Symposium SB06—Bringing Mechanobiology to Materials—From Molecular Understanding to Biological Design

The actin cytoskeleton plays a fundamental role in giving cells their shape and mediating interactions between the cell and the extracellular environment. Specifically, actin filaments transmit the forces generated in the cytoskeleton by myosin motors to the extracellular matrix. It has been shown previously that proteins that contain multiple LIM domains (e.g. zyxin, paxillin, etc…) specifically recognize sites of strain in the cytoskeleton and localize to stress fiber tears. Using these proteins as markers in conjunction with an optogenetic approach to induce local contractions in the cytoskeleton, we have recently shown that strain in actin filaments is distributed non-uniformly. We find that in addition to random failures in the network, strain in the actin cytoskeleton preferentially concentrates at the interface between focal adhesions and stress fibers. We also surprisingly find that these proteins localize to sites of potential compression. Here we use the LIM family protein testin to explore these mechanisms of mechanosensing behavior. Using a combination of laser ablations and optogenetics, we find that full length testin localizes to the cytosol and is not mechanosensitive. Truncations of the protein that include just its three LIM domains (LIM1-3), however, localize to focal adhesions and sites of strain. Stress fibers that are fully severed show no recruitment of the LIM1-3 construct, suggesting that LIM domain proteins recognize a change in the conformation of actin, and not just free barbed ends. Further truncation constructs reveal that the first LIM domain is required for mechanosensitivity. Finally, using various phosphomutants, we find that we can drive testin to localize at adhesions, but that only a subset of them retain their mechanosensitivity. Together these results indicate a potential role for phosphorylation in regulating the conformation of testin, thereby controlling its mechanosensitivity and its ability to recognize local strain in actin filaments.

Tandem repeat motifs are ubiquitous across the central dogma of biology and regulate a variety of physiological processes including gene regulation, membrane functionalization etc. For example, in eukaryotic cells, the ends of linear chromosomes are capped by 50-200 nt long telomeric DNA, consisting of tandem hexanucleotide guanine (G)-rich repeats. Four of such telomeric, G-rich motifs can spontaneously associate and fold into thermodynamically stable structures, known as G-Quadruplexes (GQs). Interplay between mechanical forces and structural transformations is fundamental to several biological processes, such as, transcription, replication, cellular locomotion etc. We integrated the unique abilities of single molecule fluorescence and optical traps to demonstrate unprecedented heterogeneity in GQs, with at least six interconvertible species which differ in mechanical responses, but are largely resistant to unfolding by cellular motor proteins associated with transcription, replication etc. Additionally, our studies revealed direct mechanical regulation of a GQ-core in presence of unassociated G-rich motifs. The extreme conformational and mechanical diversity of GQs may induce differential protein binding in cells and serve as cornerstones for engineering novel anti-cancer therapeutics. The conformational versatility of G repeats have been harnessed for development of molecular tools called “light-up” aptamers used for visualization and localization of RNA in cells. We evaluated the mechanical stabilities of two classes of such RNA aptamers, known as Mango and Spinach. Our results illustrated that despite a common GQ core, the aptamers have characteristic mechanical signature and in general, with respect to Mango, Spinach is mechanically weaker and susceptible to unraveling via helicases and polymerases. Thus, the RNA aptamers can be leveraged as molecular platforms for simultaneous visualization and prediction of forces involved in transcription. We next extended our assay to demonstrate linear spring-like mechanical response of a malarial circumsporozoite protein (CSP). CSP being a surface protein, this facilitates gliding motility of the sporozoites in the host. Moreover, the repeat peptides can be used for engineering cellular tension sensors which are sensitive to forces between 1 and > 30 pN.

Biomaterial scaffolds that are designed to incorporate dynamic, spatiotemporal information have the potential to interface with cells and tissues to direct behavior. Here, I will describe a bioinspired, programmable nanotechnology–based platform that harnesses cellular traction forces to activate growth factors, eliminating the need for exogenous triggers (e.g., light), spatially diffuse triggers (e.g., enzymes, pH changes), or passive activation (e.g., hydrolysis)¹. Flexible aptamer technology is used to create modular, synthetic mimics of the Large Latent Complex that restrains transforming growth factor–β1 (TGF–β1). I will show that this nanotechnology–based approach works with multiple growth factors, integrates with both 2D and 3D substrates and scaffolds, and enables fundamentally new capabilities such as selective activation of growth factors by differing cell types (e.g., activation by smooth muscle cells but not fibroblasts).

1. Stejskalová, A., Oliva, N., England, F. J., Almquist, B. D., Adv. Mater. 2019, 31, 1806380. DOI: 10.1002/adma.201806380

A major unanswered question in biology is how mechanical forces are generated, detected and transduced by cells to impact biochemical and genetic programs. Our work is aimed at uncovering the mechanical principles at play in cells and tissues using novel molecular, imaging and bioengineering tools. Here we present insights gleaned from non-invasive approaches to measure and manipulate mechanical cues in native cellular conditions. We find that the mechanically-activated ion channel Piezo1 transduces cell-generated traction forces to regulate lineage choice of neural stem cells. We show that actomyosin-based cellular traction forces generate spatially-restricted Piezo1 Ca²⁺ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their Ca²⁺ flicker activity is enriched in regions proximal to force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca²⁺ flickers allow spatial segregation of mechanotransduction events, and that mobility allows channel molecules to efficiently respond to local mechanical stimuli.

The key influence of the microenvironment on cell behavior and fate is increasingly recognized. It follows that new techniques to control the 3D environment around cells are essential to understand the processes by which cells probe and respond to the cues received by their microniches. Here, we present an approach that allows transforming microwells into artificial microniches where the chemical coating, the rheological properties and the topographical properties can be differentially controlled on the top, sides and bottom of the wells and assembled in a combinatorial way. This technique is also compatible with high and super resolution imaging that allows probing the dynamics of cell cytoskeleton and regulatory proteins with unprecedented resolution down to the single molecule level in 3D. We exemplify how these bone fide artificial microniches can be used to induce full apico-basal polarization of single epithelial cells as well as to control intercellular stresses driving the anisotropic growth of intercellular lumens. We will detail our recent studies on the role of mechanical forces in the development of bile canaliculi in liver and explain our minimal organ approach.

Regulating the emergence of leading individuals is a central problem to collectively migrating biological entities. For example, leaders in the mobile animal groups arise through collective decision making of the followers. However, the fundamental control of leader selection remains unclear in the physiologically relevant collective migration of epithelial cells. Here we present that the selective emergence of leader cells at the epithelial wound-margin depends on the dynamics of the follower cells and is spatially limited by the length-scale of collective force transduction. Owing to the dynamic heterogeneity of the monolayer, cells behind the prospective leaders manifest locally increased traction and monolayer stresses much before these leaders display any phenotypic traits. Once formed, the territory of a leader can extend only to the length up-to which cells can pull on their neighbors. These findings provide a novel mechanobiological-insight into the hierarchy in cell collectives during epithelial wound healing.
Lit.: A molecular mechanotransduction pathway regulates collective migration of epithelial cells; Nature Cell Biology 2015; DOI 10.1038/NCB3115
Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics; Nature Materials 2018; DOI 10.1038/NMAT5005
Mechanical interactions among followers determine the emergence of leaders in migrating epithelial cell collectives, Nature Communication 2018; DOI 10.1038/S41467-018-05927-6

Crosslinked semi-flexible polymer networks are ubiquitous to both the internal cytoskeleton and the extracellular matrix. The viscoelasticity of these networks is therefore presumed to dominate tissue mechanics. However, the responses of soft tissues and semiflexible polymer gels to uniaxial loading differ from each other in many respects. Tissues stiffen in compression but not in extension, whereas semiflexible polymer networks soften in compression and stiffen in extension. In shear deformation, semiflexible polymer gels stiffen with increasing strain, but tissues do not. Tissue rheology emerges from an interplay between strain-stiffening polymer networks and volume-conserving cells within them. Polymer networks that soften in compression but stiffen in extension, can be converted to materials that stiffen in compression, but not in extension, by including within the network either cells or inert particles to restrict the relaxation modes of the fibrous networks that surround them

Integrins are heterodimeric transmembrane adhesion proteins that link the cytoskeleton to the extracellular matrix (ECM), and hence play a central role in the construction of complex, multicellular tissues. Although integrins are required for both cellular traction generation and for sensing mechanical cues such as substrate rigidity, the magnitude of the forces born by integrin heterodimers was unclear. We used FRET-based molecular tension sensors to determine the magnitude and origins of the forces experienced by individual integrins in living cells. We found that this distribution was highly skewed, with the majority of integrins bearing loads of ~2 pN, while a small subpopulation experienced forces >11 pN. Further experiments revealed that this distribution was controlled in a modular manner: integrin heterodimer usage controlled the number and stability of cellular connections to the ECM, while the proteins that link integrins to the cytoskeleton regulated the distribution of loads borne by individual integrin complexes. These and other observations support a general model for how cells create the regulated and dynamic adhesion complexes that are a defining feature of multicellular life.

A Multiscale approach (similar to the FE2 method) is developed to investigate the mechanics of the facet capsule ligament (FCL). The multiscale analysis technique allows for discrete micromechanical representations of the collagen tissue, coupled to a macroscale model which can use geometries obtained from segmented image stacks of biologically relevant domains. These complex biological geometries are coupled with other data sets, such as initial network orientation fields, to obtain models which can output biologically relevant mechanics information such as network orientation trajectories and local stresses not accessible by direct measurement.
One of the challenges with the multiscale analysis is choosing an adequate sized representative volume element (RVE) at the microscale. This talk will discuss size effects on the RVE fiber network, and tools which can be used to ameliorate the large RVE sizes which are needed.

The mechanical properties of cells, such as cell stiffness, are important biomarkers in both clinical applications (e.g. detecting circulating tumor cells) and fundamental biological studies. Despite the importance of cell stiffness in mechanobiology, there are relatively few facile techniques that have the temporal resolution for basic research, or the throughput required for clinical applications. Limitations of current technologies is that they rely on optical equipment, which can be bulky and expensive, or indirect measurements of force, such as calcium labeling. Here, we describe a new technique for monitoring the properties of particles and cells within an elastic, microfluidic channel. As a particle flows inside the channel, it deforms the walls in a characteristic manner. This fluid-induced deformation (i.e., elastohydrodynamics) is measured by using ultra-sensitive piezoresistive strain sensors. By transducing the wall deformation into voltage, we are able to monitor analytes within the channel without the use of any optical equipment. We discuss our validation of using elastohydrodynamic phenomena to monitor analytes and the future directions for the platform.

