poly (vinyl alcohol) nanocomposite film with enhanced mechanical properties for application in bone tissue regeneration

Journal Pre-proof TiO2 doped chitosan/poly (vinyl alcohol) nanocomposite film with enhanced mechanical properties for application in bone tissue regeneration

Sarim Khan, Muskan Garg, S. Chockalingam, P. Gopinath, Patit Paban Kundu PII:

S0141-8130(19)36379-2

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.246

Reference:

BIOMAC 14027

To appear in:

International Journal of Biological Macromolecules

Received date:

11 August 2019

Revised date:

20 November 2019

Accepted date:

30 November 2019

Please cite this article as: S. Khan, M. Garg, S. Chockalingam, et al., TiO2 doped chitosan/ poly (vinyl alcohol) nanocomposite film with enhanced mechanical properties for application in bone tissue regeneration, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.11.246

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© 2019 Published by Elsevier.

Journal Pre-proof TiO2 doped Chitosan/Poly (vinyl alcohol) nanocomposite film with enhanced mechanical properties for application in Bone tissue regeneration Sarim Khan1, Muskan Garg1, S. Chockalingam2, P Gopinath2,3, Patit Paban Kundu 1 *

1 Department of Chemical Engineering, Indian Institute of Technology Roorkee, 247667, India 2 Department of Biotechnology, Indian Institute of Technology Roorkee, 247667, India

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3 Centre of Nanotechnology, Indian Institute of Technology Roorkee, 247667, India

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* Corresponding Author: Email: [email protected], Telephone: +91-7251040403

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Abstract

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Here, TiO2 nanoparticles have been doped into the polymer film-construct of Chitosan/poly (vinyl alcohol)/ Nano-hydroxyapatite (CPHT I – III) to enhance the mechanical and biological properties of the film so as to mimic the human bone extracellular matrix for application in human bone regeneration. The synthesized films are highly porous in nature along with the presence of macrovoids. Significantly enhanced mechanical properties were obtained upon the addition of TiO2 in comparison to previous literature. Increasing content of n-HAP-TiO2 increased the elasticity, tensile strength of the films and the antibacterial efficacy against both Gram-Positive and Gram-Negative Bacteria. The pH of CPHT I-III films in saline remained in the low alkalinity range of (7.48 – 7.53) on day 14. CPHT I-III films were compatible with the human erythrocytes as their hemolysis was well below the limit of acute hemolysis. The in-vitro studies revealed the highly cytocompatible nature of CPHT III (15% n-HAP-TiO2) for osteoblast-like MG – 63 cell attachment and proliferation. The study has revealed that CPHT III has the potential to be used for bone tissue regeneration, our future studies will be focused on the in-vivo investigations to establish its use in clinical settings.

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Keywords

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Nanocomposite; Titanium dioxide; Bone Tissue Engineering

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Introduction

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Bone tissue engineering (BTE) is an emerging field wherein bionanocomposite films and scaffolds are being employed for human bone tissue regeneration [1],[2]. Briefly, polymers are blended with nanofillers or natural fibers to enhance their mechanical or biological properties to mimic the human bone extracellular matrix. The shortcomings associated with the industry standards namely allografting and autografting entails the development of biomaterials which can be used for bone regeneration [3],[4]. Autografting is usually associated with donor site morbidity, higher risk of infection, insufficient sealing of the gaps and higher cost of the two surgeries required at the donor as well as the host site. Allografting is also entangled with certain shortcomings such as the immunogenic rejection, higher risk of infection and the increased risk of disease transmission, these complications limit their use as an implant. The human bone comprises majorly of collagen and hydroxyapatite which are the organic and inorganic components respectively. An ideal bone tissue regeneration template should possess multiple functionalities such as high porosity, favorable mechanical properties, antibacterial efficacy, hemocompatibility and cytocompatibility [5]. From the literature, it is observed that all these functionalities cannot be afforded by a polymer itself, so bionanocomposites are synthesized to impart multiple functionalities to the polymer-nanofiller templates.

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In the attempt to develop an ideal bionanocomposite film, amongst a number of polymerceramic composites, chitosan (CTS) and nano-hydroxyapatite (n-HAP) have been established to be good bioactive entities for applications in bone tissue engineering. Chitosan (CTS) is synthesized from the exoskeleton of crustaceans and is a deacetylated form of chitin which is found in the exoskeletons of crustaceans and insects [6]. Chitosan has been found to be highly biocompatible with the human tissues and is also biodegradable with its monomeric products being excreted from the body [7]. It is osteoconductive in nature and possesses a good antibacterial action [8]. The introduction of n-HAP in the chitosan polymer matrix increases its osteoconductivity, stiffness and enhances osteoblast proliferation on its surface [9]. But the addition of n-HAP also increases the brittle nature of the composite, hence resulting in poor mechanical strength of the film [10]. To overcome such complications, several attempts have been made at adding synthetic polymers to these polymer constructs [11][12][13][14]. Amongst these synthetic polymers, poly (vinyl alcohol) (PVA) is a good choice owing to its water-soluble nature and biodegradability [15]. PVA is also found to enhance the mechanical properties and the biocompatible nature of the film-construct [16]. Recently, the composites employing the use of TiO2 nanoparticles (Titania) have gained a lot of attention in the field of tissue engineering due to the enhancement in the mechanical strength and bioactivity of the composites. This enhancement is attributed to the addition of TiO2 nanoparticles to these composites[34][35][36]. Titania acts as an inorganic substrate that draws over the molecules, proteins, and cells to adhere to its surface, this results in bone formation[37]. To mimic the mechanical strength of the

