Modeling collagen fibril self-assembly from extracellular medium in embryonic tendon.

Collagen is a key structural component of multicellular organisms and is arranged in a highly organized manner. In structural tissues such as tendons, collagen forms bundles of parallel fibers between cells, which appear within a 24-h window between embryonic day 13.5 (E13.5) and E14.5 during mouse embryonic development. Current models assume that the organized structure of collagen requires direct cellular control, whereby cells actively lay down collagen fibrils from cell surfaces. However, such models appear incompatible with the time and length scales of fibril formation. We propose a phase-transition model to account for the rapid development of ordered fibrils in embryonic tendon, reducing reliance on active cellular processes. We develop phase-field crystal simulations of collagen fibrillogenesis in domains derived from electron micrographs of inter-cellular spaces in embryonic tendon and compare results qualitatively and quantitatively to observed patterns of fibril formation. To test the prediction of this phase-transition model that free protomeric collagen should exist in the inter-cellular spaces before the formation of observable fibrils, we use laser-capture microdissection, coupled with mass spectrometry, which demonstrates steadily increasing free collagen in inter-cellular spaces up to E13.5, followed by a rapid reduction of free collagen that coincides with the appearance of less-soluble collagen fibrils. The model and measurements together provide evidence for extracellular self-assembly of collagen fibrils in embryonic mouse tendon, supporting an additional mechanism for rapid collagen fibril formation during embryonic development.

Couple stresses and discrete potentials in the vertex model of cellular monolayers.

The vertex model is widely used to simulate the mechanical properties of confluent epithelia and other multicellular tissues. This inherently discrete framework allows a Cauchy stress to be attributed to each cell, and its symmetric component has been widely reported, at least for planar monolayers. Here, we consider the stress attributed to the neighbourhood of each tricellular junction, evaluating in particular its leading-order antisymmetric component and the associated couple stresses, which characterise the degree to which individual cells experience (and resist) in-plane bending deformations. We develop discrete potential theory for localised monolayers having disordered internal structure and use this to derive the analogues of Airy and Mindlin stress functions. These scalar potentials typically have broad-banded spectra, highlighting the contributions of small-scale defects and boundary layers to global stress patterns. An affine approximation attributes couple stresses to pressure differences between cells sharing a trijunction, but simulations indicate an additional role for non-affine deformations.

Notch controls the cell cycle to define leader versus follower identities during collective cell migration.

Coordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, such as cancer. How cell cycle progression affects migration, and vice versa, remains largely unknown. We address these questions by combining in silico modelling and in vivo experimentation in the zebrafish trunk neural crest (TNC). TNC migrate collectively, forming chains with a leader cell directing the movement of trailing followers. We show that the acquisition of migratory identity is autonomously controlled by Notch signalling in TNC. High Notch activity defines leaders, while low Notch determines followers. Moreover, cell cycle progression is required for TNC migration and is regulated by Notch. Cells with low Notch activity stay longer in G1 and become followers, while leaders with high Notch activity quickly undergo G1/S transition and remain in S-phase longer. In conclusion, TNC migratory identities are defined through the interaction of Notch signalling and cell cycle progression.

Cell surface fluctuations regulate early embryonic lineage sorting.

In development, lineage segregation is coordinated in time and space. An important example is the mammalian inner cell mass, in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the fetus). While the molecular requirements have been well studied, the physical mechanisms determining spatial segregation between EPI and PrE remain elusive. Here, we investigate the mechanical basis of EPI and PrE sorting. We find that rather than the differences in static cell surface mechanical parameters as in classical sorting models, it is the differences in surface fluctuations that robustly ensure physical lineage sorting. These differential surface fluctuations systematically correlate with differential cellular fluidity, which we propose together constitute a non-equilibrium sorting mechanism for EPI and PrE lineages. By combining experiments and modeling, we identify cell surface dynamics as a key factor orchestrating the correct spatial segregation of the founder embryonic lineages.

Collagen fibril assembly: New approaches to unanswered questions.

Collagen fibrils are essential for metazoan life. They are the largest, most abundant, and most versatile protein polymers in animals, where they occur in the extracellular matrix to form the structural basis of tissues and organs. Collagen fibrils were first observed at the turn of the 20th century. During the last 40 years, the genes that encode the family of collagens have been identified, the structure of the collagen triple helix has been solved, the many enzymes involved in the post-translational modifications of collagens have been identified, mutations in the genes encoding collagen and collagen-associated proteins have been linked to heritable disorders, and changes in collagen levels have been associated with a wide range of diseases, including cancer. Yet despite extensive research, a full understanding of how cells assemble collagen fibrils remains elusive. Here, we review current models of collagen fibril self-assembly, and how cells might exert control over the self-assembly process to define the number, length and organisation of fibrils in tissues.