A number of cell functions rely on the formation of macromolecular platforms in the plasma membrane. While the functional role of these assemblies has been intensively investigated over the years, little is known about the mechanisms underlying their formation. In this presentation, several possible pathways will be discussed, including the role of the membrane elasticity, composition fluctuations, and the interactions with the cytoskeleton. Furthermore, cooperative attachments of proteins with different length, flexibility and affinities will be explored, allowing the development of a hypothesis regarding the simultaneous repellent and catalytic roles of the glycocalyx in the formation of membrane-associated macromolecular assemblies.

Cell fate decisions are influenced by many cues, which together constitute the cell microenvironment. One critical regulator is the extracellular matrix (ECM), which varies not only in composition, but also in physical properties such as stiffness. The impact of matrix stiffness on cell spreading and differentiation has been studied intensively on 2D surfaces using synthetic hydrogels, but very little is known about stiffness sensing within more complex 3D matrices.
Here, a major hurdle is to isolate the role of ECM stiffness from other matrix properties, in particular degradability. If cells are fully encapsulated, changes in bulk stiffness also influence the amount of matrix crosslinks that a cell has to cleave in order to spread and interact with its surroundings, impacting cell shape and function. Here, we have developed a sugar-based hydrogel system that offers independent control over mechanical properties, adhesive ligand density and matrix degradation rates. The material can be processed under physiologic conditions rendering it suitable for cell encapsulation. Matrix metalloproteinase (MMP) cleavable peptides as crosslinking units enable cellular matrix remodeling and variation of their sequence gives access to a range of degradation rates. Using this system, we study the impact of matrix stiffness and degradability on cell spreading, mesenchymal stem cell differentiation and angiogenic sprouting. In particular, we demonstrate that matrix degradability, mechanics and adhesivity jointly control the multicellularity of 3D endothelial cell invasion.

A technique for tracking the longitudinal rotation of nanoscale objects has the potential to serve as a powerful experimental tool to researchers across a variety of fields. Longitudinal rotation (i.e. rolling about a long axis) is a subtle movement that requires less free space than other types of movements such as translation and lateral rotation. Therefore, longitudinal rotation is particularly sensitive to small forces and does not significantly alter the local environment. This is especially true for long cylindrical objects since they require no additional space to rotate along their long axis. As such, tracking the longitudinal rotation of nanowires can serve as a sensitive and minimally perturbing method to probe forces in nanoscale environments. In this work, we present a simple and versatile method of tracking longitudinal rotation using a kinked silicon nanowire. By imaging the changes in the length of the kinked nanowire’s arm projected on the detection plane, we can measure the nanowire’s longitudinal rotation. To precisely measure the projected length of the nanowire’s arm, we developed a sub-pixel particle detection algorithm. To improve efficiency in the data processing pipeline, we incorporated a statistical procedure (principal component analysis) to automate the detection process. We applied this method to study the longitudinal rotation of nanowires in the presence and absence of endothelial cells. We found that nanowire rotational dynamics were significantly affected by the nanowire’s environment. While the free-floating nanowires underwent consistent and fast sub-diffusive rotation, the cell-interacting nanowires exhibited a greater variety of behaviors, including slow sub-diffusive random rotation and super-diffusive unidirectional rotation. The nanowires underwent this type of unidirectional rotation when the cell actively extended its boundary to cover the nanowire or when the cell contracted significantly. Our ability to detect cellular behavior via conspicuous changes in the nanowire’s longitudinal rotation showcases the sensitivity of this technique. Furthermore, the detection algorithm and the statistical method developed for this work can be applied to tracking the movements of other nanoscale objects with various geometries.

Mechanical and chemical signals from the extracellular milieu are crucial to cell differentiation, morphology, and function. New materials are needed that can send precise, multivariate cues on cellular length- and timescales. For this purpose we are synthesizing novel near-IR actuators and incorporating them in new composite materials. The resultant biocompatible structures can change modulus and 3D morphology in response to tissue-penetrating wavelengths of light on multiple length scales, and we are now incorporating protein and drug functionalities onto the materials' surface. We are currently tailoring these composite materials for use as venous stents and stem cell differentiation scaffolds.

A long recognized tenet of biological systems is that structure gives rise to function. Mechanical force in contrast has emerged only recently as a critical dimension that links form and function, providing the central effector to sculpt the body plan during morphogenesis, as well as a mechanism for cells to sense and respond to local changes in tissue structure and mechanics. Despite the realization that forces, form, and function permeate all living systems, we as a research community sorely lack methods to control the mechanics of the environment, the spatial organization of cells, or the architecture of cell-matrix and cell-cell interfaces, which collectively define the boundary conditions for how forces are transmitted into cells. Here, I will describe our efforts to design and build physical microenvironments that explicitly manipulate and monitor the physical (structural and mechanical) interactions between cells and their surroundings. Using these approaches, we demonstrate that mechanical forces generated either internally by the cytoskeleton or externally regulate cell, matrix, and tissue structure, signaling, and function, and begun to use these insights to build in vitro organotypic models of the cardiovascular system that mimic native tissue functions. I will use our studies to illustrate how deeper insights into the structure-function links are critical to our ability to engineer stem cells to recapitulate differentiated function, and how engineered systems ultimately could have a major impact on biomedical research.

Biohybrid soft robotics is an emerging field of research that offers new concepts for the current challenges of soft robotics. This includes the ability to self-heal, soft touches and miniaturization possibilities, as well as situational and autonomous adaptations. Here, living beings have an unprecedented variety of possibilities to manipulate and interact with objects and the combination of elements from soft robotics with living cells opens up new opportunities. As an example, skeletal muscle cells can be used to actuate biohybrid, hydrogel-based actuators but they require electrical stimulation in order to contract. However, most currently used biomaterials, e.g. in cardiac tissue engineering, lack electrical conductivity and appropriate mechanical properties. Both parameters are important for regulating skeletal muscle cell behavior. In this work, we present conductive and structured hydrogel scaffolds where the conductivity is achieved by incorporating reduced graphene oxide or carbon nanotubes in the material. Further, the stiffness of the scaffolds can be tailored to match the stiffness of native muscle tissue. MTT-assays have shown that the scaffolds have no negative impact on the cell viability. Apart from this, the samples are biofunctionalized with collagen to enhance cell adhesion. The presented work illustrates the potential of conductive and structured hydrogels as a first step towards the development of a biohybrid, hydrogel-based actuator.

Microtubule (MT)-kinesin gliding systems are known as the biomolecules to cause cell motility cytokinesis and cellular transport [1]. Previously, we reported the local detaching control of a living cell from the SiN membrane and investigated spatio-temporal distributions of intracellular elastic strain as mechanical strain microscopy by using a fine virtual cathode (VC) on the VC display [2]. In addition, to examine the biomolecules control using the VC display [3], we reported the pause of the target gliding MTs [4]. In the same way as the former report, we considered that the pause mechanics of the gliding MTs are caused by detachments between the biomolecules (= MTs and kinesins) bindings. In this report, we examined to cause the detachments between the target MT and kinesins on the VC. As the results, some target MTs were confirmed to move to the directions different from the each MTs axial direction by applying the VC. These results showed that the MTs detached from the kinesins and showed the detach between the biomolecules can be controlled on the VC. In summary, the detachments of the biomolecules binding forces include the mechanical function could provide the novel tool to molecule mechanobiology.

References
[1] H. Carlotte, et al., Current Biology. 25, R677-R691 (2015).
[2] T. Hoshino, et al., Sensors and Actuators B: Chemical. 236, 659-667 (2016).
[3] T. Hoshino, et al., Biochemical and Biophysical Research Communications. 432, 345-349 (2013).
[4] K. Hatazawa, et al., Biochemical and Biophysical Research Communications. 514, 821-825 (2019).

Cells of all types react in response to changes in their mechanical environment typically to return their surroundings to homeostatic conditions. In cancer cells this response is varied in ways which also vary based on the cancer type or even the phenotype within one type of cancer. These are some of the reasons why mechanical stiffness and the extracellular matrix (ECM) play key roles in tumor formation/progression and cancer cell phenotype. Cancer cells originating from different tissues, possessing different stiffnesses, will respond to their environment differently, which could yield important information in better understanding cancer cell physiology. Gelatin methacrylate (GelMA) is a UV-crosslinkable hydrogel that has been shown to be effective in the 2D and 3D culturing of cells in a wide variety of applications including cancer models. Cells can easily bind to GelMA 2D surfaces and within 3D structures, and can proliferate, elongate, and remodel their surroundings due to the presence of natural binding and enzymatic degradation sites in the gelatin backbone. GelMA is a highly elastic material with tunable mechanical stiffness through variation of gel concentration and degree of methacrylation. It has been established that normal breast tissue has a compressive modulus of roughly 200 Pa, whereas precancerous regions have a modulus of roughly 600 Pa and cancerous regions can be as high as 1-2 kPa. Analogous studies are underway to create similar stiffness ranges for other cancer types such as prostate, as well as to control stem cell differentiation via similar principles. The major aims in these studies were to create robust GelMA hydrogels in the 3 mechanical ranges (normal, precancerous, tumor) for breast and other tissues, and once validated to investigate the differences in behavior, morphology and gene expression of cancer cells encapsulated in these hydrogels. Validation studies with NIH 3T3 cells determined the parameters and conditions (exposure time, lamp power, photoinitiator concentration) where cells could be 3D encapsulated with a high degree of early viability that was maintained for 1-2+ weeks. Preliminary results suggest evidence that different cancer cell types react differently to varied levels of mechanical stiffness. Further experiments will aim to further elucidate these effects and the factors driving these phenomena through RNA isolation and analysis using NextGen sequencing.

Rhythmic movements in biology are abundant, across a number of length scales from the beating of a heart to digestion, locomotion, and breathing. These simple yet vital functions are controlled by patterns of signals created by a series of neurons, known as central pattern generators. Neurons within these central pattern generators are capable of robustly maintaining these complex oscillations without the benefit of inputs from higher brain functions, yet they are also able to modify their oscillatory behavior in response to stimuli.
The Belousov-Zhabotinsky reaction is a fluid which acts as a chemical oscillator, which obeys reaction-diffusion dynamics. This chemical oscillator periodically oxidizes and reduces, causing a periodically spike in voltage. Using microfluidic techniques, we can embed pockets of this chemical oscillator within a gel network which allows us to control the connections between these pockets. It has been shown that using this technique, we can create artificial central pattern generators.
In this work, we will investigate how individual artificial neurons respond to changes in local environment in order to examine how these artificial neurons change their behavior in response to changes in stimuli. Additionally, we will examine the communication between pairs of these artificial neurons in order to understand how the interactions give rise to robust complex patterns.

*We acknowledge financial support from the U. S. Army Research Laboratory and the U. S.
Army Research Office under contract/ grant number W911NF-16-1-0094, and the microfluidics
facility of the NSF MRSEC DMR-1420382.

