Long-Term Culture of Fibr oblasts in Contracted Collagen Gels: Effects on Cell Growth and Biosynthetic Activity

Shigenori Nakagawa, M.D., Ph.D., Pamela Pawelek, B.S., and Frederick Grinnell, Ph.D. Departme.nt of Cdl Biology and Anatomy. UT Southwestern Medio.l Ce nter. D.20112.s, Te~as. U.S.A.

The purpose of these studies was to walyze the consequences of long-term collagen gel contraction on fibroblast growth and metabolic activity. After 4 weeks, floating gcls were 98% contracted, and attached gels were 94% contracted. During this culture period, fibroblasts in floating gels regressed sig­nificantly compared to fibroblasts in attached gels, although the cells remaining in the floa ting gels were viable. In at­tached gels, fibroblasts were bipolar; whereas in floating gels, fibroblasts wefe stellate. Therefore, differences between sur­vival of fibroblasts in attached and floating collagen gels might depend on cell shape. Similarly, extracellular matrix organization and its influence on cell shape might control

During connective tissue repair, 6brobl,asts exh.ibil several diffcrenr activities. At first. they migrate from adjacem [Jssues intO the wound region. In the wound regIOn, they prolife.rate and synthesize a col· lagen-rich exuaceUuIar matrix that fitls the wound

defect. The fibroblasts also contribute ro the remodeling of newly synthesized extracellular matrix.. Finally, the expanded fibroblast population regresses (1 ,2J. Depending on the siloe and location of the wound, contraction of granulation tissue plays a role in wound healing 11,2J. A subpopulation of wound fibroblasts cailed myofi­broblasts may participate in the wound contraction phenomenon [3,4J

To learn more about the regulation of fibroblast functlon during wound repair, we focused our studies on fibroblasts cuJrured in collagen gels \5]. This modd is of particular interest because tht cdls rapidly reorganize and contract the collagen gels 16- 8), analo­gous to wound contraction in vivo [9,10]. Reorganization of coUa· gen gels by fibroblasts requ.ires celJ motile forces f11] propagated throughout the interconnected collagen fibril network. Disruption of the interconnected nerwork inhibits contraction (12J. During the initial phase of contraction, cells mechanically hold the reorganized collagen fibrils in place. Later, incerfibrillar collagen bonds stabilize the reorganized collagen fibrils without cells (13).

Substantial differences in collagen gel contraction by fibroblasts occur , depending on geometrical constraints. Contraction of gels

M.anuscript received J anu:ary 18. 1989: accepted for publ ication July 7. 1989.

Reptint requests to: Frederick Grinnell. Ph.D. , Dep<l.rtJT,ltnt of eel! Bioi· ogy :md Anatomy. UT Southwestern Med.ical Center. Dall:as. T eX3s 75235.

Studies supported by::l contract from the Kendall Wound Care Co. and by NIH gr2nt GMJ1321.

Abbreviation: FRS: FeuJ bovine serum

fibroblast proliferation in granulation tissue. During long­term culture of fibroblasts in contracted collagen gels, 70%-80% of the starting collagen was degraded. Collagen synthe­sized by cells in 4-d cultures was mostly procollagen secreted into the medium. On the other hand. collagen synthesized in 4-week cultures was processed to a(l ) chains and incorpo­rated into the matrix. There also were other differences be­tween the proteins synthesized by fibroblasts after shon-term and long-term culture in contracted gels. These findings show that fibroblasts in long-term coll agen gel cultures ex­press unique growth a.nd biosynthetic characteristics. ) !rlllest DermatoI93:792-798, 1989

attached to underlying culture dishes results in decreased gel thick­ness withour a change in gel diameter [7,14]' Contraction of Aoar­iug gels results in decreased gel thickness and diameter [6]. During contraction, collagen fibrils in attached collagen gels become al igned in the plane of cell spreadi.:n.g, and fibroblasts spread with a bipolar morphology. In floating collagen gels, on the other h3nd, collagen fibrils remain unorgani2cd, and fibroblasts spread with a stellate morphology !I5]. DNA synthesis by cells in Aoating gels decreases significantly after gel contract ion 116,17}, unlike DNA synthesis in contracted, attached collagen gels [1 5J. T herefore, in collagen gels comracted for sbort periods (1- 4 d), ex tracellular matrix organization and cell shape regulate fibroblast growth rather than gel contraction per se.

