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The objective of this study was to enhance ectopic bone formation in a three-dimensional (3-D) hybrid scaffold in combination with bioreactor perfusion culture system. The hybrid scaffold consists of two biomaterials, a hydrogel formed through self-assembly of peptide–amphiphile (PA) with cell suspensions in media, and a collagen sponge reinforced with poly(glycolic acid) (PGA) fiber incorporation. PA was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH₂ terminus of the peptide. A 3-D network of nanofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. Osteogenic differentiation of mesenchymal stem cells (MSC) in the hybrid scaffold was greatly influenced by the perfusion culture …