Development and characterisation of a low-concentration sodium dodecyl sulphate decellularised porcine dermis.

The aim of this study was to adapt a proprietary decellularisation process for human dermis for use with porcine skin. Porcine skin was subject to: sodium chloride (1 M) to detach the epidermis, trypsin paste to remove hair follicles, peracetic acid (0.1% v/v) disinfection, washed in hypotonic buffer and 0.1% (w/v) sodium dodecyl sulphate in the presence of proteinase inhibitors followed by nuclease treatment. Cellular porcine skin, decellularised porcine and human dermis were compared using histology, immunohistochemistry, GSL-1 lectin (alpha-gal epitope) staining, biochemical assays, uniaxial tensile and in vitro cytotoxicity tests. There was no microscopic evidence of cells in decellularised porcine dermis. DNA content was reduced by 98.2% compared to cellular porcine skin. There were no significant differences in the biomechanical parameters studied or evidence of cytotoxicity. The decellularised porcine dermis retained residual alpha-gal epitope. Basement membrane collagen IV immunostaining was lost following decellularisation; however, laminin staining was retained.

Evaluation of Copper and Hydrogen Peroxide Treatments on the Biology, Biomechanics, and Cytotoxicity of Decellularized Dermal Allografts.

Decellularized tissue allografts are paving the way as an alternative to cellular tissue transplantation. Effective sterilization or decontamination of tissue allografts is paramount for the safety of the allograft; however, some of the current sterilization procedures have a detrimental effect on the tissue scaffold. The bactericidal and virucidal activity of copper (II) ions and hydrogen peroxide (H2O2) have been widely reported, however, their effect on the biology, biochemistry, and biocompatibility of decellularized tissue have yet to be elucidated. In this study, decellularized human dermis (dCELL human dermis) was treated with copper (II) chloride (CuCl2) and H2O2; both singly and in combination, and parameters, including concentration, pH, and synergy between CuCl2 and H2O2, were evaluated to identify conditions where any detrimental effects on the tissue scaffold were observed. Skin from 13 human donors was retrieved with appropriate consent and processed into dCELL human dermis. The dCELL human dermis was then treated for 3 h with 0.1 mg/L-1 g/L (w/v) CuCl2 and 0.01-7.5% (v/v) H2O2 and combinations of both of these in the same concentration range. dCELL human dermis treated with solutions of 0.1 mg/L-1 g/L CuCl2 or 0.01-7.5% H2O2 caused no detrimental effects on gross histology, collagen denaturation, collagen orientation, and biomechanical properties of the tissue or cytotoxicity. The highest combined concentration of CuCl2 and H2O2 demonstrated an increase in ultimate tensile strength, loss of collagen type IV immunostaining at the dermal-epidermal junction, and in vitro cytotoxicity. Combinations within the range of up to 10 mg/L CuCl2 with up to 0.5% H2O2 had no effect. The data identify the concentrations of CuCl2 and H2O2 solutions that have no effect on the biological, biomechanical, and biochemical properties of dCELL human dermis, while retaining biocompatibility. These treatments may be suitable for use as sterilization/decontamination agents on human decellularized tissues.

Development of an improved bone washing and demineralisation process to produce large demineralised human cancellous bone sponges.

Shaped demineralised bone matrices (DBM) made from cancellous bone have important uses in orthopaedic and dental procedures, where the properties of the material allow its insertion into confined defects, therefore acting as a void filler and scaffold onto which new bone can form. The sponges are often small in size, <1.0 cm(3). In this study, we report on an improved bone washing and demineralisation process that allows production of larger DBM sponges (3.375 or 8.0 cm(3)) from deceased donor bone. These sponges were taken through a series of warm water washes, some with sonication, centrifugation, 100 % ethanol and two decontamination chemical washes and optimally demineralised using 0.5 N hydrochloric acid under vacuum. Demineralisation was confirmed by quantitative measurement of calcium and qualitatively by compression. Protein and DNA removal was also determined. The DBM sponges were freeze dried before terminal sterilisation with a target dose of 25 kGy gamma irradiation whilst frozen. Samples of the sponges were examined histologically for calcium, collagen and the presence of cells. The data indicated lack of cells, absence of bone marrow and a maximum of 1.5 % residual calcium.

