1. FGF-7 Expression Enhances the Performance of Bioengineered Skin
Gulsun Erdag, Daniel A. Medalie, Hinne Rakhorst, Gerald G. Krueger, Jeffrey R. Morgan 
Molecular Therapy 
Volume 10, Issue 1, Pages (July 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Gene-modified bioengineered skin secretes FGF-7 and increased levels of VEGF. A time course of FGF-7 secretion was determined by removing portions of the culture medium of confluent cultures of control or genetically modified keratinocytes grown (A) on tissue culture plastic or (B) as bioengineered skin. Levels of FGF-7 protein were determined by ELISA. (C) Culture media from bioengineered skin were also assayed for levels of VEGF by ELISA (P < 0.01, FGF-7 vs control). (D) To determine cell number, bioengineered skin was treated with dispase, followed by trypsin and Hoechst staining to facilitate the counting of nucleated cells (*P < 0.001, FGF-7 vs control).
Molecular Therapy  , 76-85DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
FGF-7 expression enhances cell migration and self-healing of bioengineered skin. To measure cell migration, control (unmodified) or FGF-7-modified keratinocytes were seeded into PDMS stencils. Stencils were removed after the cells attached and image analysis was used to quantify cell migration at 12 and 24 h. (A) Shown are photos of the PDMS stencil of squares with 2-mm sides, control cells, and FGF-7-expressing cells after 24 h of migration. The means ± SEM of a total of 18 samples from two experiments are plotted (*P < 0.01 FGF-7 vs control). (B) To measure self-healing, 1.5-mm circular wounds were created in the center of bioengineered skin. The wounded skins were placed on a second layer of acellular dermis and cultured for additional 5 days. Samples were stained every 24 h with SYTO 13 and observed by fluorescence microscopy (original magnification ×4). (C) Self-healing was quantified by measuring the remaining unclosed wound area of seven or eight skins for each time point in duplicate experiments (*P < FGF-7 vs control). (D) After image analysis, samples were fixed in formalin and paraffin samples were stained with H&E (original magnification ×10).
Molecular Therapy  , 76-85DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
FGF-7 expression enhances the antibacterial activity of bioengineered skin. Bioengineered skins were inoculated with (A) 3.2 × 104 E. coli, (B) 4.6 × 104 S. aureus, or (C) 6.5 × 104 P. aeruginosa. The number of viable bacteria, in colony-forming units (CFU), was determined after 24 h. Results are expressed as means ± SEM of triplicates from two separate experiments (*P < 0.01, FGF-7 vs control). (D) RNA was isolated from bioengineered skin and RT-PCR was used to determine mRNA levels of HBD-1, HBD-2, LL-37, ALP, lysozyme, BPI, and PLA2 in composite grafts of control and FGF-7 keratinocytes. Expression levels were normalized to G3PDH mRNA expression in control and FGF-7 skin. Data are consistent with results obtained in two separate experiments repeated three times (REL, relative expression level).
Molecular Therapy  , 76-85DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Grafts of bioengineered skin expressing FGF-7 are hyperproliferative. Bioengineered skin of (A, C, E, G) control unmodified and (B, D, F, H) modified keratinocytes seeded onto acellular dermis was grafted to full-thickness excisional wounds on the back of athymic mice. (A, B, E, F) One week and (C, D, G, H) 6 weeks after transplantation, samples were taken and stained for hematoxylin and eosin (A, B, C, D) or immunostained for the proliferation marker, Ki67 (E, F, G, H).
Molecular Therapy  , 76-85DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
FGF-7 expression speeds revascularization. (A) Bioengineered skin with either control or FGF-7-expressing keratinocytes was transplanted to full-thickness skin wounds on athymic mice and samples were harvested at 1, 2, and 3 weeks. The underside of each graft along with the surrounding mouse tissue was photographed (original magnification ×25). (B) Image analysis was used to compute the total graft area and the area revascularized, which was plotted as a function of time (*P < 0.01 FGF-7 vs control).
Molecular Therapy  , 76-85DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions