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Pectin mediated synthesis of nano hydroxyapatite-decorated poly(lactic acid) honeycomb membranes for tissue engineering
Accepted Manuscript Title: Pectin mediated synthesis of nano hydroxyapatite-decorated poly(lactic acid) honeycomb membranes for tissue engineering Authors: A. Shebi, S. Lisa PII: DOI: Reference:
S0144-8617(18)30910-X https://doi.org/10.1016/j.carbpol.2018.08.012 CARP 13909
To appear in: Received date: Revised date: Accepted date:
12-5-2018 28-7-2018 4-8-2018
Please cite this article as: Shebi A, Lisa S, Pectin mediated synthesis of nano hydroxyapatite-decorated poly(lactic acid) honeycomb membranes for tissue engineering, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.08.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pectin mediated synthesis of nano hydroxyapatite-decorated
A. Shebi a and S. Lisa*a
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Soft Materials Research Laboratory, Department of Chemistry, National Institute of Technology, Calicut, India 673601. E-mail: [email protected]
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Graphical abstract
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poly(lactic acid) honeycomb membranes for tissue engineering
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Highlights
Pectin from bitter gourd as template for the synthesis of nanohydroxyapatite
Fabrication of breath figure patterned H-PLA/nHAp nanocomposites
Nanocomposites were anti-cancerous and cytocompatible
H-PLA/nHAp - efficient for bone regeneration
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Abstract The fabrication of porous films with bioactive nanoparticles has been arousing interest in nanobiotechnology. The biocompatible nanocomposite membrane could mimic the functions of 1
basement membrane besides the augmented cell functions including adhesion, spreading, proliferation, and differentiation. We have reported the green template synthesis of nano hydroxyapatite (nHAp) using pectin from bitter gourd fruits followed by the fabrication of nHAp (2 wt%, 4 wt%, and 6 wt%) incorporated honeycomb-like poly(lactic acid) (PLA) films to evaluate the effect of nHAp on the surface patterning of PLA films. The use of naturally
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available bitter gourd with pectin content could efficiently stabilize or envelop the nanoparticles after nucleation process, resulting in reduced particle size. The cell viability over normal cells and cancer cells were evaluated for H-PLA/nHAp films. The present work recommends the biofriendly surface engineering of PLA films with the assistance of nHAp particles for bone tissue engineering.
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Keywords: Pectin, Chelating agent, Hydroxyapatite, Poly(lactic acid), Honeycomb-like, tissue
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engineering.
1. Introduction
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Polymeric biomaterials have been extensively used over the past few years and are found to have
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a scope to build on the novelty in different disciplines (Sahoo, Praveen, Panda, 2007; Nicolas, Peter, 2013). The modulation of their morphology and properties by incorporating various chemical structures and functional molecules evaluates the performance of the biopolymer,
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especially in biomedical field. The biodegradable, biocompatible, mechanically and thermally stable biopolymers such as poly (glycolic acid) (PGA), poly (ethylene glycol) (PEG), poly(lactic
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acid) (PLA) (Yang et al., 2004; Kim, Lee & Knowles, 2006) and poly (caprolactone) (PCL) (Armentano et al., 2010; Rochina, Vidaurre, Cortazar & Lebourg, 2015) enables the
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decomposition by microorganisms (in the presence of suitable fillers) contributing towards environment friendliness. Among this group of polymers, PLA was widely used in bone fixation, surgical suture, tissue engineering and drug delivery (Zhang & Ma, 2004; Hong et al., 2005, Yang et al., 2009; Nasongkla et al., 2004). The surface properties, morphology and mechanical strength greatly influence the application of biomaterials in tissue engineering. The surface topography on substrates like honeycomb pores 2
can tune the cell functions like cell adhesion and proliferation (Wu & Wang, 2012). The impact of honeycomb pores on cell functions was known to depend on the hydrophilicity, size and distribution of pores (McMillan et al., 2007; Wang et al., 2010). Surface patterning of PLA can be done through various techniques to create pores for better cell properties. Breath figure technique, a facile and low cost approach can be implemented for the fabrication of honeycomb
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patterned membranes (Bunz, 2006). The process involves the evaporation of a highly volatile solvent such as chloroform, dichloromethane and carbon disulphide from the polymer solution. The rapid evaporation of solvent causes a subsequent reduction in atmospheric temperature, leading to the condensation of water vapor in the humid atmosphere. The water droplets condensed on the polymer surface get ordered to form arrays and the complete evaporation of solvent and water droplets form porous textured membranes. The stabilization of condensed
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water droplets could be achieved by the incorporation of hydrophilic materials into the PLA
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(Rarima, Asaletha & Unnikrishnan, 2018). The integration of hydrophilic nano hydroxyapatite (nHAp), which has similarity towards bone composition, can enhance the cell functions (Chen et
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al., 2014).
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Hydroxyapatite is known to be a ceramic component with excellent biodegradability and biocompatibility. It is widely used in tissue engineering, as the Ca2+ ions present in the nHAp can
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react with the carboxy group in aminoacids, proteins etc. in the human body (Vila, Salcedo &
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Regi, 2012; Fernandez-Yague et al., 2014). The synthetic methods for nHAp involve sol-gel method, hydrolysis, precipitation, co-precipitation method and can also be extracted from sea shells, animal bones and so on. The synthetic methods utilize toxic organic solvents for obtaining
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nanosized nHAp. The nHAp synthesized in plant extract medium are eco-friendly and can be undoubtedly used in biomedical field. The bio-mediated synthesis is advantageous over the
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chemical methods as they involve low cost, eco friendly synthesis and bulk production, without toxic influence. There are many recent reports on the green template assisted synthesis of
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hydroxyapatite (Christopher, George, Won & Jonathan, 2013; Sundrarajan et al., 2015). But we emphasized a novel synthesis route for nHAp by using renewable pectin extract from bitter gourd fruit, in which pectin act as a chelating agent and stabilize the nHAp particles. Bitter gourd, being a food and medicine contain pectin, vitamins, minerals, flavanoids, saponins, peptides and phenolic groups. Among this, pectin is a biodegradable polysaccharide that could promote wound healing at tissue sites. The pectin content could improve the cell proliferation 3
and are widely used for the medicinal applications (Kokkonen et al., 2008). In extracellular mineralization, the pectin with functional sites such as carboxyl and hydroxyl groups could promote the binding of calcium ions (Ca2+) from the solution to form carboxylate ions, which initiates the crystal nucleation and growth (Gilbert, Abrecht & Frazer, 2005). Gopi et al. (Gopi, Bhuvaneshwari, Indira & Kavitha, 2013) utilized sucrose and tartaric acid as green templates for
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the synthesis of nHAp nanoparticles.
The cell response by the honeycomb textured pure polymers (without fillers) has been reported recently. Abdal-hay et al. (Abdal-hay, Pant & Lim, 2013) proposed electrospun hydroxyapatite/nylon-6 scaffolds for bone tissue engineering. Zhang et al. (Zhang, Liao & Cui, 2003) have successfully prepared a nanohydroxyapatite/collagen nanocomposite that could
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mimic the natural bone. The response of metal nanoparticles, ceramics, polymers and nanocomposites towards osteoblast was reviewed by Tran et al. (Tran & Webster, 2009). A
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novel route was adopted by Sinha et al. (Sinha, Mishra & Ravishankar, 2009) to synthesise
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nanohydroxyapatite/poly(vinyl alcohol) microspheres for biomedical applications. There are
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plenty of other reports on the biological or cell reponse of hydroxyapatite, which could mimic the natural bone composition (Rogel, Qiu & Ameer, 2008). The size of hydroxyapatite in the
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current work, were efficiently controlled by pectin extract, however the size reduction was not
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much effective in previous reports (Gergley et al., 2010; Han et al., 2004). In the present work, we aimed to prepare PLA composites with porous texture controlled by pectin-stabilized nanohydroxyapatite. To the best of our knowledge, there are no reports on the
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synthesis of nHAp nanoparticles using pectin derived from bitter gourd fruits as a green template. The present work mainly focuses on the incorporation of hydrophilic nHAp
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(synthesized with the assistance of pectin) into hydrophobic PLA for the generation of ordered pores on the membrane with the aid of breath figure method. The cell response by nHAp has
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been investigated using both normal cells and cancer cells. The honeycomb patterned PLA membrane with biocompatible nHAp was evaluated for the application as a scaffold in bone tissue engineering. 2. Materials & Methods 2.1 Materials
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Polylactide (PLA) (molecular weight = 160,000), CaCl2.2H2O (99.98%) and (NH4)2HPO4 were purchased
from
Sigma-Aldrich
(USA),
isopropyl
alcohol,
hydrochloric
acid
and
dichloromethane (DCM, 99.0%) was procured from Merck (Germany). Bitter gourd fruits were collected from a local area in Calicut, Kerala. The obtained chemicals were of analytical grade and were used without further purification.
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2.2. Extraction of pectin from plant extracts (GP).
