British Journai of Dermatology (1992) 127. 365-371,

Peripheral anchorage of dermal equivalents

C . A . L O P E Z VALLE.*t F.A.AUGER.*$ P.ROMPRE.*§ VERONIQUH BOUVARD* ANDLUCIE GERMAIN*i:*Skin Culture iMboratory. Saint-Sacrement Hospital, Quebec. Quebec. Canada. CIS 4L8

•f Department of Microbioioffij. Lava! University. Sainte-Foy. Queiwc, Canada. GIK 7F4

^Department of Surgery. iMval University, Sainte-i-oy. Quebec, Canada, GIK 7P4

>$ Department of Chemical F.ngineering. Uiva! University. Sainle-Foii, Quebec. Canada. GIK 7P4

Accepted for publication 1 7 April 1992

Summary Human fibroblasts can induce collagen gel contraction with different kinetics depending on thenumber ofcells and on the collagen concentration within this lattice, which has been considered as adermal equivalent. Skin equivalent is a combined culture of dermo-epidermal layers which may be oftherapeutic value in the treatment of burn patients. However, the current production ofthe dermalequivalent component gives results that present many drawbacks for their eventual clinical use as afirst step in obtaining a skin equivalent. These include: (i I final surfaces which are very small; less than20% ofthe initial size Iii) excessive thickness which may hamper successful graft take (iii) fibroblaststhat do not have an arrangement comparable with normal dermal tissue.

We propose, as a solution to these problems, the utilization of a 5-mm-wide fibre-glass filter ringperipherally attached to the surface ofthe Petri dishes to prevent inordinate contraction while thefibroblasts reorganize the collagen gel. Using this technique the initial surface was preserved and thedermal equivalent contracted only in thickness. Histological analysis of these anchored equivalentsconfirmed an alignment of fibroblasts and collagen fibres resembling normal derma! tissue. Weconsider this method useful in the development of dermo-epidermal sheets for clinical purposes.

The various techniques developed for the culture ofepidermal sheets have allowed an important break-through in the treatment of large burn wounds.' 'However these grafts, although they provide adequateand permanent coverage, are incomplete, because theylack the dermal component of skin. 'I'he absence ofdermis may be an important drawback because thedermal layer adds mechanical properties such as con-stant tension, shock resistance, elasticity and extensi-bility '^ to the protective barrier quality of the epidermis.

Histological analysis of transplanted epidermal graftshas shown that 'neodermis' formation under these graftsis not present during the first year after grafting.'' Thisdelay of at least 1 year in tissue reorganization may beobviated by grafting dermo-epidermal sheets, thus even-tually reducing the scar tissue formation stage.

The utilization of collagen gel as a matrix for growingcells was first described in 195f>.'' Since then, modifica-tions of this culture technique have been suggested toallow cell growth in a 3-dimensional environment.

Correspondenee: Dr F.Auger. Laboratoire de recherche des gr;inds

brules. Hopital du Saint-Sucrement. IOSO chemin .Sainte-Foy, Quebec.

Canada. t]lS4L«,

which could be experimentally altered to resemblenatural and pathological conditions.^ In 1979 a methodwas reported of using collagen to recreate a dermis-Iikestructure in vitro.'' Subsequently, the culture of skinequivalents (SE) has been extensively described.** "

The SE consists of two components: the dermalequivalent (DE) which is prepared first, and the epider-mal equivalent which is recreated by applying a suspen-sion of keratinocytes on to the DE. The critical step in theSE technique is the DE formation, which is obtained bymixing fibroblasts with a collagen solution. This solutionwill gel after a few minutes, according to the physico-chemical conditions.'-'" The incorporated fibroblastsactively reorganize the collagen fibres and induce adramatic contraction."'" The final suriace ofthe DE isinversely proportional to the number of cells, anddirectly proportional to the collagen concentration.'Thus, some authors have proposed that a judicious ratioof cell to collagen concentration in the DE may beselected in order to limit the amount of contraction.However, our own experience has proved otherwise, i.e.that DE contraction is quite severe. Furthermore, theaddition of keratinocytes to obtain a SE will alwaysfurther reduce the final surface in a concentration-

365

C.LOPEZ-VALLE et al

dependent manner.' •*"' ^ All keratinocyte concentrationsgiving histologically acceptable SF lead to an importantadditional contraction (final surface < 20% of the initialsurface).''* The mechanisms by which the keratinocytesinduce such a contraction are not completely under-stood, although these cells can interact with nativecollagen fibrils and reorganize the network in theabsence of fibroblasts'' independently from serum andfibronectin.'^

Thus the production of a clinically useful hilayered SEpresents a formidable challenge. Therefore, we havedeveloped a new technique for obtaining DE with aminimal level of contraction. The addition to the culturedishes of a peripheral ring made of filter material enableda firm anchorage of these DE. The contraction phenom-enon induced by the fibroblasts contained in the collagengel reduced the thickness but not the DE surface.Furthermore, histological analysis of these anchoreddermal equivalents (ADE) demonstrated a cellular-fibrillar arrangement much closer to normal skin his-

than any previous technique.

