title: 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.
abstract: 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.
  
  
  OBJECTIVE
  (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.
authors.affiliation: Plastic & Reconstructive Surgery Research, Dermatological Sciences, School of Translational Medicine, Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: F
authors.initials: F
authors.lastname: Syed
authors.forename: E
authors.initials: E
authors.lastname: Ahmadi
authors.forename: S A
authors.initials: SA
authors.lastname: Iqbal
authors.forename: S
authors.initials: S
authors.lastname: Singh
authors.forename: D A
authors.initials: DA
authors.lastname: McGrouther
authors.forename: A
authors.initials: A
authors.lastname: Bayat
mesh_terms: D000293:Adolescent; D000328:Adult; D001706:Biopsy; D015153:Blotting, Western; D024042:Collagen Type I / Q000096:biosynthesis*; D024061:Collagen Type III / Q000096:biosynthesis*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007627:Keloid / Q000473:pathology*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D012867:Skin / Q000378:metabolism* / Q000473:pathology; D055815:Young Adult
chemical_list: D024042:Collagen Type I; D024061:Collagen Type III; D012333:RNA, Messenger