Growth factor-free chondrogenesis and immunomodulation of Wharton’s jelly MSCs on chitosan-hyaluronic acid films

This study explores a surface modification strategy that mimics the native cellular environment through the layer-by-layer assembly of natural polyelectrolytes. Specifically, we developed a multilayer matrix composed of 10 alternating layers of chitosan (a polycation) and hyaluronic acid (a polyanion), seeded with Wharton’s jelly mesenchymal stem cells (WJ-MSCs) derived from human umbilical cords. These cells are attractive for cartilage regeneration due to their accessibility, robust differentiation potential, and low immunogenicity. WJ-MSCs were cultured on the multilayer films at a density of 3000 cells/cm 2 in standard growth medium. Positive controls included cells on multilayer films supplemented with transforming growth factor-beta (TGF-β), while negative controls were cells cultured on glass in standard medium. Cell morphology, proliferation, matrix formation, and expression of key chondrogenic markers were assessed. The WJ-MSCs adhered well, exhibited fibroblast-like morphology, and expressed characteristic MSC markers (CD44, CD90, CD73) while lacking hematopoietic markers (CD34, CD45), as defined by ISCT guidelines. The chitosan–hyaluronic acid (CHI-HA) films supported spontaneous chondrogenic differentiation, as demonstrated by upregulation of chondrogenic genes and proteins, and positive staining for chondroitin sulfate. Notably, chondrogenic differentiation on CHI-HA films enhanced the immunomodulatory profile of WJ-MSCs, as shown by upregulation of IL-10 and selective modulation of TLR expression. Despite increased TNF-α, this was attributed to TGF-β signaling rather than inflammation. Overall, CHI-HA films promoted both chondrogenic and immunoregulatory functions, offering a promising platform for cartilage tissue engineering.