Open Access
Add to BibSonomyAbstract
The production of 3D scaffolds with well-controlled architecture at the micrometer-scale is a fundamental issue for the advancement of tissue engineering towards applications in health care. Stereolithography is a highly versatile and accurate technique to fabricate 3D scaffolds with controlled architectures. Here, a scalable stereolithography method combining mask projection with excimer laser is reported. Its capability is showcased by a variety of mm-sized 3D biodegradable scaffolds patterned with a spatial resolution well-suited for tissue engineering applications. The presented method offers a concrete possibility to scale-up stereolithography-based production of 3D scaffolds to be used in regenerative medicine with potentially high-impact on health care.
© 2014 Optical Society of America
1. Introduction
In 1993, a seminal paper introduced Tissue Engineering (TE) as the discipline applying the principles of biology and engineering to the development of functional substitutes for damaged tissues [1]. TE has since emerged as an interdisciplinary field that encompasses elements of medicine, biology, physics, chemistry, and materials science with the goal of growing tissues or organs directly on controlled three-dimensional (3D) environments called scaffolds which are used as biomaterials alone or as carriers for cells.
3D scaffolds for TE applications have to possess some general characteristics, such as biocompatibility, porosity, and mechanical properties adjusted to properly support the engineered tissue [2]. The control of these characteristics strongly depends on the fabrication method used, which has to be also reliable and scalable in order to be considered for large-scale production necessary for concrete applications in health-care. Therefore, the demand for accurate and scalable 3D scaffold fabrication methods is crucial for the further advancement in TE.
Stereolithography (SL) is a highly versatile and accurate technique to fabricate 3D scaffolds with controlled micro-architectures. It is based on a layer-by-layer spatially controlled light-induced crosslinking, i.e., photocuring, of a polymeric liquid resin composed of a crosslinkable monomer/polymer and a photoinitiator molecule (PI). Indeed, many SL methods in combination with photocurable resins have been reported so far for the production of 3D biocompatible scaffolds [3–6]. SL can be performed in two different ways depending on the irradiation method: i) in the direct laser writing method the resin is cured either by single photon absorption while scanning a focused laser beam on its surface [7, 8], or directly into 3D space by exploiting two-photon excitation of the PI molecules [9, 10]; ii) in the image projection method a single layer is exposed in parallel to produce a specific pattern simultaneously over an extended area [11–16].
In micro-fabrication there is a clear relationship between the overall size of the structure, the resolution with which a structure is built, and the time needed for the fabrication: the higher the resolution with which a part can be built, the smaller will be its maximum size fabricated within a reasonable time scale. Direct laser writing can achieve sub-100 nm spatial resolution, yet the image projection method is more suitable to be scaled up due to its parallel processing capability. Among micro-fabrication processes, micro-molding replication technique also offers the possibility to achieve high-yield production. Micro-molds may be replicated many times to produce micro-structures as for example biomimetic adhesive [17], carbon MEMS [18], PDMS micro-structures [19], and TE scaffolds [20, 21]. However, even if such approach can be successfully applied to fabricate micro/nano-structures with large overhangs and high aspect ratio it cannot be used to replicate many times fully porous and interconnected 3D scaffolds with a large volume due to basic geometrical limitations in the de-molding process [22, 23]. In fact, fully interconnected porous 3D structures with mm to cm scale dimension and micrometer scale features cannot be de-molded without disrupting the 3D pattern unless the mold is scarified, but then the yield is greatly reduced since a new mold has to be produced for each replica.
From the material point of view, a number of biodegradable photocurable resins have been developed and used to fabricate 3D scaffolds that will degrade in the physiological environment [24]. A promising and versatile biodegradable and photocurable material is poly(propylene fumarate) (PPF), a linear polyester with: i) an unsaturated backbone, that allows the crosslinking; ii) cytocompatible degradation products based on propylene glycol and fumaric acid, an intermediate of Krebs cycle in glucose metabolism [25]. Fumarate-based scaffolds have been tested for example in bone regeneration [7, 26] and nervous system repair [27–29]. The PI concentration in the PPF-based resin can be used to tune the mechanical properties of the produced scaffolds according to the specific requirements needed for tissue regeneration [30].
It must be noted that although SL works with photocurable materials, its versatility allows indirect methods to be applied for the fabrication of 3D scaffolds with a wide range of biomaterials. This can be achieved by combining SL with the replication method mentioned above [31]. In practice, SL can be applied to fabricate a fully interconnected porous 3D mold which can be replicated using a variety of non-photocurable biomaterials to produce a scaffold that can be used for example in prototyping and research level applications.
Biodegradable scaffolds have to provide a temporary environment to promote tissue regeneration. Therefore, it is the capacity to integrate with the hosting environment that will finally determine the effectiveness of a biodegradable scaffold. This requires a certain degree of resolution and fidelity in the scaffold production concerning the macro-shape and microarchitecture. Nonetheless, nanoscale and molecular level functionalization of biodegradable scaffolds are important to enhance tissue growth [7,8,14] as well as to achieve controlled drugs release [32]. Previous studies proved that 2D PPF-based scaffolds fabricated by photocuring with XeCl excimer laser at 308 nm are biocompatible [33], and can be functionalized with nanoparticles [34, 35] and elastin fibers [36]. These results demonstrate the appropriateness of excimer photocured PPF scaffolds for TE applications and provide the basis for the development of an accurate and scalable 3D fabrication method. In the following, this novel 3D scaffold fabrication method, namely Mask Projection Excimer laser StereoLithography (MPExSL), is presented. This method has been specifically developed aiming at a reliable and scalable production of 3D biodegradable scaffolds with spatial resolution adequate for TE applications. A variety of 3D PPF-based scaffolds are presented to illustrate the fabrication capability of the MPExSL method and an outlook towards higher fabrication yield is also given.
2. Mask Projection Excimer laser StereoLithography system
MPExSL relies on a layer-by-layer building up process where one layer is fabricated by image projection using pulsed excimer laser radiation [37, 38]. The schematic of the system and a photo of the actual apparatus are shown in Figs. 1(a) and 1(b) respectively. The output beam of a XeCl excimer laser at 308 nm is coupled to a customized mask projection optical system with a demagnification of 4, a numerical aperture of 0.1, and comprising a telescope and a motorized variable attenuator. A motorized mask holder selects the image to be projected on the resin surface in order to control the scaffold internal architecture for each layer. The mask selector, shown on the left side of the photo in Fig. 1(b), comprises a precision linear stage with micrometer resolution/repeatability and a holder plate that can host up to 5 masks of 25 mm×25 mm dimensions. It is noted that in perspective the total number of masks available can be increased using a compact 2-axis motorized stage or a motorized holder wheel, for example 20 masks of 25 mm-size can be loaded on a wheel of about 20 cm in diameter. In the MPExSL technique presented hereby, lithographic photo-masks (chromium on quartz) are used, but stencil masks may also be applied. The macro-shape of the scaffold (e.g., the scaffold diameter in the horizontal plane) can be further adapted by means of an iris diaphragm placed right in front of the mask.
Fig. 1 (a) Schematic of the MPExSL system; (b) photo of the system. Inset in (a) shows the curing depth in PPF:DEF (7:3) as a function of the laser pulse dose using a fluence of 20 mJ/cm2, and the continuous lines are the results of a logarithmic fit on the data points.
