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A Serum-Free Study

Elbert T. Cheng, MD; Kenneth C. Nowak, MD; R. James Koch, MD

Arch Facial Plast Surg. 2001;3:252-257.

ABSTRACT
Background  A serum-free in vitro model was used to determine the effect of combined carbon dioxide (CO₂) and erbium (Er):YAG laser (Derma K; ESC/Sharplan Medical Systems, Yokneam, Israel) irradiation on keloid-producing fibroblasts (KFs) from 2 distinct facial sites. Transforming growth factor ß1 (TGF-ß1) and basic fibroblast growth factor (bFGF) play an integral part in wound healing and were assayed using this model. It has always been a clinical impression that fibroblasts from different regions of the face behave differently. This is exemplified by patients prone to lobule keloid formation after ear piercing, who heal normally after a facial incision.

Design  Laboratory-based wound healing.

Methods  Human KF cell lines were established from operative specimens using standard explant techniques. At 48 hours after seeding, 20% of each well was exposed to 1.7 J/pulse of Er:YAG laser energy and CO₂ delivered at 3 or 5 W and at a duty cycle of 25%, 50%, or 100%. Using a quantitative enzyme-linked immunosorbent assay, TGF-ß1 and bFGF were assayed from collected supernatants.

Results  Laser-treated ear lobule KFs demonstrated decreased TGF-ß1 production when compared with preauricular KFs. Statistical significance (P<.005) was seen in the 3-W CO₂ 25% duty cycle; a trend was seen in the 3-W CO₂ 50% duty cycle (P<.08). Preauricular KFs secreted increased bFGF when compared with lobule KFs. Significance was seen in the 3-W CO₂ 25% and 50% duty cycles (P<.05). Laser-treated preauricular KFs had increased bFGF secretion when compared with non–laser-treated preauricular KFs in the 3-W CO₂ 25%, 50%, and 100% duty cycles.

Conclusions  Combined CO₂ and Er:YAG laser treatment decreases the production of TGF-ß1 in preauricular and ear lobule KFs. This laser may have clinical promise in the treatment of keloids. Finally, the different growth factor profiles obtained suggest that KFs from the ear lobule and preauricular regions are different.

INTRODUCTION

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REGIONAL VARIABILITY exists in the body in its response to wound healing. Clinical experience has shown a predilection for keloid or hypertrophic scar formation after trauma in certain regions of the body. As physicians, we commonly see ear lobule keloids, but we uncommonly see keloids in the midportion of the face. Beauty and vanity have led people to induce trauma to the face in search of a youthful effect. Since ancient times, exfoliation of the skin has been used to improve its quality and texture. Ancient Egyptians used salts and animal oils, whereas Turks used fire to wound the skin with the goal of aesthetic improvement.¹ In the 20th century, chemical peels, dermabrasion, and laser treatments have allowed the surgeon specializing in facial aesthetics a controlled means of ablating skin. A fine line exists between inducing normal wound healing vs wound healing gone awry.

Keloids remain a perplexing problem for the surgeon. Differing from a hypertrophic scar that grows within the scar borders, the keloid extends beyond like a crab claw and produces an excess of collagen. Despite being described as far back as 3000 BC in the Smith papyrus, the treatments for keloids have been moderately effective at best.¹ These have ranged from simple excision and closure, injection with intralesional corticosteroids, and closure with local flaps, to cryotherapy, systemic chemotherapy, ionizing radiation, and pressure. The fact that no single treatment is universally accepted is a testament to the lack of a successful treatment modality for this benign tumor.

Lasers have been increasingly used in the field of facial aesthetic surgery. Since the mid to late 1980s, the indications for the carbon dioxide (CO₂) laser have grown to include keloid excision, rhinophyma and actinic cheilitis ablation, excision of benign and malignant lesions, and facial resurfacing to treat facial rhytids and elastosis.²

In our own laboratory, the superpulsed CO₂ laser has shown promising results in increasing the secretion of basic fibroblast growth factor (bFGF) and decreasing the release of transforming growth factor ß1 (TGF-ß1) from keloid-producing and normal dermal fibroblasts.³

We used a new laser delivering combined erbium (Er):YAG and CO₂ irradiation (Derma K; ESC/Sharplan Medical Systems, Yokneam, Israel) to the same tissue area simultaneously. In the Er mode, the 2940-nm energy can be used to precisely ablate thin layers (20-30 µm) of tissue with minimal thermal damage.^4-5 In the CO₂ mode, the 10 600-nm irradiation produces coagulation of exposed capillaries without producing necrosis. Ultimately, this instrument provides precise ablation of tissue in a dry, hemostatic tissue bed.

