Hydroxyapatite Plates Research Articles

A Pliocene boxfish (Tetraodontiformes, Ostraciidae) from New Zealand – a preview of future environmental change?

ABSTRACT We report on an articulated fossil boxfish (Tetraodontiformes, Ostraciidae) recently recovered from the Pliocene of the North Island of New Zealand. The specimen was collected from the Tangahoe Formation, a mid-Pliocene (c. 3.0-3.4 Ma) shallow marine deposit, at Waihi Beach, South Taranaki. The fossil boxfish measures 10.7 cm in standard length, with an estimated total length of c. 13–14 cm (the caudal fin is not preserved). The fish is preserved in right lateral view, lying on its side, and has an intact body covering of fused hydroxyapatite plates that rigidly encase the fish, as is characteristic of boxfishes. The plates are hexagonal to subhexagonal in shape and largest close to the dorsal midline. Fossil boxfish have previously been recorded from Northern Hemisphere sites ranging in age from Palaeocene to Quaternary, but not from the Southern Hemisphere. Recent reports note that boxfishes and several other tropical Pacific fish species are now being seen off of northern New Zealand – the Pliocene boxfish from Taranaki, as well as an intriguing addition to New Zealand’s paleohistory, may also reflect how the ongoing impact of climate change will return New Zealand to a warmer marine ecosystem – similar to what prevailed during the Pliocene.

Growth of hydroxyapatite plate-like nanoparticles by additive free precipitation for the deposition of aligned coatings

In recent years, nanoparticles' morphology has been tailored to tune nanoparticle specific properties for several applications. In particular, plate-like particles have been synthesized to create an analog of nacre microstructure exhibiting outstanding mechanical performance attributed to nacre's micro and nano hierarchy. However, the synthesis of equiaxed hydroxyapatite (HA) plates for constructing an oriented architecture has remained as an astounding challenge. In this study, we designed an additive-free chemical precipitation route in which the pH was increased stepwise to maintain the particles' equiaxed 2d shape. Our results showed that the produced particles exhibited HA's crystal structure and highly sustained the equiaxed/rectangular plate morphology of the octacalcium phosphate (OCP) phase, formed as an interphase during the precipitation. The reaction time, reactant concentration, and heat treatment were found essential in controlling the particles' morphology, size, and crystal symmetry. The crystal features of the particles were dissected via high-resolution scanning and transmission electron microscopes (HRSEM and HRTEM) and X-ray diffraction (XRD) analysis. Moreover, the Williamson-Hall plot and Rietveld Size-strain algorithm were implemented to calculate the crystallite size and strain of the particles. HRSEM and HRTEM micrographs showed that ultrathin (10–20 nm) 2d HA particles with mean lengths of 140–150 nm grew through the c-axis. In the last stage, we attempted to use the particles to deposit oriented coatings by electrophoretic deposition (EPD).

Analysis of reaction products on hydroxyapatite created by tooth etchants with different compositions.

The purpose of this study was to investigate the reaction products formed by application of three tooth etchants to hydroxyapatite. Tooth etchants with three different compositions, designed for application to teeth before dental adhesive - " K-etchant GEL" (containing phosphoric acid), "Enamel Conditioner" (containing organic acids), and "Multi Etchant" (containing acidic monomer) - were applied to hydroxyapatite plates. Atomic force microscopy measurements revealed that Multi Etchant formed nano-sized particles on the hydroxyapatite. X-ray diffraction and Fourier transform infrared spectrometer analyses of the powdered hydroxyapatite indicated that Enamel Conditioner produced calcium tartrate whereas K-etchant GEL generated monetite. These results indicated that each etchant reacted with hydroxyapatite in a different way. Not only differences among the etching ability of etchants, but also differences in the reaction compounds they produce may influence bonding performance in clinical practice.

Podosome-Driven Defect Development in Lamellar Bone under the Conditions of Senile Osteoporosis Observed at the Nanometer Scale.

