Enabling tendon regeneration through the application of platelet rich plasma

Introduction: The differences in the matrix composition, organisation and structure of the tendinous extracellular and interfascicular matrices give rise to the differences in the mechanical properties between energy-storing and positional tendons. Animal models have revealed differences in processes such as the expression and amount of collagen, glycosaminoglycans and matrix metalloproteinases, indicating the requirement of different extracellular environments between energy-storing and positional tendons. The ability of tendons to heal, including energy-storing and positional tendons, is limited, often resulting in a healed construct that is functional and mechanically inferior to the tendon pre-injury. Platelet Rich Plasma (PRP) is an approached used in the treatment of tendon pathologies, showing mixed results. Therefore, the aims of this thesis were to investigate the differences in the extracellular matrix gene expression and cellular processes including metabolic activity between energy-storing and positional tendons and whether the application of PRP altered these profiles in 2D and 3D models. Method: Firstly, individual PRP profiles were created from 26 participants, 13 male and 13 female (male - mean age (+SD) = 41.31+14.43 years; female - mean age (+SD) = 37.69+12.98 years). The whole blood cell concentrations, platelet concentration in PRP and concentration of 8 growth factors were obtained for each of the 26 participants. In a novel approach, paired human Achilles (energy-storing) and tibialis anterior (positional) tendon samples were obtained from amputated limbs of 3 tendon donors (mean age (+SD) = 25.68+18.78 years). Tendon derived cells were explanted and expanded, and the metabolic activity, morphology, viability and gene expression of extracellular matrix genes of paired Achilles and tibialis anterior tendon cells were measured with and without the application of PRP in 2D cell culture and 3D (time intervals 1- 7 days). Results: Age, gender and intervals between venepuncture did not affect PRP content between male and female participants. The major finding was the differential effect of PRP on paired Achilles and tibialis anterior tendon derived cells. Cells derived from the tibialis anterior possessed a significantly greater metabolic activity than Achilles cells when cultured in control conditions (2% FCS, experimental day 3, p < 0.0001, day 7, p = 0.031, and the low platelet concentration [plasma control] 10% PPP, day 3, p < 0.0001, day 7 p = 0.0019). The expression of collagens - I, III and V were also highest in cells derived from the tibialis anterior tendons, more so than Achilles, at experimental days 1 and/or 3 when cultured in 2% FCS or 10% PPP. However, the metabolic activity and gene expression of tibialis anterior tendon cells did not increase upon the application of PRP compared to controls. There appeared to be phenotypic drifting of tendon cells, suggested by a reduction in their morphology (aspect ratio) which may have also been a factor in the reduced expression of extracellular matrix related genes. Attempts to recapitulate these results in a 3D PRP gel were largely unsuccessful. The stabilisation of PRP gels could not be achieved due to rapid degradation. PRP gels lost their solid, gel-like appearance before measurements could be taken at the specified timed intervals. Conclusion – These results indicate differences between the metabolic activity and extracellular composition of energy-storing and positional tendon derived cells. Due to the lack of a suitable 3D PRP gel model in this study, it was not possible to conclusively accept or reject the central hypothesis of this thesis. However, this study does present results that further pre-clinical studies such as an appropriate 3D model and/or serum-free cultured conditions could develop