Optimized demineralization of human cancellous bone by application of a vacuum.

Human demineralized bone matrix derived from cortical bone is used by surgeons due to its ability to promote bone formation. There is also a need for shaped demineralized bone matrices made from cancellous bone, where the properties of the material allow its insertion into defects, therefore acting as a void filler and scaffold onto which new bone can form. In this study, we report that demineralized bone sponges were prepared by dissecting and cutting knee bone into cancellous bone cubes of 1 cm(3) . These cubes were then taken through a series of warm water washes, some with sonication, centrifugation, and two decontamination chemical washes. The cubes were optimally demineralized into sponges with 0.5N hydrochloric acid under vacuum with constant pH measurement. Demineralization was confirmed by quantitative measurement of calcium and qualitatively by compression. The sponges were freeze dried before terminal sterilisation with a target dose of 25 kGy gamma radiation whilst frozen. Samples of the sponges were histologically examined for calcium and collagen and also tested for osteoinductivity. Data showed well defined collagen staining in the sponges, with little residual calcium. Sponges from two out of three donors demonstrated osteoinductivity when implanted into the muscle of an athymic mouse.

Development of a decellularised dermis.

The purpose of this investigation was to develop a decellularised human dermis suitable for allografting. Samples of human skin were obtained from deceased donors and taken through a series of steps to remove all cellular material. The steps were: chemical removal of the epidermis, disinfection, lysing of cells in hypotonic buffer, a detergent treatment and a nuclease buffer to remove residual nuclear material. Histological preparations of the decellularised dermis produced were then investigated. In addition residual DNA content, structural strength, collagen denaturation, cytotoxicity and in vivo tissue reactivity following implantation in a murine model were examined. For all donors tested there was no change in morphology as viewed by light microscopy. Mean DNA removal was evaluated at 92.1%. There were no significant changes in structural strength or evidence of collagen degradation. The tissue did not appear to be cytotoxic or elicit an immune response when implanted in the mouse model. A decellularised tissue has been developed that would appear to be suitable for a range of surgical procedures.

Development of methods for studying the differentiation of human mesenchymal stem cells under cyclic compressive strain.

Human mesenchymal stem cells (hMSC) have numerous potential advantages over terminally differentiated cells and embryonic stem cells for use in tissue engineering applications. The aims of this study were to develop methods to test the hypothesis that hMSC could be differentiated using cyclic compressive strain alone. hMSC were successfully isolated, purified using D7-FIB antibody, cloned, and characterized. The cells were subsequently analyzed using fluorescence-activated cell sorting using a panel of antibodies and differentiation into multiple cell lineages. D7FIB-positive cells were then seeded into collagen-alginate scaffolds and subjected to 10% or 15% cyclic compressive strain for 4 out of 24 hours for up to 21 days in a bespoke servo-assisted displacement-controlled device. Cells were analyzed using adenosine triphosphate assay to determine cell number, live-dead cell assay, and quantitative real-time polymerase chain reaction at 7 and 21 days. Cloned D7-FIB-positive hMSCs showed evidence of differentiation to an osteogenic lineage under 10% cyclic compressive strain alone (core binding factor alpha 1 (CBFA-1) was significantly upregulated at 7 and 21 days by a factor of 18.3 and 32.2, respectively) and to an osteo-chondrogenic lineage under 15% cyclic compressive strain alone (increased expression of CBFA-1, Sox9, and aggrecan). A combination of a composite viscoelastic scaffold and controlled cyclic compressive strain may be useful for study of the differentiation of MSC.

Development and characterization of acellular allogeneic arterial matrices.

