Nano-biocomposite scaffolds of chitosan ...

International Journal of Biological Macromolecules 111 (2018) 923–934

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International Journal of Biological Macromolecules journal homepage: https://www.journals.elsevier.com/ijbiomac

Nano-biocomposite scaffolds of chitosan, carboxymethyl cellulose and silver nanoparticle modified cellulose nanowhiskers for bone tissue engineering applications Abshar Hasan, Gyan Waibhaw, Varun Saxena, Lalit M. Pandey ⁎ Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam, 781039, India

a r t i c l e

i n f o

Article history: Received 31 July 2017 Received in revised form 5 January 2018 Accepted 13 January 2018 Available online xxxx Keywords: Chitosan Cellulose nanowishkers Carboxymethyl cellulose Antimicrobial scaffolds Silver nanoparticles Bone tissue engineering

a b s t r a c t In the present work, we aimed to synthesize highly efficient nano-composite polymeric scaffolds with controllable pore size and mechanical strength. We prepared nanocomposite (CCNWs-AgNPs) of silver nanoparticles (AgNPs) decorated on carboxylated CNWs (CCNWs) which serves dual functions of providing mechanical strength and antimicrobial activity. Scaffolds containing chitosan (CS) and carboxymethyl cellulose (CMC) with varying percent of nanocomposite were fabricated using freeze drying method. XRD and FESEM analysis of nanocomposite revealed highly crystalline structure with AgNPs (5.2 nm dia) decorated on ~200 nm long CCNWs surface. FTIR analysis confirmed the interaction between CCNWs and AgNPs. Incorporation of nanocomposite during scaffolds preparation helped in achieving the desirable 80–90% porosity with pore diameter ranging between 150 and 500 μm and mechanical strength was also significantly improved matching with the mechanical strength of cancellous bone. The swelling capacity of scaffolds decreased after the incorporation of nanocomposite. In turn, scaffold degradation rate was tuned to support angiogenesis and vascularization. Scaffolds apart from exhibiting excellent antimicrobial activity, also supported MG63 cells adhesion and proliferation. Incorporation of CCNWs also resulted in improved biomineralization for bone growth. Overall, these studies confirmed excellent properties of fabricated scaffolds, making them self-sustained and potential antimicrobial scaffolds (without any loaded drug) to overcome bone related infections like osteomyelitis. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Any foreign material, particularly biomaterials in tissue engineering, when comes into contact with biological environment (e.g. body fluids) serves as a potential site for microbial invasion causing infection [1]. There has been huge loss in terms of implant failure and deterioration due to such microbial contamination. Various researchers are actively working to counteract such implant associated infections by applying different techniques such as surface modification, anti-fouling polymeric coatings, usage of antibiotics etc. [2–4]. Many researchers have reported the good antifouling performance of polyethylene glycol (PEG) coatings. However, significant densities of bacteria may still attach on PEG, and the polymer may undergo oxidative degradation at physiological conditions [2]. New antifouling polymer coatings with improved antifouling properties are actively being sought. Moreover, long term antibiotic treatments have their own adverse side effects of creating antibiotic resistant bacteria. Hence, development of alternative antimicrobial agents such as metallic (zinc, copper, and silver etc.) based agents, plant derived antimicrobial compounds etc. are the need of the hour [5–7]. ⁎ Corresponding author. E-mail address: [email protected] (L.M. Pandey).

https://doi.org/10.1016/j.ijbiomac.2018.01.089 0141-8130/© 2018 Elsevier B.V. All rights reserved.

Silver (Ag) is widely known for its antimicrobial activity and can be used against broad spectrum of infectious agents, both in ionic as well as metallic forms [8]. Oxidised form of Ag (Ag+) disrupts bacterial cell membrane, inhibits enzymes and DNA replication, causing death of many clinically relevant strains [9]. Usage of such antimicrobial agents in preparing tissue engineering scaffolds/hydrogels can answer many implant associated problems without producing any deleterious effect on recipient health. Various synthetic and natural polymers such as polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), carboxymethyl cellulose (CMC), chitosan (CS), collagen, gelatin etc. have been widely investigated for tissue engineering applications [10]. Collagen and HAp are the major constituents of human bone and hence are widely used as a composite material for bone regeneration applications [11]. PLA is a biocompatible and biodegradable polymer, exhibiting moderate mechanical and thermal properties, which can be improved by making its composite with other polymers/ ceramics for biomedical applications [12]. CS is of great interest as it is naturally occurring second most abundantly polymer (after cellulose) that possess excellent biodegradable, biocompatible, bioactive and antimicrobial properties [7]. It is a deacetylated product of chitin, composed of repeated units of 1–4 linked d-glucosamine and N-acetyl-d-glucosamine residues, which can easily

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A. Hasan et al. / International Journal of Biological Macromolecules 111 (2018) 923–934

