Laser fabrication of porous silicon-based platforms for cell culturing n-J. Pela ez,1 Carmen-N. Afonso,1 Fidel Vega,2* Gonzalo Recio-Sa nchez,3 Ramo 3 3 4 l-J. Martın-Palma3 Vicente Torres-Costa, Miguel Manso-Silvan, Josefa-P. Garcıa-Ruiz, Rau Laser Processing Group, Instituto de Optica, CSIC, Serrano 121, 28006 Madrid, Spain , 37, 08222 Terrasa, Spain Departament d’Optica i Optometrıa, UPC, Violinista Vellsola 3 noma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain Departamento de Fısica Aplicada, Universidad Auto 4 noma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain Departamento de Biologıa Molecular, Universidad Auto 1 2
Received 9 January 2013; revised 14 March 2013; accepted 10 April 2013 Published online 7 June 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbmb.32966 Abstract: In this study, we explore the selective culturing of human mesenchymal stem cells (hMSCs) on Si-based diffractive platforms. We demonstrate a single-step and flexible method for producing platforms on nanostructured porous silicon (nanoPS) based on the use of single pulses of an excimer laser to expose phase masks. The resulting patterns are typically 1D patterns formed by fringes or 2D patterns formed by circles. They are formed by alternate regions of almost unmodified nanoPS and regions where the nanoPS surface has melted and transformed into Si nanoparticles. The patterns are produced in relatively large areas (a few square millimeters) and can have a wide range of periodicities and
aspect ratios. Direct binding, that is, with no previous functionalization of the pattern, alignment, and active polarization of hMSCs are explored. The results show the preferential direct binding of the hMSCs along the transformed regions whenever their width compares with the dimensions of the cells and they escape from patterns for smaller widths suggesting that the selectivity can be tailored through the patC 2013 Wiley Periodicals, Inc. J Biomed Mater Res tern period. V Part B: Appl Biomater, 101B: 1463–1468, 2013.
Key Words: cell culturing, cell alignment, laser patterning, porous silicon, Si nanoparticles
ez R-J, Afonso C-N, Vega F, Recio-Sa nchez G, Torres-Costa V, Manso-Silva n M, Garcıa-Ruiz J-P, How to cite this article: Pela Martın-Palma R-J. 2013. Laser fabrication of porous silicon-based platforms for cell culturing. J Biomed Mater Res Part B 2013:101B:1463–1468.
INTRODUCTION
Human mesenchymal stem cells (hMSCs) are a promising source for cell transplantation therapies. Thus, they are increasingly being used for bone, cartilage, and fat transplantation and repair,1,2 for which it is essential to control mesenchymal stem cells adhesion and migration. Cells respond to synthetic nanoscale and microscale topographic surfaces in a wide array of responses, which depend on many factors, including cell type, feature size, and geometry as well as the physical properties of the bulk substrate material.3 The use of patterned substrates for hMSCs culturing has shown that the cells within the pattern behave more like a tissue than like individual cells, and it was the pattern geometry and not the cell population what influences differentiation.4 Nanostructured porous silicon (nanoPS) is very attractive for biosensing applications,5 the sensing mechanism relying on the variation of the refractive index on either infiltration6,7 or adsorption8 of the bio species into the porous structure. Patterned nanoPS is being used for a number of novel applications9 such as biomedical platforms for drug delivery10 or electrical biosensors.11 One- and
two-dimensional micropatterns of nanoPS have been recently exploited to control the surface distribution and shape of hMSCs and to study cell adhesion and migration characteristics.12 In addition, patterned structures on a number of materials, including nanoPS,13 are currently being exploited for diffraction-based biosensors.14 Generally, there are two main approaches for fabricating patterns on nanoPS. The first approach consists of microstructuring the c-Si substrate with the desired pattern12 followed by the porosification of the pattern. The second approach consists of creating the pattern directly on the nanoPS layer. This has been done either by dry soft lithography to selectively remove regions of nanoPS15 or by stamp pressing.13 A more flexible approach is direct laser writing of fringes or grids,16,17 but the procedure is especially time consuming to cover large areas. None of these methods has the capability to offer flexibility in the pattern design in a time-efficient process, in large areas, and in a single-step process. In this study, we report a single-step and flexible method for producing tailored diffraction-based platforms in
Correspondence to: F. Vega (e-mail: [email protected]) Contract grant sponsor: MAT2011-28345-C02-01 and MAT2011-28345-C02-02 (Spain) Contract grant sponsor: European Social Fund (JAE-doc programme; to R. J. P.)
