Development of Bioresorbable HydrophilicâHydrophobic Electrospun

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Development of bioresorbable hydrophilic-hydrophobic electrospun scaffolds for neural tissue engineering Luanda Chaves Lins, Florence Wianny, Sébastien Livi, Idalba Andreina Hidalgo, Colette Dehay, Jannick Duchet-Rumeau, and Jean-Francois Gerard Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00820 • Publication Date (Web): 15 Sep 2016 Downloaded from http://pubs.acs.org on September 16, 2016

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Development

of

bioresorbable

hydrophilic-

hydrophobic

electrospun scaffolds for neural

tissue engineering Luanda Chaves LINSa*, Florence WIANNYb, Sébastien LIVIa*, Idalba Andreina HIDALGOa, Colette DEHAYb, Jannick DUCHET-RUMEAUa, Jean-François GÉRARDa. a

Université de Lyon, Ingénierie des Matériaux Polymères CNRS, UMR 5223 ; INSA Lyon, F69621, Villeurbanne, France. b

Univ Lyon, Université Claude Bernard Lyon 1, Inserm, Stem Cell and Brain Research Institute U1208, 69500 Bron, France.

*Corresponding to: Sébastien LIVI. E-mail address: [email protected] Luanda C. Lins. E-mail address: [email protected] .

Keywords : Bioabsorbable polymers; Electrospun fiber; Neural Tissue Engineering; Threedimensional scaffolds.

ABSTRACT In this study, electrospun fiber scaffolds based on biodegradable and bioabsorbable polymers and showing a similar structure to that of extracellular matrix (ECM) present in the neural tissues were prepared. The effects of electrospun-based scaffolds processed from poly(lactic acid) (PLA)/poly(lactide-b-ethylene glycol-b-lactide) block copolymer (PELA) and PLA/polyethylene glycol (PEG) (50:50 by wt) blends on the morphology, wettability, and

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mechanical properties, as well as on neural stem cell (NSC) behavior, were investigated. Thus, PLA/PELA and PLA/PEG fiber mats composed of PEG with different chain lengths were evaluated for optimal use as tissue engineering scaffolds. In both cases, the hydrophilic character of the scaffold surface was increased from the introduction of PEG homopolymer or PEG-based block copolymer compared with neat PLA. A microphase separation and a surface erosion of PLA/PEG blend-based electrospun fibers were highlighted, whereas PLA/PELA blend-based fibers displayed a moderate hydrophilic surface and a tunable balance between surface erosion and bulk degradation. Even if the mechanical properties of PLA fibers containing PEG or PELA decreased slightly, an excellent compromise between stiffness and the ability to sustain large deformation was found for PLA/PELA(2k), which displayed a significant increase in strain at break, i.e., up to 500%. Our results suggest that both neat PLA and PLA/PELA blends supplemented with growth factors may mimic neural-like constructs and provide structural stability. Nonetheless, electrospun PLA/PELA blends have a suitable surface property, which may act synergistically in the modulation of biopotential for implantable scaffolding in neural tissue engineering.

INTRODUCTION Neural tissue engineering (NTE) is a multidisciplinary and emerging field that seeks a deep understanding of the synergy between neuroscience and engineering materials. Recently, three dimensional (3D) electrospun-based scaffolds from biocompatible and biodegradable polymers have been broadly demonstrated as a good substrate for tailoring cell function

1-4

.

The result of the process is a 3D network of interconnected fibers deposited on the collector’s surface. This membrane presents an open, fully interconnected geometry with a high area/volume ratio, conveniently mimicking the topology of the ECM and thus allowing cell ingrowth, uniform cell distribution, and neovascularization of the scaffold. Electrospinning is

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a one-step method, requiring an inexpensive and relatively easy to use setup, adjustable to the laboratory environment to produce small- and large-scale electrospun scaffolds 1, 4-6. Scaffolds can be easily designed in order to tailor the key parameters governing the interactions of cells with the matrix such as its pattern architecture and surface chemistry 5. Suitable hydrophilic-hydrophobic patterned surfaces, mechanical properties, and degradation profiles of scaffolds are critical to support cell attachment, proliferation, and differentiation 710

. The increasing clinical demand for implants has focused a great deal on

biorebsorbable polymers and their degradation behavior 11, 12. Poly(hydroxy acid)-type, homoand copolymers, constitute the greatest promising bioresorbable aliphatic polyesters in the field of tissue engineering and pharmaceutical applications 13-15. Poly(lactic acid) (PLA), which can be easily spun, has been identified as an appropriate polymer for biomedical applications due to its hydrolytic degradation kinetics leading to lactic acids that could be subsequently eliminated as carbon dioxide and water via the Krebs’ cycle

16, 17

. The large interest in PLA and its copolymers was further explained by

the fact that the FDA (Food and Drug Administration) has approved these polymers for a number of clinical applications 16. Due to its thermoplastic processability and their biological properties as well as its biodegradability and lack of immunogenicity, the PLA may be promising for neural tissues.

18

. L-lactic acid is an intermediate metabolite of anaerobic

glycolysis, the principal degradation product of PLA-based scaffolds.

19

. Although not

present in this work, several studies have proven that lactic acid is an alternative energy source for neural and glial cells 5, 19, 20. Nevertheless, the physico-chemical properties of PLA-based scaffolds, such as their slow biodegradation kinetics, their high stiffness, and their hydrophobic surface, do not fit the requirements for some biomedical applications. Moreover, PLA is a very brittle material,


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showing less than 10% strain at break. The poor toughness of PLA can be a limitation for neural tissue engineering application.

21

. Biomechanical signals may be transferred to cells

via the properties of micro/ nanoscale of a substrate. To introduce spatiotemporal controlling parameters in the cells, in order to influence a positive behavior of the neural cell, a scaffold should mimic the mechanical properties of the ECM 22, 23. The brain tissues are provided of fibrillar soft ECM, this fibrillar material are surrounded mainly with hyaluronan and glycosaminoglycans which have a low modulus

23

. Keeping this in mind,

for an fabricated support resemble a native of the brain tissues, the materials-based scaffolds can be designed to consist on a smaller mechanical properties 24. There is great interest focused on the increase in hydrophilicity and the reduction of the brittleness of PLA fiber-based scaffolds for tissue engineering purposes. It has been reported that blending PLA films with hydrophilic and soft poly(ethylene glycol) (PEG) is an effective strategy to increase surface hydrophilicity. In biomedical applications, the use of PEG is generally accepted due to its good biocompatibility and very low toxicity; blending PEG with PLA may also increase the biocompatibility of L-lactide polymers

25, 26

. Cell

survival is envisaged by the balance of hydrophilicity and rate degradation 27. PEG has been investigated for use in the treatment of injury to neuronal membranes and death of neurons and their processes after traumatic spinal cord injury 26. The hydrophilic character of this polymer can accelerate and enhance the neuronal membrane resealing process, avoiding deficits and disabilities in traumatic brain and spinal cord injury. In addition, the PEG smooth feature allows the neural cells deform the external environment effortlessly, which can generate stimulus for cell survival and differentiation 23. However, PEG and PLA have limited miscibility due to differences in surface energies, which induce a repulsive interaction between the two polymers, leading to a phase separation at the fiber surface

28-30

. Actually, the copolymerization of PEG with lactides has


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been used as a source of hydrophilicity because the terminal hydroxyl groups reacts readily with lactones to yield ABA-type copolymers. Poly(L-lactide)-b-poly(ethylene glycol)-bpoly(L-lactide) (PELA) is a friendly block copolymers for tissue engineering application, because it shows physico-chemical intermediate characteristics, furthermore has low toxicity and absence of antigenicity 31-33. According to the literature, PELA copolymers clearly exhibit a good balance between degradation rate and hydrophilicity 8, 34, 35. Nonetheless, there may be some limitations to the intended uses of PELA copolymer in the electrospinning process because of their low molecular weight. It is known that higher-molecular-weight polymers have a greater number of entanglements per chain, which is a prerequisite for the formation of a stable fiber jet. To resolve this problem, PELA copolymers can be substantially blended by electrospinning with higher-molecular-weight polymers, for example, PLA. This is related to poor molecular-level interactions leading to a thermodynamically nonstable morphology after mixing and poor interfacial adhesion. In a recent work by Hendrick and Frey, it was reported that the use of a PELA copolymer in the PLA blends was more effective than adding PEG homopolymer at fine-tuning of PLA's hydrophilicity properties 13. Often, interactions are considered even more important in blends than in other heterogeneous polymeric materials in determining the mutual solubility of the phases and the interphase thickness formed during blending as well as the structure and properties of the blend

36

. The model proposed by Hendrick and Frey implies that interactions between the

blend PLA homopolymer/PEG homopolymer blend were not as important as in the case of the PLA homopolymer/PELA copolymer blend

13

. Good miscibility of the PLA homopolymer

with the PLA block of the PELA copolymer and limited compatibility between the PLA homopolymer and the PEG homopolymer are assumed. Thus, all of this results in the


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improvement of the wetting properties of fibers, while keeping their morphology relatively constant 13, 37, 38. The mechanism of degradation in PEG, PLA, and PELA membranes and sutures in aqueous media has been investigated as a function of pH level, crystallinity, temperature, and time, by studying the changes that occur in molar mass and tensile strength

16, 33, 39, 40

.

However, the degradation behavior of the same polymers processed by electrospinning have not yet been widely studied. Therefore, this paper focuses on the comparison between binary blends of PLA with PELA block copolymers or PEG homopolymer, varying the molecular weight of PEG. We intended to demonstrate that, considering the combination of bi-component polymer blends and the electrospinning process, electrospun scaffolds could be obtained with tunable mechanical properties, surface wettability, and degradation kinetics. The in vitro degradation behavior of the electrospun PLA, PLA/PELA, and PLA/PEG fiber membranes was monitored by gravimetric analysis, morphological changes, and molecular-weight variations. Furthermore, we studied the impact of the variation of the scaffold-based system characteristics set out above on the survival and proliferation of monkey NSCs in vitro, a parameter that has not been explored up to now.

EXPERIMENTAL Materials Table 1 summarizes the materials employed for the synthesis of PELA block copolymers as well as the preparation of solutions for processing electrospun scaffolds.

Table 1. Materials used for PELA copolymers synthesis and membranes processing by electrospinning.


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Molar mass (g.mol-1)

Supplier Poly(ethylene glycol) 2,000 and 20,000 Sigma-Aldrich PEG Poly(D-L-lactic acid) 100,000 PLA 2002D NatureWorks® PDLLA* L-lactide (LLA) 144 Tokyo Chemical Industry Stannous octoate 405.12 Sigma-Aldrich (Sn(Oct)2) *The notation “PLA” from now on only refers to “PDLLA” unless otherwise indicated separately. PLA 2002D was purchased from NatureWorks® LLC Co. (USA), and consists of 98% L-lactide and 2% D-lactide units. The molecular weight is 100,000 g.mol-1 with polydispersity index of 2 and a density of 1.24 g cm-3.

All solvents (toluene, chloroform, diethyl ether, ethanol, tetrahydrofuran, N,Ndimethylformamide) were supplied by Sigma-Aldrich and used as received.

Synthesis of PELA block copolymers The selected block copolymers were synthesized by ring opening polymerization of Llactide in the presence of poly(ethylene glycol) with different molecular weights i.e. 2,000 (PEG2k) and 20,000 (PEG20k) g.mol-1, using non-toxic catalyst stannous octoate denoted (Sn(Oct)2). Among these catalysts, this catalyst is the most frequently mentioned in literature because it leads to high yields and high molecular weights

41

. L-lactide was dried into

anhydrous toluene and sodium hydride (NaH) under stirring overnight

35, 42

. Then, this

solution was filtered and the solvent was removed by evaporation under vacuum. The purified reaction products were analyzed by 1H-NMR, Thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), and their molecular weights were measured by sizeexclusion chromatography (SEC) in THF solvent (see ESI). Scheme 1 represents the chemical route based on the insertion-coordination mechanism of the L-lactide/PEG/Sn(Oct)2 system 43

.


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Scheme 1. Synthesis of poly(lactide-b-ethylene glycol-b-lactide) (PELA) triblock copolymers.

The molar compositions of the copolymers were determined by the integral peak ratio of 3.65 ppm (IPEG) and to PLA blocks at 1.46 ppm (IPLA) almost agreed with those expected from the feed composition. This results was supported by the following spectroscopic 1H NMR (CDCl3) data (see ESI). The compositional data describing the PELA copolymers synthesized are summarized in Table 2.

Table 2. PLAx/PEG y/PLAx triblock copolymers obtained from polymerization of l-lactide in the presence of PEG using (Sn(Oct)2) as catalyst. Nomenclature

Copolymer

PELA2k

Initial Final MnPEG DPPEGb DPPLAc MnPLAd Mncopoe LA/EG LA/EGa 1 0.7 2,000 45 19 1,368 3,34

PLA684/PEG2k/PLA684 PLA1,440/PEG20k/ PELA20k 1 0.14 20,000 455 40 2,880 22,90 PLA1,440 a Determined by using the integration ratio of resonances due to PEG blocks at 3.65 ppm (IPEG) and to PLA blocks at 1.46 ppm (IPLA) in the 1H NMR spectra. b DPPEG =2,000/44 or 20,000/44 c DPPLA = DPPEG X (IPLA / IPEG)x4 d MnPLA = (144/2) x DPPLA e Mn = 44 x DPPEG + MnPLA

Electrospinning processing


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In order to prepare block copolymer solutions for membranes by electrospinning process, solubility tests were performed in different solvents. The goal was to qualitatively identify the Critical Micelle Concentration (CMC) in order to determine the maximum value of concentration (%w/w) which could to be employed in the electrospinning process. The copolymers present an intermediate solubility behavior relative to their PEG and PLA constitutive blocks. In both cases, homogeneous solutions were obtained in chloroform (CHCl3) and N,N-dimethylformamide (DMF). In the opposite, in water (H2O), ethanol (CH3CH2OH), and tetrahydrofuran (THF) both copolymers form a cloudy solution (PELA20k to a greatest extent than that of PELA2k, due to the large segments of PEG 20K). According to these results, 15 % w/w solutions based on an 80:20 mixture of CHCl3 (non-polar solvent) and DMF (polar solvent). Indeed, chloroform has been widely used for the processing of PLA by electrospinning 43. Nevertheless, the polarity of the PEG segments of the copolymer aiming to form a homogeneous solution must be taken into account. In addition, solvents with a high dielectric constant (k) must be preferred because they facilitate the electrospinning process. In the case of chloroform this constant is very low (k = 4.8), compared to DMF, where k= 38 44. Electrospun membranes were obtained using the NANON-01A Electrospinning Setup from MECC. 20 mL of each polymer solution were prepared as detailed above, including PLA, PLA/PELA2k, PLA/PELA20k, PLA/PEG2k, and PLA/PEG20k (summarized in Table 3). The electrospinning process was carried out at 25 kV, a tip-collector distance of 15 cm, and a steady flow rate of 1 mL.h-1 (spinneret with a hole diameter of 0.838 ± 0.0015 mm). The electrospun fiber was collected on a metal drum rotating speed at 50 rpm. The resulting scaffold contained interconnected webs of thin fibers, which was dried at room temperature for a week prior to usage.


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Table 3. Nomenclature of copolymer/homopolymer and homopolymer/homopolymer blends. Nomenclature PLA PLA/PELA2k PLA/PELA20k PLA/PEG2k PLA/PEG20k

Blends Homopolymer/Copolymer PLA(100k)/PELA (2k or 20k) 50:50 Homopolymer/Homopolymer PLA(100k)/PEG (2k or 20k) 50:50

Scaffolds morphology The electrospun memnbranes were imaged using a Philips XL30 scanning electron microscope (SEM) to study the surface and the morphology of the samples. Prior to observation, all samples were coated by gold sputtering in a Bal-Tec Sputter Coater SCD005, for 30 seconds at a current of 30 mA. Average fiber diameter was determined from measurements taken perpendicular to the long axis of the fibers (500 measurements per field). The fiber diameters of the electrospun mats were investigated using scanning electron microscope and ImageJ 1.48v software. Thus, five images per sample were used for each fibers sample. From each image, at 400 different segments were randomly selected and the variance was analyzed by one-way normal distribution using R program language for statistical computing and graphics (Version 0.99.473, copyright 2009 −2015 RStudio, Inc.). An R script was written to perform the analysis. All the measurements were expressed as mean standard deviation (SD). The porous size in scaffolds was measured from scanning electron microscopy (SEM) images using ImageJ software. The porosity can be obtained using the helium gas pycnometer in order to obtain the matrix volume (model AccuPycTM II1340, Micromeritics). Helium Pycnometer working principle is based on the gas law 45.


