Facilitated vascularization and enhanced bone regeneration by manipulation hierarchical pore structure of scaffolds

Sufficient vascularization is quite important for preventing cell death and promoting host integration during the repair of the critical sized bone defects. Porous structure providing enough space for the ingrowth of vessels is an essential consideration during the scaffold's development. In this study, we designed and fabricated three kinds of porous structured scaffolds based on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), such as mono-structured PHBHHx scaffolds with macro pores (PH-1), di-structured PHBHHx scaffolds with macro-meso pores (PHS-2), and tri-structured PHBHHx scaffolds with macro-micro-meso pores (PHS-3), respectively. In vitro effects of the hierarchical porous scaffolds on human umbilical vein endothelial cells (HUVECs), such as cell attachment, glucose and lactate detection, relative gene expressions of endothelial markers were investigated. The PHS-3 scaffolds exhibited preferential potency of inducing better angiogenesis in vitro. Consequently, the hierarchical porous scaffolds were applied to load rhBMP-2 and repair the critical sized bone defect (15 mm) in rabbits. Microangiography analysis by three dimensional micro-computed tomographic (micro-CT) demonstrated that the volume of blood vessels within the defect area was higher in the rhBMP-2 loaded PHS-3 (PHS-3/rhBMP-2) than that in other rhBMP-2 loaded porous scaffolds with simplex or double scaled pores (PH-1/rhBMP-2 or PHS-2/rhBMP-2) at 4 weeks and 8 weeks, which implied that multi-level porous structure was conducive to nutrition transmission and revascularization. Further investigations of orthotopic bone formation by micro-CT, histological and immunohistochemistry analysis confirmed the most accelerated new bone formation rate in the PHS-3/rhBMP-2 group. The maximum load value of the regenerated bone induced by PHS-3/rhBMP-2 at 12 weeks was 258.47 ± 14.77 N which did not show significant difference from the normal bone of 268.81 ± 12.05 N. These results highlighted that introducing multi-level pores into the biocompatible scaffolds may be an effective approach to promote angiogenesis and bone regeneration.