An Advancing Allografts Brief October Sterilization of Allografts. Advancing Allografts

Transcription

1 An Advancing Allografts Brief October 24 Sterilization of Allografts Advancing Allografts

2 Ensuring Safety in Tissue Transplantation: The Sterilization of Allografts By Lloyd Wolfinbarger, Jr., PhD Tissue Safety A Major Concern in Transplantation Disease transmission and bacterial infection in musculoskeletal transplantation continue to raise significant concerns despite efforts to reduce health risks to transplant recipients. Nonetheless, allograft usage among orthopedic surgeons and neurosurgeons has risen dramatically over the past two decades, resulting in impressive life-enhancing benefits. In 22 alone, nearly 1 million allografts were distributed in the United States. Recently, an urgent concern about allograft safety was raised when an implant contaminated with Clostridium sordellii caused the death of a 21-year old man. The Food and Drug Administration (FDA) responded by proposing requirements for Good Tissue Practices (GTPs) covering procedures, facilities, personnel, equipment, supplies, reagents, process and labeling controls, process changes and validation, storage, receipt and distribution, records, tracking, and handling of complaints. 1 Current preparation methods, including screening for disease, bacterial testing and aseptic processing, substantially reduce risk, but do not completely eliminate the possibility of allograft-associated infections. A more certain way to prevent infection and preserve function is needed. Sterilization has been proposed as one definitive method for eliminating microorganisms without adversely affecting the structure of transplanted tissue 1. This paper explores the current state of tissue transplantation safety and offers an in-depth look at such new sterilization methods as included in Allowash XG. First, a brief exploration of the current risk of allograftrelated infection will be helpful to understanding the challenges involved in safe tissue transplantation. Estimated Rates of Viremia Demonstrate Safety Limitation Hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and human T-lymphotropic virus (HTLV) have all been transmitted by tissue transplantation. 2,3,4 According to a recent study using data from the various review and testing procedures of tissue-banking organizations, the estimated incidence of viremia at the time of donation is 1 in 55, for HBV, 1 in 34, for HCV, 1 in 42, for HIV, and 1 in 128, for HTLV. 5 Actual incidence rates were estimated to be 3.118, , 12.38, and 5.586, respectively. Thus, prevalence rates of HBV, HCV, HIV, and HTLV infections are lower among tissue donors than in the general population. This finding is not surprising, since tissue donors are chosen carefully based on medical history, physical examination and interviews with the next of kin. This process, however, is not as effective as face-to-face interviews conducted with blood donors. Not surprisingly, the estimated probability of undetected viremia at the time of tissue donation is higher among tissue donors than among firsttime blood donors. The fact that the probability of collecting products from a viremic donor is low, but not negligible, remains a primary safety concern in transplantation. In blood donation, the implementation of nucleic acidamplification testing of minipools (pools of 16 to 24 1

3 blood donations) has markedly reduced the residual risk of viremia and transfusion-transmitted infection. Currently, efforts are underway to implement such testing on cadaveric samples. Allograft Sustained Susceptibility to Contamination Utilizing allograft tissues can increase the inherent risk of bacterial and fungal contamination. The surgical results of contamination can be serious, but often go unreported. 6 Thus, the FDA has recently increased inspection and enforcement of tissue donation, including the implementation of a regulatory framework for tissues. Even so, it has been widely proposed that more stringent, comprehensive steps be taken to promote and enhance tissue safety. 7 Current Tissue Bank Methods Major Safety Limitations The goal of allograft tissue processing is to provide the safest possible material to the surgeon while preserving the inherent tissue characteristics of the graft. Even with adequate donor screening, however, there remains a risk of allograft contamination. Oversight of tissue-banking practices has become more stringent to include monitoring by the FDA, the American Association of Tissue Banks (AATB) and individual state agencies. The FDA now requires preparation, validation and written procedures to reduce the probability of contamination during processing. The AATB publishes quality standards for procuring tissue, processing tissue (including time limits for retrieval), and for screening donors, as well as publishing strict recommendations for preservation, sterilization, preparation, evaluation and labeling of tissues. Individual tissue banks can apply for voluntary accreditation by meeting AATB standards, which include aseptic techniques, microbiologic testing (i.e., aerobic, anaerobic, and fungal preprocessing and postprocessing cultures) and adverse outcomes reporting. Despite these safety guides, allograft preparation procedures could be enhanced for safety. For instance, not all tissue banks apply for AATB accreditation. In 22, approximately 1% of musculoskeletal allografts were processed by nonaccredited banks. 1 Further, a recent investigation by Kainer and colleagues demonstrated that infections acquired through bacterial contamination of allografts are underreported and have the potential for resulting in substantial complications or death. 6 The study suggests that the current standards for processing and testing allograft tissue need to be enhanced to prevent such life-threatening allograft-associated infections. As a general rule, implanted tissues are not processed with sporicidal methods. Moreover, current regulations do not require tissue banks to eliminate bacteria present on tissues at the time of recovery or to use processing methods that guarantee tissue sterility. 6 Most tissue banks process musculoskeletal allografts aseptically by treating the tissue using various chemical, mechanical and detergent steps. Aseptic processing is defined by the AATB as the processing of tissue using methods to prevent, restrict or minimize contamination with microorganisms from the environment, processing personnel or equipment. These methods reduce the inherent microbial bioburden present in incoming cadaveric tissue without entirely eradicating microorganisms that sometimes remain after aseptic processing. The specific problem with aseptic processing is that it only minimizes bacteria without eradicating organisms and spores, especially in tissue that is heavily contaminated at the time of recovery. 8 To reduce bacterial contaminants, some tissue banks suspend tissue in antimicrobial solutions. However, these solutions may not eradicate spores, as demonstrated by in vitro studies. 6 Two sterilization methods that can be used to eliminate spores are gamma irradiation and treatment with ethylene oxide. However, both methods have the potential to create technical problems with allografts, limiting their usefulness in tissue processing. 8 Further, high doses of gamma irradiation may adversely affect the biomechanical properties of allografts. 9,1,11,12 Ethylene oxide has a limited ability to penetrate tissue and has been associated with adverse outcomes such as synovitis 13 or damage to musculoskeletal tissue, resulting in an unacceptably high rate of mechanical failure. 14 2

4 To investigate true eradication of risk, several banks have developed low-temperature sterilization approaches to kill spores, while preserving allograft biomechanical integrity and function. Notably, such a sterilization technique is best served when it can be validated. Using allografts, Moore and colleagues developed a viable adaptation of sterilization for medical devices. Specifically, these investigators adapted AAMI/ISO 1137 Method 2B terminal sterilization validation to musculoskeletal grafts, both soft tissue and bone grafts, using gamma radiation. This sterilization method determines the minimum absorbed dose of radiation necessary to achieve a sterility assurance level (SAL) of 1-6 for products with consistently low levels of microbial bioburden. By achieving a validated sterility assurance level (SAL) of 1-6 for an allograft, the FDA permits the product to be labeled sterile. Since the destruction of microorganisms by gamma irradiation follows an exponential rule, the probability of survival is a function of the following: The number and species of microorganisms present on the allograft The lethality of the gamma irradiation process (i.e., radiosensitivity), and inflammation when implanted and the osteoconductive, biomechanical or structural properties of such tissues are not affected or altered. Genuine Sterility Confidence Using Allowash XG LifeNet introduced Allowash in 1995, when it was a revolutionary process in cleaning and disinfection. Allowash Solution is a combination of three detergents, which have demonstrated superiority in solubilization of bone marrow. These detergents include Brij 35, Nonoxynol 9, and Nonidet P4 for solubilizing bone marrow cells. In addition, during the Allowash process, hydrogen peroxide in a 3% solution is used as a scrubber and disinfectant, and a 7% isopropanol alcohol solution is used as a disinfectant and drying agent. In the traditional Allowash process, bone marrow and other cellular elements associated with bone are removed by detergents in cleaning steps. The hydrogen peroxide and alcohol processing steps further reduce potential bioburden by acting as disinfectants. See Figure 1 for a schematic of the cut graft protocol design. "Cut Graft" Protocol Design The environment the organisms are in during the irradiation process SAL can be mathematically derived to define the probability of viable microorganism on an individual graft following a Preprocessing/ Debridement 1 Dry Spin 2 3 Wet Spin Allowash with H 2 O 2 Solution/ Ultrasonication specified gamma irradiation dose. 3 Gamma Irradiation Sterilization A New Standard for Allograft Safety Gamma irradiation sterilization is the only method available that has been proven to eliminate viruses, bacteria, fungi and spores from tissue without affecting the structural or biomechanical attributes of tissue grafts. 15,16,17 The efficacy of allograft sterilization is supported by the absence of bacterial or viral allograft-associated infections in tissue processed by this method. 6 LifeNet offers a new technology, Allowash XG, which results in sterile tissue with no residual processing agents left to complicate clinical use. Allowash XG processed tissue does not produce 6 Ultrasonication in H 2 O 2 Dry Spin Dry Spin Ultrasonication in IPA Soak in Water 5 Ultrasonication in Antibiotic Solution Dry Spin Step in Protocol 1 Soak in Water 4 Ultrasonication in H 2 O 2 Figure 1. Cut graft protocol design for Allowash Technology. 11 Dry Spin

5 Allowash XG is LifeNet s proprietary comprehensive and validated process, which begins by controlling incoming bioburden, reduces bioburden through a controlled (patent protected) cleaning and disinfecting process, and ends with terminally sterilized allograft tissue. Allowash XG-associated terminal sterilization ensures that all allograft tissue is free of bacteria and other viable and detectable organisms, including mycobacteria, viruses, fungi and spores. It is important to note that Allowash XG offers sterility without compromising the biomechanical or biochemical properties of allografts needed for surgical applications. The following steps ensure sterilization: Step 1: Bioburden Control This meticulous and rigorous screening routine is employed for all donors and tissue recovery samples. Screening mirrors the requirements set forth by the FDA and AATB. This first step allows for stringent control of bioburden on incoming donor tissues before entering LifeNet s processing facilities. Step 2: Bioburden Assessment At the time of recovery, all tissue is sampled to assess for microbiological contamination. Standard microbiological methods, employing both aerobic and anaerobic culture media, are employed to culture and identify bacteria and fungi. Donor blood samples are also used for required infectious disease testing and evaluated for acceptability regarding the potential for hemodilution. This extensive serological testing exceeds industry standards with the latest NAT advanced testing techniques, allowing LifeNet to control and virtually eliminate incoming bioburden on tissues. state and federal requirements, including cgxp for medical devices. LifeNet facilities maintain cleanliness levels that minimize or eliminate environmentally mediated graft contamination. Step 4: Rigorous Cleaning, Blood and Marrow Removal Through flushing, centrifugation, hypotonic processes and ultrasonication, blood elements (i.e., marrow and lipids) are solubilized and removed. Key solutions are forced into and through the bone matrix and then directed to waste, resulting in the lysis of cells and solubilization and removal of greater than 99% of the cellular and humoral elements of bone. A key advantage of this step in the Allowash process is the potential for an approximate 3-log removal of disease elements associated with emerging (unknown) infectious diseases. The Allowash process accomplishes significant bioburden reduction through simple cleaning of tissues and thus anticipates the need for reducing problems associated with emerging infectious diseases. Step 5: Disinfection and Rinsing Regimen Freed from over 99% of marrow and lipids, the tissue is then subjected to an intensive decontamination, disinfection and scrubbing regimen designed to eliminate viruses and bacteria. Failure to remove such tissue elements prior to chemical or physical disinfection results in preferential reaction of these processes with the tissue elements rather than the contaminant microorganisms. However, Allowash processed tissues are freed of these tissue elements; thus, any residual contaminating micro-organisms are immediately accessible to the disinfecting reagents. As final steps in the process, tissues undergo water soak mediation (rinsing) to remove processing reagents, followed by centrifugation and/or microabsorption to remove residual water. Step 3: Minimizing Contamination This step utilizes state-of-the-art processing and serves to further reduce any already-present low bioburden on tissues, as they are prepared for cleaning and disinfection. Due to the need for facilities designed for the processing and preservation of both musculoskeletal and cardiovascular tissue allografts, all tissue handling and processing areas have been designed to allow for compliance with FDA, 4

6 Step 6: Terminal Sterilization The Allowash XG process concludes with a controlled, low-level dose of gamma irradiation, which is administered at low temperatures after packaging. Due to extremely low bioburden levels on tissues post-allowash processing, gamma irradiation doses as low as 8.3 kgy (absorbed dose) result in sterile tissue grafts. This final step produces sterilization levels of 1-6 SAL without compromising the biomechanical or biochemical properties needed for surgical implementation. See Table 1 for summary. Steps to Sterilization Description Summary 1. Bioburden Meticulous and rigorous Control screening routine; based on FDA and AATB guidelines; strict donor exclusions. 2. Bioburden Extensive serologic testing for Assessment microbiological contamination, including bacteria, fungi and infectious diseases. 3. Minimizing State-of-the-art processing to Contamination maintain or further reduce an already low bioburden. 4. Rigorous Cleaning, Flushing, centrifugation, Blood and Marrow hypotonic processes and Removal ultrasonication to solubilize and remove blood elements, (i.e., marrow and lipids). 5. Disinfection and Intensive decontamination, Rinsing Regimen disinfection and scrubbing regimen to remove and eliminate viruses and bacteria, and centrifugation or microabsorption to remove residual water. 6. Terminal Sterilization Low-level dose of gamma irradiation at low temperatures. Table 1. Allowash XG sterilization steps. Validation of Allowash XG and Gamma Irradiation The AAMI/ISO 1137 Method 2B terminal sterilization validation was undertaken in a study by LifeNet R&D to validate sterilization of musculoskeletal grafts, both soft tissue and bone grafts, using gamma irradiation. 18 Method 2B utilizes the incremental dosing approach, allowing the calculation of a SAL of 1x1-2 to constitute the Verification Dose, which is used to validate the sterilization process on a regular (approximately quarterly) basis. According to this method, the microbiological results from the Verification Dose experiments are then used to calculate the sterilization dose that provides a SAL of 1x1-6. Therefore, the probability of a viable microorganism being present on an allograft post-gamma irradiation is one in a million at the calculated sterilization dose. Samples of bone and soft tissue allografts were from donors for which research consent had been granted. The allograft tissue was aseptically processed utilizing LifeNet s patent-protected Allowash Technology in environmentally controlled suites. Each incremental batch tested had 2 allografts exposed to each dose of irradiation (1-5 kgy). Thus, each batch contained 1 allografts that resulted in 2 samples for sterility testing, since each graft was bisected for aerobic and anaerobic microbiological testing. A total of three batches of tissues were tested and resulted in 1% culture negative test results. Because all tissue samples tested were culture negative, the verification dose was, by standard protocol, set to be 1 kgy. By Method 2B, the minimum sterilization dose (absorbed dose) needed to achieve an SAL of 1-6 was calculated to be 8.3 kgy. Batch 1 kgy 2 kgy 3 kgy 4 kgy 5 kgy 1 1. kgy 1.9 kgy 2.8 kgy 3.7 kgy 4.8 kgy 2 1. kgy 1.9 kgy 3.1 kgy 4.1 kgy 5.1 kgy 3 1. kgy 2. kgy 2.8 kgy 3.7 kgy 4.8 kgy Verification.9 kgy N/A N/A N/A N/A Dose Table 2. Average absorbed dose of gamma irradiation for each incremental dose experiment and verification dose experiment. 5