As described above, expression of different fibroblast funcrions during woond repair usually occurs duri.ng a period of weeks rather than days [1,2]. Therefore, we investigated the long-rerm. in vitro consequences of collagen gel contraction by fibroblasts. We found rhat during 4 weeks in contracted. floating collagen gels, the nbro· blast population regressed 90%. In parallel experiments, the fibro­blast populat ion in conrracted. attached collagen gels expanded. Severalacrivi ties nor usually observed in short-term cultures, such as collagenase- activity and procollagen processing. were evident dur· ing the long·term cultures. Derails of these observations follow.

MATERIALS AND METHODS

CeU Culture in Hydrated Collageo Gels Human skin fibro­blast tUonolayerculwres were established from foreskins obt,ajned at circumcis ions and maintained and harvested as before [20]. Prepara­rion of hydrated collagen gels from Virrogen " 100" collagen (Col­lagen Corp, Palo Alto, CAl has been described previously lI2,13J. Fibroblasts were added to the neutralized collagen solutions (1.5 mg/ ml) at a concentration of 1 0' cells/0.2 ml. Aliquots (0.2 ml) of t.he ce li/coJ.lagen mixtures were warmed to rOom temperature and placed in Costar 24-wdl culture places. Each aliquot occupied an area outlined by a 12-mm-diameter circular score within a well.

0022.202X/ 89/S03.50 Copyrighr © 1989 by The Society for Investigative Dermatology, Inc.

792

VOL. 9), NO. 6 DECEMBER 1989

Gels were polymerized by raising the temperature to 37°C. The samples were incubated for 60 min, after which 1.0 ml of culture medium [Dulbecco's modified Eagle's medium supleme.nced with 10% fcral bovine serum (FBS) and 50 pg/m1 ascorbic acid] was added to each well. After polymerization, the fibroblasts were dis­persed throughout the gels. To obtain cultures of floating collagen gels, the attached gels were gently lifted off" the bottom of the wells with a spatula.

Measurement of Gel Contraction Gel contraction was deter­mined by measuring gel volume 115.17]. We added 1 pCi of JH-HzO (1 mCiJg. New England Nuclear) to the culture medium. After 1 h, the medium was removed, and the gels were rinsed quickly and dissolved in 0.5 ml of 1M NaOH. The samples were neutralized with HCI. mixed with 10 ml Budget Solve (RPI Corp.). and cOllllted ill a Beckman Scintillation spectrometer. Initial gel volume was determined in gels withom fibroblasts.

Microscopy Microscopic analysis was done as described pre­viously 18]. Samples were fixed for 2 h at 4"C with 2% glutaralde­hyde. 1 % paraformaldehyde. and 1 % tannic acid, in O.l M Na caco­dylate (pH 7.4). postfixed for 30 min at 22"C wi'" 1% aqueous OsO ... and stained en bloc with 1 % aqueous uranyl acetate for 30 min at 4°C. After dehydration. specimens were embedded in Epon 812. Thick sections were stained with 1 % toluidine blue and exam­ined and photographed using a Zeiss Pholomicroscope Ill. Thin sections were examined and photographed with a Philips 300 e1ec­trOll microscope.

For :mached gels. multiple thick sections from central a.nd mar­ginal regions of the gels were prepared and examined. No differ­ences in cell morphology were evident in the different regioru. For floating gels. thick sections contained both the margins and central region of the gels. The thick sections photographed were selected based on their typical appearance. and thin sections were prepared from the same block faces.

Cell Recovery Collagen gels were solubilized by incubation for 140 min at 37°C with 0.2 ml of150 roM NaCl, 10 mM Ca acetate. and 20 rnM Hepes (ph 7.2). containing 2 mg/ m1 collagenase (Type I. Sigma Chem Co). Subsequently. 0.1 ml of 0.25% trypsin (GIBCO) and 0.02 mI 0.3M EDT A were added to the samples and incubated for an additional 20 min at 37 °. This combination of treatments dissolved the gels and dissociated any cell clumps. Ali­quots of the samples were mixed with trypan blue. and cell number was measured with a hemocytometer.

Collagen Synthesis Collagen synthesis was measured by the pro­tease~free collagenase method [18J. Culrures were radiolabeled metabolically for 24 h with 10 pCijml >H-L-proline (126.9 Cij mmole, New England Nuclear) added in fresh culrure medium. At the end of the incubations. the medium was removed. The gels were dissolved by incubating them for 90 min at 3rc with 0.4 m! of 10 mM HCI, and then the solubil ized gds were neutralized with NaOH. One-half of each medium and solubilized gel sample was brought to t ml containing 30 mM Tris, 6 mM Ca acetate, pH 7.2. and treated with collagenase (5 BTC units/mI, Form III, Advanced Biofactures Corp) for 3 b at 37°C. Bovine serum albumin (0.5 mg/ mJ) was added to collagenase-treated and untreated samples to act as carrier protein. after which all the samples were subjected to three precipitations with 10% TCA :U 4°C. The final precipitates were dissolved with Protosol, and radioactiviry was measured as above.

Cultures to be analyzed by 5DS~PAGE were radiolaheled meta­bolically [or 4 d with 30 pCi/ ml "S-methionine (1072 Ci/mmole. N ew England Nuclear) added in methionine-deficient culture me­dium. At the end of tbe incubations. rhe medium was removed. and the gels were solubilized as above. Medium and gel samples were treated with or without collagenase Fonn Ul and TCA precipitated as above.

Collagen Degradation Radiolabeled collagen was prepared by acetylation with JH~acetic anhydride (2.5 mei, 50 mCi/mmole. New England Nuclear Corp). as described [19]. The specific radio-

100

• E 75 " '0

> .. " 50 .. c

" <3 25

" 0

0

FIBROBLASTS IN CONTRACTED COLLAGEN GELS 793

\\

7

.L

0.-0. Attached gel. - cells

0-0 Allac hed gel. + cell s

0 - 0 F'lollltmg gel. ..;. cells

14 21 Da ys o f Cul lure

28

Figure 1. Colbgen gel contraction during 4 weeks of culture. Collagen gel s were attached to culture dishes or floating in cuJture medium and eonu.ined fibroblasts as indicated. At the times shown, the gd volume was measured. Dau. are averages ± S.D. from duplicate samples. O ther details are in MClttriQis Clnd Mtlhods.

activity of the radiolabeled collagen preparation was 1.5 X 1O' cpm/ mg. Gels containing radiolabeled collagen and fibroblasts were polymerized as described above for non-radiolabeled collagen. A{ the rimes when collagen degradation was measured, the culrure medium was removed from the incubations. The gels were rinsed briefly and dissolved with 1M NaOH, neutralized with 1M HCI, and radioactivity was counted with a scintillation spectrophotome­ter as above.

Electrophoresis and Fluorograpby Samples for 50S-PAGE were dissolved in reducing sample buffer (62.5 mM Tris, 2% SDS, 10% glycerol. 0 .01 % bromophenol blue, 5% mercaptoethanol. pH 6.8). Electrophoresis was performed using 4% - 16% acrylamide gradient slab gels containing a gradient of 3 -8 M urea (20). Acryl­Aide (FMC Corp) was used as the crosslinker, and gels were sup­ported on GeiBond-PAG film (FMC Corp). After electrophoresis. gels were fixed and stained with Coomassie blue. Gels were im­pregnated for 1.5 h at 22°C with EnlHance (New England Nuclear Corp) containing 2% glycerol. Gel films were dried in a 60°C oven and exposed to Kodak XAR-5 film on a Cronex intensifying screen (E.!. Dupont De Nemours & Co.).

