Effects of Comano Spring Water-derived Bacterial Lysates on Skin Regeneration: An Ex-vivo Study.

BACKGROUND/AIM: A native non-pathogenic bacterial microflora was identified in Comano (TN, Italy) spring water. The aim of this study was to investigate the regenerative effects of some of the bacterial lysates extracted from this water in a human ex-vivo skin experimental wound model. MATERIALS AND METHODS: Bacterial lysates were extracted from four new isolates: lysate 1 (L1) - closest relative Rudaea cellulosilytica, phylum Proteobacteria; lysate 2 (L2) - closest relative Mesorhizobium erdmanii, phylum Proteobacteria; lysate 3 (L3) - closest relative Herbiconiux ginseng, phylum Actinobacteria; lysate 4 (L4) - closest relative Fictibacillus phosphorivorans, phylum Firmicutes. Their regenerative effects were investigated in a human ex-vivo skin experimental wound healing model at 3 (T1), 5 (T2), and 10 days (T3). RESULTS: The samples cultured with the L2 lysate displayed both an earlier and complete restoration of all the skin layers and their features were the closest to the normal skin. The regenerated epidermis demonstrated a complete maturation as the normal epidermis. The papillary dermis appeared mature, and the reticular dermis displayed both collagen and elastic fibres regularly parallel to the skin surface. An anti-inflammatory effect was displayed by the L1 lysate, but this action did not constitute a regenerative effect, suggesting that pathways for inflammation and regeneration might be different. CONCLUSION: The therapeutic power of spring waters is not exclusively related to their mineral composition, but it may also be attributable to their native non-pathogenic bacterial microflora.

Platelet Rich Plasma Enhancement of Skin Regeneration in an ex-vivo Human Experimental Model.

This study reports on the development of an original, ex-vivo wounded skin culture protocol using autologous Platelet Rich Plasma (PRP) and enriched Dulbecco's Modified Eagle's Medium (DMEM). Human skin samples obtained from specimens harvested during reduction mammoplasty procedures, were injured in their central portion-to create a standard wound-and cultured under three different conditions: - enriched DMEM with saline solution in the central wound (control)- enriched DMEM with the same medium in the central wound- enriched DMEM plus 2.5% autologous PRP, with the same PRP added medium in the central wound. Morphological analysis was carried out at 0 h (T0) and on days 1, 3, 5 and 10 (T1-T3-T5-T10) using Hematoxylin and Eosin; Masson's trichrome staining; Weigert staining and Ki-67 staining to identify the skin histological features in the different experimental conditions. The combination of DMEM and PRP allowed a favorable modulation of the epithelial cells and fibroblasts proliferation, and a relevant anti-inflammatory action. PRP also demonstrated an inhibitory effect on both the collagen and elastic fibers' de-structuration and a favorable modulation of the re-organization of these fibers. The step by step histological and immune-histo-chemical regenerative effects of PRP on human skin wound repair and regeneration process was observed over a period of 10 days.

Unrecognized Cell Torpidity as a Risk Factor in Elective Plastic Surgery.

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Ex vivo regenerative effects of a spring water.

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.

hASC and DFAT, Multipotent Stem Cells for Regenerative Medicine: A Comparison of Their Potential Differentiation In Vitro.

Adipose tissue comprises both adipose and non-adipose cells such as mesenchymal stem cells. These cells show a surface antigenic profile similar to that of bone-marrow-derived MSC. The cells derived from the dedifferentiation of mature adipocytes (DFAT) are another cell population with characteristics of stemness. The aim of this study is to provide evidence of the stemness, proliferation, and differentiation of human adipose stem cells (hASC) and DFAT obtained from human subcutaneous AT and evaluate their potential use in regenerative medicine. Cell populations were studied by histochemical and molecular biology techniques. Both hASC and DFAT were positive for MSC markers. Their proliferative capacity was similar and both populations were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. DFAT were able to accumulate lipids and their lipoprotein lipase and adiponectin gene expression were high. Alkaline phosphatase and RUNX2 gene expression were greater in hASC than in DFAT at 14 days but became similar after three weeks. Both cell populations were able to differentiate into chondrocytes, showing positive staining with Alcian Blue and gene expression of SOX9 and ACAN. In conclusion, both hASC and DFAT populations derived from AT have a high differentiation capacity and thus may have applications in regenerative medicine.

Novel perspectives on the role of the human microbiota in regenerative medicine and surgery.

Plastic surgery is transitioning from a fine craftsmanship to a regenerative science. In wound healing, the role of microorganisms is no longer considered to be just counteracting, but also promoting. Furthermore, host-microbe interactions are essential for numerous aspects of normal mammalian physiology, from metabolic activity to immune homeostasis. Each area of the human body hosts a unique microbial community, and the composition of microbiota is dependent on the host, age and the anatomical area, and it changes according to the characteristics of the microenvironment. Every squared centimeter of skin contains ~1 billion bacteria. The majority of microorganisms of the skin are commensal or temporary passing members. Skin flora mechanisms interacting or influencing the human physical skin barrier are not well defined. Resident skin bacteria provide the first line of defence against potentially dangerous pathogens and produce small molecules that influence their microbial neighbours. Furthermore, the microbiota activates and assists innate immunity and influences adaptive immunity. Various types of immune and non-immune cells contribute to wound healing. The proliferative phase of wound healing is inversely proportional to the extent of the post-traumatic inflammatory reaction. Topical bacterial lipopolysaccharide application markedly affects wound healing by accelerating the resolution of inflammation, increasing macrophage infiltration, enhancing collagen synthesis and altering the secretion of mediators involved in skin regeneration. Various studies have investigated the biological contents of thermal spring waters, and their anti-inflammatory and immune protective roles. In addition, the regenerative properties of thermal spring waters were analysed in an experimental animal wound model. The areas treated with thermal water healed faster than the areas treated with conventional dressings, and exhibited a collagen and elastic fiber network comparable with the normal skin. Thus, the microbial environment may be considered as a potential tool in regenerative medicine and surgery.