John N. Kearney

Guidelines on processing and clinical use of skin allografts

Abstract 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 techiques 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.

Impaction allografting in revision total hip replacement

Impaction allografting of bone has been used successfully as a technique to reconstitute bone loss in both the femur and acetabulum in revision total hip replacement (THR) since the pioneering work of the groups in Nijmegen,[1][1] The Netherlands, and from Exeter,[2][2] England. The procedure

Processing of Whole Femoral Head Allografts: A Method for Improving Clinical Efficacy and Safety

Femoral heads removed during primary hip replacement surgery are widely utilised as a source of allograft bone. Despite evidence that processing these grafts to remove blood and marrow elements improves both the clinical performance and safety of these allografts, many are transplanted without any processing being applied at all. The goal of this study was to investigate the efficiency of an allograft processing protocol which incorporates pasteurisation, (3 h, 56–60°C) centrifugation, (1850g, 2 × 15 min, 40°C) sonication, and repeated washing in warm (56–60°C, 19 h) distilled, sterile water to remove blood and marrow elements from the graft. The protocol also involves applying heat treatment to the grafts which has been demonstrated to inactivate many pathogenic viruses. Following the processing procedure, the grafts are lyophilised and sterilised with ethylene oxide gas. The amount and rate of removal of 4 different components of blood and marrow from 6 whole femoral head allografts were measured. These were lipid, soluble protein, elastase and chloride ions. Lipid removal was assessed gravimetrically by solvent extraction of dried samples, soluble protein by the Bradford assay, elastase by radioimmunoassay and choride ion content by a modified commercially available colorimetric assay. Removing lipid from grafts has been shown to increase the rate of incorporation when the graft is used clinically. Elastase was studied as a marker of leukocyte removal, as evidence suggests the majority of potentially infective transmissible spongiform encephalopathy (TSE) activity resides in a sub-population of leukocytes. Soluble protein was studied as a marker of plasma removal, as a smaller amount of TSE infectivity resides here. Chloride removal was measured as this is a necessary pre-requisite to terminal sterilisation with ethylene oxide. The results showed that the protocol removed 74.5% (range: 68.0–90.8) of the lipid content, 96.4% (range: 94.8–98.4) of the soluble protein content, 97.7% (range: 97.1–100) of the elastase content and 98.8% (range: 98.0–99.2) of the chloride ion content. We have shown that processing designed to improve the clinical efficiency and safety of bone allografts can be accomplished without compromising the structural and biological properties of the graft.

An evaluation of the capacity of differently prepared demineralised bone matrices (DBM) and toxic residuals of ethylene oxide (EtOx) to provoke an inflammatory response in vitro.

Demineralised bone matrix (DBM) is a form of allogeneic tissue graft widely used in oral and maxillofacial procedures. There is a long history of controversy relating to the suitability of ethylene oxide gas (EtOx) as a terminal sterilisation agent for this graft, relating to its effects on the clinical performance of the grafts. Furthermore, the generation of a toxic residual chemical (ethylene chlorohydrin, ECl) during the ethylene oxide sterilisation of patellar tendon allografts has been implicated in the failure of these grafts owing to the induction of a localised inflammatory response. In this study we have investigated the capacity of a range of different DBM preparations, and ECl dilutions, to induce the production of three pro-inflammatory cytokines, interleukin-6 (IL-6), interleukin-1beta (IL-1beta), and tumour necrosis factor alpha (TNF-alpha) from human peripheral blood mononuclear cells (PBMNCs). The levels of EtOx and ECl in EtOx terminally sterilised DBM and mineralised bone grafts were measured by gas chromatography. It was found that the only factor capable of rendering DBM pro-inflammatory was the presence of small (<20 micrometre diameter) DBM particles. No other processing or sterilisation technique resulted in the DBM becoming pro-inflammatory. Although it was also found that DBM, when EtOx-sterilised, retained more ECI than mineralised bone grafts following a standard EtOx sterilisation protocol, ECl did not provoke an inflammatory response in vitro at levels up to and including those which are cytotoxic to PBMNCs.

