Matthew B. Applegate

Laser-based three-dimensional multiscale micropatterning of biocompatible hydrogels for customized tissue engineering scaffolds

Significance In this paper we present results on 3D, multiscale laser machining of soft, transparent biomaterials suited for cellular growth and/or implantation. We use an ultrafast laser to generate high-resolution, 3D structures within the bulk of a transparent soft-biomaterial formulation that can support cell growth and allow cells to penetrate deep within the material. The structure is created by multiphoton absorption which, thanks to the clarity of the silk gels, is possible nearly 1 cm below the surface of the material. This depth represents an ∼10× improvement over other materials. The ability to create micrometer-scale voids over such a large volume has promising applications in the biomedical field and its efficacy was demonstrated both in vitro and in vivo. Light-induced material phase transitions enable the formation of shapes and patterns from the nano- to the macroscale. From lithographic techniques that enable high-density silicon circuit integration, to laser cutting and welding, light–matter interactions are pervasive in everyday materials fabrication and transformation. These noncontact patterning techniques are ideally suited to reshape soft materials of biological relevance. We present here the use of relatively low-energy (< 2 nJ) ultrafast laser pulses to generate 2D and 3D multiscale patterns in soft silk protein hydrogels without exogenous or chemical cross-linkers. We find that high-resolution features can be generated within bulk hydrogels through nearly 1 cm of material, which is 1.5 orders of magnitude deeper than other biocompatible materials. Examples illustrating the materials, results, and the performance of the machined geometries in vitro and in vivo are presented to demonstrate the versatility of the approach.

Directed assembly of bio-inspired hierarchical materials with controlled nanofibrillar architectures

In natural systems, directed self-assembly of structural proteins produces complex, hierarchical materials that exhibit a unique combination of mechanical, chemical and transport properties. This controlled process covers dimensions ranging from the nano- to the macroscale. Such materials are desirable to synthesize integrated and adaptive materials and systems. We describe a bio-inspired process to generate hierarchically defined structures with multiscale morphology by using regenerated silk fibroin. The combination of protein self-assembly and microscale mechanical constraints is used to form oriented, porous nanofibrillar networks within predesigned macroscopic structures. This approach allows us to predefine the mechanical and physical properties of these materials, achieved by the definition of gradients in nano- to macroscale order. We fabricate centimetre-scale material geometries including anchors, cables, lattices and webs, as well as functional materials with structure-dependent strength and anisotropic thermal transport. Finally, multiple three-dimensional geometries and doped nanofibrillar constructs are presented to illustrate the facile integration of synthetic and natural additives to form functional, interactive, hierarchical networks.

Biocompatible silk step-index optical waveguides

Biocompatible optical waveguides were constructed entirely of silk fibroin. A silk film (n=1.54) was encapsulated within a silk hydrogel (n=1.34) to form a robust and biocompatible waveguide. Such waveguides were made using only biologically and environmentally friendly materials without the use of harsh solvents. Light was coupled into the silk waveguides by direct incorporation of a glass optical fiber. These waveguides are extremely flexible, and strong enough to survive handling and manipulation. Cutback measurements showed propagation losses of approximately 2 dB/cm. The silk waveguides were found to be capable of guiding light through biological tissue.

Photocrosslinking of Silk Fibroin Using Riboflavin for Ocular Prostheses

A novel method to photocrosslink silk fibroin protein is reported, using riboflavin (vitamin B2) as a photoinitiator and the mechanism of crosslinking is determined. Exposure of riboflavin-doped liquid silk solution to light results in the formation of a transparent, elastic hydrogel. Several applications for this new material are investigated including corneal reshaping to restore visual acuity and photolithography.

Silk: A Different Kind of “Fiber Optics”

Like a moth emerging from a cocoon, silk-based devices—optically versatile and friendly to biological systems—are poised to take flight in a range of new, innovative applications.

Determination of multiphoton absorption of silk fibroin using the Z-scan technique

The multiphoton absorption of silk fibroin at 810 nm was determined by open-aperture Z-scan. Three-photon absorption was confirmed at this wavelength and the three-photon cross section of silk fibroin was measured. Silk fibroin of varying molecular weight was tested and the cross section was found to increase exponentially with increasing molecular weight. The confirmation of a relatively large three-photon absorption cross section in silk will help lay the groundwork for future investigation of direct laser writing of three-dimensional structures within a silk matrix.

Evaluation of Silk Inverse Opals for “Smart” Tissue Culture

Visually tracking the subtle aspects of biological systems in real time during tissue culture remains challenging. Herein, we demonstrate the use of bioactive, cytocompatible, and biodegradable inverse opals from silk as a multifunctional substrate to transduce both the optical information and cells during tissue culture. We show that these substrates can visually track substrate degradation in various proteases during tissue digestion and protein deposition during the growth of mesenchymal stem cells. Uniquely, these substrates can be integrated in multiple steps of tissue culture for simple-to-use, visual, and quantitative detectors of bioactivity. These substrates can also be doped, demonstrated here with gold nanoparticles, to allow additional control of cell functions.

Stomatopods detect and assess achromatic cues in contests

Lay SummaryStomatopods (mantis shrimp) may disclose their fighting ability to opponents when they display a colored patch, the meral spot. We used an ultrafast laser to increase meral spot brightness of a Caribbean stomatopod species. Fights over ownership of a refuge were shorter and more active when the resident stomatopod had a brightened meral spot. This suggests that stomatopods are more willing to engage in contests when their opponents have brighter meral spots.

A simple model of multiphoton micromachining in silk hydrogels

High resolution three-dimensional voids can be directly written into transparent silk fibroin hydrogels using ultrashort pulses of near-infrared (NIR) light. Here, we propose a simple finite-element model that can be used to predict the size and shape of individual features under various exposure conditions. We compare predicted and measured feature volumes for a wide range of parameters and use the model to determine optimum conditions for maximum material removal. The simplicity of the model implies that the mechanism of multiphoton induced void creation in silk is due to direct absorption of light energy rather than diffusion of heat or other photoproducts, and confirms that multiphoton absorption of NIR light in silk is purely a 3-photon process.

Silk Fibroin: Photocrosslinking of Silk Fibroin Using Riboflavin for Ocular Prostheses (Adv. Mater. 12/2016)

Dissolved silk protein mixed with riboflavin can be crosslinked to form an elastic hydrogel in the presence of blue/violet light. Here, a photomask is used by F. G. Omenetto and co-workers, as described on page 2417, to illuminate the solution, and the unpolymerized silk is rinsed away. These gels have tremendous potential to be used as corneal prostheses.