Defective Bone Repair in C57Bl6 Mice With Acute Systemic Inflammation
Defective Bone Repair in C57Bl6 Mice With Acute Systemic Inflammation
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DOI：
10.1007/s11999-016-5159-7
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发表时间：
2017-03-01
影响因子：
4.2
通讯作者：
Martineau, P. A.
中科院分区：
文献类型：
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作者：
Behrends, D. A.;Hui, D.;Martineau, P. A.
骨修复是由对损伤的局部炎症反应开始的。全身炎症的存在会损害骨愈合并经常导致畸形愈合，尽管其潜在机制仍不清楚。我们的研究目的是利用小鼠皮质骨修复模型来确定全身炎症对骨愈合微环境中细胞的影响。（1）脂多糖（LPS）诱导的全身炎症是否会影响初次骨愈合中再生骨的数量和质量？ (2)全身炎症是否会改变骨愈合微环境中的血管化以及炎症细胞、成骨细胞和破骨细胞的数量或活性？在5至9个月大的雌性和雄性C57BL/6小鼠的股骨骨干中钻孔皮质缺损，每天全身注射LPS或生理盐水作为对照，连续7天。小鼠在术后 1 周（对照，n = 7；LPS，n = 8）、2 周（对照，n = 7；LPS，n = 8）和 6 周（对照，n = 9；LPS，n = 8）处死。使用 microCT 对缺损处骨的数量（每组织体积的骨体积 [BV/TV]）和微结构（小梁分离和厚度、孔隙率）随时间进行量化。使用组织化学分析评估血管内皮细胞 (CD34)、巨噬细胞 (F4/80)、成骨细胞（碱性磷酸酶 [ALP]）和破骨细胞（抗酒石酸酸性磷酸酶 [TRAP]）的存在或活性。手术后 6 周，注射 LPS 的小鼠骨骼中只有八个缺陷之一完全桥接，而对照小鼠骨骼中的九个缺陷中有七个（比值比） [OR]，0.04；95% CI，0.003-0.560；p = 0.007）。 LPS治疗小鼠皮质骨的减少反映在BV/TV降低（21% +/- 4% vs 39% +/- 10%；p < 0.01）、小梁分离增加（240 +/- 36 μm vs 171 +/- 29 μm；p < 0.01）、小梁厚度减少（81 +/- 18 μm vs 110 +/- 22 μm） m；p = 0.02），以及术后 6 周的孔隙率（79% +/- 4% vs 60% +/- 10%；p < 0.01）。愈合缺陷伴随着 CD34（1.1 +/- 0.6 vs 3.4 +/- 0.9；p < 0.01）、ALP（1.9 +/- 0.9 vs 6.1 +/- 3.2；p = 0.03）和 TRAP（3.3 +/- 4.7 vs 7.2 +/- 4.0；p = 0.01）活性下降以及 F4/80 增加(13 +/- 2.6 vs 6.8 +/- 1.7; p < 0.01) 术后 2 周的活动。结果表明，LPS 诱导的全身炎症减少了小鼠股骨再生的数量并损害了骨再生的质量。这些影响与血运重建受损、成骨细胞和破骨细胞骨转换减少以及巨噬细胞分解代谢活性增加有关。这项临床前研究的结果支持全身炎症患者初次骨愈合受损的临床观察结果。根据我们的数据，在愈伤组织中局部施用 VEGF 以刺激血运重建，或移植干细胞以增强骨转换，是改善临床实践结果的潜在可行方法。
Bone repair is initiated with a local inflammatory response to injury. The presence of systemic inflammation impairs bone healing and often leads to malunion, although the underlying mechanisms remain poorly defined. Our research objective was to use a mouse model of cortical bone repair to determine the effect of systemic inflammation on cells in the bone healing microenvironment.(1) Does systemic inflammation, induced by lipopolysaccharide (LPS) administration affect the quantity and quality of regenerating bone in primary bone healing? (2) Does systemic inflammation alter vascularization and the number or activity of inflammatory cells, osteoblasts, and osteoclasts in the bone healing microenvironment?Cortical defects were drilled in the femoral diaphysis of female and male C57BL/6 mice aged 5 to 9 months that were treated with daily systemic injections of LPS or physiologic saline as control for 7 days. Mice were euthanized at 1 week (Control, n = 7; LPS, n = 8), 2 weeks (Control, n = 7; LPS, n = 8), and 6 weeks (Control, n = 9; LPS, n = 8) after surgery. The quantity (bone volume per tissue volume [BV/TV]) and microarchitecture (trabecular separation and thickness, porosity) of bone in the defect were quantified with time using microCT. The presence or activity of vascular endothelial cells (CD34), macrophages (F4/80), osteoblasts (alkaline phosphatase [ALP]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP]) were evaluated using histochemical analyses.Only one of eight defects was bridged completely 6 weeks after surgery in LPS-injected mouse bones compared with seven of nine defects in the control mouse bones (odds ratio [OR], 0.04; 95% CI, 0.003-0.560; p = 0.007). The decrease in cortical bone in LPS-treated mice was reflected in reduced BV/TV (21% +/- 4% vs 39% +/- 10%; p < 0.01), increased trabecular separation (240 +/- 36 mu m vs 171 +/- 29 mu m; p < 0.01), decreased trabecular thickness (81 +/- 18 mu m vs 110 +/- 22 mu m; p = 0.02), and porosity (79% +/- 4% vs 60% +/- 10%; p < 0.01) at 6 weeks postoperative. Defective healing was accompanied by decreased CD34 (1.1 +/- 0.6 vs 3.4 +/- 0.9; p < 0.01), ALP (1.9 +/- 0.9 vs 6.1 +/- 3.2; p = 0.03), and TRAP (3.3 +/- 4.7 vs 7.2 +/- 4.0; p = 0.01) activity, and increased F4/80 (13 +/- 2.6 vs 6.8 +/- 1.7; p < 0.01) activity at 2 weeks postoperative.The results indicate that LPS-induced systemic inflammation reduced the amount and impaired the quality of bone regenerated in mouse femurs. The effects were associated with impaired revascularization, decreased bone turnover by osteoblasts and osteoclasts, and by increased catabolic activity by macrophages.Results from this preclinical study support clinical observations of impaired primary bone healing in patients with systemic inflammation. Based on our data, local administration of VEGF in the callus to stimulate revascularization, or transplantation of stem cells to enhance bone turnover represent potentially feasible approaches to improve outcomes in clinical practice.