Advances in meniscus repair of children
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摘要:
儿童及青少年半月板损伤通常较成人少见，但随着儿童参加体育锻炼的数量及锻炼强度的增加，半月板损伤的频率也在逐步增加。目前，成人半月板撕裂诊疗方面研究已经很成熟，儿童半月板损伤修复研究也取得了一定进展，但关于儿童半月板损伤的修复缺乏共识。本研究参考近年来国内外有关儿童半月板损伤治疗方面的文献，从半月板血管分布、损伤分型、损伤机制及修复方式等方面进行综述，以期为儿童半月板损伤的修复提供参考依据。
Abstract:Meniscus injury is usually less common in children than in adults. However, with the increase of the number and intensity of physical exercise in children, the frequency of meniscus injury in this group is gradually increasing. At present, the diagnosis and treatment of adult meniscus tear have been well studied, while the repair of children's meniscus injury has also made some progress, but there is no consensus on the repair of meniscus injury in children. This study referred to domestic and foreign literature on meniscus injury treatment in children in recent years and reviewed the research progress in meniscus vascular distribution, classification, injury mechanism, and repair methods, hoping to provide a reference for the repair of meniscus injury in children.
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Keywords:
- meniscus injury /
- children /
- repair /
- research advances /
- vascular distribution /
- physical exercise
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创面修复包括机体自身组织的再生、修复、重建以及后期的人为干预，影响因素较多，易引发局部和全身感染[1]。创面的愈合，尤其是外科术后的创面修复一直是困扰临床医生的难题。复方甘菊利多卡因凝胶(简称甘美达凝胶)的主要成分为盐酸利多卡因和洋甘菊花酊，其对口腔炎症与疼痛具有显著的疗效，但关于其在外科手术创面愈合中作用的研究仍较少[2-5]。本研究建立皮肤创面大鼠动物模型，观察甘美达凝胶对创面修复的效果，并探讨其作用机制，现将结果报告如下。
1. 材料与方法
1.1 实验动物及分组
SD大鼠18只[北京华阜康生物科技股份有限公司提供，动物许可证号为SCXK(京)2019-0008], 体质量180~220 g, 性别相同。饲养条件为自由进食和饮水，室温20 ℃, 相对湿度约50 ℃。皮肤创面模型建成后，将18只大鼠随机分为实验组(甘美达凝胶)、阳性药组(多黏菌素B)和模型组(生理盐水冲洗)。治疗时间观察点分别为治疗7、14 d时, 7 d时每组为6只, 14 d时每组为3只。
1.2 主要药物及试剂
复方甘菊利多卡因凝胶(Kamistad, STADA Consumer Health Deutschland GmbH), 复方多黏菌素B软膏(孚诺，浙江孚诺医药股份有限公司)，生理盐水。肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)酶联免疫吸附测定(ELISA)试剂盒(Fankew, Shanghai FANKEL Industrial Co., Ltd)。
1.3 建立皮肤创面模型
