Improvements in Resolution of Additive Manufacturing: Advances in

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Review

Improvements in resolution of additive manufacturing: advances in twophoton polymerization and direct-writing electrospinning techniques Laura Bourdon, Jean-Christophe Maurin, Kerstin Gritsch, Arnaud Brioude, and Vincent Salles ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00810 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 11, 2018

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ACS Biomaterials Science & Engineering

Improvements in resolution of additive manufacturing: advances in two-photon polymerization and direct-writing electrospinning techniques Laura Bourdon†, Jean-Christophe Maurin†‡, Kerstin Gritsch†‡, Arnaud Brioude†, Vincent Salles*† Univ Lyon, Université Claude Bernard Lyon 1, CNRS, Laboratoire des Multimatériaux et Interfaces, F-69622 Villeurbanne, France

‡ Conservative Dentistry Department – Dentistry Faculty of Lyon, 11 rue Guillaume Paradin 69372 Lyon *Corresponding author: Vincent Salles, PhD Assistant-Professor Laboratoire des Multimatériaux et Interfaces 6 rue Grignard, 69622 Villeurbanne Tel: +33 (0)4 72 43 16 08 Email: [email protected]

Keywords: 3-D Printing; Tissue Engineering; Highly resolved scaffolds; two-photon polymerization; Direct writing electrospinning.

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Table of Contents 1.

Introduction .........................................................................................................................4

2.

3D printing using TPP .........................................................................................................6 2.1 Principle of photopolymerization 3D-printing techniques................................................6 2.2 Benefits of complex geometries produced by TPP for tissue engineering .......................8 2.3 Materials used in TPP for tissue engineering..................................................................12

3.

Emerging technologies of Direct Writing Electrospinning ...............................................14 3.1 Principle of techniques based on DWES.........................................................................16 3.2 Scaffold geometry produced by NFES............................................................................17 3.3 Materials used in NFES...................................................................................................21 3.4 FFES: resolution versus working distance ......................................................................23

4.

Summary............................................................................................................................25


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ABBREVIATIONS AM: Additive manufacturing TE: tissue engineering 3D: three dimensional ECM: extra cellular matrix STL: stereolithography CLIP: continuous liquid interface production MSTL: microstereolithography TPP: two-photon polymerization DW: direct-writing ES: electrospinning DWES: direct-writing electrospinning NFES: near-field electrospinning FFES: far-field electrospinning



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ABSTRACT In recent years, additive manufacturing (AM) technologies have attracted significant interest in many industrial and research fields, particularly in tissue engineering. Printed structures used as physical and bioactive supports for tissue regeneration are becoming increasingly complex so as to mimic natural tissues in order to answer future medical needs. Reproducing the biological environment of a native tissue from the microscopic to the macroscopic scale appears to be the best strategy for effective regeneration. Recent advances in AM have led to the production of scaffolds designed with a high precision. This review presents results concerning two AM technologies which enable the highest accuracy of scaffold design to be obtained, with a precision down to the nanoscale. The first technique is based on a two-photon polymerization (TPP) process while the other on a direct-writing electrospinning (DWES) system. Here, we present an overview of the fabrication mechanisms, the final scaffold properties and their applications in tissue engineering. The production of highly-resolved structures offers new possibilities for studying cell behavior in a controlled environment, and also for adjusting the desired scaffold properties to address current and future needs in tissue engineering. The current technical limitations and future challenges are thus also discussed in this review.


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1. Introduction Nowadays, tissue engineering (TE) is considered a promising strategy for the cure or replacement of affected tissue or organs. 1 TE consists in growing biological tissue from the patient’s cells which renders the building of organs possible without any donor or incompatibility issues. This TE approach relies on the use of biomolecules and an ephemeral three-dimensional (3D) scaffold as a bioactive support to help cells grow and organize themselves in three dimensions.

2

It is a very

challenging technique because many parameters concerning the cell environment can affect the cell’s biological behavior. To produce a functional organ or tissue, the environment created during 3D cell-culture needs to be as close as possible to that of the natural extracellular matrix considered in terms of chemical and physical properties and cell stimuli. 3 The bioactivity and geometry of the biomaterial therefore have to respond to strict requirements. In the past, numerous biomaterials including natural and synthetic polymers have been used for TE according to their compatibility, biodegradation kinetics and mechanical properties.

4

In fact,

natural polymers are very interesting for promoting cell activity, while synthetic polymers are more often used for their physicochemical and mechanical properties and their manufacturing flexibility. In order to promote and control the biological activity of the synthetic scaffold, bioactive molecules are usually employed 5 and among them, growth factors are used. These are biomolecules known for their capacity to initiate a specific biological activity leading to cell proliferation and specific differentiation. The nature of the intracellular signals can be controlled by adjusting both the type and amount of growth factor in the cell environment. Thus, in order to have a more targeted action, these soluble biomolecules can be introduced into a biodegradable scaffold which as the name suggests degrades over time and releases the growth factor in a controlled manner. Other 5 ACS Paragon Plus Environment


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biomolecules like fibronectin or laminin are also interesting in order to tailor cell-cell and cellECM (Extra Cellular Matrix) interactions that regulate tissue properties. The latter can be deposited on the scaffold surface to increase cell adhesion to the scaffold surface. However, whatever the considered bioactive molecules, they have to be compatible with the material(s) and the shaping process used for scaffold fabrication. Moreover, from a design point of view, it has been proved that cell activity can be influenced by the pore interconnectivity, morphology and pore size of the scaffold.

6,7

For instance, on a macroscopic scale, the structural geometry should enhance cell

diffusion, invasion and organization, have good mechanical properties and, in the case of implants, an external shape which fits the defective site dimensions well. In addition, the high surface-tovolume ratio is an important parameter to favor the interaction between cells and the scaffold and induce major activity for the regeneration processes such as cell adhesion, differentiation and proliferation.

8–10

Therefore, the main challenges for fabrication of functional scaffolds for TE

applications are to manufacture these specific and complex structures with a design precision down to the micro- and nanoscales. The manufacturing technique must fulfill different criteria such as the ability to shape many kinds of material in order to adjust the scaffold properties, and also a high resolution in order to produce nano-objects which are important in inducing cell activity. In previous studies, many techniques have been used to manufacture scaffolds for TE applications including phase separation electrospinning (ES)

15.

11,

gas foaming

12,

self-assembly

13,

particulate-leaching

14

and

Structures formed using those techniques are very porous with a large

specific surface area which helps stimulate cell adhesion on the scaffold. However, they do not allow precise control of the morphology or pore size, which are two key parameters in adapting cell activity.

16

AM techniques have therefore attracted much interest in the production of 3D

scaffolds as they partially solve these problems and are capable of processing many materials and 6 ACS Paragon Plus Environment

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complex architectures in a reasonable time. It is also worth noting that AM is considered to be the formalized term for rapid prototyping and 3D printing, corresponding to techniques producing 3D objects layer-by-layer, with a 3D Computer-Aided Design (3D CAD) system. 17 Nowadays, seven different families of AM are referenced

17:

photo-polymerization processes,

powder-bed fusion processes, extrusion-based systems, material jetting, binder jetting, sheet lamination and directed-energy deposition processes. Resolution of the printed object is very important and usually guides the selection of the type of AM machine and the kind of raw material that has to be employed. X and Y are considered to be the planar dimensions and usually present a resolution which is different to that of the Z-axis. It is now assumed that the “minimum feature size” and the “layer height” correspond to the resolution in the XY plane and along the Z-axis, respectively. The AM techniques known to obtain small feature size (XY plane resolution) use lasers. Among them, the process based on the two-photon technology gives the highest resolution with feature sizes of about 0.2µm, as demonstrated in the 1970s.

18

In parallel, technologies of

direct-writing (DW) have been developed and recent advances highlight the possibility of fabricating 3D scaffolds with a precision lower than the micro-scale. This is possible thanks to a technique based on electrohydrodynamics system: the direct-writing electrospinning. 19 In response to current and future needs in tissue engineering, and due to the fast evolution of both nanotechnologies and medical needs, this review aims to give an overview of recent advances of the two AM techniques leading to the fabrication of scaffolds with the highest resolution, i.e. twophoton photopolymerization (TPP) and direct-writing electrospinning (DWES).



