Biomimetic, Osteoconductive Non-mulberry Silk Fiber Reinforced

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Biomimetic, Osteoconductive Non-mulberry Silk Fiber Reinforced Tricomposite Scaffolds for Bone Tissue Engineering Prerak Gupta, Mimi Adhikary, Joseph Christakiran M., Manishekhar Kumar, Nandana Bhardwaj, and Biman B. Mandal ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11366 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on October 27, 2016

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ACS Applied Materials & Interfaces

Biomimetic,

Osteoconductive

Non-mulberry

Silk

Fiber

Reinforced

Tricomposite Scaffolds for Bone Tissue Engineering Prerak Gupta1, Mimi Adhikary1, Joseph Christakiran M1, Manishekhar Kumar1, Nandana Bhardwaj2, Biman B. Mandal1*

1

Biomaterial

and

Tissue

Engineering

Laboratory,

Department

of

Biosciences

and

Bioengineering, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India 2

Life Sciences Division, Institute of Advanced Study in Science and Technology (IASST),

Guwahati- 781035, Assam, India

*Corresponding author E-mail: [email protected], [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT Composite biomaterials as artificial bone graft materials are pushing the present frontiers of bioengineering. In this study, a biomimetic, osteoconductive tricomposite scaffold made of hydroxyapatite (HA) embedded in non-mulberry Antheraea assama (A. assama) silk fibroin fibers and its fibroin solution is explored for osteogenic potential. Scaffolds were physicochemically characterized for morphology, porosity, secondary structure conformation, water retention ability, biodegradability and mechanical property. The results revealed ~5 fold increase in scaffold compressive modulus on addition of HA and silk fibers to liquid silk as compared to pure silk scaffolds while maintaining high scaffold porosity (~90%) with slower degradation rates. X-ray diffraction (XRD) results confirmed deposition of HA crystals on composite scaffolds. Furthermore, the crystallite size of HA within scaffolds was strongly regulated by the intrinsic physical cues of silk fibroin. Fourier Transform Infrared (FTIR) spectroscopy studies indicated strong interactions between HA and silk fibroin. The fabricated tricomposite scaffolds supported enhanced cellular viability and function (ALP activity) for both MG63 osteosarcoma and human bone marrow stem cells (hBMSCs) compared to pure silk scaffolds without fiber or HA addition. In addition, higher expression of osteogenic gene markers such as collagen-I (ColI), osteocalcin (OCN), osteopontin (OPN) and bone sialoprotein (BSP) further substantiated the applicability of HA composite silk scaffolds for bone related applications. Immunostaining studies confirmed localization of Col-I and BSP and were in agreement with real time gene expression results. These findings demonstrate the osteogenic potential of developed biodegradable tricomposite scaffolds with the added advantage of the affordability of its components as bone graft substitute materials.


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Keywords: Non-mulberry silk; Bone tissue engineering; Hydroxyapatite; Silk fiber; Composite scaffold.

1. INTRODUCTION Bone fracture resulting in non-unions and defects due to trauma or degenerative conditions has escalated into a major area of concern in orthopedic care. According to a recent study, nearly half million patients annually avail bone defect repairs in the United States alone costing more than $2.5 billion.1 The number of patients is expected to double by 2020 globally as an unwelcome fallout of increased life expectancy due to better medical care. Autografts and allografts remain the most preferable methods for bone repair. Donor site morbidity associated with autografting, risk of disease transmission and reported cases of immune rejections associated with allografting limits the clinical implementation of these procedures.2-3 In this increasing predicament, tissue engineering has advanced better alternatives in biobased functional substitutes and implants for repair of defective sites. It involves delivery of precursor cells to the damaged site with the help of an appropriate scaffolding structure having similar form and composition to that of natural bone. An ideal bone scaffolding matrix must provide temporary mechanical support capable of withstanding in vivo mechanical loading; adequate pore size to enable optimum diffusion of nutrients and oxygen and interconnected pores to encourage cellular infiltration. Further, it is expected that scaffold would mediate matrix-host integration (osseo-integration) and maintain either osteogenic commitment of cells (osteo-conduction) or stimulate osteogenic differentiation of progenitor cells (osteo-induction) or both; degrade and resorb with time matching the remodeling kinetics of host tissue.4-5



