Geoffrey Punshon

Development of a hybrid cardiovascular graft using a tissue engineering approach

Tissue engineering of endothelial cells (EC) and chemical engineering with anticoagulant moieties has been undertaken in order to improve prosthetic graft patency and thrombogenicity. This was done by covalently bonding a compliant poly(carbonate‐urea)urethane graft (MyoLink™) with with arginine‐glycine‐aspartate (RGD) or/and heparin (Hep) to ascertain whether EC retention could be improved. The retention of these moieties and EC was assessed after exposure to pulsatile flow. We covalently bonded RGD, Hep, and RGD/Hep onto the luminal surface of MyoLink using spacer arm technology. Narrow‐beam X‐ray photoelectron spectroscopy was carried out to check the efficiency of the bonding. EC were radiolabeled and seeded onto native MyoLink and with 1) RGD‐, 2) Hep‐, and 3) RGD/Hep‐bonded grafts and exposed to shear stress in a physiological flow circuit for 6 h, which reproduces femoral artery flow waveforms and pulsatility. Results were recorded on a gamma camera imaging system. Viability of cells was tested with a modified Alamar Blue assay (ABA) and scanning electron microscopy for morphological appearance of seeded cells. Experiments were repeated (n=6). RGD, Hep, and RGD/Hep were bonded together in a uniform distribution on the luminal surface of each graft type, and bioactivity of each moiety covalently bonded was very high. In the flow circuit, there was exponential cell retention for the first 60 min of flow for all the grafts, but after 6 h of exposure to pulsatile flow the RGD/Hep‐bonded graft had a significantly better cell retention rate than native MyoLink (75.7%±2.3 vs. 60.5±10.1, P<0.05). ABA test showed that all the seeded cells postexposure to flow were viable, and significantly higher metabolic activity was recorded on a RGD/Hep‐bonded graft than with MyoLink‐seeded graft (P<0.01). Using RGD/Hep covalently bonded onto graft surfaces improves cell retention and provides an antithrombogenic surface for initial blood flow in vivo until full EC activity develops postseeding. This would allow the development and further improvement of hybrid grafts.—Tiwari, A., Salacinski, H. J., Punshon, G., Hamilton, G., Seifalian, A. M. Development of a hybrid cardiovascular graft using a tissue engineering approach. FASEB J. 16, 791–796 (2002)

Magnetic beads (Dynabead™) toxicity to endothelial cells at high bead concentration: Implication for tissue engineering of vascular prosthesis

Magnetic beads (Dynabeads™) have been used for the purification of endothelial cells. One application for this procedure may be for single-stage seeding of bypass grafts. The number of endothelial cells (EC) isolated is crucial and therefore to increase the number of cells extracted, a higher number of Dynabeads™ per cell may need to be used. The effect of large numbers of CD31 Dynabeads™ on cell proliferation/metabolism is unknown. We undertook this study using CD31-coated Dynabeads™ and EC from human umbilical vein. EC were coated at concentrations of 4, 10, or 50 beads per cell. The cells were cultured for 6 days with control being normal EC. Cellular proliferation was assessed by trypsinization of cells and metabolism assessed with an Alamar blue™ viability assay. In a further experiment a compliant polyurethane graft was single-stage seeded with both coated Dynabeads™ and normal EC. The results showed that using a higher number of beads per cell resulted in a reduction in cell proliferation and a reduction in cell metabolism. The total number of Dynabeads™-coated cells in culture compared to controls (%) by day 6 were 30.7±2.56, 41.3±9.8 and 59.2±7.3 for 50, 10, and 4 beads per cell, respectively. The corresponding results for Alamar blue were 43.7±1.2, 61.8±1.4, and 72.1±4.3. The seeded grafts showed reduced metabolism with the Dynabeads™-coated EC. In conclusion, high numbers of beads per cell have a late detrimental effect on cell proliferation and metabolism. Therefore for single-stage seeding lower numbers of Dynabeads™ will need to be used with resultant reduction in the number of available EC.

Assessment of the potential of progenitor stem cells extracted from human peripheral blood for seeding a novel vascular graft material.

Abstract.  Objective: A novel nanocomposite has recently been developed based on polyhedral oligomeric silsesquioxane attached by direct reaction onto a urethane segment, as a potential vascular graft material; its trade name is UCL‐Nano. The UCL‐Nano has been demonstrated to have similar viscoelastic properties to the walls of a natural artery, to be resistant to degradation and to be able to sustain endothelial cell seeding. Human peripheral blood contains both circulating endothelial cells and endothelial progenitor cells, which may be suitable for conduit seeding. The aim of this study was to develop a system with the potential to deliver an endothelial cell‐seeded bypass graft in a realistic time frame. Materials and methods: Endothelial progenitor cells and circulating endothelial cells were isolated from human peripheral blood and were characterized by fluorescent‐activated cell sorting, reverse transcriptase‐polymerase chain reaction and immunohistochemistry. Isolated cells were seeded on nanocomposite and were maintained in culture for 35 days. Results: The UCL‐Nano was successfully seeded with cells and a confluent cell layer was achieved after 14‐day culture. Cells remained viable and confluent on the nanocomposite for 35 days. Conclusion: In conclusion, these results suggest that this process has potential both for a realistic and achievable two‐stage seeding process for vascular bypass grafts and for the potential development of a device, with the aim of achieving in situ seeding once implanted.

