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Matthew M. Hanasono, MD; Magdalena Kita, BS; Anthony A. Mikulec, MD; Devon Lonergan, BS; R. James Koch, MD, MS

Arch Facial Plast Surg. 2003;5:26-30.

ABSTRACT
Objective  To evaluate differences in fibroblast autocrine growth factor production by human fetal, keloid, and normal adult dermal fibroblasts.

Design  Serum-free cell lines of fetal, keloid, and normal adult dermal fibroblasts were established. Cell counts were performed and supernatants collected at 4, 24, and 72 hours. Cell-free supernatants were quantitatively assayed for transforming growth factor 1 (TGF-1) and basic fibroblast growth factor (bFGF).

Results  Population doubling times for fetal, keloid, and normal adult fibroblasts were 120.0, 88.1, and 128.4 hours, respectively. Differences in population doubling times did not reach statistical significance. Statistically significant differences between TGF-1 levels from fetal and normal adult fibroblasts were seen at 24 and 72 hours. Significant differences between TGF-1 levels from keloid and normal adult fibroblasts were also seen at 24 and 72 hours. Fetal fibroblasts demonstrated higher levels of bFGF than normal adult fibroblasts at each time point, but these differences were not statistically significant. No significant differences were observed between keloid and normal adult bFGF levels.

Conclusions  Both fetal and keloid fibroblasts produce significantly more TGF-1 than normal adult fibroblasts. Our data and the data of others suggest that fetal fibroblasts produce more bFGF than adult fibroblasts. The serum-free model we describe can be used to quantitatively measure autocrine growth factor production by cells that underlie clinically different types of wound healing. This model provides information that may allow us to better treat and prevent undesirable scarring.

INTRODUCTION

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FETAL WOUNDS heal without histologic evidence of scarring.¹ Fibroblasts are the main effectors of scarless healing in fetal tissue and such healing can occur outside the fetal environment.^2-5 Several studies suggest that differences in autogenously produced growth factors exist between adult and fetal wounds.^6-11 The most promising and best studied of these growth factors include transforming growth factor 1 (TGF-1) and basic fibroblast growth factor (bFGF).

In contrast to fetal wound healing, keloids are characterized by the formation of exuberant scar tissue that does not flatten over time. They are associated with an abnormal proliferation of fibroblasts as well as an overproduction of extracellular matrix and collagen.^12-13 Treatment for keloid scars is problematic because no single modality produces uniformly satisfactory results. Evidence suggests that keloid formation may be caused, in part, by deranged growth factor activity.

Transforming growth factor 1 is a key cytokine involved in the initiation and termination of tissue repair.¹⁴ It is also secreted by multiple cells, including fibroblasts. Its sustained production underlies the development of tissue fibrosis. Younai et al¹⁴ investigated the in vitro effects of TGF-1 on the rate of collagen synthesis in keloid fibroblasts, hypertrophic scar fibroblasts, and normal skin fibroblasts. In response to exogenous TGF-1, keloid fibroblasts produced 12 times more collagen than normal fibroblasts and 4 times more than hypertrophic scar fibroblasts.

Basic fibroblast growth factor is secreted by multiple cells, including dermal fibroblasts, and its target cells are of mesodermal and neuroectodermal origin.¹⁵ In general it is mitogenic, encourages cell survival, inhibits collagen production, and stabilizes cellular phenotype. Tan et al¹⁵ evaluated the effects of bFGF on keloid fibroblast cultures. They found that bFGF causes a dose-dependent inhibition of hydroxyproline biosynthesis, an index of collagen production.

This study evaluates autocrine TGF-1 and bFGF production by dermal fibroblasts from tissues that span the range of the wound-healing phenomenon: fetal fibroblasts for scar-free healing; normal adult fibroblasts for normal healing; and keloid fibroblasts for exuberant, aberrant healing. It is possible that keloids could be treated or prevented by correcting locally insufficient or excessive concentrations of growth factors. Likewise, by understanding the scar-free healing mechanism exhibited by fetal wounds, it may be possible to manipulate the growth factor milieu to simulate this process in wounds of the mature dermis.

