DECELLULARIZED AND ENGINEERED TENDONS WITH SHEEP AMNIOTIC EPITHELIAL STEM CELLS ALLO-TRANSPLANTED IN EXPERIMENTALLY INDUCED SHEEP TENDON RESECTION

Achilles tendinopathy affects athletes, recreational exercisers and even inactive people. The tendon has a poor ability to regenerate, and even after healing the breaking point results in the scar tissue formation that impairs its mechanical properties. To overcome to the poor results of the current therapies, stem cell based treatments appears to be an innovative solution. In this context, amniotic epithelial stem cells (AECs) are receiving growing interest from scientist because of their pluripotency, easy accessibility, low immunogenicity and immunomodulatory properties making them ideal candidates in allo and xenotransplantation setting for stem cell therapy approaches. Preclinical model studies on experimentally tendon defects confirmed ovine AECs (oAECs) direct and indirect role in tendon regeneration also modulating the inflammatory response during tendon healing. In order to improve the partial or total tendon resection with the use of oAECs, this research aims to investigate the allo-transplantation of biological decellularized tendons or biocompatible electrospun scaffolds, both engineered with oAECs. Before starting with the above aims, to confirm oAECs in vitro capabilities to modulate the inflammatory response, this research focused on their in vivo immunomodulatory ability when transplanted into an allogeneic recipient. In particular, we investigated the allotransplanted oAECs immunomodulatory role during the early regeneration phase of sheep tendon experimentally induced defects. Specifically, it was evaluated the oAECs role exerted on macrophages (Mφ), in particular on M1Mφ pro-inflammatory and M2Mφ anti-inflammatory subpopulations involved in tissue regeneration. The in vivo experiments results indicate the oAECs modulate the M1Mφ pro-inflammatory switching toward the M2Mφ regenerative phenotype in the tendon lesion areas. Then, in this project, it has been developed a decellularization technique to obtain decellularized tendons that keep the extracellular matrix (ECM) bioactive components for their use as biological scaffolds for oAECs colonization. We conducted comparative studies of decellularization techniques either using detergents or decellularization of fetal tendon explants cultured in incubator, allowing cells to abandon the tissue and colonize the petri dish. The results on biological scaffolds indicated that the detergent decellularization method was effective for the complete cell components elimination but need to be improved in order to maintain the ECM bioactive components, while the decellularization culture method adopted has demonstrated to achieve a good conservation of the ECM components, but tendons were not completely decellularized. Thus, only the technique with detergents allowed obtaining decellularized tendons that soon will be cultured with oAECs obtaining biological engineered scaffolds. We also evaluated oAECs in vitro biocompatibility on biodegradable polymers, in particular on three electrospun biomaterials used for medical devices, Polylactic acid (PLA), Polycaprolactone (PCL), polylactic acid-co-glycolic (PLGA). This phase of the research was possible thanks to the collaboration of Dr. Tammaro (ENEA Research Center- Brindisi Italy).These scaffolds were treated with 3 different sterilization methods: 70% ethanol, ethylene oxide, ethanol 70% rehydrated for 24 hours, in order to verify which one was the best technique for the analysed scaffolds in terms of ultrastructural morphology. Ovine AECs biocompatibility on PLGA sterilized in ethanol 70% rehydrated revealed to be the best in terms of scaffolds ultrastructural stability, cell colonization and spatial organization around the microfibers. Tissue engineering represent a promising approach in regenerative medicine based on the synergy between biocompatible scaffolds miming ECM and oAECs, although the realization of the ideal scaffold should consider several factors, such as chemical and ultrastructural composition prior their use in allo- and xeno-transplantation setting. Next research will be focused on using aligned fibers PLGA scaffolds (in collaboration with Dr. Tammaro and INNOVENT enterprise), that mimic tendon structure, taking advantage of oAECs capacity to differentiate in tenocyte like cell and subsequently using PLGA engineered scaffolds on experimental of partial or total tendon resection preclinical studies to verify their augmented regenerative potential.