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利用成像技术评估脐带间充质干细胞疗法对犬膝关节炎的疗效

Evaluation of the Curative Effect of Umbilical Cord Mesenchymal Stem Cell Therapy for Knee Arthritis in Dogs Using Imaging Technology.

作者信息

Zhang Bei-Ying, Wang Bing-Yun, Li Shao-Chuan, Luo Dong-Zhang, Zhan Xiaoshu, Chen Sheng-Feng, Chen Zhi-Sheng, Liu Can-Ying, Ji Hui-Qin, Bai Yin-Shan, Li Dong-Sheng, He Yang

机构信息

School of Life Science and Engineering, Foshan University, Foshan, Guangdong 528231, China.

School of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.

出版信息

Stem Cells Int. 2018 May 15;2018:1983025. doi: 10.1155/2018/1983025. eCollection 2018.

Abstract

OBJECTIVE

The aim of this study was to assess the efficacy of canine umbilical cord mesenchymal stem cells (UC-MSCs) on the treatment of knee osteoarthritis in dogs.

METHODS

Eight dogs were evenly assigned to two groups. The canine model of knee osteoarthritis was established by surgical manipulation of knee articular cartilage on these eight dogs. UC-MSCs were isolated from umbilical cord Wharton's jelly by 0.1% type collagenase I and identified by immunofluorescence staining and adipogenic and osteogenic differentiation . A suspension of allogeneic UC-MSCs (1 × 10) and an equal amount of physiological saline was injected into the cavitas articularis in the treated and untreated control groups, respectively, on days 1 and 3 posttreatment. The structure of the canine knee joint was observed by magnetic resonance imaging (MRI), B-mode ultrasonography, and X-ray imaging at the 3rd, 7th, 14th, and 28th days after treatment. Concurrently, the levels of IL-6, IL-7, and TNF- in the blood of the examined dogs were measured. Moreover, the recovery of cartilage and patella surface in the treated group and untreated group was compared using a scanning electron microscope (SEM) after a 35-day treatment.

RESULTS

Results revealed that the isolated cells were UC-MSCs, because they were positive for CD44 and negative for CD34 surface markers, and the cells were differentiated into adipocytes and osteoblasts. Imaging technology showed that as treatment time increased, the high signal in the MRI T2-weighted images decreased, the echo-free space in B ultrasonography images disappeared basically, and the continuous linear hypoechoic region at the trochlear sulcus thickened. On X-ray images, the serrate defect at the ventral cortex of the patella improved, and the low-density gap of the ventral patella and trochlear crest gradually increased in the treated group. On the contrary, the high signal in the MRI T2-weighted images and the echo-free space in B ultrasonography images still increased after a 14-day treatment in the untreated control group, and the linear hypoechoic region was discontinuous. On the X-ray images, there was no improvement in the serrate defect of the ventral cortex of the patella. Results for inflammatory factors showed that the blood levels of IL-6, IL-7, and TNF- of the untreated control group were significantly higher than those of the treated group ( < 0.05) 7-14 days posttreatment. The result of SEM showed that the cartilage neogenesis in the treated group had visible neonatal tissue and more irregular arrangement of new tissue fibers than that of the untreated control group. Furthermore, more vacuoles but without collagen fibers were observed in the cartilage of the untreated control group, and the thickness of the neogenetic cartilage in the treated group (65.13 ± 5.29, 65.30 ± 5.83) and the untreated control group (34.27 ± 5.42) showed a significant difference ( < 0.01).

CONCLUSION

Significantly higher improvement in cartilage neogenesis and recovery was observed in the treated group compared to the untreated control group. The joint fluid and the inflammatory response in the treated group decreased. Moreover, improved recovery in the neogenetic cartilage, damaged skin fascia, and muscle tissue around the joints was more significant in the treated group than in the untreated control group. In conclusion, canine UC-MSCs promote the repair of cartilage and patella injury in osteoarthritis, improve the healing of the surrounding tissues, and reduce the inflammatory response.

摘要

目的

本研究旨在评估犬脐带间充质干细胞(UC-MSCs)对犬膝关节骨关节炎的治疗效果。

方法

将8只犬平均分为两组。通过手术操作这8只犬的膝关节软骨建立犬膝关节骨关节炎模型。用0.1%Ⅰ型胶原酶从脐带华通氏胶中分离UC-MSCs,并通过免疫荧光染色及成脂和成骨分化进行鉴定。在治疗后第1天和第3天,分别将异体UC-MSCs悬液(1×10)和等量生理盐水注入治疗组和未治疗对照组的关节腔。在治疗后第3、7、14和28天,通过磁共振成像(MRI)、B型超声和X线成像观察犬膝关节结构。同时,检测受试犬血液中IL-6、IL-7和TNF-的水平。此外,在治疗35天后,用扫描电子显微镜(SEM)比较治疗组和未治疗组软骨和髌骨表面的恢复情况。

结果

结果显示分离出的细胞为UC-MSCs,因为它们的CD44表面标志物呈阳性,CD34呈阴性,且细胞可分化为脂肪细胞和成骨细胞。成像技术显示,随着治疗时间的增加,MRI T2加权图像中的高信号降低,B超声图像中的无回声区基本消失,滑车沟处的连续线性低回声区增厚。在X线图像上,治疗组髌骨腹侧皮质的锯齿状缺损改善,髌骨腹侧和滑车嵴的低密度间隙逐渐增大。相反,未治疗对照组在治疗14天后,MRI T2加权图像中的高信号和B超声图像中的无回声区仍增加,线性低回声区不连续。在X线图像上,髌骨腹侧皮质的锯齿状缺损无改善。炎症因子结果显示,未治疗对照组在治疗后7 - 14天血液中IL-6、IL-7和TNF-的水平显著高于治疗组(<0.05)。SEM结果显示,治疗组的软骨新生有可见的新生组织,且新组织纤维的排列比未治疗对照组更不规则。此外,在未治疗对照组的软骨中观察到更多的空泡但无胶原纤维,治疗组(65.13±5.29,65.30±5.83)和未治疗对照组(34.27±5.42)新生软骨的厚度有显著差异(<0.01)。

结论

与未治疗对照组相比,治疗组在软骨新生和恢复方面有显著更高的改善。治疗组的关节液和炎症反应减少。此外,治疗组新生软骨、受损皮肤筋膜和关节周围肌肉组织的恢复改善比未治疗对照组更显著。总之,犬UC-MSCs促进骨关节炎中软骨和髌骨损伤的修复,改善周围组织的愈合,并减少炎症反应。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a579/5976915/9183519eb235/SCI2018-1983025.001.jpg

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