Nanoceria Can Act as the Cues for Angiogenesis in Tissue

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Tissue Engineering and Regenerative Medicine

Nanoceria can act as the cues for angiogenesis in tissue engineering scaffolds: Towards next generation in situ tissue engineering Robin Augustine, Yogesh Dalvi, Pan Dan, Nebu George, Debora Helle, Ruby Varghese, Sabu Thomas, Patrick Menu, and Neelakandapillai Sandhyarani ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01102 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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ACS Biomaterials Science & Engineering

Nanoceria can act as the cues for angiogenesis in tissue engineering scaffolds: Towards next generation in situ tissue engineering Robin Augustine1*, Yogesh B. Dalvi2, Pan Dan3, Nebu George2, Debora Helle3, Ruby Varghese2, Sabu Thomas4, Patrick Menu3, Neelakandapillai Sandhyarani1 1Nanoscience

Research Laboratory, School of Nano Science and Technology, National Institute of Technology

Calicut, Kozhikode, Kerala 673601, India. 2Pushpagiri 3Ingénierie

Research Centre, Pushpagiri Institute of Medical Sciences, Tiruvalla, Kerala 689101, India. Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS - Université de Lorraine, Vandoeuvre-lès

Nancy, F54500, France. 4International

and Inter University Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University,

Kottayam, Kerala 686560, India. *Corresponding authors [email protected] (R. Augustine)

Abstract The next generation tissue engineering, exploits the body's own regenerating capacity by providing an optimum niche by means of a scaffold for the migration and subsequent proliferation endogenous cells to the site of injury, enhance regeneration or healing and bypass laborious in vitro cell culture procedures. Such systems also required to have enough angiogenic capacity for the subsequent patency of implanted scaffolds. Exploitation of nano dimensional ceria’s (nCeO2) redox properties in in situ tissue engineering to promote cell angiogenesis is poorly understood. As a novel strategy, electrospun polycaprolactone (PCL) based tissue engineering scaffolds loaded with nCeO2 were developed and evaluated for the morphological and physicomechanical features. In addition, in vitro and in vivo studies were performed to show the ability of nCeO2 containing scaffolds to enhance cell adhesion and angiogenesis. These studies confirmed that nCeO2 containing scaffolds supported cell adhesion and angiogenesis better than bare scaffolds. Gene expression studies had shown that angiogenesis related factors like HIF1α and VEGF were upregulated. Overall results show that incorporation of nCeO2 plays a key role in scaffolds to enhance angiogenesis, cell adhesion and cell proliferation and can produce successful outcome in in situ tissue engineering. Key words: Nanoceria, in situ tissue engineering, angiogenesis, polycaprolactone

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Introduction The field of tissue engineering has produced many achievements in producing novel tools and translating various biomaterial approaches towards the development of functional tissue engineered products. Although the conventional tissue engineering approaches, which are based on the use of autologous cells and pre-seeded scaffolds for implantation at the injury, has been well established, they are time consuming and laborious1. The commercialization of such products are limited due to the difficulty to transport and store that make them less convenient and clinically less viable. In order to bypass the bottlenecks of cell based tissue engineering, a new concept referred to as in situ tissue engineering that utilizes the body's own regenerating capacity2. This method exploits target-specific tissue engineering scaffolds that can effectively control the microenvironment at the implantation site and attract, mobilize and facilitate the proliferation of host stem/progenitor cells to desired tissues3 Rapid development of an adequate vasculature is a major prerequisite for the survival of the engineered construct after their implantation and during the long-term function. In conventional tissue engineering, prevascularization has been recognized as a promising approach focusing at the generation of a preformed microvasculature in tissue engineered constructs prior to their application4. However, this approach is not feasible in in situ tissue engineering. Although incorporation of angiogenic growth factors is considered as promising for vascularization5, the poor stability of the growth factors reduce the potential of such interventions in in situ approach6. On account of these drawbacks, it is most important to explore new strategies to provide vascularization in scaffolds. Cerium oxide nanoparticles (nCeO2) has the potential to induce angiogenesis, as evident from both in vitro and in vivo model systems7. The major mechanism of nCeO2 induced angiogenesis is the existence of cerium in a +3 (reduced) or +4 (oxidized) forms at the particle surface8. Specifically, nCeO2 trigger angiogenesis by stabilizing hypoxia inducing factor 1α (HIF- 1α) by regulating the intracellular oxygen environment7. Noteworthy to mention, Xiang et al., successfully used nCeO2 as an activator of calcium channels of mesenchymal stem cells to induce angiogenesis in bone grafts9. However, to best of our knowledge, there are no reports describing 2 ACS Paragon Plus Environment

