Sterilization of HIV with irradiation: relevance to infected bone allografts.

BACKGROUND: Bone allograft banks commonly sterilize frozen bone by irradiation. The dose-response relationship for HIV is calculated and the dose required to inactivate the bioburden of virus that may be present in allograft bone is determined. METHODS: A virus titre experiment is performed using irradiated frozen HIV. The virus is maintained on dry ice (approximately -70 degrees C) and is exposed to a cobalt 60 source with 0-40 kGy irradiation at 5 kGy intervals. Lymphocyte cell cultures are exposed to serial dilutions of the irradiated virus. The virus titre is quantified by cytological changes of HIV infection and p24 immunofluorescence. RESULTS: There is a linear relationship between the virus titre and the radiation dose delivered. The inactivation rate of irradiated virus was 0.1134 log10 tissue culture infective doses 50/mL per kGy (95% confidence intervals, 0.1248-0.1020). The irradiation dose required to inactivate the HIV bioburden in allograft bone is 35 kGy. The irradiation dose required to achieve a sterility assurance level of 10(-6) is 89 kGy. This dose exceeds current recommendations for sterilizing medical products and the current practice of many bone banks. CONCLUSIONS: It is concluded that gamma irradiation should be disregarded as a significant virus inactivation method for bone allografts.     

"Amnion Bank"--the use of long term glycerol preserved amniotic membranes in the management of superficial and superficial partial thickness burns

With about 2 million cases of burns in India of which about 90% are superficial and superficial partial thickness burns, burn injuries present a major challenge to the scarce medical resources available. The main requirement in treating these burns is an economical, easy to apply, readily available dressing that will provide good pain relief, protect the wound from infection, promote healing, prevent heat and fluid loss, be elastic and non-antigenic and adhere well to the wound. It was in the quest of such an ideal dressing that the Burn Centre of the Army Hospital (Research and Referral) New Delhi, India has been using human amniotic membranes preserved long term in 85% glycerol for treating these wounds. The results of this quest have resulted in a protocol where these long term glycerol preserved membranes (some of which have now been preserved for about 2.5 years) are used for treating superficial and superficial thickness burns presenting at our center. This paper shows the excellent results obtained with the use of this extremely economical dressing and emphasizes the importance of establishing such "Amnion Banks" in all hospitals especially in developing countries.     

STERILIZATION OF HIV WITH IRRADIATION: RELEVANCE TO INFECTED BONE ALLOGRAFTS

Background : Bone allograft banks commonly sterilize frozen bone by irradiation. The dose–response relationship for HIV is calculated and the dose required to inactivate the bioburden of virus that may be present in allograft bone is determined. Methods : A virus titre experiment is performed using irradiated frozen HIV. The virus is maintained on dry ice (approximately –70°C) and is exposed to a cobalt 60 source with 0–40 kGy irradiation at 5 kGy intervals. Lymphocyte cell cultures are exposed to serial dilutions of the irradiated virus. The virus titre is quantified by cytological changes of HIV infection and p24 immunofluorescence. Results : There is a linear relationship between the virus titre and the radiation dose delivered. The inactivation rate of irradiated virus was 0.1134 log₁₀ tissue culture infective doses ₅₀/mL per kGy (95% confidence intervals, 0.1248–0.1020). The irradiation dose required to inactivate the HIV bioburden in allograft bone is 35 kGy. The irradiation dose required to achieve a sterility assurance level of 10^–6 is 89 kGy. This dose exceeds current recommendations for sterilizing medical products and the current practice of many bone banks. Conclusions : It is concluded that gamma irradiation should be disregarded as a significant virus inactivation method for bone allografts.     

Validation of 11 kGy as a Radiation Sterilization Dose for Frozen Bone Allografts

A radiation sterilization dose (RSD) of 25 kGy is deleterious to bone allografts. This study aimed to establish a lower RSD for bone allografts using method 1 of International Standard Organisation 11137.2:2006. This provides a database to select an RSD corresponding to an allograft's bioburden, given that the bioburden's gamma resistance is equal to or less than the standard. This can be verified by irradiating 100 allografts at a dose selected to provide a sterility assurance level of 10⁻². The bioburden of our allografts was 0, which prescribed a verification dose of 1.3 kGy. After irradiating 100 allografts, sterility tests returned no positive cultures. We therefore validated an RSD of 11 kGy for allografts with that bioburden. According to the standard, this RSD provides a sterility assurance level of 10⁻⁶ for bone allografts.     