Basic FGF is a single-chain polypeptide (16.5-18.2 kd) known for its mitogenic, chemoattractant, regulatory, and angiogenic abilities.^6-7 Basic FGF is found in a wide variety of cell types and, of all known growth factors, has the broadest range of target cells by influencing all of the diverse cells in wound healing, ie, capillary endothelial cells, fibroblasts, glial cells, neuronal cells, osteoblasts, and myoblasts.⁷ Basic FGF is part of a large gene family composed of 7 members, including acidic FGF (aFGF) and products of HSTF-1/KS3, INT-2, and FGF-5 oncogenes.^7-8

The fibroblast growth factor family consists of 2 closely related isoforms, basic (bFGF) and acidic (aFGF). In general, most cells are much more sensitive to bFGF than aFGF. Basic FGF has been found to be 10- to 30-fold more active than aFGF and can maximally stimulate proliferation of some cells at 1 ng/mL.^7-8 Basic FGF is stored as an inactive peptide in the extracellular matrix, integrated within the basement membrane. It functions locally and is released when injury has occurred.^{3, 6, 8}

Basic FGF is a potent modulator of collagen production by keloid-producing fibroblasts (KFs). Tan et al³ have shown that this cytokine inhibits and down-regulates type I collagen gene expression by KFs. Others have shown that the altered collagen metabolism results from stimulating the expression of collagenase in these cells.⁹

Transforming growth factor ß1 is a homodimeric structure with 2 disulfide-linked polypeptide chains of 12.5 kd. It is a member of a family of pleiotropic growth factors and is produced by platelets, macrophages, fibroblasts, and smooth muscle cells.¹⁰ One of 2 transforming growth factors (the other being TGF-), TGF-ß1 was originally characterized by its ability to reversibly induce the transformed phenotype in certain nontumorigenic cells.¹¹ Now known to elicit a variety of biological activities, TGF-ß1 has been shown to dramatically increase the expression of several collagen types in cultured fibroblasts.¹⁰ This growth factor is now postulated to play an active role in keloid formation.

The purpose of this study was to examine the effects of blended Er:YAG and CO₂ laser energy on KFs from 2 different facial regions in a serum-free in vitro model. Our laboratory has shown in previous studies the importance of a serum-free medium for analysis of growth factor production by fibroblasts.^{2, 12} This laser is believed to produce less intense erythema of a shorter duration resulting from facial laser skin resurfacing as well as excellent clinical results.¹³ The laser's effect on KF proliferation and production of bFGF and TGF-ß1 may provide insights into the following: (1) this laser's ability to prevent aberrant wound healing during facial laser resurfacing; (2) its efficacy in treating facial keloids; and (3) different behavior of preauricular KFs compared with ear lobule KFs.

MATERIALS AND METHODS

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FIBROBLAST CULTURES

The outlined method was approved by the Institutional Review Board at Stanford University Medical Center, Stanford, Calif. Fibroblasts were obtained directly from operative specimens and established as primary cell culture lines. Keloid-producing fibroblasts were obtained from preauricular and ear lobular tissue. With the use of sterile technique under a laminar flow hood, the dermis of the gross specimen was isolated and minced into 1- to 2-mm³ fragments. An antimicrobial wash was performed using 5% penicillin, streptomycin sulfate, and amphotericin B (Gibco, Grand Island, NY) in Dulbecco phosphate-buffered saline solution (PBS) (Gibco). The specimens were then placed in 2.5 mL of culture medium (10% fetal calf serum in Dulbecco Modified Eagle Medium with 1% L-glutamine and 1% penicillin, streptomycin sulfate, and amphotericin B) (Gibco) in crosshatched 25-cm² tissue culture flasks (T25) (Falcon; Becton-Dickinson, Franklin Lakes, NJ). Flasks were then incubated at 37°C in a humidified 5% CO₂ atmosphere. The medium was changed every 2 days.