采用10%水合氯醛对大鼠进行麻醉，剂量为30 mg/kg。将大鼠背部脱毛消毒，备皮面积为2 cm×2 cm。将大鼠麻醉后固定于手术台上，在背部脊柱一侧头尾两端平行于脊柱标记1个圆形切口，用手术刀沿标记线切除全层皮肤至深筋膜，形成创面。
1.4 治疗过程
创面模型形成后，实验组、阳性药组和模型组分别在局部涂抹药物，每天2次。模型组采用生理盐水冲洗溃疡面。分别在治疗1、7、14 d时记录创面愈合情况，并用游标卡尺测量溃疡直径。
1.5 观察指标
分别于治疗7、14 d时将各组大鼠脱颈处死，切除溃疡组织，分为2块，其中一块置于-80 ℃冰箱冻存，留待TNF-α、IL-6和IL-1β检测使用; 另一块放入10%甲醛溶液中固定24 h, 行常规脱水、包埋、切片，采用苏木精-伊红染色(HE染色)观察创面的组织病理变化和免疫组织化学染色结果。采用ELISA试剂盒检测创面组织中TNF-α、IL-6和IL-1β的含量，方法参照试剂盒说明书。
1.6 统计学方法
采用SPSS 25.0进行统计分析，计量数据采用(x±s)表示，结果比较采用单因素方差分析，并进行方差齐性检验，多重比较采用LSD-t检验或T2(M)检验。P < 0.05为差异有统计学意义。
2. 结果
2.1 各组大鼠治疗7、14 d时创面愈合情况
各组大鼠建模时溃疡面积基本一致(图 1)。治疗7 d时，实验组、阳性药组创面均较模型组缩小，但差异无统计学意义(P>0.05); 治疗14d时，阳性药组创面仍大于模型组，但差异也无统计学意义(P>0.05)。见表 1。
表 1 各组大鼠不同时点皮肤创面横径比较(x±s)cm 时点 模型组 实验组 阳性药组 术后1 d 2.10±0.09 2.02±0.08 2.05±0.14 治疗7 d 1.35±0.19 1.22±0.04 1.33±0.10 治疗14 d 0.87±0.15 0.73±0.06 1.03±0.15 2.2 各组大鼠创面的组织形态学观察
治疗7 d时，模型组创面组织中可见明显的炎性渗出层，肉芽组织水肿，毛细血管丰富且扩张，充血明显，大量炎症细胞浸润; 与模型组相比，实验组、阳性药组炎性渗出层明显变薄或消失，肉芽组织水肿减轻，毛细血管密度减少且扩张不明显，炎症细胞密度减少，同时可见较明显的成纤维细胞出现。见图 2。
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图 2 治疗7 d时各组大鼠创面的组织形态学HE染色情况A、D: 模型组创面组织形态学可见较厚的炎性渗出层，肉芽组织中血管丰富，炎症细胞密度较大; B、E: 实验组创面组织形态学可见炎性渗出层较薄，肉芽组织中血管减少，炎症细胞密度降低，成纤维细胞增多; C、F: 阳性药组创面组织形态学可见肉芽组织的成熟度低于实验组。A、B、C放大倍数为40倍, D、E、F放大倍数为400倍。治疗14 d时，模型组创面仍可见少量炎性渗出，但肉芽组织中血管数量明显减少，扩张不明显，炎症细胞数量减少，成纤维细胞增加并可见胶原形成; 与模型组相比，实验组血管减少更加明显且呈闭塞状态，炎症细胞数量明显减少，成纤维细胞数量明显增加，胶原排列有序; 与模型组相比，阳性药组创面有改善，但炎症细胞数量和血管密度仍略多。见图 3。
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图 3 治疗14 d时各组大鼠创面的组织形态学HE染色情况A、D: 模型组创面组织形态学可见肉芽组织中血管狭长，炎症细胞密度降低，成纤维细胞和胶原出现; B、E: 实验组创面组织形态学可见血管数量明显减少，成纤维细胞核胶原较模型组增多; C、F: 阳性药组创面组织形态学可见肉芽组织成熟度低于实验组。A、B、C放大倍数为40倍，D、E、F放大倍数为400倍。2.3 各组创面组织中TNF-α、IL-6和IL-1β的表达
治疗7 d时，实验组大鼠创面组织中TNF-α、IL-6和IL-1β的表达水平均低于模型组、阳性药组，差异有统计学意义(P < 0.05或P < 0.01)。治疗14 d时，实验组大鼠创面组织中TNF-α、IL-6和IL-1β的表达水平仍低于模型组，但差异无统计学意义(P>0.05)。见表 2。
表 2 各组大鼠不同时点创面组织中TNF-α、IL-6和IL-1β的表达(x±s)ng/mg 组别 治疗7 d 治疗14 d TNF-α IL-6 IL-1β TNF-α IL-6 IL-1β 模型组 53.58±5.83* 17.13±2.01* 5.68±0.69* 61.85±5.72 21.30±1.60 6.34±0.52 实验组 31.24±5.25 8.48±0.36 2.99±0.28 57.92±5.95 17.37±2.18 5.42±0.58 阳性药组 39.90±4.61* 12.38±0.32** 4.51±0.36* 60.51±2.72 19.18±1.65 6.70±0.22 TNF-α: 肿瘤坏死因子-α; IL-6: 白细胞介素-6; IL-1β: 白细胞介素-1β。与实验组比较, *P < 0.05, **P < 0.01。 2.4 各组创面组织中胶原含量的苦味酸-天狼猩红染色情况