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natural bone for load-bearing applications, ceramic particles such as TiO2 nanoparticles are an excellent choice. Their addition is expected to result in higher tensile strength, antibacterial action and higher cell proliferation on the surface of the films without introducing any toxicity upon their addition in small amounts [17][18]. Recently, Neel at al., 2014 reported that the addition of TiO2 modified glass regulated the differentiation of osteoblast-like human osteosarcoma cells (HOS) on its surface[38]. The addition of TiO2 nanoparticles to a nanocomposite is also reported to result in an increase in apatite formation during immersion in SBF over a prolonged period[35]. Their addition is also expected to suppress the immunogenic response which transpires post-implantation of the graft[39]. These studies support the potential exploitation of TiO2 nanoparticles in the field of bone tissue engineering.

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We hypothesized that the polymer film-construct of CTS/PVS/n-HAP-TiO2 would mimic the extracellular matrix of the natural human bone resulting in human bone regeneration. The bionanocomposite films are expected to be highly porous with interconnected pores due to the low miscibility between the CTS and PVA [19]. The addition of PVA and TiO2 is expected to enhance the mechanical properties of the films such as ultimate tensile strength, elongation at break (%) and elasticity [19]. The antibacterial action of CTS and TiO2 Nanoparticles is expected to augment the antibacterial efficacy of the films. Finally, the films are expected to be highly biocompatible towards the osteoblast proliferation due to the biocompatibility of all the components in the construct.

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To date, no attempt has been made at synthesizing a nanocomposite construct containing Chitosan (CTS), poly (vinyl alcohol) (PVA), Nano-Hydroxyapatite (n-HAP) and TiO2 nanoparticles. In this work, we synthesized the bionanocomposite films using the solvent casting method [20]. The films have haven characterized using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), field emission scanning electron microscope (FESEM) and atomic force microscopy (AFM). The mechanical properties of the films were established using a Universal testing machine (UTM), Thermogravimetric analyzer (TGA) and Dynamic mechanical analyzer (DMA). The water absorption studies and the pH study were also carried out for the films. The antibacterial efficacy has also been investigated using the Liquid culture method. To establish the hemocompatibility of the films, the hemolytic assay was carried out using human blood samples. The bioactivity of the films was confirmed by the in vitro biomineralization study. The protein adsorption study was carried out to assess the effect of nanofiller addition on the protein attachment at the surface of the film. Finally, to assess the osteoblast proliferation and attachment on the film surface, MTT assay was carried out using osteoblast-like MG-63 cells to establish the use of these films as a template targeted for human bone regeneration.

2. Experimental Procedure 2.1 Synthesis

Journal Pre-proof 2.1.1 Synthesis of nano-HAP-TiO2 Nano-Hydroxyapatite (n-HAP) was synthesized using the wet precipitation method [21]. The amount of TiO2 Nanoparticles (0.2 %, 0.4 %, 0.6 %; w/w) added in the composite was minimal as a higher amount could be cytotoxic. To form a fused salt with n-HAP, 4 wt % of TiO2 (Sigma Aldrich) nanoparticles were added to an aqueous solution containing 96 wt % of n-HAP. The solution was stirred at 800 rpm for 2 h at room temperature. The resulting solution was filtered and the precipitate was heated overnight in a hot air oven at 60°C. The resulting fused salt is soluble in aqueous solutions.

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2.1.2 Synthesis of CTS/PVA/n-HAP-TiO2 nanocomposites (CPHT I-III)

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The solvent casting method was employed to synthesize thin films of the CTS/PVA/n-HAP-TiO2 nanocomposites [20]. The films were developed with Chitosan (55%; w/w), PVA (30-40%; w/w) (Thomas Baker, Molecular weight of 125,000) and n-HAP-TiO2 (5-15 %; w/w). First, the calculated amount of Chitosan (HiMedia Private Ltd, Degree of deacetylation >=75%) polymer was added to a dilute aqueous solution of 2.5% (v/v) glacial acetic acid solution and then stirred at 600 rpm for 2 h to get a clear solution of the polymer formulation. Subsequently, the required amount of PVA polymer and n-HAP-TiO2 (Table 1) were added to the solution and stirred at 600 rpm for 8 h. The solution was partially neutralized using 0.1 N NaOH solution (HiMedia Private Ltd). The polymer formulation was then ultrasonicated at 200 W for 1 h to get a homogenous blend. The formulation was then poured into a Petri-dish which was kept at room temperature. The solvent was allowed to evaporate at room temperature for 2 days and then the dry film was kept in a hot air oven at 60°C for another 24 h.

