title: Effect of fibroblasts on epidermal regeneration.
abstract: BACKGROUND
  There is little information on specific interactions between dermal fibroblasts and epidermal keratinocytes. The use of engineered skin equivalents consisting of organotypic cocultures of keratinocytes and fibroblasts offers an attractive approach for such studies.
  
  
  OBJECTIVE
  To examine the role fibroblasts play in generation and maintenance of reconstructed epidermis.
  
  
  METHODS
  Human keratinocytes were seeded on collagen matrices populated with increasing numbers of fibroblasts and cultured for 2 weeks at the air-liquid interface.
  
  
  RESULTS
  In the absence of fibroblasts, stratified epidermis with only three or four viable cell layers was formed. In the presence of fibroblasts, keratinocyte proliferation was stimulated and epidermal morphology was improved. Epidermal morphogenesis was also markedly improved in epidermis generated in organotypic keratinocyte monocultures grown in medium derived from dermal equivalents or from organotypic keratinocyte-fibroblast cocultures. These observations clearly indicate the proliferation-stimulating activity of soluble factors released from fibroblasts. Under all experimental conditions, onset of keratinocyte differentiation was shown by the expression of keratin 10 in all suprabasal cell layers. With increasing numbers of fibroblasts incorporated into the collagen matrix, the expression of markers associated with keratinocyte activation, e.g. keratins 6, 16 and 17 and the cornified envelope precursor SKALP decreased, and involucrin localization shifted toward the granulosum layer. This fibroblast-mediated effect was even more pronounced when the fibroblasts were precultured in the collagen matrices for 1 week instead of overnight. The basement membrane proteins collagen VII and laminin 5 were present at the epithelial-matrix border. The expression of integrin alpha 6 beta 4 and of E-cadherin was comparable with that seen in native skin and was not significantly modulated by fibroblasts. Under all experimental conditions the expression of integrin subunits alpha 2, alpha 3 and beta 1 was upregulated, indicating keratinocyte activation.
  
  
  CONCLUSIONS
  Our results illustrate that numbers of fibroblasts in the collagen matrix and their functional state is a critical factor for establishment of normal epidermal morphogenesis.
authors.affiliation: Department of Dermatology, Leiden University Medical Centre, Sylvius Laboratory, PO Box 9503, 2300 RA Leiden, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: el-Ghalbzouri
authors.forename: S
authors.initials: S
authors.lastname: Gibbs
authors.forename: E
authors.initials: E
authors.lastname: Lamme
authors.forename: C A
authors.initials: CA
authors.lastname: Van Blitterswijk
authors.forename: M
authors.initials: M
authors.lastname: Ponec
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002454:Cell Differentiation; D002455:Cell Division; D018920:Coculture Techniques / Q000379:methods; D003094:Collagen / Q000378:metabolism; D024143:Collagen Type VII / Q000032:analysis / Q000378:metabolism; D000078404:Epidermal Cells; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D016023:Integrins / Q000032:analysis / Q000378:metabolism; D015603:Keratinocytes / Q000378:metabolism / Q000502:physiology*; D007797:Laminin / Q000032:analysis / Q000378:metabolism; D009024:Morphogenesis; D012038:Regeneration; D017768:Skin, Artificial; D013997:Time Factors
chemical_list: D015415:Biomarkers; D024143:Collagen Type VII; D016023:Integrins; D007797:Laminin; C097043:laminin alpha5; D003094:Collagen