Genotypic and functional characterization of fibroblasts derived from pressure sores

Abstract

IntroductionPressure sores are a major health problem in people with spinal cord injury resulting in ischaemic tissue lesions caused by prolonged pressure against a bony surface. Conventional therapies are often defective and fundamental researches on the healing process of pressure sores must be enriched in order to understand any novel therapies that may be applied. We focalize on pressure sores fibroblasts as dermal fibroblasts perform a critic role in wound healing by populating the wound site to produce extracellular matrix. After characterizing morphological and the genetic profile of healthy fibroblasts and fibroblasts from pressure ulcers, we conducted an analysis of fibroblast proliferation, migration and myofibroblastic differentiation capacity. Materials and Methodsafter acquisition of dermal explants and fibroblasts culture, we conducted histological analysis, an evaluation of gene expression by RT-qPCR and an assessment of fibroblasts proliferation and migration capacity through IncuCyte. A study of the differentiation of fibroblasts into myofibroblasts through the detection of Alpha-Smooth Muscle Actin (-SMA) expression by immunofluorescence was also conducted. Resultshistological analysis showed histological analysis showed dermal disorganization in pressure sore compared with health skin, differences in morphological aspects and density of fibroblasts. Pressure sore fibroblasts express less genes coding for ECM proteins, metalloproteases, collagen III, Connective tissue growth factor (CTGF) and ACTA2 coding for -SMA. Pathological fibroblasts appear to proliferate less quickly than healthy fibroblasts but no differences in migration capacity were found. After stimulation under TGF-{beta}, pressure sore fibroblasts lose their ability to differentiate into myofibroblasts compared to healthy fibroblasts and this could be in relation with a less expression of ACTA2. ConclusionAll of our results highlight a morphological, genetic and functional difference between healthy and pathological fibroblasts which have a modified phenotype, less effective for skin repair. This suggests that new therapies for chronic wounds must take into account the environment in which they are applied and that pathological cells do not necessarily respond to treatments in the same way as healthy cells. Our results are not statistically significant, although several trends emerge. This is explained by the heterogeneity of the patients medical history and requires repetition of the experiments.