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CTS (wt %) PVA (wt %) CPHT I 55 40 CPHT II 55 35 CPHT III 55 30 Table 1: Classification of CPHT I-III on the basis of the content

HAP-TiO2 (wt %) 5 10 15

2.2 XRD The X-ray patterns of CPHT I-III were recorded using a Glancing Angle XRD (Bruker Model- D8Advance), which was operating at 40 kV and 30 mA. The samples were scanned in the range of 2θ = 5-70 °. Results for all the samples were recorded in triplicates and the representative figures are presented for the samples CPHT I-III. 2.3 FTIR The FTIR studies of CPHT I-III films were carried out using an FT-IR spectrometer (PerkinElmer) in Attenuated Total Reflection (ATR) mode. Fourier transform infrared absorption spectra of the

Journal Pre-proof thin films were recorded in the wavenumber range of 400-4000 cm-1 with a resolution of 4 cm-1. The samples were scanned in triplicates, representative spectra have been presented for each sample as no significant variations were encountered. 2.4 Morphological Analysis

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To assess the surface morphology and the dimensions of the pores present in CPHT I-III, Field Emission Scanning Electron Microscopy (FESEM) was employed. It was also used for measuring the shape and dimensions of n-HAP and TiO2 nanoparticles. First, the samples were coated with gold ions using an ion sputter (BAL-TEC; SCD 005). Then, the sample films were subsequently imaged using FE-SEM Quanta 200 FEG and the n-HAP and Nano- TiO2 were imaged using ZEISS Gemini SEM 300. The images were captured in triplicates and no significant variations were encountered. The size range of n-HAP was analyzed using Fiji - ImageJ software.

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2.5 AFM

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Atomic force microscopy (AFM) was used to investigate the nano-topographic features such as surface roughness of the CPHT I-III dry films. To carry out the studies, a scanning probe microscope (NT-MDT-INTEGRA) with AFM module was used. The assessment was carried out in contact mode using a soft cantilever at room temperature. The images were captured in duplicates and no significant variations were encountered.

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2.6 TGA

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To establish the thermal stability of the CPHT I-III nanocomposites, their Thermogravimetric analysis (TGA) was performed in a thermo-gravimetric and differential thermal analyzer (TGDTA, SII 6300 EXSTAR). Samples were gradually heated from 35°C to 600°C at a controlled heating rate of 20°C/min in a nitrogen atmosphere. The variation of the residual weight of the sample with the temperature was recorded for their due thermogravimetric analysis 2.7 Mechanical Properties

To assess the mechanical properties such as Young’s modulus, ultimate tensile strength, elongation at break and stiffness of the nanocomposite films, CPHT I-III films were tested in the tensile mode in the universal testing machine for biomaterials (Bose ElectroForce 3200 Series III). The testing was carried out at ambient conditions and at a crosshead speed of 5 mm/min. All the films had a gauge length of 20 mm, a width of 5 mm and a uniform thickness of 0.06 mm. 2.8 Rheological Measurements

Journal Pre-proof In order to study the effect of the content of PVA and n-HAP-TiO2 in the films on their elasticity, dynamic mechanical analysis (TA Q800) was carried out in a frequency range of 0.1 – 5 Hz at room temperature in the tensile mode at the constant strain of 0.1. The variation of storage modulus (E’) and loss modulus (E”) with the frequency was recorded for CPHT I – III. 2.9 pH study

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To determine the pH variation of CPHT I-III, 10 mg of each film sample was mixed with 40 mL solution of freshly prepared physiological saline (0.9% NaCl). The pH values were recorded using a pH meter after 1, 2, 4, 7 and 14 days. The solution was constantly stirred at 400 rpm and 37°C.

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2.10 Water absorption

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Water absorption or swelling studies were carried out to measure the ease of cell infiltration into the films. The dry weights (W0) of CPH I-III were recorded, and then the samples were immersed in distilled water for 24 h. The samples were taken out periodically from the water and gently pressed against filter paper to absorb the water present at the surface of the film. The samplers were displaced after 1, 4, 8 and 24 h of first immersion, their respective weights were recorded as Wt. The following equation was used to measure the extent of water absorption Ea:

2.11 Hemolytic assay

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Ea = (Wt-W0)/W0*100

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The hemolytic assay was carried out to establish the compatibility of CPHT I-III films with human erythrocytes. Approval was granted by the Institute human ethics committee, IIT Roorkee for carrying the tests. Before drawing out the blood, due consent was taken from the adults for the subsequent use of their blood samples. A standard procedure was followed for the experiment (ASTM F756 - 00). The lysis of the Red Blood Cells actuates the release of hemoglobin and ensuing centrifugation segregates the cell debris and the intact cells. The number of cells lysed by the respective sample solutions of CPHT I-III is proportional to the amount of hemoglobin present in the supernatant. Using a micro-centrifuge, the freshly collected human blood samples were centrifuged at 4200 rpm and 4°C, to separate the blood samples into plasma and erythrocytes (RBC). Erythrocytes aggregated at the bottom of the tube were then washed with phosphate buffer solutions (PBS) at a pH of 7.4 and re-suspended in the PBS solution making the final volume equal to the initial amount of human blood sample. Subsequently, each sample solution (50 μL) was taken in a 1 mL centrifuge tube, which was then diluted with 900 μL of PBS and finally, erythrocyte solution (50 μL) was added to the above tube. This was followed by a 10 min period of incubation in dark at 4°C and the tubes were then subsequently

