Mineralized Hydroxyapatite

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Carbon nanofiber/polycaprolactone/mineralized hydroxyapatite nanofibrous scaffolds for potential orthopedic applications Shinyjoy Elangomannan, Kavitha Louis, Bhagya Mathi Dharmaraj, K. Venkata Saravanan, Kannan Soundarapandian, and Dhanaraj Gopi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13058 • Publication Date (Web): 27 Jan 2017 Downloaded from http://pubs.acs.org on January 28, 2017

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ACS Applied Materials & Interfaces

Carbon nanofiber/polycaprolactone/mineralized hydroxyapatite nanofibrous scaffolds for potential orthopedic applications Shinyjoy Elangomannan †, Kavitha Louis⊗, Bhagya Mathi Dharmaraj ⊗, Venkata Saravanan⊗, Kannan Soundarapandian§, Dhanaraj Gopi †,‡,* †

Department of Chemistry, Periyar University, Salem 636 011, Tamilnadu, India Department of Physics, School of Basic and Applied Sciences, Central University of Tamilnadu, Thiruvarur 610 101, Tamilnadu, India § Proteomics and Molecular Cell Physiology Laboratory, Department of Zoology, Periyar University, Salem 636 011,Tamilnadu, India ‡ Centre for Nanoscience and Nanotechnology, Periyar University, Salem 636 011, Taminadu, India * Corresponding author. Tel.: +91 427 2345766; fax: +91 427 2345124. E-mail address: [email protected] (D. Gopi). ⊗

Abstract Hydroxyapatite (Ca10 (PO4)6(OH)2, HAP), a multi-mineral substituted calcium phosphate is one of the most substantial bone mineral component that has been widely used as bone replacement materials because of its bioactive and biocompatible properties. However the use of HAP as bone implants is restricted due to its brittle nature and poor mechanical properties. To overcome this defect and to generate suitable bone implant material, HAP is combined with biodegradable polymer (Polycaprolactone (PCL)). In order to enhance the mechanical property of the composite, carbon nanofibers (CNF) is incorporated to the composite, which has long been considered for hard and soft tissue implant due to its exceptional mechanical and structural properties. It is well known that nanofibrous scaffold are the most are prominent material for the bone

reconstruction,

we

have

developed

a

new

remarkable

CNF/polycaprolactone

(PCL)/mineralized hydroxyapatite (M-HAP) nanofibrous scaffolds on titanium (Ti). The as developed coatings were characterized by various techniques. The results indicate the formation and homogenous distribution of components in the nanofibrous scaffolds. Incorporation of CNF into PCL/M-HAP composite significantly improves the adhesion strength and elastic modulus of the scaffolds. Furthermore, the responses of Human osteosarcoma (HOS MG63) cells cultured 1 ACS Paragon Plus Environment


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onto the scaffolds demonstrate that the viability of cells were considerably high for CNF incorporated PCL/M-HAP than PCL/M-HAP. In vivo analysis show the presence of soft fibrous tissue growth without any significant inflammatory signs which suggests that incorporated CNF did not counteract the favorable biological roles of HAP. For load bearing applications, research in various bone models is needed to substantiate the clinical availability. Thus, from the obtained results we suggest that CNF/PCL/M-HAP nanofibrous scaffolds can be considered as potential candidate for orthopedic applications. Keywords: Hydroxyapatite, Biodegradable polymers, CNF, nanofibrous scaffold, bone implant applications 1.

INTRODUCTION Biomaterials are those which help to enhance the lifespan of human beings and quality of

life and therefore, show a rapid development to keep with the demands of people of advanced age. The evolution of biomaterials for hard tissue repair and replacement is a challenging and promising research area. In particular, bone tissue engineering is a rapidly growing research which provides an innovative and promising approach for the repair and regeneration of traumatized bone tissues.1,2 In general, any material that is to be used for tissue regeneration must possess biocompatible surfaces and favourable mechanical properties. Titanium implants is one of the most used metallic materials for various types of bone anchored reconstructions because of its mechanical properties, biocompatibility and resistance to corrosion.3,4 However, generally these metallic surfaces bear adequately low bioactivity, therefore they necessitate a quality surface treatment in order to improve the bioactivity which enhances the osseointegration with hard bone tissues. These metallic implants need to shorten bone healing period in vivo. However the new bone formation on the implant surface needs a

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long time. Since these reactions lead to unexpected risk in patients it is very significant to assess and optimize the biocompatibility of implants. For this purpose, an enhanced bioactive layer is developed over the metallic implant surface in order to support bone bonding ability. Considering these, Ti implants are most generally coated with HAP, a bioceramic material with chemical and crystallographic similarity to the inorganic minerals of human hard tissues. This is currently being used in hard tissue engineering for bone repair or regeneration. This bioactive coating on Ti implants is beneficial to improve the implant-bone tissue integration.5,6 Since HAP is a multi-mineral substituted calcium phosphate, it can be further substituted by various mineral ions so as to enhance the biological properties. The substitution of mineral ions can be tuned in relevance with the application of the implant material. There are numerous studies on the fabrication of mineralized HAP and mainly, the favorable effect of mineral ions such as zinc (Zn2+), silver (Ag2+), magnesium (Mg2+), gallium (Ga3+), silicon (Si2+), manganese (Mn2+), strontium (Sr2+), etc., in HAP based coatings on metal substrates has been reported.7-11 For enhancing the bioactivity of a material, it is crucial that the substituted minerals must promote cell attachment and proliferation to induce bone regeneration. The substitution of manganese into HAP (Mn-HAP), results with an attractive characteristic of enhancing cellular function and thereby finds its application in the field of bone tissue engineering.12,13 The divalent manganese is an essential trace metal that influences the metabolism of bone by regulating the osteoblast differentiation and bone resorption.14,15 Manganese plays a crucial role in activating integrins, receptors mediating cellular interactions with the extracellular matrix and cell surface ligands. The substitution of Mn ions in HAP, favours osteoblasts proliferation and increases the biocompatibility.16



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Gallium, at present is widely used in the area of biomedical applications because of its strong affinity to bone tissue and inhibits bone resorption. In addition, gallium ions are clinically effective in dealing with the osteoporosis treatment and cancer related hypercalcemias.17,18 Since gallium ions show excellent antibacterial activity, it is believed to play a positive role in the acceptance of implants. In addition, gallium as a diagnostic and therapeutic agent, also finds its application in treating the metabolic disorders in soft and hard tissues.19,20 Though HAP and mineralized HAP have shown to induce bone growth, they exhibit poor mechanical properties. However, the brittle nature and poor strength of HAP, unable compete with the mechanical behaviour of natural bone which would restrict its clinical usage in load-bearing devices.21 The limitation of poor mechanical property of HAP has paved the way for many material researchers to hunt for the polymer composites. Though there are many methods available for the enhancement of mechanical property of HAP and mineralized HAP, the most common and widespread method is to incorporate a suitable polymer into HAP that no unfavorable effects on the human body. Polycaprolactone, a semi-crystalline aliphatic polymer is one of the most important biodegradable polymer with significant toughness, excellent biocompatibility, well determined biodegradability, good mechanical strength and higher fracture energy than most of the other biocompatible polymers.22 In addition, PCL has drawn significant consideration because it can be easily made-up into complicated shapes, has good porosity, and excellent biocompatibility. All these properties makes PCL to be more attractive for long-term implant application.23-25 The incorporation of bone-bioactive inorganic component like HAP into the polymer matrix has shown better interaction and improved cell adhesion with the biological environment.26 These polymer/HAP composite when used at high loadings, exhibit adverse effect on the mechanical


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properties (i.e., the composite shows poor mechanical properties which may not be suitable for direct-loading applications).27 The poor mechanical properties are mainly owing to the brittleness of the HAP and also its lack of interaction with polymer. To overcome this weakness and to provide mechanical rigidity and strength for development of orthopedic load-bearing implants with exceptional biocompatibility, incorporation of suitable of reinforcing materials like CNTs, CNF, TiO2, ZrO2 is adopted.28-31 In addition to superior mechanical and biocompatibile properties, an ideal reinforcing material for bone implant applications must promote cell attachment, proliferation, differentiation and tissue regeneration.32 The reinforcement ability of CNTs in the CNTs/HAP composites is noticeable, but the ultrashort length (up to several micrometers) of CNTs restricts it as reinforcing fillers. In comparison to natural bones, the reinforcing ability and mechanical properties of CNTs are not sufficient for major load-bearing applications.33 Among the reinforcing materials, CNF is less expensive, less toxic, can be easily functionalized and is better dispersible than CNTs.34 CNF relatively of longer length (up to several centimeters) with chemical structures identical to that of CNTs would be a prior choice. The reinforcement of CNF provides additional structural stability, good conductivity and excellent mechanical stability to the matrix.35 Due to strong mechanical strength as well as excellent biocompatibility, CNF have become known to be promising materials for bone implant applications.36,37 They can be uniaxially oriented,38 and are less likely to curve in matrices. For the formation of CNF composites, the surface of CNF is treated with acid, oxidizing agents and surfactants which produce carboxylic, hydroxyl and ketonic groups. The presence of functional groups on the surface modifies the properties of CNF and improves dispersion and interactions with HAP matrix.39 Functionalized CNF can easily form various chemical bonds and thereby improves its compatibility with matrix.40-44 All these properties would make CNF an exceptional



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reinforcing fillers for the HAP matrix. Therefore efficient utilization of CNF within the composite is requisite for understanding their prospective reinforcing ability. Since, HAP and CNF as composite exhibit strong interfacial bonding with enhanced mechanical strength, they can be used as a bone replacement material. The incorporation of CNF itself will show a tremendous improvement in the mechanical properties of the composite. Though there are several coating methods available for the development of the composite over the implant surface, electrochemical deposition technique has exceptional advantages because of its comparatively low deposition temperature, simple procedure, helps in the formation of a uniform coating on a porous and/or complex-shaped substrate, easy accessibility and low cost of equipment. Hence, it is wholly accepted as one of the most suitable and convenient methods for the development of coating of Ti metal. In general, the perfect material for bone tissue regeneration must exhibit superior biocompatibility, biodegradability with controllable degradation kinetics, simple production and adequate mechanical properties. In this article, the development and characterization of a nanofibrous scaffold (CNF/PCL/M-HAP), is reported. The combination of CNF with PCL and M-HAP appeared to be a reasonable strategy for mimicking natural bone, as this resulted in a remarkable improvement of their corrosion resistance, mechanical properties and the biocompatibility. Thus, the as developed CNF/PCL/M-HAP nanofibrous scaffold will be potentially suitable for orthopedic applications. 2.

MATERIALS AND METHODS

2.1.

Chemicals Calcium nitrate dihydrate (Ca(NO3)2.2H2O), gallium nitrate (Ga(NO3)3), manganese

nitrate hexahydrate (Mn(NO3)2.6H2O), dipotassium hydrogen phosphate (K2HPO4), CNF, PCL, nitric acid and sulphuric acid, acquired from Aldrich Chemical Co. (Aldrich, India), were 6 ACS Paragon Plus Environment

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utilized for the fabrication of composite coating on Ti by electrochemical

deposition.

All the chemicals were of analytical grade and used as received. Deionized water was used throughout the experiment. 2.2.

Oxidation of CNF As received CNF was refluxed with 300 mL of a concentrated nitric acid in round-

bottomed flask fitted with a reflux condenser and a thermometer for 48 h at 120 °C. The obtained mixture was washed with 500 mL of deionised water and then vacuum-filtered through a filter paper (3 µm porosity). The washing procedure was repeated until the pH of filtrate reached a value of 7 and vacuum dried at 100 °C. Such oxidized CNF were coded as O-CNF. 2.3.

Specimen preparation Titanium specimens (99.9% purity) of the size 10×10×3 mm were cut and embedded in

epoxy resin by leaving area of 1 cm2 for exposure to the solution. Before the electrodeposition process, the specimens were roughened with different grades of SiC emery papers from 400 to 1200 grit and washed with distilled water and ultrasonically cleaned in 100% acetone for 10 min, in order to remove any surface residues. All the specimens received a final rinse in deionized (DI) water and then immediately dried in flowing air and then used for further purpose. 2.4.

Electrolyte preparation The electrolyte for M-HAP, PCL/M-HAP and CNF/PCL/M-HAP coatings on Ti was

prepared in three series as shown below: Series 1: Preparation of M-HAP electrolyte Analytical grade Ca(NO3)2.2H2O, Mn(NO3)2.6H2O and Ga(NO3)3, were dissolved separately in deionized water and were mixed in the ratio of 8:1:1, respectively.



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The K2HPO4 was dissolved in deionized water and mixed with the above solutions to produce the target (Ca+Mn+Ga)/P ratio of 1.67. The electrolyte solution was prepared in the N2 gas atmosphere and the pH of the electrolyte was maintained to 4.7 using NaOH or HCl at 28 °C using thermostat. Finally, the obtained electrolyte for M-HAP deposition was stirred in magnetic stirrer for 2 h at 180 rpm in order to maintain uniform distribution of the mineral ions in the electrolyte. Series 2: Preparation of PCL/M-HAP electrolyte PCL (0.5 g) was dissolved in chloroform under magnetic stirring for 3 h at room temperature. The PCL solution is then added to the M-HAP electrolyte solution (prepared as mentioned in series 1) drop-wise, under constant stirring. The obtained solution is exposed to strong ultrasonic treatment for effective dispersion. Series 3: Preparation of CNF/PCL/M-HAP electrolyte To the electrolyte of PCL/M-HAP (series 2), different concentrations of (1, 2 and 3 wt.%) of oxidized CNF (O-CNF) was added by maintaining pH of 4.7 and then stirred for 2 h. Further, the electrolyte for O-CNF/PCL/M-HAP deposition must be exposed to strong ultrasonic treatment to ensure the effective dispersion of CNF in the matrix for about 30 min. 2.5.

Electrodeposition on Ti `The electrochemical deposition of M-HAP, PCL/M-HAP and O-CNF (different

concentrations)/PCL/M-HAP was performed in a three-electrode system in which the platinum electrode was used as a counter electrode, a saturated calomel electrode (SCE) and Ti as a reference and working electrode, respectively. Prior to electrodeposition process the Ti substrates were polished with different grades of SiC abrasive paper, washed with acetone, de-ionized water and then dried in air. The M-HAP, PCL/M-HAP and CNF/PCL/M-HAP was


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electrodeposited on Ti at -1.4 V vs. SCE using the electrochemical workstation CHI 760 C (CH instruments, USA). After electrodeposition process, the substrates were gently rinsed with deionised water and then dried at room temperature for 24 h. 2.6.

Characterization techniques

2.6.1. Surface characterizations Fourier transform infrared spectroscopy (FT-IR, Impact 400 D Nicholet Spectrometer) was employed to substantiate the presence of functional groups in the as-developed (PCL/MHAP and at different concentrations of CNF in CNF/PCL/M-HAP composite) coatings on Ti samples. The analysis was performed in the frequency range from 4000 to 400 cm−1 with 32 scans and spectral resolution of 4 cm−1. Before analysis, the coatings were scraped carefully from the surface of Ti substrate and were then mixed with KBr and pressed into discs. Phase analysis of the above said coatings were carried out by the X-ray diffraction (XRD) technique using a diffractometer (Bruker D8 Advance diffractometer) in the 2θ range of 20-60° at a scan rate of 0.02°. The high resolution SEM (HRSEM, JSM 840A Scanning microscope operating with accelerating voltage of 30.0kV) was performed to examine the morphological nature of the M-HAP, PCL/M-HAP and different concentrations of CNF in CNF/PCL/M-HAP composite coatings. Elemental analysis and elemental mapping was carried out using energy dispersion spectroscopy (EDX) to examine presence of different elements present in 2 wt.% CNF/PCL/MHAP composite coating. Further, the microstructure of the different concentrations of CNF in CNF/PCL/M-HAP composite coating was examined by high resolution transmission electron microscopic analysis (HRTEM, JEOL JEM 2100 Co., Tokyo, Japan). Samples for the HRTEM analysis were prepared by the scraping the as-developed composite coatings from the Ti



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substrate and dispersed in ethanol which is further ultrasonicated for 15 min to ensure complete dispersion. Subsequently droplets of suspension were put on carbon grid (200 meshes) coated with copper and the solvent was allowed to evaporate. 2.6.2. Electrochemical evaluation of the composite coatings To assess the corrosion resistance of uncoated and various composite coated Ti, electrochemical

characterizations

involving

potentiodynamic

polarization

studies

and

electrochemical impedance spectroscopy (EIS) were performed in simulated body fluid (SBF) solution. The SBF solution for performing the electrochemical studies, was prepared according to Kokubo’s protocol.45 For this purpose, a three electrode cell configuration is used and the electrochemical measurements were performed using the CHI 760 electrochemical workstation (USA), in which, Ti and platinum serves as the working and counter electrodes, respectively whereas, saturated calomel electrode (SCE) was used as the reference electrode. All the potential values in the text are related to the SCE. The potential range of -1 and 1 V vs. SCE was used in this polarization study with a scan rate of 1 mV/s. Also, the frequency for the electrochemical impedance spectroscopic measurements were obtained in the range of 10-1 Hz to 105 Hz with 5 mV of perturbation amplitude.

