Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul, Korea.
Bone. 2010 Mar;46(3):673-9. doi: 10.1016/j.bone.2009.10.018. Epub 2009 Oct 22.
Distraction osteogenesis (DO) is a unique postnatal bone formation process, which is characterized by a profuse increase in vascularization. Recently endothelial progenitor cells (EPCs) have been reported to circulate in substantial numbers under physiologic conditions and to contribute to bone regeneration. The authors investigated EPC mobilization in patients undergoing limb lengthening.
Thirteen of 24 consecutive patients who underwent limb-lengthening procedures on weight-bearing long bones (femur and tibia) were included in this study. Peripheral blood samples were taken at four different time points from each patient, that is, before operation (pre-Op), 2 or 3 days after osteotomy (early-PO), before the start of distraction (pre-Dist), and at 7 to 14 days after the start of distraction (during-Dist). Numbers of leukocytes and levels of plasma C-reactive protein (CRP) were determined. After isolating mononuclear cells (MNCs) by centrifugation, we performed FACS analysis on freshly isolated MNCs using antibodies to the cell surface markers; CD34, CD133, vascular endothelial growth factor receptor 2 (VEGFR2), and alkaline phosphatase. MNCs were also cultured in endothelial cell growth medium and numbers of EPC colony-forming units were counted. Plasma levels of EPC-mobilizing cytokines, such as, VEGF, SDF-1, and MCP-1, were determined by ELISA.
Numbers of leukocytes and CRP plasma levels increased significantly during the early-PO period (p<0.01) but were maintained within normal range in the during-Dist period. FACS analysis of freshly isolated MNCs showed that EPC-enriched cell fractions increased after distraction, but that alkaline phosphatase-positive cell numbers were unchanged. Numbers of EPC colony-forming units significantly increased in the during-Dist period (p<0.01). Plasma levels of VEGF and SDF-1 significantly increased in the during-Dist period (p<0.05). In femoral lengthening patients whose healing index was less than 30 days/cm, the number of CFUs was 46.8 in the during-Dist period, whereas it was 12.7 in patients whose healing index was more than 30 days/cm (p=0.088).
This study demonstrates a mobilization of EPC population during distraction osteogenesis in human limb-lengthening patients. Distraction strain provoked increases in the plasma levels of EPC-mobilizing cytokines, such as, VEGF and SDF-1. These findings suggest a possibility that therapeutical approaches which modulate EPC mobilization may speed bone healing by angiogenesis-osteogenesis coupling during distraction osteogenesis.
骨牵引成骨术（DO）是一种独特的后天骨形成过程，其特征是血管生成大量增加。最近，内皮祖细胞（EPC）在生理条件下大量循环，并有助于骨再生。作者研究了在接受肢体延长术的患者中 EPC 的动员情况。
本研究纳入了 24 例连续接受承重长骨（股骨和胫骨）肢体延长术的患者中的 13 例。每位患者在四个不同时间点采集外周血样本，即手术前（术前）、截骨后 2 或 3 天（早期 PO）、开始牵引前（术前 Dist）和开始牵引后 7 至 14 天（期间 Dist）。测定白细胞数量和血浆 C 反应蛋白（CRP）水平。通过离心分离单核细胞（MNC）后，我们使用针对细胞表面标志物的抗体对新鲜分离的 MNC 进行流式细胞术分析；CD34、CD133、血管内皮生长因子受体 2（VEGFR2）和碱性磷酸酶。MNC 也在内皮细胞生长培养基中培养，并计数 EPC 集落形成单位的数量。通过 ELISA 测定 EPC 动员细胞因子（如 VEGF、SDF-1 和 MCP-1）的血浆水平。
白细胞数量和 CRP 血浆水平在早期 PO 期间显著升高（p<0.01），但在期间 Dist 期间保持在正常范围内。对新鲜分离的 MNC 进行的 FACS 分析表明，牵引后 EPC 富集细胞分数增加，但碱性磷酸酶阳性细胞数量不变。期间 Dist 期间 EPC 集落形成单位数量显著增加（p<0.01）。期间 Dist 期间 VEGF 和 SDF-1 的血浆水平显著增加（p<0.05）。在愈合指数小于 30 天/cm 的股骨延长患者中，期间 Dist 期 CFU 数为 46.8，而愈合指数大于 30 天/cm 的患者为 12.7（p=0.088）。
本研究在人类肢体延长患者的骨牵引成骨术中证明了 EPC 群体的动员。牵伸应变引起 EPC 动员细胞因子（如 VEGF 和 SDF-1）的血浆水平升高。这些发现表明，通过调节 EPC 动员的治疗方法可能通过牵张成骨过程中的血管生成-成骨耦联加速骨愈合。
