ABSTRACT
Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.
ABSTRACT
BACKGROUND: Numerous clinical studies have shown that biofilm formation by Staphylococcus epidermidis on the outer surface of a silicone breast implant is strongly associated with capsular contracture formation. Traditional administration of systemic antibiotics and antiseptic washing are not necessarily the most effective methods for the prevention of initial biofilm formation on implants in the clinical scenario. In this study an alternative or supplement was sought for preventing or delaying bacterial colonisation and adherence to the outer surface of a breast implant, by establishing an in vitro model for investigating this complex problem. The in vitro antimicrobial activity of several antimicrobial agents was investigated for inhibitory effects on biofilm formation by S. epidermidis. METHODS: The study consisted of two experiments. The first experiment consisted of two groups (A and B) of seven discs each whilst the second experiment was divided into three groups (C, D and E) of 14 discs each. Each group of 14 consisted of seven smooth and seven textured discs. Discs (biopsies) of each implant were individually coated with one of six different antimicrobial agents. Controls that received no agent were included in the various experimental groups. In the first experiment disc diffusion sensitivity testing was performed and inhibition zone sizes were measured. In the second experiment the discs were cultured in broth. The degree of biofilm formation was evaluated by scanning electron microscopy (SEM). RESULTS: In the first in vitro experiment, all six agents showed a measurable antimicrobial effect against the biofilm-forming strain of S. epidermidis when compared to the effect against the American Type Culture Collection strain. In the second in vitro experiment, discs coated with Chloramex, Fucidin and Terramycin did not allow biofilm formation to take place for at least 7 days. CONCLUSIONS: Staphylococcus epidermidis biofilm formation on the outer surface of a silicone breast implant was prevented in vitro for at least 7 days by coating with an appropriate antimicrobial agent. Further evaluation of the interaction between antimicrobial coating agents and S. epidermidis biofilm formation needs to be made before conclusions regarding the clinical scenario can be drawn.