be chemically modified and functionalized [13]. It had been widely used as wound dressings, drug delivery vehicles, packing materials, coating of biomedical devices and for various tissue engineering biomaterials [10,14]. Although CS is biocompatible, bioactive and biodegradable but its scaffolds used in tissue engineering applications, particularly bone tissue engineering suffer from low mechanical strength. This problem was resolved by making composite scaffolds of synthetic polymers, ceramic materials like hydroxyapatite and cellulose nanocrystals [10,14,15]. Carboxymethylcellulose (CMC) is a highly hydrophilic, semi-synthetic, and a promising biopolymer used in various biomedical applications and can be potentially functionalised for improving its existing performances [16]. Being anionic in nature and having structural similarity with CS, they form polyelectrolyte complex via strong ionic cross linking which in turn enhances the hydrophilicity and swelling behavior of CS [17,18]. A hydrogel of CMC-hydroxyapatite (HAp) composite was used for enhancing biological applications in bone tissue engineering [19]. Here, we report the application of bifunctional cellulose nanowhiskers (CNWs) decorated with AgNPs refereed as CCNWsAgNPs nanocomposite, incorporated inside the polymeric matrix for bone tissue engineering applications. CNWs are nanosized cellulose fibers synthesized by acid hydrolysis of cellulose and are used as nanofillers in polymeric matrices for making biosensors substrates [20], improving thermal and mechanical properties of food packaging materials and for sustained drug delivery etc. [21,22] CCNWs-AgNPs nanocomposite provides dual function of providing mechanical and thermal strength due to CNWS and antimicrobial property due to AgNPs. The synergistic effect of CS and CCNWs-AgNPs against both Gram positive and negative bacteria has helped us to develop a highly efficient antimicrobial scaffold that was able to completely eradicate microbial population and hence can overcome bone related infections such as osteomyelitis. 2. Materials and methods 2.1. Materials Chitosan (CS, Mw = 50–190 kDa and degree of deacetylation = 75– 85%, Cat. No. 448869), silver nitrate (AgNO3, Cat. No. 209139), 2,2,6,6Tetramethyl-1-piperidinyloxy radical (TEMPO, Cat. No. 214000) and sodium bromide (NaBr, Cat. No. S4547) were procured from Sigma-Aldrich, India. Cellulose (Cat. No. MB 132), carboxymethyl cellulose (CMC) sodium, and other chemicals like MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide], sodium borohydride (NaBH4) potassium bromide (KBr), dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) were purchased from HiMedia, India. Sodium chloride (NaCl), potassium chloride (KCl), monobasic potassium phosphate (KH2PO4), dibasic sodium phosphate (Na2HPO4), sulfuric acid, sodium hypochlorite (NaClO) and acetic acid were procured from Merck, India. Double distilled water (MiliQ, 18 mΩ, Millipore systems) was used throughout the experiments. 2.2. Methods 2.2.1. Preparation and carboxylation of cellulose nanowhiskers (CNWs) Acid hydrolysis method reported elsewhere [23,24] was used for preparing cellulose nanowhiskers from microcrystalline cellulose powder. Briefly, cellulose powder (1:10 w/v) was acid hydrolyzed in sulfuric acid (62%) at 50 °C under constant stirring for 2 h. Hydrolysis was stopped by adding ice cold water and the resulting solution was centrifuged (10,000 rpm for 10 min), washed several times with water until neutral pH and lyophilized (Scanvac, coolsafe) to get cellulose nanowhiskers. The carboxylation of CNWs was carried out by oxidising hydroxyl groups present on CNWs surface to carboxylic acid groups using

NaClO/NaBr/TEMPO oxidation system [25,26]. Briefly, carboxylated CNWs (CCNWs) was prepared by slowly stirring 100 mL of 1% CNWs suspension with TEMPO (70 mg/g of CNWs) and NaBr (180 mg/g of CNWs). NaClO (9 wt%) was added dropwise with slow stirring and its pH was maintained at 10.5 for a period of 10 h. Finally, 20 mL of absolute ethanol was added to quench the reaction followed by washing with water and later filtered, lyophilized and stored at 4 °C. 2.2.2. Synthesis of CCNWs-AgNPs nanocomposite Scheme 1 shows the detailed illustration of CCNWs-AgNPs nanocomposite synthesis. CCNWs-AgNPs nanocomposite was synthesized using CCNWs suspension during metallic reduction method reported previously by Liu et al. [20,25]. Briefly, 1% CCNWs (w/v) suspension was gently stirred with 5 mL AgNO3 (1.0 × 10−2 M) solution followed by reduction with NaBH4 (1.0 × 10−2 M) solution under constant stirring for 1 h at 25 °C to yield 1.766 wt% silver in nanocomposite. Post reduction, CCNWs-AgNP nanocomposite was washed several times with MiliQ water, centrifuged, lyophilized overnight and stored at 4 °C until further use. 2.2.3. Fabrication of CS/CMC/CCNWs-AgNPs composite scaffold Composite scaffolds of CS, CMC and nanocomposite were fabricated by varying percentage of nanocomposite in the final mixture. Briefly, 1% (w/v) CS solution was first prepared in acetic acid (1% v/v) solution to which 1% (w/v) CMC was added and left for stirring for 3 h. CS/CMC solution was later mixed with different percentage of CCNWs-AgNPs nanocomposite (0, 1%, 2.5%, 5%, 10%) and gently stirred for another 1 h to make a homogenous mixture. Mixtures were frozen at −20 °C for 12 h before freeze drying. Microporous scaffolds were later neutralized with 1 N NaOH and washed repeatedly with water to achieve neutral pH. Washed scaffolds were again freeze dried and stored in a desiccator under vacuum. Nanocomposite scaffolds containing 0, 1, 2.5, 5 and 10% of CCNWs-AgNPs were termed as SCA-0, SCA-1, SCA-2.5, SCA-5 and SCA-10, respectively. 2.2.4. Characterization Synthesized CNWs, CCNWs, CCNWs-AgNPs nanocomposite and nanocomposite scaffolds were characterized using various techniques. Field emission scanning electron microscopy (FESEM, Zeiss, Model: Sigma) was used for size and morphology analysis of CNWs, CCNWs and CCNWs-AgNPs as well as for the microporous structure of scaffolds. Transmission electron microscopy (TEM) was employed to determine the size and shape of CCNWs-AgNPs using JEOL instrument (JEM 2100). Optical characterization of CCNWs and CCNWs-AgNPs was done by scanning in 300 to 600 nm range (Infinite 200 Pro, Tecan). Fourier transform infrared (FTIR, Thermo, Nicolet iS10) spectroscopy was used at a scanning speed of 32 scans per second at resolution 2 cm−1 for determining interactions between different components and functional groups. X-ray diffraction (XRD, Bruker, Model: D8-Advance) equipped with a Cu-Kα radiation source (λ = 0.154) at 30 kV and 15 mA was used for samples analysis at diffraction angle (2θ) in the range 5° to 40° at a scanning step time of 2 s and step size of 0.02°. Thermal properties of CCNWs and CCNWs-AgNPs nanocomposite were analyzed using NETZSCH STA 449F3 instrument operated at a heating rate of 10 °C/min up to 600 °C. 2.2.5. Scaffold porosity and mechanical strength Liquid displacement method reported by Cao et al. [27] was used to measure the porosity of the scaffolds. Briefly, known weight of lyophilized scaffold (Wd) was immersed in hexane and was subjected to vacuum for 10 min to force hexane into scaffold's pores. Scaffold was then immediately transferred to weighing bottle containing hexane of known volume (V1) and weight (W1). After immersion of scaffold in weighing bottle, the final volume (V2) and weight (W2) were recorded to calculate the porosity (φ) of the scaffold using following expression


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Scheme 1. Detailed illustration of CCNWs-AgNPs nanocomposite synthesis.

