Cationic polyelectrolyte hydrogel fosters fibroblast spreading, proliferation, and extracellular matrix production: Implications for tissue engineering

JOURNAL OF CELLULAR PHYSIOLOGY 198:133–143 (2004)

Cationic Polyelectrolyte Hydrogel Fosters FibroblastSpreading, Proliferation, and Extracellular MatrixProduction: Implications for Tissue Engineering

MARIO DE ROSA,1 MARIA CARTENI’,1 ORSOLINA PETILLO,2,3 ANNA CALARCO,3

SABRINA MARGARUCCI,2 FRANCESCO ROSSO,1 ALFREDO DE ROSA,1 ERNESTO FARINA,1

PASQUALE GRIPPO,1 AND GIANFRANCO PELUSO3*1Department of Experimental Medicine, II University of Naples,

School of Medicine, Naples, Italy2Institute of Protein Biochemistry, CNR, Naples, Italy

3Department of Experimental Oncology, National Cancer Institute,Naples, Italy

Fibrous encapsulation is known to occur tomany prosthetic implants and is thoughtto be due to the cells not adhering adequately to the surface. For developing newmaterials able to enhance cellular adhesion by mimicking extracellular matrixcomponents, polyelectrolyte polymers, characterized by tunable surface charges,have been proposed. Here we demonstrate that panoply of cell functions over atwo-dimensional substratum is influenced by surface charge. We have at firstgenerated structurally related polyelectrolyte substrata varying in their positivesurface charge amount and subsequently evaluated a variety of behaviors of humanprimary fibroblasts seeded on these polymers. The proportion of adherent, spread-ing, and proliferating cells was increased significantly on cationic hydrophilic sur-faces when compared with the neutral base surface. The extent of cell spreadingcorrelated with cytoskeleton organization as assessed using immunofluoresc-ence techniques. In the key experiment, the presence of cationic charges on celladhesion-resistant neutral surface increased the synthesis of collagen I and III, therelease of their metabolites, and the expression of their mRNA by fibroblasts.Interestingly, the scarce collagen deposits on neutral polymer consisted, for themost part, of collagen I while collagen III was present only in traces probably due tothe secretion of metalloproteinase-2 by non-adherent fibroblasts. Taken together,these results show that polyelectrolyte films may promote the attachment offibroblast cells as well as their normal secretory phenotype. Both effects could bepotentially useful in integrating soft connective tissue to the implant, decreasing thechance of its fibrous encapsulation. J. Cell. Physiol. 198: 133–143, 2004.� 2003 Wiley-Liss, Inc.

Cell spreading over a substratum plays a pivotal rolein many biological processes such as organogenesis,wound healing, and malignancy (Folch and Toner,2000). Examples are the interactions between cells andtheir extracellular matrix (ECM), which have beenshown to be the major determinants of cell shape(Goldmann, 2002). Many ECM components facilitateattachment, spreading, and formation of focal adhesionsboth by presenting multiple sites for cell interactionswith many different types of cell surface receptors, andby promoting clustering of integrins and perhaps of non-integrin adhesion receptors such as glycosaminoglycansreceptors as well (Bornstein and Sage, 2002). Under-standing the process of cell spreading is critically im-portant also for the emerging field of tissue engineeringand for the future use of biomaterial scaffolds for tissueor organ regeneration (Langer, 2000). Because spread-ing of cells over a synthetic material must come through

the interactions between cells and material surface, it isimportant to elucidate the mechanism(s) by which a cellcan bind to a foreign artificial substratum and, thus,activate a cascade of events leading to attachment andspreading (Anderson, 1988). Several studies have con-sidered the ability of various biocompatible polymersto encourage cell adhesion and spreading (Webb et al.,2000; Boraldi et al., 2003). The process is complex and

� 2003 WILEY-LISS, INC.

*Correspondence to: Gianfranco Peluso, INT-Naples, Via P.Castellino 111, 80131 Naples, Italy.E-mail: [email protected]

Received 17 March 2003; Accepted 9 June 2003

DOI: 10.1002/jcp.10397

involves the interaction of a number of factors includingsurface charge, wettability (hydrophilicity/hydrophobi-city), porosity, and roughness (Folch and Toner, 2000).There are several clinical evidences that the absence ofcell attachment can induce many biological adversereactions against prosthetic materials. For example,poly(hydroxyethyl methacrylate) polymer (pHEMA), anuncharged non-adhesive hydrogel, has been used in anumber of biomedical applications including the man-ufacture of soft contact lenses and intraocular lenses(Versura et al., 1999; Hoffman, 2002). Nevertheless, thisbioinert hydrogel would not appear to offer considerablepromise in down-regulating in vivo some of the deleter-ious biological reactions associated with conventionalpolymeric materials such as fibrosis, although fibroblastadhesion to pure pHEMA in vivo is limited and cells donot tend to spread or exhibit normal morphologicalfeatures (D’Hermies et al., 1998). Thus, zero reaction isoften an inappropriate biological reaction to implant-ed materials. This is particularly true in situationswhere good tissue integration or tissue regeneration isparamount.

Success in creating materials, which encourage celladhesion, tissue integration, and tissue regeneration,has been derived from mimicking the natural interfacebetween cells and extracellular matrix (Healy et al.,1999). Bioactive materials upregulate specific elementsof the biological response at the tissue/material interfacesuch as the presence of specific residues able to improvecell adhesion (Hubbell, 1999). Since hydrophilic materi-als, which incorporate positive charges tend to favorcell adhesion and spreading, we developed a new syn-thetic pHEMA, derived-polymer that presents posi-tively charged trimethyl ammonium residues on itssurface against a background inert to cell spreading. Tovary the cell–substratum adhesion, six structurallypHEMA-related copolymers differing in 2-methacryloy-loxyethyltrimethyl ammonium chloride (METAC) con-tent have been prepared. This novel approach addressesthe specific contributions of surface charge to the pro-cess of cell spreading, while eliminating other typesof receptor-mediated adhesive interactions. Further,given that both the concentration of positive charges aswell as its spatial arrangement, (on the material sur-face or in the bulk), potentially influence observed cellbehaviors, we extensively studied the new polymers toevaluate the average charge surface density in order toassess the relative contributions of trimethyl ammo-nium residues on cell spreading. Additionally, func-tional assays were carried out to monitor proliferationof fibroblasts on the charged and uncharged scaffolds.Microscopy analysis confirmed increased cell spreadingon charged versus uncharged polymers, spreading beingaccompanied by the development of pronounced actinstress fibers (Richards et al., 2001).

