Polysaccharide-based polyampholyte complex formation: Investigating the role of intra-chain interactions.

The difference in inter-chain and intra-chain electrostatic attraction was investigated in polyelectrolyte and polyampholyte electrostatic complex formation. Three polymers with similar backbone molecular structures including chitosan (Ch) polycation, carboxymethyl cellulose (CMCe) polyanion, and carboxymethyl chitosan (CMCh) polyampholyte were used for this purpose. The turbidimetric, water content, and rheological measurements for polyampholyte self-complex showed more dependence on the ionic strength rather than the polyelectrolyte one. The degree of dissociation (α), dissociation constant (pKa), and intrinsic persistence length were calculated by applying the Katchalsky-Lifson model to potentiometric data. We studied the gyration radii as a function of Debye length and observed the polyampholyte chain contractions due to the intra-chain electrostatic attractions, which minimize the entropic gain of the inter-chain complex formation. This is in accordance with the decrease in pKa by αc for CMCh which is the opposite of that for the Ch and CMCe samples. We also found that the polyampholyte has less intrinsic and electrostatic persistence length compared with both polyanion and polycation with similar chain structures indicating the impact of the inter-chain electrostatic interaction on the complex properties. This study deepens our insight about the behavior of CMCh and the nature of difference between CMCh and Ch/CMCe electrostatic complexes.

Shape memory injectable cryogel based on carboxymethyl chitosan/gelatin for minimally invasive tissue engineering: In vitro and in vivo assays.

Shape-memory cryogels have drawn attention as an injectable system to minimize the risks associated with surgical implantation in tissue engineering. To achieve shape memory behavior with hydration as an external stimulus, it is necessary to have a porous elastic network. To achieve this, it is crucial to control the crosslinking process at the time of pore formation, especially for natural-based polymers. In this study, a versatile method using a cryogelation method in the presence of chemical and physical crosslinkers is investigated to obtain an injectable super macroporous elastic structure based on a poly(ampholyte) (carboxymethyl chitosan) and a protein (gelatin). Mechanical, swelling, shape memorizing behavior, injectability, and in vitro and in vivo behavior of cryogels were studied. Cryogelation in a subzero temperature led to the formation of scaffolds with interconnected pores of the size of 350 µm which swelled completely after 3 min. Cryogels had crosslink density up to 22% and elastic modulus in the hydrated state up to 0.054 and 1.733 MPa at low and high strains, respectively, and low hysteresis (<30 kPa). Injectability studies confirmed the ability of the cryogels to be injected through a 16G needle. In vitro studies demonstrated good cellular penetration, cell adhesion, and high cell viability (>100%). In vivo studies using mice showed that the body's response was befitting without inflammation and any side effect for the liver and kidneys.

Injectable hydrogels for bone and cartilage tissue engineering: a review.

Tissue engineering, using a combination of living cells, bioactive molecules, and three-dimensional porous scaffolds, is a promising alternative to traditional treatments such as the use of autografts and allografts for bone and cartilage tissue regeneration. Scaffolds, in this combination, can be applied either through surgery by implantation of cell-seeded pre-fabricated scaffolds, or through injection of a solidifying precursor and cell mixture, or as an injectable cell-seeded pre-fabricated scaffold. In situ forming and pre-fabricated injectable scaffolds can be injected directly into the defect site with complex shape and critical size in a minimally invasive manner. Proper and homogeneous distribution of cells, biological factors, and molecular signals in these injectable scaffolds is another advantage over pre-fabricated scaffolds. Due to the importance of injectable scaffolds in tissue engineering, here different types of injectable scaffolds, their design challenges, and applications in bone and cartilage tissue regeneration are reviewed.

Fe3O4 nanoparticles coated with carboxymethyl chitosan containing curcumin in combination with hyperthermia induced apoptosis in breast cancer cells.

