Optimization of a tissue engineering scaffold for use in bone tissue engineering requires control of many factors such as pore size, porosity, permeability and, as this study shows, the composition of the matrix. The collagen-glycosaminoglycan (GAG) scaffold variants were fabricated by varying the collagen and GAG content of the scaffold. Scaffolds were seeded with MC3T3 osteoblasts and cultured for up to 7 days. During the culture period, osteoblastic activity was evaluated by measuring metabolic activity, cell number, and spatial distribution. Collagen and GAG concentrations both affected osteoblast viability, proliferation, and spatial distribution within the scaffold. Scaffolds containing 1% collagen (w/v) and 0.088% GAG (w/v) were found to have a porosity of approximately 99%, high cell metabolic activity and cell number, and good cell infiltration over the 7 days in culture. Taken together, these results indicate the need to tailor the parameters of a biological substrate for use in a specific tissue application, in this case bone tissue engineering.