Finally, to study the influence of polymer surface-charge on the production and secretion of ECM proteins,collagen synthesis was examined during attachment ofhuman primary fibroblasts onto charged and uncharg-ed scaffolds. We analyzed the three major regulatorymechanisms that control collagen turnover, which are(1) synthesis, secretion, and deposition of collagens,(2) expression of matrix metalloproteinases (MMP), and(3) expression of tissue inhibitors of metalloprotein-

ases (TIMP). In this respect, fibroblasts cultured onuncharged pHEMA polymer, compared with those seed-ed on charged pHEMA-co-METAC copolymers, show adecrease in secretion and deposition of total collagens,an increase in the collagen I/collagen III ratio, an in-crease in pro-MMP2 concentration and active MMP2level, and a normal TIMP2 concentration. Thus, celladhesion to cationic polyelectrolytes triggers signalsthat regulate cell survival, cell cycle progression, ex-pression of tissue-specific phenotypes and secretion ofmolecules able, in turn, to maintain the normal ECMstructure and integrity.

MATERIALS AND METHODSChemicals

2-hydroxyethyl methacrylate (HEMA) and 2,20-azoi-sobutyronitrile (AIBN), were supplied by Fluka, Milan,Italy. METAC, sodium chloride, sodium citrate, mono-basic, dibasic sodium phosphate, and gluteraldehydewere purchased by Sigma-Aldrich, Milan, Italy. Petro-leum and diethyl ethers were supplied by Carlo Erba,Milan, Italy. Activated charcoal was supplied by ServaFeinbiochemical, Heidelberg, Germany. All reagents forcell cultures were obtained from Hyclone, Milan, Italy.Plastic tissue culture were from Falcon, Milan, Italy.[3H]-proline and [3H]-thymidine were bought fromAmersham, Milan, Italy.

Polymers synthesis and characterization

Commercial HEMA contains about 200 ppm ofhydroquinone monomethylether (IQ) as quencher and0.4% of ethylene glycol dimethacrylate (EGDMA, abifunctional agent) as contaminant. To eliminate theseimpurities, we carried out a purification step based onsilica gel adsorption chromatography (granulometry0.040–0.073 mm).

Purification of HEMA monomer was confirmed by GasChromatography, using a Hewlett-Packard instrumentMod. 5971 A, equipped with a HP-5 column and a UVdetector.

METAC was purified with activated charcoal, ac-cording to a previously described method (Salamoneet al., 1985).

Copolymers of HEMA and METAC monomers wereprepared by radical chain polymerization using differ-ent monomer molar ratio, and AIBN was used asinitiator at a concentration of 0.1% w/w respect to mono-mer mixture. Monomers were mixed together undermagnetic stirring before polymerization. The mixturewas degassed for about 15 min under nitrogen flow,loaded on sealed glass chambers equipped with a vul-canized silicon rubbers, and cured in a stove according tothe following thermal program: 2 h at 608C, 4 h at 708C,and 1 h at 858C (Odian, 1991). After the polymerization,the gels were washed extensively with sterile distilledwater. Differential scanning calorimetry (DSC) wasemployed to optimize the polymerization thermal con-ditions and to evaluate the Glass Transition of synthe-sized materials. The tests were carried out by using aDuPont calorimeter under nitrogen flow.

X-ray photoelectron spectrometer (XPS)

The surfaces of synthesized polymers were examinedwith an XPS (Physical Electronics Mod. 5500-PHI), in

134 DE ROSA ET AL.

which the takeoff angle of the photoelectron was 688(about 10 nm of width). The METAC mole fractions ofthe copolymers, was calculated as previously reported(Ishihara et al., 1999).

Specimens

Twelve skin biopsies were obtained from patients(seven men and five women; mean age, 31.9 years;range, 23–36 years) undergoing subcutaneous cystablation. Preoperative evaluation of these patientsshowed an absence of skin abnormalities. All patientswere informed and consented to the procedure.

Cell cultures

Explants of skins were prepared by carefully puttingthe epidermis at the top. Cells were grown into collagen-precoated petri dishes in Dulbecco’s modified Eaglemedium (DMEM) supplemented with 10% fetal calfserum (FCS), 10% horse serum, 2 mmol/L L-glutamine,105 U/L penicillin, and 100 mg/ml streptomycin at 378Cin a 95% air, 5% CO2 atmosphere. Cell growth beganwithin 3–5 days, and cells reached confluence after2 weeks. Cells were then trypsinized, seeded at a densityof 10,000 cells/cm2 (first passage), and subcultured to beused at passage 3 or 4 after 8–10 population doublings inthe same culture medium described above without horseserum.

Cytotoxicity assay

Detection of cytotoxicity effects was performed indir-ectly by quantification of mitochondrial dehydrogenaseactivity via the enzymatic conversion of MTT tetra-zolium (Sigma) to a colored formazan product. After thefibroblasts were cultured in 24-well plates for 2 days inthe presence or absence of the different polymers, thecell viability was evaluated using the MTT assay inwhich 100 ml of MTT (5 mg/ml) were added to each welland incubated at 378C for 4 h. At the end of the assay, theblue formazan reaction product was dissolved by adding0.2 ml of DMSO and transferred to a 96-well plate. Theabsorbance was measured at 570 nm using a Bio-Rad500 spectrophotometric microplate reader.