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The bulk volume of a sample is given by:

= + Where: = Bulk Volume (Bulk volume is the apparent volume of the sample) = Matrix Volume (True matrix volume of sample was defined by experimental values of pycnometer) = Porous Volume The porosity was calculated using the equation 46: % =

−

Mechanical behavior of scaffolds The mechanical performances through static tensile tests were performed on a MTS 2/M electromechanical testing machine at room temperature (22±1°C) and under a relative humidity of 30±5%. All membranes were cut to dimensions of 4mm × 10mm × thickness (several tens of microns). In addition, a crosshead speed of 5 mm.min-1 was used.

Water absorption To determine the spontaneous water uptake of the scaffolds, the demand wettability test was performed using wilhelmy wetting method 47. The scaffolds was emerged in distilled water at room temperature to obtain the wet weight Wwet. The measurements were carried out until the initial equilibrium was reached and the relative water uptake in percent of dry weight Wdry was calculated according to: % =

− 100 11


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Structural characterization DSC analyses were performed on a Q10 (from TA Co. Ltd., USA) from – 80°C to 200°C with a heating rate of 5 °C.min-1 and under nitrogen flow of 50 mL. min-1. In all cases, the weight of samples was between 5 and 10 mg. Melting temperature (Tm), glass transition temperature (Tg) and the melting temperature (∆Hm) were measured and determined during the first heating scan. The crystallinity relative to crystalline PLA and PEG is calculated from Xc (%)=((1/∆H0f)*( ∆Hf/w)) *100. Where ∆Hf is the melting enthalpy of PLA and/or PEG, w represents the weight fraction of PLA or PEG in the polymer mixtures and ∆H0f is the melting enthalpy corresponding to a 100% crystalline sample, 196.8 J/g and 93 J/g, for PEG and PLA, respectively 48, 49.

In vitro hydrolytic degradation Electrospun membranes were cut into rectangles (10 x 40 x ~0.1 mm3) for in vitro degradation testing. Each cut specimen was measured for initial weight, and was then placed in a test tube containing 20 ml of phosphate-buffered saline (PBS, 0.1 M, pH 7.4) for in vitro degradation study. The tubes were placed in an incubator that was maintained at 37 °C during 12 weeks. At predetermined intervals (every week), triplicate samples for each kind of membrane were recovered, rinsed with distilled water to remove residual buffer salts, and dried to constant weight in a vacuum desiccator. The morphological changes were evaluated by SEM (as mentioned above). The weight loss was determined gravimetrically by comparing the dry weight remaining at a specific time with the initial weight. % =

(" − ) 100 " 12


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Where: " = initial weight and = residual weight

The molecular-weight of recovered matrix polymer was determined using SEC as described above. The membranes were dissolved in THF and filtered to remove insoluble residues. Both eluents were used with a flow rate of 1 mL.min-1. Columns were connected to an RI detector. The sample concentration was 5 mg.mL-1 and the injection volume was 100 µL.

Neural stem cell culture PLA, PLA/PELA2k and PLA/PELA20k membranes were punched into 6-mm diameter disks, UV treated for 30 minutes, and placed at the bottom of 96-well dishes. Membranes were pre-equilibrated in “NSC medium” (described below) overnight at 37°C, in 5% CO2, 5% O2. Two types of NSCs were used in this study: (1) ESC-NSCs were derived from monkey ESCs previously isolated in our laboratory 50. Briefly, monkey ESCs were cultured to confluency for 15 days with daily medium change. Neuroepithelial-like cells spontaneously emerging in culture were selected manually and transferred into gelatin coated dishes (0.1%; Sigma), and cultured in “NSC medium” (DMEM/F12, N2 supplement, 1% nonessential amino

acids,

2mM

L-glutamine,

0.1mM

β-mercaptoethanol

https://www.thermofisher.com/fr/fr/home/brands/invitrogen.html)

;

Invitrogen

supplemented

with

; 20

ng/ml FGF2 (Millipore; http://www.merckmillipore.com) and 20 ng/ml EGF (Millipore). ESC-NSCs were trypsinized (trypsin 0,025%, EDTA, 0,1g/l) (Invitrogen), seeded on top of the membranes, and cultured for 1- 5 days in NSC medium, supplemented with FGF2 and


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EGF. Medium was changed every other day. ESC-NSCs used in this study stably express the TAU-green fluorescent fusion protein (TAU-GFP), which binds the GFP to microtubules. (2) Fetal-NSCs were derived from the germinal zones of a cynomolgus macaca fetal brain (embryonic day E79), as previously described

51, 52

. Briefly, germinal zones were

manually isolated, dissociated by incubation in Trypsin/EDTA (trypsin 0,025%, EDTA, 0,1g/l) (Invitrogen), and seeded on top of the membranes in NSC medium supplemented with FGF2 (20ng/ml), EGF (20ng/ml) and Leukemia inhibitory Factor (LIF; 1000IU/ml) for 5 days. Experiments were performed in accordance with national and European laws and institute guidelines for animal experimentation (protocol C2EA42-12-11-0402-003 approved by the Animal Care and Use Committee CELYNE; C2EA 42). Surgical procedures conform to European requirements 2010/63/UE.

Immunofluorescence Membranes were fixed by immersion in 2% paraformaldehyde in cold phosphatebuffer at 4°C for 30 min and permeabilized with Triton X-100 (0.5% in Tris-buffered saline (TBS)). Aspecific binding was blocked by incubation in normal goat serum or normal donkey serum (10% in TBS) (Jackson Immunoresearch Laboratories, West Grove, PA, USA; http://www.jacksonimuno.com) for 20 min at room temperature (RT). Membranes were incubated overnight at 4°C with primary antibodies diluted in antibody diluent (Dako, Glostrup, Denmark; http://www.dako.fr) as follows: monoclonal anti-Nestin (1:100, MAB5326, Chemicon, Temecula, CA, USA: http://www.chemicon.com), polyclonal goat anti-Sox2, (1/100, sc17320, Santacruz Biotechnology, Inc., http://www.scbt.com), polyclonal rabbit anti Cleaved-Caspase 3 (Asp175; 1/500; Cell Signaling), mouse anti Ki67 (1/100; Menarini diagnostics; http://www.menarinidiag.co.uk). Membranes were rinsed with TBS, and incubated with affinity-purified goat or donkey anti-mouse, anti-rabbit or anti-goat IgG or


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IgM conjugated to Alexa488, 555 or 647 (Invitrogen) for 1h at RT. Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) (Invitrogen; D103; 2µg/ml) for 3 min. Membranes were mounted on slides in Fluoromount G (Clinisciences). Images were acquired by confocal microscopy (Leica, TCS SP at 20X objective; http://www.leicamicrosystems.com). The total number of cells (DAPI+ cells) and number of Sox2+ cells were counted using ImageJ (1.46r) software. 4 to 6

RESULTS AND DISCUSSION

Membrane morphology Because scaffolds can recapitulate 3D structure of the extracellular matrix (ECM), they hold good promise for tissue engineering applications. The objective is to prepare uniform membranes from the various polymer blends having suitable characteristics for the development of the cellular process, i.e., the fiber network homogeneity and porosity, the fiber diameter, and the nature of the fiber-fiber connections. The morphology of the membranes from the various polymer blends was observed by SEM to determine their mean fiber diameter, distribution, and pore size area of the 3D fibrous network, establishing comparisons between the films obtained from different blends. PLA, PLA/PELA, and PLA/PEG-based membranes were prepared by electrospinning. We adjusted the electrospinning parameters to produce uniform fibers (flow rate = 1 mL.h-1, rotation speed = 50 rpm, voltage applied = 25 kV, solution concentration = 15% w/w, and amount of solution used = 20 mL). SEM micrographs in Figure 1 show the morphology of the processed membranes and Figure 2 reports the distributions of fiber diameters. Threedimensional interconnected fibers with smooth and round shapes were formed. In general, the


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mats prepared from PLA/PELA2k and PLA/PEG2k blends displayed more homogeneous morphologies than PLA, PLA/PELA20k, and PLA/PEG20k blend-based ones.

Figure 1. SEM micrographs of electrospun fibers. The images on the left have a magnification of 500X (scale bar 50 µm), and the images on the right have a magnification of 1000X (scale bar 20 µm).


