In our earlier work, a flexible fibermat consisting of a biodegradable composite with soluble silicate species, which has been reported to enhance bone formation, was prepared successfully using poly(
Many types of scaffolds have been investigated for use in bone regeneration [
The present authors have developed a novel composite consisting of poly(
Three-dimensional scaffolds are common in biomaterials. Electrospinning is a process for forming fibermats with a high porosity and a high flexibility [
A naturally occurring hydrated aluminum silicate, (HO)3Al2O3Si(OH), which is called imogolite, has a nanotubular structure of ~2.2 nm external and ~1.0 nm internal diameters [
In the present work, SiPVH fibermats were coated with INTs to improve their hydrophilicity to enhance cellular compatibility at the early stage after implantation.
SiV particles of 1
SiPVH was prepared by kneading PLLA (Mitsui Chemicals, LACIA; molecular weight: Mw = ~140 kDa) with SiV particles at 200°C for 10 min in a weight ratio of PLLA/SiV = 7/3.
SiPVH was dissolved in chloroform for electrospinning to prepare 10 wt% PLLA solution. The fibermats were prepared by electrospinning at a voltage of 20 kV using the conditions and compositions previously found to be optimal for preparing microfibers of ~10
INTs were synthesized, essentially following a method described by Suzuki et al. [
Sodium hydroxide (NaOH; Wako Pure Chemicals) aqueous solution of 1 M was slowly added to the above-described sodium silicate/aluminum chloride solution at a rate of 2 mL·min−1 until the pH of the solution reached 6.8. The sample was separated by centrifugation and the obtained precipitates were rinsed in DW with stirring. After the centrifugation-rinsing process was repeated three times, the resulting aluminum silicate precursors were dispersed again in 12 L of DW. Subsequently, the solution was acidified again by the addition of 12 mL of hydrochloric acid (HCl; Wako Pure Chemicals) with a concentration of 5 M and then heated at 95°C for 1 or 4 d for INT formation. Consequently, the resulting INTs were dispersed in an aqueous solution with a concentration of 0.087 wt%. The INTs were observed by atomic force microscopy (AFM) in the tapping mode. Figure
AFM images of products after heating at 95°C for (a) 1 d and (b) 4 d.
The INT coating of SiPVH fibermats was achieved using an electrophoretic deposition (EPD) method.
Each fibermat was cut into 15 mm × 15 mm × 0.2 mm dimensions with scissors and then placed on 15 mm × 60 mm × 0.25 mm aluminum foil. One hundred microliters of ethanol were dropped on the foil to fix each fibermat on the cathode electrode.
The objective of the present work is to prepare a hydrophilic coating with an extremely thin layer of INTs on skeletal fibers. Our preliminary work showed the EPD conditions for the preparation. The point of zero charge (PZC) in an INT-containing solution has been reported to be pH 6.0 [
The samples were coated with amorphous osmium using plasma chemical vapor deposition (CVD) and then observed morphologically using a field emission scanning electron microscopy (SEM) system (JSM-6301F, JEOL, Japan), with an energy-dispersive spectrometer (EDS).
The static contact angle of water on the fibermats was measured using a CCD camera and SImage mini ver. 5.01 software. The average contact angle was determined from the measurements at ten random points per sample.
The fibermats were cut into disk-shaped pieces of 15 mm in diameter with scissors for cell culture tests, and the resulting samples were sterilized using ethylene oxide gas. Murine osteoblast-like cells (MC3T3-E1 cells) were seeded onto the samples in 24-well plates at a density of 50,000 or 80,000 cells
The number of murine osteoblast-like cells (MC3T3-E1 cells) was evaluated using Cell Counting Kit-8 (Dojindo, Japan). The cells were rinsed with
The cells cultured on the samples for 3 h were fixed in 2.5% glutaraldehyde for 40 min at 4°C, dehydrated through a series of increasing concentrations of ethanol, and finally dried with hexamethyldisilazane. The dried samples were observed by SEM after they were coated with amorphous osmium.
To evaluate the initial adhesion of the cells on the fibers, they were cultured on the samples for 3 h and then fixed in phosphate buffer solution (PBS) containing 4% paraformaldehyde for 30 min at 4°C. The cells were then treated with PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X for 25 min at 4°C. Finally, they were fluorescence-stained with 50
In our earlier work, PLLA fibermats consisting of skeletal fibers of ~10
SEM images and EDS spectra of (a) noncoated and (b, c) INT-coated SiPVH fibermats. (b) INT220-coated and (c) INT570-coated SiPVH fibermats. Center: magnified views; right: EDS spectra.
There are almost no significant differences in the appearance between the SiPVH fibermat coated with INT220 and the noncoated one, as shown in Figures
The EDS spectrum in Figure
To discuss the EPD effect, a dip-coating method was used: after the SiPVH fibermat was dipped in the INT-dispersed aqueous solution (0.087%), it was drawn up at a speed of ~1 mm
The noncoated fibermat exhibited hydrophobicity of a contact angle of 121°. In contrast, a drop of water immediately penetrated the fibermats coated with INTs, which indicated that the hydrophilicity of these fibermats was improved markedly after INT coating. This is because INTs are hydrophilic owing to the hydroxyl groups on their surfaces and have a high water absorbency, attributed to their nanotubular structure. In particular, the fibermat coated with INT220 showed excellent hydrophilicity as well as that coated with INT570, although it is difficult to morphologically distinguish them from the noncoated one. A very small number of INTs seem to be sufficient for improving the hydrophilicity of fibermats.
The cellular proliferation behavior of the INT-coated fibermats in comparison with that of the noncoated one was evaluated using MC3T3-E1 cells. Figure
Numbers of cells attached to noncoated and INT-coated fibermats after 3 days of culture.
The doubling time (DT) of the number of cells for discussing proliferation behavior is determined by
Figure
Numbers of cells attached to noncoated and INT-coated fibermats at 3 h after seeding.
Figures
SEM images of cells on (a) noncoated and (b, c) INT-coated SiPVH fibermats at 3 h after seeding. (b) INT220-coated and (c) INT570-coated SiPVH fibermats.
Fluorescence micrographs of cells on (a) noncoated and (b, c) INT-coated SiPVH fibermats at 3 h after seeding. (b) INT220-coated and (c) INT570-coated SiPVH fibermats.
Watari and coworkers reported that protein adsorption on nanofibrous scaffolds contributes to excellent cell adhesion and growth [
Furthermore, the INT nanostructure may also be related to cell adhesion. Hirata et al. reported that the nanostructure of multiwalled carbon nanotubes might be effective in cell entrapment [
In the present work, a trace amount of INT was coated on SiPVH fibers: no existence could be observed by SEM. The measurement of the INT amount on the fibers is not easy and the investigation is in progress. Although there might be almost no need to consider the influence of the Al3+ ion dissolved from the trace amount of INT on living body, we would have to discuss its behavior in future.
There are almost no reports on the solubility of aluminum silicates in living body or under its simulated environment, to the best of our knowledge. Although imogolite has been reported to dissolve slightly in alkaline solutions of pH > 10 [
Aluminum silicate INTs were coated successfully on an electrospun fibermat, consisting of poly(
The authors are indebted to Dr. Yoshio Ota, Dr. Xianfeng Yao, and Dr. Kazuya Oribe of Orthorebirth Co., Ltd., for their helpful discussions. This work was supported in part by Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP; Seeds actualization type: AS2311416F) from Japan Science and Technology Agency.