poly (lactic acid) nanofibrous scaffolds with potential applications in tissue engineering

Journal Pre-proof Development of poly (mannitol sebacate)/poly (lactic acid) nanofibrous scaffolds with potential applications in tissue engineering

Mahya Rahmani, Mohammad-Mehdi Khani, Shahram Rabbani, Alireza Mashaghi, Farsad Noorizadeh, Reza Faridi-Majidi, Hossein Ghanbari PII:

S0928-4931(19)32876-0

DOI:

https://doi.org/10.1016/j.msec.2020.110626

Reference:

MSC 110626

To appear in:

Materials Science & Engineering C

Received date:

16 August 2019

Revised date:

14 December 2019

Accepted date:

1 January 2020

Please cite this article as: M. Rahmani, M.-M. Khani, S. Rabbani, et al., Development of poly (mannitol sebacate)/poly (lactic acid) nanofibrous scaffolds with potential applications in tissue engineering, Materials Science & Engineering C (2018), https://doi.org/10.1016/j.msec.2020.110626

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© 2018 Published by Elsevier.

Journal Pre-proof

Development of poly (mannitol sebacate)/Poly (lactic acid) nanofibrous scaffolds with potential applications in tissue engineering

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Mahya Rahmani 1, Mohammad-Mehdi Khani 2,3, Shahram Rabbani 4, Alireza Mashaghi 5,6

Department of Medical Nanotechnology, School of Advanced Technologies in Medicine,

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Farsad Noorizadeh 7, Reza Faridi-Majidi 1, Hossein Ghanbari 1,4*

Tehran University of Medical Sciences (TUMS), Tehran, Iran Department of Tissue Engineering and Applied Cell Sciences, School of Advanced

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2

Medical Nanotechnology and Tissue Engineering Research Center, Shahid Beheshti

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3

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Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

University of Medical Sciences , Tehran, Iran Research Center for Advanced Technologies in Cardiovascular Medicine, Tehran Heart

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4

Center, Tehran University of Medical Sciences (TUMS), Tehran, Iran 5

Medical Systems Biophysics and Bioengineering, Leiden Academic Centre for Drug

Research, Faculty of Science, Leiden University, Leiden, Netherlands 6

Harvard Medical School, Harvard University, Boston, USA

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Basir Eye Health Research Center, Tehran, Iran

* Corresponding Author: Hossein Ghanbari, MD, PhD Tehran University of Medical Sciences, Italia Street, Quds Avenue, Keshavarz Blv, Tehran, Iran Postal Code: 1417755469 Email: [email protected], Tel: (+9821)-43052139; Fax: (+9821)-88991117

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Highlights: 

A series of novel nanofibrous scaffolds based on poly (mannitol sebacate) and poly (lactic acid) were fabricated using electrospinning method.



The physicochemical, mechanical and cytocompatibility properties of hybrid PMS:PLA nanofibers were then evaluated. The

hybrid

nanofibers

revealed

appropriate

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biocompatibility.

and

enhanced

These novel PMS:PLA nanofibrous scaffolds could be considered as potential

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characteristics

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Graphical abstract:

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candidates for tissue engineering applications.

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Abstract Developing a biomimetic substrate with intrinsic potential for cell attachment and growth has always been a tissue engineering challenge. In the present research, we successfully fabricated PMS:PLA nanofibrous scaffolds for the first time using electrospinning process by adjusting blending ratios, feed rates and polymer concentrations. A desirable composition was found when homogenous nanofibers with an average fiber diameter of 235±38nm were

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achieved at 10% w/v for PMS:PLA 60:40. The scaffolds were then characterized for their

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microstructure, mechanical strength and elasticity, degradation rate, porosity, wettability and

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cell/tissue compatibility. Mechanical analysis and degradation behavior of PMS:PLA

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nanofibrous scaffolds revealed appropriate elasticity, stiffness and strength, as well as degradation rate appropriate for soft tissues. Nitrogen adsorption-desorption analysis

in

vitro

and

in

vivo

biocompatibility

evaluations

revealed

enhanced

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Further

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discovered that mesoporous nanofibers with enhanced specific surface area were fabricated.

cytocompatibility, proliferation and tissue responses of PMS:PLA nanofibrous scaffolds with

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desirable cell-scaffold interactions. Moreover, PMS:PLA nanofibrous scaffolds exhibited negligible inflammatory responses with significantly thinner fibrotic capsule formation and minor infiltration of inflammatory cells compared to PLA nanofibers. These findings suggest that PMS/PLA nanofibrous scaffolds could be introduced as potential candidates with improved properties for soft tissue engineering applications.

Keywords: Tissue engineering, Electrospinning, Nanofiber, Scaffold, Poly (Mannitol Sebacate), Poly (Lactic Acid).

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1. Introduction Considering the fact that the chemical composition and the microenvironment of the cellsubstrate have great impacts on the fate of the exposed cells, several new materials and methods have been established for fabricating nanofibrous scaffolds to mimic native extracellular matrix (ECM) [1-5]. Natural ECM is a structure composed mainly of collagen and elastin fibers with length scales ranging from nanometers to a few micrometers [6].

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Hence nanofibrous scaffolds could potentially mimic natural ECM better than bulk

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biomaterials[7-9].

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Poly (polyol sebacate) (PPS) aliphatic polyesters have been proposed as good candidates with tunable properties for biomedical applications [10-14]. These elastomeric polymers,

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including poly mannitol sebacate (PMS), are composed of biocompatible sugar alcohol and a

lP

diacid, whereas sebacic acid is the natural intermediate in the oxidation of fatty acids and mannitol is metabolized in an insulin-independent metabolic pathway[14-16]. The physico-

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chemical characteristics of PPS are absolutely versatile due to the length of alcohol

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substitution [16]. Bruggeman et al. [16] have developed a class of biodegradable PPS films with adjustable mechanical properties and biodegradation rates, which could be designed for a wide range of applications in tissue engineering. The PPS characteristics can be altered by tuning the polyol/diacid ratios. Several reports were found on poly(glycerol sebacate) (PGS) and Poly (xylitol sebacate) (PXS) fibers as hybrid or core-shell structures that confirm the diversity

of

chemical,

mechanical

and

cell-tissue

responses

[17-23].

