Scanning electron microscopic assessment on surface morphology of preserved human amniotic membrane after gamma sterilisation

Human amniotic membrane that has been processed and sterilised by gamma irradiation is widely used as a biological dressing in surgical applications. The morphological structure of human amniotic membrane was studied under scanning electron microscopy (SEM) to assess effects of gamma radiation on human amniotic membrane following different preservation methods. The amniotic membrane was preserved by either air drying or submerged in glycerol before gamma irradiated at 15, 25 and 35 kGy. Fresh human amniotic membrane, neither preserved nor irradiated was used as the control. The surface morphology of glycerol preserved amnion was found comparable to the fresh amniotic membrane. The cells of the glycerol preserved was beautifully arranged, homogonous in size and tended to round up. The cell structure in the air dried preserved amnion seemed to be flattened and dehydrated. The effects of dehydration on intercellular channels and the microvilli on the cell surface were clearly seen at higher magnifications (10,000×). SEM revealed that the changes of the cell morphology of the glycerol preserved amnion were visible at 35 kGy while the air dried already changed at 25 kGy. Glycerol preservation method is recommended for human amniotic membrane as the cell morphological structure is maintained and radiation doses lower than 25 kGy for sterilization did not affect the appearance of the preserved amnion.     

Radiation Processed Amniotic Membranes in the Treatment of Non-Healing Ulcers of Different Etiologies

The amniotic membranes were collected from the placentae of selected and screened donors. Processing was done by washing the fresh amniotic membrane successively in sterile saline, 0.05% sodium hypochlorite solution and sterile distilled water until it was completely cleared of blood particles. The membranes were sterilized by gamma irradiation at 25 kGy. The processed amniotic membranes were applied to 50 open wounds comprising of 42 full thickness defects and eight partial thickness defects. These included leprotic, diabetic, traumatic, gravitational ulcers and superficial burn in the form of scald and corrosive burn. The radiation processed amniotic membranes favoured healing of unresponsive and non-healing ulcers of different etiologies. Ulcers with duration of minimum 3 weeks to maximum 12 months were found to heal in 2-6 weeks by the application of amniotic membranes.     

The solution and solid state stability and excipient compatibility of parthenolide in feverfew

The objectives of this research were to evaluate the stability of parthenolide in feverfew solution state and powdered feverfew (solid state), and explore the compatibility between commonly used excipients and parthenolide in feverfew. Feverfew extract solution was diluted with different pH buffers to study the solution stability of parthenolide in feverfew. Powdered feverfew extract was stored under 40 degrees C/0% approximately 75% relative humidities (RH) or 31% RH/5~50 degrees C to study the influence of temperature and relative humidity on the stability of parthenolide in feverfew solid state. Binary mixtures of feverfew powered extract and different excipients were stored at 50 degrees C/ 75% RH for excipient compatibility evaluation. The degradation of parthenolide in feverfew solution appears to fit a typical first-order reaction. Parthenolide is comparatively stable when the environmental pH is in the range of 5 to 7, becoming unstable when pH is less than 3 or more than 7. Parthenolide degradation in feverfew in the solid state does not fit any obvious reaction model. Moisture content and temperature both play important roles affecting the degradation rate. After 6 months of storage, parthenolide in feverfew remains constant at 5 degrees C/31% RH. However, approximately 40% parthenolide in feverfew can be degraded if stored at 50 degrees C/31% RH. When the moisture changed from 0% to 75% RH, the degradation of parthenolide in feverfew increased from 18% to 32% after 6-month storage under 40 degrees C. Parthenolide in feverfew exhibits good compatibility with commonly used excipients under stressed conditions in a 3-week screening study.     

