The topographical features and material composition of scaffolds have a powerful influence on cell behaviors such as proliferation and differentiation. Here, scaffolds consisting of aligned fibers with incorporated: bioactive collagen I were tested for their ability to enhance osteogenesis in vitro. Rat adipose, derived mesenchymal stem cells (ADSCs) were seeded on the scaffolds and their morphology, proliferation, and osteogenic differentiation ware examined. Aligned scaffolds with collagen I showed the best osteogenic properties. Also, adhesion-related genes showed the higher expression on aligned scaffolds with collagen I. Our findings indicate that fiber alignment combined with incorporation of collagen I increases the capacity of electrospun scaffolds to induce enhanced and directed osteogenesis. Such scaffolds may, therefore, have potential for improving guided oral bone regeneration.