Influence of fluorine substitution on the morphology and structure of hydroxyapatite nanocrystals prepared by hydrothermal method

Hydroxyapatite (HAp) nanocrystals with different levels of fluorine substitution (P/F = 0, 6, 4 and 2) on the OH sites were produced via hydrothermal method. The fluorine substitution was found to alter the morphology of crystals appreciably. The aspect ratio and the crystallinity of HAp crystals increased with increasing fluorine substitution. The presence of broad ring and hallow ring patterns in electron diffraction suggests the low-crystalline nature of HAp crystals. With increasing fluorine substitution, the diffraction patterns exhibited discrete rings and numerous diffraction spots, implying the increased crystallinity. Raman spectra from the HAp nanoparticles also support the less-crystalline nature of the pristine HAp and the enhanced crystallization by fluorine substitution. In HAp crystals processed with no fluorine substitution, surface energy and planar Ca²⁺ density are less sensitive to the crystallographic orientation because of its low-crystalline nature, favoring equi-axed or slightly elongated particles. The addition of fluorine apparently increased the crystallinity, enhancing the orientation dependent growth and accordingly the aspect ratio. Osteoblast proliferation was observed to be enhanced by fluorine substitution in HAp. In vitro biological data support that the excellent osteoblastic cell viability and functional activity of the fluoridated apatite.     

Poly(lactide-co-glycolide)/hydroxyapatite nanofibrous scaffolds fabricated by electrospinning for bone tissue engineering

Poly(lactide-co-glycolide) (PLGA) nanofibrous composite scaffolds having nano-hydroxyapatite particles (HAp) in the fibers were prepared by electrospinning of PLGA and HAp with an average diameter of 266.6 ± 7.3 nm. Microscopy and spectroscopy characterizations confirmed integration of the crystalline HAp in the scaffolds. Agglomerates gradually appeared and increased on the fiber surface along with increase of the HAp concentration. In vitro mineralization in a 5 × simulated body fluid (SBF) revealed that the PLGA/HAp nanofibrous scaffolds had a stronger biomineralization ability than the control PLGA scaffolds. Biological performance of the nanofibrous scaffolds of the control PLGA and PLGA with 5 wt% HAp (PLGA/5HAp) was assessed by in vitro culture of neonatal mouse calvaria-derived MC3T3-E1 osteoblasts. Both types of the scaffolds could support cell proliferation and showed sharp increase of viability until 7 days, but the cells cultured on the PLGA/5HAp nanofibers showed a more spreading morphology. Despite the similar level of the cell viability and cell number at each time interval, the alkaline phosphatase secretion was significantly enhanced on the PLGA/5HAp scaffolds, indicating the higher bioactivity of the as-prepared nano-HAp and the success of the present method for preparing biomimetic scaffold for bone regeneration.     

Ultrastructural Analysis on the Osteogenesis and Transformation of Calcium Phosphate Ceramics in Vivo

To study the osteogenesis and transformation process of calcium phosphate bioceramic in vivo, biodegradable porous β-tricalcium phosphate ceramics (β-TCP, φ5×8 mm) were implanted in the tibia of rabbits. β-TCP ceramics with surrounding bone tissue were retrieved and observed by SEM, TEM and EPMA every month after implantation.The results showed that osteogenesis was active and β-TCP ceramics bonded to bones directly. The new bones were forming and maturing as materials were continuously degrading, and materials were finally replaced by new bone. Parts of the materials were degraded, absorbed and recrystallized, while the rest were dispersed to the spongy bone and the Haversian lamella in an irregular arrangement, becoming incorporated into bone formation directly by remodeling the structure. Some β-TCP crystals cleaved along its (001) rhombohedral plane and formed lath-like crystals in vivo.     

Prolonged release from PLGA/HAp scaffolds containing drug-loaded PLGA/gelatin composite microspheres

Porous scaffolds that can prolong the release of bioactive factors are urgently required in bone tissue engineering. In this study, PLGA/gelatin composite microspheres (PGMs) were carefully designed and prepared by entrapping poly(l-lactide-co-glycolide) (PLGA) microspheres (PMs) in gelatin matrix. By mixing PGMs with PLGA solution directly, drug-loaded PLGA/carbonated hydroxyapatite (HAp)/PGMs composite scaffolds were successfully fabricated. In vitro release of fluorescein isothiocyanate-dextran (FD70S) as a model drug from the scaffolds as well as PMs and PGMs was studied by immersing samples in phosphate buffered saline (pH = 7.4) at 37°C for 32 days. Compared with PMs, PGMs and PLGA/HAp/PGMs scaffolds exhibited slow and steady release behavior with constant release rate and insignificantly original burst release. The swelling of PGMs, diffusion of drugs, and degradation of polymer dominated the release behaviors synergistically. The PLGA/HAp/PGMs scaffold offers a novel option for sequential or simultaneous release of several drugs in terms of bone regeneration.     