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2. 3D printing using TPP 2.1 Principle of photopolymerization 3D-printing techniques AM techniques using lasers were developed between the 1970’s and ‘80’s by several teams. 18,20,21 Due to the interesting capabilities of this new technique, a commercial system was produced in 1987 by 3D System Inc., using a process called stereolithography (STL). This technique is capable of producing structures by photopolymerization of a photo-curable liquid resin (Error! Reference source not found.). It generally uses a UV laser beam to illuminate the surface of a resin vat which simultaneously solidifies. The first layer, which is the base structure, is formed on a support. The next step consists in repeating the fabrication of the other layers by lowering the support into the vat and solidifying the thin resin layer with the laser. This liquid resin consists of both a photoinitiator and a mixture of monomers and oligomers. The photoinitiator converts the laser’s physical energy into chemical energy used for the polymerization of these monomers and oligomers. To illuminate the resin, vector scanning or mask projection techniques can be used. For the former, a line of resin is cured by scanning whereas for the latter, a 2D pattern is cured by projection illumination. This strategy called “Digital Light Processing” allows a higher fabrication speed since an entire layer can be processed at one time. Moreover, unlike the scanning process, the resin is polymerized on the bottom of the vat which allows oxygen inhibition to be limited.


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Figure 1. (a) STL process with an optical device used for MSTL. (b) The difference between single-photon polymerization used in STL, MSTL, and TPP. Reproduced with permission from ref 22. Copyrights 2015 Hindawi.

More recently, a new photopolymerization 3D printing technique called “Continuous liquid interface production” (CLIP) using the same configuration was presented as an ultra-fast STL process.

23

Indeed, it was shown that the production time for the same scaffold by Digital Light

Processing and by CLIP was increased from 16 hours to only 70 minutes and with a better surface finish. This improvement was possible thanks to continuous production having replaced the layerby-layer one. The CLIP device consists of an oxygen-permeable film between the UV illumination and the resin which inhibits photopolymerization at the bottom of the vat. This thin surface called the “dead zone” means the resin can be more easily replaced in a continuous fashion, facilitating continuous production. A displacement speed of 30 cm.h-1 can be obtained for a z-axis resolution of 100 µm. 24



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Microstereolithography (MSTL) is a technique also based on STL but which integrates optical devices in order to improve the resolution. The UV laser beam can be focused to 1-2 µm and thus solidifies a very small volume of photo-curable resin. A 3D microstructure with submicron resolution can be thus produced. 25 TPP STL, also called “direct laser writing lithography”, 26 is a fabrication process similar to that of conventional STL. However, instead of linear one-photon absorption, this technique is based on a non-linear two-photon absorption. 27 This difference leads to polymerization activation in the very small volume of the laser’s focal point where the photon density is high enough to exceed the TPP threshold (Error! Reference source not found.). The TPP technique therefore has a resolution close to the nanoscale, beyond the diffraction limit of laser radiation. In general, powerful femtosecond-pulsed lasers are used with a radiation wavelength in the near-infrared (NIR) to induce two-photon absorption. 28 The use of this kind of laser allows deep penetration in transparent material and therefore requires no support to build the structure because it is formed in the vat volume. Generally, a viscous resin is used to keep the structure suspended in the vat while it is being fabricated. Furthermore, the depth penetration of the laser is sufficiently high to build structures with millimeter dimensions. Nowadays, TPP is the most accurate 3D-printing technique. The resolution is about 100-200 nm

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and can be as low as 52 nm

30

by adjusting the threshold

photoinitiator, laser intensity and scanning speed. The latter can vary between 1000 mm.s-1 and 100 µm.s-1 depending on the spatial resolution. 24


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2.2 Benefits of complex geometries produced by STL and TPP for tissue engineering Generally, laser-based additive manufacturing offers the main advantage of producing complex structural geometries, very interesting for TE applications. It renders possible the study of the relationship between geometry and induced biological response. TPP technique could complete the range of capabilities offered by the other laser-based additive manufacturing techniques by producing geometries controlled down to the nanoscale. It was revealed that structure with different dimensions could induce a different cell behavior. 8–10 Those techniques are thus complementary for inducing an efficient tissue regeneration. In order to review this important aspect, several studies referring to structures produced by STL technique will be presented first, followed by the studies referring to structures produced by TPP with a higher resolution. Melchels et al.

31

highlighted the possibility of producing structures by STL with different pore

morphologies such as cubic, diamond or gyroid and with a gradient in pore size. Although, these structures have not yet been tested in cell-cultures, they could be a useful tool in determining and studying the optimized architecture for enhancing the regeneration of specific biological tissues. In another study 32, eight structures with both different pore morphologies and surface curvatures were produced by STL (Error! Reference source not found.). The water permeability and surface area were given for each structure. No cell cultures have yet been made but the author suggests that this kind of analysis could lead to a good trade-off between the permeability of the structure induced by high surface curvature (which allows regeneration in oxygen and nutriments, and the removal of cellular waste) and the surface area induced by a lower curvature (which promotes cell adhesion). 11 ACS Paragon Plus Environment


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The effect of microstructure on cell behavior have been studied thanks to TPP technique. In the study by Cha et al. 33 for instance, micropatterns formed on the surface structure by TPP were tested in a cell culture with a mouse pre-osteoblast cell line (Error! Reference source not found.). Surface structures with micropatterns like pillars or ridges induced a greater cell activity than a flat structure: cell attachment and differentiation increased with a rough surface. Once again, this study highlighted the importance of micrometric structures in tissue regeneration, and also showed that TPP can be useful in adapting and promoting specific regeneration.

Figure 2. SEM images of the eight different scaffold geometries of poly (trimethylene carbonate) manufactured by TPP: (a) Diamond; (b) Gyroid; (c) Schwarz P; (d) Fisher-Koch S; (e) Double Diamond; (f) Double Gyroid; (g) Double Shwarz P; and (h) F-RD (scale bars represent 1 mm). Reproduced with

permission from ref 32. Copyrights 2017 IOPscience. Due to the ease of making complex shapes, another advantage is the better control of mechanical properties with respect to the structure. Mechanical stimuli are indeed an important parameter for TE because they can promote specific cell activity. 34 By tailoring the composition of the structure, the effect of structural elasticity has been studied.

35

However, for more specific mechanical 12

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properties, the geometry also needs to be adjusted. For instance, TPP has been used to study the effect of another important physical property of the scaffold, the Poisson’s ratio. 36 This is the ratio of transverse contraction to axial extension strain in the direction of the stretching force. Scaffolds with a positive (PPR) and a negative (NPR) Poisson’s ratio were made and studied with 10T1/2, an embryonic fibroblast cell line. In both scaffolds, cells showed good proliferation and adhesion. It was also shown that cells applied forces to the structure until its deformation. Abnormal cell division was also observed because of the spatial distribution of adhesive contacts that cells make during movement on the NPR scaffold. This study highlights the fact that TPP can be used to build complex geometries and contribute to the study of mechanobiology for TE applications.



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Figure 3. (A) SEM images of three different scaffolds manufactured by TPP where two have surface micro-patterns: from left to right- structure with micro-pillars, micro-ridges and a flat structure, respectively. (B) Images taken of confocal microscopy of actin-stained pre-osteoblast cells on the three types of TPP structures after 1 day incubation. Reproduced with permission from ref 33. Copyrights 2012 IOPscience.


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Mimicking the extracellular environment of native biological tissue to enhance regeneration is the key principle of TE and to apply it, the scaffolds used were porous and sometimes fibrous structures. Marino et al. 37 went further by reproducing the trabecular architecture of bones by TPP from micro-tomography images. Those structures, called an “Osteoprint”, were formed from a hybrid photocurable resin (OrmoComp) and were tested with SaOS-2, a human osteosarcomaderived cell line. The results revealed that the Osteoprint has a significant impact on cell behavior, for example, by decreasing cell proliferation and enhancing osteogenic differentiation. The latter was confirmed by the change in cytoskeleton arrangement and the cell and nucleus shapes. Thus, TPP is also very interesting in producing architectures very similar to those of natural extracellular matrices and then tailoring cell behavior to the native tissue.

2.3 Materials used in TPP for tissue engineering TPP has several advantages in terms of high resolution manufacturing of complex architectures. The main drawback of this technique is a limited number of raw materials that can be used. Indeed, the number of commercially-available photo-curable resins is still currently limited, but a lot of efforts are put to solve this lack of materials diversity. The first resins used with STL were based on acrylic and epoxy macromers. In order to compensate for the limited range of photo-sensitive resins, the strategy of combining TPP and micro-molding techniques was used. For example, Koroleva et al. 38 used this technique to produce a scaffold composed of fibrin gel. The first step consisted in producing the 3D structure by TPP from a simple acrylic-based resin which was then encapsulated in a PDMS matrix to form the micro-mold. The final step consisted in reproducing the original TPP structure by incorporating any interesting biomaterial in the mold, in this case, fibrin. This strategy is interesting since there is no restriction concerning the material. However,



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the need for different steps in the process and the presence of structural defects that may occur 39 make it less advantageous than conventional TPP. At the same time, many studies were conducted to develop a wider range of photo-curable resins. In order to produce biodegradable and biocompatible structures from TPP, a resin was developed in 2000 from ε-caprolactone and trimethyl carbonate. 40 Since then, other resins have been made such as those based on synthetic polymers: poly-L-lactide acid 41 or poly(propylene fumarate) 42. Hybrid organic-inorganic biomaterials have also been developed to benefit from the good tradeoff between properties like hardness, elasticity, chemical and thermal stability.

