Three-Dimensional Polydopamine Functionalized Coiled

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3D polydopamine functionalized coiled microfibrous scaffolds enhance human mesenchymal stem cells colonization and mild myofibroblastic differentiation Mehmet Berat Taskin, Ruodan Xu, Hans Vejersøe Gregersen, Jens Vinge Nygaard, Flemming Besenbacher, and Menglin Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02994 • Publication Date (Web): 06 Jun 2016 Downloaded from http://pubs.acs.org on June 9, 2016

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ACS Applied Materials & Interfaces

3D polydopamine functionalized coiled microfibrous scaffolds enhance human mesenchymal stem cells colonization and mild myofibroblastic differentiation Mehmet Berat Taskina, Ruodan Xua, Hans Gregersenb, Jens Vinge Nygaardb, Flemming Besenbachera, Menglin Chena,b* a

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000

Aarhus C, Denmark b

Department of Engineering, Aarhus University, DK‐8000 Aarhus C, Denmark

Abstract Electrospinning has been widely applied for tissue engineering as its versatility of fabricating extracellular matrix (ECM) mimicking fibrillar scaffolds. Yet there are still challenges such as that these two-dimensional (2D) tightly packed, hydrophobic fibers often hinder cell infiltration and cellscaffold integration. In this study, polycaprolactone (PCL) was electrospun into grounded coagulation bath collector, resulting in 3D coiled microfibers with in situ surface functionalization with hydrophilic, catecholic polydopamine (pDA). The 3D scaffolds showed biocompatibility and well integration with human bone marrow derived human mesenchymal stem cells (hMSCs), with significantly higher cell penetration depth compared to that of the 2D PCL microfibers from traditional electrospinning. Further differentiation of human mesenchymal stem cells (hMSCs) into fibroblast phenotype in vitro indicates that, compared to the stiff, tightly packed, 2D scaffolds which aggravated

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myofibroblasts related activities, such as upregulated gene and protein expression of α-smooth muscle actin (α-SMA), 3D scaffolds induced milder myofibroblastic differentiation. The flexible 3D fibers further allowed contraction with the well-integrated, mechanically active myofibroblasts, monitored under live cell imaging, whereas the stiff 2D scaffolds restricted that. Keywords: wet electrospinning, 3D, polydopamine, hMSC, myofibroblasts

Introduction Electrospinning technology has been widely recognized for fabricating tissue engineering biomaterials which renders bulk polymers and composites into submicron fibers that are structurally similar to fibrillary structure of the natural extracellular matrix (ECM). Apart from its merits in ease of use and applicability in vast selection of materials, it has been reported mainly for fabricating 2D scaffolds, due to the densely packed fibers. Solutions including using porogens (such as salt leaching1, sacrificial polymers2-3, 3D melt electrospinning,4 near-field electrospinning5 and modifying collectors to control electric field6, have been developed to overcome this issue. Among them, a few attempts on wet electrospinning,7 where electrospun fibers are collected in a non-solvent liquid bath with low surface tension, acting as a dispersant and coagulant8 instead of a solid collector, have been reported to be able to produce 3D constructs with lightly packed fibers. On the other hand, rendering the fiber surface to promote cellular adhesion is critical for the successful application of the electrospinning technology in biomedical applications. Inspired by the adhesive proteins secreted by mussels for attachment to wet surfaces, a straightforward, substrate-independent, surface coating strategy based on catecholic chemistry9 has emerged since Lee et al. discovered the


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polymerization of catecholic amine, dopamine.10-11 Polydopamine (pDA) has previously been demonstrated to promote cell adhesion and further regulate stem cells differentiation on various substrates.12-14 The pDA surface was found to not only stabilize mesenchymal stem cell adhesion and multipotency13, but also support reprogramming of human somatic cells and long-term self-renewal of human pluripotent stem cells under defined conditions.15 pDA modified 3D printed PLA scaffold, electrospun poly(lactic acid)16 and layer-by-layer stacking electrospun PCL/gelatin fibers14 enhanced osteogenic differentiation of human adipose-derived stem cells (hADSCs).17 pDA coated poly(εcaprolactone) diacrylate (PCLDA) also enhanced osteogenesis of BMSC.18 Although the mechanism is still under investigation, Lee et al19 reported that the pDA stimulate oesteogenic differentiation of periodontal ligament stem cells via activation of the integrin α5/β1 and PI3K signaling pathways. Li et al20

reported

calcium

phosphate

apatite/PDA

hybridized−polyethylene

terephthalate

(APA/PDA−PET)20 grafts support the attachment of bone marrow stromal cells (BMSCs) and stimulate the in vitro proliferation and osteogenic/angiogenic differentiation of BMSCs via activation of the PKC/p-ERK1/2 signaling pathway. Furthermore, pDA also facilitated highly efficient, simple immobilization of growth factors, adhesion peptides and nucleic acids onto polymer substrates. The neurotrophic growth factor or peptide-immobilized substrates greatly enhance differentiation and proliferation of human NSCs (human fetal brain-derived NSCs and human induced pluripotent stem cell-derived NSCs) at a level comparable or greater than currently available animal-derived coating materials (Matrigel).12 The pDA coated electropspun PCL fibers attached with RE-1 silencing transcription factor (REST) siRNA were found to be able to enhance neuronal commitment of mouse neural stem/progenitorcells (NPCs).21



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Mesenchymal stem cells (MSCs) are multi-potent cells capable of differentiating into multi-lineages such as, osteocytes, adipocytes, chondrocytes, myocyte and vascular endothelial cells22-23. Being a key player for tissue regeneration and homeostasis24-25, MSCs are found in a wide spectrum of presence, such as bone marrow, adipose tissue, blood, adult muscle tissue, etc26. Their fate can be regulated via both solubles (e.g growth factors) and biomechanics. While functions of growth factors are reported to be potent and well-defined 27-28, the responses of cells to environmental mechanical signals with downstream regulation of various pathways, such as differentiation

29

, proliferation30 or apoptosis31, are

rather more recent findings. Engler et al. and Nakayama et al. reported that stiff (25-40 kPa) substrates promote osteogenesis, intermediate stiffnesses (8–17 kPa) enhance myogenesis, while soft (0.1 - 1 kPa) substrates favor neural lineages 29, 32. Fibroblast is an omnipresent cell type in almost all tissues, which is known to contribute to wound healing process via their ECM modelling ability that promote migration of both cellular fluids and solubles to the injury site to maintain tissue integrity

33

. Despite being essential, fibroblasts

scientifically remain in a highly ambiguous area due to its characteristic diversity in different tissues 34. In the wounded tissues, quiescent fibroblasts get activated by various external factors such as inflammation and mechanical stress which induce regulation of signaling molecules such as transforming growth factor beta 1(TGF-β1) to regulate myofibroblast differentiation for contraction of granulation tissue

35

. However, persistent myofibroblastic activity in the wounded tissue can result in

pathological conditions known as fibrosis, which may result in reduced tissue functions, organ failure or even death36-37. Myofibroblasts are commonly identified by their α-smooth muscle actin (α-SMA) expression together with other stress actin fibers

38-39

. The resulted increased matrix stiffness increase

contractility of the myofibroblasts, which in return further upregulates connective tissue growth factor(CTGF) and TGF-β1 expressions, two prominent components in wound healing as well as


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fibrosis39. This increased tension is often resulted from the stiffness of the wound dressing scaffold and/or overly expressed ECM

32, 40

. During ECM remodeling, matrix metalloproteinases (MMPs), on

the other hand, are responsible to degrade excess ECM

41

to allow cell migration for proper wound

healing. Together with tissue inhibitor of metalloproteinases (TIMPs), ECM turnover is delicately regulated as seen in fetal tissues, which could contribute to their scarless healing process 42-44. Herein, we have for the first time fabricated 3D coiled microfibrous scaffolds by electrospinning polycaprolactone (PCL) into grounded bath collector, with in-situ surface functionalization of polydopamine (pDA) in one single step.

In comparison to 2D conventional electrospun fibers,

morphology and mechanical properties of the 3D fibers were characterized by Scanning electron microscopy (SEM), Contact angle measurement (CA) and Dynamic mechanical analysis. Their biocompatibility to hMSCs was evaluated by toxicity/viability assays, and cell infiltration was monitored for 4 weeks. The differentiation of hMSCs into fibroblast/myofibroblast phenotype was investigated by real-time polymerase chain reaction (qPCR) quantifying gene expressions, immunochemical staining for protein expressions and live-cell imaging for contractions.

