Effect of gamma rays and accelerated electron beam on medullary lipids decomposition: influence of dose and irradiation temperature.

Ionizing radiation sterilization of non-defatted bone grafts has been found to deteriorate their quality and biocompatibility due to induction of lipid peroxidation products toxic for osteoblast-like cells. Therefore, the purpose of our study was to evaluate the effect of two types of ionizing radiation-gamma rays (G) or accelerated electron beam (EB) applied with two doses at different temperature conditions on hydrocarbons production, resulting from decomposition of palmitic and oleic acids-most abundant fatty acids in medullary lipids. Bone marrow samples isolated from femoral shafts of 6 male donors (aged 46-67 years) were irradiated with G or EB with doses of 25 or 35 kGy at different temperature conditions (ambient or deep freezing temperature). Fresh-frozen, non-irradiated samples served as control. Marrow lipids were extracted with n-hexane (Soxhlet's method), hydrocarbons fraction isolated on Florisil column chromatography, separated by gas chromatography and detected by mass spectrometry. Irradiation of bone marrow with sterilization doses of ionizing radiation (G and EB) was found to induce lipid radiolysis as measured by resulting hydrocarbons production. The effect was dose-dependent, whereas no marked influence of radiation type was observed. In contrast, irradiation temperature had a profound effect on lipids decomposition which was partially prevented while irradiation was performed in deep frozen state. Defatting of bone grafts prior to ionizing radiation sterilization seems essential for their biocompatibility, whereas irradiation in a deep-frozen state might compromise the effectiveness of sterilization and needs further studies.

Chitosan-Human Bone Composite Granulates for Guided Bone Regeneration.

The search for the perfect bone graft material is an important topic in material science and medicine. Despite human bone being the ideal material, due to its composition, morphology, and familiarity with cells, autografts are widely considered demanding and cause additional stress to the patient because of bone harvesting. However, human bone from tissue banks can be used to prepare materials in eligible form for transplantation. Without proteins and fats, the bone becomes a non-immunogenic matrix for human cells to repopulate in the place of implantation. To repair bone losses, the granulate form of the material is easy to apply and forms an interconnected porous structure. A granulate composed of ß-tricalcium phosphate, pulverized human bone, and chitosan-a potent biopolymer applied in tissue engineering, regenerative medicine, and biotechnology-has been developed. A commercial encapsulator was used to obtain granulate, using chitosan gelation upon pH increase. The granulate has been proven in vitro to be non-cytotoxic, suitable for MG63 cell growth on its surface, and increasing alkaline phosphatase activity, an important biological marker of bone tissue growth. Moreover, the granulate is suitable for thermal sterilization without losing its form-increasing its convenience for application in surgery for guided bone regeneration in case of minor or non-load bearing voids in bone tissue.

Structural mechanical properties of radiation-sterilized human Bone-Tendon-Bone grafts preserved by different methods.

To avoid the risk of infectious disease transmission from donor to recipient, allografts should be terminally sterilized. In the previous paper (Kaminski et al. in Cell Tissue Bank 10:215-219, 2009) we presented the effect of various methods of preservation (deep fresh freezing, glycerolization, lyophilization), followed by irradiation with different doses of electron beam (EB), on material (intrinsic) mechanical properties of human patellar tendons cut out as for anterior cruciate ligament reconstruction, obtained in failure tensile test. As structural mechanical properties are equally important to predict the behaviour of the graft as a whole functional unit, the purpose of the present paper was to show the results for failure load and elongation, obtained in the same experiment. Paired Bone-Tendon-Bone grafts (BTB) were prepared from cadaveric human patella tendons with both patellar and tibial attachments. They were preserved by deep freezing, glycerolization or lyophilization and subsequently EB-irradiated with the doses of 25, 35, 50 or 100 kGy (fresh-frozen grafts) or a single dose of 35 kGy (glycerolized and lyophilized grafts). Each experimental (irradiated) group was provided with control (non-irradiated), donor-matched group. The specimens from all groups were subjected to mechanical failure tensile test with the use of Instron system in order to measure their structural properties (failure load and elongation). All lyophilized grafts were rehydrated before mechanical testing. In our study we did not observe significant deterioration of structural mechanical properties of BTB grafts processed by fresh-freezing and then terminal sterilized with growing doses of EB up to 100 kGy. In contrast, BTB grafts processed by glycerolization or lyophilization and irradiated with 35 kGy showed significant decrease of failure load. Obtained results suggest that deep-frozen irradiated grafts retain their initial mechanical properties to an extent which does not exclude their clinical application. However, biomechanical investigations constitute only the first step to evaluate the potential clinical usefulness of such allografts and further extensive in vivo studies are needed.

Effect of gamma radiation and accelerated electron beam on stable paramagnetic centers induction in bone mineral: influence of dose, irradiation temperature and bone defatting.

