, DNA from the ETO group bones did not have distinct DNA bands; it was smeared throughout the length of the gel, appearing denatured and suggestive of reduction or elimination of intact virus or provirus. FeLV provirus in donor bone All negative-control allografts implanted into cats in the negative-control group had negative results when tested for FeLV provirus. Three (range, 2,157 to 20,318 copies; mean = 9,555 copies) of 5 (range 0 to 20,318 copies; mean i SE, 5,733 i 3,400 copies) ETO—treated bones had positive results for FeLV pmoviral DNA" .All five glycerol-treated bones had positive results for FeLV proviral DNA (range, 4,459 to 80,464 copies; mean i SE, 47,120 1 11,690 copies). In contrast, blood samples from only 1 of the recipient cats in the glycerol group had positive results when tested for FeLV proviral DNA. All untreated positive-control bones 71 had positive results for FeLV proviral DNA (range, 7,322 to 87,113 copies; mean i SE, 49,459 i 15,760 copies), as did recipient blood samples. The untreated kxxma graft stored at ambient temperature for 4 weeks had less FeLV proviral DNA (3,750 copies) than glycerol—treated or untreated bones (Table 3). Radiography High—resolution. radiographs taken cu? implanted. limbs after cats were euthanatized revealed moderate callus formation an: proximal enui distal host-graft interfaces in all cats. Host bone appeared incorporated into graft bone at. all host-graft interfaces, and. graft. bone was almost indistinguishable in rmxfl: cats. Subjectivelyy differences in healing were not observed among groups (Figure 6). Histologic examination Good incorporation of the donor graft by host bone was observed histologically, and no appreciable differences were seen between groups. Active remodeling and incorporation was detected in all bones along proximal and distal host—graft interfaces. At the graft interfaces, dead 72 graft bone was surrounded by new woven host bone. Bone grafts were identified by empty lacunae within bone surrounded by new woven host bone (Figures 7 and 8). Moderate periosteal proliferation was observed bridging the host-graft interfaces. Moderate infiltration of neutrophils was evident at the graft sites, but differences in inflammatory or toxic changes were not observed. between groups. 73 Figure 6: High-resolution radiographs of a harvested radius and ulna 8 weeks after implantation with a cortical bone allograft. Moderate callus formation was present around the graft site in all cats. a) Negative control, b) Positive control, c) Glycerol group, d) ETO group. 74 Photomicrograph of the host-graft interface in the ulna of a cat that received a cortical bone allograft. e I Figure 7 lacunae as empty host visualized be new can graft Dead bone (10X) bone. woven by surrounded 75 Figure 8: A magnified photomicrograph of the host-graft interface as seen in Figure 7.(20X) 76 VIABILITY OF RETROVIRUS (FELINE LEUKEMIA VIRUS) IN CORTICAL BONE GRAFTS AFTER ETHYLENE OXIDE STERILIZATION OR 98% GLYCEROL PRESERVAT ION MATERIALS AND METHODS Harvesting and preparation of bone allografts Four weeks before cell inoculation, 3 groups of bones (n = 5 bones/group) from 5 SPF cats (3 bones from each cat) infected with the Rickard strain of FeLV were aseptically harvested and stored fresh—frozen at -700 C in a sterile plastic conical tube. One of the 3 metacarpal bones from each of the FeLV- infected cats was assigned to 1 of 2 FeLV-infected treatment groups (ETO-treatment or glycerol-preservation) or to the FeLV-infected positive-control group (no treatment). After thawing, intact bones for the ETO group were separately double-wrapped by use of heat-sealed plastic and sterilized with 100% ETO. After ETO sterilization, bone samples were aerated at 500 C for 12 hours (relative humidity, 30%) and refrozen at —700 C. Bones in the glycerol group were placed separately into 77 conical centrifuge tubes that contained a 98% solution of glycerol, then were stored in the dark at room temperature (220 C) for 4 weeks.9l7 The positive-control group of bones de not undergo treatment and were placed separately into sterile plastic conical tubes and refrozen at —700C. Cell culture Confluent cells from ea feline fibroblast cell line (FEA) were passaged. at £1 1:3 dilution into 25 separate sterile flasks containing 10 mls of media the day before test samples were added. Media consisted of Dulbecco’s modified eagle medium with 15% fetal bovine serum, 2% glutamine, 2% NaHCO3, 1% Na-Pyruvate, 10 ug/ml gentocin, and 10 ug/ml enrofloxacin. Samples of stock media and FEA cells were saved prior to inoculation to confirm FeLV negative status. Media was aspirated from the cells and replaced with 10 ml of FEA cell media containing a 0.03 mg/ml diethylaminoethyl dextran for 30 minutes, then replaced with 10 ml of FEA cell media when test samples were added. Each untreated or treated bone was individually minced using lembert rongeurs, and 250 mg of minced bone was immediately introduced into a separate flask of FEA cells. 78 Each flask was then placed into a 370C, 5% C02, humidified incubator. An additional 0.5 1n1 cu? media was added. to five flasks of FEA cells (negative control). In addition, five flasks of FEA cells were inoculated with 0.5 ml of supernatant. fluid. from 51 productively FeLV-infected. cell line, FL-74 (positive control) cells. Cells were allowed to grow until confluency, seen as a homogeneous layer of FEA cells covering the dependent wall of the flask when viewed using en1 inverted light ndcroscope. When cellular confluency was reached, media was individually saved from each flask. Cells in each flask were trypsinized and centrifuged to pellet the cells. The supernatant fluid was discarded, and the remaining cells were resuspended in 1 m1 of media. A 1:15 dilution of resuspended cells was then introduced into new flasks containing 10 mls of media. The remaining resuspended cells were saved for DNA extraction and quantification of FeLV provirus. This process was repeated for a total of four passages. 79 FeLV p27 antigen The ELISA was used to test for FeLV p27 antigen in culture media samples obtained prior to inoculation and at each passage. An ELISA microplate reader was used to quantify antigen on the basis of optical density (OD). The sample-to-positive control ratio (S/P) was calculated, using the following formula: (OD of sample) — (OD of negative-control sample) (OD of positive-control sample) — (OD of negative-control sample). Calculated S/P values of 2 0.1 were considered positive. Extraction and quantification of DNA A DNA extraction kit was used to extract the DNA from FEA cells obtained preinoculation and at each of the 4 cell passages. The DNA extracted from the cells was quantified, using a DNA flourometer. Samples of DNA were digested with the restriction endonuclease EcoRl, euxi digested DNA samples were stored at 4°C in sterile microfuge tubes. 80 FeBV'provirus A. real-time» quantitative jpolymerase chain reaction (QPCR) assay was used to quantify FeLV proviral DNA in 100 ng 'of digested DNA extracted from FEA cell samples. Negative and positive calibration standards were assayed in parallel with test samples. Statistical Analysis To compare the mean amount of DNA provirus measured between groups preinoculation and at passages 1, 2, 3, and 4, a one-way ANOVA was performed. A Bonferroni multiple comparisons test was performed at each passage to test for significance between groups. Differences were considered statistically significant at p<0.05. RESULTS FeLV p27 antigen All media samples in the negative control, ETO- Sterilized, and glycerol—preserved groups were negative for FeIAV p27 antigen throughout the entire study. Due to 81 laboratory error, one sample from the ETO-sterilized group from the second passage was excluded. Four of five media samples from the positive bone control group were positive for FeLV p27 antigen at the first and second passages. At the third and fourth passages, all samples in this group had positive results. All samples in the positive virus (FL-74 cell) control group were positive for FeLV p27 antigen at all four passages (Table 4). FeLV provirus .All cell culture samples in 13KB negative control and ETO groups had negative results throughout the study for FeLV proviral DNA by QPCR. Four of 5 samples in the glycerol group had positive FeLV proviral DNA at the first passage (range, 42.6 to 581.5 copies). At the second passage, 3 of 5 samples in this group were positive for FeLV provirus (range, 1.58 to 27.97 copies). All of the glycerol group samples were negative for FeLV provirus at the third and fourth passages. One sample in this group was negative at all 4 passages. All samples in the positive bone and positive virus (FL-74 cell) control groups were positive for FeLV provirus 82 throughout the study. The mean for the positive bone control group at the first passage was 8,142 copies. The mean for this group peaked at the second passage (254,487 copies) and declined at the third (107,019 copies) and fourth (104,486 copies) passages. The highest