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nanoPS for bioapplications using ultraviolet (UV) laser interference. As a proof of concept of the culturing capabilities of the patterns, we explore direct culturing of hMSCs, that is, with no previous functionalization, on patterns fabricated by this method. Preliminary results show that the tailoring of the pattern period is essential for selective culturing. EXPERIMENTAL
Preparation of nanoPS The nanoPS layers are fabricated by electrochemical etching of low resistivity (0.01–0.05 Xcm) p–type silicon wafers with (100) orientation.18 Etching current was 80 mA/cm2, and two different etching times were used, 7.5 and 10 s. The porosity of the layers has been determined by measuring the spectral reflectance and fitting the experimental data using a three-layer model on c-Si. The layers were assumed to be a mixture of Si and air (% porosity). A good agreement was achieved for a first thin layer (6–8 nm) of low porosity (57%) on top of the c-Si substrate followed by an intermediate thin layer (19–29 nm) of 67%–70% porosity and a top thick layer (293–335 nm) having 81% of porosity. Thus, the total thicknesses is 320/370 nm, where the first and second number, respectively, correspond to the 7.5/10 s etching time. Fabrication of patterns The patterning method is an interferential process that uses an excimer laser (k 5 193 nm, s 5 20 ns) to expose fringe phase masks optimized for high efficiency in the 61 diffraction orders. These orders are made to overlap and interfere at the nanoPS surface by using a pair of lenses in telescope configuration. Thus, the nanoPS surface is exposed to a modulated intensity formed by the maxima and minima of interference.19 By using different combinations of lenses, the period of the modulation can be modified easily. We have used either one phase mask to create 1D pattern of fringes or a pair of phase masks with 90 of relative orientation to create 2D patterns of circles. A CCD camera is used to record in situ the diffraction pattern of the irradiated areas by illuminating the patterned region with a blue (k 5 405 nm) diode laser beam. Characterization and cell culture The structural properties of the patterns are characterized by field emission scanning electron microscopy (SEM) in planar (PV) and cross-section (CS) views. HMSCs were directly cultured with no previous functionalization of the nanoPS substrates, on some of the patterns following a procedure similar to that reported elsewhere.20 Human bone marrow samples (2–4 ml) from healthy donors were provided by Hospital Universitario La Princesa (Madrid, Spain). Cells were collected by centrifugation on 70% Percoll gradient and seeded at 200,000/cm2 in Dulbecco’s modified Eagle’s medium with low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS). The medium was replaced twice per week. Before cell culture, surfaces of both the patterns and the surrounding nanoPS were exposed to UV light for 10 min, thoroughly washed with
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phosphate-buffered saline (PBS), placed on a 24-multiwell plate, and seeded with 15,000 cells. Cells were then incubated for 72 h with DMEM-LG adjusted to 10% FBS at 37 C in 5% CO2. After washing twice with PBS, cells were fixed in 3.7% formaldehyde in PBS for 30 min at room temperature (RT). For the immune staining, hMSCs were permeated in 0.5% Triton X-100 in cytoskeleton (CSK) buffer (100 mM NaCl, 10 mM Pipes pH 6.8, 3 mM MgCl2, 3 mM EGTA, and 0.3 M sucrose) for 30 min RT. Samples were blocked with 1% bovine serum albumin in PBS for 1 h at RT. Primary reactions took place with sera from autoimmune mice during 1 h. After washing, the surfaces were incubated in dark conditions for 1 h with Alexa 488 Phalloidin (1:500, Invitrogen) and 40 ,6-diamidino-2-phenylindole (1:5000, Calbiochem). After incubation, the surfaces were washed, dehydrated with absolute ethanol, and mounted with Mowiol/Dabco (Calbiochem). Cells were finally visualized in a fluorescence inverted microscope (Olympus IX81, Olympus Corporation, Shinjuku, Tokyo, Japan) coupled to a CCD color camera.
RESULTS
Figure 1 shows optical images of different patterns recorded in reflection configuration. The 1D patterns [Figure 1(a-c)] have been produced using different projection optics leading to periods varying more than one order of magnitude. The contrast of the gray regions of the images is identical to that of the as-grown sample and thus are most likely nontransformed or barely transformed regions. All patterns extend over a relatively large area, the extension of which depends on the projection optics factor. For instance, for the projection optics used in Figure 1(b), the exposed area was of 4 mm2, which could be further increased by removing a rectangular aperture set prior to the phase mask to save sample space. All the patterns exhibit diffractive features as seen in the insets of Figure 1, which are consistent with the 1D [Figure 1(a-c)] and 2D [Figure 1(d)] character of the patterns as well as their periodicities. The method gives access to many other types of patterns by adjusting the orientation and separation of two masks21 or by using two consecutive exposures of a single mask, the mask being rotated between exposures.22 The reproducibility of the laser patterning process is excellent and is only limited by the laser fluence variation among pulses, which is typically below 5%. Once formed, the pattern features (periodicity, roughness, etc.) are very stable. Figure 2(a) shows a PV-SEM image of the 1D pattern whose optical image was shown in Figure 1(b) showing it is similarly formed by dark and bright contrast regions with the same period. Figure 2(b) is a magnified image centered in one of the dark regions and shows a porous network morphology similar to that of the as-grown sample [Figure 2(c)]. This result further supports that these regions are barely transformed on irradiation, and thus, they must correspond to regions exposed to the laser intensity minima. Figure 2(b) also shows the transition region between the dark and bright contrast regions where the laser action induces the formation of brighter contrast areas whose size
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ORIGINAL RESEARCH REPORT
referred to from now on as nanoparticles (NPs). Interestingly, the structure of the nanoPS can be still recognized in the regions among the NPs. Since the bright contrast regions in Figure 2(a) are those where the largest transformation with respect to the as-grown sample [Figure 2(c)] has taken place, they must correspond to the regions exposed to the laser intensity maxima. Complementary information about the transformation occurring on laser exposure has been obtained by cleaving
FIGURE 1. Optical images of 1D fringe (a–c) and 2D circle (d) patterns produced, respectively, in the 320- and 370-nm thick nanoPS layer film using different projection optics and thus the laser fluences used and the periods achieved are different: (a) 198 mJ/cm2 and 1.7 lm, (b) 50 mJ/cm2 and 6.3 lm, (c) 11 mJ/cm2 and 31 lm, and (d) 19 mJ/cm2 and 6.3 lm. The insets show the corresponding experimental diffraction patterns. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
increases toward the center of the brighter regions of Figure 2(a). Figure 2(d) shows the center of the bright contrast region where round isolated features are seen that will be
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FIGURE 2. PV-SEM images of a 1D fringe pattern with a period of 6.3 lm produced in the 370-nm-thick nanoPS film using 44 mJ/cm2: (a) overall image of the pattern and magnified images of: (b) the dark contrast region of the pattern, (c) the as-grown sample, and (d) the bright contrast region of the pattern. The arrow in (a) approximately indicates the cleaving direction for the cross-section observations.