采用苦味酸-天狼猩红染色分析创面组织中胶原含量的变化，治疗7、14 d时，实验组创面组织中的胶原含量均高于模型组，且胶原较为粗大，见图 4。
2.5 各组创面组织的免疫组织化学染色情况
本研究以灰度值表示免疫组化的表达, CCR7/CD68表示经典活化(M1)型巨噬细胞的比例, CD163/CD68表示替代活化(M2)型巨噬细胞的比例，结果以累积光密度(IOD)表示。结果显示，治疗7、14 d时，各组M1型和M2型巨噬细胞的比例差异均无统计学意义(P>0.05)。见表 3。
表 3 各组大鼠不同时点创面组织中CCR7/CD68和CD163/CD68的表达(x±s)IOD 组别 治疗7 d 治疗14 d CCR7/CD68 CD163/CD68 CCR7/CD68 CD163/D68 模型组 1.15±0.38 1.42±0.27 0.93±0.07 1.06±0.10 阳性药组 0.91±0.14 0.99±0.05 1.03±0.08 1.00±0.08 实验组 1.09±0.04 1.08±0.17 1.00±0.18 1.03±0.11 3. 讨论
皮肤创面的愈合一般需要经过凝血期、炎症期、增殖期和重塑期。在创面形成初期，毛细血管内的血液流入创面，与其他生物学分子形成凝血块覆盖于创面表面; 炎症期时，中性粒细胞在创面聚集并开始吞噬病原体等微生物，发挥清除作用; 在损伤后48~72 h, 巨噬细胞开始发挥吞噬功能，随着创面愈合的过程，巨噬细胞的表型也发生相应的改变，为成纤维细胞提供大量的生物因子; 在创伤后72 h, 炎症因子也会被招募至创面发挥相应的作用，伴随大量肉芽组织形成，成纤维细胞开始迁移，胶原逐渐增多[6]。
肛肠疾病手术后病原微生物的控制尤为重要，这是因为肛肠术后的创面多为开放性创面，加之粪便的残留和刺激，极易受细菌感染，导致伤口愈合缓慢。目前，促进创面愈合的方法有很多，包括各种敷料、组织工程皮肤、生长因子、干细胞治疗、天然药物和复方药物等，其中综合应用多种方法是临床常用的选择。甘美达凝胶是一种复方外用凝胶，其主要成分为洋甘菊花酊、盐酸利多卡因。洋甘菊花酊是甘美达凝胶的主要成分，具有抗炎、促进伤口愈合的作用，已被证实对多种细菌，尤其是金黄色葡萄球菌具有明显的抑制作用[7-8]; 盐酸利多卡因的主要作用是阻断痛觉传入的产生和传导，具有快速的止痛作用，且局部应用不影响手术创面愈合[9]。本实验以大鼠皮肤创面模型为研究对象，治疗7、14 d时，病理形态学显示采用甘美达凝胶的实验组创面炎性渗出层消退时间提前，肉芽组织更早进入成熟期，胶原含量也明显增多; 创面缩小的差异无统计学意义可能与观察时间偏短有关。
TNF-α是一种促炎症细胞因子，主要参与机体的炎症反应和免疫反应，可以激活中性粒细胞，增强其吞噬能力，也可以促进巨噬细胞的活化[10]。IL-6和IL-1β也是重要的促炎症介质，在全身性感染的发生发展中起着重要的作用，是启动炎症反应的因子，可以触发炎性反应瀑布效应[11]。研究[12]表明，炎症因子含量过高不仅可以导致溃疡组织的炎症反应，还会抑制创面的愈合，降低炎症因子的水平，进而促进溃疡的愈合。本实验采用ELISA法检测不同时点创面组织中炎症因子的含量，在用药7 d时，采用甘美达凝胶的实验组创面组织中炎症因子TNF-α、IL-6和IL-1β均较模型组显著降低，但在用药14 d时差异无统计学意义，说明甘美达凝胶在创面早期抗炎治疗中可以发挥重要的作用，可以促使肉芽组织更趋于成熟，推测可能与炎症因子水平的控制有关。
巨噬细胞是由外周血中的单核细胞分化而来，是机体重要的免疫细胞，具有吞噬、杀伤病原微生物的功能，也可以分泌炎性因子或细胞因子来调节机体的免疫反应，参与组织的修复[13-14]。在不同的微环境中，巨噬细胞可以分化为M1型和M2型, M1型主要发挥抗原提呈、吞噬杀灭病原微生物的作用, M2型则可抑制局部炎症反应，促进组织的再生修复和创面的愈合[15]。有研究[16]认为巨噬细胞可以促进创面愈合，也有研究[17]认为巨噬细胞只是引发了炎症反应，并不利于创面愈合。本研究结果显示，在创面形成后7、14 d, 各组创面组织中的巨噬细胞数量差异无统计学意义，表型也无明显变化。
综上所述，甘美达凝胶对皮肤创面具有较好的抗炎和促进愈合的作用，创伤早期抑制炎症因子释放可能是其主要作用机制之一。
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