Proton micromachining of substrate sca�olds forcellular and tissue engineering

Jose Luis Sanchez a, G. Guy b, J.A. van Kan a, T. Osipowicz a, F. Watt a,*

a Department of Physics, Research Centre for Nuclear Microscopy, National University of Singapore, Singapore 119260, Singaporeb Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609, Singapore

Abstract

Three dimensional patterns (grooves and ridges) were micromachined in PMMA using a 600 keV proton beam from

the nuclear microscopy facility at the Research Centre for Nuclear Microscopy, National University of Singapore.

Swiss 3T3 ®broblasts (ATCC CCL92, Rockville, MD) have been seeded onto these patterns, and the following ob-

servations have been made: (a) Cells were not found in the grooves (depth 9 lm, width 6.6 lm); (b) Cells were highly

aligned and elongated on narrow ridges (4.2 lm wide), with the degree of alignment and elongation reduced for wider

ridges. The underlying mechanism responsible of this cellular behaviour is assumed to be induced by the mechanical

restrictions imposed by the topographic features on cellular migration, cell adhesion and concomitant changes in the

cytoskeletal. The use of topographical stimuli to regulate cell function is an area of high potential, with implications in

the engineering of tissue for spare-part surgery. Proton micromachining, which has the unique advantage of being the

only technique capable of direct-write 3D micromachining at sub-cellular dimensions has unique advantages in this area

of research. Ó 1999 Elsevier Science B.V. All rights reserved.

1. Introduction

Tissue engineering is a rapidly developing andhighly interdisciplinary ®eld that applies the prin-ciples of cell biology, engineering and materialsscience to the culture of tissue. The arti®ciallygrown tissue then can be implanted directly intothe body, or it can form part of a device that re-places organ functionality.

In natural tissues, the cells are arranged with athree dimensional organisation which provides the

appropriate functional, nutritional and spatialconditions. The culture of tissues Ôin vitroÕ there-fore requires bio-compatible tissue sca�olds to givethe cells not only physical support, but also tomimic the conditions of natural tissue therebyenhancing cell proliferation and di�erentiationnecessary for the regeneration and replacement ofdamaged cells. Three dimensional amorphousmesh structures made from collagen or polymer®bres are the most common types of tissue scaf-folds currently used [1]. These types of structuresare aimed at providing the cells with the sameorganisation and mechanical properties of thenatural tissue they replace. Although this ap-proach has had some success in skin and bone

Nuclear Instruments and Methods in Physics Research B 158 (1999) 185±189

www.elsevier.nl/locate/nimb

* Corresponding author. Fax: +65-7776162; e-mail: phy-

wattf@nus.edu.sg

0168-583X/99/$ - see front matter Ó 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 8 - 5 8 3 X ( 9 9 ) 0 0 5 2 8 - 5

production, mesh sca�olds do not exhibit the well-de®ned geometrical environment necessary for thestrict geometrical guidance and con®nement re-quired by most cells.

Tissue sca�olds of speci®c 3D geometry mayhave the ability to promote and control, via to-pographic stimuli, tissue di�erentiation and cre-ation of an extra cellular matrix (ECM), therebyincreasing the quality and range of tissues suitablefor graft. Current work has centred on the e�ectsof surface topography on cell adhesion, motility,cell guidance and cell shape characteristics, andthere is a growing body of evidence to suggest thatthese characteristics can be in¯uenced by surfacetopography [2±4]. Alterations to the morphologi-cal behaviour of cells have major functional con-sequences, in that changes in shape are closelyassociated with the cytoskeleton, which in turn arelinked to transmembrane proteins. Contact medi-ated processes regulate cell migration and prolif-eration, and these in turn are coupled to themechanisms which control gene activity andtherefore cell function. Cell function regulationthrough contact with speci®c surface topographyis a new ®eld with exciting possibilities.

Proton micromachining is a new techniquewhich can produce three-dimensional high aspectratio micro-machined surfaces of di�erent shapesand patterns by altering a resist structure using afocused beam of MeV protons in a fast, directwrite process [5]. 3D microstructures with wellde®ned geometrical shapes have been produced atsizes down to 100 nm, well below cell dimensions.By utilising a bio-compatible resist material (e.g.,PMMA), the technique of proton micromachininghas extremely high potential for the production ofsubstrates for cell and tissue engineering. Thispaper describes some preliminary results on theuse of proton micromachined sca�olds for cellalignment.

2. Experimental

2.1. Substrate manufacture

A piece of PMMA, approximately 1 cm square,thickness 2 mm, was exposed to a 0.6 MeV proton

beam using the nuclear microscope facility at theResearch Centre for Nuclear Microscopy, Na-tional University of Singapore. The beam spot wasfocused to approximately 1 lm diameter at acurrent of 1 pA, and scanned over the PMMA in aseries of patterns consisting of nine di�erent typesof ridge/groove structures. Each of the nine ridge/groove patterns was exposed over an area of400 ´ 400 lm using a scan resolution of 512 by 512pixels [6]. To achieve a homogeneous exposure,multiple repeated exposures of the patterns werecarried out until the optimum dose of 60 nC/mm2

was achieved [7]. The width of all grooves wasconstant at 6.6 lm, and the depth of all thegrooves was maintained at 9 lm, corresponding tothe range of 0.6 MeV protons in PMMA. Theridge width was constant within each of the ninepatterns, but varied from pattern to pattern from4.3 to 20 lm in steps of about 2 lm. Fig. 1 showsan electron micrograph of the structure with aridge width of 14 lm. After the exposure thesample was developed using the procedures de-scribed in [5].

2.2. Cell culture and sterilization procedures

The micromachined PMMA substrate wassterilized by soaking in 70% ethanol for 10±20 minfollowed by aseptic transfer to a culture media for20 min prior to seeding with cells. Although thissimple sterilisation procedure was adequate for thecell type used in this experiment, more investiga-tions are required to see if this procedure is suit-able for all cells. The substrate was placed in a 45mm culture dish and 5 ml of cell suspension wasadded. Swiss 3T3 ®broblasts (ATCC CCL92,Rockville, MD) were grown and maintained inDulbecco's modi®ed Eagle's medium supplement-ed with 10% fetal bovine serum (FBS) (HycloneLaboratories, Logan, UT), 2 mM glutamine, 10mM HEPES, pH 7.4 and 100 units/ml penicillinand streptomycin. Cells were seeded sparsely sothat there were only one or two cells at most on thegrids. The cells were allowed to grow into the gridsover the next ®ve days. Using the above procedure,the substrate can be re-used after cleaning withethanol and cleaning out debris in a sonicatorbath.

186 J.L. Sanchez et al. / Nucl. Instr. and Meth. in Phys. Res. B 158 (1999) 185±189

Cell behaviour (i.e., movement, alignment etc.)on the PMMA substrate was monitored using anoptical microscope.

3. Results

The cells were observed to grow readily on thePMMA substrate, and several interesting featureswere noticed.

(a) Cells grown on the smooth non-patternedsurface of the PMMA exhibited normalgrowth behaviour, i.e., that of plating to anoptimum density with no obvious degree ofalignment (see Fig. 2a).(b) No cells were found in any of the grooves;indicating that the constraints of 9 lm depthand 6.6 lm groove width were not suitablefor cell growth.(c) Cells were present on the ridges: The cellspresent on the narrow ridges (see Fig. 2band c) were elongated and aligned in the direc-tion of the ridges, whereas the cells present onthe wide ridges showed a certain degree ofalignment which reduced as the ridges becamewider (not shown). In several cases, cellsfound on the widest ridges spanned the nar-row grooves separating them (see Fig. 2d).

4. Discussion and conclusion

PMMA cell sca�olds, made up of a series of 3Dgroove/ridge patterns of increasing ridge widthfrom 4 to 20 lm, have been manufactured usingproton micromachining. Swiss 3T3 ®broblasts(ATCC CCL92, Rockville, MD) seeded onto thesemicrostructures exhibited di�erent morphologicalbehaviour, which depended on the width of theridges. In general the narrower the ridge width, themore aligned and elongated the cells became.

The main e�ect induced by the ridges is thephenomenon of contact guidance: The cells haveundergone substantial elongation and spreadingwith the long axis aligning to the direction of theridges. The underlying mechanism responsible forthis cellular behaviour is still not completely un-derstood although it is assumed to be induced bythe mechanical restrictions imposed by the topo-graphic features on the cellular cytoskeletal. Theuse of topographical stimuli to regulate cell func-tion is an area of high potential, with implicationsin the engineering of tissue for spare-part surgery.Proton micromachining, which has the uniqueadvantage of being the only technique capable ofdirect-write 3D micromachining at sub-cellulardimensions [7,8], has unique advantages in thisarea of research.

Fig. 1. Electron micrographs of the micromachined pattern in PMMA, 24 lm groove depth, 6.6 lm groove width, 14 lm ridge width.

J.L. Sanchez et al. / Nucl. Instr. and Meth. in Phys. Res. B 158 (1999) 185±189 187

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References

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