Persistent HSc can result from a prolonged inflammatory process in wound healing, involving the activation of fibrogenic cytokines or GFs, such as TGF-β1, FGF, platelet-derived GF, and IL-1.1 Fibroblasts are important cells that are integrally involved in this process.16,17 They are activated through the effects of the above-mentioned cytokines and GFs, resulting in increased cell proliferation, collagen production, and collagen contraction (Fig. 10).18,19 In the clinical setting, certain drugs are available for the inhibition of scar formation, such as steroids, CCBs, and IFNs.20 Recently, TGF-β1 was shown to be a potential antiscar therapeutic agent.21 Each of these agents has a different mechanism of scar inhibition (Fig. 10),22–24 and each involves its advantages and disadvantages. For example, steroids can block fibroblast proliferation, collagen formation, and contraction, and increase the effect of collagenase, but may cause adverse effects, such as injection pain, skin atrophy, depigmentation, and telangiectasias.24,25 Calcium channel blockers can induce a change in the phenotype of fibroblasts and increase the activity of collagenase26 but may not be as effective as steroids, although their side effects have seldom been reported.24 Interferons may inhibit fibroblast proliferation, collagen production, and contraction but can lead to several side effects, such as fever, chills, fatigue, malaise, and headache.27 Some authors suggest combinations of these agents with other drugs, such as steroids, for clinical use.10,23 New potential antiscarring approaches involve the use of basic fibroblast GF, TGF-β3, and interleukin-10,28 as well as antimitotic drug injections.29 Improving the efficacy of these drugs and reducing their side effects were the purposes of this study.
The animal model used in this study has been previously validated. Human scar tissue implanted into a subcutaneous pocket in nude (athymic) mice can retain the same histological characteristics and sustain viability and morphology for at least 60 days.30 The synthesis of glycoaminoglycans continues in HSc implanted in control animals. Using such an animal model to test drug efficacy is therefore considered a reliable method.
Because the experimental animals were small and could not tolerate a large amount of scar tissue, the implanted scar tissue measured only 0.5 g in size. Accordingly, the dose of the drug used for the injection was a reduced dose ranging from 0.016 mL to 0.05 mL/g. The concentration of each drug in the same injected dose may not have been the same; however, it was a fixed ratio of the clinical dosage and would thus reflect clinical efficacy.
By weight, the scar size reduced maximally in group C compared with that in groups B and A (78.6%, 82.3%, and 86% of the original size, respectively; Fig. 3). Therefore, the combination of steroid, CCB, and IFN inhibited HSc development significantly effective than did a single drug. This phenomenon could be explained by the fact that different drugs have different inhibition mechanisms but no detectable adverse interactions. In addition, the reduction in the steroid dosage in combination therapy was also expected to decrease its side effects. Some of the efficacy of the combination drug therapy can be attributed to CCB, which causes fewer side effects in general.
Regarding the evaluation of cell (fibroblast) count, 0.05 mL of steroid exhibited greater inhibition of fibroblast proliferation than did the control (A > B, Fig. 4). The same dose of the combined drug regimen (0.016 mL of steroid, CCB, and INF each, totaling approximately 0.5 mL) produced greater fibroblast inhibition than did a single dose (0.5 mL) of steroid (B > C, Fig. 4). These results support our hypothesis that combination therapy increases efficacy, and possibly decreases the side effects of high doses of a single drug.
The role of CCB in the combination regimen should be emphasized. Upon reviewing the literature,31 there are no obvious side effects for local treatment with verapamil hydrochloride injection at a dose of 0.02 to 0.1 mL per scar. The response rate was approximately 55%.32 The CCB used in this study, verapamil, has been shown to induce changes in fibroblast gene expression, resulting in decreased collagen synthesis and increased collagenase production.33 These effects appear to be mediated by interruption of the basic cellular G-protein signal transduction pathway that is critical to the regulation of fibroblast behavior. Verapamil induces scar degradation when injected in the skin, fascia, and periocular tissue.34 The effects of verapamil on scars have been demonstrated in experimental animal models as well as in humans.11 Therefore, we chose CCB as a component of our combination regimen.
Intralesional IFN-γ injection in patients with steroid and HSc may cause a 30% to 50% reduction in scar size.35 This may be because IFN-γ acts as a regulator of collagenase expression and tissue inhibitor of matrix metalloproteinase-1 mRNA in human hypertrophic and normal dermal fibroblasts,23 and as a regulator of collagen synthesis and mRNA expression in normal and HSc fibroblasts.22 Because of the possible adverse side effects, IFN-γ is seldom used clinically in antiscar therapy. However, some authors have attempted to combine the use of IFN-γ with other drugs, as was done in the current study.4
The quantification of collagen (type I) in the implanted scars at 4 weeks after implantation of scars treated with different regimens showed that fibroblast collagen formation in group C was inhibited to a greater extent than that in group B significantly and to an even greater extent than that in group A significantly too (Fig. 5). In nonimplanted scars, the concentration of type I collagen secreted into the media from the scar fibroblasts after treatment was significantly the least in group C, followed by group B and group A (Fig. 6). These results not only confirmed that the combination therapy was more effective than a single drug but also revealed that combination therapy reduced the amount of collagen to a significantly greater extent than the control group. This may be because combination therapy can inhibit almost every step of the wound healing process. The reason we chose a steroid for comparison is because the steroid is currently the most popular drug used clinically to control persistent HSc. The fibroblasts from HSc tissue when cultured in the presence of hydrocortisone demonstrated growth inhibition.36 Steroids also show inhibitory effects on collagen biosynthesis and enhance collagen breakdown.37 They also inhibit the action of TGF-β, a fibrogenic cytokine, and reduce scar formation.38 Although the effect of the steroid was greater than that of the other agents used, it is known to have many side effects mentioned earlier in this manuscript; therefore, reduction in the dose of steroid component of the combination regimen is preferable. In this study, only collagen type I was used for study. It is more convenient for comparison. However, the type of collagen in scar tissue including type I and III and the ratio of collagen components in HSc and normal scar should be considered as a variation of scar character.39 It is worthwhile to do a further study.
Histological findings from HE staining in this study showed considerably lesser disorganization of collagen fibers in scar tissues in group C than in groups B and C. This result is consistent with the effect of the drugs, as mentioned previously (Fig. 7).
The results of the FPCL study showed that the order of collagen contraction inhibition was group C-group B-group A. That lower doses of single drugs in combination are more effective than a higher dose of steroid in the inhibition of collagen contraction is an interesting finding. These results may be due to the roles played by CCB and IFN in altering mechanical tension through mechanotransduction pathways.40,41 As observed in our study, a combination of reduced doses of each antiscar drug can be more effective than a large dose of a single drug, for example, steroid, in controlling HSc and possibly reducing the side effects of each drug.
Although this study revealed several characteristics of scar inhibition therapy, the underlying mechanisms require further study, such as why CCB alone inhibits collagen contraction greater than combination therapy and steroid monotherapy. Further, the dose of each drug used in this study was equal. To reduce the side effects of certain drugs, such as steroids and IFN, the effects of an adjusted dose ratio (e.g., lower doses of steroid and IFN and a higher dose of CCB) need to be explored.
In this study, by implanting human scar tissues in a suitable animal model, we found that combination therapy was significantly effective in decreasing scar volume (weight), fibroblast count, the amount and contraction rate of collagen, and the disorganization of collagen bundles compared to steroid monotherapy and control group. Thus, there are possible advantages to using a combination therapy, including a reduction in the side effects of each single drug, particularly the steroids and IFN.11
1. Armour A, Scott PG, Tredget EE. Cellular and molecular pathology of HTS: basis for treatment. Wound Repair and Regeneration: Official Publication of the Wound Healing Society [and] the European Tissue Repair Society
. 2007; 15: 6–17.
2. Mrowietz U, Seifert O. Keloid scarring: new treatments ahead. Actas Dermosifiliogr
. 2009; 100: 75–83.
3. Al-Attar A, Mess S, Thomassen JM, et al. Keloid pathogenesis and treatment. Plast Reconstr Surg
. 2006; 117: 286–300.