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the potential of nCeO2 to induce cell proliferation or angiogenesis in in situ tissue engineering approach. In this study, we developed a biomaterial scaffold to produce higher cell proliferation and angiogenesis by utilizing the angiogenic property of nCeO2, as well as the favourable biomaterial properties of PCL, for in situ tissue engineering applications. The present study aimed to incorporate nCeO2 in PCL scaffolds, characterize this novel nanocomposite biomaterial by various physicochemical techniques, and investigate the biocompatibility and angiogenic potential using various in vitro and in vivo biological characterizations. Experimental Materials used Polycaprolactone (PCL) was obtained from Sigma-Aldrich, USA. Cerium nitrate hexahydrate (Ce(NO3)3·6H2O) was procured from Alfa Aeser. Aqueous ammonia was purchased from HIMEDIA, Mumbai. Gramashri variety of fertilized hen’s eggs was purchased from Regional Poultry Farm, Chathamangalam, Kozhikode, Kerala, India. Histopaque, polyethyleneimine (PEI), paraformaldehyde and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) and 4',6-Diamidino-2-Phenylindole Dihydrochloride (DAPI) were purchased from Sigma-Aldrich (USA). Phalloidin was purchased from Thermo Fisher Scientific (France). The lactate dehydrogenase (LDH) assay kit was obtained from Roche (Switzerland). Dulbecco’s Modified Eagle’s Medium (DMEM) (Lonza) was used for fibroblast cell culture. Endothelial basic medium (Lonza) was used for human umbilical vein endothelial cell (HUVEC) culture. Media were supplemented with 10% fetal bovine serum (FBS, Lonza), 100 mg ml-1 Fungizone (Fisher), 100 IU ml-1 penicillin (Sigma-Aldrich) and 200 mM L-glutamine (Sigma-Aldrich) on culture plates. All other reagents/solvents used in this study were from standard manufacturers and of analytical grade quality. Synthesis and characterization of nCeO2 nCeO2 are synthesized using cerium nitrate and aqueous ammonia in the presence of gelatine. For the nCeO2 synthesis, 0.5 M of cerium nitrate hexahydrate (Ce(NO3)3·6H2O) was dissolved in 25 ml of ultrapure water and stirred for 30 min. Similarly, in another beaker, 50 mL of 2% w/v gelatine aqueous solution was prepared. After complete dissolution, both solutions were mixed together 3 ACS Paragon Plus Environment


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and magnetically stirred for 30 min. Then, about 25 ml of aqueous ammonia solution was added dropwise to the above solution. This solution was stirred for 10 h at room temperature for the formation and stabilization of nCeO2. Finally, light yellow suspension was obtained due to the formation of nCeO2 with excess Ce4+ in the presence of oxygen. This suspension was centrifuged to remove relatively larger particles (at 1000 rpm). The supernatant was further centrifuged at 8000 rpm and the centrifugate was washed several times with ultrapure water and then in absolute alcohol. The obtained nanoparticles were dried in oven at 80C for overnight. The dried powders were thoroughly grinded using mortar and pestle and calcined at 600C for 10 h. Several supportive analyses were performed to characterize the properties of the synthesized nCeO2. Fourier transform infrared (FT-IR) spectra are measured with a Perkin Elmer Frontier MIR with PIKE Gladi ATR (USA) attachment and DTGS detector on a diamond crystal between 550– 1500 cm-1 with 15 scans at 4 cm−1 resolution using Spectrum 400 software 62 (version 6.3) in the range 4000–400 cm−1. The samples were characterized for their purity and crystallinity by X-ray diffraction (XRD) analysis. XRD was recorded in the 2θ range of 10◦–90 using a MiniFlex X-ray diffractometer. Crystallite size was obtained by using the Debye’s Scherrer equation (1) D = K/ βcos(θ)

