Cost-effectiveness of decellularised bone allograft compared with fresh-frozen bone allograft for acetabular impaction bone grafting during a revision hip arthroplasty in the UK.

OBJECTIVES: Fresh-frozen allograft is the gold-standard bone graft material used during revision hip arthroplasty. However, new technology has been developed to manufacture decellularised bone with potentially better graft incorporation. As these grafts cost more to manufacture, the aim of this cost-effectiveness study was to estimate whether the potential health benefit of decellularised bone allograft outweighs their increased cost. STUDY DESIGN: A Markov model was constructed to estimate the costs and the quality-adjusted life years of impaction bone grafting during a revision hip arthroplasty. SETTING: This study took the perspective of the National Health Service in the UK. PARTICIPANTS: The Markov model includes patients undergoing a revision hip arthroplasty in the UK. INTERVENTION: Impaction bone grafting during a revision hip arthroplasty using either decellularised bone allograft or fresh-frozen allograft. MEASURES: Outcome measures included: total costs and quality-adjusted life years of both interventions over the lifetime of the model; and incremental cost-effectiveness ratios for both graft types, using base case parameters, univariate sensitivity analysis and probabilistic analysis. RESULTS: The incremental cost-effectiveness ratio for the base case model was found to be £270 059 per quality-adjusted life year. Univariate sensitivity analysis found that changing the discount rate, the decellularised bone graft cost, age of the patient cohort and the revision rate all had a significant effect on the incremental cost-effectiveness ratio. CONCLUSIONS: As there are no clinical studies of impaction bone grafting using a decellularised bone allograft, there is a high level of uncertainty around the costs of producing a decellularised bone allograft and the potential health benefits. However, if a decellularised bone graft was manufactured for £2887 and lowered the re-revision rate to less than 64 cases per year per 10 000 revision patients, then it would most likely be cost-effective compared with fresh-frozen allograft.

P22-A142†Validation of an improved closed system for dispensing serum eye drops.

PURPOSE: NHS Blood and Transplant Tissue and Eye Services provide a serum eye drop (SED) service to patients suffering from severe dry eye syndrome. Currently SED are dispensed using an automatic closed filling system (TF) manufactured by Meise Medizintechnik (Germany). An improved version (ATS) has recently been introduced by Meise, based on patient feedback on the TF system. ATS vials are easier to open, with a more secure, tamper evident closure and a better quality nozzle.To evaluate the suitability of ATS vials, a validation protocol, previously developed for TF vials, was repeated. It comprised assessment of their integrity following simulated storage and transport, and the stability and sterility of SED stored in them. METHOD: Firstly, a process simulation assessment was performed using bovine serum. Vials were filled, and frozen to -80oC. They were then removed from frozen storage and checked for damage, before being put into transport containers and shipped on a round-trip journey to simulate delivery to patients. On return the vials were thawed and the integrity of each vial checked visually and by application of a standard force.Subsequently a shelf-life study was carried out using three batches of human SED. The vials were initially frozen to -80oC, then stored for set time points of 1, 3, 6 and 12 months in a standard domestic freezer set at 20oC (to mimic a home freezer). At each time point, 10 vials were thawed and examined for integrity, and the sterility and stability of the contents. Stability was assessed by measuring serum albumin concentrations and sterility by testing for presence of microbial contamination, under aerobic and anaerobic conditions. RESULTS: No vial damage or leakage was found at any time point in the ATS vials. No microbial contamination was detected, and no change in albumin levels was detected in SED throughout the storage period. CONCLUSION: This study has demonstrated that the ATS vials are suitable for provision of SED for clinical use to patients. Feedback is now being gathered from a patient focus group relating to usability of the vials.

P21-A141†Quantification of bioactive factors in human serum eyedrops.

