-y-

.::-<-"~-,

He'"fLlbA;

f,.J L¿.!l- 4.c.a- ...¡;:. ~-= C-6 -

p,'.J ¡.h ~ .-.J.-:. ~OL- .4<Oc.:C4~

-f- T 6 ~ -c...:cc«.~

Richard Y. Lin, 8.S., Kerry M. Sullivan, M.O., Peter A. Argenta, 8.S., Martin Meuli, M.O.,H. Peter Lorenz, M.O., and N. Scott Adzick, M.O.

From the Department of Surgery, University of California-San Francisco,San Francisco, California

ObjectiveFetal skin wounds heal without scarring. To determine the raje of TGF-fJ, in fetal wound healing,mANA express ion of TGF-fJ, was analyzed in human fetal and adult ski n wounds.

MethodsHuman fetal skin transplanted to a subcutaneous location on an adult athymic mouse that wassubsequently wounded heals without scar, whereas human adult skin heals with scar formation inthat location. In situ hybridization for TGF-f3, mANA expression and species-specificimmunohistochemistry for fibroblasts, macrophages, and neutrophils were performed in humanadult wounds, fetal wounds, and fetal wounds treated with a TGF-f3, slow release disk.

ResultsTransforming growth factor-{3, mANA expression was induced by wounding adult ~k~

ConclusionsTransforming growth factor-fJ, is an important modulator in scar formation. Anti- TGF-fJ, strategiesmay promote scarless healing in adult wounds.

cytokine transforming growth factor-befa (TGF-,B) pro-motes inflammatory cell recruitment and collagen depo-sitian in healing wounds and may play an important rolein scar formation. Previous studies on adult rats haveshown that wound application ofneutralizing antibodiesto TGF-,B results in markedly reduced scar formation.1

Unlike the adult, the wounded midgestational humanfetus has the remarkable ability to regenerate normalskin architecture without scarring. Currently, the un-derlying mechanisms of scarless wound repair remainunknown. Scarless healing may result from a unique cellpopulation, extracellular matrix, or cytokine milieu. The

146

1,/1~t'6, ANNALS OF SURGERY

Vol. 222, No. 2,146-154@ 1995 Lippincott-Raven PubJishers

147TGF-fJ Induces Scar in Fetal WoundsVoi 222.No 2

lmmunostaining for TGF-fJ, protein has demonstrated adeficiency of TGF-fJ, in fetal wounds when comparedwith adult wounds.2 Furthermore, addition of TGF-fJIto fetal wounds induces scar formation.3,4 Human fetalskin transplanted to a subcutaneous positÍon on an adultathymic mouse that is subsequently wounded heals with-out scar, whereas human adult skin heals with scar for-mation in that location.5 By using ibis model of woundrepair, we examined human TGF-fJI mRNA expressionby in situ hybridization in adult wounds, fetal wounds,and fetal wounds treated with exogenous TGF-fJ,. Spe-cies-specific cell types in the wound environment alsowere determined by immunohistochemistry.

Figure 1. Translorming growth laclor-{J1 slow release disks were placedbenealh Ihe grafted fetal lissue al Ihe lime 01 wounding.MATERIALS AND METHODS

Animals

Female adult athymic (nu/nu) nude mice (CharlesRiver Laboratories, Wilmington, MA) at 6 to 7 weeks ofage were housed in groups of four to six in sterile cagesat the University of California, San Francisco (UCSF)animal care facility and red food and water ad libitum.Animal management was in accordance with the policiesofthe UCSF Animal Care Committee and the NationalInstitutes ofHealth guidelines for the caTe of experimen-tal animals.

Transplantation

Skin samples were transplanted onto athymic mice re-cipients as previously described. Briefly, skin was washedtwo times in sterile phosphate-buffered saline solution,trimmed into 0.5 cm X 0.5-cm squares, and trans-planted. AII procedures were performed in a laminarflow hood under aseptic conditions with inhalation etheror metophane anesthesia.

Skin grafts were transplanted into subcutaneous poc-kets on the flanks ofthe nude mice. Each mouse receivedtwo grafts, one on each flank. The subcutaneous pocketswere created by incising a l -cm length of skin obliquelyalong the mouse flank and forming a l -cm XI-cm cavityunderneath the panniculus carnosus. The grafts wereplaced, with dermis directed down, onto mouse fascia.Tl1e pockets were closed with metal clips.

Human Skin

Human fetal scalp skin from 18-week gestational agefetuses (n = 12) was obtained from therapeutic abortionmaterial after signed consent for the use of this tissue forresearch purposes under an approved UCSF Committeeon Human Research protocolo Human fetal tissue collec-tion conformed to the current recommendations of theNational Institutes of Health. Gestational age was deter-mined by fetal foot length. Adult human skin (n = 8) fromthe head and chest was obtained after signed consent [rompatients undergoing surgery for noninfectious, non-neo-plastic conditions. Skin was placed in cooled serum-freeRoswell Park Memorial Institute-1640 media (RPMI-1640; Gibco, Grand Island, NY) with 25 mmol/L Hepes,0.3 g/L L-glutamine, 2.0 g/L NaHCO3, and 1 % penicil-lin/streptomycin. Skin was maintained in these condi-tions at 4 C for 12 to 24 hours, until transplantation.

Wounding

Seven days after transplantation, full-thickness linearincisions were made in the grafts. To gain access to thesubcutaneous grafts, pockets were opened through a sep-arate incision. Wounds were marked with India ink to aidin localizing the wound during analysis. In one mouse,a cellulose disk designed to release TGF-{:JI ayer 14 days(Innovative Research, Toledo, OH) was placed directlyunder the wound, between the human graft's dermis andthe mouse's fascia (Fig. 1). The disk placed under the graftin the mouse's right flank contained 10,ug ofTGF-{:J1 in acellulose/cholesterol¡lactose vehicle. A control disk withan equivalent volume of vehicle was placed under thewounded graft on the mouse's left flank. Mouse skinpockets were closed with clips and the wounds were left toheal.

Supported by the Howard Hughes Research institute Medical StudentFeIlowship (Dr. Lin), the American CoIlege of Surgeons Scholar-ship (Dr. SuIlivan), and NIH grants HD 25505 and GM 27345

(Dr. Adzick).Address reprint requests to N. Scott Adzick, M.O., Children's Hospital

ofPhiladelphia, 34th Sto and Civic Center Boulevard, Philadelphia,

PA 19104.Accepted for publication September 14, 1994.

148 Un and Others Ann Surg .August 1995

Table 1. CHARACTERIZA TION OF ANTIBODIES USED IN THIS S'TUDY*

AnimalSourc~ of Antibody Manufacturer

Dilution

(in Phosphate-Buffered Saline)Antigen

1:2001:2001:2001:10001:2001:10001:2501:801:80

MouseGoa!Rabbi!Rabbi!Rabbi!RabbitGoa!Goa!Rabbit

Boehringer; Westbury NYICN; Costa Mesa CAChemicon; Temcula, CAAccurate; Westbury, NYChemicon; Temcula, CAAccurate; Westbury, NYSigma; SI. Louis, MOSigma; Si. Louis MO

Sigma; Si. Louis, MO

Human vimentinMouse vimentinHuman macrophagejmonocyteMouse macrophagejmonocyte*Human neutrophilMouse neutrophil*Mouse immunoglobulin G*Rabbit immunoglobulin G*Goat immunoglobuJin G*

.Antibody conjugated w~h fluorescein isothiocyanate.

sequences were selected to avoid cross-hybridizationwith mouse TGF-fJ¡. Antisense oligonucleotide probes(one 26 base-pair sequence from exon 7, one 27 base-pair sequence from exon 7, and one 28 base-pair se-quence from exon 6) were synthesized (R&D Systems,Minneapolis, MN) and end-Iabeled with biotin. Probeswere purified by polyacrylamide gel electrophoresis andsupplied in an equimolar mixture.

Harvesting of Grafts

The mice were killed 1, 6, and 12 hours and 1, 3, 7,and 14 days after wounding, and the grafts were removed.Grafts with the slow-release disks were harvested 14 daysafter wounding. Wounded fetal skin grafts were snap-fro-zen using precooled isopentane in liquid nitrogen.Wounds were cut in 8-~m sections on a Reichert-Jungcryostat at -20 C and mounted on polysialic acid glassslides in preparation for in situ hybridization and immu-nostaining. AII tissue and slides were stored at -80 C. Probe Specificity

Dot-blot hybridization was performed to determinespecificity and sensitivity of the probe, using standardtechniques.6 The human-specific TGF-fJ. probes fromR&D Systems were hybridized against 0.01 to 5 ng ofTGF-fJ. cloned into the pUC18 plasmid to determinesensitivity ofthe probe for TGF-fJl, against 0.01 to 5 ngof pUC18 alone to rule out the possibility of the probeactually detecting the pUC 18 plasmid but not TGF-fJ 1,and against 5 ng of platelet-derived growth factor A(PDGF A), platelet-derived growth factor B (PDGF B),acidic fibroblast growth factor (aFGF), basic fibroblastgrowth factor (bFGF), TGF-fJ, insulin-like growth factor1 (IGF-l), endothelial-cell growth factor beta (ECGF-fJ),and nerVe growth factor beta (NGF-fJ) cloned intopUC18 to determine the sensitivity ofthe probe. Filterswere washed twice for 15 minutes at 42 C and exposedfor 3 days.

Preparation of Probes

The human TGF-(:J) sequence was compared withGenBank, and specific sequences for human TGF-(:J,were isolated. These sequences were then compared withthe mouse TGF-(:J¡ sequence. Human-specific TGF-(:J,

Do! 8101 wi1h TGF-beta Probe

TGF-beta clonedínto pUC18

., 5ngpUC18 Alone Specilicity Test

PDGFA

PDGF B

aFGF

bFGF

TGF-beta

IGF-1

ECGF-bela

NGF.beta

5 n9

n9 , n9

5 n9 5 n9

.

~,1 ng , "9

.05 n9 05 ng

In Situ Hybridization

Slides were warmed to room temperature in a moistchamber. The slides were successively washed in phos-phate-buffered saline (2 X 5 minutes, 20 C), 2X SSC (10minutes, 60 C), H2O (rinse, 20 C), 50 mmol/L Tris pH7.6 (5 minutes, 20 C), and then incubated in 5 ¡Lg ofpro-teinase K (Sigma, Sto Louis, MO) per milliliter ofO.05 M

01 "9 01 ngI

Figure 2. Dot bicI of human TGF-{3, probe against 0.01-5.0 ng of TGF-{3, cloned into pUC18, against 0.01-5.0 ng of pUC18 alone, and against 5ng of PDGF A, PDGF B, aFGF, bFGF, TGF-{3, IGF-1, ECGF{3, and NGF-{3

cloned into pUC18.

149TGF-ij Induces Scar in Fetal WoundsVOl 222. No 2

: F

'. '".S

,:.~-.

" ~~~,:~

--~-,. i ...L-- 1- -

Figure 3. (A) (top le't) Trichrome histology 01 a healed human adult skin gralt placed in the subcutaneous position 01 the nude mouse Trichromestalns collagen a deep blue The adult skin heals wlth disorganized colla gen lormatlon characteristic 01 scar (arrows) (8) (top right) In situ hybrid-ization lor TGF.¡3, mRNA was perlormed on adult skin 7 days alter \"Jounding Cells prodUClng TGF.¡3, mRNA are lound throughout the wound(stralght arrows) and In the basallayer 01 the epidermis (curved arrows) (E = epidermis: scale bar = 100 ¡Jm)

