Purpose: To optimize mineralization conditions for fabricating bone biomimetic HA-collagen scaffolds. These biomimetic scaffolds will be tested in later experiments for their osteoinductive potential in differentiating MSCs into more mature osteoblasts compared to non-biomimetic HA-collagen scaffolds. Methods: To create the scaffolds, fibrillar collagen self-assembly was induced by slowly raising the pH of dissolved collagen. The scaffolds were fixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). After fixation, scaffolds were biomimetically mineralized by exposure to a supersaturated, metastable solution of calcium and phosphorus stabilized with L poly-aspartic acid (pAsp). As controls, scaffolds mineralized in the same calcium and phosphorus solution without pAsp served as non-biomimetic HA-collagen scaffolds. Scaffolds were mineralized for 2, 4, 6, and 8 days for later experiments that will assess the effect that the extent of mineralization has on osteoblast differentiation. Scanning electron microscopy (SEM) assessed mineral morphology and location. In particular, backscattered electrons from SEM were used to detect heavier elements from mineral such as Ca and P in the scaffolds. Transmission electron microscopy (TEM) was used to determine the presence of HA and alignment of mineral with selected area electron diffraction (SAED). Results: Biomimetic scaffolds mineralized for longer amounts of time (6 and 8 days) exhibit excess, unaligned extrafibrillar mineral compared to those mineralized for 4 days (Fig. 1). Scaffolds mineralized 2 days exhibited no detectable mineralization with SEM and backscatter SEM (Fig. 1 insets).Fig. 2TEM demonstrated the presence of aligned, intrafibrillar mineral, as shown by the preferentially aligned 002 arcs from the c-axis reflection of HA in the direction of the collagen fibrils (Fig. 3; diffraction pattern inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Yellow circle denotes positioning of selected area aperture used to generate the diffraction pattern (inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusions: Mineralization for 4 days with pAsp is the optimal time point for “biomimetic”, intrafibrillarly mineralized collagen scaffolds for future studies regarding osteogenic differentiation. Ultimately, this bone tissue engineering project is expected to contribute to engineering bone-cartilage tissue interfaces in osteoarthritis.