Impaction allografting in revision total hip replacement.
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Acetabulum↗
Biomedical subjects
J N Kearney
Publications and source records attributed to J N Kearney.
Validation of radiation dose received by frozen unprocessed and processed bone during terminal sterilisation.
Fresh frozen femoral heads (FH) and frozen processed bone (FP) are widely used as a source of allograft bone. The FP bone and some of the FH are terminally sterilised by the National Blood Service Tissue Services (NSBTS), via application of a minimum 25 kGy gamma radiation dose. To comply with the Guidelines for the Blood Transfusion Services in the United Kingdom (2002), frozen musculoskeletal tissue must be maintained below -40 degrees C during storage and transit. In practice, NBSTS stores bone long-term in -80 degrees C freezers. During transport for irradiation, a temperature of circa -79 degrees C is maintained by packing the bone in dry ice. An evaluation of the radiation dose received by bone has previously been made via dosimeters located within the tissue and dry ice, however, some evidence suggests that low temperature can influence the accuracy of the dosimeter readings. The aim of this study was to determine the actual radiation dose received by FH and FP bone during the irradiation process. This was accomplished by comparing radiation dose readings from dosimeters placed in dry ice with dosimeters placed in a dry ice substitute of similar dimensions and density i.e., polytetrafluoroethylene (PTFE) at ambient temperature. New packing formats were developed for both FH and FP bone such that 15 FH or 3 kg of FP bone could be irradiated in one transport box at any given time in a standardised fashion. The data show that low temperature consistently increased dosimeter readings 10--27%, and that radiation dose always fell within the range of 25--40 kGy (FH=25.1--35.7 kGy; FP bone=25.2--32.4 kGy).
Bone Transplantation↗
Clinical effectiveness of processed and unprocessed bone.
Bone allografts have been used clinically for a number of years. Understanding the biology of bone healing and the impact that bone banking has on this helps to improve the methodologies used in increasing the quality and safety of banked bone. Banked bone in its various forms has been used in a variety of surgical procedures, and although there is no doubt that it is clinically effective, most of the studies have been retrospective and non-randomized. The review attempts to summarize some of the data in this area and highlights some of the difficulties encountered in such work. Although there is no doubt that bone banking is nowadays better controlled, there are ever-increasing pressures to produce bone that is as safe as possible with the least impact on its effectiveness. This can only be achieved if the requirements of the providers and users of bone are better understood.
Bone Banks↗
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.
Anti-Bacterial Agents↗
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 Young's modulus of the tendons, and that ultimate strain was significantly higher in PAA treated tendons.
Adult↗
Assessment of the biological properties of human split skin allografts disinfected with peracetic acid and preserved in glycerol.
Skin allografts derived from cadaveric human donors are widely used in the treatment of serious burn injuries and other conditions, such as ulcers. In order to render these allografts safe for clinical use, and to enable them to be preserved and banked for long periods, effective methods of decontamination and preservation are required. These methods must not adversely affect graft properties essential for clinical performance. We have investigated the application of a peracetic acid (PAA) disinfection protocol, coupled with preservation in either glycerol or propylene glycol to achieve these goals. An effective decontamination procedure, comprising of a 3h exposure to 0.1% (v/v) PAA in phosphate buffered saline (PBS) at pH 7.0, was developed and had no significant detrimental effects on the structure of skin. Cadaveric skin allografts were then treated with this disinfection protocol and subsequently preserved in either 85% (v/v) glycerol or propylene glycol in PBS, and the biological properties of the allografts thought to be essential to successful clinical performance were 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. Neither the disinfection protocol nor either of the preservation techniques rendered the grafts cytotoxic or pro-inflammatory. The PAA disinfection and glycerol preservation protocol had no effects on collagenase susceptibility, whereas the disinfection protocol in combination with propylene glycol rendered some of the test samples significantly more susceptible to collagenase digestion. Therefore, this study has demonstrated that PAA disinfection combined with glycerol preservation is suitable for skin allografts. The use of propylene glycol as a preservation agent for skin requires further development.
Bacillus subtilis↗
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.
Bone Demineralization Technique↗
Clinical evaluation of skin substitutes.