A large proportion of patients with Alzheimer's disease or Parkinson's disease may have symptoms of both diseases at the same time. Such diseases overlapping Alzheimer’s disease and Parkinson’s disease have been attempted to explain with the hetero-oligomer hypothesis associated with Amyloid-beta (Aβ) peptides and Alpha-synuclein (α-syn) peptides. However, neither presence nor structure of the hetero-oligomers have not been clarified.
Herein, we have investigated surface structure of protein oligomers at the single peptide level. We employed Atomic Force Microscopy (AFM) with a liquid cell to characterize the hetero-oligomers generated in vitro. In particular, a probe tethering an antibody recognizing the N-terminal of Aβ and another probe tethering N-terminal of α-syn were prepared, and one-to-one interaction was realized through utilizing dendron coating of the probes before the conjugation with the antibodies.^{1, 2} High resolution force maps of the oligomers were obtained with thus-prepared AFM probes.
It revealed that specific unbinding events with respect to two different antibodies were observed within an oligomer, and such coexistence of specific pixels was persistent for all sizes under investigation. Because homo-oligomers were not observed at all, it can be said that formation of the hetero-oligomers is strongly favored. It is intriguing to note that the percentage of positive pixels for the hetero-oligomer is higher than that for the homo-oligomer, suggesting a different mode of aggregation for the hetero-peptide oligomerization. We suggest that this approach is useful for understanding the overlap of neurodegenerative brain disorders.

References
1. Y. J. Jung, B. J. Hong, W. Zhang, S. J. Tendler, P. M. Williams, S. Allen, J. W. Park, J. Am. Chem. Soc. 129, 9349 (2007).
2. D. H. Kim, J.-E. Lee, Z.Y. Xu, K. R. Geem, Y. Kwon, J. W. Park, I. Hwang, Nat. Commun. 6, 6843 (2015).

Acknowledgement
This work is supported by the KGPF (Korea Global Ph.D. Fellows) from NRF (National Research Foundation of Korea).

Significant progress has been made in the de novo design of protein-based biomaterials at the atomic-level.¹ But, to date, there has not been a significant advance in the de novo design of macroscopic biomaterials that match the extraordinary mechanical properties of some natural materials such as silk. Self-organising materials are commonly observed in biological systems and the ability to control this self-organising behaviour in optimally designed systems opens up new approaches for the creation of advanced materials. Synthetic biology provides the tool to build artificial proteins that can be ideal precursors for such advanced materials as their structure can be controlled at the atomic level and synthesised with monodispersed precision. In this work, we show that de novo synthetic proteins spontaneously display liquid crystalline behaviour at critical concentrations. These liquid crystalline synthetic proteins were successfully wet-spun into aligned tough continuous fibres without disruption of the protein back-bone.


1. J. T. MacDonald, B. V. Kabasakal, D. Godding, S. Kraatz, L. Henderson, J. Barber, P. S. Freemont and J. W. Murray, Proceedings of the National Academy of Sciences, 2016, 113, 10346-10351.

Water-responsive (WR) materials that swell and shink in response to changes in relative humidiy (RH) show a great potential as building blocks for developing better actuators and artificial muslces. Bacillus (B.) subtilis spore has demonstrated a WR energy denstiy of ~10 MJ m^-3 that is significantly higher than that of reported actuator materials. However, the origin of such a powerful actuation of spores remains unclear. Here, we present that cortex layers within spore exhibit dramatic water-responsiveness that could dominate spores’ WR actuation. We isolated cortex from B. subtilis spores and found that its WR strain and stiffness reach ~38.7 % and 1 GPa, respectively, which suggests a WR energy density of 74.9 MJ m^-3. To correlate WR properties of cortex to that of spores, we characterized the distribution of cortex within spores by 3D reconstructing serial cross-secional images of spores obtained from an FIB-SEM. We have analyzed 85 spore samples and found cortex’s average thickness and volume ratio to be 122.4 nm and 52.4 %, respectively. These results, together with the measured WR strain and energy density, suggest that spores’ water-responsiveness is largely contributed by its cortex layer rather than other components. Our finding of the extreme water-responsiveness of spores’ cortex also provides insight to its important fucntions in spores’ biological processes.

Biomechanical forces play a critical role in our body. Collagenous tissues such as cartilage and bone are smart materials that can adapt their properties in response to mechanical forces through altering their structures from the molecular level up. They are able to convert mechanical forces into biochemical signals that control many biological and pathological processes such as wound healing and tissue remodeling. However, how collagenous tissues convert the biomechanical forces into signaling that regulates the cells during tissue development and remodeling remains unclear.
Here we employed a bottom-up computational approach to investigate the joint mechanobiology from two aspects: the molecular mechanisms of the extracellular matrix degradation and the molecular mechanisms of how point mutations affect the constitutive activity of transmembrane cation channel. We find that specific residues in the vicinity of the catalytic site play crucial roles in forming a stable binding pose in cartilage, which is vital for cartilage degradation. Our results also show that point mutations alter the molecular structure of the transmembrane protein and thus affect the channel size, which is one of the essential factors that alter the constitutive activity of the channel. Understanding the molecular mechanisms from the molecular level is crucial for the development of novel skeletal regenerative medicine or preventative strategies for related diseases.

As one of the most exciting biomaterials nature has to offer, spider silk has long fascinated researchers in various disciplines because of its both superior mechanical properties and biocompatibility. In order to replicate these properties in synthetic materials, an accurate and detailed structural description of silk is necessary. Though protein nanofibrils have been proposed to be the key structural element in natural silk fibers, experimental evidence about their existence, dimension, and structure is insufficient. Many important details of these nanofibrils, including their formation and spatial organization within a silk thread, are still missing.
Recently, we showed that the ribbon-like silk of the brown recluse spider is entirely composed of 20-nm thin nanofibrils using atomic force microscopy (AFM) and scanning electron microscopy (SEM). This is the first time that the fibrillar percentage and the complete structure down to the nanofibril level can be determined experimentally for any spider silk. First-ever evidence of individual isolated spider silk nanofibrils was also obtained. We also studied fibers from other spider species and observed nanofibrils with similar morphologies. Hence, we suggest that nanofibrils are the dominating structural element in all silk fibers.
Consequently, the formation of nanofibrils will also be important in novel synthetic materials inspired by spider silk. For the first time, we achieved in-vitro self-assembly of nanofibrils from the native silk dope of the golden silk orb-weaver spider (Nephila clavipes), one of the most studied species. In the presence of shear, silk protein molecules form individual 20-nm thin nanofibrils longer than 1 mm, similar with what we have observed in natural silk fibers. This unprecedented observation suggests an intrinsic tendency to form a linear fibrillar structure for the silk molecules. Furthermore, based on our spectroscopy study about the protein secondary structures within spider silk, and with the help of force modulation microscopy (FMM), we can characterize the mechanical behaviors of these nanofibrils at the molecular level.
Thus, our exhaustive investigation of silk nanofibrils from multiple angles points to nanofibrils as the basic and necessary building blocks of all silk fibers. This puts a complete structural description for natural silk materials within reach, and benefits efforts to develop protein- and polymer-based high-performance materials and biocompatible medical devices.

In nature, cells are exposed to various environmental conditions that can influence their behavior. How chemical cues influence cell behavior has been studied for decades, whereas the sensing and reaction of cells to external mechanical stimuli still contains a vast number of open questions.

A very well-known aspect, however, is that integrin-based adhesion clusters can grow upon externally applied shear forces. Furthermore, many cell types respond to the stiffness of their underlying substrate, and it has even been shown that cells can feel a stiff substrate underneath a soft material layer if the soft layer is thin. For example, mesenchymal stem cells sense an underlying hard substrate if the thickness of the soft layer is below 5 µm. This ability of cells to feel through soft material layers is essential in the design and application of implants, particularly in soft environments like the brain. Hence, the understanding of the underlying principles of these mechanosensory cell mechanisms is vital for biomedical research. In addition, the manipulation of cells with mechanical stimuli has great potential for biomedical applications.

However, external mechanical cues might affect single cells and cells in ensembles in different ways as cells in collective ensembles have the ability to distribute the applied stress over several cells via, for example, rearrangement processes. Consequently, they can bear mechanical stress better than single cells. To investigate how single cells and collective cell ensembles react to external stress, we built a setup to induce different mechanical stimuli. We will here present results on how such induced mechanical stimuli influence the cell behavior of cell ensembles and furthermore show how the thickness of soft layers affects the adhesion of single cells.

Quantum emitters placed in resonant optical cavities have shown modified spontaneous emission rates and frequency when they are coupled in the weak coupling regime. In the so-called strong coupling regime, however, the coupled oscillators, i.e., emitters and optical modes, exchange energies inextricably, creating new hybrid states called polaritons. Recently, this half-light half-matter quasi-state has been demonstrated in a system where molecular vibrations are coherently coupled to optical cavity modes. In this talk, we will demonstrate our recent results on the vibrational strong coupling, with a particular focus on its ability to modify chemical reactivity. We monitor transmission spectra of the Fabry-Pérot microcavity filled with species that cause a simple addition reaction. Both reactants and products have strong molecular vibrations that couple to the optical cavity modes, resulting in quantifiable vacuum Rabi splittings. We examine the reaction rates in and out of the cavity to elucidate the influence of vibrational strong coupling on the chemical reactivity. Our results will extend the potential of cavity-modified material properties, which will have important implications for controlling chemical reactivity and biological activity by light.

The demand for tissues and organs is unmet by the current supply. Engineering solutions are desired to create 3D cell structures to help fulfill this need. Using a unique bottom-up approach, we can rapidly form 3D cell assemblies by exploiting the diamagnetic property of cells. Most mammalian cells are weakly attracted to a magnet. This is also true of their culture medium, which is an aqueous solution of proteins, sugars, and nutrients to maintain their growth. The addition of a paramagnetic salt hydrate, such as gadopentatic acid (Gd-DTPA), transforms the culture medium to become paramagnetic. This establishes a difference between the magnetic susceptibility of cells and their liquid counterpart. The presence of a magnetic field gradient displaces the suspended cells towards regions of relatively lower magnetic field in the direction of where H is the magnetic field. This movement of cells followed by the movement of the culture medium seeds the formation of 3D cell cluster in a contactless, label-free manner within hours. Using this method, 3D cell structures can be formed on an ultra-low attachment surface, and 2.5D cell structures on a tissue-culture treated surface. The presence of a co-culture introduces new printing abilities and cell morphologies. This engineering solution has the potential to overcome limitations in bioprinting to expedite efforts in drug discovery, tissue engineering and regenerative medicine.

