PCL based bilayer membranes for guided bone regeneration

Accepted Manuscript Nanosized CaP-silk fibroin-PCL-PEG-PCL/PCL based bilayer membranes for guided bone regeneration

Sibel Türkkan, A. Engin Pazarçeviren, Dilek Keskin, Nesrin E. Machin, Özgür Duygulu, Ayşen Tezcaner PII: DOI: Reference:

S0928-4931(17)30432-0 doi: 10.1016/j.msec.2017.06.016 MSC 8143

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

3 February 2017 26 May 2017 16 June 2017

Please cite this article as: Sibel Türkkan, A. Engin Pazarçeviren, Dilek Keskin, Nesrin E. Machin, Özgür Duygulu, Ayşen Tezcaner , Nanosized CaP-silk fibroin-PCL-PEGPCL/PCL based bilayer membranes for guided bone regeneration, Materials Science & Engineering C (2017), doi: 10.1016/j.msec.2017.06.016

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Nano sized CaP-silk fibroin-PCL-PEG-PCL/PCL based bilayer membranes for guided bone regeneration

Sibel Türkkana, A. Engin Pazarçevirenb, Dilek Keskina,b,c, Nesrin E. Machind, Özgür

Department of Biomedical Engineering, Middle East Technical University, Ankara,

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Duygulue, Ayşen Tezcaner a,b,c*

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06800 Turkey

Department of Engineering Sciences, Middle East Technical University, Ankara, 06800

BIOMATEN Center of Excellence in Biomaterials and Tissue Engineering, Middle East

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c

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Turkey

Department of Chemical Engineering and Applied Chemistry, Atılım University, Ankara

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Technical University, Ankara, 06800 Turkey

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06836, Turkey

Turkey

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Materials Institute, TUBITAK Marmara Research Center, Gebze-Kocaeli, 41470,

*Corresponding author e-mail: [email protected]

Tel: +903122104452

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ABSTRACT Guided bone regeneration (GBR) concept has been developed to prevent the formation of non-functional scar tissue layer on defect site by undertaking barrier role. In this study, a new bilayer membrane which consisted of one layer of electrospun silk fibroin/PCL-

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PEG-PCL incorporating nanocalcium phosphate (SPCA)1 and one layer of PCL membrane was developed for GBR. To improve the osteoconductivity of membranes,

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nano sized calcium phosphates synthesized by Flame Spray Pyrolysis method were

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incorporated into membranes at 10% (wt) (SPCA10) and 20% (wt) (SPCA20) of the polymer content. The structural and chemical analyses revealed the well-integrated two

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layers of membranes with a total thickness of ca 100 µm. In the regenerative layer, the

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highly porous mesh structure had a thickness of 12.6 µm with randomly oriented fibers having diameters around 760 nm, and nanoparticles dispersed homogenously. The

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mechanical test results showed remarkable improvement on the tensile strength of

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membranes with incorporation of nanoparticles. Higher water affinity of nanoCaP included membranes was proved by lower contact angle values and higher percent water uptake capacity. Biomineralization assay revealed that nucleation and growth of apatites

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around fibers of SPCA10 and SPCA20 were apparent while on SPCA0 apatite minerals

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were barely detected after 10 days. Human dental pulp stem cells (DPSC) were seeded on electrospun layer of the bilayer membranes for biocompatibility and osteocompatibility study. Increasing nanoCaP amount resulted in higher cell adhesion, proliferation, ALP activity and calcium deposition on membranes. These overall results confirmed the biocompatibility and potential applicability of proposed membranes for GBR treatments.

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silk fibroin/PCL-PEG-PCL incorporating nanocalcium phosphate (SPCA)

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KEYWORDS Guided bone regeneration, Electrospinning, fibroin, PCL-PEG-PCL, Nanocalcium

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phosphate, Flame Spray Pyrolysis

1. Introduction

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Guided bone regeneration (GBR) therapy concept has been developed primarily on dental

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operations for posterior implants [1]. In such operations membranes are utilized in restoration of alveolar bone tissue and mandible defect sites. The clinical studies of these

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membranes showed that their use resulted with higher intrabony regeneration [2].

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Although there are promising results for GBR membranes which are mostly from preclinical studies and preliminary human trials, there is need for developing better barrier

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membranes with improved functionality.

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GBR membranes are developed to stabilize the wound and substantiate both soft and hard tissues by forming a secluded space for cells to recruit and regenerate the functional

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tissue. As a biomaterial, membranes should show good biocompatibility, have structural and mechanical stability and reasonable degradation rate that matches with newly formed

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new tissue. Besides these properties, GBR membranes are required to suffice the induction of bone regeneration on the surface facing bone tissue while prevent the invasion penetration and migration of fibroblasts from the other face towards bone scar. Therefore, one side of membrane is designed as less porous to resist fibroblast migration and other side is fabricated as more porous to increase surface interaction with bone cells, as well as providing bioactive agents (e.g growth factors, ceramics) that are included for osteoinduction and osteoconduction. There are numerous studies on the development of 3

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GBR membranes involving in vitro and/or in vivo studies [3–5]. In the light of these progresses, GBR membranes have evolved as a candidate biomaterial for bone tissue engineering applications (i.e., long bone defect reconstructions of sizes larger than 4-5 cm in clinics), and for covering bone substitutes to enhance bone healing process [6].

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Poly (ε-caprolactone) (PCL) is one of the widely applied synthetic polymers for tissue

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engineering applications. It is well-known by mechanical strength, flexibility, proper

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degradation profile. There has been several studies of PCL on guided bone regeneration; however, it is combined with nanohydroxyapatite, gelatin, collagen or PLLA in order to

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increase the hydrophilicity and biological activity of membranes [4, 7, 8]. Poly(εcaprolactone)–poly(ethylene glycol)–poly(ε-caprolactone) (PCL-PEG-PCL) is linear

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triblock polymer that has gained interest owing to its hydrophilic residues, and related increased biocompatibility and biodegradability [9]. It has been studied as

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nanohydroxyapatite incorporated electrospun membrane for guided bone regeneration,

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and the biocompatibility of electrospun PCL-PEG-PCL membranes has been reported [10]. Silk fibroin is one of the mostly used natural polymers in tissue engineering. Silk

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fibroin is already used for GBR technique either alone, functionalized or drug loaded. The remarkable mechanical properties, reasonable degradation rate, good biocompatibility

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and hydrophilicity, processability and cost efficiency of fibroin improve the applicability of material for tissue engineering [11]. Besides that characterization studies of silk fibroin as GBR membrane material revealed efficacy of protein on cellular responses as natural extracellular matrix collagen [12, 13] The attempts on improving osteoinductivity and osteoconductivity of resorbable membranes were conducted by the incorporation of bioactive ceramics, like hydroxyapatite (HA) [14], tricalcium phosphate (TCP) [15] and calcium phosphate (CaP) minerals [16]. Particularly nano sized calcium phosphate 4

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particles were favored for mimicking natural bone tissue and enhancing mechanical and biological responses [17]. Recently, industrially applied aerosol derived system, Flame Spray Pyrolysis method, has been utilized for the synthesis of nanoceramics. In the last few years, it has been applied in biomedical studies in order to get nanosized calcium

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phosphate particles. By spraying process, particle size significantly decreases with respect to conventional methods and by flame supply the combustion reaction is conducted in

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which phase and crystallinity of calcium phosphates can be manipulated. It is a highly

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promising, versatile, cost and time effective method [18].

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In this study we aimed to fabricate a novel bilayer composite membrane that is composed of nanoCaP incorporated silk fibroin-PCL-PEG-PCL (SPCA) electrospun layer and PCL

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membrane layer for guided bone regeneration applications. Structural difference on two sides of bilayer membrane was aimed to construct an occlusive barrier against connective

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tissue by hydrophobic PCL layer and to induce bone tissue regeneration by hydrophilic,

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nanoCaP incorporated electrospun SPCA layer. NanoCaP particles were synthesized by Flame Spray Pyrolysis (FSP) method as in our previous study and were sintered at 700oC

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for 30 min before incorporation [19]. Fabricated bilayer membranes were characterized by mechanical tests, scanning electron microscopy (SEM) and Fourier Transform infrared

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spectroscopy (FTIR) analyses. In vitro degradation, hydrophilicity, water uptake capacity, and bioactivity tests were also conducted to characterize further the biomaterials properties of the membranes. Cytocompatibility of the membranes was tested in terms of cell adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells (DPSCs) seeded on electrospun SPCA layers of the bilayer membranes.

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2. Materials The cocoons of Bombyx mori silkworm were obtained from Akman İpek Company (Bursa, Turkey). Calf thymus DNA, Sodium bicarbonate (NaHCO3), lithium bromide (LiBr), ε-caprolactone, PEG (Mn: 4000 Da) and HFIP (1,1,1,3,3,3-Hexafluoro-2-

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propanol) were purchased from Sigma Aldrich. All other chemicals used in the study

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were of reagent grade and used as purchased.

3.1.

Synthesis of PCL-PEG-PCL (PCEC)

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3. Experimental

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PCEC triblock copolymer was synthesized by ring opening polymerization of εcaprolactone (ε-CL) initiated by PEG as explained in reported study [20]. Briefly, PEG

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was kept in 100°C for 30 min under N2 flow to remove moisture. Then, ε-CL with PEG/εCL (1:24 (w/w) feed ratio) and dibutyltin dilaurate as catalyst (with a concentration of

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0.5% of total reactants) were added and the mixture was stirred at 140°C for 24 h under

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N2 atmosphere. Synthesized copolymer was dissolved in dichloromethane and precipitated by adding cold ethanol to remove the catalyst and residual ε-CL. The

Preparation of bilayer membranes

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3.2.

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precipitate was then filtered and dried at 40°C in vacuum oven.

The layers were prepared with solvent casting and electrospinning methods. PCL (10%, w/v) was dissolved in chloroform and casted on petri glass and dried at room temperature. The dry PCL membrane surface was wetted with 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) prior to electrospinning of second layer.

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NanoCaP synthesis and characterization Nanocalcium phosphate particles were synthesized by flame spray pyrolysis synthesis as stated in our previous work [19]. Briefly, calcium acetate hydrate (99. 99% SigmaAldrich) was dissolved in propionic acid (for synthesis ≥ 99%, Merck) at 60oC for 1 hour,

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and tributyl phosphate precursor (assay ≥ 99%, Sigma-Aldrich) was added and stirred until a clear solution was observed. The liquid precursors were pumped through nozzle at

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7 rpm (2.4 ml/min), dispersed by oxygen gas (9 L/min) and ignited with methane (2

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L/min) and oxygen (2 L/min) premixed gases. The formed nanoparticles were collected on a glass fiber filter above the flame, with the aid of a vacuum pump. Before membrane

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fabrications, CaP nanoparticles were sintered at 700oC for 30 min starting from room

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temperature with a 10oC/min heating rate in order to remove the impurities in phase variations.

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The structural and morphological characterizations of synthesized sintered nanoparticles

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were analyzed by using FTIR. The samples were pelleted with potassium bromide (KBr) (Bruker Platinum ATR-IR spectrometer, Germany) (at spectral region 400-4000 cm-1),

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XRD (X-Ray Diffractometer, D/MAX2200/PC, Rigaku Co., Japan) scanning electron microscopy (JEOL JSM-6335F FEG/SEM operating at 20 kV equipped with an Oxford

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Instruments X-Max80,AZtec software), transmission electron microscopy-energy dispersive spectrometry (JEOL-2100 HRTEM operating at 200 kV (LaB6 filament) Images were taken by Gatan Model 694 Slow Scan CCD Camera

Preparation of electrospun layer by electrospinning Firstly, silk fibroin was isolated from cocoons of Bombyx mori [21]. Briefly, cocoons were cut into small pieces and boiled with 0.02 M NaHCO3 for 30 min and washed with 7

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deionized water several times to remove the sericin proteins from the silk fibers. The degummed silk fibers were then dried overnight at 37oC. Dried fibers were dissolved in 9.3 M LiBr at 60oC for 4 h and the solution was centrifuged at 9000 rpm for 20 min at 4oC in order to remove undissolved fibers or debris. The regenerated silk fibroin solution

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was dialyzed against deionized water for 48 h with several changes of water to remove the lithium bromide. The obtained aqueous silk fibroin solution was frozen and

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lyophilized. The dried silk sponge was then dissolved in HFIP to obtain a final

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concentration of 5% (w/v).

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Electrospinning conditions were optimized according to the ambient temperature at 25⁰C and 20% humidity. Silk fibroin/PCL-PEG-PCL (SP) blend was filled into 2 mL sterile

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polypropylene syringe with blunt 18G needle (Hayat Siringa, Turkey) and connected to syringe pump (New Era, USA). PCL film was placed on the aluminum collector and the

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surface of the membrane was moistened with HFIP prior to electrospinning. During

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electrospinning the collector was rotated at 100 rpm in clockwise direction. Then, SP blend was electrospun with 1.5 mL/h flow rate under 20 kV at a distance of 15 cm from

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needle tip to collector. The fabricated bilayer membrane groups are listed in Table 1.

Table 1. Compositions of the bilayer membranes used in the study. Abbreviation

Polymer Blend

nanoCaP Content

SPCA0

5% (w/v) SF and PCEC in HFIP

0

SPCA10

5% (w/v) SF and PCEC in HFIP

10% (w/w) to polymer blend

SPCA20

5% (w/v) SF and PCEC in HFIP

20% (w/w) to polymer blend

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3.3.

Membrane characterization

3.3.1.

Structural analysis

Morphology of the bilayer membranes was investigated by SEM analysis (FEI

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NanoSEM, USA). Thickness of nonporous PCL layer and electrospun fiber diameters

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were determined with ImageJ software (NIH, USA) by at least 250 measurements from

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each sample. For in vitro degradation study, each membrane was cut in an area of 5 mm x 5 mm by titanium blades, and incubated in 1 ml lipase solution (110 U/L in PBS, 0.1

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M, pH 7.4) under 37°C for a week (n=4). The lipase solution was refreshed each day. In addition, the chemical composition of bilayer membranes was characterized by FT-IR

Water uptake and contact angle measurements

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3.3.2.

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(IFS 66/S, Bruker, Germany) analysis.

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Membranes were cut at dimensions; 5 mm x 5 mm for water uptake analysis. Samples were dipped in PBS for various time intervals (3, 24, 48 and 72 h). Afterwards, they were

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weighed until no weight change was observed (n=4). Equation 1 was used to determine

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the water uptake capacity of the bilayer membranes:

𝑊𝑎𝑡𝑒𝑟 𝑈𝑝𝑡𝑎𝑘𝑒 (%) = ((𝑊𝑤 − 𝑊𝑖 )/𝑊𝑖 )𝑥100

(1)

where ww refers to the weight of the samples after awaiting in PBS for defined periods of time, and wi was the initial weight of the samples.

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Surface wettability of the bilayer membranes was analyzed by water contact angle measurements using goniometer (Attension, Biolin Scientific, Sweden) at 25°C. Sessile drop method was employed on both layer surfaces of bilayer membranes for up to 3 sec

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(n=4). The contact angles were measured applying Young-Laplace formula (Equation 2):

(2)

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𝛾𝑠𝑣 = 𝛾𝑠𝑙 + 𝛾𝑙𝑣 𝑐𝑜𝑠𝜃𝛾

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where γsv, γsl and γlv refer to solid-vapor, solid-liquid, and liquid-vapor interfacial

3.3.3.

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tensions respectively, and cosθγ was the wetting angle. In vitro bioactivity study

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In vitro bioactivity of the bilayer membranes was analyzed through incubation in stimulated body fluid (SBF) for predetermined time periods (1, 5 and 10 days). SBF (1X)

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was prepared as previously reported [22]. Bilayer membranes were punched to obtain

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samples having 12 mm diameter. The samples were sterilized in 70% ethanol for 2 h prior to incubation in SBF. Morphology of apatite-like structures on fibers was investigated by

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SEM analysis (FEI NanoSEM, USA) and quantified by SEM-energy dispersive

3.3.4.

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spectrometry (EDS) analysis

Mechanical study

In order to determine mechanical strength of the bilayer membranes, pull to break analysis was employed (n=4). Briefly, dry membranes were cut by 5 mm x 10 mm and confined between jaws connected to a universal mechanical testing machine (LR50 K Lloyd Instruments, UK). To prevent jaw slippage, coarse sandpaper was fixed on the jaws. Then, samples were tested under uniaxial tension with a crosshead speed of 1 mm/min until

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break. Ultimate tensile strength (UTS) and tensile modulus of the membranes were determined at break when maximum tensile loading occurred on the specimens [23]. Four specimens were tested for each group. 3.4.

Cell culture studies

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3.4.1. Cell viability Cytocompatibility study of the bilayer membranes was conducted with human dental pulp

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stem cells (DPSCs, 3rd passage). DPSCs were cultured in cell culture medium consisting

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Dulbecco’s MEM supplemented with 10% FBS and 100 units penicillin/streptomycin at 37°C in 5% CO2 atmosphere in carbon dioxide incubator (5215 Shel Lab., Cornelius, OR,

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USA). After reaching 80% confluency, cells were subcultured using 0.1% of

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Trypsin/EDTA solution. Bilayer membranes were punched in 12 mm diameter samples and sterilized in 70% ethanol containing PBS for 2 h in 12 well tissue culture plates

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(TCPS). Then, DPSCs were seeded on the electrospun layer of the bilayer membranes at

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an initial seeding density of 25 000 cell/sample in 25 µL to improve cellular attachment on samples. After seeding samples were kept in CO2 incubator for 1 h and 10 µL cell culture medium was added every 15 min to prevent drying. After 2 h, 2 mL cell culture

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medium was added and samples were incubated for defined time periods (1, 4 and 7 days).

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At the end of each time point, Prestoblue viability reagent (Invitrogen, USA) was added to samples in cell culture medium at a ratio of 1/9 (v/v) and incubated for 6 h. At the end of incubation period, aliquots were taken from media and colorimetric measurements were performed at 570 nm as main wavelength and 600 nm as reference wavelength. Viability of DPSCs on samples were determined as percent reduction of reagent using supplier’s protocol. The morphology of cells seeded on membrane 1st and 7th day incubation was examined by SEM. The samples were rinsed with PBS and fixed by 4%

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paraformaldehyde solution. Cell viability on membranes was also assessed by confocal microscopy analysis. Fixed cells on samples were permeabilized with 1% (v/v) Triton X100 in dH2O and were stained by FITC for cytoskeleton (green) and by DRAQ5 for DNA (red). To prevent non-specific staining, samples were incubated in 1% (w/v) bovine serum

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albumin (BSA) in PBS for 30 min under 37°C prior to staining of cellular components. 3.4.2. Osteogenic differentiation

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Initial seeding density of cells on bilayer membranes was 25,000 cells/membrane and

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after seeding, cells were incubated for 2h to attach. In order to ensure cell proliferation

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on the samples, membranes were incubated in cell culture medium for 3 days. Then, cell culture medium was replaced with osteogenic differentiation medium (DMEM/F12

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supplemented with 10% FBS, 100 units Penicillin/Streptomycin and 10-7 M dexamethasone, 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid). Samples were

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incubated for 7, 14 and 21 days. At the end of each time point, samples were rinsed with

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PBS and kept in cold ALP lysis buffer prepared by 0.1% Triton X-100, 0.1% (w/v) sodium azide and 1% protease inhibitor cocktail in PBS for 30 min. Lysates were then

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incubated with p-nitrophenyl phosphate solution in a ratio of 1:5 (v/v) at 37⁰C for 30 min. 4-nitrophenol was measured spectrophotometrically at 405 nm in microplate reader and

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its amount was determined using the calibration curve constructed in the range of 0-250 µM concentration of 4-nitrophenol. Moreover, lysates were incubated in Hoechst DNA dye in a ratio of 1:8 (v/v) for 10 min at 25⁰C, and subsequently fluorescence readings were performed with fluorometer (Modulus, Turner Biosystems, USA). Calf thymus genomic unsheared DNA at different concentrations (0-1000 ng/mL) was used to construct the calibration curve for Hoechst DNA dye in order to determine the DNA amounts in the cell lysates.

