University of Groningen Self-assembling nanofiber hydrogels to attenuate epithelial mesenchymal transition in lens epithelial cells da Cruz Barros, Raquel Sofia

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Self-assembling nanofiber hydrogels to attenuate epithelial mesenchymal transition in lens

epithelial cells

da Cruz Barros, Raquel Sofia

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Publication date:

2018

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da Cruz Barros, R. S. (2018). Self-assembling nanofiber hydrogels to attenuate epithelial mesenchymal

transition in lens epithelial cells. University of Groningen.

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[103] Auffarth GU, Golescu A, Becker KA, Völcker HE. Quantification of posterior capsule opacification with round and sharp edge intraocular lenses. Ophthalmology. 2003;110:772-80. [104] Nishi O, Nishi K, Akura J, Nagata T. Effect of round-edged acrylic intraocular lenses on preventing posterior capsule opacification. Journal of Cataract & Refractive Surgery.

2001;27:608-13.

CHAPTER 2

Self-assembled nanofiber

coatings for controlling cell

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Re-production with permission of Journal of Biomedical Materials Research – part A: Barros RC, Gelens E, Bulten E, Tuin A, de Jong MR, Kuijer R, van Kooten TG. Self-assembled nanofiber coatings for controlling cell responses. Journal of Biomedical Materials Research Part A. 2017;105:2252-65.

Nanofibers are thought to enhance cell adhesion, growth and function. We demonstrate that the choice of building blocks in self-assembling nanofiber systems can be used to control cell behavior. The use of 2D-coated, self-assembled nanofibers in controlling lens epithelial cells, fibroblasts and mesenchymal stem cells was investigated, focusing on gene and protein expression related to the fibrotic response. To this end three nanofibers with different characteristics (morphology, topography and wettability) were compared to two standard materials frequently used in culturing cells, TCPS and a collagen type I coating. Cell metabolic activity, cell morphology and gene and protein expression were analyzed. The most hydrophilic nanofiber with more compact network consisting of small fibers proved to provide a beneficial 2D environment for cell proliferation and matrix formation while decreasing the fibrotic/stress behavior in all cell lines when compared with TCPS and the collagen type I coating. This nanofiber demonstrates the potential to be used as a biomimetic coating to study the development of fibrosis through epithelial-to-mesenchymal transition. This study also shows that nanofiber structures do not enhance cell function by definition, because the physico-chemical characteristics of the nanofibers influence cell behavior as well and actually can be used to regulate cell behavior towards sub-optimal performance.

KEYWORDS

self-assembled nanofibers, coating, fibrosis, cellular stress, epithelia-mesenchymal transition

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2

Re-production with permission of Journal of Biomedical Materials Research – part A: Barros RC, Gelens E, Bulten E, Tuin A, de Jong MR, Kuijer R, van Kooten TG. Self-assembled nanofiber coatings for controlling cell responses. Journal of Biomedical Materials Research Part A. 2017;105:2252-65.

ABSTRACT

Nanofibers are thought to enhance cell adhesion, growth and function. We demonstrate that the choice of building blocks in self-assembling nanofiber systems can be used to control cell behavior. The use of 2D-coated, self-assembled nanofibers in controlling lens epithelial cells, fibroblasts and mesenchymal stem cells was investigated, focusing on gene and protein expression related to the fibrotic response. To this end three nanofibers with different characteristics (morphology, topography and wettability) were compared to two standard materials frequently used in culturing cells, TCPS and a collagen type I coating. Cell metabolic activity, cell morphology and gene and protein expression were analyzed. The most hydrophilic nanofiber with more compact network consisting of small fibers proved to provide a beneficial 2D environment for cell proliferation and matrix formation while decreasing the fibrotic/stress behavior in all cell lines when compared with TCPS and the collagen type I coating. This nanofiber demonstrates the potential to be used as a biomimetic coating to study the development of fibrosis through epithelial-to-mesenchymal transition. This study also shows that nanofiber structures do not enhance cell function by definition, because the physico-chemical characteristics of the nanofibers influence cell behavior as well and actually can be used to regulate cell behavior towards sub-optimal performance.

KEYWORDS

self-assembled nanofibers, coating, fibrosis, cellular stress, epithelia-mesenchymal transition

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INTRODUCTION

Fibrosis [1], cancer [2] and auto-immune diseases [3] can be considered the final stage of cell alterations/differentiations in the affected tissues. For instance, fibrosis is an integral part of organ failure in liver, lungs, kidney, heart and eye. Epithelial mesenchymal transition (EMT) is a process present in embryogenesis, but also in organ fibrosis and cancer metastasis [4]. It actually is a transformation of epithelial cells to mesenchymal-like cells. This process can occur in any organ that can be affected by stress stimuli, as for example, continuously high levels of cortisol or high levels of UV radiation which can generate an inflammatory response [5]. Many organs are constituted of several cell types, the most prevalent cells being epithelial cells, mesenchymal cells and fibroblasts. It is known that these three cell types can be involved in the development of EMT [4] and consequently in fibrosis [6]. Studies of new biomaterials that can influence cell function and differentiation are becoming an important tool in creating in vitro model systems for studying these processes.

The studies of in vitro 2D culture models to support cell proliferation and cell function have obtained attention in the scientific community for many years. One of the most frequently used techniques is the coverage of different surfaces with compounds that can elicit this cell behavior. Collagen type I has been investigated [7] [8] and currently is one of the most popular coatings in the market. Collagen type I is a constituent of the extracellular matrix in many if not all tissues [9], and improves cell adhesion, proliferation and viability [10]. However, collagen of either human or animal origin does not have a constant quality, and varies between the different suppliers. Alternatives can be found in (self-assembled) nanofiber systems that present similar nanostructures to the cells. These fibers are extensively studied because of their wide use in in vitro, ex vivo and in vivo biomedical applications. Hydrogels of low-molecular-weight gelators (LMWGs) are produced by self-assembly of low molecular weight molecules into a network of fibers, in which water remains entrapped, connected by non-covalent interactions such as hydrophobic interactions, π-π interactions, electronic interactions, hydrogen bonding or combinations of them [11]. LMWGs exhibit particular features as fibers assemble in a well ordered way, being thermo-reversible owing to their non-covalent linkages, gelation already with low concentrations and good clearance by the body [12]. LMWGs show a high potential as a scaffold for drug delivery [13] and cell storage [14]. Jan van Esch et al. developed LMWGs based on a 1,3,5-triamide cis,cis-cyclohexane core [12, 13, 15]. This structure was based on a 1D core connected with L-amino acid moieties providing additional forces for self-assembly. Nanofibers made of LMWGs can be used either as a surface coating resembling collagen coatings, applicable on virtually any surface, or as a 3D (injectable) gel [16]. These synthetic compounds offer advantages over biological coatings such as collagen type I due to their improved consistency, the lack of animal components, and the relative ease of further development and application of modifications in their chemistry.