(1): φð%Þ ¼

degradation) period was also calculated. W 2 −W 1 −W d ρðV 2 −V 1 Þ

ð1Þ

Where ρ is the density (0.655 gcm−3) of hexane, (W2 − W1 − Wd) is the weight of hexane penetrated into scaffold and (V2 − V1) is the apparent volume of scaffold. The mechanical strength of scaffolds was determined in terms of the compressive performance using UTM (Universal Testing Machine) instrument (Instron 5944 U.S.) equipped with 100 N load cell with the cross head speed of 1 mm/min. Compression experiments were performed in PBS (pH 7.4) at ambient atmospheric condition (20 °C and 55% relative humidity) [27,28]. 2.2.6. Swelling and degradation studies Lyophilized scaffolds of known initial weights (Wi) were immersed in PBS (pH 7.4) for different time interval at 37 °C. After each time point, the scaffolds were removed and weighed (Wf) immediately after removal of excess PBS from scaffolds with the aid of blotting paper. The swelling percentage was evaluated using following expression (2): %Swelling ¼

W f −W i 100 Wi

ð2Þ

Enzymatic degradation of lyophilized scaffolds (of known weight, Wi) was carried out in PBS containing lysozyme (2 mg/mL, in PBS, pH 7.4) at 37 °C in orbital shaker (Orbitek, Scigenics), under constant agitation at 60 rpm, for different time intervals (4, 8, 12, 16 and 20 days). Spent solution was replaced everyday with fresh lysozyme solution. After each time point, scaffolds were removed from lysozyme solution and lyophilized overnight to get final dry weight as (Wt). The percentage degradation as a function of time was calculated using following expression: Degradation ð%Þ ¼

W i −W t 100 Wi

ð3Þ

The degradation kinetic study was performed by using first order kinetics [29] as shown below and the 50% scaffold degradation (T50

M ¼ M0 1−e−kt

ð4Þ

Where M0 was initial scaffold mass, M represents remaining mass after t days and k is degradation rate constant (day−1).

2.2.7. Protein adsorption studies Protein adsorption capacity of scaffolds was analyzed using method described by Sainitya et al. [18] with slight modifications. Briefly, equal volume of scaffolds was prepared and pretreated with 70% ethanol for 10 min followed by hydration with PBS (1×, pH 7.4). Hydrated scaffolds were immersed in 10% fetal bovine serum (FBS) solution prepared in PBS for 2 h to ensure completion of adsorption process. Later scaffolds were rinsed in PBS (2 times) to remove unbound proteins. Post washing, scaffolds were immersed in 5% SDS (w/v) solution for 1 h at 37 °C under constant agitation (60 rpm) to detach adsorbed proteins. Finally desorbed protein concentration was estimated by the bicinchoninic acid (BCA) assay [22,30].

2.2.8. Antibacterial activity Antibacterial assay of the scaffolds was performed using protocol based on ASTM G 21–09 and is widely reported by other researchers [25,31,32] Briefly, circular scaffolds of 15 mm diameter were washed in 70% ethanol to remove surface bacteria followed by washing with sterilized water. Antibacterial activity was tested against Escherichia coli (E. coli, Gram negative) and Enterococcus hirae (E. hirae, Gram positive) bacteria. Bacterial culture (100 μL) of 1.0 × 106 colony forming units per mL (CFU/mL) with 100 μL of media was dropped onto scaffolds and left for incubation in sterilized petri dish at 37 °C for 24 h. Each scaffold was later transferred to fresh petri dish and washed with 20 mL of 0.87 wt% NaCl solution containing 0.03 wt% Tween 80 at pH 7. This washing solution was diluted 20 times with PBS followed by spreading of 100 μL of this diluted solution on LB (Luria Bertani)-agar plates for counting bacterial colonies after incubation at 37 °C for 24 h. Counted number of colonies were multiplied with dilution factor to calculate CFU per sample. Finally, antibacterial ratio (%) was calculated using

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3. Results and discussion

following expression: Antibacterial ratio ð%Þ ¼

Nc −N s 100 Nc

ð5Þ

Where NC and NS are mean number of bacterial colonies on blank (SCA0) and scaffold (SCA-1 and SCA-2.5) samples, respectively. 2.2.9. Cytotoxicity and cell attachment assay MG-63, a human osteosarcoma cell line, procured from NCCS, Pune (India), was used for investigating the in vitro cytotoxicity and cell attachment assays. Cell line was maintained in CO2 incubator at 37 °C, 5% CO2 and 85% humidity and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% antibiotic (Pen Strep, Invitrogen). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed for assessing cytotoxicity as described by Saravanan et al. [7]. Initially, ‘conditioned media’ was prepared by soaking the calculated amount of UV sterilized scaffold in serum free DMEM media for 24 h at 37 °C. Later, supernatant was collected, filtered (0.22 μm) and referred as ‘conditioned media’. Conditioned media (100 μL) prepared from SCA-0 and SCA-2.5 was added separately in triplicate to 24 well plate (Corning, Costar) already seeded with 1 × 104 cells/mL/ well in complete growth media. Experiment was run for 6 days and assay was performed on 2nd, 4th and 6th day. Complete growth media without conditioned media served as control. Spent media was replaced after every 24 h. After each specified time interval, media was replaced with fresh media containing 20 μL of MTT solution (5 mg/mL, PBS pH 7.4) and incubated for 4 h at 37 °C. Later, optical density (OD) was measured at 570 nm (Infinite 200 Pro, Tecan) after incubating in DMSO (150 μL) for 10 min. In vitro cell attachment assay was performed on SCA-0 and SCA-2.5 scaffolds after seeding cells at a concentration of 1 × 105 cells/mL. Scaffolds along with MG-63 cells were incubated for 5 days in DMEM media. Post incubation, scaffolds were harvested and unattached cells were removed by washing with PBS followed by staining of cells with calceinAM dye (Sigma-India, 4 μM in PBS) for 30 min and visualized under fluorescent microscope with excitation filter of 450–490 nm. 2.2.10. In vitro bioactivity in SBF In vitro bioactivity test was carried out to determine the capabilities of scaffolds for apatite mineralization in bone tissue engineering. Briefly, the synthesized scaffold samples were immersed in simulated body fluid (SBF) solution which has concentration and composition similar to human body fluid. SBF was prepared in double distilled water by dissolving the different chemicals in the following sequence: NaCl (8.026 g/L), NaHCO3 (0.352 g/L), KCl (0.225 g/L), K2HPO4·3H2O (0.230 g/L), MgCl2·6H2O (0.311 g/L), CaCl2 (0.293 g/L) and Na2SO4 (0.072 g/L). Then the liquid was buffered to pH of 7.4 with tri-hydroxymethyl aminomethane [(CH2OH)3CNH2] (6.063 g/L) and hydrochloric acid (1 M, 40 mL) at 37 °C [33]. CCNWs were used instead of CCNWs-AgNPs for the fabrication of scaffolds to be tested for apatite deposition in SBF assay. Triplicate samples of SCA-0, SCA-1, SCA-2.5, SCA-5 and SCA10 were immersed in the SBF solution in polypropylene bottles for 14 days at 37(± 0.5)°C and with the solution refreshed every 24 h to maintain 7.4 pH [34]. The samples following SBF immersion were rinsed gently with ethanol and distilled water and then dried in hot air oven at 45 °C overnight to remove any entrapped water molecules. Deposition of apatite layer after immersion in SBF for 14 days was studied by FESEM and energy dispersive spectroscopy (EDS) analysis for their microstructure and elemental analysis. 2.2.11. Statistical analysis All the experiments were carried out in triplicate (n = 3) and statistical analysis was carried out by OriginPro 8.5 with the values presented as mean ± standard deviation.

3.1. Characterization Acid hydrolysis (using concentrated sulfuric acid) is a well-known method of deconstruction of cellulose microfibrils for formation of highly crystalline nanosized particles and are called as cellulose nanowhiskers and sometimes referred as nanocrystals [35]. Detailed characterizations of synthesized CNWs are reported in our previous work [22]. Briefly, the synthesized CNWs were 20–30 nm in diameter and approximately 200 nm in length with high crystallinity of 92.8% and crystallite size of 4.1 nm. Fig. 1(a) shows the FESEM image of CCNWs of approximately 200–250 nm length and 20 nm diameter. TEMPO mediated conversion of CNWs surface's primary OH− to COO− groups served as adsorption sites for Ag+ metallic ions, which were further reduced to AgNPs from AgNO3 in presence of reducing agent (NaBH4). Strong interaction between OH− and COO− groups and AgNPs not only helped in stabilizing AgNPs but also prevented their aggregation [25]. Fig. 1(b) shows the TEM image of AgNPs dispersed in the presence of CCNWs with the average particle size of 5.2 (±0.4) nm in diameter, calculated using ImageJ software. Nanocomposite showed well-contrasted AgNPs because CCNWs give little contrast against the nanoparticles. High affinity of AgNPs towards carboxyl and hydroxyl groups of CCNWs resulted in strong immobilization and stabilization of AgNPs particles on CCNWs [25,36]. UV–vis adsorption spectra were recorded to determine the optical properties of CCNWs and CCNWs-AgNPs as shown in Fig. 1(c). An adsorption band between 350 and 500 nm is attributed to Surface Plasmon Resonance due to AgNPs, indicating AgNPs formation in CCNWs suspension during CCNWs-AgNPs nanocomposite formation. Whereas no such band was seen in the given range for CCNWs samples and similar observation was also reported previously by Liu et al. [25]. Fig. 1(d) shows XRD characterization CCNWs-AgNPs exhibiting characteristic peaks of Ag at 2θ = 37.1°, 43.5°, and 64.8° that corresponds to crystallographic planes from (111), (200), and (220) of cubic silver [20] along with 14.7°, 16.4°, 22.4° and 34.6° which are attributed to cellulose I [22]. Incorporation of new 2θ peaks due to Ag clearly depicts the formation of AgNPs supported on CCNWs surfaces. Fig. 2(a) shows the FTIR spectra of cellulose powder, CNWs, CCNWs and CCNWs-AgNPs. Previously, we have reported acid hydrolysis of cellulose powder to form CNWs which was confirmed by the appearance of an additional peak at 1205 cm−1 , attributed to sulfate groups (due to esterification) [22]. Peak at 1642 cm−1 region corresponds to carbonyl (C=O) group which is assumed to be formed due to partial oxidation of carbon atoms present in the cellulose molecules [25]. Formation of carboxylic groups in CCNWs due to oxidation of primary alcohols (present in CNWs) via TEMPO mediated mechanism results in appearance of a new peak at 1699 cm−1. Attachment of AgNPs to carbonyl groups of CCNWs resulted in the shifting of peak to 1600 cm−1 and the similar result was also reported by Liu et al. [25]. Thermal decomposition analysis describes the physico-chemical properties of material and is analyzed using TGA graphs as shown in Fig. 2(b). CNWs being more flexible, formed entanglements of the nanowhiskers and, hence were more thermally stable as compared to its precursor cellulose powder [37]. Initial weight loss in all the samples between 50 and 150 °C is attributed to moisture loss which was chemisorbed and/or intermolecularly hydrogen bonded water molecules [38]. CNWs were most stable as their degradation started at around 320 °C, leaving 32% of decomposed product at 500 °C. Thermal decomposition point of CCNWs (TEMPO oxidised) and CCNWs-AgNPs reduced significantly to 200 °C due to formation of sodium and metallic-carboxylate groups, respectively, from the C6 primary hydroxyls of cellulose microfibril surfaces [38,39]. Binding of silver metal although


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Fig. 1. Characterization of CCNWs and CCNWs-AgNPs nanocomposite. (a) FESEM analysis of CCNWs at 100 KX magnifications, (b) TEM analysis of CCNWs-AgNPs nanocomposite along with particle size distribution (inset) showing average particle size of 5.2 nm in diameter of AgNPs, (c) UV–vis absorbance spectra and (d) XRD pattern of CCNWs (inset) and CCNWsAgNPs nanocomposite.