Structural changes in plasma membranes prepared from irradiated Chinese hamster V79 cells as revealed by Raman spectroscopy

The effect of gamma irradiation on the integrity of plasma membranes isolated from Chinese hamster V79 cells was investigated by Raman spectroscopy. Plasma membranes of control V79 cells show transitions between (-) 10 and 5 degrees C (low-temperature transition), 10 and 22 degrees C (middle-temperature transition), and 32 and 40 degrees C (high-temperature transition). Irradiation (5 Gy) alters these transitions markedly. First, the low-temperature transition shifts to higher temperature (onset and completion temperatures 4 and 14 degrees C). Second, the middle-temperature transition shifts up to the range of about 20-32 degrees C, but the width remains unchanged. Third, the higher temperature transition broadens markedly and shifts to the range of about 15-40 degrees C. Protein secondary structure as determined by least-squares analysis of the amide I bands shows 36% total helix, 55% total beta-strand, and 9% turn plus undefined for control plasma membrane proteins. Plasma membrane proteins of irradiated V79 cells show an increase in total helix (40 and 45% at 5 and 10 Gy, respectively) and a decrease in the total beta-strand (48 and 44% at 5 and 10 Gy, respectively) structures. The qualitative analysis of the Raman features of plasma membranes and model compounds in the 1600 cm-1 region, assigned to tyrosine groups, revealed that irradiation alters the microenvironment of these groups. We conclude that the radiation dose used in the survival range of Chinese hamster V79 cells can cause damage to plasma membrane proteins without detectable lipid peroxidation, and that the altered proteins react differently with lipids, yielding a shift in the thermal transition properties.     

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.     

Kinetic properties and storage stability of catalase immobilized on to florisil

The covalent immobilization of bovine liver catalase (CAT) on to florisil via glutaraldehyde was investigated. Optimum immobilization pH and temperature were determined as pH 6.0, 10 degrees C respectively, while the amount of initial CAT per g of carrier and immobilization time was determined as 5 mg g(-1) and 120 min, respectively. The Vmax values for free and immobilized CAT were found to be 1.7 x 10(5) and 2.0 x 10(4) micromol H2O2 min(-1) mg protein(-1), respectively, whereas KM values were 33.3 mM and 1722.0 mM respectively. Operational stability was determined by using a stirred batch-type column reactor. Immobilized CAT retained about 40% of its initial activity after 50 uses. It showed higher storage stability than free CAT at 4 degrees C and 25 degrees C. Its storage stability increased with increasing relative humidity (RH) from 0 to 20% of the medium. The highest storage stability was obtained in 20% RH, however, further increase in RH from 40 to 100% significantly decreased the storage stability.     

Declining viability and lipid degradation during pollen storage

Declining viability of pollen during storage at 24° C in atmospheres of 40% relative humidity (RH) and 75% RH was studied, with special emphasis on lipid changes. Pollens of Papaver rhoeas and Narcissus poeticus, characterized by a high linolenic acid content, were compared with Typha latifolia pollen which has a low linolenic acid content. The rationale behind this was to answer the question of whether lipid peroxidation is involved in the rapid viability loss and reduced membrane integrity of, in particular, the unsaturated-lipid pollen types. Viability and membrane integrity degraded more rapidly at 75% RH than at 40% RH. All pollen species showed deesterification of acyl chains of lipids but no detectable peroxidation at both RH levels. Considerable amounts of lipid-soluble antioxidants were detected that did not degrade during storage. Free fatty acids and lysophospholipids were formed during storage, the effects of which on membranes are discussed. These degradation products were very prominent in the short-lived Papaver pollen. The loss of viability does coincide with phospholipid deesterification. A significant decrease of the phospholipid content occurred at 75% RH, but not at 40% RH. Based on compositional analyses of phospholipids and newly formed free fatty acids, it was concluded that the deesterification of acyl chains from the lipids occurred at random. We suggest that, due to the low water content of the pollen, free radicals rather than unspecific acyl hydrolases are involved in the deesterification process.     

Secretions of interleukin-1beta and tumor necrosis factor alpha by whole fetal membranes depend on initial interactions of amnion or choriodecidua with lipopolysaccharides or group B streptococci

