Chris A. Bashur

Nerve Growth Factor-Immobilized Electrically Conducting Fibrous Scaffolds for Potential Use in Neural Engineering Applications

Engineered scaffolds simultaneously exhibiting multiple cues are highly desirable for neural tissue regeneration. To this end, we developed a neural tissue engineering scaffold that displays submicrometer-scale features, electrical conductivity, and neurotrophic activity. Specifically, electrospun poly(lactic acid-co-glycolic acid) (PLGA) nanofibers were layered with a nanometer thick coating of electrically conducting polypyrrole (PPy) presenting carboxylic groups. Then, nerve growth factor (NGF) was chemically immobilized onto the surface of the fibers. These NGF-immobilized PPy-coated PLGA (NGF-PPyPLGA) fibers supported PC12 neurite formation (28.0±3.0% of the cells) and neurite outgrowth (14.2 μm median length), which were comparable to that observed with NGF (50 ng/mL) in culture medium (29.Oil.3%, 14.4 μm). Electrical stimulation of PC12 cells on NGF-immobilized PPyPLGA fiber scaffolds was found to further improve neurite development and neurite length by 18% and 17%, respectively, compared to unstimulated cells on the NGF-immobilized fibers. Hence, submicrometer-scale fibrous scaffolds that incorporate neurotrophic and electroconducting activities may serve as promising neural tissue engineering scaffolds such as nerve guidance conduits.

Incorporation of photo-carbon monoxide releasing materials into electrospun scaffolds for vascular tissue engineering.

Hyper-proliferation of smooth muscle cells (SMCs) and a reduction in endothelial cell function are reasons for poor patency rates of current tissue engineered small-diameter vascular grafts. The controlled delivery of carbon monoxide (CO), a gasotransmitter involved in cell signaling, could improve vascular cell function in these grafts. Current CO releasing molecules (CORMs) can improve endothelialization of injured vessels with appropriate doses, but they still have limitations. The goal of this project was to generate a novel tissue engineered scaffold that includes a non-toxic and photoactivatable CORM. This is the first use of a CORM for tissue engineering. The results demonstrated that CORM-loaded, electrospun poly(ɛ-caprolactone) scaffolds can be photo-activated and release CO. The fluorescence that develops after CO release can be used to non-destructively track the extent of reaction. Further, activation can occur when both dry and incubated in cell culture conditions. However, incubation in serum protein-containing media decreases the time frame for activation, demonstrating the importance of testing the release profile in culture conditions. Rat SMCs were able to attach, grow, and express contractile SMC markers on activated CORM-loaded meshes and controls. Overall, these findings demonstrate that CORM-loaded electrospun scaffolds provide a promising delivery system for vascular tissue engineering.

Impact of elastin incorporation into electrochemically aligned collagen fibers on mechanical properties and smooth muscle cell phenotype

Application of tissue-engineered vascular grafts (TEVGs) for the replacement of small-diameter arteries is limited due to thrombosis and intimal hyperplasia. Previous studies have attempted to address the limitations of TEVGs by developing scaffolds that mimic the composition (collagen and elastin) of native arteries to better match the mechanical properties of the graft with the native tissue. However, most existing scaffolds do not recapitulate the aligned topography of the collagen fibers found in native vessels. In the current study, based on the principles of isoelectric focusing, two different types of elastin (soluble and insoluble) were incorporated into highly oriented electrochemically aligned collagen (ELAC) fibers and the effect of elastin incorporation on the mechanical properties of the ELAC fibers and smooth muscle cell (SMC) phenotype was investigated. The results indicate that elastin incorporation significantly decreased the modulus of ELAC fibers to converge upon that of native vessels. Further, a significant increase in yield strain and decrease in Youngs modulus was observed on all fibers post SMC culture compared with before the culture. Real-time polymerase chain reaction results showed a significant increase in the expression of α-smooth muscle actin and calponin on ELAC fibers with insoluble elastin, suggesting that incorporation of insoluble elastin induces a contractile phenotype in SMCs after two weeks of culture on ELAC fibers. Immunofluorescence results showed that calponin expression increased with time on all fibers. In conclusion, insoluble elastin incorporated ELAC fibers have the potential to be used for the development of functional TEVGs for the repair and replacement of small-diameter arteries.