Likewise,

phisicochemical properties of PMS are considerably different from PGS or PXS film[16]. The tensile Young‟s moduli and elongation of PPS polyesters were reportedly ranging from 0.37 ±0.08 to 378 ± 33 MPa and 10.90 ± 1.37% to 205.16 ± 55.76%, respectively[17]. Among synthesized PPS polymers, PMS 1:2 exhibited considerably different and enhanced

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Journal Pre-proof mechanical properties in comparison with widely-studied PGS[10, 16, 18, 19], but to the best of our knowledge, PMS has never been studied as a fibrous scaffold. In the same way, in vitro and in vivo biocompatibility assays confirmed that PMS had an acceptable tissue response with significantly less inflammation compared to poly(lactide-co-glycolide) (PLGA) which has been widely used for biomedical applications[16]. One of the most challenging drawbacks of PPS polyesters for fabricating nanofibrous scaffolds using electrospinning process is the lack of sufficient chain entanglement due to low viscosity[20, 22].

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Subsequently, several natural and synthetic polymers, including Poly(lactic acid) (PLA)[23],

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poly(Ɛ-caprolactone) (PCL)[21], collagen[24] and gelatin[25] have been investigated for

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electrospinning of PGS. Although PLA has received considerable attention due to the Food

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And Drug Administration (FDA) approval, the degradation rate and particularly tissue responses have not been outstanding for tissue engineering applications [26, 27]. Hence, it

lP

should be improved with different strategies such as blending [28, 29] or copolymerization

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profile.

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[30] with new alternative elastomers with adequate mechanical properties and degradation

In this study, in order to overcome the limitations of the PMS electrospinning process and enhance the tissue responses of PLA nanofibers, PMS was blended for the first time with PLA to fabricate elastomeric nanofibrous scaffolds. PLA as a carrier could facilitate the nanofibers formation due to increasing the solution viscosity and chain entanglement. PLA has been extensively electrospun for tissue engineering applications with a wide range of solvent choices [31, 32]. However, PLA presented poor cell behavior and moderate inflammatory responses due to its hydrophobicity and low degradation rate in previous studies [9, 26, 27, 33]. Next, we characterized the electrospun nanofibers for their mechanical and cellular properties and tissue responses. Therefore in this paper, we report the results of

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Journal Pre-proof in vitro and in vivo evaluation of these novel nanofibrous PMS:PLA scaffolds with enhanced mechanical properties, appropriate for tissue engineering applications.

2. Materials and Methods: 2.1. Materials All chemicals were purchased from Sigma Aldrich Company except as noted below. PLA

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with 130 kDa molecular weight and electrospinning instrument were provided by FNM

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Company. All cell culture media such as high glucose Dulbecco‟s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), penicillin/streptomycin, trypsin, and Phosphate Buffer

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Saline (PBS) were purchased from Gibco Company. LDH Assay Kit and F-actin Staining Kit

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were also provided by Roche Applied Science and Abcam, respectively. NIH-3T3 cell line

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was purchased from Bank of human and animal cells, IBRC.

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2.2. Synthesis of PMS polymer

The PMS polymer was prepared according to previously described Methods by Bruggeman

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et al.[16] with some modifications. Briefly, PMS was synthesized by polycondensation process of mannitol and sebacic acid with a 1:2 molar ratio. 0.05 mol (~9g) mannitol and 0.1 mol (~20g) sebacic acid were mixed and melted in a three-necked flask. The synthesis flask was placed in an oil heating bath at 150 ±5 °C under argon gas bubbling through the reaction mixture. The synthesized polymer was collected and used for electrospinning procedure. 2.3. Fabrication of PMS/PLA nanofibrous scaffolds The electrospun scaffolds were fabricated by dissolving PMS:PLA and pure PLA in Hexafluoroisopropanol (HFP) solvent. The overall polymer concentration was in the range of 8 to 12% w/v. Pure PLA solution was prepared at 8% w/v for electrospinning. The PMS:PLA

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Journal Pre-proof weight ratios were adjusted at 70:30, 60:40, 50:50 and 40:60. The homogenous polymer solutions were obtained after 24 hours of stirring at room temperature before electrospinning process. Electrospinning was carried out by loading polymer solution in a 5ml-syringe with a 23G blunt needle. The flow rate of injection was 0.5 and 1 ml/h under adjusted constant voltage (21 kV). The distance between the collector and needle was set to 20 cm in all experiments. Random fibers were gathered on a circular collector covered with aluminum foil rotating with 400 rpm. The nanofibrous mats were dried under chemical flow hood to remove

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2.4. Nanofibrous scaffold characterization

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residual solvent and then stored in a desiccator until used for further studies.

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H-NMR. Proton Nuclear Magnetic Resonance (H-NMR) spectra of PMS 1:2 was recorded by INOVA 500 MHz spectrometer after dissolving the polymer in DMSO-d6 solvent. The

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composition of prepared PMS could be estimated from the integral ratios of the characteristic

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peaks of alcoholic and acidic moieties.

GPC. Gel Permeation Chromatography (GPC) was obtained to determine the molecular

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weight range of the prepared polymer. The weight average molecular weight (Mw), the number average molecular weight (Mn) and polydispersity index (PDI) were measured (GC 7890 -MSD (5975C), Agilent Technologies). SEM. The surface morphology and microstructure of electrospun nanofibers were evaluated by Scanning Electron Microscope (SEM) (AIS2100) at an accelerating voltage of 20 kV. Nanofibers were mounted and coated with a thin layer of gold using a sputter coater (SC7620). At least n=50 fibers per each sample were randomly chosen to measure the fiber diameter using Image J software (NIH).

