The scaffold is an essential component in tissue engineering. By assessing a novel method, in which paraffin wax and starch particles with various sizes were placed in a growing culture of Acetobacter xylinum, three-dimensional nanofibril network of bacterial cellulose (BC) scaffolds with defined micropores were developed. The resulting BC had different morphologies and pore interconnectivity. Paraffin particles were incorporated throughout the scaffold, while starch particles were found only in the outermost area of the resulting scaffold. The porogens were successfully removed after culture with bacteria and no residues were detected with Electron Spectroscopy for Chemical Analysis (ESCA) or Fourier Transform Infra Red Spectroscopy (FT-IR). The main purpose of use for the resulting scaffolds is tissue engineering of blood vessels, therefore SMC were selected as the cell type to culture in the scaffolds. The scaffolds were placed in Boyden chambers and seeded with smooth muscle cells (SMC). Platelet-derived growth factor was used as an attractant for cell ingrowth. Smooth muscle cell ingrowth and collagen production were investigated using histology and fluorescence procedures. Furthermore, organ bath techniques were used to evaluate the mechanical activity by stimulating the cells with relaxatory and contractile compound. The SMC attached to and proliferated on and partly into the scaffolds. Smooth muscle cells were found throughout the BC scaffolds that were constructed using paraffin particles. Collagen production was detected microscopically after 2 weeks of culturing. There was no response to either relaxatory (papaverine) or contractile (adrenaline, noradrenaline, KCl or acetylcholine) stimulating compounds.