Development of a Novel Protein-based Hydrogel for 3D Bioplotting

Abstract

Organ failure is a major health problem, and annually thousands of people die or develop long-term morbidity from a lack of available donor organs and tissues. Tissue engineering is an emerging field that focuses on methods of creating functional tissues with the goal to repair or replace damaged and failing tissue and organs. Finding suitable materials for tissue engineering, especially for three-dimensional (3D) plotting, is an important area of research in tissue engineering. In natura, cells are supported by the extracellular matrix (ECM), which provides the biochemical and biophysical cues that cells need for maintaining the appropriate phenotype and healthy physiology. Much of the research in tissue engineering has focused on developing bioartificial ECM to create a hospitable environment for cells by constructing a scaffold using natural and synthetic materials, but the complexity of the ECM is difficult to replicate. The objective of this present research was to develop a novel protein-based hydrogel composed of ECM and silk fibroin (SF) for 3D bioplotting in tissue engineering. Literature reveals that ECM has many favourable properties for tissue engineering. Cells need an ECM to develop and proliferate, and natural ECM is ideal for this. The ECM is very complex, with hundreds of biomolecules that are highly conserved across evolutionary lines that improves their xenogeneic compatibility. Decellularization of ECM is a straightforward process that leaves behind a protein skeleton, and ECM can be solubilized in a manner that allows for custom moulding and 3D bioplotting. However, ECM often has poor mechanical properties and degrades rapidly in vivo, which necessitates combining ECM with other polymers to better tune the degradation rate and provide structural support. Another common protein that has found use in tissue engineering is silk fibroin (SF). Silk cocoons can be processed to extract and solubilize the SF, and SF has favourable properties for tissue engineering, including high biocompatibility. One of the drawbacks of SF is that cells have difficulty attaching to it because it lacks the amino acid sequences that cells recognize for attachment. Another drawback is that SF degrades very slowly in vivo. These necessitate combining SF with other materials to optimize SF for in vivo use. This thesis explores the development of a protein-based hydrogel that is composed of ECM and SF. Porcine urinary bladders were decellularized, enzymatically digested, and lyophilized to achieve a soluble urinary bladder ECM (UBECM) powder that can be rehydrated. Using a combination of methods, a high concentration SF suspension was prepared from silk cocoons. Silk cocoons were processed by degumming to remove sericin proteins, solubilized using Ajisawa’s reagent, and dialysed against a series of urea solutions to promote careful refolding of the SF proteins before being dialysed again distilled water. The rehydrated UBECM powder was carefully mixed with the solubilized SF (SF-UBECM), and some experiments also cross-linked SF and UBECM using a combination of sonication and 500 U/ml of Mushroom Tyrosinase (MT) (SF-UBECM-MT). The SF-UBECM-MT mixture was assessed for mechanical properties, swelling ratio, and in 3D plotting with different ratios of SF and UBECM. The SF-UBECM was prepared and tested at all possible permutations of 1%, 3%, and 5% SF with 2%, 4%, 6%, 8%, and 10% ECM. Permutations with 5% SF were found to undergo spontaneous gelation of the SF and were thus discarded from testing. Measurement of the Young’s modulus values of the remaining permutations ranged from 3.3 – 16.4 kPa, which is comparable to native human tissues. The rheological testing demonstrated that the SF-UBECM behaves as a shear-thinning non-Newtonian fluid, and it was found that the 1% SF – 6% UBECM, 1% SF – 8% UBECM, and 1% SF – 10% UBECM permutations also undergo structural breakdown under shearing. As viscosity was very high in the 1% SF – 10% UBECM permutation, this permutation was discarded from further testing. During swelling ratio tests of the 1% SF – 6% UBECM and 1% SF – 8% UBECM permutations, there was evidence to suggest diffusion both into and out of the hydrogel, though overall there was more diffusion out of the scaffold. Three-dimensional plotting of the SF-UBCEM-MT proved challenging, though preliminary experiments demonstrated that 3D plotting is possible. This research demonstrates a potential new protein-based hydrogel for use in tissue engineering with the potential to be used for creating 3D plotted scaffolds. Preparation of the UBECM produced a powder that could easily be rehydrated to desired concentration for use with SF. Extracting SF from silk cocoons successfully produced high-concentration suspensions that mixed well with rehydrated UBECM. The two protein suspensions are able to be cross-linked using a combination of sonication and the addition of MT. The SF-UBECM-MT was found to have favourable mechanical properties that are comparable to native tissues and is capable of being 3D plotted for fabricating scaffolds for tissue engineering. This hydrogel can potentially be used to fabricate scaffolds for growing tissues and organs.