Compositions Including Synthetic and Natural Blends for Integration and Structural Integrity: Engineered for Different Vascular Graft Applications

Tissue engineering approaches for small-diameter arteries require a scaffold that simultaneously maintains patency by preventing thrombosis and intimal hyperplasia, maintains its structural integrity after grafting, and allows integration. While synthetic and extracellular matrix-derived materials can provide some of these properties individually, developing a scaffold that provides the balanced properties needed for vascular graft survival in the clinic has been particularly challenging. After 30 years of research, there are now several scaffolds currently in clinical trials. However, these products are either being investigated for large-diameter applications or they require pre-seeding of endothelial cells. This progress report identifies important challenges unique to engineering vascular grafts for high pressure arteries less than 4 mm in diameter (e.g., coronary artery), and discusses limitations with the current usage of the term small-diameter. Next, the composition and processing techniques used for generating tissue engineered vascular grafts (TEVGs) are discussed, with a focus on the benefits of blended materials. Other scaffolds for non-tissue engineering approaches and stents are also briefly mentioned for comparison. Overall, this progress report discusses the importance of defining the most critical challenges for small diameter TEVGs, developing new scaffolds to provide these properties, and determining acceptable benchmarks for scaffold responses in the body.

Collagen incorporation within electrospun conduits reduces lipid oxidation and impacts conduit mechanics.

Modulating the host response, including the accumulation of oxidized lipid species, is important for improving tissue engineered vascular graft (TEVG) viability. Accumulation of oxidized lipids promotes smooth muscle cell (SMC) hyper-proliferation and inhibits endothelial cell migration, which can lead to several of the current challenges for small-diameter TEVGs. Generating biomaterials that reduce lipid oxidation is important for graft survival and this assessment can provide a reliable correlation to clinical situations. In this study, we determined the collagen to poly(ε-caprolactone) (PCL) ratio required to limit the production of pro-inflammatory species, while maintaining the required mechanical strength for the graft. Electrospun conduits were prepared from 0%, 10%, and 25% blends of collagen/PCL (w/w) and implanted in the rat peritoneal cavity for four weeks. The results showed that adding collagen to the PCL conduits reduced the accumulation of oxidized lipid species within the implanted conduits. In addition, the ratio of collagen had a significant impact on the recruited cell phenotype and construct mechanics. All conduits exhibited greater than 44% yield strain and sufficient tensile strength post-implantation. In conclusion, these results demonstrate that incorporating collagen into synthetic electrospun scaffolds, both 10% and 25% blend conditions, appears to limit the pro-inflammatory characteristics after in vivo implantation.

Peritoneal pre-conditioning reduces macrophage marker expression in collagen-containing engineered vascular grafts

Engineered vascular grafts have shown promise as arteriovenous shunts, but they have not yet progressed to clinical trials for coronary arteries <4u202fmm in diameter such as the coronary arteries. Control over initial biomaterial properties and remodeling are necessary to generate viable grafts. In this study, we blended collagen with a synthetic material, poly(ε-caprolactone), to modulate the post-grafting inflammatory response while avoiding aneurysmal-like dilation and failure that can occur with pure collagen grafts. We also used pre-implantation in an in vivo bioreactor to recruit autologous cells and improve patency after grafting. Electrospun conduits were pre-implanted within rat peritoneal cavities and then grafted autologously into abdominal aortae. Conduit collagen percentages and pre-implantation were tested for their impact on graft remodeling and patency. Burst pressures >2000u202fmmHg, reproducible expansion with systole/diastole, and maintenance of mechanical integrity were observed. More importantly, peritoneal pre-implantation reduced overall lipid oxidation, intimal layer thickness, and expression of an M1 macrophage marker. The condition with the most collagen, 25%, exhibited the lowest expression of macrophage markers but also resulted in a thicker intimal layer. Overall, the 10% collagen/PCL with peritoneal pre-implantation condition appeared to exhibit the best combination of responses, and may result in improved clinical graft viability.nnnSTATEMENT OF SIGNIFICANCEnThis manuscript describes a rodent study to systematically determine the benefits of both pre-implantation in the peritoneal cavity and specific ratios of collagen on engineered vascular graft viability. We show that pre-implantation had a significant benefit, including decreasing the expression of macrophage markers and reducing lipid oxidation after grafting. This study is the first time that the benefits of peritoneal pre-implantation have been compared to an off the shelf, directly grafted control condition. We also demonstrated the importance of specific collagen ratio on the response after grafting. Overall, we feel that this article will be of interest to the field and it has the potential to address a significant clinical need: a graft for coronary arteries <4u202fmm in diameter.

