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Effects of lyophilization on the infectivity of enveloped and non-enveloped viruses in bone tissue
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Biomaterials 26 (2005) 6558–6564 www.elsevier.com/locate/biomaterials
Effects of lyophilization on the infectivity of enveloped and non-enveloped viruses in bone tissue Christine Uhlenhauta,b, Thomas Do¨rnerb, Georg Paulia, Axel Prussb, a
b
Center for Biological Safety, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany Tissue Bank, Institute for Transfusion Medicine, Charite´—University Medicine, Schumannstr. 20/21, 10117 Berlin, Germany Received 25 February 2005; accepted 15 April 2005 Available online 6 June 2005
Abstract Recently reported qualitative experiments proved that retroviral infectivity is not destroyed by lyophilization performed on systemically infected bone and tendon. The now accomplished quantitative determination of residual infectivity for enveloped and non-enveloped viruses allows a validation of the production process regarding viral safety in freeze-dried bone transplants. The lyophilization effect on the infectivity of two non-enveloped viruses (Maus Elberfeld virus, MEV; Porcine parvovirus, PPV) and one enveloped virus (Vesicular Stomatitis virus, VSV) was examined for virus-spiked bone material in comparison to lyophilized viruses, original virus stock, and air-dried viruses. All experiments were carried out with both cell-free and cell-associated virus. Significant differences were observed regarding the reduction of virus titers (TCID50). Infectivity of VSV was reduced by about 3–4 log10 using lyophilization in presence of bone matrix and of MEV by 6–7 log10, while no substantial reduction in virus titers was observed for PPV. Lyophilization of cell-free or cell-associated virus is not sufficient to inactivate viruses completely. However, lyophilization could have an additive effect in line with other production steps used in the manufacturing process. r 2005 Elsevier Ltd. All rights reserved. Keywords: Bone transplants; Lyophilization; Freeze-drying; Virus safety; Tissue bank
1. Introduction In spite of increasing efforts to develop alternative materials and procedures, allogeneic bone transplantation remains an indispensable tool for reconstructing extensive bone defects. The number of allogeneic bone grafts transplanted in the US amounts to 650,000– 900,000 [1–3] and in Germany to approx. 35,000/yr. In comparison to artificial bone substitution or material Abbreviations: EMCV, Encephalomyocarditis virus; FeLV, Feline Leukemia virus; HAV, Hepatitis A virus; HBV, Hepatitis B virus; HCV, Hepatitis C virus; HIV, Human immunodeficiency virus; MEV, Maus Elberfeld virus; moi, Multiplicity of infection; PPV, Porcine parvovirus; TCID50, Tissue culture infectious dose (50%); TSE, Transmissible spongiform encephalopathy; VSV, Vesicular Stomatitis virus Corresponding author. Fax: +49 0 30 450 525976. E-mail address: [email protected] (A. Pruss). 0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.04.049
derived from animals, allogeneic material has the major advantage of being able to act osteoconductively or in part osteoinductively due to its physiological structure. Furthermore, the potential ‘en bloc’ replacement is another advantage of allogeneic bone transplantation; even large defects of up to 10 cm in length heal mostly without intricacies [4]. Autologous bone transplants are considered as ‘gold standard’ but their use is limited due to the confined reservoir. In addition to these limitations, the necessary secondary operation on the iliac crest could lead to considerable neurological complications [5], whereas xenotransplantation could involve the risk of TSE transmission. Only a limited amount of bones from living donors is available, i.e. femoral heads after total hip replacements, while an adequate supply of bone tissue (e.g. femora, tibiae, vertebrae, os ilium) could be obtained from cadaveric sources.
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Whereas living donors can be tested for relevant infection markers (i.e. HBV surface antigen and antibodies against HBV core, HIV and HCV or the genomes of these viruses) at the time of tissue donation and a second time after a reasonable quarantine storage of the bones, the safety of cadaveric bones can only be assessed by testing the donors at the time of death or by testing recipients of solid organs from deceased donors 6 months after transplantation [6]. Therefore by testing for known pathogens, the safety of a bone tissue transplant can not be fully guaranteed. It is virtually impossible to perform reliable tests for every pathogen. In spite of rigorous donor screening, transmissions of HIV, HBV, and HCV have occurred via transplantation of (deep-frozen) bone and tendon allografts [7–13]. The transmission of HTLV-1 by a deep-frozen allogeneic bone transplant has been described [14]. Rare or zoonotically transmitted pathogens remain undetectable. Furthermore, infections within a diagnostic window can not be detected, even though a reduction of the diagnostic window can be achieved by application of PCR [15]. Hence it is crucial to apply sterilization methods that inactivate all potential pathogens, including viruses, bacteria, fungi and their spores. In addition to their reconstructive efficacy, a central issue in quality assessment must be the validation of the microbial safety of the tissues [16,17]. Various sterilization techniques of tissues have been developed to meet the requirements of obtaining pathogen-free transplants while maintaining the biological properties. For example, an effective inactivation of HIV has been demonstrated using sterilization techniques such as moist heat, gamma irradiation, peracetic acid [18–21] and ethylene oxide. However, these treatments have detrimental effects on the biological and mechanical properties of the treated tissues [22–33]. Lyophilization has been used to preserve musculoskeletal tissues over long periods. The method does not require elaborate treatment but there are conflicting data regarding changes of the physical properties’ of the transplant [34,35]. Cocultivation of lyophilized tendon and bone segments derived from systemically infected donors with susceptible cells demonstrated the presence of infectious Feline Leukemia virus (FeLV) and proved that retroviral infectivity is not destroyed by lyophilization [36]. This study by Crawford et al. used an indirect method (p27 antigen assay) and determination of proviral load by quantitative real-time PCR. The results demonstrated the presence of infectious FeLV after lyophilization. However, the cell cultures were challenged with an unknown amount of FeLV particles (derived from five FeLV-infected donor cats) and only proviral load was determined. Thus, it was shown evidentiary that infectivity was not destroyed but the degree to which lyophilization reduced virus infectivity was not determined.
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The objective of our study is the quantitative evaluation of inactivating effects of lyophilization on relevant and model viruses when used for the production of allograft bone transplants in order to enhance microbial safety of bone tissue transplantation. Although lyophilization of bone allografts does not guarantee virus safety, a considerable reductive effect on the infectivity of some viruses due to freeze-drying could be used in combination with other sterilization methods. Three relevant and model viruses were selected and appropriate test conditions developed to evaluate the effects of lyophilization on virus infectivity. Maus Elberfeld virus was used as non-enveloped model virus and Porcine parvovirus (PPV) was used as a virus which is known to be resistant to physico-chemical inactivation procedures. The Vesicular Stomatitis virus (VSV) was used to examine possible effects of the virus envelope in the context of the lyophilization procedure.
