Biopolymers with tunable sizes and morphologies for developmental engineering
Developmental engineering (DE) has been proposed to imitate natural developmental biology processes by implementing modular scaffolds cultured with corresponding mammalian cell types for gradual tissue assembly to manufacture functional tissues via the bottom-up approach. DE may overcome the limitations of the top-down approach such as limited mass transfer, lack of essential cells and tissues, and difficulty to standardise process. However, the mechanistic insights of cell-cell and cell-material interactions have yet to be fully understood. Cell behaviour is related to scaffold size and morphology. Polymeric particles were used as microcarriers for modular scaffolds in this research. Consequently, it is necessary to study how to prepare particles with tunable size and morphology and to investigate the interaction between cell and modular scaffolds as well as bacterial contamination risk due to long-term cell/tissue culture.
In this research, polymeric particles, including poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(lactic acid) (PLA), with various sizes and morphologies were prepared by using different preparation techniques. Solid nanoparticles (~50–200 nm) were obtained via nanoprecipitation when solvent has high affinity for water. Slight reduction of nanoparticle size was observed when using low volumetric ratio of organic and aqueous phase, low polymer concentration and slow stirring speed within the Ouzo region. Microspheres (~5–150 μm) were obtained by emulsion and solvent evaporation method which involved surfactants to stabilise organic and aqueous phases. The particle size and distribution affected by solvent removal methods resulting in larger particle size with broader distribution by evaporation whilst smaller particle size with narrower distribution by distillation. A decrease in particle size and distribution was found when using high volumetric ratio of organic and aqueous phase, low polymer concentration, fast stirring speed and high surfactant concentration. Porous nanoparticles and microspheres were prepared by nanoprecipitation with the assistance of hydrolysis and modified single/double emulsion with addition of porogen, respectively. The inter-channel structure was created under efficient homogenisation in primary emulsion, relatively high polymer concentration and sufficient surfactant as well as was promoted by hydrolysis. The final particle size increased when using high polymer concentration and slow stirring speed in secondary emulsion.
After seeding human dermal fibroblasts (HDFs) on the prepared modular scaffolds (polymeric particles), it was found that HDFs were cultured on the flat tissue culture plates (TCPs) fully covered by particles. With the decrease of the amount of particles, HDFs could be directly seeded onto large (diameter: 30–100 μm) PMMA particles, but not small (diameter: 5–20 μm) PMMA, nor all the PLA and PS particles due to charged surface and relatively high hydrophilicity of PMMA. During tissue cultures, HDFs migration from the TCPs surfaces onto all the particles and colonisation on the clustered PMMA or PLA particles into modular tissues with varying sizes via cell bridging and stacking were observed. Moreover, PLA post-treated by hydrolysis and poly-L-lysine (PLL) coating promoted cell initial attachment significantly.
Further comparisons of PLA discs and cellulosic scaffolds revealed that HDFs utilised the same cell bridging and stacking strategies to colonise the open pores, corners and gaps on 3D-printed PLA discs and irregular porous areas on cellulosic scaffolds. With the aid of simulation via different power law models based on 2D cell cultures as well as specific cell seeding experimental validations, it was found the effects of actual cell densities on the HDFs infiltrations into porous scaffolds.
In addition, the bacterial contamination risk in cell/tissue culture was noticed due to long culture period in the assembly process. The bacteriophage addition was considered as one alternative as Escherichia coli bacteriophage T4 (T4) and Escherichia coli bacteriophage M13 (M13) were evaluated and no detectable impact on the 2D, 3D, and modular tissue culture whilst their anti-infection effects maintained. T4 and M13 also exhibited the potential for coating modular scaffolds to prevent bacterial contamination in DE as they were able to coat on particles and stand multiple deliberate rinses and thermal treatment below 80 °C.
- Aeronautical, Automotive, Chemical and Materials Engineering
Rights holder© Yu Xiang
NotesA Doctoral Thesis. Submitted in partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy of Loughborough University.
Supervisor(s)Xujin Bao ; Tao Sun
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