Mechanisms and Microenvironment Investigation of Cellularized High Density Gradient Collagen Matrices via Densification

Biological tissues and biomaterials are often defined by unique spatial gradients in physical properties that impart specialized function over hierarchical scales. The structure of these materials forms continuous transitional gradients and discrete local microenvironments between adjacent (or within) tissues, and across matrix–cell boundaries, which is difficult to replicate with common scaffold systems. Here, the matrix densification of collagen leading to gradients in density, mechanical properties, and fibril morphology is studied. High‐density regions form via a fluid pore pressure and flow‐driven mechanism, with increased relative fibril density (10×), mechanical properties (20×, to 94.40 ± 18.74 kPa), and maximum fibril thickness (1.9×, to >1 μm) compared to low‐density regions, while maintaining porosity and fluid/mass transport to support viability of encapsulated cells. Similar to the organization of the articular cartilage zonal structure, it is found that high‐density collagen regions induce cell and nuclear alignment of primary chondrocytes. Chondrocyte gene expression is maintained in collagen matrices, and no phenotypic changes are observed as a result of densification. Collagen densification provides a tunable platform for the creation of gradient systems to study complex cell–matrix interactions. These methods are easily generalized to compression and boundary condition modalities useful to mimic a broad range of tissues.     

Self‐Assembled Hydrogel Fiber Bundles from Oppositely Charged Polyelectrolytes Mimic Micro‐/Nanoscale Hierarchy of Collagen

Fiber bundles are present in many tissues throughout the body. In most cases, collagen subunits spontaneously self‐assemble into a fibrilar structure that provides ductility to bone and constitutes the basis of muscle contraction. Translating these natural architectural features into a biomimetic scaffold still remains a great challenge. Here, a simple strategy is proposed to engineer biomimetic fiber bundles that replicate the self‐assembly and hierarchy of natural collagen fibers. The electrostatic interaction of methacrylated gellan gum with a countercharged chitosan polymer leads to the complexation of the polyelectrolytes. When directed through a polydimethylsiloxane channel, the polyelectrolytes form a hierarchical fibrous hydrogel demonstrating nanoscale periodic light/dark bands similar to D‐periodic bands in native collagen and align parallel fibrils at microscale. Importantly, collagen‐mimicking hydrogel fibers exhibit robust mechanical properties (MPa scale) at a single fiber bundle level and enable encapsulation of cells inside the fibers under cell‐friendly mild conditions. Presence of carboxyl‐ (in gellan gum) or amino‐ (in chitosan) functionalities further enables controlled peptide functionalization such as Arginylglycylaspartic acid (RGD) for biochemical mimicry (cell adhesion sites) of native collagen. This biomimetic‐aligned fibrous hydrogel system can potentially be used as a scaffold for tissue engineering as well as a drug/gene delivery vehicle.     

Engineering Functional Collagen Scaffolds: Cyclical Loading Increases Material Strength and Fibril Aggregation

Variation in collagen fibril diameter in nature is a major factor determining biological material properties. However, the mechanism resulting in this fibril diameter difference is not clear and generally assumed to be cell‐dependent. It is certainly not possible so far to engineer this into implantable scaffold materials. This gap in our knowledge is crucial for the fabrication of truly biomimetic tissue‐like materials. We have tested the idea that fibril diameter can be regulated directly without cell involvement, using cyclical mechanical loading to promote fibril fusion. Specific loading regimes increased collagen fibril diameter (> 2 fold) in direct relation to cycle number, whilst thin fibrils disappeared. Tensile properties increased, producing a 4.5 fold rise in break strength. This represents the first demonstration of direct cyclical load‐promoted fibril fusion and provides a direct relation with material properties. The ability to control material properties in this way makes it possible to fabricate truly biomimetic collagen materials without cells.     

