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The Effect of Copper Tripeptide and Tretinoin on Growth Factor Production in a Serum-Free Fibroblast Model

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The Effect of Copper Tripeptide and Tretinoin on Growth Factor Production in a Serum-Free Fibroblast Model ORIGINAL ARTICLE The Effect of Copper Tripeptide and Tretinoin on Growth Factor Production in a Serum-Free Fibroblast Model Matthew C. McCormack, BA; Kenneth C. Nowak, MD; R. James Koch, MD, MS Objective: To evaluate the effect of copper tripeptide Tretinoin-treated normal fibroblasts secreted more and tretinoin on normal and keloid-producing dermal fi- bFGF than did controls at 24 hours (P,.05). Tret- broblasts in a serum-free in vitro model. The cellular re- inoin-treated keloid-producing fibroblasts secreted sponse was described in terms of viability and secretion more TGF-b1 than did controls at 120 hours (P,.05). of basic fibroblast growth factor (bFGF) and transform- Keloid-producing fibroblasts treated with copper tri- ing growth factor-b1 (TGF-b1). peptide secreted less TGF-b1 than did controls at 24 hours (P,.05); a similar trend was observed in normal Methods: Primary cell lines were established from pa- fibroblasts. tient facial skin samples obtained during surgery and plated in serum-free media. At 0 hour, copper tripep- Conclusions: Normal fibroblasts treated with tretinoin tide (1310 −9 mol/L), tretinoin (1310 −5 mol/L), or ap- produced more bFGF than did controls, and this might propriate control vehicle was added. Cell counts and vi- partially explain the clinically observed tightening ef- ability were established at 24, 72, and 120 hours. fects of tretinoin. Normal and keloid-producing dermal Supernatants were collected at the same intervals and were fibroblasts treated with copper tripeptide secreted less assessed for bFGF and TGF-b1 concentrations using the TGF-b1 than did controls, suggesting a possible clinical enzyme-linked immunosorbent assay technique. use for decreasing excessive scar formation. Results: Cell lines showed viability between 86% and 96% (mean, 92%) throughout the experiment. Arch Facial Plast Surg. 2001;3:28-32 BERRANT WOUND healing is duce substantially more procollagen and a significant problem for fibronectin than do normal dermal fibro- many surgical patients. blasts (NFs) in culture. The current re- Inadequate healing is of- search indicates that at the core of keloid ten due to an underlying etiology are wound-healing cytokines— Amedical condition such as diabetes, pre- growth factors that regulate production of vious radiation therapy, poor nutritional extracellular matrix (ECM) components. status, or malignancy. Other patients, at An imbalance in the levels of vari- the opposite end of the spectrum, may ous cytokines generated in the wound- form hypertrophic scars or keloid tissue. healing process may lead to keloid for- Keloids do not represent a more severe mation. In addition, it may be the proper form of hypertrophic scars. There is no balance of cytokines in the wound envi- simple continuum from normal skin scar ronment that allows for normal wound to hypertrophic scar to keloid tissue. Col- healing. Such growth factors, which pro- lagen bundles in hypertrophic scars re- mote cell growth, division, and migra- main parallel in orientation (as in normal tion in wounded tissue, are secreted by skin), whereas keloids have randomly or- dermal fibroblasts. Two of these key ganized sheets of collagen. Also, whereas growth factors were considered in the contractile myofibroblasts are common in present study. hypertrophic scars, they are relatively ab- Transforming growth factor-b1 From the Wound Healing sent in keloids. (TGF-b1) is a key cytokine in the initia- and Tissue Engineering Laboratory, Division of Multiple treatment modalities have at- tion and termination of tissue repair. In Otolaryngology–Head and tempted to reduce such excess scarring, relation to other known tissue repair cy- Neck Surgery, Stanford yet none have established long-standing tokines, it strongly stimulates synthesis of University Medical Center, results.1 Keloid-producing dermal fibro- the major ECM proteins, namely, colla- Stanford, Calif. blasts (KFs) have been shown2,3 to pro- gen, proteoglycan, and fibronectin.4,5 You- (REPRINTED) ARCH FACIAL PLAST SURG/ VOL 3, JAN-MAR 2001 WWW.ARCHFACIAL.COM 28 ©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 MATERIALS AND METHODS commercially available serum-free media (UltraCulture; Bio- whittaker, Walkersville, Md) to achieve a final concentra- tion of 1.0310 −5 mol/L. This concentration has proven to CELL CULTURES be most effective for in vitro studies of procollagen inhi- bition.21,24 Total concentration of solvent was less than 0.1%. Primary cultures of dermal fibroblasts were established from This concentration has been shown to be nontoxic to fi- excisional biopsies of 3 different keloid and 3 different nor- broblasts and has no effect on collagen metabolism by these mal facial skin specimens using a standard explant tech- cells.21 The corresponding amount of ethanol was added nique. All cell lines were directly established from opera- to the serum-free media in control samples. tive specimens. Keloid specimens were from the lobule and normal skin samples were from the preauricular and men- CELL PLATING IN SERUM-FREE MEDIA tal region from 5 different patients. The described method was approved by the institutional review board at Stan- At the time of experimentation, fibroblasts were released ford University Medical Center. from flask walls using 0.05% trypsin solution. The trypsin The dermis was isolated from the specimens and was inactivated using trypsin soybean inhibitor (GIBCO) minced. Antimicrobial treatment consisted of washing the in a 1:1 ratio. Cells were suspended in UltraCulture and specimens in Dulbecco phosphate-buffered saline solu- then transferred into 24-well culture plates (Falcon; Becton- tion (PBS) with 5% penicillin, streptomycin, and ampho- Dickinson) at a concentration of 63104 cells/well. Ultra- tericin (GIBCO, Grand Island, NY). The minced speci- Culture was selected for its ability to sustain dermal fibro- mens were placed in scored 25-cm2 tissue (T25) flasks blast growth to at least 7 days with greater than 90% (Falcon; Becton-Dickinson, Franklin Lakes, NJ) with a viability.25 Cells were counted in duplicate using phase- 2.5-mL solution of primary culture media (20% fetal bo- contrast microscopy and a hemocytometer. Viable cells were vine serum in Dulbecco modified Eagle medium; 1% peni- determined using trypan blue exclusion. The plates were cillin, streptomycin, and amphotericin; and 1% L- incubated for 48 hours to allow for adequate settling. Af- glutamine) (GIBCO). The dermal specimens were stored ter this time, cells were washed in PBS and fresh UltraCul- and maintained at 37°C in a humidified 5% carbon diox- ture was added, this time with the appropriate modulator ide atmosphere. or vehicle included (0 hour). After 24 hours, the media were changed with 5.0 mL of primary culture media. The media were then changed MEASUREMENT OF GROWTH FACTOR every 2 days until fibroblasts were visualized under light CONCENTRATIONS microscope to be growing outward from the explanted tis- sue. At this time the tissue was removed. With sufficient The supernatant was drawn from the culture wells at 24, outgrowth of fibroblasts, cells were passed into 75-cm2 tis- 72, and 120 hours and stored at –70°C until the time of sue (T75) flasks using 0.05% trypsin (GIBCO) in PBS. Pri- assay. Each sample was assayed using the enzyme-linked mary culture media were changed every third day, and suc- immunosorbent assay technique. Growth factors bFGF and cessive cultures were passed at confluence. Cells from TGF-b1 were assayed using Quantikine High Sensitivity passages 4 and 5 were used for experimentation. All work and Quantikine assay kits (R&D Systems, Minneapolis, was performed under a laminar flow hood using a sterile Minn), respectively. Assays were read using an automated technique. plate reader (Elx800; Bio-Tek Instruments Inc, Winooski, Vt). Optical densities were analyzed with KC4 software (Bio- MODULATORS Tek Instruments Inc). Assays were read with the specified filter for each assay with application of a reference filter to A concentrate was prepared by dissolving the GHK-Cu (Pro- correct for optical imperfections in the plate. cyte Corp, Kirkland, Wash) in PBS just before experimen- tation, which was then diluted in serum-free media to a con- STATISTICAL ANALYSIS centration of 1.0310 −9 mol/L. Previous studies9,10 have shown this concentration to be effective in stimulating col- Each data point represents duplicate cell counts with as- lagen and glycosaminoglycan production by fibroblasts. says performed in duplicate. Statistical differences were as- Tretinoin (Sigma-Aldrich Corp, St Louis, Mo) was dis- sessed using 2-sample and paired t tests. Differences at the solved in ethanol immediately before use, then diluted in 5% level were considered statistically significant. nai et al6 investigated the in vitro effects of TGF-b1on crease aberrant scar or keloid formation by reducing the fibroblasts and found that KFs produce many times more amount of collagen deposited during wound healing. collagen than do NFs when stimulated by TGF-b1. Cul- The modulators selected for evaluation were the cop- tured KFs also secrete more TGF-b1 than do NFs— per tripeptide complex glycyl-L-histidyl-L-lysine-Cu2+ further evidence that excessive amounts of this cyto- (GHK-Cu) and all-trans retinoic acid (tretinoin). A natu- kine in the wound environment may be central to keloid rally occurring tripeptide, GHK-Cu has been shown to formation and growth.7 have significant clinical application in the field of wound The second growth factor assayed in our study, ba- healing and tissue repair. It has been demonstrated that sic fibroblast growth factor (bFGF), has been shown to GHK-Cu stimulates cultured NFs to synthesize colla- inhibit hydroxyproline biosynthesis, an index of colla- gen and induces a dose-dependent increase in the syn- gen production, in cultured KFs.8 Methods that in- thesis of glycosaminoglycans.9-11 It also has been shown crease or stabilize bFGF secretion might therefore de- that ECM accumulation increases in the rat wound model (REPRINTED) ARCH FACIAL PLAST SURG/ VOL 3, JAN-MAR 2001 WWW.ARCHFACIAL.COM 29 ©2001 American Medical Association.
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