All the electrochemical parameters are

standardized with respect to the 1 cm2 area of the Ti electrode and the electrochemical datas obtained were recorded using internally available software. 2.6.3. Mechanical characterization The mechanical properties of coating on the metallic implant suggests helpful information about the adhesion strength and hardness of the coating and underlying implant. The adhesion strength of the PCL/M-HAP and different concentrations of CNF in CNF/PCL/M-HAP composite coated specimens were studied by pull-out test using a Universal Instron Mechanical


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Testing system (Instron 5565, Instron Co.), according to ASTM F 1044-05 standard. The

as

developed

samples

were

measured

at

steady

cross-head

speed.

The samples were then kept in oven at 100 ºC for about 50 min and the pull-out test was performed

using

a

universal

testing

machine

at

a

crosshead

speed

of

1 mm min-1 with at least five measurements for each sample. Vickers hardness test for the same composite coated Ti was calculated over the surface of PCL/M-HAP and CNF/PCL/M-HAP using Akashi AAV-500 series hardness tester. The load used was 490 mN with a dwell time of 20 s. The obtained hardness value for the samples is an average of 5 different hardness test measurements. 2.6.4. Biological characterizations

In vitro antibacterial activity

The in vitro antibacterial property of the M-HAP, PCL/M-HAP and different concentrations of CNF in CNF/PCL/M-HAP composite coated Ti was evaluated against Staphylococcus aureus (S. aureus) (ATCC 25923) and Escherichia coli (E. coli) (ATCC 25922) by disc diffusion method. It is reported that, S. aureus and E. coli were the most common pathogens associated with biomaterial centered infections and hence they are found suitable for estimating

the

antibacterial

properties

of

the

composite

coatings

on

Ti.46

Fresh overnight broth cultures (Trypton soy broth with 0.6% yeast extract) which were incubated at 37 °C were used for the preparation of the inoculums of the microorganisms. The as-prepared cultures were utilized for the estimation of anti-bacterial activity of the samples. Muller Hinton agar was used for the agar diffusion test which was performed by forming 4 mm thick layers of agar in the petri dishes. In order to obtain semi confluent growth, dense inoculums of tested microorganisms were added into the petri dishes and were left for 10 min in the laminar air flow. Discs of size 6 mm were made from Whatman no:3 filter paper and then immersed into different 11 ACS Paragon Plus Environment


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concentration of (25, 50, 75, 100, 125 µL) composite coated samples and kept at equivalent distances and then incubated in a bacteriological incubator at 37 ˚C overnight. The width of zone of inhibition (mm) around the disc of the as-developed samples were measured to evaluate the the anti-bacterial activity against the two bacterial strains.

In vitro cytotoxicity

Human osteosarcoma MG63 cells (HOS MG63, ATCC CRL-1427TM) procured from National Centre for Cell Sciences (NCCS), Pune, India, were cultured using standard culture medium, Dulbecco’s Modified Eagle Medium (DMEM, GIBCO). For every two days the medium was renewed and the cultures were preserved in a humidified atmosphere of 5% CO2 and 95% humidified air, at 37 °C. The cultures were then incubated with 0.1% trypsin and 0.1% ethylene diamine tetra acetic acid (EDTA) for 5 min and then detached from the culture flask. Modified MTT 3-(4,5-dimetyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide) assay was used for the evaluation of viability of cells on different concentrations of CNF in CNF/PCL/M-HAP composite coated sample and control sample (without composite coating). HOS MG63 Cells with the density of 104 cells/well were seeded on the composite coated samples in 12 well plates. After 24 h of incubation, MTT assay solution was added to the samples and then kept in incubation for 4 h at 37 °C in a humidified 5% CO2 atmosphere as a function of incubation time for 1, 4 and 7 days. Finally the MTT reagent was then detached, followed by the addition of dimethyl sulfoxide to dissolve the formazan crystals and the absorbance at 570 nm was measured on ELISA microplate reader after shaking the plate for 15 min. % cell viability was calculated with respect to control as follows, % Cell viability = [A] Test / [A] Control x 100


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Animal study and surgical procedure

The main aim of this study is to implant Ti with and without coatings inside the femur bone of Wistar rats and to evaluate its tissue, cell behavior around the implants using histological observations. Animal care and the surgical method were adopted as per the strategy of the Institutional Animal Ethics Committee (IAEC) at Kovai Medical Center Research and Educational Trust, Coimbatore, India (KMCRET/Ph.D/3./2013-2014). About 15 male Wistar rats of average weight 200-250 g were used for this study and the rats were housed under customary conditions at a controlled temperature (20 °C) and a light/dark cycle (12/12 h). All the rats were grouped into 3 groups as follows: Group I as uncoated (5 nos), Group II as PCL/M-HAP (5 nos) and Group III as CNF/PCL/M-HAP composite coated Ti (5 nos). All the rats were separately anesthetized through intra peritoneal injections of ketamine (20 mg/kg) and xylazine (2 mg/kg) and were given a mixture of 20% v/v isoflurane and propylene glycol for inhalation. The surgical site was shaved first, rubbed with iodine and the muscles were separated over the femur to expose the periosteum. It is also very important that that the implantation must be accomplished under aseptic condition. An opening was made at the femur bone by making a 2 mm hole using the drilling machine. The drilling must be done with adequate irrigation with saline solution all through the drilling process in order to minimize the temperature rise in the bone. All the animals were given penicillin as antibiotic for three postoperative days. The rats were set aside on nurturing for 14 and 28 days and were supervised twice a day, particularly for the duration of the first week after surgery. After 14 and 28 days of implantation, the rats were sacrificed and the implants specimens were carefully harvested for the histological evaluation.



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For histological observations, the rat bones with the implants were sliced to a thickness of 1-2 mm with a low speed diamond saw and were fixed in neutral formalin solution of 20%, decalcified inside acetic acid solution of 10% for 4 days and fixed in paraffin. After the removal of the implants from the rats bones, ultrathin sections were cut at 70 nm and were stained with Mallory and hematoxylineosin staring solution and were histologically analyzed by light microscopy. 2.6.5. Statistical analysis In the present study all the experimental data were conveyed as average ± standard deviation and a statistically significant differentiation were considered to be present at p˂0.05 as significant. All the experimental groups of the study were carried out in triplicate and the results were analyzed using ANOVA statistical study. 3.

RESULTS AND DISCUSSION

3.1.

FTIR spectra Fourier transform infrared spectroscopic analysis was carried out to analyze the structural

modifications that occurred in the formation of the composite. Figure 1(a-d) illustrates the FTIR spectra of PCL/M-HAP and different concentrations (1 wt.%, 2 wt.% and 3 wt.%) of CNF reinforced PCL/M-HAP composite coating. Several peaks were observed for the pure PCL/MHAP coating (Fig. 1(a)), in which the peaks for M-HAP were supported by the presence of functional groups like hydroxyl and phosphate (PO43-) group, respectively. The absorption peaks appear at 962 and 472 cm−1 (υ1 and υ2 vibration mode), 1036 and 1095 cm−1 (υ3 mode of P–O symmetric stretching vibration), 568 and 603 cm−1 (υ4 P–O bending vibration) corresponds to the PO43− group of M-HAP. The above peaks seemed to agree with the presence of vibrational modes of phosphate group. The peaks at 3570 and 632 cm−1 corresponds to the hydroxide (OH−)


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stretching mode.47 The characteristics peaks at 2927, 2860 and 1718 cm-1 are attributed to the presence of CH2 and –C=O groups, whereas the peak at 1168 cm-1 is assigned to C–O stretching of PCL in the PCL/M-HAP composite.48 All the characteristics peaks obtained at Fig. 1(a), clearly evidences the development of PCL/M-HAP composite coating and no other peaks corresponding to any trace of impurities were detected. The spectra obtained at different concentrations (1 wt.%, 2 wt.% and 3 wt.%) of CNF reinforced PCL/M-HAP composite coating is shown in Fig. 1(b-d). These spectra also show the peaks attributable to PCL/M-HAP composite and in addition to that, the characteristic peaks at 1736 cm−1 and 1584 cm−1 were also found which corresponds to the carboxyl group and carboxylate group on the surface of CNF obtained as a result of oxidation of CNF (Fig. 1(d)).49,50 All the other characteristic peaks of PCL/M-HAP were found to have shifted slightly to a lower wave number compared with PCL/M-HAP coating which might be due to the reinforcement of CNF in PCL/M-HAP coating. These FT-IR peaks indicate that a strong interaction exists between the PCL/M-HAP coating and CNF in CNF/PCL/M-HAP composite coating on Ti substrates. Thus, in this study, the presence of the characteristic peaks like phosphate and hydroxyl groups of hydroxyapatite, polymer and also carboxyl and carboxylate groups supports the formation of CNF/PCL/M-HAP composite nanofibrous scaffolds. 3.2.