METHODS

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FIBROBLAST PRIMARY CULTURES

Normal fibroblast and keloid fibroblast primary cell lines were established from skin obtained from operative specimens. The keloid scar tissue was obtained from the auricular lobule of an 18-year-old white woman. The normal skin sample was obtained from the neck of a 38-year-old white woman. Exemption to use operative specimens that would otherwise be discarded was obtained from the Human Subjects Committee of Stanford University. Fetal fibroblasts derived from the ear skin of a white human embryo in its 14th gestational week were obtained from a cell line repository (Coriell Laboratories, Camden, NJ).

Cell lines from each specimen were established and propagated first in a serum-containing medium, then in a serum-free environment. Using sterile technique under a laminar flow hood, the dermal specimens were minced into approximately 1-mm³ fragments on a Petri dish with a sterile scalpel blade. They were then washed in Dulbecco phosphate-buffered saline solution (PBS) with 5% penicillin-streptomycin-amphotericin (PSA) (Gibco, Grand Island, NY), and placed in scored 75-cm² tissue culture flasks (Falcon, Becton-Dickinson, Franklin Lakes, NJ) with 10 mL of culture medium (10% fetal calf serum in Dulbecco Modified Eagle Medium with 1% L-glutamine and 1% PSA) (Gibco). The specimens were then stored in a humidified incubator at 37°C with a 5% carbone dioxide atmosphere.

After 24 hours, the medium was changed to 5 mL of primary culture medium. The medium was then changed every 2 days until fibroblasts growing outward from the explanted tissue were observed under light microscopy. At that time, the tissue was removed from the medium. With sufficient outgrowth of fibroblasts, cells were subcultured in 75-cm² culture flasks. The primary culture medium was changed every third to fourth day. Successive cultures were passed at confluence.

CELL PLATING IN SERUM-FREE MEDIUM

Experiments were performed with early passage cells (second through ninth passages). At the time of experimentation, confluent cells were released from the flask wall using 0.05% trypsin. The trypsin was then inactivated using trypsin soybean inhibitor (Gibco) in a 1:1 ratio, and cells were suspended in UltraCULTURE (Biowhittaker, Walkersville, Md)—a commercially available serum-free medium that we have shown to sustain fibroblast cell cultures for durations similar to those used in this study.¹⁶ Cells were counted in duplicate using phase contrast microscopy and a hemocytometer. Viable cells were determined using trypan blue dye exclusion. Fetal, keloid, and normal adult fibroblasts were then seeded at a density of 6 x 10⁴ cells/mL in each well of a 24-well plate (Falcon, Becton-Dickinson). Each cell line was cultured in triplicate.

CELL COUNTS

Cell counts were performed using the WST-1 assay (Boehringer Mannheim, Indianapolis, Ind) at 4, 24, and 72 hours after initiation for growth curve generation. The WST-1 assay is a colorimetric assay used in the quantification of cell proliferation and cell viability based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. It is a nonradioactive alternative to the [³H]-thymidine incorporation assay. Assays were read using an automated plate reader (E1x800; Bio-Tek Instruments, Inc, Winooski, Vt). Optical densities were analyzed with KC4 software (Bio-Tek Instruments, Inc). Cell counts were determined by comparison with a standard curve derived from known cell quantities and corrected based on the initial seeding density of 6 x 10⁴ cells/mL. We had previously performed control studies with this assay under the culture conditions used in this study and found the results to be consistent with cell counts achieved by counting with a hemocytometer after trypan blue staining to exclude nonviable cells (unpublished data).

GROWTH FACTOR ASSAYS

Expression of TGF-1 and bFGF was evaluated for each of the cell cultures by solid phase enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minn) at 3 representative time points: 4, 24, and 72 hours. Finally, cell-free samples of UltraCULTURE were also assayed for TGF-1 and bFGF at each time point to confirm that there was no growth factor contribution from the growth media. Assays were read using an automated plate reader and optical densities were analyzed with KC4 software.

STATISTICAL ANALYSIS

Each cell line was cultured in triplicate. Mean cell counts were used to determine population doubling times (PDTs). The PDTs of fetal and keloid fibroblasts were compared with the PDT of normal adult fibroblasts using the paired t test. Mean TGF-1 and bFGF levels obtained from fibroblasts from each of the 3 cell lines (fetal, keloid, and normal adult) were also evaluated using the paired t test. Differences at the .05 level were considered significant.