Many studies have demonstrated that curcumin has potential anticancer properties. This research aims to study the effect of iron (II, III) oxide (Fe3O4) nanoparticles coated with carboxymethyl chitosan containing curcumin combination with hyperthermia on breast cancer cells. Magnetic nanoparticles coated with carboxymethyl chitosan containing curcumin (MNP-CMC-CUR) were prepared and specified. MCF-7, MDA-MB-231, and human fibroblast cells were treated with free curcumin and MNP-CMC-CUR at concentrations of 0-60 µM and at different time points. A combined therapy of MNP-CMC-CUR and hyperthermia was performed on MCF-7 cells. The cytotoxicity of curcumin and MNP-CMC-CUR combined with hyperthermia was assessed by MTT. The changes in TP53 and CASPASE3 gene expression were evaluated using real-time PCR. Both cell apoptosis and cell cycle were studied by Annexin/PI staining. The results of MTT showed that the IC50 amount of MNP-CMC-CUR has significantly decreased compared to free curcumin (p < 0.05) and MNP-CMC-CUR in combination with the hyperthermia, and significantly reducing the metabolic activity of the cells (p < 0.05). Real-time PCR results revealed the up-regulation of TP53 and CASPASE3 (p < 0.05). The combinational therapy-induced cell apoptosis (64.51%) and sub-G1 cell cycle were arrested in MCF-7 cells. Based on these observations, a combination of MNP-CMC-CUR with hyperthermia could inhibit the proliferation of MCF-7 cells.

Injectable and reversible preformed cryogels based on chemically crosslinked gelatin methacrylate (GelMA) and physically crosslinked hyaluronic acid (HA) for soft tissue engineering.

Hydrogels are a promising choice for soft tissue (cartilage, skin and adipose) engineering and repair. However, lack of interconnected porosity and poor mechanical performance have hindered their application, especially in natural polymer-based hydrogels. Cryogels with the potential to overcome the shortcomings of hydrogels have drawn attention in the last few years. Thus, in this study, highly porous and mechanically robust cryogels based on interpenetrating polymer network (IPN) of gelatin methacrylate (GelMA) and hyaluronic acid (HA) were fabricated for soft tissue engineering application. Cryogels have a constant amount of GelMA (3% wt) with different concentrations of HA (from 5% to 20 % w/w). In fact, crosslinking through cryogelation in subzero temperature facilitates the formation of interconnected pores with 90 % porosity percentage without external progen. On the other hand, high mechanical stability (no failure up to 90 % compression) was achieved due to the cryogelation and chemical crosslinking of GelMA as well as physical crosslinking of HA. Furthermore, the porous and hydrophile nature of the cryogels resulted in shape memory properties under compression, which can reverse to initial shape after retaining the water. Although increasing the HA concentration followed by the density of physical crosslinking boosted the mechanical performance of cryogels under compression, it limited the reversibility properties. Nevertheless, all cryogels with different HA concentrations showed acceptable gel strength and Young's modulus (G-H-20, E = 6kPa) and had appropriate pore size for cell infiltration and nutrient transportation with good cell adhesion and high cell viability (more than 90 %). The unique property of fabricated cryogels that facilitate less invasive delivery makes them a promising alternative for the soft tissue application.

Fabrication of nanocomposite/nanofibrous functionally graded biomimetic scaffolds for osteochondral tissue regeneration.

One of the main challenges in treating osteochondral lesions via tissue engineering approach is providing scaffolds with unique characteristics to mimic the complexity. It has led to application of heterogeneous scaffolds as a potential candidate for engineering of osteochondral tissues, in which graded multilayered-structure should promote bone and cartilage growth. By designing three-dimensional (3D)-nanofibrous scaffolds mimicking the native extracellular matrix's nanoscale structure, cells can grow in controlled conditions and regenerate the damaged tissue. In this study, novel 3D-functionality graded nanofibrous scaffolds composed of five layers based on different compositions containing polycaprolactone(PCL)/gelatin(Gel)/nanohydroxyapatite (nHA) for osteoregeneration and chitosan(Cs)/polyvinylalcohol(PVA) for chondral regeneration are introduced. This scaffold is fabricated by electrospinning technique using spring as collector to create 3D-nanofibrous scaffolds. Fourier-transform infrared spectroscopy, X-ray diffraction, energy dispersive X-ray spectroscopy, scanning electron microscopy, mechanical compression test, porosimetry, and water uptake studies were applied to study each layer's physicochemical properties and whole functionally graded scaffold. Besides, biodegradation and biological studies were done to investigate biological performance of scaffold. Results showed that each layer has a fibrous structure with continuous nanofibers with improved pore size and porosity of novel 3D scaffold (6-13 µm and 90%) compared with two-dimensional (2D) mat (2.2 µm and 19.3%) with higher water uptake capacity (about 100 times of 2D mat). Compression modulus of electrospun scaffold was increased to 78 MPa by adding nHA. The biological studies revealed that the layer designed for osteoregeneration could improve cell proliferation rate in comparison to the layer designed for chondral regeneration. These results showed such structure possesses a promising potential for the treatment of osteochondral defects.

