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Artificial bone materials are in great need due to a lot of bone injuries. Herein, collagen/nano-hydroxyapatite (Col/nHA, C-H) composite nanospheres were obtained by in-situ mineralization, and poly L-lactic acid/collagen/nano-hydroxyapatite (PLLA/Col/nHA, P-C-H) was further prepared by high-speed shear emulsification method. The interfacial properties and structure between PLLA and nHA are regulated by the adhesive property of Col. The morphology, structure and properties of P-C-H microsphere were characterized in detail by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brunauer-Emmett-Teller (BET) and simulated degradation of PBS in vitro. The results show that C-H is uniformly distributed in P-C-H microspheres, and a mesoporous material with a "pomegranate" structure and a particle size of 5–30 μm is self-assembled based on C-H multiple composite microspheres. It is beneficial to the sustained-release degradation of P-C-H and the retention/release of Ca2+. The 60-day PBS degradation shows that PLLA delays the degradation of nHA, making the degradation rate of P-C-H basically consist with the human bone healing cycle. The co-culture of P-C-H with MC3T3-E1 cells shows that P-C-H has high biocompatibility and no cytotoxicity. The cell viability is higher than 100 % in 72 h, indicating P-C-H has a proliferation effect on cell growth. Alkaline phosphatase and quantitative real-time PCR test show a positive promotion of P-C-H in cell proliferation and differentiation. The multi-layered P-C-H microspheres have an application potential in bone tissue engineering. [ABSTRACT FROM AUTHOR]