The present study evaluated the secretions of interleukin (IL)-1beta and tumor necrosis factor (TNF) alpha by fetal membranes stimulated with group B streptococci (GBS) and lipopolysaccharide (LPS). The aim was to evaluate the initial response of full-thickness membranes to the microbial insult using an in vitro experimental model that allowed testing of the individual contributions of amnion and choriodecidua to stimulation. Full-thickness membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation without evidence of active labor. The membranes were mounted in Transwell devices, physically separating the upper and lower chambers. The LPS (500 ng/ml) or GBS (1 x 10(6) colony-forming units/ml) was added to either the amniotic or choriodecidual surface, and accumulation of IL-1beta and TNFalpha were measured in both compartments using a specific ELISA. Fetal membranes followed different patterns of secretion of proinflammatory cytokines that depended on the side to which the stimulus was added or the nature of the stimulus itself. The TNFalpha was secreted by amnion and choriodecidua in the presence of LPS or GBS, and stimulation with GBS induced a greater synthesis of IL-1beta than did stimulation with LPS. Choriodecidual tissue was more responsive than amniotic tissue, and this response tended to be higher even when the stimulation was only on the amniotic side. However, the amnion plays an active role in recognizing LPS or GBS, contributing a significant amount of TNFalpha. Thus, cooperative and bidirectional communications occur between amnion and choriodecidua in response to bacterial products, which include intermembranous cytokine traffic and signaling between tissues.     

The cholinergic reaction of the amnion in the chick embryo

Studies have been made of the spontaneous contractions of the amnion and acetylcholine sensitivity of amniotic membrane in 8--14-day chick embryos. In 12--14-day embryos, the spontaneous rhythmic contractions were rather rare as compared to those in 8--9-day ones, their frequency being also lower. On the basis of kinetic analysis, it was concluded that both the dissociation constant (K) and the value of Pmax do not exhibit significant changes for tonotropic reaction from the 8th to the 14th day and for chronotropic reaction--from the 8th to the 10th day of incubation. After the 10th day of incubation, dose-effect chronotropic reaction not expressed. The spontaneous activity of the amnion and acetylcholine sensitivity of the amniotic membrane depend on the temperature being maximal at 38 degrees C. Possible regulatory mechanisms of contractile activity in chick amnion are discussed.     

Rhythmic contractions in chick amnio-yolk sac and snake amnion during embryogenesis

The rhythmic movements of fetal membranes in chick and reptile embryos were studied to explore the developmental role of the extra-embryonic motor activity. In the snakes Lamprophis fuliginosus and Elaphe radiata, rhythmic contractions of amnion inside the developing egg were recorded from the 11th incubation day until pre-hatching stages (ca. day 60-72). The duration of these contractions averaged 2.02+/-0.27 min. The frequency ranged from 2 to 6 per 10 min and averaged 4.61+/-0.57 per 10 min. A tendency of frequency to increase toward the end of embryogenesis was observed. Lowering the temperature from 28 to 20 degrees C significantly decreased the frequency of amnion contractions to 2.85+/-0.91 per 10 min. The isolated snake amnion retained its capacity for spontaneous contraction. Noradrenaline inhibited, acetylcholine stimulated and serotonin did not affect the rhythmic activity of the isolated snake amnion. Similar effects were found when these agents were applied into the snake amniotic cavity. In the chick, yolk sac rhythmic contractions were recorded from the fifth until the 12th incubation days. The duration of these contractions ranged from 15 to 60 s, their frequency averaged 11.8+/-3.18 per 10 min and depended on temperature. The low temperature threshold was approximately 30 degrees C. After surgical removal of the amnion and embryo, the yolk sac continued contracting inside the egg. The yolk sac rhythmic contractions likely participate in the space movement of the embryo inside the egg during embryogenesis.     

Physiological and morphological characteristics of the rhythmic contractions of the amnion in veiled chameleon (Chamaeleo calyptratus) embryogenesis

A morpho-functional study of the veiled chameleon (Chamaeleo calyptratus) amnion was performed as part of a general comparative study of amnion rhythmic contractions (ARC) in reptile and bird embryogenesis. Eggs incubated at 27.5 degrees C were used at different developmental stages from day 80 (D80) to D184 for the recording of ARC using a force transducer. Slow ARC, about 1 min in duration, were revealed from D88 (stage 31) to the near-hatching stages (incubation time was 183-198 days). The frequency and amplitude of slow ARC increased significantly towards the end of incubation. This ARC enhancement correlated with the differentiation of smooth muscle elements in the amniotic membrane from single spindle-shaped cells to complex "star-like" structures and with the development of the actin fibers, revealed by phalloidin, in the amniotic muscle layer. Short-term changes in temperature influenced ARC and heart rate (HR). Cooling to 25 degrees C from the control (27.5 degrees C) resulted in a significant decrease in both ARC frequency and HR. Heating to 30 degrees C significantly increased the embryonic HR, but not ARC frequency. Within the temperature range from 25 to 30 degrees C the temperature effect on ARC frequency and HR was reversible.     