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When moving from the hills to the center of the trenches, there is a gradual decrease of the nanoPS layer thickness as a result of the formation of the NPs and the reduction of the porous layer thickness. At the trenches [Figure 3(d)], it is seen that the NPs accumulate and are in contact with the underneath c-Si substrate, meaning that the fluence at the intensity maxima was high enough to melt the whole original porous layer. As a proof of concept of the biofunctionality of the patterns, we have attempted to culture hMSCs in two patterns, namely the ones having the smallest [1.7 lm, Figure 1(a) and largest, 31 lm, Figure 1(c)] periodicities. Figure 4 shows blue fluorescence images of the cultured patterns. It is seen [Figure 4(a)] that the hMSCs bind out of the rectangular region (area of 0.2 mm2) whose underlying pattern (not visible in the image) has the smallest periodicity. A significant cell accumulation at the edges of the pattern is observed. Instead, the hMSCs bind and align to the trenches
FIGURE 3. (a) CS-SEM image along the cleaving direction indicated by the arrow in Figure 2(a). CS-SEM images of (b) hills, (c) the asgrown sample, and (d) trenches. Images in (a) and (b) have been slightly tilted to visualize in addition the sample surface.
the samples along a direction approximately perpendicular to the fringes [arrow in Figure 2(a)]. A CS-SEM image taken with the sample slightly tilted to also visualize the surface is shown in Figure 3(a). It evidences that the pattern is formed by hills and trenches. Figure 3(b) shows a magnification of one of the hills, and the comparison with the asgrown sample [Figure 3(c)] evidences that the structure and thickness of the hills are almost identical to those of the as-grown sample, thus confirming what was deduced from the PV-SEM images, that is, the nanoPS remains practically unaffected in these regions and thus they correspond to the laser intensity minima. Figure 3(c) also shows that the as-grown nanoPS layer has a thickness of 370 nm in good agreement with the thickness determined by spectral reflectance. Moreover, the low-porosity first layer of 6–8 nm thickness used in the optical model to fit the reflectance spectra is consistent with the thickness of the c-Si–nanoPS interface.
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FIGURE 4. Blue fluorescence images of hMSCs on patterns having (a) the smallest (1.7 lm) and (b) the largest (31 lm) periodicities. The underlying patterns cannot be seen in the images but the pseudo clear rectangle of 0.2 mm2 in (a) corresponds to the patterned region, and the sketch in the bottom of (b) makes easier the identification of the position of the trenches in the pattern. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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of the pattern where nanoPS has converted into NPs in the pattern having the largest periodicity [Figure 4(b)]. In this case, the cells show extended podia and decentralized nucleus suggesting an active polarization.20
DISCUSSION
To explain the features of the patterns, especially the formation of NPs, we have to consider that as we move away from the dark regions into the bright ones in Figure 2(a), the laser intensity increases from a minimum value below transformation threshold to a maximum value that must be above the melting threshold of nanoPS. Because of the rough and discontinuous character of the nanoPS surface, melting is heterogeneously nucleated at discrete points of the surface leading to small “islands” of molten silicon. The significant lateral gradient at the transition region induces deeper melting in the regions exposed to the laser intensity maxima. Thus, larger NPs are observed in these regions [see Figure 2(b,d)]. We have immersed the patterned sample in hydrofluoric acid (HF) solution to dissolve any SiO2 and observed it again in the SEM. We found that the NPs remain unaffected, which suggests that they are formed by silicon and any significant formation of SiO2 in the transformed regions can be discarded. This result is in contrast with the patterns created with the scanning technique using a continuous wave (CW) blue laser diode that induced melting and formation of oxidized regions.17 The long irradiation time of this early work (s 5 0.5 ls) and even longer melt duration allow significant diffusion of oxygen in the molten silicon and thus facilitates oxide formation. On the contrary, Si oxidation upon a single and short pulse exposure under conditions similar to those in this study (s 5 20 ns) is negligible.23 By comparing the dimensions of the Si NPs as measured in the PV-SEM [Figure 2(b,c)] and CS-SEM [Figure 3(d)] images, it becomes evident that they are almost spherical and have diameters in the range of 100–250 nm. It is worth pointing out that some of these NPs are also observed out of the trenches [Figure 2(a,b)] and even at the hills [Figures 2(a) and 3(b)]. However, although the NPs at the trenches show generally a low contact angle and thus wet the substrate [Figure 3(d)], they have a very high contact angle at the hills.24 This suggests that the NPs at the trenches were formed by melting followed by rapid solidification on the rough c-Si surface, whereas the latter should have a different origin that requires further investigation. In addition, the results show that the height and width of the trenches are controlled by local fluence. This opens the possibility of fabricating diffractive patterns with different hill to trench width ratio by tuning the laser fluence. Finally, it is worth to point out that the laser patterning process introduces no new element or chemical product that may induce a toxic response because the products are basically based on Si and O, and furthermore, the biocompatibility of nanoPS has been documented in literature since very long.25 To discuss the preliminary results of direct hMSCs culturing on the laser patterned samples, two facts have to be taken into account to make significant statistics. First, although circa
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5000 cells were cultured over an area of 1 cm2, not many of them were constrained to adhere in the area of interest. Second, the presence of the laser-induced pattern may favor a dispersion of cells on nonpatterned areas. This is observed in our statistics because the number of adhered cells per surface area on patterned areas reaches approximately 20% of the ones adhered on nonpatterned nanoPS. However, in spite of this relatively low rate of adhesion, 100% of the hMSCs bound to the patterns are aligned and polarized whenever the width of trenches are large enough to accommodate the cells [Figure 4]. This result suggests that the laser-induced trenches act as microfluidic wells for the cells during cell colonization of the surface. Further work is in progress to enhance the statistics relative to the number of cells that align and polarize as a function of the features of the pattern. The fact that the hMSCs bind preferentially to the trenches where nanoPS has converted into Si NPs [Figure 3(d)] may indicate that the physical and chemical properties of these areas also play a role on the cell culturing process such as influencing the wettability and adhesive properties of cells.26 Earlier work has reported cell culturing on patterns fabricated by ion beam irradiation of a c-Si substrate through a mask followed by galvanostatic etching in HF.12 These patterns were formed by alternating flat stripes of crystalline Si and nanoPS and exhibited no noticeable relief. The cultured hMSCs had no preference to adhere on the nanoPS stripes, and this result was explained in terms of the antifouling properties of the nanoPS and differences in wettability between the flat Si and the nanoPS stripes. The patterns reported in this study show instead a clear topography of hills and trenches formed by nanoPS (as in Ref. 12) and silicon NPs, respectively, rather than flat Si. Overall, our results might represent a step forward in developing tools for hMSCs isolation and single-cell analysis.27
CONCLUSIONS
Single-pulse UV laser interference is a versatile and time efficient means to fabricate diffractive platforms in nanoPS with different shapes and a wide range of periodicities in relatively large areas (up to a few square millimeters). They are formed by alternate regions of almost unaltered regions, both in thickness and structure, of the nanoPS and regions where the nanoPS has melted and converted into Si NPs and thus have a reduced thickness. The hMSCs bind directly and align along the transformed regions of the pattern whenever the width of the trenches on these regions compares with the dimensions of the hMSCs. The morphology of the bound hMSCs is consistent with their active polarization, and thus, the results suggest that the fabricated platforms are promising for cell selectivity and discrimination. REFERENCES 1. Tuan RS, Boland G, Tuli R. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther 2003;5:32–45. 2. Mauney JR, Volloch V, Kaplan DL. Matrix-mediated retention of adipogenic differentiation potential by human adult bone marrow-derived mesenchymal stem cells during ex vivo expansion. Biomaterials 2005;26:6167–6175.
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3. Bettinger CJ, Langer R, Borenstein JT. Engineering substrate topography at the micro- and nanoscale to control cell function. Angew Chem Int Ed Engl 2009;48:5406–5415. 4. Luo W, Jones SR, Yousaf MN. Geometric control of stem cell differentiation rate on surfaces. Langmuir 2008;24:12129–12133. 5. Song M, Yun D, Jin J, Min N, Hong S. Comparison of effective working electrode areas on planar and porous silicon substrates for cholesterol biosensor. Jpn J Appl Phys 2006;45:7197–7202. 6. Saarinem JJ, Weiss SM, Fauchet PM, Sipe JE. Optical sensor based on resonant porous silicon structures. Opt Express 2005;13:3754–3764. 7. Martın-Palma RJ, Manso M, Torres-Costa V. Optical biosensors based on semiconductor nanostructures. Sensors 2009;9:5149– 5172. 8. Vashpanov Y, Son J, Kwack K, Shing S. Electronic parameters of mesoporous silicon upon adsorption of plant viruses. Jpn J Appl Phys 2008;47:5100–5102. n M, Torres-Costa V. Biomedical 9. Martın-Palma RJ, Manso-Silva applications of nanostructured porous silicon: A review. J Nanophoton 2010;4:042502–20. 10. Anglin EJ, Cheng L, Freeman WR, Sailor MJ. Porous silicon in drug delivery devices and materials. Adv Drug Deliv Rev 2008;60:1266–1277. ~ izares G, Manso M, Preda I, 11. Recio-Sanchez G, Dominguez-Can rrez A, Soriano L, Martin-Palma R. Surface Torres-Costa V, Gutie functionalization of nanostructured porous silicon by APTS: Toward the fabrication of electrical biosensors of bacterium Escherichia coli. Curr Nanosci 2011;7:178–182. ~ oz G, Sa nchez-Vaquero V, Mun ~ oz12. Torres-Costa V, Martınez-Mun Gonza lez-Me ndez L, Punzo n-Quijorna E, Gallach-Pe rez D, Noval A, n M, Climent-Font A, Garcıa-Ruiz JP, Martın-Palma Manso-Silva RJ. Engineering of silicon surfaces at the micro- and nanoscales for cell adhesion and migration control. Int J Nanomed 2012;7:623–630. 13. Ryckman JD, Liscidini M, Sipe JE, Weiss SM. Porous silicon structures for low-cost diffraction-based biosensing. Appl Phys Lett 2010;96:171103–171106. 14. Liscidini M, Sipe JE. Enhancement of diffraction for biosensing applications via Bloch surface waves. Appl Phys Lett 2007;91:253125–3.
