Development of angiogenic models to investigate neovascularisation for tissue engineering applications

The aim of this project was to develop in vitro models for angiogenesis that have the capability of combining pro-angiogenic cells with an extracellular matrix (ECM) component that can be monitored under flow conditions to learn more about the ‘rules’ of angiogenesis. Significant advancement has been made in the field of tissue engineering in recent years, however one of the current obstacles limiting progression is the production of thick, complex tissues due to the lack of rapid neovascularisation of the constructs upon implantation. Blood vessel formation is tightly regulated and relies on the chronologically precise adjustment of vessel growth, maturation and suppression of endothelial cell growth - all of which are controlled by a large number of factors which influence each other. To induce vascularisation within tissue-engineered (TE) substitutes the same processes need to occur. A number of different vascularisation strategies have been investigated in an attempt to overcome this issue but as yet there is no unified solution to this problem. The most promising attempts have used scaffolds with vascular architectures, perfusion conditions and relevant cell types. Although it is recognised that perfusion conditions, the cell type and scaffold architecture are important with regards to vascularisation strategies many of the techniques fail to consider them in combination. It is therefore important to take a step back and understand how these factors work together to i promote angiogenesis in order to advance this crucial area. This lack of understanding is further compounded by the deficiencies of current angiogenesis models. Current in vitro models fail to combine the use of supporting cells, the extracellular matrix and fluid flow in 3D. Although this complexity exists within in vivo models such assays are primarily limited by the species used, organ sites available and complicated analysis techniques. In this project two in vitro angiogenesis models were developed. The first was derived from the decellularisation of a rat jejunum. Characterisation showed the retention of key extracellular matrix (ECM) components and the removal of almost all cellular material. Re-endothelialisation with human dermal endothelial cells (HDMECs) of the patent vascular network showed enhanced results when co-cultured with human dermal fibroblasts (HDFs). In an attempt to induce angiogenesis, vascular endothelial growth factor (VEGF) loaded gels were placed on top of the scaffold whilst being continuously perfused with media. Placing VEGF loaded gels onto the recellularised jejunum led to the expression of the Notch ligand Delta- like-4 (DLL4) by HDMECs indicating their transformation into tip cells which are synonymous with sprouting angiogenesis. The second was produced through the combination of robocasting and electrospinning. Nanofibrous poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) scaffolds with hollow channels capable of perfusion were produced that could be re-endothlialised with HDMECs. Again the addition of HDFs enhanced cellular distribution in the channels. Placing VEGF loaded gels onto the surface of the scaffolds led to the outgrowth of HDMECs into the gel, forming perfusable tubules. Overall these two models overcome limitations of current in vitro models since they offer the capability of combining pro-angiogenic cells with ECM components that can be monitored under flow conditions. With further development they could provide more sophisticated platforms upon which to investigate the angiogenic process

Age and frailty are independently associated with increased COVID-19 mortality and increased care needs in survivors: results of an international multi-centre study

INTRODUCTION: Increased mortality has been demonstrated in older adults with COVID-19, but the effect of frailty has been unclear.METHODS: This multi-centre cohort study involved patients aged 18years and older hospitalised with COVID-19, using routinely collected data. We used Cox regression analysis to assess the impact of age, frailty, and delirium on the risk of inpatient mortality, adjusting for sex, illness severity, inflammation, and co-morbidities. We used ordinal logistic regression analysis to assess the impact of age, Clinical Frailty Scale (CFS), and delirium on risk of increased care requirements on discharge, adjusting for the same variables.RESULTS: Data from 5,711 patients from 55 hospitals in 12 countries were included (median age 74, IQR 54-83; 55.2% male). The risk of death increased independently with increasing age (>80 vs 18-49: HR 3.57, CI 2.54-5.02), frailty (CFS 8 vs 1-3: HR 3.03, CI 2.29-4.00) inflammation, renal disease, cardiovascular disease, and cancer, but not delirium. Age, frailty (CFS 7 vs 1-3: OR 7.00, CI 5.27-9.32), delirium, dementia, and mental health diagnoses were all associated with increased risk of higher care needs on discharge. The likelihood of adverse outcomes increased across all grades of CFS from 4 to 9.CONCLUSIONS: Age and frailty are independently associated with adverse outcomes in COVID-19. Risk of increased care needs was also increased in survivors of COVID-19 with frailty or older age

