Modified hydroxyethylmethacrylate hydrogels as a modelling tool for the

study of cell-substratum interactions

PETER R. BERGETHON, VICKERY TRINKAUS-RANDALL and CARL FRANZBLAU

Department of Biochemistry, Boston University School of Medicine, SO East Concord Street, Boston, MA 02118, USA

Summary

The interactions between cells and their extra-cellular substratum environment are complex anddifficult to study. Defined, synthetic substrata arevaluable tools for experimentally determining therole of ionic and receptor-specific interactions be-tween cells and their substrata. Hydrogels havebeen modified to contain stoichiometrically definedquantities of both positive and negative charge aswell as specific proteins. These synthetic surfacesare water-rich matrices that possess hydroxylgroups, positive and negative ionized charges andnative proteins, and can be considered as models ofextracellular matrices on which an assessment ofcharge contribution and macromolecular contentand specificity can be addressed with respect tocell-matrix interactions. This study shows that

simple gels made of polyhydroxyethylmethacrylatedo not support the spreading of cells but that thegeneration of copolymers by the addition of mono-mers that contain ionizable functional groups, willpermit cell spreading. These simple modificationsdo not lead to cellular proliferation, yet whencollagen is entrapped in the hydrogel substratum,proliferation occurs. The proliferative rate of cellsgrown on collagen-containing surfaces may bemodified by altering the stoichiometry of the ioniz-able polymers used to make the surface. This studydescribes a synthetic, definable model for the studyof cell-substratum interactions and control.

Key words: cell-substratum interaction,hydroxyethylmethacrylate, hydrogels.

Introduction

The interaction of cells with their substrata has beenimplicated in the normal processes of cell attachment,spreading, proliferation and differentiation (Grinnell,1978; Trinkaus, 1984; Ruoslahti et al. 1985). In addition,it is likely that the interaction between cells and thesurfaces they grow on is involved in numerous pathologi-cal processes including neoplasm, atherogenesis andwound healing. Much research has been focused on therole of various cell—substratum specific interactionsthrough the extracellular matrix proteins such as col-lagen, fibronectin, laminin (Yamada, 1983), and to somedegree, elastin (Faris et al. 1983). The interactionsbetween the cell and its substratum surface is one of veryclose proximity, i.e. 45-100 nm (Izzard & Lochner,1976), and therefore will be influenced greatly by thestructure of the interphase region, especially in terms ofthe orientation of aqueous dipoles, counterions andorganic adsorbants. This view is valuable when thecomplete makeup of the extracellular matrix is consideredas being composed of a non-homogeneous (Freeman,1987) array of functional species that includes the hydro-philic moieties contributed by the hydroxyl groups of

Journal of Cell Science 92, 111-121 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

polysaccharides and glycoproteins, the positive andnegative charges associated with sulphated glycosamino-glycans, the acidic and basic residues of amino acid side-chains, and the highly ordered arrangement of aminoacids represented in the tertiary and quaternary struc-ture. The proteins provide microenvironments whoseproperties depend both on charge (indeed, one of theroles of proteins can be crudely considered as one of semi-rigid charge fixing) and on highly non-polar hydrophobicregions in molecules such as elastin and collagen.

Great uncertainty exists in the actual surface structurein natural substratum systems because of the heterogen-eity and dynamic aspects of the surface processes. Atheoretical treatment of the surfaces is extremely difficultdue to the lack of definition. A carefully defined matrixmodel would therefore be valuable in studying the role ofthe various interactions at the cell-substratum interfaceempirically. The use of modified hydroxyethylmethacry-late (HEMA) hydrogels to support cell growth in vitrohas been previously described (Civerchia-Perez et al.1980). These modifications included the incorporation ofextracellular matrix proteins into the hydrogel structure,thus permitting cellular spreading and proliferation onsurfaces otherwise inhospitable to anything more than

111

cell attachment (Folkman & Moscona, 1978; Lydon et al.1985). To investigate the role that ionizable surfacegroups play in the regulation and modulation of cellularspreading and proliferation, we further modified HEMAhydrogels by copolymerizing negatively charged moieties(methacrylic acid) and positively charged moieties {N,N-dimethylaminoethylmethacrylate) into these surfaces.Experiments with a variety of cells, including IMR-90fibroblasts, rabbit stromal fibroblasts and corneal epi-thelial cells all confirmed that the presence of ionizablecharges permitted spreading, while in the absence ofionizable charges, significant spreading did not occur.Proliferation occurs when collagen-containing surfacesare used. If these collagenous surfaces are further modi-fied by the incorporation of charge-containing mono-mers, the proliferation response is altered. This studyshows that cells respond to the surface characteristicscontributed in part by ionizable charge and that questionsabout cell-substratum interactions may be studied invitro in a system of defined synthetic substrata.

