DEVELOPMENTALBIOLOGY147, 207-215 (1991)
Immunohistochemical Localization of Growth Factors in Fetal Wound Healing DAVID J. WHITBY*'t AND MARK W. J. FERGUSON *'1 *Department of Cell and Structural Biology, School of Biological Sciences, University of Manchester, Coupland 3 Building, Manchester, M13 9PL, United Kingdom; and t Department of Plastic Surgery, St James' University Hospital, Leeds, United Kingdom Accepted May 24, 1991
Fetal wound healing occurs rapidly, in a regenerative fashion, and without scar formation, by contrast with adult wound healing, where tissue repair results in scar formation which limits tissue function and growth. The extracellular matrix deposited in fetal wounds contains essentially the same structural components as that in the adult wound but there are distinct differences in the spatial and temporal distribution of these components. In particular the organization of collagen in the healed fetal wound is indistinguishable from the normal surrounding tissue. Rapidity of healing, lack of an inflammatory response, and an absence of neovascularization also distinguish fetal from adult wound healing. The mechanisms controlling these differing processes are undefined but growth factors may play a critical role. The distribution of growth factors in healing fetal wounds is unknown. We have studied, by immunohistochemistry, the localization of platelet-derived growth factor (PDGF), transforming growth factor # (TGFfl), and basic fibroblast growth factor (bFGF), in fetal, neonatal, and adult mouse lip wounds. TGFfl and bFGF were present in neonatal and adult wounds, but were not detected in the fetal wounds, while PDGF was present in fetal, neonatal, and adult wounds. This pattern correlates with the known effects in vitro of these factors, the absence of an inflammatory response and neovascularization in the fetal wound, and the patterns of collagen deposition in both fetal and adult wounds. The results suggest that it may be possible to manipulate the adult wound to produce more fetal-like, scarless, wound healing. © 1991 Academic Press, Inc. INTRODUCTION In both fetus and adult, wound healing occurs by a process involving cell m i g r a t i o n , proliferation, a n d diff e r e n t i a t i o n , r e m o v a l of d a m a g e d tissue, and s y n t h e s i s of an e x t r a c e l l u l a r m a t r i x (ECM). However, in the fetus, in incised skin wounds, healing occurs rapidly, w i t h o u t an i n f l a m m a t o r y response, leading to r e s t o r a t i o n of n o r m a l tissue a r c h i t e c t u r e and function (Robinson and Goss, 1981; Rowsell, 1984; Hallock, 1985; L o n g a k e r et al., 1990; W h i t b y a n d Ferguson, 1991), while in the a d u l t the end result is tissue r e p a i r by scar f o r m a t i o n , w i t h subseq u e n t loss of function and r e s t r i c t i o n of growth. Defining the m e c h a n i s m s controlling t h e s e differing processes m a y indicate w a y s to a l t e r the adult w o u n d so t h a t healing occurs in a m o r e fetal-like m a n n e r , w i t h o u t scar f o r m a t i o n . The ECM deposited in f e t a l and a d u l t w o u n d s h a s m a n y c o m p o n e n t s in c o m m o n (glycoproteins, collagen t y p e s I, III, IV, V, VI, and proteoglycans), b u t the t i m i n g and p a t t e r n of deposition of t h e s e e l e m e n t s differs ( W h i t b y and Ferguson, 1991; W h i t b y et al., 1991). As the functional p r o p e r t i e s (and p r o b l e m s ) of scar tissue in the a d u l t are a reflection of its collagenous s t r u c t u r e , 1To whom reprint requests and correspondence should be addressed.
the p a t t e r n of collagen deposition in f e t a l and a d u l t wounds is of p a r t i c u l a r interest. In the f e t a l wound, collagen is deposited in a r e t i c u l a r p a t t e r n indistinguishable f r o m the adjacent, n o r m a l , tissue, while in the adult, large, parallel bundles of collagen are laid down p e r p e n d i c u l a r to the w o u n d s u r f a c e to f o r m a s c a r ( W h i t b y a n d Ferguson, 1991; L o n g a k e r et al., 1990). The m e c h a n i s m s controlling ECM s y n t h e s i s d u r i n g w o u n d h e a l i n g a r e unknown, but the effects, in vivo and i n vitro, of several g r o w t h f a c t o r s suggest t h a t t h e y p l a y an i m p o r t a n t role in tissue r e p a i r (for review see H u a n g et al., 1988; Fox, 1988; Assoian, 1988; K s a n d e r , 1989; M c G r a t h , 1990; Kovacs, 1991). P l a t e l e t - d e r i v e d g r o w t h f a c t o r ( P D G F ) is c h e m o t a c t i c and a p o t e n t m i t o g e n for fibroblasts, c h e m o t a c t i c for m o n o c y t e s a n d n e u t r o p h i l s (Ross et al., 1986), a n d can r e g u l a t e collagen s y n t h e s i s ( N a r a y a n a and Page,1983). T r a n s f o r m i n g g r o w t h f a c t o r # (TGF#) can s t i m u l a t e or inhibit cell p r o l i f e r a t i o n , induce ECM synthesis, p a r t i c u l a r l y collagen and fibronectin (Ignotz and Massague, 1986; R o b e r t s et al., 1986; Ignotz et al., 1987), and i n h i b i t m a t r i x d e g r a d a t i o n (Laiho et al., 1986). Basic fibroblast g r o w t h f a c t o r ( b F G F ) is s t r o n g l y angiogenic a n d also s t i m u l a t e s fibroblast prol i f e r a t i o n and ECM s y n t h e s i s (Rifkin and Moscatelli, 1989). T h e r e are m a n y differences b e t w e e n the f e t a l a n d the a d u l t w o u n d which m a y a l t e r the h e a l i n g p r o c e s s - sterile aqueous f e t a l e n v i r o n m e n t , the absence of an in-
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0012-1606/91 $3.00 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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TABLE 1 PRIMARY (1-3) AND FITC-CoNJUGATED SECONDARY (4, 5) ANTIBODIES
Antibody raised against
Antibody raised in
Source
Dilution (in PBS)
1
PDGF (Human platelets)
Goat
British Biotechnology, Oxford (BDA 16)
1:40
2
TGFfi (Porcine platelets) (detects TGFfil and ~2)
Rabbit
British Biotechnology, Oxford (BDA 5)
1:5
3
bFGF (bovine brain)
Rabbit
British Biotechnology, Oxford (BDA 4)
1:40
4
Rabbit IgG
Sheep
Serotec
1:160
5
Goat IgG
Rabbit
Northeast Biomedicals
1:40
flammatory response in the fetal wound, the state of differentiation of the fetal cells, biomechanical forces at the wound site, and tissue oxygenation and p H - - a n d some of these differences may exert an effect by altering the growth factors present in the fetal wound; amniotic fluid contains growth factors (Haigh et aL, 1989); whilest macrophages, as part of an inflammatory response, release growth factors. However, the growth factor profile of healing fetal wounds is unknown. We have therefore carried out an immunohistochemical study to localize the distribution of PDGF, TGF/~, and bFGF in lip wounds in fetal, neonatal, and adult mice. MATERIALS AND METHODS
Upper lip wounds in Day 16 fetal mice, neonatal mice, and adult mice were studied, from 1 to 72 hr postwounding in the fetus and from 1 to 120 hr postwounding in the neonate and adult. This wound is reproducible, can be made under direct vision, without significant fetal loss, and the healing of two differing epithelial surfaces (skin and oral mucosa) can be compared. F e t a l Wounds
At Day 16 of gestation, time-mated, MF1 mice were anesthetized using a halothane/oxygen/nitrous oxide mixture. A 2-mm, vertical, full thickness wound was created in the left upper lip of the fetus using techniques described by Whitby and Ferguson (1991). The wound surfaces lay in apposition without a suture. Three fetuses were wounded in each pregnant animal. Following
recovery from anesthesia the pregnancy continued until the wounds were harvested. Neonatal and A d u l t Wounds
Under halothane/oxygen/nitrous oxide anesthesia 3mm, vertical, full thickness wounds were created in the left upper lip of neonatal mice (within 12 hr of birth) and adult mice, age 6-8 weeks. A single 6 / 0 nylon suture was placed to appose the wound surfaces. Wound H a r v e s t and Tissue Preparation
Animals were killed by an overdose of chloroform at multiple time points following wounding. Fetal wounds were harvested 1, 6, 12, 18, 24, 36, 48 and 72 hr postwounding. In the adult and neonate, wounds were harvested 1, 6, 12, 18, 24, 36, 48, 72 and 120 hr postwounding. Following excision of the lower lip and mandible, the fetal and neonatal heads were snap frozen in precooled isopentane over liquid nitrogen, and the tissue was erabedded in OCT compound (Miles Inc., Elkart. IN). The adult upper lips were excised intact, snap frozen, and embedded in OCT. Seven-micrometer transverse sections were cut at -20°C in a Leitz cryostat, acetone fixed, and air dried. Sections were stored at -70°C prior to immunostaining. I m m u no h istoc h e m ist ry
The primary and secondary (FITC-conjugated) antibodies used for indirect immunostaining are detailed in
FIG. 1. Transverse lip sections. Stained for PDGF. (A) 16-day fetus 1 h r postwounding (pw). PDGF is present at the margins of the central part of the wound. (B) 16-day fetus 20 hr pw. PDGF is present at the margins of the wound and within the fibrillar clot at the skin surface. (C) 16-day fetus 24 hr pw. Wound reepithelialized and mesenchymal defect closed. Residual staining for PDGF beneath reformed epidermis. (D) Neonate i hr pw. PDGF is present throughout the clot filling the wound and at the margins of the wound. (E) Neonate 48 hr pw. Reepithelialization complete and dermal wound closed. PDGF is only present beneath the epidermis at the skin surface of the wound and within the clot on the surface of the wound. (F) Neonate 48 hr pw. Control section substituting PBS for primary antibody. Note faint nonspecific staining of the clot on the wound surface only. (G) Adult 12 hr pw. PDGF is present at the margins of the wound. (H) Adult 72 h r pw. PDGF is present at the margins of the wound, e, epidermis; m , oral mucosa; arrow: site of wound; bar: 100 #m.
WHITBY AND FERGUSON
Growth Factors in Fetal Wounds
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©
?
Q
WHITBYANDFERGUSON Growth Factors in Fetal Wounds Table 1. Characterization and specificities of these antibodies have been published and are summarized in the product information sheets. Nevertheless, for each antibody specificity was tested by preincubation of the prim a r y antibody with purified antigen, prior to staining. In tissues containing receptors for the antigen under investigation, prior incubation of the antibody with the antigen usually increases the area and intensity of staining (Van Noorden, 1986): such was the case in preliminary controls of this design, in this study. Accordingly, the antibody was preincubated with the growth factor adsorbed onto Sepharose beads (Van Noorden, 1986) in an Epindorf tube, for 1 h at room t e mperat ure with continuous agitation on a Luckham 'rock and roll' machine. The resulting solution (minus the beads) was then used in place of the primary antibody in the following protocol. Control sections at each time point were also stained, substituting phosphate buffered saline (PBS) for the primary antibody. Specifically, for the anti-TGF~ antibody, prior incubation of 1 ml of the diluted antibody with 10 ng of natural porcine TGF~-I.2 (British Biotechnology BDP7) completely abolished staining. Prior incubation of 1 ml of the antibody with 10 ng of porcine TGF~-I (British Biotechnology BDP3) abolished the majority of the staining, but not all of it, suggesting t hat other TGF~ isoforms were recognized by the antibody. Equally, prior incubation of 1 ml of the antibody with 10 ng of porcine TGFfl-2 (British Biotechnology BDP5) did not abolish staining: it merely reduced the intensity. The inference from these preincubation studies is t ha t the TGF/~ antibody detects both ~-1 and ~-2 isoforms, and the predominant isoform at the wound site seems to be TGF~-I. It is also possible th at TGF~-3 is recognized by the antibody. Clearly the situation is complex, as the polyclonal antibody may recognise shared epitopes in TGF~-I, 2, and 3, and detection of all of these isoforms may be blocked by prior incubation with one of the isoforms. It is not known whether this antibody recognizes isoform-specific epitopes with the same affinity as isoform-conserved epitopes. For the purpose of this study therefore, we will describe the pattern of staining under the generic term of TGF~: the precise details of the isoforms
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present at the wound are awaiting studies with completely isoform-specific detecting antibodies. For the PDGF antibody, prior incubation of 1 ml of the diluted antibody with 10 ng of human PDGF (British Biotechnology BDP9) completely abolished staining. It should be noted t h a t this antibody detects both a and b chains of PDGF. In the case of the bFG F antibody, prior incubation of 1 ml of the diluted antibody with 10 ng of bovine basic FG F (British Biotechnology BDP13) completely abolished staining. By contrast, incubation of the antibody with 10 ng of bovine acidic F G F (British Biotechnology BDP12) had no effect on staining and this antibody is therefore specific for bFGF. Incubation with p r i m a r y antisera was for 24 hr at 4 C. This was followed by three, 5-min rinses in PBS. Incubation with secondary antisera was for 1 hr at 20 C followed by three 5-min rinses in PBS. Sections were mounted in a nonfading medium, DABCO (1,4-diazobicyclo-(2,2,2}-octane), and photographed using a Leitz Dialux microscope and Kodak Ektachrome 160 ASA film corrected for tungsten light. Black and white prints were made from the color slides. RESULTS PDGF was present at the wound surface, and within the clot filling the wound, 1 hr postwounding in fetal, neonatal, and adult wounds (Figs. 1A and 1D). As the mesenchymal or dermal wound was infiltrated by cells the clot was removed and staining for PDGF became patchy. The clot within fetal wounds appeared markedly less dense than t h a t in the adult wounds and P D G F was present only at the skin side of the fetal wound by 24 hr (Figs. 1B and 1C). PD G F was no longer detectable in the fetal wound by 48 hr. In the neonatal wound by 48 hr there was patchy staining for PDGF, again predominantly on the skin side of the wound (Fig. 1E) and P D G F was no longer present within the wound at 72 hr. P D G F was present within the adult wound until 72 h r postwounding (Figs. 1G and 1H), but not at 120 h r postwounding. In both fetal and adult wounds the staining for PDGF was predominantly extracellular, within the
FIG.2. (A and D-H) Transverselip sections. Stainedfor TGF~.(A) 16-dayfetus I hr pw. TGF~is present around developinghair folliclesbut not present in the wound. (B) Normal skin of 16-dayfetus. Pericellular distribution of TGF/~in basal epidermis of unwounded skin. (C) Normal tongue of 16-dayfetus. Pericellullar staining for TGF~ in suprabasal epithelium of posterior tongue. (D) Neonate 6 hr pw. TGFflis present at the margins of the wound and within the clot predominantlyon the skin side of the wound. (E) Neonate 12 hr pw. TGF~ staining is confinedto the margins of the wound and the clot on the skin side of the wound. (F) Neonate 48 hr pw. Wound reepithelializedand dermal defect closed. TGF~ is present around developinghair folliclesand basal epidermis. No staining at the site of the wound. Note the few, scattered, single cells staining positivefor TGF/~within the dermis. (G) Adult 12 hr pw. Skin side of the wound. TGF~is present at the margins of the wound. Intense staining for TGF~within the clot. (H) Adult 120 hr pw. Wound reepithelializedand dermal defect closed. TGF~ is present in basal epidermis and, faintly, around hair follicles.No staining for TGF~within the site of the wound. (I) Adult 12 hr pw. Skin side of the wound. Controlsection following preincubation of antibody with TGF~-I.2. All staining is abolished, cl, clot; e, epidermis; h, hair follicle;o, oral mucosa; t, tongue; arrow: site of wound; bar: 100 #m.
