Browsing by Author "Davies, Neil"

Now showing 1 - 18 of 18
  • Thumbnail Image
    Item
    Open Access
    Angiopoietin-2 and platelet-derived growth-BB factor cooperatively affect peripheral blood monocyte fibrinolysis
    (2007) Bezuidenhout, Louise; Davies, Neil
    Includes bibliographical references.
  • Thumbnail Image
    Item
    Open Access
    Computational biomechanics in the remodelling rat heart post myocardial infarction
    (2016) Masithulela, Fulufhelo James; Franz , Thomas; Davies, Neil; Dubuis, Laura
    Cardiovascular diseases account for one third of all deaths worldwide, more than 33% of which are related to ischemic heart disease, including myocardial infarction (MI). This thesis seeks to provide insight and understanding of mechanisms during different stages of MI by utilizing finite element (FE) modelling. Three-dimensional biventricular rat heart geometries were developed from cardiac magnetic resonance images of a healthy heart and a heart with left ventricular (LV) infarction two weeks and four weeks after infarct induction. From these geometries, FE models were established. To represent the myocardium, a structure-based constitutive model and a rule-based myofibre distribution were developed to simulate both passive mechanics and active contraction.
  • Thumbnail Image
    Item
    Open Access
    Computational biomechanics of acute myocardial infarction and its treatment
    (2015) Sirry, Mazin Salaheldin; Franz, Thomas; Davies, Neil
    The intramyocardial injection of biomaterials is an emerging therapy for myocardial infarction. Computational methods can help to study the mechanical effect s of biomaterial injectates on the infarcted heart s and can contribute to advance and optimise the concept of this therapy. The distribution of polyethylene glycol hydrogel injectate delivered immediately after the infarct induction was studied using rat infarct model. A micro-structural three-dimensional geometrical model of the entire injectate was reconstructed from histological micro graphs. The model provides a realistic representation of biomaterial injectates in computational models at macroscopic and microscopic level. Biaxial and compression mechanical testing was conducted for healing rat myocardial infarcted tissue at immediate (0 day), 7, 14 and 28 days after infarction onset. Infarcts were found to be mechanically anisotropic with the tissue being stiffer in circumferential direction than in longitudinal direction. The 0, 7, 14 and 28 days infarcts showed 443, 670, 857 and 1218 kPa circumferential tensile moduli. The 28 day infarct group showed a significantly higher compressive modulus compared to the other infarct groups (p= 0.0055, 0.028, and 0.018 for 0, 7 and 14 days groups). The biaxial mechanical data were utilized to establish material constitutive models of rat healing infarcts. Finite element model s and genetic algorithms were employed to identify the parameters of Fung orthotropic hyperelastic strain energy function for the healing infarcts. The provided infarct mechanical data and the identified constitutive parameters offer a platform for investigations of mechanical aspects of myocardial infarction and therapies in the rat, an experimental model extensively used in the development of infarct therapies. Micro-structurally detailed finite element model of a hydrogel injectate in an infarct was developed to provide an insight into the micromechanics of a hydrogel injectate and infarct during the diastolic filling. The injectate caused the end-diastolic fibre stresses in the infarct zone to decrease from 22.1 to 7.7 kPa in the 7 day infarct and from 35.7 to 9.7 kPa in the 28 day infarct. This stress reduction effect declined as the stiffness of the biomaterial increased. It is suggested that the gel works as a force attenuating system through micromechanical mechanisms reducing the force acting on tissue layers during the passive diastolic dilation of the left ventricle and thus reducing the stress induced in these tissue layers.
  • Thumbnail Image
    Item
    Open Access
    A computational study of post-infarct mechanical effects of injected biomaterial into ischaemic myocardium
    (2012) Miller, Renee; Franz, Thomas; Davies, Neil
    Cardiovascular diseases account for one third of all deaths worldwide, more than 33% of which are related to ischaemic heart disease, involving a myocardial infarction (MI). Emerging MI therapies involving biomaterial injections have shown some benefits; the underlying mechanisms of which remain unclear. Computational models offer considerable potential to study the biomechanics of a myocardial infarction and novel therapies. Geometrical data of a healthy human left ventricle (LV) obtained from magnetic resonance images (MRI) was used to create a finite element (FE) mesh of the LV at the end-systolic time point using Continuity® 6.3 (University of California in San Diego, US). A mesh of 96 hexahedral elements with high order basis functions was employed to adequately describe the geometry of the LV. Simulations of diastolic filling and systolic contraction were performed using a transversely isotropic exponential strain energy function and a model for active stress based on contraction at the cellular level. An anterior apical, transmural MI was modelled in the LV encompassing 16% of the LV wall volume. The infarct was modelled at acute and fibrotic stages of post-infarct LV remodelling by altering the constitutive and active stress models to best describe passive and active behaviour of the ischaemic myocardium at each time point. The geometry of the LV with the fibrotic infarct was adjusted to represent the wall thinning that occurs during LV post-MI remodelling. Hydrogel injection was modelled as layers with material properties differing from those of the surrounding myocardium while accounting for thickening of the LV wall at the injection site. The study design comprised a healthy case and two infarcted cases with and without hydrogel injection. The end-diastolic volume (EDV) increased in the acute infarct model compared to the healthy case due to the reduced stiffness in the infarct wall. An EDV increase was not observed in the fibrotic infarct model compared to the healthy case. This was partially attributed to the increase in infarct stiffness and partially due to the fact that remodelling-related dilation of the LV was not implemented in the model. Inclusion of hydrogel lowered EDV in both the acute and fibrotic models. The predicted ejection fraction (EF) decreased from 41.2% for the healthy case to 28.5% and 33.0% for the acute and fibrotic infarct models, respectively. Inclusion of hydrogel layers caused an improvement in EF in the acute model only.
  • Thumbnail Image
    Item
    Open Access
    Determination of Cross-Directional and Cross-Wall Variations of Passive Biaxial Mechanical Properties of Rat Myocardia
    (2022-03-24) Ngwangwa, Harry; Nemavhola, Fulufhelo; Pandelani, Thanyani; Msibi, Makhosasana; Mabuda, Israel; Davies, Neil; Franz, Thomas
    Heart myocardia are critical to the facilitation of heart pumping and blood circulating around the body. The biaxial mechanical testing of the left ventricle (LV) has been extensively utilised to build the computational model of the whole heart with little importance given to the unique mechanical properties of the right ventricle (RV) and cardiac septum (SPW). Most of those studies focussed on the LV of the heart and then applied the obtained characteristics with a few modifications to the right side of the heart. However, the assumption that the LV characteristics applies to the RV has been contested over time with the realisation that the right side of the heart possesses its own unique mechanical properties that are widely distinct from that of the left side of the heart. This paper evaluates the passive mechanical property differences in the three main walls of the rat heart based on biaxial tensile test data. Fifteen mature Wistar rats weighing 225 ± 25 g were euthanised by inhalation of 5% halothane. The hearts were excised after which all the top chambers comprising the two atria, pulmonary and vena cava trunks, aorta, and valves were all dissected out. Then, 5 × 5 mm sections from the middle of each wall were carefully dissected with a surgical knife to avoid overly pre-straining the specimens. The specimens were subjected to tensile testing. The elastic moduli, peak stresses in the toe region and stresses at 40% strain, anisotropy indices, as well as the stored strain energy in the toe and linear region of up to 40% strain were used for statistical significance tests. The main findings of this study are: (1) LV and SPW tissues have relatively shorter toe regions of 10–15% strain as compared to RV tissue, whose toe region extends up to twice as much as that; (2) LV tissues have a higher strain energy storage in the linear region despite being lower in stiffness than the RV; and (3) the SPW has the highest strain energy storage along both directions, which might be directly related to its high level of anisotropy. These findings, though for a specific animal species at similar age and around the same body mass, emphasise the importance of the application of wall-specific material parameters to obtain accurate ventricular hyperelastic models. The findings further enhance our understanding of the desired mechanical behaviour of the different ventricle walls.
  • Thumbnail Image
    Item
    Open Access
    Development of a multi-stage purification process for serum-derived exosomes and evaluation of their regenerative capacity
    (2020) de Boer, Candice; Davies, Neil
    Exosomes are secreted membrane vesicles (30-100 nm) found in tissue culture media and various body fluids that have potential as therapeutics and disease biomarkers. Current literature has reported regenerative benefits for blood-derived exosomes but the majority of these studies purified exosomes using ultracentrifugation (UC), a method that has been found to have high levels of protein contamination. Here the regenerative capacity of exosomes isolated by size exclusion chromatography (SEC), a method shown to reduce protein contamination, from human serum was assessed. SEC isolates were found to contain suitably sized vesicles and exosomal markers (CD9, CD81 and TSG101). These isolates allowed for cellular uptake of a range of fluorescent labels and enhanced cellular fibroblast proliferation and endothelial sprout formation in a 3D spheroid-based angiogenesis assay. Further to this, functionality was shown to be retained after incubation of the isolates for 21 days at 37°C. Though a promising indication of regenerative potential, it was found that the isolates contained significant levels of ApoB containing lipoproteins (up to 15 µg ApoB/ml). It was shown that these lipoproteins were predominately the very low and intermediate density lipoproteins. It was found that low-density lipoprotein can impact exosome uptake studies that use fluorescent nucleic acid, protein and lipid dyes. As a substantial extraneous lipoprotein content could also interfere with other downstream applications and analyses such as proteomic analysis, a multistep purification method was developed. A simple 3-step density gradient (DG) UC was introduced prior to SEC that incorporated a high-density iodixanol cushion overlaid by a 18% iodixanol step containing UC concentrated human serum that was then overlaid with 6% iodixanol. This DG relied on flotation to remove lipoproteins. After the multi-step purification (UC DG SEC) ApoB and ApoA1 were not detectable by enzyme-linked immunosorbent assay and western blotting respectively. The UC DG SEC isolates were positive for CD9 and TSG101 and morphologically, as viewed by transmission electron microscopy, had the canonical exosome shape and size. Nanoparticle tracking analysis showed that though exosome marker levels were similar, there were 100 times more particles in SEC purified isolates relative to those from UC DG SEC, emphasising the extent of lipoprotein removal. Proteomic analysis identified 224 proteins in UC DG SEC isolates relative to the 135 from SEC, with substantial increases in exosome-associated proteins and reductions in lipoproteins. The UC DG SEC exosomes still elicited a significant increase in cell proliferation of human dermal fibroblasts but no increase in endothelial sprout formation. After subcutaneous implantation in a rat model, the highly purified exosomes potentially increased an angiogenic response. In conclusion, we show that serum SEC-derived exosomes with much reduced protein content do have regenerative properties but contain contaminating lipoproteins. Our new isolation technique isolated purer serum exosomes that retained cell proliferation stimulation and potentially enhanced an in vivo angiogenic response. This approach should render the isolated exosomes more suitable for biomarker discovery, molecular composition determination and biological function analysis.
  • Thumbnail Image
    Item
    Open Access
    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.
  • Thumbnail Image
    Item
    Open Access
    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.
  • Thumbnail Image
    Item
    Open Access
    The in vivo characterisation of a C-domain specific ACE inhibitor
    (2013) Sharp, Sarah-Kate; Davies, Neil
    The ACE protein is a zinc-dependent dipeptidyl carboxypeptidase comprised of two homologous domains termed the C- and N-domain. The C-domain is primarily responsible for the catalytic production of Ang II, while the tetrapeptide acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is predominantly cleaved by the N-domain, and both domains play a role in the metabolism of vasodilatory peptide bradykinin. In the event of myocardial infarction (MI), cardiac output and blood pressure decreases, resulting in activation of the RAS and an increase in both Ang II production and bradykinin metabolism. While initially compensatory, prolonged RAS activation has been shown to have long-term detrimental effects, and pharmaceutical intervention in the form of ACE inhibition is the first line treatment following an MI event. The ACE inhibitors currently in clinical use target both domains equally, and it has been suggested that the major side-effects of this drug class are largely attributable to the inhibition of bradykinin breakdown. A novel C-domain selective ACE inhibitor lisinopril-Trp (lisW-S) incorporates a tryptophan moiety into the P2' position of the clinically available ACE inhibitor lisinopril.
  • Thumbnail Image
    Item
    Open Access
    Investigation of synthetic hydrogels as therapy for myocardial infarction
    (2011) Kadner, Karen; Davies, Neil
    This thesis investigated the potential of synthetic polyethylene glycol (PEG) hydrogels for restoration of biomechanical integrity and for controlled cardiac release of drugs. ... The aim of this study was to directly compare the effect of injecting an enzymatically degradable polyethylene glycol (PEG) hydrogel into the myocardium immediately or seven days after permanent ligation of the left anterior descending artery in rats on pathological remodeling.
