Browsing by Author "Bezuidenhout Deon"

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    Open Access
    Development and characterisation of a heparinised fibrin hydrogel as a delivery vehicle for regenerative medicine
    (2024) Ngcobo, Silindile; Davies, Neil; Bezuidenhout Deon
    Fibrinogen is an attractive hydrogel candidate for regenerative medicine due to its inherent biocompatibility, biodegradability, and clinical approval. It is the precursor protein cleaved by the protease thrombin that polymerises into a fibrous network known as fibrin. The present study sought to modify fibrinogen through covalent attachment of heparin to form heparinised fibrin hydrogels. Heparin is known to bind a significant number of growth factors and its anti-coagulative characteristic plays a role in minimising thrombotic events. We therefore developed a novel method for attaching heparin to fibrinogen that allowed for retention of thrombin-based polymerisation. Additionally, the potential of co-modification with polyethylene glycol (PEG) enhancing heparinisation and improving fibrin resistance to proteolytic degradation was assessed. The modified forms of fibrin hydrogel were then assessed for their potential as regenerative hydrogels. To conjugate heparin to fibrinogen, we explored the utility of an acrylated form of heparin (developed in Professor Bezuidenhout's Polymer Laboratory) that preferentially binds to thiol groups (such as cysteine side chains in proteins) via a Michael-type addition reaction. As additional free thiols might increase heparin binding, we also modified the fibrinogen with a n-hydroxysuccinimide-PEG-thiol (NHSPEG-SH) molecule, and a maleimide based assay confirmed that 0.75 free thiols were successfully bound to fibrinogen to form modified fibrinogen-PEG-SH (FP). This represented a greater than 100-fold increase in free thiols present in native fibrinogen. This binding was further confirmed with a Fourier-transform infrared spectroscopy. Fibrinogen and FP were then assessed for hep-acr binding. Two assays were used for heparin quantification, namely calorimetric 3-Methyl-2- Benzothiazolinone Hydrazone assay modified for quantification of protein bound heparin, and a commercial fluorescent probe-based assay. Both assays showed that hep-acr was successfully conjugated to fibrinogen and FP at 0.2-0.3 molecules of heparin bound per either molecule to form fibrin(ogen)-heparin (FH) and fibrin(ogen)- peg-heparin (FPH) respectively. Binding through the acrylate moiety was confirmed with non-acr hep binding at 10 times lower levels. All modified forms of fibrinogen retained the ability to polymerise via the action of thrombin. Though not amplifying heparinisation, PEGylation and heparinisation combined strongly protected fibrin from spontaneous in-vitro degradation at 37ºC compared to all other hydrogel formulations (fibrin and FH were 100% degraded by day 5; FP by day 6 and FPH only by 57% at day 15). Micrographs from scanning electron microscopy (SEM) revealed a more fibrillar structure after polymerisation for FH closer in morphology to that of fibrin, while FP and FPH both formed more sheet like structures. Rheology showed a reduction in storage modulus/ mechanical stiffness for FP (18.3 ± 1.3 Pa) and FPH (17.6 ± 2.7 Pa) compared to normal fibrin (42.1 ± 1.5 Pa) and FH (36.7 ± 1.9 Pa). Despite these differences, both FH and FPH were antithrombotic as determined by the attached heparin completely impeding clot formation for the duration of analysis (2 hours) with thromboelastography, as compared to whole blood, fibrinogen, and FP which formed clots within 1.4 ± 0.1, 1.5, and 1.3 ± 0.1 min respectively. All the hydrogels were biocompatible, with viability percentages not less than 70% after three days of 3D culture for all three cell lines evaluated, namely human umbilical vein endothelial cells (HUVECs), human dermal fibroblasts and adipose tissue-derived stem cells (ADSCs) as indicated by live/dead assays. A 3D HUVEC spheroid-based angiogenesis assay found that FH substantially stimulated formation of capillary-like structures relative to fibrin. When evaluated for growth factor release in-vitro, the burst release in the first day was reduced by 5-fold for both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the presence of heparin. Both FH and FPH further prolonged and sustained bFGF and VEGF release in-vitro relative to their unheparinised counterparts. The FH and FPH released 16 ± 0.7 and 16 ± 0.7 ng bFGF per day, and 20.5 ± 2.2 ng and 18.4 ±1.6 ng VEGF per day up to 15 days respectively with bioactivity maintained. An initial in-vivo subcutaneous rat model