The degradation mechanism of human trabecular bone harvested from the central part of the femoral head of a patient with a fragility fracture of the femoral neck under conditions of senile osteoporosis was investigated by high-resolution electron microscopy. As evidenced by light microscopy, there is a disturbance of bone metabolism leading to severe and irreparable damages to the bone structure. These defects are evoked by osteoclasts and thus podosome activity. Podosomes create typical pit marks and holes of about 300–400 nm in diameter on the bone surface. Detailed analysis of the stress field caused by the podosomes in the extracellular bone matrix was performed. The calculations yielded maximum stress in the range of few megapascals resulting in formation of microcracks around the podosomes. Disintegration of hydroxyapatite and free lying collagen fibrils were observed at the edges of the plywood structure of the bone lamella. At the ultimate state, the disintegration of the mineralized collagen fibrils to a gelatinous matrix comes along with a delamination of the apatite nanoplatelets resulting in a brittle, porous bone structure. The nanoplatelets aggregate to big hydroxyapatite plates with a size of up to 10 x 20 μm2. The enhanced plate growth can be explained by the interaction of two mechanisms in the ruffled border zone: the accumulation of delaminated hydroxyapatite nanoplatelets near clusters of podosomes and the accelerated nucleation and random growth of HAP nanoplatelets due to a nonsufficient concentration of process-directing carboxylated osteocalcin cOC.

Functionalized Graphene Oxide Shields Tooth Dentin from Decalcification

This in vitro study assessed the efficacy of functionalized graphene oxide (f-GO) nanocomposites on the decalcification of dentin, because dental caries of the root surface is becoming one of the new problems in aged society. Hydroxyapatite plates (HAP) and dentin slices were coated with f-GO nanocomposites by comparing them to silver diamine fluoride as a positive control, then treated with decalcification solutions such as ethylenediaminetetraacetic acid and citrate at 37°C for 24 h. Scanning electron microscopy (SEM) revealed significant protection of the surface morphology of HAP and dentin. On the other hand, a cariogenic Streptococcus mutans growth was inhibited by f-GO nanocomposites. In addition, cytotoxicity of them to epithelial cells was much less than that of povidone-iodine, which is commonly used for oral disinfectant. We synthesized 5 different f-GO nanocomposites such as GO–silver (Ag), GO-Ag–calcium fluoride (CaF2), GO-CaF2, GO-zinc, and GO–tricalcium phosphate (Ca3(PO4)2). They were standardized by evaluating under SEM, transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetry analysis (TGA), and Raman spectra after being synthesized in an aseptic technique. The abilities of GO-Ag, GO-Ag-CaF2, and GO-CaF2 nanocomposites were most preventive for decalcification. In addition, GO-Ag and GO-Ag-CaF2 almost completely inhibited S. mutans growth. However, they did not exhibit cytotoxicity to epithelial cells except at the highest concentration (0.1 w/v%) of GO-Ag and GO-Ag-CaF2. Furthermore, these f-GO nanocomposites exhibited less or no discoloration of dentin, although commonly used silver diamine fluoride causes discoloration of dentin to black. Thus, these f-GO nanocomposites are useful to protect dental caries on the tooth root that becomes a social problem in aged society.

From monetite plate to hydroxyapatite nanofibers by monoethanolamine assisted hydrothermal approach

Calcium phosphates offer outstanding biological adaptability. Thanks to their specific physico-chemical properties they are one of the most widely used materials in bone tissue engineering applications. The search for an innovative and economic strategy of synthesizing their different forms has been drawing considerable attention in the field. Herein, we report on a facile hydrothermal process in the presence of ethylenediamine tetraacetic acid and monoethanolamine to obtain various forms of calcium phosphates. The monoethanolamine served as an alkaline source and crystal growth modifier, while ethylenediamine tetraacetic acid was used to control the Ca2+ supersaturation level under high temperature and high pressure conditions. The obtained inorganic compounds were examined for their elemental composition, morphology, and structure using scanning electron microscopy, Raman spectroscopy, and powder x-ray diffraction. We were able to selectively synthesize monetite plate-like microcrystals as well as hydroxyapatite plates and nanofibers by simply varying the concentration of monoethanolamine.

Size Control Synthesis of Hydroxyapatite Plates and Their Application in the Preparation of Highly Oriented Films

Hydroxyapatite (HAp) plates were successfully synthesized using dodecanedioic acid (DDDA). Following the preparation of an ethanolic solution of DDDA, solutions of calcium and phosphate were added in succession to produce a white slurry upon mixing. Subsequent processing of this slurry under hydrothermal conditions provided HAp plates of various sizes. Indeed, the plate size could be changed by varying the quantity of DDDA employed in the synthesis, with larger particles being obtained in the presence of larger quantities of DDDA. It was confirmed that a calcium–DDDA complex (Ca-DDD, calcium dodecanedioate) was formed upon mixing of the DDDA and calcium solutions. The observed morphological control could potentially be accounted for by the formation of Ca-DDD. More specifically, during crystal growth, Ca-DDD plays the role of a nucleation substrate (template), a pH buffer, and a calcium reservoir. Finally, we demonstrated that the obtained HAp plates could be employed to prepare a HAp film with high cryst...

Self-assembled sponge-like hydroxyapatite induced by modified articular cartilage membrane template

Using modified pig bone articular cartilage membrane as template, a sponge-like hydroxyapatite (HA) scaffold material was in-situ synthesized by self-assembly in simulated body fluid. Its crystallographic structure and composition were studied by SEM, XRD, FTIR and TEM, which showed that the synthesized sample was carbonated HA crystals and the three-dimensional interconnected sponge-like structure consists of massive nano hydroxyapatite plates. The physicochemical properties of the samples were tested and the results indicated that the sponge-like HA had high density, porosity and specific surface. Experimental cell culture results also showed that sponge-like HA greatly increases the bioactivity, osteoconductivity and differentiation of human osteosarcoma MG-63 cells.

Enhancement of calcification by osteoblasts cultured on hydroxyapatite surfaces with adsorbed inorganic polyphosphate.

Inorganic polyphosphate has been expected to accelerate bone regeneration. However, there are limited evidences to prove that polyphosphate adsorbed on the surface of a hydroxyapatite plate enhances calcification of cultured osteoblasts. In this study, we examined the effect of polyphosphate adsorbed onto the surface of a hydroxyapatite plate on the attachment, proliferation, differentiation, and calcification of osteoblasts. After hydroxyapatite plates were soaked in solutions of polyphosphate, the plate surfaces were analyzed by scanning electron microscopy and toluidine blue staining to confirm adsorption of polyphosphate. The hydroxyapatite plates were further subjected to the measurements of surface roughness, water contact angle, and the binding capacity of calcium ions. Cell culture experiments were carried out using MC3T3-E1 pre-osteoblastic cells. It was found that soaking a hydroxyapatite plate in a polyphosphate solution gave rise to an increase in surface roughness and reduction in water contact angle in a concentration-dependent manner, suggesting the adsorption of polyphosphate onto the surface of a hydroxyapatite plate. It was further observed that surface-adsorbed polyphosphate exhibited an inhibitory effect on cell adhesion and proliferation. In contrast, cell differentiation was promoted on hydroxyapatite plates with adsorbed polyphosphate, when assessed from expression of differentiation marker genes including alkaline phosphatase, osteopontin, and osteocalcin. In addition, calcification of the culture was enhanced on hydroxyapatite plates with relatively low density of adsorbed polyphosphate. Our results as a whole provided an evidence to show that there is a narrow window with regard to the surface density of adsorbed polyphosphate for the enhancement of osteoblast calcification.

Biocompatible nanostructured solid adhesives for biological soft tissues.

The present study indicates a new application of inorganic biomaterials (bioceramics) as a soft tissue adhesive. Organic adhesives such as fibrin glues or cyanoacrylate derivatives have been commonly used clinically. However, their limited biocompatibility and/or low adhesion strength are some drawbacks that impair their clinical application. In this study, we synthesized a novel solid adhesive with biocompatible and biodegradable HAp nanoparticles without the aid of organic molecules, and showed a rapid and strong adhesion of mouse soft tissues compared to conventional fibrin glues. Given the importance of wet adhesion in biomedicine and biotechnology applications, our results will help not only in developing an efficient approach to close incised soft tissues, but also in finding novel ways to integrate soft tissues with synthetic hydrogels (such as drug reservoirs).

Hydroxyapatite formation from calcium carbonate single crystal under hydrothermal condition: Effects of processing temperature

Hydrothermal processing has been used for synthesis of hydroxyapatite (HAp) crystals with unique morphologies. Our previous studies reported that the morphology of HAp was affected by dissolution behavior of starting materials under a hydrothermal condition. In this study we evaluated the effects of processing temperature on formation and morphology of HAp crystals under a hydrothermal condition. Calcite (CaCO3) single crystal as a starting material was hydrothermally treated in a phosphate solution at various temperatures. After hydrothermal processing for 24h, needle-shaped HAp crystals formed on the surfaces of all the samples. The size of HAp crystals increased with increasing the processing temperature. Plate-shaped HAp was observed on the cross-section of the starting material treated at 200°C. The structure of HAp plates were generated by arrangement of needle-shaped HAp crystals during their crystal growth. The difference in the processing temperature affects the morphology and structure of HAp formed on the starting material under the hydrothermal condition.

