Tuning Myoblast and Pre-osteoblast Cell Adhesion Site, Orientation

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Tuning Myoblast and Pre-osteoblast Cell Adhesion Site, Orientation and Elongation through Electroactive Micropatterned Scaffolds Teresa Marques-Almeida, Vanessa F. Cardoso, Sylvie Ribeiro, Francisco M. Gama, Clarisse Ribeiro, and Senentxu Lanceros-Méndez ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00020 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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Tuning Myoblast and Pre-osteoblast Cell Adhesion Site, Orientation and Elongation through Electroactive Micropatterned Scaffolds

Teresa Marques-Almeidaa, Vanessa F. Cardosoa,b*, Sylvie Ribeiroa,c, Francisco M. Gamad, Clarisse Ribeiroa,d,*, Senentxu Lanceros-Mendeza,e,f

aCF-UM-UP,

Centro de Física das Universidades do Minho e Porto, Universidade do Minho,

Campus de Gualtar, 4710-057 Braga, Portugal bCMEMS-UMinho,

Universidade do Minho, Campus de Azurém, Guimarães 4800-058,

Portugal cCBMA,

Centro de Biologia Molecular e Ambiental, Universidade do Minho, Campus de

Gualtar, 4710-057 Braga, Portugal dCEB,

Centro de Engenharia Biológica, Universidade do Minho, Campus de Gualtar, 4710-057

Braga, Portugal eBCMaterials,

Basque Center for Materials, Applications and Nanostructures, UPV/EHU

Science Park, 48940 Leioa, Spain fIKERBASQUE,

Basque Foundation for Science, 48013-Bilbao, Spain

*corresponding author: [email protected], [email protected]

1 ACS Paragon Plus Environment

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Abstract: Electroactive polymers are being increasingly used in tissue engineering applications. Together with the electromechanical clues, morphological ones have been demonstrated to determine cell proliferation and differentiation. This work reports on the micro patterning of poly(vinylidene fluoride-co-trifluoroethylene) - P(VDF-TrFE) scaffolds - and their interaction with myoblast and pre-osteoblasts cell lines, selected based on their different functional morphology. The scaffolds were obtained by soft lithography and obtained in the form of arrays of lines, intermittent lines, hexagons, linear zigzags and curved zigzags with dimensions of 25, 75 and 150 µm. Moreover, the scaffolds were tested in cell adhesion assays of myoblasts and pre-osteoblasts cell lines. The results show that more linear surface topographies and dense morphology have a large potential in the regeneration of musculoskeletal tissue while non-patterned scaffolds or more anisotropic surface microstructures present largest potential to promote the growth and regeneration of bone tissue. In this way, cell adhesion site, orientation and elongation can be controlled by choosing properly the topography and morphology of the scaffolds, indicating their suitability and potential for further proliferation and differentiation assays.

Keywords: Poly(vinylidene fluoride-co-trifluoroethylene); topography; morphology; cell adhesion; myoblasts; pre-osteoblasts



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1. Introduction The loss or dysfunction of an organ or tissue still represents a challenge to biomedicine. To address these needs, strategies such as transplants, prostheses or pharmacological therapies have been employed so that patients can improve quality of life. Nevertheless, the lack of compatible donors, transmission of diseases and high susceptibility to rejections or infections represent serious risks associated with these approaches.1 Thus, tissue engineering (TE) emerges as alterative or complementary to these strategies in order to regenerate/develop new tissues through the integration of temporary support structures – the scaffolds – that act as substitutes of the natural extracellular matrix (ECM) and thus promote and guide correct tissue pattern formation.2-3 Biocompatible polymeric materials, both natural and synthetic, are being strongly investigated for this purpose. Those of natural origin, such as collagen or chitosan have been widely used in TE4-5 as they are well tolerated and show structural similarity to molecules present in biological tissues. On the other hand, their physico-chemical and mechanical properties are not versatile,6 hindering their tailoring to specific applications. As alternative, synthetic biomaterials have been also used, especially polymeric ones, as their physicochemical properties are easily adaptable and reproducible.7 More recently, smart and functional materials, in particular electroactive polymers (EAPs) have been recognized as a promising choice for the TE field, for mimicking natural cell’s environment.8 In fact, EAPs are not only passive structural materials, but they also play a functional role in the systems to which they are integrated9 by actively stimulating cell growth and differentiation through the recreation of the in vivo environment. In addition, those polymers can be processed in various shapes and morphologies, may present biodegradability and suitable mechanical properties.10 Within the class of EAPs, piezoelectric polymers show the ability of 3 ACS Paragon Plus Environment

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convert electrical energy into mechanical energy, and vice versa, which is promising for applications that involve mechanoelectrical sensitive tissues such as bone,11 muscle12 or neurons13. Examples of piezoelectric polymers are poly(vinylidene fluoride) (PVDF), poly (L-lactic acid) (PLLA), and polyhydroxybutyrate (PHB). PVDF and its copolymers are the ones with the best electroactive performance, including pyro-, piezoand ferroelectric responses. Depending on processing conditions, PVDF can present five crystalline phases known as α, β, δ, γ and ε, being the β-phase the most technologically relevant for having the highest electroactive response.14-15 From all its copolymers,

poly(vinylidene

fluoride-co-trifluoroethylene)