Apart from providing an optimal 2D environment for supporting cells, it is recognized that the culturing itself can be associated with stress and even a tendency towards fibrotic responses, for example induced by molecular sources (e.g. TGF-βs), by the seeding on

different surfaces, by multiple cell passaging or even by the use of serum in culture media, all of which elements often are fundamental to a cell culture theater [17, 18]. Therefore, maintaining cell lines or primary cells in vitro poses a real challenge with respect to their natural behavior. Although 3D environments seem to be promising, 2D environments often are more practical to work with [[19] [20]].

Human lens epithelial cells (LEC) are present as a monolayer in the anterior eye lens and show a well-defined polarity that can quickly change with molecular stimuli [21, 22] or when not cultured on appropriate surfaces [23], resulting in a poor control of the LEC phenotype. Most of the studies to control LEC are based on blocking cell proliferation without paying attention to its phenotype or genotype. Fibroblasts are widely used to study cell behavior in the presence of different biomaterials due to their importance in wound healing [24] and the foreign body response to implanted biomaterials [25]. They also contribute to fibrotic reactions in most of the organs because the fibrotic tissue is constituted also by fibroblast like cells [26]. Mesenchymal stem cells can differentiate into a variety of cell types such as, myofibroblasts, fibroblasts, chondrocytes, osteoblasts and adipocytes [27]. These cells also have the ability to migrate into an injured site and contribute to a tissue healing response [28, 29]. Fibroblasts and mesenchymal cells were the first cell lines to be cultured on glass and plastic, which make them notable cell lines to include in studying interactions with materials in general [30, 31]. As mentioned, all these cell types have been amply studied on different surfaces due their importance in most tissues, however these cells are also present at different stages of the fibrotic process, which makes them very suitable for studying the fibrotic process and associated cell differentiation. Epithelial cells [32], mesenchymal stem cells [33] and fibroblasts [34] under fibrotic/stress stimuli can change their phenotype and genotype to an intermediate stage characterized by the presence of fibroblast-like cells, also known as myofibroblasts [4]. Myofibroblasts are well characterized by the reorganization of actin filaments, increase of fibronectin [35] and de novo synthesis of α-SMA [36]. The cells produce a fibrotic extracellular matrix, which together with the changed cell morphology (fiber morphology) results in the fibrotic tissue. Transforming growth factor β (TGF-β) appears to play an important role in the EMT process. Receptors for TGF-βs are denoted activin receptor-like kinases (ALKs) and the increase of their expression is associated with the change of epithelial cells towards mesenchymal cells, a signal of EMT activation [37]. Also, upregulation of ALK1, 2 and 5 are mentioned as inducers or at least signatures of EMT [38-40]. Altogether, EMT seems to involve signaling within the TGF-β growth factor family, including both the growth factors and their receptors, resulting in the changed extracellular matrix containing fibronectin, collagen types III and VI, and α-SMA [41-43]. In the inflammatory process pro-inflammatory cytokines are released such as tumor necrosis factor (TNF‐α) and interleukin‐1 (IL‐1) that induce TGF-βs secretion [44]. This makes inflammation the major process contributing to the initiation of EMT in adults [5].

We investigated the use of 2D-coated, self-assembled nanofibers in controlling behavior of lens epithelial cells, fibroblasts and mesenchymal stem cells. To this end three nanofibers with different physico-chemical characteristics (morphology, topography and wettability) were compared to two standard materials frequently used in culturing cells,

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2

INTRODUCTION

Fibrosis [1], cancer [2] and auto-immune diseases [3] can be considered the final stage of cell alterations/differentiations in the affected tissues. For instance, fibrosis is an integral part of organ failure in liver, lungs, kidney, heart and eye. Epithelial mesenchymal transition (EMT) is a process present in embryogenesis, but also in organ fibrosis and cancer metastasis [4]. It actually is a transformation of epithelial cells to mesenchymal-like cells. This process can occur in any organ that can be affected by stress stimuli, as for example, continuously high levels of cortisol or high levels of UV radiation which can generate an inflammatory response [5]. Many organs are constituted of several cell types, the most prevalent cells being epithelial cells, mesenchymal cells and fibroblasts. It is known that these three cell types can be involved in the development of EMT [4] and consequently in fibrosis [6]. Studies of new biomaterials that can influence cell function and differentiation are becoming an important tool in creating in vitro model systems for studying these processes.

The studies of in vitro 2D culture models to support cell proliferation and cell function have obtained attention in the scientific community for many years. One of the most frequently used techniques is the coverage of different surfaces with compounds that can elicit this cell behavior. Collagen type I has been investigated [7] [8] and currently is one of the most popular coatings in the market. Collagen type I is a constituent of the extracellular matrix in many if not all tissues [9], and improves cell adhesion, proliferation and viability [10]. However, collagen of either human or animal origin does not have a constant quality, and varies between the different suppliers. Alternatives can be found in (self-assembled) nanofiber systems that present similar nanostructures to the cells. These fibers are extensively studied because of their wide use in in vitro, ex vivo and in vivo biomedical applications. Hydrogels of low-molecular-weight gelators (LMWGs) are produced by self-assembly of low molecular weight molecules into a network of fibers, in which water remains entrapped, connected by non-covalent interactions such as hydrophobic interactions, π-π interactions, electronic interactions, hydrogen bonding or combinations of them [11]. LMWGs exhibit particular features as fibers assemble in a well ordered way, being thermo-reversible owing to their non-covalent linkages, gelation already with low concentrations and good clearance by the body [12]. LMWGs show a high potential as a scaffold for drug delivery [13] and cell storage [14]. Jan van Esch et al. developed LMWGs based on a 1,3,5-triamide cis,cis-cyclohexane core [12, 13, 15]. This structure was based on a 1D core connected with L-amino acid moieties providing additional forces for self-assembly. Nanofibers made of LMWGs can be used either as a surface coating resembling collagen coatings, applicable on virtually any surface, or as a 3D (injectable) gel [16]. These synthetic compounds offer advantages over biological coatings such as collagen type I due to their improved consistency, the lack of animal components, and the relative ease of further development and application of modifications in their chemistry.

Apart from providing an optimal 2D environment for supporting cells, it is recognized that the culturing itself can be associated with stress and even a tendency towards fibrotic responses, for example induced by molecular sources (e.g. TGF-βs), by the seeding on

different surfaces, by multiple cell passaging or even by the use of serum in culture media, all of which elements often are fundamental to a cell culture theater [17, 18]. Therefore, maintaining cell lines or primary cells in vitro poses a real challenge with respect to their natural behavior. Although 3D environments seem to be promising, 2D environments often are more practical to work with [[19] [20]].

Human lens epithelial cells (LEC) are present as a monolayer in the anterior eye lens and show a well-defined polarity that can quickly change with molecular stimuli [21, 22] or when not cultured on appropriate surfaces [23], resulting in a poor control of the LEC phenotype. Most of the studies to control LEC are based on blocking cell proliferation without paying attention to its phenotype or genotype. Fibroblasts are widely used to study cell behavior in the presence of different biomaterials due to their importance in wound healing [24] and the foreign body response to implanted biomaterials [25]. They also contribute to fibrotic reactions in most of the organs because the fibrotic tissue is constituted also by fibroblast like cells [26]. Mesenchymal stem cells can differentiate into a variety of cell types such as, myofibroblasts, fibroblasts, chondrocytes, osteoblasts and adipocytes [27]. These cells also have the ability to migrate into an injured site and contribute to a tissue healing response [28, 29]. Fibroblasts and mesenchymal cells were the first cell lines to be cultured on glass and plastic, which make them notable cell lines to include in studying interactions with materials in general [30, 31]. As mentioned, all these cell types have been amply studied on different surfaces due their importance in most tissues, however these cells are also present at different stages of the fibrotic process, which makes them very suitable for studying the fibrotic process and associated cell differentiation. Epithelial cells [32], mesenchymal stem cells [33] and fibroblasts [34] under fibrotic/stress stimuli can change their phenotype and genotype to an intermediate stage characterized by the presence of fibroblast-like cells, also known as myofibroblasts [4]. Myofibroblasts are well characterized by the reorganization of actin filaments, increase of fibronectin [35] and de novo synthesis of α-SMA [36]. The cells produce a fibrotic extracellular matrix, which together with the changed cell morphology (fiber morphology) results in the fibrotic tissue. Transforming growth factor β (TGF-β) appears to play an important role in the EMT process. Receptors for TGF-βs are denoted activin receptor-like kinases (ALKs) and the increase of their expression is associated with the change of epithelial cells towards mesenchymal cells, a signal of EMT activation [37]. Also, upregulation of ALK1, 2 and 5 are mentioned as inducers or at least signatures of EMT [38-40]. Altogether, EMT seems to involve signaling within the TGF-β growth factor family, including both the growth factors and their receptors, resulting in the changed extracellular matrix containing fibronectin, collagen types III and VI, and α-SMA [41-43]. In the inflammatory process pro-inflammatory cytokines are released such as tumor necrosis factor (TNF‐α) and interleukin‐1 (IL‐1) that induce TGF-βs secretion [44]. This makes inflammation the major process contributing to the initiation of EMT in adults [5].

We investigated the use of 2D-coated, self-assembled nanofibers in controlling behavior of lens epithelial cells, fibroblasts and mesenchymal stem cells. To this end three nanofibers with different physico-chemical characteristics (morphology, topography and wettability) were compared to two standard materials frequently used in culturing cells,

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TCPS and a collagen type I coating. Cell metabolic activity, cell morphology and gene and protein expression were analyzed.

MATERIAL AND METHODS Materials

For cell cultures were used Minimum Essential Media (MEM), fetal bovine serum (FBS), GlutaMax, penicillin, streptomycin, actinomycin, ascorbic acid, sodium pyruvate and trypsin-EDTA (all Invitrogen™-Gibco® (Bleiswijk, The Netherlands)). For cell seeding T75 tissue culture polystyrene plates (TCPS) and collagen type I plates were used (Greiner (Alphen aan den Rijn, The Netherlands)). Human recombinant transforming growth factor β1 (rhTGF-β1 from R&D systems (Abingdon, UK)) was used to stimulate human fibroblasts (positive control). For cell analysis, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromid), crystal violet and acetic acid were used from Sigma-Aldrich (Zwijndrecht, The Netherlands) and 2-propanol from Merck-Europe (Amsterdam, The Netherlands). Immunohistochemistry was performed with triton X-100, bovine serum albumin (BSA), rabbit-α-human fibronectin antibody, mouse-α-human α-smooth muscle actin antibody, DAPI (4',6-diamidino-2-phenylindole) and phalloidin-TRITC (tetramethylrhodamine isothiocyanate), all from Sigma-Aldrich. The secondary antibodies, FITC (fluorescein isothiocyanate)-labelled donkey-α-rabbit IgG and goat-α-mouse IgG were from Jackson Immunoresearch Europe (Suffolk, UK). For RNA extraction and cDNA production we used the InviTrap® Spin Cell RNA mini kit from Invitrek and IQ™SYBR® Green Super Mix from BioRad (Veenendaal, The Netherlands), respectively. For the nanofiber production, all chemicals were purchased from Aldrich, Fluka, Bachem or TCI and used without further purification.

Methods

Nanofiber production

Characterization

After each reaction step products were characterized using proton NMR. For the final gelators: characterization of the three compounds consisted of proton NMR and HPLC-MS. NMR experiments were performed using a Varian Gemini NMR spectrometer operating at 200 MHz, or a Varian VXR NMR spectrometer operating at 300 MHz. All spectra were recorded in DMSO-d6 unless stated otherwise. Final products were characterized by HPLC-MS on a Zorbax SB C18 column (50 x 2.1 mm, 1.8 µm).

