Application of a high-level peracetic acid disinfection protocol to re-process antibiotic disinfected skin allografts

Skin allografts, derived from cadaveric donors, are widely used for the treatment of burns and ulcers. Prior to use in clinical situations, these allografts are disinfected using a cocktail of antibiotics and then cryopreserved. Unfortunately, this antibiotic disinfection procedure fails to decontaminate a significant proportion and these contaminated grafts can not be used clinically. We have investigated whether it is possible to apply a second, more potent disinfection procedure to these contaminated grafts and effectively to re-process them for clinical use. Cadaveric skin grafts, treated with antibiotics and cryopreserved, were thawed and a peracetic acid (PAA) disinfection protocol applied. The grafts were then preserved in a high concentration of glycerol or propylene glycol, and properties thought to be essential for successful clinical performance assessed. The cytotoxicity of the grafts was assessed using both extract and contact assays; damage to the skin collagen was assessed using a collagenase susceptibility assay and the capacity of the grafts to elicit an inflammatory response in vitro was assessed by quantifying the production of the pro-inflammatory cytokine TNF-alpha by human peripheral blood mononuclear phagocytes. PAA disinfection, in conjunction with either glycerol or propylene glycol preservation, did not render the grafts cytotoxic, pro-inflammatory, or increase their susceptibility to collagenase digestion. The rates of penetration of glycerol and propylene glycol into the re-processed skin were comparable to those of fresh skin. This study has demonstrated that PAA disinfection combined with immersion in high concentrations of either glycerol or propylene glycol was an effective method for re-processing contaminated skin allografts, and may justify their clinical use.     

The measurement of water activity in allogeneic skin grafts preserved using high concentration glycerol or propylene glycol

In the presence of free water, many degradation reactions can occur within stored tissues including enzymatic digestion, oxidation (peroxidation) and hydrolytic reactions, as well as the detrimental effects of microbial growth, therefore most long-term banking techniques are designed to avoid free water. One method currently used for banking of skin grafts is the use of high concentration (85%) glycerol as a preservative. In this case, the glycerol was assumed to dehydrate the skin by osmosis and diffusion out of the cells and skin matrix respectively. We have recently shown that this assumption is incorrect and the converse occurs, i.e. glycerol enters the skin and sequesters the water. It was therefore essential to determine whether enough water had been immobilised to prevent degradation of the tissue. Using an instrument (Pawkit) designed to measure water activity (aw) it was shown that a stepwise reduction in aw was achieved when the skin was immersed in 50 and 85% glycerol or propylene glycol, respectively. At the end of the glycerolisation process, the final aw was shown to be circa 0.3. An aw of 0.3 is known to minimise lipid peroxidation and reduce other degradation reaction rates to very low levels. It was concluded that the current glycerolisation protocol results in effective sequestration of water avoiding degradation of the skin during storage. The method presented should be used as a quality control step to confirm adequacy of preservation for each batch of glycerolised skin.     

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO+10% PG+10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6+/-16.7% (mean+/-S.D.) and 77.8+/-15.5%, were achieved by the straw vitrifying method (20.5% DMSO+15.5% acetamide+10% PG, thawing at 43 degrees C and washing in 0.5M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.     

Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient females

Four experiments were conducted to define a system for the direct transfer of frozen-thawed bovine embryos to recipient females. In Experiment I, nonsurgically recovered embryos were frozen in 1.5 M ethylene glycol (EG), 1.5 M propylene glycol (PG), 1.5 M DMSO or 1.4 M glycerol (GLY), and then thawed and placed directly into holding medium. Viability at 72 hours of post-thaw culture was 70, 11, 25 and 30% for the four groups, respectively. In Experiments II and III, 1.0, 1.5 and 2.0 M concentrations of EG were compared; a concentration of 1.5 M appeared to provide optimal cryopreservation and survival after direct rehydration. In Experiment IV, embryos were packaged in straws containing only 1.5 M EG, in straws containing a column of 1.5 M EG and the embryo and two columns of PB1 in a 1:3 ratio of volumes (EG PB1 ), or were frozen in 1.4 M glycerol. After thawing, embryos in EG and EG PB1 treatments were transferred directly to recipient females, while embryos frozen in GLY were rehydrated using a three-step procedure. In the first trial, pregnancy rates at approximately 60 days of gestation for embryos frozen in EG and GLY groups were 39 and 62%, respectively (P<0.10). In the second trial, the pregnancy rate for embryos frozen in EG PB1 was equal to that of embryos frozen in GLY (50% in both groups). These experiments demonstrate the potential for using ethylene glycol as a cryoprotectant for bovine embryos, thus permitting direct transfer of frozen-thawed embryos to recipient females.     

In vitro survival of murine morulae after quick freezing in the presence of chemically defined macromolecules and different cryoprotectants

We studied the ability of frozen-thawed mouse morulae to develop in vitro when the cryoprotectant proteins were substituted with one of the following nonorganic macromolecules: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and ficoll. We also determined how these agents interacted with 3 different cryoprotectants: glycerol (GLY), propylene glycol (PG), and ethylene glycol (EG). The influence of both of the above factors was measured on the basis of post-thaw morphological appearance, the percentage of development to the expanded blastocyst stage and the total cell count. Morulae (n=950) were collected from superovulated mice. Those classified as good or excellent were distributed among the 12 different freezing solutions, obtained by combining the 3 cryoprotectants with the 4 macromolecules (the 3 mentioned above, plus a control of 5% fetal calf serum) in phosphate buffered saline (PBS). Embryos frozen in PVA, PVP and ficoll tended to be a little difficult to recover from the straws. Development to the expanded blastocyst stage was significantly lower (P<0.05) in propylene glycol (43.6%) than in ethylene glycol (79.5%) or in glycerol (76.1%). Polyvinyl alcohol provided a higher survival rate when combined with glycerol (90.3) or ethylene glycol (95.0), but when it was combined with propylene glycol, only 56.5% of embryos survived after thawing. A positive interaction was observed between glycerol and PVA and between ethylene glycol and PVA or ficoll. The results indicate that fetal serum could be successfully substituted for any of the 3 chemically defined macromolecules. However, our findings also suggest that the use of PG as a cryoprotectant should be avoided when mouse morulae are frozen using the quick freezing method.     

Using Raman Spectroscopy in Studying the Effect of Propylene Glycol,Oleic Acid,and Their Combination on the Rat Skin

The permeability enhancement effect of oleic acid (OA) and propylene glycol (PG) as well as their (1:1 v/v) combined mixture was studied using rat skin. The percutaneous drug administration is a challenge and an opportunity for drug delivery. To date, there is limited research that illustrates the mechanism of penetration enhancers and their combinations on the skin. This project aims to explore the skin diffusion and penetration enhancement of PG, OA, and a combination of PG-OA (1:1 v/v) on rat skin and to identify the potential synergistic effect of the two enhancers utilizing Raman spectroscopy. Dissected dorsal skin was treated with either PG or OA or their combination for predetermined time intervals after which the Raman spectra of the treated skin were collected with the enhancer. A spectrum of the wiped and the washed skin were also collected. The skin integrity was tested before and after exposure to PG. The skin histology proved that the skin integrity has been maintained during experiments and the results indicated that OA disrupted rat skin lipid as evident by changes in the lipid peak. The results also showed that PG and OA improved the diffusion of each other and created faster, yet reversible changes of the skin peaks. In conclusion, Raman spectroscopy is a potential tool for ex vivo skin diffusion studies. We also concluded that PG and OA have potential synergistic reversible effect on the skin.     

Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae)

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me2SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at five concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me2SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: > 20% for EG, > 30% for Me2SO and methanol, and > 40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modified cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus.     

Direct transfer of bovine embryos frozen-thawed in the presence of propylene glycol or ethylene glycol under on-farm conditions in an integrated embryo transfer program

An integrated bovine embryo transfer program was conducted in collaboration with 11 Japanese prefectural livestock experiment stations. The program was conducted to evaluate the practicability of the direct transfer method for bovine embryos frozen-thawed in the presence of propylene glycol (PG) or ethylene glycol (EG) under on-farm conditions. Embryos at the compacted morula to expanded blastocyst stages were collected from superovulated donors on Day 7 or 8 after estrus and equilibrated in 1.6 M PG or 1.8 M EG in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 20% heat-inactivated calf serum. Embryos were then loaded individually into a 0.25-ml straw and placed directly into a cooling chamber of a programmable freezer precooled to -7 degrees C. After 2 min, the straw was seeded, maintained at -7 degrees C for 8 min more, and then cooled to -30 degrees C either at 0.3 degree C/min or 0.5 degree C/min before being plunged into liquid nitrogen. Embryos at the same stages were also frozen in the presence of 1.4 M glycerol (GLY) by a conventional method, which served as a control. The frozen embryos were thawed by allowing the straws to stand in air for 5 to 10 sec and then immersing them in a 30 degrees C water bath. Embryos frozen-thawed in the presence of PG or EG were nonsurgically transferred into the uterine horn without diluting the cryoprotectant. Embryos frozen-thawed in the presence of GLY were nonsurgically transferred after removing GLY either by the stepwise method (GLY-I) or by in situ dilution with 0.3 M sucrose solution (GLY-II). A total of 1,273 (PG: 400, EG: 418, GLY-I: 177, GLY-II; 278) frozen-thawed embryos was transferred into recipients, yielding 545 pregnancies (overall: 42.8%, PG: 36.0%, EG; 44.7%, GLY-I; 48.6%, GLY-II; 46.0%). The pregnancy rate with PG was significantly lower than that with EG or GLY-II (P < 0.05). The pregnancy rate was affected by the type of cryoprotectant, the region where the embryo transfer program was carried out, the developmental stage of the embryos, the parity of the recipients, and corpus luteum (CL) quality of the recipients. There were no differences in rates of abortion and stillbirth among the 3 cryoprotectants. The present study demonstrates that EG can be effectively used as a cryoprotectant for freezing and direct transfer of bovine embryos, and that the direct transfer method is applicable under on-farm conditions.     

Permeation of glycerol and propane-1,2-diol into human platelets

The permeability of human platelets to glycerol (GLY) and propane-1,2-diol (propylene glycol, PG) has been determined by measuring the time course of their change in volume following abrupt immersion in solutions of these solutes. A simple light-scattering method, and its calibration to measure mean platelet volume is described. The data are analyzed by means of the Kedem-Katchalsky (K-K) equations, modified to take into account the nonideal behavior of both intracellular and extracellular solutes. The values of the K-K parameters at 2, 21, and 37 degrees C, respectively, were as follows: the hydraulic conductivities (Lp) were 1 x 10(-7), 7 x 10(-7) and 3 x 10(-6) cm.sec-1.atm-1; the solute permeabilities for PG (omega RTPG) were 1.9 x 10(-6), 2.8 x 10(-5), and 1.3 x 10(-4) cm.sec-1; the solute permeabilities for GLY (omega RTGLY), at 21 and 37 degrees C only, were 2.6 x 10(-7) and 1.4 x 10(-6) cm.sec-1. The reflection coefficient (sigma) was 1 throughout. The relevant activation energies were -Lp, 16.5 kcal.mol-1; omega RTPG, 20.5 kcal.mol-1; and omega RTGLY, 17.9 kcal.mol-1. The use of these data is illustrated by computing schedules for the addition and removal of GLY and PG so that the amplitudes of changes in platelet volume are held within predetermined limits.     

Membrane permeabilization of phosphatidylcholine liposomes induced by cryopreservation and vitrification solutions

Membranes are the primary site of freezing injury during cryopreservation or vitrification of cells. Addition of cryoprotective agents (CPAs) can reduce freezing damage, but can also disturb membrane integrity causing leakage of intracellular constituents. The aim of this study was to investigate lipid-CPA interactions in a liposome model system to obtain insights in mechanisms of cellular protection and toxicity during cryopreservation or vitrification processing. Various CPAs were studied including dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), dimethyl formamide (DMF), and propylene glycol (PG). Protection against leakage of phosphatidylcholine liposomes encapsulated with carboxyfluorescein (CF) was studied upon CPA addition as well as after freezing-and-thawing. Molecular interactions between CPAs and phospholipid acyl chains and headgroups as well as membrane phase behavior were studied using Fourier transform infrared spectroscopy. A clear difference was observed between the effects of DMSO on PC-liposomes compared to the other CPAs tested, both for measurements on CF-retention and membrane phase behavior. All CPAs were found to inhibit membrane leakiness during freezing. However, exposure to high CPA concentrations already caused leakage before freezing, increasing in the order DMSO, EG, DMF/PG, and GLY. With DMSO, liposomes were able to withstand up to 6 M concentrations compared to only 1 M for GLY. Cholesterol addition to PC-liposomes increased membrane stability towards leakiness. DMSO was found to dehydrate the phospholipid headgroups while raising the membrane phase transition temperature, whereas the other CPAs caused an increase in the hydration level of the lipid headgroups while decreasing the membrane phase transition temperature.     

Cryopreservation of sperm from Atlantic halibut (Hippoglossus hippoglossus, L.) for commercial application

Development of Atlantic halibut (Hippoglossus hippoglossus) aquaculture will be enhanced with cryopreservation of halibut sperm by ensuring a reliable supply of sperm of desired quality and quantity. To assist in its commercial application, the cryopreservation of large volumes of halibut sperm was investigated. Three cryoprotectants were compared: dimethyl sulfoxide (DMSO), polyethylene glycol (PG) and glycerol (GLY) at two concentrations (10% or 15%). Two salt solutions, Hanks’ balanced salt solution (HBSS) and 0.1 M KHCO₃ with 0.125 M sucrose solution (KS) were tested as diluents. Both factors were examined in 1.6 mL volumes. A cryopreservation volume of 4 mL and a low dilution ratio (1:1) were examined separately. Based on motility and fertilization rate, 10% and 15% DMSO diluted with HBSS or KS solution proved to be effective extenders with mean fertilization rates ranging from 52.2 ± 27.2% to 65.8 ± 26.1%; none of which were significantly different from that of the control. Four other extenders, 10% PG or 10% GLY with HBSS or KS, resulted in significantly lower fertilization rates. Use of a 4 mL cryopreservation volume did not exhibit a significant effect on fertilization rate or motility of post-thawed sperm compared to a 1.6 mL volume (P > 0.05); while the use of a dilution ratio of one part sperm with three parts cryopreservation solution (1:3 v/v with sperm concentration of 0.51 ± 0.11 × 10¹⁰ cells/ml) had a significantly better preservation effect than using a ratio of 1:1 with sperm concentration of 1.02 ± 0.21 × 10¹⁰ cells/ml (P < 0.05). From these results, an optimized protocol for the cryopreservation of Atlantic halibut sperm using a volume as large as 4 mL has been established.     

