Lee Dong Yeon, Cho Tae-Joon, Kim Jin A, Lee Hye Ran, Yoo Won Joon, Chung Chin Youb, Choi In Ho
Department of Orthopaedic Surgery, Seoul National University College of Medicine, South Korea.
Bone. 2008 May;42(5):932-41. doi: 10.1016/j.bone.2008.01.007. Epub 2008 Jan 26.
Fracture healing and distraction osteogenesis (DO) are unique postnatal bone formation processes, and neovascularization is critically required for successful bone regeneration. We investigated endothelial progenitor cell (EPC) mobilization during bone regeneration, and the possible contribution of EPCs to increased vascularization and new bone formation, especially in DO.
Mouse tibia fracture and rat tibia DO models were used in this study. The proportion of EPCs among the peripheral and splenic mononuclear cells (MNCs) was determined by examining the endothelial lineage staining characteristics and EPC cell surface markers. Messenger RNA expression of molecules related to EPC mobilization and homing at the fracture site were analyzed by ribonuclease protection assay and reverse-transcription polymerase chain reaction. In the rat tibia DO model, we measured blood flow during DO, and determined the distribution of ex vivo-expanded and intravenously-infused EPCs.
The proportion of EPCs among the peripheral and splenic MNCs increased after fracture, peaked on post-fracture day 3, and returned to basal levels during the healing period. Messenger RNA expression of EPC mobilizing cytokines such as vascular endothelial growth factor (VEGF), stem cell factor, monocyte chemoattractant protein-1, and stromal cell-derived factor-1, were upregulated at the fracture callus. The plasma VEGF levels peaked prior to the increase in the EPC proportion. Adhesion molecules involved in EPC homing were expressed at the fracture callus. In the DO model, the temporal pattern of the increase in the EPC proportion was similar to that in the fracture healing model, but the EPC proportion increased again during the distraction and consolidation phases. The distraction gap was relatively ischemic during the distraction phase and blood flow increased profusely later in the consolidation phase. The number of EPCs homing to the bone regeneration site in the DO model correlated with the number of transplanted EPCs in a dose-dependent manner.
These findings suggest that signals from the bone regeneration site mobilize EPCs from the bone marrow into the peripheral circulation. Increased EPC mobilization and homing may contribute to neovascularization and thus to new bone formation in fracture healing and DO.
骨折愈合和牵张成骨（DO）是出生后独特的骨形成过程，而新生血管形成是骨再生成功的关键要求。我们研究了骨再生过程中内皮祖细胞（EPC）的动员情况，以及EPC对增加血管化和新骨形成的可能贡献，尤其是在牵张成骨中。
本研究使用小鼠胫骨骨折和大鼠胫骨牵张成骨模型。通过检查内皮谱系染色特征和EPC细胞表面标志物来确定外周血和脾单核细胞（MNC）中EPC的比例。通过核糖核酸酶保护分析和逆转录聚合酶链反应分析骨折部位与EPC动员和归巢相关分子的信使核糖核酸表达。在大鼠胫骨牵张成骨模型中，我们测量了牵张成骨过程中的血流量，并确定了体外扩增并静脉注射的EPC的分布情况。
骨折后外周血和脾MNC中EPC的比例增加，在骨折后第3天达到峰值，并在愈合期恢复到基础水平。骨折痂处EPC动员细胞因子如血管内皮生长因子（VEGF）、干细胞因子、单核细胞趋化蛋白-1和基质细胞衍生因子-1的信使核糖核酸表达上调。血浆VEGF水平在EPC比例增加之前达到峰值。参与EPC归巢的黏附分子在骨折痂处表达。在牵张成骨模型中，EPC比例增加的时间模式与骨折愈合模型相似，但在牵张期和巩固期EPC比例再次增加。牵张期牵张间隙相对缺血，而在巩固期后期血流量大量增加。在牵张成骨模型中归巢到骨再生部位的EPC数量与移植的EPC数量呈剂量依赖性相关。
这些发现表明，来自骨再生部位的信号将EPC从骨髓动员到外周循环中。EPC动员和归巢的增加可能有助于新生血管形成，从而促进骨折愈合和牵张成骨中的新骨形成。
