Gene Expression Profile of Tissue Engineered Skin Subjected to Acute Barrier Disruption  Piyush Koria, Daniel Brazeau, Keith Kirkwood, Patrick Hayden, Mitchell Klausner, Stelios T. Andreadis  Journal of Investigative Dermatology  Volume 121, Issue 2, Pages 368-382 (August 2003) DOI: 10.1046/j.1523-1747.2003.12364.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Acetone treatment decreased the metabolic activity of tissue-engineered skin. Skin equivalents were wounded by application of acetone in the tissue culture inserts for the indicated times. The acetone was removed, fresh medium was added in the lower compartment, and the tissues were returned to culture at the air–liquid interface. At the indicated time after wounding, the tissues were incubated with MTT for 3 h and then lyzed in extractant solution overnight. The next day the optical density values were measured at 570 nm and corrected for nonspecific background by subtracting the optical density at 650 nm. The absorbance of wells containing lysis buffer was subtracted as background. Each point shows the mean and standard deviation of duplicate samples in a representative experiment (n=2). Journal of Investigative Dermatology 2003 121, 368-382DOI: (10.1046/j.1523-1747.2003.12364.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Acetone treatment disrupted the epidermal barrier. The tissues were wounded by application of absolute acetone to the apical surface for 10 min. At the end of the exposure time, the remaining acetone was removed and the tissues were placed in fresh culture medium. At the indicated time points, transepidermal electrical resistance was measured with an EVOM Epithelial Voltohmmeter equipped with an Endohm Tissue Resistance Measurement Chamber as described in Materials and Methods. Journal of Investigative Dermatology 2003 121, 368-382DOI: (10.1046/j.1523-1747.2003.12364.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Acetone affected skin equivalents in a time- and position-dependent fashion. Skin equivalents were wounded by application of acetone in the tissue culture inserts for 10 min. The acetone was removed, fresh medium was added in the lower compartment, and the tissues were returned to culture at the air–liquid interface. Hematoxylin and eosin staining of paraffin-embedded wounded and unwounded tissue sections at 4 h (A), 8 h (B), and 24 h (C) postwounding (magnification 40×). Journal of Investigative Dermatology 2003 121, 368-382DOI: (10.1046/j.1523-1747.2003.12364.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Gene clusters using SOM. The 154 genes that passed the criteria were grouped into nine groups. The ratios are represented on the y axis and the time points on the x axis. The middle line (black, solid) represents the average expression of a gene belonging to each cluster and the other two lines (gray, dashed) indicate the standard deviation. The numbers in each panel show the number of genes belonging to the corresponding cluster. Journal of Investigative Dermatology 2003 121, 368-382DOI: (10.1046/j.1523-1747.2003.12364.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Comparison of the expression ratios of selected genes measured by real-time PCR and microarrays. Real-time PCR was performed for four genes: FRA-1 (A), EGR-1 (B), thymosin β10 (C), and PLA2 (D). The ratios were calculated as described in Materials and Methods and plotted as a function of time. Each point corresponds to the mean of triplicate samples in a representative experiment (n=2). Journal of Investigative Dermatology 2003 121, 368-382DOI: (10.1046/j.1523-1747.2003.12364.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Acetone induced hyperproliferation in the basal layer of skin equivalents. Skin equivalents were wounded by application of acetone in the tissue culture inserts for 10 min. The acetone was removed, fresh medium was added in the lower compartment, and the tissues were returned to culture at the air–liquid interface. At 24 h paraffin sections were stained for the proliferation antigen Ki67. The fraction of proliferating basal cells (Ki67+) was counted in six randomly selected fields of view. The difference between wounded and unwounded tissues was statistically significant (p=0.0001). The results are representative of three independent experiments. Journal of Investigative Dermatology 2003 121, 368-382DOI: (10.1046/j.1523-1747.2003.12364.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Schematic of the kinetics of gene expression in response to acetone treatment. Genes that increase or decrease at early and late times after barrier disruption are grouped according to their function. Tissue engineered skin displays a two wave dynamic response to acute barrier disruption. An early phase of survival and inflammation is followed by differentiation phase to restore the lost barrier. Journal of Investigative Dermatology 2003 121, 368-382DOI: (10.1046/j.1523-1747.2003.12364.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions