Chemical and Ultrastructural Characterization of Dentin Treated with Remineralizing Dentifrices.

The aim of this study is to investigate dentin chemical and ultrastructural changes upon exposure to remineralizing dentifrices. Dentin disks were obtained from permanent human molars and treated for 7 days with the dentifrices: (1) C group-control (no dentifrice); (2) S group-Sensodyne Repair & Protect; (3) D group-Dentalclean Daily Regenerating Gel; and (4) DB group-D group + Dentalclean regenerating booster. Afterwards, samples were submitted to an additional 7 days of toothbrushing associated with daily acidic challenge. Samples were imaged and analyzed (days 1, 7, and 14) for Young's modulus by atomic force microscopy (AFM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). SEM and AFM revealed precipitate deposition on dentin surfaces in groups S, D, and DB, formed as early as day 1. Surface elemental analysis showed a Si increase on all brushed surfaces. Similar surface morphology was maintained after the acidic challenge period. Bright-field TEM/SAED revealed the formation of nanocrystalline hydroxyapatite inside the dentin tubules of groups S, D, and DB after day 7. Group C presented a gradual reduction of Young's modulus from days-1-14, whereas all remaining groups had increased values. All evaluated dentifrices led to successful formation of hydroxyapatite and increased dentin stiffness.

Focusing on the Native Matrix Proteins in Calcific Aortic Valve Stenosis.

Calcific aortic valve stenosis (CAVS) is a widespread valvular heart disease affecting people in aging societies, primarily characterized by fibrosis, inflammation, and progressive calcification, leading to valve orifice stenosis. Understanding the factors associated with CAVS onset and progression is crucial to develop effective future pharmaceutical therapies. In CAVS, native extracellular matrix proteins modifications, play a significant role in calcification in vitro and in vivo. This work aimed to review the evidence on the alterations of structural native extracellular matrix proteins involved in calcification development during CAVS and highlight its link to deregulated biomechanical function.

DNA hydrogels for bone regeneration.

DNA-based biomaterials have been proposed for tissue engineering approaches due to their predictable assembly into complex morphologies and ease of functionalization. For bone tissue regeneration, the ability to bind Ca2+ and promote hydroxyapatite (HAP) growth along the DNA backbone combined with their degradation and release of extracellular phosphate, a known promoter of osteogenic differentiation, make DNA-based biomaterials unlike other currently used materials. However, their use as biodegradable scaffolds for bone repair remains scarce. Here, we describe the design and synthesis of DNA hydrogels, gels composed of DNA that swell in water, their interactions in vitro with the osteogenic cell lines MC3T3-E1 and mouse calvarial osteoblast, and their promotion of new bone formation in rat calvarial wounds. We found that DNA hydrogels can be readily synthesized at room temperature, and they promote HAP growth in vitro, as characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, atomic force microscopy, and transmission electron microscopy. Osteogenic cells remain viable when seeded on DNA hydrogels in vitro, as characterized by fluorescence microscopy. In vivo, DNA hydrogels promote the formation of new bone in rat calvarial critical size defects, as characterized by micro-computed tomography and histology. This study uses DNA hydrogels as a potential therapeutic biomaterial for regenerating lost bone.

Peptide-Decorated DNA Nanostructures Promote Site-Specific Hydroxyapatite Growth.

The guiding principle for mineralized tissue formation is that mineral growth occurs through the interaction of Ca2+ and phosphate ions with extracellular matrix (ECM) proteins. Recently, nanoengineered DNA structures have been proposed as mimics to ECM scaffolds. However, these principles have not been applied to mineralized tissues. Here, we describe DNA nanostructures, namely, a DNA nanotube and a DNA origami rectangle that are site specifically functionalized with a mineral-promoting "SSEE" peptide derived from ECM proteins present in mineralized tissues. In the presence of Ca2+ and phosphate ions (mineralizing conditions), site-specific calcium phosphate formation occurred on the DNA nanostructures. Amorphous calcium phosphate or hydroxyapatite was formed depending on the incubation time, shape of the DNA nanostructure, and amount of Ca2+ and phosphate ions present. The ability to design and control the growth of hydroxyapatite through nanoengineered scaffolds provides insights into the mechanisms that may occur during crystal nucleation and growth of mineralized tissues and can inspire mineralized tissue regeneration strategies.

