Tissue Concentrations of Zinc, Iron, Copper, and Magnesium During the Phases of Full Thickness Wound Healing in a Rodent Model

Abstract

Wound healing is a complex orchestration of processes involving cell proliferation, migration, differentiation, anabolism, and catabolism in order to restore skin continuity. Within these processes, elements such as metallic ions are involved due to their implications in cell behavior and enzymatic activity regulation. This study analyzed the kinetics of zinc, iron, copper and magnesium concentrations in a full thickness open wound rat model over 14 days. We made wounds with a diameter of 6 mm on the back of Lewis rats and let them heal naturally prior to analysis by histology and inductively coupled plasma mass spectrometry analysis. Histological and immunofluorescence analysis confirmed an inflammation phase until 7 days, epithelial proliferation phase from 16 h to 10 days, and remodeling phase from 7 days onward. These defined phases were correlated with the measured metal element kinetics. Zinc concentrations showed an inverted parabolic progression between 30.4 and a maximum of 39.9 μg/g dry weight. Magnesium values had a similar pattern between 283 and 499 μg/g dry weight. Copper concentrations, on the other hand, followed an inverted sigmoid trend with a decrease from 9.8 to 1.5 μg/g dry weight. Iron had a slight decrease in concentration for 24 h followed by an increase to a maximum of 466 μg/g dry weight. In conclusion, zinc, iron, and copper, even though differing in their total mass within the wound, exhibited concentration curve transitions at day 3. Interestingly, this time point correlates with the maximum proliferating keratinocyte rate during the proliferation phase.

Keywords: Copper; Iron; Magnesium; Metal; Skin wound healing; Zinc.

Fig. 1

Histologic measurement of wound diameter development. Data were calculated by measurements of wound margins of H&E staining of 6-μm thin cut of wounds taken at the median part of the wound ± SD (n = 3 rats in each group; one rats data is the mean of 3 wounds)

Fig. 2

Histologic measurement of epidermis development. Data were calculated by measurement of growing epidermis from the margins of H&E staining of 6-μm thin cut of wounds taken at the median part of the wound ± SD (n = 3 rats in each group; one rats data is the mean of 3 wounds)

Fig. 3

Histologic measurement of reepithelialization relative to the wound diameter. Data were calculated by measurement of growing epidermis from the margins normalized to the wound diameter of H&E staining of 6-μm thin cut of wounds taken at the median part of the wound ± SD (n = 3 rats in each group; one rats data is the mean of 3 wounds)

Fig. 4

Immunofluorescence measurement of number of Ki-67-positive keratinocytes at the wound margins in time in relation to the observed epidermis length. Dot line represents healthy skin baseline (0.03 U ± 0.02). All data were displayed as mean ± SD of three rats (n = 3 rats in each group; one rat’s data is the mean of 3 wounds)

Fig. 5

Development of zinc concentration in healing wound. Zinc element was determined by ICP-MS. Dot line represents healthy skin baseline. All data were displayed as mean ± SD of three rats (n = 3 rats in each group; one rat’s data is the mean of 3 wounds)

Fig. 6

Development of iron concentration in healing wound. Iron element was determined by ICP-MS. Dot line represents healthy skin baseline. All data were displayed as mean ± SD of three rats (n = 3 rats in each group; one rat’s data is the mean of 3 wounds)

Fig. 7

Development of magnesium concentration in healing wound. Magnesium element was determined by ICP-MS. Dot line represents healthy skin baseline. All data were displayed as mean ± SD of three rats (n = 3 rats in each group; one rat’s data is the mean of 3 wounds)

Fig. 8

Development of copper concentration in healing wound. Copper element was determined by ICP-MS. Dot line represents healthy skin baseline. All data were displayed as mean ± SD of three rats (n = 3 rats in each group; one rat’s data is the mean of 3 wounds)