Kelly A. Burke

Dynamic cell behavior on shape memory polymer substrates

Cell culture substrates of defined topography have emerged as powerful tools with which to investigate cell mechanobiology, but current technologies only allow passive control of substrate properties. Here we present a thermo-responsive cell culture system that uses shape memory polymer (SMP) substrates that are programmed to change surface topography during cell culture. Our hypothesis was that a shape-memory-activated change in substrate topography could be used to control cell behavior. To test this hypothesis, we embossed an initially flat SMP substrate to produce a temporary topography of parallel micron-scale grooves. After plating cells on the substrate, we triggered shape memory activation using a change in temperature tailored to be compatible with mammalian cell culture, thereby causing topographic transformation back to the original flat surface. We found that the programmed erasure of substrate topography caused a decrease in cell alignment as evidenced by an increase in angular dispersion with corresponding remodeling of the actin cytoskeleton. Cell viability remained greater than 95% before and after topography change and temperature increase. These results demonstrate control of cell behavior through shape-memory-activated topographic changes and introduce the use of active cell culture SMP substrates for investigation of mechanotransduction, cell biomechanical function, and cell soft-matter physics.

Soft shape memory in main-chain liquid crystalline elastomers

The field of shape memory polymers (SMPs) has been dominated by polymeric network systems whose fixing mechanism is based on crystallization or vitrification of the constituent chains, rendering such systems stiff in comparison to elastomers, gels, and living tissues. In this report, we describe the synthesis and characterization of liquid crystalline elastomers that exhibit both bulk and surface shape memory effects with compositionally dependent transition temperatures that determine the shape fixing and shape recovery critical temperatures. Main-chain, segmented liquid crystalline elastomers were synthesized using hydrosilylation linking of poly(dimethylsiloxane) oligomers with mesogenic dienes of two compositions and a tetrafunctional crosslinker. Calorimetric and dynamic mechanical analyses revealed two composition-determined thermal transitions for the LCEs, including a glass transition event at low temperatures (33 °C < Tg < 48 °C) and a first-order isotropization transition at higher temperatures (57 °C < Ti < 71 °C), each increasing with an increase in the concentration of the more slender, unsubstituted mesogenic diene, 5H. Despite the existence of a glass transition event, the materials remain soft at low temperature, a finding explained by vitrification of only the mesogen-rich layers within the smectic phase. Shape memory behavior was evaluated quantitatively and revealed excellent shape fixing and shape recovery values, generally in excess of 98%, with the recovery temperature depending on composition in a manner determined by the LCE phase behavior, particularly Tg. The temperature-dependent kinetics of shape memory were analyzed for a selected LCE composition, revealing exponential time dependence with rate constants that depended on temperature in an Arrhenius manner. Finally, the softness of the LCE SMPs was exploited to fix an embossed, micron-scale pattern on the surface and then recover the equilibrium flat state quite completely. We envision application of this surface shape memory phenomenon in the areas of soft lithography, especially microcontact printing and microfluidics.

Multifunctional silk-heparin biomaterials for vascular tissue engineering applications.

Over the past 30 years, silk has been proposed for numerous biomedical applications that go beyond its traditional use as a suture material. Silk sutures are well tolerated in humans, but the use of silk for vascular engineering applications still requires extensive biocompatibility testing. Some studies have indicated a need to modify silk to yield a hemocompatible surface. This study examined the potential of low molecular weight heparin as a material for refining silk properties by acting as a carrier for vascular endothelial growth factor (VEGF) and improving silk hemocompatibility. Heparinized silk showed a controlled VEGF release over 6 days; the released VEGF was bioactive and supported the growth of human endothelial cells. Silk samples were then assessed using a humanized hemocompatibility system that employs whole blood and endothelial cells. The overall thrombogenic response for silk was very low and similar to the clinical reference material polytetrafluoroethylene. Despite an initial inflammatory response to silk, apparent as complement and leukocyte activation, the endothelium was maintained in a resting, anticoagulant state. The low thrombogenic response and the ability to control VEGF release support the further development of silk for vascular applications.

Lyophilized Silk Sponges: A Versatile Biomaterial Platform for Soft Tissue Engineering

We present a silk biomaterial platform with highly tunable mechanical and degradation properties for engineering and regeneration of soft tissues such as, skin, adipose, and neural tissue, with elasticity properties in the kilopascal range. Lyophilized silk sponges were prepared under different process conditions and the effect of silk molecular weight, concentration and crystallinity on 3D scaffold formation, structural integrity, morphology, mechanical and degradation properties, and cell interactions in vitro and in vivo were studied. Tuning the molecular weight distribution (via degumming time) of silk allowed the formation of stable, highly porous, 3D scaffolds that held form with silk concentrations as low as 0.5% wt/v. Mechanical properties were a function of silk concentration and scaffold degradation was driven by beta-sheet content. Lyophilized silk sponges supported the adhesion of mesenchymal stem cells throughout 3D scaffolds, cell proliferation in vitro, and cell infiltration and scaffold remodeling when implanted subcutaneously in vivo.

