Artificial Cells, Nanomedicine, and Biotechnology An International Journal

ISSN: 2169-1401 (Print) 2169-141X (Online) Journal homepage: http://www.tandfonline.com/loi/ianb20

Differentiation of human endometrial stem cells into endothelial-like cells on gelatin/chitosan/ bioglass nanofibrous scaffolds Atefeh Shamosi, Davood Mehrabani, Mahmoud Azami, Somayeh EbrahimiBarough, Vahid Siavashi, Hossein Ghanbari, Esmaeel Sharifi, Reza Roozafzoon & Jafar Ai To cite this article: Atefeh Shamosi, Davood Mehrabani, Mahmoud Azami, Somayeh EbrahimiBarough, Vahid Siavashi, Hossein Ghanbari, Esmaeel Sharifi, Reza Roozafzoon & Jafar Ai (2016): Differentiation of human endometrial stem cells into endothelial-like cells on gelatin/chitosan/ bioglass nanofibrous scaffolds, Artificial Cells, Nanomedicine, and Biotechnology To link to this article: http://dx.doi.org/10.3109/21691401.2016.1138493

Published online: 16 Feb 2016.

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Date: 16 February 2016, At: 09:54

ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2016 http://dx.doi.org/10.3109/21691401.2016.1138493

Differentiation of human endometrial stem cells into endothelial-like cells on gelatin/chitosan/bioglass nanofibrous scaffolds Atefeh Shamosia, Davood Mehrabanib,c, Mahmoud Azamia, Somayeh Ebrahimi-Barougha, Vahid Siavashid, Hossein Ghanbarie, Esmaeel Sharifia, Reza Roozafzoona and Jafar Aia,f a

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Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran; bStem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; cDepartment of Regenerative Medicine, University of Manitoba, Winnipeg, Canada; dDepartment of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran; eDepartment of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran; fBrain and Spinal Injury Research Center, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran

ABSTRACT

ARTICLE HISTORY

The capacity of gelatin/chitosan/bioactive glass nanopowders (GEL/CS/BGNPs) scaffolds was investigated for increasing human endometrial stem cells (hEnSCs) differentiation into the endothelial cells in the presence of angiogenic factors. GEL/CS nanofibrous scaffold with different contents of BGNPs were fabricated and assessed. Expression of endothelial markers (CD31, vascular endothelial cadherin (VE-cadherin), and KDR) in differentiated cells was evaluated. Results showed the diameter of nanofiber increases with decreasing the BG content in GEL/CS scaffolds. Moreover, in vitro study indicated that the GEL/CS/BGNPs scaffold with 1.5% BGNPs content provided a suitable three-dimensional structure for endothelial cells differentiation. Thus, the GEL/CS/BGNPs scaffold can be recommended for blood vessels repair.

Received 21 November 2015 Accepted 2 January 2016 Published online 11 February 2016

Introduction One of the major obstacles to promote repair and regeneration during injury is the lack of neovascularization resulting into absence of new and sufficient blood vessel formation to transfer oxygen and nutrients to the injured cells and to remove the metabolic wastes (Walthers et al. 2014). A variety of cytokines and growth factors play significant roles in controlling and inhibiting capillary network development in tissue regeneration (Soulitzis et al. 2006). Vascular grafts with56 mm internal diameter were shown to be thrombogenic and lead to an incomplete vascular healing, so application of vascular grafts with smaller internal diameter is still a challenge in vascular regeneration (Lovett et al. 2010). In this approach, a biodegradable scaffold is required to support as an adhesive substrate for the seeded cells and a physical preservation to facilitate the formation of the angiogenesis when vascular grafts with small internal diameter (56 mm) cannot be applied (Heyligers et al. 2005, Hoerstrup et al. 2001, Meinhart et al. 1997). Indeed, to develop a capillary network with the capability to deliver adequate nutrients to migrated cells within the scaffold is crucial for the cell survival and the healing improvement after implantation (Rouwkema et al. 2008). Moreover, the distribution of natural tissue cells is generally limited to a distance of 150 mm away from the nearest capillary vessel, which is the efficient diffusion distance of nutrients and oxygen (Lovett et al. 2009). Thus, as in the formation of native tissue, angiogenesis following scaffold implantation is of critical

