Photo-induced formation of hydroxyapatite on TiO2 synthesized by a chemical–hydrothermal treatment

The formation of hydroxyapatite (HAp) on TiO₂ surfaces under continuous ultraviolet (UV) irradiation was investigated. Pure Ti substrates were chemically treated with H₂O₂/HNO₃ at 353 K for 20 min to form a TiO₂ gel layer. The specimens were then hydrothermally treated with an aqueous NH₃ solution in an autoclave at 453 K for 12 h. An adhesive and sufficiently crystallized anatase-type TiO₂ film could be synthesized on the Ti surface. The specimens were immersed in simulated body fluid in darkness or under UV irradiation with a centered wavelength of λ = 365 nm. Under dark conditions, a thin homogeneous HAp film was formed, with just a few spherical clusters of HAp. The UV illumination promoted the formation of HAp clusters, which may be due to the generation of functional Ti–OH or Ti–O groups on the TiO₂ surface. On the other hand, the UV light produced electron-hole pairs in the TiO₂, and the photogenerated holes that migrated to the surface repelled the Ca²⁺ ions in the solution. As a consequence, the UV irradiation suppressed the formation of a HAp thin film.     

Affinity adsorption of human vitamin K-dependent coagulation factor IX onto heparin-like poly (styrene sodium sulfonate) adsorbent

Adsorption of proteins on polymer material plays an important role in a number of fields, particularly in separation of biomolecules by chromatographic methods. The work reports here the synthesis of modified cross-linked polystyrene gel beads as a stationary phase in liquid chromatography for the purification of factor IX. Suitable chemical groups, such as sulfonate which confer this polymer heparin-like adsorbing property, were grafted on the aromatic ring of the hydrophobic matrix. This functional group was chosen on the basis of the biospecific molecular interactions between factor IX and its ligand particularly heparin in such manner to enhance its binding ability and efficacy. Adsorption of factor IX on to this functional polymer was performed under physiological conditions according two modes: non-competitive adsorption (adsorption of factor IX alone) and competitive adsorption (adsorption of factor IX in the presence of another vitamin-K dependent coagulation factors). The adsorbed factor IX content at the interface allows to establish the chemisorption isotherm curves. The adsorption rate in both cases was found to be significantly high and the affinity constants, estimated by the Langmuir model, were: 4.7 × 10⁸ and 4.1 × 10⁸ l/M respectively. Affinity chromatography on column using this functional polymer as a stationary phase confirms its high ability to adsorb factor IX at low ionic strength. Thus, the synthesized packing material gel functionalised by sulfonate group can be used advantageously as a heparin-like adsorbent in purification of factor IX.     

Surface modification of poly(l-lactide) electrospun fibers with nanocrystal hydroxyapatite for engineered scaffold applications

The hydrophobicity of the poly(l-lactide) (PLLA) surface was modified by incorporating hydroxyapatite (HAp) nanocrystalline particles during the electrospinning process for the engineered scaffold applications. The HAp nanocrystals were synthesized with 30 nm in diameter and 100–120 nm in length, which subsequently formed micrometer-sized agglomerates in the range of 2.5 μm. The synthesized HAp agglomerates were electrospun in the PLLA solution, and the HAp nanocrystals were desirably exposed on the surface of the electrospun PLLA fibers to give higher surface energy and lower contact angles with water. The surface-exposed hydrophilic HAp nanocrystals substantially increased the precipitation of various salts on the HAp/PLLA fiber surfaces in a buffer solution due to the hydrophilic nature and ionic affinity of HAp. Finally, the developed HAp/PLLA fibers desirably sustained the fibrous structural integrity during the accelerated-aging test in water, which was not the case with the pristine PLLA fibers.     

