Development of in situ hydrogel for biomedical applications and delivery of blood stage malaria vaccine Al-Abboodi, Aswan 10.4225/03/58868a61d81c0 https://bridges.monash.edu/articles/thesis/Development_of_in_situ_hydrogel_for_biomedical_applications_and_delivery_of_blood_stage_malaria_vaccine/4559152 Since rates of tissue growth vary significantly between tissue types, and also between individuals due to differences in age, dietary intake and lifestyle-related factors, engineering a scaffold system that is appropriate for personalized tissue engineering remains a significant challenge. In this study, we developed a gelatin-hydroxyphenylpropionic acid/carboxylmethylcellulose-tyramine (Gtn-HPA/CMC-Tyr) porous hydrogel system that allows the pore structure of scaffolds to be altered in vivo after implantation. Crosslinking of Gtn-HPA/CMC-Tyr hydrogels via horseradish peroxidase oxidative coupling was examined both in vitro and in vivo. Post-implantation, further alteration of the hydrogel structure was achieved by injecting cellulase enzyme to digest the CMC component of the scaffold; this treatment yielded a structure with larger pores and higher porosity than hydrogels without cellulase injection. Using this approach, the pore sizes of scaffolds were altered in vivo from 32–87 m to 74–181 m in a user-controlled manner. The mechanical properties of the hydrogel are similar to those of soft tissues. Biocompatibility of the hydrogel was demonstrated using African green monkey kidney (COS-7) cells, and faster cell growth was observed in hydrogels after cellulase digestion. The new hydrogel system developed in this work provides clinicians with the ability to tailor the structure of scaffolds post-implantation depending on the growth rate of a tissue or an individual’s recovery rate, and could thus be ideal for personalized tissue engineering. Porous hydrogels provide an excellent environment for cell growth and tissue regeneration, with high permeability for oxygen, nutrients, and other water-soluble metabolites through their high water-content matrix. The ability to image three-dimensional (3D) cell growth is crucial for understanding and studying various cellular activities in 3D context, particularly for designing new tissue engineering scaffold, but it is still challenging to study cell biomaterial interfaces with high resolution imaging. We demonstrate using focused ion beam (FIB) milling, electron imaging, and associated microanalysis techniques that novel 3D characterizations can be performed effectively on cells growing inside 3D hydrogel scaffold. With FIB-tomography, the porous microstructures were revealed at nanometer resolution, and the cells grown inside. The results provide a unique 3D measurement of hydrogel porosity, as compared with those from porosimetry, and offer crucial insights into material factors affecting cell proliferation at specific regions within the scaffold. We also proved that high throughput correlative imaging of cell growth is viable through a silicon membrane based environment. The proposed approaches, together with the protocols developed, provide a unique platform for analysis of the microstructures of novel biomaterials, and also for exploration of their interactions with the cells. The ability to induce an immune response with recombinant Plasmodium yoelii, 19-kDa C-terminal fragment of merozoite surface protein 1 (PyMSP119) encapsulated within a Gelatin-hydroxyphenylpropionic acid/carboxylmethylcellulose-tyramine (Gtn-HPA/CMC-Tyr) porous hydrogel network and the efficacy of such an immune response was investigated as a new method of improving vaccination against malaria. The theory behind this work is that the slow, sustained release of the malaria antigen may induce and maintain a high level of immune response for a prolonged period. In vitro protein release studies by immunoblot were performed firstly to determine the PyMSP119 release profile for three different concentrations of Gtn-HPA/ CMC-Tyr hydrogel: 10%, 15% and 20%. It was found that the release profile followed biphasic kinetics, with slow, close to first order release for the first 8 hours followed by a faster release. These results showed that Gtn-HPA/ CMC-Tyr hydrogels could be used for the controlled release of antigens. In vivo studies were performed to measure the antibody responses elicited in mice by different hydrogel vaccine complexes which involved flagellin adjuvant (FljB). Higher serum antibody titers against PyMSP119 were observed with the administration of a complex of hydrogel/PyMSP119/FljB in three injections compared to other complexes. Robust IgG1 and total IgG response were observed after 3 injections of the hydrogel/PyMSP119/FljB complex as a result of the combination of the slow sustained release of the antigen from the hydrogel, the presence of the FljB adjuvant and the antigen boost injections. A complex of hydrogel/PyMSP119 in three injections (without adjuvant) generated moderate total IgG responses against PyMSP119 antigen, indicating the role of the hydrogel in maintaining the immune responses that lead to appropriate sustained responses. These findings support the utility of porous hydrogel-based antigen vaccine systems to induce the antibody responses that may be suitable for a diversity of diseases including malaria. An anisotropic matrix that allows users to alter its properties and structure in situ after synthesis can better mimic the dynamic in vivo microenvironment such as tissues undergoing morphogenetic processes or wounds undergoing tissue repair compared to preformed anisotropic matrices. In this study, porous gradients are generated in situ in a hydrogel comprising enzymatically crosslinked gelatin hydroxyphenylpropionic acid (GTN-HPA) conjugate and carboxylmethyl cellulose tyramine (CMC-TYR) conjugate. The hydrogel is then used to immobilise human fibrosarcoma cells HT1080 in a microfluidic chamber. The GTN-HPA component acts as the backbone of the hydrogel, while CMC-TYR acts as a biocompatible sacrificing polymer in the hydrogel. By allowing the diffusion of a biocompatible cellulase enzyme through the hydrogel in a spatially controlled manner, the selective digestion of the CMC component of the hydrogel by the cellulase then gives rise to a porosity gradient in situ prior to use instead of during the synthesis of the hydrogel. The influence of this dynamic porosity gradient on the chemotactic response of cancer cell was studied both in the absence and in the presence of chemoattractant. The platform illustrates the potential of hydrogel-based microfluidics to mimic the three-dimensional in vivo microenvironment for cell-based screening applications. 2017-01-23 22:57:35 Restricted access and full embargo 1959.1/977987 Injectable hydrogel thesis(doctorate) Malaria vaccine monash:130408 Porosity 2014 Microfluidic channels ethesis-20140729-110734