reduced the degradation point significantly but the degradation rate was massively slowed down giving ~44% degraded product even at 500 °C. Fig. 3 shows the FTIR spectra of CS, CMC and SCA-2.5. Characteristic peaks in CS at 1030, 1076, and 1655 cm−1, corresponds to C\\O stretching, O\\H blending, and amide I, respectively. CMC shows characteristic peak at 1592 cm−1 which is attributed to asymmetrical

COO− stretching. Peaks at 1600 cm−1 due to amide II of CS and at 1592 cm−1 due to CMC disappeared and a common peak was seen at 1575 cm−1 resulting due to interaction between CS and CMC [18]. Furthermore, electrostatic interaction was confirmed between NH2 group of CS and COO− groups of CCNWs and CMC due to shifting of amide I peak from 1655 cm−1 to 1645 cm−1 in SCA-2.5. Appearance of peak at 1700 cm−1 in SCA-2.5 corresponds to C_O stretching of COOH group

Fig. 2. Characterization of cellulose powder, CNWs, CCNWs and CCNWs-AgNPs using (a) FTIR and (b) TGA.

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Table 1, increase in CNWs contents (1, 2.5, 5 and 10%) results in increase in the average pore diameter. The pore diameter of SCA-0 was found to be in the range 30–60 μm which increased to 50–100 μm due to incorporation of 1% CCNWs-AgNPs in SCA-1 and up to 250–400 μm for SCA-5. This phenomenon can be attributed to hydrogen bond formation between nanofillers (CCNWs-AgNPs) and polymeric matrix, resulting in restricted motion of backbone chain. Moreover, repulsive forces between COO− groups of nanofiller and of CMC further cause hindrance in backbone chain mobility. Combined effect of such interactions causes reduced covalent cross-linking between polymeric precursors which ultimately results in increased pore diameter [35]. As a requirement of an ideal scaffold, the pore size should be at least 100 μm for the diffusion of essential nutrients and oxygen but pore sizes in the range between 200 and 350 μm are found optimum for bone in-growth [42]. We were able to yield the similar pore sizes by increasing the CCNWs-AgNPs content in SCA-5 scaffold. Moreover, it is reported that vascularization and bone ingrowth into implant requires osteointegration and osteoconduction which preferably takes place when the interconnected porosity lies in the range 70–90% [9,43]. Notably, porosity of cancellous bone lies in the range between 60 and 80% [44], which was very well obtained in the synthesized scaffolds (shown in Table 1). 3.2. Scaffold porosity and mechanical strength

Fig. 3. FTIR characterization of CS, CMC and SCA-2.5 in the range 900–1800 cm−1.

present on CCNWs. We could not distinguish the characteristic peak of CCNWs-AgNPs nanocomposite at 1600 cm−1 (as shown in Fig. 2(a)) in the SCA-2.5 spectra due to overlapping peaks of NH2 bending of CS and asymmetrical COO− stretching of CMC. Pore size plays a vital role in scaffolds used in bone tissue engineering for regulating cell infiltration and behavior, angiogenesis, nutrient exchange and oxygen diffusion during bone regeneration [40–42]. FESEM analysis of synthesized scaffolds was carried out to determine the microporous structure and difference in the pore size due to incorporation of CCNWs. Fig. 4 shows images with disorganized interconnected porous structures which are formed due to solid–liquid phase separation [27]. CCNWs-AgNPs nanocomposites were evenly distributed within the scaffold's pore walls hence no large aggregates were seen in any SEM image. As shown in Fig. 4(a–e) and mentioned in

Scaffold porosity was evaluated using well established liquid displacement method [27]. Porosity of scaffolds plays an important role in regulating diffusion of nutrients and oxygen. As shown in Table 1, porosity of scaffolds reduced with increasing nanofiller concentration possibly due to reduction of water content in polymeric matrix. The mechanical strength of the prepared scaffolds was investigated by measuring the compressive strength [27]. As mentioned in Table 1, the compressive strength increased from SCA-0 (0.37 MPa) to SCA-1 (1.07 MPa), SCA-2.5(2.14 MPa), SCA-5 (3.84 MPa), SCA-10 (3.95 MPa) with the increase in the percentage of nanocomposite from SCA-1 to SCA10. Slight difference between compressive strength of SCA-5 and SCA10 indicated lesser effect of adding nanocomposite (from 5 to 10%) on mechanical strength of scaffold. It is believed that the compact packing of CCNWs-AgNPs nanocomposites due to interaction between carboxylic groups of CCNWs and amino groups of CS plays an important role in enhancing mechanical properties of the porous scaffolds. The chemical interactions between functional groups of different polymers and Ca2+ and PO4− of hydroxyapatite (HAp) resulted in the compressive

Fig. 4. FESEM images of scaffolds (a) SCA-0, (b) SCA-1, (c) SCA-2.5, (d) SCA-5, and (e) SCA-10. Scale bar 100 μm.


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Table 1 Pore size, percent porosity and mechanical strength of prepared scaffolds. Sample

Pore size (μm)

Porosity (%)

Compressive strength (MPa)

Degradation constant (k, day−1)

T50 degradation (dt50, days)

SCA-0 SCA-1 SCA-2.5 SCA-5 SCA-10

30–60 50–100 150–200 250–400 350–550

96.1 ± 1.8 92.6 ± 2.9 89.6 ± 2.5 84.7 ± 2.3 76.8 ± 2.1

0.35 1.07 2.14 3.84 3.95

0.04819 0.03186 0.02846 0.01658 0.01264

14.4 21.8 24.3 41.8 54.8

strength ranging between 0.27 MPa to 6 MPa [45,46]. In the present study, incorporation of varying content of CCNWs-AgNPs nanocomposite from SCA-0 to SCA-10 resulted in the enhanced compressive strength from 0.35 MPa to 3.95 MPa, which is similar to that of cancellous bone. Similar mechanical strength was reported by Zhou et al. upon reinforcing of CNWs in electrospun maleic anhydride grafted poly(lactic acid) scaffolds [47]. 3.3. Swelling and degradation studies Swelling and fluid retention properties of scaffolds are essential to study as they play vital role in regulating cell infiltration and adhesion, nutrient and metabolite transportation [18]. Swelling behavior of scaffold also regulates pore size and can be helpful in achieving required pore diameter by carefully altering composite composition. The percent swelling of films was observed in PBS (pH = 7.4) at 37 °C for different time interval. Effect of different concentration of CCNWs-AgNPs on the swelling behavior with respect to time was evaluated and is shown in Fig. 5(a). Swelling of polymeric matrix due to PBS uptake is responsible for reduction in degree of intermolecular crosslinking between CS-CMCCCNWs-AgNPs by weakening hydrogen bonds between them [48].