Tough Lessons From Bone: Extreme Mechanical Anisotropy at the Mesoscale

Bone is mechanically and structurally anisotropic with oriented collagen fibrils and nanometer‐sized mineral particles aggregating into lamellar or woven bone.[1] Direct measurements of anisotropic mechanical properties of sublamellar tissue constituents are complicated by the existence of an intrinsic hierarchical architecture. Methods such as nanoindentation provide insight into effective modulus values; however, bulk material properties cannot sufficiently be characterized since such measurements represent properties of near‐surface volumes and are partially averaged over fibril orientations.[2–5] In this study, we focus on the material properties of bone at one single level of hierarchy. By measuring properties of individual parallel‐fibered units of fibrollamellar bone under tension under controlled humidity conditions, an unusually high anisotropy is found. Here, we clearly demonstrate ratios as large as 1:20 in elastic modulus and 1:15 in tensile strength between orientations perpendicular and parallel to the main collagen fiber orientation in native wet bone; these ratios reduce to 1:8 and 1:7, respectively, under dry conditions. This extreme anisotropy appears to be caused by the existence of periodic, weak interfaces at the mesoscopic length scale. These interfaces are thought to be relevant to the proper mechanical and physiological performance of bone.     

Complementarity and Uncertainty in Intrafibrillar Mineralization of Collagen

Biomineralization in vertebrates is a ubiquitous and tightly regulated process which creates hierarchical structures for the skeleton. Because of the lack of understanding and applicability of cell‐based or biological systems to achieve intrafibrillar mineralization, scientists adopted various in vitro methods to elucidate the mechanism of intrafibrillar mineralization. In this article, biomimetic intrafibrillar mineralization of collagen in its wide ramifications is reviewed. It is intriguing how prevailing intrafibrillar mineralization mechanisms derived from two potentially discordant crystallization philosophies were equally adept, depending on the experimental context, at theorizing the formation of calcium phosphate within a fibrillar template. This complementarity is not unique to biomineralization and has precedence in other fundamental physical interpretations. A new intrafibrillar mineralization process based on the use of polycationic process‐directing agent added uncertainty to the use of existing mechanisms in accounting for the observations.     

Scalable Synthesis of Multifunctional Epidermis‐Like Smart Coatings

Bioinspired self‐healing materials provide a sustainable solution for mitigating catastrophic failure and prolonging service time, but such potential may be largely offset by the tedious fabrication process that can hardly be scaled up. It is shown that a scalable doctor blade coating process may be used for the synthesis of multifunctional epidermis‐like smart coatings. The industry‐compatible technique not only preserves the biomimetic bilayer design with largely improved film production efficiency but also allows readily adjusted film compositions and structures for optimizing the synergetic healing performance and mechanical properties. Variable amounts of montmorillonite clay nanosheets can be incorporated in the top hard layer for achieving a high modulus (34.3 ± 1.4 GPa) and hardness (1.65 ± 0.09 GPa), which also affect the number of scratch‐healing cycles by controlling the decay rate of upward polymer diffusion from the soft bottom layer. These high‐performance smart coatings are transparent, bactericidal, and show good adhesions to various substrates including glass, plastics, and fiberboard. When used as a surface protection for fiberboard, excellent flame‐retardant properties are demonstrated thanks to the dominant presence of clay nanosheets. It is believed that this work may provide important guidance for transforming interesting biomimetic design into practical manufacturing.     

Oriented Strontium Carbonate Nanocrystals within Collagen Films for Flexible Piezoelectric Sensors

Bone, assembled by mineralized collagen fibrils, displays piezoelectric properties under external stimulation to affect tissue growth. The mineralized collagen fibrils consist of collagen and oriented inorganic nanocrystals. Inspired from the unique structures and piezoelectric effect of mineralized collagen fibrils, the intrafibrillar mineralization of oriented strontium carbonate nanocrystals is achieved in vitro, which also exhibits good piezoelectric properties. The amorphous strontium carbonate precursors penetrate from the gap zones and fill gradually into the whole space within the collagen fibrils, and transform into a co-oriented crystalline phase. Isolated mineralized collagen fibrils with organized SrCO₃ nanocrystals acquire good flexible properties and inverse piezoelectric responses with an effective piezoelectric coefficient of 3.45 pm V⁻¹, much higher than individual collagen (1.12 pm V⁻¹) and SrCO₃ crystals (0.092 pm V⁻¹). These results may indicate that the organic and inorganic components synergistically contribute to the piezoelectric effect of bone. Furthermore, devices of flexible piezoelectric thin films assembled by SrCO₃ mineralized collagen fibrils exhibit a regular open-circuit voltage of 1.2 V under compressive stress and a stable cycling short-circuit current of 80 nA under a bending mode. It can also facilitate the development of promising piezoelectric sensors.     

