Growth Of Apatite Crystals Research Articles

Human genetic diseases of phosphate and pyrophosphate metabolism

In humans, physiological bone and tooth mineralization is a complex cell-mediated process. Prerequisites for proper mineralization include sufficient amounts of minerals (calcium and phosphate [Pi]) to initiate the formation and the growth of apatite crystals and adequate amounts of mineralization inhibitors, such as pyrophosphate (PPi), to prevent uncontrolled extraskeletal mineralization.In this review, we provide an overview of the genetics of human disorders of mineralization, focusing on Pi and PPi metabolism and transport diseases, as the Pi/PPi ratio is an important determinant of crystal production in vivo. Variants in genes implicated in the homeostasis of this ratio may lead to a systemic or local increased Pi/PPi ratio, either by increasing the Pi concentration or by decreasing the PPi concentration, resulting in ectopic calcifications; conversely, variants may lead to a decreased Pi/PPi ratio, resulting in defective mineralization.Owing to the implication of common pathways and, occasionally, to some extent of clinical overlap, an accurate diagnosis and understanding of the pathophysiology of these disorders may be challenging. However, precise molecular characterization of these conditions not only facilitates their diagnosis, but also helps to gather evidence regarding the pathophysiology and phenotype–genotype correlation to improve medical care and develop innovative therapeutics.

Diffusion mechanisms between La2SiO5 and SiO2 during formation of textured lanthanum silicate oxyapatite crystals

Lanthanum silicate oxyapatite (LSO) is a promising alternative electrolyte for solid oxide fuel cell (SOFC) applications. They exhibit anisotropic oxide-ionic conduction; therefore, a preferential crystal orientation along the c-axis is necessary to achieve optimal conductivity. This study is performed to understand the reaction mechanisms involved in the formation of an LSO layer at the interface of the La2SiO5/SiO2 diffusion pair during reactive sintering at high temperatures. Different La2SiO5/SiO2 bilayers were fabricated in sandwich-type structures using silica-type quartz or La2SiO5 supports obtained via electrophoresis and uniaxial pressing, respectively. These supports were pre-sintered and then coated with a suspension of the opposite component. The diffusion pairs were isothermally heated at 1500 °C at different holding times (10, 20, and 40 h) and at 1600 °C for 10 h. Their surfaces and cross-sections were investigated via X-ray diffraction, scanning electron microscopy (SEM) observations, energy-dispersive X-ray spectroscopy (EDS), and Raman spectroscopy.Experimental results show that two different driving forces were involved in the nucleation and growth of apatite crystals. The first is the chemical potential gradient in lanthanum between the components of the layers, and the second is the electric field created to preserve the electroneutrality of the system. The predominant diffusing species in this bilayer system were La+III and O-II, and their diffusion is enhanced by the formation of a glassy phase on the silica grains through the transformation of silica-type quartz into cristobalite. The formation of a La2Si2O7 intermediate phase was detected, which is vital to the interdiffusion of species and the nucleation and growth of oriented apatite crystals. The porosity gradient observed in the cross-section after reactive diffusion suggests the possibility of achieving an SOFC half-cell device via this process.

Highly acidic pH facilitates enamel protein self-assembly, apatite crystal growth and enamel protein interactions in the early enamel matrix.

Tooth enamel develops within a pH sensitive amelogenin-rich protein matrix. The purpose of the present study is to shed light on the intimate relationship between enamel matrix pH, enamel protein self-assembly, and enamel crystal growth during early amelogenesis. Universal indicator dye staining revealed highly acidic pH values (pH 3-4) at the exocytosis site of secretory ameloblasts. When increasing the pH of an amelogenin solution from pH 5 to pH 7, there was a gradual increase in subunit compartment size from 2nm diameter subunits at pH 5 to a stretched configuration at pH6 and to 20nm subunits at pH 7. HSQC NMR spectra revealed that the formation of the insoluble amelogenin self-assembly structure at pH6 was critically mediated by at least seven of the 11 histidine residues of the amelogenin coil domain (AA 46-117). Comparing calcium crystal growth on polystyrene plates, crystal length was more than 20-fold elevated at pH 4 when compared to crystals grown at pH 6 or pH 7. To illustrate the effect of pH on enamel protein self-assembly at the site of initial enamel formation, molar teeth were immersed in phosphate buffer at pH4 and pH7, resulting in the formation of intricate berry tree-like assemblies surrounding initial enamel crystal assemblies at pH4 that were not evident at pH7 nor in citrate buffer. Amelogenin and ameloblastin enamel proteins interacted at the secretory ameloblast pole and in the initial enamel layer, and co-immunoprecipitation studies revealed that this amelogenin/ameloblastin interaction preferentially takes place at pH 4-pH 4.5. Together, these studies highlight the highly acidic pH of the very early enamel matrix as an essential contributing factor for enamel protein structure and self-assembly, apatite crystal growth, and enamel protein interactions.