The capping agent for the nanoparticle synthesis, i.e., green template, pectin (GP) was isolated from the fruit extract by the reported method (Khule et al., 2012). The fresh bitter gourd fruits (10g) were washed to remove impurities and were dried at 37 oC overnight. The dried fruits were suspended in 50 mL of deionized water (solid–liquid ratio 1:30, w/v), and the pH (around 2) was adjusted by conc. HCl. The mixture was refluxed for 1 h and the suspension was centrifuged and
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the supernatant solution was filtered and collected. To the filtrate, 50 mL isopropyl alcohol was
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added (filtrate–isopropyl alcohol ratio 1:1, v/v) dropwise to precipitate out pectin. The pectin was collected, washed and dried in vacuum to utilize as stabilizing agent in the synthesis of PLA
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nanoparticles. The functional groups of pectin extracted from the bitter gourd fruits were
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identified by FT-IR & NMR characterization and the spectra is shown in Fig.S1 & Fig.S2. 2.3. Synthesis of crystalline nHAp nanoparticles using GP as a green template
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For the synthesis of nHAp particles, the precursors CaCl2.2H2O and (NH4)2HPO4 in the molar
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ratio of 1.667 (Ca2+/PO43-) was mixed with 50 mL of 0. 2 wt% GP in a 250 mL beaker and was stirred for about 1 h at RT. The pH 10 of the extract-precursor mixture was adjusted using aqueous ammonia solution. The solution was initially heated at 90 oC for about 2 h under
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constant stirring. Further, the reaction was brought to 80 oC and was kept overnight for 12 h. The milky white precipitate was obtained as a sign of completion of the reaction. The product was
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dispersed in ethanol to avoid agglomeration. The residue was collected by centrifugation and was washed multiple times with ethanol and distilled water to avoid impurities. The as-synthesized
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particles were calcined at 750 ºC for 8 h to obtain pure nHAp particles. For the sake of comparison, the nHAp particles were synthesized also in the absence of pectin. 2.4. Fabrication of honeycomb-like porous films For the preparation of membranes, the formulations are as follows: (i) neat PLA-2.5 wt% PLA(w/v) was dissolved in DCM (ii) H-PLA/nHAp nanocomposites- 4 different samples by varying nHAp amount(w/w-0 wt%, 2 wt%, 4 wt% and 6 wt%) were separately dispersed in 5
DCM solution by using probe sonicator for 30 min. To the solution (DCM/nHAp), PLA-2.5 wt% was added and was stirred for 6 h at room temperature to obtain the PLA/nHAp/DCM solution. The honeycomb morphology was obtained on the membranes by breath figure method at a RH of 90% and an air flow rate of 50 mLmin-1. The porous membranes were vacuum dried to completely remove the solvent and water droplets.
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2.5. Characterization
The pectin extracted from bitter gourd was characterized by using a Fourier transform infrared spectrometer (JASCO 4700) in ATR-FTIR mode. The pectin was further confirmed by 1H NMR technique. About 5 mg of dry pectin sample was dissolved in 99.8% D2O (Sigma) at 10 mg/mL concentration and analysed using a Bruker proton NMR (500 MHz) at 80 °C. The particle size
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and morphology of nHAp was initially optimized using field-emission scanning electron microscope (FE-SEM; Hitachi Su 66000) and further characterized by transmission electron
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microscopy (TEM, JEM-2010) to obtain the average particle size. The pores obtained by breath
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figure method on H-PLA and H-PLA/nHAp nanocomposite films were analysed using FE-SEM.
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An image J analysis software (image J 1.50i) was used to measure the pore diameter from different zones of the micrographs (SEM). The elemental composition in nHAp was estimated by
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energy-dispersive spectroscopy (EDS) after coating the sample with gold. Average particle size of nHAp in aqueous suspension was measured using dynamic light scattering technique
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(Malvern Instruments, Southborough, MA, USA). The confirmation of homogeneity in the composition of nHAp was done by monitoring the X-ray diffraction pattern and further the
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crystallinity was also analysed from X-ray diffraction (Rigaku Miniflex 600) technique performed with a 2-theta scan from 10° to 80°. Schimadzu Autograph, AG-Xplus series was
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used to obtain the tensile strength and % of elongation of H-PLA and H-PLA/nHAp nanocomposites with an applied load of 10 N and a crosshead speed of about 10 mm/min. The static water contact angles of the H-PLA and H-PLA/nHAp films were measured at room
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temperature and 60% relative humidity, using standard goniometer (DIGIDROP Hamburg, Romans sur Isere, France) for evaluating the hydrophilicity. 2.6. Hemolysis of honeycomb PLA nanocomposites. The hemolytic assay was carried using fresh human blood to detect the amount of released hemoglobin on direct contact with the drug loaded systems. The blood was initially diluted with 6
0.9% saline to prepare a stock solution. The honeycomb-like mats with 6mm diameter was immersed in 10 mL of 0.9% saline and was incubated for 30 min at 37 °C. The content was again incubated for 60 min at 37 °C, after adding 0.2 mL of the stock solution. 0.9% saline solution was considered as the negative control and distilled water as positive control. All the solutions were then incubated in a shaker incubator at 37 oC for 2 h. Followed by incubation, the samples
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were centrifuged 3000 rpm for 10 min and the absorbance of supernatant solution at 540 nm was determined (Shebi, Sanjeev & Lisa, 2017). The extent of hemolysis by the samples on direct contact with blood was determined by the following equation: OD of test sample−OD of (−Ve) control
HP = OD of (+Ve)control−OD of (–Ve) control 100……………(1)
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Where OD stands for optical density or absorbance. 2.7. Cytotoxicity (normal cell)
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The cytotoxicity of the membranes was evaluated using L929 mouse fibroblast cell culture as per
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ISO10993-5. Initially the sterilized membranes were placed on cells seeded on a 48 well plate.
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The surviving cells percentage was evaluated by MTT assay and the morphology of cells after treatment was observed using phase contrast microscopy. During the MTT assay, the tetrazolium salt (MTT) gets reduced to formazan crystals in the presence of enzyme succinate dehydrgenase,
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which can be visualized by a colour change from yellow to purple. The intensity of purple colour
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is proportional to the cell viability. Followed by this reaction, the culture was again sterilized and was treated with 200 µL MTT solution per mL culture. The culture was then incubated at 37 oC
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for 3 h and was treated with 300 µL DMSO (for each well). After the cell lysis was completed, the centrifuged solution was checked for the absorbance. The absorbance (A) at 540 nm of the
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the cell treated MTT solution with sample (test) and without sample (control) was measured (Shebi & Lisa, 2018).
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The % viability was calculated as follows, A of test
% Viability = A of control 100…………….(2)
2.8. Cytotoxicity (Cancer cell) The toxicity towards cancer cells were examined using HeLa (Cervical carcinoma) cells procured from National Centre for Cell Sciences (NCCS), Pune, India. 100 µL of cell suspension 7
(5x104 cells/well) was seeded on 96 well plate and was incubated. The MTT assay was carried out as per the procedure mentioned before. The increase in intensity of purple colour obtained after reduction shows the enhanced cell viability. The absorbance (A) at 540 nm for the test solution and control (without sample) was measured. The % viability was calculated using equation (1). The change in morphology of cells on contact with material (with respect to
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control) was evaluated under inverted phase contrast microscope (Olympus CKX41) attached with an imaging camera (Suga et al., 2017).
3. Results & discussion 3.1. Possible mechanism
The preparation of GP stabilized nHAp involves the following mechanism and is demonstrated in Fig.1. Initially, calcium ions (Ca2+) were chelated with carboxyl group of GP at pH 10. The
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reactive carboxyl group in pectin molecules electrostatically interact with the Ca2+ ions to form
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calcium pectinate (Ca2+-- COO--) complex (Gopi et al., 2014). The Ca2+ ions get immobilized in
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the pectin surface and promote nucleation process. The slower addition of (NH4)2HPO4 solution to the chelate complex causes the nucleation of GP/nHAp, through the ionic interaction of
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phosphate (PO43-) ions with Ca2+ ions in calcium pectinate. The pure nHAp particles were obtained by the calcinations of nHAp/GP composite at 750 ºC for 8 h. The mechanism provides
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an insight into the formation of nHAp on the polymeric backbone of pectin extracted from bitter
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gourd fruits.
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Fig.1. Mechanistic pathway for pectin assisted synthesis of nHAp. 3.2. Field emission-scanning electron microscope analysis The effect of GP in the extract on the formation of nHAp was studied by morphological comparison of the samples synthesized in the absence and presence of GP and are shown in Fig.2. The sample, nHAp/GP had fine aggregated particles with irregular morphology and
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reduced size of about 35–55 nm (Fig.2b). However, the bare nHAp particles had a highly agglomerated and an unresolved morphology. This observation reveals the morphological effect by GP, arising from the interaction of carboxyl group in GP with Ca2+ ions in the solution that can effectively control the growth of nHAp by forming a protective layer even after nHAp formation. The obtained morphology obtained in Fig.2a for nHAp might be due to the possible agglomeration in the absence of GP. The DLS results indicate that the average particle size of
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nHAp synthesized in the presence and absence of GP were found to be 65 nm and 418 nm
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respectively (Fig.S3). The nano dimension of the particles contributes large surface area for the
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cell growth.
chelating agent (GP).
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Fig.2. SEM micrograph of nHAp nanoparticles prepared (a) without chelating agent (b) with
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Fig.3. EDX plot of biosynthesized nHAp using GP as a chelating agent.
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The elemental mapping of the sample was done using EDAX analysis and the spectrum obtained is shown in Fig.3. The elements C,O,P and Ca was successfully detected, validating the presence
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of pectin along with the hydroxyapatite particles in nHAp/GP. The Ca/P weight ratio was
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calculated and was found to 1.66 for the sample nHAp/GP, which is consistent with the previous
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reports (Tlotleng, Akinlabi, Shukla & Pityana, 2014), and was found to be close to the Ca/P ratio in natural bone.
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3.3. Nanoscale surface analysis (HRTEM)
Further morphological analysis using TEM validated that the GP assisted synthesis of nHAp
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particles had no agglomeration, due to the chelating effect of the former. Fig.4a shows the magnified image of GP modified nHAp dried at 100 °C. The average particle size for
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nHAp/GP was found to be 43.21±9.59 nm. The GP was found to control the size and growth of
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nanoparticles (Gopi et al., 2014).
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Fig.4. (a) TEM images of hydroxyapatite used in this study: (b) corresponding size distribution histogram.
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3.4. X-ray diffraction analysis of nHAp/GP
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The synthesized nHAp particles in the absence and presence of GP were subjected to XRD
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analysis and the respective patterns are shown in Fig.5a. The XRD patterns of extract free nHAp are in good agreement with the ICDD card No. 09-0432. The 2θ values at 17.0º, 27.8º and 34.4º
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reveals the existence of tricalcium phosphate (TCP) according to ICDD card No. 09-0169. Mixed phases of calcium phosphate were observed in nHAp particles synthesized in the absence
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of fruit extract, as evident from the spectrum.
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The nHAp/GP crystallinity was verified using XRD analysis and the patterns confirm that GP does not cause any crystalline phase transformation. The GP restricts the crystal growth as well
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as prevents the aggregation at initial stages and forms a stable complex, leading to a transformation from amorphous phase to crystalline phase. The peaks for nHAp were at 2θ:
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25.85, 28.81, 31.57, 32.85, 34.16, 39.88, 46.83, 48.35, 50.38 and 53.05, respectively corresponds to the crystalline planes (002), (210), (211), (300), (202), (310), (333), (312), (321) and (004),which is congruent with the International Centre for Diffraction DataNo.09 0432 (ICDD)
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for nHAp (Mobasherpour, Heshajin, Kazemzadeh & Zakeri, 2007; Ejaz, Joachim, Friedrich & Michael, 2012; Sych et al., 2014).
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Fig.5 (a) XRD pattern and (b) FTIR spectra of nHAp synthesized in the presence and absence of GP.
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The observation signifies that the GP has a crucial role in improving the purity of nHAP
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particles. The XRD pattern in Fig.5a confirms that the crystallinity of nHAp particles was not
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altered on capping with GP. 3.5. FT-Infrared spectroscopic analysis
The FT-IR analysis was performed to identify the functional groups present in the samples. The
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vibrational frequencies of phosphate, carbonate and amide groups were monitored for the
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confirmation of produced nHAp particles. The FT-IR spectra of nHAp synthesized in the presence and absence of GP is shown in Fig.5b. The pectin peaks were not found in the spectrum
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for nHAp/GP particles, showing the effectiveness of calcination to obtain pure nHAp. The tricalcium phosphate (TCP) was normally found in the nHAp particles synthesized without GP
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(Gopi et al., 2008). The extract has obtained vibrational peaks corresponding to C-N stretching for aliphatic amines, C-H aromatic, C=C stretching and O-H stretching at 1050, 1380, 1638 and 3350 cm -1 (David et al., 2014). A careful investigation was on the peaks obtained for synthesized
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nHAp particles. For pure nHAp synthesized without GP, the hydroxyl stretching and bending frequencies are 3560 and 630 cm-1. The triply degenerate asymmetric and non degenerate symmetric stretching vibrational modes of phosphate groups are obtained at (1093 cm-1, 1036 cm-1) and 961 cm-1 respectively. The peaks at 605 and 568 cm-1 could be attributed to the doubly degenerate bending mode of the P-O bond and 475 cm-1 is also due to the doubly degenerate bending mode of the same group. All the observed vibrational frequencies confirm the formation 12
of nHAp. The peaks at 870 and 2358 cm-1 corresponds to the stretching frequency of phosphate group, indicating the presence of TCP in nHAp synthesized in the absence of GP. The retention of same pattern with minor changes was obtained for nHAp/GP and the absence of peak at 870 and 2358 cm-1 shows that the TCP was not formed (Gopi et al., 2010). 3.6. Morphology of honeycomb-like PLA/nHAp nanocomposites.
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The TEM image and the size distribution of the green synthesized nHAp particles displayed an average particle size around 43 nm and were found to have good dispersing ability in solvents. Further, the nHAp/GP particles are dispersed in PLA-DCM solution to fabricate honeycomb-like structured films. The influence of nHAp for the formation of pores were studied by varying the
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amount of nHAp (0 wt%, 2 wt%, 4 wt%, and 6 wt%) in PLA.
Fig.6. SEM images of honeycomb PLA films with incorporation of various nHAp contents
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(0 wt%, 2 wt%, 4 wt%, and 6 wt%) and the average pore size corresponding to the SEM images in the same row.
It was observed that a significant influence was exerted by nHAp for the pore formation. A
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regular distribution of pores on PLA film was attained on increasing the content of nHAp from 0 wt% to 4 wt% and the pattern quality was lowered on increasing the content to 6 wt%. The
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morphological images scanned using SEM shows that the incorporation of nHAp into PLA contributes porous patterning on the PLA membrane as observed in Fig.6. The distribution diagram (Fig.S4) shows that the average pore diameter of the PLA composites H-PLA, H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp are 2.5±1.7 μm, 6.2±3.5 μm 12.3±2.5 μm and 8.5±5.9 μm respectively. The % of porosity of H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp composites are 15%, 33%, 48% and 25% respectively. The porosity can 13
improve cell growth on the composite membranes. The homogeneous dispersion of nHAp particles plays an inevitable role in stabilizing the water droplets condensed on the polymer surface during breath figure formation (Wu & Wang, 2012; Zhao, Zhang, Wang & Li, 2006). The optimized amount for uniform dispersion of the particles was 4%, on further increase in content to 6%, nHAp precipitation was observed even on ultrasonication. This excess content of
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nHAp would result in self agglomeration rather than stabilizing the condensed water droplets. This might affect the regularity in pore patterning as well as the pore size that is clearly evident in the Fig.6d. The agglomeration of nHAp leading to uneven pores on the membrane with excess 6% nHAp inclusion is shown in Fig.S5. The higher density of water droplets compared to that of DCM, allows the penetration of droplets deep into the membranes resulting in multi-layer pores. 3.7. Mechanical property
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The extent to which the mechanical properties are altered for a polymer, PLA on incorporation
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with nHAp is examined. The tensile strength and % of elongation of the samples: H-PLA, H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp are shown in Fig.7. Tensile strength of
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F-PLA and H-PLA reference were 38.93±1.45 and 27.32±1.58 MPa and that for H-PLA/2-
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nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp composites were 30.94±1.66, 33.56±1.71 and 30.81±1.58 MPa, respectively. It is obvious that the tensile strength of F-PLA to be higher than
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that of nanocomposites. However, the reinforcing effect for the polymer is observed only upto a
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certain filler content, here it is upto 4%. On account of poor filler-polymer interaction with further addition of nHAp (filler) causes retardation in mechanical strength (Garoushi, Lassila & Vallittu, 2011). As a result of such poor interaction of polymer with excess nHAp, H-PLA/6-
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nHAp composite had reduced mechanical properties, when compared to H-PLA/2-nHAp and H-PLA/4-nHAp composites. The crystallinity could be suggested as a factor to contribute the
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mechanical strength, i.e., high crystallinity is positive towards imparting tensile strength to PLA (Zhao et al., 2016). The increase in tensile strength of H-PLA/nHAp composites could be due to
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their enhanced hardness with the increment of nHAp content. The elongation at break was found to be 4.91± 0.42% for H-PLA/2-nHAp, 8.18±0.39% for H-PLA/4-nHAp and 2.18±0.32% for H-PLA (Fig.7b), which implies that the ductility of H-PLA/nHAp composite is significantly improved on comparison with H-PLA. It is known that the improvement in ductility causes the delay in fracture, which declines the abrupt failure for the H-PLA/nHAp nanocomposites.
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Fig.7. (a) Stress-strain curves (b) Tensile strength-percentage of elongation of F-PLA, H-PLA, H-PLA/2-nHAp, H-PLA/4-nHAp, and H-PLA/6-nHAp.
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The presence of excess nHAp particles in the PLA matrix may generate voids during the
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elongation that leads to easy breakage and rupture of bonds, resulting in the reduction of
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elongation at break to 7.23± 0.41% for H-PLA/6-nHAp. 3.8. Wettability of films surface (Contact angle) For the application of samples in biomedical field, the hydrophilic behaviour was evaluated by
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water contact angle measurements. Fig.8. shows the contact angle images of the samples and the
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results are summarized in Table 1 (Fig.S6). The wettability is greatly influenced by the surface topography of the membrane.
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Even though H-PLA/nHAp films and bare PLA films had similar topography, a hydrophilic contact angle of about 49º was attained for nHAp incorporated (Gong et al., 2017) PLA films.
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This was due to the presence of hydrophilic nHAp particles in the matrix. The water droplets are evenly distributed and stabilized by the nHAp particles in the films instead of improving the formation of air pockets to induce hydrophobicity. During contact angle measurement, the water
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dropped on the surface penetrates into the pores to reduce the contact angle between membrane surface and the water droplet. The intention to create hydrophilic membranes was mainly to enhance the adhesion, spreading and proliferation of cells and the hydrophilicity could be achieved by adding hydrophilic inorganic materials.
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Fig.8. Contact angle images of the samples (a) F-PLA; (b) H-PLA (c) H-PLA/2-nHAp (d) H-PLA/4-nHAp and (e) H-PLA/6-nHAp.
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In addition to the hydrophilicity, film roughness can also cause better cell adhesion and the
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results claim that all the membranes pose a surface with better cell attachment in the bone tissue.
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3.9. Hemolysis
Hemolysis assay is considered as a complementary study towards the biocompatibility of the membranes. The blood compatability of biomaterials proposing for use in tissue engineering is a
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critical issue to be addressed. The hemocompatibilty of the scaffolds was evaluated with respect
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to a negative control (normal saline) and a positive control (double distilled water). Hemocompatibility of the porous nanocomposites were investigated and are shown in Fig.S7.
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The hemolytic percentage (HP) of each scaffold was evaluated from the absorbance obtained at 540 nm. For bare F-PLA and H-PLA film, HP% was found to be 0.38±0.11% and 0.59±0.15%
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respectively. It was hiked sequentially on increasing the nHAp content and the values were respectively 1.55±0.22%, 2.25±0.24% and 3.56±0.25% for H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp systems. All the membranes were found to be non-hemolytic with HP less
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than 5% as defined by American Society for Testing and Materials International (ASTM Standard, F756-08) (Radha, Balakumar, Venkatesan & Vellaichamy, 2015).
3.10. Cytotoxicity Cytotoxicity analysis for H-PLA/nHAp composites has been opted to compare the cell proliferation by the systems with normal cells and cancer cells. MTT assay was carried out using the human cervical cancer cell line (HeLa) and human normal cell line–Fibroblast (L929).The 16
studies show that the presence of n-HAp particles inhibited the growth of cancer cells and was found to increase in inhibition rate with increase in nHAp particles. The cervical cancer inhibitions for H-PLA, H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp were 4±1.2%, 47±2.4%, 63±2.5% and 56±2.2%, respectively. This observation indicates that the inhibition by nHAp depends upon the morphology of nHAp. The apoptosis of HeLa cells depends on the
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concentration of nHAp and the treatment time. The influence of particle size and dosage of nHAp particles for the inhibition of cancer cells are shown in Fig.9a. The nHAp particles contains positively charged Ca2+ ions to bind acid groups and negatively charged PO43- ions to attract basic groups of biomolecules. The sialic acid residues in plasma membranes render more negative charge on cancer cells than normal cells which could be adsorbed by nHAp nanoparticles. This high adhering capacity of nHAp derives from the electrostatic attraction
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between negative charges on cancer cells with positive binding sites in nHAp. The cancer cell
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inhibition after binding with nHAp could be attributed to the depletion of proteins required for
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cell cycle progression (Han, Wang, Dai & Li, 2012; Lieske, Norris & Toback, 1997).
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Fig.9. Effects of HAp on cell viability of (a) Cancer cell (HeLa) and (b) Normal cell (L929).
Intracellular response is greatly influenced by the morphology of scaffolds. The extent of cellscaffold interaction such as cell attachment and proliferation effects the cell differentiation. Calcium phosphate in nanophase with high surface area enhances the cell adhesion, proliferation and differentiation. Hence, the porous membranes containing nanostructured hydroxyapatite were also checked for its efficiency to use as substrates for the cell growth using L929 cells. The
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cells and ECM secreted by cells were found to be evenly spread on the scaffold surfaces and also penetrated inside the pores on the films. The MTT assay results (Fig.9b.) conveys that the cell adhesion improved on incorporation of nHAp particles in PLA matrix. The cell viability % of porous membranes: H-PLA, H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp with L929 cells were 78.9±3.06%, 84.1±3.5%, 90.8±2.1% and 84.2±3.2%. The composites does not
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cell proliferation by nHAp incorporated PLA membranes.
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indicate any remarkable toxicity towards mice fibroblast cells (L929), indicating the supportive
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Fig.10. Phase contrast image of H-PLA (a,e), H-PLA/2-nHAp (b,f), H-PLA/4-nHAp (c,g) and H-PLA/6-nHAp (d,h) nanocomposites cultured on (a-d) normal and (e-h) cancer cells for 3 days
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(Marked arrow indicate the nuclei fragmentation or apoptotic body formation). The porous surface contributed better cell adhesion and cell proliferation as demonstrated in
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Fig.10. The nanocomposite with 4 wt% nHAp loading (H-PLA/4-nHAp) induced better cell growth, when compared to the other two composites on evaluating for about 72 h. Fig.10. shows
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the phase contrast optical microscope images of H-PLA/4-nHAp nanocomposites seeded on L929 and HeLa cells. The images indicate that the H-PLA/4-nHAp composites inhibited the growth of cancer cells and exhibit higher viability towards the normal L929 cells. To conclude,
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the H-PLA/4-nHAp system suppresses the proliferation of cancer cells and promotes the spreading and proliferation of normal cells (Han et al., 2014). By careful investigation of the obtained results, H-PLA/4-nHAp was found to be highly biocompatible and could be employed as a feasible scaffold for the substitute of bone in the cancer patients.
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Conclusion The work was focused on the surface engineering of PLA membranes by nHAp particles synthesized novely by using pectin from bitter gourd extract medium. The honeycomb-like morphology obtained through breath figure patterning largely influenced the biological
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performance of the nanocomposites. The presence of GP controlled the size range of hydroxyapatite in nanoregime. The Ca/P weight ratio was calculated from EDAX analysis and was found to be 1.66. The XRD analysis confirms the formation of nHAp/GP without any crystalline phase transformation, but reveals the presence of tricalcium phosphate for nHAp synthesized in the absence of GP. The GP had a significant role in the formation of pure nHAp without any mixed phase of calcium phosphate. A regular distribution of pores on PLA film was attained on increasing the content of nHAp from 0 wt% to 4 wt% and the pattern quality was
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lowered on increasing the content to 6 wt%. A narrow pore size distribution was exhibited by the
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PLA/4-nHAp composite. The reinforcing effect for the polymer is observed only upto 4wt%
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filler incorporation; further addition resulted in poor polymer-filler interaction. The cytotoxicity studies using the human cervical cancer cell line – (HeLa) and human normal cell line–Fibroblast
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(L929) showed that the nanocomposites were highly viable towards the normal cells, while inhibiting the growth of cancer cells by 63±2.5%. The higher cell viability of the composite,
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PLA/4-nHAp was evident from the cytotoxicity test using normal cell line. The present study
Acknowledgements
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proposes the implementation of PLA/4-nHAp composite as a scaffold for bone regeneration.
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This research was financially supported by Ministry of Human Resource Development (MHRD), India. The authors thankfully acknowledge Dr. G. Unnikrishnan, Dr. A. Sujith, R. Rarima, P.K.
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Adnan and Jithin Raj, Department of Chemistry, NIT Calicut and K. Ramshad, Department of Chemistry, IIT Madras for their timely help. We extend our gratitude to Dr. Shibu and
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Krishnakumar (STIC, CUSAT) for their cooperation in recording TEM.
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Stability, 127(5), 2-10.
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5. Contact angle Fig.S6. Table 1 Contact angle of water on F-PLA, H-PLA and H-PLA/nHAp composite films and results are presented as mean for n = 3.
Sample
Contact
code
name
angle
F- PLA
72±7
b
H-PLA
124±11
c
H-PLA/2-nHAp
66±6
d
H-PLA/4-nHAp
53±4
e
H-PLA/6-nHAp
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Sample
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49±5
S0144-8617(18)30910-X https://doi.org/10.1016/j.carbpol.2018.08.012 CARP 13909
To appear in: Received date: Revised date: Accepted date:
12-5-2018 28-7-2018 4-8-2018
Please cite this article as: Shebi A, Lisa S, Pectin mediated synthesis of nano hydroxyapatite-decorated poly(lactic acid) honeycomb membranes for tissue engineering, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.08.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pectin mediated synthesis of nano hydroxyapatite-decorated
A. Shebi a and S. Lisa*a
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Soft Materials Research Laboratory, Department of Chemistry, National Institute of Technology, Calicut, India 673601. E-mail: [email protected]
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Graphical abstract
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poly(lactic acid) honeycomb membranes for tissue engineering
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Highlights
Pectin from bitter gourd as template for the synthesis of nanohydroxyapatite
Fabrication of breath figure patterned H-PLA/nHAp nanocomposites
Nanocomposites were anti-cancerous and cytocompatible
H-PLA/nHAp - efficient for bone regeneration
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Abstract The fabrication of porous films with bioactive nanoparticles has been arousing interest in nanobiotechnology. The biocompatible nanocomposite membrane could mimic the functions of 1
basement membrane besides the augmented cell functions including adhesion, spreading, proliferation, and differentiation. We have reported the green template synthesis of nano hydroxyapatite (nHAp) using pectin from bitter gourd fruits followed by the fabrication of nHAp (2 wt%, 4 wt%, and 6 wt%) incorporated honeycomb-like poly(lactic acid) (PLA) films to evaluate the effect of nHAp on the surface patterning of PLA films. The use of naturally
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available bitter gourd with pectin content could efficiently stabilize or envelop the nanoparticles after nucleation process, resulting in reduced particle size. The cell viability over normal cells and cancer cells were evaluated for H-PLA/nHAp films. The present work recommends the biofriendly surface engineering of PLA films with the assistance of nHAp particles for bone tissue engineering.
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Keywords: Pectin, Chelating agent, Hydroxyapatite, Poly(lactic acid), Honeycomb-like, tissue
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engineering.
1. Introduction
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Polymeric biomaterials have been extensively used over the past few years and are found to have
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a scope to build on the novelty in different disciplines (Sahoo, Praveen, Panda, 2007; Nicolas, Peter, 2013). The modulation of their morphology and properties by incorporating various chemical structures and functional molecules evaluates the performance of the biopolymer,
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especially in biomedical field. The biodegradable, biocompatible, mechanically and thermally stable biopolymers such as poly (glycolic acid) (PGA), poly (ethylene glycol) (PEG), poly(lactic
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acid) (PLA) (Yang et al., 2004; Kim, Lee & Knowles, 2006) and poly (caprolactone) (PCL) (Armentano et al., 2010; Rochina, Vidaurre, Cortazar & Lebourg, 2015) enables the
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decomposition by microorganisms (in the presence of suitable fillers) contributing towards environment friendliness. Among this group of polymers, PLA was widely used in bone fixation, surgical suture, tissue engineering and drug delivery (Zhang & Ma, 2004; Hong et al., 2005, Yang et al., 2009; Nasongkla et al., 2004). The surface properties, morphology and mechanical strength greatly influence the application of biomaterials in tissue engineering. The surface topography on substrates like honeycomb pores 2
can tune the cell functions like cell adhesion and proliferation (Wu & Wang, 2012). The impact of honeycomb pores on cell functions was known to depend on the hydrophilicity, size and distribution of pores (McMillan et al., 2007; Wang et al., 2010). Surface patterning of PLA can be done through various techniques to create pores for better cell properties. Breath figure technique, a facile and low cost approach can be implemented for the fabrication of honeycomb
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patterned membranes (Bunz, 2006). The process involves the evaporation of a highly volatile solvent such as chloroform, dichloromethane and carbon disulphide from the polymer solution. The rapid evaporation of solvent causes a subsequent reduction in atmospheric temperature, leading to the condensation of water vapor in the humid atmosphere. The water droplets condensed on the polymer surface get ordered to form arrays and the complete evaporation of solvent and water droplets form porous textured membranes. The stabilization of condensed
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water droplets could be achieved by the incorporation of hydrophilic materials into the PLA
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(Rarima, Asaletha & Unnikrishnan, 2018). The integration of hydrophilic nano hydroxyapatite (nHAp), which has similarity towards bone composition, can enhance the cell functions (Chen et
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al., 2014).
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Hydroxyapatite is known to be a ceramic component with excellent biodegradability and biocompatibility. It is widely used in tissue engineering, as the Ca2+ ions present in the nHAp can
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react with the carboxy group in aminoacids, proteins etc. in the human body (Vila, Salcedo &
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Regi, 2012; Fernandez-Yague et al., 2014). The synthetic methods for nHAp involve sol-gel method, hydrolysis, precipitation, co-precipitation method and can also be extracted from sea shells, animal bones and so on. The synthetic methods utilize toxic organic solvents for obtaining
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nanosized nHAp. The nHAp synthesized in plant extract medium are eco-friendly and can be undoubtedly used in biomedical field. The bio-mediated synthesis is advantageous over the
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chemical methods as they involve low cost, eco friendly synthesis and bulk production, without toxic influence. There are many recent reports on the green template assisted synthesis of
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hydroxyapatite (Christopher, George, Won & Jonathan, 2013; Sundrarajan et al., 2015). But we emphasized a novel synthesis route for nHAp by using renewable pectin extract from bitter gourd fruit, in which pectin act as a chelating agent and stabilize the nHAp particles. Bitter gourd, being a food and medicine contain pectin, vitamins, minerals, flavanoids, saponins, peptides and phenolic groups. Among this, pectin is a biodegradable polysaccharide that could promote wound healing at tissue sites. The pectin content could improve the cell proliferation 3
and are widely used for the medicinal applications (Kokkonen et al., 2008). In extracellular mineralization, the pectin with functional sites such as carboxyl and hydroxyl groups could promote the binding of calcium ions (Ca2+) from the solution to form carboxylate ions, which initiates the crystal nucleation and growth (Gilbert, Abrecht & Frazer, 2005). Gopi et al. (Gopi, Bhuvaneshwari, Indira & Kavitha, 2013) utilized sucrose and tartaric acid as green templates for
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the synthesis of nHAp nanoparticles.
The cell response by the honeycomb textured pure polymers (without fillers) has been reported recently. Abdal-hay et al. (Abdal-hay, Pant & Lim, 2013) proposed electrospun hydroxyapatite/nylon-6 scaffolds for bone tissue engineering. Zhang et al. (Zhang, Liao & Cui, 2003) have successfully prepared a nanohydroxyapatite/collagen nanocomposite that could
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mimic the natural bone. The response of metal nanoparticles, ceramics, polymers and nanocomposites towards osteoblast was reviewed by Tran et al. (Tran & Webster, 2009). A
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novel route was adopted by Sinha et al. (Sinha, Mishra & Ravishankar, 2009) to synthesise
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nanohydroxyapatite/poly(vinyl alcohol) microspheres for biomedical applications. There are
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plenty of other reports on the biological or cell reponse of hydroxyapatite, which could mimic the natural bone composition (Rogel, Qiu & Ameer, 2008). The size of hydroxyapatite in the
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current work, were efficiently controlled by pectin extract, however the size reduction was not
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much effective in previous reports (Gergley et al., 2010; Han et al., 2004). In the present work, we aimed to prepare PLA composites with porous texture controlled by pectin-stabilized nanohydroxyapatite. To the best of our knowledge, there are no reports on the
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synthesis of nHAp nanoparticles using pectin derived from bitter gourd fruits as a green template. The present work mainly focuses on the incorporation of hydrophilic nHAp
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(synthesized with the assistance of pectin) into hydrophobic PLA for the generation of ordered pores on the membrane with the aid of breath figure method. The cell response by nHAp has
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been investigated using both normal cells and cancer cells. The honeycomb patterned PLA membrane with biocompatible nHAp was evaluated for the application as a scaffold in bone tissue engineering. 2. Materials & Methods 2.1 Materials
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Polylactide (PLA) (molecular weight = 160,000), CaCl2.2H2O (99.98%) and (NH4)2HPO4 were purchased
from
Sigma-Aldrich
(USA),
isopropyl
alcohol,
hydrochloric
acid
and
dichloromethane (DCM, 99.0%) was procured from Merck (Germany). Bitter gourd fruits were collected from a local area in Calicut, Kerala. The obtained chemicals were of analytical grade and were used without further purification.
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2.2. Extraction of pectin from plant extracts (GP).
The capping agent for the nanoparticle synthesis, i.e., green template, pectin (GP) was isolated from the fruit extract by the reported method (Khule et al., 2012). The fresh bitter gourd fruits (10g) were washed to remove impurities and were dried at 37 oC overnight. The dried fruits were suspended in 50 mL of deionized water (solid–liquid ratio 1:30, w/v), and the pH (around 2) was adjusted by conc. HCl. The mixture was refluxed for 1 h and the suspension was centrifuged and
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the supernatant solution was filtered and collected. To the filtrate, 50 mL isopropyl alcohol was
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added (filtrate–isopropyl alcohol ratio 1:1, v/v) dropwise to precipitate out pectin. The pectin was collected, washed and dried in vacuum to utilize as stabilizing agent in the synthesis of PLA
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nanoparticles. The functional groups of pectin extracted from the bitter gourd fruits were
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identified by FT-IR & NMR characterization and the spectra is shown in Fig.S1 & Fig.S2. 2.3. Synthesis of crystalline nHAp nanoparticles using GP as a green template
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For the synthesis of nHAp particles, the precursors CaCl2.2H2O and (NH4)2HPO4 in the molar
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ratio of 1.667 (Ca2+/PO43-) was mixed with 50 mL of 0. 2 wt% GP in a 250 mL beaker and was stirred for about 1 h at RT. The pH 10 of the extract-precursor mixture was adjusted using aqueous ammonia solution. The solution was initially heated at 90 oC for about 2 h under
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constant stirring. Further, the reaction was brought to 80 oC and was kept overnight for 12 h. The milky white precipitate was obtained as a sign of completion of the reaction. The product was
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dispersed in ethanol to avoid agglomeration. The residue was collected by centrifugation and was washed multiple times with ethanol and distilled water to avoid impurities. The as-synthesized
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particles were calcined at 750 ºC for 8 h to obtain pure nHAp particles. For the sake of comparison, the nHAp particles were synthesized also in the absence of pectin. 2.4. Fabrication of honeycomb-like porous films For the preparation of membranes, the formulations are as follows: (i) neat PLA-2.5 wt% PLA(w/v) was dissolved in DCM (ii) H-PLA/nHAp nanocomposites- 4 different samples by varying nHAp amount(w/w-0 wt%, 2 wt%, 4 wt% and 6 wt%) were separately dispersed in 5
DCM solution by using probe sonicator for 30 min. To the solution (DCM/nHAp), PLA-2.5 wt% was added and was stirred for 6 h at room temperature to obtain the PLA/nHAp/DCM solution. The honeycomb morphology was obtained on the membranes by breath figure method at a RH of 90% and an air flow rate of 50 mLmin-1. The porous membranes were vacuum dried to completely remove the solvent and water droplets.
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2.5. Characterization
The pectin extracted from bitter gourd was characterized by using a Fourier transform infrared spectrometer (JASCO 4700) in ATR-FTIR mode. The pectin was further confirmed by 1H NMR technique. About 5 mg of dry pectin sample was dissolved in 99.8% D2O (Sigma) at 10 mg/mL concentration and analysed using a Bruker proton NMR (500 MHz) at 80 °C. The particle size
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and morphology of nHAp was initially optimized using field-emission scanning electron microscope (FE-SEM; Hitachi Su 66000) and further characterized by transmission electron
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microscopy (TEM, JEM-2010) to obtain the average particle size. The pores obtained by breath
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figure method on H-PLA and H-PLA/nHAp nanocomposite films were analysed using FE-SEM.
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An image J analysis software (image J 1.50i) was used to measure the pore diameter from different zones of the micrographs (SEM). The elemental composition in nHAp was estimated by
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energy-dispersive spectroscopy (EDS) after coating the sample with gold. Average particle size of nHAp in aqueous suspension was measured using dynamic light scattering technique
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(Malvern Instruments, Southborough, MA, USA). The confirmation of homogeneity in the composition of nHAp was done by monitoring the X-ray diffraction pattern and further the
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crystallinity was also analysed from X-ray diffraction (Rigaku Miniflex 600) technique performed with a 2-theta scan from 10° to 80°. Schimadzu Autograph, AG-Xplus series was
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used to obtain the tensile strength and % of elongation of H-PLA and H-PLA/nHAp nanocomposites with an applied load of 10 N and a crosshead speed of about 10 mm/min. The static water contact angles of the H-PLA and H-PLA/nHAp films were measured at room
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temperature and 60% relative humidity, using standard goniometer (DIGIDROP Hamburg, Romans sur Isere, France) for evaluating the hydrophilicity. 2.6. Hemolysis of honeycomb PLA nanocomposites. The hemolytic assay was carried using fresh human blood to detect the amount of released hemoglobin on direct contact with the drug loaded systems. The blood was initially diluted with 6
0.9% saline to prepare a stock solution. The honeycomb-like mats with 6mm diameter was immersed in 10 mL of 0.9% saline and was incubated for 30 min at 37 °C. The content was again incubated for 60 min at 37 °C, after adding 0.2 mL of the stock solution. 0.9% saline solution was considered as the negative control and distilled water as positive control. All the solutions were then incubated in a shaker incubator at 37 oC for 2 h. Followed by incubation, the samples
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were centrifuged 3000 rpm for 10 min and the absorbance of supernatant solution at 540 nm was determined (Shebi, Sanjeev & Lisa, 2017). The extent of hemolysis by the samples on direct contact with blood was determined by the following equation: OD of test sample−OD of (−Ve) control
HP = OD of (+Ve)control−OD of (–Ve) control 100……………(1)
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Where OD stands for optical density or absorbance. 2.7. Cytotoxicity (normal cell)
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The cytotoxicity of the membranes was evaluated using L929 mouse fibroblast cell culture as per
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ISO10993-5. Initially the sterilized membranes were placed on cells seeded on a 48 well plate.
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The surviving cells percentage was evaluated by MTT assay and the morphology of cells after treatment was observed using phase contrast microscopy. During the MTT assay, the tetrazolium salt (MTT) gets reduced to formazan crystals in the presence of enzyme succinate dehydrgenase,
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which can be visualized by a colour change from yellow to purple. The intensity of purple colour
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is proportional to the cell viability. Followed by this reaction, the culture was again sterilized and was treated with 200 µL MTT solution per mL culture. The culture was then incubated at 37 oC
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for 3 h and was treated with 300 µL DMSO (for each well). After the cell lysis was completed, the centrifuged solution was checked for the absorbance. The absorbance (A) at 540 nm of the
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the cell treated MTT solution with sample (test) and without sample (control) was measured (Shebi & Lisa, 2018).
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The % viability was calculated as follows, A of test
% Viability = A of control 100…………….(2)
2.8. Cytotoxicity (Cancer cell) The toxicity towards cancer cells were examined using HeLa (Cervical carcinoma) cells procured from National Centre for Cell Sciences (NCCS), Pune, India. 100 µL of cell suspension 7
(5x104 cells/well) was seeded on 96 well plate and was incubated. The MTT assay was carried out as per the procedure mentioned before. The increase in intensity of purple colour obtained after reduction shows the enhanced cell viability. The absorbance (A) at 540 nm for the test solution and control (without sample) was measured. The % viability was calculated using equation (1). The change in morphology of cells on contact with material (with respect to
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control) was evaluated under inverted phase contrast microscope (Olympus CKX41) attached with an imaging camera (Suga et al., 2017).
3. Results & discussion 3.1. Possible mechanism
The preparation of GP stabilized nHAp involves the following mechanism and is demonstrated in Fig.1. Initially, calcium ions (Ca2+) were chelated with carboxyl group of GP at pH 10. The
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reactive carboxyl group in pectin molecules electrostatically interact with the Ca2+ ions to form
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calcium pectinate (Ca2+-- COO--) complex (Gopi et al., 2014). The Ca2+ ions get immobilized in
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the pectin surface and promote nucleation process. The slower addition of (NH4)2HPO4 solution to the chelate complex causes the nucleation of GP/nHAp, through the ionic interaction of
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phosphate (PO43-) ions with Ca2+ ions in calcium pectinate. The pure nHAp particles were obtained by the calcinations of nHAp/GP composite at 750 ºC for 8 h. The mechanism provides
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an insight into the formation of nHAp on the polymeric backbone of pectin extracted from bitter
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gourd fruits.
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Fig.1. Mechanistic pathway for pectin assisted synthesis of nHAp. 3.2. Field emission-scanning electron microscope analysis The effect of GP in the extract on the formation of nHAp was studied by morphological comparison of the samples synthesized in the absence and presence of GP and are shown in Fig.2. The sample, nHAp/GP had fine aggregated particles with irregular morphology and
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reduced size of about 35–55 nm (Fig.2b). However, the bare nHAp particles had a highly agglomerated and an unresolved morphology. This observation reveals the morphological effect by GP, arising from the interaction of carboxyl group in GP with Ca2+ ions in the solution that can effectively control the growth of nHAp by forming a protective layer even after nHAp formation. The obtained morphology obtained in Fig.2a for nHAp might be due to the possible agglomeration in the absence of GP. The DLS results indicate that the average particle size of
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nHAp synthesized in the presence and absence of GP were found to be 65 nm and 418 nm
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respectively (Fig.S3). The nano dimension of the particles contributes large surface area for the
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cell growth.
chelating agent (GP).