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Calcium phosphate and bioresorbable bioactive glass based micro-porous ceramic scaffolds have been explored extensively in the past few decades for use in bone tissue engineering (BTE). These ceramic scaffolds have the innate ability to induce biologic response similar to that of bone but are impaired by brittleness and low mechanical strength.6 In contrast, several natural or synthetic polymers exhibit better mechanical strength and bioactivity which contribute to enhanced functionality in fabrication of various bone grafts.7 On the other hand, as these polymer based matrices exhibit rapid strength deterioration in vivo which makes them ostensibly non-durable for load bearing applications.6 Thus composite materials combining the advantages of both polymers and ceramics with far improved mechanical properties and cellmatrix interactions have carved a niche in BTE applications in the recent years.1 Bone in fact is a composite material and made up of 65% inorganic mineral phase, 25% organic phase primarily composed of collagen-I. The major constituent of the inorganic phase of bone is hydroxyapatite (HA) which has been widely investigated as bone replacement substitute for its high regeneration ability due to its osteoconductive properties.8-9 The clinical implementation of HA alone, has certain limitations including its intrinsic brittleness and slow degradation rate.10 The former property causes dislocation from the site of transplantation while the latter causes prolonged retention.11 Therefore incorporation of bone-like mineral HA into porous biodegradable polymers such as gelatin, collagen, poly (lactic-co-glycolic acid) (PLGA), chitosan, alginate etc. has proved to be a promising alternative for bone tissue engineering.9, 12-15 Natural silk fibroin has been extensively explored for its osteogenic potential owing to its similarity to collagen-I which is the main structural protein in bone composing the organic phase. Silk fibroin (SF) primarily is obtained from two major groups which includes domesticated mulberry and wild non-mulberry varieties. SF obtained from Bombyx mori (mulberry) silkworm


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is the most investigated silk for a host of tissue engineering applications.16-18 However, the less explored non-mulberry SF, particularly Antheraea mylitta and Antheraea assama is reported to have additional advantages over mulberry silk in terms of enhanced physicochemical, thermal and mechanical stability; with its improved cell-substrate interaction attributed to presence of intrinsic RGD cell binding motif within its genome.19-22 In addition, previous reports suggested superior osteogenic and mineralization potential of non-mulberry silk (Antheraea mylitta) when compared to its mulberry counterpart (Bombyx mori).23-24 Silk continues to hold out promise as a biomaterial in bone tissue engineering application due to its superior properties of high mechanical strength, biocompatibility and tunable biodegradability.17,25 Moreover the combinatorial effect of silk protein and HA has shown to enhance surface roughness and osteoconductivity which lead to human bone marrow stem cell (hBMSCs) differentiation. Apart from improving the mineral content, HA incorporation into silk scaffolds has been reported to enhance 8-fold compressive modulus compared to pure silk scaffold. 26 As a part of processing, incorporation of HA within silk scaffolds improves its mechanical properties however, it needs further tuning in order to meet the practical requirement of load bearing application and avoid excessive brittleness. Silk scaffolds reinforced with raw fibers have demonstrated significant improvement in compressive strength of up to 13 MPa in hydrated conditions. For composite preparation, finely chopped and degummed (post sericin removal) silk fibers are reported to be highly beneficial.17 The raw silk fibers present in silkworm cocoons consists primarily of silk fibroin in the inner layer and sericin, a glue like protein in the outer layer, in 75 to 25 wt% ratio, respectively. These fibers are known to be highly thermo stable, biodegradable and highly crystalline with well-aligned structure and are reported to be used as reinforcements to improve mechanical properties of scaffolds. Further, the length of