1074 Ottawa Ankle rules cannot be safely used to rule out ankle fractures in patients who present ≥10 days post-injury

Aims/Objectives/BackgroundThe Ottawa ankle rules (OAR) have been validated as a highly sensitive tool to rule out ankle fractures and reduce need for radiography. However, datasets validating OAR to date have excluded patients presenting ≥10 days post-injury and there is a need to ascertain if OAR can be safely used to rule out ankle fractures in this population.Methods/DesignPatients presenting with ankle injuries to an emergency department (ED) in England between June 2015 and November 2020 were identified retrospectively through a clinical-coding search. Patient records were used to confirm the number of days between injury and presentation; those who presented ≥10 days post-injury were included for further analysis. Data was collected from ED documentation including region of pain, bony tenderness and weight-bearing status. OAR were used to categorise patients as ‘Ottawa-positive’, ‘Ottawa-negative’ or insufficient documentation. It was recorded whether the patient underwent radiography and whether the formal radiograph report confirmed a clinically-significant fracture. Patients who didn’t undergo radiography and didn’t subsequently re-present were deemed not to have a fracture. Data collected for each patient was checked and agreed by two authors.Results/Conclusions6782 patients presented with ankle injuries, of which 126 patients presented ≥10 days post-injury. Of these 126 patients, 9 were Ottawa-positive, 90 were Ottawa-negative and 27 patients had insufficient documentation. 85 patients underwent radiography and 19 were found to have clinically-significant fractures. Of these fracture patients, 4 were Ottawa-positive and 15 were Ottawa-negative.Within our dataset, OAR demonstrated a sensitivity of 21.05%, specificity 93.75%, PPV 44.40% and NPV 83.30%. Using Fishers exact test, p=0.0658. OAR demonstrate poor sensitivity and cannot be safely used to rule out ankle fractures in patients who present ≥10 days post-injury. However, due to the p-value and low power there may be a risk of type 2 error and a larger study may prove otherwise.</jats:sec

Vascularization strategies for tissue engineers

All tissue-engineered substitutes (with the exception of cornea and cartilage) require a vascular network to provide the nutrient and oxygen supply needed for their survival in vivo. Unfortunately the process of vascular ingrowth into an engineered tissue can take weeks to occur naturally and during this time the tissues become starved of essential nutrients, leading to tissue death. This review initially gives a brief overview of the processes and factors involved in the formation of new vasculature. It then summarizes the different approaches that are being applied or developed to overcome the issue of slow neovascularization in a range of tissue-engineered substitutes. Some potential future strategies are then discussed

Thiolene- and Polycaprolactone Methacrylate-Based Polymerized High Internal Phase Emulsion (polyhipe) Scaffolds for Tissue Engineering

Highly porous emulsion templated polymers (PolyHIPEs) provide a number of potential advantages in the fabrication of scaffolds for tissue engineering and regenerative medicine. Porosity enables cell ingrowth and nutrient diffusion within, as well as waste removal from, the scaffold. The properties offered by emulsion templating alone include the provision of high interconnected porosity, and, in combination with additive manufacturing, the opportunity to introduce controlled multiscale porosity to complex or custom structures. However, the majority of monomer systems reported for PolyHIPE preparation are unsuitable for clinical applications as they are nondegradable. Thiol-ene chemistry is a promising route to produce biodegradable photocurable PolyHIPEs for the fabrication of scaffolds using conventional or additive manufacturing methods; however, relatively little research has been reported on this approach. This study reports the groundwork to fabricate thiol- and polycaprolactone (PCL)-based PolyHIPE materials via a photoinitiated thiolene click reaction. Two different formulations, either three-arm PCL methacrylate (3PCLMA) or four-arm PCL methacrylate (4PCLMA) moieties, were used in the PolyHIPE formulation. Biocompatibility of the PolyHIPEs was investigated using human dermal fibroblasts (HDFs) and human osteosarcoma cell line (MG-63) by DNA quantification assay, and developed PolyHIPEs were shown to be capable of supporting cell attachment and viability