The use of silver nitrate-incorporated amniotic membrane as a temporary dressing

Human amniotic membrane is one of the most effective biological dressings used in burn treatment. Since 1978, we have been using human amniotic membrane incorporating 0.5 per cent silver nitrate. Studies in 96 patients using this method showed that the silver-incorporated membranes gave a better therapeutic effect than plain amniotic membranes. It was noted that 0.5 per cent silver nitrate incorporated into the membranes increased their manageability, provided easier application to the burned area and created a bactericidal effect, therefore reducing the risk of contamination and infection. One of the main advantages of wound coverage with amniotic membrane is that it does not appear to discourage re-epithelization, reduces fluid, protein, heat and energy loss, increases mobility and most important this may be the ideal wound cover next to the patient's own skin. Therefore, we highly recommend the use of 0.5 per cent silver nitrate-incorporated amniotic membrane, since it is readily available and freely obtainable, has low preparation and storage costs that make it an ideal dressing to use, especially in countries where economic factors prevent the purchase of other types of dressings.     

Amniotic membrane as a biological dressing in the management of burns

This report details observations in 90 patients with dermal depth burns treated using amniotic membrane. The patients were divided into three subgroups: superficial dermal, intermediate dermal and deep dermal burns diagnosed clinically. All patients were dressed with amniotic membrane which was changed daily. The amniotic membrane relieved the discomfort of dressing changes, postoperative pain and oozing and allowed rapid epithelialization and early healing in superficial and intermediate depth dermal burns. In deep dermal burns the membrane was dissolved because of slough in the burn wound. After removal of the slough the amniotic membrane helped in rapid regeneration of epithelium and early healing.     

Fracture resistance of gamma radiation sterilized cortical bone allografts.

Gamma radiation is widely used for sterilization of human cortical bone allografts. Previous studies have reported that cortical bone becomes brittle due to gamma radiation sterilization. This embrittlement raises concern about the performance of a radiation sterilized allograft in the presence of a stress concentration that might be surgically introduced or biologically induced. The purpose of this study was to investigate the effect of gamma radiation sterilization on the fracture resistance of human femoral cortical bone in the presence of a stress concentration. Fracture toughness tests of specimens sterilized at a dose of 27.5 kGy and control specimens were conducted transverse and longitudinal to the osteonal orientation of the bone tissue. The formation of damage was monitored with acoustic emission (AE) during testing and was histologically observed following testing. There was a significant decrease in fracture toughness due to irradiation in both crack growth directions. The work-to-fracture was also significantly reduced. It was observed that the ability of bone tissue to undergo damage in the form of microcracks and diffuse damage was significantly impaired due to radiation sterilization as evidenced by decreased AE activity and histological observations. The results of this study suggest that, for cortical bone irradiated at 27.5 kGy, it is easier to initiate and propagate a macrocrack from a stress concentration due to the inhibition of damage formation at and near the crack tip.     

The effect of gamma radiation sterilization on the fatigue crack propagation resistance of human cortical bone