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3.4.3. Statistical analysis One way analysis of variance (ANOVA) and Tukey’s tests for multiple comparisons were done to assess the statistical significance using SPSS Statistics software (ver. 23.0; IBM Corporation, NY, USA). Differences were considered statistically significant at p ≤ 0.05.

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4. Results and discussion In this study, we incorporated previously synthesized nanocalcium phosphates [19] and

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examined their structural and biological effects in SP based bilayer GBR membranes.

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Before membrane fabrication, particles with different calcium to phosphate ratios (Ca/P:

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1.54, 1.91, 2.19) were sintered at 700oC and 900oC for 30 min, 60 min and 120 min in order to examine the crystal phase and crystallinity. The effects of sintering process of

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particles were examined by XRD, SEM and TEM analyses (Data not presented). Among the results, 700oC 30 min sintered particles with Ca/P: 2.19 ratio formed pure crystal

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structures and least agglomerates and was chosen for membrane studies. In samples

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treated at higher temperature, extra necking and agglomeration leaded higher grain sizes were observed (Data not presented). After 30 min treatment at 700 oC, SEM and TEM

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analyses revealed that the morphology and the crystal structure of the particles were

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conserved. (Figure 1b,c).

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Figure 1 SEM image (A), TEM image (B) and crystalline regions of particles (C), FTIR

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spectrum (D) and XRD patterns (E) of nanoCaP (Ca/P: 2.19) sintered at 700oC for 30

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min. The labels on XRD patterns indicate the hydroxyapatite specific peaks.

XRD analysis was performed to examine the crystal phase of sintered particles, and

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patterns were compared with ICDD reference patterns of hydroxyapatite (Figure 1d). The

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samples with different sintering time and temperatures, gave similar XRD patterns. In Figure 1d, XRD spectrum of least agglomerated sample sintered at 700oC for 30 min is presented. Observed sharp peaks were correlated with characteristic diffraction peaks of hexagonal Ca10(PO4)6(OH)2. Particularly, observed peaks around 25.9°, 31.8°, 32.9°, and 34.1° corresponded to the reflections of 002, 211, 300 and 202 of an apatite structure respectively [24].

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The chemical composition of particles was examined by FTIR- ATR analysis (Figure 1e). The spectrum was compared with literature, and weak peak around 3640 cm-1 was assigned to OH stretching of H2O in crystal lattice. Broad and weak peak around 1456 cm-1 and weak peak around 878 cm-1 were assigned as CO3 in crystal lattice which could

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be due to remaining organic groups in Ca or P precursors. The strong peaks around 1041 cm-1 and 1073 cm-1 were as triply degenerated vibration (v3) of P-O bonds and the peaks

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around 634 cm-1 and 571 cm-1 were assigned as triply degenerated bending mode (v4) of

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O-P-O bonds [24, 25].

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To accomplish the functional composite structure of GBR membranes, proposed bilayer membrane model was developed by combining solvent casting and electrospinning

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fabrication methods. For this, nanocalcium phosphate particles containing silk fibroin, and PCL-PEG-PCL (SPCA) blend was electrospun on solvent casted PCL membrane. In

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solvent casting method, solvent aeration creates a rough and semi porous top surface

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compared to more smooth and less porous glass facing one. This aerated nonpolar PCL surface was used to hold nanofibers of polar polymer blend during electrospinning. To

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increase the conductivity, the membrane surface was moistened with solvent of the polymer. The surface morphology of the bilayer membranes was evaluated with SEM

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analysis (Figure 2). The SEM micrographs revealed that interconnected and open porous networks were developed by randomly oriented fiber formation via electrospinning in the presence or absence of CaP. The average diameter of CaP excluded fibers was found as 266 nm from SEM micrographs (Figure 2a). In CaP included networks, the average diameter of fibers increased about 3 folds and recorded as 756 nm (Figure 2d). This was an expected result since incorporation of inorganic particles into electrospinning blend can decrease the conductivity of the mixture and form thicker fibers [26]. Thicknesses of 15

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the electrospun layer and PCL membrane layer in bilayer membrane were around 12.60 µm and 90.55 µm, respectively (Figure 2b, c). The diameter and thickness values were in agreement with other electrospun GBR membrane samples in literature. The average diameters of fibers were reported between 200 and 800 nm, and total thickness of

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membrane was reported to be 100 µm [27]. The integration of nanoCaP particles was verified by observing nodules through fibers in SEM images (Figure 2e). The EDS

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analysis of these agglomerates was performed, and it was elicited that they were

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incorporated CaP particles (Figure 2f) The nanoparticles dispersed through fibers were

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visualized by Ca and P element specific on spot EDS mapping (Figure 2g,h,j).

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Figure 2 SEM electrographs of electrospun layer without nanoCaP (A), cross-section of PCL layer of the GBR membrane (B), electrospun layer cross-section (C), electrospun layer w/ nanoCaP (D), close-up view of nanoCaP in fibers (E) and EDS result of nanoCaP (Ca/P ratio = 2.19) (F). EDS mapping micrographs of SEM image of the fibers (G), Ca element specific (H), C element specific (I), P element specific (J), O element specific (K) visualization.

