Recently, the development and the application of 3D scaffold able to promote stem cell differentiation represented an essential field of interest in regenerative medicine. In particular, functionalized scaffolds improve bone tissue formation and promote bone defects repair. This research aims to evaluate the role of ascorbic acid (AS) supplementation in an in vitro model, in which a novel 3D-scaffold, bovine pericardium collagen membrane called BioRipar (BioR) was functionalized with human Gingival Mesenchymal Stem Cells (hGMSCs). As extensively reported in the literature, AS is an essential antioxidant molecule involved in the extracellular matrix secretion and in the osteogenic induction. Specifically, hGMSCs were seeded on BioR and treated with 60 and 90 μg/mL of AS in order to assess their growth behavior, the expression of bone specific markers involved in osteogenesis (runt-related transcription factor 2, RUNX2; collagen1A1, COL1A1; osteopontin, OPN; bone morphogenetic protein2/4, BMP2/4), and de novo deposition of calcium. The expression of COL1A1, RUNX2, BMP2/4 and OPN was evaluated by RT-PCR, Western blotting and immunocytochemistry, and proved to be upregulated. Our results demonstrate that after three weeks of treatment AS at 60 and 90 μg/mL operates as an osteogenic inductor in hGMSCs. These data indicate that the AS supplementation produces an enhancement of osteogenic phenotype commitment in an in vitro environment. For this reason, AS could represent a valid support for basic and translational research in tissue engineering and regenerative medicine.