Introduction: Cultured keratinocytes (CK) can be used to resurface massive burn wounds, but the long-term fate of these grafts is difficult to assess without formal biopsy. To facilitate graft evaluation, we have recently labeled CKs with green fluorescent protein (GFP), via adenoviral transfection. The purpose of this study is to determine if fluorescent CKs can be successfully grafted, maintain fluorescence for non-invasive monitoring, and provide stable wound coverage. Methods: Human keratinocytes harvested from discarded skin were plated with growth-arrested 3T3 fibroblasts, allowed to reach confluence, and transfected with a GFP adenoviral vector at a multiplicity of infection of 10. CK sheets were liberated with dispase, grafted onto flanks of anesthetized athymic mice (n=24), and protected with an occlusive, hydrocolloid dressing. CK grafts were assessed in situ via epifluorescent microscopy 3, 4, 5, 7, and 10 days after transfer. CK biopsies (n=8) obtained 14 days after grafting were stained with FITC-labeled antibody to human MHC Class I antigen and counterstained with H&E to assess graft architecture. Results: CK grafts provided stable wound coverage, despite mild to moderate contraction by 14 days. H&E revealed normal epithelial architecture, and viable human keratinocytes were detected by FITC staining. Initial fluorescence of GFP-transfected CK grafts was similar to that of in vitro controls (which express GFP > 4 weeks) but rapidly decreased in intensity over time. Standardized mean fluorescence 3, 4, 5, 7, and 10 days after transfer was 80.7, 61.5, 17.3, 3.8, and 1.9 units, respectively (p<0.05). Conclusions: CK sheets transfected with GFP adenoviral vector can be successfully grafted to provide stable wound coverage. The persistence of human keratinocytes 14 days after grafting, combined with attenuated fluorescence, implies that in vivo GFP expression is transient in this model. Although the temporary expression of GFP limits the usefulness of graft evaluation, transient expression of transfected genetic material may prove to be beneficial in the controlled delivery of growth factors for wound healing.
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