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.

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability.

One of the main challenges in limbal stem cell (LSC) biology and transplantation is the lack of definitive cell surface markers which can be used to identify and enrich viable LSCs. In this study, expression of 361 cell surface proteins was assessed in ex vivo expanded limbal epithelial cells. One marker, CD200 was selected for further characterization based on expression in a small subset of limbal epithelial cells (2.25% ± 0.69%) and reduced expression through consecutive passaging and calcium induced differentiation. CD200 was localized to a small population of cells at the basal layer of the human and mouse limbal epithelium. CD200+ cells were slow cycling and contained the majority of side population (SP) and all the holoclone forming progenitors. CD200+ cells displayed higher expression of LSCs markers including PAX6, WNT7A, CDH3, CK14, CK15, and ABCB5 and lower expression of Ki67 when compared to CD200- . Downregulation of CD200 abrogated the ability of limbal epithelial cells to form holoclones, suggesting an important function for CD200 in the maintenance and/or self-renewal of LSCs. A second marker, CD109, which was expressed in 56.29% ± 13.96% of limbal epithelial cells, was also found to co-localize with ΔNp63 in both human and mouse cornea, albeit more abundantly than CD200. CD109 expression decreased slowly through calcium induced cell differentiation and CD109+ cells were characterized by higher expression of Ki67, when compared to CD109- subpopulation. Together our data suggest that CD200 expression marks a quiescent population of LSCs with holoclone forming potential, while CD109 expression is associated with a proliferative progenitor phenotype. Stem Cells 2018;36:1723-1735.

Clinical evaluation of Roche COBAS® AmpliPrep/COBAS® TaqMan® CMV test using nonplasma samples.

Cytomegalovirus (CMV) infection is a leading cause of loss of hearing, vision, and mental retardation in congenitally infected children. It is also associated with complications of organ transplant and opportunistic HIV coinfection. The Roche COBAS® AmpliPrep/COBAS® TaqMan® CMV test is an FDA-approved test that measures CMV DNA viral load in plasma for the diagnosis and management of patients at risk of CMV-associated diseases. Besides plasma, CMV is often found in bronchoalveolar lavage (BAL), cerebrospinal fluid (CSF), and urine. Thus, monitoring of CMV for critical care of patients in these nonplasma samples becomes necessary. The objective of this study was to conduct an analytic and clinical feasibility study of the Roche CMV test in BAL, CSF, and urine. The lower limit of detection, analytic measurement range, assay sensitivity, specificity, and precision were determined. Results of this study showed that the lower limit of detections were 50, 100, and 300 IU/mL for BAL, CSF, or urine, respectively. The analytic measurement ranges were from log10 2.48 to log10 5.48. The assay specificity was 94.4% for BAL and 100% for CSF and urine. The assay precision was all within the acceptable range. The performance of Roche test was further compared with 2 comparators including the RealTime CMV assay (Abbott Molecular) and a CMV Quantitative Polymerase Chain Reaction test (Vela Diagnostics). There was a general positive correlation between the Roche method and the Abbott or the Vela method. Overall, this study suggests that the Roche CMV test is suitable for the quantification of CMV viral load DNA in the described nonplasma samples.

Antibiotics versus no antibiotics in the treatment of acute uncomplicated diverticulitis - a systematic review and meta-analysis.

BACKGROUND: Acute uncomplicated diverticulitis (AUD) is common and antibiotics are the cornerstone of traditional conservative management. This approach lacks clear evidence base and studies have recently suggested that avoidance of antibiotics is a safe and efficacious way to manage AUD. The aim of this systematic review is to determine the safety and efficacy of treating AUD without antibiotics. METHODS: A systematic search of Embase, Cochrane library, MEDLINE, Science Citation Index Expanded, and ClinicalTrials. gov was performed. Studies comparing antibiotics versus no antibiotics in the treatment of AUD were included. Meta-analysis was performed using the random effects model with the primary outcome measure being diverticulitis-associated complications. Secondary outcomes were readmission rate, diverticulitis recurrence, mean hospital stay, requirement for surgery and requirement for percutaneous drainage. RESULTS: Eight studies were included involving 2469 patients; 1626 in the non-antibiotic group (NAb) and 843 in the antibiotic group (Ab). There was a higher complication rate in the Ab group however this was not significant (1.9% versus 2.6%) with a combined risk ratio (RR) of 0.63 (95% CI, 0.25 to 1.57, p=0.32). There was a shorter mean length of hospital stay in the Nab group (standard mean difference of -1.18 (95% CI, -2.34 to -0.03 p= 0.04). There was no significant difference in readmission, recurrence and surgical intervention rate or requirement for percutaneous drainage. CONCLUSION: Treatment of AUD without antibiotics may be feasible with outcomes that are comparable to antibiotic treatment and with potential benefits for patients and the NHS. Large scale randomised multicentre studies are needed. This article is protected by copyright. All rights reserved.

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.

Watch-and-wait approach versus surgical resection after chemoradiotherapy for patients with rectal cancer (the OnCoRe project): a propensity-score matched cohort analysis.

BACKGROUND: Induction of a clinical complete response with chemoradiotherapy, followed by observation via a watch-and-wait approach, has emerged as a management option for patients with rectal cancer. We aimed to address the shortage of evidence regarding the safety of the watch-and-wait approach by comparing oncological outcomes between patients managed by watch and wait who achieved a clinical complete response and those who had surgical resection (standard care). METHODS: Oncological Outcomes after Clinical Complete Response in Patients with Rectal Cancer (OnCoRe) was a propensity-score matched cohort analysis study, that included patients of all ages diagnosed with rectal adenocarcinoma without distant metastases who had received preoperative chemoradiotherapy (45 Gy in 25 daily fractions with concurrent fluoropyrimidine-based chemotherapy) at a tertiary cancer centre in Manchester, UK, between Jan 14, 2011, and April 15, 2013. Patients who had a clinical complete response were offered management with the watch-and-wait approach, and patients who did not have a complete clinical response were offered surgical resection if eligible. We also included patients with a clinical complete response managed by watch and wait between March 10, 2005, and Jan 21, 2015, across three neighbouring UK regional cancer centres, whose details were obtained through a registry. For comparative analyses, we derived one-to-one paired cohorts of watch and wait versus surgical resection using propensity-score matching (including T stage, age, and performance status). The primary endpoint was non-regrowth disease-free survival from the date that chemoradiotherapy was started, and secondary endpoints were overall survival, and colostomy-free survival. We used a conservative p value of less than 0·01 to indicate statistical significance in the comparative analyses. FINDINGS: 259 patients were included in our Manchester tertiary cancer centre cohort, 228 of whom underwent surgical resection at referring hospitals and 31 of whom had a clinical complete response, managed by watch and wait. A further 98 patients were added to the watch-and-wait group via the registry. Of the 129 patients managed by watch and wait (median follow-up 33 months [IQR 19-43]), 44 (34%) had local regrowths (3-year actuarial rate 38% [95% CI 30-48]); 36 (88%) of 41 patients with non-metastatic local regrowths were salvaged. In the matched analyses (109 patients in each treatment group), no differences in 3-year non-regrowth disease-free survival were noted between watch and wait and surgical resection (88% [95% CI 75-94] with watch and wait vs 78% [63-87] with surgical resection; time-varying p=0·043). Similarly, no difference in 3-year overall survival was noted (96% [88-98] vs 87% [77-93]; time-varying p=0·024). By contrast, patients managed by watch and wait had significantly better 3-year colostomy-free survival than did those who had surgical resection (74% [95% CI 64-82] vs 47% [37-57]; hazard ratio 0·445 [95% CI 0·31-0·63; p<0·0001), with a 26% (95% CI 13-39) absolute difference in patients who avoided permanent colostomy at 3 years between treatment groups. INTERPRETATION: A substantial proportion of patients with rectal cancer managed by watch and wait avoided major surgery and averted permanent colostomy without loss of oncological safety at 3 years. These findings should inform decision making at the outset of chemoradiotherapy. FUNDING: Bowel Disease Research Foundation.

