Non-mulberry silk fibroin grafted PCL nanofibrous scaffold: Promising ECM for bone tissue engineering

Accepted Manuscript Non-mulberry silk fibroin grafted PCL nanofibrous scaffold: Promising ECM for bone tissue engineering Promita Bhattacharjee, Deboki Naskar, Hae-Won Kim, Tapas K. Maiti, Debasis Bhattacharya, Subhas C. Kundu PII: DOI: Reference:

S0014-3057(15)00442-5 http://dx.doi.org/10.1016/j.eurpolymj.2015.08.025 EPJ 7049

To appear in:

European Polymer Journal

Received Date: Revised Date: Accepted Date:

16 June 2015 17 August 2015 22 August 2015

Please cite this article as: Bhattacharjee, P., Naskar, D., Kim, H-W., Maiti, T.K., Bhattacharya, D., Kundu, S.C., Non-mulberry silk fibroin grafted PCL nanofibrous scaffold: Promising ECM for bone tissue engineering, European Polymer Journal (2015), doi: http://dx.doi.org/10.1016/j.eurpolymj.2015.08.025

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Non-mulberry silk fibroin grafted PCL nanofibrous scaffold: Promising ECM for bone tissue engineering Promita Bhattacharjee1,Deboki Naskar2, Hae-Won Kim3, Tapas K. Maiti2, Debasis Bhattacharya1*, Subhas C. Kundu2** 1

Materials Science Centre, 2Department of Biotechnology,Indian Institute of Technology Kharagpur, West Bengal-721302, India 3

Institute of Tissue Regeneration Engineering (ITREN) and Department of Nanobiomedical Science BK21 Plus NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 330-714, South Korea.

*Corresponding authors: *Professor Debasis Bhattacharya

Telephone: +91 - 3222 – 283976;Fax: +91-3222-278707 E-mail: [email protected] (D. Bhattacharya)

**Professor S. C. Kundu Telephone: +91-3222- 283764;Fax: +91-3222-278707 E-mail: [email protected] (S. C. Kundu)

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Abstract A comparative study of nanofibrous scaffolds with inclusion of nonmulberry silk protein fibroin is presented for application in bone tissue engineering. Introduction of silk fibroin into the scaffolds is carried out in two ways: by electrospinning blend of poly(Є-caprolactone) (PCL) and by grafting fibroin on aminolyzed electrospun nanofibrous PCL. Verification of aminolysis was provided by confocal laser microscopy of rhodamine B isothiocyanate tagged substrates. Absorbance spectroscopy of the products of the reaction between NH2 groups and ninhydrin was used fo quantification of aminolysis. Presence of nitrogen on the substrates was established using energy dispersive X-ray while scanning electron microscopy was used to substantiate their nanofibrous morphology. Evaluation of ATR-FTIR results showed that secondary structure of fibroin was preserved in the respective substrates. Presence of fibroin improves hydrophilicity, measured by dynamic contact angle, and surface roughness, topography viewed by atomic force microscopy.

These characteristics support cell growth

and proliferation. The mechanical strength of the scaffolds is enhanced due to presence of fibroin. Different biophysical characterizations indicate better hydrophilicity, higher nitrogen content, and higher surface roughness of the fibroin grafted scaffolds.

Both fibroin-grafted

and fibroin-blended scaffolds successfully support activity and viability of human osteoblast like cells. Cell cycle analysis, alkaline phosphatase assay and Alizarin red S staining are used to substantiate cell cycle pattern, proliferation and resultant neo-matrix generation on the scaffolds respectively. The results show that fibroin grafted matrices are better at supporting cell adhesion, growth, and proliferation. The findings demonstrate advantages of fibroin blended and grafted matrices for use in bone tissue engineering applications. Keywords: Nonmulberry silk, poly (Є-caprolactone), aminolysis, nanofibers, bone tissue engineering

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Introduction: Certain synthetic polymers, like poly(Є-caprolactone) (PCL), poly(lactic acid) (PLA) and poly(lactide-co-glycotide) (PLGA), are used extensively as scaffolds for regenerative medicine research, tissue engineering of blood vessel, skin, cartilage and bone1. Use of these polymers follows due to their ease of fabrication, suitable mechanical properties, tractable degradation

kinetics,

and

reasonable

biodegradability2.

However,

the

deficient

cytocompatibility of such synthetic materials means that they are unable to create an ideal environment for the cells. Among biodegradable polymers, PCL – an aliphatic polyester – is proposed for multifaceted applications like drug delivery carriers,3 scaffolds for growing osteoblasts and fibroblasts, and grafts for skin tissue4. PCL is hydrophobic in nature and lacks any biological recognition sites. Thus, by itself, PCL provides a less than optimal environment for the development and growth of cells5. To overcome this shortcoming of PCL, attempts are being made to infuse it with bioactive proteins such as chitosan,6 collagen7 and gelatin8.

Electrospinning provides a simple and efficient method for fabricating nanofibrous substrates that are similar in nature to native tissuematrix9. The nano-composites produced by electrospinning blends of PCL with natural polymers10 have improve cytocompatibility. The interaction between cells and implants primarily takes place on the surface layers of the implants. Surface modification of PCL substrates to improve cytocompatibility without treating the bulk is hence a lucrative option. Modifying the surface of electrospun PCL nanofibers to instil a natural polymer/protein can improve hydrophilicity and compatibility with cells. Several techniques, including but not limited to, γ-ray irradiation, end-grafting, laser induced ozone oxidization, and plasma treatment 11-15 have been successfully tried for modifying the surface of nanofibrous PCL matrices. Such modification leads to better suitability for cell growth16. Amongst the available surface modification techniques, aminolysis stands out as a simple and dependable solution17. Amino groups introduced during this process onto the surface of a PCL substrate are harmless to living tissue. They impart hydrophilicity to the surface and provide active spots for fixing other biomolecules (like collagen or RGD peptides) on the surface. Further they can neutralize any acidic components generated when the scaffold eventually degrades18. PCL molecules pose abundant ester groups (-COO-). Consequently, a diamine like ethylenediamine (EDA) or hexanediamine (HDA) can be used in such a way that one amine 3