The Effect of Material Surface Microstructure on the Enhancement of Bone-Bonding Ability of a Hydroxyapatite-Implant by Low-Intensity Pulsed Ultrasound (LIPUS)

Excellent firm bonding between the biomaterials and bone tissue (osseointegration and osteo-conductivity) has been desired for the stability in vivo of dental implants and artificial joints. Much has been learned about this concept, which has led to significant improvements in the design and surface modification of implants in the field of implant dentistry, orthopedic surgery. We have already reported that low-intensity pulsed ultrasound (LIPUS) irradiation can accelerate the bone bonding ability of the bio-conductive materials such as bioactive titanium and hydroxyapatite implant. However, it is still unclear whether the LIPUS could have same effect to different types of the bioactive-materials. Therefore, in this study, the differences of bone-like hydroxyapatite formation on some kind of hydroxyapatite surface in simulated body fluid (SBF) under the LIPUS irradiation were investigated. Two kinds of hydroxyapatite samples immersed in SBF was exposed to ultrasound waves, the bone-like apatite on the surface was analyzed by Scanning electron microscopy and X-ray diffraction. As a result, the enhancement of hydroxyapatite formation on the surface by LIPUS was confirmed, the initial epitaxial nucleation and crystal growth of apatite depended on crystal structure of the surface of matrix materials.

Influence of bacterial nanocellulose surface modification on calcium phosphates precipitation for bone tissue engineering

Bacterial nanocellulose (BNC) is a nano fibrillar polymer, which is biostable and non-resorbable when inside the human body. It has excellent biocompatibility and a microstructure with high mechanical strength, and if processed correctly, can mimic the extra-cellular matrix architecture. BNC, modified with bone-like minerals such as calcium phosphates, can improve cell adhesion and promote the formation of new bone tissues. As a result of the need for three-dimensional (3D) porous scaffolds for bone tissue regeneration, this study evaluated the effect of calcium phosphate mineralization process on BNC and (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO)-oxidized BNC scaffolds, to understand the influence of hydroxyl or carboxylate groups on the nucleation and growth of apatite crystals. The results showed 3D scaffolds with controlled microporosity, between 50 and 350 µm, and interconnected pores. The porous morphology of the TEMPO-oxidized BNC scaffolds varied significantly with the oxidation time and only remained preserved after 60 min of the TEMPO-mediated oxidation. BNC and TEMPO-oxidized BNC scaffolds were used to compare two different mineralization treatments. The growth of homogeneously distributed microcrystals was observed in the unmodified BNC scaffolds, whereas heterogeneously distributed microcrystals were observed in the TEMPO-oxidized BNC scaffolds because of the oxidation treatment which affected the continuity of the surface by fracturing some fibers. Also, in vitro cell studies revealed good cellular adhesion and high cell viability in the modified and unmodified BNC scaffolds. Most of the modifications seemed adequate for cellular adhesion.

Osteopontin regulates biomimetic calcium phosphate crystallization from disordered mineral layers covering apatite crystallites