An important role for adenine, cholera toxin, hydrocortisone and triiodothyronine in the proliferation, self-renewal and differentiation of limbal stem cells in vitro.

The cornea is a self-renewing tissue located at the front of the eye. Its transparency is essential for allowing light to focus onto the retina for visual perception. The continuous renewal of corneal epithelium is supported by limbal stem cells (LSCs) which are located in the border region between conjunctiva and cornea known as the limbus. Ex vivo expansion of LSCs has been successfully applied in the last two decades to treat patients with limbal stem cell deficiency (LSCD). Various methods have been used for their expansion, yet the most widely used culture media contains a number of ingredients derived from animal sources which may compromise the safety profile of human LSC transplantation. In this study we sought to understand the role of these components namely adenine, cholera toxin, hydrocortisone and triiodothyronine with the aim of re-defining a safe and GMP compatible minimal media for the ex vivo expansion of LSCs on human amniotic membrane. Our data suggest that all four components play a critical role in maintaining LSC proliferation and promoting LSC self-renewal. However removal of adenine and triiodothyronine had a more profound impact and led to LSC differentiation and loss of viability respectively, suggesting their essential role for ex vivo expansion of LSCs. Replacement of each of the components with GMP-grade reagents resulted in equal growth to non-GMP grade media, however an enhanced differentiation of LSCs was observed, suggesting that additional combinations of GMP grade reagents need to be tested to achieve similar or better level of LSC maintenance in the same manner as the traditional LSC media.

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.

Abdominal wall reconstruction with components separation and mesh reinforcement in complex hernia repair.

BACKGROUND: Abdominal closure in the presence of enterocutaneous fistula, stoma or infection can be challenging. A single-surgeon's experience of performing components separation abdominal reconstruction and reinforcement with mesh in the difficult abdomen is presented. METHODS: Medical records from patients undergoing components separation and reinforcement with hernia mesh at Royal Liverpool Hospital from 2009 to 2012 were reviewed. Patients were classified by the Ventral Hernia Working Group (VHWG) grading system. Co-morbidities, previous surgeries, specific type of reconstruction technique, discharge date, complications and hernia recurrence were recorded. RESULTS: Twenty-three patients' (15 males, 8 females) notes were reviewed. Median age was 57 years (range 20-76 years). Median follow-up at the time of review was 17 months (range 2-48 months). There were 13 grade III hernias and 10 grade IV hernias identified. Synthetic mesh was placed to reinforce the abdomen in 6 patients, cross-linked porcine dermis was used in 3, and a Biodesign® Hernia Graft was placed in 14. Complications included wound infection (13%), superficial wound dehiscence (22%), seroma formation (22%) and stoma complications (9%). To date, hernias have recurred in 3 patients (13%). CONCLUSIONS: Components separation and reinforcement with biological mesh is a successful technique in the grade III and IV abdomen with acceptable rate of recurrence and complications.

An investigation of donor and culture parameters which influence epithelial outgrowths from cultured human cadaveric limbal explants.

Limbal stem cell deficiency is a blinding disease which affects the cornea at the front of the eye. The definitive cure involves replacing the corneal epithelial (limbal) stem cells, for example by transplanting cultured limbal epithelial cells. One method of performing cultures is to grow a sheet of epithelial cells from a limbal explant on human amniotic membrane. The growth of limbal tissue can be variable. The aim of this study is to investigate how different donor and culture factors influence the ex vivo growth of cadaveric limbal explants. Limbal explant cultures were established from 10 different cadaveric organ cultured corneo-scleral discs. The growth rate and the time taken for growth to be established were determined. Statistical analysis was performed to assess correlation between these factors and donor variables including donor age, sex, time from donor death to enucleation, time from enucleation to organ culture storage and duration in organ culture. Growth curves consistently showed a lag phase followed by a steeper linear growth phase. Donor age, time between death and enucleation, and time between enucleation and organ culture were not correlated to the lag time or the growth rate. Time in organ culture had a significant correlation with the duration of lag time (P = 0.003), but no relationship with the linear growth rate. This study shows that an important factor correlating with growth variation is the duration of corneo-scleral tissue in organ culture. Interestingly, donor age was not correlated with limbal explant growth.