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The presence of nanoparticles and integration of the two layers of membranes were also confirmed by FTIR analysis (Figure 3a). In FTIR scan of top electrospun surface of constructed membranes, the absorption bands observed at 1632 cm-1 (amide I), 1506 cm1

(amide II) and 1232 cm-1 (amide III) represented the specific stretching of silk fibroin

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protein which is in agreement with literature [28]. In order to examine these absorption bands, FTIR spectrum of only silk fibroin was also included in Figure 3a. PCEC specific

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strong peaks were marked at 2940 cm-1, 2868 cm-1 and 1723 cm-1. The C=O stretching

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vibrations of ester carbonyl group of PCL and PCEC were notably appeared at 1723cm . It was noted that separately PCEC should have peaks at region 1240-1106 cm-1 which

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belong to C-O-C stretching vibrations of repeated –OCH2CH3 units of PEG [10].

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However, PCEC was examined as electrospinning blend and mixed with fibroin so that in this region, fibroin has its relatively prevailed amide III stretching which dominated

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PCEC peaks. As stated above, CaP particles have characteristic strong -PO4 vibrations at

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1041 cm-1 and when electrospinning blend with and without particles are compared, the difference in this peak was clearly seen. The FTIR scan of bottom PCL cast layer exhibited peaks specific to pure PCL (Figure 3a). The bands at 1723cm-1 belong to

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stretching vibrations of the carboxyl groups (C=O), bands at 1180 cm-1 belong to

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stretching vibrations of the ether groups (C–O–C), and the bands at 2940 cm-1 and 2868 cm−1 belong to symmetric C–H stretching.

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Figure 3 FTIR ATR spectra of silk fibroin membrane, CaP included and excluded bilayer membranes (A). Water contact angle measurements of PCL (81.53°) (B), SPCA0 (80.35°) (C), SPCA10 (39.74°) (D) and SPCA20 (48.18°) (E) Weight loss ratio (%) (F) Water

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Uptake (%) (G) and Stress strain curve (H) of composite bilayered membranes.

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The wettability tests were performed in order to gain an insight on the surface activity of membranes for initial cell attachment. The decrease in contact angle values of electrospun fibers showed their improved hydrophilicity with the increasing amount of nanoCaP (Figure 3c-e). In addition, GBR membranes with a top electrospun layer had lower contact angle values compared to pure PCL film (Figure 3b-e). The pure PCL film had a contact angle of 81° (Figure 3b). The surfaces having contact angle of less than 90° are assigned as hydrophilic surfaces [10]. Meanwhile, in the same study, the contact angle of 19

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electrospun PCEC surface has been defined as a hydrophilic layer and the contact angle was reported as 82.5± 2.3° [10]. Here, HFIP is used as the main solvent during the preparation of solvent casted PCL film as well as electrospinning the fibrous layer of the bilayered GBR membranes. HFIP is a water miscible organic solvent and having alcohol

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side groups displaying higher hydrophilicity than other organic solvents like chloroform, tetrahydrofuran and dichloromethane [29]. Therefore HFIP can form extensive hydrogen

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bonding than other solvents exemplified previously. Consequently, the use of HFIP might

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have led to the rearrangement of polymer chains resulting in a decrease in the water contact angle. This could be explained by the fact that the surface properties of the final

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product are in direct relationship with the solvent used [30]. In addition, the hydrophilicity

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of electrospun layer of fabricated membranes was proved by contact angle values being less than 90°. Furthermore, in our membranes, hydrophilic amino groups and carboxylic

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groups of silk fibroin also had an effect on slight decrease in the contact angle value of

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bilayer membranes (80.35°±0.05). There was a slight difference between contact angle values of SPCA10 (39.74°) and SPCA20 (48.18°) samples. The observed difference could be as a consequence of the change in the surface properties of membranes like roughness.

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As stated previously, SEM examinations revealed that fiber sizes increased and fibers

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become rougher with the addition of more nanoparticles. The water drop could not penetrate on rough CaP containing fibers as well as on smooth fibers [10, 31]. In vitro degradation was conducted in lipase solution. Lipase specifically attacks on ester groups of PCL or peptide bonds in silk fibroin. The degradation profile of bilayer membranes revealed that 11% wt loss occured by enzymatic cleavage within a week. There was no significant difference between weight loss values (%) of CaP included and excluded membranes (Figure 3f). Correspondingly, there was no significant difference between

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water uptake capacity of bilayered membranes. Indeed there was a slight increase in water affinity of SPCA10 (380%) and SPCA20 (540%) due to increase in CaP content (Figure 3g). To evaluate mechanical stability of membranes, tensile tests were applied. Tensile test in dry conditions was done to assess the handling flexibility of membranes. This test

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gives an indication of bending ability and ease of shaping, or cutting of membranes when periodontists or orthopedists need to apply on a scar tissue. The analysis revealed that,

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with increasing nanoCaP content of membranes, higher ultimate tensile strength values

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were observed. The additional nanoCaP related energy dissipating mechanism and plasticization were not detected (Figure 3h). The strain-stress curves of membranes

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revealed that samples obeyed Hooke's law up to 8% strain and then continued with elastic