Uniaxial Hydroxyapatite Growth on a Self-Assembled Protein Scaffold.

Biomineralization is a crucial process whereby organisms produce mineralized tissues such as teeth for mastication, bones for support, and shells for protection. Mineralized tissues are composed of hierarchically organized hydroxyapatite crystals, with a limited capacity to regenerate when demineralized or damaged past a critical size. Thus, the development of protein-based materials that act as artificial scaffolds to guide hydroxyapatite growth is an attractive goal both for the design of ordered nanomaterials and for tissue regeneration. In particular, amelogenin, which is the main protein that scaffolds the hierarchical organization of hydroxyapatite crystals in enamel, amelogenin recombinamers, and amelogenin-derived peptide scaffolds have all been investigated for in vitro mineral growth. Here, we describe uniaxial hydroxyapatite growth on a nanoengineered amelogenin scaffold in combination with amelotin, a mineral promoting protein present during enamel formation. This bio-inspired approach for hydroxyapatite growth may inform the molecular mechanism of hydroxyapatite formation in vitro as well as possible mechanisms at play during mineralized tissue formation.

DNA nanostructures as templates for biomineralization.

Nature uses extracellular matrix scaffolds to organize biominerals into hierarchical structures over various length scales. This has inspired the design of biomimetic mineralization scaffolds, with DNA nanostructures being among the most promising. DNA nanotechnology makes use of molecular recognition to controllably give 1D, 2D and 3D nanostructures. The control we have over these structures makes them attractive templates for the synthesis of mineralized tissues, such as bones and teeth. In this Review, we first summarize recent work on the crystallization processes and structural features of biominerals on the nanoscale. We then describe self-assembled DNA nanostructures and come to the intersection of these two themes: recent applications of DNA templates in nanoscale biomineralization, a crucial process to regenerate mineralized tissues.

Nanostructure of mouse otoconia.

Mammalian otoconia of the inner ear vestibular apparatus are calcium carbonate-containing mineralized structures critical for maintaining balance and detecting linear acceleration. The mineral phase of otoconia is calcite, which coherently diffracts X-rays much like a single-crystal. Otoconia contain osteopontin (OPN), a mineral-binding protein influencing mineralization processes in bones, teeth and avian eggshells, for example, and in pathologic mineral deposits. Here we describe mineral nanostructure and the distribution of OPN in mouse otoconia. Scanning electron microscopy and atomic force microscopy of intact and cleaved mouse otoconia revealed an internal nanostructure (~50 nm). Transmission electron microscopy and electron tomography of focused ion beam-prepared sections of otoconia confirmed this mineral nanostructure, and identified even smaller (~10 nm) nanograin dimensions. X-ray diffraction of mature otoconia (8-day-old mice) showed crystallite size in a similar range (73 nm and smaller). Raman and X-ray absorption spectroscopy - both methods being sensitive to the detection of crystalline and amorphous forms in the sample - showed no evidence of amorphous calcium carbonate in these mature otoconia. Scanning and transmission electron microscopy combined with colloidal-gold immunolabeling for OPN revealed that this protein was located at the surface of the otoconia, correlating with a site where surface nanostructure was observed. OPN addition to calcite growing in vitro produced similar surface nanostructure. These findings provide details on the composition and nanostructure of mammalian otoconia, and suggest that while OPN may influence surface rounding and surface nanostructure in otoconia, other incorporated proteins (also possibly including OPN) likely participate in creating internal nanostructure.

Homochirality in biomineral suprastructures induced by assembly of single-enantiomer amino acids from a nonracemic mixture.

