Hydroxyapatite Microparticles Research Articles

Design of novel three-phase PCL/TZ–HA biomaterials for use in bone regeneration applications

The design of bioactive scaffold materials able to guide cellular processes involved in new-tissue genesis is key determinant in bone tissue engineering. The aim of this study was the design and characterization of novel multi-phase biomaterials to be processed for the fabrication of 3D porous scaffolds able to provide a temporary biocompatible substrate for mesenchymal stem cells (MSCs) adhesion, proliferation and osteogenic differentiation. The biomaterials were prepared by blending poly(epsilon-caprolactone) (PCL) with thermoplastic zein (TZ), a thermoplastic material obtained by de novo thermoplasticization of zein. Furthermore, to bioactivate the scaffolds, microparticles of osteoconductive hydroxyapatite (HA) were dispersed within the organic phases. Results demonstrated that materials and formulations strongly affected the micro-structural properties and hydrophilicity of the scaffolds and, therefore, had a pivotal role in guiding cell/scaffold interaction. In particular, if compared to neat PCL, PCL-HA composite and PCL/TZ blend, the three-phase PCL/TZ-HA showed improved MSCs adhesion, proliferation and osteogenic differentiation capability, thus demonstrating potential for bone regeneration.

Sustained Efficacy of Erythropoietin with a Hydroxyapatite Carrier Administered in Mice

For chronic kidney disease patients with renal anemia, recombinant human erythropoietin (rHuEPO) is a very effective drug; however, the treatment regime is troublesome, requiring multiple administrations each week. In the present study, we examined the efficiency of hydroxyapatite (HAp) as a drug delivery carrier for the sustained release of erythropoietin (EPO) to reduce the frequency of administration. Spray-dried HAp microparticles, formed from zinc-containing HAp (Zn-HAp) and Zn-HAp calcined at 400 degrees C, were used as carriers of EPO, and five Zn-HAp formulation samples incorporating EPO were prepared; no formulation, poly-L-lactic acid (PLA) formulation, zinc (Zn) formulation, Zn/PLA formulation, and calcined/Zn/PLA formulation. ICR mice were administered these samples or commercial rHuEPO (Epogin) as a control from dorsal neck subcutaneous, and hematological and histopathological analyses, including enzyme-linked immunosorbent assay for plasma EPO concentration, were performed. An increase in the blood EPO level was detected on days 3 and 8 post-administration. Peak hematopoiesis was delayed and higher hematological values were obtained on day 14 post-administration with no serious adverse reactions compared with the control. The Zn/PLA formulation sample was found to be most effective in reducing the initial peak while sustaining the delayed release of EPO. In conclusion, the Zn-HAp formulation samples were considered to be useful carriers for the sustained release of EPO, and the Zn/PLA formulation appears to be the most effective of five Zn-HAp formulation samples in sustaining EPO release.

Adsorption and Sustained Release of Insulin from Zinc Hydroxyapatite Microparticle with Poly (Lactic Acid) Coating

Insulin delivery carriers using low crystalline porous zinc hydroxyapatite (ZnHAp) microparticles with different crystal sizes and novel formulation method of poly (lactic acid) (PLA) have been developed to achieve the sustained-release of insulin. The adsorption isotherm curves of insulin dissolved into acetic acid of pH4.5 onto ZnHAp nanocrystals show non-Langmuir type due to the dissolution of the ZnHAp microparticles; the adsorption amounts were increased with the increase of crystalline sizes. The PLA formulation, coating the outer layer of microparticles, showed the apparent reduction of initial burst for insulin. The amounts of initial release of insulin decreased with the increase of crystalline sizes of ZnHAp, which could be attributed to the different meso-porous structure depending on its crystal sizes.

Rigid Hydroxyapatite-Alginate Beads for Sustained Release of Paclitaxel

Rigid hydroxyapatite (HAp)-alginate beads were prepared as drug delivery carriers for an anti-cancer drug, paclitaxel (Taxol). Paclitaxel was loaded into the HAp microparticle in process of a spray-drying technique. The HAp-alginate beads including paclitaxel were obtained by a droplet method into barium solution as ionic cross-linkage and dehydration. Cross-sectional analyses indicated the homogeneity of HAp microparticles and barium ions inside the bead. The ratio of alginate to HAp in the beads dominated both mechanical and swelling properties. Drug-release experiment demonstrated the sustained release of paclitaxel from the beads cross-linked with barium ion for 7 days.

Laser technologies for fabricating individual implants and matrices for tissue engineering

This paper presents the results of work on the development and modification of laser methods of rapid prototyping and fabrication of individual implants and matrices for tissue engineering. A technology has been developed for shaping mineral-polymer bioactive structures, based on laser photopolymerization of a liquid mixture of polyfunctional acrylic oligomers with microparticles of hydroxyapatite. A method is proposed and developed for surface-selective laser sintering, making it possible to sinter polymeric microparticles without melting them completely, but only submelting their surface. This made it possible to obtain bioactive polymeric matrices based on bioresorbable aliphatic polyesters.

Synthesis and Characterization of Metal Ions Containing Hydroxyapatite Microparticles with High Specific Surface Area

Zinc and/or iron ions containing hydroxyapatite (HA) microparticles, which had twice as high specific surface area and higher pore volumes compared with the pure HA, were fabricated by a spray drying method; the zinc and/or iron ions were incorporated into the constitutional nanocrystals with below 20 nm in size in preparing. The small amount of carbonate ions was substituted in the phosphate group. The particle sizes were distributed in the range of 1 to 20 microm in diameter and its averaged size was approximately 6 microm. The microparticles with a spherical shape showed a single phase of HA and, after treated at higher temperature than 800 degrees C, had the high crystallite HA and the other phase of metal oxide such as ZnO or Fe2O3. The microparticles with higher specific surface area will be applicable for the drug delivery carriers of proteins.

Drug-Supported Microparticles of Calcium Carbonate Nanocrystals and Its Covering with Hydroxyapatite

Drug-supported spherical microparticles with below 3 microm in diameter of calcium carbonate (CC) nanocrystals were precipitated by a complex decomposition method from Na2CO3 and CaCl2 solutions with hydrocortisone phosphate (HyC). The HyC was completely incorporated into the microparticles and was adsorbed on the surface of nanocrystals. The crystal phases of vaterite and calcite were controlled by the addition of magnesium ions. Spherical microparticles of calcite cores and petal-shaped hydroxyapatite (HAp) outer layers were fabricated by soaking calcite into the SBF, or a supersaturated solution of phosphate ions to enhance the crystal growth of HAp.

Preparation of Hydroxyapatite-Alginate Gels as a Carrier for Controlled Release of Paclitaxel

Hydroxyapatite (HAp)-alginate gels were developed as drug delivery carriers of the anti-cancer drug, water-insoluble paclitaxel (Taxol). The spray-drying technique was employed for loading the paclitaxel into spherical HAp microparticles with 1 to 20 μm in diameter. The microparticles loaded with 2.4 or 7.3wt% of paclitaxel were then mixed with sodium alginate, which was followed by Ca2+-mediated gelation. The compressive strength of the HAp-alginate gels and the release of paclitaxel from the gels in a medium were investigated in vitro. The alginate matrix was effective for the achievement of controlled release of anti-cancer drugs.

Method of fabrication of submicron composite microparticles of hydroxyapatite and ferromagnetic nanoparticles for a protein drug carrier

Composite microparticles of hydroxyapatite (HAp) and ferromagnetic nanoparticles were prepared using algenic acid gel cross-linked by bivalent calcium ions. The resultant size of the composite microparticle could be reduced to the submicron scale using extremely vigorous stirring by a homogenizer at 16 000 r.p.m. The instantaneous gellation induced by the bivalent ion cross-linking was revealed to be advantageous for combining the constituent nanoparticles of HAp and ferromagnetic nanoparticles. Since the structural arrest occurs instantly at the moment of cross-linking by the bivalent calcium ion, the added constituent HAp/ferromagnetic nanoparticles do not have time to be segregated apart from the binding gel; consequently, the submicron-scale composite state of these constituents could be instantaneously immobilized as in the dispersed state in liquid by the applied high shear. Thus, the instantaneous gellation characteristic of the bivalent ion cross-linking is preferable for incorporating different nanoparticles. The retention ability of the test protein (cytochrome c) was examined using the bincinchoninic assay method. Comparison of the protein retention ability with the control sample without HAp nanoparticles revealed that the incorporated HAp predominantly contributes to the retention ability of cytochrome c.