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Figure 2 reports the average fiber diameter after optimization of the electrospinning process. PLA shows a fiber morphology towards thicker (3.10 ± 0.89 µm), almost plate-like fibers and evidence of fibers flattening and coalescing on collection

7, 13

. As previously

mentioned, homogeneous morphologies were observed for PLA/PELA2k and PLA/PEG2k, with a consistent fiber diameter all along their extension. It has previously been shown that the amount of low-molecular-weight PEG presented in the blends could increase adherence among the fibers due to their increased hydrophilicity and hydroxyl groups compared with that of PLA53. However, the existence of hydrogen bonding interactions among PEG molecules and carboxyl groups of the PLA did not cause fiber coalescence. As shown in Figure 1, PLA/PELA and PLA/PEG blends have interconnected fibers, but with well distributed pores in the electrospun membrane. By considering the values of mean fiber diameter, it appears that an increase in the molar mass of PEG used in PLA/PEG leads to a loss of the homogeneity in the morphology of electrospun membranes.. Using higher molecular weight PEG (PEG20k) leads to the formation of fibers with non-uniform size diameter with a bimodal distribution, which could be a consequence of poor interactions between the polymers. In addition, compatibilization of PLA is impeded when the length chain of PEG is increasedn ,due to difficult access to the side blocks of the hydroxyl groups in PLA/PEG blends, which are supposed to interact with the chains of PLA in the blends. The poor compatibility between PLA and PEG homopolymer has already been reported 13, 54, 55. Thus, using a diblock copolymer with low molecular weights appears to be the most productive method of increasing PEG content. Figure 2 shows that PLA/PELA2k was the only blend that showed a unimodal distribution. Di- and triblock copolymers behave like compatibilizers in immiscible PLA/PEG blends, allowing a very uniform morphology 13.


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Figure 2. Distribution curves of diameter size for electrospun membranes with different compositions (mean ± standard deviation n ≥ 400 fibers).

Porosity between fibers and fiber surface properties affect moisture transport in electrospun membranes

53

. Pores in a tissue-engineered membrane make up the space in

which cells reside, and this parameter is directly related to the success of a scaffold 56. During spinning, random deposition layer by layer of the fibers on the collector, generates the different morphologies. The porosity of electrospun membranes may be related to repulsive electrostatic forces, surface tension, or the viscoelasticity of the polymeric jet. Based on volume measurements, electrospun scaffolds showed a variable porosity including (between 78% and 89%). Concerning PLA membrane, a porosity of 78% was obtained, which can be explained by their morphology where the PLA fibers are fused or bonded together at their contact sites, leading to a decrease in spacing in the threedimensional network (Figure 1). As shown in Table 4, the porosity of the electrospun scaffold


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increases in the presence of the PELA and PEG in the blend. This suggests that blend composition is an important factor affecting porosity.

Table 4. Porosity and pores size of electrospun scaffolds as a function of the composition. Composition

Porosity %

PLA PLA/PELA2k PLA/PELA20k PLA/PEG2k PLA/PEG20k

78 88 89 82 85

Pores size (µm2) 3.89 ±1.50 1.94 ±0.19 1.45 ±0.89 1.15 ±0.25 1.89 ±0.76

For scaffolds containing PEG homopolymers or PELA copolymers, pore size area can be correlated with fiber diameter, as shown in the work of Hendrick et al. and Pham et al.

13, 57

. This can be verified in the SEM images and values of pore size area shown in

Table 4. An increase or decrease in pore size area is a consequence of the increase/decrease in fiber diameter. However, fiber formation is dependent on material interactions. The presence of plate-like fibers along the electrospun membranes increase the pore size area, as observed in the case of membranes produced using the neat PLA. On neat PLA membranes, the average pore area and the dispersion of the pore size are significantly higher compared with those of fibers blends. The coalescence of the neat PLA fibers resulted in a wide space between adjacent fibers in the membrane.

Mechanical properties of membranes Because scaffolds must sustain the forces generated during tissue growth, i.e., physiological activities and related biomechanics, the mechanical behavior of the electrospun films is a key parameter

54

. Indeed, many parameters can strongly influence the mechanical

behavior of the final electrospun material such as the molecular structure of the fiber,


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porosity, entanglement between the fibers, diameter, anisotropy of the fibers, and the fiber– fiber interactions in the membranes 58, 59. Due to its biodegradability and its ability to be bioresorbable, PLA is a good candidate for biomedical applications and tissue engineering. However, one of the limitations of this polymer is its poor elongation at break (from 10% to 30%) 60, 61. As reported previously, there are different ways to increase the mechanical performance of the PLA-based scaffolds. the use of biocompatible polymer such as PEG was studied for tissue engineering applications. For example, Sheth et al. have highlighted the influence of PEG amount on the final properties in the blends composed of PLA/PEG, and they have observed a significative improvement of the strain at break when a great amount of PEG was used

55

. Other authors

have studied the combination of acetyl tri-n-butyl citrate with PEG having various molar masses to plasticize PLA. Their results showed an improvement of the strain at failure of PLA coupled with decreases of the stiffness 62. The chemical nature of the material as well as the architecture of the fiber network play a crucial role on the mechanical performances of the electrospun membranes

55

. To

determine the influence of PEG homopolymer or PELA copolymer in PLA into the mechanical behavior of PLA, uniaxial tension mode was used (see Figure 3).


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4,5 4,0

PLA

3,5 3,0

Stress (MPa)

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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PLA/PELA2k 2,5

PLA/PELA20k

2,0 1,5 1,0

PLA/PEG2k

0,5

PLA/PEG20k

0,0 0

100

200

300

400

500

Strain (%)

Figure 3. Static tensile tests of neat PLA and PLA containing PEG homopolymer or PELA copolymer.

Table 5. Mechanical properties of electrospun fibrous scaffolds of neat-PLA, and PLA/PELA and PLA/PEG blends. Samples PLA PLA/PELA2k PLA/PELA20k PLA/PEG2k PLA/PEG20k

Tensile Modulus (MPa) 38.0 ± 3.4 7.6 ± 0.1 11.7 ± 0.8 11.5 ± 1.3 10.8 ± 0.4

Strain (%) 12.9 ± 2.7 513.4 ± 6.8 170.5 ± 12.1 97.1 ± 12.5 79.6 ± 16.1

Stress (MPa) 4.3 ± 0.8 2.7 ± 0.1 1.9 ± 0.2 1.3± 0.2 0.6 ± 0.2

In all cases, the Young’s modulus of the membranes decreased markedly in the presence of PEG or PELA in the blends. This drop was considerable and yielded a reduction in the tensile modulus by approximately 3 times smaller, e.g., from 38 MPa for neat PLA fibers to 7.6, 11.7, 11.5, and 10.8 MPa for PLA/PELA2k, PLA/PELA20k, PLA/PEG2k, and PLA/PEG20k electrospun blends, respectively (Table 4). These results can be explained by the addition of flexible PEG segments compared with neat PLA matrix, which results in 21 ACS Paragon Plus Environment

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decreased tensile modulus and provides flexibility to the polymer blends. In fact, PEG act as plasticizing agent of PLA matrix. Sheth et al. have reported that an increase of PEG amounts into PLA/PEG blends reduced the Young Modulus

59

. Contrarily, other studies have

confirmed that the introduction of PEG into PLA can also lead to a significant reduction of the strain at break which was attributed to non-miscibility of the PEG and PLA blend 8. In our case, the elongation at break increased when PELA and PEG were blended with PLA. The blend with PLA matrix generate an additional free volume inside the polymer leading to a better flexibility of the polymeric chains

63

. However, different results were

obtained between PLA containing PELA or PEG electrospun blends. In the first case, PELA copolymers led to significant increases in strain at break with values of 513% and 170% for PLA/PELA2k and PLA/PELA20k, respectively. In the second case, the addition of PEG into PLA also induced more moderate increases, i.e., 97% and 80% for PEG2k and PEG20k, respectively. These results clearly demonstrate the superior affinity of the PLA/PELA blends with PLA copolymer matrix. The chain end groups in binary PLA/PEG blends have a significant role on the mechanical behavior. An improvement in the failure strain in PLA/PELA blends is due to the interaction between the PLA bulk chains with the PLA side blocks of the PELA copolymer. For example, various authors demonstrated that the chain ends of PEG (hydroxyl or/and methyl) have a strong influence on the compatibility and the crystallinity of PEG/PLA blends 64

. In addition, the stress is smaller for homopolymer-homopolymer (PLA/PEG) blends

compared to homopolymer-copolymer (PLA/PELA) blends which are caused by better mobility of the copolymer chains (PELA) but also by a decrease of the intermolecular interaction. One can notice that the increase in strain at break in the PLA/PELA blend is very high as PEG blocks in the copolymer are short, i.e., the PELA2k-based blend displays a higher


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elongation at break than the PELA20k-based one. It is generally known that short chain polymers can serve as plasticizers when mixed with polymers of large chains. Moreover, there is a clear relationship between strain at break and crystallinity. As will be discussed below, blends with longer PEG chains have a greater degree of crystallinity, which directly influences mechanical behavior. The crystalline phase is ordered and with high cohesion, contrary to the amorphous phase, which is in disorder and less cohesive. Considering a larger scale, the poorer mechanical properties of PLA membranes can also be attributed to non-uniform fiber morphology. PLA/PELA membranes display a good compromise between stiffness and the ability to sustain a large deformation. Finally, neural

cells responds to lower stiffness regimes compared with cells from other tissues

24

In

scaffold membranes, a soft component can be added to the initial polymer material in order to fit the neural tissue requirements.