Perfusion of rabbit kidneys with solutions containing propane-1,2-diol

Propane-1,2-diol (propylene glycol, PG) permeates more rapidly than glycerol, has a strong glass-forming tendency, and appears to have a low toxicity. It is therefore attractive as a potential cryoprotectant for renal preservation. In this paper we compared the effect on subsequent function, of exposing rabbit renal cortical slices to 1 M PG or glycerol in a range of vehicle solutions and we demonstrated a remarkably low toxicity of PG at this concentration. Rabbit kidneys were then perfused with solutions containing PG up to a maximum concentration of 3 M, after which the cryoprotectant was removed and the function of cortical slices prepared from the perfused kidneys was assessed. Marked differences in perfusion characteristics were found between PG and glycerol and between different vehicle solutions for PG, but the two most suitable perfusates, both containing about 100 mM mannitol, permitted normal function in slices prepared after removal of PG. These results indicate that, with an appropriate vehicle perfusate, exposure to PG up to a concentration of 3 mol/liter has remarkably little effect upon vascular resistance and the renal cortical functions measured.     

Decrease of transport of some polyols in sickle cells

This paper reports the results of kinetic studies on the inward net-flux of small non-electrolytes (ethylene glycol, glycerol and erythritol) in sickle cells as compared to normal erythrocytes. Net transport rates were evaluated by turbidimetric measurements for ethylene glycol and glycerol and by hematocrit monitoring for erythritol. A 2-fold and 4-fold reduction in the permeability coefficient for ethylene glycol and glycerol, respectively, were found in sickle cells as compared to normal erythrocytes. In contrast, no significant changes in erythritol transport kinetics were observed. The dependence of glycerol permeability on temperature, pH and oxygenation is the same in both types of cells. A significant correlation was observed between glycerol permeability and cell density only for sickle cells. The results indicate that irreversible modifications of membrane proteins, responsible for the glycerol and ethylene glycol transport, do occur in sickle cells.     

Design and <Emphasis Type="Italic">In Vitro</Emphasis> Evaluation of Capsaicin Transdermal Controlled Release Cubic Phase Gels

The purpose of this study was to design and investigate the transdermal controlled release cubic phase gels containing capsaicin using glycerol monooleate (MO), propylene glycol (1,2-propanediol, PG), and water. Three types of cubic phase gels were designed based on the ternary phase diagram of the MO–PG–water system, and their internal structures were confirmed by polarizing light microscopy (PLM) and small-angle X-ray scattering (SAXS). Release results showed the cubic phase gels could provide a sustained system for capsaicin, while the initial water content in the gels was the major factor affecting the release rate. Release kinetics was determined to fit Higuchi’s square-root equation indicating that the release was under diffusion control. The calculated diffusion exponent showed the release from cubic phase gels was anomalous transport. The unique structure of the cubic phases, capsaicin distributed in the lipid bilayers, and cubic phase gel swelling contributed to the release mechanism. The cubic phase gel may be an interesting application for transdermal delivery system of capsaicin in alleviating the post-incision pain.     

Effect of different cryoprotectants on the structural preservation of follicles in frozen zebu bovine (Bos indicus) ovarian tissue

Cryopreservation of ovarian tissue is a new and promising technique for germ-line storage. The objective of this study was to evaluate the effect of four cryoprotectants (at two concentrations each) on the preservation of zebu bovine preantral follicles after ovarian cryostorage. Strips of ovarian cortex were cryopreserved using glycerol (GLY; 10 or 20%), ethylene glycol (EG), propanediol (PROH) or dimethylsulphoxide (DMSO; 1.5 or 3M). In addition, a toxicity test was performed for each cryoprotectant by exposing the ovarian tissue to them without freezing. Tissues were analyzed by histology and transmission electron microscopy. Ovarian tissue frozen in either concentration of DMSO or PROH or in 10% GLY retained a higher percentage of morphologically normal follicles (73-88%) than tissue frozen in 20% GLY or in either concentration of EG (16-52%). In the toxicity test, exposure of tissues to DMSO, PROH or GLY resulted in higher percentages of normal follicles (80-97%) than exposure to EG (49%). Electron microscopy revealed damage to the ultrastructure of follicles frozen in 10% GLY, while follicles cryopreserved in DMSO and PROH at either concentration exhibited normal ultrastructure. In conclusion, DMSO and PROH were the most effective cryoprotectants for zebu ovarian tissue, preserving the structural integrity of somatic and reproductive cells within the ovary.     

