electrospn 9 nedjari-full

The International Istanbul Textile Congress 2013 May 30th to June 1th 2013, Istanbul, Turkey

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MICRO-WEAVING OF HONEYCOMB NANOFIBROUS MEMBRANES: APPLICATION TO BONE REGENERATION

S. NEDJARI1, S. EAP2, A. HEBRAUD1, N. BENKIRANE-JESSEL2, G. SCHLATTER1

1ICPEES Institut de Chimie et Procédés pour l’Energie, l’Environnement et la Santé UMR 7515, CNRS, Université de Strasbourg, 25 Rue Becquerel, 67087 Strasbourg Cedex, France

2INSERM Nanomédecine Régénérative Ostéoarticulaire et Dentaire UMR 1109, Université de Strasbourg, 11 Rue Humann, F-67085 Strasbourg Cedex, France

[email protected] Abstract: Controlling the deposition of the nanofibres is crucial issue in novel strategies for tissue engineering because electrospun structured scaffolds can mimic the complex structures of the body. Electrostatic forces can be controlled at the vicinity of the collector by using structured collectors with different shapes and dielectric properties [1]. By using this strategy, honeycomb electrospun nanofibrous scaffolds with pattern diameters of 40 to 360 microns were obtained. Fibres structuration and osteoblastic cell growth on electrospun scaffolds of two FDA approved polymers, poly(ε-caprolactone) and poly(D, L-lactic acid), were studied : optimum structuration was achieved by playing on processing parameters (solution conductivity, distance, voltage). The fibre morphologies, the scaffold roughness and the honeycomb patterns size of the membrane were perfectly controlled. Keywords: honeycomb electrospun scaffolds; bone regeneration; micro-weaving; organised nanofibres; 1. Introduction Nerves, heart, bones are among the most complex structured tissues of the body. Researchers are developing techniques to create new biomaterials that can enhance the tissue reconstruction. Electrospinning is one of the most promising ways to generate scaffolds for tissue engineering: nanofibres provide a good substrate for cells adhesion and proliferation because they can mimic the extra cellular matrix. Moreover, as electrospinning is a very versatile method, many natural polymers (gelatin, silk fibroin chitosan, collagen) and synthetic (PCL, PLA, PGA) can be electrospun and a large variety of nanofibrous scaffolds can be achieved [2]. Although, electrospun nanofibres are generally obtained in random way, organised nanofibres (aligned nanofibres, nanofibres meshes) [3] can be generated by changing the nature of the collector and therefore the electrostatic forces applied on the jet. Indeed, at the vicinity of the collector, the electrostatic forces are driven by the local structuration and dielectric properties of the collector [1]. By controlling the organisation of the fibres, the scaffold’s performances are improved and the regeneration is enhanced: as an example, for nerve regeneration application, when neurites are cultured on a parallel array of aligned nanofibres, they preferentially extend along the long axis of the fibres [4]. Following this strategy, different honeycomb collectors were made by lithographic processes in order to create honeycomb membranes for bone reconstruction. The range of the honeycomb pattern size is from 40 µm to 360 µm. Using this honeycomb micro-patterned collectors, PCL and PLA honeycomb nanofibres membranes are successfully obtained (Figure1). By playing on the processing parameters, control of the structuration can be achieved. Morphology of the scaffolds (fibre diameter, alignment degree, shrinkage) is analysed and the differences of behaviour between PCL and PLA are shown. Finally, the influence of the structuration on osteoblasts cells proliferation and cells morphology will be studied.


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Figure 1: Electrospinning setup using a honeycomb collector.

2. Materials and Methods Honeycomb micro-patterns were obtained thanks to photolithography process on silicon wafers. Photoresist SU-8 2050 was used to obtain patterns with H = 60 µm in height. A conductive layer (Al-120 nm and Au-30 nm) was deposited on the wafers to make them conductive. Internal diameters of the honeycomb are DHC = 40 µm; 80 µm; 160 µm and 360 µm. The width of the honeycomb wall is fixed to 20 µm. Poly-(ε-caprolactone) (PCL) (Mw = 80 kg/mol-1,Perstrop, commercial name: CAPA 6806) was dissolved into dichloromethane (DCM )/N,N-dimethyl-formamide (DMF) (60/40 V/V) at a concentration of 15 % w/w. 5% of formic acid was added to the solution to increase its conductivity . The flow rate was at 1.1 mL/h and the tip to-collector distance was 13 cm. The voltage was 25 kV. After 10 minutes of electrospinning, the PCL honeycomb scaffold was immerged into ethanol to peel the membrane from the collector. Poly (D, L-lactic acid) (PLA), (Mw = 180 kg/mol-1, Natureworks, commercial name: 7000D) was dissolved into (50/50 V/V) DCM/DMF, at a concentration of 8.2% w/w. The flow rate was also 1.1 mL/h and the distance was 18 cm. Two power supplies were used: at the needle, the voltage was Vneedle = +12 kV and at the collector, it was Vcollector = -5 kV. After 15 minutes of electrospinning, the membrane was peeled from the collector in isopropanol. The morphology of the honeycomb nanofibres scaffolds were observed with a scanning electron microscopy (SEM) with an accelerating voltage of 5 kV (VEGA 3 LMV, TESCAN, Czech Republic). 3. Results