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Journal Pre-proof ATR-FTIR. The functional groups in the chemical composition of the nanofibrous scaffolds were characterized by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. ATR-FTIR spectra were recorded on Spectrometer (Bomem MB-102) from 400 to 4000 cm−1 wavenumber range. All spectra were obtained after 20 scans at the spectral resolution of 4 cm−1. Mechanical testing. A uniaxial tensile test was carried out to disclose the mechanical

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properties of nanofibers in dry and wet conditions. The test samples were prepared as the

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rectangular strips (15mm×5mm) stretching with the Instron 5566 at the crosshead speed of 5 mm.min-1. The instrument was equipped with a 50 N load cell and a 15mm gauge width. For

C overnight and then subjected to the tensile test. Samples were stretched until failure, and

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0

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the tensile test of wet fibrous scaffolds, all the samples were soaked in PBS (pH  7.4) at 37

lP

stress-strain curves were obtained. Young‟s Modulus (E) was derived from the slope of stress-strain curves in the range of 5–10% strain. The toughness of nanofibrous scaffolds was

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calculated by area under the stress-strain curve up to ultimate tensile strength (UTS). At least

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five specimens were subjected to a tensile test for each scaffold. BET surface area and pore size studies. The Brunauer–Emmett–Teller (BET) and Barrett– Joyner–Halenda (BJH) techniques are used to determine the specific surface area and pore size distribution of PMS:PLA nanofibers. The BET method is established on physical adsorption and desorption of N2 gas on the surface of the sample. PLA and PMS:PLA nanofibrous scaffolds were degassed under vacuum for 48 h at room temperature. N2 adsorption-desorption isotherms were recorded using a BELSORP-mini II apparatus. In vitro degradation studies. Circular nanofibrous scaffolds (10 mm in diameter) weighed and soaked in 5 ml PBS (37 0C, pH7.4) containing sodium azide (Sigma Aldrich, 0.05% w/v) [16]. Scaffolds were agitated continuously in a shaker incubator at 37 0C with 100 rpm

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Journal Pre-proof and removed at each time points, washed with distilled water, then dried at room temperature under chemical hood for 72h. Five samples were weighted. The mass loss of each scaffold was measured with Eq. 1:

Eq. 1 and

represent the dry mass before and after soaking, respectively.

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Where

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Contact Angle. The hydrophilicity of the nanofibrous scaffolds was evaluated by measuring

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the contact angle of the water droplet. The wettability was characterized by depositing a 4μldroplet of distilled water on the surface of PMS:PLA and pure PLA fibrous scaffolds. The

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experiments were done based on a static drop technique by a video contact angle system

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(CA-500A). Droplet images were analyzed by Image J contact angle plugin (Drop Analysis).

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2.5. In vitro cytocompatibility assays

cytotoxicity”

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Cell Seeding. The cytotoxicity assays were carried out according to the “Tests for in vitro standard

assessment

published

by the

International

Standardization

Organization (ISO 10993-5:2009. The pure PLA and PMS:PLA nanofibers were washed with a serial dilution of ethanol and deionized water and then dried under a laminar flow hood. Nanofibers were sterilized by UV radiation at least 20 min for each side for further use. The 3T3 fibroblast was cultured in high glucose-DMEM, supplemented with 10% FBS, and 1% antibiotic (100 IU/mL penicillin,100 mg/mL streptomycin) while the incubator was adjusted at 37 0C with 5% of CO2. The 3T3 cells were seeded on 96-well tissue culture plates (TCP) at a final concentration of 1 × 104 cell/well. After 24 hours at about 80% confluency, the extract and direct assay were carried out on sterilized nanofibers. The liquid extract was prepared by soaking nanofibrous scaffold in complete cell culture media in an incubator at 370C overnight

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Journal Pre-proof (n=7). After 24 hours of culturing 3T3 cells, the culture medium was removed, and each wellreceived 150 µl of the extract dilution of nanofibers. The direct contact assay was performed by placing individual nanofibers on the semi-confluent cell layer in each well. The cell culture plates were incubated for the next 48 hours before extract and direct assay. Alamar Blue Assay. The metabolic activity and cell viability status of seeded fibroblasts were investigated by Alamar Blue assay. Briefly, the culture media of each well was replaced

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with 10% v/v Alamar Blue reagent was added to complete fresh medium (without phenol red)

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and the culture plates incubated in a light-protected cover for the next 6 h. The absorbance of reduced supernatants was read at 570 and 600 nm as a reference using a plate reader

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(Cytation 5, Biotek). The quantitative evaluation of cell viability was performed by

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calculating the percentage difference between nanofibers and control cells seeding on TCP

Eq. (1)

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using Eq. (1):

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Where 80586 and 117216 are molar extinction coefficient of oxidized Alamar Blue at 570 and 600 nm, respectively. AT570 and AT600 are the absorbances of test wells (each well contains nanofiber or the extract) at 570 nm and 600nm. AC570 and AC570 are also the absorbances of untreated control cells at 570 nm and 600nm. LDH assay. LDH detection was used to monitor cytoplasmic LDH activity released from damaged 3T3 cells to the extracellular environment. For this purpose, the serum-free supernatant of each well was removed to react with the LDH reaction mixture prepared according to Rosh protocol (version 11). The absorbance was read at 490 and 600 nm as a reference after about 30 min. To calculate the LDH release, low and high controls were

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Journal Pre-proof determined the spontaneous and maximum LDH release from test wells with untreated cells and Triton X-100 (2% v/v) treated cells, respectively. The proliferation assay was also carried out after 1, 3, 5 and 7 days of 3T3 culture by Alamar Blue and LDH assay. At each time point, the supernatant of seeded scaffolds was collected to react with LDH reaction mixture and the nanofibers were incubated with media supplemented with 10 vol% Alamar Blue. TCP and PLA were chosen as positive and scaffold controls,

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respectively.

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Cell attachment. The cell attachment properties were assessed by seeding 3T3 fibroblast cell

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line on the PMS:PLA with selected weight ratios (60:40, 50:50 and 40:60) and pure PLA.

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Cells seeded on nanofibers were fixed with formaldehyde solution (4%) after three days. Subsequently, experimental samples were treated with serial dilutions of absolute ethanol

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(35% ,50%,70%,80%,90%,100%) and dried under the chemical flow hood overnight. Cell

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attachment and cell spreading were observed using SEM. Cell-seeded scaffolds were stained with Phalloidin (green) and DAPI (blue) to evaluate the F-actin in cytoskeletal structure and

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nuclei 24 h after seeding cells. The fixed cells on scaffolds permeabilized with 0.1% Triton X-100 for 5 min. Then Phalloidin working solution and DAPI were added. 2.6. In vivo assay:

PMS:PLA and pure PLA nanofibers were prepared to discover the biocompatibility of subcutaneously implanted nanofibers in approximately 250 g weight male Lewis rats (Pasteur Institute of Iran). Twenty Lewis male rats were randomly divided into four groups (n = 5) for subcutaneously implanting PMS:PLA 60:40, 50:50, 40:60 and PLA nanofibrous scaffolds. Scaffolds were cut with a diameter of 10 mm in a circular shape. All samples were washed with ethanol 70% and sterilized by UV radiation for at least 20 min for each side. The sterilized scaffolds were implanted at the back of each rat which was shaved and washed with 11