Structural blend characterization PLA and PEG electrospun fibers are potential carriers for biomedical applications for surgical resorbable sutures, and their physical and structural properties can have a direct influence on the cellular response 65. The morphology and crystalline structure of a crystalline polymer have high impact on its chemical stability 66. In this sense, we analysed the thermal properties of the polymers using differential scanning calorimetry (DSC). The thermal behavior of PLA, PEG2k, PEG20k, PELA2k, PELA20k, and their respective electrospun blends are summarized in Figure 4. From the literature and DSC data reported herein, the thermal behaviors indicate that PEG and PLA can both melt and crystallize in separate domains when the PEG and PLA blocks are both long enough 33, 67. For neat PLA fibers, the temperature at glass transition was approximately 53 °C. The absence of crystallization during the cooling for PLA has already been reported by Miyata et


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al. (Figure 4C and D, for neat PLA) For the PLA/PEG and PLA/PELA blends, the melting endotherm of PEG and the glass transition of PLA overlapped, due to the presence of PEG 68. In addition, two melting peaks are observed in neat PLA fibers, and can be deconvoluted by a Gaussian fitting temperature around 150 °C (Figure 4, black line). Their presence is due to the co-existence of the β phase and α phase

69

. PLA shows two crystal

modifications, depending on the conditions used for their formation. At the lowest temperatures, the melting peak represents fibrillar β crystal created under elongational deformation, also known as oriented phase. At the highest temperatures, the melting peak represents α crystal 66. During the electrospinning process, the polymer solution jet is highly stretched, which leads to the orientation of the polymer chain along the fiber length, and to the drawing of polymer chains 70. Depending on of the molecular weight of the PEG used, one can see a difference in the PLA melting peak when it was blended with the homopolymer and copolymer (i.e., PEG and PELA, respectively). In this work, the use of PEG2k and PELA2k promotes the formation of β phase and the use of PEG20k and PELA20k leads to α phase. Indeed, the incorporation of PEG with larger chains may hinder the orientation of the crystalline structure of the PLA due to the increase in the degree of entanglement. Regarding PEG2k and PEG20k, melting temperatures at 54 °C and 64 °C, respectively, were obtained (Figure 4A and B, dashed purple and green lines for PEG2k and PEG20k, respectively). This difference was attributed to the molar mass of PEG (PEG2k PEG20k). In the case of PELA2k (pink dashed line) and PELA20k (green dashed line), melting endotherms at 34.2 °C and 62.0 °C were observed corresponding to the PEG segment. After copolymerization, the intermingling of the PLA blocks with the PEG chains leads to a shift of the melting peak at lower temperatures., and to a lack of PLA crystallinity 11.


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Homopolymer-homopolymer (PLA/PEG) and homopolymer-copolymer (PLA/PELA) blends show different physical properties. In fact, the presence of the PLA in PLA/PEG blends does not modify the melting temperature of the PEG, i.e., 54 °C and 64 °C for PLA/PEG2k (purple full line) and PLA/PEG20k (green full line), respectively. For PLA/PELA, the incorporation of PELA2k (pink full line) and PELA20k (red full line) leads to a decrease in the Tm attributed to PEG segments, i.e., 46.5 and 52.2 °C, respectively. It should be stressed that measurable phase mixing has occurred. The variation in melting temperature may indicate the magnitude of the polymer-copolymer interaction, resulting in a certain degree of phase compatibility

59

. Sun et al. have shown that this is

because the earlier crystallization of the PLA polymer strongly restricted the crystallization of the PEG blocks, leading to lower Tm and Tc values 67. Figure 4 clearly shows that the Tm of PEG segments is more disturbed in the shorter the chain (PELA2K) for a given PLA/PELA blend. Younes and Cohn explain that this occurrence is due to the dispersion of small chains of PEG in the amorphous parts of the PLA 29. Moreover, the Tm of PEG segments decreases by more when PLA is added to PLA/PELA than to PEG/PLA blends, as previously observed by Pannu et al., who examined the miscibility of PELA copolymer with PDLA homopolymer

38

. Nakafuku elucidated the

relationship between the crystallization behavior of PLA and PEO in the binary blend

71, 72

.

According to the above results, the depression of the melting temperature is indicative of interactions between the polymers. Park et al. observed that the blends of PLA and triblock copolymers of poly(ethylene oxide) (PEO)/poly(propylene oxide) (PPO)/PEO exhibited phase separation. They suggested that amorphous PLLA is miscible with copolymers

73

.


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A

PEG2k

H eat Flo w (W /g) E xo U p

H eat Flow (W /g) Exo U p

34.2°C

145.1°C

(β phase)

Tg= 53°C

151.2°C

PLA

(α phase)

PLA-PEG2k PLA-PELA2k

B

PEG20k PELA20k

119.2°C

PELA2k

36.8°C

145.1°C

(β phase)

Tg= 53°C

151.2°C

(α phase)

PLA PLA-PEG20k PLA-PELA20k 52.2°C

54.3°C 46.5°C

64.0°C

62.0°C 5mW

5mW

-40

-20

0

20

40

60

80

100

120

140

160

-40

-20

0

20

C 31.1°C

PLA-PELA2k

80

100

120

140

160

D 42.7°C

90.4°C PLA-PEG2k PLA

15.0°C

60

39.4°C

90.4°C

H ea t Flow (W /g ) E xo U p

10.9°C

40

Temperature (°C)

Temperature (°C)

Heat Flow (W /g) E xo Up

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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54.3°C

PELA2k

30.6°C 84.0°C PLA-PELA20k 89.1°C

PLA-PEG20k PLA PELA20k

PEG2k 5mW

5mW -40

-20

0

20

40

60

80

100

120

140

160

PEG20k -40

-20

0

20

40

60

80

100

120

140

160

Temperature (°C)

Temperature (°C)

Figure 4. DSC thermograms. In (A) and (C), scanning was from −40 °C to 160 °C under N2 at 5 °C/min. In (B) and (D), the sample was cooled in the DSC from 180 °C to −40 °C under N2 at 5 °C/min.

Table 6. Thermal properties of neat-PLA, PLA/PELA, and PLA/PEG blends.

PLA PEG2k PEG20k PELA2k PELA20k PLA/PELA2k PLA/PEG2k

∆HmPEG (J/g) 149.5 156.3 13.9 92.2 16.5 57.1

∆HmPLA (J/g) 22.6 4.9 15.2 17.4

∆HcPEG (J/g) 135.3 156.2 31.3 104.4 19.7 47.3

∆HcPLA (J/g) 4.6 15.5 15.3

χ%PLA χ%PEG 49 13 23 37

76 79 11 52 27 57 26


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PLA/PELA20k PLA/PEG20k

28.6 61.9

14.4 15.9

24.7 77.7

8.4 10.4

27 34

32 61

The melting endotherm is composition-dependent: in a miscible blend, it usually decreases for both components. In contrast, it remains constant in an immiscible system exhibiting fully separated phases. In this case, each crystallizable components display the Tm of its corresponding pure homopolymer. Semi-miscible blends represent an intermediate dispersion condition between those of fully miscible and immiscible blends, and comprise separated phases with interwining of the different polymer chains leading to a diffuse interface

64, 71

. It is shown that in the blends with PLA, PEG blocks for PELA copolymers

exhibit a lower Tm and Tc, than those of PEG homopolymers with similar lengths of PEG blocks. Tm and Tc increase slightly when increasing the chain length of the PLA blocks. These results indicate that PLA/PEG blends are semimiscible or immiscible and PLA/PELA blends are miscible. One of the possible explanations for the higher miscibility of PLA/PELA compared with PLA/PEG systems can be that both blends were processed by electrospinning, i.e., the temperature is below the melting temperature of PEG (25 °C). At this temperature, both PLA and PEG in the homopolymer/homopolymer blends may produce a crystalline/crystalline system exhibiting a more complex behavior than that of crystalline/amorphous system. In contrast, the crystallinity and Tm of the PEG blocks are much lower for the copolymerhomopolymer blends, facilitating the crystalline lamellae of the PEG in the amorphous region of the PLA. Another possible explanation for the higher miscibility of the PLA/PELA blend, is that the PLA lateral block copolymer assists the interaction of the PLA bulk chains with PEG blocks 64.


