Biomineralization Studies Research Articles

Collagen mineralization and its applications in hard tissue repair

Mineralized collagen is an organic–inorganic composite material, which is the building unit of bones and teeth. This review aims to provide an in-depth study of the collagen biomineralization and applications relating to hard tissue regeneration.

Biomimetic Mineralization of Hierarchical Nanofiber Shish-Kebabs in a Concentrated Apatite-Forming Solution

Recently, hierarchical polymer nanostructures called nanofiber shish-kebabs (NFSKs) have shown great potential in directing the distribution of minerals for biomineralization. In this work, we report controlled biomimetic mineralization in block copolymer-containing NFSKs. NFSKs were formed by crystallizing polycaprolactone-block-poly(acrylic acid) (PCL-b-PAA) kebabs on PCL fiber backbones. The block copolymer kebabs were periodically distributed on PCL nanofibers with a spacing of ∼100–300 nm. Free ions could infiltrate into anionic PAA nanodomains and initiate the NFSK-templated biomimetic mineralization process. A concentrated calcium phosphate solution was used in the present study, which led to significantly accelerated mineralization kinetics compared with the previously studied simulated body fluid systems. Electron microscopy, Fourier-transform infrared spectroscopy, and wide-angle X-ray diffraction were used to characterize the structure and morphology of the mineralized NFSKs. A three-stage mineralization process was identified, and carbonated nonstoichiometric hydroxyapatite was observed after stage 2 with the mineral location templated within kebabs. This accelerated mineralization process yielded a periodic mineral distribution of a biomimetic calcium phosphate phase along the fibers, and the decrease in mineral formation time allows for more efficient biomineralization study and composite formation.

The Iron-Responsive Genome of the Chiton Acanthopleura granulata.

Molluscs biomineralize structures that vary in composition, form, and function, prompting questions about the genetic mechanisms responsible for their production and the evolution of these mechanisms. Chitons (Mollusca, Polyplacophora) are a promising system for studies of biomineralization because they build a range of calcified structures including shell plates and spine- or scale-like sclerites. Chitons also harden the calcified teeth of their rasp-like radula with a coat of iron (as magnetite). Here we present the genome of the West Indian fuzzy chiton Acanthopleura granulata, the first from any aculiferan mollusc. The A. granulata genome contains homologs of many genes associated with biomineralization in conchiferan molluscs. We expected chitons to lack genes previously identified from pathways conchiferans use to make biominerals like calcite and nacre because chitons do not use these materials in their shells. Surprisingly, the A. granulata genome has homologs of many of these genes, suggesting that the ancestral mollusc may have had a more diverse biomineralization toolkit than expected. The A. granulata genome has features that may be specialized for iron biomineralization, including a higher proportion of genes regulated directly by iron than other molluscs. A. granulata also produces two isoforms of soma-like ferritin: one is regulated by iron and similar in sequence to the soma-like ferritins of other molluscs, and the other is constitutively translated and is not found in other molluscs. The A. granulata genome is a resource for future studies of molluscan evolution and biomineralization.

Synthesis and Evaluation of a Zinc Eluting rGO/Hydroxyapatite Nanocomposite Optimized for Bone Augmentation.

Repair of critical size bone defects is a clinical challenge that usually necessitates the use of bone substitutes. For successful bone repair, the substitute should possess osteoconductive, osteoinductive, and vascularization potential, with the ability to control post-implantation infection serving as an additional advantage. With an aim to develop one such substitute, we optimized a zinc-doped hydroxyapatite (HapZ) nanocomposite decorated on reduced graphene oxide (rGO), termed as G3HapZ, and demonstrated its potential to augment the bone repair. The biocompatible composite displayed its osteoconductive potential in biomineralization studies, and its osteoinductive property was confirmed by its ability to induce mesenchymal stem cell (MSC) differentiation to osteogenic lineage assessed by in vitro mineralization (Alizarin red staining) and expression of osteogenic markers including runt-related transcription factor 2 (RUNX-2), alkaline phosphatase (ALP), type 1 collagen (COL1), bone morphogenic protein-2 (BMP-2), osteocalcin (OCN), and osteopontin (OPN). While the potential of G3HapZ to support vascularization was displayed by its ability to induce endothelial cell migration, attachment, and proliferation, its antimicrobial activity was confirmed using S. aureus. Biocompatibility of G3HapZ was demonstrated by its ability to induce bone regeneration and neovascularization in vivo. These results suggest that G3HapZ nanocomposites can be exploited for a range of strategies in developing orthopedic bone grafts to accelerate bone regeneration.