A highly efficient microwave-assisted synthesis of an LED-curable methacrylated gelatin for bio applications.

This study deals with the development of an LED-curable methacrylated gelatin (GelMA) synthesis via microwave (MW) irradiation with a reaction and purification time-, energy-, and methacrylation reagent-saving approach. To investigate the efficiency of MW irradiation in GelMA synthesis, characteristics of the GelMAs prepared by using glycidyl methacrylate (GMA) or methacrylic anhydride (MA) via the MW-assisted (MWA) method were compared comprehensively with those synthesized via the conventional heating method. Moreover, MWA reaction conditions were optimized in terms of methacrylation reagent concentrations (C), reaction time (t), and MW power (P). Characterization and assessment of the GelMAs were conducted with 1H NMR, FT-IR, and Raman spectroscopy along with physical-mechanical, thermal, and hydrophilicity analysis. The results demonstrated that the MWA synthesized GMA-GelMA hydrogels were possessed of increased methacrylation degree (MD), gel fraction (GF), tensile strength (TS), elongation at break (EB), glass transition temperature (T g), and water contact angle (WCA) as well as decreased swelling degree (SD) values in comparison to those of MA-GelMA and GMA-GelMA hydrogels prepared via the MWA and conventional method, respectively. Enhanced properties of the MWA synthesized GMA-hydrogels suggested an effective methacryloyl conjugation leading to a greater amount of covalent crosslinking density justified by the dipolar moment calculations. Optimal GMA C, t, P, and purification time for a highly crosslinked GelMA hydrogel (MD: 96.1%, GF: 98.3%, SD: 10.11%, TS: 6.7 MPa, EB: 175.2%, T g: 75.34 °C, and WCA: 72.22°) were found to be a 5 times molar excess over the primary amine groups of gelatin, 5 min, 500 W, and 24 h, respectively. Thus, the optimized MW conditions offer a promising green method to prepare GelMAs for bio applications.

Chitosan/PVA/Doxycycline Film and Nanofiber Accelerate Diabetic Wound Healing in a Rat Model.

In this study, we evaluated the effects of nanofiber and film polymers with doxycycline for treating a wound in a diabetic rat model. 108 male rats were divided into six groups, the control group, the diabetic control, and the groups were diabetic rats receiving different wound dressing. At the 3rd, 7th, and 14th days, macroscopic/histologic imaging and tissue sampling were performed. Tissues were analyzed for IL-1ß, TNF-α, IL-10, TIMP-1, and MMP-2 by using ELISA. Dressings of chitosan, polyvinyl alcohol, and doxycycline increased the rate of wound closure, the volume of collagen, dermal, and epidermis. In addition, it increased the number of fibroblasts and basal cell epidermis cells, vascular length, and decreased the number of neutrophil cells. Inflammatory cytokines and MMP-2 were decreased, and anti-inflammatory IL-10 and TIMP-1 were increased. It was ultimately attained that the combination of chitosan/ polyvinyl alcohol /doxycycline could be a useful dressing for the healing of diabetic wounds.

Mechanical Characteristics of SPG-178 Hydrogels: Optimizing Viscoelastic Properties through Microrheology and Response Surface Methodology

