Twisted-plywood-like tissue formation in vitro. Does curvature do the twist?

Little is known about the contribution of 3D surface geometry to the development of multilayered tissues containing fibrous extracellular matrix components, such as those found in bone. In this study, we elucidate the role of curvature in the formation of chiral, twisted-plywood-like structures. Tissues consisting of murine preosteoblast cells (MC3T3-E1) were grown on 3D scaffolds with constant-mean curvature and negative Gaussian curvature for up to 32 days. Using 3D fluorescence microscopy, the influence of surface curvature on actin stress-fiber alignment and chirality was investigated. To gain mechanistic insights, we did experiments with MC3T3-E1 cells deficient in nuclear A-type lamins or treated with drugs targeting cytoskeleton proteins. We find that wild-type cells form a thick tissue with fibers predominantly aligned along directions of negative curvature, but exhibiting a twist in orientation with respect to older tissues. Fiber orientation is conserved below the tissue surface, thus creating a twisted-plywood-like material. We further show that this alignment pattern strongly depends on the structural components of the cells (A-type lamins, actin, and myosin), showing a role of mechanosensing on tissue organization. Our data indicate the importance of substrate curvature in the formation of 3D tissues and provide insights into the emergence of chirality.

On shape forming by contractile filaments in the surface of growing tissues.

Growing tissues are highly dynamic, and flow on sufficiently long timescales due to cell proliferation, migration, and tissue remodeling. As a consequence, growing tissues can often be approximated as viscous fluids. This means that the shape of microtissues growing in vitro is governed by their surface stress state, as in fluid droplets. Recent work showed that cells in the near-surface region of fibroblastic or osteoblastic microtissues contract with highly oriented actin filaments, thus making the surface properties highly anisotropic, in contrast to what is expected for an isotropic fluid. Here, we develop a model that includes mechanical anisotropy of the surface generated by contractile fibers and we show that mechanical equilibrium requires contractile filaments to follow geodesic lines on the surface. Constant pressure in the fluid forces these contractile filaments to be along geodesics with a constant normal curvature. We then take this into account to determine equilibrium shapes of rotationally symmetric bodies subjected to anisotropic surface stress states and derive a family of surfaces of revolution. A comparison with recently published shapes of microtissues shows that this theory accurately predicts both the surface shape and the direction of the actin filaments on the surface.

Curvature in Biological Systems: Its Quantification, Emergence, and Implications across the Scales.

Surface curvature both emerges from, and influences the behavior of, living objects at length scales ranging from cell membranes to single cells to tissues and organs. The relevance of surface curvature in biology is supported by numerous experimental and theoretical investigations in recent years. In this review, first, a brief introduction to the key ideas of surface curvature in the context of biological systems is given and the challenges that arise when measuring surface curvature are discussed. Giving an overview of the emergence of curvature in biological systems, its significance at different length scales becomes apparent. On the other hand, summarizing current findings also shows that both single cells and entire cell sheets, tissues or organisms respond to curvature by modulating their shape and their migration behavior. Finally, the interplay between the distribution of morphogens or micro-organisms and the emergence of curvature across length scales is addressed with examples demonstrating these key mechanistic principles of morphogenesis. Overall, this review highlights that curved interfaces are not merely a passive by-product of the chemical, biological, and mechanical processes but that curvature acts also as a signal that co-determines these processes.

Wall Shear Stress Analysis and Optimization in Tissue Engineering TPMS Scaffolds.

When designing scaffolds for bone tissue engineering (BTE), the wall shear stress (WSS), due to the fluid flow inside the scaffold, is an important factor to consider as it influences the cellular process involved in new tissue formation. The present work analyzed the average WSS in Schwartz diamond (SD) and gyroid (SG) scaffolds with different surface topologies and mesh elements using computational fluid dynamics (CFD) analysis. It was found that scaffold meshes with a smooth surface topology with tetrahedral elements had WSS levels 35% higher than the equivalent scaffold with a non-smooth surface topology with hexahedral elements. The present work also investigated the possibility of implementing the optimization algorithm simulated annealing to aid in the design of BTE scaffolds with a specific average WSS, with the outputs showing that the algorithm was able to reach WSS levels in the vicinity of 5 mPa (physiological range) within the established limit of 100 iterations. This proved the efficacy of combining CFD and optimization methods in the design of BTE scaffolds.

Challenges in computational fluid dynamics applications for bone tissue engineering.