group reacts with an ester group on a PCL molecule to form –CONH– and the other amine group may be crosslinked with a biopolymer. Using this process, researchers have introduced bioactive molecules like collagen, chitosan,18 and gelatin19 onto PCL and fibronectin on PLL20. Silk, a natural polymer, is biodegradable and biocompatible, shows nominal immune reactivity, and has excellent permeability for water and oxygen21. Additionally, silk has good formability for fabrication purposes and can also be blended with other natural or synthetic polymers to enhance any advantageous characteristics22. The silk fibroin from Indian tropical tasar silkworm, Antheraea mylitta, apart from possessing the afore-mentioned desirable properties of silk,23 is also known to enhance osseointegration in vitro24. This fibroin has tripeptide (Arg-Gly-Asp) integrin binding RGD sequences inherent to it25 and these sequences augment cell adhesion and proliferation26. This work deals with the grafting of non-mulberry silk protein fibroin onto aminolyzed PCL nanofibrous matrices. Simultaneously, nanofibrous matrices are also fabricated by electrospinning non-mulberry silk fibroin/PCL blend. A comparative study is carried out between the above two methods of introducing non-mulberry silk fibroin in PCL nanofibrous matrices are explored. Biophysical characterizations, using energy dispersive x-ray diffraction, absorption spectroscopy, scanning electron microscopy, ATR-FTIR and confocal microscopy of these nanocomposite matrices are conducted. The cytocompatibility of the fabricated matrices are carried out using human osteoblast-like cells. The results show nonmulberry tasar silk fibroin grafted aminolyzed PCL nanofibrous matrices to be possibly an ideal scaffold material for osteogenesis. 2. Materials and methods 2.1. Materials Principal materials used in this study are listed here off: poly(Є-caprolactone) (Mol. wt.= 80,000) (Sigma, St. Louis, USA), chloroform (Sigma, St. Louis, USA), glutaraldehyde (Sigma, St. Louis, USA), 1, 6-hexanediamine (TCI, Japan), Thiazolyl Blue Tetrazolium Bromide (MTT, Sigma, USA), Rhodamine B isothiocyanate (RBITC), Thiazolyl Blue Tetrazolium Bromide (MTT, Sigma, USA), ninhydrin (Sigma, St. Louis, USA); sodium dodecyl sulfate (Mol. Wt. = 288.38) (J. T. Baker, NJ, USA); polyethylene glycol (Mol. Wt. = 6000) (Merck, India); cellulose dialysis tubing with cut off 12,000 and 3500 kDa (Pierce, USA); tissue culture grade polystyrene plastic flasks and plates (Tarsons, India); Dulbecco’s

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modified eagle medium (DMEM, Gibco BRL, USA), fetal calf serum, trypsin, EDTA, penicillin-streptomycin antibiotics (Gibco BRL, USA); and alamar blue (Invitrogen, USA). Silkworms: Indian non-mulberry tropical tasar, Antheraea mylitta, silkworms were reared at our IIT Kharagpur Silk Farm till they reached their late fifth instar and were about to start spinning. Cell lines: Human osteoblast-like cells (MG 63) were purchased from National Centre for Cell Science (NCCS), Pune, India. 2.2. Isolation of silk protein fibroin from nonmulberry silk glands Silkworm larvae at the late fifth instar were dissected and the protein fibroin was extracted from their posterior silk glands. Prior to extracting fibroin, the silk glands were washed using de-ionized water, thus removing any traces of sericin27. Fibroin was squeezed out from the glands using fine forceps and solubilized in aqueous solution of 1 %w/v sodium dodecyl sulfate, 10 mM Tris (pH 8.0), and 5 mM EDTA at room temperature. Dialysis of the solution was carried out to eliminate the remnants of the excess of surfactant. Final concentration of the fibroin solution was adjusted to 2 wt%. 2.3. Electrospinning (e-spinning) of blends into nanofibrous matrices 10 wt% poly (Є-caprolactone) (PCL) solution was prepared by continued stirring for 3 h in chloroform, at 37 °C. This solution was blended with equal volume of 2 wt% silk fibroin at 37 °C by gentle stirring over 2 h. E-spinning was carried out using a 5 ml glass syringe with 22G stainless steel blunt tip needle (ID = 0.413 mm). The needle in turn was connected to a 0.8 mm ID capillary, which was linked to the 11 kV DC supply. A copper net collector was placed 15 cm from the capillary tube and bore the grounded counter electrode. To control outflow through the capillary, a syringe pump was used with flow rate set at 0.1 ml/h. The e-spun matrices were collected upon aluminum foils. These matrices were stripped off the foils when they reached a thickness of about 8 µm. For spinning matrices of such thickness, approximately 8 h was required. The matrices were treated in ethanol for 2 mins before further investigations.

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2.4. Non-mulberry silk fibroin immobilized PCL nanofibrous scaffolds 2.4.1. Preparation of PCL nanofibrous matrices and aminolysis of the scaffolds

Nanofibrous PCL matrices were fabricated following the procedure outlined in Section 2.3. These matrices were cut in 2 cm × 2 cm sections and were cleaned by submerging in absolute alcohol-water solution (1:1 v/v) for 2-3 h followed by washing with copious amounts of deionized water. The matrices were subsequently immersed in a 10 wt% solution of 1, 6hexanediamine in 2-propanol solution at 37 °C for a predetermined length of time18.The matrices were then rinsed in deionized water, at room temperature, for 24 h, followed by vacuum drying at 30 °C for another 24 h to remove any free 1,6-hexanediamine.

2.4.2. Immobilization of silk fibroin

Aminolyzed matrices were kept immersed for 3h in 1% (w/v) glutaraldehyde (GA) solution at room temperature. The matrices were rinsed using deionized water 8-10 times to remove any free GA. The matrices were then put in 2 wt% silk fibroin protein solution, maintained at 2-4 °C, for 24 h. Post this incubation, silk fibroin immobilized nanofibrous matrices were immersed in deionized water for 24 h to rinse off excess silk fibroin. These matrices were finally dried off and treated with ethanol for a couple of minutes before subsequent studies. 2.5. Determination of amino groups of aminolyzed PCL matrices Amino groups on PCL membranes subjected to aminolysis were labelled with Rhodamine B isothiocyanate (RBITC) by immersing in 0.1 mg/mL RBITC solution for 24 h at 2-4 °C. The matrices were then immersed in deionized water for 24 h, at room temperature, to rinse off free RBITC. Fluorescence of RBITC labelled matrices at 580 nm was measured by confocal laser scanning microscopy (Olympus FV 1000, Olympus, Japan). 2.6. Quantitative determination of –NH2 groups on aminolyzed PCL matrices The ninhydrin analysis method was employed to quantitatively detect the amount of NH 2 groups on the aminolyzed PCL matrix. The aminolyzed matrices were immersed for 1 min in 1.0 mol/L ninhydrin/ethanol solution, were then placed in a glass tube and heated for 15 min at 80 °C to accelerate the ninhydrin – amino group reaction to evaporate any adsorbed ethanol. Subsequently 5 ml 1, 4-dioxane was added in the tube. This dissolved the matrix and 6

gave the contents a characteristic blue colour. To stabilize this blue compound, 5 ml of 2propanol was added. The absorbance of the tube contents was measured at 538 nm using a microplate reader (Thermo Scientific Multiskan Spectrum, Japan). A standard calibration curve of absorbance was constructed using known concentrations of 1, 6-hexanediamine in 1, 4-dioxane/isopropane [Figure 2(a)] to get a quantitative value from the absorbance. 2.7. Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was carried out for characterizing the surface properties of the different nanofibrous matrices using ATRFTIR equipment from Nexus- 870, Thermo Nicolet Corporation, USA.

2.8. Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX)

An analytical SEM (A-SEM, ZEISS EVO 60 Scanning Electron Microscope, Carl ZEISS SMT, Germany) was used to investigate the nanofibrous matrices. The presence/absence of nitrogen on the different matrices was corroborated through EDX. Analysis of the SEM images was performed with Image J® (release 1.47 for Windows) to determine fiber diameter. The fiber diameter was measured at 40 arbitrary sites on the SEM image frame with Image J®. The values were averaged out to get the representative fiber diameter for any particular nanofibrous matrix. 2.9. Atomic force microscopy (AFM) Surface roughness of the different matrix samples, 10 × 10 µm2 in area, was examined by atomic force microscope (AFM; Model 5100, Agilent Technologies, USA). The topographic images were obtained using intermittent contact mode and silicon cantilevers (PPPNCL, Nanosensors, Inc., USA). The silicon cantilevers had force constant of ~40 Nm-1 and resonating frequency of ~169.52 kHz. Pico Image Basic Software (Agilent Technologies) was used to analyse the AFM images and obtained the surface roughness of the nanofibrous matrices in terms of a surface root mean square (RMS) roughness (Rq).

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2.10. Dynamic contact angle measurement The hydrophilicity of the matrices was determined from advancing and receding angle of water using a goniometer (Data Physics Instruments, Filderstadt, Germany). 2.11. Characterization of mechanical properties Tensile tests were performed on a universal testing machine (UTM; Instron Electroplus, E1000) on dumbbell shaped samples made following ASTM 638-5 standard. The samples were 50 mm long and 10 mm wide at 25 °C and 50% relative humidity. To obtain stressstrain curve and ultimate tensile strength of the matrices, the test was run with a 5 kg loadcell and at an extension rate of 3 mm/min. 2.12. Cell culture on the nanofibrous scaffolds The cell culture medium, for the human osteoblasts like cells was made of DMEM, 10% fetal calf serum, and 1% penicillin/streptomycin. The cultures were kept till they reached 90% confluence in a humidified environment of 5% CO2, at 37 °C. Following confluence, cells were trypsinized, centrifuged, and suspended back in media for counting. The scaffolds (1 × 1 cm2) were sterilized for 30 min with 70% ethanol and UV light, washed repeatedly with sterile PBS (pH 7.4) followed by treatment with DMEM medium for 4 h to create a better environment for the cells. Just prior to cell seeding, to ensure better penetration of cells, scaffolds were partially dried for 2 h. Fifteen micro-litres of the cell suspension in medium, containing 105 cells, was added dropby-drop on to each nanofibrous matrix. Following seeding, to boost cell adhesion in the initial hour, the matrices were maintained in a humidified environment, at 37 °C, 5% CO 2. The matrices were kept in medium for 14 days while medium was replaced every alternate day. 2.12.1. Cell adhesion assay Cell adhesion onto the scaffolds was measured using concentration of the unattached cells in the culture media after two, four, and six hours of initial seeding. At each time point, the culture medium was flushed onto the culture dish using a pipette. This dislodged any cells that might have attached to the culture dish. This was carried out with care not to disturb the cell laden scaffolds. Ten microliter of the cell suspension was then transferred to a chamber of the haemocytometer to get the concentration of the unattached cells. The number of

8

unattached cells thus determined was then subtracted from the density of cells initially seeded. This residual was taken as the number of cells adhering to each scaffold28.

2.12.2. Cell viability assay The MTT assay was performed at different time points over the full cell culture duration to investigate cell viability on the matrices. The matrices were incubated in 5 mg/ml MTT stock solution that was diluted at a ratio of 1:10 using PBS (pH 7.4). Formazan crystal formed post incubation was dissolved in dimethyl sulfoxide and the absorbance of the resulting solutions was measured using the Manufacturer’s protocol with spectrophotometer (Bio-Rad, iMark). 2.12.3. Cell proliferation by alamar blue assay Cell proliferation on matrices over 14 days was assessed by alamar blue dye-reduction24. The alamar blue dye was diluted in the culture medium with a 1:10 dye-to-media ratio. The scaffolds were incubated in the dye solution for 4 h in the dark. The dye reduction was determined spectrophotometrically in a microplate reader (Thermo Scientific Multiskan Spectrum, Japan) at 570 and 600 nm. The percentage of dye reduction was calculated from Equation (1), % AB reduction = [(εoxλ2)(Aλ1)- (εoxλ1)(Aλ2)/ (εredλ1)(A’λ2)- (εredλ2)(A’λ1)] x 100

(1)

where ελ1 and ελ2 were molar extinction coefficient of alamar blue at 570 and 600 nm respectively, εox in oxidized and εred in reduced form; Aλ1 and Aλ2 were absorbance of the test wells; and A’λ1 and A’λ2 were the absorbance of the negative control wells. All given pairs are values at 570 and 600 nm. 2.12.4. Cytotoxicity assay To estimate the proliferation of cells, a modified lactate dehydrogenase (LDH) assay was performed using the WST-8 kit (BioCat, Heidelberg, Germany)29. After 1, 3 and 6 days of culture, the medium from the wells was removed and the samples were gently washed twice with PBS. The attached cells were lysed for 30 min at 37 °C with 100 µl 0.5% Triton X-100/ sample to release LDH from the cytosol of all viable cells. The plate was then centrifuged at 1300 rpm for 10 min to separate if any cell debris. Ten microliter of the cell lysis solution was transferred into a 96 well plate. For the reference, 0.5% Triton X-100 solution was used. Hundred microliters of LDH reaction reagent was added to the lysates followed by 30 min of incubation at room temperature. The OD value at 450 nm was recorded. This reading was taken as a measure for the quantity of cells.

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2.12.5. Cellular morphology and distribution within the construct Laser confocal and SEM images were examined for the cell morphology and the dispersion of the cells on the nanofibrous scaffolds. The scaffolds with the cells on them were fixed using 4% paraformaldehyde for 1 h. The scaffolds examined under SEM were then dehydrated with ethanol gradients (50 to 100% v/v H2O, increasing in steps of 10% v/v). The constructs were placed for 20 min in each solution and at the end, briefly exposed to isoamyl acetate and completely dried using vacuum drier. The dried samples were sputtered with gold and SEM was carried out at 20 kV (JEOL JSM-5800 scanning electron microscope). For confocal laser microscopy the scaffolds after 7 days culture were fixed using 4% paraformaldehyde and blocked with 1% bovine serum albumin (BSA) for 1 h. The cells were then permeabilized over 5 min by use of 0.1% Triton X-100, prepared in BSA. The actin filaments were stained using Alexa Fluor® 488 and the nuclei with Hoechst 33342. The confocal laser microscopy was performed on Olympus FV 1000 (Olympus, Japan) and postprocessing was carried out with Olympus FV 1000 Advanced software version 4.1 (Olympus, Japan).

