Gary G. Leisk

GoQBot: a caterpillar-inspired soft-bodied rolling robot

Rolling locomotion using an external force such as gravity has evolved many times. However, some caterpillars can curl into a wheel and generate their own rolling momentum as part of an escape repertoire. This change in body conformation occurs well within 100 ms and generates a linear velocity over 0.2 m s(-1), making it one of the fastest self-propelled wheeling behaviors in nature. Inspired by this behavior, we construct a soft-bodied robot to explore the dynamics and control issues of ballistic rolling. This robot, called GoQBot, closely mimics caterpillar rolling. Analyzing the whole body kinematics and 2D ground reaction forces at the robot ground anchor reveals about 1G of acceleration and more than 200 rpm of angular velocity. As a novel rolling robot, GoQBot demonstrates how morphing can produce new modes of locomotion. Furthermore, mechanical coupling of the actuators improves body coordination without sensory feedback. Such coupling is intrinsic to soft-bodied animals because there are no joints to isolate muscle-generated movements. Finally, GoQBot provides an estimate of the mechanical power for caterpillar rolling that is comparable to that of a locust jump. How caterpillar musculature produces such power in such a short time is yet to be discovered.

Bone Tissue Engineering with Premineralized Silk Scaffolds

Silk fibroin biomaterials are being explored as novel protein-based systems for cell and tissue culture. In the present study, biomimetic growth of calcium phosphate on porous silk fibroin polymeric scaffolds was explored to generate organic/inorganic composites as scaffolds for bone tissue engineering. Aqueous-derived silk fibroin scaffolds were prepared with the addition of polyaspartic acid during processing, followed by the controlled deposition of calcium phosphate by exposure to CaCl(2) and Na(2)HPO(4). These mineralized protein-composite scaffolds were subsequently seeded with human bone marrow stem cells (hMSC) and cultured in vitro for 6 weeks under osteogenic conditions with or without BMP-2. The extent of osteoconductivity was assessed by cell numbers, alkaline phosphatase and calcium deposition, along with immunohistochemistry for bone-related outcomes. The results suggest increased osteoconductive outcomes with an increase in initial content of apatite and BMP-2 in the silk fibroin porous scaffolds. The premineralization of these highly porous silk fibroin protein scaffolds provided enhanced outcomes for the bone tissue engineering.

Silk-Based Electrospun Tubular Scaffolds for Tissue-Engineered Vascular Grafts

Electrospinning was used to fabricate non-woven nanofibrous tubular scaffolds from Bombyx mori silk fibroin using an all aqueous process. Cell studies and mechanical characterization tests were performed on the electrospun silk tubes to assess the viability of their usage in bioengineering small-diameter vascular grafts. Human endothelial cells and smooth muscle cells were successfully cultured on the electrospun silk. Mechanical characterization tests demonstrated burst strength sufficient to withstand arterial pressures and tensile properties comparable to native vessels. These cellular and mechanics outcomes demonstrate potential utility of these electrospun silk scaffolds for small-diameter vascular grafts.

Spider silks and their applications

Spider silks are characterized by remarkable diversity in their chemistry, structure and functions, ranging from orb web construction to adhesives and cocoons. These unique materials have prompted efforts to explore potential applications of spider silk equivalent to those of silkworm silks, which have undergone 5,000 years of domestication and have a variety of uses, from textiles to biomedical materials. Recent progress in genetic engineering of spider silks and the development of new chimeric spider silks with enhanced functions and specific characteristics have advanced spider silk technologies. Further progress in yields of expressed spider-silk proteins, in the control of self-assembly processes and in the selective exploration of material applications is anticipated in the future. The unique features of spider silks, the progress and challenges in the cloning and expression of these silks, environmentally triggered silk assembly and disassembly and the formation of fibers, films and novel chimeric composite materials from genetically engineered spider silks will be reviewed.

Dynamic Culture Conditions to Generate Silk-Based Tissue-Engineered Vascular Grafts

Tissue engineering is an alternative approach for the preparation of small-diameter (<6mm) vascular grafts due to the potential to control thrombosis, anastomotic cellular hyperplasia and matrix production. This control also requires the maintenance of graft patency in vivo, appropriate mechanical properties and the formation of a functional endothelium. As a first step in generating such tissue-engineered vascular grafts (TEVGs), our objective was to develop a tissue-engineered construct that mimicked the structure of blood vessels using tubular electrospun silk fibroin scaffolds (ESFSs) with suitable mechanical properties. Human coronary artery smooth muscle cells (HCASMCs) and human aortic endothelial cells (HAECs) were sequentially seeded onto the luminal surface of the tubular scaffolds and cultivated under physiological pulsatile flow. The results demonstrated that TEVGs under dynamic flow conditions had better outcome than static culture controls in terms of cell proliferation and alignment, ECM production and cell phenotype based on transcript and protein level assessments. The metabolic activity of HCASMCs present in the TEGs indicated the advantage of dynamic flow over static culture in effective nutrient and oxygen distribution to the cells. A matrigel coating as a basement membrane mimic for ECM significantly improved endothelium coverage and retention under physiological shear forces. The results demonstrate the successful integration of vascular cells into silk electrospun tubular scaffolds as a step toward the development of a TEVG similar to native vessels in terms of vascular cell outcomes and mechanical properties.

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).

Effect of Hydration on Silk Film Material Properties

Effects of hydration on silk fibroin film properties were investigated for water-annealed and MeOH-treated samples. Hydration increased thickness by 60% for MeOH-immersed films, while water-annealed samples remained constant. MeOH-immersed films showed an 80% mass loss due to water, while water-annealed lost only 40%. O(2) permeability was higher in MeOH-immersed films with Dk values of 10(-10) (mL O(2) x cm) x (cm(-1) x s(-1) x mmHg(-1)), while those of water-annealed films reached only one fifth of this value. All films showed a decrease in Youngs modulus and increased plastic deformation by two orders of magnitude when submerged in saline solution. FT-IR showed that beta-sheet content in water-annealed films increased with increasing water vapor pressure, while MeOH-immersed films showed no change.

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.