43

The most used

hybrid material in TPP is ORMOCER (Organically-MOdified CERamic). It is composed of a silica O-Si-O backbone and organic functions, and is currently very much used in dentistry composites. Functionalized natural materials based on chitosan 44, hyaluronic acid 45 and proteins 46 have also been produced from TPP. Another challenging biomaterial has been manufactured using TPP: hydrogels. These gels are very interesting for TE applications in terms of cell adhesion, proliferation, and the ability to biomimic the water-content of soft tissue. The challenges, as for the other cases, are having a chemical structure which can be cross-linked but especially having an efficient water-soluble photoinitiator with biocompatible properties. 47,48 Hydrogels based on poly(ethylene glycol) diacrylate (PEGDA) 49,

chitosan 44, hyaluronic acid 45 and collagen type I 50 have been made. In addition, biocompatible

and water-soluble photoinitiators like riboflavin (vitamin B2) have been used in TPP for producing hydrogels. 50 To promote biological activity, it is also possible to cross link biomolecules with the resin. For instance, in the study by Kufelt et al. 44, the chitosan was covalently cross-linked with a photosensitive modified vascular endothelial growth factor (VEGF). Ovsianikov’s team went 16 ACS Paragon Plus Environment

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further by using TPP to fabricate a 3D hydrogel structure containing cells.

51

They used a

photosensitive gelatin and a hydrophilic photoinitiator whose cytocompatibility was considered high after an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cells located in the vicinity of the TPP structure survived the process and even proliferated. Unfortunately, the cells located in the laser-exposed region were damaged. They concluded that this damage was not caused by the laser radiation but by reactive species that might be generated during the TPP process. TPP is a promising technique for the fabrication of scaffolds for TE. The possibility of producing complex structures with nanometer resolution allows the study of optimized conditions for regenerating specific biological tissue. As it has been shown, TPP technique can be used to adapt the surface topography with micro-structuration 33 or finely adjust the structure morphology as the natural extra-cellular matrix for suitable regeneration

37.

It has also been used to study cellular

behavior in response to resolute structure which presented specific properties.

36

Moreover, as a

result of many studies, the range of materials that can be used has expanded. Biocompatible materials known to be efficient for tissue regeneration such as poly-L-lactide, polycaprolactone, natural biomaterials and hydrogels can also be manufactured by TPP. It is even possible to produce bioactive material by introducing cells or biomolecules inside the structure. The first preclinical study was led with a scaffold made for cartilage regeneration in a rabbit.

26

The scaffold was

fabricated in 2.5 h, with geometrical dimensions of 2.1 x 2.1 x 0.21 mm3. Small features of about 10µm were produced. One of the main disadvantages of laser-based technique like the STL and the TPP techniques is the difficulty in producing a structure with a complex composition. Since fabrication is done in a resin 17 ACS Paragon Plus Environment


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vat, only one kind of polymer can be produced at a time. Therefore, to produce a multi-material structure requires fabricating the structure step by step with different resin vats. Even though this manufacturing process is not easy, Ritcher et al 52 used it to study the cell response of a structure containing different materials on which proteins were specifically bound e.g. fibronectin and biotinylated vitronectin. This work brings out limitations regarding fabrication of highly resolved 3D objects with a complex composition. In this context, other DW technologies merged in the last years in order to develop processes which are capable to tackle this issue of designing scaffolds with gradients of compositions, at the micro- and even nano-scale. This purpose can be addressed with processes based on extrusion under electric field.

3. Emerging technologies of Direct Writing Electrospinning The term “Direct Writing” (DW) corresponds to directed energy, material jetting and material extrusion processes. If we consider AM processes which add material to a surface, the several subcategories are defined: ink-based DW, laser transfer DW, thermal spray DW, beam deposition DW and liquid-phase direct deposition. 17 Ink-based DW is the most promising technique regarding the ability of a process to produce affordable 3D objects in an acceptable time. With these specifications, a technique combining 3D printing and polymer extrusion under an electric field has emerged in the recent years. Electrohydrodynamics corresponds to the study of the dynamics of fluids submitted to an electric field. Electrospraying, electro-jetting and electrospinning (ES) are three shaping processes using electrohydrodynamics to accelerate the extruded material toward a surface on which the deposition occurs, layer by layer. Among them, the fabrication of 3Dstructured micro-objects dedicated to TE applications offers the best compromise between resolution and fabrication speed when a combination of 3D printing and ES is carried out. 18 ACS Paragon Plus Environment

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For decades, ES has been widely studied because of its industrial scalability and ability to produce nanometer features in a continuous way with a high speed of production. Indeed, electrospun filaments can be produced at a rate of several meters per second. 53 Those advantages are also very interesting for TE applications.

15

The large specific surface area induced by the production of

submicronic objects leads to a good interaction with the biological environment. Moreover, the nano-objects produced can be used to enhance specific biological activities. For techniques based on direct-writing electrospinning (DWES), the principle is comparable to the ES process. The technique produces submicron filaments from a polymer solution (or a fused polymer) subjected to an electric field. 54 In a typical ES process, a polymer droplet is formed at the end of a metallic capillary and a high voltage is applied between this capillary and a conductive collector inducing a concentration of free electric charges at the droplet surface. When electric attraction exceeds the surface tension of the liquid, the droplet is distorted into a conical shape called a Taylor’s cone. Then, at a certain voltage threshold, material is ejected toward the collector. If the liquid presents suitable viscoelastic properties, the ejected liquid can form a filament; otherwise, it breaks into droplets leading to electrospraying or electro-jetting (depending on the trajectory of the droplets). Parameters such as solution concentration, polymer molecular weight, surface tension and electric field have to be monitored to control the jet morphology (droplet or filament). 55 In a conventional ES process, the ejected filament is distributed over a conical space (whipping effect) before reaching the collector. The material is then randomly deposited on the disk surface of the collector. The difference between conventional ES and DW techniques lies in the precision of the material deposition. The latter usually integrate a movable needle and/or a movable collector along the X and Y axes allowing the ejected material to be drawn onto the collector (Error! Reference source not found.). 19 ACS Paragon Plus Environment


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Figure 4. Different configurations used in EHD techniques. (a) Configuration of conventional ES. The needle-collector distance, H, is usually between several and tens of centimeters (Far-field). (b) Configuration used for Near-field ES where H is between several and hundreds of millimeters. (c) Configuration used in drop-on-demand techniques where H is in the order of hundreds of micrometers. Reproduced with permission from ref 19. Copyrights 2016 The Royal Society of Chemistry.

Recently, DW techniques have been studied for AM, making them even more promising for TE applications. Studies on DWES techniques are generally based on how to obtain localized deposition of the filament.

3.1 Principle of techniques based on DWES Several studies dealing with DWES have been published in the last few years, especially for TE purposes. Electrospun structures are indeed very useful for these applications because the fibrous scaffolds produced are similar to natural fibrous extracellular matrices. For DW techniques and in order to localize the filament deposition, different strategies have been proposed. In a conventional ES process, the flight trajectory of the filament between the nozzle and the collector adopts two different behaviors.

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The first part of the trajectory which begins at the end of the capillary is

stable and straight and has a typical length of about 3 to 20 mm. The second part of the filament 20 ACS Paragon Plus Environment

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trajectory then adopts a whipping motion because of electrostatic instabilities of the electric charges located in the filament (Error! Reference source not found.-b). The most used strategy, developed in order to better control the filament deposition area, is a technique called “Near-field Electrospinning” (NFES). It consists in reducing the needle-tip-to-collector distance down to the millimeter scale to keep a rectilinear trajectory between the jet and the substrate (Error! Reference source not found.). 57 Another emerging strategy combines different techniques aiming to suppress jet instabilities. One approach, where the working distance is the same order of magnitude (centimeter scale) as that used for conventional ES, justifies the name of “Far-field” ES (FFES). 58 The NFES technique, first proposed in 2006 57, uses the stable filament trajectory to control the deposition location. Indeed, by decreasing the needle-tip-to-collector distance to the millimeter scale and with an X,Y motion of the collector, a controlled and oriented deposition of filament is possible. Sun et al.

