Design, fabrication and characterization of light-responsive functionalized hydrogel for tissue engineering applications

Stimuli-responsive hydrogels have gained immense consideration due to the unique properties such as spectacular volume transition in response to a variety of physical and chemical stimuli. These hydrogels have been particularly effective, considering exceptional degree of authority over material properties because of external signals. The precise control over the hydrogel properties resulted in significant advancements in medical devices and improved methodologies for tissue engineering applications. In this thesis, we aimed to develop biocompatible and biodegradable stimuli-responsive hydrogels for tissue engineering applications. Different methods of hydrogel fabrication were studied throughout the study such as free radical photopolymerization and photodimerization reactions. In the first part of the thesis, we pursued the conventional method of hydrogel formation where polymers were functionalized with light-sensitive anthracene. Glycan-based alginate hydrogels have great potential in creating new vehicles with responsive behavior and tunable properties for biomedicine. However, precise control and tunability in properties present major barriers for clinical translation of these materials. We reported the synthesis of pH responsive anthracene modified glycan-based hydrogels for selective release of therapeutic molecules. Hydrogels were crosslinked through simultaneous photopolymerization of vinyl groups and photodimerization of anthracene. Incorporation of anthracene into these gels lead to reversible control on crosslinking and transition between gel/sol states through dimerization/dedimerization of anthracene groups. Chemotherapeutic drug doxorubicin-loaded hydrogels were then tested in a cancer mimetic microenvironment where 85% of the drug was released from anthracene-conjugated hydrogels at pH 2 for 6 days. Control on gelation with anthracene incorporation was observed through alterations in modulus, where storage modulus was increased two- and five-fold with anthracene conjugation during photopolymerization and photodimerization, respectively. Furthermore, cell survival analysis revealed that anthracene conjugation could selectively compromise cancer cell viability without inducing significant toxicity on healthy fibroblasts. This study combines light-induced control of crosslink density due to the anthracene and pH-triggered therapeutics delivery with alginate. The approach would be applicable for systems where multiple control is required with high precision. Further, we reported the synthesis of single and dual-crosslinked anthracene-functional chitosan-based hydrogels in the absence of toxic initiators. Single crosslinking was achieved through dimerization of anthracene, whereas dual-crosslinked hydrogel was formed through dimerization of anthracene and free radical photopolymerization of methacrylated-chitosan in the presence of non-toxic initiator riboflavin, a well-known vitamin B2. Both single and dual-crosslinked hydrogels were found to be elastic, as was determined through rheological analysis. We observed that the dual-crosslinked hydrogels exhibited higher Youngs modulus than the single-crosslinked hydrogels, where the modulus for single and dual-crosslinked hydrogels were measured as 9.2±1.0 kPa and 26±2.8 kPa, respectively resulting in significantly high volume of cells in dual-crosslinked hydrogel (2.2x107 µm3) compared to single-crosslinked (4.9x106 µm3). Furthermore, we investigated the cytotoxicity of both hydrogels towards 3T3-J2 fibroblast cells through CellTiter-Glo assay. Finally, immunofluorescence staining was carried out to evaluate the impact of hydrogel modulus on cell morphology. This study comprehensively presents functionalization of chitosan with anthracene, uses nontoxic initiator riboflavin, modulates the degree of crosslinking through dimerization of anthracene and free radical photopolymerization, and further modulates cell behavior through the alterations of hydrogel properties. In the second part of the thesis, a novel technique was introduced and photocrosslinked hydrogels were synthesized from natural and synthetic polymers in the absence of any photoinitiator or coinitiator. Very simple and facile method was developed to synthesize biocompatible and non-toxic hydrogels through free-radical polymerization. The potential of the developed procedure is shown against natural and synthetic polymers both. For this, acrylated forms of alginate, chitosan, gelatin, hyaluronic acid, and polyethylene glycol (PEG) were used and mechanically robust hydrogels were developed under UV (365 nm) and visible (430 nm) light as determined through rheological analysis. We also observed that the UV light-crosslinked hydrogels exhibited higher crosslinking and showed high Youngs modulus as compared to visible light-crosslinked hydrogels. Cell viability analysis shows that the developed hydrogels are non-toxic towards fibroblast cells. The biomedical potential of these hydrogels was further explored by encapsulating the two model cell lines where NIH-3T3 fibroblast cells and bone-marrow derived mesenchymal stem cells (rBM-MSCs) were encapsulated in the hyaluronic acid (HA)-based hydrogel. The enhanced growth rate of the cells shows that the crosslinking procedure is non-toxic to the cells. Scope of this study was further expanded to sustained release of therapeutics. DOX was successfully loaded into HA-based hydrogel. The maximum loading was observed as 97% and an accelerated release rate was seen in visible light-based crosslinked hydrogel due to the open pore structure compared to the UV-based hydrogel. Another potential application for such initiator-free hydrogel might be the vascularization which was also explored in this part of the thesis. Engineering of organized vasculature is a key step in the improvement of functional and clinically relevant tissue constructs. Various hydrogel-based culture systems are used to make in vitro models for angiogenesis. Here, we developed initiator-free photocrosslinked gelatin-based hydrogels under UV (365 nm) and visible light (430 nm) for the tubular formation of functional vascular networks. Detailed rheological analysis revealed that Gel-MA hydrogels were robust and viscoelastic in nature. We tuned the mechanical properties of Gel-MA hydrogel depending on the type of exposed light. By this way, we obtained stiff and soft Gel-MA hydrogels. Immunofluorescence labeling was used to track the impact of hydrogel modulus over the cell shape and tubular formation. We found higher tubular formation in soft Gel-MA hydrogel compared to the stiff hydrogel. Further, we also investigated the tubular formation in the presence and absence of growth factors and found that tubular formation was much higher in the presence of growth factors as expected. The cytotoxicity of the developed hydrogel was evaluated towards HUVECs by using the CellTiter-Glo assay and found to be non-toxic.