Journal Pre-proof ethanol 70% and Betadine. The animals were humanely sacrificed by CO2 inhalation after a week post-implantation and samples were harvested. Tissue samples from implantation sites were obtained consisting of skin with surrounding tissues and fixed with formalin 10% for 48h then processed and embedded in paraffin. From each implanted nanofibrous scaffold four histological sections with a thickness of 5μm were prepared and stained with Hematoxylin and Eosin (H&E) and the Masson trichrome (MT) methods. The acute inflammatory responses were assessed by an independent pathologist. Ten microscopic fields (High power

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field, X400) of implanted area were randomly selected and evaluated for counting

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inflammatory cells, using computer software Image-Pro Plus® V.6 (Media Cybernetics, Inc.,

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Silver Spring, USA). The average score of each criterion from these four sections was then

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recorded and used for comparison. Moreover, inflammatory cells and fibrosis capsules were studied using a semi-quantitative scoring system as described in ISO 10993-6 in order to

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examine potential inflammatory response adjacent to the nanofibrous implants. The number

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of inflammatory cells present at surrounding tissues scored with 0-4 according to the number of cells per HPF. A semi-quantitative scoring system described as 0 = 0 cells, 1= rare, 1–5

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cells, 2= 5–10 cells and 3= heavy infiltrate, and 4= densely packed infiltrate for the number of inflammatory cells and other parameters were scored as 0=absent, 1=minor, 2=mild, 3=moderate, 4=extensive inflammation. All animal studies were conducted following the regulations and approval of the Research Ethics committee of TUMS (approval number: IR.TUMS.VCR.REC.1395.886). Statistical analysis. All data were expressed as mean ± standard deviation (SD). Statistical comparisons to determine the significance was performed by one way and two way ANOVA followed by Holm-Sidak's test. The level of statistical significance was set at P<0.05, except otherwise noted. All error bars in diagrams were presented as SD.

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Journal Pre-proof 3. Result and discussion: 3.1. PMS synthesis and characterization The PMS aliphatic polyester was fabricated using a polycondensation reaction of 1:2 theoretical molar composition of mannitol and sebacic acid. The chemical composition of PMS polyester in theory and by H-NMR spectrum was reported in Table 1. The integral ratio of the H-NMR characteristic peaks represented the chemical ratio of sebacic acid to mannitol

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[34]. Moreover, the proton peaks observed at 3.5-5.5 ppm labeled „a-b‟ in Fig. 1a are

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coordinated with methylene units of mannitol[35]. Furthermore, the signals appeared at

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1.3,1.5 and 2.3 ppm labeled „c-e‟ in chemical formula and spectrum are related to sebacic

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acid.

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The residual water and DMSO peaks were also considered at 3.3 and 2.5 ppm, respectively. It seems that the water signals is probably due to the moisture absorbed by DMSO. The area

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under the peaks corresponding to mannitol and sebacic acid revealed approximately twofold of sebacic acid involved in polymer compared with a polyol [16, 34-36]. The average

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molecular weights (Mw, Mn) of synthesized PMS are also reported in Table 1. As it is expected, low molecular weight PMS was obtained while the reaction stopped before gelation time [35, 37].

1.1. Nanofibrous scaffold characterization Chemical functional groups on nanofibrous scaffolds were assessed by ATR-FTIR spectra. As it is shown in Fig. 1b, five regions are defined in the absorbance ATR-FTIR spectra. There was typical broadband in PMS spectrum located in the range of 3500–3200 cm−1. Table 1. PMS polyester theoretical composition and average molecular weight after polycondensation reaction with 1:2 molar ratio of mannitol to sebacic acid at 150 ±5 °C

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Composition of polycondensation Composition by H NMR

0.88:2

Mw(g/mol) a

5287

Mn (g/mol) b

2340

PDI c

2.26

a

Mw: Weight Average Molecular Weight, b Mn: Number Average

Molecular Weight, c PDI: Poly Dispersity

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Index (Mw /Mn)

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Journal Pre-proof Fig. 1 (a) A representative H-NMR spectrum of PMS 1:2 polymer. Signal intensities of the mannitol at 3.5–5.5 ppm were identified on hydrogens of chemical formula and H-NMR spectrum by labels „a‟ and „b‟, respectively. Signal intensities of sebacic acid were identified at the peaks of 1.3, 1.5 and 2.3 ppm labeled on hydrogens of the carbons „c‟, „d‟ and „e,‟ respectively. (b) FTIR spectra of the electrospun scaffold for pure PMS, PLA and different ratios (60:40, 50:50 and 40:60) of PMS:PLA. The main peaks of PMS:PLA scaffolds (60:40,

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50:50 and 40:60) are comparable with pure PMS and PLA samples

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It is indicated to O-H stretch bond of hydroxyl groups which is related to mannitol moiety in the structure of PMS [38]. C-H bonds stretch in the range of 3000–2850 cm−1 and C-H bonds

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bend at 1500–1400 cm−1 also corresponds to methylene groups of PMS. The intense peaks

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presented at about 1800-1600 cm−1 and 1100 cm−1 confirms the formation of ester bonds in

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the backbone of PMS polymer due to the polycondensation process of mannitol and sebacic acid. These two regions attributed to double bond C=O and single bond C-O stretching of the

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carboxylate functional groups which are represented in low and high molecular weight PPS

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[16, 35, 39]. The PMS spectrum presents peaks at 1730 cm-1 and about 1442 cm−1 could be attributed to unreacted acidic groups of sebacic acid. The appearance of the split peaks at 1700-1800 cm-1 is most likely attributed to the low crosslinking condition of PMS which leads to fabricating low molecular weight polymer [21, 30, 35, 40]. Concerning the PLA spectrum, peaks at 1750 and 1090 cm-1 are mostly assigned to the stretching of C=O and C–O bonds. Characteristic frequencies of symmetric and asymmetric stretching in methyl bonds were revealed at 2940, 2992 cm-1 and 1364,1453 cm-1, respectively. Peaks at 1364 and 1453 cm-1 were Also observed, which corresponds to symmetric and asymmetric bending of methyl groups in the PLA chain[40]. The comparison among the principal peaks of PMS and PLA with different ratios (60:40, 50:50 and 40:60) is

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Journal Pre-proof comparable with pure PMS and PLA samples. It confirms that the physical mixing of PMS and PLA with the different weight ratios successfully occurred. As it is summarized in Table 2, the polymer concentration in the range of 8-12%w/v was examined in order to optimize the fibers‟ diameter and morphology. It was found that PMS:PLA blends could be electrospun into homogeneous and smooth nanofibers (Fig 2.a, b, c) with a concentration of 10%w/v. In comparison with other concentrations, lower polymer

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contents lead to the formation of beads (8% w/v) or fused junction between fibers (9% w/v)

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in all blending composition. In contrast, increasing concentrations (up to 12 %w/v) resulted in significantly thicker fibers. Consequently, the 10 %w/v overall polymer concentration was

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distinguished as the appropriate concentration for obtaining desirable fibers at the nanoscale.