XRD analysis Figure 2(a-d) illustrates the X-ray diffraction patterns of PCL/M-HAP and

CNF/PCL/M-HAP composites with different concentration (1 wt.%, 2 wt.% and 3 wt.%) of CNF. Fig. 2(a) shows the XRD pattern of PCL/M-HAP composites in which the XRD peaks for PCL, a semi-crystalline polymer can be identified at 2θ=21.5°, 22.1° and 23.7° which can be assigned to (110) (111) and (200), respectively (ICDD Card No.: 74-566). Besides the



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characteristic peaks of PCL, the X-ray diffraction patterns of PCL/M-HAP (Fig. 2(a)) show the crystalline peaks of M-HAP at 26.0º, 28.1º, 29.3º, 31.9º, 33.1º, 34.1º, and 40.0º which are in good agreement with database of International Centre for Diffraction Data (ICDD card No. 090432). The acquired peaks confirm the incorporation of M-HAP with PCL in the coating. Moreover, the peaks become slightly broader when compared to the peaks for HAP, which implies the low crystalline nature of the composite. Fig. 2(b-d) presents the XRD patterns for different concentrations of CNF in CNF reinforced PCL/M-HAP composite coating. These patterns present the peaks corresponding to both PCL and M-HAP. Besides the above characteristic diffraction peaks belonging to PCL/M-HAP coating, the XRD patterns of CNF/PCL/M-HAP (with 1 wt.%, 2 wt.% and 3 wt.% of CNF) composite coated samples, exhibits the diffraction peaks close to 26o which can be assigned to the diffraction of (0 0 2) planes of graphite (ICDD card number: 13-0148). The addition of different concentrations of CNF did not considerably affect the XRD patterns instead a mild decrease in the peak intensity is observed which indicates a slight decrease in the crystallinity of the CNF/PCL/M-HAP composites. This low crystalline nature of the composite favours enhanced bone generation. From the literature it is well know that new bone could not be formed in highly crystalline nature of the composite material.51 Thus, in Fig. 2(b-d) characteristic peaks for CNF, PCL and M-HAP were found which suggests the successful formation of CNF/PCL/M-HAP composite coatings on Ti. From the XRD patterns it is also well supported that no obvious structural change is induced in the PCL/M-HAP coatings by reinforcement of CNF in the composite. 3.3.

HRSEM and EDAX analysis The morphology of the minerals substituted HAP and PCL incorporated minerals

substituted HAP is shown in Fig. 3(a-d). The HRSEM micrograph of Mn substituted HAP (Fig.


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3(a)) shows the presence of small granular particles with irregular pores in between them. Similarly the Ga substituted HAP (Fig. 3(b)) presented a highly distributed uniform bead like particles. Figure 3(c) corresponds to the morphology obtained for the PCL coating. The formation of needle like structure with major agglomeration in the form of cluster is obtained. The nature of the polymer, PCL, is clearly evident from the figure. For the PCL/M-HAP coating (Fig. 3(d)), the typical needle-like agglomerated structure of PCL still dominated the same. Along with that, the small minute granules and beads were present which supports the presence of M-HAP in PCL/M-HAP coatings. Figure 4(a-d) shows the HRSEM images of CNF/PCL/M-HAP composite coatings developed with different concentration of CNF (1 wt.%, 2 wt.% and 3 wt.%). It can be noted from the figure that, all the composite coatings were inter connected with each other. The microscopic images of Fig. 4(b) provide clear evidence that CNF (2 wt.%) were uniformly covered with PCL and mineralized hydroxyapatite. The oxidation of CNF will allow the surface of CNF to have the sufficient hydrophilicity and innumerable functional groups as nucleation sites. Consequently, the minerals substituted HAP could be fast deposited on the O-CNF evenly with the PCL molecules along the surface of CNF. The CNF/PCL/M-HAP composite coating seemed to be dense and hard. The HRSEM micrographs of CNF/PCL/M-HAP composite coatings revealed a slight difference with varying CNF concentrations in CNF/PCL/M-HAP composite coating (Fig. 4(a-c)). It is inferred that the occurrence of CNF in PCL/M-HAP matrix caused the fabrication of a uniform composite coating and as the concentration of CNF was increased, the composite coating was obtained with porous and finer morphology, leading to increasing compactness of the composite coatings (Fig. 4(d)). The electrodeposition of the CNF, PCL, M-HAP led to the development of CNF/PCL/M-HAP nanofibrous scaffold with



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interconnected network like structure. Thus, the surface morphological evaluation of the CNF/PCL/M-HAP composite coating exhibited a complete surface coverage and homogeneous arrangement. It is evident that the interconnected network like nanofibrous CNF/PCL/M-HAP structure can provide a considerable enhancement in the mechanical strength of the coated substrate. Moreover, the interconnected network like nanofibrous scaffold, is seen to be adequate for various biomedical applications. The fibrous scaffold with minute open pore structure is good for blood supply and cell attachment.52 The development of CNF/PCL/M-HAP composite nanofibrous scaffold achieves a better representation of natural bone. Elemental distribution in the composite coating was executed by EDAX analysis which shows the existence of chemical elements like calcium and phosphorous characteristic of ceramic phase, oxygen representative of polymeric compound and carbon for CNF. Fig. 5 shows the EDAX mapping images of 2 wt.% CNF/PCL/M-HAP composite coating that contains the elements like Ca, Ga, Mn, P, O and C which supports for the presence of CNF/PCL/M-HAP composite coating. Transmission Electron Microscopic images of different concentrations of CNF reinforced PCL/M-HAP coating is presented in Fig. 6(a-c). Comparing the different concentrations, the 1 wt.% of CNF in the composite coating exhibits scattered morphology of the composite material. Whereas on increasing the concentration of CNF to 2 wt.% (Fig. 6(b)), it is observed that very few particles are present around the tubular CNF which might support the presence of polymer and minerals substituted HAP in the composite coating. Further, on increasing the concentration to 3 wt.% some aggregated particles adheres on the surface of nanofibers. The formation of composite could not be identified clearly at this increased concentration. From the TEM observations it is obvious that the oxidation of CNF helped to generate composite material


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with polymer and M-HAP. It has been determined from the TEM image that, CNF molecules are uniformly dispersed in a polymer/M-HAP matrix at low concentration which means that any excess accumulation of CNF may cause unfavorable effects due to the production of agglomerates or lumps. Hence, from this study CNF taken at optimum concentration of 2 wt.% can be successful in strengthening the composite coating both mechanically and biologically. 3.4.

Electrochemical characterisation

3.4.1. Potentiodynamic polarisation studies Corrosion resistance is one of the main significant properties of the metallic implants which are used in the orthopedic application. The corrosion protection performance of the uncoated Ti and as-developed composite coatings on Ti are examined for long-term biomedical applications in SBF solution. Figure 7 explains the potentiodynamic polarisation curves of uncoated Ti, PCL/M-HAP, CNF/PCL/M-HAP with three different CNF concentrations (1, 2 and 3 wt.%) coated Ti samples, respectively in the potential range of -1 and 1 V vs. SCE at an OCP condition in SBF solution. Table 1 shows the corrosion potential (Ecorr) and corrosion current density (Icorr) values obtained from the polarization curves. The Ecorr and Icorr values for uncoated Ti sample obtained potentiodynamic polarization curves, were found to be -0.57±0.005 V vs. SCE and 0.54±0.003 µA/cm2, respectively. While the polarisation curve recorded for PCL/MHAP coated Ti sample showed Ecorr, and Icorr values as 0.07±0.003 V vs. SCE and 0.08±0.006 µA/cm2, respectively. An investigation of polarization curves showed a positive shift in the polarization values (Ecorr, and Icorr) of the CNF/PCL/M-HAP composite coatings (with three different CNF concentrations (1, 2 and 3 wt.%)) on Ti, which strongly reveals the corrosion protection efficiency of the composite coating. The polarization values of coating increased on increasing concentrations of CNF (upto 2 wt.%) in the composite. The polarisation curve of the



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CNF/PCL/M-HAP (at 2 wt.% of CNF) coated Ti samples which revealed the Ecorr and Icorr values

as 0.210±0.004 V vs. SCE and 0.006±0.006 µA/cm2respectively. Among the coatings, the 2 wt.% CNF in CNF/PCL/M-HAP composite coating showed superior corrosion protection efficiency in SBF solution (Table 1) owing to its more uniform with compact arrangement of nanofibrous scaffold and the results are in good concurrence with HRSEM results. The following order shows the the Ecorr and Icorr values of the coatings PCL-M-HAP < 1 wt.% CNF/PCL/M-HAP < 3 wt.% CNF/PCL/M-HAP < 2 wt.% CNF/PCL/M-HAP

As the concentration of CNF is increased further to 3 wt.% of CNF in composite coating, the Ecorr slightly decreased and Icorr increased when compared to that of 2 wt.% CNF/PCL/M-HAP (Table 1). The maximum shift of Ecorr and Icorr values towards the noble direction in SBF solution

is

evident

from

2

wt.%

CNF/PCL/M-HAP

which

indicates

that

the

2 wt.% CNF/PCL/M-HAP coated Ti specimen revealed the maximum corrosion protection

efficiency. Thus, the 2 wt.% CNF/PCL/M-HAP coating on Ti can act as a corrosion protective barrier layer and increase the long term duration of the implant for load bearing applications. 3.4.2. Electrochemical impedance spectroscopy studies The EIS is one of the predominant methods that provides mechanistic information through the electrochemical impedance profile. This technique also offers more useful information on capacitive and resistive behavior and its measurements were also performed in SBF solution. Figure 8(a-b) explains the representative equivalent circuits of uncoated and composite coated Ti specimen in SBF solution. As it can be seen from the Fig. 8(a) that the equivalent circuit for uncoated Ti consisted of Rs which corresponds to the solution resistance, Rp1 represents the polarisation resistance and Cdl1 represents double layer capacitance, respectively. Rp2 and Cdl2 in the second subsystem of circuit relates to resistance and capacitances for the composite coated Ti (Fig. 8(b)). 20 ACS Paragon Plus Environment

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The EIS spectrum observed for uncoated and composite coated Ti shown in Fig. 8(c) was fitted using the circuit model represented as in Fig. 8(a,b) and the obtained values are given in Table 1. On comparison between the uncoated and composite coated Ti, the acquired polarization resistance (Rp) value for the 2 wt.% CNF/PCL/M-HAP composite coating is found higher (257.6±0.02 kΩ cm2) than that obtained for the PCL/M-HAP and other concentration of CNF in CNF/PCL/M-HAP composite coatings. The Rp value for the uncoated and PCL/M-HAP coated Ti was found at 52.4±0.06 kΩ cm2 and 115.7±0.04 k Ω cm2, whereas for the 1 and 3 wt.% of CNF in CNF/PCL/M-HAP composite coating, the Rp value was observed to be 230.6±0.02 kΩ cm2 and 245±0.03 kΩ cm2, respectively. A highest Rp value of 257.4±0.02 kΩ cm2 for 2 wt.% CNF/PCL/M-HAP composite coating on Ti, propose that the as developed composite coating obstructs the ion diffusion process from the electrolyte and thereby enhances the corrosion resistance of Ti. Hence from the EIS studies, it can be accomplished that the 2 wt.%CNF/PCL/M-HAP composite coating on Ti is superior in corrosion resistance which can be used in orthopedic applications for enhancing the longevity of the implants. 3.5.

Mechanical characterization of the coatings

3.5.1. Adhesion strength The adhesion strength of the as-developed PCL/M-HAP and different concentrations of CNF in CNF reinforced PCL/M-HAP composite coatings on Ti was evaluated using the ASTM F 1044-05 adhesion test and the results of the adhesion strength are portrayed in Fig. 9. The adhesive strength of the CNF (2 wt.%)/PCL/M-HAP coated Ti was 27.6 ± 0.5 MPa which is higher than that of PCL/M-HAP (21.3± 0.4) and 1 and 3 wt.% of CNF reinforced PCL/M-HAP composite coatings 24.2± 0.5, 26.1± 0.7 MPa, respectively. Thus from this results, it is obvious that incorporation of CNF in the PCL/M-HAP coatings possesses excellent adhesion to the Ti



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surface. This high adhesion strength of CNF/PCL/M-HAP composite coatings will make it highly suitable for load bearing applications. 3.5.2. Vickers micro-hardness Vickers micro-hardness (Hv) is the most significant parameter for a metallic implant to be used

in

load

bearing

applications.

The

Hv

values

of

the

as-developed

PCL/M-HAP and different concentrations of CNF in PCL/M-HAP composite coatings on Ti samples obtained by means of the Vickers micro hardness test are depicted in Fig. 9. The hardness values of PCL/M-HAP, 1 wt.% CNF/PCL/M-HAP coatings were found to be 8.10± 0.34 and 9.23 ± 1.2 GPa, respectively. From the results it is clear that as the concentration of CNF in the CNF/PCL/M-HAP coatings is increased to 2 and 3 wt.%, the Hv value was found to be 9.5 ± 1.4 Hv and 10.17 ± 1.3 Hv, respectively. Thus, the presence of CNF in the composite coatings improved the hardness of the coating on Ti surface making it suitable for load bearing orthopedic applications. The mechanical property (adhesion strength, elastic modulus and hardness) obtained for the composite coated Ti are comparable to those of natural bones. This mechanical properties thus obtained is greater than the required mechanical strength of human bone tissue. 3.6.

Biological characterizations

3.6.1. Antibacterial activity The antibacterial activity of the bioceramic coated metallic implants is to be evaluated in order to avoid surgical infections in the field of orthopedic surgery. The composite coatings were investigated for its antibacterial activities according to the disc diffusion method against two prokaryotic strains such as E. coli and S. aureus, respectively, which are frequently responsible for post surgical infections associated with the implants. Fig. 10 shows the inhibition zones for


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the composite coated sample against S. aureus (Fig. 10(a)) and E. coli (Fig. 10(b)) at various concentrations such as 25, 50, 75, 100 and 125 µL, respectively. It should be noted from the figure that for CNF/PCL/M-HAP composite coatings, the inhibitory zone against E. coli and S. aureus is larger than the other composite coated samples. The inhibition zones for the CNF/PCL/M-HAP composite coated sample against E. coli and S. aureus strains were obtained as 15.5 & 14.3 mm for 25µL, 16.8 & 16.4 mm for 50 µL, 16.9 & 16.7 mm for 75 µL, 17.2 & 17.0 mm for 100 µL and 18.1 & 17.9 mm for 125 µL volumes, respectively. At the highest concentration (125 µg /ml), the CNF/PCL/M-HAP composite coated sample confirms the maximum level of antimicrobial activity by demonstrating the highest inhibition zone diameter against E. coli. The in vitro antibacterial activity of the CNF/PCL/M-HAP coating against E. coli was slightly greater when compared to that of activity against S. aureus bacteria is attributed to the variation found in the structure of cell wall. These data revealed that the substitution of minerals like Ga3+ and Mn2+ plays a very significant role in improving the antibacterial activity of the CNF/PCL/M-HAP composite which is also clearly evident from the images of plates exhibiting the zone of inhibition (Fig. 10(c)). 3.6.2. In vitro biocompatibility The composite coatings (different concentrations of CNF reinforced PCL/M-HAP composite) were prone to cytotoxic studies with HOS MG 63 cells. The viability of cells on the composite

coatings

was

estimated

using

MTT

assay

at

1, 4 and 7 days of incubation and is presented as bar diagram in Fig. 11. From the MTT assay test the non-toxic nature of all the coatings is clearly revealed. In particular, the cells cultured on the CNF/PCL/M-HAP composite coated sample showed excellent viability after 4 and 7 days of incubation. The viability of the cells on 1 and 2 wt.% CNF/PCL/M-HAP composite coated



