Fabrication of three-dimensional interconnected nanoporous hydroxyapatite by freeze-thaw process of amorphous calcium phosphate-poly(vinyl alcohol) gel
Graphical abstract
Introduction
Hydroxyapatite (HAp; Ca10(PO4)6(OH)2; stoichiometric Ca/P molar ratio of 1.67) is chemically similar to the inorganic component of bone matrix. Porous HAp is expected to be used as bone substitutes, scaffolds, separation filters, and catalyst supports. Many types of porous HAp are prepared by sintering HAp powder with a polymeric binder. A typical method for the preparation of macroporous HAp scaffolds is freeze-drying [1], [2], [3], [4], [5]. This method produces only macroporous HAp since the sizes of the pore-forming agents (ice crystals) and the HAp powder particles are limited to the micrometer-scale. Nanostructured HAp materials (e.g., nanoparticles and nanoporous materials) are attractive for tissue engineering owing to their improved biocompatibility and bioactivity [6], [7], [8], [9]. Nanoporous structures enhance cell adhesion, proliferation, and differentiation. Furthermore, interconnected pores are accessible to gases, liquids, and particulate suspensions and provide a means of cell distribution and migration. However, it is difficult to form porous HAp with both nanopores and interconnected pores by a conventional templating method.
Amorphous calcium phosphate (ACP) nanoparticles, which are the precursor of HAp, are obtained by a wet chemical precipitation method, which is generally used to prepare HAp nanoparticles owing to its simplicity. ACP nanoparticles are partially sintered during their calcination (HAp formation) process [10], [11], [12]. The sintering of ACP nanoparticles is possible to form a nanoporous HAp structure. Layrolle et al. [13] reported the preparation of microporous HAp with an average pore size of 0.2 μm by sintering an ACP green specimen at 1100 °C. Okada et al. [14], [15], [16] prepared nanoporous plates by the assembly of low-crystallinity HAp (ACP) nanoparticles using a casting method on an oil substrate.
We have developed an aggregation process starting from ACP nanoparticles to fabricate three-dimensional (3D) interconnected nanoporous HAp. We focused on the self-assembling ability of poly(vinyl alcohol) (PVA), which is a water-soluble polymer and has a good affinity to ACP, as the driving force for assembling ACP nanoparticles. PVA has an interesting property: PVA chains in aqueous solution are networked by a freeze-thaw process [17], [18]. Upon freezing, the water is frozen and PVA chains aggregate. In polymer-rich regions, intermolecular hydrogen bonds are formed, which act as crosslinking points, and thus a continuous network structure is formed. The freeze-thaw process of PVA aqueous solution is worthy of note for the fabrication of 3D interconnected nanoporous HAp from ACP nanoparticles. The network formation ability of PVA chains produces an ACP network structure owing to the interaction between ACP nanoparticles and PVA chains, resulting in a 3D interconnected HAp structure.
In this study, we fabricated 3D interconnected nanoporous HAp in bulk by assembling ACP nanoparticles with the control of the partially sintered structure. A network of ACP nanoparticles was generated by the freeze-thawing of an ACP-PVA gel. The effects of the preparation conditions of the freeze-thaw process and the ACP-PVA gel on the interconnectivity and the pore and skeleton sizes of the fabricated nanoporous HAp were investigated.
Section snippets
Chemicals
Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, 99.9%, Kanto Chemical Co., Inc., Japan), diammonium hydrogen phosphate ((NH4)2HPO4, 99.0%, Wako Pure Chemical Industries, Ltd., Japan), 10% ammonia solution (Wako), and acetone (99.5%, Wako) were used as received. Two types of PVA powder with different degrees of polymerization (DP; 300 and 1700) were supplied by Kuraray Co., Ltd., Japan, for which the degree of hydrolysis was 98.6 mol%. Deionized water was used in all experiments.
Preparation of ACP
ACP was prepared by
Fabrication of 3D interconnected nanoporous HAp
Fig. 1 shows XRD patterns of the products prepared under different conditions (samples S0, S3, and S6). Only peaks corresponding to HAp crystal were observed for all products (samples S0-S12), indicating that the products were single-phase HAp regardless of the preparation conditions. The effects of the addition of PVA aqueous solution and the freeze-thaw process for ACP on the morphology of the HAp were clarified. Fig. 2 shows SEM images of the products (samples S0-S4) obtained under different
Conclusions
We prepared 3D interconnected nanoporous HAp by assembling ACP nanoparticles. ACP nanoparticles were assembled by the addition of PVA aqueous solution and a subsequent freeze-thaw process. The interaction between ACP nanoparticles and PVA chains induced the homogeneous arrangement of ACP nanoparticles, and the freeze-thaw process induced the segregation and compaction of ACP nanoparticles. Furthermore, the addition of acetone enhanced homogeneous segregation and the formation of continuous
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