BACKGROUND: Clinical evidence has suggested that the rate of fracture in allografts sterilized with gamma radiation may be higher than that in controls. Gamma radiation sterilization has been shown to affect the post-yield properties of bone but not the elastic modulus. Since most allograft fractures occur with subcritical loads during activities of daily living, it may be that the fatigue properties of irradiated allografts are diminished. In this study, the fatigue crack propagation behavior of cortical bone sterilized with gamma radiation was compared with that of gender and age-matched controls. We hypothesized that gamma radiation significantly reduces the resistance of cortical bone to fatigue crack growth. METHODS: Specimens for fatigue crack propagation testing were machined from four pairs of fresh-frozen human femora obtained from four individuals (a younger male, younger female, older male, and older female donor). Half of the specimens were sterilized with 31.7 kGy of gamma radiation. The specimens were cyclically loaded to failure in a servohydraulic testing system, and crack growth was monitored. The cyclic stress intensity factor and the fatigue crack growth rate were calculated to examine the kinetics of fatigue crack growth. Following testing, the damage zone around the fracture plane was analyzed histologically. RESULTS: The morphology and kinetics of crack growth in irradiated specimens differed from the control data. Overall, the irradiated bone was significantly less resistant to fatigue crack growth than was control tissue (p < 0.05). There was less microdamage associated with fracture in the irradiated specimens than in the control specimens, with the exception of the bone from the older female donor. CONCLUSIONS: Gamma radiation sterilization significantly reduces the fatigue crack propagation resistance of cortical bone. Irradiated specimens also demonstrate a smaller amount of microdamage along the fracture plane. These findings may be due to ultrastructural alterations in the collagen matrix caused by radiation. CLINICAL RELEVANCE: This study suggests that, despite having pre-yield mechanical properties that are similar to those of nonirradiated bone, gamma-radiation-sterilized allograft may be more predisposed to fracture even under the subcritical loads that occur during the activities of daily living.     

In vitro and in vivo investigation of a novel amniotic‐based chitosan dressing for wound healing

It is more than a decade that amniotic membrane has been used as a wound dressing because of its anti‐inflammatory, anti‐microbial, anti‐fibrotic, anti‐scarring properties, as well as its pain relieving and epithelialization promoting features. However, amniotic membrane had limited applications because it needs to suture in surgery, is highly fragile, firmly adhere to the wound and may cause bleeding and pain when changing the bandage. This study investigated the possibility of development of a novel amniotic‐based chitosan gel dressing as a potential wound repair substrate with marked efficacy. In this experiment, amniotic gel prepared based on chitosan/PVP gel containing human amniotic membrane extract (AME‐Gel) was investigated in terms of wound‐healing efficacy and scar preventive effects in a rat burn model. The levels of re‐epithelialization and dermal regeneration were examined by histological assessment using H&E and Masson's trichrome staining. Also, we clarified the mechanism of healing and cytokine‐releasing activities of AME as well as its effect on epithelization, angiogenesis, and fibroblast growth and migration. Our results revealed that AME‐Gel induces epidermal and dermal regeneration at a shorter time through formation of granulation tissue, enhancement of fibroblast proliferation, and improvement of blood capillary formation concomitant with developing collagen bundles. Therefore, AME‐Gel could be considered a simple and easy to be used as a biological dressing for any type of superficial burn wounds, without any adverse effects.     

