Abstract The ultimate goal of bone tissue engineering is to develop bony tissues on tissue engineered constructs that mimic the native bone. Nanoscale characterization of in vitro generated bony tissues on engineered scaffolds is essential to understanding both the physical and mechanical characteristics of the engineered bone. Bone nodule formation, a typical early indicator of bone formation was observed on chitosan–polygalacturonic acid–hydroxyapatite (Chi–PgA–HAP) nanocomposite films without the use of differentiating media. Thus, the Chi–PgA–HAP substrates designed are osteoinductive and provide an appropriate microenvironment for cell organization and tissue regeneration. Imaging using atomic force microscopy revealed several levels of hierarchical structures of bone in the bone nodules, consisting of mineralized collagen fibers, fibrils and mineral deposits in extrafibrillar spaces. The nanoscale elastic properties of the collagen and mineral crystals were found to be in close agreement with the experimental and simulations results on natural bone reported in the literature. Fourier transform infrared spectroscopy experiments indicate the presence of collagen and biological apatite in bone nodules exhibiting the characteristics of newly precipitated, immature bone. Collectively, our structural, chemical, and mechanical analyses support the conclusion that synthetic bone nodules mimic the hierarchy of natural bone.