Modification of deproteinized bovine bone matrices by cold atmospheric plasma: surface characterization and in vitro biological responses in murine pre-osteoblasts
Bone substitutes; Cold atmospheric plasma; Surface modification; Pre-osteoblasts; Osteogenic differentiation.
Bovine xenogeneic bone grafts are widely used in maxillomandibular reconstruction; however, industrial processing may reduce their wettability and surface bioactivity. In this context, cold atmospheric plasma has emerged as a surface modification strategy capable of increasing biomaterial hydrophilicity and chemical reactivity without necessarily altering their structural properties. This project will evaluate the effects of cold atmospheric plasma on the surface properties of deproteinized bovine bone matrices and on the biological responses of MC3T3-E1 murine pre-osteoblasts. Block and particulate matrices will be investigated in separate studies. Initially, the operating parameters of a helium-fed cold atmospheric plasma jet generated by dielectric barrier discharge will be optimized. For each matrix format, untreated, gas-flow-only, and plasma-treated groups will be compared. Surface characterization will include analyses of wettability, surface free energy, chemical composition by XPS and ATR-FTIR, morphology and elemental composition by scanning electron microscopy (SEM/EDS), nanotopography by AFM, mineral structure by X-ray diffraction, optical emission spectroscopy, temperature monitoring during treatment, and hydrophobic recovery. The biological responses of murine pre-osteoblasts will be assessed through cell adhesion and organization assays, including vinculin immunolocalization and SEM; metabolic activity by MTT and Alamar Blue; viability by Live/Dead staining; cell death (apoptosis and necrosis) by Annexin V/PI; proliferation by Ki-67; cell-cycle analysis; oxidative stress; and osteogenic differentiation through alkaline phosphatase activity, osteogenic marker expression, and quantification of mineralized matrix by Alizarin Red staining. Plasma treatment is expected to increase matrix wettability and surface bioactivity without compromising composition or structure, thereby promoting cell adhesion, viability, and osteogenic differentiation. These findings may support the development of a rapid and operationally simple strategy for the bioactivation of xenogeneic bone grafts used in bone regeneration.