Fibroblasts from the Growing Margin of Keloid Scars Produce Higher Levels of Collagen I and III Compared With Intralesional and Extralesional Sites: Clinical Implications for Lesional Site-directed Therapy

F. Syed; E. Ahmadi; S.A. Iqbal; S. Singh; D.A. McGrouther; A. Bayat


The British Journal of Dermatology. 2011;164(1):83-96. 

In This Article

Abstract and Introduction


Background Overproduction of collagen and its abnormal assembly are hallmarks of keloid scars. Type I/III collagen ratios are altered in keloids compared with normal skin. Fibroblasts from different sites in keloid tissue, perilesional compared with intralesional and extralesional sites, show differential apoptosis and contraction. Additionally, early vs. later cell culture passages display differential collagen expression. We therefore hypothesize that keloid fibroblasts from the growing margin of the keloid express higher levels of collagen type I and III, and that collagen production is altered by extended cell culture passage.
Objectives (i) To measure collagen I and III at mRNA and protein levels quantitatively in keloid fibroblasts, growth media and tissue sections; and (ii) to perform tissue staining for collagen I and III expression in different lesional sites.
Methods Keloid fibroblast cultures from intralesional, perilesional and extralesional sites (n = 8 separate keloid cases, yielding 64 biopsy samples) were established from passage 0 to passage 3. Collagen I and III mRNA was quantified using quantitative reverse transcription–polymerase chain reaction. We also measured the protein levels quantitatively by developing a highly specific and sensitive capture sandwich enzyme-linked immunosorbent assay. A novel in-cell Western blotting was carried out in addition to haematoxylin and eosin and Herovici staining on keloid tissue sections for collagen I and III.
Results Collagen types I and III were significantly higher (P < 0·03) in fibroblasts from the growing margin (perilesional site) compared with extralesional and intralesional keloid biopsy sites. As the passage number increased, the amount of collagen I significantly (P < 0·05) decreased and the collagen I/III ratio also decreased.
Conclusions Our data show that cells from the growing margin of keloid scars have a higher production of collagen I and III compared with other lesional sites. Additionally, temporal extension of cell passage affects collagen production. Clinically these findings may influence selection and interpretation of extended cell passage and provide future direction for lesional site-specific therapy in keloid scars.


Baron Jean-Louis Alibert (1768–1837) identified keloid as a clinical disease entity in 1806.[1] He named these lesions cancroide, later changing the name to cheloide to avoid confusion with cancer. The word is derived from the Greek chele, meaning crab's claw, and the suffix -oid, meaning like.[1] Keloid scars are fibroproliferative quasineoplastic lesions which are characterized by increased collagen deposition and growth beyond the boundaries of the original wound margin with the propensity for invasion into adjacent normal skin.[1–6] Several mechanisms have been proposed for keloid formation and the high proliferation rate of keloid fibroblasts, such as a high expression of certain cytokines including transforming growth factor-β[7] and insulin-like growth factor-1.[8,9] In addition, there is an imbalance between proliferation and apoptotic cell death postulated to be due to p53 gene mutations, downregulation of certain apoptotic related genes and unfolded protein response regulation in keloid cells.[2,10–13] Keloids are also shown to be associated with the HLA group of antigens.[14]

Keloids present a therapeutic challenge as these lesions can cause significant pain, pruritus and psychosocial problems as well as physical disfigurement.[15] The management of keloid scars has remained a clinical challenge despite recent advances in new pharmaceutical therapeutics.[15,16] Current treatments which include surgical excision and/or corticosteroid injection[17–20] do not eradicate keloids completely and these scars frequently tend to recur.[21,22] Other potential treatment options include radiotherapy, cryotherapy, topical silicone and application of antineoplastic agents, even though none has been shown to be curative of this disease.[23–32] Recent literature suggests that combinations of treatments may be more efficacious in prevention of recurrence. Table S1 (see Supporting Information) gives details of modalities of therapy used for prevention of keloids in predisposed patients in addition to various treatment options available at present for the management of keloid scars.[23–32] Interestingly, none of these therapies has addressed the treatment of specific lesional sites within keloid scars (i.e. centre vs. margin) to date.

The metabolism of collagen and its regulation are significant to many diseases characterized by excessive accumulation or loss of collagen.[33,34] Excessive accumulation of collagen is known not only in keloids but also in hypertrophic scars, scleroderma and in skin tumour growth.[35] It has been reported that type I/III collagen ratios are altered in keloid[36,37] and in hypertrophic scars[38–40] compared with normal skin. In our previous study, we reported that different lesional sites in keloid scars regulate gene expression differently,[41,42] and heterogeneity within the keloid lesion may exist in terms of gene expression.[43,44] This notion of site differences in disease progression has been strengthened by the observation that keloid recurrence as well as prognosis seems to be affected by total excision or retention of the growing margin (perilesion) of the keloid scar.[45] More importantly in relation to our work, different passage numbers of primary fibroblasts behave differently in their gene expression and growth kinetics of collagenase gene expression.[46,47] Therefore, the aim of this study was to quantify the amount of collagen type I and III and their ratio in early vs. later passages of keloid fibroblasts extracted from different keloid lesional sites, comparing perilesional with intralesional and extralesional sites. In addition, we measured the gene and protein levels and performed tissue staining for collagen I and III. For this purpose we developed and validated a sensitive and specific capture sandwich enzyme-linked immunosorbent assay (ELISA) (previously unreported) to measure and quantify collagen type I and III in the secreted media as well as in fibroblasts.


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