Cell adhesion and spreading assays

Sterile discs of pHEMA-co-METAC or pHEMA poly-mers were held on the bottom of multiwell dishes(96-well plate), and rinsed twice with phosphate buf-fered saline (PBS). The polymers were soaked overnightin growth medium without FCS, prior to cell seeding.Independent experiments were undertaken for eachtested material using the cell lines derived from primaryhuman fibroblasts. For the assessment of the cell adhe-sion, the different polymers placed in 96-well plateswere incubated in serum free culture medium sup-plemented with 2% (TCH) serum substitute (ICN,Milan, Italy). On each plate, five columns of wells werecoated with the different polymers, ranging from 0 (purepHEMA) to 20% METAC in 5% increments. The wells inthe sixth column were coated with polymers without celladdition, and wells in the seventh column were leftuntreated. A single-cell suspension was prepared bytrypsin/EDTA treatment. Wells were seeded in dupli-cate with 100 ml of a fibroblast cell suspension (7�104 cells/ml), and the plates were incubated under cell

culture conditions for different periods of time. At eachtime point, plates were removed, and each well was filledwith medium, sealed with adhesive film (LMT-Seal-Ex;Phoenix Research Products), and inverted for 5 min,permitting unattached cells to fall away. Sealing filmwas then removed, and the plates were blotted on papertowels to remove excess liquid. Plates were gentlywashed with medium, excess liquid was removed asdescribed above, and the adherent cells were fixed to thesubstratum with 5% formaldehyde in PBS. The cellswere then stained with an aqueous solution of crystalviolet (5 g/L) followed by elution of the dye with33% aqueous acetic acid. Absorbance at 570 nm wasdetermined with a Tecan Spectrafluor Plus microplatereader (Tecan Italia, Milan, Italy) and the number ofcells was determined from a standard curve of absor-bance against cell numbers calculated from a mean of sixexperiments.

The polymers incubated in parallel in culturemedium, but without cells, were stained, washed, andserved as background. The background was subtractedout for the determination of optical density.

Measurement of spread cell surface area

The cell spreading was evaluated at indicated inter-vals by phase-contrast microscopy. An individual cellwas counted as ‘‘spread’’ when diameter was minimallytwice that of the nucleus. The percentage of spread cellswas evaluated as (number of spread cells/total of num-ber cells in field)� 100¼percentage of spreading. Datafrom three to seven assays were analyzed for statisticalsignificance by analysis of variance (ANOVA), and areshown graphically as mean�SEM.

Cells plated on the different polymers, were photo-graphed at 120 min post-plating under a phase-contrastmicroscope with the use of an Olympus camera andKodak T-Max 400 film, at the same magnification for allcultures. Scanned negatives were digitized at identicalresolutions, and three random fields were selected foreach polymer containing minimally 200 cells/field. Meancell surface area was determined with the use of SigmaScan Pro image analysis software (Jandel Scientific,Erkrath, Germany), and normalized as percentage ofcontrol spread cell surface area.

Analysis of cytoskeleton

Cytoskeleton of the fibroblast cells was analyzed usingrodhamine-conjugated phalloidin to specifically stainfilamentous actin. Cells seeded on the two differentpolymers for 24 h were fixed with 3.5% formaldehydePBS for 10 min on ice, permeabilized with 0.1% TritonX-100 and incubated for 40 min with 0.1 mg/mlrodhamine-conjugated phalloidin (Sigma). After exten-sively washing with PBS, the slides were mounted using50% glycerol. Finally, cell cytoskeleton of at least200 cells was examined by a fluorescence microscopeand photographs were taken at �1,000 magnification.

Scanning electron microscopy (SEM) preparation

Cells, seeded onto different polymers, were fixed by2.5%gluteraldehydesolution incacodylatebuffer (0.1M,pH¼ 7.4) for 1 h at 48C. The samples were then rinsedthree times with the same buffer, slowly dehydrated

FIBROBLAST BEHAVIOUR ON CATIONIC HYDROGELS 135

using increasing concentration of ethanol, critical point-dried using CO2, mounted on aluminum specimen stubs,and coated with gold–palladium. Finally, a HitachiScanning Electron Microscope was used to view thesurfaces of the various samples.

Cell proliferation

For determination of cell proliferation, cell types weresubcultured at passage 3 at a density of 8,000 cells/cm2.The proliferation assay was measured by using [3H]-thymidine incorporation assay. Briefly, quiescent cellswere plated at 2�104 cells/ml in uncoated or polymercoated 24-well plates containing complete DMEM, andincubated at 378C for 48 h. At the last 4 h of incubation,cells were pulsed with [3H]-thymidine (0.2 mCi/ml) andharvested. DNA synthesis was measured by quantitat-ing [3H]-thymidine incorporation into trichloraceticacid-precipitable material.

Determination of collagen secretionand deposition onto the material surfaces

Collagen secretion and deposition onto the materialsurfaces as well as total secreted and total depositedproteins were assessed by proline incorporation assays(Peterkofsky and Diegelmann, 1971). Confluent, serum-deprived cultures of primary human fibroblasts wereseeded into 12-well plates coated with pHEMA or withpHEMA-co-METAC polymers (at the indicated concen-trations of METAC) in the presence of 0.5 mCi/ml of [3H]-proline and 10 mg/ml of ascorbic acid. Determination oftotal secreted proteins or collagens was performed inthe supernatants from fibroblasts collected after 40 hculture. Briefly, 400ml aliquots of supernatant from eachwell were incubated with 100 ml of collagenase assaybuffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, and2.5 mM N-ethylmaleimide) containing 30 U/ml of col-lagenase (from Clostridium histolyticum, Sigma) for 4 hat 378C. In parallel, a second 400 ml aliquot was incubat-ed in assay buffer without collagenase. Then, 50 ml ofFCS and 100ml of TCA were added to the samples, beforeincubating on ice for 30 min, to precipitate protein frac-tions. Precipitates were applied onto filter units (What-man, Milan, Italy) and washed three times with 2 ml ofTCA and two times with 2 ml of 80% ethanol. Each filterwas placed into 4 ml of liquid scintillation fluid, andradioactivity was determined in a scintillation counter.Amounts of total secreted proteins were calculated asdisintegrations per minute (dpm) in supernatants with-out collagenase. Secreted collagens were calculated asdisintegrations per minute (dpm) in supernatants with-out collagenase–dpm in supernatants with collagenaseas previously described (Peterkofsky and Diegelmann,1971). Determination of de novo deposition of total pro-teins and collagens on polymer surfaces was performedafter the culture supernatants were removed and thefibroblasts were lysed with 25 mM NH4OH for 10 min atroom temperature (RT). The polymers were ethanolfixed (70% ethanol, two times for 15 min at RT) andwashed twice with 50 mM Tris HCl, 1 mM CaCl2, and1 mM proline, pH 7.5, and then incubated for 4 h at 378Cin assay buffer either with or without collagenase asdescribed above. The supernatants were removed after4 h and residual proteins were solubilized by overnightincubation in 0.3 M NaOH–1% SDS. Equal aliquots of

