Extracellular matrix assembly during chondrogenic differentiation of amniotic fluid- and adipose-derived mesenchymal stromal cells under TGF-β3 induction

Abstract Osteoarthritis is a major degenerative joint disease often triggered by traumatic injuries, yet current treatment options remain limited in restoring cartilage integrity. Mesenchymal stromal cells (MSCs) represent a promising source for chondrogenic regeneration, but their differentiation potential may vary according to tissue of origin. Here, we compared the chondrogenic differentiation capacity of human amniotic fluid–derived MSCs (hAF-MSCs, CD117⁺ selected) and human adipose-derived MSCs (hAD-MSCs) when cultured within porous chitosan–xanthan gum (CX) scaffolds under TGF-β3 stimulation. Both MSC types were expanded, phenotypically characterized, and induced toward chondrogenesis. Robust adhesion and proliferation within CX scaffolds were observed, accompanied by deposition of extracellular matrix enriched in glycosaminoglycans and collagen fibers. Histological staining (hematoxylin–eosin, alcian blue, picrosirius red, and Masson’s trichrome) and immunohistochemistry confirmed the synthesis of cartilage-specific macromolecules, including collagen type II and aggrecan, while immunofluorescence highlighted a strong collagen II signal. Scanning electron microscopy further revealed extensive cell–matrix interactions and abundant fibrillar organization. These findings demonstrate that both hAF-MSCs and hAD-MSCs undergo effective chondrogenic differentiation within CX scaffolds under TGF-β3 stimulation. Notably, hAF-MSCs exhibited a superior integration and an enhanced extracellular matrix assembly compared to hAD-MSCs, providing new biochemical insights into their extracellular matrix assembly and supporting their potential application in cartilage tissue engineering.