中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (23): 3646-3652.doi: 10.12307/2023.579

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

手术诱导膝骨关节炎兔模型的制备方法

张传成1,沈美花1,陈利锋2,王华松2,项  阳1,谭张奎2   

  1. 1武汉科技大学,湖北省武汉市  430065;2中国人民解放军中部战区总医院,湖北省武汉市  430070
  • 收稿日期:2022-06-28 接受日期:2022-10-24 出版日期:2023-08-18 发布日期:2023-01-16
  • 通讯作者: 陈利锋,博士,副主任医师,中国人民解放军中部战区总医院,湖北省武汉市 430070
  • 作者简介:张传成,男,1994年生,山东省淄博市人,汉族,武汉科技大学在读硕士,主要从事风湿免疫方面的研究。 沈美花,女,1997年生,黑龙江省大庆市人,汉族,2022年武汉科技大学医学院毕业,硕士。
  • 基金资助:
    湖北省医学青年拔尖人才(第一层次)(1371),项目负责人:陈利锋

Preparation of rabbit models of surgically induced knee osteoarthritis

Zhang Chuancheng1, Shen Meihua1, Chen Lifeng2, Wang Huasong2, Xiang Yang1, Tan Zhangkui2   

  1. 1Wuhan University of Science and Technology, Wuhan 430065, Hubei Province, China; 2Central Theater Command General Hospital of the Chinese PLA, Wuhan 430070, Hubei Province, China
  • Received:2022-06-28 Accepted:2022-10-24 Online:2023-08-18 Published:2023-01-16
  • Contact: Chen Lifeng, MD, Associate chief physician, Central Theater Command General Hospital of the Chinese PLA, Wuhan 430070, Hubei Province, China
  • About author:Zhang Chuancheng, Master candidate, Wuhan University of Science and Technology, Wuhan 430065, Hubei Province, China Shen Meihua, Master, Wuhan University of Science and Technology, Wuhan 430065, Hubei Province, China
  • Supported by:
    Young Top-notch Medical Talents of Hubei Province (the First Level), No. 1371 (to CLF)

摘要:

文题释义:

膝骨关节炎:是一种以膝关节软骨退变和骨质增生为特征的疾病,主要表现为膝关节疼痛肿胀及活动障碍,多发于中老年人群,早期常需保守治疗,晚期则行手术治疗,如不及时治疗则会引起关节畸形,甚至残疾。在膝关节部位还常患有膝关节滑膜炎、韧带损伤、半月板损伤、膝关节游离体等关节疾病。
关节软骨:属于透明软骨,表面光滑,能减少相邻两骨的摩擦,缓冲运动时产生的震动。关节软骨主要的功能是缓解压力的作用,它的结构非常精细和科学,以适应不同的功能需要。在压力作用下,软骨被压缩,解除压力,又可伸展,起到保护关节作用。老年人关节软骨弹性、延伸能力减弱,因此老年人的关节软骨易受损伤,易发生退行性骨关节病。

背景:目前常用的膝骨关节炎动物模型制备方法主要有手术创伤和关节腔内注射蛋白酶诱导两大类。关节腔内注射药物诱导模型并不稳定,传统的手术造模方法对膝关节损伤较大,且成模过程与临床膝骨关节炎的发展过程差异较大。
目的:通过不同手术方式损伤膝关节制备兔膝骨关节炎模型,探讨膝骨关节炎动物模型的最佳制备方法。
方法:将16只雄性五六月龄新西兰大白兔随机分为假手术组、模型A组、模型B组、模型C组,每组4只。假手术组切开右侧膝关节内侧后立即缝合;模型A组磨损右侧膝关节半月板、磨损前交叉韧带;模型B组摘除右侧膝关节半月板、磨损前交叉韧带;模型C组摘除右侧膝关节半月板、切断前交叉韧带。术后连续3 d注射抗生素,记录一般情况。4周后通过X射线片观察关节情况,苏木精-伊红染色观察软骨组织变化,酶联免疫吸附法检测右侧膝滑膜液和血清中炎症因子水平。

