Our animal implantation studies have demonstrated that, after osteogenic processing, cultured human periosteal sheets form osteoid tissue ectopically without the aid of conventional scaffolding materials. To improve the osteogenic activity of these periosteal sheets, we have tested the effects of including a scaffold made of salmon collagen-coated ePTFE mesh. Periosteal sheets were produced with minimal manipulation without enzymatic digestion. Outgrown cells penetrated into the coated mesh fiber networks to form complex multicellular layers and increased expression of alkaline phosphatase activity in response to the osteoinduction. In vitro mineralization was notably enhanced in the original tissue segment regions, but numerous micro-mineral deposits were also formed on the coated-fiber networks. When implanted subcutaneously into nude mice, periosteal sheets efficiently form osteoid around the mineral deposits. These findings suggest that the intricate three-dimensional mesh composed of collagen-coated fibers substantially augmented the osteogenic activity of human periosteal sheets both in vitro and in vivo.