Figure 1: Chemical structure of compounds 1-3.

The utilized coatings were prepared with compounds 1, 2 and 3 (Fig.1) using procedures that were described in detail by Ikonen et al. [16]. The synthesis of compound 1 was first described by van Bommel et al. [12]. The synthesis of compounds 2 and 3 is described in supplementary information.

Biomaterial characterization

For scanning electron microscopy (SEM) nanofiber coatings were prepared onto coverslips. Samples were subsequently dried with a Baltec CPD 030 critical point dryer. Sample coating was done with Au/Pd ∼3 nm (Sputtercoater Balzers 120B), and samples were analyzed using a JEOL JSM-6301F cold field emission scanning electron microscope at 5000x and 25000x magnification (University Medical Center Groningen, The Netherlands).

Wettability was measured by sessile contact angle measurements using liquids with different polarities (water, formamide, diiodomethane and a-bromonaphthalene) contacting the surface of each biomaterial and the contact angle was measured using a Matlab algorithm. From the obtained data, the surface free energy including the LW (δLW), AB (δAB), the electron-donating and accepting components were calculated using the equations given below. Triplicate measurements were made.

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2

TCPS and a collagen type I coating. Cell metabolic activity, cell morphology and gene and protein expression were analyzed.

MATERIAL AND METHODS Materials

For cell cultures were used Minimum Essential Media (MEM), fetal bovine serum (FBS), GlutaMax, penicillin, streptomycin, actinomycin, ascorbic acid, sodium pyruvate and trypsin-EDTA (all Invitrogen™-Gibco® (Bleiswijk, The Netherlands)). For cell seeding T75 tissue culture polystyrene plates (TCPS) and collagen type I plates were used (Greiner (Alphen aan den Rijn, The Netherlands)). Human recombinant transforming growth factor β1 (rhTGF-β1 from R&D systems (Abingdon, UK)) was used to stimulate human fibroblasts (positive control). For cell analysis, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromid), crystal violet and acetic acid were used from Sigma-Aldrich (Zwijndrecht, The Netherlands) and 2-propanol from Merck-Europe (Amsterdam, The Netherlands). Immunohistochemistry was performed with triton X-100, bovine serum albumin (BSA), rabbit-α-human fibronectin antibody, mouse-α-human α-smooth muscle actin antibody, DAPI (4',6-diamidino-2-phenylindole) and phalloidin-TRITC (tetramethylrhodamine isothiocyanate), all from Sigma-Aldrich. The secondary antibodies, FITC (fluorescein isothiocyanate)-labelled donkey-α-rabbit IgG and goat-α-mouse IgG were from Jackson Immunoresearch Europe (Suffolk, UK). For RNA extraction and cDNA production we used the InviTrap® Spin Cell RNA mini kit from Invitrek and IQ™SYBR® Green Super Mix from BioRad (Veenendaal, The Netherlands), respectively. For the nanofiber production, all chemicals were purchased from Aldrich, Fluka, Bachem or TCI and used without further purification.

Methods

Nanofiber production

Characterization

After each reaction step products were characterized using proton NMR. For the final gelators: characterization of the three compounds consisted of proton NMR and HPLC-MS. NMR experiments were performed using a Varian Gemini NMR spectrometer operating at 200 MHz, or a Varian VXR NMR spectrometer operating at 300 MHz. All spectra were recorded in DMSO-d6 unless stated otherwise. Final products were characterized by HPLC-MS on a Zorbax SB C18 column (50 x 2.1 mm, 1.8 µm).

Figure 1: Chemical structure of compounds 1-3.

The utilized coatings were prepared with compounds 1, 2 and 3 (Fig.1) using procedures that were described in detail by Ikonen et al. [16]. The synthesis of compound 1 was first described by van Bommel et al. [12]. The synthesis of compounds 2 and 3 is described in supplementary information.

Biomaterial characterization

For scanning electron microscopy (SEM) nanofiber coatings were prepared onto coverslips. Samples were subsequently dried with a Baltec CPD 030 critical point dryer. Sample coating was done with Au/Pd ∼3 nm (Sputtercoater Balzers 120B), and samples were analyzed using a JEOL JSM-6301F cold field emission scanning electron microscope at 5000x and 25000x magnification (University Medical Center Groningen, The Netherlands).

Wettability was measured by sessile contact angle measurements using liquids with different polarities (water, formamide, diiodomethane and a-bromonaphthalene) contacting the surface of each biomaterial and the contact angle was measured using a Matlab algorithm. From the obtained data, the surface free energy including the LW (δLW), AB (δAB), the electron-donating and accepting components were calculated using the equations given below. Triplicate measurements were made.

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∆𝑮𝑮𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳 = −𝟐𝟐𝟐𝟐𝜸𝜸𝜸𝜸𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳

∆𝑮𝑮𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨 = −𝟐𝟐𝟐𝟐𝜸𝜸𝜸𝜸𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨

∆𝑮𝑮𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝒂𝒂𝒂𝒂𝑻𝑻𝑻𝑻= ∆𝑮𝑮𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳 + ∆𝑮𝑮𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨

The surfaces were analyzed by Atomic Force Microscopy (AFM, Veeco NanoScope) in the hydrated mode, i.e. in PBS. AFM images were obtained with the tapping mode.

Cell culture

Three different cell lines were used. All of them were cultured at 37 °C in a 5%CO2 atmosphere. Human lens epithelial cells (LEC-B3, ATCC) were cultured in MEM supplemented with 17% FBS, and 1% of Pen/Strep, 1% of GlutaMAX and 1% of sodium pyruvate. Human skin fibroblasts (hSkF) were cultured with MEM supplemented with 20% FBS, 1% of GlutaMax, penicillin and streptomycin. Human mesenchymal stem cells (isolated from patients) were cultured in MEM alpha Medium (1X)+ GlutaMAX supplemented with 10% heat-inactivated FBS, 2% APS and 0.1% ascorbic acid, as described before [45]. On 90% confluence cells were trypsinized and propagated in T75 flasks. Cell culture medium was exchanged every two days. For each experiment, cells were seeded at a density of 20 000 cells/well (corresponding to 1.0x104/cm2) in 24 wells plates coated with compounds 1, 2 and 3 or on the controls: commercialized plates of collagen type I and TCPS. After 2 and 5 days in culture cells were analyzed.