(1)

where, λ is the X-ray wavelength, K is the shape factor, β is the line broadening at half the maximum intensity (FWHM) in radians and θ is the Bragg angle. Morphology and size of the nanoparticles were analysed by transmission electron microscopy (TEM) (JEOL JEM 2100). Specimens for TEM were prepared by ultrasonic dispersion of nCeO2 sample in isopropyl alcohol and a drop of the suspension was then put on a copper specimen grid coated with ultrathin carbon film. Selected area electron diffraction (SAED) was used to determine the crystallinity of nCeO2 and to identify the crystallographic planes. Fabrication and characterization of PCL/nCeO2 scaffolds


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We used electrospinning for the fabrication of bare PCL and PCL/nCeO2 nanocomposites scaffolds. Prior to the addition of PCL into acetone, nCeO2 were weighed and dispersed by ultrasonication for 15 min in the solvent. Then, the PCL pellets was added to the above dispersions to get a final concentration of 15% w/v and stirred until completely dissolved. The prepared solutions were taken in 10 mL syringes with an 18-gauge needle. Finally, prepared suspensions (10 mL) were electrospun with different percentages of nCeO2 nanoparticles. We maintained 10cm distance between the needle tip and the collector. An applied voltage of 18 kV was also maintained. The solution flow rate was 2.5 mL·h–1. The deposited fibrous scaffolds were carefully cut and removed using a surgical blade. Electrospun PCL scaffolds without any nCeO2 were designated as PCL. Scaffolds containing 0.5%, 1%, 2% and 3% (w/w) of nCeO2 were named as PCL/nCeO2-0.5, PCL/nCeO2-1, PCL/nCeO2-2 and PCL/nCeO2 -3, respectively. Physico-mechanical Characterization of scaffolds SEM analysis of scaffolds Specimens were characterized using field emission scanning electron microscopy (FE-SEM, Hitachi SU6600) at an accelerating voltage of 15 kV. Prior to the SEM analysis, samples were sputter coated with gold for 15 seconds to make them conducting. FTIR and XRD analysis of scaffolds FTIR analysis (in ATR-FTIR mode) and XRD analysis were performed to confirm the presence of nCeO2 in PCL scaffolds. Instruments and method used were similar to that were used for the characterization of nCeO2. Differential Scanning Calorimetry (DSC) DSC analysis (TA Instruments, DSC Q-20) was performed to determine the thermodynamic and crystalline nature of the nanocomposite scaffolds. The samples (5 mg) were heated from -80°C to +150°C at 10°C/min with a 1 min hold at +150°C. Subsequently, samples were cooled at 10°C/min to -80°C. Various properties such as crystallization temperature (Tc), melting temperature (Tm), enthalpy of melting (ΔHm), enthalpy of crystallization (ΔHc) and degree of crystallinity (Xc) were measured. The percentage of crystallinity was determined using equation (2). % Crystallinity = [ΔHm]/ΔHm° X 100%

(2) 5

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ΔHm and ΔHm° are the enthalpy of melting of the samples and enthalpy of melting of 100% crystalline PCL respectively (ΔHm° = 139.5 J/g)10. Porosity of the scaffolds Percentage of bulk porosity of PCL and PCL/nCeO2 scaffolds was measured using alcohol displacement method11. Tensile properties of PCL and PCL/nCeO2 scaffolds Mechanical behavior of a tissue engineering scaffold should be relatively similar to the adjacent tissue to avoid mechanical incompatibility and the pain during muscular movement. Tensile measurements of the PCL and PCL/nCeO2 scaffolds were performed with a Tinus Olsen H50 KT Universal Testing Machine according to the ASTM D 882 standard. PCL and PCL/nCeO2 scaffolds were cut into rectangles with dimensions of 6×1 cm2 and placed between the grips of the tester. A 3 cm gauge length was maintained for mechanical loading. The load was 100-N and crosshead speed of stretching was 1 mm/min. The representative values of tensile property were obtained from five independent measurements and expressed as the mean ± standard deviation (SD). In vitro biocompatibility studies Haemolysis assay and blood cell aggregation study About two millilitres of human blood was collected in VACUETTE® blood collection tubes and centrifuged at 1000 rpm for 5 min to separate the plasma. The RBC pellet was washed twice with PBS and diluted with PBS (1:4 ratio for RBC and PBS respectively). The scaffolds were kept in 2 mL sterile PBS for 48 h and the extract was collected. 100 μl of this extract was mixed with 100 μl dilute RBC suspension and incubated at 37°C for 3 h. In this experiment, normal saline was used as the negative control (no haemolysis) and ultrapure water as the positive control (100% haemolysis). After incubation, the samples were removed from RBC suspensions and were centrifuged at 1000 rpm for 5 min to remove the RBCs. The optical density (OD) of supernatant was measured at 541 nm using a UV-Vis spectrophotometer (Shimadzu, Model 1700, Japan). From the OD values, the % haemolysis was calculated12. One millilitre of RBC was diluted to 10 ml with saline. All the PCL and PCL/nCeO2 scaffolds were extracted in 2 ml PBS for 48 h; 100 µl of this extract was added to 100 µl diluted RBC. After 6 ACS Paragon Plus Environment

