Abnormal Mechanical Loading Induces Cartilage Degeneration by Accelerating Meniscus Hypertrophy and Mineralization After ACL Injuries In Vivo.
Abnormal Mechanical Loading Induces Cartilage Degeneration by Accelerating Meniscus Hypertrophy and Mineralization After ACL Injuries In Vivo.
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体内 ACL 损伤后，异常机械负荷通过加速半月板肥大和矿化而诱导软骨退化
DOI：
10.1177/0363546515621285
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发表时间：
2016-03
期刊：
影响因子：
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通讯作者：
Wei L
中科院分区：
文献类型：
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作者：
Du G;Zhan H;Ding D;Wang S;Wei X;Wei F;Zhang J;Bilgen B;Reginato AM;Fleming BC;Deng J;Wei L
背景：尽管前交叉韧带(ACL)损伤患者发生创伤后骨关节炎(PTOA)的风险很高，但半月板肥大和矿化在前交叉韧带损伤后PTOA中的作用尚不清楚。目的/假设：本研究的目的是确定半月板是否对异常负荷有反应，以及前交叉韧带损伤是否导致半月板肥大和钙化。假说：(1)前交叉韧带损伤后异常的机械负荷导致半月板肥大和矿化，这与体内关节软骨的损伤有关；(2)在体外，异常的机械负荷导致牛半月板组织肥大和矿化标志物的过度表达。研究设计：对照实验室研究。方法：在体豚鼠实验(假设1)：3月龄雄性Hartley豚鼠(n=9)行右膝前交叉韧带横断术(ACLT)，左膝作为对照。术后5.5个月取膝关节，在取膝关节前1天和5天用钙素标记法检测半月板钙化情况。软骨和半月板的损伤和矿化分别用骨关节炎研究会国际评分和半月板分级来量化。用免疫组织化学法和酶联免疫吸附试验检测大鼠血清中印度刺激素(IHH)、基质金属蛋白酶-13(MMP13)、X型胶原(Col X)、进行性强直同系物(ANKH)、胞外核苷酸焦磷酸酶/磷酸二酯酶-1(ENPP1)、碱性磷酸酶(ALP)、无机焦磷酸盐(PPI)和无机磷酸盐(PI)的含量。体外牛半月板外植体研究(假设2)：将牛半月板外植体置于25%的0.3赫兹应变中1、2、3小时。活/死染色检测细胞存活率。用实时定量逆转录聚合酶链式反应和Western印迹法检测细胞培养24、48、72小时后的mRNA表达水平和蛋白水平。收集条件培养液，用于硫酸氨基葡聚糖(GAG)的释放和PI/PPI测定。结果：在体豚鼠实验中，ACLT组半月板大小、面积及钙化强度均较对照组明显增大。半月板钙化面积：r=0.925，P<0.0001；半月板钙化强度：r=0.944，P<0.0001。ACLT组IHH、MMP13、Col X、AnkH、ENPP1和ALP的表达均高于对照组。与对照组相比，ACLT组PI水平和PI/PPI比值分别升高63%和42%。牛半月板组织块体外培养研究：加载3h后，牛半月板组织块表层可见细胞死亡。3小时负荷后，基质金属蛋白酶-13、血管紧张素转换酶、ENPP1和碱性磷酸酶的mRNA表达和蛋白水平均上调。3h负荷组培养上清液中PI/PPI比值和硫酸氨基半乳糖含量均增加。结论：半月板肥大和矿化与前交叉韧带损伤后软骨退变有关。临床意义：研究数据表明，抑制半月板肥大和钙化可能会降低前交叉韧带损伤后PTOA的风险。
Background: Although patients with an anterior cruciate ligament (ACL) injury have a high risk of developing posttraumatic osteoarthritis (PTOA), the role of meniscus hypertrophy and mineralization in PTOA after an ACL injury remains unknown. Purpose/Hypothesis: The purpose of this study was to determine if menisci respond to abnormal loading and if an ACL injury results in meniscus hypertrophy and calcification. The hypotheses were that (1) abnormal mechanical loading after an ACL injury induces meniscus hypertrophy and mineralization, which correlates to articular cartilage damage in vivo, and (2) abnormal mechanical loading on bovine meniscus explants induces the overexpression of hypertrophic and mineralization markers in vitro. Study Design: Controlled laboratory study. Methods: In vivo guinea pig study (hypothesis 1): Three-month-old male Hartley guinea pigs (n = 9) underwent ACL transection (ACLT) on the right knee; the left knee served as the control. Calcification in the menisci was evaluated by calcein labeling 1 and 5 days before knee harvesting at 5.5 months. Cartilage and meniscus damage and mineralization were quantified by the Osteoarthritis Research Society International score and meniscus grade, respectively. Indian hedgehog (Ihh), matrix metalloproteinase–13 (MMP-13), collagen type X (Col X), progressive ankylosis homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase–1 (ENPP1), alkaline phosphatase (ALP), inorganic pyrophosphate (PPi), and inorganic phosphate (Pi) concentrations were evaluated by immunohistochemistry and enzyme-linked immunosorbent assay. In vitro bovine meniscus explant study (hypothesis 2): Bovine meniscus explants were subjected to 25% strain at 0.3 Hz for 1, 2, and 3 hours. Cell viability was determined using live/dead staining. The levels of mRNA expression and protein levels were measured using real-time quantitative reverse transcription polymerase chain reaction and Western blot after 24, 48, and 72 hours in culture. The conditioned medium was collected for sulfated glycosaminoglycan (GAG) release and Pi/PPi assay. Results: In vivo guinea pig study: Meniscus size and area as well as intensity of meniscus calcification were significantly increased in the ACLT group compared with the control group. Both calcified area and intensity were correlated with cartilage damage in the ACLT group (meniscus calcified area: r = 0.925, P < .0001; meniscus calcified intensity: r = 0.944, P < .0001). Ihh, MMP-13, Col X, ANKH, ENPP1, and ALP expression were increased in the ACLT group compared with the control group. The Pi level and Pi/PPi ratio increased by 63% and 42%, respectively, in the ACLT group compared with the control group. In vitro bovine meniscus explant study: Cell death was found in the superficial zone of the bovine meniscus explants after loading for 3 hours. The mRNA expression and protein levels of MMP-13, ANKH, ENPP1, and ALP were up-regulated in all 3-hour loaded samples. The Pi/PPi ratio and sulfated GAG content in the culture medium were increased in the 3-hour loaded group. Conclusion: Meniscus hypertrophy and mineralization correlated to cartilage degeneration after ACL injuries. Clinical Relevance: The study data suggest that the suppression of meniscus hypertrophy and calcification may decrease the risk of PTOA after ACL injuries.