supernatants and solubilized residual proteins weresubjected to liquid scintillation counting. Calculationswere made following a previously described method(Peterkofsky and Diegelmann, 1971; Agelli and Wahl,1988). Radiolabeled collagen chains were resolved bySDS–PAGE as previously described (Sansilvestri-Morel et al., 2001). The bands corresponding to 1(V),1(Iþ III), and 2(I) labeled chains were cut off, and radio-activity was counted. Because the number of cells cul-tured on charged and uncharged polymers could varyafter 40 h culture and, thus, affect the total amount ofprotein and collagen production, all the data werenormalized to the cell number seen in parallel culturesat the same period of time.

RNA isolation and northern blot analysis

Fibroblasts were grown at confluence and lysed in4 mol/L guanidinium isothiocyanate, 0.97% 2-mercap-toethanol, 2% SDS, and 0.01 mol/L Tris-HCl at pH 7.5.Total RNA was then isolated by the SV total RNAIsolation System (Promega, Milan, Italy). For Northernblot analysis, denatured RNAs (15mg per lane) were sub-mitted to electrophoresis through denatured agarosegels and then transferred to membranes. The mem-branes were prehybridized, hybridized, and then wash-ed. Autoradiographic bands were quantified by a gelanalysis software (Quantity One, Bio-Rad, Milan, Italy).The probes included a 1.8 kb EcoRI fragment of human1(I) collagen cDNA (ATCC), a 1.3 kb EcoRI fragment ofhuman 1(III) collagen cDNA (ATCC), and a 2 kb fullhuman b-actin cDNA (Clontech, BD Biosciences, Milan,Italy). The results are expressed as relative height of thepeak between collagen I or III and b-actin mRNA bands.

Telopeptides and propeptidesof collagen I and III

Fibroblasts seeded on the different substrates wereincubated for 40 h in DMEM supplemented with 2 mmol/L L-glutamine, 105 U/L penicillin, 100 mg/ml streptomy-cin, and 50 mg/ml of L-ascorbic acid. Culture media werethen collected. The metabolites of collagen I and III weremeasured using commercially available radioimmu-noassays (Orion Diagnostica, Dasit, Milan, Italy). Forthe normalization of the results see the ‘‘determinationof collagen secretion and deposition onto the materialsurfaces’’ section.

MMPs and tissue inhibitorsof metalloproteinases

Fibroblasts seeded on the different substrates wereincubated for 40 h in DMEM supplemented with 2 mmol/L L-glutamine, 105 U/L penicillin, 100 mg/ml streptomy-cin, and 50 mg/ml L-ascorbic acid. Culture media werethen collected. Pro-MMP1, pro-MMP2, pro-MMP7, pro-MMP8, pro-MMP9, and pro-MMP13 and their inhi-bitors in free or complexed form (tissue inhibitor ofmetalloproteinase (TIMP)1, MMPx-TIMP1, TIMP2,and MMPx-TIMP2) were quantified in supernatantsusing commercially available enzyme immunoassayskits (Amersham Pharmacia Biotech). The active form ofMMP2 was quantified in culture media using the MMP2activity assay system kit (Amersham Pharmacia Bio-tech). Because the number of cells cultured on chargedand uncharged polymers could vary after 40 h culture

136 DE ROSA ET AL.

and, thus, affect the amount of MMPs and TIMPssecreted, all the data were normalized to the cell numberpresent in the cultures.

Statistical analysis

Data are presented as mean�SEM. Student’s t-testfor unpaired observations was used to compare resultsobtained in the cultures of cells seeded on the neutral orcharged polymer. Statistical significance was assumedat P<0.05.

RESULTSPolymer synthesis and characterization

The synthesis of pHEMA and pHEMA-co-METACcopolymers was performed according to the literature(Salamone et al., 1985).

The thermal program employed for copolymer synth-esis was the best between tried ones. Indeed, in the firstphase of the polymerization, performed at low tempera-ture, the system stabilized, freeing the mixture from thenitrogen molecules produced by AIBN homolysis, thus,allowing to obtain more homogeneous polymers. Theglass transition (Tg) of pHEMA and pHEMA-co-METACwere showed in Table 1. A significant Tg variationwas demonstrated between pHEMA and pHEMA-co-METAC polymers.

A high-resolution surface analysis of the pHEMA-co-METAC copolymers was performed by XPS to detect thechemical composition of the material surface. The analy-sis carried out on the different spectra showed thatMETAC mole fraction present on the surface of thedifferent copolymers was a function of METAC repeatunit in the feed composition (Table 1).

Cell spreading kinetics and morphology

All the materials used were characterized by anelevated biocompatibility. Cell viability, as determinedby MTT assay, was not affected by incubation on thedifferent polymers.

In the pHEMA-co-METAC polymers, METAC incor-poration resulted in a change from a minimally adhesive(METAC content¼0 mol%) to a highly adhesive sub-stratum (METAC content¼10 mol%). Cell attachmentto the polymers was monitored by two criteria: the rateof cell attachment and the degree of cell spreading overtime. Rate of attachment of fibroblast cells markedlydeclined with decreasing METAC content of the polymersubstrata. This was evident at all time points examinedand it is illustrated in Figure 1. Fibroblasts seeded on

the pHEMA substratum tended to aggregate ratherthan spread and in all cases were easily removed upongentle washing of the plate. Optical microscopy andSEM analysis, demonstrated that cells cultured oncharged and uncharged polymers may differ in theirculture morphology, adopting a rounder shape onpHEMA substratum (Fig. 2a,b) than on pHEMA-co-METAC polymers (Fig. 2d,e).