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to plastic transitional patterns. Stiffer bilayer membrane structures were observed with increasing nanoCaP amounts. The ultimate tensile strength (UTS) was significantly higher for the highest amount of CaP incorporated group when compared with no CaP

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added one;(p ≤ 0.05); 11.1±0.4MPa (20% wt. CaP) vs 4.9±1.0 MPa (0% wt. CaP). SPCA20 membranes showed the highest UTS value among all samples. However, no statistical difference was observed between SPCA10 and SPCA20 membranes (Figure The

reported

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3h).

composite

electrospun

GBR

membranes

of

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PLGA/collagen/nanohydroxyapatite (5%wt) recorded as 7.3±0.6 MPa ultimate tensile strength value [33]. In another reported multilayered GBR membrane (nanohydroxyapatite/collagen/poly(lactic-co-glycolic-acid) (nCHAC/PLGA) and PLGA) showed higher tensile strength value (9.7±1.7 MPa) compared to neighboring interosseous ligament (0.14±0.05 MPa) [32]. It was deduced that these membranes can be well integrated into tissue site under the force from neighboring ligament. In our two layered membrane model, SPCA20 (11.1±0.4MPa) and SPCA10 (7.1 ±0.8 MPa) were in

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closer values and in agreement with literature. Our membranes showed improved elastic behavior with nanoCaP particles incorporation into the fibers than excluded membranes. The corresponding increase in tensile strength and preserving elasticity suggest that our bilayered CaP included membranes can be considered as prospective GBR membranes.

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To evaluate the in vitro bioactivity of membranes with different CaP amounts (SPCA0, SPCA10, and SPCA20), samples were soaked into simulated body fluids (SBF).

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Membranes were examined by SEM micrographs at 1, 5 and 10 days of incubation in

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SBF (1X) (Error! Reference source not found.a). As shown in 5µm scale was clearly observed that in nanoCaP containing bilayer membranes, SPCA10 and SPCA20, fibers

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were covered with cauliflower-like apatatite-like formations and there was no significant

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difference in the distribution of these apatite-like structures. Precipitation on SPCA10 and SPCA20 membranes started as early as the 1st day of incubation in SBF and the increase

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in apatite amount was noticed by coverage of the fibers during 10 days of examination

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(Figure 4d-f, g-I). Meanwhile SPCA0 samples showed mineral precipitation on surface after 10 days of SBF immersion (Figure 4a-c). SEM images revealed that fibers were not always covered homogenously with nanoparticles. However it was interpreted that

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coverage was comparable to findings in the literature and clearly nanoCaP acted as

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nucleation sites and initiated precipitation on surface [33]. In bone mineralization metabolism, initially amorphous calcium phosphate minerals nucleate on extracellular matrix and in dynamic ionic environment eventually precipitated minerals hydrolyze to hydroxyapatite in Ca/P ratio: 1.67 [34]. SEM-EDS analysis revealed that in SBF solution incorporated nano CaP particles in SPCA10 and SPCA20 have undergone biomimetic mineralization process and Ca/P ratio of minerals on surface varied (Supplementary Figure 1). After 10 days of incubation, Ca/P ratio recorded (1.68) for apatite-like

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precipitations on SPCA10 bilayered membranes was closer to the ratio in natural bone mineral (Figure 4f). This finding was in correlation with the literature and minerals were in similar surface ionic interaction and change in Ca/P atomic ratio for the growth of bone apatite minerals [34]. SPCA10 and SPCA20 membranes showed better bioactivity

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potential than SPCA0 by the surface activating effect of nanoCaP that observed in water affinity tests by dramatic decrease in contact angle and notable increase in percent water

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uptake capacity. Similar results were presented in another multilayered membrane study

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in which as prepared nanoCaP particles synthesized by flame spray pyrolysis (FSP) were blended with collagen and PLGA matrix in different weight ratios. Increasing nanoCaP

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amount resulted in more apatite-like precipitates on electrospun fibers [16]. It was

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deduced that incorporation of nanoapatites served as a surface to new apatite like

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precipitates that would provide good bone bonding behavior for the fabricated structure.

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Figure 4 SEM images of bioactivity study of membranes in SBF (1X). SPCA0 from

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day 1 to day 10 (A-B-C), SPCA10 from day 1 to day 10, respectively (D-E-F) and SPCA20 from day 1 to day10 respectively (G-H-I) Elemental analysis results (SEM-

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EDS) of the precipitates are included on the SEM images

For biocompatibility studies, human dental pulp stem cells (DPSCs) were used. DPSCs are gaining an attention in guided bone or tissue regeneration especially for periodontal tissue engineering. by their osteogenic origin, proliferation profile and differentiation capacity [35]. Cells were seeded on both sides of bilayered membranes and morphology of cells was examined by SEM at different time points (Figure 5). After day1 incubation, SEM images revealed that DPSCs had attached on both sides. After 5 days of incubation,

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it was observed that electrospun side of membranes was more favorable for cell attachment and proliferation. The osteogenic cells have distinct morphologies and cytoskeletal reorganization on distinct biomaterial surfaces. [36]. Randomly oriented fibers, fiber diameter and roughness provided required mesh structure for cells to attach.

