Acta Biomaterialia 6 (2010) 1248–1257

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Acta Biomaterialia

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Electrospun microfiber meshes of silicon-doped vaterite/poly(lactic acid) hybridfor 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, Japanb Yamahachi Dental Manufacturing Co., 54-1 Ochigara, Nishiura Cho, Gamagori, Aichi 443-0105, Japanc Yabashi Industries Co. Ltd., 188-1 Akasaka Cho, Ogaki, Gifu 503-2213, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 May 2009Received in revised form 23 October 2009Accepted 9 November 2009Available online 11 November 2009

Keywords:Polylactic acidCalcium carbonateSiliconMembraneOsteoblast

1742-7061/$ - see front matter � 2009 Acta Materialdoi:10.1016/j.actbio.2009.11.013

* Corresponding author. Tel./fax: +81 52 735 7250.E-mail address: obata.akiko@nitech.ac.jp (A. Obata

Silicon-releasable microfiber meshes consisting of silicon-doped vaterite (SiV) particles and poly(lacticacid) (PLA) hybrids were prepared by electrospinning. Due to their flexibility and porosity they formedideal membranes or scaffolds for guided bone regeneration. In addition, a trace amount of silicon specieshas been reported to stimulate osteogenic cells to mineralize and enhance bone formation. We propose anew method of preparation of silicon-releasing microfiber meshes by electrospinning. Their structure andhydroxyapatite (HA)-forming abilities in simulated body fluid were examined. In addition, we studiedtheir stimulatory effects on osteoblast-like cells in vitro and bone-forming ability in vivo, with a specialemphasis on their ability to release silicon. The meshes consisted of a hybrid of carboxy groups in PLA andamino 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 released0.2–0.7 mg l�1 silicon species into the culture medium over 7 days. Enhanced proliferation of osteo-blast-like cells was observed using the meshes and new bone formed on the meshes when implanted intothe calvaria of rabbits. These meshes, therefore, provide an excellent substrate for bone regeneration andexhibit enhanced bone-forming ability under both in vitro and in vivo conditions.

� 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Biodegradable polymer scaffolds have been developed for use inbone reconstruction, tissue engineering and drug delivery systems[1,2]. In addition, three-dimensional scaffolds are common in bio-materials. Electrospinning is a process that can generate polymerfiber meshes with high porosity and flexibility, both essential com-ponents of tissue engineering [3–5]. In general, the process of elec-trospinning is affected by both system parameters and processparameters. System parameters include polymer molecular weight(Mw) and polymer solution properties such as viscosity and con-ductivity. Process parameters, on the other hand, involve flow rateof the polymer solution, electric potential and the distance be-tween the capillary and collector, among others [3]. It is possibleto control the fiber diameter and the thickness of fiber mesh scaf-folds by optimizing these parameters. To use electrospun fibermeshes in bone tissue engineering applications, their various prop-erties, including material, fiber orientation, porosity and surfacemodification, must be considered.

To optimize the mechanical and biomimetic properties of a fibermesh it is necessary to choose the appropriate materials, since the

ia Inc. Published by Elsevier Ltd. A

).

orientation and diameter of the fibers in electrospun scaffolds caninfluence cellular compatibility [6–8]. The fiber diameter in elec-trospun scaffolds has, for example, been reported to influenceosteoblast proliferation and mineralization. Electrospun fibermeshes have been fabricated to contain DNA and proteins, whichenhance cellular compatibility and bone formation upon their re-lease [8–11]. The DNA and proteins are released by degradationof polymer matrices such as poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA) and poly(glycolide acid) (PGA).

Despite their stimulatory effects on osteoblasts and bone mar-row-derived stem cells, electrospun fiber scaffolds consisting ofpolymer/ceramic composites have not been widely researchedas biomaterials. Polymer and ceramic composites have beendeveloped for bone reconstruction and showed good mechanicaland bone-forming properties [12–14]. We hypothesize that elec-trospun fiber scaffolds consisting of PLA/calcium carbonate com-posites would also have good mechanical and bone-formingproperties. Calcium carbonate, for example, is well known as abioresorbable material, shows excellent bone-forming abilityand has been used clinically as a bone filler [15,16]. In our pre-vious research PLA/calcium carbonate composites (vaterite)showed excellent hydroxyapatite (HA)-forming ability, cellularcompatibility and biocompatibility in in vitro and in vivo tests[17,18].

ll rights reserved.

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Guided bone regeneration (GBR) is a useful method of bonereconstruction in vivo. GBR membranes require flexibility to adaptto the shape of the bone defect and strength to maintain the spacefor bone formation and to connect with the soft tissues. An electro-spun fiber mesh is a good candidate for a GBR membrane due tothe flexibility inherent in the electrospinning process. Release ofcalcium and silicon species from an electrospun fiber mesh is anadditional attribute which promotes bone regeneration. Calciumspecies and a trace amount of silicon species have been reportedto enhance bone formation by osteoblasts [19–23]. Previously wedeveloped silicon-doped vaterite (SiV) powders and their PLA com-posites and tested their cellular compatibility using mouse osteo-blast-like cells [24]. A film of the composites was prepared bydip coating the composite solution on a coverglass and drying atroom temperature. The film showed release of calcium and siliconinto the culture medium through degradation of the PLA and SiVmatrix. Cell proliferation and mineralization on the composite filmwas enhanced compared with an undoped vaterite and PLA com-posite film, which showed no silicon release.

Our preliminary experiments demonstrated that a flexible fibermesh consisting of PLA composites with a large amount of SiVcould be successfully prepared by the electrospinning method,while a fiber mesh consisting of a PLA composite with vaterite (un-doped type) was brittle. We propose to further characterize the SiVand PLA hybrid (Si–PVH) and compare its chemical structure withthe undoped vaterite and PLA hybrid (PVH). To enhance cellularadhesion the composite fiber meshes were coated with HA usingsimulated body fluid (SBF). The cellular and tissue compatibilitiesof both prepared fiber meshes were evaluated by in vitro testsusing mouse osteoblast-like cells (MC3T3-E1 cells) and in vivotests using rabbit calvaria.

2. Materials and methods

2.1. Preparation of SiV and vaterite powders

Silicon-doped vaterite (SiV) powders were prepared by a car-bonation process with methanol. A sample of 150 g Ca(OH)2 wasmixed with 60 ml aminopropyltriethoxysilane (APTES) and2000 ml methanol with the addition of CO2 gas for 75 min at a rateof 2000 ml min�1. The resulting slurry was dried at 110 �C, result-ing in the SiV powder. The content of silicon in SiV was estimatedto be approximately 3 wt.% by X-ray fluorescence analysis(RIX3000, Rigaku, Japan) (50 kV, 60 mA). A calibration curve (X-ray intensity vs. SiO2 content) was established by measuring lime-stone containing various amounts of SiO2. Vaterite powders werealso prepared by the carbonation process without APTES. SiV andvaterite powders were characterized by X-ray diffractometry(XRD) (RAD-B, Rigaku, Japan) (CuKa, 40 kV, 20 mA). The specificsurface areas of SiV and vaterite were measured according to aBET theory using nitrogen gas adsorption (NOVA3000, Quanta-chrome Corp., USA). The powders were deaerated at 150 �C for1 h before measurement. The structure of silicon in SiV was exam-ined by 29Si magic angle spinning nuclear magnetic resonance (29SiMAS-NMR) (UNITY plus, Varian, USA), operating at pulses of 6.3 lsand at recycle delays of 15 s. The sample spinning speed was3.0 kHz. Chemical shifts were measured relative to polydime-thylsiloxane.

2.2. Preparation of microfiber meshes

The Si–PVH and PVH microfiber meshes were prepared by anelectrospinning method. SiV or vaterite powder was mixed withPLA (Purasorb�; molecular weight 260 kDa, PURAC, The Nether-lands) 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 preparethe solution for electrospinning. In our preliminary experiments,this ratio was found to be optimal for preparing microfiber meshesof approximately 10 lm diameter. The PLA microfiber mesh wasalso prepared by an electrospinning method. The PLA was dis-solved in chloroform at 9 wt.% to prepare the solution for electros-pinning. The prepared solutions were loaded into a syringe pump(FP-W-100, Melquest, Japan) with a syringe needle (22 gauge forSi–PVH and PVH, 19 gauge for PLA), which was set at0.05 ml min�1. The distance between the needle and an aluminumcollector was 150 mm. Electrospinning was carried out at 20 kV ofimpressed voltage and �100 lA of applied current (Power SupplyHARb-40 P0.75, Matsusada Precision Inc., Japan) at room tempera-ture and approximately 55% relative humidity.

The SiV powders and resultant electrospun fibers were coatedwith amorphous osmium using plasma chemical vapor deposition(CVD) equipment and then morphologically observed by fieldemission scanning electron microscopy (SEM) (JSM-6301F, JEOL,Japan) (accelerating voltage 5 keV). Structural analyses of Si–PVHwere performed using 13C cross-polarization magic angle spinningnuclear magnetic resonance (13C CP/MAS-NMR) (UNITY plus, Var-ian, USA) and Fourier transform infrared (FTIR) spectroscopy (FT-IR-460 Plus, JASCO, Japan). 13C CP/MAS-NMR spectra were takenwith 4.7 ls and 8.0 s recycle delays. The sample spinning speedwas 3.0 kHz. Chemical shifts were measured relative toadamantane.

The molecular weights of PLA matrix in the prepared mesheswere measured by gel permeation chromatography (GPC) (Shima-dzu, Japan) using two columns (KF-604, Shodex, Japan). Themeshes were dissolved in chloroform at a concentration of 10 mgPLA per 5 ml chloroform and the solutions filtered. The measure-ments were carried out at 40 �C at a flow rate of 0.6 ml min�1. Poly-styrene standards (SM-105, Shodex, Japan) were used to establish acalibration curve.

2.3. Coating of the fibers with HA

The prepared microfiber mesh was immersed in 10 ml of anaqueous solution containing ion concentrations 1.5 times thoseof SBF (1.5 SBF) at 37 �C for 24 h. The 1.5 SBF solution consistingof 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 HPO42�, 0.75 mM SO4

2�,50 mM (CH2OH)3CNH2 and 45.0 mM HCl was prepared using re-agent grade NaCl, NaHCO3, KCl, K2PO4�3H2O, MgCl2�6H2O, HCl,CaCl2, NaSO4 and NH2C(CH2OH)3. After immersion in 1.5 SBF solu-tion the mesh was washed with distilled water and then dried atroom temperature. The dried samples were coated with amor-phous osmium using plasma CVD equipment and observed bySEM.