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20 min of incubation at 37ºC, cell fractions were isolated by centrifugation (1000 rpm, 5 min). The pellets of RBCs were resuspended in PBS, and images were taken using an inverted microscope (Leica DMIRB, Germany). In vitro cell adhesion and cell viability studies Human mesenchymal stem cells (hMSCs), human fibroblasts and HUVECs were used to evaluate cytocompatibility and cell adhesion behavior of the scaffolds. Human umbilical cords were collected with informed consent after full-term births using the guidelines approved by the University Hospital center of Nancy (France). hMSCs and HUVECs were isolated and expanded as previously reported13. Human neonatal foreskin fibroblast cells (BJ – ATCC – CRL-2522) were obtained from American Type Cell Culture (ATCC). Prior to the cell seeding, we sterilized all the scaffolds (1 x 1 cm size) by 70% alcohol treatment for 20 minutes and subsequent UV irradiation for 20 minutes on each side. All the scaffolds were kept in 24 well cell culture plates containing DMEM medium overnight in the CO2 incubator. Fibroblasts, hMSCs, HUVECs were marked with a membrane marker 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbo-cyanine perchlorate (DiI, Invitrogen, D282, France) prior to the seeding on the prewetted scaffolds at a density of 50,000 cells

per scaffold in 100 µL of appropriate media. After cell seeding, plates containing scaffolds were kept in CO2 incubator (37°C, 5% CO2 supply) for 2h for the adhesion of cells on the scaffolds. Controls without samples were also maintained. Then, 400 µL of media were added to every well and incubated for 24 h in CO2 incubator (37°C, 5% CO2 supply). To visualize cell adhesion, samples were fixed with 4% paraformaldehyde for 15 min at room temperature, after 3 wash, cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Images were taken with a fluorescent microscope (Leica DMI 3000B, Germany). To assess the viability of cells cultured on scaffolds, MTT was performed. We used the respective protocol provided by the manufacturer of the assay kit (M2003, Sigma-Aldrich). Briefly, the media were removed after 24 h of cell culture and MTT (0.5mg/mL) was added to the wells and incubated for 4 h in CO2 incubator. Then, 300

µL of DMSO was added to the cells to solubilize the formazan crystals and 100 µL of the solutions were transferred to a 96-well plate. The absorbance (Optical density, OD) of the plate was measured at 570 nm using an automated plate reader (Varioskan Flash, Thermo scientific, France). The cell viability (%) was calculated using equation (3). Cell viability = Mean OD of Treatment/Mean OD control X100%


(3)


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To determine the cytotoxicity of scaffold to the cells, LDH assay was carried out according to the respective protocols provided by the manufactures (Roche, Switzerland). After the incubation of cells with scaffolds, 100 μl of culture medium was taken from the wells, mixed with 100 μl of LDH reaction solution and incubated for 30 minutes (Room temperature). Absorbance was measured at 492 nm using an automated plate reader (Varioskan Flash, Thermo scientific, France). Untreated cells (low control) and cells treated with 1% (v/v) Triton-X100 (high control) were used to determine spontaneous and maximum release of LDH, respectively. The percentage of cell cytotoxicity was calculated using equation (4).