Ionizing radiation has been found to induce stable defects in the crystalline lattice of bone mineral hydroxyapatite, defined as CO(2) (-) radical ions possessing spins. The purpose of our study was to evaluate CO(2) (-) radical ions induced in non-defatted or defatted human compact bone by gamma radiation (G) and accelerated electron beam (EB), applied with two doses at different temperatures. Moreover, the potential effect of free radical ion formation on mechanical parameters of compact bone, tested under compression in the previous studies, was evaluated. Bone rings from femoral shafts of six male donors (age 51 ± 3 years) were collected and assigned to sixteen experimental groups according to different processing methods (non-defatted or defatted), G and EB irradiation dose (25 or 35 kGy), and irradiation temperature [ambient temperature (AT) or dry ice (DI)]. Untreated group served as control. Following grinding under LN2 and lyophilization, CO(2) (-) radical ions in bone powder were measured by electron paramagnetic resonance spectrometry. We have found that irradiation of bone with G and EB induces formation of enormous amounts of CO(2) (-) radical ions, absent from native tissue. Free radical ion formation was dose-dependent when irradiation was performed at AT, and significantly lower in EB as compared to G-irradiated groups. In contrast, no marked effect of dose was observed when deep-frozen (DI) bone samples were irradiated with G or EB, and free radical ion numbers seemed to be slightly higher in EB-irradiated groups. Irradiation at AT induced much higher quantities of CO(2) (-) radical ions then on DI. That effect was more pronounced in G-irradiated bone specimens, probably due to longer exposure time. Similarly, bone defatting protective effect on free radical ion formation was found only in groups irradiated for several hours with gamma radiation at ambient temperature. Ambient irradiation temperature together with exposure time seem to be key parameters promoting CO(2) (-) radical ion formation in bone mineral and may mask the opposite effect of defatting and the possible effect of irradiation type. Significant weak negative correlations between CO(2) (-) radical ion number and some mechanical properties of compact bone rings (Young's modulus and ultimate stress) were found.

Tissue banking training courses: Polish experience.

Personnel directly involved in the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells should be appropriately qualified and provided with timely and relevant training according to EU directives. In the time of new tissue and cells regulations implementation such a training system existed in Poland only at a local level. The first training programme outlines for various groups of health professionals engaged in tissue banking practice was created in co-operation with the Institute for LifeLong Learning at University of Barcelona in 2006. This initial training courses were financially supported by EU Transition Facility Programme 2004. Then, starting from 2006, based on previous experience, system of advanced training courses was created. This training programme was financially supported by the National Programme for the Development of Transplantation Medicine 2006-2009-POLGRAFT financed by Polish Ministry of Health. During 2006 and 2007 first set of tissue banking initial training courses were provided according to TF 2004 project. Over 200 pathologists, forensic medicine specialists and other medical doctors responsible for donor screening and classification, medical directors of tissue establishments, technical staff; tissue graft users: orthopaedic surgeons, neurosurgeons, cardiosurgeons and ophthalmologists were trained. Between 2006 and 2009 there were organized 8 advanced tissue banking training courses according to POLGRAFT programme. There were organized both theoretical and practical courses on various aspects of tissue for over 350 persons. We present our experience in organisation of international and national tissue banking training courses.

Effect of accelerated electron beam on mechanical properties of human cortical bone: influence of different processing methods.

Accelerated electron beam (EB) irradiation has been a sufficient method used for sterilisation of human tissue grafts for many years in a number of tissue banks. Accelerated EB, in contrast to more often used gamma photons, is a form of ionizing radiation that is characterized by lower penetration, however it is more effective in producing ionisation and to reach the same level of sterility, the exposition time of irradiated product is shorter. There are several factors, including dose and temperature of irradiation, processing conditions, as well as source of irradiation that may influence mechanical properties of a bone graft. The purpose of this study was to evaluate the effect e-beam irradiation with doses of 25 or 35 kGy, performed on dry ice or at ambient temperature, on mechanical properties of non-defatted or defatted compact bone grafts. Left and right femurs from six male cadaveric donors, aged from 46 to 54 years, were transversely cut into slices of 10 mm height, parallel to the longitudinal axis of the bone. Compact bone rings were assigned to the eight experimental groups according to the different processing method (defatted or non-defatted), as well as e-beam irradiation dose (25 or 35 kGy) and temperature conditions of irradiation (ambient temperature or dry ice). Axial compression testing was performed with a material testing machine. Results obtained for elastic and plastic regions of stress-strain curves examined by univariate analysis are described. Based on multivariate analysis, including all groups, it was found that temperature of e-beam irradiation and defatting had no consistent significant effect on evaluated mechanical parameters of compact bone rings. In contrast, irradiation with both doses significantly decreased the ultimate strain and its derivative toughness, while not affecting the ultimate stress (bone strength). As no deterioration of mechanical properties was observed in the elastic region, the reduction of the energy absorption capacity of irradiated bone rings apparently resulted from changes generated by irradiation within the plastic strain region.

Effect of gamma irradiation on mechanical properties of human cortical bone: influence of different processing methods.