Journal Pre-proof centrifuged at 4200 rpm for 10 minutes. Then, optical density (OD) of the supernatant was recorded using a spectrophotometer at 540 nm. The negative control sample (50 μL erythrocytes solution + 950 μl PBS; no lysis) and positive control sample (50 μL erythrocytes solution + 950 μL DI water; complete lysis) were prepared to compare their OD values with those of the supernatant of the CPHT I-III sample solutions. The percentage of hemolysis (%) for CPHT I-III was calculated using the following equation: Hemolysis (%) = 100*(OD(sample)- OD(Negative Control))/(OD(sample)- OD(Positive Control))

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The hemolysis study was carried out in triplicates and all the values reported were the means of triplicate count ± standard deviation (SD).

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2.12 Antibacterial properties

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The antibacterial properties of CPHT I-III were established following the standard procedure of the liquid culture method [22], the tests were carried out against the bacterial strain of Escherichia coli XL1B (Gram-Negative) and Staphylococcus aureus (Gram-Positive). The microbes were acquired from NCCS, Pune (India). Briefly, S. aureus was inoculated in Nutrient Broth (NB) while E. coli was inoculated in Luria-Bertani broth (LB), for their respective exponential growth. 0.04 mg of sample films of CPHT I-III were each added into 50 mL of bacterial suspension and then incubated in a vibrator at 37°C and 200 rpm for 18 h. Suspension aliquots (500 μL) were periodically displaced from each suspension solution at pre-defined time intervals of 1, 4, 8, 16, 24 h to measure their optical density (OD) at 600 nm. The respective increase in OD signifies bacterial growth in the sample film and bacterial suspension. The experiments were carried out in triplicates and a representative figure has been presented here. The structure of E. coli and S. aureus were examined before and after treatment with CPHT II. Briefly, respective bacterial suspensions were drop-fixed on a square glass film (1 mm X 1 mm), which was then taken for surface imaging to a Field Emission Scanning Electron Microscope (FE-SEM Quanta 200 FEG). The images were captured in triplicates and no significant variation was encountered. 2.13 In-vitro cell attachment and proliferation studies To establish the cytocompatibility of the CPHT I-III nanocomposite films with the human bone osteoblast cells, MTT assay was carried out with the osteoblast-like MG-63 cell line using a standard protocol. The MG-63 cells have the ability to maintain a differentiated phenotype in culturing conditions and they proliferate competitively faster than the osteoblast cells. For the said purpose, MG – 63 cells were procured from NCCS, Pune, India. All the cultures were regularly maintained in Dulbecco's Modified Eagle Medium (DMEM) (Procured from HiMedia). For the MTT assay, each film sample was cut into a square with a side of 4 mm. The film samples were then placed into the polystyrene 96-well plates. To each well containing the

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films, 1 x 103 cells were seeded along with a standard media volume of 100 µl. The plate was incubated at 37°C in a humidified incubator with 5% CO2. Then at Day 3 and Day 7 after the initial seeding, the cell proliferation was studied by adding 10 μL of PBS (1X) diluted solution of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT)(Procured from Sigma Aldrich) to each well, followed by a 2 h incubation in the humidified incubator. In order to dissolve the formazan crystals formed, the culture media having the MTT solution was removed and replaced with a 200 μL solution of dimethyl sulfoxide (DMSO). Finally, the absorbance at 570 nm was recorded along with the background measurement at 690 nm for all the wells. A film of Chitosan (CTS) was used as the control for the experiment as the amount of CTS was constant in CPHT I-III. Cell viability (%) was calculated relative to the control cells.

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To assess the cell adhesion and attachment on the surface of the film, live images were captured on Day 3 after the initial seeding using an Inverted microscope (EVOS FL-C cell imaging system, Life Technologies) and FESEM (ZEISS Gemini SEM 300).

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2.14 Protein adsorption study

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The uniform film samples of CPHT I-III were incubated with cell culture media (DMEM) containing 10 % fetal bovine serum (FBS) in a 96 well plate at 37°C in a humidified incubator with 5% CO2 for 4 h. Following the incubation, the media was removed, and the film samples were washed thrice with PBS to remove the unadhered proteins. The film samples were then moved to a fresh 96 well plate, 200 μL of 2 % (w/v) sodium dodecyl sulfate (SDS) solution was then added to elute the protein and left for 30 mins under shaking conditions. The concentration of the protein on the films was evaluated by using the BCA assay kit (Sigma Aldrich). The optical density at 562 nm was recorded after 30 min of adding bicinchoninic acid (BCA) reagent to each well. A nanocomposite film of chitosan (100 % CTS) was used as a control for the experiment. The total protein adsorbed on the films was calculated by plotting a standard curve of FBS standards against their concentration. 2.15 In vitro biomineralization The nanocomposite films were immersed in a 1X simulated body fluid (SBF) solution for 7 days at 37°C with constant shaking. The SBF solution was prepared as per the standard protocol [40]. The SBF solution was replaced every 2 days. On day 7, after removing the films from the SBF solution, they were dried in a hot air oven at 45°C for 2 h. The samples were then subsequently taken for imaging and EDX analysis using FESEM (ZEISS Gemini SEM 300). The experiment was carried out in triplicates and no significant variation was encountered. So, the presented images are representative of the triplicates. 2.16 Statistical analysis

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All the quantitative results are expressed as means with standard deviations of at least triplicate measurements. Except for TGA, which was only carried out once. Statistical analysis was carried out on GraphPad Prism 5 software using Analysis of variance (ANOVA) test with Turkey post hoc test for multiple comparisons and P < 0.05 was considered statistically significant.

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3. Results and Discussion

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3.1 X-Ray Diffraction

Figure 1: XRD Patterns of CPHT I (a), CPHT II (b) and CPHT III (c). The X-ray diffraction patterns of CPHT I-III (Fig 1) were analyzed to confirm the presence of various entities in the polymer matrix. Patterns of CPHT I-III represent amorphous materials with homogenous natures. The presence of chitosan polymer can be characterized by the presence of peaks at 2θ = 9.46°, 11.86° and a broad peak at 20° signifying the amorphous nature of the polymer [11]. Poly (vinyl alcohol) was characterized due to the presence of diffraction peaks at 19.34° and 40.06° [12]. In addition, due to the presence of Nanohydroxyapatite in CPHT I-III, diffraction peaks could be observed at 2θ = 25.86° and 31.68 ° (SI 2), which correspond to (002) and (211) crystal planes of n-HAP [12]. Finally, small peaks at 2θ = 47.98° (SI 2) corresponding to (200) plane, confirmed the presence of TiO2 in CPHT I-III [23]. It

Journal Pre-proof can be observed that the peaks for n-HAP and nano-TiO2 are very weak in intensity, this can be attributed to their presence in smaller amounts and their homogenous distribution in the polymer matrix.

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3.2 Fourier transform infrared spectroscopy

Figure 2: FTIR spectra of CPHT I-III From the FTIR spectra of CPHT I-III (Fig 2), the stretching C=O vibrations of the amide group present in Chitosan can be characterized by the presence of absorption bands at 1736, 1740 and 1740 for CPHT I-III respectively [25]. Broad absorption bands are observed in the range of 3150-3480 cm-1, which are attributed to the overlapping absorption bands from O-H stretching in PVA and n-HAP [12]. The phosphate stretching vibration bands of n-HAP were present at 1113-950 cm-1, 1138-945 cm-1 and 1133-940 cm-1 for CPHT I-III respectively [12]. The presence of absorption bands at 1410, 1405 and 1420 for CPHT I-III respectively is attributed to the Ti-O modes in the TiO2 nanoparticles [24], the intensity of these peaks increased with increasing TiO2 content in CPHT I-III.

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3.3 Surface and Size morphology

Figure 3: FESEM Images of CPHT I-III (a)-(c) and n-HAP (d) Upon investigation, SEM images of TiO2 nanoparticles(SI 1) revealed that they are spherical in shape and their size lies in the range of 25 – 50 nm (Fig 3.d). The synthesized nanohydroxyapatite particles were spherical in shape with their size varying in the range of 15-40 nm (Fig 3(d)). The morphological study of CPHT I – III revealed the highly porous nature of these nanocomposite films. The pores were uniformly sized in CPHT I and CHPT II (Fig 3(a), 3(b)), with a size range of 0.5 – 2 μm. While the pores were non-uniformly sized in CPHT III (Fig 3(d)), with their sizes varying from 0.1 – 2 μm. Macrovoids are abundantly present in CPHT I and CPHT II, which is attributed to the fact that CTS and PVA are not highly miscible with each other [19]. Also, these macrovoids are scarce in CPHT III, which is due to the fact that the amount of PVA is

Journal Pre-proof lesser in CPHT III. This resulting surface morphology is indicative of favorable fibroblast cell attachment and proliferation on its surface [19]. 3.4 Atomic Force Microscopy

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AFM studies revealed the presence of uniform micropores on the surface of CPHT I and CPHT II, with their sizes and distribution being more uniform in nature (Fig 4). The distribution and size of micropores are observed to be non-uniform on the surface of CPHT III. From the 3D topography analysis, the average surface roughness (Sa) values of the CPHT I-III were found to be 10.9323 nm, 16.1953 nm, and 4.44548 nm respectively, establishing that CPHT II has the roughest surface amongst the three variants. The higher surface roughness of CPHT II can be attributed to the presence of a larger number of macrovoids whose walls are rough in nature. The surface roughness will also play an important role during human bone osteoblast cell adhesion and proliferation, which is discussed later [19].

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3.5 Thermogravimetric Analysis

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From the thermogravimetric analysis, it can easily be observed that CPHT I-III follows a fourstep degradation (SI 3). Firstly, in the range of 100 – 200 °C due to the loss of solvent molecules trapped in the polymer matrix, then in the range of 250-300 °C due to the chain-scission reactions of Chitosan polymer. Finally, in the ranges of 350 – 500°C and 500 – 600 °C due to the chain-scission reactions and continual elimination of PVA, which generally requires a higher temperature [26]. From SI 3 and Table 2, it can be observed that the residual weight of CHPT III and CPHT II is higher than CPHT I throughout the temperature range (SI 3). This indicates that the CPHT II and CPHT III films have higher thermal resistance than CPHT I film. This higher thermal resistance can be attributed to the presence of a higher amount of crystalline n-HAPTiO2 in CPHT III and CPHT II over CPHT I [27].

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Journal Pre-proof Figure 4: Nanotopographical features of CPHT I (a), CPHT II (b) and CPHT III (c)

Temperature(°C) at 20 % Residual weight Residual weight (%) at 350°C Residual Weight (%) at 500°C Table 2: Analysis of Thermal resistances of CPHT I-III

CPHT I 258.545

CPHT II 266.55

CPHT III 260.84

48.95 35.06

50.86 37.16

52.74 39.73

3.6 Mechanical Properties

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Ultimate tensile strength Elongation at Break Stiffness (N/m) (MPa) (%) CPHT I 36.315 + 2.17 23.143 + 1.06 76.32 + 5.98 CPHT II 47.450 + 3.24 32.720 + 2.48 101.39 + 9.87 CPHT III 62.898 + 3.49 39.41 + 2.11 135.07 + 12.52 Table 3: Mechanical Properties of CPHT I-III. Data here is represented as means of triplicate values ± SD.

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The mechanical properties were recorded for CPHT I – III in order to analyze the reinforcing effect n-HAP-TiO2 in CPHT I – III on their physical strength. Mechanical strength is a crucial parameter in the design of bone tissue templates as the template should be mechanically strong to bear the mechanical load experienced by the template during walking or mild jumping. It was observed that the Ultimate tensile strength (UTS) increased gradually from CPHT I to CPHT III (Table 3), with CPHT III having an ultimate tensile strength of 62.898 MPa, these values are higher than the ones reported in similar literature [11][13][14][28]. Bhowmick et al., 2017 doped ZrO2 into a polymer construct with a similar composition, they achieved a maximum ultimate tensile strength of 13.23 MPa [12]. The UTS of natural trabecular bone is around 50 MPA [33], so CPHT III can be safely applied in-vivo without undergoing any expected mechanical failure. The elongation at break and stiffness also gradually increased with increasing n- HAP- TiO2 content from CPHT I to CPHT III. The increase in mechanical properties from CPHT I to CPHT III is attributed to the increasing content of the metallic oxide (TiO2) in the nanocomposites. 3.7 Rheological measurement analysis The rheological measurement trends for CPHT I-III in the frequency range of 0.1-5 Hz confirms their viscoelastic nature (Fig 5). As the amount of nanofiller (n-HAP-TiO2) is increased in the films, the storage modulus increased as well, indicating better intermolecular interactions with increasing content of n-HAP-TiO2. This increase in intermolecular interactions gives rise to better mechanical properties. As the frequency is increased, the storage modulus also increases for CPHT I-III, indicating that films are spending lesser time for re-orientation at larger

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frequencies [29]. With an increasing trend of storage modulus and decreasing loss modulus with an increase in frequency, it establishes that the energy conserved is more than the energy lost due to a frequency stimulus at higher frequencies. Thus, it can be concluded that these films are elastic in nature at higher frequencies and their viscous nature decreases with increasing frequency.

Journal Pre-proof Figure 5: Trend of Storage Modulus (Top) and Loss Modulus (Bottom) with frequency for CPHT I-III. Data here is represented as means of triplicate values ± SD.

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3.8 pH study

Figure 6: pH studies for CPHT I-III over 14 days. Data here is represented as means of triplicate values ± SD. The pH of CPHT I-III was monitored over the duration of 14 days in the presence of physiological saline, the study revealed that the pH at Day 1 was mildly acidic for all the variants (Fig 6). The Day 1 pH for CPHT I -III were 6.42, 6.80 and 6.58 respectively. But with the passage of time, the pH values started to increase and reached a plateau by Day 7 and there were small increases in pH until Day 14 for CPHT I- III. The final pH of CPHT I – III at the end of Day 14 were 7.59, 7.51 and 7.54 respectively. Day 14 pH values are less than 7.6, so the nanocomposite films can be safely applied in the human body, as an increase in the pH of human blood or serum above 7.6 could lead to alkalemia [30].

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3.9 Water Absorption study

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Figure 7: Water Absorption studies for CPHT I-III. Data here is represented as means of triplicate values ± SD. The aqueous swelling studies were carried out for CPHT I – III for 24 h to establish the water uptake capacities of the films (Fig 7). The water uptake capacity is directly proportional to the flux of cell infiltration upon seeding, so a higher capacity indicates good cell infiltration inside the 3D structure. The uptake capacity increased for CPHT I -III with time and attained an equilibrium value after 8 h. CPHT I had the highest water uptake, which can be attributed to the highest amount of PVA and the least amount of n-HAP present in CPHT I amongst CPHT I – III, as PVA is hydrophilic in nature and the stiff nature of n-HAP -TiO2 increases the hydrophobicity of the composite [31]. As a result, CPHT III had the least water uptake amongst CPHT I - III.

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The hemolytic assay was carried out for CPHT I – III and a control sample of CTS film to establish the hemocompatibility of the nanocomposite films (Fig 8). The results revealed that the hemolysis for all the films was well below 5 %, which is regarded as the limit of acute hemolysis. The CPHT III sample had the lowest hemolysis of 2.49 %, which is remarkably low in comparison to the similar previously published literature [11][28]. CPHT II had higher hemolysis than the control sample of CTS. The erythrocytes were completely lysed when they came in contact with water in the positive control while they did not undergo any lysis when they were in contact with PBS in the negative control. The results have established that these films can be safely applied in humans without significantly damaging the erythrocytes during their interaction

Figure 8: Hemolytic assay for CPHT I-III. Data here is represented as means of triplicate values ± SD, wherein * indicates P ≤ 0.05 when compared with Chitosan (Used for comparison).

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3.11 Antibacterial efficacy studies

Figure 9: Bacterial growth in E. coli suspensions (top) and S. aureus (bottom) suspensions. Data here is represented as means of triplicate values ± SD.

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Figure 10: FESEM images of Untreated E. coli (a), CPHT II treated E. coli (b), untreated S. aureus (c) and CPHT II treated S. aureus (d).

From the results of the liquid culture method, we can observe the antimicrobial efficacy of CPHT I – III against both the gram-positive and gram-negative bacterial strains. The difference in the efficacy amongst CPHT I – III arises from the difference in the amount of leachable content. Since PVA doesn’t have antibacterial action and the amount of chitosan (CTS) is the same in all the three variants. The leachable contents in the films are HAP and TiO2 nanoparticles. So, in theory, the nanocomposite films with the highest n-HAP- TiO2 content would have the highest antimicrobial efficacy. In the study against E. coli, CPHT I – III had significantly lower OD than the control E. coli suspension. Amongst CPHT I – III, CPHT III had the highest antimicrobial action against E. coli (Fig 9). The study against S. aureus revealed that the antimicrobial action against it is higher than that against E. coli, as OD even dropped in value between 16 h and 24 h of study (Fig 9). Here again, CPHT III had the highest action which can be attributed to the highest amount of leachable n-HAP-TiO2 present in it amongst CPHT I – III.

Journal Pre-proof From the FESEM images of untreated and CPHT II treated E. coli, it was established that treatment with CPHT II destabilized the tubular structure of E. coli, causing the attrition of the cell walls (Fig 10 (b)). The treatment of S. aureus with CPHT II led to necrosis of the cells which could be established from the fragmentation of the microbes into smaller fragments (Fig 10 (d)).

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3.12 In vitro cell attachment and proliferation (MTT assay)

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Figure 11: Cell Viability studies on CPHT I-III using MTT assay. Data here is represented as means of triplicate wells ± SD, wherein * indicates P ≤ 0.05 and ** indicates P ≤ 0.01 when compared with Cells only (control).

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The cell proliferation studies were carried out on Day 3 and Day 7 to investigate the cell viability of osteoblast-like MG-63 cells on the CPHT I – III nanocomposite films. It was observed that the addition of small amounts of TiO2 in CPHT I-III resulted in no cytotoxic effect on MG-63 cells. CPHT III film had the highest cell proliferation of all the films on both Day 3 and Day 7, this is possibly due to the presence of a larger amount of osteoconductive n-HAP-TiO2 (15 %) [17] in CPHT III as PVA is biologically inert (Fig 11). Moreover, the cell proliferation wasn’t restrained after Day 3 in any of the films, the cells continued to proliferate till Day 7. From the surface images of the nanocomposite films on Day 3 (Fig 12), we could observe cell attachment at the surface of the films. From the FESEM images (Fig 13) it could be seen that the MG-63 cells were well spread with a flattened morphology on the surface of CPHT III (Fig 3.(c)) signifying an advanced stage of cell attachment[32], the polygonal cells were observed to be elongated in all the directions with their filopodia attached to the CPHT III surface (Fig 13). These features established that the surface of the CPHT III films encourage human bone osteoblastic cell

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adhesion. The images in Figure 12, are representatives of three individual film samples of the same type of film.

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Figure 12: Inverted Micrograph images of MG-63 cell attachment on CPHT I-III ((a)-(c)).

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Figure 13: FESEM images of MG-63 cell attachment on the surface of CPHT III ((a) & (b)).

