Poly(ethylene oxide) (PLGA:PEO) - ACS Publications - American

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Hybrid randomly electrospun PLGA:PEO fibrous scaffolds enhancing myoblast differentiation and alignment Olivera Evrova, Vahid Hosseini, Vincent Milleret, Gemma Palazzolo, Marcy Zenobi-Wong, Tullio Sulser, Johanna Buschmann, and Daniel Eberli ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11291 • Publication Date (Web): 11 Oct 2016 Downloaded from http://pubs.acs.org on October 14, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

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

ACS Applied Materials & Interfaces

Hybrid randomly electrospun PLGA:PEO fibrous scaffolds enhancing myoblast differentiation and alignment Olivera Evrova,a,b Vahid Hosseinic, Vincent Milleretd, Gemma Palazzoloe, Marcy ZenobiWonge, Tullio Sulserb, Johanna Buschmanna and Daniel Eberli*b a

Division of Plastic Surgery and Hand Surgery, University Hospital Zürich, Sternwartstrasse

14, 8091 Zürich, Switzerland b

Laboratory for Tissue Engineering and Stem Cell Therapy, Department of Urology and

Zürich Center for Integrative Human Physiology (ZIHP), University of Zürich, University Hospital Zürich, Frauenklinikstrasse 10, 8091 Zürich, Switzerland c

Laboratory of Applied Mechanobiology, ETH Zürich, Vladimir-Prelog-Weg 1-5/10, 8093

Zürich, Switzerland. d

Laboratory for Cell and Tissue Engineering, Department of Obstetrics, University Hospital

Zürich, Schmelzbergstrasse 12/PF 125, 8091 Zürich, Switzerland e

Cartilage Engineering and Regeneration, ETH Zürich, Otto-Stern-Weg 7, 8093 Zürich,

Switzerland

* To whom correspondence should be addressed: [email protected] Phone: + 41 44 255 9549; Fax: +41 44 255 4555

ACS Paragon Plus Environment


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

Page 2 of 43

ABSTRACT Cellular responses are regulated by their microenvironments and engineered synthetic scaffolds can offer control over different microenvironment properties. This important relationship can be used as a tool to manipulate cell fate and cell responses for different biomedical applications.

We show for the first time in this study how blending of

poly(ethylene oxide) (PEO) to poly(lactic-co-glycolic acid) (PLGA) fibers to yield hybrid scaffolds changes physical and mechanical properties of PLGA fibrous scaffolds and in turn affects cellular response. For this purpose we employed electrospinning to create fibrous scaffolds mimicking the basic structural properties of the native extracellular matrix. We introduced PEO to PLGA electrospun fibers by spinning a blend of PLGA:PEO polymer solutions in different ratios. PEO served as a sacrificial component within the fibers upon hydration, leading to pore formation in the fibers, fiber twisting, increased scaffold disintegration and hydrophilicity, decreased Young’s modulus and significantly improved strain at break of initially electrospun scaffolds. We observed that the blended PLGA:PEO fibrous scaffolds supported myoblast adhesion and proliferation and resulted in increased myotube formation and self – alignment, when compared to PLGA-only scaffolds, even though the scaffolds were randomly oriented. The 50:50 PLGA:PEO blended scaffold showed the most promising results in terms of mechanical properties, myotube formation and alignment, suggesting an optimal microenvironment for myoblast differentiation from the PLGA:PEO blends tested. The explored approach for tuning fiber properties can easily extend to other polymeric scaffolds and provide a valuable tool to engineer fibrillar microenvironments for several biomedical applications. Keywords: hybrid scaffolds, electrospinning, PLGA, PEO, myoblast differentiation, muscle tissue engineering 1 ACS Paragon Plus Environment

Page 3 of 43

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


INTRODUCTION

How cells interact and respond to physical cues in their microenvironments has been shown to play an essential role in cell differentiation, change in cell fate and tissue formation.1-3 This is important in basic and applied cell studies in tissue engineering and regenerative medicine, where scaffold design, biomaterial and biological cues greatly affect specific cellular and tissue responses. For instance, in skeletal muscle tissue engineering, improving myoblast differentiation to myotubes and their proper alignment is still considered a major challenge.4

Nano- or microfibrous scaffolds and other fiber based templates have been widely studied for tissue engineering and cell studies and have showed promising results.5, 6 Using either natural or synthetic polymers, electrospinning allows for production of scaffolds which resemble the morphology and topography of natural extracellular matrix (ECM), providing topographical and required chemical cues for cell growth and tissue formation. Moreover, recent advances in electrospinning techniques allow for production of scaffolds from a variety of polymers, so that different morphologies and properties can be obtained by changing the fabrication parameters or polymer chemistry.7-10

Though mechanical properties of fibrillar

microenvironments have not been considered to play a major role in cell response, recent work has shown that individual and network fiber stiffness can alter human mesenchymal stem cells’ early response by a mechanism of fiber recruitment. In softer fibrillar microenvironments using cellular forces, cells were able to spatially displace the fibers towards them and thus remodel the structure of the scaffolds, similarly as pulling and pushing on native fibrillar ECM components.11

2 ACS Paragon Plus Environment


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

Page 4 of 43

In this study, we introduce a novel approach to tune the physical and mechanical properties of electrospun PLGA fibrous scaffolds, one of the most extensively studied polymers for synthetic scaffold production, without any chemical modification of PLGA.12,13 To achieve this we have incorporated poly(ethylene oxide) (PEO) inside PLGA fibers to act as a porogen that dissolved away from the fibers upon hydration of the scaffold. This resulted in changes to individual fibers distinct from the previously described role of PEO as sacrificial element on the scale of the whole scaffold.14 PEO inclusion led to changes in the architecture and morphology of the fibers upon scaffold hydration and eventually changed also how fibers can be recruited by nearby cells. Furthermore, by varying the PEO amount within the PLGA polymeric fibers, the physical properties of PLGA could be tuned in terms of hydrophilicity, wettability and disintegration rate, while still maintaining a structurally stable scaffold that allows cell attachment, proliferation and differentiation.

We studied how these scaffold changes can be translated to changes in cells’ response and differentiation, by assessing myoblast proliferation, differentiation, myotube formation and self-alignment on the different PLGA:PEO scaffolds and compared to PLGA-only scaffolds.

EXPERIMENTAL SECTION Materials Poly(lactic-co-glycolic acid) (PLGA, Resomer® Sample MD Type RG 85:15, Mw = 280 kDa) was purchased from Boehringer Ingelheim, Germany. Poly(ethylene oxide) (PEO) (Mw = 300 kDa), chloroform ≥ 99.8 % (0.5-1.0% ethanol as stabilizer), ethanol ≥ 99.8 %, fluorescein isothiocyanate labeled phalloidin (phalloidin-FITC), anti-sarcomeric-α-actinin antibody, Cy3 sheep anti-mouse-conjugated IgG and 4’6-diamidino-2-phenylindole dilactate (DAPI) were purchased from Sigma Aldrich. Dulbecco’s modified Eagle’s medium (DMEM, 3 ACS Paragon Plus Environment

Page 5 of 43

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


catalogue number: 11995), phosphate buffered saline (PBS), fetal bovine serum (FBS), horse serum (HS), penicillin/streptomycin (P/S), Live/Dead Assay kit and trypsin/EDTA were purchased from Life Technologies. 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (MTS) assay was obtained from Promega. Anti-desmin antibody was purchased from BD Biosciences.

Preparation of PLGA:PEO polymer blends

PLGA:PEO polymer blends with final polymer concentration of 6 wt % were prepared by dissolving respective amounts of PLGA and PEO (Table 1) in a mixture of chloroform and ethanol (75:25 w/w). Three PLGA:PEO polymer blends were prepared, namely: 30:70, 50:50, 70:30. PLGA-only, i.e. 100 (w/w) PLGA was used as a polymer solution control. The prepared polymer solutions were dissolved overnight at room temperature and were stirred for 30 minutes before use in order to obtain homogenous solutions.

Table 1. Composition and names of polymer blends used as experimental groups for scaffold production

Ratio of PLGA:PEO (w/w) in a final polymer blend of 6 wt % in 75:25 (w/w) ratio of chloroform/ethanol Name of polymer blend (scaffold) 100 70:30 50:50 30:70

PLGA (wt %)

PEO (wt %)

6 4.2 3 1.8

/ 1.8 3 4.2

Scaffold production by electrospinning

For the production of electrospun scaffolds, an in-house assembled electrospinning device was used consisting of a spinning head with a blunt end made of stainless steel tube (1 mm inner diameter and 0.3 mm wall thickness, Angst & Pfister AG, Zürich Switzerland), a DC 4 ACS Paragon Plus Environment


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

Page 6 of 43

high voltage supply (Glassman High Voltage Inc., High Bridge, NJ, USA), a hollow cylindrical rotating aluminum mandrel as a collector and a syringe pump (AL1000 Aladdin, World Precision Instruments, Inc., Germany). The polymer solution of interest was loaded into a 5 mL syringe (B.Braun Melsungen AG, Germany) and pumped into the spinning head with 0.7 mL/h flow rate. A voltage of 11 kV was applied and the distance between the spinning head and the collector was fixed to 15 cm. Electrospinning was performed at a relative humidity of 30 – 40 %. The produced scaffolds were collected on an aluminum foil and dried under vacuum overnight at room temperature.

Scanning electron microscopy

Samples of electrospun scaffolds were mounted on metal stubs with conductive double-sided tape. Samples were sputter coated (SCD500, Bal-tec) with platinum in order to obtain 10 nm coating and then examined by scanning electron microscopy (SEM) (Zeiss SUPRA 50 VP, Zeiss, Cambridge, UK) at an accelerating voltage of 5 kV. Fiber diameters of each sample were measured from SEM images that were analyzed by the analysis software Fiji. First a diagonal line was drawn on the image, and the fiber diameter of the fibers was measured perpendicular to the fiber length at the points where the diagonal line crossed the fibers. The measurement was done manually with the measurement tool in Fiji, after calibration with the scale bar of the microscope image. 3 images for each scaffold were analyzed, with the average of 30 counts per scaffold in order to calculate the average fiber diameter.

Scaffold degradation, weight loss, hydrophilicity, shrinkage and porosity

Samples of each PLGA:PEO scaffold (n = 3) with a diameter of 12 mm were cut, placed in 4 mL of phosphate buffered saline (1xPBS, pH 7.4), and incubated at 37°C and 5 % CO2. At specific time points (1, 2, 3, 7, 10, 21 and 30 days), samples from each scaffold were taken 5 ACS Paragon Plus Environment

Page 7 of 43

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


out, rinsed with 1xPBS (pH 7.4) first and distilled water after, and then dried under vacuum overnight at room temperature. Afterwards the samples were analyzed by SEM. In order to determine the weight loss of each PLGA:PEO scaffold, samples with a diameter of 12 mm were cut, their weight was measured and then immersed in 3 ml of ddH2O and incubated at 37°C and 5 % CO2. At each time point (1, 2, 3, 7, 10, 21 and 30 days), samples were rinsed with ddH2O, dried under vacuum overnight, and their weight was recorded again. The remaining weight of each scaffold was calculated according to equation 1:

ℎ % =

× 100;

(1) where Wi is the initial weight of the scaffolds before degradation and Wf is the final weight of the scaffolds after degradation.

The hydrophilicity of the PLGA:PEO scaffolds was assessed by performing dynamic water contact angle measurements.

Measurements were performed with a contact angle

goniometer, DSA 100 (Krüss GmbH, Germany). Dynamic water contact angle measurements were carried out with drop volumes from 10 to 20 µL and with a dosing speed of 30 µL/min, at room temperature. Analysis of the series of images recorded was performed using the DropSnake15 plugin for Fiji. Results are presented as change in water contact angle over time. 4 samples per type of scaffold were measured.

The shrinkage of the electrospun scaffolds was determined by measuring scaffold’s dimensions after production and after 3 days in an aqueous environment. Briefly, 2 cm x 2 cm samples from each scaffold were cut and their length and width were measured. Afterwards, the scaffolds were immersed in 3 mL 1xPBS (pH 7.4) and incubated at 37°C and 5 % CO2 for 3 days. After the specific time, the scaffolds were rinsed with ddH2O, dried



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

Page 8 of 43

under vacuum overnight, and their dimensions (length and width) were recorded again. The shrinkage of each scaffold was calculated according to equation 2: ℎ % =

× 100;

(2)

where Ai is the initial area of the scaffolds before immersion in 1xPBS and Af is the final area of the scaffolds after 3 days in 1xPBS.

The bulk densities of the electrospun scaffolds were determined gravimetrically by taking out 1 cm x 1 cm samples from each scaffold and measuring their weight and thickness. The overall scaffold porosity (P) was calculated by the following equation 3: !

P = 1 − # × 100; !

(3)

"

where ρ is the bulk density of the scaffolds and ρ0 is the polymer density of PLGA or the mixture of PLGA and PEO. The ρ0 of PLGA and PEO used for calculation were 1.26 g/cm3 and 1.21 g/cm3, respectively. Three samples per condition (n = 3) were measured.

Mechanical testing

The mechanical properties of all PLGA:PEO scaffolds were obtained from stress/ strain curves measured using a uniaxial load test machine (model 5864, Instron tensile tester, High Wycombe, Buck, UK) equipped with a 10 N load cell. Standard dog-bone shaped samples with a testing region of 12 x 2 mm2 and thickness range of 50 – 120 µm were punched out and an elongation rate of 12 mm/ min was applied until failure. The Young’s modulus [MPa], strain at break [%] and ultimate tensile stress [MPa] were determined for each condition. Mechanical testing was performed on differently treated PLGA:PEO scaffolds, namely dry scaffolds (electrospun and dried in a desiccator, without dissolution of the PEO component, 7 ACS Paragon Plus Environment

Page 9 of 43

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


i.e. not treated), dry UV treated PLGA:PEO scaffolds, degraded PLGA:PEO scaffolds after 3 days with dissolved PEO component (dried before measurements) and wetted PLGA:PEO scaffolds with 15 µL of water after mounting them on the test machine. The measurements were averaged over three samples (n = 3).

FTIR characterization of scaffolds

Hybrid PLGA:PEO scaffolds and PLGA-only scaffolds were also characterized by Fourier transform infrared spectroscopy (FTIR). Experiments were carried out on a Cary 670 Fourier transform infrared spectrometer (Agilent technologies) equipped with a SPECAC attenuated total reflection (ATR) diamond accessory. Dry, dry UV treated scaffolds (60 minutes) and degraded (3 days) (dried) scaffolds were characterized. Spectra were collected within the wavenumber range of 500 – 4000 cm-1 and the interferometer was scanned with an acquisition rate of 37.5 kHz at 2 cm-1 resolution. The results presented here focus on the region 650 – 3500 cm-1. A total of 64 scans were averaged to obtain one spectrum, while three regions of the scaffolds per condition were scanned. The spectral data was preprocessed by applying rubber band baseline correction in Python. For the presentation of the results, spectra were normalized either to the C=O peak at 1750 cm-1 or to the C-O (C-O-C) peak at 1085 cm-1.

Cell culture and cell seeding on scaffolds

For direct assessment of myotube formation on the PLGA:PEO scaffolds, C2C12 myoblasts expressing cytoplasmic eGFP were used. The transgenic cell line was kindly donated by Prof. 8 ACS Paragon Plus Environment


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

Page 10 of 43

Mary K. Baylies, Sloan-Kettering Institute, New York, USA. For live/dead assays, proliferation assays and immunocytochemistry experiments, non-transfected C2C12 myoblasts were used. Upon thawing, eGFP expressing C2C12 cells and non-transfected C2C12 cells were seeded separately at the density of 104 cells/cm2 and cultured in growth medium (DMEM containing high Glucose, L-Glutamine and pyruvate, supplemented with 20 % FBS and 1% P/S) in a humidified incubator with 5 % CO2 in air, at 37°C. The myoblasts were trypsinized using 0.05% trypsin/EDTA when they reached 70-80 % confluency and used for experiments.

PLGA:PEO scaffolds, once produced and dried under vacuum overnight, were cut in 15 x 15 mm sized samples, placed in 12-well tissue culture plates and UV sterilized for 60 minutes. eGFP expressing C2C12 myoblasts and non-transfected C2C12 myoblasts were harvested and seeded at a density of 1.5 x 104 cells/scaffold, respectively. After 2 hours of cell seeding, sterile stainless steel O-rings (outer diameter: 12 mm, inner diameter: 8 mm) were placed on top of the scaffolds to ensure their stability in culture and prevent scaffold floating. After 1 day of culture, the growth medium was replaced with differentiation medium (DMEM containing high glucose, L-Glutamine and pyruvate, supplemented with 5 % horse serum and 1 % P/S). The differentiation medium was changed every two days during the culture period.

