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Endocrinology Vol. 138, No. 9 3987-3996
Copyright © 1997 by The Endocrine Society

ARTICLES

The Mechanism of Excisional Fetal Wound Repair in Vitro Is Responsive to Growth Factors

David A. Belford

Cooperative Research Centre for Tissue Growth and Repair, Child Health Research Institute, Woman’s and Children’s Hospital, North Adelaide 5006, Australia

Address all correspondence and requests for reprints to: David A. Belford, Cooperative Research Centre for Tissue Growth and Repair, Child Health Research Institute, Woman’s and Children’s Hospital, 72 King William Road, North Adelaide 5006, Australia. E-mail: david.belford{at}dhn.csiro.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the ability of fetal rat skin to heal an excisional wound in vitro. Skin from the backs of E17–E19 rats was wounded using a 1-mm diameter cutting needle and suspended in culture on a 6-pin cradle for 72 h. Neither contraction nor epithelial closure was observed within wounds created in skin from E19 embryos. In contrast, wounds in E17 skin contracted to 35–50% of their original area over 72 h, although, in the absence of serum, complete wound closure was not observed. Addition of FBS at the time of culture resulted in the movement of the epithelium over the dermal margins of the wound to effect complete closure. Histological sections through these healed wounds revealed an epithelial bridge spanning the dermal margins of the wound. A similar mechanism of repair was observed in the presence of day 14 adult wound fluid. The response of wounds in E17 skin to a range of growth factors was then assessed in an attempt to reproduce the serum response under defined conditions. Insulin-like growth factor I or epidermal growth factor did not significantly affect wound closure. Basic fibroblast growth factor, transforming growth factor-ß, or platelet-derived growth factor did promote wound closure although, in contrast to the serum-induced response, wound histology revealed repair had been achieved by dermal fibroblasts that occupied the space between the epithelial margins of the healed wound. We have therefore shown that the epithelial component of fetal wound repair proceeds in organ-cultured fetal skin in the absence of an adhesive substrate over which to migrate and is dependent on the source of trophic factors. The inability of skin taken from the E19 embryo to heal in vitro suggests a developmental switch in the mechanism of wound epithelialization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ABILITY of the fetal wound to heal without scar formation is well documented (1, 2, 3). To date most studies have focused on the biology of the fetal skin fibroblast (4, 5), the fetal wound matrix (6, 7, 8), and the role of inflammation at the wound site (9, 10). Significantly, several studies have shown that the ability of the fetal wound to heal without scarring is an intrinsic property of early to midgestation skin. Thus, human fetal skin transplanted to an sc pocket in an adult nude mouse heals an incisional wound by reconstitution of dermal structure and appendages (11). Similarly, the neonatal marsupial is able to repair the wounded dermis in an adult environment without scarring, an ability lost after pouch day 9 (12). In contrast, an incisional wound in adult or late gestation sheep skin transplanted back onto the early gestation fetus heals in the fetal environment with formation of a scar (13). An in utero switch from a fetal to an adult mechanism of repair in late gestation has been described in the sheep (14), primate (15), and rat (16). Interestingly, this ability of the surface wound to heal without scar formation is not a general property of all fetal tissues, as both the diaphragm and the stomach heal with scar formation (17, 18).

Less is known about the mechanism of repair of the fetal excisional wound. The ability of the fetal excisional wound to heal in vivo has been shown to be species dependent (19, 20, 21) and, in the case of the rabbit, inhibited by exposure to amniotic fluid (22). Fetal rat skin is able to heal an excisional wound in serum-supplemented suspension culture (16), a response attributed to centripetal spreading of the mesenchymal layer (23). This healing response was not a property of skin from later stage embryos. Martin and colleagues (24, 25) have shown that that an excisional wound on the fetal chick or rodent heals by both mesenchymal contraction and active movement of the epithelium over the dermal margins of the wound. The presence of an actin-cable surrounding the wound suggested that this movement was the result of the coordinated expression and action of actin filaments to effect a purse-string mechanism of wound closure.

The aim of the current study was to use an in vitro suspension-culture model (16) to determine 1) whether the epithelial component of fetal excisional wound repair is an intrinsic property of isolated fetal skin, and 2) the trophic requirements of this response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Insulin-like growth factor-I (IGF-I) was purchased from GroPep (Adelaide, Australia) and epidermal growth factor (EGF) from Auspep (Parkville, Australia). Platelet-derived growth factor (PDGF-BB), basic fibroblast growth factor (bFGF; FGF-2) and transforming growth factor-ß1 (TGF-ß1) were obtained from Austral Biologicals (San Ramon, CA).