2.4. Measurement of silicon release from a Si–PVH microfiber meshbefore and after HA coating

The amount of silicon released from the Si–PVH microfibermeshes before and after HA coating was evaluated by immersingthem in 4 ml of a culture medium (alpha minimum essential med-ium, a-MEM) containing 10% fetal bovine serum (FBS) and incu-bated at 37 �C in a humidified atmosphere of 95% air, 5% CO2 for5 days. The sample coated with HA is denoted Si–PVH/HA mesh.The size of the sample used was 10 � 10 mm. The culture mediumwas changed after either 1 or 3 days soaking, according to the pro-cess of the cell culture test. The sample was taken out of the culturemedium and the medium filtered. The level of silicon species in theresulting medium was measured by inductively coupled plasmaatomic emission spectroscopy (ICP-AES) (ICPS-500, Shimadzu, Ja-

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pan). A calibration curve was established using an aqueous solu-tion of Si4+ ions at concentrations of 1, 10 and 20 ppm.

2.5. Cell culture tests

The microfiber mesh used for the cell culture tests was steril-ized using ethylene oxide gas. Mouse osteoblast-like cells(MC3T3-E1 cells) were seeded onto the prepared mesh (Si–PVH/HA or PLA mesh) in 24-well plates at a density of 30,000cells well�1. A Termanox� plastic plate, which has often been usedas a control sample, was used as a control sample and a-MEM con-taining 10% FBS was used as the culture medium. The medium waschanged after 1 day of culture and then changed every other day.The number of MC3T3-E1 cells was evaluated after treatment withCell Counting Kit-8 (Dojindo, Japan). One reagent in the kit is awater soluble tetrazolium salt which is reduced by dehydrogen-ases in live cells to generate water soluble formazan. Water solubleformazan has an absorption maximum at about 460 nm. The num-bers of live cells in the samples were counted by measuring theabsorbance of the resulting medium at 450 nm. Student’s t-testwas used for the cell counts. Differences between the samples weredetermined by Student’s t-test, with p < 0.05 considered to be sta-tistically significant. The cells cultured on the samples for 3 dayswere fixed in 2.5% glutaraldehyde for 40 min at 4 �C, dehydratedthrough a series of increasing concentrations of ethanol and, final-ly, dried with hexamethyldisilazane. The dried samples werecoated with amorphous osmium using plasma CVD equipmentand observed by SEM.

Fig. 1. Schematic images of (A) preparation of the bilayered mesh and (B) protocol of taccomplished by pressing with a stainless steel mesh at 150 �C and soaking in 1.5 SBF forPLA mesh. An 8 mm diameter hole was drilled into the front midline of the animal’s ca

2.6. Preparation of a bilayered microfiber mesh consisting of Si–PVH/HA and PLA meshes

A microfiber mesh having large spaces is not an appropriatecandidate for a GBR membrane because the mesh may allowfibrous cells and soft tissues to permeate through them [25]. Toaddress this issue we prepared a bilayered microfiber mesh usingSi–PVH/HA mesh and the dense PLA mesh, which was not expectedto allow any cell permeation. In addition, the strength of the Si–PVH/HA mesh was improved by combination with the dense PLAmesh. Details of the preparation method have been reported byWakita et al. [26]. It was found that the combination induced nofracture or fragmentation of Si–PVH/HA and PLA.

The PLA mesh was pressed at 15 MPa for 1 min to increase itsdensity. Adherence between the Si–PVH mesh and the dense PLAmesh was accomplished by pressing at 15 MPa using a stainlessmesh (No. 40) at 150 �C and soaking in 1.5 SBF for 1 day. The resultwas a bilayered mesh consisting of Si–PVH/HA mesh and the densePLA 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 pur-chased from Kitayama Labes Co. Ltd. (Japan). The animals werehoused in vivaria at 23 ± 3 �C, 50 ± 20% humidity and a 12 h on/off light cycle and ventilation was cycled approximately 12 times

he in vivo test. Adherence between the Si–PVH mesh and the dense PLA mesh was1 day. The result was a bilayered mesh consisting of Si–PVH/HA mesh and the denselvaria and then covered with the prepared bilayered mesh.

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per hour. The animals were maintained according to the NationalInstitutes of Health (NIH) guide for care and use of laboratory animals.

After allowing the animals to adapt to the new environment for12 days they were stratified into three groups of five animals each.After anesthetizing the animals an 8 mm diameter hole was drilledinto the front midline of the calvaria using a bone cutter (BL-30 2A,Osada Electric Co. Ltd., Japan) and then covered with the preparedbilayered meshes (Fig. 1B). The bilayered mesh was fixed by beingplaced between the skin and bone. The meshes were implantedwith the Si–PVH/HA mesh in contact with the bone and the densePLA mesh in contact with the skin. The implanted meshes wereharvested with the surrounding tissues for histological examina-tion after 4, 8 and 12 weeks. The samples were dehydrated,embedded in methyl methacrylate and then sliced, resulting inslides of approximately 20 lm thickness. The slides were stainedwith Villanueva-Goldner and observed by optical microscopy(CH-2, Olympus, Japan). Villanueva-Goldner stains mineralized tis-sues green.

Fig. 2. SEM photographs of (A) vaterite and (B) SiV. The SiV and vaterite particleshave diameters of �1 lm and �500 nm, respectively. The SiV particles are largerthan the vaterite particles. This suggests that the conditions for aggregation of theprimary particles in the suspension were influenced by adding APTES.

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

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 ofaggregates of nano-sized primary particles. The sizes of the SiVparticles were larger than those of the vaterite particles. The re-sults of the nitrogen gas adsorption method revealed that the spe-cific surfaces of SiV and vaterite were 67.2 and 30.7 m2 g�1,respectively. These results suggest that the conditions for aggrega-tion of the primary particles in the suspension were influenced bythe addition of APTES.

Fig. 3 shows the XRD patterns of SiV and vaterite. A weak peakcorresponding to calcite appeared at approximately 29� in the pat-tern of the vaterite sample. A trace amount of calcite was foundwith a predominant vaterite phase, because the calcite phase ismore stable than the other polymorphs of calcium carbonate, vate-rite and aragonite. In the present work the sample is denoted‘‘vaterite”. The peaks of the SiV pattern are broader than those ofthe vaterite pattern. SiV was found to consist predominantly ofvaterite.

The structure of APTES in SiV was further characterized by 29SiMAS-NMR spectroscopy. Fig. 4 shows the 29Si MAS-NMR spectrumof SiV. A peak at �50 to �80 ppm can be seen. The peak may besuperimposed on those corresponding to T2 [NH2(CH2)3Si(OSi)2

(OH, OC2H5)] and T3 [NH2(CH2)3Si(OSi)3] [27]. This indicates thatno APTES in SiV existed as the monomer and that siloxane wasformed by condensation of APTES during the preparation of SiV.

Mesh flexibility was felt to be a reflection of its structural prop-erties, such as the molecular weight of the PLA matrix and struc-tural changes in the interface between the matrix and thevaterite or SiV powders. Table 1 shows the weight average molec-ular weight (Mw), number average molecular weight (Mn) andpolydispersity (Mw/Mn) of PLA and the PLA matrix in PVH andSi–PVH. Although the molecular weight of PLA decreased slightlyafter kneading with the SiV powders, it decreased significantlyafter kneading with vaterite powder. The prepared Si–PVH meshshowed flexibility without breaking, whereas the PVH mesh wasbrittle and was easily broken by hand.

The ratios Mw/Mn for PVH and Si–PVH were higher than thatfor PLA. This indicates that the molecular weights of these compos-ites were disperate. Fig. 5 shows various peaks corresponding tothe carboxy groups in the 13C CP/MAS-NMR spectra of the preparedcomposites. The band of the carboxy peak shifted slightly and a

new peak appeared in the low magnetic field 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.

Fig. 4. 29Si MAS-NMR spectrum of SiV. The peak at �50 to �80 ppm may besuperimposed 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 andsiloxane was formed.

Table 1Weight average molecular weight (Mw), number average molecular weight (Mn) andpolydispersity (Mw/Mn) of PLA, PVH and Si–PVH.

Mw (kDa) Mn (kDa) Mw/Mn

PLA 259 168 1.5PVH 17 7 2.4Si–PVH 185 86 2.1

Fig. 5. 13C CP/MAS-NMR spectra of (A) PVH and (B) Si–PVH. The peaks furtherseparated into two peaks, A and B. The intensity of peak B in the spectrum of PVH islarger than that of Si–PVH. This indicates that more carboxy groups formedcoordinate bonds with calcium ions in PVH than Si–PVH.

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Peak A was defined at one magnetic field in the band and peak B asone magnetic field lower in the band. The intensity of peak B in thespectrum of PVH was larger than in the spectrum ofSi–PVH. This indicates that more carboxy groups formedcoordinate bonds with calcium ions in PVH than Si–PVH and thatthe formation of coordinate bonds caused the decrease in molecu-lar weight. Fig. 6 shows the FTIR spectra of Si–PVH and PVH. In thespectrum of Si–PVH a new weak peak appears at around1650 cm�1, as well as the peaks corresponding to PLA and CO3

2�.This peak appears to originate from the amide bond, which indi-cates that carboxy groups on PLA form chemical bonds with aminogroups on APTES.

Fig. 7A demonstrates the greater flexibility of the Si–PVH mesh,which could be folded without fracture or fragmentation using apair of tweezers. Microfibers approximately 10 lm in diameterwere observed on the Si–PVH mesh surface and were found totwine around one another. The size of the gaps between themicrofibers varied between 10 lm and several hundred microme-ters, as shown in Fig. 7B. The SiV particles observed on the micro-fiber surface were coated with a thin film of the PLA matrix, asshown in Fig. 7C. The fracture face of the microfibers showed thatthe particles were closely packed in the microfibers (Fig. 7D).

The particles were embedded in the PLA matrix and afterimmersion in 1.5 SBF solution for 1 day the surfaces of the microf-ibers were coated with leaf-shaped deposits, as shown in Fig. 7E.No significant change was observed in the gap sizes between themicrofibers before and after immersion (Fig. 7F). After HA coatingfurther testing demonstrated several cracks, but the coating didnot peel off. The deposits were confirmed by XRD to be HA ofapproximately 1 lm thickness (Fig. 7G). On the other hand, thePVH mesh consisted of short microfibers approximately 7 lm indiameter (Fig. 7H).