Cell viability and proliferation

Cell viability of C2C12 myoblasts on each PLGA:PEO scaffold was assessed using Live/ Dead viability assay for mammalian cells. The cell viability was assessed after 3 and 21 days, at which point samples were shortly rinsed with PBS and the staining was performed with 2 µM Calcein AM-4 µM ethidium homodimer solution for 30 minutes at room temperature. The samples were imaged with a fluorescent microscope and structured illumination (Zeiss Axio Observer with Apotome II, Zeiss, Germany). Each condition was done in triplicate 9 ACS Paragon Plus Environment

Page 11 of 43

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


(n=3) and three random images were taken per sample. The images were analyzed with Fiji and the cell viability is represented in % of live cells/ total cells for day 3 and 21. The proliferation of C2C12 myoblasts onto the different scaffolds was assessed at 3, 7 and 21 days, using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium inner salt (MTS) assay according to the manufacturer’s protocol.

C2C12 myotube characterization and quantification of myotube alignment by immunostaining

To assess myotube formation, PLGA:PEO scaffolds seeded with eGFP expressing C2C12 myoblasts were analyzed after 3, 7 and 14 days of culture. Briefly, at each time point, scaffolds were shortly rinsed with PBS and fixed with 3.6 % paraformaldehyde for 15 minutes, followed by a wash with PBS and 10 minutes incubation with 5 µg/mL DAPI. The samples were rinsed 5 times with PBS in order to eliminate any background from DAPI staining the scaffold. Imaging of the samples was done with the fluorescent microscope.

Scaffolds seeded with non-transfected C2C12 myoblasts were cultured for 14 days, after which the scaffolds were immunostained for myotube markers. For this, scaffolds were shortly rinsed with PBS and fixed with 3.6 % paraformaldehyde for 15 minutes, followed by a wash with PBS. Permeabilization was performed with 0.5 % Triton X – 100 for 10 minutes at room temperature. Afterwards, the samples were incubated with 5 % bovine serum albumin and 0.1 % Triton X – 100 for 45 minutes. A primary mouse monoclonal IgG antibody against sarcomeric α-actinin or primary mouse IgG antibody against desmin was added to the samples at 1:200 dilution or 1:50 dilution, respectively, and incubated at 4°C overnight. The samples were then washed three times with PBS and incubated with Cy3 sheep anti mouse IgG antibody at a dilution of 1:1000 in 1 % BSA, for 1 hour at room temperature, washed 3 times and imaged. 10 ACS Paragon Plus Environment


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

Page 12 of 43

The image analysis was done with Fiji and the fusion index (%), number of myotubes/field of view, myotube length and width on each PLGA:PEO scaffold were determined. Cells with more than 2 nuclei were counted as a myotube. The fusion index was defined as the ratio of the number of nuclei forming the myotube over the total number of nuclei, converted into percentage (%).16, 17

The alignment of myotubes per field of view was quantified at day 14. Shortly, the angle of orientation of each myotube in a field of view was determined and histograms of the distribution of angles (0-180°) were plotted for each image, for each condition respectively. For determining whether there is a preferred myotube alignment/field of view on each scaffold, the standard deviation of the histograms for each image was calculated assuming normal distribution. The standard deviations were averaged for each condition. For each condition, equivalent normal distribution curves with the calculated average standard deviation were generated. The narrowness of the distribution correlates with the preferred alignment of myotubes on each scaffolds condition.

Statistical analysis

Data were analyzed with Origin (OriginLab, Northampton, MA). Values are reported as means ± standard deviation. One-way analysis of variance (one-way ANOVA) was performed to test the significance of differences between the different conditions in all the experiments. The comparison probabilities (p) were calculated using Bonferroni test for comparison and statistical significance was accepted for p-values lower than 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).


Page 13 of 43

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


RESULTS

Fiber diameter and morphology of hybrid PLGA:PEO scaffolds

Scaffolds consisting of different blends of PLGA and PEO with increasing PEO content (70:30, 50:50 and 30:70) were prepared and compared to PLGA-only scaffold, referred to as 100 PLGA. The effect of PEO addition in the polymer blend was tested by comparing the fiber morphology and diameter of the obtained scaffolds. During the production it was noticed that lower flow rates, 0.5 – 1 mL h-1, promote fiber formation of PLGA:PEO scaffolds and eliminate blob formation within the fibers (data not shown). Thus, a flow rate of 0.7 mL h-1 for every condition was chosen. The humidity during the electrospinning process was also kept between 30 – 40 % due to the fact that increase in humidity affected the morphology of the electrospun fibers (Figure S1 and S2) and could lead to different properties of the scaffolds.

Proper fibers with smooth, defect-free surface and homogenous structure were obtained from all polymer blends (Figure 1). The fiber diameters of the resulting scaffolds ranged from 1.48 ± 0.28 µm, 1.75 ± 0.17 µm and 1.59 ± 0.30 µm for 30:70, 50:50 and 70:30 PLGA:PEO, respectively. The PLGA-only scaffold showed an average diameter of 2.04 ± 0.17 µm



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

Page 14 of 43

(Figure 1). A narrow distribution of fiber diameter within each scaffold was observed, thus resulting in homogenous scaffold morphology.

Figure 1. PLGA:PEO initial fiber morphology and characterization. SEM micrographs of PLGA-only and PLGA:PEO hybrid scaffolds with corresponding fiber diameter distribution for each condition . Fibers with smooth surfaces were obtained, blob and defect free for all conditions. Scale bars: 10 µm.

Degradability, shrinkage and hydrophilicity of hybrid PLGA:PEO scaffolds

The hydrophilicity of the hybrid PLGA:PEO scaffolds was tested by measuring the dynamic water contact angle. The changes in water contact angle overtime are shown in Figure 2A, 13 ACS Paragon Plus Environment

Page 15 of 43

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


while the calculated advancing and receding angles for each scaffold are shown in Table S1. Results show that PLGA-only scaffold is highly hydrophobic in nature with an advancing water contact angle of 117.87 ± 7.55 and a receding contact angle of 111.25 ± 8.18 degrees, thus not changing significantly over the time course of the measurement and exhibiting somewhat small hysteresis. With the incorporation of PEO in the PLGA:PEO scaffolds, the water contact angle was significantly decreased compared to PLGA-only scaffold (Table S1). 30:70 PLGA:PEO and 50:50 PLGA:PEO experienced higher hysteresis than PLGA – only scaffolds. Their advancing contact angles were 88.70 ± 1.81 and 76.99 ± 6.75 degrees, respectively, while the receding ones were measured to be 60.55 ± 2.12 and 50.18 ± 15.93 degrees, respectively (Table S1). 70:30 PLGA:PEO showed the highest significant decrease in water contact angle when compared to the other scaffolds, down to an advancing angle of 10.89 ± 2.01 degrees and a receding one of 7.38 ± 1.07 degrees. Interestingly, even though some of the PLGA:PEO scaffolds showed large hysteresis, the hydrophilicity of PLGA:PEO scaffolds decreased with the increase of PEO concentration inside the fibers, where 70:30 PLGA:PEO was the most hydrophilic scaffold. The hybrid PLGA:PEO scaffolds didn’t show changes in scaffold

porosity compared to the

PLGA-only

scaffold (Figure 2B).



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

Page 16 of 43

Figure 2. Physical properties of hybrid PLGA:PEO electrospun scaffolds. (A) dynamic water contact angle [°] measurements for each hybrid PLGA:PEO scaffold. The dash-dot lines represent the error bars (standard deviation) of the line graphs for each condition. (B) Porosity of each hybrid PLGA:PEO scaffold.

