Microwave Sterilization of Femoral Head Allograft

The potential shortage of allograft bone has led to the needto investigate other sources of bone for allografts. Some allograftbone donated from primary total hip arthroplasty recipientsmust be discarded or treated to become useable as a result ofbacterial contamination. Femoral head allografts were contaminatedwith Staphylococcus aureus and Bacillus subtilis. A domesticmicrowave oven was used. The contaminated bone was exposed tomicrowave irradiation for different time periods. The sampleswere then cultured to attempt to grow the two bacterial species.The contaminated bone samples failed to grow any organisms after2 min of exposure to microwave irradiation. This study showsthat sterilization of femoral head allografts contaminated withS. aureus and B. subtilis can be achieved with microwave irradiationin a domestic microwave oven. This method of sterilization ofbone allografts is cheap, easily used, and an effective wayto process contaminated bone.

INTRODUCTION

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Introduction

Femoral head allografts are collected from patients undergoingprimary total hip arthroplasty. After the bone is frozen andconfirmed to be free from bacteriological and viral infectiveagents, it can be released for use in operations such as revisionhip surgery or spinal fusion. The increasing use of bone allografts,in particular, in association with impaction grafting for revisionhip surgery, has increased the demand for bone allografts. Thisincreased use of allograft bone has led Galea et al. (13) tosuggest that the demand will soon outstrip the present supplyfrom primary total hip replacements. Part of the shortfall occursbecause femoral heads contaminated with bacteria are discarded.Bacterial contamination is found in between 10 and 15% of allavailable bone donations (13; R. Frame [Scottish National BloodTransfusion Service, Law Hospital, Lanarkshire, Scotland], personalcommunication, 1999). Before this bone can be used, it mustbe processed with gamma irradiation or ethylene dioxide. Theseexpensive treatments are known to affect the quality of thebone (1, 4).

A simple, reliable, and cheap alternative for decontaminationof donor bone would allow the use of previously contaminatedbone and would help to address the shortage in allograft bonestocks. The project described here further examines the possiblerole of microwaves in decontamination of femoral head allograftbone.

MATERIALS AND METHODS

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Materials and Methods

Femoral head allografts were obtained from the bone bank facilityof the Blood Transfusion Service at Law Hospital, Carluke, Scotland.On harvesting of each of the femoral heads, bone chips werecollected and swabs from the periosteal surface were taken andtested for contamination. All specimens were clear of bacterialcontamination. The blood of all donors was tested and foundto be negative for markers of infection with hepatitis B orC virus, human immunodeficiency virus type 1 or 2, or Treponemapallidum. To minimize the risk from Creutzfeldt-Jakob disease(CJD), none of the donors had a family history of CJD; had receivedgrowth hormone, human pituitary extracts, or gonadotrophic hormones;had undergone brain or spinal surgery to remove a tumor or cystprior to 1993; or had any degenerative neurological condition.The bone was frozen to -80°C. In all aspects the bone usedwas considered sterile by the Blood Transfusion Service at LawHospital but was considered unfit for release because of a pasthistory of neoplasia. Bones were stored before use at -30°C.

Each allograft specimen was thawed at 4°C in its sterilecontainer prior to use. Under sterile conditions, remnants ofthe femoral neck were resected with a power saw to leave onlythe sphere of the femoral head. Four cores of bone were removedfrom the cut surface with a sterile trephine to give cores ofbone approximately 2 cm long. The femoral heads were replacedin their sterile containers and stored at 4°C. Two coresof bone were then incubated in broth of Staphylococcus aureusand two cores of bone were incubated in broth with a Bacillussp. for 24 h. After incubation one bone core from each of theincubation broths was replaced into the appropriate femoralhead. The other core was immediately cultured. The femoral headwas placed in a microwave oven (model M1713; Samsung) operatingat 2,450 MHz. The specimens were irradiated for 0 (controls),1, and 2 to 6 min at 800 W. The cores were then removed andcultured (see below). Three samples incubated with each specieswere irradiated for 1, 2, 3, and 4 min; and two samples incubatedwith each species were irradiated for 5 and 6 min (64 samples).

Microbiology. Two organisms were selected to test the effectiveness of microwaveactivity on sterilization of femoral bone cores. The organismswere S. aureus NCTC 6571 and Bacillus subtilis var. globigii(B. subtilis).

Overnight stock cultures of the two control organisms were preparedin 20 ml of Schaedler's broth. Optimum inoculation levels wereestablished and consisted of 50 µl of stock culture forB. subtilis and 200 µl of stock culture for S. aureusNCTC 6571. Each of the organisms from the stock cultures wasinoculated into 16 broth cultures (volume, 5 ml; two separategroups of broth cultures). All broth cultures were given a uniqueidentifier to allow tracking of the individual femoral cores.Two femoral head cores were then placed in each broth cultureand incubated aerobically at 37°C for 24 h to allow eachcore to become colonized with the appropriate organism. Twogrowth control broth cultures and a sterile control broth culturewere also prepared and incubated as described above.

Following incubation, the femoral head cores were removed fromthe broth cultures. One core was immediately placed in 5 mlof sterile Schaedler's broth and the other was replaced in theoriginal femoral head. Thus, two bone cores were inserted intoeach femoral head: one core inoculated with S. aureus and theother core inoculated with B. subtilis. At this stage Gram stainingof each Schaedler's broth culture was performed to ensure thepresence of spore-forming organisms and the presence of S. aureus.In addition, the Bacillus species chosen produced a distinctivered floccular appearance in the broth, which was evidence ofgrowth of the organism in the broth.

The femoral head was then microwaved as described above. Afterirradiation both cores were removed from the femoral head andcultured separately in 5 ml of Schaedler's broth. The growthcontrol and sterile control were subcultured onto Columbia bloodagar (CBA) plates by using 300-µl sterile loops. All brothcultures containing the cores and the CBA plates were then incubatedaerobically at 37°C for 24 h.

Following 24 h of incubation, all 64 broth cultures were subculturedonto CBA plates by using 300-µl sterile plastic loopsto allow identification of growth. These plates were then incubatedaerobically at 37°C for 24 h, and the broth cultures werereincubated. The broth cultures producing growth (identifiedby visible turbidity of the broth) were subcultured to confirmthe presence of the relevant organism and then discarded. Allother broth cultures were reincubated for up to 10 days, withdaily inspection and subculturing of those cultures showingturbidity. The remainder of the broth cultures were terminallysubcultured at 10 days. There was evidence of contaminationin 1 of the 64 broth cultures.

RESULTS

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Results

Gram staining of the Schaedler's broth cultures after initialincubation but before irradiation identified S. aureus and aspore-forming Bacillus sp. in each of the cultures.

The results showed that no growth could be obtained in specimenssubjected to microwave irradiation for 2 min or longer. No differencewas noted for the two species of bacterium used as controls.All control (nonirradiated) specimens were positive for growthafter 24 h, as determined by the method outlined above (Table1). There was no growth in the sterile control. This suggeststhat no contaminants were present in the Schaedler's broth culturesbefore inoculation with the femoral head bone cores. The growthcontrol for each species was positive. This suggests that theinocula from the stock specimens of the two species were sufficientto produce growth under the conditions used in this experiment.

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TABLE 1. Occurrence of growth in femoral head bone cores inoculated with bacteria and subjected to microwave irradiation^a

DISCUSSION

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Discussion

The expected shortfall in allograft bone predicted by Galeaet al. (13) presents a problem for all orthopedic surgeons,not just revision hip surgeons. Allograft bone is widely used.Therefore, every effort should be expended to maximize the retrievalof femoral heads from primary total hip replacements.

Even though femoral head allograft donations clear a rigorousscreening program, up to 15% of donations are contaminated withbacteria (Frame, personal communication). This bone can stillbe processed for use. However, this processing is expensive,time-consuming, and not without potential risks to those workingwith and receiving the bone. In some centers the bone is discarded.A cheap, reliable, safe, easily used, and fast method of sterilizingallograft bone would be advantageous. Microwave irradiationcould be such a technique. The effect of microwave irradiationhas frequently been studied in the past. However, only one report(20) to the European Federation of National Associations ofOrthopaedics and Traumatology has suggested the use of microwavesto sterilize bone allografts. Microwave irradiation has beenshown to be effective in the destruction of bacteria (2, 9,11, 14, 15, 19-25), viruses (18, 21, 25), fungi (3, 8, 21),and parasites (6). To date most studies have examined the useof microwaves in the sterilization of laboratory equipment (2,15, 19, 21-25) and surgical instruments (21, 22, 24). Douglas-Joneset al. (9) looked at the sterilizing effects of microwaves onMycobacterium tuberculosis and their use for tissue fixation.All these studies showed that microwave irradiation sterilizescontaminated materials. The use of microwaves to sterilize boneallografts has not been reported in peer-reviewed journals.