(P(VDF-TrFE))

was

specifically designed to crystallize in the electroactive β-phase, independently of the processing conditions.16-17 Besides its active properties, PVDF-based polymers are easily tailored in terms of structure and morphology,18 which allow new and challenging applications in TE in order to induce specific cell responses.19 The performance of a scaffold is determined by several factors. In fact, cell adhesion to a scaffold comprises a series of physic and chemical reactions, which directly influence cell proliferation and differentiation that are essential for an adequate tissue-specific regeneration.20 Different cell types have distinct adhesion mechanisms when in contact with a particular biomaterial; on the other hand, the same cell type can answer distinctly to different biomaterials.21-22 Several recent works have been devoted to discover the more suitable kind of scaffold morphology and/or surface topography for each kind of tissue regeneration.23-24 It has been report that aligned β-PVDF fibers-based scaffolds promote elongation and directional growth of myoblasts, while non-oriented fibers generates random adhesion of cells.25 Since skeletal muscle is constituted by long and parallel bundles of multinucleated myotubes formed by the differentiation and fusion of myoblast, aligned



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fibers or aligned surface structure capable of promoting myoblast elongation is an added-value for musculoskeletal engineering.21,

26-27

In turn, bone tissue evidences a

diverse microenvironment, constituted by non-oriented collagen fibers to cortical (compact) and trabeculae (porous) matrices. Therefore, precursor bone cells like preosteoblasts are naturally inserted in different microenvironment, which provides them resilience when in contact with different scaffolds topographies and morphologies.28 Nevertheless, unlike myoblasts, this kind of cells is not favored by aligned matrices, since bone morphology do not present any aligned feature. Furthermore, it has been demonstrated that porous morphologies stimulate osteogenesis, enable transport of oxygen and nutrients and enhance osseointegration, influencing directly cells proliferation and differentiation.29-30 The processing of three dimensional (3D) patterned scaffolds with specific surface topographies allows to mimic cell’s microenvironment in a more reliable way. Fashioned polystyrene (PS) scaffolds patterned with pillars were fabricated and evaluated for osteogenic differentiation in human mesenchymal stem cells (hMSC), without any osteogenic factor.31 The scaffolds allowed an increased rate of osteogenic differentiation by proper control of the diameter and inter-pillars distances, showing its capability to define cell behavior only by topographic physical stimulus. Patterned polydimethylsiloxane (PDMS) scaffolds with linearly aligned microgrooves with various heights were fabricated to promote myoblast alignment for musculoskeletal regeneration.32 The results demonstrated that the topography dimensions influence substantially cell behavior. SU-8 scaffolds with surfaces composed by hexagons with diameter in the range of the micrometers were fabricated to study the impact of the size in Chinese hamster Ovary (CHO) cell growth.33 Once again, cell growth density proved to depend directly in the microcavity radius, providing adequate distance for cells communication. Thus, a series of factors must be taken into


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account during the design and fabrication of a scaffold, which include not just the material but also its morphology, topography and dimensions. The present work seeks to innovate by taking advantage of the physicochemical and mechanical properties of P(VDF-TrFE) (henceforward designated simply the “scaffolds”, for sake of simplicity) and producing patterned scaffolds with various and relevant surface topographies and morphologies for application in two distinct cells cultures: myoblasts and pre-osteoblasts. The scaffolds were produced using PDMS molds obtained by soft-lithography. The objective of this systematic study is to understand how the surface topography, dimension and morphology of the scaffolds influence cell behavior, with the final goal of developing a solid base for further proliferation and differentiation studies of myoblast and pre-osteoblast cells under static and dynamic electroactive stimuli.

2. Experimental procedures SU-8 microstructures with an area of 1.6×1.3 cm2 were fabricated and used to produce low-cost, flexible and chemically resistant PDMS molds by soft lithography. The latter were repeatedly used to fabricate scaffolds by replica molding.16 SU-8 microstructures consisting of arrays of lines, intermittent lines, hexagons, linear zigzags and curved zigzags, with widths and spacing of 25, 75 and 150 µm, were fabricated. These dimensions were selected according to the osteoblast and myoblast cells that feature mean dimensions of 20-30 µm. In particular, 150 µm was selected because it is twice as much as 75 μm and to verify if the cells prefer the surface of the structure or its interior and how they react to larger environments. The microstructures were manufactured with a thickness twice the size of the patterned features. These microstructures, replicated on the scaffolds using the PDMS molds, were chosen to study the behavior of pre6 ACS Paragon Plus Environment


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osteoblasts and myoblasts cells to differently patterned surfaces. Moreover, porous and non-porous patterned scaffolds were produced by properly controlling the crystallization rate and tested.

2.1.

Materials

Epoxy-based negative photoresist (SU-8 types 25 and 100) and Sylgard® 184 Silicone Elastomer (PDMS) were obtained from Microchem and Dow Corning, respectively. Standard microscope glass slides (76 × 26 mm) from Labbox were used for the fabrication of the SU-8 molds. Aluminum adhesive tape (cat. no. 56223) was acquired from Tesa. P(VDF-TrFE) 70/30 (70 mol% vinylidene fluoride monomer; 30 mol% trifluoroethylene) powder and N,N-dimethylformamide (DMF) were supplied from Solvay and Merk, respectively. All chemicals and solvents were used as received.