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Burns↗
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 degrees C) centrifugation, (1850g, 2 x 15 min, 40 degrees C) sonication, and repeated washing in warm (56-60 degrees 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.
Journal Article↗
Quality issues in skin banking: a review.
Over the last decade the concept of quality in healthcare has gained increasing prominence, not least in the field of skin and tissue banking. This was brought into sharp focus by the appearance of HIV, however, over the decade other viruses have also gained notoriety, e.g. Hepatitis C. The risk of cross infection has been the major factor driving tissue and blood banking towards ever improving standards of quality, which have been reinforced by national legislation and regulations, and professional standards. This has also provided an opportunity to consider other aspects of skin banking that might also affect quality and to offer standards or guidelines to optimise these aspects. The purpose of this review is to highlight these various quality issues in order to allow surgeons to make informed choices and decisions regarding their sources and uses of skin allografts.
Burns↗
Evaluation of proteinase inhibitors and free radical inhibitors/scavengers in reducing post-thaw viability loss of cryopreserved skin.
Cryopreserved skin must be used immediately after thawing or discarded owing to rapid post-thaw deterioration in viability. This is inconvenient and wasteful. The purpose of this study was to evaluate whether release of protease enzymes from cryogenically damaged cells or the action of free radicals on skin cells, is the cause of this deterioration. Following thawing. skin was incubated for 24 h at 4 degrees C in a range of protease inhibitors and free radical inhibitors/scavengers. The rate of deterioration was significantly reduced by using complex treatments including addition of serum, egg white and raised pH. These treatments are known to inhibit various groups of protease enzymes but would clearly have additional effects on the cells. Of the remaining treatments most of the specific protease inhibitors improved viability although not significantly. Treatments designed to inhibit or scavenge free radicals had little or no effect.
Animals↗
Inflammation in acne vulgaris: failure of skin micro-organisms to modulate keratinocyte interleukin 1 alpha production in vitro.
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Acne Vulgaris↗
An investigation into the potential of extracellular matrix factors for attachment and proliferation of human keratinocytes on skin substitutes.
The purpose of this investigation was to assess the potential of commercially available extracellular matrix proteins, as enhancers of human keratinocyte attachment and proliferation, with a view to their incorporation into a skin equivalent. The following substrates were studied: type I and type IV collagen, fibronectin, gelatin and laminin. Human keratinocytes were cultured in low-calcium, serum-free medium. The number of cells attached to each substrate, observed under phase-contrast inverted microscopy in randomly selected fields of view, were counted 2 h afterseeding. Measurements of growth rate and colony-forming efficiency were made at 24-h intervals. None of the substrates tested were found to have an effect significant enough to warrant further investigation or inclusion into skin equivalent.
Analysis of Variance↗
Effect of burn patient serum on fibroblast and keratinocyte cell morphology.
The effect of burn patient serum on fibroblast and keratinocyte cell morphology in culture was investigated using the scanning electron microscope. Serum was taken from five patients with burn injuries ranging from 8 to 65 per cent TBSA (10-65 per cent full-thickness). One patient had superficial burns. Pooled serum from 23 volunteers was used as the control serum. The cells were seeded onto collagen-coated glass coverslips and incubated for 5 days with culture medium containing 10 per cent (v/v) control serum or patient serum taken during the early postburn period. Scanning electron micrographs demonstrated a reduction in fibroblast cell density with serum from patients with full-thickness burns. Furthermore, the spindle shape of the fibroblast cell was greatly exaggerated compared with control cultures. The integrity of the keratinocyte sheet was destroyed when keratinocyte cells were incubated with serum fron patients with full-thickness burns. Globular-like structures or membrane protrusions were present in concentrated areas on keratinocyte cells which were not present in control cultures. This study demonstrated the vulnerability of cutaneous cells to systemic factors present in the early postburn serum. The extent of the effect appears to be related to the presence of full-thickness injury. This effect may further explain the frequent aberrant wound healing response to burn injury.
Adult↗
Donor valves as substitutes for heart valve replacement.