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The most critical features which have crucial roles in obtaining bead-less homogenous

lP

nanofibers at constant voltage are the concentration and blending ratios of polymers. The formation of electrospun nanofibers highly require chain entanglements of polymers in the

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polymeric solution that are supplied at higher concentrations with convenient viscosity.

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Increasing polymer concentration leads to thickening of the fiber diameter and decreasing the homogenecity of nanofibers. By increasing the concentration from a certain limit, the increased viscosity of polymer solution had the opposite effect and prevented jet formation. Therefore, further increasing of the concentrations leads to droplet formation due to high viscosity and surface tension of the solution [31, 41-43]. Table 2. The diameter of PMS:PLA electrospun nanofibers from solutions with different polymer concentrations and ratios for two determined feed rates (0.5 and 1 ml/h) PMS:PLA 70:30 Fiber Diameter (nm) Concentration (%w/v)

Rate = 0.5 ml/h

PMS:PLA 60:40 Fiber Diameter (nm)

PMS:PLA 50:50 Fiber Diameter (nm)

PMS:PLA 40:60 Fiber Diameter (nm)

Rate = 1 ml/h

Rate = 0.5 ml/h

Rate = 0.5 ml/h

Rate = 1 ml/h

Rate = 1 ml/h

Rate = 0.5 ml/h

Rate = 1 ml/h

228 ± 55

220 ± 37

207 ± 41

227 ± 41

275 ± 44

290 ± 48

302 ± 39

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210 ± 42

Beaded fibers Beaded fibers Beaded fibers Beaded fibers Beaded fibers Beaded fibers Beaded fibers

Beaded fibers

9

10

196 ± 53

287±70

Beaded fibers Beaded fibers

253 ± 42

254 ± 56

303 ± 57

414 ± 51

368 ± 76

397 ± 121

379 ± 57

399 ± 85

235 ± 38

246 ± 41

421 ± 68

440 ± 89

438 ± 62

483 ± 70

Beaded fibers Beaded fibers

275 ± 60

325 ± 41

349 ± 82

467 ± 61

482 ± 79

467 ± 54

499 ± 61

553 ± 102

12

439 ± 98

497 ± 103

510 ± 62

548 ± 72

614 ± 137

633 ± 128

650 ± 87

666 ± 101

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Fig. 2 SEM images of electrospun PMS:PLA nanofibers at the concentration of (a) 9% , (b) 10% , (c) 11%.w/v for optimizing the morphology and microstructure of the nanofibers, different blending ratios of PMS and PLA (70:30, 60:40, 50:50 and 40:60) at two feed rates (0.5 and 1ml/h) were presented. Gaussian distribution of nanofiber diameters was presented below of each SEM image. SEM images scale bar:10 μm. (d) The effects of feed rate and PMS content on the nanofibers average diameter (Mean ± SD).

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Fig. 3 The typical stress-strain curves PMS/PLA and pure PLA nanofibrous scaffolds

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exhibited in dry and wet conditions

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The minimum feed rate of the polymer solution is required to maintain the Taylor Cone through capillary forces. By increasing the feed rate (Fig. 2d) mostly the mean diameter of

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nanofibers increased due to the excessive polymer provided in solution for nanofibers

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forming. This is in consistence with other findings which reported similar trends [41, 44].

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PLA nanofibers with 773±188-nm diameter were electrospun as a control sample with

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distinctly thicker nanofibers in comparison with PMS:PLA blends (data not shown). In other words, as it is shown in Fig. 2d and Table 2 by increasing PLA content; the formation of

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beads on nanofibers was remarkably decreased while the mean diameter of fibers was reasonably increased. This fibers thickening due to PLA addition is in agreement with those results established in the literature [45]. As a result, the optimized concentration and ratio for obtaining homogenous nanofibers without junctions or beads were at 10% w/v polymer concentration with selected 60:40, 50:50 and 40:60 PMS: PLA blends ratios. The uniaxial tensile test was carried out to characterize the mechanical properties of PMSbased nanofibrous scaffolds. The typical stress-strain curves for selected ratios (60:40, 50:50, 40:60 and pure PLA are exhibited in dry and wet conditions in Fig. 3. the linear region followed by an elastic behavior was revealed approximately up to 10% strain. The extracted mechanical characteristics such as yield strength (σy), Young‟s modulus (E), ultimate tensile

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Journal Pre-proof strength (σUTS), failure strain (Ɛfailure) and toughness (MJ/m3) were reported in Table 3. Although selected compositions of nanofibers showed a comparable Young‟s modulus (E) of about 50 MPa in a dry condition, wet nanofibers exhibited a significant decrease in all polymeric ratios except pure PLA which remained without notable change. This most likely occurred in pure PLA nanofibers because of the hydrophobic nature of PLA which could inhibit water absorption. Thus, no significant difference in mechanical properties was observed in dry and wet conditions for pure PLA nanofibers as reported in the literature. In

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addition, increasing the weight percentage of PLA in the composition of nanofibers lead to a

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significant increase in E, σUTS, and toughness but Ɛfailure did not change remarkably. Table 3. Comparison of mechanical properties for PMS-based and pure PLA nanofibrous

Tensile strength, σuts (MPa )

Failure strain, Ɛfailure (%)

Toughness (MJ/m3)

