Nondestructive Microstructural Analysis of Porous Bioceramics by Microfocus X-ray Computed Tomography (μCT): A Proposed Protocol for Standardized Evaluation of Porosity and Interconnectivity Between Macro-pores

Microfocus X-ray computed tomography (μCT) has now become widely available for the nondestructive evaluation of porous bioceramics suitable for use as a bone substitute in orthopedic surgery. As part of an official Japanese working committee, we recently participated in the preparation of a proposed standard protocol for the quantitative μCT analysis of porous bioceramics sent to the International Organization for Standardization (ISO). In this protocol, the recommended basic conditions for analysis were [field of view (XY plane): 3.0 mm, spatial resolution: 6 μm/pixel (or the closest minimal values available for both parameters on a particular μCT system), matrix size: 512 pixels], and we have now further determined the optimal values for more detailed parameters (e.g., threshold determination). To validate the utility of the complete protocol, three different types of ceramic sample [a ceramic of β-tricalcium phosphate (β-TCP) and two types of hydroxyapatite (HAp) with different porosities] were evaluated with three different types of cone-beam μCT scanner (the Shimadzu SMX-100CT, Shimadzu inspeXio-90CT, and Skyscan-1174 scanners). Acquired images were quantified using 3D-reconstruction software, VGStudio MAX (version 1.2). After comparing data obtained from these three μCT scanners, we have found that determinations of both porosity and pore-interconnectivity were very similar from one system to the other although the total number of measured pores did vary between scanners. The present data indicate that our protocol for μCT analysis is reliable enough to quantify the porosity and interconnectivity of porous bioceramics and would therefore facilitate both large-scale screening and quality control of porous bioceramic samples.     

Preparation of microstructured hydroxyapatite microspheres using oil in water emulsions

Hydroxyapatite (HAP) microspheres with peculiar spheres-in-sphere morphology were prepared by using oil-in-water emulsions and solvent evaporation technique. Ethylene vinyl acetate co-polymer (EVA) was used as the binder material. Preparation of HAP/EVA microspheres was followed by the thermal debinding and sintering at 1150°C for 3 h to obtain HAP microspheres. Each microsphere of 100–1000 μm was in turn composed of spherical hydroxyapatite granules of 2–15 (μm size which were obtained by spray drying the precipitated HAP. The parameters such as percentage of initial HAP loading, type of stabilizer, concentration of stabilizer, stirring speed and temperature of microsphere preparation were varied to study their effect on the particle size and geometry of the microspheres obtained. It was observed that these parameters do have an effect on the size and shape of the microspheres obtained, which in turn will affect the sintered HAP microstructure. Of the three stabilizers used viz. polyoxyethylene(20) sorbitan monopalmitate (Tween-40), sodium laurate and polyvinyl alcohol (PVA), only PVA with a concentration not less than 0.1 wt% showed controlled stabilization of HAP granules resulting in spherical microspheres of required size. Morphologically better spherical microspheres were obtained at 20°C. Increasing the stirring speed produced smaller microspheres. Smaller microspheres having size < 50 μm were obtained at a stirring speed of 1500 ±50 rpm. A gradual decrease in pore size was observed in the sintered microspheres with increase in HAP loading.     

Preparation of hollow hydroxyapatite microspheres

The preparation of hollow hydroxyapatite (HA) microspheres as potential drug-delivery vehicles was investigated. A lithium-calcium-borate (10Li₂O-15CaO-75B₂O₃) (mol%) glass, made by fusing the components at 1100°C for 1 h, was ground to a powder and passed through a flame at ∼1400°C to spheroidize the particles. The resulting glass microspheres (106–125 μm in diameter) were reacted in 0.25 M K₂HPO₄ solution for 5 days at 37°C and pH 10–12, resulting in the formation of porous, hollow microspheres of a calcium phosphate (Ca-P) material with external diameters similar to those of the original glass particles. Heat treatment at 600°C for 4 h partially converted the Ca-P material to HA, as confirmed by X-ray diffraction, and also increased the strength of the hollow microspheres.     

Synthesis of aligned porous poly(ε-caprolactone) (PCL)/hydroxyapatite (HA) composite microspheres

We synthesized poly(ε-caprolactone) (PCL)/hydroxyapatite (HA) composite microspheres with an aligned porous structure and evaluated their potential applications in bone tissue engineering. A range of HA particles (0, 5, 10 and 20 wt.% in relation to the PCL polymer) were added to a PCL solution in order to improve the biocompatibility of the porous PCL/HA composite microspheres. All the synthesized microspheres showed that the HA particles were distributed well in the PCL matrix, while preserving their aligned porous structure. The average size of the PCL/HA composite microspheres increased from 62 ± 7 to 179 ± 95 μm with increasing HA content from 0 to 20 wt.%. The incorporation of the HA particles to the PCL polymer led to a considerable improvement in in vitro bioactivity, which was assessed by immersing the PCL/HA composite microspheres in simulated body fluid (SBF). A number of apatite crystals could be precipitated on the surface of the aligned porous PCL/HA composite microspheres after soaking in the SBF for 7 days.     

Fabrication and characterization of porous poly(lactic-<Emphasis Type="Italic">co</Emphasis>-glycolic acid) (PLGA) microspheres for use as a drug delivery system

In this work, Simvastatin (SIM) loaded porous poly(lactic-co-glycolic acid) (PLGA) microspheres were fabricated using the W/O/W1/W2 double emulsion and solvent evaporation method. The optimal conditions for fabricating porous PLGA microspheres were determined to be 20% distilled water (v/v), 10% PLGA (m/v), and a 4:1 ratio of internal polyvinyl alcohol (PVA) to dichloromethane (DCM). The pores size distribution of porous PLGA microspheres was varied from 0.01 to 40 μm, while their particle displayed a bimodal size distribution that had two diameter peaks at around 100 μm and 500 μm. The SIM encapsulation efficacy was found to be very high with a yield near 80% and the porous PLGA microspheres showed the excellent biocompatibility. In addition, the drug release profile was found to be significantly different from a temporal basis. Base on the combined results of this study, SIM loaded PLGA microspheres holds great promise for use in biomedical applications, especially in drug delivery system or tissue regeneration.     

Cancellous bone from porous T{i}6Al4V by multiple coating technique

A highly porous T{i}6Al4V with interconnected porous structure has been developed in our previous study. By using a so-called “Multiple coating” technique, the porous T{i}6Al4V can be tailored to resemble cancellous bone in terms of porous structure and mechanical properties. A thin layer of T{i}6Al4V slurry was coated on the struts of base porous T{i}6Al4V to improve the pore structure. After two additional coating, pore sizes ranged from 100 μm to 700 μm, and the porosity was decreased from ∼90% to ∼ 75%, while the compressive strength was increased from 10.3 ± 3.3 MPa to 59.4 ± 20.3 MPa and the Young's modulus increased from 0.8 ± 0.3 GPa to 1.8 ± 0.3 GPa. The pore size and porosity are similar to that of cancellous bone, meanwhile the compressive strength is higher than that of cancellous bone, and the Young's modulus is between that of cancellous bone and cortical bone. Porosity, pore size and mechanical properties can be controlled by the parameters in such multiple coating processes. Therefore the porous T{i}6Al4V with the characteristics of cancellous bone is expected to be a promising biomaterial for biomedical applications. Author to whom all correspondence should be addressed.     

Preparation of uniform Silicalite-1 microspheres with large secondary pore architecture using monodisperse porous polystyrene particles as template

A new method was developed for preparation of silicalite-1 microspheres with uniform diameter about 2.5 μm using monodispersed micron-sized poly- styrene-co-divinylbenzene (PSD) porous particles as template. In this method, the PSD particles were impregnated first with tetraethoxysilane (TEOS) and then with tetrapropylammonium hydroxide (TPAOH). After crystallization and calcination, the silicalite-1 microspheres, possessing a regularly spherical morphology similar to the original silicalite-1, were successfully synthesized. More importantly, the silicalite-1 microspheres have large secondary pores in the range of 40 to 120 nm and high pore volume up to 0.7 cm³g^− 1. All these would facilitate the applications of microspheres in catalysis avoiding diffusion limitation.     

Hydrothermal preparation of tailored hydroxyapatite

Hydrothermal vapor treatment method was applied for preparation of ceramic biomaterials. Hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂; HA) ceramics prepared by sintering with random crystal surface have already been used as bone-repairing materials which can directly bond to natural bones. If materials of HA could have the tailored specific crystal surface, they should have the advantage of adsorptive activity and osteoconductivity in comparison with the sintered HA. In the present study, porous HA sheets of about 50 μm to 1 mm in thickness and porous HA granules of about 50 μm to 1 mm in size with tailored crystal surface were prepared by the hydrothermal vapor exposure method at temperatures below 200°C. Porous sheets and porous granules of HA with controlled crystal surface should be suitable for scaffold of cultured bone, for bone graft material and for drug delivery system (DDS).     

Preparation of 3-D regenerated fibroin scaffolds with freeze drying method and freeze drying/foaming technique

Although three-dimensional fibroin scaffolds have been prepared with freeze drying method, the porosity and pore sizes still can not satisfy the requirement of tissue engineering. In this article, fibroin porous scaffold with high porosity and > 100μm diameter interconnected pores was firstly prepared with freeze drying method through adjusting fibroin concentration. The morphology of different scaffolds lyophilized from different fibroin concentration was observed by SEM. A novel freeze drying improved method, freeze drying/foaming technique, was also devised to prepare fibroin scaffolds at different fibroin concentrations. Using the said method, the porosity and pore size of fibroin scaffolds prepared from 12% concentration were 85.8 ± 4% and 109 ± 20 μm respectively with yield strength up to 450 ± 6 KPa while the porosity and pore size of fibroin scaffolds prepared from 8% concentration were 96.9 ± 3.6% and 120 ± 30 μm respectively with yield strength up to 30 ± 1 KPa. The freeze drying/foaming technique produced scaffolds with a useful combination of high yield strength, interconnected pores, and pore sizes greater than 100 μm in diameter. Through adjusting fibroin concentration and thawing time, the porosity, pore sizes and mechanical properties could be controlled to satisfy the different requirements of tissue engineering. The results suggested that fibroin scaffolds prepared with the above methods could be formed for utility in biomaterial application.     

Sustained release optimized formulation of anastrozole-loaded chitosan microspheres: in vitro and in vivo evaluation

The purpose of this study was to develop sustained release formulation of anastrozole-loaded chitosan microspheres for treatment of breast cancer. Chitosan microspheres cross-linked with two different cross-linking agents viz, tripolyphosphate (TPP) and glutaraldehyde (GA) were prepared using single emulsion (w/o) method. A reverse phase HPLC method was developed and used for quantification of drug in microspheres and rat plasma. Influence of cross-linking agents on the properties of chitosan microspheres was extensively investigated. Formulations were characterized for encapsulation efficiency (EE), compatibility of drug with excipients, particle size, surface morphology, swelling capacity, erosion and drug release profile in phosphate buffer pH 7.4. EE varied from 30.4 ± 1.2 to 69.2 ± 3.2% and mean particle size distribution ranged from 72.5 ± 0.5 to 157.9 ± 1.5 μm. SEM analysis revealed smooth and spherical nature of microspheres. TPP microspheres exhibited higher swelling capacity, percentage erosion and drug release compared to GA microspheres. Release of anastrozole (ANS) was rapid up to 4 h followed by slow release status. FTIR analysis revealed no chemical interaction between drug and polymer. DSC analysis indicated ANS trapped in the microspheres existed in amorphous form in polymer matrix. The highest correlation coefficients (R ²) were obtained for Higuchi model, suggesting a diffusion controlled mechanism. There was significant difference in the pharmacokinetic parameters (AUC_0−∞, Kel and t_1/2) when ANS was formulated in the form of microspheres compared to pure drug. This may be attributed to slow release rate of ANS from chitosan microspheres and was detectable in rat plasma up to 48 h which correlates well with the in vitro release data.     

Effect of drying conditions during synthesis on the properties of hydroxyapatite powders

The effect of the drying conditions during the hydroxyapatite (HAp) powder synthesis on the size and microstructure was studied. The starting materials were agitated in water, dried at 60–150 °C, and heat-treated at 720 °C. The heat-treated HAp powders were crystalline, and their particle sizes decreased with an increase in the drying time, but were independent of the drying temperature. For a 3 day drying period, it was 1.61 μm, and 0.55 μm for 21 days. The surface zeta potential of the HAp powder with a long drying period was more negatively charged than that from the short drying period. The average primary particle sizes of the HAp powders seem to be almost equivalent, and so the drying period may be related to the dispersibility of the primary particles, and may lead to a small HAp particle size.     

Fluorinated polyimide–silica films with low permittivity and low dielectric loss

A sol–gel process was used to prepare polyimide–silica hybrid films from the fluorinated polyimide precursors (6FDA-ODA) and tetraethylorthosilicate (TEOS) in N,N-dimethyl acetamide. The hybrid film was then treated with hydrofluoric acid to remove the dispersed silica particles, leaving inside the film pores with diameters ranged from 80 nm to 1 μm, which depended on the size of the silica particles. The chemical structures and morphology of the hybrid and porous films were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy, respectively. The synthesized porous fluorinated polyimide films show low relative dielectric permittivity of 1.9, rendering them promising for microelectronic packaging materials.     

Fabrication and characterization of porous hydroxyapatite microspheres by spray-drying method

In the present paper, porous hydroxyapatite (HA) microspheres were fabricated using gelatin as a pore-forming agent by spray-drying method. The mean particle size of the microspheres is about 7 μm and the surface area is about 53.4 m²/g. The experimental results showed that the porosity of the prepared microspheres is higher and the pores are more interconnected compared with the microspheres obtained without any additives.     

Evaluation of hydroxyapatite microspheres made from a borate glass to separate protein mixtures

A hydroxyapatite (HA, Ca₁₀(PO₄)₆(OH)₂), transformed from a calcium-containing borate glass, has been investigated for its protein adsorption and chromatographic characteristics. Microspheres of the borate glass were transformed into HA by reacting them with a 0.25 M phosphate (K₂HPO₄) solution for 24 h at 37 °C (pH 9.0). The HA microspheres with a diameter of 45–90 μm were hand packed into a steel column (4.6 mm × 80 mm) and used to separate a binary protein mixture of bovine serum albumin (BSA) and lysozyme. HA microspheres, with a diameter <45 μm, were used for separating a protein mixture of BSA, myoglobin, and lysozyme. These microspheres had a diameter that was 20–30 times larger than commercial HA column packing spherical particles, 2–3 μm, but these microspheres had a six times larger surface area and a more uniform spherical shape. These advantages compensated for their larger size and the separation results were comparable to those commercially available HA columns in the separation of the proteins studied. These unique HA microspheres, made from microspheres of a borate glass, are considered to be useful as packing materials for protein separation in chromatography.     

Indirect rapid prototyping of biphasic calcium phosphate scaffolds as bone substitutes: influence of phase composition,macroporosity and pore geometry on mechanical properties

While various materials have been developed for bone substitute and bone tissue engineering applications over the last decades, processing techniques meeting the high demands of scaffold shaping are still under development. Individually adapted and mechanically optimised scaffolds can be derived from calcium phosphate (CaP-) ceramics via rapid prototyping (RP). In this study, porous ceramic scaffolds with a periodic pattern of interconnecting pores were prepared from hydroxyapatite, β-tricalcium phosphate and biphasic calcium phosphates using a negative-mould RP technique. Moulds predetermining various pore patterns (round and square cross section, perpendicular and 60° inclined orientation) were manufactured via a wax printer and subsequently impregnated with CaP-ceramic slurries. Different pore patterns resulted in macroporosity values ranging from about 26.0–71.9 vol% with pore diameters of approximately 340 μm. Compressive strength of the specimens (1.3–27.6 MPa) was found to be mainly influenced by the phase composition as well as the macroporosity, both exceeding the influence of the pore geometry. A maximum was found for scaffolds with 60 wt% hydroxyapatite and 26.0 vol% open porosity. It has been shown that wax ink-jet printing allows to process CaP-ceramic into scaffolds with highly defined geometry, exhibiting strength values that can be adjusted by phase composition and pore geometry. This strength level is within and above the range of human cancellous bone. Therefore, this technique is well suited to manufacture scaffolds for bone tissue engineering.     