Bone injuries or defects that require invasive surgical treatment are a serious clinical issue, particularly when it comes to treatment success and effectiveness. Accordingly, bone tissue engineering (BTE) has been researching the use of computational fluid dynamics (CFD) analysis tools to assist in designing optimal scaffolds that better promote bone growth and repair. This paper aims to offer a comprehensive review of recent studies that use CFD analysis in BTE. The mechanical and fluidic properties of a given scaffold are coupled to each other via the scaffold architecture, meaning an optimization of one may negatively affect the other. For example, designs that improve scaffold permeability normally result in a decreased average wall shear stress. Linked with these findings, it appears there are very few studies in this area that state a specific application for their scaffolds and those that do are focused on in vitro bioreactor environments. Finally, this review also demonstrates a scarcity of studies that combine CFD with optimization methods to improve scaffold design. This highlights an important direction of research for the development of the next generation of BTE scaffolds.

Rubbing Powders: Direct Spectroscopic Observation of Triboinduced Oxygen Radical Formation in MgO Nanocube Ensembles.

Powder compaction-induced surface chemistry in metal oxide nanocrystal ensembles is important for very diverse fields such as triboelectrics, tribocatalysts, surface abrasion, and cold sintering of ceramics. Using a range of spectroscopic techniques, we show that MgO nanocube powder compaction with uniaxial pressures that can be achieved by gentle manual rubbing or pressing (p ≥ 5 MPa) excites energetic electron-hole pairs and generates oxygen radicals at interfacial defect structures. While the identification of paramagnetic O- radicals and their adsorption complexes with O2 point to the emergence of hole centers, triboemitted electrons become scavenged by molecular oxygen to convert into adsorbed superoxide anions O2 - as measured by electron paramagnetic resonance (EPR). By means of complementary UV-photoexcitation experiments, we found that photon energies in the range between 3 and 6 eV produce essentially the same EPR spectroscopic fingerprints and optical absorption features. To provide insights into this effect, we performed density functional theory calculations to explore the energetics of charge separation involving the ionization of low-coordinated anions and surface-adsorbed O2 - radicals at points of contact. For all selected configurations, charge transfer is not spontaneous but requires an additional driving force. We propose that a plausible mechanism for oxygen radical formation is the generation of significant surface potential differences at points of contact under loading as a result of the highly inhomogeneous elastic deformations coupled with the flexoelectric effect.

Advanced materials design based on waste wood and bark.

Trees belong to the largest living organisms on Earth and plants in general are one of our main renewable resources. Wood as a material has been used since the beginning of humankind. Today, forestry still provides raw materials for a variety of applications, for example in the building industry, in paper manufacturing and for various wood products. However, many parts of the tree, such as reaction wood, branches and bark are often discarded as forestry residues and waste wood, used as additives in composite materials or burned for energy production. More advanced uses of bark include the extraction of chemical substances for glues, food additives or healthcare, as well as the transformation to advanced carbon materials. Here, we argue that a proper understanding of the internal fibrous structure and the resulting mechanical behaviour of these forest residues allows for the design of materials with greatly varying properties and applications. We show that simple and cheap treatments can give tree bark a leather-like appearance that can be used for the construction of shelters and even the fabrication of woven textiles. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'.

Surface tension determines tissue shape and growth kinetics.

The collective self-organization of cells into three-dimensional structures can give rise to emergent physical properties such as fluid behavior. Here, we demonstrate that tissues growing on curved surfaces develop shapes with outer boundaries of constant mean curvature, similar to the energy minimizing forms of liquids wetting a surface. The amount of tissue formed depends on the shape of the substrate, with more tissue being deposited on highly concave surfaces, indicating a mechano-biological feedback mechanism. Inhibiting cell-contractility further revealed that active cellular forces are essential for generating sufficient surface stresses for the liquid-like behavior and growth of the tissue. This suggests that the mechanical signaling between cells and their physical environment, along with the continuous reorganization of cells and matrix is a key principle for the emergence of tissue shape.

Protecting Offspring Against Fire: Lessons From Banksia Seed Pods.