The photocurable resin is contained in a transparent cup supported by a multi-axis motorized stage. A CCD camera is used to on-line image the resin surface and in situ monitor the fabrication process. Positioning of the resin surface on the image plane is achieved by moving the sample with an XYZ stage. A fourth motorized stage (W) controls the vertical position of the scaffold-supporting platform immersed in the resin container cup in order to implement the layer-by-layer fabrication, i.e., after a layer is photocured the W stage moves downwards into the resin and allows the recast of a fresh liquid resin layer on top of the previously cured layer. The applied light dose determines the actual photocured depth, i.e., layer thickness, while the magnitude of the downwards step of the W stage determines the overlap between two adjacent layers. For example, to produce a layer of 120 μm thickness with a 20 μm overlap the following procedure consisting of 4 steps is currently implemented starting from an already cured layer: i) the W stage moves downwards by 2000 μm to immerse the cured part into the resin; ii) the W stage moves upwards by an amount of (2000-120+20) μm; iii) a time interval is waited to allow the resin layer to properly recast; iv) a shot dose is applied to cure a 120 μm-thick resin film. In addition, the XY stages can be moved during the fabrication procedure, increasing the system flexibility. During the actual fabrication process, since the W stage moves downwards the resin surface in the cup will move upwards by a specific amount proportional to the volume of the platform’s supporting posts being immersed in the resin. This accumulating vertical shift of the resin surface is taken into account when significantly high structures, i.e., with many layers, are produced. At the same time, the resin surface moves out from the preset image plane. This correction is also taken into account by moving the Z stage downwards, keeping the resin surface within the depth of focus of the optical system. The entire process, comprising laser pulsing, pulse energy setting, mask selection, and stage movements, is controlled with a PC using a simple and easily programmable routine. It is noted that in the current procedure described above a passive recasting method is used, i.e., steps i) to iii). The implementation of an active recasting method would speed up the fabrication process by decreasing the time needed for single layer photocuring as discussed in the “Conclusions and outlook” section.
The MPExSL fabrication process is graphically illustrated by the animated video Media 1 and schematically reported in Fig. 2: (a) conceptual digital rendering of the shape of the scaffold to be fabricated; (b) 3D layered internal micro-structure determined by the applied mask, as shown in the upper left inset; (c) further fabrication flexibility achieved by the iris opening/closing which allows to select a sub portion of a mask, as shown in the upper left inset, and movement of XY stages, as indicated by the arrow.
Fig. 2 Schematic description of the MPExSL process ( Media 1): a) conceptual digital rendering of the shape of the scaffold to be fabricated (the reported scale bar is indicative of the overall exposed dimension achievable with the present MPExSL set-up); b) internal layered micro-structure with a porosity determined by the applied mask as shown in the inset (the reported scale bar is indicative of the achievable resolution with the present MPExSL set-up); c) further system flexibility achieved by the mask iris opening, as shown in the inset, and movement of XY stages, as highlighted by the arrow.
Diethyl fumarate (DEF) is applied as diluent to reduce the resin viscosity as needed for proper resin recast [39]. When using 308 nm light and PPF:DEF (7:3 w:w) resin with 0.6 wt% or 1.0 wt% PI (BAPO, Irgacure 819), the curing depth can be very well controlled in the 20 to 170 μm range by varying the applied laser pulse dose as shown in the inset of Fig. 1(a). In this way, the thickness of layers building up the 3D scaffold can be well controlled with a micrometer resolution avoiding over-curing, which is essential for a reliable and precise scaffold fabrication. Indeed, the UV light at 308 nm is absorbed to some extent also by the PPF polymer [33] resulting in a self-limited light penetration into the resin. When using longer wavelength light, i.e., near-UV/visible, the use of absorbers/scatterers into the resin, e.g., dyes or nanoparticles, is often necessary in order to limit the light penetration depth, control the curing depth, and avoid over-curing [15, 16].
3. 3D scaffolds
All scaffolds presented here are fabricated at 50 Hz laser repetition rate and with a single pulse laser fluence of 20 mJ/cm2 on the resin surface. The laser pulse dose, i.e., number of delivered laser pulses, is 336 and 700 for 0.6% and 1% PI concentration, respectively, corresponding to a curing depth of 120 μm for both resin mixtures, and the layer overlap is 40 μm. For imaging with scanning electron microscopy all presented scaffolds are coated with a thin gold layer.
Woodpile scaffolds - Scaffolds with a woodpile architecture are standard structures applied, for example, to test the effect of porosity and interconnectivity, but can also be used as building blocks for more sophisticated structures. Figure 3(a) presents an image of a woodpile scaffold fabricated from PPF:DEF with 1% PI using 2 striped masks with orthogonal orientation, and applying the same exposure pattern for two consecutive layers. Figures 3(b) and 3(c) present the top and side views of the scaffold, respectively, showing open pores of tens of μm.
Multi-conduit scaffolds - Cylindrical multi-conduit scaffolds are ideal as implants for the nervous system regeneration, and the possibility to thoroughly control their design and to fabricate such scaffolds with channel diameter in the few hundred micrometer range is very attractive [40]. MPExSL is properly suited for the scalable production of such multi-conduit scaffolds. A 3 mm-high cylindrical scaffold of 5 mm external diameter and with conduits diameter of about 600 μm is shown in Fig. 4(a). Figure 4(b) shows a multi-conduit scaffold with modulated external diameter controlled by adjusting the iris aperture for each exposure, i.e., layer. The conduit diameter in this scaffold is 50 μm, as shown in the inset of Fig. 4(b). A photo of these multi-conduit scaffolds is reported in Fig. 4(c). The multi-conduits scaffolds shown in Fig. 4 are produced from PPF:DEF with 1% PI.
Fig. 4 (a) a 3 mm-high, 5 mm external diameter cylindrical scaffolds with conduits of 600 μm; (b) multi-conduit scaffold with modulated external diameter and 50 μm conduit diameter (shown in inset); (c) photo of the two multi-conduit scaffolds.
Multiple exposure scaffolds - Performing multiple exposures per layer, i.e., moving the XY stages between each exposure on the same layer, 3D scaffold with increased dimensions or an array of separate scaffolds can be produced. To achieve increased dimensions, a partial overlap between subsequent photocured part of the same layer has to be ensured. This approach enables to produce scaffolds with more sophisticated geometry using a limited set of masks. In Fig. 5(a) a 3D scaffold obtained with a single exposure per layer and using a star-shaped mask is shown.
Fig. 5 (a) scaffold obtained with a star-shaped mask; (b) scaffold obtained with the star-shaped mask using multiple exposures; (c) scaffold obtained with a square-shaped mask using multiple exposures.
Figures 5(b) and 5(c) present multiple exposure scaffolds obtained by using 4 exposures with the star-shaped mask, and 8 exposures with a square-shaped mask, respectively, demonstrating the feasibility of this aforementioned fabrication approach. The scaffolds shown in Fig. 5 are produced from PPF:DEF with 0.6% PI.
Similarly, by moving the XY stages during exposures arbitrary scaffold geometry can be obtained when using single-spot masks, eventually of various diameters and shapes. This approach allows the use of computer-generated files in a manner similar to the direct laser writing approach, further expanding the system flexibility, albeit at the expenses of production time.
4. Conclusions and outlook
With layer-by-layer MPExSL at 308 nm the production of scaffolds with tunable pore size, controlled micro-architecture, and mm-size dimensions is achieved. The method is illustrated by using a PPF:DEF resin resulting in 3D biodegradable scaffolds. The present apparatus is capable of exposing an area of a several mm2, easily expandable through the multiple exposure approach. The reported results demonstrate that MPExSL is an efficient tool for the fabrication of biocompatible structures with defined patterns in all three spatial dimensions and with a spatial resolution well-suited for TE applications.
Given the fast advance in TE and regenerative medicine it is foreseen that in the near future state-of-the-art biodegradable scaffolds will have to be produced on a daily basis and routinely used in implants’ manufacturing. Therefore, a scalable fabrication method combining flexibility and adequate spatial resolution is indispensable to satisfy the increasing demand of 3D biodegradable scaffolds. As outlook, MPExSL can indeed be scaled up for example through the combination of large area projection systems, high-power excimer lasers (both already widely and routinely used in the industry, e.g., semiconductor and photovoltaics), and the implementation of an active recasting system for the resin (already implemented in standard direct writing stereolithography systems used for rapid prototyping). As an example, let’s consider a medium power XeCl excimer laser delivering 0.6 J laser pulses at 50 Hz repetition rate (average power 30 W), a MPExSL system with 1:1 projection, and 20 mJ/cm2 fluence on the resin, which corresponds to a total irradiated area on the resin surface of 30 cm2, i.e., a circle of 6 cm in diameter. Given that 100 μm-think layer of PPF:DEF with 1% PI is photocured in about 4 seconds of irradiation (see graph in Fig. 1(a)) and assuming a 20 μm-overlap between layers, i.e. a final pore size of 60 μm in the vertical direction, and a conservative 2 second-time period needed to recast a fresh resin layer with an active recasting system, e.g., a blade sweeping on the resin surface, it results in a volumetric production yield of 144 cm3 per hour, i.e. a 4.8 cm-high scaffold. It is noted that the resolution of the present MPExSL system is about 10 μm in both vertical and horizontal directions. Higher horizontal resolution can be achieved by implementing aberration corrected optics widely available for excimer laser systems. The irradiation conditions, i.e., fluence, repetition rate, and total irradiated surface, can be tuned according to the specific need, and parallel production of many smaller scaffolds over an extended area is also feasible. Moreover, different excimer laser systems are nowadays available and used in (bio-medical) industry. From one side, excimer laser systems with hundreds of watts of average power can allow even larger surfaces to be irradiated with a single exposure or can be used with few MPExSL systems in parallel. On the other hand, compact air-cooled high-repetition rate excimer laser with average power up to several watts can be used to efficiently expose smaller areas or to implement direct laser writing irradiation.
