Immunohistochemical analysis on MMP-2 and MMP-9 for wound age determination

We performed immunohistochemical study combined with morphometrical analyses in order to examine the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 using 55 human skin wounds of different ages: group I, 0–3 days (n = 16); II, 4–7 days (n = 11); III, 9–14 days (n = 16); and IV, 17–21 days (n = 12). Immunopositive reactions for MMP-2 were observed in all human skin specimens including uninjured skin as control. The number of MMP-2⁺ macrophages was significantly increased in accordance with wound ages. In contrast to MMP-2, no MMP-9⁺ signals were detected in uninjured and wound specimens aged less than 1 day. However, the number of MMP-9⁺ macrophages profoundly appeared in groups II and III. Morphometrically, in all of wound samples aged 9–12 days, MMP-2⁺ cell number was more than 20. On the contrary, most of the remaining samples had <20 positive cells. However, only one sample (a 7-day-old wound) showed 21 positive cells. Thus, with regard to practical applicability with forensic safety, MMP-2⁺ macrophages of >20 would indicate a wound age of 7–12 days. Additionally, 10 out of 12 wound specimens aged 9–12 days showed the MMP-2⁺ cell number of >25, implying that MMP-2⁺ cell number of >25 would indicate the wound age of 9–12 days. On the contrary, all wound samples aged 3–14 days except for only one sample had MMP-9⁺ cell number of >30, indicating that MMP-9⁺ cell number of >30 would indicate the wound age of 3–14 days. Collectively, MMP-2 seemed to be more distinct marker, compared with MMP-9.