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Water absorption Many researchers have demonstrated that cell adhesion, spreading, and growth are dependent on the surface chemistry of fibers

8, 27

. Usually, the hydrophilic/hydrophobic

balance of the scaffold surface is very important in tissue culture and can influence initial cell adhesion and survival

8, 74

. Thus, the hydrophilic character of PLA/PEG and PLA/-PELA

scaffolds was assessed looking to water wettability. The transport of liquid in a membrane is governed by interactions between the liquid and the membrane and by fiber geometry, as well as the spacing between the fibers

13, 75

. Figure 5 illustrates the influence of PEG and PELA

copolymers on the wettability of PLA fibers. According to the literature, the wetting of a membrane occurs in two phases. In the first step (phase I), the uptake displays a high initial rate of water absorption, followed by Phase II, called the equilibrium phase, where a prolonged period of slow absorption occurs 76, 77

. The amount of water absorbed during phase I is commonly defined as the amount of water

required to hydrate the specimen. Further water absorption (Phase II) would be driven by chain expansion (Figure 5). In the case of PLA, PLA/PEG2k, and PLA/PELA2k, it is of interest to note that water absorption equilibrium is reached in only a few seconds. The water content estimates did not vary substantially: there is only a variation of approximately 0.20 g in water-uptake values. On the other hand, PLA/PELA20k and PLA/PEG20k had good absorption in phase I, reaching 0.4 g of water. However, in phase II, a similar behavior of PLA/PELA2k and PLA/PEG20k can be observed. After a rapid water absorption phase, these membranes are still absorbing water continuously and did not achieves an equilibrium.


Page 29 of 53

0,7

Phase II PLA/PEG20k

0,6

Phase I Water-uptake (g)

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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0,5

0,4

PLA/PELA20k PLA/PELA2k

0,3

PLA/PEG2k

0,2

PLA

0,1

0

50

100

150

200

250

Time (sec) Phase I

Figure 5. Relative water-uptake during the imbibition period for hydrated of PLA, PLA/PELA, and PLA/PEG blends.

The results show that the addition of PEG sequences considerably improves the hydrophilicity of the blends as compared with PLA membranes 76. Therein, insofar as in the water absorption rate is related to hydrophilicity, the length of the PEG segments is an important factor

31-33

. In this work, it was consistently observed that the more hydrophilic

surfaces are those with the greatest content of PEG. The chain expansion with water absorption increases almost linearly for PLA/PELA20k and PLA/PEG20k blends, reflecting the higher water-uptake compared with PLA/PELA2k and PLA/PEG2k blends. Hendrick and


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Frey have suggested that using a lengthy chain of the PEG polymer or copolymer in the blend assists in driving more PEG to the fiber surface 13. The PLA membrane acts as a material with a hydrophobic character, which explains the low absorption of water by the membrane compared with membranes containing PEG or PELA. Bhattarai et al. also have shown that the hydrophobic character of PLA films could be decreased adding less than 10 wt% of PEG. In their study, neat PLA membranes had contact angles on the order of 80°, and after addition of PEG the contact angle decreases to 68 ° 55. Cui et al. have also reported a significant decrease in water angle measurement for PLA electrospun with the addition of PEG 8. Hendrick and Frey also showed that water wettability enhanced significantly with the addition of PELA copolymer to the PLA electrospun membranes 13. Thus, it is verified that the addition of PEG in the form of copolymers or homopolymers is an efficient way of fine-tuning hydrophilicity properties in PLA-based scaffolds, enhancing not only fiber morphology and fiber diameter distribution but mechanical properties as well. Generally, the hydrophilic/hydrophobic feature of the scaffold is of paramount importance for tissue engineering. The matrix degradation, along with surface wettability, can influence cell adhesion, cell infiltration, and cell survival on electrospun membranes 8, 27.

Hydrolytic degradation The degradation profile of scaffolds is one of the most important facets for the end application as tissue engineering. Morphological controls, such as weight variations, the degree of crystallinity, physical aging, and chain orientation, may be useful for controlling the degradation behavior of a biodegradable polymer 78. It is possible to modulate the degradation of electrospun fibers modifying the surface wettability and bulk properties of scaffolds 8.


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Towards this end, the chemical nature of the polymer matrix, i.e., its composition and its water uptake are key parameters that influence the rate of polymer degradation

79

.

Understanding the polymer's degradation mechanisms and identifying degradation products are crucial to selecting and designing polymers for tissue engineering applications. In the current study, hydrolytic degradation was carried out on PLA, PLA/PELA, and PLA/PEG blends over 12 weeks. The effects with regard to the morphological changes in fibers, mass loss of the fiber membranes, and molecular weight reduction of the polymer were determined. Figure 6 shows the plot of weight loss (wt%) as a function of time for membrane scaffolds. At the same time, Figure 6 shows the morphological changes in fibers after incubation for 3, 6, 9, and 12 weeks. A simple but rather effective technique to characterize the degradation of polymeric membranes is the recording of weight loss during the degradation period. This control of weight loss profiles allows for an assessment of the type of degradation that a polymer matrix is undergoing. The weight loss of the PLA electrospun scaffolds remained virtually unchanged for the first 3 weeks (2 wt%). Beyond 6 weeks, the weight loss increased to reach 4% and 16%, respectively, at the end of 12 weeks. In contrast, for PLA/PELA and PLA/PEG electrospun blends, an increase in weight was observed due to increased hydrophilicity as shown in the wettability test (Figure 5). Changes in composition have an influence on the degradation profiles of PLA/PELA and PLA/PEG fibers. However, PLA/PEG2k, PLA/PELA20k, and PLA/PEG20k showed a rapid loss of their initial weight until week 3 (13, 14, and 20 wt%, respectively), while a constant loss was detected for the PLA/PELA2k (4 wt%). A possible explanation is that the initial weight loss is attributed to the diffusion of water and these membranes have a greater hydrophilicity on the fiber's surface (this region is sometimes called the induction period) 12.


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Indeed, the faster water absorption must favor the dissolution of PEG on the surface and consequently accelerates the initial hydrolytic degradation of the membranes 8, 32, 39. Although PLA/PELA blends (2k and 20k) showed a different initial degradation profile, after 3 weeks, these membranes exhibited a similar behavior, with a fast weight-loss rate until the end of 12 weeks. Notably, PLA/PEG blends (2k and 20k) also showed a similar degradation profile up to 12 weeks, regardless of the PEG chain length. At week 12, there was a nearly 30 and 70 wt% mass loss for the PLA/PELA and PLA/PEG, respectively. The greatest weight loss, in the case of copolymers blends (PLA/PELA) compared with homopolymer blends (PLA/PEG), mainly resulted from the existence of PEG segments in the PLA middle chains. Various authors have suggested that during this period the chain scission occurs at the ester linkage connecting PEG-PLA blocks

12, 14, 80

. The cleavage of

ester-ether junctions occurs more quickly in the case of PLA copolymers because the preferential cleavage is exactly in the LA-EG junctions 14. Hu and Liu have proved by NMR the formation of hydroxyl end groups connected to PEG blocks and carboxyl end groups connected to PLA blocks after hydrolysis of PELA triblock copolymers 81.

In addition, the inter-block binding with PEG causes some local interference in the PLA crystalline matrix, causing greater vulnerability in the end LA units to water molecule attack.

34

. As a result, PLA oligomers of the short copolymer segments are released

accelerating the cleavage of PEG blocks and PLA bulk.

12

.