Even if the MPExSL method is here demonstrated using PPF:DEF polymeric resin, it is important to note that in principle all UV-curable materials can be used for 3D scaffold fabrication, as for example other fumarate-based materials, like poly(caprolactone fumarate) (PCLF) [41, 42] or oligo(poly(ethylene glycol) fumarate) (OPF) [43], blends of non-photocurable polymers with PPF, like PPF-PCL blend [44], organically modified ceramics (ORMOCERs) [45], and acrylated bio-polymers, like collagen-based gelatin methacrylate (GelMA) [13], poly(ethylene glycol) diacrylate (PEGDA) [15,46], Methacrylated Glycol Chitosan (MGC) [47] and poly(d,l-lactide) dimethacrylate [14, 48]. The actual fabrication parameters (e.g., laser pulse dose, fluence per laser pulse and PI concentration) as well as the resulting scaffolds properties (e.g., photocured layer thickness, mechanical stability and biocompatibility) will have to be determined by performing systematic experimental tests for each photocurable resin of interest.
In conclusion, given the flexibility and the overall simple approach of excimer laser photocuring, the MPExSL technique offers a concrete possibility to translate SL fabrication method from the rapid prototyping arena to the actual production of 3D microstructures, as for example biodegradable scaffolds to be used in regenerative medicine.
Acknowledgments
The authors gratefully thank Alice Scarpellini for SEM imaging. This work has received funding support from the EC (FP7-NMP-2013-EU-China), grant agreement n. 604263 ( NEUROSCAFFOLDS).
References and links
1. R. Langer and J. P. Vacanti, “Tissue engineering,” Science 260, 920–926 (1993). [CrossRef] [PubMed]
2. S. J. Hollister, “Porous scaffold design for tissue engineering,” Nature Mater. 4, 518–524 (2005). [CrossRef]
3. S. A. Skoog, P. L. Goering, and R. J. Narayan, “Stereolithography in tissue engineering,” J Mater Sci: Mater Med 25, 845–856 (2014).
4. M. J. Sawkins, K. M. Shakesheff, L. J. Bonassar, and G. R. Kirkham, “3D cell and scaffold patterning strategies in tissue engineering,” Recent Pat. Biomed. Eng. 6, 3–21 (2013). [CrossRef]
5. F. P. W. Melchels, J. Feijen, and D. W. Grijpma, “A review on stereolithography and its applications in biomedical engineering,” Biomaterials 31, 6121–6130 (2010). [CrossRef] [PubMed]
6. A. M. Greiner, B. Richter, and M. Bastmeyer, “Micro-engineered 3-D scaffolds for cell culture studies,” Macromol. Biosci. 12, 1301–1314 (2012). [CrossRef] [PubMed]
7. J. W. Lee, K. S. Kang, S. H. Lee, J.-Y. Kim, B.-K. Lee, and D.-W. Cho, “Bone regeneration using a microstereolithography-produced customized poly(propylene fumarate)/diethyl fumarate photopolymer 3-D scaffold incorporating BMP-2 loaded PLGA microspheres,” Biomaterials 32, 744–752 (2011). [CrossRef]
8. P. X. Lan, J. W. Lee, Y.-J. Seol, and D.-W. Cho, “Development of 3D PPF/DEF scaffolds using micro-stereolithography and surface modification,” J. Mater. Sci.: Mater Med. 20, 271–279 (2009).
9. M. T. Raimondi, S. M. Eaton, M. Laganà, V. Aprile, M. M. Nava, G. Cerullo, and R. Osellame, “Three-dimensional structural niches engineered via two-photon laser polymerization promote stem cell homing,” Acta Biomater. 9, 4579–4584 (2013). [CrossRef]
10. V. Melissinaki, A. Gill, I. Ortega, M. Vamvakaki, A. Ranella, J. W. Haycock, C. Fotakis, M. Farsari, and F. Claeyssens, “Direct laser writing of 3-D scaffolds for neural tissue engineering applications,” Biofabrication 3, 045005 (2011). [CrossRef]
11. D. Dean, J. Wallace, A. Siblani, M. O. Wang, K. Kim, A. G. Mikos, and J. P. Fisher, “Continuous digital light processing (cDLP): highly accurate additive manufacturing of tissue engineered bone scaffolds,” Virtual. Phys. Prototyp. 7, 13–24 (2012). [CrossRef] [PubMed]
12. A. P. Zhang, X. Qu, P. Soman, K. C. Hribar, J. W. Lee, S. Chen, and S. He, “Rapid fabrication of complex 3D extracellular microenvironments by dynamic optical projection stereolithography,” Adv. Mater. 24, 4266–4270 (2012). [CrossRef] [PubMed]
13. R. Gauvin, Y.-C. Chen, J. W. Lee, P. Soman, P. Zorlutuna, J. W. Nichol, H. Bae, S. Chen, and A. Khademhosseini, “Microfabrication of complex porous tissue engineering scaffolds using 3-D projection stereolithography,” Biomaterials 33, 3824–3834 (2012). [CrossRef] [PubMed]
14. A. Ronca, L. Ambrosio, and D. W. Grijpma, “Preparation of designed poly(d,l-lactide)/nanosized hydroxyapatite composite structures by stereolithography,” Acta Biomater. 9, 5989–5996 (2013). [CrossRef]
15. L.-H. Han, G. Mapili, S. Chen, and K. Roy, “Projection microfabrication of three-dimensional scaffolds for tissue engineering,” Journal of Manufacturing Science and Engineering 130, 021005 (2008). [CrossRef]
16. J. Wallace, M. O. Wang, P. Thompson, M. Busso, V. Belle, N. Mammoser, K. Kim, J. P. Fisher, A. Soblani, Y. Xu, J. F. Welter, D. P. Lennon, J. Sun, A. I. Caplan, and D. Dean, “Validating continuous digital light processing (cDLP) additive manufacturing accuracy and tissue engineering utility of a dye-initiator package,” Biofabrication 6, 015003 (2014). [CrossRef] [PubMed]
17. D. Sameoto and C. Menon, “A low-cost, high-yield fabrication method for producing optimized biomimetic dry adhesives,” J. Micromech. Microeng. 19, 115002 (2009). [CrossRef]
18. Y. Daicho, T. Murakami, T. Hagiwara, and S. Maruo, “Formation of three-dimensional carbon microstructures via two-photon microfabrication and microtransfer molding,” Opt. Mat. Express 3, 875–883 (2013). [CrossRef]
19. A. Schaap and Y. Bellouard, “Molding topologically-complex 3D polymer microstructures from femtosecond laser machined glass,” Opt. Mat. Express 3, 1428–1437 (2013). [CrossRef]
20. A. Koroleva, A. Gill, I. Ortega, J. W. Haycock, S. Schlie, S. D. Gittard, B. N. Chichkov, and F. Claeyssens, “Two-photon polymerization-generated and micromolding-replicated 3-D scaffolds for peripheral neural tissue engineering applications,” Biofabrication 4, 025005 (2012). [CrossRef]
21. A. Koroleva, S. Gittard, S. Schlie, A. Deiwick, S. Jockenhoevel, and B. Chichkov, “Fabrication of fibrin scaffolds with controlled microscale architecture by a two-photon polymerization-micromolding technique,” Biofabrication 4, 015001 (2012). [CrossRef] [PubMed]
22. C. N. LaFratta, T. Baldacchini, R. A. Farrer, J. T. Fourkas, M. C. Teich, B. E. A. Saleh, and M. J. Naughton, “Replication of two-photon-polymerized structures with extremely high aspect ratios and large overhangs,” J. Phys. Chem. B 108, 11256–11258 (2004). [CrossRef]
23. M. Malinauskas, M. Farsari, A. Piskarskas, and S. Juodkazis, “Ultrafast laser nanostructuring of photopolymers: A decade of advances,” Physics Reports 533, 1–31 (2013). [CrossRef]
24. J. L. Ifkovits and J. A. Burdick, “Review: photopolymerizable and degradable biomaterials for tissue engineering applications,” Tissue Eng. 13, 2369–2385 (2007). [CrossRef] [PubMed]
25. F. K. Kasper, K. Tanahashi, J. P. Fisher, and A. G. Mikos, “Synthesis of poly(propylene fumarate),” Nat. Protoc. 4, 518–525 (2009). [CrossRef] [PubMed]
26. C. Wei, L. Cai, B. Sonawane, S. Wang, and J. Dong, “High-precision flexible fabrication of tissue engineering scaffolds using distinct polymers,” Biofabrication 4, 025009 (2012). [CrossRef] [PubMed]
27. B. K. Chen, A. M. Knight, N. N. Madigan, L. Gross, M. Dadsetan, J. J. Nesbitt, G. E. Rooney, B. L. Currier, M. J. Yaszemski, R. J. Spinner, and A. J. Windebank, “Comparison of polymer scaffolds in rat spinal cord: a step toward quantitative assessment of combinatorial approaches to spinal cord repair,” Biomaterials 32, 8077–8086 (2011). [CrossRef] [PubMed]
28. S. Wang, M. J. Yaszemski, A. M. Knight, J. A. Gruetzmacher, A. J. Windebank, and L. Lu, “Photo-crosslinked poly(ε-caprolactone fumarate) networks for guided peripheral nerve regeneration: Material properties and preliminary biological evaluations,” Acta Biomater. 5, 1531–1542 (2009). [CrossRef] [PubMed]
29. S. Wang and L. Cai, “Polymers for fabricating nerve conduits,” Int. J. Polym. Sci. 2010, 138686 (2010). [CrossRef]
30. K.-W. Lee, S. Wang, B. C. Fox, E. L. Ritman, M. J. Yaszemski, and L. Lu, “Poly(propylene fumarate) bone tissue engineering scaffold fabrication using stereolithography: effects of resin formulations and laser parameters,“ Biomacromolecules 8, 1077–1084 (2007). [CrossRef] [PubMed]
31. H.-W. Kang, J. H. Park, and D.-W. Cho, “Development of an indirect stereolithography technology for scaffold fabrication with a wide range of biomaterial selectivity,“ Tissue Engineering 18, 719–729 (2012). [CrossRef] [PubMed]
32. J. Choi, K. Kim, T. Kim, G. Liu, A. Bar-Shir, T. Hyeon, M. T. McMahon, J. W. M. Bulte, J. P. Fisher, and A. A. Gilad, “Multimodal imaging of sustained drug release from 3-D poly(propylene fumarate) (PPF) scaffolds,” J. Control. Release 156, 239–245 (2011). [CrossRef] [PubMed]
33. S. Beke, F. Anjum, H. Tsushima, L. Ceseracciu, E. Chieregatti, A. Diaspro, A. Athanassiou, and F. Brandi, “Towards excimer-laser-based stereolithography: a rapid process to fabricate rigid biodegradable photopolymer scaffolds,” J. R. Soc. Interface 9, 3017–3026 (2012). [CrossRef] [PubMed]
34. S. Beke, L. Kőrösi, A. Scarpellini, F. Anjum, and F. Brandi, “Titanate nanotube coatings on biodegradable photopolymer scaffolds,” Mat. Sci. Eng. C 33, 2460–2463 (2013). [CrossRef]
35. S. Beke, R. Barenghi, B. Farkas, I. Romano, L. Kőrösi, S. Scaglione, and F. Brandi, “Improved cell activity on biodegradable photopolymer scaffolds using titanate nanotube coatings,” Mat. Sci. Eng. C, DOI: [CrossRef] .