Methods

Medium preparation

A combination of Dulbecco-Vogt modification of Eagle smedium (DME) with Ham's F12 in a 3:1 proportion(Flow Labs, Mississauga. Ont.. Canada) was used as thebase medium. To each litre of medium 24-3 mg ofadenine (Sigma Chemical, St Louis. MO. U.S.A.) wasadded. The medium was sterilized through 0-22-pmfilters. Millipak 40 (Millipore. Bedford. MA. U.S.A.) andbrought to pH 7 3 5-7-45. Before use this medium wassupplemented with 5 /ig/ml bovine crystallized insulin(Sigma). 5 /ig/ml human transferrin (Sigma). 0-4 /ig/mlhydrocortisone (Calbiochem. CA. U.S.A.). 10" '" M cho-lera toxin {Schwarz/Mann. Cleveland. OH. U.S.A.). 10%fetal calf serum (Sigma). 10 ng/ml human EGF (ChironCorp.. Emeryville. CA. U.S.A.). 100 IU/ml penicillin G(Sigma) and 100 /^g/ml streptomycin sulphate (Sigma).This complete medium formulation is based on con-ditions developed for serial cultivation of keratino-

Fibroblast cultures

Fibroblast cultures were established from skin obtainedat the time of reductive breast surgery. The cells wereseeded at 1 x lO*" per flask and propagated in thepreviously described supplemented medium. These cells

were inoculated in 75 cm- Falcon tissue culture flasks(Becton Dickinson, Mississauga. Ont.. Canada) and werekept at 37°C in an 8% CO, atmosphere.

Cells were resuspended just before confluence with asolution of 0 01% FDTA and ()-()5% trypsin in PBS. Asample was taken to determine the number of cellsobtained. The suspension was centrifuged at 300 g for10 min and a final count performed before bringing thesuspension, in DME-HAM, to the desired concentration.

Collagen solution

Acid-soluble type I collagen, from calfskin (Sigma typeIII) was dissolved overnight at 4°C in 1:1000 acetic acidsolution to a final concentration of 7 • 11 mg/ml or 4 - 2 2mg/ml.

Preparation of anchoring Petri dishes

Two 5 mm-wide rings were cut from a glass microfibrefilter 934-AH (Whatman. Maidstone. U.K.). These wereglued together and attached to the bottom of 81-cm^square bacteriological Petri dishes using epoxy cement.The dishes were then resterilized in ethylene oxide.

Control fioating dermal equivalents were prepared instandard bacteriological Petri dishes.'

Preparation of dermal equivalents

Fibroblast-populated collagen gels were prepared in 50-ml centrifugation tubes (Sarstedt. Ville St-Laurent. QC.Canada) with various collagen and cell ratios. Thesemixtures were poured into S1 -cm^ LabTek bacteriologi-cal dishes, with or without the fibre-glass filter ring. Eachdish contained 5-6 ml of x2-7 concentrated DMEmedium with penicillin and streptomycin. 3 7 ml of fetalcalf serum. 9 5 ml of collagen solution. 0-1 5 ml of 0 1 NNaOH. and 1 ml of fibroblasts suspended in x 1 DME-HAM. Dishes were incubated at 37°C in an 8% CO2/92%air atmosphere. Gels were completely set after 10 min.Complete medium was used for media changes. Mediachanges were initiated on the third day after DEpreparation and subsequently performed each third day.

Evahiation of dermal equivalent dimensions

To calculate dermal equivalent dimensions, all Petridishes were placed on a glass surface 7 cm above an X-ray viewbox, A piece of black cardboard was used on theviewbox's screen to obtain indirect transillumination.Square fioating dermal equivalents (FDE) showed slight

PERIPHERAL ANCHORAGE OF DERMAL EQUIVALENTS 367

differences in side length. Photographs of each equiva-lent were taken daily. The standardized pictures pro-duced were read with planimetric scales to obtain thedaily surface of each equivalent. Peripherally anchoreddermal equivalent surfaces did not change. All resultsare given as a percentage of the initial surface.

