Mean normal dermal fibroblast growth curves in the presence and absence of silicone gel in serum-free growth media.
Mean keloid fibroblast growth curves in the presence and absence of silicone gel in serum-free growth media.
Mean fetal dermal fibroblast growth curves in the presence and absence of silicone gel in serum-free growth media.
Mean basic fibroblast growth factor (bFGF) levels found in serum-free supernatants collected from normal dermal fibroblast cell cultures.
Mean basic fibroblast growth factor (bFGF) levels found in serum-free supernatants collected from keloid fibroblast cell cultures.
Mean basic fibroblast growth factor (bFGF) levels found in serum-free supernatants collected from fetal dermal fibroblast cell cultures.
Hanasono MM, Lum J, Carroll LA, Mikulec AA, Koch RJ. The Effect of Silicone Gel on Basic Fibroblast Growth Factor Levels in Fibroblast Cell Culture. Arch Facial Plast Surg. 2004;6(2):88-93. doi:10.1001/archfaci.6.2.88
From the Wound Healing and Tissue Engineering Laboratory, Division of Otolaryngology/Head and Neck Surgery, Stanford University School of Medicine, Stanford, Calif.
Copyright 2004 American Medical Association. All Rights Reserved.
Applicable FARS/DFARS Restrictions Apply to Government Use.2004
Background Topical silicone gel has shown promise in the treatment of hypertrophic and keloid scars. However, its mechanism of action remains undetermined.
Objective To investigate whether the presence of silicone alters the secretion of basic fibroblast growth factor (bFGF), a key cytokine involved in the scar formation process.
Design Serum-free fibroblast cell cultures were established from normal, keloid, and fetal skin, which heals without scarring, and exposed to silicone gel. Serial cell counts were performed, and supernatants were collected for bFGF quantification by enzyme-linked immunosorbent assay at 4, 24, 72, and 120 hours.
Results Growth curves were similar and no statistically significant differences in population doubling times were observed between treated and untreated specimens. Statistically significant differences in bFGF levels between treated and untreated normal fibroblasts were observed at 24, 72, and 120 hours after cell culture initiation. Differences in bFGF levels between treated and untreated fetal fibroblasts that approached statistical significance were observed at 72 and 120 hours.
Conclusions These results suggest that silicone gel is responsible for increased bFGF levels in normal and fetal dermal fibroblasts. We postulate that silicone gel treats and prevents hypertrophic scar tissue, which contains histologically normal fibroblasts, by modulating expression of growth factors such as bFGF. Our data support the hypothesis that substances that favorably influence wound healing do so by correcting a deficiency or overabundance of the growth factors that orchestrate the tissue repair process.
Aberrant wound healing in the form of keloid and hypertrophic scar formation is a significant problem that affects millions of patients yearly. Keloids and hypertrophic scars are associated with an abnormal proliferation of fibroblasts as well as overproduction of extracellular matrix and collagen.1 This pathologic process is believed to be mediated, at least in part, by abnormal levels of growth factors.2 Treatment for keloid and hypertrophic scars is problematic with no single modality producing uniformly satisfactory results.
Topical silicone gel has been successfully used for the treatment of aberrant scar tissue that results from thermal burn wounds, surgical procedures, and traumatic events. The mechanism of action has not been conclusively proven and remains a subject of controversy. It has been suggested that hydration, rather than an inherent property of silicone itself, modulates the effect on wound healing.3-4 Other authors believe that low-molecular-weight silicone oil is responsible for the effects of silicone on wound healing.5-6
We evaluated the effect of silicone gel on dermal fibroblasts grown in serum-free media from tissues that span the range of the wound healing phenomenon: fetal fibroblasts for scar-free healing; normal fibroblasts for normal healing; and keloid-producing fibroblasts for exuberant, aberrant wound healing. It may be that substances that favorably influence wound healing, such as silicone, do so by correcting a deficiency or overabundance of growth factors. The significance of this study involves ascertaining whether modulating the growth factor milieu will allow us to achieve the goal of optimal wound healing with minimal or no scar formation.
