BIOPRESERVATION AND BIOBANKING Volume 10, Number 6, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2012.0048
Simple Novel Bone Bank Storage: The Singapore General Hospital Experience Chay-You Ang, Danny Shi-Theng Loh, Hoe-Wai Chaw, and Pak-Lin Chin
Introduction: Since 1981, the Singapore General Hospital (SGH) bone bank has proven to be a safe, reliable, and economical source of bone allografts. Femoral heads are used exclusively and are procured from patients undergoing hip arthroplasty. Screening for HIV, hepatitis B, and hepatitis C is carried out prior to surgery. Patients with ongoing infection and past history of malignancy are excluded. The bone graft procured is washed and autoclaved to 134C for 3 minutes. It is then stored in saline solution containing penicillin and streptomycin at - 80C. Based on our experience, such a system can be readily duplicated in developing nations with minimal cost and equipment. This article presents our experience in the procurement and storage of femoral head allografts with clinical results to support the safety profile. Methods: Ten femoral heads were harvested from patients who underwent hip arthroplasty. The femoral heads were autoclaved and stored at - 80C in an antibiotic solution. Bone chips were sent for culture immediately after autoclaving and at 3 and 6 months. Results: All specimens passed the initial sterility testing and remained sterile up to 6 months. A retrospective study of 9 patients who had 13 allografts implanted between 2008 and 2010 showed that none of the recipients acquired an infection or transmissible disease due to the allografts. Conclusion: This study showed that our protocol allows for procurement of femoral head allografts with minimal contamination and that they can maintain sterility for up to 6 months. This finding is further supported by our clinical results. Hence, this protocol will be useful for bone banks in developing nations where sterility conditions are suboptimal and cost is an issue.
Introduction
I
n 1981, the Singapore General Hospital (SGH) Bone Bank was founded by Professor Tan Ser Kiat to meet the rising demand for allogenic bone in increasingly complex orthopedic surgeries. At that time, the acceptable practice was to harvest suitable allografts and store them at - 80C without further processing.1 However, the contamination rate from such practices was high, and contaminated allografts had to be discarded.1,3–6 Hence, when the SGH protocol was developed, autoclaving was performed prior to storage in order to reduce contamination of allografts and to decrease wastage. Over the next 3 decades, the bank collected unwanted femoral heads from patients who underwent hip arthroplasty and preserved them for future use after autoclaving. Femoral heads are exclusively banked due to the high volume of hip arthroplasty performed at our center, the ease of storage, and their relatively predictable shape and size. Throughout the years, the SGH bone bank has proven to be a safe, reliable, and economical source of bone allo-
grafts. Other than the addition of compulsory HIV testing in the 1990s, the protocol has remained largely unchanged. Based on our experience, such a system can be readily duplicated in developing nations with minimal cost and equipment. This article presents our experience in the procurement and storage of femoral head allografts with clinical results to support the safety profile.
Methods During February 2012, femoral heads were harvested from 10 different patients for this study. Of the 10 patients, 6 had neck of femur fractures, 3 had primary osteoarthritis of the hip, and 1 had avascular necrosis. Patients were screened prior to surgery for hepatitis B, hepatitis C, and HIV, and all were negative. Femoral heads of poor quality (determined intraoperatively by the surgeon) or from patients who have ongoing infection or active malignancy, are excluded from our bone bank. After retrieval, the femoral heads were stripped of any remaining soft tissue and passed out to the circulating scrub
Singapore General Hospital, Singapore.
526
BONE BANKING IN SINGAPORE
527
nurse for autoclaving at 134C for 3 min in a sterile container. Small bone chips were taken from the autoclaved bone and sent for sterility testing while the rest of the bone and the remaining bone chips were immersed in a solution of 1 mega unit crystalline penicillin and 1 g streptomycin. The container was then sealed, labeled, dated, and kept frozen at - 80C. Should any microorganisms be detected during the sterility testing, the femoral head is discarded. The remaining bone chips from each specimen were sent for sterility testing again at 3 and 6 months. The specimens were discarded after 6 months at the end of the study.
Results All specimens passed the initial sterility testing and remained sterile up to 6 months. Hence, none of the allografts were discarded as a result of contamination by microorganisms. In order to further substantiate the safety profile of our bone bank, a retrospective study of the outcomes in allograft recipients was performed. The presence of wound infection or acquisition of transmissible illness (as a result of the allograft) was used as a primary endpoint. Between 2008 and 2010, a total of 13 femoral head allografts were implanted in 9 patients. One patient required 3 allografts, and another two patients used 2 allografts each. Four patients required allografts for reconstruction of Hill-Sach’s deformity, another 4 patients underwent revision arthroplasties, and the last patient had revision surgery for non-union of a right tibia fracture. Eight patients were followed for a minimum of 1 year; one patient was lost to follow-up after discharge. There were no reports of wound infection in the eight patients. Although the patients were not tested for hepatitis B, hepatitis C, or HIV after surgery, none of them developed any signs or symptoms suggestive of such infections. One patient underwent a revision surgery for non-union of his tibia fracture after implantation of the allograft. No microorganisms were grown from his intraoperative cultures.
Discussion While the National University Hospital in Singapore functions as the national bone bank,2 the SGH bone bank Table 1. Hospital AZ St. Jozef Hospital
Donors Living
Laboratory Tests
operates as an in-house bank that caters mainly to its own Orthopedic Department. Although the SGH bone bank allows for storage of up to 40 allografts, in recent years only 5 to 10 allografts have been used annually. This decline in usage is largely due to the availability of commercial allografts and graft substitutes. Most hospitals that run an in-house bone bank do not perform additional sterilization procedures on the allografts after procurement.1,3–5 The SGH bone bank takes the additional step of autoclaving the allografts in an attempt to reduce the risk of disease transmission and contamination. Table 1 shows the different protocols adopted by several other bone banks. According to our knowledge, no other bone banks include autoclaving as part of their protocol. Since contamination of the exterior surface of allografts can occur in up to 22% of allografts without secondary sterilization,6 sterilization of bone allografts by gamma irradiation is recommended.6,7 However, gamma irradiation is very expensive and is beyond the capability of many small institutions. As such, we recommend the use of autoclave which is commonly available and economical. Autoclaving sterilizes the exterior surface of allografts and should theoretically eliminate any risk of contamination. In our study, all the allografts were successfully decontaminated after autoclaving and remained so up to 6 months of storage. None of the allografts were discarded due to contamination and thus there was minimal wastage. Furthermore, our clinical results showed that none of the recipients developed symptoms of wound infection or transmissible illnesses during 1 year of follow-up. As such, autoclaving of allografts may be a useful antiseptic option for orthopedic surgeons in developing nations where the sterility conditions are suboptimal and availability of allografts may be scarce. Fresh allografts carry the highest risk of rejection by the host and although frozen allografts have reduced rejection rates, the risk of complications remains high.8 Autoclaving further reduces the risk of rejection by the recipient presumably due to thermal inactivation of the antigenic components in the allografts.8 Our clinical results support this theory since none of our patients developed symptoms of graft-versus-host disease. Several authors have shown that autoclaving of bone at 134C for 3 minutes does not significantly alter its
Bone Bank Protocols Type of Grafts
Hep B, Hep C, CMV, Femoral heads syphillis, HIV, CRP and ESR Leicester Living and Hep B, Hep C, syphillis, Femoral heads (living). Bone Bank cadaveric HIV. Blood cultures Long bones and and CMV (cadaveric) osteochondral grafts (cadaveric) VU University Living Hep B, Hep C, HTLV, Femoral heads Medical Center syphillis, HIV, ESR Living and Hep B, Hep C, syphillis, Long bones and soft National cadaveric HIV tissues University Hospital
Processing
Storage
Swab cultures
- 86C
Swab cultures, Irradiated at 25 kGy, wash in antimicrobials Tissue cultures, histology Irradiated at 25 kGy, lyophilization, tissue cultures, antibiotic wash
- 80C
- 80C - 80C or room temperature (lyophilized)
CMV, cytomegalovirus; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; HIV, human immunodeficiency virus; HTLV, human T-lymphotropic virus.
528 mechanical properties.9,10 The osteoconductive properties are conserved as well.11 But it has been shown in animal studies that there is a loss of osteoinduction in autoclaved bone grafts, which leads to an impairment in healing.12,13 However, autoclaving at 120C for 20 min was used in both studies and therefore it is possible that a shorter autoclaving period will allow preservation of some osteoinductive properties. Further studies are required to determine the effects of different autoclaving protocols on the osteoinductive properties of allografts.
Conclusion This pilot study shows that the SGH bone bank protocol is a safe and economical method of preserving femoral heads for establishing an in-house bone bank. Through autoclaving, the rates of graft contamination and post-transplant infection were reduced. However, larger and longer-term studies are required to affirm the results of this study, and possible alteration in the biological properties of bone from autoclaving needs to be investigated further.
Author Disclosure Statement No competing financial interests exist. All funding for this study was provided by the Department of Orthopaedic Surgery, Singapore General Hospital, Singapore.
References 1. Tomford WW, Ploetz JE, Mankin HJ. Bone allografts of femoral heads: Procurement and storage. J Bone Joint Surg Am 1986;68: 534–537. 2. Nather A. Musculoskeletal tissue banking in Singapore: 15 years of experience (1988–2003). J Orthop Surg (Hong Kong) 2004;12: 184–190. 3. Nielsen HT, Larsen S, Andersen M, Ovesen O. Bone bank service in Odense, Denmark. Audit of the first 10 years with bone banking at the Department of Orthopaedics, Odense University Hospital. Cell Tissue Bank 2001;2:179–183. 4. Meermans G, Roos J, Hofkens L, Cheyns P. Bone banking in a community hospital. Acta Orthop Belg 2007;73:754–759.
ANG ET AL. 5. Zwitser EW, Jiya TU, George Licher H, van Royen BJ. Design and management of an orthopaedic bone bank in The Netherlands. Cell Tissue Bank 2012;13:63–69. 6. Sommerville SM, Johnson N, Bryce SL, Journeaux SF, Morgan DA. Contamination of banked femoral head allograft: Incidence, bacteriology and donor follow up. Aust N Z J Surg 2000;70: 480–484. 7. Chapman PG, Villar RN. The bacteriology of bone allografts. J Bone Joint Surg Br 1992;74:398–399. 8. Shegarfi H, Reikeras O. Review article: Bone transplantation and immune response. J Orthop Surg (Hong Kong) 2009;17:206–211. 9. Voggenreiter G, Ascherl R, Fr.uh HJ, Blumel G, SchmitNeuerburg KP. Konservierung und Sterilisation von Kortikalisbiomechanische Untersuchungen an der Ratte. Unfallchir 1995; 98:53–58. 10. Moreno J, Forriol F. Effects of preservation on the mechanical strength and chemical composition of cortical bone: An experimental study in sheep femora. Biomaterials 2002;23: 2615–2619. 11. Taguchi Y, Pereira BP, Kour AK, Pho RW, Lee YS. Autoclaved autograft bone combined with vascularized bone and bone marrow. Clin Orthop Relat Res 1995;320:220–230. 12. Vural R, Akesen B, Karakayalı M, Yalc¸ınkaya U, Aydınlı U. The comparison of the negative effect of autoclaving and pasteurization on bone healing. Acta Orthop Traumatol Turc 2010;44: 322–327. 13. Zoricic S, Bobinac D, Lah B, Maric I, Cvijanovic O, Bajek S, Golubovic V, Mihelic R. Study of the healing process after transplantation of pasteurized bone grafts in rabbits. Acta Med Okayama 2002;56:121–128. 14. Ivory JP, Thomas IH. Audit of a bone bank. J Bone Joint Surg Br 1993;75:355–357.
Address correspondence to: Dr. Chay-You Ang Department of Orthopaedic Surgery Singapore General Hospital Outram Road Singapore 169609 Singapore E-mail: [email protected]
Simple Novel Bone Bank Storage: The Singapore General Hospital Experience Chay-You Ang, Danny Shi-Theng Loh, Hoe-Wai Chaw, and Pak-Lin Chin
Introduction: Since 1981, the Singapore General Hospital (SGH) bone bank has proven to be a safe, reliable, and economical source of bone allografts. Femoral heads are used exclusively and are procured from patients undergoing hip arthroplasty. Screening for HIV, hepatitis B, and hepatitis C is carried out prior to surgery. Patients with ongoing infection and past history of malignancy are excluded. The bone graft procured is washed and autoclaved to 134C for 3 minutes. It is then stored in saline solution containing penicillin and streptomycin at - 80C. Based on our experience, such a system can be readily duplicated in developing nations with minimal cost and equipment. This article presents our experience in the procurement and storage of femoral head allografts with clinical results to support the safety profile. Methods: Ten femoral heads were harvested from patients who underwent hip arthroplasty. The femoral heads were autoclaved and stored at - 80C in an antibiotic solution. Bone chips were sent for culture immediately after autoclaving and at 3 and 6 months. Results: All specimens passed the initial sterility testing and remained sterile up to 6 months. A retrospective study of 9 patients who had 13 allografts implanted between 2008 and 2010 showed that none of the recipients acquired an infection or transmissible disease due to the allografts. Conclusion: This study showed that our protocol allows for procurement of femoral head allografts with minimal contamination and that they can maintain sterility for up to 6 months. This finding is further supported by our clinical results. Hence, this protocol will be useful for bone banks in developing nations where sterility conditions are suboptimal and cost is an issue.
Introduction
I
n 1981, the Singapore General Hospital (SGH) Bone Bank was founded by Professor Tan Ser Kiat to meet the rising demand for allogenic bone in increasingly complex orthopedic surgeries. At that time, the acceptable practice was to harvest suitable allografts and store them at - 80C without further processing.1 However, the contamination rate from such practices was high, and contaminated allografts had to be discarded.1,3–6 Hence, when the SGH protocol was developed, autoclaving was performed prior to storage in order to reduce contamination of allografts and to decrease wastage. Over the next 3 decades, the bank collected unwanted femoral heads from patients who underwent hip arthroplasty and preserved them for future use after autoclaving. Femoral heads are exclusively banked due to the high volume of hip arthroplasty performed at our center, the ease of storage, and their relatively predictable shape and size. Throughout the years, the SGH bone bank has proven to be a safe, reliable, and economical source of bone allo-
grafts. Other than the addition of compulsory HIV testing in the 1990s, the protocol has remained largely unchanged. Based on our experience, such a system can be readily duplicated in developing nations with minimal cost and equipment. This article presents our experience in the procurement and storage of femoral head allografts with clinical results to support the safety profile.
Methods During February 2012, femoral heads were harvested from 10 different patients for this study. Of the 10 patients, 6 had neck of femur fractures, 3 had primary osteoarthritis of the hip, and 1 had avascular necrosis. Patients were screened prior to surgery for hepatitis B, hepatitis C, and HIV, and all were negative. Femoral heads of poor quality (determined intraoperatively by the surgeon) or from patients who have ongoing infection or active malignancy, are excluded from our bone bank. After retrieval, the femoral heads were stripped of any remaining soft tissue and passed out to the circulating scrub
Singapore General Hospital, Singapore.
526
BONE BANKING IN SINGAPORE
527
nurse for autoclaving at 134C for 3 min in a sterile container. Small bone chips were taken from the autoclaved bone and sent for sterility testing while the rest of the bone and the remaining bone chips were immersed in a solution of 1 mega unit crystalline penicillin and 1 g streptomycin. The container was then sealed, labeled, dated, and kept frozen at - 80C. Should any microorganisms be detected during the sterility testing, the femoral head is discarded. The remaining bone chips from each specimen were sent for sterility testing again at 3 and 6 months. The specimens were discarded after 6 months at the end of the study.
Results All specimens passed the initial sterility testing and remained sterile up to 6 months. Hence, none of the allografts were discarded as a result of contamination by microorganisms. In order to further substantiate the safety profile of our bone bank, a retrospective study of the outcomes in allograft recipients was performed. The presence of wound infection or acquisition of transmissible illness (as a result of the allograft) was used as a primary endpoint. Between 2008 and 2010, a total of 13 femoral head allografts were implanted in 9 patients. One patient required 3 allografts, and another two patients used 2 allografts each. Four patients required allografts for reconstruction of Hill-Sach’s deformity, another 4 patients underwent revision arthroplasties, and the last patient had revision surgery for non-union of a right tibia fracture. Eight patients were followed for a minimum of 1 year; one patient was lost to follow-up after discharge. There were no reports of wound infection in the eight patients. Although the patients were not tested for hepatitis B, hepatitis C, or HIV after surgery, none of them developed any signs or symptoms suggestive of such infections. One patient underwent a revision surgery for non-union of his tibia fracture after implantation of the allograft. No microorganisms were grown from his intraoperative cultures.
Discussion While the National University Hospital in Singapore functions as the national bone bank,2 the SGH bone bank Table 1. Hospital AZ St. Jozef Hospital
Donors Living
Laboratory Tests
operates as an in-house bank that caters mainly to its own Orthopedic Department. Although the SGH bone bank allows for storage of up to 40 allografts, in recent years only 5 to 10 allografts have been used annually. This decline in usage is largely due to the availability of commercial allografts and graft substitutes. Most hospitals that run an in-house bone bank do not perform additional sterilization procedures on the allografts after procurement.1,3–5 The SGH bone bank takes the additional step of autoclaving the allografts in an attempt to reduce the risk of disease transmission and contamination. Table 1 shows the different protocols adopted by several other bone banks. According to our knowledge, no other bone banks include autoclaving as part of their protocol. Since contamination of the exterior surface of allografts can occur in up to 22% of allografts without secondary sterilization,6 sterilization of bone allografts by gamma irradiation is recommended.6,7 However, gamma irradiation is very expensive and is beyond the capability of many small institutions. As such, we recommend the use of autoclave which is commonly available and economical. Autoclaving sterilizes the exterior surface of allografts and should theoretically eliminate any risk of contamination. In our study, all the allografts were successfully decontaminated after autoclaving and remained so up to 6 months of storage. None of the allografts were discarded due to contamination and thus there was minimal wastage. Furthermore, our clinical results showed that none of the recipients developed symptoms of wound infection or transmissible illnesses during 1 year of follow-up. As such, autoclaving of allografts may be a useful antiseptic option for orthopedic surgeons in developing nations where the sterility conditions are suboptimal and availability of allografts may be scarce. Fresh allografts carry the highest risk of rejection by the host and although frozen allografts have reduced rejection rates, the risk of complications remains high.8 Autoclaving further reduces the risk of rejection by the recipient presumably due to thermal inactivation of the antigenic components in the allografts.8 Our clinical results support this theory since none of our patients developed symptoms of graft-versus-host disease. Several authors have shown that autoclaving of bone at 134C for 3 minutes does not significantly alter its
Bone Bank Protocols Type of Grafts
Hep B, Hep C, CMV, Femoral heads syphillis, HIV, CRP and ESR Leicester Living and Hep B, Hep C, syphillis, Femoral heads (living). Bone Bank cadaveric HIV. Blood cultures Long bones and and CMV (cadaveric) osteochondral grafts (cadaveric) VU University Living Hep B, Hep C, HTLV, Femoral heads Medical Center syphillis, HIV, ESR Living and Hep B, Hep C, syphillis, Long bones and soft National cadaveric HIV tissues University Hospital
Processing
Storage
Swab cultures
- 86C
Swab cultures, Irradiated at 25 kGy, wash in antimicrobials Tissue cultures, histology Irradiated at 25 kGy, lyophilization, tissue cultures, antibiotic wash
- 80C
- 80C - 80C or room temperature (lyophilized)
CMV, cytomegalovirus; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; HIV, human immunodeficiency virus; HTLV, human T-lymphotropic virus.
528 mechanical properties.9,10 The osteoconductive properties are conserved as well.11 But it has been shown in animal studies that there is a loss of osteoinduction in autoclaved bone grafts, which leads to an impairment in healing.12,13 However, autoclaving at 120C for 20 min was used in both studies and therefore it is possible that a shorter autoclaving period will allow preservation of some osteoinductive properties. Further studies are required to determine the effects of different autoclaving protocols on the osteoinductive properties of allografts.
Conclusion This pilot study shows that the SGH bone bank protocol is a safe and economical method of preserving femoral heads for establishing an in-house bone bank. Through autoclaving, the rates of graft contamination and post-transplant infection were reduced. However, larger and longer-term studies are required to affirm the results of this study, and possible alteration in the biological properties of bone from autoclaving needs to be investigated further.
Author Disclosure Statement No competing financial interests exist. All funding for this study was provided by the Department of Orthopaedic Surgery, Singapore General Hospital, Singapore.
References 1. Tomford WW, Ploetz JE, Mankin HJ. Bone allografts of femoral heads: Procurement and storage. J Bone Joint Surg Am 1986;68: 534–537. 2. Nather A. Musculoskeletal tissue banking in Singapore: 15 years of experience (1988–2003). J Orthop Surg (Hong Kong) 2004;12: 184–190. 3. Nielsen HT, Larsen S, Andersen M, Ovesen O. Bone bank service in Odense, Denmark. Audit of the first 10 years with bone banking at the Department of Orthopaedics, Odense University Hospital. Cell Tissue Bank 2001;2:179–183. 4. Meermans G, Roos J, Hofkens L, Cheyns P. Bone banking in a community hospital. Acta Orthop Belg 2007;73:754–759.
ANG ET AL. 5. Zwitser EW, Jiya TU, George Licher H, van Royen BJ. Design and management of an orthopaedic bone bank in The Netherlands. Cell Tissue Bank 2012;13:63–69. 6. Sommerville SM, Johnson N, Bryce SL, Journeaux SF, Morgan DA. Contamination of banked femoral head allograft: Incidence, bacteriology and donor follow up. Aust N Z J Surg 2000;70: 480–484. 7. Chapman PG, Villar RN. The bacteriology of bone allografts. J Bone Joint Surg Br 1992;74:398–399. 8. Shegarfi H, Reikeras O. Review article: Bone transplantation and immune response. J Orthop Surg (Hong Kong) 2009;17:206–211. 9. Voggenreiter G, Ascherl R, Fr.uh HJ, Blumel G, SchmitNeuerburg KP. Konservierung und Sterilisation von Kortikalisbiomechanische Untersuchungen an der Ratte. Unfallchir 1995; 98:53–58. 10. Moreno J, Forriol F. Effects of preservation on the mechanical strength and chemical composition of cortical bone: An experimental study in sheep femora. Biomaterials 2002;23: 2615–2619. 11. Taguchi Y, Pereira BP, Kour AK, Pho RW, Lee YS. Autoclaved autograft bone combined with vascularized bone and bone marrow. Clin Orthop Relat Res 1995;320:220–230. 12. Vural R, Akesen B, Karakayalı M, Yalc¸ınkaya U, Aydınlı U. The comparison of the negative effect of autoclaving and pasteurization on bone healing. Acta Orthop Traumatol Turc 2010;44: 322–327. 13. Zoricic S, Bobinac D, Lah B, Maric I, Cvijanovic O, Bajek S, Golubovic V, Mihelic R. Study of the healing process after transplantation of pasteurized bone grafts in rabbits. Acta Med Okayama 2002;56:121–128. 14. Ivory JP, Thomas IH. Audit of a bone bank. J Bone Joint Surg Br 1993;75:355–357.
Address correspondence to: Dr. Chay-You Ang Department of Orthopaedic Surgery Singapore General Hospital Outram Road Singapore 169609 Singapore E-mail: [email protected]