2.12.6. Alkaline phosphatase assay (ALP) Protocol given by Kim et al.30 was used to spectrophotometrically measure the alkaline phosphatase produced by MG-63 cultured on the different matrices. At specific day points, the cell laden constructs were washed with PBS (pH 7.4), homogenized with 1 ml Tris buffer (1 M, pH 8.0), and sonicated for 4 min on ice. Of this suspension, 20 µl was incubated with 1 ml of 16 mM p-nitrophenyl phosphate (Sigma) solution for 5 min, at 30 °C. To measure pnitrophenol produced in presence of ALP, the absorbance at 405 nm was evaluated and ALP activity was reported as p-nitrophenol produced, normalized by incubation duration and cell count: µmole/min/105 cells. For cell count, cells were extracted from the constructs using trypsin–EDTA solution and counted with a hemocytometer. 2.12.7. Alizarin red-S staining for mineralization To measure calcification produced by the osteoblast like cells on the scaffolds, Alizarin Red S staining was used after 14 days of cell culture. The cell cultured matrices were fixed over 1 h in 4% paraformaldehyde, washed in PBS (pH 7.4), and stained by using 1 wt% alizarin red S (Sigma Aldrich, St Louis, USA) for 8 to 10 min. Afterwards, to get rid of excess dye, the matrices were repeatedly washed with PBS (pH 7.4) till the PBS finally remained colourless. The images were taken using ECLIPSE TS100 (Nikon, Japan). The calcium 10

deposition on the matrices was also examined using a colorimetric quantification after 14 days of culture31. Each matrix was incubated for 30 min, with shaking, in 800 µl acetic acid (10 %v/v) at room temperature. The cells were then scraped off the matrix and vortexed for 30 s in a 1.5 ml microcentrifuge tube. The slurry from vortexing was ensconced in 500 µl mineral oil. This combination was heated for 10 min at 85 °C, transferred onto ice for 5 min, followed by centrifuging at 20,000 g for 15 min. Of the supernatant formed, 500 µl was taken in a fresh 1.5 ml microcentrifuge tube and 200 µl ammonium hydroxide (10 %v/v) was mixed to neutralize the acid. The absorbance of the supernatant was then measured at 405 nm, in triplicate. 2.12.8. Cell viability, Live/dead assay Manufacturer’s protocol was followed (Molecular Probes, USA) to perform live/dead assay. 40 nM calcein AM and 20 nM ethidiumhomodimer were taken and dye solution was prepared in DMEM, without FBS. Cell laden matrices were collected after 7 days of culture and rinsed thrice with 1X PBS. The dye solution was used to stain the matrices, for 30 min in dark. After that the matrices were cleansed with PBS. The matrices were then examined through confocal microscopy (FV 1000 Advance software v. 4.1, Olympus), excitation of 488 nm and 543 nm lasers, to detect live cells stained green from calcein and dead cells stained red from ethidiumhomodimer. 2.12.9. Cell cycle analysis MG-63 cells were seeded onto the different nanofibrous matrices, at a density of 1×10 5 cells per matrix, and were incubated for 1 and 3 days. After incubation period, the cells were extracted from the matrices by applying trypsin–EDTA solution. The activity of trypsin in cell suspension was deactivated by adding 5 ml of the complete medium and the suspension was then centrifuged for 10 min at 1000 rpm to get a cell pellet. This cell pallet was suspended in cold PBS (pH 7.4) and again centrifuged at 1000 rpm, over 10 min. Subsequently, the chilled 70% ethanol was added to the pallet while shaking. The cells were incubated at 4 °C in the ethanol for 45 min and then washed twice with PBS. The final cell pallet was then suspended in 200 µl PBS with 0.1 mg RNAseA (Sigma, USA) added per ml solution to be incubated at 37 °C for 30 min. The cells were then washed with PBS, centrifuged, and suspended in 0.5 ml PBS that had 20 µl propidium iodide (PI, Sigma, USA) solution (1 mg ml-1 PI). Propidium iodide is an intercalating, blue excited dye and was used for analysis of cell cycles. After keeping the cells suspended for 25 min in dark and at 4 °C, 11

the nuclei suspension resulted. This suspension was analysed at an excitation of 488 nm using a flow cytometer (FACS Calibular, B-D using Cell Quest Pro software). 2.13. Statistical analysis Unless otherwise specified, data for all cases are presented as mean ± standard deviation (SD) with sample size (n) of 3. One way ANOVA, followed by Tukey’s HSD test, was used to compare the results obtained for different matrix compositions. Significant differences were categorised as *** p < 0.001; ** p < 0.01; * p < 0.05. The statistical analysis was carried out using R statistical environment.

3. Results 3.1. Incorporation of amino groups via aminolysis For confirming presence of free NH2 groups in the aminolyzed matrices, Rhodamine B isothiocyanate (RBITC) was used to label these groups. The fluorescence emission was checked with confocal laser microscopy. Figure 1(a) shows effect of aminolyzing time on surface concentration of amino groups when using 10 wt% 1, 6-hexanediamine for aminolysis. With aminolyzing time of one hour, the PCL nanofibrous matrix labelled using RBITC is given in Figure 1 (b). From the variation of fluorescence intensity, an increase in the free amino groups with the aminolyzing time was observed. This was peaked at about 1h, and then dropped off. This trend might be due to free amino groups reacting further with carboxyl groups on the terminal chain or because longer aminolyzing time might have degraded the surface of the matrices18. 3.2. Quantitative measurement of NH2 concentration by ninhydrin analysis The ninhydrin assay for quantification of NH2 concentration on PCL nanofibrous matrices also showed that a peak concentration of about 2.3×10-6 mol/cm2 [Figure 2(b)] was reached at 1 h of aminolyzing time. The concentrations were estimated using the standard curve in Figure 2(a), which was constructed using known quantities of 1, 6-hexanediamine. Both fluorescence and quantitative studies showed that beyond 1 h of aminolysis, the -NH2 concentration on nanofibrous PCL substrates declined. Hence 1 h was taken as the optimal aminolysis duration for the present study.

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3.3 ATR-FTIR Spectroscopy analysis ATR-FTIR spectra (500–4000 cm-1) of PCL and

aminolyzed PCL are presented in

Figure 3(a) while those of 2% silk fibroin/PCL (2SF/PCL) and 2% silk fibroin grafted aminolyzed PCL (aminolyzed PCL-silk) are presented in Figure 3(b). From the major vibration peaks detected for PCL nanofibrous matrices, the following strongest bands associated with PCL were identified: 2949 cm-1, asymmetric CH2 stretching; 2865 cm-1, symmetric CH2 stretching; 1727 cm-1, carbonyl stretching(C=O); 1293 cm-1, C–O and C–C stretching in the crystalline phase; 1240 cm-1 asymmetric C-O-C stretching; 1190 cm-1, OC– O stretching; 1170 cm-1, symmetric C-O-C stretching; 1157 cm-1, C–O and C–C stretching in the amorphous phase. Some of the bands were observed only due to de-convolution of bands, e.g. in the 1150–1200 cm−1 region, only 2 bands could be distinguished on the spectrum, but due to de-convolution 3 bands were seen [Figure 3(a)]32. The spectrum data of aminolyzed PCL nanofibrous matrix did not show any –NH2 or –CONH– stretching [Figure 3(a)]. This could be due to the amino groups, which did not react with the ester groups. It was more likely because this particular characterization method lacked the required sensitivity. For this reason labelling NH2 groups with RBITC, a more sensitive method, was used for verifying presence of free NH2 groups. In the FTIR spectra of the nonmulberry silk fibroin protein, three vibration peaks associated with amide groups were distinguished: 1650–1630 cm-1 for amide I (C = O stretching), 1540– 1520 cm- 1 for amide-II (secondary NH bending, due to β-sheet structure), and 1270–1230 cm- 1 for amide III (C–N and N–H functionalities)33. Absorption peaks at ~1535 cm- 1 and ~1648 cm- 1 corresponded

to

β-sheet structure (amide II). The spectra of both 2% silk

fibroin/PCL (2SF/PCL) blended nanofibrous matrix and 2% silk fibroin grafted aminolyzed PCL nanofibrous matrix presented peaks of silk fibroin as well as PCL without any considerable deviation. An absorbance peak at ~3283 cm-1, not present in 2SF/PCL spectra, was observed for 2% silk fibroin grafted aminolyzed PCL nanofibrous matrix [Figure 3(b)]. This peak, in all likelihood, corresponds to the amide A band of silk fibroin (associated with the NH-stretching vibration)34. The peak could have broadened in the current spectra due to hydrogen bonding associated with the silk fibroin. Thus, the spectra observed in Figure 3(b) clearly point towards effective blending and immobilization of silk fibroin onto aminolyzed PCL nanofibers by crosslinking using glutaraldehyde 13

