A three-dimensional (3D) printed biomimetic hierarchical scaffold with a covalent modular release system for osteogenesis.

Hydroxyapatite (HA) ceramics are well known for their biocompatibility, bioactivity, and osteoconductive nature. However, limited hierarchical structure and lack of ease in modularity hinder the widespread application of conventional HA ceramics. By using three-dimensional printing (3DP) techniques with multiple materials, including HA, complex biological and mechanical architecture of natural organisms can be achieved through biomimetics. In this study, we designed an osteoid, biomimetic, hierarchical, porous HA ceramic 3D printed scaffold (3DPs). Further incorporation of a covalent, modular, controlled release system (CMR), based on Watson-Crick's complementary oligonucleotides, and was added to carry a bone morphogenetic protein-2 (BMP2) peptide. The choice of a HA biomimetic scaffold housing BMP2 protein fragments was selected to successfully promote osteogenesis both in vitro and in vivo. Scanning electron microscopy, micro-computed tomography analysis and computer fluid dynamics simulations of the 3DPs showed a uniform biomimetic hierarchical structure and an effective interior permeability. Active molecules were found bound with high stability and modular to the scaffold surface via the CMR system. After 7 days of incubation under physiological conditions, approximately 90% of active factors remained bound. Compared to control groups, the 3DPs-CMR-BMP2 group significantly enhanced cell proliferation and adhesion. Moreover, the 3DPs-CMR-BMP2 group exhibited more extensive and sustained osteogenic effects through upregulated expression of osteogenic factors and enhanced calcium deposition, as compared to study and control groups. Furthermore, ectopic osteogenesis and a critical calvarial defect model confirmed that the 3DPs-CMR-BMP2 group significantly promoted in vivo bone healing versus control. Thus, our results showed that biomimetic hierarchical 3DPs with a CMR system successfully promote cell proliferation, adhesion, differentiation and osteogenesis, on a continuous cycle. The biomimetic hierarchical 3DPs with a CMR system offers a promising multi-functional, bone substitute material for treatment of patients with bone defects.