Elise Spedden

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies

We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.

Extracellular matrix structure and nano-mechanics determine megakaryocyte function.

Cell interactions with matrices via specific receptors control many functions, with chemistry, physics, and membrane elasticity as fundamental elements of the processes involved. Little is known about how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate hemopoiesis into the bone marrow-matrix environment. To address this hypothesis, in this work we focus on the regulation of MK development by type I collagen. By atomic force microscopy analysis, we demonstrate that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human MKs through the activation of integrin-α2β1-dependent Rho-ROCK pathway and MLC-2 phosphorylation. Most importantly, this mechanism seemed to mediate MK migration, fibronectin assembly, and platelet formation. On the contrary, a decrease in mechanical tension caused by N-acetylation of lysine side chains in type I collagen completely reverted these processes by preventing fibrillogenesis.

Neuron Biomechanics Probed by Atomic Force Microscopy

Mechanical interactions play a key role in many processes associated with neuronal growth and development. Over the last few years there has been significant progress in our understanding of the role played by the substrate stiffness in neuronal growth, of the cell-substrate adhesion forces, of the generation of traction forces during axonal elongation, and of the relationships between the neuron soma elastic properties and its health. The particular capabilities of the Atomic Force Microscope (AFM), such as high spatial resolution, high degree of control over the magnitude and orientation of the applied forces, minimal sample damage, and the ability to image and interact with cells in physiologically relevant conditions make this technique particularly suitable for measuring mechanical properties of living neuronal cells. This article reviews recent advances on using the AFM for studying neuronal biomechanics, provides an overview about the state-of-the-art measurements, and suggests directions for future applications.

Neuronal alignment on asymmetric textured surfaces

Axonal growth and the formation of synaptic connections are key steps in the development of the nervous system. Here, we present experimental and theoretical results on axonal growth and interconnectivity in order to elucidate some of the basic rules that neuronal cells use for functional connections with one another. We demonstrate that a unidirectional nanotextured surface can bias axonal growth. We perform a systematic investigation of neuronal processes on asymmetric surfaces and quantify the role that biomechanical surface cues play in neuronal growth. These results represent an important step towards engineering directed axonal growth for neuro-regeneration studies.

Effects of surface asymmetry on neuronal growth.

Detailed knowledge of how the surface physical properties, such as mechanics, topography and texture influence axonal outgrowth and guidance is essential for understanding the processes that control neuron development, the formation of functional neuronal connections and nerve regeneration. Here we synthesize asymmetric surfaces with well-controlled topography and texture and perform a systematic experimental and theoretical investigation of axonal outgrowth on these substrates. We demonstrate unidirectional axonal bias imparted by the surface ratchet-based topography and quantify the topographical guidance cues that control neuronal growth. We describe the growth cone dynamics using a general stochastic model (Fokker-Planck formalism) and use this model to extract two key dynamical parameters: diffusion (cell motility) coefficient and asymmetric drift coefficient. The drift coefficient is identified with the torque caused by the asymmetric ratchet topography. We relate the observed directional bias in axonal outgrowth to cellular contact guidance behavior, which results in an increase in the cell-surface coupling with increased surface anisotropy. We also demonstrate that the disruption of cytoskeletal dynamics through application of Taxol (stabilizer of microtubules) and Blebbistatin (inhibitor of myosin II activity) greatly reduces the directional bias imparted by these asymmetric surfaces. These results provide new insight into the role played by topographical cues in neuronal growth and could lead to new methods for stimulating neuronal regeneration and the engineering of artificial neuronal tissue.

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.

A new path to platelet production through matrix sensing

Megakaryocytes (MK) in the bone marrow (BM) are immersed in a network of extracellular matrix components that regulates platelet release into the circulation. Combining biological and bioengineering approaches, we found that the activation of transient receptor potential cation channel subfamily V member 4 (TRPV4), a mechano-sensitive ion channel, is induced upon MK adhesion on softer matrices. This response promoted platelet production by triggering a cascade of events that lead to calcium influx, β1 integrin activation and internalization, and Akt phosphorylation, responses not found on stiffer matrices. Lysyl oxidase (LOX) is a physiological modulator of BM matrix stiffness via collagen crosslinking. In vivo inhibition of LOX and consequent matrix softening lead to TRPV4 activation cascade and increased platelet levels. At the same time, in vitro proplatelet formation was reduced on a recombinant enzyme-mediated stiffer collagen. These results suggest a novel mechanism by which MKs, through TRPV4, sense extracellular matrix environmental rigidity and release platelets accordingly.

Neuronal growth as diffusion in an effective potential.

Current understanding of neuronal growth is mostly qualitative, as the staggering number of physical and chemical guidance cues involved prohibit a fully quantitative description of axonal dynamics. We report on a general approach that describes axonal growth in vitro, on poly-D-lysine-coated glass substrates, as diffusion in an effective external potential, representing the collective contribution of all causal influences on the growth cone. We use this approach to obtain effective growth rules that reveal an emergent regulatory mechanism for axonal pathfinding on these substrates.