3D printing provides a novel approach to repair bone defects using customized biomimetic tissue scaffolds. To make a bone substitute closest to natural bone structure and composition, two different types of hydroxyapatite, Nano hydroxyapatite (nHA) and deproteinized bovine bone (DBB), were dispersed into collagen (CoL) to prepare the bio-ink for 3D printing. In doing so, a porous architecture was manufactured with 3D printing technology. The physical and chemical properties of the materials were evaluated, including biocompatibility and effect on the osteogenic differentiation of the human bone marrow-derived mesenchyme stem cells (hBMSCs). The XPS, XRD, FTIR, and the mechanical analysis of the material indicated that the two HA were consistent in their elements, but different in their chemical bonds and crystal phases. The SEM results showed the different surface morphologies of the HA crystals as well as the scaffolds, which would be the main factors affecting the internal porous structure of the scaffold. There were no differences between the two composite scaffolds in cell proliferations. FITC-phalloidine/vinculin /DAPI staining indicated hBMSCs can adhere well to the 3D printed surfaces. Alkaline phosphatase (ALP) staining reflected ALP expressed on both of the osteogenic-induction medium (OM) group but not on proliferation medium (PM) group. The RT-PCR results showed the expression levels of osteogenesis-related genes RUNX2, SOX9, OCN and CoL1A1 in OM group were significantly increased after 7 days compared with the PM group (P＜0.01). The expression of SOX9, OCN and CoL1A1 in nHA/CoL scaffolds were higher than that in CoL scaffolds (p<0.05). The expression of OCN and CoLIA1 in DBB/CoL scaffolds were higher than that in CoL scaffolds (p<0.05). In conclusion, the physicochemical and biological properties of 3D bio-printed scaffolds consisting of nHA/CoL or DBB/CoL would be well suited for the scaffolds to being a porous customized bone substitute, 3D printing scaffolds would be a prospective candidate for clinical application in future.