Bone cell transfection in tissue culture using hydroxyapatite microparticles

Coprecipitates of calcium phosphate and DNA have been used in vitro for several decades for cell transfection. We evaluated the efficiency of calcium phosphate ceramics associated to plasmid DNA in the transfection of bone cells in vitro when they are grown in tissue culture. Newborn rat calvariae and tibia epiphyses were grown on an agar surface for a period of 48 h to 30 days. The hydroxyapatite (HA)-particles were loaded with a plasmid bearing a galactosidase reporter gene by incubation of the plasmid solution in PBS with the particles. One milligram of HA-particles was then placed in contact with the bone explants for 8 and 30 days. Histological sections were then performed and the galactosidase activity was revealed using an X-gal solution. At eight days, very few cells expressing the galactosidase activity were detected. By 30 days, however, the explants appeared uniformly stained blue. The staining of sections showed that the osteoblasts, chondroblasts, perichondroblasts, and perisoteal cells all expressed the lacZ gene while the number of cells stained in the control was negligible. The time dependence of the transfection suggests that transfection using ceramics is linked to the degradation of the ceramic by the cells. Furthermore, the cells are stained remote from the particles suggesting that the particles induce a coprecipitate of DNA in the explant.

Particle Size of Hydroxyapatite Granules Calcified from Red Algae Affects the Osteogenic Potential of Human Mesenchymal Stem Cells in vitro

Hydroxyapatite (HA) microparticles as a carrier in an injectable tissue-engineered bone filler are considered promising candidates for the treatment of small bone defects in the craniomaxillofacial region. HA granules calcified from red algae, varying in size, were evaluated in vitro for their suitability to be used as a carrier for human mesenchymal stem cells (hMSCs). Three groups of granules were produced in grain sizes of 10–100, 200–500 and 600–1,000 µm. After seeding and culturing hMSCs under osteogenic differentiation conditions onto HA particles for 3, 6 and 9 days, cellular proliferation (tetrazolium salt, XTT), alkaline phosphatase (ALP)-specific activity and total protein synthesis were investigated. The osteoblastic phenotype of the cells was evaluated by assaying the bone-specific genes osteocalcin, osteopontin and collagen type I. XTT assay revealed significantly higher (p < 0.01) proliferation of cells grown on the smallest grain size after 9 days of culture. Regarding ALP-specific activity, significantly higher levels of activity were detected in cells grown on the smallest grain size. Different grain sizes had no significant effects on the secretion of osteocalcin and osteopontin. Collagen type I production was significantly higher (p < 0.05) in cells grown on the biggest grain size in comparison with the two other grain sizes. These results show that the particle size of HA microparticles affects the osteogenic potential of cultured hMSCs and lead to the conclusion that particle size has differential effects on ALP-specific activity and collagen type I production.

Protein Adsorption Ability of Porous Hydroxyapatite Microparticle with Metal Ions

Hydroxyapatite (HAp) also shows the high absorption ability for proteins with low degradations. In this study, to modify the absorption ability and to obtain high surface area, metal ions (Zn, Mg, Fe) were introduced in preparing the HAp nanocrystals, and porous microparticles were subsequent fabricated by a spray drying method. HAp microparticles with Zn, Mg, Fe ions had a single phase of low crystalline HAp. The specific surface area of the HAp, Mg-HAp, Fe-HAp and Zn-HAp were 87, 150, 152, 167 m2/g, respectively. In the adsorption experiment of a lysozyme as a basic proteine, Zn-HAp showed the highest adsorption ability. On the other hand, the HAp microparticles witn metal ions showed higher adsorption ability for BSA as a acitic protein than pure HAp microparticles.

Controlled Release of Protein Drugs Using Porous Hydroxyapatite Microparticles

Controlled Release of Protein Drugs Using Porous Hydroxyapatite Microparticles

Injectable porous hydroxyapatite microparticles as a new carrier for protein and lipophilic drugs

Hydroxyapatite (Ca10 (PO4)6(OH)2) is a biodegradable material that forms a major component of bones and teeth. We prepared injectable spherical porous hydroxyapatite microparticles (SP-HAp) as a drug carrier by the spray-drying method. We then examined the usefulness of SP-HAp as a carrier for drugs such as interferon alpha (IFNα), testosterone enanthate (TE), and cyclosporin A (CyA). SP-HAp had an average diameter of 5 μm and a porosity of approximately 58%. It could be injected subcutaneously through a 27-gauge needle. SP-HAp was observed to be biodegradable. The speed of degradation of SP-HAp could be regulated by altering the calcination temperature. IFNα was adsorbed well to SP-HAp particles, but INFα was released faster from the particles, than the particles could degrade in both in vitro and in vivo experiments. Addition of human serum albumin and zinc (reinforcement) to IFNα-adsorbed SP-HAp caused marked prolongation of release in vivo. The in vivo release of testosterone enanthate and CyA from SP-HAp preparation, which was easily injectable, was similarly prolonged to that from the oil preparation. In conclusion, the SP-HAp seems to be useful as a biodegradable and subcutaneously injectable drug carrier. It is suggested that the reinforcement of the SP-HAp is very effective on the sustained release of drugs.

Investigation of Apatite Deposition onto Charged Surfaces in Aqueous Solutions Using a Quartz‐Crystal Microbalance

Hydroxyapatite (HAp) deposition onto positively charged surfaces (i.e., self‐assembled monolayers (SAMs) terminated with NH2 head groups) and negatively charged surfaces (i.e., OH‐SAMs (weak) and COOH‐SAMs (strong)) soaked at 50°C in aqueous supersaturated solutions (1.5 SBF, pH 7.0–7.6; SBF = simulated body fluid) was investigated using a quartz‐crystal microbalance. The results revealed that the solution conditions greatly influenced the formation of HAp on the charged surfaces. In a stable supersaturated solution of simulated body fluid (1.5 SBF, pH &lt;7.4), more strongly negative surfaces had a more powerful induction capability for the heterogeneous nucleation of HAp (COOH &gt; OH), whereas nucleation was obviously prohibited on a positive surface (NH2‐SAM). On the other hand, after the calcium phosphate particles had nucleated homogeneously in an unstable soaking solution (1.5 SBF, pH ≧7.4), adhesion of the HAp microparticles to the NH2‐SAM was observed. A two‐step interaction is conceivable to describe the formation of HAp on the positive NH2‐SAM: At the first stage, electrostatic interaction dominates the adhesion of HAp microparticles; at the second stage, hydrogen bonds possibly form between the HAp microparticles and the amino head groups of the NH2‐SAM, for a firm bonding with the substrate, and the microparticles grow progressively into a thin film. The electrophoretic behaviors of the HAp microparticles confirmed this hypothesis.

Physical and biological evaluations of sintered hydroxyapatite/silicone composite with covalent bonding for a percutaneous implant material

A composite (HA/silicone) of hydroxyapatite (HA) microparticles with an average diameter of 2.0 micro m covalently linked to a silicone substrate has been developed, and its physical and biological properties as a percutaneous soft-tissue-compatible material have been evaluated. In tensile property measurement, samples of HA/silicone and the original silicone were similar in tensile strength, ca. 7.8 MPa, and elongation at break, ca. 570%. It was found that chemical surface modification with HA particles presented no mechanical disadvantage. In an adhesive-tape peeling test, scanning electron microscopic (SEM) observation showed that HA particles coupled directly to the substrate were not removed. HA particles may bond strongly with the substrate. In human periodontal ligament fibroblast attachment and proliferation experiments, the number of cells attached to HA/silicone was 14 times greater than that attached to the original silicone after 24 h of incubation. The value on HA/silicone was ca. 80% versus that on a tissue-culture plastic used as a positive control. After 72 h of incubation, the number of cells grown on HA/silicone increased to the level of the positive control. In observation of fluorescence microscopy stained by Hoechst 33342, cells appeared to tightly adhere to HA particles coupled to the silicone sheet due to intact nuclear morphology. Observation of cells by fluorescence dye with rhodamin phalloidin showed an extensive F-actin cytoskeleton on HA/silicone. In a 4-week animal implant test, force required to pull out the HA/silicone sheet was 15 times that of the original silicone. HA-particle coating on silicone with covalent linkage gave the inert surface bioactivity. The HA composite thus effectively prevents germ infection percutaneously.