3.4. Morphology and elemental analysis of nanofibrous matrices The SEM images of PCL nanofibers, aminolyzed PCL nanofibers, 2% silk fibroin/PCL (2SF/PCL) blended nanofibers and 2% silk fibroin grafted aminolyzed PCL nanofibers (aminolyzed PCL-silk) are shown in Figure 4(a-d), respectively. The electrospun nanofibrous scaffolds have a smooth and uniform appearance. No beads formation was observed, and pore distribution showed good interconnectivity. Using Image J® software to analyse the SEM images, diameter of the PCL nanofibers were estimated to be between 150 and 500 nm (Table I). Post aminolysis and grafting of silk fibroin did not significantly change the fiber diameters. The EDX data of 2% silk fibroin grafted aminolyzed PCL nanofibrous matrix surface showed maximum wt% of nitrogen (Table I). 3.5. Surface topography and surface roughness of nanofibrous matrices Surface topology of the nanofibrous matrices was characterized by AFM [Figure 5(a-d)]. The surface RMS roughness (Rq) was determined by processing AFM images with Pico Image® software and the values for different matrices are given in Table I. 2% silk fibroin grafted aminolyzed PCL showed maximum surface roughness (293±0.04nm) and PCL nanofibrous matrix had the minimum surface roughness (99.8±0.14nm), and the difference was statistically significant at p < 0.01. The enhanced surface roughness is likely to have provided more surface area for cell adhesion. This could have also improved availability of medium and serum proteins. This can lead to the better cellular growth as compared with matrices of less surface irregularity.35, 36 3.6. Dynamic contact angle The hydrophilicity has an important effect on biocompatibility of substrates 37. The contact angles of different nanofibrous matrices were measured (Table II). With a measured water contact angle of 107.87±9.21°, the unmodified PCL nanofibrous matrix is clearly hydrophobic. Following the aminolysis and grafting of silk fibroin the contact angle measurements showed increased hydrophilicity. The results from post aminolysis measurements verified the enhancement of hydrophilicity due to introduction of NH 2

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groups38,

39

. The results show that protein grafting, through aminolysis, reduces the

hydrophobicity and thus can improve biocompatibility and cell proliferation.

3.7. Mechanical properties of nanofibrous scaffolds Data from tensile testing of the substrates is summarized in Table I and Figure 6. During aminolysis, if the reaction with diamine cleaves too many ester groups in the PCL chain, the mechanical properties of PCL scaffold may be adversely affected. To ensure that this had not occurred, tensile strength of the aminolyzed PCL scaffold was measured and compared with that of control. Stress-strain curves for PCL scaffold (control) and PCL scaffold subjected to 1 h aminolysis are presented in Figure 6(a). While both scaffolds have similar ultimate elongation (UE) (~50%), the aminolyzed PCL scaffold has a higher ultimate tensile strength (UTS) than pure PCL – 3.5±0.32 MPa vis-à-vis 2.3±0.44MPa. The UE of aminolyzed PCL scaffolds was much higher than those of scaffolds made by electrospinning of natural proteins like collagen, gelatin, elastin, and tropoelastin (8-10% UE) and synthetic polymers like poly(lactide) (PLA) (30% UE)40,41. As shown in Figure 6 (b), the scaffolds of PCL blended with 2% silk fibroin (2SF/PCL) had a slightly improved tensile strength (4.89±0.68 MPa) while grafting of 2% silk fibroin improved scaffold strength dramatically (10.1±0.31 MPa). The blending might have developed inter-fiber bonds in the nanofibrous matrix, thus increasing tensile strength. On the other hand, grafting fibroin seems to have been more effective in imparting the superior strength of silk onto scaffolds. The important aspect to note is that irrespective of aminolysis, blending with SF or grafting, SF enhanced mechanical strength significantly. The scaffolds thus had desirable mechanical properties for application in bone tissue engineering. 3.8. Cell culture 3.8.1. Cellular adhesion on scaffolds MG 63 cells more promptly attached onto silk fibroin blended and grafted scaffolds as compared to the controls (Figure 7(a)). Within the first hour, 76, 79, and 92 percentages of cells had attached onto aminolyzed PCL, SF blended and SF grafted PCL scaffolds respectively. These values increased to 90, 95, and 97% over the next four hours. Nanoscale surface roughness of the scaffolds is likely to have led to greater adsorption of adhesive molecules42 and hence primarily improved cell adherence.

15

3.8.2. Viability, proliferation and cytotoxicity assay of the cells Day 14 results of viability (MTT assay) [Figure 7(b)] and cell proliferation (alamar blue assay) [Figure 7(c)] were higher for SF blended and SF grafted scaffolds as compared to PCL and aminolyzed PCL scaffolds. Between SF blended and SF grafted scaffolds, better results were noted for SF grafted scaffolds. Compared with their values from days 1, 5 and 7, both metabolic activity and cell proliferation had increased by day 14. Quantification of MG63 growth on the scaffolds being analysed was performed with a modified LDH assay. As shown in Figure 7(d), the highest number of viable cells was detected on aminolyzed PCL-silk followed by 2SF/PCL after a culture period of 24 h. The pure PCL scaffold presented the least number of cells. Analysis was carried out on day points 1, 3, and 6. Number of viable MG63 cells remained consistently lower on pure PCL and aminolyzed PCL, as compared to that of 2SF/PCL and aminolyzed PCL-silk surfaces, respectively. The results show that the nanofibrous matrices do not have any associated cytotoxicity. 3.8.3. Cell morphology within constructs The cells upon pure PCL and aminolyzed PCL nanofibrous scaffolds had round to oval forms and had no characteristic orientation [Figure 8 (a), (b)]. On the SF blended matrices, the cell morphology appeared to be similar with spindle morphology but has slightly improved filopodia-like extensions when compared to pure PCL or aminolyzed PCL matrices [Figure 8 (c)]. However, on the SF grafted structures, most cells showed a nearly flat form and were integrated well with each other, occupying almost the entire examined area [Figure 8(d)]. Abundant cell secreted neo-matrix deposition was observed mainly on the SF grafted scaffolds, where the matrix deposition was so extensive that the underlying fibrous matrix structure was not readily discernable. While the nanofibrous structure and randomness in arrangement could provide topological cues for cellular adhesion43, the integrin binding motif of silk fibroin implemented on the nanofiber surface might be effective in improving the subsequent cellular processes. 3.8.4. Observations under confocal microscope Since cell cytoskeleton organization is important for cell attachment and morphology, the actin filaments were stained using Alexa Fluor® 488 (green), nuclei were stained with Hoechst 33342 (blue), and the constructs were examined under confocal microscope [Figure 9]. The 3D constructs were Z-scanned during confocal microscopy to examine cell growth upon different layers. Scans across multiple layers were then merged into the final image. Images showed extensive and uniformly distributed actin filaments on SF blended and SF grafted constructs. Maximum number of cells was seen on SF grafted construct 16