57

highlighted the importance of collector speed. In fact, one of the main

advantages of ES is the high production speed due to the electrically forced stretching. In the case of the DW technique, parameters like collector speed need to be synchronized with this high speed production in order to prevent the filament from curling in on itself when it reaches the collector. By avoiding filament instability, it is possible to obtain a straight jet to the target and design a controlled and oriented 2D filament pattern. Much work has been published on scaffolds prepared by the NFES process, relying on either a melt or wet fabrication route.

3.2 Scaffold geometry produced by NFES As previously mentioned, the quality of the scaffold designed by DWES is strongly dependent on the relative speed between the needle and collector. The relative displacement must be comparable to the filament linear speed which is mainly governed by the force induced by the electrical field. 21 ACS Paragon Plus Environment


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The speed is usually between 10 and 200 mm.s-1. This means that the deceleration required for direction changing during printing has to be considered when designing the structure as the filament would not be sufficiently stretched. The same consideration is taken for designing structures with a high curvature.

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The other main challenge for controlling structural design is to have high

resolution printing in order to stack filaments on top of one another. This requires devices with sufficiently high displacement resolution. Taking into consideration the above specifications, design of different structural geometries like walls, grids or triangles is possible thanks to speed printing, electrical voltage and solution viscosity optimization.

59-60-61

Usually, solution viscosity is monitored by optimizing (i) the polymer

concentration and molecular weight for the wet-electrospinning technique, or (ii) the spinning temperature in case of melt-electrospinning. For instance, complex structures have been produced with wet NFES using a 70% PCL solution in acetic acid. Firstly, for a meniscus regeneration application

62,

the structure was composed of 300 layers disposed in a circular arc and crossed-

linked with radial layers. This structure underwent a complete in vitro test with mesenchymal stem/stromal cells (MSCs). The results were encouraging because the test showed that the scaffold has a low cytotoxicity and sGAG, Aggrecan, collagen type I and II production were promoted. Moreover, cells proliferated well throughout the structure, attached and aligned along the filaments with elongated actin filaments. Secondly, for a tendon regeneration application 63, the 3D structure was made with a rectangular mesh pattern and layers containing different filament diameters in order to ensure both good mechanical properties and promote cell adhesion. Once the structure was printed, the mesh was rolled to make a tubular shaped like a tendon (Error! Reference source not found.5). The in vitro test of the structure was interesting because the cell activity was influenced


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in two ways. First, the rectangular pattern oriented along the tube’s length could promote cell shape elongation, and also the structure promoted collagen type I expression.

Figure 5. (a) Image of the scaffold structure for the regeneration of a tendon. (b) SEM image of the sideview of the scaffold. (c) SEM image showing the stacking of the different thick and thin filaments. (d) SEM image of the cross-section of the scaffold. Reproduced with permission from ref 63. Copyrights 2015 The Society For Biomaterials.

The reduction in filament diameter also focused the attention of researchers on continuously reducing the scaffold dimension, down to the nanoscale. Some work showed the importance of 23 ACS Paragon Plus Environment


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parameters such as heating temperature 59, collector motion speed 60, voltage 64 as well as the inner diameter of the needle 65. Recently, very fine filaments of about 800 nm have been produced by NFES via a melted polymer and thus permitted PCL grids to be built. Deposition resolution was sufficient to stack filaments leading to box patterns of 90µm and 150 µm width (Figure 6 - 7). 65 These grids of 80µm in height were deposited onto NCO-sP(EO-stat-PO)-coated glass slides and tested in vitro with primary human mesenchymal stromal cells (MSCs) from trabecular bone. After 4 and 10 days of incubation, fluorescence microscopy showed good cell adhesion to the structure and migration through the device (Figure 7).

Figure 6. (A) Images of the scaffold printed by melt NFES on a microscope slide. (B) and (C) SEM images of the box-structure demonstrating the good reproducibility and precision of the manufacturing technique. Reproduced with permission from ref 65. Copyrights 2015 IOPscience.


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Figure 7. Fluorescence microscopy images of HMSCs seeded on 50 stacked filament structures produced by NFES. (A)-(C) Structure of 90µm mesh-widths incubated for 4 and 10 days, respectively. (B)-(D) Structure of 150 µm mesh-widths incubated for 4 and 10 days, respectively. Reproduced with permission from ref 65. Copyrights 2015 IOPscience.

3.3 Materials used in NFES A large range of materials can be processed by ES; the only condition is that the material must have sufficient viscoelastic properties. In the review by Thenmozhi et al. 66, an overview was made of the different materials already electrospun for biomedical applications, which include synthetic and natural organic polymers and hybrid organic-inorganic materials. In NFES, PCL is the most used material as much for the melt as the wet process. Indeed, this polymer has high mechanical



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properties and can be easily electrospun at a relatively low temperature (80°C), or after solubilization in several organic solvents or in acetic acid. 61,67 In 2017, hybrid inorganic-organic filaments were printed using the NFES technique for bone regeneration. 68 A solution of 8wt% PCL and a fraction up to 70wt% of α-TCP was electrospun on a collector supplemented with ethanol. They explain that an open porosity is generated in the filament as the latter is deposited on liquid ethanol. Biological assays were performed with mouse preosteoblast cells and showed excellent cellular activity including high attachment, proliferation and differentiation thus highlighting the interest of forming porous filaments. A study showed the possibility of producing PCL scaffolds integrating hydroxyapatite nanoparticles to mimic the composition of natural bone. 69 Filaments were produced and stacked to form very porous structures. Cell cultures made with MC3T3-E1 showed proliferation and good cell viability throughout the structure. It would be interesting to do further research in order to study the effect of hydroxyapatite nanoparticles on cell behavior. A recent study also shows the possibility of integrating biomolecules in a polymer solution during the NFES process. 70 PCL/PVP scaffolds were produced integrating tetracycline hydrochloride, a therapeutic reagent known for its anti-inflammatory and antibacterial action. No cell-culture was made on the scaffold but the drug release was investigated relative to the filament spacing, polymer mixture composition and drug concentration. It is interesting to observe that drug release can be monitored with those parameters. For instance, compared to a PCL structure, a mixture of PCL/PVP accelerates the drug release thanks to the hydrophilicity of PVP. There is more and more interest in studying alginates as they are biosourced and easy to shape. In addition, it has been shown that alginate hydrogels are interesting materials for NFES. For example, 26 ACS Paragon Plus Environment

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in the study by Yeo et al. 71, alginate was shaped by NFES supplemented with an aerosol using a CaCl2 solution which spontaneously crosslinked the surface of the jet so keeping its cylindrical shape. By using a hydrogel containing cells, they also demonstrated that cells can survive the electric-field-based process. Indeed, a high viability (87%) of human adipose stem cells was obtained which was close to the viability obtained with an extruded-based process. Unfortunately, the structure’s height was limited to only four layers. NFES has proved to be efficient in building scaffolds. Structures with straight or curved filaments allow the production of different scaffold geometries characterized by promising regenerative applications. For instance, they enhance the alignment and attachment of cells along the filaments, as well as the distribution and growth of cells. Moreover, when solutions are used (wet process), biomolecules which promote a therapeutic or regenerative action, can be integrated into and released from the structure. This is a good strategy for favoring different biological activities and thus tailoring tissue regeneration. Concerning the filament diameter, it is indeed important to mimic the sub-micron fibrous collagen of ECM native tissue to enhance its regeneration. Even though Hochlertner et al. 65 showed that it is possible to obtain finer filaments of about 800 nm, it is difficult to obtain sub-micron filaments with NFES, contrary to conventional ES. This is mainly due to the low working distance which dramatically reduces the stretching length of the filament. Another issue is the limited height of the structure: about 200 µm. This can arise from two phenomena. The first could be an electrostatic destabilization created by an electric charge accumulation on the insulating filament after deposition. The second explanation could be the high Z sensitivity of the electric field for small working distances. To overcome this, the working distance could be increased to extend the scaffold dimensions. For instance, FFES is similar to NFES, but with a working distance on a centimeter scale. 27 ACS Paragon Plus Environment


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3.4 FFES: resolution versus working distance A tip-to-collector working distance on the centimeter scale implies electrostatic instabilities on the jet, as observed in conventional ES. A few studies deal with strategies to limit them and thus obtain a localized deposition. In fact, the longer time of flight helps solvent evaporation and thus filament solidification, and so the greater the distance, the larger the filament stretching. Theoretically, DW techniques related to the FFES process should achieve a resolution on the nanoscale. In the literature, two different strategies have been employed to obtain scaffolds by FFES. One of them uses one or more auxiliary electrodes in order to confine and localize the jet via electric repulsion. 72

For instance, a metallic cylinder placed around the jet confines the filament deposition area. 73

By inducing a linear collector movement, they showed that the line width of this area is inversely proportional to the collector movement speed. From 2mm/s to 50mm/s they obtained a line width of 950 µm to 641 nm, respectively, the latter corresponding to a rectilinear deposition of the filament. Even though they found good conditions to stretch the filament and make it straight, they chose to use a collector speed lower than 50 mm/s to stack several layers. Thus, filaments were randomly deposited over a limited circular area of about 300 µm in diameter. Different layers were stacked on one another but limited in number because the resulting 3D line shape is a flat Gaussian. Those PCL scaffolds were tested with a mouse embryonic fibroblast cell line (NIH3T3). The results showed that the scaffolds induced low cytotoxicity and act as a support to attach the cells. However, no results were given regarding the morphological evolution of the scaffold after its contact with the culture medium.


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Another strategy highlighted by Zhou et al. 74 consists in optimizing the solution properties to limit jet instability where the nature of the solvent, polymer molecular weight and concentration and viscoelasticity of the solutions are influential parameters. They found out that in ES, a lower dielectric constant of solvent and a higher viscoelasticity of solutions (allowed by a high polymer molecular weight and concentration) could increase the stable jet length until 25cm with a poly(Llactic acid) solution. From their findings, a study produced a scaffold from a solution of poly(Llactid acid)/poly(ethylene oxide) in trifluoroethanol. 75 Micronic filaments (1µm in diameter) were produced and precisely deposited, although this precision was insufficient to stack the filaments on one another. However, the in vitro assay made with human umbilical arterial smooth muscle cells showed good proliferation as well as good penetration of cells in the structure compared to an electrospun mat scaffold. FFES has also been studied with a melt polymer. In 2013, Farrugia et al. 76 produced a PCL scaffold with a 7.5 µm filament diameter. These scaffolds were tested with human dermal fibroblast cells. A very good cell proliferation with a total recovery of the structure by the fibroblasts was observed after 14 days of cell culture. Moreover, an ECM dermal protein such as fibronectin or collagen type I has been produced by fibroblasts during the cell culture showing the capacity for these cells to produce a new ECM. Another study highlighted the interest of using different fabrication techniques to build scaffolds for cartilage regeneration. In order to increase the mechanical properties of electrospun printed scaffolds and hydrogels, the two were combined. 77 The resulting 1 mm high PCL scaffolds made using an FFES technique were first produced and then filled with a gelatin methacrylamide hydrogel. The mechanical tests performed on the scaffold reveal that the properties are very similar 29 ACS Paragon Plus Environment


to those of the native cartilage. The cell-culture assay made with human chondrocytes showed that the scaffold can upregulate the matrix mRNA expression in comparison to a simple hydrogel. These results prove that a more specific investigation of the scaffold’s mechanical properties helps find the right conditions to obtain appropriate tissue regeneration. As a conclusion, FFES is an interesting technique for producing finer filaments for tissue regeneration as it can lead to scaffolds with similar structures to those of ECM native tissue. However, the main drawback of this technique is that the larger distance between the needle and collector during processing reduces the localization accuracy of the deposited filament. Consequently, scaffolds produced by this method are often not composed of perfectly stacked filaments. This implies a low reproducibility of the fabrication process. Even though some studies have been realized to improve deposition accuracy 78–81, improvements still need to be made in this field.

4. Summary Through this review, we can see that two-photon stereolithography (TPP) and direct-writing electrospinning (DWES) are promising techniques for the fabrication of scaffolds for tissue engineering. TPP offers the possibility of producing complex structures with nanometer scale resolution and opens new horizons for the optimization of conditions for regenerating specific biological tissue. In addition, recent investigations on the chemistry of photosensitive molecules have contributed to widening the range of materials that can be shaped with this process. For example, those known to be efficient for tissue regeneration such as poly-L-lactide, polycaprolactone, natural biomaterials and hydrogels can be manufactured through two-photon 30 ACS Paragon Plus Environment

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stereolithography. The main drawback of this additive manufacturing technique may be the difficulty in producing structures composed of different materials, which is an obstacle for the regeneration of organs composed of several tissues. Concerning DWES techniques, recent studies have reported the fabrication of fibrous scaffolds which are very interesting for tissue regeneration applications. These techniques can produce filaments with micrometric diameters using many different biomaterials via the melt or wet method. Near-field electrospinning, where the needle-to-collector distance is on the mm scale, has proven to be efficient in obtaining a localized and precise deposition both with a polymer solution and a melted polymer. However, it is difficult to produce filament diameters below several µm. On the contrary, far-field electrospinning is a technique using larger working distances resulting in finer filaments, although the deposition accuracy is lower than that obtained with near-field electrospinning, and thus an effort must be made by researchers to improve this point. In the future, strategies to improve the stacking quality of the filaments while keeping a reasonable fabrication speed should be found in order to obtain scaffolds with appropriate dimensions. The combination of different techniques to build those structures is perhaps a solution for the different issues mentioned. This strategy is still little studied but it seems very promising. It is thought that a better mimicking of the extracellular matrix could be found by designing the scaffold on a multiscale approach. Future work should therefore go in this direction, knowing that the successful technique will be the one allowing high production speed, reproducibility and versatility in terms of type of material used.



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Laser-based AM techniques promise to lead to fast production in the near future thanks to the CLIP strategy which has already led to a production speed almost 10 times greater than conventional stereolithography. However, the accuracy of software and machine displacement could limit high resolution AM scale up. Nevertheless, TPP and DWES are two AM techniques that can be considered as precious tools in the field of cell biology research. The capacity to produce microand nanostructures mimicking the natural extra-cellular matrix is a real challenge and the information collected on the behavior of cells placed in contact with them may represent a precious advance for future regenerative medicine.

ACKNOWLEDGEMENTS The authors would like to thank the French National Agency for Research and Claude Bernard University of Lyon for financial support. AUTHOR DISCLOSURE STATEMENT No competing financial interests exit

REFERENCES (1)

Place, E. S.; Evans, N. D.; Stevens, M. M. Complexity in Biomaterials for Tissue Engineering. Nat. Mater. 2009, 8 (6), 457–470 DOI: 10.1038/nmat2441.

(2)

Rustad, K. C.; Sorkin, M.; Levi, B.; Longaker, M. T.; Gurtner, G. C.; Rustad, K. C.; Sorkin, M.; Levi, B.; Longaker, M. T.; Rustad, K. C.; et al. Strategies for Organ Level Tissue Engineering. 32 ACS Paragon Plus Environment

Page 33 of 53 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


Organogenesis 2010, 6 (3), 151–157 DOI: 10.4161/org.6.3.12139. (3)

Charras, G.; Sahai, E. Physical Influences of the Extracellular Environment on Cell Migration. Nat. Rev. Mol. Cell Biol. 2014, 15, 813–824 DOI: 10.1038/nrm3897.

(4)

Ozdil, D.; Aydin, H. M. Polymers for Medical and Tissue Engineering Applications. J. Chem. Technol. Biotechnol. 2014, 89 (12), 1793–1810 DOI: 10.1002/jctb.4505.

(5)

Bishop, C. J.; Kim, J.; Green, J. J. Biomolecule Delivery to Engineer the Cellular Microenvironment for Regenerative Medicine. Ann. Biomed. Eng. 2014, 42 (7), 1557–1572 DOI: 10.1007/s10439-013-0932-1.Biomolecule.

(6)

Perez, R. A.; Mestres, G. Role of Pore Size and Morphology in Musculo-Skeletal Tissue Regeneration. Mater. Sci. Eng. C 2016, 61, 922–939 DOI: 10.1016/j.msec.2015.12.087.

(7)

Zhang, Z. Z.; Jiang, D.; Ding, J. X.; Wang, S. J.; Zhang, L.; Zhang, J. Y.; Qi, Y. S.; Chen, X. S.; Yu, J. K. Role of Scaffold Mean Pore Size in Meniscus Regeneration. Acta Biomater. 2016, 43, 314–326 DOI: 10.1016/j.actbio.2016.07.050.

(8)

Dvir, T.; Timko, B. P.; Kohane, D. S.; Langer, R. Nanotechnological Strategies for Engineering Complex Tissues. Nat. Nanotech. 2010, 6 (1), 13–22 DOI: 10.1038/nnano.2010.246.

(9)

Krishna, L.; Dhamodaran, K.; Jayadev, C.; Chatterjee, K.; Shetty, R.; Khora, S. S.; Das, D. Nanostructured Scaffold as a Determinant of Stem Cell Fate. Stem Cell Res. Ther. 2016, 7 (1), 188 DOI: 10.1186/s13287-016-0440-y.