Wildfires are a natural component in many terrestrial ecosystems and often play a crucial role in maintaining biodiversity, particularly in the fire-prone regions of Australia. A prime example of plants that are able to persist in these regions is the genus Banksia. Most Banksia species that occur in fire-prone regions produce woody seed pods (follicles), which open during or soon after fire to release seeds into the post-fire environment. For population persistence, many Banksia species depend on recruitment from these canopy-stored seeds. Therefore, it is critical that their seeds are protected from heat and rapid oxidation during fire. Here, we show how different species of Banksia protect their seeds inside follicles while simultaneously opening up when experiencing fire. The ability of the follicles to protect seeds from heat is demonstrated by intense 180 s experimental burns, in which the maximum temperatures near the seeds ranged from ∼75°C for B. serrata to ∼90°C for B. prionotes and ∼95°C for B. candolleana, contrasting with the mean surface temperature of ∼450°C. Many seeds of native Australian plants, including those of Banksia, are able to survive these temperatures. Structural analysis of individual follicles from these three Banksia species demonstrates that all of them rely on a multicomponent system, consisting of two valves, a porous separator and a thin layer of air surrounding the seeds. The particular geometric arrangement of these components determines the rate of heat transfer more than the tissue properties alone, revealing that a strong embedment into the central rachis can compensate for thin follicle valves. Furthermore, we highlight the role of the separator as an important thermal insulator. Our study suggests that the genus Banksia employs a variety of combinations in terms of follicle size, valve thickness, composition and geometric arrangement to effectively protect canopy-stored seeds during fire.

Organic Molecule-Driven Polymeric Actuators.

Inspired by the motions of plant tissues in response to external stimuli, significant attention has been devoted to the development of actuating polymeric materials. In particular, polymeric actuators driven by organic molecules have been designed due to their combined superiorities of tunable functional monomers, designable chemical structures, and variable structural anisotropy. Here, the recent progress is summarized in terms of material synthesis, structure design, polymer-solvent interaction, and actuating performance. In addition, various possibilities for practical applications, including the ability to sense chemical vapors and solvent isomers, and future directions to satisfy the requirement of sensing and smart systems are also highlighted.

Comparative in situ analysis reveals the dynamic nature of sclerenchyma cell walls of the fern Asplenium rutifolium.

Background and Aims: A key structural adaptation of vascular plants was the evolution of specialized vascular and mechanical tissues, innovations likely to have generated novel cell wall architectures. While collenchyma is a strengthening tissue typically found in growing organs of angiosperms, a similar tissue occurs in the petiole of the fern Asplenium rutifolium. Methods: The in situ cell wall (ultra)structure and composition of this tissue was investigated and characterized mechanically as well as structurally through nano-indentation and wide-angle X-ray diffraction, respectively. Key Results: Structurally the mechanical tissue resembles sclerenchyma, while its biomechanical properties and molecular composition both share more characteristics with angiosperm collenchyma. Cell wall thickening only occurs late during cell expansion or after cell expansion has ceased. Conclusions: If the term collenchyma is reserved for walls that thicken during expansive growth, the mechanical tissue in A. rutifolium represents sclerenchyma that mimics the properties of collenchyma and has the ability to modify its mechanical properties through sclerification. These results support the view that collenchyma does not occur in ferns and most probably evolved in angiosperms.

Tensile forces drive a reversible fibroblast-to-myofibroblast transition during tissue growth in engineered clefts.

Myofibroblasts orchestrate wound healing processes, and if they remain activated, they drive disease progression such as fibrosis and cancer. Besides growth factor signaling, the local extracellular matrix (ECM) and its mechanical properties are central regulators of these processes. It remains unknown whether transforming growth factor-ß (TGF-ß) and tensile forces work synergistically in up-regulating the transition of fibroblasts into myofibroblasts and whether myofibroblasts undergo apoptosis or become deactivated by other means once tissue homeostasis is reached. We used three-dimensional microtissues grown in vitro from fibroblasts in macroscopically engineered clefts for several weeks and found that fibroblasts transitioned into myofibroblasts at the highly tensed growth front as the microtissue progressively closed the cleft, in analogy to closing a wound site. Proliferation was up-regulated at the growth front, and new highly stretched fibronectin fibers were deposited, as revealed by fibronectin fluorescence resonance energy transfer probes. As the tissue was growing, the ECM underneath matured into a collagen-rich tissue containing mostly fibroblasts instead of myofibroblasts, and the fibronectin fibers were under reduced tension. This correlated with a progressive rounding of cells from the growth front inward, with decreased α-smooth muscle actin expression, YAP nuclear translocation, and cell proliferation. Together, this suggests that the myofibroblast phenotype is stabilized at the growth front by tensile forces, even in the absence of endogenously supplemented TGF-ß, and reverts into a quiescent fibroblast phenotype already 10 µm behind the growth front, thus giving rise to a myofibroblast-to-fibroblast transition. This is the hallmark of reaching prohealing homeostasis.

Scaffold curvature-mediated novel biomineralization process originates a continuous soft tissue-to-bone interface.

