# Biomimetic three-dimensional scaffolds with aligned electrospun nanofibers and enlarged pores for enhanced cardiac cell colonization

Florence Flaig, Anne Hébraud, Emeline Lobry, Damien Favier, Antoine Egele, Patrick Kékicheff, Pierre Joanne, Onnik Agbulut, Guy Schlatter

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Florence Flaig, Anne Hébraud, Emeline Lobry, Damien Favier, Antoine Egele, et al., Biomimetic three-dimensional scaffolds with aligned electrospun nanofibers and enlarged pores for enhanced cardiac cell colonization. Materials & Design, 2024, 248, pp.113481. 10.1016/j.matdes.2024.113481. hal-05224923

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# Biomimetic three-dimensional scaffolds with aligned electrospun nanofibers and enlarged pores for enhanced cardiac cell colonization

Florence Flaig<sup>a</sup>, Anne Hébraud<sup>a</sup>, Emeline Lobry<sup>a</sup>, Damien Favier<sup>b</sup>, Antoine Egele<sup>b</sup>, Patrick Kékicheff<sup>b</sup>, Pierre Joanne<sup>c</sup>, Onnik Agbulut<sup>c</sup>, Guy Schlatter<sup>a,*</sup>

## ARTICLE INFO

Keywords:

Electrospinning

Aligned nanofibers

Biomimetic scaffolds

Tissue engineering

## ABSTRACT

Among the wide range of biomimetic scaffolds, those made of aligned nanofibers represent an important class for tissue engineering applications, and particularly for cardiac repair. Electrospinning is a powerful technique allowing the fabrication of scaffolds with aligned nanofibers. However, the resulting morphology is characterized by low porosity and small pore size due to the formation of a compact structure of aligned fibers preventing cell colonization inside the scaffold. In the present work, supported by numerical simulation, it is demonstrated that when optimal electrostatic interactions occur during the simultaneous deposition of electrospun nanofibers and electrosprayed microparticles on a collector rotating at high speed, a 3D structured scaffold is obtained. Thanks to a self-organization mechanism, islands of microparticles intercalate inside a network of loosely packed and highly aligned nanofibers. In the case of poly(lactic acid) electrospinning and poly(glycerol sebacate)/cyclo-dextrin electrospraying, the resulting 3D biomimetic scaffold, deeply investigated by X-ray microtomography and mechanical characterization, shows an open porosity with enlarged interconnected pores with a mechanical behavior mimicking that of cardiac tissue. Finally, after functionalizing the fiber surface using laminin, it is shown an efficient 3D colonization of cardiac cells through a network of aligned nanofibers.

## 1. Introduction

Tissue engineering is an approach allowing the replacement or repair of tissues or organs. The principle of this method mainly relies upon the promotion of cells growth on a bioinspired scaffold that mimics the extracellular matrix (ECM) of the tissue which has to be repaired. The structure of the ECM is fibrous, 3D, composite and hierarchically organized with characteristic length scales ranging from nm to mm. Tissue engineering has been identified for the repair of a wide range of organs and among them, the heart, for which the myocardial tissue shows a hierarchical structure with aligned cells embedded into 3D honeycomb-like micro-patterns formed by undulated perimysial collagen fibers [1,2]. Thus, a scaffold having a fibrous structure is crucial for cell organization, survival and function of the seeded cells. In addition, in order to respect the anisotropic organization of the cardiac tissue and promote the development of functional cardiac syntium, the nanofibers need to be aligned while favoring an efficient cell colonization inside the scaffold [3].

Electrospinning is a well-known process allowing the fabrication of non-woven nanofibrous scaffolds. The principle relies on the processing of a polymer solution which is subjected to a high electric field in the range of few kV/cm. The electrified polymeric jet exiting the emission electrode experiences electro-hydrodynamic instabilities in the form of whipping movements which result in the elongation of the jet, the decrease of its diameter, the evaporation of the solvent through convection exchanges with the surrounding atmosphere and finally, the deposition on a grounded electrode, the so-called collector, of a continuous dry polymer fiber having a diameter of several hundreds of nm [4–6]. Due to the whipping movements, the nanofibers are randomly deposited in the form of a non-woven scaffold. Moreover, by simply decreasing the number of polymer chain entanglements in the processed solution, the morphology of the deposited material can be varied from regular nanofibers to microparticles [7]. Under these conditions the process is named electrospraying. Playing with the material parameters, the processing and atmospheric conditions [8] as well as the way the fibers are deposited on the collector, it is possible

<footnote><sup>a</sup> ICPEES, Institut de Chimie Et Procédé Pour L'Energie, L'Environnement Et La Santé, CNRS, UMR 7515, Université de Strasbourg, 25 Rue Becquerel, 67087 Strasbourg Cedex 2, France</footnote>

<footnote><sup>b</sup> ICS, Institut Charles Sadron, Université de Strasbourg, CNRS, UPR 22, F-67000 Strasbourg, France</footnote>

<footnote><sup>c</sup> Sorbonne Université, Institut de Biologie Paris-Seine (IBPS), CNRS UMR 8256, Inserm ERL U1164, Biological Adaptation and Ageing, 75005 Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: guy.schlatter@unistra.fr (G. Schlatter).</footnote>
to tune the final structure of the scaffold at the scale of the nanofiber up to the scale of the fibrous assembly [9]. In particular, 3D microstructured scaffolds can been obtained thanks to the fact that the electrospun jet is electrically charged and can thus be manipulated using the Coulomb force acting at the vicinity of the collector just before its landing [10,11]. For example, when electrospinning is carried out using a grounded conducting collector with protrusions regularly distributed on its surface, the deposited nanofiber forms a surface which is non-uniformly charged. Indeed, in such a process, the fiber portions deposited in direct contact with the top of the protrusions are immediately discharged whereas the suspended portions between the protrusions can retain the charges for long times: an electrostatic template is formed [12]. Depending on the geometry of the collector (shape and size of the protrusions, distance between the protrusions) [13,11] and the electric conductivity of the deposited nanofiber [14], the electrostatic template with regularly distributed attractive and repulsive fibrous areas is formed with varying intensity, leading to the electrostatic guidance of the nanofiber during its landing and finally to the fabrication of a microstructured scaffold. Moreover, it has been shown that simultaneous electrospinning and electrospraying allows the building of 3D microstructured composite scaffolds with chosen polymers for the fibers and the particles as a function of the targeted application [15–17]. For such process, the deposited electrospun fibers allow the formation of the repulsive areas whereas the electrosprayed particles, being influenced by the electrostatic template, are preferentially deposited on the attractive domains, i.e. over the top of the protrusions where the charges can be efficiently dissipated towards the ground. Beyond the fact that the electrosprayed particles can provide additional properties and functions for the final material, they enhance the electric contact between the fibers, improving the efficient formation of the electrostatic template during long time of production and enhancing thus the 3D structuration through larger thickness than what is achieved when electrospinning is carried out alone [15]. Moreover, although such structuration process is guided by the design of the patterned collector (i.e. shape, size and distance between the protuberances), it has been shown that a structuration can also be initiated with a flat collector. For example, when electrospinning of poly(lactic acid) (PLA) is simultaneously carried out with electrospraying of poly (ethylene oxide) (PEO), the deposited PLA nanofibers and PEO microparticles self-organize in the form of honeycomb-like structures [18]. The resulting scaffolds exhibit a three-dimensional structure with higher porosity and larger pore size than those obtained by simple electrospinning (i.e. without electrospraying).