4. Gupta S, Sharma VK. Standard guidelines of care: keloids and hypertrophic scars. Indian J Dermatol Venereol Leprol
. 2011; 77: 94–100.
5. Patel PA, Bailey JK, Yakuboff KP. Treatment outcomes for keloid scar management in the pediatric burn population. Burns
. 2012; 38: 767–771.
6. Sadeghinia A, Sadeghinia S. Comparison of the efficacy of intralesional triamcinolone acetonide and 5-fluorouracil tattooing for the treatment of keloids. Dermatol Surg
. 2012; 38: 104–109.
7. Wu XL, Gao Z, Song N, et al. Clinical study of auricular keloid treatment with both surgical excision and intralesional injection
of low-dose 5-fluorouracil and corticosteroids. Zhonghua Yi Xue Za Zhi
. 2009; 89: 1102–1105.
8. Davison SP, Dayan JH, Clemens MW, et al. Efficacy of intralesional 5-fluorouracil and triamcinolone in the treatment of keloids. Aesthet Surg J
. 2009; 29: 40–46.
9. Darougheh A, Asilian A, Shariati F. Intralesional triamcinolone alone or in combination with 5-fluorouracil for the treatment of keloid and hypertrophic scars. Clin Exp Dermatol
. 2009; 34: 219–223.
10. Lee JH, Kim SE, Lee AY. Effects of interferon
-alpha2b on keloid treatment with triamcinolone acetonide intralesional injection
. Int J Dermatol
. 2008; 47: 183–186.
11. Yang JY, Huang CY. The effect of combined steroid
and calcium channel blocker injection on human hypertrophic scars in animal model
: a new strategy for the treatment of hypertrophic scars. Dermatol Surg
. 2010; 36: 1942–1949.
12. Polo M, Kim YJ, Kucukcelebi A, et al. An in vivo model of human proliferative scar. J Surg Res
. 1998; 74: 187–195.
13. Brem H, Golinko MS, Stojadinovic O, et al. Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF. J Transl Med
. 2008; 6: 75.
14. Ruangpanit N, Chan D, Holmbeck K, et al. Gelatinase A (MMP-2) activation by skin fibroblasts: dependence on MT1-MMP expression and fibrillar collagen form. Matrix Biol
. 2001; 20: 193–203.
15. Ehrlich HP. The fibroblast
-populated collagen lattice. A model of fibroblast
collagen interactions in repair. Methods Mol Med
. 2003; 78: 277–291.
16. Wang J, Dodd C, Shankowsky HA, et al. Wound Healing Research G. Deep dermal fibroblasts contribute to hypertrophic scarring. Lab Invest
. 2008; 88: 1278–1290.
17. Supp DM, Hahn JM, Glaser K, et al. Deep and superficial keloid fibroblasts contribute differentially to tissue phenotype in a novel in vivo model of keloid scar. Plast Reconstr Surg
. 2012; 129: 1259–1271.
18. Cui Q, Wang Z, Jiang D, et al. HGF inhibits TGF-beta1-induced myofibroblast differentiation and ECM deposition via MMP-2 in Achilles tendon in rat. Eur J Appl Physiol
. 2011; 111: 1457–1463.
19. Xie JL, Bian HN, Qi SH, et al. Basic fibroblast
growth factor (bFGF) alleviates the scar of the rabbit ear model in wound healing. Wound Repair Regen
. 2008; 16: 576–581.
20. Tredget EE, Nedelec B, Scott PG, et al. Hypertrophic scars, keloids, and contractures. The cellular and molecular basis for therapy. Surg Clin North Am
. 1997; 77: 701–730.
21. Sadick H, Herberger A, Riedel K, et al. TGF-beta1 antisense therapy modulates expression of matrix metalloproteinases in keloid-derived fibroblasts. Int J Mol Med
. 2008; 22: 55–60.
22. Harrop AR, Ghahary A, Scott PG, et al. Regulation of collagen synthesis and mRNA expression in normal and hypertrophic scar
fibroblasts in vitro by interferon
-gamma. J Surg Res
. 1995; 58: 471–477.
