Development of tissue engineered blood vessels using cell-seeded acellular porcine arterial scaffolds

The patency rate of small diameter synthetic bypass grafts remains poor. The aim of this study was to develop a biocompatible, acellular, arterial scaffold, assess the scaffolds regenerative capacity using ovine vascular cells and begin preliminary studies of a vascular tissue bioreactor for the development of a tissue engineered graft for peripheral and coronary arterial bypass. Porcine carotid arteries were decellularised using a protocol developed at the University of Leeds. Arteries were incubated sequentially in disodium ethylenediaminetetraacetic acid, hypotonic solution, sodium dodecyl sulphate [0.1% w/v], DNAse and RNAse, hypertonic solution and 0.1% (v/v) peracetic acid. To ensure decellularisation, representative arterial histological sections were stained using haematoxylin and eosin and 4’,6-diamidino-2-phenylindole to confirm removal of cells and cell nuclei. The total DNA content of treated arteries was also determined. Biocompatibility of the acellular scaffolds was assessed using contact and extract cytotoxicity assays using both primary cells (porcine and ovine endothelial cells and smooth muscle cells) and two distinct cell lines (murine 3T3 and BHK cells). Ovine endothelial cells were harvested from the femoral arteries of sheep following digestion with collagenase. Ovine smooth muscle cells were isolated from ovine arterial explant cultures. To determine correct cell phenotype, immuno-staining was performed using a variety of primary antibodies to vascular cell markers by indirect immunofluorescence. Ovine vascular cells were then seeded onto the luminal surface of the decellularised vessels in both a two-dimensional and three-dimensional manner. A cell viability assay (Live / Dead Stain ®) was performed to confirm the viability of seeded cells. A vascular bioreactor was successfully assembled and preliminary sterility runs were performed in preparation for future scaffold pre-conditioning. The decellularisation protocol resulted in porcine carotid arteries that were free from cells with >90% of the total DNA being removed. The decellularised porcine carotid artery was not cytotoxic to any test cells. Indirect immunofluorescence performed on the harvested cells confirmed correct ovine endothelial cell and smooth muscle cell phenotype after cell isolation using magnetic bead separation. Ovine vascular cells were successfully seeded onto the luminal matrix of decellularised arteries in both two-dimensional and three-dimensional experiments. Seeded cells were viable at 48 hours post incubation. A vascular bioreactor was successfully assembled and was kept free from macroscopic microbial contamination for a maximum of fourteen days. In conclusion, porcine carotid arteries were successfully decellularised using an established decellularisation protocol. The remaining acellular scaffolds demonstrated capability in allowing the attachment and proliferation of xenogeneic vascular cells onto the scaffold surface. Further work developing a vascular bioreactor in order assess the functionality and ongoing cell viability of the seeded scaffold will be needed to assess the efficacy of decellularised porcine carotid artery as a viable conduit for arterial bypass

The NeST (Neoadjuvant systemic therapy in breast cancer) study: National Practice Questionnaire of United Kingdom multi-disciplinary decision making

Abstract: Background: Neoadjuvant systemic therapy (NST) is increasingly used in the treatment of breast cancer, yet it is clear that there is significant geographical variation in its use in the UK. This study aimed to examine stated practice across UK breast units, in terms of indications for use, radiological monitoring, pathological reporting of treatment response, and post-treatment surgical management. Methods: Multidisciplinary teams (MDTs) from all UK breast units were invited to participate in the NeST study. A detailed questionnaire assessing current stated practice was distributed to all participating units in December 2017 and data collated securely usingREDCap. Descriptive statistics were calculated for each questionnaire item. Results: Thirty-nine MDTs from a diverse range of hospitals responded. All MDTs routinely offered neoadjuvant chemotherapy (NACT) to a median of 10% (range 5–60%) of patients. Neoadjuvant endocrine therapy (NET) was offered to a median of 4% (range 0–25%) of patients by 66% of MDTs. The principal indication given for use of neoadjuvant therapy was for surgical downstaging. There was no consensus on methods of radiological monitoring of response, and a wide variety of pathological reporting systems were used to assess tumour response. Twenty-five percent of centres reported resecting the original tumour footprint, irrespective of clinical/radiological response. Radiologically negative axillae at diagnosis routinely had post-NACT or post-NET sentinel lymph node biopsy (SLNB) in 73.0 and 84% of centres respectively, whereas 16% performed SLNB pre-NACT. Positive axillae at diagnosis would receive axillary node clearance at 60% of centres, regardless of response to NACT. Discussion: There is wide variation in the stated use of neoadjuvant systemic therapy across the UK, with general low usage of NET. Surgical downstaging remains the most common indication of the use of NAC, although not all centres leverage the benefits of NAC for de-escalating surgery to the breast and/or axilla. There is a need for agreed multidisciplinary guidance for optimising selection and management of patients for NST. These findings will be corroborated in phase II of the NeST study which is a national collaborative prospective audit of NST utilisation and clinical outcomes