Effect of urea on formation of hydroxyapatite through double-step hydrothermal processing

The effect of urea on the formation of hydroxyapatite (HAp) was studied by employing the double-step hydrothermal processing of a powder mixture of beta-tricalcium phosphate (β-TCP) and dicalcium phosphate dihydrate (DCPD). Co-existence of urea was found to sustain morphology of HAp crystals in the compacts under an initial concentration of 2 mol dm^-3 and less. Homogenous morphology of needle-like crystals was observed on the compacts carbonated owing to decomposition of urea. Carbonate ions (CO₃^2-) was found to be substituted in both the phosphate and hydroxide sites of HAp lattice. The synthesized HAp was calcium deficient, as it had a Ca/P atomic ratio of 1.62 and the phase was identified as calcium deficient hydroxyapatite (CDHA). The release of CO₃^2- ions from urea during the hydrothermal treatment determined the morphology of the CDHA in the compacts. The usage of urea in the morphological control of carbonate-substituted HAp (CHAp) employing the double-step hydrothermal method is established.     

Bone tissue response to titanium implant surfaces modified with carboxylate and sulfonate groups

The present study assessed in vivo new bone formation around titanium alloy implants chemically grafted with macromolecules bearing ionic sulfonate and/or carboxylate groups. Unmodified and grafted Ti–6Al–4V exhibiting either 100% carboxylate, or 100% sulfonate, or both carboxylate and sulfonate groups in the percent of 50/50 and 80/20 were bilaterally implanted into rabbit femoral condyle. Neither toxicity nor inflammation were observed for all implants tested. After 4 weeks, peri-implant new bone formation varied as a function of the chemical composition of the titanium surfaces. The percent bone-implant contact (BIC) was the lowest (13.4 ± 6.3%) for the implants modified with grafted carboxylate only. The value of BIC on the implants with 20% sulfonate (24.6 ± 5.2%) was significantly (P < 0.05) lower than that observed on 100% sulfonate (38.2 ± 13.2%) surfaces. After both 4 and 12 weeks post-implantation, the BIC value for implants with more than 50% sulfonate was similar to that obtained with the unmodified Ti–6Al–4V. The grafted titanium alloy exhibiting either 100% sulfonate or carboxylate and sulfonate (50% each) groups promoted bone formation. Such materials are of clinical interest because, they do not promote bacteria adhesion but, they support new bone formation, a condition which can lead to osseointegration of bone implants while preventing peri-implant infections.     

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.     

Nano-scaled hydroxyapatite/polymer composite III. Coating of sintered hydroxyapatite particles on poly(4-methacryloyloxyethyl trimellitate anhydride)-grafted silk fibroin fibers

A novel composite coupling between nano-scaled hydroxyapatite (HAp) particles and poly[4-methacryloyloxyethyl trimellitate anhydride (4-META)]-grafted silk fibroin (SF) through ionic interaction was synthesized. The weight gain of poly(4-META) by graft-polymerization increased with increasing the reaction time, eventually reaching a plateau value of about 20 wt%. The HAp nano-particles were adsorbed equally and dispersively on the treated SF fiber surface. The HAp content in the composite was 4.554 wt% ± 0.098 (n = 4), confirmed by thermogravimetry (TG). This synthetic system requires no heat to connect HAp to SF and is useful when applying to non-heat-resistant polymers. The L-929 cell-adhesion test shows that the HAp/SF composite improves bioactivity compared to the original SF.     

Hydroxyapatite formation on porous ceramics of alpha-tricalcium phosphate in a simulated body fluid

Alpha-tricalcium phosphate (α-TCP) ceramic is a bioresorbable material that degrades in bone tissue after implantation, since it exhibits higher solubility than beta-tricalcium phosphate (β-TCP) ceramics. The high solubility of α-TCP in an aqueous solution causes its transformation into hydroxyapatite (HAp) through hydrolysis. While one expects the formation of hydroxyapatite after exposure to an aqueous solution mimicking a body environment, we occasionally find variation in HAp formation in the simulated body fluid (SBF). In the present study, HAp formation resulting from exposure to SBF was investigated for some types of α-TCP ceramics with different porosities and specific surface area. Reduced porosity and large surface area of porous specimens may increase the local density of Ca²⁺ in the surrounding SBF to increase the degree of supersaturation with respect to HAp. Thus, the porosity and specific surface area are significant parameters for determining not only bioabsorbability but also the ability to form HAp.     

Photoluminescence and doping mechanism of theranostic Eu3+/Fe3+ dual-doped hydroxyapatite nanoparticles

Theranostic nanoparticles currently have been regarded as an emerging concept of ‘personalized medicine’ with diagnostic and therapeutic dual-functions. Eu³⁺ doped hydroxyapatite (HAp) has been regarded as a promising fluorescent probe for in vivo imaging applications. Additionally, substitution of Ca²⁺ with Fe³⁺ in HAp crystal may endow the capability of producing heat upon exposure to a magnetic field. Here we report a preliminary study of doping mechanism and photoluminescence of Eu³⁺ and Fe³⁺ doped HAp nanoparticles (Eu/Fe:HAp). HAp with varied concentration of Eu³⁺ and Fe³⁺ doping are presented as Eu(10 mol%):HAp, Eu(7 mol%)-Fe(3 mol%):HAp, Eu(5 mol%)-Fe(5 mol%):HAp, Eu(3 mol%)-Fe(7 mol%):HAp, and Fe(10 mol%):HAp in the study. The results showed that the HAp particles, in nano-size with rod-like morphology, were successfully doped with Eu³⁺ and Fe³⁺, and the particles can be well suspended in cell culture medium. Photoluminescence analysis revealed that particles have prominent emissions at 536 nm, 590 nm, 615 nm, 650 nm and 695 nm upon excitation at a wavelength of 397 nm. Moreover, these Eu/Fe:HAp nanoparticles belonged to B-type carbonated HAp, which has been considered an effective biodegradable and biocompatible drug/gene carrier in biological applications.     

Synthesis of organic derived hydroxyapatite scaffold from pig bone waste for tissue engineering applications

Micro porous hydroxyapatite (HAp) had drawn great attention in the field of tissue engineering due to its numerous applications such as tissue regeneration, dental, drug delivery, and adsorption and desorption of substances etc. The chemical synthesis of HAp is often faced with the high cost of starting materials and often lacks the presence of beneficial ions which can promote biological reactions. This paper examined a novel application of pig bone waste for the synthesis of HAp via heat treatment between 600 and 1000 °C. Thus synthesized powder was characterized by X-ray diffraction (XRD), Fourier transform infra-red spectroscopy (FT-IR), Thermogravimetric analysis (TGA), Scanning electron microscopy (SEM), Energy dispersive X-ray analysis (EDX) and Transmission electron microscopy (TEM). XRD results revealed the main characteristic peaks of single phase HAp powder, while the presence of various functional groups such as PO₄^3?, CO₃^2? and OH^? corresponding to HAp were observed by FT-IR analysis. The elemental composition of as-synthesized HAp powder as observed by EDX showed the presence of Ca and P in addition to some beneficial metals such as Na, K, Mg and Si which plays vital roles in biological applications. SEM and TEM observation confirmed the microscopic sctructure of the as-synthesized HAp to be rod-like morphology with 38–52 nm in length. Porous HAp scaffold up to 65% porosity could be prepared using ammonium bicarbonate as pore forming agent. Therefore, bio-waste such as pig bones can be utilized in the synthesis of porous hydroxyapatite scaffold which can serve as an alternative for the conventional chemical method.     

Hydrothermal synthesis of composites of well-crystallized hydroxyapatite and poly(vinyl alcohol) hydrogel

Composites of hydroxyapatite (HAp) and poly(vinyl alcohol) (PVA) hydrogel were fabricated by the hydrothermal treatment of calcium phosphate powder. Alpha-tricalcium phosphate (α-TCP) or beta-tricalcium phosphate (β-TCP) powder was dispersed in PVA hydrogel and exposed to water vapor at 120 °C, 140 °C or 160 °C for 6 h. Low crystallinity HAp was formed in specimens prepared from α-TCP and PVA hydrogel prior to hydrothermal treatment, which was caused by hydrolysis of α-TCP. This allowed specimen shape to be retained after hydrothermal treatment. β-TCP showed less reactivity in forming HAp in the PVA hydrogel, which led to the formation of large rod-shaped crystals approximately 15 μm in length. Specimens from β-TCP and PVA were too soft to retain their shape after hydrothermal treatment. HAp with controlled morphology was prepared using different types of tricalcium phosphate precursor. The application of α-TCP allowed the in situ fabrication of HAp/PVA composites.     

Adsorption property and affinity chromatography of polystyrene derivative sorbent towards cow's milk xanthine oxidase

On the basis of the biospecific molecular recognition between complementary chemical groups of xanthine oxidase (XO) and their ligands particularly sulphated glycoaminoglycans and heparin. Poly (styrene chlorosulfonyl) particles modified by sulfonate sodium groups was synthesized and its adsorption property towards cow's milk XO was established. The adsorption of XO onto this functional polymer was performed in batch at 4 °C and at pH 6.0 during 30 min. of incubation. The adsorbed XO content at the interface allows establishing the chemisorption isotherm curve. The affinity association estimated from this adsorption isotherm according to the Langmuir equation was found to be significantly high in the magnitude of 1.25 × 10⁶ M^? 1. Affinity chromatography on column using this functional polymer as a stationary phase confirms its high ability to adsorb XO at low ionic strength. In fact, the xanthine oxidase of the crude extract is strongly adsorbed onto the sorbent and is eluted at high ionic strength with out any significant loss of its biological activity. The purified enzyme possesses a protein flavin ratio (PFR) of 6.05 with a specific activity of 1.78 UI/mg. On the other hand, the electrophoresis of XO fraction showed a single band with a molecular weight of about 150 kDa. Thus, the synthesized beads functionalized by sulfonate group could be used efficiently and advantageously in the purification of XO instead of other conventional chromatographic methods which need several steps.     

Adsorption of gum Arabic on bioceramic nanoparticles

Surface modification agents can be used to tailor the surface chemistry and biological activity of bioceramic nanoparticles in very intriguing ways. However, the specific modes of interactions between macromolecules and nanoparticles can be difficult to characterize. The aim of this study was to investigate the adsorption of gum Arabic on hydroxyapatite (HAp) and magnetic nanoparticles (MNP) using the bicinchoninic acid (BCA) test. Gum Arabic (GA) is a natural gum that has been widely used as an emulsifying agent and shows promise for dispersing nanoparticles in aqueous solutions. The adsorption of GA onto HAp nanoparticles followed a Langmuir isotherm with an adsorption plateau occurring at 0.2 g GA/g HAp. The adsorption of GA onto MNP attained a maximum value of 0.6 g GA/g MNP, after which it decreased to approximately 0.2 g GA/g MNP. The maximum adsorption density of GA on both MNP and HAp is equivalent when normalized to the specific surface area (4 × 10^− 3 g GA/m²). Adsorbed GA molecules were displaced from the surface of HAp and MNP in the presence of phosphate ions.     

Effect of collagen fibril formation on bioresorbability of hydroxyapatite/collagen composites

Porous hydroxyapatite/collagen (HAp/Col) composite is a promising biomaterial and a scaffold for bone tissue engineering. The effect of fibril formation of Col in the porous composite on bioresorbability and mechanical strength was investigated. The fibril formation, in mixing a self-organized HAp/Col nanocomposite and sodium phosphate buffer at a neutral condition, occurred during incubation at 37 °C, resulting in gelation of the mixture. The porous composites with and without the incubation were obtained by freeze-drying technique, in which macroscopic open pores were formed. The compressive strength of the porous composite with the incubation (34.1 ± 1.6 kPa) was significantly higher than that without the incubation (28.0 ± 3.3 kPa) due to the fibril formation of Col. The implantations of the porous composites treated with a dehydrothermal treatment in bone holes revealed that bioresorption was clearly depended on the fibril formation. The bioresorbability in vivo was almost matched to the in vitro test using enzymatic reaction of collagenase.     