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 eyelid 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, Stanford, Calif. Fetal fibroblasts derived from ear skin of a 14th-gestational-week white human embryo were obtained from a cell line repository (Coriell Laboratories, Camden, NJ).
Cell lines from each specimen were established and propagated in a serum-free environment. Using sterile technique under a laminar flow hood, the dermal specimen was minced into approximately 1-mm3 fragments on a Petri dish with a sterile scalpel blade. The specimens were washed in Dulbecco phosphate-buffered saline solution with 5% penicillin/streptomycin/amphotericin (GIBCO, Grand Island, NY). The specimens were then placed in scored 75-cm2 tissue culture flasks (T75) (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% penicillin/streptomycin/amphotericin) (GIBCO). The specimens were then stored in a humidified incubator at 37°C with a 5% carbon dioxide atmosphere.
After 24 hours, the media was changed with 5 mL of primary culture media. The media was then changed every 2 days until fibroblasts were visualized under light microscopy to be growing outward from the explanted tissue. At that time, the tissue was removed. With sufficient outgrowth of fibroblasts, cells were subcultured into 75-cm2 culture flasks. Primary culture media was changed every third to fourth day. Successive cultures were passed at confluence. Fetal fibroblasts, which were obtained from an already established line, were also passed at confluence.
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 inactivated using trypsin soybean inhibitor (GIBCO) in a 1:1 ratio. Cells were then suspended in UltraCULTURE (BioWhittaker, Walkersville, Md), a commercially available serum-free media, which we have previously shown to sustain fibroblast cell cultures for durations similar to those used in this study.7 Cells were counted in duplicate using phase contrast microscopy and a hemocytometer. Viable cells were determined using trypan-blue dye exclusion. Normal, keloid, and fetal fibroblasts were then seeded at a density of 6 × 104 cells/mL in each well of a 24-well plate (Falcon).
Scarfade silicone gel (a gift from Hansen Medical, Tacoma, Wash) was placed into selected wells within the 24-well plates. Approximately 0.05 mL of silicone gel was placed on the side walls of the wells completely immersed in serum-free media but in minimal contact with the floor of the wells where fibroblasts attached. Untreated cells from each cell line were used for controls. Normal, keloid, and fetal fibroblasts were incubated with and without silicone gel in serum-free media for 4, 24, 72, and 120 hours.
At each predetermined time point, cell-free supernatant was collected from the testing wells. One-milliliter samples were stored at −20°C in microcentrifuge tubes for later growth factor assays (see section titled "Growth Factor Assays"). At the time of each cell culture initiation, 1 mL of UltraCULTURE was also placed in wells containing and not containing silicone gel. One milliliter of this sample was also collected and stored at –20°C in a microcentrifuge tube for later growth factor assay.
Cell counts were performed using the water-soluble tetrazolium salt (WST)-1 assay (Boehringer Mannheim, Indianapolis, Ind) at 4, 24, 72, and 120 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. 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 × 104 cells/mL.
Expression of basic fibroblast growth factor (bFGF) was evaluated for each of the postmodulation cell cultures by solid phase enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minn) at 4 representative time points: 4, 24, 72, and 120 hours. Unmodulated samples from each source were also evaluated by ELISA at the 4 representative time points. Again, samples were analyzed by ELISA in triplicate. Finally, samples of UltraCULTURE exposed to silicone and not exposed to silicone were also assayed for bFGF at each time point. Assays were read using an automated plate reader and optical densities were analyzed with KC4 software. Enzyme-linked immunosorbent assays were performed in a blinded fashion with respect to the cell line, whether the cells were exposed to silicone, and growth time of each sample studied to minimize observer bias.
