Effects of ionizing radiation and preservation on biomechanical properties of human costal cartilage

Tissue banks around the world store human cartilage obtained from cadaveric donors for use in diverse reconstructive surgical procedures. To ensure this tissue is sterile at the time of distribution, tissues may be sterilized by ionizing radiation. In this work, we evaluate the physical changes in deep frozen costal cartilage (?70 °C) or costal cartilage preserved in high concentrations of glycerol (>98 %) followed by a terminal sterilization process using ionizing radiation, at 3 different doses (15, 25 and 50 kGy). Tension and compression tests were carried out to determine the mechanical changes related both to the different preservation methods and irradiation doses. For both methods of preservation, tension strength was increased by about 24 %, when cartilage tissue was irradiated with 15 kGy. Deep frozen samples, when irradiated with 25 or 50 kGy, had a decrease in their mechanical performance, albeit to a lesser extent than when tissues were preserved in high concentration of glycerol and equally irradiated. In conclusion, processing in high concentration of glycerol did not increase tissue protection against radiation damage; while cartilage preserved in high concentrations of glycerol withstands radiation up to 25 kGy, deep frozen human costal cartilage may be sterilized with a doses up to 50 kGy without significant mechanical impact.     

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.     

Quantifying the ultrastructure changes of air-dried and irradiated human amniotic membrane using atomic force microscopy: a preliminary study

Air-dried and sterilized amnion has been widely used as a dressing to treat burn and partial thickness wounds. Sterilisation at the standard dose of 25 kGy was reported to cause changes in the morphological structure as observed under the scanning electron microscope. This study aimed to quantify the changes in the ultrastructure of the air-dried amnion after gamma-irradiated at several doses by using atomic force microscope. Human placentae were retrieved from mothers who had undergone cesarean elective surgery. Amnion separated from chorion was processed and air-dried for 16 h. It was cut into 10?×?10 mm, individually packed and exposed to gamma irradiation at 5, 15, 25 and 35 kGy. Changes in the ultrastructural images of the amnion were quantified in term of diameter of the epithelial cells, size of the intercellular gap and membrane surface roughness. The longest diameter of the amnion cells reduced significantly after radiation (p?<?0.01) however the effect was not dose dependent. No significant changes in the shortest diameter after radiation, except at 35 kGy which decreased significantly when compared to 5 kGy (p?<?0.01). The size of the irradiated air-dried amnion cells reduced in the same direction without affecting the gross ultrastructure. At 15 kGy the intercellular gap decreased significantly (p?<?0.01) with Ra and Rq, values reflecting surface roughness, were significantly the highest (p?<?0.01). Changes in the ultrastructure quantified by using atomic force microscope could complement results from other microscopic techniques.     

Properties of Air Dried Radiation Processed Amniotic Membranes under Different Storage Conditions

Amniotic membranes collected from the placentae of screened donors were processed, air dried and sterilized by gamma irradiation at 25 kGy. Effect of storage under different temperature and humidity conditions (10 degrees C, RH 80-90%; 10 degrees C, RH 40-50%; 40 degrees C, RH 50-60% and 40 degrees C, RH 10-20%) on the properties of the membrane were examined. Infrared (IR) spectral scanning was carried out to examine degradation or change if any in the tissue under different storage conditions. The degradation of amnion on irradiation with gamma rays or during storage after irradiation would tend to produce the relative variation in IR absorption troughs. This kind of addendum was absent in all the samples indicating no qualitative change in the material property of amnion. Water absorption and water vapour transmission rate (WVTR) of the membrane remained unchanged even after 6 months. No effect on the microbial permeability of membrane was observed during storage. The amniotic membranes were found to be impermeable to different strains of bacteria - Bacillus, Escherichia coli, Pseudomonas, Citrobacter, Flavimonas and Staphylococcus. The results indicate that amniotic membranes processed by air-drying are stable and can be stored under different environmental conditions without compromise to their clinical performance.     