The percentage of fibroblasts that spread versus timewas determined on pHEMA-co-METAC plated cells, andcompared with cells seeded on pHEMA, which is notpermissive to spreading. Under the conditions usedherein, cells attached to pHEMA-co-METAC 10% poly-mers within 5 min of plating but remained rounded,although they exhibited distinct refractive changes in-dicative of initiation of early spreading. Partial spread-ing of 20–25% of the plated cells was observed at�15 min; full spreading of 90–95% of the cell populationwas seen at �60 min and was characterized by a flat-tened ‘‘fried-egg’’ appearance. Fibroblast cells assumeda typically fibroblast pyramidal shape, which was asso-ciated with onset of migration �2 h after plating, withwell formed stress fibers (Fig. 2d–f). Cells plated on thepHEMA substrate remained rounded, did not spreadover the time of the assay, and did not show well-formedstress fibers (Fig. 2a–c). Decreasing METAC content ofthe polymer substrata as depicted after 2 h in Figure 3also markedly reduced cell population spreading. Thereduction in adhesion was most marked in assays, asMETAC content was lower than 10% (Fig. 3). ThepHEMA-plated cells remained viable and spread if col-lected and replated on tissue culture polystyrene (TCP).

To determine whether the effects of METAC contentupon cell attachment and spreading are due to directcell–polymer interactions or are mediated by compo-nents deposited upon the polymer from the serum in theculture medium, fibroblast cell suspensions were cul-tured for 24 h on pHEMA-co-METAC 10% in mediumsupplemented with either FCS or TCH, a commercialserum substitute. In the presence of serum, cells attach-ed completely in 24 h. Again, in the absence of serum, celladhesion occurred on this most-preferred substratumsurface at a degree slightly lower than in presence ofserum.

Cell proliferation

To determine whether the different polymers wereable to modulate cell-proliferation in anchorage-depen-dent fibroblasts, we subjected cells, seeded on pHEMA

TABLE 1. Surface characteristics and glass transition of the synthesized materials

Polymers

METAC mole fraction

Glass transition (Tg8C)In monomer mixtures At surface

pHEMA 0.000 0.000 87.5�0.8pHEMA-co-METAC 5% 0.050 0.136 104�1.0*pHEMA-co-METAC 10 % 0.100 0.283 113�1.1*pHEMA-co-METAC 15 % 0.150 0.315 115�1.0*pHEMA-co-METAC 20 % 0.200 0.321 116�1.1*

The surfaces of synthesized polymers were examined with an X-ray photoelectron spectrometer. Differential scanningcalorimetry was employed to optimize the polymerization thermal conditions and to evaluate the glass transition ofsynthesized materials (n¼ 3). Data are expressed as mean�SD.*P< 0.001 poly(hydroxyethyl methacrylate) (pHEMA) versus pHEMA-co-methacryloyloxyethyltrimethyl ammoniumchloride (METAC).

FIBROBLAST BEHAVIOUR ON CATIONIC HYDROGELS 137

or on pHEMA-co-METAC polymers, to DNA synthesisassays. As Figure 4 shows, cultivation of cells onpHEMA-co-METAC induced their growth in a waydependent by the amount of trimethyl ammonium pre-sent on material surface. The growth response in cellscultured on pHEMA hydrogel was practically abolished.Notably, the proliferation response in cells cultured onpHEMA-co-METAC with a low METAC concentra-tion (5%) was not strongly increased, while culture ofcells on copolymers with concentrations of METAChigher than 10% did not augment the growth stimula-tion obtained by using METAC 10%.

Thus, pHEMA-co-METAC caused not only profoundmorphological changes on cells consistent with their

Fig. 1. Fibroblast adhesion on charged and uncharged polymers.Human fibroblasts were seeded on poly(hydroxyethyl methacrylate)polymer (pHEMA) and on pHEMA-METAC hydrogels and allowed togrowth for different periods of time. At the end of the incubation time,cell numbers were measured using the crystal violet technique. Valuesare mean�SEM from three independent experiments with one cellline. Similar results were obtained using the different cell lines ofprimary human fibroblasts. *Significant difference for cultures onpHEMA versus cultures on pHEMA-co-METAC or on tissue culturepolystyrene (TCP), *P< 0.001; **P< 0.05.

Fig. 2. Charge surface polymer affects cell spreading and cytoske-leton organization. Cell attachment and spreading are greatly reducedfor fibroblasts cultured on pHEMA polymer (a, b) versus pHEMA-co-METAC 10% polymer (d, e). Actin stress fibers were visualized atvarying polymer charge surface densities. Shown here are typicalstains from cells plated on pHEMA (c) and on pHEMA-co-METAC10% (f) hydrogels.

Fig. 3. Morphology of fibroblasts seeded on charged and unchargedsubstrata. Cells were allowed to adhere to pHEMA and to pHEMA-co-METAC polymers. After 120 min, non adherent cells were washed offwith phosphate buffered saline (PBS) and attached and spread (solidbars) versus non spread cells (open bars) were counted by imageanalysis. Values are mean�SEM from three independent experi-ments with one cell line. Similar results were obtained using thedifferent cell lines of primary human fibroblasts. *Significant differ-ence for cultures on pHEMA versus cultures on pHEMA-co-METAC10% or on TCP, *P< 0.001.

Fig. 4. Graphical representation of the [3H]-thymidine incorporationby six fibroblast cell lines grown on charged and uncharged hydrogels.Radioactivity counted (CPM) in each well after 72 h is taken as anindex of proliferation; the values are expressed as percentage of pro-liferation with respect to the cell seeded on TCP. Values are mean�SEM from six independent experiments.

138 DE ROSA ET AL.

spreading, but also promoted cell proliferation. The lasteffect was much more robust at those concentrationsof pHEMA-co-METAC able to induce the highest cellattachment, suggesting that the fibroblast proliferationwas dramatically modulated by cell shape through themagnitude or the duration of anchorage dependentsignaling mediators.

Proteins and collagens secreted by cellscultured on different substrates

All experiments analyzing the impact of differentsubstrates on protein and collagen secretion by [3H]-proline incorporation were done on cultures of primaryhuman fibroblasts. As depicted in Figure 5A,B, the in-crease of METAC concentration in the polymers inducedin the fibroblasts an increase of total secreted proteins(Fig. 5A) and secreted collagens (Fig. 5B), in a dose-dependent manner over a METAC concentration rangefrom 5 to 10%. After 40 h, the fibroblasts cultured onpHEMA-co-METAC 10% hydrogels had significantlyincreased total secreted proteins compared with those

cultured on pure pHEMA, (17,700� 500 vs. 7,000�800 dpm; P<0.005), while no effect was seen whenMETAC was used at concentrations lower than 10%(Fig. 5A).

