Biocompatibility testing of UV-crosslinked and thermo-responsive hydrogels for 3D bioprinting
Ho, Davin Wei Ern
Date of Issue2017-05-30
School of Mechanical and Aerospace Engineering
Gelatin Methacrylate (GelMA) hydrogels have the innate capability of cell compatibly, but performs poorly in printability when used as an ink in bio printing. In the interest of fabricating a hydrogel to be used in tissue engineering, Gelatin Methacrylate is often combined with synthetic or natural polymers, in order to encompass broader mechanical properties, whilst still tapping on the cell compatibility capability of the Gelatin Methacrylate. A man made polymer Pluronic-Monocarboxylate (PluMP) possess favourable printing properties. Introducing GelMA with PluMP might lead to a discovery of a potential bio material for bio printing – One that eliminates the poor printability of Gelatin Methacrylate whilst still possessing favourable cell compatibility conditions. The objective of the following project is to determine and study how cell compatibility (Bio compatibility) is affected by the concentration of GelMA in a hydrogel composite with PluMP. A potential bio material for tissue engineering where cells are seeded onto the printed scaffold after three-dimensional bio printing, was fabricated and tested on. Bio materials for scaffolds place bio compatibility, bio degradability, mechanical properties, scaffold morphology and porosity as characteristic priorities. The two individual components namely GelMA and PluMP were mixed together in different ratios, to fabricate a GelMA hydrogel composite. PluMP is used as the buffer for varying the concentration with GelMA as it provides greater printability of the scaffolds, without affecting the biological properties of the GelMA in the composite. The individual components were fabricated based on weight by volume (w/v); GelMA synthesised at 15% w/v, and PluMP synthesised at 30% w/v. The concentration ratios created from mixing GelMA with PluMP were: 2:1, 1.5:1, 1:1, 1:1.5, and 1:2. These samples of varying concentrations were put through a series of different biocompatibility tests to determine its repercussions on its biological properties. Rate of degradation, a favourable biological property for the bio material - printed scaffolds must maintain its size and shape for a sufficient amount of time first, in order to be used for purposes such as tissue engineering – Natural biological degradation eliminates complications arising from the need for physical removal of the scaffold. Rate of degradation was determined by submerging the samples in a 0.02% collagenase enzyme solution, and weighing the individual samples over 16 days. Samples which had higher GelMA concentrations displayed lower rates of degradation, when compared to mixtures with lower GelMA concentrations. All concentrations of GelMA, were not able to maintain their structural integrity beyond day 16. Observation of the samples under a scanning electron microscope revealed a degree of porosity with respect to its concentration. Samples with a lower GelMA concentration displayed greater porosity. Porosity is an important physical property for the bio material as it firstly affects the exposure of cell media to the seeded cells in the scaffold, which determines if the cells in the scaffold are able to receive sufficient nutrients from tissue media. Secondly, porosity would allow the movement of seeded cells on the surface, into the scaffold. Cell viability of the bio material is the mortality rate of the cells after seeding. Calcein AM (Component A) and Ethidium homodimer-1 (Component B) live and dead staining solutions were used to soak and highlight the live cells and the dead cells. Observation of the fluorescence under a microscope revealed similar cell viability for all concentrations. Concluding that GelMA concentration has negligible effect on cell viability. Cell proliferation measures the rate of cell multiplication and growth on the bio material after seeding. Seeded samples were first observed via Scanning Electron Microscopy. Samples which had lower concentrations of GelMA displayed greater cell attachment on the surface, as compared to samples of greater GelMA concentrations. The increase in porosity of the former samples led to an increase in surface area, which ultimately granted an increased surface area for the attachment of cells. The seeded samples were also soaked in Presto-Blue solution (To quantitatively analyse the proliferation of cells) and together with a fluorescence machine, cell numbers per sample were calculated over 7 days. Samples which had lower GelMA concentrations had higher cell number counts, concluding that lower concentrations of GelMA is inversely proportional to rate of cell proliferation. In conclusion, the increase in GelMA concentration does not see an absolute increase in all cell viability factors (Degradation, porosity, viability, proliferation and cell attachment morphology).
Final Year Project (FYP)
Nanyang Technological University