Revista EIA, ISSN 1794-1237 Número 4p. 113-122. Noviembre 2005Escuela de Ingeniería de Antioquia, Medellín (Colombia)

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DnNrm Gelleco , DeREr HANsToRD

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It has been clear that the surface properties of the materials used as substrates for cell culture can in-

fluence the cell behavior affecting different factors such as cell orientation, adhesion, growth, differentiation,

and apoptosis. In the last fewyears, several micro and nanofabrication techniques have emerged as important

tools used to modify the surface properties of those materials at the micro or nano scale, topologically or

chemically. This paper reüews the most used microfabrication techniques to introduce modifications on the

materials surlaces and its implications on cell behaüor. This reüew ends up showing the possible medical

applications derived as a source of future work in the field.

KEY WORDS: cell adhesion; cell proliferation; materials surfaces; microfabrication; surface topogra-

phy; surface chemistry.

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Ha quedado claro que las propiedades srperficiales de los materiales utilizados como sustratos para

cultivo celular pueden influir en el comportamiento de las células afectando diferentes factores tales como la

orientación celular, adhesión, crecimiento, diferenciación y apoptosis. En los últimos años, varias técnicas de

microfabricación y nanofabricación han emergido como herramientas importantes usadas para modificar las

propiedades superficiales de esos materiales en la escala micrométrica y nanométrica, ya sea topográfica o

químicamente. Este artículo revisa las técnicas de microfabricación mas usadas para introducir modificaciones

en las uperficies de los materiales y sus implicaciones en el comportamiento celular. Esta reüsión termina mos-

trando las posibles aplicaciones médicas derivadas como una fuente de futuros desarrollos en este campo.

PALABRAS CLAVE: adhesión celular; proliferación celular; superficie de los materiales; microfabricación;

topografia de la superficie; química de la superficie.

" Ingeniero Biomédico EIA-CES. Grupo de Investigación en Ingenieía Biomédica EIA-CES, Línea de Biomateriales yBiotecnología en Salud. bmdagal@eia.edu.co

** PhD Materials Science and Mineral Engineering (UC Berkeley). Biomedical Engineering Center, The Ohio StateUniversit5l The Ohio McroMD Laboratory. hansford.4@osu.edu

Artículo recibido 9-lX-2005. Aprobado con revisión 9-)0-2005

Discusión abierta hasta mayo 2oo6

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Lately a great variety of microfabrication te-chniques have been used to micropattern materialssurfaces in order to regulate cell functions. This te-chnologyis a powerful tooi to study different factorssuch as cell attachment and growth, and to createorganized cell cultures for applications in tissue en-gineering. Patterned substrates are used principallyin cell culürres, but the medical implants sciencehas also shown interest for these approaches sincethe interaction of cells with the materials surfaces isclearly important in the rejection or effectiveness ofanyimplant. Variations on the surface chemistry andtopology have been done with different techniqueslike photolithography, micro contact printing, andlift-off. This paper reüews the most used and recenttechniques to micropattern surfaces, and describespotential medical applications trying to open a doorfor future work in the field.

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Figure 1. A. Schematized photolithographic process.B. SEM micrograph of the mold (silicon wafer)

obtained by photolithography.

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Figure 2. A. Schematized soft lithography process.B. SEM micrograph of the PDMS mold obtained by

soft lithography.

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Topographical cues play a crucial role in me-diating not onlycell orientarion and biocomparibiliry,but also factors such as protein (and gene) expressionand cell differentiation. The semiconductor industryallows us to fabricate well-def,ined surface features inthe 100's of micrometers down to a fewnanometers;the most common technique used to introduce topo-graphicai changes ro the surfaces is photolithography(figure 1), which consists of selectively exposing aphotoresistance to Wlight through a deñned mask.Soft lithography represents a non-photolithographicstrategybased on self-assembly and replica moldingfor carrying out micro- and nanofabrication; it is anefficient and low-cost method for the formation andmanufacturing of micro and nanostructures rangingfrom 30 nm ro I00 ¡rm (figure 2) tt-31.

