Glucose diffusivity and spreading experiments with porous scaffolds and membranes for tissue engineering bioreactor

Shuai Wang (109515)

In three dimensional (3D) bone tissue engineering, the nutrient transfer process is one of the main issues that should be resolved so as to avoid the lack of nutrients (e.g., glucose and oxygen) feeding the cells in extracapillary space (ECS) region, also known as scaffold in an in vitro static culture. For example, hollow fibre membrane bioreactor (HFMB) has been built to resolve this diffusion limitation and make it possible to produce 3D bone tissues under laboratory conditions. However, in previous study, the glucose diffusivity in CCM is assumed to be same as in water, leading to inaccurate prediction of nutrient transport process in HFMB mathematical models. Hence, the overall aim of this study is to analyse and link the fluid spreading performance and glucose diffusivity across both commercial and in-house membranes and scaffolds saturated with water (reference fluid) and cell culture media (CCM) in HFMB with their microstructure with a view to carry out more accurate modelling of nutrient transport in HFMB.
As a critical role in HFMB, various types of membranes and scaffolds have been used in this project, including commercial scaffolds and membranes (cellulose nitrate membrane (CN), polyvinylidene fluoride (PVDF) membrane, polycaprolactone (PCL) scaffold, collagen scaffold and poly-l-lactide acid (PLLA) scaffold), self-made 3D printed and electrospun scaffolds. The pore-morphologies of all scaffolds are analysed by scanning electronic microscope (SEM) images with a view to investigate their effects on glucose diffusivity. 3D printed scaffolds are examined in this study, as these produced scaffolds have uniform microstructural properties and precise pore morphologies. However, despite the small pore size of these scaffolds is suitable for bone tissue, their level of uniformity and regularity was found to be low. This is one of the main reasons why the scaffolds are made with electrospinning technique in this study. In the electrospinning process, the effects of operating parameters on fiber-fiber space and fiberdiameter of polycaprolactone (PCL) scaffolds on scaffolds morphologies are investigated. Given that the electrospinning process is repeatable, the fiber-fiber space and fiber diameter can be controlled, both of which increase with higher flow rate.
Wettability of the tissue engineering scaffold is a critical parameter for cell attachment. But, there are limited data about the relationship between scaffolds morphology characteristics and spreading behaviour, which is focused in this study. In this study, Kruss DSA100 is used to measure the contact angle of water/ cell culture media (CCM) drop on both dry/pre-wetted varying pore size 3D printed scaffolds and electrospun PCL scaffolds. The results show that all scaffolds have low wettability. With increasing fiber-fiber space of scaffolds, the static contact angle decreases. Furthermore, compared to water drop, the contact angle is higher and it takes longer time to achieve balance with CCM drops, which show worse wettability, due to relative higher viscosity. Pre-wetted scaffolds shows better results with significantly reduced static contact angle owing to lower surface roughness and surface tension between wet surface and droplet. Hence, a conclusion can be drawn that wetting of electrospun PCL scaffolds is a prerequisite for attachment of cells.
The glucose diffusivities of the scaffolds in both water (as reference) and cell culture media (CCM) with and without all membrane and scaffolds are measured, where pore size distribution, porosity and tortuosity are determined and correlated to the effective diffusivity, using a diaphragm cell method. As expected, the effective diffusivity increases correspondingly with the pore size of the materials. It is also observed that the effective glucose diffusivity through the pores of these materials in CCM is smaller than in water.
It is well recognized however that it is impossible to obtain results from cell culture experiments due to its sensitivity, which makes it prominent to use other methods to predict these results. For this reason, effective glucose diffusivity is determined to predict in CCM and water by processing scanning electronic microscope (SEM) images. It can be seen that results obtained from image processing are in agreement with the experimentally obtained diffusion coefficient results as well as its porosity values. With image processed and experimtenal diffusivities, the glucose transport process in HFMB is simulated based on the Krogh cylinder assumption. The simulation shows diffusion process across membrane is the limiting step of mass transfer in HFMB. The minimum glucose concentration in the reactor is also investigated, which is found a linear relationship with glucose diffusivity across scaffold. The diffusivity across porous material measured in this study optimise the modelling and help us to have a better understanding of nutrient transfer in HFMB. </p

Glucose diffusivity and spreading experiments with porous scaffolds and membranes for tissue engineering bioreactors

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