PMS:PLA 60:40

2.53±0.20 *

57.10±9.52 *

3.12±0.18 *

84.64±15.21

185.08 ±22.27*

PMS:PLA 50:50

1.76±0.34 *

47.88±4.82 *

2.51±0.12 *

80.86±16.05

144.90±10.74*

PMS:PLA 40:60

2.75±0.41 *

47.92±5.78 *

85.96±15.64

262.24±62.14*

6.28±0.66

112.02±0.06

7.18±0.81

60.15±3.74

387.28±61.42

3.88±0.76 *

PMS:PLA 60:40

1.45±0.17 *

30.66±1.88# *

1.91±0.22*

63.21±11.33

99.07±8.87 #

PMS:PLA 50:50

1.52±0.08 *

33.47±2.82*

2.13±0.14*

91.00±2.67

127.27±16.38*

PMS:PLA 40:60

2.04±0.27 *

41.46±4.88*

3.06 ± 0.10*

76.85±24.63

180.00±46.56*

PLA

6.10±0.77

94.68±0.12

7.14±0.86

63.61±5.22

379.05±0.637

PLA

Wet

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Young's modulus , E (MPa)

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Dry

a

Yield Strength, σy (MPa)

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Scaffold Composition

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scaffolds in dry and wet conditions

a

Young's modulus was calculated from the initial slope of the stress-strain curve approximately up to 10% strain. (n = 5; One-way ANOVA with Holm-Sidak multiple comparison tests, #p < 0.05 compared to each corresponding scaffold in dry condition and *p < 0.05 compared to pure PLA nanofibrous scaffolds in dry/wet state)

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Journal Pre-proof PMS-based nanofibres exhibited lower stiffness, strength and higher elasticity due to the reduction of E, σUTS, and rise of Ɛfailure which is most likely attributed to the elastomeric features of PMS, affecting mechanical properties of the blended nanofibers[46]. To the best of our knowledge, there is no report on the fabrication of PMS nanofibers and their mechanical properties in the literature. Comparative research was carried out on PMS:PLA nanofiber as a filler up to 15 wt% to PMS substrate displayed similar elongation but

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enhanced E, σUTS and toughness. It is mentioned that it‟s due to effective interaction and adhesion of PMS matrix to PLA nanofibers. The effective stress distribution in elastomeric

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polymers resulted in increasing mechanical strength[37]. However, in PGS:PCL blended

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nanofibers, the addition of PGS up to 75% exhibited the lower stiffness, strength, and

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elasticity whereas increasing the amount of PGS up to 83% showed remarkable higher

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stiffness and strength [21].

With regard to the core/shell PGS/PLLA nanofibers, it has been reported that the addition of

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less than 5 wt% PLLA indicated no noteworthy enhancement in mechanical properties.

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However, without considering the statistical analysis, the increment of PLLA resulted in the reduction of Ɛfailure and increase in E on average[23]. In fact, the mechanical evaluation of PGS/PLA blends published in the literature is suffering from the lack of interpretable data on the higher blending weight percentage of PLA[23]. In general, the more PGS content presented in the blending composition of the nanofibers resulted in the reduction of young modulus and σUTS with higher Ɛfailure which is in agreement with the results obtained in this research [21, 47]. It is worth to note that the diameter and thickness of the nanofibrous scaffolds are two crucial factors playing an important role in determining the mechanical properties of nanofibers. Accordingly, thicker scaffolds with larger fiber diameters lead to lower stiffness and strength[48]. As it can be observed in Fig. 2 and Table 2 at 10% w/v the average diameter of nanofibers rose up from 235±38 to 483±70 nm with the presence of more

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Journal Pre-proof PMS in the blending composition. Although the statistical analysis revealed no significant differences among the mechanical properties of three blending ratios of PMS-based nanofibers, PMS:PLA 40:60 with higher PLA content on average revealed less stiffness, strength, and toughness compared to PMS:PLA 60:40 nanofibers. This could be attributed to the fiber's thickening due to the addition of more PLA to the structure. It is required to mention that the scaffold thickness was not varied between the samples and kept PMS in the range of 120-150 nm. Thus it seems that the thickness of the nanofibrous mat was not a major

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factor in our research in altering the mechanical properties. The obtained mechanical

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properties of PMS:PLA nanofibrous scaffolds seems to be similar to collagen fibers,

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cartilage, and aortic heart valve [10].

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The N2 adsorption-desorption isotherms and the pore size distribution curves of PLA and

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PMS:PLA nanofibers with different weight ratios are presented in Fig. 4. According to International Union of Pure and Applied Chemistry (IUPAC) classification[49], the resultant

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PMS:PLA nanofibers followed from the shape of the type II isotherm which refers to

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mesoporous (2-50 nm pore diameter) structures. These findings are in agreement with previous reports about the porosity of nanofibers[50, 51]. According to the BJH method (Fig. 4b), the PMS:PLA electrospun nanofiber mostly contained the pores sizes in the range of 2-4 nm. BET specific surface area and BJH pore volume of electrospun nanofiber were reported in Table. 4. The specific surface areas of 6.48, 10.61, 8.91 m2.g−1 was enhanced for the PMS:PLA 60:40, 50:50, 40:60 respectively, in comparison with for PLA nanofibers (2.41 m2.g−1 ). Based on findings on the BET and BJH evaluations the specific surface areas obtained for PMS:PLA nanofibers were in the range which is found advisable for biomedical application [50, 51].

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Journal Pre-proof The degradation rate of the electrospun PLA and PMS:PLA nanofibers was assessed in Fig 4. The in vitro degradation studies of PLA and PMS:PLA nanofibers with weight ratios of

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60:40, 50:50 and 40:60 were performed in PBS solution (37 0C, pH7.4) for up to 35 days.

Fig. 4 a) The N2 adsorption-desorption isotherms b) Pore size distribution of PLA and PMS:PLA nanofibrous scaffold with different ratios (60:40, 50:50, 40:60) Table 4. Specific surface area and pores volume of PLA and PMS:PLA nanofibrous scaffold Sample (Nanofiber)

BET Specific surface area (m2.g-1 )

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BJH pores volume (m3.g-1)

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6.48

0.012

PMS:PLA 50/50

10.61

0.017

PMS:PLA 40/60

8.91

0.019

PLA

2.41

0.003

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PMS:PLA 60/40

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Fig. 5 In vitro degradation rate of PLA and PMS:PLA nanofibres (60:40, 50:50 and 40:60) in PBS solution at 37 0C for 35 days

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The weight loss of PMS:PLA 60:40, 50:50 and 40:60 nanofibers were approximately 43.99

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%, 43.46%,32.92% in 35 days, respectively, while the degradation rate of PLA nanofibers was measured as 4.10% within the same period. The higher weight loss of PMS:PLA nanofibers was revealed that blending PMS and PLA polymer could improve the biodegradability of PLA nanofibers. As a result of faster degradation rate of PMS compared with PLA, this biodegradable polyester could be used to modify PLA degradation rate. These results are in agreement with previous studies and demonstrated that PMS:PLA 40/60 nanofibers with the lower degradation rate corresponded to nanofibers with the higher strength and lower strain[35]. It seems that the higher amounts of hydrophobic PLA in the microstructure of nanofibers lead to relatively decreasing the hydrolysis of the ester bonds in PMS backbone [16, 52, 53].