Activation of phospholipase C in rabbit brain membranes by carbachol in the presence of GTP gamma S; effects of biological detergents

Rabbit brain cortical membranes incubated with carbachol in the presence of GTP gamma S show a marked increase in the degradation of exogenous phosphatidylinositol 4,5-bisphosphate. This activation of phospholipase C is dependent on the presence of deoxycholate and maximal at 0.8-1 mM deoxycholate. There is negligible activation by carbachol alone but in the presence of GTP gamma S a carbachol effect can be readily demonstrated. Optimal activation of phospholipase C by carbachol was seen at 10 to 100 nM free Ca2+. Washing cortical membranes with hypertonic buffer extracted 60% of the membrane protein yet the carbachol and GTP gamma S coupling remained intact. Incubation of the membranes with lysophosphatidylcholine, Nonidet P-40, sodium deoxycholate or digitonin at concentrations considerably less than those frequently used to solubilize membrane proteins abolished the carbachol response. Octyl glucoside and sodium cholate also uncoupled receptor regulation of phospholipase C but only at concentrations where solubilization of membrane proteins occurred. Prior exposure of membranes to carbachol did not prevent the uncoupling observed as a result of detergent treatment. Incubation of the membranes with carbachol and GTP gamma S did not appear to be accompanied by specific release of either active phospholipase C or inhibitors of phospholipase C activity.     

Influence of environmental storage relative humidity on biological indicator resistance, viability, and moisture content.

The effect of environmental storage relative humidity (RH) on the moisture content, viability, and moist heat and gaseous ethylene oxide (EO) resistance of biological indicators (BIs) was evaluated. No statistically significant difference was observed between the initial Bacillus stearothermophilus spore population and the spore population of BIs stored at 20 degrees C and 0, 20, 44, of 55% RH or under ambient, 4 degrees C, or -20 degrees C conditions after 12 months. A statistically significant decrease in moist heat resistance from initial starting levels was found for BIs stored at 20 degrees C and either 0 or 20% RH. There was a statistically significant decrease in the B. subtilis BI spore population, compared with initial levels, when the BIs were stored at 20 degrees C and 0% RH concomitant with a significant increase in their EO resistance. BI storage at 20 degrees C and 20 or 44% RH, or under ambient, 4 degrees C, or -20 degrees C conditions, had no significant effect on EO resistance. BIs stored at 20 degrees C and 66% RH demonstrated a significantly lower EO resistance compared with starting levels.     

The effect of ultrahigh frequency electromagnetic fields on the reduction of ferricyanide by human erythrocytes in the presence of methylene blue

Effect of microwave radiation with the frequency of 1000 +/- 10 MHz and specific absorption rate of 220-580 mV/g on the ferricyanide reduction by human red blood cells in the presence of methylene blue (carrier of oxidation-reduction equivalents through the membrane) was studied at different temperatures in the region of 23-34 degrees C. The temperature dependence of the ferricyanide reduction rate in Arrhenius plots shows two sharp "anomalous" sites with apparently negative activation energy at 26-27 and 29-30 degrees C. Broadness and expression of the "anomalous" sites increased with an increase of the blood storage time. The increase of the ferricyanide reduction rate under microwave irradiation was observed only in the temperature regions corresponding to the "anomalous" sites of the temperature dependence.     

Expression of inflammatory markers in pig amnion after intraamniotic infection with nonpathogenic or enteropathogenic<Emphasis Type="Italic">Escherichia coli</Emphasis>

The pig amnion was in vivo intraamniotically infected with E. coli for 10 h at 80-85 d of gestation either with the nonpathogenic O86 strain or enteropathogenic O55 strain. TNF-alpha, IL-10, IL-1beta and IFN-gamma were determined in amniotic fluids by ELISA, the expression of cytokines and some other inflammatory markers was determined by immunohistochemistry. Intraamniotic infection induced high levels of TNF-alpha in amniotic fluids which correlated with bacterial virulence whereas IL-10 was induced only by O86. The IL-1beta level did not increase significantly and was expressed in all infected membranes. IFN-gamma was negligible or absent. TNF-alpha, IL-12p40, calprotectin, HSP65 and gp91phox were found by immunohistochemistry only in amnion membranes infected with the enteropathogenic strain 055.     

