Ethylene oxide gas sterilization: a simple technique for storing explanted skull bone. Technical note

The authors evaluated the effectiveness of a simple technique using ethylene oxide (EtO) gas sterilization and room temperature storage of autologous bone grafts for reconstructive cranioplasty following decompressive craniectomy. The authors retrospectively analyzed data in 103 consecutive patients who underwent cranioplasty following decompressive craniectomy for any cause at the University of Illinois at Chicago between 1999 and 2005. Patients with a pre-existing intracranial infection prior to craniectomy or lost to follow-up before reconstruction were excluded. Autologous bone grafts were cleansed of soft tissue, hermetically sealed in sterilization pouches for EtO gas sterilization, and stored at room temperature until reconstructive cranioplasty was performed. Cranioplasties were performed an average of 4 months after decompressive craniectomy, and the follow-up after reconstruction averaged 14 months. Excellent aesthetic and functional results after single-stage reconstruction were achieved in 95 patients (92.2%) as confirmed on computed tomography. An infection of the bone flap occurred in eight patients (7.8%), and the skull defects were eventually reconstructed using polymethylmethacrylate with satisfactory results. The mean preservation interval was 3.8 months in patients with uninfected flaps and 6.4 months in those with infected flaps (p = 0.02). A preservation time beyond 10 months was associated with a significantly increased risk of flap infection postcranioplasty (odds ratio [OR] 10.8, p = 0.02). Additionally, patients who had undergone multiple craniotomies demonstrated a trend toward increased infection rates (OR 3.0, p = 0.13). Data in this analysis support the effectiveness of this method, which can be performed at any institution that provides EtO gas sterilization services. The findings also suggest that bone flaps preserved beyond 10 months using this technique should be discarded or resterilized prior to reconstruction.     

Ethylene oxide sterilization of autologous bone flaps following decompressive craniectomy

Summary ¶Introduction. In patients undergoing decompressive craniectomy, the bone flap is temporarily preserved either in the subcutaneous tissue of the patient or frozen. However, there are some drawbacks related to these methods. Material and methods. In 16 patients in whom the bone flap was removed for decompressive craniectomy, the bone was firstly washed in hydrogen peroxide and then placed in hermetically-sealed bags and sterilized using ethylene oxide. The bone was repositioned after an average period of 4.3 months. Results. One patient sustained an infection of the surgical wound which required permanent exclusion of the bone flap. In all the others, esthetic and functional results were good after an average follow-up of 20 months. Control CT-scan of the bone flap demonstrated preservation of its structural features with fusion of the bone margins and revitalization of the flap. On MRI a subdural space was again visible. Conclusions. Sterilization of the bone flap with ethylene oxide in patients undergoing decompressive craniectomy avoids some of the drawbacks related to the techniques currently used. The easiness, low cost, good aesthetic and functional results of this procedure make it a valid alternative to other techniques for preservation of autologous bone in decompressive craniectomies.Published online September 26, 2003     

Effects of preservation and sterilization on cortical bone grafts

The effects of preservation and sterilization on the structural properties of cortical bone were investigated. Specimens of cortical bone from rat tibiae were frozen (–70°C for 28 days), freeze-dried, irradiated (1, 5, 25 and 50 kGy) or autoclaved (at 134°C for 3 or 5 min), and examined by scanning electron microscopy. Cryopreservation and irradiation had no deleterious effects on the surface structure of the cortical bone. Freeze-drying caused microcracks running parallel to the mineralized fiber bundles. After autoclaving, a time-dependent distension, swelling and amalgamation of the fibrillary matrix was observed. This denaturation of the organic matrix was more pronounced after 5 min than 3 min autoclaving. The alterations of the fibrillary structure described above might be due to a preservation- and sterilization-induced decrease of the biological and biomechanical potential of bone grafts.Supported by DFG grant     

Ethylene oxide does not extinguish the osteoinductive capacity of demineralized bone: A reappraisal in rats

We examined the influence of ethylene oxide (EO) and gamma irradiation on the osteoinductive capacity of demineralized bone. Demineralized bone powder prepared from Wistar rats was exposed to EO (55 °C or 40 °C) or gamma irradiation (25 KGy) or was preserved in ethanol. Sterilely-prepared bones served as controls. The powder was packed in a gelatin capsule and implanted for 6 weeks in muscles of 6-week-old female rats. Exposure of demineralized bone particles to EO 55 °C resulted in an almost complete loss of osteoinductivity. Irradiated bones lost about 40% of their osteoinductive capacity, while sterilization with EO at 40 °C resulted in only a slight alteration of the osteoinductivity, as assessed by the recovered weight ratio, calcium content, alkaline phosphatase activity measurements and histo-morphometry. Ethanol treatment had no influence on the new bone yield when compared to controls.

As EO exposure at 40 °C is a true sterilization procedure, it can be recommended in a clinical setting for its small effect on osteoinductive capacity as assessed experimentally in rats.     

Influence of ethylene oxide sterilization on the activity of native reindeer bone morphogenetic protein

We studied the effects of ethylene oxide sterilization (Steri-Vac 4XL, temperature 29°C, exposure time 4 h 10 min, ethylene oxide concentration 860 mg/l) on the osteoinductivity of partially purified native reindeer bone morphogenetic protein (BMP) in a hind leg muscle pouch model of male NMRI mice. BMP was administered in implants containing 3 mg in a collagen carrier. Implants without sterilization and without BMP served as controls. New bone formation was evaluated based on the calcium yield, radiographic and histological examination 3 weeks after implantation. The implants without BMP were not able to induce new bone visible in radiographs. In the sterilized BMP group, the mean area of new bone was 35% (p=0.004) and density 32% (p=0.000) smaller than in the nonsterilized group. Calcium yield was 20% lower in the sterilized group than in the nonsterilized group, but this difference was not significant (p=0.22). It was many times lower in the group without BMP than in the above-mentioned groups (p=0,001). We conclude that ethylene oxide gas sterilization reduces the bone-forming activity of native reindeer BMP by one third.

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Résumé Nous avons étudié les effets de la stérilisation à loxyde déthylène (Steri-Vac 4XL, température 29ºC, temps dexposition 4 h, concentration de loxyde déthylène 860 mg/l) sur losteoinductivité de la proteine morphogénétique osseuse (BMP) du renne partiellement purifié dans un modèle fait par une bourse du muscle de la patte postérieure de la souris NMRI virile. La BMP a été administré par des implants qui contiennent 3 mg dans un porteur de collagène. Des implants sans stérilisation et sans BMP ont servi de contrôle. La néo- formation osseuse a été évaluée sur la formation de calcium, radiographiquement et histologiquement trois semaines après implantation. Les implants sans BMP nétaient pas capables dinduire un nouvel os visible sur les radiographies. Avec la BMP stérilisé la surface moyenne dos nouveau était 35% (p=0.004) et la densité 32% (p=0.000) plus petite que dans le groupe BMP non—stérilisé. La production de calcium était 20% inférieure dans le groupe BMP stérilisé que dans le groupe BMP non—stérilisé, mais cette différence nétait pas significative (p=0.22). Elle était plusieurs fois plus faible dans le groupe sans BMP que dans les groupes susmentionnés (p=0,001). Nous concluons que la stérilisation à loxyde déthylène réduit dun tiers lactivité ostéoformatrice de la BMP de renne.

     

Incorporation of nonviable bone grafts: Autoclaved autogeneic and frozen allogeneic bone grafts compared in the rabbit

In 14 adult rabbits the middle third of the ulna was resected bilaterally followed by reimplantation of resected bone after autoclaving on one side and transplantation of allogeneic bone on the other. In 7 animals the bilateral implants were supplemented with allogeneic bone matrix. The reconstructions were studied in vivo by serial radiography, scintigraphy, and bone mineral determination. The animals were killed at 16 weeks, and the ulnar reconstructions further studied by high resolution radiography, ⁴⁵Ca autoradiography, and histology. In both types of nonsupplemented reconstructions, new bone formation was poor; nonunion occurred in three out of seven autoclaved reimplants and in five out of seven allogeneic transplants. Supplemented with allogeneic bone matrix, both types of reconstructions exhibited abundant new bone formation and complete incorporation of all implants.

Enhancement of new bone formation is probably more important than the type of nonviable bone graft chosen for reconstruction of large skeletal defects.     

Allogeneic grafts for bone tumor: 21 cases of osteoarticular and segmental grafts

We treated 21 aggressive and malignant bone tumors by wide resection and replacement with deep-frozen osteoarticular and segmental (intercalary and block) allografts. Radiologic and histologic studies showed a gradual accretion of new bone on the graft trabeculae, sometimes with total creeping substitution. Substantial resorption of grafted condylar bone occurred in 3 of 14 cases. One of them ended with arthrodesis; in the other 2 the result after augmentation autografts was fair. Radiographically, a gradual joint surface destruction was observed in all the osteoarticular grafts after 5 years, not correlating with joint function, however. Biopsies showed some cartilage regeneration. Each patient underwent, on an average, two operations. Function after osteoarticular grafts at 3-16 years was excellent in 1 case, good in 4, fair in 6, and poor in 1 case; 2 cases were too recent for evaluation. Function 3-12 years after segmental grafts was excellent in 3 cases and poor in 3 cases (1 amputation due to nonunion, 1 amputation due to recurrence, and 1 prosthetic replacement due to recurrence); 1 case was too recent for evaluation. We conclude that an allograft is an acceptable alternative in the reconstruction of large tumor defects. However, it still presents unsolved immunologic and preservation problems, which make the prognosis guarded.     

Gamma irradiation and ethylene oxide in the sterilization of native reindeer bone morphogenetic protein extract.

BACKGROUND AND AIMS: For human use, it is necessary to sterilize bone morphogenetic proteins (BMPs), in order to reduce the risk of infections and associated complications. We compared the effects of ethylene oxide and gamma irradiation in the sterilization of native reindeer BMP extract with regard to bone induction in the Balb/C mouse thigh muscle pouch model. MATERIALS AND METHODS: BMP extract, sterilized with ethylene oxide gas (Steri-Vac 4XL, temperature 29 degrees C, exposure time 4 h, ethylene oxide concentration 860 mg/l), or gamma irradiation at doses of 3.15 MRad was administered in implants containing 5 or 10 mg of BMP extract with collagen carrier. Non-sterilized collagen implants served as controls. New bone formation was evaluated based on the incorporation of Ca45 and radiographically three weeks after implantation. RESULTS: The collagen was not able to induce new bone visible in radiographs. The mean Ca45 incorporation in the gamma sterilized group containing 5 mg of BMP extract was 30% (p = 0.04) and that containing 10 mg of BMP extract was 60% (p = 0.02) higher than seen in the corresponding ethylene oxide sterilized groups. The mean new bone areas were 45% higher in the gamma sterilized groups than in the corresponding ethylene oxide sterilized groups, but the differences were not significant. The mean optical density of new bone in the gamma sterilized group containing 5 mg of BMP extract was 75% (p = 0.00) and in that containing 10 mg of BMP extract was 70% (p = 0.00) higher than seen in the corresponding ethylene oxide sterilized groups. CONCLUSION: Native reindeer BMP extract is more sensitive to the effects of ethylene oxide gas sterilization than gamma irradiation. These results suggest that gamma irradiation is recommendable for the sterilization of BMP extracts.     

Autogenous bone grafts: nonvascular and vascular.

Nonvascularized autogenous bone grafts have been used for at least 150 years. Vascularized bone grafts were first done only 17 years ago. We now know that bone grafts act through osteoconductive and osteoinductive mechanisms to produce new bone. Autogenous bone grafts remain the gold standard against which allografts and artificial graft materials should be measured.     

Extracorporeal irradiation and incorporation of bone grafts: Autogeneic cortical grafts studied in rats

The incorporation of resected, extracorporeally irradiated (1, 5, 25 and 50 kGy) and orthotopically reim-planted autogeneic cortical bone was investigated in 116 adult Wistar rats. 7 mm-long diaphyseal segments of the tibia were resected, irradiated and re-implanted using K-wire osteosynthesis. Autogeneic fresh grafts served as controls. Graft healing was evaluated by radiography and histomorphometric study at 3, 6, 9, and 12 weeks. At 3 weeks, two thirds of the 50 kGy irradiated grafts were fractured and therefore the series with this dose was interrupted because of mechanical graft insufficiency. After 3 and 6 weeks there were no statistically significant differences among the control group and 1 or 5 kGy irradiated grafts. The healing of 25 kGy irradiated grafts was delayed from the sixth week onwards and continued until the end of the experiment at 12 weeks (50% reduction of incorporation). The incorporation of 1 and 5 kGy irradiated grafts showed a 16% (1 kGy) to 24% (5 kGy) delay at 12 weeks, compared to autogeneic fresh grafts. 1 and 5 kGy irradiated autogeneic bone grafts retain most of their biological potential. Resection, extracorporeal irradiation and reimplantation of bone tumors may therefore be a possible alternative to allografting.     

Neovascularized bone grafts: experimental investigation.

Vascularized bone grafts are standardized procedures in reconstructive surgery but there are some disadvantages: donor site morbidity, limited number of "natural" donor sites, and complex technique. In this study, we test the possibility of creating a "neovascularized" bone graft utilizing a vascular implantation procedure in a rabbit model. Sixteen New Zealand adult white rabbits were used. In each animal, two iliac crest bone grafts (7 x 7 x 10 mm) were harvested. Vascular implantation of the right superficial femoral vessels was performed in one of the two grafts, which was wrapped in a silicone envelope to avoid neovascularization from the surrounding tissues and positioned in a subcutaneous pocket in the right medial thigh. On the left side, the bone block, wrapped in the silicone envelope, was buried subcutaneously without vascular implantation. The operated animals were divided into two groups: Group I included eight rabbits explanted 4 weeks postoperatively and Group II included eight rabbits explanted 8 weeks postoperatively. Tetracycline injection was performed 72 hours preexplantation to evaluate new bone formation. Selective colloidal ink injection in the axial artery was performed to investigate the neovascularization before inclusion in poly-methyl-methacrylate (PMMA). Histological examination was performed in all explanted specimens comparatively. Histological examination 8 weeks after surgery showed a marked neovascularization, with normal bone cells. Tetracycline labeling showed new bone formation with a normal pattern. In all nonvascularized specimens, no viable cells or neovascularization and no bone formation were found. The vascular implantation procedure can induce a good neovascularization with new bone formation in a small bone graft. The possibility of neovascularization induction by the simple vascular implantation procedure has several clinical implications in reconstructive surgery.     

Contaminated anterior cruciate ligament grafts: the efficacy of 3 sterilization agents.

PURPOSE: A study was undertaken to determine the incidence of positive cultures resulting from an anterior cruciate ligament (ACL) specimen dropped on the operating room floor and the efficacy of sterilizing the specimen by soaking in 1 of 3 antimicrobial solutions: an antibiotic solution of neomycin and polymyxin B, 10% providone-iodine solution, and standard chlorhexidine gluconate solution. TYPE OF STUDY: Randomized trial. MATERIALS AND METHODS: Fifty ACL specimens removed from patients undergoing total knee arthroplasty were used as the test group. The specimens were longitudinally sectioned into 4 equal pieces. The 4 pieces were dropped on the floor and left for a period of 15 seconds. Cultures were taken from each specimen after immersion in 1 of the 3 sterilization solutions for a period of 90 seconds. One of the 4 specimens was cultured without being exposed to any solution, thereby establishing these specimens as the control group. Cultures of a floor swab were taken at the same time and place that the ACL was dropped. RESULTS: The floor swab cultures were positive in 48 of the 50 specimens (96%). The ACL control group (untreated dropped grafts) had 29 of 50 specimens positive (58%). The grafts soaked in antibiotic solution had 3 of 50 specimens positive (6%). The grafts soaked in providone-iodine solution had 12 of 50 specimens positive (24%). The grafts soaked in chlorhexidine gluconate solution had 1 of 50 specimens positive (in broth only) (2%). CONCLUSION: This study shows that significant contamination occurs when dropping specimens on the floor, as 58% of the dropped grafts had positive cultures. Of the 3 sterilization techniques used, chlorhexidine gluconate seems to be the most efficient with only a single broth culture (2%) found to be positive. The antibiotic solution was second best (6%), although there is no statistically significant difference between these 2 groups. The 10% providone-iodine solution under these test conditions was the least effective of all the 3 sterilization agents with 24% cultures positive after immersion.