The degradation of the hybrid PLGA:PEO scaffolds was analyzed in an aqueous environment for a period of 30 days at 37°C. The change in fiber morphology and influence of PEO presence during degradation is shown in the SEM micrographs in Figure 3. The incorporation of PEO in the polymer blend formulation led to pore and crack formation in the fibers of PLGA:PEO scaffolds that was after visible 3 days (Figure 3). The changes appear as either circular pores or more longitudinal cracks along the fiber length. At this point, 50:50 and 30:70 PLGA:PEO scaffolds show more visible crack formation and scaffold degradation, compared to 70:30 PLGA:PEO. In addition, increasing PEO concentration within the scaffold led to loss of fiber structure, resulting in more flexible and twisted fibers. Morphological changes like pore formation in individual fibers and loss of structure were not visible for the PLGA-only scaffold, i.e. 100 PLGA. (Figure 3). Scaffold degradation was also determined in terms of weight loss over a period of 30 days. From Figure 4A it can be observed that major weight loss happened between 1 and 3 days of the scaffold’s degradation period. At 7 days, the remaining scaffold weight was around 29.61 %, 49.77 % and 71.53 % for 30:70, 50:50 and 70:30 PLGA:PEO scaffolds, respectively, which suggests complete dissolution of the 15 ACS Paragon Plus Environment

Page 17 of 43

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


PEO content. After 7 days, no major changes in terms of remaining weight of PLGA:PEO scaffolds are observed. The difference in weight loss at every time point (Figure 4A) was significant among all the conditions, with 30:70 (PLGA:PEO) exhibiting the largest weight loss of around 70 %. The weight loss of the pure PLGA scaffold was the lowest, with 93.75 % remaining weight after 7 days in aqueous environment.

Due to the weight loss exhibited in a short time, the scaffolds were further characterized in terms of size changes, i.e. scaffold shrinkage. 30:70 PLGA:PEO scaffold exhibited the highest degree of shrinkage, with around 86 % shrinkage. 50:50 PLGA:PEO and 70:30 PLGA:PEO scaffolds showed 78.63 % and 74.14 %, shrinkage, respectively (Figure 4B). Interestingly, PLGA-only (100 PLGA) scaffolds also experienced average shrinkage of 65.48 %. However, the shrinkage of PLGA:PEO scaffolds was significantly higher when compared to PLGA-only scaffolds.



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

Page 18 of 43

Figure 3. Degradation of hybrid PLGA:PEO electrospun scaffolds. SEM micrographs tracking scaffold disintegration and pore formation on fibers. Scale bars: (upper) 5 µm and (lower) 2 µm.


Page 19 of 43

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


Figure 4. Degradation and shrinkage of hybrid PLGA:PEO electrospun scaffolds. (A) Remaining weight [%] of PLGA:PEO scaffolds upon degradation at 37°C. (B) Shrinkage [%] of PLGA:PEO scaffolds after 3 days incubation at 37°C. (*p < 0.05, **p < 0.01, ***p < 0.001.)

Mechanical properties of hybrid PLGA:PEO scaffolds

Uniaxial mechanical testing was performed on PLGA:PEO and PLGA-only scaffolds in order to determine the Young’s modulus [MPa], strain at break [%] and ultimate tensile stress [MPa] of the different scaffolds. Moreover, the mechanical properties of differently treated scaffolds, namely, dry scaffolds (PEO not dissolved away), dry UV treated scaffolds, 3 days degraded scaffolds (dried before measurement) and wetted scaffolds were investigated. The results for all the conditions are presented in terms of stress/strain profiles in Figure 5 (results for all replicates are included in Figure S4), while the results of the different mechanical properties are summarized in Table 2. For the dry scaffolds, before UV treatment and PEO 18 ACS Paragon Plus Environment


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

Page 20 of 43

dissolution, a decrease in Young’s modulus in PLGA:PEO scaffolds with increasing concentration of PEO, when compared to PLGA-only scaffold was observed. The decrease from 46.6 ± 7.9 MPa for PLGA to 19.6 ± 2.6 MPa for 30:70 PLGA:PEO was significant (Table 2, Figure 5A). Introduction of PEO led to significant increase in the strain at break [%] of the scaffolds. With increasing PEO concentration, the strain at break increased to 237 ± 19 %, 402 ± 91 % and 672 ± 37 % for 70:30, 50:50 and 30:70 PLGA:PEO scaffolds, respectively, when compared to PLGA-only scaffold (83 ± 7 %). A small, but not significant decrease in the ultimate tensile stress [MPa] of the PLGA:PEO scaffolds when compared to PLGA – only scaffold was measured (Table 2, Figure 5A).

UV treatment affected the mechanical properties of all the dry scaffolds by slightly reducing the Young’s modulus, strain at break and ultimate tensile stress of all the scaffolds (Table 2, Figure S5). The strain at break of the different hybrid PLGA:PEO scaffolds was mostly affected by the UV treatment, while the one of PLGA did not change (Table 2) These differences are also visible in the stress/strain profiles presented in Figure 5B.

The mechanical properties of the degraded scaffolds in an aqueous environment and after PEO dissolution were inferior to the ones of dry scaffolds, with or without UV treatment. Namely, significant decrease in Young’s modulus, strain at break and ultimate tensile stress was observed for all the scaffolds (Table 2, Figure S5). Most significant changes were observed for 30:70 PLGA:PEO scaffold, as visible also in the stress/strain profile in Figure 5C.

In addition, the mechanical properties of wetted scaffolds were also measured. Once PLGA:PEO scaffolds were wetted, they lost the mechanical properties reported as for dry or UV treated samples (Figure S5), especially the 30:70 PLGA:PEO scaffold, for which some of the mechanical properties could not be measured (Table 2, Figure 5D). The PLGA – only 19 ACS Paragon Plus Environment

Page 21 of 43

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


scaffold exhibited some decrease Young’s modulus and ultimate tensile stress, however it retain

its

property

terms of strain

at break.

in

Figure 5. Mechanical properties of the hybrid PLGA:PEO electrospun scaffolds under different treatments. (A) Full and initial stress/strain profile of hybrid scaffolds in a dry state (no treatment). (B) Full



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

Page 22 of 43

and initial stress/strain profile of UV treated dry hybrid scaffolds. (C) Full and initial stress/strain profile of 3 days degraded hybrid scaffolds (dried), with PEO component dissolved away. (D) Full and initial stress/strain profile of scaffolds wetted at measurement.

Table 2. Calculated average ± standard deviation of Young’s modulus [MPa] (E), ultimate tensile stress [MPa] (UTS) and strain at break [%] (SB) for all hybrid PLGA:PEO scaffolds under different treatments, namely dry scaffolds, dry UV treated scaffolds, 3 days degraded scaffolds (dried) and scaffolds wetted at measurement. (*p

< 0.05,

** p < 0.01, *** p < 0.001; NA – not measurable)

Dry scaffolds (not treated)

Dry UV treated scaffolds

Degraded scaffolds (Day 3) (dried)

Wetted Scaffolds (at measurement)

E [MPa]

UTS [MPa]

SB [%]

E [MPa]

UTS [MPa]

SB [%]

E [MPa]

UTS [MPa]

SB [%]

E [MPa]

UTS [MPa]

SB [%]

100

46.6 ± 7.9

2.2 ± 0.1

83 ± 7

43.2 ± 6.9

1.9 ± 0.05

84 ± 10

33.7 ± 4.6

1.6 ± 0.15

73 ± 21

25.6 ± 8.9

1.2 ± 0.11

82 ±

70:30

40 ± 3.3

1.8 ± 0.2

237 ± 19

36.7 ± 2.9

1.3 ± 0.04

156 ± 26

2.2 ± 0.4 *

1.3 ± 0.04 *

214 ± 30 *

6.7 ± 1.9 *

0.3 ± 0.05 *

120 ±

50:50

33.6 ± 7.1

1.7 ± 0.5

402 ± 91 **

24 ± 3.1 *

1.6 ± 0.15 *

273 ±

1± 0.13 *

236 ± 81 *

2.7 ± 0.5 *

0.24 ± 0.01 *

20 ±

67

1.1 ± 0.1 *

30:70

19.6 ± 2.6 *

1.8 ± 0.1

672 ± 37***

17.4 ± 1.3 *

1.8 ± 0.09

512 ± 32

13.2 ± 9.5 *

1± 0.24

24 ± 7

NA

NA

NA

7

35

3*


Page 23 of 43

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


FTIR characterization of hybrid PLGA:PEO scaffolds

Figure 6. FTIR absorbance spectra for hybrid PLGA:PEO scaffolds under different treatments. (A) FTIR spectra for all dry scaffolds. (B) FTIR spectra for all dry scaffolds after UV treatment. (C) FTIR spectra for all scaffolds after 3 days degradation in aqueous environment and thus PEO dissolution from the scaffolds.