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sample was higher with longer incubation period (4 and 7 days), which indicates the non-toxic nature of CNF. As the concentration of CNF is increased after 2 wt.% the viability of the cells were decreased, which clearly shows the optimum concentration of CNF as 2 wt.% in the composite. Also, the substitution of mineral ions like Gallium, Manganese and the biopolymer in the composite coatings promoted healthy growth of the cells. These comparative results suggest that CNF/PCL/M-HAP composite coated Ti would be a capable candidate in search of a scaffold to be used as bone implant for orthopedic applications. Figure 12 presents the fluorescent microscopic images in the form of live/dead cells over the composite coated Ti samples after exposure for incubation period of 1, 4 and 7. The cells spread well above the surface of the sample and only very rounded cells were observed on the CNF (2 wt.%) /PCL/M-HAP at 1 day of incubation compared to 4 and 7 days of incubation. But after 1 day of incubation, higher numbers of viable cells were found on the samples and no dead cells were seen. From the images, it can be observed that the incubation period of 7 days was found to be more suitable for cell adhesion and proliferation. The enhanced viability of the cells over the CNF(2 wt.%)/PCL/M-HAP composite coated samples are mainly due to the existence of minerals substituted in HAP and the biopolymer. The MTT assay results show that the CNF/PCL/M-HAP composite coatings are non-toxic to the cells, and thus favor cell attachment and proliferation. 3.6.3. In vivo biocompatibility To evaluate biocompatible nature of the composite coated implant in vivo study was performed on male Wister rats. Surgical sites were completely healed in all the groups and all the animals that received composite coated implants quickly recovered. In particular, the rats that received CNF/PCL/M-HAP composite coated implants (Group III) showed no signs of infection


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on clinical examination during the observation period and neither any signs of infection were observed in the preparations. Histological examination showed no occurrence of inflammation in the composite coated Ti implants (Fig. 13). To study the former, the uncoated, PCL/M-HAP coated and CNF/PCL/M-HAP composite coated Ti implants were harvested after implantation for 2 and 4 weeks together with surrounding tissue. From Fig. 13(e), the formation of fibrous tissue around the implants was found which contains the fibroblastic cells along with the rim of newly formed trabecular bone. The group III implants exhibited most favorable healing response by the presence of newly formed trabecular bone (Fig. 13(f)). On comparison with the implantation of 2 weeks, the most favorable condition for the trabecular bone formation could be achieved with the 4 weeks of implantation period. Along with that, the ingrowth of abundant osteoblast-like cells is observed around the implants which suggest that CNF/PCL/M-HAP composite coated material provide the platform for the formation of bone tissues. Thus, from the result excellent bone formation and osseointegration was noticed in group III implants. These findings verify the positive effects of reinforcement of CNF in polymer and minerals substituted HAP which also proves the non-toxic nature of CNF and in good accord with the results reported in the literature.53,54 These findings further supports that the CNF/PCL/M-HAP composite coated HAP has positive effects in the orthopedic applications as bone replacement material. 4.

CONCLUSION This study aims to introduce a novel nanofibrous scaffolds as potential implants for

orthopedic

applications.

Reinforcement

of

CNF

of

different

concentrations

(1, 2 and 3 wt.%) with minerals substituted HAP and polymer matrix has resulted in the considerable improvements in the physiochemical, morphological, electrochemical, mechanical



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Page 26 of 48

and biological properties. The FTIR, XRD, SEM-EDX and TEM clearly confirms the presence of CNF/PCL/M-HAP composite material on Ti. The corrosion resistance of the as developed coatings is well evident from the electrochemical characterizations. For the biomedical applications, the implants need to satisfy multiple functional requirements such as mechanical strength and biocompatibility. The reinforcement of CNF in the PCL/M-HAP matrix would serve most properly for multiple requirements. As the CNF concentration increases till a particular concentration, the adhesion strength and the hardness also increases. The viability and proliferation activity of the osteoblasts cells on the composite coatings is well supported by MTT assay results. Among the coatings, the CNF/PCL/M-HAP composite is biocompatible with a enhanced cell proliferation rate in contrast to all the other coatings. Success of the as developed coatings in orthopedic applications requires a good understanding of the bone to implant relationship with respect to the specific tissue regeneration. This new material can establish direct bonds with bone tissue after implantation. This could be well evident from the in vivo study. Further researches of CNF/PCL/M-HAP composite in large animals are required to evaluate their prospective use as bone replacement materials. Acknowledgements One of the authors D. Gopi acknowledges major financial support from the Department of Science and Technology, New Delhi, India (DST-TSD, Ref. No.: DST/TSG/NTS/2011/73), DST-EMEQ, Ref. No.: SB/EMEQ-185/2013) and Defence Research and Development Organisation, New Delhi, India, (DRDO, Ref. No.: ERIP/ER/1103949/M/01/1513). D. Gopi also acknowledges UGC, New Delhi, India for the Research Award (Ref. No.: F. 30-1/ 2013(SAII)/RA-2012-14-NEW-SC-TAM-3240 and Council of Scientific and Industrial Research, New Delhi, India (CSIR, Ref. No.:01(2859)/16/ EMR-II).


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References 1. Petite, H.; Viateau, V.; Bensaid, W.; Meunier, A.; de Pollak, C.; Bourguignon, M.; Oudina, K.; Sedel, L.; Guillemin, G. Tissue Engineered Bone Regeneration. Nat. Biotechnol., 2000, 18, 959-963. 2. Stevens, M. M. Biomaterials for Bone Tissue Engineering. Mater. Today, 2008, 11, 1825. 3. Alghamdi, H. S.; Bosco, R.; van den Beucken, J. J. P.; Frank Walboomers, X.; Jansen, J. A. Osteogenicity of Titanium Implants Coated with Calcium Phosphate or Collagen type-I in Osteoporotic Rats. Biomaterials, 2013, 34, 3747-3757. 4. Prodanov, L.; Lamers, E.; Domanski, M.; Luttge, R.; Jansen, J. A.; Walboomers, X. F.; The Effect of Nanometric Surface Texture on Bone Contact to Titanium Implants in Rabbit Tibia. Biomaterials, 2013, 34, 2920-2927. 5. Wijesinghe, W. P. S. L.; Mantilaka, M. M. M. G. P. G.; Chathuranga Senarathna, K. G.; Herath, H. M. T. U.; Premachandra, T. N.; Ranasinghe, C. S. K.; Rajapakse, R. P. V. J.; Rajapakse, R. M. G.; Edirisinghe, M.; Mahalingam, S.; Bandara, I. M. C. C. D.; Singh, S. Preparation of Bone Implants by Coating Hydroxyapatite Nanoparticles on Self Formed Titanium dioxide Thin Layers on Titanium Metal Surfaces. Mater. Sci. Eng. C, 2016, 63, 172-184. 6. Raphel, J.; Holodniy, M.; Goodman, S. B.; Heilshorn, S. C. Multifunctional Coatings to Simultaneously Promote Osseointegration and Prevent Infection of Orthopaedic Implants. Biomaterials, 2016, 84, 301-314.



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Page 28 of 48

7. Gopi, D.; Nithiya, S.; Shinyjoy, E.; Kavitha, L. Spectroscopic Investigation on Formation and Growth of Mineralized Nanohydroxyapatite for Bone Tissue Engineering Applications. Spectrochimi. Acta Part A, 2012, 92, 194-200. 8. Gopi, D.; Shinyjoy, E.; Kavitha, L. Synthesis and Spectral Characterization of Silver/Magnesium

Co-substituted

Hydroxyapatite

for

Biomedical

Applications.