The main problems of heart valve replacement in Sri Lanka are the cost of prosthetic valves and anticoagulant related complications. The use of human donor heart valves (homografts; allogeneic heart valves [AHV]) will alleviate these shortcomings. Recipients of AHV do not require anticoagulant therapy. Moreover, cryopreservation of AHV offers the opportunity for the storage of valves for an extended length of time with the preservation of valve integrity which is essential for their function after implantation. A donor valve bank can potentially provide diameter matched valves for recipients. Current research suggests that the adverse immunological reactions initiated by AHV cause tissue degeneration in a proportion of these implants. However, the grafts may be improved before implantation during the disinfection and storage of the valves. In this essay an overview on the advantages of using AHV, current concepts of valve banking, recent advances in the understanding of AHV immunogenicity, emerging techniques for immunomodulation of AHV and the possibility of setting up a donor heart valve bank in Sri Lanka are discussed.
Heart Valves↗
The effect of cryopreservation on the immunogenicity of allogeneic cardiac valves.
A proportion of implanted cryopreserved allogeneic cardiac valves (ACV) fail due to tissue degeneration initiated by immunological reactions. This study was carried out in a rat model system [Brown Norway (BN; RT1(n)) to Lewis (RT1(l))] to determine the possibility of cryoimmunomodulation of ACV. The immunogenicity of fresh and cryopreserved (1 degrees, 5 degrees, 10 degrees, 30 degrees, and >100 degrees C/min) BN aortic valve conduits (AVC) was assessed using a mixed AVC cell/responder lymphocyte reaction. No significant differences (p > 0.05) in immunogenicity between fresh and cryopreserved (1 degrees C/min and 5 degrees C/min) AVC were observed between 120 and 168 h of co-culture. A significant reduction in immunogenicity was observed with AVC cryopreserved using cooling rates of 10 degrees, 30 degrees, and >100 degrees C/min. The viability of fresh and cryopreserved AVC was determined by a [3H]proline uptake assay. A decrease in viability was observed at cooling rates of more than 1 degrees C/min. The feasibility of cryoimmunomodulation of ACV with the maintenance of viability of a proportion of cells was demonstrated.
Animals↗
Human keratinocyte isolation and cell culture: a survey of current practices in the UK.
A survey was conducted to establish current techniques for isolation and culture of human keratinocytes. A questionnaire was sent to all units thought to be involved in keratinocyte culture, a total of 34 individuals; 62 per cent of those surveyed responded to the questionnaires. The proportion of individuals using high-calcium medium to culture keratinocytes was 53 per cent, while 47 per cent used low-calcium serum-free medium. The majority of replies followed trends dependent on the culture method employed. Details of anatomical donor skin site, keratinocyte isolation and culture were compared. In particular, the problems associated with the use of commercially prepared low-calcium, serum-free medium were reported. The basic principles of keratinocyte culture reported in the literature were seen amongst all the replies received. It is interesting to note the variations in methods adopted as techniques are passed on and continuously modified to suit the requirements of the individual worker. This survey also highlights the difficulties that can occur when using mass-produced complex media.
Adolescent↗
Mechanical properties of tendons: changes with sterilization and preservation.
Tendon allografts are commonly used to replace damaged anterior cruciate ligaments (ACL). Some of the sterilization and preservation techniques used by tissue banks with tendon allografts are thought to impair the mechanical properties of graft tissues. The tensile mechanical properties of porcine toe extensor tendons were measured using a dynamic testing machine following either freezing, freeze-drying, freezing then irradiation at 25 kGy (2.5 MRad), freeze-drying then irradiation, or freeze-drying then ethylene oxide gas sterilization. There was a small but significant difference in Young's modulus between the frozen group (0.88 GPa + 0.09 SD) and both the fresh group (0.98 GPa 1 0.12 SD) and the frozen irradiated group (0.97 GPa 1 0.08 SD). No values of Young's modulus were obtained for the freeze-dried irradiated tendons. The ultimate tensile stress (UTS) of the freeze-dried irradiated group (4.7 MPa 1 4.8 SD) was significantly different from both the fresh and the frozen irradiated groups, being reduced by approximately 90 percent. There were no significant changes in UTS or Young's modulus between any of the other groups. If irradiation is to be used to sterilize a tendon replacement for an ACL it must take place after freeze-drying to maintain mechanical properties.
Animals↗