2 Development of Advanced Technologies for Manufacturing of Tissue Engineering Scaffolds

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2 Development of Advanced Technologies for Manufacturing of Tissue Engineering Scaffolds

Carlos Mota

¹

, Dario Puppi

¹

, Federica Chiellini

¹

, Emo Chiellini

¹

1

Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOLab), Department of Chemistry and Industrial Chemistry, University of Pisa, via Vecchia Livornese 1291, 56010 San Piero a Grado (Pi), Italy.

Abstract

The development of new computer-aided manufacturing technologies, for the production of scaffolds with customized internal structure and external shape, allows to minimize the handiwork and to enhance the reproducibility and control over scaffold morphology. Within the framework of the present PhD thesis, a melt extrusion-based additive manufacturing (AM) equipment was developed and combined with electrospinning technique for the fabrication of dual-scale micro/nanostructured scaffolds. This system was further modified to produce melt-electrospun three-dimensional scaffolds. Changes in this equipment were performed to study the feasibility for the production of wet-spinning scaffolds with a layer-by-layer approach. Moreover, a new computer controlled wet-spinning apparatus was specifically developed for the production of scaffolds by AM, starting from polymer solutions.

2.1 Introduction

The complexity of scaffolds, in terms of composition, microarchitecture, and external shape and

size, designed for the engineering of different types of tissues and organs requires the introduction

of new fabrication technologies capable of satisfying a great variety of prerequisites. Researchers in

the Tissue Engineering (TE) field started to explore manufacturing technologies developed for

different industrial applications and soon realized their potential use in scaffolds fabrication. As an

example, electrospinning (ES) was originally developed for the production of fibres for other

applications (e.g. filtration membrane) [Dersch et al., 2007; Gopal et al., 2006; Teo and

Ramakrishna, 2006] and it is nowadays investigated for the production of nano/microfibrous

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scaffolds for TE [Boudriot et al., 2006; Pham et al., 2006; Puppi et al., 2010b; Sill and von Recum, 2008; Teo et al., 2011]. Additive manufacturing (AM) techniques were initially developed for the production of prototypes for automotive and aerospace industries and in the last ten years have been investigated as tools to automatically produce TE scaffolds. For instance, stereolithography was studied to produce moulds to cast hydroxyapatite (HA) scaffolds [Levy et al., 1999] while Fused Deposition Modeling (FDM) was investigated for the production of poly(ε-caprolactone) (PCL) and PCL/HA scaffolds [Hutmacher, 2000]. A number of studies have reported the development of customized AM equipments capable of processing a wide range of materials for the fabrication of scaffolds with customized internal and external structure. The developed AM technologies have gained popularity due to reproducibility, reliability and capability to design precise porous structures for TE applications. Another advantage is the possibility of producing anatomically-shaped, clinically-sized scaffolds on the basis of three-dimensional (3D) models acquired from the patient using medical imaging techniques (e.g. magnetic resonance imaging, X- ray computed tomography). Within the present PhD, the development of novel techniques and equipments for the fabrication by AM of complex structures with different morphologies and loaded with bioactive agents was investigated.

2.2 Development of Hybrid Equipment Combining Additive Manufacturing (AM) and Electrospinning (ES) Technologies

A new hybrid system combining ES and a melt extrusion-based AM technique was designed, assembled and employed to produce multi-scale scaffolds composed of structural elements with different chemical composition and size scale (Figure 1a-c).

Figure 1 - Novel additive hybrid apparatus (ES + AM): a) simplified scheme, b) 3D model of the equipment

and c) photo of the assembled equipment.

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The AM system is based on the principles of a previous manufacturing apparatus named Bioextruder [Domingos et al., 2010; Domingos et al., 2009; Mateus et al., 2007; Mota et al., 2009], composed of a screw extrusion head and allowing the controlled deposition of polymer melt filaments with a layer-by-layer approach. Moreover, this system allows the processing of a wide range of synthetic and natural polymers with the possibility of incorporating bioceramics or other bioactive agents into the produced filaments. The AM system and the controlling software were developed in collaboration with the Centre for Rapid and Sustainable Product Development of the Polytechnic Institute of Leiria (Portugal).

