Increased Skin Collagen Extractability and Proportions of Collagen Type III Are Not Normalized after 6 Months Healing of Human Excisional Wounds Simon.

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Increased Skin Collagen Extractability and Proportions of Collagen Type III Are Not Normalized after 6 Months Healing of Human Excisional Wounds Simon P. Robins, George Milne, Alexander Duncan Journal of Investigative Dermatology Volume 121, Issue 2, Pages 267-272 (August 2003) DOI: 10.1046/j.1523-1747.2003.12373.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Electrophoresis of collagens solubilized by pepsin digestion of human skin biopsies. Delayed reduction with 2-mercaptoethanol was performed after 1 h and the proportions of collagen I and III were estimated by densitometric scanning of the α1(I) and α1(III) bands. The upper panel shows a typical stained gel with baseline biopsy samples run in lanes 1–5, with the 3, 6, 8, 12, and 16 wk time-points in lanes 6–10, respectively. Control standards containing 10%, 20%, and 30% collagen III relative to collagen I+III were run in lanes 11–13, respectively, and lane 14 contains a collagen V standard. The α1(III) band in the unfractionated samples often occurred as a doublet as indicated. The lower panel shows a standard curve constructed by electrophoresis of a series of collagen I:III mixtures. Journal of Investigative Dermatology 2003 121, 267-272DOI: (10.1046/j.1523-1747.2003.12373.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Chromatography of the mature skin collagen cross-link, HHL. Hydrolysates of skin biopsies were prefractionated as described in Materials and Methods and submitted to ion exchange chromatography with postcolumn derivatization using ortho-phthaldialdehyde and monitoring fluorescence at excitation 355 nm, emission 450 nm. The three overlaid chromatograms are of 550 pmol HHL standard (dotted line), a baseline biopsy sample (broken line), and a 24 wk re-excision biopsy (full line), the last two containing similar amounts of tissue hydroxyproline. Journal of Investigative Dermatology 2003 121, 267-272DOI: (10.1046/j.1523-1747.2003.12373.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 HPLC separation of pyridinium cross-links of elastin and collagen. Acid hydrolysates of skin biopsy samples were prefractionated on cellulose columns using an automated Gilson ASPEC system followed by reversed-phase HPLC with dual monitoring. The upper panel shows the coelution of Des and Ide detected by ultraviolet absorption and the lower panel shows the positions of Pyd and Dpd detected by fluorescence. A fluorescent internal standard (IS), O-acetyl-pyridinoline, was used to quantify both the collagen- and elastin-derived cross-links. Journal of Investigative Dermatology 2003 121, 267-272DOI: (10.1046/j.1523-1747.2003.12373.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Changes in the collagen cross-link ratio, Pyd/Dpd, in skin biopsy samples. The measured Pyd/Dpd ratios in five initial biopsies and six re-excision samples are shown as the mean±SD (n=8, except for 24 wk where n=6). The significance of the difference between the relevant group and groups at all subsequent time-points is shown by asterisks (p<0.001). Journal of Investigative Dermatology 2003 121, 267-272DOI: (10.1046/j.1523-1747.2003.12373.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Changes in the extractability of skin collagen with pepsin in relation to mature cross-link content. (a) The extractability of skin collagen by pepsin digestion for five initial biopsy sites and re-excision samples at six time-points up to 24 wk expressed as mean±SD (n=8, except for 24 wk where n=6) is shown in comparison with values for HHL concentrations expressed similarly for the corresponding samples. Statistically significant differences are omitted for clarity but are shown in Table I and Table II. (b) Linear regression of individual values for pepsin extractability and HHL concentration for all biopsy samples with the equation y=-0.017×+1.71 and significant correlation coefficient r2=0.89 (p<0.0001). Journal of Investigative Dermatology 2003 121, 267-272DOI: (10.1046/j.1523-1747.2003.12373.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions