Sterilization of HIV with irradiation: relevance to infected bone allografts.

BACKGROUND: Bone allograft banks commonly sterilize frozen bone by irradiation. The dose-response relationship for HIV is calculated and the dose required to inactivate the bioburden of virus that may be present in allograft bone is determined. METHODS: A virus titre experiment is performed using irradiated frozen HIV. The virus is maintained on dry ice (approximately -70 degrees C) and is exposed to a cobalt 60 source with 0-40 kGy irradiation at 5 kGy intervals. Lymphocyte cell cultures are exposed to serial dilutions of the irradiated virus. The virus titre is quantified by cytological changes of HIV infection and p24 immunofluorescence. RESULTS: There is a linear relationship between the virus titre and the radiation dose delivered. The inactivation rate of irradiated virus was 0.1134 log10 tissue culture infective doses 50/mL per kGy (95% confidence intervals, 0.1248-0.1020). The irradiation dose required to inactivate the HIV bioburden in allograft bone is 35 kGy. The irradiation dose required to achieve a sterility assurance level of 10(-6) is 89 kGy. This dose exceeds current recommendations for sterilizing medical products and the current practice of many bone banks. CONCLUSIONS: It is concluded that gamma irradiation should be disregarded as a significant virus inactivation method for bone allografts.     

STERILIZATION OF HIV WITH IRRADIATION: RELEVANCE TO INFECTED BONE ALLOGRAFTS

Background : Bone allograft banks commonly sterilize frozen bone by irradiation. The dose–response relationship for HIV is calculated and the dose required to inactivate the bioburden of virus that may be present in allograft bone is determined. Methods : A virus titre experiment is performed using irradiated frozen HIV. The virus is maintained on dry ice (approximately –70°C) and is exposed to a cobalt 60 source with 0–40 kGy irradiation at 5 kGy intervals. Lymphocyte cell cultures are exposed to serial dilutions of the irradiated virus. The virus titre is quantified by cytological changes of HIV infection and p24 immunofluorescence. Results : There is a linear relationship between the virus titre and the radiation dose delivered. The inactivation rate of irradiated virus was 0.1134 log₁₀ tissue culture infective doses ₅₀/mL per kGy (95% confidence intervals, 0.1248–0.1020). The irradiation dose required to inactivate the HIV bioburden in allograft bone is 35 kGy. The irradiation dose required to achieve a sterility assurance level of 10^–6 is 89 kGy. This dose exceeds current recommendations for sterilizing medical products and the current practice of many bone banks. Conclusions : It is concluded that gamma irradiation should be disregarded as a significant virus inactivation method for bone allografts.     

Allograft tissue irradiation and failure rate after anterior cruciate ligament reconstruction:A systematic review

AIM:To evaluate whether anterior cruciate ligament(ACL) allograft irradiation is effective for sterility without compromising graft integrity and increasing failure rate.METHODS:A literature search was conducted using Pub Med,Cochrane,and Google.The following search terms were used:"Gamma irradiation AND anterior cruciate ligament AND allograft" with a return of 30 items.Filters used included:English language,years 1990-2015.There were 6 hits that were not reviewed,as there were only abstracts available.Another 5 hits were discarded,as they did not pertain to the topic of interest.There were 9 more articles that were excluded:Three studies were performed on animals and 6 studies were meta-analyses.Therefore,a total of 10 articles were applicable to review.RESULTS:There is a delicate dosing crossover where gamma irradiation is both effective for sterility without catastrophically compromising the structural integrity of the graft.Of note,low dose irradiation is considered less than 2.0 Mrad,moderate dose is between 2.1-2.4 Mrad,and high dose is greater than or equal to 2.5 Mrad.Based upon the results of the literature search,the optimal threshold for sterilization was found to be sterilization at less than 2.2 Mrad of gamma irradiation with the important caveat of being performed at low temperatures.The graft selection process also must include thorough donor screening and testing as well as harvesting the tissue in a sterile fashion.Utilization of higher dose(≥ 2.5 Mrad) of irradiation causes greater allograft tissue laxity that results in greater graft failure rate clinically in patients after ACL reconstruction.CONCLUSION:Allograft ACL graft gamma irradiatedwith less than 2.2 Mrad appears to be a reasonable alternative to autograft for patients above 25 years of age.     

Effect of sterilization on bone morphogenetic protein

Demineralized bone matrix and bone morphogenetic protein have been used clinically to accelerate bone regeneration. However, the best method of sterilization has been the subject of controversy. Some investigators have used ethylene oxide, but others have reported that doses adequate for sterilization destroyed the osteoinductivity of demineralized bone matrix and that gamma irradiation was less harmful in this respect. We used partially purified bone morphogenetic protein and type-I collagen to investigate the effects of sterilization by ethylene oxide and gamma irradiation on the activity of bone morphogenetic protein. Osteoinductivity was reduced considerably after sterilization by gamma irradiation at 2.5 Mrad and by ethylene oxide at 37°C for 4 hours and at 55°C for 1 hour; however, the reduction induced by ethylene oxide at 29°C for 5 hours was about half of the control values. This study showed that ethylene oxide at 29°C for 5 hours can be used clinically for sterilization of bone morphogenetic protein. We also investigated the effect of gamma irradiation on bone morphogenetic protein and the collagen carrier separately and found that collagen was far more labile than bone morphogenetic protein.     

Irradiation has no effect on the incorporation of impacted morselized bone: a bone chamber study in goats

Background Gamma irradiation has been widely used for sterilization of bone allografts. However, gamma irradiation alters proteins. This is favorable when it reduces immunogenicity, but is undesirable when osteoinductive proteins are damaged. Although the effect of gamma irradiation on BMPs has been studied, the effect of irradiation on the process of incorporation of morselized bone chips remains unclear. We studied the effects of sterilization by gamma irradiation on the incorporation of impacted morselized allografts.

Methods Bone chambers with impacted allografts, rinsed impacted allografts, allografts that were rinsed and subsequently irradiated, and an empty control were implanted in proximal medial tibiae of goats. Incorporation was evaluated using histology and histomorphometry.

Results Histology revealed evidence of bone graft incorporation, which proceeded in a similar way in unprocessed, rinsed, and both rinsed and irradiated bone grafts. After 12 weeks, no difference in bone and tissue ingrowth was found between the unprocessed, the rinsed, and the rinsed and subsequently irradiated allografts. The amount of unresorbed graft remnant was highest in the unprocessed bone grafts.

Interpretation We conclude that sterilization with gamma irradiation does not influence the incorporation of impacted rinsed bone allografts.     

Demineralization for inactivation of infectious retrovirus in systemically infected cortical bone: in vitro and in vivo experimental studies

BACKGROUND: Clinical and experimental studies have demonstrated viral transmission through the transplantation of fresh-frozen infected bone. While sterilization methods sufficient to inactivate the human immunodeficiency virus (HIV) have been shown to markedly alter osteoconductive and osteoinductive properties of bone allografts, the ability of a process for creating demineralized bone matrix to abrogate transmission of a retrovirus has not been investigated, to our knowledge. We hypothesized that a clinically accepted demineralization procedure would alter the nucleic acids of the feline leukemia virus (FeLV, a retrovirus with a structure and replication cycle similar to those of HIV), inactivating the virus in infected bone and rendering it noninfectious. METHODS: Bone infected with FeLV was demineralized with a method employed for creating demineralized bone matrix powder. The effects of demineralization on cellular and (pro)viral nucleic acids were examined with use of gel electrophoresis and quantitative polymerase chain reaction, respectively. To compare the infectivity of the demineralized bone matrix with that of mineralized bone particles in cell cultures and in animals in which they had been implanted, we measured FeLV p27 antigen and (pro)viral nucleic acids as well as antiviral antibodies. RESULTS: Demineralization of FeLV-infected bone appeared to inactivate the virus by degradation and fragmentation of the DNA, rendering it noninfectious in both in vitro and in vivo test systems. In contrast, untreated mineralized FeLV-infected bone contained intact nucleic acids and readily transmitted the virus in both test systems. CONCLUSIONS: The demineralization process inactivated infectious retrovirus in infected cortical bone, thereby preventing disease transmission.     

Experimental study of the thermic effect on bone at 60 degrees C, as applied to bone allograft]

Bone reconstruction methods increasingly often require using bank bone. These massive bone fragments can be taken only from dead subjects or those in irreversible coma. Surgical sterility of the samples does not always guarantee the absence of an HIV infection, for which seroconversion often occurs very late. B. Spire's work has shown the effectiveness of a 30-minute heat treatment at 56 degrees C to inactivate HIV in blood products. Our study has therefore evaluated the effectiveness of a heat treatment method for bone to inactivate HIV on one hand, and the mechanical and histologic consequences of this treatment on rabbit bone. All bone fragments in this study were frozen at -80 degrees C to reproduce the same conditions of use as in current bone banks. Heating deep-frozen fragments of spongious tissue and of bone marrow from seropositive subjects in a 60 degrees C humid heat allowed confirming thermal sterilization of HIV, but a greater number of case is required to support this technique, as well as a verification with cortical bone. Thermal sterilization of bone allografts does not alter the mechanical properties nor the possibilities of bone regrowth in allografts. Application to human bone allografts should be confirmed by a greater number of cases, but it appears as a simple means to suppress the current disadvantage of late seroconversion control.     

High-dose gamma irradiation for soft tissue allografts: High margin of safety with biomechanical integrity.

Screening and processing methods currently in place have made the risk of bacterial and viral infections from allograft tissues extremely low. However, the development of a terminal sterilization method that does not adversely affect tissue function would provide an added safety to tissues for transplantation. We assessed whether high-dose gamma irradiation could be used as an effective terminal sterilization method for allografts without impairing the preimplantation mechanical integrity of the tissues. Semitendinosus tendons were pretreated with a radioprotectant solution and then irradiated to 50 kGy under well-defined conditions that included a tight dose range and maintained low temperatures. Maximum force, strain, stress, modulus, and strain energy density for tendons irradiated to 50 kGy were compared to nonirradiated control tendons and tendons irradiated to 18 kGy by a commercial tissue bank using their existing method. The preimplantation biomechanical properties of the 50-kGy group compared favorably to the nonirradiated and 18 kGy groups. A study to evaluate the postimplantation mechanical and biological performance of grafts irradiated to 50 kGy is ongoing. Pathogen inactivation was also quantified following 50 kGy of irradiation, with > or =4.5 logs of Sindbis virus and 4.9 logs of parvovirus kill achieved. Analysis of Clostridium sordellii inactivation kinetics indicated that a 16 log10 reduction is predicted with 50 kGy of irradiation. A high dose of gamma irradiation using the described conditions can reduce infectious risks associated with soft tissue allografts while maintaining the preimplantation biomechanical performance of the tissues.     

Polyethylene cross-linking by two different methods reduces acetabular liner wear in a hip joint wear simulator.

Advances in cross-linking have led to the development of wear resistant ultrahigh molecular weight polyethylene for total joint replacement. This study compared wear reduction by two different cross-linking methods as measured in a hip wear simulator. One highly cross-linked polyethylene was treated with 7.5 Mrad gamma irradiation with post-irradiation annealing and a sterilization dose of 2.5 Mrad (10 Gamma), while the other used 9.5 Mrad warm irradiation with 10 MeV electron-beam (9.5 EB). Liners of the same design, made from nominally cross-linked (gamma sterilized) polyethylene were also tested. Gravimetric wear analysis was performed every 500,000 cycles for 5,000,000 cycles. After correcting for weight gain due to water absorption, the nominally cross-linked liners demonstrated mean wear rates of 15.7 (+/-1.7) and 12.5 (+/-1.0) mg/million cycles. Both highly cross-linked polyethylene liners demonstrated significantly less wear than their respective controls (with mean wear rates of 1.5 (+/-1.2) and -1.4 (+/-1.5) mg/million cycles). The 9.5 EB liners gained weight presumably due to increased fluid absorption, in addition to that measured in loaded-soaked control implants. Any wear occurring was therefore assumed to have been more than offset by weight gain. Highly cross-linked polyethylene was significantly more wear resistant than non- or nominally cross-linked polyethylene. The differences in wear rates between the two highly cross-linked polyethylene designs (9.5 EB or 10 Gamma) are probably too small to be clinically significant.     

The effects of various cleaning and sterilization processes on allograft bone incorporation

Although bone allografts have a long history of safe and effective clinical use, there is great variability in tissue processing and sterilization processes. Chemical and gamma radiation sterilization are commonly performed on allografts, and numerous animal and clinical studies have examined their integration into host bone. The objective of this literature review was to assess the effects of various cleaning and sterilization processes on the incorporation of allograft bone.     

Fracture resistance of gamma radiation sterilized cortical bone allografts.

Gamma radiation is widely used for sterilization of human cortical bone allografts. Previous studies have reported that cortical bone becomes brittle due to gamma radiation sterilization. This embrittlement raises concern about the performance of a radiation sterilized allograft in the presence of a stress concentration that might be surgically introduced or biologically induced. The purpose of this study was to investigate the effect of gamma radiation sterilization on the fracture resistance of human femoral cortical bone in the presence of a stress concentration. Fracture toughness tests of specimens sterilized at a dose of 27.5 kGy and control specimens were conducted transverse and longitudinal to the osteonal orientation of the bone tissue. The formation of damage was monitored with acoustic emission (AE) during testing and was histologically observed following testing. There was a significant decrease in fracture toughness due to irradiation in both crack growth directions. The work-to-fracture was also significantly reduced. It was observed that the ability of bone tissue to undergo damage in the form of microcracks and diffuse damage was significantly impaired due to radiation sterilization as evidenced by decreased AE activity and histological observations. The results of this study suggest that, for cortical bone irradiated at 27.5 kGy, it is easier to initiate and propagate a macrocrack from a stress concentration due to the inhibition of damage formation at and near the crack tip.     

Effect of gamma irradiation on the osteoinductivity of morphogenetic protein extract from reindeer bone

BACKGROUND: Bone morphogenetic proteins (BMPs), which are capable of stimulating the production of new bone, must be sterilized before preclinical and clinical use to reduce the risk of infections and associated complications. In this study, we investigated the effects of gamma sterilization on the osteoinductivity of native reindeer BMP extract in the Balb/C mouse thigh muscle pouch model. METHODS: 5 mg of native reindeer BMP extract and 5 mg of bovine serum albumin were administered separately either in gelatine capsules or mixed with gelatine as injections. The dose of gamma irradiation was 4.1 Mrad. Unsterile capsules and injections served as controls. New bone formation was evaluated based on the incorporation of Ca45 and also radiographically 3 weeks after implantation. RESULTS: Albumin-containing implants and injections did not induce new bone formation, as monitored in radiographs. Gamma sterilization did not reduce the osteoinductivity of native BMP extract in capsules, but a significant decrease in osteoinductivity--measured as area (50%) and Ca45 incorporation of new bone (27%)--was seen after injection. Gamma sterilization had no effect on the optical density of new bone induced by native BMP extract administered in capsules or by injection. INTERPRETATION: We conclude that, as gamma irradiation did not reduce the osteoinductivity of reindeer BMP extract in gelatine capsules, this method appears to be suitable for sterilization of BMPs to be given in capsule form. Native reindeer BMP extract was more sensitive to irradiation in soluble collagen (gelatine) than BMP in gelatine capsules. This finding must be given serious consideration regarding treatment of patients, but the remaining activity may be sufficient for the induction of bone formation in preclinical and clinical situations.     

Sterilization of HIV by gamma irradiation

Summary A human immuno-deficiency virus (HIV) infected bone allograft model has been created using HTLV-IIIB virus in a concentration simulating a massively HIV infected bone allograft donor [HTLV-III is the denomination initially given to the human immuno-deficiency by the american team of Prof. Gallo. It represent the virus HIV 1 of the present international nomenclature]. 5×10⁴ tissue culture infective doses per ml. of virus were placed within the medullary cavity of bovine femora and tibiae with a radiation dosimeter, and the ends sealed with lead. The bone/virus model was maintained at –70° C while being irradiated with 1 to 4 megarads of gamma irradiation in increments of 0.5 megarads. The study showed that the HTLV-IIIB virus is a relatively radio-resistant organism, a property common to most viruses. The results suggest that HTLV-IIIB can be inactivated in bone infected with a clinically significant viral load, as may be found in donors who are initially negative when screened for HIV. It is recommended that bone allografts which are secondarily sterilized by gamma irradiation receive at least 2.5 megarads. The amount of radiation absorbed by the bone cortex was minimal.

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Résumé Un modèle d'allogreffe osseuse infectée par le virus de l'immuno-déficience humaine (VIH) a été créé en utilisant le virus HTLV-III B [HTLV-III est l'appellation donnée initialement au virus de l'immuno-déficience humaine par l'équipe américaine du Pr Gallo. Il équivaut donc au virus HIV 1 de l'actuelle nomenclature internationale.] à une concentration simulant le cas le plus grave: correspondant à un donneur d'allogreffe osseuse infecté de façon massive par le VIH, 5×10⁴ doses de culture de tissus infectés par ml de virus ont été placées dans le canal médullaire d'un fémur et d'un tibia bovins avec un dosimètre à radiations, les extrémités du modèle étant scellées par du plomb. Le modèle os/virus a été maintenu à une température de –70° Celsius tout en étant soumis à une irradiation gamma de 1 à 4 mégarads par paliers de 0,5 mégarads. Cette étude a montré que le virus HTLV-III B est un organisme relativement résistant aux radiations, propriété commune à la plupart des virus. Les résultats laissent à penser que le virus HTLV-III B pourrait être inactivé dans un os infecté par une charge virale cliniquement significative, comme on peut en trouver chez les donneurs qui réagissent initialement de façon négative aux tests VIH courants. Il est recommandé que les allogreffes osseuses stérilisées par radiations gamma reçoivent au moins 2,5 mégarads. La quantité de radiation absorbée par la corticale osseuse était minimale.

     

Mechanical strength of cortical allografts with gamma radiation versus ethylene oxide sterilization

We investigated the effects of gamma irradiation versus ethylene oxide (ETO) sterilization on the mechanical strength of cortical bone grafts. Tibias were collected from cadavers of mature goats. Sixty test specimens were randomized into four groups: fresh (no processing), frozen (freezing at -70 degrees C), gamma-irradiated, and ETO-sterilized specimens. Torsion, three-point bending, and compression testing were separately performed with a material testing machine. Parameters studied included maximum stress, strain, deflection, extension, load, shear modulus, and E-modulus. Compared with findings for the fresh specimens, findings were as follows for gamma-irradiated specimens: maximal shear modulus, reduced by 48%; shear stress, by 55%; deflection, by 71%; bending stress, by 51%; bending strain, by 74%; extension, by 60%; and compression strain, by 50%. However, there were no reductions in those parameters for the frozen specimens or the ETO-sterilized specimens. These findings confirm that shear, bending, and compression strength of cortical allografts are weakened by gamma irradiation at room temperature. To maintain optimum mechanical properties, ETO sterilization of allografts is better than gamma sterilization, especially for cortical bone, because it is usually used in load-bearing settings.     

Free radical scavenging alleviates the biomechanical impairment of gamma radiation sterilized bone tissue.

Terminal sterilization of bone allografts by gamma radiation is often essential prior to their clinical use to minimize the risk of infection and disease transmission. While gamma radiation has efficacy superior to other sterilization methods it also impairs the material properties of bone allografts, which may result in premature clinical failure of the allograft. The mechanisms by which gamma radiation sterilization damages bone tissue are not well known although there is evidence that the damage is induced via free radical attack on the collagen. In the light of the existing literature, it was hypothesized that gamma radiation induced biochemical damage to bone's collagen that can be reduced by scavenging for the free radicals generated during the ionizing radiation. It was also hypothesized that this lessening of the extent of biochemical degradation of collagen will be accompanied by alleviation in the extent of biomechanical impairment secondary to gamma radiation sterilization. Standardized tensile test specimens machined from human femoral cortical bone and specimens were assigned to four treatment groups: control, scavenger treated-control, irradiated and scavenger treated-irradiated. Thiourea was selected as the free radical scavenger and it was applied in aqueous form at the concentration of 1.5 M. Monotonic and cyclic mechanical tests were conducted to evaluate the mechanical performance of the treatment groups and the biochemical integrity of collagen molecules were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native mechanical properties of bone tissue did not change by thiourea treatment only. The effect of thiourea treatment on mechanical properties of irradiated specimens were such that the post-yield energy, the fracture energy and the fatigue life of thiourea treated-irradiated treatment group were 1.9-fold, 3.3-fold and 4.7-fold greater than those of the irradiated treatment group, respectively. However, the mechanical function of thiourea treated and irradiated specimens was not to the level of unirradiated controls. The damage occurred through the cleavage of the collagen backbone as revealed by SDS PAGE analysis. Irradiated specimens did not exhibit a noteworthy amount of intact alpha-chains whereas those irradiated in the presence of thiourea demonstrated intact alpha-chains. Results demonstrated that free radical damage is an important pathway of damage, caused by cleaving the collagen backbone. Blocking the activity of free radicals using the scavenger thiourea reduces the extent of damage to collagen, helping to maintain the mechanical strength of sterilized tissue. Therefore, free radical scavenger thiourea has the potential to improve the functional life-time of the allograft component following transplantation.     

Tendon allograft sterilized by peracetic acid/ethanol combined with gamma irradiation

Background

Research and clinical applications have demonstrated that the effects of tendon allografts are comparable to those of autografts when reconstructing injured tendons or ligaments, but allograft safety remains problematic. Sterilisation could eliminate or decrease the possibility of disease transmission, but current methods seldom achieve satisfactory sterilisation without affecting the mechanical properties of the tendon.

Hypothesis

Peracetic acid-ethanol in combination with low-dose gamma irradiation (PE-R) would inactivate potential deleterious microorganisms without affecting mechanical and biocompatible properties of tendon allograft.

Study design

Controlled laboratory design.

Methods

HIV, PPV, PRV and BVDV inactivation was evaluated. After verifying viral inactivation, the treated tendon allografts were characterised by optical microscopy, scanning electron microscopy and tensile testing, and the cytocompatibility was assessed with an MTT assay and by subcutaneous implantation.

Results

Effective and efficient inactivation of HIV, PPV, PRV and BVDV was observed. Histological structure and ultrastructure were unchanged in the treated tendon allograft, which also exhibited comparable biomechanical properties and good biocompatibility.

Conclusion

The preliminary results confirmed our hypothesis and demonstrated that the PE-R tendon allograft has significant potential as an alternative to ligament/tendon reconstruction.

Clinical relevance

Tendon allografts have been extensively used in ligament reconstruction and tendon repair. However, current sterilisation methods have various shortcomings, so PE-R has been proposed. This study suggests that PE-R tendon allograft has great potential as an alternative for ligament/tendon reconstruction.

What is known about this subject

Sterilisation has been a great concern for tendon allografts. However, most sterilisation methods cannot inactivate viruses and bacteria without impairing the mechanical properties of the tendon allograft.

What this study adds to existing knowledge

Peracetic acid/ethanol with gamma irradiation can effectively inactivate viruses and bacteria. Meanwhile, tendon allografts sterilised by this method maintain their physiological tendon structure, biomechanical integrity and good compatibility.     

Is it safe to reuse disposable laparoscopic trocars?rid=""

BACKGROUND: The reuse of disposable laparoscopic instruments carries a risk of transmitting infectious diseases such as hepatitis and HIV. We evaluated the safety of reusing disposable trocars by studying the chances of their harboring infectious viruses after resterilization in an in vitro setting. METHODS: Disposable laparoscopic trocars were exposed to horse blood contaminated with high or low viral concentrations of herpes simplex virus type 1 (HSV1) and attenuated polio virus type 1 at room temperature for 2 h. HSV1 was chosen as the surrogate for lipid viruses that include hepatitis B and HIV virus; polio virus represented the nonlipid viruses that cause infections in immunocompromised patients and are more resistant to sterilization. The trocars were subsequently cleaned and resterilized by low-temperature steam and formaldehyde at 80 degrees C for 3 h. Viral cultures were then repeated after sterilization. RESULTS: A cytopathic effect (CPE) was demonstrated at both concentrations for HSV1 in all trocars before but not after sterilization. For the polio virus, CPE was evident in 50% of the trocars (two of four) exposed to high viral concentration after sterilization. CONCLUSION: Disposable trocars are difficult to resterilize and may harbor infectious viruses after their initial use. Therefore, the reuse of disposable trocars in laparoscopic surgery cannot be recommended.     

Viruses adsorbed on musculoskeletal allografts are inactivated by terminal ethylene oxide disinfection.