KEYWORDS

Endothelial cells; gelatin/chitosan/bioglass; human endometrial stem cells; nanofiber scaffold

significance for the development, functionality, and integration of engineered tissue (Rouwkema et al. 2008). In the field of nanotechnology in tissue engineering, the electrospinning processing technique has been receiving extensively increasing attention in vascular tissue engineering because of its attractive properties, such as fabricating ultrafine polymer fibers with diameters in the range of micrometers to nanometers that mimic the natural extracellular matrices (ECM) (Jegal et al. 2011). The architecture and surface morphology of these nanofibers can be balanced by modifying the content or composition of the components (Grafahrend et al. 2011, Lee et al. 2008, Zhang et al. 2010). Various composite scaffolds have been fabricated by combining natural biopolymer or synthetic polymer with inorganic bioactive materials (Jie and Yubao 2004, Li et al. 2001, Liao et al. 2007, Petricca et al. 2006). Bioactive glass (BG) has commonly been used for its osteoinductive and osteoconductive capabilities; recent studies have showed BG exhibit angiogenic effect in vitro (Day 2005, Leu and Leach 2008, Shamosi et al. 2015) and in healthy animal models (Day et al. 2004, Leach et al. 2006). Polymeric biomaterials used to fabricate Ebrous scaffolds usually include natural biopolymers and chemically prepared polymers such as collagen, chitosan (CS), and gelatin (GEL). Gelatin is a biocompatible and biodegradable protein with a variety of applications in multiple fields of science (Kathuria et al. 2009, Hoveizi et al. 2015). As chemical composition of gelatin is very similar to natural collagen, it can be a suitable candidate as

CONTACT Jafar Ai [email protected] Professor of Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, [email protected] Professor of Stem Cell and Transgenic Technology Research Tehran University of Medical Sciences, Tehran, Iran; Davood Mehrabani Center, Shiraz University of Medical Sciences, Shiraz, Iran ß 2016 Taylor & Francis

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biomimetic scaffolds for tissue engineering. Chitosan is a natural, biocompatible, and biodegradable polysaccharide material derived from chitin which is structurally similar to glycosaminoglycans (Chen et al. 2010), a main component of ECM that shows antimicrobial activity, good cell adhesion, and proliferation properties (Kong et al. 2010). Endometrial stem cells (EnSCs) have been determined in humans (Ghobadi et al. 2015) and were shown to have critical roles in uterine function and cyclic renewal and share a prominent contribution in regeneration and repair of a normal endometrium (Sakr et al. 2014). This regenerative capacity is due to presence of EnSCs that were demonstrated to be immuno-privileged in comparison with other cell types, shedding a new light for cell-based therapies and rendering these cells as a promising resource in regenerative medicine (Shamosi et al. 2015, Ulrich et al. 2013). Their differentiation potential to mesodermal and ectodermal cellular lineages, such as hepatocyte (Khademi et al. 2014, Niknamasl et al. 2014), neural cells (Ai and Mehrabani 2010b, Asmani et al. 2013, Ebrahimi-Barough et al. 2015), osteoblasts (Ai and Mehrabani 2010c) and heart muscles were previously shown (Ai and Mehrabani 2010a). This study aimed to evaluate the effect of the nanofibrous scaffolds on human EnSCs (hEnSCs) differentiation into endothelial cells lineage. Human EnSCs were induced to differentiate into endothelial-like cells in the presence of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) in vitro on tissue culture plate (TCP) and scaffolds. Then, differentiated endothelial-like cells were investigated morphologically and expression of endothelial markers was studied using real-time PCR and immunocytochemical analysis for GEL/ CS, GEL/CS/BGNPs (bioactive glass nanopowders), and TCP groups.

Materials and methods GEL/CS solution preparation, electrospinning process, and SEM analysis GEL/CS solution was prepared through a process that was previously reported (Jafari et al. 2011). CS (575% deacetylated, Sigma-Aldrich, St. Louis, MO) and gelatin A (GEL, Sigma, Type A from porcine Skin) were dissolved in 10 ml aqueous acetic acid (80% v/v) (Merck), respectively to make a CS solution of 2% (w/ v) and a GEL solution of 18% (w/v). The 18% GEL solution (w/v) was mixed with the 2% CS solution (w/v) at different w/w ratios (GEL/CS: A1: 30/70, A2: 40/60, A3: 50/50, A4: 60/40, and A5: 70/ 30) and were stirred at 60  C for 2 h. Each prepared solution was filled into a 5 ml plastic syringe with an 18-gauge needle and connected to a high voltage power supply. The electrospinning was performed under voltages of 15 kV, distance of 15 cm and the flow rate of 0.4 ml/h at room temperature. The morphology of GEL/CS nanofibers was assessed using a scanning electron microscope (SEM, Philips, XL30 microscope) at an accelerating voltage of 15 kV after samples were coated with a homogeneous gold layer by using a sputter coater.

(TEOS, Merck, Kenilworth, NJ) was added into 30 ml of 0.1 M nitric acid (HNO3, Merck, Kenilworth, NJ), the mixture was allowed to react for 45 min for the acid hydrolysis of TEOS. The following reagents were added sequentially, allowing 2 h for each reagent to react completely: 0. 91 g of triethyl phosphate (TEP, Merck, Kenilworth, NJ), 6.14 g of calcium nitrate tetrahydrate (Ca (NO3)2_4H2O, Merck, Kenilworth, NJ), and 1.28 g of magnesium nitrate hexahydrate (Mg (NO3)2_6H2O, Merck, Kenilworth, NJ). The solution was maintained in a cylindrical Teflon container for 10 d at room temperature to allow a polycondensation reaction to occur until the gel was formed. The gel was heated at 70  C for 3 d, the water was removed and a hole was inserted in the lid of container to allow the leakage of gases while the gel was heated to 120  C for 2 d to remove all the water. To stabilize the glass structure, the powder was heated at 600  C for 24 h. The product was grinded by ball milling (SVD15IG5–1, LG Company) for 10 h to get homogeneous powder. Finally, its crystal structure of BGNPs was examined by using X-ray diffraction (XRD, Philips PW 3710, Netherlands) analysis to identify the amorphous structure of BGNPs.