Microwave irradiation has been shown to cause cell death inosteocytes, whose use in bone tumor surgery has been explored(10, 16). However, it appears that cell death occurs with exposuresto microwave irradiation of durations shorter than those requiredto destroy bacteria.

This study has shown that femoral head allografts contaminatedwith bacteria can be rendered sterile by microwave irradiation.

The choice of S. aureus and B. subtilis was deliberate. S. aureusis a common pathogen and a common contaminant of femoral headallografts (5, 17). It is also relatively easy to grow fromcontaminated materials. B. subtilis is a spore-forming organism.It is commonly used in laboratories to determine the effectivenessof the sterilizing procedures used in hospitals and industry.Spores are more difficult to destroy than vegetative bacteria.Therefore, at the power settings used in this study, short exposuresto microwaves were effective at destroying both forms of bacteriaused in the experiment.

The method by which the microwaves destroy the organisms isnot clear. The study clearly shows that short exposure to microwaveirradiation achieves sterilization of experimentally contaminatedfemoral head allografts. Microwaves act on water, which absorbsmicrowaves. This presumably leads to heating of the water inthe cytoplasm of the bacteria. This heating is presumed to killthe organism or render it incapable of cell division (7). Otherssuggest that microwaves have a direct lethal effect on the organisms(12). How the effect is produced is not clear, but it does appearto be real.

We wanted to perform the study with the same type of bone usedfor allografts. In our hospitals, this donor bone comes mainlyfrom primary total hip replacement patients. We had consideredthe use of bone from hip fracture patients requiring hemiarthroplasty.However, we believed that the quality of the bone from theseosteoporotic patients would be different and that the effectsof microwaves on bone from these types of patients might notaccurately represent the effects of microwaves on osteoarthriticbone. The bone samples used in the study were from our regionalbone bank. They had been rejected because at the 6-month reviewthe donor had indicated that he or she had a past history ofone of the conditions highlighted in Materials and Methods thatexcluded the use of that particular bone specimen. In all otherrespects the bone samples used are exactly the same as thoseused for allograft bone. The selection process for bone donationis very strict and, therefore, this type of rejected bone becomesavailable very infrequently. We therefore had to wait a considerabletime to obtain the specimens that we used in this study. Weaccept that larger numbers of samples must be examined at eachtime interval to allow better investigation of the phenomenonthat we have described in this report. However, the presentstudy gives a clear indication that this intervention couldprove to be useful for bone graft procurement in the future.

Further studies need to be performed with larger numbers ofbone specimens to determine if the phenomenon that we observedis real. The biomechanical effects of microwave irradiationon bone were not addressed in this study. From the results ofexperiments with rat femurs, Liebergall et al. (16) have suggestedthat microwave irradiation of bones has minimal biomechanicaleffect. Whether this effect is consistent for larger human bonesis not known but will be the subject of future experimentation.

The process of microwave sterilization of bone allografts ischeap and easy to use and appears to be effective for sterilizationof contaminated femoral head allografts. If these preliminarydata are confirmed on a larger scale, the use of microwave irradiationin this way could help to reduce some of the shortfalls in allograftstocks predicted by Galea et al. (13).

ACKNOWLEDGMENTS

We thank the Bone Bank at the Blood Transfusion Laboratory ofLaw Hospital for supplying the femoral head allografts. We alsothank the staff of the Bacteriology Department at Southern GeneralHospital, Glasgow, Scotland, for advice on the microbiologicalaspects of the project. We thank D. Wyper of the Research andDevelopment Department at Southern General Hospital for adviceand funding for the project.

FOOTNOTES

* Corresponding author. Present address: Department of Orthopaedics, St. James' University Hospital, Beckett St., Leeds LS9 7TF, United Kingdom. Phone: 0113 206 4732. Fax: 0113 206 5156. E-mail: Robert.dunsmuir{at}leedsth.nhs.uk.

REFERENCES

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