Biopsies of dermal equivalents

At least two punch biopsies of 1 cm diam. were takenfrom different sites on each dermal equivalent. Biopsieswere fixed in Bouin's solution and embedded in paraffinin a standard manner, followed by staining with haema-toxylin and eosin for cell analysis. Masson's trichromewas employed for collagen pattern visualization.

Thickness measurements

The thickness measurements of all dermal equivalentswere performed by optical microscopy of histoiogicalslides. A Leitz Wetzlar (Germany) eyepiece with crossreticulation (TO mm/100 divisions) was employed. Alldermal equivalents showed thickness variations. There-fore, we performed five measurements on each equiva-lent and the mean result with the SD is included inTable 1.

started more or less rapidly, depending on cell numberand collagen concentration.

The initial contraction speed varied in relation to cellnumber. Gels with 18,500 fibroblasts/cm- showed areduction to 4% of the original surface area at 24 h whilethose containing 245 cells/cm~ showed a mean reduc-tion to 85% of the original surface area during the sameperiod. By the fourth day most of the contraction processhad occurred, and the original surface area was reducedto 2 and 45%. respectively, in the previously describedconditions. No further contraction was observed afterthe sixteenth day in any experimental condition, and themean final surfaces were less than 1 and 3 5% for theabove mentioned cell numbers. The results showingthe relationship between fioating DE surfaces andvarious cell concentrations are summarized in Figure 1.

As was shown previously.' the optical properties ofcollagen gels change during contraction. They becomeprogressively more opaque and eventually whitish-yellow. Furthermore, the consistency of dermal equiva-lents varied depending on cell numbers and collagenconcentration.

Increase of the collagen concentration was accompa-nied by a reduction in the contraction rate and by anaugmentation of the final surface. Figure 2 shows anexample of the influence of collagen concentration.

Results

Floating dermal equivalent formation

Under phase-contrast microscopy, round, trypsinizedfibroblasts began to show cell processes a few minutesafter gel setting. Two hours later the majority of cellsbecame stellate. After a short time-lag, contraction

Peripherally anchored dermal equivalent formation

Changes in cell morphology were simiiar to thoseobserved in floating dermal equivalent formation asobserved by phase-contrast microscopy. After gelation.cells were evenly sprinkled throughout the DE. Refrin-gent fibres appeared within 24 h connecting ceils in acircular pattern. Our observations suggest that daughter

Table 1. Thickness of relHted FDR and ADE in

FDEADEFDEADE

Collagenconcentration

(mg/ml)

2 1 1

4 224 22

18,000

1499 ±168

Fibroblasts/cm-

1235

1 J45±9I30 7 ±58

Nl)NI)

617

1169 ±109i26±65

NDND

555

782±I17286 ±J6

NDND

"Expressed as mean±SD.ND = not determined.I-'DK were Ihicker than ADI' in related experimental conditions, Riindom analysis showed thai Iheanchoring method induced formation of thinner dermal equivalents than the floating method.Initial volume was 20 ml and initial surface was 81 cm* for both PDE and ADE,

ibS C.LOPEZ-VALLE et aL

Fibroblasts numbers

-m- 1,5X10^- • - 5,0X10''- 0 - 4.5X10''- * - 3,6X10'*- * - 2.0X10'*

, i ^^*—A—

E0a£

ace

SOi

70 -

60 -

50 -

40 -

30 .

10 -

Days

Figure 1. Influence of fibrohlasts numbers on final surface, 'I'hc initialcontraction speed varied in relation to cell numbers. By the fourth daymosi ofthe contraction process had occurred. No further contractionwas observed after the sixteenth day in any experimental amdition.Collagen concentration was 2'11 mg/ml.

20 .

10 -

Collagen concentrationO 4,22 mg/ml• 2,11 mg/ml

H12 16

Days

Figure 2. Influence of collagen concentration on final surface. Increaseofthe collagen concentration was accompanied by a reduction in thecontraction rale and by an augmentation of the final surface. Totallibroblast number was constant, 4'5 x 10'' (initial concentration of555 cells/cm-). Bars represent SEM from three replicates.

cells formed bundles in this original arrangement, andthis increasing number of fibroblasts began outwardmigration in all directions (Fig. 3). At the final stage, cellswere arranged in a complex three-dimensional circulararray, with most cells parallel to the surface ofthe Petridish. No surface reduction was noted when the fibre-glass filter ring was used.