A myriad of shapes are found in biological tissues, often naturally evolved to fulfill a particular function. In the field of tissue engineering, substrate geometry influences cell behavior and tissue formation in vitro, yet little is known how this translates to an in vivo scenario. Here we investigate scaffold curvature-induced tissue growth, without additional growth factors or cells, in an ovine animal model. We show that soft tissue formation follows a curvature-driven tissue growth model. The highly organized endogenous soft matrix, potentially under mechanical strain, leads to a non-standard form of biomineralization, whereby the pre-existing organic matrix is mineralized without collagen remodeling and without an intermediate cartilage ossification phase. Micro- and nanoscale characterization of the tissue microstructure using histology, backscattered electron (BSE) and second-harmonic generation (SHG) imaging and synchrotron small angle X-ray scattering (SAXS) revealed (i) continuous collagen fibers across the soft-hard tissue interface on the tip of mineralized cones, and (ii) bone remodeling by basic multicellular units (BMUs) in regions adjacent to the native cortical bone. Thus, features of soft tissue-to-bone interface resembling the insertion sites of ligaments and tendons into bone were created, using a scaffold that did not mimic the structural or biological gradients across such a complex interface at its mature state. This study provides fundamental knowledge for biomimetic scaffold design in the fields of bone regeneration and soft tissue-to-bone interface tissue engineering. STATEMENT OF SIGNIFICANCE: Geometry influences cell behavior and tissue formation in vitro. However, little is known how this translates to an in vivo scenario. Here we investigate the influence of scaffold mean surface curvature on in vivo tissue growth using an ovine animal model. Based on a multiscale tissue microstructure characterization, we show a seamless integration of soft tissue into newly formed bone, resembling the insertion sites of ligaments and tendons into bone. This interface was created using a scaffold without additional growth factors or cells that did not recapitulate the structural or biological gradients across such a complex tissue interface at its mature state. These findings have important implications for biomimetic scaffold design for bone regeneration and soft tissue-to-bone interface tissue engineering.

Curvature-controlled defect dynamics in active systems.

We have studied the collective motion of polar active particles confined to ellipsoidal surfaces. The geometric constraints lead to the formation of vortices that encircle surface points of constant curvature (umbilics). We have found that collective motion patterns are particularly rich on ellipsoids with four umbilics where vortices tend to be located near pairs of umbilical points to minimize their interaction energy. Our results provide a perspective on the migration of living cells, which most likely use the information provided from the curved substrate geometry to guide their collective motion.

Materials Nanoarchitecturing via Cation-Mediated Protein Assembly: Making Limpet Teeth without Mineral.

Teeth are designed to deliver high forces while withstanding the generated stresses. Aside from isolated mineral-free exception (e.g., marine polychaetes and squids), minerals are thought to be indispensable for tooth-hardening and durability. Here, the unmineralized teeth of the giant keyhole limpet (Megathura crenulata) are shown to attain a stiffness, which is twofold higher than any known organic biogenic structures. In these teeth, protein and chitin fibers establish a stiff compact outer shell enclosing a less compact core. The stiffness and its gradients emerge from a concerted interaction across multiple length-scales: packing of hydrophobic proteins and folding into secondary structures mediated by Ca2+ and Mg2+ together with a strong spatial control in the local fiber orientation. These results integrating nanoindentation, acoustic microscopy, and finite-element modeling for probing the tooth's mechanical properties, spatially resolved small- and wide-angle X-ray scattering for probing the material ordering on the micrometer scale, and energy-dispersive X-ray scattering combined with confocal Raman microscopy to study structural features on the molecular scale, reveal a nanocomposite structure hierarchically assembled to form a versatile damage-tolerant protein-based tooth, with a stiffness similar to mineralized mammalian bone, but without any mineral.

Flexible and Actuating Nanoporous Poly(Ionic Liquid)-Paper-Based Hybrid Membranes.

Porous and flexible actuating materials are important for the development of smart systems. We report here a facile method to prepare scalable, flexible actuating porous membranes based on a poly(ionic liquid)-modified tissue paper. The targeted membrane property profile was based on synergy of the gradient porous structure of a poly(ionic liquid) network and flexibility of a tissue paper. The gradient porous structure was built through an ammonia-triggered electrostatic complexation of a poly(ionic liquid) with poly(acrylic acid), which were previously impregnated inside the tissue paper. As a result, these porous membranes undergo deformation by bending in response to organic solvents in the vapor or liquid phase and can recover their shape in air, which demonstrates their ability to serve as solvent sensors. Besides, they show enhanced mechanical properties due to the introduction of mechanically flexible tissue paper that allows the membranes to be designed as new responsive textiles and contractile actuators.

