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Collagen type I, the main component of the extracellular matrix in vertebrates, is widely used in tissue engineering applications. This is on account that collagen molecules can self‐assemble under certain conditions into 3D fibrillar hydrogels. Although there is an extensive body of literature studying collagen self‐assembly, there is a lack of systematic understanding on how different experimental factors, such as pH and temperature, and their cumulative effects guide the self‐assembly process. In this work, a comprehensive workflow to study the interactive effects of several assembly parameters on the collagen self‐assembly process is implemented. This workflow consists of: 1) efficient statistical sampling based on Design of Experiments, 2) high‐throughput and automated data collection and 3) automated data analysis. This approach enables to screen several parameters simultaneously and derive a set of mathematical equations that link parameters with the kinetics and morphological aspects of collagen self‐assembly, and can be used to design collagen constructs with predefined characteristics. [ABSTRACT FROM AUTHOR]

Author(s): Alexander E. S. Van Driessche (corresponding author) [1]; Nani Van Gerven [5, 6]; Paul H. H. Bomans [2, 3]; Rick R. M. Joosten [2, 3]; Heiner Friedrich [2, 3]; [...]

Investigating and understanding the intrinsic material properties of biogenic materials, which have evolved over millions of years into admirable structures with difficult to mimic hierarchical levels, holds the potential of replacing trial-and-error-based materials optimization in our efforts to make synthetic materials of similarly advanced complexity and properties. An excellent example is biogenic silica which is found in the exoskeleton of unicellular photosynthetic algae termed diatoms. Because of the complex micro- and nanostructures found in their exoskeleton, determining the intrinsic mechanical properties of biosilica in diatoms has only partly been accomplished. Here, a general method is presented in which a combination of in situ deformation tests inside an SEM with a realistic 3D model of the frustule of diatom Craspedostauros sp. (C. sp.) obtained by electron tomography, alongside finite element method (FEM) simulations, enables quantification of the Young's modulus (E = 2.3 ± 0.1 GPa) of this biogenic hierarchical silica. The workflow presented can be readily extended to other diatom species, biominerals, or even synthetic hierarchical materials. [ABSTRACT FROM AUTHOR]

Determination of mechanical properties of (silica) thin films is important for the development of new applications. However, a versatile approach for small‐scale sample fabrication and in situ mechanical testing of these materials is currently lacking which can overcome the existing difficulties in conventional testing approaches. Here, it is shown that by combining focused ion beam scanning electron microscopy (FIB‐SEM) with micromanipulators free‐standing silica beams of the desired dimensions can be fabricated. Using in situ bending tests on such beams inside the SEM and finite element method simulations, the Young's modulus of thin‐film silica and the effects of gallium ion beam radiation on Young's modulus are determined. Furthermore, the effects of controlled relative humidity on the mechanical behavior of silica beams are investigated. The demonstrated FIB‐SEM approach can be extended to other materials and preparation conditions to measure, model, and optimize mechanical properties of thin films for a wide range of (coating) applications. [ABSTRACT FROM AUTHOR]

The mineralization of collagen in vitro has been extensively investigated for hydroxyapatite, silica, calcium carbonate and lepidocrocite (γ-FeOOH). Henceforth, it is interesting to investigate whether collagen also could serve as a generic mineralization template for other minerals, like magnetite. To this end, and inspired by the partial oxidation approach, first a ferrous hydroxide (Fe(OH)2) intermediate is synthesized via the titration of base to a solution of Fe2+. Subsequently, the Fe(OH)2 is mixed with collagen fibrils and poly(aspartic acid) is added to promote the formation of intrafibrillar crystals. Platelet-shaped lepidocrocite crystals being present throughout the entire thickness of the collagen fibrils can be realized, as was confirmed with electron tomography. The formation of lepidocrocite, which is an Fe3+ compound, is hypothesized to be induced via oxidation of the Fe2+ species and, therefore, the oxygen concentration during titration, TEM sample preparation and drying of TEM samples are investigated. Although the reaction is sensitive to small changes in experimental conditions, highly mineralized collagen fibers can be realized. [ABSTRACT FROM AUTHOR]

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three‐dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top‐down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D‐spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two‐dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis. [ABSTRACT FROM AUTHOR]

Transmission electron microscopy (TEM) is an important analysis technique to visualize (bio)macromolecules and their assemblies, including collagen fibers. Many protocols for TEM sample preparation of collagen involve one or more washing steps to remove excess salts from the dispersion that could hamper analysis when dried on a TEM grid. Such protocols are not standardized and washing times as well as washing solvents vary from procedure to procedure, with each research group typically having their own protocol. Here, we investigate the influence of washing with water, ethanol, but also methanol and 2‐propanol, for both mineralized and unmineralized collagen samples via a protocol based on centrifugation. Washing with water maintains the hydrated collagen structure and the characteristic banding pattern can be clearly observed. Conversely, washing with ethanol results in dehydration of the fibrils, often leading to aggregation of the fibers and a less obvious banding pattern, already within 1 min of ethanol exposure. As we show, this process is fully reversible. Similar observations were made for methanol and propanol. Based on these results, a standardized washing protocol for collagenous samples is proposed. [ABSTRACT FROM AUTHOR]