study of fibrin formulations loaded with 1 µg of VEGF showed pronounced vessel formation into all forms of fibrin independent of VEGF after 10 days. A significant increase in vessel ingrowth for either heparinised form of fibrin was not observed. This dosage of VEGF has previously been seen to stimulate neovascularisation into heparinised PEG hydrogels in our laboratory and it was hypothesised that the high levels of vascularisation stimulated by fibrin alone may have obscured the VEGF induced angiogenesis. Further studies exploring VEGF dosage and concentration of fibrin hydrogels should be carried out. As delivery of stem cells within hydrogels is a key present function of regenerative hydrogels, the impact of the heparinised fibrinogens on ADSC differentiation underwent preliminary investigation. The impact of heparin on mesenchymal stem cell differentiation is presently unclear with several contradictory reports in the literature. Predictably the soft fibrin hydrogels in all formulations strongly promoted adipogenesis relative to stiff tissue culture plastic (TCP). More surprisingly, osteogenesis was also increased on the fibrin hydrogels relative to TCP. Additionally, heparinisation altered differentiation, where osteogenic differentiation in FH was significantly increased against all groups, while this increase was observed in FH when compared to the FP group for adipogenic differentiation. In conclusion, this study presents two methods of conjugating heparin to fibrinogen to form anti-thrombotic heparinised fibrin hydrogels where polymerisation via thrombin activity was retained. The bound heparin in both hydrogel systems substantially reduced burst release of bFGF and VEGF and allowed for sustained release of bioactive growth factors. The FPH was advantageous in that it reduced inherent fibrin degradation, and provided a transparent physical appearance that was useful in viewing encapsulated cells. The FH did not significantly reduce mechanical stiffness compared to unmodified fibrin, and further encouraged more HUVEC invasion in 3D. All formulations of fibrin hydrogels proved to be significantly better matrices for ADSC adipogenic and osteogenic differentiation as compared to TCP. These findings are crucial for applications in stem cell and, growth factor delivery and ultimately tissue regeneration.
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    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.
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    Open Access
    The design and prototyping of a Transcatheter Aortic Valve Implantation training system specific for aortic valve regurgitation.
    (2023) Nkoma, Fakih Fadi; Ofoegbu, Chima; Bezuidenhout Deon
    Aortic regurgitation (AR) caused by rheumatic heart disease is a significant cause of mortality in developing countries. The replacement of the regurgitant aortic valve was historically performed by surgical insertion of a bioprosthetic or mechanical heart valve. Transcatheter Aortic Valve Implantation (TAVI) has become a common surgical procedure to replace defective aortic valves. The balloon-expandable (BE) transapical TAVI technique involves, a replacement valve crimped onto an inflatable medical balloon and delivered in the aortic valve by making a small incision in the chest and going through the apex of the heart. Once the crimped replacement valve is in position the balloon is inflated, expanding the replacement valve in the native aortic valve, and pushing the native leaflet onto the side. The balloon is then deflated and removed from the heart leaving the prosthetic valve in position. Strait Access Technologies (SAT), a South-African start-up has developed an in innovative lowcost BE transapical TAVI for people suffering from AR. The delivery device consists of a nonocclusive balloon, allowing blood to flow freely during the entire procedure, and uses three locating arms called trunks for easy implantation through temporary anchoring tactile feedback. Compared to majority of the delivery system in the market the heart of the patient doesn't need to be stopped when the SAT delivery device is being used and minimal imaging system is required during the implantation procedure. After determining the optimal orthogonal projection of the aortic root using fluoroscopy and following the right cusps rule, each trunk is positioned in an aortic leaflet cusp to keep relative motion between the delivery device and the heart, and to locate the valve axially and rotationally. Once the trunks are in position the surgeons apply a small force and get tactile feedback to confirm positioning. The force applied