An explant technique was used to establish cell lines. The flasks were monitored for fibroblast cellular outgrowth by means of phase-contrast microscopy. Once fibroblasts were visualized, the gross tissue specimens were removed. When sufficient outgrowth of fibroblasts occurred, the flasks were washed with PBS to remove nonadherent cells. The remaining adherent fibroblasts were released using 0.05% trypsin (Gibco) PBS, subcultured, and passed into 75-cm² culture flasks (T75) (Falcon; Becton-Dickinson). Primary culture medium was changed every fourth day, and successive cultures were passed at confluence.

CELL PLATING IN SERUM-FREE MEDIA

Experiments were performed with cells in their seventh passage. At the time of experimentation, 0.05% trypsin was used to release the confluent cells from the flask wall. Trypsin soybean inhibitor (Gibco) in a 1:1 ratio inactivated the trypsin. Cell culture viability was determined by means of trypan-blue dye exclusion, and cells counts were performed in duplicate using a hemocytometer and phase-contrast microscopy. Cells were then seeded at a density of 2 x 10⁴ cells/mL in each well of a 24-well plate (Falcon; Becton-Dickinson) using a commercially available serum-free medium (UltraCULTURE; Biowhittaker, Walkersville, Md). This medium has been shown to sustain dermal fibroblast growth to at least 7 days with greater than 90% viability.^{2, 11}

COMBINED CO₂ AND Er:YAG LASER TREATMENT

Forty-eight hours after seeding (time, 0 hours), cells were prepared for laser treatments. Each cell well was washed 3 times with PBS, then aspirated before laser treatments. Cell wells were exposed to laser energy using 1.7 J of Er:YAG and either 3 W of CO₂ at 25%, 50%, or 100% duty cycle or 5 W of CO₂ at 25%, 50%, or 100% duty cycle. Control wells received no irradiation. The scanning system (DermaScan; ESC/Sharplan Medical Systems) was set to the smallest parallelogram (6.0 x 4.8 mm). This pattern irradiated 18% of each cell well. All trays were treated in duplicate. After irradiation, 1 mL of serum-free medium was added to each well and placed in the incubator for the appropriate time. The supernatant from each well was collected at the appropriate time intervals for later growth factor assays. Cell counts and viability were obtained from each well using a hemacytometer, phase-contrast microscope, and the trypan-blue dye exclusion method.

ENZYME-LINKED IMMUNOSORBENT ASSAY

Human growth factors TGF-ß1 and bFGF were assayed from each well using commercially available enzyme-linked immunosorbent assay kits (Quantikine and Quantikine High Sensitivity, respectively; R&D Systems, Minneapolis, Minn). Optical densities were analyzed using commercially available software (KC4; Bio-Tek Instruments, Inc, Winooski, Vt). Assays were read using an automated plate reader (Elx800; Bio-Tek Instruments, Inc) The TGF-ß1 and bFGF enzyme-linked immunosorbent assay sensitivities are 7 pg/mL and 0.50 pg/mL, respectively.

STATISTICAL ANALYSIS

Each data point represents duplicate cell counts with assays performed in duplicate. Cell population-doubling times (PDT) were calculated from logarithmic best-fit curves. Data analysis and statistics were performed using commericially available software (Microsoft Excel for Windows 97, Version 7.0; Microsoft Corp, Redmond, Wash). Statistical differences between groups were assessed using the paired t test. Differences at the 5% level were considered statistically significant.

RESULTS

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All populations of cells exhibited exponential growth in the serum-free medium (Figure 1 and Figure 2). Viability at 0 hours for all populations of cells ranged from 85% to 98%. The PDT was calculated for each population of cells. The mean PDT of preauricular KFs (44.3 hours) was faster than that of ear lobule KFs (51.2 hours). This difference was not statistically significant. The control groups, which received no laser irradiation, had shorter PDTs than all other groups that received laser treatment (Figure 3).

View larger version (16K): [in this window] [in a new window] Figure 1. A sample preauricular keloid fibroblast cell line growth curve. Percentages indicate carbon dioxide laser duty cycles.