PURPOSE: NHS Blood and Transplant supply serum eye drops (SED) for the treatment of severe dry eye syndrome, however, understanding of what components of SED contribute to their activity is limited. SEDs are produced from a patient's own blood or from an allogeneic donor source. The serum component is separated from the whole blood which is then diluted 50/50 with sterile saline, and contains bioactive molecules that are believed to help heal and maintain the ocular surface. The objective of this study is to quantify the amount of bioactive molecules in donor serum, and to understand how processing variables effects these factors. METHODS: Samples of SEDs from 28 male allogenic donors were taken from ultra-low temperature storage and thawed. They were then centrifuged at 13,000 rpm at 4oC to remove potential contaminants such as residual red blood cells. Duplicate test samples were analysed for epidermal growth factor (EGF) and fibroblast growth factor (FGF) using ELISA kits. Analysis was carried out using Excel. RESULTS: The age range of the donors was 17 to 79 years (mean 47.9).Mean time from venepuncture to refrigerated storage was 6 hours 12 minutes with time ranging from 2 hours 40 minutes to 9 hours 35 minutes.The concentration of EGF found in the diluted serum ranged from 0.048 to 1.90 ng/ml (mean 0.87 ng/ml), and FGF concentration ranged from 4.88 to 39.50 pg/ml (mean 12.37 pg/ml).Analysis showed that there was no correlation between either age of the donor, or sample transfer time and growth factor concentration. CONCLUSION: Our study demonstrated that with both types of growth factors measured in the SED, a wide range of concentrations were found in the donor samples. Compared to published data EGF was at higher range while FGF was lower. Further analysis of other factors present in the donor serum is being undertaken to determine if any pattern can be found.

Repopulation of decellularised porcine pulmonary valves in the right ventricular outflow tract of sheep: Role of macrophages.

The primary objective was to evaluate performance of low concentration SDS decellularised porcine pulmonary roots in the right ventricular outflow tract of juvenile sheep. Secondary objectives were to explore the cellular population of the roots over time. Animals were monitored by echocardiography and roots explanted at 1, 3, 6 (n = 4) and 12 months (n = 8) for gross analysis. Explanted roots were subject to histological, immunohistochemical and quantitative calcium analysis (n = 4 at 1, 3 and 12 months) and determination of material properties (n = 4; 12 months). Cryopreserved ovine pulmonary root allografts (n = 4) implanted for 12 months, and non-implanted cellular ovine roots were analysed for comparative purposes. Decellularised porcine pulmonary roots functioned well and were in very good condition with soft, thin and pliable leaflets. Morphometric analysis showed cellular population by 1 month. However, by 12 months the total number of cells was less than 50% of the total cells in non-implanted native ovine roots. Repopulation of the decellularised porcine tissues with stromal (α-SMA+; vimentin+) and progenitor cells (CD34+; CD271+) appeared to be orchestrated by macrophages (MAC 387+/ CD163low and CD163+/MAC 387-). The calcium content of the decellularised porcine pulmonary root tissues increased over the 12-month period but remained low (except suture points) at 401 ppm (wet weight) or below. The material properties of the decellularised porcine pulmonary root wall were unchanged compared to pre-implantation. There were some changes in the leaflets but importantly, the porcine tissues did not become stiffer. The decellularised porcine pulmonary roots showed good functional performance in vivo and were repopulated with ovine cells of the appropriate phenotype in a process orchestrated by M2 macrophages, highlighting the importance of these cells in the constructive tissue remodelling of cardiac root tissues.

Conjunctival epithelial cells resist productive SARS-CoV-2 infection.

Conjunctival epithelial cells, which express viral-entry receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine type 2 (TMPRSS2), constitute the largest exposed epithelium of the ocular surface tissue and may represent a relevant viral-entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of basal, suprabasal, and superficial epithelial cells, and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA sequencing (RNA-seq), with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-κB activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplantation.

One threat, different answers: the impact of COVID-19 pandemic on cornea donation and donor selection across Europe.