Materials and methods

MaterialsTns-(hydroxymethyl)aminomethane, sodium chloride, am-monium persulphate, sodium bisulphite and sodium bicarbon-ate were purchased from Sigma Chemical Co., St Louis, MO.Dulbecco's modified media, Ham's F-12 nutrient medium,sodium pyruvate, penicillin-streptomycin solution, non-essential amino acid solution and EDTA-trypsin solution wereobtained from Gibco Laboratory, Grand Island, NY. Foetalcalf serum was bought from Biofluids, Rockville, MD or Sigma.2-Hydroxyethylmethacrylate, methacrylic acid and NJ\'-dimethylaminoethylmethacrylate were obtained from Poly-sciences, Warrington, PA. Ethylene glycol, sodium hydroxideand hydrochloric acid were from Fisher Scientific Co. DispaseII was purchased from Boehringer-Mannheim Biochemicals,Indianapolis, IN. ITS (insulin, transferrin, selenium) wassupplied by Collaborative Research, Bedford, MA. New Zea-land rabbits were obtained from Pine Acres, Norton, MA.Tritiated valine was bought from New England Nuclear,Boston, MA. Untreated 24-well tissue culture plastic trays wereobtained by arrangement with Corning Glass Works, Corning,NY.

Preparation of hydtvgelsHydrogels were prepared by a modification of the method ofCiverchia-Perez (1980). Simple hydrogels of hydroxyethyl-methacrylate were prepared by preparing a 1:1:1 (by vol.)mixture of ethylene glycol, 2-hydroxyethylmethacrylate and anaqueous buffer, usually 0-05 M-Tris-HCl, 0-lSM-NaCl. Afterdegassing under low pressure, and while on ice, 0-1 ml of 12%sodium bisulphite and Olml of 6% ammonium persulphatesolution were added to the mixture. Gels in the shape of flatsheets were prepared by using a form that consisted of asandwich of two pre-cleaned glass microscope slides spaced by 2no. 1 coverslips. This sandwich was held in position with analligator clip. The separation of the slides was approximately0-5 mm. The polymer mixture was injected in between the glassslides with a Pasteur pipette and was drawn into the space bycapillary action. Following injection of the slides with polymer,the slides were polymerized at 37CC for 2h. When polymeriz-ation was complete, the sandwich form was separated and the

sheet of polymer was peeled from the form. These sheets werethen exhaustively dialysed against 0-05 M-Tris-HCl, 0-15M-NaCl buffer solution, pH7-4. To prevent the polymer sheetsfrom being damaged during the dialysis process, they wereplaced in 50 ml plastic centrifuge tubes that had multiple 0-5 cmholes bored into them. These containers ensured the free flow ofdialysis medium while protecting the gel material. When all ofthe ethylene glycol had been removed from the polymerized gel,the gels became crystal clear and were then ready to be preparedfor cell culture experiments. Discs or buttons of polymer werecut from the polymerized sheets with a no. 10 cork borer. Theradius of the resultant buttons was 140 mm. The buttons werethen placed, one per well, into untreated Corning 24-well tissueculture trays. To sterilize the buttons, 1 ml of a 10% penicil-lin-streptomycin antibiotic solution made in Puck's Ca - andMg2+-free saline was added and the trays were exposed for 2hto u.v. germicidal irradiation. The antibiotic solutions wereremoved and the gels were incubated for 30min with mediaprior to cell seeding.

Hydrogels that incorporated collagen were prepared in asimilar fashion except that a solution containing solubilizedcollagen in 0-05 M-Tris-HCl, 0- IS M-NaCl solution was used inplace of the aqueous buffer. Solubilized collagen was preparedeither from lyophilized rat-tail tendon collagen or calf-skincollagen, by solubilizing the protein in 3 % acetic acid at 4°C.The collagen preparations were shown to be essentially purecollagen by both amino acid analysis and by polyacrylamide gelelectrophoresis. This was followed by exhaustive dialysisagainst 0-05 M-Tns-HCl, 0-15M-NaCl buffer (Tris buffer), allat 4°C. The final concentration of the collagen in the solutionused in the preparation of the hydrogels was determined byamino acid analysis on a Beckman amino acid analyser model6300 after hydrolysis in 6 M - H C 1 at 110°C for 24h.

The copolymer gels were prepared by mixing a (v: v) dilutionof either methacrylic acid (MAA) or Ar^V-dimethylaminoethyl-methacrylate (NDAM) to the HEMA polymer prior to mixing.The copolymer solution was then mixed at the same 1:1:1 ratioas cited above. Typically the copolymer concentrations weremixed at 0-99 ml of HEMA to 0-01 ml of MAA or NDAM (1 %)or 0-999ml of HEMA to 0-001 ml of MAA or NDAM (0-1 %) .