7 Soft Soft Cortico- Tissue Tissue Cortical cancellous Cancellous with without Bone Bone Bone Bone Bone B. SUBTILIS Pass Pass Pass Pass Pass ATCC 6633 C. ALBICANS Pass Pass Pass Pass Pass ATCC 1231 A. NIGER Pass Pass Pass Pass Pass ATCC 1644 S. AUREUS Pass Pass Pass Pass Pass ATCC 6538 C. SPOROGENES Pass Pass Pass Pass Pass ATCC P. AERUGINOSA Pass Pass Pass Pass Pass ATCC 927 Table 3. Bacteriostasis/fungistasis (B/F) results for six families of Allowash -treated allografts. Batch 1 kgy 2 kgy 3 kgy 4 kgy 5 kgy 1 /2 /2 /2 /2 /2 grafts grafts grafts grafts grafts 2 /2 /2 /2 /2 /2 grafts grafts grafts grafts grafts 3 /2 /2 /2 /2 /2 grafts grafts grafts grafts grafts Verification /1 N/A N/A N/A N/A Dose grafts Table 4. Positive (non-sterile) grafts per total grafts assessed at each incremental irradiation dose and verification dose. The investigators concluded that Method 2B terminal sterilization validation can readily be transferred from the medical device industry to tissue banking. Valid, reliable results are produced when appropriate considerations are taken into account. Low Dose Gamma Irradiation No Impact on Biomechanical Tissue Properties Gamma irradiation has been shown to provide complete tissue sterilization when used in sufficiently high doses. 19 However, using high dose irradiation (>4kGy) has a negative impact on the biomechanical and biologic properties of the tissue and cannot be recommended for processing of allogenic tissue. Block and colleagues set about to determine a recommended low irradiation dose that achieves sterilization without compromising biomechanical properties. 2 These investigators found that an irradiation dose in the range of 1 to 15 kgy provides effective bactericidal coverage with minimal impact on allograft integrity and function. The temperature at which irradiation is used in allograft processing appears to be crucial. Most studies show that administering irradiation at room temperature in freeze-dried or hydrated samples is particularly problematic, negatively affecting biomechanical tissue properties. Several studies have further demonstrated the safety of low-dose gamma irradiation on allograft tissue. One study performed on ovine by Wheeler and colleagues was designed to evaluate the effects of irradiation dose on the structural properties of the tendon-bone junction (insertion site). 21 The study investigators found that there were no statistical differences between the 15 kgy treatment and the nonirradiated controls for any biochemical properties tested. However, the 25 kgy irradiated specimens were statistically different from the non-irradiated controls in stiffness, ultimate load and ultimate stress. Specifically, the 25 kgy irradiated samples had reductions of 24% in stiffness, 27% in ultimate strength, 29% in ultimate load compared to the contralateral non-irradiated control. High-dose irradiation significantly reduces structural and material properties of patellar tendon, whereas low-dose irradiation has no significant effect on tendon properties. 6

8 LifeNet Studies For the LifeNet Soft Tissue Study, data was collected from 15 Allowash processed tibialis tendons, which served as experimental controls, and 15 experimental tibialis tendons, which received 18 kgy of irradiation at dry-ice temperatures. The actual delivered dose for the tendons was kgy. Two methods were used to assess the effects of irradiation on the tendons: biomechanical testing to assess changes in the tensile strength or Young s modulus, a ratio of stress to strain; and enzyme susceptibility testing. The two methods provide a macroscopic functional assessment of the tendons and a microscopic molecular assessment of the tendons, respectively. The following three figures illustrate the results of the study: 22 Stress (MPa) Figure 2. Tensile strength of gamma irradiated tendons compared to non-gamma irradiated tendons. 6 Control 18 kgy % Hydroxyproline in the Pellet % Hydroxyproline in the Supernatant Control 18 kgy 5 kgy ambient Figure 4. Chymotrypsin sensitivity of Allowash -processed tendons compared to Allowash-processed and gamma irradiated tendons. These data demonstrate that using 18 kgy of gamma irradiation at dry-ice temperatures does not cause a statistically significant decrease in the tensile strength, elastic modulus or enzyme susceptibility of Allowash XG processed allograft tendon tissue. Hard Tissue in Iliac Crest Wedges and Cloward Dowels The purpose of the LifeNet Hard Tissue Study was to determine the effects of gamma irradiation on the compressive strength of traditional weight-bearing cut grafts (iliac crest wedges and Cloward dowels) 23. It was hypothesized that the gamma irradiation treatment would not result in any statistically significant alteration in the compressive strength of traditional cut grafts as assessed by biomechanical testing. Young's Modulu s Control 18 kgy The traditional cut grafts tested were 9 mm iliac crest wedges and 14 mm Cloward dowels. The experimental group was Allowash treated, freeze-dried, and then gamma irradiated on dry ice with an absorbed dose of kgy. The control group was Allowash treated, freeze-dried and received no gamma irradiation treatment. Prior to testing, the samples were measured and the cross-sectional area was calculated. The samples were then loaded to failure and the compressive strength was calculated. 7 Figure 3. Young s Modulus for gamma irradiated tendons compared to non-gamma irradiated tendons. The average compressive strength for the non-irradiated, Allowash-treated iliac crest wedges was 21.6 ± 6.9 MPa. The average compressive strength for the irradiated, Allowash-