Microsphere-based scaffolds encapsulating tricalcium phosphate and hydroxyapatite for bone regeneration

Bioceramic mixtures of tricalcium phosphate (TCP) and hydroxyapatite (HAp) are widely used for bone regeneration because of their excellent cytocompatibility, osteoconduction, and osteoinduction. Therefore, we hypothesized that incorporation of a mixture of TCP and HAp in microsphere-based scaffolds would enhance osteogenesis of rat bone marrow stromal cells (rBMSCs) compared to a positive control of scaffolds with encapsulated bone-morphogenic protein-2 (BMP-2). Poly(d,l-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds encapsulating TCP and HAp mixtures in two different ratios (7:3 and 1:1) were fabricated with the same net ceramic content (30 wt%) to evaluate how incorporation of these ceramic mixtures would affect the osteogenesis in rBMSCs. Encapsulation of TCP/HAp mixtures impacted microsphere morphologies and the compressive moduli of the scaffolds. Additionally, TCP/HAp mixtures enhanced the end-point secretion of extracellular matrix components relevant to bone tissue compared to the “blank” (PLGA-only) microsphere-based scaffolds as evidenced by the biochemical, gene expression, histology, and immunohistochemical characterization. Moreover, the TCP/HAp mixture groups even surpassed the BMP-2 positive control group in some instances in terms of matrix synthesis and gene expression. Lastly, gene expression data suggested that the rBMSCs responded differently to different TCP/HAp ratios presented to them. Altogether, it can be concluded that TCP/HAp mixtures stimulated the differentiation of rBMSCs toward an osteoblastic phenotype, and therefore may be beneficial in gradient microsphere-based scaffolds for osteochondral regeneration.     

Formation and properties of composites comprised of calcium-deficient hydroxyapatites and ethyl alanate polyphosphazenes

Composites comprised of calcium-deficient hydroxyapatite (HAp) and biodegradable polyphosphazenes were formed via cement-type reactions at physiologic temperature. The composite precursors were produced by blending particulate hydroxyapatite precursors with 10 wt% polymer using a solvent/non-solvent technique. HAp precursors having calcium-to-phosphate ratios of 1.5 (CDH) and 1.6 (CDS) were used. The polymeric constituents were poly[bis(ethyl alanato)phosphazene] (PNEA) and poly[(ethyl alanato)(1) (p-phenylphenoxy)(1) phosphazene] (PNEA(50)PhPh(50)). The effect of incorporating the phenyl phenoxy group was evaluated as a means of increasing the mechanical properties of the composites without retarding the rates of HAp formation. Reaction kinetics and mechanistic paths were characterized by pH determination, X-ray diffraction analyses, scanning electron microscopy, and infrared spectroscopy. The mechanical properties were analyzed by compression testing. These analyses indicated that the presence of the polymers slightly reduced the rate HAp formation. However, surface hydrolysis of polymer ester groups permitted the formation of calcium salt bridges that provide a mechanism for bonding with the HAp. The compressive strengths of the composites containing PNEA(50)PhPh(50) were superior to those containing PNEA, and were comparable to those of HAp produced in the absence of polymer. The current composites more closely match the structure of bone, and are thus strongly recommended to be used as bone cements where high loads are not expected.     

Low temperature synthesis of a self-assembling composite: hydroxyapatite-poly[bis(sodium carboxylatophenoxy)phosphazene]