Figure 4. (A) (bottom left) Trlchrcme histology demonstrates that the human fetal skin healed ~ithout scar.lndia ink (1) demarcates the imtlal site of

Injury (B) (bottom ,ight) In situ hybrldlzation for TGF-/1' mANA was performed on fetal skin 3 days alter woundlng Transformíng growth factor./1,was detected In the basallayer of the epidermis (curved arrows) General background staimng was seen in the dermis but no cells producing TGF./1, mANA were found (scale bar = 100 ¡1m)

(v/v) formamide (2 X 5 minutes, 37 C), and 0.2X SSC/30% (v/v) formamide (2 X 5 minutes, 37 C). To demon-strate that the target nucleic acid was RNA. control tissuesections were pretreated with 100 #lg ofRNase/mL of2XSSC and 10 mmol/L MgCI2 for I hour at 37 C before

hybridization.

Tris pH 7.6 at 37 C for 1 hour. Sections were then rinsedin 1 X phosphate-buffered saline, incubated in 4% para-formaldehyde in 1 X phosphate-buffered saline at 4 C for20 minutes, and then rinsed in H2O. Slides were prehy-bridized with 30% (v/v) formamide, 4X SSC, and 150Jlg/mL sonicated salmon sperm DNA for 1 hour at 37 Cin a humid hybridization chamber and then hybridizedin a solution containing 30% (v/v) formamide, 4X SSC.10% (w/v) dextran sulfate, 150 Jlg/mL of sonicatedsalmon sperm DNA, and 200 ng/mL ofthe probe for 24hours at 37 C in a moist chamber. After hybridization.the sections were successively washed in 4X SSC/30%(v/v) formamide (2 X 5 minutes, 37 C). 2X SSC/30%

Detection 01 Biotin-Labeled Probes

Tissue sections were blocked for 30 minutes with a so-lution containing 3% protease-free bovine serum albu-min (Sigma), 0.1 mol/L Tris-CI pH 7.5,0..2 mol/L NaCI,and 0.05% Triton-X-1 00 (Sigma). Slides \\'ere then incu-

151Vol. 222. No 2 TGF-¡3lnduces Scar in Fetal Wounds

Figure 5. (A) (top left) Trichrome histology 01 the letal graft with the control disk demonstraies the typicalscarless healing seen in letal skin. No disorganized collagen lormation or scar is seen India ink (1) particlesmark the wound site. (B) (top right) Trichrome histology 01 the wounded letal skin graft with a 10-p;g slowrelease TGF-{3, disk demonstrat65 a disorganized collagen pattern characteristic 01 scar (arrows). In situ hy-bridization lor TGF.{3, mANA on a serial section Irom the letal skin with the TGF-{3, slow release disk was thenperformed (C) (middle left). The area 01 scar is denoted by the large arrows. Cells producing TGF-{1, mANA (C)are lound throughout the wound (indicated by small arrows) and the surrounding unwounded dermis (indicatedby curved arrows). (D) (bottom right) Under higher magnilication at the scar site, cells producing TGF-{1, mANA(C) are seen. (E) (bot/om left) Immunohistochemistry lor human vimentin showed that these cells are humanlibroblasts (F). (5A-5C: scale bar = 100 p;m; 5D-5E: scale bar = 30 p;m)

specific background binding. Sections were stained withprimary antibody for 1 hour, washed with phosphate-buffered saline, and stained with fluorescein-linked im-munog1obulin G secondary antibody. Sections wereviewed and photographed with a Zeiss fluorescent mi-croscope (Zeiss, Berlin, Germany). Negative control sec-tions had nonimmune goat serum (Cedarlane Labora-tories Limited, Homby, Canada) substituted for primaryantibody. Mouse and human dermis sections that werenot wounded were stained separately with their corre-sponding species-specific primary antibody and counter-specific antibody to function as both positive and nega-tive controls.

bated in 10 #lg of streptavidin alkaline phosphatase permilliliter of 0.1 mol/L Tris-Cl pH 7.5, 0.2 mol/L NaCl,and 0.05% Triton-X-lOO for 25 minutes. Three 10 min-utes washes in 0.1 mol/L Tris-Cl pH 7.5, 0.2 mol/LNaCl, and 0.05% Triton-X-100 were followed by a 10-minute wash in 0.1 mol/L Tris-Cl pH 9.5 and 0.1 mol/LNaCl. The color development was carried out by incu-bating the slides for 30 minutes at room temperature ina substrate solution containing 0.66 mg/mL of nitrobluetetrazolium and 0.33 mg/mL of 5-bromo-4-chloro-indo-lyl phosphate in 0.1 mol/L Tris-Cl pH 9.5 and 0.1 mol/L NaCl. The reaction was stopped by incubating the slidein 1 mmol/L of edetic acid.

RESULTSImmunohistochemistry

Primary antibodies used were specific for human andmouse vimentin (a fibroblast marker), human andmouse macrophages. and human and mouse neutrophils(Table 1). Sections were fixed in acetone for 1 to 2 min-utes at room temperature. Serum from the species oftheprimary and secondary antibody was used to block non-

Oot-blot hybridization demonstrated that the TGF-,B.probe was able to detect up to 0.05 ng/ml of TGF-f3.clonedinto pUC18. The TGF-,B. probe did not detectthe pUC 18 plasmid alone. The TGF-f3¡ probe hybridizedwith the cONA of TGF-,BI' but did not hybridize with

Figure 6. (A) (/eft) Immunohistochemistry with species-specilic antibodies demonstrated an influx 01 mousemacrophages (M) into the wound. (8) (right) Immunohistochemistry also revealed a recruitment 01 mouseneutrophils (N) into the wound. No human macrophages, human neutrophils, or mouse libroblasts were de.tected in the wound. (scale bar = 30 ¡¡.m)

153TGF-fJ Induces Scar in Fetal WoundsVol 222.No 2

taneous fetal graft wounds, there is a lack of an inflam-matory cell infiltrate during scarless fetal repair. How-ever, exogenously added TGF-fJ. during wounding pro-motes an adult-like inflammatory response, and thewound heals with scarring. The presence of adult mouseneutrophils and adult mouse macrophages that are re-cruited to the subcutaneous human fetal graft woundmay be another reason why scar formation occurs.

Strategies to block TGF-fJ may prevent scarring. Injec-tion of anti- TGF-fJ polyclonal neutralizing antibodiesinto adult rat wounds markedly diminishes scarring.1Subsequent studies have revealed that specific neutral-ization ofboth TGF-fJI and TGF-fJ2 isoforms has a muchgreater synergistic antiscarring effect than neutralizationof either isoform alone.31 Injection of the TGF-fJ3 iso-forro at the time of wounding downregulates TGF-fJ.and TGF-fJ2 levels and leads to a pronounced antiscar-ring result.32 Not only do anti- TGF-fJ strategies seemeffective in reducing scar formation in adult wounds,they also may be effective in ameliorating the effects ofother fibrotic processes. For instance, administration ofeither TGF-fJ. antiserum or decorin (a polysaccharidethat binds and inactivates TGF-fJ.) suppresses the patho-logical increase in matrix synthesis that occurs in an ani-mal model of gIomerulonephritis.33,34

Future fetal wound healing studies will help us un-derstand the biology of scarless healing and may yieldinsight into the prevention of scar formation. Recent ad-vances in fetal wound healing research suggest a numberofways in which the matrix and cellular response ofthehealing adult wound might be manipulated to reducescarring.35 Rigorous testing of anti- TGF-fJ strategies maylead to clinical induction of scarless healing in adults and

children.