Force-based three-dimensional model predicts mechanical drivers of cell sorting.

Many biological processes, including tissue morphogenesis, are driven by cell sorting. However, the primary mechanical drivers of sorting in multicellular aggregates (MCAs) remain controversial, in part because there is no appropriate computational model to probe mechanical interactions between cells. To address this important issue, we developed a three-dimensional, local force-based simulation based on the subcellular element method. In our method, cells are modelled as collections of locally interacting force-bearing elements. We use the method to investigate the effects of tension and cell-cell adhesion on MCA sorting. We predict a minimum level of adhesion to produce inside-out sorting of two cell types, which is in excellent agreement with observations in several developmental systems. We also predict the level of tension asymmetry needed for robust sorting. The generality and flexibility of the method make it applicable to tissue self-organization in a myriad of other biological processes, such as tumorigenesis and embryogenesis.

A Physics-Inspired Mechanistic Model of Migratory Movement Patterns in Birds.

In this paper, we introduce a mechanistic model of migratory movement patterns in birds, inspired by ideas and methods from physics. Previous studies have shed light on the factors influencing bird migration but have mainly relied on statistical correlative analysis of tracking data. Our novel method offers a bottom up explanation of population-level migratory movement patterns. It differs from previous mechanistic models of animal migration and enables predictions of pathways and destinations from a given starting location. We define an environmental potential landscape from environmental data and simulate bird movement within this landscape based on simple decision rules drawn from statistical mechanics. We explore the capacity of the model by qualitatively comparing simulation results to the non-breeding migration patterns of a seabird species, the Black-browed Albatross (Thalassarche melanophris). This minimal, two-parameter model was able to capture remarkably well the previously documented migration patterns of the Black-browed Albatross, with the best combination of parameter values conserved across multiple geographically separate populations. Our physics-inspired mechanistic model could be applied to other bird and highly-mobile species, improving our understanding of the relative importance of various factors driving migration and making predictions that could be useful for conservation.

Chondroitinase ABC treatment results in greater tensile properties of self-assembled tissue-engineered articular cartilage.

Collagen content and tensile properties of engineered articular cartilage have remained inferior to glycosaminoglycan (GAG) content and compressive properties. Based on a cartilage explant study showing greater tensile properties after chondroitinase ABC (C-ABC) treatment, C-ABC as a strategy for cartilage tissue engineering was investigated. A scaffold-less approach was employed, wherein chondrocytes were seeded into non-adherent agarose molds. C-ABC was used to deplete GAG from constructs 2 weeks after initiating culture, followed by 2 weeks culture post-treatment. Staining for GAG and type I, II, and VI collagen and transmission electron microscopy were performed. Additionally, quantitative total collagen, type I and II collagen, and sulfated GAG content were measured, and compressive and tensile mechanical properties were evaluated. At 4 wks, C-ABC treated construct ultimate tensile strength and tensile modulus increased 121% and 80% compared to untreated controls, reaching 0.5 and 1.3 MPa, respectively. These increases were accompanied by increased type II collagen concentration, without type I collagen. As GAG returned, compressive stiffness of C-ABC treated constructs recovered to be greater than 2 wk controls. C-ABC represents a novel method for engineering functional articular cartilage by departing from conventional anabolic approaches. These results may be applicable to other GAG-producing tissues functioning in a tensile capacity, such as the musculoskeletal fibrocartilages.

Success rates and immunologic responses of autogenic, allogenic, and xenogenic treatments to repair articular cartilage defects.

This review examines current approaches available for articular cartilage repair, not only in terms of their regeneration potential, but also as a function of immunologic response. Autogenic repair techniques, including osteochondral plug transplantation, chondrocyte implantation, and microfracture, are the most widely accepted clinical treatment options due to the lack of immunogenic reactions, but only moderate graft success rates have been reported. Although suspended allogenic chondrocytes are shown to evoke an immune response upon implantation, allogenic osteochondral plugs and tissue-engineered grafts using allogenic chondrocytes exhibit a tolerable immunogenic response. Additionally, these repair techniques produce neotissue with success rates approaching those of currently available autogenic repair techniques, while simultaneously obviating their major hindrance of donor tissue scarcity. To date, limited research has been performed with xenogenic tissue, although several studies demonstrate the potential for its long-term success. This article focuses on the various treatment options for cartilage repair and their associated success rates and immunologic responses.

