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The purpose of this study was to prepare a 3D nanocomposite bone substitute prepared from gelatin (GEL) and synthesized hydroxyapatite (HAP) in order to mimic natural bone feathers through a novel layer solvent casting combined freeze-drying technique. The adhesion between superimposed layers was ensured by applying a GEL solution. Furthermore, samples were cross-linked with 1% glutaraldehyde (GA). Many investigators have studied the biocompatibility of GEL/HAP composite; however, little attention has been paid to improve mechanical properties of such systems and to determine influence of fabrication parameters systematically. Therefore, in this work, we decided to emphasize on pore size and morphology-compressive mechanical properties relationships and in vitro cytotoxicity by changing GEL/HAP content. The chemical bonding and the microstructure of HAP nanopowder and the nanocomposites features i.e. pore size and morphology were investigated by FT-IR, XRD, TEM, optical microscope and SEM, respectively. A comparative mechanical study in compression has been performed between porous GEL/HAP nanocomposite and natural bone. Results showed that with the technique used, it was possible to synthesize HAP nanopowder with grain size of less than 10 nm. The scaffolds prepared had an open, interconnected porous structure with pore sizes ranging from 100 nm to 1 mum and densities from 75 to 93%. It was also found that increasing initial GEL concentration and HAP content enhances the elastic modulus (E) and affects pore size and morphology. The stress-strain behavior in compression was very similar to natural spongy bone where the compressive modulus obtained was about 180 MPa. With applying a mathematic model, predictions of porosity and E values has been made for higher percentage of HA in view of preparing samples with optimized resistance. In addition, the biological response of scaffolds was evaluated by L929 fibroblast cells for cytotoxicity evaluation.