Spectrochimi. Acta part A, 2014, 127, 286-291. 9. Cox, S. C.; Jamshidi, P.; Grove, L. M.; Mallick, K. K. Preparation and Characterisation of Nanophase Sr, Mg, and Zn Substituted Hydroxyapatite by Aqueous Precipitation. Mater. Sci. Eng. C, 2014, 35, 106-114. 10. Bakin, B.; Koc Delice, T.; Tiric, U.; Birlik, I.; Ak Azem, F. Bioactivity and Corrosion Properties of Magnesium Substituted CaP Coatings Produced via Electrochemical Deposition. Surf. Coat. Technol., 2016, 301, 29-35. 11. Deliorman, A. M. Electrospun Cerium and Gallium Containing Silicate Based Bioactive Glass Fibers for Biomedical Applications. Ceram. Inter., 2016, 42, 897-906. 12. Moreira, M. P.; Dulce de Almeida Soares, G.; Dentzer, J.; Anselme, K.; Ágata de Sena, L.; Kuznetsov, A.; Araujo dos Santos, E. Synthesis of Magnesium and Manganese Doped Hydroxyapatite Structures Assisted by the Simultaneous Incorporation of Strontium. Mater. Sci. Eng. C, 2016, 61, 736-743. 13. Shin, E.; Yong Kim, I.; Baek Cho, S.; Ohtsuki, C. Hydroxyapatite Formation on TitaniaBased Materials in a Solution Mimicking Body Fluid: Effects of Manganese and Iron Addition in Anatase. Mater. Sci. Eng. C, 48, 2015, 279-286. 14. Huang, Y.; Qiao, H.; Nian, X.; Zhang, X.; Zhang, X.; Song, G.; Xu, Z.; Zhang, H.; Han, S. Improving the Bioactivity and Corrosion Resistance Properties of


Page 29 of 48

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Electrodeposited Hydroxyapatite Coating by Dual Doping of Bivalent Strontium and Manganese Ion. Surf. Coat. Technol., 2016, 291, 205-215. 15. Fujitani, W.; Hamada, Y.; Kawaguchi, N.; Daito, K.; Uchinaka, A.; Matsumoto, T.; Kajima, Y.; Daito, M.; Nakano, T. Synthesis of Hydroxyapatite Containing Manganese and its Evaluation of Biocompatibility. Nano Biomed., 2010, 2, 37-46. 16. Miola, M.; Vitale Brovarone, C.; Maina, G.; Rossi, F.; Bergandi, L.; Ghigo, D.; Saracino, S.; Maggiora, M.; Angela Canuto, R.; Muzio, G.; Verne, E. In Vitro Study of Manganese Doped Bioactive Glasses for Bone Regeneration. Mater. Sci. Eng. C, 2014, 38, 107-118. 17. Verron, E.; Bouler, J. M.; Scimeca, J. C. Gallium as a Potential Candidate for Treatment of Osteoporosis. Drug Discovery Today, 2012, 17, 1127-1132. 18. Melnikov, P.; Teixeira, A. R.; Malzac, A.; de B. Coelho, M. Gallium Containing Hydroxyapatite for Potential Use in Orthopedics. Mater. Chem. Phys., 2009, 117, 86-90. 19. Franchini, M.; Lusvardi, G.; Malavasi, G.; Menabue, L. Gallium Containing Phospho Silicate Glasses: Synthesis and In Vitro Bioactivity. Mater. Sci. Eng. C, 2012, 32, 14011406. 20. Valappil, S. P.; Ready, D.; Abou Neel, E. A.; Pickup, D. M.; O’Dell, L. A.; Chrzanowski, W.; Pratten, J.; Newport, R. J.; Smith, M. E.; Wilson, M.; Knowles, J. C. Controlled Delivery of Antimicrobial Gallium Ions from Phosphate Based Glasses. Acta Biomater., 2009, 5, 1198–1210. 21. Neelgund, G. M.; Olurode, K.; Luo, Z.; Oki, A. A Simple and Rapid Method to Graft Hydroxyapatite on Carbon Nanotubes. Mater. Sci. Eng. C, 2011, 31, 1477–1481.



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22. Chastain, S. R.; Kundu, A. K.; Dhar, S.; Calvert, J. W.; Putnam, A. J. Adhesion of Mesenchymal Stem Cells to Polymer Scaffolds Occurs via Distinct ECM Ligands and Controls their Osteogenic Differentiation. J. Biomed. Mater. Res., 2006, 78 A, 73-85. 23. Xin, X.; Hussain, M.; Mao, J. J. Continuing Differentiation of Human Mesenchymal Stem Cells and Induced Chondrogenic and Osteogenic Lineages in Electrospun PLGA Nanofiber Scaffold. Biomaterials, 2007, 28, 316-325. 24. Choi, S. H.; Park, T. G. Synthesis and Characterization of Elastic PLGA/PCL/PLGA TriBlock Copolymers. J. Biomater. Sci. Polym. Ed., 2002, 13, 1163-1173. 25. Holland, S. J.; Jolly, A. M.; Yasin, M.; Tighe, B. J. Polymers for Biodegradable Medical Devices: II. Hydroxybutyrate Hydroxyvalerate Copolymers: Hydrolytic Degradation Studies. Biomaterials, 1987, 8, 289-295. 26. Miller, N. D.; Williams, D. F. On the Biodegradation of Poly-β-hydroxybutyrate (PHB) Homopolymer and Poly-β-hydroxybutyrate-Hydroxyvalerate Copolymers. Biomaterials, 1987, 8, 129-137. 27. Bianco,

A.;

Federico,

E.D.;

Moscatelli,

I.;

Camaioni,

A.;

Armentano,

I.;

Campagnolo, L.; Dottori, M.; Kenny, J. M.; Siracusa, G.; Gusmano, G. Electrospun Poly(ε-caprolactone)/Ca-deficient

Hydroxyapatite

Nanohybrids:

Microstructure,

Mechanical Properties and Cell Response by Murine Embryonic Stem cells. Mater. Sci. Eng. C, 2009, 29, 2063–2071. 28. Khanal, S. P.; Mahfuz, H.; Rondinone, A. J.; Leventouri, Th. Improvement of the Fracture Toughness of Hydroxyapatite (HAp) by Incorporation of Carboxyl Functionalized Single Walled Carbon Nanotubes (CfSWCNTs) and Nylon. Mater. Sci. Eng. C, 2016, 60, 204-210.


Page 31 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


29. Yang, Q.; Sui, G.; Shi, Y.Z.; Duan, S.; Bao, J.Q.; Cai, Q.; Yang, X.P. Osteocompatibility Characterization of Polyacrylonitrile Carbon Nanofibers Containing Bioactive Glass Nanoparticles. Carbon, 2013, 56, 288-295. 30. Pushpakanth, S.; Srinivasan, B.; Sreedhar, B.; Sastry, T. P. An In Situ Approach to Prepare Nanorods of Titania Hydroxyapatite (TiO2–HAp) Nanocomposite by Microwave Hydrothermal Technique. Mater. Chem. Phys., 2008, 107, 492-498. 31. Samanipour, F.; Bayati, M. R.; Zargar, H. R.; Golestani-Fard, F.; Troczynski, T.; Taheri, M. Electrophoretic Enhanced Micro Arc Oxidation of ZrO2–HAp–TiO2 Nanostructured Porous Layers. J. Alloys Compd., 2011, 509, 9351-9355. 32. Hutmacher, D. W. Scaffolds in Tissue Engineering Bone and Cartilage. Biomaterials, 2000, 21, 2529-2543. 33. White, A. A.; Best, S. M.; Kinloch, I. A.; Hydroxyapatite Carbon Nanotube Composites for Biomedical Applications: A Review. Int. J. Appl. Ceram. Technol., 2007, 4, 1-13. 34. Kuilla, T.; Bhadra, S.; Yao, D. H.; Kim, N. H.; Bose, S.; Lee, J. H. Recent Advances in Graphene Based Polymer Composites. Prog. Polym. Sci., 2010, 35 (11), 1350-1375. 35. Tibbetts, G. G.; Lake, M. L.; Strong, K. L.; Rice, B. P. A Review of the Fabrication and Properties of Vapor-Grown Carbon Nanofiber/Polymer Composites. Compos. Sci. Technol., 2007, 67 (7−8), 1709-1718. 36. Yang, L.; Zhang, L.; Webster, T. J. Carbon Nanostructures for Orthopedic Medical Applications. Nanomedicine, 2011, 6, 1231–1244. 37. Ormsby, R.; McNally, T.; Mitchell, C.; Dunne, N. Incorporation of Multiwalled Carbon Nanotubes to Acrylic Based Bone Cements: Effects on Mechanical and Thermal Properties. J. Mater. Sci. Mater. Med., 2010, 21, 2287–2292.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 48

38. Zhou, Z.; Lai, C.; Zhang, L.; Qian, Y.; Hou, H.; Reneker, D.H.; Fong, H. Development of Carbon Nanofibers from Aligned Electrospun Polyacrylonitrile Nanofiber Bundles and Characterization of their Microstructural, Electrical, and Mechanical properties. Polymer, 2009, 50, 2999-3006. 39. Tran, P. A.; Zhang, L.; Webster, T. J. Carbon Nanofibers and Carbon Nanotubes in Regenerative Medicine. Adv. Drug Delivery Rev., 2009, 61, 1097-1114. 40. Wang, D. H., Sihn, S., Roy, A. K., Baek, J. B., Tan, L. S. Nanocomposites Based on Vapor Grown Carbon Nanofibers and an Epoxy: Functionalization, Preparation and Characterization. Eur. Polym. J., 2010, 46(7), 1404-1416. 41. Ahn, S. N., Lee, H. J., Kim, B. J., Tan, L. S., Baek, J. B. Epoxy/Amine Functionalized Short

Length

Vapor

Grown

Carbon

Nanofiber

Composites.