Biomimetic synthesis of enamel-like hydroxyapatite on self-assembled monolayers

Hydroxyapatite (HAp) crystals mimicking tooth enamel in chemical composition and morphology were formed on sulfonic-terminated self-assembled monolayer (SAM) in 1.5SBF with F⁻ at 50 °C for 7 days. F⁻ ions showed a marked effect on the composition and morphology of deposited HAp crystals. In the absence of F⁻ ions, HAp containing CO₃²⁻ were formed on SAM, and worm-like crystals of 200–300 nm in length aggregated to form a spherical morphology. When F⁻ was added, HAp crystals containing both CO₃²⁻ and F⁻ were formed on SAM. Needle-shaped crystals of high aspect ratio and 1–2 μm in length grew elongated along the c-axial direction. In addition, these needle-shaped crystals grew in bundles, mimicking HAp crystals in tooth enamel. After the process of ripening, the needles in bundle grew to large size of up to 10 μm in length, and still kept no crystal–crystal fusion like enamel HAp crystals. The formation of enamel-like HAp can be attributed to the substitute of F⁻ for OH⁻ by disturbing the normal progress of HAp formation on SAM. The results suggest potential applications in preparing a novel dental material by a simple method.     

Electrolytic deposition of magnesium-substituted hydroxyapatite crystals on titanium substrate

Electrolytic deposition of magnesium-substituted hydroxyapatite (Mg-HAp) coatings on titanium was investigated and Mg-HAp crystals with up to 2 wt.% Mg²⁺ were deposited in electrolytes with various Mg²⁺ concentration. The incorporation of Mg substantially changed the morphology of the HAp crystals and decreased the crystal size and crystallinity of the HAp. The similarity with natural dentin and bone in composition and the increased specific surface of the Mg-HAp coatings on the Ti substrates were believed to benefit the bioactivity and the drug-carrying properties of the coatings.     

Nano-hydroxyapatite reinforced polyhydroxybutyrate composites: A comprehensive study on the structural and in vitro biological properties

Nanocomposites based on polyhydroxybutyrate (PHB) and hydroxyapatite (HAp) have recently been proposed for application in bone repair and regeneration, but very limited studies have investigated the effect of HAp on the rheological and thermal behavior of PHB. More important, the efficiency of a biomaterial depends greatly on its ability to interact with cells, but little is known about this interaction for this kind of nanocomposite. Hence, this paper dealt with some of the characteristics of solution-casted PHB/HAp nanocomposite films, and tried to explore the effect of HAp nanoparticles on cellular responses. The results showed that both rheological and thermal properties can be tailored by incorporating appropriate amounts of nanoparticles. In vitro studies showed a significant increase in proliferation and differentiation of MC3T3-E1 on nanocomposites compared to the neat polymer. Surface examination indicated that topography and chemistry of surface are important factors influencing cellular processes; while no cell differentiation was found on the neat polymer, nanocomposite with 15 wt.% filler content exhibited a pronounced differentiation resulting from high surface roughness and large amount of exposed HAp. These results suggest that HAp particles play a much more important role in determining the biological performance of PHB than has previously been supposed.     

Unidirectionally oriented hydroxyapatite/collagen composite fabricated by using a high magnetic field

A unidirectionally oriented hydroxyapatite (HAp)/collagen composite was prepared by using a high magnetic field. In the present study, a calcium-containing collagen solution was mixed with a phosphate-containing solution and kept at a temperature of 37 °C. During this process, the heat-induced self-assembling of collagen molecules and precipitation of HAp crystals took place simultaneously. Both collagen fibrils and HAp crystals have magnetic susceptibility anisotropy, which makes it possible for them to be oriented by using a high magnetic field. In order to prepare a unidirectionally oriented structure, samples were rotated when the high magnetic field was imposed perpendicularly to the rotation axis. The results indicated that collagen fibrils have been unidirectionally oriented with weakly crystallized HAp nanocrystals closely attached along the surfaces of the collagen fibrils. A biocomposite with similar structural and composition to natural bones has been prepared.     