Each silicone modulated and unmodulated cell line was cultured in triplicate. Mean cell counts were used to determine population doubling times (PDTs). Population doubling times of cells treated and not treated with silicone were compared using the paired t test. Mean bFGF levels obtained from treated and untreated fibroblasts from each of the 3 cell lines (normal, keloid, and fetal) were also evaluated using the paired t test. Differences at the 5% level were considered significant.
Each of the cell lines (normal, keloid, and fetal) exhibited growth in serum-free media. Of note, it was observed that an oily film disseminated from the silicone gel, into the medium bathing the cells within the first 24 hours. We suspect that this was most likely silicone oil, although this was not tested. Average growth curves of fibroblasts grown in the presence of silicone and in the absence of silicone were plotted (Figure 1, Figure 2, and Figure 3).
The shapes of the growth curves were similar regardless of the presence of silicone, but differed between fibroblast cell type. All 3 cell lines showed an initial decline in cell population after seeding with a proliferative recovery after the third day. The decline in fetal fibroblast cell population occurred later (72 hours) than was observed for normal and keloid fibroblasts (24 hours).
From logarithmic best-fit curves, PDTs were calculated and are given in Table 1. Keloid fibroblasts grown in the presence or absence of silicone exhibited the fastest PDTs, followed by normal fibroblasts with or without silicone. Paired t tests performed on the various cell lines showed no statistically significant differences between PDTs of the same cell type in the presence or absence of silicone gel.
Enzyme-linked immunosorbent assays for bFGF were performed for each of the triplicated cell lines. Levels of bFGF were quantified by ELISA, and the results are shown in Figure 4, Figure 5, and Figure 6. Paired t tests were used to compare differences in bFGF levels between fibroblasts from the same type of skin (Table 2). Statistically significant (P<.05) differences in bFGF levels between normal fibroblasts treated with silicone and not treated with silicone were observed at 24, 72, and 96 hours. Differences in bFGF levels between fetal fibroblasts treated with silicone and not treated with silicone approached significance (P = .05) at 72 and 96 hours. No statistically significant differences in bFGF levels were seen in keloid fibroblasts at any of the time points. As expected, there was no bFGF found in serum-free media alone, regardless of whether silicone was present.
It is believed that abnormal levels of tissue growth factors play a major role in the pathogenesis of keloid and hypertrophic scars. Basic fibroblast growth factor is secreted by multiple cells including dermal fibroblasts, with its target cells being of mesodermal and neuroectodermal origin.2 In general, it is mitogenic, encourages cell survival, inhibits collagen production, and stabilizes cellular phenotype. Tan et al2 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.
In contrast to keloids and hypertrophic scars, fetal wounds heal without histologic evidence of scarring.8 Fibroblasts are the main effector of scarless healing in fetal tissue, and this healing can occur outside the fetal environment.9-10 Lee et al11 found an increase in expression of acidic and bFGFs, and transforming growth factor β1 (TGF-β1) in fetal fibroblasts compared with adult fibroblasts. Their work suggests that differences in cytokine production may contribute to the suboptimal wound healing seen in adult wounds compared with the scarless healing of fetal wounds.
The use of topical silicone products, including silicone gel and silicone gel sheeting, for the treatment of burn scars was first described in 1982 and has since gained widespread popularity in reducing and preventing keloid and hypertrophic scars.12 The safety and efficacy of topical silicone products has become generally accepted based on numerous reports.13-15 In a study by Fulton,13 complete resolution of hypertrophic or keloidal scars was reported in 6 of 20 patients, and 9 of 20 patients were judged to have a good response to therapy. In another study, postoperative use of silicone gel sheeting resulted in a 12.5% recurrence rate for excised keloids vs a 37.5% recurrence rate in keloids treated by excision only.14
The mechanism of action for topical silicone remains unknown. It was shown by Quinn et al5 and Quinn6 that silicone gel sheeting exerts negligible pressure compared with the 15 to 40 mm Hg required by pressure garments for efficacy. Changes in temperature and oxygen tension were also investigated, and no differences were found between treated scars and normal skin. The water vapor transmission rate of silicone gel was found to be half that of skin, and, when removed, the loss of water increases dramatically for 15 to 20 minutes. However, this effect on water vapor transmission was mimicked with an occlusive dressing that did not demonstrate any therapeutic activity. Without evidence that topical silicone sheeting produced significant pressure, temperature, oxygen tension changes as well as no meaningful effects due to moisture, Quinn et al5 and Quinn6 concluded that an in vitro release of low-molecular-weight silicone fluid from a silicone gel was most likely responsible for its efficacy.
More recent studies have suggested that static electricity generated by silicone gel and silicone gel sheeting plays a role in the antiscarring effects. Hirshowitz et al16 speculated that static-electric field induction is responsible for the response of hypertrophic and keloidal scars to silicone products, based on good results with a silicone gel cushion that generates 3 to 4 kV/in of negatively charged static electricity. Other investigators maintain that hydration and occlusion are responsible for the beneficial effects of silicone.3-4
Fulton13 looked for the presence of silica in the tissue after application of silicone gel sheeting in 4 patients and reported that no silica from the dressing was found at the wound site after biopsy of treated skin. More recently, Shigeki et al17 found that silicone gel sheeting released silicone in a time and pH-dependent manner when placed in aqueous solution. Additionally, they found that silicone could be detected in excised normal and hypertrophic scar skin to which silicone gel sheeting was applied ex vivo under hydrated conditions. Although we did not specifically test for the presence of silicone in the media, we observed an oil dispersing from the gel likely containing silicone.
Silicone gel appears to have no effect on cellular proliferation rates. In serum-free media, differences in growth rates were observed between different cell types, as expected, but no statistically significant differences in PDT or in the configuration of the growth curves were found, regardless of the presence of silicone at the dosages and duration examined in the present study. Since cell counts were nearly equal between cell cultures treated with silicone and those not treated, the quantitative differences in bFGF concentrations were likely due to an effect of silicone rather than differences in proliferation or cell death.
This study suggests that silicone gel works at least in part by increasing the levels of bFGF. An increased level of bFGF would be expected to reduce collagen proliferation based on other studies of its effects.2 This study links the clinically observed benefit of silicone scar treatment with a quantifiable biochemical effect observed in an isolated cell culture system. A prior investigation by Ricketts et al18 also supports the findings of the present study. In that study, messenger RNA (mRNA) for several cytokine growth factors was isolated by performing reverse transcription–polymerase chain reaction on digested hypertrophic scar biopsy specimens. Treatment of hypertrophic scars by either occlusive dressing or silicone gel resulted in increased mean levels of interleukin 8, bFGF, and granulocyte-macrophage colony-stimulating factor compared with untreated hypertrophic scar specimens. In addition, decreased levels of TGF-β and fibronectin were observed. None of these differences observed with silicone gel were statistically significant, however. A decrease in mean bFGF mRNA from hypertrophic scar tissue treated with occlusive dressing compared with untreated hypertrophic scar tissue was the only statistically significant difference found.
Unlike the study by Ricketts et al,18 the present study demonstrates a difference in actual protein product rather than indirect evidence of increased growth factor obtained by assaying for mRNA expression. Prior in vitro studies of fibroblast growth factor production have been confounded by the presence of serum-containing tissue culture media because serum contains growth factors. The senior investigator (R.J.K.) recently developed a serum-free in vitro fibroblast model.19 Since the only growth factors present are products of the fibroblasts themselves, autocrine products are assayed without exogenous contributions, increasing the confidence in the findings achieved.
It may also be that both chemical properties and physical properties of silicone gel are responsible for its therapeutic action. Sawada and Sone3 found that scar hydration and occlusion without the addition of silicone resulted in significant improvement in scar redness, elevation, induration, itching, and pain compared with petroleum jelly control after 5 months. It has been shown indirectly that growth factors such as platelet-derived growth factor and bFGF accumulated under occlusive dressings by antibody assays of wound fluid retained in an occluded wound.20 As previously mentioned, Ricketts et al18 found differences in cytokine expression caused by both silicone gel and occlusive wound dressing.