Effects of a Peracetic Acid Disinfection Protocol on the Biocompatibility and Biomechanical Properties of Human Patellar Tendon Allografts

Patellar tendon allografts, retrieved from cadaveric human donors, are widely used for replacement of damaged cruciate ligaments. In common with other tissue allografts originating from cadaveric donors, there are concerns regarding the potential for disease transmission from the donor to the recipient. Additionally, retrieval and subsequent processing protocols expose the graft to the risk of environmental contamination. For these reasons, disinfection or sterilisation protocols are necessary for these grafts before they are used clinically. A high-level disinfection protocol, utilising peracetic acid (PAA), has been developed and investigated for its effects on the biocompatibility and biomechanics of the patellar tendon allografts. PAA disinfection did not render the grafts either cytotoxic or liable to provoke an inflammatory response as assessed in vitro. However, the protocol was shown to increase the size of gaps between the tendon fibres in the matrix and render the grafts more susceptible to digestion with collagenase. Biomechanical studies of the tendons showed that PAA treatment had no effect on the ultimate tensile stress or Youngs modulus of the tendons, and that ultimate strain was significantly higher in PAA treated tendons.

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.

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) disinfecton 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 cytototoxicity 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-α 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.

Evaluation of NaOH treatment of human dura mater implants to obviate Creutzfeldt- Jakob Disease transmission

One of the few disinfectants known to inactivate the causative agent of Creutzfeldt-Jakob Disease is NaOH. In this study, NaOH was evaluated as a possible routine treatment for human dura mater alloimplants. Use of high concentration NaOH (1 M) resulted in protein loss and macroscopic changes to the tissue. Lower concentrations (0.1 M), although exhibiting little direct detrimental effect, greatly increased the susceptibility of the tissue to collagenase digestion. The use of NaOH treated commercial or institutionally prepared human dura mater should be approached with caution.

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 ϕ×174, 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 >6.1log10 of the model virus, and gamma irradiation (at 25–40 kGy and applied to frozen allografts) inactivated 3.6–4.0log10 of the model virus and >4log10 of the bacterial spores. Gamma irradiation at this dosage is therefore not in itself a sterilisation process with respect to viruses.

Yorkshire Regional Tissue Bank—Circa 50 Years of Tissue Banking

banking began in the early 1950’s, and the US Naval Tissue Bank at Bethesda is well known to be one of the earliest large-scale banks to be established in the world. The impetus for the establishment of this bank was the return to the US of military casualties from the Korean conflict, requiring extensive replacement and reconstructive surgery. Ironically, it was an earlier conflict, the Second World War, and the horrific burn injuries suffered by servicemen in that War, that had led Sir Peter Medawar to attempt to overcome the problems with grafting of skin allografts and, in the process, he laid the foundations for transplantation immunology for which he received the Nobel Prize. Nevertheless, until the advent of immunosuppressive therapy and extensive tissue typing, the transplantation of viable allografts was rarely successful and the Bethesda bank stored tissues largely as non-viable biological implants. Although rightly famed for its pioneering achievements in tissue banking, the US Naval Hospital was not the only centre developing methods for the banking of human donor tissue. The City of Leeds, Yorkshire, was also the focus for research on tissue preservation methodology. The close proximity between the University of Leeds and the Leeds General Infirmary no doubt facilitated many collaborations between the two institutions. The Medical School, housing the University Department of Microbiology, was to the Southern edge of the campus, only yards from the Infirmary. It was natural, therefore, for the surgeons wanting to preserve artery grafts by freeze drying, to approach the microbiologists, not only because of their proximity but also because of their 11 year experience of preserving bacteria by freeze drying (Flewett et al. 1955). Although arterial grafts had been previously preserved in a solution advocated by Gross in 1949 (Gross et al. 1949), these grafts deteriorated after 3 or 4 weeks or storage. In order to freeze dry the arteries a special sample tube was designed to fit onto a modified freeze drier manifold (Fig. 1). The manifold led to a condenser which comprised of a condensing tube immersed in a freezing mixture of dry ice/ acetone within a thermos flask ()79 C). Secondary drying was achieved with a phosphorus pentoxide trap just prior to the vacuum pump. This whole apparatus cost £130 to build, at the time. The original intention was to acquire the arteries aseptically in the operating theatre and to J. N. Kearney (&) National Blood Service Tissue Services, Deansbrook Road, Middlesex, Edgware HA8 9BD, UK e-mail: john.kearney@nbs.nhs.uk Cell Tissue Banking (2006) 7:259–264 DOI 10.1007/s10561-006-9006-z