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

PURPOSE: 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.

METHODS

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.

RESULTS

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).

CONCLUSION

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.