The ruptured anterior cruciate ligament does not heal spontaneously as it has a low capacity for healing. Therefore, the development of new healing techniques employing tissue engineering is vital. As a potentially new approach for ligament regeneration, this study used a highly oriented fiber scaffold made of elastin and collagen (the mean diameters were 1.7 ± 0.4 μm and 0.5 ± 1.4 μm, respectively), which comprise the extracellular matrix of the ligament. In addition, a multiple-type dynamic culture consisting of a combination of pressure and twist stimulation was performed to examine the influence of mechanical force on the functional maintenance of ligament cells and on the differentiation of ligament cells to osteoblast-like cells. Our results show that a pressure stimulation and elastin A upregulated the expression of alkaline phosphatase (ALP) (a marker of osteogenic differentiation) and promoted the osteogenic differentiation of ligament cells. In addition, the twist stimulation upregulated the expression of type III collagen (the main component of ligament tissue). Furthermore, the combination of pressure and twist stimulation promoted the expression of type III collagen and ALP protein depending on the portion of scaffold.