Tim Jia-Ching Lo

Electrogelation for Protein Adhesives

Adv. Mater. 2010, 22, 711–715 2010 WILEY-VCH Verlag Gm IC A T IO N Adhesives are common in biology as critical elements in motility, adhesion, and survival for many land and sea creatures. Despite many attempts to mimic such features with natural or synthetic polymers, this has proven to be challenging due to the subtle and metastable state of the polymeric material properties that are required to control the functional attributes of such systems including during storage, processing, adhesion, and release. The viscoelastic behavior also limits the types of material systems that can be exploited for biomimetic approaches to this important material behavior. Most often, modified polysaccharides are found associated with mucoadhesives from biological systems, due to their hydration and charge density. We report the discovery of a novel, electrically mediated adhesive formed from silkworm silk. This process, termed electrogelation provides a protein-based adhesive that offers biomimetic features when used in conjunction with devices. Further, we report on the solution behavior, morphology, and structural features of electrogels (e-gels), to demonstrate the mechanisms involved in the process. The adhesion can be controlled via electrical inputs. Most importantly, and quite unexpectedly, this is a reversible process, depending on voltage, time, and conditions used. This finding is very novel, as silkbased protein systems in particular are usually considered irreversible in terms of polymer transitions from the solution to solid state, mediatedmost often by solvents andmechanical shear forces. The basis for the current discovery comes from recent observations where aqueous solutions of silkworm silk were exposed to direct current (DC). Under certain electric fields, the solution began to gel on the positive electrode (Fig. 1). This observation prompted further investigation into the conditions and responses of the solution under different electric fields. While electrospinning of polymers, including silks, is performed at voltage potentials as high as>30 kV, the utilization of low DC voltages to generate a controlled volume of silk gel is novel. In the basic setup (Fig. 1), electrodes are immersed in an aqueous solution of silk protein and 25 VDC is applied over a 3min period to a pair of mechanical pencil leads. As the process progresses, bubbles evolve on both electrodes. Since the silk solution has a high water content, electrolysis occurs during electrogelation. The bubbles reflect the generation of oxygen gas at the positive electrode and hydrogen gas at the negative electrode during electrolysis. Within seconds of the application of the voltage, a visible gel forms at the positive electrode, locking in some oxygen bubbles at the electrode surface as the gel emanates outward. While the gel appears to have formed symmetrically about the electrode in Figure 1, it is typical that the forming gel front is directed toward the negative electrode. When silk electrogelation is executed in a voltage-controlled format, the current draw in the process follows a repeated trend; initially high current draw drops exponentially to a minimal milliampere level. The actual current amplitudes depend on many factors, including applied voltage level, electrode area and spacing, and conductivity of the silk solution. The decay in current draw is likely related to the electrical insulating effect of the growing volume of silk gel and the bubbles that become trapped near the positive electrode surface. Silk gel formed through electrogelation has a highly viscous (soft) consistency and is very tacky, bearing a resemblance to thick mucus. Remarkably, we observed that after the electric field was turned off, the adhesive gel state was retained, thus, the structural state of the protein formed under e-gel conditions was sufficiently stable to retain material functions in the absence of the applied electric field. Yet, the gel formed can be returned to the solution state through a reverse electrical process (Fig. 1). If the electrode polarity is reversed and 25 VDC reapplied, the gel disappears, while fresh gel is formed on the newly created positive electrode. Electrogelation and reversal back to silk solution can be cycled many times. If an electrogelation process is performed for an extended period of time, or at high voltage, the gel nearest the positive electrode persists and reversal of the electric field no longer drives the gel back to solution form. Alternatively, after intermediate electrogelation times, heating the gel up to ca. 60 8C led to the disappearance of the gel-like material and transitioning back to the solution state. When cooled back to room temperature, e-gel reformed as a highly viscous and tacky material, thus, the gelation is reversible over several cycles. Consequently, the process is controllable in terms of gel features, reversibility, or permanency, such as by temperature. The changes in mechanical characteristics due to electrogelation of silk solutions were investigated by dynamic oscillatory shear rheology. For silk solutions, liquidlike, viscous behavior measured by the loss modulus (G00) dominated the mechanical response within the probed frequency range (v) with G00 v (Fig. 2A). On the other hand, the mechanical response of e-gels resembled that of a soft–solidlike, physical gel. There was a significant increase in the elastic response, measured by the storage modulus (G0). The frequency dependence of G0 was weak but finite (G0 v), while the apparent minimum in G00 suggested a possible G0, G00 crossover at even lower frequencies due to eventual relaxation of temporary, physical crosslinks. To investigate the significance of increased proton concentration in the mechanism of e-gel formation at the positive electrode, we titrated the solution to control the pH, termed pH-gels (Fig. 2A).