23. Ghahary A, Shen YJ, Nedelec B, et al. Interferons gamma and alpha-2b differentially regulate the expression of collagenase and tissue inhibitor of metalloproteinase-1 messenger RNA in human hypertrophic and normal dermal fibroblasts. Wound Repair Regen
. 1995; 3: 176–184.
24. Hochman B, Locali RF, Matsuoka PK, et al. Intralesional triamcinolone acetonide for keloid treatment: a systematic review. Aesthetic Plast Surg
. 2008; 32: 705–709.
25. Tang YW. Intra- and postoperative steroid
injections for keloids and hypertrophic scars. Br J Plast Surg
. 1992; 45: 371–373.
26. Boggio RF, Freitas VM, Cassiola FM, et al. Effect of a calcium-channel blocker (verapamil) on the morphology, cytoskeleton and collagenase activity of human skin fibroblasts. Burns
. 2011; 37: 616–625.
27. Duncan MR, Berman B. Gamma interferon
is the lymphokine and beta interferon
the monokine responsible for inhibition of fibroblast
collagen production and late but not early fibroblast
proliferation. J Exp Med
. 1985; 162: 516–527.
28. Liu W, Wu X, Gao Z. New potential antiscarring approaches. Wound Repair Regen
. 2011; 19: 22–31.
29. Wang XQ, Liu YK, Qing C, et al. A review of the effectiveness of antimitotic drug injections for hypertrophic scars and keloids. Ann Plast Surg
. 2009; 63: 688–692.
30. Kischer CW, Pindur J, Shetlar MR, et al. Implants of hypertrophic scars and keloids into the nude (athymic) mouse: viability and morphology. J Trauma
. 1989; 29: 672–677.
31. Gaspar R, Hajagos-Toth J. Calcium channel blockers as tocolytics: principles of their actions, adverse effects and therapeutic combinations. Pharmaceuticals
. 2013; 6: 689–699.
32. Lee RC, Doong H, Jellema AF. The response of burn scars to intralesional verapamil. Report of five cases. Arch Surg
. 1994; 129: 107–111.
33. Yue H, Uzui H, Shimizu H, et al. Different effects of calcium channel blockers on matrix metalloproteinase-2 expression in cultured rat cardiac fibroblasts. J Cardiovasc Pharmacol
. 2004; 44: 223–230.
34. Doong H, Dissanayake S, Gowrishankar TR, et al. The 1996 Lindberg Award. Calcium antagonists alter cell shape and induce procollagenase synthesis in keloid and normal human dermal fibroblasts. J Burn Care Rehabil
. 1996; 17: 497–514.
35. Granstein RD, Rook A, Flotte TJ, et al. A controlled trial of intralesional recombinant interferon
-gamma in the treatment of keloidal scarring. Clinical and histologic findings. Arch Dermatol
. 1990; 126: 1295–1302.
36. Russell JD, Russell SB, Trupin KM. Differential effects of hydrocortisone on both growth and collagen metabolism of human fibroblasts from normal and keloid tissue. J Cell Physiol
. 1978; 97: 221–229.
37. Cohen IK, Diegelmann RF, Johnson ML. Effect of corticosteroids on collagen synthesis. Surgery
. 1977; 82: 15–20.
38. Stojadinovic O, Lee B, Vouthounis C, et al. Novel genomic effects of glucocorticoids in epidermal keratinocytes: inhibition of apoptosis, interferon
-gamma pathway, and wound healing along with promotion of terminal differentiation. J Biol Chem
. 2007; 282: 4021–4034.
39. Cheng W, Yan-hua R, Fang-gang N, Guo-an Z. The content and ratio of type I and III collagen in skin differ with age and injury. Afr J Biotechnol
. 2011; 10: 2524–2529.
40. Sokabe M, Sachs F. The structure and dynamics of patch-clamped membranes: a study using differential interference contrast light microscopy. J Cell Biol
. 1990; 111: 599–606.
41. Ogawa R. Mechanobiology of scarring. Wound Repair Regen
. 2011; 19: 2–9.
Keywords:Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
hypertrophic scar; fibroblast; steroid; calcium channel blocker (verapamil); interferon; animal model; combined therapy; intralesional injection