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FIG. 3. (A and D-F) transverse lip sections. Stained for bFGF. (A) 16-day fetus 1 hr pw. No staining for bFGF within the fetal wound. (B) Transverse section through the tongue of 16-day fetus, bFGF is present within the skeletal muscle of the developing tongue. (C) Mandibular (Meckel's) cartilage of 16-day fetus, bFGF is present within cartilage. (D) Neonate I hr pw. bFGF is present within the dermis adjacent to the wound. (E) Neonate 6 hr pw. b F G F is present at the surface of the wound, within the clot filling the wound and in the dermis adjacent to the wound. (F) Adult 6 hr pw. bFGF is present at the surface of the wound and within the clot filling the wound, e, epidermis; m, oral mucosa; t, tongue; arrow: site of wound; bar: 100 ttm.
Grawth Factors in Fetal Wounds
WHITBY AND FERGUSON TABLE 2 GROWTH FACTOR DISTRIBUTIONIN WOUNDS Time postwounding PDGF Fetus Neonate Adult TGF~ (1 and 2) Fetus Neonate Adult bFGF Fetus Neonate Adult
1 hr
6 hr
12 h r
24 h r
++
++
++ ++ ++
++ ++ ++
++ ++
+ +
+ +
++
_
m
+ +
+ +
48 h r
-
+ ++
72 h r
_
+
213
(Fig. 3C). In the adult and neonatal wounds at I and 6 hr postwounding bFGF was present extracellularly at the surface of the wound and within the dermis adjacent to the wound (Figs. 3D-3F). Precise identification of cell types showing cellular staining for bFGF at the wound margin was not possible. The pattern of staining for growth factors within the three wound groups is summarized in Table 2. DISCUSSION
m
+
fibrin clot and in the ECM of the dermis immediately adjacent to the wound. In the fetal wounds, staining of fibroblasts and macrophages adjacent to the wound was apparent, with the latter cell type being scarce. In the adult wound there was staining of macrophages which were much more profuse, as well as staining of the epithelia and hair follicles immediately adjacent to the wound. This pattern of staining suggests that most of the PDGF present in the wound is released from degranulating platelets and binds to the ECM. TGF~ localized in a pericellular distribution around the basal cells of the undamaged epidermis (Fig. 2B) and was prominent in the superficial layer of the mucosa of the posterior tongue (Fig. 2C). There was also marked pericellular staining around the hair follicles of fetal skin but TGF~ was not detected within or at the margins of the wound (Fig. 2A). Similarly, both neonatal and adult tissues showed the presence of TGF~ around hair follicles and in the basal epidermis (Figs. 2F and 2H). Additionally at 1, 6, and 12 hr TGF~ was also detected at the wound surface and within the clot of both neonatal and adult wounds (Figs. 2D, 2E and 2G). This staining appeared to be largely extracellular at the wound margins, but in the adjacent dermis there was staining of a few scattered cells which were predominantly macrophages (Fig. 2F). At the wound margins in the neonate and adult there was a significant macrophage and monocyte infiltrate from 6 hr postwounding but the limitations of cryosections did not allow precise identification of cell types with intracellular staining for TGF~. bFGF was not present in relation to the fetal wound (Fig. 3A) although it was present within the skeletal muscle of the tongue (Fig. 3B) and localized in a pericellular distribution around the chondrocytes within the mandibular (Meckel's) cartilage adjacent to the wound
PDGF was present at the wound surface and within the clot of fetal, neonatal and adult wounds, from 1 hr postwounding. PDGF is released from the c~granules of platelets activated by coagulation (Kaplan et al., 1979 a,b), and the presence of this factor within fetal, neonatal, and adult wounds might therefore be anticipated. The period during which PDGF was present within the wound varied from 24 hr in the fetus to 72 hr in the adult and this may reflect differences in the continued release in the wound by other cells which synthesize a PDGFlike factor, such as macrophages and endothelial cells (Huang et al., 1988). Binding of the highly cationic PDGF to components of the ECM such as glycosaminoglycans may also prolong the local activity of the factor at the wound and this is thought to be the reason for the very short half life of circulating PDGF (Bowen-Pope et al., 1984). The a granules of platelets also contain TGFB (Wakefield et al., 1988); although this factor was clearly demonstrated in neonatal and adult wounds, from 1 to 12 hr postwounding, it was not detected in the fetal wound. This may reflect a relative decrease in the number of degranulating platelets at the fetal wound and/or an absence of the factor in the fetal platelet. The absence of an inflammatory response in the fetal wound may also influence the level of TGFfl: macrophages are a potent source of TGFB (Assoian et aL, 1987) so t h a t an absent or reduced monocyte infiltrate in the fetal wound would be expected to result in lower levels of TGF~. Moreover, TGF~ is autoinductive (Van Obberghen-Schilling et al., 1988), binding at its own gene promoter to increase its synthesis, and is chemotactic to monocytes and macrophages, resulting in their accumulation at the wound site, thus further raising the growth factor levels (Roberts and Sporn, 1990; Massague, 1990). TGFfl also stimulates the synthesis of other growth factors and synergizes with PDGF (Roberts and Sporn, 1990; Massague, 1990). Differences in the kinetics and organization of the ECM in fetal and adult wound healing, the presence of TGF~ in the adult wound and its absence from the fetal wound, correlate with the known effects of TGF~ in vitro and in vivo. TGF~ causes collagen accumulation by stimulating its synthesis (Ignotz and Massague, 1986;
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Roberts et al., 1986; Ignotz et al., 1987) and inhibiting its degradation (Laiho et al., 1986). In an animal model of glomerulonephritis the pathological increase in ECM synthesis can be suppressed by an antiserum to TGFB1 (Border et al., 1990), and conversely, the addition of TGF~ to polyvinyl alcohol sponges implanted in fetal rabbits produces fibrosis when compared to control sponges (Krummel et al., 1988). The densely packed collagen fibrils and relative abundance of collagen in adult wounds may thus be due in part to the presence of TGFfl in the adult wound, whilest the absence or low level of TGF/~ in the fetal wound may influence the pattern of collagen deposition in the wound, with subsequent restoration of a normal tissue architecture. bFGF is present in a wide variety of tissues, and a recent study of the rat fetus (Gonzalez et al., 1990) has localized bFGF in basement membranes, with strongly positive intracellular staining of skeletal muscle and cartilage. The staining for bFGF in the mouse fetus in this study concurs with this pattern. One unusual aspect of the biology of bFGF is that it lacks a normal signal sequence to mediate release from the cell via the normal secretory pathway, and the method of secretion into the ECM is unclear. The presence of bFGF within the neonatal and adult wounds may simply be due to cell death but this would not explain its absence from the fetal wound. bFGF localizes in basement membranes (Folkmann et aL,1988; Gonzalez et a/.,1990) and has been extracted from subendothelial ECM (Vlodasky et al., 1987) where it is bound to heparan sulfate proteogtycan, bFGF can be released from this "storage depot" by heparanase, expressed by activated platelets and neutrophils (IshaiMichaeli et a/.,1990), suggesting a mechanism whereby bFGF can be activated at a wound by coagulation or by an inflammatory response. The absence of bFGF in the fetal wounds may thus be secondary to an absence of an inflammatory response in the fetal wound, bFGF is a potent angiogenic factor (Folkman and Klagsbrun, 1987) and neovascularization is a normal component of the adult wound healing process. Profuse new capillary formation can be clearly demonstrated in the adult wound by staining for type IV collagen and laminin, localized in the endothelial basement membranes. However, in the same study (Whitby and Ferguson, 1991) staining for type IV collagen and laminin in the fetal wounds did not reveal significant new capillary formation, but rather a vascular structure similar to the adjacent normal fetal tissue. The profuse neovascularization of adult wounds may thus be due to the angiogenic effects of bFGF and TGFB, whilest the restoration of a normal vascular pattern in the fetal wound may be a reflection of the absence or reduced level of bFGF and TGF~ in the fetal wound.