Since Pasteur first successfully separated right-handed and left-handed tartrate crystals in 1848, the understanding of how homochirality is achieved from enantiomeric mixtures has long been incomplete. Here, we report on a chirality dominance effect where organized, three-dimensional homochiral suprastructures of the biomineral calcium carbonate (vaterite) can be induced from a mixed nonracemic amino acid system. Right-handed (counterclockwise) homochiral vaterite helicoids are induced when the amino acid L-Asp is in the majority, whereas left-handed (clockwise) homochiral morphology is induced when D-Asp is in the majority. Unexpectedly, the Asp that incorporates into the homochiral vaterite helicoids maintains the same enantiomer ratio as that of the initial growth solution, thus showing chirality transfer without chirality amplification. Changes in the degree of chirality of the vaterite helicoids are postulated to result from the extent of majority enantiomer assembly on the mineral surface. These mechanistic insights potentially have major implications for high-level advanced materials synthesis.

Nanostructure, osteopontin, and mechanical properties of calcitic avian eggshell.

Avian (and formerly dinosaur) eggshells form a hard, protective biomineralized chamber for embryonic growth-an evolutionary strategy that has existed for hundreds of millions of years. We show in the calcitic chicken eggshell how the mineral and organic phases organize hierarchically across different length scales and how variation in nanostructure across the shell thickness modifies its hardness, elastic modulus, and dissolution properties. We also show that the nanostructure changes during egg incubation, weakening the shell for chick hatching. Nanostructure and increased hardness were reproduced in synthetic calcite crystals grown in the presence of the prominent eggshell protein osteopontin. These results demonstrate the contribution of nanostructure to avian eggshell formation, mechanical properties, and dissolution.

Chiral acidic amino acids induce chiral hierarchical structure in calcium carbonate.

Chirality is ubiquitous in biology, including in biomineralization, where it is found in many hardened structures of invertebrate marine and terrestrial organisms (for example, spiralling gastropod shells). Here we show that chiral, hierarchically organized architectures for calcium carbonate (vaterite) can be controlled simply by adding chiral acidic amino acids (Asp and Glu). Chiral, vaterite toroidal suprastructure having a 'right-handed' (counterclockwise) spiralling morphology is induced by L-enantiomers of Asp and Glu, whereas 'left-handed' (clockwise) morphology is induced by D-enantiomers, and sequentially switching between amino-acid enantiomers causes a switch in chirality. Nanoparticle tilting after binding of chiral amino acids is proposed as a chiral growth mechanism, where a 'mother' subunit nanoparticle spawns a slightly tilted, consequential 'daughter' nanoparticle, which by amplification over various length scales creates oriented mineral platelets and chiral vaterite suprastructures. These findings suggest a molecular mechanism for how biomineralization-related enantiomers might exert hierarchical control to form extended chiral suprastructures.

New insights into the chemical and isotopic composition of human-body biominerals. I: Cholesterol gallstones from England and Greece.

We have analyzed gallstones from four patients of Europe and particularly from England (including samples from a mother and a daughter) and Greece. According to the XRD, FTIR, NMR and laser micro-Raman results the studied materials correspond to typical cholesterol monohydrate (ChM). The micro-morphology of cholesterol microcrystals was investigated by means of SEM-EDS. The XRF results revealed that Ca is the dominant non-organic metal in all gallstones (up to ∼1.95wt.%) together with Fe, Cu, Pb and Ni (up to ~19ppm for each metal). Gallstones from England contain additional Mn (up to ~87ppm) and Zn (up to ∼6ppm) while the sample of the mother contains negligible Zn and Mn, compared to that of her daughter, but significant As (~4.5ppm). All cholesterol gallstones examined are well enriched in potentially toxic metals (Pb, as well as Ni in one case) and metalloids (As also in one case) as compared to the global average. The position of Zn, which is a characteristic biometal, in the structure of cholesterol, was investigated by molecular simulation using the Accelrys Materials Studio(®) software. On the basis of IRMS results, all gallstones examined exhibit a very light δ(13)C signature (average δ(13)C ~-24‰ PDB). Gamma-ray spectrometry measurements indicate the presence of (214)Pb and (214)Bi natural radionuclides due to the (238)U series as well as an additional amount of (40)K.