The integration of micro and nanotechnologytechniques with the biological field has led resear-chers to make cell growth experiments on topogra-phically modified surfaces. For long time experimentshave reported that comparing with smooth surfaces;slightly roughened surfaces could improve the os-teointegration, reduce fibrous encapsulation andincrease the integration of percutaneous implants.These eüdences could lead to the development ofnew prosthetic and medical implant deüces.

Van Kooten et al. [4] showed that using microtextured PDMS (rubber) substrates to culture fibro-blast could reduce signiñcantly capsule formation.In this study the interaction of human fibroblastswith rubber surfaces was observed using a cell cycleanalysis. PDMS substrates were textured with 2 ¡rm,5 ¡rm, and 10 pm wide grooves or kept smooth. Cellson 10 ¡rm showed less proliferation than cells with2 pm and 5 pm grooves.

Cheroundi et al. [S] went into in-üvo experi-ments, studying the performance of percutaneousmicro machined materials. They found that theirimplantswere able to induce epithelial downgrowtharound the implant. Finally the response of fibroblastwas greatlyaffected bythe dimensions of the structu-

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res fibroblasts oriented along 3 pm and 10 pm deepgrooves whereas they inserted obliquely into 22 pm

deep grooves).

The cellular migration (speed and orientationof the movement) is also affected by the surface to-pography. Jian and Saltzman [6] studied the effect ofmicropatterned surfaces on neutrophil cells. In thisstudy, parallel ridgelgrooves were micropatternedon glass surfaces using a photosensiúve polyimide.The width (z pm) and length (400 pm) of the ridgeswere kept constant, the height (5 pm or 3 pm) andthe repeat spacing (6 ¡tm -14 pm) of the ridges werechanged to investigate the effect of micro topogra-phy on neutrophil migration. They found that morethan 95olo of neutrophils moved in the direction ofthe long axis of ridges/grooves according with aphenomenon termed "contact guidance"; the rateof cell movement was strongly dependent on themicro topography of the ridges, the highest speed ofmovement was obtained at a ridge height of 5 pmand spacing of 10 pm, indicating that the movementwas about 10 times faster than that on smooth glass

surfaces.

The conhguration ofgroovelridges has beenwidely explored by the researchers. Von Recum and

Van Kooten as well as Walboomer et al. have deeplystudied the effects of this confrguration on differentcell lines such as fibroblasts, epithelial cells, and en-

dothelial cells U, 81. These studies showed that thecells aligned along with the groove's axis; and thatthe groove depth and width play crucial roles in thedegree of alignment.

A few studies are focused on different confi-gurations. Hansford et al. [9] studied the effect of agrid pattern (45 pm side squares and 1o pm deep) onthe cell alignment. Human osteosarcoma cells (HOS)

were cultured on PLGA micropatteined substrates;comparing with flat films (controls), the cells on themicropatterned surfaces were truly affected by themicro topography (figure 3), influencing factors ascell alignment, orientation, proliferation, and mor-phology.

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Figure 3. Observing the nucleus (PlRNAasestained) of HOS cells cultured on both micro and

non micropatterned substrates. A and B show cellscultured on a grid patterned PLGA substrate (cells

aligning with the surface features). C. Cells culturedon a flat PLGA substrate (control).

Craighead et al. [10] studied the effects ofmicro-pillars geometry on nervous system cells. Sili-con pillars were 0.5 ¡rm in width, 1.0 pm or 2.7 pmin height, and separated by 1.0 pm. Results showedthatboth, the LRM55 cells and the primaryastrocytes

demonstrated a preference for the pillars rather than

the flat silicon surface, and no discernable preference

for either pillar height. On the other hand, Wilkinsonet al. have looked at the attachment of rat epitenonceils to nanometer-sized polystS,rene pillars; resultsshowed a significant decrease in the number of cellsattached to the pillars configuration, in comparisonwith the smooth control surfaces; they proposedthat such nanopatterned surfaces could be useful as

non-adhesive layers for implant devices.

Not only the cellular migration, but also the ce-

llular activation is affected by the microphotography.

Changes in the cytoskeleton condensation produced

by the contact of a cell with the micro-topographydetermine the cellular activation. Wojciak-Stothard

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et al. [11] studied the activation of a macrophagesline and rat peritoneum by topographic features ona nanometric scale; theyfound that cells culh:red onnanogrooves showed a higher phagocytotic actiütythan cells culflrred on plain substrata. In other study,Chou et al. [12] showed that the expression of mRNAfor fibronectin was improved by culturing the fibro-blast on microgrooved substrates.

Both micro and nano-scale topographicalpatteming have been shown to influence cell interac-

tionswith the material surface. Regulation of differentcell actiüties such as proliferation, adhesion, andapoptosis, etc. could be veryuseful for the design ofmedical deüces, implants, and biomaterials.

3" §§_§§§,§&fr§ fi§§§es§sYñYSome microfabrication techniques allow

producing chemically modified surfaces. Chemicalpatterning has been widelyused to study the interac-

tions of the cells with their substrates. The most used

techniques are: microcontact printing GCP), drylift-off

and chemical photo-immobilizing. These techniqueshave been exploited for producing micro pattems ofbiomoleculesin order to str,rdyand manipulate cellular

interactions üth different substrates.

The method of microcontact printing was ini-tially developed by Kumar et al. [13] and Hidber et

aI. [14] to pattem surfaceswith self-assembled mono-layers (SAMs) of alkanethiolates and colloids, to use

them as masks in dry etching and chemical process.

It involves two of the microfabrication techniquesused for topographical patterning (photolithography

and soft lithography). Briefly, the desired pattern isprinted onto a silicon wafer using a standard photoli-

thography technique. An inverse replica of this mold

is made of polydimethylsiloxane (PDMS, thermallycured silicone elastomer) using soft lithography. Af-

ter that, the PDMS mold is covered with the desiredmolecule and it is put in contact with the surfaceintended to pattern (figure 4).

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Figure 4. Schematized microcontact printingprocess.

Shinghü et al., Chen et al. and Mrksich etal. [15-17] have deeply applied the Microcontactprinting technique for cell patterning. The techni-que was successfully used to imprint gold surfaceswith specific patterns of alkanethiols SAMs, creating"islands" of defined shape and size that supportedextracellular matrix protein adsorption, and therefore

cell attachment. Results showed that it was possible

to place cells in defined locations and arrays, and todictate their shape.

Defining the degree of cell extension proüdedcontrol over cell growth and protein secretion. In adifferent experiment, Chen et al. [16] controlled thegrowth and apoptosis of human and boüne capillaryendothelial cells by using micropatterned substratesthat contained extracellular matrix-coated adhesiveislands of decreasing size, to progressively restrict cell

extension. Using pCP, fibronectin adhesive squareislands were patterned onto a single substrate. Afterthe cell culture, they found out that square-shapedcells were produced almost matching the size andshape of the adhesive island (figure 5A). Apoptosisas well as DNA synthesis were affected by the size ofthe island (ñgure 5B).

¡iCP has been exploited specifrcally to controlneuron cell attachment and neurite morphologyU8-201. Random growth of axons and dendrites incultures makes diffrcult to control slmapse formationand development of neural network. The group ofKam et al. [20] used microcontact printing to define

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an interconnected lattice network of polylysine-conjugated laminin (a protein-polypeptide ligandthat is an effective promoter of neuron outgrowthon material surfaces). Rat hippocampal neuronsselectively adhered to the patterned regions. Longaxonal processes were extended by adhered neurons;

they did not deviate from the prescribed patterns,

demonstrating that neurons respond to this protein

with high selectiüty and that these techniques couldproüde long-range guidance of axonal outgrowth.(figures 6 and 7).

Drylift-offis a more recent technique appliedto pattern biological active molecules. Ilic andCraighead [21] described the lift-off process usingparylene as photosensitive poll,rner. The parylene ispattemed onto the substrate byusing a combinationof photolithography and reactive ion etching. Thesubstrate is incubated with a solution of the desiredmolecule to allow its adsorption. After that, theparylene is peeled offof the substrate mechanically,leaüng the molecule selectively adsorbed on thesurface (figure 8). Using this method, theypattemedaminopropyltriethoxysilane (APTS), antibodies, andpoly-L-lysine. Results showed that there were bindingof E. Coli and rat basophilic cells to antibodies andpoly-L-lysine, respectively, indicating that biologicalactivity is maintained with this pattern technique.

Bhatia et al. Q2) proposed a variation of thelift-off technique. A photoresist is patterned on aglass substrate by photolithography. After the deve-lopment, the desirable molecule is deposited onto

the pattemed glass surface, making chemical bonds

with the exposed glass region. Finally, the photoresist

is selectivelyremoved with acetone, leaving the mo-

lecule pattern on the glass surface. The main goal ofthe research was to make organized cell co-cultures(hepatocltes and 3T3 ñbroblast). The results werequite satisfactory demonstrating that this technique

is also useful to make zuccessful chemical patterning,

therefore controlling cell adhesion.

Figure 5. A. Micrograph of the adhesive islandspatterned onto the substrate. B. Plot showing

the variation of the DNA synthesis and apoptosisdepending on the adhesive island area. Figure

adapted from reference# 12.

Figure 6. Micrograph illustrating the geometryand dimensions of the micro-scale patterns.

Substrate-bound polylysine-conjugated laminin wasimmunofluorescently labeled with Alexa-488. Figure

adapted form reference # 19.

Figure 7. Rat hippocampal neurons seeded ontopatterned substrates after 1 day. A. dw =7.5 pm,

dl=13 pm. B dw=6.7 pm, dl=43 pm andC. dw=2.6 pm dl=43 pm. Substrate-bound laminin

was labeled with Alexa-488, and appears red.Neurons were identified by immunochemical stainingfor a neuron-specific subunit of tubulin with QuantumRed, and are shown in green-yellow. Figure adapted

from reference # 16.

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Figure 8. Schematization of the dry lift-off process.A. Combination of photolithography and reactive ionetching techniques to pattern both the parylene and

the substrate.

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Figure 9. Schematic process of thephotoimmobilization technique described by

Matsuda's group.

Chen et al. 126l used Matsuda's method topattern immobilized sulphated hyaluronic acid on apolystyrene plate and examine the interaction withblood cells. Platelet adhesion was reduced on theregions immobilized with sulfated hyaluronic acid.In the same way, Park and lto 127) micropatternedheparin onto polystyrene substrates; and in the pre-

sence of fibroblast growth factor (FGF), the growth

of mouse fibroblast STO cells was enhanced onlyon the heparin-immobilized regions. These resultsindicated that micropatterned-immobilized heparinactivated FGF for cell growth actiüty.

Micropatterning with cell growth factors hasbeen of great interest for the researchers, since itwas demonstrated that immobilized growth factorproteins could regulate cell functions without thecellular intemalization [28-30]. Chen et al. [31] foundthat immobilized insulin had an extremely high mi-togenic effect, which was much higher than that ofnative insulin. Similar results were obtained usingimmobilized epidermal growth factor (EGF) t321.Most of the researchers in the field agree that the

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Figure 8 B. Methodology used to selectivelyimmobilize the molecule of interest.

Photo-immobilization of molecules is also awidelyused technique to perform chemical patterns.

Matsuda ef- al. [23-25] reported the development ofa micropatteming technology for culrured cells, byprecise surface regional modification üa photoche-mical ñxation of phenyl azido-derivatized polymers

on polymer surfaces. The process consists in coupling

the molecule of interest with a photoreactive group,finally, this complex (molecule and photoreactivegroup) is patterned on a substrate by using a con-ventional photolithographic process (figure 9). This

technique was used to pattern hydrophilic regionsonto a hydrophobic polymer surface; then, endothe-lial cell behaüor was evaluated, showing that thesecells adhered, spread, and proliferated only on the

hydrophobic regions.

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ENCTNTTNINC MATERIALS SURFACES BY MICRoFABRICATIoN TECHNIQUES, To REGULATE IN vITRo CELL FUNCTIoNS: REVIEw

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immobilization inhibits the down-regulation induced

by the cellular internalization, thus continuing thestimulation for a long time.

Ito et al. [33] and Chen et al. [34] photo-immobilized insulin and EGF on a poly (ethylene

terephthalate) or polystyrene. Chinese hamsterovary (CHO) cells overexpressing the cognate re-ceptors were cultured on the patterned surfaces.After 8 hours, phosphorylation (activation) was in-

vestigated by staining with an anti-phosphotyrosineantibody. The signaling proteins were activated only

in cells cultured on the areas with the immobilizedbiomolecules. The important result was that the cellproliferation was accelerated only in the photo-im-

mobilized areas.

A relatively new approach is worked withstimuli-responsive polymers. Ito et al. [3S] and Chen

et al. [36] immobilized a thermo-responsive polymer

onto a polystyrene substrate using the method descri-

bed by Matsuda. The irradiated regions cross-linked

the polymers to the polysryrene film. The non-expo-

sed regions were washed away with water. Mousefibroblast STO cells were cultured on this pattemed

film at 37 "C. There was no significant difference ofcell attachment at this temperature, but when thefilm was maintained at 10'C, cells on the photo-im-

mobilized regions detached (figure 10).

Figure 10. A. Cells adhered to the entire substrateat 37"C. B. Cells adhered only to the non-patterned

regions at 10'C.

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As we could see, surface micropatterning toregulate cell functions has been deeplyinvestigated;this field may have a strong impact on the medicalscience, specihcally in areas such as implants, drugdelivery systems (using cell therapy), tissue enginee-

ring, and biomaterials.

There are several disorders related to alte-rations on the retinal pigment epithelium (RPE),these are: Stargardt's disease, age-related maculardegeneration and some forms of retinitis pigmen-tosa. Recent studies propose cell transplantation ofRPE as a successful therapy for diseases like retinaldegeneration; the problem is that RPE cells are polar,

they have an apical and a basal domain, so randomplacement of these cells into the subretinal spacedoes not always result in RPE regeneration.

Lu et al. [37] sudied a way to culture RPEcells, proposing a transplantation of an organizedsheet of cells with appropriate orientation as the-rapy. The experiments were focused on how couldbe afected the shape and function of RPE cells bychanging the surfaces properties of the substratewith micro-scale patterns of octadecyltrichlorosaline(OTS, hydrophobic organosilane). The sudied con-ñguration was circles of 1o pm or 50 pm in diameter(borosilicate glass-hydrophilic region), surroundedby OTS (hydrophobic region). Cells were seeded,and after 4 h theyfound that most of the cells on the

10 pm pattern had round morphology the spread ce-

lls were few and the average size was approximately

10 pm; cells on the 50 pm pattern were organizedinclusters conhned within the hydrophilic region, theaverage size was 20 ¡rm; cells on the control substra-

teswere spread and more elongated. When the cells

reached confluence, they were closely packed and

formed a continuous monolayer; the cytoskeletonorganization was similar for the two micropatterned

surfaces (actin organized into circumferential ringsat the apical side and short filaments at the basalside); this array of the actin filaments has been found

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ENCiXEERTNC MATERTALS SURFACES By MTCROFABRTCATTON TECHNIaUE¡ TO-'1I*9-L:!ArE lN VITRO CELL FUNCTIONS: REVIEW

in differentiated epithelial cells, and it is associated

with tight junctions that are crucial to maintain cell

polarity. These results demonstrated that is possible to

modulate RPE cell shape and phenorypic expression

using micropatterned surfaces.

Several sportsmen and people with degenerati-

ve (or autoimmune) diseases have problems with the

articular cartilage. Petersen et al' [38] worked on the

design of micro-engineered scaffolds for cartilage tis-

sue engineering applications. Chondrocites behaüor

was studied when culflrred on micropattemed aga-

rose substrates. Immunofluorescent assay for type II

collagen was used to check the chondrocite's actiüty'

Results showed that the cell adhesion was higher on

the micropattemed surfaces than that on the smooth

controls; the chondrocites grewup within the squares,

and factors such as cell adhesion and proliferation

were directlyrelated to the square dimensions' Immu-

nofluorescence assay§ demonstrated the production

of type II collagen in the matrix.

Nerve regeneration is one of the biggest

issues related to patient's rehabilitation. Leng et al'

[39] proposed the use of microcontact printing as a

prototype üsual prosthesis interface. Micropatterned

regions of growth factors were created with a final

resolution of 5 pm (the substratesused were glass and

a hydrophobic polymer) ' Stimulation electrodes were

used to improve the growth of the rat retinal ganglion

cells (RGCs). Results showed that using microcon-

tact printing they were able to direct the growth of

isolated rat RGCs on both glass and hydrophobicplastic surfaces. Finally, the authors concluded that

the ability to direct the growth of RGCs opens the

possibility of a üsual prosthesis interface based on

the regeneration of retinal cells.

In the same way, there are a lot of researches

showing that micro and nanotechnology are an

option to solve different problems on the medical

science. A few are briefly mentioned below

- Bhadriraju and Chen [40] say that the facilitiesproüded by different microfabrication techniques

to spatially organize cells and their adhesion has

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enhanced our ability to engineer cell functionsex üvo. They propose the use of these tools tocreate more in üvo-like cultures. This could be

useful for cell-based drug discovery and targetvaiidation.

- The group of Lee [4t] proposes microcontactprinting as an effective tool to pattern inhibitory

molecules in order to regulate the growth ofretinal pigment epithelial cells or iris pigment

epithelial cells on human lens capsule for trans-

plantation.

- Hongchun and Yoshihito 142) studied how couldbe controlled the celi attachment and detachment

on micropattern-immobilized poly(N-isopropyla-

crylamide) with gelatin. Research on this field has

great impacts on any implantable device.

s. üffi§{üh{§s§q}§{§Now it is clear that the materials surface

properties affect the cell behaüor. Both, chemical

and topographical surface features have effects on

different cell biologyfactors such as attachment, pro-

liferation, differentiation, activation, gene expression

and apoptosis. Semiconductors industry became an

important tool to modify those features at the nano

or micro scale allowing the scientific community to

engineer materials surfaces with determined micro

features (chemically or topologically) in order to find

a way to control the cell behavior' Regulation of cell

functions by micropatterning surfaces has several

applications in Medicine. Controlling the amount of

proteins secreted by the cells (gene expression) could

be useful for cell therapy (drug delivery system); regu-

lating the migration of the cells (direction and speed)

could be useful for nerve regeneration; controlling

the attachment and detachment of the cells could

be useful for any medical implant (maybe avoiding

some immune response or fibroblast encapsulation);

organized cell co-cultures could open the door for

in-ütro reconstruction of organs. In general, all these

are new fields that should be investigated to get a

better understanding on this area and to frnally find

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[1] Whitesides G. M., Ostuni E, Takayama S, JiangX, IngberDE. Soft lithography in biology and biochemistry. AnnuRev Biomed Eng. 2001; 3:335-73. Reüew

[2] Yu T., Ober C. K. Methods for the topographical pat-teming and pattemed surface modification of hydrogelsbased on hydroxyethyl methacrylate. Biomacromol-ecules. 2003 Sep-Oct; 4(5):1126-3f .

[3] Wang C., Madou M. From MEMS to NEMS with carbon.Biosens Bioelectron. 2005 Apr I 5; 2O(IO) :218 l -7 .

[4] Van Kooten T. G., Whitesides J. F., von Recum A. In-fluence of silicone (PDMS) sudace texture on humanskin fibroblast proiiferation as determined bycell cycleanalysis. J Biomed Mater Res 1998;43:1-14.

[5] Chehroudi B., McDonnel D., Brunette D. M. The effectsof micromachined surfaces on formation of bone{iketissue on subcutaneous implants as assessed by radi-ography and computer image processing. J BiomedMater Res 1997 ; 34:279-90.

[6] Tan J., Saluman W M. Topographical control of hu-man neutrophil motility on micropattemed materialswith various surface chemistry. Biomaterials. 2002Aug;23(15):3215-25.

[7] Von Recum A., van Kooten T. G. The influence ofmicro-topography on cellular response and the impli-cations for silicone implants. J Biomater Sci Poll,rn Ed.r99 5;7 (2) : t8 1-98. Reüew.

[8] Walboomers X. F., Monaghan W, Cunis A. S., JansenJA. Attachment of frbroblasts on smooth and micro-grooved polysryrene. J. J. Biomed Mater Res. 1999Aug;46Q),ztz'zo.

[9] Hansford D., Gallego D., Patiño D., Calderón D., Fer-rell N., Chakrapani A. Microfabrication and in ütroevaluation of Polylactic-co- glycolic Acid (PLGA) TissueEngineering Scaffoids. First Colombian Congress ofBioengineering and Biomedical Engineering, Medellín- Colombia. October, 2003.

[10] Craighead H. G., Tumer S. W, Daüs R. C., James C.D., Perez A. M., St. John P. M., Isaacson M. S., KamL., Shain Wi, Tumer J. N., Banker Q. Chemical andtopographical zurface modificationfor control of centralnervous system cell adhesion. J Biomed Microdev 1998;1(1):4944.

[11] Wojciak-Stothard 8., Cunis 4., Monaghan W, Mac-donald K. and Wilkinson C. Guidance and activation ofmurine macrophages by nanometric scale topography.Exp Cell Res. 1996 Mar 15;223(2):426-35.

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[12] Chou L., Firth J. D., Uitto V J., Brunerte D. M. Substra-tum sudace topography alters cell shape and regulatesfibronectin mRNA level, mRNA stabiliry secretion andassemble in human fibroblast. J. Cell Sci. 1 995 Apr; I08(ft4:1563-73.

ll3l KumarA., Biebuyck H. A., Whitesides G. M. Pattemingself-assembled monolayers: applications in materialsscience. Langrnuir 1994; lO(9 :1498-51 l.

ll4l Hidber P. O., Helbig W, Kim E., Whitesides G. M.Microcontact printing of palladium colloids: micron-scale patteming by electroless deposition of copper.Langmuir 1996; 12(6) :1375-80.

[15] Shinghü R., Kumar A.,Lopez G. P., StephanopoulosG. N., Wang D. I., Whitesides G. M., Ingber D. E.Engineering cell shape and function. Science 1994;264:296-8.

[I6] Chen C. S., Mrkich M., Huang S., Whitesides G. M.,Ingber D. E. Geometric control of cell life and death.Science. 1997 May 30; 27 6(5317) :1 425-8..

ltTl Mrkich M., Chen C. S., Xia Y., Dike L. E., Ingber D.E., Whitesides G. M. Controlling cell attachment oncontoured surfaces with self-assembled monolayersof alkanethiolates on gold. Proc Natl Acad Sci U S A.199ó Oct t; 93Qo):Ío77 5-8.

[18] LengT., Huie P., Mehenti N.2., PetermanM. C., Lee C.J., Marmor M. F. Sanislo S. R., Bent S. F., BlumenkranzM. S., Fishman H. A. Directed ganglion cell growth andstimulation with microcontact printing as a protoqpevisual prosthesis interface. Annual Meeting AbstractSearch and Program Planner 2o02. Abstract No4454.

[19] Scholl M., Sproessler C., Denyer M., Krause M.,Nakajima K., Maelicke A., Knoll W, OffenhaeusserA. Ordered networks of rat hippocampal neurons at-tached to silicon oxide surfaces. J Neurosci Meüods.2000 Dec t5; 104(1):65-75.

[20] Kam L., Shain W, Tumer J. N., Bizios R. Axonal out-growth of hippocampal neurons on micro-scale net-work of polylysine-conjugated laminin. Biomaterials.2oo f May;Z2(f O) : 1049 - 54.

[21] Ilic B., Craighead H. G. Patteming of chemicallysensi-tive biological materials using a polyirer-based dry liftoff. J Biomed Microdev 2oOO;2(4):317-22.

[22] Bhatia S. N., Yarmush M. L., Toner M. Controllingcell interactions by micropatteming in co-cultures:Hepatocytes and 3T3 fibroblasts. J Biomed Mater Res.1997 Feb;34Q):189-99.

I ü::1

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[23] Matsuda T., Inoue K., Sugawara T. Development ofmicropatteming technology for cultured cells. TransAm Soc Artif Intem Organs 1990; 36:M559-63

[24] Matsuda T., Inoue K. Novel photoreactive surfacemodifi cation technology for fabricated deüces. ASAIOTrans. 1 990 Jul-Sep;36(3) :M5 59-62.

[25] Matsuda T., Sugawara T. Development of surfacephotochemical modifi cation method for micropattem-ing of cultured cells. J Biomed Mater Res. 1995 Jun;29(6):749'56.

[26] Chen G., Ito Y., Imanishi Y., Magnani A., Lamponi S.,Barbucci R. Photoimmobilization of sulphated hyal-uronic acid for antithrombogenicity. Bioconjug Chem.1997 Sep-Oct; 8(9:7 3o-4.

[27] Park YS., Ito Y. Micropattem-immobilization of heparinto regulate cell growth with fibroblast gromh factor(FGF). Cytotechnology, 33(r -3) July 2000. tt7 -r22.

[28] Ito Y. Intelligent materials for tissue engineering. In:Akaike T., Okano T., Akashi M., Terano T., Yui N., edi-tors. Advances in biomaterials science. Tokyo: CMC1997. p. 421-32.

[29] Ito Y. Tissue engineering with immobilized growthfactors. Mater Sci Eng 1998; C6:267-74.

[30] Ito Y., Chen G., Imanishi Y. Artificial juxtacrine stimu-lation for tissue engineering. J Biomater Sci Polym Ed.1998; 9(8):879-90. Reüew.

[31] Chen G., Ito Y., Imanishi Y. Mitogenic activity ofwater-soluble and water-insoluble insulin conjugates.Bioconjug Chem. 1997 Mar-Apr;8(2): 106- I0.

[32] Ito Y., U J. S., Takahashi T., Imanishi Y., Okabayashi Y.,Kido Y., Kasuga M. Enhancement of the mitogenic ef-fect by artificial juxtacrine stimulation using immobilizedEGF. J Biochem (Tokyo). 1997 Mari7213):514-2o.

[33] Ito Y., Chen G., Imanishi Y. Photo-immobilization ofinsulin onto polystyrene dishes for protein-free cellculture. Biotechnol Prog I996; l2:7OO-3.

[34] Chen G., Ito Y., Imanishi Y. Photo-immobilization ofepidermal growth factor enhances its mitogenic effectby artifrcial juxtacrine signaling. Biochim Biophys Acta.1997 Sep 1 1; 1358(2):200-8.

[35] Ito Y., Chen G., Guan Y., Imanishi Y Partemed im-mobilization of thermoresponsive polymer. Langmuir1997;13:2756-9.

[36] Chen G., Imanishi Y., Ito Y. Effect of protein and cell be-haüor on pattem- graft ed thermoresponsive pol¡.'rner.J Biomed Mater Res. 1998 Ocr;42(1):38-M.

[37] Lu L., Kam L., Hasenbein M., Nyalakonda K., Bizios R.,GoKpferich A, Young J. F., Mikos AG. Retinal pigmentepithelial cell function on substrates with chemicallymicropattemed zudaces. Biomaterials. 1999 Deci2}(23-24):2351-61.

[38] Petersen E. F., Spencer R. G., McFarland E. W. Micro-engineering Neocartilage Scaffolds. Biotechnol Bioeng.2ooZ Jun 30;78(7):801-4.

[39J Leng T., Huie P., Mehenti N. 2., Peterman M. C., LeeC. J., Marmor M. F., Sanislo S. R., Bent S. F., Blumen-kranz M. S., Fishman H. A. Directed ganglion cellgrowth and stimulation with microcontact printing asa prototlpe üsual prosüesis interface. ARVO AnnualMeeting Abstract Search and Program Planner. 2002.Abstract No 4454.

[40] Bhadriraiu K., Chen C. S. Engineering cellular micro-environments to improve cell-based drug testing. DrugDiscov Today. 2OO2 Jun I ;Xl l):612-20. Reüew.

[41] Lee C. J., Philip H., Leng T., Peterman M. C., Mar-mor M. F., Blumenkranz M. S., Bent S. F., FishmanH. A. Microcontact printing on human tissue forretinal cell transplantation. Arch Ophthalmol. 2002Dec;t2o(tz):17r4-8.

[42] Hongchun L., Yoshihiro I. Cell attachment anddetachment on micropattern-immobilized poly(N-isopropylacrylamide) with gelatin. Lab Chip. 2002Aug;2(3):175-8. Epub 2oo2 Aug t3.

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