Composite hydrogels of bio-inspired protein polymers : mechanical and structural characterization

In this thesis we presented various combinations of custom-designed protein polymers that formed composite hydrogels. In chapter 2, composite hydrogels were prepared by mixing silk-like block copolymers (CP2SE48CP2) with collagen-like block copolymers (T9CR4T9). We found that by adding the collagen-like protein polymer the storage modulus, the critical stress and critical strain values of the composite hydrogels were significantly improved in comparison to the single networks. With cryo-transmission electron microscopy (cryo-TEM) we observed that the silk-like fibers were bundled in the presence of the collagen-like protein polymer, probably due to depletion attraction interactions. In follow-up research on these composite hydrogels in chapter 3, we tried to get more insight into the exact toughening mechanism and self-healing capabilities of the composite network by performing cyclic loading/unloading tests. We found that mechanical hysteresis occurred in these composite hydrogels. The energy that was dissipated could be split into two contributions: a part belonging to the permanent rupture of silk-like fibers, and a viscoelastic part belonging to the assembly and disassembly of collagen-like triple helices. Both these contributions increased as the concentration of the collagen-like protein polymer in the composite network was increased, resulting in toughening of the composite network. Furthermore, it was observed that the silk-like fiber network was not able to recover, while the composites could recover up to 70% of the original storage modulus after failure. In chapter 4 we studied composite networks of silk-like block copolymers (CP2SE48CP2) and a FMOC-functionalized dipeptide (FMOC-LG) which could both form fibers. With cryo-TEM and atomic force microscopy (AFM) we found that two different types of fibers were formed, indicating that orthogonal self-assembly occurred in this system. We found with rheology that the storage moduli of the composite fiber networks were significantly higher (75 kPa vs. 400 kPa) than that of the single networks. Strain-hardening present in the FMOC-LG fiber network disappeared when the silk-like protein polymer was present. In chapter 5 hydrogels with both physical and chemical crosslinks were prepared from collagen-like protein polymers (T9CRT9). The chemical crosslinks were introduced by crosslinking lysine residues present in the random-coil middle blocks with glutaraldehyde. We found with rheology that the order in which the physical and chemical networks were formed did not influence the final storage modulus of the hydrogel. Depending on the amount of glutaraldehyde added we found an increase of up to an order of magnitude in the storage modulus for the collagen-like hydrogels. To investigate effects on the nonlinear rheological properties cyclic loading/unloading tests were performed. It was observed that before hydrogel failure occurred no hysteresis was observed between consecutive cycles. Both physical and chemical crosslinks ruptured when the hydrogel was fractured. In chapter 6 we studied hydrogels formed by the co- assembly of an asymmetric silk-collagen-like protein polymer (SH8CR4T9) with a symmetric oppositely charged silk-like protein polymer (CP2SE48CP2). This was done in a step-wise approach: (1) the S blocks were co-assembled into silk-like fibers. (2) the T blocks were assembled into triple helical nodes by reducing the temperature. We confirmed with confocal laser scanning microscopy and NMR that both monomers were present in the same fibers. With rheology we found that these composite hydrogels did respond in a reversible manner to temperature changes, with which the mechanical strength of the hydrogel can be tuned. In chapter 7 hydrogel formation of a modified silk-like protein polymer with a cysteine-residue attached to the C-terminal side (CP2SH48CP2-Cys) was studied. With rheology we showed that hydrogels that were formed in oxidizing conditions, where disulfide-bridges could form, were much stronger than those formed in reducing conditions. Both hydrogels formed in oxidizing and reducing conditions showed a scaling of modulus versus concentration consistent with entangled semi-flexible networks. This result implied that the disulfide-bridges formed between cysteine-residues formed loops in the coronae of the fibers. The increase in mechanical strength of the fibers was related to the increase in persistence length of the fibers in oxidizing conditions observed with AFM. With self-consistent field theory-simulations it was shown that the formation of loops in the corona resulted in a slight reduction of the lateral pressure in the corona of the fibers. However, this effect is by itself not sufficient to cause a significant change in persistence length. Due to the reduction in lateral pressure, the stacking of monomers into fibers is probably influenced: fibers with a more crystalline structure and with less detects are formed, resulting in improved mechanical properties of the hydrogels. In the general discussion in chapter 8, we reflect on our work, discuss about future directions of research, and possible applications of protein polymers.