Abnormal grain growth enhanced densification of liquid phase-sintered WC–Co in support of the pore filling theory

This study examines the effect of grain growth on densification during liquid phase sintering of compacts with faceted grains. Two kinds of WC powders with different sizes were used to produce WC–Co alloys. Large pores of ~5 μm size were generated in 95WC–5Co (wt%) using spherical Co particles of the same size. The overall sintering behavior was observed by measuring grain growth and densification as a function of sintering time at a sintering temperature of 1350 °C. When the WC powder was fine (0.4 μm), large pores disappeared upon filling of pores by liquid with the formation of abnormal grains. On the contrary, when the WC powder was large (4.2 μm), grain growth is not observed, and large pores remained intact even after a long period of sintering (24 h). These observations confirm that densification during final stage liquid phase sintering occurs via filling of pores by liquid as a result of grain growth. This finding is consistent with the model of densification predicted by the pore filling theory.     

Direct visualization and quantification of bone growth into porous titanium implants using micro computed tomography

The utility of porous metals for the integration of orthopaedic implants with host bone has been well established. Quantification of the tissue response to cementless implants is laborious and time consuming process requiring tissue processing, embedding, sectioning, polishing, imaging and image analysis. Micro-computed tomography (μCT) is a promising three dimensional (3D) imaging technique to quantify the tissue response to porous metals. However, the suitability and effectiveness of μCT for the quantification of bone ingrowth remains unknown. The purpose of this study was to evaluate and compare bone growth within porous titanium implants using both μCT and traditional hard-tissue histology techniques. Cylindrical implants were implanted in the distal femora and proximal tibiae of a rabbit. After 6 weeks, bone ingrowth was quantified and compared by μCT, light microscopy and backscattered electron microscopy. Quantification of bone volume and implant porosity as determined by μCT compared well with data obtained by traditional histology techniques. Analysis of the 3D dataset showed that bone was present in the pores connected with openings larger 9.4 μm. For pore openings greater than 28.2 μm, the size of the interconnection had little impact on the bone density within the porosity for the titanium foams.     

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.     

Fabrication of chitosan/poly(ε-caprolactone) composite hydrogels for tissue engineering applications

The aim of this study was to fabricate three-dimensional (3D) porous chitosan/poly(ε-caprolactone) (PCL) hydrogels with improved mechanical properties for tissue engineering applications. A modified emulsion lyophilisation technique was developed to produce 3D chitosan/PCL hydrogels. The addition of 25 and 50 wt% of PCL into chitosan substantially enhanced the compressive strength of composite hydrogel 160 and 290%, respectively, compared to pure chitosan hydrogel. The result of ATR–FTIR imaging corroborated that PCL and chitosan were well mixed and physically co-existed in the composite structures. The composite hydrogels were constructed of homogenous structure with average pore size of 59.7 ± 14 μm and finer pores with average size of 4.4 ± 2 μm on the wall of these larger pores. The SEM and confocal laser scanning microscopy images confirmed that fibroblast cells were attached and proliferated on the 3D structure of these composite hydrogels. The composite hydrogels acquired in this study possessed homogeneous porous structure with improved mechanical strength and integrity. They may have a high potential for the production of 3D hydrogels for tissue engineering applications.     

Inflammatory reaction in rats muscle after implantation of biphasic calcium phosphate micro particles

Several studies have shown that macro micro porous bioceramics ectopically implanted promote bone tissue formation. This study aims at investigating the inflammatory response towards biphasic calcium phosphate (BCP) ceramic micro particles. BCP composed of hydroxyapatite (HA) and beta-tricalcium phosphate, HA/β -TCP ratio of 50/50, were prepared by sintering at 1200°C for 5 h. After crushing, 3 fractions of BCP micro particles < 20, 40–80 and 80–200 μ m were sieved. The micro particles were carefully characterized by using X-ray diffraction (XRD), scanning electron microscopy (SEM) and laser scattering. The inflammatory reactions induced by BCP micro particles implanted in quadriceps muscles of rats for 7, 14 and 21 days were studied by histology (n = 8/group). A fibrous tissue encapsulation of the BCP micro particles implanted in muscle tissue was observed and fibrosis was similar for the 3 groups of micro particles. The comparison of the cellular response indicated that the total number of cells was significantly higher for BCP < 20 μ m than for 40–80 and 80–200 μ m (p < 0.0001). The number of macrophages was relatively higher for the smallest than for the intermediate and largest fractions (p < 0.0001). The relative percentage of giant cells was higher for the intermediate and largest size of particles than for the smallest. The number of lymphocytes was comparable for the 3 fractions and after the 3 delays. Therefore, the BCP micro particles < 20 μ m initiated an inflammatory response which might play an important role in osteogenesis.