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

Page 24 of 43

All scaffolds showed homogenous composition and no differences in the FTIR spectra of different areas scanned within the scaffold (Figure S6-S9). In the FTIR spectra shown in Figure 6, strong band at 1750 cm-1 can be observed for all scaffolds, due to the presence of the carbonyl group (C = O stretch) in PLGA.18 In addition there are some bands in the region from 1300 to 1150 cm-1 (specifically at 1186 cm-1) which can be attributed to the symmetric and asymmetric vibrations of C – O (esters), thus also highlighting the presence of PLGA.18 In addition to these peaks, in the PLGA-only scaffolds there is a small signal in the region between 2992 and 2944 cm-1 coming from the CH2 and CH3 stretching vibrations.19 With the introduction and increase in PEO content within the scaffolds, appearance of two characteristic peaks for poly(ethylene) oxide can be observed, at 2848 cm-1 and 1085 cm-1. The band at 2848 cm-1 is the broadest for 30:70 PLGA:PEO and can be assigned to the symmetrical stretching of the C-H bond (from the aliphatic CH2 groups in PEO).20 The signal at 1085 cm-1 also increased with the increase in PEO content (Figure 6) and can be attributed to the asymmetric stretching mode of C-O (C-O-C) bonds of PEO, together with the band at 960 cm-1,20,21 while the band at 840 cm-1 comes from the rocking vibrations of the methylene groups.21 The UV treatment of dry scaffolds did not lead to any major changes in any of the scaffolds (Figure 6). The obtained FTIR spectra for UV treated scaffolds are overlapping with the spectra obtained for dry (not treated) scaffolds (Figure S10 – S13).

Furthermore, FTIR characterization allowed for determining the dissolution of PEO after scaffold degradation in aqueous environment within 3 days. In Figure 6C, the resulting FTIR spectra for all PLGA:PEO blends after 3 days degradation overlap with the spectra of PLGA after degradation, thus confirming the PEO dissolution. Once the scaffolds have degraded for some time, the characteristic peaks of PEO, present in the spectra in Figure 6A and 6B, can no longer be observed.


Page 25 of 43

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


In addition, DSC analysis was performed to investigate the properties of the PLGA:PEO blends and the effect of electrospinning on the degree of phase separation between the two components. The PLGA here used is amorphous and is characterized with a glass transition temperature (Tg), while PEO being crystalline is represented by a melting temperature (Tm). Results show (Figure S14, Table S2) that the blending of the two components had influence on the Tg of PLGA and Tm of PEO. Addition of PEO in the blends led to a decrease in the Tg of PLGA from 51.46 °C (PLGA only) to 43.06 °C (30:70 PLGA:PEO), as well as decrease in the Tm of PEO from 71.35 °C (PEO only) to 63.80 °C (70:30 PLGA:PEO).

C2C12 viability and proliferation on fibrillar PLGA:PEO scaffolds

We further evaluated the ability of the PLGA:PEO scaffolds to support cell viability and proliferation of C2C12 myoblasts. Cell viability was analyzed after 3 and 21 days in culture. All the scaffolds showed high cell viability without significant difference among the conditions (Figure 6A and 6B) at day 3 and maintained high viability up to 21 days. In particular, cell viability at day 3 was 86.11 %, 71.97 %, 77.63 % and 73.3 % for PLGA-only (100 PLGA), 70:30, 50:50 and 30:70 PLGA:PEO scaffolds, respectively (Figure 7A). At day 21 the cell viability was 84.59 %, 97.12 %, 86.19 % and 82.74 % for the same scaffolds (Figure 7A).



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

Page 26 of 43

Figure 7. C2C12 cell viability and proliferation on different hybrid PLGA:PEO electrospun scaffolds. (A) Cell viability on different scaffolds at day 3 and 21. (B) MTS assay to test C2C12 cell proliferation (metabolic activity) on PLGA:PEO scaffolds at day 3, 7 and 21. (*p < 0.05).

The proliferation (metabolic activity) over time of C2C12 myoblasts on the scaffolds was measured at 3, 7 and 21 days in culture. Initially, at day 3, pure PLGA scaffolds showed significantly increased cell proliferation when compared to PLGA:PEO scaffolds, however this difference was not significant at later time points of 7 and 21 days (Figure 7B). The increasing PEO concentration in the initial scaffolds did not led to major or significant effect on the cell proliferation over time on the different PLGA:PEO scaffolds.

C2C12 myotube formation and alignment on hybrid PLGA:PEO scaffolds

One key challenge in skeletal muscle tissue engineering is myoblast fusion and eventual differentiation into myotubes. For easier assessment of the myoblast fusion on the PLGA:PEO scaffolds, C2C12 myoblasts expressing cytoplasmic eGFP were used. Upon dissolution of the PEO component from the fibers, morphological changes take place in terms of global scaffold porosity and fiber morphology that can lead to changes in cell morphology, ease of fiber recruitment and downstream cell response. Figure 8 depicts a schematic of proposed changes in scaffold and fiber architecture, as well as cell response. In Figure 9, the 25 ACS Paragon Plus Environment

Page 27 of 43

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


sequential differentiation of myoblasts into myotubes over time is visible. The eGFP expression is shown in green. By day 14, myotubes were formed on each PLGA:PEO scaffold and on PLGA-only scaffolds too (Figure 9A). Moreover, the cells fused to myotubes in an aligned manner by themselves regardless of the fiber alignment below them. Some myotubes were already visible at day 7 on 30:70 (PLGA:PEO) scaffolds.

Figure 8. Scheme of obtaining hybrid PLGA:PEO scaffolds and changes following PEO dissolution from the fibers. Changes in fiber/scaffold morphology and architecture are shown (pore formation on fibers, fiber twisting), as well as changes in cell morphology and cell response for each PLGA:PEO hybrid scaffold,

respectively.

The differentiation of myoblasts into multinucleated myotubes can be monitored with staining for specific differentiation markers that are only expressed once myoblasts are differentiated. Muscle structural proteins like α-actinin and desmin are myogenic differentiation markers.22 The expression of α-actinin and desmin in the myotubes formed on 26 ACS Paragon Plus Environment


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

Page 28 of 43

all the scaffolds was tested. Immunostaining showed expression of these two markers and proper myotube formation in all hybrid PLGA:PEO scaffolds and the PLGA-only scaffold after 14 days in culture (Figure 9B). The immunostaining for desmin in formed myotubes for 50:50 and 30:70 PLGA:PEO scaffolds also shows self-alignment of myotubes on the scaffolds. On all the scaffolds, myotubes with fiber-like morphology can be observed.

Figure 9. C2C12 myoblast proliferation and differentiation (myotube formation) on hybrid PLGA:PEO electrospun scaffolds. (A) eGFP fluorescently labelled C2C12 myoblasts (cytoplasmic eGFP expression is shown in green) on PLGA:PEO scaffolds, stained with DAPI and imaged after 3, 7, 14 and 21 days. (B) Immunostaining of α-actinin and desmin, in C2C12 myotubes obtained on the different PLGA:PEO scaffolds


Page 29 of 43

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


after 14 days in culture. In addition, samples were stained for F-actin (green) and nuclei (blue) (B). Scale bars: (A) and (B) 100 µm.

Quantification of differentiated myoblasts was performed using low magnification (10x) images from both, eGFP expressing C2C12 myotubes and immunostained C2C12 myotubes formed on the scaffolds. In addition, the average number of myotubes per field of view, myotube length and width were measured and the results of the quantification are shown in Figure 10. The graph in Figure 10A shows that after 14 days in culture there is a significant increase in cell differentiation on 50:50 PLGA:PEO and 30:70 PLGA:PEO, when compared to PLGA-only or 70:30 PLGA:PEO scaffolds. The fusion index on 50:50 PLGA and 30:70 PLGA:PEO was 24.32 % and 36.37 %, respectively. From the graph (Figure 10B) it can be seen that all hybrid scaffolds caused an increased number of myotubes per field of view compared to PLGA-only, but only the 50:50 PLGA:PEO condition showed a significant increase. The myotubes obtained on PLGA-only (100 PLGA) scaffolds were shorter and wider when compared to myotubes formed on all PLGA:PEO scaffolds, which were longer and thinner (Figure 9C and 9D).

Data suggests that hybrid scaffolds allowed significantly higher self – alignment of myotubes per field of view when compared to PLGA-only scaffolds (Figure 10E and 10F). PLGA-only scaffolds resulted in a wide distribution of myotube angle orientation, as depicted by the equivalent normal distribution curves shown in Figure 10F. The narrowest distribution of myotube orientation was observed for 30:70 PLGA:PEO, but this was not significantly different than the other hybrid PLGA:PEO scaffolds.