Bone repair is known to be a complex process affected by many parameters such as fracture size. 3D composite porous tissue scaffolds loaded with bioactive molecules and cells are being developed to offer a better solution for bone fracture healing. Accordingly, an ideal bone tissue scaffold should provide the following functions: a. mechanical support for the growth and functioning of new tissue, b. adequate porosity and permeability for nutrients and oxygen supply, waste removal and growth factors release, c. suitable surface for cell attachment, differentiation and growth, and d. controlled degradation. Therefore, an optimal bone tissue scaffold should have a multi-functional structure with desired mechanical, biological and chemical properties. However, it is still unclear what kind of properties optimal bone scaffolds should provide leading to effective tissue repair. Thus, there is a critical need to find methods suitable for designing novel bone scaffolds and the investigation of structure-function relationships for optimal tissue regeneration. Existing experimental studies are usually very time and cost ineffective.

Most of the computational design studies in literature that target ideal scaffold geometries with desired functional properties do not account for dynamic effects within the scaffold-tissue-cell environment. Among these, topology optimization based studies offer promise to design novel architectures of bone tissue scaffolds¹ but mostly have neglected the dynamic mechano-biological nature of regenerative healing². Unlike topology optimization, scaffold design studies based on parametric size optimization³ and mechano-biological regeneration models offer limited design freedom, hence resulting designs are not necessarily the best candidates to ensure effective healing. Moreover, angiogenesis - a critical part of the regeneration process- and scaffold degradation are not considered in any of these studies together despite their known coupled effect in healing. To address these limitations, we propose, for the first time, a computational framework to design the optimal microstructure of scaffolds that maximize bone formation considering mechano-biology, angiogenesis and scaffold degradation simultaneously. Thus, our model includes the following 4 parts in an integrated fashion: 1. Mechano-biological model describing tissue differentiation, 2. Angiogenesis, 3. Topology optimization targeting maximum bone formation, and 4. Time dependent degradation of the bone scaffold.

The proposed computational design framework is developed in a MATLAB GUI as an integration of above computational modules with a FEA software, namely COMSOL Multiphysics, a SIMP based topology optimization method and a random walk approach for angiogenesis modeling. The computationally designed scaffolds are compared with existing designs and fabricated based on a recently introduced phase separation technique. Morphological and mechanical characterization techniques will be performed to validate bone regeneration performance of designed bone tissue scaffolds. Integration of the proposed computational framework to existing experimental studies should pave the way for more efficient and low-cost solutions in bone tissue engineering.

Keywords: Tissue engineering, bone tissue scaffold, topology optimization, mechano-biology, angiogenesis, scaffold degradation.

References
[1] Wang, Y., Luo, Z., Zhang, N., & Qin, Q. 2016, Structural and Multidisciplinary Optimization, 54(2), 333-347.
[2] Geris, L., Vander Sloten, J., & Van Oosterwyck, H. 2010, Biomechanics and modeling in mechanobiology, 9(6), 713-724.
[3] Boccaccio, A., Uva, A. E., Fiorentino, M., Lamberti, L., & Monno, G. 2016, International journal of biological sciences, 12(1), 1.

Cells sense their environment by transducing mechanical stimuli into biochemical signals. Commonly used tools to study cell mechanosensing provide limited spatial and force resolution. To overcome this limitation, we recently reported a novel platform for monitoring cell forces, which was based on nanowires functionalized with ligands for cell immunoreceptors, and used this platform to explore mechanosensitivity of Natural Killer (NK) cells [1]. We found that nanowires stimulate cell contraction, and, by assessing the nanowire deflection, detected forces of down to 10 pN applied by cells. Furthermore, we found that the combination of (i) nanowire topography and (ii) activating produced enhanced activation of NK cells. Thus, we proposed a mechanosensing mechanism of NK cells, by which they integrate biochemical and mechanical stimuli into a decision-making machinery analogous to AND logic gate, whose output is the immune activation. This mechanosensing platform allowed unprecedented integration of biochemical and mechanical cues at the nanometric length-scale. Still, this platform lacked the ability to control another important signaling aspect – the spatial distribution and clustering of extracellular ligands. Indeed, in the physiological immune synapse, ligand-receptor pairs form highly regulated nanometric clusters, whose exact role is still obscure. On the contrary, in our reported nanowire-platforms the activating ligands were immobilized all over the nanowires surface, providing continuous medium for biochemical stimulation of NK cells.
Motivated to investigate the role spatial ligand clustering in cell mechano-sensation, we engineered a new nanowire-based platform for mechanical stimulation of NK cells, in which extracellular ligands are spatially confined within specific regions on nanowires. To that end, we grew Si nanowires from Au catalytic nanoparticles, and selectively functionalized the Au nanowire tips, whose size was about 30-50 nm, with biotinylated thiols, to which we conjugated biotinylated anti-NKP30 (activating ligands for NK cells)vianeutravidin bridge. We verified that the functionalization is specific to the nanowire tips using fluorescent imaging of the attached avidin. We then stimulated primary NK cells onto the functionalized nanowires, which we grew either as a continuous array, or as ordered separated clusters with a diameter of 5 microns. Interestingly, we detected two sub-populations within the cells stimulated on the clustered nanowires. The first sub-population consisted of cells on the surface among the nanowire clusters. These cells had elongated shape, and expressed very low degree of activation, as assessed by the expression of degranulation marker CD107a. The second sub-population consisted of cells laying on the top of the nanowire clusters. These cells showed high circularity, low spreading area, and enhanced degree of activation, most probably due to the combination of (i) the high compliance of nanowires to the mechanical forces applied by the cells and (ii) the contact formed by the cell with the antigen-functionalized nanowire tips. These newly results confirmed the importance the integration of the mechanical and chemical cues in the immune signaling. Furthermore, the demonstrated nanowire platform for the cell stimulation presented two new, unprecedented features. On the molecular level, it allows and exquisite control over spatial positioning and clustering of extracellular ligands on the nanowires. On the cell level, it allows three-dimensional spatio-mechano-chemical guidance of the cell motility and immune activity, and paves the way to numerous studies aimed at understanding the integration of mechanical and biochemical signals in cells.
[1] G. Le Saux, N. Bar Hanin, A. Edri, U. Hadad , A. Porgador, and M. Schvartzman , “Nanoscale Mechanosensing of Natural Killer Cells is Revealed by Antigen-Functionalized Nanowires”, Adv. Mater. 31,1805954 (2019)

Mammalian cell morphology is a key determinant of function, igniting significant interest in understanding how cell spreading is regulated by microenvironmental cues. These cues include cell-cell interactions, soluble signaling molecules, and adhesion to the extracellular matrix (ECM) via transmembrane receptors; the physical properties of the ECM, particularly its stiffness and rate of stress relaxation are known to regulate the ability of a variety of cell types to spread in 2D and 3D culture. In this report, we explore the role of matrix plasticity in controlling the spread of mesenchymal stem cells (MSCs). First, we describe a hydrogel system in which plasticity can be decoupled from elastic modulus and stress relaxation behavior, and then demonstrate that plasticity has broad impact on expression of genes related to matrix interactions, MSC spreading, and focal adhesion formation, with greatest spreading at an intermediate level of substrate plasticity. Kinetic Monte Carlo (KMC) simulations of a molecular clutch model predict the cell spreading rate to be a similar function of the plasticity of the matrix. Matrix plasticity also affects Yes-associated protein (YAP) translocation, and both spreading behavior and YAP translocation can be tuned by altering actomyosin contractility. Altogether, these findings demonstrate a key role for matrix plasticity in MSC biology and spreading, and we anticipate this will have ramifications in the design of biomaterials to control cell function and fate and in numerous biological contexts where matrix properties are altered.

Collective cell migration is the migration of cells in a group rather than individually. It plays a pivotal role not only in physiological processes, including morphogenesis, wound healing, and immune responses, but also in pathological ones, such as cancer invasion, metastasis, and tissue fibrosis. In addition to such involvements in various biological reactions, collective migration draws interests of researchers as it includes emergent collective characteristics that cannot be expected from simple summation of multiple single cells. For example, at the leading edges of migrating epithelial cell sheets, boundary cells are divided into actively migrating “leader cells” with frequent extension of lamellipodia and “follower cells” following the leaders. Also, some of cell collective exhibit autonomous group rotation with preferential asymmetry (left or right). All of these phenomena indicate that surrounding cells are not mere physical hindrance, rather those cells chemically and mechanically communicate with each other and against their extracellular matrices (ECMs) to function as an active soft matter. To decipher such complex regulation mechanisms of collective phenotypes from the reductionism viewpoint, our group is developing photoactivatable substrates based on photocleavable poly(ethylene glycol), where collective migration can be analyzed spatiotemporally under controlled chemical, mechanical, and geometrical cues (1-3). In this presentation, I will represent some of our recent progresses in the development and applications of the photoactivatable substrates, with special focus on the impact of chemical and mechanical cues on leader cell formation. I will also represent our new photoresponsive materials that can be used for analyzing collective behaviors on dissipative matrices.

[Reference]
(1) S. Yamamoto, K. Okada, N. Sasaki, A. Chang, K. Yamaguchi, J. Nakanishi,* "Photoactivatable hydrogel interfaces for resolving the interplay of chemical, mechanical, and geometrical regulation of collective cell migration", Langmuir, in press.
(2) S. A. Abdellatef and J. Nakanishi,* "Photoactivatable substrates for systematic study of the impact of an extracellular matrix ligand on appearance of leader cells in collective cell migration." Biomaterials, 169: 72-84 (2018).
(3) J. Nakanishi,* "Photoactivatable Substrates: A Material-based Approach for Dissecting Cell Migration" Chem. Rec. 17: 611-621 (2017) (Review).

The ability to dynamically control the mechanical properties of biomaterials is critical for regenerative medicine applications. For example, matrix stiffness controls the differentiation of stem cells, or the metastasis of cancer cells. However, most methods for tuning the mechanical properties of 3D hydrogels are not reversible, or involve potentially harmful stimuli like UV light. Here we use DNA as a programmable crosslinker to reversibly change the stiffness of gelatin hydrogels. We modified DNA with a methacrylate moiety, allowing it to be crosslinked along with methacrylated gelatin (GelMA). By tuning the amount of pre-formed DNA crosslinks relative to GelMA, we were able to tune the stiffness of the hydrogels obtained from 0.5 – 2 kPa, as measured by an AFM-based method. The crosslinkers contained single-stranded toehold sequences, allowing them to be broken by adding fully complementary displacement strands. The stiffness of the formed hydrogels could be tuned through the amount of displacement strand added, and the process was reversible: adding more crosslinker restored the stiffness to the original value. We explored various crosslinker lengths, designs, and geometries (including branched and more rigid multi-helical DNA tiles), demonstrating the straightforward tunability of DNA. The use of GelMA allows for photopatterning of hydrogels into micron-sized patterns, and cells could be incorporated into the materials and responded to the reversible changes in stiffness. DNA-based crosslinking also enables spatial patterning of stiffness, and will allow for incorporation of multiple ligands in a programmable manner to impart the gels with bioactivity. We will also outline chemical strategies for incorporating DNA into other biomaterial scaffolds, such as hyaluronic acid or PEG, demonstrating the versatility of our approach. We envision that DNA-based control of stiffness will allow for programmable and reversible control of hydrogel properties, and lead to advanced biomaterials for regenerative medicine, tissue engineering, and fundamental biological studies.