Cell metabolic activity assays

MTT was added at 0.5 mg/mL to the wells plate. Cells were incubated for 4 hours at 37° C in a 5%CO2 environment. The blue crystals formed after incubation were dissolved with 2-propanol and absorbance was measured at 590nm using a Fluostar OPTIMA plate reader.

After the MTT assay, wells were washed 3 times in PBS and 500μL/well of 0.1% crystal violet solution was added. After 20 minutes of incubation at RT while shaking, plates were washed with water until the dark blue color disappeared. The cell-bound stain was extracted with 400μL/well of 33% acetic acid and the supernatants were measured at 600 nm. For each material and time point 8 wells were used and full growth medium was used as a control in both assays.

Immunohistochemistry and image acquisition

On day 2 and day 5 cells were washed with PBS, fixed with 3.7% paraformaldehyde for 15 minutes and washed again with PBS. Membrane permeabilization was achieved in 3 minutes with 0.5% triton X-100 in PBS followed of washing with PBS. Blocking solution, 5% BSA in PBS, was added and incubated at RT for 20 minutes. Primary antibody were applied at dilutions of 1:400 (fibronectin) and 1:100 (α-SMA), diluted in PBS containing

1%BSA (1%PBSA) and allowed to incubate at RT for 1 hour. After cells were washed 3 times with 1%PBSA the secondary antibodies were applied for 2 hour at RT. DAPI (2μg/mL f.c.) was used for nucleic staining and TRITC-labelled phalloidin (2μg/mL) for staining of the actin cytoskeleton.

All samples were visualized with a confocal laser scanning microscope (LEICA TCS SP2) equipped with a UV laser, Argon laser and Helium laser. For acquiring images a 40x water-immersion objective lens was used allowing a continued hydration of the samples.

Real-time PCR

Cultures of human skin fibroblasts were supplemented with 10 ng/mL of activated rhTGF-β1 [46] and cultured for 2 and 5 days in TCPS 24 well plates. These stimulated cells

were used as a positive control for all other PCR measurements.

All cells were extracted from the bottom of the plates using a cell scraper and the lysis buffer from the InviTrap® Spin Cell RNA mini kit. All procedures of mRNA extraction were performed according to the manufactures instructions. Primers were designed using the online Primer-3 software at the NCBI website and were synthesized by Biolegio (Nijmegen, The Netherlands) (Table 1).

Table 1: Forward and reverse primers sequences and annealing temperatures.

The RNA yield and quality were checked using the Nanodrop system, genomic DNA was removed using a DNA-freeTM kit (Ambion), and cDNA was synthesized using the iScript cDNA synthesis kit (BioRad). Quantitative real time PCR was performed in duplicate in 384 wells plates using IQ™SYBR® Green Super Mix on a CFX 384™ Real

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2

∆𝑮𝑮𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳 = −𝟐𝟐𝟐𝟐𝜸𝜸𝜸𝜸𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳

∆𝑮𝑮𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨 = −𝟐𝟐𝟐𝟐𝜸𝜸𝜸𝜸𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨

∆𝑮𝑮𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝒂𝒂𝒂𝒂𝑻𝑻𝑻𝑻= ∆𝑮𝑮𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳 + ∆𝑮𝑮𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝑨𝑨𝑨𝑨𝑨𝑨𝑨𝑨

The surfaces were analyzed by Atomic Force Microscopy (AFM, Veeco NanoScope) in the hydrated mode, i.e. in PBS. AFM images were obtained with the tapping mode.

Cell culture

Three different cell lines were used. All of them were cultured at 37 °C in a 5%CO2 atmosphere. Human lens epithelial cells (LEC-B3, ATCC) were cultured in MEM supplemented with 17% FBS, and 1% of Pen/Strep, 1% of GlutaMAX and 1% of sodium pyruvate. Human skin fibroblasts (hSkF) were cultured with MEM supplemented with 20% FBS, 1% of GlutaMax, penicillin and streptomycin. Human mesenchymal stem cells (isolated from patients) were cultured in MEM alpha Medium (1X)+ GlutaMAX supplemented with 10% heat-inactivated FBS, 2% APS and 0.1% ascorbic acid, as described before [45]. On 90% confluence cells were trypsinized and propagated in T75 flasks. Cell culture medium was exchanged every two days. For each experiment, cells were seeded at a density of 20 000 cells/well (corresponding to 1.0x104/cm2) in 24 wells plates coated with compounds 1, 2 and 3 or on the controls: commercialized plates of collagen type I and TCPS. After 2 and 5 days in culture cells were analyzed.

Cell metabolic activity assays

MTT was added at 0.5 mg/mL to the wells plate. Cells were incubated for 4 hours at 37° C in a 5%CO2 environment. The blue crystals formed after incubation were dissolved with 2-propanol and absorbance was measured at 590nm using a Fluostar OPTIMA plate reader.

After the MTT assay, wells were washed 3 times in PBS and 500μL/well of 0.1% crystal violet solution was added. After 20 minutes of incubation at RT while shaking, plates were washed with water until the dark blue color disappeared. The cell-bound stain was extracted with 400μL/well of 33% acetic acid and the supernatants were measured at 600 nm. For each material and time point 8 wells were used and full growth medium was used as a control in both assays.

Immunohistochemistry and image acquisition

On day 2 and day 5 cells were washed with PBS, fixed with 3.7% paraformaldehyde for 15 minutes and washed again with PBS. Membrane permeabilization was achieved in 3 minutes with 0.5% triton X-100 in PBS followed of washing with PBS. Blocking solution, 5% BSA in PBS, was added and incubated at RT for 20 minutes. Primary antibody were applied at dilutions of 1:400 (fibronectin) and 1:100 (α-SMA), diluted in PBS containing

1%BSA (1%PBSA) and allowed to incubate at RT for 1 hour. After cells were washed 3 times with 1%PBSA the secondary antibodies were applied for 2 hour at RT. DAPI (2μg/mL f.c.) was used for nucleic staining and TRITC-labelled phalloidin (2μg/mL) for staining of the actin cytoskeleton.

All samples were visualized with a confocal laser scanning microscope (LEICA TCS SP2) equipped with a UV laser, Argon laser and Helium laser. For acquiring images a 40x water-immersion objective lens was used allowing a continued hydration of the samples.

Real-time PCR

Cultures of human skin fibroblasts were supplemented with 10 ng/mL of activated rhTGF-β1 [46] and cultured for 2 and 5 days in TCPS 24 well plates. These stimulated cells

were used as a positive control for all other PCR measurements.

All cells were extracted from the bottom of the plates using a cell scraper and the lysis buffer from the InviTrap® Spin Cell RNA mini kit. All procedures of mRNA extraction were performed according to the manufactures instructions. Primers were designed using the online Primer-3 software at the NCBI website and were synthesized by Biolegio (Nijmegen, The Netherlands) (Table 1).

Table 1: Forward and reverse primers sequences and annealing temperatures.

The RNA yield and quality were checked using the Nanodrop system, genomic DNA was removed using a DNA-freeTM kit (Ambion), and cDNA was synthesized using the iScript cDNA synthesis kit (BioRad). Quantitative real time PCR was performed in duplicate in 384 wells plates using IQ™SYBR® Green Super Mix on a CFX 384™ Real

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Time PCR System (BioRad). Each well contained 10 µL of an aqueous solution consisting of 5 μL SybrGreen, 0.4 μL primer mix (reverse+forward), 3 μL cDNA and 1.6 μL water. The PCR amplification protocol was one cycle of 95°C for 3 minutes and 95°C for 10 seconds, followed by 39 cycles at 95°C for 2 seconds, 60°C (for all primers except α-SMA) or 57.5°C (for α-SMA primer) for 30 seconds, 60°C for 5 seconds and finally 90°C for 5 seconds. 18S RNA was used as a reference working at 60°C. Genomic data was normalized with samples from the same cell type grown on TCPS without any stimulus using 2-ΔΔCt method [47].

Statistical analysis

For crystal violet and MTT analysis ANOVA repeated measures were applied to analyze the influence of the different coatings on the well plates and time of incubation versus control groups. If F-values were larger than α=0.05 the Holm-Sidak method was applied as post hoc analysis.

For RT-PCR analysis ANOVA was performed. In order to determine the influence of the (nanofiber) coating and culturing time (days) on gene expression, the Holm-Sidak method was applied.

RESULTS

The commercialized collagen type I coating showed a very smooth, compact and uniform surface when visualized on SEM (data not shown). This coating with a ΔG value of 26.7mJ/m2 was the least hydrophilic compound followed by compound 3 (ΔG = 53.4mJ/m2). Different from collagen, small nanofibers were clearly visible on SEM and AFM (Fig. 2 C, F, I) for this compound, that make it a rougher and semi-compact surface. Compound 1 was the most hydrophilic coating with ΔG = 83.4mJ/m2. Its compact and rough surface showed the smallest nanofibers without free spaces between them (Fig.2 A, D, G). Due to its compact surface this coating was the most similar to collagen type I. The hydrophobic compound 2 had a ΔG = -54.2mJ/m2 and its surface contained the largest nanofibers with free spaces between them, that made this coating also the most spacious coating (Fig.2 B, E, H).

Figure 2: SEM (A-F), AFM (G-I) images and surface free energy values (ΔG) for the three nanofibers coatings,

compound 1, 2 and 3. Low (A-C) and high (D-F) magnification of SEM are shown. Bars represent 1 µm (A-C, E, F) or 100 nm (D) and 20x20 µm (G-I).

The MTT assay was used to analyze cell metabolic activity, sometimes referred to as viability, while crystal violet assay was used to measure cell mass as a measure for cell proliferation or survival. In general, compound 2 produced the lowest values for metabolic activity. Cells on collagen type I and compound 1 showed larger values than on TCPS for both assays. LEC showed a similar activity on collagen type I and compound 1 (Δabs=0.168 and 0.200 respectively). At day 2 these two coatings had an increase of 37% and 33% for collagen type I and compound 1 respectively, but these values became similar to TCPS at day 5 (Fig.3A). On collagen type I cells had their largest proliferation at day 2 but showed a decrease from day 2 to 5 (Δabs=-0.304) while on compound 1 they demonstrated an increase (Δabs=0.615). LEC on compound 2 had developed a very low

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2

Time PCR System (BioRad). Each well contained 10 µL of an aqueous solution consisting of 5 μL SybrGreen, 0.4 μL primer mix (reverse+forward), 3 μL cDNA and 1.6 μL water. The PCR amplification protocol was one cycle of 95°C for 3 minutes and 95°C for 10 seconds, followed by 39 cycles at 95°C for 2 seconds, 60°C (for all primers except α-SMA) or 57.5°C (for α-SMA primer) for 30 seconds, 60°C for 5 seconds and finally 90°C for 5 seconds. 18S RNA was used as a reference working at 60°C. Genomic data was normalized with samples from the same cell type grown on TCPS without any stimulus using 2-ΔΔCt method [47].

Statistical analysis

For crystal violet and MTT analysis ANOVA repeated measures were applied to analyze the influence of the different coatings on the well plates and time of incubation versus control groups. If F-values were larger than α=0.05 the Holm-Sidak method was applied as post hoc analysis.

For RT-PCR analysis ANOVA was performed. In order to determine the influence of the (nanofiber) coating and culturing time (days) on gene expression, the Holm-Sidak method was applied.

RESULTS

The commercialized collagen type I coating showed a very smooth, compact and uniform surface when visualized on SEM (data not shown). This coating with a ΔG value of 26.7mJ/m2 was the least hydrophilic compound followed by compound 3 (ΔG = 53.4mJ/m2). Different from collagen, small nanofibers were clearly visible on SEM and AFM (Fig. 2 C, F, I) for this compound, that make it a rougher and semi-compact surface. Compound 1 was the most hydrophilic coating with ΔG = 83.4mJ/m2. Its compact and rough surface showed the smallest nanofibers without free spaces between them (Fig.2 A, D, G). Due to its compact surface this coating was the most similar to collagen type I. The hydrophobic compound 2 had a ΔG = -54.2mJ/m2 and its surface contained the largest nanofibers with free spaces between them, that made this coating also the most spacious coating (Fig.2 B, E, H).