Toxicity tests of cryoprotecting agents for Mytilus galloprovincialis (Lamark, 1819) early developmental stages

Global aquaculture production of blue mussel has increased over last years. This work reaffirms the great potential of cryopreservation technique on mussel industry and overcome economic barriers a cause of a traditional and rudimentary management and continue growing. The aim of this work is to set some preliminary basis attending to toxicity of cryoprotecting agents (CPAs) on different development stages of Mytilus galloprovincialis as a start point to develop a stable cryopreservation protocol. Toxicity tests were carried out by using common CPAs (dimethyl-sulfoxide (Me₂SO), glycerol, (GLY), propylene glycol (PG) and ethylene glycol (EG)) in a range from 0.5 to 3 M on fertilized egg, trochophore larva, and D-larva of Mytilus galloprovincialis. Results evidenced more resistance of older development stages to toxicity. Of all CPAs tested, toxicity testing highlights PG or EG as suitable CPAs for cryopreservation of early development stages; whereas D-larva was unaffected by any of the CPAs tested. Preliminary cryopreservation trials were developed to obtain information into cell cryoprotection. Further research should be focused on membrane permeability and other parameters, such as the balance between toxicity and cryoprotective effect of CPAs.     

Propylene Glycol Liposomes as a Topical Delivery System for Miconazole Nitrate: Comparison with Conventional Liposomes

Propylene glycol (PG)-phospholipid vesicles have been advocated as flexible lipid vesicles for enhanced skin delivery of drugs. To further characterize the performance of these vesicles and to address some relevant pharmaceutical issues, miconazole nitrate(MN)-loaded PG nanoliposomes were prepared and characterized for vesicle size, entrapment efficiency, in vitro release, and vesicle stability. An issue of pharmaceutical importance is the time-dependent, dilution-driven diffusion of propylene glycol out of the vesicles. This was addressed by assessing propylene glycol using gas chromatography in the separated vesicles and monitoring its buildup in the medium after repeated dispersion of separated vesicles in fresh medium. Further, the antifungal activity of liposomal formulations under study was assessed using Candida albicans, and their in vitro skin permeation and retention were studied using human skin. At all instances, blank and drug-loaded conventional liposomes were included for comparison. The results provided evidence of controlled MN delivery, constant percent PG uptake in the vesicles (≈45.5%) in the PG concentration range 2.5 to 10%, improved vesicle stability, and enhanced skin deposition of MN with minimum skin permeation. These are key issues for different formulation and performance aspects of propylene glycol-phospholipid vesicles.     

Physicochemical characterization of bioactive polyacrylic acid organogels as potential antimicrobial implants for the buccal cavity

This study describes the formulation and physicochemical characterization of poly(acrylic acid) (PAA) organogels, designed as bioactive implants for improved treatment of infectious diseases of the oral cavity. Organogels were formulated containing a range of concentrations of PAA (3-10% w/w) and metronidazole (2 or 5% w/w, representing a model antimicrobial agent) in different nonaqueous solvents, namely, glycerol (Gly), polyethylene glycol (PEG 400), or propylene glycol (PG). Characterization of the organogels was performed using flow rheometry, compressional analysis, oscillatory rheometry, in vitro mucoadhesion, moisture uptake, and drug release, methods that provide information pertaining to the nonclinical and clinical use of these systems. Increasing the concentration of PAA significantly increased the consistency, compressibility, storage modulus, loss modulus, dynamic viscosity, mucoadhesion, and the rate of drug release. These observations may be accredited to enhanced molecular polymer entanglement. In addition, the choice of solvent directly affected the physicochemical parameters of the organogels, with noticeable differences observed between the three solvents examined. These differences were accredited to the nature of the interaction of PAA with each solvent and, importantly, the density of the resultant physical cross-links. Good correlation was observed between the viscoelastic properties and drug release, with the exception of glycerol-based formulations containing 5 and 10% w/w PAA. This disparity was due to excessive swelling during the dissolution analysis. Ideally, formulations should exhibit controlled drug release, high viscoelasticity, and mucoadhesion, but should flow under minimal stresses. Based on these criteria, PEG 400-based organogels composed of 5% or 10% w/w PAA exhibited suitable physicochemical properties and are suggested to be a potentially interesting strategy for use as bioactive implants designed for use in the oral cavity.     

