Syntheses of Single-Crystal Apatite Particles with Preferred Orientation to the a- and c-Axes as Models of Hard Tissue and Their Applications

Abstract

Hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂; HAp] is the mineral component of vertebrate hard tissues and an important raw material for biomaterials. The HAp crystal belongs to a hexagonal system and has two types of crystal plane with different atomic arrangements: positively charged calcium ions are mainly present in the a(b)-planes, while negatively charged phosphate ions and hydroxyl groups are mainly present in the c-planes. In vertebrate long bone surfaces, HAp crystals have a c-axis orientation, which leads to the development of the a(b)-plane; while in tooth enamel surfaces, they have an a(b)-axis orientation, which leads to the development of the c-plane. However, it is not clear why the orientations of long bone and tooth enamel are in different crystal planes. In order to clarify this question, we have synthesized single-crystal apatite particles with preferred orientation to the a- and c-axes as models for bone and teeth enamel. This review first describes the syntheses process of single-crystal apatite particles with preferred orientation to a(b)- and c-axes and then discusses specific protein adsorption to the crystal surface of the resulting plate- and fiber-shaped apatite particles with different surface charges. In addition, porous apatite-fiber scaffolds (AFSs) fabricated using the fiber-shaped apatite particles and their application to tissue engineering of bone are described on the basis of the three-dimensional cell culture of mesenchymal stem cells derived from rat bone marrow using the AFS settled into a radial-flow bioreactor.

1. INTRODUCTION

Hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂; HAp] is an inorganic compound that has a composition similar to that of human bone and teeth.^1–3) It is an osteoconductive material that can be used as a biomaterial for artificial bone, cement, and teeth roots.^4–9) HAp can also be used as an adsorbent for bio-related substances such as proteins and amino acids because it does not cause degeneration of bio-related substances during the adsorption process.^10–14)

HAp typically has a hexagonal crystal structure with a P6₃/m space group, and its crystal unit cell is characterized as a=b=0.942 nm, c=0.688 nm, α=β=90°, and γ=120°.^15,16) There are two major crystal planes, the a(b)-plane (ac and bc crystal faces) and c-plane (ab crystal face), on the HAp crystal. The a(b)-plane is rich in calcium ions and positively charged, while the c-plane is rich in phosphate and hydroxide ions and negatively charged.^2,10,11)

Depending on the hard tissue type, the HAp crystal exhibits different orientations, as illustrated in Fig. 1. For example, in a living bone, the c-axes of HAp crystallites 50 nm×25 nm×4 nm in size are parallel to the extending collagen fibers, resulting in exposure of their a(b)-planes on the bone surface.^17–22) On the other hand, in dental enamel, HAp crystallites 100 µm×50 nm×50 nm in size form enamel prisms extending from the dentino-enamel junction, resulting in their c-planes being preferentially parallel to the enamel surface.^22–26) Moreover, the a(b)-plane and c-plane exhibit anisotropic characteristics, such as resolvability, biocompatibility, and absorptive activity.^{2,10,11,27–29)} However, it is not clear why the orientations of living bone and dental enamel are in different crystal planes.

Fig. 1. Single-Crystal Apatite Particles with Preferred Orientation to a- and c-Axes as Models for Hard Tissue: Bone and Tooth Enamel

Well-controlled orientation is considered a key factor in the development of high-performance materials. Numerous studies on orientation-controlled HAp particles, coating layers, and dense ceramics have been reported.^26–47) c-Axis-oriented fiber-like and a(b)-axis-oriented plate-like HAp particles can be synthesized using the homogeneous precipitation method,^30–33) hydrothermal method,^34–36) and electrochemical method.^37,38)

This review first describes the syntheses process of single-crystal apatite particles with preferred orientation to the a(b)- and c-axes as models for tooth enamel and bones, respectively, together with characterization of the resulting apatite particles. Specific protein adsorption to the crystal surfaces of the resulting plate- and fiber-shaped apatite particles with different charges is discussed. In addition, we describe porous apatite-fiber scaffolds (AFSs) fabricated using the fiber-shaped apatite particles and their application to tissue engineering of bone on the basis of the three-dimensional (3D) cell culture of rat bone marrow cells (RBMCs) as a model of mesenchymal stem cells using the AFS settled into a radial-flow bioreactor (RFB).

2. SYNTHESES OF SINGLE-CRYSTAL APATITE PARTICLES WITH PREFERRED ORIENTATION TO THE a(b)- AND c-AXES AS MODELS FOR HARD TISSUES AND THEIR CHARACTERIZATION

Fiber-shaped HAp particles with preferred orientation to the c-axis (aHAp) can be easily synthesized by the homogeneous precipitation method using an aqueous solution containing 0.167 mol·dm⁻³ calcium nitrate tetrahydrate [Ca(NO₃)₂·4H₂O], 0.100 mol·dm⁻³ ammonium hydrogen phosphate [(NH₄)₂HPO₄], 0.50 mol·dm⁻³ urea [(NH₂)₂CO], and 0.1 mol·dm⁻³ nitric acid (HNO₃) as a starting solution.^30,31) The starting solution (750 cm³) is refluxed at 80°C for 24 h to form octacalcium phosphate [Ca₈H₂(PO₄)₆·5H₂O; OCP], and then the OCP is converted into HAp by further heating at 90°C for 72 h.

Plate-shaped HAp particles with preferred orientation to the a(b)-axis (cHAp) can be also synthesized from the air-liquid interface of the starting solution via the enzyme reaction of urea with urease.⁴⁷⁾ Briefly, the starting solution with a Ca/P ratio of 1.67 is first prepared by mixing 5.0 mmol·dm⁻³ calcium carbonate (CaCO₃), 3.0 mmol·dm⁻³ phosphoric acid (H₃PO₄), and 1.0 mol·dm⁻³ (NH₂)₂CO, with the pH adjusted to 3.0 by the addition of aqueous HNO₃. The starting solution is dispensed into glass Petri dishes immediately after adding 2.73 cm³ of urease (activity: 127 unit·mg⁻¹) solution (0.1 mass%), and the mixture is heated in an incubator at 50°C for 96 h. After reaction, the interface product is collected and processed by heat treatment at 600°C for 2 h in an electric furnace at a heating rate of 10°C·min⁻¹.

Figure 2 shows X-ray diffraction (XRD) patterns of aHAp and cHAp particles, together with isotropically oriented HAp (iHAp) particles as a control. The iHAp particles can be obtained by heating commercially available HAp (HAp-100; Taihei Chemical Industrial Co., Ltd., Osaka, Japan) at 600°C for 2 h. All of the product particles are identified as HAp single phase. The diffraction pattern for iHAp in Fig. 2c has reflections that correspond well to standard HAp with an isotropic orientation. For aHAp particles (Fig. 2a), the diffraction intensities of the (100), (200), and (300) reflections, which correspond to the a-plane of HAp crystals, were more intense than those of iHAp. For the cHAp particles (Fig. 2b), the diffraction intensities of the (002) and (004) reflections, which correspond to the c-plane, were more intense. These results indicate that the three types HAp particle have different orientations: aHAp has a preferred orientation to the c-axis direction of the hexagonal crystal, which leads to the development of the a- or b-plane; cHAp has a preferred orientation to the a- or b-axis, which leads to the development of the c-plane; and iHAp has an isotropic orientation, so that the a(b)- and c-planes are randomly exposed.

Fig. 2. XRD Patterns of (a) aHAp, (b) cHAp, and (c) iHAp

Figure 3 shows particle morphology of the three types HAp particle observed using a scanning electron microscope (SEM). aHAp particles (Fig. 3a) have uniform fiber-like morphology with particle sizes ca. 20–60 µm long and 0.5–3.5 µm wide. cHAp particles (Fig. 3b) have hexagonal plate-like morphology with an average size of ca. 10 µm. iHAp particles (Fig. 3c) are agglomerated with irregular morphology. The specific surface areas (SSAs) of aHAp, cHAp, and iHAp are 4.28, 17.51, and 30.59 m²·g⁻¹, respectively.

Fig. 3. SEM Images of (a) aHAp, (b) cHAp, and (c) iHAp

Figure 4 shows bright-field images of aHAp, cHAp, and iHAp particles observed using a transmission electron microscope (TEM), together with their selected area electron diffraction (SAED) patterns. The low-magnification TEM images of aHAp (Fig. 4a) and cHAp (Fig. 4b) particles reveal the same morphologies as the SEM observations. The iHAp (Fig. 4c) sample is composed of randomly shaped, fine particles that were ca. 20–60 nm wide and 30–150 nm long.

Fig. 4. Low-Magnification TEM Images and SAED Patterns of (a) aHAp, (b) cHAp, and (c) iHAp Particles

High-magnification HR-TEM images of (d) aHAp, (e) cHAp, and (f) iHAp.

The SAED observation of aHAp particles was performed at several areas along the long axis of one fiber-like particle; all of the measured SAED patterns have a similar geometry to that shown as an inset in Fig. 4a. This SAED pattern has clear spots arranged in rectangles, and the spots aligned along the long and short axes of the fiber can be indexed as (00l) and (h10) spots, respectively, from the [110] zone axis of the highly crystalline hexagonal structure. More detailed analysis was conducted using the high-resolution (HR)-TEM lattice image, and the results are shown in Fig. 4d. Two types of orthogonal linear lattice are evident, and the average lattice spacings were determined to be at 0.34 and 0.47 nm, which correspond to d₍₀₀₂₎ and d₍₁₁₀₎, respectively.

The SAED pattern of cHAp particles (inset in Fig. 4b) exhibits clear, hexagonally arranged spots. The lattice constants were determined to be a=b=0.94 nm and γ=120°, indicating that the observation direction is along the [001] zone axis of the HAp crystal. The HR-TEM lattice image of cHAp shown in Fig. 4e reveals a highly periodic arrangement of atoms. The lattice fringes have a spacing of 0.82 nm, which corresponds to d₍₁₀₀₎. However, no lattice fringes in other directions were observed.

The SAED pattern of iHAp particles (inset in Fig. 4c) shows Debye rings, which correspond to a polycrystal. Figure 4f shows the HR-TEM lattice image of iHAp, where the linear lattices are arranged in random directions. Compared with the flattened surfaces of aHAp and cHAp, the iHAp surface was very irregular.

Thus, the properties of these different HAp particles can be summarized as follows: aHAp particles are fiber-shaped HAp single crystals with a c-axis orientation parallel to the long axis; cHAp particles are plate-like HAp single crystals with a preferred orientation to the a(b)-axis; and iHAp particles are irregularly shaped HAp polycrystals with an isotropic orientation.

The zeta-potentials of the HAp particles were measured in phosphate-buffered saline (PBS(−)) solution. All of the HAp particles have negative zeta-potentials in the order of cHAp (−26.16±1.41 mV)>iHAp (−20.80±1.30 mV)>aHAp (−15.62±2.22 mV). The difference in the zeta-potentials can be explained by the different atomic arrangements on the two types of crystal plane. Phosphate ions form the bulk of the HAp structure, and hydroxide ions are all stacked above each other in hexagonal channels along the c-axis, where each hydroxide ion is coordinated to the three surrounding calcium ions that lie in the same a(b)-plane. The c-plane is perpendicular to the hexagonal channels in the c-axis, which contain hydroxide and phosphate ions, whereas the a(b)-plane has hydroxide ion channels lying parallel to the surface and the surface is terminated by phosphate ions.^{2,16,48–50)} Thus, the a(b)-plane is rich in positively charged calcium ions, while the c-plane is rich in negatively charged phosphate and hydroxide ions.