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Fig.2. SEM micrograph of nHAp nanoparticles prepared (a) without chelating agent (b) with
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Fig.3. EDX plot of biosynthesized nHAp using GP as a chelating agent.
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The elemental mapping of the sample was done using EDAX analysis and the spectrum obtained is shown in Fig.3. The elements C,O,P and Ca was successfully detected, validating the presence
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of pectin along with the hydroxyapatite particles in nHAp/GP. The Ca/P weight ratio was
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calculated and was found to 1.66 for the sample nHAp/GP, which is consistent with the previous
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reports (Tlotleng, Akinlabi, Shukla & Pityana, 2014), and was found to be close to the Ca/P ratio in natural bone.
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3.3. Nanoscale surface analysis (HRTEM)
Further morphological analysis using TEM validated that the GP assisted synthesis of nHAp
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particles had no agglomeration, due to the chelating effect of the former. Fig.4a shows the magnified image of GP modified nHAp dried at 100 °C. The average particle size for
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nHAp/GP was found to be 43.21±9.59 nm. The GP was found to control the size and growth of
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nanoparticles (Gopi et al., 2014).
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Fig.4. (a) TEM images of hydroxyapatite used in this study: (b) corresponding size distribution histogram.
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3.4. X-ray diffraction analysis of nHAp/GP
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The synthesized nHAp particles in the absence and presence of GP were subjected to XRD
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analysis and the respective patterns are shown in Fig.5a. The XRD patterns of extract free nHAp are in good agreement with the ICDD card No. 09-0432. The 2θ values at 17.0º, 27.8º and 34.4º
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reveals the existence of tricalcium phosphate (TCP) according to ICDD card No. 09-0169. Mixed phases of calcium phosphate were observed in nHAp particles synthesized in the absence
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of fruit extract, as evident from the spectrum.
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The nHAp/GP crystallinity was verified using XRD analysis and the patterns confirm that GP does not cause any crystalline phase transformation. The GP restricts the crystal growth as well
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as prevents the aggregation at initial stages and forms a stable complex, leading to a transformation from amorphous phase to crystalline phase. The peaks for nHAp were at 2θ:
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25.85, 28.81, 31.57, 32.85, 34.16, 39.88, 46.83, 48.35, 50.38 and 53.05, respectively corresponds to the crystalline planes (002), (210), (211), (300), (202), (310), (333), (312), (321) and (004),which is congruent with the International Centre for Diffraction DataNo.09 0432 (ICDD)
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for nHAp (Mobasherpour, Heshajin, Kazemzadeh & Zakeri, 2007; Ejaz, Joachim, Friedrich & Michael, 2012; Sych et al., 2014).
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Fig.5 (a) XRD pattern and (b) FTIR spectra of nHAp synthesized in the presence and absence of GP.
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The observation signifies that the GP has a crucial role in improving the purity of nHAP
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particles. The XRD pattern in Fig.5a confirms that the crystallinity of nHAp particles was not
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altered on capping with GP. 3.5. FT-Infrared spectroscopic analysis
The FT-IR analysis was performed to identify the functional groups present in the samples. The
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vibrational frequencies of phosphate, carbonate and amide groups were monitored for the
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confirmation of produced nHAp particles. The FT-IR spectra of nHAp synthesized in the presence and absence of GP is shown in Fig.5b. The pectin peaks were not found in the spectrum
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for nHAp/GP particles, showing the effectiveness of calcination to obtain pure nHAp. The tricalcium phosphate (TCP) was normally found in the nHAp particles synthesized without GP
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(Gopi et al., 2008). The extract has obtained vibrational peaks corresponding to C-N stretching for aliphatic amines, C-H aromatic, C=C stretching and O-H stretching at 1050, 1380, 1638 and 3350 cm -1 (David et al., 2014). A careful investigation was on the peaks obtained for synthesized
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nHAp particles. For pure nHAp synthesized without GP, the hydroxyl stretching and bending frequencies are 3560 and 630 cm-1. The triply degenerate asymmetric and non degenerate symmetric stretching vibrational modes of phosphate groups are obtained at (1093 cm-1, 1036 cm-1) and 961 cm-1 respectively. The peaks at 605 and 568 cm-1 could be attributed to the doubly degenerate bending mode of the P-O bond and 475 cm-1 is also due to the doubly degenerate bending mode of the same group. All the observed vibrational frequencies confirm the formation 12
of nHAp. The peaks at 870 and 2358 cm-1 corresponds to the stretching frequency of phosphate group, indicating the presence of TCP in nHAp synthesized in the absence of GP. The retention of same pattern with minor changes was obtained for nHAp/GP and the absence of peak at 870 and 2358 cm-1 shows that the TCP was not formed (Gopi et al., 2010). 3.6. Morphology of honeycomb-like PLA/nHAp nanocomposites.
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The TEM image and the size distribution of the green synthesized nHAp particles displayed an average particle size around 43 nm and were found to have good dispersing ability in solvents. Further, the nHAp/GP particles are dispersed in PLA-DCM solution to fabricate honeycomb-like structured films. The influence of nHAp for the formation of pores were studied by varying the
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amount of nHAp (0 wt%, 2 wt%, 4 wt%, and 6 wt%) in PLA.
Fig.6. SEM images of honeycomb PLA films with incorporation of various nHAp contents
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(0 wt%, 2 wt%, 4 wt%, and 6 wt%) and the average pore size corresponding to the SEM images in the same row.
It was observed that a significant influence was exerted by nHAp for the pore formation. A
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regular distribution of pores on PLA film was attained on increasing the content of nHAp from 0 wt% to 4 wt% and the pattern quality was lowered on increasing the content to 6 wt%. The
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morphological images scanned using SEM shows that the incorporation of nHAp into PLA contributes porous patterning on the PLA membrane as observed in Fig.6. The distribution diagram (Fig.S4) shows that the average pore diameter of the PLA composites H-PLA, H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp are 2.5±1.7 μm, 6.2±3.5 μm 12.3±2.5 μm and 8.5±5.9 μm respectively. The % of porosity of H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp composites are 15%, 33%, 48% and 25% respectively. The porosity can 13
improve cell growth on the composite membranes. The homogeneous dispersion of nHAp particles plays an inevitable role in stabilizing the water droplets condensed on the polymer surface during breath figure formation (Wu & Wang, 2012; Zhao, Zhang, Wang & Li, 2006). The optimized amount for uniform dispersion of the particles was 4%, on further increase in content to 6%, nHAp precipitation was observed even on ultrasonication. This excess content of
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nHAp would result in self agglomeration rather than stabilizing the condensed water droplets. This might affect the regularity in pore patterning as well as the pore size that is clearly evident in the Fig.6d. The agglomeration of nHAp leading to uneven pores on the membrane with excess 6% nHAp inclusion is shown in Fig.S5. The higher density of water droplets compared to that of DCM, allows the penetration of droplets deep into the membranes resulting in multi-layer pores. 3.7. Mechanical property
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The extent to which the mechanical properties are altered for a polymer, PLA on incorporation
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with nHAp is examined. The tensile strength and % of elongation of the samples: H-PLA, H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp are shown in Fig.7. Tensile strength of
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F-PLA and H-PLA reference were 38.93±1.45 and 27.32±1.58 MPa and that for H-PLA/2-
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nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp composites were 30.94±1.66, 33.56±1.71 and 30.81±1.58 MPa, respectively. It is obvious that the tensile strength of F-PLA to be higher than
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that of nanocomposites. However, the reinforcing effect for the polymer is observed only upto a
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certain filler content, here it is upto 4%. On account of poor filler-polymer interaction with further addition of nHAp (filler) causes retardation in mechanical strength (Garoushi, Lassila & Vallittu, 2011). As a result of such poor interaction of polymer with excess nHAp, H-PLA/6-
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nHAp composite had reduced mechanical properties, when compared to H-PLA/2-nHAp and H-PLA/4-nHAp composites. The crystallinity could be suggested as a factor to contribute the
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mechanical strength, i.e., high crystallinity is positive towards imparting tensile strength to PLA (Zhao et al., 2016). The increase in tensile strength of H-PLA/nHAp composites could be due to
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their enhanced hardness with the increment of nHAp content. The elongation at break was found to be 4.91± 0.42% for H-PLA/2-nHAp, 8.18±0.39% for H-PLA/4-nHAp and 2.18±0.32% for H-PLA (Fig.7b), which implies that the ductility of H-PLA/nHAp composite is significantly improved on comparison with H-PLA. It is known that the improvement in ductility causes the delay in fracture, which declines the abrupt failure for the H-PLA/nHAp nanocomposites.
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Fig.7. (a) Stress-strain curves (b) Tensile strength-percentage of elongation of F-PLA, H-PLA, H-PLA/2-nHAp, H-PLA/4-nHAp, and H-PLA/6-nHAp.