Improvement of human skin cell growth by radiation-induced modifications of a Ge/Ch/Ha scaffold

Gelatin-/chitosan-/hyaluronan-based biomaterials are used in tissue engineering as cell scaffolds. Three gamma radiation doses (1, 10 and 25 kGy) were applied to scaffolds for sterilization. Microstructural changes of the irradiated polymers were evaluated by using scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). A dose of 25 kGy produced a rough microstructure with a reduction of the porosity (from 99 to 96 %) and pore size (from 160 to 123 μm). Radiation also modified the glass transition temperature between 31.2 and 42.1 °C (1 and 25 kGy respectively). Human skin cells cultivated on scaffolds irradiated with 10 and 25 kGy proliferated at 48 h and secreted transforming growth factor β3 (TGF-β3). Doses of 0 kGy (non-irradiated) or 1 kGy did not stimulate TGF-β3 secretion or cell proliferation. The specific growth rate and lactate production increased proportionally to radiation dose. The use of an appropriate radiation dose improves the cell scaffold properties of biomaterials.     

Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiation

An important part of the preparation of biological material for transplantation is sterilization. The aim of our study was to assess the impact of ionizing radiation on three types of biological tissues and the impact of different doses on cells and extracellular matrix. Three types of frozen tissues (porcine skin xenografts, human skin allografts and human amnion) were divided into five groups, control and groups according to the dose of radiation to which these samples were exposed (12.5, 25, 35 and 50 kGy). The tissue samples were fixed by formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid schiff reaction and silver impregnation. The staining with hematoxylin and eosin showed hydropic degeneration of the cells of epidermis in xenografts by the dose of 12.5 kGy, in human skin it was observed by the dose of 35 kGy. The staining for elastic fibers revealed damage of fine elastic fibers in the xenografts dermis by the dose of 12.5 kGy, in the allografts by 35 kGy. Another change was the disintegration of basement membrane of epithelium, especially in the human amnion at the dose of 50 kGy. The silver impregnation visualized nuclear chromatin condensation mainly in human amnion at the dose of 12.5 kGy. Our results have shown that the porcine xenografts and human amnion were more sensitive to irradiation than the human skin. In the next phase of the project we will focus at more detailed changes in the tissues using immunohistochemical techniques.     

Validation and Substantiation of 25 kGy as Sterilization Dose for Lyophilized Human Amnion Membrane

The validation and substantiation of sterilization dose for lyophilized human amnion membrane by gamma irradiation delivered by Co⁶⁰ source were investigated. The validation experiments were conducted according to ISO 13409 method B. A total of 120 human amnion membranes were collected. Of these, 10 membranes were used for estimation of bioburden and 20 membranes were used for the individual sterility test at verification dose. The average bioburden per product unit with sample item portion (SIP = 1) for lyophilized human amnion membrane was 572 cfu. The verification dose experiments were done at dose of 8.1 kGy and the results of sterility tests showed that human amnion membrane got one positive. Consequently, the sterilization dose of 25 kGy was confirmed and substantiated.     

Study of tensiometric properties,microbiological and collagen content in nile tilapia skin submitted to different sterilization methods

Tissue bioengineering development is a global concern and different materials are studied and created to be safe, effective and with low cost. Nile Tilapia skin had shown its biological potential as covers for the burn wound. This study evaluates the tilapia skin histological, collagen properties and tensiometric resistance, after treatment by different sterilization methods. Tilapia skin samples were submitted to two sterilization processes: (1) chemical, which consisted in two 2% chlorhexidin baths, followed by sequential baths in increasing glycerol concentrations; and (2) radiation, when glycerolized skin samples were submitted to gamma radiation at 25, 30 and 50 kGy. Microscopic analyzes were performed through Haematoxylin–eosin and Picrosirius Red under polarized light. For tensiometric analysis, traction tests were performed. Glycerol treated skin presented a discrete collagen fibers disorganization within the deep dermis, while irradiated skin did not show any additional change. Throughout the steps of chemical sterilization, there was a higher proportion of collagen with red/yellow birefringence (type I) in the skin samples up to the first bath in chlorhexidin, when compared to samples after the first two glycerol baths (P < 0.005). However, there was no difference in relation to total collagen between groups. In irradiated skin, there was a larger total collagen preservation when using until 30 kGy (P < 0.005). Tensiometric evaluation did not show significant differences in relation to maximum load in the groups studied. We concluded that chemical and radiation (25 and 30 kGy) are efficient methods to sterilize Nile Tilapia skin without altering its microscopic or tensiometric characteristics.     