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Journal Pre-proof The wettability behavior of nanofibrous scaffolds had a significant impact on cell adhesion and spreading. Not only this feature could alter protein-nanofibers interactions, but it could also affect cell affinity to the scaffolds ref. According to Fig. 6, it can be seen that the water

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contact angle of PLA nanofibers was 133.9±1.6 0 without any significant change after a

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Fig. 6 Contact angle represented for pure PLA and PMS:PLA nanofibers of varying

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concentrations (60:40, 50:50 and 40:60) after a period of 10s. (n = 5, One way ANOVA followed by Holm-Sidak's multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001,

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****p < 0.0001, Mean ± SD)

period of time (10s). This confirms the hydrophobic nature of PLA nanofibers with angles higher than 900 which is in agreement with the previous studies[20]. Although similar trends existed in PLA-rich scaffolds (PMS:PLA 40:60), there was a notable difference between contact angles of PLA and PMS/PLA 40:60 at 10s. It can be seen that however, the contact angles decreased approximately 300, the scaffold possessed hydrophobic nature with angles higher than 90°. The nanofibers containing 60% and 50% PMS exhibited a statistically major reduction in contact angle at 5s and 10s. This result shows that the addition of aliphatic PMS polyester to the composition of PLA nanofibers lead to appear hydrophilic nature in blended nanofibers [35] and the surface of PMS-based nanofibers thoroughly absorbed water droplet

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Journal Pre-proof after 10s. This is mostly due to the presence of hydroxyl groups of mannitol moieties in the polymer chain [35, 54]. 1.2. In vitro cell cultures studies on PMS:PLA nanofibrous scaffolds PPS family is a well-known elastomeric polyester with a wide range of applications in tissue engineering and regenerative medicine[10, 12, 16]. The cytocompatibility of the PMS:PLA

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nanofibers were evaluated by the assessment of metabolic activities and the damage of the cell membrane. As it is shown in Fig. 7a, the cytocompatibility of PMS:PLA nanofibers was

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acceptable in different weight ratios. Quantitative analysis of cell viability in direct and

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extract assay after 48h clearly demonstrated that the metabolic activity of fibroblast was not

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considerably different in PMS:PLA nanofibers in all weight ratios compared to TCP and pure

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PLA scaffolds with the exception of 50:50 ratio. Moreover, the percentage of released LDH enzyme into the extracellular space was in the acceptable range in all ratios compared with

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positive control. According to the results, there were no significant differences among the LDH release from fibroblast cells seeded on nanofibres containing PMS. In direct assay, the

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LDH release in PMS based nanofibers was considerably less than PLA and TCP while there was no significant difference between PLA and TCP. Fig. 7b showed that the cell proliferation on PMS-based nanofibers compared with PLA was statistically enhanced after 5 and 7days on 50:50 and after 3day on 40:60 ratio, whereas there were no considerable differences between PLA and blended scaffolds on the other time points. The similarity of Alamar Blue reduction in all scaffolds and TCP on the first day could indicate effective cell adaptation on PMS-based nanofibers. This could be attributed to the cells favored to attach and spread over the PMS in the microstructure of nanofibers to achieve a confluent fibroblastic cell layer [16]. Although PLA nanofibers reported as a compatible scaffold for cell adhesion, proliferation and spreading, its poor hydrophilicity is 27

Journal Pre-proof still challenging [26, 28, 33]. In fact, there were various studies on the improvement of the cell attachment on hydrophobic nanofibers such as PLA and PCL by blending with hydrophilic polymers[55-57]. Hence, it was reported that synthetic PGS containing films or nanofibers enhanced cell attachment and proliferation due to higher hydrophilicity compared

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to pure PLA or PCL[21, 23, 25].

Fig. 7 (a) Quantitative evaluation of 3T3 viability by Alamar Blue and LDH assay after 48h cell seeding. Cytotoxicity assays were performed under the direct and extract protocol of ISO 10993-5. (b) Cell proliferation on 1 day and 3, 5 and 7 days post-seeding 3T3 cells Control:(Cell +Media + Lysis) (c) Evaluation of cell attachment and morphology of 3T3 cells on PMS:PLA nanofibers (60:40, 50:50,40:60) and pure PLA 3 days after seeding. statistical analysis was evaluated using ANOVA

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Journal Pre-proof with Tukey's multiple comparisons test (*compared to PLA nanofibers, n = 7, *p < 0.05, Mean ± SD, scale bar: 30μm)

These cytocompatibility and proliferation results were confirmed with the SEM images obtained from 3T3-seeded scaffolds. As it is illustrated in Fig. 5c, it was found that 3T3 cells attached and spread over the hydrophilic surface of PMS-based nanofibres whereas there was a considerable amount of rounded cells on PLA nanofibers after three days. As it is clearly

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seen in these SEM images, not only fibroblasts revealed desirable interactions and adhesion connections on PMS-based nanofibers with enhanced proliferation on PMS-based nanofibers,

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but also they effectively maintained their elongated morphology in comparison with PLA

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nanofibers. This trend is compatible with cell behaviors on PGS scaffolds due to the

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improvement of hydrophilicity on the surface of each nanofiber [48]. The presence of PMS in

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the microstructure of nanofibers improved the interaction of fibroblast with PMS:PLA nanofibrous scaffold. This process would be mediated by the absorption of plasma membrane

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proteins on the surface of the nanofibers. As chemical composition, nanotopography and stiffness of the substrate could have major impacts on cell attachment, retention and spread,

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the addition of hydrophilic PMS component to the hydrophobic PLA polymer leads to enhanced cell behavior on these scaffolds [25].