RESULTS

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Fibroblasts from the 3 cell lines exhibited growth in serum-free medium. The 3 cell lines showed no obvious growth for the first 24 hours after seeding (lag phase), then rapid proliferation (log phase) after the first 24 hours. Growth curves were plotted for each cell line (not shown). Population doubling times were calculated for comparative purposes. The PDTs for fetal, keloid, and normal adult fibroblasts were 120.0, 88.1, and 128.4 hours, respectively. Although keloid fibroblasts had the shortest PDT, differences between the PDTs of the 3 cell lines did not reach statistical significance.

The average TGF-1 concentration was divided by the average number of viable cells to yield the TGF-1 concentration per cell at each time point (Figure 1). Statistically significant differences between the TGF-1 concentration per cell of fetal and normal adult fibroblasts were observed at 24 and 72 hours (P<.05 and P<.01, respectively). Statistically significant differences between the TGF-1 concentration per cell of keloid and normal adult fibroblasts were also observed at 24 and 72 hours (P<.05 for both time points).

View larger version (21K): [in this window] [in a new window] Figure 1. The average transforming growth factor 1 (TGF-1) concentration per cell in serum-free fetal, keloid, and normal adult dermal fibroblast cell culture.

The average bFGF concentration per cell is shown in Figure 2. Fetal fibroblasts exhibited slightly greater bFGF concentrations per cell than normal adult fibroblasts at 4, 24, and 72 hours. However, none of these differences reached statistical significance. Likewise, no statistically significant differences were observed between bFGF concentrations per cell in keloid fibroblasts and the normal adult fibroblasts serving as controls.

View larger version (20K): [in this window] [in a new window] Figure 2. The average basic fibroblast growth factor (bFGF) concentration per cell in serum-free fetal, keloid, and normal adult dermal fibroblast cell culture.

No TGF-1 or bFGF was detected in the serum-free medium, as expected.

COMMENT

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Controversy in the literature exists regarding the production of TGF-1 by fetal fibroblasts relative to adult fibroblasts.^6-9 Most prior studies comparing growth factor production in the fetal and adult fibroblast are limited in that they use a rabbit- or mouse-derived model of wound healing. Broker et al,¹⁰ however, found a decrease in messenger RNA (mRNA) expression of acidic and basic fibroblast growth factors and TGF-1 in human fetal fibroblasts compared with human adult fibroblasts. As measurement of mRNA is indirect evidence of differences in growth factor production, the next logical step is to directly assay for secreted growth factors.

Lee et al¹¹ recently used Western blot analysis to demonstrate a qualitatively greater expression of TGF-1, acidic fibroblast growth factor, and bFGF by human fetal fibroblasts compared with adult fibroblast cell lines. Broker et al¹⁰ reported contradictions between relative production of mRNA and protein products detected by Western blot analysis for the same growth factors. Moreover, they¹⁰ put forward the hypothesis that the contradictions could be caused by posttranscriptional regulation, and that the latter may be due either to inhibited translation of adult fibroblast growth factors into protein or to faster degradation of fetal growth factor proteins. The data of Broker et al agree with our finding reached by ELISA, that TGF-1 concentrations per cell are 5 to 7 times higher in fetal fibroblasts than in normal adult fibroblasts.

Although the average bFGF concentration per cell was found to be slightly higher in fetal fibroblasts than in normal adult fibroblasts, none of the differences for each time point reached statistical significance in this study. Lee et al¹¹ noted that differences between fetal and adult expression of both bFGF and TGF-1 were more difficult to appreciate in fibroblasts grown in a serum-free medium and were more marked when grown in a medium containing 15% fetal bovine serum. It is possible that the lack of significant differences in bFGF secretion between fetal fibroblasts and normal adult fibroblasts is due to our method's inability to detect small differences in growth factor levels. However, results obtained from cells grown in a typical serum-containing medium may be less accurate because serum contains exogenous growth factors (see below).

The production of TGF-1 by keloids was previously studied. Peltonen et al¹⁷ demonstrated that TGF-1 mRNA and TGF-1 protein are associated with excessive collagen synthesis and extracellular matrix accumulation in keloids. Lee et al¹⁸ performed a semiquantitative Western blot analysis of TGF-1 expression and found increased expression of TGF-1 in keloid compared with normal human dermal fibroblasts. These results agree with the findings in the present study.