The Effect of Sterilization on Silk Fibroin Biomaterial Properties

The effects of common sterilization techniques on the physical and biological properties of lyophilized silk fibroin sponges are described. Sterile silk fibroin sponges were cast using a pre-sterilized silk fibroin solution under aseptic conditions or post-sterilized via autoclaving, γ radiation, dry heat, exposure to ethylene oxide, or hydrogen peroxide gas plasma. Low average molecular weight and low concentration silk fibroin solutions could be sterilized via autoclaving or filtration without significant loses of protein. However, autoclaving reduced the molecular weight distribution of the silk fibroin protein solution, and silk fibroin sponges cast from autoclaved silk fibroin were significantly stiffer compared to sponges cast from unsterilized or filtered silk fibroin. When silk fibroin sponges were sterilized post-casting, autoclaving increased scaffold stiffness, while decreasing scaffold degradation rate in vitro. In contrast, γ irradiation accelerated scaffold degradation rate. Exposure to ethylene oxide significantly decreased cell proliferation rate on silk fibroin sponges, which was rescued by leaching ethylene oxide into PBS prior to cell seeding.

Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins.

Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form β-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants.

Long term perfusion system supporting adipogenesis

Adipose tissue engineered models are needed to enhance our understanding of disease mechanisms and for soft tissue regenerative strategies. Perfusion systems generate more physiologically relevant and sustainable adipose tissue models, however adipocytes have unique properties that make culturing them in a perfusion environment challenging. In this paper we describe the methods involved in the development of two perfusion culture systems (2D and 3D) to test their applicability for long term in vitro adipogenic cultures. It was hypothesized that a silk protein biomaterial scaffold would provide a 3D framework, in combination with perfusion flow, to generate a more physiologically relevant sustainable adipose tissue engineered model than 2D cell culture. Consistent with other studies evaluating 2D and 3D culture systems for adipogenesis we found that both systems successfully model adipogenesis, however 3D culture systems were more robust, providing the mechanical structure required to contain the large, fragile adipocytes that were lost in 2D perfused culture systems. 3D perfusion also stimulated greater lipogenesis and lipolysis and resulted in decreased secretion of LDH compared to 2D perfusion. Regardless of culture configuration (2D or 3D) greater glycerol was secreted with the increased nutritional supply provided by perfusion of fresh media. These results are promising for adipose tissue engineering applications including long term cultures for studying disease mechanisms and regenerative approaches, where both acute (days to weeks) and chronic (weeks to months) cultivation are critical for useful insight.

Electrodeposited gels prepared from protein alloys

AIM Silk-tropoelastin alloys, composed of recombinant human tropoelastin and regenerated Bombyx mori silk fibroin, are an emerging, versatile class of biomaterials endowed with tunable combinations of physical and biological properties. Electrodeposition of these alloys provides a programmable means to assemble functional gels with both spatial and temporal controllability. MATERIALS & METHODS Tropoelastin-modified silk was prepared by enzymatic coupling between tyrosine residues. Hydrogel coatings were electrodeposited using two wire electrodes. RESULTS & DISCUSSION Mechanical characterization and in vitro cell culture revealed enhanced adhesive capability and cellular response of these alloy gels as compared with electrogelled silk alone. CONCLUSION These electro-depositable silk-tropoelastin alloys constitute a suitable coating material for nanoparticle-based drug carriers and offer a novel opportunity for on-demand encapsulation/release of nanomedicine.

Silk-Based Injectable Biomaterial as an Alternative to Cervical Cerclage An In Vitro Study

Objective: New therapies to prevent preterm birth are needed. Our objective was to study an injectable biomaterial for human cervical tissue as an alternative to cervical cerclage. Study Design: Human cervical tissue specimens were obtained from premenopausal gynecological hysterectomies for benign indications. A 3-part biomaterial was formulated, consisting of silk protein solution blended with a 2-part polyethylene glycol gelation system. The solutions were injected into cervical tissue and the tissue was evaluated for mechanical properties, swelling, cytocompatibility, and histology. Results: The stiffness of cervical tissue more than doubled after injection (P = .02). Swelling properties of injected tissue were no different than native tissue controls. Cervical fibroblasts remained viable for at least 48 hours when cultured on the biomaterial. Conclusions: We report a silk-based, biocompatible, injectable biomaterial that increased the stiffness of cervical tissue compared to uninjected controls. Animal studies are needed to assess this biomaterial in vivo.

Evaluation of the Spectral Response of Functionalized Silk Inverse Opals as Colorimetric Immunosensors.

Regenerated silk fibroin is a high molecular weight protein obtained by purifying the cocoons of the domesticated silkworm, Bombyx mori. This report exploits the aqueous processing and tunable β sheet secondary structure of regenerated silk to produce nanostructures (i.e., inverse opals) that can be used as colorimetric immunosensors. Such sensors would enable direct detection of antigens by changes in reflectance spectra induced by binding events within the nanostructure. Silk inverse opals were prepared by solution casting and annealing in a humidified atmosphere to render the silk insoluble. Next, antigen sensing capabilities were imparted to silk through a three step synthesis: coupling of avidin to silk surfaces, coupling of biotin to antibodies, and lastly antibody attachment to silk through avidin-biotin interactions. Varying the antibody enables detection of different antigens, as demonstrated using different protein antigens: antibodies, red fluorescent protein, and the beta subunit of cholera toxin. Antigen binding to sensors induces a red shift in the opal reflectance spectra, while sensors not exposed to antigen showed either no shift or a slight blue shift. This work constitutes a first step for the design of biopolymer-based optical systems that could directly detect antigens using commercially available reagents and environmentally friendly chemistries.