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Fig. 8 Representative fluorescence microscopy images of nuclei and F-actin filaments

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staining with Phalloidin (green) and DAPI(blue) respectively after 24h seeding of 3T3 cells

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on nanofibers. Cells were observed at 200× magnification (scale bar:100μm). The interactions of 3T3 fibroblast cells with PMS-based and pure PLA nanofibers one day

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after seeding of fibroblast cells are shown in Fig. 8. It can be seen from F-actin and nucleus staining of seeded 3T3 cells, fibroblasts adhere and spread on nanofibrous scaffolds in all samples. However, the attachment and mainly spreading with elongated morphology were more prominent on PMS containing nanofibers (PMS:PLA 60:40). The key factors playing essential roles in adhering fibroblasts onto the scaffold could be ECM-mimickingcharacteristics of PMS-based nanofibrous scaffolds. Hydrophilic features of these scaffolds, together with desirable stiffness and mechanical properties and nanoscale topography might be involved in better cell affinity of these scaffolds[22].In fact, the wetting properties of nanofibers are enhanced in nanofibers containing PMS polyester. Previous studies

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Journal Pre-proof determined the deep cell penetration and ECM growth in porous PGS-PLLA scaffold as a result of enhanced hydrophilicity and pore sizes [29]. According to the previous reports on the cytocompatibility of low crosslinked PGS-PCL blend structures [48], it seems that the cell responses to the PLA nanofibrous scaffolds could be extensively improved by adding low-crosslinked PMS with abundant free hydroxyl groups. This modification confers hydrophilic nature to the inherently hydrophobic PLA. In

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other words, free hydroxyl functional groups could facilitate the cell-matrix interactions

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which could lead to spreading cells with appropriate morphology and more physiological characteristics [58]. Furthermore, it seems that the similarity of stiffness and strength could

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induce cell morphology from rounded to naturally elongated [59, 60]. Similar results were

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also reported for electrospun scaffolds with more PGS contents [21].

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1.3. In vivo assessment of PMS: PLA nanofibrous scaffolds

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The in vivo biocompatibility of PMS-based nanofibers was examined via subcutaneous implantation of nanofibrous scaffolds at the back of the rat models. Following implantation,

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the surgical wounds of the rats healed well in all groups after one-week post-implantation. No gross acute inflammatory reactions or rejection was observed by observation of the wounds in all groups (Fig. 9 a-d). Considering the histological H&E and MT stained cross-sections in Fig. 9 e-l, the implanted nanofibers which were surrounded by connective tissue induced an acute inflammatory response with different grades which scored in Fig. 9m. Numerous macrophages, lymphocytes, and multinucleated giant cell existed around PLA nanofibers at the implantation site, demonstrating the moderate acute inflammatory responses which are similar to the reported literature [27, 61]. According to pathological scores related to the fibrous capsule, it was found that PMS:PLA 60:40 nanofibers had significantly thinner capsules than other scaffolds. A dense layer of 31

Journal Pre-proof fibrotic connective tissue along with numerous macrophages, lymphocytes, and polymorphonuclear inflammatory cells encapsulated implanted PMS:PLA 40:60 and 50:50. On the other hand, the nanofibrous scaffolds in all groups were not degraded and remained intact without the formation of scar or granulation tissues. Although the addition of PMS to the composition of nanofibers lead to less infiltration of inflammatory cells especially in PMS:PLA 60:40, there were no remarkable statistical differences among 50:50, 40:60 and pure PLA nanofibrous scaffolds. As a consequence, the inflammatory responses of PMS-

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based nanofibrous scaffolds were revealed enhanced biocompatibility in vivo compared to

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PLA nanofibers, considering due to a thinner fibrotic capsule and minor infiltration of

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inflammatory cells.

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m)

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Inflammatory cells

Fibrosis

Group

(N=4)

(N=4)

PMS:PLA 60:40

1,2,1,1

1,2,1,1

PMS:PLA 50:50

2,3,3,2*

3,2,4,2*

PMS:PLA 40:60

2,1,2,1

4,3,2,3**

4,2,4,3*

3,3,4,3**

PLA

Fig. 9 In vivo evaluation of biocompatibility by subcutaneously implanted PMS:PLA and pure PLA nanofibrous scaffolds at the back of the rat one-week post-implantation (n=5, four

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Journal Pre-proof groups). Gross observation of the implant zones for (a) PMS:PLA 60:40, (b) PMS:PLA 50:50 (c) PMS:PLA 40:60 (d) PLA nanofibrous scaffold at explanation. Red Circle and arrows point to the implantation site. Representative images of H&E stained sections of implanted (e) PMS:PLA 60:40, (f) PMS:PLA 50:50 (g) PMS:PLA 40:60 (h) PLA nanofibrous scaffold with surrounding tissues in 10× and more details at 40× demonstrating the acute inflammatory response. The magnified area is represented (in e-h images) by the black rectangular. (i-l) Histological stained images by MT. Thin black arrows:

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polymorphonuclear inflammatory cells, arrowheads: mononuclear inflammatory cells and

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thick arrows: multinucleated giant cells in H&E and MT staining. Sc: nanofibrous scaffold,

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M: skin muscles. (m) The scores of infiltration of inflammatory cells and fibrosis in different

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experimental groups. (n=5, one-way ANOVA followed by Tukey post hoc comparisons, *

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2. Conclusion:

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P<0.05, ** P<0.01 compared with a control group with no fibrous scaffolds)

In this research, PMS-based nanofibrous scaffolds with different ratios were successfully

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developed by electrospinning method. In order to overcome the limitations of electrospinning process for PMS and facilitate nanofibers formation, it was blended with PLA as a carrier to increase the solution viscosity and chain entanglement. Nanofibrous scaffolds based on PMS:PLA were successfully fabricated and characterized. The mechanical properties of PMS-based nanofibers were in the range of soft human tissues, making these scaffolds good candidates for tissue engineering applications. The degradation behavior of PMS/PLA nanofibrous scaffolds was appropriate for soft tissue engineering applications. In vitro biocompatibility evaluations confirmed the effective cell–scaffolds interaction between 3T3 and PMS based nanofibers. At the same time, PMS-based nanofibers exhibited negligible inflammatory responses compared to the FDA approved PLA nanofibers. In conclusion,

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Journal Pre-proof PMS-based nanofibres could be fabricated by electrospinning process and our outcomes also indicated that PMS:PLA nanofibrous scaffolds can be applied as a potentially well-qualified candidate for soft tissue engineering applications. Acknowledgments: The authors would like to acknowledge Tehran University of Medical Sciences for providing

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financial support (grant number: 95-03-87-33089) for this work.

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