(10)

Narayan, R. J.; Doraiswamy, A.; Chrisey, D. B.; Chichkov, B. N. Medical Prototyping Using 33 ACS Paragon Plus Environment


Page 34 of 53

Two Photon Polymerization. Mater. Today 2010, 13 (12), 42–48 DOI: 10.1016/S13697021(10)70223-6. (11)

Akbarzadeh, R.; Yousefi, A. M. Effects of Processing Parameters in Thermally Induced Phase Separation Technique on Porous Architecture of Scaffolds for Bone Tissue Engineering. J. Biomed. Mater. Res. Part B Appl. Biomater. 2014, 102 (6), 1304–1315 DOI: 10.1002/jbm.b.33101.

(12)

Ji, C.; Annabi, N.; Hosseinkhani, M.; Sivaloganathan, S.; Dehghani, F. Fabrication of Poly-DLLactide/polyethylene Glycol Scaffolds Using the Gas Foaming Technique. Acta Biomater. 2012, 8 (2), 570–578 DOI: 10.1016/j.actbio.2011.09.028.

(13)

Luo, J.; Tong, Y. W. Self-Assembly of Collagen-Mimetic Peptide Amphiphiles into Biofunctional Nanofiber. ACS Nano 2011, 5 (10), 7739–7747 DOI: 10.1021/nn202822f.

(14)

Reignier, J.; Huneault, M. A. Preparation of Interconnected Poly(ε-Caprolactone) Porous Scaffolds by a Combination of Polymer and Salt Particulate Leaching. Polymer. 2006, 47 (13), 4703–4717 DOI: 10.1016/j.polymer.2006.04.029.

(15)

Agarwal, S.; Wendorff, J. H.; Greiner, A. Use of Electrospinning Technique for Biomedical Applications. Polymer. 2008, 49 (26), 5603–5621 DOI: 10.1016/j.polymer.2008.09.014.

(16)

Hollister, S. J. Porous Scaffold Design for Tissue Engineering. Nat. Mater. 2005, 4, 518–524 DOI: 10.1038/nmat1421.

(17)

Gibson, I.; Rosen, D.; Stucker, B. Additive Manufacturing Technologies: 3D Printing, Rapid 34 ACS Paragon Plus Environment

Page 35 of 53 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


Prototyping and Direct Digital Manufacturing, Second Edi.; Springer: New York Heidelberg Dodrecht London, 2015. (18)

Wilson, J. E. Radiation Chemistry of Monomers, Polymers, and Plastics. Phys. Today 1974, 27 (7), 50–51 DOI: 10.1063/1.3128702.

(19)

Zhang, B.; He, J.; Li, X.; Xu, F.; Li, D. Micro/nanoscale Electrohydrodynamic Printing: From 2D to 3D. Nanoscale 2016, 8 (34), 15376–15388 DOI: 10.1039/C6NR04106J.

(20)

Charles W. Hull. Apparatus for Production of Three-Dimensional Objects by Stereolithography, 1984.

(21)

Kodama, H. Automatic Method for Fabricating a Three-Dimensional Plastic Model with Photo-Hardening Polymer. Rev. Sci. Instrum. 1981, 52 (11), 1770–1773 DOI: 10.1063/1.1136492.

(22)

Lee, J. W. 3D Nanoprinting Technologies for Tissue Engineering Applications. J. Nanomater. 2015, 2015, 14 p DOI: 10.1155/2015/213521

(23)

Tumbleston, J. R.; Shirvanyants, D.; Ermoshkin, N.; Janusziewicz, R.; Johnson, A. R.; Kelly, D.; Chen, K.; Pinschmidt, R.; Rolland, J. P.; Ermoshkin, A.; et al. Continuous Liquid Interface of 3D Objects. Science (80-. ). 2015, 347 (6228), 1349–1352 DOI: 10.1126/science.aaa2397.

(24)

Ligon, S. C.; Liska, R.; Stampfl, J.; Gurr, M.; Mülhaupt, R. Polymers for 3D Printing and Customized Additive Manufacturing. Chem. Rev. 2017, 117 (15), 10212–10290 DOI: 10.1021/acs.chemrev.7b00074. 35 ACS Paragon Plus Environment


(25)

Page 36 of 53

Zhang, X.; Jiang, X. N.; Sun, C. Micro-Stereolithography of Polymeric and Ceramic Microstructures. Sensors Actuators, A Phys. 1999, 77 (2), 149–156 DOI: 10.1016/S09244247(99)00189-2.

(26)

Mačiulaitis, J.; Deveikytė, M.; Rekštytė, S.; Bratchikov, M.; Darinskas, A.; Šimbelytė, A.; Daunoras, G.; Laurinavičienė, A.; Laurinavičius, A.; Gudas, R.; et al. Preclinical Study of SZ2080 Material 3D Microstructured Scaffolds for Cartilage Tissue Engineering Made by Femtosecond Direct Laser Writing Lithography. Biofabrication 2015, 7 (7), 15015 DOI: 10.1088/1758-5090/7/1/015015.

(27)

WeiB, T.; Hildebrand, G.; Schade, R.; Liefeith, K. Two-Photon Polymerization for Microfabrication of Three-Dimensional Scaffolds for Tissue Engineering Application. Eng. Life Sci. 2009, 9 (5), 384–390 DOI: 10.1002/elsc.200900002.

(28)

Sugioka, K.; Cheng, Y. Femtosecond Laser Three-Dimensional Micro- and Nanofabrication. Appl. Phys. Rev. 2014, 1 (4), 41303 DOI: 10.1063/1.4904320.

(29)

Kawata, S.; Sun, H.-B.; Tanaka, T.; Takada, K. Finer Features for Functional Microdevices. Nature 2001, 412 (6848), 697–698 DOI: 10.1038/35089130.

(30)

Zongsons Gan; Yaoyu Cao; Richard A. Evans; Min Gu. Three-Dimensional Deep SubDiffraction Optical Beam Lithography with 9 Nm Feature Size. Nat. Commun. 2013, 4, 2061 DOI: 10.1088/0960-1317/21/12/125014.

(31)

Melchels, F. P. W.; Bertoldi, K.; Gabbrielli, R.; Velders, A. H.; Feijen, J.; Grijpma, D. W. Mathematically Defined Tissue Engineering Scaffold Architectures Prepared by 36 ACS Paragon Plus Environment

Page 37 of 53 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


Stereolithography.

Biomaterials

2010,

31

(27),

6909–6916

DOI:

10.1016/j.biomaterials.2010.05.068. (32)

Blanquer, S. B. G.; Werner, M.; Hannula, M.; Sharifi, S.; Lajoinie, G. P. R.; Eglin, D.; Hyttinen, J.; Poot, A. A.; Grijpma, D. W. Surface Curvature in Triply-Periodic Minimal Surface Architectures as a Distinct Design Parameter in Preparing Advanced Tissue Engineering Scaffolds. Biofabrication 2017, 9 (2), 25001 DOI: 10.1088/1758-5090/aa6553.

(33)

Cha, H. Do; Hong, J. M.; Kang, T.-Y.; Jung, J. W.; Ha, D.-H.; Cho, D.-W. Effects of MicroPatterns in Three-Dimensional Scaffolds for Tissue Engineering Applications. J. Micromechanics Microengineering 2012, 22 (12), 125002 DOI: 10.1088/09601317/22/12/125002.

(34)

Vogel, V.; Sheetz, M. Local Force and Geometry Sensing Regulate Cell Functions. Nat. Rev. Mol. Cell Biol. 2006, 7 (4), 265–275 DOI: 10.1038/nrm1890.

(35)

Lo, C.-M.; Wang, H.-B.; Dembo, M.; Wang, Y. Cell Movement Is Guided by the Rigidity of the Substrate. Biophys. J. 2000, 79 (1), 144–152 DOI: 10.1016/S0006-3495(00)76279-5.

(36)

Zhang, W.; Soman, P.; Meggs, K.; Qu, X.; Chen, S. Tuning the Poisson’s Ratio of Biomaterials for Investigating Cellular Response. Adv. Funct. Mater. 2013, 23 (25), 3226–3232 DOI: 10.1002/adfm.201202666.

(37)

Marino, A.; Filippeschi, C.; Genchi, G. G.; Mattoli, V.; Mazzolai, B.; Ciofani, G. The Osteoprint: A Bioinspired Two-Photon Polymerized 3-D Structure for the Enhancement of Bone-like Cell Differentiation. Acta Biomater. 2014, 10 (10), 4304–4313 DOI: 37 ACS Paragon Plus Environment


Page 38 of 53

10.1016/j.actbio.2014.05.032. (38)

Koroleva, A.; Gittard, S.; Schlie, S.; Deiwick, A.; Jockenhoevel, S.; Chichkov, B. Fabrication of Fibrin Scaffolds with Controlled Microscale Architecture by a Two-Photon Polymerization–micromolding Technique. Biofabrication 2012, 4 (1), 15001 DOI: 10.1088/1758-5082/4/1/015001.

