Effect of Adenovirus-mediated Decorin Gene Therapy on Fibrotic Remodeling in Tissue-engineered Skin

  • Author / Creator
    Sanon, Saahil
  • Background: Hypertrophic scar (HTS) is a fibroproliferative disorder of the skin that results in the development of painful, red, raised scar lesions. Transforming growth factor beta 1 (TGF-β1) has been implicated as a key driver of this disorder. Primarily, these effects include an increased deposition of collagen and matrix proteoglycans, as well as the suppressed expression of matrix-remodeling proteins like matrix metalloproteinase 1 (MMP-1). The proteoglycan decorin (DCN) has been shown to bind to and inactivate TGF-β1. Moreover, DCN plays a key role during collagen fibrillogenesis by regulating collagen fibril thickness and organization. Therefore, adenoviral (Ad5)-DCN gene therapy may have therapeutic effects for HTS. Objectives: This project used a co-cultured tissue-engineered in vitro model to explore anti-fibrotic matrix remodeling after treating HTS fibroblasts with Ad5-DCN for 18 hours. Anticipated results included increased DCN protein levels, reduced contraction, normalized orientation of the extracellular matrix, and elevated gene expression of MMP-1. Methods: Tissue-engineered skin was prepared by co-culturing fibroblasts and keratinocytes in collagen-glycosaminoglycan scaffolds for 17 days. Keratinocytes isolated from normal skin were used for all scaffolds while dermal fibroblasts were from one of four groups: 1) normal skin fibroblasts (n=12), 2) HTS fibroblasts (n=12), 3) HTS fibroblasts + Ad5-DCN (n=12), 4) HTS fibroblasts + Ad5-GFP (n=12). Contraction during culture was evaluated using digital analysis of matrix area. Histological sections were stained with picrosirius red and analyzed using fast Fourier analysis to determine collagen organization. mRNA levels of TGF-β1, collagen 1 alpha 1 (COL1A1), MMP-1, and DCN were measured via RT-qPCR. Total collagen levels were evaluated using LC/MS for the amount of 4-hydroxyproline, while DCN protein was measured using ELISA and immunofluorescent staining. Repeated measures ANOVA with Holm-Bonferroni post-hoc analysis was used to assess significance, with p<0.05 being significant. Results: After day 17, tissue-engineered skin constructs created using Ad5-DCN-treated HTS fibroblasts were less contracted than those with untreated HTS fibroblasts. The Ad5-DCN-treated group showed elevations in both soluble and matrix-bound DCN protein levels, as well as increased mRNA expression of DCN. Normalized extracellular matrix remodeling was observed, as indicated by collagen orientation index and collagen bundle packing index values. This may be the result of elevated MMP-1, which was found to be increased 5-fold in mRNA expression by the Ad5-DCN-treated group as compared to the untreated HTS group. Additionally, COL1A1 mRNA and total collagen protein were both reduced in the Ad5-DCN group as compared to the HTS Fb group. No significant difference was found with respect to TGF-β1 mRNA expression between groups. Conclusions: Treatment of HTS fibroblasts with Ad5-DCN leads to improvement in contraction and matrix remodeling in a tissue-engineered in vitro model of the skin. Further in vivo study may lead to improved tissue-engineered skin for treatment of acute injuries or to assist in the reconstruction of HTS post-burn, possibly through systemic effects of by reducing scarring when used for skin resurfacing of local injuries.

  • Subjects / Keywords
  • Graduation date
    2016-06:Fall 2016
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Surgery
  • Specialization
    • Experimental Surgery
  • Supervisor / co-supervisor and their department(s)
    • Tredget, Edward (Surgery)
  • Examining committee members and their departments
    • Adesida, Adetola (Surgery)
    • Churchill, Thomas (Surgery)
    • Tredget, Edward (Surgery)
    • Ding, Jie (Surgery)