Stem cells are widely known in the state of the art of cell-based therapies. Recently, ADSCs are becoming a popular resource of adult stem cells across different fields, and latest publications show its wide application for the treatment of soft tissue injuries like tendon injuries, which represent a high percentage of the consultations in orthopaedic practitioners. Molecular-based therapies and local deliveries are necessary for an effective treatment of chronic tendon injuries. In this study, human ADSCs were selected to investigate its differentiation potential into the tendon phenotype. Customised cell culture media was used as the differentiation factor. In the present study, ADSCs were used in passage 3 to ensure pluripotency in vitro. Using the customised cell culture media, its time, concentration and frequency of refreshment effects were investigated. On the selected time points different techniques were performed: 1,) cells were harvested, and messenger RNA (mRNA) was examined by Real Time Polymerase Chain Reaction (RT-PCR), analysing the expression of common tendon and extracellular matrix (ECM) markers. Protein expression was determined by Western Blotting. 2) Collagen content was analysed by tissue digestion and colorimetric techniques. 3) Deoxyribonucleic Acid (DNA) was stained, and fluorescent imaging was used to characterise nuclear roundness. 4) Metabolic activity of the cultures was assessed using CellTiter 96® Aqueous One Solution (MTS). 5) Cell proliferation was evaluated using CyQuant® Cell Proliferation Assay.Introduction
Materials and Methods