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fibers within reinforced scaffolds has been shown to influence porosity, interconnectivity, compressive modulus and biodegradability.17 The purpose of this study is to fabricate a silk fiber reinforced tricomposite multifaceted scaffold (consisting of silk fibroin solution, silk fibroin fiber and HA) that would have beneficial properties of each individual added component while complementing their shortcomings thus yielding a superior scaffold for bone tissue engineering. To impart osteoconductivity to the composite, HA deposition was carried out in a bio-inspired fashion. Taken together, the current strategy envisages developing a biomimetic, mechanically strong, tricomposite silk based scaffold with synergistic properties. Herein, the composite scaffolds were physico-chemically characterized and evaluated as matrices using MG63 osteosarcoma and human bone marrow stem cells (hBMSCs) to analyze cellular viability, proliferation, extracellular matrix production by histomorphology and osteogenic genes expression in a 3D culture system. For comparison, pure silk fibroin freeze dried scaffolds were utilized as control. 2. EXPERIMENTAL SECTION 2.1 Preparation of silk fibroin (SF) solution and fibers Silk fibroin was obtained from 5th instar mature larvae of A. assama sourced from local silk farms following previously reported protocol.19 Briefly, silk glands were collected and washed extensively with MilliQ water to remove any soluble traces of sericin. Collected silk glands were squeezed with fine forceps and the extracted silk fibroin was dissolved in 1% (w/v) sodium dodecyl sulfate (SDS, Himedia, India). Subsequently, SDS was removed by extensive dialysis against water using a dialysis membrane (MWCO 12 kDa, Sigma Aldrich, U.S.A.). Similarly, silk fibroin fiber was obtained from the cocoons of A. assama by degumming the cocoons in


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boiling 0.02 M sodium carbonate solution for 30 min to remove sericin. Degummed fibers were thoroughly washed with MilliQ water and dried at 37 ºC. 2.2 Scaffold fabrication and mineral deposition Dried silk fibers were finely chopped with scissors and packed into cylindrical Teflon molds (diameter 1.5 cm). Silk fibroin solution (2% w/v) was then poured onto the fiber layer in a ratio of 1:2 w/w and kept at -20 ºC for pre-freezing. The scaffolds were further freeze dried for 30 h in a lyophilizer (Alpha 1-4 LD plus - Martin Christ, Germany) followed by treatment with ethanol (80% v/v in water) to induce insolubility in water (Figure 1A, 1B). Obtained fiber reinforced scaffolds were subsequently mineralized with HA using previously reported alternate dipping method.27 The scaffolds were first dipped into 0.5 M CaCl2 (pH 7.2, 37 ºC, Himedia, India) followed by rinsing with deionized water. The scaffolds were further immersed in 0.2 M Na2HPO4 (pH 8.96, 37 ºC, Himedia, India) and rinsed with deionized water (Figure 1C). These steps were considered as the 1st cycle of Ca–P treatment. All the scaffolds were treated for 3 subsequent cycles at 37 ºC followed by overnight lyophilization. The compositions of different silk scaffolds used in this study are reported in Table 1.



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Figure 1. Schematic representation of silk fibroin composite scaffold fabrication. (A) silk fibroin freeze dried scaffolds, (B) fiber reinforced silk fibroin freeze dried scaffolds and (C) scheme representing HA deposition onto silk fibroin composite scaffolds by alternate dipping method. Table 1: Composition of various scaffold types. Scaffold type MSMF HA MSMF MS HA MS

A. assama fiber + + -

2% silk fibroin silk solution + + + +

Hydroxyapatite + + -

+ represents presence and – represents absence of particular component

2.3 Morphological analysis Surface morphology and architecture of fabricated scaffolds were studied using field emission scanning electron microscopy (FESEM, Zeiss, Germany). Briefly, cross-sectioned scaffolds were sputter coated with gold (Au) for 60 s. The samples were imaged at an operating voltage of 2.5


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kV. Captured images were further processed with NIH ImageJ software and at least 30 pores were processed for pore size determination. 2.4 Scaffold porosity and estimation of water retention ability The porosity of composite silk scaffolds was assessed following liquid displacement method.28 The scaffolds were cut into equal sized disc (6 mm diameter x 2 mm thickness). A known volume (V1) of hexane was poured into graduated measuring cylinder and scaffold discs were carefully dipped. Entrapped air within scaffolds was carefully removed by gentle pressure against cylinder wall. The volume after scaffold immersion was recorded (V2). Final hexane volume was recorded after removal of scaffold (V3). In order to estimate change in volume due to evaporation during measurement, a second measuring cylinder without scaffold was used as control to normalize the final volume. Porosity was calculated using the following formula: Scaffold Porosity (%) = [(V1-V3)/(V2-V3)]x100……………………………..[1] Water retention ability of scaffolds was studied using conventional gravimetric procedure. Lyophilized scaffolds were weighed and recorded in the dry state (Wd) followed by immersion in phosphate buffered saline (PBS, pH 7.4). At pre-determined time points scaffolds were carefully taken out, excess surface liquid was wiped by filter paper, followed by recording swollen weight (Ws). The swelling ratio was calculated using the equation: Swelling Ratio = (Ws-Wd)/Wd………………….……………… [2] Where, Wd is the dry weight of the construct and Ws is the swollen weight of the construct. 2.5 Enzymatic degradation and mechanical properties of scaffolds Enzymatic degradation of 3D silk scaffolds was evaluated using protease XIV derived from S. griseus (Sigma Aldrich, U.S.A.) with an activity of 3.5 U/mg following a predefined protocol.29 Briefly, silk scaffolds were immersed in PBS (pH 7.4) containing 2 U/mL protease and incubated



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at 37 °C. Enzyme solution was replaced every 3 days with freshly prepared solution. Scaffolds immersed in PBS without enzyme under similar conditions were used as control. On every 7th day, scaffolds were taken out, rinsed in distilled water, air dried and weighed to calculate weight loss over time for 28 days. Mechanical property of scaffolds was analyzed using a Universal Testing Machine (UTM, Instron 5944, U.S.A.) fitted with 100 N load cell. Compression tests were conducted under hydrated conditions (BioPuls bath) using PBS (pH 7.0) at 37 ºC. For recording, displacement control mode was used, with a crosshead displacement rate of 1 mm/min following ASTM standard D1621-04a (standard test method for compressive properties of rigid cellular plastics). The compressive stress-strain curves were plotted and the compressive modulus was determined for each sample using Bluehill version 3.66 software. 2.6 X-Ray Diffraction (XRD) The crystal phase of scaffold was characterized by wide angle X-ray diffraction analysis (XRD, RIGAKU, Japan). The working condition of XRD was CuK0 radiation via a rotating anode at 40 kV and 50 mA. Data was recorded with 0.05 °/s step size and in the range of scattering angles (2θ) between10° to 60°. The lattice spacing (d) was calculated using Bragg’s equation, d = λ / 2 sinθ, and the crystallite size was calculated using the Scherrer’s equation, D = 0.9 λ / ß cosθ; where λ is the wavelength of the X-ray (0.154 nm) and ß represents the full width half maxima of the diffracting angle (θ) used. 2.7 Fourier Transform Infrared spectroscopy (FTIR) Fourier transform infrared spectroscopy (FTIR, Nicolet 5700, U.S.A.) was performed under absorbance mode within the spectral range of 4000 to 400 cm-1 to characterize the samples. All spectra were recorded with a resolution of 4 cm-1 and an average of 32 scans.


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2.8 Cell culture and maintenance Human osteosarcoma cells (MG63) were procured from NCCS (National Centre for Cell Science, Pune, India) and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, U.S.A.) supplemented with 10% fetal bovine serum (FBS, Gibco, U.S.A.) and 1% antibioticantimycotic solution (Himedia, India). Human bone marrow stem cells (hBMSC) were isolated from bone marrow aspirate (Lonza, Basel, Switzerland)30 and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, U.S.A.) supplemented with 10% fetal bovine serum (FBS, Gibco, U.S.A.) and 1 ng/mL basic fibroblast growth factor (bFGF, Invitrogen, U.S.A.) at 37 ºC, 5% CO2 in a humidified incubator (95% humidity). Cells were passaged after reaching 80% confluence. For further studies, hBMSCs were used at early passages (P3-P5). 2.9 Cell seeding on composite silk scaffolds Scaffolds sized 6 mm diameter and 2 mm thickness were used for cell seeding. Before cell seeding, scaffolds were conditioned overnight in complete culture media followed by seeding with 3×105 MG63 cells suspended in 10 µL of growth medium (DMEM supplemented with 10% FBS). The seeded constructs were placed in a 37 °C humidified incubator with 5% CO2 for 3 h to allow cell adherence. After 3 h, 1 mL of culture media was gently added to each well. Fresh medium was replaced every alternate day and scaffolds were finally harvested on day 14. Similarly, scaffolds were evaluated for their osteogenic potential to differentiate hBMSCs in presence of osteogenic media. Approximately 3×105 cells were seeded onto each scaffold on day 0 and incubated for 28 days at 37 ºC, 5% CO2. After the initial 3 h of cell attachment on preconditioned scaffolds, they were transferred to 24 well culture plates. Following 3 days of culture in normal complete growth medium, the cell seeded scaffolds were transferred to osteogenic medium comprising of DMEM supplemented with 10% FBS, 0.1 mM non-essential amino acids,