Preparation of GEL/CS/BGNPs solutions and electrospinning process CS (575% deacetylated, Sigma-Aldrich, St. Louis, MO) and GEL (Sigma-Aldrich, St. Louis, MO, Type A from porcine Skin) were dissolved in 10 ml aqueous acetic acid (80% v/v), respectively to make a CS solution of 2% (w/v) and a GEL solution of 18% (w/v). GEL and CS polymers were mixed in a ratio of 70/30 (w/w) (GEL/ CS). The loadings of BGNPs in GEL/CS solutions were set as 0.5, 1.5, and 3 wt% (GEL/CS/BGNPs solutions: B1: 70:30:0.5, B2: 70:30:1.5, B3: 70:30:3 (w/w/w)). For electrospinning, polymer blend of GEL/CS/BGNPs solution was Elled in a 5 ml plastic syringe with an 18-gauge flat-tip needle. The Fow rate of polymer solution was adjusted at 1.2 ml/h using a syringe pump. A high applied voltage was regulated at 21 kV to dominate the surface tension, and a distance of 13 cm between collector and needle tip was maintained during the electrospinning process. The GEL/CS/BGNPs nanofibers were then cross-linked with glutaraldehyde vapor for 12 h and soaked in deionized water for 48 h.

Scaffold characterization The morphology and fiber uniformity of nanofibrous scaffolds (B1, B2, and B3) were observed by SEM. Energy-dispersive X-ray spectroscopy (EDX, Bruker, Carl Zeiss, Germany) attached to SEM was employed to study the atomic elemental analysis of composite nanofibrous scaffolds (B1, B2, and B3). Chemical band structure of nanofibrous scaffolds were assessed by using Fourier transform infrared (FTIR) spectroscopy (Thermo-Nicolet). The diameter of each fiber was computed by 10 random measurements using image J software (NIH, Bethesda, MD).

Preparation of BGNPs and characterization

HEnSC isolation and characterization

The sol-gel derived BG consisting of SiO2, MgO, CaO, and P2O5 was synthesized as follows: 13.13 g of tetraethyl orthosilicate

Written informed consent (according to instruction of ethics committee of Tehran University of Medical Sciences) was

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obtained from each donor after a full explanation of the procedures and aims of the study. EnSCs isolation were obtained from endometrial biopsies sampled during the proliferative phase of 19- to 25-year-old women who had not received a hormonal exogenous treatment for at least three months before surgery. HEnSCs were isolated according to the previously published report (Ebrahimi-Barough et al. 2013). The obtained biopsy was maintained in Hanks balanced salt solution (HBSS; Invitrogen, Carlsbad, CA) with 100 IU/ml penicillin (GIBCO Laboratories, Grand Island, NY), and 100 IU/ml streptomycin (GIBCO Laboratories, a Island, NY). The sample was enzymatically digested in 0.3% (w/v) collagenase type I (GIBCO Laboratories, a Island, NY) at 37  C for 60 min. The suspension was then neutralized by Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, Gibco) containing 10% (v/v) fetal bovine serum (FBS, GIBCO Laboratories, a Island, NY), 100 IU/ml penicillin, and 100 IU/ml streptomycin, passed through 70 and 40 mm filters and centrifuged at 1500 rpm for 5 min. The pellet of hEnSCs was resuspended in DMEM/F12 supplemented with 10% FBS, 100 IU/ml penicillin, and 100 IU/ml streptomycin. Cultures were incubated in 5% (v/v) CO2 at 37  C for 10 d. The medium was first changed 24 h after plating and thereafter regularly changed twice a week. After passage three, hEnSCs were characterized using flow cytometry for cell surface markers including CD105, CD29, CD73, CD45, CD34, and CD31.

Cell seeding on scaffolds Nanofibrous scaffolds were sterilized by UV radiation for 3 h, washed two times with PBS for 1 h and incubated in DMEM/F12 with 10% FBS for 24 h before cell seeding. After the hEnSCs reached to 80–90% confluency, they were detached by incubation with trypsin/EDTA (0.25% trypsin and 0.1% EDTA) solution for 5 min at 37  C to create a single cell suspension. Then, 5  104 cells were seeded onto the top of the nanofibrous scaffold and incubated in DMEM/F12, 10% FBS, 100 IU/ml penicillin, and 100 IU/ml streptomycin for 3 h to allow hEnSCs to attach onto the surface of the nanofibrous scaffold, and fresh complete medium was added to each well for further incubation.

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dehydrogenases enzymes of living and healthy cells to oxidize a tetrazolium salt (3–(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into an insoluble purple formazan product. HEnSCs was cultured in DMEM with 10% FBS, 100 IU/ml penicillin, and 100 IU/ml streptomycin at 37  C under a 5% CO2 incubator. All nanofibrous scaffolds were punched into 18 mm and then sterilized with UV for 2 h both side each. The sterilized scaffolds were transferred in the 24-well tissue culture plate and incubated in DMEM/F12 with 10% FBS overnight to improve cell attachment on scaffolds’ surfaces. Then, hEnSCs at a density of 5  104 cells/scaffold were added in each well and incubated for 1, 3, and 5 d. A quantity of 40 ml of 5 mg/ml stock MTT solution was added to each well containing 400 ml of medium, and the plates were incubated at 37  C for 4 h. The medium was discarded and the formazan crystals were dissolved in 400 ml of dimethyl sulfoxide (DMSO). Subsequently, the formazan solution was transferred to a 96-well microplate plate in triplicate, and MTT assay was detected with measuring the absorbance of each well at 570 nm by a spectrophotometric plate reader (Rayeto).

Endothelial-like cells induction of hEnSCs on scaffolds and TCP For endothelial induction, hEnSCs were treated with induction media containing angiogenic growth factors. 2  104 hEnSCs of the 2th passage were seeded on both GEL/CS and GEL/CS/ 1.5%BGNPs nanofibrous scaffolds and TCP. The cells were cultured in DMEM/F12, 10% (v/v) FBS and 100 U/ml penicillin, and 100 U/ml streptomycin for 24 h. For inducing differentiation in hEnSCs toward endothelial cells, the DMEM/F12 was removed and substituted with angiogenic medium for 17 d. Angiogenic medium consists of DMEM/F12 supplemented with 2% FBS, 10 ng/ml FGF-2 (Sigma-Aldrich, St. Louis, MO), and 1% penicillin/streptomycin for 3 d; and DMEM/F12, 2% FBS, 10 ng/ ml FGF-2 and 50 ng/ml VEGF (Sigma-Aldrich, St. Louis, MO), and 1% penicillin/streptomycin for another 14 d. During this time, cells were observed under a phase-contrast microscope every day and assessed their morphological changes post treatment. The media were changed every 2 d.

Immunocytochemical analysis Evaluation of cell attachment and morphology of hEnSCs on nanofibrous scaffolds Morphological assessment of the cultured hEnSCs after 3 d post seeding on GEL/CS and GEL/CS/BGNPs scaffolds were performed using SEM. After 3 d, cell-containing scaffolds were fixed with 300 ml glutaraldehyde (2.5% in PBS) for 1 h at room temperature, washed with PBS for 5 min and dehydrated in ascending ethanol series (30, 50, 70, 80, 90, and 100% at 37  C for 5 min each). Finally, the samples were sputter-coated with thin layer of gold and studied using a SEM, operated at 15 kV.

Cell viability and proliferation assay MTT assay was performed to investigate the cell viability of hEnSCs on nanofibrous scaffolds for 1, 3, and 5 d of cell culture. MTT assay indicates the capacity of mitochondrial

After 17d of induction, cells were fixed with 4% (w/v) paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in PBS for 20 min at 4 C and permeabilized using 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature. After washing with PBS (three times, 5 min each), all samples were blocked by 5% bovine serum albumin (BSA, SigmaAldrich, St. Louis, MO) for 30 min at room temperature, incubated overnight at 4  C with the primary antibodies against CD31/PECAM1 (mouse monoclonal anti-human antibody; Abcam, 1:200), VE-Cadherin (vascular endothelial cadherin; mouse monoclonal anti-human antibody; R & D systems, Abingdon, UK, 1:100), and KDR (mouse monoclonal antihuman; Abcam, Cambridge, UK, 1:200) diluted in 5% BSA in PBS. Secondary antibodies included Alexa Fluor 488 Goat AntiMouse IgG (1:500) and Alexa Fluor 594 Goat Anti–Mouse IgG (1:700) for 1 h at room temperature, and nuclei were

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counterstained with DAPI (49, 6-diamidino-2- phenylindole, Sigma-Aldrich, St. Louis, MO). For negative control specimens, only the secondary antibodies were applied. No specific positive staining pattern was observed in either case. Cells were then studied under fluorescence microscope (Olympus BX51, Japan). For cell counting, at least four microscopic fields were studied randomly for each well; the number of DAPIstained nucleus (at least 200 cells) and the number of Alexa Fluor 488-labeled VE cadherin+ and KDR+ and Alexa Fluor 594labeled CD31/PECAM1+ in the same fields were counted. The ratio of the positive cells (stained cells) was calculated by dividing the number of positive cells over the total number of cells (DAPI-stained nucleus). For each used antibody, three independent experiments were used and results were determined as percentage of positive stained cells in each group.

