Ultrasound-assisted Ice Nucleation, Fibroblast Migration and Oxygen Delivery in Collagen Scaffolds for Tissue Engineering

Abstract

Three-dimensional, porous collagen tissue engineering scaffolds can be produced using freeze-drying. However, the stochastic nature of ice nucleation may lead to architectural variability between structures created using nominally similar preparation conditions and this can impact subsequent tissue ingrowth and function. Another common problem in tissue engineering is inadequate nutrient supply to the scaffold centre, which can lead to imbalanced cell distribution and tissue necrosis. The aim of the thesis was to address these issues by investigating three different aspects of ultrasound application on the performance of collagen-based tissue engineering scaffolds. The work has addressed underpinning research questions, covering scaffold fabrication, the stimulation of cell migration within the structure, and the ability to provide targeted oxygen delivery. Collagen scaffolds were fabricated by freeze-drying and crosslinking using N-(3-Dimethyl-aminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) with N-Hydroxysuccinimide (NHS). Scaffolds were freeze-dried at -20 °C and crosslinked with a 15:6:10 molar ratio of EDC, NHS and collagen carboxylic acid groups respectively. Micro-computed tomography (MicroCT) was used to characterise scaffold architecture and to quantify the pore dimensions. Pore size variability was noted through the thickness of the scaffold which, for consistency, required sample trimming before further testing. For this reason, a possible method to reduce the stochastic nature of the ice nucleation was sought. Ultrasound was applied to supercooled collagen suspensions (40 kHz frequency, 0.2 W cm−2 acoustic intensity) to investigate whether ice nucleation could be stimulated. It was found that the technique was an effective trigger, as characterised by an instantaneous increase in temperature to the equilibrium freezing temperature. It was also noted that the nucleation temperature correlated with pore diameter. The addition of 0.25 mg ml−1 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/polyethylene glycol-40 (PEG-40) stearate phospholipid microbubbles in collagen slurry produced significantly smaller pore sizes (p ≤ 0.05) with mean diameter of 84 ± 4 µm, compared to 102 ± 6 µm without microbubbles. The presence and impact of cavitation were confirmed through imaging of ultrasonic nucleation with microbubbles. The work confirmed that ice nucleation could be controlled, as hypothesised, but it was noted that the concomitant reduction in pore size would not be advantageous for cellular infiltration into the scaffold. However, the cavitation caused by ultrasound offered further possibilities to influence cellular behaviour within the scaffold. The effects of cavitation and local pressures produced by high-intensity focused ultrasound (HIFU) on directional migration in human dermal fibroblast (HDF) culture were investigated by measuring cavitation response and cell distributions. HIFU was applied (0.5 MHz) to HDF cultures with 0.25 mg ml−1 polystyrene cavitation agents in collagen scaffolds for one, three or five days consecutively to investigate the impact of repeated HIFU exposure. HDF culture was seeded at one end of the scaffold, with ultrasound applied parallel to the length at the same end. After 5 days, HDF cells were distributed 15% further along the scaffold length, parallel to the direction of ultrasound, relative to the control samples (p ≤ 0.05). It was also noted that, as HIFU exposure increased, total cell counts decreased (p ≤ 0.01). For this reason, methods were considered to increase cell viability via targeted oxygen delivery using ultrasound. The feasibility was explored of using DSPC/PEG-40 stearate phospholipid microbubbles as oxygen delivery vehicles. Oxygen release was measured during ultrasound exposure (40 kHz, 0.3 W cm−2) and the effects on cell metabolism and cell viability in HDF and HT1080 populations cultured in collagen scaffolds were investigated. Oxygen-loaded microbubbles were delivered to the centre of the cell cultures, using either 10 minutes or 30 seconds of exposure to ultrasound, daily, for 10 days. While the metabolism of both HT1080 cells and HDF cells was reduced, and the viability of HT1080 cells also decreased, the viability of HDF cells was enhanced. Scanning electron microscopy (SEM) revealed that HT1080 cells exhibited less cell binding with ultrasound, and cavities were produced in the collagen structure due to cavitation pressures. This work established the use of an oxygen-releasing biomaterial with targeted characteristics to improve cell viability of 3D HDF culture and inhibit viable HT1080 development. The work carried out in this thesis developed three distinct approaches with ultrasound to improve current techniques used in collagen-based tissue engineering. The results from experiments using different acoustic frequencies and intensities have advanced knowledge across the areas of scaffold production, cell migration and oxygen delivery.