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.

足够的血管形成对于防止细胞死亡和促进修复关键大小的骨缺损期间的宿主整合非常重要。为支架的向内生长提供足够的空间的多孔结构是脚手架开发过程中的重要考虑因素。在这项研究中，我们设计并制造3种基于聚（3-羟基丁酸酯多孔结构支架的共-3-羟基己酸酯）（PHBHHx），诸如与大孔（PH-1）的单结构PHBHHx支架，二具有大介孔的结构化PHBHHx支架（PHS-2）和具有大介孔的三结构化PHBHHx支架（PHS-3）。体外研究了分级多孔支架对人脐静脉内皮细胞（HUVEC）的影响，如细胞附着，葡萄糖和乳酸的检测，以及内皮标记的相关基因表达。PHS-3支架表现出优先诱导体外更好的血管生成的能力。因此，分层多孔支架被应用于加载rhBMP-2并修复兔子的临界大小骨缺损（15毫米）。通过三维显微计算机断层扫描（micro-CT）进行的微血管造影分析表明，缺陷区域内的血管体积在装有rhBMP-2的PHS-3（PHS-3 / rhBMP-2）中高于在其他rhBMP中-4负载多孔支架在第4周和第8周具有单孔或双比例孔（PH-1 / rhBMP-2或PHS-2 / rhBMP-2），这表明多级多孔结构有利于营养传递和血运重建。通过微CT，组织学和免疫组织化学分析对原位骨形成的进一步研究证实，PHS-3 / rhBMP-2组中新骨形成速度最快速。