Matrix development in self-assembly of articular cartilage.

BACKGROUND: Articular cartilage is a highly functional tissue which covers the ends of long bones and serves to ensure proper joint movement. A tissue engineering approach that recapitulates the developmental characteristics of articular cartilage can be used to examine the maturation and degeneration of cartilage and produce fully functional neotissue replacements for diseased tissue. METHODOLOGY/PRINCIPAL FINDINGS: This study examined the development of articular cartilage neotissue within a self-assembling process in two phases. In the first phase, articular cartilage constructs were examined at 1, 4, 7, 10, 14, 28, 42, and 56 days immunohistochemically, histologically, and through biochemical analysis for total collagen and glycosaminoglycan (GAG) content. Based on statistical changes in GAG and collagen levels, four time points from the first phase (7, 14, 28, and 56 days) were chosen to carry into the second phase, where the constructs were studied in terms of their mechanical characteristics, relative amounts of collagen types II and VI, and specific GAG types (chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronan). Collagen type VI was present in initial abundance and then localized to a pericellular distribution at 4 wks. N-cadherin activity also spiked at early stages of neotissue development, suggesting that self-assembly is mediated through a minimization of free energy. The percentage of collagen type II to total collagen significantly increased over time, while the proportion of collagen type VI to total collagen decreased between 1 and 2 wks. The chondroitin 6- to 4- sulfate ratio decreased steadily during construct maturation. In addition, the compressive properties reached a plateau and tensile characteristics peaked at 4 wks. CONCLUSIONS/SIGNIFICANCE: The indices of cartilage formation examined in this study suggest that tissue maturation in self-assembled articular cartilage mirrors known developmental processes for native tissue. In terms of tissue engineering, it is suggested that exogenous stimulation may be necessary after 4 wks to further augment the functionality of developing constructs.

Effects of initial cell seeding in self assembly of articular cartilage.

Current forays into tissue engineering of articular cartilage in vitro using the self-assembling method have produced constructs possessing significant extracellular matrix and resulting mechanical properties. However, large numbers of native articular chondrocytes are necessary to produce functional engineered cartilage; all previous work with the self-assembling process has used 5.5 x 10(6) cells/construct. In this study, the effects of initial cell seeding (0.25-11 x 10(6) cells/construct) on tissue quality were investigated. Results showed that tissue engineered articular cartilage was formed, when using at least 2 million cells/construct, possessing dimensional, compositional, and compressive properties approaching those of native tissue. It was noted that higher seeding contributed to thicker constructs with larger diameters and had a significant effect on resulting biochemical and biomechanical properties. It was further observed that aggregate modulus increased with increased seeding. By combining gross morphological, histological, biochemical, and biomechanical results, an optimal initial seeding for the self-assembling process of 3.75 x 10(6) cells/construct was identified. This finding enhances the translatability of this tissue engineering process by reducing the number of cells needed for tissue engineering of articular cartilage by 32% while maintaining essential tissue properties.

Characterization of fibroblast morphology on bioactive surfaces using vertical scanning interferometry.

Tissue donor scarcity is a major hindrance to articular cartilage tissue engineering. Previous research shows that dermal fibroblasts express chondrocytic markers after seeding on aggrecan-coated surfaces. Since cell roundness appears to correlate with chondrocytic behavior of dermal fibroblasts, this study quantified roundness by measuring cell height and surface area-volume ratio. In addition to aggrecan as a surface coating, collagen type II and decorin, two other major extracellular matrix components of articular cartilage, were examined. Aggrecan, collagen type II, and decorin were coated onto a glass substrate using three application techniques: static drying, airbrush, and painting. Vertical scanning interferometry (VSI) is a novel technique that allows for the expedient morphological determination of single cells. Interferometry was used for the characterization of protein-coated surfaces in addition to characterizing the morphology of single dermal fibroblasts after 24 h of seeding. Fibroblast height was found to vary from 1.0 to 4.0 microm and protein coating, application technique, and seeding position were significant factors (p < 0.002). The largest cell heights were observed on aggrecan and collagen type II coated surfaces using the air brush and static applications. Additionally, variations were observed for surface area-volume ratio, ranging from 1.75 to 11.94 microm(-1) with decorin resulting in the lowest ratio, followed by collagen type II and aggrecan. This study identifies optimal coating conditions for stimulating morphology in dermal fibroblasts that is characteristic of the chondrocytic phenotype. These conditions can be employed to attempt articular cartilage regeneration and bypass difficulties due to a paucity of donor tissue.