Mechanical environment around cells, not only physiological chemistry surrounding cells, strongly affects cell behaviors such as spreading, proliferation, and differentiation. Here, we describe a novel platform of cell culture scaffold that possesses tunable mechanical property. The design strategy for the materials includes self-assembly of block copolymers, photo-responsiveness, and solvent engineering of polymer gels. We show a 3D cell scaffold gel as one example that can change stress relaxation surrounding cells on demand. The gel is made by self-assembly of a well-defined coumarin containing ABA triblock copolymer. We demonstrated that the cell spreading embedded the 3D scaffold dynamically changes corresponding stress relaxation of gels.

Some engineers build with steel, copper, polymer composites, and wood. Bioengineers build with cells and because these cells are alive, they have a vote in our successes or failures. Our team has spent the last 15 years understanding how cells build themselves, how tissues assemble themselves, and the emergence of structure-function relationships in organs. These hierarchal organizations span from nanometer to meter length scale and have provided insight, and useful tools for drug discovery, toxicology, food, and regenerative medicine. This presentation will discuss how we have tried to understand the architecture and function of biological pumps, both in marine lifeforms and human organs, to understand the fundamental design principles that allow cells to serve as engineering materials.

The extracellular matrix surrounding cells in tissue is a dynamic composite material, where the presentation of biophysical and biochemical information directs functional bioactivities. We are interested in how the properties of the extracellular matrix guides tissue form and function and have developed a suite of engineered extracellular matrices to probe matrix structure-cell function relationships. Microengineered hydrogels that control tissue geometry, mechanics and composition have served to model how the microenvironment may exert an influence on cellular processes. Here we demonstrate how integrating dynamic chemistry into these hydrogels can further elucidate dynamic processes in vivo. For instance, we will present a new concept for remodelling hydrogels where the mechanics and chemistry can be changed through applied forces, akin to how natural matrices are remodelled with dynamic tension and compression. Using bioprinting we can spatially define soluble signals in precise architectures to further augment dynamic signals within the materials. These and other hydrogel-based model systems are changing the way in which fundamental biological questions can be probed, which will aid our understanding of morphogenesis while revealing new design parameters for regenerative biomaterials.

Cells have developed several mechanisms to protect themselves from physical damage. One of these mechanisms is the cell's ability to form stress-fibers as a response to mechanical deformation. This can for example be observed in epithelial cells that form stress-fibers to withstand the cyclic deformation of a blood vessel. Stress-fibers are cross-linked bundles of actin-filaments that show a higher stiffness than the underlying unlinked filaments. This ability to actively increase the stiffness as a response to deformation was the archetype for the development of our innovative strain-stiffening structures. A material behaves strain-stiffening if its stiffness is increased with increasing deformation. This behavior is desired for engineering applications like damping systems or shock absorbers where all requirements are met with already available materials. But these materials have disadvantages that make them unsuitable for special applications like novel orthotic devices or artificial blood vessels. The stiffening is either not reversible, highly dependent on the rate of deformation, can only be achieved with special materials or only occurs when the material is compressed. By taking advantage of the strain-stiffening mechanism we observed in cells, we were able to come up with structures that do not show any of these disadvantages. Our structures contain parallel slats, as a resemblance of cytoskeletal fibers, that touch each other upon deformation. This mimics the cross-linking of cytoskeletal fibers. The touching of the slats also leads to a change of mechanical properties of the entire material and its stiffness is increased. This effect is completely reversible, does not depend on the rate of deformation and every elastic material can be brought into the developed geometry. By variations of the geometry and the underlying material we are able to finely tune the mechanical properties of the structure and define, if the stiffening occurs upon compression or elongation. The influence of several geometrical factors on the strain-stiffening behavior was determined via finite element simulations and tensile tests. With the help of our novel strain-stiffening structures more comfortable orthotic devices and more compliant artificial blood vessels are now in development.

Engineered Living Materials (ELMs) that incorporate genetically modified cells to actively adjust the expression and organization of biomacromolecules are excellent candidates for applications in bioelectronics, biosensing, enzyme biocatalysis, and smart materials. Here we report a new strategy to create an ELM using Caulobacter crescentus. The surface layer (S-layer) protein of C. crescentus has been engineered to display a 2D array of functional peptide, i.e. SpyTag, over the entire cell body. A layer of closely packed nanoparticles is attached to the recombinant protein lattice through formation of SpyCatcher-SpyTag iso-peptide bond. The functionalized bacterial cells are further crosslinked into hierarchically ordered 3D assemblies. Stiffness of the self-assembled bacterial assemblies increases more than 30 times compared to non-crosslinked case. Cleavage of a disulfide bond inserted between SpyCatcher and the nanoparticles results melting of the bacterial assemblies, indicating the specific covalent linkage between S-layer proteins and nanoparticles is responsible for the enhanced mechanical properties. In the presence of excess nanoparticles, the crosslinked bacterial assemblies can autonomically self-regenerate after being damaged due to the continuous growth of C. crescentus cells and the expression of new S-layer proteins. Furthermore, the bacterial that coated with magnetic nanoparticles can form cohesive assemblies guided by external magnetic field, and the shape of the assemblies maintains when the magnetic field is removed due to the crosslinking. The ubiquity of S-layers in almost all archaea and many bacteria indicates the current crosslinking method could be applied to other microbe systems to create new ELMs with tunable mechanical properties.

Lipid vesicles are aqueous volumes surrounded by a bilayer of lipid molecules, which are amphiphilic molecules with their head groups facing water and tail groups facing oil. These vesicles are simple models that mimic cell membranes and can be used for drug delivery. Similarly, block copolymers are amphiphilic molecules that form vesicles by themselves or when mixed with lipids. Like lipid vesicles, polymer vesicles can also be used for cell membrane mimicry and drug delivery. One interesting type of lipid/polymer vesicle is the asymmetric vesicle, in which its bilayer is composed of two dissimilar lipid monolayers or a lipid monolayer and a polymer monolayer. Importantly, all eukaryotic cell membranes exhibit this type of asymmetry and asymmetry is also proposed to enhance mechanical properties of the membrane. Here, we use microfluidics to fabricate mono disperse and highly controllable asymmetric vesicles, which unlike the conventional methods that often end up with highly poly disperse samples. To achieve this, asymmetric vesicles are produced using water/oil1/oil2/water emulsions in a glass capillary device, with different lipids/polymers immersed in two different volatile oil phases. Using the asymmetric vesicles, we are trying to measure how mechanical properties are affected by this asymmetry and also how to improve the degree of asymmetry in our vesicles even more. In future, we envision asymmetric lipid/polymer vesicles could open a new door in the field of drug delivery.

Mechanophores are force sensitive molecules that undergo productive chemical transformations under a mechanical force including color change, small molecule release, and cross-linking. Mechanophore reactivity depends on the intrinsic molecular structure as well as the extrinsic environment of the mechanophore. This presentation summarizes the extrinsic, force-focusing effects of mechanochemical activation at an interface. New methodologies are developed to characterize interfacial mechanochemical transformations for two different color-changing mechanophores. Maleimide–anthracene (MA) mechanophores covalently anchored at a fused silica–polymer interface are activated using laser-induced stress waves. In contrast to activation in solution or bulk polymers, whereby a proportional increase in mechanophore activity is observed with applied stress, interfacial activation occurs collectively with spallation of the polymer film. MA mechanophores located at the interface between poly-(glycidyl methacrylate) (PGMA) polymer brushes and Si wafer surfaces were also activated locally using atomic force microscopy (AFM) probes. In a separate set of experiments, the reaction of spiropyran (SP) mechanophores is demonstrated at nanoparticle/polymer interfaces. A new SP mechanophore containing a pentene and alkyl bromide group at each pulling point was designed and synthesized for functionalization of silica nanoparticles. The mechanical activity of the SP functionalized at the interface between silica particles and a polymer matrix is compared with that of SP linked directly into bulk polymers. As anticipated, the interfacial mechanophores exhibit more efficient mechanical activation under tensile loading.

Nature has effectively revised engineering designs of elastomeric biopolymers through years of evolutionary history. In this work, we examine the molecular and multiscale mechanisms of elasticity of biopolymers exhibiting exceptional elasticity in vivo to deduce design principles and mechanisms that can be used to develop novel elastic biopolymers for medical and engineering applications. We consider two examples from nature: resilin, a biopolymer found in insect cuticles and elastin, a key component within the extracellular matrix of elastic tissues in mammals. We use molecular models to compare effects of sequence and structural hierarchy, hydration, librational effects and temperature on elasticity. Through this comparison, we identify unifying principles that can be used for rational design of elastomeric biopolymers.

Complexity in biological systems spontaneously emerges from an initial state of symmetry through coupled reaction and diffusion, a process known as morphogenesis. In this work, we explore the coupled reaction and thermal diffusion inherent to frontal ring-opening metathesis polymerization (FROMP) of dicyclopentadiene (DCPD) as a synthetic mimic to biological morphogenesis. Propagation instabilities arise from an initial state of symmetry and generate patterns of thermal fluctuations on multiple length scales. Incorporation of thermo-active small molecules enables spontaneous patterning of the optical, chemical, and mechanical properties of structural thermosets. Material stiffness and glass transition temperatures were found to vary by up to two-fold and 30 °C, respectively. Control over patterns by tuning reaction and diffusion rates will also be discussed.

Embodied Intelligence is the principle that describes autonomous mechanical responses to external environmental inputs (e.g., stress causes strain). This concept has been explored in depth previously and has resulted in a variety of compliant mechanisms and smart materials that combine, for example, the functions of structure and actuation. Energy (e.g., Chemical, Mechanical, Electrical, Optical) sources can also be put to multifunctional use; for example, a lead-acid battery being used a counterweight in a forklift. A more principled approach to Embodied Energy, however, does not presently exist. This talk will, for the first time, introduce this concept and focus on specific examples of forming these energy sources at a finer scale, into structural and other functional components and composite materials that provide benefits in the form of reduced SWaP tradeoffs. Examples of how these energetic composites and their chemo-electro-opto-mechanical responses can be tuned to perform work autonomously or in cooperation with higher level sensing and control for augmenting, supplementing, or replacing some robotic systems, including morphing structures, will be presented, and identification of gaps and future needs will also be discussed.

