Bioactive LbL-assembled multilayer nanofilms upregulate tenogenesis and angiogenesis enabling robust healing of degenerative rotator cuff tendons in vivo.

Degenerative changes to rotator cuff tendons are often diagnosed in elderly patients. Spontaneous healing of degenerative tendons is rather inefficient as a result of the lack of a suitable microenvironment for tendon stem cell differentiation and vascularization. Herein, multilayer hyaluronic acid/chitosan (HA/CS) nanofilms were assembled by a layer-by-layer assembly method onto aligned poly(epsilon-caprolactone) (PCL) nanofibers for use in healing degenerative tendons. Materials testing showed that the number of layers of HA/CS nanofilms could adjust the hydrophilicity and wettability of the nanofibrous membranes. In vitro, the optimal 8-layer (termed as 8LP) membrane afforded aligned morphology of tendon stem/progenitor cells (TSPCs) and up-regulated mRNA expression of tenogenic markers (SCX, BGN). In a rabbit model with disorganized rotator cuff tendons, the 8LP group up-regulated mRNA levels of collagen I/III and tenascin (TNC) at 6 weeks, but not 12 weeks, post-surgery as compared to the native PCL group. Next, vascular endothelial growth factor-loaded 8LP (termed as 8LP-V) was prepared. Compared to 8LP, 8LP-V produced higher levels of angiogenesis in the tendons at 6 or 12 weeks post-surgery, thus supplying endogenous pre-tendon growth factors (TGF-beta, IGF-1) to further enhance tenogenic transcriptional factors. As a result, 8LP-V yielded thicker collagen fibers and/or higher tendon stiffness as compared to the 8LP and clinical pericardial patch groups. This study highlights the rational design of LbL-assembled multilayer HA/CS films to upregulate tenogenesis for robust healing of degenerative rotator cuff tendons.