To estimate the surface occupancy of the negative andpositive charges in the copolymer gels, models of the copolymercomponents were built using Cochrane's molecular modellingkits. Because no crosslinking agent is used in the polymerizationof these gels, the assumption was made that there is essentiallyno crosslinking of one linear chain to another but rather thecomplex entanglement of long linear chains of poly(HEMA). Itwas assumed that the MAA and NDAM were uniformlydistributed throughout the volume of the gel and that there wasno preferential adsorption of the charge-containing polymerchains to the glass slides during the casting process.

Cell cultureIMR-90 cells. IMR-90 neonatal human lung fibroblast cells

were obtained from the N.I.A. Aging Cell Culture Repository,Institute for Medical Research, Camden, NJ. All cells used inthese experiments were taken from PDL 21, a large pool ofwhich had been established and then frozen in liquid nitrogen insamples of 1-5X106. Prior to experiments, a sample of cells wasthawed and seeded into a Corning T-75 tissue culture flask.After 1 week in passage, cells were harvested. Harvesting wascarried out by washing twice in Puck's Ca2+- and Mg2+-freesaline and then treating the cell layer with 3 ml of 1 %trypsin-EDTAsolution for 5 min at 37°C. Trypsin was neutral-ized and cells were washed in 10 ml of total of Dulbecco'sModified media, supplemented with 3-7gl~' NaHCG"3, 1%sodium pyruvate, 1 % non-essential amino acids, 1 % penicillin-

112 P. R. Bergethon et al.

-streptomycin solution and 10% foetal calf serum (DV 3.7Complete Media). After counting in a Coulter counter, cellswere diluted to a concentration of 20 000 cells ml"1 and wereplaced on the copolymer gels. Generally 75-80% of the cellswere viable after trypsinization as assessed by exclusion dyetreatment. To examine the potential role played by the adsorp-tion of serum proteins to the surfaces, experiments wereconducted with 10, 5 and 1 % and no serum protein in the cellculture media. In these experiments, cells were washed com-pletely free of serum and were seeded onto gels in a serum-freeor lower percentage supplemented media. In these serum-manipulated experiments, gels were pre-treated with serum-free media.

Cornea I epithelial and stromal fibmblast cells. New Zealandrabbits were killed with 5 ml of sodium pentobarbital (325 mg)administered intravenously. Corneas were excised and Desce-met's membrane and the endothelia were removed with forceps.The remaining cornea (epithelium, basement membrane andstroma) was incubated in Dulbecco's modified Eagle's mediumcontaining 10^M-calcium and l-2unitsml~' dispase II for 1 h.Small sheets of epithelium were teased from the underlyingstroma (Trinkaus-Randall & Gipson, 1985) and placed apicalside up on Falcon tissue culture plastic containing Dulbecco'smodified Eagle's medium (1:1) with Hani's F-12 nutrientmedium supplemented with 1 % non-essential amino acids, 1 %penicillin-streptomycin, 5% calf serum and 0-5% ITS. Onceconfluence was achieved, the cells were subcultured usingtrypsin-EDTA (1:4, split). The cell suspension was seededonto polymers at a seeding density of 1-5 X104 cells/disc.

Stromal fibroblasts were cultured from New Zealand rabbitstromas. Corneas were excised and both the epithelium andendothelium were removed. The stroma was cut into smallpieces with a tissue chopper and the tissue was placed on thebottom of Falcon T-25 flasks. The explants were cultured inDulbecco's modified Eagle's medium, supplemented as for thecorneal epithelial cells, until confluence was achieved. The cellswere subcultured using trypsin—EDTA in a 1:6 split and thecells were seeded at a plating density of 2-9X104cells/disc. Atotal of 70-75 % of the cells were found to be viable followingtrypsinization, for both the epithelial cells and fibroblasts.

Cell countsTo follow the pattern of growth on the copolymer hydrogels,cell numbers were determined over the first week in culture.Cells were counted by removing each hydrogel button from the24-well culture plate and after washing in Tris buffer with ashear rate that averaged 60cms" and was estimated to be atmaximum 100cms" , and then placed in an Isoton Coultercounter vial. A 0-5 ml sample of 1 % trypsin solution was addedand the gel was incubated at 37 °C with shaking for 20min.After enzymic removal of the cells, Isoton diluent was added tothe vials and the cells and diluent were mixed vigorously.Subsequently each vial was counted in triplicate in a Coultercounter. Time points were chosen at 4 h after seeding (this timepoint defined the seeding density), 24, 48, 96 and 144h afterseeding.

Protein productionBecause on certain surfaces cells remained rounded and did notspread, new protein production was used as a measure of cellviability. Cells were pulsed with 25 ,uCi of L[3,4(«)-3H]valine(sp. act. 42Cimmol~1) per well for periods of 4-24 h, 24-48 hor 48-72 h. After the pulse period was completed, the cells wereharvested, the medium being separated from the cell layer. Thecell layer was homogenized. Both homogenized cell layer andmedium fraction were lyophilized and then exhaustively dia-lysed against deionized water at 4°C. Following dialysis the

samples were resuspended in water and portions were taken forcounting in an LKB 1218 RackBeta liquid scintillation counterand for total protein determination by a Bradford dye bindingassay (Bradford, 1976). Results were expressed as radioactivectsjfg" of total new protein.

Photography and spreading detenninationConcurrent with cell counting, photographs of cells were takenwith an inverted phase-contrast Nikon Diaphot microscope torecord variations in cell morphology and spreading. Photo-graphs were taken of a representative field after the entire gelwas scanned. Cell spreading was graded by two independentinvestigators at the first two time points (4h and 24 h). Cellswere evaluated for spreading in the following manner. Gelswere removed from the 24-well tissue culture tray and vigor-ously washed in Tris buffer as described in the section on cellcounting. Cells on each gel type were given a grade from 0 to 3depending on the degree of spreading; 0 was assigned whencells were rounded and less than 10% of the cells that wereadherent after washing were spread. Grade 1 indicated that10—25 % of the cells were spread. A grade of 2 was used in caseswhere between 25 and 70 % of the cells were spread and grade 3was assigned in cases where more than 70 % of the populationon the gels were spread. Fibroblasts normally undergo filo-podial spreading while corneal epithelial cells spread in alamellipodial manner.

Results

Sutface characteiistics of the copolymer gelsThe preparation of all copolymer hydrogels yieldedtransparent, non-toxic surfaces. These surfaces would allsupport cell attachment and remained transparentthroughout the experiments, thus permitting visual ob-servation at any point in the experiments. On somesurfaces cells remained rounded. To establish that thecells on all surfaces were viable and able to synthesize newproteins, regardless of morphology, incorporation ofradiolabelled valine was followed from 4-24, 24-48 and48-72h. For all cell types and on all surfaces, includingsimple HEMA gels, cells were able to synthesize newproteins. While the rates of synthesis did vary dependingon cell type, surface and time, all cells regardless ofmorphology produced new proteins during the times ofthe analysis (data not shown). Previous experience withprotein-modified HEMA hydrogels as well as thesestudies have shown that they may be used in experimentsinvolving electron microscopy, time-lapse video-photography, radioisotope and biochemical studies(Franzblau et al. 1988). Model building and calculationsusing the simplifying assumption that the polymer back-bone occupied the surface in a flat linear fashion with themethyl and ester side-chains arranged orthogonally to thebackbone (Fig. 1) gave the information found in Table 1.In the calculation of these data it was assumed thatorientation of the ionizable functional groups was notsignificantly affected by interaction with the glass slidesused as casts. This was based on the estimated thicknessof the Gouy-Chapman layer, given the ionic strength ofthe aqueous phase of the polymerization. This thicknesswould be expected to be between 0-5 and 0-7 nm and,given the residue size of the HEMA side-chain as

Hydmgels in cell-substratum inteiactions 113

l"3

1C-0101

I21 2

OH

C C

1C-01 —'01

T2OH

-c-Ic

• c —IC-0I0I

™ 2

OH

-c —IC-0I -0I

2OH

Table 1. Surface occupancy of functional gmups permm2

-c —IC-0I0

I

I 2

CH2

Oil

-C —IC-0I -0

I

I 2

CH_OH H,

-C —IC-0I

N

CH,

-c —IC-0I0I

2OH

-c —IC-0I -0I» 2

OH

Fig. 1. Proposed structure of the HEMA-copolymerhydrogels. In A and B the linear backbone that is producedby the polymerization of the acrylic moieties of the monomershas repeating side-chains of either methyl groups or the ester-linked group. It has been shown that in aqueousenvironments, the polar groups rotate to point toward thesurface (Ratner, 1986). Monomer A is the polymerizedstructure of HEMA, B is the structure of MAA, C is thestructure of NDAM. The repeat number of the HEMA units(A,,), depends on the copolymer mixture, i.e. for a 1 %HEMA-MAA gel, n = 65, while for a 1 % HEMA-NDAMmixture, n = 133. n can be calculated by finding the ratio ofhydroxyl groups to ionizable moieties as listed in Table 1. InC, an idealized representation of the size of the modelledmolecules is shown. Given the orthogonal arrangement of theside-chains in the polymerized molecules, the rectangulardimensions of the 'space-filled' molecule are approximately asgiven. Hydrogens on the polymer chain have been left out forsimplicity.

087 nm, it was assumed that significant repulsion of thenegative groups or attraction of the positive groups didnot occur. The residue size of a single HEMA in the

Polymer

mixture -OH -COOH

HEMA only

HEMA-MAA

HEMA-NDAM

1-000%0-100%0-010%0-001%

1-000%0-100%0-010%0-001%

1-OOxlO'3

9-75X10'2

9-85X10'2

9-99X10'2

9-99X10'2

9-92X10'2

9-99X10'2

9-99X1012

9-99X10'2

1-5x10"1-5x10'°1-5X109

1-5X108

7-425X10'°7-425X109

7-425X108

7-425X107

Number of functional groups calculated to be present at thesurface of a hydrogel, based on the proportions, molarity andmodelled structure as shown in Fig. 1.

polymer is approximately 0-33 nm X 0-33 nm X 087 nm,NDAM is 0-33 nm X 0-33 n m x 0-118 nm and MAA is0-33 nm X 0-33 nm X 0-33 nm. It can be seen that for acopolymer mixture of 0 1 %, a charge density of the orderof 10 mm"2 negative or positive charges for MAA andNDAM, respectively, is expected to be present. For agiven percentage (v:v) of the MAA versus the NDAM,there are roughly twice the number of negatively chargedcarboxyl groups compared with the positively chargednitrogens.

Spreading and proliferation of cells on hydrogel surfacesOn surfaces without collagen, plating efficiencies were inthe range of 10-20%. On collagen-containing surfacesthe plating efficiencies were somewhat increased andin the range of 25-40%. On all surfaces and cell typestested, alterations in polymer stoichiometry had littleeffect on the plating efficiency, though the platingefficiency on the hydrogels was always lower than ontissue-culture plastic. Surfaces were assessed for theirability to support cell spreading by independent grading(by two observers), by phase-contrast microscopy andthese results are summarized in Fig. 2A,B. In exper-iments done in the presence of serum, all cells spread onsurfaces that contained ionizable copolymers. This is incontrast to the lack of spreading found by all cells on theHEMA hydrogels alone. When hydrogels containingvarying percentages of MAA or NDAM were studied at24 h, spreading was judged to be optimal (filopodial forfibroblasts and lamellopodial spreading for corneal epi-thelial cells) when a 0-01% to 0-1% gel was the cellgrowth surface. All of the surfaces that contained col-lagen, with or without the ionizable polymers, were ableto support spreading well, regardless of the concentrationof the copolymers.

When IMR-90 and stromal fibroblasts were plated ontogels that contained collagen, regardless of the copolymercomposition, filopodial spreading was found to be exten-sive within 4h (Fig. 3). In contrast, surfaces that did notcontain collagen, but did contain ionizable copolymers,showed only minimal flattening or spreading at 4h.However, by 24 h fibroblasts on these gels showed

114 P.R. Bergethon et al.

Spreading versus polymer composition

0 MAA-fibroblastNDAM-fibroblast

0001 001 01 1 5Copolymer (%)

Spreading versus polymer composition

o.GO

0 MAA-epithelialH NDAM-epithelial

0 0-001 0-01 01 1 5Copolymer (%)

Fig. 2. Cell spreading on various percentage copolymer gels.The spreading index of corneal epithelial cells and stromalfibroblasts as described in the text is shown for differentpercentage combinations of HEMA-copolymer gelscontaining MAA or NDAM. A. Spreading for fibroblasts;and B, spreading for epithelial cells.

widespread filopodial spreading. Experiments were con-ducted with IMR-90 fibroblasts in serum-free media toinvestigate the potential role of the serum proteins in thespreading process. Interestingly, in the absence of serum,on gels containing ionizable copolymers only, fibroblastsshowed lamellopodial spreading within 2h, with earlyfilopodial spreading occurring at 4h. Later time pointsshowed extensive filopodial spreading. Fig. 4 illustratesthe spreading morphologies of the IMR-90 fibroblasts inthe presence and absence of serum.

Corneal epithelial cells are characterized by lamellopo-dial spreading when grown on tissue-culture plastic. Thespreading of these cells is generally found after 24 hon tissue-culture plastic. When these cells were grown oncollagen-containing gels, spreading was found at 24 h. Onthe MAA-containing copolymer gels, slight lamellopodialspreading was found to occur between 24 and 48 h. Incontrast, on the NDAM-containing gels, the epithelialcells began spreading by 24 h. In the gels that containedboth collagen and NDAM, the spreading was markedlyenhanced at 24 h (see Fig. 5).

