Ex vivo regenerative effects of a spring water

Abstract

Previous experiments by our group have indicated the regenerative effects of a spring water (Comano), which was possibly associated with the native non-pathogenic bacterial flora. The present study aimed to confirm these regenerative properties in a human ex vivo experimental model in the context of physiological wound healing. Human 6-mm punch skin biopsies harvested during plastic surgery sessions were injured in their central portion to induce skin loss and were cultured in either conventional medium (controls) or medium powder reconstituted with filtered Comano spring water (treated samples). At 24, 48 and 72 h the specimens were observed following staining with hematoxylin and eosin, Picrosirius Red, orcein and anti-proliferating cell nuclear antigen. Compared with the controls, the treated samples exhibited reduced overall cell infiltration, evidence of fibroblasts, stimulation of cell proliferation and collagen and elastic fiber regeneration. In the spring water, in addition to 12 resident non-pathogenic bacterial strains exhibiting favorable metabolic activities, more unknown non-pathogenic species are being identified by genomic analysis. In the present study, the efficacy of this 'germ-free', filtered spring water in wound regeneration was indicated. Thus, the Comano spring water microbiota should be acknowledged for its regenerative properties.

Keywords: human cell culture; microbiota; regeneration; spring water.

Figure 1.

Hematoxylin and eosin staining. Representative images of the US sample at T₀; Ctrl samples cultured in DMEM with saline solution applied to the central wound; and CW-treated samples cultured in DMEM powder reconstituted with filtered CW and with filtered CW applied to the central wound at T₁, T₂ and T₃. Histological features are indicated as follows: Black circle, loose dermis; black triangle, new tissue; black arrow, inflammatory perivascular cellular infiltration; black star, papillary dermis; dotted arrow, fibroblasts; black cross, re-epithelialization. Magnification, ×5 (US); scale bar, 200 µm; and ×10 (CW and Ctrl); scale bar, 100 µm. US, untreated skin; Ctrl, control; DMEM, Dulbecco's modified Eagle's medium; CW, Comano spring water; T_0–3, 0–72 h.

Figure 2.

Picrosirius Red staining. Representative images of the US sample at T₀; Ctrl samples cultured in DMEM with saline solution applied to the central wound; and CW-treated samples cultured in DMEM powder reconstituted with filtered CW and with filtered CW applied to the central wound at T₁, T₂ and T₃. White ovals indicate newly formed collagen fibers. Magnification, ×10; scale bar, 100 µm. US, untreated skin; Ctrl, control; DMEM, Dulbecco's modified Eagle's medium; CW, Comano spring water; T_0–3, 0–72 h.

Figure 3.

Orcein staining. Representative images of the US sample at T₀; Ctrl samples cultured in DMEM with saline solution applied to the central wound; and CW-treated samples cultured in DMEM powder reconstituted with filtered CW and with filtered CW applied to the central wound at T₁, T₂ and T₃. Black drumsticks indicate elastic fibers; black triangles indicate newly formed tissue. Magnification, ×5 (US); scale bar, 200 µm and ×10 (CW and Ctrl); scale bar, 100 µm. US, untreated skin; Ctrl, control; DMEM, Dulbecco's modified Eagle's medium; CW, Comano spring water; T_0–3, 0–72 h.

Figure 4.

Immunohistochemistry for proliferating cell nuclear antigen (brown). Representative images of the US sample at T₀; Ctrl samples cultured in DMEM with saline solution applied to the central wound; and CW-treated samples cultured in DMEM powder reconstituted with filtered CW and with filtered CW applied to the central wound at T₁, T₂ and T₃. Magnification, ×10; scale bar, 100 µm. US, untreated skin; Ctrl, control; DMEM, Dulbecco's modified Eagle's medium; CW, Comano spring water; T_0–3, 0–72 h.

Figure 5.

Ratio of mean PCNA-positive nuclei count between CW, Ctrl and US samples at different time points. A ratio >1 indicated a higher mean value for the condition of interest compared with the reference condition at the same time point. The vertical dashed line corresponds to the condition of no difference between the samples (value=1); each bar depicts the mean ratio (reported at the top of each bar). PCNA, proliferating cell nuclear antigen; CW, Comano spring water; Ctrl, control; US, untreated skin; T_0–3, 0–72 h.