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15. Sirbuly BDJ, Lowman GM, Scott B, Stucky GD, Buratto SK. Patterned microstructures of porous silicon by dry-removal soft lithography. Adv Mater 2003;9510:149–152. 16. Khung Y-L, Graney SD, Voelcker NH. Micropatterning of porous silicon films by direct laser writing. Biotechnol Progr 2006;22:1388–1393. 17. De Stefano L, Rea I, Arcangela-Nigro M, Della Corte FG, Rendina I. A parametric study of laser induced ablation-oxidation on porous silicon surfaces. J Phys Condens Matter 2008;20:265009–265005. 18. Canham LT. Silicon quantum wire array fabrication by electrochemical and chemical dissolution of wafers. Appl Phys Lett 1990;57:1046–1048. 19. Huster J, M€ uller J, Renner H, Brinkmeyer E. Scanning phasemask DUV inscription of short-period large-area photoresist gratings. J Lightwave Tech 2011;29:2621–2628. rez DG, Me ndez 20. Noval AM, Vaquero VS, Quijorna EP, Costa VT, Pe n M. LG, Montero I, Palma RJM, Font AC, Ruiz JPG, Manso-Silva Aging of porous silicon in physiological conditions: Cell adhesion modes on scaled 1D micropatterns. J Biomed Mater Res A 2012;100:1615–1622. 21. Klein-Wiele J-H, Simon P. Fabrication of periodic nanostructures by phase-controlled multiple-beam interference. Appl Phys Lett 2003;83:4707–4709. ez RJ, Afonso CN, Bulır J, Lancˇok J, Novotn y M. Diffractive 22. Pela and coloured films by laser interferometry patterning. Surf Coating Tech 2012;211:205–208. 23. Orlowski TE, Mantell Da. Ultraviolet laser-induced oxidation of silicon: The effect of oxygen photodissociation upon oxide growth kinetics. J Appl Phys 1988;64:4410–4414. 24. Campbell CT. Ultrathin metal films and particles on oxide surfaces: Structural, electronic and chemisorptive properties. Surf Sci Rep 1997;27:1–111. 25. Buckberry L, Bayliss S. Porous silicon as a biomaterial. Mater World 1999;7:213–215. 26. Berry CC, Campbell G, Spadiccino A, Robertson M, Curtis ASG. The influence of microscale topography on fibroblast attachment and motility. Biomaterials 2004;25:5781–5788. 27. Gothard D, Tare RS, Mitchell PD, Dawson JI, Oreffo RO. In search of the skeletal stem cell: Isolation and separation strategies at the macro/ micro scale for skeletal regeneration. Lab Chip 2011;11:1206–1220.
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Received 9 January 2013; revised 14 March 2013; accepted 10 April 2013 Published online 7 June 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbmb.32966 Abstract: In this study, we explore the selective culturing of human mesenchymal stem cells (hMSCs) on Si-based diffractive platforms. We demonstrate a single-step and flexible method for producing platforms on nanostructured porous silicon (nanoPS) based on the use of single pulses of an excimer laser to expose phase masks. The resulting patterns are typically 1D patterns formed by fringes or 2D patterns formed by circles. They are formed by alternate regions of almost unmodified nanoPS and regions where the nanoPS surface has melted and transformed into Si nanoparticles. The patterns are produced in relatively large areas (a few square millimeters) and can have a wide range of periodicities and
aspect ratios. Direct binding, that is, with no previous functionalization of the pattern, alignment, and active polarization of hMSCs are explored. The results show the preferential direct binding of the hMSCs along the transformed regions whenever their width compares with the dimensions of the cells and they escape from patterns for smaller widths suggesting that the selectivity can be tailored through the patC 2013 Wiley Periodicals, Inc. J Biomed Mater Res tern period. V Part B: Appl Biomater, 101B: 1463–1468, 2013.
Key Words: cell culturing, cell alignment, laser patterning, porous silicon, Si nanoparticles
ez R-J, Afonso C-N, Vega F, Recio-Sa nchez G, Torres-Costa V, Manso-Silva n M, Garcıa-Ruiz J-P, How to cite this article: Pela Martın-Palma R-J. 2013. Laser fabrication of porous silicon-based platforms for cell culturing. J Biomed Mater Res Part B 2013:101B:1463–1468.