Cytotoxicity = [(OD of Experimental – OD of Low control)/(OD of High control – OD of Low control) x 100%

(4)

Both MTT and LDH assays were performed in triplicates and the results were expressed as mean ± S.D. In vivo studies Angiogenesis assay in chicken chorioallantoic membrane (CAM) The CAM assay was performed according to our reported protocol14. Fertilized and incubated chicken eggs of the Gramasri variety were procured from the Regional Poultry Farm, Chathamangalam, Kerala, India, and used for the experiments. The angiogenic response was observed for a period of 0-8h. In vivo implantation studies Male Sprague Dawley rats weighing 180-260g were selected from inbred group for the study. The animals were purchased from Kerala Veterinary College, Mannuthy, India. All the animal experiments were carried with the prior permission from Institutional animal ethics committee and were conducted strictly adhering to the guidelines of CPCSEA (No.602/PO/Re/S/2002/CPCSEA) constituted by the Animal Welfare Division of Government of India in Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla. Animals were provided with standard environmental controlled conditions of 23 ± 5ºC, 12h light-dark cycle, have free access to standard food (Krish Scientist’s Shoppe, Bangalore) and UV sterile water. Biocompatibility and biological performance of the nanocomposite scaffolds was determined using murine model. Based on the in vitro cell culture studies, PCL, PCL/nCeO2-1, PCL/nCeO2-2


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and PCL/nCeO2-3 were selected for animal studies. A total of 12 rats were divided into four groups containing three rats each. Animals were anaesthetised by the intraperitoneal injection of combination of ketamine hydrochloride (50 mg/kg) and xylazine (5 mg/kg). All the scaffolds for implantation were cut into 1 × 1 cm pieces and pre-treated by sterilizing it with 70% alcohol for 20 min and then with UV radiation for 30 min. Scaffolds were implanted subcutaneously in the abdominal region of rats. At the end of each week of implantation (for a period of 4 weeks), the implantation site was reopened and the scaffolds were taken out and visually inspected for inter group variations in inflammation and angiogenesis. Scaffolds with surrounding subcutaneous tissue were then surgically removed for histological evaluation. Samples were stained with hematoxylin and Eosin (H & E) and observed under microscope to determine in vivo biocompatibility and angiogenesis. Gene expression analysis on the tissues attached to the implanted scaffolds The mRNA expressions of VEGF (Vascular endothelial growth factor), EGF-R (Endothelial growth factor), TNF-α (Tumor necrosis factor), COX-2 (Cycloxygenase-2) and HIF (Hypoxia Inducing factor) in wound tissues were determined with real-time quantitative PCR (qRT-PCR). Total RNA was isolated from subcutaneous tissue attached on the implanted scaffolds for cDNA synthesis. RNA isolated from the subcutaneous tissue of untreated healthy rats were used as controls (calibrator samples). We used β-actin housekeeping genes as internal standard for normalization. An aliquot (9 μL) of cDNA was used as a template for the subsequent RT-PCR. The RT-PCR assay was performed with 2×SYBR green (Takara, Japan) in the Applied Biosystem step one plus thermal cycler according to the standardised protocol. The following thermal cycling profile was used (45 cycles): 95 °C for 10 min, 95 °C for 15 s, 58 °C for 1 min (depending on the primers used) and 95 °C for 15 s. Details of primers used in the study are given in supplementary information, Table S1. The 2−ΔΔCT method proposed by Livak and Schmittgen15 was used to determine the fold change in expression by normalizing the resulting threshold cycle (Ct) values of the target mRNAs to the Ct values of the house keeping gene (internal control) β-actin (ΔCT = Ct (Experiment) − Ct (β-actin)). The fold change in expression was then calculated as 2−ΔΔCT. Statistical analysis



