Modulating the Viscoelastic Properties of Covalently Crosslinked Protein Hydrogels.

Protein engineering allows for the programming of specific building blocks to form functional and novel materials with customisable physical properties suitable for tailored engineering applications. We have successfully designed and programmed engineered proteins to form covalent molecular networks with defined physical characteristics. Our hydrogel design incorporates the SpyTag (ST) peptide and SpyCatcher (SC) protein that spontaneously form covalent crosslinks upon mixing. This genetically encodable chemistry allowed us to easily incorporate two stiff and rod-like recombinant proteins in the hydrogels and modulate the resulting viscoelastic properties. We demonstrated how differences in the composition of the microscopic building blocks change the macroscopic viscoelastic properties of the hydrogels. We specifically investigated how the identity of the protein pairs, the molar ratio of ST:SC, and the concentration of the proteins influence the viscoelastic response of the hydrogels. By showing tuneable changes in protein hydrogel rheology, we increased the capabilities of synthetic biology to create novel materials, allowing engineering biology to interface with soft matter, tissue engineering, and material science.

Chemically cross-linked hydrogels from repetitive protein arrays.

Biomaterials for tissue regeneration must mimic the biophysical properties of the native physiological environment. A protein engineering approach allows the generation of protein hydrogels with specific and customised biophysical properties designed to suit a particular physiological environment. Herein, repetitive engineered proteins were successfully designed to form covalent molecular networks with defined physical characteristics able to sustain cell phenotype. Our hydrogel design was made possible by the incorporation of the SpyTag (ST) peptide and multiple repetitive units of the SpyCatcher (SC) protein that spontaneously formed covalent crosslinks upon mixing. Changing the ratios of the protein building blocks (ST:SC), allowed the viscoelastic properties and gelation speeds of the hydrogels to be altered and controlled. The physical properties of the hydrogels could readily be altered further to suit different environments by tuning the key features in the repetitive protein sequence. The resulting hydrogels were designed with a view to allow cell attachment and encapsulation of liver derived cells. Biocompatibility of the hydrogels was assayed using a HepG2 cell line constitutively expressing GFP. The cells remained viable and continued to express GFP whilst attached or encapsulated within the hydrogel. Our results demonstrate how this genetically encoded approach using repetitive proteins could be applied to bridge engineering biology with nanotechnology creating a level of biomaterial customisation previously inaccessible.

Silk fibroin nanoscaffolds for neural tissue engineering.

The nervous system is a crucial component of the body and damages to this system, either by injury or disease, can result in serious or potentially lethal consequences. An important problem in neural engineering is how we can stimulate the regeneration of damaged nervous tissue given its complex physiology and limited regenerative capacity. To regenerate damaged nervous tissue, this study electrospun three-dimensional nanoscaffolds (3DNSs) from a biomaterial blend of silk fibroin (SF), polyethylene glycol (PEG), and polyvinyl alcohol (PVA). The 3DNSs were characterised to ascertain their potential suitability for direct implant into the CNS. The biological activity of 3DNSs was investigated in vitro using PC12 cells and their effects on reactive astrogliosis were assessed in vivo using a photothrombotic model of ischaemic stroke in mice. Results showed that the concentration of SF directly affected the mechanical characteristics and internal structure of the 3DNSs, with formulations presenting as either a gel-like structure (SF ≥ 50%) or a nanofibrous structure (SF ≤ 40%). In vitro assessment revealed increased cell viability in the presence of the 3DNSs and in vivo assessment resulted in a significant decrease in glial fibrillary acidic protein (GFAP) expression in the peri-infarct region (p < 0.001 for F2 and p < 0.05 for F4) after stroke, suggesting that 3DNSs could be suppressing reactive astrogliosis. The findings enhanced our understanding of physiochemical interactions between SF, PEG, and PVA, and elucidated the potential of 3DNSs as a potential therapeutic approach to stroke recovery, especially if these are used in conjunction with drug or cell treatment.

Current and novel polymeric biomaterials for neural tissue engineering.

The nervous system is a crucial component of the body and damages to this system, either by of injury or disease, can result in serious or potentially lethal consequences. Restoring the damaged nervous system is a great challenge due to the complex physiology system and limited regenerative capacity.Polymers, either synthetic or natural in origin, have been extensively evaluated as a solution for restoring functions in damaged neural tissues. Polymers offer a wide range of versatility, in particular regarding shape and mechanical characteristics, and their biocompatibility is unmatched by other biomaterials, such as metals and ceramics. Several studies have shown that polymers can be shaped into suitable support structures, including nerve conduits, scaffolds, and electrospun matrices, capable of improving the regeneration of damaged neural tissues. In general, natural polymers offer the advantage of better biocompatibility and bioactivity, while synthetic or non-natural polymers have better mechanical properties and structural stability. Often, combinations of the two allow for the development of polymeric conduits able to mimic the native physiological environment of healthy neural tissues and, consequently, regulate cell behaviour and support the regeneration of injured nervous tissues.Currently, most of neural tissue engineering applications are in pre-clinical study, in particular for use in the central nervous system, however collagen polymer conduits aimed at regeneration of peripheral nerves have already been successfully tested in clinical trials.This review highlights different types of natural and synthetic polymers used in neural tissue engineering and their advantages and disadvantages for neural regeneration.