Abstract
Introduction
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.
Materials and Methods
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.
Results
In this work, we systematically evaluated the doses and time effect of the customised media on the differentiation potential of ADSCs. Our results showed significant differences in the cell performance between the conditions investigated. Interestingly, ADSCs presented enhanced tendon marker expression (mRNA and protein level) and collagen content. The different tendon and ECM markers analysed by RT-PCR showed doses and time-dependent effect, establishing a connection with
its role in the tissue. We believe this could offer a possible regenerative treatment without overstimulation. Despite the condition, ADSCs presented 95%–100% viability and proliferation values, demonstrating the non-toxic effect of the media.
Conclusion
This study contributes to the knowledge of differentiation potential of ADSCs in tendon repair. Furthermore, the tendon phenotype generated in the 2D cultures changes when different variables are investigated. Knowing the molecular basis and conformations of the tendon phenotype is key in tendon research. Hence we believe these results can show a new paradigm in tendon repair, making possible to select more suitable treatments depending on the status of the injury on the patients.
Acknowledgements
This work was supported by Rosetrees Trust, Arthritis Research UK and the Universityof East Anglia.