2.2.

Fabrication of the SU-8 molds

SU-8 25 was used to fabricate SU-8 microstructures with thickness of 50 µm, while SU-8 100 was used for those 150 and 300 µm thick. Details on the different processing steps and the corresponding parameters used to obtain the different SU-8 molds can be found in reference34. All patterns were first designed and printed by Microlitho in photolithographic materials. After the final development step, the samples were properly cleaned with isopropyl alcohol and dried gently with compressed air. All glass substrates with SU-8 microstructures were surrounded by aluminum adhesive tape to allow further pouring of the PDMS solution into a confined space.

2.3.

Fabrication of the PDMS molds


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Approximately 5 g of degassed PDMS mixture composed of 1/10 wt% curing agent/base were poured in each SU-8 mold, left to rest during 10 min to obtain a flat surface and cured at 100 ºC during 30 min by means of a hot-plate (Präzitherm PZ23-2). Alongwise, flat PDMS films were fabricated using SU-8 free glass slides and the same protocol previously described. The latter were cut with proper dimensions to further create walls (with 3 mm wide) around the PDMS microstructures. A very small and homogenous film of uncured PDMS was swab on the PDMS walls, glue to the corners of each PDMS microstructures carefully peel off from the SU-8 molds and cured onto a hot-plate at 100 ºC during 1 h.

2.4.

Fabrication of the P(VDF-TrFE) scaffolds

A solution of P(VDF-TrFE) was prepared using DMF as solvent in a copolymer volume fraction of 8 %. The dissolution was performed under magnetic stirring until a homogenous and transparent solution is obtained, which take approximately 1 h. A slight heating at 30 ºC was performed in the first 15 min to prevent aggregates and accelerate the dissolution process. Before filling with P(VDF-TrFE) solution, the PDMS molds were first treated by oxygen plasma (Electronic Diener Plasma-SurfaceTechnology, Zepto) at a plasma power of 100 W for 20 min under an oxygen pressure of 0.8 mbar. This optimized process is required to decrease the natural hydrophobic surface of PDMS (≈100 º)35 and thus guarantee the proper spread of the copolymer solution through the microstructures. Two crystallization temperatures were used to obtain scaffolds with different morphologies. In fact, the porosity of the scaffolds can be controlled by the evaporation rate of the solvent DMF, which depends directly on the crystallization temperature.36 Thus, porous patterned scaffolds were obtained when crystallized at 25 ºC, which take approximately 1 week. In turn, dense patterned



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scaffolds were obtained when crystallized at 100 ºC during 3 h.37 Additionally, nonpatterned scaffolds with both morphologies, porous and dense, were also processed to work as controls. An air oven (JP Selecta 200208) was used for this purpose, to guarantee a homogeneous and controlled crystallization through time. After complete crystallization, the scaffolds were easily peel off from the PDMS molds, showing white color or being transparent according to their porous or dense morphologies, respectively. The PDMS molds were washed with acetone and distilled water and properly stored for further reuse. Eight scaffolds of each microstructure and morphology were fabricated.

2.5.

Physico-chemical characterization

A Leica M80 stereomicroscope and a Veeco Dektak 150 Surface profilometer were used to visualize the microstructures and dimensions of the SU-8 and PDMS molds. A scanning electron microscope (SEM) Quanta 650 FEG from FEI was employed to characterize the topography and morphology of scaffolds. For evaluation of the surface hydrophobicity, contact angle measurements were performed using a Data-Physics OCA20 and ultrapure water (drop volume of 3 µL and rate of 0.5 µL.s-1). Six measurements were carried out in each scaffold, the contact angles being presented as the average and standard deviation. The crystalline phase of the samples was confirmed by Fourier transformed infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) using a Spectrum Two™ from Perkin-Elmer, with 64 scans in the range between 400 and 4000 cm−1 and a resolution of 4 cm−1. Differential scanning calorimetry (DSC) was performed with a DSC 6000 from Perkin-Elmer. Pieces of approximately 6 mg were cut and placed into a 40 µL aluminum pans. The samples were heated between 30 and 200 ºC at a scanning rate of 10 ºC.min.-1. In turn, pieces of


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approximately 10 mg were cut and analyzed by thermogravimetry (TGA) using a TGA 4000 from Perkin-Helmer heating the samples between 30 and 650 ºC at a heat flow of 10 ºC.min.-1.

2.6.

Cell culture and evaluation

2.6.1. Scaffolds sterilization For in vitro assays, circular films with 6 mm diameter were cut and placed in standard 48-well cell culture plates, exposed to ultraviolet light (UV) for 1 h on each side and then washed 5 times (5 min each time) with sterile phosphate buffer saline (PBS) 1 × solution.

