Acta Biomaterialia 6 (2010) 12481257

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Electrospun microber meshes of silicon-doped vaterite/poly(lactic acid) hybrid for guided bone regeneration
Akiko Obata a,*, Toshiki Hotta a, Takashi Wakita a,b, Yoshio Ota c, Toshihiro Kasuga a
a

Graduate School of Engineering, Nagoya Institute of Technology, Gokiso Cho, Showa ku, Nagoya 466-8555, Japan Yamahachi Dental Manufacturing Co., 54-1 Ochigara, Nishiura Cho, Gamagori, Aichi 443-0105, Japan c Yabashi Industries Co. Ltd., 188-1 Akasaka Cho, Ogaki, Gifu 503-2213, Japan
b

a r t i c l e

i n f o

a b s t r a c t
Silicon-releasable microber meshes consisting of silicon-doped vaterite (SiV) particles and poly(lactic acid) (PLA) hybrids were prepared by electrospinning. Due to their exibility 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 microber meshes by electrospinning. Their structure and hydroxyapatite (HA)-forming abilities in simulated body uid 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 0.20.7 mg l1 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. 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Article history: Received 29 May 2009 Received in revised form 23 October 2009 Accepted 9 November 2009 Available online 11 November 2009 Keywords: Polylactic acid Calcium carbonate Silicon Membrane Osteoblast

1. Introduction Biodegradable polymer scaffolds have been developed for use in bone reconstruction, tissue engineering and drug delivery systems [1,2]. In addition, three-dimensional scaffolds are common in biomaterials. Electrospinning is a process that can generate polymer ber meshes with high porosity and exibility, both essential components of tissue engineering [35]. In general, the process of electrospinning is affected by both system parameters and process parameters. System parameters include polymer molecular weight (Mw) and polymer solution properties such as viscosity and conductivity. Process parameters, on the other hand, involve ow rate of the polymer solution, electric potential and the distance between the capillary and collector, among others [3]. It is possible to control the ber diameter and the thickness of ber mesh scaffolds by optimizing these parameters. To use electrospun ber meshes in bone tissue engineering applications, their various properties, including material, ber orientation, porosity and surface modication, must be considered. To optimize the mechanical and biomimetic properties of a ber mesh it is necessary to choose the appropriate materials, since the
* Corresponding author. Tel./fax: +81 52 735 7250. E-mail address: obata.akiko@nitech.ac.jp (A. Obata).

orientation and diameter of the bers in electrospun scaffolds can inuence cellular compatibility [68]. The ber diameter in electrospun scaffolds has, for example, been reported to inuence osteoblast proliferation and mineralization. Electrospun ber meshes have been fabricated to contain DNA and proteins, which enhance cellular compatibility and bone formation upon their release [811]. The DNA and proteins are released by degradation of polymer matrices such as poly(lactic acid) (PLA), poly(lactideco-glycolide) (PLGA) and poly(glycolide acid) (PGA). Despite their stimulatory effects on osteoblasts and bone marrow-derived stem cells, electrospun ber scaffolds consisting of polymer/ceramic composites have not been widely researched as biomaterials. Polymer and ceramic composites have been developed for bone reconstruction and showed good mechanical and bone-forming properties [1214]. We hypothesize that electrospun ber scaffolds consisting of PLA/calcium carbonate composites would also have good mechanical and bone-forming properties. Calcium carbonate, for example, is well known as a bioresorbable material, shows excellent bone-forming ability and has been used clinically as a bone ller [15,16]. In our previous research PLA/calcium carbonate composites (vaterite) showed excellent hydroxyapatite (HA)-forming ability, cellular compatibility and biocompatibility in in vitro and in vivo tests [17,18].

1742-7061/$ - see front matter 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.11.013

