A fluorophore-tagged RGD peptide to control endothelial cell adhesion to micropatterned surfaces

The long-term patency rates of vascular grafts and stents are limited by the lack of surface endothelialisation of the implanted materials. We have previously reported that GRGDS and WQPPRARI peptide micropatterns increase the endothelialisation of prosthetic materials in vitro. To investigate the mechanisms by which the peptide micropatterns affect endothelial cell adhesion and proliferation, a TAMRA fluorophore-tagged RGD peptide was designed. Live cell imaging revealed that the micropatterned surfaces led to directional cell spreading dependent on the location of the RGD-TAMRA spots. Focal adhesions formed within 3 h on the micropatterned surfaces near RGD-TAMRA spot edges, as expected for cell regions experiencing high tension. Similar levels of focal adhesion kinase phosphorylation were observed after 3 h on the micropatterned surfaces and on surfaces treated with RGD-TAMRA alone, suggesting that partial RGD surface coverage is sufficient to elicit integrin signaling. Lastly, endothelial cell expansion was achieved in serum-free conditions on gelatin-coated, RGD-TAMRA treated or micropatterned surfaces. These results show that these peptide micropatterns mainly impacted cell adhesion kinetics rather than cell proliferation. This insight will be useful for the optimization of micropatterning strategies to improve vascular biomaterials

Multifunctional surface modifications to capture and expand flowing endothelial colony- forming cells

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Design of a 3D printer head for additive manufacturing of sugar glass for tissue engineering applications

Additive manufacturing is now considered as a new paradigm that is foreseen to improve progress in many fields. The field of tissue engineering has been facing the need for tissue vascularization when producing thick tissues. The use of sugar glass as a fugitive ink to produce vascular networks through rapid casting may offer the key to vascularization of thick tissues produced by tissue engineering. Here, a 3D printer head capable of producing complex structures out of sugar glass is presented. This printer head uses a motorized heated syringe fitted with a custom made nozzle. The printer head was adapted to be mounted on a commercially available 3D printer. A mathematical model was derived to predict the diameter of the filaments based on the printer head feed rate and extrusion rate. Using a 1 mm diameter nozzle, the printer accurately produced filaments ranging from 0.3 mm to 3.2 mm in diameter. One of the main advantages of this manufacturing method is the self-supporting behaviour of sugar glass that allows the production of long, horizontal, curved, as well as overhanging filaments needed to produce complex vascular networks. Finally, to establish a proof of concept, polydimethylsiloxane was used as the gel matrix during the rapid casting to produce various “vascularized” constructs that were successfully perfused, which suggests that this new fabrication method can be used in a number of tissue engineering applications, including the vascularization of thick tissues

A Multi-Parameter, High-Content, High-Throughput Screening Platform to Identify Natural Compounds that Modulate Insulin and Pdx1 Expression

Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements

CK19+ cell expansion in medium containing bFGF, EGF, HGF and KGF compared to control media.

<p>A) Growth curve based on the number of CK19+ cells enumerated at different time points in the presence of 20 ng/mL each of bFGF, EGF, HGF and KGF or control media (*p<0.05 for two-way comparisons with the 10% FBS condition). B) Phase contrast images of the cultures taken on day 6. N = 1 pancreas with 6 replicate cultures.</p

Phenotype of dispersed unsorted islet-depleted pancreatic cells cultured in serum-containing medium.

<p>On day 8, mixed cultures of CK19+ duct-like cells and vimentin+ fibroblast-like cells with very rare amylase+ cells are obtained. After a single passage, cultures consisted mainly of vimentin+ cells.</p

Fractions of cells obtained before and after sorting on day 1.

<p>Fractions of cells obtained before and after sorting on day 1.</p

In vitro oxygen imaging of acellular and cell-loaded beta cell replacement devices

Type 1 diabetes (T1D) is an autoimmune disease that leads to the loss of insulin-producing beta cells. Bioartificial pancreas (BAP) or beta cell replacement strategies have shown promise in curing T1D and providing long-term insulin independence. Hypoxia (low oxygen concentration) that may occur in the BAP devices due to cell oxygen consumption at the early stages after implantation damages the cells, in addition to imposing limitations to device dimensions when translating promising results from rodents to humans. Finding ways to provide cells with sufficient oxygenation remains the major challenge in realizing BAP devices’ full potential. Therefore, in vitro oxygen imaging assessment of BAP devices is crucial for predicting the devices’ in vivo efficiency. Electron paramagnetic resonance oxygen imaging (EPROI, also known as electron MRI or eMRI) is a unique imaging technique that delivers absolute partial pressure of oxygen (pO2) maps and has been used for cancer hypoxia research for decades. However, its applicability for assessing BAP devices has not been explored. EPROI utilizes low magnetic fields in the mT range, static gradients, and the linear relationship between the spin–lattice relaxation rate (R1) of oxygen-sensitive spin probes such as trityl OX071 and pO2 to generate oxygen maps in tissues. With the support of the Juvenile Diabetes Research Foundation (JDRF), an academic-industry partnership consortium, the “Oxygen Measurement Core” was established at O2M to perform oxygen imaging assessment of BAP devices originated from core members’ laboratories. This article aims to establish the protocols and demonstrate a few examples of in vitro oxygen imaging of BAP devices using EPROI. All pO2 measurements were performed using a recently introduced 720 MHz/25 mT preclinical oxygen imager instrument, JIVA-25™. We began by performing pO2 calibration of the biomaterials used in BAPs at 25 mT magnetic field since no such data exist. We compared the EPROI pO2 measurement with a single-point probe for a few selected materials. We also performed trityl OX071 toxicity studies with fibroblasts, as well as insulin-producing cells (beta TC6, MIN6, and human islet cells). Finally, we performed proof-of-concept in vitro pO2 imaging of five BAP devices that varied in size, shape, and biomaterials. We demonstrated that EPROI is compatible with commonly used biomaterials and that trityl OX071 is nontoxic to cells. A comparison of the EPROI with a fluorescent-based point oxygen probe in selected biomaterials showed higher accuracy of EPROI. The imaging of typically heterogenous BAP devices demonstrated the utility of obtaining oxygen maps over single-point measurements. In summary, we present EPROI as a quality control tool for developing efficient cell transplantation devices and artificial tissue grafts. Although the focus of this work is encapsulation systems for diabetes, the techniques developed in this project are easily transferable to other biomaterials, tissue grafts, and cell therapy devices used in the field of tissue engineering and regenerative medicine (TERM). In summary, EPROI is a unique noninvasive tool to experimentally study oxygen distribution in cell transplantation devices and artificial tissues, which can revolutionize the treatment of degenerative diseases like T1D