Site-Selective Adhesion of Hydroxyapatite Microparticles on Charged Surfaces in a Supersaturated Solution

Site-selective adhesion of hydroxyapatite (HAp) microparticles to NH2-terminated self-assembled monolayers (NH2–SAM, positive surface) and OH–SAM (negative) was investigated in the present paper. HAp microparticles were measured to be negatively charged, and homogeneously formed at 50°C in the simulated body fluid (SBF) with high supersaturation degree (1.5 SBF; the concentrations of Ca2+ and PO43− alone are 1.5 times that in 1.0 SBF) buffered carefully at 7.6∼7.7. The zeta potential of microparticles decreased from −11.5 mV to neutrality owing to the absorption of cations accompanied by the simultaneous growth of microparticles. The micropattern of HAp particulate films was successfully fabricated by the use of site-selective adhesion of HAp microparticles on the positive NH2–SAM owing to the attractive electrostatic interactions. The quantitative analysis using quartz crystal microbalance also confirmed the identical phenomena.

A hydroxyapatite coating covalently linked onto a silicone implant material.

A novel composite consisting of hydroxyapatite (HAp) microparticles covalently coupled onto a silicone sheet was developed. Initially, an acrylic acid (AAc) -grafted silicone sheet with a 16.7 microg/cm(2) surface graft density was prepared by corona-discharge treatment. The surface of sintered, spherical, carbonated HAp particles with an average diameter of 2.0 microm was subsequently modified with amino groups. The amino group surface density of the HAp particles was calculated to be approximately one amino molecule per 1.0 nm(2) of particle surface area. These samples were characterized with Fourier transform infrared spectrometry and X-ray photoelectron spectroscopy. After the formation of ammonium ionic bonds between both samples under aqueous conditions, they were reacted at 180 degrees C for 6 h in vacuo to form covalent bonds through a solid-phase condensation. The HAp particles were coupled to the AAc-grafted silicone surface by a covalent linkage. Further improvements in the adhesive and bioactive properties of the HAp-coated silicone material are expected.

Hydroxyapatite microcarriers for biocontrolled release of protein drugs

It is demonstrated that hydroxyapatite microcarriers suitable for biocontrolled release of protein drugs can be obtained by allowing hydroxyapatite to crystallize in the presence of protein. From tiny nuclei, radially grown star-like hydroxyapatite particles were formed at 100°C that were allowed to crystallize further at room temperature in the presence of BSA. After incubation for several weeks, the hydroxyapatite microparticles contained 5–10% BSA; 1–3% was incorporated into the crystalline hydroxyapatite microcarrier. It was impossible to release the incorporated BSA from the microcarriers even by extensive desorption procedures unless the hydroxyapatite crystals were dissolved. In vitro release of the incorporated BSA in a hydroxyapatite dissolving EDTA buffer of pH 5.9 proceeded gradually and reached completion in 3 weeks. At pH 6.9 the release rate of the incorporated BSA was 4-times lower, which is in line with the slower rate of dissolution of the hydroxyapatite at that pH. In vivo a hydroxyapatite microcarrier suspension was not detectable 1 month after injection in rabbit muscle, confirming the assumption that in soft tissue hydroxyapatite is fully resorbed.

A unique opportunity to upgrade genitourinary medicine.

<i>BACKGROUND AND PURPOSE:</i> Our newly developed biocompatible embolic materials, hydroxyapatite ceramic microparticles, have good visibility during injection control and were shown to be capable of producing effective occlusion of the distal arteriocapillary bed in an experimental animal study. The purpose of this present study was to evaluate hydroxyapatite ceramic microparticles for use in human meningioma embolization. <i>METHODS:</i> Thirteen patients with meningiomas underwent preoperative superselective embolization with the use of hydroxyapatite microparticles. Radiologic and histopathologic studies of the surgical specimens were performed. <i>RESULTS:</i> During embolization, no microcatheter clogging was observed and angiographic devascularization was consistently obtained without unexpected proximal occlusions. Histopathologic findings showed that there was mild inflammatory response in the thrombosed lumen. <i>CONCLUSION:</i> Hydroxyapatite microparticles are excellent embolic materials for the treatment of human meningioma. They have excellent biocompatibility and good injection control, which produces occlusion of the distal arteriocapillary bed.