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Guided bone regeneration (GBR) is a useful method of bone reconstruction in vivo. GBR membranes require exibility to adapt to the shape of the bone defect and strength to maintain the space for bone formation and to connect with the soft tissues. An electrospun ber mesh is a good candidate for a GBR membrane due to the exibility inherent in the electrospinning process. Release of calcium and silicon species from an electrospun ber mesh is an additional attribute which promotes bone regeneration. Calcium species and a trace amount of silicon species have been reported to enhance bone formation by osteoblasts [1923]. Previously we developed silicon-doped vaterite (SiV) powders and their PLA composites and tested their cellular compatibility using mouse osteoblast-like cells [24]. A lm of the composites was prepared by dip coating the composite solution on a coverglass and drying at room temperature. The lm showed release of calcium and silicon into the culture medium through degradation of the PLA and SiV matrix. Cell proliferation and mineralization on the composite lm was enhanced compared with an undoped vaterite and PLA composite lm, which showed no silicon release. Our preliminary experiments demonstrated that a exible ber mesh consisting of PLA composites with a large amount of SiV could be successfully prepared by the electrospinning method, while a ber mesh consisting of a PLA composite with vaterite (undoped type) was brittle. We propose to further characterize the SiV and PLA hybrid (SiPVH) and compare its chemical structure with the undoped vaterite and PLA hybrid (PVH). To enhance cellular adhesion the composite ber meshes were coated with HA using simulated body uid (SBF). The cellular and tissue compatibilities of both prepared ber meshes were evaluated by in vitro tests using mouse osteoblast-like cells (MC3T3-E1 cells) and in vivo tests using rabbit calvaria. 2. Materials and methods 2.1. Preparation of SiV and vaterite powders Silicon-doped vaterite (SiV) powders were prepared by a carbonation process with methanol. A sample of 150 g Ca(OH)2 was mixed with 60 ml aminopropyltriethoxysilane (APTES) and 2000 ml methanol with the addition of CO2 gas for 75 min at a rate of 2000 ml min1. The resulting slurry was dried at 110 C, resulting in the SiV powder. The content of silicon in SiV was estimated to be approximately 3 wt.% by X-ray uorescence analysis (RIX3000, Rigaku, Japan) (50 kV, 60 mA). A calibration curve (Xray intensity vs. SiO2 content) was established by measuring limestone containing various amounts of SiO2. Vaterite powders were also prepared by the carbonation process without APTES. SiV and vaterite powders were characterized by X-ray diffractometry (XRD) (RAD-B, Rigaku, Japan) (CuKa, 40 kV, 20 mA). The specic surface areas of SiV and vaterite were measured according to a BET theory using nitrogen gas adsorption (NOVA3000, Quantachrome Corp., USA). The powders were deaerated at 150 C for 1 h before measurement. The structure of silicon in SiV was examined by 29Si magic angle spinning nuclear magnetic resonance (29Si MAS-NMR) (UNITY plus, Varian, USA), operating at pulses of 6.3 ls and at recycle delays of 15 s. The sample spinning speed was 3.0 kHz. Chemical shifts were measured relative to polydimethylsiloxane. 2.2. Preparation of microber meshes The SiPVH and PVH microber meshes were prepared by an electrospinning method. SiV or vaterite powder was mixed with PLA (Purasorb; molecular weight 260 kDa, PURAC, The Netherlands) at 200 C for 10 min with a kneader, resulting in the

