Christian Grasl

Electrospinning of aligned fibers with adjustable orientation using auxiliary electrodes

Abstract A conventional electrospinning setup was upgraded by two turnable plate-like auxiliary high-voltage electrodes that allowed aligned fiber deposition in adjustable directions. Fiber morphology was analyzed by scanning electron microscopy and attenuated total reflection Fourier transform infrared spectroscopy (FTIR-ATR). The auxiliary electric field constrained the jet bending instability and the fiber deposition became controllable. At target speeds of 0.9 m s−1 90% of the fibers had aligned within 2°, whereas the angular spread was 70° without the use of auxiliary electrodes. It was even possible to orient fibers perpendicular to the rotational direction of the target. The fiber diameter became smaller and its distribution narrower, while according to the FTIR-ATR measurement the molecular orientation of the polymer was unaltered. This study comprehensively documents the feasibility of directed fiber deposition and offers an easy upgrade to existing electrospinning setups.

Healing characteristics of electrospun polyurethane grafts with various porosities.

Pore size and porosity control the rate and depth of cellular migration in electrospun vascular fabrics and thus have a strong impact on long-term graft success. In this study we investigated the effect of graft porosity on cell migration in vitro and in vivo. Polyurethane (PU) grafts were fabricated by electrospinning as fine-mesh, low-porosity grafts (void fraction (VF) 53%) and coarse-mesh, high-porosity grafts (VF 80%). The fabricated grafts were evaluated in vitro for endothelial cell attachment and proliferation. Prostheses were investigated in a rat model for either 7 days, 1, 3 or 6 months (n=7 per time point) and analyzed after retrieval by biomechanical analysis and various histological techniques. Cell migration was calculated by computer-assisted morphometry. In vitro, fine-pore mesh favored early cell attachment. In vivo, coarse mesh grafts revealed significantly higher cell populations at all time points in all areas of the conduit wall. Biomechanical tests indicated sufficient compliance, tensile and suture retention strength before and after implantation. Increased porosity improves host cell ingrowth and survival in electrospun conduits. These conduits show successful natural host vessel reconstitution without limitation of biomechanical properties.

Biodegradable, thermoplastic polyurethane grafts for small diameter vascular replacements

Biodegradable vascular grafts with sufficient in vivo performance would be more advantageous than permanent non-degradable prostheses. These constructs would be continuously replaced by host tissue, leading to an endogenous functional implant which would adapt to the need of the patient and exhibit only limited risk of microbiological graft contamination. Adequate biomechanical strength and a wall structure which promotes rapid host remodeling are prerequisites for biodegradable approaches. Current approaches often reveal limited tensile strength and therefore require thicker or reinforced graft walls. In this study we investigated the in vitro and in vivo biocompatibility of thin host-vessel-matched grafts (n=34) formed from hard-block biodegradable thermoplastic polyurethane (TPU). Expanded polytetrafluoroethylene (ePTFE) conduits (n=34) served as control grafts. Grafts were analyzed by various techniques after retrieval at different time points (1 week; 1, 6, 12 months). TPU grafts showed significantly increased endothelial cell proliferation in vitro (P<0.001). Population by host cells increased significantly in the TPU conduits within 1 month of implantation (P=0.01). After long-term implantation, TPU implants showed 100% patency (ePTFE: 93%) with no signs of aneurysmal dilatation. Substantial remodeling of the degradable grafts was observed but varied between subjects. Intimal hyperplasia was limited to ePTFE conduits (29%). Thin-walled TPU grafts offer a new and desirable form of biodegradable vascular implant. Degradable grafts showed equivalent long-term performance characteristics compared to the clinically used, non-degradable material with improvements in intimal hyperplasia and ingrowth of host cells.