The free oligomers will

accelerate the hydrolysis of other ester bonds and consequently speed up the degradation process. This mechanism is determined as autocatalysis. 78.


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Figure 6. Percentage weight loss versus time and morphological changes of in vitro degradation of electrospun scaffolds of PLA, PLA/PELA, and PLA/PEG blends. 33 ACS Paragon Plus Environment

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In all cases, matrix based on PLA/PELA or PLA/PEG blends were swollen between 6 and 9 weeks compared with undegraded fibers, which is due to chain relaxation of the polymer after incubation of the scaffold in the PBS medium (Figure 6). At 9 weeks, fibers seemed partially adhering to one another and be breaking down. At the end of 12 weeks, it was found that for all membranes, most of the fibers were broken down. The fibrous structure with PELA copolymer scaffold disappeared, and a membrane-like structure, which agglomerated from fragmented chunks, was formed. The hydrolytic degradation of all scaffolds is carried out in 4 steps as represented in Figure 7: I) water diffusion, II) formation of hydroxyl end groups connected to PEG and carboxyl end groups connected to PLA, III) oligomers with acidic end-groups catalyze the hydrolysis reaction and water molecules diffuse into the fibers created by the removal of the oligomers and IV) a critical weight loss is reached due to the presence of oligomers entrapped into polymer contributing totally to the autocatalytic effect. Meanwhile, soluble oligomers that are close to the surface start to diffuse out from the polymer, which in turn encourages the breakdown of the fibers.

Figure 7. Schematic representation of hydrolytic degradation of fibers containing PLA. 34 ACS Paragon Plus Environment

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Hydrolysis is evidenced by two stages of degradation: surface erosion and bulk degradation. The degradation mechanism for the scaffolds is dependent on the water speed penetration into the fiber (surface erosion) and chemical bond breakdown of the polymer (bulk degradation). For biodegradable polymers, the degradation mechanism can be

determined by the difference between the breakage rate of chemical bonds in the molecular chains and distribution of water penetration into the polymeric matrix 8. As shown in Figure 8, the decrease in MW (see ESI) is much smaller even though of the greater weight loss of these membranes, suggesting that the erosion of fibers proceeds mainly via surface dissolution. Furthermore, PLA and PLA/PEG blend fibers show a greater surface erosion than PLA/PELA blends, and the weight reduction was relatively higher than the molar mass loss for these membranes. For instance, for PLA and PLA/PEG membranes, the molar mass dropped down to 10% of its initial value compared to a loss of 30%−40% for PLA/PELA up to 12 weeks.

100 90 80

Molar Mass (Mw)

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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70 60 50 40

PLA PLA-PELA2k PLA-PELA20k PLA-PEG2k PLA-PEG20k

30 20 10 0 0

1

2

3

4

5

6

7

8

9

10

11

12

Degradation Time (Weeks)

Figure 8. Percentage of PLA (100k) degradation products based on SEC traces of PLA, PLA/PELA, and PLA/PEG membranes.


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The addition of PEG in both blends allows a greater hydrophilicity on the fibers surface resulting in the dissolution of PEG chains and faster water diffusion into the chains. The results demonstrate that the hydrolytic degradation of the scaffolds with PELA copolymer is influenced by the amount of degradable ester linkages between PEG and PLA blocks.

Degradation based on the surface erosion mechanism occurs when the dissolution of the polymer is faster than the breaking of the bonds in the matrix

8, 82

. For scaffolds

containing PEG homopolymers, the fibers degradation occurs by erosion due to the availability of the hydrophilic polymer on the fibers surface. The non- or semi-miscible PLA/PEG blends, allows that regions containing PEG dissolve more easily in the aqueous surrounding fluid, occurring the erosions in the fibers. The use of PELA copolymers may accelerate the formation of lactide acid turns out to be an effective way to increase the degradation ratio of the electrospun PLA-based scaffold. For PLA/PELA scaffolds, in addition to catalytic degradation due PLA oligomer release, amorphous regions are more prevalent in these blends. The increase of PLA oligomers, and consequently lactic acid, causes an accelerated hydrolysis of the ester bond between PLA-PEG segments resulting in further degradation. However, the lactate degradation product is also easily removed from the fibers therefore occurs both types of degradation in this type of blend. In conclusion, fibers containing PLA/PEG displayed a surface erosion, while fibers with middle levels of PLA/PELA showed a profile between surface erosion and bulk degradation.

The slow degradation rate of the neat PLA fibers through hydrolysis may be due to a slow water diffusion into the polymer chains. The PLA-based scaffold displays a more hydrophobic character, the fibers are coalescing with each other, and pore sizes are small


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which hinders the water diffusion, and consequently, the breakage of the backbone ester groups 21. The degradation rate is often considered to be an important selection condition for implantable scaffolds

21

. Thus, the degradation rate of the PLA-based scaffold can be too

slow for neural tissue engineering. A PLA-based implant can leads to a long in vivo life time when these scaffolds have a slow degradation rate 83, 84_ENREF_76. One goal of tissue engineering is to allow the body’s own cells, over time, to eventually substitute the implanted scaffold 85. Ideally, a scaffold must have a degradation rate combined with the formation of new-tissue in order to make a smooth transition of the artificial cell-support for a natural tissue. 86.

Effect of hydrophilic-hydrophobic systems on NSC behavior The use of NSC–polymer constructs may be a powerful technique for in vitro study and in vivo treatment of neurodegenerative diseases. In neural tissue engineering, the first step in developing a good polymer-cell interaction is the construct of the scaffold with a suitable surface for the intended application. In this section, monkey embryonic stem cells (ESCs) and NSCs were used as model cells to test the cell affinity of the electrospun blends. Monkey

ESCs were previously derived from Rhesus monkey embryos, and exhibit all of the characteristic features of primate ESCs

50

. Two types of monkey NSCs were used: NSCs

derived from monkey ESCs (ESC-NSCs) (Figure 9) and fetal NSCs derived from fetal monkey brain (Figure 10A). Monkey ESCs and NSCs share the same properties as human ESCs and NSCs, which make them the most appropriate cell types to pre-validate the behavior of primate cells on the different scaffolds. Furthermore, monkey NSCs can be grafted on the monkey brain with a reduced risk of immune rejection, and represent


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invaluable tools for preclinical studies involving neural tissue engineering for the treatment of neurodegenerative diseases. We first characterized NSCs upon culture on PLA, PLA/PELA, and PLA/PEG blend based-scaffolds (Figure 9 and 10A,B). We found that when seeded on the neat PLA, PLA/PELA2K, and PLA/PELA20K blend substrates, ESC-NSCs expressed the NSC markers Nestin and SOX2 (Figure 9 A,B), as observed with fetal-NSCs (Figure 10B). ESC-NSCs also expressed the proliferating marker Ki67 and rarely expressed the apoptotic marker Cleaved-Caspase3 (Figure 9 A,B). On neat PLA and PLA/PELA20k, a high proportion of ESC-NSCs expressed SOX2 (71% and 67%, respectively) (Figure 9C), and the cell density was similar (on average 541 and 494 cells/mm2, respectively after 2-day culture). On PELA/PLA2K, the cell density was reduced (339 cells/mm2), and the proportion of Sox2 was slightly weaker (57%). Taking advantage of TAU-GFP labeling, which allows the visualization of cell morphology in exquisite details 45, we also observed that ESC-NSCs exhibited typical NSC morphology when cultured on PLA and PLA/PELA2k and 20k membranes, extending nice processes from the cell bodies. The culture of ESCs on PLA and PLA/PELA20K induced neural differentiation of ESCs, as judged by the reduced OCT4 expression, and presence of numerous Nestin+ neuroepithelial rosettes. In contrast, we noted cell death and decreased OCT4 and Nestin expression on PLA/PELA2k (Figure 10C), suggesting that PLA/PELA2k substrate is not as favorable as PLA and PLA/PELA20k for the maintenance and neural differentiation of monkey ESCs.


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Figure 9. Characterization of ESC-NSCs upon culture on PLA, PLA/PEG2K, PLA/PEG20K, PLA/PELA2K, and PLA/PELA 20K scaffolds. A) Live imaging of TAU-GFP expressing 39 ACS Paragon Plus Environment

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NSCs and immunostaining for Nestin and Cleaved-Caspase3 in NSCs cultured for 1DIV Scale bar, 50 µm (Caspase3 staining); 20 µm (Nestin staining). B) Immunostaining for TAU-GFP, SOX2, and Ki67 in ESC-NSCs cultured for 2 and 5DIV on the different scaffolds. Scale bars, 50 µm (2DIV); 20 µm (5DIV). C) Cell density and percentage of SOX2-positive cells after 2DIV; mean +/- SEM; n = 2. Abbreviations: DIV, days in vitro.