Figure 2: A) SEM image of a PCL honeycomb scaffold. B) SEM images of PLA honeycomb scaffold

Thanks to architectured collectors, electrospun PCL and PLA scaffolds present honeycomb electrospun shape. However, the morphology of architectured PCL scaffolds is different from architectured PLA ones (Figure 2A and 2B). For PCL scaffolds, a bimodal distribution of fibre diameters is observed, with thin fibres of diameter around 250 nm and thicker fibres of diameters between 1 and 2 µm. The thicker fibres, probably still wet and more charged are attracted by the conductive walls of the collector. Therefore, thicker fibres are preferentially situated on the hillside of the wall, forming high walls, while thinner fibres are located both in the centre and on the walls of the honeycomb patterns. Only fibres on the hillside align along the sides of the hexagon shape.

B) A)


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In contrary, for the PLA scaffolds, the fibre diameter distribution is monomodal and the average diameter is around 500 nm. In this case, all the fibres located on the wall present high degree of alignment along the sides of the hexagon shape. Due to the differences in fibre diameter distribution and fibre deposition, the PCL scaffolds present a higher roughness than PLA ones with higher honeycomb walls. Besides, the height H of the scaffold is also increasing with the diameter of the honeycomb pattern. Moreover, shrinkage happens during the peeling (in ethanol for the PCL and in isopropanol for the PLA scaffolds) of the scaffolds from the patterned collector. Thus, and the honeycomb pattern size of the final membrane is generally inferior to the collector one. This shrinkage is more important for PLA than for PCL. Fibre diameter, alignment degree and the roughness are parameters that will influence osteoblasts behaviour. In vitro studies on these architectured scaffolds will show the impact of the honeycomb organisation the osteoblasts proliferation and morphology. The comparison between PCL and PLA will help us in choosing the best biocompatible polymer for structured electrospun scaffold for bone regeneration. 4. Conclusion The lithographic process is a good way to produce structured collectors with specific shapes. Coupled with electrospinning, honeycomb electrospun scaffolds in PCL and PLA can be made with a good control of their morphology. By using the same collector, differences are observed between the final structures of PCL and PLA scaffolds. These differences will affect osteoblasts cell behaviour, known to be very sensitive to the topography and the roughness of the substrate. Acknowledgements This study was supported by the Agence Nationale de la Recherche (NeoTissage), France. We also thank the cleanroom STNANO of Strasbourg: Sabine Siegwald, Romain Bernard for their technical support. We also thank Christophe Mélart, Christophe Sutter and Thierry Djekriff for their help in the electrospinning set up. References [1] Lavielle N., Hébraud A., Mendoza C., Ferrand A., Benkirane-Jessel N., Schlatter G., Structuring and molding of electrospun nanofibres: Effect of electrical and topographical local properties of micro-patterned collectors, Macromolecular Materials and Engineering, Vol. 297 (2012), No 10, pp. 958-968 [2] Goh Y.-F., Shakir I., Hussain R., Electrospun fibres for tissue engineering, drug delivery, and wound dressing, Journal of Materials Science, Vol. 48 (2013), No 8, pp. 3027-3054 [3] Teo WE, Ramakrishna S., A review on electrospinning design and nanofibre assemblies, Nanotechnology, Vol. 17 (2006), No 14, pp. R89-R106 [4] Xie J., MacEwan MR., Li X., Sakiyama-Elbert SE., Xia Y., Neurite Outgrowth on Nanofibre Scaffolds with Different Orders, Structures, and Surface Properties, ACS Nano, Vol. 3 (2009), No 5, pp.1151-1159

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