In 1987 it was anticipated that unsterilized tissues would transmit virus diseases such as hepatitis and HIV-1 from infected donors so a freeze-drying process for musculoskeletal tissue was developed to include terminal ethylene oxide (EO) exposure for 14 h. We found no studies of EO efficacy when viruses were associated with human allografts so we studied the antiviral effect of terminal EO disinfection using all but the final freeze-drying phase of this clinical processing protocol (CPP). Specifically we looked at EO inactivation of HIV-1, a human hepatitis B surrogate and test viruses known to be highly resistant to disinfecting agents, including irradiation. Freeze-drying, ordinarily required after EO disinfection and part of the CPP, was not done. Suspensions of HIV-1, Bovine viral diarrhea, Reovirus type 3, Duck hepatitis B, Poliomyelitis and Canine parvovirus were adsorbed on glass, demineralized bone powder, and preprocessed strips of femoral cortex, iliac wedges, cancellous blocks and patellar bone-tendon-bone preparations and subjected to EO disinfection. Test viruses were inactivated at the end of 7 h of EO disinfection, providing a safety factor in the CPP of at least 100%. Because allografts can transmit viruses, terminal EO disinfection should provide safer musculoskeletal allografts than non-disinfected tissues or those irradiated with a standard irradiation dose. New spontaneously appearing viruses would probably be inactivated with this terminal EO disinfection but they and viral bioweapons will require individual validation to assure viral inactivation.     

11 kGy gamma irradiated demineralized bone matrix enhances osteoclast activity

Background

Demineralized bone matrix (DBM) allografts are widely used in orthopaedic clinics. However, the biological impact on its osteoinductivity after its sterilization process by gamma irradiation is not well studied. Furthermore, little is known about the relationship between residual calcium levels on osteoinductivity.

Hypothesis

We hypothesize that low-dose gamma irradiation retains the osteoinducitivity properties of DBM and causes ectopic bone formation.

Materials and methods

A randomised animal trial was performed to compare tissue and molecular responses of low-dose (11 kGy) gamma irradiated and non-irradiated human DBM at 6 weeks post-intramuscular implantation using an athymic rat model. In addition, we correlated residual calcium levels and bone formation in gamma irradiated DBM.

Results

A modified haematoxylin and eosin stain identified ectopic bony capsules at all implanted sites with no significant difference on the amount of new bone formed between the groups. Statistically significantly lower ratio of alkaline phosphatase expression over tartrate-resistant acid phosphatase and/or cathepsin K expressions was found between the groups.

Discussion

This study found that low-dose gamma irradiated DBM, which provides a sterility assurance level of 10^?6 for bone allografts, retained osteoinductivity but exhibited significantly enhanced osteoclastic activity. Furthermore, this is the first study to find a positive correlation between residual calcium levels and bone formation in gamma irradiated DBM.     

In vitro study of the role of various preservation methods on mechanical properties of allografts of the patellar tendon]

Tendon allografts are presently used for surgical reconstruction of the anterior cruciate ligament. The aim of this study was the in vitro evaluation of the modification of the mechanical properties of conserved human patellar tendons, deep-frozen or freeze-dried and sterilized with gamma rays. Thirty pairs of patellar tendons (central third) with their bony attachments were removed from fresh corpses, frozen in liquid nitrogen, then conserved at -80 degrees C. Out of each pair, one tendon was submitted to an additional treatment and the other was used as a control. Three types of treatment were studied: 2.5 Mrad irradiation (groupe I), freeze-drying with 1% residual humidity (groupe II), consecutive freeze-drying and irradiation (group III). After warming up (controls and group I treated tendons), the specimens were submitted to mechanical tests on the pulling machine with a suitable arrangement. Two types of tests were performed: a creep test (80 cycles at 1 Hz between 0 and 50 N) then an ultimate tensile strength test (0.25 mm/s). Freeze-drying causes macroscopic morphological alterations, which are even more marked if irradiation is applied in addition. The three types of treatment induce an increase in creep and a decrease in the ultimate, tensile strength and in Young's module, all being statistically significant. The three types of treatment alter the mechanical properties of the allograft. Freeze-drying maintains a better resistance of the graft. The combination of freeze-drying and irradiation is the most damaging method, and freeze-drying even seems to potentialize the harmful effects of irradiation resistance (group II freeze-dried grafts), the treated allografts present with changes in their viscoelastic properties that are incompatible with their clinical use. This study has also shown that some parameters influence the extent of the damaging effects of these three treatments, and experiments are being continued to optimize the conservation and sterilization procedure.