The ability of the various surfaces to support a

proliferating cell population was found to vary dependingon the cell type, the surface components and stoichi-ometry of the copolymer mixture. Fig. 6 shows charac-teristic growth curves for the cell types on the six types ofsurfaces. There is a clear separation of the slope of growthcurves depending on whether or not collagen was in-cluded in the hydrogel. For IMR fibroblasts grown ongels without collagen, there was a slight but consistentincrease in cell number on the gels that contained MAA.For the stromal fibroblasts there was no significantincrease in cell number in gels that contained only thepolymers. Corneal epithelial cells also showed no signifi-cant increase in cell number on the polymer surfacesalone.

When collagen was included in the hydrogel mixtures,the presence of the ionizable copolymers played a muchmore significant role. While IMR-90 cells grown onHEMA-collagen showed a 4-5-fold increase in cell num-ber in 96 h, the collagen-containing HEMA-MAA gelssupported a 25-fold increase, while the collagen-HEMA-NDAM gels supported a 17-fold increase in cellnumber. In the case of the stromal fibroblasts, theHEMA-collagen surface supported an increase of ap-proximately 33-fold in contrast to a 77-fold increase forthe MAA and 100-fold increase for the NDAM-collagenmixture hydrogels. The corneal epithelial cells demon-strated a significantly different response to the varioushydrogels. The HEMA-collagen gels showed a fivefoldincrease in cell number in contrast to a 2-8-fold increasein the case of the HEMA-MAA-collagen gel. TheHEMA-NDAM-collagen gel showed a 17-fold increasein cell number.

The role played by serum proteins on the proliferationand spreading of cells was assessed by varying theconcentration of serum present in the media. Whetherserum was 10 or 5%, cells spread and proliferatedequally. When cells were grown in 1 % serum, spreadingof the cells was also equivalent to that observed at 10%serum concentrations. However, cells were quiescent anddid not proliferate. Proliferation of cells grown in theabsence of serum was not assessed.

Discussion

Hydrogels are convenient and interesting models that canbe used in the study of cell-substratum interactions.Hydrogels, composed of HEMA, are hydrophilic becauseof the presence of vast numbers of hydroxyl groups.These hydrogels characteristically imbibe water and swelluntil water accounts for between 30 and 40 % of the finalvolume of the matrix (Refojo & Yasuda, 1965). Thischaracteristic is shared by natural extracellular matrixthat contains proteoglycans. Previously, HEMA hydro-gels have been employed to study the effects of sub-stratum-relevant proteins presented to cells in culture bytheir entrapment in the polymer matrix. These definedprotein-hydrogel polymers have been used to study cellgrowth on collagen substrata with varying collagen con-centrations (Civerchia-Perez et al. 1980), altered syn-thesis of procollagen types (Faris et al. 1983), and the

Hydrogels in cell-substratum interactions 115

Fig. 3. Morphology of IMR-90 cells grown on HEMA or HEMA-collagen gels. Photographs were taken through a NikonDiaphot microscope with phase-interference optics (X40). A. IMR-90 fibroblasts at 24h on HEMA only gel, cells on all gels ofthis composition whether in the presence or absence of serum had this appearance; B, spreading at 4h in serum-containingmedium on HEMA-collagen gels, all of the collagen containing gels had this morphology.

116 P. R. Bergethon et al.

Fig. 4. Spreading of IMR-90 fibroblasts in the absence or presence of serum. A. Early spreading (2h) of IMR-90 fibroblasts onHEMA-MAA gels plated in the absence of serum versus; B, which shows the lack of early spreading (4h) when plated in thepresence of serum; for comparison at 24h; C shows speading in the absence of serum; while D shows spreading in presence ofserum.

substratum-dependent effects on nerve fibre growth andneuronal differentiation (Carbonetto et al. 1983). In thisstudy, through the stoichiometric addition of functionalgroups that contribute ionizable charge to the matrix, themodel has been enhanced to examine the role of thesecharges in cell—substratum interaction.

The role that ionizable charge plays in mediating theadhesion and spreading of cells has been of interest toinvestigators for many years. In numerous studies, thepresence of charge, whether polycations such as poly-lysine coating (McKeehan & Ham, 1976) or variousanions including sulphonates and carboxyl groups (Mar-oudas, 1975, 1976) have been shown to play a role in theadhesion and spreading of cells in culture. Study has beendirected at discovering which functional groups areresponsible for adhesion of anchorage-dependent cells.Often the starting surfaces are highly hydrophobic sub-stances and treatments to induce hydrophilicity have

been found to improve cell adhesion significantly. Thereis good evidence that it is the presence and concentrationof hydroxyl groups that is most important in adherence(Curtis et al. 1986a; Brandley et al. 1987). However, thefunctional groups that lead to adhesion of cells seem to bedifferent from those responsible for spreading. Thisconcept is supported by Brandley et al. (1987), whoseresults suggest that while short-term adhesion is moststrongly mediated by hydroxyl groups, the presence ofcarboxyl moieties is preferred for long-term growth.Brandley also reports that growth of Balb/3T3 fibro-blasts occurred on hydroxyl-derived polyacrylamide sur-faces. Lydon et al. (1985) used 6-h assays in whichspreading of cells was examined and found that withpolymer surfaces composed of greater than 90 % HEMA,cell spreading did not occur. In those studies, theaddition of hydrophobic copolymers was associated withspreading. Our studies showed that both the fibroblasts

Hydrogels in cell-substratum interactions 117

Fig. 5. Spreading morphology of corneal epithelial cells grown on copolymer gels. A. Minimal spreading on HEMA-MAAcopolymer gel; B, significantly greater spreading on the HEMA-NDAM copolymers; C, comparison of the spreading onHEMA-collagen gels with the enhanced spreading on the HEMA-NDAM collagen gels (D).

and epithelial cell lines were adherent to and remainedviable on the simple HEMA hydrogels, whose majorfunctional group is the hydroxyl moiety, but did notspread unless ionizable functional groups were alsopresent. Spreading in our studies was sensitive to thestoichiometry of the copolymer composition. On thebasis of the dose-response assessment of spreading andmodelled charge densities, it appears that spreading doesoccur even when the charge density is as low asl-5xl08mm~ carboxylic groups or 7-4xl07mm~ ter-tiary amino groups.

The question of whether sera or serum proteins arenecessary for cell adhesion and spreading has also beenthe subject of much study. The need for the adsorption ofmaterials such as fibronectin before adherence andspreading can occur has been advanced (Yamada, 1983;Grinnell, 1978). While Brandley et al. (1987) reported anabsolute requirement for serum in their studies of theadherence and spreading of Balb/3T3 fibroblasts on

derivatized polyacrylamide gels, Curtis et al. (1983,19866) showed that BHK cells adhere and spread onmodified polystyrene surfaces in the complete absence ofserum or serum components. Our studies with IMRfibroblasts reported here and previous studies byTrinkaus-Randall & Franzblau (1987) with corneal epi-thelial cells on tissue-culture plastic indicate that thesecells will adhere and spread in the absence of serum. It isof interest that in the case of the IMR-90 fibroblasts, thepresence of serum slowed the spreading process on thehydrogels compared to serum-free experiments. Theability of the cells, cultured in serum-free conditions, tobecome adherent and spread may be secondary to theelaboration of microexudative products as described byGrinnell (1978), or dependent on the modification of thesubstratum as suggested by Curtis and co-workers(1986a). It is interesting to suggest that the time delay inthe spreading of the IMR-90 cells in the presence ofserum is due to the modification or removal of one or

118 P. R. Bergethon et al.

8000A. Corneal epithelial cells, no collagen B. Corneal epithelial cells, with collagen

00 20 40 60 80 100 120

. Time (h)

0 20 40 60 80 100 120Time (h)

C. IMR-90 fibroblasts, no collagen10000 100000T

D. IMR-90 fibroblasts, with collagen

100Time (h)

100Time (h)

Fig. 6. Representative growth curves for cell types on each surface tested. All copolymer mixtures are 0-1 % gels. A. Cornealepithelial cells grown on gels of copolymer only ( • ) HEMA; (A) HEMA-MAA; ( • ) HEMA-NDAM; B, corneal epithelialcells grown on gels of collagen and copolymer composition ( • ) HEMA; (D) HEMA-collagen; (A) HEMA-collagen-MAA;( • ) HEMA-collagen-NDAM; C, IMR-90 fibroblasts grown on gels of copolymer only (symbols as in A); D, IMR-90fibroblasts grown on gels of collagen and copolymer composition ( • ) HEMA; ( • ) HEMA-collagen;(A) HEMA-collagen-MAA; (O) HEMA-collagen-NDAM.

more adsorbed serum components before spreading canproceed. The presence of ionizable functional groupsseems to be necessary for spreading to occur on thesegels. If the cells are able to modify the substratum bysecreting a microexudate or through the oxidation ofsubstratum hydroxyl groups, it is puzzling that thesimple HEMA gels are not modified, thus permittingspreading. A possible explanation may lie in the work ofHolly & Refojo (1975), who showed that side-chains ofthe HEMA polymers are capable of reorienting, depen-ding on the nature of an adjacent phase. When anaqueous solution is the adjacent phase the side-chainswith the hyroxyl groups would be turned toward thesurface, but it is possible that the presence of the 'cellularphase' would restructure the water and the other com-ponents of the interphase and lead to a turning of themethyl side-chains towards the surface. Thus modifi-cation of the hydroxyl group would be prevented.

The addition of collagen to the matrices early had theeffect of enhancing cell spreading and permitting pro-liferation to occur. The role collagen plays in cell functionhas been well reviewed by Hays (1981). Cells have been

shown to interact both directly with collagen and in-directly through molecules such as fibronectin and lam-inin (reviewed by Yamada, 1983). Previously the interac-tion of cells with collagen-containing HEMA hydrogelshas been studied in this laboratory (Franzblau et al.1988). The addition of the ionizable copolymers to thecollagen-containing hydrogels was thought to be anenhanced model of the natural extracellular matrix inwhich extracellular matrix proteins are found along withthe charged components of proteoglycans. Cells spreadand adhered to the collagen-containing gels easily.Though even cells adherent to the HEMA were viable (asjudged by de novo protein synthesis), surface factorsbeyond those necessary for adherence and spreading werenecessary to support proliferation. The role that collagenplays in the proliferative response of the cells is likely tobe a receptor-mediated response. Surprisingly, the ad-dition of ionizable groups to the matrix had a modulatingeffect on the proliferative rate of the cells studied. Whileno clear mechanism for this effect can be described at thistime, it is possible that the added surface charge of thematrix has the effect of ordering both water dipoles as

Hydrogels in cell-substratum interactions 119

well as affecting the ionic and organic adsorbents in theinterphase between the cell and the matrix. For example,the addition of cationic charge will lead to an increasedtendency for the adsorption of cations such as Ca2+ andMg2"1" at the inner Helmholtz plane. The resultingrestructuring of the interphase region that is contributedto by both the matrix and the cells would be likely to haveprofound effects on the signals transduced to the cellsthat carry information about the extracellular matrix.This explanation of interphase restructuring is attractivebecause it can also account for the seeming incongruitybetween the results of Lydon et al. (1985), who enhancedspreading with the addition of hydrophobic copolymersand our addition of hydrophilic copolymers. Both ofthese modifications would affect the water structure ofthe hydration sheaths of the surface, with an increase inthe clathrate nature of the sheath in the case of thehydrophobic copolymers and an increase in irrotationallybound water to the anionic and cationic moieties withionizable copolymer addition.

The differential response of the various cell types to thematrices in these experiments are an example of how thecombination of forces derived from matrix components iscrucial in understanding the behaviour of differentiatedcells. It is not surprising that the naturally occurringmatrices for corneal epithelial cells and fibroblast celllines have different compositions of extracellular proteinsand glycoproteins. However, as our data indicate, differ-ent cells are sensitive to the presence of other forces at thesurface. Corneal epithelial cells clearly prefer the hydro-gel surfaces in which cationic functional groups areadded, though the data also indicate that either ionizablegroup plus collagen-containing matrices is preferred overthe other surfaces tested. Since the likely effect of theaddition of cationic and anionic functional groups is areordering of the interphase region, it seems probablethat the different cells respond to the altered environmentwith modified spreading and proliferative activities. Aconsequence of this view is that both the differentiatingcell, and the cell involved in pathobiological processessuch as injury and wound healing, will respond toextracellular matrix signals arising not only from theidentity and conformation of the macromolecules atthe surface but also to the net forces experienced in theinterphase and to the potential reorganization of thewater, electrolytes and organic adsorbents in the regionduring growth and pathological events.

The use of synthetically formed defined surfaces isuseful in the study of cell-substratum interaction. Inves-tigating the interactions of cells with specific carbo-hydrate moieties and glycoproteins has been approachedthrough derivatized polyacrylamide gels (Schnaar et al.1978; Pless et al. 1983) and, recently, derivatized poly-acrylamide gels have been used to study the effects ofimmobilized carboxylic, hydroxyl and tertiary aminegroups on fibroblast cells (Brandley, 1987). The modifiedHEMA hydrogels we have described may be consideredas a model extracellular matrix that is composed ofcomplex sugars (hydroxyl groups), ionic groups (positivecharge from amino groups and negative charge fromsulphonated proteoglycans) and the proteins that make

up the extracellular matrix. One of the most excitingaspects of this system is that each component of thehydrogel can be varied, to change the functional groupsin the surface simultaneously. For example, hydroxylgroups can be varied by using a methylmethacrylatepolymer that is free of hydroxyl groups. The copolymeri-zation of MAA and NDAM can be mixed to give variousratios of positive and negative charges, to mimic thebiochemical components of the matrix, and finally vari-ous proteins can be embedded to examine their inter-relationship to the total picture. Because the model can becarefully defined in all aspects, these modified hydrogelswill be useful in studying the interaction between cellsand their substrata.

This work was supported by N1H grants HL-01759 (PRB),EY-06000 (VTR) and HL-19717 and HL-13262 (CF). Specialthanks to Valerie Verbitsky and Rosemarie Moscaritolo for theirexcellent technical assistance.

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(Received 13June 19SS -Accepted 13 October 19SS)

Hydrogels in cell-substratum interactions 121