Bioprocess‐Inspired Microscale Additive Manufacturing of Multilayered TiO2/Polymer Composites with Enamel‐Like Structures and High Mechanical Properties

Natural structure‐forming processes found in biological systems are fantastic and perform at ambient temperatures, in contrast with anthropogenic technologies that commonly require harsh conditions. A new research direction “bioprocess‐inspired fabrication” is proposed to develop novel fabrication techniques for advanced materials. Enamel, an organic–inorganic composite biomaterial with outstanding mechanical performance and durability, is formed by repeating the basic blocks consisting of columnar hydroxyapatite or fluorapatite and an organic matrix. Inspired by the enamel formation process, a microscale additive manufacturing method is proposed for achieving a multilayered organic–inorganic columnar structure. In this approach, rutile titanium dioxide (TiO₂) nanorods, polymers, and graphene oxide (GO) are sequentially assembled in a layer‐by‐layer fashion to form an organic–inorganic structure. In particular, GO serves as a substrate for TiO₂ nanorods and interacts with polymers, jointly leading to the strength of the composites. Impressively, this enamel‐like structure material has hardness (1.56 ± 0.05 GPa) and ultrahigh Young's modulus (81.0 ± 2.7 GPa) comparable to natural enamel, and viscoelastic property (0.76 ± 0.12 GPa) superior to most solid materials. Consequently, this biomimetic synthetic approach provides an in‐depth understanding for the formation process of biomaterials and also enables the exploration of a new avenue for the preparation of organic–inorganic composite materials.     

Biomimetic Mineralized Organic–Inorganic Hybrid Macrofiber with Spider Silk‐Like Supertoughness

Spider silk fibers (SSF) have a hierarchical structure composed of proteins with highly repetitive sequences and biomineralization is sophisticated in hierarchical organic–inorganic constructions. By using inorganic hydroxyapatite (HAP) and organic polyvinyl alcohol (PVA) to simulate the rigid crystalline and flexible amorphous protein blocks of SSF, respectively, biomimetic mineralization is herein attempted for the large‐scale preparation of SSF‐like macrofibers with a hierarchical ordered structure, a superhigh tensile strength of 949 ± 38 MPa, a specific toughness of 296 ± 12 J g^?1, and a stretch ability of 80.6%. The hybrid macrofibers consist of microfibers, and their outstanding performance (e.g., extreme tolerance to temperatures ranging from ?196 to 80 °C and superior ability to inhibit the transverse growth of cracks) is attributed to the hierarchical arrangement as well as the organic–inorganic integrated structure within the oriented mineralized polymer chains. The biomineralization‐inspired technique provides a promising tactic that can be used to synthesize functional organic–inorganic fibers that are structurally complex and, furthermore, industrially manufacture SSF‐like artificial fibers with a supertoughness.     

