Kie Fujikura

Cellular Migration to Electrospun Poly(Lactic Acid) Fibermats

Abstract Nonwoven fabrics prepared via an electrospinning method, so-called electrospun fibermats, are expected to be promising scaffold materials for bone tissue engineering. In the present work, poly(L-lactic acid) (PLLA) fibermats, consisting of fibers with diameters ranging from 1 to 10 μm, were prepared by electrospinning. Mouse osteoblast-like cells (MC3T3-E1) were seeded on the fibermats with various fiber diameters (10, 5 and 2 μm; they are denoted by samples A, B and C, respectively) and cultured in two different directions in order to compare the migration behaviours into the scaffold of the normal condition and the anti-gravity condition. The cells in/on the fibermats were observed by laser confocal microscopy to estimate the cellular migration ability into them. When the MC3T3-E1 cells were cultured in the normal direction, the thickness of their layer increased to approx. 90 μm in the sample A, consisting of 10-μm fibers after 13 days of culture, while that in the sample C, consisting of 2-μm fibers, did not increase. When the MC3T3-E1 cells were cultured in the anti-gravity condition, the thickness of the cell layer in the sample A increased to approx. 60 μm. These results mean that the MC3T3-E1 cells migrated into the inside of sample A in either the normal direction or the anti-gravity one. The cellular proliferation showed no significant difference among the fibermats with three different fiber diameters; MC3T3-E1 cells on the fibermat with 2 μm fiber diameter grew two-dimensionally, while they grew three-dimensionally in the fibermat with 10 μm fiber diameter.

Effective encapsulation of laccase in an aluminium silicate nanotube hydrogel

Aluminium silicate nanotubes (ASNT), with a length to width ratio of four, were synthesized from aluminium chloride and sodium silicate, and the ASNT hydrogel was easily prepared by adjusting the pH to 7. The hydrogel nanotube concentration was 1.5 wt%. Laccase, a type of oxidase, was encapsulated during ASNT hydrogel formation. This encapsulation method will have fewer negative effects on the relatively unstable enzyme because of the milder conditions used, which are different from sol–gel silica formation by acidic catalysis using strong acids such as hydrochloric acid. The obtained hydrogels were fully characterized by various methods such as field-emission scanning electron microscopy and Fourier transform infrared spectroscopy. The ASNT-hydrogel encapsulated enzyme worked well; notably, laccase–ASNT hydrogels prepared from the shortest nanotubes exhibited a higher activity than the free laccase in solution because of an improvement in the substrate affinity of the encapsulated enzyme. Tryptophan fluorescence spectroscopy indicated that the highly ordered structure of laccase was not altered once bound to the nanotubes within the hydrogel. Notably, after nine repeated reactions, laccase encapsulated in the ASNT hydrogel retained its activity. The cycle performance of the encapsulated enzyme indicates that no enzyme was released from the ASNT hydrogel. In addition, the laccase–ASNT hydrogel was easily used to prepare transparent thin films on glass cover slips, while still maintaining the enzyme activity. Another oxidase, myoglobin, was also encapsulated in the same type of ASNT hydrogel. Although free myoglobin in solution demonstrated oxidation activity, the activity of the bound protein was remarkably decreased due to changes in its tertiary structure when inside the ASNT hydrogel.

Preparation of electrospun fiber mats using siloxane-containing vaterite and biodegradable polymer hybrids for bone regeneration.

An electrospun fiber mat using a new composite consisting of siloxane-containing vaterite (SiV) and poly(lactic-co-glycolic acid) (PLGA) (denoted by SiPLGVH) was prepared with the aim of applying it as a membrane for use in a guided bone regeneration (GBR) system. Another electrospun fiber mat using a previously described composite consisting of SiV and poly(L-lactic acid) (denoted by SiPVH) was also prepared as a comparative sample. SiPLG VH fiber mats showed superior results in terms of mechanical tensile properties and cellular behavior. Their elongation before failure was about eight times higher than that of SiPVH. The numbers of osteoblast-like cells that proliferated on the SiPLGVH fiber mats, regardless of the hydroxyapatite coating, were comparable to that of SiPVH. The cells spread more, two dimensionally, on the SiPLGVH fiber mats, since the pores between fibers were narrowed down because of swelling of the PLGA matrix during cell culture. This two-dimensional cellular proliferation quality on the SiPLGVH fiber mats is expected to be suitable for materials used in GBR, leading to control of infiltration of the soft tissue and great tissue integration with the surrounding tissue.

Preparation of electrospun poly(lactic acid)-based hybrids containing siloxane-doped vaterite particles for bone regeneration.

Abstract Siloxane/poly(L-lactic acid)/vaterite hybrid (SiPVH) fibremats constantly release calcium ions and ionic silicon species that have the potential to promote bone regeneration. In order to improve the mechanical properties of the SiPVH fibremats, the effect of various silixane-containing vaterite (denoted by SiV) content (10-60 wt%) on tensile properties was assessed. SiPVH fibremats with 30 wt% SiV content showed the highest tensile strength of 2.87 ± 0.39 MPa. Based on the energy-dissipation mechanism, failure initiated at the stress concentration points such as pores on the fibre surfaces or filler particles. In the case of the SiPVH fibremats with 20 and 30 wt% SiV, stress concentration occurred around the filler particles, where the applied energy was directly converted to small volume dilatation around the filler particles during failure of the fibres. This mechanism can be applied only when the material contains the polymer and the filler particles in specific ratios; 20 and 30 wt% filler content in the fibremat in this work. To coat the fibre surfaces with bone-like apatite SiPVH fibremats were soaked in modified simulated body fluid (1.5 SBF). Bone-like apatite formed on the surfaces of SiPVH fibremats with more than 30 wt% of SiV content in 1 day of soaking. These results reveal that the SiPVH fibremats containing 30 wt% SiV have suitable mechanical properties for bone filler materials.

Preparation and rheological characterization of imogolite hydrogels

Imogolite, one of the aluminium silicates, has a nanotube structure and has been known to form gel under alkaline condition. Imogolite nanotubes were synthesized in an acidic solution with various tube lengths by controlling the aging time from 1 d to 14 d. The length of the nanotubes grew from 100 nm to several µm as the aging time. Pure imogolite hydrogels were prepared by applying a salting-outmethod and centrifugation fromits dispersed solutions with various tube lengths and solution pH. Imogolite hydrogel can be classified as the physically cross-linked one; the structure of the gel network is considered to be the entanglements and hydrogen bonding among nanotubes. The theoretical water contents of the prepared hydrogels were calculated as ∼99.7% in average. Gelation percentage significantly increased as the length of imogolite nanotubes. Whereas hydrogel prepared from 4 d aging sample showed the highest storage modulus of ∼970 Pa, it was found that the hydrogel could be prepared in the pH range from 6 to 10. The gel strength reached the highest value of 1000 Pa when the gel was prepared from the imogolite dispersed solution of pH 8. It could be explained by the surface charge variation of the imogolite.

Aligned electrospun siloxane-doped vaterite/poly(l-lactide) composite fibremats: evaluation of their tensile strength and cell compatibility

Siloxane-doped vaterite (SiV)/poly(l-lactide) hybrid-composite (SiPVH) has been developed in our group as the bone repair material and successfully fabricated into a non-woven electrospun fibremat. The aim of this work is to prepare aligned electrospun SiPVH fibremats with varied SiV content and compare their tensile properties and cell compatibilities using mouse osteoblast-like cells. It was observed that the maximum stress exhibited some non-linear trend as a function of SiV content: the highest stress value was reached with 30 wt.% SiV and decreased significantly with more than 40 wt.% SiV. Cellular morphology and proliferation were taken under examination on both aligned and random electrospun SiPVH fibremats. The cells started to orient themselves only 3 h after seeding on the aligned fibremat and they continued to elongate along the fibres. The number of the cells cultured up to seven days on both random and aligned fibremats was well comparable; therefore the alignment did not show negative effect on the cellular proliferation.