Protonation and ionization of amino and carboxyl groups, respectively, causes CS swelling in aqueous medium. CMC is hydrophilic in nature due to presence of carboxylic group and swells rapidly in presence of PBS, resulting in increased weight of scaffolds. As shown in graph (Fig. 5(a)), decrease in swelling behavior with increased amount of CCNWs-AgNPs can be attributed to CCNWs-CCNWs, CCNWs-AgNPsCS, and CCNWs-AgNPs-CMC interactions. Overall increase in hydrogen bonding between CCNWs-AgNPs-CMC-CS in the polymer matrix is also responsible for increased intramolecular heterogeneous interactions, and may also be considered for decreased water binding capacity of scaffolds [48,49]. Schott's second order swelling kinetics model (shown below) was used and fitted well with scaffold's swelling data to demonstrate swelling behavior of scaffolds. The model is expressed as: t

Qt

¼1

Kis

þ1

Q∞

t

ð6Þ

Where, Qt, represents swelling capacity of scaffolds (g g−1) at time interval ‘t’, Kis is initial swelling rate constant and Q∞ is theoretical equilibrium swelling capacity (g g−1), respectively. Fig. 5(b) shows kinetic fitting to obtain swelling parameters (Kis and Q∞), which are presented in Table 2. Kis and Q∞ values decreased with increasing CCNWs-AgNPs

Fig. 5. (a) Swelling behavior of scaffolds containing different amount of CCNWs-AgNPs at various time interval, (b) t/Qt Vs time graph for different composite scaffolds, (c) In vitro enzymatic degradation of composite scaffolds with respect to time, and (d) Average amount of adsorbed protein after 2 h of incubation on different scaffolds.

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Table 2 The kinetic parameters for swelling of different nanocomposite scaffolds. Scaffolds

Kis (g g−1)

Q∞ (g g−1)

R2

SCA-0 SCA-1 SCA-2.5 SCA-5 SCA-10

33.01 9.19 7.39 9.35 8.40

1084.59 1007.05 813.00 769.23 595.238

0.99973 0.99974 0.99937 0.99958 0.99986

amount which signifies low PBS uptake capacity due to the incorporation of nanocomposites that formed strong intramolecular network within polymeric matrix. Enzymatic degradation is a process that starts as soon as any biodegradable polymeric implant comes in contact with body fluids. Concentration of circulating enzymes, particularly lysozyme is higher at the implant site so as to prevent any microbial infection. It is actively secreted by the immune cells, as a first line of defense against microbial invasion [50]. Lysozyme degrades chitosan by hydrolysing β-1,4 glycosidic bond present between N-acetylglucosamine units via SN1 mechanism [18]. Hence, it becomes important to study the effect of lysozyme on the stability of chitosan based scaffolds. Fig. 5(c) shows the in vitro degradation profile of scaffolds over a period of time. CMC is hydrophilic in nature, and swells rapidly in presence of PBS resulting in the decrease in the interactions between CMC and CS due to loosening of physical crosslinking between them. Faster swelling of scaffold exposed more incision sites for the action of lysozyme which resulted in higher degradation rate, as seen in SCA-0. Results indicated that the degradation rate decreased sequentially with the increase in the percentage of CCNWs-AgNPs nanocomposite. Incorporation of nanocomposite is considered responsible for the formation of intermolecular polymeric interactions that masked incision sites for lysozyme activity, hence lesser degradation was observed for SCA-1, SCA-2.5, SCA-5 and SCA-10 in comparison to SCA-0. Additionally, inclusion of nanocomposite in scaffolds provide diffusion barrier for PBS, which restricted swelling and consequently reduced matrix degradation. Enzymatic degradation data agreed well with the swelling results, indicating that the swelling of matrix caused exposure of more incision sites for lysozyme which resulted in higher degradation rate. The degradation data was fitted using first order kinetics, M = M0(1 − e−kt) and the kinetic parameters are shown in Table 1. T50 degradation time (dt50, days) was evaluated using kinetic parameters to establish a relationship between weight loss due to degradation and incubation time. We found that by varying the content of nanocomposite, we can regulate the degradation of scaffold and can tune it according to a particular tissue regeneration time. The great challenges in synthesizing tissue engineering scaffolds are to maintain the mechanical strength, sufficient porosity, and degradation rate. Biodegradable scaffolds synthesized from natural polymers are favorable since (a) they do not produce any immunogenic responses, (b) their by-products are non-toxic, and exit from body without interfering with other organs [51]. Biodegradable scaffolds degrade over a period of time at physiological conditions allowing host cells to proliferate and synthesize extracellular matrix [52]. Degradation time of the tissue engineering biomaterial should match the tissue regeneration time in order to support tissue formation at damage and degeneration site. Based on tissue type and age, the desirable degradation rate may vary. For example, healing of bone fracture in young individuals can take up to six weeks for repair, while in elderly it can extend up to several months [51]. High degradation rate of scaffolds can adversely alter the porosity, water absorption and morphology of scaffolds thereby effecting the cell proliferation and vitality and even provoking host responses [52]. Sung et al. studied the effect of degradation rate of scaffolds prepared from poly lactic-glycolic acid co-polymer (PLGA) and polycaprolactone (PCL) on cell growth and angiogenesis and reported