" ~ c •

Allac hed gel. - c el b E 75 • '" c • " 50 ~ '0 u .. c 25 '!>

<3 Att.ached gel . + cells

" 0 0 I. 21 28

Days 01 Culture

Figure 2. Collagen degradation during 4 weeks of culture. Collagen gels prepared with radiolabded collagen were amched [0 cWturc dishes or float­ing in culture med.ium and contained fibroblasts as indicated. At the rimes shown, the percent angina] collagen remaining in the gels was measured. Data are the averages ± S.D. from duplicate samples. Other details are in Mtltr.riQis Clrld Mt.htHh.

79. NAK. .. \GA WA IT AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 3. Morphologic fearureJi of contractcd collagen gcls observed by ligh t microscopy. Fibroblasts in aruched collagen gels after 24 h of contraction were bipot...r celts. dongated parallel to the c.uhure di~h surface (A). In contracted . floatin g gels, on the other hand, ftbroblasts had a srell.ue appearance and random arrangement (B) . The bipolar rnorphology of fibroblasts in attached collagen gels was similar after 2 (C) and 4 weeks (£) of culture. but the cells became more closely packed together. In flo:.lting col l.:rogen g('is. fibroblast ret.:roined their srell.:r.te morphology. but rhe cells were somc:what more dendritic .:roffer 2 (D) and 4 weeks (F) of culture. Other details are in Maftrials and Mdhods (Bar: 10 J-lm) .

RESULTS

Contraction of Collagen Gels During. weeks of C ulture After 2 d of culture. Hoating collagen gds containing fibroblasts con.tracted to a slightly greater extem than attached collagen gels (-90% vs 80%) (Fig 1). Afcer 4 weeks. the extent of conr"ction of the floating gels and att<lched gels was 98% and 94%, respecrively. Therefore, the volume of anached gels was about 3 times greater than the volume of floating gds. Non-specific contraction, i.e .. decrease in gel volume without cells. was -10% after 2 weeks and - 20% ~ f(er 4 weeks.

Collagen gel contraction couJd have occurred in part because of collagen degradation. There was only a slight loss of collagen from the gels during the initial 1-2 d of culture (Fig 2). After 1 week of culture, however. collagen degradation became quite noticeable. By the end of 4 weeks, - 80% of the starting col lagen was degraded in

the attached gels and -70% in the floating gds (Fig 2). Considering the differences in gel volume (Fig') and col lagen degradation (Fig 2), it could be calculated thal after 4 weeks the collagen density (1.5 mgj ml in the original gels) was about 20 mgj ml in the float­ing gels and 4.5 mg/ ml in the attached gels. These values ignore new collagen synthesi s. By way of comparison, the collagen con­centration in skin exceeds 50 mg/ ml.

Some collagen loss from the gels also occurred withouf added fibrobl asts (- 30% during the first 2 weeks of culture) . Tllis non­speci fic loss did not increase further during the first 2 weeks of culture. Therefore. a subfraction of the colla.gen apparently incor­porated into the gels in an unstable manner. The non-specific de­crease in gel volume (Fig 1) could be accountt'd for by the non-spe­cific loss of collagen from the gels (Fig 2). Based on cell recovery (sec Fig 5 below), collagen loss normalized on a per cell b2sis was much higher 1n fio;)ri ng gels than in attached gels.

VOL 93. NO.6 DECEMBER 1989 FIBROBLASTS IN CONTRACTED COLLAGEN GELS 795

Fi~e", Morphologic features of contracted collagen gds observed by electron microscopy. A signi6cant decrease in collagen fibrils visible by electron nllcroseap)' occurred between 2 :lnd 4 weeks (C compared witb A). In tbe floating collagen gels. rhe densiry of extracellular matrix and diStribution of colbgen fibrils did flot chang!" notice:lbly between 2 (B) and 4 weeks (D). Other deatils are in Mattn'als and MnhoJs (Bar: 2.urn).

Morphologic Features of Collagen Gels contracted for up to .. Weeks Fibroblasts in attached collagen gels after 24 h of con­traction were bipolar ce ll s, elongated parallel co rhe cul ture dish surface (Fig 3A). In contracted, floating gels. fibroblasts had a stel­late appearance and random arrangement (Fig 3B) [15]. The bipolar morphology offibroblasts in attached collagen gels was similar aftet 2 weeks (Figs 3C and 4A) and 4 weeks (Figs 3£ and 4C) of culture. During chis time, the cells became more closely packed together. In floating collagen gels, distribution of fibroblasts was unchanged after 2 (Figs 3D and 4B) and 4 weeks (Figs 3F and 4D) of culrure. edl morphology remained stdlate, bur the cdl pseudopodia be­came more prominent relative to the celJ bodies.

In attached collagen gels. the extracellular matrix developed an uneven distribution during long-term culrure (Fig 3E). Also. a sig­nificant decrease in collagen fibrils visible by electron microscopy occurred between 2 and 4 weeks (compare Fig 4C and A). In the floating collagen gels. the densiry of extracellular matrix and distri­bution of collagen fibrils did nO{ change noticeably berween 2 (Figs 3D and 4B) and 4 week, (Fig, 3F and 4D).

Cell Recovery from Contracted Collagen Ge.ls Although floating collagen gels contracted more than attached collagen gels. the density of fibroblasts in attached gels appeared greater than in

fl oa ting gels (Fig 3). This observation was quantified by dissolving the gels and counting the cells thar could be recovered. In the experiment described in Fig 5, attached gels were contracted by fibroblasts for 1 d and subsequently cultured ei ther attached to dishes or floating in medium. Berween 2 and 14 d, cells in attached gels underwenr at least tv.'o doublings to - 4.8 X lOs/gei. During the samc pcriod , cells in fl oating gels decreased by > 50% to

- 0.35 X l OS/ gel. Although there was no further increase in cell number in aruched ge ls between 2 and 4 weelu. rhe cells in floating gels decreased by another 50% during that period.

T o learn if the fibroblasrs in collagen gels cuIrured for 4 weeks were viable. cells harvested from the gels were re~pla[ed in tissue cul ture dishes. Mosr of the cells from attached gels were viable and sta rted ro divide after a l -d lag period (Fig 6) . Only abour one-half rhe cells from the floating gels were viable. These cells started to divide after 1 d at the same rate as the cells harvested from attached gel ,.

Collagen Synthesis in Contracted Collagen Gels Figure 7 shows rhe rare of collagen synthesis, measured by incorporation of radiolahded proline into coll agenase~sensitive extracellular matrix and medium protei~. at severa] times during the 4-week culture period. Consistent with the cdl recovery data. there was a signili-

796 NAKAG" w" IT Al

6

.0 0

"" , Attac hed g e l ~

" • > 0 0 • '" !E 2 -;; '-'

Floa.Ling gel

0 0 7 J' 2 1 28

Days of Cu I lure

Figure 5. Rl::covery of cells (rom contracted ge.l.s. Collagen gels containing fibroblasts were attached to culturt' dishes for 1 d and then cultured further. eirhl::r attached or floating in cwture medium. At th.e times shown. thl:: gels were dissolved and the number of recovl::rl::d cells determined. Dara are the averages ± S.D. from dup\i.cate samples. Other details are in Maltrials and Mt'thrxis.

cam increase in collagen synthesis in attached gels and a decrease in collagen syn thcsis in fl oating gels. Nonnalized by cell number. collagcn syntltesis in 4-week. atuched gds was twice ~ high as collagen synthesis in 4-week f10aring ge.ls.