Results SEM analysis of GEL/CS scaffolds

Real-time PCR analysis

The diffraction pattern of the BG was obtained by XRD analysis and is demonstrated in Figure 2(A). The lack of any peaks in XRD pattern shows that sol–gel procedure is able to form noncrystalline amorphous structure of BG at 600  C. SEM images of electrospun GEL/CS in different content of BGNPs can be seen in Figure 2(B). It was shown that there was marked difference in the diameter of nanofibers with different contents of BGNPs. As indicated in Figure 2(B), increasing the content of BGNPs up to 1.5% was revealed to maintain the electrospinnability and uniform fiber morphology, whereas fiber diameter was shown to decrease. Micro-beads were found to increase exponentially (from 185 nm to 6.05 mm) with increase in the content of BGNPs. The EDAX analysis of GEL/CS/BGNPs nanofibers shows the presence of Ca, O, Mg, P, Si (atomic composition of BG), and C, O, N (atomic composition of GEL and CS) elements (Figure 2B). Figure 2(C) (obtained results from image J) indicates that the most suitable ratio for endothelial differentiation was GEL/ CS to BGNPs (70:30:1.5 wt%) and the reason was diameter, uniformity, and consistency of fibers. The result revealed that the percent content of BGNPs in GEL/CS/BGNPs scaffolds had clear impact on the homogeneity and diameters of the fiber.

Total RNAs were extracted from differentiated cells by RNeasy plus Mini Kit (Qiagen, Japan) according to the manufacturer’s instructions and complementary DNA (cDNA) was made by PrimeScript First Strand cDNA Synthesis Kit (Takara Bio Inc., Japan). The SYBR1 Premix Ex TaqTM II (Tli RNaseH Plus) was used for evaluating the expression of FLT1, PECAM, VwF, VECAD, and GAPDH as a housekeeping gene and monitored in Rotor-gene Q real-time analyzer (Corbett). The oligo nucleotide primer pairs used for real-time PCR reaction are listed in Table 1. The investigations were conducted on 2D and 3D culture samples. RT–PCR data were analyzed using comparative threshold cycle method, and each amplification was repeated in triplicate. Relative gene expression was analyzed by REST-384 software (Pfaffl et al. 2002).

Statistical analysis SPSS software version 11.5 (SPSS Inc., Chicago, IL) was used for statistical analysis. Data were expressed as the mean ± standard deviation (SD). Analytical comparisons were assessed by oneway ANOVA test. The P values were considered significant at P50.05, P50.01, and P50.001.

Different ratios of GEL/CS (A1, A2, A3, A4, and A5) were electrospinned and uniformity and morphology of nanofibers were studied by SEM (Figure 1). Fiber morphology and diameter of GEL/CS nanofibrous scaffolds were affected by ratio of GEL to CS. The average diameter and uniformity of GEL/ CS nanofibers increased due to the decrease in viscosity of the blended solutions by increasing the content of GEL component. SEM results revealed that bead-free uniform fibers were formed when the GEL/CS ratio equals to 70/30 (A5) (weight ratio), as shown in Figure 1(E).

Characterization of BGNPs and nanofibrous scaffolds

Table 1. Primer sequences used for real-time PCR. Gene

Accession no.

FLT1 PECAM VWF VECAD Gapdh

NM_002019 NM_000442 NM_000552 NM_004360 NM_002046

primer sequence (50 –30 ) F F F F F

ACCATAATCATTCCG AAG CAAG G R TAGAGTCAGCCACAACCAAGG AGCGTCATTGGCGTGTTG R CTCCTTCCCGTTTACCTTTCC CTTGGTCACATCTTCACATTCAC R AAGTCATTGGCTCCGTTCTC TGAAGGTGACAGAGCCTCTGGAT R TGGGTGAATTCGGGCTTGTT ACATCATCCCTGCCTCTACTG R CCTG CTTCACCA CCTTCTTG

Figure 1. SEM images of electrospun fibers of GEL/CS: (A) 30/70, (B) 40/60, (C) 50/50, (D) 60/40, and (E) 70/30(wt./wt.).

Size (bp)

Annealing ( C)

168 100 125 151 180

60 60 60 60 60

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Figure 2. (A) X-ray diffraction of BGNPs. (B) FTIR spectra showed the presence of chemical band groups related to GEL/CS/BGNPs nanofibrous scaffolds. (C) SEM images revealing the topography of B1, B2, and B3 nanofibrous scaffolds. EDX analysis showing atomic composition of BGNPs ions (Ca, P, Si, and Mg) peaks were incorporated into GEL/CS nanofibrous scaffolds. (D) The average diameter of fibers with different contents of BGNPs in GEL/CS/ BGNPs nanofibrous scaffolds. Error bars show the mean ± SD.