Another implication of our findings involves the issue of whether silicone products are truly inert within the body. Ahn et al21 found no histologic evidence of inflammation or foreign body reaction in treated scars nor could they detect a difference in vascularity, presence of rete pegs, or number of fibroblasts or inflammatory cells between treated and untreated specimens. Another study by Wong et al15 confirmed no inflammation or foreign body reaction histologically in skin treated with silicone gel for 1 year. In contrast, Ladin et al22 demonstrated a clonotypic T-cell response in the pericapsular infiltrate from patients with silicone implants suggesting that silicone can stimulate proliferation of T lymphocytes. However, numerous clinical studies on silicone gel-filled breast implants have failed to find an association with increased rates of systemic disease despite early anecdotal reports of increased rates of collagen vascular disease, and it is believed the biological effect of implants, if any, are limited.23
Interestingly, no silicone-related effects were seen with keloid cells in our study. It may be that silicone is more effective on hypertrophic scars than on keloidal scars. While the clinical distinction between hypertrophic scars and keloids can be unclear, there are specific morphologic and functional differences between the 2 types of aberrant scars.24-25 Hypertrophic scars are composed of normal fibroblasts, while keloid fibroblasts have a distinct appearance and behavior. In addition, the histologic arrangement of collagen in hypertrophic scars resembles that of normal tissue, while collagen in keloids is larger, less regular, and has a higher interfibrillar distance. The next logical step in the elucidation of silicone's effect on scar reduction is to perform serum-free growth factor studies on hypertrophic scar tissue. Effects on normal fibroblast bFGF levels observed in the present study suggests that silicone should be used in hypertrophic scar treatment as well as preventively on clean, well-coated wounds when there is a concern for hypertrophic scar formation.
It is unclear whether silicone increases bFGF levels by inducing de novo production of bFGF or by stabilizing bFGF produced by cells in usual quantities. It is also not known whether silicone affects levels of other growth factors involved in the wound healing process, such as TGF-β and platelet-derived growth factor. Serum-free studies to explore these questions are ongoing in our laboratory.
To our knowledge, this is the first study that demonstrates a biochemical rather than a physical effect induced by the presence of silicone. Our data support the hypothesis that substances that favorably influence wound healing do so by correcting a deficiency or overabundance of growth factors. By defining the changes induced by treatments that enhance wound healing, we hope to gain increased understanding of the healing process and an ability to manipulate the wound environment to achieve the clinical goal of scarless healing.
Serum-free media can sustain growth of fibroblast cell culture, providing a model to test fibroblast autocrine growth factor production without exogenous contributions from serum-containing media. Silicone gel does not appear to affect the proliferation rate of normal, keloid, and fetal fibroblasts. Normal and fetal fibroblasts exhibit higher levels of bFGF when exposed to silicone gel. The increase in bFGF level associated with silicone treatment of normal fibroblasts, which are found in normal and hypertrophic scar tissue, suggests a possible mechanism of action for the clinically observed response of hypertrophic scar to silicone gel. There was no increase in bFGF level by keloid fibroblasts, suggesting less efficacy in scar treatment or a different mechanism of action.
Corresponding author and reprints: Matthew M. Hanasono, MD, Division of Plastic Surgery, New York Presbyterian Hospital-Weill Cornell Medical Center, 525 E 68th St, Box 115, New York, NY 10021 (e-mail: email@example.com).
Accepted for publication August 5, 2003.
This study was presented at the American Academy of Facial Plastic and Reconstructive Surgery (AAFPRS) Fall Meeting; September 5, 2001; Denver, Colo (winner of the AAFPRS Resident's Travel Award).