2.6.2. Cell culture Two different cell lines were used and grown in 75 cm2 cell-culture flasks at 37 °C in a 95 % humidified air containing 5 % CO2 incubator. MC3T3-E1 pre-osteoblast cells (Riken bank) were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 1 g.L-1 glucose, 1 % penicillin/streptomycin (P/S, Biochrom) and 10 % Fetal Bovine Serum (FBS, Biochrom); C2C12 myoblast cells (ATCC) were grown in DMEM with 4 g.L-1 glucose, 1 % P/S and 10 % FBS. Culture media were changed every two days until reaching 60-70 % confluence. For cell adhesion assays, 2500 cells were added to each P(VDF-TrFE) scaffolds and incubated for 24 h. After this time, the P(VDF-TrFE) scaffolds were washed with PBS 1 x solution, fixated with 4 % formaldehyde (Panreac) for 10 min at 37 ºC in a 5 % CO2 incubator and observed as described on section 2.6.3.



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2.6.3. Cell morphology After

fixation,

two

replicates

of

each

kind

of

samples

were

used

for

immunofluorescence and SEM assays. For the immunofluorescence assay, the cells were washed three times with PBS 1x solution and then incubated in 1 µg.mL-1 of phalloidin tetramethylrhodamine (TRITC, Sigma Aldrich) solution for 45 min at room temperature. Finally, the samples were washed with PBS 1 x and incubated in 1 μg.mL-1 of a 4,6- diamidino-2phenylindole (DAPI, Sigma Aldrich) solution for 5 min and washed again with PBS 1 x solution for visualization in a fluorescence microscopy (Olympus BX51 Microscope). Regarding SEM analysis, each sample was dehydrated in a graded series of ethanol (10%, 30%, 50%, 60%, 70%, 80%, 90% and 99%) for 20 min each. After that, the samples were added to aluminium pin stubs with electrically conductive carbon adhesive tape (PELCO Tabs™), then placed (without coating) inside a Phenom Charge Reduction Sample Holder (CHR) and finally they were characterized using a desktop SEM coupled with energy-dispersive X-ray spectroscopy analysis (Phenom ProX with EDS detector (Phenom-World BV, Netherlands). Images were taken at 10 kV and a size spot of 3.3.

3. Experimental Results and Discussion 3.1.

Physico-Chemical characterization

Successful fabrication of PDMS molds and micropatterned P(VDF-TrFE) material (the “scaffold”) directly relies on the proper fabrication of the SU-8 molds. Thus, stereomicroscopy and profilometry were used to characterize the molds during the different processing stages (data not shown). Although some SU-8 microstructures with 11 ACS Paragon Plus Environment

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larger thickness feature slightly sloping walls that will be reflected in the scaffolds, as will be shown latter, all of them present microstructures with good resolution and the intended dimensions. Figure 1 presents surface and cross-section SEM images of non-patterned scaffolds.

Figure 1: Surface SEM images and corresponding transversal cross section in the inset of a) dense and b) porous non-patterned P(VDF-TrFE) scaffolds.

As expected, a thermal treatment at 100 ºC promptly after the deposition increases the solvent evaporation rate avoiding the formation of pores and leading to dense transparent films with smooth and flat surfaces (Figure 1a). In turn, at 25 ºC the evaporation rate of DMF is slower, inducing the formation of porous white films (Figure 1b). The morphologies were replicated into patterned scaffolds. Figure 2 shows SEM images of two different microstructures, namely hexagons and lines. These microstructures were selected as representative of all produced specimens, since they bear all the relevant geometrical features.



Lines

75 µm

25 µm

Hexagons

150 µm

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

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Figure 2: SEM images of porous patterned P(VDF-TrFE) scaffolds of hexagons and lines microstructures with dimensions of 25, 75 and 150 µm. The corresponding dense morphology counterparts are shown in the inset images.

When crystallized at 100 ºC, dense scaffolds with well-defined microstructures are successfully obtained in all dimensions. The same conclusion can be drawn for the samples crystallized at 25 ºC, except for the 25 µm dimension where the microstructures appear compromised. In fact, in this last case, the SEM images show total absence of hexagons and discontinuities in the lines, due to the overlapping of the pores formed during crystallization. Indeed, the pores present diameters of 31±19 µm and 19±12 µm


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respectively, thus being larger than the microstructures (25 µm). It can also be observed that the hexagonal microstructures feature sloping walls, resulting in inverted hexagonal pyramids instead of hexagonal prisms, similar to those obtained in the SU-8 molds. Contact angle measurements, using the circle fitting mode, were performed in all scaffolds to evaluate the surface wettability. The contact angle on a smooth surface is a function of the fluid and the surface materials. However, the contact angle for nonsmooth surfaces, being strongly dependent on the roughness, further improves hydrophilicity in hydrophilic surfaces and hydrophobicity in hydrophobic ones.38 A turning point between hydrophilic and hydrophobic surfaces of 65º was used as reference, being hydrophilic for contact angles lower than 65º and hydrophobic for higher values.39 P(VDF-TrFE), as a fluorinated polymer, generally presents a hydrophobic surface, i.e., a low surface energy.40 The results presented in Figure 3 support this statement. All samples show contact angles higher than 100º, except for the dense non-patterned scaffolds (control), which present a lower contact angle of 76.3 ± 2.4º. In turn, the porous non-patterned scaffolds exhibit a contact angle of 107.8 ± 2.6º, much higher than the dense non-patterned ones, which is explained by the increase of the surface roughness associated with the presence of pores.41 This behavior is further enhanced when the surface of the scaffolds is micropatterned due to the increase of the effective surface area,42 with contact angles between 98.7 ± 1.4º and 137.8 ± 2.1º, with no significant difference between morphologies.