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Clearly we cannot be certain that TGF~ or bFGF is absent from fetal wounds; their levels could simply be below the detecting threshold of our immunohistochemical procedures. Nonetheless the staining patterns observed are suggestive of at least major differences in the relative levels of the factors between fetal and adult wounds. Measurement of the actual levels of such factors within incised healing wounds is currently impossible; even measuring levels within artificial wound chambers is difficult. Neither immunohistochemical techniques nor measurement techniques can currently distinguish between the levels of active versus inactive growth factors, which are important for the healing process. Notwithstanding such caveats, we believe t h a t our data reveal for the first time important differences between the growth factor profiles of healing fetal and adult wounds. The distribution of TGFf3 and bFGF correlates with our present understanding of the differences between fetal and adult wound healing and suggests that an absence, or reduction in levels, of these growth factors may have a significant effect on wound healing. Manipulating adult wound healing to become more fetal-like may be possible by altering the growth factors present in the adult wound. This study was funded by a grant from the North West Regional Health Authority. We are most grateful for additional financial support provided by a grant from the Streatfeild & Mackenzie Mackinnon Research Fund. REFERENCES ASSOIAN, R. K., FLEURDELYS,B. E., STEVENSON,H. C., MILLER, P. J., MADTES, D. K., RAINES, E. W., ROSS, R., and SPORN, M. B. (1987). Expression and secretion of type beta transforming growth factor by activated human macrophages. Proc. Natl. AcacL Sci. USA 84, 6020-6024. ASSOIAN, R. K. (1988). The role of growth factors in tissue repair. IV. Type ~-transforming growth factor and stimulation of fibrosis. I n "The Molecular and Cellular Biology of Wound Repair" (R. A. F. Clark and P. M. Henson, eds.), pp. 273-280. Plenum, New York. BORDER, W. A., OKUDA,S., LANGUINO,L. R., SPORN, M. B., and RUOSLAHTI, E. (1990). Suppression of experimental glomerulonephritis by antiserum against transforming growth factor t~l. Nature 346, 371-374. BOWEN-POPE, D. F., MALPASS, T. W., FOSTER, D. M. and ROSS, R. (1984). Platelet derived growth factor in vivo: Levels, activity, and rate of clearance. Blood 64, 458-469. FOLKMAN,J., and KLAGSBRUN,M. (1987). Angiogenic factors. Science 235, 442-447. FOLKMAN, J., KLAGSBRUN,M., SASSE, J., WADZINSKI,M., INGBER, D., and VLODASKY,I. (1988). A heparin-binding angiogenic factor--basic fibroblast growth factor--is stored within basement membrane. Am. J. PathoL 130, 393-400. FOX, G. M. (1988). The role of growth factors in tissue repair. III. Fibroblast growth factor. In "The Molecular and Cellular Biology of Wound Repair" (R. A. F. Clark and P. M. Henson, Eds.), pp. 265-271. Plenum, New York.
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GONZALEZ,A., BUSCAGLIA,M., ONG, M., and BAIRDA. (1990). Distribution of basic fibroblast growth factor in the 18-day rat fetus: Localization in the basement membranes of diverse tissues. J. Cell Biol. ll0, 753-765. HAIGH, R., D'SOVZA,S. W., MICKLEWRIGHT,L., GREGORY,H., BUTLER, S. J., HOLLINGSWORTH,M., DONNAI, P., and BOYD, R. D. H. (1989). Human amniotic fluid urogastrone (epidermal growth factor) and fetal lung phospholipids. Br. J. Obst. GynaecoL 96, 171-178. HALLOCK, G. G. (1985). In utero cleft lip repair in A / J mice. Plast. Reconstr. Surg. 75, 785-788. HUANG, J. S., OLSEN, T. J., and HUANG, S. S. (1988). The role of growth factors in tissue repair. I.Platelet-derived growth factor. In 'The Molecular and Cellular Biology of Wound Repair', (R. A. F. Clark and P. M. Henson, Eds.), pp.243-251. Plenum, New York. IGNOTZ, R. A., and MASSAGUE,J. (1986). Transforming growth factorstimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. Biol. Chem. 261, 43374345. IGNOTZ, R. A., ENDO, T. and MASSAGUE,J. (1987). Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta. J. Biol. Chem. 262, 6443-6446. ISHAI-MICHAELI,R., ELDOR, A., and VLODASKY,I. (1990). Heparanase activity expressed by platelets, neutrophils, and lymphoma cells releases active fibroblast growth factor from extracellular matrix. Cell Reg. 1, 833-842. KAPLAN, D. R., BROEKMAN,M. J., CHERNOFF,A., LESZNIK, G. R., and DRILLINGS, M. (1979a). Platelet alpha-granule proteins: studies on release and subcellular localization. Blood 53, 604-618. KAPLAN D. R., CHAO, F. C., STILES, C. D., ANTONIADES,H. N., and SCHER, C. D. (1979b). Platelet alpha-granules contain a growth factor for fibroblasts. Blood 53, 1043-1052. KOVACS,E. J. (1991). Fibrogenic cytokines: the role of immune mediators in the development of scar tissue. Immunol. Today. 12, 17-23. KRUMMEL,W. M., MICHNA,B. A., THOMAS,B. L., SPORN, M. B., NELSON, J. M., SALZBERG,A. M., COHEN, I. K., and DIEGELMANN,R. F. (1988). Transforming growth factor beta induces fibrosis in a fetal wound model. J. Pediatr. Surg. 23, 647-652. KSANDER, G. A. (1989). Exogenous growth factors in dermal wound healing. Annu. Rep. Medicinal Chem. 24, 223-232. LAIHO, M., SAKESELA,0., ANDREASEN,P. A., and KESKI-OJA, J. (1986). Enhanced production and extracellular deposition of the endothelial-type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-beta. J. Cell Biol. 103, 2403-2410. LONGAKER,M. T., WHITBY,D. J., ADZICK,N. S., CROMBLEHOLME,T. M., LANGER, J. C., DUNCAN,B. W., BRADLEY,S. M., STERN, R., FERGUSON, M. W. J., and HARRISON, M. R. (1990). Studies in fetal wound healing. VI. Second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation. J. Pediatr. Surg. 25, 63-69.
