Facile synthesis of carbon fiber reinforced polymer-hydroxyapatite ternary composite: A mechanically strong bioactive bone graft

Accepted Manuscript Facile synthesis of carbon fiber reinforced polymerhydroxyapatite ternary composite: A mechanically strong bioactive bone graft

Chandrani Sarkar, Sumanta Kumar Sahu, Arvind Sinha, Jui Chakraborty, Subhadra Garai PII: DOI: Reference:

S0928-4931(18)32127-1 https://doi.org/10.1016/j.msec.2018.12.064 MSC 9174

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

20 July 2018 2 November 2018 18 December 2018

Please cite this article as: Chandrani Sarkar, Sumanta Kumar Sahu, Arvind Sinha, Jui Chakraborty, Subhadra Garai , Facile synthesis of carbon fiber reinforced polymerhydroxyapatite ternary composite: A mechanically strong bioactive bone graft. Msc (2018), https://doi.org/10.1016/j.msec.2018.12.064

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Facile synthesis of carbon fiber reinforced polymer-hydroxyapatite ternary composite: A mechanically strong bioactive bone graft Chandrani Sarkar1,2,3*, Sumanta Kumar Sahu2, Arvind Sinha1, Jui Chakraborty4, Subhadra

Advanced Materials and Processes Division, CSIR-National Metallurgical Laboratory,

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Garai1*†

Department of Applied Chemistry, Indian Institute of Technology (Indian School of

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Jamshedpur 831007, India

Mines), Dhanbad 826004, Jharkhand, India

Department of Chemistry, Mahila College, Kolhan University, Chaibasa 833201,

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Jharkhand, India

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CSIR-Central Glass & Ceramic Research Institute, 196, Raja S.C. Mullick Road,

Jadavpur, Kolkata-700 032

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Corresponding authors:

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*

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Chandrani Sarkar, [email protected], Contact no.- +91-6201119480 Subhadra Garai, [email protected]; Tel: +91-657-2345067; Fax: +91-657-2345213 On 03 Aug 2018, Dr. Subhdara Garai passed away

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†

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ABSTRACT Carbon fiber reinforced carboxymethyl cellulose-hydroxyapatite ternary composites have been synthesized by a simple wet precipitation method for weight bearing orthopedic

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application. Composites were synthesized with the incorporation of chemically

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functionalized carbon fibers. The functional groups onto the surface of fibers induced the

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formation of hydroxyapatite at the bridging position through which fibers were effectively bound with matrix. Consequently, the flexural strength and compressive strength of

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composite have reached to 140 MPa and 118 MPa, respectively. The flexural modulus of

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the composite is in the range of 9-22 GPa. In-vitro cell study showed that the composite possesses excellent cell proliferation and differentiation ability. With these excellent

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mechanical and biological properties, synthesized composite exhibits potential to be used as

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a mechanically compatible bioactive bone graft.

graft

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1. Introduction

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Keywords: Carbon fiber; Carboxymethyl cellulose; Hydroxyapatite; Composite; Bone

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For decades, carbon fiber reinforced composite acquired wide attention from both academic and industrial sectors due to its outstanding mechanical properties. Generation wise the composition, design and fabrication process of carbon fiber reinforced composite have been modified to develop lightweight load-bearing structures. In this sense, polymers are considered as most attractive matrix materials due to their exceptional properties such as light weight, low price and ease of shapeability. Commonly, the functional composites are fabricated by combining the strength of carbon fibers (CFs) with the ductility of 2

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polymer matrix [1, 2]. These composites have been extensively used in different areas such as automotive, aerospace, construction industries etc. In recent years, carbon fiber reinforced polymer (CFs/P) composites have also been studied in the biomedical field [3, 4]. Nowadays, they are being widely used to manufacture medical equipment such as

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stabilizers, bolds and hip joints [5, 6]. Moreover, FDA (Food and Drug Administration)

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approved CFs/P composite as most suitable implant for load bearing purpose. But the bioinactive nature of CFs/P composite limits its clinical applications, due to the formation of

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fibrous capsule around implants that restricts its integration to host bone [7]. Therefore, the

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bioactivity of CFs/P composite needs to be improved.

Hydroxyapatite (HA) is a mineral component of natural bone, having excellent

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biological properties. It shows osteogenic potential that helps in bone growth and tissue

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adhesion [7]. For this, researchers have incorporated HA in CFs/P composites to develop bioactive load-bearing implants. Shen et.al. have prepared carbon fiber reinforced

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hydroxyapatite/polylactide (CF/PLA/HA) biocomposite [8]. Zhang et.al. have synthesized a

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carbon fiber reinforced nano HA/polyamide66 (CF/HA/PA) ternary biocomposite [9]. Xu et.al. have designed PEEK/CF/n-HA ternary biocomposite as bioactive bone implant [7].

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Similarly, Deng et.al. have also reported a PEEK/n-HA/CF ternary composite as bioactive bone graft [10]. Recently, Deng et.al. have fabricated a CF/HA/PA46 biocomposite for load-bearing orthopedic application [11]. In these reports, researchers have used compounding-molding

process

to

synthesize

carbon

fiber

reinforced

polymer-

hydroxyapatite ternary composites. Unfortunately, this method has few demerits such as

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attrition of fibers, non-uniform dispersion of fiber and lack of interfacial interaction that reduce the quality of the composites [12]. Concerning the difficulties and disadvantages of aforementioned methods, we devised a

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simple wet precipitation route to synthesize a three dimensional carbon fiber reinforced

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polymer-HA ternary composite for weight bearing orthopedic application. In this study, we

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have chosen a derivative of cellulose i.e. carboxymethyl cellulose (CMC) as matrix material due to its low price, high availability, hydrophilic and biocompatible nature.

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Moreover, CMC has dispersing property, and generally use to disperse carbon fiber [13].

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Previously, we have synthesized a structurally stable carboxymethyl cellulosehydroxyapatite composite (CMC-HA) [14]. In the present work, we have efficiently

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incorporated CFs in CMC-HA system by a facile way, and developed a mechanically

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strong bone substitute. Structural, mechanical and biological properties of synthesized composites were thoroughly studied. Study demonstrated that the functional groups onto

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the surface of CFs facilitate the deposition of HA on its surface; and these HA act as

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bridging molecule between fiber and matrix. As a result, the mechanical property of CMCHA-CFs composite has been enhanced significantly with the addition of small amount of

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CFs. In-vitro cell interaction with MC3T3 cells showed that the CMC-HA-CFs composite possess cell proliferation, adhesion and differentiation ability. The objective of this work is to demonstrate a facile viable approach for the synthesis of three dimensional carbon fiber reinforced carboxymethyl cellulose-hydroxyapatite (CMC-HA-CFs) ternary composite for weight bearing orthopedic application, particularly as a mechanically compatible bioactive bone graft. 4

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2. Experimental Method 2.1. Material Carbon fiber (CFs) [diameter~7-10 µm, tensile strength~ 3.5 GPa, density~ 1.77 g cm-1] was procured from Nickunj Group, Mumbai, India. Carboxymethyl cellulose (CMC),

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di-ammonium hydrogen phosphate [(NH4)2HPO4], calcium nitrate tetra hydrate

experiments, deionized (DI) water has been used.

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2.2. Functionalization of carbon fiber

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[Ca(NO3)2.4H2O] and liquid ammonia were purchased from Merck, India. In all the

The surface of CFs was functionalized according to reported method [15]. Firstly,

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fibers were chopped into 1-2 mm size with scissor, and were washed in acetone. Washed

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fibers were oven dried at 60 °C. For functionalization, 1 g dried fibers were immersed in 500 mL acid mixture (H3PO4/HNO3/H2SO4, VH3PO4:VHNO3:VH2SO4= 1:2:6) for 2 h. After 2 h,

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fibers were filtered and repeatedly washed with DI water, until the pH~7.

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2.3. Synthesis of carbon fiber reinforced carboxymethyl cellulose-hydroxyapatite

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ternary composite (CMC-HA-CFs)

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In the present study, three systematically different CMC-HA-CFs ternary composites have been synthesized with 0.25 wt%, 0.5 wt% and 0.75 wt% CFs and labeled as CF-0.25, CF-0.5 and CF-0.75, respectively. For the synthesis of each composite, 10 g CMC was dissolved in 1000 mL DI water with gentle heating. After that, the desired content of functionalized CFs were added into polymer solution and stirred vigorously with hand. Afterwards, 300 mL calcium aqueous solution (0.99M) was slowly added to fiber containing polymer solution. The mixture was made alkaline (pH ~10-11) by supplying 5

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ammonia and aged overnight at room temperature. Next day, 600 mL phosphate aqueous solution (0.56 M) was slowly added to the mixture and stirred. Immediately, ammonia was supplied to the mixture (pH ~10-11) and stirred vigorously. The mixture was aged at room temperature for 7 days. After aging, it was washed with DI water and allowed to dry by

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solvent loss at 55 °C. As a result of drying and physical gelation, the three dimensional

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CMC-HA-CFs composites were obtained. For comparative study, CMC-HA composite without CFs was also prepared (CF-0) [14].

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2.4. Characterization

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The chemical structure of CFs and functionalized CFs were examined using Nicolet, AlmegaXR dispersive raman spectrometer (using a Nd:YAG Laser source, λ=532 nm). FTIR

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(Fourier transform infrared) spectra of CFs, functionalized CFs and synthesized composites

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were acquired (JASCO-FTIT, Model 410) by KBr method over a wavelength range 400

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cm-1 to 4000 cm-1. The surface morphology of CFs, functionalized CFs and CMC-HA-CFs composite were captured using Field emission scanning electron microscope (FESEM)

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[Supra 55, Carl Zeiss, Germany]. FESEM equipped with the analyzer of energy dispersive

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spectroscopy (EDS) and elemental mapping was used to investigate the presence of elements and their distribution on the surface of CFs. The X-ray diffraction (XRD) patterns of synthesized composites were recorded on Bruker X-ray diffractometer (D8 Discover). The micro structural features of composites were observed using JEOL 2100 Transmission Electron Microscope (TEM).

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The compressive strength of synthesized composites [~16 mm × 8 mm × 8 mm] was measured using Universal testing machine [Tinius Olsen (H25KS), USA] followed by ASTM D695, where load has been applied in 0.2 mm min-1 rate. The flexural property of synthesized composite [width (b)~2 mm, thickness (d)~1.5

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mm and length~30 mm] was measured by three-point bending using Universal testing

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machine [Hounsfield test equipment (H10KS-0290), UK] followed by ASTM C1161. In this test, load has been applied in 0.2 mm min-1 rate. The flexural strength, flexural strain

3𝐹𝐿 6𝐷𝑑 𝐿3 𝑚 ; 𝐹𝑙𝑒𝑥𝑢𝑟𝑎𝑙 𝑠𝑡𝑟𝑎𝑖𝑛 = ; 𝐹𝑙𝑒𝑥𝑢𝑟𝑎𝑙 𝑚𝑜𝑑𝑢𝑙𝑢𝑠 = 2𝑏𝑑 2 𝐿2 4𝑏𝑑 3

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𝐹𝑙𝑒𝑥𝑢𝑟𝑎𝑙 𝑠𝑡𝑟𝑒𝑛𝑔𝑡ℎ =

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and flexural modulus of composites were calculated by using the given equations:

Where, F is the maximum flexural load, L is the support span (25 mm), b= width of the

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sample, d= thickness of the sample, D= maximum deflection, m= slope of the linear portion

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of the flexural load-deflection curve, respectively. To check the reproducibility, each nanocomposite was tested three times.

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2.5. In-vitro cell survivability, adhesion and differentiation study

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For in-vitro cell study, we have chosen preosteoblast MC3T3 cells because it is a

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suitable cell model of osteogensis [16]. The cytocompatibility of composites was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) assay. As per the guidelines of ISO 10993-5, composites extracts were carefully dropped on pre-seeded (3×104 cells/well in 6-well plate) cells and incubated for a week in humidified incubator [14, 17, 18]. At day 1, day 4 and day 7, MTT solution was added into each well and incubated for 3 h. After that, MTT solution was replaced with dimethyl sulphoxide (DMSO) and the absorbance was measured at 595 nm using micro plate reader (i-Mark, 7

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Bio-Rad, USA). For cell adhesion study, MC3T3 cell suspension (1 × 104 cells mL-1) was carefully dropped on CMC-HA-CFs composite (~2 mm × 2 mm × 0.2 mm) and incubated for a week. At day 1, day 4 and day 7, images of cells around the samples were captured using MOTIC AE31 phase contrast microscope. After that, cells were fixed with 4% para-

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formaldehyde for 10 minute and dehydrated with ethanol solutions. The cells morphology

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on the surface of composites was observed using SEM.

In-vitro cell differentiation study namely alkaline phosphatase (ALP) activity and

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matrix mineralization potential of MC3T3 cells were evaluated by direct method, where

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CMC-HA-CFs (~2 mm × 2 mm × 0.2 mm) composite was cautiously placed over the monolayer of cells and cultured for desired period [17, 18]. In these studies, osteogenic

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media [DMEM containing 50 μg/mL ascorbic acid, 10 mM β-glycerophosphate, 10 mM

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dexamethasone] and basal media (DMEM) were set as positive control and negative control, respectively. ALP activity of preosteoblast MC3T3 cells was measured after 7 and

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14 days of culture, using ALP Kit (HiMedia, CCK035). Extracellular mineralization

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potential of MC3T3 cells was studied at 7, 14 and 21 days of culture, by alizarin red staining (ARS) and von kossa staining (VKS). Images of newly deposited mineral were

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captured using MOTIC AE31 phase contrast microscope. 2.6. Statistical analysis Collected data were presented as mean and standard deviation (n=3). One way analysis of variance (ANOVA) was performed using Graph Pad Prism to determine statistical significance between the samples at p < 0.05. 8

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3. Result and discussion Our study demonstrates a viable wet precipitation method for the synthesis of three dimensional carbon fiber reinforced carboxymethyl cellulose-hydroxyapatite ternary

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composite for weight bearing orthopedic application. Fig. 1 represents the synthetic

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procedure of CMC-HA-CFs composite.

Fig. 1. Scheme of synthetic procedure of three dimensional carbon fiber reinforced carboxymethyl cellulose-hydroxyapatite ternary composite (CMC-HA-CFs).

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3.1. Characterization of functionalized carbon fiber In this study, we have chemically functionalized the CFs to have effective bonding between fiber and matrix. Raman spectra of fiber before and after functionalization are

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given in Fig. 2a. Each spectrum consists of two main bands, characteristic graphitic G-

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band and disordered D -band at 1350 cm-1 and 1600 cm-1 respectively. The ratio of

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intensities between the D and G bands (ID/IG) was increased in functionalized CFs (~1.09) compared to unmodified CFs (~0.94), indicating that the graphitic structure of CFs was

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disturbed due to functionalization. It showed that some functional groups were covalently

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introduced onto the surfaces of CFs [19]. The surface functional groups of CFs after chemical treatment were identified by FTIR spectroscopy [Fig. 2b]. In the spectrum of

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functionalized CFs, we have found a broad band in the region 3600-3000 cm-1 assigned the

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-OH stretching of newly generated hydroxyl groups onto the surface of CFs after chemical treatment. The position of this band at lower wavenumbers indicates the presence of strong

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hydrogen bond. Surprisingly, we have found two new bands at 1295 cm-1 and 925 cm-1 in

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the spectrum of functionalized CFs. It confirmed the formation of new functional units i.e. P=O and P-O-C respectively onto the surface of fiber after chemical treatment [20]. Surface

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morphology of CFs before and after functionalization was characterized by FESEM. Before functionalization [Fig. 2c], the surface of CFs was clean and smooth. After functionalization the surface became irregular and few pits are found [Fig. 2d], which may be due to oxidation of surface carbon. It can be further confirmed from EDS analysis where the content of oxygen is higher in the spectrum of functionalized CFs as compared to CFs. Furthermore, we have found an additional element phosphorous (P) with very slight 10

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amount 1.22%. From these results, we have concluded that the phosphorous containing functional groups (P=O, P-O-C) were formed on the surface of CFs after chemical

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treatment, which has also been proved by Feng et.al. [15].

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Fig. 2. (a) Raman and (b) FTIR spectra of carbon fiber and functionalized carbon fiber;

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FESEM images of (c) carbon fiber and (d) functionalized carbon fiber (inset: corresponding EDS spectra).

3.2. Identification of mineral phase of the synthesized composites XRD and FTIR are two widely used techniques to identify the different phases of calcium phosphate minerals. XRD pattern of composites [Fig. S4 (a)] reveals (002), (102), (202), (211), (213), (222), (310), (004), (511) reflections of HA (JCPDS file: 09-0432). In FTIR spectra [Fig. S4 (b)], the characteristic vibrational bands of phosphate group of HA 11

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are present at 1041-1030 cm-1 (P-OH bend.), 600-605 cm-1 (O-P-O bend.) and 560-580 cm-1 (P-O-P str.), indicating the successful formation of hydroxyapatite composites [14]. Furthermore, the hydroxyl group of HA and CMC gave an overlapped band at 3600-3100 cm-1. In all the composites, the carboxylic band of CMC [Fig. S4 (c)] was shifted from its

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original position (1622 cm-1) to higher wavenumber at 1642cm-1. It indicates that the

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mineralization of HA in the composites has been occurred at the carboxylic end of CMC [14]. However, the characteristic bands of CFs were not identified in the spectra of CMC-

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HA-CFs composites. Those bands may be overlapped with other bands which cannot be

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distinguishable.

3.3. Microstructure of synthesized CMC-HA-CFs composite

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The surface morphology of CMC-HA-CFs composite is shown in Fig. 3. In Fig. 3a,

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we observed that the CFs were protruded from bulk area of matrix, and partially oriented in

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a particular direction. The effective interaction of fiber with matrix can be visualized in Fig. 3b, where no gap is found at the junction of fiber and matrix. Even the layer of mineralized

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matrix adhered on the surface of CFs that represents the strong bonding between fiber and

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matrix. For deeper understanding, we have captured the magnified image of surface part of CFs [Fig. 3c]. Surprisingly, we have noticed very small spherical particles (10-20 nm size) are entirely covered the surface of fiber, which are absent in both the magnified image of CFs and functionalized CFs [Fig. S5(a, b)]. This indicates that the spherical nanoparticles were grown on the surface of CFs during composite formation stage. The elemental analysis of surface part of CFs was presented in Fig. 3d, e. In the EDS spectrum [Fig. 3d] Carbon (C), Oxygen (O), Calcium (Ca) and Phosphorous (P) peaks are found, confirming 12

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the presence of those elements on the surface of the CFs. The atomic Ca/P ratio of this part is 1.63, indicating the formation of HA on the surface of fiber [21]. In elemental mapping [Fig. 3e], we observed C, O, Ca and P elements are uniformly distributed on the entire surface of CFs. From this observance, we assumed that the formation of spherical

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nanoparticles on the surface of CFs is occurred during composite formation stage, and those

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nanoparticles are mainly composed of calcium phosphate i.e. hydroxyapatite. Huang et. al. have grown the similar type of spherical HA particles on the surface of CFs by in-situ

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mineralization process, and proved that single type of functional group onto the surface of CFs induced the formation of HA in arranged and ordered structure [22]. Here, it can be

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concluded that the phosphorous containing functional groups (P=O, P-O-C) induce the

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orderly deposition of ions on the surface of fiber, and grow as spherical HA nanoparticles.

Fig. 3. (a) Surface morphology of carbon fiber reinforced carboxymethyl cellulosehydroxyapatite composite; (b) image of fiber-matrix inter phase; (c) magnified image of 13

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surface part of carbon fiber; (d) EDS spectrum and (e) element mapping of the surface part of carbon fiber in the composite. The microstructures of matrix part of composites are given in Fig. S6; which are same in all composites. In these images, agglomerated HA nanoparticles were found which

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are fairly knitted in CMC matrix. This type of observation was also found in Kumar et. al.

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report [23]. It is due to the formation of in-situ HA nanoparticles which were mineralized in CMC matrix through physical interactions [14, 23]. Because of these interactions between

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macromolecular chain of cellulose and HA, the composites became less porous; and only few micro-pores were found in the surfaces of composites.

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The HRTEM images of CMC-HA-CFs composite are shown in Fig. 4. In Fig. 4a,

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we observed the layer of matrix adhered with the surface of CFs, which is in good agreement with FESEM image [Fig. 3b]. In matrix part [Fig. 4b], we found the

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systematically arranged HA particles of size 30-40 nm embedded in CMC. From magnified

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image [Fig. 4c] of interfacial zone, we have indexed the (211), (201) and (110) planes of HA. The SAED (selected area electron diffraction) pattern of interfacial zone is given in

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Fig. 4d, where the ring pattern depicts the nanocrystalline HA and the brightest one

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represents the (211) plane of HA. It shows that the HA not only present in the matrix part, but also occupied the interfacial zone and formed bridged HA between fiber and matrix. This type of observation has also been proved by Liu et.al. where HA acted as intermediate layer and showed good adherence at the interface [24].

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Fig. 4. HRTEM image of (a) carbon fiber reinforced carboxymethyl cellulosehydroxyapatite ternary composite; (b) magnified image of matrix part and (c) magnified

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image of interfacial zone; (d) SAED pattern of interfacial area.

3.4. Formation mechanism of CMC-HA-CFs ternary composite Based on the experimental findings, a possible formation mechanism of three dimensional CMC-HA-CFs ternary composite was proposed. In the composite formation stage, the Ca2+ ions get adsorbed on the surface of phosphate functionalized CFs via 15

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electrostatic attraction and trigger the initial step of HA nucleation [25]. With time more deposition (Ca2+ and PO43- ions) occur and grow as tiny spherical particles of HA which are entirely covered the surface of CFs. Meantime, formation of hydroxyapatite (HA) is also occurred at the carboxylate (COO-) sites of CMC and develop HA embedded CMC matrix

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[14]. We know that the HA effectively binds with polar groups (COO-, OH) of CMC via

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ionic/ H- bonds. Thus, it can be assumed that the HA nanoparticles on the surface of CFs attract the mineralized matrix towards CFs and strongly bind them via ionic/ H- bonds. At

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the end, three dimensional CMC-HA-CFs composite is successfully formed. In brief, there are three key aspects for the formation of CMC-HA-CFs ternary composite- (i)

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mineralization of HA at the surface of the phosphate functionalized CFs and COO- ionic

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sites of CMC by electrostatic interaction; (ii) The HA nanoparticles on the surface of CFs acts as bridged molecule between fiber and CMC matrix, and effectively bind them; (iii)

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CFs hierarchical composite.

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overall interactions lead the formation of mechanically strong three dimensional CMC-HA-

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3.5. Mechanical properties of synthesized composites The mechanical properties of synthesized composites were measured by means of

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compression and flexural test, and tabulated in Table S1 (supplementary information). It can be seen that compressive strengths of CF-0, CF-0.25, CF-0.5 and CF-0.75 are almost 10 MPa, 49 MPa, 88 MPa and 118 MPa, respectively. It is observed that the compressive strength of composites was increased with the addition of high strength CFs. The compressive stress-strain curves of composites are given in Fig. 5a. The CMC-HA and CMC-HA-CFs composites showed viscoelastic deformation under compressive load, 16

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whereas CF-0 and CF-0.25 deformed at higher strain; and CF-0.5 and CF-0.75 deformed at lower strain. This is due to the higher content of CFs in CF-0.5 and CF-0.75, and its uniform dispersion in the composites [9]. Another factor may be bridged HA that increased the stiffness of interface and restricted the molecular movement along the interface under

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stress, which leads the less strain [26, 27].

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The flexural strength of composites was also increased with CFs content. The flexural strength of CF-0, CF-0.25, CF-0.5 and CF-0.75 are almost 21 MPa, 37 MPa, 60

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MPa and 141 MPa, respectively. The flexural modulus of CF-0, CF-0.25, CF-0.5 and CF0.75 are nearly 7 GPa, 9 GPa, 12 GPa and 22 GPa respectively. In Fig. 5b, CMC-HA (CF-

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0) composite shows strain softening behaviour after achieving maximum stress. CMC-HA-

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CFs composites exhibit elastic deformation, where CF-0.75 shows catastrophic failure. This is due to the presence of high content of CFs and its better interaction with matrix [9, 26,

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27].

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Fig. 5. (a) Compressive and (b) Flexural stress-strain curves of synthesized composites.

From the above observation, it is expected that matrix cracking and fiber delamination or fiber breakage are the dominant failure modes in CMC-HA-CFs ternary composite under load. In future, detail study on failure mechanism will be carried out.

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The results of current study and some prior studies on carbon fiber reinforced polymer-hydroxyapatite ternary composites are summarized in Table 1 and compared with cortical bone. It can be observed that the most of the previously-studied ternary composites have higher mechanical properties compared to the human bone [8-9, 11]. It is known that

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the ideal bone grafts should possess mechanical strength in the range of human bone for

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their possible weight bearing application. The results of present study are in the range of human bone. The study does not perfectly correlate with available results in the literatures

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because of using different fiber weight fraction and dissimilar test methods. In this study, we have used very less amount (0.25 -0.75 wt%) of CFs compared to other literatures, and

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developed a mechanically compatible CMC-HA-CFs bonegraft. It signifies that the

Fiber content (wt%)

Compressive strength (MPa)

Flexural strength (MPa)

Flexural Modulus (GPa)

Ref.