Silica-coated gold nanostars for surface-enhanced resonance Raman spectroscopy mapping of integrins in breast cancer cells

Surface-Enhanced Resonance Raman Spectroscopy (SERRS) has great potential for improving cancer research and diagnosis. Capable of sub-femtomolar detection, and a high degree of multiplexing, SERRS is an attractive new technique for studying cancer biology. We have developed PEGylated silica-coated gold nanostars that can be tuned to match the Raman laser-light source wavelength, providing high-level SERRS/SERS enhancement when combined with various reporter molecules. Furthermore, the particles were conjugated with cyclo-RGDf/k peptide to investigate integrin expression of breast cancer cells using high-speed Raman mapping. We propose that this may provide a better understanding of the role of integrins in breast cancer invasiveness.

Temporal changes in peritoneal cell phenotype and neoelastic matrix induction with hyaluronan oligomers and TGF-β1 after implantation of engineered conduits

The neoassembly and maturation of elastic matrix is an important challenge for engineering small‐diameter grafts for patients with peripheral artery disease. We have previously shown that hyaluronan oligomers and transforming growth factor‐β (elastogenic factors or EFs) promote elastogenesis in smooth muscle cell (SMC) culture. However, their combined effects on macrophages and inflammatory cells in vivo are unknown. This information is needed to use the body (e.g., peritoneal cavity) as an “in vivo bioreactor” to recruit autologous cells to implanted EF‐functionalized scaffolds. In this study, we determined if peritoneal fluid cells respond to EFs like smooth muscle cells and if these responses differ between cells sourced during different stages of inflammation triggered by scaffold implantation. Electrospun poly(ε‐caprolactone)/collagen conduits were implanted in the peritoneal cavity prior to peritoneal fluid collection at 3–42 days postimplantation. Cells from the fluid were cultured in vitro with and without EFs to determine their response. Their phenotype/behaviour was assessed with a DNA assay, quantitative real‐time PCR, and immunofluorescence. The EFs reduced peritoneal cell proliferation, maintained cell contractility, and unexpectedly did not exhibit proelastic effects, which we attributed to differences in cell density. We found the greatest elastin deposition in regions containing a high cell density. Further, we found that cells isolated from the peritoneal cavity at longer times after conduit implantation responded better to the EFs and exhibited more CD31 expression than cells at an earlier time point. Overall, this study provides information about the potential use of EFs in vivo and can guide the design of future tissue‐engineered vascular grafts.

Rapid generation of three-dimensional microchannels for vascularization using a subtractive printing technique

The development of tissue-engineered products has been limited by lack of a perfused microvasculature that delivers nutrients and maintains cell viability. Current strategies to promote vascularization such as additive three-dimensional printing techniques have limitations. This study validates the use of an ultra-fast laser subtractive printing technique to generate capillary-sized channels in hydrogels prepopulated with cells by demonstrating cell viability relative to the photodisrupted channels in the gel. The system can move the focal spot laterally in the gel at a rate of 2500 mm/s by using a galvanometric scanner to raster the in plane focal spot. A Galilean telescope allows z-axis movement. Blended hydrogels of polyethylene glycol and collagen with a range of optical clarities, mechanical properties and swelling behavior were tested to demonstrate that the subtractive printing process for writing vascular channels is compatible with all of the blended hydrogels tested. Channel width and patterns were controlled by adjusting the laser energy and focal spot positioning, respectively. After treatment, high cell viability was observed at distances greater than or equal to 18 μm from the fabricated channels. Overall, this study demonstrates a flexible technique that has the potential to rapidly generate channels in tissue-engineered constructs.

Delivery of Antioxidant and Anti-inflammatory Agents for Tissue Engineered Vascular Grafts

The treatment of patients with severe coronary and peripheral artery disease represents a significant clinical need, especially for those patients that require a bypass graft and do not have viable veins for autologous grafting. Tissue engineering is being investigated to generate an alternative graft. While tissue engineering requires surgical intervention, the release of pharmacological agents is also an important part of many tissue engineering strategies. Delivery of these agents offers the potential to overcome the major concerns for graft patency and viability. These concerns are related to an extended inflammatory response and its impact on vascular cells such as endothelial cells. This review discusses the drugs that have been released from vascular tissue engineering scaffolds and some of the non-traditional ways that the drugs are presented to the cells. The impact of antioxidant compounds and gasotransmitters, such as nitric oxide and carbon monoxide, are discussed in detail. The application of tissue engineering and drug delivery principles to biodegradable stents is also briefly discussed. Overall, there are scaffold-based drug delivery techniques that have shown promise for vascular tissue engineering, but much of this work is in the early stages and there are still opportunities to incorporate additional drugs to modulate the inflammatory process.