Figure 2(D) reveals the FTIR spectra for cross-linked GEL/CS nanofibrous composite containing BGNPs. FTIR spectra of GEL/ CS/BGNPs nanofibrous composite showed a number of chemical spectral bands including C¼O stretching vibration at 1663 cm1 for the protein amide I band, C–H stretch and N–H

bend at 1546 cm1 for amide II, N–H bending and C–N stretch at 1244 cm1 for amide III groups, and N–H stretch at 3300 cm1 for the amide band which are attributing to chemical characteristics of GEL. The vibrational modes at 3340 cm1 (O–H vibration), 2920 cm1 (CH3 vibration),

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1560 cm1 (C¼O) are characteristic of CS structure (Gylien_e et al. 2013, Mohamed and Mostafa 2008). The other vibrations that can be attributed to BG are found at 1042 cm1 of Si–O–Si stretching, and 1024 cm1 of po4 stretching, respectively (Mozafari et al. 2010, Mukundan et al. 2013).

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HEnSCs culture and characterization After culturing for 2 d, some adherent cells appeared in the bottom of the flask. After nearly 10 d of plating, these cells proliferated into numerous clusters, and could be used for the first subculture. After at least three passages, hEnSCs became relatively homogeneous, elongated, or spindle-shaped in appearance. After three passages, specific surface markers of these cells were characterized using flow cytometric analysis in order to ensure the purity of the stem cell population. Flow cytometric analysis indicated that the hEnSCs were positive for CD105+(95.11), CD29+(95.95), and CD73+(61.34) (mesenchymal stem cell markers) and negative for CD34, CD45 (hematopoietic markers), and CD31 (endothelial marker) (Figure 3).

Evaluation of cell adhesion and viability SEM images of cell adhesion on GEL/CS (70:30) and GEL/CS/ BGNPs (70:30:1.5%) electrospun nanofibrous scaffolds after 3 d of culture was shown in Figure 4(A) and (C). After seeding the same number of hEnSCs on nanofibrous scaffolds, SEM images

revealed attachment and spread of hEnSCs on both of the electrospun scaffolds, but higher hEnSC attachment and proliferation on GEL/CS/BGNPs scaffolds compared to GEL/CS nanofibers were seen. These results show a better interaction of cells with GEL/CS/BGNPs nanofibrous scaffolds. DAPI staining of cultured cells on GEL/CS and GEL/CS/BGNPs nanofibrous scaffolds also revealed the better attachment of hEnSCs on the GEL/CS/BGNPs scaffolds (Figure 4B and D) that was in accord with the obtained SEM results.

SEM results MTT assay was performed to determine the viability and the rate of hEnSCs proliferation on GEL/CS and GEL/CS/BGNPs nanofibrous scaffolds for 1, 3, and 5 d of culture as shown in Figure 4(E). The MTT results after 24 h showed that hEnSCs were not only alive but also proliferated in the presence of GEL/CS/ BGNPs nanofibrous scaffold, which confirms the biocompatibility of the prepared scaffold during cell culturing period. At day 3 of culture, GEL/CS nanofibrous scaffold also indicated the increased cell proliferation and cell viability rate of hEnSCs during the culture period, but GEL/CS/BGNPs scaffold showed the higher cell proliferation and viability rate compared with other cell cultures (TCP and GEL/CS scaffold). The results of MTT assay showed that GEL/CS/BGNPs scaffold is a suitable nanofibrous substrate than GEL/CS nanofibers and TCP for cell attachment and proliferation.

Figure 3. Flow cytometric analysis of the isolated hEnSCs for mesenchymal stem cell markers (CD105, CD29, and CD73), hematopoietic markers (CD34 and CD45) and endothelial marker(CD31). As shown, the isolated cells are positive for CD105, CD29, and 73 and are negative for CD34, CD45, and CD31.

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Figure 4. SEM micrographs and DAPI staining of hEnSCs after 3 days of cell culture on GEL/CS (A and B) and GEL/CS/BGNPs nanofibrous scaffolds (C and D). (E) MTT assay of hEnSCs was seeded on GEL/CS and GEL/CS/BGNPs scaffolds and TCP after 1, 3, and 5 days. Data are presented as mean ± SD of three independent experiments (*P50.05; **P50.01, ***P50.001), (n ¼ 3).

Figure 5. Morphological characterization of endothelial-like cells induced from hEnSCs. Phase-contrast images of hEnSCs before and after the treatment in TCP group. During the inductions, the morphology of cells changed from a spindle cell shape to a polygonal-shaped endothelial cell on day 17.

Evaluation of hEnSCs differentiation into endothelial-like cells on nanofibrous scaffolds and TCP To induce endothelial-like cell differentiation of hEnSCs on TCP and nanofibrous scaffolds, the cells were treated with signaling molecules including FGF-2 and VEGF for 17 d. To identify the endothelial differentiation morphologically, the cells were observed daily by using a phase-contrast microscope in TCP group (Figure 5). After 24 h post treatment, cells adherent to TCP showed spindle-shaped appearance. Seven days after induction by signaling molecules, cells were elongated and flattened, exhibiting endothelial cell-like shape. These endothelial cell-like cells proceeded to develop, endothelial cell morphology appeared in cell culture and cells became polygonal-shaped endothelial cells at the end of the induction period (Day 17).