Novel biomimetic hydroxyapatite/alginate nanocomposite fibrous scaffolds for bone tissue regeneration

Hydroxyapatite/alginate nanocomposite fibrous scaffolds were fabricated via electrospinning and a novel in situ synthesis of hydroxyapatite (HAp) that mimics mineralized collagen fibrils in bone tissue. Poorly crystalline HAp nanocrystals, as confirmed by X-ray diffractometer peak approximately at 2θ = 32° and Fourier transform infrared spectroscopy spectrum with double split bands of PO₄(v ₄) at 564 and 602 cm^?1, were induced to nucleate and grow at the [–COO^?]–Ca²⁺–[–COO^?] linkage sites on electrospun alginate nanofibers impregnated with PO₄ ^3? ions. This novel process resulted in a uniform deposition of HAp nanocrystals on the nanofibers, overcoming the severe agglomeration of HAp nanoparticles processed by the conventional mechanical blending/electrospinning method. Preliminary in vitro cell study showed that rat calvarial osteoblasts attached more stably on the surface of the HAp/alginate scaffolds than on the pure alginate scaffold. In general, the osteoblasts were stretched and elongated into a spindle-shape on the HAp/alginate scaffolds, whereas the cells had a round-shaped morphology on the alginate scaffold. The unique nanofibrous topography combined with the hybridization of HAp and alginate can be advantageous in bone tissue regenerative medicine applications.     

New application of poly(butylene succinate) (PBS) based ionomer as biopolymer: a role of ion group for hydroxyapatite (HAp) crystal formation

Sodium sulfonate ionic group bearing PBS ionomer (PBSi) and hydroxyapatite (HAp) composites were prepared by soaking in the simulated body fluid (SBF) solution, which is the biomimetic method, as well as the effects of the ionic group on the HAp crystal formation and growth were investigated. The introduced sodium sulfonate ionic groups operated as the functional groups with negative charge densities, which can bind plentiful Ca²⁺ ions efficiently, consequently serving as active sites allowing HAp crystals to grow on the surfaces of the PBSi matrix. By SEM analysis, it was observed that HAp became growing as the shape with the porous holes. These holes are thought to be very suitable for the ingrowths of the surrounding tissue and the assistance to the bone formation. Based on this finding, it can be clearly concluded that the ionic groups in the PBSi may be decisive factors in growing HAp, and it is anticipated that this novel materials can contribute to excellent biopolymer.     

Self-assembly phenomenon of hydroxyapatite nanocrystals on chondroitin sulfate

Self-assembly phenomenon of hydroxyapatite (HAp) nanocrystals on chondroitin sulfate (ChS) templates was investigated. A HAp/ChS hybrid was synthesized through a precipitation method with a calcium hydroxide suspension and phosphoric acid solution containing the ChS. The preferential alignment of the crystallographic c-axis of the HAp crystals parallel to the long axis direction of the ChS templates was observed to occur with the chemical interactions between the HAp crystals and the functional groups of the ChS. This phenomenon was interpreted by the crystallochemical specific nucleation and growth of the HAp crystals regulated by the pre-organized functional groups of the ChS template. Practical implication may involve a biomimetic artificial bone or cartilage can be made through a precipitation method with suitable bio-organics.     

Design and in vitro evaluation of novel γ-PGA/hydroxyapatite nanocomposites for bone tissue engineering

In order to improve biocompatibility and cell growth, poly-γ-glutamic acid (γ-PGA) was used as an additive, and a series of γ-PGA/hydroxyapatite (γ-PGA/HAp) nanocomposites were prepared. Then the morphology, water uptake and retention abilities, in vitro degradation properties in the simulated medium, and cytotoxicity of these γ-PGA/HAp nanocomposites were investigated. The results show that the γ-PGA/HAp nanocomposites have homogenous nano-sized grains, hydrophilicity, biocompatibility, and controlled in vitro degradation, suggesting that the γ-PGA/HAp nanocomposites are novel nanostructure composites with great potential application in the field of bone tissue engineering.     

