The extracellular matri ofx lip wounds in fetal, neonatal ... · Key words: extracellular matrix, fetal wound healing, fibronectin, tenascin, collagen, chondroitin sulphate. Introduction

Development 112, 651-668 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

651

The extracellular matrix of lip wounds in fetal, neonatal and adult mice

DAVID J. WHITBY12 and MARK W. J. FERGUSON1*

1 Department of Cell and Structural Biology, School of Biological Sciences, University of Manchester, Coupland 3 Building, Manchester,M13 9PL, UK2Department of Plastic Surgery, University Hospital of South Manchester, Manchester, M20 9LR, UK

* Author for correspondence

Summary

Wound healing in the fetus occurs rapidly, by aregenerative process and without an inflammatoryresponse, resulting in complete restitution of normaltissue function. By contrast, in the adult, wounds healwith scar formation, which may impair function andinhibit further growth. The cellular mechanisms under-lying these differing forms of wound healing areunknown but the extracellular matrix (ECM), throughits effects on cell function, may play a key role. We havestudied the ECM in upper lip wounds of adult, neonataland fetal mice at days 14, 16 and 18 of gestation. Thespatial and temporal distribution of collagen types I, HI,IV, V and VI, fibronectin, tenascin, laminin, chondroi-tin and heparan sulphates were examined immunohisto-chemically. Results from the fetal groups were essen-tially similar whilst there were distinct differencesbetween fetus, neonatc and adult. Fibronectin waspresent at the surface of the wound in all groups at 1 hpost-wounding. Tenascin was also present at the woundsurface but the time at which it was first present differedbetween fetus ( lh), neonate (12 h) and adult (24 h). Thetime of first appearance paralleled the rate of woundhealing which was most rapid in the fetus and slowest inthe adult. Tenascin inhibits the cell adhesion effect offibronectin and during development the appearance of

tenascin correlates with the initiation of cell migration.During wound healing the appearance of tenascinpreceded cell migration and the rapid closure of fetalwounds may be due to the early appearance of tenascinin the wound. Collagen types I, ID, IV, V and VI werepresent in all three wound groups but the timing andpattern of collagen deposition differed, with restorationof the normal collagen pattern in the fetus and a scarpattern in the adult. This confirms that lack of scarringin fetal wounds is due to the organisation of collagenwithin the wound and not simply lack of collagenformation. The distribution of chondroitin sulphatediffered between normal fetal and adult tissues andbetween fetal and adult wounds. Its presence in the fetalwound may alter collagen fibril formation. No inflam-matory response was seen in the fetal wounds. Thedifferences in the ECM of fetal and adult woundssuggests that it may be possible to alter the adult woundso that it heals by a fetal-like process without scarformation, loss of tissue function or restriction ofgrowth.

Key words: extracellular matrix, fetal wound healing,fibronectin, tenascin, collagen, chondroitin sulphate.

Introduction

Wound healing is a complex process involving cellularmigration, proliferation and differentiation with extra-cellular matrix (ECM) synthesis and matrix remodelling(for review see Clark, 1988a). Many of these mechan-isms are common to development and wound healing.Recent studies have demonstrated significant differ-ences between adult and fetal wound healing (Adzick etal. 1985; Dixon, 1960; Hallock, 1985; Robinson andGoss, 1981; Rowsell, 1984). In the fetus, healing occursrapidly, by a regenerative process without scar forma-tion, while in the adult it leads to scar formation, oftenlimiting tissue function and restricting further growth.The fetal response to a wound may be related to its

developmental stage, the sterile aqueous fetal environ-ment or a combination of both factors.

These observations and the advent of surgery in uterofor lethal congenital anomalies (Harrison et al. 1990a,b)have stimulated interest in the possibility of in uterocorrection of craniofacial anomalies such as cleft lip andcleft palate (Christ, 1986; Dado et al. 1990). Theanticipated benefits of fetal repair are scarless healingwith normal midfacial growth and normal palatalfunction but further understanding of the mechanismsunderlying fetal wound healing is essential. Further-more knowledge of fetal wound healing may also allowmanipulation of the adult wound so that it heals in afetal-like manner without scar formation, with enor-mous benefits for all types of reconstructive surgery,

652 D. J. Whitby and M. W. J. Ferguson

where scar formation restricts tissue function andgrowth.

Collagen is the principal component of the ECM anda scar is loosely defined as an abnormal collection ofcollagen following wound repair. Recent studies of fetalwound healing have concentrated on collagen forma-tion (DePalma et al. 1989; Longaker et al. 1990; Merkelet al. 1988; Siebert et al. 1990), utilizing differentspecies, at varying gestational ages and several differentwound models. The multiplicity of models has pro-duced conflicting results, although there is agreementthat, in incised fetal skin, wound healing takes placerapidly, in a regenerative manner, without scar forma-tion and without an inflammatory response. Somestudies using wound implants have shown no collagendeposition, whilst others have found collagen within thefetal wound. However, because the implant alters thehealing process, it is difficult to relate these results tothe scarless healing seen in an incisional wound and therole of collagen in fetal wounds is still disputed.

The dermal ECM is a complex aggregate ofglycoproteins and glycosaminoglycans which in con-junction with collagen determine the physiologicalproperties of the tissue (Uitto et al. 1989). The roleplayed by these other components of the ECM in fetalwound healing is unknown but is likely to be significant.

The nature and time during development when thetransition from fetal to adult wound healing occurs isunknown, but it may relate to the developmental stageof the fetus, maturity of the fetal immune system (as aninflammatory response is not seen in the fetal wound) orthe change from fetal to neonatal environment.

To address these problems, in the least artificialmodel, we have carried out a detailed study of the ECMof incisional upper lip wounds in fetal mice at threegestational ages, in neonatal mice and in adult mice.Using indirect immunostaining, the spatial and tem-poral localisation of collagen types I, III, IV, V and VI,fibronectin, tenascin, laminin, chondroitin and heparansulphates during wound healing has been established.Direct immunostaining of endogenous immunoglobu-lins was used as a marker of an inflammatory responseat the wound.

Materials and methods

Five groups of wounds were studied: fetal wounds created atdays 14, 16 or 18 of gestation, neonatal wounds createdbetween 6 and 12 h post-parturition and adult wounds made inyoung mice of 6-8 weeks age. An upper lip wound was used inthis study for two reasons. (1) A wound of reproducible sizecan be made under direct vision after exteriorising the fetalsnout from the uterus, without significant fetal loss. (2) Thewound allows study of the healing of two epithelial surfaces(skin and oral mucosa) and mesenchyme including skeletalmuscle.