3.2. Ease of silicon release

Fig. 8 shows the dynamics of the release of silicon ions fromboth the Si–PVH mesh and Si–PVH/HA mesh into the culture med-ium over 5 days. Closed bars indicate the ion amount in the culturemedium and open bars indicates the total amount of ions releasedup to each measurement point. After 1 day of immersion a concen-tration of 8.2 mg l�1 ionic silicon species had been released fromSi–PVH, while this amount was reduced to 1.2 mg l�1 on day 3and 0.59 mg l�1 on day 5. The Si–PVH mesh was then soaked in cul-ture medium after being incubated in 1.5 SBF for 1 day and coatedwith HA. The amount of the silicon species released from it wassignificantly reduced, with only 0.68 mg l�1 released from themesh after 1 day of soaking in culture medium. The amount re-leased was even smaller on days 3 and 5 of soaking, at 0.39 mg l�1

on day 3 and 0.41 mg l�1 on day 5.

3.3. Proliferation of MC3T3-E1 cells on the prepared meshes

The cellular compatibility of the Si–PVH/HA mesh comparedwith the PLA mesh was evaluated in culture using MC3T3-E1 cells.Fig. 9 shows the numbers of cells after culture on Si–PVH/HA mesh,PLA mesh and Thermanox� for 2 weeks. The number of cells afterculture on the Si–PVH/HA mesh was higher than that on the PLA

Fig. 6. FTIR spectra of (A) PVH and (B) Si–PVH. A weak peak appears at around 1650 cm�1 in the spectrum of Si–PVH. This peak appears to originate from the amide bondbetween carboxy groups in PLA and amino groups in APTES.

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mesh at all time points. The PVH mesh could not be used as a con-trol mesh due to its extreme brittleness.

Fig. 10 shows SEM micrograph of cells cultured on the Si–PVH/HA mesh for 3 days. The cells elongated on the fiber surfaces andextended over several fibers. Some of them entered the fiber meshand extended within it.

3.4. Bone formation in the prepared meshes

An 8 mm diameter hole in bone is generally not regenerated ifnothing is done to stimulate it. In our preliminary experiment aPLGA mesh was implanted in the same position as that in thisstudy for 12 weeks and no new bone was formed in/on the meshat the center of the hole (data not shown). Bone growth into thedefect from the margin was observed in all of the samples. There-fore, we tested the effects of the bilayered meshes on bone forma-tion by observing new bone formation at the center of the 8 mmholes. This animal test was a fundamental study. Although this ani-mal test was not a load-bearing model, we believe that it was use-ful in evaluating the bone-forming ability of the bilayered meshes.Fig. 11 shows the in vivo response to the bilayered meshes andnew bone formation at the center of the holes. Villanueva-Goldnerstain showed that new bone formed in the mesh 4 weeks afterimplantation. Bone began to form around the pressed pointsformed during layering of the two types of mesh, the Si–PVH/HAmesh and the dense PLA mesh. The bone area expanded in theSi–PVH/HA mesh after 8 weeks. After 12 weeks new bone forma-tion was observed over almost the entire area of implanted Si–PVH/HA mesh. There was no tissue inflammation observedhistologically.

4. Discussion

Earlier work showed that composite films which release siliconcan stimulate MC3T3-E1 cells to proliferate and mineralize [17,24].These composite films were prepared by dip coating a solution ofSiV and PLA composites on a coverglass, drying at room tempera-ture and pealing the dried composite from the glass. The preparedcomposite film had no pores to allow cells to enter, therefore, cellsproliferated only on the film surface. When applying this techniqueto a GBR membrane the composites should have porosity in orderto induce adhesion and proliferation of cells and to provide nutri-tion to the cells.

To address this issue we developed a fiber mesh using PLA andSiV formed by electrospinning. We anticipated that the electrospunfiber mesh would contain a large volume of pores and would showflexibility, both essential components of a GBR membrane. The Si–PVH mesh showed greater flexibility and a higher weight averagedmolecular weight PLA than the PVH mesh, in the case of SiV due tosiloxane on the vaterite surface, which resulted in the formation ofa flexible mesh containing a large amount of SiV powder (60 wt.%).

There has been increasing interest in the use of electrospun fi-brous meshes as scaffolds in medicine. In particular, biocompatiblepolymers such as PLGA and poly(caprolactone) have been appliedas materials for meshes, incorporating various growth factors orDNA [3]. On the other hand, very few studies have been performedon fibrous meshes consisting of polymer/ceramic composites. Pre-viously, PLGA and HA composite scaffolds fabricated by electros-pinning contained only 5–10 wt.% HA nanocrystals [29]. Webelieve that a larger amount of calcium is needed to enhance thebone-forming ability of electrospun fibrous meshes and vateritepowders are effective materials for providing calcium. In the pres-ent work a fibrous mesh containing a large amount of vaterite (upto 60 wt.%) was successfully prepared by kneading and subsequentelectrospinning. Vaterite or SiV particles were embedded in a PLAmatrix, with very thin PLA layers on the particle surfaces, as shownin Fig. 7C and D. Calcium ions are supposed to be released throughdegradation of the thin PLA layer. The PVH mesh, however, wasbrittle, without flexibility, due to a drastic decrease in the PLAmolecular weight after kneading with vaterite powders, as shownin Table 1.