significant angiogenesis within 14–28 days of implantation [53]. Material scientists are investigating broad range of polymers and their composites with ceramics and synthetic polymers to develop an ideal scaffold that can fulfill tissue engineering requirements [54,55]. Qasim et al. recently studied the in vitro and in vivo degradation stability of chitosan-hydroxyapatite scaffolds and reported that complete degradation take place in vivo in 30 days [56]. Sharma et al. reported the challenges and advantage of incorporating nanohydroxyapatite (nHAp) in the composite scaffold of chitosan-gelatin-alginate [15]. The nanobiocomposite scaffold underwent 35% degradation after 28 days due to incorporation of nHAp and was reported suitable for bone tissue engineering with controlled degradation rate. Similarly, Dan et al. reported around 50% degradation of chitosan-gelatin-nHAp scaffold in 30 days [57]. Controlled degradation rate in the present synthesized scaffolds showed better response in terms of stability and agreed well with the reported scaffolds for bone tissue engineering [15,53,56,57]. dt50 degradation time for SCA-1 and SCA-2.5 was in between 22 and 24 days whereas it was higher for SCA-5 and SCA-10 due to increased content of nanocomposite and was in between 42 and 55 days. Our reported dt50 degradation time in all the scaffolds was sufficient enough for angiogenesis and development of proper vascularized system for tissue regeneration at fracture/damage site.

3.4. Protein adsorption studies Cell adhesion and proliferation is a complex process that involves cascade of reaction which takes place at bio-interface. Protein adsorption is the initial most step that occurs when any foreign material comes in contact with body fluid. Adsorbed proteins serve as cushions and help cells in adhesion, proliferation and differentiation [1]. Scaffolds being porous in structure allow more protein adsorption on its surface in comparison to 2D constructs, thereby providing enhanced biocompatibility for cell proliferation and migration [58]. Protein adsorption study was performed using 10% FBS (fetal bovine serum, Invitrogen) solution (prepared in PBS, pH 7.4) and adsorbed protein mass was quantified using BCA kit. As shown in Fig. 5(d), adsorbed protein mass increased from 10.2 (±0.2) mg/g (in SCA-0) to 12.8 (±0.2) mg/g with the incorporation of CCNWs-AgNPs in SCA-1. Notably, protein adsorption reduced on increasing the content of nanocomposite in SCA-2.5, SCA-5 and SCA-10. It can be attributed to the decrease in the porosity of the scaffolds on increasing CCNWs content which is directly related to the surface area of the scaffold. The scaffolds exhibited considerable amount of protein adsorption and was sufficient enough for promoting cell adhesion and proliferation as reported in literature [58].

3.5. Antibacterial activity Antibacterial activity of fabricated nanocomposite scaffolds, SCA-2.5 and SCA-5 was tested against Gram negative (E. coli, MTCC 1610) and Gram positive (E. hirae, MTCC 3612) bacteria and compared with SCA0 scaffold. Previous studies showed that progressive release of Ag+ ions from embedded Ag nanoparticles into aqueous medium is mainly responsible for antimicrobial activity of AgNPs based materials [59,60]. Released Ag+ ions in medium enter inside the bacteria cells and disturb the bacterial respiratory chain by binding to thiol groups of the involved enzymes. They also interacts with bacterial membrane and causes structural changes and further intercalates with DNA, causing cell death [61]. As shown in Fig. 6, the densely packed colony formation takes place for SCA-0 samples for both E. coli and E. hirae strains, whereas very few colonies appear in SCA-2.5 and SCA-5 samples, at the same 20 dilutions. Notably, the colony counting was difficult even at higher dilution (20 times diluted) for SCA-0 sample for both the strains in comparison to SCA-2.5 and SCA-5 samples, we consider ~100% antibacterial efficiencies for both scaffold samples.


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Fig. 6. Antibacterial activity of SCA-0, SCA-2.5 and SCA-5 against (a–c) E. coli and (d–f) E. hirae. Cell spreading showed here was done after diluting the cultures 20 times in PBS.

3.6. Cytotoxicity and cell attachment assay Biomaterial's 3D structures such as scaffolds and hydrogels used in tissue engineering should be inert and biocompatible i.e. support cellular adhesion, growth and proliferation without showing any detrimental effects. The fabricated nanocomposite scaffolds were assessed using different techniques such as MTT, FESEM, and live/dead staining. We investigated the feasibility of using these fabricated scaffolds on

human bone-like cell line, MG-63. In bone tissue engineering, osteoblasts play vital role in bone formation and are solely responsible for bone matrix synthesis, secretion, and mineralization [58]. High concentration of conditioned media, i.e. in 1:10 ratio (conditioned media: DMEM), was used for determining cytotoxicity. As shown in Fig. 7(a), the cell biocompatibility on MG-63 cells was tested for 2, 4 and 6 days. After every incubation period, the concentrations of live cells in samples were comparable to control cell concentration as evaluated the MTT

Fig. 7. Analysis of biocompatibility and cell adhesion towards fabricated scaffolds. (a) MTT assay showing cell viability (%) with respect to different incubation time, (b) FESEM image of adhered MG-63 cells on surface and porous structure of SCA-2.5 scaffold, (c) and (d) Fluorescence imaging of attached cells on SCA-0 and SCA-2.5 scaffolds using calcein AM dye, respectively.

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assay. MTT results revealed no significant change in the cell growth pattern in sample as well as in control due to non-toxic properties of chitosan, CMC and CCNWs. Hence, antibacterial and highly biocompatible properties of these scaffolds make them highly applicable for using in bone tissue engineering applications. FESEM imaging was done for visualizing cell attachment and penetration inside the porous structures of scaffold. In all the scaffolds, MG-63 cells were able to adhere onto the surface as well as penetrate inside the 3D structures. The representative images of adhered MG-63 cells after 24 h of cell seeding on SCA-2.5 is shown in Fig. 7(b). Cells adhered onto the surface and penetrated inside the structures and can be easily distinguished as shown with the arrow mark in the Fig. 7(b). Fluorescence imaging was carried out to differentiate between the dead and live cells after staining with Calcein-AM dye. Post 5 days of incubation, cells were captured using inverted fluorescent microscope. The bright green spots on the surface of SCA-0 and SCA-2.5 scaffold as shown in Fig. 7(c and d) represent clumps of attached live cells, indicating healthy interactions between cells and scaffolds. The fluorescence images were analyzed using ImageJ software to find the adhered cell density, which was found to be ~400 and ~250 cells at SCA-2.5 and SCA-0, respectively in a 0.2 mm2 image area.