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.     

The influence of various storage conditions on cell viability in amniotic membrane

Up to now freeze-dried, gamma-sterilised or glycerol-preserved amniotic membranes (AMs) have widely been used in the field of ophthalmology and wound care (e.g. leg ulcers, burns). After some preservation processes in use, like freeze-drying or glycerol-preserving, the cells in the AM are no longer viable. Within this study we evaluated the influence of different short-term and long-term storage conditions on cell viability in AM. Therefore AMs from cesarean section placentae were washed and biopsied to evaluate the microbiological status and to determine the viability of the tissue. Additionally, viability under various storage conditions was examined by assessment of mitochondrial activity. Preservation included temperatures above and below 0°C as well as various media compositions. As expected, cell viability in amnion decreases during storage, in fact the effect was more pronounced when stored frozen, but the higher viability of amnion obtained by storage above 0°C with medium is associated with the limitation to a short period of storage of about 28 days. The evaluated preservation methods are the basis for future non-clinical in-vivo studies in which the possible benefit of amnion as a viable biomaterial in wound healing will be investigated. A part of this work was presented at the World Congress on Tissue Banking in Rio in May 2005 and was honoured by the Poster Award Commission.     

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.     

Stability of Benzocaine Formulated in Commercial Oral Disintegrating Tablet Platforms

Pharmaceutical excipients contain reactive groups and impurities due to manufacturing processes that can cause decomposition of active drug compounds. The aim of this investigation was to determine if commercially available oral disintegrating tablet (ODT) platforms induce active pharmaceutical ingredient (API) degradation. Benzocaine was selected as the model API due to known degradation through ester and primary amino groups. Benzocaine was either compressed at a constant pressure, 20 kN, or at pressure necessary to produce a set hardness, i.e., where a series of tablets were produced at different compression forces until an average hardness of approximately 100 N was achieved. Tablets were then stored for 6 months under International Conference on Harmonization recommended conditions, 25°C and 60% relative humidity (RH), or under accelerated conditions, 40°C and 75% RH. Benzocaine degradation was monitored by liquid chromatography–mass spectrometry. Regardless of the ODT platform, no degradation of benzocaine was observed in tablets that were kept for 6 months at 25°C and 60% RH. After storage for 30 days under accelerated conditions, benzocaine degradation was observed in a single platform. Qualitative differences in ODT platform behavior were observed in physical appearance of the tablets after storage under different temperature and humidity conditions.     

Infectivity of Echinococcus granulosus protoscolices under different conditions of temperature and humidity

The aim of this study was to examine the effect of different temperatures and humidities on the infectivity of Echinococcus granulosus protoscolices. Eighteen dogs (6 groups, n = 3 each) were fed with offal mince harbouring approximately 20,000 protoscolices of E. granulosus of different viabilities. Dogs were infected with E. granulosus protoscolices of: (1) 5% viability at -10 degrees C and 50% relative humidity (RH); (2) 30% viability at 0 degrees C and 60% RH; (3) 20% viability at +10 degrees C and 65% RH; (4) 15% viability at +30 degrees C and 75% RH; (5) 11% viability at +40 degrees C and 80% RH; (6) 68% viability (control group). Dogs in each group were necropsied at 29-49 days post-infection. Mean intensities of E. granulosus recovered from dogs were 256.7 +/- 60.3 in the second group; 32.7 +/- 7.1 in the third group; 40.3 +/- 15.5 in the fourth group and 1533 +/- 513 in the control group. However, no parasites were recovered from the first and fifth groups. Results obtained in the present study show that larval stages could be infective for 1 to 4 weeks during spring, autumn or winter months when maximal temperatures are approximately 0-10 degrees C. In conclusion, cold-storage depots in slaughterhouses and abattoirs where sheep carcasses might be discarded should be kept at -20 degrees C for 2-3 days, dogs should be properly controlled and adequate control programmes must be established in areas where the disease is endemic.