Anchored dermal equivalents with a collagen concen-tration of 2 1 1 mg/mi and containing 18.500 fibro-blasts/cm' had a tendency to tear in their centralportion. This phenomenon disappeared when the colla-gen concentration was raised to 4-22 mg/ml for thesame cell number.

A progressive opacity and solidification ofthe gel werealso observed macroscopically. according to culturetime.

Floating dermal equivalents (PDF)

Histology of fioating dermal equivalents synthesizedwith different cell numbers and collagen concentrationspresented some common characteristics.1. A fibrobiast multilayer enveloped both surfaces offloating equivalents. This phenomenon did not occurwhen the cell number was lower than 444 fibroblasts/

2. Fibroblasts within the FDE were orientated perpen-dicular to tissue length, whereas those present at the FDEsurface were parallel to tissue length (Fig. 4b).3. Collagen fibres were orientated perpendicular to the.surface (Fig. 4b). This histological feature was notobserved when the fibrobiast numbers were < 617 cells/cm^.4. The surface of floating dermal equivalents containingmore than 1235 cells/cm^ showed many folds. Thesefolds were filled with colonies of fibroblasts (Fig. 4al.

Anchored dermal equivalents (ADF.)

Histology of peripherally anchored dermal equivalentsdiffered from the floating equivalents.1. All experimental conditions showed an absence ofafibrobiast multilayer envelope around the matrix.2. Fibroblasts were orientated longitudinally to tissuelength (Fig. 5b).3. Collagen fibre orientation was mostly longitudinal inalignment (Fig. 5b).4. No folds were observed.A microscopic study of the fibre-glass filter ring in theADE showed penetration of the collagen into the inter-stices of this material. The anchoring phenomenonseemed to be related to its entrapment in the porous

PERIPHERAL ANCHORAGE OF DERMAL EQUIVALENTS 569

Figure 3. Living peripherally anchored dermal equivalent conlaininnI S x l O ' ' fibrohlasts at 48 h in culture (18,000 cells/cm^). Cellsarranged in a circular pattern. Daughter cells began migrulionoutward in all directions. View from below using phase contraslmicroscopy ( x 1 J 5).

Structure, rather than to any cellular binding, as verylew cells were seen within the fibre-glass ring (Fig. 5c).

Stability of peripherally anchored equivalents

A set of experiments was carried out to evaluate thestability of these ADK after their detachment from thetilter ring. A contraction of approximately 10% occurredwithin 5 days after detachment in dermal equivalentswith 18.500 libroblasts/cnr (Fig. 61.

Thickness measurements

Fifty-two fioating dermal equivalents were comparedwith 44 anchored dermal equivalents. Random analysisshowed a mean thickness of 673 fim for FDE. with arange of 151-1560 }im. Mean thickness for ADE was548 ^m with a range of 100-496 ^m. Comparison of

some related experimental conditions are presented inTable 1.

Discussion

This study was initiated to evaluate whether a peripheralanchoring technique might solve the inherent deficien-cies in the usual methods of recreating a DE in vitro. Theresults obtained with the FDE technique illustrate thesepitfalls.

The FDE technique has invariably produced iinalsurfaces that were so small that they precluded their useas clinically significant grafts for burn therapy. Loweringfibrobiast numbers in these collagen lattices lessened thisshrinkage phenomenon.' '"'•''' However, the resultantDE was then so poorly organized that it was very fragile.Furthermore, the histological resemblance to normaldermis was lost.

One proposed solution to circumvent the shrinkageproblem is the use of much larger tissue culture tlasks.There are. however, practical limits to this approach,and the initial surface would have to be extremely large.For example, to obtain a S x 8 cm graft, considering aconservative 9 5% shrinkage level, an initial culture dishof 100 X 1 60 cm would be necessary. These conditionsare impractical and preclude the possibility of anyscaling up of operations. In addition, if a solution ofkeratinocytes is then 'plated' on these DE to obtain SE. afurther reduction of the final surface will ensue whichwill greatly compound the problem.'"*'''

The resultant FDE are relatively thick, and this mayhave significant effects on the process of angiogenesiswhich normally occurs after transplantation. As showedby Yannas. using cell-free polymeric membranes, thisingrowth process could take between 10 and 46 daysafter application, depending mainly on thickness, chemi-cal composition and type of polymerization.-"' -' Interfer-ence with vaseularization lessens the viability of the