Biocomposite structures are difficult to characterize by bulk approaches due to their morphological complexity and compositional heterogeneity. Therefore, a versatile method is required to assess, for example, the mechanical properties of geometrically simple parts of biocomposites at the relevant length scales. Here, it is demonstrated how a combination of Focused Ion Beam Scanning Electron Microscopy (FIB‐SEM) and micromanipulators can be used to isolate, transfer, and determine the mechanical properties of frustule constituents of diatom Thalassiosira pseudonana (T.p.). Specifically, two parts of the diatom frustule, girdle bands and valves, are separated by FIB milling and manipulated using a sharp tungsten tip without compromising their physical or chemical integrity. In situ mechanical studies on isolated girdle bands combined with Finite Element Method (FEM) simulations, enables the quantitative assessment of the Young's modulus of this biosilica; E = 40.0 GPa. In addition, the mechanical strength of isolated valves could be measured by transferring and mounting them on top of premilled holes in the sample support. This approach may be extended to any hierarchical biocomposite material, regardless of its chemical composition, to isolate, transfer, and investigate the mechanical properties of selected constituents or specific regions. [ABSTRACT FROM AUTHOR]

Biodegradable porous calcium phosphate (CaP) ceramics are widely used as synthetic graft substitutes for bone regeneration, owing to their chemical and structural similarity to bone and associated bioactivity in terms of bone‐bonding, osteoconductive, and even osteoinductive properties. Nevertheless, the intrinsic brittleness and poor processability of porous CaP ceramics strongly impair their clinical applicability. Herein, a biphasic calcium phosphate (BCP) sponge is developed that consists of a self‐supporting network of seamlessly interwoven hydroxyapatite nanowires and β‐tricalcium phosphate nanofibers and possesses a highly interconnected porous structure with open cell geometry and ultrahigh porosity. Owing to its unique properties, the ceramic sponge can be easily processed into various shapes and dimensions, such as cylindrical scaffolds and thin, flexible membranes. Moreover, the BCP sponge can be introduced into a bone defect in a compacted or folded state from a syringe and, upon wetting, expand to its original shape, thereby filling the cavity. The nanofibrous sponge gradually degrades in vitro and rapidly mineralizes when immersed in simulated body fluid. Moreover, it adsorbs significantly more proteins than a conventional porous BCP ceramic. Finally, the nanofibrous sponge supports the attachment, proliferation, and osteogenic differentiation of human mesenchymal stromal cells comparable to the conventional porous BCP ceramic. [ABSTRACT FROM AUTHOR]

Liquid‐Phase (Scanning) Transmission Electron Microscopy (LP‐(S)TEM) has become an essential technique to monitor nanoscale materials processes in liquids in real‐time. Due to the pressure difference between the liquid and the microscope vacuum, bending of the silicon nitride (SiNx) membrane windows generally occurs. This causes a spatially varying liquid layer thickness that makes interpretation of LP‐(S)TEM results difficult due to a locally varying achievable resolution and diffusion limitations. To mediate these difficulties, it is shown: 1) how to quantitatively map liquid layer thickness for any liquid at less than 0.01 e− Å−2 total dose; 2) how to dynamically modulate the liquid thickness by tuning the internal pressure in the liquid cell, co‐determined by the Laplace pressure and the external pressure. It is demonstrated that reproducible inward bulging of the window membranes can be realized, leading to an ultra‐thin liquid layer in the central window area for high‐resolution imaging. Furthermore, it is shown that the liquid thickness can be dynamically altered in a programmed way, thereby potentially overcoming the diffusion limitations towards achieving bulk solution conditions. The presented approaches provide essential ways to measure and dynamically adjust liquid thickness in LP‐(S)TEM experiments, enabling new experiment designs and better control of solution chemistry. [ABSTRACT FROM AUTHOR]

Hexamethylenetetramine (HMTA) is commonly used as a base releasing agent for the synthesis of ZnO under mild aqueous conditions. HMTA hydrolysis leads to gradual formation of a base during the reaction. Use of HMTA, however, does have limitations: HMTA hydrolysis yields both formaldehyde and ammonia, it provides no direct control over the ammonia addition rate or the total amount of ammonia added during the reaction, it results in a limited applicable pH range and it dictates the accessible reaction temperatures. To overcome these restrictions, this work presents a direct base titration strategy for ZnO synthesis in which a continuous base addition rate is maintained. Using this highly flexible strategy, wurtzite ZnO can be synthesized at a pH >5.5 using either KOH or ammonia as a base source at various addition rates and reaction pH values. In situ pH measurements suggest a similar reaction mechanism to HMTA-based synthesis, independent of the varied conditions. The type and concentration of the base used for titration affect the reaction product, with ammonia showing evidence of capping behaviour. Optimizing this strategy, we are able to influence and direct the crystal shape and significantly increase the product yield to 74% compared to the ∼13% obtained by the reference HMTA reaction. [ABSTRACT FROM AUTHOR]