180

a)

140 120 100 80 60 40

Control 25 µm 75 µm 150 µm

160 140 120 100 80 60 40 20

20 0

180

Contact Angle (º)

Contact Angle (º)

b)

Control 25 µm 75 µm 150 µm

160

0 Ctrl Lines Intermittent

Line

Curve

zigzag

zigzag

Hex

lines

Ctrl Lines Intermittent Hex lines

Line

Curve

zigzag

zigzag

Figure 3: Contact angle measurements: a) dense P(VDF-TrFE) scaffolds and b) porous P(VDF-TrFE) scaffolds. The controls correspond to non-patterned surfaces.

The physicochemical properties of the scaffolds are influenced by the molecular conformation and crystallinity of the copolymer. Thus, FTIR and thermal analysis of the scaffolds were performed to ascertain whether their properties are influenced by the processing conditions. The results are presented in Figure 4.

a)

Dense Porous

b)

100

Weight loss (%)

Transmitance (u.a.)

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

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10

1600

1400

1200

510

840

1400 1287

1000

800 -1

Wavelenght (cm )

600

400

Dense Porous

80 60 40 20 0

0

100

200

300

400

500

600

700

Temperature (ºC)


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c)

Dense Porous

Heat Flow (W.g-1)

0.5

d)

Morphology

Dense

Porous

TFP (ºC)

99 ± 2

96 ± 2

TM (ºC)

143 ± 3

143 ± 3

ΔHM (J.g-1)

29 ± 0.6

24 ± 0.5

χC (%)

32 ± 0.6

26 ± 0.5

endo

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


40

60

80

100 120 140 160 180 200

Temperature (ºC)

Figure 4: Representative a) FTIR-ATR spectra; b) TGA thermograms; c) DSC thermographs and d) corresponding TFP, Tc, ΔHM and χC of porous and dense P(VDFTrFE) scaffolds.

The FTIR-ATR spectra demonstrate that independently of the experimental conditions all samples feature the same infrared spectra with the main adsorption bands corresponding to the all trans TTT´ polymer conformation (Figure 4a). In fact, the electroactive and polar β-phase was identified by the specific vibration modes at ~510, 840, 1287 and 1400 cm-1 that represent CF2 bending; CF2 symmetric stretching; CF2 and CC symmetric stretching and CCC bending; and CH2 wagging and CC antisymmetric stretching, respectively.8, 43-44 Regarding the TGA thermograms presented in Figure 4b, both porous and dense scaffolds present the same behavior with a single degradation step between 430 and 500 ºC, characteristic of this copolymer, which is explained by the chain-stripping where the carbon-hydrogen scissions occur, leading to the formation of hydrogen fluoride.45 In turn, the DSC thermograms are characterized by two endothermic peaks that are associated with the ferroelectric-paraelectric transition (TFP) between 90 and 100 ºC and the melting temperature of the paraelectric phase (TM) between 130 and 16 ACS Paragon Plus Environment


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150 ºC, as presented in Figure 4c. According to these results, the melting enthalpies (Δ HM) were obtained and the degree of crystallinity (χC) calculated using the equation 1.

ΔHM

χC (%) = ΔH100 × 100

(Eq. 1)

where ΔH100 is the melting enthalpy for a 100 % crystalline P(VDF-TrFE) 70/30 sample, taken as 91.5 J.g-1.36,

46

The results, summarized in Figure 4d, show that the

processing conditions do not affect significantly the main characteristics of the scaffolds. Both TFP and TM show small variation, within experimental error. In turn, the degree of crystallinity increases slightly from 26 to 32% for the porous and dense scaffolds, respectively, which can be associated with the increase of the solvent evaporation rate and therefore quicker polymer crystallization at 100 ºC instead of 25 ºC.47 Nevertheless, it can be concluded that the processing conditions do not significantly influence the thermal characteristics of the copolymer P(VDF-TrFE).

3.2.

Cell adhesion evaluation

Cell adhesion assays using C2C12 myoblasts and MC3T3-E1 pre-osteoblasts were performed in the scaffolds to study the influence of the material´s microstructure on the cell behavior, once these cells present different morphology and both as subjected to different electromechanical clues during its function. For example, the myotube is an unusually elongated cell48 in comparison to other cell types, such as bone cells, which are not elongated.

3.2.1. Influence of surface topography


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Cell adhesion assays were first conducted on non-patterned scaffolds (controls). Representative immunofluorescent images are presented in Figure 5.

Figure 5: Cell adhesion of C2C12 myoblasts and MC3T3-E1 pre-osteoblasts on dense and porous non-patterned P(VDF-TrFE) scaffolds.