Differentiation analysis of hEnSCs-derived endothelial cells by immunocytochemistry and real-time PCR To investigate endothelial differentiation, the expression of endothelial-specific markers in differentiated endothelial-like cells was assessed by using immunocytochemical staining and real-time PCR analysis in TCP and nanofibrous scaffolds. After

17 d post treatment, immunocytochemistry analysis showed that endothelial biomarkers (CD31, KDR, and VE Cad) were expressed in angiogenic-induced cells in TCP, GEL/CS, and GEL/ CS/BGNPs nanofibrous scaffolds groups (Figure 6A). Control group was negative for these markers post treatment (17th day). Immunocytochemical study results indicated higher expression of these endothelial markers in GEL/CS/ BGNPs compared with cell samples differentiated in GEL/CS and TCP (Figure 6B). For the analysis of expression of endothelial cell markers at the level of messenger RNA (mRNA), real-time PCR was performed in 2D control and 3D cell culture. The expression of FLT1, PECAM, VECAD, and VwF markers was assessed after 17 d of angiogenesis induction. Our results revealed that expression of endothelial-specific markers significantly increased (P50.001) in cells differentiated on GEL/ CS/BGNPs compared with TCP and GEL/CS groups. Our result showed that GEL/CS/BGNPs nanofibrous scaffolds could provide a suitable 3D structure for differentiation of hEnSCs to endothelial cells (Figure 6C).

Discussion The main purpose of this study was to evaluate the capability of hEnSCs cultured on TCP, GEL/CS, and GEL/CS/BGNPs

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Figure 6. (A) Immunocytochemical images of cells stained with KDR, VE-Cadherin, and CD31 antibodies on 17th day post-treatment in TCP, GEL/CS, and GEL/CS/BGNPs scaffolds. Immunocytochemical analysis indicated that differentiated hEnSCs were positive for KDR, VE-Cadherin, and CD31 after 17 days post-treatments (scale bar, 50 mm). (B) Percentages of cells expressing biomarkers KDR, VE-Cadherin, and CD31 compared with TC P, GEL/CS, GEL/CS/BGNPs nanofibrous scaffolds. (P50.001, n ¼ 3, mean ± SD). (C) Real-time PCR of endothelial-like cell markers in differentiated hEnSCs. Differentiated cells were evaluated for RNA expression of endothelial biomarkers 17 days post induction. Results revealed that differentiated cells could highly express FLT1, CD31, VECAD, and VwF. Expression of these biomarkers is higher in differentiated cells treated with BGNP compared with GEL/CS and TCP. These data indicated positive role of BG and 3D culture in endothelial stimulation. GAPDH is the housekeeping gene control (***P50.001; *P50.05). Error bars show the mean ± SD (n ¼ 3).

nanofibrous scaffolds to differentiate into endothelial-like cells in the presence of FGF-2 and VEGF signaling molecules for 17 d. Expression of endothelial-specific markers such as FLT1, CD31, VECAD, and VwF in mRNA levels and protein were analyzed using real-time PCR and immunocytochemistry, respectively. The results of real-time PCR and immunocytochemistry for endothelial markers revealed that differentiation ability of cultured cells on GEL/CS/BGNPs scaffold were higher than those on the TPC and GEL/CS surfaces. This study is the first to report on endothelial -like cell differentiation of hEnSCs in 2D and 3D cell culture.

Suitable cells for tissue engineering should be easily harvested, adhered, and proliferated. Mesenchymal stem cells (MSCs) are the most common sources of cells for vascular tissue engineering (Riha et al. 2005). Endometrial stem cells are a new population of MSCs present in the basal layer of endometrium that play a dynamic remodeling role in cyclic endometrial renewal (Wolff et al. 2007). In contrast with embryonic stem cells, transplanted hEnSCs do not have tumorigenic capacity and do not have political and ethical agenda for clinical trials. Also, isolation of MSCs from adipose and bone marrow is invasive procedure, requires local anesthesia and can cause

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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY

chronic pain, infection, and bleeding whereas the endometrial layer is one source of MSCs that can be easily isolated by uterine biopsy sample with access through the cervix. HEnSCs are known as multipotent types of cells with immunomodulatory properties and self-renewal ability that were used as stem cell sources in this study (Ai and Davood 2010). Nano-scale environment was reported to control the adhesion, proliferation, and differentiation of cells through an increment in countercurrent exchanges between cell and ECM (Panda et al. 2010). In recent years there has been great interest in the fabrication of nanofibers via electrospinning in vascular tissue engineering applications. Polymeric nanofibrous scaffolds can be used for a broad spectrum of tissue engineering applications. Scaffolds made of natural polymer become most promising substrates because of their biodegradable and biocompatible properties. GeL nanofiber (Montero et al. 2012) and GEL–CS nanofibrous scaffolds were found to improve the cell viability and angiogenesis (Qian et al. 2011). Grafting of GEL on electrospun poly-nanofibers (caprolactone) may be a suitable scaffold in expansion of endothelial cells, and their proliferation and orientation (Ma et al. 2005). In order to fabricate suitable nanofibers for endothelial differentiation, we first studied the effect of the different ratios of the BGNPs on the fiber diameter and fiber morphology and then investigated the effect of the scaffolds on differentiation of the hEnSCs into endothelial lineage. The results of SEM indicated that the ratio of the BGNPs had an effect on the homogeneity and diameters of the nanofibers. Fiber diameter of GEL/CS in our study was 180–200 nm, which is in agreement with results obtained by Jafari et al. (2011). In order to promote the angiogenesis with exogenous agent, BGNPs were entrapped in the GEL/CS nanofibrous scaffolds. The nanofiber diameter range increased with the reduction of BGNPs content in GEL/CS solutions (53– 235 nm) (Figure 2C). Furthermore, the results showed that increasing the flow rate to 1.2 ml/h (Figure 2B) led to the formation of bead-free uniform fibers in GEL/CS/BGNPs nanofibrous scaffolds, whereas optimum flow rates were 0.4 ml/h in GEL/CS scaffolds (Figure 1). FGF-2 and VEGF are known to induce MSC differentiation into endothelial-like cells (Konno et al. 2010). Previous studies have indicated that FGF-2 and VEGF possessed an angiogenic synergistic effect on the induction of angiogenesis, and FGF-2 promoted VEGF and KDR expression in the endothelial cells (Holmes and Zachary 2008) and MSCs (Park et al. 2015). VE-cadherin is an endothelial intercellular adherence junction that is related to the complex formation with VEGFR-2 (Carmeliet et al. 1999). In this study, hEnSCs were cultured in the angiogenic media containing FGF-2 and VEGF; cells revealed morphology of endothelial cells and expressed CD31/PECAM, VE-cadherin, and KDR, indicating that they have differentiated to mature endothelial cells. Cao et al. used FGF-2 and VEGF to evaluate whether adipose derived adult stem cells (ADAS cells) could be differentiated into endothelial-like cells. They showed that ADAS cells not only revealed the endothelial morphology but also expressed the specific markers of mature endothelial cells such as CD31/PECAM and VE-cadherin (Cao et al. 2005). Our results are in accordance with this study, and differentiated cells were expressed by endothelial gene markers such as CD31 and VE-cadherin as well as other transcription factors such

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as VEGFR2 in treatment groups during the angiogenic induction period. To compare the endothelial differentiation of hEnSCs on TCP, GEL/CS, and GEL/CS/BGNPs scaffolds, expression of endothelial markers were studied in mRNA level and protein by real-time PCR and immunocytochemistry, respectively. The findings showed that topographic characteristic of the surface significantly influences differentiation of hEnSCs into endothelial-like cells. HEnSCs revealed an increase in endothelial differentiation on GEL/CS/BGNPs scaffold in comparison with TCP and GEL/CS surfaces. We compare the expression findings between GEL/CS and GEL/CS/BGNPs nanofibrous scaffolds. Our results also confirmed the positive role of BG in the stimulation of FGF-2 and VEGF secretion in cultured MSCs (Day 2005), and could clearly indicate the effect of BG on increasing quality of hEnSCs differentiation into endothelial-like cells in vitro. Consequently, our data showed that GEL/CS/BGNPs nanofibers could provide better nano-environment for differentiation of hEnSCs to endothelial cells. The results of our study opened up new promising opportunities for development of new nanofibrous scaffolds with improved properties for use as 3D structure in vascular tissue engineering, and moreover, the use of stem cells with nanofibrous material expected to be an ideal strategy for treating endothelial dysfunction diseases such as chronic ischemic heart disease or peripheral ischemic disorders.

Conclusions The GEL/CS/BGNPs nanofibrous scaffold in our study was shown to have a good effect on angiogenesis and endothelial cell differentiation. The nanoscale and nanofiber GEL/CS/BGNPs mat based on their large surface area and composition as well as the enhancement of the cell viability were demonstrated to be advantageous scaffolds for angiogenesis and tissue regeneration, and healing. So, GEL/CS/BGNPs nanofibrous scaffolds can be recommended for proliferation and differentiation of hEnSCs and would be a promising scaffold to regenerate endothelial cell needed in blood vessels with 56 mm internal diameter.

Disclosure statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Funding information This research has been supported by Iranian National Science Foundation (INSF) grant number 91042417.

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bioglass nanofibrous scaffolds.

The capacity of gelatin/chitosan/bioactive glass nanopowders (GEL/CS/BGNPs) scaffolds was investigated for increasing human endometrial stem cells (hE...
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