mean copy number for the positive virus (FL-74 cell) control group was at the first passage (87,064 copies). The means steadily decreased at the second (85,677 copies), third (59,8945 copies), and fourth (38,357 copies) passages (Table 5). No statistically significant differences were observed between the negative control and ETO groups throughout the study. A significant difference was present between the glycerol group and the negative control and ETO groups at time first. passage. However, rm) significant. differences between these groups throughout the remainder of the study were seen. No statistically significant differences between the positive bone and positive virus (FL-74 cell) control groups were present throughout the study. 83 Table 4—Positive FeLV p27 antigen results for cell culture media from negative control, ETO-sterilized, glycerol- treated, and positive bone and virus control groups. Cell Culture Media Samples with Positive FeLV p27 Antigen Results Passage Group 1 2 3 4 Negative - conno101=5) O O O O ETO (n = 5) 0 0‘ 0 0 glycerol (11 = 0 0 O O Positive-control 4 4 5 5 bones (n = 5) (0.22) (0.39) (0.34) (0.50) Positive-control 5 5 5 5 virusQi=5) (0.55) (0.45) (0.31) (0.44) See Table 1 for key. Positive-control virus group = inoculated with supernatant fluid from a FeLV-infected cell line (FL-74). Values reported as number of samples within a group with positive results (and mean S/P ratio). Due to laboratory error, one sample from the ETO- sterilized group from the second passage was excluded. 84 Table 5—Results of a quantitative polymerase chain reaction test to detect the number of copies of FeLV provirus in negative control, ETO-sterilized, glycerol-treated, and positive bone and virus control group cell cultures. Copies of FeLV Provirus Cell samples Group (Passage) 1 2 3 4 Negative Control 0 0 O 0 (n=5) ETO Group (n=5) 0 0 0 0 8:13?“ 4 3 ‘ 0 * * (n=5) (187) (9) (0-63) Positive 5 5 5 5 Contml 8142 254 486) (107 019) (104 486) Bone(n=5) (’ ) ( , , , Positive Control 5 5 5 5 Virus(n=5) (87,064) (85,677) (59,895) (38,357) See Table 4 for key. Values reported indicate the number of cell cultures with positive copy numbers for FeLV provirus (mean copies). * Considered negative. Copy numbers of 22 or greater were considered positive. 85 DISCUSSION The feline leukemia virus cat system has been used as an effective model to investigate transmission of a retrovirus via bone and connective tissue allografts.104’105 Although extrapolations must be rmxka to determine whether sterilization methods exhibiting virucidal activity against FeLV would be effective against HIV, FeLV is a safe and efficient model. The SIV is the lentivirus most closely related to HIV. However, purchase, housing, and maintenance costs as well as the risk of injury to investigators are higher for monkeys than cets. Surgical procedures on cats are relatively inexpensive, yet it is possible to perform intricate treatment and transplant operations that would not be possible using a murine system. There are limitations with other lentiviruses as a model for HIV’ as well as advantages for using FeLV to investigate transmission of retroviruses following transplantation. Many serologic tests to detect FeLV infection are available. Investigators can detect early and late stages of FeLV infection using assays to detect FeLV 86 antigen, antibody, and provirus. Similar assays are available for FIV, but detection of provirus is limited to specific research strains. Cell types that FIV reportedly infects are restricted to lymphocytes, macrophages, astrocytes, microglial cells, and. endothelial cells, and have not been shown to include bone tissue.108 In contrast, transmission of FeLV through bone and connective tissue allotransplantation has been documented, making FeLV the best model.104'105 A limitation of an in vivo study is the expense and availability of animals, which in turn restricts the number of animals per group. A larger number of cats per group may have increased our power of confidence for results of the in vivo study. Importantly, transmission of FeLV occurred in one cat in the glycerol group, but was not documented in any of the cats that received ETO-treated bone grafts. Various techniques have been reported for preservation and sterilization of canine cortical bone 27'33'73'119 However, their use in most veterinary allografts. practices has been restricted by the cost and inconvenience of allograft collection, processing, anui storage. Typical 87 methods utilized include sterile