Figure 2: SEM (A-F), AFM (G-I) images and surface free energy values (ΔG) for the three nanofibers coatings,

compound 1, 2 and 3. Low (A-C) and high (D-F) magnification of SEM are shown. Bars represent 1 µm (A-C, E, F) or 100 nm (D) and 20x20 µm (G-I).

The MTT assay was used to analyze cell metabolic activity, sometimes referred to as viability, while crystal violet assay was used to measure cell mass as a measure for cell proliferation or survival. In general, compound 2 produced the lowest values for metabolic activity. Cells on collagen type I and compound 1 showed larger values than on TCPS for both assays. LEC showed a similar activity on collagen type I and compound 1 (Δabs=0.168 and 0.200 respectively). At day 2 these two coatings had an increase of 37% and 33% for collagen type I and compound 1 respectively, but these values became similar to TCPS at day 5 (Fig.3A). On collagen type I cells had their largest proliferation at day 2 but showed a decrease from day 2 to 5 (Δabs=-0.304) while on compound 1 they demonstrated an increase (Δabs=0.615). LEC on compound 2 had developed a very low

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viability and did not show signals of survival at day 5. Also on compound 3 LEC exhibited low metabolic activity and proliferation.

The same pattern of growing from day 2 to day 5 was present for HSkF for both viability and proliferation measurements (Fig. 3 C and D). Cells on collagen type I and compound 1 had a similar augmentation from day 2 to day 5, Δabs=0.745 and Δabs=0.812. Inversely to LEC, HSkF on compound 2 showed a significant increase between days, however its values were much lower than on TCPS or on the other coatings. HSkF on compound 3 showed the largest variation (Δabs=0.824) from day 2 to 5. The same pattern was expressed for the crystal violet values, however cells on compounds 2 and 3 appeared with a small Δabs of 0.120 and 0.196 respectively.

hMSC was the cell line with the lowest values for both assays (Fig. 3 E and F). The variation in cell proliferation between days remained low in all coatings. Once more, cells on collagen type I and compound 1 showed similar values on cell viability and proliferation. For this last assay, hMSC on compound 1 had a large increase of 80.44% compared to TCPS at day 5. The compound 2 was the worst coating regarding hMSC viability and proliferation with values near zero, similar to the behavior of LEC. hMSCs on compound 3 demonstrated low viability and proliferation at both time points.

Figure 3: MTT measurements for LEC (A), HSkF (C) and hMSC (E); and crystal violet measurements for LEC

(B), HSkF (D) and hMSC (F) on day 2 and day 5 of culture in collagen type I surfaces or on compounds 1, 2 and 3. The red bold lines represent the TCPS values on day 2 and the dotted lines the values for day 5. The percentage of increase or decrease between each material and the TCPS is expressed in the tables above each graphic. The variation between day 2 and day 5 for each material is showed by Δabs. Error bars depict standard deviations. Full statistical analysis of MTT and crystal violet is in the supplementary information.

Fluorescently stained cells demonstrating nuclei, actin cytoskeleton and fibronectin were visualized by confocal microscopy for all cell-material combinations (Fig. 4). LEC was the cell line that expressed the lowest quantity of fibronectin. In these cells the most impressive results came from the compound 2 where cell clusters were observed from day 2 onward. These clusters of LEC had a large increase in size on day 5 because cells were trying to survive making connections within the clusters but expressing low quantity of fibronectin. Compound 3 also showed a low quantity of cells but the presence of clusters was not detected, instead few LEC attempted to spread on the coating. At day 5 these

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2

viability and did not show signals of survival at day 5. Also on compound 3 LEC exhibited low metabolic activity and proliferation.

The same pattern of growing from day 2 to day 5 was present for HSkF for both viability and proliferation measurements (Fig. 3 C and D). Cells on collagen type I and compound 1 had a similar augmentation from day 2 to day 5, Δabs=0.745 and Δabs=0.812. Inversely to LEC, HSkF on compound 2 showed a significant increase between days, however its values were much lower than on TCPS or on the other coatings. HSkF on compound 3 showed the largest variation (Δabs=0.824) from day 2 to 5. The same pattern was expressed for the crystal violet values, however cells on compounds 2 and 3 appeared with a small Δabs of 0.120 and 0.196 respectively.

hMSC was the cell line with the lowest values for both assays (Fig. 3 E and F). The variation in cell proliferation between days remained low in all coatings. Once more, cells on collagen type I and compound 1 showed similar values on cell viability and proliferation. For this last assay, hMSC on compound 1 had a large increase of 80.44% compared to TCPS at day 5. The compound 2 was the worst coating regarding hMSC viability and proliferation with values near zero, similar to the behavior of LEC. hMSCs on compound 3 demonstrated low viability and proliferation at both time points.

Figure 3: MTT measurements for LEC (A), HSkF (C) and hMSC (E); and crystal violet measurements for LEC

(B), HSkF (D) and hMSC (F) on day 2 and day 5 of culture in collagen type I surfaces or on compounds 1, 2 and 3. The red bold lines represent the TCPS values on day 2 and the dotted lines the values for day 5. The percentage of increase or decrease between each material and the TCPS is expressed in the tables above each graphic. The variation between day 2 and day 5 for each material is showed by Δabs. Error bars depict standard deviations. Full statistical analysis of MTT and crystal violet is in the supplementary information.

Fluorescently stained cells demonstrating nuclei, actin cytoskeleton and fibronectin were visualized by confocal microscopy for all cell-material combinations (Fig. 4). LEC was the cell line that expressed the lowest quantity of fibronectin. In these cells the most impressive results came from the compound 2 where cell clusters were observed from day 2 onward. These clusters of LEC had a large increase in size on day 5 because cells were trying to survive making connections within the clusters but expressing low quantity of fibronectin. Compound 3 also showed a low quantity of cells but the presence of clusters was not detected, instead few LEC attempted to spread on the coating. At day 5 these

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cells were expressing long fibronectin fibers and the nuclei were larger than cells on TCPS. The collagen and the compound 1 coatings had shown a cell response that was much similar to TCPS. For both coatings LEC appeared as a confluent layer of cells maintaining their natural behavior [48]. Inversely to TCPS, the production of fibronectin was increased from day 2 to day 5, and its production was well organized.