Characterisation, corrosion resistance and in vitro bioactivity of manganese-doped hydroxyapatite films electrodeposited on titanium

This work elucidated the corrosion resistance and in vitro bioactivity of electroplated manganese-doped hydroxyapatite (MnHAp) film on NaOH-treated titanium (Ti). The NaOH treatment process was performed on Ti surface to enhance the adhesion of the MnHAp coating on Ti. Scanning electron microscopy images showed that the MnHAp coating had needle-like apatite crystals, and the approximately 10 μm thick layer was denser than HAp. Energy-dispersive X-ray spectroscopy analysis revealed that the MnHAp crystals were Ca-deficient and the Mn/P molar ratio was 0.048. X-ray diffraction confirmed the presence of single-phase MnHAp, which was aligned vertically to the substrate. Fourier transform infrared spectroscopy indicated the presence of phosphate bands ranging from 500 to 650 and 900 to 1,100 cm^?1, and a hydroxyl band at 3,571 cm^?1, which was characteristic of HAp. Bond strength test revealed that adhesion for the MnHAp coating was more enhanced than that of the HAp coating. Potentiodynamic polarisation test showed that the MnHAp-coated surface exhibited superior corrosion resistance over the HAp single-coated surface. Bioactivity test conducted by immersing the coatings in simulated body fluid showed that MnHAp coating can rapidly induce bone-like apatite nucleation and growth. Osteoblast cellular tests revealed that the MnHAp coating was better at improving the in vitro biocompatibility of Ti than the HAp coating.     

Distribution of hydroxyapatite crystallite orientation and ultrasonic wave velocity in ring-shaped cortical bone of bovine femur

At the nanoscopic level, bone consists of calcium phosphate, which forms incomplete hydroxyapatite (HAp) crystals. The preferred orientation of the c-axis of HAp crystallites induces anisotropy and inhomogeneity of elastic properties in bone. In this study, the effect of the preferred orientation of HAp crystallites on the spatial distribution of ultrasonic wave velocity was experimentally investigated, considering bone mineral density (BMD) and microstructure. Three ring-shaped cortical bone samples were made from a 36-month-old bovine femur. Longitudinal wave velocity was measured by a conventional ultrasonic pulse system, using self-made polyvinylidene fluoride transducers. The integrated intensity of the (0002) peak obtained using X-ray diffraction was estimated to evaluate the amount of preferred orientation. The velocity distribution pattern was similar to the distribution of integrated intensity of (0002). The effect of the preferred orientation of HAp crystallites on velocity was clearly observed in the plexiform structure, despite the fact that the BMD value was almost independent of the preferred orientation of HAp crystallites. Velocity measurement of cortical bone can reveal information about HAp crystallite orientation.     

Mineralised tissues as nanomaterials: analysis by atomic force microscopy

Mineralised tissues, such as bone, consist of two material phases: collagen protein fibrils that form the structural models upon which the mineral, calcium hydroxyapatite, is subsequently deposited. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair, and matrix proteins are replaced by the synthetic activity of osteoblasts and then calcify. The resolution of atomic force microscopy and use of unmodified, fully calcified samples has enabled the imaging of the overall bone and dentine structure, including collagen and mineral phases. Mineral crystals, in the diameter size range of 225 nm up to 1.4 microm, were found in unmodified bone and dentine respectively. D-banded collagen is observed in dentine after acid treatment and in bone after osteoclast-mediated matrix resorption; axial periodicity values of approximately 67 and 69 nm are observed, respectively. These experimental approaches have enabled the structure of mineralised tissues to be examined in native samples and will facilitate the study of bone structure in important clinical disorders of the skeleton, such as osteoporosis.     

Bioceramic coating of hydroxyapatite on titanium substrate with Nd-YAG laser

The ability to bond to bone tissue is a unique property of bioactive ceramics. Hydroxyapatite (HAp) is one of the potential bioceramics candidates due to its superior bio-compatibility. Significant effort has been devoted to coat HAp ceramics on metallic substrates. Most of these processes, such as ion-beam sputter coating, thermal spraying, and flame spraying, are high temperature line of sight processes, which suffer from undesirable phase formation and weak metal/HAP bonding strength. This paper presents a unique process to coat HAp powders on titanium substrates at low temperature and enhance the coating/substrate interface by laser surface engineering. Nd-YAG laser transmits HAp powders and the laser power is absorbed by titanium substrate to produce a thin layer of molten region. During coating process, HAp powders are kept at low temperature before they are entrapped in metallic layer. Scanning electron microscope (SEM) was used to investigate the microstructure of coating; the chemical composition of the coating is determined by energy dispersive spectrometry (EDS). Mechanical properties of the interface between coating and Ti substrate were investigated by nanoindentation.     