Day 16 and day 18 fetal woundsTime-mated, female, MF1 mice were anaesthetised withhalothane/oxygen/nitrous oxide on day 16 or day 18 ofgestation. A midline laparotomy exposed the pregnantuterus. Using an operating microscope (Wild), one horn of

the uterus was exteriorised and a purse-string suture of 10/0nylon placed through all layers of the uterine wall on its anti-mesenteric surface. A cruciate incision through the myome-trium and membranes allowed the snout of the fetus to bemanipulated out of the uterus (Fig. 1A-C,G). Placement ofthe suture superior to the visible position of the snout andextension of the fetal neck to exteriorise the snout preventedescape of the placental vessels or upper limbs, which areimpossible to return to the uterus. Use of the purse stringsuture restricts amniotic fluid loss and prevents expulsion ofthe fetus. A 2 mm, full thickness, vertical incision in the leftupper lip of the fetus was made with microsurgical scissors(Fig. ID and H). The edges of the wound lie in apposition andwere left unsutured. The fetus was returned to the uterus, thepurse string suture closed (Fig. IE and I) and the abdominalwound was repaired. Three fetuses were wounded in eachanimal. The pregnancies then continued until the fetuses wereharvested or birth occurred, on day 19 of gestation. For thefetal study, sham operations, where the above procedure wascarried out except for the lip incision, were used as controls.

Day 14 fetal woundsIn day 14 fetuses, this technique required modification as itproduced a high abortion rate and, in the remaining viablefetuses, there were marked deformities of the hind limbs andlower trunk. These appeared to be due to amniotic fluid lossand subsequent compression of the fetus by the myometrium.Day 16 and day 18 fetuses were more resistant to compressionand did not show these deformities. Mouse embryos developnormally and survive surgical manipulation if released fromthe myometrium, as long as the uterine membranes remainintact and the fetus is attached to the placenta (Muneoka et al.1986). In the day 14 fetus, following exposure of the uterus,the myometrium was incised longitudinally along its anti-mesenteric border, releasing the developing fetuses from thecompressive forces of the myometrium. A purse string suturewas placed through the uterine membranes only, which werethen incised to allow exteriorisation of the fetal snout. Theprocedure continued as described for the day 16 and day 18fetuses, with closure of the uterine membranes but withoutrepair of the myometrium.

Neonatal and adult woundsUnder anaesthesia 3-4mm, vertical, full thickness, upper lipwounds were made in neonatal mice between 6 and 12 h afterbirth and in adult mice at age 6-8 weeks. A single 6/0 nylonsuture was placed through the lower margin of the wound toappose the wound surfaces and the animals were allowed torecover from anaesthesia.

Wound harvest and sectioningAnimals were killed by an overdose of chloroform at multipletime points post-wounding (pw). In the fetal wounds, thesetime points were at 1, 6, 12,16, 20, 24, 48, 72 h and 5 days pwand, in both neonate and adult at 1, 6, 12, 24, 48, 72 h, 5, 7,and 12 days pw. With the 16 and 18 day fetal wounds, thelatter time points were during the neonatal period. It waspossible to recognise those neonatal animals wounded as afetus by the presence of a narrow furrow visible on the upperlip at the site of the original wound (Fig. 1J).

For the fetal and neonatal wounds, the head was snapfrozen in precooled isopentane, embedded in OCT compound(Miles Inc., Elkart, IN) and 7/an transverse sections cut at—20°C in a Leitz cryostat. For the adult wounds, the upper lip

ECM of lip wounds in fetal mice 653

H

Fig. 1. Operative procedure. (A) Midline abdominal incision. (B) Part of one uterine horn lifted from abdomen and sutureplaced in antimesenteric surface of uterus. (C) Uterus incised and fetal snout manipulated out of uterus. (D) Upper lipincision. (E and F) Uterine and abdominal incisions closed. (G) Fetus visible through uterine wall. (H) Upper lip incision.(I) Uterine suture closed. (J) SEM of 16 day fetal head 24 h post-wounding. The wound (arrow) is re-epithelialised. e, ear;5, snout; u, upper limb. Bar: lmm.

Table 1. Primary antibodies

Primary Ab raised against

Type I collagenType III collagenType IV collagenType V collagenType VI collagenFibronectinTenascinLamininChondroitin sulphateHeparan sulphate

MF 20 (light meromyosin)

Primary Ab raised in

RabbitGoatRabbitGoatRabbitSheepRabbitMouse (IgG) monoclonalMouse (IgM) monoclonalRabbit

Mouse (IgG) monoclonal

Source

Institut Pasteur de LyonSeralabInstitut Pasteur de LyonSeralabDr Shirley Ayad, ManchesterSerotecDr R. Chiquet-Ehnsman, BaselICN BiomedicalsICN BiomedicalsDr P. Bletchley and Dr J. Anderson

ManchesterDevelopmental Biology Hybridoma Bank,

Iowa

Dilution(in PBS)

1:401:1001:801:1001:1001:1001:2001:10001:4001:100

1:50

Secondary Ab(see Table 2)

1212131451

4

was excised, snap frozen and embedded as above and 7/antransverse sections cut.

ImmunohistochemistryPrimary and secondary antibodies used for indirect immuno-

staining are shown in Tables 1 and 2. Incubation with primaryantisera was for lh followed by three rinses in phosphate-buffered saline (PBS). Incubation with FITC-conjugatedsecondary antisera was for 1 h followed by a further threerinses with PBS. The sections were mounted in a non-fadingmedium, DABCO (l,4-diazobicyclo-{2,2,2}-octane), and


Table 2. FITC-conjugated secondary antibodies

Table 1 reference Secondary Ab raised against Secondary Ab raised in Source Dilution (in PBS)

RabbitGoatSheepMouse IgGMouse IgM

SheepRabbitDonkeyGoatGoat

SerotecNortheast BiomedicalsSerotecZymed LaboratoriesZymed Laboratories

1:1601:401:401:301:30

photographed using a Leitz Dialux microscope on KodakEktachrome 160 ASA film corrected for tungsten light. Blackand white prints were made from these colour slides. All ofthe antibodies used have previously published characterisa-tion and specificities. None the less, in this study, for eachantibody, specificity was tested by pre-absorption of theprimary antibody with biochemically purified antigen prior toimmunostaining by the above protocol. This abolished allstaining by the antibody. For each primary antibody and eachtime point, control sections were stained, substituting PBS forthe primary antibody.

Results

Overall 81.4% of the fetuses operated on survived(149/183). The majority of the failures were due toplacental disruption at the time of surgery or accidentalocclusion of the placental vessels whilst closing theuterus. 5 or 6 wounds were available for study at each ofthe time points in each fetal group. All neonatal (27)

and adult (27) animals survived providing 3 wounds forstudy at each time point. All control staining pro-cedures were negative.

The results from the three fetal groups wereessentially similar and will be presented as one group.The same elements of the ECM were present in eachwound group but there were distinct spatial andtemporal differences between the fetal and adultwounds. The neonatal wound had features in commonwith fetal and adult wounds, suggesting that a gradualshift occurs from fetal type to adult type wound healing,rather than an abrupt transition.

Tables 3 and 4 summarise the results.

Epithelialisation, tenascin and fibronectin depositionThe rate of re-epithelialisation and closure of themesenchymal defect varied between the three groups.In the fetal wounds epithelial migration was apparent at12 h pw and by 20 h pw re-epithelialisation of the skin

FN

TN

Fetus

Neonate

Adult

Fetus

Neonate

Adult

Epithelial-isauon

Table 3.1 hour

3Present at wound

margins andwithin clot

Present at wound

0

0

Epithelialisation,6 hours

3

1

0

0

12 hours

3

1

Present at wound

0

fibronectin (FN) and tenascin (TN) deposition in24 hours

3

3Within adjacent

dermis4

At epidermal-dermal junction

3Within adjacent

dermis4


Present at wound

margins

Complete in fetus

48 hours

3Patchy staining at

wound site

3

3

3Patchy staining at

wound site4


3

Within adjacentdermis

4At epidermal-

dermal junction

3Within adjacent

dermis4

At epidermal-dermaJ junction

Complete inneonate

72 hours

2Normal pattern

3Patchy staining at

wound site

3Patchy staining at

wound site

2Normal pattern

3

Patchy withinwound site

4At epidermal-dermal junction

3

Within adjacentdermis

4At epidermal-dermal junction

Complete in adult

5 days

2

3Patchy staining at

wound site

3

Patchy staining atwound site

2

2

Essentiallynormal pattern-reflecting loss of

hair follicles

3Within adjacent

dennis4


wound7 days

2

2Pattern reflects

scar

2

Pattern reflectsscar

2

2

3

Patchy withinwound site

4Al epidermal-dermal junction

12 days

2

2

2

2

2

3

At epidermal-dermal junction(Absent from

dennis)

Relative intensity of staining at wound site in comparison to normal tissue: 0, absent. 1, <normal; 2, normal (not normal pattern): 3. >normal; 4, ••normal.


Table 4. Collagen (C) and chondroitin sulphate (CS) deposition at site of wound

CI, III,V, VI

CS

civ

Fetus

Neonate

Adult

Fetus

Neonate

Adult

Fetus

Neonate

Adult

1-12 hours

0

0

0

0

0

0

0

0

0

24 hours

0

0

0

1

0

0

2Present in epithelialbasement membrane

0

0

48 hours

1All collagen types

present

1All collagen types

present

0

2 Normal pattern

0

0

2

2Present in epithelialbasement membrane

0

72 hours

2Regeneration of

normal tissuearchitecture

1All collagen types

present

0

2

0

0

2

2

2Epithelial BM

1Neovasculansation

5 days

2

2Normal pattern at

oral mucosa.Minimal scar at skin

1All collagen types

present

2

0

0

2

2

2Epithelial BM

3Neovasculansation

7 day's

2

2

3Parallel bundles of

collagen withinwound

2

0

0

2

2

2Epithelial BM

3Neovasculansation

12 days

2

2

3Parallel bundles of

collagen withinwound

2

0

0

2

2

2Epithelial BM

4Ncovasculansation

Relative intensity of staining at wound site in comparison to normal tissue: 0, absent; 1, <normal; 2, normal (not normal pattern), 3, >normal; 4, *• normal

wound was complete. Re-epithelialisation of the oralmucosal wound was virtually complete by 16 h and in allof the wounds re-epithelialisation of the oral mucosaproceeded more rapidly than closure of the skin wound.Re-epithelialisation preceded closure of the mesenchy-mal wound which was complete by 48 h pw. In theneonatal wounds, re-epithelialisation of both skin andoral mucosa was complete by 48 h pw and themesenchymal wound closed by 72 h. Re-epithelialisa-tion was complete in the adult wound by 72 h and, as inthe fetal and neonatal wounds, healing of the oralmucosa was quicker than healing of the skin wound.The connective tissue defect had closed by 5 d pw.

Fibronectin (Fn) was diffusely present in the mes-enchymal ECM of the normal tissues adjacent to thewound with a linear staining pattern at the level of thebasement membrane. In the fetal wounds at lh pw, alayer of Fn was present at the wound surface(Fig. 2A,D and F). Increased staining for Fn in theregion of the wound was present at all time points up to72 h pw in the 16 and 18 d fetal wounds. At 48 h theenhanced staining for Fn within the wound was patchy(Fig. 2B). In the 14 d fetal wounds at 48 h, the stainingpattern for Fn in the region of the wound wasindistinguishable from the adjacent normal tissue(Fig. 2E). Similarly in the 16 and 18 d wounds by 72 h anormal staining pattern for Fn was present and theoriginal site of the wound was not detectable (Fig. 2C).At 1 h pw in both neonatal and adult wounds, a layer ofFn was present at the surface of the wound (Fig. 2G and

H) and increased staining for Fn within the wound waspresent in both wound groups up to 7 d pw.

By contrast to Fn, tenascin (Tn) was very limited inits distribution. In the undamaged fetal tissues Tn waspresent in and below the basement membranes of theoral mucosa and the dermal-epidermal junction,particularly around the developing hair follicles(Fig. 3D). This pattern was also present in the normalneonatal and adult tissues although the staining wasdiscontinuous and less intense at the dermal-epidermaljunction.

At 1 h pw in the fetal wounds, tenascin was presenton the surface of the wound (Fig. 3A,E and F). By 24 hpw the staining for Tn was more prominent and Tn waspresent within the mesenchyme adjacent to the wound(Fig. 3B). The most intense staining at this time wasnear the basement membranes of the epidermis andoral mucosa adjacent to the wound. Tn persisted withinthe wound at 48 h pw (Fig. 3C) and at 72 h pw waspatchily present within the mesenchyme although stillprominent at the dermal-epidermal junction. By 5 d pwa normal pattern of staining for Tn had been restored(Fig. 3D) and the site of the wound was onlydistinguishable by a narrow epithelial furrow.

Tn was first present at the surface of the neonatalwounds 12 h pw (Fig. 3G). At the outer margin of thewound, where the dermis was exposed, Tn staining waspresent within the mesenchyme but not directly at thesurface of the wound (Fig. 3G - indicated by doublearrow). The margin of this staining precisely mirrored


Fig. 2. Transverse lip sections, skin uppermost, oral mucosa facing downwards. Stained for fibronectin (Fn). (A) 16d fetuslh post-wounding (pw). Fn widely distributed in mesenchymal ECM, with linear staining at basement membrane ofepidermis and oral mucosa. Layer of Fn seen at wound surface and within clot. (B) 16 d fetus 48 h pw. Wound re-epithelialised, mesenchymal defect closed. Patchy increased staining for Fn in wound area still visible. (C) 16d fetus 5d pw.Normal Fn pattern, no scar formation. (D) 14d fetus lh pw. Fn at wound surface. (E) 14d fetus 48h pw. Normal Fnpattern, no scar pattern. (F) 18d fetus lh pw. Fn at wound surface and within clot. (G) Neonate lh pw. Fn at woundsurface and within clot. (H) Adult lh pw. One edge of wound shown, with Fn at wound surface, h, hair follicle; m, oralmucosa; o, oral mucosal salivary gland; s, skin; t, tongue. Arrow: site of wound. Bar: 100im\.

the pattern of staining for endogenous immunoglobu-lins in the same wound (Fig. 7J). This precise congruityof staining patterns for Tn and endogenous immuno-globulins was only seen in the neonatal wounds at thistime point. In a similar manner to the fetal wounds, thestaining for Tn was more prominent within themesenchyme at 24 h and 48 h pw (Fig. 3H) with themost intense staining at the dermal-epidermal junction.By 5 d pw an essentially normal pattern of staining hadbeen restored although the site of the wound wasobvious because of the loss of hair follicles (Fig. 31).

In the adult wounds, Tn was first, faintly, visible atthe surface of the wound at 24h pw (Fig. 3J). Stainingwas more intense, adjacent to the original wound, at

48 h pw and by 5d pw Tn was diffusely present at thesite of the wound (Fig. 3K). This staining was lessmarked at 7d pw and at 12 d pw there was limitedpatchy staining within the healing connective tissue(Fig. 3L). At this time, intense staining for Tn waspresent at the dermal-epidermal junction where epi-thelial projections into the dermis (enhanced rete pegs)were prominent.

Collagen type TV, laminin and heparan sulphateIn undamaged fetal, neonatal and adult tissues,collagen type IV (CIV) localised in the epithelialbasement membranes of the skin and oral mucosa andin endothelial basement membranes (Fig. 4G). No new


staining for CIV was demonstrated in relation to thewound surface in any of the wound groups.

In the fetal wounds, by 5d pw a normal pattern ofCIV staining was restored and the pattern at the site ofthe wound was indistinguishable from the surroundingtissue (Fig. 4G and H). Re-epithelialisation was com-plete before CIV was present at the site of the reformedbasement membrane beneath the new epithelium. Inthe neonate, by 5 d pw on the oral side of the wound anormal pattern of CIV was restored. On the skin side ofthe wound staining for CIV highlighted the loss of hairfollicles although the pattern of staining was otherwiseessentially normal (Fig. 41). In the adult wound at 72 hpw re-epithelialisation was complete and CIV stainingwas present in the reformed basement membranes.

In the adult wound, at 5d (Fig. 4J) pw a band ofgranulation tissue containing Fn, CI, CIII, CV and CVI(see below) is present in the wound separating thenormal tissues on either side of the wound. A fewcapillaries are present within this tissue demonstratedby the staining of CIV within endothelial basementmembranes. By 7d pw, new capillary formation withinthe wound is more obvious (Fig. 4K) and, by 12 d pw,profuse neovascularisation with multiple capillary loopsis apparent (Fig. 4L). New capillary formation is anintegral part of adult wound healing and is welldemonstrated by staining of endothelial basementmembranes. Exuberant new vessel formation was notseen in the healing fetal or neonatal wounds.

Staining for laminin (Ln) showed a similar pattern tothe staining seen with CIV, as Ln co-localises inepithelial and endothelial basement membranes. Nostaining for Ln was seen in relation to the woundsurface and re-epithelialisation was complete beforestaining for Ln was visible in the reformed basementmembrane (Fig. 4A and B). Neovascularisation of theadult wound and its absence in the fetal wound was alsodisplayed by Ln staining.

Ln was present in the endomysium of myotubuleswhich in fetal wounds could be seen crossing the site ofthe original wound (Fig. 4C). Muscle regeneration inthe fetal wounds was also demonstrable by using anantibody against light meromyosin, MF 20, whichshowed myotubules crossing the original wounded area(Fig. 4D). Muscle regeneration was not seen inneonatal or adult wounds.

Heparan sulphate (HS) localised only at the base-ment membranes of the epidermis and oral mucosa. HSstaining at the wound was restricted to the reformingepithelial basement membranes and, in a similarpattern to CIV and Ln, re-epithelialisation was com-plete before HS was present in the basement membrane(Fig. 4E and F).

Collagen types I, III, V and VIType III collagen (CIII) was widely localised in themesenchymal and connective tissues of fetus, neonateand adult (Fig. 5). In the fetal wounds CIII was notpresent within the wound at 24 h pw (Fig. 5A). By 48hpw, collagen was visible within the wound (Fig. 5B)and, at 72h, the reticular pattern of CIII staining was

restored at the site of the wound (Fig. 5C). This patternwas indistinguishable from the pattern of CIII stainingin the tissues adjacent to the wound and the onlyindication of the site of the wound was an epithelialfurrow at the skin surface. Muscle regeneration wasalso demonstrated by CIII staining (Fig. 5D). In the14 d fetus, collagen deposition was seen earlier than in16 and 18 d fetuses (Fig. 5E) and regeneration of anormal reticular pattern was present at 48 h pw(Fig. 5F).

CIII was first deposited in the neonatal wounds at48 h pw (Fig. 5G), with more intense staining at 72 h pw(Fig. 5H) and, at 5 d pw on the oral side of the wound, anormal reticular pattern of CIII had been restored(Fig. 51). On the skin side, loss of hair follicles wasapparent but the pattern of CIII deposition still shows areticular configuration (Fig. 51), unlike the parallelbundles of collagen fibres seen in the adult wound(Fig. 5L). In the adult wound, CIII deposition is notseen until after.72 h pw (Fig. 5J). At 5 d, CIII is presentin the wound (Fig. 5K) and, at 12 d pw, bundles of CIIIlying parallel to the wound surface are seen disruptingthe normal tissue structure (Fig. 5L).

