Hyaluronic Acid Hydrogels for Minimally Invasive and Controlled Delivery of Extracellular Vesicles in Tendon Injuries

Statement of Purpose: Tendinopathy is a common disease, encompassing a multitude of conditions (from chronic inflammation to acute rupture) with scarce curative therapy available. The modulation of inflammation, regeneration of injured tissue, and the functional recovery represent clinical challenges. Recently, platelet-derived extracellular vesicles (EVs) have shown great potential to induce regenerative pathways in tendon due to their encapsulated biomolecules. However, the controlled delivery of EVs using minimally invasive techniques is necessary to avoid rapid tissue clearance and maximize the therapeutic effects. Two types of EVs were isolated from lysed (PL-EVs) and Ca2+-activated platelets (PRP-EVs). Hyaluronic acid (HA) of two different MW (40 kDa and 200 kDa) functionalized with adipic acid dihydrazide (adh-HA) together with an aldehyde-modified HA (a-HA) were used as precursors for two hydrogel formulations with EVs-delivery capacity. Rheology was used for hydrogel characterization. EVs release was studied along a period of 30 days at physiological conditions, with and without hyaluronidase (1 IU/mL). EVs were characterized by WB, NTA and flow cytometry. Bioactivity of EVs was studied on human tendon-derived stem cells (hTDCs) and primary macrophages, performing cell viability, mRNA expression and protein expression assays. Cellular uptake of EVs was also studied by flow cytometry and microscopy. Hydrogels showed injectability and self-healing characteristics, independently of the EVs loading, given the dynamic character of the hydrazone bond. The release of EVs was slower in hydrogels made with high MW HA. Biodegradability did not significantly vary between formulations but a trend for quicker initial degradation for 40 kDa adh-HA was noted. Release was heavily dependent on the degradation of the hydrogel by hyaluronidase. Both PL- and PRP-derived EVs had an average size around 100 nm, and were positive for CD9, CD63 and ApoB48. hTDCs stimulated with PRP-EVs showed higher cellular activity, improved tendon markers (scleraxis, tenomodulin) but no significant reduction of fibrosis markers. PRP-derived EVs were uptake quicker than PL-derived EVs, yet the final signal was identical in PRP-derived and PL-derived EVs in 72h treated cells. Also, PL- and PRP-derived EVs were able to polarize macrophages. We developed an injectable hydrogel able to control the release of EVs for up to 7 days. PRP-derived EVs show higher bioactivity and quicker uptake than PL-derived EVs, and both EVs fractions were more bioactive than their crude counterparts. The system hold potential in the field of controlled EVs release using minimally invasive strategies.