Enhanced 3D-printed bredigite scaffolds with osteoconductivity and angiogensis for bone tissue engineering

Abstract

Critical bone defects resulting from congenital anomalies, severer trauma or osteosarcoma resection do not heal spontaneously over the natural lifetime. The ideal bone substitutes for treating bone defects should meet certain requirements regarding mechanical properties, osteoconductivity, angiogenesis, and osteogenesis. Although many studies have developed various scaffolds as bone substitutes, most of them were focused on partial functions, leaving the multi-functional scaffolds as well as the synergistic actions within them to be developed. In this study, bredigite (Ca7Mg(SiO4)4), a biocompatible material with high mechanical strength, was 3D printed with customized structures, which fully satisfy the ingrowth demands of vessels and bones. Further, to enhance the osteoconductivity of the scaffold, DOPA-Nylon3, a new type of functional polymer with similar effects as extracellular matrix peptides, was coated on the scaffold surfaces using an impregnation method. Moreover, because vascularization plays a crucial role in the bone regeneration, deferoxamine (DFO), a well-accepted angiogenic compound, was modified covalently by conjugation with iminodiacetic acid to impart the calcium affinity, thereby being able to chelate on the calciferous bredigite scaffolds. Ultimately, the 3D-printed bredigite scaffolds were enhanced by the osteoconductive DOPA-Nylon3 and angiogenic SF-DFO to become multi-functional scaffolds for bone defects. The individual characteristics and functions of DOPA-Nylon3 and SF-DFO were first evaluated. The results indicated that DOPA-Nylon3 not only promoted cell adhesive behavior in vitro but also improved the cell survival rate in vivo. Calcium affinity assays indicated that SF-DFO exhibited a calcium-seeking property due to the calcium-chelation effect of iminodiacetic acid, and was demonstrated to promote the expression of angiogenesis-related genes and drive the vascularization of HUVECs. Examinations of the scaffold coatings that included contact angle measurements and SEM-EDS analysis revealed that both DOPA-Nylon3 and SF-DFO were successfully grafted. Subsequently, comprehensive assessments were conducted to study the mechanical and biological functions of the double-coated scaffolds in vitro. According to the mechanical analysis, the novel structure of the scaffold exhibited much better performance than the most commonly used cross-structured scaffold. In addition, the robust growth of cells and their well-spread morphology demonstrated that the scaffold satisfied the requirements of cytocompatibility and bioactivity. Furthermore, improved angiogenesis and osteogenesis activities were also demonstrated through vessel formation assays, ALP activities, and the expression of osteogenesis-related genes. For the in vivo evaluation, a novel fixation system, comprising six self-tapping titanium screws and a bridge-like PEEK plate, was developed to treat the critical-sized defect in the rat. The system underwent detailed mechanical analysis for the optimized parameters to avoid any unexpected failure in vivo. The radiographic and histomorphology assessments of the excised scaffolds revealed that, compared with single-coated scaffolds, the double-coated scaffolds exhibited wider vascular networks (angiogenesis) and more extensive bone formation (osteogenesis). In conclusion, due to the customized microstructure, the 3D-printed bredigite scaffold reached a balance between porosity and mechanical properties. When coated with DOPA-Nylon-3 and SF-DFO, the scaffolds exhibited excellent osteoconductivity and angiogenesis in vitro and satisfying osteogenesis in the CSD rat model, therefore, as a qualified tissue engineering bone scaffold, it has a promising prospect in clinical practice.