For HSkF, once more, the compound 2 demonstrated the formation of clusters since day 2. At day 5 cells seemed to try to elongate and interact with the surface but the core of the clusters still increase in cell number with high levels of fibronectin. HSkF on compound 3 had an increase of cell number from day 2 to 5 with a large increase of fibronectin at the last day. The cells were spread on the coating very differently from LEC. Cell on collagen and compound 1 have showed a similar behavior with TCPS. The cell cytoskeleton was well organized and fibronectin was expressed in fibers as expected for this cell line [49]. The fibronectin fibers at day 5 on the compound 1 had the same shape as fibers on TCPS at day 2 (Fig.4B).

hMSC on compound 2 showed again the creation of small clusters at day 2 with posterior augmentation on day 5 but low production of fibronectin. However, the presence of nuclei with a low quantity of actin filaments indicated that hMSC failed to connect with the coating. As on LEC, the compound 3 did not create a favorable environment for cell proliferation. The cell number was very low on both days but at day 2 the quantity of actin was low and the fibronectin high. Contrarily at day 5 where the quantity of actin increased and fibronectin decreased. Collagen and compound 1 have shown similar number of cells as TCPS, however both the actin and fibronectin expression were lower. At day 5 fibronectin became well organized into fibers on the three different compounds and the quantity of this protein on compound 1 was similar to TCPS while on collagen it was lower (Fig.4C).

Figure 4: Confocal images of fluorescent staining of LEC (A), HSkF (B) and hMSC (C) cultured for 2 and 5

days on TCPS, collagen type I, and the three nanofibers coatings. Blue: DAPI; red: TRITC-phalloidin; green: FITC-fibronectin. Scale bar=75μm. More details for fibronectin staining in supplementary information (Figure S1).

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2

cells were expressing long fibronectin fibers and the nuclei were larger than cells on TCPS. The collagen and the compound 1 coatings had shown a cell response that was much similar to TCPS. For both coatings LEC appeared as a confluent layer of cells maintaining their natural behavior [48]. Inversely to TCPS, the production of fibronectin was increased from day 2 to day 5, and its production was well organized.

For HSkF, once more, the compound 2 demonstrated the formation of clusters since day 2. At day 5 cells seemed to try to elongate and interact with the surface but the core of the clusters still increase in cell number with high levels of fibronectin. HSkF on compound 3 had an increase of cell number from day 2 to 5 with a large increase of fibronectin at the last day. The cells were spread on the coating very differently from LEC. Cell on collagen and compound 1 have showed a similar behavior with TCPS. The cell cytoskeleton was well organized and fibronectin was expressed in fibers as expected for this cell line [49]. The fibronectin fibers at day 5 on the compound 1 had the same shape as fibers on TCPS at day 2 (Fig.4B).

hMSC on compound 2 showed again the creation of small clusters at day 2 with posterior augmentation on day 5 but low production of fibronectin. However, the presence of nuclei with a low quantity of actin filaments indicated that hMSC failed to connect with the coating. As on LEC, the compound 3 did not create a favorable environment for cell proliferation. The cell number was very low on both days but at day 2 the quantity of actin was low and the fibronectin high. Contrarily at day 5 where the quantity of actin increased and fibronectin decreased. Collagen and compound 1 have shown similar number of cells as TCPS, however both the actin and fibronectin expression were lower. At day 5 fibronectin became well organized into fibers on the three different compounds and the quantity of this protein on compound 1 was similar to TCPS while on collagen it was lower (Fig.4C).

Figure 4: Confocal images of fluorescent staining of LEC (A), HSkF (B) and hMSC (C) cultured for 2 and 5

days on TCPS, collagen type I, and the three nanofibers coatings. Blue: DAPI; red: TRITC-phalloidin; green: FITC-fibronectin. Scale bar=75μm. More details for fibronectin staining in supplementary information (Figure S1).

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LEC and hMSC were stained for α-SMA protein (Fig. 5). From the confocal images it can be seen that LEC were expressing α-SMA on all surfaces with an increase on day 5. TCPS, collagen and compound 1 promoted α-SMA expression in cells in a granular shape mainly located near the nuclei. Cells on both collagen and compound 1 appeared with a larger expression of this protein than on TCPS. The clusters of cells present on compound 2 were full of α-SMA with a large increase of its expression at day 5. LEC on compound 3 also had a large increase in α-SMA production between both days and this protein was detected not only in a granular pattern but also as stress fibers (arrows Fig.5A).

For hMSC (Fig.5B) α-SMA expression increased from day 2 to 5 on all surfaces. On TCPS, collagen and compound 1 α-SMA become more mature in fibers at day5 and well aligned with the cytoskeleton. On compound 2 the α-SMA was detected only in a granular shape inside the clusters of cells. On the other hand, cells on compound 3 expressed fibers of α-SMA since day 2 with a substantial increase of long stress fibers on day 5 (arrows Fig.5B).

Figure 5: Confocal images for α-SMA staining in LEC (A) and hMSC (B) cultured for 2 and 5 days on TCPS,

collagen type I, and the three nanofibers coatings. Blue: DAPI, red: TRITC-phalloidin, green: FITC-α-SMA. Scale bar=75μm . More details for α-SMA staining in supplementary information (Figure S2).

Gene expression of ALK1, ALK2, ALK5, TGF-β1, -β2, -β3, collagen type III, VI and

α-SMA was measured by real-time RT-PCR (Fig.6) after 2 and 5 days of cell culture. Fibroblasts cultured with TGF-β1 have been reported to change to myofibroblasts [50-52],

for this reason HSkF cultured on TCPS and stimulated with TGF- β1 were used as the

positive control for fibrosis-related gene expression. For LEC, the compound 1 was the only coating that induced a decrease from day 2 to 5 for ALK1, the gene upregulated most in the positive control. LEC on compound 3 showed upregulations in all ALKs (Fig. 6 A, D). TGF-βs exhibited, in both days, low values for LEC on compound 1, while on collagen at day 2 TGF-β3 was upregulated as well as TGF-β2 on compound 3 at day 5 (Fig. 6 B,E).

The different compounds did not significantly changed LEC regulation for collagen type III. Only LEC on compound 3 were slightly decreased with collagen type VI mRNA and on

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