In sharp contrast with the results obtained with PLA and PLA/PELA scaffolds, very few ESC-NSCs survived on PLA/PEG2K and 20K (Figure 9A), resulting in a very low cell density at 2 DIV (6 and 107 cells/mm2, respectively). Cells exhibited a rounded shape and grew as compact cell clumps, two morphological features that are not characteristically found in NSCs. After 5DIV, no ESC-NSCs could be observed on either PLA/PEG2K or 20K membranes. These results suggest that upon seeding on PLA/PEG membranes, cells rounded up and rapidly detached from the membranes, possibly following apoptosis. Similarly, fetal NSCs and ESCs did not survive upon culture on PLA/PEG2K and 20K (Figure 10B,C). These results indicate that PLA/PEG2K and 20K membranes are not appropriate for the maintenance of monkey NSCs and ESCs.


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Figure 10. Characterization of fetal-NSCs and ESCs upon culture on PLA, PLA/PEG2K, PLA/PEG20K, PLA/PELA2K and PLA/PELA 20K scaffolds. A) Phase contrast of fetal NSCs. Scale bar, 20 µm. (B) Nuclear staining of NSCs (DAPI) revealed that PLA/PEG2K and 20K do not sustain NSC maintenance, in contrast to PLA and PLA/PELA scaffolds, left 41 ACS Paragon Plus Environment

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panels; Immunostaining for Nestin and Sox2 in fetal NSCs cultured for 6DIV, right panels. Scale bars, 50µm (DAPI); 20µm (Nestin/Sox2). C) Immunostaining for OCT4, Nestin and TAU-GFP in ESCs cultured for 6DIV on the different scaffolds. Left panels: Scale bars, 50µm. On PLA, shown are Nestin+-neuroepithelial rosettes surrounding a lumen (white dotted line), characteristic of neural differentiation. These structures are also observed on PLA/PELA20K. On PLA/PELA2K, ESCs did not express Nestin and Oct4, and rounded cells with fragmented nuclei were observed at the periphery of the colonies (yellow arrow-heads), suggestive of cell death. On PLA/PELA20K, few OCT4+ cells were observed in the colonies (white arrow head). Right panels: Scale bars, 10µm. Abbreviations: DIV, days in vitro.

Intriguingly, the results revealed that the PLA membrane was more effective in sustaining NSC maintenance. We expected better results with electrospun PLA/PEG and PLA/PELA blends, due to the hydrophilic-hydrophobic characteristics of these membranes. However, the NSC density was almost the same in the PLA/PELA blend as in neat PLA (Figure 9B). Two explanations can be given for the following results. The first one is that the rapid PEG dissolution in PLA/PEG membranes in cell culture medium can result in the obliteration of the three-dimensional porous structure, and shuts the interconnecting pores. Secondly, it is generally thought that a very hydrophilic surface can result in a suppression of cell adhesion and absorption of proteins. Cui et al. have reported that in PLA/PEG blends, the cells survived and proliferated in a smaller number when the scaffolds had a high wettability or a low wettability 8. The objective of tissue engineering is to proliferate the endogenous cells to replace the implanted scaffold and eventually renew the tissue over time. The scaffold must have a


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controlled decay rate so as to allow cells to produce their own extracellular matrix

85

. The

basic requirements for an implantable scaffold are thus its capacity and its speed of degradation. As previously discussed, the hydrophilic PEG segments into the PELA copolymer speeds up the water penetration within the scaffold and a bulk degradation pattern was found (Figure 8). The rate of degradation was influenced by the chemical composition (lactic acid subunits), the molecular weight, and the crystallinity of the polymers. We showed that despite their good cellular activity, PLA membranes do not exhibit a high degradation rate.

A crucial factor in neural tissue engineering is to understand the mechanosensitivity between cells and their substrate. In general, mechanosensitivity requires the conversion of mechanical forces applied to cells from the outside or of an active measurement of stiffness of the surroundings by the cells themselves into intracellular biochemical signals 22, 23. During brain development, NSCs will differentiate into mature neural cells and glial cells. The NSCs respond with different mechanical behavior of the membrane during cell

differentiation.

Biomechanical

signals

that

a

scaffold

transmits

to

the

microenvironment of neural tissues and their cellular constituents is extremely important because glial and neuronal cells respond to these stimuli to proliferate and differentiate 87. George et al. have reported that in a mixed environment, astrocytes exhibit better behavior in a soft matrix, whereas neuronal cells respond to a broader range of mechanical properties 88. The low elastic modulus of the adult brain and spinal cord coincides with the near absence of the kind of ECM that forms scaffolds in most other soft tissues. Thus, a scaffold for neural tissue engineering must have a balance between tensile modulus and strain. Although the PLA membrane has shown a decrease in modulus, this also showed a significant improvement in strain. Thus, the membranes containing the copolymer PELA


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had a better compromise between flexibility and stiffness, allowing NSCs to survive and differentiate in vivo and in vitro. High porosity in PLA/PELA membranes compared with neat-PLA membranes also constitutes an important feature in order to enable an efficient circulation of nutrients and wastes, but also to provide enough of a void for ECM regeneration. In this context, PLA/PELA20k membranes may thus represent attractive alternatives to PLA membranes.

CONCLUSION In this work, electrospun PLA-based scaffolds were developed by combining as blends PLA with PELA copolymer or PEG homopolymer. Thus, an easy way for the development of PLA-based membranes having improved hydrophilicity was demonstrated. PLA-based electrospun membranes exhibit fibers having diameters of 310 nm, whereas the ones based on blends have fibers with much lower diameters, i.e. ranging from 110 to 125 nm. Scaffolds also showed improved porosity after blending, this means 78% for neat PLA fibers, as compared to 82-89% for the fibers blends. Likewise, the data obtained highlight that the mechanical properties as well as the hydrolytic degradation kinetics of the fibers could be improved with a mixture of biopolymers, mainly for blending with PELA copolymer. A decrease of the tensile modulus was determined for all blends while the opposite phenomenon was observed concerning the elongation at break with PELA blend. According to the thermal properties this result is due to a more effective blend with copolymers. We conclude that the presence PELA copolymer improves compatibility between the hydrophilic-hydrophobic segments of the blend.


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In vitro degradation and the hydrophilicity of the membranes can be modified by varying the chemical composition of the blend. Our results have shown that the best way to produce PLA based scaffolds having suitable properties is to use the PELA2k copolymer for a blend. Our results revealed the PLA electrospun fiber constitute a good scaffold for the survival and proliferation of monkey NSCs and for inducing neural differentiation of ESCs. The increase in hydrophilicity did not greatly improved NSC and ESC maintenance. However, PLA/PELA20k and PLA exhibited similar neural inducing activity on ESCs, and sustained the maintenance of NSCs, regardless of their origin (derived from ESCs or from fetal brain). Although the electrospun blends did not improve cell maintenance as compared to PLA fiber, they have the advantage to exhibit a suitable hydrolytic degradation and a better elongation at break, which is required for an implantable scaffold.

ACKNOWLEDGEMENTS This work was supported by the National Council for Scientific and Technological Development – CNPq, Brazil; LABEX CORTEX (ANR-11-LABX-0042) and LABEX DEVweCAN (ANR-10-LABX- 061) of Université de Lyon, within the program ‘Investissements d'Avenir’ (ANR-11-IDEX-0007), operated by the French National Research Agency (ANR); IHU CESAME (ANR-10-IBHU-0003).

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bioresorbable

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Development

of

hydrophilic-

hydrophobic

electrospun scaffolds for neural

tissue engineering Luanda Chaves LINSa*, Florence WIANNYb, Sébastien LIVIa*, Idalba Andreina HIDALGOa, Jannick DUCHET-RUMEAUa, Jean-François GÉRARDa.

Influences of hydrophilic-hydrophobic bioabsorbable scaffolds on monkey neural stem cells.


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Biomacromolecules

Influences of hydrophilic-hydrophobic bioabsorbable scaffolds on monkey neural stem cells. 254x190mm (96 x 96 DPI)


Development of Bioresorbable HydrophilicâHydrophobic Electrospun

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