Surgeons have used cryopreserved vascular allografts successfully for many years to treat arterial occlusive disease and to repair arterial aneurysms. Vascular allografts demonstrate high patency rates but contain viable cells, which may evoke a rejection response following implantation. Removing the cells could prevent such a response and negate the need for cryopreservation and ultra-low temperature storage. The objectives of the study were to characterize human common femoral arteries and develop a decellularization protocol with a view to the generation of biocompatible and biomechanically functional vascular grafts for use in vascular bypass and arteriovenous access. The arteries were decellularized by subjecting the tissue to a single freeze-thaw cycle followed by sequential incubation in hypotonic tris buffer and low concentration sodium dodecyl sulphate. Each artery was disinfected using 0.1% (v/v) peracetic acid. Histological analysis demonstrated a lack of cells following decellularization and confirmed the integrity of the tissue histioarchitecture and retention of major structural proteins. There was a >95% reduction in DNA levels. The acellular tissues and extracts were not cytotoxic to either mouse 3T3 or baby hamster kidney cells. Biomechanical properties were determined by burst pressure, compliance, and tensile tests, which confirmed the retention of biomechanical properties following decellularization. In conclusion the study has developed a suitable protocol for the removal of cells from human common femoral arteries without adversely affecting the biochemical or biomechanical properties. These properties indicate the potential use for acellular human common femoral arteries for vascular bypass or arteriovenous access.

The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells.

Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (< or =4% in both intact and acellular groups) and ABCG2 (< or =3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (< or =6%) and ABCG2 (< or =8%). HAM frozen at -80 degrees C in Hank's Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (< or =68%) and ABCG2 (< or =62%) expression (p<0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs this ability whereas simple frozen HAM appears to work extremely well for this purpose.

Biocompatibility of acellular human pericardium.

BACKGROUND: Previous studies have shown successful decellularization of human pericardium without affecting the major structural components and strength of the matrix. The aim of this study was to assess the biocompatibility and reseeding potential of the acellular human pericardial scaffold. MATERIALS AND METHODS: Pericardia were treated sequentially with hypotonic buffer, sodium dodecyl sulfate, and a nuclease solution. The presence of cellular attachment factors after decellularization was evaluated using immunohistochemistry. The scaffold was seeded with dermal fibroblasts and cellular attachment to and numbers of cells penetrating were assessed over time. Biocompatibility was also evaluated following subcutaneous implantation into a mouse model for three months. RESULTS: After decellularization, the scaffold stained positively for fibronectin, but collagen IV and laminin staining was reduced. Seeded fibroblasts attached to the mesothelial surface and were visualized in the tissue within a week of seeding. The majority of fibroblasts in the tissue were viable and there was evidence of remodeling of the matrix. Analysis of the explanted tissues from mice showed that fresh/frozen and glutaraldehyde-fixed pericardia were encapsulated with a thick layer of inflammatory cells and fibrous tissue. In contrast, the decellularized scaffold was infiltrated with myofibroblasts, CD34+ cells and macrophages, indicating a healthy repair process. Compared with the glutaraldehyde-fixed tissue, the calcium content of the fresh/frozen and decellularized pericardia was negligible. CONCLUSIONS: The pericardial scaffold was biocompatible in vitro and in the mouse model in vivo.

Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering.

The aim of this study was to produce a natural, acellular matrix from porcine bladder tissue for use as a scaffold in developing a tissue-engineered bladder replacement. Full-thickness, intact porcine bladders were decellularised by distention and immersion in hypotonic buffer containing 0.1% (w/v) SDS and nuclease enzymes. Histological analysis of the resultant matrices showed they were completely acellular; that the major structural proteins had been retained and that there were some residual poorly soluble intracellular proteins. The amount of DNA per mg dry weight of fresh porcine bladder was 2.8 (+/-0.1) microg/mg compared to 0.1 (+/-0.1) microg/mg in decellularised bladder and biochemical analysis showed proportional differences in the hydroxyproline and glycosaminoglycan content of the tissue before and after decellularisation. Uniaxial tensile testing indicated that decellularisation did not significantly compromise the ultimate tensile strength of the tissue. There was, however, an increase in the collagen and elastin phase slopes indicating decreased extensibility. Cytotoxicity assays using porcine smooth muscle cell cultures excluded the presence of soluble toxins in the biomaterial. In summary, a full-thickness natural acellular matrix retaining the major structural components and strength of the urinary bladder has been successfully developed. The matrix is biocompatible with bladder-derived cells and has potential for use in urological surgery and tissue-engineering applications.

Development of a bacteriophage model system to investigate virus inactivation methods used in the treatment of bone allografts.

Bone allografts are commonly used in a variety of surgical procedures, to reconstruct lost bone stock and to provide mechanical support during the healing process. Due to concerns regarding the possibility of disease transmission from donor to recipient, and of contamination of grafts during retrieval and processing procedures, it is common practice to sterilise bone allografts prior to issue for clinical use. It is vital that the sterilisation processes applied to allografts are validated to demonstrate that they achieve the required level of bioburden reduction, and by extension that validated models are used for these studies. Two common sterilisation protocols applied to bone allografts are gamma irradiation and ethylene oxide gas sterilisation, and there are currently no validated models available for measuring the anti-viral efficacy of ethylene oxide treatment with regard to bone allografts or readily useable models for assessing the anti-viral efficiency of gamma irradiation treatment. We have developed and validated models for both these sterilisation processes, using the bacteriophage varphix174, and utilised the models to measure the antiviral activity of the standard ethylene oxide and gamma irradiation sterilisation processes applied to bone allografts by the National Blood Service. For the irradiation model, we also utilised bacterial spores (Bacillus pumilus). Our results show that ethylene oxide sterilisation (which can only be applied to lyophilised grafts) inactivated > 6.1 log(10) of the model virus, and gamma irradiation (at 25 -40 kGy and applied to frozen allografts) inactivated 3.6 - 4.0 log(10) of the model virus and > 4 log(10) of the bacterial spores. Gamma irradiation at this dosage is therefore not in itself a sterilisation process with respect to viruses.

Production of an acellular amniotic membrane matrix for use in tissue engineering.

A clinical need exists for an immunologically compatible surgical patch with a wide range of uses including soft tissue replacement, body wall repair, cardiovascular applications, and as a wound dressing. This study aimed to produce an acellular matrix from human amniotic membrane for future assessment as a surgical patch and a delivery system for epithelial cells. A novel detergent-based protocol was modified to remove all cellular components from amnion to render it non-immunogenic. Amnion was harvested within 24 h after elective caesarean section (n = 12). One sample group remained fresh, whereas the other was treated with 0.03% (w/v) sodium dodecyl sulphate, with hypotonic buffer and protease inhibitors, nuclease treatment, and terminal sterilization, using peracetic acid (0.1% v/v). Fresh and treated amnion was analyzed histologically for the presence of cells, deoxyribonucleic acid (DNA), collagen, glycosaminoglycans (GAGs), and elastin. Quantitative analysis was performed to determine levels of GAGs, elastin, hydroxyproline, denatured collagen, and DNA. The biomechanical properties of the membrane were determined using uniaxial tensile testing to failure. Histological analysis of treated human amnion showed complete removal of cellular components from the tissue; the histoarchitecture remained intact. All major structural components of the matrix were retained, including collagen type IV and I, laminin, and fibronectin. Differences were observed between fresh and decellularized amnion in matrix hydroxyproline (34.7 microg/mg vs 49.7 microg/mg), GAG (42.5 microg/mg vs 85.4 microg/mg), denatured collagen (2.2 microg/mg vs 1.7 microg/mg), and elastin (359.2 microg/mg vs 490.8 microg/mg) content. DNA content was diminished after treatment. Acellular matrices were biocompatible, cells grew in contact, and there was no decrease in cell viability after incubation with soluble tissue extracts. In addition, no significant reduction in ultimate tensile strength, extensibility, or elasticity was found after decellularization. Removal of the cellular components should eliminate immunological rejection. The resulting matrix was biocompatible in vitro and exhibited no adverse effects on cell morphology or viability.

Development and characterization of an acellular human pericardial matrix for tissue engineering.

This study aimed to produce an acellular human tissue scaffold with a view to recellularization with autologous cells to produce a tissue-engineered pericardium that can be used as a patch for cardiovascular repair. Human pericardia from cadaveric donors were treated sequentially with hypotonic buffer, SDS in hypotonic buffer, and a nuclease solution. Histological analysis of decellularized matrices showed that the human pericardial tissue retained its histioarchitecture and major structural proteins. There were no whole cells or cell fragments. There were no significant differences in the hydroxyproline (normal and denatured collagen) and glycosaminoglycan content of the tissue before and after decellularization (p > 0.05). There were no significant changes in the ultimate tensile strength after decellularization (p > 0.05). However, there was an increased extensibility when the tissue strips were cut parallel to the visualized collagen bundles (p = 0.005). No indication of contact or extract cytotoxicity was found when using human dermal fibroblasts and A549 cells. In summary, successful decellularization of the human pericardium was achieved producing a biocompatible matrix that retained the major structural components and strength of the native tissue.