The results demonstrated that, contrary to dense scaffolds that present clean images, porous scaffolds have the particularity of scatter fluorescent light creating background and making their analysis more difficult. Further, cells can also penetrate the pores, leading to cells in different focal planes and, therefore, contributing to blurred images. Nonetheless, it is possible to conclude that both C2C12 and MC3T3-E1 cells are adhered in a totally unordered way and feature characteristic elongated and rounded shapes, respectively, independently of the scaffold morphology, which is in agreement with previous studies.19 Then, cell assays were performed on all patterned scaffolds with a dimension of 150 µm and both morphologies, i.e dense and porous, to study the influence of the surface 18 ACS Paragon Plus Environment


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topography on cell behavior. This dimension was selected since it allows an easier cell evaluation in larger and well-defined microstructures. The results are presented in Figure 6.

Figure 6: Cell adhesion of C2C12 myoblasts and MC3T3-E1 pre-osteoblasts on dense and porous patterned P(VDF-TrFE) scaffolds with dimension of 150 µm. Inset represents focus in the depth of the microstructure. All immunofluorescence images show good cell density, meaning that the increased surface hydrophobicity of patterned scaffolds has no effect on cell adhesion.


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Regarding myoblast cells, it is possible to observe that cells adhere both on the “peaks” and “valleys” of the microstructures, in all scaffolds. Moreover, in the case of dense scaffolds the cells are oriented along the microstructures. This trend suggests that the scaffold topography has an influence on the directionality of cells adhesion. This is more accentuated in the microstructures constituted by lines, intermittent lines, linear zigzag and curved zigzag, which evidence the elongation of myoblast through the scaffold surface, characteristic phenotype of musculoskeletal cells. This result is in agreement with the literature, which states that linear topographies (oriented fibers) show the ability to guide cell alignment and stimulate the elongation of myoblasts.25 Nonetheless, a deeper analysis shows that the myoblast cells are elongated at the peaks of the microstructures, whereas in the valleys cells appear more rounded and with a smaller cytoskeleton (inset in Figure 6). The same is observed in all the samples (data not shown). This is attributed to the more confined environment in the valleys.10 In fact, as previously stated, the walls of the microstructures appear to be slightly inclined, reducing the area available in the valleys of the structures. In porous scaffolds, the analysis is more difficult due to the lower quality of the images, as stated earlier. However, the cytoskeleton of the myoblasts appears to be smaller, probably because cells are entering the pores on this type of surface, hindering the observation of the cellular elongation and orientation. This hypothesis will be latter confirmed by SEM (Figure 9). Regarding pre-osteoblasts cells, the literature does not report, to date, any preference in terms of scaffolds topography for these cells. The fact that bone has diverse microenvironments (porous - trabecular bone - and dense - cortical bone - but not aligned structures)49 makes osteoblastic cells easily adaptable to different surrounding environment.28 Nonetheless, contrary to myoblasts that adhered on both peaks and



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valleys of the microstructures, the immunofluorescent images show that the preosteoblast cells are well adhered essentially at the top of the P(VDF-TrFE) microstructures, covering and defining accurately the peaks geometry. Thus, the topography of the scaffolds demonstrates to play an important role in the growth direction of pre-osteoblasts cells, as observed with myoblasts.

3.2.2. Influence of surface topography dimensions In order to evaluate the influence of the microstructures dimensions, cells adhesion assays were performed on the scaffolds with hexagons and lines 25, 75 and 150 µm wide. Results of immunofluorescent images of C2C12 myoblast cells are presented in Figure 7.


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Figure 7: Cell adhesion of C2C12 myoblasts on dense and porous patterned P(VDFTrFE) scaffolds with hexagons and lines topographies and with dimensions of 25, 75 and 150 µm.

A general analysis of the immunofluorescent images allows to conclude that myoblast cells adhered both to the peaks and valleys of the microstructures, although some variations and adhesion site preferences may be identified, as will be discussed below. Regarding the scaffolds with hexagonal topography, it is observed that microstructures with a dimension of 25 µm do not exert any influence on the myoblasts behavior. In fact, myoblast cells adhered randomly and with no orientation to the scaffolds, both in porous and dense morphologies. As previously discussed (Figure 2), the 25 µm 22 ACS Paragon Plus Environment


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hexagonal porous topography was not well defined due to the overlap of pores, which explains this lake of influence. Nonetheless, although the dense counterpart was obtained, featuring the same size with good resolution, the smaller hexagonal topography did not lead to any effect on the directionality of myoblasts adhesion. This is probably because myoblast has a diameter of ~10 µm, in the same size range of the microstructural features. In turn, the same topography with peaks and valleys 75 and 150 µm wide and dense morphology induces the adhesion of myoblast cells mostly around the hexagonal boundaries. Although the preferential adhesion site of myoblasts does not change with dimension of the microstructure, the morphology of the scaffolds seems to be more relevant. Cells adhere preferably on the top of the porous microstructures. This trend seems to increase with the height of the peaks, being more accentuated at the 150 µm microstructures, although cells are present also within the hexagons, as referred previously. It should be noted that along with the increase of the hexagons sizes (25, 75 and 150 µm), the height of the microstructures also increases linearly (50, 150 and 300 µm, respectively), which may influence the myoblasts adhesion site and cells communication. Besides, the characteristic elongated phenotype of myoblast cells seems to be compromised because of the microstructures shape and also probably by the confinement given by the hexagons that difficult cells elongation, essential in further phases of regeneration 50. The scaffolds with linear topography and dense morphology demonstrates a clear influence on myoblasts behavior. In fact, myoblast cells adhere well and with an elongated phenotype in the dense topographies with different dimensions. In particular, the 25 µm linear topography evidence a high density of cells considerably elongated and oriented along the lines. That density of cells seems to decrease with increasing size of the microstructures. Furthermore, in the scaffolds with 75 µm wide features, myoblast