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MASSAGUE,J. (1990). The transforming growth factor/~ family. Annu. Rev. Cell Biol. 6, 597-641. MCGRATH, M. H. (1990). Peptide growth factors and wound healing. Clin. Plast. Surg. 17, 421-432. NARAYANA,A. S., and PAGE, R. C. (1983). Biosynthesis and regulation of type V collagen in diploid human fibroblasts. J. BioL Chem. 258, 11694-11699. RIFKIN, D. B., and MOSCATELLI,D. (1989). Recent developments in the cell biology of basic fibroblast growth factor. J. Cell Biol. 109, 1-6. ROBERTS, A. B., SPORN, M. B., ASSOIAN, R. K., SMITH, J. M., ROCHE, N. S., WAKEFIELD,L. M., HEINE, U. I., LIOTTA,L. A., FALANGA,V., KEHRL, J. H., and FAUCI, A. S. (1986) Transforming growth factorbeta: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. NatL Acad Sci. USA 83, 4167-4171. ROBERTS, A. B., and SPORN, M. B. (1990). The transforming growth factor ~s. In 'Peptide Growth Factors and Their Receptors' (M. B. Sporn and A. B. Roberts, Eds.), pp. 419-472. Springer-Verlag, Berlin, Heidelberg New York. ROBINSON,B. W., and GOSS, A. N. (1981). Intrauterine healing of fetal rat cheek wounds. Cleft Palate J. 18, 251-255. ROSS, R., RAINES, E. W., and BOWEN-POPE, D. F. (1986). The biology of platelet derived growth factor. Cell 46, 155-169. ROWSELL, A. R. (1984). The intra-uterine healing of foetal muscle wounds: Experimental study in the rat. Br. J. Plast. Surg. 37, 635642. VAN NOORDEN, S. (1986). Tissue preparation and immunostaining techniques for light microscopy. In 'Immunocytochemistry: Modern Methods and Applications', (J. M. Polak and S. Van Noorden Eds.), 2nd ed., pp. 26-53. Wright, Bristol. VAN OBBERGHEN-SCHILLING, E., ROCHE, N. S., FLANDERS, K. C., SPORN, M. B., and ROBERTS,A. B. (1988). Transforming growth factor E1 positively regulates its own expression in normal and transformed cells. J. Biol. Chem. 263, 7741-7746. VLODASKY, I., FOLKMAN, J., SULLIVAN, n., FRIDMAN, R., ISHAI-MICHAELI, R., SASSE, J., and KLAGSBRUN,M. (1987). Endothelial cellderived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix. Proc. Natl. Acad. Sci. USA 84, 2292-2296 WAKEFIELD, L. M., SMITH, D. M., FLANDERS,K. C., and SPORN, M. B. (1988). Latent transforming growth factor-B from human platelets. J. Biol. Chem. 263, 7646-7654. WHITBY, D. J., and FERGUSON,M. W. J. (1991). The extracellular matrix of lip wounds in fetal, neonatal and adult mice. Development 112, 651-668. WHITBY, D. J., LONGAKER,M. T., HARRISON,M. R., ADZICK,N. S. and FERGUSON, M. W. J. (1991). Rapid epithelialisation of fetal wounds is associated with early deposition of tenascin. J. Cell Sci. in press.
Immunohistochemical Localization of Growth Factors in Fetal Wound Healing DAVID J. WHITBY*'t AND MARK W. J. FERGUSON *'1 *Department of Cell and Structural Biology, School of Biological Sciences, University of Manchester, Coupland 3 Building, Manchester, M13 9PL, United Kingdom; and t Department of Plastic Surgery, St James' University Hospital, Leeds, United Kingdom Accepted May 24, 1991
Fetal wound healing occurs rapidly, in a regenerative fashion, and without scar formation, by contrast with adult wound healing, where tissue repair results in scar formation which limits tissue function and growth. The extracellular matrix deposited in fetal wounds contains essentially the same structural components as that in the adult wound but there are distinct differences in the spatial and temporal distribution of these components. In particular the organization of collagen in the healed fetal wound is indistinguishable from the normal surrounding tissue. Rapidity of healing, lack of an inflammatory response, and an absence of neovascularization also distinguish fetal from adult wound healing. The mechanisms controlling these differing processes are undefined but growth factors may play a critical role. The distribution of growth factors in healing fetal wounds is unknown. We have studied, by immunohistochemistry, the localization of platelet-derived growth factor (PDGF), transforming growth factor # (TGFfl), and basic fibroblast growth factor (bFGF), in fetal, neonatal, and adult mouse lip wounds. TGFfl and bFGF were present in neonatal and adult wounds, but were not detected in the fetal wounds, while PDGF was present in fetal, neonatal, and adult wounds. This pattern correlates with the known effects in vitro of these factors, the absence of an inflammatory response and neovascularization in the fetal wound, and the patterns of collagen deposition in both fetal and adult wounds. The results suggest that it may be possible to manipulate the adult wound to produce more fetal-like, scarless, wound healing. © 1991 Academic Press, Inc. INTRODUCTION In both fetus and adult, wound healing occurs by a process involving cell m i g r a t i o n , proliferation, a n d diff e r e n t i a t i o n , r e m o v a l of d a m a g e d tissue, and s y n t h e s i s of an e x t r a c e l l u l a r m a t r i x (ECM). However, in the fetus, in incised skin wounds, healing occurs rapidly, w i t h o u t an i n f l a m m a t o r y response, leading to r e s t o r a t i o n of n o r m a l tissue a r c h i t e c t u r e and function (Robinson and Goss, 1981; Rowsell, 1984; Hallock, 1985; L o n g a k e r et al., 1990; W h i t b y a n d Ferguson, 1991), while in the a d u l t the end result is tissue r e p a i r by scar f o r m a t i o n , w i t h subseq u e n t loss of function and r e s t r i c t i o n of growth. Defining the m e c h a n i s m s controlling t h e s e differing processes m a y indicate w a y s to a l t e r the adult w o u n d so t h a t healing occurs in a m o r e fetal-like m a n n e r , w i t h o u t scar f o r m a t i o n . The ECM deposited in f e t a l and a d u l t w o u n d s h a s m a n y c o m p o n e n t s in c o m m o n (glycoproteins, collagen t y p e s I, III, IV, V, VI, and proteoglycans), b u t the t i m i n g and p a t t e r n of deposition of t h e s e e l e m e n t s differs ( W h i t b y and Ferguson, 1991; W h i t b y et al., 1991). As the functional p r o p e r t i e s (and p r o b l e m s ) of scar tissue in the a d u l t are a reflection of its collagenous s t r u c t u r e , 1To whom reprint requests and correspondence should be addressed.