SDS-PAGE profiles of collagens and ocher proteins synthesized b)' fibroblasts differed for 5-d and 4-weck attached gel cultures. We focused our .mention on attached gels because cell recovery from the Hoa.ring gel culrures was so low. Fibroblasts in the attached gels were radiolabeled with 35$-methionine. Medium and gel samples from 5-d cul tures (Fig 8, lanes 1 and 3, respectively) contained 10 - 20 ndiolabeled polypeptides. most of which were insensitive to collagenase (Fig 8, lon6 2 and 4). Two major collagenase-sensitive components around 180 and 150 kDa and a col lagenase-insensitive component at 220 kDa were id.entified previously as procoHagens <>1 (I) and a2(I) and fibronecrin [21 ,22J. These three components were [he Jru.jor polypeptides found in medium from fibroblast monolayer culturC'i radio labeled with proline. After overnight pc-p­sin treatment in the cold. the 180- and 150-kDa components shifted to molecular masses 145 and 122 kDa 'a1(1) and <>2(1) collagen chains], and the 220-kDa component disappeared (data nOt shown).

100

.n 5 0

~

~ "' E

" z .. '-' 1 0

05 0

Cells Harvest.ed from Alla c hed Gel

2

Cells Harves ted from flo alm& eel

3

Da)'s of Cullure

Figure 6. Growrh of cells recovered from 4-weelc contncta! gels. Colla­gen gels wl::re attached to cultu~ dishes or fl02ting in cultur<" me<li.um for 4 wl::cks. Then the gels were dissolved. and l OS recovered cells were plated in culture flasks in growth medium. At the times shown. the cdls were har­vested and cell number was dl::fennilled. Data are the averages ± S.D. &om duplicate sample!.. Other details are in Matuials and Mt'lhods.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

15 .0

E fr 120

'" " Alt.a c hed gel

• 90 ;; • '" c 6 0 ~ U> c • " 30 !E -0 u

Floatmg gel

00 0 7 14 2J 28

D6)'s o f Cu i lure

Figure 7. Coll.agen synthesis in contracta! gels. Collagen gels containing fibroblasn were anachcd to culture dishes for I d and then cuJtured further either attached or floatin g in culture medium. Cultures were incubated with ndiolabeled proline for 24 h prior to th~ times shown. and incorporation of radioactivity into collage.n was measured. O:au are the avcr:.gcs± S.D. from triplicate samples. Other dl::tail s art' in Alluvials a"d M~tlloJs.

In contrast to the 5-d samples. cel lular processing of procollagen [0 collagen a chains occurred in gels contracted for 4 weeks (Fig 8, Latl(S 7 and 8). Besides (he chang~s in collagen processing, there wert' several ocher major changes in proteins synthesized by fibro­blasts in 5-d and 4-week cultures. A prominent poJypepcidt' of - 65 kDa appeared in the medium samples from 5-d culrures (Lanes 1 and 2, asterisk) bur not in medium samples from 4-week cultures (Lanes 5 and 6). On [he other hand, a pair of pepcides -1 16 kDa were

Fn­

Pro <l1(I)­Pro a2(1)-

*

1 234

-116-97

-66 -

_43 -

5 678

-01 (I) -a2 (I )

Figure 8. Pattern of collagen and protein synthesis in attached collagen gel cultures. Fibroblasts in att:ached collagen gel cultures were radiolabeled with l5S-methionine between t and 5 d of culture or 24 and 28 d of culture. untS

J - 4: 5~d cultures: una 5 - 8: 28-d cultures. Samples were medium (Ianr,s 1 and 5); medium + collagenase (Iallts 2 and 6); gel (Iants J and 7); and gel + collagen2se (lalllS 4 and 8). Five-day cultures con tained procollagens but nOt collagen Q chains, but collagen a: ch~ns were" m:t.jor component in the gel from 28-<1 cultures. Most 6bronectin (Fn) was found in the culttlre medium, not in tht geh. It. m:tior - 66-kDa component (D$ltrisk) in the medium from 5-<1 cultures was not observed in 28-d cultures. A major - 116-kDa pair of componentS (aslrrisk) in the medjum from 28-d cuJtures waS not observed in the 5-d cultures. Poola! sample1i &om twO culture well .. were loaded onto each lane. Other derails are in Malerials and Mtthrxls.

VOL. 93. NO. 6 DECEMBER 1989

barely visible in medium from 5-d cultures (Lanes 1 and 2, asterisk) but were major constituents in 4-week cul tures. Also striking was the gre:ate:r accumulation ofFN in the: medium sampl,es compared to the gel samples. Apparently. most of the FN syntbesized within comr.lcted gels did nor incorporate into the extracellular matrix.

DISCUSSION

The purpose of these studies was to analyze the consequences of long-tenn collagen gel contraction on fibroblast growth and meta­bolic activity. Fibroblasts ill :mached gels and in floacing gels dif­fered signj fi cantly in their responses to long-term contraction as summarized in Table 1. After 4 weeks. floating gels were 98% contracted and attached gels were 94% contracted. During [his culture period. fibroblasts in floating gels regressed - fivefold . while fibroblast!i in attached gels increased -fivefold. The cells remainin g in the floating gels were viable, however. When re­turned to routine culture cooditions, they divided at the same rate as cells removed from attached collagen gels.

Previous studies showed that DNA synthesis by nbroblasts in floating collagen gels decreased after gel contraction [16, 17}, but a similar decrease did not occur after conrraction of anached collagen gels 115J. Therefore. gel contraction per se and the increased density of the matrix did not cause cessation of DNA synthesis. After short­term conrraction of attached gels (1-4 d), fibroblasts were bipolar. On the other hand. afte r contr.lction of floatin g gels. fibroblasts were s{cllate (15J. The difference in cell shape can be understood in terms of asymmetry and the deve,lopment of tension in the gels. In the case of attached collagen gels, the contractile force exened by fibroblasts is distributed asymmetrically. Tension develops as indi­catC'd by the alignmenr of collagen fibrils parallel to the culture dish surface. Tht" fibroblasts spread along the collagen fibrils and have a bipolar morphology. In the case of floating collagen gels. the con­tractile force exerted by fibroblasts is distributed symmetrically. The matrix remains relaxed as indicated by the nonorganized ar­rangement of collagcn nbril s. The fibroblasts spread by extension of pst"udopodia without adopting a particular polarity. In the present work, we observed a similar morphologic difference during the 4-week culture period; that is, bipolar cells in attached gels and stellate cells in floating gels. Therefore, growth of fibroblasts in anached collagen gels and regression of fib roblasts in floating colla­gen gels might depend on cell shape.

Growth of normal fibroblasts on planar surfaces [23J and growth of normal endothelial cens on exrracdlular matrix-coated surfaces [24J requires cell spreading, although the mechanism by which cel l spread.ing controls DNA synthesis is poorly understood. One as.r:.ct of control might be at the level of cellular oncogene expression l25J. Significantly, dddition of platelet derived growth fdctor, fibroblast growth factor. or transforming growth factor-p did not restore nor­mal levels of DNA synthesis to fibroblasts in floating collagen gels that had been contracted for 2 d (15).

The for~going results suggest the novel idea that the regulation of fibroblast prolife ration ill granulation tissue by growth factors might depend on ex tracellular matrix organization and its influence on ce ll shape. In tht' f":lrly phases of wound healing, during granula-

Table t. Changes in Contracted Collagen Gels During Long-Term Culturea

Attached gds FI~ring gds

Feature 2d 4 w«ks 2d 4 weeks

Gel conrr2crion (% 81 94 91 98 decrease in gd volume)

Cell number (105) 0.9 4.61 1.2 0.2 Cdlsh:tpe 8 ipolar Bipolar Stelb.te Stellate Coll.agc n dcgrad.ation (% 17 83 18 73

original collagen IOSf) Collagen synthesis pro a(I) a(I) not dOlle

• ScI'; teXI for an cxpt:mation.

