Structural evolution of photocrosslinked silk fibroin and silk fibroin-based hybrid hydrogels: A small angle and ultra-small angle scattering investigation.

Regenerated Bombyx mori silk fibroin (RSF) is a widely recognized protein for biomedical applications; however, its hierarchical gel structure is poorly understood. In this paper, the hierarchical structure of photocrosslinked RSF and RSF-based hybrid hydrogel systems: (i) RSF/Rec1-resilin and (ii) RSF/poly(N-vinylcaprolactam (PVCL) is reported for the first time using small-angle scattering (SAS) techniques. The structure of RSF in dilute to concentrated solution to fabricated hydrogels were characterized using small angle X-ray scattering (SAXS), small angle neutron scattering (SANS) and ultra-small angle neutron scattering (USANS) techniques. The RSF hydrogel exhibited three distinctive structural characteristics: (i) a Porod region in the length scale of 2 to 3nm due to hydrophobic domains (containing ß-sheets) which exhibits sharp interfaces with the amorphous matrix of the hydrogel and the solvent, (ii) a Guinier region in the length scale of 4 to 20nm due to hydrophilic domains (containing turns and random coil), and (iii) a Porod-like region in the length scale of few micrometers due to water pores/channels exhibiting fractal-like characteristics. Addition of Rec1-resilin or PVCL to RSF and subsequent crosslinking systematically increased the nanoscale size of hydrophobic and hydrophilic domains, whereas decreased the homogeneity of pore size distribution in the microscale. The presented results have implications on the fundamental understanding of the structure-property relationship of RSF-based hydrogels.

Engineering DN hydrogels from regenerated silk fibroin and poly(N-vinylcaprolactam).

The development of novel hydrogels that possess adequate elasticity and toughness to withstand mechanically active environments, along with being biocompatible, remains a significant challenge in the design of materials for tissue engineering applications. In this study, a family of regenerated silk fibroin (RSF) based double network (DN) hydrogels were fabricated for the first time using a rapid one-pot method. The DN hydrogels combine the rigid covalently crosslinked RSF with the softer poly(N-vinylcaprolactam) (PVCL) through strong physical interactions. The formation of these DN hydrogels resulted in an improvement of the water uptake capacity, elasticity and toughness compared to the individual RSF hydrogel. The elasticity of the RSF/PVCL DN hydrogels was closer to that of native cartilage, which makes them promising materials for cartilage regeneration applications. An in vitro study on adhesion, proliferation and differentiation of a mouse pre-chondrocyte cell line (ATDC5) conducted using microscopic analysis, a cell proliferation assay and RT-PCR confirmed the cells cultured on the less stiff hydrogels demonstrated the most favourable chondrogenic response. Thus, this study demonstrates the potential of RSF-based hybrid hydrogels for cartilage tissue engineering applications.

Tunable Thermoresponsiveness of Resilin via Coassembly with Rigid Biopolymers.

The ability to tune the thermoresponsiveness of recombinant resilin protein, Rec1-resilin, through a facile coassembly system was investigated in this study. The effects of change in conformation and morphology with time and the responsive behavior of Rec1-resilin in solution were studied in response to the addition of a rigid model polypeptide (poly-l-proline) or a hydrophobic rigid protein (Bombyx mori silk fibroin). It was observed that by inducing more ordered conformations and increasing the hydrophobicity the lower critical solution temperature (LCST) of the system was tuned to lower values. Time and temperature were found to be critical parameters in controlling the coassembly behavior of Rec1-resilin in both the model polypeptide and more complex protein systems. Such unique properties are useful for a wide range of applications, including drug delivery and soft tissue engineering applications.

Facile and rapid ruthenium mediated photo-crosslinking of Bombyx mori silk fibroin.

A facile and rapid ruthenium catalysed photo-crosslinking method was employed to prepare Bombyx mori silk fibroin hydrogels for the first time that may be used for biomedical applications. The gels have been characterised by differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), photo-acoustic Fourier transform infrared spectroscopy (PA-FTIR), and X-ray diffraction (XRD). We have reported the tuning of the properties of the chemically crosslinked gels through hydration level. DSC and DMA measurements have shown that the molecular chain dynamics and mechanical properties are dependent on the moisture/water content. The structural changes upon gelation were examined through the use of DSC, PA-FTIR and XRD, which confirmed that the gels were less crystalline than the original silk powder. Finally, the biocompatibility and hence, the potential tissue engineering application of the silk fibroin hydrogel was also assessed by culturing chondrocyte progenitor cells for up to one week on the engineered fibroin surfaces.