Background: Self-assembling peptides (SApeptides) have growing applications in tissue engineering and regenerative medicine. The application of SApeptide-based hydrogels depends strongly on their viscoelastic properties. Optimizing the properties is of importance in tuning the characteristics of the hydrogels for a variety of applications. Methods: In this study, we employed statistical modeling, conducted with the response surface methodology (RSM) and particle tracking microrheology, to investigate the effects of self-assembling SPG-178 peptide and added NaCl salt concentrations and milieu type (deionized water or blood serum) on the viscoelastic properties of SPG-178 hydrogels. A central composite RSM model was employed for finding the optimum value of the parameters to achieve the highest storage modulus and the lowest tan δ. Results: Viscoelastic properties of each sample, including storage modulus, loss modulus, and tan δ, were determined. Storage modulus and tan δ were modeled, accounting for the impact of the SPG-178 peptide and NaCl concentrations and milieu type on the viscoelastic properties. It was found that the SPG-178 hydrogel storage modulus was positively influenced by the SPG-178 peptide concentration and the serum. Conclusion: A combination of microrheology and RSM is a useful test method for statistical modeling and analysis of rheological behavior of solid-like gels, which could be applied in various biomedical applications such as hemostasis.

3D in vitro cancerous tumor models: Using 3D printers.

Recently, magnetic Hyperthermia is one of the promising methods for cancer treatments. In this method by applying magnetic fields and generating heat, cancerous tissues are eliminated. The degree and pattern of generated heat in cancerous and adjacent non-cancerous tissues plays an important role on the outcome of the treatment. it is mainly affected by diffusion and distribution pattern of magnetic nanoparticles within the cancerous and non-cancerous tissues. Study the diffusion and distribution patterns of magnetic nanoparticle in vivo is difficult and costly in many cases and in some cases evaluating the amount of generated heat at cancer site is almost impossible. In vitro models for cancer tissues are alternatives for in vivo models. However, usual in vitro models could not resembling all the characteristics of a cancer tumor. In this hypothesis we propose that using 3D printers can provide a platform to fabricate a personalized in vitro cancer model which could simulate the most important features of the cancerous tissues (including shape and vascular network) and can be used to study magnetic hyperthermia in a simulated media of compatible to in vivo conditions.

Semi-IPN Films and Electrospun Nanofibers Based on Chitosan/PVA as an Antibacterial Wound Dressing.

The antimicrobial activity of a wound dressing is a key factor for preventing and treating wound infection. The current study evaluated the physiochemical properties and antimicrobial activities of semi-IPNs (interpenetrating polymer networks) based on chitosan/polyvinyl alcohol (PVA) films and nanofibers as candidates for wound dressings and investigated the effects of morphologies (nanofibrous mats and films), crosslinking conditions of chitosan chains (uncrosslinked and crosslinked with genipin), and the presence of antibacterial drug (doxycycline) on their physicochemical and antibacterial properties. The morphology, chemical structure, fluid uptake, water vapor transmission rate, antimicrobial activity, and doxycycline release profile were assayed using SEM, FTIR spectroscopy, swelling test, permeation test, agar diffusion antibiogram, and dissolution test, respectively. The results demonstrated that crosslinking chitosan with genipin reduced the diameter of nanofibers, fluid uptake, and drug release from both nanofiber mats and film samples. According to the results, wound dressings with film morphology have better antimicrobial activity than those with nanofiber. The chitosan/PVA/Doxycycline 1% film has the potential for use as an antimicrobial wound dressing.

On the analysis of microrheological responses of self-assembling RADA16-I peptide hydrogel.

This work aims to obtain a hydrogel based on self-assembling RADA16-I with proper rheological properties for hemostasis application. Response surface methodology (RSM) was performed to predict the gelation and stiffness of the hydrogel in different concentrations of peptide and NaCl in water and blood serum milieus. Particle tracking microrheology technique was used to evaluate Brownian motion of polystyrene particles in the peptide solutions to obtain their trajectories and measure the viscoelastic properties (G'', G″, and tan δ). Formation of gel was influenced by the concentrations of peptide and salt and their interactions. Optimum response for maximizing elastic modulus was obtained in the presence of blood serum in comparison with water. Negative effect of excess amount of NaCl was predicted by RSM model and confirmed by animal study. Circular dichroism (CD) analysis showed formation of ß-sheet secondary structure in water. On the other hand, in the presence of blood serum, tertiary structure was formed. Dimensional characterization of peptide fibers was performed by means of AFM. Peptide self-assembly in blood serum (pH around 7) which contains different ions, led to enhancing bonds between fibers, caused increasing the fiber diameter and length by 20 and 10 times, respectively. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 330-338, 2019.

Chitosan/polyethylene glycol fumarate blend films for wound dressing application: in vitro biocompatibility and biodegradability assays.

