From lignin to living systems: Carbon dots modulate early cytoskeletal organization and phenotypic marker expression in undifferentiated mesenchymal stem cells

Carbon dots (CDs) are emerging bioactive nanomaterials for tissue engineering applications. This study aimed to reproduce softwood lignin-derived carbon dots (LCDs) and nitrogen-doped LCDs (N@LCDs) and evaluate their effects on early human adipose-derived mesenchymal stem cell (hMSC) behavior in 2D monolayer and 3D culture systems.

LCDs/N@LCDs were reproduced and characterized. CDs were ultrasonically sprayed onto 2D substrates and polyethylenimine (PEI)-treated electrospun poly(lactic-co-glycolic acid) (PLGA, 65:35) 3D scaffolds for in vitro analyses. Distribution after drop-casting and ultrasonic spray deposition (USD) was quantified using nearest-neighbor distance (NND). Native versus cell media-exposed LCD/N@LCD functionalities were compared by Fourier-transform infrared spectroscopy (FTIR). Scaffolds were characterized for structural and compositional properties. Cell attachment, morphology, and biomarker expression were evaluated.

USD reduced LCD/N@LCD concentration of short NND relative to drop-casting. Following media exposure, LCD/N@LCDs exhibited unique changes in FTIR spectra, supporting distinct media-associated interfacial functionalities. The F-actin aspect ratio differed among groups. (p = 0.0287); LCD/N@LCD increased hMSC elongation relative to control (p = 0.0587 and 0.0371, respectively). 2D cultures showed increased fibronectin-182, MAP2, and GFAP in LCD/N@LCD groups (p < 0.05) compared to untreated substrates. Compared to untreated scaffolds, hMSCs on N@LCD-treated scaffolds displayed increased vinculin (p = 0.015), MAP2 (p = 0.032), GFAP (p > 0.05), and CD31 expression (p = 0.044). Collagen type I and fibronectin-182 increased on LCD/N@LCD-treated scaffolds (p > 0.05).

LCDs/N@LCDs regulate early hMSC cytoskeletal organization and biomarker expression. Exposure of LCDs/N@LCDs to the biological media exhibited variations in FTIR spectra, suggesting differences in adsorbed proteins, potentially consequential of variant corona composition. Results demonstrate LCD/N@LCDs functioning as bioactive cues, supporting their potential in tissue engineering applications.