Production of bFGF in keloids has not been as well studied. We reported previously that levels of bFGF autogenously produced by keloid and normal adult fibroblasts are not significantly different, although both cell types increase production of bFGF after treatment with carbon dioxide laser in a fluence-dependent manner.¹⁹ This suggests that autogenous bFGF may not play a key role in the formation of keloids, but that it may be a potential modulator in their treatment or prevention. The level of exogenous bFGF, which is produced by cells of many other types, including endothelial cells, smooth muscle cells, and chondrocytes, may be more important in the mechanism of keloid formation.²⁰

Surprisingly, our data and the results of the authors mentioned above suggest that both fetal and keloid fibroblasts produce greater amounts of TGF-1 than normal adult fibroblasts. Although reproducible differences in growth factor production suggest that TGF-1 and bFGF are, at least in part, responsible for differences in scar formation, the mechanisms by which they affect the complex process of wound healing are still unclear. While these growth factors undoubtedly play a major role in scar formation or lack thereof, they are probably only one piece of the puzzle. Other growth factors, fundamental differences in fibroblasts, and paracrine effects from inflammatory cells present during wound healing are likely to contribute to the healing phenomenon in ways that are just beginning to be understood.

In vitro studies of fibroblast autocrine characteristics have been confounded by the presence of serum-containing tissue culture media because serum contains growth factors. The senior investigator of this study (R.J.K.) has developed a serum-free in vitro fibroblast model.²¹ Since the only growth factors present in this model are products of the fibroblasts themselves, autocrine products may be assayed without exogenous contributions.

Exponential growth was supported by the serum-free conditions of this study. Although keloid fibroblasts exhibited the shortest doubling time (88.1 hours), no statistically significant differences in PDTs were noted between fetal, keloid, and normal adult fibroblasts. Other studies using serum-supplemented culture media have also not shown significant differences in growth rates between keloid and normal fibroblasts.^22-24 We showed previously that growth rates and viability for keloid fibroblasts propagated in a commercially available serum-free medium are comparable to those propagated in serum-containing media.¹⁶

As the fibroblasts in the present study replicated they were bathed only in their own autocrine growth factors, not in serum as had been the case in previous studies.¹⁸ The quantitation method used to evaluate cell growth at each time point, WST-1 assay, measures only viable cells. This allowed us to determine the average TGF-1 concentration per viable cell at each time point, and improved the accuracy of our results. Use of ELISA to detect TGF-1 and bFGF allowed us to make a quantitative comparison of growth factor expression between cell types.

We present here a serum-free model that can be applied to dermal fibroblasts from sources that take into account variables such as skin site, ethnicity, and age. A wider range of fibroblast samples may reveal a site-specific variation in growth factor production, explaining why some skin areas, such as the chest, have a predisposition to hypertrophic and keloid scarring, while others, such as the midface, tend to heal with minimal scarring. We acknowledge that in this study, fibroblasts from normal ear skin, which were not available, would have been preferred for comparison with fibroblasts from keloidal ear skin and fetal ear skin. Autocrine production of other growth factors, such as insulin growth factor, epidermal growth factor, and platelet-derived growth factor can also be investigated with this model.

CONCLUSIONS

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1. No statistically significant differences in PDTs were observed between fetal, keloid, and normal adult fibroblast cell cultures.

2. At 24 and 72 hours after cell culture initiation, fetal and keloid cells both demonstrated significantly higher levels of TGF-1 concentration per cell than normal adult fibroblasts serving as controls.

3. No statistically significant differences in bFGF concentration per cell between fetal and normal adult fibroblasts or between keloid and normal adult fibroblasts were observed at 4, 24, 72 hours, although at all time points fetal fibroblasts exhibited higher levels of bFGF than normal adult fibroblasts.

4. The serum-free model we describe can be used to quantitatively measure autocrine growth factor production by cells that underlie clinically different types of wound healing, and is likely to provide information that will allow us to better treat and prevent undesirable scarring.

AUTHOR INFORMATION 

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Corresponding author: Matthew M. Hanasono, MD, Division of Plastic and Reconstructive Surgery, New York–Presbyterian Hospital, Weill-Cornell University Medical Center, 525 E 68th St, Box 115, New York, NY 10021.

Accepted for publication October 31, 2001.

This study was supported by the 2000 American Academy of Facial Plastic and Reconstructive Surgery Resident Research Grant.

This study was presented at the American Academy of Facial Plastic and Reconstructive Surgery Spring Meeting, Palm Desert, Calif, May 12, 2001.

From the Wound Healing and Tissue Engineering Laboratory, Division of Otolaryngology–Head and Neck Surgery, Stanford University Medical Center, Stanford, Calif.

REFERENCES

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