The present study was undertaken to investigate the low temperature formation of a hydroxyapatite-polyphosphazene polymer composite likely to be biocompatible. The temperature range studied (25 to 60°C) was selected to bracket physiological temperatures. The composite precursors consisted of CaHPO₄·2H₂O, Ca₄(PO₄)₂O, and poly[bis(sodium carboxylatophenoxy)phosphazene]. The results indicate that a synergistic relationship exists in the formation of a polyphosphazene network and hydroxyapatite (HAp) matrix phase during composite synthesis. Calcium from the HAp precursors participates in the formation of a Ca crosslinked polymeric network which influences the rate of HAp formation and its morphology. The mechanistic paths taken during composite formation were followed by determining variations in the concentration of species in solution (at physiological temperature), rates of heat evolution, and microstructural development. These analyses indicate that the polymer controls the kinetics of hydroxyapatite formation and the composite microstructure. Low reaction temperatures and a high proportion of polymer facilitate the formation of a highly interconnected composite. The presence of the polyphosphazene allows a metastable calcium phosphate solution to persist for extended periods prior to the formation of hydroxyapatite. The degree of supersaturation and the length of the induction period increase with an increase in polyphosphazene content. The temperature dependence of these induction periods obeyed an Arrhenius relationship.     

Mechanochemical synthesis of hydroxyapatite from Ca(OH)2-P2O5 and CaO-Ca(OH)2-P2O5 mixtures

Dry grinding of Ca(OH)₂-P₂O₅ and CaO-Ca(OH)₂-P₂O₅ mixtures was conducted by a planetary ball mill to investigate the mechanochemical solid-phase reaction for the synthesis of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂, HAp). HAp was synthesized by grinding of the two sets of mixtures. The formation of HAp from the Ca(OH)₂-P₂O₅ mixture was more advantageous than that from the CaO-Ca(OH)₂-P₂O₅ one. This synthesis reaction from the former mixture was almost completed within 30 min of grinding. The presence and amount of H₂O contained in the starting mixtures played a key role to promote the formation of HAp. Especially, in the former mixture, the prolonged grinding assisted the solid-phase reaction of the intermediate DCPD and Ca(OH)₂ to produce HAp more effectively.     

Generation of hydroxyapatite patterns by electrophoretic deposition

Hydroxyapatite (HAp) patterns with distinct boundaries were generated by electrophoretic deposition (EPD) utilizing an insulating mask that partially blocks the electric field. For the EPD process, we selected two types of mask: a polytetrafluoroethylene (PTFE) board with holes and a resist pattern. A porous PTFE film, which differed from the mask PTFE, was employed as a substrate and attached to the mask. EPD was performed with a suspension of wollastonite particles in acetone, which were deposited on the substrate in the form of the patterned mask. The deposited wollastonite particles induced HAp patterns during a soak in simulated body fluid (SBF). As a result, minute HAp patterns, such as dots, lines, and corners were fabricated on the porous PTFE substrate with a minimum line width of about 100 μm.     

Bioactive ceramic composites sintered from hydroxyapatite and silica at 1200∘C: preparation, microstructures and in vitro bone-like layer growth

Bioceramic composites were synthesized by sintering the powders of hydroxyapatite (HAp) mixed directly with additive of 0.5, 1.0, 2.0, 5.0 and 10 wt.%SiO₂, respectively, at 1200^∘C. X-ray diffraction (XRD) analysis indicated that the phase transformation from HAp to tricalcium phosphate (TCP) comprising α-TCP and Si-TCP occurred and became more prominent with the addition of SiO₂ and the increase in SiO₂ content. The observations of their surface microstructures showed that the addition of SiO₂ suppressed the grain growth and promoted the formation of crystalline-glassy composites denoted HAp + TCP/Bioglass. As the SiO₂ content is as high as 5 wt.%, the composite made a feature of crystalline clusters with different sizes consisting of HAp and TCP grains surrounded by the matrix of glassy phase. Furthermore, the dependence of in vitro bioactivity of these composites on the SiO₂ content was biomimetically assessed by determining the changes in surface morphology, i.e., bone-like apatite layer growth, after soaking in an acellular stimulated body fluid (SBF) for 3 days at 36.5^∘C. It was found that the HAp-SiO₂ composites showed a much faster bone-like layer growth than pure HAp, and the propensity of composites to exhibit a better bioactivity was getting more notable with increasing SiO₂ content, except for the case of the highest content of 10 wt.%. It was believed that the formation of the bone-like layer on the surfaces of these bio-composites is closely related to the increasingly provided silanol groups and transformed TCP phase in materials associated with the content of SiO₂ added.