  • Thumbnail Image
    Item
    Open Access
    Investigations into the stability of growth factor induced-vasculature and the effects of synthetic biomaterials on heart remodelling after myocardial infarction
    (2011) Dobner, Stephan; Davies, Neil
    This work was based on the hypothesis that optimization of growth factor delivery rate and duration, combined with a biomaterial scaffold, could lead to an improved strategy for therapeutic neovascularization. To test this hypothesis, a novel in vivo model system that allows for characterization of stability and mural cell investment of newly created vessels was designed.
  • Thumbnail Image
    Item
    Open Access
    Methods and adaptations required to perform small-animal MRI scanning using a large bore clinical MRI
    (2012) Saleh, Muhammad G; Meintjies, Ernesta; Davies, Neil; Franz, Thomas
    Small-animal imaging has been widely implemented to study succession of disease, therapeutic treatments and the effects of environmental insults. The gold standard noninvasive technique for following progression of heart failure in small-animal models is magnetic resonance imaging (MRI). The aim of this project was to adapt a clinical MRI system to perform small-animal cardiac MRI. The first part of the thesis describes the adaptations required, which included design and construction of a small-animal radiofrequency (RF) coil, physical support (cradle), a core body temperature regulation system, and optimization of pulse sequences. The system was validated using a phantom and in-vivo in 5 healthy rats. The signal-to-noise ratio (SNR) in the phantom was 91% higher using the small-animal coil compared to the standard head coil. SNRs of 7 ± 2 and 18.9 ± 0.6 were achieved in myocardium and blood, respectively, in healthy rats and MR left ventricular mass (LVM) was highly correlated with (r=0.87) with post-mortem mass. In the second part of the study, left ventricular remodeling (LVR) was investigated in a nonreperfused model of myocardial infarction (MI) in 5 sham and 7 infarcted rats. Rats were scanned at 2 and 4 weeks post surgery to allow for global and regional functional and structural analyses of the heart. Images were of sufficient quality to enable semi-automatic segmentation using Segment. Significant increase in end-systolic volume (ESV) was observed in MI rats at 2 weeks post surgery. At 4 weeks post surgery, end-diastolic volume (EDV) and ESV of MI rats were significantly higher than in sham rats. Ejection fraction (EF) of MI rats dropped significantly at 2 weeks and a further significant drop was observed at 4 weeks indicating contractile dysfunction. Wall thickness (WTh) analyses in MI rats at 4 weeks revealed significant reduction in end-diastolic (ED) wall thickness in the anterior region due to necrosis of myocytes. In the posterior region, WTh was significantly higher due to LV hypertrophy. At end-systole (ES), the MI rats revealed significant decrease in WTh in the anterior and lateral regions. MI rats suffered reduction in systolic wall thickening in all regions of the heart, indicating global contractile dysfunction.
  • Thumbnail Image
    Item
    Open Access
    Modified polyethylene glycol hydrogels for growth factor delivery and controlled tissue invasion
    (2019) Gustafsson, Carla Astrid; Davies, Neil
    The prevalence of cardiovascular disease and myocardial infarction-induced heart failure has risen significantly over recent years, emphasising the need for new, effective therapeutic strategies. A promising alternative approach is the cardiac delivery of potentially cardioprotective and regenerative growth factors from biomaterial scaffolds. One hydrogel system that has promise in this area is an injectable enzymatically degradable polyethylene glycol (PEG) hydrogel. Two modifications aimed at further optimising this system as a regenerative medicine scaffold were explored. Firstly, the covalent addition of heparin into the PEG backbone was assessed for its ability to stimulate angiogenesis by assessing the controlled release of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and placental growth factor 2 (PlGF-2), and also assaying endothelial cell sprouting in an in vitro 3D spheroid angiogenesis assay. The second modification involved overlaying an increasingly hydrolytic degradability on top of the enzymatically degradable background of the hydrogel. The potential of this modification to regulate the rate of hydrogel replacement by invading tissue was assessed in the 3D spheroid assay and a subcutaneous implant study in a rat model. The covalent coupling of heparin was found to substantially increase the rate of release of bFGF, VEGF and PlGF-2 over 20 days by 23%, 42% and 19%, respectively, relative to nonheparinised PEG hydrogels (p<0.01). A 3D spheroid-based angiogenesis assay was modified for use in quantifying endothelial cell sprouting in PEG hydrogels. bFGF and VEGF were shown to elicit a significant increase (2.3 – 2.4-fold increase) in average cumulative sprout lengths relative to that seen in the control spheroids (p<0.01). However, PlGF-2 did not stimulate a significant response (1.4-fold increase, p=NS). In follow up studies with heparinised hydrogels, it was found that the 3D angiogenesis was not rigorously established and ways forward are discussed. Enzymatically degradable PEG hydrogels that retained their enzymatic degradability with increasing levels of potential for hydrolysis were formed by increasing the proportion of PEGacrylate (PEG-Ac) and correspondingly decreasing the portion of PEG-vinyl sulfone (PEG-VS) monomers. PEG-Ac forms hydrolytically unstable bonds with the peptide crosslinker whilst 4 PEG-VS forms stable linkages. This approach was shown through swelling studies to be capable of generating a range of hydrolytic degradation rates. Sprouting of endothelial cells from PEG hydrogel embedded spheroids was shown to increase as the PEG-AC concentration increased. Importantly, the rate of tissue invasion in vivo was also shown to be positively correlated with the PEG-Ac concentration. The increased utility of these hydrogels to act as delivery vehicles for therapeutic agents, through covalent coupling of heparin, is promising for their use as regenerative medicine scaffolds. Additionally, so is the ability to finely tune tissue invasion by manipulating their hydrolytic degradability.
  • Thumbnail Image
    Item
    Open Access
    Plasma and leukocyte gelatinases in health and disease
    (2004) Marillier, Reece Gerrad; Marais, Adrian; Davies, Neil
  • Thumbnail Image
    Item
    Open Access
    The regulation of matrix metalloproteinase (MMP) secretion in human vascular cells exposed to extracellular and wound matrices
    (2002) Bracher, Mona; Davies, Neil; Zilla, Peter
    Proteolytic degradation is a central feature of the cellular remodelling in wounds during healing. The regulation of matrix metalloproteinases (a key superfamily of proteases involved in the above process) by wound healing components is an area of ongoing investigation. However, a systematic study of the exposure of the human derived vascular cells - endothelial cells, fibroblasts, smooth muscle cells and macrophages - to well defined ECM (collagen type I, III and IV and laminin) and wound healing matrix proteins (fibrin and plasma fibronectin) has not yet beeen carried out.
  • Thumbnail Image
    Item
    Open Access
    Role of extracellular environment in mechanical properties of human cardiac fibroblasts and myofibroblasts
    (2021) Haasbroek, Pieter Daniël; Franz, Thomas; Davies, Neil
    Acute MI results in adverse remodelling of the myocardium, eventually leading to contractile dysfunction and chronic heart failure. Collectively, the formation of fibrotic scar tissue in the left ventricle inhibits its contractile function and leads to a cardiac output loss. Cardiac fibroblast and myofibroblast cells are integral in the remodelling of injured tissue and are the most anticipated therapeutic target for cardiac fibrosis. To date, there is no anti-fibrotic therapy, which we argue, is due to the lack of knowledge on the functionality of the cardiac fibroblast. It was hypothesised that a change in stiffness of the remodelling extracellular environment associated with fibrosis leads to a change of the mechanical properties and contractile forces of cardiac fibroblasts and myofibroblasts. Hence, this dissertation aimed to investigate the effects of extracellular stiffness on mechanical properties of cardiac fibroblasts and myofibroblasts using engineered in vitro microenvironments with tuneable physical properties. Polyethylene Glycol (PEG) hydrogels offer ideal properties to serve as an extracellular matrix (ECM) mimicking biomaterial. Different techniques of mechanical characterisation used in this study established that the elastic modulus of 20 kDa 8-arm PEG-VS hydrogel crosslinked with dithiothreitol (DTT) reagent, is directly proportional to the concentration of PEG precursor used to form the material. Rheological measurements indicated an elastic shear modulus of 500 ± 13 Pa and 2721 ± 39 Pa for PEG gels of 4% (m/v) and 10% (m/v), respectively. Specialised micro-indentation and uniaxial tensile testing found PEG gels of 7%, 10%, 14% and 18% concentration to have elastic moduli of 42.0 ± 2.8 kPa, 65.3 ±8.0 kPa, 78.2 ± 8.4 kPa, and 121.5 ± 11.9 kPa, respectively. The volumetric swelling ratio was shown to be inversely proportional to the precursor concentration. The addition of cell adhesion proteins, such as RGD peptides, resulted in a decrease in the shear modulus (G') of the hydrogel, as the peptides take up PEG arms which are potential crosslinks. This slight compromise to the structural integrity is relevant when tuning in vitro 3D extracellular environments to desired conditions. Cardiac fibroblasts (CFs) and cardiac myofibroblasts (MFs), the latter obtained through stimulation with transforming growth factor-beta 1 (TGF-β1) of CF, were cultured and embedded in 3D PEG hydrogel matrices. PEG was supplemented with RGD peptides to provide cellular adhesion points, imitating the native myocardial environment. The intracellular stiffness of the embedded cells was quantified through passive mitochondrial particle tracking. We investigated the isolated effect of soft (4% m/v) and stiff (10% m/v) matrices on the stiffness of CFs and differentiated MFs. An increase in cell stiffness with was observed with an increase in matrix stiffness from soft to stiff PEG gel, with α = 0.19-0.36 through all delay times for cells in soft matrices and α = 0.09-0.18 throughout all delay times for cells in stiff matrices (p ≤ 0.05) for both standard CFs as well as for maladaptive MFs. This direct proportionality confirms that CFs behave according to the principles of mechanotransduction, using cytoskeleton-based rigidity-sensing mechanisms between themselves and their environment, and as a consequence, their inherent stiffness is regulated by the rigidity of their environment. No considerable difference of cell fluidity was found between cell phenotypes (CF & MF) in soft matrices. In stiff matrices, MFs seemed to become somewhat stiffer across the delay times, although with no significant effect. The knowledge of the extent to which the mechanics of cardiac fibroblasts and myofibroblasts and their ECM are interrelated is essential for the understanding of pro-fibrotic mechanotransduction and ECM production to prevent, attenuate and reverse cardiac fibrosis.
  • Thumbnail Image
    Item
    Open Access
    Stimulation of angiogenesis through growth factor delivery from synthetic heparinised hydrogels
    (2017) Chokoza, Cindy; Davies, Neil; Bezuidenhout, Deon
    Objectives: Vascular diseases are one of the leading causes of death. Due to minimal regenerative capability of the heart, alternative therapies have been sought after with engineered biomaterials being extensively investigated in this area. In this study, enzymatically degradable heparinised polyethylene glycol (PEG-Hep) hydrogels were synthesized and characterised for the binding and controlled release of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), as well as their bioactivity and angiogenic potential in vivo. Methodology: VEGF and bFGF were combined into 4% (m/v) PEG-Hep hydrogels. The binding and release rates of VEGF and BFGF were analysed via an immunosorbent assay. Released growth factor bioactivity was measured using an XTT metabolic assay on human saphenous vein endothelial cells and human dermal fibroblasts. Neovascularisation was quantified in a subcutaneous rat angiogenesis model in which hydrogel growth factor combinations were implanted within porous polyurethane discs and analysed after a 4 week period. A novel hybrid hydrogel able to degrade proteolytically and hydrolytically was further developed, characterised and analysed as above. Results: PEG-Hep hydrogels demonstrated substantial growth factor binding ability (500-600 ng) and allowed sustained release (10-20 ng/day) for up to 28 days. Bioactivity of the growth factors was retained throughout the release period. The degradation rate of the hydrogels could be controlled in vivo by varying the ratio of monomers capable of forming either hydrolytically or proteolytically degradable crosslinks. Qualitative and quantitative analysis demonstrated a pronounced and significant angiogenic response in vivo (p<0.05). Conclusion: Heparinised PEG hydrogels show significant promise as controlled release vehicles for growth factors and warrant further examination in a myocardial infarction model.