OBJECTIVES: To assess to which extent the COVID-19 pandemic affected corneal transplantation by virtue of donor selection algorithms in different European countries. DESIGN: Survey. SETTING: 110 eye banks in 26 European countries. PARTICIPANTS: 64 eye banks covering 95% of European corneal transplantation activity. INTERVENTIONS: A questionnaire listing the number of corneas procured and distributed from February to May 2018-2020 was circulated to eye banks. MAIN OUTCOME MEASURES: The primary outcome was the number of corneal procurements. Additional outcomes were national algorithms for donor selection, classified according to their stringency (donors with COVID-19 history, suspected for COVID-19, asymptomatic, PCR testing) and the pandemic severity in each country. We calculated Spearman's correlation coefficient to determine, two by two, the relationship between the 3-month decline in eye banking activity (procurement), the stringency of donor selection algorithm and the grading of pandemic severity (cases and deaths). A partial correlation was run to determine the relationship between decline and stringency while controlling for pandemic severity. RESULTS: Procurements decreased by 38%, 68% and 41%, respectively, in March, April and May 2020 compared with the mean of the previous 2 years, while grafts decreased, respectively, by 28%, 68% and 56% corresponding to 3866 untreated patients in 3 months. Significant disparities between countries and the decrease in activity correlated with stringency in donor selection independent of pandemic severity. CONCLUSIONS: Our data demonstrate significant differences between countries regarding donor screening algorithms based on precautionary principles and, consequently, a decrease in the donor pool, already constrained by a long list of contraindications. Fundamental studies are needed to determine the risk of SARS-CoV-2 transmission by corneal transplantation and guide evidence-based recommendations for donor selection to justify their substantial medical and economic impact.

Decellularised human bone allograft from different anatomical sites as a basis for functionally stratified repair material for bone defects.

Tissue engineered bone solutions aim to overcome the limitations of autologous and allogeneic grafts. Decellularised tissues are produced by washing cellular components from human or animal tissue to produce an immunologically safe and biocompatible scaffold, capable of integration following implantation. A decellularisation procedure utilising low concentration sodium dodecyl sulphate (0.1% w/v) was applied to trabecular bone from human femoral heads (FH) and tibial plateaus (TP). Biological (histology, DNA quantification), biomechanical (compression testing) and structural (µCT) comparisons were made between decellularised and unprocessed cellular tissue. Total DNA levels of decellularised FH and TP bone were below 50 ng mg-1 dry tissue weight and nuclear material was removed. No differences were found between cellular and decellularised bone, from each anatomical region, for all the biomechanical and structural parameters investigated. Differences were found between cellular FH and TP and between decellularised FH and TP. Decellularised FH had a higher ultimate compressive stress, Young's modulus and 0.2% proof stress than decellularised TP (p = 0.001, 0.002, 0.001, Mann Whitney U test, MWU). The mineral density of cellular and decellularised TP bone was significantly greater than cellular and decellularised FH bone respectively (cellular: p = 0.001, decellularised: p < 0.001, MWU). The bone volume fraction and trabecular thickness of cellular and decellularised FH bone were significantly greater than cellular and decellularised TP bone respectively (cellular: p = 0.001, 0.005; decellularised: p < 0.001, <0.001, MWU). Characterisation of decellularised trabecular bone from different anatomical regions offers the possibility of product stratification, allowing selection of biomechanical properties to match particular anatomical regions undergoing bone graft procedures.

26†Development of human amniotic membrane products for regenerative medicine applications.

INTRODUCTION: Human amniotic membrane (HAM) has important biological properties that make this tissue an ideal substrate for regenerative medicine applications, including treatment of ocular diseases and wound healing. NHSBT can successfully decellularise HAM for promoting enhancement of limbal stem cell expansion in vitro more efficiently than the cellular HAM.1 In this study we present new formulations of decellularised HAM as freeze-dried powder and derived natural hydrogel. The aim was to develop a variety of GMP-compliant allografts to treat ocular diseases. MATERIALS AND METHODS: Six HAM, obtained from elective caesarian deliveries, were dissected, decontaminated and subjected to an in-house developed decellularisation protocol including a mild SDS concentration as detergent and nuclease steps. Following decellularisation, the tissue was placed in a sterile tissue culture flask and freeze dried. The freeze-dried tissue was cut into pieces of ~1g each, dipped into liquid nitrogen, then ground with a pulverisette. Ground tissue was solubilised using porcine pepsin and 0.1M HCl (stirred for 48 hours, 25oC). At the end of solubilisation, the pre-gel solution was kept on ice to adjust the pH back to 7.4. Gelation was induced when the temperature of the solution was increased to 25oC and aliquots were used for both in vitro cytotoxicity (up to 48 hours) and biocompatibility (up to 7 days) testing (MG63 and HAM cells). Cells were added into the solution before gelling and on top after gelling. RESULTS: The pre-gel solution obtained from decellularised HAM appear homogenous without undigested powder, and it was able to gel within 20 minutes at RT. Gels with a concentration of 4-8mg/mL tissue powder retained shape (including in an aqueous environment). When added on top of gels, cells were observed to attach and proliferate over time. When added into gels, the cells were observed throughout the gels and appeared to be migrating through the gel. CONCLUSION: Acellular HAM can be successfully freeze dried and converted into new formulations for topical application (powder and hydrogel). The new formulations could improve HAM delivery and provide a better scaffold for tissue regeneration. To our knowledge, this is the first time an amnion hydrogel formulation has been developed in GMP compliant setting for tissue banking purpose. Further studies will also investigate the ability of amnion hydrogel to promote stem cells differentiation into the three lineages (adipogenic, chondrogenic, osteogenic) in and/or on the gels. REFERENCES: Figueiredo GS et al. Acta Biomater 2017;61, 124-133.