3D printing of Pickering emulsion inks to construct poly(D,L-lactide-co-trimethylene carbonate)-based porous bioactive scaffolds with shape memory effect

Biocompatible and bioactive scaffolds with multiscale porosity, shape memory effect and drug delivery performance are highly promising in bone tissue engineering applications within the approach of minimally invasive surgical implantation. In this study, we report an effective method to fabricate multiscale porous bioactive (MPB) scaffolds with shape memory effect by freeze drying of 3D printed high internal phase Pickering emulsion (Pickering HIPE) templates stabilized by hydrophobically modified hydroxyapatite and silica nanoparticles. MPB scaffolds containing a shape memory polymer matrix of poly(D,L-lactide-co-trimethylene carbonate) display high porosity and fully interconnected filament networks with multiscale pore structures. The in vitro biomineralization study verifies that MPB scaffolds are bioactive with the apatite formation ability. The thermal property testing shows that the glass transition temperature of MPB scaffolds is easily tuned to fall between 45.9 and 49.1 °C by varying the HIPE composition. Moreover, MPB scaffolds exhibit observable shape memory properties with fast shape recovery. The antibacterial drug enrofloxacin can be efficiently loaded into MPB scaffolds and then released in a sustained manner displaying effective antimicrobial effect. Furthermore, cell culture assays confirm that MPB scaffolds could sustain the cell growth, proliferation and osteogenic differentiation, indicating the cytocompatibility and osteoinduction activity of MPB scaffolds. Therefore, combining freeze drying with 3D printing of Pickering HIPE templates paves the way towards MPB scaffolds with shape memory effect, which are promising as minimally invasive implantation scaffolds in bone tissue engineering applications.

ProminTools: shedding light on proteins of unknown function in biomineralization with user friendly tools illustrated using mollusc shell matrix protein sequences

Biominerals are crucial to the fitness of many organism and studies of the mechanisms of biomineralization are driving research into novel materials. Biomineralization is generally controlled by a matrix of organic molecules including proteins, so proteomic studies of biominerals are important for understanding biomineralization mechanisms. Many such studies identify large numbers of proteins of unknown function, which are often of low sequence complexity and biased in their amino acid composition. A lack of user-friendly tools to find patterns in such sequences and robustly analyse their statistical properties relative to the background proteome means that they are often neglected in follow-up studies. Here we present ProminTools, a user-friendly package for comparison of two sets of protein sequences in terms of their global properties and motif content. Outputs include data tables, graphical summaries in an html file and an R-script as a starting point for data-set specific visualizations. We demonstrate the utility of ProminTools using a previously published shell matrix proteome of the giant limpet Lottia gigantea.

Polycarboxylated Eggshell Membrane Scaffold as Template for Calcium Carbonate Mineralization

Biomineralization is a process in which specialized cells secrete and deliver inorganic ions into confined spaces limited by organic matrices or scaffolds. Chicken eggshell is the fastest biomineralization system on earth, and therefore, it is a good experimental model for the study of biomineralization. Eggshell mineralization starts on specialized dispersed sites of the soft fibrillar eggshell membranes referred to as negatively charged keratan sulfate mammillae. However, the rest of the fibrillar eggshell membranes never mineralizes, although 21% of their amino acids are acidic. We hypothesized that, relative to the mammillae, the negatively charged amino acids of the fibrillar eggshell membranes are not competitive enough to promote calcite nucleation and growth. To test this hypothesis, we experimentally increased the number of negatively charged carboxylate groups on the eggshell membrane fibers and compared it with in vitro calcite deposition of isolated intact eggshell membranes. We conclude that the addition of poly-carboxylated groups onto eggshell membranes increases the number of surface nucleation sites but not the crystal size.