graft harvesting followed by low-temperature storage (-20 to —40 C) or lyophilization (freeze—drying). Frozen bone grafts have a shelf life of only 6 to 12 months. Freeze-drying grafts has the advantage of allowing storage at ambient temperature, but this technique reportedly decreases the mechanical strength of bone.33 Secondary sterilization methods can be used to eliminate the need for aseptic harvesting of bone grafts, thereby reducing cost and increasing convenience. Gamma irradiation is an effective sterilization technique for deep penetration of thick tissues, but requires instruments that may not be available in most veterinary hospitals. In addition, high doses of irradiation have been associated with decreased osteoinductive and biomechanical properties of bone grafts.33 Ethylene oxide has been widely used for sterilization and reportedly is effective for removing surface contamination, but decreases bone induction and incorporation properties as well as reducing mechanical bone strength . 3'142'152 An optimal storage and sterilization technique would eliminate the need for special equipment, sterile harvesting of grafts, or both. 88 Reports of viral transmission to people following transplantation of soft-tissue or bone grafts have raised concerns regarding sterilization and storage of these grafts."'21'23’25 Although ETO reportedly is an effective bactericidal agent, its virucidal properties for tissues have not been documented. In contrast, virucidal activity of an 85% solution of glycerol against HSV-l and polioviruses was reported.97 Soft-tissue grafts stored in an 85% solution of glycerol at 200 C had complete inactivation of HSV—1 and poliovirus after 8 or 22 days of storage, respectively. Because that was an in vitro study, data was not available regarding the effects of transmission of these viruses in an1.in vivo system. Investigations of the virucidal properties of glycerol on transplanted bone grafts have not been described. However, treatment of canine femoral cortical bone allografts with a 98% solution of glycerol at ambient temperature was adequate for storage and resulted in good healing when a bone plate was used for 7 Complete periosteal bridging was seen at stabilization.2 the graft sites with a continuity of cortices at the host— graft interfaces 90 days after graft implantation. 89 In the studies reported. here, results of IETO sterilization. were promising, because all recipient cats had negative results when tested for viral antigen and provirus with no evidence of Viral transmission. Although 3 of 5 ETO-treated donor bone grafts had positive results for FeLV provirus, none of the recipient cats in this group became infected after transplantation. Lack of viral transmission to recipients of ETO-treated bone grafts may be attributable to a decrease in the infectious viral load. Additionally, the 3 bones from the ETO group that had positive results (mean, 9,555 copies) had less FeLV provirus than bones for the glycerol (mean, 47,120 copies) or positive—control (mean, 49,459 copies) groups (Table 3). Antibody titers to FOCMA 2 1:16 may result from exposure to infectious virus or, possibly, viral antigens. A low-positive antibody titer to FOCMA was detected in 1 cat in the ETO group despite lack of viral antigenemia. This may have resulted from a viral antigen(s) in the bone graft stimulating an1 antibody' response 1J1 the recipient, despite lack of intact infectious virus. Alternatively, antigen concentrations may have been too low to detect a positive reaction in blood samples. 90 Cats in this study did not have evidence of ETO toxicosis. Toxic byproducts of ETO sterilization include ethylene glycol and ethylene chlorhydrin. Apart from the toxic effects of these ethylene oxide residuals, another possible explanation for reduced incorporation may be ETO- induced alkylation of amino acids.142 In the in vivo study reported here, equivalent bone incorporation was observed in cats in the ETO group, compared with cats in the untreated. negative- and.jpositive-control groups. Although there were undoubtedly toxic byproducts, lack of difference in incorporation 1J1 this model Hey 1x3 partly attributable to the young age of the cats and their high propensity for healing. A study that uses adult cats would likely reveal differences in incorporation. Transmission of FeLV to one of the cats in the glycerol group may have been associated with immunocompetence of the recipient, infectious virus titer of the graft, or both. Because all donor bones