The armored carapace of the boxfish

The boxfish (Lactoria cornuta) has a carapace consisting of dermal scutes with a highly mineralized surface plate and a compliant collagen base. This carapace must provide effective protection against predators as it comes at the high cost of reduced mobility and speed. The mineralized hydroxyapatite plates, predominantly hexagonal in shape, are reinforced with raised struts that extend from the center toward the edges of each scute. Below the mineralized plates are non-mineralized collagen fibers arranged in through-the-thickness layers of ladder-like formations. At the interfaces between scutes, the mineralized plates form suture-like teeth structures below which the collagen fibers bridge the gap between neighboring scutes. These sutures are unlike most others as they have no bridging Sharpey’s fibers and appear to add little mechanical strength to the overall carapace. It is proposed that the sutured interface either allows for accommodation of the changing pressures of the boxfish’s ocean habitat or growth, which occurs without molting or shedding. In both tension and punch testing the mineralized sutures remain relatively intact while most failures occur within the collagen fibers, allowing for the individual scutes to maintain their integrity. This complex structure allows for elevated strength of the carapace through an increase in the stressed area when attacked by predators in both penetrating and crushing modes.

Continuous synthesis of dispersant-coated hydroxyapatite plates

A continuous flow hydrothermal synthetic route which allows the direct “in situ” capping/coating of hydroxyapatite nanoplates with functional dispersants in a single stage is reported.

Fabrication, characterization, and biocompatibility of ethyl cellulose/carbonated hydroxyapatite composite coatings on Ti6Al4V.

In order to improve the biocompatibility of metallic implants, bioactive components are often used as coatings so that a real bond with the surrounding bone tissue can be formed. We prepared ethyl cellulose/carbonated hydroxyapatite composite coatings (ECHCs) on Ti6Al4V substrates with carbonated hydroxyapatite coatings (CHACs) without ethyl cellulose as controls. The inorganic constituent on the CHACs and ECHCs is calcium-deficient carbonated hydroxyapatite with a flaky texture and a low degree of crystallinity. The flaky carbonated hydroxyapatite plates aggregate to form macropores with an aperture size of around 0.5-2.0μm. The presence of ethyl cellulose provides superior morphology, contact angle, and biocompatibility characteristics. In comparison to CHACs, ECHCs exhibit a smoother, crack-free surface because the cracks are filled by ethyl cellulose. Moreover, the contact angle of ECHCs is 37.3°, greater than that of CHACs (13.0°). Surface biocompatibility was investigated by using human bone mesenchymal stem cells (hBMSCs). The attachment, spreadability, viability and proliferation of hBMSCs on ECHCs are superior to those on CHACs. Thus, the crack-free ECHCs have excellent biocompatibility and are appropriate for use as biological implants.

Chitosan/carbonated hydroxyapatite composite coatings: Fabrication, structure and biocompatibility

Chitosan/carbonated hydroxyapatite composite coatings (CCHCs) were fabricated according to the following steps: (i) preparation of calcium carbonate coatings (CCCs) on Ti6Al4V substrates by electrophoretic deposition; (ii) transformation of CCCs into carbonated hydroxyapatite coatings (CHACs) in a phosphate buffer solution (PBS); and (iii) formation of CCHCs by modification of CHACs with chitosan. Inorganic constituents in CCHCs are plate-like carbonated hydroxyapatite particles with a low crystallinity. Interestingly, macropores with a pore size of 0.5–2μm still remain among the carbonated hydroxyapatite plates even after the chitosan deposits homogeneously on the coatings. Moreover, CCHCs have moderately hydrophilic surfaces with a contact angle of 29.4° due to the presence of the chitosan. Biocompatibility tests have been carried out by using human bone mesenchymal stem cells (hBMSCs) as cell models. The hBMSCs show better cell morphology, adhesion, spreading and proliferation on CCHCs than on CHACs. The excellent biocompatibility of CCHCs is mainly attributed to the organic/inorganic compositions, macroporous structure and moderately hydrophilic surfaces. Hence, CCHCs have great potentials for bone implants.

Preparation and characterization of low-crystallized hydroxyapatite nanoporous plates and granules

We examined the conditions for preparing nanoporous hydroxyapatite (HAp) plates and granules by assembling low-crystallinity HAp nanoparticles without template/binder molecules or high-temperature/pressure treatments. We first prepared HAp nanoparticles with different particle sizes and then the nanoporous plate or granule was prepared by drying an aqueous dispersion of HAp nanoparticles on an oil substrate. The hydrophobic oil substrate was preferable to prevent crack formation in HAp plates. The size of the HAp plate could be controlled by the concentration of HAp dispersion, and millimeter-sized granules were obtained by drying a small volume of HAp dispersion on/in the oil substrate. The nanoparticle-assembled HAp had nanosized pores, and the pore size could be changed by adjusting the size of the HAp nanoparticles used. It was found that both hydrophilic and hydrophobic liquids could infiltrate inside the nanopores. The nanoporous HAp granules also could adsorb and release proteins, and the protein adsorption and release delayed in the presence of the nanopores.