29†Production of ultra-thin decellularised dermis to treat severe ocular diseases.

INTRODUCTION: The ocular surface may be damaged by several ocular conditions such as chemical trauma, infection, neoplasia or autoimmune disease causing a loss of tissue and function leading to a painful loss of vision. Tissue regeneration is needed to re-establish homeostasis of the ocular surface and to preserve vision. Present replacement strategies have limitations ranging from availability of the same type of tissue to long-term stability. NHSBT currently produces decellularised dermis (DCD) for clinical allografting; comprising a "thin" (up to 1.0 mm) and a thick (>1.2 mm) DCD, used to treat non-healing leg ulcers or in rotator cuff repair. Even the thin DCD, however, is too thick for ophthalmic purposes. The objective of this study was to develop a new ultra-thin DCD for ocular allografting. MATERIALS AND METHODS: Skin was retrieved, with consent for non-clinical use, from the back, front and back of the thighs of 3 different deceased donors, within 48 hours post-mortem. The tissue was cut into 5x5 cm squares and decellularised over 5 days as follows: decontamination with antimicrobials, de-epidermalisation (1M NaCl), hypotonic washes, detergent washes (with 0.01% SDS) and nuclease incubation. The DCD obtained was examined for integrity, handleability, residual remaining DNA and potential ultra-structural changes (by histology, DAPI and hematoxylin and eosin staining). RESULTS: We obtained an intact ultra-thin DCD using the same standard GMP protocol, regularly used to decellularise skin for clinical use. Tissue handleability was comparable to amniotic membrane, as evaluated by the ophthalmic surgeons as well as tissue bank assistants. The mean thickness of the tissue was 0.25 mm (±0.11) at the end of processing (total N=18 samples from 3 donors). Histology confirmed successful removal of epithelial cells and integrity of the extracellular matrix. CONCLUSION: We have successfully validated standard operating procedures for the production of ultra-thin DCD, in the attempt to obtain a valid alternative to amnion for the reconstruction of specific ocular regions (fornix, eye lids), where increased strength may be required. The thickness measurements at the end of processing suggest ultra-thin DCD obtained could represent a promising scaffold for regeneration of conjunctival tissue.

28†Validation of a closed system for dispensing serum eye drops.

INTRODUCTION: NHS Blood and Transplant Tissue and Eye Services (TES) offer a serum eyedrop (SE) service to patients suffering from severe ocular surface disease. SE are prepared from serum collected at blood donation sessions; the serum is diluted 1:1 with physiological saline. Formerly, 3ml aliquots of diluted serum were aliquoted into glass bottles in a Grade B clean room. Since this service was started, Meise Medizintechnik have developed an automatic closed filling system consisting of tubing-linked chains of squeezable vials. They can be heat-sealed closed, under sterile conditions, after the vials have been filled. MATERIALS AND METHODS: TES R&D were asked to validate the Meise system to increase the efficiency and speed of SE production. Validation of the closed system consisted of a process simulation assessment, using bovine serum and simulating each step of the filling process, freezing to -80oC, checking the integrity of each vial and packing the vials into storage containers. They were then put into transport containers and shipped on a round-trip journey to simulate delivery to patients. On return the vials were thawed and the integrity of each vial re-checked visually and by squeezing in a plasma expressor.Subsequently a shelf-life study was carried out on three batches of fully consented human allogeneic SE. The serum was dispensed into vials, frozen as above and stored for set time points 0, 1, 3, 6 and 12 months in a standard domestic freezer set at -15-20oC to mimic a patient's freezer. At each time point, 10 random samples of vials were removed, and the outer containers were tested for damage or deterioration, the vials for integrity and their contents for sterility and stability. Stability was assessed by measuring serum albumin concentrations and sterility by testing for microbial contamination. RESULTS: No structural damage or leakage was found in any of the vials, or the tubing evaluated, after thawing, at any time point. In addition, all samples tested negative for microbial contamination and serum albumin levels were always within the expected range (3 - 5 Dg/L) at each set time point. CONCLUSION: These results demonstrate that Meise closed system vials can successfully dispense SE drops and the vials can be stored frozen without affecting integrity, sterility or stability. These vials have been in use in TES for 3 years saving clean room space and greatly increasing the numbers of patients that can use the SE service.

3†Emergency salvage of time expired clinical corneas during the COVID-19 pandemic.

INTRODUCTION: Corneas for clinical use can be stored for a maximum of 28 days in organ culture medium after death. At the beginning of the COVID-19 pandemic in 2020 it became apparent that; a rare situation was arising in that clinical operations were being cancelled and that there would be a surplus of "clinical grade" corneas. Consequently, when the corneas reached the end of the storage period, if the tissue had appropriate consent, they were transferred to the Research Tissue Bank (RTB). However, University research had also stopped due to the pandemic and there was a situation where the RTB had good quality tissue without any users. Rather than discarding the tissue, a decision was made to store the tissue for future use by cryopreservation. MATERIALS AND METHODS: An established protocol for cryopreserving heart valves was adapted. Individual corneas were placed into wax histology cassettes then inside a Hemofreeze heart valve cryopreservation bag with 100 ml cryopreservation medium (10% Dimethyl sulphoxide)). They were frozen in a controlled rate freezer (Planer, UK) to below -150oC and stored in vapour phase over liquid nitrogen (VPLN) below -190oC. To assess morphology, six corneas were cut in half, one half was processed for histology whilst the other half was cryopreserved, stored for 1 week then thawed and processed for histology. The stains used were Haematoxylin and Eosin (H&E) and Miller's with Elastic Van Gieson (EVG). RESULTS: Comparative histological examination indicated that there were no visible, major, detrimental changes in morphology in the cryopreserved group as compared to the controls. Subsequently, a further, 144 corneas were cryopreserved. Samples were assessed for handling properties by eye bank technicians and ophthalmologists. The eye bank technicians felt that the corneas may be suitable for training purposes such a DSAEK or DMEK. The ophthalmologists said that they had no preference between the fresh or cryopreserved corneas, and both would be equally suitable for training purposes. CONCLUSION: Time expired, organ-cultured corneas, can be successfully cryopreserved using an established protocol by adapting the storage container and conditions. These corneas are suitable for training purposes and may prevent discard of corneas in future.

9†Supply of non-clinical ocular tissue from a tissue and eye services research tissue bank.

INTRODUCTION: NHS Blood and Transplant Tissue and Eye Services (TES) is a human multi-tissue, tissue bank supplying tissue for transplant to surgeons throughout the UK. In addition, TES provides a service to scientists, clinicians and tissue bankers by providing a range of non-clinical tissue for research, training and education purposes. A large proportion of the non-clinical tissues supplied is ocular tissue ranging from whole eyes, to corneas, conjunctiva, lens and posterior segments remaining after the cornea is excised. The TES Research Tissue Bank (RTB) is based within the TES Tissue Bank in Speke, Liverpool and is staffed by two full-time staff. Non-clinical tissue is retrieved by Tissue and Organ Donation teams across United Kingdom. The RTB works very closely with two eye banks within TES, the David Lucas Eye Bank in Liverpool and the Filton Eye Bank in Bristol. Non-clinical ocular tissues are primarily consented by TES National Referral Centre Nurses. METHODS AND RESULTS: The RTB receives tissue via two pathways. The first pathway is tissue specifically consented and retrieved for non-clinical use and the second pathway is tissue that becomes available when tissue is found to be unsuitable for clinical use. The majority of the tissue that the RTB receives from the eye banks comes via the second pathway. In 2021, the RTB issued more than 1000 samples of non-clinical ocular tissue. The majority of the tissue, ~64% was issued for research purposes (including research into glaucoma, COVID-19, paediatrics and transplant research), ~31% was issued for clinical training purposes (DMEK and DSAEK preparation, especially after COVID-19 cessation of transplant operations, training for new eye bank staff) and ~5% was issued for in-house and validation purposes. One of the findings was that corneas are still suitable for training purposes up to 6-months after removal from the eye.In 2021, the RTB received 43 applications for ocular projects from new customers and supplied to 36 different projects, meeting 95% of all orders placed this year. DISCUSSION: The RTB works to a partial cost-recovery system and in 2021 became self-sufficient. The supply of non-clinical tissue is crucial for advancement in patient care and has contributed to several peer-reviewed publications.

Effects of Chemical and Radiation Sterilisation on the Biological and Biomechanical Properties of Decellularised Porcine Peripheral Nerves.

There is a clinical need for novel graft materials for the repair of peripheral nerve defects. A decellularisation process has been developed for porcine peripheral nerves, yielding a material with potentially significant advantages over other devices currently being used clinically (such as autografts and nerve guidance conduits). Grafts derived from xenogeneic tissues should undergo sterilisation prior to clinical use. It has been reported that sterilisation methods may adversely affect the properties of decellularised tissues, and therefore potentially negatively impact on the ability to promote tissue regeneration. In this study, decellularised nerves were produced and sterilised by treatment with 0.1% (v/v) PAA, gamma radiation (25-28 kGy) or E Beam (33-37 kGy). The effect of sterilisation on the decellularised nerves was determined by cytotoxicity testing, histological staining, hydroxyproline assays, uniaxial tensile testing, antibody labelling for collagen type IV, laminin and fibronectin in the basal lamina, and differential scanning calorimetry. This study concluded that decellularised nerves retained biocompatibility following sterilisation. However, sterilisation affected the mechanical properties (PAA, gamma radiation), endoneurial structure and basement membrane composition (PAA) of decellularised nerves. No such alterations were observed following E Beam treatment, suggesting that this method may be preferable for the sterilisation of decellularised porcine peripheral nerves.

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells.

PURPOSE: Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. METHODS: Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). RESULTS: scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks. CONCLUSIONS: Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.

Co-expression of SARS-CoV-2 entry genes in the superficial adult human conjunctival, limbal and corneal epithelium suggests an additional route of entry via the ocular surface.

PURPOSE: The high infection rate of SARS-CoV-2 necessitates the need for multiple studies identifying the molecular mechanisms that facilitate the viral entry and propagation. Currently the potential extra-respiratory transmission routes of SARS-CoV-2 remain unclear. METHODS: Using single-cell RNA Seq and ATAC-Seq datasets and immunohistochemical analysis, we investigated SARS-CoV-2 tropism in the embryonic, fetal and adult human ocular surface. RESULTS: The co-expression of ACE2 receptor and entry protease TMPRSS2 was detected in the human adult conjunctival, limbal and corneal epithelium, but not in the embryonic and fetal ocular surface up to 21 post conception weeks. These expression patterns were corroborated by the single cell ATAC-Seq data, which revealed a permissive chromatin in ACE2 and TMPRSS2 loci in the adult conjunctival, limbal and corneal epithelium. Co-expression of ACE2 and TMPRSS2 was strongly detected in the superficial limbal, corneal and conjunctival epithelium, implicating these as target entry cells for SARS-CoV-2 in the ocular surface. Strikingly, we also identified the key pro-inflammatory signals TNF, NFKß and IFNG as upstream regulators of the transcriptional profile of ACE2+TMPRSS2+ cells in the superficial conjunctival epithelium, suggesting that SARS-CoV-2 may utilise inflammatory driven upregulation of ACE2 and TMPRSS2 expression to enhance infection in ocular surface. CONCLUSIONS: Together our data indicate that the human ocular surface epithelium provides an additional entry portal for SARS-CoV-2, which may exploit inflammatory driven upregulation of ACE2 and TMPRSS2 entry factors to enhance infection.

Single-stage repair of contaminated hernias using a novel antibiotic-impregnated biologic porcine submucosa tissue matrix.