in the glycerol group had more FeLV provirus (mean, 47,120 copies) than the untreated bone stored at ambient temperature for 4 weeks (3,750 copies), it is possible that the 98% solution of glycerol enhanced viral preservation. Unfortunately, infectivity could not be assessed in the untreated bone 91 because it was not implanted in a cat. Despite the fact that ambient temperature storage of untreated FeLV-infected bone appeared to decrease viral load, the resultant putrefaction precludes its use. Donor bones in the positive-control group had the highest amount of FeLV provirus (mean, 49,459 copies). All cats in the positive-control group had positive results when tested for FeLV p27 antigen 2 weeks after transplantation. Although 22 cats in tflma positive-control group subsequently had negative results for FeLV p27 antigen (Table 1), these cats had positive results for FeLV provirus at weeks 4 and 8 (Table 3). Transient antigenemia has been reported in cats with natural FeLV infection or infection resulting from experimental implantation of FeLV- infected bone and connective tissues.10“'105 However, investigators in those studies did not assess FeLV status by use of the more sensitive quantitative PCR technique. All cats in the positive-control group were seropositive to FOCMA, confirming exposure tx> FeLV. Furthermore, all cats had positive results for FeLV provirus in blood samples, documenting infection with FeLV. 92 In the in vitro study, a higher proviral load in glycerol-preserved compared with ETO—treated bone grafts was observed at passage 1. However, cultures containing both glycerol-preserved and ETO-treated bones were negative for FeLV provirus at passages 2 through 4. Results for FeLV p27 antigen were consistently negative in media samples from cultures containing both glycerol-preserved and ETO-treated bones. It appeared that FeLV viral particles in these bone grafts were noninfectious. This was in contrast with results of the in vivo study, where one of the recipient cats that received a glycerol- preserved bone graft became infected with FeLV. During the first passage, cells in the glycerol group took approximately' 5 days longer to achieve confluency. Following the first passage and a change in media, cells in the glycerol group achieved confluency at time same rate as cells in other groups. It is likely that residual glycerol present on the bone samples affected the media environment by' lyophilization. The resultant slowing (If cell replicaticmi was less than. optimal for 'viral replication. Detection of the FeLV provirus segment in the glycerol group at passage 1 may have been due to viral particles that were either noninfectious or unable to replicate in 93 this environment. In contrast, tine environment presented to the glycerol-preserved bones in the in Vivo study was highly cellular with diverse cell types and good. blood flow, providing optimal opportunities for replication. Differences observed in transmission of retrovirus in glycerol-preserved bones to cells versus cats highlight the importance of conducting both in vitro and in vivo studies, respectively. Whole, intact FeLV provirus or smaller amplifiable regions of FeLV provirus may be detected in DNA samples analyzed by QPCR. Detection of the small segment of provirus amplified by QPCR does not establish infectivity of donor bone grafts. In contrast, detection of provirus or antigen in blood following allotransplantation or passage in cell culture does prove transmissibility of the retrovirus. The fact that all cats in the positive—control group had positive results for FeLV antigen and provirus in blood samples confirms that freezing does not effectively impair viability of the retrovirus or prevent transmission after implantation. This was substantiated by the in vitro study that showed. positive results for FeLV’ provirus in cultures that contained untreated bone from FeLV-infected cats at all 4 passages. 