Maeda et al. reported that in vaterite and PLA composites cal-cium ions on vaterite particles formed coordinate bonds with car-boxy groups in the PLA structure [30]. The polymer chains in theSi–PVH mesh, however, were not broken by the formation of coor-dinate bonds between calcium ions and carboxy groups (Fig. 5) dueto siloxane derived from APTES on the vaterite surface, which pre-vents the formation of coordinate bonds. This resulted in the for-mation of a flexible mesh containing a large amount of SiVpowder (60 wt.%).

The specific surface areas of SiV and vaterite were estimated tobe 67.2 and 30.7 m2 g�1, respectively, by nitrogen gas adsorption.The peaks corresponding to vaterite in the XRD pattern of SiV werebroader than that of vaterite (Fig. 3). This result indicates that thediameter of the primary particles of SiV is smaller than that ofvaterite. Crystal growth of the primary particles of vaterite ap-

Fig. 7. Photographs of the prepared meshes. (A) Photograph of the Si–PVH mesh in a flexibility test. The Si–PVH mesh was folded without fracture or fragmentation using apair of tweezers. (B–D) SEM photographs of the Si–PVH mesh. Microfibers approximately 10 lm in diameter are found to twine around one another. The SiV particlesobserved at the microfiber surface are coated with a thin film of the PLA matrix. (D) Fracture surface of a fiber of the mesh. The particles are closely packed in the microfiber.(E and F) SEM photographs of the Si–PVH/HA mesh. The surfaces of the microfibers are coated with leaf-shaped deposits. (G) Crack in the HA coating on the mesh after theflexibility 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 microfibersapproximately 7 lm in diameter.

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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, wasvery small, as shown in Fig. 6.

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

Fig. 9. Numbers of MC3T3-E1 cells after culture on Si–PVH/HA mesh, PLA mesh andThermanox� 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 PVHmesh could not be used as a control mesh due to its extreme brittleness.

Fig. 10. SEM micrograph of the Si–PVH/HA mesh after culturing MC3T3-E1 cells for3 days. Arrows indicate the cells. The cells elongate on the fiber surfaces and extendover several fibers. Some of them enter the fiber mesh and extend within it.

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In general, when organic agents such as growth factors and DNAare applied as additives in scaffold materials they are denatured.Most organic agents suffer damage to their structure on exposureto solvents or heat treatment during preparation of their compositescaffolds. On the other hand, the effects of the silicon species in SiVon 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 ofMC3T3-E1 cells were enhanced on a dense film consisting of SiVand PLA composites [24]. In addition, human osteoblasts and mes-enchymal stem cells were stimulated to mineralize and differenti-ate on siloxane-doped PLA and vaterite hybrid membranes whichreleased a trace amount of silicon species [31,32]. Therefore, thesilicon species appear to be good candidates for the enhancementof cell activation and bone formation. In addition, since the APTESin SiV forms siloxane (Fig. 4), no monomer would be expected to beincluded in SiV. The C–Si and Si–O bonds in siloxane are too strongto be hydrolyzed. The silicon species may, therefore, be releasedwith the siloxane.

Since a large volume of pores is an essential component of aGBR membrane, Si–PVH fibers between 0.5 and 20 lm diameterwere prepared in our laboratory. The diameter was controlled bychanging the conditions during electrospinning, such as the PLAconcentration in the composite solution and the solvent. In thepresent work composite microfibers of 10 lm diameter were usedfor cell culture and the animal tests, because we hypothesized thatthe spaces between the microfibers provide the scaffold for cellproliferation. However, when considering application as a GBRmembrane a microfiber mesh with large spaces may not be anappropriate candidate because the mesh may allow cells and softtissue to permeate through the scaffold. These issues were solvedby preparing the bilayered microfiber mesh using a Si–PVH/HAmesh and a dense PLA mesh.

Hydroxycarbonate apatite, the HA formed in SBF, was reportedto show higher cellular compatibility than pure HA [33]. The pro-liferation of MC3T3-E1 cells on a silicon-doped PLA/vaterite hybridmembrane was reportedly enhanced by an HA coating on themembrane, prepared in SBF [17]. The HA formation in SBF occurredin the presence of functional groups needed to induce the nucle-ation of HA, such as Si–OH, Ti–OH and carboxy groups on the mate-rial surface, and supersaturation of the HA [34,35]. To increase thesupersaturation of HA in SBF it is important to release a large num-ber of Ca2+ ions from the samples. HA formation on the Si–PVHmesh was induced by carboxy groups in PLA and calcium ions re-leased from SiV. The microfiber surfaces of the Si–PVH mesh werecompletely covered with HA after 1 day of immersion in 1.5 SBF

Fig. 11. Histology of in vivo response to the bilayered meshes. Villanueva-Goldnerstaining shows new bone formed in the meshes at the center of 8 mm diameterholes drilled into the frontal midline of rabbit calvaria. Asterisks indicate miner-alized tissues. Arrows indicate pressed points formed during preparation of thebilayered meshes. Bars indicate the mesh area. Bone begins to form around thepressed points formed through layering of the two types of mesh, Si–PVH/HA meshand dense PLA mesh. New bone formation was observed over almost the entire areaof implanted Si–PVH/HA mesh after 12 weeks.

1256 A. Obata et al. / Acta Biomaterialia 6 (2010) 1248–1257

(Fig. 7E and F). The HA formed on the microfibers tightly contactedtheir surfaces (Fig. 7G). The HA layer may contain silicon speciesreleased from the Si–PVH mesh. Maeda et al. reported that siliconspecies released from siloxane-doped PLA and vaterite hybridmembranes were trapped in the HA layer precipitated in SBF [17].