Blending is one of the effective approaches in preparing tailored materials with a wide range of properties. Thus, chitosan-based polymers have been fabricated and used as wound dressings since they possess better properties than those of the constituent materials. The objective of this work was to evaluate the biocompatibility and biodegradability of biodegradable blend films based on polyethylene glycol-co-fumarate (PEGF) and chitosan (Ch). The blend films of Ch/PEGF were prepared by solution casting/solvent evaporation method. Degradation behavior of these blend films was evaluated in a simulated fluid at physiological pH supplemented with lysozyme at a concentration similar to that in human serum by weight loss of the films and changes in the pH of media. When the pH of incubation media was analyzed, with an increase of PEGF content in the blend films, the degradation rate increased accordingly. The pH of the media of samples was not significantly changed at any measured time point and all films kept their integrities during 28 days. The biocompatibility of the films and cell behavior on the surface of these films were investigated by in vitro tests. Biological assessment using mouse fibroblast cell line L929 on the blend films of Ch/PEGF indicated that films supported the attachment, spreading and proliferation of cells. Since the Ch/PEGF films are biocompatible with the tailored biodegradation rate, they might have a great prospective position in the application of wound dressings.

Combinational drug delivery using nanocarriers for breast cancer treatments: A review.

Breast cancer (BC) is the most common cancer in women that requires special attention due to low response to conventional treatments. The common method for treating cancer (especially BC) is applying a single anticancer agent, however, due to some disadvantages including cytotoxicity, side effects, and multidrug resistance, the efficiency and application of this method are limited. To overcome these challenges, the combinational delivery of anticancer drugs (including chemical agents, genetic materials, etc.) has been introduced. To increase the efficacy of this new method, several nanocarriers including inorganic nanoparticles (such as, magnetic nanoparticles, silica nanoparticles, etc.) and organic ones (e.g., dendrimers, liposomes, micelles, and polymeric nanoparticles) have been used. Based on the literature, combinational delivery using nanocarriers showed promising results in the treatment of BC. In this review, combination regimens for the treatment of BC, nanocarriers containing combinations of pharmaceutical agents (including small molecule chemotherapeutic, biological, and gene therapy agents) as an opportunity to overcome chemotherapy challenges and, finally, examples of these formulations have been presented. This review aims to provide a better understanding of these increasingly important new methods of cancer treatment and the main issues and key considerations for a rational design of nanocarriers used in combinational delivery of different synergistic anticancer agents. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2272-2283, 2018.

Effect of electrospinning parameters on morphological properties of PVDF nanofibrous scaffolds.

Smart materials like piezoelectric polymers represent a new class of promising scaffold in neural tissue engineering. In the current study, the fabrication processing parameters of polyvinylidine fluoride (PVDF) nanofibrous scaffold are found as a potential scaffold with nanoscale morphology and microscale alignment. Electrospinning technique with the ability to mimic the structure and function of an extracellular matrix is a preferable method to customize the scaffold features. PVDF nanofibrous scaffolds were successfully fabricated by the electrospinning technique. The influence of PVDF solution concentration and other processing parameters like applied voltage, tip-to-collector distance, feeding rate, collector speed and the solvent were studied. The optimal parameters were 30 w/v% PVDF concentration, 15 kV applied voltage, 18 cm tip-to-collector distance, 0.5 ml/h feeding rate, 2500 rpm collector speed and N,N'-dimethylacetamide/acetone as a solvent. The mean fiber diameter of the obtained scaffold was 352.9 ± 24 nm with uniform and aligned morphology. Finally, the cell viability and morphology of PC-12 cells on the optimum scaffold indicated the potential of PVDF nanofibrous scaffold for neural tissue engineering.

Biodegradable microrobots for targeting cell delivery.

These days, cell delivery is considered a potential method for treatment of many genetic diseases or tissue regeneration applications. In conventional cell delivery methods, cells are encapsulated in or cultured on biocompatible polymers. However, the main problem with these carriers is their lack of targeting ability. For tissue regeneration or many cell treatments, it is needed to deliver cells to a specific site of action. Magnetic microrobots based on industrial photoresists have been studied in literature for magnetically controllable carriers. However, there are some issues about biodegradation and removal of these microrobots from the body. In this paper, we hypothesis fabrication of new generation of biodegradable magnetic microrobots based on additive manufacturing methods to overcome this problem and to bring this evolving field to a new level.