Guidelines on processing and clinical use of skin allografts.

Processing methods used for banking of skin for subsequent therapeutic use depend on whether the skin is to retain viability or not. For viable skin grafts, sterilisation techniques cannot be applied, however antibiotics and antimycotics may be used to disinfect the tissue with respect to bacteria and fungi. Nevertheless, strict standards are applied to avoid disease transmission from donor to recipient involving donor medical history, donor testing for viral diseases, aseptic retrieval and processing, and control of storage temperature. Cryopreservation is the preferred method for long term storage of viable skin grafts. If viability is not required, then additional long term preservation methods may be used including deep-freezing, freeze-drying or high concentration solute preservation. All three methods work by reducing water activity. In addition it is possible to apply certain sterilisation techniques that have been shown not to damage the tissue. It is important that sterilisation methods are validated in accordance with precise definitions of sterilisation, and for the initial levels of "bioburden" expected to be present immediately prior to application of the sterilisation method. The application of improved and refined methodologies in accordance with defined standards has ensured improved graft performance while reducing risk to the recipient.

In-vitro assessment of the functional performance of the decellularized intact porcine aortic root.

BACKGROUND AND AIMS OF THE STUDY: Tissue-engineered heart valves offer the potential to deliver a heart valve replacement that will develop with the young patient. The present authors' approach is to use decellularized aortic heart valves reseeded in vitro or in vivo with the patient's own cells. It has been reported that treatment of porcine aortic valve leaflets with 0.1% (w/v) sodium dodecyl sulfate (SDS) in hypotonic buffer produced complete leaflet acellularity without affecting tissue strength. The present study aim was to investigate the effect of an additional treatment incorporating 1.25% (w/v) trypsin and 0.1% (w/v) SDS on the biomechanics and hydrodynamics of the aortic root. This treatment has been shown to produce decellularization of both the aorta and valve leaflets. METHODS: Fresh porcine aortic roots were treated to reduce the thickness of their aortic wall, and incubated in hypotonic buffer for 24 h. The leaflets were masked with agarose gel, and the aorta was treated with 1.25% (w/v) trypsin for 4 h at 37 degrees C. The trypsin and agarose were removed and the roots incubated with 0.1% (w/v) SDS in hypotonic buffer for 24 h. Fresh and treated circumferential and axial aortic specimens were subjected to uniaxial tensile testing, while intact porcine aortic roots were subjected to dilation and pulsatile flow testing. RESULTS: Decellularized aortic wall specimens demonstrated significantly decreased elastin phase slope and increased transition strain compared to the fresh control. However, the treatment did not impair tissue strength. Decellularized intact roots presented complete leaflet competence under systemic pressures, increased dilation and effective orifice areas, reduced pressure gradients, physiological leaflet kinematics and reduced leaflet deformation. CONCLUSION: The excellent leaflet kinematics and hydrodynamic performance of the decellularized roots, coupled with the excellent biomechanical characteristics of their aortic wall, form a promising platform for the creation of an acellular valve scaffold with adequate mechanical strength and functionality to accommodate dynamic cell repopulation in vitro or in vivo. This approach can be used for both allogeneic and xenogeneic tissue matrices.

Biocompatibility and recellularization potential of an acellular porcine heart valve matrix.