Materials and methods Scaffold fabrication 17.5% (w/v) PCL(Mw = 70 000‐90 000, Sigma‐Aldrich) solution was prepared by directly dissolving it in chloroform. Then the polymer solution was dispensed at a feeding rate of 3ml/h from a syringe with a metallic needle (20 gauge) situated vertically on a pump system (New Era Pump Systems, Model: Ne-300, NY, USA). A grounded metal container filled with absolute ethanol was situated 15 cm away



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from the needle to collect the polymer jet. 10-12 kV is applied by a high voltage supplier to the metallic needle to withdraw polymer solution from needle to the collector. For pDA coating, same polymer solution was spun on 95% ethanol bath containing 1 mM NaOH and 5 mg/ml dopamine. Collected pDA-3D fibers were first air-dried then vigorously washed with phosphate buffered saline (PBS). For conventional electrospun 2D fibers, same polymer solution was horizontally electrospun with a feeding rate of 3.6 ml/h. 15 kV was applied to the needle and the polymer fiber were collected on an a aluminum sheet wrapped around a grounded mandrel with a rotation speed of 120 RPM, placed 15 cm away from the needle. Fibers were freezedried before the following cellular experiments. Scaffolds were punched out with a Ø12mm biopsy puncher and sterilized under 254 nm UV source for 30 minutes prior to cell seeding. Scanning electron microscopy Scaffolds were visualized under scanning electron microscope (FEI, Nova 600 NanoSEM) without any metal coating. Low vacuum mode was selected to reduce charging. 10 kV acceleration rate was used to capture images. Energy Dispersive X-ray (EDX) analysis was done by Quantax 70 (Bruker, Germany) mounted on TM-3030(Hitachi, Japan) scanning electron microscope. Contact angle measurement Contact angle measurements were done by placing a drop of water on PCL fiber meshes at room temperature. Drop shape analysis was done by recording the drops’ behavior for 30 seconds at a recording speed of 25 frames per second. (Krüss Drop Shape Analysis System DSA100). Dynamic mechanical analysis


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To determine the mechanical properties of the scaffolds, the scaffolds of 1 cm by 2.5 cm were examined with a Dynamic mechanical analysis; this was done on the Bose Electroforce 3200. The frequency was set to go from 1-50s-1, in steps of 1, with dynamic amplitude of 0.1 mm. The cross section area of the scaffold was measured using liquid nitrogen to cut the scaffold and measure it by using SEM. The e-modulus was set up as result over frequency. To get the stress strain curve, we used the data from the 49th frequency and one single sinus wave. Cell seeding Human bone marrow mesenchymal cells (hMSC) (Lonza) were maintained and expanded in growth medium (Dulbecco's Modified Eagle Medium + 10% Fatal bovine serum + 1% Penicillin/Streptavidin) in tissue culture flask until passage 4. Cells then were seeded on scaffolds by a drop seeding method. Briefly, 25 µl of cell suspension of 40 000 cells, were placed as a drop on each fiber samples in 48 well plate and left in the incubator for the initial cell attachment. After 1 hour of incubation, culture media was filled up to 500 µl. Cells were maintained in growth media for 24 hours to ensure cellular adhesion. Cytotoxicity Analysis Cytotoxic properties scaffolds were analyzed by quantifying lactate dehydrogenase (LDH), a cytosolic enzyme present in all type of cells. Membrane disrupted cells release LDH in culture media which was collected a day after culturing with culture media for LDH activity analysis. Following manufacturer’s protocol (Roche Diagnostics) calorimetrically obtained LDH activities at 490 nm were plotted using the equation below. Low control was the cells seeded on TCP whereas high control was the cells treated with 1% triton upon seeding.