Biomimetic Structures: Biological Implications of Dipeptide‐Substituted Polyphosphazene–Polyester Blend Nanofiber Matrices for Load‐Bearing Bone Regeneration

Successful bone regeneration benefits from three‐dimensional (3D) bioresorbable scaffolds that mimic the hierarchical architecture and mechanical characteristics of native tissue extracellular matrix (ECM). A scaffold platform that integrates unique material chemistry with nanotopography while mimicking the 3D hierarchical bone architecture and bone mechanics is reported. A biocompatible dipeptide polyphosphazene‐polyester blend is electrospun to produce fibers in the diameter range of 50–500 nm to emulate dimensions of collagen fibrils present in the natural bone ECM. Various electrospinning and process parameters are optimized to produce blend nanofibers with good uniformity, appropriate mechanical strength, and suitable porosity. Biomimetic 3D scaffolds are created by orienting blend nanofiber matrices in a concentric manner with an open central cavity to replicate bone marrow cavity, as well as the lamellar structure of bone. This biomimicry results in scaffold stress–strain curve similar to that of native bone with a compressive modulus in the mid‐range of values for human trabecular bone. Blend nanofiber matrices support adhesion and proliferation of osteoblasts and show an elevated phenotype expression compared to polyester nanofibers. Furthermore, the 3D structure encourages osteoblast infiltration and ECM secretion, bridging the gaps of scaffold concentric walls during in vitro culture. The results also highlight the importance of in situ ECM secretion by cells in maintaining scaffold mechanical properties following scaffold degradation with time. This study for the first time demonstrates the feasibility of developing a mechanically competent nanofiber matrix via a biomimetic strategy and the advantages of polyphosphazene blends in promoting osteoblast phenotype progression for bone regeneration.     

Bioinspired Assembly of Hierarchical Light‐Harvesting Architectures for Improved Photophosphorylation

Molecular assembly offers a bottom‐up way to construct biomimetic architectures with unique structures and properties. Although artificial photophosphorylation systems have long been developed, their microstructures have yet to achieve the sophisticated order and hierarchy of natural organisms. Herein, by utilizing principles in the natural plant leaves, it is shown that a biomimetic system with hierarchically ordered and compartmentalized structures, combining photosystem II (PSII) and adenosine triphosphate (ATP) synthase, can be obtained through template‐directed layer‐by‐layer assembly. Under light illumination, PSII in such a highly ordered light‐harvesting array, splits water to produce protons and electrons. Furthermore, a remarkable proton gradient is created across the covering ATP synthase‐reconstituted lipid membrane. As a consequence, highly efficient photophosphorylation is achieved. Outstandingly, the rate of ATP production in this hierarchical light‐harvesting architecture is enhanced 14 times, compared to that in the nature. This study paves a new way to assemble bioinspired systems with enhanced solar‐to‐chemical energy conversion efficiency.     

Tenogenic Induction of Human MSCs by Anisotropically Aligned Collagen Biotextiles

A novel biofabrication modality, electrophoretic compaction with macromolecular alignment, is utilized to make collagen threads that mimic the native tendon's structure and mechanical properties. A device with kinematic electrodes is designed to fabricate collagen threads in continuous length. For the first time, a 3D‐biotextile is woven purely from collagen. Mechanical properties and load‐displacement behavior of the biotextile mimic those of the native tendon while presenting a porosity of 80%. The open pore network facilitates cell seeding across the continuum of the bioscaffold. Mesenchymal stem cells (MSCs) seeded in the woven scaffold undergo tenogenic differentiation in the absence of growth factors and synthesize a matrix that is positive for tenomodulin, COMP and type I collagen. Up‐regulation of tenomodulin, a tendon specific marker, is 11.6 ± 3.5 fold, COMP is up‐regulated 16.7 ± 5.5 fold, and Col I is up‐regulated 6.9 ± 2.7 fold greater on ELAC threads when compared to randomly oriented collagen gels. These results demonstrate that a bioscaffold woven using collagen threads with densely compacted and anisotropically aligned substrate texture stimulates tenogenesis topographically, rendering the electrochemically aligned collagen as a promising candidate for functional repair of tendons and ligaments.     

