Browsing by Author "Bezuidenhout, Deon"

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    Open Access
    Coaxially electrospun heparin-eluting scaffolds for vascular graft application
    (2022) Krause, Gerhard Jaco; Bezuidenhout, Deon
    The use of electrospun scaffolds for small diameter vascular grafts (< 6 mm) has shown promise in the search for alternative solutions, as current synthetic grafts have high failure rates. The inclusion of heparin into such scaffolds can be beneficial for vascular graft applications as it could prevent mid-graft thrombosis, stabilise and potentiate growth factors as well as subdue undue proliferation of smooth muscle cells. Previous attempts at including heparin by chemical modification or inclusion into the bulk of the electrospun fibres (blend or emulsion electrospinning) were successful but resulted in burst release, reduced bioactivity and rapid elution of the heparin due to bulk degradation of the polymers. This project aimed to develop scaffolds comprising electrospun degradable polyurethane fibres with coaxially incorporated heparin sodium (HepNa+ ) with improved release kinetics and heparin activity for application in the tissue engineering of blood vessel substitutes. Scaffold sheets were cut from tubes (ID = 25 mm) produced on a rotating mandrel by coaxial, conventional and blend electrospinning of a degradable polyurethane, DegraPol® (DP30). Three coaxially electrospun groups were produced with DP30 (CHCl3) shells and polyethylene oxide, PEO (H2O/EtOH) cores containing either low (0.3 wt%), high (0.6 wt%) or no HepNa+ . Blend electrospinning was achieved by incorporating heparin (after modification to its tributylamine (TBA) salt for solubility) into DP30 solution (in CHCl3). Lastly, a control group was produced by conventional electrospinning of DP30 (CHCl3). The morphological (fibre diameter, fibre orientation, pore size and porosity), mechanical (tensile stress and strain, suture retention) and thermal (glass transition, melting and crystallization temperature) properties of the scaffolds were characterised and the corresponding in vitro drug release (heparin quantification and activity) and degradation response over 6 weeks in PBS (37 °C) were determined. Subsequently, conditions were optimised in a pilot study to electrospun small diameter (ID = 2.6 mm) tubular grafts and their morphological and mechanical properties (hoop stress, burst pressure and compliance) were determined. Coaxial electrospinning of DP30 with a water core and especially the addition of HepNa+ resulted in a decrease in fibre diameter (40 %), OI (23 %), pore size (39 %) and porosity (20 %) (all P < 0.05), most likely due to increased conductivity and dielectric constant. With one exception, there was no difference in the directional tensile properties between scaffold groups (ultimate tensile stress > 0.9 MPa, maximum strain > 100 %, suture retention > 2.4 N) or within groups between the longitudinal and circumferential tensile properties. After 6 weeks of in vitro degradation, all groups exhibited similar mechanical losses of approximately 40 % in ultimate tensile stress and 80 % in maximum elongation in circumferential and longitudinal directions. The smaller vascular grafts had burst pressures superior to native vasculature and compliances approximating those of healthy arteries. Thermal analyses (DSC) of the different groups showed similar thermograms with little intergroup variation and indicated that the electrospinning process did not unduly affect the thermal properties or crystallinity, of DP30. There was also no major variations in thermograms of degraded samples. Blend electrospun scaffolds showed the expected initial burst release of HepTBA (47.7 %, 3 days) followed by a sustained release (56.1 %, 6 weeks). Coaxially incorporated HepNa+ also exhibited initial burst release (67.5-69.7 %, 3 days) for both the low and high heparin content groups followed by improved sustained release (81.9 - 97.7%, 6 weeks). Coaxial incorporation had a 2× higher heparin encapsulation efficiency than blend incorporation (approaching 100 %). Heparin, post-TBA-modification, did not fully retain its antithrombotic properties (54.9 % reduction), which was further reduced after incorporation and release (24.2 % reduction). HepNa+ , however, retained its full antithrombotic activity post coaxial incorporation and elution. Coaxial electrospinning of heparin in DP30 shows potential for producing small diameter vascular grafts with mechanical properties comparable to small blood vessels. Although some initial burst release occurred, the sustained release over 6 weeks, incorporation of heparin without the need for modification at improved efficiency, and the retained activity of the heparin after electrospinning incorporation and elution; holds promise for vascular graft applications. Future work should aim for the production of continuous cores within fibre morphology and evaluating graft performance in an in vivo model to determine whether an appropriate and sufficient amount of heparin has been included to affect the desired response.
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    Open Access
    Design and development of an electrospun polymeric transcatheter heart valve for tissue engineering
    (2022) Guess, Rosslee Christie; Bezuidenhout, Deon; Appa, Harish
    The growing global burden of valvular heart disease (VHD) remains a significant challenge to overcome. Despite the advancements in heart valve replacements and the ground-breaking introduction of transcatheter valve technologies, current solutions offer suboptimal therapy as they either require lifelong anticoagulation or have limited durability. The ideal replacement valve should mimic the functionality of its native counterpart, comprise of viable living tissue, and offer lifelong durability, also for young patients. The central aims of this project were to design, manufacture, and evaluate two types of electrospun trileaflet transcatheter heart valve (THV) substitutes, one with non-degradable Pellethane® leaflets (PEL valves) and the other type using degradable DegraPol15® leaflets (DP valves). PEL and DP scaffolds were successfully electrospun after implementing a humidity control system and optimising input (ambient and processing) parameters; and were characterised for their morphological (fiber geometry and architecture), mechanical (static and dynamic), and hydraulic (wettability and permeability) properties. Six distinct valve manufacturing concepts (leaflet suturing, leaflet bonding, sandwich spinning, trileaflet preforming, inverted trileaflet preforming and submould assembly) were designed and prototyped for feasibility screening and selection. The selected concept was further employed to produce PEL and DP valves that were subsequently assessed for hydrodynamic function and durability by means of pulsatile flow analyses and accelerated fatigue testing. The fibres of DP scaffolds were approximately three times thicker than those in PEL scaffolds (5.2 ± 1.3 vs 1.8 ± 0.5 µm) with correspondingly larger pores (16.3 ± 4 vs 8.0 ± 1.7 µm). All scaffolds were essentially randomly oriented, and porosities averaged at 75 ± 5%. PEL scaffolds were approximately three times stronger than DP counterparts with slightly lower extensibility (550 vs 730%). From the six valve manufacturing concepts, the method involving the stitching of pre-cut leaflets onto the expandable stent (CoCr, 23 mm) after heat bonding an electrospun PEL skirt was selected. Four groups of valves, differing in average leaflet thickness (n = 2–4 per group), were produced for each material type. Under simulated aortic conditions, the PEL valves (N = 10) achieved remarkably high accelerated fatigue life of up to 260 million cycles (equivalent to 2.5 years), with a positive correlation between leaflet thickness and durability. The hydrodynamic parameters obtained for the PEL valves were well within ISO standards (EOA > 1.25 cm2 , mean pressure gradient < 10mmHg, and RF < 20% at a cardiac output of 5 l/min). In the current embodiments, DP valves (N=14) were not able to withstand aortic pressures but demonstrated good hydrodynamic function at nominal pulmonary pressures. Although much further study is required, the concepts described in this project contribute valuable insight into the manufacturing, performance, and durability of devices intended for transcatheter heart valve tissue engineering.
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    Open Access
    Design of an adventitial type reinforcement of prosthetic vascular grafts through mechanically affirmed material and structure modulation
    (2001) Millam, Ross David Alexander; Zilla, Peter; Bezuidenhout, Deon
    The high occurrence of vascular disease in the 20th century has been the driving force for researchers to produce a successful small diameter synthetic graft. Large diameter synthetic grafts remain patent for extended periods due to high flow rate, while smaller diameter grafts occlude more readily. Mechanical property mismatch between graft and host artery has been cited as one of the major factors that contribute to graft occlusion. It has thus been important to develop a readily available graft that is accepted by the body and does not cause flow abnormalities and stress-concentrations at graft-artery junctions. The object of this project was to ascertain the effect of an adventitial reinforcement on elastic compliance of synthetic porous polyurethane grafts.
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    Open Access
    Development of a fatigue tester and material model for flexible heart valve applications
    (2019) Van Breda, Braden; Mouton, Hennie; Bezuidenhout, Deon
    The leaflet material in heart valve prostheses is required to be both flexible and durable to eliminate the need for chronic anticoagulation medication and accommodate younger patients with longer life expectancies. This investigation aims to provide two of the necessary tools to design and test suitably flexible and durable materials for heart valve replacement. These tools address the question of how to model the stress-strain behaviour of polymer networks and thermoplastic polyurethanes in particular, as well as how to practically evaluate the durability of the proposed material. A model for polyurethane stress-strain behaviour is proposed, whereby the number of monomers between crosslinks is suggested to evolve with macroscopic strain. Following the polymer chain entanglement theory, the increase in the number of monomers between crosslinks is further extended to be a function of strain rate, incorporating the viscous effect observed in polyurethanes. A multistation, micro-tensile specimen fatigue tester was developed to evaluate material durability. The proposed equilibrium polyurethane model accurately predicts the experimental data across the full material strain range. The proposed model extension sufficiently captures the rate dependence of polyurethane, however, fails to account for the raised specimen temperatures at high strain rates. The developed fatigue tester is verified to successfully feature selectable variables including test frequency (1 - 20 Hz), amplitude (1 - 6 mm), waveform (Triangular, Sinusoidal, Square and Custom) and environmental temperature control (23 - 50 oC). Less than 10% error in measured force is observed when compared to a commercial tensile tester. The proposed model successfully provides a platform to aid the design of flexible materials suitable for heart valve leaflets. The developed fatigue tester enables the assessment of material durability across a range of test conditions, successfully providing a tool for leaflet material durability analysis and verification.
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    Drug eluting electrospun scaffolds for tissue regeneration
    (2018) Van den Bergh, Willem Johannes Wian; Bezuidenhout, Deon; Franz, Thomas
    The desired healing response to electrospun scaffolds in tissue engineering is often limited by poor ingrowth due to insufficient porosity, thrombogenicity, lack of vascularisation and/or excessive inflammation. This study aimed at increasing structural porosity and incorporating/delivering anti-thrombotic/angiogenic (heparin) and anti-inflammatory (dexamethasone) agents. Porosity enhancement techniques were explored using two different approaches i) electrospinning of biostable polymer (Pellethane® , Pel) with concomitant electrospraying of soluble microparticles, which were subsequently removed to increase scaffold interconnectivity and ii) electrospinning of biodegradable polymer (DegraPol® , DP) at low collecting temperatures. Dexamethasone (Dex) was incorporated by simple admixture, while heparin (Hep) required chemical modification (heparin tributylammonium, HepTBA) to achieve solubility. Release rates were determined in vitro, followed by thrombogenicity (thromboelastography) and cytotoxicity (cell viability) assessments of modified/unmodified heparin prior to incorporation and after elution. Finally, in vivo responses were evaluated in a subcutaneous model (24 rats) for up to 12 weeks. Porosity was enhanced (P0.1). At 12 weeks of implantation, high-porosity Pel scaffolds allowed for full tissue ingrowth (>98%) while conventional scaffolds were limited (0.3). High-porosity scaffolds produced by combined electrospinning/spraying have the potential to enhance healing. Dex or HepTBA can be incorporated and eluted from degradable electrospun scaffolds, and localised delivery of HepTBA improves implant vascularisation. This study may contribute towards tissue engineered vascular graft development where anti-thrombogenicity and increased vascularisation are desired.
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    Drug eluting hydrogels design, synthesis and evaluation
    (2012) Ahrenstedt, Lage; Bezuidenhout, Deon; Hult, Anders
    Hydrogels have successfully proved themselves useful for drug delivery applications and several delivery routes have been developed over the years. The particular interest in this work was to design, synthesise and evaluate in situ forming drug eluting hydrogels, which have the potential to ameliorate the healing of cardiovascular diseases.
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    Electrospun Tissue Engineered Vascular Grafts
    (2019) Hülk, Vincent Martin; Bezuidenhout, Deon
    ePTFE and Dacron vascular grafts are successful in large diameter applications but provide poor outcomes in small diameter (<6 mm) applications. Occlusion, poor cell ingrowth and a mismatch of compliance compared to the native vessel, cause failure to the current synthetic grafts. Spontaneous transmural endothelialisation can be enhanced in non-degradable scaffolds with heparin-mediated growth factor delivery via heparin surface modification, as well as induce an anti-thrombotic lumen. Electrospinning of vascular grafts offer a means to mimic the natural extracellular matrix (ECM) with improved porosity and pore for increased cell ingrowth, incorporation of drugs for sustained release, and tailoring mechanical properties match that of the native vessel. This study aims to produce a small diameter biodegradable vascular graft with sufficient porosity/pore size and incorporated heparin as an angiogenic/anti-thrombotic agent. DegraPol® (DP30), a degradable polyurethane was dissolved in different ratios of chloroform/HFIP (24%w/w) and electrospun at 30%, 40%, and 50% rH to obtain a small diameter vascular graft with improved porosity/pore size and mechanical properties. The sodium salt of heparin (HepNa+ ) was modified to heparin tributylamine (HepTBA) to ensure the solubility in organic solvents and incorporated into the electrospinning solution at 3% and 5% wt/wt (w HepTBA/w polymer). The grafts were analysed for morphological (fibre diameter, porosity/pore size, fibre alignment) and mechanical (hoop stress, strain, burst pressure, compliance) properties. After in vitro elution and degradation studies, grafts (DP30 and DP30+3%HepTBA) were evaluated in an in vivo pilot study using a rat infrarenal aortic interposition model (28 days). Relative humidity did not significantly influence the scaffold morphology or the mechanical properties, for solvent systems used. However, the addition of HFIP to the solution had on average a 2.9-fold decrease in the circumferential UTS and strain from 0.63±0.16 MPa and 90±16%, respectively. The grafts showed theoretical compliance in the physiological range of 6-8%/100mmHg and showed a significant amount of drug release in the first 5 days and a cumulative release of 62% and 36% (respectively) by day 28. The DP30 and DP30+3%HepTBA grafts lost 46% (P> 0.5) and 50% (P< 0.01) of its circumferential UTS respectively, whereas the loss in maximum strain for the same groups was 66% (P< 0.0001) and 76% (P< 0.01). A porosity of 64.5±2.7% and 54.9±2.2% (P< 0.01) was achieved for the DP30 and DP30+3%HepTBA with 52% and 29% of the pore sizes larger than 10 µm. The in vivo pilot study showed patent grafts with tissue ingrowth and endothelium on the lumen for DP30 as well as the DP30+3%HepTBA group. The DP30 grafts show promise for the replacement of small diameter vessels. Heparin-eluting grafts will be further evaluated in long-term isolated loop models to determine their capacity for spontaneous transmural endothelialisation.
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    Open Access
    Electrospun Tissue Engineered Vascular Grafts
    (2019) Hülk, Vincent Martin; Bezuidenhout, Deon
    ePTFE and Dacron vascular grafts are successful in large diameter applications but provide poor outcomes in small diameter (<6 mm) applications. Occlusion, poor cell ingrowth and a mismatch of compliance compared to the native vessel, cause failure to the current synthetic grafts. Spontaneous transmural endothelialisation can be enhanced in non-degradable scaffolds with heparin-mediated growth factor delivery via heparin surface modification, as well as induce an anti-thrombotic lumen. Electrospinning of vascular grafts offer a means to mimic the natural extracellular matrix (ECM) with improved porosity and pore for increased cell ingrowth, incorporation of drugs for sustained release, and tailoring mechanical properties match that of the native vessel. This study aims to produce a small diameter biodegradable vascular graft with sufficient porosity/pore size and incorporated heparin as an angiogenic/anti-thrombotic agent. DegraPol® (DP30), a degradable polyurethane was dissolved in different ratios of chloroform/HFIP (24%w/w) and electrospun at 30%, 40%, and 50% rH to obtain a small diameter vascular graft with improved porosity/pore size and mechanical properties. The sodium salt of heparin (HepNa+ ) was modified to heparin tributylamine (HepTBA) to ensure the solubility in organic solvents and incorporated into the electrospinning solution at 3% and 5% wt/wt (w HepTBA/w polymer). The grafts were analysed for morphological (fibre diameter, porosity/pore size, fibre alignment) and mechanical (hoop stress, strain, burst pressure, compliance) properties. After in vitro elution and degradation studies, grafts (DP30 and DP30+3%HepTBA) were evaluated in an in vivo pilot study using a rat infrarenal aortic interposition model (28 days). Relative humidity did not significantly influence the scaffold morphology or the mechanical properties, for solvent systems used. However, the addition of HFIP to the solution had on average a 2.9-fold decrease in the circumferential UTS and strain from 0.63±0.16 MPa and 90±16%, respectively. The grafts showed theoretical compliance in the physiological range of 6-8%/100mmHg and showed a significant amount of drug release in the first 5 days and a cumulative release of 62% and 36% (respectively) by day 28. The DP30 and DP30+3%HepTBA grafts lost 46% (P> 0.5) and 50% (P< 0.01) of its circumferential UTS respectively, whereas the loss in maximum strain for the same groups was 66% (P< 0.0001) and 76% (P< 0.01). A porosity of 64.5±2.7% and 54.9±2.2% (P< 0.01) was achieved for the DP30 and DP30+3%HepTBA with 52% and 29% of the pore sizes larger than 10 µm. The in vivo pilot study showed patent grafts with tissue ingrowth and endothelium on the lumen for DP30 as well as the DP30+3%HepTBA group. The DP30 grafts show promise for the replacement of small diameter vessels. Heparin-eluting grafts will be further evaluated in long-term isolated loop models to determine their capacity for spontaneous transmural endothelialisation.
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    Evaluation of cellular interactions with functionalized scaffolds for cardiovascular tissue engineering
    (2021) Wimberley, Olivia; Bezuidenhout, Deon; Davies, Neil
    Introduction: Cardiovascular disease is a leading cause of death worldwide, with heart valve disease in particular becoming a rising problem in developing countries. Tissue engineering offers the next step in heart valve tissue replacement surgery. Synthetic scaffolds used in tissue engineering often do not have sufficient cell adhesion, thus the addition of biosignals is crucial. Surface modifications can be used to improve desired cell adhesion and proliferation. To covalently attach cell adhesion peptides/biosignals, synthetic hydrogel spacers are often used in an interim grafting step. Poly (acrylic acid) (PAA) is a non-toxic, FDA approved hydrogel that has been shown in preliminary studies (previous research and own experience) to improve cell adhesion even without the addition of specific cell-binding compounds. This project aims to show the effect of systematically increasing the concentration of PAA modified 2D and 3D polyurethanes (biodegradable and biostable) on cell adhesion, persistence, and proliferation. Experimental Methods: In Part 1, 2D nondegradable Pellethane® films were surface modified by varying the poly (acrylic acid-co-acrylamide) (P(AA-co-AM)) comonomer feed ratio from 0 to 100 % PAA in 20 % increments, using poly (acrylamide) (PAM) as a copolymer (unmodified and collagen coated controls). Surface properties were analysed using SEM imaging, staining, energy-dispersive X-ray spectrometry (SEM-EDS) and toluidine blue carboxyl assays (TBCA). Endothelial cells were isolated from human saphenous veins using an enzymatic digestion method, and identified by staining with DAPI and Cy3 against CD31. Isolated endothelial cells and human dermal fibroblasts (Cell bank: R039/2016) were seeded onto Pellethane® films (8 000 cells/film). Live/dead staining and XTT cell viability assays were performed over 24 and 72 hrs, respectively. Following this, XTT cell viability assays were performed at 7 and 24 hrs post-seeding on endothelial cells cultured under serum-free conditions (20 000 cells/film; unmodified and 80 % PAA). In Part 2, both Pellethane® and DegraPol® (degradable) 2D films and 3D electrospun scaffolds were used. The polymer samples were surface modified with 0, 40, and 80 % PAA. All samples were imaged using SEM prior to in vitro cell culture evaluation. Endothelial cells were seeded (8 000 cells/film) onto surface modified polymer samples, and XTT cell viability assays were performed over 72 hrs. Three-dimensional scaffolds seeded with endothelial cells (20 000 and 50 000 cells/film; unmodified and 80 % PAA) were immunocytochemically stained (Hoechst and CD31) at Day 1, 3, and 7 post-seeding. Results and discussion: In Part 1, SEM imaging and preliminary staining confirmed the addition of PAA to polymer surfaces. Systematically increasing [AA] in the P(AA-co-AM) comonomer ratio resulted in the expected increase in surface-COOH functional groups (TBCA and SEM-EDS). The number of COOH groups increased as [PAA] increased from 0-40 % (R 2=0.76; P 0.0001) before plateauing (TBCA). This was further confirmed by a decreasing N/O ratio with increasing [AA] monomer (R2=0.70; P< 0.001) (SEM-EDS). An increase in [PAA] resulted in a linear increase in endothelial cell adhesion and persistence (R 2=0.92 (live/dead staining) and 0.96 (XTT cell viability assays); P< 0.05). Endothelial cell viability on surfaces modified with 80 and 100 % PAA was comparable to that achieved on the collagen positive control. High concentrations of PAA also showed improved fibroblast adhesion (R2= 0.71 (live/dead staining) and 0.54 (XTT cell viability assays); P< 0.05) but did not display any persistence or viability close to that obtained on the collagen. Collagen coated surfaces displayed the highest cell adhesion and proliferation for both cell types (XTT cell viability assays and live/dead staining). Endothelial cell adhesion was improved by both the addition of PAA to the polymer surface, and FBS to the cell culture medium (P< 0.05) (cells cultured under serum-free conditions). In Part 2, the improvement of endothelial cell adhesion on PAA modified 2D Pellethane® films was confirmed and additionally shown on 2D DegraPol® (P ≤0.05) (XTT cell viability assays). However, the endothelial cell persistence seen in earlier assays was not observed. The positive effect of increasing [PAA] did not translate to 3D scaffolds, and cell behaviour was improved on unmodified surfaces in comparison to any of the PAA modified groups (XTT cell viability assays and immunocytochemical staining). This discrepancy is proposed to be a difference in grafting efficiency on the degradable materials and the 3D structure of the electrospun scaffolds. Conclusions: An increase in PAA surface modification on polyurethane can improve endothelial cell adhesion and persistence on nondegradable 2D polyurethane scaffolds. These results did not translate to electrospun scaffolds, probably due to the complex 3D cell environment. Further investigation is required for use in TEHV and other applications.
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    Heparanoid hydrogels for cardiovascular tissue regeneration
    (2015) Janse van Rensburg, Aliza; Bezuidenhout, Deon; Davies, Neil
    Heparin (Hep) and heparan sulfate (HS) have been shown to possess anticoagulative properties, inhibit smooth muscle cell proliferation, moderate inflammation and control angiogenesis by stabilization and potentiation of growth factors (GF). These properties are potentially very useful for the treatment of cardiovascular diseases, especially when delivered as injectable hydrogels that can form in situ. This project focused on developing Hep and HS hydrogels for localized GF delivery. Hep and HS were acrylated, characterized and crosslinked with PEG tetra-thiols, either directly (10m% Hep/HS-Ac, Type 1) or by copolymerization with 20PEG8Ac or 20PEG8VS (4m% copolymer; 1.5% Hep/HS-Ac) to form degradable (Type 2D) or non-degradable (Type 2N) gels, respectively. Gelation times, viscoelasticity, swelling, mesh size, Hep/HS elution and activity, as well as GF incorporation and release were studied in vitro. Type 2D gels with covalently incorporated (CI) Hep and GFs were evaluated in vivo as ingrowth matrices in porous polyurethane (PU) scaffolds for healing response in a rat subcutaneous model.
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    High porosity electrospun scaffolds for small diameter vascular graft applications
    (2015) Voorneveld, Jason Dirk; Bezuidenhout, Deon; Bezuidenhout, Deon; Franz, Thomas
    Porosity, pore size and pore interconnectivity have been shown to be critical factors for cellular infiltration into vascular grafts. While electrospinning has been shown to produce many promising characteristics for the fabrication of vascular graft scaffolds, it has yet to create sufficient porosity for transmural endothelial in-growth. This study was aimed at using dual electrospinning with sacrificial fibre extraction to produce scaffolds with controllable porosity characteristics while maintaining sufficient structural strength to resist deformation during implantation. Scaffolds were subsequently covalently grafted with heparin, a known anti-coagulant with growth-factor binding properties.
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    Hybrid Electrospun Transcatheter Aortic Heart Valves for Tissue Engineering Applications
    (2022) Taylor, Jade; Bezuidenhout, Deon
    Bioprosthetic and mechanical heart valves are suboptimal due to both calcific degeneration and the need for lifelong anticoagulation treatment. Tissue engineering (TE) has the potential to overcome these and other shortcomings by providing viable valves capable of growth, remodelling, and repair. Electrospinning has gained popularity as one of the methods used to create porous, threedimensional TE scaffolds that mimic the extracellular matrix (ECM) and allow for cellular ingrowth into the construct. The aim of the current study was to investigate the feasibility of producing electrospun scaffolds with both degradable and reinforcing, non-degradable, fibres to improve mechanical properties and allow for improved control over degradation rates to avoid catastrophic valve failure due to premature valve degradation and inadequate tissue ingrowth. Degradable (DegraPol15®; DP15), non-degradable (Pellethane®), and hybrid (dual) scaffolds (± 1:1 mass ratio of DP15 and Pellethane®) were electrospun and characterised morphologically and mechanically before and after various periods of hydrolytic degradation. Transcatheter aortic heart valves (23 mm) with DegraPol®, Pellethane®, and hybrid leaflets respectively, were fabricated by means of bonding and suturing into balloon expandable transcatheter stents and tested in vitro at left heart conditions to determine hydrodynamic performance and durability. Ultimate tensile strength (UTS) decreased as electrospun scaffolds degraded, with DegraPol® exhibiting a more rapid decrease in UTS than hybrid scaffolds after six weeks of incubation (57 ± 5% vs 24 ± 10%). The same trend was seen in the distinct reduction of maximum elongation of the scaffolds (35 ± 5% and 18 ± 8 %) . Pellethane® exhibited a reduction in both UTS and % elongation close to that of hybrid scaffolds (27 ± 6% and 13 ± 6% respectively) with the Young's modulus of all scaffolds remaining nearly unaffected by the hydrolytic degradation. Hybrid scaffolds had suture retention strength similar to DegraPol®, however, hybrid scaffolds unexpectedly had the lowest tear strength (4.7 ± 0.29 N/mm) with Pellethane® scaffolds displaying superior strength in both tear strength (13.53 ± 0.35 N/mm) and suture retention strength (16.13 ± 0.8 N/mm longitudinally). In terms of valve hydrodynamic performance, all valves had an average effective orifice area (EOA) larger than 2 cm2 and pressure gradients lower than 8mmHg with no significant difference between the groups in either of these measurements. Pellethane® and hybrid valves both had lower regurgitation fractions than DegraPol® valves (30.3 ± 5.3% 28.6 ± 1.5% and 56.4 ± 4.4% for Pellethane®, hybrid and DegraPol® valves respectively), with high (>20%) values resulting from inherent material porosity shown by water permeability tests. With regards to valve durability, DegraPol® valve leaflets failed before adequate testing conditions could be reached while Pellethane® valves lasted for ±40 million cycles and were removed before failure. Hybrid valves lasted for an intermediate average of 7.0 ± 2.3 million cycles at 100 mmHg. The study shows that the polymers can be dual spun from degradable and non-degradable polymers to produce hybrid scaffolds which can potentially be used, with further development, in tissue engineering applications such as heart valves when the degradable component may lack sufficient physical strength.
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    Isolating and optimising transmural endothelialization as an independent mode of spontaneous vascular graft healing
    (2014) Pennel, Timothy Charles; Bezuidenhout, Deon; Zilla, Peter
    Background: Poorly designed animal models with short, non-isolated grafts have led researchers to exclusively investigate transanastomotic endothelialization(TAE), which is known to be limited to the perianastomotic region in humans. Alternative spontaneous forms of endothelialization (fallout and transmural) need to be independently investigated and optimised to redirect future conduit research. Methods: All implants were performed in infra-renal rat aorta. TAE was determined at 2, 4, and 6 weeks (n=6/time-point) (ePTFE;ID1.7mm;IND15-25µm). High-porosity polyurethane (internal diameter 1.7mm- 150mm pore size) grafts were interposed between ePTFE grafts for 2, 4, 6, and 8 weeks (n=6/time point). Grafts were looped to increase their length and were implanted for 6, 8, 12, and 24 weeks (n=8/time-point). Perigraft isolation included (PU skin, 50µm, or 350µm wall thickness wrap, (IND30µm;ePTFE) to prevent transmural ingrowth; 12-weeks. The mid-graft was further surface-treated with heparin or heparin-VEGF/PDGF and implanted for 3 weeks (n=10/group). Explanted samples were analysed by light, immunofluorescence and scanning electron microscopy for vascularization and endothelialization. Results: TAE occurred at 0.6±0.4mm/wk., which slowed from 0.8±0.4 to 0.5±0.3mm/wk., [p=0.039] from 2 to 6 weeks. Straight composite grafts separation zones were too short to isolate transmural ingrowth beyond four weeks. However, a broad endothelial-free zone was preserved in all looped composite grafts (23.6±10.1, 23.7±8.2, 13.4±11.0, 10.5±5.6mm for 6, 8, 12, and 24 weeks respectively), [p=0.0031]. Mid-graft pre-confluence was reached by 6 weeks (55±45%) and confluence between week 12 and 24 (95.0±10.0% and 84.0±30.13%). The subintimal thickness did not increase (91.8±93.9 mm vs. 71.4±59.4 mm at 6 and 24 weeks, respectively; NS). All sealed grafts occluded. Mid-graft endothelialization was not influenced by 50µmFilmwrap (93±8.3%), but the 350µm-Wrap significantly reduced coverage (16±30%, pre Conclusions: Transmural endothelialization can be clearly distinguished from TAE in a high throughput rat model. A looped interposition graft model provides sufficient isolation length to separate the two events for up to half a year without an increase in intimal hyperplasia. Growth factor surface modification further enhances this form of healing, while perigraft isolation confirms that fallout endothelialization is insufficient to heal the mid-graft with confluence. These findings may be useful for the development of clinical peripheral vascular (including endoluminal) grafts, as TM endothelialization remains a viable healing mode in humans.
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    Optimization of structural and mechanical properties of electro-spun biodegradable scaffolds for vascular vissue regeneration
    (2013) Khatib, Rodaina Omar; Franz, Thomas; Krynauw, Hugo; Bezuidenhout, Deon
    Current replacements for diseased arteries include autologous and artificial grafts. The availability of autologous grafts is limited and artificial grafts tend to fail when applied to small calibre vessels (<;6 mm) due to graft thrombosis and mechanical mismatch between artery and graft. Tissue engineering offers a promising approach to overcome these shortcomings. With porosity as a fundamental prerequisite for tissue ingrowth, several techniques have been introduced for producing porous scaffolds including electro-spinning. This study involved the development and optimisation of electro-spun biodegradable scaffolds for vascular tissue regeneration by tailoring parameters of the electro-spinning process and investigating the change in mechanical and physical properties of the scaffolds associated with hydrolytic in vitro degradation.
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    Paravalvular sealing pf percutaneous heart valves
    (2019) Conradie, David Gideon; Bezuidenhout, Deon; De Villiers, Jandre
    Paravalvular regurgitation (PVR), which frequently occurs after transcatheter aortic valve replacements (TAVR) can lead to adverse clinical consequences and has been shown to correlate to an increased late mortality and morbidity. Quantification, graduation and testing for PVR has proven challenging and a standardized method for pre-clinical testing is still sought. Commercial transcatheter heart valves (THV's) rely on sealing skirts made from treated pericardium or polyethylene terephthalate (PET) fabrics. The current study was aimed at developing novel electrospun skirts for the minimization of PVR in THV's. Thermoplastic polyurethane (TPU) was electrospun onto mandrels and several techniques (CO2 - laser, ultra-sonic, solvent and conductive heat bonding) used to attach the scaffolds to pre-coated TAVR stents. Attachment strength was modelled by finite element analysis (FEA) of stents in the crimped and expanded conditions and empirically determined by physical pull-off tests. PVR was evaluated for four different skirt designs (1× single layer “FLAT” and 3× double layers: Forward Flow Filling “FFF”, No Filling “NF” and Back Flow Filling “BFF”) using a pulse duplicator fitted with perforated mounting rings. Optimization of solution, process and environmental parameters yielded scaffolds with average fibre diameters of 3.17 ± 0.64 μm and average pore sizes 9.52 ± 6.90 μm. Tensile strength was found to be similar in the direction perpendicular to collector rotation [Abstract incomplete due to DSpace NOT being able to accommodate some formulas and equations in the abstract, BS]
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    Patient-specific thoracic endovascular aoratic repair (TEVAR)
    (2019) Lin, Andrew; Bezuidenhout, Deon; Thierfelder, Nikolaus
    Endovascular aortic repair (EVAR) is a minimally invasive procedure to treat aortic aneurysms. Current off-the-shelf devices may not fit the patient perfectly, potentially increasing the chance of post-operative complications. This project aims to provide proof of concept for rapidly creating inexpensive patient-specific EVAR stent-grafts, conforming to the unique anatomy of the patient. After investigating the range of electrospinning shape capabilities on idealised stent-graft geometries (straight, tapered, elliptical, and curved), CT scans was used to create blood and aortic models of an abdominal aortic aneurysm. The former was used to design a patientspecific stent-graft geometry, 3D print a conductive electrospinning mandrel, and electrospin (290 mm, +18 kV, -3 kV, 5 ml/hr, 5 mm/s, 750 rpm) Polyurethane (PU). Sinusoidal Nitinol reinforcement segments were subsequently incorporated into the graft. Various geometries were successfully spun. Electrospun PU scaffolds had a mean ultimate tensile strength of 7.3 MPa, mean Young’s Modulus of 1.9 MPa, and a mean maximum strain of 571%. Fibre morphology analysis showed a mean orientation index of 0.25 (750 rpm) and 0.35 (1000 rpm), mean fibre diameter of 2.3 µm, and a mean pore size of 7.5 µm; pore size indicates possibility of endothelialisation. Nitinol reinforced patient-specific graft was successfully made and stent-grafts of various stent patterns had radial forces between 1.3 to 5.8 N (comparable to 2.8 N from a commercial example). FEA simulation highlighted various advantages of customised stent-grafts that conform to the anatomy over standard cylindrical devices such as better seal and contact traction. Simulation results (25 mm Ø, cylindrical, electrospun stent-graft) showed close approximations to experimental results; its use for future stent-graft design optimisations is promising. Mock insertion of an electrospun patient-specific stent-graft was performed in a 3D-printed transparent-PLA hollow aortic model with good conformity, albeit subpar visibility without a backlight and inflexibility. Although further improvements can be made to the individual steps, proof of principle was achieved. This process is very promising for the manufacturing of patient-specific devices that could offer better long term outcomes.
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    The performance of cross-linked acellular arterial scaffolds as vascular grafts; pre-clinical testing in direct and isolation loop circulatory models
    (2016) Pennel, Timothy; Bezuidenhout, Deon; Zilla, Peter
    There is a significant need for small diameter vascular grafts to be used in peripheral vascular surgery; however autologous grafts are not always available, synthetic grafts perform poorly and allografts and xenografts degenerate, dilate and calcify after implantation. We hypothesized that chemical stabilization of acellular xenogenic arteries would generate off-the-shelf grafts resistant to thrombosis, dilatation and calcification. To test this hypothesis, we decellularized porcine renal arteries, stabilized elastin with pentagalloyl glucose and collagen with carbodiimide/activated heparin and implanted them as trans- position grafts in the abdominal aorta of rats as direct implants and separately as indirect, isolation-loop implants. All implants resulted in high patency and animal survival rates, ubiquitous encapsulation within a vascularized collagenous capsule, and exhibited lack of lumen thrombogenicity and no graft wall calcification. Peri-anastomotic neo-intimal tissue overgrowth was a normal occurrence in direct implants; however this reaction was circumvented in indirect implants. Notably, implantation of non- treated control scaffolds exhibited marked graft dilatation and elastin degeneration; however PGG significantly reduced elastin degradation and prevented aneurismal dilatation of vascular grafts. Overall these results point to the outstanding potential of crosslinked arterial scaffolds as small diameter vascular grafts.
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    Polymeric Transcatheter Heart Valves
    (2019) Coetzee, Johan; Bezuidenhout, Deon; de Villiers, Jandre
    Rheumatic heart disease (RHD) is one of the main causes of heart disease in the emerging world. Once the disease has become symptomatic valve repair or replacement is the only treatment currently available. Commercial bioprosthetic valves (surgical and transcatheter) suffer from calcification and decreased durability, especially when implanted into younger patients, while mechanical surgical valves require life-long anticoagulation. Polymeric transcatheter aortic valve insertion (TAVI) is proposed as a solution to provide long-term durability without the need for anticoagulation. A manufacturing method involving the spray coating of polyurethane solutions onto valve moulds, pre-coating a TAVI stent with a polyurethane of higher durometer, and subsequently spraying the combination of the stent and the mould to form integral attachments of the leaflets, is described. Valves were tested for leaflet thickness distribution, hydrodynamic function and accelerated durability, and subsequently implanted in an acute ovine TAVI model as proof of concept. Effective Orifice Area (EOA) >1.7 cm2, regurgitation < 10% and transvalvular pressure gradient < 10 mmHg were achieved. Leaflet thickness correlated indirectly with the EOA and directly with transvalvular pressure gradient, but not regurgitation. After iterative improvements in the manufacturing process, valves with average thicknesses ranging from 140 to 160 μm showed highest durability (>150 million, and up to 600 million cycles). Surface roughness was reduced by applying a final pure solvent coat. Implanted valves showed good function, with no apparent central or paravalvular regurgitation, perfusion of coronaries, and EOA greater than1.6 cm2. In conclusion, a polymeric TAVI valve made by a new manufacturing method showed durability up to 15 years equivalent in vitro, and good hydrodynamic function in vitro and in vivo. The devices hold many potential advantages in terms of automation and cost of manufacturing, as well as function and longevity.
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    Pulsatile Electropolishing of Nitinol Stents
    (2019) Cloete, Jeran Andre; Bezuidenhout, Deon; Levecque, Pieter
    Alloys that oxidize easily such as those containing titanium or chromium present a challenge to electropolishing because the polarization that dissolves the metal species produces positive ions, these oxidize and form stable surface layers of metallic oxides that prevent further dissolution. This is usually overcome with the use of acid solutions that dissolve the metallic oxide. This thesis aims to shift the primary control of the electropolishing e_ect from electrolyte variables to a combination of potential variation and hydrodynamic interference. Traditionally this is achieved with one continuous mass removal process that operates after a steady state of dissolution is established, generally requiring hydro_uoric or phosphoric acid to achieve titanium dioxide breakdown. The resulting concentration gradient is heavily a_ected by electrolyte variables such as viscosity and electrical resistance, while the electrical polarization is constrained by the metallic oxide reaction rate which creates a complex net of interdependent variables that can be di_cult to tune. A rapidly changing electric _eld was applied to modulate the alloying element dissolution rates. In tandem with the electropolishing development, stages prior to the electropolishing step were selectively removed to simplify the process. Utilizing a three electrode system and an external potentiostat controller to permit greater _exibility, a variety of alternating current pulsatile waveforms were investigated and the resulting e_ect on surface topology was observed using SEM and AFM microscopes. Di_erential pulse voltammogram yielded a feedback parameter on surface composition, and various pulse parameters were adjusted to optimize for surface smoothness, and identify the primary control variable. An electropolishing method is presented which achieves a :50% reduction in the Sa surface roughness value to an area average of 45 nm on a laser cut tubular stent geometry. It is shown that this method can be adapted to eliminate the need for chemical etching or mechanical polishing prior to electropolishing. The resulting polished surface displays corrosion resistance equivalent or better than other electropolished Nitinol surfaces from literature with a breakdown potential >1V vs SCE, and a similarly high repassivation potential. Balancing the charge in the anodic and cathodic pulses was the key to minimizing the resulting surface roughness, and eliminating micropits. Nitinol is a nearly binary alloy of NiTi and a charge transfer ratio of 1 yielded the smoothest surfaces at current densities around :1 A/cm2. The initial surface condition was found to be irrelevant to electropolishing control with respect to oxide composition, provided enough mass was removed to fully dissolve the initial layers of mixed composition.
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    Stabilised decellularised vascular grafts in an ovine carotid model
    (2018) Mentor, Keno; Bezuidenhout, Deon; Pennel, Timothy
    Background: There is an urgent clinical need for an alternative vascular graft, especially for smaller artery applications such as in below-knee and coronary artery bypass. Currently available synthetic grafts have unacceptably low patency rates, while autologous saphenous vein grafts are not feasible in one third of patients. Decellularised vascular grafts have been investigated as alternative conduits, but this chemical treatment results in degradation of the extracellular matrix. Chemical stabilization of elastin with penta-galloyl glucose (PGG) combined with collagen stabilisation during covalent heparinisation was previously investigated by our group in a small animal model and shown to be effective and safe. The current study describes their evaluation in a large animal (ovine) model. Methods: Porcine mammary arteries were harvested, decellularised according to an established protocol involving rinsing with sodium hydroxide, alcohol (ETOH), treatment with DNAse/RNAse enzymes, immersion in PGG and subsequently surface modified with covalently bound heparin. Samples of the grafts were also tested for radial and suture retention strength. The prepared grafts were implanted as interposition grafts into the carotid arteries of 6 sheep, using industry standard 6mm expanded polytetrafluoroethylene (ePTFE) on the contralateral side of each animal as control. In-situ patency was determined by ultrasound and angiography at two months, following which the grafts were explanted for macro- and microscopic analysis. Results: In-vitro evaluation: Grafts showed significant levels of bound heparin (14.56 mg/g vs 0.69mg/g in untreated tissue) and demonstrated similar mechanical properties to those of human carotid arteries. Survival: Five out of six sheep survived the full 2-month implant period, while the remaining animal developed sepsis shortly after implantation and was euthanized on day 4. Patency: None of the decellularised grafts were patent at explant, as assessed by ultrasound, angiography and macroscopic examination. Two of the five control (ePTFE) grafts were patent. Microscopic analysis: An inflammatory cell infiltrate with vascularised granulation tissue was found encasing the decellularised xenografts with little or no sign of endothelial cell infiltration. Signs of early occlusion, likely due to technical factors, was noted at the sites of anastomosis. Conclusion: Although demonstrating similar mechanical properties to human carotid arteries, and promising results in the small animal model, the stabilised decellularised vascular grafts failed to achieve endothelialisation or patency in this sheep carotid model. Significant calibre mismatch between the test graft and the native artery is thought to be the primary factor in the failure of these grafts, highlighting the potential difficulty in acquiring grafts of an appropriate size from animal sources.