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Cytotoxicity(%)= (experimental value-low control)/(high control-low control) x100 Live/Dead Staining To analyze viability of the cells, cell viability kit (Ready Probes, ThermoFisher Scientific) was used. One day after the cell seeding, NucBlue® and NucGreen® for all cells and dead cells respectively were introduced to the cell culture media. Fluorescent images were taken after 15 minutes of incubation with dyes using a microscope, EVOS FL Auto (ThermoFisher Scientific) at 10x magnification. Acquired images were analyzed using imageJ software. Randomly selected areas(n≥3) were used to count number of live and dead cells(n≥170). Viability/Proliferation Studies Long term cellular viability/proliferation is determined using CCK-8(Dojindo) analysis. After culturing cells in differentiation media for 1, 14 and 28 days, cell media were treated with manufacturer protocol. Briefly, CCK-8 reagent having soluble tetrazolium salt was diluted 1-20 ratio with culture media. Cells were incubated with this mixture for an hour before collecting the media. Tetrazolium salt in CCK-8 is reduced by dehydrogenase in cytoplasm giving out yellow tetrazolium dye. Intensity of yellow color analyzed by a plate reader at 450 nm corresponds to the number of the viable cells. For calculation of proliferation index, absorbance values at a time point were normalized against day 1 absorbance values for the same group (Ndayx/Nday1) Cellular infiltration Nucleus (Hoechst 33342) and f-actin (atto 488-phalloidin) stained scaffolds at day 1, 14 and 28 were visualized by confocal microscope. The scaffolds were scanned at 10x magnification in Z direction and


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the distance from first observed cell to the last is recorded. Representative 3D reconstructions were prepared in ImageJ. Mean infiltration values were calculated (n≥6) and plotted in GraphPad Differentiation Cells were maintained in fibroblast differentiation media (Dulbecco's Modified Eagle Medium + 10% Fatal bovine serum + 1% Penicillin/Streptavidin + 50 µg/ml ascorbic acid + CTGF (100 ng/ml)) for 4 weeks. For the further myofibroblastic differentiation, cells were cultured in Dulbecco's Modified Eagle Medium + 10% Fatal bovine serum + 1% Penicillin/Streptavidin+ TGF-β1(10ng/ml). Half of the cell medium is refreshed every 3rd day. qPCR RNA isolation from samples was carried out by TRIzol (Sigma Aldrich, Schnelldorf,Germany) treatment following company’s protocol. Isolated RNAs were quantified with Nanodrop ND100(NanoDrop Technologies, Inc.) at 260 and 280 nm. cDNA synthesis was done using High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA) with a total RNA of 250 ng for each reaction at 37 °C for 1 hour. A Lightcycler 480H (Roche Diagnostics, Mannheim, Germany) was used to quantify mRNA content using their corresponding cDNA samples amplified by real-time quantitative PCR technique. mRNA folds were calculated using standard ∆∆Ct method with GAPDH assigned as a housekeeping gene and plotted against the reference as 2∆∆Ct . Obtained values for each marker were normalized against the values in 2D samples at 2 weeks. Primer sequences used in qPCR can be found in Table S1. Immunochemical staining and confocal microscopy



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Cells constructs were rinsed with PBS and fixed with 4% formaldehyde for 15 minutes at 4 °C. Cells were permeabilized by 0.05 % Triton X-100 incubation for 15 minutes. Then, a blocking buffer, 1% BSA in PBS applied for 1 hour at room temperature. Primary antibodies were diluted in 3% BSA in PBS and cells were incubated with primary antibodies overnight at 4 °C on a shaker. Then, secondary antibody staining is done by incubating samples for 2 hours at room temperature. Cells were counter stained with Atto-488 Phalloidin and Hoechst. In every step, samples were washed 3 times with PBS. Cell constructs were soaked with an antifade agent (Sigma) before visualization under a confocal microscope (Zeiss LSM 700 – Carl Zeiss Micro-Imaging- Germany). Live cell imaging Live cell imaging was carried out for 2D, 3D and pDA-3D thin scaffolds seeded with enhanced green fluorescent protein(EGFP) expressing human mesenchymal cells(300 000 cells/cm2). Images were taken during the TGF-β1 treatment for myofibroblast differentiation after 2 weeks of incubation with fibroblast differentiation medium. Culture plates were placed on EVOS FL Auto Imaging System (Thermofisher Scientific, USA) equipped with an incubation chamber to maintain standard cell culture environment (5% CO2, 37 °C and relative humidity of 95%). Briefly, z-stack bright field images and green fluorescent protein images were acquired every 4 hours in the course of 3 days at 10X magnification. Images were analyzed and combined into short video clips(9 frame per second) using ImageJ software. Statistical analysis


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All the data presented are plotted as mean values ± standard error of the mean. Statistical analysis between the groups was done by either one-way Analysis of Variance test (ANOVA) with Tukey’s test using Prism 5 software (GraphPad, San Diego, CA, USA). Statistical significance was set as p

Three-Dimensional Polydopamine Functionalized Coiled

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