Effect of dermal fibroblasts conditioned medium (DFCM) on IN VITRO skin re- epithelialization

Abstract

A key event in wound healing is re-epithelialization, which is accomplished by the proliferation, migration, and differentiation of epidermal keratinocytes. These processes are mainly regulated via paracrine signaling of cytokines, chemokines and growth factors secreted by dermal fibroblasts. Impaired epithelialization, which is prevalent in chronic wounds and aging skin, is caused by lack of these essential factors. In vitro cell culture and protein extraction techniques enables collection of fibroblasts secreted factors from the waste medium as dermal fibroblast conditioned medium (DFCM), which could facilitate re-epithelialization during wound healing. The aim of the current study was to elucidate the effect of DFCM on in vitro re- epithelialization process i.e. keratinocyte attachment, proliferation, migration, and differentiation. Skin samples were collected from consented donors to isolate fibroblasts and keratinocytes. DFCM was prepared by culturing confluent fibroblasts with serum-free keratinocyte-specific (DFCM-KM) and fibroblast-specific (DFCM- FM) medium. Keratinocytes supplemented with DFCM were observed under time- lapse imaging to evaluate attachment, proliferation, and migration. The scratch assay was performed to assess keratinocytes rate of healing. DFCM collected with 3-day incubation of confluent fibroblasts (DFCM-KM-3 and DFCM-FM-3) were resulted in enhancement of keratinocyte re-epithelialization process compared to 1-day and 2-day incubation. It was also found that DFCM-KM-3 significantly enhanced keratinocyte attachment (77.66×10215.15×102 cells/cm2) compare to control (64.36×102+10.31×102 cells/cm3) and DFCM-FM-3 (50.58×10219.83×10 cells/cm2) (p<0.05). While, DFCM-FM-3 significantly increased keratinocyte wound healing (155.4×10 +5.24×102 um2/h) compare to control (115.5×10243.25×102um3/h) and DFCM-KM-3 (120.1x10 +5.24×102um2/h) (p<0.05). Moreover, supplementation of DFCM-FM-3 resulted in the enlargement of keratinocyte area and demonstrated collective migration during healing, which was distinctly different from keratinocytes supplemented with DFCM-KM-3 and on control condition. Further analysis confirms that the presence of high calcium in DFCM-FM-3 facilitated the changes. Also, to understand the effect of DFCM on re-epithelialization of aging skin, DFCM was supplemented to keratinocytes from 3 different age groups (≥18-35 years, 36-54 years and ≥55 years). It was found that irrespective of donor age, DFCM-KM-3 enhances keratinocyte attachment while DFCM-FM-3 enhances keratinocyte healing rate, indicating the potential application of DFCM for impaired re-epithelialization.