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50 µg⁄mL L-ascorbic acid, 100 nM dexamethasone and 10 mM β-glycerolphosphate. Every alternate day, half of the spent media in each well was replaced with fresh osteogenic medium for the next 28 days. 2.10 Cell viability and proliferation Cell proliferation study was performed using alamar blue dye reduction assay (ThermoFisher Scientific, U.S.A.) according to manufacturers’ protocol. Alamar blue is a non-toxic dye and does not affect viability of cells. Briefly, scaffolds with and without cells were incubated at 37 ºC in DMEM medium containing 10% (v/v) alamar blue dye (Invitrogen, U.S.A.). At the end of 3 h incubation, 100 µL of medium from each sample was read at 570/600 nm in a multiplate reader (Tecan Infinite Pro 200). Non-seeded scaffolds supplemented with 10% alamar blue dye was used as a negative control. Proliferation of MG63 cells and hBMSCs within silk composite scaffolds were analyzed at predefined time points. In vitro cell attachment and viability was studied by staining MG63 cells with calceinAM (Sigma Aldrich, U.S.A.). Briefly, the cell-seeded scaffolds, harvested after 14 days of cell culture, were first washed with PBS to remove any non-adherent cell and cell debris. Cell seeded scaffolds were further stained with calcein-AM working solution (4 µM in PBS) for 20 min and visualized under fluorescence microscope (EVOS FL, Life technologies, U.S.A.) with excitation filter of 450–490 nm (green, calcein-AM). In a separate batch, hBMSC seeded scaffolds were processed differently, where scaffolds were incubated in a staining solution containing 0.5% (v/v) Triton X-100, 1% (w/v) Bovine Serum Albumin (BSA), 0.1% (v/v) Hoechst 33342 (Sigma Aldrich, U.S.A.) and AdipoRed (AdipoRed Assay Reagent, Lonza, U.S.A.). AdipoRed was used to stain silk and Hoechst 33342 (Sigma Aldrich, U.S.A.) to counterstain cell nucleus. Post staining, scaffolds were washed with PBS thrice and visualized under all-in-one fluorescence


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microscope BZ-X700 (KEYENCE, U.S.A.). BZ-X Analyzer software was used to perform Zstacking. 2.11 Alkaline phosphatase (ALP) activity and differentiation of hBMSCs Alkaline phosphatase activity was assayed using colorimetric ALP kit (Abcam, U.K.) according to manufacturer’s protocol. Briefly, spent media from cell seeded scaffolds was collected at different time points. 80 µL of the collected spent media was incubated with 50 µL of pnitrophenyl phosphate (5 mM) solution at 37 ºC for 1h in dark. At the end of the incubation period, enzyme activity was terminated by adding 20 µL of stop solution. Alongside, a standard curve was plotted using para-Nitrophenylphosphate (pNPP) concentration ranging between 0 and 0.02 µmol/well. The amount of p-nitrophenol produced was measured by monitoring absorbance at 405 nm which corresponds to the alkaline phosphatase activity. Results are presented as (U/L)/DNA content. DNA content was measured fluorometrically using PicoGreen assay kit (Molecular Probes, Life technologies, U.S.A.) according to manufacturer's protocol. Briefly, after 14 days of culture, scaffolds were finely chopped using scissors and cells were further lysed in 0.5% (v/v) Triton X-100 (Sigma Aldrich, U.S.A.), 5 mM MgCl2 (Himedia, India), 20mM Tris-HCl (pH 7.5, Himedia, India) and 150 mM NaCl (Himedia, India for 3h. 25 µL of the extract was added to assay microplate wells containing PicoGreen pre-mixed with 175 µL TE buffer (10 mM TrisHCl, pH 7.5, 1 mM EDTA) and incubated for 5 min. The PicoGreen–DNA complex was finally detected at a fluorescence excitation/emission wavelength of 490/520 nm using a multiwell plate reader (Tecan Infinite Pro 200). The total amount of DNA in the sample was determined using standard curve.