The statistical analysis was performed using the un-paired Student’s t-test and “One way ANOVA” with Dunnett’s post-test performed using Graph- Pad Prism Version 6.04, San Diego, California, USA (*significantly different from control at P < 0.05; **significantly different from control at P < 0.001; ***significantly different from control at P < 0.0001, N.SNo significant difference). Results Characterization of synthesized nCeO2 As the first step in this study, we synthesized nCeO2 nanoparticles by means of wet-chemical precipitation method and the synthesized nanoparticles were characterized by various physicochemical techniques. The XRD pattern of nCeO2 is shown in Fig. 1A. The XRD pattern was collected from 10 to 90 2 degrees. It clearly indicated the polycrystalline structure of the nCeO2. Well distinguishable characteristic peaks corresponding to the miller indices (111), (200), (220), (311),(222), 400), (331), (420) and (422) were observed at 2θ = 28.49°, 33.04°, 47.45° , 56.38°, 59.11°, 69.42°, 76.71°,79.17° and 88.51°, respectively. All Bragg peaks with miller indices were in agreement with the cubic lattice of pure CeO2 and were comparable with JCPDS DATA (898436). The diffraction peaks in the XRD pattern was very close to the face centered cubic fluorite structure of CeO2 crystal16. Crystallite size was calculated from the XRD results using the Debye’s Scherrer equation. The crystallite size was found to be in the range from 9-16 nm.


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Fig. 1. XRD pattern (A), FTIR spectrum (B), TEM images (C & D) of synthesized nCeO2 particles. D shows the lattice fringes of a single nCeO2 particle. Inset shows the SAED pattern of nCeO2. FTIR spectrum of the synthesized nCeO2 is given in Fig. 1B. The bands at 3378 cm-1 and 1641 cm-1 represents the ν (O–H) mode of (H-bonded) bound water molecules and δ (OH), respectively17. As a general trend, residual water and hydroxyl groups are detected on nCeO218. A small intense peak at 1641 cm-1 can be due to the presence of carbonyl group in ammonium carbonate on the surface of CeO2 nanomaterials19. This can be also from the HOH bending vibration of adsorbed water molecules20. The bands at 1537 cm-1 and 1328 cm-1 represent the 11 ACS Paragon Plus Environment


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presence of nitro groups from the precursors21. The band at 848 cm-1 and below 750 cm−1 with a maxima near 400 cm−1 is due to the Ce-O stretching band of the nCeO222,23. Fig. 1C displays the transmission electron micrograph of prepared nCeO2. A slightly agglomerated quasi-spherical nanoparticles with diameter in the range 4–12 nm can be observed from the TEM micrograph. However, HRTEM is very useful in revealing the atomic structures of individual nCeO2 and their lattice fringes (Fig. 1D). All the HRTEM images show that the crystal orientations of nCeO2 in a particle were uniform. The d spacing of the lattice fringes of approximately 0.32 nm for the nCeO2 sample from the HRTEM images correspond to the (111) plane of nCeO224,25. This is in good agreement with the standard data (JCPDS 34–0394). The monocrystalline property of the particles was confirmed by the SAED pattern (Inset of Fig. 1D), which was recorded from a single agglomerate consists of many nanoparticles. The high crystallinity of the powder leads to its corresponding well-pronounced Debye–Scherrer diffraction rings in the selected area electron diffraction (SAED) pattern that can be assigned to the reflections (111), (200), (220), (311), (222), (400), (331) and (420) of cubic CeO2 (There were no additional rings in the SAED pattern stemming from any other crystalline impurities). Noteworthy to mention, the overall structure which was determined by SAED was also comparable with that of XRD results. General characterization of PCL/nCeO2 scaffolds As Fig. 2A indicates, all the fabricated scaffolds showed highly porous interconnected network of polymeric fibres composed of individual fibres with various diameters. Scaffolds showed heterogeneity in fibre morphology and diameter. We could not observe any beaded morphology among fibres. X-ray diffraction (XRD) analysis was performed to determine the effect of nCeO2 on the crystalline behaviour of PCL scaffolds and to confirm the presence of nCeO2 in the PCL matrix. The XRD patterns of the scaffolds are presented in Fig. 2B. XRD patterns of PCL and PCL/nCeO2 scaffolds showed that the main diffraction peak of the PCL was at the Bragg angles 21.5° and 23.9° which can be assigned to (110) and (200) planes respectively

26,27.

