Akiko Obata

Electrospun microfiber meshes of silicon-doped vaterite/poly(lactic acid) hybrid for guided bone regeneration

Silicon-releasable microfiber meshes consisting of silicon-doped vaterite (SiV) particles and poly(lactic acid) (PLA) hybrids were prepared by electrospinning. Due to their flexibility and porosity they formed ideal membranes or scaffolds for guided bone regeneration. In addition, a trace amount of silicon species has been reported to stimulate osteogenic cells to mineralize and enhance bone formation. We propose a new method of preparation of silicon-releasing microfiber meshes by electrospinning. Their structure and hydroxyapatite (HA)-forming abilities in simulated body fluid were examined. In addition, we studied their stimulatory effects on osteoblast-like cells in vitro and bone-forming ability in vivo, with a special emphasis on their ability to release silicon. The meshes consisted of a hybrid of carboxy groups in PLA and amino groups in siloxane, derived from aminopropyltriethoxysilane or calcium ions on the SiV surface. This hybrid exhibited an enhanced ability to form HA. The meshes coated with HA released 0.2-0.7 mg l(-1) silicon species into the culture medium over 7 days. Enhanced proliferation of osteoblast-like cells was observed using the meshes and new bone formed on the meshes when implanted into the calvaria of rabbits. These meshes, therefore, provide an excellent substrate for bone regeneration and exhibit enhanced bone-forming ability under both in vitro and in vivo conditions.

Enhanced in vitro cell activity on silicon-doped vaterite/poly(lactic acid) composites

A biodegradable composite with silicon-species releasability was prepared using poly(l-lactic acid) (PLLA) and silicon-doped vaterite (SiV) particles. SiV with particle diameters of approximately 1 mum was prepared using aminopropyltriethoxysilane (APTES) as the silicon species by a carbonation process and then mixed with PLLA in methylene chloride according to a SiV to PLLA weight ratio of 1:2, resulting in the preparation of composite slurry. A composite film was prepared by dipping a cover glass in the slurry. The composite films were incubated in a culture medium for 7 days and the silicon concentration of the medium was measured to estimate the species releasability of the composites. A trace amount of silicon species was continuously released from the composites for 7 days, the amount depending on the content of APTES in SiV. On the composite releasing silicon species, mouse osteoblast-like cells (MC3T3-E1 cells) were significantly stimulated to proliferate and differentiate in comparison with those on a composite containing no silicon species. The proliferation of the cells on the composites releasing larger amounts of silicon species (0.51mgl(-1)day(-1)) was higher than that on the composites releasing smaller amount of the species (0.21mgl(-1)day(-1)). The silicon species in the composites were effective in enhancing the cellular functions. The composites were expected to be useful as a scaffold material for bone tissue engineering.

Effects of niobium ions released from calcium phosphate invert glasses containing Nb2O5 on osteoblast-like cell functions.

The effects of niobium ions released from 60CaO-30P(2)O(5)-(10-x)Na(2)O-xNb(2)O(5) (mol %, x = 0-10) glasses on MC3T3-E1 cell functions were evaluated by culture tests with two systems; cell culture on glass plates, or in culture media containing glass extracts. Alkaline phosphatase (ALP) activity in the cells cultured on the glass plates containing 3 and 5 mol % of Nb(2)O(5) was significantly higher than that on the Nb(2)O(5)-free glass, although proliferation was not enhanced on all glasses containing Nb(2)O(5). Cells cultured in the medium containing 3 × 10(-7) M niobium ions showed the highest ALP activity in comparison with other Nb-containing media or normal medium, regardless of the presence of osteogenic factors (ascorbic acid, β-glycerophosphate and dexamethasone) in the media. Calcium deposition by the cells cultured in the medium containing 3 × 10(-7) M niobium ions was twice as high as those cultured in medium containing no niobium ions. The effects of niobium ions were thought to depend on ion concentration, and to enhance differentiation and mineralization of osteogenic cells rather than their initial adhesion or proliferation.

Stimulation of human mesenchymal stem cells and osteoblasts activities in vitro on silicon-releasable scaffolds.

Cellular activities of human osteoblasts (HOBs) and mesenchymal stem cells (MSCs) on a silicon-releasable scaffold, siloxane-doped poly(lactic acid) and vaterite composite coated with hydroxycarbonate apatite (SPV-H), were estimated using a medium with or without organic factors, such as dexamethasone (Dex) and beta-glycerophosphate (beta-GP), for inducing mineralization or differentiation. As a control, a composite film containing no silicon (denoted by PV-H) was prepared using poly(lactic acid) and vaterite. HOBs cultured on SPV-H formed some agglomerates, bone nodules, after a 21-day culture in a medium without the organic factors, whereas no agglomerate was observed on PV-H. Laser Raman spectra implied that calcium phosphate precipitated in HOBs on the SPV-H. The silicon species in SPV-H stimulated HOBs to mineralization. The culture tests using MSCs show that the level of alkaline phosphatase (ALP) activity in the cells cultured on SPV-H increased during the 21-day culture in a medium without Dex and beta-GP. The level was unchanged in MSCs cultured on PV-H. In the case of supplementing Dex and beta-GP to the medium, the level of ALP activity in MSCs cultured on SPV-H was higher than that on PV-H at all time points during the 21-day culture. The silicon species in SPV-H were regarded to induce and enhance the osteogenic differentiation of MSCs.

Preparation of poly(lactic acid)/siloxane/calcium carbonate composite membranes with antibacterial activity

A poly(lactic acid) (PLA)/siloxane/calcium carbonate composite membrane containing mercapto groups (PSC-SH) with antibacterial ability and excellent bone-forming ability was prepared using 3-mercaptopropyltrimethoxysilane for application in guided bone regeneration. Mercapto groups were reported to adsorb silver ions, which are well known to show antibacterial activity. Ionic silicon species were reported to stimulate the proliferation of osteoblasts. A PSC-SH membrane with a thickness of about 10 microm shows high flexibility. The PLA in PSC-SH was converted from the crystalline phase to the amorphous phase due to dispersion of condensed siloxane clusters. The amount of mercapto group on PSC-SH surface was estimated to be about 55 nmol mm(-2) by quantitative analysis using the thiol-disulfide exchange reaction. PSC-SH adsorbed silver ions on its surface after being soaked in 6 microM silver acetate aqueous solution for 1 min. The adsorbed silver ions were seen by X-ray photoelectron spectroscopy to form SAg and SO3Ag bonds. A trace amount of ionic silicon species was released from the membrane after soaking in culture medium. PSC-SH with adsorbed silver ions showed good antibacterial activity and cellular compatibility in tests conducted with Staphylococcus aureus and mouse osteoblast-like cells, respectively. Antibacterial activity is expected to occur during the implantation operation by the silver ions but not to remain in the body for a long period, as the ions were present on the surface of the membrane but not inside the structure. The membrane should be useful as a biodegradable material with antibacterial activity and bone-forming ability.

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.

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.

Aluminum silicate nanotube coating of siloxane-poly(lactic acid)-vaterite composite fibermats for bone regeneration

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(L-lactic acid) and siloxane-containing calcium carbonate particles by electrospinning. The fibermat showed enhanced bone formation in an in vivo test. In the present work, to improve the hydrophilicity of skeletal fibers in a fibermat, they were coated with nanotubular aluminum silicate crystals, which have a hydrophilic surface that has excellent affinity to body fluids and a high surface area advantageous for pronounced protein adsorption. The nanotubes were coated easily on the fiber surface using an electrophoretic method. In a conventional contact angle test, a drop of water rapidly penetrated into the nanotube-coated fibermat. The culture test using murine osteoblast-like cells (MC3T3-E1) showed that the cell attachment to the nanotube-coated fibermat at an early stage after seeding was enhanced in comparison with that to the noncoated one. This approach may provide a new method of improving the surface of polymer-based biomaterials.

Enhanced polarization of hydroxyapatite using the design concept of functionally graded materials with sodium potassium niobate

The present work aims to enhance the electrical activities of hydroxyapatite (HA) without affecting its bioactivity through the development of functionally graded materials (FGM) using biocompatible sodium potassium niobate (NKN) piezoelectrics as an intermediary layer. The NKN layer was sandwiched between HA layers via buffer interlayers (abbreviated as HA–NKN–HA) and optimally processed using the spark plasma sintering route. The dielectric and electrical properties were studied over a wide range of temperatures (25–500 °C) and frequencies (10−1 to 106 Hz). In vitro cellular response in terms of initial cell adhesion and proliferation on the FGM as well as the corresponding monoliths was assessed using human osteoblast-like SaOS2 cells. A reasonably good combination of dielectric and electrical properties, such as dielectric constant (38), AC conductivity [5.5 × 10−9 (ohm cm)−1], piezoelectric strain coefficient (4.2 pC N−1), electromechanical coupling coefficient (0.17), mechanical quality factor (81) and remnant polarization (0.06 μC cm−2) in reference to natural bone has been achieved with the developed FGM. The mechanism of conduction remains similar in the FGM to that in pure HA. Impedance analyses suggest the occurrence of two polarization processes in HA and NKN monoliths, whereas more than two polarization processes are observed in the FGM. The significant increase in cell proliferation with culture duration of up to 5 days suggests that the developed FGM favor the cell growth and proliferation. In addition, the present study also establishes the superior cytocompatibility of the perovskite NKN phase. The developed FGM can be a potential substitute for electro-active orthopedic prosthetic implant applications.

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.