<200

180

7-25

[5,27]

5-20

-

159-223

-

[11]

5-20

116-212

89-138

-

[9]

CF/HA/PLA

20

-

220-430

12-22

[8]

PEEK/CF/nHA

15

-

-

-

[7]

PEEK/nHA/CF

20

-

-

-

[10]

CMC-HA-CF

0.250.75

49-118

35-140

9-22

present study

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Composite

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interfacial interaction between fiber and matrix has been improved in the process.

CF/HA/PA46

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CF/HA/PA66

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Human bone

Table 1: Comparison of our CMC-HA-CFs composite with other carbon fiber reinforced hydroxyapatite-polymer composites as well as with human bone

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3.6. In-vitro cell survivability, proliferation and differentiation study The survivability and proliferation of preosteoblast MC3T3 cells cultured with CMC-HA and CMC-HA-CFs extracts were assessed using MTT-assay. The absorbance

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produced by metabolically active live cells in test samples and control are shown in Fig. 6a.

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None of the samples showed any significant toxicity towards MC3T3 cells after a week.

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The attachment and proliferation of MC3T3 cells on the surface of CMC-HA-CFs composite after different days of culture are shown in Fig. 6b. In these images, we

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observed that the MC3T3 cells attached and spread well on the surface of composite.

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Moreover, the cell density was increased with incubation time and almost covered the entire surface at day 7, indicating that the surface of composite provides good environment

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for cell growth and proliferation. The SEM images of cell seeded CMC-HA-CFs composite

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are in consistence with corresponding optical images of surrounding cells, where the cell

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density was also increased with incubation time.

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Fig. 6. (a) MTT assay of MC3T3 cells cultured with composites extract for 1, 4 and 7 days;

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(b) Morphology of MC3T3 cells onto (in SEM images) and around (in Phase contrast images) the CMC-HA-CFs composite at day 1, day 4 and day 7. (in phase contrast images,

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magnification = 10×);

In this report, the ALP enzyme activity of MC3T3 cells in basal media (negative control), osteogenic media (positive control) and test sample (CMC-HA-CFs) were measured at day 7 and day 14. The ALP enzyme activity [Fig. 7a] of MC3T3 cells in test sample (CMC-HA-CFs composites) was remarkably higher at day 14, compared to negative control (basal media). However, the ALP activity of MC3T3 cells is highest in 21

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osteogenic media (positive control). The in-vitro mineralization of MC3T3 cells in positive control, in negative control and in presence of CMC-HA-CFs at different time points (day 7, day 14 and day 21) are presented in Fig. 7b, c. After 14 days and 21 days of culture, we found significant calcium phosphate deposits in the well-plate of positive control and test

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sample. In ARS (Fig. 7b) and VKS (Fig. 7c), these deposits are red and deep-brown colour,

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respectively. No such deposition was obtained in negative control (Fig. 7b, c) during the whole study. Because, preosteoblast MC3T3 cells are unable to express a fully

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differentiated osteoblast phenotype i.e. the formation of mineralized extracellular matrix in normal media, [16, 28]. Interestingly, it was observed that CMC-HA-CFs composite

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accelerates MC3T3 cells for high ALP activity, and also helps in extracellular

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mineralization without any osteogenic supplements. This may be due to the presence of hydroxyapatite nanoparticles (size- 30-40 nm) which is closely resemblance with inorganic

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component of natural bone. Due to this intrinsic property of HA, cells rapidly recognize the

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existence of nano-HA in the extracellular environment and modulate genes expression

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accordingly to induce osteogenic differentiation [29].

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Fig. 7. (a) ALP enzyme activity of MC3T3 cells at day 7 and day 14; Matrix mineralization

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of MC3T3 cells in osteogenic media (positive control), basal media (negative control) and

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in presence of CMC-HA-CFs composite at day 7 , day 14 and day 21, measured by (b) ARS and (c) VKS.

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4. Conclusions

Present work offers a new viable wet precipitation method for the synthesis of carbon fiber reinforced polymer-hydroxyapatite ternary composite for weight bearing orthopedic application. Compared with the existing methods, this method has several advantages- (i) simple and cost effective; (ii) less amount of carbon fiber has been used; (iii) easy and uniform distribution of carbon fibers in the matrix; (iv) facilitate effective 23

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fiber-matrix interactions with the formation of bridged HA at the interface. Hence, the flexural and compressive properties of synthesized composites have been significantly enhanced with addition of carbon fibers. The synthesized CMC-HA-CFs ternary composite not only possess good mechanical properties, but also has favorable biological properties

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for preosteoblast MC3T3 cells to proliferate and differentiate. These results showed the

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viability of CMC-HA-CFs ternary composite as mechanically compatible bioactive

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bonegraft particularly in weight bearing site.