  • Thumbnail Image
    Item
    Open Access
    Sustained hydrogel-based delivery of RNA interference nanocomplexes for gene knockdown
    (2019) Ngarande, Ellen; Davies, Neil; Bezuidenhout Deon
    Scaffold based delivery of RNA interference (RNAi) molecules such as free small interfering RNA (siRNA) and microRNA has recently begun to be employed towards treatment of diseases such as cancer, bone regeneration, muscular dystrophy and cardiovascular disease. Effective translation from bench side to clinical use of RNAi has been limited in part because upon systemic delivery the RNAi molecules are degraded by RNases and flushed by excretory organs causing an inefficient duration of gene silencing effect at target tissues. These challenges can potentially be minimised by delivering RNAi molecules via non-viral nanoparticle carriers encapsulated in biocompatible, biodegradable and injectable scaffolds such as hydrogels. Various scaffolds have been shown to aid in sustained localised delivery of RNAi molecules and improve gene silencing. This research focused on optimising and establishing such an RNAi hydrogel-siRNA-nanoparticle (hydrogel-nanocomplex) system for targeted and sustained gene knockdown both in vitro and in vivo using dendrimer and lipid based nanoparticles in combination with synthetic polyethylene glycol (PEG) and natural fibrin hydrogel scaffolds. Four siRNA nanocarriers were investigated for siRNA delivery, that is, fourth generation dendrimer nanoparticles poly(amidoamine) (D) and its modified version (MD) with PEG and a lipid 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) molecule, commercial lipid based Lipofectamine® RNAiMax and Invivofectamine® 3.0 nanoparticles. D and MD achieved better RNase protection compared to lipid nanocomplexes though Invivofectamine® 3.0 nanocomplexes protected a small percentage of siRNA over 10 days. The MD nanoparticle displayed improved siRNA release and transfection efficacy compared to D but efficacy of the dendrimers was lower than the lipid particles. Four hydrogels that have not been investigated for RNAi were assessed for sustainability. Namely, hydrolytically and proteolytically degradable PEG-acrylate (PEGAC), proteolytically degradable PEG - vinyl sulfone (PEG-VS) hydrogels, unmodified fibrin and PEGylated fibrin hydrogel. The nanocomplex release rate in vitro from the various hydrogels showed minimal release from PEGylated hydrogels, burst release from unmodified fibrin and sustained release from PEGylated fibrin. Invivofectamine® 3.0 nanocomplexes retained efficacy optimally after release from PEGylated fibrin hence this hydrogel was utilised for downstream analysis. For in vivo sustained delivery to be effective, determination of hydrogel persistence in vivo was required. After injection in the mouse tibialis anterior (TA) muscle PEG-AC and PEGylated fibrin gels degraded within 2 days. The efficacy of the various nanocomplexes was assayed in a 3D assay that more closely resembled delivery in soft tissue. PEGylated fibrin containing nanocomplexes with cell death siRNA sequences was polymerised around a preformed PEGylated fibrin cell containing droplet. Invivofectamine® 3.0 nanocomplex consistently achieved the highest gene knockdown effect with no evidence of cytotoxicity whilst Lipofectamine® RNAiMax was ineffective. MD showed signs of cytotoxicity when delivered in a sustained fashion. Thus Invivofectamine® 3.0 nanocomplexes in PEGylated fibrin hydrogel were found to be the optimal gel-nanocomplex system to proceed to in vivo assessment. BALB/c GFP transgenic injected in their TA muscle with Invivofectamine® 3.0 nanocomplexes made with siRNA targeting GFP or myostatin (siGFP/siMSTN) in the presence or absence of PEGylated fibrin gel were analysed 7 days post treatment for siRNA retention and GFP and Mstn gene knockdown. Increased retention of siRNA after encapsulation in PEGylated fibrin was observed at 7 days. A non-significant reduction in GFP protein was seen for limbs injected with siGFP- fibrin after 7 days. A substantial and significant reduction in Mstn mRNA levels was elicited by delivery of siMstn–fibrin. Furthermore, only siMstn-fibrin resulted in significant increase in muscle mass. In this study, dendrimer based nanoparticles were found to effectively protect siRNA against RNases however lipid based nanocomplexes were the most efficacious at gene knockdown. The combination of Invivofectamine® 3.0 and PEGylated fibrin was shown to be the most effective in 3D assays and as an injectable controlled release scaffold into soft tissue suggesting that this approach has therapeutic potential.