辐照中脱矿骨基质粒径对胶原结构影响的实验研究

目的探讨不同粒径大小对γ辐照中脱钙骨基质(demineralized bone matrix,DBM)中胶原结构的影响以及辐照保护剂的有效性。方法取同一供体的冻干皮质骨,依据Urist改良法制备不同粒径的(0.5~1.0 mm、1.2~2.8 mm、3.3~4.7 mm及5.7~7.0 mm)DBM样品,按照不同剂量分为:0 kGy、15 kGy、25 kGy及25 kGy(辐照保护剂),真空密封后储存于-80℃冰箱待用。通过扫描电镜观察胶原表面形态,大体观察胶原表面结构损伤的程度;将样品按照0.2 g/ml生理盐水比例在50℃条件下72 h,利用浸提液颜色深度观察胶原被辐照损伤的程度;使用2,4-二硝基苯肼(吸光光度计法)测定样品中羰基含量;十二烷基硫酸钠聚丙烯酰胺钠凝胶电泳法(Sodium dodecyl sulfatepolyacrylamide gel electrophoresis,SDS-PAGE)测定样品中胶原分子量的变化;利用差示热量扫描法(differential scanning calorimetry,DSC)检测样品热变性温度以观察胶原热稳定性。结果样品浸提液颜色与γ辐照剂量相关度较高,未辐照样品浸提液颜色清亮,而在同粒径下随辐照剂量加大浸提液黄色逐渐加深,5.7~7.0 mm粒径组颜色相对较浅;25 kGy组相比于25 kGy+保护剂组浸提液颜色加深。扫描电镜观察到γ辐照导致胶原结构紊乱,纤维断裂,随着辐照剂量增大损伤区域增多,当粒径增大时,损伤区域有减少的趋势;相比于25 kGy组,25 kGy+保护剂组胶原结构性破坏减少。差示热量扫描法得出样品热交换曲线,随着粒径增大,热变性温度有增高的趋势,粒径间对比有统计学差异(F=189.4,P<0.001);同粒径间差异不明显。SDS-PAGE发现同粒径下γ辐照剂量愈大,胶原分子量愈小;同辐照条件下随粒径较小,高分子量胶原含量减少明显;225 kGy+保护剂组相比于25 kGy组,高分子量增多。羰基含量结果显示在同一粒径下,γ辐照使羰基含量增多,0.5~1.0 mm组(F=13.631,P=0.002),1.2~2.8 mm组(F=6.390,P=0.016),3.3~4.7 mm组(F=5.630,P=0.023),5.7~7.0 mm组(F=4.150,P=0.048)的差异均有统计学意义,不同粒径间随着粒径增大羰基含量逐渐减小但差异统计学意义(F=0.560,P=0.650)。结论γ辐照与胶原的氧化损伤具有明显的剂量反应关系,随着γ辐照剂量的增加,胶原损伤程度逐渐增加;DBM的粒径大小影响着胶原对γ辐照的敏感度,随着粒径的减小,DBM颗粒更易被γ辐照损伤;辐照保护剂在辐照过程中对胶原有一定程度的保护作用。     

Benefits of cryopreserved human amniotic membranes in association with conventional treatments in the management of full‐thickness burns

The use of split‐thickness skin autografts (STSA) with dermal substitutes is the gold standard treatment for third‐degree burn patients. In this article, we tested whether cryopreserved amniotic membranes could be beneficial to the current treatments for full‐thickness burns. Swines were subjected to standardised full‐thickness burn injuries, and then were randomly assigned to treatments: (a) STSA alone; (b) STSA associated with the dermal substitute, Matriderm; (c) STSA plus human amniotic membrane (HAM); and (d) STSA associated with Matriderm plus HAM. Clinical and histological assessments were performed over time. We also reported the clinical use of HAM in one patient. The addition of HAM to classic treatments reduced scar contraction. In the presence of HAM, skin wound healing displayed high elasticity and histological examination showed a dense network of long elastic fibres. The presence of HAM increased dermal neovascularization, but no effect was observed on the recruitment of inflammatory cells to the wound. Moreover, the use of HAM with classical treatments in one human patient revealed a clear benefit in terms of elasticity. These results give initial evidence to consider the clinical application of HAM to avoid post‐burn contractures and therefore facilitate functional recovery after deep burn injury.     

028 Clinical Trials of Allogenic Cultured Dermal Substitute for the Treatment of Burn Wounds

Aim: Recently, various types of cultured skin substitutes have been developed and some of them are used clinically. This study was designed to evaluate the efficacy of allogenic cultured dermal substitute which was applied to burn injuries in clinical trials such as deep dermal burns and dermal burns. Methods: Allogenic cultured dermal substitute (CDS) was simply applied to the burn wound, over which covering materials were applied to protect CDS. Results: The application of CDS to deep dermal burns was proved to facilitate healthy granulation tissue formation at early stage and epithelialization from the outer margins. When CDS applied to the debrided wound surface of dermal burns, an excellent wound bed was generated which was suitable for the graft take of an autologous patch. Conclusion: CDS provides an excellent epithelialization and granulation for burn wounds.     

Clinical practice of glycerol preserved allograft skin coverage

This retrospective study examines the use and advantages/disadvantages of glycerol preserved human allograft skin in our burn care facility between February 1997 and December 1999. Three hundred and twenty patients were included into the study, 85 of whom were treated with human cadaver skin. The usage of allograft slightly increased the number of operative procedures per percent of the total body surface area burn. There were no adverse effects noted from the use of allograft. The group of patients with allograft use had a significantly larger burn size, ABSI score and length of ICU stay. Demographically the groups were comparable. The considerably easier handling and storage of glycerol preserved allograft skin make it preferable to cryopreserved allograft skin in all indications where it is used as a temporary wound closure. We recommend the usage of cryopreserved skin in cases where the integration of a dermal component as a permanent part of wound closure is desired.     

