FABRICATION OF 3D MICRO-TEXTURED SCAFFOLDS FORTISSUE ENGINEERING

Alvaro Mata, Aaron J. Fleischman, Shuvo RoyDepartment of Chemical and Biomedical Engineering, Cleveland State University

Department of Biomedical Engineering, Lerner Research InstituteThe Cleveland Clinic Foundation, Cleveland, Ohio

INTRODUCTIONSpecific micro- and macro-scale features within a three-dimensional (3D) scaffold have significant

effects on cells and tissues, and play an important role in complex tissue function.1 Traditional fabricationtechniques for tissue engineering 3D scaffolds such as phase separation, fiber bonding, and solvent castinghave limited reproducibility and control of the micro-architecture (pore size, geometry, and distribution).1,2,3

Newer methods that achieve precise micro-architectures such as fused deposition modeling, selective lasersintering, 3-D printing, and micro-stereolithography have limited control of the scaffold surface topography1,which has been demonstrated to affect cell characteriztics.4,5 Microfabrication-based techniques have alsobeen used to construct precise micro-architectures for tissue engineering scaffolds6,7, but withoutincorporation of surface micro-textures. The current study presents the design, assembly, and validation of amechanical jig for dual-sided molding of Polydimethylsiloxane (PDMS) layers. The stacking of these layersform a 3D scaffold with both precise micro-architecture and surface micro-textures that can be designed forspecific tissue engineering applications.8

DESIGN AND ASSEMBLY OF MECHANICAL JIGThe mechanical jig was designed and assembled to provide precise alignment of the molds during

dual-sided molding of PDMS layers. The molds were either silicon wafers, silicon wafers patterned with SU-8 photoresist, or cured PDMS layers with surface micro-textures. The mechanical jig consisted of variouscomponents that were either purchased from Newport Corporation (Irvine, CA) or built in the PrototypeLaboratory in the Lerner Research Institute at The Cleveland Clinic Foundation (Cleveland, OH) (Figure 1).The custom pieces and assembly of the jig were designed using AutoCAD 2004 (Autodesk, Inc., San Rafael,CA) (Figure 2a).

Figure 1 Components of the mechanical jig for precise alignment of molds prior to assembly. Somecommercially available components were also custom machined by Newport Corporation to fitspecifications of the overall assembly (*).

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Part Newport Cat. # Quantity1 Bottom Plate M-SA-08X08 * 12 Top Plate M-SA-08X08 * 13 Z-Stage 271 14 X-Y-Stage M-406 15 Rotational Stage M-UTR120A 16 Posts M-P-3 127 Adaptor Plate M-PBN12 * 18 Adaptor Plate Built at CCF 2

Part Newport Cat. # Quantity1 Bottom Plate M-SA-08X08 * 12 Top Plate M-SA-08X08 * 13 Z-Stage 271 14 X-Y-Stage M-406 15 Rotational Stage M-UTR120A 16 Posts M-P-3 127 Adaptor Plate M-PBN12 * 18 Adaptor Plate Built at CCF 2

The major components of the device included a Z-stage for vertical motion control, an X-Y-stage forlateral motion control, and a rotation stage for angular motion control. These three parts were assembled oneon top of the other and positioned within two plates and four columns that provided stability and unificationof the different parts (Figure 2b). In addition, adaptor plates were machined at The Cleveland ClinicFoundation to serve as attachment pieces between the three stages. Construction of the jig started with theassembly of four columns of three M-P-3 posts each. These columns were formed by three posts in order toachieve a height to accommodate all the different stages, including the completely extended Z-stage. Thecolumns were positioned on the corners of the bottom plate (M-SA-08X08). Afterwards, the 271 Z-stage wasscrewed in the middle of the bottom plate. A custom adaptor plate was subsequently screwed to the top of the271 Z-stage and to the bottom of the M-406 X-Y-stage. Then, the second custom adaptor plate was firstscrewed to the bottom of the M-UTR120A rotational-stage, and then to the top of the M-406 X-Y-stage. TheM-PBN12 adaptor plate was screwed on the top of the M-UTR120A rotational-stage, and served as the platethat supported one of the two molds. Finally, the top plate (M-SA-08X08) to hold the other mold was screwedto the top of the four columns, and provided stability to the whole structure.

DUAL-SIDED MOLDING OF PDMS WITH MECHANICAL JIG In order to test the alignment capabilities of the mechanical jig, various patterned silicon wafers andmicro-textured PDMS layers were used as molds for dual-sided molding of PDMS. The molds werepatterned silicon wafers and cured PDMS layers with 10 µm high and 10 µm diameter posts separatedby 10 µm (Figure 3a), and 11 µm high and 45 µm wide curved channels (Figure 3b). These molds werecoated with 1H,1H,2H,2H-Perfluoro-decyltrichlorosilane (Lancaster, Pelham, NH) to aid the release of themolded PDMS layer. PDMS Sylgard 184 (Dow Corning, Midland, MI) was mixed as previously described9,poured on top of both molds, distributed to cover all the patterned areas of the molds, and degassed for 15min. Then, both molds (containing uncured PDMS) were placed on the mechanical jig, aligned, and broughtto contact (with the patterned sides facing each other) while squeezing the uncured PDMS (Figure 4). The jigwas then placed inside of an oven at 75° C for 2 hours to cure the PDMS. After curing, the two molds wereremoved from the jig, allowed to cool to room temperature, and immersed in methanol to remove (separate)both molds from the squeezed PDMS.

Bottom Plate

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Figure 2 Image illustrates (a) the AutoCAD drawing and (b) layout of the final assembly of thedifferent components of the mechanical jig. Dimensions are in millimeters.

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The mechanical jig controlled the lateral, vertical, and rotational motion of the molds used duringdual-sided molding of PDMS layers. This device achieved a relative alignment of the molds within ~±10 µmin the X-Y plane and ~±50 µm in the Z direction. The resulting PDMS layers were between 5 - 100 µm thickand exhibited micro-textures on both sides of the PDMS layers (Figure 5). When the two molds were incontact, the areas of contact produced through holes on the PDMS layer (Figure 5c,d).

Figure 4 Photographs of mechanical jig depicting the position of the SU-8 molds with theuncured PDMS (a); and the two molds brought in contact to squeeze the PDMS (PDMS“sandwich”) showing the horizontal, vertical, and rotational motion control knobs of the jig (b).

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Figure 3 SEM images illustrate the geometrical features of the two molds used for validation ofthe jig including (a) 10 µm high and 10 µm diameter posts separated by 10 µm; and (b) 11 µmhigh and 45 µm wide curved channels.

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Figure 5 SEM images of PDMS layers thatwere dual-sided molded with the mechanicaljig. PDMS layers were between 5 - 100 µmthick, and exhibited either micro-textures onboth sides of the layer (a, b), or through holes(c, d). Special care was given to ensuringuniform contact between the two molds.Otherwise, uneven contact due to waferbowing and edge bead formation could resultin blocked through holes or non-uniformthickness of the PDMS layers.

Si SU-8 2100 SU-8 2010

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The fabrication of the 3D scaffold consists of three basic steps that are multi-level SU-8 moldfabrication, dual-sided molding of PDMS layers, and stacking of PDMS layers. The details of the fabricationof the 3D scaffold have been published elsewhere (Figure 6).8,10 The resulting 3D PDMS scaffold comprised66% porosity by volume with 300 µm diameter meandering vertical pores, 200 µm x 400 µm horizontalpores, and 71% of the surfaces within the scaffold covered with 10 µm diameter and 10 µm high posts(Figure 7).

Figure 6 Fabrication of 3D PDMS scaffolds. The first step is the processing of multi-level SU-8 moldsstarting with patterning (spin coating, baking, exposing, and post-exposure baking) of a 200 µm thicklayer of SU-8 2100 (a1) followed by patterning of a 10 µm thick layer of SU-8 2010 (a2), andpatterning of a 100 µm thick layer of SU-8 2100 (a3). All three layers were developed simultaneouslywith SU-8 Developer to dissolve the un-exposed regions (a4). The second step consists of dual-sidedmolding of PDMS. Images illustrate the two aligned molds in contact with each other while squeezingand molding PDMS (b1); and the cured PDMS layer released from the molds (b2). The third step in thefabrication is stacking of the PDMS layers. Images depict stamping of a PDMS layer over a 10 µmthick uncured PDMS film (adhesive) to wet the tips of the 200 µm diameter columns (c1); andsubsequent stacking of layers to achieve a meandering pore geometry (c2). Curing of the adhesivePDMS resulted in adhesion of all the PDMS layers to realize a 3D scaffold with 66% porosity byvolume and 71% of surfaces covered with 10 µm diameter and 10 µm high posts.

Figure 7 SEM images of (a) a five-layer PDMS scaffold on a penny depicting the through holeson each layer; (b) the scaffold cross-section showing the alignment between adjacent layers thatresulted in a meandering pore geometry; and (c) a closer view depicting the 10 µm diameter and10 µm high posts (inset) present on all horizontal surfaces (71% of all surfaces within the 3Dscaffold).

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CONCLUSIONMicrofabrication-based techniques have great potential to control micro- and macro-geometrical

features within 3D scaffolds. This paper presented the design, development, and validation of a mechanicaljig that allows dual-sided molding of PDMS layers, which is a critical step in the fabrication of the 3DPDMS scaffolds. The mechanical jig was successfully constructed from the assembly of custom made andpurchased parts that were integrated to support an X-Y-stage, a Z-stage, and a rotational stage. Thecombination of these three stages allowed the motion control of molds within ~±10 µm in the X-Y plane and~±50 µm in the Z direction. Validation of the mechanical jig produced PDMS layers that were between 5 -100 µm thick and exhibited micro-textures on both sides of the PDMS layers or through holes.

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