during the tactile feedback procedure causes the native leaflets to droop. The droop can be defined as the displacement of the leaflet in the axial direction due to the pull force of the trunks. The amount of droop affects the axial positioning of the valve which is critical as is can led to regurgitation or valve embolization which can potentially be fatal. It has been shown that the complication rate during TAVI reduces drastically after the first 100 implantation, hospitals with high volumes of TAVI have better outcomes. Surgeon proficiency in TAVI procedures can be achieved in multiple ways including workshops, course, simulation and animal trial. Training is important when surgeons are introduced to new technologies. The training methods and the repeat practice can drastically improve their clinical outcome. Most training systems currently available do not indicate the droop of the leaflet based on the force applied. This study aims to define the relationship between the force applied during anchoring of the trunks and the native leaflet droop, to determine and quantify the force required to accurately position the valve, and, to develop a training rig to train surgeons in the correct use of the device. v To do so a pull-force tool replicating the trunks of the delivery device was designed and coupled to a force gauge. Three radiopaque markers were inserted into cadaveric pig hearts connected to a pulsatile pump replicating the blood flow. The pull-force tool was inserted in the pumping heart anchored in the native leaflet and pulled to cause droop. The forces applied on the native leaflet and the droops were recorded under fluoroscopy. Three equations defining the relationship between the droop and the force applied per leaflet were then defined. The force required to position the valve accurately was calculated. A training rig was also developed to train surgeons on the use of the delivery device and was divided into three main parts: an imaging system, a circulatory loop, and a force indication device. The imaging system was designed based on the Phillips BV Pulsera C-arm. It was designed to have similar dimensions, and the same degrees of movement as the image needs to be in a specific orientation for follow the right cusps rule, and to hold a standard security camera that projects the images on a screen to replicate the fluoroscopy imaging the surgeon will see during the procedure. The circulatory loop was designed using 3D printed clear materials and replicating the native anatomy dimensions. The data from the first part of the study was used to develop a force indicator, designed to be a replica of the TAVI delivery device, and uses a colour light system to indicate to the surgeon if adequate force is being applied to accurately position the valve. The three equations derived after measuring the droop test of all leaflets are FL1 = -3.4831 x DroopL1 - 1.94, FL2 = -4.5872 x DroopL2 - 1.88, and FL3 = -4.7007 x DroopL3 – 1.70, fot the Right Coronary Cusps (RCC), Non-Coronary Cusps (NCC) and the Left Coronary Cusps (LCC) respectively. The forces required to position the valve accurately was calculated to be between 7.39N and 17.96N, for a minimum droop of 1.2mm and a maximum droop of 4.6mm. The C section of the C-arm is made of aluminium, the C arm can move and be locked into position when the adequate image is obtained using 2 different locking mechanisms. It is designed to hold a camera that can project images into a screen, replicating fluoroscopy images. The circulation loop was 3D printed, using parts made of clear silicone and clear rigid material, a valve was deployed inside using a delivery device de-aired with a solution mixed with food colouring to confirm visibility during the procedure without the use of X-ray. Images were taken during the deployment and compared to the X-ray images and the images obtains could be used as substitute of fluoroscopy images to train surgeons. The force indicator was designed with a light that shines blue when the force applied is less than 7.39N the light shines green when the force applied is between 7.39N and 17.96N and the light shines red when the force applied is greater than 17.39N. majority of the parts were 3D printed. The force indicator was built and tested to confirm that the light shined as designed. This will give a good tactile feedback indication to the surgeon to know when adequate force is applied. The three systems designed, used in conjunction, can be used to train surgeons on the TAVI without being exposed to any radiation, obtain adequate imaging, and apply the appropriate force to accurately position the valve.