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cells adhered on the peaks of the microstructures and seem to spread beyond the edges of the lines evidencing interconnections, while in the structures 150 µm wide they are totally separated by the valleys and mostly adhered at the peaks of the microstructures. These results may indicate a preferential and thus larger adhesion of myoblast cells in the presence of minor spaces so they can be closer, than microstructures with higher height and wider valleys that hinder or even prevent cells communication 33. In the case of porous line topographies, the preferential adhesion site and elongation of the myoblast cells are not so clear as in the denser ones; maybe the porous morphology makes the elongation of this kind of cells more difficult. In the case of the smaller lines, the discontinuities of the lines (SEM images of Figure 2) may also be responsible for the lack of influence of the microstructures on the elongation of the cells. Nonetheless, it is observed that although the cell density seems to be higher in the 25 µm microstructures, as in the denser scaffolds, the orientation of cells is more evident in the lines 150 µm wide. Thus, it is possible to state that all line scaffolds, of all dimensions and morphologies, do influence myoblast adhesion site, orientation and elongation. As previously mentioned, myoblast elongation has been shown previously to be promoted by linear parallel topographies 25-26. Thus, the obtained results are in agreement with the literature, justifying the use on this type of scaffolds topographies, in particular the denser ones, for musculoskeletal tissue engineering. The results of cell adhesion assays of pre-osteoblasts MC3T3-E1 on dense and porous scaffolds with hexagons and lines topographies are presented in Figure 8.



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Figure 8: Cell adhesion of MC3T3-E1 pre-osteoblasts on dense and porous patterned P(VDF-TrFE) scaffolds with hexagons and lines topographies and with dimensions of 25, 75 and 150 µm.

Pre-osteoblast cells seems to adhere preferably at the peaks of the microstructures, except for the 75 µm hexagonal topography where they adhere mostly inside the hexagons. Furthermore, no influence on cell behavior due to both the dense and porous hexagonal microstructures with dimension of 25 µm was noticed: cells were found dispersed though the scaffold surfaces with no preferential orientation. The preosteoblast cell diameter (~30 µm), larger than the hexagons (~25 µm), results in cell


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adhesion across the entire surface of the scaffolds, with no influence of the topography (which is not well defined in the case of the porous scaffold). However, it is possible to observe a good cell distribution, as occurred with the dense non-patterned scaffolds. Dense hexagonal topographies with dimensions of 75 and 150 µm influence preosteoblasts behavior in distinct ways, as previously stated. In fact, while cells adhered preferably inside the hexagons of 75 µm, they are mainly present in the peaks of the 150 µm ones and also on the porous hexagons scaffolds. With regard to dense linear topographies, pre-osteoblast cells present well adhered to the top of the microstructures, following properly the lines. The peculiar morphology of these samples imposes strong limitations on the focal adhesion formation that will influence their morphology, and consequently also cell behavior. The smaller the lines, the more elongated is the cytoskeleton, evidencing cellular interconnections across consecutive lines. Thus, cells communication appears to decrease with wider spaces between lines. A similar cell behavior is observed in the porous linear topographies. It should be noted that cell communication is imperative for normal development of all type of cells.51 In fact, although it was proved under this study that it is possible to control and tailor site adhesion, orientation and elongation of cells through the control of the scaffold microstructure (shape, features size and porosity), it is important to conceive scaffolds that support proper orientation and effective communication. Hereupon and according to the results obtained, dense scaffolds with more parallel linear topography seems more adequate for musculoskeletal cells, while non-patterned scaffolds or with anisotropic microstructures, such as hexagons or curved zigzags, are more suitable to proceed with bone proliferation and differentiation assays. In this last case, porous morphology are recommended for favoring bone growth, nutrient diffusion and osseointegration.29



The main results regarding cell adhesion in the different samples are summarized in the table 1.

Table 1: Cell adhesion of C2C12 myoblasts and MC3T3-E1 pre-osteblasts on the different patterned P(VDF-TrFE) scaffolds. Preferential Cell Preferred