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After 10 days of incubation confocal microscopy images revealed that there were more well spread alive cells with extended cytoplasmic attachment on electrospun layers

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compared to PCL layer. Higher cell number was observed on SPCA10 and SPCA20

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layers than on SPCA0 layer (Figure 5 i, l, o). Viability tests confirmed that with increasing nanoCaP, cell proliferation increased. SPCA20 membranes showed highest viability

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values (p ≤ 0.05) (Figure 5a). As in correlation with water uptake results, surface

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hydrophilicity of fibers increased with nanoCaP addition which would contribute to access of cells to nutrition [27]. Calcium phosphate particles were reported to have a high

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protein adsorption capacity by ionic interactions [37]. Previously, adsorbed amount of

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adhesion molecules fibronectin and vitronectin on fibers of PCL, collagen and nanohydroxyapatite based electrospun scaffolds were revealed extra interaction of nanohydroxyapatite with serum proteins, and akin mesenchymal stem cell (MSC)

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viability arised upon seeding [8]. Nanosized calcium phosphate particles (20 to 80 nm)

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mimic natural bone minerals and induce differentiation of osteogenic cells [38]. In order to elucidate the biological response towards electrospun layer of bilayer membranes, osteogenic metabolism of cells was examined (Figure 5b,c). One of the osteogenic markers, ALP enzyme is responsible from the initiation of biomineralization through the biological production of inorganic phosphate groups in extracellular environment and intracellular storage of calcium ions [39]. In metabolism, by initial intracellular ALP activity increase, cellular differentiation to osteogenic phenotype initiates. A decrease in

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intracellular ALP activity in following time points is a sign for the influx of inorganic phosphate to intracellular environment. ALP activity and Ca2+ storage studies revealed that nanoCaP presence improved ALP synthesis and intracellular calcium storage of cells (Figure 5Error! Reference source not found.b,c).Among samples, cells on SPCA20

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membranes showed significantly highest ALP enzyme expression at 2nd week (p ≤ 0.05). Lowest ALP enzyme activity on SPCA0 membranes proved the osteoinductive effect of

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nano CaP. Similarly, intracellular calcium storage of cells on SPCA10 and SPCA20

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samples showed significantly higher amounts compared to cells seeded on SPCA0 samples (Figure 5c). NanoCaP provided significant increase in calcium intake and storage

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of cells and thus it enhanced osteogenic differentiation of cells. Consequently, in vitro

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cell culture studies showed that the electrospun layer of membranes designed for interfacing bone surface had high porosity and proper surface activity that satisfies the

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requirements for cellular attachment and functionality of osteogenic cells. Meanwhile,

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nonporous PCL layer prevents infiltration of fibroblasts and forms a secluded space for

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bone tissue formation within porous electrospun layer.

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Figure 5 Proliferation (A), ALP activity (B) and intracellular calcium deposition (C)

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of human dental pulp stem cells (DPSCs) seeded on electrospun layer of bilayer membranes. Representative SEM and confocal microscopy images of DPSCs on PCL

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(D-E-F) SPCA0 (G-H-I) SPCA10 (J-K-L) and SPCA20 (M-N-O) from day1 to day10 respectively (Significant differences between samples were designated by # and &, p<0.05)

5. Conclusion A composite bilayer membrane composed of nanoCaP incorporated silk fibroin-PCLPEG-PCL (SPCA) electrospun layer and solvent cast PCL membrane layer was fabricated for the first time for guided bone regeneration applications. Structural and chemical analyses proved that two different phase formed a stable coherent structure. The 27

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mechanical reinforcing effect of CaP particles were observed with increasing tensile strength of membrane. Increasing CaP content of the electrospun SPCA layer increased the hydrophilicity and water uptake ability of the membranes which, in turn, resulted in improved dental pulp stem cell attachment, proliferation and migration within pores of

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the electrospun layer. Additionally, CaP nanoparticles incorporated membranes served as nucleation sites for biological minerals and triggered further mineralization which

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improved osteoinductive and osteoconductive properties of membranes. Our preliminary

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results suggest that CaP incorporated bilayer membranes are good candidates for GBR

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applications after being tested in vitro.

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Acknowledgments

The authors would like to acknowledge with thanks the financial support from the METU

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BAP-07-02- 2013-00, and The Scientific and Technological Research Council of Turkey

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(TUBITAK, Grant No 106M232). Sibel Türkkan (Ataol) was supported financially by TUBITAK (Grant No 112T749). The authors would also like to express their appreciation

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by Dr. Arda Büyüksungur (BIOMATEN) for the technical support and Reza Moonesi

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Rad for providing DPSCs.

Conflict of interest There is no conflict of interest for the study.

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Highlights 

Fabricated bilayer membrane was proposed for guided bone regeneration applications

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Silk Fibroin/PCL-PEG-PCL electrospun and PCL casted bilayer was fabricated

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for the first time Synthesized nano calcium phosphates assisted fiber integrity in electrospun layer

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Structural and chemical analyses proved that there is a stable and coherent

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Nanoparticles included in the GBR bilayer membrane improved mechanical

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properties and biological properties significantly

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

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structure of composite GBR bilayer membrane

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

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