Inside Front Cover: Thermally Responsive Biomineralization on Biodegradable Substrates (Adv. Funct. Mater. 16/2007)

Biomineralization offers an elegant example of how nature can design complex, hierarchical, and structurally/morphologically controllable materials. João Mano and co‐workers of the University of Minho, Portugal report on p. 3312 that modifying the surface of bioactive substrates, together with the effect of temperature, triggers the formation of apatite upon immersion in simulated body fluid. This “smart” biomineralization concept could be combined with patterning methodologies by controlling the microstructure of the surface. Such a concept could be extended in the biomimetic production of other minerals, being triggered by other kind of stimuli (e.g., pH or ionic strength), in substrates with more complex geometries. Biomineralization offers an elegant example of how nature can design complex, hierarchical, and structurally/morphologically controllable materials. In this work, the surface of bioactive substrates prepared from poly(L ‐lactic acid) and reinforced with Bioglass are modified by the graft polymerization of poly(N‐isopropylacrylamide), (PNIPAAm) after plasma activation. It is found that such treatment, together with temperature, could trigger the formation of apatite on the biodegradable substrate upon immersion in simulated body fluid above the PNIPAAm lower critical solution temperature (LCST); in contrast, no apatite is formed at room temperature. A control experiment on a material that is not subjected to surface treatment does not show any evidence of mineral deposition at the two analyzed temperatures. This “smart” biomineralization concept is combined with patterning methodologies to control the microstructure of the surface onto which PNIPAAm is grafted. In this case, the apatite is formed at 37 °C in the modified regions. We suggest that this concept could be extended in the biomimetic production of other minerals, where it would be triggered by another kind of stimulus (e.g., pH or ionic strength) in substrates with more complex geometries.     

A 3D Cell-Free Bone Model Shows Collagen Mineralization is Driven and Controlled by the Matrix

Osteons, the main organizational components of human compact bone, are cylindrical structures composed of layers of mineralized collagen fibrils, called lamellae. These lamellae have different orientations, different degrees of organization, and different degrees of mineralization where the intrafibrillar and extrafibrillar minerals are intergrown into one continuous network of oriented crystals. While cellular activity is clearly the source of the organic matrix, recent in vitro studies call into question whether the cells are also involved in matrix mineralization and suggest that this process could be simply driven by the interactions of the mineral with extracellular matrix. Through the remineralization of demineralized bone matrix, the complete multiscale reconstruction of the 3D structure and composition of the osteon without cellular involvement are demonstrated. Then, this cell-free in vitro system is explored as a realistic, functional model for the in situ investigation of matrix-controlled mineralization processes. Combined Raman and electron microscopy indicate that glycosaminoglycans (GAGs) play a more prominent role than generally assumed in the matrix–mineral interactions. The experiments also show that the organization of the collagen is in part a result of its interaction with the developing mineral.     

Modulation of Mechanical Interactions by Local Piezoelectric Effects

Piezoelectricity is a well‐established property of biological materials, yet its functional role has remained unclear. Here, a mechanical effect of piezoelectric domains resulting from collagen fibril organisation is demonstrated, and its role in tissue function and application to material design is described. Using a combination of scanning probe and nonlinear optical microscopy, a hierarchical structuring of piezoelectric domains in collagen‐rich tissues is observed, and their mechanical effects are explored in silico. Local electrostatic attraction and repulsion due to shear piezoelectricity in these domains modulate fibril interactions from the tens of nanometre (single fibril interactions) to the tens of micron (fibre interactions) level, analogous to modulated friction effects. The manipulation of domain size and organisation thus provides a capacity to tune energy storage, dissipation, stiffness, and damage resistance.     

Synthetic Bone-Like Structures Through Omnidirectional Ceramic Bioprinting in Cell Suspensions

The integration of hierarchical structure, chemistry, and functional activity within tissue-engineered scaffolds is of great importance in mimicking native bone tissue. Bone is a highly mineralized tissue which forms at ambient conditions by continuous crystallization of the mineral phase within an organic matrix in the presence of bone residing cells. Despite recent advances in the biofabrication of complex engineered tissues, replication of the heterogeneity of bone microenvironments has been a major challenge in constructing biomimetic bone scaffolds. Herein, inspired by the bone biomineralization process, the first example of bone mimicking constructs by 3D writing of a novel apatite-transforming ink in a supportive microgel matrix with living cells is demonstrated. Using this technique, complex bone-mimicked constructs are made at room temperature without requiring invasive chemicals, radiation, or postprocessing steps. This study demonstrates that mineralized constructs can be deposited within a high density of stem cells, directing the cellular organization, and promoting osteogenesis in vitro. These findings offer a new strategy for fabrication of bone mimicking constructs for bone tissue regeneration with scope to generate custom bone microenvironments for disease modeling, multicellular delivery, and in vivo bone repair.     