adhesion on stretching

Samples adhesion sites

peaks or (Y/N) valleys

25 µm

Dispersed around

Peaks

N

Valleys

N

Peaks

N

Peaks

N

Valleys

N

Peaks

N

the scaffold 75 µm

Mostly around the hexagonal

Dense boundaries

Pre-osteoblast cells

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

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150 µm

Mostly around the hexagonal boundaries

Hexagons 25 µm

Dispersed around the scaffold

75 µm Porous


150 µm



Page 29 of 39

boundaries

Dense

25 µm

Along the lines

Peaks

Y

75 µm

Along the lines

Peaks

Y

150 µm

Along the lines

Peaks

Y

25 µm

Along the lines

Peaks

Y

75 µm

Along the lines

Peaks

Y

150 µm

Along the lines

Peaks

Y

25 µm

No influence,

Both

N

Valleys

N

Valleys

N

Both

N

Peaks

N

Peaks

N

Lines

Porous

Randomly adhesion 75 µm Dense


150 µm

Myoblast cells

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



Hexagons 25 µm

No influence, Randomly adhesion

75 µm

Mostly around the

Porous hexagonal boundaries 150 µm




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boundaries

Dense

25 µm

Along the lines

Peaks

Y

75 µm

Along the lines

Peaks

Y

150 µm

Along the lines

Peaks

Y

25 µm

Close to the lines

Both

N

Both

N

Peaks

Y

but without defined

Lines

orientation Porous

75 µm

Close to the lines but without defined orientation

150 µm

Along the lines

3.2.3. Analysis of cell adhesion by SEM In addition to the fluorescent microscopy analysis of cell adhesion behavior, representative samples were selected for visualization by SEM. Thus, cell adhesion assays in hexagonal and linear scaffolds with dimensions of 150 µm were selected and analyzed. The results are presented in Figure 9.


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Figure 9: SEM micrographs of C2C12 myoblasts and MC3T3-E1 pre-osteoblasts cell adhesion on dense and porous hexagons and lines P(VDF-TrFE) scaffolds topographies with a dimension of 150 µm.

Similarly to the previously discussed and analyzed by fluorescence microscopy, the SEM images show that myoblast cells adhered to both the peaks and valleys of the microstructures, independently of the scaffold morphology, but more clearly in the case of the hexagonal microstructures. On the dense linear scaffolds, myoblasts exhibit a higher elongation of the cytoskeleton at the top of the microstructures, while in the valleys they are smaller and less elongated. In the case of porous linear scaffolds, myoblasts penetrate the scaffold pores, which is demonstrated by some cytoskeleton connections across the bottom of two consecutive lines (see marking in the figure 9). Regarding to the bone cells and dense scaffolds, cells are mainly at the top of the structures in both topographies, but they also adhere to the side walls, which could not be observed in the fluorescence images. On porous scaffolds, pre-osteoblast cells seem avoid the inside of both topographies, which is in agreement with the data of fluorescence microscopy. As for myoblasts, these cells exhibit a cell elongation along 30 ACS Paragon Plus Environment


Page 32 of 39

the lines, following properly the lines. In the hexagons structures, the cells adhered around these structures both in denser and porous structures.

4. Conclusions The present work reports on the processing and evaluation for tissue engineering applications of porous and dense microstructured P(VDF-TrFE) scaffolds. The physicochemical analysis demonstrates that the processing technique and conditions do not change the intrinsic properties of the material. Thus, adhesion cell assays using myoblasts C2C12 and pre-osteoblasts MC3T3-E1, selected due to their different morphological features and for being exposed to electromechanical solicitations within the human body, were performed to study the influence of P(VDF-TrFE) topography and morphology on cells behavior. It was proven that difference microstructure geometries, dimensions and morphologies do influence directly cells behavior. Of all evaluated P(VDF-TrFE) scaffolds, the results demonstrated that more linear surface topographies and dense morphology have a large potential in the regeneration of musculoskeletal tissue, for promoting orientation, elongation and parallel positioning of myoblast cells, which is important for subsequent fusion in myotubes during differentiation assays. In turn, although pre-osteoblasts show similar behavior on different

topographies,

non-patterned

scaffolds

or

more

anisotropic

surface

microstructures presenting porous morphology seem to present largest potential to promote the growth and regeneration of bone tissue. Thus, the produced scaffolds shown to be very promising for future cell proliferation and differentiation assays of this kind of cells. Moreover, the use of the electroactive polymers can also be combined to mimic more properly their natural electromechanical microenvironment under dynamic cell culture. 31 ACS Paragon Plus Environment

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Acknowledgements This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding UID/FIS/04650/2013 and UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and projects POCI-01-0145FEDER-028237 and POCI-01-0145-FEDER-028159 funded by national funds through Fundação para a Ciência e a Tecnologia (FCT) and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI); and also under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors

also

thank

the

FCT

for

the

SFRH/BPD/98109/2013

(V.F.C.),

SFRH/BD/111478/2015 (S.R.) and SFRH/BPD/90870/2012 (C.R.) grants. The authors thank F. Vaz, University of Minho, for providing the equipment for the surface treatment of the samples. The authors acknowledge funding by the Spanish Ministry of Economy and Competitiveness (MINECO) through the project MAT2016-76039-C4-3R (AEI/FEDER, UE) and from the Basque Government Industry and Education Department under the ELKARTEK, HAZITEK and PIBA programs. The SEM measurements have been conducted at Center of Biological Engineering (CEB), Braga, Portugal. The authors thank CEB for offering access to their instruments and expertise. Moreover, the authors also thank the Centre for Microelectromechanical Systems (CMEMS), Guimarães, Portugal, for providing the conditions for the manufacture and characterization of the SU-8 and PDMS moulds.



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