Recellularization and Integration of Dense Extracellular Matrix by Percolation of Tissue Microparticles.

Cells embedded in the extracellular matrix of tissues play a critical role in maintaining homeostasis while promoting integration and regeneration following damage or disease. Emerging engineered biomaterials utilize decellularized extracellular matrix as a tissue-specific support structure; however, many dense, structured biomaterials unfortunately demonstrate limited formability, fail to promote cell migration, and result in limited tissue repair. Here, we developed a reinforced composite material of densely packed acellular extracellular matrix microparticles in a hydrogel, termed tissue clay, that can be molded and crosslinked to mimic native tissue architecture. We utilized hyaluronic acid-based hydrogels, amorphously packed with acellular articular cartilage tissue particulated to ~125-250 microns in diameter and defined a percolation threshold of 0.57 (v/v) beyond which the compressive modulus exceeded 300kPa. Remarkably, primary chondrocytes recellularized particles within 48 hours, a process driven by chemotaxis, exhibited distributed cellularity in large engineered composites, and expressed genes consistent with native cartilage repair. We additionally demonstrated broad utility of tissue clays through recellularization and persistence of muscle, skin, and cartilage composites in a subcutaneous in vivo mouse model. Our findings suggest optimal strategies and material architectures to balance concurrent demands for large-scale mechanical properties while also supporting recellularization and integration of dense musculoskeletal and connective tissues. TABLE OF CONTENTS ENTRY: We present a new design framework for regenerative articular cartilage scaffolds using acellular extracellular matrix particles, packed beyond a percolation threshold, and crosslinked within chondroinductive hydrogels. Our results suggest that the architecture and the packing, rather than altering the individual components, creates a composite material that can balance mechanics, porosity to enable migration, and tissue specific biochemical interactions with cells. Moreover, we provide a technique that we show is applicable to other tissue types.

Enhanced diffusion by reversible binding to active polymers.

Cells are known to use reversible binding to active biopolymer networks to allow diffusive transport of particles in an otherwise impenetrable mesh. We here determine the motion of a particle that experiences random forces during binding and unbinding events while being constrained by attached polymers. Using Monte-Carlo simulations and a statistical mechanics model, we find that enhanced diffusion is possible with active polymers. However, this is possible only under optimum conditions that has to do with the relative length of the chains to that of the plate. For example, in systems where the plate is shorter than the chains, diffusion is maximum when many chains have the potential to bind but few remain bound at any one time. Interestingly, if the chains are shorter than the plate, we find that diffusion is maximized when more active chains remain transiently bound. The model provides insight into these findings by elucidating the mechanisms for binding-mediated diffusion in biology and design rules for macromolecular transport in transient synthetic polymers.

Helical growth during the phototropic response, avoidance response, and in stiff mutants of Phycomyces blakesleeanus.

The sporangiophores of Phycomyces blakesleeanus have been used as a model system to study sensory transduction, helical growth, and to establish global biophysical equations for expansive growth of walled cells. More recently, local statistical biophysical models of the cell wall are being constructed to better understand the molecular underpinnings of helical growth and its behavior during the many growth responses of the sporangiophores to sensory stimuli. Previous experimental and theoretical findings guide the development of these local models. Future development requires an investigation of explicit and implicit assumptions made in the prior research. Here, experiments are conducted to test three assumptions made in prior research, that (a) elongation rate, (b) rotation rate, and (c) helical growth steepness, R, of the sporangiophore remain constant during the phototropic response (bending toward unilateral light) and the avoidance response (bending away from solid barriers). The experimental results reveal that all three assumptions are incorrect for the phototropic response and probably incorrect for the avoidance response but the results are less conclusive. Generally, the experimental results indicate that the elongation and rotation rates increase during these responses, as does R, indicating that the helical growth steepness become flatter. The implications of these findings on prior research, the "fibril reorientation and slippage" hypothesis, global biophysical equations, and local statistical biophysical models are discussed.

Separating the contributions of zona pellucida and cytoplasm in the viscoelastic response of human oocytes.

The successful characterization of the mechanical properties of human oocytes and young embryos is of crucial relevance to reduce the risk of pregnancy arrest in in-vitro fertilization processes. Unfortunately, current study has been hindered by the lack of accuracy in describing the mechanical contributions of each structure (zona pellucida, cytoplasm) due to its high heterogeneity. In this work, we present a novel approach to model the oocyte response taking into account the effect of both zona and cytoplasm, as well as different loading conditions. The model is then applied to develop an experimental protocol capable of accurately separating the viscoelastic contribution of zona and cytoplasm by simply varying the loading condition. This new protocol has the potential to open the door to improving our understanding the mechanical properties of oocytes at different stages, and provide a quantitative predictive ability to the evaluation of oocyte quality. STATEMENT OF SIGNIFICANCE: Assisted reproductive technologies, such as in vitro fertilization, often rely on identifying high quality oocytes or female egg cells. The viscoelastic properties of these cells, such as stiffness and stress relaxation time, have been identified as potential objective indicators of cell quality. However, their characterization has proven difficult due to the structural heterogeneity of the cell and inconsistent loading conditions. This paper presents a new model that, although simple, addresses the above difficulties to provide accurate estimations of the cell's mechanical properties. Learning from this model, we then propose a novel non-invasive testing protocol to allow oocyte characterization with increased accuracy. We believe this effort would improve consistency in measurements and enhance our knowledge on the mechanics of oocytes.

How do fire ants control the rheology of their aggregations? A statistical mechanics approach.

Active networks are omnipresent in nature, from the molecular to the macro-scale. In this study, we explore the mechanical behaviour of fire ant aggregations, closely knit swarms that display impressive dynamics culminating with the aggregations' capacity to self-heal and adapt to the environment. Although the combined elasticity and rheology of the ant aggregation can be characterized by phenomenological mechanical models (e.g. linear Maxwell or Kelvin-Voigt model), it is not clear how the behaviour of individual ants affects the aggregations' emerging responses. Here, we explore an alternative way to think about these materials, describing them as a collection of individuals connected via elastic chains that associate and dissociate over time. Using our knowledge of these connections-e.g. their elasticity and attachment/dissociation rates-we construct a statistical description of connection stretch and derive an evolution equation for the corresponding stretch distribution. This time-evolving stretch distribution is then used to determine important macroscopic measures, e.g. stress, energy storage and energy dissipation, in the network. In this context, we show how the physical characteristics and activities of individual ants can explain the elasticity, flow and shear thinning of the aggregation. In particular, we find that experimental results are matched if the detachment rate between two individuals increases with tension in the connection.

* Understanding the Spatiotemporal Degradation Behavior of Aggrecanase-Sensitive Poly(ethylene glycol) Hydrogels for Use in Cartilage Tissue Engineering.

Enzyme-sensitive hydrogels are promising cell delivery vehicles for cartilage tissue engineering. However, a better understanding of their spatiotemporal degradation behavior and its impact on tissue growth is needed. The goal of this study was to combine experimental and computational approaches to provide new insights into spatiotemporal changes in hydrogel crosslink density and extracellular matrix (ECM) growth and how these changes influence the evolving macroscopic properties as a function of time. Hydrogels were designed from aggrecanase-sensitive peptide crosslinks using a simple and robust thiol-norbornene photoclick reaction. To study the influence of variations in cellular activity of different donors, chondrocytes were isolated from either juvenile or adult bovine donors. Initial studies were performed to validate and calibrate the model against experiments. Through this process, two key features were identified. These included spatial variations in the hydrogel crosslink density in the immediate vicinity of the cell and the presence of cell clustering within the construct. When these spatial heterogeneities were incorporated into the computational model along with model inputs of initial hydrogel properties and cellular activity (i.e., enzyme and ECM production rates), the model was able to capture the spatial and temporal evolution of ECM growth that was observed experimentally for both donors. In this study, the juvenile chondrocytes produced an interconnected matrix within the cell clusters leading to overall improved ECM growth, while the adult chondrocytes resulted in poor ECM growth. Overall, the computational model was able to capture the spatiotemporal ECM growth of two different donors and provided new insights into the importance of spatial heterogeneities in facilitating ECM growth. Our long-term goal is to use this model to predict optimal hydrogel designs for a wide range of donors and improve cartilage tissue engineering.

Local Heterogeneities Improve Matrix Connectivity in Degradable and Photoclickable Poly(ethylene glycol) Hydrogels for Applications in Tissue Engineering.

Hydrolytically degradable poly(ethylene glycol) (PEG) hydrogels are promising platforms for cell encapsulation and tissue engineering. However, hydrolysis leads to bulk degradation and a decrease in hydrogel mechanical integrity. Despite these challenges, hydrolytically degradable hydrogels have supported macroscopic neotissue growth. The goal of this study was to combine experimental methods with a multiscale mathematical model to analyze hydrogel degradation concomitant with neocartilage growth in PEG hydrogels. Primary bovine chondrocytes were encapsulated at increasing densities (50, 100, and 150 million cells/mL of precursor solution) in a radical-mediated photoclickable hydrogel formed from 8-arm PEG-co-caprolactone end-capped with norbornene and cross-linked with PEG dithiol. Two observations were made in the experimental system: (1) the cell distribution was not uniform and cell clustering was evident, which increased with increasing cell density and (2) a significant decrease in the initial hydrogel compressive modulus was observed with increasing cell concentration. By introducing heterogeneities in the form of cell clusters and spatial variations in the network structure around cells, the mathematical model explained the drop in initial modulus and captured the experimentally observed spatial evolution of ECM and the construct modulus as a function of cell density and culture time. Overall, increasing cell density led to improved ECM formation, ECM connectivity, and overall modulus. This study strongly points to the importance of heterogeneities within a cell-laden hydrogel in retaining mechanical integrity as the construct transitions from hydrogel to neotissue.