Animals
Time-mated pregnant Sprague-Dawley rats were obtained from the CSIRO Division of Human Nutrition (Adelaide, Australia). Rats were smear-positive on day 0. All experiments were approved by the Animal Care and Ethics Committee, CSIRO Division of Human Nutrition, following the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Wound fluid was obtained from Hunt-Schilling chambers placed in the backs of adult male rats (250–300 g) as previously described (26). Wound fluid was collected percutaneously at 14 days, centrifuged to remove cells and tissue debris, and stored frozen at -20 C until use.

Fetal skin cultures
Wounding and culture of fetal rat skin was undertaken according to the method of Ihara et al. (16), with minor changes. Pregnant rats were killed by CO2 asphyxiation, the fetuses removed from the uterus, weighed, and placed in HBSS. All further procedures were undertaken in a laminar flow hood under sterile conditions. Fetal rats were pinned on a dissecting board and a 1-cm x 1-cm piece of skin was dissected from the back of each fetus using fine scissors and forceps. A full thickness, 1-mm diameter wound was created in each explant using a squared-off, sharpened 19G needle. The wound margin was marked by dipping the needle into a sterile solution of India ink before wounding. The wounded skin was then floated onto a 6-pin cradle, taking care to preserve the natural tension of the skin. Each explant was washed before placement in a 12-well tissue-culture dish (Corning, NY) containing DMEM (Flow Laboratories, Irvine, Scotland, UK) alone or DMEM plus 10% FBS (Cytosystems, Castle Hill, Australia) to a final volume of 2 ml. Experiments using individual recombinant growth factors were conducted according to the same protocol except the factors were added in DMEM containing 0.1% BSA (RIA grade, Sigma Chemical Co., St. Louis, MO). FBS and BSA-containing control cultures were included on each plate. Cultures were maintained in a humidified atmosphere at 37 C in 5% CO2-air for 72 h. The wounds were photographed using a standard focal length jig at the time of culture, and at 24, 48, and 72 h.

Histology
Cultures were fixed in methacarn for 2 h before storage in 70% alcohol and processing by graded dehydration. Skin pieces were embedded in wax blocks, and 3 µm sections were taken through the wound, which was identified by the ink particles. Care was taken to section through each wound. Sections were stained with hematoxylin and eosin, viewed, and photographed.

Planimetry and statistical analysis
The wound margin was traced from photographs onto acetate sheets that were then scanned into a computer (Apple Macintosh Onescanner connected to an Apple Macintosh LCIII). Wound areas were calculated using an image analysis system (Prismview, Dapple Systems Inc., Sunnyvale, CA) and are expressed as the percentage of the original wound area. Wound closure data in response to recombinant growth factors were analyzed by Kruskal-Wallis one-way ANOVA on ranks, with posthoc comparisons to the DMEM plus 0.1% BSA group undertaken using Dunn’s test. Data were considered significant with P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A series of preliminary experiments were conducted to assess the viability of organ cultured fetal skin. Histological sections through the wounded skin after 72 h culture in either the presence or absence of serum showed no evidence of cell death or subepidermal liquefaction. Excisional wounds in skin harvested from E19 embryos or later did not heal in either the presence or absence of serum (Fig. 1Go). After 72 h in culture, all wounds were only slightly contracted. Histology showed no evidence of either dermal or epidermal movement into the wound defect (not shown). All further studies were therefore undertaken using the E17 fetus (0.6–0.8 g). Skin from fetal rats of 0.5 g or less lacked the tensile strength to allow consistent placement on the cradles and often pulled off the pegs during culture.



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  		Figure 1. Response of excisional wounds in organ cultured E19 rat skin. Skin was dissected from the dorsum of day 19 embryos and wounded using a 1-mm diameter cutting needle. Wounds are shown at the time of culture (A, B) and at 72 h (C, D) in the presence (A, C) or absence (B, D) of 10% FBS. Bar, 1 mm.