Ultrastrong Hierarchical Porous Materials via Colloidal Assembly and Oxidation of Metal Particles

Porous materials are useful as lightweight structures, bone substitutes, and thermal insulators, but exhibit poor mechanical properties compared to their dense counterparts. Biological materials such as bone and bamboo are able to circumvent this trade‐off between porosity and mechanical performance by combining pores at multiple length scales. Inspired by these biological architectures, a manufacturing platform that allows for the fabrication of Al₂O₃ foams and Al₂O₃/Al composites with hierarchical porosity and enhanced mechanical properties is developed. Macroscale pores are formed through the assembly of aluminum particles around templating air bubbles in wet foams, whereas the thermal oxidation of the metal particles above 800 °C generates porosity at the micrometer scale. After elucidating the mechanism of pore formation under different sintering conditions at the microscale, the mechanical performance of the resulting hierarchical foams using compression experiments and finite element simulations is evaluated. Porous materials manufactured via this simple approach are found to reach unparalleled mechanical properties with near‐zero sintering shrinkage and minimum loss in mechanical strength. The ability to produce macroscopic objects with ultrahigh strength at porosities up to 95% makes this an attractive manufacturing technology for the fabrication of high‐performance lightweight structures or advanced thermal and acoustic insulators.     

Mechanical Characterization of Amyloid Fibrils Using Coarse‐Grained Normal Mode Analysis

Recent experimental studies have shown that amyloid fibril formed by aggregation of β peptide exhibits excellent mechanical properties comparable to other protein materials such as actin filaments and microtubules. These excellent mechanical properties of amyloid fibrils are related to their functional role in disease expression. This indicates the necessity of understanding how an amyloid fibril achieves the remarkable mechanical properties through self‐aggregation with structural hierarchy. However, the structure‐property–function relationship still remains elusive. In this work, the mechanical properties of human islet amyloid polypeptide (hIAPP) are studied with respect to its structural hierarchies and structural shapes by coarse‐grained normal mode analysis. The simulation shows that hIAPP fibril can achieve the excellent bending rigidity via specific aggregation patterns such as antiparallel stacking of β peptides. Moreover, the length‐dependent mechanical properties of amyloids are found. This length‐dependent property has been elucidated from a Timoshenko beam model that takes into account the shear effect on the bending of amyloids. In summary, the study sheds light on the importance of not only the molecular architecture, which encodes the mechanical properties of the fibril, but also the shear effect on the mechanical (bending) behavior of the fibril.     

An Organoid for Woven Bone

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel, both cellular and matrix formation processes. Stem cell-based organoids are promising as functional, self-organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self-organizing co-culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in in vivo osteocytes, the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under the influence of external triggers (mechanical stimulation, drug administration).     

Adopting the Principles of Collagen Biomineralization for Intrafibrillar Infiltration of Yttria‐Stabilized Zirconia into Three‐Dimensional Collagen Scaffolds

In this paper, a process for generating collagen‐yttria‐stabilized amorphous zirconia hybrid scaffolds by introducing acetylacetone‐inhibited zirconia precursor nanodroplets into a poly(allylamine)‐coated collagen matrix is reported. This polyelectrolyte coating triggers intrafibrillar condensation of the precursors into amorphous zirconia, which is subsequently transformed into yttria‐stabilized zirconia after calcination. These findings represent a new paradigm in the synthesis of non‐naturally occurring collagen‐based hybrid scaffolds under alcoholic mineralizing conditions.