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The purpose of this study is to investigate the potential of tissue engineered heat-treated porcine trabecular bones as a bone substitute through three dimensional culture system with bone marrow stromal cells (BMSCs). Structural characteristics, mechanical stiffness and chemical composition were investigated. BMSCs were dynamically seeded into the block and cultured by either static or dynamic culturing condition. The cell-seeded bone blocks were examined histologically starting from 3 days up to 21 days after culture. The effects of seeding and culturing conditions on the tissue generation were examined. The effect of the collagen coating on the cell adhesion at initial stage was also investigated. The heat-treated porcine bone block was found to be hydroxyapatite (HA)-based material. It had high porosity (pore size: 300-500 m) and had similar strength to the human trabecular bones (Young’s modulus=346.33 MPa). Observation by scanning electron microscopy (SEM), H&E staining and transmission electron microscopy (TEM) revealed bony tissue regeneration such as the cell adhesion, the formation of osteoids, and generation of lamellar bone and woven bone. The result showed that the dynamic culture was superior to static culture. Although there was no significant effect of the collagen coating on the cell activities, the attachment was improved in collagen coated bone blocks. Based on all the results obtained from this study, we could conclude this heat-treated porcine trabecular bone had the potential of the xenograft for bone regeneration.