Acceleration of wound healing by a live yeast cell derivative

Acceleration of the normal rate of burn wound healing would serve to decrease the morbidity and possibly the mortality of burn victims. A live yeast cell derivative (LYCD) has previously been reported to stimulate wound epithelialization and this study was designed to evaluate that hypothesis. Twenty-six human skin graft donor sites in nine patients were compared in a double-blind, randomized, single-center inpatient study. Thin donor sites were used as a model for superficial wound healing. Statistically significant earlier angiogenesis and epithelialization occurred in donor sites treated with LYCD ointment as compared with donor sites in the same patients treated simultaneously with ointment base. Stinging pain was noted by seven patients, but in all cases the pain was mild and required no analgesia.     

Cryopreserved cultured epidermal allografts achieved early closure of wounds and reduced scar formation in deep partial-thickness burn wounds (DDB) and split-thickness skin donor sites of pediatric patients

Burn treatment in children is associated with several difficulties, e.g. available skin replacement is small, donor area could expand, and subsequent hypertrophic scar and contracture could become larger along with their physical growth. In order to have better clinical results, the authors prepared cryopreserved cultured epidermal allografts from excess epidermal cells of other patients, and applied the epidermal allografts to 55 children, i.e. 43 cases of deep partial-thickness burn wounds (DDB) due to scald burn and 12 cases with split-thickness skin donor sites. In the 43 DDB patients, epithelialization was confirmed 9.1+/-3.6 days (mean+/-S.D.) after treatment. In 10 of the 43 patients, epithelialization was comparable between the area which received the epidermal allografts (grafted area) and the area which did not receive the epidermal allografts but was covered with usual wound dressing (non-grafted area). As a result, epithelialization day was 7.9+/-1.7 in grafted areas and 20.5+/-2.3 in non-grafted areas. In the 12 patients with split-thickness skin donor sites, epithelialization was confirmed 6.3+/-0.9 days after treatment. Epithelialization of the grafted and non-grafted areas was comparable in 8 of the 12 patients, and it was 6.5+/-1.1 days and 14.1+/-1.6 days, respectively. In these 10 DDB patients and 8 split-thickness skin donor site patients, redness and scar formation were also milder in the grafted area. The 55 patients have been followed up for 1-8 years (mean, 4.75 years), and scar formation was suppressed in both DDB and split-thickness skin donor sites. These findings showed that cryopreserved cultured epidermal allografts achieve early closure of the wounds and good functional outcomes.     

Uses and abuses of a biosynthetic dressing for partial skin thickness burns

The following report reviews 851 applications of Biobrane on partial skin thickness burn wounds awaiting epithelialization. After the patients had been evaluated and resuscitated as needed, the burn wounds were cleansed and debrided. Those evaluated as shallow were treated with Biobrane application. Joint surfaces were splinted for immobilization. The wound was inspected at 24 and 48 h and if any fluid had accumulated it was aspirated and the wound was redressed. When the Biobrane was adherent, the wound was covered with a light dressing and joint immobilization was discontinued. Treatment with Biobrane dressing provided certain advantages over other topical wound care. As the dressing changes were performed less frequently outpatient care was possible, with a resultant decrease in both the length of hospital stay and the ultimate cost of burn care. Wound desiccation is prevented and pain is decreased. Accurate diagnosis of wound depth is crucial if Biobrane is to be used. Very deep wounds will not allow Biobrane adherence, neither will it occur if the wound has a high bacterial count. If joint surfaces are not splinted, the Biobrane will shear and not adhere to the wound. Convex and concave surfaces can be treated with Biobrane, which may need to be meshed.     