Among the wide range of microstructured scaffolds that were proposed in the literature, those built from aligned nanofibers have numerous applications of interest in tissue engineering [19]. One can cite nerve guidance [20,21], tendon and ligament [22] repair as well as muscle and cardiac tissue engineering [23–30]. Two strategies can be used to obtain scaffolds with aligned electrospun nanofibers. The first one relies on the use of patterned collectors with parallel grooves [31,32]. In this case, the formed electrostatic template leads to the deposition of the nanofibers along a direction preferentially perpendicular to the grooves. However, the quality of the fiber alignment is in correlation with the capacity the suspended nanofibers can keep their charges after their deposition. For example, this strategy is not adapted to align nanofibers made from polysaccharide, polyanions, polycations or a polymer formulation containing a salt [14]. The second strategy relies upon the deposition onto a collector moving at a higher velocity than the whipping movements of the electrospun jet [21,33]. In general, a rotating drum is used to collect the nanofibers. A critical tangential velocity of the drum, in the range of 10 m.s⁻¹ to few tens of m.s⁻¹, is necessary to overpass the velocity of the landing nanofiber jet and align efficiently the fiber perpendicularly to the rotation axis as it has been demonstrated in the case of PLA nanofibers [34,35]. The advantage of this strategy is that the nanofibers can be aligned independently of their electrostatic properties (i.e. even for the nanofibers for which an electrostatic template cannot be induced). However, both electrostatic and mechanical alignment strategies lead to the fabrication of compact scaffolds with low porosity and small pore size due to the dense

fiber alignment. As a result, these compact scaffolds can only interact in 2D at the interface between their upper surface and cells: a whole 3D cell colonization is thus not possible [36]. Inspired from our previous results [18], the present work aims to answer this issue thanks to the elaboration of 3D microstructured scaffolds with both aligned electrospun nanofibers and large pores allowing improved 3D cell colonization. The strategy consists in the simultaneous electrospinning of nanofibers and electrospraying of microparticles on a cylindrical collector rotating at high velocity. The mechanical alignment of the electrospun nanofibers is thus expected simultaneously with the self-organization of the electrosprayed microparticles in the form of large aggregates inserted between the nanofibers enlarging the pore size of the resulting scaffold. Our goal being the fabrication of biomimetic scaffolds for cardiac tissue engineering, it is proposed to elaborate a composite scaffold made of poly(lactic acid) (PLA) and poly(glycerol sebacate) (PGS), a bioelastomer being extensively studied these recent years. Indeed, PGS shows complementary properties such as biocompatibility, biodegradability and mechanical properties (especially Young modulus and strain at breaks) making this elastomer very attractive for wound healing, controlled drug release and tissue engineering [37]. However, because PGS is an elastomer, the elaboration of PGS-based electrospun scaffolds necessitates an additional crosslinking step after the electrospinning of prepolymer of PGS (pPGS) in order to form an elastomeric 3D network. In a previous work, we demonstrated that the addition of PGS with PLA increases the hydrophilicity of the scaffold and improves its surface functionalization by Matrigel or Laminin, a commonly used cell culture matrix [38]. Furthermore, it was shown in this previous work that a scaffold of randomly oriented PLA/PGS nanofibers supports the efficient development of cardiomyocytes showing great potential for cardiac application.

Thus, the elaboration of biomimetic scaffolds with either PLA or PLA/PGS aligned nanofibers and PGS/hydroxypropyl-β-cyclodextrin (HPβCD) microparticles was targeted in the frame of the present work. The addition of HPβCD, a cyclic oligosaccharide, was chosen to stabilize the production of microparticles by electrospraying and because this compound is also able to encapsulate drug, which could be an interesting option for further developments [39]. In a first part, the whole structuration mechanism allowing the formation of 3D biomimetic scaffolds with aligned nanofibers and enlarged pores is deeply studied with the help of numerical simulation. In a second part, the 3D internal structure of a crosslinked PGS-based biomimetic scaffolds is thoroughly investigated thanks to scanning electron microscopy and X-ray microtomography [40]. The mechanical properties of the scaffolds are then studied along and perpendicular to the fiber alignment and discussed in relation to the mechanical behavior of cardiac muscle. Finally, cell colonization using either normal human ventricular cardiac fibroblasts or newborn rat primary cardiomyocytes is studied in Laminin functionalized 3D scaffolds made of aligned PLA nanofibers and PGS/HPβCD microparticles. These results are compared and discussed with the cell colonization of scaffolds made of aligned PLA nanofibers alone. It is worth noting that in the present article, in vivo assessment has been deliberately omitted. Indeed, this work addresses one of the major limitations of cardiac tissue engineering, i.e. scaffold’s porosity and pore size. This is why our work dealt with 3D structuring of the scaffold, with the ultimate aim of improving cell colonization. It is believed that validation at animal level will not provide any additional information at this stage. Therefore, in line with the principles of the 3Rs (Replacement, Reduction and Refinement), we have decided not to include animal validation in this work.

## 2. Materials and methods

### 2.1. Materials and solution preparation

Poly(glycerol sebacate) prepolymer (pPGS, degree of esterification = 85 %) was synthesized under a microwave procedure from a glycerol:sebacic acid (G/SA) molar ratio of 1/1 as described in our previous works [38,41].
Poly(lactic acid) (PLA, Ingeo 7000D, NatureWorks, M<sub>n</sub> = 124 kg/mol, M<sub>w</sub> = 227 kg/mol measured by size exclusion chromatography), dimethylformamide (DMF, > 99.8 %, Sigma-Aldrich), dichloromethane (DCM, > 99.8 %, Carlo Erba), hydroxypropyl-β-cyclodextrin (HPβCD, 97 %, Acros Organics) and absolute anhydrous ethanol (≥ 99.9 %, Carlo Erba) were used as received.

## 2.2. Electrospinning process

To align the fibers in one direction, a grounded collector (diameter = 13 cm, height = 3 cm) was used with a rotational speed of 3000 rpm using a dedicated motor (EUROSTAR 20 high speed digital, IKA). The speed of 3000 rpm led to a tangential speed of 20 m/s at the collector surface which was enough to align the nanofibers, while too high speed (4000 rpm i.e. tangential speed of 27 m/s) led to the breaking of some fibers. 18-gauge needles were fed by the electrospinning or electro-spraying solutions through silicone pipes by syringe pumps (Fisher Scientific) and connected to high-voltage power supplies (SL10, Spellman). All concentrations are given as weight percentages relative to the total weight of the solution.

PLA fibers were electrospun from solutions concentrated at 10 wt% in DCM/DMF 6/4 wt/wt injected at a flow rate of 1.5 mL/h and a voltage of 16 kV was applied with a needle-to-collector distance of 16 cm.

PLA/PGS fibers were electrospun from solutions concentrated at 7 wt % of PLA and 3 wt% of pPGS in DCM/DMF 6/4 wt/wt injected at a flow rate between 1.2 mL/h and a voltage of 15 kV was applied to the needle with a needle-to-collector distance of 16 cm.

pPGS/HPβCD electrosprayed solutions were concentrated at 15 wt% of pPGS and 15 wt% of HPβCD in DMF/ethanol 3/7 wt/wt. They were injected at 2 mL/h and 15 kV was applied to the needle with a needle-to-collector distance of 18 cm.

For the scaffolds embedding both fibers and particles, two needles were placed on either side of the continuously rotating collector, as already described in our previous work (see the scheme of Fig. 2d) [18]. The collector was covered by an aluminum foil. Samples were fabricated by continuous electrospinning of PLA fibers and electrospraying of pPGS/HPβCD particles for 60 min. The resulting scaffolds were then peeled from the aluminum foil and cured 2 days at 120 °C in order to crosslink the PGS.

Thinner samples were also prepared to compare the structure behavior between scaffolds elaborated either with PLA or PLA/PGS nanofibers. For that, samples were fabricated from three superimposed bilayers of electrospun fibers (PLA or PLA/PGS) deposited during 2 min on the cylindrical collector rotating at 3000 rpm followed by electro-sprayed pPGS/HPβCD particles deposited during 1 min at the same speed of rotation.

In order to characterize the way PLA and PLA/PGS electrospun fibers are able to store electric charges during their deposition, the surface potential was measured at the top surface of the deposited fibers during electrospinning alone. For that, a non-contacting electrostatic voltmeter ESVM (Trek Model 347-3-H-CE) connected to a computer for data acquisition was used. The measurement is based on a field-nulling technique for non-contacting voltage measurement achieving direct current stability and high accuracy, with no need for fixed probe-to-surface spacing. The technique allows an accurate measurement of the surface potential of stationary or moving surfaces. The measurement range is 0 to ± 3 kV with accuracy of 0.1 % of the full range. The probe of the voltmeter (end-view probe model 6000B-7C with a speed of response of 4.5 ms for 1 kV step) having a disc surface of 11.2 mm diameter was placed 2 mm from the surface of the collector at the opposite side of the electrospinning needle. An acquisition rate of 30 measurements/s was chosen. With this set-up, it was possible to follow the accumulation of charges, resulting in voltage, during electrospinning as already shown in a previous article [42]. PLA and PLA/PGS fibers were electrospun during 10 min with this set-up.

## 2.3. Morphological and mechanical characterization

The scaffolds were observed by scanning electron microscopy (SEM) after coating by a thin layer of gold. The SEM (Vega 3, Tescan) was used in high vacuum mode with a voltage of 5 kV. The mean diameter of the fibers was calculated over 100 diameters measurements using ImageJ software. The mean values are given with the standard deviation. The degree of alignment of PLA and PLA/PGS fibers produced by high speed rotation of the collector was also evaluated using ImageJ. The angle formed by the fibers and the horizontal border of the picture was compared with the mean angle value. SEM observations were also done to show the internal organization of the structured scaffolds. For this, the scaffolds were immersed in liquid nitrogen and then directly cut with a razor blade. The cut section was then observed.

Uniaxial tensile tests were conducted with the Linear Tension Geometry of a rheometer (Discovery HR3, TA Instruments) on samples made of aligned fibers with or without particles. The tests were done on the longitudinal and transversal direction, before and after crosslinking. Rectangular samples were cut with 5 mm width and about 20 mm long. The samples were fixed in the clamps with an initial gap of 10 mm and stretched at 100 µm/s until breaking. 5 measurements were performed for each type of samples. Young's modulus, ultimate tensile stress (UTS) and maximal strain were calculated from the stress-strain curves. The mean values are given with their standard deviation.

The internal structure of a membrane composed of aligned fibers and particles was investigated by X-ray microtomography (EasyTom 150/ 160, RXSolutions). A rectangular sample of approximatively 3x20x0.2 mm³ was fixed on the holder. Tomographical reconstruction was carried out with the software myVGL by a filtered back projection. Beam power and energy ranged from 4 W and 70 kV for lowest resolution (10 µm) to 0.8 W and 50 kV for highest resolution (0.5 µm). The images were reconstructed after one rotation, from 2016 projections at 6 fps for the lowest resolution and 1600 projections at 0.6 fps for the higher resolution. A 3D reconstruction of the sample was analyzed through slices perpendicular to the tangential velocity (axis Z, see Fig. 6). The parallel slices have all equal thickness set to 50 µm (a value corresponding to 100 times the voxel size of the µ-CT reconstruction) to provide data with sufficient statistics. The local orientation of the elongated nanofibers has been inferred by application of grayscale-to-grayscale filters to increase the visibility of the entities based upon their X-ray attenuation. By enhancing the object edges the objects can be isolated, labelled, and their boundaries can be located with a good accuracy essential for a reliable structural analysis. However, the thresholding methods through the exploitation of voxel intensity histogram shape information, is rendered difficult when spheroidal particles stick to the elongated nanofibers as the topological operators based on object connectivity are lured. The segmentation process that should partition the volume into groups of voxels of each phase of interest (the microparticles and the nanofibers respectively) may become ineffective, and may give the impression that the local fiber segments have a different orientation. This is likely the explanation for the observed spreading out effect over the θ-range 45°-90°. A lower bound for the contribution of those spheroidal microparticles has been estimated to ~0.1 % in the frequency level. As a result, the orientational order parameter *S* is certainly under-evaluated by ~0.025.

## 2.4. Numerical simulations

Numerical simulations using Comsol® software were carried out in order to compute the electric field and, using the particle tracing module, to model the trajectories of electrosprayed microparticles before their landing on the scaffold during its fabrication by electrospinning-electrospraying.
## 2.5. Biological characterization

### 2.5.1. Cell culture

Normal Human ventricular cardiac fibroblasts (NHVCF, #CC-2904, Lonza, Basel, Switzerland) were cultured in Cardiac Fibroblast Growth Medium-3 (FGM-3, #CC-4526, Lonza, Basel, Switzerland) at 37 °C and 5 % CO₂. Before confluence, cells were detached using 0.1 % trypsin-EDTA (Thermo Fisher Scientific, Saint-Herblain, France). The cells were rinsed and suspended in FGM-3 before use for subsequent experiments. Newborn rat primary cardiomyocytes were obtained from 1-day-old Wistar RjHan rats (Janvier Labs, Saint-Berthevin, France). Hearts were cut in small pieces using scissors and cardiac cells were isolated using the neonatal heart dissociation kit (Miltenyi Biotec, Paris, France) according to the manufacturer's instructions. Using this kit, cardiomyocytes were purified by depletion of non-cardiomyocytes (such as fibroblasts, endothelial cells, blood cells). Before seeding the cells on the scaffolds, they were sterilized by UV at a wavelength of 254 nm and an energy of 200 mJ for 10 min. Subsequently, to increase the cellular attachment, they were functionalized through preincubation with 0.7 µg Laminin (L2020, Sigma-Aldrich, France) diluted in DMEM/F12 (Thermo Fisher Scientific, Saint-Herblain, France) overnight at 37 °C and then washed twice with phosphate-buffered saline (PBS) followed by a rinse with culture medium. 1x10⁶ cells were seeded on a scaffold fixed in a CellCrown™ (Scaffdex, Tampere, Finland) placed inside a 48-well plate and cultured with FGM-3 for normal human ventricular cardiac fibroblasts and with DMEM medium (with 4.5 g/L D-Glucose and without Na Pyruvate) supplemented with L-glutamine, penicillin-streptomycin, 10 % horse serum and 5 % foetal bovine serum for newborn rat primary

cardiomyocytes for 3 days.

### 2.5.2. Immunostaining

After 3 days of culture, scaffolds were washed with PBS and fixed with 1 % paraformaldehyde solution. After 2 washing steps, they were incubated with 5 % bovine serum albumin (BSA) during 1 h. Then Alexa Fluor-488 Phalloidin (Life technologies) was used to stain the cells. After 3 washes in PBS, scaffolds were mounted with mowiol. All images were captured using a motorized confocal laser scanning microscope (TCS SP5, Leica Microsystems, Nanterre, France). y-projection images in xz dimensions were processed using ImageJ software to calculate cell colonization.

### 2.5.3. Statistics

Analyses were conducted using GraphPad Prism 8.00 (GraphPad Software Inc., SD, USA). For one-way analysis of variance, the nonparametric Mann-Whitney test was used. A *p*-value < 0.05 was considered significant. Values are given as the means ± standard error of the mean.

## 3. Results

### 3.1. Initiation of the building of 3D scaffolds with aligned nanofibers and enlarged pores

When either PLA or PLA/PGS nanofibers are electrospun alone (i.e. without electrospraying) on a cylindrical collector rotating at 3000 rpm leading to a tangential speed of 20 m/s, a compact fibrous structure of

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Aligned fibers obtained from electrospinning alone. a) PLA fibers after electrospinning. b) PLA fibers after the curing step. c) PLA/PGS fibers after electrospinning. d) PLA/PGS fibers after the curing step.</figcaption></figure>
aligned fibers is obtained (Fig. 1a and b). This compact structure is even more pronounced after the curing of these scaffolds (Fig. 1c and d) and especially in the case of PLA/PGS due to the melting of PGS before its complete crosslinking. In the case of PLA/PGS fibers, a mean diameter of 516 ± 315 nm was measured whereas a value of 454 ± 125 nm was achieved for PLA fibers. PLA/PGS fibers are thicker and their diameter distribution is broader than pristine PLA ones probably because the electrospun PLA/PGS solution is less homogeneous with a broader overall molar mass distribution due to the presence of both pPGS oligomer and PLA. Moreover, although the alignment quality is favored by the high speed of the collector, alignment defects are observed and especially in greater numbers for PLA/PGS than for PLA fibers. Indeed, a better degree of fiber alignment was measured in the case of PLA for which 74 % of the fiber axis form an angle < 15° with the perpendicular direction to the collector rotation axis than in the case of PLA/PGS fibers for which only 54 % of the fibers form an angle < 15° with the expected theoretical direction of alignment. A rotation speed of 4000 rpm (i.e. tangential speed of 27 m/s at the collector surface) instead of 3000 rpm was tested to improve the fiber alignment, however, for such a speed, fiber breakage was observed.

Since scaffolds composed of aligned fibers are compact, it is proposed

to intercalate particles into the fibrous structure in order to increase the inter-fiber distance and thus the overall pore size. To study how electrosprayed microparticles are intercalated, samples made of 3 superimposed bilayers, each corresponding to a layer of aligned fibers (PLA or PLA/PGS) coated by a layer of electrosprayed pPGS/HPβCD particles, were fabricated (Fig. 2). Better fiber alignment is measured for fibers embedding particles: 85 % of the fibers form an angle < 15° in the case of PLA/PGS fibers whereas highly aligned fibers were obtained in the case of PLA fibers for which 92 % of the fibers form an angle < 5° with the expected theoretical direction of alignment. Beyond the fiber alignment itself, a tremendous difference is observed between the samples prepared from PLA/PGS fibers and those from PLA ones. Indeed, electrosprayed particles are homogeneously deposited on PLA/PGS fibers (Fig. 2a) whereas particle aggregation is observed when electrospraying is carried out on a layer of PLA fibers (Fig. 2b). In this latter case, after the deposition of the first bilayer composed of PLA electrospun fibers and pPGS/HPβCD electrosprayed particles, an electrostatic template with repulsive and attractive domains is formed. Indeed, the portions of fibers being in direct contact with particles can efficiently dissipate their electric charges whereas the fibers strands hanging between the particles are able to store residual charges [15].

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Top surface of samples elaborated from 3 superimposed bilayers, each corresponding to a layer of nanofibers (PLA or PLA/PGS) deposited during 2 min followed by 1 min of electrospraying pPGS/HPβCD microparticles. a) Scaffold with PLA/PGS nanofibers. b) Scaffold with PLA nanofibers. c) Surface potential measured during 10 min of electrospinning alone (i.e. without electrospraying) of PLA (green upper curve) and PLA/PGS (red lower curve). d) Scheme of the electrospraying set-up. e) Scheme of the deposited fibers and particles in the case of conducting fibers (e.g. PLA/PGS). f) Scheme of the deposited fibers and particles in the case of insulating fibers (e.g. PLA).</figcaption></figure>
Thus, an electrostatic template is formed with charged areas corresponding to the suspended fiber portions and discharged areas located above the particles. Then, during the deposition of the second bilayer, the charged electrosprayed particles, being subjected to the electrostatic template, are preferentially deposited on the previously deposited ones because of attractive electric forces. The deposition of electrospun PLA fibers in the second bilayer allows to maintain the electrostatic template ensuring thus a self-organization of PLA fibers and pPGS/HPβCD particles (Fig. 2b and f). The development of the 3D fibrous structure will be deeply explained in the next section with the help of numerical simulations. Furthermore, PLA fibers are deposited with almost no alignment defect (92 % of the fibers forming an angle < 5°) as compared to the samples prepared without electrosprayed particles (74 % of the fiber forming an angle < 15°). These overall differences in the scaffold structuration may be explained by the fact that, unlike PLA, the deposited PLA/PGS layer is not able to generate an electrostatic template leading to the self-organization of electrosprayed particles (Fig. 2e). Since the formation of an electrostatic template is only possible when electrospun fibers are able to store efficiently the electric charges [14], this property was compared between PLA and PLA/PGS fibers. For this purpose, the surface potential generated over deposited fibers was measured during electrospinning alone (i.e. without electrospraying) of either PLA or PLA/PGS (Fig. 2c). A linear increase of the surface potential of 570 ± 110 mV s⁻¹ was measured in the case of electrospun PLA fibers whereas a lower value of only 45 ± 15 mV s⁻¹ was measured for PLA/PGS. Such a difference can be explained by the fact that the addition of pPGS in the solution of electrospinning introduces a high number of hydroxyl and carboxylic acid functions which enhance the electric conductivity of the fibers leading to rapid charge dissipation that does not allow the formation of an electrostatic template (Fig. 2e).

## 3.2. Morphological characterization and mechanisms of the 3D structuration

As the 3D morphology (i.e. fiber alignment, pore size distribution) is of first importance for cardiac tissue engineering, a deep morphological investigation using SEM and 3D X-ray microtomography has been carried out in the case of scaffolds obtained after one hour of simultaneous PLA electrospinning and pPGS/HPβCD electrospraying on a cylindrical collector rotating at 3000 rpm (i.e. tangential speed of 20 m.s⁻¹, Fig. 2d for the scheme of the set-up). A thick membrane is fabricated with a structuration visible to the naked eyes, as shown on Fig. 3. SEM observations show that the structuration on the top of the scaffold (i.e. fibers and particles deposited at the end of the fabrication process) is due to clusters of particles (encircled in red on Fig. 4a and 4d) interconnected by highly-aligned fibers confirming that fibers and particles are able to self-organize (Fig. 4c) during the whole process in spite of the high rotation speed of the collector and the long production time.