 
Closure of fetal wounds in serum-supplemented culture
Photomicrographs and planimetry data for wounds in E17 fetal rat skin cultured in the presence and absence of serum are shown in Figs. 2Go and 3Go. In serum-free medium, wounds contracted to 35–50% of the wounded area over the 72 h culture period (Fig. 2Go, B and D; Fig. 3Go), although never closed. Addition of FBS (10% vol/vol) to the medium at the time of culture promoted complete wound closure (Fig. 2Go, A and C; Fig. 3Go). After 48–72 h culture in the presence of serum, healed wounds could be identified only by a small pimple-like raised area on the epidermal surface, and to accurately locate the wound on histological sections it was necessary to tattoo the wound margin with India ink at the time of wounding. Observation of the movement of the ink particles also indicated the mechanism of wound closure. After 72 h in the presence of serum, the healed wounds often exhibited a target-like appearance, with an outer ring of ink enclosing a concentration of ink in the centre of the wound. A dermal view of the closed wound after 72 h in serum-supplemented culture clearly showed that the outer ring of ink particles resided on the mesenchymal layer (Fig. 2EGo). Movement of the ink particles with time in culture was clearly shown on explants heavily labeled with India ink (Fig. 4Go). Particles originally at the wound margin at the time of culture (Fig. 4AGo) were observed to progress inwards by 24 h (Fig. 4BGo), to form a concentration of ink in the centre of the wound at 48 and 72 h (Fig. 4Go, C and D). A dermal view of the healed wound again showed the presence of a marginal ring (Fig. 4EGo), suggesting epithelial movement over the dermal layer.



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  		Figure 2. Response of excisional wounds in organ cultured E17 rat skin. Fetal skin was dissected from the dorsum of E17 rats and wounded using a 1-mm diameter cutting needle. The wound margin was marked by dipping the cutting needle into India ink immediately before wounding. Wounds are shown at the time of culture (A, B) and at 72 h (C, D, E, F) in the presence (A, C, E) or absence (B, D, F) of 10% FBS. E and F, Dermal surface of the wound. Bar, 1 mm.

 


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  		Figure 3. Time course of excisional wound closure in organ cultured E17 rat skin. Fetal skin was dissected from the dorsum of E17 rats, wounded using a 1 mm cutting needle, and cultured in the presence ({circ}) or absence (•) of 10% FBS. Wounds were photographed at the time of culture and at 24, 48, and 72 h, the wound margin traced onto acetate sheets, and wound areas calculated by scanning serial tracings into a image analysis program. Data are expressed as a percentage of the original wounded area, each point representing the mean ± SEM of 10 wounds.

 


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  		Figure 4. Movement of ink particles during repair of an excisional wound in E17 rat skin cultured in the presence of 10% FBS. Fetal skin was dissected from the dorsum of E17 rats and wounded using a 1-mm diameter cutting needle that had been dipped into India ink. The wound is shown at the time of culture (A) and at 24 h (B), 48 h (C) and 72 h (D). The dermal surface is shown at 72 h (E). Bar, 1 mm.

 
Wound histology is shown in Fig. 5Go. Wounded E17 skin maintained in serum-free medium showed no evidence of epidermal movement into the wound defect (Fig. 5AGo). In contrast, histological sections through the wound after 72 h in serum-supplemented medium revealed an epidermal bridge spanning the dermal margins of the wound, confirming that the epithelial layer had migrated over the dermal margins of the wound to effect final closure. Ink particles remaining in the dermal margin of the wound are visible.



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  		Figure 5. Histology of excisional wounds in E17 fetal rat skin after 72 h culture. Cultures were maintained in DMEM alone (A) or DMEM plus 10% FBS (B) for 72 h before fixation and processing (H + E; x 10).

 
Response of wounds in E17 skin to growth factors
The healing of excisional wounds in skin from E17 embryos was examined in the presence of IGF-I, EGF, PDGF-BB, bFGF, or TGF-ß1 added individually to the explants. To determine the optimal concentration of each factor, preliminary experiments were conducted over a dilution series between 1 µg/ml and 0.3 ng/ml. Control cultures in DMEM plus BSA (0.1%) or FBS (10%) were included on all plates. Representative photomicrographs of organ cultures are shown in Fig. 6Go, and the decrease in wound area for all cultures in the presence of the optimal concentrations of the indicated factors is shown in Fig. 7Go. When compared with cultures in serum-free medium alone, neither IGF-I nor EGF significantly enhanced wound closure (Fig. 6Go, A–D). The optimal concentration of these factors (100 ng/ml) produced only a slight, nonsignificant, enhancement of wound contraction (Fig. 7Go). Higher concentrations of either factor did not further enhance wound closure. A slight protrusion of the dermal layer into the wound space was a consistent feature of wounds cultured in the presence of EGF (Fig. 6DGo and Fig. 8BGo). The appearance of histological sections through the wound margin after 72 h in the presence of IGF-I were identical to those taken after culture in DMEM plus BSA alone, and showed no evidence of epidermal or dermal movement into the wound space (Fig. 8AGo).



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  		Figure 6. Response of excisional wounds in organ cultured E17 rat skin to recombinant growth factors. Wounds are shown at the time of culture (A, C, E, G, I) and at 72 h (B, D, F, H, J) in the presence of IGF-I (100 ng/ml; A, B), EGF (100 ng/ml; C, D), bFGF (50 ng/ml; E, F), TGF-ß1 (100 ng/ml; G, H) and PDGF-BB (1 µg/ml; I, J). Bar, 1 mm.