[Figure 9 (d)], followed by SF blended construct [Figure 9 (c)]. However, for aminolyzed PCL matrix [Figure 9 (b)], actin distribution was sparse and isolated to just around the cell nuclei. For the pure PCL construct [Figure 9 (a)], cell numbers and actin filaments were both minimal. 3.8.5. Alkaline phosphatase (ALP) assay As an established marker of osteogenic differentiation, ALP was analysed during cell culture. The activity was presented [Figure10 (b)] as the value of p-nitrophenol produced when normalized to the cell number [Figure10 (a)]. The SF grafted constructs showed maximum ALP activity while SF blended constructs were next. These findings affirm the ability of SF blended and grafted matrices to bolster differentiation among the seeded osteogenic cells. 3.8.6. Mineralization of the constructs by Alizarin red S staining Alizarin Red S stained constructs after 14 days of culture are shown in Figure 11(a). As can be seen, staining was limited to certain patches in PCL and aminolyzed PCL, implying nominal mineralization. On the other hand, SF blended and SF grafted constructs showed intense staining over their entire surface, attesting to pervasive mineralization. To establish a control for the matrix backgrounds, acellular SF blended and SF grafted matrices were also stained and presented. Figure 11(b) shows the colorimetric quantification of calcium deposits on different nanofibrous matrices on day 14. This test showed significantly higher calcium content for aminolyzed PCL-silk fibroin scaffolds compared to the others.

3.8.7. Cell viability assay Viability and growth of cells on different nanofibrous matrices were examined using Live/Dead staining [Figure 12]. From the distribution of green colored cells in confocal images taken on day 7, the SF grafted matrix appeared to have the most viable cells, followed by SF blended matrix. 3.8.8. Cell cycle analysis Representative graphs from cell cycle analysis following 1 and 3 days of culture are presented in Figure 13. Seeded cells adhered well on the nanofibrous matrices and proliferated fast. Results from pure PCL scaffolds were taken as control. Gated percentage of cells in Sub G0, G0/G1, S, and G2/M phases is denoted as M1, M2, M3, and M4, respectively. On Day 1, M2 phase showed ~69% cells in control, 68.5% in aminolyzed PCL, 73.2% in SF blended, and 65.6% in SF grafted scaffolds. M4 phase showed 17% cells in control, 17.5% in 17

aminolyzed PCL, 18% in SF blended, and 20% in SF grafted scaffolds. M3 phase of control had about 3.01% cells as compared to 3.4%, 3.8%, and 5.29% of cells in aminolyzed PCL, SF blended, and SF grafted scaffolds, respectively. Since, after initial seeding, cells would have required some time to adapt to their new surroundings, proliferation on Day 1 was slow and this accounted for the small number of cell in M3 phase. Even so, it may be noted that cell percentage in M3 phase was slightly higher for SF grafted scaffolds. This may be taken as evidence that SF grafted matrices were slightly better at supporting cell proliferation. SF grafted scaffolds also showed the smallest percentage of dead cells, i.e., cells in M1 phase (1.9%). The amount of dead cells (M1 phase) did not increase on day 3. This suggests that all scaffolds used in the cell culture had good biocompatibility. M3 phase showed 6.15% cells in control, 6.78% in aminolyzed PCL, 14% in SF blended, and 18% in SF grafted scaffolds. This increase in percentage of cells in M3 phase from Day 1 to Day 3 further supported that the low percentages on Day 1 were due to the time required by cells to adapt to their changed environment early on. Also, distinctly larger percentage of M3 phase cells were detected for SF blended and SF grafted scaffolds as compared to the control and aminolyzed PCL scaffold suggesting better cellular proliferation. The control and aminolyzed PCL scaffolds instead show something akin to a cell cycle arrest in M2 phase and cells did not proceed to their normal M3 phase. For both scaffolds, over 60% cells remained in M2 phase. 4. Discussion Nanofibrous matrices are made by electrospinning a blend of 2 wt% SF with PCL and by grafting 2 wt% SF on aminolyzed PCL nanofibrous matrices. PCL itself is reasonably biocompatible and biodegradable5, can support a wide range of cells including marrow stromal cells44, and can be conveniently electrospun into nanofibrous matrices. From our previous investigations, use of 2 wt% of non-mulberry SF has been identified as a better choice for cell proliferation45. As PCL has an inherently inert surface for peptide conjugation, aminolysis is used to introduce functional NH2 groups onto this surface.18,46 The ester group on the PCL chain reacts with one amino group of 1,6- hexanediamine to form a covalent bond while the other amino group remains free. Using RBITC labelling and quantitative analysis with ninhydrin, it is confirmed that NH2 groups have been successfully introduced on the surface of the nanofibrous matrices. Introducing NH2 groups on surface of the matrices has two beneficial aspects. One is that it improves hydrophilicity of the surface. The other is that it can provide active sites for immobilizing biocompatible molecules like proteins, polysaccharides, 18

peptides, or cell growth factors. Aminolyzed matrices are treated with glutaraldehyde (GA) and thus the NH2 group forms a bond with OHC–CHO to yield a –N=CH-CHO bond. The remaining aldehyde group can then form a covalent coupling with NH2 groups available on most biomacromolecules. In this particular case, silk fibroin protein is grafted and immobilized on the matrices thus treated. Presence of the amide I band, which primarily embodies amide group’s C=O stretching vibration, was detected on the FTIR spectra to verify conformation of protein.47 Strength of hydrogen bonds between C=O and N-H groups affects the vibration frequency of this band, and conformational structure of the protein’s backbone determines strength of the bonds. 48 FTIR spectra from both the SF blended and SF grafted matrices show absorption peaks, without any major transformations, that correspond to β-sheet structure of protein. Thus, it may be deduced that these matrices are structurally stable49. Fiber diameter has been known to affect behaviour of MG-63 cells; in particular, large fiber diameters may reduce cellular migration50. In the current study, all matrices have average fiber diameters in nanometric range. The SF blended matrices has smaller fiber size (avg. dia. = 170 nm) as compared to the SF grafted matrices (avg. dia. = 493 nm). Even so, SF grafted matrices show better cell proliferation and migration, as observed from MTT and Alamar blue assay data. This may be ascribed to a combination of factors, as given below. SF grafted matrices have greater percentage of nitrogen on their surface than SF blended matrices (16.97% compared to 10.93%). Since presence of nitrogen has been known to aid osteoblast differentiation and proliferation, this may have been one of the contributing factors. Similarly, SF grafted matrices are more hydrophilic than SF blended matrices (Table II)51 and since surfaces with better hydrophilicity provide better cellular compatibility37, this may be taken as a second contributing factor. As PCL has a hydrophobic nature, others have also devised surface treatments to tackle this problem18. Our results show that hydrophilicity improved, as indicated from reduced contact angles, both for SF grafted and SF blended constructs. It is noteworthy that while fibroin is made up of mostly hydrophobic amino acids, like glycine, alanine, serine and others, its inherent structure makes it hydrophilic52. Thus, addition of fibroin, whether by blending or grafting, causes increase of hydrophilicity. The grafting ensured that the entire surface of the matrix is affected by SF and hence SF grafted matrices are more hydrophilic in nature than SF blended ones. Surface roughness, in nanometre range, has been shown to impact adhesion and proliferation of osteoblasts42. Deligianni et al.53 found that with increased surface roughness, about 40% more human bone marrow cells adhered to hydroxyapatite, while Webster et al.54 have 19

reported significant rise in osteoblast adhesion by fabricating surfaces with large nanometer range surface roughness using small grain size of ceramics (specifically, alumina, titania, and hydroxyapatite). Studies have also shown that osteoblasts cultured on surfaces with nanorange roughness gave greater deposition of calcium containing minerals and alkaline phosphatase synthesis compared to traditional ceramic surfaces.55 Topography analysis using AFM shows surface roughness is highest for SF grafted matrices, followed by SF blended matrices (RMS surface roughness of 293 nm and 210 nm, respectively). The SF grafted constructs show maximal cell adhesion and ALP activity, while values of SF blended constructs are second highest. Thus the higher surface roughness of SF grafted matrices also acted as a third contributing factor for the better performance in cell behaviours. Silk, due to oriented β-sheet crystals at nanoscale and due to shear-alignment between fiber chains, has remarkable mechanical strength56. Consequently, inclusion of SF improves tensile strength of the nanofibrous matrices. With ultimate tensile strength considerably larger than the 1.2 MPa associated with naive trabecular bones, 57 the present constructs can also suitably support the mechanical strength requirements in bone tissue engineering. Effective adherence of cells onto scaffolds leads to formation of ordered ECM 58 and presence of ECM is a requirement for successful tissue reconstruction. Nano-scaled structure, in terms of surface roughness and fiber diameter, and the inherent integrin binding peptide (RGD) sequences in the SF of this particular silkworm species conceivably contributes to the favourable cyto-compatibility of the matrices with SF inclusion. It is also likely that the nanoscaled structure of the matrices could have exposed cryptic integrin binding peptide (RGD) sequences in SF, 59 inducing even better cellular adhesion and growth. Compared to matrices of pure PCL and aminolyzed PCL, the SF blended and SF grafted matrices have greater number of live cells at the end of Day 14, as confirmed using live/dead assay. Progression of cell proliferation from Day 5 to Day 7 is a little slow for all the matrices [Figure 7(c)]. This slightly restricted progress may be ascribed to the cells requiring some time to adjust and adapt to the 3D matrix upon being transferred from the 2D cultures 60. The cell cycle analysis demonstrates that SF blended and SF grafted constructs support MG-63 cell growth with a normal cell cycle pattern and no arrests. Since a categorical attribute of osteoblasts is mineralization as osteogenesis matures, 61 ALP activity and Ca mineralization assay are used to quantify such mineralization upon scaffolds. Substantial levels of mineralization lead to cells having an orderly, sheet-like structure58. Increasing ALP activity over the culture duration, intensity of Alizarin Red staining, and SEM micrographs that show sheet-like arrangement of cells upon the matrices, all these 20

observations establish commendable nature of these scaffolds. The encouraging results are found for matrices particularly with SF grafting. Similarly, images from confocal laser microscopy show random actin-stress fibers along with dense cell colony deposits across the SF grafted nanofibrous matrices and to a slightly less extent on the SF blended matrices. Overall results show that the inclusion of silk fibroin leads to significant improvement of scaffold properties in terms of biocompatibility, mechanical strength, cell adhesion and proliferation, and osteoconductivity. At the same time, fibroin grafted nanofibrous matrices are a notably better choice than fibroin blended matrices. 5. Conclusion Silk fibroin from the non-mulberry tropical tasar silkworm Antheraea mylitta acts as a bioactive polymer. Inclusion of this fibroin, whether by blending or by grafting, into nanofibrous matrices, effected superior osteoconductive scaffolds as against controls made of PCL and aminolyzed PCL. The results show that the grafting of 2 wt% silk fibroin onto the aminolyzed PCL nanofibrous matrices presents better cell adhesion, growth, and formation of ECM than the scaffolds made by blending 2 wt% fibroin and PCL. Additionally, the mechanical properties of the scaffolds also match suitably to those of naïve bone tissue. The scaffolds are indicative of a promising material for use in bone regeneration. The current in vitro study serves as a starting point for design and testing of clinically relevant orthopaedic grafts in vivo.

Acknowledgements Department of Biotechnology and Indian Council of Medical Research, Govt. of India supported the work. SCK wishes to acknowledge the facilities provided by the Institute of Tissue Regeneration Engineering, Dankook University, South Korea during his short visit to their laboratories.

Funding Sources Department of Biotechnology (BT/PR10941/MED/32/333/2014), and Indian Council of Medical Research (5/13/12/2010/NCD-III), Govt. of India.

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Legends of Tables and Figures Table I: Mean fiber diameter, element weight%, surface roughness and tensile properties of the different (pure poly (Є-caprolactone) (PCL), aminolyzed PCL, 2% silk fibroin blended PCL (2SF/PCL) and 2% silk fibroin grafted aminolyzed PCL (aminolyzed PCL-silk)) nanofibrous matrices. Table II: Dynamic contact angle measurements of nanofibrous matrices (pure poly (Єcaprolactone) (PCL), aminolyzed PCL,2% silk fibroin (SF) blended PCL (2SF/PCL) and 2% silk fibroin grafted aminolyzed PCL (aminolyzed PCL-silk)) (n=3,mean ± SD) Figure 1: (a) Variation of fluorescence intensity of RBITC-immobilized PCL nanofibrous matrices, with aminolyzing time. Aminolysis took place at 37 °C, using 10 wt % solution of 1,6-hexanediamine in 2-propanol, over different time intervals (b) RBITC labeled aminolyzed PCL nanofibrous matrix (aminolyzing time 1h) taken by confocal laser scanning microscopy. Figure 2: (a) Variation of absorbance levels at 538 nm for products of the reaction between ninhydrin and 1,6-hexanediamine, with NH2 concentration. (b) Variation of absorbance levels at 538 nm for ninhydrin treated aminolyzed PCL matrices, with aminolyzing time. Figure 3: FTIR spectra of (a) poly (Є-caprolactone) (PCL) and aminolyzed PCL (b) silk fibroin (SF) blended PCL (2SF/PCL) and 2% silk fibroin grafted aminolyzed PCL(aminolyzed PCL-silk) nanofibrous matrices. The blended and grafted nanofibrous matrices reveal peaks of both PCL and SF without any major alterations. Figure 4: Scanning electron micrographs of randomly aligned electrospun pure PCL (a) aminolyzed PCL (b), silk fibroin blended (2SF/PCL) (c) and grafted (aminolyzed PCL-silk) (d) nanofibrous matrices. Scale bar = 1 μm. Figure 5: The AFM pictographs of nanofibrous matrices (pure PCL, aminolyzed PCL, 2% silk fibroin blended PCL (2SF/PCL) and 2% silk fibroin grafted aminolyzed PCL (aminolyzed PCL-silk)) show the surface topography (10µm×10µm scan area). Surface roughness (Rq) was calculated from AFM images using Pico Image Basic Software and presented in Table I. Aminolyzed PCL-silk matrices exhibit the highest surface roughness.