Excessive silicon intake has been regarded as causing kidneydamage, but silicon species have been reported to be one of theessential trace elements for healthy bone and to stimulate osteo-genic cells to proliferate and mineralize [36]. The proliferationand differentiation of MC3T3-E1 cells on a membrane consistingof a Si–PVH (not electrospun) mesh were enhanced compared with

that of PVH [24]. Proliferation of the cells on a HA-coated mem-brane was better than that on an uncoated one [17]. MC3T3-E1cells proliferated well on the Si–PVH/HA mesh, as shown inFig. 9. This may be due to the effects of the silicon species on cellfunction and the HA coating, which shows high cellular compati-bility. In addition, the large pore spaces in the Si–PVH/HA meshare expected to cause the cells to proliferate, since they can enterthe mesh and elongate on the fiber surfaces, as shown in Fig. 10.

With regard to the in vivo portion of the study, histologicalstudies revealed new bone formation in the bilayered mesh atthe center of holes drilled in rabbit calvaria (Fig. 11). Bone forma-tion started from inside the Si–PVH/HA mesh, specifically at thepressed points, and not from the edge of the hole. Mineralizationof osteoblasts in the mesh appeared to be induced at the pressedpoints, where the microfibers were dense. Although this is aninteresting phenomenon, the reason behind it is not clear. Furtherwork to clarify this mechanism is in progress. The mechanicalstrength of the Si–PVH/HA mesh was improved by layering witha PLA mesh, which made available a sufficient amount of spacefor new bone formation in the hole after 12 weeks. The densePLA mesh showed high strength and blocked new bone formationat its interface with the Si–PVH/HA mesh. Thus, the dense PLAmesh worked to maintain the space and the Si–PVH/HA meshworked as a scaffold to induce bone formation by osteoblasts.

5. Conclusion

Silicon-releasing microfiber meshes have been prepared by anelectrospinning technique for application as a GBR membrane.The meshes consisted of a hybrid between silicon-doped vateriteand PLA. The carboxy groups on PLA formed amide bonds withamino groups in the siloxane phase derived from APTES and coor-dinated with calcium ions on the surface of the silicon-doped vate-rite. The meshes showed enhanced HA formation in 1.5 SBF andcontinuously released a trace amount of silicon species into theculture medium, which facilitated bone regeneration. The meshesalso enhanced proliferation of osteoblast-like cells in vitro and in-duced bone formation in vivo.

Acknowledgements

This work was supported in part by a Grant-in-Aid for YoungScientists (B) (No. 21700487) from The Ministry of Education, Cul-ture, Sports, Science and Technology.

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figs. 1, 5, 6, 10 and 11,are difficult to interpret in black and white. The full colour imagescan be found in the on-line version, at doi: 10.1016/j.actbio.2009.11.013.

References

[1] Bramwell VW, Eyles JE, Oya Alpar H. Particulate delivery systems forbiodefence subunit vaccines. Adv Drug Deliv Rev 2005;57:1247–65.

[2] Santerre JP, Woodhouse K, Laroche G, Labow RS. Understanding thebiodegradation of polyurethanes: from classical implants to tissueengineering materials. Biomaterials 2005;26:7457–70.

[3] Still TJ, von Recum HA. Electrospinning: applications in drug delivery andtissue engineering. Biomaterials 2008;29:1989–2006.

[4] Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. Electrospun nanofibrousstructure: a novel scaffold for tissue engineering. J Biomed Mater Res2002;60:613–21.

[5] Xin X, Hussain M, Mao JJ. Continuing differentiation of human mesenchymalstem cells and induced chondrogenic and osteogenic lineages in electrospunPLGA nanofiber scaffold. Biomaterials 2007;28:316–25.

A. Obata et al. / Acta Biomaterialia 6 (2010) 1248–1257 1257

[6] Badami AS, Kreke MR, Thompson MS, Riffle JS, Goldstein AS. Effect of fiberdiameter on spreading, proliferation, and differentiation of osteoblastic cellson electrospun poly(lactic acid) substrates. Biomaterials 2006;27:596–606.

[7] Yang F, Murugan R, Wang S, Ramakrishna S. Electrospinning of nano/microscale poly(L-lactic acid) aligned fibers and their potential in neural tissueengineering. Biomaterials 2005;26:2603–10.

[8] Zeng J, Xu X, Chen X, Liang Q, Bian X, Yang L, et al. Biodegradable electrospunfibers for drug delivery. J Control Release 2003;92:227–31.

[9] Xu X et al. Ultrafine medicated fibers electrospun from W/O emulsions. JControl Release 2005;108:33–42.

[10] Kenawy ER, Bowlin GL, Mansfield K, Layman J, Simpson DG, Sanders EH, et al.Release of tetracycline hydrochloride from electrospun poly(ethylene-co-vinylacetate), poly(lactic acid), and a blend. J Control Release 2002;81:57–64.

[11] Jiang H, Hu Y, Li Y, Zhao P, Zhu K, Chen W. A facile technique to preparebiodegradable coaxial electrospun nanofibers for controlled release ofbioactive agents. J Control Release 2005;108:237–43.

[12] Kim SS, Park MS, Jeon O, Choi CY, Kim BS. Poly(lactide-co-glycolide)/hydroxyapatite composted scaffolds for bone tissue engineering.Biomaterials 2006;27:1399–409.