2. Materials and methods 2.1. Preparation of bone matrix Native cortical bones derived from a single donor were ground with a rotor speed mill (Pulverisette 14, Fritsch, IdarOberstein, Germany) to particles sized between 0.08 and 0.8 mm. The bone matrix was irradiated with 15 kGy (GammaService GmbH, Radeberg, Germany). This treatment was introduced to inactivate potentially present microbes such as bacteria, fungi and spores. The donor was tested negative for the following infection markers: anti-HIV-1/2, HBsAg, antiHBc, anti-HCV, and TPHA. Vials containing irradiated bone matrix were opened and handled aseptically under BSL2 conditions. 2.2. Viruses A non-enveloped virus, Encephalomyocarditis virus (EMCV) strain Maus Elberfeld virus (MEV), genus Cardiovirus, family Picornaviridae, was used as model virus for Hepatitis A virus, Rhinoviruses, and Enteroviruses such as Poliovirus. MEV virions have a diameter of approximately 30 nm and contain the positive-strand ssRNA genome. VSV, genus Vesiculovirus, family Rhabdoviridae, was used as general model for enveloped viruses, especially as model for Lyssavirus, the causing agent of Rabies. The VSV negativestrand ssRNA genome has a genome length of approximately 11 kb. The virions are approximately 65 nm in diameter and 180 nm in length. The PPV, genus parvovirus, family Parvoviridae, a non-enveloped virus, was used as model virus for the human-pathogenic parvovirus B19. PPV, like other animal parvoviruses, was chosen for its well-known resistance to physico-chemical inactivation methods [37]. PPV virions are 25 nm in diameter and have icosahedral symmetry. The genome consists of a single molecule of linear ssDNA, 5.2 kb in size. The effect on virus infectivity was tested for all viruses
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with and without cell association in comparison to control virus stocks. 2.3. Cell lines and cell culture media MEV was grown and titrated on HEp2 cells, a human cell line established via HeLa contamination (ATCC CCL-23). VSV was grown and titrated on BHK21 cells (baby hamster kidney cells; ATCC CCL-10). PPV was grown and titrated on PK13 cells (porcine kidney cell line, obtained from Cornelia Schmitt, Robert Koch-Institut). Dulbecco’s modified Eagle’s medium (D-MEM) was used, supplemented with glutamine (2 mg/ml), 10% fetal calf serum (FCS) for PK13, and 5% FCS for HEp2 and BHK21 cells. All cells were cultivated at 37 1C in a humidified atmosphere of 5% carbon dioxide (CO2). 2.4. Virus preparation and titration All virus stocks were prepared from the supernatant of infected cells and virus titer was determined as described elsewhere [38]. Briefly, virus infectivity was titrated by standard microtitration assay. Cell suspension (100 ml per well) was seeded into 96-well microtiter plates at a concentration of 104 cells per well for HEp2 and BHK21, and 2.5 103 cells for PK13, respectively. Cells were cultivated at 37 1C over night. The samples were serially diluted 1:3 in medium. Eight wells were inoculated with 100 ml aliquots of each dilution. Each 96-well plate included eight wells with non-infected control cells. The microtiter plates were incubated at 37 1C and evaluated daily for cytopathogenic effect (CPE) by light microscope. Cultures showing the characteristic CPE were considered to be positive. Infection of cells leads to their detachment and visible changes in cell morphology; these CPEs were recorded on day 2 post infection for VSV, on day 3 post infection for MEV, and on day 6 post infection for PPV. The detection limit of the virus titration is 3.00 100 TCID50. Due to the interference of bone particles with the cell culture, the detection limit for the matrix adherence is higher than for the titration of other samples (23 compared to 3 TCID50). The infectivity titers were calculated as TCID50. TCID50 is defined as that dilution of a virus required to infect 50% of a given number of inoculated cell cultures (50% endpoint). The TCID50 was calculated according to Spearman and Ka¨rber [39]: TCID50 =ml ¼ Dðn=p 0:5Þ=D0 D V (D: dilution factor; n: number of wells displaying CPE; p: number of wells tested with the same dilution in parallel; D0 : reciprocal of the first dilution; V: volume (ml)). Cellular debris was removed by centrifugation and aliquots were stored at 70 1C until use. Virus stocks with an appropriate titer ranging from 5 to 8 log10 TCID50 for the respective viruses were used. 2.5. Controls Since native bone material was used, possible toxic effects of residual fat and other components on cells were investigated. Native bone matrix of 2 ccm was mixed with 3 ml of D-MEM without FCS and L-glutamine. The suspension was incubated at 37 1C on a shaker water bath for 1 h. FCS and L-glutamine were added. Three different tests for toxicity were performed.
(1) After washing the cells, suspension of bone matrix was added undiluted, 1:3 diluted, and 1:6 diluted. After 1 hour the cells were washed twice with phosphate buffered saline (PBS) and growth medium was added. (2) The suspension was added to washed HEp-2 cells undiluted, 1:3 diluted, and 1:6 diluted, and the cells were incubated with the matrix–D-MEM suspension. (3) The suspension was centrifuged for 3 min at 2000 g. The undiluted supernatant and the supernatant diluted in D-MEM 1:3 and 1:6, respectively, were used as growth medium for cells. After 3 days of cultivation, cells incubated with bone matrix followed by washing steps showed no significant differences compared to untreated cells (1). However, higher concentrations of bone particles remaining within the cell culture suffocated and mechanically damaged cells (2). Thus, the lowest titer in such experiment for matrix titration can be calculated X2.3 101 TCID50. The dilutions of supernatant showed a mild toxic effect with the undiluted samples and the samples diluted 1:3. Cells incubated with a 1:6 dilution of the supernatant were confluent after 3 days and showed no signs of CPE (3). Therefore, titrations started with a sample dilution 1:5. Original virus stocks were freeze-dried without bone matrix and titrated to assess the influence of native bone components on virus titers and the reduction of TCID50 due to lyophilization. All virus stocks were air-dried at room temperature (20–22 1C): VSV 24 h, MEV 26 h, PPV 27 h in a safety cabinet. After resuspension in cell culture medium the TCID50 was determined by titration. 2.6. Spiking of bone matrix Two cubic centimeters (ccm) of native bone matrix were spiked with the same volume of virus stock under sterile conditions. Samples were stored at 70 1C at least overnight and subsequently subjected to freeze-drying. 2.7. Cell-associated experiments Infection kinetics were obtained independently (for VSV, MEV) in order to perform cell-associated experiments. These experiments were carried out with a multiplicity of infection (moi) of 1. Cells were harvested at different time points. The cells were subjected to three cycles of freeze-thawing (70 1C/ 20 1C) in order to release the viruses and the titer was determined. The infected cells used for spiking the bone matrix were harvested when a maximum of virus could be detected within cells while CPE was not recorded for a majority of cells. It was determined to be 4–5 h for both viruses. Infected cells were cultivated in 25 cm2 flasks. Cells were harvested when cells started showing CPE. The virus-containing supernatant was removed and cells were washed twice with PBS. Cells were harvested using a cell-scraper and resuspended in 2 ml of cell culture medium. This suspension was immediately mixed with 2 ccm of native bone matrix. The virus-spiked bone matrix was immediately stored at 70 1C until lyophilization. Control samples of infected cells which were infected and harvested in parallel to the samples mixed with bone matrix were stored at 70 1C. The spiked and freeze-dried bone matrix and the control samples were resuspended in 2 ml of cell culture
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Table 1 Comparison of residual infectivity for cell-associated viruses (VSV, MEV) Infectivity (TCID50/ml)
VSVa
VSV controlb
MEVa
MEV controlb
Virus and bone matrix, lyophilized (supernatant) Standard deviation Virus and bone matrix, lyophilized (particles) Standard deviation
7.5 101 2.6 101 1.1 102 5.0 101
9.3 103 1.8 103 7.2 103 1.5 103
4.3 100 0.28 100 p2.3 101 Not applicable
1.4 105 7.6 104 2.4 105 3.5 104
All samples were treated alike, except for control samples that were not subjected to lyophilization. The cell-associated experiments were carried out three-fold and the data for titration of infected, freeze-dried cells were carried out as double values. No residual infectivity was detected above the detection limit for particles adhering to the bone matrix. Therefore, no standard deviation is given. a Infected cells and matrix, lyophilized, resuspended, 3 freeze-thawed. b Infected cells and matrix, resuspended, 3 freeze-thawed.
medium and subjected to three cycles of freeze-thawing (70 1C/20 1C) in order to release virus particles from cells prior to titration. The comparison of TCID50 for bone matrix samples and controls showed the loss of infectivity due to freeze-thawing versus the effect of lyophilization (Table 1). 2.8. Lyophilization Lyophilization (lyophilizator TG 5.4 Vakutec, Heidenau, Germany) was carried out using routine tissue bank procedures for 24–30 h. Conditions: product: start: 26 1C, end +34 1C, condenser: start 65 1C, end 75 1C; pressure: start 85%, end 37% (related to the barometric pressure, 760 Torr). Residual moisture was determined independently in three samples as double value, using an electronic moisture analyzer (MA 40, Sartorius, Go¨ttingen, Germany). 2.9. Resuspension and titration The lyophilized samples were resuspended and washed in 8 ml of cell culture medium for 30 min at room temperature, corresponding to a 1:5 dilution of the original virus stock. Matrix and supernatant were separated by centrifugation for 10 min at 1000 g. The supernatant was removed and titrated as described above. The matrix was resuspended in fresh cell culture medium and titrated separately to determine whether virus adhered to the matrix. Lyophilized and air-dried virus stocks were resuspended in cell culture medium and titrated as described above. After evaluation of cell cultures for CPE by light microscope, the cells were fixated with 4% formaldehyde and stained with Giemsa.
3.2. Vesicular Stomatitis virus (VSV) The titer (TCID50) of the original virus stock was determined as 2 107/ml. Lyophilization of virus spiked onto bone matrix reduced the infectious titer by 2 log10. It could be shown that virus adhered to the bone matrix, although cortical bone material was used that has no cavities. The titer determined for the virus adhering to the bone matrix after lyophilization was determined to be 3–4 log10 lower compared to the original virus stock. Lyophilization of the virus stock without bone matrix resulted in an about two-fold reduction of the virus titer. However, air-drying of virus stock reduced the titer by 5 log10 (Fig. 1). 3.3. Encephalomyocarditis virus (EMCV) strain Maus Elberfeld virus (MEV) The titer of the original MEV stock was determined as 2.4 108 TCID50/ml. Drying of MEV lowered the virus titer considerably (Fig. 2). Lyophilization of virus stock reduced the titer by 5 log10 and of virus-spiked bone matrix by 6–7 log10. Air-drying of MEV stock resulted in a reduction by about 6 log10. No adherence of virus to the bone matrix could be detected (detection limit p2.3 101 TCID50). 3.4. Porcine parvovirus (PPV) PPV stock showed no significant reduction in virus titer in any of the assays (Fig. 3). The reduction of virus infectivity was o1 log10.
3. Results
3.5. Cell-associated viruses
3.1. Residual moisture
Titers of cell-associated virus were reduced by mixing infected cells with bone matrix prior to lyophilization. Original virus stock subjected to three cycles of freezethawing showed no significant loss of infectivity compared to the untreated virus stock (1.2 107 compared to 1.5 107). For MEV the infectious titer
Residual moisture was determined to be between 1% and 3% for the lyophilized samples. It was not determined for air-dried samples (estimation: 20% moisture).
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10 9
PPV 10 7 10 6
TCID50
TCID50
10 6
10 3
10 5 10 4 10 3 10 2
10 0
Stock
Stock and matrix lyophilized, supernatant
Stock and Lyophilized matrix stock lyophilized, adhering to matrix particles
Air-dried stock
Fig. 1. Effect of lyophilization on the infectivity (TCID50/ml) of VSV. The TCID50 of the virus stock was determined in four independent titrations. Results for supernatant and matrix-adherent infectious titer was determined as six-fold values (two independent lyophilizations). The TCID50 for lyophilized virus-stock was determined in two independent titrations, the infectious titer for air-dried virus stock was determined once.
10 1 Stock
Stock and matrix lyophilized, supernatant
Stock and Lyophilized matrix stock lyophilized, adhering to matrix particles
Air-dried stock
Fig. 3. Effect of lyophilization on the infectivity (TCID50/ml) of PPV. The infectious titer of the virus stock was determined in two independent experiments. The TCID50 for supernatant and matrixadherent samples was performed as six-fold values (using two independent lyophilizations). The TCID50 for lyophilized virus-stock was determined in two independent titrations, the infectious titer for air-dried virus stock was determined once.
MEV 10 12
TCID50
10 8
10 4
* 10 0
Stock
Stock and matrix lyophilized, supernatant
Stock and Lyophilized matrix stock lyophilized, adhering to matrix particles
Air-dried stock
* Detection limit ≤ 2.3x101 TCID50 Fig. 2. Effect of lyophilization on the infectivity (TCID50/ml) of MEV. The infectious titer of the virus stock was determined in five independent experiments. The TCID50 for supernatant and matrixadherent samples was performed as six-fold values (using two independent lyophilizations). The TCID50 for lyophilized virus-stock was determined in two independent titrations, the infectious titer for air-dried virus stock was determined as single experiment. Due to the interference of bone particles with the cell culture, the detection limit for the matrix adherence is higher than for the titration of other samples (23 compared to 3 TCID50). No residual infectivity was detected above this detection limit for particles adhering to the bone matrix.
was reduced by 4–5 log10. Cell-associated MEV showed a stronger decrease of virus titer in comparison to the control. Lyophilization of MEV-infected cells without matrix particles resulted in a reduction of virus titers by approximately 4 log10. MEV-infected cells spiked onto bone matrix particles showed a decrease of titers by a factor of about 5 log10 compared to controls of
infected cells that were subjected to freeze-thawing cycles. The titer of cell-associated PPV showed no difference when comparing spiked and lyophilized cell-free virus (2.2 104) and virus adhering to the matrix (2.3 104). PPV infectivity was not significantly reduced by freezedrying with or without matrix, either by freeze-thawing (three cycles, 70 1C to room temperature) or airdrying.
4. Discussion Recently, it was demonstrated that freeze-drying does not inactivate retrovirus infectivity completely [36]. Although these findings are of outstanding importance, published data regard only qualitative aspects, using serological and nucleic acid testing techniques. The infectious titer of the samples was not assessed. In contrast, we determined the effect of freeze-drying on the infectivity titers of selected model viruses under routine tissue bank production conditions. Not only cell-free but also cell-associated viruses were tested. Thus, the selected model viruses and experimental conditions allow a sound validation of the production process regarding virus safety in lyophilized bone grafts. Virus infectivity was not efficiently inactivated by lyophilization although MEV and VSV titers were considerably lower in lyophilized samples compared to the original virus stock and controls. Air-drying of MEV and VSV resulted in lower infectious titers than lyophilization. Thus, the relatively gentle drying by lyophilization, even without adding specific stabilizers, is
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reflected in these experiments. PPV titers were not significantly reduced by lyophilization or air-drying. This finding is consistent with previously published data regarding the stability of parvoviruses [40]. The results indicate that freeze-drying could have a reducing effect on the infectivity of some viruses, but these findings can not be generalized. While the residual infectivity of MEV after lyophilization was about 5 log10 lower compared to the original virus stock, little or no effect on PPV titers was observed by lyophilization (o1 log10). These findings are not surprising because lyophilization is also used for the preservation of microorganisms (e.g. surveys, drugs). It is important to bear in mind that (1) donors are only screened for a selected number of pathogens, and it is virtually impossible to screen every transplant for a broad spectrum of viruses including rare pathogens, e.g. zoonotically transmitted viruses, (2) that diagnostic windows could pose further diagnostic problems, and (3) that although musculoskeletal transplants are of extremely high value for the individual recipient, improving the quality of life, they are usually not indispensable to survival. Therefore it is desirable to establish potent inactivation steps into the production process that efficiently inactivate virus infectivity, since some viruses could show rates of manifestation of up to 100%, e.g. measles virus [41]. Nevertheless, reduction of infectivity through lyophilization could be used in combination with other methods such as moist-heat inactivation [18], gamma irradiation [42], or peracetic acid-ethanol treatment [43]. A combination of different physico-chemical inactivation methods could provide relatively high virus safety while maintaining the biological properties of musculoskeletal transplants.
5. Conclusions Lyophilization is widely used as a gentle preservation method for bone tissue transplants. In contrast to solid organ transplants, bone allografts are not indispensable to life. Thus, pathogen inactivation procedures included into the production process should provide the highest biological safety. Our experiments revealed that lyophilization is not sufficient as sole inactivation method regarding virus safety of musculoskeletal transplants, since considerable residual infectivity was detected in experimental models for enveloped as well as for nonenveloped viruses.
Acknowledgements This work was supported by the Musculoskeletal Transplant Foundation (MTF), Edison, NJ, USA, and
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the DIZG—German Institute for Cell and Tissue Replacement, Berlin, Germany, as well as by SFB 421 of the Charite´, Berlin, Germany. We thank Ursula Erikli for copy-editing. We are grateful to Frank Schweiger and Sven Schurig for skilful technical assistance. References [1] CDC. Update: allograft-associated bacterial infections. MMWR 2002;51:207–10. [2] Eastlund T, Strong DM. Infectious disease transmission through tissue transplantation. In: Philips GO, editor. Advances in tissue banking 7. Singapore, London: World Scientific Publishing; 2004. p. 51–131. [3] American Association of Tissue Banks. 2002 Annual registration survey. AATB newsletter 2004;27:8. [4] Regel G, Sudkamp NP, Illgner A, Buchenau A, Tscherne H. 15 years allogeneic bone transplantation. Indications, treatment and results. Unfallchirurg 1992;95:1–8. [5] Wippermann BW, Schratt HE, Steeg S, Tscherne H. Complications of spongiosa harvesting of the ilial crest. A retrospective analysis of 1,191 cases. Chirurg 1997;68:1286–91. [6] Wissenschaftlicher Beirat der Bundesa¨rztekammer. Richtlinien zum Fu¨hren einer Knochenbank. Dt A¨rzteblatt 2001;98: A1011–116. [7] Buck BE, Resnick L, Shah SM, Malinin TI. Human immunodeficiency virus cultured from bone. Implications for transplantation. Clin Orthop 1990;251:249–53. [8] Asselmeier MA, Caspari RB, Bottenfield S. A review of allograft processing and sterilization techniques and their role in transmission of the human immunodeficiency virus. Am J Sports Med 1993;21:170–5. [9] Casey JW, Roach A, Mullins JI, Burck KB, Nicolson MO, Gardner MB, Davidson N. The U3 portion of feline leukemia virus DNA identifies horizontally acquired proviruses in leukemic cats. Proc Natl Acad Sci USA 1981;78:7778–82. [10] Simonds RJ, Holmberg SD, Hurwitz RL, Coleman TR, Bottenfield S, Conley LJ, Kohlenberg SH, Castro KG, Dahan BA, Schable CA. Transmission of human immunodeficiency virus type 1 from a seronegative organ and tissue donor. N Engl J Med 1992;326:726–32. [11] Wilmes E, Gu¨rtler L, Wolf H. Transmissibility of HIV infections via allogeneic transplants. Laryngol Rhinol Otol (Stuttg) 1987;66:332–4. [12] CDC. Hepatits C transmission from an antibody-negative organ and tissue donor. MMWR 2003;52:273–4. [13] Shutkin NM. Homologous-serum hepatitis following the use of refrigerated bone-bank bone. J Bone Jt Surg Am 1954; 36-A:160–2. [14] Sanzen L, Carlsson A. Transmission of human T-cell lymphotrophic virus type 1 by a deep-frozen bone allograft. Acta Orthop Scand 1997;68:72–4. [15] Zou S, Dodd RY, Stramer SL, Strong DM. Tissue safety study group. Probability of viremia with HBV, HCV, HIV, and HTLV among tissue donors in the United States. N Engl J Med 2004; 351:751–9. [16] Kakaiya R, Miller WV, Gudino MD. Tissue transplant-transmitted infections. Transfusion 1991;31:277–84. [17] Pruss A, Go¨bel UB, Pauli G. Infections associated with musculoskeletal-tissue allografts. (Correspondence). N Engl J Med 2004;351:1358–9. [18] Pruss A, Kao M, von Garrel T, Frommelt T, Gu¨rtler L, Benedix F, Pauli G. Virus inactivation in bone tissue transplants (femoral heads) by moist heat with the ‘‘Marburg bone bank system’’. Biologicals 2003;31:75–81.
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human bone-patellar tendon-bone allografts. Am J Sports Med 1995;23:643–6. Roberts TS, Drez Jr. D, McCarthy W, Paine R. 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;19:35–41. Nather A, Thambyah A, Goh JC. Biomechanical strength of deep-frozen versus lyophilized large cortical allografts. Clin Biomech (Bristol Avon) 2004;19(5):526–33. Bettin D, Rullko¨tter V, Polster J, Fuchs S. Primary biomechanical influence of different sterilization methods on a freeze-dried boneligament transplant. Arch Orthop Trauma Surg 1999;119:236–40. Crawford MJ, Swenson CL, Arnoczky SP, O’Shea J, Ross H. Lyophilization does not inactivate infectious retrovirus in systemically infected bone and tendon allografts. Am J Sports Med 2004;32:580–6. Blu¨mel J, Schmidt I, Willkommen H, Lo¨wer J. Inactivation of parvovirus B19 during pasteurization of human serum albumin. Transfusion 2002;42:1011–8. Scheidler A, Rokos K, Reuter T, Ebermann R, Pauli G. Inactivation of viruses by beta-propiolactone in human cryo poor plasma and IgG concentrates. Biologicals 1998;26:135–44. Spearman A, Ka¨rber G, Cavalli-Sforza L, editors. Biometrie. Grundzu¨ge biologisch-medizinischer Statistik. Stuttgart: Gustav Fischer; 1974. p. 171–3. Roberts PL, Hart H. Comparison of the inactivation of canine and bovine parvovirus by freeze-drying and dry-heat treatment in two high purity factor VIII concentrates. Biologicals 2000;28:185–8. Atkinson W, Wolfe C, Humiston S, Nelson R, editors. Measles. Epidemiology and prevention of vaccine-preventable diseases. (The Pink Book.). 6th ed. Atlanta: Centers for Disease Control and Prevention; 2000. Pruss A, Kao M, Gohs U, Koscielny J, von Versen R, Pauli G. Effect of gamma irradiation on human cortical bone transplants contaminated with enveloped and non-enveloped viruses. Biologicals 2002;30:125–33. Pruss A, Baumann B, Seibold M, Kao M, Tintelnot K, von Versen R, Radtke H, Do¨rner T, Pauli G, Go¨bel UB. Validation of the sterilization procedure of allogeneic avital bone transplants using peracetic acid-ethanol. Biologicals 2001;29:59–66.
Biomaterials 26 (2005) 6558–6564 www.elsevier.com/locate/biomaterials
Effects of lyophilization on the infectivity of enveloped and non-enveloped viruses in bone tissue Christine Uhlenhauta,b, Thomas Do¨rnerb, Georg Paulia, Axel Prussb, a
b
Center for Biological Safety, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany Tissue Bank, Institute for Transfusion Medicine, Charite´—University Medicine, Schumannstr. 20/21, 10117 Berlin, Germany Received 25 February 2005; accepted 15 April 2005 Available online 6 June 2005
Abstract Recently reported qualitative experiments proved that retroviral infectivity is not destroyed by lyophilization performed on systemically infected bone and tendon. The now accomplished quantitative determination of residual infectivity for enveloped and non-enveloped viruses allows a validation of the production process regarding viral safety in freeze-dried bone transplants. The lyophilization effect on the infectivity of two non-enveloped viruses (Maus Elberfeld virus, MEV; Porcine parvovirus, PPV) and one enveloped virus (Vesicular Stomatitis virus, VSV) was examined for virus-spiked bone material in comparison to lyophilized viruses, original virus stock, and air-dried viruses. All experiments were carried out with both cell-free and cell-associated virus. Significant differences were observed regarding the reduction of virus titers (TCID50). Infectivity of VSV was reduced by about 3–4 log10 using lyophilization in presence of bone matrix and of MEV by 6–7 log10, while no substantial reduction in virus titers was observed for PPV. Lyophilization of cell-free or cell-associated virus is not sufficient to inactivate viruses completely. However, lyophilization could have an additive effect in line with other production steps used in the manufacturing process. r 2005 Elsevier Ltd. All rights reserved. Keywords: Bone transplants; Lyophilization; Freeze-drying; Virus safety; Tissue bank
1. Introduction In spite of increasing efforts to develop alternative materials and procedures, allogeneic bone transplantation remains an indispensable tool for reconstructing extensive bone defects. The number of allogeneic bone grafts transplanted in the US amounts to 650,000– 900,000 [1–3] and in Germany to approx. 35,000/yr. In comparison to artificial bone substitution or material Abbreviations: EMCV, Encephalomyocarditis virus; FeLV, Feline Leukemia virus; HAV, Hepatitis A virus; HBV, Hepatitis B virus; HCV, Hepatitis C virus; HIV, Human immunodeficiency virus; MEV, Maus Elberfeld virus; moi, Multiplicity of infection; PPV, Porcine parvovirus; TCID50, Tissue culture infectious dose (50%); TSE, Transmissible spongiform encephalopathy; VSV, Vesicular Stomatitis virus Corresponding author. Fax: +49 0 30 450 525976. E-mail address: [email protected] (A. Pruss). 0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.04.049
derived from animals, allogeneic material has the major advantage of being able to act osteoconductively or in part osteoinductively due to its physiological structure. Furthermore, the potential ‘en bloc’ replacement is another advantage of allogeneic bone transplantation; even large defects of up to 10 cm in length heal mostly without intricacies [4]. Autologous bone transplants are considered as ‘gold standard’ but their use is limited due to the confined reservoir. In addition to these limitations, the necessary secondary operation on the iliac crest could lead to considerable neurological complications [5], whereas xenotransplantation could involve the risk of TSE transmission. Only a limited amount of bones from living donors is available, i.e. femoral heads after total hip replacements, while an adequate supply of bone tissue (e.g. femora, tibiae, vertebrae, os ilium) could be obtained from cadaveric sources.
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Whereas living donors can be tested for relevant infection markers (i.e. HBV surface antigen and antibodies against HBV core, HIV and HCV or the genomes of these viruses) at the time of tissue donation and a second time after a reasonable quarantine storage of the bones, the safety of cadaveric bones can only be assessed by testing the donors at the time of death or by testing recipients of solid organs from deceased donors 6 months after transplantation [6]. Therefore by testing for known pathogens, the safety of a bone tissue transplant can not be fully guaranteed. It is virtually impossible to perform reliable tests for every pathogen. In spite of rigorous donor screening, transmissions of HIV, HBV, and HCV have occurred via transplantation of (deep-frozen) bone and tendon allografts [7–13]. The transmission of HTLV-1 by a deep-frozen allogeneic bone transplant has been described [14]. Rare or zoonotically transmitted pathogens remain undetectable. Furthermore, infections within a diagnostic window can not be detected, even though a reduction of the diagnostic window can be achieved by application of PCR [15]. Hence it is crucial to apply sterilization methods that inactivate all potential pathogens, including viruses, bacteria, fungi and their spores. In addition to their reconstructive efficacy, a central issue in quality assessment must be the validation of the microbial safety of the tissues [16,17]. Various sterilization techniques of tissues have been developed to meet the requirements of obtaining pathogen-free transplants while maintaining the biological properties. For example, an effective inactivation of HIV has been demonstrated using sterilization techniques such as moist heat, gamma irradiation, peracetic acid [18–21] and ethylene oxide. However, these treatments have detrimental effects on the biological and mechanical properties of the treated tissues [22–33]. Lyophilization has been used to preserve musculoskeletal tissues over long periods. The method does not require elaborate treatment but there are conflicting data regarding changes of the physical properties’ of the transplant [34,35]. Cocultivation of lyophilized tendon and bone segments derived from systemically infected donors with susceptible cells demonstrated the presence of infectious Feline Leukemia virus (FeLV) and proved that retroviral infectivity is not destroyed by lyophilization [36]. This study by Crawford et al. used an indirect method (p27 antigen assay) and determination of proviral load by quantitative real-time PCR. The results demonstrated the presence of infectious FeLV after lyophilization. However, the cell cultures were challenged with an unknown amount of FeLV particles (derived from five FeLV-infected donor cats) and only proviral load was determined. Thus, it was shown evidentiary that infectivity was not destroyed but the degree to which lyophilization reduced virus infectivity was not determined.
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The objective of our study is the quantitative evaluation of inactivating effects of lyophilization on relevant and model viruses when used for the production of allograft bone transplants in order to enhance microbial safety of bone tissue transplantation. Although lyophilization of bone allografts does not guarantee virus safety, a considerable reductive effect on the infectivity of some viruses due to freeze-drying could be used in combination with other sterilization methods. Three relevant and model viruses were selected and appropriate test conditions developed to evaluate the effects of lyophilization on virus infectivity. Maus Elberfeld virus was used as non-enveloped model virus and Porcine parvovirus (PPV) was used as a virus which is known to be resistant to physico-chemical inactivation procedures. The Vesicular Stomatitis virus (VSV) was used to examine possible effects of the virus envelope in the context of the lyophilization procedure.
2. Materials and methods 2.1. Preparation of bone matrix Native cortical bones derived from a single donor were ground with a rotor speed mill (Pulverisette 14, Fritsch, IdarOberstein, Germany) to particles sized between 0.08 and 0.8 mm. The bone matrix was irradiated with 15 kGy (GammaService GmbH, Radeberg, Germany). This treatment was introduced to inactivate potentially present microbes such as bacteria, fungi and spores. The donor was tested negative for the following infection markers: anti-HIV-1/2, HBsAg, antiHBc, anti-HCV, and TPHA. Vials containing irradiated bone matrix were opened and handled aseptically under BSL2 conditions. 2.2. Viruses A non-enveloped virus, Encephalomyocarditis virus (EMCV) strain Maus Elberfeld virus (MEV), genus Cardiovirus, family Picornaviridae, was used as model virus for Hepatitis A virus, Rhinoviruses, and Enteroviruses such as Poliovirus. MEV virions have a diameter of approximately 30 nm and contain the positive-strand ssRNA genome. VSV, genus Vesiculovirus, family Rhabdoviridae, was used as general model for enveloped viruses, especially as model for Lyssavirus, the causing agent of Rabies. The VSV negativestrand ssRNA genome has a genome length of approximately 11 kb. The virions are approximately 65 nm in diameter and 180 nm in length. The PPV, genus parvovirus, family Parvoviridae, a non-enveloped virus, was used as model virus for the human-pathogenic parvovirus B19. PPV, like other animal parvoviruses, was chosen for its well-known resistance to physico-chemical inactivation methods [37]. PPV virions are 25 nm in diameter and have icosahedral symmetry. The genome consists of a single molecule of linear ssDNA, 5.2 kb in size. The effect on virus infectivity was tested for all viruses
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with and without cell association in comparison to control virus stocks. 2.3. Cell lines and cell culture media MEV was grown and titrated on HEp2 cells, a human cell line established via HeLa contamination (ATCC CCL-23). VSV was grown and titrated on BHK21 cells (baby hamster kidney cells; ATCC CCL-10). PPV was grown and titrated on PK13 cells (porcine kidney cell line, obtained from Cornelia Schmitt, Robert Koch-Institut). Dulbecco’s modified Eagle’s medium (D-MEM) was used, supplemented with glutamine (2 mg/ml), 10% fetal calf serum (FCS) for PK13, and 5% FCS for HEp2 and BHK21 cells. All cells were cultivated at 37 1C in a humidified atmosphere of 5% carbon dioxide (CO2). 2.4. Virus preparation and titration All virus stocks were prepared from the supernatant of infected cells and virus titer was determined as described elsewhere [38]. Briefly, virus infectivity was titrated by standard microtitration assay. Cell suspension (100 ml per well) was seeded into 96-well microtiter plates at a concentration of 104 cells per well for HEp2 and BHK21, and 2.5 103 cells for PK13, respectively. Cells were cultivated at 37 1C over night. The samples were serially diluted 1:3 in medium. Eight wells were inoculated with 100 ml aliquots of each dilution. Each 96-well plate included eight wells with non-infected control cells. The microtiter plates were incubated at 37 1C and evaluated daily for cytopathogenic effect (CPE) by light microscope. Cultures showing the characteristic CPE were considered to be positive. Infection of cells leads to their detachment and visible changes in cell morphology; these CPEs were recorded on day 2 post infection for VSV, on day 3 post infection for MEV, and on day 6 post infection for PPV. The detection limit of the virus titration is 3.00 100 TCID50. Due to the interference of bone particles with the cell culture, the detection limit for the matrix adherence is higher than for the titration of other samples (23 compared to 3 TCID50). The infectivity titers were calculated as TCID50. TCID50 is defined as that dilution of a virus required to infect 50% of a given number of inoculated cell cultures (50% endpoint). The TCID50 was calculated according to Spearman and Ka¨rber [39]: TCID50 =ml ¼ Dðn=p 0:5Þ=D0 D V (D: dilution factor; n: number of wells displaying CPE; p: number of wells tested with the same dilution in parallel; D0 : reciprocal of the first dilution; V: volume (ml)). Cellular debris was removed by centrifugation and aliquots were stored at 70 1C until use. Virus stocks with an appropriate titer ranging from 5 to 8 log10 TCID50 for the respective viruses were used. 2.5. Controls Since native bone material was used, possible toxic effects of residual fat and other components on cells were investigated. Native bone matrix of 2 ccm was mixed with 3 ml of D-MEM without FCS and L-glutamine. The suspension was incubated at 37 1C on a shaker water bath for 1 h. FCS and L-glutamine were added. Three different tests for toxicity were performed.
(1) After washing the cells, suspension of bone matrix was added undiluted, 1:3 diluted, and 1:6 diluted. After 1 hour the cells were washed twice with phosphate buffered saline (PBS) and growth medium was added. (2) The suspension was added to washed HEp-2 cells undiluted, 1:3 diluted, and 1:6 diluted, and the cells were incubated with the matrix–D-MEM suspension. (3) The suspension was centrifuged for 3 min at 2000 g. The undiluted supernatant and the supernatant diluted in D-MEM 1:3 and 1:6, respectively, were used as growth medium for cells. After 3 days of cultivation, cells incubated with bone matrix followed by washing steps showed no significant differences compared to untreated cells (1). However, higher concentrations of bone particles remaining within the cell culture suffocated and mechanically damaged cells (2). Thus, the lowest titer in such experiment for matrix titration can be calculated X2.3 101 TCID50. The dilutions of supernatant showed a mild toxic effect with the undiluted samples and the samples diluted 1:3. Cells incubated with a 1:6 dilution of the supernatant were confluent after 3 days and showed no signs of CPE (3). Therefore, titrations started with a sample dilution 1:5. Original virus stocks were freeze-dried without bone matrix and titrated to assess the influence of native bone components on virus titers and the reduction of TCID50 due to lyophilization. All virus stocks were air-dried at room temperature (20–22 1C): VSV 24 h, MEV 26 h, PPV 27 h in a safety cabinet. After resuspension in cell culture medium the TCID50 was determined by titration. 2.6. Spiking of bone matrix Two cubic centimeters (ccm) of native bone matrix were spiked with the same volume of virus stock under sterile conditions. Samples were stored at 70 1C at least overnight and subsequently subjected to freeze-drying. 2.7. Cell-associated experiments Infection kinetics were obtained independently (for VSV, MEV) in order to perform cell-associated experiments. These experiments were carried out with a multiplicity of infection (moi) of 1. Cells were harvested at different time points. The cells were subjected to three cycles of freeze-thawing (70 1C/ 20 1C) in order to release the viruses and the titer was determined. The infected cells used for spiking the bone matrix were harvested when a maximum of virus could be detected within cells while CPE was not recorded for a majority of cells. It was determined to be 4–5 h for both viruses. Infected cells were cultivated in 25 cm2 flasks. Cells were harvested when cells started showing CPE. The virus-containing supernatant was removed and cells were washed twice with PBS. Cells were harvested using a cell-scraper and resuspended in 2 ml of cell culture medium. This suspension was immediately mixed with 2 ccm of native bone matrix. The virus-spiked bone matrix was immediately stored at 70 1C until lyophilization. Control samples of infected cells which were infected and harvested in parallel to the samples mixed with bone matrix were stored at 70 1C. The spiked and freeze-dried bone matrix and the control samples were resuspended in 2 ml of cell culture
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Table 1 Comparison of residual infectivity for cell-associated viruses (VSV, MEV) Infectivity (TCID50/ml)
VSVa
VSV controlb
MEVa
MEV controlb
Virus and bone matrix, lyophilized (supernatant) Standard deviation Virus and bone matrix, lyophilized (particles) Standard deviation
7.5 101 2.6 101 1.1 102 5.0 101
9.3 103 1.8 103 7.2 103 1.5 103
4.3 100 0.28 100 p2.3 101 Not applicable
1.4 105 7.6 104 2.4 105 3.5 104
All samples were treated alike, except for control samples that were not subjected to lyophilization. The cell-associated experiments were carried out three-fold and the data for titration of infected, freeze-dried cells were carried out as double values. No residual infectivity was detected above the detection limit for particles adhering to the bone matrix. Therefore, no standard deviation is given. a Infected cells and matrix, lyophilized, resuspended, 3 freeze-thawed. b Infected cells and matrix, resuspended, 3 freeze-thawed.
medium and subjected to three cycles of freeze-thawing (70 1C/20 1C) in order to release virus particles from cells prior to titration. The comparison of TCID50 for bone matrix samples and controls showed the loss of infectivity due to freeze-thawing versus the effect of lyophilization (Table 1). 2.8. Lyophilization Lyophilization (lyophilizator TG 5.4 Vakutec, Heidenau, Germany) was carried out using routine tissue bank procedures for 24–30 h. Conditions: product: start: 26 1C, end +34 1C, condenser: start 65 1C, end 75 1C; pressure: start 85%, end 37% (related to the barometric pressure, 760 Torr). Residual moisture was determined independently in three samples as double value, using an electronic moisture analyzer (MA 40, Sartorius, Go¨ttingen, Germany). 2.9. Resuspension and titration The lyophilized samples were resuspended and washed in 8 ml of cell culture medium for 30 min at room temperature, corresponding to a 1:5 dilution of the original virus stock. Matrix and supernatant were separated by centrifugation for 10 min at 1000 g. The supernatant was removed and titrated as described above. The matrix was resuspended in fresh cell culture medium and titrated separately to determine whether virus adhered to the matrix. Lyophilized and air-dried virus stocks were resuspended in cell culture medium and titrated as described above. After evaluation of cell cultures for CPE by light microscope, the cells were fixated with 4% formaldehyde and stained with Giemsa.
3.2. Vesicular Stomatitis virus (VSV) The titer (TCID50) of the original virus stock was determined as 2 107/ml. Lyophilization of virus spiked onto bone matrix reduced the infectious titer by 2 log10. It could be shown that virus adhered to the bone matrix, although cortical bone material was used that has no cavities. The titer determined for the virus adhering to the bone matrix after lyophilization was determined to be 3–4 log10 lower compared to the original virus stock. Lyophilization of the virus stock without bone matrix resulted in an about two-fold reduction of the virus titer. However, air-drying of virus stock reduced the titer by 5 log10 (Fig. 1). 3.3. Encephalomyocarditis virus (EMCV) strain Maus Elberfeld virus (MEV) The titer of the original MEV stock was determined as 2.4 108 TCID50/ml. Drying of MEV lowered the virus titer considerably (Fig. 2). Lyophilization of virus stock reduced the titer by 5 log10 and of virus-spiked bone matrix by 6–7 log10. Air-drying of MEV stock resulted in a reduction by about 6 log10. No adherence of virus to the bone matrix could be detected (detection limit p2.3 101 TCID50). 3.4. Porcine parvovirus (PPV) PPV stock showed no significant reduction in virus titer in any of the assays (Fig. 3). The reduction of virus infectivity was o1 log10.
3. Results
3.5. Cell-associated viruses
3.1. Residual moisture
Titers of cell-associated virus were reduced by mixing infected cells with bone matrix prior to lyophilization. Original virus stock subjected to three cycles of freezethawing showed no significant loss of infectivity compared to the untreated virus stock (1.2 107 compared to 1.5 107). For MEV the infectious titer
Residual moisture was determined to be between 1% and 3% for the lyophilized samples. It was not determined for air-dried samples (estimation: 20% moisture).
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10 9
PPV 10 7 10 6
TCID50
TCID50
10 6
10 3
10 5 10 4 10 3 10 2
10 0
Stock
Stock and matrix lyophilized, supernatant
Stock and Lyophilized matrix stock lyophilized, adhering to matrix particles
Air-dried stock
Fig. 1. Effect of lyophilization on the infectivity (TCID50/ml) of VSV. The TCID50 of the virus stock was determined in four independent titrations. Results for supernatant and matrix-adherent infectious titer was determined as six-fold values (two independent lyophilizations). The TCID50 for lyophilized virus-stock was determined in two independent titrations, the infectious titer for air-dried virus stock was determined once.
10 1 Stock
Stock and matrix lyophilized, supernatant
Stock and Lyophilized matrix stock lyophilized, adhering to matrix particles
Air-dried stock
Fig. 3. Effect of lyophilization on the infectivity (TCID50/ml) of PPV. The infectious titer of the virus stock was determined in two independent experiments. The TCID50 for supernatant and matrixadherent samples was performed as six-fold values (using two independent lyophilizations). The TCID50 for lyophilized virus-stock was determined in two independent titrations, the infectious titer for air-dried virus stock was determined once.
MEV 10 12
TCID50
10 8
10 4
* 10 0
Stock
Stock and matrix lyophilized, supernatant
Stock and Lyophilized matrix stock lyophilized, adhering to matrix particles
Air-dried stock
* Detection limit ≤ 2.3x101 TCID50 Fig. 2. Effect of lyophilization on the infectivity (TCID50/ml) of MEV. The infectious titer of the virus stock was determined in five independent experiments. The TCID50 for supernatant and matrixadherent samples was performed as six-fold values (using two independent lyophilizations). The TCID50 for lyophilized virus-stock was determined in two independent titrations, the infectious titer for air-dried virus stock was determined as single experiment. Due to the interference of bone particles with the cell culture, the detection limit for the matrix adherence is higher than for the titration of other samples (23 compared to 3 TCID50). No residual infectivity was detected above this detection limit for particles adhering to the bone matrix.
was reduced by 4–5 log10. Cell-associated MEV showed a stronger decrease of virus titer in comparison to the control. Lyophilization of MEV-infected cells without matrix particles resulted in a reduction of virus titers by approximately 4 log10. MEV-infected cells spiked onto bone matrix particles showed a decrease of titers by a factor of about 5 log10 compared to controls of
infected cells that were subjected to freeze-thawing cycles. The titer of cell-associated PPV showed no difference when comparing spiked and lyophilized cell-free virus (2.2 104) and virus adhering to the matrix (2.3 104). PPV infectivity was not significantly reduced by freezedrying with or without matrix, either by freeze-thawing (three cycles, 70 1C to room temperature) or airdrying.
4. Discussion Recently, it was demonstrated that freeze-drying does not inactivate retrovirus infectivity completely [36]. Although these findings are of outstanding importance, published data regard only qualitative aspects, using serological and nucleic acid testing techniques. The infectious titer of the samples was not assessed. In contrast, we determined the effect of freeze-drying on the infectivity titers of selected model viruses under routine tissue bank production conditions. Not only cell-free but also cell-associated viruses were tested. Thus, the selected model viruses and experimental conditions allow a sound validation of the production process regarding virus safety in lyophilized bone grafts. Virus infectivity was not efficiently inactivated by lyophilization although MEV and VSV titers were considerably lower in lyophilized samples compared to the original virus stock and controls. Air-drying of MEV and VSV resulted in lower infectious titers than lyophilization. Thus, the relatively gentle drying by lyophilization, even without adding specific stabilizers, is
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reflected in these experiments. PPV titers were not significantly reduced by lyophilization or air-drying. This finding is consistent with previously published data regarding the stability of parvoviruses [40]. The results indicate that freeze-drying could have a reducing effect on the infectivity of some viruses, but these findings can not be generalized. While the residual infectivity of MEV after lyophilization was about 5 log10 lower compared to the original virus stock, little or no effect on PPV titers was observed by lyophilization (o1 log10). These findings are not surprising because lyophilization is also used for the preservation of microorganisms (e.g. surveys, drugs). It is important to bear in mind that (1) donors are only screened for a selected number of pathogens, and it is virtually impossible to screen every transplant for a broad spectrum of viruses including rare pathogens, e.g. zoonotically transmitted viruses, (2) that diagnostic windows could pose further diagnostic problems, and (3) that although musculoskeletal transplants are of extremely high value for the individual recipient, improving the quality of life, they are usually not indispensable to survival. Therefore it is desirable to establish potent inactivation steps into the production process that efficiently inactivate virus infectivity, since some viruses could show rates of manifestation of up to 100%, e.g. measles virus [41]. Nevertheless, reduction of infectivity through lyophilization could be used in combination with other methods such as moist-heat inactivation [18], gamma irradiation [42], or peracetic acid-ethanol treatment [43]. A combination of different physico-chemical inactivation methods could provide relatively high virus safety while maintaining the biological properties of musculoskeletal transplants.
5. Conclusions Lyophilization is widely used as a gentle preservation method for bone tissue transplants. In contrast to solid organ transplants, bone allografts are not indispensable to life. Thus, pathogen inactivation procedures included into the production process should provide the highest biological safety. Our experiments revealed that lyophilization is not sufficient as sole inactivation method regarding virus safety of musculoskeletal transplants, since considerable residual infectivity was detected in experimental models for enveloped as well as for nonenveloped viruses.
Acknowledgements This work was supported by the Musculoskeletal Transplant Foundation (MTF), Edison, NJ, USA, and
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the DIZG—German Institute for Cell and Tissue Replacement, Berlin, Germany, as well as by SFB 421 of the Charite´, Berlin, Germany. We thank Ursula Erikli for copy-editing. We are grateful to Frank Schweiger and Sven Schurig for skilful technical assistance. References [1] CDC. Update: allograft-associated bacterial infections. MMWR 2002;51:207–10. [2] Eastlund T, Strong DM. Infectious disease transmission through tissue transplantation. In: Philips GO, editor. Advances in tissue banking 7. Singapore, London: World Scientific Publishing; 2004. p. 51–131. [3] American Association of Tissue Banks. 2002 Annual registration survey. AATB newsletter 2004;27:8. [4] Regel G, Sudkamp NP, Illgner A, Buchenau A, Tscherne H. 15 years allogeneic bone transplantation. Indications, treatment and results. Unfallchirurg 1992;95:1–8. [5] Wippermann BW, Schratt HE, Steeg S, Tscherne H. Complications of spongiosa harvesting of the ilial crest. A retrospective analysis of 1,191 cases. Chirurg 1997;68:1286–91. [6] Wissenschaftlicher Beirat der Bundesa¨rztekammer. Richtlinien zum Fu¨hren einer Knochenbank. Dt A¨rzteblatt 2001;98: A1011–116. [7] Buck BE, Resnick L, Shah SM, Malinin TI. Human immunodeficiency virus cultured from bone. Implications for transplantation. Clin Orthop 1990;251:249–53. [8] Asselmeier MA, Caspari RB, Bottenfield S. A review of allograft processing and sterilization techniques and their role in transmission of the human immunodeficiency virus. Am J Sports Med 1993;21:170–5. [9] Casey JW, Roach A, Mullins JI, Burck KB, Nicolson MO, Gardner MB, Davidson N. The U3 portion of feline leukemia virus DNA identifies horizontally acquired proviruses in leukemic cats. Proc Natl Acad Sci USA 1981;78:7778–82. [10] Simonds RJ, Holmberg SD, Hurwitz RL, Coleman TR, Bottenfield S, Conley LJ, Kohlenberg SH, Castro KG, Dahan BA, Schable CA. Transmission of human immunodeficiency virus type 1 from a seronegative organ and tissue donor. N Engl J Med 1992;326:726–32. [11] Wilmes E, Gu¨rtler L, Wolf H. Transmissibility of HIV infections via allogeneic transplants. Laryngol Rhinol Otol (Stuttg) 1987;66:332–4. [12] CDC. Hepatits C transmission from an antibody-negative organ and tissue donor. MMWR 2003;52:273–4. [13] Shutkin NM. Homologous-serum hepatitis following the use of refrigerated bone-bank bone. J Bone Jt Surg Am 1954; 36-A:160–2. [14] Sanzen L, Carlsson A. Transmission of human T-cell lymphotrophic virus type 1 by a deep-frozen bone allograft. Acta Orthop Scand 1997;68:72–4. [15] Zou S, Dodd RY, Stramer SL, Strong DM. Tissue safety study group. Probability of viremia with HBV, HCV, HIV, and HTLV among tissue donors in the United States. N Engl J Med 2004; 351:751–9. [16] Kakaiya R, Miller WV, Gudino MD. Tissue transplant-transmitted infections. Transfusion 1991;31:277–84. [17] Pruss A, Go¨bel UB, Pauli G. Infections associated with musculoskeletal-tissue allografts. (Correspondence). N Engl J Med 2004;351:1358–9. [18] Pruss A, Kao M, von Garrel T, Frommelt T, Gu¨rtler L, Benedix F, Pauli G. Virus inactivation in bone tissue transplants (femoral heads) by moist heat with the ‘‘Marburg bone bank system’’. Biologicals 2003;31:75–81.
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