结果与结论:①模型组右侧膝关节明显出现红肿热痛情况,皮温明显高于假手术组;②术后模型组右侧膝关节皮温较假手术组明显升高,差异有显著性意义(P < 0.05);③X射线片显示假手术组膝关节未见异常,模型组出现不同程度的关节面破坏及关节间隙狭窄,其中模型C组最为严重;④苏木精-伊红染色显示假手术组为软骨组织正常形态;模型组出现软骨细胞数量减少,层次紊乱,其中模型C组软骨结构损坏严重,可见血管生成;⑤酶联免疫吸附实验显示,模型组兔血清炎症因子水平较假手术组明显升高,差异有显著性意义(P < 0.001);模型组兔右侧膝关节滑膜液炎症因子水平较假手术组明显升高,差异有显著性意义(P < 0.001);⑥提示通过对各组兔膝关节一般情况、影像学及相关因子改变分析发现,以上3种手术损伤膝关节方法均能成功构建兔膝骨关节炎模型,模型C组已达到膝骨关节炎中晚期改变,模型A、B组符合早中期膝骨关节炎改变;此外实验证实膝骨关节炎病情进展速度与膝关节损伤程度呈正相关,血清中炎性因子水平相比于滑膜液对膝骨关节炎病情变化更敏感。

https://orcid.org/0000-0002-6373-5392(张传成)

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程

关键词: 膝骨关节炎, 动物模型, 炎症, 关节积液, 软骨

Abstract: BACKGROUND: The two methods commonly used for preparing animal models of knee osteoarthritis are surgical trauma and intra-articular injection of protease. However, the models induced by intra-articular injection of drugs are not stable, the traditional surgical modeling method has great damage to the knee joint, and the modeling process differs greatly from the development of clinical knee osteoarthritis.
OBJECTIVE: To evaluate the best preparation method for the animal model of knee osteoarthritis through establishing a rabbit knee osteoarthritis model by injuring the knee joint in different surgical ways. 
METHODS: Sixteen male New Zealand white rabbits aged 5-6 months were randomly divided into sham operation group, model group A, model group B and model group C, with four rats in each group. In the sham operation group, the medial side of the right knee joint was incised and immediately sutured. In the model group A, the meniscus and anterior cruciate ligament of the right knee were worn. In the model group B, the meniscus of the right knee joint was removed and the anterior cruciate ligament of the right knee was worn. In the model group C, the right knee meniscus was removed and the anterior cruciate ligament of the right knee joint was transected. Antibiotics were injected for 3 consecutive days after operation, and the general conditions were recorded. After 4 weeks, the knee joints were observed by X-ray, the changes of cartilage tissue were observed by hematoxylin-eosin staining, and the levels of inflammatory factors in the synovial fluid and serum of the right knee were detected by enzyme-linked immunosorbent assay.
RESULTS AND CONCLUSION: Redness, swelling and heat pain were evident in the right knee joint of the model group, and the skin temperature in the model groups was significantly higher than that in the sham operation group. Postoperative skin temperature of the right knee joint in the model groups was significantly higher than that in the sham operation group (P < 0.05). The X-ray observation showed no abnormalities in the knee joint in the sham operation group, while the model groups showed varying degrees of articular surface destruction and joint space narrowing, with the model group C being the most severe. Hematoxylin-eosin staining showed normal cartilage tissue in the sham-operated group, while the model groups showed a reduction in the number of chondrocytes with disordered structural levels. In the model group C, the cartilage structure was seriously damaged with angiogenesis being visible. Enzyme-linked immunosorbent assay showed that serum inflammatory factor levels in the model groups were significantly higher than those in the sham operation group (P < 0.05). The level of inflammatory factors in the synovial fluid of the right knee joint was significantly higher in the model groups than the sham operation group (P < 0.05). To conclude, through the analysis of the general condition, imaging and related factors of the knee joint of each group, it was found that the above three surgical methods of the knee injury could successfully construct the rabbit knee osteoarthritis model. The model group C reached the middle and late stage of knee osteoarthritis and the model groups A and B were consistent with the changes in early and middle knee osteoarthritis. In addition, this experiment confirmed that the speed of knee osteoarthritis progression is positively correlated with the degree of knee injury, and the level of inflammatory factors in serum is more sensitive to the changes of knee osteoarthritis than that in the synovial fluid.

Key words: knee osteoarthritis, animal model, inflammation, joint effusion, cartilage

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