The Israel National Skin Bank: Quality Assurance and Graft Performance of Stored Tissues

The Israel National Skin Bank (INSB) was founded jointly by the Israel Defense Forces (IDF) Medical Corps and the Ministry of Health in 1986. The prime purpose of the Skin Bank is to treat burn victims incurred at war or during mass casualty incidences. The INSB Protocol is comprised of international skin bank protocols and our previous and present research results. They provide the framework for selecting optimal guidelines for procurement, processing, preservation, storage and evaluation of transplantation performance of viable skin grafts. For evaluation and direct comparison of graft performance of glycerolized or cryopreserved skin stored for long periods, we have applied a mouse recipient model developed by us. This model assesses graft performance before the rejection process takes place. The in vivo design has inherent clinical relevance, which is especially appealing. Cryopreserved skin performed better than glycerolized skin (p > 0.027), but fresh skin performed significantly better than cryopreserved skin (p > 0.003), as analyzed by the Mann–Whitney non-parametric test. Then graft performance of skin specimens were cryopreserved by programmed or stepwise freezing and stored at -80°C or in liquid nitrogen for 1 and 6–10 months was evaluated. The average score of skin preserved by programmed freezing and stored in liquid nitrogen is the highest for both storage periods. This method has a highly significant advantage (p < 0.007) over the others for 6–10 months storage, evaluated by graft adherence. Several interaction factors determine the quality of cryopreserved skin. Highly significant is the interaction factor/'combined effect' of sample variability with the method of cryopreservation or with the storage period. Finally, the results of paired comparison of selected histology criteria of cryopreserved to fresh skin indicated that storage of skin for up to 5 years did not impair significantly its performance compared to fresh skin, whereas, after six years of storage, there was a highly significant (p < 0.001) impairment in skin quality. We offer a simplified in vivo model and analysis for cryopreserved skin graft performance, suggesting that the evaluation procedures, which are issues of great interest in skin banking, may help future skin banks to make informed choices and decisions regarding quality issues.     

Development and Evaluation of Ethyl Cellulose-Based Transdermal Films of Furosemide for Improved In Vitro Skin Permeation

Transdermal films of the furosemide were developed employing ethyl cellulose and hydroxypropyl methylcellulose as film formers. The effect of binary mixture of polymers and penetration enhancers on physicochemical parameters including thickness, moisture content, moisture uptake, drug content, drug–polymer interaction, and in vitro permeation was evaluated. In vitro permeation study was conducted using human cadaver skin as penetration barrier in modified Keshary–Chein diffusion cell. In vitro skin permeation study showed that binary mixture, ethyl cellulose (EC)/hydroxypropyl methylcellulose (HPMC), at 8.5:1.5 ratio provided highest flux and also penetration enhancers further enhanced the permeation of drug, while propylene glycol showing higher enhancing effect compared to dimethyl sulfoxide and isopropyl myristate. Different kinetic models, used to interpret the release kinetics and mechanism, indicated that release from all formulations followed apparent zero-order kinetics and non-Fickian diffusion transport except formulation without HPMC which followed Fickian diffusion transport. Stability studies conducted as per International Conference on Harmonization guidelines did not show any degradation of drug. Based on the above observations, it can be reasonably concluded that blend of EC–HPMC polymers and propylene glycol are better suited for the development of transdermal delivery system of furosemide.