formation of PLA composites containing 60 wt.% of the powder. The composites were dissolved in chloroform at 10 wt.% to prepare the solution for electrospinning. In our preliminary experiments, this ratio was found to be optimal for preparing microber meshes of approximately 10 lm diameter. The PLA microber mesh was also prepared by an electrospinning method. The PLA was dissolved in chloroform at 9 wt.% to prepare the solution for electrospinning. The prepared solutions were loaded into a syringe pump (FP-W-100, Melquest, Japan) with a syringe needle (22 gauge for SiPVH and PVH, 19 gauge for PLA), which was set at 0.05 ml min1. The distance between the needle and an aluminum collector was 150 mm. Electrospinning was carried out at 20 kV of impressed voltage and $100 lA of applied current (Power Supply HARb-40 P0.75, Matsusada Precision Inc., Japan) at room temperature and approximately 55% relative humidity. The SiV powders and resultant electrospun bers were coated with amorphous osmium using plasma chemical vapor deposition (CVD) equipment and then morphologically observed by eld emission scanning electron microscopy (SEM) (JSM-6301F, JEOL, Japan) (accelerating voltage 5 keV). Structural analyses of SiPVH were performed using 13C cross-polarization magic angle spinning nuclear magnetic resonance (13C CP/MAS-NMR) (UNITY plus, Varian, USA) and Fourier transform infrared (FTIR) spectroscopy (FTIR-460 Plus, JASCO, Japan). 13C CP/MAS-NMR spectra were taken with 4.7 ls and 8.0 s recycle delays. The sample spinning speed was 3.0 kHz. Chemical shifts were measured relative to adamantane. The molecular weights of PLA matrix in the prepared meshes were measured by gel permeation chromatography (GPC) (Shimadzu, Japan) using two columns (KF-604, Shodex, Japan). The meshes were dissolved in chloroform at a concentration of 10 mg PLA per 5 ml chloroform and the solutions ltered. The measurements were carried out at 40 C at a ow rate of 0.6 ml min1. Polystyrene standards (SM-105, Shodex, Japan) were used to establish a calibration curve. 2.3. Coating of the bers with HA The prepared microber mesh was immersed in 10 ml of an aqueous solution containing ion concentrations 1.5 times those of SBF (1.5 SBF) at 37 C for 24 h. The 1.5 SBF solution consisting of 3.75 mM Ca2+, 213.0 mM Na+, 2.25 mM Mg2+, 7.5 mM K+, 222.45 mM Cl, 6.3 mM HCO3 , 1.5 mM HPO4 2 , 0.75 mM SO4 2 , 50 mM (CH2OH)3CNH2 and 45.0 mM HCl was prepared using reagent grade NaCl, NaHCO3, KCl, K2PO43H2O, MgCl26H2O, HCl, CaCl2, NaSO4 and NH2C(CH2OH)3. After immersion in 1.5 SBF solution the mesh was washed with distilled water and then dried at room temperature. The dried samples were coated with amorphous osmium using plasma CVD equipment and observed by SEM. 2.4. Measurement of silicon release from a SiPVH microber mesh before and after HA coating The amount of silicon released from the SiPVH microber meshes before and after HA coating was evaluated by immersing them in 4 ml of a culture medium (alpha minimum essential medium, a-MEM) containing 10% fetal bovine serum (FBS) and incubated at 37 C in a humidied atmosphere of 95% air, 5% CO2 for 5 days. The sample coated with HA is denoted SiPVH/HA mesh. The size of the sample used was 10 10 mm. The culture medium was changed after either 1 or 3 days soaking, according to the process of the cell culture test. The sample was taken out of the culture medium and the medium ltered. The level of silicon species in the resulting medium was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (ICPS-500, Shimadzu, Ja-

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pan). A calibration curve was established using an aqueous solution of Si4+ ions at concentrations of 1, 10 and 20 ppm. 2.5. Cell culture tests The microber mesh used for the cell culture tests was sterilized using ethylene oxide gas. Mouse osteoblast-like cells (MC3T3-E1 cells) were seeded onto the prepared mesh (SiPVH/ HA or PLA mesh) in 24-well plates at a density of 30,000 cells well1. A Termanox plastic plate, which has often been used as a control sample, was used as a control sample and a-MEM containing 10% FBS was used as the culture medium. The medium was changed after 1 day of culture and then changed every other day. The number of MC3T3-E1 cells was evaluated after treatment with Cell Counting Kit-8 (Dojindo, Japan). One reagent in the kit is a water soluble tetrazolium salt which is reduced by dehydrogenases in live cells to generate water soluble formazan. Water soluble formazan has an absorption maximum at about 460 nm. The numbers of live cells in the samples were counted by measuring the absorbance of the resulting medium at 450 nm. Students t-test was used for the cell counts. Differences between the samples were determined by Students t-test, with p < 0.05 considered to be statistically signicant. The cells cultured on the samples for 3 days were xed in 2.5% glutaraldehyde for 40 min at 4 C, dehydrated through a series of increasing concentrations of ethanol and, nally, dried with hexamethyldisilazane. The dried samples were coated with amorphous osmium using plasma CVD equipment and observed by SEM.

2.6. Preparation of a bilayered microber mesh consisting of SiPVH/ HA and PLA meshes A microber mesh having large spaces is not an appropriate candidate for a GBR membrane because the mesh may allow brous cells and soft tissues to permeate through them [25]. To address this issue we prepared a bilayered microber mesh using SiPVH/HA mesh and the dense PLA mesh, which was not expected to allow any cell permeation. In addition, the strength of the Si PVH/HA mesh was improved by combination with the dense PLA mesh. Details of the preparation method have been reported by Wakita et al. [26]. It was found that the combination induced no fracture or fragmentation of SiPVH/HA and PLA. The PLA mesh was pressed at 15 MPa for 1 min to increase its density. Adherence between the SiPVH mesh and the dense PLA mesh was accomplished by pressing at 15 MPa using a stainless mesh (No. 40) at 150 C and soaking in 1.5 SBF for 1 day. The result was a bilayered mesh consisting of SiPVH/HA mesh and the dense PLA mesh (Fig. 1A). 2.7. Evaluation of in vivo bone-forming ability The in vivo study was carried out at the laboratory of Hamri Co. Ltd. (Japan). Fifteen male New Zealand rabbits (Kbl:NZW), 14 weeks of age and between 2.50 and 3.20 kg in weight, were purchased from Kitayama Labes Co. Ltd. (Japan). The animals were housed in vivaria at 23 3 C, 50 20% humidity and a 12 h on/ off light cycle and ventilation was cycled approximately 12 times

Fig. 1. Schematic images of (A) preparation of the bilayered mesh and (B) protocol of the in vivo test. Adherence between the SiPVH mesh and the dense PLA mesh was accomplished by pressing with a stainless steel mesh at 150 C and soaking in 1.5 SBF for 1 day. The result was a bilayered mesh consisting of SiPVH/HA mesh and the dense PLA mesh. An 8 mm diameter hole was drilled into the front midline of the animals calvaria and then covered with the prepared bilayered mesh.

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per hour. The animals were maintained according to the National Institutes of Health (NIH) guide for care and use of laboratory animals. After allowing the animals to adapt to the new environment for 12 days they were stratied into three groups of ve animals each. After anesthetizing the animals an 8 mm diameter hole was drilled into the front midline of the calvaria using a bone cutter (BL-30 2A, Osada Electric Co. Ltd., Japan) and then covered with the prepared bilayered meshes (Fig. 1B). The bilayered mesh was xed by being placed between the skin and bone. The meshes were implanted with the SiPVH/HA mesh in contact with the bone and the dense PLA mesh in contact with the skin. The implanted meshes were harvested with the surrounding tissues for histological examination after 4, 8 and 12 weeks. The samples were dehydrated, embedded in methyl methacrylate and then sliced, resulting in slides of approximately 20 lm thickness. The slides were stained with Villanueva-Goldner and observed by optical microscopy (CH-2, Olympus, Japan). Villanueva-Goldner stains mineralized tissues green.

3. Results 3.1. Materials characterization Fig. 2 shows SEM micrographs of the SiV and vaterite particles. The SiV and vaterite particles had diameters of $1 lm and $500 nm, respectively. These two types of particles consisted of aggregates of nano-sized primary particles. The sizes of the SiV particles were larger than those of the vaterite particles. The results of the nitrogen gas adsorption method revealed that the specic surfaces of SiV and vaterite were 67.2 and 30.7 m2 g1, respectively. These results suggest that the conditions for aggregation of the primary particles in the suspension were inuenced by the addition of APTES. Fig. 3 shows the XRD patterns of SiV and vaterite. A weak peak corresponding to calcite appeared at approximately 29 in the pattern of the vaterite sample. A trace amount of calcite was found with a predominant vaterite phase, because the calcite phase is more stable than the other polymorphs of calcium carbonate, vaterite and aragonite. In the present work the sample is denoted vaterite. The peaks of the SiV pattern are broader than those of the vaterite pattern. SiV was found to consist predominantly of vaterite. The structure of APTES in SiV was further characterized by 29Si MAS-NMR spectroscopy. Fig. 4 shows the 29Si MAS-NMR spectrum of SiV. A peak at 50 to 80 ppm can be seen. The peak may be superimposed on those corresponding to T2 [NH2(CH2)3Si(OSi)2 (OH, OC2H5)] and T3 [NH2(CH2)3Si(OSi)3] [27]. This indicates that no APTES in SiV existed as the monomer and that siloxane was formed by condensation of APTES during the preparation of SiV. Mesh exibility was felt to be a reection of its structural properties, such as the molecular weight of the PLA matrix and structural changes in the interface between the matrix and the vaterite or SiV powders. Table 1 shows the weight average molecular weight (Mw), number average molecular weight (Mn) and polydispersity (Mw/Mn) of PLA and the PLA matrix in PVH and SiPVH. Although the molecular weight of PLA decreased slightly after kneading with the SiV powders, it decreased signicantly after kneading with vaterite powder. The prepared SiPVH mesh showed exibility without breaking, whereas the PVH mesh was brittle and was easily broken by hand. The ratios Mw/Mn for PVH and SiPVH were higher than that for PLA. This indicates that the molecular weights of these composites were disperate. Fig. 5 shows various peaks corresponding to the carboxy groups in the 13C CP/MAS-NMR spectra of the prepared composites. The band of the carboxy peak shifted slightly and a

Fig. 2. SEM photographs of (A) vaterite and (B) SiV. The SiV and vaterite particles have diameters of $1 lm and $500 nm, respectively. The SiV particles are larger than the vaterite particles. This suggests that the conditions for aggregation of the primary particles in the suspension were inuenced by adding APTES.

Fig. 3. XRD patterns of (A) vaterite and (B) SiV. A weak peak corresponding to calcite appears at approximately 29 in the pattern of the vaterite sample, but the vaterite sample and SiV were found to consist predominantly of vaterite phase. The peaks for SiV were broader than those of vaterite.

new peak appeared in the low magnetic eld area of the band, where carboxy groups form coordinate bonds with bivalent ions [28]. The peaks further separated into two peaks, peak A and B.

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Fig. 4. 29Si MAS-NMR spectrum of SiV. The peak at 50 to 80 ppm may be superimposed on those corresponding to T2 [NH2(CH2)3Si(OSi)2 (OH, OC2H5)] and T3 [NH2(CH2)3Si(OSi)3]. This indicates that no APTES in SiV exists as the monomer and siloxane was formed.

Table 1 Weight average molecular weight (Mw), number average molecular weight (Mn) and polydispersity (Mw/Mn) of PLA, PVH and SiPVH. Mw (kDa) PLA PVH SiPVH 259 17 185 Mn (kDa) 168 7 86 Mw/Mn 1.5 2.4 2.1

Peak A was dened at one magnetic eld in the band and peak B as one magnetic eld lower in the band. The intensity of peak B in the spectrum of PVH was larger than in the spectrum of SiPVH. This indicates that more carboxy groups formed coordinate bonds with calcium ions in PVH than SiPVH and that the formation of coordinate bonds caused the decrease in molecular weight. Fig. 6 shows the FTIR spectra of SiPVH and PVH. In the spectrum of SiPVH a new weak peak appears at around 1650 cm1, as well as the peaks corresponding to PLA and CO3 2 . This peak appears to originate from the amide bond, which indicates that carboxy groups on PLA form chemical bonds with amino groups on APTES. Fig. 7A demonstrates the greater exibility of the SiPVH mesh, which could be folded without fracture or fragmentation using a pair of tweezers. Microbers approximately 10 lm in diameter were observed on the SiPVH mesh surface and were found to twine around one another. The size of the gaps between the microbers varied between 10 lm and several hundred micrometers, as shown in Fig. 7B. The SiV particles observed on the microber surface were coated with a thin lm of the PLA matrix, as shown in Fig. 7C. The fracture face of the microbers showed that the particles were closely packed in the microbers (Fig. 7D). The particles were embedded in the PLA matrix and after immersion in 1.5 SBF solution for 1 day the surfaces of the microfibers were coated with leaf-shaped deposits, as shown in Fig. 7E. No signicant change was observed in the gap sizes between the microbers before and after immersion (Fig. 7F). After HA coating further testing demonstrated several cracks, but the coating did not peel off. The deposits were conrmed by XRD to be HA of approximately 1 lm thickness (Fig. 7G). On the other hand, the PVH mesh consisted of short microbers approximately 7 lm in diameter (Fig. 7H).

Fig. 5. 13C CP/MAS-NMR spectra of (A) PVH and (B) SiPVH. The peaks further separated into two peaks, A and B. The intensity of peak B in the spectrum of PVH is larger than that of SiPVH. This indicates that more carboxy groups formed coordinate bonds with calcium ions in PVH than SiPVH.

3.2. Ease of silicon release Fig. 8 shows the dynamics of the release of silicon ions from both the SiPVH mesh and SiPVH/HA mesh into the culture medium over 5 days. Closed bars indicate the ion amount in the culture medium and open bars indicates the total amount of ions released up to each measurement point. After 1 day of immersion a concentration of 8.2 mg l1 ionic silicon species had been released from SiPVH, while this amount was reduced to 1.2 mg l1 on day 3 and 0.59 mg l1 on day 5. The SiPVH mesh was then soaked in culture medium after being incubated in 1.5 SBF for 1 day and coated with HA. The amount of the silicon species released from it was signicantly reduced, with only 0.68 mg l1 released from the mesh after 1 day of soaking in culture medium. The amount released was even smaller on days 3 and 5 of soaking, at 0.39 mg l1 on day 3 and 0.41 mg l1 on day 5. 3.3. Proliferation of MC3T3-E1 cells on the prepared meshes The cellular compatibility of the SiPVH/HA mesh compared with the PLA mesh was evaluated in culture using MC3T3-E1 cells. Fig. 9 shows the numbers of cells after culture on SiPVH/HA mesh, PLA mesh and Thermanox for 2 weeks. The number of cells after culture on the SiPVH/HA mesh was higher than that on the PLA