94 The studies reported here documented that ETO sterilization appeared to denature DNA and had effective virucidal activity against the retrovirus FeLV. In contrast, use of a 98% solution of glycerol was inadequate for viral sterilization of cortical bone allografts. Comparison of the virucidal effects of glycerol-preserved with untreated. positive—control grafts did. not reveal a reduction in the quantity of amplifiable FeLV provirus in donor grafts treated with a 98% solution of glycerol. Although transmission of FeLV was decreased in recipients of glycerol-treated bone grafts, suggesting decreased infectivity, a 4-week duration of glycerol treatment for bone allografts cannot Ema recommended. for ‘virucidal sterilization. Additional studies may be warranted to examine the effect of prolonged (eg, 6 months) glycerol treatment on virus-infected bone grafts. Histologically, no differences in incorporation at the host-graft interface were observed between groups. It may be concluded from this study that bone allografts sterilized with ETO or a 98% solution of glycerol had comparable incorporation of host bone, compared with untreated-control groups in this model using young cats. 95 However, ETO sterilization had superior virucidal activity against the retrovirus FeLV. Ethylene oxide sterilization abrogated transmission of FeLV infection, possibly by denaturing the DNA. However, quantities of provirus detected by QPCR in ETO—treated donor bone grafts were reduced but not eliminated. Additional studies to determine whether provirus in ETO- treated bone was intact infectious virus or smaller noninfectious segments of proviral DNA are warranted. Lack of transmissLmi of FeLV to recipients of ETO-treated bone grafts suggests that the veterinary community may be cautiously optimistic regarding the safety and efficacy of this widely available treatment. 96 CONCLUSIONS The in vitro and in vivo studies reported here documented that ETO sterilization appeared to denature DNA and had effective virucidal activity against the retrovirus FeLV. Quantities of amplifiable provirus detected by QPCR in ETO—treated donor bone grafts were reduced but not eliminated. We hypothesized that virus particles and provirus in bone were rendered noninfectious following ETO treatment. In contrast, although treatment of cortical bone allografts with a 98% solution of glycerol appeared effective in in vitro studies, it was inadequate for viral sterilization in an in vivo model. Comparison of the virucidal effects of glycerol-preserved with untreated positive-control grafts ciui not reveal ea reduction 1J1 the quantity of amplifiable FeLV provirus in donor grafts treated with a 98% solution of glycerol. Although transmission of FeLV was decreased in glycerol-treated compared. with untreated. bone graft recipients suggesting decreased infectivity, 4-week glycerol treatment for tmme allografts cannot be recommended for virucidal sterilization. 97 While the glycerol—preservation protocol used in this study does not show adequate antiviral effects, additional studies may be warranted using different concentrations (eg, 85% glycerol solution), storage temperatures, or prolonged. preservation times “my 6 months). Moreover, additional ETO sterilization protocols may be investigated to further document its antiviral effect. It also may be concluded from these studies that bone allografts sterilized with ETO or a 98% solution of glycerol luni comparable incorporation cfif host bone, compared with that for untreated-control groups in this model using young cats. These paired studies are a good example of the use of both in vitro and in vivo studies to investigate the same question. iResults of the ;U1 vitro study were encouraging for both ETO and glycerol and warranted additional investigation. However, the in vivo study was a more complete model to test transmisshm1 of the retrovirus and demonstrated that although ETO appeared to have adequate antiviral activity, 98% glycerol was ineffective. This points out that both in vitro .and in vivo studies are 98 required to reach a reliable conclusion regarding antiviral efficacy of treatment protocols. Results of these studies may be applied to both human and veterinary medicine. The orthopedic community should continue 11) be vigilant regardimg the potential of infectious viral particles present in bone sterilized with ETO. Screening procedures of candidates for donation of allografts are the most important aspect of preventing implantation. of infected. Ibone. Dogs are frequent recipients of bone transplants, and results of these studies may be used as a starting point for investigations of the efficacy of ETO for preventing transmission of canine diseases through allotransplantation. 99 RECOMMENDATIONS Lack of transmission of FeLV to recipients of ETO- treated bone grafts suggests that veterinary and human communities may be cautiously optimistic regarding the safety and efficacy' of this widely available treatment. Intense screening of Emmential allograft donors should be continued. 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