After the curing step allowing the crosslinking of PGS inside the particles, the same organization is still visible (Fig. 4d and e). Curing

also causes the particles to fuse together (Fig. 4f) resulting in a more cohesive scaffold that is much easier to handle. Thanks to that, it was possible to cut it in liquid nitrogen in order to observe them in the thickness. These observations allowed to confirm the low density and the high thickness of such samples. Indeed, they are about 300 µm thick as spun compared to only about 150 µm for aligned PLA fibers produced during the same time without particles. Furthermore, Fig. 5 shows that the scaffold is composed of large pores surrounded by aligned fibers mimicking thus the structure found in the heart tissue.

To get more insight into the 3D internal structure of a cured scaffold composed of aligned PLA fibers and PGS/HPβCD particles, a deep investigation using X-ray tomography [43] was carried out. Despite the similar atomic composition of fibers and particles, the contrast was enough to distinguish the domains made of particles and those of fibers (Fig. 6). This may be explained by the high density of oxygen atoms in cyclodextrins (about one over three) compared to PLA (one over four). It is shown (Fig. 6a) that fibers and particles deposited at the end of the process (i.e. at the vicinity of the scaffold’s top surface) form larger pores (surrounded by red dashed lines) than at the first time of the fabrication (i.e. near the bottom of the scaffold having been in contact with the collector surface, Fig. 6b). Along transversal xy (Fig. 6c) and a longitudinal xz (Fig. 6d) planes, domains with few particles (dark areas) surrounded by clusters of particles (surrounded by yellow dashed lines) can be observed. As shown in Fig. 6f and the video (Movie-Fig-6e-f) in supplementary files and as represented in the scheme of Fig. 6, the scaffold shows a hierarchical 3D structure with highly aligned PLA fibers separated by clusters of particles and with large pores with a lateral size increasing from few tens of microns to few hundreds of microns along the thickness of the scaffold (x direction, i.e. the pores are larger on Fig. 6a than on Fig. 6b).

As illustrated in the three-dimensional rendering view of the specimen (Fig. 7a), the nanofibers appear to be aligned along the Z axis as defined in Fig. 6. This qualitative observation can be supplemented through a statistical analysis of their orientational alignment. The alignment of the nanofibers along the Z axis is quantified using the 3D Herman’s orientational order parameter [44] given by:

$$S = \left\langle \frac{3\cos^2\theta - 1}{2} \right\rangle \qquad (1)$$

where the mean-square cosine is calculated by integrating all the configurations over the azimuthal angle θ with respect to the axis Z. For perfect vertical orientation S = 1, while for isotropic orientation S = 0. The orientational order parameter of the entire specimen amounts to S = 0.621 ± 0.025 suggesting a very good alignment of the nanofibers along the Z axis. The local change in nanofibers direction can be quantified by an analysis of the orientation θ distribution through each slab position along Z (Fig. 7c-e). All the histograms appear with the same features, namely a pronounced peak located around θ ~ 7° surrounded by two valleys. The fact that the presence of the maximum is located around θ ~ 7° rather than 0°, may be explained by the interactions

<figure><img src="image_5.png" /><figcaption>Fig. 3. A) photography of a scaffold made of aligned PLA fibers and pPGS/HPβCD particles showing the roughness at the scaffold surface induced by the self-organization of particles and fibers. b) Low mag SEM picture of the same 3D scaffold.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 4. SEM pictures of 3D scaffold with aligned nanofibers and enlarged pores before and after thermal curing. The domains highlighted in red correspond to islands of particle aggregates.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 5. Cross-sectional view of a scaffold made of aligned PLA fibers and PGS/HPβCD particles, with a zoom on two pores.</figcaption></figure>

between charged elongated objects that moreover have a local rigidity (persistence length). Indeed, not only electrostatic interactions are repulsive, but orientational depletion combine an attractive interaction of short-range [45] and also a structural steric repulsion due to the local rigidity of the elongated objects [46]. As a result, the nanofibers would well distribute in a cone of half vertical angle of a few degrees. Thus, the orientational order parameter value $S = 0.621 \pm 0.025$, being equivalent to what is observed for both thermotropic and lyotropic liquid crystals in their nematic phase [47], reflects a very good alignment with

nanofibers which can be considered as semi-flexible chains that meander back and forth along the Z axis.

In order to show how particles are deposited during their electro-spraying and to explain the increase of the pore size through the scaffold's thickness, numerical simulations have been carried out. For this purpose, electrospraying of particles is modeled after a certain time of processing during which a layer of aligned fibers and aggregated particles have been already deposited. The first step of the simulation consists in the computing of the electric field in the vicinity over the
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** X-ray microtomography of a biomimetic 3D scaffold after thermal treatment with aligned PLA nanofibers and enlarged pores and its schematic 3D view (top right) showing clusters of pPGS/HPβCD microparticles and aligned nanofibers. a) A slice parallel to the surface of the sample (yz plane) close to the top surface of the scaffold (resolution: 2 µm). b) A (yz) slice close to the collector surface (resolution: 2 µm). c) Transversal (xy) view (resolution: 0.5 µm). d) Longitudinal (xz) view (resolution: 0.5 µm). e) Current transversal (xy) view (resolution: 0.5 µm). f) 3D view of the scaffold and the highlighted (xy) plane shown in figure e) (resolution: 0.5 µm). A movie of this 3D view can be uploaded as supplementary files.</figcaption></figure>

layer of the already deposited fibers and particles. Two cases have been considered: for the first one, the size of the pores (i.e. the distance between the aggregates of particles) is equal to 75–100 µm (case of Fig. 8a); for the second one, the presence of a larger pore corresponding to the merging of two smaller ones is considered (case of Fig. 8d). The pore sizes taken for these simulations have been chosen in regards with SEM pictures taken from the first moments of the fibers and particles deposition which is equivalent to the situation of SEM picture shown in Fig. 1b. For both cases, it is assumed that at a distance $h = 300$ µm over the deposited layer of fibers and particles, the electric field is homogeneous and thus, the potential $V_{at h=300 \mu m}$ can be calculated from the distance $d_{nc}$ and the difference of potential $V_{nc}$ imposed between the needle and the collector (respectively $d_{nc} = 16$ cm and $V_{nc} = 15$ kV, $V_{at h=300 \mu m} = \frac{V_{nc}}{d_{nc}}h = 28.1$ V). Furthermore, it is assumed that the aggregates of particles are directly connected to the collector and thus, an imposed potential of 0 V is considered on these areas. And finally, as the portions of aligned fibers are suspended between the aggregated particles (i.e. these portions of fibers are not in direct contact with the collector), a surface charge density $\sigma$ is considered on the top of these domains. Since the length of the suspended portions of fibers is only in the order of several tens of µm, they are not able to store all the charges