 


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  		Figure 7. Wound closure in organ cultured E17 rat skin after 72 h in the presence of recombinant growth factors. Data are expressed as a percentage of the original wounded area, each bar representing mean ± SEM for the indicated number of cultures. Growth factors were diluted in DMEM-0.1% BSA and added at the following concentrations: IGF-I (100 ng/ml); EGF (100 ng/ml); bFGF (50 ng/ml); TGF-ß1 (100 ng/ml); PDGF-BB (1 µg/ml). *, P < 0.05 vs. DMEM-BSA.

 


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  		Figure 8. Histology of excisional wounds in E17 fetal rat skin after 72 h culture in the presence of recombinant growth factors. Cultures were maintained in DMEM-0.1% BSA plus IGF-I (100 ng/ml; A), EGF (100 ng/ml; B), bFGF (50 ng/ml; C), TGF-ß1 (100 ng/ml; D) or PDGF-BB (1 µg/ml; E) for 72 h before fixation and processing (H + E; x 10).

 
Addition of bFGF, TGF-ß, or PDGF to the fetal skin explants resulted in a significant decrease in wound area after 72 h culture. At the optimal concentration, complete wound closure was seen in 8/11 wounds in bFGF (50 ng/ml), 8/12 wounds in TGF-ß (100 ng/ml), and 8/8 wounds in the presence of PDGF (1 µg/ml). The mechanism of closure in response to these growth factors was clearly different to that observed in serum-supplemented cultures. While ink particles associated with the epidermal margin moved into the wound defect, they did not meet in the centre of the wound (Fig. 6Go, E–J), and no ring of ink was observed on the dermal surface (not shown). Moreover, wound histology after 72 h culture showed the presence of dermal fibroblasts between the epidermal margins of the wound (Fig. 8Go, C–E). In particular, a dense, highly cellular wound infiltrate was characteristic of all cultures after 72 h in the presence of PDGF (Fig. 8EGo). In contrast, fibroblasts within the TGF-ß treated wounds were more sparsely distributed and noted to assume a more rounded morphology (Fig. 8DGo). The effect of these factors was dose dependent. Concentrations of TGF-ß greater than 100 ng/ml and bFGF greater than 50 ng/ml were found to be inhibitory to wound closure. Lower concentrations of TGF-ß (0.5, 5 or 50 pg/ml) did not enhance wound closure above that seen in DMEM plus BSA alone. Addition of PDGF-BB at concentrations less than 500 ng/ml was ineffective in promoting wound repair.

Response to adult wound fluid
To determine whether the pattern of wound repair observed in FBS was reproduced in the adult wound environment, wounded E17 skin was cultured in the presence of fluid aspirated from Hunt-Schilling chambers 14 days after placement in adult rats. A serum-like response was seen in the presence of 5 or 10% (vol/vol) wound fluid. Wound closure was rapid, with 11/11 wounds 100% healed at 48 h culture, leaving only a small dimple at the centre of the wound site (Fig. 9BGo). Wound histology was similar to that observed in FBS-supplemented cultures showing epithelium bridging the dermal wound margins (Fig. 9CGo).



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  		Figure 9. Wound closure in organ cultured E17 rat skin after 72 h in the presence of adult rat wound fluid. Fetal skin was dissected from the dorsum of E17 rats and wounded using a 1-mm diameter cutting needle. Wound fluid was collected from Hunt-Shilling chambers 14 days after sc placement in adult rats and added to cultures at 10% vol/vol. Wounds are shown at the time of culture (A) and at 72 h (B). C, Histological section through the wound after 72 h culture in 10% wound fluid.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vitro investigation of wound repair using organ culture systems allows direct manipulation of the healing environment without interference by systemic factors (27, 28). Such an approach would seem valid in the context of fetal wound repair given the intrinsic ability of the skin to heal a wound outside the fetal environment (11, 16). While the ability of the fetal dermis to restore collagen architecture within a healing incisional wound has been the subject of significant research effort, repair of excisional wounds by fetal skin has received less attention. The current report demonstrates that skin isolated from the E17 rat fetus retains the ability to heal an excisional wound by a combined process of dermal contraction and, importantly, by movement of the epithelium over the dermal margins of the wound. This epithelial response was observed in suspension culture and therefore could not proceed by the migratory processes that characterize adult wound repair, which require an adhesive substrate (29). Epithelial closure was dependent on the presence of serum or wound fluid and was not promoted by IGF-I, EGF, PDGF, bFGF, or TGF-ß.