the p a t t e r n of collagen deposition in f e t a l and a d u l t wounds is of p a r t i c u l a r interest. In the f e t a l wound, collagen is deposited in a r e t i c u l a r p a t t e r n indistinguishable f r o m the adjacent, n o r m a l , tissue, while in the adult, large, parallel bundles of collagen are laid down p e r p e n d i c u l a r to the w o u n d s u r f a c e to f o r m a s c a r ( W h i t b y a n d Ferguson, 1991; L o n g a k e r et al., 1990). The m e c h a n i s m s controlling ECM s y n t h e s i s d u r i n g w o u n d h e a l i n g a r e unknown, but the effects, in vivo and i n vitro, of several g r o w t h f a c t o r s suggest t h a t t h e y p l a y an i m p o r t a n t role in tissue r e p a i r (for review see H u a n g et al., 1988; Fox, 1988; Assoian, 1988; K s a n d e r , 1989; M c G r a t h , 1990; Kovacs, 1991). P l a t e l e t - d e r i v e d g r o w t h f a c t o r ( P D G F ) is c h e m o t a c t i c and a p o t e n t m i t o g e n for fibroblasts, c h e m o t a c t i c for m o n o c y t e s a n d n e u t r o p h i l s (Ross et al., 1986), a n d can r e g u l a t e collagen s y n t h e s i s ( N a r a y a n a and Page,1983). T r a n s f o r m i n g g r o w t h f a c t o r # (TGF#) can s t i m u l a t e or inhibit cell p r o l i f e r a t i o n , induce ECM synthesis, p a r t i c u l a r l y collagen and fibronectin (Ignotz and Massague, 1986; R o b e r t s et al., 1986; Ignotz et al., 1987), and i n h i b i t m a t r i x d e g r a d a t i o n (Laiho et al., 1986). Basic fibroblast g r o w t h f a c t o r ( b F G F ) is s t r o n g l y angiogenic a n d also s t i m u l a t e s fibroblast prol i f e r a t i o n and ECM s y n t h e s i s (Rifkin and Moscatelli, 1989). T h e r e are m a n y differences b e t w e e n the f e t a l a n d the a d u l t w o u n d which m a y a l t e r the h e a l i n g p r o c e s s - sterile aqueous f e t a l e n v i r o n m e n t , the absence of an in-
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0012-1606/91 $3.00 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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TABLE 1 PRIMARY (1-3) AND FITC-CoNJUGATED SECONDARY (4, 5) ANTIBODIES
Antibody raised against
Antibody raised in
Source
Dilution (in PBS)
1
PDGF (Human platelets)
Goat
British Biotechnology, Oxford (BDA 16)
1:40
2
TGFfi (Porcine platelets) (detects TGFfil and ~2)
Rabbit
British Biotechnology, Oxford (BDA 5)
1:5
3
bFGF (bovine brain)
Rabbit
British Biotechnology, Oxford (BDA 4)
1:40
4
Rabbit IgG
Sheep
Serotec
1:160
5
Goat IgG
Rabbit
Northeast Biomedicals
1:40
flammatory response in the fetal wound, the state of differentiation of the fetal cells, biomechanical forces at the wound site, and tissue oxygenation and p H - - a n d some of these differences may exert an effect by altering the growth factors present in the fetal wound; amniotic fluid contains growth factors (Haigh et aL, 1989); whilest macrophages, as part of an inflammatory response, release growth factors. However, the growth factor profile of healing fetal wounds is unknown. We have therefore carried out an immunohistochemical study to localize the distribution of PDGF, TGF/~, and bFGF in lip wounds in fetal, neonatal, and adult mice. MATERIALS AND METHODS
Upper lip wounds in Day 16 fetal mice, neonatal mice, and adult mice were studied, from 1 to 72 hr postwounding in the fetus and from 1 to 120 hr postwounding in the neonate and adult. This wound is reproducible, can be made under direct vision, without significant fetal loss, and the healing of two differing epithelial surfaces (skin and oral mucosa) can be compared. F e t a l Wounds
At Day 16 of gestation, time-mated, MF1 mice were anesthetized using a halothane/oxygen/nitrous oxide mixture. A 2-mm, vertical, full thickness wound was created in the left upper lip of the fetus using techniques described by Whitby and Ferguson (1991). The wound surfaces lay in apposition without a suture. Three fetuses were wounded in each pregnant animal. Following
recovery from anesthesia the pregnancy continued until the wounds were harvested. Neonatal and A d u l t Wounds
Under halothane/oxygen/nitrous oxide anesthesia 3mm, vertical, full thickness wounds were created in the left upper lip of neonatal mice (within 12 hr of birth) and adult mice, age 6-8 weeks. A single 6 / 0 nylon suture was placed to appose the wound surfaces. Wound H a r v e s t and Tissue Preparation
Animals were killed by an overdose of chloroform at multiple time points following wounding. Fetal wounds were harvested 1, 6, 12, 18, 24, 36, 48 and 72 hr postwounding. In the adult and neonate, wounds were harvested 1, 6, 12, 18, 24, 36, 48, 72 and 120 hr postwounding. Following excision of the lower lip and mandible, the fetal and neonatal heads were snap frozen in precooled isopentane over liquid nitrogen, and the tissue was erabedded in OCT compound (Miles Inc., Elkart. IN). The adult upper lips were excised intact, snap frozen, and embedded in OCT. Seven-micrometer transverse sections were cut at -20°C in a Leitz cryostat, acetone fixed, and air dried. Sections were stored at -70°C prior to immunostaining. I m m u no h istoc h e m ist ry
The primary and secondary (FITC-conjugated) antibodies used for indirect immunostaining are detailed in
FIG. 1. Transverse lip sections. Stained for PDGF. (A) 16-day fetus 1 h r postwounding (pw). PDGF is present at the margins of the central part of the wound. (B) 16-day fetus 20 hr pw. PDGF is present at the margins of the wound and within the fibrillar clot at the skin surface. (C) 16-day fetus 24 hr pw. Wound reepithelialized and mesenchymal defect closed. Residual staining for PDGF beneath reformed epidermis. (D) Neonate i hr pw. PDGF is present throughout the clot filling the wound and at the margins of the wound. (E) Neonate 48 hr pw. Reepithelialization complete and dermal wound closed. PDGF is only present beneath the epidermis at the skin surface of the wound and within the clot on the surface of the wound. (F) Neonate 48 hr pw. Control section substituting PBS for primary antibody. Note faint nonspecific staining of the clot on the wound surface only. (G) Adult 12 hr pw. PDGF is present at the margins of the wound. (H) Adult 72 h r pw. PDGF is present at the margins of the wound, e, epidermis; m , oral mucosa; arrow: site of wound; bar: 100 #m.
WHITBY AND FERGUSON
Growth Factors in Fetal Wounds
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©
?
Q
WHITBYANDFERGUSON Growth Factors in Fetal Wounds Table 1. Characterization and specificities of these antibodies have been published and are summarized in the product information sheets. Nevertheless, for each antibody specificity was tested by preincubation of the prim a r y antibody with purified antigen, prior to staining. In tissues containing receptors for the antigen under investigation, prior incubation of the antibody with the antigen usually increases the area and intensity of staining (Van Noorden, 1986): such was the case in preliminary controls of this design, in this study. Accordingly, the antibody was preincubated with the growth factor adsorbed onto Sepharose beads (Van Noorden, 1986) in an Epindorf tube, for 1 h at room t e mperat ure with continuous agitation on a Luckham 'rock and roll' machine. The resulting solution (minus the beads) was then used in place of the primary antibody in the following protocol. Control sections at each time point were also stained, substituting phosphate buffered saline (PBS) for the primary antibody. Specifically, for the anti-TGF~ antibody, prior incubation of 1 ml of the diluted antibody with 10 ng of natural porcine TGF~-I.2 (British Biotechnology BDP7) completely abolished staining. Prior incubation of 1 ml of the antibody with 10 ng of porcine TGF~-I (British Biotechnology BDP3) abolished the majority of the staining, but not all of it, suggesting t hat other TGF~ isoforms were recognized by the antibody. Equally, prior incubation of 1 ml of the antibody with 10 ng of porcine TGFfl-2 (British Biotechnology BDP5) did not abolish staining: it merely reduced the intensity. The inference from these preincubation studies is t ha t the TGF/~ antibody detects both ~-1 and ~-2 isoforms, and the predominant isoform at the wound site seems to be TGF~-I. It is also possible th at TGF~-3 is recognized by the antibody. Clearly the situation is complex, as the polyclonal antibody may recognise shared epitopes in TGF~-I, 2, and 3, and detection of all of these isoforms may be blocked by prior incubation with one of the isoforms. It is not known whether this antibody recognizes isoform-specific epitopes with the same affinity as isoform-conserved epitopes. For the purpose of this study therefore, we will describe the pattern of staining under the generic term of TGF~: the precise details of the isoforms
211
present at the wound are awaiting studies with completely isoform-specific detecting antibodies. For the PDGF antibody, prior incubation of 1 ml of the diluted antibody with 10 ng of human PDGF (British Biotechnology BDP9) completely abolished staining. It should be noted t h a t this antibody detects both a and b chains of PDGF. In the case of the bFG F antibody, prior incubation of 1 ml of the diluted antibody with 10 ng of bovine basic FG F (British Biotechnology BDP13) completely abolished staining. By contrast, incubation of the antibody with 10 ng of bovine acidic F G F (British Biotechnology BDP12) had no effect on staining and this antibody is therefore specific for bFGF. Incubation with p r i m a r y antisera was for 24 hr at 4 C. This was followed by three, 5-min rinses in PBS. Incubation with secondary antisera was for 1 hr at 20 C followed by three 5-min rinses in PBS. Sections were mounted in a nonfading medium, DABCO (1,4-diazobicyclo-(2,2,2}-octane), and photographed using a Leitz Dialux microscope and Kodak Ektachrome 160 ASA film corrected for tungsten light. Black and white prints were made from the color slides. RESULTS PDGF was present at the wound surface, and within the clot filling the wound, 1 hr postwounding in fetal, neonatal, and adult wounds (Figs. 1A and 1D). As the mesenchymal or dermal wound was infiltrated by cells the clot was removed and staining for PDGF became patchy. The clot within fetal wounds appeared markedly less dense than t h a t in the adult wounds and P D G F was present only at the skin side of the fetal wound by 24 hr (Figs. 1B and 1C). PD G F was no longer detectable in the fetal wound by 48 hr. In the neonatal wound by 48 hr there was patchy staining for PDGF, again predominantly on the skin side of the wound (Fig. 1E) and P D G F was no longer present within the wound at 72 hr. P D G F was present within the adult wound until 72 h r postwounding (Figs. 1G and 1H), but not at 120 h r postwounding. In both fetal and adult wounds the staining for PDGF was predominantly extracellular, within the
FIG.2. (A and D-H) Transverselip sections. Stainedfor TGF~.(A) 16-dayfetus I hr pw. TGF~is present around developinghair folliclesbut not present in the wound. (B) Normal skin of 16-dayfetus. Pericellular distribution of TGF/~in basal epidermis of unwounded skin. (C) Normal tongue of 16-dayfetus. Pericellullar staining for TGF~ in suprabasal epithelium of posterior tongue. (D) Neonate 6 hr pw. TGFflis present at the margins of the wound and within the clot predominantlyon the skin side of the wound. (E) Neonate 12 hr pw. TGF~ staining is confinedto the margins of the wound and the clot on the skin side of the wound. (F) Neonate 48 hr pw. Wound reepithelializedand dermal defect closed. TGF~ is present around developinghair folliclesand basal epidermis. No staining at the site of the wound. Note the few, scattered, single cells staining positivefor TGF/~within the dermis. (G) Adult 12 hr pw. Skin side of the wound. TGF~is present at the margins of the wound. Intense staining for TGF~within the clot. (H) Adult 120 hr pw. Wound reepithelializedand dermal defect closed. TGF~ is present in basal epidermis and, faintly, around hair follicles.No staining for TGF~within the site of the wound. (I) Adult 12 hr pw. Skin side of the wound. Controlsection following preincubation of antibody with TGF~-I.2. All staining is abolished, cl, clot; e, epidermis; h, hair follicle;o, oral mucosa; t, tongue; arrow: site of wound; bar: 100 #m.
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FIG. 3. (A and D-F) transverse lip sections. Stained for bFGF. (A) 16-day fetus 1 hr pw. No staining for bFGF within the fetal wound. (B) Transverse section through the tongue of 16-day fetus, bFGF is present within the skeletal muscle of the developing tongue. (C) Mandibular (Meckel's) cartilage of 16-day fetus, bFGF is present within cartilage. (D) Neonate I hr pw. bFGF is present within the dermis adjacent to the wound. (E) Neonate 6 hr pw. b F G F is present at the surface of the wound, within the clot filling the wound and in the dermis adjacent to the wound. (F) Adult 6 hr pw. bFGF is present at the surface of the wound and within the clot filling the wound, e, epidermis; m, oral mucosa; t, tongue; arrow: site of wound; bar: 100 ttm.
Grawth Factors in Fetal Wounds
WHITBY AND FERGUSON TABLE 2 GROWTH FACTOR DISTRIBUTIONIN WOUNDS Time postwounding PDGF Fetus Neonate Adult TGF~ (1 and 2) Fetus Neonate Adult bFGF Fetus Neonate Adult
1 hr
6 hr
12 h r
24 h r
++
++
++ ++ ++
++ ++ ++
++ ++
+ +
+ +
++
_
m
+ +
+ +
48 h r
-
+ ++
72 h r
_
+
213
(Fig. 3C). In the adult and neonatal wounds at I and 6 hr postwounding bFGF was present extracellularly at the surface of the wound and within the dermis adjacent to the wound (Figs. 3D-3F). Precise identification of cell types showing cellular staining for bFGF at the wound margin was not possible. The pattern of staining for growth factors within the three wound groups is summarized in Table 2. DISCUSSION
m
+
fibrin clot and in the ECM of the dermis immediately adjacent to the wound. In the fetal wounds, staining of fibroblasts and macrophages adjacent to the wound was apparent, with the latter cell type being scarce. In the adult wound there was staining of macrophages which were much more profuse, as well as staining of the epithelia and hair follicles immediately adjacent to the wound. This pattern of staining suggests that most of the PDGF present in the wound is released from degranulating platelets and binds to the ECM. TGF~ localized in a pericellular distribution around the basal cells of the undamaged epidermis (Fig. 2B) and was prominent in the superficial layer of the mucosa of the posterior tongue (Fig. 2C). There was also marked pericellular staining around the hair follicles of fetal skin but TGF~ was not detected within or at the margins of the wound (Fig. 2A). Similarly, both neonatal and adult tissues showed the presence of TGF~ around hair follicles and in the basal epidermis (Figs. 2F and 2H). Additionally at 1, 6, and 12 hr TGF~ was also detected at the wound surface and within the clot of both neonatal and adult wounds (Figs. 2D, 2E and 2G). This staining appeared to be largely extracellular at the wound margins, but in the adjacent dermis there was staining of a few scattered cells which were predominantly macrophages (Fig. 2F). At the wound margins in the neonate and adult there was a significant macrophage and monocyte infiltrate from 6 hr postwounding but the limitations of cryosections did not allow precise identification of cell types with intracellular staining for TGF~. bFGF was not present in relation to the fetal wound (Fig. 3A) although it was present within the skeletal muscle of the tongue (Fig. 3B) and localized in a pericellular distribution around the chondrocytes within the mandibular (Meckel's) cartilage adjacent to the wound