2.2.1 Additive manufacturing hardware

The novel hybrid AM equipment is based on a modular assembly, allowing an easy customization

of the fabrication system configuration. It is composed of four axis X, Y, Z and A (platform rotary

axis) controlled by individual stepper motors. With this equipment it is possible to use translation

speeds of the needle up to 600 mm·min

^-1

. The control of each axis is performed by an ad-hoc

controller console (Figure 2a) directly connected to a line print terminal 1 (LPT1) port of the

desktop computer. The screw extruder head is attached to the X and Y axis allowing a controlled

deposition of the melt filament in the X-Y plane. The screw extruder head incorporates two hot

runner coil heating elements of 250W, connected to the pre-melting reservoir and to the extrusion

chamber, that are controlled independently by means of proportional–integral–derivative (PID)

temperature controllers (ESM-9430, EMKO). These PID controllers allow a good stability of the

temperature throughout the whole process and minimize the residence time of the melt at high

temperature, thus avoiding the risk of polymer thermal degradation. Moreover, the temperature

console allows to control a further third zone. The pre-melting reservoir, with a capacity of 50 ml,

is connected to a N

2

gas line to force polymer melt flowing to the screw. The screw extruder is

driven by a direct current (DC) motor connected to a DC power supply that allows the variation of

screw rotation speed until a maximum of 29 r.p.m. for an electrical voltage of 24Vdc. The

construction platform is actuated by two stepper motors: one on the Z axis and another on the A

axis (rotation). The last one allows the automatic positioning of the construction platform on the

horizontal direction for the collection of AM filaments, and on the vertical direction for the

collection of electrospun fibres. The properties of the assembled equipment are resumed in Table 1.

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Table 1 - Hybrid system: technical data

Number of axis Four: X, Y, Z and A (rotary axis)

Working volume 150 x 150 x 100 mm

Rotary axis range 0º to 90º

Working temperature Room temperature to 250°C

Nozzle size 0.1 mm to 1mm

Reservoir pressure range 1 bar to 10 bar

Reservoir capacity 100 ml

Temperature controller P.I.D (three zones) Screw Rotation Range 0 – 29 r.p.m.

Communication port Serial Port

Communication language NC-code

High voltage power supplies

N°2 SL60PN300 – 60kV (maximum voltage) and reversible polarity;

N°1 SL70P60 - 30 kV (maximum voltage).

Syringe pump BSP-99M ( feed rate from 0.145 to 140 ml·h

^-1

)

2.2.2 Additive manufacturing software

The filament deposition patterns designed and employed during this research activity were

calculated on an algorithm developed in Matlab (The MathWorks, Inc.) [Bártolo et al., 2004]. This

algorithm was modified according to the equipment used and to the experiments performed. The

pattern code used in all equipments is based on NC-code language, a standard language used in

computer numerical control machines. In the case of the hybrid system the patterns, calculated

using Matlab, were loaded into the controlling software. This system is controlled in real-time

using the EMC2 free license software (GNU GPLv2, General Public License version 2)

[http://www.linuxcnc.org/]. This software runs under Linux operative system and it was adapted to

control the AM prototype hybrid system.

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Figure 2 – a) Command and control station of the hybrid equipment composed of temperature controller, four axis console, computer and DC power supply, b) ES power supplies.

2.2.3 Electrospinning (ES)

The AM system previously described was coupled to an ES apparatus in order to produce multi-

scale and multi-material scaffolds. The ES system [Puppi et al., 2010a; Puppi et al., 2010b; Puppi

et al., 2011] is composed of three high voltage power supplies (Spellman High Voltage Electronic

Corporation, United Kingdom): two with reversible polarity (SL60PN300) and 60 kV of maximum

output voltage, and one with positive polarity (SL70P60) and 30 kV of maximum output voltage

(Figure 2b). A programmable logic controller (PLC) allows the remote control of the high voltage

power supplies, and the equipment includes interlock safety features. A syringe pump (BSP-99M,

Braintree Scientific Inc, MA) allows the control of the solution feed rate (Figure 1a).

