No abstract

Electrochemically controlled, reversible assembly of biopolymers into hydrogel structures is a promising technique for on-demand cell or drug encapsulation and release systems. An electrochemically sol-gel transition has been demonstrated in regenerated Bombyx mori silk fibroin, offering a controllable way to generate biocompatible and reversible adhesives and other biomedical materials. Despite the involvement of an electrochemically triggered electrophoretic migration of the silk molecules, the mechanism of the reversible electrogelation remains unclear. It is, however, known that the freshly prepared silk electrogels (e-gels) adopt a predominantly random coil conformation, indicating a lack of crosslinking as well as thermal, mechanical and morphological stabilities. In the present work, the tuning of covalent and physical β-sheet crosslinks in silk hydrogels was studied for programming the structural properties. Scanning electron microscopy (SEM) revealed delicate morphology, including locally aligned fibrillar structures, in silk e-gels, preserved by combining glutaraldehyde-crosslinking and ethanol dehydration. Fourier transform infrared (FTIR) spectroscopic analysis of either electrogelled, vortex-induced or spontaneously formed silk hydrogels showed that the secondary structure of silk e-gels was tunable between non β-sheet dominated and β-sheet dominated states. Dynamic oscillatory rheology confirmed the mechanical reinforcement of silk e-gels provided by controlled chemical and physical crosslinks. The selective incorporation of either chemical or physical or both crosslinks into the electrochemically-responsive, originally unstructured silk e-gel should help in the design for electrochemically-responsive protein polymers.

As part of a study of elicited angiogenesis, hyaluronan (HA)-based hydrogels crosslinked by polyethylene glycol diacrylate (PEGDA) were loaded with combinations of the cytokine growth factors vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor (KGF) and platelet derived growth factor (PDGF). GF release in vivo was controlled by covalent incorporation of thiol-modified heparin into thiolated HA hydrogels, which were injected into the ear pinnae of mice and allowed to crosslink in situ. GF release in vivo was controlled by covalent incorporation of thiol-modified heparin in the gels. The ears were harvested at 7 or 14 days post implantation, and vascularization evaluated via a Neovascularization Index (NI). The study demonstrates that in situ gelling implants produced no gross inflammation, redness or swelling, and an improved tolerance compared to HA-based dry film implants. All treatments showed significantly more vascularization than either contralateral ears or ears receiving a sham surgery. The maximum response was observed after 14 days in the ears receiving 0.3% Hp, gelatin-containing gels loaded with VEGF+KGF (NI = 3.91). The study revealed injected growth factor-loaded HA-based hydrogels can successfully produce localized controllable vascularization, while minimizing tissue necrosis, polymorphonuclear leukocytes and inflammation. The ability to target and controllably release growth factors can prove a useful tool in specific diseased tissue/organ angiogenesis.

A new, biocompatible hyaluronic acid (HA)-silk hydrogel composite was fabricated and tested for use as a securable drug delivery vehicle. The composite consisted of a hydrogel formed by cross-linking thiol-modified HA with poly(ethylene glycol)-diacrylate, within which was embedded a reinforcing mat composed of electrospun silk fibroin protein. Both HA and silk are biocompatible, selectively degradable biomaterials with independently controllable material properties. Mechanical characterization showed the composite tensile strength as fabricated to be 4.43 ± 2.87 kPa, two orders of magnitude above estimated tensions found around potential target organs. In the presence of hyaluronidase (HAse) in vitro, the rate of gel degradation increased with enzyme concentration although the reinforcing silk mesh was not digested. Composite gels demonstrated the ability to store and sustainably deliver therapeutic agents. Time constants for in vitro release of selected representative antibacterial and anti-inflammatory drugs varied from 46.7 min for cortisone to 418 min for hydrocortisone. This biocomposite showed promising mechanical characteristics for direct fastening to tissue and organs, as well as controllable degradation properties suitable for storage and release of therapeutically relevant drugs.

Concentration gradients of guidance molecules influence cell behavior and growth in biological tissues and are therefore of interest for the design of biomedical scaffolds for regenerative medicine. We developed an electrospining method to generate a dual-gradient of bioactive molecules and fiber density along electrospun nanofibers without any post spinning treatment. Functionalization with fluorescent molecules demonstrated the efficiency of the method to generate a discontinuous concentration gradient along the aligned fibers. As a proof of concept for tissue engineering, the silk nanofibers were functionalized with increasing concentrations of nerve growth factor (NGF) and the biological activity was assessed and quantified with rat dorsal root ganglion (DRG) neurons cultures. Protein assays showed the absence of passive release of NGF from the functionalized fibers. The results demonstrated that the NGF concentration gradient led to an oriented and increased growth of DRG neurons (417.6 ± 55.7 μm) compared to a single uniform NGF concentration (264.5 ± 37.6 μm). The easy-to-use electrospinning technique combined with the multiple molecules that can be used for fiber functionalization makes this technique versatile for a broad range of applications from biosensors to regenerative medicine.