responsible to the total jet current $I$. [14] Thus, only a fraction $\alpha$ of the initial linear charge density $\lambda = I/V$ is kept along the suspended fiber portions (where $V$ is the jet velocity). Considering that the distance between two neighboring parallel fibers is $d$, a value of $\sigma = \alpha\lambda/d$ should be considered over the suspended PLA fibers. However, the estimation of $\alpha$ at this level is not possible. Thus, considering the data from previous studies [14,15], a surface charge density of $\sigma = 2.10^{-6}$ C/m$^2$ is a good order of magnitude and has been chosen in the present numerical simulation. The electric potential and the streamlines are shown on Fig. 8a for the case of the small pores and on Fig. 8d for the case with the larger pore. It is shown that repulsive areas are located over the suspended fibers and that the streamlines converge to the aggregates of particles. Moreover, a highly positive potential is obtained in the case of the presence of the large pore (Fig. 8d) showing that the larger the pore, the larger the repulsive potential. In a second step, starting from these two situations of electric field, the trajectory during landing of freshly electrosprayed particles has been modeled. The trajectory of a particle of mass $m$, diameter $d_p$ and charge $q$ subjected to an external electric field $E$ is governed by the following momentum equation:
<figure><img src="image_9.png" /><figcaption>Fig. 7. Nanofibers as seen by X-ray microtomography in a 3D rendering view (top left) of a specimen sub-volume at high resolution (voxel size: 0.5 µm); the plane (Y, Z) at X=0 defines the interface with the collector onto which the fibers are deposited. The X-ray absorbing material is represented on a grey scale in which white and black colors correspond to highly and least absorbing materials, respectively (the air appears in uniform black). From the orientation distributions of the nanofibers (lower plots, where θ is the angle between the director of a nanofiber element and the axis Z) through slabs of equal 50 µm thickness across the specimen depth, orientational order parameters S = (3cos²θ-1/2) are inferred as a function of Z (top right). The uncertainty is discussed in the main text.</figcaption></figure>

$$m \frac{\partial \mathbf{v}}{\partial t} = \mathbf{F}_E + \mathbf{F}_D + \mathbf{F}_q + \mathbf{F}_g \qquad (2)$$

where **v** is the velocity of the particle, **F**<sub>E</sub> = q**E** is the electrostatic force, **F**<sub>D</sub> is the drag force, **F**<sub>g</sub> = mg is the gravitational force and **F**<sub>q</sub> is the particle-particle electrostatic interaction force induced by the surrounding cloud of charged particles. For all simulations, only **F**<sub>E</sub> and **F**<sub>D</sub> were taken into account since the weight of micron size particles is negligible and it was assumed that particle-particle interactions are negligible compared to **F**<sub>E</sub>. The drag force is computed thanks to the following equation:

$$\mathbf{F}_D = -0.5C_D\pi(d_p/2)^2 \rho_{air} \|\mathbf{v}\| \qquad (3)$$

where ρ<sub>air</sub> is the density of air which is considered static and C<sub>D</sub> is the drag coefficient. Considering that particle velocity is in the range of 5 to 40 m.s<sup>-1</sup> [48,49] and that the particle diameter is ranging between 1 and 3 µm (see Fig. 4), the drag coefficient can thus be estimated [50] in the range of 0.5 < C<sub>D</sub> < 5. We therefore, use these two extreme values of C<sub>D</sub> in order to model the effect of air friction on the particle trajectory. Considering the electric field E calculated in the first step of both cases of Fig. 8a and d, we used the particle tracing module of Comsol© in order to compute the trajectory of electrosprayed particles. Fig. 8b and 8c show the particle trajectories over small pores in the case of C<sub>D</sub> = 0.5 and C<sub>D</sub> = 5 respectively (see also the movie in the supp. info for C<sub>D</sub> = 5). Even

for lower friction condition (i.e. for the lowest C<sub>D</sub> value), electrosprayed particles are selectively deposited on the attractive domains made of the particle aggregates (Fig. 8b). However, for the highest C<sub>D</sub> value, the particles are slowed down more effectively, which makes it possible to enhance the selectivity of deposition on the electrostatically attractive areas (Fig. 8c). Due to height fluctuations, electrospun fibers may jump over an aggregate of particles resulting in the incremental increase in pore size, as shown in Fig. 8d. As a consequence, this newly formed pore induces a local increase of the repulsive potential modifying the trajectory of electrosprayed particles which are thus selectively deposited in a very efficient manner, with an effect that is all the more pronounced the higher the C<sub>D</sub> value (Fig. 8e, f and the movie in the supp. info). Thus, when the deposition is made on a drum rotating at high velocity, the self-organization mechanism forming islands of aggregated particles embedded in a loosely packed network of aligned fibers is self-sustained, and even enhanced, during the process of electrospinning and electrospraying. A three-dimensional scaffold with aligned nanofibers and enlarged pores with a gradient in pore size is consequently obtained.

### 3.3. Mechanical behavior of scaffolds

Mechanical properties of scaffolds made of aligned fibers with and without particles were evaluated before and after the curing step in the direction of the fibers and in the perpendicular direction.
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Numerical simulation by the finite elements method using Comsol® software for two kinds of 3D structures before (a, b and c) and after (d, e and f) the formation of a larger pore size of 195 µm due to the jump of nanofibers over two smaller pores of respectively 100 µm and 75 µm. The planes of symmetry are represented by the thick dotted black lines. A homogeneous potential of 28.1 V is imposed at the upper horizontal dotted green line. Field of electric potential in color and streamlines in black (a, d). Trajectories of electrosprayed microparticles in colors and streamlines in black for a drag coefficient $C_D=5$ (b, e). Trajectories of electrosprayed microparticles in colors and streamlines in black for a drag coefficient $C_D=0.5$ (c, f). Movies showing microparticles flying over the electrostatic template for the conditions of figures b and e are available in the [supplementary files](#).</figcaption></figure>

### 3.3.1. Mechanical behavior of scaffolds made of aligned fibers alone

In the case of scaffolds made of fibers alone (i.e. without electro-sprayed microparticles), results are given for both directions in Fig. 9. In the longitudinal direction, there is very little contribution of interaction between the fibers to the measured stress. In this case, the real mechanical properties of the material (PLA or PLA/PGS) are mainly measured. In the transversal direction, the stress is mainly due to the interactions (adhesion, friction) between the fibers.

In the longitudinal direction, the first observation that can be done is the high values of modulus and ultimate tensile stress (UTS). They can be compared to the values found for randomly deposited fibers as already published in our previous work [38]: As spun, the Young's modulus and UTS are one order of magnitude higher when the fibers are aligned. For example, for aligned PLA fibers, the Young's modulus is 43 MPa versus 4 MPa for randomly deposited PLA fibers. The alignment offers a high mechanical strength in the direction of the fibers. As explained above, in the anisotropic case, almost no friction at contact points contributes to the initial modulus. It corresponds exclusively to the deformation of the material the fibers are made of. On the other hand, in the case of randomly deposited fibers, it is mainly the reorientation of the fibers in the tensile direction and the interactions between the fibers that contribute to the initial modulus. From the typical stress-strain curves given in Fig. 9A, it should be noticed that the rupture is rarely abrupt: the fracture spreads progressively while the fibers break successively. It is particularly visible for as-spun PLA fibers. PLA/PGS fibers, especially after curing, form more cohesive membranes that break more abruptly. After curing, Young's modulus and UTS increase, as observed for randomly deposited fibers. This can be explained by changes in the material, namely crystallization of PLA and cross-linking of PGS [38]. The presence of the soft PGS in the fibers decreases the rigidity of the membranes: The Young's modulus and UTS are lower for PLA/PGS fibers. This difference is particularly clear after cross-linking. The same tendency was not observed for randomly deposited PLA and PLA/PGS fibers [38] where the contribution of the interaction

between fibers was more important. PGS increases the cohesion between fibers, as already explained, which increases the strength of the fibrous material. But it has no effect for aligned fibers stretched in their longitudinal direction.

The force measured in the transversal direction was measurable but very low; the data comprise thus a lot of noise. It corresponds to interaction forces (adhesion and friction) rather than to the deformation of the polymer itself. Under the action of friction forces, fibers are dragged during stretching of the membrane, which allows high strains before the break point, where no fibers are in contact anymore. As in the longitudinal direction, the Young's modulus and the UTS increase after curing. The strain at break tends to decrease. Here, crystallization of PLA and crosslinking of PGS should play a very minor role. These results are however easy to justify for PLA/PGS fibers because of the merging of the fibers during curing (Fig. 1d). The same evolution of the properties in the pure PLA case indicates that some strong interactions appear also between PLA fibers during curing. In the transversal direction, the mechanical behavior is not very dependent on the presence of PGS. The only noticeable difference relies on the strain at break for as spun fibers, which is much higher for PLA fibers. Indeed, there are no strong interactions at contact points in as spun PLA membranes. Thus, the fibers interact together only by weak friction. Finally, the sample can be largely deformed before the ultimate tensile stress is reached.

### 3.3.2. Mechanical behavior of composite scaffolds made of aligned fibers and particles

As for aligned fibers without particles, mechanical properties of such materials are very different in the longitudinal and in the transversal direction: both were tested before and after curing, and the results are given in Fig. 10. The Young's moduli and UTS are much lower in the transversal direction, while high ultimate strains can be reached. Again, this is due to the low contribution of interactions between fibers in the longitudinal direction, whereas there are dominant in the transversal direction. The curing has also the same effect than for the case without
<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Mechanical properties of aligned PLA and PLA/PGS fibers. (A) Typical stress–strain curves in the longitudinal and transversal directions before and after curing. (B) Results after tensile tests in the longitudinal and transversal directions before and after curing. The error bars correspond to the standard deviation.</figcaption></figure>

particles: it tends to increase the Young's modulus and the UTS in the direction of the fibers because of the crystallization of PLA. Moreover, the same increase is observed in the perpendicular direction because of the crosslinking of PGS located in the microparticle clusters, which improve the cohesion of the membrane.

The Young's modulus and UTS are globally lower for the samples with particles than for aligned PLA fibers alone (see Table 1 for comparison purposes). This tendency is stronger after curing. This could be explained

by the lower density of samples with particles. Indeed, the same total fiber's length should be deposited in both cases during the electrospinning-electrospraying process because the final production time was equivalent, but the thickness is much higher when particles are included among the fibers (300 µm versus 150 µm without particles). After curing, this difference is more pronounced because while PLA fibers collapse under the effect of the temperature, the structure is well conserved when particles are present. Since aligned fibers are the

<figure><img src="image_12.png" /><figcaption>**Fig. 10.** Typical stress–strain curves in the longitudinal and transversal directions of aligned PLA fibers structured with pPGS/HPβCD particles.</figcaption></figure>
components responsible for the longitudinal mechanical strength of scaffolds, the ones made of aligned fibers and particles being less dense in terms of fibers than those made of aligned fibers alone. Thus, the apparent stress, which takes into account the intrinsic behavior of the fibers and the voids between them, is necessarily lower for the scaffolds embedding particles. However, this observation is not sufficient to explain the discrepancy after curing. Indeed, the presence of the particles increases the interaction between fibers. Friction forces located in the particles areas contribute to the stress. Moreover, the particles clusters provide a better cohesion of the material, especially in the transversal direction, that allows higher strains at break. This is mostly true after crosslinking of PGS.

Finally, the inclusion of particles among the aligned PLA fibers has an effect on the mechanical properties of the scaffolds. A strong anisotropy is still observed, but higher strains can be reached for lower moduli, an interesting characteristic for soft tissue engineering: after curing, the modulus in the longitudinal direction is about 70 MPa and the strain at break 30 % versus about 100 kPa and 150 % in the transversal direction. The fabrication of scaffolds made of aggregated microparticles embedded among aligned fibers led to a mechanical behavior mimicking that of cardiac tissue. Moreover, the formulation of the particles was chosen to combine the advantageous properties of PGS and the possibility of encapsulation offered by the cyclodextrin, making these scaffolds promising for cardiac tissue engineering applications.

### 3.4. Cardiac cell colonization

The proposed scaffolds show open, interconnected porous networks with aligned fibers being potentially suitable for cardiac cell colonization. To assess the cytocompatibility (i.e. cell adhesion), anisotropy as well as cell colonization capacity, normal human ventricular cardiac fibroblasts or newborn rat primary cardiomyocytes were seeded on Laminin-coated scaffolds made of aligned PLA fibers alone (namely PLA scaffolds) and those made of aligned PLA fibers and PGS/HPβCD particles (namely PLA:PGS/HPβCD scaffolds). Confocal microscopy over the all thickness of scaffolds was performed using phalloidin labelling, that stains actin filaments, to evaluate cell adhesion, growth and cell penetration after 3 days of culture on these mats. As clearly visible in Fig. 11 (z-projection images in xy dimensions, upper panel), cells were able to adhere on the PLA or PLA:PGS/HPβCD mats and perfectly aligned along the axis of the electrospun fibers. Regarding cell penetration, the y-projection images in xz dimensions showed a 2-fold superior penetration with PLA:PGS/HPβCD compared to PLA mats (20.12 ± 1.74 vs. 42.19 ± 1.26, *p* < 0.0001) for normal human ventricular cardiac fibroblasts and 3-fold superior penetration with PLA:PGS/HPβCD compared to PLA mats (18.41 ± 0.59 vs. 64.26 ± 0.57, *p* < 0.0001) for newborn rat primary cardiomyocytes. These preliminary results clearly demonstrate the

<caption>**Table 1**</caption>

Mechanical properties in the longitudinal direction of scaffolds made of aligned fibers alone and of particles and aligned fibers.

<table><thead><tr><th></th><th></th><th>Thickness (μm)</th><th>E (MPa)</th><th>UTS (MPa)</th><th>ε<sub>break</sub> (%)</th></tr></thead><tbody><tr><td rowspan="2">Aligned PLA fibers</td><td>as spun</td><td>118 ± 26</td><td>44 ± 20</td><td>4 ± 1</td><td>31 ± 4</td></tr><tr><td>cured</td><td>50 ± 11</td><td>746 ± 194</td><td>19 ± 5</td><td>35 ± 9</td></tr><tr><td rowspan="2">Aligned PLA/PGS fibers</td><td>as spun</td><td>43 ± 17</td><td>72 ± 20</td><td>2 ± 1</td><td>6 ± 1</td></tr><tr><td>cured</td><td>28 ± 17</td><td>493 ± 169</td><td>7 ± 3</td><td>2 ± 1</td></tr><tr><td rowspan="2">Aligned PLA fibers + PGS/CD particles</td><td>as spun</td><td>240 ± 18</td><td>29 ± 9</td><td>3 ± 0.4</td><td>25 ± 5</td></tr><tr><td>cured</td><td>254 ± 75</td><td>69 ± 27</td><td>3 ± 1</td><td>34 ± 6</td></tr></tbody></table>

efficacy of the 3D scaffolds made of loosely packed PLA fibers regarding cardiac cell colonization.