Controlling Enamel Remineralization by Amyloid-Like Amelogenin Mimics

In situ regeneration of the enamel-like structure of hydroxyapatite (HAp) crystals under oral conditions is significant for dental caries treatment. However, it is still a challenge for dentists to duplicate the elegant and well-aligned apatite structure bonding to the surface of demineralized enamel. A biocompatible amelogenin-inspired matrix, a phase-transited lysozyme (PTL) film mimicking an N-terminal amelogenin with central domain (N-Ame) combined with synthetic peptide (C-AMG) based on the functional domains of C-terminal telopeptide (C-Ame) is shown here, which is formed by amyloid-like lysozyme aggregation at the enamel interface through a rapid one-step aqueous coating process. In the PTL/C-AMG matrix, C-AMG facilitated the oriented arrangement of amorphous calcium phosphate (ACP) nanoparticles and their transformation to ordered enamel-like HAp crystals, while PTL served as a strong interfacial anchor to immobilize the C-AMG peptide and PTL/C-AMG matrix on versatile substrate surfaces. PTL/C-AMG film-coated enamel induced both of the in vivo and in vitro synthesis of HAp crystals, facilitated epitaxial growth of HAp crystals and recovered the highly oriented structure and mechanical properties to levels nearly identical to those of natural enamel. This work underlines the importance of amyloid-like protein aggregates in the biomineralization of enamel, providing a promising strategy for treating dental caries.     

Mechanical and photocatalytic properties of hydroxyapatite/titania nanocomposites prepared by combined high gravity and hydrothermal process

Hydroxyapatite/titania nanocomposites of different ratios have been successfully synthesized by combined high gravity and hydrothermal methods. SEM and TEM observations showed that small spheres of TiO₂, identified as anatase crystals of 10–15 nm, were deposited on HAp rod-like crystals. EDAX analysis confirmed the presence of Ca, P, Ti and O. X-ray diffraction patterns indicated the presence of hydroxyapatite and anatase phase. More number of anatase peaks appeared in the XRD patterns with higher colloidal concentration of TiO₂ in the HAp/TiO₂ compound. Mechanical stability of the HAp/TiO₂ nanocomposites was determined by reinforcing them with high molecular weight polyethylene (HMWPE) and the tensile strength of the samples was analyzed. Photocatalytic activity of the HAp/TiO₂ particles was examined by decomposition of methyl orange (MO). The results showed that photocatalytic properties of HAp/TiO₂ composites are more effective than that of individual HAp and TiO₂ which implied that the HAp improved the photocatalytic activity of well known photocatalyst TiO₂.     

Hydroxyapatite nanorod-reinforced biodegradable poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactic acid) composites for bone plate applications

Novel PLLA composite fibers containing hydroxyapatite (HAp) nanorods with or without surface lactic acid grafting were produced by extrusion for use as reinforcements in PLLA-based bone plates. Fibers containing 0–50% (w/w) HAp nanorods, aligned parallel to fiber axis, were extruded. Lactic acid surface grafting of HAp nanorods (lacHAp) improved the tensile properties of composites fibers better than the non-grafted ones (nHAp). Best tensile modulus values of 2.59, 2.49, and 4.12 GPa were obtained for loadings (w/w) with 30% lacHAp, 10% nHAp, and 50% amorphous HAp nanoparticles, respectively. Bone plates reinforced with parallel rows of these composite fibers were molded by melt pressing. The best compressive properties for plates were obtained with nHAp reinforcement (1.31 GPa Young’s Modulus, 110.3 MPa compressive strength). In vitro testing with osteoblasts showed good cellular attachment and spreading on composite fibers. In situ degradation tests revealed faster degradation rates with increasing HAp content. To our knowledge, this is the first study containing calcium phosphate–polymer nanocomposite fibers for reinforcement of a biodegradable bone plate or other such implants and this biomimetic design was concluded to have potential for production of polymer-based biodegradable bone plates even for load bearing applications.