Enhancing Biocompatibility and Biophysical Properties of Three-Dimensional Collagen Scaffolds Using Nonthermal Plasma Treatment

Collagen–glycosaminoglycan (CG) scaffolds are widely used in regenerative medicine due to their biocompatibility but often suffer from low stiffness and limited structural integrity, motivating post-processing strategies to improve performance without introducing cytotoxic residues. This study evaluated radio-frequency tailored nitrogen nonthermal plasma (NTP) treatment as a rapid, solvent-free approach to enhance the biophysical properties and cellular performance of 3D CG scaffolds. Scaffolds were treated for 2 or 5 minutes and compared with untreated controls using microstructural analyses (SEM with pore morphometrics; histology), chemistry verification (FTIR; EDS elemental profile), surface wettability (contact angle), absorption kinetics, enzymatic degradation, and cell studies using human adipose-derived stem cells (ADSCs). NTP treatment altered pore architecture (thicker pore struts and increased pore size/porosity) while preserving chemical composition. Surface hydrophilicity increased markedly (reduced contact angle) and liquid absorption accelerated. Mechanical testing showed statistically significant increases in dry compressive modulus after NTP exposure, indicating improved scaffold stiffness. Biologically, ADSC attachment and metabolic activity increased on NTP-treated scaffolds, with significantly higher cell numbers by DAPI quantification. Under osteogenic culture for 21 days, NTP-treated scaffolds promoted stronger osteogenic differentiation, including significantly elevated RUNX2 (≈9–11×) and increased osteocalcin expression relative to controls. Overall, the work demonstrates that brief nitrogen NTP treatment can improve mechanical stiffness, wettability, and cell response of collagen-based scaffolds without chemical crosslinkers, supporting broader use in regenerative medicine and bone-related applications.