Lithography-based additive manufacture of ceramic biodevices with design-controlled surface topographies

The possibility of manufacturing textured materials and devices, with surface properties controlled from the design stage, instead of being the result of machining processes or chemical attacks, is a key factor for the incorporation of advanced functionalities to a wide set of micro- and nano-systems. High-precision additive manufacturing (AM) technologies based on photopolymerization, together with the use of fractal models linked to computer-aided design tools, allow for a precise definition of final surface properties. However, the polymeric master parts obtained with most commercial systems are usually inadequate for biomedical purposes and their limited strength and size prevents many potential applications. On the other hand, additive manufacturing technologies aimed at the production of final parts, normally based on layer-by-layer melting or sintering ceramic or metallic powders, do not always provide the required precision for obtaining controlled micro-structured surfaces with high-aspect-ratio details. Towards the desired degree of precision and performance, lithography-based ceramic manufacture is a remarkable option, as we discuss in the present study, which presents the development of two different micro-textured biodevices for cell culture. Results show a remarkable control of the surface topography of ceramic parts and the possibility of obtaining design-controlled micro-structured surfaces with high-aspect-ratio micro-metric details.


Introduction
Material (and device) surface topography has a direct influence on several relevant properties, linked to its final performance, such as friction coefficient [1], wear resistance [2], self-cleaning ability [3], biocompatibility [4], optical response [5], touch perception, overall esthetic aspect, and even flavor [6]. Therefore, it also plays a determinant role in material selection in engineering design, especially in the field of micro-and nano-system development, in which the effects of topography on the incorporation of advanced properties are even more remarkable.
Normally a device surface topography is a consequence of its material's natural state or the result of machining processes, chemical attacks, or post-processes used for the manufacture of a device or product. Several strategies for modifying material topographies and surface properties have taken advantage of conventional surface micro-machining [7], laser ablation [8], micro-molding [9], biomimetic templating [10], physical and chemical vapor deposition processes [11], sol-gel procedures [12], and molecular self-assembly [13]. All these processes require enormous hands-on expertise and final result depends on several control parameters, whose interdependencies are normally complex to understand, characterize, model, and master [14]. As can be seen from the previously cited documents, top-down and bottom-up approaches for controlling surface properties co-exist and in many cases complement each other [15], the former being more focused on mass-production (as it derives from the micro-electronic industry), the latter providing remarkable geometrical versatility. Combinations of top-down and bottom-up approaches are frequent and have usually focused on manufacturing the larger micro-metric features by means of top-down processes (micro-machining, etching…) and the smaller nano-metric details by using bottom-up techniques (CVD, PVD, sol-gel…).
Advances in computer-aided design and in additive manufacturing (AM) technologies are opening new horizons for controlling surface topography, of materials and devices, from the design stage and in a very direct, rapid, and easy way. Even though conventional computer-aided design is only capable of handling Euclidean geometries and mainly rely on simple operations (sketch-based operations, extrusions, pads, holes, circular grooves…) for obtaining "soft" solids and surfaces, recent approaches relying on the use of matrix-based programming have already proved to be useful for designing rough surfaces and textured objects adequately described by fractal geometries [16,17]. In parallel, the continued progress on additive manufacturing technologies (also called "solid free-form fabrication" due to the complex geometries attainable), especially during the last decade, has increased the range of materials capable of being additively processed and greatly promoted their precision, even down to nano-metric features, with implications in the development of advanced materials, metamaterials, and devices based on them [18,19].
In spite of the aforementioned potentials, the polymers used in lithography-based AMT, are frequently of limited biocompatibility or inadequate for biomedical applications, what limits enormously the span of final applications. For instance, common thermoplastics used for the mass production of medical devices, including poly(methyl methacrylate) (PMMA) or polycarbonate (PC), cannot be processed using conventional additive manufacturing technologies, and multi-stage processes are required for linking free-form fabrication with mass production [20]. Recent research has also achieved groundbreaking improvements in the bio-compatibility of AM materials [21,22] and dramatically helped to increase the manufacture speed and the attainable precision of these technologies [23]. On the other hand, additive manufacturing technologies aimed at the production of final parts for demanding applications, normally based on layer-by-layer melting or sintering ceramic or metallic powders, do not provide the required precision for obtaining controlled micro-textures and for enabling related advanced functionalities.
Towards the desired degree of precision and performance, LCM technology is a good alternative in terms of detail and attainable aspect ratios. In short, LCM is based on the layer-by-layer selective curing of a photosensitive resin which contains homogeneously dispersed ceramic particles. The photopolymer acts as binder between the ceramic particles and makes the precise shaping of the part possible. The shaped form is produced as a green body that has to be further processed to obtain a part with higher relative density (99.4 % T.D.). These post-processing steps include the debinding, i.e., the thermal decomposition of the binder and the subsequent sintering into a compact ceramic part. This technology was developed at TU Wien [24][25][26] and is currently industrially developed by Lithoz GmbH.
In this study, we present the development of two different micro-textured biodevices: a textured cell culture platform and a textured micro-device for studying cell motility. Results show a valuable alternative for precisely defining and controlling the surface topography of ceramic parts from the design stage and for obtaining high-aspect-ratio micro-metric details in ceramic components with complex geometries. Final parts can be directly used for cell culture purposes, due to the adequate properties of the ceramic materials used, without requiring post-processes such as CVD-PVD depositions for promoting biocompatibility or additional replications steps using biomaterials. The strength, toughness, and hardness of the additively obtained ceramic parts make them even candidates for the manufacture of mold inserts for mass production. The following section explains the methods and materials used, before paying attention to the main results obtained, proposing some future directions and detailing our concluding remarks.  The first application example is a set of micro-textured fractal surface-based cell culture platforms, with an improved level of detail, for more adequate interactions at a cellular level than previous preliminary prototypes [27]. The upper images of Fig. 1 show a couple of micro-textured platforms with different roughness levels. These circular platforms are 10 mm in diameter. In both cases, the upper surfaces include a fractal texture. Typical peak heights lay between 50 and 250 μm for the left model and between 350 and 550 μm for the right model.
The second application example (shown in the lower image of Fig. 1) corresponds to a micro-system for studying cell motility and addressing the effect of surface texture on cell migration and overall behavior, as previously detailed [28]. The system includes a couple of micro-chambers connected by several micro-channels, for guiding cell movement, each of them with a different texture at its bottom. A typical cell motility experiment should begin with the incorporation of cells to one of the chambers and of growth factors to the other one, so as to promote cell movement from one chamber to another. Channels are 200 μm in width and 6 mm in length. The roughness of the different channels (maximum peak height) reaches values around 5, 5, 100, and 150 μm, respectively.
Both micro-systems are based on a design procedure firstly described by our team [27] combining different steps, including (a) the generation of fractal textures using fractional Brownian surface models with the help of Matlab (The Mathworks Inc.), (b) the conversion of the fractal surfaces into .stl format for further manipulation with computer-aided design programs, (c) the incorporation of thickness to the fractal surfaces using conventional CAD modeling tools, and (d) the combination, by means of Boolean operations, of the textured zones with other solids previously designed, in order to obtain more complex micro-systems or to adjust final size and external shape. Our preliminary in vitro trials with both microsystems were carried upon rapid prototypes adequately coated with diamond-like carbon, to avoid the toxic effects of the acrylic resin, and upon some rapid copies obtained using PDMS casting, as previously detailed [27,28], and showed promising results.  Figure 2 shows a design adaptation, eliminating the excess of material in the lateral walls of the micro-system by means of Boolean operations, for controlling over-exposure to blue light, which may limit precision, and for minimizing material contraction and related stresses during sintering, as further discussed in the manufacturing section.