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The differentiation ability of hBMSCs towards osteogenic lineage was tested to determine its stemness. Accordingly, hBMSCs were cultured in standard tissue culture plate for 7 days in presence of osteogenic media and further stained with Alizarin Red dye (Sigma Aldrich, U.S.A.) to visualize calcium deposition. 2.12 Gene expression analysis using Real Time PCR Real Time PCR was used to determine expression of collagen I (Col-I), osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA transcripts. After 14 days of culture, MG63 cell seeded scaffolds were treated with TRIzol reagent (Sigma Aldrich, U.S.A.) for 30 min and centrifuged at 13,000 rpm (4 ºC, 10 min). Supernatant was collected in fresh tubes followed by chloroform treatment. Further chloroform treated samples were centrifuged at 13,300 rpm for 15 min at 4 ºC. Subsequently, the upper aqueous phase containing RNA was added with isopropanol to obtain RNA pellet. Following RNA isolation, 1 µg of isolated RNA was used for the reverse-transcriptase reaction to synthesize cDNA using reverse transcription kit (Applied Biosystems, U.S.A.) and PCR equipment (Takara, Japan). Real-time PCR reactions were performed and monitored using SYBR Green PCR Mastermix on a 7500 Fast Real Time System (Applied Biosystems, U.S.A.). Expression of each target gene was normalized with GAPDH. Primer sequences used are listed in Table 2. Table 2: Primer sequences used for Real Time PCR S. No. Gene Collagen I (Col-I) 1 2

Osteopontin (OPN)

3

Osteocalcin (OCN)

Primer sequence F: 5'-CGGAGGAGAGTCAGGAAG-3' R: 5'-CAGCAACACAGTTACACAAG-3' F: 5'-TGAAACGAGTCAGCTGGATG-3' R: 5'-TGAAATTCATGGCTGTGGAA-3' F: 5'-CAGCGAGGTAGTGAAGAGAC-3' R: 5'-GCCAACTCGTCACAGTCC-3' 14 ACS Paragon Plus Environment

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4

GAPDH

F: 5'-GACCTGACCTGCCGTCTA-3' R: 5'-GTTGCTGTAGCCAAATTCGTT-3'

For hBMSCs, RNA was isolated and reverse transcribed after 28 days of culture following above mentioned

protocol.

Following

genes

were

tested:

Col-I

(Hs00164004_m1),

BSP

(Hs00173720_m1), OPN (Hs00167093_m1). Results were plotted upon normalization with human GAPDH (Hs99999905_m1) (ThermoFisher Scientific, U.S.A.). 2.13 Histological analysis 3D silk scaffolds seeded with MG63 cells following 14 days of culture were fixed in neutral buffered formalin (NBF, Sigma Aldrich, U.S.A.), dehydrated in graded ethanol and finally embedded in paraffin. The scaffolds were cut into 5 µm thick sections using a manual rotary microtome (Leica biosystems, U.S.A.) and mounted on microscope slides. Scaffold sections were analyzed by immunostaining to qualitatively localize and evaluate the extent of collagen type I (Col-I) and bone sialoprotein (BSP) deposition. During preparation, sections were incubated with 0.1% (v/v) blocking serum (Vectastain Elite Universal ABC kit, Vectors lab, U.K.) for 20 min in order to stop endogenous peroxidase activity. Constructs were further incubated with monoclonal primary rabbit antibody (Abcam, U.K.) against Col-I and BSP (1:300) for 30 min, followed by incubation with biotinylated universal secondary antibody (Vectastain Elite Universal ABC kit, Vectors lab, U.S.A.). Following incubation the sections were dipped into ABC reagent containing avidin-horseradish peroxidase. Peroxidase substrate 3, 3′, 5, 5′-Tetramethylbenzidine (TMB Peroxidase (HRP) Substrate kit, Vectors lab, U.S.A.) was used to develop a bright blue reaction product. The positive reactivity of staining was documented by photo-microscopy using bright field illumination under an inverted microscope (EVOS FL, Life technologies, U.S.A.).



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2.14 Statistical analysis All experiments were performed at least in triplicates and data represented as mean ± standard deviation unless otherwise stated. Data was statistically processed using one way analysis of variance (ANOVA) to assess significant differences amongst various groups followed by Tukey’s test using OriginPro 8.0 software. #p≤0.05 was considered significant and

##

p≤0.01 as

highly significant for reported results. 3. RESULTS 3.1 Morphological analysis of composite silk scaffolds 3-D porous scaffolds were fabricated using freeze drying process (Figure 1). Fiber length exhibited a heterogeneous distribution and ranged between 0.5 to 2.5 mm (calculated by processing microscopic images of 50 different fibers using ImageJ software, Figure S1). Gross morphology of scaffolds is depicted in Figure 2 and amount of HA deposited within silk composite scaffolds is listed in Table 3.