Electrospun polyurethane vascular grafts: In vitro mechanical behavior and endothelial adhesion molecule expression

Engineered small diameter vascular grafts must closely match mechanical characteristics of native vessels and exhibit stimulus-responsive bioactivity. In this study, mechanical homogeneity of electrospun small diameter polyurethane grafts as well as spontaneous attachment, proliferation, and adhesion molecule expression of endothelial cells (EC) in their presence was studied in vitro. Axial and circumferential tensile strengths were measured and found to be twofold higher in the circumferential direction. EC attachment was easily achieved without precoating the fiber matrix. Stimulation of EC with interleukin-1beta (IL-1beta) led to a statistically significant upregulation of the adhesion molecules E-Selectin, ICAM-1, and VCAM-1. Quantification of adhesion molecule expression by means of energy-dispersive X-ray microanalysis revealed no differences in the stimulatory responses of EC cultured on electrospun polyurethane when compared with cells grown on tissue culture-treated cover slips. Summarizing, highly uniform small diameter polyurethane grafts were fabricated and shown to allow spontaneous EC attachment. The synthetic graft surface neither impaired the endothelial response toward IL-1beta stimulation nor did it adversely affect the regulation of expression of endothelial adhesion molecules.

Electrospun Small‐Diameter Polyurethane Vascular Grafts: Ingrowth and Differentiation of Vascular‐Specific Host Cells

No small-diameter synthetic graft has yet shown comparable performance to autologous vessels. Synthetic conduits fail due to their inherent surface thrombogenicity and the development of intimal hyperplasia. In addressing these shortcomings, electrospinning offers an interesting alternative to other nanostructured, cardiovascular substitutes because of the close match of electrospun materials to the biomechanical and structural properties of native vessels. In this study, we investigated the in vivo behavior of electrospun, small-diameter conduits in a rat model. Vascular grafts composed of polyurethane were fabricated by electrospinning. Prostheses were implanted into the abdominal aorta in 40 rats for either 7 days, 4 weeks, 3 months, or 6 months. Retrieved specimens were evaluated by histology, immunohistochemical staining, confocal laser scanning microscopy, and scanning electron microscopy. At all time points, we found no evidence of foreign body reaction or graft degradation. The overall patency rate of the intravascular implants was 95%. Within 7 days, grafts revealed ingrowth of host cells. CD34+ cells increased significantly from 7 days up to 6 months of implantation (P < 0.05). Myofibroblasts and myocytes showed increasing cell numbers up to 3 months (P < 0.05). Ki67 staining indicated unaltered cell proliferation during the whole follow-up period. Besides biomechanical benefits, electrospun polyurethane grafts exhibit excellent biocompatibility in vivo. Cell immigration and differentiation seems to be promoted by the nanostructured artificial matrix.

Biomechanical Properties of Decellularized Porcine Pulmonary Valve Conduits

Tissue-engineered heart valves constructed from a xenogeneic or allogeneic decellularized matrix might overcome the disadvantages of current heart valve substitutes. One major necessity besides effective decellularization is to preserve the biomechanical properties of the valve. Native and decellularized porcine pulmonary heart valve conduits (PPVCs) (with [n = 10] or without [n = 10] cryopreservation) were compared to cryopreserved human pulmonary valve conduits (n = 7). Samples of the conduit were measured for wall thickness and underwent tensile tests. Elongation measurement was performed with a video extensometer. Decellularized PPVC showed a higher failure force both in longitudinal (+73%; P < 0.01) and transverse (+66%; P < 0.001) direction compared to human homografts. Failure force of the tissue after cryopreservation was still higher in the porcine group (longitudinal: +106%, P < 0.01; transverse: +58%, P < 0.001). In comparison to human homografts, both decellularized and decellularized cryopreserved porcine conduits showed a higher extensibility in longitudinal (decellularized: +61%, P < 0.001; decellularized + cryopreserved: +51%, P < 0.01) and transverse (decellularized: +126%, P < 0.001; decellularized + cryopreserved: +118%, P < 0.001) direction. Again, cryopreservation did not influence the biomechanical properties of the decellularized porcine matrix.

Electrodynamic control of the nanofiber alignment during electrospinning

A technique is presented to electrospin straight and aligned fibers on a stationary featureless target. Two parallel rotatable plate-like auxiliary electrodes were applied with a time-varying square wave potential. Thereby, the electrospinning jet was periodically deflected between the electrodes, which led to an aligned fiber-deposition. Straight fibers were deposited at a potential difference of 11 kV and a switching frequency of 40 Hz between the auxiliary electrodes. With this setup, freely adjustable orientations can be achieved regardless of the targets design or its movement.

