**6. Methods sterilization, infectious diseases and biosafety**

Another extremely important issue is the study of methods of sterilization of frozen and freeze-dried grafts, since there still remains controversy and need for further studies. The currently available techniques have advantages and disadvantages regarding efficacy and maintenance of mechanical and biological properties. Significant deleterious effects on the use of cobalt 60 in the sterilisation of freeze-dried grafts have been reported, demonstrating that, even at low dosage, radiation is capable of destroying the morphogenetic properties, mainly in non-demineralised bone.

The effects of radiation on the biomechanics of the grafts are dose-dependent. Fidele et al. in 1995, studied the damage caused by the application of different doses of gamma radiation exposure on seven biomechanical parameters of frozen allogenic patellar bone-grafting. The sterilization dose accepted for inactivation of HIV, for example, is about 25 kGy. However, four out of the seven parameters measured were reduced after 20 kGy and after 30 kGy all parameters have shown significant reductions. Also, Zhang, Cornu and Delloye in 1997, in an experimental study in rats, compared the ability of graft osteoinduction after gamma radiation sterilisation (25 kGy), ethylene oxide (EO) at 55°C and 40°C or preservation in ethanol showing that the OE at 40°C and ethanol have not negatively affected the osteoinductive capacity, gamma radiation has decreased 40% and OE to 55°C had an almost completely loss of this potential. Some authors indicated that other factors in sterilisation need to be observed are the toxic residues from the OE that may remain in the graft and are released when in contact with liquids. Reference is also considered to the toxicity of gamma radiation when in contact with fat present in the graft, in addition to the mechanical change that the radiation may cause.

The Biology of Bone Grafts 245

Lyophilisation, as proposed and spread in 1951 by the USA Navy Tissue Bank24, is a technique by which dehydration of the water contained in a product is removed by sublimation, i.e., starting from a previously frozen material, placed under vacuum, the water goes directly from a solid state into mist30. Before that, however, the bone graft is washed, centrifuged, decellularised, chemically degreased and subjected to physical processes of cleaning and sterilisation preserving only the protein-mineral matrix, thus

Fig. 3. Histological section of bone sample degreased, decellularized and freeze-dried. Note

Moreover, lyophilisation is a method of processing and storage of musculoskeletal tissues that allows not only the use of human bones, tendons and fascias (allogeneic), but also and mainly the bovine (xenogenic) ones, since by reducing antigenicity, provides

Currently, in major health centres different types of bone grafts are available for reconstructive orthopaedic surgery: frozen and freeze-dried autologous, allogeneic and lyophilised xenogenic (bovine). The autologous graft, though not requiring processing, has been considered from the integration viewpoint, the preferred tissue for bone replacement. There is evidence that survival of osteoblasts and osteocytes from autologous graft are dependent on the quality of the receptor bed. Moreover, the amount of graft obtained is limited, surgical time is increased and complications, either systemic or local, of the

biocompatibility, sterilisation and can be stored at room temperature.

intervention required to obtain the graft ranges from 21% to 49%.

decreasing antigenicity23 (Figure 3).

that the only remaining protein-mineral matrix.

Another way to sterilise is to autoclave the bone, although it has also limitations. According to the recommendation of the European Community, aimed at inactivating prions (infectious proteins), tissues from bovine origin should be sterilised in an autoclave at 132°C for 1h, but the biomechanical effects of this method of sterilisation has proven to reduce in approximately 70%the mechanical resistance to compression of the graft. Moreover, many authors support the safety of non-sterilised bone grafts provided by banks that adopt appropriate methodology for donor selection and quality control and such control is so important that when applying these criteria, 20% to 30% of the harvested material will be discarded due to bacteriological or serological inadequate results. Despite this severe control, to name a few, in November 2001 there were two reported cases in the U.S., by Clostridium sordellii infection in surgical patients who received bone transplants. In March 2002, the Centre for Disease Control and Prevention (CDC, Atlanta) has received 26 reports from different locations of secondary bacterial infections in transplanted musculoskeletal tissues. In these events the CDC investigated donors and confirmed the presence of those bacteria in their tissues. Several other notifications have been done and are reported every year, which provides the need for even a more strict control, revision of adopted routines and, perhaps, searching for methods of sterilisation that may minimally interfere with quality of the graft.

Taylor in 2002, stated that prions, responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalitis, unlike bacteria and viruses, are resistant to procedures such as autoclaving or exposure to sodium hydroxide, but, the association of alkaline substances and heat even at 100°C for 1 minute appears to be effective to inactivate prions. In addition, when isolated used, sodium hypochlorite also demonstrated ability to deactivate prions.

Alike prions, potentially transmissible viral diseases such as hepatitis A, B or C, HIV and HTLV, and other more recently isolated viruses and certain types of tumour cells, should also be of concern, even with current knowledge and care from the point of view of serological or other diagnostic methods, which clearly demonstrates the importance of a wide discussion about sterilisation processes.

As published by Sugihara et al. in 1999, about 1% of tumour cells in 137 femoral heads subjected to histological examination in patients undergoing THA due to primary arthrosis and Palmer et al. also found high rates of histological abnormalities in 1146 patients with hip joint arthrosis, even suggesting that pathological examinations should be included as part of screening for donor tissues.

### **7. Processing and storage**

Many ways of processing and storage bone tissue for clinical application have been proposed and used in Tissue Banks around the world. Among them, we highlight the deepfrozen (-80oC) and lyophilisation, which are the most widely used and accepted methods. The ultralow freezing temperature is reached in freezers that go as low as -80oC with graphical systems constantly monitoring the temperature, having their own power generators and emergency alarms alerting when temperature increases. So, after rigorous screening of donors (living or dead), culture and serological tests for bacteria and fungi, and processing in a surgical environment the graft is subjected to freezing at-80C that allows its storage for up to 5 years. However, it is worth of note that low temperatures do not play any role in the sterilisation of the material, but decreases antigenicity indeed, allowing more useful handling of the graft.

Another way to sterilise is to autoclave the bone, although it has also limitations. According to the recommendation of the European Community, aimed at inactivating prions (infectious proteins), tissues from bovine origin should be sterilised in an autoclave at 132°C for 1h, but the biomechanical effects of this method of sterilisation has proven to reduce in approximately 70%the mechanical resistance to compression of the graft. Moreover, many authors support the safety of non-sterilised bone grafts provided by banks that adopt appropriate methodology for donor selection and quality control and such control is so important that when applying these criteria, 20% to 30% of the harvested material will be discarded due to bacteriological or serological inadequate results. Despite this severe control, to name a few, in November 2001 there were two reported cases in the U.S., by Clostridium sordellii infection in surgical patients who received bone transplants. In March 2002, the Centre for Disease Control and Prevention (CDC, Atlanta) has received 26 reports from different locations of secondary bacterial infections in transplanted musculoskeletal tissues. In these events the CDC investigated donors and confirmed the presence of those bacteria in their tissues. Several other notifications have been done and are reported every year, which provides the need for even a more strict control, revision of adopted routines and, perhaps, searching for methods of sterilisation that may minimally interfere with

Taylor in 2002, stated that prions, responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalitis, unlike bacteria and viruses, are resistant to procedures such as autoclaving or exposure to sodium hydroxide, but, the association of alkaline substances and heat even at 100°C for 1 minute appears to be effective to inactivate prions. In addition, when isolated used, sodium hypochlorite also demonstrated ability to deactivate prions. Alike prions, potentially transmissible viral diseases such as hepatitis A, B or C, HIV and HTLV, and other more recently isolated viruses and certain types of tumour cells, should also be of concern, even with current knowledge and care from the point of view of serological or other diagnostic methods, which clearly demonstrates the importance of a

As published by Sugihara et al. in 1999, about 1% of tumour cells in 137 femoral heads subjected to histological examination in patients undergoing THA due to primary arthrosis and Palmer et al. also found high rates of histological abnormalities in 1146 patients with hip joint arthrosis, even suggesting that pathological examinations should be included as part of

Many ways of processing and storage bone tissue for clinical application have been proposed and used in Tissue Banks around the world. Among them, we highlight the deepfrozen (-80oC) and lyophilisation, which are the most widely used and accepted methods. The ultralow freezing temperature is reached in freezers that go as low as -80oC with graphical systems constantly monitoring the temperature, having their own power generators and emergency alarms alerting when temperature increases. So, after rigorous screening of donors (living or dead), culture and serological tests for bacteria and fungi, and processing in a surgical environment the graft is subjected to freezing at-80C that allows its storage for up to 5 years. However, it is worth of note that low temperatures do not play any role in the sterilisation of the material, but decreases antigenicity indeed, allowing more

quality of the graft.

wide discussion about sterilisation processes.

screening for donor tissues.

useful handling of the graft.

**7. Processing and storage** 

Lyophilisation, as proposed and spread in 1951 by the USA Navy Tissue Bank24, is a technique by which dehydration of the water contained in a product is removed by sublimation, i.e., starting from a previously frozen material, placed under vacuum, the water goes directly from a solid state into mist30. Before that, however, the bone graft is washed, centrifuged, decellularised, chemically degreased and subjected to physical processes of cleaning and sterilisation preserving only the protein-mineral matrix, thus decreasing antigenicity23 (Figure 3).

Fig. 3. Histological section of bone sample degreased, decellularized and freeze-dried. Note that the only remaining protein-mineral matrix.

Moreover, lyophilisation is a method of processing and storage of musculoskeletal tissues that allows not only the use of human bones, tendons and fascias (allogeneic), but also and mainly the bovine (xenogenic) ones, since by reducing antigenicity, provides biocompatibility, sterilisation and can be stored at room temperature.

Currently, in major health centres different types of bone grafts are available for reconstructive orthopaedic surgery: frozen and freeze-dried autologous, allogeneic and lyophilised xenogenic (bovine). The autologous graft, though not requiring processing, has been considered from the integration viewpoint, the preferred tissue for bone replacement. There is evidence that survival of osteoblasts and osteocytes from autologous graft are dependent on the quality of the receptor bed. Moreover, the amount of graft obtained is limited, surgical time is increased and complications, either systemic or local, of the intervention required to obtain the graft ranges from 21% to 49%.

The Biology of Bone Grafts 247

Fig. 4b. Sample graft ½ lyophilized human femoral head.

\*CI, Confidence Interval

**Readings Human (%) CI\* 95% Bovine (%) CI\* 95%** 

Water 7.93 - 7.75 - Fat 0.06 0.1 0.1 0.1 Nitrogen 4.3 0.1 4.3 0.1 Proteins 27.5 0.2 27.2 0.2 Phosphorus 11.9 0.1 11.9 0.2 Total P2O5 27.1 0.2 27.2 0.7 Calcium 24.6 0.7 23.7 0.6 Ca/P 2.06 - 1.99 - Total sodium 0.57 0.01 0.46 0.01 Ashes 64.8 0.6 64.3 0.1 Chlorides 1.3 0.06 1.3 0.2

Table 1. Physical and Chemical analysis of lyophilised bones HCPATB

The frozen allogeneic graft is the most widely used and accepted nowadays, but due to the reduced number of Tissue Banks in our country and the small number of donations is not always available to a wider use. Moreover, despite of all care and biosafety standards adopted by the banks there is still some risk of transmission of infectious diseases and tumours.

The lyophilised allogeneic graft, produced by the majority of U.S. tissue banks, is still not widespread among us, and few studies have reported the use of this graft in orthopaedic surgery and with a small number of cases and limited methods for assessing osteointegration, further studies are needed to better define its use (Figures 4a and 4b).

Fig. 4a. Sample of lyophilized bovine graft.

The xenogenic grafts, commonly used in dentistry, although controversial, are beginning to be used in orthopaedic surgeries, especially those of bovine origin, due to its easy preparation, availability and similarity. The bovine bone has chemical composition, porosity, size, shape and biological behaviour similar to its human counterpart as it can be clearly observed in Table 1. Besides those features, bovine graft provides structural support, osteoconduction and a high content of calcium and phosphorus, all of them essential factors for the newly formed bone tissue.

The most important issue, regardless of the type of graft chosen by the surgeon, is the guarantee that the graft has been processed and stored according to the standards of graft management as established by the Associations of Tissue Banks and the national and international health authorities.

The frozen allogeneic graft is the most widely used and accepted nowadays, but due to the reduced number of Tissue Banks in our country and the small number of donations is not always available to a wider use. Moreover, despite of all care and biosafety standards adopted by the banks there is still some risk of transmission of infectious diseases and

The lyophilised allogeneic graft, produced by the majority of U.S. tissue banks, is still not widespread among us, and few studies have reported the use of this graft in orthopaedic surgery and with a small number of cases and limited methods for assessing osteointegration, further studies are needed to better define its use (Figures 4a and 4b).

The xenogenic grafts, commonly used in dentistry, although controversial, are beginning to be used in orthopaedic surgeries, especially those of bovine origin, due to its easy preparation, availability and similarity. The bovine bone has chemical composition, porosity, size, shape and biological behaviour similar to its human counterpart as it can be clearly observed in Table 1. Besides those features, bovine graft provides structural support, osteoconduction and a high content of calcium and phosphorus, all of them essential factors

The most important issue, regardless of the type of graft chosen by the surgeon, is the guarantee that the graft has been processed and stored according to the standards of graft management as established by the Associations of Tissue Banks and the national and

tumours.

Fig. 4a. Sample of lyophilized bovine graft.

for the newly formed bone tissue.

international health authorities.

Fig. 4b. Sample graft ½ lyophilized human femoral head.


\*CI, Confidence Interval

Table 1. Physical and Chemical analysis of lyophilised bones HCPATB

The Biology of Bone Grafts 249







hyperimmune globulin, rabbis vaccine (prior animal bites),




a. bone tissue: below 18 and over 70 years b. tendon tissue: below 18 and over 55 years c. osteochondral tissue: below 18 and over 45 years

**CONDITIONS YES NO** 

parathyroid, yellow fever and Sabin poliomyelitis)



contaminated blood by HIV, hepatitis B or C?

sexual partners within the past 12 months?


in the past 48h prior to blood sample


months?




serum non-derived B hepatitis)


passive immunizations

sample analysis?



analysis?