Piezoelectric electrospun nanocomposite comprising Au NPs/PVDF for nerve tissue engineering.

In this study, gold nanoparticles/Polyvinylidenefluoride (PVDF) composite electrospun mat with enhanced piezoelectricity were fabricated and characterized. Gold colloidal nanoparticles (Au NPs) were prepared via laser ablation of metallic targets in liquid media. The active Q-switched Nd:YAG laser was used as an irradiation source. Then, PVDF was dissolved in Au NPs colloidal solution at 30% wt for the synthesis of Au NPs/PVDF composite nanofibers by electrospinning. The optical absorbance spectra of Au NPS and the polymeric solutions were obtained by the UV-Visible spectroscopy. Moreover, the morphology of Au NPS, nanostructures of fibers and diameter size distribution of nanofibers were analyzed by Scanning Electron Microscopy, Field Emission Scanning Electron Microscopy, and Transmitted Electron Microscopy methods. The crystallinity and piezoelectricity of PVDF and Au NPs/PVDF composite nanofibers mats were measured by X-Ray Diffraction and Fourier Transform Infrared methods. Subsequently, in vitro cytocompatibility was evaluated by MTT assay and the attachment and morphology of PC-12 cells cultured on scaffolds were studied. It was found that laser ablated Au NPs can be used in electrospun nanofibers of PVDF with adequate structural properties and increase piezoelectricity of nanofibers which might be suitable for applying as nerve tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1984-1993, 2017.

Fabrication of cancellous biomimetic chitosan-based nanocomposite scaffolds applying a combinational method for bone tissue engineering.

The aim of this study was to mimic the specific structure of bone and fabricate a biomimetic nano-hydroxyapatite (HA)/chitosan (Cs)/gelatin scaffolds using combination of particle leaching and freeze drying methods eliminating mold effects. To achieve an optimum structure, scaffolds with different gelatin/Cs weight ratio were fabricated. Morphological characterization of scaffolds by scanning electron microscopy method showed highly interconnected porous structures similar to cancellous bone with mean pore size ranging from 140 to 190 µm. Nano-HA crystals were dispersed homogeneously in the polymer matrix according to the energy-dispersive X-ray spectroscopy and transmission electron microscopy images. Fourier transform infrared and X-ray diffraction results disclosed that chemical interactions were formed between nano-HA, Cs, gelatin and crystallinity of each material decreased with blending. It was found that increasing the gelatin content significantly improved water uptake, degradation rate as well as attachment, infiltration and proliferation of Saos2 cells to the scaffolds. The presented results confirm that the designed biomimetic nano-HA /Cs/gelatin scaffolds can be used as promising substitutes for bone tissue engineering.

Particle size modeling and morphology study of chitosan/gelatin/nanohydroxyapatite nanocomposite microspheres for bone tissue engineering.

In this study, nanocomposite microspheres based on chitosan/gelatin/nanohydroxyapatite were fabricated, and effects of the nanohydroxyapatite/biopolymer (chitosan/gelatin) weight ratio (nHA/P), stirring rate, chitosan concentration and biopolymer concentration on the particle size, and morphology of nanocomposite microspheres were investigated. Particle size of microspheres was modeled by design of experiments using the surface response method. Particle size, morphology of microspheres, and distribution of nanoparticles within the composite microspheres were evaluated using an optical microscope, scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. X-ray diffraction and Fourier transform infrared spectroscopy were applied to study the physical and chemical characteristics of microspheres. Results showed that by modulating the nHA/P ratio, chitosan concentration, polymer concentration, and stirring rate, it is possible to fabricate microspheres in wide rages of particle size (5-150 µm). Analysis of variance confirmed that the modified quadratic model can be used to predict the particle size of nanocomposite microspheres within the design space. SEM studies showed that microspheres with different compositions had totally different morphologies from dense morphologies to porous ones. TEM images demonstrated that nanoparticles were distributed uniformly within the polymeric matrix. MTT assay and cell culture studies showed that microspheres with different compositions possessed good biocompatibility. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.