Our findings support the observations of Martin and colleagues using the whole embryo (24, 25). These investigators observed that although some 50% of the healing of an excisional wound on the fetal chick or rodent was a result of mesenchymal contraction, basal epidermal cells at the wound margin were able to coordinate actin filaments to effect a contractile, purse-string mechanism of wound closure. Epithelial cells were observed to lack lamellipodia, orientate along the axis of the wound, and migrate over the mesenchymal wound margins. A pile-up of epithelial cells in the centre of the healed wound was observed, possibly reflecting the ongoing action of the contractile response (cf Fig. 4Go, C and D). A similar coordinated contractile mechanism of epithelial repair has been reported in wounded monolayers of cultured Caco-2 cells (30). The role of contractile proteins in repair of excisional wounds in isolated fetal skin is currently under investigation in our laboratory.

In the absence of serum, wounds in suspension-cultured E17 fetal skin contracted to some 35–50% of the wounded area, although epidermal movement over the dermal wound margin was not seen. This contractile response made a variable contribution to wound closure in the presence of serum, as the size of the ring of ink particles remaining on the dermal margin was always less than the size of the original wound (Figs. 2EGo and 4EGo compared with Fig. 2AGo and 4AGo, respectively). Whether this response represents a fetal equivalent of adult wound contraction is unknown. A previous in vivo study noted that the appearance of {alpha}-smooth muscle actin-positive myofibroblasts in the fetal sheep excisional wound coincided with scar formation in late gestation (2).

In contrast to wounds in skin from E17 embryos, neither wound contraction nor epithelial closure was observed by wounds in E19 skin. Ihara et al. (16) have shown that, while wounds on the E16 rat fetus heal rapidly with reconstitution epidermal structure, repair of the day 18 wound resembles postnatal healing, with invasion by inflammatory cells, angiogenesis, and only thin undifferentiated epidermal coverage. Thus, the rat embryo undergoes a fetal to adult switch in healing mechanism over this gestational period. The ability of wounds on postnatal rats to heal in vitro has been investigated by Greenwald et al. (27), who found that optimal healing could only be achieved if wounds were left in situ for 48 h before culture, suggesting that cells of adult wound repair require activation by systemic factors. Together, this data would suggest that a component of the fetal to adult transition is a loss of the ability of the fetal epidermal cell to respond to wounding by movement over the mesenchyme according to a substrate-independent purse-string mechanism.

As adult wound fluid was able to replace FBS as a trophic source, the epithelial response to wounding was not dependent on a fetal environment. Similarly, Lorenz and co-workers have shown that wounded human fetal skin heals by a scar free mechanism in an sc pocket on an adult athymic mouse (11). However, our attempts to reproduce the in vitro response to serum using individual growth factors were unsuccessful. High concentrations of PDGF, bFGF, or TGF-ß did promote closure of the fetal wound in vitro, although the mechanism was clearly different to that observed in the presence of serum. Histology showed that the epidermal margins of the closed wound were separated by fibroblasts, suggesting a primary site of action for these factors within the dermis to promote wound contraction and fibroblast division. In this regard, TGF-ß has been shown to promote the reorganization of a collagen fibers by fibroblasts to contract a 3-dimensional collagen gel (31, 32), although bFGF and PDGF were ineffective in these studies. Current data would suggest that these factors play only a minor role in fetal wound repair. Thus, while PDGF and TGF-ß have been detected in the fetal wound, they are cleared more rapidly than in the adult wound (33, 34, 35). Basic FGF is undetectable in the fetal wound (33). Moreover, in vivo exposure of the fetal wound to supraphysiological concentrations of either PDGF or TGF-ß results in a more adult-like picture of wound repair with increased inflammatory cell and fibroblast infiltrate and increased collagen deposition and scar formation (36, 37, 38). Conversely, administration of anti-TGF-ß to adult wounds results in a more fetal, regenerative pattern of repair with fewer inflammatory cells and blood vessels and collagen fibre orientation more resembling unwounded dermis (39).

In summary, this report demonstrates that an excisional wound in isolated, suspension-cultured E17 rat skin heals by a combined process of wound contraction and the substrate-independent movement of epithelium over the dermal margins of the wound. The epidermal component of this response was dependent on the presence of either serum or wound fluid, although not promoted by the individual growth factors tested. The molecular events that underlie the ability of E17 but not E19 epithelium to respond to wounding in this manner are currently the subject of investigation in our laboratory.


   Acknowledgments
 
Ingrid Liepe, Kaylene Pickering, and Anna Seamark are thanked for their technical help and expertise.

Received January 22, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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