The other interstitial collagens studied, types I, Vand VI, showed essentially similar patterns to CIII withregeneration of a normal pattern in the fetal wounds,scar formation in the adult and a mixed pattern in theneonate. In each wound group these collagens were allpresent from the same time point that CIII was firstseen.

Type VI collagen (CVI) was distributed diffuselythroughout the ECM in an identical pattern to CIII. Itwas first seen in the fetal wound at 48 h pw (Fig. 6A andB) and a normal pattern was present at 72 h pw (Fig. 6C- compare with Fig. 5C). In the neonate, CVI waspresent at 48 h pw and a similar pattern to CIII was seenat 5 d pw with loss of hair follicles but an otherwiseessentially normal, reticular pattern of CVI. In theadult, CVI was present at 5d and at 12 d pw fibrils ofCVI are seen within the wound parallel to the woundsurface (Fig. 6D).

Type V collagen (CV) is diffusely present throughoutthe ECM but also localises in the epithelial andendothelial basement membranes. Staining for CI wasfaint but discernable in all wound groups. As with CIIIand CVI, staining for these collagens showed regener-ation of a normal pattern in the fetal wounds (Fig. 6E -compare with Figs 5C and 6C), a virtually normalpattern in the neonatal wounds, apart from the loss ofhair follicles, (Fig. 6F and H - compare with Fig. 51)and scar formation in the adult (Fig. 6G). As CVlocalises in the endothelial basement membranes, theneovascularisation of the adult wound was also appar-ent.

Chondroitin sulphateChondroitin sulphate (CS) was diffusely present in theECM of the fetal mesenchyme. The homogenousstaining pattern of CS was distinctly different from thefibrillar pattern seen with the interstitial collagens andFn. CS was not initially present within the fetal wound


Fig. 3. For legend see p. 660


Fig. 4. For legend see p. 660


Fig. 3. Transverse lip sections, skin uppermost, oralmucosa facing downwards. Stained for tenascin (Tn).(A) 16 d fetus lh pw. Tn present in the basementmembranes of the dermal-epidermal junction and oralmucosa. Tn staining present at surface of wound. (B) 16dfetus 24 h pw. Tn staining present in mesenchyme adjacentto wound, particularly at the interface with the dermal-epidermal junction. (C) 16 d fetus 48 h pw. Wound re-epithelialised and mesenchymal defect closed. Staining fortenascin present at site of wound. (D) 16d fetus 5d pw.Near normal pattern of Tn - no staining within wound.(E) 14d fetus 1 h pw. Tn present at surface of wound.(F) 18 d fetus 1 h pw. Tn present at surface of wound.(G) Neonate 12 h pw. Tn present at surface of wound.Where the dermis to the left of the wound is exposed, Tnis present within the dermis but not at the wound surface(indicated by double arrow). The limits of the staining forTn mirror the extent of the inflammatory response at thesurface of the wound demonstrated by staining forendogenous immunoglobulins (see Fig. 7J). (H) Neonate48 h pw. Intense staining for Tn within healing wound.(I) Neonate 5 d pw. Tn staining restricted to epithelialbasement membranes - note absence of hair follicles inskin side of wound but normal pattern of staining beneathoral mucosa. (J) Adult 24h pw. Faint staining for Tn atbasement membrane of epidermis and in hair follicles butno staining at wound surface. (K) Adult 5d pw. Intensestaining for Tn within healing wound. (L) Adult 12 d pw.Increased staining for Tn adjacent to basement membraneof epithelial ingrowths. Patchy staining for Tn still presentwithin healing wound, b, basement membrane; h, hairfollicle; m, oral mucosa; n, nasal cavity; o, oral mucosalsalivary glands; s, skin; t, tongue. Arrow: site of wound.Bar: 100/an.Fig. 4. Transverse lip sections, skin uppermost, oralmucosa facing downwards. (A-C) stained for laminin (Ln).

(A) 18 d fetus 24 h pw. Ln present in basement membranesof epidermis and hair follicles, oral mucosa andendothelium. Epithelialisation of wound complete, Lnabsent beneath healed epithelium. No staining for Lnrelated to wound. (B) 18 d fetus 24h pw. Highermagnification of oral mucosa from Fig. 4A. Note stainingof Ln in mucosal basement membrane and absence ofstaining beneath re-epithelialised wound. (C) 16d fetus 5dpw. Ln staining outlines myotubules regenerating at thesite of the wound.(D) Staining pattern with MF 20(meromyosin). 16 d fetus 5d pw. Staining of MF20highlights myotubule regeneration. (E-F) Stained forheparan sulphate (HS). (E) 16 d fetus 20 h pw. HS presentin basement membrane of undamaged oral mucosa, butabsent beneath migrating epithelium of the wound. (F) 16dfetus 24 h pw. Basement membrane beneathreepithelialised wound has regenerated, showing presenceof HS. (G-L) Stained for type IV collagen (CIV). (G) 16dfetus 5 d pw. CIV localised in epithelial and endothelialbasement membranes. Staining pattern within woundindistinguishable from normal surrounding tissues - no scarformation and no neovascularisation. (H) 14 d fetus 72 hpw. Normal pattern of CIV, no scar formation. (I) Neonate5d pw. Regeneration of normal pattern of staining for CIVat oral mucosal aspect of wound. CIV shows normalvascular pattern in skin aspect of wound but loss of hairfollicles. (J) Adult 5d pw. Band of scar tissue, containingCI, CHI, CV, and CVI (see Fig. 5), separating normaltissues. Within this scar tissue scanty neovascularisation isapparent, with CIV present in the basement membrane ofnew capillaries. (K) Adult 7d pw. Furtherneovascularisation present within site of wound. (L) Adult12 d pw. Profuse neovascularisation with multiple capillaryloops present in wound site, b, basement membrane; m,oral mucosa; mt, myotubules; s, skin; t, tongue. Arrow:site of wound. Bar: 100 pcm.

(Fig. 7A) but from 20 h pw CS was present in the wound(Fig. 7B-D).

In the neonate, the distribution of CS within themesenchyme is less diffuse and more marked on theoral side of the wound (Fig. 7E). CS was not presentwithin the wound at any of the time points studied(Fig. 7F) and by 5 d pw CS is largely localised aroundthe hair follicles and oral mucosal glands (Fig. 7G).This is similar to the pattern seen in the adult tissueswhere CS was only localised around the hair folliclesand was not present within the wound except for a very

small area immediately below the regenerated base-ment membrane (Fig. 7H).