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

Page 30 of 43

Figure 10. Characterization of myotubes formed on hybrid scaffolds after 14 days in culture. (A) Fusion index [%] of myotubes on scaffolds. (B) Number of myotubes per field of view on each scaffold condition. (C) Myotube width [µm] for each scaffold condition. (D) Myotube length [µm] for each scaffold condition. (E) Differences in averaged standard deviation of histograms (σ) for preferred myotube alignment in different fields of view on each scaffold condition. (F) Equivalent normal distributions for degree of preferred alignment in field of view per scaffold, with standard deviation (σ) as the averaged σ from all histograms per condition. (*p < 0.05, **p < 0.01, ***p < 0.001.)


Page 31 of 43

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


4. Discussion Electrospinning is one method to produce fibrous scaffolds that closely resemble the morphology of native ECM. It is widely used to produce natural or synthetic polymeric scaffolds, with desired topography27-29 and different fiber diameters.23-26 Electrospun scaffolds should be biocompatible, possess adequate physical and mechanical properties and eventually be biodegradable without any undesirable side effects.30 The work presented illustrates an easy method to obtain hybrid scaffolds with tunable physical and mechanical properties of electrospun PLGA scaffolds. By tuning the PEO content within the fibers, the dynamics (flexibility), structure and morphology of the fibers within the scaffolds were changed and we explored how these changes affected myoblast differentiation. Hybrid PLGA:PEO scaffolds, where PLGA served as a mechanically stable component, were reproducibly fabricated with homogenous fiber diameters, smooth outer surface and no bead formation within the scaffolds (Figure 1). Once hydrated, the blended PEO dissolved away within the initial 3 days, without further changes after 7 days (Figure 3 and 4), which was further confirmed by the FTIR analysis (Figure 6).This caused circular or longitudinal crack formation along the individual PLGA fibers in the range of few hundred nanometers. Such pore (crack) formations led to faster fiber disintegration, loss of initial fiber morphology, changes in global scaffold porosity (Figure 3) and resulted in more twisted or wrinkled fibers, thus creating different fibrous microenvironments. Similar results were reported before in a study on a drug delivery application of PCL/PEG electrospun scaffolds, where changing the PEG ratio allowed for selective pore formation and thus altered drug delivery properties of the scaffolds.31 However, an approach like this was never used for cell studies or tissue engineering purposes.



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

Page 32 of 43

Even though 30:70 and 50:50 PLGA:PEO experienced large hysteresis in the water contact angle, differences in behavior were visible, when compared to 70:30 PLGA:PEO scaffolds. Namely, we observed that the hydrophilicity of the hybrid electrospun scaffolds decreased with increasing PEO concentration. One reason for this might be phase separation and migration of the minor polymer component in the blend towards the surface of the fibers. For instance, in the case of 70:30 PLGA:PEO the core might be formed of PLGA, while PEO being more distributed at the surface, thus obtaining very hydrophilic surface. In the case of 30:70 PLGA:PEO, PLGA may be more accessible at the surface. Additional fact that might account for this is also small differences in surface roughness, however this was not measured for the scaffolds. DSC analysis showed that the two polymers exhibited some degree of miscibility, which led to changes in the Tm of PEO and Tg of PLGA (Figure S14, Table S2). Although partial phase separation is still present, blending of PLGA and PEO resulted in retention of the fiber shape after PEO dissolution from the scaffolds. The fibers containing the highest amount of PEO (30:70 PLGA:PEO) did not completely disintegrate within time, suggesting that phase separated PLGA and PEO domains are not very large within the electrospun fibers. This can be a result of fast ejection of the blend solution and a quick evaporation of the solvent at room temperature during electrospinning. Similar observation has been reported for PCL/PEO blended scaffolds.32 Inclusion of PEO within the PLGA scaffolds altered the mechanical properties of the dry hybrid scaffolds by increasing strain at break [%],and decreasing Young’s modulus and tensile stress, when compared to PLGA-only scaffold. The increase in strain at break [%] was directly dependent on PEO concentration where it acted as a plasticizer (Figure 5), which has been shown before in combination with other polymers.33-35 UV treatment of dry scaffolds was investigated as the scaffolds were UV treated before cell experiments, and this resulted in slight decrease in the overall mechanical properties of the scaffolds, as described before.36 31 ACS Paragon Plus Environment

Page 33 of 43

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


The mechanical properties were lost to a large extent after PEO dissolution from the PLGA:PEO scaffolds after 3 days, but not for PLGA-only scaffold, as expected. However, characterizing the mechanical properties of the PLGA:PEO scaffolds in a dry state before or after PEO dissolution does not reflect the exact properties that the cells are sensing in the fibrillar microenvironments in culture. Taking this into account, the mechanical properties of wet scaffolds were assessed, which is more relevant for cellular responses. Wetted PLGA:PEO scaffolds were much softer, easy to deform and break with the applied force during the measurement. This sheds light on the fact that the scaffolds can behave differently in culture. Cellular forces are exerted at the cell’s focal adhesion points and are within very low range (several nN per focal adhesion) and are varying with cell type.37,

38

Thus, wet

PLGA:PEO scaffolds might undergo spatial rearrangements more easily than wet PLGA-only scaffolds, where cells more easily recruit and rearrange fibers. Dependent on the rate of PEO dissolution the PLGA:PEO fibers are more easily deformable, with a successive decrease in elastic modulus (E) and increased flexibility, thus providing dynamically changing microenvironment that affects cellular responses. The hybrid PLGA:PEO scaffolds showed good cell compatibility, like the PLGA-only scaffold and C2C12 myoblasts were able to attach and proliferate on the hybrid scaffolds with excellent viability, at both early and late time points. The lower cell proliferation observed on the hybrid scaffolds (Figure 7B) might be due to two reasons. Firstly, it can be due to lower initial cell attachment, leading to different initial cell number on each scaffold. Secondly, for the longer time point (21 days), increased differentiation of the C2C12 myoblasts, on the hybrid PLGA:PEO scaffolds, compared to PLGA-only scaffold, can decrease cell proliferation which is in agreement with the measured myoblast differentiation on the scaffolds (Figure 9 and 10).39



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

Page 34 of 43

Another difference between the hybrid scaffolds and the PLGA-only scaffold was their shrinkage on the macroscale and fiber twisting, thus resulting in global porosity loss. Nanopore formation in the fibers could have led to more flexible fibers and easier remodeling of the scaffolds, thus increasing cell- cell contacts (Figure 8). It has been shown that lower fiber stiffness permits active cellular forces to recruit nearby fibers, dynamically increasing ligand density at the cell surface and promoting focal adhesion formation and related signaling.11 The hybrid PLGA:PEO scaffolds showed increased myoblast differentiation as measured by formation of multinucleated myotubes, compared to PLGA-only scaffolds (Figure 9 and 10). Higher myoblast fusion index was proportional to increased PEO content, which can be a result of increased myoblast contact and fusion and could have been mediated by the newly proposed mechanism of fiber recruitment by which cells probe and respond to softer fibrillar microenvironments differently than stiffer ones.11, 40 Formation of longer myotubes formed can be a result of increased end-to-end cell fusion. Lateral cell fusion also takes place during myotube formation, resulting in thicker and shorter myotubes.41,42 PEO modified PLGA scaffolds led to significantly longer and thinner myotubes, which suggests a dominant role of end-to-end myoblast fusion, whereas shorter and thicker myotubes were observed on PLGAonly scaffolds (Figure 10). Scaffold stiffness, as measured by the Young’s modulus (E), has previously been shown to play a role in cell differentiation.1,43 A stiff film (> 320 kPa) can make longer and thinner myotubes compared to a soft film.44 However, the Young’s modulus of all scaffolds studied here is much higher than 320 kPa (in the range of MPa, Table 2) On the other hand, a smooth surface of a film or a hydrogel is different than a fibrillar network11 and cells can respond differently to the same stiffness. Regardless, we think that the change in Young’s modulus of the hybrid scaffolds plays no critical role in cellular response and all the scaffolds presented have modulus to allow myoblast differentiation. However, fiber flexibility can affect the 33 ACS Paragon Plus Environment

Page 35 of 43

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


cellular response, since cells are able to recruit nearby fibers, pull on them, and with that sense different microenvrionments.40 In our modified scaffold in the first instances, the PEO component is still within the fibers, completely being released only within few days. Afterwards, the porous PLGA fibers can be more easily recruited by the cells, thus resulting in different cellular responses. Interestingly, we observed increase in local myotube self – alignment on the hybrid scaffolds even though the scaffolds were randomly oriented. Myotubes on the PLGA-only scaffolds had a wide range of myotube orientation (Figure 10) as expected on a randomly electrospun scaffold. However, hybrid PLGA:PEO scaffolds resulted with increased self-alignment within field of view, but with no large differences between each other. This observation can be a result of the general changes in these microenvironments. It is suggested that C2C12 myotubes are able to self-align along wide range of surface.45 At the cellular level, guidance can be felt by either scaffold topography or neighboring cells. Following scaffold twisting and change in fiber morphology some of the primary random topology is lost in the hybrid scaffolds, which is not the case with PLGA-only scaffolds. Thus, PLGA-only scaffolds provide a more rigid platform that cells can sense and follow because fibers retained their shape better compared to the softer hybrid scaffolds, thus cells are guided by the randomly oriented scaffold. In the latter case, cells have more degree of freedom to self-organize and self-align as the PEO component dissolves away and lead to fiber twisting, deformability and global porosity loss (Figure 8). Therefore, it is possible that better myotube self-organization and self-alignment could be induced by providing more cell deformable environment with the hybrid scaffolds.