Substantiation of 25 kGy radiation sterilization dose for banked air dried amniotic membrane and evaluation of personnel skill in influencing finished product bioburden

Preparation of amniotic membrane (AM) by air drying method followed by radiation sterilization is simple and valuable approach; sterility and quality of the final AM product are depending on the quality management system at the tissue bank. Validation and substantiation of radiation sterilization dose (RSD) for tissue allografts is an essential step for the development and validation of the standard operating procedures (SOP). Application of SOP is perfectly relying on trained staff. Skills differences among personnel involved in AM preparation could have an effect on microbiological quality of the finished product and subsequently on the RSD required. AM were processed by four different couples of the tissue bank technicians. The AM grafts were randomly selected and subjected to bioburden test to validate and substantiate the 25 kGy RSD. Bioburden test for AM grafts were also useful to evaluate the skill of the tissue bank technicians and thus, to validate the current SOP for air dried AM. Moreover, the effect of placental source on bioburden counts on AM grafts was assessed. Substantiation of the 25 kGy RSD at a sterility assurance level of 10^?1, and sample item portion = 1, was carried out using Method VD _max ²⁵ of the International Organization for Standardization, document no. 11137-2 (ISO in Sterilization of healthcare products—radiation—part 2: establishing the sterilization dose, Method VDmax—substantiation of 25 kGy or 15 kGy as the sterilization dose, International Standard Organization, 2006). The results showed that there were no significant differences in the bioburdens of the four batches (α = 1 %), this means no significant differences in the skill of the four couples of the tissue bank technicians in terms of their ability to process AM according to the air dried AM SOP. The 25 kGy RSD was validated and substantiated as a valid sterilization dose for the AM prepared with the current established SOP at the Biotechnology Research Center experimental tissue bank. The donor’s type of delivery, normal or caesarean, showed no significant effect on the levels of microbial counts on the tested AMs (α = 1 %).     

Silica gel dessication of amniotic membrane with related epithelium cells for ocular surface reconstruction

Cornea reparative regeneration when in various pathological states needs creating certain conditions to intensify the potential of regional stem cells mitotic activity. AIMS OF THE RESEARCH: To find out the degree of the epithelium AM preservation after preliminary processing and conservation by means of dehydration over silica gel with further sterilization; to study the effectiveness of clinical treatment of AM conserved in the surroundings with vital ability epithelium and AM dried over silica gel. MATERIALS AND METHODS: There was carried out an investigation of 18 samples of native amnion treated with antibiotics and 18 total surface amnion samples conserved by drying over silica gel and then sterilized by gamma rays. Clinical experiments were carried out on patients with severe chemical and thermal burns--18 people (21 eyes). After the burn trauma all the patients underwent the standard procedure of necrectomy, the covering of the eyeball with amniotic membrane dried over silica gel. CONCLUSION: The drying out of the amniotic membrane over silica gel on frames without being fixed on nitrocellulose paper makes the process of the amniotic membrane conservation simpler and makes it possible to preserve its unique biological qualities. The effectiveness of the regeneration of epithelium tissue of the eyeball surface with amniotic membrane dried over silica gel without the vital capacity cells of the epithelium layer is analogous to the regeneration of epithelium cells with amniotic membrane with vital capacity cells. With eye burns AM coverage hinders the formation of rough conjunctiva cicatrix, provides a favorable out-of-cell matrix substrate for epithelium migration and leads to quicker regeneration of one's own epithelium, makes further visual rehabilitation simpler.     