Figure 6: Stress–strain curves of (a) PCL and aminolyzed PCL, and (b) silk fibroin (SF) blended PCL (2SF/PCL) and 2% silk fibroin grafted aminolyzed PCL (aminolyzed PCL-silk) nanofibrous matrices. A substantial difference in tensile stress (MPa) observed between silk fibroin blended and grafted nanofibrous matrices. Figure 7: The response of osteoblast like cells (MG-63) seeded on the nanofibrous matrices and cultured for 14 days at 37 oC and 5% CO2 humidified atmosphere. (a) Initial cell 28

attachment efficiency on nanofibers measured up to 5 hrs by counting the cells from suspension at each time point. Pure PCL served as control. (b) Viability and (c) proliferation of the cells during culture for 14 days, indicating superior cell response on the silk-blended and -grafted matrices compare to PCL and aminolyzed PCL. *** p < 0.001, ** p < 0.01 and * p < 0.05, n=3 at each time point (One way ANOVA followed by Tukey’s HSD test) Figure 8: Scanning electron micrographs of the osteoblast-like cells (MG-63) seeded on the nanofibrous matrices after 14 days of culture. The cells formed continuous multilayer sheets on silk fibroin blended (c) and grafted scaffolds (d) compared to a few isolated cells on the PCL (a) and aminolyzed PCL (b) scaffolds. Scale bar = 10 μm. Figure 9: The cytoskeletal actin organization and distribution of MG-63 cells grown on nanofibrous constructs at day-point 7. The confocal images were taken after staining the actin filaments with Alexa Fluor® 488 (green) and counterstaining with Hoechst 33342 (blue) for nuclei. Better actin organization, cell-cell contiguity, and larger cell numbers were observed in 2% silk fibroin blended (c) and grafted (d) matrices compared to PCL (a) and aminolyzed PCL (b) matrices. Magnification = 20X. Scale bar = 100μm. Figure 10: (a) Cell count and (b) Alkaline phosphatase (ALP) activity osteoblast-like cells (MG-63) seeded on nanofibers after 14 days in cell culture. ALP activity was reported as pnitrophenol produced, normalized by incubation duration and cell count: µmole/min/10 5 cells. ALP activity of all the constructs increased with time, the highest activity observed on 2% silk fibroin grafted PCL constructs. *** p < 0.001; ** p < 0.01; * p < 0.05, n=3 at each time point (One way ANOVA followed by Tukey’s Honest significant difference test). Figure 11: (a) Mineralization of the extracellular matrix indicated by the intensity of alizarin red S staining; more homogenous coloration in silk fibroin blended (2SF/PCL) and grafted (aminolyzed PCL - silk) constructs compared to only patches in pure PCL and aminolyzed PCL. Acellular (scaffolds without cells) nanofibrous scaffolds of aminolyzed PCL and aminolyzed PCL-silk stained to serve as reference templates for comparison. Scale bar = 100 μm. Magnification=10X. (b) Quantification of mineral deposition using colorimetric assay of different nanofibrous matrices after 14 days of culture (pure PCL, aminolyzed PCL, 2% silk fibroin blended PCL (2SF/PCL) and 2% silk fibroin grafted aminolyzed PCL (aminolyzed PCL-silk)). Figure 12: The viability of MG-63 on different nanofibrous scaffolds (pure PCL, aminolyzed PCL, 2% silk fibroin blended PCL(2SF/PCL) and 2% silk fibroin grafted aminolyzed PCL 29

(aminolyzed PCL-silk)) using live/dead assay (live cells are green and dead cells appear as red) with confocal microscopy on day 14. High viability was maintained for silk fibroin blended and grafted matrices. Magnification=20X. Scale bar = 50μm. Figure 13: Cell cycle analysis of MG-63 cells grown on different nanofibrous matrices; (a,c,e,g) and (b,d,f,h) represent data sets for 1 and 3 days growth, respectively; where (a,b) control PCL; (c,d) aminolyzed PCL (e,f) 2% silk fibroin blended PCL(2SF/PCL) and (g,f) 2% silk fibroin grafted aminolyzed PCL (aminolyzed PCL- silk) nanofibrous matrices for 1 and 3 days respectively.

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Table I: Mean fiber diameter, element weight%, surface roughness and tensile properties of the different (pure poly (Є-caprolactone) (PCL), aminolyzed PCL,2% silk fibroin blended PCL(2SF/PCL) and 2% silk fibroin grafted aminolyzed PCL (aminolyzed PCL-silk)) nanofibrous matrices.

Types of Scaffold

Mean fiber diameter (nm)

Element weight %

Surface roughness (Rq) (nm)

Ultimate tensile strength (MPa)

Elongation at break (%)

Carbon (CK)

Nitrogen Oxygen (NK) (OK)

PCL

498±10

67.24

-

28.88

99.8±0.14

2.3±0.44

49±5%

aminolyzed PCL

495±18

35.19

8.92

38.89

167±0.23

3.5±0.32

46.5±7.8%

2SF/PCL

170±20

10.45

10.93

56.66

210±0.18

4.89±0.68

54.63±6.21%

Aminolyzed PCL-silk

493±12

12.25

16.97

58.17

293±0.04

10.1±0.31

55.9±6.28%

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Table II: Dynamic contact angle measurements of nanofibrous matrices (pure poly (Єcaprolactone)(PCL), aminolyzed PCL,2% silk fibroin(SF) blended PCL(2SF/PCL) and 2% silk fibroin grafted aminolyzed PCL(aminolyzed PCL-silk))(n=3,mean ± SD) Types of Scaffold

Advancing contact angle (º)

Receding contact angle(º)

Mean contact angle (º)

PCL

110.42

105.32

107.87±0.51

Aminolyzed PCL

102.83

94.53

98.68±1.11

2SF/PCL

96.32

93.22

94.77±0.47

Aminolyzed PCL-silk

36.38

31.98

Goniometer images

34.18±2.88

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Highlights:  Silk fibroin from Antheraea mylitta was included in nanofibrous PCL matrices  Inclusion was carried out either by grafting or by blending the fibroin  Inclusion of fibroin improved strength and biocompatibility of the matrices  Fibroin grafted matrices were comparatively more biocompatible

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Non-mulberry silk fibroin grafted PCL nanofibrous scaffold: Promising ECM for bone tissue engineering Promita Bhattacharjee,Deboki Naskar, Tapas K. Maiti, Hae-Won Kim, Debasis Bhattacharya, Subhas C. Kundu

Actin organization

Silk fibroin grafted nanofibrous matrix

Mineralization 2% SF grafted aminolyzed PCL nanofibrous matrix

Tensile stress (MPa)

10

8

6

4

2SF/PCL nanofibrous matrix

2

0 0

10

20

30

40

Strain (%)

50

60

70

Mechanical property

12