5. Conclusions



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

Page 36 of 43

We have shown how addition of a hydrophilic component, in this case PEO, to a hydrophobic component, PLGA, can affect and tune the scaffold’s physical and mechanical properties. Acting as a sacrificial element, PEO addition allowed for tuning PLGA scaffolds, changing its physical properties and allowing fiber twisting and wrinkling, as well as scaffold shrinkage. With this approach, hybrid fibrillar scaffolds were obtained which led to different cellular responses and enhanced myoblast differentiation. The hybrid PLGA:PEO scaffolds allowed myoblast attachment, proliferation and enhanced myoblast fusion and myotube formation. In light of the recent findings, that fibrillar microenvironments undergo spatial rearrangements when in the presence of cells11, we think that by blending PLGA and PEO we created hybrid scaffolds offering dynamically changing environments that affected myoblasts response and enhanced their differentiation. Moreover, our newly designed fibrillar scaffolds not only improved myotube formation, but by being more flexible and deformable also allowed myotube self-alignment which has not been achieved by previous randomly electrospun scaffolds. We consider this study to be further evidence that cell – material interactions play an important role in modulating cellular responses. Finally, we believe that the explored method here can be modified and applied to other polymeric fibrillar scaffolds for basic cell studies and tissue engineering applications. SUPPORTING INFORMATION Effect of humidity on electrospun fibers (SEM images); additional data on dynamic water contact angle measurements, mechanical testing and FTIR spectra; thermal analysis (DSC) ACKNOWLEDGMENTS This work was supported by departmental funds of University Hospital Zurich, as well as a funding from the EMDO foundation (grant no.815). We would like to thank Sarah Nötzli for her advice in the electrospinning technique. Dr. Kirill Feldman is highly acknowledged for


Page 37 of 43

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


his help with mechanical measurements and DSC analysis. We thank Prof. Mary K. Baylies for kindly providing eGFP expressing C2C12 myoblasts for the experiments. We would like to thank Dr. Rok Simic for his help and assistance in FTIR measurements.

REFERENCES

(1)

Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E., Matrix Elasticity Directs Stem

Cell Lineage Specification. Cell 2006, 126, 677-689. (2)

Trappmann, B.; Gautrot, J. E.; Connelly, J. T.; Strange, D. G. T.; Li, Y.; Oyen, M. L.;

Cohen Stuart, M. A.; Boehm, H.; Li, B.; Vogel, V.; Spatz, J. P.; Watt, F. M.; Huck, W. T. S., Extracellular-matrix Tethering Regulates Stem-cell Fate. Nat. Mater. 2012, 11, 642-649. (3)

Wen, J. H.; Vincent, L. G.; Fuhrmann, A.; Choi, Y. S.; Hribar, K. C.; Taylor-Weiner,

H.; Chen, S.; Engler, A. J., Interplay of Matrix Stiffness and Protein Tethering in Stem Cell Differentiation. Nat. mater. 2014, 13, 979-987. (4)

Ostrovidov, S.; Hosseini, V.; Ahadian, S.; Fujie, T.; Parthiban, S. P.; Ramalingam,

M.; Bae, H.; Kaji, H.; Khademhosseini, A., Skeletal Muscle Tissue Engineering: Methods to Form Skeletal Myotubes and Their Applications. Tissue Eng., Part B 2014, 20, 403-436. (5)

Hosseini, V.; Kollmannsberger, P.; Ahadian, S.; Ostrovidov, S.; Kaji, H.; Vogel, V.;

Khademhosseini, A., Fiber-Assisted Molding (FAM) of Surfaces with Tunable Curvature to Guide Cell Alignment and Complex Tissue Architecture. Small 2014, 10, 4851-4857. (6)

Wolf, M. T.; Dearth, C. L.; Sonnenberg, S. B.; Loboa, E. G.; Badylak, S. F., Naturally

Derived and Synthetic Scaffolds for Skeletal Muscle Reconstruction. Adv. Drug Delivery

Rev. 2015, 84, 208-221. (7)

Flemming, R. G.; Murphy, C. J.; Abrams, G. A.; Goodman, S. L.; Nealey, P. F.,

Effects of Synthetic Micro- and Nano-structured Surfaces on Cell Behavior. Biomaterials 36 ACS Paragon Plus Environment


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

Page 38 of 43

1999, 20, 573-588. (8)

Badami, A. S.; Kreke, M. R.; Thompson, M. S.; Riffle, J. S.; Goldstein, A. S., Effect

of Fiber Diameter on Spreading, Proliferation, and Differentiation of Osteoblastic Cells on Electrospun Poly(lactic acid) Substrates. Biomaterials 2006, 27, 596-606. (9)

Luwang Laiva, A.; Venugopal, J. R.; Sridhar, S.; Rangarajan, B.; Navaneethan, B.;

Ramakrishna, S., Novel and Simple Methodology to Fabricate Porous and Buckled Fibrous Structures for Biomedical Applications. Polymer 2014, 55, 5837-5842. (10)

Munj, H. R.; Nelson, M. T.; Karandikar, P. S.; Lannutti, J. J.; Tomasko, D. L.,

Biocompatible Electrospun Polymer Blends for Biomedical Applications. J. Biomed. Mater.

Res., Part B 2014, 102, 1517-1527. (11)

Baker, B. M.; Trappmann, B.; Wang, W. Y.; Sakar, M. S.; Kim, I. L.; Shenoy, V. B.;

Burdick, J. A.; Chen, C. S., Cell-mediated Fibre Recruitment Drives Extracellular Matrix Mechanosensing in Engineered Fibrillar Microenvironments. Nat. Mater. 2015, 14, 12621268. (12)

Gentile, P.; Chiono, V.; Carmagnola, I.; Hatton, P. V., An Overview of Poly(Lactic-

co-Glycolic) Acid (PLGA)-based Biomaterials for Bone Tissue Engineering. Int. J. Mol. Sci. 2014, 15, 3640-3659. (13)

Danhier, F.; Ansorena, E.; Silva, J. M.; Coco, R.; Le Breton, A.; Préat, V., PLGA-

based Nanoparticles: An Overview of Biomedical Applications. J. Controlled Release 2012, 161, 505-522. (14)

Baker, B. M.; Shah, R. P.; Silverstein, A. M.; Esterhai, J. L.; Burdick, J. A.; Mauck,

R. L., Sacrificial Nanofibrous Composites Provide Instruction Without Impediment and Enable Functional Tissue Formation. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 14176-14181. (15)

Stalder, A. F.; Kulik, G.; Sage, D.; Barbieri, L.; Hoffmann, P., A Snake-based

Approach to Accurate Determination of both Contact Points and Contact Angles. Colloids


Page 39 of 43

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


Surf., A 2006, 286, 92-103. (16)

Goudenege, S.; Pisani, D. F.; Wdziekonski, B.; Di Santo, J. P.; Bagnis, C.; Dani, C.;

Dechesne, C. A., Enhancement of Myogenic and Muscle Repair Capacities of Human Adipose-derived Stem Cells with Forced Expression of MyoD. Mol. Ther. 2009, 17, 10641072. (17)

Krause, R. M.; Hamann, M.; Bader, C. R.; Liu, J. H.; Baroffio, A.; Bernheim, L.,

Activation of Nicotinic Acetylcholine Receptors Increases the Rate of Fusion of Cultured Human Myoblasts. J. Physiol. 1995, 489, 779-790. (18)

Erbetta, C. D. C.; Alves, R. J.; Resende, J. M.; Freitas, R. F. d. S.; de Sousa, R. G.,

Synthesis and Characterization of Poly(D,L-Lactide-co-Glycolide) Copolymer. J. Biomater.