### 4. Discussion

This study opens very interesting perspectives, particularly in the field of tissue engineering and cardiac repair. The modalities of fabrication of scaffolds for such application are very diverse as well as the used materials. Here, we propose a combination of techniques (electrospinning and electrospraying) together with the use of PLA/PGS to promote the fabrication of a highly porous fibrous scaffold with aligned nanofibers. It was demonstrated that anisotropy enhanced the function of cardiomyocytes [51]. Most interestingly, the different cardiac cells that populate the heart display similar behavior when cultured on aligned synthetic scaffolds made of PCL/gelatin [52], a finding that has been also demonstrated in our study using different materials. However, the effect of porosity was less systematically addressed, especially using identical materials. Here, we demonstrated that increasing porosity does not seem to modify substantially cell adhesion, orientation and survival even if it increases drastically the infiltration of these cells inside the scaffold. Thus, our strategy can clearly support a better cellularization of electrospun aligned scaffolds which is the main limit in the field as it was discussed, for example, in a recent article [53,54] dealing with 3D scaffolds mimicking the heart's helical structure and obtained using focused rotary jet spinning. Our approach seems to be a credible alternative to fiber-infused gel scaffolds that have been developed to address cell penetration [55].

The ability to promote efficient cell infiltration inside a scaffold is a major hurdle in tissue engineering, particularly for applications in cardiac tissue. Through our approach, we have successfully designed and

<figure><img src="image_13.png" /><figcaption>**Fig. 11.** Cell colonization of scaffolds with only aligned PLA fibers (left) and 3D biomimetic scaffolds with aligned PLA fibers and PGS/HPβCD microparticles (right) with either normal human ventricular cardiac fibroblasts (A) or newborn rat primary cardiomyocytes (B). After 3 days of culture, cells were stained with alexa-488-phalloidin and images were taken using a confocal microscope. Upper panels show the results of z-projection images in xy dimensions and lower panels show the results of y-projection images in xz dimensions. Scale bars: Horizontal = 100 µm; Vertical = 50 µm.</figcaption></figure>
manufactured a scaffold that mimics the four fundamental characteristics of the native extracellular matrix, i.e. stiffness, elasticity, anisotropy, and porosity. These properties are essential for guiding cellular behavior, ensuring structural integrity and maintaining tissue-specific functions. Stiffness and elasticity are critical for replicating the biomechanical environment of cardiac tissues, while anisotropy, reflecting the directional organization of fibers, helps replicate the alignment of cells found in native cardiac muscle. The high porosity of the scaffold further facilitates nutrient exchange, vascularization and the migration of cells throughout the construct, which are essential for sustained tissue growth.

The versatility of this scaffold opens up the opportunity for it to be used in a variety of biomedical applications. In disease modeling, it can be used to build 3D in vitro models of cardiac tissue, enabling researchers to study disease mechanisms under physiologically relevant conditions. This is particularly useful for understanding the pathophysiology of cardiovascular diseases as well as for screening and testing the efficacy of new therapeutic molecules in a more accurate tissue-like environment. Additionally, this scaffold holds potential as a tissue substitute in regenerative medicine, where it can be applied to restore lost or damaged tissue. It could be used to replace or repair damaged heart tissue, providing structural support and promoting functional recovery. Finally, the presence of hydroxypropyl-β-cyclodextrin (HPβCD), a biocompatible oligosaccharide, in the scaffold can be exploited to encapsulate drugs by host-guest inclusion complexes.

## 5. Conclusion

Biomimetic 3D scaffolds made of aligned electrospun nanofibers are of paramount interest for their applications in cardiac tissue engineering. A novel approach has been proposed in order to enlarge efficiently the scaffold's pore size and the distance between the fibers with the aim of improving cardiac cell colonization as compared to what has been reported in the literature. The strategy relies on the mechanical alignment of electrospun fiber through the deposition on a collector rotating at high velocity in parallel with the electrospraying of microparticles allowing a structuration of the fabricated scaffold through an electrostatic self-organization mechanism. Scaffolds made of PLA and PGS have been considered since these compounds have demonstrated their potential for cardiac tissue engineering application. Thus, the elaboration of scaffolds comprising particles of PGS and cyclodextrin and fibers made of PLA alone or a mixture of PLA and PGS was studied.

When a mixture of PLA and PGS is electrospun, PGS/HPβCD particles which are simultaneously electrosprayed are randomly dispersed between the fibers that are poorly aligned perpendicularly to the rotation axis of the cylindrical collector. In this case, no specific structuration of the scaffold can be obtained and moreover, the fibers closely packed together resulting in scaffolds with a reduced average pore size.

When only PLA is electrospun in combination with PGS/HPβCD electrospraying, the situation is radically different. Indeed, much better alignment is measured with 92 % of the fibers forming an angle < 5° with the expected theoretical direction of alignment (i.e. the direction perpendicular to the axis of rotation of the collector). Furthermore, as opposed to PLA/PGS electrospinning, the electrosprayed particles are deposited in the form of isolated islands of particle aggregates. These islands are interconnected with loosely packed highly aligned PLA fibers. Thanks to the measurement of the surface potential during electrospinning, the capacity allowing this specific structuration was corroborated with the fact that PLA fibers are able to store electric charges favoring the self-organization of fibers and particles through an electrostatic mechanism. Numerical simulation has also shown that the mechanism of self-organization is self-sustained and favors the increase of the pore size in the thickness of the scaffold. Using X-ray tomography, the 3D internal structure of the scaffolds was investigated and highlighted the open porosity with enlarged interconnected pores with sizes in the range of few tens to few hundreds of microns. Tensile

test measurements highlighted the strong anisotropy of the scaffolds and that after thermal curing, the modulus in the longitudinal direction is about 70 MPa and the strain at break 30 % versus about 100 kPa and 150 % in the transversal direction, a mechanical behavior mimicking that of cardiac tissue. Finally, normal human ventricular cardiac fibroblasts as well as newborn rat primary cardiomyocytes were seeded on Laminin-coated structured 3D scaffolds. It was demonstrated that each kind of cardiac cells adhere and perfectly align along the axis of the electrospun fibers. Furthermore, the interconnected network of large pores allows cell colonization and penetration through the thickness of the scaffolds.

In conclusion, the materials proposed in the present article represent a new class of biomimetic 3D scaffolds for potential use in cardiac tissue engineering as well as other applications such as nerve guidance or tendon and ligament repair.

## CRediT authorship contribution statement

**Florence Flraig:** Writing – original draft, Investigation, Formal analysis. **Anne Hébraud:** Writing – review & editing, Supervision, Investigation, Formal analysis, Conceptualization. **Emeline Lobry:** Writing – review & editing, Investigation. **Damien Favier:** Investigation. **Antoine Egele:** Investigation. **Patrick Kékicheff:** Writing – original draft, Investigation. **Pierre Joanne:** Investigation. **Onnik Agbulut:** Writing – review & editing, Supervision, Investigation, Formal analysis, Conceptualization. **Guy Schlatter:** Writing – review & editing, Writing – original draft, Supervision, Software, Project administration, Investigation, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgment

The authors are grateful to the French National Research Agency for the financial support of this work (Project MimHeart project ANR-15-CE08-0010-02). We also thank Christophe Sutter, Christophe Mélart and Thierry Djekriff for their help in the electrospinning set up and Alexandre Simon and Jean-Sebastien Vartanian-Grimaldi for their help in the cell culture.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biotades.2024.113481.

## Data availability

Data will be made available on request.

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