Figure 5B depicts the effects of different artificialsubstrates on secreted collagens as assayed by collage-nase digestion of cell supernatants. Charged polymersinduced an increased secretion of collagens by humanfibroblasts in comparison with pure pHEMA (from1,050� 300 to 5,900� 600 dpm; P< 0.005).

Total proteins and collagens depositedby cells on different substrates

Secreted ECM molecules such as collagens are eitherrapidly degraded or deposited and cross-linked intoexisting ECM. We assessed the actual deposition of totalproteins and collagens on polymer substrata by analyz-ing protein deposit on charged and uncharged polymersafter selective removal of culture supernatants andcell layers. The cell response to pHEMA and pHEMA-co-METAC substrates, in terms of deposition of totalproteins, is similar to the cell response observed forsecretion of total proteins. Deposited total proteinaceousECM increased in cells cultured on pHEMA-co-METACcompared with that in cells cultured on pHEMA(15,900�600 vs. 3,500� 200 dpm; P< 0.005) (Fig. 5A).

Again, we analyzed deposition of collagens within thetotal protein deposit by collagenase digestion of ECM.Using this method, we demonstrated that collagendeposition increased in presence of pHEMA-co-METACsubstrate (4,400�100 vs. 590�70 dpm; P<0.001)(Fig. 5B).

Synthesis of collagen I, III, and V

Although fibroblasts cultured on pure pHEMA secret-ed a lower amount of collagen than fibroblast cultureson 10% pHEMA-co-METAC, they synthesized, in per-centage, significantly less collagen III but more col-lagen I than fibroblasts seeded on pHEMA-co-METAC10% polymer (Fig. 6). These decreased amounts of col-lagen III were found both in the culture medium and cell

Fig. 5. A: Effect of charged and uncharged substrata on total secret-ed (open bars) or deposited proteins (solid bars). Human fibroblastswere cultured on pHEMA and on pHEMA-co-METAC polymers inpresence of 0.5 mCi/ml of [3H]-proline and protein secretion in culturesupernatants, or protein deposition on material surfaces was assessedafter 40 h culture. Values (dpm, disintegrations per minute) aremean�SEM from three independent experiments with one cell line.Similar results were obtained in the different cell lines of primaryhuman fibroblasts. *Significant difference for cultures on pHEMAversus cultures on pHEMA-co-METAC, *P<0.005; B: Effect ofcharged and uncharged substrata on secreted (open bars) or depositedcollagens (solid bars). Human fibroblasts were cultured on pHEMAand on pHEMA-co-METAC polymers in presence of 0.5 mCi/ml of [3H]-proline and collagen secretion in culture supernatants, or collagendeposition on material surfaces was assessed after 40 h culture.Values (dpm), are mean�SEM from three independent experimentswith one cell line. Similar results were obtained in the different celllines of primary human fibroblasts. *Significant difference for cultureson pHEMA versus cultures on pHEMA-co-METAC, *P< 0.005.

Fig. 6. Differences between types of collagen product by humanfibroblasts cultured on charged and uncharged polymers. Fibroblastsseeded on pHEMA (solid bars) and on pHEMA-co-METAC 10% (openbars) were incubated in the presence of [3H]-proline. Radiolabeledcollagen I, III, and V were resolved by SDS–PAGE. The relativeproportions of radioactivity recovered in each collagen were deter-mined. The total content of collagen (Iþ IIIþV) is 100%. Values aremean�SEM from six independent experiments with one cell line.Similar results were obtained in the different cell lines of primaryhuman fibroblasts. *P<0.01, **P< 0.001 for cells cultured on pHEMAversus cells cultured on pHEMA-co-METAC 10%.

FIBROBLAST BEHAVIOUR ON CATIONIC HYDROGELS 139

layer extract; no significant difference was observed inthe synthesis of collagen V.

Northern blot experiments were performed to evalu-ate the expression of collagen I and III mRNA in fibro-blasts cultured on charged and uncharged polymers.The increase or the decrease in collagen I synthesis byfibroblast cells was correlated with the expression ofthe mRNA of collagen I (Fig. 7). On the contrary, a com-parable expression of collagen III mRNA was found infibroblasts cultured either on pHEMA or on pHEMA-co-METAC substrates.

To estimate more in detail the level of secretedcollagen I and III in the culture media, the N- andC-terminal propeptides of procollagen I, as well as theN-terminal propeptides of procollagen III were quanti-fied. The augmented level of collagen I synthesis wasconfirmed by a significant increase of collagen I meta-bolites in the culture media of fibroblasts seeded onpHEMA-co-METAC, and, accordingly, the decreasedproduction of collagen I in the culture media of fibro-blasts seeded on pHEMA was related to a decrease ofcollagen I metabolites. The level of amino-terminalpropeptides of procollagen III was similar in all thefibroblast cultures (data not shown).

Production of MMPs and TIMPs

Quantification of pro-MMP1, pro-MMP2, pro-MMP7,pro-MMP8, pro-MMP9, pro-MMP13, TIMP1, TIMP2,and the complex MMP1/TIMP1 was performed in thesupernatants of fibroblasts cultures. The concentrations

of pro-MMP7, pro-MMP8, pro-MMP9, and pro-MMP13were lower than the cut off value of the assays forall the cultures, but pro-MMP1, pro-MMP2, TIMP1,TIMP2, and the complex MMP1/TIMP1 were detectablein all culture supernatants. No significant difference inthe concentrations of pro-MMP1, TIMP1, TIMP2, andthe complex MMP1/TIMP1 was observed between cellsseeded on charged and uncharged polymers, whereas asignificant increase of pro-MMP2 was detected in fibro-blasts cultured on pHEMA (Table 2). Thus, in presenceof pHEMA the level of pro-MMP2 was higher than theconcentration of its major inhibitor, TIMP2. Quanti-fication of the active form of MMP2 was thereforeperformed, and a significant difference was observedbetween cells growth on pHEMA and on pHEMA-co-METAC 10% substrates (9.78�1.17 and 3.85�1.09 ng/ml, respectively).

