Wound healing and skin regeneration on FT-skin
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Presented by: Marisa Meloni
Introduction: Skin regeneration is a physiological and extremely complex process involving the reorganization of different cell types to restore tissue continuity after wound: activated keratinocytes migrate from the wound edges to generate a provisional wound bed matrix where they can proliferate, differentiate and stratify into a new epidermis. The complete skin repair process involves fibroblasts activation and ECM remodeling.
The aim of this research project was to develop an in vitro experimental model on 3D reconstructed Full thickness skin that in a relatively short experimental window was able to recapitulate the 3 essential steps of wound repair: re-epithelization, fibroblasts activation and ECM remodeling by following the localization and expression of well known biomarkers.
Methods: Commercial available Full-Thickness Skin model was injured with 1-mm biopsy punch by a standardized and reproducible procedure. The wound bed evolution was followed by a time course approach: 24h and 3-8 days.
2-O-α-D-Glucopyranosyl-L-Ascorbic Acid was used dosed in the culture medium as positive reference.
The immuno-histochemical analysis (CK 10, 14, a-SMA, fibronectin, Integrin b1) was used to qualitatively and quantitatively capture cell migration, polarization and differentiation and ECM remodeling. TGFb1 and KGF genes were quantified by qRT-PCR.
Results: Within the mechanisms of skin repair investigated the following results seems to be of interest and they globally recapitulate in a convenient and relatively short experimental window the main phases of skin regeneration.
Keratinocytes migration and nuclei orientation: the migration of keratinocytes was confirmed by analyzing CK14 and ITG b1 expression and localization in the wound bed and cell polarization thanks to nuclei orientation in a time-dependent manner: the process was fully completed after 3 day.
Fibroblasts activation and newly ECM components in the wound bed
The role of activated fibroblasts-myofibroblasts as major players in the ECM remodeling process, has been demonstrated by a-smooth muscle actin and fibronectin localization by immunofluorescence analysis: a fully restored skin integrity was observed at day 8.
Conclusion: We have demonstrated that by using standardized injury procedure an in vitro reconstructed 3D FT-skin was able to recapitulate the main steps of the skin regeneration and healing process in the two skin compartments and the results obtained are in good agreement with literature data and clinical findings. The approach described seems promising to assess dermo-pharmaceutical and cosmetic formulations efficacy and to elucidate aspects of myofibroblast biology in healthy and disease conditions.
The aim of this research project was to develop an in vitro experimental model on 3D reconstructed Full thickness skin that in a relatively short experimental window was able to recapitulate the 3 essential steps of wound repair: re-epithelization, fibroblasts activation and ECM remodeling by following the localization and expression of well known biomarkers.
Methods: Commercial available Full-Thickness Skin model was injured with 1-mm biopsy punch by a standardized and reproducible procedure. The wound bed evolution was followed by a time course approach: 24h and 3-8 days.
2-O-α-D-Glucopyranosyl-L-Ascorbic Acid was used dosed in the culture medium as positive reference.
The immuno-histochemical analysis (CK 10, 14, a-SMA, fibronectin, Integrin b1) was used to qualitatively and quantitatively capture cell migration, polarization and differentiation and ECM remodeling. TGFb1 and KGF genes were quantified by qRT-PCR.
Results: Within the mechanisms of skin repair investigated the following results seems to be of interest and they globally recapitulate in a convenient and relatively short experimental window the main phases of skin regeneration.
Keratinocytes migration and nuclei orientation: the migration of keratinocytes was confirmed by analyzing CK14 and ITG b1 expression and localization in the wound bed and cell polarization thanks to nuclei orientation in a time-dependent manner: the process was fully completed after 3 day.
Fibroblasts activation and newly ECM components in the wound bed
The role of activated fibroblasts-myofibroblasts as major players in the ECM remodeling process, has been demonstrated by a-smooth muscle actin and fibronectin localization by immunofluorescence analysis: a fully restored skin integrity was observed at day 8.
Conclusion: We have demonstrated that by using standardized injury procedure an in vitro reconstructed 3D FT-skin was able to recapitulate the main steps of the skin regeneration and healing process in the two skin compartments and the results obtained are in good agreement with literature data and clinical findings. The approach described seems promising to assess dermo-pharmaceutical and cosmetic formulations efficacy and to elucidate aspects of myofibroblast biology in healthy and disease conditions.