Characterization of a highly cross-linked ultrahigh molecular-weight polyethylene in clinical use in total hip arthroplasty

This article reports on a commercially available extensively cross-linked ultrahigh molecular-weight polyethylene (HXPE) produced by subjecting molded GUR 1050 ultrahigh molecular-weight polyethylene (UHMWPE) to 100 ± 10 kGy of electron beam radiation followed by melt annealing and sterilization by gas plasma. When compared to contemporary conventional molded GUR 1050 UHMWPE sterilized by 37 kGy of gamma radiation, the HXPE material has enhanced wear properties, has no detectable free radicals, and is resistant to oxidation and oxidative-related material property changes. The relative wear improvement of the HXPE is maintained in the presence of bone cement or alumina particles. The HXPE produced greater than 90% fewer wear particles in all size ranges and statistically significantly (P < .0001) smaller average-size particles than did the conventional UHMWPE.     

Chitin membrane for wound dressing application – preparation,characterisation and toxicological evaluation

Chitin, a unique biopolymer based on the N‐acetyl‐glucosamine monomer is envisioned to promote rapid dermal regeneration and accelerate wound healing. It has many useful and advantageous biological properties for its application as a wound dressing. Chitin membranes were prepared using lithium chloride/dimethylacetamide solvent system and evaluated for use as a wound dressing. Swelling behaviour, moisture vapour transmission rate, microbial impermeability and antimicrobial efficacy of the dressings was evaluated. The chitin dressing provided an effective barrier to microbial penetration and exerted a broad bacteriostatic action against Gram‐positive and Gram‐negative organisms. Gamma irradiation at 25 kGy was found suitable for sterilisation of the dressings. The thermal decomposition of unirradiated and irradiated chitin membranes was investigated. No significant change in the thermal behaviour because of irradiation at 25 kGy was observed. In vitro biodegradation of unirradiated and irradiated chitin membranes showed the susceptibility of the chitin dressing to lysozyme. Irritant effect of the chitin membrane dressings on skin was tested. Subcutaneous and scarification test in guinea pigs showed no signs of inflammation. This was further supported by the Finkelstein’s test performed in rabbits. The chitin membranes were found to have optimal performance characteristics of a wound dressing and showed no toxicity or possible adverse reactions. The study shows the chitin dressings as useful adjunct in wound care.     

Treatment of dermal depth burn wounds with an antimicrobial agent-releasing silicone gel sheet

Silicone gel sheets containing 0.02 per cent Ofloxacin were used in the treatment of 24 patients with a total of 27 dermal depth burn wounds. The gel provided a continuing drug delivery system from the dressing to the wound. Clinically the silicone gel sheets did not adhere to the wound and could be removed easily without pain. No infection developed in any of the dermal depth burn wounds treated with the gel sheets. The silicone gel sheets were found to promote prompt epithelialization in 16 burn wounds of superficial dermal depth (mean 8.4 days) compared with ointment-impregnated gauze dressings (mean 14 days, P less than 0.01). There was less pain and discharge by macroscopic observation of the absorbent materials from both dressings. In nine wounds of mixed superficial and deep dermal burn, the silicone gel also provided prompt epithelialization (mean 12 days) compared to the control wounds (mean 22 days, P less than 0.01).     

Effects of ionizing radiation on the mechanical properties of human bone

Allogeneic bone grafts are frequently sterilized by means of ionizing radiation. We investigated the effects of ionizing radiation on both quasistatic and impact mechanical properties of human bone. Specimens from four paired femora of four donors received doses of 29.5 kGy (“Standard” frequently used by tissue bandks), 94.7 kGy (“high”) or 17 kGy (“low”) of ionizing radiation. Young's modulus was unchanged by any level of radiation. Radiation significantly reduced bending strength, work to fracture, and impact energy absorption; in each case, the severity of the effect increased from low to standard to high doses of radiation. Work to fracture was particularly severely degraded; specimens irradiated with the high dose absorbed only 5% of the energy of the controls. Radiation. even at relatively low doses, makes the bone more brittle and thereby reduces its energy-absorbing capacity. We suggest that because the level of radiation required to produce an acceptable level of viral inactivation (90 kGy) produces an unacceptable reduction in the mechanical integrity of the bone. low levels of radiation, sufficient to produce bacterial safety, should be used in conjunction with biological tests to ensure viral safety.