DISCUSSION

Surgical strategies that have evolved to deal withtissue loss include organ transplantation from one indi-vidual to another, tissue transfer from a healthy site toaffected site in the same individual, and replacement oftissue function with mechanical devices such as pros-thetic valves (Papadaki et al., 2001). Tissue engineeringseeks to provide a new solution to tissue loss that is,replacement or restoration of tissue or organ functionwith constructs that contain specific population ofliving cells (Ochoa and Vacanti, 2002). Indeed, althoughsome techniques of guided tissue regeneration rely onmatrices alone, and other approaches rely on cellsalone, most investigators in tissue engineering use cellscombined with matrices to achieve new tissue forma-tion (Hoffman, 2002). In this system, cells are seededonto synthetic polymer matrices, proliferated/expandedin vitro, and then implanted. The polymer serves as ascaffold or a template to guide cell organization andgrowth and allows diffusion of nutrients to the trans-planted cells. Because adhesion of cells over a syntheticmaterial must come through the interactions betweencells and material surface, it is important to facilitatethe mechanism(s) by which a cell can bind to a foreignartificial substratum and, thus, activate a cascade ofevents leading to attachment and spreading (Brunetteand Chehroudi, 1999). Ideally, materials used as scaf-folds must possess some biological properties able toenhance cell adhesion by mimicking extracellularmatrix components. (Dillow and Tirrell, 1998; Healyet al., 1999).

The most important step toward the control of celladhesion, and spreading concerns, therefore, the im-

Fig. 7. Collagen I and collagen III mRNA expression in humanfibroblasts cultured on charged and uncharged substrata. Collagen Ia1 mRNA (open bars) and collagen III a1 mRNA (solid bars) expres-sion were quantified. Total RNA of fibroblasts cultured on pHEMA orpHEMA-co-METAC were extracted and hybridized to human collagenI, III, and b-actin probes. All results were normalized to b-actin andexpressed as relative height of the peak of mRNA bands. Values arethe mean�SEM of six independent experiments.*P<0.005 forpHEMA versus pHEMA-co-METAC.

TABLE 2. Analysis of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in culture media of fibroblastsgrowth on pHEMA (A), pHEMA-co-METAC 10% (B), or tissue culture polystyrene (TCP) (C)

Polymers(ng/ml) Pro-MMP1 Pro-MMP2 Pro-MMP7 Pro-MMP8 Pro-MMP9 Pro-MMP13

Pro-MMP1/TIMP1 TIMP1 TIMP2

A 8.3�0.9 8,173�921* ND ND ND ND 11.2� 2.7 1,492� 294 25.6�4.5B 8.5�1.9 4,327�503 ND ND ND ND 12.5� 1.9 1,383� 385 24.0�4.3C 8.7�1.3 4,514�634 ND ND ND ND 13.4� 1.8 1,470� 349 27.5�3.9

Fibroblasts were cultured and supernatants were collected. Production of MMPs and TIMPs was quantified by immunoassay.Data are expressed as mean�SEM; ND, non detectable.*P< 0.05 for A versus B and C.

140 DE ROSA ET AL.

provement at the nanometer to micrometer scales ofsome specific material surface properties (Angelova andHunkeler, 1999). It has been shown that different para-meters such as hydrophobicity and hydrophilicity(Sagvolden et al., 1999), surface charge (Qiu et al.,1998), roughness (Dufrene et al., 1999), and free energycan affect cellular adhesion (Kapur and Rudolph, 1998).The definition of general rules is not straightforward,and the observed cellular behaviors probably dependnot only on a single parameter but also on a complexcombination of different factors.

Recently, a new type of tunable surface biomaterialhas been proposed (Decher, 1997). It consists ofpolyanions and/or polycations polyelectrolyte films thathave shown several homologies with charged ECMmacromolecules such as complex carbohydrates (Picartet al., 2001). The natural polyelectrolytes are able tointeract with the lipid bilayer portion of cellular mem-branes, these electrostatic interactions seem to beintegral to the biological functioning of charged macro-molecules. As a consequence, it has been hypothesizedsimilar cell-interacting and biological properties also forartificial polyelectrolytes (Cevc, 1990; Serizawa et al.,2000; Berth et al., 2002). After these preliminaryremarks it could assume that the interactions of cellswith synthetic polyelectrolytes have been extensivelystudied. On the contrary, the influence of the polymersurface charges on cell adhesion and on the biologicalfunctions of attached cells has not been rigorouslyinvestigated. At the cellular scale, only a few studiesare concerned with the direct evaluation of cellularinteractions with synthetic polyelectrolytes, althoughthey have already widely been used to render anartificial substrate either adhesive, as for poly(L-lysine),or nonadhesive, as for polyacrylyc acid coupled to poly(ethylene glycol) (Amirpour et al., 2001). Elbert et al.(1999) used alginate polyelectrolyte films in order tobuild a nonadhesive barrier. More recently, it wasshown that cells can be deposited on a poly(L-lysine)/poly-(L-glutamic acid) substrate and grown for severaldays in culture in a serum-free medium.

Here we demonstrate that panoply of cell func-tions over a two-dimensional substratum is influenc-ed by surface charge. We have at first generatedstructurally related copolymeric polyelectrolyte sub-strata varying in their positive surface charge andsubsequently evaluated a variety of behaviors, such asattachment, spreading, proliferation and differentia-tion, of primary dermal fibroblasts seeded on thesepolymers. Cell–substratum adhesion as well as cellproliferation was increased by the METAC content onthe polymer surface. Enhanced cell spreading andincreased actin stress fiber organization was observ-ed on positive charged surface in comparison withneutral surface. Since preferential attachment andgrowth of cells has been demonstrated on hydrophilicsurfaces, it might possible speculate that hydro-philicity, rather than the polyelectrolyte nature ofthe pHEMA-co-METAC substrates, is the cause of theselective cell binding to charged surfaces. In the presentstudy, the cellular responses of primary human fibro-blasts did not display a consistent trend of attachment inrelation to surface wettability, but rather varied as afunction of METAC residues present on polymer surface.