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The presence of excess nHAp particles in the PLA matrix may generate voids during the
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elongation that leads to easy breakage and rupture of bonds, resulting in the reduction of
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elongation at break to 7.23± 0.41% for H-PLA/6-nHAp. 3.8. Wettability of films surface (Contact angle) For the application of samples in biomedical field, the hydrophilic behaviour was evaluated by
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water contact angle measurements. Fig.8. shows the contact angle images of the samples and the
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results are summarized in Table 1 (Fig.S6). The wettability is greatly influenced by the surface topography of the membrane.
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Even though H-PLA/nHAp films and bare PLA films had similar topography, a hydrophilic contact angle of about 49º was attained for nHAp incorporated (Gong et al., 2017) PLA films.
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This was due to the presence of hydrophilic nHAp particles in the matrix. The water droplets are evenly distributed and stabilized by the nHAp particles in the films instead of improving the formation of air pockets to induce hydrophobicity. During contact angle measurement, the water
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dropped on the surface penetrates into the pores to reduce the contact angle between membrane surface and the water droplet. The intention to create hydrophilic membranes was mainly to enhance the adhesion, spreading and proliferation of cells and the hydrophilicity could be achieved by adding hydrophilic inorganic materials.
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Fig.8. Contact angle images of the samples (a) F-PLA; (b) H-PLA (c) H-PLA/2-nHAp (d) H-PLA/4-nHAp and (e) H-PLA/6-nHAp.
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In addition to the hydrophilicity, film roughness can also cause better cell adhesion and the
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results claim that all the membranes pose a surface with better cell attachment in the bone tissue.
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3.9. Hemolysis
Hemolysis assay is considered as a complementary study towards the biocompatibility of the membranes. The blood compatability of biomaterials proposing for use in tissue engineering is a
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critical issue to be addressed. The hemocompatibilty of the scaffolds was evaluated with respect
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to a negative control (normal saline) and a positive control (double distilled water). Hemocompatibility of the porous nanocomposites were investigated and are shown in Fig.S7.
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The hemolytic percentage (HP) of each scaffold was evaluated from the absorbance obtained at 540 nm. For bare F-PLA and H-PLA film, HP% was found to be 0.38±0.11% and 0.59±0.15%
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respectively. It was hiked sequentially on increasing the nHAp content and the values were respectively 1.55±0.22%, 2.25±0.24% and 3.56±0.25% for H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp systems. All the membranes were found to be non-hemolytic with HP less
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than 5% as defined by American Society for Testing and Materials International (ASTM Standard, F756-08) (Radha, Balakumar, Venkatesan & Vellaichamy, 2015).
3.10. Cytotoxicity Cytotoxicity analysis for H-PLA/nHAp composites has been opted to compare the cell proliferation by the systems with normal cells and cancer cells. MTT assay was carried out using the human cervical cancer cell line (HeLa) and human normal cell line–Fibroblast (L929).The 16
studies show that the presence of n-HAp particles inhibited the growth of cancer cells and was found to increase in inhibition rate with increase in nHAp particles. The cervical cancer inhibitions for H-PLA, H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp were 4±1.2%, 47±2.4%, 63±2.5% and 56±2.2%, respectively. This observation indicates that the inhibition by nHAp depends upon the morphology of nHAp. The apoptosis of HeLa cells depends on the
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concentration of nHAp and the treatment time. The influence of particle size and dosage of nHAp particles for the inhibition of cancer cells are shown in Fig.9a. The nHAp particles contains positively charged Ca2+ ions to bind acid groups and negatively charged PO43- ions to attract basic groups of biomolecules. The sialic acid residues in plasma membranes render more negative charge on cancer cells than normal cells which could be adsorbed by nHAp nanoparticles. This high adhering capacity of nHAp derives from the electrostatic attraction
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between negative charges on cancer cells with positive binding sites in nHAp. The cancer cell
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inhibition after binding with nHAp could be attributed to the depletion of proteins required for
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cell cycle progression (Han, Wang, Dai & Li, 2012; Lieske, Norris & Toback, 1997).
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Fig.9. Effects of HAp on cell viability of (a) Cancer cell (HeLa) and (b) Normal cell (L929).
Intracellular response is greatly influenced by the morphology of scaffolds. The extent of cellscaffold interaction such as cell attachment and proliferation effects the cell differentiation. Calcium phosphate in nanophase with high surface area enhances the cell adhesion, proliferation and differentiation. Hence, the porous membranes containing nanostructured hydroxyapatite were also checked for its efficiency to use as substrates for the cell growth using L929 cells. The
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cells and ECM secreted by cells were found to be evenly spread on the scaffold surfaces and also penetrated inside the pores on the films. The MTT assay results (Fig.9b.) conveys that the cell adhesion improved on incorporation of nHAp particles in PLA matrix. The cell viability % of porous membranes: H-PLA, H-PLA/2-nHAp, H-PLA/4-nHAp and H-PLA/6-nHAp with L929 cells were 78.9±3.06%, 84.1±3.5%, 90.8±2.1% and 84.2±3.2%. The composites does not
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cell proliferation by nHAp incorporated PLA membranes.
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indicate any remarkable toxicity towards mice fibroblast cells (L929), indicating the supportive
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Fig.10. Phase contrast image of H-PLA (a,e), H-PLA/2-nHAp (b,f), H-PLA/4-nHAp (c,g) and H-PLA/6-nHAp (d,h) nanocomposites cultured on (a-d) normal and (e-h) cancer cells for 3 days
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(Marked arrow indicate the nuclei fragmentation or apoptotic body formation). The porous surface contributed better cell adhesion and cell proliferation as demonstrated in
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Fig.10. The nanocomposite with 4 wt% nHAp loading (H-PLA/4-nHAp) induced better cell growth, when compared to the other two composites on evaluating for about 72 h. Fig.10. shows
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the phase contrast optical microscope images of H-PLA/4-nHAp nanocomposites seeded on L929 and HeLa cells. The images indicate that the H-PLA/4-nHAp composites inhibited the growth of cancer cells and exhibit higher viability towards the normal L929 cells. To conclude,
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the H-PLA/4-nHAp system suppresses the proliferation of cancer cells and promotes the spreading and proliferation of normal cells (Han et al., 2014). By careful investigation of the obtained results, H-PLA/4-nHAp was found to be highly biocompatible and could be employed as a feasible scaffold for the substitute of bone in the cancer patients.
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Conclusion The work was focused on the surface engineering of PLA membranes by nHAp particles synthesized novely by using pectin from bitter gourd extract medium. The honeycomb-like morphology obtained through breath figure patterning largely influenced the biological
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performance of the nanocomposites. The presence of GP controlled the size range of hydroxyapatite in nanoregime. The Ca/P weight ratio was calculated from EDAX analysis and was found to be 1.66. The XRD analysis confirms the formation of nHAp/GP without any crystalline phase transformation, but reveals the presence of tricalcium phosphate for nHAp synthesized in the absence of GP. The GP had a significant role in the formation of pure nHAp without any mixed phase of calcium phosphate. A regular distribution of pores on PLA film was attained on increasing the content of nHAp from 0 wt% to 4 wt% and the pattern quality was
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lowered on increasing the content to 6 wt%. A narrow pore size distribution was exhibited by the
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PLA/4-nHAp composite. The reinforcing effect for the polymer is observed only upto 4wt%
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filler incorporation; further addition resulted in poor polymer-filler interaction. The cytotoxicity studies using the human cervical cancer cell line – (HeLa) and human normal cell line–Fibroblast
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(L929) showed that the nanocomposites were highly viable towards the normal cells, while inhibiting the growth of cancer cells by 63±2.5%. The higher cell viability of the composite,
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PLA/4-nHAp was evident from the cytotoxicity test using normal cell line. The present study
Acknowledgements
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proposes the implementation of PLA/4-nHAp composite as a scaffold for bone regeneration.
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This research was financially supported by Ministry of Human Resource Development (MHRD), India. The authors thankfully acknowledge Dr. G. Unnikrishnan, Dr. A. Sujith, R. Rarima, P.K.
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Adnan and Jithin Raj, Department of Chemistry, NIT Calicut and K. Ramshad, Department of Chemistry, IIT Madras for their timely help. We extend our gratitude to Dr. Shibu and
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Krishnakumar (STIC, CUSAT) for their cooperation in recording TEM.
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5. Contact angle Fig.S6. Table 1 Contact angle of water on F-PLA, H-PLA and H-PLA/nHAp composite films and results are presented as mean for n = 3.
Sample
Contact
code
name
angle
F- PLA
72±7
b
H-PLA
124±11
c
H-PLA/2-nHAp
66±6
d
H-PLA/4-nHAp
53±4
e
H-PLA/6-nHAp
A
CC
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TE
D
M
A
N
a
U
SC RI PT
Sample
25
49±5