The Role of Titanium Surface Nanostructuring on Preosteoblast Morphology, Adhesion, and Migration.

Surface structuring of titanium-based implants is known to modulate the behavior of adherent cells, but the influence of different nanotopographies is poorly understood. The aim is to investigate preosteoblast proliferation, adhesion, morphology, and migration on surfaces with similar surface chemistry but distinct nanotopographical features. Sonochemical treatment and anodic oxidation are employed to fabricate disordered, mesoporous titania (TMS) and ordered titania nanotubular (TNT) topographies on titanium, respectively. Morphological evaluation reveals that cells are polygonal and well-spread on TMS, but display an elongated, fibroblast-like morphology on TNT surfaces, while they are much flatter on glass. Both nanostructured surfaces impair cell adhesion, but TMS is more favorable for cell growth due to its support of cell attachment and spreading in contrast to TNT. A quantitative wound healing assay in combination with live-cell imaging reveals that cell migration on TMS surfaces has a more collective character than on other surfaces, probably due to a closer proximity between neighboring migrating cells on TMS. The results indicate distinctly different cell adhesion and migration on ordered and disordered titania nanotopographies, providing important information that can be used in optimizing titanium-based scaffold design to foster bone tissue growth and repair while allowing for the encapsulation of drugs into porous titania layer.

Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation.

Signals from the microenvironment around a cell are known to influence cell behavior. Material properties, such as biochemical composition and substrate stiffness, are today accepted as significant regulators of stem cell fate. The knowledge of how cell behavior is influenced by 3D geometric cues is, however, strongly limited despite its potential relevance for the understanding of tissue regenerative processes and the design of biomaterials. Here, the role of surface curvature on the migratory and differentiation behavior of human mesenchymal stem cells (hMSCs) has been investigated on 3D surfaces with well-defined geometric features produced by stereolithography. Time lapse microscopy reveals a significant increase of cell migration speed on concave spherical compared to convex spherical structures and flat surfaces resulting from an upward-lift of the cell body due to cytoskeletal forces. On convex surfaces, cytoskeletal forces lead to substantial nuclear deformation, increase lamin-A levels and promote osteogenic differentiation. The findings of this study demonstrate a so far missing link between 3D surface curvature and hMSC behavior. This will not only help to better understand the role of extracellular matrix architecture in health and disease but also give new insights in how 3D geometries can be used as a cell-instructive material parameter in the field of biomaterial-guided tissue regeneration.

Hierarchically Arranged Helical Fiber Actuators Derived from Commercial Cloth.

The first hygroscopically tunable cloth actuator is realized via impregnation of a commercial cloth template by a three dimensionally (3D) nanoporous polymer/carbon nanotube hybrid network. The nanoporous hybrid guarantees diffusion of water into the cloth actuator and amplifies the deformation scale. The cloth actuators are mechanically stable with high tensile strength. Because the commercial cotton cloth is inexpensive, such actuators capable of complex motions can be produced in a large size and scale for a wide variety of utilities (e.g. electric generators and "smart" materials).

Relation between the Macroscopic Pattern of Elephant Ivory and Its Three-Dimensional Micro-Tubular Network.

Macroscopic, periodic, dark and bright patterns are observed on sections of elephant tusk, in the dentin part (ivory). The motifs-also called Schreger pattern-vary depending on the orientation in the tusk: on sections perpendicular to the tusk axis, a checkerboard pattern is present whereas on sections longitudinal to it, alternating stripes are observed. This pattern has been used to identify elephant and mammoth ivory in archeological artifacts and informs on the continuous tissue growth mechanisms of tusk. However, its origin, assumed to be related to the 3D structure of empty microtubules surrounded by the ivory matrix has yet to be characterized unequivocally. Based on 2D observations of the ivory microtubules by means of a variety of imaging techniques of three different planes (transverse, longitudinal and tangential to the tusk axis), we show that the dark areas of the macroscopic pattern are due to tubules oblique to the surface whereas bright areas are related to tubules parallel to it. The different microstructures observed in the three planes as well as the 3D data obtained by SR-µCT analysis allow us to propose a 3D model of the microtubule network with helical tubules phase-shifted in the tangential direction. The phase shift is a combination of a continuous phase shift of π every 1 mm with a stepwise phase shift of π/2 every 500 µm. By using 3D modeling, we show how the 3D helical model better represents the experimental microstructure observed in 2D planes compared to previous models in the literature. This brings new information on the origin of the unique Schreger pattern of elephant ivory, crucial for better understanding how archaeological objects were processed and for opening new routes to rethink how biological materials are built.