In vitro small intestinal epithelial cell growth on a nanocomposite polycaprolactone scaffold

Tissue engineering of the small intestine remains experimental despite worldwide attempts to develop a functional substitute for short bowel syndrome. Most published studies have reported predominant use of PLLA (poly‐L‐lactide acid)/PGA (polyglycolic acid) copolymer as the scaffold material, and studies have been limited by in vivo experiments. This lack of progress has inspired a fresh perspective and provoked further investigation and development in this field of tissue engineering. In the present paper, we exploit a relatively new nanocomposite of POSS (polyhedral oligomeric silsesquioxane) and PCL [poly(caprolactone‐urea)urethane] as a material to develop porous scaffolds using a solvent casting/particulate leaching technique to fabricate porous scaffolds in different pore sizes and porosities. Scaffolds were characterized for pore morphology and porosity using scanning electron microscopy and micro‐computed tomography. Rat intestinal epithelial cells were then seeded on to the polymer scaffolds for an in vitro study of cell compatibility and proliferation, which was assessed by Alamar Blue™ and lactate dehydrogenase assays performed for 21 days post‐seeding. The results obtained demonstrate that POSS–PCL nanocomposite was produced as a macroporous scaffold with porosity over the range of 40–80% and pore size over the range of 150–250 μm. This scaffold was shown to support epithelial cell proliferation and growth. In conclusion, as a further step in investigating small intestinal tissue engineering, the nanocomposite employed in this study may prove to be a useful alternative to poly(lactic‐co‐glycolic acid) in the future.

A new technique for measuring the cell growth and metabolism of endothelial cells seeded on vascular prostheses

For the improvement of vascular graft patency, an endothelial cell (EC) lining is desirable. It is essential that the EC remains viable after being seeded onto the prosthetic graft. The aim of this study was to adapt an Alamar redox assay (ABRA) as a technique to monitor the viability of ECs seeded on prosthetic grafts. To test the graft types, we seeded human umbilical vein ECs on compliant polyurethane (CPU), expanded polytetrafluoroethylene, and Dacron at a density of 2 x 10(5) cell/cm(2). After 24 h of incubation, ABRA was added, and the absorbance was measured at 4, 8, and 24 h. To assess seeded cell concentrations on grafts, we seeded CPU at densities ranging from 1 x 10(5) to 8 x 10(5) cell/cm(2). The validity of the test was assessed with sodium azide and mitomycin C, known physiological perturbators. ABRA reduction demonstrated that ECs were viable and functional postseeding on the prosthetic grafts. A significant correlation was observed with ABRA reduction and cell concentrations (p < 0.001). The acid phosphatase assay demonstrated enzyme activity in the cells, but they were not maintained under normal physiological conditions. The ABRA bioreduced product was soluble, stable, and noncytotoxic over 24 h. The assay is independent of the geometry or physiochemistry of the graft type. The technique allows the continuous assessment of the metabolism and viability of seeded cells, is simple to perform, and does not destroy the cells or graft materials.

UV surface modification of a new nanocomposite polymer to improve cytocompatibility

A novel modified nanocomposite was studied for the adhesion and proliferation of the human umbilical vein endothelial cell (HUVEC) line EA.hy926. The nanocomposite under investigation was poly(carbonate-urea)urethane with silsesquioxane nano-cages, here in the form of a mixture of two polyhedral oligomeric silsesquioxanes. The nanocomposite surfaces were exposed to ultraviolet (UV) light of a Xe* 2-excimer lamp at a wavelength of 172 nm in an ammonia atmosphere. The effects of the irradiation were characterized by atomic force and scanning electron microscopy (AFM, SEM), X-ray photo-electron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FT-IR) using an attenuated total reflection (ATR) device and measurements of advancing water contact angle (CA). The irradiation resulted in the introduction of new hydrophilic N- and O-containing groups into the surface, which was initially amphiphilic, while surface morphology remained mainly unchanged. Slight chemical changes were also observed for the silsesquioxane nano-cages at the surface. Onto the untreated and irradiated samples HUVECs were seeded and grown for various durations in culture. Standard tissue-culture polystyrene (PS) was employed as a positive control to check the efficiency of the cell-culture methods. Viability and proliferation of the cells were then assessed using a non-radioactive assay. Compared to the untreated nanocomposite polymer, irradiation times of at least 5 min resulted in a significantly increased cell proliferation between 3 and 8 days after seeding with the HUVEC line EA.hy926.