[13] Zang K, Wang Y, Hillmyer MA, Francis LF. Processing and properties of porouspoly(L-lactide)/bioactive glass composites. Biomaterials 2004;25:2489–500.

[14] Kasuga T, Maeda H, Kato K, Nogami M, Hata K, Ueda M. Preparation ofpoly(lactic acid) composites containing calcium carbonate (vaterite).Biomaterials 2003;24:3247–53.

[15] Liao H, Mutvei H, Sjöström M, Hammarström L, Li J. Tissue responses to naturalaragonite (Margaritifera shell) implants in vivo. Biomaterials 2000;21:457–68.

[16] Vago R, Plotquin D, Bunin A, Sinelnikov I, Atar D, Itzhak D. Hard tissueremodeling using biofabricated coralline biomaterials. J Biochem BiophysMethods 2002;50:253–9.

[17] Maeda H, Kasuga T, Hench LL. Preparation of poly(L-lactic acid)–polysiloxane–calcium carbonate hybrid membranes for guided bone regeneration.Biomaterials 2006;27:1216–22.

[18] Maeda H, Kasuga T, Nogami M. Bonelike apatite coating on skeleton ofpoly(lactic acid) composite sponge. Mater Trans 2004;45:989–93.

[19] Jones JR, Tsigkow O, Coates EE, Stevens MM, Polak JM, Hench LL. Extracellularmatrix formation and mineralization on a phosphate-free porous bioactiveglass scaffold using primary human osteoblast (HOB) cells. Biomaterials2007;28:1653–63.

[20] Xynos ID, Edgar AJ, Buttery LDK, Hench LL, Polak JM. Ionic products of bioactiveglass dissolution increase proliferation of human osteoblasts and induceinsulin-like growth factor II mRNA expression and protein synthesis. BiochemBiophys Res Commun 2000;276:461–5.

[21] Xynos ID, Edgar AJ, Buttery LDK, Hench LL, Polak JM. Gene-expression profilingof human osteoblasts following treatment with the ionic products of Bioglass�

45S5 dissolution. J Biomed Mater Res 2001;55:151–7.

[22] Gough JE, Jones JR, Hench LL. Nodule formation and mineralization of humanprimary osteoblasts cultured on a porous bioactive glass scaffold. Biomaterials2004;25:2039–46.

[23] Reffitt DM et al. Orthosilicic acid stimulates collagen type 1 synthesis andosteoblastic differentiation in human osteoblast-like cells in vitro. Bone2003;32:127–35.

[24] Obata A, Tokuda S, Kasuga T. Enhanced in vitro cell activity on silicon-dopedvaterite/poly(lactic acid) composites. Acta Biomater 2009;5:57–62.

[25] Pham QP, Sharma U, Mikos AG. Electrospun poly(e-caprolactone) microfiberand multilayer nanofiber/microfiber scaffolds: characterization of scaffoldsand measurement of cellular infiltration. Biomacromolecules2006;7:2796–805.

[26] Wakita T, Obata A, Kasuga T. New fabrication process of layered membranesbased on poly(lactic acid) fibers for guided bone regeneration. Mater Trans2009;50:1737–41.

[27] Yabuta T, Tsuru K, Hayakawa S, Ohtsuki C, Osaka A. Synthesis of bioactiveorganic–inorganic hybrids with c-methacryloxypropyltrimethoxysilane. J Sol–Gel Sci Technol 2000;19:745–8.

[28] Asada M, Asada N, Toyoda A, Ando I, Kurosu H. Side-chain structure ofpoly(methacrylic acid) and its zinc salts in the solid state as studied by high-resolution solid-state 13C NMR spectroscopy. J Mol Struct 1991;244:237–48.

[29] Nie H, Wang CH. Fabrication and characterization of PLGA/HAp compositescaffolds for delivery of BMP-2 plasmid DNA. J Control Release2007;120:111–21.

[30] Maeda H, Kasuga T, Nogami M, Ota Y. Preparation of calcium carbonatecomposites and their apatite-forming ability in simulated body fluid. J CeramSoc Jpn 2004;112:S804–8.

[31] Obata A, Kasuga T. Cellular compatibility of bone-like apatite containingsilicon species. J Biomed Mater Res 2008;85A:140–4.

[32] Obata A, Kasuga T. Stimulation of human mesenchymal stem cells andosteoblasts activities in vitro on silicon-releasable scaffolds. J Biomed MaterRes 2009;91A:11–7.

[33] Landi E et al. Biomimetic Mg- and Mg, CO3-substituted hydroxyapatites;synthesis characterization and in vitro behaviour. J Eur Ceram Soc2006;26:2593–601.

[34] Takadama H, Kim H-M, Kokubo T, Nakamura T. Mechanism ofbiomineralization of apatite on a sodium silicate glass: TEM–EDX studyin vitro. Chem Mater 2001;13:1108–13.

[35] Takadama H, Kim H-M, Kokubo T, Nakamura T. An X-ray photoelectronspectroscopy study of the process of apatite formation on bioactive titaniummetal. J Biomed Mater Res 2001;55:185–93.

[36] Carlisle EM. Silicon as an essential trace element in animal nutrition. In:Evered D, O’Connor M, editors. Silicon biochemistry. Chichester: Wiley; 1986.p. 123–39.