3. PROTEIN ADSORPTION ON SINGLE-CRYSTAL HAp PARTICLES WITH PREFERRED ORIENTATION TO THE a(b)- AND c-AXES

This section discusses the specific protein adsorption on the resulting single-crystal HAp particles with preferred orientation to the a(b)- and c-axes, i.e., cHAp and aHAp particles.⁵¹⁾ Two types of protein with different charges, bovine serum albumin (BSA; pI 4.7) and lysozyme (LSZ; pI 11.2), were selected for this study. BSA at concentrations of 0 to 3000 ppm and LSZ solutions at 0 to 5000 ppm were prepared by dissolving the protein powders in PBS(−) solution. Each HAp particle sample was placed into a 5-cm³ polypropylene centrifugation tube and 2.5 cm³ of protein solution was added. The amounts of aHAp, cHAp, and iHAp particles used were 150, 50, and 50 mg, respectively. The protein adsorption experiment was performed at 25°C for 48 h. After adsorption, the slurry was centrifuged (2000 rpm, 2.5 min), and the amount of protein in the supernatant was measured with a UV spectrophotometer using the UV adsorption band at 280 nm.

aHAp, cHAp, and iHAp with c-axis-oriented HAp single crystals, a(b)-axis-oriented HAp single crystals, and isotropically oriented HAp polycrystals, respectively, were used as model particles to investigate the influence of different HAp crystal planes on the selective adsorption behaviors of acidic BSA and basic LSZ proteins. Figure 5 shows adsorption isotherms for (a) BSA and (b) LSZ on aHAp, cHAp, and iHAp, respectively. These profiles are representative of a typical Langmuir plot. The amount of adsorbed BSA (Fig. 5a) was normalized for the SSA (n_BSA) and was in the order of aHAp (2.10±0.23 mg·m⁻²)>iHAp (0.78±0.08 mg·m⁻²)>cHAp (0.45±0.10 mg·m⁻²). For LSZ (Fig. 5b), the order of the normalized adsorbed amount (n_LSZ) was the opposite: cHAp (0.75±0.07 mg·m⁻²)>iHAp (0.26±0.05 mg·m⁻²)≒aHAp (0.25±0.05 mg·m⁻²).

Fig. 5. Adsorption Isotherms for BSA (a) and LSZ (b) on aHAp, cHAp, and iHAp (n=5; Error Bars: Standard Deviation)

The adsorption coverage ratios of BSA (θ_BSA) and LSZ (θ_LSZ) adsorbed on HAp particle surfaces can be calculated according to Eqs. (1) and (2), where the theoretical values are estimated to be 2.52 and 2.02 mg·m⁻², respectively, assuming face-on-face adsorption of globular BSA (14×4-nm²) and LSZ (3.0×4.5-nm²) molecules, which have prolate ellipsoidal shapes.^52,53) For BSA adsorption, θ_BSA was 83.3% for aHAp, 31.0% for iHAp, and 17.9% for cHAp. For LSZ adsorption, θ_LSZ was 12.4% for aHAp, 12.9% for iHAp, and 37.1% for cHAp.

  

(1)

  

(2)

These results indicate that BSA has a high affinity for aHAp particles, whereas LSZ has a high affinity for cHAp particles. The reason for this is the different orientation of the two types of HAp particle. The a(b)- and c-planes of HAp crystals have different atomic arrangements, which result in different surface zeta-potentials. Therefore, aHAp particles with developed a(b)-planes easily adsorb negatively charged acidic proteins by the electrostatic force of attraction, whereas cHAp particles with developed c-planes easily adsorb positively charged basic proteins.

4. CREATION OF A NOVEL AFS USING SINGLE-CRYSTAL HAp PARTICLES WITH PREFERRED ORIENTATION TO THE c-AXIS AND ITS APPLICATION TO TISSUE ENGINEERING

Tissue engineering is an important technology that encourages regeneration of defective tissue utilizing scaffolds, cells, and growth factors. It is still problematic to repair miscellaneous tissue defects in clinical practice because only limited suitable autologous tissue is available, and allografts still put recipients at potential risk of infections. Therefore, regeneration of the desired tissue via the tissue engineering route is an important alternative. In bone tissue engineering, porous calcium-phosphate ceramics are generally used as scaffolds.

We succeeded in developing a scaffold with interconnected macropores, which enables 3D cell culture using fiber-like apatite (aHAp) and carbon beads.^54,55 The AFS has excellent biocompatibility in vitro^54,56) and in vivo.^57,58) Using the AFS, we attempted to create tissue-engineered bone^54,55,59,60) as well as tissue-engineered liver.^61–63)

Figure 6 shows the fabrication process of the AFS, together with SEM images of its microstructure. First, the aHAp particles are mixed with spherical carbon beads with a diameter of ca. 150 µm in mixed solvent ethanol : water=1 : 1 (v/v). As previously reported,⁵⁴⁾ the carbon beads are added to aHAp in the HAp/carbon ratio of 1 : 20 (w/w). The resulting compacts are fired at 1300°C for 5 h in a steam atmosphere to fabricate the AFS; we refer to the scaffold derived from HAp : carbon=1 : 20 (w/w) as “AFS2000.”

Fig. 6. Fabrication Process of AFS Derived from aHAp Particles, together with the SEM Images of the Microstructure

The XRD pattern revealed that single-phase HAp was present in AFS2000. SEM observations showed that the AFS2000 scaffold is composed of large pores 100–300 µm in diameter and smaller pores formed by intertwining of individual fibers and that the pores are interconnected in the structure. As compared with the microstructure of the carbon-free derived scaffold (AFS0), the pore sizes of the scaffolds AFS1000 and AFS2000 became significantly enlarged as the amount of carbon beads added increased.

Recently, we have carried out 3D cell culture of mesenchymal stem cells derived from RBMCs using the AFS settled into an RFB to create tissue-engineered bone with a 3D structure.^59,60) Figure 7 shows an overview of the system used. First, 3D cell culture using the RFB was performed for 0, 1, or 2 weeks using normal medium α-MEM with 10% FBS [α-MEM(+)], and then for 0, 1, or 2 weeks using α-MEM(+) containing dexamethasone, ascorbic acid, and β-sodium glycerophosphate. Tissue-engineered bones were fabricated under 12 different 3D cell culture conditions, including flow rates of circulating medium (0.4 to 16.5 cm³·min⁻¹). The level of differentiation of osteoblasts in tissue-engineered bone was examined by determining the content of two types of differentiation marker into osteoblasts, alkaline phosphatase for the initial/middle stage and osteocalcin for the late stage. The tissue-engineered bone with various differentiation levels into osteoblasts could be reconstructed through 3D cell cultures of RBMCs using the AFS settled into the RFB. In addition, we clarified that the optimized flow rate (6.3 cm³·min⁻¹) of circulating medium promoted the differentiation into osteoblasts. Therefore, the present AFS and RFB system is effective for the creation of tissue-engineered bone with a 3D structure.

Fig. 7. Reconstraction of Tissue-Engineered Bone with 3D Structure through 3D-Cell culture of RBMC Cells Using the AFS Settled into the RFB

5. CONCLUSION

HAp is the mineral component of vertebrate hard tissues and an important raw material in biomaterials. In vertebrate long bone surfaces, HAp crystals have a c-axis orientation, which leads to the development of the a(b)-plane; while in tooth enamel surfaces, they have an a(b)-axis orientation, which leads to the development of the c-plane. However, it is not clear why the orientations of long bone and tooth enamel are in different crystal planes. To clarify this question, we successfully synthesized single-crystal apatite particles with preferred orientation to the a- and c-axes as models for bone and teeth enamel.

The selective adsorption behaviors of negatively charged BSA and positively charged LSZ to the surfaces of the three types of HAp particle (aHAp, cHAp, iHAp) were clarified in PBS(−) solution at pH 7.3 and 25°C for 48 h. The amount of BSA adsorbed, normalized for SSA, was in the order of aHAp>iHAp>cHAp; however, the order for LSZ was reversed to cHAp>iHAp≒aHAp. These results indicate that the a(b)- and c-planes of HAp crystal have high specificity for the adsorption of acidic or basic proteins.

In addition, we described porous AFSs fabricated using fiber-shaped apatite particles and their application to tissue engineering of bone on the basis of the 3D cell culture of mesenchymal stem cells derived from RBMCs using the AFS settled into the RFB. The present AFS and RFB system is effective for the creation of tissue-engineered bone with a 3D structure.

Finally, we hope that the findings from the present study will help expand and deepen our knowledge of HAp and provide new insights into future R&D of biomaterials.

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