Extraction of cells for single-stage seeding of vascular-bypass grafts

Experimental data are reported for the seeding of prosthetic vascular grafts with either mesothelial or endothelial cells as part of a research strategy in tissue engineering with the aim of improving graft patency and developing new techniques for single‐stage cell extraction and seeding that would give a step reduction in surgery time. New data are reported for two different sources of cells, peritoneal lavage and subcutaneous fat. All experiments were undertaken in patients undergoing abdominal aortic aneurysm repair. Cells extracted from peritoneal lavage were insufficient for a single‐stage seeding process. Subcutaneous fat was processed using either a positive cell‐extraction method using CD31 Dynabeads or by a negative extraction method using CDw90‐coated magnetic beads. Only positive cell extraction gave reliably sufficient numbers of endothelial cells as a source for single‐stage seeding of vascular grafts.

The effect of shear stress on human endothelial cells seeded on cylindrical viscoelastic conduits: an investigation of gene expression.

The present study assesses the effect of physiological shear stress on gene expression from human ECs (endothelial cells) seeded on a small‐diameter cylindrical bypass graft constructed from nanocomposite based on poly(carbonate‐silsesquioxane‐bridge‐urea)urethane. ECs were seeded on to 5‐mm‐diameter conduits, placed in a physiological flow circuit and exposed to 1 or 4 h of shear stress at 1.4±0.3 Pa. Subsets of conduits were incubated at 37 °C and 5% CO2/95% O2 for a further 4 h to determine if gene expression returned to basal levels. PCR was conducted for glyceraldehyde‐3‐phosphate dehydrogenase, TGFβ‐1 (transforming growth factor β‐1), COL‐1 (collagen‐1) and PECAM‐1 (platelet/EC adhesion molecule‐1). Increases in gene expression were seen following flow in nanocomposite conduits. These were significant at 4 h for TGFβ‐1, COL‐1 and PECAM‐1. After a 4 h recovery period, there were no significant differences in gene intensity, suggesting that this change is transient. These data prove that mRNA can be obtained from ECs seeded on tubular conduits and exposed to shear stress and that gene‐expression studies can be successfully carried out. We believe this is a substantial improvement on studies based on flat sheets.

Is there an alternative to systemic anticoagulation, as related to interventional biomedical devices?

To reduce the toxic effects, related clinical problems and complications such as bleeding disorders associated with systemic anticoagulation, it has been hypothesized that by coating the surfaces of medical devices, such as stents, bypass grafts, extracorporeal circuits, guide wires and catheters, there will be a significant reduction in the requirement for systemic anticoagulation or, ideally, it will no longer be necessary. However, current coating processes, even covalent ones, still result in leaching followed by reduced functionality. Alternative anticoagulants and related antiplatelet agents have been used for improvement in terms of reduced restenosis, intimal hyperphasia and device failure. This review focuses on existing heparinization processes, their application in clinical devices and the updated list of alternatives to heparinization in order to obtain a broad overview, it then highlights, in particular, the future possibilities of using heparin and related moieties to tissue engineer scaffolds.

An analysis of use of crowd medical services at an English football league club.

AIM Although crowd medical services in English league football are common, there is little data examining the use of such services. The aim of this study was to provide detailed data by examining the usage of the Crowd Medical Service at a First Division Football Club (Millwall FC) over six Football League seasons. This usage has implications in terms of resources, provision of level of service and training needs of staff. METHODS A prospective data collection form was designed and used to capture all episodes of care over the length of six football league seasons, each approximately 9 months in duration which resulted in a census of activity. This included episodes that did not normally require detailed documentation for statutory purposes, such as informal advice. RESULTS There were 445 episodes of care recorded over 146 home games. Over six years presentation was consistent. This gives a mean of 3.14 episodes per game (2.7-4.1). There is little research in this area and so it is difficult to compare uptake with other clubs. The episodes of care were broadly grouped into medical (67.0%) or injury (33.0%). Most of those presenting did so with non-emergent (pre-existing) medical problems or minor injuries. Staff (for example Stewards) made up 44% of consultations. One hundred and eight people had a primary and secondary reason for presenting. Such episodes are more time consuming and in turn have an impact on resources. Some stated they had presented because of ease of access to the service, they found access to their General Practitioner (GP) difficult or were not registered with a GP at all. CONCLUSION The principal focus of a crowd medical service is to manage a major incident and medically emergent spectators. The majority of presentations were minor injuries and pre-existing medical conditions. In addition staff make up a significant proportion of the users. Some re-focussing of guidance is therefore necessary if these results are typical of English League Football. In turn this reflects a need for more education in the management of minor injuries and chronic disease management and recognition in training and service provision.