Epithelial cells are the body’s front-line defenders against foreign objects and environmental challenges and could be considered the biological equivalent of surface engineers. These cells continuously secrete high water content mucin gels to protect approximately 400 square meters of underlying cells, tissues, and organs from damage. Surfaces involving intentional and frequent sliding contact – such as the cornea, the digestive and reproductive tracts, and many distal interfaces for organs and tissues – adopt a stratified epithelial tissue approach. Epithelial cells are sophisticated sensors of mechanical stresses; friction and the resulting direct contact shear stresses have been shown to increase gene expression of pro-inflammatory cytokines and pro-apoptotic markers in vitro. In this work, a suite of experiments was performed under acute and chronic conditions to explore the onset and progression of apoptosis in corneal epithelial cell monolayers using in situ fluorescence microscopy and a custom-built microtribometer with aqueous gel probes. Recent results suggest that the onset of apoptosis occurs near physiological shear stresses (<100 Pa), although direct contact pressures in the absence of sliding did not initiate apoptosis, even at contact pressures approaching 3,000 Pa. These findings may help inform future designs of soft implants to mitigate friction-induced inflammation.

The mechanical properties of different mammal tissues vary within the body. Starting with the structural components of the body like bones with a Young’s modulus of several hundreds of kPa down to soft tissue like the brain with a Young’s modulus of 1 kPa and below.¹ This knowledge is important for the development of synthetic material for biomedical applications. It is assumed, that a mismatch between implant material and tissue can lead to scar tissue formation and tissue remodeling. Consequently, the understanding and investigation of the link between the mechanical properties and the structure of an implant material and the tissue response are essential. Another critical point is that cells in the body are often situated in a 3D structured and porous environment. Therefore, it is important to mimic such environments in implant materials to investigate questions such as how single cells migrate through different 3D constrictions and the consequences on the cells.
A step towards studying these questions is the use of a synthetic and 3D structured material, which closely mimics the natural tissue and its environment with similar hydration and mechanical properties. We developed a 3D environment consisting of a polyacrylamide hydrogel with interconnected and hollow microchannels. ² It enables to vary the Young’s modulus by changing the chemical composition of the crosslinker to match elasticity values within a certain (soft) tissue regime up to 120 kPa.
Using such materials, we are investigating different hydrogel stiffness (1 kPa – 50 kPa) and the consequences of compliant and incompliant material mechanics towards cell behavior in 3D environments. With the possibility of using different adhesion ligands for the biofunctionalization such as collagen or fibronectin, we are able to specifically induce cell adhesion of different cell types. These cell types include pathogenic species, such as Acanthamoebae castellanii, for which we can use the hydrogel microchannels as biomedical capture device. Another possibility is the usage of cells with a preference to soft tissue surroundings such as fibrosarcoma cells, which act as an excellent robust role model for cells of the soft tissue region.
In conclusion, we show an artificial 3D interconnected environment with designed Young’s moduli and our investigation towards the influences on the cellular behavior of fibrosarcoma cells such as adhesion and migration on and within interconnected microchannels.


[1] R. J. Pelham et al. PNAS (1997)
[2] S. B. Gutekunst et al., ACS Applied Biomaterials (2019)

Silicone gels are commonly used for the encapsulation and construction of implantable medical devices. Viscous silicone “oils” are also commonly used for lubrication of current-generation pre-fillable syringes, which are known to expel silicone oil droplets into patients during administration of aqueous drug formulations. While silicones are generally viewed as relatively inert to the cellular milieu, they can mediate a variety of inflammatory responses and other deleterious effects, but the mechanisms underlying the bioactivity of silicones remain unresolved.
Cells physically interrogate their extracellular environment to make decisions related to cell proliferation, migration and other critical processes. In addition to biochemical signals, physical properties of the extracellular matrix, including its stiffness, are key regulators for cell behaviors. Typically, on stiff substrates, cells display large spreading areas, assemble robust integrin-based adhesion complexes, whereas on soft substrates these functions are suppressed. However, cell behaviors that defy expectations based on substrate rigidity alone have been observed.

Here, we report that silicone liquids and gels have high surface stresses that can strongly resist deformation at cellular length scales. We demonstrate that cells interacting with soft materials with high surface tension primarily sense and respond to surface tension and not the bulk elastic moduli of the materials. Our results are consistent with theory that predicts that solid surface tension can dominate over elasticity at cellular length scales. On silicone materials with appreciable surface tension, cells assemble robust adhesion complexes and cytoskeleton stress fibers, spread over large areas, upregulate canonical integrin-based signal transduction, proliferate and migrate efficiently. Grown directly on the interior materials of silicone breast implants, cells are well spread and show nuclear localization of gene transcriptional factor YAP. In 3D culture models, liquid silicone droplets support robust cellular adhesion and the formation of multinucleated monocyte derived cell masses that recapitulate phenotypic aspects of granuloma formation in the foreign body response.

Together, our results indicate that material surface tension is a cellular stimulant that should be considered in application of silicones for biomedical purposes.

The hematopoietic stem cell (HSC) niche in the bone marrow is a unique microenvironment, which controls HSC maintenance and differentiation throughout the entire lifespan. HSCs are the source of all blood cells that are produced in billions on a daily basis. The niche is the only place, where HSCs can expand while keeping their stem cell properties. Thus, recreating the HSC niche in the lab for producing HSCs for clinical applications is an intriguing research objective that is pursued since the 1960s, when HSC transplantation became a life-saving treatment option for patients with hematological diseases. In this endeavor, researchers have concentrated for a long time on the influence of biological and chemical factors that are naturally found in the niche on HSCs. Only during the last decade, the importance of physical parameters – including 3D architecture, nanostructure and mechanical stimuli – became evident. We and others could show in vitro that human hematopoietic stem and progenitor cells are mechanosensitive and we proposed a model, how mechanical properties in the HSC niche might change and thus influence HSC behavior under different physiological conditions. Nevertheless, most in vitro experiments to test the mechanosensitive responses of cells still rely on hydrogels, in which the crosslinking degree is modulated to tune the mechanical properties of the resulting polymer network. Changing the crosslinking degree, however, does not only change the E modulus of hydrogels, but also their porosity, hydrophilicity or other parameters at the same time, which makes it impossible to conclude on the influence of one particular parameter from these experiments. To overcome this challenge, we developed a new platform-technology allowing to investigate mechanotransduction in a hydrogel-independent way. In a proof-of-principle study we could show that this platform allows to decouple mechanical from biochemical properties of biomolecules and thus to investigate the influence of both parameters on cells independently from each other. The results of these studies will help us to gain a fundamental understanding of the role of mechanobiology in the HSC niche, which might be an important step towards the goal of an artificial niche for HSC production for clinical applications.

Cell invasion into the surrounding matrix from non-vascularized primary tumors is the main mechanism by which cancer cells migrate to nearby blood vessels and metastasize to eventually form secondary tumors. This process is mediated by an intricate coupling between intracellular and extracellular forces that depend on the stiffness of the surrounding stroma and the alignment of matrix fibers. A multiscale model is used to elucidate the two-way feedback loop between stress-dependent cell contractility and matrix fiber realignment and strain stiffening, which enables the cells to polarize and enhance their contractility to break free from the tumor and invade into the matrix. Importantly, our model can be used to explain how morphological and structural changes in the tumor microenvironment, such as elevated rigidity and fiber alignment prior to cell invasion, are prognostic of the malignant phenotype. The model also predicts how the alignment of matrix fibers can recruit macrophages, which are among the first responders of the innate immune system following organ injury and are crucial for repair, resolution, and re-establishing homeostasis of damaged tissue. I will discuss how the deformation of the nucleus during migration can lead to changes in the spatial organization of chromosomes and their intermingling which can result in genetic mutations and genomic instability. I will also discuss how targeting extracellular matrix mechanics, by preventing or reversing tissue stiffening or interrupting the cellular response in cancer and fibrosis, is a therapeutic approach with clinical potential.

BIO: Vivek Shenoy is the Eduardo D. Glandt President’s Distinguished Professor in the School of Engineering and Applied Sciences at the University of Pennsylvania. Dr. Shenoy's research focuses on developing theoretical concepts and numerical methods to understand the basic principles that control the behavior of both engineering and biological systems. He has used rigorous analytical methods and multiscale modeling techniques, ranging from atomistic density functional theory to continuum methods, to gain physical insight into a myriad of problems in materials science and biomechanics. Dr. Shenoy's honors include a National Science Foundation CAREER Award (2000), the Richard and Edna Solomon Assistant Professorship (2002-2005) and the Rosenbaum Visiting Fellowship from the Isaac Newton Institute of Mathematical Science, University of Cambridge and the Heilmeier award for excellence in faculty research (2019). He is the principal investigator and director of the NSF-funded Science and Technology Center for Engineering Mechanobiology established in 2016. He also serves the editor of the Biophysical Journal and is a fellow of the American Institute for Medical and Biological Engineering.

Sculpting of structure and function of three-dimensional multicellular tissues depend critically on the spatial and temporal coordination of cellular physical properties. Yet the organizational principles that govern these events, and their disruption in disease, remain poorly understood. Here, I will introduce our recent progress performing biomechanical imaging to quantify cell and extracellular matrix (ECM) mechanics, as well as their mechanical interaction. By integrating confocal microscopy with optical tweezers, we have developed a platform to map in three dimensions the spatial and temporal evolution of positions, motions, and physical characteristics of individual cells throughout a growing mammary cancer organoid model. Compared with cells in the organoid core, cells at the organoid periphery and the invasive front are found to be systematically softer, larger and more dynamic. These mechanical changes are shown to arise from supracellular fluid flow through gap junctions, suppression of which delays transition to an invasive phenotype. Together, these findings highlight the role of spatiotemporal coordination of cellular physical properties in tissue organization and disease progression.

Epithelial tumors exhibit dysregulated cell-cell and cell-matrix adhesions as they invade into the surrounding extracellular matrix. In particular, the epithelial-mesenchymal transition (EMT) is associated with weakened cell-cell adhesions and strengthened cell-matrix adhesions, resulting in multicellular dissemination. Traction force microscopy enables new insights into the cell-generated forces that mediate these behaviors, but has primarily been applied to individual cells in 3D. Here, we elucidate the collective tractions of multicellular clusters in 3D matrix after induction of the EMT master regulator Snail. We find that multicellular clusters exhibit characteristic spatial signatures that can be used for mechanophenotypic profiling. In particular, EMT results in highly localized “hotspots” of strong cell-matrix adhesion, associated with high contractility and front / back polarization. We further show that chemotherapeutics and targeted inhibitors can perturb clusters towards more epithelial or mesenchymal-like mechanophenotype. We envision that this 3D culture assay will enable high content preclinical screening of targeted anticancer compounds as well as to predict the clinical response of human patient samples.

Carcinoma cells tend to migrate in collective strands, ducts, sheets or clusters (Friedl, & Gilmour, Nat. Rev. Mol. Cell. Bio. 2009). To migrate collectively, the epithelial collective has been argued to overcome geometric constraints attributable to cell jamming (Atia et al., Nat. Phys. 2018). If so, then the greater is the degree of cellular jamming, then the more would be the extent to which each individual cell becomes caged by its neighbors, and therefore, the less rapidly it would be able to migrate (Park et al., Nat Mat. 2015). The jamming hypothesis, however, has never been tested in the context of cancer cell invasiveness. Using classical in vitro cultures of six breast cancer models, here we investigate structural signatures of jamming, dynamical signatures of jamming, and the relationship between them. In order of increasing invasiveness, the cell lines examined included MCF10A, MCF10A.vector; MCF10A.14-3-3z; MCF10.Erb2, MCF10AT; and MCF10CA1a. Across all models tested, cell shape and shape variability from cell-to-cell conformed well to structural signatures of cell layer jamming. In all cases but one, migratory dynamics changed roughly in concert with expectations based on structural signatures –as the strength of structural signatures of unjamming increased, the rapidity of migratory dynamics tended to progressively increased. The exception was the case of MCF10CA1a, wherein structure signified a moderately jammed state whereas migratory dynamics were excessively rapid and therefore discordant with structure. Closer examination of migratory dynamics of MCF10CA1a showed anomalously large migratory persistence, but the mechanism of discordance in this case remains unclear. A hallmark of cancer is multiple dimension of heterogeneity. Nevertheless, each of the diverse cases examined here reveals that cell jamming imposes an overriding geometric constraint.

Progression to metastatic breast cancer requires cancer cells to invade from a solid tumor into the surrounding stroma and escape into a lymphatic or blood vessel. To understand the biophysical and biochemical parameters that define the kinetics of invasion and escape, we engineered a three-dimensional model of human breast microtumors embedded within native extracellular matrix. We previously found that interstitial fluid pressure (IFP) determines the invasive response of human breast microtumors: specifically, interstitial hypertension (i.e., elevated IFP) prevents invasion, whereas interstitial hypotension (i.e., lowered IFP) promotes invasion. We have now used this system to examine the effects of matrix density, proteolysis, proliferation, and IFP on the kinetics of tumor cell escape into an empty cavity. Our data suggest that the physical microenvironment of a tumor dictates the rates of two early steps in the metastatic cascade, namely, invasion of the surrounding interstitium and escape into an open space. These physical features dictate whether escape results from a ballistic or diffusive invasion process. Furthermore, acute changes in interstitial pressure can suppress tumor cell escape after invasion has already occurred. Our results point to the possibility of using physical therapies to delay or prevent metastatic progression in breast cancer.

Cancer metastasis, i.e., the spreading of cells from the primary tumor to distant organs, is responsible for more than 80% of all cancer deaths. During cancer cell invasion and metastasis, tumor cells migrate through interstitial spaces and transendothelial openings substantially smaller than the diameter of the cell. Recent research has made it apparent that cells migrating in such confined three-dimensional (3D) environments face substantially physical challenges. In particular, the cell nucleus is the largest and stiffest organelle, making nuclear deformation a rate-limiting factor in the passage of cells through confined 3D environments. We have used micro- and nano-fabrication approaches to generate microfluidic devices that closely mimic the physical constraints of physiological interstitial environments, while providing precise control over the constriction geometry and enabling live-cell imaging at high spatial and temporal resolution. Using these devices, we demonstrated the importance of available pore size and nuclear deformability on the ability of cells to move through 3D environments. We combined these devices with fluorescent reporters for nuclear envelope rupture and DNA damage to assess the functional consequences of the physical forces exerted on the nucleus during confined migration. In addition, we developed a microfluidic micropipette aspiration device to rapidly measure nuclear stiffness in large numbers of cells. We found that highly metastatic breast cancer cells had decreased levels of the nuclear envelope proteins lamin A/C, which determine nuclear deformability, compared to less aggressive tumor cells, and that the increased nuclear deformability promoted migration through tight spaces. Increasing expression of lamin A in breast cancer cells with normally low levels of lamin A/C significantly impaired their invasive properties, while depletion of lamin A/C increased invasive potential through micron-scale microfluidic constrictions and dense collagen matrices. Importantly, analysis of human breast tumor tissue microarrays showed that low levels of lamin A/C correlated with reduced disease-free survival, demonstrating the clinical relevance of our findings. Taken together, these studies indicate that downregulation of lamin A/C could promote both cancer cell invasion and metastasis in breast cancer while highlighting the appeal of engineered materials and microenvironments to study tumor cell mechanobiology. Insights gained from this work could improve prognostic approaches; ultimately, targeting regulator pathways associated with altered lamin expression may offer novel therapeutic avenues to control metastatic disease in breast cancer.

The U.S. National Cancer Institute (NCI) leads, conducts, and supports cancer research across the nation to advance scientific knowledge and help people live longer, healthier lives. Many advancements in cancer research in areas of progression, metastasis, and treatment response have been enabled by the development of innovative technologies, including novel biomaterials, microfluidics, and biomimetic engineered technologies. Several programs at the NCI have helped foster cancer technology development in these areas as well as the overall convergence of approaches and perspectives from the physical sciences and engineering into cancer research. Over the past decade, the NCI has supported the NCI Physical Sciences – Oncology Network (PS-ON), which is comprised of nearly 30 transdisciplinary teams that integrate physical sciences perspectives with cancer research to complement and expand on our current understanding of cancer across many biological length- and time-scales. Thematic areas under investigation in the PS-ON include transcriptional dynamics and genomic architecture, modeling evolutionary dynamics of treatment response, cancer mechanobiology and the physical microenvironment, and multi-scale computational modeling approaches to integrate data across length scales. PS-ON investigators and those in the affiliated Cancer Tissue Engineering Collaborative (TEC) research program are utilizing biomaterials and biofabrication for experimental model systems of cancer that recapitulate the tumor microenvironment and tumor-stromal interactions. There is also sophisticated incorporation of bioreactors and microfluidic culture to mimic perfusion, lymphatics, interstitial pressure, and molecular gradients. Other research initiatives supported by the NCI to promote convergent, cross-disciplinary research that include projects incorporating novel biomaterials are the Cancer Systems Biology Consortium (CSBC) and the Innovative Molecular Analysis Technologies (IMAT). The NCI demonstrates its interest in supporting materials science and engineering in cancer research by investment in these areas through investigator-initiated research and the targeted programs. The continued investigation of the physical dynamics of cancer and incorporation of novel biomaterials and biomimetic engineered technologies will be important, focusing on understanding the complex and dynamic multiscale interactions of the tumor, host, and immune system. Innovative technology development will continue to be critical for unprecedented measurements and discoveries in cancer research.

The nucleus links physically to cytoskeleton, adhesions, and extracellular matrix – all of which are subject to stresses and strains. We have taken various materials-intensive approaches to study nuclear rupture in tumors [1], embryonic organs [2], and various in vitro models, and we find rupture results from high nuclear curvature, leading to cytoplasmic mis-localization of multiple DNA repair factors and transcription factors that impact cell fate and function. Curvature is imposed by external probe [1], by migrating quickly (not slowly) through constricting micropores [3,4], or by cell attachment to either aligned matrix or stiff matrix [1], and theory indicates rupture pores from by a heterogeneous nucleation mechanism [5]. Mis-localization of nuclear factors is greatly enhanced by nucleoskeleton depletion (soft nuclei), requires many hours for nuclear re-entry, and correlates with pan-nucleoplasmic foci of DNA damage and with electrophoretic breaks. Excess DNA damage is rescued in ruptured nuclei by co-overexpression of multiple DNA repair factors as well as by soft matrix or inhibition of either actomyosin tension or oxidative stress – with combination treatments needed to rescue a cell cycle checkpoint [4]. Increased contractility has the opposite effect, and stiff tumors with softened nuclei indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Normal differentiation processes of myogenesis and ostoegenesis are also affected by migration through constricting pores, suggesting general effects on cell fates [6]. Mis-repair of DNA is further suggested by two cancer lines that, after constricted migration, exhibit greater genome variation [1,3]. References: [1] Y Xia, … DE Discher. Nuclear rupture at sites of high curvature compromises retention of DNA repair factors J Cell Biol (2018). [2] S Cho … Discher DE. Mechanosensing by the lamina protects against nuclear rupture, DNA damage, and cell cycle arrest. Dev Cell (2019). [3] J Irianto … DE Discher. DNA damage follows repair factor depletion and portends genome variation in cancer cells after pore migration. Curr Biol (2017). [4] Y Xia … Discher DE. Rescue of DNA damage after constricted migration reveals bimodal mechano-regulation of cell cycle. J Cell Biol (2019). [5] D Deviri … Discher DE, Safran SA. Scaling laws indicate distinct nucleation mechanisms of holes in the nuclear lamina. Nature Physics (2019). [6] LR Smith … Discher DE. Constricted migration modulates stem cell differentiation. Mol Biol of the Cell (2019).

Tumors show increased tissue level forces and a present with a chronically stiffened extracellular matrix (ECM), and transformed cells exhibit a perturbed oncogene-stimulated and ECM-tuned mechanophenotype. We have been studying how these aberrant cell and tissue level forces promote malignant transformation and drive tumor metastasis, and how they modulate tumor recurrence and treatment resistance in breast and pancreatic cancer and glioblastoma. We use two and three dimensional culture models with tuned extracellular matrix stiffness, as well as transgenic and syngeneic mouse models, human PDX models and human biospecimens, in which ECM crosslinking and stiffness and integrin mechanosignaling can be quantified and modified. Our studies have thus far revealed that the ECM in all tumors is progressively remodeled and stiffened by stromal fibroblasts and that this occurs prior to malignant transformation. We determined that ECM remodeling and stiffening is mediated very early during malignancy by stromal fibroblasts that are activated by factors including TGFb that are secreted by infiltrating pro inflammatory macrophages. The stromal-fibroblast stiffened ECM disrupts tissue organization, promotes cell growth and survival and drives cell invasion. A chronically stiffened tissue stroma drives angiogenesis, and activates STAT3 to induce key cytokines and chemokines that promote pro-tumor immunity to foster tumor growth and dissemination and impede tumor treatment. The stiffened ECM also drives an epithelial to mesenchymal transition and primes the metastatic niche to foster metastasis. I will discuss the dynamic and reciprocal interplay between tissue tension and innate and acquired immunity and how this can not only force tumor aggression and metastasis but may also initiate tumor progression.

