SUPPORTING INFORMATION Hybrid 3D-2D printing methods for bone scaffolds fabrication V A Seleznev and V Ya Prinz Rzhanov Institute of Semiconductor Physics, Siberian Branch of the Russian Academy of Sciences (ISP SBRAS), pr. Lavrentieva 13, Novosibirsk, Russia, 630090; E-mail:
[email protected]
Materials: Poly(L-lactide) PURASORB PL 65 with a molecular weight of 65000 (Corbion, Netherlands), PDMS Sylgard 184 (Dow Corning, USA), photopolymerizable UV-PDMS X-34-4184 (Shin-Etsu, Japan), photocurable resin 3DKA83G (CARIMA, Korea). Polymer films were used to fabricate intermediate polymer stamps (IPS®), and TU2-90 resist was used to perform a combined UV and thermal NIL process (STU® process). Si master nanostamps were purchased from Obducat, Sweden. The nanostructured Si master nanostamp was a periodic array of extended nanogrooves 100 nm wide (array pitch 190±5 nm, groove depth 155±5 nm) covering the entire surface of a 150-mm diameter Si substrate.
Fabrication of PLA films PLA films were prepared from PLA powders dissolved in chloroform. Full dissolution of powders yielding a homogeneous solution was achieved after several days under permanent agitation. The prepared PLA solutions were applied onto 100-μm thick polytetrafluorethylene film substrates using a K Hand Coater (RK Print Coat Instruments Ltd, UK), which facility permitted preparation of thickness-uniform (in the range of thicknesses from 4 to 500 μm) large-area (over 100 cm2) PLA films. In our study, a 200-μm thick layer of PLA solution was applied onto polytetrafluorethylene film substrates. The solwent was removed from the applied films by drying them at room temperature during 48 hours. As a result, homogeneous PLA films 10 or 20 μm thick prepared respectively from 5- or 10-% PLA solutions were formed on the polytetrafluorethylene substrates. Due to the high chemical inertness of the polytetrafluorethylene substrates, the PLA films could easily be detached from them. The thickness of the films was controlled using a micrometer gauge.
Fabrication of silicon master stamps The fabrication of silicon master stamps with micro- and nanostructured regions was performed by means of photolithography and nanoimprint lithography. Initially, nanoimprint lithography was used to form a nanorelief presenting a nanogroove array on a Si substrate. In the latter process, a nanostructured Si master stamp with a periodic array of extended nanogrooves of depth 155±5 nm and width 100 nm (array pitch 190±5 nm) that covered the entire surface of a 150-mm diameter Si substrate, was used. Technologies developed by Obducat (Sweden) were employed [1]. Initially, an intermediate polymer stamp (IPS®) was fabricated. That polymer stamp was subsequently used to perform a combined UV and thermal NIL (STU® process) treatment of an 80-nm thick TU-2-90 resist layer applied onto a Si substrate with the help of a spincouter. Anisotropic reactive ion
etching of Si substrate in CF6 plasma through a mask prepared on a PlasmaLab System 100 facility («Oxford Instruments», Great Britain) resulted in the formation of nanogrooves of depth 150 nm and width 80 nm located at 190-nm period (Fig. 2b). The resist remnants were removed during 20 minutes in a piranha solution (3 parts H2SO4 +1 part H2O2). The next step was the preparation of a microrelief by means of optical lithography. To this end, the nanostructured surface was protected with a 200-nm thick layer of aluminum obtained using ion-beam sputtering deposition. A microrelief with open Haversian canals communicating with canaliculi with horizontally arranged lacunas (Fig. 2.а) was prepared using a two-level lithography and 5-8 μm deep reactive ion etching of substrate in CF6 plasma. The Al layer was removed, together with the resist, using a 20-min treatment of the samples in a piranha solution. For diminishing their adhesion properties, Si stamps were covered with an аnti-sticking and anti-adhesion tridecafluoro-(1,1,2,2)-tetrahydrooctyl-trichlorosilane (F13 -TCS) coating in an evacuated chamber during 2 hours [2, 3].
Fabrication of PDMS stamps The materials for the preparation of transparent stamps were enhanced-rigidity thermally polymerizable dimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) [4] and photocurable UV-PDMS X-34-4184 [5]. The stamps were micro- and nanostructured PDMS layers 6 μm to 500 μm thick bonded to 76-mm diameter, 800-μm thick Borofloat glass wafers. The fabrication procedure of the stamps comprised the following steps. In the case of Sylgard 184, the pre-polymer base was mixed with the curing agent at a ratio of 5:1, and in the case of UVPDMS, the two components of the material (А and В) were mixed together in a proportion of 1:1. Then, the mixtures were degassed in a vacuum chamber for 30 min. Thin (6 to 500 μm) layers of prepared PDMS mixtures were applied onto Si stamps using a K Hand Coater (RK Print Coat Instruments Ltd, UK). The PDMS layers were then degassed in vacuum for 15 min.; during this time, the PDMS material flew into the recessed areas of the master stamp. A clean Borofloat glass wafer activated in oxygen plasma was carefully applied onto the Si stamp with the PDMS layer so that to prevent air trapping in the PDMS layer, and a stamping procedure on a nanoimprint lithography facility «Eitre 6 Nano Imprint Lithography System» (Obducat, Sweden) was performed. In the case of PDMS Sylgard 184, stamping was performed at a pressure of 10 bar during 10 minutes at temperature 195 °С. In the case of UV-PDMS X-34-4184, stamping was made at room temperature with subsequent UV polymerization that lasted for 10 minutes. After the polymerization of the hybrid glass-PDMS stamp was carefully detached from the Si master stamp. Afterwards, the fabricated stamps were used for stamping the PLA material or they were installed into the vat window of the stereolithography facility.
Shrinkage of polymer models Immediately after the formation of a model, the built-in PLA films were planar. After several days, bulging of the films and, in some cases, even their detachment from the supporting pillars occured (Fig. S1). That phenomenon was related with the shrinkage of the 3-D model. During the 3D printing process, resin photopolymerization proceeded, and the products of the photopolymerization reaction kept staying in the volume of the formed 3D scaffold. With the passage of time, outdiffusion of those products to scaffold surface and their volatilization occurred, and shrinkage took place. In our opinion, the bulging of the built-in films is not a very critical factor for
Figure S1. Bulging of PLA films and their shrinkage-induced detachment from support pillars. The detachment of PLA films from the supporting pillars occurs from the smooth surface of PLA film.
the formation of bone scaffolds since the width of the gaps between the structured films is additionally defined by the supporting pillars. If obtaining of precise scaffold dimensions is required, then corrections for shrinkage are to be introduced while designing the CAD model. Moreover, polymers exhibiting a smaller post-polymerization shrinkage can be developed.
Cell growth results By now, first biological experiments with micro- and nanostructured PLA 2D bone matrices have been performed. Below, we report a new result gained in our immunocyte-chemical study of the osteogenic properties of the 2D bone matrices. Osteogenic cells were cultivated onto micro- and nanostructured PLA 2D bone matrices. In Figure S2, a coloration demonstrating the collagen expression and an early stage of bone tissue formation is clearly seen.
Figure S2. Expression of type I collagen antigen (yellow region) with differentiated osteogenic cells observed by the 28th day of expression on the surface of a PLA 2D bone matrix (cytoplasmic and extracytoplasmatic localizations); staining of DAPI nuclei (blue spots). The image was obtained by means of fluorescence microscopy.
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