In order to achieve successful wound repair by regenerative tissue engineering using mesenchymal stem cells (MSCs), it is important to understand the response of stem cells in the scaffold matrix to mechanical stress. To investigate the clinical effects of mechanical stress on the behavior of cells in scaffolds, bone marrow-derived mesenchymal stem cells (MSCs) were grown on a type-I collagen-glycosaminoglycan (GAG) scaffold matrix for one week under cyclic stretching loading conditions. The porous collagen-GAG scaffold matrix for skin wound repair was prepared, the harvested canine MSCs were seeded on the scaffold, and cultured under three kinds of cyclic stretching loading conditions (0%: control, 5% strain, 15% strain). After 7 days incubation, MSCs were evaluated histologically and immunohistochemically regarding the proliferation and differentiation. Cultured MSCs in the high strain (15% strain) group showed active alpha-smooth muscle actin (alpha-SMA) expression and poor differentiation into type-I collagen-positive cells, whereas enhanced differentiation into type-I collagen positive cells and a lack of alpha-SMA expression where shown in the lower stress (5% strain) group. These results suggest that mechanical stress may affect the proliferation and differentiation of stem cells, and subsequently the wound healing process, through attachment interactions between the stem cells and scaffold matrix. Our findings provide an additional consideration for clinical treatment of wound repair using regenerative tissue engineering.