Kigure 4. (a| Histological appearance of fullthickness FDE showing an envelope offibroblasts mainly on the surface, and oneIbid tilled by u colony of fibroblasts (FC).Initial collagen concentration was 2-1 I mg/ml and fibrobiast number was l8.5()O/cm'(Haematoxylin and eosin. Taken at x 20 andenlarged photographically to x 4().|(b) FnlargemenE of the indicated portionshowing fibroblasts (arrowheads) andcollagen Iibres orienlated perpendicular totissue length, (Taken at x 80 and enlargedphotographically to x 160,)

370 C.LOPEZ-VALLE et al

(b)

Figure 5. (al Histological appearance of fullthickness ADE. Comparison with h'igurt 4|a)shows that the thicicncss of the ADI-; is lessfor the siime conditions (initial cell andcollagen concentrations). Initial collagenconcentration was 2-\\ mg/ml and fibroblastnumber was 18,5()()/cm-. (Haematoxylinand eosin. Taken at x 20 and enlargedphotographically to x40.) (b) Fnlargementof tbe indicated portion of Figure 5(a)showing fibroblasts (arrowheads) andcollagen fibres orientated longitudinally inrelation to tissue length, (Taken at x 4U andenlarged photographically to x 160.)(c| Histology of the libre-giass ring (F)interface witb the ADE (arrowbead),Haematoxylin and eosin staining. (Taken atX 20 and enlarged photographically tox40,)

overlying epidermis and results in a lower take level.However, this was not noted in previously describedexperiments using SE.^'"' but it is possible that the smallsize of these grafts permitted their lateral nourishment.

Lastly, our histological analysis of these FDE hasshown that their cellular and fibrillar arrangement,mostly perpendicular to the surface, is not comparablewith normal human dermis.''* It seems logical to strivefor DE with histological features as close as possible to thenormal dermis.

80

24

Figure ft. Comparative final surfaces of floating and anchored dermalequivalents. I'ioating dermal equivalent surface: 0-74%. anchoreddermal equivalent surface after freeing: 87-8%, Collagen concentra-tion 2 1 I mg/ml, fibroblasts numbers: 18.S(H)/cm^. Arrow indicatesthe freeing day. Bars represent SHM from tbree replicates.

Our results show that the anchoring of the dermalequivalent is a practical solution to the drawbacks of theEDE. In their studies of tissue morphogenesis. Harris et al.described a related technique for stabilizing collagen gelsdevoid of incorporated cells. Eibroblasts were subse-quently added to the surface of these collagen gels. Theobjective of these studies was the analysis of thegeneration of fibrillar patterns created by variousmechanical instabilities.-*^ '" whereas we wished toobtain large and stable DE. We have shown that theperipheral anchoring technique can achieve such resultswithout complicated manipulations of the culture condi-tions. Anchorage transformed the contraction processinto a unidimensional rather than a three-dimensionalphenomenon. The only axis of shrinkage involved thethickness of the ADK because it was the only axis alongwhich the fibroblasts could act freely. As previouslydiscussed, we fee! that a thinner DE may facilitate grafttake and the survival of the keratinocytes contained inthe SE. This cultured SE has to be nourished by osmosisduring the first few days after grafting, as are standardgrafts."

The histological features of the ADE are also muchcloser to normal dermis.'** The longitudinal arrange-ment of cells and fibrils in ADE is quite remarkable.

Our observations suggest that the attachment of theADE to the filter ring is a fibrillar entrapment phenome-non rather than a cellular binding mechanism. Thus, itis possible that any porous material will permit effica-cious anchoring of dermal equivalents. The choice of anoptimal material would be dictated by its biocompati-bility. by its ease of use and its resistance to sterilization,allowing repeated utilization.

PERIPHERAL ANCHORAGE OF DERMAL EQUIVALENTS 371

We helicve that the ADE model presented in this reportis a first step in the production of new skin suhstitutes fortreatment of burn patients by tissue culture methods.The addition of other molecules in the initial mixturecould be helpful in obtaining a more complex andstructured dermal equivalent. Such an anchoring tech-nique could be readily amenable to large-scale produc-tion of skin equivalent for wound coverage of burns.

Acknowledgments

This work was supported by Grant No. MA-1022 i fromthe Medical Research Council of Canada. Award B-2committee 01A from the Fonds FCAR (Quebec) toC.A.Lopez Valle and Award PCJS 1 from Natural Sciencesand Engineering Research Council of Canada toP.Rompre, and Fellowships from the Fonds de laRecherche en Sante du Quebec (FRSQ) to F.A.Auger andL.Germain,

We thank Dr George Nascimento from Chiron Corp-oration for graciously supplying F̂ GF, Celine D.Fugere forassistance with histology and jo-Anne Masse for helpwith technical work.

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