An in vitro evaluation of zinc silicate fortified chitosan scaffolds for bone tissue engineering

Tissue engineering aims at replacement, repair, and regeneration of tissues by a combination of scaffolds, growth factors, and cells. In this study, we report the synthesis of biodegradable composite scaffolds fortified with mesoporous zinc silicate (mZS) and assessment of in vitro properties for bone tissue engineering (BTE) applications. The scaffolds consisted of chitosan (CS) incorporated with mZS at 0.1, 0.2, 0.3, and 0.5 wt%. The bio-composite scaffolds were visualized using Field Emission Scanning Electron Microscopy (FE-SEM). The incorporation of mZS was confirmed using Energy dispersive x-ray (EDS) analysis. Biomineralization studies were conducted in simulated body fluid (SBF) and indicated bioactivity of fabricated scaffolds. The scaffolds also displayed antibacterial action against Staphylococcus aureus. Cellular attachment within the scaffold network established biocompatibility of the material. Incorporation of mZS within the chitosan scaffolds matrix improved properties such as porosity, degradation rate, and biomineralization. Therefore, fabricated scaffolds exhibit exceptional features and have the potential to serve as an implant for BTE applications.

The Study of the Quartz Sand Bio Consolidation Processes as a Result of Carbonate Mineralization by Urolithic Bacteria

The complexity and a large number of variable factors of the bacterial carbonate mineralization process in the cement stone system, which is a polylineal and polydisperse system, has led to the question of each component’s role in bio consolidation processes. For carbonate biomineralization occurring in cement concrete during its restoration, it is impossible to fully describe the bio consolidation mechanism without studying the effect on individual components of the cement system (aggregate and binder). In the environment of quartz sand (the most common type of aggregate in cement concrete) there is no direct source of calcium, which is found in cement stone, which excludes the influence of abiotic factors (alkalinity of the medium) on the calcium carbonate production by bacterial cultures. This article presents the results of the study of carbonate biomineralization by urolithic bacterial cultures in a filler medium, in which quartz sand of three fractions (1.25–0.63; 0.63–0.315; 0.315–0.16) was used to assess degree and intensity of induction processes. As a result of the study, the physical and chemical factors of the bacterial carbonate mineralization process intensification in quartz sand samples were established. The time boundaries of the appearance of a consolidated layer were revealed within two fractions (0.63–0.315 and 0.315–0.16), which are directly related to the penetration depth of the solution with precursors and the bacterial inoculum by the percolation method. The analysis of the cemented layers’ microstructure is carried out, the features of neoplasms are considered. The type of bacterial culture that exhibits the most active cementing properties - Bacillus pumilus (VKM B-23).

Two Metagenome-Assembled Genome Sequences of Magnetotactic Bacteria in the Order Magnetococcales.

Magnetotactic bacteria represent a valuable model system for the study of microbial biomineralization and magnetotaxis. Here, we report two metagenome-assembled genome sequences of uncultivated magnetotactic bacteria belonging to the order Magnetococcales These genomes contain nearly complete magnetosome gene clusters responsible for magnetosome biomineralization.

Chitosan/polypropylene glycol hydrogel composite film designed with TiO2 nanoparticles: A promising scaffold of biomedical applications

The present study explores the preparation and characterization of chitosan/poly (propylene glycol)/titanium dioxide (CH/PPG/TiO2) composite hydrogels in view of their developing applications such as antimicrobial packaging, wound dressing and antibacterial materials. The prepared CH/PPG/TiO2 films were comprehensively characterized by several methods. The size distribution showed the average size of the TiO2 nanoparticles (NPs) was about 40 nm. Additionally, other properties including swelling ratio, water retention, water contact angle, porosity, water uptake, in vitro enzymatic degradation, water vapor transmission rate, in vitro biomineralization studies, and mechanical tests were evaluated in detailed. Besides these characterizations, the antimicrobial activity of CH/PPG/TiO2 composite film against Staphylococcus aureus, Escherichia coli, and Candida lipolytica was evaluated by using disc diffusion method. Based on the obtained results, the CH/PPG/TiO2 composite hydrogels showed enhanced water vapor permeability, porosity, water retention, and swelling ratio. An improvement was observed in the examined mechanical and thermal properties with the addition of TiO2 NPs. The tensile strength and elongation at break values of CH/PPG/TiO2 were 3.0 MPa and 31%, respectively. Most importantly, the CH/PPG/TiO2 composite hydrogels showed strong antimicrobial properties. Finally, the developed composite scaffold prepared in this study may possess potentially useful in biomedical applications.

Prism substructures in the shell of Pinna nobilis (Linnaeus, 1758), Mollusca – Evidence for a three-dimensional pulsed-growth model

In the shells of the Pelecypods belonging to the Pinnidae family, the calcareous prismatic units of the outer layer are long-standing references for biomineralization studies. To elucidate how the mechanism of prism formation enables both shell elongation and thickness increase, a top-down structural analysis of these classical “simple prisms” has been carried out, taking advantage of shell sampling on actively mineralizing animals. Particular attention was paid to the morphological and structural patterns of the calcareous units sequentially produced at the margins of the growth lamellae. This pre-prismatic part of the shell allows for studying the mineralizing stages not taken into account in prism reconstructions based on samples taken from older areas of the shell. Examination of the microstructural sequence shows that within the actively mineralizing area of the shell, a step-by-step structuring process is continuously running, providing a renewed view of prism formation as it makes obvious the progressive occurrence of their specific patterns. Given the critically endangered status of the species, a better knowledge of the mineralization process associated to shell growth may become handy for future studies aimed at understanding the health status of individuals based on their shell records.

Oxygen isotope composition of waters recorded in carbonates in strong clumped and oxygen isotopic disequilibrium

Abstract. Paleoenvironmental reconstructions, which are mainly retrieved from oxygen isotope (δ18O) and clumped isotope (Δ47) compositions of carbonate minerals, are compromised when carbonate precipitation occurs in isotopic disequilibrium. To date, knowledge of these common isotopic disequilibria, known as vital effects in biogenic carbonates, remains limited, and the potential information recorded by δ18O and Δ47 offsets from isotopic equilibrium values is largely overlooked. Additionally, in carbonates formed in isotopic equilibrium, the use of the carbonate δ18O signature as a paleothermometer relies on our knowledge of the paleowaters' δ18O value, which is often assumed. Here, we report the largest Δ47 offsets observed to date (as much as −0.270 ‰), measured on microbial carbonates that are strongly linked to carbonate δ18O offsets (−25 ‰) from equilibrium. These offsets are likely both related to the microorganism metabolic activity and yield identical erroneous temperature reconstructions. Unexpectedly, we show that the δ18O value of the water in which carbonates precipitated, as well as the water–carbonate δ18O fractionation dependence on temperature at equilibrium, can be retrieved from these paired δ18O and Δ47 disequilibrium values measured in carbonates. The possibility to retrieve the δ18O value of paleowaters, sediments' interstitial waters or organisms' body water at the carbonate precipitation loci, even from carbonates formed in isotopic disequilibrium, opens long-awaited research avenues for both paleoenvironmental reconstructions and biomineralization studies.

Supramolecular Hydrogels with Properties Tunable by Calcium Ions: A Bio-Inspired Chemical System.