(39)

Koroleva, A.; Gill, A. A.; Ortega, I.; Haycock, J. W.; Schlie, S.; Gittard, S. D.; Chichkov, B. N.; Claeyssens, F. Two-Photon Polymerization-Generated and Micromolding-Replicated 3D Scaffolds for Peripheral Neural Tissue Engineering Applications. Biofabrication 2012, 4 (2), 25005 DOI: 10.1088/1758-5082/4/2/025005.

(40)

Matsuda, T.; Mizutani, M.; Arnold, S. C. Molecular Design of Photocurable Liquid Biodegradable Copolymers. 1. Synthesis and Photocuring Characteristics. Macromolecules 2000, 33 (3), 795–800 DOI: 10.1021/ma991404i.

(41)

Kuznetsova, D.; Ageykin, A.; Koroleva, A.; Deiwick, A.; Shpichka, A.; Solovieva, A.; Kostjuk, S.; Meleshina, A.; Rodimova, S.; Akovanceva, A.; et al. Surface Micromorphology of CrossLinked Tetrafunctional Polylactide Scaffolds Inducing Vessel Growth and Bone Formation. Biofabrication 2017, 9 (2), 25009 DOI: 10.1088/1758-5090/aa6725.

(42)

Cooke, M. N.; Fisher, J. P.; Dean, D.; Rimnac, C.; Mikos, A. G. Use of Stereolithography to Manufacture Critical-Sized 3D Biodegradable Scaffolds for Bone Ingrowth. J. Biomed. Mater. Res. 2003, 64B (2), 65–69 DOI: 10.1002/jbm.b.10485.

(43)

Farsari, M.; Vamvakaki, M.; Chichkov, B. N. Multiphoton Polymerization of Hybrid 38 ACS Paragon Plus Environment

Page 39 of 53 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


Materials. J. Opt. 2010, 12 (12), 124001 DOI: 10.1088/2040-8978/12/12/124001. (44)

Kufelt, O.; El-Tamer, A.; Sehring, C.; Meißner, M.; Schlie-Wolter, S.; Chichkov, B. N. WaterSoluble Photopolymerizable Chitosan Hydrogels for Biofabrication via Two-Photon Polymerization. Acta Biomater. 2015, 18, 186–195 DOI: 10.1016/j.actbio.2015.02.025.

(45)

Kufelt, O.; El-Tamer, A.; Sehring, C.; Schlie-Wolter, S.; Chichkov, B. N. Hyaluronic Acid Based Materials for Scaffolding via Two-Photon Polymerization. Biomacromolecules 2014, 15 (2), 650–659 DOI: 10.1021/bm401712q.

(46)

Chan, B. P.; Ma, J. N.; Xu, J. Y.; Li, C. W.; Cheng, J. P.; Cheng, S. H. Femto-Second Laser-Based Free Writing of 3D Protein Microstructures and Micropatterns with Sub-Micrometer Features: A Study on Voxels, Porosity, and Cytocompatibility. Adv. Funct. Mater. 2014, 24 (3), 277–294 DOI: 10.1002/adfm.201300709.

(47)

Huang, X.; Wang, X.; Zhao, Y. Study on a Series of Water-Soluble Photoinitiators for Fabrication of 3D Hydrogels by Two-Photon Polymerization. Dye. Pigment. 2017, 141, 413– 419 DOI: 10.1016/j.dyepig.2017.02.040.

(48)

Williams, C. G.; Malik, A. N.; Kim, T. K.; Manson, P. N.; Elisseeff, J. H. Variable Cytocompatibility of Six Cell Lines with Photoinitiators Used for Polymerizing Hydrogels and Cell

Encapsulation.

Biomaterials

2005,

26

(11),

1211–1218

DOI:

10.1016/j.biomaterials.2004.04.024. (49)

Jan Torgerse, Aleksander Ovsianikov, Vladimir Mironov, Niklas Pucher, Xiaochua Qin, Zhiquan Li, Klaus Cicha, Thomas Machacek, Robert Liska, Verena Jantsch, J. S. Photo39 ACS Paragon Plus Environment


Page 40 of 53

Sensitive Hydrogels for Three-Dimensional Laser Microfabrication in the Presene of Whole Organisms. JBO Lett. 2012, 17 (10), 15–18 DOI: 10.1117/1.JBO.17.10. (50)

Bell, A.; Kofron, M.; Nistor, V. Multiphoton Crosslinking for Biocompatible 3D Printing of Type I Collagen. Biofabrication 2015, 7 (3), 35007 DOI: 10.1088/1758-5090/7/3/035007.

(51)

Ovsianikov, A.; Mühleder, S.; Torgersen, J.; Li, Z.; Qin, X. H.; Van Vlierberghe, S.; Dubruel, P.; Holnthoner, W.; Redl, H.; Liska, R.; et al. Laser Photofabrication of Cell-Containing Hydrogel Constructs. Langmuir 2014, 30 (13), 3787–3794 DOI: 10.1021/la402346z.

(52)

Richter, B.; Hahn, V.; Bertels, S.; Claus, T. K.; Wegener, M.; Delaittre, G.; Barner-Kowollik, C.; Bastmeyer, M. Guiding Cell Attachment in 3D Microscaffolds Selectively Functionalized with Two Distinct Adhesion Proteins. Adv. Mater. 2017, 29 (5), 1–6 DOI: 10.1002/adma.201604342.

(53)

Choi, S. J.; Kong, C. S.; Han, D. H.; Kim, H. S. Online Measurement of Electrospinning Jet Velocity of Polyvinyl Alcohol. Int. Polym. Process. 2016, 3 (3), 285–291 DOI: 10.3139/217.3123.

(54)

Doshi, J.; Reneker, D. H. Electrospinning Process and Applications of Electrospun Fibers. J. Electrostat. 1995, 35 (2–3), 151–160 DOI: 10.1016/0304-3886(95)00041-8.

(55)

Yu, J. H.; Fridrikh, S. V.; Rutledge, G. C. The Role of Elasticity in the Formation of Electrospun Fibers. Polymer. 2006, 47 (13), 4789–4797 DOI: 10.1016/j.polymer.2006.04.050.

(56)

Rutledge, G. C.; Fridrikh, S. V. Formation of Fibers by Electrospinning. Adv. Drug Deliv. Rev. 40 ACS Paragon Plus Environment

Page 41 of 53 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


2007, 59 (59), 1384–1391 DOI: 10.1016/j.addr.2007.04.020. (57)

Sun, D.; Chang, C.; Li, S.; Lin, L. Near-Field Electrospinning. Nano Lett. 2006, 6 (4), 839–842 DOI: 10.1021/nl0602701.

(58)

Li, X.; Li, Z.; Wang, L.; Ma, G.; Meng, F.; Pritchard, R. H.; Gill, E. L.; Liu, Y.; Huang, Y. Y. S. Low-Voltage Continuous Electrospinning Patterning. ACS Appl. Mater. Interfaces 2016, 8 (47), 32120–32131 DOI: 10.1021/acsami.6b07797.

(59)

He, J.; Xia, P.; Li, D. Development of Melt Electrohydrodynamic 3D Printing for Complex Microscale Poly ( ε -Caprolactone ) Scaffolds. Biofabrication 2016, 8 (3), 1–11 DOI: 10.1088/1758-5090/8/3/035008.

(60)

Brown, T. D.; Dalton, P. D.; Hutmacher, D. W. Direct Writing by Way of Melt Electrospinning. Adv. Mater. 2011, 23 (47), 5651–5657 DOI: 10.1002/adma.201103482.

(61)

Li, J. L.; Cai, Y. L.; Guo, Y. L.; Fuh, J. Y. H.; Sun, J.; Hong, G. S.; Lam, R. N.; Wong, Y. S.; Wang, W.; Tay, B. Y.; et al. Fabrication of Three-Dimensional Porous Scaffolds with Controlled Filament Orientation and Large Pore Size via an Improved E-Jetting Technique. J. Biomed. Mater. Res. Part B Appl. Biomater. 2014, 102 (4), 651–658 DOI: 10.1002/jbm.b.33043.

(62)

Li

Jinlan.

Biofabrication

and

Characterization

of

Meniscal

Scaffolds

via

Electrohydrodynamic Jet Printing Technique, National university of Singapore, 2014. (63)

Wu, Y.; Wang, Z.; Ying Hsi Fuh, J.; San Wong, Y.; Wang, W.; San Thian, E. Direct E-Jet Printing of Three-Dimensional Fibrous Scaffold for Tendon Tissue Engineering. J. Biomed. Mater. 41 ACS Paragon Plus Environment


Page 42 of 53

Res. Part B Appl. Biomater. 2015, 105 (3), 616–627 DOI: 10.1002/jbm.b.33580. (64)

Hochleitner, G.; Youssef, A.; Hrynevich, A.; Haigh, J. N.; Jungst, T.; Groll, J.; Dalton, P. D. Fibre Pulsing during Melt Electrospinning Writing. BioNanoMaterials 2016, 17 (3–4), 159– 171 DOI: 10.1515/bnm-2015-0022.