FIUROHLASTS IN CONTRACTED COLLAGEN GELS 797

tion tissue formation, the wound bed would be asymmetric because of the fi~ronecrin-rich wound interface ft .26]. Cells producing and contracung new exrracellular matrix in this environment would be bipolar and responsive to growth factors. As production of new extracellular matrix replaces the wound interface. the tissue would regain its symmerricai organization. Then cells would become stel­late and regress, returning to the state of fibroblasts in resting dermis 11.27). The analogy is imperfect but provides a reasonable account for the cycle of growth activation and cessation during wound healing.

An unexpected feature of long-term cell cultures was the loss of starting collagen from both the attached gels and floating gels (70% - 80%) during the 4.week culture period. Because the loss is cel! ~ependenr, it is likely to have occurred because of collagenase activ ity. Expression of collagenase activity i.n monolayer cell culture does not occur without modifying the cdls or adding procollagen­asC' acti vators [28,29]' Even in organ cultures, little collagenase ac­tivity was detectable in serum-containing medium [29]. The colla­genase level normalized to cell number was much higher in floating gels than in attached gds, consistent with the increased expression of procollagenase message by fibroblasts in floating gels 130]. W e have not attempted to isolate collagenase from the long-term cul­tures. and the enzyme might be bound to matrix r:aher than released in soluble form .

Another change in the iong-tenn cultures was collagen process­ing. After 5 d, fibroblasts in attached collagen gels secreted pro­collagen into the medium. After 4 weeks, fibroblasts in attached collagen gels incorporated collagen a{I) chains into the matrix. ~roba~ly, collagen .processing enzymes accumulated in the gds dur­lIlg thIS culture penod [31.32J. That secreted proteins could dilfuse out of the gels was shown by release of mosr fibronectin into the medium. both in 5-d cultures and in 4-week cultures. In fact, almost 110 fibronectin accumulated in the extracellular matrix of 4-week c.ultures. Perhaps there are a limited number of fibronectin binding S1tcs on the coBagen matrix, which werc saturated early in the culture period.

There also were a variety of other differences between (he pro­teins. synthesized by fibroblasts after shoft#cerm and long-tenn cul­ture III contracted gels. For instance. a major -66 kDa componenr found in the medium of S-d cultures was no longer synthesized in 4·week cultures . .A.. pair of -1 16 kDa components found in the medium of 4·week cultures was absent in 5-d cultures. Whether cells ffOm 4--week cultures can revert to the earlier phenotype after release from the gels is an important quesrion about the conrrol of fibroblast differentiation by the exrraceJlul;t.r matrix and one that we now are investigating.

Finally, it is important to note that al l of the studies in this report were" done with neonatal foreskin fibroblasts. Possibly, fibroblasts from adults or from other anatomical locations would behave dif­fereurly in colbgen ge,ls.

W,, art' illdt:btal f!l Dr. Tam Gilma n arlJ Dr. Jim McNight oJtill' KtllJall W~lInJ Mana.l!emt:1I1 Division oJKr1,dall WOllnd Cart Corp.Jor interestillg us i" fong-term C ~lltll ra, alld to Dr. William Snell ond Dr. Georgt Bloom Jar thtir helpJul sugges­tlot/s.

REFERENCES

1. Peacock EE: Wound Repair. 3rdEd. w.s. Saunders Co., Phihadelphi:a, 1984

2. Cbrk RAF: Cutaneo us tissue repa.ir; Basic biological considerations. J Am Aod Demurol 13:701-725, 1985

3. Gabbiani G, Hi rscheJ BJ. Ryan GE. St:ukov PRo Majno G: Granubtion tissu(' as a contnlCtile organ. A study of struCfUr(' and function. J Exp Med 135,719- 733. 1972

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798 N AKAGA WA 1::T AL

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