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2.3 Development of Screw Extrusion-based Melt-electrospinning (Melt-ES) Apparatus

In order to study the processability of PCL polymer into 3D melt-electrospun scaffolds (see chapter 8) the hybrid system was further modified. To produce melt-electrospun fibres, an electrical field was applied to the AM instrument between the nozzle and the construction platform. A conductive copper plate (10x10 cm) functioning as collector for the melt-electrospun fibres was fixed onto the construction platform. The melt-electrospinning (melt-ES) experiments were performed with and without the movement of the extruder head in the X-Y plane. The head motion allowed fabricating 3D meshes exploiting a layer-by-layer concept. For the melt-ES experiments the Z axis was only used for the positioning of the platform to the desired distance from the extrusion nozzle.

The electrical field was created by means of a high voltage power supply (SL70P60, Spellman High Voltage Electronic Corporation, United Kingdom) connected to the construction platform while the screw extruder was electrically grounded in order to prevent electrical discharges.

The temperature and humidity on the spinning chamber were controlled by means of an auxiliary infrared lamp (Philips, 150W) and monitored using a thermo-hygrometer.

2.4 Modification of the AM Apparatus for Computer-controlled Wet-spinning

The development of a computer-controlled wet-spinning technique, that exploits layer-by-layer deposition of 3D scaffolds for TE, was investigated modifying the AM equipment (Figure 3) as reported by following.

Figure 3 - Computer-controlled wet-spinning apparatus: a) Simplified scheme; b) Image of the modified

equipment.

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A syringe pump NE-1000 (New Era Pump Systems Inc., Wantagh, NY, USA) was used to control the feeding rate of the polymeric solution to the tip of the needle. The desired solution was loaded into a syringe and connected to the extrusion needle by means of a plastic tube. The extrusion nozzle was fixed to the inactivated extrusion head allowing the movement in the X-Y plane as previously described.

A beaker containing the desired non-solvent was attached on the top of the construction platform and the needle was immersed into the non-solvent at the desired distance to the bottom of the beaker. The needle was kept immersed along the fabrication process and the platform was lowered after the production of each layer in order to keep constant the distance between the needle tip and the last layer. The good results obtained with this novel approach (see chapter 5), allowed the development of a new AM equipment for the fabrication of wet-spun scaffolds.

2.5 Development of a New Computer-controlled Wet-spinning Apparatus

An ad-hoc computer-controlled wet-spinning system was developed by adapting a subtractive Rapid Prototyping (RP) equipment (MDX-40A, ROLAND DG Mid Europe Srl, Italy) to allow the deposition of polymeric solutions with a controlled 3D pattern (Figure 4).

Figure 4 - Computer-controlled wet-spinning equipment.

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The milling head unit of the Roland machine was replaced by a syringe pump system (NE-1000, New Era Pump Systems Inc., Wantagh, NY, USA). The syringe pump was placed in vertical position eliminating the use of a transferring tube as described in section 1.4. Moreover, the high accuracy of the MDX-40A equipment allowed a better control over the deposition of the fibres.

With this equipment it is also possible to use translation speeds of the needle up to 1800 mm·min

^-1

, thus reducing the production times of each scaffold. Moreover, the big platform that equips the MDX-40A equipment (X=305mm, Y=305mm, Z=105 mm) allows consecutive production of several scaffolds in the same production set.

The lay-down pattern for the production of 3D scaffolds was calculated using an algorithm developed in Matlab software as previously described. The generated pattern was uploaded into the Roland equipment through the software Vpanel for MDX-40A. Scaffolds with different internal and external morphology can be produced.

2.6 Conclusions

The production of scaffolds using computer-controlled technologies can allow to enhance the control over internal and external structures and to enhance the production rate. An advanced melt extrusion-based AM apparatus was developed and combined with an ES equipment for the production of multi-scale and multi-material TE scaffolds. Such equipment can be easily adapted for the production of scaffolds with melt-ES and computer-controlled wet-spinning principles.

Furthermore, a dedicated apparatus for the production of wet-spun scaffolds with high control over microarchitecture of pores and high production rate was developed. By means of this system it is possible to produce scaffolds from a wide range of polymers and with the possibility of including bioactive agents and/or cells.

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