The secondary sterilisation by irradiation reduces the risk of infectious disease transmission with tissue allografts. Achieving sterility of bone tissue grafts compromises its biomechanical properties. There are several factors, including dose and temperature of irradiation, as well as processing conditions, that may influence mechanical properties of a bone graft. The purpose of this study was to evaluate the effect of gamma irradiation with doses of 25 or 35 kGy, performed on dry ice or at ambient temperature, on mechanical properties of non-defatted or defatted compact bone grafts. Left and right femurs from six male cadaveric donors aged from 46 to 54 years, were transversely cut into slices of 10 mm height, parallel to the longitudinal axis of the bone. Compact bone rings were assigned to the eight experimental groups according to the different processing method (defatted or non-defatted), as well as gamma irradiation dose (25 or 35 kGy) and temperature conditions of irradiation (ambient temperature or dry ice). Axial compression testing was performed with a material testing machine. Results obtained for elastic and plastic regions of stress-strain curves examined by univariate analysis are described. Based on multivariate analysis it was found that defatting of bone rings had no significant effect on any mechanical parameter studied, whereas irradiation with both doses decreased significantly the ultimate strain and its derivative toughness. The elastic limit and resilience were significantly increased by irradiation with the dose 25 kGy, but not 35 kGy, when the time of irradiation was longer. Additionally, irradiation at ambient temperature decreased maximum load, elastic limit, resilience, and ultimate stress. As strain in the elastic region was not affected, decreased elastic limit resulted in lower resilience. The opposite phenomenon was observed in the plastic region, where in spite of the lower ultimate stress, the toughness was increased due to the increase in the ultimate strain. The results of our study suggest that there may be an association between mechanical properties of bone tissue grafts and the damage process of collagen structure during gamma irradiation. This collagen damage in cortical bone allografts containing water does not depends on the temperature of irradiation or defatting during processing if dose of gamma irradiation does not exceed 35 kGy.

An evaluation of sterilisation processes.

A sterile environment is one of the basic elements of in vitro cell culture. When choosing an appropriate sterilisation method, the possibility that the physical and chemical properties of the sterilised material could be altered by the sterilisation process itself, should be considered. Avoiding any potential problems of toxicity arising as a consequence of the sterilisation process is essential, not only in in vitro cell culture procedures, but especially in the case of the sterilisation of medical devices which come into contact with human tissue (e.g. catheters, surgical tools, and containers used for transplant preparation and storage). As it is not possible to predict the potential effects of every combination of test material and sterilisation process, we have designed a simple test, which can be easily performed to ensure the absence of cytotoxicity. The test involves the culturing of a non-adherent cell line in direct contact with the test material, in micro-wells attached to the surface of the test device. By using this novel test method, three sterilisation procedures were compared for each material. The results indicated that, neither ionising irradiation nor ethylene oxide left toxic residues on the surface of polystyrene; and that, in the case of steel, neither steam sterilisation nor ethylene oxide left toxic residues on the metal. The cold plasma system, which left toxic residues on the surface of both materials, required a post-sterilisation period of 24 hours in the case of steel, and 10 days in the case of polystyrene, in order to eliminate toxic residues prior to their use.

Bladder squamous metaplasia of the urothelium - introductory report.

Seven female patients hospitalized in the Surgery Ward of Warsaw's Hospital for Children are described. Nonspecific abdominal pains, recurrent urinary tract infections, and voiding disorders (difficulty in voiding commencement) were the primary causes of hospitalization. Urodynamics and cystoscopy with a histopathology section of the urothelium achieved the diagnosis of nonkeratizing squamous cell metaplasia of the urothelium. The topic is not new, although the diagnosis is particularly rare in the pediatric patient. Until now, the existing world literature has described only a few cases of this kind of metaplasia in children. These findings may be helpful in differential diagnosis of urinary bladder disease. It is postulated that nonkeratizing squamous cell metaplasia of the urothelium, in the future, may lead to malignant changes in the urothelium. Therefore, the therapeutic process merits close monitoring.

ESTIV questionnaire on the acquisition and use of primary human cells and tissue in toxicology.

The ability to use human cells and tissues in toxicology research and testing has the benefit that it obviates the need to undertake species extrapolation when assessing human hazard. However, obtaining and using human cells and tissues is logistically difficult, ethically complex and is a potential source of infections to those coming into contact with human cell material. The issue is also controversial, with the recent EU legislation draft on tissue engineering, and also due to some instances of human material being obtained and used without informed consent. There are also varying regulations and attitudes relating to the use of human cells and tissues throughout Member States of the EU, and there is a need for harmonisation. The European Society of Toxicology in Vitro (ESTIV) Executive Board and the European Network of Human Research Tissue Banks (ENRTB) have conducted a survey to ascertain the extent to which human cells and tissues are used by its members, how these are obtained, what local regulations are in force, how the material is used, and the advantages and disadvantages experienced by members in using such material, as opposed to cell lines. The results obtained have been compared with the results from a previous survey conducted in 2000. It is hoped that this information will help to facilitate the process of acquiring and using human cells and tissues in a safe and effective way to promote the use of non-animal approaches for investigating the mechanisms of toxicity, and for predicting the toxic hazard of substances.