Boc-L-DOPA(OBn)2-OH is a simple synthetic molecule that promotes hydrogelation through electrostatic and π-π stacking interactions. Hydrogelation can occur in alkaline conditions by the use of triggers. Four hydrogels were prepared varying the base, NaOH or Na2CO3, and the trigger, GdL or CaCl2. When the hydrogel formed in the presence of Na2CO3 and CaCl2, the concomitant production of CaCO3 crystals occurred, generating an organic/inorganic composite material. It was observed that the hydrogel once self-assembled preserved its status even if the trigger, the calcium ions, was removed. The viscoelastic behavior of the hydrogels was analyzed through rheological experiments, which showed a solid-like behavior of the hydrogels. The corresponding xerogels were analyzed mainly by scanning electron microscopy (SEM) and synchrotron X-ray diffraction analysis (XRD). They showed differences in structure, morphology, and fiber organization according to their source. This research presents a hydrogel system that can be applied as a soft biomaterial for tissue engineering, cosmetics, food, and environmental science. Moreover, it represents a model for biomineralization studies in which the hydrogel structure can act as an analogue of the insoluble matrix that confines the calcification site, provides Ca2+, and preserves its structure.

Tissue-specific evaluation of suitable reference genes for RT-qPCR in the pond snail, Lymnaea stagnalis.

Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression. To obtain reliable results with this method, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail (Lymnaea stagnalis) is a common research organism, particularly in the areas of learning and memory, and is an emerging model for the study of biological asymmetry, biomineralization, and evolution and development. However, no systematic assessment of qPCR reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from several commonly studied tissues in L. stagnalis as well as across the entire body. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, beta-tubulin, ubiquitin, prenylated rab acceptor protein 1, and a voltage gated potassium channel. These genes exhibited a wide range of expression levels among tissues. The tissue-specific stability of each of the genes was consistent when measured by the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Our data indicate that the most stable reference genes vary among the tissues that we examined (central nervous system, tentacles, lips, penis, foot, mantle). Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we provide suggestions for strong pairs of reference genes for single- and multi-tissue analyses of RT-qPCR data in L. stagnalis.

Atom Probe Tomography (APT) Characterization of Organics Occluded in Single Calcite Crystals: Implications for Biomineralization Studies

Occlusion of organic components in synthetic calcite crystals has been recently used as a model to understand the role of intra-crystalline organics in biominerals. However, the characterization of the distribution of both types of organics inside these calcite crystals is very challenging. Here, we discuss the potential of using the technique of atom probe tomography (APT) for such characterization, focusing on the analysis of chitin incorporation in single crystals. Additionally, APT has at least the same spatial resolution as TEM tomography, yet with the advantage of obtaining quantitative chemical data. Results show that chitin, either after degradation with yatalase or in the form of nanofibers, forms discrete clusters (2 to 5 nm) in association to water and hydronium molecules, rather than forming a 3D network inside crystals. Overall findings indicate that APT can be an ideal technique to characterize intra-crystalline organic components in abiogenic and biogenic carbonates to further advance our understanding of biomineralization.

Fabrication and characterization of poly (ethylenimine) modified poly (l-lactic acid) nanofibrous scaffolds

Bone tissue engineering aims to construct biological substitutes for repairing bone defects. Nanofibrous (NF) scaffolds are commonly utilized to mimic the extracellular matrix (ECM) environment and promote tissue regeneration in tissue engineering process. Poly (lactic acid) (PLA) has attracted much attention in the field of tissue engineering because of its biocompatibility, biodegradability and so on. However, the intrinsic hydrophobicity and the lacking of active functional groups limit its practical application to some extent. In this study, poly(ethylenimine) (PEI) modified PLLA nanofibrous scaffolds were fabricated in a one step process by aminolysis combined with thermally induced phase separation technique for introducing more functional groups, PEI acting as the modifier. The morphology of PEI-modified PLLA scaffolds prepared under different experimental conditions was analyzed by scanning electron microscope (SEM). The suitable conditions to fabricate scaffolds with a homogeneous nanofibrous structure, good hydrophilicity and excellent mechanical properties were determined according to the results of SEM, water contact angle (WCA) and mechanical properties testing. Besides, Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy (1H NMR), X-ray Photoelectron Spectroscopy (XPS) and gel permeation chromatography (GPC) were used to confirm the occurrence of the ammonolysis reaction between PLLA and PEI. The in vitro biomineralization study showed that the PEI-modified PLLA scaffolds had a greater ability to induce the formation of apatite in 1.5SBF than PLLA scaffolds, indicating that the bone-bioactivity of PLLA scaffolds was significantly improved after modification with PEI. Furthermore, cell culture assay revealed that MC3T3-E1 osteoblasts exhibited better proliferation performance on the PEI-modified PLLA scaffolds. All the results implied that the synthesized modified PLLA nanofibrous scaffolds may provide promising applications in bone tissue engineering.

