Immunohistochemical Evaluation of Non-Thermal Plasma-Treated Xenogenic Bone Graft for Enhanced Bone Regeneration in a Rabbit Calvarial Defect Model

Bone graft materials may undergo biological aging during storage, potentially reducing surface hydrophilicity and early cellular responses. Non-thermal plasma treatment has been investigated as a surface modification strategy to improve the biological performance of graft materials. This study evaluated osteogenic and angiogenic responses associated with non-thermal plasma-treated xenogenic bone grafts using tissues obtained from an 8-mm rabbit calvarial defect model. Twenty-four adult male New Zealand white rabbits were assigned to 2-, 4-, and 8-week healing periods, and defects were filled with untreated or plasma-treated xenografts. Immunohistochemical expressions of PCNA, VEGF, PECAM-1, collagen I, collagen III, ALP, BMP2, BMP4, BMP7, osteocalcin, and osteopontin were assessed semiquantitatively. Most markers showed greater expression in the experimental group during the early and intermediate healing stages. VEGF and PECAM-1 showed significant intergroup differences at 2 and 4 weeks but not at 8 weeks, whereas several osteogenic markers showed lower expression in the experimental group at 8 weeks. These temporal expression patterns suggest that non-thermal plasma treatment is associated with earlier activation of biological responses related to cell proliferation, angiogenesis, and osteogenesis, followed by changes consistent with later tissue maturation and remodeling.