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Fig. 6. FTIR spectra of (A) PVH and (B) SiPVH. A weak peak appears at around 1650 cm1 in the spectrum of SiPVH. This peak appears to originate from the amide bond between carboxy groups in PLA and amino groups in APTES.

mesh at all time points. The PVH mesh could not be used as a control mesh due to its extreme brittleness. Fig. 10 shows SEM micrograph of cells cultured on the SiPVH/ HA mesh for 3 days. The cells elongated on the ber surfaces and extended over several bers. Some of them entered the ber mesh and extended within it. 3.4. Bone formation in the prepared meshes An 8 mm diameter hole in bone is generally not regenerated if nothing is done to stimulate it. In our preliminary experiment a PLGA mesh was implanted in the same position as that in this study for 12 weeks and no new bone was formed in/on the mesh at the center of the hole (data not shown). Bone growth into the defect from the margin was observed in all of the samples. Therefore, we tested the effects of the bilayered meshes on bone formation by observing new bone formation at the center of the 8 mm holes. This animal test was a fundamental study. Although this animal test was not a load-bearing model, we believe that it was useful in evaluating the bone-forming ability of the bilayered meshes. Fig. 11 shows the in vivo response to the bilayered meshes and new bone formation at the center of the holes. Villanueva-Goldner stain showed that new bone formed in the mesh 4 weeks after implantation. Bone began to form around the pressed points formed during layering of the two types of mesh, the SiPVH/HA mesh and the dense PLA mesh. The bone area expanded in the SiPVH/HA mesh after 8 weeks. After 12 weeks new bone formation was observed over almost the entire area of implanted Si PVH/HA mesh. There was no tissue inammation observed histologically. 4. Discussion Earlier work showed that composite lms which release silicon can stimulate MC3T3-E1 cells to proliferate and mineralize [17,24]. These composite lms were prepared by dip coating a solution of SiV and PLA composites on a coverglass, drying at room temperature and pealing the dried composite from the glass. The prepared composite lm had no pores to allow cells to enter, therefore, cells proliferated only on the lm surface. When applying this technique to a GBR membrane the composites should have porosity in order to induce adhesion and proliferation of cells and to provide nutrition to the cells.

To address this issue we developed a ber mesh using PLA and SiV formed by electrospinning. We anticipated that the electrospun ber mesh would contain a large volume of pores and would show exibility, both essential components of a GBR membrane. The Si PVH mesh showed greater exibility and a higher weight averaged molecular weight PLA than the PVH mesh, in the case of SiV due to siloxane on the vaterite surface, which resulted in the formation of a exible mesh containing a large amount of SiV powder (60 wt.%). There has been increasing interest in the use of electrospun brous meshes as scaffolds in medicine. In particular, biocompatible polymers such as PLGA and poly(caprolactone) have been applied as materials for meshes, incorporating various growth factors or DNA [3]. On the other hand, very few studies have been performed on brous meshes consisting of polymer/ceramic composites. Previously, PLGA and HA composite scaffolds fabricated by electrospinning contained only 510 wt.% HA nanocrystals [29]. We believe that a larger amount of calcium is needed to enhance the bone-forming ability of electrospun brous meshes and vaterite powders are effective materials for providing calcium. In the present work a brous mesh containing a large amount of vaterite (up to 60 wt.%) was successfully prepared by kneading and subsequent electrospinning. Vaterite or SiV particles were embedded in a PLA matrix, with very thin PLA layers on the particle surfaces, as shown in Fig. 7C and D. Calcium ions are supposed to be released through degradation of the thin PLA layer. The PVH mesh, however, was brittle, without exibility, due to a drastic decrease in the PLA molecular weight after kneading with vaterite powders, as shown in Table 1. Maeda et al. reported that in vaterite and PLA composites calcium ions on vaterite particles formed coordinate bonds with carboxy groups in the PLA structure [30]. The polymer chains in the SiPVH mesh, however, were not broken by the formation of coordinate bonds between calcium ions and carboxy groups (Fig. 5) due to siloxane derived from APTES on the vaterite surface, which prevents the formation of coordinate bonds. This resulted in the formation of a exible mesh containing a large amount of SiV powder (60 wt.%). The specic surface areas of SiV and vaterite were estimated to be 67.2 and 30.7 m2 g1, respectively, by nitrogen gas adsorption. The peaks corresponding to vaterite in the XRD pattern of SiV were broader than that of vaterite (Fig. 3). This result indicates that the diameter of the primary particles of SiV is smaller than that of vaterite. Crystal growth of the primary particles of vaterite ap-

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Fig. 7. Photographs of the prepared meshes. (A) Photograph of the SiPVH mesh in a exibility test. The SiPVH mesh was folded without fracture or fragmentation using a pair of tweezers. (BD) SEM photographs of the SiPVH mesh. Microbers approximately 10 lm in diameter are found to twine around one another. The SiV particles observed at the microber surface are coated with a thin lm of the PLA matrix. (D) Fracture surface of a ber of the mesh. The particles are closely packed in the microber. (E and F) SEM photographs of the SiPVH/HA mesh. The surfaces of the microbers are coated with leaf-shaped deposits. (G) Crack in the HA coating on the mesh after the exibility test. The coating of approximately 1 lm thickness does not peel off. (H) SEM photograph of the PVH mesh. The PVH mesh consists of short microbers approximately 7 lm in diameter.

peared to be prevented by the siloxane, which may cover them. Amino groups on the siloxane on the vaterite surface formed amide

bonds with carboxy groups. The number of bonds, however, was very small, as shown in Fig. 6.

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Fig. 8. Silicon ion concentration in culture medium after incubation of (A) SiPVH mesh and (B) SiPVH/HA mesh. Closed bars indicate the ion concentration in the culture medium. Open bars indicates the total amount of ions released until each measurement point.

Fig. 9. Numbers of MC3T3-E1 cells after culture on SiPVH/HA mesh, PLA mesh and Thermanox for 2 weeks (*p < 0.05). The numbers of cells after cultured on the Si PVH/HA mesh are higher than that on the PLA mesh at all time points. The PVH mesh could not be used as a control mesh due to its extreme brittleness.

Fig. 10. SEM micrograph of the SiPVH/HA mesh after culturing MC3T3-E1 cells for 3 days. Arrows indicate the cells. The cells elongate on the ber surfaces and extend over several bers. Some of them enter the ber mesh and extend within it.

In general, when organic agents such as growth factors and DNA are applied as additives in scaffold materials they are denatured. Most organic agents suffer damage to their structure on exposure to solvents or heat treatment during preparation of their composite scaffolds. On the other hand, the effects of the silicon species in SiV on cellular activity depend on silicon and not an organic structure. Silicon is usually more stable than organic agents. In our previous work, the proliferation and differentiation of MC3T3-E1 cells were enhanced on a dense lm consisting of SiV and PLA composites [24]. In addition, human osteoblasts and mesenchymal stem cells were stimulated to mineralize and differentiate on siloxane-doped PLA and vaterite hybrid membranes which released a trace amount of silicon species [31,32]. Therefore, the silicon species appear to be good candidates for the enhancement of cell activation and bone formation. In addition, since the APTES in SiV forms siloxane (Fig. 4), no monomer would be expected to be included in SiV. The CSi and SiO bonds in siloxane are too strong to be hydrolyzed. The silicon species may, therefore, be released with the siloxane. Since a large volume of pores is an essential component of a GBR membrane, SiPVH bers between 0.5 and 20 lm diameter were prepared in our laboratory. The diameter was controlled by changing the conditions during electrospinning, such as the PLA concentration in the composite solution and the solvent. In the present work composite microbers of 10 lm diameter were used for cell culture and the animal tests, because we hypothesized that the spaces between the microbers provide the scaffold for cell proliferation. However, when considering application as a GBR membrane a microber mesh with large spaces may not be an appropriate candidate because the mesh may allow cells and soft tissue to permeate through the scaffold. These issues were solved by preparing the bilayered microber mesh using a SiPVH/HA mesh and a dense PLA mesh. Hydroxycarbonate apatite, the HA formed in SBF, was reported to show higher cellular compatibility than pure HA [33]. The proliferation of MC3T3-E1 cells on a silicon-doped PLA/vaterite hybrid membrane was reportedly enhanced by an HA coating on the membrane, prepared in SBF [17]. The HA formation in SBF occurred in the presence of functional groups needed to induce the nucleation of HA, such as SiOH, TiOH and carboxy groups on the material surface, and supersaturation of the HA [34,35]. To increase the supersaturation of HA in SBF it is important to release a large number of Ca2+ ions from the samples. HA formation on the SiPVH mesh was induced by carboxy groups in PLA and calcium ions released from SiV. The microber surfaces of the SiPVH mesh were completely covered with HA after 1 day of immersion in 1.5 SBF