References

wounds are at least relatively TGF-(:JI protein and mRNAdeficiento

In vivo studies have demonstrated that exogenouslyapplied TGF-(:J promotes scar formation and increasescollagen deposition in both adult and fetal woundso Inadult wound models, low-dose subcutaneous injection ofTGF-(:JI stimulates fibrosis and accumulation of fibro-blasts, macrophages, and granulocytes.17 Furthermore,wound repair studies using subcutaneous chambers havedemonstrated that TGF-(:JI induces rapid formation ofconnective tissue.14 In fetal wounds, the addition ofTGF-(:J to polyvinyl alcohol sponges implanted in fetalrabbits produces fibrosis,3 and TGF-(:J. slow release disksinduce dose-dependent scarring in human fetal skin afterwounding.4 In vitro studies have shown that TGF-(:JI en-hances the synthesis of collagen, fibronectin, elastin, hy-aluronate, and other matrix components.18-20 Exposureof fetal dermal fibroblasts to TGF-fJI results in markedupregulation of collagen gene expression.21 Thus, the cel-lular and matrix machinery that is necessary for scar for-mation exists in fetal wounds.

The cellular response to TGF-(:J¡ administration dur-ing wound repair is unclearo In situ hybridization studiesof an adult pig skin excisional model demonstrated thatTGF-(:JI injections enhance mRNA content of collagentype lo collagen type III, fibronectin, and TGF-fJI itselfwhile stromelysin mRNA expression decreases.22 Ourstudy shows that exogenous TGF-(:JI actually induces hu-man fetal fibroblasts to produce TGF-fJI mRNA at thewound si te and in the surrounding dermis. In vitro stud-ies have demonstrated that TGF-(:J. enhances transcrip-tion of its own mRNA and thus amplifies its effects.23Mediators released from inflammatory cells also havebeen shown to increase TGF-fJ. expression in adult fi-broblasts.14.21 Thus, the induction ofTGF-(:J1 mRNA infetal fibroblasts is probably the result of both the directeffect of exogenous TGF-(:JI and the recruitment of in-flammatory cells with their subsequent release of cyto-kines. Fetal fibroblasts have also been shown in vitro tobe capable of producing TGF-fJ 1.24 The presence of "ac-tivated" TGF-(:JI cells in the wound site appears to beimportant for the induction of scarring in fetal skinoTransforming growth factor-fJ. may have induced the fe-tal fibroblasts to become more adult-like.

Recent fetal wound healing studies have correlated theabsence of scarring with a sparse inflammatory response,as evidenced by markedly reduced neutrophil, macro-phage, and monocyte infiltrates,2S.26 absence of endoge-nous immunoglobulins at the wound site,27.28 reducedangiogenesis, and altered levels of peptide growth fac-tors.29 The transition of the fetal healing phenotype toa scarring adult phenotype in the marsupial correlatesdirectly with the amount ofinflammatory reaction at thewound site.30 Our study demonstrates that, in the subcu-

l. Shah M, Foreman DM, Ferguson MWJ. Control ofscarring in theadult wounds by neutralising antibody to transforming growth fac-

tor{3.Lancet 1992;339:213-214.2. Su\\ivan KM, Lorenz HP, Adzick NS. The Tole of transforming

growth factor beta in human fetal wound healing. Surg Forum

1993; 79:625-626.3. Krummel TM, Michna BA, Thomas BL, et al. Transforming

growth factor beta in a fetal wound modelo J PediatrSurg 1988; 23:

647-652.4. Su\\ivan KM, Lorenz HP, Meuli M, et al. A model of scarless hu-

man fetal wound repair is deficient in transforming growth factor

beta. J Pediatr Surg 1995; 30: 198-203.5. Lorenz HP, Longaker MT, Perkocha LA, et al. Scarless wound

repair: a human fetal skin modelo Development 1992; 124:253-

259.6. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Labo-

ratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Lab-

oratory Press, 1989, pp 7.53-7.57.7. Lane AT, Scott GA, Day KH. Development ofhuman fetal skin

transplanted to Dude mice. J Invest Dermatol1989; 93:787- 791.