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The search for alternative therapies to improve bone regeneration continues to be a major challenge for the medical community. Here we report on the enhanced mineralization, osteogenesis, and in vivo bone regeneration properties of a bioactive elastin-like recombinamer (ELR) membrane. Three bioactive ELRs exhibiting epitopes designed to promote mesenchymal stem cell adhesion (RGDS), mineralization (DDDEEKFLRRIGRFG), and both cell adhesion and mineralization were synthesized using standard recombinant protein techniques. The ELR materials were then used to fabricate membranes comprising either a smooth surface (Smooth) or channel microtopographies (Channels). Mineralization and osteoblastic differentiation of primary rat mesenchymal stem cells (rMSCs) were analyzed in both static and dynamic (uniaxial strain of 8% at 1 Hz frequency) conditions. Smooth mineralization membranes in static condition exhibited the highest quantity of calcium phosphate (Ca/P of 1.78) deposition with and without the presence of cells, the highest Young's modulus, and the highest production of alkaline phosphatase on day 10 in the presence of cells growing in non-osteogenic differentiation medium. These membranes were tested in a 5 mm-diameter critical-size rat calvarial defect model and analyzed for bone formation on day 36 after implantation. Animals treated with the mineralization membranes exhibited the highest bone volume within the defect as measured by micro-computed tomography and histology with no significant increase in inflammation. This study demonstrates the possibility of using bioactive ELR membranes for bone regeneration applications. [ABSTRACT FROM AUTHOR]