3.7. In vitro bioactivity in SBF Bio-mineralization property of scaffolds indicates the ability of formation of new bones, hence, plays an important role in designing biomaterials which are applied in bone tissue engineering. Cellulose nanocrystals exhibit bio-mineralization property due to presence of OH and SO2− groups distributed on nanocrystals surface which pro4 motes deposition of calcium and phosphate ions [62]. Recently, Ohtsuki et al. reported the effect and optimum percentage of carboxyl groups (COO−) of carboxylated cellulose nanofibers for hydroxyapatite deposition on nanofibers [63]. Carboxyl groups are involved in electrostatic interactions between nanowhiskers and Ca2+ ions present in solution, inducing apatite nucleation. We too investigated the in vitro bio-mineralization of SCA-0, SCA-2.5 and SCA-5 to study the effect of incorporation of CCNWs on apatite deposition. Substantial mineralization was observed in SCA-2.5 and SCA-5 scaffolds as compared to SCA-0 due to incorporation of CCNWs. Post mineralization, the morphology of scaffolds changes from smooth to rough due to deposition of mineral ions as shown in FESEM images in Fig. 8. EDS spectra also clearly depict the presence of Ca, P and O elements in the deposited minerals. The weight percentage of deposited Ca and P in SCA-0 was 0.1% each which

Fig. 8. FESEM and EDS images of (a) SCA-0, (b) SCA-2.5, and SCA-5. FESEM images of SCA-2.5 and SCA-5 show apatite deposition as reflected by surface roughness and confirmed by EDS analysis.


increased to 1% (Ca) and 0.1% (P) in SCA-2.5 and to 1.9% (Ca) and 0.5% (P) in SCA-5. Hence, we can strongly conclude that the presence of CCNWs in our nanocomposite scaffolds were majorly responsible for apatite deposition. 4. Conclusion In the present work, we have reported the fabrication, characterization and application of nano-composite polymeric scaffolds for bone tissue engineering with controllable pore size and mechanical strength. We prepared nanocomposite of silver nanoparticles (AgNPs) decorated on carboxylated CNWs (CCNWs) which serves dual functions of providing mechanical strength and antimicrobial activity due to CNWs and AgNPs, respectively. Nanocomposite was well characterized by various techniques like FESEM, XRD, TEM, FTIR and TGA. Scaffolds containing chitosan (CS) and carboxymethyl cellulose (CMC) with varying percent of CCNWs-AgNPs nanocomposite were fabricated using freeze drying method. Incorporation of CCNWs-AgNPs in scaffolds improved mechanical strength, scaffold porosity, and swelling properties with enhanced resistance for enzymatic degradation. Scaffolds exhibited sufficient protein adsorption and mineralization capacity that is essentially required for osteoblast cell adhesion, proliferation and bone tissue regeneration. Scaffolds apart from exhibiting excellent antimicrobial activity, also supported cells growth as revealed by MTT and live-dead staining assays. To our best knowledge, this works reports for the first time the potential application of CCNWs-AgNPs nanocomposite in the preparation of highly efficient antimicrobial scaffolds with improved mechanical strength and degradation properties for bone tissue engineering, which can overcome bone related infections like osteomyelitis. Acknowledgements The authors would like to thank IIT Guwahti for financial assistance (Sanction no: BSBESUGIITG00993) and also the Department of Science and Technology, Government of Indiafor financial assistance (Sanction nos: DST/INSPIRE/04/2014/002020, ECR/2016/001027) for this work. Authors highly acknowledge the Department of Biosciences and Bioengineering and Central Instruments Facility (CIF) of Indian Institute of Technology Guwahati (IITG), India for providing all necessary instrumentation facilities to carry out this research work. Authors are also thankful to Dr. Biman Mandal and Ms. Shreya Mehrotra for helping with UTM facility. References [1] A. Hasan, L.M. Pandey, Review: polymers, surface-modified polymers, and self assembled monolayers as surface-modifying agents for biomaterials, Polym.-Plast. Technol. Eng. 54 (13) (2015) 1358–1378. [2] I. Banerjee, R.C. Pangule, R.S. Kane, Antifouling coatings: recent developments in the design of surfaces that prevent fouling by proteins, bacteria, and marine organisms, Adv. Mater. 23 (6) (2011) 690–718. [3] K.H.A. Lau, C. Ren, T.S. Sileika, S.H. Park, I. Szleifer, P.B. Messersmith, Surface-grafted polysarcosine as a peptoid antifouling polymer brush, Langmuir 28 (46) (2012) 16099–16107. [4] A. Hasan, L.M. Pandey, Kinetic studies of attachment and re-orientation of octyltriethoxysilane for formation of self-assembled monolayer on a silica substrate, Mater. Sci. Eng. C 68 (2016) 423–429. [5] Y. Miyasaki, J.D. Rabenstein, J. Rhea, M.-L. Crouch, U.M. Mocek, P.E. Kittell, M.A. Morgan, W.S. Nichols, M. Van Benschoten, W.D. Hardy, Isolation and characterization of antimicrobial compounds in plant extracts against multidrug-resistant Acinetobacter baumannii, PLoS One 8 (4) (2013), e61594. [6] A. Stanković, S. Dimitrijević, D. Uskoković, Influence of size scale and morphology on antibacterial properties of ZnO powders hydrothemally synthesized using different surface stabilizing agents, Colloids Surf. B: Biointerfaces 102 (2013) 21–28. [7] S. Saravanan, S. Nethala, S. Pattnaik, A. Tripathi, A. Moorthi, N. Selvamurugan, Preparation, characterization and antimicrobial activity of a bio-composite scaffold containing chitosan/nano-hydroxyapatite/nano-silver for bone tissue engineering, Int. J. Biol. Macromol. 49 (2) (2011) 188–193. [8] H.H. Lara, E.N. Garza-Treviño, L. Ixtepan-Turrent, D.K. Singh, Silver nanoparticles are broad-spectrum bactericidal and virucidal compounds, J. Nanobiotechnol. 9 (1) (2011) 30.

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