View larger version (16K): [in this window] [in a new window] Figure 2. A sample ear lobule keloid fibroblast cell line growth curve. Percentages indicate carbon dioxide laser duty cycles.

View larger version (44K): [in this window] [in a new window] Figure 3. Preauricular and ear lobule keloid fibroblast population-doubling times. Differences were not significant. CO2 indicates carbon dioxide.

Laser-treated preauricular KFs secreted more bFGF than did laser-treated ear lobule KFs, except when comparing the non–laser-treated groups (Figure 4). The preauricular KFs treated with 3 W of CO₂ at a duty cycle of 25%, 50%, and 100% all secreted more bFGF than did the non–laser-treated preauricular KF group. Statistical significance was not reached.

View larger version (55K): [in this window] [in a new window] Figure 4. Preauricular and ear lobule keloid fibroblast secretion of basic fibroblast growth factor (bFGF). Differences were not significant. CO2 indicates carbon dioxide.

Secretion of TGF-ß1 increased progressively up to 96 and 120 hours for preauricular and ear lobule KFs, respectively. Preauricular KFs secreted more TGF-ß1 than did ear lobule KFs in all groups. Statistical significance (P<.005) was seen in the 3-W CO₂ 25% duty cycle, whereas a trend was seen in the 3-W CO₂ 50% duty cycle (P<.08). Laser-treated preauricular and ear lobule KFs demonstrated decreased TGF-ß1 secretion when compared with non–laser-treated KFs (Figure 5). In the preauricular KFs, statistical significance was seen in the 3-W CO₂ 25% and 50% duty cycle groups when compared with the non–laser-treated preauricular KFs (P = .04 and P = .05, respectively). In the ear lobule KFs, statistical significance was also seen in the 3-W CO₂ 25% duty cycle group (P = .04), whereas a trend was observed in the 3-W CO₂ 100% duty cycle group (P = .09).

View larger version (50K): [in this window] [in a new window] Figure 5. Preauricular and ear lobule keloid-producing fibroblast (KF) secretion of transforming growth factor ß1 (TGF-ß1). Significance was reached in preauricular and ear lobule KFs when comparing laser-treated with non–laser-treated keloid groups given 3 W of carbon dioxide (CO2) energy at a duty cycle of 25% and 50% (P = .04). Significance was also seen when comparing laser-treated preauricular KFs with non–laser-treated KFs when given 3 W of CO2 energy at a duty cycle of 50% (P = .05). A trend was seen when comparing the laser-treated ear lobule KFs with non–laser-treated KFs at 3 W of CO2 energy at a duty cycle of 100% (P = .09).

COMMENT

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The incidence of keloid formation is equal between men and women, and occurs in anywhere from 5% to 15% of wounds in high-risk populations, ie, dark-skinned individuals and those aged 2 to 40 years.¹ Keloids appear to run in families,¹⁴ and despite some assumptions that inheritance is autosomal dominant, the form has not been clearly determined.¹⁵ There are numerous theories as to the cause of keloids, but none has been proven. A few of these are ischemia-related proliferation of perivascular myofibroblasts and endothelial cells; tension-induced excess collagen production by fibroblasts; inflammatory-, immunity-, and autoimmunity-induced antigen and antibody responses; and induction of fibroblasts by estrogen-, androgen-, and melanocyte-stimulating hormones and other hormonally related processes.¹

Keloids seem to occur with different frequencies in different regions of the body. They almost never appear on eyelids, central portion of the face, palms of the hand, soles of the feet, or genitalia.¹⁶ On the other hand, they are commonly seen on earlobes, especially after ear piercing; presternal and deltoid regions; wounds that cross skin-tension lines; wounds that are closed under tension; and wounds that develop in thicker skin.¹

Normal fibroblasts and KFs appear to have no difference in cellular morphology but have vastly different cellular function.¹⁷ Keloids are histologically characterized by an abundance of extracellular matrix of connective tissue. Increased production of elastin, chondroitin sulfate proteoglycans, fibronectin, and especially collagen occurs in KFs compared with their normal counterparts.^{3, 10-11,18} Collagen is the predominent extracellular matrix component of keloids with an excess of type I collagen production.¹⁹ Keloids contain randomly organized sheets of thick collagen fibers, compared with discrete, organized collagen bundles in normal fibroblasts.

Light amplification by the stimulated emission of electrons (or LASER) is a tool invented in the 1960s. The ability to increase precision and uniformity of tissue ablation was very attractive, and the search for other treatment modalities for keloids and hypertrophic scars was begun. Castro et al²⁰ discovered that the Nd:YAG laser selectively suppressed collagen production without affecting cell proliferation; they concluded that this laser treatment could be used to reduce collagen deposition in conditions such as keloids and hypertrophic scars. The work of Abergel et al²¹ continued that of Castro et al. They confirmed that the Nd:YAG laser selectively suppresses collagen production in fibroblast cultures and in healthy skin in vivo irrespective of a thermal effect.²¹ Thus, both conclusions were similar, ie, that this laser may be useful for the treatment of fibrotic conditions, such as keloids and hypertrophic scars. Abergel et al²¹ were also able to show that 2 low-energy lasers, helium-neon and gallium-arsenide, stimulated collagen production in human skin fibroblast cultures, suggesting that these lasers could be used for enhancement of wound-healing processes. Apfelberg et al²² worked with the argon and CO₂ lasers on trunk and earlobe keloids. Only 1 patient of 13 responded to their multiple bore-hole argon technique, followed by total excision with the CO₂ laser. Alster and Williams²³ discovered that the 585-nm flashlamp-pumped pulsed-dye laser improved erythema, scar height, skin surface texture, and pruritus on hypertrophic or keloidlike median sternotomy scars, with effects lasting 6 months. Nowak et al² have shown that the superpulsed CO₂ laser stimulates the release of bFGF and inhibits the release of TGF-ß1 in keloids. These profiles of growth factors imply that this laser may have beneficial effects in the treatment of keloids. Recently, we have also shown that simultaneous Er:YAG and CO₂ laser irradiation also produces a favorable profile of growth factors when delivered to facial keloids (E.T.C. and R.J.K., unpublished data, October 1999).

We used combined Er:YAG and CO₂ irradiation to the same tissue area simultaneously by means of a collimated hand piece with a 3-mm spot diameter and a scanning system manufactured to be used with the described laser system. The laser system consists of a 0.1– to 1.7–J/pulse Er:YAG laser that delivers a wavelength of 2.94 µm and a pulse duration of 350 microseconds combined with a 0- to 10-W CO₂ laser that delivers a wavelength of 10.6 µm and a pulse duration of up to a 100% duty cycle. Since the system delivers both laser energies simultaneously, the CO₂ pulsing is dependent on the rate of the Er delivery. The high energy of the Er:YAG laser allows precise ablation, whereas the subablative CO₂ heats the tissue and provides laser energy for hemostasis and tissue tightening. The laser system combines a pulsed (350-microsecond) Er:YAG system, operable at a fluence of 5 to 25 J/cm², and a continuous-wave low-power CO₂ system. The CO₂ laser component delivers pulses of variable duration, operating mostly at 30 to 50 milliseconds.¹⁴ The laser has a spot size of 3 mm, and with an integrated scanning pattern generator has the ability to irradiate an area as large as 20 mm. The early results using this laser system appear to be positive, with shorter healing times secondary to less thermal damage and consistent results. Healing generally occurs within 5 days, and the resulting erythema completely disappears within 4 to 6 weeks.¹³ At present, there are no published reports in the literature on the efficacy of this laser on regional keloid differences.

In vitro studies have shown the cellular function of fibroblasts and keloids to be altered by growth-inhibitory and growth-stimulatory factors, such as TGF-ß1 and bFGF.^{3, 8, 10-11,17} Basic FGF alters cellular function in keloids by stabilizing cell morphology, increasing cell migration and proliferation, and cell survival. Our results showed that laser-treated preauricular KFs secreted more bFGF than did ear lobule KFs. This difference in growth factor profile may be our first bit of data showing a regional difference between the 2 keloid groups. The increased rate of proliferation of preauricular compared with ear lobule KFs and the mitogenic role of this growth factor may explain the increased bFGF secretion.

Laser-irradiated preauricular KFs produced more bFGF when exposed to 3 W of laser energy compared with the control group. The ability of bFGF to repress the synthesis of type I collagen in fibroblasts has been known since the early 1980s.⁸ The work of Tan et al³ confirmed that bFGF down-regulates type I collagen production by KFs. The laser's ability to increase the release of bFGF at 3 W in the preauricular KFs implies a possible protective and preventative role in the development of keloids during facial laser resurfacing. It also implies a role for the treatment of preauricular keloids.

The concentration of TGF-ß1 was decreased in laser-treated preauricular KFs when compared with that of non–laser-treated preauricular KFs. Prolonged or excessive TGF-ß1 secretion in fibroblast cell culture has been postulated to contribute to the development of keloids.¹⁴ Transforming growth factor ß1 selectively increases collagen production in keloid cell culture as opposed to normal fibroblasts in culture.^{10, 20} Statistical significance was reached when preauricular KFs underwent laser irradiation with 3 W of CO₂ at a duty cycle of 25% and 50%.

The concentration of TGF-ß1 was also decreased in ear lobule KFs when compared with that of non–laser-treated ear lobule KFs. Statistical significance was reached when ear lobule KFs underwent laser irradiation with 3 W of CO₂ at a duty cycle of 25%, and a definite trend was seen with 3 W of CO₂ at a duty cycle of 100%.

Skin resurfacing by means of the CO₂ laser for the treatment of facial elastosis has become a standard modality of treatment in clinical practice. The beneficial role of the CO₂ laser has been thought to result from thermal contraction of collagen leading to tightening of the dermal layer.^21-23 The combined Er:YAG and CO₂ laser is a new-generation laser that has the capability of combining 2 laser modalities in precisely ablating thin layers of tissue with minimal thermal damage under a hemostatically controlled environment.^4-5

CONCLUSIONS

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To our knowledge, this is the first study in the literature examining the effects of this laser on regional keloid variability in a serum-free in vitro model. The ability of this laser to treat facial elastosis safely without excessive scar formation is very significant. Our study demonstrates that this laser increases the release of bFGF, which has been shown to promote tightly organized collagen bundles, and decreases the concentration of TGF-ß1, which has also been shown to promote fibrosis formation, in preauricular KFs.

This study demonstrates the following important key points: (1) The Er:YAG and CO₂ laser set at 3 W at a duty cycle of 25% or 50% may help prevent excessive scar formation during facial laser resurfacing. (2) Through its increase in bFGF and decrease in TGF-ß1 secretion, the Er:YAG and CO₂ laser may provide a viable treatment modality for preauricular keloids. (3) Regional differences exist between preauricular and ear lobule keloids and are based on the growth factor profiles.

AUTHOR INFORMATION

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Accepted for publication February 21, 2001.

Presented at the 2000 Spring Meeting of the American Academy of Facial Plastic & Reconstructive Surgery, Orlando, Fla, May 13, 2000.

Corresponding author: R. James Koch, MD, Facial Plastic and Reconstructive Surgery, Division of Otolaryngology–Head and Neck Surgery, R-135, Stanford University Medical Center, Stanford, CA 94305-5328 (e-mail: rjk{at}stanford.edu).

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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1. Lawrence WT. In search of the optimal treatment of keloids: report of a series and a review of the literature. Ann Plast Surg. 1991;27:164-178. FULL TEXT | ISI | PUBMED 2. Nowak KC, Koch RJ, McCormack M. The effect of superpulsed CO₂ laser energy on keloid and normal dermal fibroblast secretion of growth factors: a serum-free study. Plast Reconstr Surg. 2000;105:2039-2048. ISI | PUBMED 3. Tan EM, Rouda S, Greenbaum SS, Moore JH Jr, Fox IV JW, Sollberg S. Acidic and basic fibroblast growth factors down-regulate collagen gene expression in keloid fibroblasts. Am J Pathol. 1993;142:463-470. ABSTRACT 4. Hughes PS. Skin contraction following erbium:YAG laser resurfacing. Dermatol Surg. 1998;24:109-111. FULL TEXT | ISI | PUBMED 5. Weinstein C. Computerized scanning erbium:YAG laser for skin resurfacing. Dermatol Surg. 1998;24:83-89. FULL TEXT | ISI | PUBMED 6. Brew EC, Mitchell MB, Harken AH. Fibroblast growth factors in operative wound healing. J Am Coll Surg. 1995;180:499-504. ISI | PUBMED 7. Schweigerer L. Basic fibroblast growth factor as a wound healing hormone. Trends Pharmacol Sci. 1988;9:427-428. FULL TEXT | PUBMED 8. Gospodarowicz D. Biological activities of fibroblast growth factors. Ann N Y Acad Sci. 1991;638:1-8. ISI | PUBMED 9. Buckley-Sturrock A, Woodward SC, Senior RM, Griffin GL, Klagsburn M, Davidson JM. Differential stimulation of collagenase and chemotactic activity in fibroblasts derived from rat wound repair tissue and human skin by growth factors. J Cell Physiol. 1989;138:70-78. FULL TEXT | ISI | PUBMED 10. Bettinger DA, Yager DR, Diegelmann RF, Cohen IK. The effect of TGF-beta on keloid fibroblast proliferation and collagen synthesis. Plast Reconstr Surg. 1996;98:827-833. ISI | PUBMED 11. Raghow R, Postlethwaite AE, Keski-Oji J, Moses HL, Kang AH. Transforming growth factor-ß increases steady state levels of type I procollagen and fibronectin messenger RNAs posttranscriptionally in cultured human dermal fibroblasts. J Clin Invest. 1987;79:1285-1288. 12. Hong R, Lum J, Koch RJ. Growth of keloid-producing fibroblasts in commerciallly available serum-free media: a comparative study. Otolaryngol Head Neck Surg. 1999;121:469-473. FULL TEXT | ISI | PUBMED 13. Greene D, Egbert BM, Utley DS, Koch RJ. In vivo model of histologic changes after treatment with the superpulsed CO₂ laser, erbium:YAG laser, and blended lasers: a 4- to 6-month prospective histologic and clinical study. Lasers Surg Med. 2000;27:362-372. FULL TEXT | ISI | PUBMED 14. Koch RJ, Goode RL, Simpson GT. Serum-free keloid fibroblast cell culture: an in vitro model for the study of aberrant wound healing. Plast Reconstr Surg. 1997;99:1094-1098. ISI | PUBMED 15. Tredget EE, Nedelec B, Scott PG, Ghahary A. Hypertrophic scars, keloids, and contractures: the cellular and molecular basis for therapy. Surg Clin North Am. 1997;77:701-730. FULL TEXT | ISI | PUBMED 16. Datubo-Brown DD. Keloids: a review of the literature. Br J Plast Surg. 1990;43:70-77. FULL TEXT | ISI | PUBMED 17. Crockett DJ. Regional keloid susceptibility. Br J Plast Surg. 1964;17:245-253. 18. Babu M, Diegelmann R, Oliver N. Keloid fibroblasts exhibit an altered response to TGF-beta. J Invest Dermatol. 1992;99:650-655. FULL TEXT | ISI | PUBMED 19. Younai S, Nichter LS, Wellisz T, Reinisch J, Nimni ME, Tuan TL. Modulation of collagen synthesis by transforming growth factor-beta in keloid and hypertrophic scar fibroblasts. Ann Plast Surg. 1994;33:148-151. FULL TEXT | ISI | PUBMED 20. Castro DJ, Abergel RP, Meeker C, Dwyer RM, Lesavoy MA, Uitto J. Effects of the Nd:YAG laser on DNA synthesis and collagen production in human skin fibroblast cultures. Ann Plast Surg. 1983;11:214-222. FULL TEXT | ISI | PUBMED 21. Abergel RP, Meeker CA, Lam TS, Dwyer RM, Lesavoy MA, Uitto J. Control of connective tissue metabolism by lasers: recent developments and future prospects. J Am Acad Dermatol. 1984;11:1142-1150. ISI | PUBMED 22. Apfelberg DB, Maser MR, Lash H, White D, Weston J. Preliminary results of argon and carbon dioxide laser treatment of keloid scars. Lasers Surg Med. 1984;4:283-290. ISI | PUBMED 23. Alster TS, Williams CM. Treatment of keloid sternotomy scars with 585 nm flashlamp-pumped pulsed-dye laser. Lancet. 1995;345:1198-2000. FULL TEXT | ISI | PUBMED

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