Hydrothermal synthesis of hydroxyapatite plates prepared using low molecular weight heparin (LMWH)

Materials with enhanced physical and biological properties have been used for biomedical applications and can be developed by functionalizing them using various components. Hydroxyapatite (HAP), among other available synthetic material, serves as one of the best tools in orthopaedics and ceramic coatings. The porous structure of HAP helps in bone cell regeneration, chemical integration of bone and also favours the interaction between bone and tissues. Herein, we have demonstrated a simple procedure for the synthesis of HAP using low molecular weight heparin (LMWH), a structural analogue of bone heparan sulphate proteoglycan. The presence of small sized HAP plates with well-defined structures was revealed using electron microscopic analysis. The phase purity of the synthesized HAP was evaluated using X-ray diffraction pattern obtained before and after immersion in simulated body fluid (SBF).

Geometry as a Factor for Tissue Growth: Towards Shape Optimization of Tissue Engineering Scaffolds

Scaffolds for tissue engineering are usually designed to support cell viability with large adhesion surfaces and high permeability to nutrients and oxygen. Recent experiments support the idea that, in addition to surface roughness, elasticity and chemistry, the macroscopic geometry of the substrate also contributes to control the kinetics of tissue deposition. In this study, a previously proposed model for the behavior of osteoblasts on curved surfaces is used to predict the growth of bone matrix tissue in pores of different shapes. These predictions are compared to in vitro experiments with MC3T3-E1 pre-osteoblast cells cultivated in two-millimeter thick hydroxyapatite plates containing prismatic pores with square- or cross-shaped sections. The amount and shape of the tissue formed in the pores measured by phase contrast microscopy confirms the predictions of the model. In cross-shaped pores, the initial overall tissue deposition is twice as fast as in square-shaped pores. These results suggest that the optimization of pore shapes may improve the speed of ingrowth of bone tissue into porous scaffolds.

How Linear Tension Converts to Curvature: Geometric Control of Bone Tissue Growth

This study investigated how substrate geometry influences in-vitro tissue formation at length scales much larger than a single cell. Two-millimetre thick hydroxyapatite plates containing circular pores and semi-circular channels of 0.5 mm radius, mimicking osteons and hemi-osteons respectively, were incubated with MC3T3-E1 cells for 4 weeks. The amount and shape of the tissue formed in the pores, as measured using phase contrast microscopy, depended on the substrate geometry. It was further demonstrated, using a simple geometric model, that the observed curvature-controlled growth can be derived from the assembly of tensile elements on a curved substrate. These tensile elements are cells anchored on distant points of the curved surface, thus creating an actin “chord” by generating tension between the adhesion sites. Such a chord model was used to link the shape of the substrate to cell organisation and tissue patterning. In a pore with a circular cross-section, tissue growth increases the average curvature of the surface, whereas a semi-circular channel tends to be flattened out. Thereby, a single mechanism could describe new tissue growth in both cortical and trabecular bone after resorption due to remodelling. These similarities between in-vitro and in-vivo patterns suggest geometry as an important signal for bone remodelling.

Effect of pH and temperature on the morphology and phases of co-precipitated hydroxyapatite

This paper reports a high-yield process to fabricate biomimetic hydroxyapatite nano-particles or nano-plates. Hydroxyapatite is obtained by simultaneous dripping of calcium chloride and ammonium hydrogen phosphate solutions into a reaction vessel. Reactions were carried out under various pH and temperature conditions. The morphology and phase composition of the precipitates were investigated using scanning electron microscope and X-ray diffraction. The analyses showed that large plates of calcium hydrophosphate are formed at neutral or acidic pH condition. Nanoparticles of hydroxyapatite were obtained in precipitates prepared at pH 9–11. Hydroxyapatite plates akin to seashell nacre were obtained at 40 °C and pH 9. This material holds promise to improve the strength of hydroxyapatite containing composites for bone implant or bone cement used in orthopaedic surgeries. The thermodynamics of the crystal growth under these conditions was discussed. An assembly mechanism of the hydroxyapatite plates was proposed according to the nanostructure observations.