PDGF was present at the wound surface and within the clot of fetal, neonatal and adult wounds, from 1 hr postwounding. PDGF is released from the c~granules of platelets activated by coagulation (Kaplan et al., 1979 a,b), and the presence of this factor within fetal, neonatal, and adult wounds might therefore be anticipated. The period during which PDGF was present within the wound varied from 24 hr in the fetus to 72 hr in the adult and this may reflect differences in the continued release in the wound by other cells which synthesize a PDGFlike factor, such as macrophages and endothelial cells (Huang et al., 1988). Binding of the highly cationic PDGF to components of the ECM such as glycosaminoglycans may also prolong the local activity of the factor at the wound and this is thought to be the reason for the very short half life of circulating PDGF (Bowen-Pope et al., 1984). The a granules of platelets also contain TGFB (Wakefield et al., 1988); although this factor was clearly demonstrated in neonatal and adult wounds, from 1 to 12 hr postwounding, it was not detected in the fetal wound. This may reflect a relative decrease in the number of degranulating platelets at the fetal wound and/or an absence of the factor in the fetal platelet. The absence of an inflammatory response in the fetal wound may also influence the level of TGFfl: macrophages are a potent source of TGFB (Assoian et aL, 1987) so t h a t an absent or reduced monocyte infiltrate in the fetal wound would be expected to result in lower levels of TGF~. Moreover, TGF~ is autoinductive (Van Obberghen-Schilling et al., 1988), binding at its own gene promoter to increase its synthesis, and is chemotactic to monocytes and macrophages, resulting in their accumulation at the wound site, thus further raising the growth factor levels (Roberts and Sporn, 1990; Massague, 1990). TGFfl also stimulates the synthesis of other growth factors and synergizes with PDGF (Roberts and Sporn, 1990; Massague, 1990). Differences in the kinetics and organization of the ECM in fetal and adult wound healing, the presence of TGF~ in the adult wound and its absence from the fetal wound, correlate with the known effects of TGF~ in vitro and in vivo. TGF~ causes collagen accumulation by stimulating its synthesis (Ignotz and Massague, 1986;
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DEVELOPMENTALBIOLOGY
Roberts et al., 1986; Ignotz et al., 1987) and inhibiting its degradation (Laiho et al., 1986). In an animal model of glomerulonephritis the pathological increase in ECM synthesis can be suppressed by an antiserum to TGFB1 (Border et al., 1990), and conversely, the addition of TGF~ to polyvinyl alcohol sponges implanted in fetal rabbits produces fibrosis when compared to control sponges (Krummel et al., 1988). The densely packed collagen fibrils and relative abundance of collagen in adult wounds may thus be due in part to the presence of TGFfl in the adult wound, whilest the absence or low level of TGF/~ in the fetal wound may influence the pattern of collagen deposition in the wound, with subsequent restoration of a normal tissue architecture. bFGF is present in a wide variety of tissues, and a recent study of the rat fetus (Gonzalez et al., 1990) has localized bFGF in basement membranes, with strongly positive intracellular staining of skeletal muscle and cartilage. The staining for bFGF in the mouse fetus in this study concurs with this pattern. One unusual aspect of the biology of bFGF is that it lacks a normal signal sequence to mediate release from the cell via the normal secretory pathway, and the method of secretion into the ECM is unclear. The presence of bFGF within the neonatal and adult wounds may simply be due to cell death but this would not explain its absence from the fetal wound. bFGF localizes in basement membranes (Folkmann et aL,1988; Gonzalez et a/.,1990) and has been extracted from subendothelial ECM (Vlodasky et al., 1987) where it is bound to heparan sulfate proteogtycan, bFGF can be released from this "storage depot" by heparanase, expressed by activated platelets and neutrophils (IshaiMichaeli et a/.,1990), suggesting a mechanism whereby bFGF can be activated at a wound by coagulation or by an inflammatory response. The absence of bFGF in the fetal wounds may thus be secondary to an absence of an inflammatory response in the fetal wound, bFGF is a potent angiogenic factor (Folkman and Klagsbrun, 1987) and neovascularization is a normal component of the adult wound healing process. Profuse new capillary formation can be clearly demonstrated in the adult wound by staining for type IV collagen and laminin, localized in the endothelial basement membranes. However, in the same study (Whitby and Ferguson, 1991) staining for type IV collagen and laminin in the fetal wounds did not reveal significant new capillary formation, but rather a vascular structure similar to the adjacent normal fetal tissue. The profuse neovascularization of adult wounds may thus be due to the angiogenic effects of bFGF and TGFB, whilest the restoration of a normal vascular pattern in the fetal wound may be a reflection of the absence or reduced level of bFGF and TGF~ in the fetal wound.
VOLUME147, 1991
Clearly we cannot be certain that TGF~ or bFGF is absent from fetal wounds; their levels could simply be below the detecting threshold of our immunohistochemical procedures. Nonetheless the staining patterns observed are suggestive of at least major differences in the relative levels of the factors between fetal and adult wounds. Measurement of the actual levels of such factors within incised healing wounds is currently impossible; even measuring levels within artificial wound chambers is difficult. Neither immunohistochemical techniques nor measurement techniques can currently distinguish between the levels of active versus inactive growth factors, which are important for the healing process. Notwithstanding such caveats, we believe t h a t our data reveal for the first time important differences between the growth factor profiles of healing fetal and adult wounds. The distribution of TGFf3 and bFGF correlates with our present understanding of the differences between fetal and adult wound healing and suggests that an absence, or reduction in levels, of these growth factors may have a significant effect on wound healing. Manipulating adult wound healing to become more fetal-like may be possible by altering the growth factors present in the adult wound. This study was funded by a grant from the North West Regional Health Authority. We are most grateful for additional financial support provided by a grant from the Streatfeild & Mackenzie Mackinnon Research Fund. REFERENCES ASSOIAN, R. K., FLEURDELYS,B. E., STEVENSON,H. C., MILLER, P. J., MADTES, D. K., RAINES, E. W., ROSS, R., and SPORN, M. B. (1987). Expression and secretion of type beta transforming growth factor by activated human macrophages. Proc. Natl. AcacL Sci. USA 84, 6020-6024. ASSOIAN, R. K. (1988). The role of growth factors in tissue repair. IV. Type ~-transforming growth factor and stimulation of fibrosis. I n "The Molecular and Cellular Biology of Wound Repair" (R. A. F. Clark and P. M. Henson, eds.), pp. 273-280. Plenum, New York. BORDER, W. A., OKUDA,S., LANGUINO,L. R., SPORN, M. B., and RUOSLAHTI, E. (1990). Suppression of experimental glomerulonephritis by antiserum against transforming growth factor t~l. Nature 346, 371-374. BOWEN-POPE, D. F., MALPASS, T. W., FOSTER, D. M. and ROSS, R. (1984). Platelet derived growth factor in vivo: Levels, activity, and rate of clearance. Blood 64, 458-469. FOLKMAN,J., and KLAGSBRUN,M. (1987). Angiogenic factors. Science 235, 442-447. FOLKMAN, J., KLAGSBRUN,M., SASSE, J., WADZINSKI,M., INGBER, D., and VLODASKY,I. (1988). A heparin-binding angiogenic factor--basic fibroblast growth factor--is stored within basement membrane. Am. J. PathoL 130, 393-400. FOX, G. M. (1988). The role of growth factors in tissue repair. III. Fibroblast growth factor. In "The Molecular and Cellular Biology of Wound Repair" (R. A. F. Clark and P. M. Henson, Eds.), pp. 265-271. Plenum, New York.
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Growth Factors in Fetal Wounds
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