Inflammatory responseStaining for endogenous immunoglobulins was used asan indicator of an inflammatory response in thewounds. The fetal wounds did not show any stainingspecific to the wound using anti-mouse IgG or IgMantibodies (Fig. 71). Both neonatal and adult woundsshowed an inflammatory response mainly around theskin wound and within the clot filling the wound. This

Fig. 5. Transverse lip sections, skin uppermost, oralmucosa facing downwards. Stained for type III collagen(CIII). (A) 16 d fetus 24 h pw. CIII widely distributed inECM of mesenchyme, no staining for CIII within wound.(B) 16 d fetus 48 h pw. Wound re-epithelialised and CIIIpresent within wound. (C) 16d fetus 72h pw. Normalpattern of CIII staining regenerated at site of wound,without scar formation. Epithelial furrow of skin is theonly remaining indicator of the original wound. (D) 16 dfetus 72 h pw. Regenerating myotubules on oral side ofwound outlined by CIII present in endomysium. (E) 14dfetus 24 h pw. First appearance of CIII within healingwound. (F) 14d fetus 48 h pw. Regeneration of normalpattern of CIII staining, no scar formation. (G) Neonate48 h pw. Widespread distribution of CIII within connective

tissue of lip. First appearance of CIII within wound.(H) Neonate 72 h pw. Dense staining for CIII withinwound. (I) Neonate 5d pw. On the oral side of wound, anormal pattern of CIII has regenerated. On the skin sideof the wound, loss of hair follicles is apparent. Apart fromthis loss, the staining for CIII appears almost normal andthe prominent parallel bundles of collagen seen in the scarformed in the adult wound (see Fig. 5L) are not seen inthe neonatal wound. (J) Adult 72 h pw. CIII present inconnective tissue of lip, not present within wound.(K) Adult 5d pw. CIII present within wound. (L) Adult12 d pw. Parallel bundles of CIII forming scar tissuedisrupting normal tissue structure, h, hair follicle; m, oralmucosa; mt, myotubules; s, skin. Arrow: site of wound.Bar: 100 ym.


persisted in the outer part of the wound afterepithelialisation was complete. In the neonate, this waspresent from 12 h pw (Fig. 7J) to 48 h pw and in theadult from 12h pw to 72h pw (Fig. 7K-L).

Discussion

Fibronectins are dimeric glycoproteins, which undergopost-transcriptional splicing to form at least 20 isotypes


Fig. 6. Transverse lip sections, skin uppermost, oral mucosa facing downwards. (A-D) Stained for collagen type VI (CVI).(A) 16 d fetus 48 h pw. CVI diffusely present in ECM of mesenchyme and present within wound. (B) 16d fetus 48 h pw.High power view showing faint staining for CVI within wound. (C) 16 d fetus 5d pw. Regeneration of normal pattern forCVI without scar formation. (D) Adult 12 d pw. Fibres of CVI parallel to wound surfaces forming scar tissue. (E-G)Stained for collagen type V (CV). (E) 16d fetus 5d pw. CV diffusely present within ECM and also present as linearstaining of basement membranes. Normal pattern of staining for CV at site of wound without scar formation. (F) Neonate5 d pw. Normal pattern of CV staining except for loss of hair follicles on skin side of wound. Regeneration of normalpattern beneath oral mucosa. (G) Adult 12 d pw. Band of scar tissue containing numerous new capillaries with CV stainingof endothelial basement membrane (cf. Fig. 4L). (H) stained for collagen type I (CI). Neonate 5d pw. Faint staining forCI. Similar pattern to CIII (Fig. 5 I), m, oral mucosa; s, skin. Arrow: site of wound. Bar: 100 fan.

(Hynes, 1985), present in many connective tissues and,as a soluble form, in plasma. Fibronectin is part of theECM early in embryonic development (for review seeDuband et al. 1987) where its primary role is mediatingcell adhesion and cell migration (Duband et al. 1988).During wound healing, fibronectin has many potentialroles (for review see Clark, 19886): it acts as asubstratum for cell migration (Donaldson and Mahan,1983; Clark et al. 1982; Knox et al. 1986), as an opsonin(Martin et al. 1988) and as a provisional matrix for ECMassembly (Grinnell et al. 1981; Kurkinen et al. 1980;McDonald, 1988). The pattern of fibronectin splicing in

some areas of a wound resembles an embryonic pattern(ffrench-Constant et al. 1989), which raises the possi-bility that different isoforms may have varying functionswithin the wound. In this study the timing and patternof deposition of fibronectin within the wound wassimilar in the three wound groups, suggesting that itsfunction may be similar in adult and fetal wounds.

Tenascin is a glycoprotein with a very limiteddistribution in adult connective tissues (Chiquet-Ehrismann, 1990; Erickson and Bourdon, 1989). It ispresent in the mesenchyme during development ofperipheral nerves (Wehrle and Chiquet, 1990), mam-


mary gland (Chiquet-Ehrismann et al. 1986), tooth(Thesleff et al. 1987; Vainio et al. 1989), kidney(Auferheide et al. 1987), cartilage and bone (Mackie etal. 1987), and is cyclically regulated in the stroma ofnormal human breast as a function of the menstrualcycle (Ferguson et al. 1990). It has also been found inthe stroma of mammary carcinomas (Chiquet-Ehris-mann et al. 1986; Ferguson et al. 1990), basal cellcarcinomas (Stamp, 1989) and endometrial carcinomas(Vollmer et al. 1990).

In this study, the distribution of tenascin inunwounded skin and oral mucosa was similar in adultand fetus and concurred with the recent studies ofLightner etal. (1989) and Sloan etal. (1990). The spatialdistribution of tenascin in the wound was similar in fetusand adult, but the time post-wounding at which tenascinwas first present varied from fetus to neonate to adult,as did the period of time during which tenascin waspresent in the wound. Tenascin was first detected at thewound margins in the fetus at 1 h, in the neonate at 12 hand in the adult, the first faint staining for tenascin wasat 24h. In the fetus and the neonate, the pattern fortenascin staining had reverted to normal by 5d pw,while in the adult tenascin was still present within themesenchyme at 12 d pw. The time at which tenascin isfirst seen parallels the rate of epithelialisation andclosure of the mesenchymal wound, which was mostrapid in the fetus (complete by 24 h), slower in theneonate (complete by 48 h) and slowest in the adult(complete by 72 h).