INTRODUCTION
Human mesenchymal stem cells (hMSCs) are a promising source for cell transplantation therapies. Thus, they are increasingly being used for bone, cartilage, and fat transplantation and repair,1,2 for which it is essential to control mesenchymal stem cells adhesion and migration. Cells respond to synthetic nanoscale and microscale topographic surfaces in a wide array of responses, which depend on many factors, including cell type, feature size, and geometry as well as the physical properties of the bulk substrate material.3 The use of patterned substrates for hMSCs culturing has shown that the cells within the pattern behave more like a tissue than like individual cells, and it was the pattern geometry and not the cell population what influences differentiation.4 Nanostructured porous silicon (nanoPS) is very attractive for biosensing applications,5 the sensing mechanism relying on the variation of the refractive index on either infiltration6,7 or adsorption8 of the bio species into the porous structure. Patterned nanoPS is being used for a number of novel applications9 such as biomedical platforms for drug delivery10 or electrical biosensors.11 One- and
two-dimensional micropatterns of nanoPS have been recently exploited to control the surface distribution and shape of hMSCs and to study cell adhesion and migration characteristics.12 In addition, patterned structures on a number of materials, including nanoPS,13 are currently being exploited for diffraction-based biosensors.14 Generally, there are two main approaches for fabricating patterns on nanoPS. The first approach consists of microstructuring the c-Si substrate with the desired pattern12 followed by the porosification of the pattern. The second approach consists of creating the pattern directly on the nanoPS layer. This has been done either by dry soft lithography to selectively remove regions of nanoPS15 or by stamp pressing.13 A more flexible approach is direct laser writing of fringes or grids,16,17 but the procedure is especially time consuming to cover large areas. None of these methods has the capability to offer flexibility in the pattern design in a time-efficient process, in large areas, and in a single-step process. In this study, we report a single-step and flexible method for producing tailored diffraction-based platforms in
Correspondence to: F. Vega (e-mail: [email protected]) Contract grant sponsor: MAT2011-28345-C02-01 and MAT2011-28345-C02-02 (Spain) Contract grant sponsor: European Social Fund (JAE-doc programme; to R. J. P.)
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nanoPS for bioapplications using ultraviolet (UV) laser interference. As a proof of concept of the culturing capabilities of the patterns, we explore direct culturing of hMSCs, that is, with no previous functionalization, on patterns fabricated by this method. Preliminary results show that the tailoring of the pattern period is essential for selective culturing. EXPERIMENTAL
Preparation of nanoPS The nanoPS layers are fabricated by electrochemical etching of low resistivity (0.01–0.05 Xcm) p–type silicon wafers with (100) orientation.18 Etching current was 80 mA/cm2, and two different etching times were used, 7.5 and 10 s. The porosity of the layers has been determined by measuring the spectral reflectance and fitting the experimental data using a three-layer model on c-Si. The layers were assumed to be a mixture of Si and air (% porosity). A good agreement was achieved for a first thin layer (6–8 nm) of low porosity (57%) on top of the c-Si substrate followed by an intermediate thin layer (19–29 nm) of 67%–70% porosity and a top thick layer (293–335 nm) having 81% of porosity. Thus, the total thicknesses is 320/370 nm, where the first and second number, respectively, correspond to the 7.5/10 s etching time. Fabrication of patterns The patterning method is an interferential process that uses an excimer laser (k 5 193 nm, s 5 20 ns) to expose fringe phase masks optimized for high efficiency in the 61 diffraction orders. These orders are made to overlap and interfere at the nanoPS surface by using a pair of lenses in telescope configuration. Thus, the nanoPS surface is exposed to a modulated intensity formed by the maxima and minima of interference.19 By using different combinations of lenses, the period of the modulation can be modified easily. We have used either one phase mask to create 1D pattern of fringes or a pair of phase masks with 90 of relative orientation to create 2D patterns of circles. A CCD camera is used to record in situ the diffraction pattern of the irradiated areas by illuminating the patterned region with a blue (k 5 405 nm) diode laser beam. Characterization and cell culture The structural properties of the patterns are characterized by field emission scanning electron microscopy (SEM) in planar (PV) and cross-section (CS) views. HMSCs were directly cultured with no previous functionalization of the nanoPS substrates, on some of the patterns following a procedure similar to that reported elsewhere.20 Human bone marrow samples (2–4 ml) from healthy donors were provided by Hospital Universitario La Princesa (Madrid, Spain). Cells were collected by centrifugation on 70% Percoll gradient and seeded at 200,000/cm2 in Dulbecco’s modified Eagle’s medium with low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS). The medium was replaced twice per week. Before cell culture, surfaces of both the patterns and the surrounding nanoPS were exposed to UV light for 10 min, thoroughly washed with
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phosphate-buffered saline (PBS), placed on a 24-multiwell plate, and seeded with 15,000 cells. Cells were then incubated for 72 h with DMEM-LG adjusted to 10% FBS at 37 C in 5% CO2. After washing twice with PBS, cells were fixed in 3.7% formaldehyde in PBS for 30 min at room temperature (RT). For the immune staining, hMSCs were permeated in 0.5% Triton X-100 in cytoskeleton (CSK) buffer (100 mM NaCl, 10 mM Pipes pH 6.8, 3 mM MgCl2, 3 mM EGTA, and 0.3 M sucrose) for 30 min RT. Samples were blocked with 1% bovine serum albumin in PBS for 1 h at RT. Primary reactions took place with sera from autoimmune mice during 1 h. After washing, the surfaces were incubated in dark conditions for 1 h with Alexa 488 Phalloidin (1:500, Invitrogen) and 40 ,6-diamidino-2-phenylindole (1:5000, Calbiochem). After incubation, the surfaces were washed, dehydrated with absolute ethanol, and mounted with Mowiol/Dabco (Calbiochem). Cells were finally visualized in a fluorescence inverted microscope (Olympus IX81, Olympus Corporation, Shinjuku, Tokyo, Japan) coupled to a CCD color camera.
RESULTS
Figure 1 shows optical images of different patterns recorded in reflection configuration. The 1D patterns [Figure 1(a-c)] have been produced using different projection optics leading to periods varying more than one order of magnitude. The contrast of the gray regions of the images is identical to that of the as-grown sample and thus are most likely nontransformed or barely transformed regions. All patterns extend over a relatively large area, the extension of which depends on the projection optics factor. For instance, for the projection optics used in Figure 1(b), the exposed area was of 4 mm2, which could be further increased by removing a rectangular aperture set prior to the phase mask to save sample space. All the patterns exhibit diffractive features as seen in the insets of Figure 1, which are consistent with the 1D [Figure 1(a-c)] and 2D [Figure 1(d)] character of the patterns as well as their periodicities. The method gives access to many other types of patterns by adjusting the orientation and separation of two masks21 or by using two consecutive exposures of a single mask, the mask being rotated between exposures.22 The reproducibility of the laser patterning process is excellent and is only limited by the laser fluence variation among pulses, which is typically below 5%. Once formed, the pattern features (periodicity, roughness, etc.) are very stable. Figure 2(a) shows a PV-SEM image of the 1D pattern whose optical image was shown in Figure 1(b) showing it is similarly formed by dark and bright contrast regions with the same period. Figure 2(b) is a magnified image centered in one of the dark regions and shows a porous network morphology similar to that of the as-grown sample [Figure 2(c)]. This result further supports that these regions are barely transformed on irradiation, and thus, they must correspond to regions exposed to the laser intensity minima. Figure 2(b) also shows the transition region between the dark and bright contrast regions where the laser action induces the formation of brighter contrast areas whose size
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referred to from now on as nanoparticles (NPs). Interestingly, the structure of the nanoPS can be still recognized in the regions among the NPs. Since the bright contrast regions in Figure 2(a) are those where the largest transformation with respect to the as-grown sample [Figure 2(c)] has taken place, they must correspond to the regions exposed to the laser intensity maxima. Complementary information about the transformation occurring on laser exposure has been obtained by cleaving
FIGURE 1. Optical images of 1D fringe (a–c) and 2D circle (d) patterns produced, respectively, in the 320- and 370-nm thick nanoPS layer film using different projection optics and thus the laser fluences used and the periods achieved are different: (a) 198 mJ/cm2 and 1.7 lm, (b) 50 mJ/cm2 and 6.3 lm, (c) 11 mJ/cm2 and 31 lm, and (d) 19 mJ/cm2 and 6.3 lm. The insets show the corresponding experimental diffraction patterns. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
increases toward the center of the brighter regions of Figure 2(a). Figure 2(d) shows the center of the bright contrast region where round isolated features are seen that will be
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FIGURE 2. PV-SEM images of a 1D fringe pattern with a period of 6.3 lm produced in the 370-nm-thick nanoPS film using 44 mJ/cm2: (a) overall image of the pattern and magnified images of: (b) the dark contrast region of the pattern, (c) the as-grown sample, and (d) the bright contrast region of the pattern. The arrow in (a) approximately indicates the cleaving direction for the cross-section observations.
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When moving from the hills to the center of the trenches, there is a gradual decrease of the nanoPS layer thickness as a result of the formation of the NPs and the reduction of the porous layer thickness. At the trenches [Figure 3(d)], it is seen that the NPs accumulate and are in contact with the underneath c-Si substrate, meaning that the fluence at the intensity maxima was high enough to melt the whole original porous layer. As a proof of concept of the biofunctionality of the patterns, we have attempted to culture hMSCs in two patterns, namely the ones having the smallest [1.7 lm, Figure 1(a) and largest, 31 lm, Figure 1(c)] periodicities. Figure 4 shows blue fluorescence images of the cultured patterns. It is seen [Figure 4(a)] that the hMSCs bind out of the rectangular region (area of 0.2 mm2) whose underlying pattern (not visible in the image) has the smallest periodicity. A significant cell accumulation at the edges of the pattern is observed. Instead, the hMSCs bind and align to the trenches
FIGURE 3. (a) CS-SEM image along the cleaving direction indicated by the arrow in Figure 2(a). CS-SEM images of (b) hills, (c) the asgrown sample, and (d) trenches. Images in (a) and (b) have been slightly tilted to visualize in addition the sample surface.
the samples along a direction approximately perpendicular to the fringes [arrow in Figure 2(a)]. A CS-SEM image taken with the sample slightly tilted to also visualize the surface is shown in Figure 3(a). It evidences that the pattern is formed by hills and trenches. Figure 3(b) shows a magnification of one of the hills, and the comparison with the asgrown sample [Figure 3(c)] evidences that the structure and thickness of the hills are almost identical to those of the as-grown sample, thus confirming what was deduced from the PV-SEM images, that is, the nanoPS remains practically unaffected in these regions and thus they correspond to the laser intensity minima. Figure 3(c) also shows that the as-grown nanoPS layer has a thickness of 370 nm in good agreement with the thickness determined by spectral reflectance. Moreover, the low-porosity first layer of 6–8 nm thickness used in the optical model to fit the reflectance spectra is consistent with the thickness of the c-Si–nanoPS interface.
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FIGURE 4. Blue fluorescence images of hMSCs on patterns having (a) the smallest (1.7 lm) and (b) the largest (31 lm) periodicities. The underlying patterns cannot be seen in the images but the pseudo clear rectangle of 0.2 mm2 in (a) corresponds to the patterned region, and the sketch in the bottom of (b) makes easier the identification of the position of the trenches in the pattern. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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of the pattern where nanoPS has converted into NPs in the pattern having the largest periodicity [Figure 4(b)]. In this case, the cells show extended podia and decentralized nucleus suggesting an active polarization.20
DISCUSSION
To explain the features of the patterns, especially the formation of NPs, we have to consider that as we move away from the dark regions into the bright ones in Figure 2(a), the laser intensity increases from a minimum value below transformation threshold to a maximum value that must be above the melting threshold of nanoPS. Because of the rough and discontinuous character of the nanoPS surface, melting is heterogeneously nucleated at discrete points of the surface leading to small “islands” of molten silicon. The significant lateral gradient at the transition region induces deeper melting in the regions exposed to the laser intensity maxima. Thus, larger NPs are observed in these regions [see Figure 2(b,d)]. We have immersed the patterned sample in hydrofluoric acid (HF) solution to dissolve any SiO2 and observed it again in the SEM. We found that the NPs remain unaffected, which suggests that they are formed by silicon and any significant formation of SiO2 in the transformed regions can be discarded. This result is in contrast with the patterns created with the scanning technique using a continuous wave (CW) blue laser diode that induced melting and formation of oxidized regions.17 The long irradiation time of this early work (s 5 0.5 ls) and even longer melt duration allow significant diffusion of oxygen in the molten silicon and thus facilitates oxide formation. On the contrary, Si oxidation upon a single and short pulse exposure under conditions similar to those in this study (s 5 20 ns) is negligible.23 By comparing the dimensions of the Si NPs as measured in the PV-SEM [Figure 2(b,c)] and CS-SEM [Figure 3(d)] images, it becomes evident that they are almost spherical and have diameters in the range of 100–250 nm. It is worth pointing out that some of these NPs are also observed out of the trenches [Figure 2(a,b)] and even at the hills [Figures 2(a) and 3(b)]. However, although the NPs at the trenches show generally a low contact angle and thus wet the substrate [Figure 3(d)], they have a very high contact angle at the hills.24 This suggests that the NPs at the trenches were formed by melting followed by rapid solidification on the rough c-Si surface, whereas the latter should have a different origin that requires further investigation. In addition, the results show that the height and width of the trenches are controlled by local fluence. This opens the possibility of fabricating diffractive patterns with different hill to trench width ratio by tuning the laser fluence. Finally, it is worth to point out that the laser patterning process introduces no new element or chemical product that may induce a toxic response because the products are basically based on Si and O, and furthermore, the biocompatibility of nanoPS has been documented in literature since very long.25 To discuss the preliminary results of direct hMSCs culturing on the laser patterned samples, two facts have to be taken into account to make significant statistics. First, although circa
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5000 cells were cultured over an area of 1 cm2, not many of them were constrained to adhere in the area of interest. Second, the presence of the laser-induced pattern may favor a dispersion of cells on nonpatterned areas. This is observed in our statistics because the number of adhered cells per surface area on patterned areas reaches approximately 20% of the ones adhered on nonpatterned nanoPS. However, in spite of this relatively low rate of adhesion, 100% of the hMSCs bound to the patterns are aligned and polarized whenever the width of trenches are large enough to accommodate the cells [Figure 4]. This result suggests that the laser-induced trenches act as microfluidic wells for the cells during cell colonization of the surface. Further work is in progress to enhance the statistics relative to the number of cells that align and polarize as a function of the features of the pattern. The fact that the hMSCs bind preferentially to the trenches where nanoPS has converted into Si NPs [Figure 3(d)] may indicate that the physical and chemical properties of these areas also play a role on the cell culturing process such as influencing the wettability and adhesive properties of cells.26 Earlier work has reported cell culturing on patterns fabricated by ion beam irradiation of a c-Si substrate through a mask followed by galvanostatic etching in HF.12 These patterns were formed by alternating flat stripes of crystalline Si and nanoPS and exhibited no noticeable relief. The cultured hMSCs had no preference to adhere on the nanoPS stripes, and this result was explained in terms of the antifouling properties of the nanoPS and differences in wettability between the flat Si and the nanoPS stripes. The patterns reported in this study show instead a clear topography of hills and trenches formed by nanoPS (as in Ref. 12) and silicon NPs, respectively, rather than flat Si. Overall, our results might represent a step forward in developing tools for hMSCs isolation and single-cell analysis.27
CONCLUSIONS
Single-pulse UV laser interference is a versatile and time efficient means to fabricate diffractive platforms in nanoPS with different shapes and a wide range of periodicities in relatively large areas (up to a few square millimeters). They are formed by alternate regions of almost unaltered regions, both in thickness and structure, of the nanoPS and regions where the nanoPS has melted and converted into Si NPs and thus have a reduced thickness. The hMSCs bind directly and align along the transformed regions of the pattern whenever the width of the trenches on these regions compares with the dimensions of the hMSCs. The morphology of the bound hMSCs is consistent with their active polarization, and thus, the results suggest that the fabricated platforms are promising for cell selectivity and discrimination. REFERENCES 1. Tuan RS, Boland G, Tuli R. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther 2003;5:32–45. 2. Mauney JR, Volloch V, Kaplan DL. Matrix-mediated retention of adipogenic differentiation potential by human adult bone marrow-derived mesenchymal stem cells during ex vivo expansion. Biomaterials 2005;26:6167–6175.
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