Hydrogels, highly hydrated cross-linked polymer networks, have emerged as powerful synthetic analogs of extracellular matrices for basic cell studies as well as promising biomaterials for regenerative medicine applications. A critical advantage of these synthetic matrices over natural networks is that the biophysical and biochemical properties of the material can be tuned with high control and precision. For example, bioactive functionalities, such as cell adhesive sequences and growth factors, can be incorporated in precise densities. We have engineered poly(ethylene glycol) [PEG]-maleimide hydrogels that support improved pancreatic islet engraftment, vascularization and function in diabetic models. Two biomaterial strategies will be discussed. We have developed proteolytically degradable synthetic hydrogels, functionalized with vasculogenic factors, engineered to deliver islet grafts to extrahepatic transplant sites via in situ gelation. These hydrogels induce differences in vascularization and innate immune responses among subcutaneous, small bowel mesentery, and epididymal fat pad transplant sites with improved vascularization and reduced inflammation at the epididymal fat pad site. This biomaterial-based strategy improves the survival, engraftment, and function of a single pancreatic donor islet mass graft compared to the current clinical intraportal delivery technique. In a second application, we have developed a localized immunomodulation strategy using hydrogels presenting an apoptotic form of Fas ligand (SA-FasL) that results in prolonged survival of allogeneic islet grafts in diabetic mice. A short course of rapamycin treatment boosts the immunomodulatory efficacy of SA-FasL-hydrogels, resulting in acceptance and function of allografts over 200 days. Survivors generate normal systemic responses to donor antigens, implying immune privilege of the graft, and have increased T-regulatory cells in the graft. Current studies focus on evaluating this immunomodulatory strategy in a large animal model of type 1 diabetes. This localized immunomodulatory biomaterial-enabled approach may provide an alternative to chronic immunosuppression for clinical islet transplantation.

It has long been recognized that tissue mechanical properties are altered in cancer, and this can serve as the basis for early diagnosis. While the impact of changes in tissue stiffness has been the focus of research to date, the role of tissue viscoelasticity has not been widely explored. We have developed hydrogels which allow for independent control over elastic moduli and stress relaxation/creep, and can mimic the fibrillar architecture of native collagenous matrices. These material systems are being utilized in 3D cell culture models of cancer and immunotherapy, and demonstrate that the gene expression of various cell types present in tumors, including cancer cells, mesenchymal cells, and immune cells is profoundly impacted by the viscoelastic properties of their matrix.

Mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine applications due to their ability to self-renew, capacity for multipotent differentiation and secretion of a diverse array of cytokines and growth factors (the MSC secretome). These cells are frequently utilized in conjunction with biomaterial scaffolds designed to encourage cellular retention and direct the cells’ regenerative properties. However, hydrogel carriers have not yet yielded significant results in the clinic in part due to a lack of understanding of how hydrogel biophysical and biochemical properties impact cellular function. Although many synthetic hydrogels incorporate short peptides (e.g. RGD) to support integrin-mediated cell adhesion, the impact of hydrogel adhesive properties on transplanted cell function remains unknown.

We engineered integrin-specific hydrogels for the delivery of MSCs by tethering either the ubiquitous RGD cell adhesion motif or the type-1 collagen derived GFOGER adhesion motif into synthetic poly(ethylene) glycol (PEG)-based hydrogels. Integrin-specificity was confirmed using blocking antibodies and a custom spinning disk platform in which cells attached to hydrogel disks are exposed to well-defined hydrodynamic shear forces allowing for sensitive measurements of the force required to detach the cell from the substrate. The effects of integrin-specific adhesive peptide presentation on MSC secretome and MSC-macrophage interactions were evaluated in vitro using Luminex multiplex technology. Finally, integrin-specific hydrogel directed MSC bone tissue regeneration was assessed over the course of 8 weeks in a critical size radial bone defect in an NSG mouse model.

Spinning disk analysis shows that cell adhesion to hydrogels presenting the adhesive peptides RGD and GFOGER are specific to αvβ3/β1 and α2 integrins respectively. Luminex data shows that the secretome of MSCs encapsulated in integrin-specific hydrogels cluster distinctly based on peptide and secretion of IL-6, IL-8, and VEGF is increased in GFOGER functionalized gels compared to RGD and non-adhesive controls. Further, in a co-culture assay, we show that macrophage cytokine secretion is differentially modulated by MSCs encapsulated in integrin-specific hydrogels, including an increase in IL-10 secretion by macrophages interacting with GFOGER encapsulated MSCs. Finally, MSCs delivered in GFOGER functionalized hydrogels significantly enhance repair of critical size bone defects in vivo compared to RGD and non-adhesive controls. Taken together, our results demonstrate that integrin specificity can be engineered into synthetic hydrogel systems resulting in modulation of the MSC secretome, differential MSC-macrophage interactions and improved tissue healing.

Dental Pulp Stem Cells (DPSC) provide a valuable and enticing avenue for the field of regenerative medicine. Previously, we determined that DPSC cultured on monodispersed polybutadiene (PB), a biologically compatible substrate without additional coating, induced high levels of biomineralization in surfaces with a modulus over 2.3 MPa. [1] Similarly, titanium surfaces have been noticed to support osseointegration in dental implants. [2] In this study, we introduce a new method to deposit a 2~3 nm layer of titanium dioxide by Atomic Layer Deposition (ALD) on PB substrates to investigate DPSC behavior and differentiation lineages in an environment where surface chemistry has changed but substrate modulus remains the same.
ALD was employed to deposit TiO₂ on thin (20 nm) and thick (200 nm) PB substrates, which respectively formed hard and soft substrate mechanics effects. All substrates were cultured with human DPSC, with data samples taken weekly. At first week, population doubling time determined that ALD had no major effect on cell proliferation while confocal images showed similar actin density and length of DPSC on all hard and soft PB substrates, suggesting that the TiO₂ nanolayer has minimal effect on cell behaviors in the initial period. At the later stage of differentiation, biomineralization was characterized by SEM/EDS and Ramen spectroscopy, with templated, mineralized deposits observed only on ALD coated both hard and soft PB substrates. Osteocalcin (OCN) antibody staining observed by confocal also showed that ALD coating substrates favored OCN protein, suggesting that TiO₂ ALD coating promotes differentiation and biomineralization on soft PB substrates where no mineralized deposits and upregulation of OCN was found. On the other hand, on hard PB substrates, even though biomineralization and differentiation were found on both with and without ALD coating substrates, templated mineralized deposits and more evenly spread OCN protein were observed on ALD coating hard substrates, while particle-like deposits without fibers templated were presented on hard PB substrates without coating, suggesting that surface chemistry of TiO₂ coating by ALD may alter DPSC behaviors and differentiation pathway.
This ALD method provides a potential application to coat a nanolayer on titanium on any biomaterial to further promote stem cells differentiation and proliferation.
We acknowledge support from the Louis Morin Charitable Trust and the NYS Department of Economic Development.

[1] V. Jurukovski, M. Rafailovich, M. Simon, A. Bherwani, C.-C. Chan, Citation: Entangled Polymer Surface Confinement, an Alternative Method to Control Stem Cell Differentiation in the Absence of Chemical Mediators. Annals of Materials Science & Engineering, 2014.
[2] F. Iaculli, S. Di Filippo Ester, A. Piattelli, R. Mancinelli, S. Fulle, Dental pulp stem cells grown on dental implant titanium surfaces: An in vitro evaluation of differentiation and microRNAs expression, Journal of Biomedical Materials Research Part B: Applied Biomaterials 105(5) (2016) 953-965.

It is well-known that biomimetic microenvironments play an important role in governing cell function and fate. Conventionally, researchers utilize tissue culture plastic for cell culture; however, there are large and obvious differences in the biochemical and mechano-structural properties of cellular surroundings in vitro (plastic dish) and in our body. In addition, the native cellular environment incorporates several dynamic cell stimulatory factors beyond static biochemical and mechano-structural cues. To understand and bridge these gaps between in vitro and in vivo cell culture, a new research field known as ‘mechanobiology’ has emerged. Though today we have a greater understanding of the mechanobiological cellular phenomena in static systems, we have yet to fully explore the effects of ‘dynamic stimulation’ on cell behavior.
To recapitulate the complex microenvironment inside the body, spatio-temporal biomaterials have emerged as powerful tools to probe and direct active changes in cell function. In this presentation, I will briefly introduce our established dynamic cell culture platforms with ‘shape memory’ abilities. In order to evaluate the potential for cultured cells to respond to dynamic changes in their in vitro microenvironment as they do in vivo, we studied the effects of controllable anisotropic topographies on cell function. Given the importance of dynamic cues in regulating cell behaviors, investigation of such dynamic topography may have important implications to advance cellular manipulation and performance in vitro, as well as improving our understanding of cellular development in response to dynamic biophysical cues.

Epithelial organoids cultured in appropriate 3D conditions typically develop a microscopic, cystic structure lined with a polarized epithelium. Despite their great potential in research and therapy, epithelial organoids grow in heterogeneous sizes, and are too small to display physiologically relevant performance and applications. Here, we show directed assembly of mouse tracheal basal stem cell organoids towards geometrically-defined, lumenized constructs. We demonstrate perfusion of the macroscopic organoid construct; and the transplantation of the construct devoid of a carrier matrix, in a de-epithelialized explant model. Drawing parallel to liquid droplet interaction, we provide hypothesis on how epithelial organoid assembling can be achieved in a more efficient and predictable manner, of which principles could be extended to other organoid types developed form epithelial stem cells. The guided self-assembly system presented here opens up the possibility for engineering size-relevant, geometrically defined epithelial structures towards broad applications in biomimetic organoid-devices and tissue engineering.

Bio-inspired nanotechnology aspires to harness natural compounds, their chemical derivatives and formed supramolecular structures for various technological applications. In recent years, a key direction in the electronics and electro-optics technologies involves the transition from inorganic to organic components, including organic light emitting diodes (OLED), thus paving the way towards flexible and wearable electronic and light emitting devices. Bio-inspired organic materials may be the next-generation of organic optoelectronic devices based on self-organization principles, which allow facile synthesis, eco-friendliness, resistance to oxidation and no need for heavy metal doping.
We and others, have taken a reductionist approach to form new materials based on self-assembled peptide structures. We were the first to demonstrate that ultrashort peptides can form well-ordered nanostructures such as nanotubes and nanospheres. Specifically, the diphenylalanine peptide (FF) self-assemblies have been shown to display intriguing features, including mechanical, optical, semiconductive and piezoelectric properties. We thoroughly characterized mechanisms that facilitate peptide self- and co-assembly and the structural basis for the resulting physical properties. Several studies have explored the piezoelectric properties of the FF peptide. In the presence of an external electric field, vertically aligned FF microrod arrays can be organized on a substrate, resulting in enhanced piezoelectric response.
Here we show the ability of FF and other similar peptide assemblies to be used in various electronics and optics application as new bioorganic materials. FF assemblies and derivatives can act as an active optical waveguiding material, allowing locally excited states to propagate along the axis of the assemblies. In addition, Fmoc capped building blocks exhibit remarkable optical properties, such as quantum confinement and fluorescence. In addition, We based on our reductionist approach, to expand our search for minimal building blocks towards single amino acids as well as other metabolites such as nucleobases, demonstrating their self-assembly into various ordered structures. Doing this we are enlarging our library of biological building blocks which bear the potential to be novel bio-inspired supramolecular materials for optics and electronic applications.