9 treated iliac crest wedges was 22.2 ± 5.2 MPa. A T-test performed comparing the results showed that the difference between the two groups was not significant (p=.74). Figure 5 shows the compressive strength for all samples tested. Each bar depicts each sample s compressive strength and is grouped with all samples from that same donor. Compressive Strength (MPa) IIiac Crest Wedges Cloward Dowels Non-Irradiated Irradiated Figure 5. Compressive strength. These LifeNet studies show that when performed with controlled conditions on temperature, doses and setting, low-dose irradiation does not affect the biomechanical properties of tissues needed for their intended clinical applications. The DePuy Spine Study Three biomechanical tests were performed on three VERTIGRAFT allograft types at the DePuy Spine (Raynham, MA) testing lab. 24 VG2 Cervical Allograft, VG2 PLIF Allograft and VG1 ALIF Allograft, processed by LifeNet, were tested in axial compression, compressive shear and static torsion. Standard Allowash -treated controls were compared to those treated with Allowash XG. As shown in Figures 6-7, for all types (Cervical, PLIF and ALIF) tested in axial compression, no statistical differences were found between grafts that had been treated with Allowash XG versus those that had been treated with the Allowash XG without gamma irradiation (Control) (P<.5). Compressive Strength (N) Control Figure 6. Compressive strength. Allowash XG As shown in Figures 6-7, for all types (Cervical, PLIF and ALIF) tested in 45 compressive shear, no statistical differences were found between grafts that had been treated with Allowash XG versus those that had been treated with the Allowash XG without gamma irradiation (Control) (P<.5). Compressive Shear (N) VG1 ALIF VG2 Cervical VG2 PLIF VG1 ALIF Control Figure 7. Compressive shear. VG2 Cervical VG2 PLIF Allowash XG The average torsional strength of the VG2 Cervical graft was 2.5 Newton-meters. (Figure 8) The lower torsional strength of the Allowash XG treated grafts compared to the control was statistically significant. However, Panjabi et al. reported that only 1.5 Newton-meters of torque were required to produce a full range of motion in the cervical spine without damaging soft tissue structures. 25 Therefore, the torsional strength of the VG2 Cervical graft is still 1.6 times the torque required to produce a full range of motion in the cervical spine. 8

10 Torsion (N-m) Figure 8. Torsion. VG2 Cervical Control VG2 PLIF Allowash XG These grafts received a higher dose of irradiation than will be administered during normal Allowash XG processing. This safety factor also plays a part in the evaluation of these grafts and determination of their safety for use in their intended spinal application. CONCLUSION By using a validated tissue cleaning process like Allowash XG, bioburden on allografts can be reduced to extremely low levels. A key advantage of Allowash XG steps 4 and 5 is the potential for an approximate 3-log removal of disease causing elements associated with emerging (unknown) infectious diseases. Methods claiming disinfection through chemical means would need to be validated for such disinfection and such validation studies would, of necessity, occur after disease transmission had occurred. The Allowash process accomplishes significant bioburden reduction through simple cleaning of tissues and thus anticipates the need for reducing problems associated with emerging infectious diseases. Using an ISO standard methodology, a very low dose of gamma irradiation can be used to produce sterile allografts. The literature, animal testing and clinical data all indicate that allografts processed by Allowash XG exhibit no measurable detrimental effects to the properties of the tissues. While other tissue banks may claim sterility at 1-3 SAL, LifeNet and its Allowash XG deliver sterile tissue to the 1-6 SAL. Allograft users now have more options than ever in the choice of their tissue supplier. It is more than critical today that clinicians and hospital administrators rely on sterile tissue provided by well known, accredited tissue banks such as LifeNet. To date, more than 5, Allowash-processed grafts have been safely distributed and used without report of bacterial or viral allograft-associated infection directly linked to a graft screened and processed by LifeNet. With the introduction of Allowash XG, LifeNet takes tissue safety to the next level. 9

11 REFERENCES 1. Patel R and Trampuz A. Infections transmitted through musculoskeletal-tissue allografts. NEJM 35(25): Joyce MJ, Greenwald AS, Rigney R, et al. Report on musculoskeletal allograft tissue safety. Presented at the American Academy of Orthopaedic Surgeons 71st Annual Meeting, San Francisco, March 1-14, Hepatitis C virus transmission from an antibody-negative organ and tissue donor -United States, MMWR Morb Mortal Wkly Rep 24;53:273-4, Eastlund T, Strong DM. Infectious disease transmission through tissue transplantation. In: Phillips GO, Ed. Advances in Tissue Banking. Vol 7, Singapore: World Scientific, 24: Zou S, Dodd RY, Stramer SL, et al. Probability of viremia with HBV, HCV, HIV, and HLV among tissue donors in the United States. NEJM 24;251: Kainer MA, Linden JV, Whaley DN, et al. Clostridium infections associated with musculoskeletal-tissue allografts. NEJM 24;35: Goodman JL. The safety and availability of blood and tissues: progress and challenges. NEJM 351(8): Septic arthritis following anterior cruciate ligament reconstruction using tendon allografts -Florida and Louisiana, 2. MMWR, Morb Mortal Wkly Rep 21;5: Gibbons MJ, Butler DL, Grood ES, et al. Effects of gamma irradiation on the initial mechanical and material properties of goat bone-patellar tendon-bone grafts. J Orthop Res 1991;9: Rasmussen TJ, Feder SM, Butler DL, et al. The effects of 4 Mrad gamma irradiation on the initial mechanical properties of bone-patellar tendon-bone grafts. Arthroscopy 1994;1: Fideler BM, Vangsness CT Jr. Lu B, et al. Gamma irradiation: effects on biomechanical properties of human bone-patellar tendon-bone allografts. Am J Sports Med 1995;23: Loty B, Courpried JP, Tomeno B, et al. Bone allografts sterilized by irradiation: biological properties, procurement and results of 15 massive allografts. Int Orthop 199;14: Jackson DW, Windler GE, Simon TM. Intraarticular reaction associated with the use of freeze-dried, ethylene oxide-sterilized bone-patella tendon-bone allografts in the reconstruction of the anterior cruciate ligament. Am J Sports 199;18: Roberst TS, Drez D Jr., McCarthy W, et al. Anterior cruciate ligament reconstruction using freeze-dried, ethylene oxide-sterilized, bone-patellar tendon-bone allografts:two-year results in thirty-six patients. Am J Sports Med 1991;19: Bianchi JR, Ross K, James E, et al. The effect of preservation/sterilization processes on the shear strength of cortical bone. In: Vol.42 of Proceedings of the 1999 Bioengineering Conference, Big Sky, Montana, June 16-2, 1999:47. Abstract. 16. Summit MC, Bianchi JR, Keesling JE, et al. Biomechanical testing of bone treated through a new tissue cleaning process. In: Proceedings of the 25th Annual Meeting of the American Association of Tissue Banks, Washington, D.C., August 25-29, 21:55. Abstract. 17. Summit MC, Bianchi JR, Keesling JE, et al. Biomechanical testing of bone treated through a new tissue cleaning process. In: Proceedings of the 25th Annual Meeting of the American Association of Tissue Banks, Washington, D.C, August 25-29, 21:54. Abstract. 18. Moore M, Jones AL, Gaskins B. Adaptation of ANSI/AAMI/ISO 1137 Method 2B sterilization validation for medical devices to tissue banking. In Press, Bright RW, Smarsh JD, Gambill VM. Osteochondral Allografts: Biology, banking and clinical applications. Vol 1. 1st ed. Boston/Toronto:Little, Brown and Company; Block JE. The impact of irradiation on microbiological safety, biomechanical properties and clinical performance of musculoskeletal allografts. In press, Wheeler DL, Turner S, Kushner J. Effects of irradiation dose on the initial structural properties of ovine bone, patellar tendon, bone allografts. In press, Data on File, LifeNet, Data on File, LifeNet, Data on File, LifeNet, Panjabi MM, Oda T, Crisco JJ 3rd, Dvorak J, Grob D. Posture affects motion coupling patterns of the upper cervical spine. J Orthop Res 1993;11:4,

12 LifeNet 589 Ward Court Virginia Beach, VA TEL: / FAX: / VertiGraft is a registered trademark and VG1 and VG2 are trademarks of DePuy, Inc. LifeNet, Allowash and MatriGraft are registered trademarks and Allowash XG is a trademark of LifeNet Inc., Virginia Beach, VA. 24 LifeNet. All rights reserved. LNPRO1264