Details of apatite formation and development in bone below the nanometer scale remain enigmatic. Regulation of mineralization was shown to be governed by the activity of non-collagenous proteins with many bone diseases stemming from improper activity of these proteins. Apatite crystal growth inhibition or enhancement is thought to involve direct interaction of these proteins with exposed faces of apatite crystals. However, experimental evidence of the molecular binding events that occur and that allow these proteins to exert their functions are lacking. Moreover, recent high-resolution measurements of apatite crystallites in bone have shown that individual crystallites are covered by a persistent layer of amorphous calcium phosphate. It is therefore unclear whether non-collagenous proteins can interact with the faces of the mineral crystallites directly and what are the consequences of the presence of a disordered mineral layer to their functionality. In this work, the regulatory effect of recombinant osteopontin on biomimetic apatite is shown to produce platelet-shaped apatite crystallites with disordered layers coating them. The protein is also shown to regulate the content and properties of the disordered mineral phase (and sublayers within it). Through solid-state NMR atomic carbon-phosphorous distance measurements, the protein is shown to be located in the disordered phases, reaching out to interact with the surfaces of the crystals only through very few sidechains. These observations suggest that non-phosphorylated osteopontin acts as regulator of the coating mineral layers and exerts its effect on apatite crystal growth processes mostly from afar with a limited number of contact points with the crystal.

Biomimetic peptide enriched nonwoven scaffolds promote calcium phosphate mineralisation.

Cell-free translational strategies are needed to accelerate the repair of mineralised tissues, particularly large bone defects, using minimally invasive approaches. Regenerative bone scaffolds should ideally mimic aspects of the tissue's ECM over multiple length scales and enable surgical handling and fixation during implantation in vivo. Leveraging the knowledge gained with bioactive self-assembling peptides (SAPs) and SAP-enriched electrospun fibres, we presented a cell free approach for promoting mineralisation via apatite deposition and crystal growth, in vitro, of SAP-enriched nonwoven scaffolds. The nonwoven scaffold was made by electrospinning poly(ε-caprolactone) (PCL) in the presence of either peptide P11-4 (Ac-QQRFEWEFEQQ-Am) or P11-8 (Ac QQRFOWOFEQQ-Am), in light of the polymer's fibre forming capability and its hydrolytic degradability as well as the well-known apatite nucleating capability of SAPs. The 11-residue family of peptides (P11-X) has the ability to self-assemble into β-sheet ordered structures at the nano-scale and to generate hydrogels at the macroscopic scale, some of which are capable of promoting biomineralisation due to their apatite-nucleating capability. Both variants of SAP-enriched nonwoven used in this study were proven to be biocompatible with murine fibroblasts and supported nucleation and growth of apatite minerals in simulated body fluid (SBF) in vitro. The fibrous nonwoven provided a structurally robust scaffold, with the capability to control SAP release behaviour. Up to 75% of P11-4 and 45% of P11-8 were retained in the fibres after 7 day incubation in aqueous solution at pH 7.4. The encapsulation of SAP in a nonwoven system with apatite-forming as well as localised and long-term SAP delivery capabilities is appealing as a potential means of achieving cost-effective bone repair therapy for critical size defects.

Surface-transformed osteoinductive polyethylene terephthalate scaffold as a dual system for bone tissue regeneration with localized antibiotic delivery.

Multifunctional scaffolds have recently attained superior significance in tissue regeneration due to combinational activity profile that is usually accomplished by separate sequential therapy. Here, we present a dual system comprised of surface-phosphorylated PET fibrous matrix coated with ciprofloxacin-impregnated biodegradable polymer poly (hydroxyethyl methacrylate) aiming at regeneration of bone tissue deprived of bacterial infections, particularly osteomyelitis. The ATR, XPS and FESEM/EDX results provided confirmative evidences for surface phosphorylation of PET and in situ coating of the polymer. Swelling and contact angle measurements demonstrated improved hydrophilicity which is further corroborated by in vitro degradation profile in PBS. Preliminary evaluation by MTT and actin staining proved its biocompatibility while enhanced in vitro mineralization in 1.5X SBF by FESEM/EDX clearly indicate the primary nucleation and secondary growth of beautiful apatite crystals with Ca/P ratio similar to human bone. Alizarin red S and von Kossa staining validated the biomineralization in MG-63 cells. The sequential expression of early and late biomarkers -alkaline phosphatase (ALP) and osteocalcin (OSN)- of osteoblast differentiation in rat bone marrow mesenchymal cells (BMC) has demonstrated osteoinductive nature of the system. The second functionality of the scaffold has been proven by step-wise ciprofloxacin-release profile (in vitro) with ~60% release within 120h. In addition, antibacterial studies of ciprofloxacin- eluted from the scaffold have shown apparent zones of inhibition against Staphylococcus aureus (3.6±0.3cm) and Escherichia coli (3.0±0.8cm). Hence, the surface-transformed PET scaffold function as a dual system as localized antibiotic delivery vehicle against bone infections and undergo self-biomineralization leading to osteoinduction.

UHMWPE/HA biocomposite compatibilized by organophilic montmorillonite: An evaluation of the mechanical-tribological properties and its hemocompatibility and performance in simulated blood fluid

The low interaction between ultra high molecular weight polyethylene (UHMWPE) and hydroxyapatite (HA) has been one of the problems that results in a composite material with low mechanical and tribological performance due to the formation of agglomerates and microstructural defects. These properties affect the quality of the material when used for total joint implants and other applications in hard tissue engineering. This study investigated the effect of the addition of organophilic bentonite (BO) into the interface HA and UHMWPE. The composite was prepared by wet milling in a planetary mill and then by compression molding. The composites samples were characterized by XRD, FTIR, SEM and DSC. The tensile and tribological mechanical properties were also evaluated. Furthermore, in vitro degradation using simulated blood fluid (SBF) and hemocompatibility was performed. The results suggest that the addition of 10 wt% of organophilic bentonite improved the interface between the UHMWPE and HA by exfoliation/intercalation, presenting the best results of modulus of elasticity, tensile strength, coefficient of friction and rate of wear. The composite UHMWPE/HA/BO-10 wt% presented low water absorption and induced the growth of apatite crystals on its surface. Additionally, its hemocompatibility index is within normal limits and induced a low adhesion and agglomeration of platelets in contact with human blood, evidencing that the UHMWPE/HA/BO-10 wt% composite is promising for application in bone tissue engineering.

Particle-Attachment-Mediated and Matrix/Lattice-Guided Enamel Apatite Crystal Growth.

Tooth enamel is a hard yet resilient biomaterial that derives its unique mechanical properties from decussating bundles of apatite crystals. To understand enamel crystal nucleation and growth at a nanoscale level and to minimize preparation artifacts, the developing mouse enamel matrix was imaged in situ using graphene liquid cells and atomic resolution scanning transmission electron and cryo-fracture electron microscopy. We report that 1-2 nm diameter mineral precipitates aggregated to form larger 5 nm particle assemblies within ameloblast secretory vesicles or annular organic matrix subunits. Further evidence for the fusion of 1-2 nm mineral precipitates into 5 nm mineral aggregates via particle attachment was provided by matrix-mediated calcium phosphate crystal growth studies. As a next step, aggregated particles organized into rows of 3-10 subunits and developed lattice suprastructures with 0.34 nm gridline spacings corresponding to the (002) planes of apatite crystals. Mineral lattice suprastructures superseded closely matched organic matrix patterns, suggestive of a combination of organic/inorganic templates guiding apatite crystal growth. Upon assembly of 2-5 nm subunits into crystal ribbons, lattice fringes indicative of the presence of larger ordered crystallites were observed surrounding elongating crystal ribbons, presumably guiding the c-axis growth of composite apatite crystals. Cryo-fracture micrographs revealed reticular networks of an organic matrix on the surface of elongating enamel crystal ribbons, suggesting that protein coats facilitate c-axis apatite crystal growth. Together, these data demonstrate (i) the involvement of particle attachment in enamel crystal nucleation, (ii) a combination of matrix- and lattice-guided crystal growth, and (iii) fusion of individual crystals via a mechanism similar to Ostwald ripening.

Monodisperse, colloidal and luminescent calcium fluoride nanoparticles via a citrate-assisted hydrothermal route

Luminescent calcium fluoride (CaF2) nanoparticles, because of their excellent biocompatibility, excellent photostability and strong fluorescence, have received increasing attention as drug carriers and bioprobes in cell imaging. Inspired by the role of citrate in the growth of apatite crystals during natural bio-mineralization, uniform and nearly monodisperse Eu3+-doped CaF2 nanoparticles with excellent colloidal stability and high fluorescence in aqueous media have been successfully synthesized in the presence of sodium citrate using a hydrothermal method. X-ray diffraction and transmission electron microscopy show that CaF2 nanoparticles grown in the presence of sodium citrate are cubes of relatively uniform size (15 nm), and that the Eu3+ doping level has little effect on size and morphology. Zeta potentials and dynamic light scattering demonstrate that in the synthesis with sodium citrate, the colloidal stability of CaF2 nanoparticles is greatly improved upon the increase of Eu3+ doping level. Moreover, aqueous dispersions of these nanoparticles are colloidally stable and can be maintained over a wide range of pH from 5.0 to 11.0 for more than a month. Fluorescence spectra demonstrate that the doped CaF2 nanoparticles display strong red fluorescence. Fourier transform infrared spectra and thermogravimetric analyses demonstrate the adsorption of substantial quantities of sodium citrate on the surfaces of the CaF2 nanoparticles. Taken together, such colloidal behavior should be related to strong crystal inhibition of citrate ions and Eu3+ doping induced promotion thermal-decomplexing between citrate ions and calcium ions. The luminescent CaF2 nanoparticles obtained using this protocol should be promising candidates for use in many bio-related applications.

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development.

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8–16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit.

A Bioactive Hydrogel and 3D Printed Polycaprolactone System for Bone Tissue Engineering.

In this study, a hybrid system consisting of 3D printed polycaprolactone (PCL) filled with hydrogel was developed as an application for reconstruction of long bone defects, which are innately difficult to repair due to large missing segments of bone. A 3D printed gyroid scaffold of PCL allowed a larger amount of hydrogel to be loaded within the scaffolds as compared to 3D printed mesh and honeycomb scaffolds of similar volumes and strut thicknesses. The hydrogel was a mixture of alginate, gelatin, and nano-hydroxyapatite, infiltrated with human mesenchymal stem cells (hMSC) to enhance the osteoconductivity and biocompatibility of the system. Adhesion and viability of hMSC in the PCL/hydrogel system confirmed its cytocompatibility. Biomineralization tests in simulated body fluid (SBF) showed the nucleation and growth of apatite crystals, which confirmed the bioactivity of the PCL/hydrogel system. Moreover, dissolution studies, in SBF revealed a sustained dissolution of the hydrogel with time. Overall, the present study provides a new approach in bone tissue engineering to repair bone defects with a bioactive hybrid system consisting of a polymeric scaffold, hydrogel, and hMSC.

Formation of Fluorohydroxyapatite with Silver Diamine Fluoride

Silver diamine fluoride (SDF) is found to promote remineralization and harden the carious lesion. Hydroxyapatite crystallization is a crucial process in remineralization; however, the role of SDF in crystal formation is unknown. We designed an in vitro experiment with calcium phosphate with different SDF concentrations (0.38, 1.52, 2.66, 3.80 mg/mL) to investigate the effect of this additive on the nucleation and growth of apatite crystals. Two control groups were also prepared—calcium phosphate (CaCl2·2H2O + K2HPO4 in buffer solution) and SDF (Ag[NH3]2F in buffer solution). After incubation at 37 oC for 24 h, the shape and organization of the crystals were examined by bright-field transmission electron microscopy and electron diffraction. Unit cell parameters of the obtained crystals were determined with powder X-ray diffraction. The vibrational and rotational modes of phosphate groups were analyzed with Raman microscopy. The transmission electron microscopy and selected-area electron diffraction confirmed that all solids precipitated within the SDF groups were crystalline and that there was a positive correlation between the increased percentage of crystal size and the concentration of SDF. The powder X-ray diffraction patterns indicated that fluorohydroxyapatite and silver chloride were formed in all the SDF groups. Compared with calcium phosphate control, a contraction of the unit cell in the a-direction but not the c-direction in SDF groups was revealed, which suggested that small localized fluoride anions substituted the hydroxyl anions in hydroxyapatite crystals. This was further evidenced by the Raman spectra, which displayed up-field shift of the phosphate band in all the SDF groups and confirmed that the chemical environment of the phosphate functionalities indeed changed. The results suggested that SDF reacted with calcium and phosphate ions and produced fluorohydroxyapatite. This preferential precipitation of fluorohydroxyapatite with reduced solubility could be one of the main factors for arrest of caries lesions treated with SDF.

Method development for the synthesis of large crystals of pyromorphite

In the UK, water utilities add phosphate to drinking water in order to prevent lead being released from older properties that are supplied by lead water pipes. The mechanism of reduction is thought to involve the formation of insoluble lead phosphates. This research was carried out to optimise the growth of large lead apatite crystals for use in future atomic force microscopy work. In this case study, pyromorphite crystals exhibiting both prismatic hexagonal and needle-like structures with sizes up to 0.2mm were successfully grown using a caesium chloride flux. They were produced both at lower temperatures and in less time than previous studies. All samples were characterised using a variety of analytical techniques such as X-ray diffraction, infrared spectrometry, energy dispersive X-ray analysis, scanning electron microscopy and thermogravimetric analysis.

Isoexergonic Conformations of Surface-Bound Citrate Regulated Bioinspired Apatite Nanocrystal Growth.

The superior biomechanical properties of bone and dentin are dictated, in part, by the unique plate-like morphology of hydroxyapatite (HAP) nanocrysals within a hierarchically assembled collagen matrix. Understanding the mechanism of crystal growth and thus morphology is important to the rational design of bioinspired apatite nanocrystals for orthopedic and dental applications. Citrate has long been proposed to modulate apatite crystal growth, but major questions exist regarding the HAP-bound citrate conformations and the identities of the interacting functional groups and HAP surface sites. Here, we conducted a comprehensive investigation of the mechanism from the angstrom to submicrometer scale by detailed correlation of the results of high-level metadynamics simulations, employing force-fields benchmarked to experiment and density functional theory calculations with the results of high resolution transmission electron microscopy, nuclear magnetic resonance spectroscopy, solution analysis, and thermogravimetric analysis. Crystal morphology changed from needle- to plate-like with increasing citrate concentration. Citrate adsorbed more strongly on the HAP (100) face than on the (001) face, thus resulting in preferential growth in the [001] direction and the plate-like morphology. Two very different bound conformations were obtained, involving interactions of either one or both terminal carboxyl groups with three or five surface calcium ions, respectively, and a hydrogen bond between the citrate hydroxyl and the HAP surface. Remarkably, despite fewer interaction sites in the single bound carboxyl conformation, the structures were isoexergonic, so both exist at equilibrium. Identification of the former conformation is significant because it allows a greater adsorption density than is traditionally assumed and can help explain concentration-dependence of citrate in modulating crystal morphology. These unique results were enabled first by the application of advanced metadynamics, a technique necessary for the accurate simulation of ionic materials but which is rarely employed in the biomaterials and biomineralization fields and second by the detailed correlation of computational, spectroscopic, and analytical results.

Mechanistic contribution of electroconductive hydroxyapatite-titanium disilicide composite on the alignment and proliferation of cells.

We elucidate here the mechanistic contribution of a novel electroconductive hydroxyapatite-20 wt.% titanium disilicide (HA-TiSi2) composite system in favorably modulating osteoblast functions in relation to the monolithic HA. The higher electrical conductivity of HA-TiSi2(σDC ∼ 67.117 ± 3.57 S/m) in comparison to glass sample effectively guided the electroactive myoblast, leading to their significant alignment and proliferation. This favorable behavior is attributed to the formation of small electrochemical cells between HA and TiSi2phase, which produce a small electric field, directing the electroactive myoblast to migrate and grow in a particular direction. In contrast, no impact of TiSi2on osteoblast function was observed because of their inability to respond to small electric field. However, theinvitrobioactivity in simulated body fluid indicated the nucleation and growth of apatite crystals. Moreover, in the context of load-bearing capability, the presence of 20 wt.% TiSi2in HA led to increase in the fracture toughness by ∼100%. This study underscores the effectiveness of HA-TiSi2in favorably modulating the cellular activity, myoblast in particular.

Effect of organic acids on calcium phosphate nucleation and osteogenic differentiation of human mesenchymal stem cells on peptide functionalized nanofibers.