Tenascin and fibronectin are both found in themigratory pathways of neural crest cells during develop-ment and the appearance of tenascin correlates with theinitiation of cell migration (Mackie et al. 1988a).Fibronectin mediates cell-substratum adhesionthrough its interaction with cell surface receptors of theintegrin family (Pytela et al. 1985; Buck and Horwitz,1987). Tenascin interferes with integrin-mediated fibro-blast attachment to fibronectin (Chiquet-Ehrismann etal. 1988) and addition of exogenous tenascin will inhibitmesodermal cell migration during gastrulation (Riou etal. 1990). Tenascin is present in adult wounds (Mackieet al. 19886) and may have a critical role in initiating cellmigration. In this study, the appearance of tenascinpreceded keratinocyte and fibroblast migration and theearly appearance of tenascin in fetal wounds, throughits initiation of cell migration, may underlie the rapidepithelialisation seen in fetal wound healing. In relationto epithelialisation, it is of interest that the sequence fortenascin has recently been deduced and the moleculecontains thirteen consecutive epidermal-growth-factor-like repeats (Pearson et al. 1988).

Re-epithelialisation of the wounds was completebefore components of the basal lamina (Type IVcollagen, laminin and heparan sulphate) reformedbeneath the recently migrated epithelium. This patternwas seen in both fetal and adult wounds and is inagreement with previous studies showing that thebasement membrane does not reform until afterepithelial cells cease to migrate (Stanley et al. 1981).

All five types of collagen studied were present within

the fetal, neonatal and adult wounds. The timing ofcollagen deposition in the wound varied from fetus toadult - in the fetal and neonatal wounds collagen waspresent from 48 h pw, whilst in the adult all fivecollagens were seen within the wound from 5 d pw. Ineach wound group the different collagen types ap-peared concurrently (eg. in the fetus all collagen typeswere first apparent at 48 h).

For the interstitial collagens (types I, III, V, and VI),the critical difference between fetus and adult was in thesupramolecular organisation of the collagen fibrilsdeposited in the wound. In the fetus, a reticularcollagen pattern was restored and this was indis-tinguishable from the surrounding normal tissue. In theadult, large, parallel bundles of collagen, typical of scartissue, are seen running across the wound site approxi-mately at right angles to the basement membrane, thusdisrupting normal tissue architecture. One fundamentaldifference between fetal and adult wound healing istherefore the control and patterning of collagenfibrillogenesis.

Collagen fibril formation is a complex process whichis only partially understood (Burgeson, 1988a,b;Fleischmajer, 1986). Collagen types I, III and V arepresent within the same fibrils (Birk et al. 1988;Burgeson, 1988; Keene et al. 1987) and interactionsbetween different collagens will alter fibril size in vitro(Birk et al. 1990). Interactions with other macromol-ecules are also likely to influence collagen fibrillogen-esis. Proteoglycans will alter fibril size (for review seeScott, 1988; Vogel and Trotter, 1987) as will fibronectin(Speranza et al. 1987). Finally the role of the cell has tobe considered. Fibroblasts separate stages of fibrilformation in distinct cellular compartments allowingthe cell to control the final fibre orientation (Yang andBirk, 1988).

Several of the differences in the ECM of fetal andadult wounds, shown in this study, may potentially altercollagen fibril formation. Collagen types I, III, V andVI are present concurrently in both fetal and adultwounds but the relative proportions of these collagentypes is unknown. Fetal tissues contain a higherproportion of type III collagen to type I collagen(Epstein, 1974; Merkel et al. 1988) and this mayinfluence collagen fibril size. The role of glycosamino-glycans is intriguing as studies have shown high levels ofhyaluronic acid in fetal wounds (Krummel et al. 1987;Longaker et al. 1989; De Palma et al. 1989) whilstaddition of hyaluronic acid to an adult tympanicmembrane wound decreased scar formation and alteredthe degree of organisation of collagen within the wound(Hellstrom and Laurent, 1987). The present studydemonstrated the presence of chondroitin sulphatewithin fetal wounds at the time of collagen fibrilformation and its absence from the adult wound. Theinteractions of sulphated GAGs with collagen may alterfibril formation within the fetal wound. Scott andHughes (1986) demonstrated that during development,fetal bovine and chick collagen fibrils were small indiameter at a time when hyaluronic acid was abundantand a rapid increase in collagen fibril size during mid-


gestation coincided with a decrease in hyaluronic acidand chondroitin sulphate content.

The different patterning of collagen fibrils in healingfetal (regeneration of normal dermal pattern) and adult

(scar consisting of parallel, densely packed collagenfibrils at right angles to the epithelial surface) woundsmay therefore relate to differences in the cellular andextracellular milieu influencing the process of fibrillo-


Fig. 7. Transverse lip sections, skin uppermost, oralmucosa facing downwards. (A-H) Stained for chondroitinsulphate (CS). (A) 16 d fetus 12 h pw. Diffuse staining forCS in mesenchymal ECM. No staining for CS withinwound. Note the blotchy pattern of staining contrasts withthe fibrillar pattern seen with collagens and fibronectin.(B) 16 d fetus 20 h pw. CS is now present within wound.(C) 16 d fetus 20 h pw. Higher power view of (B). (D) 16 dfetus 48 h pw. (E) Neonate 6h pw. Patchy staining for CSwithin connective tissue of lip, more obviously beneath oralmucosa. No staining for CS within wound. (F) Neonate48 h pw. Unlike fetal wound CS not present within woundat later time point. (G) Neonate 5d pw. Wound healed,staining for CS largely confined to dermis surrounding hairfollicles and oral mucosal glands and connective tissuebeneath oral mucosa. (H) Adult 7d pw. Staining for CSlocalised to dermis surrounding hair follicles. CS notpresent within wound except for a very small areaimmediately adjacent to the regenerated basementmembrane. (I-L) Stained for immunoglobulin IgG andIgM. (I) 16 d fetus 12 h pw. No endogenousimmunoglobulins demonstrated in fetal wound. (J) Neonate12 h pw. Immunoglobulins present at wound surface.(Compare tenascin staining in Fig. 3G.) (K) Adult 24 h pw.Immunoglobulins present at wound surface and within clot.(L) Adult 48 h pw. Immunoglobulin staining within healingwound, h, hair follicle; m, oral mucosa; o, oral mucosalsalivary glands; s, skin. Arrow: site of wound. Bar: 100pan.

genesis, as discussed above. It may also relate todifferences in the primary wound 'scaffolding' intowhich fibroblasts migrate and lay down collagen fibrils.The fetal wound shows rapid epithelial and mesenchy-mal migration, the latter into a loose honeycomblattice, rich in (and presumably held open by)glycosaminoglyeans, proteoglycans and glycoproteins.Thus fetal fibroblasts can rapidly and easily migrate intoa loose wound lattice and rapidly deposit collagenfibres. The transmission of mechanical forces to thesefibrils by fibroblast tractioning, within a compliantmatrix, may orientate the fibrils in a reticular patternsimilar or identical to that of the normal dermis. Bycontrast, adult wounds show slow fibroblast migrationinto a denser, more resistant, wound matrix wherefibroblasts seem to migrate more easily up and down thewound margins rather than into the body of the woundscaffold itself. Within the adult wound the 'stiffer'matrix and the forces of wound contracture generatedby myofibroblasts may alter the orientation of collagenfibrils leading to the early establishment of an abnor-mal, scar like, orientation. The latter may be com-pounded by (unknown) differences in the tissue forcesbetween fetal, neonatal and adult lip wounds generatedas a result of function and movement of the lips.

Moreover, the extracellular matrix of the fetal (andneonatal) dermis is less highly ordered, has a morerapid turnover and consists of less differentiated cellscompared to the adult: these may enhance the scarlesshealing pattern of the fetus. Interestingly, studies ofwound healing in adult hair follicles have shown scarfree healing of the damaged dermal papilla (Jahoda andOliver, 19846) and the fibroblast-like cells of the papilla

retain characteristics associated with embryonic celllines, such as cell aggregation in tissue culture (Jahodaand Oliver, 1984a).

In the present study, wound healing was always fasterand more fetal like, on the oral mucosal side of theincision than on the skin side, even in the adult. Theadult oral mucosa possesses many embryonic character-istics (Sloan et al. 1990) and its structure is relativelysimple by comparison with the complex, heavilykeratinized, structure of adult epidermis. The epi-thelium of the adult oral mucosa is more fetal-like in itsstate of differentiation (as reflected in the expression ofcytokeratins), human adult oral mucosal fibroblastsexhibit numerous embryonic characteristics and ad-ditionally the oral mucosa is bathed in fluid (saliva) richin growth factors and antimicrobial agents - like fetalskin and amniotic fluid. This study has concentrated onthe ECM, as the functional properties (and problems)of a scar are largely due to its collagenous structure.However the state of epithelial differentiation andpotential epithelial-mesenchymal interactions duringwound healing requires further investigation and astudy of cytokeratins in fetal and adult wounds hasrecently been completed (Whitby and Ferguson, inpreparation).

Localisation of type IV collagen and laminin demon-strates the profuse neovascularisation present in theadult wound. The absence of neovascularisation in thefetal wounds suggests either a reduction in the level ofangiogenic factors in such wounds or an inability of thecells to respond to such factors or both. A lack ofstimuli correlates with the absence of a markedinflammatory response in the fetal wound, demon-strated in this study by the lack of endogenousimmunoglobulins. The absence of a cellular inflamma-tory response in fetal wounds is noted in several studies(Rowsell, 1984; Hallock, 1985; Burrington, 1971;Sopher, 1975) and the role of the macrophage, whichplays a key role in adult wound healing (Liebovich andRoss, 1975), in fetal wound healing is unknown.

A number of differences between the ECM of thefetal and adult wound were demonstrated in this study,but the mechanisms controlling these differing pro-cesses are undefined. The differences in the ECM, theabsence of neovascularisation in the fetal wound andthe rapid healing of oral mucosal wounds may all relateto the growth factors present in the fetal wound. Forexample TGF/3 will increase the synthesis of collagens,fibronectin and tenascin (Ignotz and Massague, 1986;Roberts etal. 1986; Pearson etal. 1988) and Krummel etal. (1988) have shown that addition of TGF to a fetalwound induces fibrosis. A separate study (Whitby andFerguson, in preparation) analysing the distribution ofvarious growth factors in fetal and adult wound healinghas demonstrated variations in TGF/J and bFGFdistribution between fetal and adult wounds, which maybe causative of some of the differences observed in thepresent study.

Finally, the present study has demonstrated that, inthe mouse, there is no sudden switch from a fetal to anadult wound healing pattern; rather there is a slow


transition in and around the neonatal period. This slowtransition is also present in long gestation animals, suchas sheep, where it occurs in the third trimester(Longaker et al. 1990). Such a slow transition isindicative of the multifactorial differences between fetaland adult wound healing: rapidity of wound closure,degree of inflammatory response, maturity and com-petence of the immune system, degree of organisationand differentiation of the wounded tissues, compositionand organisation of the extracellular matrix, presenceor absence of an aqueous sterile environment (amnioticfluid) rich in growth (and other) factors, differences intissue oxygenation, wound metabolism and pH, degreeof tissue tension from function, presence or absence ofacute inflammation. Moreover, whether or not fetalwounds scar also depends upon: the type and size of thewound (large, deep excisional wounds scar), the tissuewounded (internal tissues e.g. diaphragm, scar) (Lon-gaker et al. 1991) and the species used - rabbit fetusesdo not heal their wounds properly (Krummel et al.1987) probably due to the large amounts of maternalimmunoglobulin in rabbit amniotic fluid as a result ofthe size of the zona chorion during placentation (Wild1965). Therefore investigations searching for a magictransition point (and differences at that point) (Ihara etal. 1990) are unlikely to be productive.

The differences between fetal and adult woundhealing are multiple and subtle. This study suggests anumber of ways in which the healing adult wound mightbe experimentally manipulated to reduce scarring:enhance cell migration by addition of exogenoustenascin, develop a more open early wound scaffold tofacilitate fibroblast migration and regeneration of thenormal reticular collagen fibril orientation by additionof exogenous glycosaminoglycans and proteoglycans,clonally expand or transplant (e.g. from the oralmucosa) fibroblasts with embryonic characteristics atthe wound site. It may be possible to manipulate adultwound healing to show a more fetal-like, scarless,pattern by altering only one or two of the (major)components that are different between fetal and adulthealing.

We are grateful to Dr Ruth Chiquet-Ehrismann and DrShirley Ayad for the gifts of anti-tenascin and anti-type VIcollagen antibodies respectively. We would like to thank MrsM Poulton for technical assistance. This work was funded by agrant from the North West Regional Health Authority. Wethank the trustees of the Randall Champion Fund atWithington Hospital, Manchester, the ABC trust of BoothHall Hospital, Manchester, and the North West branch of theCleft Lip and Palate Association (CLAPA) for theiradditional financial support.

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{Accepted 21 March 1991)

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The extracellular matri ofx lip wounds in fetal, neonatal ... · Key words: extracellular matrix, fetal wound healing, fibronectin, tenascin, collagen, chondroitin sulphate. Introduction