Open Access

Show Abstract ▼

Abstract: The use of synthetic extracellular matrices (ECMs) in fundamental in vitro cell culture studies has been instrumental for investigating the interplay between cells and matrix components. To provide cells with a more native environment in vitro, it is desirable to design matrices that are biomimetic and emulate compositional and structural features of natural ECMs. Here, the supramolecular fabrication of peptide-hyaluronan (HA) hydrogels is presented as promising ECM surrogates, combining HA and rationally designed cationic amphipatic peptides [(KI)nK, lysine (K), isoleucine (I), n = 2–6] whose mechanical properties and microstructure are tunable by the peptide sequence. (KI)nK peptides adopt β-sheet configuration and self-assemble into filamentous nanostructures triggered by pH or ionic strength. The self-assembly propensity of (KI)nK peptides increases with the sequence length, forming single phase hydrogels (shorter peptides) or with phase separation (longer peptides) in presence of the anionic polyelectrolyte HA through electrostatic complexations. The gel phase formed in (KI)nK-HA complexes exhibits viscoelastic behavior and triggers the formation of human mesenchymal stem cell (MSC) spheroids which disassemble over the time. It is anticipated that these (KI)nK-HA hydrogels with tunable physical and biochemical properties offer a promising platform for in vitro applications and in stem cell therapy.

Show Abstract ▼

Abstract: Magnetite nanoparticles possess numerous fundamental, biomedical, and industrial applications, many of which depend on tuning the magnetic properties. This is often achieved by the incorporation of trace and minor elements into the magnetite lattice. Such incorporation was shown to depend strongly on the magnetite formation pathway (i.e., abiotic vs biological), but the mechanisms controlling element partitioning between magnetite and its surrounding precipitation solution remain to be elucidated. Here, we used a combination of theoretical modeling (lattice and crystal field theories) and experimental evidence (high-resolution inductively coupled plasma–mass spectrometry and X-ray absorption spectroscopy) to demonstrate that element incorporation into abiotic magnetite nanoparticles is controlled principally by cation size and valence. Elements from the first series of transition metals (Cr to Zn) constituted exceptions to this finding, as their incorporation appeared to be also controlled by the energy levels of their unfilled 3d orbitals, in line with crystal field mechanisms. We finally show that element incorporation into biological magnetite nanoparticles produced by magnetotactic bacteria (MTB) cannot be explained by crystal–chemical parameters alone, which points to the biological control exerted by the bacteria over the element transfer between the MTB growth medium and the intracellular environment. This screening effect generates biological magnetite with a purer chemical composition in comparison to the abiotic materials formed in a solution of similar composition. Our work establishes a theoretical framework for understanding the crystal–chemical and biological controls of trace and minor cation incorporation into magnetite, thereby providing predictive methods to tailor the composition of magnetite nanoparticles for improved control over magnetic properties.

Show Abstract ▼

Abstract: Biomineralization relies on the regulation of localized environments to control how minerals are formed. Through the use of confinement and specific additives, the organism is able to change the energy landscape of nucleation and growth to build single crystals with unusual morphologies. In order to better understand the environments in which biomineralization occurs, it is important to understand both the effects of specific factors on crystallization and how those factors may show up in the final mineralized tissue. Biominerals formed in intracellular vesicles have the highest level of organism-directed regulation and 2 of these types of systems are explored in this work: calcium carbonate, with which sea urchins build their skeletal components, and strontium sulfate, a rare biomineral found in the endoskeletons of marine plankton called Acantharia. In the first part of this work, the role of confinement on the crystallization is explored in the calcium carbonate system. A microfluidic assay was used to measure volume scaling of the crystallization rate in droplets filled with supersaturated calcium carbonate solution. This volume scaling predicts that on the size scale of intracellular vesicles, calcium carbonate crystallization is exceeding slow, with a 1% probability of crystallization after ~1 million years. This suggests an accelerant must be present during sea urchin embryo spiculogenesis to build their calcite skeletal components. An extension of this work to the system of barium calcium carbonate is made in the second chapter of this thesis where model selection is combined with survival analysis to make inferences in a system with a time-dependent crystallization rate. In the next section of this work, characterization of the mineralized tissue is performed in sea urchin and Acantharia spicules. In sea urchin spicule cross section, nanoscale X-ray diffraction mapping is used to measure distortions in the lattice at high resolution and sensitivity in 2 species of sea urchins which produce different types of spicules. The measured changes in d-spacing correlate with low-Z inclusions previously observed in TEM and the change in d-spacing could not be explained by fluctuations in magnesium content alone. In Acantharia, for which relatively little is known compared to sea urchins, both composition and d-spacing were characterized. X-ray fluorescence (XRF) mapping revealed compositional gradients in trace elements barium, calcium and potassium in spicule cross sections, which diffract as single crystals. D-spacing maps show features similar to those in XRF maps and TEM images. A more detailed look at composition was performed with atom probe tomography (APT) which showed even higher concentrations of sodium (on the order of 1 at%) as compared to the other trace elements. Clustering of sodium, water, and some ambiguous ion species were also observed in APT tips and implications for whether organic inclusions are present in these spicules is discussed. Biological compartments in which mineralized tissues are formed are key components to understanding crystallization pathways in biominerals and can leave traces of themselves within the mineral itself. Through the study of crystallization in confinement and characterization of features within these single crystals a clearer picture of these environments can be formed. This can provide inspiration for fabricating better and more sustainable materials as well as expand our knowledge about complex crystallization processes.

Open Access

Show Abstract ▼

Abstract: Chemically crosslinked acellular bovine pericardium (ABP) has been widely used in clinical practice as bioprostheses. To ensure its consistency and durability, crosslinkers are used in excess, with stability guided by indicators including the hydrothermal denaturation temperature, the enzymatic resistance and the degree of crosslinking. Yet, understanding of the intermolecular structure in collagen fibrils which imparts the intrinsic stability of the ABPs is lacking, and the discrepancies in the stability criteria in varied conditions are poorly explained. In this study, synchrotron small-angle X-ray scattering (SAXS) in combination with thermal and colorimetric methods are employed to investigate the changes in the structure and the stability of ABPs during crosslinking using glutaraldehyde (GA) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) at different concentrations. Based on the findings, a mechanism is proposed to explicate the crosslinking effects on collagen structure and the relationship between the structure and each stability indicator. At low crosslinker concentrations, the telopeptidyl-helical linkages are preferred which cause rearrangements in the intermolecular structure of collagen, and efficiently contribute to its stabilities. Excess crosslinking is revealed by a revert trend in structural changes and the plateauing of the stabilities, assigning to the helical-helical linkages and monovalent bindings. The former would improve thermal stability but not collagenase resistance, whereas the latter have negligible effects. Overall, this study provides mechanistic understanding of the chemical crosslinking of ABPs which will contribute to the future development of more efficient and economically viable strategies to produce bioprostheses.

Open Access

Show Abstract ▼

Abstract: The protective carapace of Skogsbergia lerneri, a marine ostracod, is scratch-resistant and transparent. The compositional and structural organisation of the carapace that underlies these properties is unknown. In this study, we aimed to quantify and determine the distribution of chemical elements and chitin within the carapace of adult ostracods, as well as at different stages of ostracod development, to gain insight into its composition. Elemental analyses included X-ray absorption near-edge structure, X-ray fluorescence and X-ray diffraction. Nonlinear microscopy and spectral imaging were performed to determine chitin distribution within the carapace. High levels of calcium (20.3%) and substantial levels of magnesium (1.89%) were identified throughout development. Amorphous calcium carbonate (ACC) was detected in carapaces of all developmental stages, with the polymorph, aragonite, identified in A-1 and adult carapaces. Novel chitin-derived second harmonic generation signals (430/5 nm) were detected. Quantification of relative chitin content within the developing and adult carapaces identified negligible differences in chitin content between developmental stages and adult carapaces, except for the lower chitin contribution in A-2 (66.8 ± 7.6%) compared to A-5 (85.5 ± 10%) (p = 0.03). Skogsbergia lerneri carapace calcium carbonate composition was distinct to other myodocopid ostracods. These calcium polymorphs and ACC are described in other biological transparent materials, and with the consistent chitin distribution throughout S. lerneri development, may imply a biological adaptation to preserve carapace physical properties. Realisation of S. lerneri carapace synthesis and structural organisation will enable exploitation to manufacture biomaterials and biomimetics with huge potential in industrial and military applications.