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Acknowledgements:

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This work was financially supported by project support group of CSIR-National

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Metallurgical Laboratory (OLP-0231). Authors Chandrani Sarkar showed her immense gratitude to University Grants Commission (UGC), India for her fellowship.

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References

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Conflict of interest: The authors declare that they have no conflict of interest.

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BIOGRAPHY

Chandrani Sarkar is a Ph. D. scholar in Advanced Materials and Processes Division, CSIR-National Metallurgical Laboratory, Jamshedpur, India. She is doing her thesis work under the guidance of Dr. Sumanta Kumar Sahu. Late Dr. Subhadra Garai was also her thesis supervisor. Her thesis work is carried out at Advanced Materials and Processes 29

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Division, CSIR-National Metallurgical Laboratory, Jamshedpur, India and Department of Applied Chemistry, Indian Institute of Technology (ISM), Dhanbad. She has received fellowship from University Grant Commission, New Delhi, India for her PhD. Currently, she is teaching undergraduate students at Department of Chemistry, Mahila College under Kolhan University, Chaibasa, Jharkhand, India. Her research work is focused on the synthesis of nanocomposite materials for possible orthopedic application.

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Dr. Sumanta Kumar Sahu received his Ph. D. in nanomedicine from Indian Institute of Technology, Kharagpur in 2011. He is currently the head of the research group, “Functional Nanomaterials for biomedical applications” at the Department of Applied Chemistry, Indian Institute of Technology (ISM), Dhanbad since 2012. He has authored over 52 scientific research papers. His research interests include nanomaterials, functional porous materials, and their applications in drug delivery, catalysis, sensing and separation.

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Dr. Arvind Sinha is a Chief Scientist at CSIR-National Metallurgical Laboratory, Jamshedpur, India. Currently, he is Head of Advanced Materials and Processes Division of this institute. He is also chairman of National Academy of Sciences (Jharkhand state chapter), India. He has received his Ph.D. award from Indian Institute of Technology (Banaras Hindu University), Varanasi in 1993 on Materials Technology. In 1999, He was among the pioneers in India to adopt ‘biomimetic’ as a research field. He has transferred six of his biomimetic technologies to different Indian Biomaterials Industries. He has published 65 papers in nationals and international journals, and has 18 patents. He has received several prestigious awards, including CSIR –Young Scientist Award, CSIR-Raman Research Fellowship, Altekar award, Nijhavan award and many more. His research interests focused on Biomimetic Materials and Biomaterials.

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Dr. Jui Chakraborty is a Senior Scientist at Bio-ceramics and Coating Division of CSIR-Central Glass and Ceramic Research Institute, Kolkata, India. Her research activity encompases application of bioactive glass coatings in healthcare and development of inorganic nanoconjugate to deliver drugs/biomolecules. She pursued her doctoral studies from National Institute of Technology, India. She has authored many papers in high impact journals along with 30

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several patents to her name. She has been conferred with several prestigious scientific research awards and serves as reviewer of many eminent international journals. She looks forward to solve healthcare problems by developing new approaches and impactful inventions.

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Dr. Subhadra Garai was Principal Scientist in Advanced Materials and Processes Division at CSIR-National Metallurgical Laboratory, Jamshedpur, India. She had received her PhD from Jadavpur University, Kolkata, India in 1998. She had published several research papers in different reputed journals. She had many patents and had transferred technology to Indian Biomaterials Industries. She had received Nijhavan award in 2015. Her research interest was to synthesize nanocomposite for orthopedic applications. She was suffering from cancer for few months. Lastly, she left for her heavenly adobe on 03 Aug 2018.

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Highlights:

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Three dimensional carbon fiber reinforced carboxymethyl cellulose-hydroxyapatite ternary composite has been synthesized by simple wet precipitation method. Bridged hydroxyapatite nanoparticles between matrix and fiber were formed during composite formation stage. Synthesized composite has high mechanical strength and modulus analogous to human bone. The ternary composite acelarates preosteoblast MC3T3 cells for ALP acitivity and extracellular mineralization.

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