BACKGROUND AND AIM OF THE STUDY: Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. The study aim was to investigate the biocompatibility and recellularization potential of an acellular porcine valve matrix. METHODS: Acellular porcine valve matrix contact and extract cytotoxicity was tested against porcine fibroblasts and smooth muscle cells (SMC). Porcine cells were incubated with decellularized aortic valve leaflets and aortic wall, and then assessed for changes in morphology and contact inhibition of growth. Soluble tissue extracts were prepared from decellularized leaflets and aortic wall, and assessed for their effect on the viability of cultured porcine cells. Acellular leaflets were seeded with either fibroblasts or SMC at 1 x 10(3) to 1 x 10(6) cells/cm2 for 24 h, or 5 x 10(4) cells/cm2 for 1-4 weeks. Cell attachment onto, and migration into, the acellular matrix was assessed by scanning electron microscopy and histology. RESULTS: No contact inhibition of growth, or changes in fibroblast or SMC morphology, were observed following contact with the acellular valve matrix. No soluble extract cytotoxicity was found. Intermediate cell-seeding densities (2.5 x 10(4) to 7.5 x 10(4) cells/cm2) of both cell types produced confluent cell attachment; at the lowest concentration (1 x 10(3) cells/cm2) cell attachment was sparse, and at the highest (1 x 10(6) cells/cm2) it was multilayered. The SMC migrated throughout the leaflet matrix over four weeks, but there was no fibroblast migration into the matrix. CONCLUSION: The absence of contact and extract cytotoxicity indicated that the acellular valve matrix was biocompatible in vitro. The failure of porcine fibroblasts to grow on, or infiltrate into, the matrix suggested that the SMC may be the preferred cell type for future leaflet recellularization studies in the development of a tissue-engineered heart valve replacement.

Use of peracetic acid to sterilize human donor skin for production of acellular dermal matrices for clinical use.

We previously reported methods for sterilizing human skin for clinical use. In a comparison of gamma-irradiation, glycerol, and ethylene oxide, sterilization with ethylene oxide after treatment with glycerol provided the most satisfactory dermis in terms of structure and its ability to produce reconstructed skin with many of the characteristics of normal skin. However, the use of ethylene oxide is becoming less common in the United Kingdom due to concerns about its possible genotoxicity. The aim of this study was to evaluate peracetic acid as an alternative sterilizing agent. Skin sterilized with peracetic acid was compared with skin sterilized using glycerol alone or glycerol with ethylene oxide. The effect of subsequently storing peracetic acid sterilized skin in glycerol or propylene glycol was also examined. Acellular dermal matrices were produced after removal of the epidermis and cells in the dermis, processed for histological and ultrastructural analysis, and the biological function was evaluated by reconstitution with keratinocytes and fibroblasts. Results showed that sterilized acellular matrices retained the integrity of dermal structure and major components of the basement membrane. There were no overall significant differences in the ability of these matrices to form reconstructed skin, but peracetic acid alone gave a lower histologic score than when combined with glycerol or propylene glycol. We conclude that peracetic acid sterilization followed by preservation in glycerol or propylene glycol offers a convenient alternative protocol for processing of human skin. It is suggested that this sterile acellular dermis may be suitable for clinical use.

Banking of non-viable skin allografts using high concentrations of glycerol or propylene glycol.

The aims of this study were to investigate the kinetics of the current glycerol banking method for the preservation of non-viable skin allografts; to improve it with respect to efficiency and microbial safety; and to investigate the possibility of using propylene glycol in place of glycerol to provide a more rapid process. Skin grafts were preserved in 98% v/v glycerol (GLY) according to the method used in the Sheffield Skin Bank. During the addition and removal processes, the amounts of GLY and water in the skin were determined using the Karl Fischer method and HPLC respectively. Propylene glycol (PG) was investigated as an alternative to glycerol with the object of shortening the process. To avoid the need for prolonged storage in glycerol to disinfect the tissue, and to improve the effectiveness of disinfection, exposure to peracetic acid (PAA) was included and its influence on the kinetics of the preservation process was evaluated. The histological and ultrastructural appearances of skin that had been banked by these methods was also investigated. It was found that the permeation of GLY in skin probably involves two processes: diffusion and binding; the rate of transport was attenuated as the GLY concentration in the skin increased. The current incubation time could be shortened, but an inconveniently prolonged washout process was required. The substitution of PG for GLY accelerated the whole process, particularly the removal process, making the method more convenient for the emergency use of skin grafts in the clinic. The penetration of PG also involved diffusion and binding, but there was no attenuation of transport as the concentration increased. The addition of PAA sterilisation did not alter the transport of GLY or PG. Structural integrity was also maintained with the new banking treatments. An improved banking method can now be proposed; it can be completed in only one working day and the risk of disease transmission is reduced.

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.