title: Use of versican variant V3 and versican antisense expression to engineer cultured human skin containing increased content of insoluble elastin.
abstract: Skin substitutes for repair of dermal wounds are deficient in functional elastic fibres. We report that the content of insoluble elastin in the dermis of cultured human skin can be increased though the use of two approaches that enhance elastogenesis by dermal fibroblasts, forced expression of versican variant V3, which lacks glycosaminoglycan (GAG) chains, and forced expression of versican antisense to decrease levels of versican variant V1 with GAG chains. Human dermal fibroblasts transduced with V3 or anti-versican were cultured under standard conditions over a period of 4 weeks to produce dermal sheets, with growth enhanced though multiple seedings for the first 3 weeks. Human keratinocytes, cultured in supplemented media, were added to the 4-week dermal sheets and the skin layer cultured for a further week. At 5 weeks, keratinocytes were multilayered and differentiated, with desmosome junctions thoughout and keratin deposits in the upper squamous layers. The dermal layer was composed of layered fibroblasts surrounded by extracellular matrix of collagen bundles and, in control cultures, small scattered elastin deposits. Forced expression of V3 and versican antisense slowed growth, decreased versican V1 expression, increased tropoelastin expression and/or the deposition of large aggregates of insoluble elastin in the dermal layer, and increased tissue stiffness, as measured by nano-indentation. Skin sheets were also cultured on Endoform Dermal Template™, the biodegradable wound dressing made from the lamina propria of sheep foregut. Skin structure and the enhanced deposition of elastin by forced expression of V3 and anti-versican were preserved on this supportive substrate. Copyright © 2014 John Wiley & Sons, Ltd.
authors.affiliation: Department of Anatomy with Radiology, School of Medical Sciences, Faculty of Medical and Health Sciences, University of Auckland, New Zealand.
authors.forename: Mervyn J
authors.initials: MJ
authors.lastname: Merrilees
authors.affiliation: Department of Anatomy with Radiology, School of Medical Sciences, Faculty of Medical and Health Sciences, University of Auckland, New Zealand.
authors.forename: Ben A
authors.initials: BA
authors.lastname: Falk
authors.affiliation: Department of Anatomy with Radiology, School of Medical Sciences, Faculty of Medical and Health Sciences, University of Auckland, New Zealand.
authors.forename: Ning
authors.initials: N
authors.lastname: Zuo
authors.affiliation: Department of Chemicals and Materials Engineering, Faculty of Engineering, University of Auckland, New Zealand.
authors.forename: Michelle E
authors.initials: ME
authors.lastname: Dickinson
authors.affiliation: Mesynthes Ltd, 2 Kingsford Smith Drive, Auckland, New Zealand.
authors.forename: Barnaby C H
authors.initials: BC
authors.lastname: May
authors.affiliation: Benaroya Research Institute, Seattle, WA, USA.
authors.forename: Thomas N
authors.initials: TN
authors.lastname: Wight
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D020405:Dermis / Q000378:metabolism; D004547:Elastic Tissue / Q000378:metabolism*; D004549:Elastin / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000166:cytology; D007633:Keratins / Q000737:chemistry; D009092:Mucous Membrane; D051381:Rats; D012756:Sheep; D012867:Skin / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*; D014333:Tropoelastin / Q000378:metabolism*; D053675:Versicans / Q000378:metabolism*; D014945:Wound Healing
chemical_list: D001672:Biocompatible Materials; D006025:Glycosaminoglycans; D014333:Tropoelastin; D053675:Versicans; D007633:Keratins; D003094:Collagen; D004549:Elastin
keywords: cultured skin; dermis; elastin; epidermis; human; versican V3