The unfolded protein response regulator GRP78 is a novel predictive biomarker in colorectal cancer.

Adjuvant fluoropyrimidine-based (5-FU) chemotherapy is a mainstay of treatment for colorectal cancer (CRC), but only provides benefit for a subset of patients. To improve stratification we examined (for the first time in CRC), whether analysis of GRP78 expression provides a predictive biomarker and performed functional studies to examine the role of GRP78 in sensitivity to 5-FU. 396 CRC patient samples were collected in a prospective uniform manner and GRP78 expression was determined by immunohistochemistry on tissue microarrays using a well-validated antibody. Expression was correlated with clinicopathological parameters and survival. The role of GRP78 in 5-FU sensitivity was examined in CRC cells using siRNA, drug inhibition and flow cytometry. GRP78 expression was significantly elevated in cancer tissue (p < 0.0001), and correlated with depth of invasion (p = 0.029) and stage (p = 0.032). Increased overall 5-year survival was associated with high GRP78 expression (p = 0.036). Patients with stage II cancers treated by surgery alone, with high GRP78 also had improved survival (71% v 50%; p = 0.032). Stage III patients with high GRP78 showed significant benefit from adjuvant chemotherapy (52% vs. 28%; p = 0.026), whereas patients with low GRP78 failed to benefit (28% vs. 32%; p = 0.805). Low GRP78 was an independent prognostic indicator of reduced overall 5-year survival (p = 0.004; HR = 1.551; 95%CI 1.155-2.082). In vitro, inhibition of GRP78 reduces apoptosis in response to 5-FU in p53 wild-type cells. GRP78 expression may provide a simple additional risk stratification to inform the adjuvant treatment of CRC and future studies should combine analysis with determination of p53 status.

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.

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.

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.

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.

UVc-irradiation sublethal stress does not alter antibiotic susceptibility of staphylococci (MRSA, MSSA, and coagulase-negative staphylococci) to ß-lactam, macrolide, and fluoroquinolone antibiotic agents.

Skin tanning, either by exposure to natural sunlight or through use of UV sunbeds, has become a popular practice in the US, where it is estimated that approximately 1 million times per day someone in the US uses UV radiation for skin tanning, equating to 30 million Americans (circa 10% of the US population) who use a tanning bed. As well as exposing the host to periods of UV radiation, such practices also expose commensal skin bacteria, including Staphylococcus aureus, to such UV radiation. Previous work has indicated that environmental stresses on bacteria may lead to an upregulation of stress responses, in an attempt for the organism to combat the applied stress and remain viable. UV light may act as an environmental stress on bacteria, and so it was the aim of this study to examine the effect of UVc light on the antibiotic susceptibility of commensal skin bacteria, to determine if UV radiation would increase the antibiotic resistance of such skin flora and thus lead to a potential skin flora with increased antibiotic resistance. Previously, it has been shown that UVc light has a greater mutational effect on bacteria compared to lower-energy UV forms, including UVa and UVb light. Therefore, we decided to employ UVc light in our study to amplify the potential for mutational events occurring in skin staphylococci organisms (n=8) including methicillin-sensitive Staphylococcus aureus (n=2), methicillin-resistant Staphylococcus aureus (n=4), and coagulase-negative staphylococci (Staphylococcus haemolyticus) (n=2) were exposed to varying degrees of sublethal radiation via UVc light, and their minimum inhibitory concentration (MIC) susceptibility was determined by broth dilution assay against three classes of commonly used antibiotics, namely ß-lactams (penicillin), macrolides (erythromycin), and fluoroquinolones (ciprofloxacin). There was no significant difference between antibiotic susceptibility before UVc exposure and until maximum sublethal stress, prior to cell death due to fatal UVc exposure with the cells. These results indicate that UV environmental stress/exposure does not upregulate antibiotic resistance, and therefore these data indicate that UVc radiation does not lead to a more antibiotic-resistant population in the staphylococci organisms post-exposure.

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.