The ESRF dark-field x-ray microscope at ID06

We present an instrument for dark-field x-ray microscopy installed on beamline ID06 of the ESRF — the first of its kind. Dark-field x-ray microscopy uses full field illumination of the sample and provides three-dimensional (3D) mapping of micro-structure and lattice strain in crystalline matter. It is analogous to dark-field electron microscopy in that an objective lens magnifies diffracting features of the sample. The use of high-energy synchrotron x-rays, however, means that these features can be large and deeply embedded. 3D movies can be acquired with a time resolution of seconds to minutes. The field of view and spatial resolution can be adapted by simple reconfiguration of the x-ray objective lens, reaching spatial and angular resolution of 30-100 nm and 0.001°, respectively. The instrument furthermore allows pre-characterization of samples at larger length scales using 3DXRD or DCT, such that a region of interest (e.g. a single grain) can be selected for high-resolution studies without the need to dismount the sample. As examples of applications we show work on mapping the subgrains in plastically deformed iron and aluminum alloys, mapping domains and strain fields in ferroelectric crystals, and studies of biominerals. This ability to directly characterize complex, multi-scale phenomena in-situ is a key step towards formulating and validating multi-scale models that account for the entire heterogeneity of materials. As an outlook, we discuss future prospects for such multi-scale characterization by combining DFXM with 3DXRD/DCT, and coherent x-ray methods for coarser and finer length-scales, respectively.

Characterization and electrochemical behavior of ZrO2/CSZ coated 316L SS before and after incubation with calcium precipitating oral bacteria

Ceria stabilized zirconia (CSZ) and zirconia (ZrO2) were deposited by electron beam physical vapor deposition (EBPVD) technique. The tetragonal and monoclinic phases of CSZ and ZrO2 were confirmed by x-ray diffraction (XRD). Smooth surface topography with equally distributed grains was observed through atomic force microscopy (AFM) observation. Calcium carbonate/phosphate precipitation by Paenibacillus sp. oral bacteria (CPOB) was isolated and identified. Biomineralization study was carried out to determine the apatite formation behavior of the samples and the results revealed that high degree of apatite formation was occurred on coated substrates rather than bare 316 L SS samples. Further, electrochemical corrosion analysis was conducted in presence and absence of oral bacteria. The results revealed that the samples containing bacteria showed improved corrosion resistance behavior due to the formation of calcite precipitation.

Surface Wetting Controls Calcium Carbonate Crystallization Kinetics

Because of the widespread presence of foreign substrates in natural settings, mineral precipitation usually occurs via heterogeneous nucleation. This process is controlled by the interplay between the fluid supersaturation and interfacial energies present between the fluid, nucleus, and substrate. Among a number of physicochemical parameters, the surface wetting properties have been shown to be a key parameter controlling heterogeneous nucleation. The present study aims at elucidating the pathway and kinetics of CaCO3 heterogeneous nucleation on a set of phlogopite micas with and without fluorine/hydroxyl substitutions, yielding substrates with contrasting hydrophilicity. Our results show that, irrespective of surface wetting properties, amorphous calcium carbonate (ACC) is formed during the early stages. The surface wetting properties have a strong effect on the crystallization kinetics: ACC precipitates persist longer on the hydrophilic (hydroxylated) surface than on the less hydrophilic (fluorinated) one. We show that this stabilization could have a thermodynamic origin because of the lower interfacial free energy between the hydrated amorphous precursor and the hydrophilic substrate. These results are highly relevant for biomineralization studies, where differences in wetting properties of organic moieties present in calcifying organisms could be used to accelerate or decelerate the crystallization of the initially formed amorphous precursor phase.