Additive manufacture of master prototypes or green parts
Once the design is prepared and optimized for 3D printing, manufacture is accomplished by means of lithography-based ceramic manufacturing (LCM) [29]. The master models or green parts are manufactured, with previously prepared alumina slurries, by digital light processing (DLP) using the CeraFab 7500 machine, Lithoz GmbH (Fig. 3). The slurries are prepared with commercial Al 2 O 3 powders. These powders are homogeneously dispersed, with the help of a dispersing agent, in a formulation containing reactive monomers and a solvent. In addition, the formulation contains a photoinitiator (typically less than 1 wt%). The photoinitiator reacts under an external energy source, in this case a LED emitting at 460 nm, which excites the initiator, creating radicals that chemically react with the monomers included in the mixture. The chain reaction forms the desired matrix of (meth)acrylate monomers that bind together the ceramic particles in the shape of the original part. The reaction occurs in a brief lapse of time, while a determinate section of the part is being projected with specific intensity and exposure time parameters. DLP uses dynamic masks, which represent an individual cross section of the part being manufactured. The light engine uses highperformance LEDs as light source and a DMD chip (digital mirror device) as dynamic mask with a resolution of 1920 × 1080 pixels and a pixel size of 40 × 40 μm [29]. The fabrication of the part is done in sequential layer-by-layer manner. For each individual layer, fresh slurry is applied on the building envelope via a dosage system and subsequent rotation of the vat. Afterwards, the building platform is lowered into the dispersion to a distance of 25 μm to the bottom of the vat, which equals the thickness of an individual layer in the green body. Then, the space-resolved exposure of the slurry is done by the projection of an image corresponding profile-generator tool, which leads to a 3D representation of the object by overlapping a set of images taken with a determinate pitch. As the result of this procedure, the final dimension could be slightly different, for that reason is understood that the heights given by the profiler are not exactly precise. However, the measurements performed help to validate the proposed approach and allow us to check that the fractal peaks manufactured with differences in height and width of less than 19 % when compared with the original CAD.
The process stands out for a manufacturing speed in the range of around 100 layers/hour with a XY plane resolution of 40 μm and a layer thickness of 25 μm, hence being among the most precise and rapid procedures for the manufacture of high-quality ceramic parts, even when compared with very recent developments [30]. In our case, shrinkage is around 19 %, well below the 30 % described by using pre-ceramic monomers [30]. The provided images show high-aspect-ratio details with lateral dimensions even lower than 40 μm and heights above 500 μm, which is well beyond an aspect ratio of 10, which is noteworthy for additively manufactured ceramic parts.

Conclusions
In this work, we have presented the development of two different micro-textured biodevices which were manufactured by lithography-based AM: a textured cell culture platform and a textured micro-device for studying cell motility. Results show a valuable alternative for precisely defining and controlling the surface topography of ceramic parts from the design stage and for obtaining high-aspect-ratio micrometric details in ceramic components with very complex geometries. The proposed process stands out for the attainable degree of detail, for the high aspect ratios, for the adequacy of final materials for biomedical testing, for the manufacturing speed and for the possibility of obtaining textured micro-systems, which are quite complex to manufacture using conventional micromachining technologies.
Regarding future studies, it will be important to focus on exploring in depth the possible applications of designcontrolled textured surfaces and related devices. We foresee relevant implications for areas including: tribology, due to the potential promotion of adhesion using fractal textures; microfluidics, due to the possibility of controlling the hydrophobicity and hydrophilicity of surfaces by acting on their topography; optics, due to the option of changing surface reflection properties and overall esthetic, and biomedical engineering, for the promotion of biomimetic designs. Currently, the design process, for enabling the introduction of controlled texture gradients and different kinds of texture variations within the surfaces of interest for additional versatility, is being improved.