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The effect of endostatin on angiogenesis and osteogenesis of steroid-induced osteonecrosis of the femoral head in a rabbit model. 内皮抑素对激素诱导的兔股骨头坏死模型血管生成和成骨的影响。
Pub Date : 2022-05-01 DOI: 10.5152/j.aott.2022.21248
Yan Zhao, Dong Li, Da-Peng Duan, Qi-Chun Song

Objective: This study aimed to investigate whether endostatin, a crucial anti-angiogenic factor, plays a negative role in angiogenesis and osteogenesis and aggravates the progression of osteonecrosis of the femoral head induced by steroid use in a rabbit model.

Methods: 66 New Zealand white rabbits were randomly divided into four groups: glucocorticoid model (GC) group (GC group, n = 18), glucocorticoid model and endostatin group (GC;ES group, n = 18), ES group (ES group, n = 18), and blank control group (CON group, n = 12). In the GC group, 10 μg/ kg lipopolysaccharide (LPS) was intravenously injected into the ear margin, and 24h after LPS injection, 20 mg/kg GC methylprednisolone (MPS) was injected into the gluteus muscle three times, each time at an interval of 24h. The animals of the GC;ES group were given as same treatment as the GC group, except for the addition of ES. MPS was not used in the ES group and CON group. ES group was only given ES, while the CON group was only given the same amount of normal saline. All animals successfully established models of femoral head necrosis, and then the difference among the Immunohistochemistry, Quantitative polymerase chain reaction (qPCR) analysis, Enzyme-linked immunosorbent assay, Biomechanical test, etracyclline-calcein double labeling, and Van Gieson staining indices were compared among the four groups.

Results: The combination of MPS and LPS was successful in establishing the femoral head necrosis model in New Zealand white rabbits. The incidence of osteonecrosis after MPS and LPS intervention was 70% (7/10), while that plus ES was 100% (10/10). At the same time, after MPS and LPS intervention, while the empty bone lacuna rate of the femoral head was significantly increased, the number of osteo- blasts was decreased. Also, the expressions of CD31 positive cells, Runx2, Osterix, COL1A1, and VEGF mRNA in the femoral head were decreased, and the levels of osteogenesis-related protein b-ALP, OCN, and angiogenic factor VEGF in the femoral head were decreased. The percentage of the trabecular bone area (%Tb.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N), labeled perimeter percent (%L.Pm), mineral apposition rate (MAR), and bone formation rate (BFR/BS) in the femoral head after MPs and LPS intervention detected by tetracycline calcein double labeling and Van Gieson staining decreased significantly, except trabecular separation (Tb.Sp) increased significantly. The compressive strength (CS), elastic modulus (EM), and strain energy (SE) of the femoral head examed by biomechanical measurement decreased significantly. All the above changes were more obvious after adding ES intervention. ES mRNA in the femoral head was undifferentiated and increased in the GC, ES, and GC;ES group compared with group CON.

Conclusion: This study has revealed that ES can inhibit angiogenesis and osteogenesis in the femoral hea

目的:本研究旨在探讨内皮抑素(一种重要的抗血管生成因子)是否在血管生成和成骨过程中发挥负面作用,并加重类固醇使用诱导的兔股骨头坏死的进展。方法:将66只新西兰大白鼠随机分为四组:糖皮质激素模型组(GC组,n=18)、糖皮质激素和内皮抑素组(GC;ES组,n=19)、ES组(ES组,n=18)和空白对照组(CON组,n=12)。GC组耳缘静脉注射脂多糖(LPS)10μg/kg,注射后24h,臀肌注射甲基强的松龙(MPS)20mg/kg,每次间隔24h。GC的动物;ES组给予与GC组相同的治疗,只是添加了ES。ES组和CON组不使用MPS。ES组只给予ES,而CON组只给予等量的生理盐水。所有动物均成功建立了股骨头坏死模型,并比较了四组动物的免疫组织化学、定量聚合酶链式反应(qPCR)分析、酶联免疫吸附试验、生物力学试验、四环素-钙黄绿素双标记和Van Gieson染色指标之间的差异。结果:MPS和LPS联合应用成功地建立了新西兰兔股骨头坏死模型。MPS和LPS干预后骨坏死的发生率为70%(7/10),而ES干预后的骨坏死发生率为100%(10/10)。同时,MPS和LPS干预后,股骨头空骨陷窝率显著增加,成骨细胞数量减少。股骨头中CD31阳性细胞、Runx2、Osterix、COL1A1和VEGF mRNA的表达降低,股骨头中成骨相关蛋白b-ALP、OCN和血管生成因子VEGF的水平降低。四环素-钙黄绿素双标记和Van Gieson染色检测的MP和LPS干预后股骨头小梁面积(%Tb.Ar)、小梁厚度(Tb.Th)、骨小梁数量(Tb.N)、标记周长百分比(%L.Pm)、矿物附着率(MAR)和骨形成率(BFR/BS)的百分比显著降低,除了小梁分离(Tb.Sp)显著增加之外。生物力学测量显示股骨头的抗压强度(CS)、弹性模量(EM)和应变能(SE)显著降低。上述变化在加入ES干预后更加明显。股骨头中的ES mRNA未分化,在GC、ES和GC中增加;结论:ES可抑制股骨头血管生成和成骨,加重股骨头坏死的发生和发展。因此,抗血管生成因子可能在ONFH的发病机制中发挥重要作用。
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Acta orthopaedica et traumatologica turcica
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