Dina S. Vara

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.

Review paper: principles and applications of surface analytical techniques at the vascular interface.

Surface properties have been found to be one of the key parameters which cause degradation and of thrombogenicity in all polymers used in biomedical devices, thus signifying the importance and the necessity for quantitative and accurate characterization of the polymer surface itself as used in the construction of the device. The characterization techniques employed generally involve thermal and spectroscopic measurements, in which class the electrochemical investigations and scanning probe microscopies can also be included. Current hypotheses on the correlations that exist between surface parameters and hemocompatibility and degradation of polymers are examined herein, but concentrating on the field of clinically utilized polymeric materials as used within medical devices themselves. Furthermore, this review provides a brief but complete synopsis of these techniques and other emerging ones, which have proven useful in the analysis of the surface properties of polymeric materials as used in the construction of cardiovascular devices. Statements and examples are given as to how specific information can be acquired from these differing methodologies and how it aids in the design and development of new polymers for usage in biomedical device construction.

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.

Endothelial Cell Retention on a Viscoelastic Nanocomposite Vascular Conduit Is Improved by Exposure to Shear Stress Preconditioning Prior to Physiological Flow

In this study, endothelial cell (EC)-seeded nanocomposite grafts were preconditioned with 1-2 dynes/cm(2) in vitro to establish whether low shear stress resulted in improved cell adherence prior to physiological shear stress (15 dynes/cm(2)). Alamar blue cell viability was assessed. Polymerase chain reaction was conducted for glyceraldehyde-3-phosphate dehydrogenase, transforming growth factor beta-1 (TGFbeta-1), vascular endothelial growth factor receptor-1 (VEGFR-1), platelet EC adhesion molecule-1, and vascular endothelial growth factor receptor-2 (VEGFR-2). The Alamar blue results demonstrated improved cellular retention following preconditioning (P < 0.001). VEGFR-2 and TGFbeta-1 expression was up-regulated, and VEGFR-1 down-regulated following preconditioning. This investigation confirms previous findings regarding the potential benefits of preconditioning, and demonstrates that these benefits can be applied to ECs seeded on the nanocomposite employed. It also demonstrates further the suitability and potential of nanocomposite for future use in tissue-engineered cardiovascular devices.

A novel method for the extraction and culture of progenitor stem cells from human peripheral blood for use in regenerative medicine

Human peripheral blood (HPB) contains both circulating endothelial cells (CECs) and endothelial progenitor stem cells (EPCs), which may be suitable for use in regenerative medicine. There has been considerable interest in using these cells, but there is no “gold standard” technique for isolating these cells. The aim of this study was to characterize and compare a number of different extraction and culture techniques to develop a system to isolate and culture cells. EPC and CEC were isolated from HPB using either Histopaque‐1077 or Lymphoprep. The two isolation methods were compared for the number of cells isolated, cell metabolism, and RNA expression. Both isolations produced viable cells and were comparable. The tissue culture method employed does have a significant effect on the cell population with regard to medium choice, fetal bovine serum concentration, and surface modification of the culture surface. In conclusion, it can be seen that although this study and previous work can suggest a basis for culture, further work to develop an optimized and agreed “gold standard” culture regime for EPC from HPB is required to maximize the potential of this source of cells for regenerative medicine and to translate its clinical use in the future.

The long-term stability in gene expression of human endothelial cells permits the production of large numbers of cells suitable for use in regenerative medicine.

Tissue engineering has been conducted in the study of cardiovascular grafts for many years. Many obstacles have been overcome in this rapidly changing field, but one difficulty has remained until now: the large number of endothelial cells (ECs) needed for seeding the inner layer of bypass graft. Recent advances in endothelial progenitor cell (EPC) isolation and culture techniques have increased the interest in genetic studies. Despite these advances in EPC studies, the “gold standard” for the seeding of tissue engineering constructs or hybrid grafts remains mature human umbilical vein endothelial cells (HUVECs). This study investigates the ability of HUVECs to be expanded in culture to provide sufficient cells for graft seeding. The levels of gene expression of key genes are then examined to ensure that these cells retain the EC phenotype. This study demonstrates that HUVECs may be cultured for up to 12 passages without alteration in phenotype. Subsequent passage numbers are sufficiently similar to those preceding them to allow cells of different passages to be mixed without gene expression anomalies.