The use of silk-based devices for fracture fixation

Metallic fixation systems are currently the gold standard for fracture fixation but have problems including stress shielding, palpability and temperature sensitivity. Recently, resorbable systems have gained interest because they avoid removal and may improve bone remodelling due to the lack of stress shielding. However, their use is limited to paediatric craniofacial procedures mainly due to the laborious implantation requirements. Here we prepare and characterize a new family of resorbable screws prepared from silk fibroin for craniofacial fracture repair. In vivo assessment in rat femurs shows the screws to be self-tapping, remain fixed in the bone for 4 and 8 weeks, exhibit biocompatibility and promote bone remodelling. The silk-based devices compare favourably with current poly-lactic-co-glycolic acid fixation systems, however, silk-based devices offer numerous advantages including ease of implantation, conformal fit to the repair site, sterilization by autoclaving and minimal inflammatory response.

Non-equilibrium Silk Fibroin Adhesives

Regenerated silkworm silk solutions formed metastable, soft-solid-like materials (e-gels) under weak electric fields, displaying interesting mechanical characteristics such as dynamic adhesion and strain stiffening. Raman spectroscopy, in situ electric field dynamic oscillatory rheology and polarized optical microscopy indicated that silk fibroin electrogelation involved intermolecular self-assembly of silk molecules into amorphous, micron-scale, micellar structures and the formation of relatively long lifetime, intermicellar entanglement crosslinks. Overall, the electrogelation process did not require significant intramolecular beta-strand or intermolecular beta-sheet formation, unlike silk hydrogels. The kinetics of e-gel formation could be tuned by changing the field strength and assembly conditions, such as silk concentration and solution pH, while e-gel stiffness was partially reversible by removal of the applied field. Transient adhesion testing indicated that the adhesive characteristics of e-gels could at least partially be attributed to a local increase in proton concentration around the positive electrode due to the applied field and surface effects. A working model of electrogelation was described en route to understanding the origins of the adhesive characteristics.

Injectable Silk Foams for Soft Tissue Regeneration

Soft tissue fillers are needed for restoration of a defect or augmentation of existing tissues. Autografts and lipotransfer have been under study for soft tissue reconstruction but yield inconsistent results, often with considerable resorption of the grafted tissue. A minimally invasive procedure would reduce scarring and recovery time as well as allow the implant and/or grafted tissue to be placed closer to existing vasculature. Here, the feasibility of an injectable silk foam for soft tissue regeneration is demonstrated. Adipose-derived stem cells survive and migrate through the foam over a 10-d period in vitro. The silk foams are also successfully injected into the subcutaneous space in a rat and over a 3-month period integrating with the surrounding native tissue. The injected foams are palpable and soft to the touch through the skin and returning to their original dimensions after pressure is applied and then released. The foams readily absorb lipoaspirate making the foams useful as a scaffold or template for existing soft tissue filler technologies, useful either as a biomaterial alone or in combination with the lipoaspirate.

Silk as a biocohesive sacrificial binder in the fabrication of hydroxyapatite load bearing scaffolds.

Limitations of current clinical methods for bone repair continue to fuel the demand for a high strength, bioactive bone replacement material. Recent attempts to produce porous scaffolds for bone regeneration have been limited by the intrinsic weakness associated with high porosity materials. In this study, ceramic scaffold fabrication techniques for potential use in load-bearing bone repairs have been developed using naturally derived silk from Bombyx mori. Silk was first employed for ceramic grain consolidation during green body formation, and later as a sacrificial polymer to impart porosity during sintering. These techniques allowed preparation of hydroxyapatite (HA) scaffolds that exhibited a wide range of mechanical and porosity profiles, with some displaying unusually high compressive strength up to 152.4 ± 9.1 MPa. Results showed that the scaffolds exhibited a wide range of compressive strengths and moduli (8.7 ± 2.7 MPa to 152.4 ± 9.1 MPa and 0.3 ± 0.1 GPa to 8.6 ± 0.3 GPa) with total porosities of up to 62.9 ± 2.7% depending on the parameters used for fabrication. Moreover, HA-silk scaffolds could be molded into large, complex shapes, and further machined post-sinter to generate specific three-dimensional geometries. Scaffolds supported bone marrow-derived mesenchymal stem cell attachment and proliferation, with no signs of cytotoxicity. Therefore, silk-fabricated HA scaffolds show promise for load bearing bone repair and regeneration needs.

Equine Model for Soft Tissue Regeneration

Soft-tissue regeneration methods currently yield suboptimal clinical outcomes due to loss of tissue volume and a lack of functional tissue regeneration. Grafted tissues and natural biomaterials often degrade or resorb too quickly, while most synthetic materials do not degrade. In previous research we demonstrated that soft-tissue regeneration can be supported using silk porous biomaterials for at least 18 months in vivo in a rodent model. In the present study, we scaled the system to a survival study using a large animal model and demonstrated the feasibility of these biomaterials for soft-tissue regeneration in adult horses. Both slow and rapidly degrading silk matrices were evaluated in subcutaneous pocket and intramuscular defect depots. We showed that we can effectively employ an equine model over 6 months to simultaneously evaluate many different implants, reducing the number of animals needed. Furthermore, we were able to tailor matrix degradation by varying the initial format of the implanted silk. Finally, we demonstrate ultrasound imaging of implants to be an effective means for tracking tissue regeneration and implant degradation.