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3.13 Protein adsorption studies

Figure 14: Protein adsorption study of CPHT I-III and CTS(control) films. Protein adsorption is a crucial initial event upon graft implantation as it plays an important role in signaling cascades that govern cellular proliferation, adhesion, and differentiation. From Fig 14, it can be concluded that the increasing content of n-HAP-TiO2 results in higher protein adsorption on the film surface. The increase in the n-HAP content leads to an increase in the available hydroxyl groups on the film surface, thus resulting in an increase of active sites for protein adsorption on the surface[9]. The addition of TiO2 facilitates increased adsorption of proteins onto the film surface as evident from the higher concentrations of protein adsorbed on the films with increasing TiO2 content from CPHT I to CPHT IIII[41]. The free hydroxyl groups

Journal Pre-proof in the films interact strongly with the polar groups present on the protein thus increasing the amount of adsorbed proteins.

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3.14 In vitro biomineralization

Journal Pre-proof Figure 15: FESEM images of CPHT III after immersion in SBF for 7 Days.

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From Fig 15, it can be observed that the immersion in SBF has led to the deposition of Ca-P mineral layers on the surface of the CPHT III film. The Ca-P minerals deposited in a regular needle-like morphology on its surface, these needle-like formations are like the natural bone. These needle-like depositions act as focal points for the osteoblast cells to adhere and proliferate. SI 4 presents the EDX analysis of a new CPHT III film while SI 5 depicts the EDX analysis of a CPHT III film which was immersed in SBF for 7 days. The EDX analysis confirmed the increase in the overall content of Ca and P on the CPHT III surface after immersion in SBF for 7 days. The presence of n-HAP and TiO2 in the nanocomposite films contributes towards the formation of these mineralized layers on their surface. The Calcium ions released from the nHAP in the films react with the phosphate ions in SBF solution to precipitate and form deposits on their surface[37]. The extensive formation of these mineralized layers on the surface of CPHT III confirms its highly bioactive nature.

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4. Conclusions

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Herein, TiO2 nanoparticles have been doped in the polymer construct of Chitosan/ poly (vinyl alcohol)/ Nano-hydroxyapatite in order to mimic the human bone extracellular matrix. Due to the addition of PVA, the CPHT I-III films are highly porous in nature, their large pores could encourage accelerated tissue ingrowth, vascularization, nutrient delivery and waste removal from the sites of bone defect healing. Increasing content of nano-HAP-TiO2 in the films, increased the mechanical properties of the films, indicating low agglomeration of the nanofillers even at 15 % loading of nano-HAP-TiO2(CPHT III). An increase in the mechanical strength upon the addition of TiO2 was higher in comparison to the addition of ZrO2 to a similar polymer construct in a previous study. CPHT III had a higher thermal resistance and elasticity over CPHT II and CPHT I, owing to the homogenous distribution of a higher filler content(n-HAPTiO2). The antibacterial efficacy of the films against both gram-positive and gram-negative strains is due to the presence of Chitosan and n-HAP-TiO2, so the efficacy increased with increasing n-HAP-TiO2 content in the films, therefore CPHT III had the highest efficacy. CPHT I-III had low hemolysis upon contact with human erythrocytes thus establishing their compatibility with human erythrocytes. Finally, due to the biocompatibility afforded by natural Chitosan and n-HAP-TiO2 present in the film, the films displayed advanced cell attachment and high proliferation with the human osteoblast-like MG-63 cells, CPHT III displayed the highest proliferation amongst CPHT I-III. The protein adsorption study revealed the high protein adsorption capacity of CPHT II and CPHT III films, which is attributed to the higher content of nHAP-TiO2 in CPHT II and CPHT III films in comparison to CPHT I films. The highly bioactive nature of the CPHT III film was confirmed from the biomineralization of the film upon immersion in

Journal Pre-proof SBF, as seen from the results of SEM and EDX analysis. Our future studies would involve the invitro molecular, cellular differentiation of human mesenchymal stem cells (hMSCs) on the CPHT III films and the effect of its in-vivo implantation in animal models. This study has established the potential use of the multi-functional CPHT III films in human bone regeneration, but further studies are required before its use can be translated into clinical settings. Conflict of Interests All authors have no competing interests to disclose.

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Acknowledgment The authors are thankful to the administration of the Institute Instrumentation Centre of IIT Roorkee for helping the authors carry out FESEM, AFM, and XRD. The authors are also thankful to Dr. Debrupa Lahiri (IIT Roorkee) and Dr. Kaushik Pal (IIT Roorkee) for helping us carry out tests on UTM and DMA respectively. Finally, the authors are grateful for the assistance of the hospital staff of IIT Roorkee for helping us collect the human blood samples. References

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Author Statement

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Sarim Khan: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - Original Draft Muskan Garg: Methodology, Investigation S. Chockalingam: Methodology, Investigation, Writing - Review & Editing P Gopinath: Supervision, Resources Patit Paban Kundu: Conceptualization, Writing - Review & Editing, Supervision, Resources

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