Carboxylate-rich organic acids play an important role in controlling the growth of apatite crystals and the extent of mineralization in the natural bone. The objective of this work was to investigate the effect of organic acids on calcium phosphate (CaP) nucleation on nanofiber microsheets functionalized with a glutamic acid peptide and osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on the CaP-nucleated microsheets. High molecular weight poly(dl-lactide) (DL-PLA) was mixed with low molecular weight L-PLA conjugated with Glu-Glu-Gly-Gly-Cys peptide, and the mixture was electrospun to generate aligned nanofiber microsheets. The nanofiber microsheets were incubated in a modified simulated body fluid (mSBF) supplemented with different organic acids for nucleation and growth of CaP crystals on the nanofibers. Organic acids included citric acid (CA), hydroxycitric acid (HCA), tartaric acid (TART), malic acid (MA), ascorbic acid (AsA), and salicylic acid (SalA). HCA microsheets had the highest CaP content at 240 ± 10% followed by TART and CA with 225 ± 8% and 225 ± 10%, respectively. The Ca/P ratio and percent crystallinity of the nucleated CaP in TART microsheets was closest to that of stoichiometric hydroxyapatite. The extent of CaP nucleation and growth on the nanofiber microsheets depended on the acidic strength and number of hydrogen-bonding hydroxyl groups of the organic acids. Compressive modulus and degradation of the CaP nucleated microsheets were related to percent crystallinity and CaP content. Osteogenic differentiation of hMSCs seeded on the microsheets and cultured in osteogenic medium increased only for those microsheets nucleated with CaP by incubation in CA or AsA-supplemented mSBF. Further, only CA microsheets stimulated bone nodule formation by the seeded hMSCs.

Amelogenin in Enamel Tissue Engineering.

In this chapter the basic premises, the recent findings and the future challenges in the use of amelogenin for enamel tissue engineering are being discoursed on. Results emerging from the experiments performed to assess the fundamental physicochemical mechanisms of the interaction of amelogenin, the main protein of the enamel matrix, and the growing crystals of apatite, are mentioned, alongside a moderately comprehensive literature review of the subject at hand. The clinical importance of understanding this protein/mineral interaction at the nanoscale are highlighted as well as the potential for tooth enamel to act as an excellent model system for studying some of the essential aspects of biomineralization processes in general. The dominant paradigm stating that amelogenin directs the uniaxial growth of apatite crystals in enamel by slowing down the growth of (hk0) faces on which it adheres is being questioned based on the results demonstrating the ability of amelogenin to promote the nucleation and crystal growth of apatite under constant titration conditions designed to mimic those present in the developing enamel matrix. The role of numerous minor components of the enamel matrix is being highlighted as essential and impossible to compensate for by utilizing its more abundant ingredients only. It is concluded that the three major aspects of amelogenesis outlined hereby--(1) the assembly of amelogenin and other enamel matrix proteins, (2) the proteolytic activity, and (3) crystallization--need to be in precise synergy with each other in order for the grounds for the proper imitation of amelogenesis in the lab to be created.

Refinement of collagen–mineral interaction: A possible role for osteocalcin in apatite crystal nucleation, growth and development

Mineralization of vertebrate tissues such as bone, dentin, cementum, and calcifying tendon involves type I collagen, which has been proposed as a template for calcium and phosphate ion binding and subsequent nucleation of apatite crystals. Type I collagen thereby has been suggested to be responsible for the deposition of apatite mineral without the need for non-collagenous proteins or other extracellular matrix molecules. Based on studies in vitro, non-collagenous proteins, including osteocalcin and bone sialoprotein, are thought to mediate vertebrate mineralization associated with type I collagen. These proteins, as possibly related to mineral deposition, have not been definitively localized in vivo. The present study has reexamined their localization in the leg tendons of avian turkeys, a representative model of vertebrate mineralization. Immunocytochemistry of osteocalcin demonstrates its presence at the surface of, outside and within type I collagen while that of bone sialoprotein appears to be localized at the surface of or outside type I collagen. The association between osteocalcin and type I collagen structure is revealed optimally when calcium ions are added to the antibody solution in the methodology. In this manner, osteocalcin is found specifically located along the a4–1, b1, c2 and d bands defining in part the hole and overlap zones within type I collagen. From these data, while type I collagen itself may be considered a stereochemical guide for intrafibrillar mineral nucleation and subsequent deposition, osteocalcin bound to type I collagen may also possibly mediate nucleation, growth and development of platelet-shaped apatite crystals. Bone sialoprotein and osteocalcin as well, each immunolocalized at the surface of or outside type I collagen, may affect mineral deposition in these portions of the avian tendon.