This can be attributed to the

semicrystalline regions of PCL polymer. As expected, when nCeO2 were incorporated in PCL scaffolds, the characteristic diffraction peaks of the nCeO2 were observed at 2 values of 28.49°,


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33.04°, 47.45° and 56.38°. However, for PCL/nCeO2-0.5 scaffolds, we were unable to distinguish any specific diffraction pattern of nCeO2 from the background. Fig. 2C shows the FTIR spectra of the electrospun PCL and PCL/nCeO2 scaffolds. The characteristic peaks of PCL and nCeO2 were observed in the spectra of nanocomposite scaffolds. PCL shows major absorption bands at 1720 cm−1, 1050 cm−1 and 1240 cm−1 corresponding to the carbonyl groups, C–O stretching and C–O–C stretching, respectively28. In addition to the above peaks, spectra of PCL/nCeO2 scaffolds showed characteristic vibrational peaks of CeO2 (Indicated by arrows in the Fig. 2C). A magnified image of the spectra is given in Fig. S1 which can give more information regarding the presence of nCeO2 signals in the spectra of nanocomposites. We can clearly see a shift of the bands at 1537 cm-1 which is due to the nCeO2 to a lower wavenumber region. The intensity of the nCeO2 signals was higher for the sample where the higher quantities of the nanoparticles were incorporated in the polymer matrix.



B Intensity (a.u.)

PCL

PCL/nCeO2-0.5 PCL/nCeO2-1 PCL/nCeO2-2 PCL/nCeO2-3 nCeO2

C

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Transmittance (a. u.)

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PCL/nCeO2-2 PCL/nCeO2-3 nCeO2

4000

D Heat Flow (W/g)

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3500

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Fig. 2. SEM micrograph of PCL (A(a)) and PCL/nCeO2-0.5 (A(b)), PCL/nCeO2-1 (A(c)), PCL/nCeO2-2 (A(d)) and PCL/nCeO2-3 (A(e)) scaffolds. B is the FTIR spectra, C is the XRD patterns and D is the DSC curves of the PCL and PCL/nCeO2 scaffolds. DSC analysis helped to identify the variation in the thermodynamic variables that occurred during the physicochemical transformations of the PCL induced by heating or cooling of the scaffolds. Fig. 2D shows the DSC thermogram of the scaffolds.

The melting enthalpies and peak

temperatures of each of them are shown in Table 2. The endothermic peaks observed between 50 and 60°C, correspond to the melting whereas the exothermic peaks observed between 25 and 35°C correspond to the crystallization of PCL. We did not observe a significant variation of melting point (Tm) and the crystallization temperature (Tc) after the incorporation of nCeO2 in the scaffolds. In contrast, melting (ΔHm) and crystallization (ΔHc) enthalpies were slightly varied for the nanocomposite scaffolds. A similar trend was also observed for the crystalline fraction (Xc%) of the nanocomposites. Table 2. Melting point (Tm), crystallization temperature (Tc), melting enthalpy (ΔHm) crystallization enthalpy (ΔHc) and crystalline fraction (Xc%) of PCL and PCL/nCeO2 scaffolds Tm (°C) PCL 54.5 PCL/nCeO2-0.5 54.9 PCL/nCeO2-1 54.7 PCL/nCeO2-2 55.2 PCL/nCeO2-3 54.6

Tc (°C) 29.1 28.3 29.2 29.3 30.0

ΔHm (J/g) 78.9 80.5 69.6 68.6 70.7

ΔHc (J/g) 65.6 74.9 68.4 69.4 73.9

Xc% 56.55 57.7 49.8 49.1 50.6

Porosity of the scaffolds Porosity of the fabricated scaffolds was measured using alcohol displacement method. The bulk porosity of PCL scaffold was about 79-86% (Table 3). PCL/nCeO2 scaffolds showed apparently the same porosity without statistically significant difference (P>0.005). Table 3: Porosity of PCL membranes in percentage. Sample PCL PCL/nCeO2-0.5 PCL/ nCeO2-1 PCL/ nCeO2-2 PCL/ nCeO2-3

Porosity (%) 85.56 ± 2.28 86.41 ± 2.56 83.65 ± 4.43 79.36 ± 7.27 81.58 ± 4.02 15 ACS Paragon Plus Environment


Tensile properties of PCL and PCL/nCeO2 scaffolds Representative tensile stress–strain curves for electrospun PCL and PCL/nCeO2 scaffolds are given in Fig. 3. The PCL membrane with 0.5% w/w nCeO2 showed significant superior tensile properties compared to neat PCL scaffolds with 3.72 ± 0.18 MPa tensile strength (P

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