(65)

Hochleitner, G.; Jüngst, T.; Brown, T. D.; Hahn, K.; Moseke, C.; Jakob, F.; Dalton, P. D.; Groll, J. Additive Manufacturing of Scaffolds with Sub-Micron Filaments via Melt Electrospinning Writing. Biofabrication 2015, 7 (3), 35002 DOI: 10.1088/1758-5090/7/3/035002.

(66)

Thenmozhi, S.; Dharmaraj, N.; Kadirvelu, K.; Kim, H. Y. Electrospun Nanofibers: New Generation Materials for Advanced Applications. Mater. Sci. Eng. B 2017, 217, 36–48 DOI: 10.1016/j.mseb.2017.01.001.

(67)

Brown, T. D.; Dalton, P. D.; Hutmacher, D. W. Melt Electrospinning Today: An Opportune Time for an Emerging Polymer Process. Prog. Polym. Sci. 2016, 56, 116–166 DOI: 10.1016/j.progpolymsci.2016.01.001.

(68)

Kim, M.; Yun, H. S.; Kim, G. H. Electric-Field Assisted 3D-Fibrous Bioceramic-Based Scaffolds for Bone Tissue Regeneration: Fabrication, Characterization, and in Vitro Cellular Activities. Sci. Rep. 2017, 7 (1), 1–13 DOI: 10.1038/s41598-017-03461-x.

(69)

Qu, X.; Xia, P.; He, J.; Li, D. Microscale Electrohydrodynamic Printing of Biobimetic PCL/nHA Composite Scaffolds for Bone Tissue Engineering. Mater. Lett. 2016, 185, 554–557 DOI: 10.1016/j.matlet.2016.09.035.


Page 43 of 53 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


(70)

Wang, J.; Zheng, H.; Chang, M.; Ahmad, Z.; Li, J. Preparation of Active 3D Film Patches via Aligned Fiber Electrohydrodynamic ( EHD ) Printing. Sci. Rep. 2017, 7, 43924 DOI: 10.1038/srep43924.

(71)

Yeo, M. G.; Ha, J. H.; Lee, H. J.; Kim, G. H. Fabrication of hASCs-Laden Structures Using Extrusion-Based Cell Printing Supplemented with an Electric Field. Acta Biomater. 2016, 38, 33–43 DOI: 10.1016/j.actbio.2016.04.017.

(72)

Deitzel, J. M.; Kleinmeyer, J. D.; Hirvonen, J. K.; Tan, N. C. B. Controlled Deposition of Electrospun Poly ( Ethylene Oxide ) Fibers. Polymer. 2001, 42 (19), 8163–8170 DOI: 10.1016/S0032-3861(01)00336-6.

(73)

Lee, J.; Lee, S. Y.; Jang, J.; Jeong, Y. H.; Cho, D. W. Fabrication of Patterned Nanofibrous Mats Using Direct-Write Electrospinning. Langmuir 2012, 28 (18), 7267–7275 DOI: 10.1021/la3009249.

(74)

Zhou, Q.; Bao, M.; Yuan, H.; Zhao, S.; Dong, W.; Zhang, Y. Implication of Stable Jet Length in Electrospinning for Collecting Well-Aligned Ultrafine PLLA Fibers. Polymer. 2013, 54 (25), 6867–6876 DOI: 10.1016/j.polymer.2013.10.042.

(75)

Yuan, H.; Zhou, Q.; Li, B.; Bao, M.; Lou, X.; Zhang, Y. Direct Printing of Patterned ThreeDimensional Ultrafine Fibrous Scaffolds by Stable Jet Electrospinning for Cellular Ingrowth. Biofabrication 2015, 7 (4), 45004 DOI: 10.1088/1758-5090/7/4/045004.

(76)

Farrugia, B. L.; Brown, T. D.; Upton, Z.; Hutmacher, D. W.; Dalton, P. D.; Dargaville, T. R. Dermal Fibroblast Infiltration of Poly ( ε -Caprolactone ) Scaffolds Fabricated by Melt 43 ACS Paragon Plus Environment


Page 44 of 53

Electrospinning in a Direct Writing Mode. Biofabrication 2013, 5 (5), 25001 DOI: 10.1088/1758-5082/5/2/025001. (77)

Visser, J.; Melchels, F. P. W.; Jeon, J. E.; Bussel, E. M. Van; Kimpton, L. S.; Byrne, H. M.; Dhert, W. J. A.; Dalton, P. D.; Hutmacher, D. W.; Malda, J. Reinforcement of Hydrogels Using Three-Dimensionally Printed Microfibres. Nat. Commun. 2015, 6 (6), 6933 DOI: 10.1038/ncomms7933.

(78)

Chen, W.; Weng, W. Continuous Aligned Poly(meta-Phenylene Isophthalamide) Fibers via Stable Jet Electrospinning. J. Appl. Polym. Sci. 2016, 133 (29), 1–7 DOI: 10.1002/app.43690.

(79)

Bisht, G. S.; Canton, G.; Mirsepassi, A.; Kulinsky, L.; Oh, S.; Dunn-Rankin, D.; Madou, M. J. Controlled Continuous Patterning of Polymeric Nanofibers on Three-Dimensional Substrates Using Low-Voltage near-Field Electrospinning. Nano Lett. 2011, 11 (4), 1831– 1837 DOI: 10.1021/nl2006164.

(80)

He, J.; Xu, F.; Cao, Y.; Liu, Y.; Li, D.; Jin, Z. Electrohydrodynamic Direct-Writing Lithography: An Alternative Maskless Technique for Microstructure Fabrication. Appl. Phys. Lett. 2014, 105 (25), 28–32 DOI: 10.1063/1.4905151.

(81)

Zheng, G.; Li, W.; Wang, X.; Wu, D.; Sun, D.; Lin, L. Precision Deposition of a Nanofibre by near-Field Electrospinning. J. Phys. D. Appl. Phys. 2010, 43, 415501 DOI: 10.1088/00223727/43/41/415501.


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(a) STL process with an optical device used for MSTL. (b) The difference between single-photon polymerization used in STL, MSTL, and TPP. 20 Copyrights 2015 Hindawi. 261x133mm (120 x 120 DPI)


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SEM images of the eight different scaffold geometries of poly (trimethylene carbonate) manufactured by TPP: (a) Diamond; (b) Gyroid; (c) Schwarz P; (d) Fisher-Koch S; (e) Double Diamond; (f) Double Gyroid; (g) Double Shwarz P; and (h) F-RD (scale bars represent 1 mm). 32 Copyrights 2017 IOPscience. 607x277mm (120 x 120 DPI)



(A) SEM images of three different scaffolds manufactured by TPP where two have surface micro-patterns: from left to right- structure with micro-pillars, micro-ridges and a flat structure, respectively. (B) Images taken of confocal microscopy of actin-stained pre-osteoblast cells on the three types of TPP structures after 1 day incubation. 33 Copyrights 2012 IOPscience. 169x153mm (150 x 150 DPI)


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Different configurations used in EHD techniques. (a) Configuration of conventional ES. The needle-collector distance, H, is usually between several and tens of centimeters (Far-field). (b) Configuration used for Nearfield ES where H is between several and hundreds of millimeters. (c) Configuration used in drop-on-demand techniques where H is in the order of hundreds of micrometers. 17 Copyrights 2016 The Royal Society of Chemistry. 333x95mm (120 x 120 DPI)



(a) Image of the scaffold structure for the regeneration of a tendon. (b) SEM image of the side-view of the scaffold. (c) SEM image showing the stacking of the different thick and thin filaments. (d) SEM image of the cross-section of the scaffold. 62 Copyrights 2015 The Society For Biomaterials. 242x115mm (120 x 120 DPI)


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(A) Images of the scaffold printed by melt NFES on a microscope slide. (B) and (C) SEM images of the boxstructure demonstrating the good reproducibility and precision of the manufacturing technique. 65 Copyrights 2015 IOPscience. 208x174mm (120 x 120 DPI)



Fluorescence microscopy images of HMSCs seeded on 50 stacked filament structures produced by NFES. (A)-(C) Structure of 90µm mesh-widths incubated for 4 and 10 days, respectively. (B)-(D) Structure of 150 µm mesh-widths incubated for 4 and 10 days, respectively. 65 Copyrights 2015 IOPscience. 193x144mm (150 x 150 DPI)


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Improvements in Resolution of Additive Manufacturing: Advances in

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