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that of PVH [24]. Proliferation of the cells on a HA-coated membrane was better than that on an uncoated one [17]. MC3T3-E1 cells proliferated well on the SiPVH/HA mesh, as shown in Fig. 9. This may be due to the effects of the silicon species on cell function and the HA coating, which shows high cellular compatibility. In addition, the large pore spaces in the SiPVH/HA mesh are expected to cause the cells to proliferate, since they can enter the mesh and elongate on the ber surfaces, as shown in Fig. 10. With regard to the in vivo portion of the study, histological studies revealed new bone formation in the bilayered mesh at the center of holes drilled in rabbit calvaria (Fig. 11). Bone formation started from inside the SiPVH/HA mesh, specically at the pressed points, and not from the edge of the hole. Mineralization of osteoblasts in the mesh appeared to be induced at the pressed points, where the microbers were dense. Although this is an interesting phenomenon, the reason behind it is not clear. Further work to clarify this mechanism is in progress. The mechanical strength of the SiPVH/HA mesh was improved by layering with a PLA mesh, which made available a sufcient amount of space for new bone formation in the hole after 12 weeks. The dense PLA mesh showed high strength and blocked new bone formation at its interface with the SiPVH/HA mesh. Thus, the dense PLA mesh worked to maintain the space and the SiPVH/HA mesh worked as a scaffold to induce bone formation by osteoblasts. 5. Conclusion Silicon-releasing microber meshes have been prepared by an electrospinning technique for application as a GBR membrane. The meshes consisted of a hybrid between silicon-doped vaterite and PLA. The carboxy groups on PLA formed amide bonds with amino groups in the siloxane phase derived from APTES and coordinated with calcium ions on the surface of the silicon-doped vaterite. The meshes showed enhanced HA formation in 1.5 SBF and continuously released a trace amount of silicon species into the culture medium, which facilitated bone regeneration. The meshes also enhanced proliferation of osteoblast-like cells in vitro and induced bone formation in vivo. Acknowledgements This work was supported in part by a Grant-in-Aid for Young Scientists (B) (No. 21700487) from The Ministry of Education, Culture, Sports, Science and Technology.
Fig. 11. Histology of in vivo response to the bilayered meshes. Villanueva-Goldner staining shows new bone formed in the meshes at the center of 8 mm diameter holes drilled into the frontal midline of rabbit calvaria. Asterisks indicate mineralized tissues. Arrows indicate pressed points formed during preparation of the bilayered meshes. Bars indicate the mesh area. Bone begins to form around the pressed points formed through layering of the two types of mesh, SiPVH/HA mesh and dense PLA mesh. New bone formation was observed over almost the entire area of implanted SiPVH/HA mesh after 12 weeks.

Appendix A. Figures with essential colour discrimination Certain gures in this article, particularly Figs. 1, 5, 6, 10 and 11, are difcult to interpret in black and white. The full colour images can be found in the on-line version, at doi: 10.1016/ j.actbio.2009.11.013. References

(Fig. 7E and F). The HA formed on the microbers tightly contacted their surfaces (Fig. 7G). The HA layer may contain silicon species released from the SiPVH mesh. Maeda et al. reported that silicon species released from siloxane-doped PLA and vaterite hybrid membranes were trapped in the HA layer precipitated in SBF [17]. Excessive silicon intake has been regarded as causing kidney damage, but silicon species have been reported to be one of the essential trace elements for healthy bone and to stimulate osteogenic cells to proliferate and mineralize [36]. The proliferation and differentiation of MC3T3-E1 cells on a membrane consisting of a SiPVH (not electrospun) mesh were enhanced compared with

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