36. S. Scaglione, R. Barenghi, S. Beke, L. Ceseracciu, I. Romano, F. Sbrana, P. Stagnaro, F. Brandi, and M. Vassalli, “Characterization of a bioinspired elastin-polypropylene fumarate material for vascular prostheses applications,” Proc. of SPIE 8792, 87920H (2013). [CrossRef]
37. F. Brandi, F. Anjum, L. Ceseracciu, A. C. Barone, and A. Athanassiou, “Rigid biodegradable photopolymer structures of high resolution using deep-UV laser photocuring,” J. Micromech. and Microeng. 21, 054007 (2011). [CrossRef]
38. S. Beke, F. Anjum, L. Ceseracciu, I. Romano, A. Athanassiou, A. Diaspro, and F. Brandi, “Rapid fabrication of rigid biodegradable scaffolds by excimer laser mask projection technique: a comparison between 248 and 308 nm,” Laser Phys. 23, 035602 (2013). [CrossRef]
39. J.-W. Choi, R Wicker, S.-H. Lee, K.-H. Choi, C.-S. Ha, and I. Chung, “Fabrication of 3-D biocompatible/biodegradable micro-scaffolds using dynamic mask projection microstereolithography,” J. Mater. Process. Tech. 209, 5494–5503 (2009). [CrossRef]
40. W. Daly, L. Yao, D. Zeugolis, A. Windebank, and A. Pandit, “A biomaterials approach to peripheral nerve regeneration: bridging the peripheral nerve gap and enhancing functional recovery,” J. R. Soc. Interface 9, 202–221 (2012). [CrossRef]
41. S. Wang, D. H. Kempen, N. K. Simha, J. L. Lewis, A. J. Windebank, M. J. Yaszemski, and L. Lu, “Photo-crosslinked hybrid polymer networks consisting of poly(propylene fumarate) (PPF) and poly(caprolactone fumarate) (PCLF): controlled physical properties and regulated bone and nerve cell responses,” Biomacromolecules 9, 1229–1241 (2008). [CrossRef] [PubMed]
42. M. B. Runge, H. Wang, R. J. Spinner, A. J. Windebank, and M. J. Yaszemski, “Reformulating polycaprolactone fumarate to eliminate toxic diethylene glycol: effects of polymeric branching and autoclave sterilization on material properties,” Acta Biomater. 8, 133–143 (2012). [CrossRef]
43. L. A. Kinard, F. K. Kasper, and A. G. Mikos, “Synthesis of oligo(poly(ethylene glycol) fumarate),” Nature Protocols 7, 1219–1227 (2012). [CrossRef] [PubMed]
44. K. Wang, L. Cai, F. Hao, X. Xu, M. Cui, and S. Wang, “Distinct cell responses to substrates consisting of poly(ε-caprolactone) and poly(propylene fumarate) in the presence or absence of cross-links,” Biomacromolecules 11, 2748–2759 (2010). [CrossRef] [PubMed]
45. J. Stampfl, S. Baudis, C. Heller, R. Liska, A. Neumeister, R. Kling, A. Ostendorf, and M. Spitzbart, “Photopolymers with tunable mechanical properties processed by laser-based high-resolution stereolithography,” J. Micromech. Microeng. 18, 125014 (2008). [CrossRef]
46. C. Cha, P. Soman, W. Zhu, M. Nikkhah, G. Camci-Unal, S. Chen, and A. Khademhosseini, “Structural reinforcement of cell-laden hydrogels with microfabricated three dimensional scaffolds,” Biomater. Sci. 2, 703–709 (2014). [CrossRef] [PubMed]
47. B. G. Amsden, A. Sukarto, D. K. Knight, and S. N. Shapka, “Methacrylated glycol chitosan as a photopolymerizable biomaterial,” Biomacromolecules 8, 3758–3766 (2007). [CrossRef] [PubMed]
48. F. P. W. Melchels, A. H. Velders, J. Feijen, and D. W. Grijpma, “Photo-cross-linked poly(d,l-lactide)-based networks. Structural characterization by HR-MAS NMR spectroscopy and hydrolytic degradation behavior,” Macromolecules 43, 8570–8579 (2010). [CrossRef]