Nanobiotechnol. 2012, 3, 208-225. (19)

Silva, A.; Cardoso, B.; Silva, M.; Freitas, R.; Sousa, R., Synthesis, Characterization,

and Study of PLGA Copolymer in Vitro Degradation. J. Biomater. Nanobiotechnol. 2015, 6, 8-19. (20)

Belleville, C.; Robert, P.; Hoebler, C.; Barry, J. L., Infrared Spectroscopic

Determination of Poly(ethylene glycol) for Nutritional Studies. J. Agric. Food Chem. 1995, 43, 2672-2677. (21)

Ratna, D.; Abraham, T. N.; Karger-Kocsis, J., Studies on Polyethylene Oxide and

Phenolic Resin Blends. J. App. Polym. Sci. 2008, 108, 2156-2162. (22)

Philippi, S.; Bigot, A.; Marg, A.; Mouly, V.; Spuler, S.; Zacharias, U., Dysferlin-

Deficient Immortalized Human Myoblasts and Myotubes as a Useful Tool to Study Dysferlinopathy. PLoS Curr. 2012, doi: 10.1371/currents.RRN1298. (23)

Wang, H. B.; Mullins, M. E.; Cregg, J. M.; Hurtado, A.; Oudega, M.; Trombley, M.

T.; Gilbert, R. J., Creation of Highly Aligned Electrospun Poly-L-Lactic Acid Fibers for Nerve Regeneration Applications. J. Neural Eng. 2009, 6, 016001.



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

(24)

Page 40 of 43

Whited, B. M.; Rylander, M. N., The Influence of Electrospun Scaffold Topography

on Endothelial Cell Morphology, Alignment, and Adhesion in Response to Fluid Flow.

Biotechnol. Bioeng. 2014, 111, 184-195. (25)

Aviss, K. J.; Gough, J. E.; Downes, S., Aligned Electrospun Polymer Fibres for

Skeletal Muscle Regeneration. Eur. Cells Mater. 2010, 19, 193-204. (26)

Yin, Z.; Chen, X.; Song, H.-x.; Hu, J.-j.; Tang, Q.-m.; Zhu, T.; Shen, W.-l.; Chen, J.-

l.; Liu, H.; Heng, B. C.; Ouyang, H.-W., Electrospun Scaffolds for Multiple Tissues Regeneration in Vivo through Topography Dependent Induction of Lineage Specific Differentiation. Biomaterials 2015, 44, 173-185. (27)

Saino, E.; Focarete, M. L.; Gualandi, C.; Emanuele, E.; Cornaglia, A. I.; Imbriani, M.;

Visai, L., Effect of Electrospun Fiber Diameter and Alignment on Macrophage Activation and Secretion of Proinflammatory Cytokines and Chemokines. Biomacromolecules 2011, 12, 1900-1911. (28)

Binder, C.; Milleret, V.; Hall, H.; Eberli, D.; Lühmann, T., Influence of Micro and

Submicro Poly(Lactic-Glycolic Acid) Fibers on Sensory Neural Cell Locomotion and Neurite Growth. J. Biomed. Mater. Res., Part B 2013, 101, 1200-1208. (29)

Erisken, C.; Zhang, X.; Moffat, K. L.; Levine, W. N.; Lu, H. H., Scaffold Fiber

Diameter Regulates Human Tendon Fibroblast Growth and Differentiation. Tissue Eng., Part

A 2013, 19, 519-528. (30)

Liu, C.; Xia, Z.; Czernuszka, J. T., Design and Development of Three-Dimensional

Scaffolds for Tissue Engineering. Chem. Eng. Res. Des. 2007, 85, 1051-1064. (31)

Liao, I. C.; Chen, S.; Liu, J. B.; Leong, K. W., Sustained Viral Gene Delivery through

Core-shell Fibers. J. Controlled Release 2009, 139, 48-55. (32)

Kim, T. G.; Lee, D. S.; Park, T. G., Controlled Protein Release from Electrospun

Biodegradable Fiber Mesh Composed of Poly(ɛ-caprolactone) and Poly(ethylene oxide). Int.


Page 41 of 43

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


J Pharm. 2007, 338, 276-283. (33)

Yoon, Y. I.; Park, K. E.; Lee, S. J.; Park, W. H., Fabrication of Microfibrous and

Nano-/Microfibrous Scaffolds: Melt and Hybrid Electrospinning and Surface Modification of Poly(L-lactic acid) with Plasticizer. BioMed Res. Int. 2013, 2013, doi:10.1155/2013/309048. (34)

Wang, B. Y.; Fu, S. Z.; Ni, P. Y.; Peng, J. R.; Zheng, L.; Luo, F.; Liu, H.; Qian, Z. Y.,

Electrospun Polylactide/Poly(ethylene glycol) Hybrid Fibrous Scaffolds for Tissue Engineering. J. Biomed. Mater. Res., Part A 2012, 100, 441-449. (35)

Wang, H.; Feng, Y.; Zhao, H.; Fang, Z.; Khan, M.; Guo, J., A Potential

Nonthrombogenic Small-diameter Vascular Scaffold with Polyurethane/Poly(ethylene glycol) Hybrid Materials by Electrospinning Technique. J. Nanosci. Nanotechnol. 2013, 13, 15781582. (36)

Shearer, H.; Ellis, M. J.; Perera, S. P.; Chaudhuri, J. B., Effects of Common

Sterilization Methods on the Structure and Properties of Poly(D,L Lactic-co-Glycolic Acid) Scaffolds. Tissue Eng. 2006, 12, 2717-2727. (37)

Schwarz, U. S.; Balaban, N. Q.; Riveline, D.; Addadi, L.; Bershadsky, A.; Safran, S.

A.; Geiger, B., Measurement of Cellular Forces at Focal Adhesions Using Elastic MicroPatterned Substrates. Mater. Sci. Eng., C 2003, 23, 387-394. (38)

Galbraith, C. G.; Sheetz, M. P., A Micromachined Device Provides a New Bend on

Fibroblast Traction Forces. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 9114-9118. (39)

G.M. Cooper, The Cell: A Molecular Approach, 2nd ed; Sinauer Associates,

Sunderland (MA), 2000. (40)

Abhilash, A. S.; Baker, Brendon M.; Trappmann, B.; Chen, Christopher S.; Shenoy,

Vivek B., Remodeling of Fibrous Extracellular Matrices by Contractile Cells: Predictions from Discrete Fiber Network Simulations. Biophysical Journal 2014, 107, 1829-1840.



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

(41)

Page 42 of 43

Clark, P.; Dunn, G. A.; Knibbs, A.; Peckham, M., Alignment of Myoblasts on

Ultrafine Gratings Inhibits Fusion in Vitro. Int. J. Biochem. Cell Biol. 2002, 34, 816-825. (42)

Nowak, S. J.; Nahirney, P. C.; Hadjantonakis, A. K.; Baylies, M. K., Nap1-mediated

Actin Remodeling is Essential for Mammalian Myoblast Fusion. J. Cell Sci. 2009, 122, 32823293. (43)

Engler, A. J.; Griffin, M. A.; Sen, S.; Bonnemann, C. G.; Sweeney, H. L.; Discher, D.

E., Myotubes Differentiate Optimally on Substrates with Tissue-like Stiffness: Pathological Implications for Soft or Stiff Microenvironments. J. Cell Biol. 2004, 166, 877-887. (44)

Ren, K.; Crouzier, T.; Roy, C.; Picart, C., Polyelectrolyte Multilayer Films of

Controlled Stiffness Modulate Myoblast Cells Differentiation. Adv. Funct. Mater. 2008, 18, 1378-1389. (45)

Junkin, M.; Leung, S. L.; Whitman, S.; Gregorio, C. C.; Wong, P. K., Cellular Self-

Organization by Autocatalytic Alignment feedback. J. Cell Sci. 2011, 124, 4213-4220.

GRAPHICAL ABSTRACT


Page 43 of 43


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


Poly(ethylene oxide) (PLGA:PEO) - ACS Publications - American

Recommend Documents