The Influence of Different Preservation and Sterilisation Steps on the Histological Properties of Amnion Allografts — Light and Scanning Electron Microscopic Studies

Despite thorough donor screening and preparation under aseptic conditions, conventional methods of preservation do not exclude the probability of a contamination with pathogenic germs. The purpose of this study was to investigate the changes of histological parameters of amnion transplants (ATs) through different methods of sterilisation and preservation. Therefore 10 different procedures for sterilisation and preservation of ATs were described. Specimens of each group were studied using different histological procedures such as light microscopy and scanning electron microscopy. General staining (Haematoxylin-eosin stain, periodic-acid-Schiff, Domack) and immunohistochemical methods have been applied in order to gain additional information concerning the structure of the amniotic epithelium and the basement membrane but also the distribution of collagens and intermediate filaments. Furthermore, the measurement of the ATs thickness was included in order to study the influence of the manufacturing procedures to this property.As a result we found that the histological appearance of the ATs is closely related to the applied sterilisation and preservation procedures.Although the basement membrane remained intact, especially the amniotic epithelium was partially destroyed by irradiation sterilisation. Further, the dissolution of the connective tissue layers into single fibre bundles was clearly visible. Procedures with and without peracetic acid sterilisation (PAA) preserved the tissue structure.Our results showed a significant variation in the tissue's thickness after different preservation procedures. Air- and freeze-dried ATs were found to be the thinnest tissues varying from 20 to 30 microm, the thickest ATs preserved in glycerol varied from 45 to 50 microm. Because ATs showed a preserved tissue structure after PAA sterilisation it can be recommended as an alternative for methods previously described in literature. Depending on the specific use of the AT one may choose from thinner or thicker allografts.     

Silica gel dissication of amniotic membrane with related epithelium cells for ocular surface reconstruction

Cornea reparative regeneration when in various pathological states needs creating certain conditions to intensify the potential of regional stem cells mitotic activity. Aims of the Research. To find out the degree of the epithelium AM preservation after preliminary processing and conservation by means of dehydration over silica gel with further sterilization; to study the effectiveness of clinical treatment of AM conserved in the surroundings with vital ability epithelium and AM dried over silica gel. Materials and methods. There was carried out an investigation of 18 samples of native amnion treated with antibiotics and 18 total surface amnion samples conserved by drying over silica gel and then sterilized by gamma rays. Clinical experiments were carried out on patients with severe chemical and thermal burns – 18 people (21 eyes). After the burn trauma all the patients underwent the standard procedure of necrectomy, the covering of the eyeball with amniotic membrane dried over silica gel. Conclusion. The drying out of the amniotic membrane over silica gel on frames without being fixed on nitrocellulose paper makes the process of the amniotic membrane conservation simpler and makes it possible to preserve its unique biological qualities. The effectiveness of the regeneration of epithelium tissue of the eyeball surface with amniotic membrane dried over silica gel without the vital capacity cells of the epithelium layer is analogous to the regeneration of epithelium cells with amniotic membrane with vital capacity cells. With eye burns AM coverage hinders the formation of rough conjunctiva cicatrix, provides a favorable out-of-cell matrix substrate for epithelium migration and leads to quicker regeneration of one's own epithelium, makes further visual rehabilitation simpler. This revised version was published online in July 2006 with corrections to the Cover Date.     

