Design of Hyaluronic Acid-based Scaffold Containing Human Endometrial Mesenchymal Stem Cells (hEnMSCs)-Derived Extracellular Vesicles for Bone Tissue Engineering: in vivo and vitro Studies

The main challenges in using stem cells (SCs) are cellular survivability, undifferentiated cells, their dose-dependent effects, or age-related deteriorating functions. In this study, it was first focused on designing a bio-substrate with suitable physicomechanical properties to provide a cell-to-cell interactive microenvironment and then on studying the role of extracellular vesicle (EV), as an alternative biologic agent to overcome the SCs limitations, and its dosage, to induce bone formation. To this end, an optimized volume ratio of polyvinyl alcohol (PVA)/chitosan (CS) solution was first selected and mixed with hyaluronic acid (HA). Accordingly, adding HA to the PVA/CS structure resulted in a more coherent network (similar to 5% decrease in fiber diameter, similar to 25% and 1.28-fold increase in porosity and modulus) and better cellular adhesion. The results of loading EV with different dosages (low and high) on the PVA/CS/HA scaffold network and implantation in the rat skull-defect model also indicated that this scaffold provides a burst release of EV; however, the higher dosage possesses the slower release with the gentler gradient in the release profile. Moreover, the in vivo studies exhibited that the high-dose treatment group possesses more ossification in line of the defect with more numerous, active osteoblasts and ossification in the osteoid, along with more symmetrical restoration (after 8 weeks) than the empty scaffold and the low-dose treatment group.