To examine other relationship between material sur-face chemistry and cell behaviors, we extended ourstudies to evaluate the production of collagens as well asthe secretion of metalloproteinases by fibroblasts seededon charged and uncharged polymers.

Indeed, achieving methods to enhance the integra-tion of a biomaterial in a tissue site of implantation,essential to promote the functional longevity of im-planted medical devices, pass from the induction of thecell–material interactions to the promotion of a normalsecretion of extracellular matrix components, such ascollagens, by biomaterial-adherent fibroblasts. Addi-tionally, other adherent fibroblast products such asmetalloproteinases play key roles in guiding the re-sponse to implanted materials by orchestrating thedegradation of the extracellular matrix at the site ofimplantation.

A previous study in our laboratory observed a signi-ficant decrease of collagen I synthesis in a model ofanchorage-dependent cells cultured on pHEMA (Pelusoet al., 1997). In the present study, we have analyzedwhether fibroblasts adherent to the hydrophilic andcationic surfaces of pHEMA-co-METAC substrates,which experience high substrate-mediated cell spread-ing, were able to produce selectively collagens, such ascollagen I and III, in comparison with cells seeded on theinert neutral pHEMA.

Because collagen I confers rigidity whereas collagenIII is involved in the extensibility of a tissue, (Parry,1988) modification of the collagen I/III ratio might con-tribute to the weakness and the decreased elasticity of atissue. For example, it has been described that anabnormal collagen I/III ratio in varicose veins leads todiminished tone and fibrosis (Sansilvestri-Morel et al.,2001). For this reason, quantification of collagen I, III,and V synthesis was performed in fibroblasts culturedon pHEMA or on pHEMA-co-METAC polymers. In addi-tion to the significant decrease in collagen production,an imbalance between collagen I and III synthesis wasobserved in fibroblasts seeded on pHEMA substrate.Moreover, whereas collagen I and its metabolites weredown produced and mRNA was down expressed in fibro-blasts obtained from cultures on pHEMA, in the samecells the decreased collagen III synthesis did not cor-relate with a reduction of both its metabolites andmRNA expression.

The reduction of collagen III secretion that wasobserved in cultures on pHEMA may be related, at leastin part, to an enzymatic degradation of the protein inthe extracellular compartment. Indeed, the content ofcollagen III could be decreased in cell layers and themedia of cells cultured on pHEMA by a metalloprotei-nase-induced degradation process. The metalloprotei-nases are particularly important as they are widelyexpressed and can degrade a wide spectrum of ECMproteins.

The production of pro-MMP1 and the inhibitorsTIMP1 and TIMP2 was similar in all fibroblast cultures,whereas the concentrations of pro-MMP7, pro-MMP8,pro-MMP9, and pro-MMP13 were under the thresholdfor detection with the assay used, so that it is difficultto make conclusions about their possible implications.However, the concentration of pro-MMP2 and of theactive form of MMP2 was increased in fibroblasts

FIBROBLAST BEHAVIOUR ON CATIONIC HYDROGELS 141

cultured on pHEMA, if compared with cells seeded onpHEMA-co-METAC. The overproduction of pro-MMP2associated with an increase of the ratio between pro-MMP2 and TIMP2, which results in an increase inMMP2 levels in the fibroblasts cultured on pHEMAcould have consequences on matrix remodeling. Metal-loproteinase-2 (MMP-2) (gelatinase A or 72 kDa type IVcollagenase) can degrade several ECM components,including collagen IV, laminin, and fibronectin and isimportant in remodeling the matrix. Fibrillar col-lagens, and particularly collagen III, are substrates forMMP2 (Benyon et al., 1999; Berton et al., 2000), and,thus, the higher MMP2 activity could be implicated inthe degradation of collagen III in cells growth onpHEMA.

The increased MMP2 content in fibroblasts may haveother consequences in vivo as factor responsible for theaugmentation of fibrogenesis. MMP-2 is secreted fromthe cell as a zymogen, designated pro-MMP-2, which isrecruited to the cell surface by interacting with tissueinhibitor of metalloproteinases-2 (TIMP-2). A number ofstudies suggest that MMP-2 contributes to the develop-ment of fibrosis in various organs. Increased expressionof this protein has been detected in fibrotic lung, liver,and kidney (Kanwar et al., 1999) and in mutant micethat over-express MMP-2 there is spontaneous develop-ment of glomerulonephritis (Schaefer et al., 1996). Arecent study indicated that MMPs might contributeto fibrosis in vivo by showing that a MMP inhibitorattenuated anti-Thy1.1-induced nephritis and glomer-ular matrix accumulation (Steinmann-Niggli et al.,1998). Collectively, these observations support theconclusion from our studies that MMP-2 expression byfibroblasts cultured on bioinert materials is both afundamental cofactor in inhibiting the attachment,spreading and matrix deposition of fibroblasts in vitro,and an important contributor to fibrosis in the site ofimplantation in vivo. The last hypothesis may explainthe fibrotic reaction against a polymer generally con-sidered inert and well tolerated as in case of pHEMA.

In addition to anchoring, cell adhesion to pHEMA-co-METAC substrates triggers signals that regulate cellsurvival, cell cycle progression, expression of tissue-specific phenotypes, and secretion of molecules able, inturn, to modulate the same ECM structure and inte-grity. On the microscopic level, it is known that poly-electrolyte coupling to plasmamembrane lipid bilayerscan induce a lateral redistribution of oppositely chargedlipids into enriched regions known as domains. Thecreation of domains is capable of influencing cellularmembrane functioning, as reviewed in several recentarticles (Welti and Glaser, 1994; Macdonald et al., 2000).They may, for example, display altered local transbi-layer permeability, thereby modifying one of the mostimportant of membrane functions. Finally, they may actto sequester enzyme substrates or activator/inhibitors,thereby controlling enzymatic activity and/or cell sig-naling processes.

Taken together, these results show that polyelectro-lyte films may promote the attachment of fibroblast cellsas well as their normal secretory phenotype. Both effectscould be potentially useful in integrating soft connectivetissue to the implant, decreasing the chance of its fibrousencapsulation.

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