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 on the immunobiological and immunochemical properties of cholera exotoxin. I. Change in the biological activity of nonpurified cholera exotoxin as affected by ionizing radiation

Crude cholera exotoxin (filtrate toxin) was irradiated with increasing doses of gamma radiation. A significant drop in enterotoxicity, in the activity of the permeation factor and a decrease in toxicity were shown to occur as radiation doses increased. Radiation doses of 50-70 kGy were found to completely inactivate enterotoxicity in liquid toxic preparations. A higher radioresistance of dried preparations in comparison with liquid ones was registered: inactivation occurred at 150-200 kGy. Different batches of the initial filtrate toxin had varying radiosensitivity. The sterilizing effect of gamma radiation was achieved at doses of 20 kGy for liquid preparations and 30 kGy for dried preparations. During the prolonged storage of the irradiated preparations of crude toxin (the term of observation being 1.5 years) at different temperatures no reversion of toxicity was found to occur, while their immunogenic properties remained unchanged.     

Cytokine expression and ultrastructural alterations in fresh-frozen,freeze-dried and γ-irradiated human amniotic membranes

The aim of this work was to compare the effects on human amniotic membrane of freeze-drying and γ-irradiation at doses of 10, 20 and 30 kGy, with freezing. For this purpose, nine cytokines (interleukin 10, platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, epidermal growth factor, transforming growth factor beta 1, and tissue inhibitors of metalloproteinase-1, -2, and -4) were titrated in 5 different preparations for each of 3 amniotic membranes included in the study. In addition, the extracellular matrix structure of each sample was assessed by transmission electron microscopy. While freeze-drying did not seem to affect the biological structure or cytokine content of the different amniotic membrane samples, γ-irradiation led to a significant decrease in the tissue inhibitors of metalloproteinase-4, basic fibroblast growth factor and epidermal growth factor, and induced structural damage to the epithelium, basement membrane and lamina densa. The higher the irradiation dose the more severe the damage to the amniotic membrane structure. In conclusion, the Authors recommend processing amniotic membrane under sterile conditions to guarantee safety at every step rather than final sterilization with γ-irradiation, thereby avoiding alteration to the biological characteristics of the amniotic membrane.     

Characterization of laminin isoforms in human amnion

Epithelial cells of the human amnion have been reported to possess similar functions to many types of cells, such as hepatocytes, neurons, and pancreatic beta-cells. We reported previously that one of the hepatocyte-like functions of human amniotic epithelial cells was reinforced by the presence of basement membrane components. Laminin is one of the main components of the basement membrane; it critically contributes to cell differentiation. Laminin has several heterotrimer isoforms composed of an alpha-, a beta-, and a gamma-chain, and each type of chain has several types of subunit chains: alpha1-5, beta1-3, and gamma1-3. In this study, we characterized the laminin subunit chains in human amnion. Laminin is produced and secreted from adjacent epithelial cells, and therefore, the gene expression of laminin subunit chains in human amniotic epithelial cells was investigated by RT-PCR. Their localization was examined by immunohistochemical staining of frozen sections. The findings suggested that the basement membrane of the human amnion contains a broad spectrum of laminin isoforms, laminin-2, -4, -5, -6, -7, -10, -11. These findings will provide clues not only for understanding the physiological roles of the amnion and hAECs, but also for applying this tissue as a source of donor cells for cell transplantation therapy.     

Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment

Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of laminins. The laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of laminins with other basement membrane components are lacking in the mature laminin 5 molecule. Therefore, the tight binding of laminin 5 to the basement membrane may occur by a unique mechanism. To examine laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the laminin 5 contained within the basement membrane of human amnion. In addition to monomeric laminin 5, we find that much of the laminin 5 isolated is covalently adducted with laminin 6 (alpha3beta1gamma1) and a novel laminin isotype we have termed laminin 7 (alpha3beta2gamma1). We propose that the association between laminin 5 and laminins 6 and 7 is a mechanism used in amnion to allow stable association of laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.     

Optimizing amniotic membrane tissue banking protocols for ophthalmic use

Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco’s modified eagle’s medium (DMEM) and glycerol at ?80 °C and at ?196 °C for a period of 6 weeks and at 4 °C in 70 % alcohol for 6 weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5 × 5 cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at ?196 and ?80 °C for 6 weeks. Similar sections were also stored at 4 °C in 70 % alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10 % FBS in humidified atmosphere of 5 % carbon dioxide at 37 °C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8 × 10⁶; 1.59, after 6 weeks in DMSO:FBS at ?196 °C 3.0 × 10⁶; 2.38 and at ?80 °C 2.1 × 10⁶; 1.60, in DMEM:Glycerol at ?196 °C 3.6 × 10⁶; 2.33 at ?80 °C 23 × 10⁶; 1.66 and at 4 °C 3.3 × 10⁶; 2.14. Histology analysis of the fresh AM showed an intact epithelial monolayer, thick basement membrane (BM) and avascular stromal matrix. Amniotic membranes stored at ?196 °C showed morphology similar to fresh AM in both preservation media and AM stored at ?80 °C showed disruption of the stromal matrix. At 4 °C the epithelial monolayer showed flattening. Fresh AM karyotype was 46XX. Analyzable spreads for karyotype were not obtained from stored AMs. Human amniotic epithelial cells were positive for both Oct-4 and G6PD genes. AM is best preserved at ?196 °C either in 1:9 DMSO:FBS or 1:1 DMEM:Glycerol. In both conditions cell viability and membrane integrity were shown to be preserved up to 6 weeks. Since analyzable chromosome spreads from cell cultures were not obtained, genomic stability could not be assessed.     

Sterilization of tendon allografts: a method to improve strength and stability after exposure to 50 kGy gamma radiation

Terminal sterilization of tendon allografts with high dose gamma irradiation has deleterious effects on tendon mechanical properties and stability after implantation. Our goal is to minimize these effects with radio protective methods. We previously showed that radio protection via combined crosslinking and free radical scavenging maintained initial mechanical properties of tendon allografts after irradiation at 50 kGy. This study further evaluates the tissue response and simulated mechanical degradation of tendons processed with radio protective treatment, which involves crosslinking in 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide followed by soaking in an ascorbate/riboflavin-5-phosphate solution. Control untreated and treated tendons were irradiated at 50 kGy and implanted in New Zealand White rabbit knees within the joint capsule for four and 8 weeks. Tendons were also exposed to cyclic loading to 20 N at one cycle per 12 s in a collagenase solution for 150 cycles, followed by tension to failure. Control irradiated tendons displayed increased degradation in vivo, and failed prematurely during cyclic processing at an average of 25 cycles. In contrast, radio protected irradiated tendons displayed greater stability following implantation over 8 weeks, and possessed strength at 59 % of native tendons and modulus equivalent to that of native tendons after cyclic loading in collagenase. These results suggest that radio protective treatment improves the strength and the stability of tendon allografts.     

Safety and efficacy of human amniotic membrane in primary pterygium surgery

Grafts made from human amniotic membrane are used to prevent recurrence of pterygium after excision. The success of the procedure can be affected by the quality of preparation and preservation of the grafts. We prospectively evaluated the safety and efficacy of cryopreserved amniotic membrane prepared at the research tissue bank of the Biotechnology Research Center in Tripoli, Libya, and used as adjunct therapy in primary pterygium excision. Twenty-six patients (15 males and 11 females) aged 21–78 years and indicated for primary pterygium excision were transplanted at the Tripoli Eye Hospital with the amniotic membrane grafts. Sixteen patients (62 %) were available for all three follow-up visits scheduled at 1, 3 and 6 months post-surgery. By the third visit, two patients (12.5 %) developed granuloma and three (18.8 %) had pterygium recurrence. The grafts were used after cryopreservation for ≤180 days or >180 days, but statistical analysis showed that the complications were not associated with the length of storage. Moreover, the high rate of complications in this study was not caused by use of cryopreserved AM. In conclusion, locally produced cryopreserved AM is safe as an adjunct therapy for treatment of primary pterygium excision.