人工马肌腱损伤病灶内治疗后通过体内磁共振成像和组织学追踪自体脂肪组织来源的间充质基质细胞——一项初步研究

Tracking of autologous adipose tissue-derived mesenchymal stromal cells with in vivo magnetic resonance imaging and histology after intralesional treatment of artificial equine tendon lesions--a pilot study.

作者信息

Geburek Florian, Mundle Kathrin, Conrad Sabine, Hellige Maren, Walliser Ulrich, van Schie Hans T M, van Weeren René, Skutella Thomas, Stadler Peter M

机构信息

Clinic for Horses, University of Veterinary Medicine Hannover, Foundation, Bünteweg 9, 30559, Hannover, Germany.

Pferdeklink Kirchheim, Nürtinger Straße 200, 73230, Kirchheim unter Teck, Germany.

出版信息

Stem Cell Res Ther. 2016 Feb 1;7:21. doi: 10.1186/s13287-016-0281-8.

DOI:10.1186/s13287-016-0281-8

PMID:26830812

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4736260/

Abstract

BACKGROUND

Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used to treat equine tendinopathies. Up to now, knowledge about the fate of autologous AT-MSCs after intralesional injection into equine superficial digital flexor tendons (SDFTs) is very limited. The purpose of this study was to monitor the presence of intralesionally injected autologous AT-MSCs labelled with superparamagnetic iron oxide (SPIO) nanoparticles and green fluorescent protein (GFP) over a staggered period of 3 to 9 weeks with standing magnetic resonance imaging (MRI) and histology.

METHODS

Four adult warmblood horses received a unilateral injection of 10 × 10(6) autologous AT-MSCs into surgically created front-limb SDFT lesions. Administered AT-MSCs expressed lentivirally transduced reporter genes for GFP and were co-labelled with SPIO particles in three horses. The presence of AT-MSCs in SDFTs was evaluated by repeated examinations with standing low-field MRI in two horses and post-mortem in all horses with Prussian blue staining, fluorescence microscopy and with immunofluorescence and immunohistochemistry using anti-GFP antibodies at 3, 5, 7 and 9 weeks after treatment.

RESULTS

AT-MSCs labelled with SPIO particles were detectable in treated SDFTs during each MRI in T2*- and T1-weighted sequences until the end of the observation period. Post-mortem examinations revealed that all treated tendons contained high numbers of SPIO- and GFP-labelled cells.

CONCLUSIONS

Standing low-field MRI has the potential to track SPIO-labelled AT-MSCs successfully. Histology, fluorescence microscopy, immunofluorescence and immunohistochemistry are efficient tools to detect labelled AT-MSCs after intralesional injection into surgically created equine SDFT lesions. Intralesional injection of 10 × 10(6) AT-MSCs leads to the presence of high numbers of AT-MSCs in and around surgically created tendon lesions for up to 9 weeks. Integration of injected AT-MSCs into healing tendon tissue is an essential pathway after intralesional administration. Injection techniques have to be chosen deliberately to avoid reflux of the cell substrate injected. In vivo low-field MRI may be used as a non-invasive tool to monitor homing and engraftment of AT-MSCs in horses with tendinopathy of the SDFT.

摘要

背景

脂肪组织来源的间充质基质细胞（AT-MSCs）常用于治疗马的肌腱病。到目前为止，关于自体AT-MSCs经皮内注射到马的指浅屈肌腱（SDFTs）后的命运的了解非常有限。本研究的目的是通过站立式磁共振成像（MRI）和组织学，在3至9周的交错时间段内监测经皮内注射的用超顺磁性氧化铁（SPIO）纳米颗粒和绿色荧光蛋白（GFP）标记的自体AT-MSCs的存在情况。

方法

四匹成年温血马接受了将10×10⁶个自体AT-MSCs单侧注射到手术造成的前肢SDFT损伤处的操作。给予的AT-MSCs表达慢病毒转导的GFP报告基因，并且在三匹马中与SPIO颗粒共同标记。在治疗后3、5、7和9周，通过对两匹马进行重复的站立式低场MRI检查以及对所有马匹进行尸检，使用普鲁士蓝染色、荧光显微镜检查以及使用抗GFP抗体的免疫荧光和免疫组织化学，来评估SDFTs中AT-MSCs的存在情况。

结果

在每个MRI的T2*加权和T1加权序列中，直到观察期结束，在治疗的SDFTs中都可检测到用SPIO颗粒标记的AT-MSCs。尸检显示，所有治疗的肌腱都含有大量的SPIO和GFP标记的细胞。

结论

站立式低场MRI有成功追踪SPIO标记的AT-MSCs的潜力。组织学、荧光显微镜检查、免疫荧光和免疫组织化学是在手术造成的马SDFT损伤处皮内注射后检测标记的AT-MSCs的有效工具。皮内注射10×10⁶个AT-MSCs会导致在手术造成的肌腱损伤及其周围存在大量的AT-MSCs，持续长达9周。注射的AT-MSCs整合到愈合的肌腱组织中是皮内给药后的一条重要途径。必须谨慎选择注射技术以避免注射的细胞基质回流。体内低场MRI可作为一种非侵入性工具来监测患有SDFT肌腱病的马中AT-MSCs的归巢和植入情况。