BACKGROUND: Single-stage repair of incisional hernias in contaminated fields has a high rate of surgical site infection (30-42%) when biologic grafts are used for repair. In an attempt to decrease this risk, a novel graft incorporating gentamicin into a biologic extracellular matrix derived from porcine small intestine submucosa was developed. METHODS: This prospective, multicenter, single-arm observational study was designed to determine the incidence of surgical site infection following implantation of the device into surgical fields characterized as CDC Class II, III, or IV. RESULTS: Twenty-four patients were enrolled, with 42% contaminated and 25% dirty surgical fields. After 12 months, 5 patients experienced 6 surgical site infections (21%) with infection involving the graft in 2 patients (8%). No grafts were explanted. CONCLUSIONS: The incorporation of gentamicin into a porcine-derived biologic graft can be achieved with no noted gentamicin toxicity and a low rate of device infection for patients undergoing single-stage repair of ventral hernia in contaminated settings. TRIAL REGISTRATION: The study was registered March 27, 2015 at www.clinicaltrials.gov as NCT02401334.

Volatile organic compounds emitted from faeces as a biomarker for colorectal cancer.

BACKGROUND: Colorectal cancer remains a leading cause of mortality and morbidity. The UK Bowel Cancer Screening Programme (BCSP) has demonstrated that detection of colorectal cancer at an earlier stage and identification of advanced pre-malignant adenomas reduces mortality and morbidity. AIM: To assess the utility of volatile organic compounds as a biomarker for colorectal neoplasia. METHODS: Faeces were collected from symptomatic patients and people participating in the UK BCSP, prior to colonoscopy. Headspace extraction followed by gas chromatography mass spectrometry was performed on faeces to identify volatile organic compounds. Logistic regression modelling and 10-fold cross-validation were used to test potential biomarkers. RESULTS: One hundred and thirty-seven participants were included (mean age 64 years [range 22-85], 54% were male): 60 had no neoplasia, 56 had adenomatous polyp(s) and 21 had adenocarcinoma. Propan-2-ol was significantly more abundant in the cancer samples (P < 0.0001, q = 0.004) with an area under ROC (AUROC) curve of 0.76. When combined with 3-methylbutanoic acid the AUROC curve was 0.82, sensitivity 87.9% (95% CI 0.87-0.99) and specificity 84.6% (95% CI 0.65-1.0). Logistic regression analysis using the presence/absence of specific volatile organic compounds, identified a three volatile organic compound panel (propan-2-ol, hexan-2-one and ethyl 3-methyl- butanoate) to have an AUROC of 0.73, with a person six times more likely to have cancer if all three volatile organic compounds were present (P < 0.0001). CONCLUSIONS: Volatile organic compound analysis may have a superior diagnostic ability for the identification of colorectal adenocarcinoma, when compared to other faecal biomarkers, including those currently employed in UK. Clinical trial details: National Research Ethics Service Committee South West - Central Bristol (REC reference 14/SW/1162) with R&D approval from University of Liverpool and Broadgreen University Hospital Trust (UoL 001098).

Surgical audit: are we not closing the loop?

PURPOSE: A clinical audit is a key component of the clinical governance framework. The rate of audit completion in general surgery has not been investigated. The purpose of this paper is to assess the rates of audit activity and completion and explore the barriers to successful audit completion. DESIGN/METHODOLOGY/APPROACH: This was a multi-centre study evaluating current surgical audit practice. A standardised audit proforma was designed. All clinical audits in general surgery during a two-year period were identified and retrospectively reviewed. Data held by the audit departments were collated, and individual audit teams were contacted to verify the data accuracy. Audit teams failing to complete the full audit cycle with a re-audit were asked to explain the underlying reasons behind this. FINDINGS: Of the six trusts approached, two refused to participate, and one failed to initiate the project. A total of 39 audits were registered across three surgical directorates. Only 15 out of 39 audits completed at least one audit cycle, with 4 deemed of no value to re-audit. Only seven audits were completed to re-audit. Achieving a publication or a presentation was the most cited reason for not completing the audit loop. ORIGINALITY/VALUE: This study demonstrates that the poor rates of audit completion rate found in other areas of clinical medicine pervade general surgery. Improved completion of an audit is essential and strategies to achieve this are urgently needed.