Tissue engineering of vascular grafts

SummaryBackgroundThere is a considerable clinical need for a sufficient prosthetic small-diameter substitute which can compete with autologous vessels. Currently used synthetic materials have a poor performance due to high thrombogeneicity and development of intimal hyperplasia. Tissue engineering is an interesting alternative approach for vascular graft fabrication.MethodsWe briefly overviewed the development of tissue-engineered vascular substitutes including endothelialized biohybrid grafts, collagen and fibrin-based scaffolds, decellularized scaffolds, cell self-assembly approaches, and biodegradable constructs based on synthetic polymers.ResultsSignificant advances have been made over the past decades in the development of tissue-engineered conduits. Biomechanical weakness, one of the major limitations of biologically based grafts has been resolved and two tissue-engineered grafts are currently under further investigation for clinical application.ConclusionsVascular tissue engineering is a promising approach to overcome the limitations of current therapies in small-diameter vascular replacement.

Biocompatibility Assessment of a New Biodegradable Vascular Graft via In Vitro Co-culture Approaches and In Vivo Model.

Following the implantation of biodegradable vascular grafts, macrophages and fibroblasts are the major two cell types recruited to the host-biomaterial interface. In-vitro biocompatibility assessment usually involves one cell type, predominantly macrophages. In this study, macrophage and fibroblast mono- and co-cultures, in paracrine and juxtacrine settings, were used to evaluate a new biodegradable thermoplastic polyurethane (TPU) vascular graft. Expanded-polytetrafluoroethylene (ePTFE) grafts served as controls. Pro/anti-inflammatory gene expression of macrophages and cytokines was assessed in vitro and compared to those of an in vivo rat model. Host cell infiltration and the type of proliferated cells was further studied in vivo. TPU grafts revealed superior support in cell attachment, infiltration and proliferation compared with ePTFE grafts. Expression of pro-inflammatory TNF-α/IL-1α cytokines was significantly higher in ePTFE, whereas the level of IL-10 was higher in TPU. Initial high expression of pro-inflammatory CCR7 macrophages was noted in TPU, however there was a clear transition from CCR7 to anti-inflammatory CD163 expression in vitro and in vivo only in TPU, confirming superior cell-biomaterial response. The co-culture models, especially the paracrine model, revealed higher fidelity to the immunomodulatory/biocompatibility behavior of degradable TPU grafts in vivo. This study established an exciting approach developing a co-culture model as a tool for biocompatibility evaluation of degradable biomaterials.

Activated Schwann Cell-Like Cells on Aligned Fibrin-Poly(Lactic-Co-Glycolic Acid) Structures: A Novel Construct for Application in Peripheral Nerve Regeneration

Tissue engineering approaches in nerve regeneration search for ways to support gold standard therapy (autologous nerve grafts) and to improve results by bridging nerve defects with different kinds of conduits. In this study, we describe electrospinning of aligned fibrin-poly(lactic-co-glycolic acid) (PLGA) fibers in an attempt to create a biomimicking tissue-like material seeded with Schwann cell-like cells (SCLs) in vitro for potential use as an in vivo scaffold. Rat adipose-derived stem cells (rASCs) were differentiated into SCLs and evaluated with flow cytometry concerning their differentiation and activation status [S100b, P75, myelin-associated glycoprotein (MAG), and protein 0 (P0)]. After receiving the proliferation stimulus forskolin, SCLs expressed S100b and P75; comparable to native, activated Schwann cells, while cultured without forskolin, cells switched to a promyelinating phenotype and expressed S100b, MAG, and P0. Human fibrinogen and thrombin, blended with PLGA, were electrospun and the alignment and homogeneity of the fibers were proven by scanning electron microscopy. Electrospun scaffolds were seeded with SCLs and the formation of Büngner-like structures in SCLs was evaluated with phalloidin/propidium iodide staining. Carrier fibrin gels containing rASCs acted as a self-shaping matrix to form a tubular structure. In this study, we could show that rASCs can be differentiated into activated, proliferating SCLs and that these cells react to minimal changes in stimulus, switching to a promyelinating phenotype. Aligned electrospun fibrin-PLGA fibers promoted the formation of Büngner-like structures in SCLs, which also rolled the fibrin-PLGA matrix into a tubular scaffold. These in vitro findings favor further in vivo testing.