Figure 2. Gross morphology of different composite silk scaffolds with and without HA. Table 3: Amount of HA deposited within silk scaffolds using alternate dipping method Types of scaffold MSMF HA MSMF MS HA MS

Weight of scaffolds before HA deposition (mg) 57.34±2.58 57.34±2.58 27.3±0.4 27.3±0.4

Weight of scaffolds after HA deposition (mg) 99.5±6.12 57.34±2.58 67.2±4.5 27.3±0.4

HA content (mg/scaffold) 42.2±7.05 0 40±4.3 0 16

ACS Paragon Plus Environment

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FESEM images revealed porous and interconnected structure within scaffolds (Figure 3A-D). The pore size ranged between 80-150 µm in all groups. Following HA deposition, formation of HA crystals within scaffold surface was observed. Silk fibroin may have helped in nucleation and deposition of these HA crystals onto its surface. Plate shaped HA crystals similar to native bone, was formed along the pore walls of composite scaffolds (MS HA and MSMF HA) and on the fiber surface of MSMF HA scaffolds (Figure 3E). Higher magnification FESEM images further confirmed HA deposition resulting in increased surface roughness and wall thickness. Fabricated 3D scaffolds showed nearly 90% porosity. Further, porous nature of the composite scaffold was maintained with minimal reduction in porosity after HA and fiber incorporation (Figure 3F). Since HA is also present in native bone, its incorporation within scaffolds is assumed to enhance osteogenic differentiation by mimicking the chemical composition of bone.



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Figure 3. Field emission scanning electron microscopic (FESEM) images of composite silk scaffolds exhibiting porous morphology and fiber reinforcement - (A) MSMF HA, (B) MSMF, (C) MS HA, (D) MS. (E) Magnified FESEM image showing deposition of plate shaped HA crystals on composite silk fibroin scaffolds. (F) Porosity of composite silk scaffolds as determined by liquid displacement method. 3.2 Water retention ability of scaffolds Composite silk scaffolds (with HA incorporation and fiber reinforcement) showed reduced swelling ratio when immersed in PBS (pH 7.4) as compared to pure silk scaffolds (MS). The scaffolds swelled and reached equilibrium within 30 min after immersing in PBS. Similarly, the swelling ratio of scaffolds with HA deposition was nearly half of the pure silk scaffolds without HA deposition (Figure 4A). In addition, scaffold with fiber reinforcement (MSMF HA and MSMF) showed lesser swelling compared to the pure silk fibroin freeze dried scaffolds (MS HA and MS). 3.3 Degradation and mechanical properties of composite silk scaffolds The degradation rate of composite silk scaffolds was determined in both protease XIV and PBS. Protease XIV enzyme is known to degrade fibroin crystalline structure.31 It was observed that MS scaffold degraded nearly 50% of its original weight in 28 days; followed by MS HA, MSMF and silk MSMF HA scaffolds, respectively (Figure 4B). In contrast, control scaffolds incubated in PBS showed minimal degradation (1-3 wt%) even after completion of 28 days. Herein, the results indicated slowing down of degradation rate following fiber reinforcement and HA deposition within silk scaffolds. The rate of degradation could possibly be tuned by manipulating the amount of HA deposition (by altering the number of cycles) and amount of silk fibers used as reinforcement. The stress-strain behavior of all silk scaffolds was found to follow similar pattern characteristic of open foamed cells or sponges. The stress-strain curves displayed an initial linear 18 ACS Paragon Plus Environment

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elastic region representing lower modulus followed by a plateau corresponding to pore wall contortion and consequently a steep increase in stress suggesting sample flattening (Figure 4C). This sponge-like behavior was observed for all the aqueous-derived silk scaffolds. It is clear from the result that both fiber reinforcement and HA incorporation resulted in increased modulus and compressive strength. The incorporation of fibers and HA in freeze dried scaffolds exhibited increment in compressive modulus. The tricomposite MSMF HA scaffold showed maximum compressive modulus (46.88±2.82 kPa, approx 5 fold higher) and compressive strength at 20% deformation (9.65±0.49 kPa, approx. 14 fold higher) in comparison to the native silk fibroin scaffold (MS) (Figure 4D, 4E). A sequential increment pattern was observed for each scaffold type with following trend: MS

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