J. Polym. Sci. A Polym. Chem., 2008, 46(22), 7473-7482. 42. Brandl, W., Marginean, G., Chirila, V., Warschewski, W. Production and Characterization of Vapour Grown Carbon Fiber/Polypropylene Composites. Carbon, 2004, 42(1), 5-9. 43. Kumar, S., Rath, T., Mahaling, R. N., Reddy, C. S., Das, C. K., Pandey, K. N., Srivastava, R.B., Yadaw, S. B. Study on Mechanical, Morphological and Electrical Properties of Carbon Nanofiber/Polyetherimide Composites. Mater. Sci. Eng. B, 2007, 141(1–2), 61-70. 44. Lozano, K., Barrera, E. V. Nanofiber Reinforced Thermoplastic Composites. I. Thermoanalytical and Mechanical Analyses. J. Appl. Polym. Sci., 2001, 79(1), 125-133. 45. Kokubo, T.; Takafama, H., How Useful is SBF in Predicting In Vivo Bone Bioactivity? Biomaterials, 2006, 27, 2907-2915.


Page 33 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


46. Gristina, A. G.; Costerton, J. W. Bacterial Adherence to Biomaterials and Tissue: The Significance of its Role in Clinical Sepsis. J. Bone Joint Surg., 1985, 67, 264-273. 47. Gopi, D.; Karthika, A.; Nithiya, S.; Kavitha, L. In Vitro Biological Performance of Minerals Substituted Hydroxyapatite Coating by Pulsed Electrodeposition Method. Mater. Chem. Phys., 144 (2014) 75-85. 48. Rezaei, A.; Mohammadi, M. R. In Vitro Study of Hydroxyapatite/Polycaprolactone (HA/PCL) Nanocomposite Synthesized by an In Situ Sol–Gel Process. Mater. Sci. Eng. C, 2013, 33, 390–396. 49. Wu, L.; Zhang, X. J.; Ju, H. X. Amperometric Glucose Sensor Based on Catalytic Reduction of Dissolved Oxygen at Soluble Carbon Nanofiber. Biosens. Bioelectron., 2007, 23, 479-484. 50. Wu, M.; Wang, Q.; Liu, X.; Liu, H. Biomimetic Synthesis and Characterization of Carbon Nanofiber/Hydroxyapatite Composite Scaffolds. Carbon, 2013, 51, 335-345. 51. Wang, P.; Zhao, L.; Liu, J.; Weir, M. D.; Zhou, X.; Xu, H. H. K. Bone Tissue Engineering via Nanostructured Calcium Phosphate Biomaterials and Stem Cells. Bone Res., 2014, 2, 14017-14030. 52. Li Loh, Q.; Choong, C. Three-Dimensional Scaffolds for Tissue Engineering Applications: Role of Porosity and Pore Size. Tissue Eng. Part B Rev., 2013, 19(6), 485– 502. 53. Brown, D. M.; Kinloch, I. A.; Bangert, U.; Windle, A. H.; Walter, D. M.; Walker, G. S.; Scotchford, C. A.; Donaldson, K.; Stone, V. An In Vitro Study of the Potential of Carbon Nanotubes and Nanofibres to Induce Inflammatory Mediators and Frustrated Phagocytosis. Carbon, 2007, 45, 1743–1756.



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54. Jensen, E. K.; Larsen, S. Y.; Nygaard, U. C.; Marioara,

C. D.; Syversen, T.

Early Combination of Material Characteristics and Toxicology is Useful in the Design of Low Toxicity Carbon Nanofiber. Materials 2012, 5, 1560-1580. Table 1 Electrochemical parameters of the uncoated Ti, PCL/M-HAP coated Ti and different concentrations of CNF/PCL/M-HAP composite coated Ti in SBF solution. Samples

Ecorr (V vs. SCE)

icorr (µA/cm2)

Rp (kΩ cm2)

Uncoated Ti

-0.57±0.005

0.54±0.003

52.4±0.06

PCL/M-HAP coated Ti

0.07±0.003

0.08±0.006

115.7±0.04

1 wt. % CNF/PCL/M-HAP coated Ti

0.188±0.006

0.01±0.002

230.6±0.02


0.210±0.004

0.006±0.006

257.4±0.02


0.198±0.001

0.007±0.002

245.3±0.03


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Fig. 1

The FT-IR spectra of (a) PCL/M-HAP and CNF/PCL/M-HAP composite coatings with different concentrations of CNF (b) 1 wt. %, (c) 2 wt. % and (d) 3 wt. %.



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Fig. 2

The XRD patterns of (a) PCL/M-HAP coating and CNF/PCL/M-HAP composite coatings on titanium at different concentrations of CNF (b) 1 wt. %, (c) 2 wt. % and (d) 3 wt. %.


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Fig. 3 HRSEM images of (a) Mn-HAP, (b) Ga-HAP, (c) PCL, (d) PCL/M-HAP coatings on Ti.



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Fig. 4

HRSEM images of CNF/PCL/M-HAP composite coatings on titanium at different concentrations of CNF (a) 1 wt. %, (b) 2 wt. %, (c) 3 wt. % and (d) enlarged image of 2 wt. % CNF/PCL/M-HAP composite coatings on Ti.


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Fig. 5 EDAX mapping images of elements Ca, Mn, Ga, P, C and O of 2 wt. % CNF/PCL/M-HAP composite coating.



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Fig. 6

HRTEM images of CNF/PCL/M-HAP composite coatings at different concentrations of CNF (a) 1 wt. %, (b) 2 wt. %, (c) 3 wt. %.


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Fig. 7 Potentiodynamic polarisation curves of uncoated Ti, PCL/M-HAP, CNF/PCL/MHAP with different CNF concentrations (1, 2 and 3 wt %) coated Ti samples in SBF solution.



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Fig. 8

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Equivalent circuits for (a) uncoated Ti, (b) composite coated Ti and (c) Nyquist plots for uncoated Ti, PCL/M-HAP, CNF/PCL/M-HAP with different CNF concentrations (1, 2 and 3 wt. %) coated Ti samples in SBF solution.


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Fig. 9

Adhesion strength and Vickers micro-hardness of PCL/M-HAP, CNF/PCL/M-HAP composite coatings on titanium at different concentrations of CNF.



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Fig. 10

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Plots showing antibacterial activity of M-HAP, PCL/M-HAP and CNF/PCL/M-HAP composite coatings at 1, 2 and 3 wt. % of CNF against (a) S. aureus, (b) E. coli bacteria and (c) photographs of zone of inhibition of 2 wt. % CNF/PCL/M-HAP composite coating against S. aureus and E. coli at different volume concentrations of the coatings.


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Fig. 11 In vitro cytotoxicity results of CNF/PCL/M-HAP composite coatings at different concentrations of CNF on HOS MG63 cells for 1, 4 and 7 days of incubation.



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Fig. 12 Fluorescent microscopic images showing the viability of HOS MG63 cells on 2 wt. % CNF/PCL/M-HAP coatings for (a) 1, (b) 4 and (c) 7 days of incubation.


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Fig. 13 Toulidene blue stained sections from the uncoated Ti-group I (a,b), PCL/M-HAP coated Ti-group II (c,d) and 2 wt. % CNF/PCL/M-HAP composite coated Ti-group III (e,f), taken after 2 and 4 weeks of implantation.



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Formation of nanofibrous scaffold (CNF/PCL/M-HAP) on titanium for improved corrosion resistance, mechanical properties and biocompatibility 177x117mm (96 x 96 DPI)

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Mineralized Hydroxyapatite

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