全膝关节置换术后Hemovac引流血作为干细胞来源

Hemovac blood after total knee arthroplasty as a source of stem cells.

作者信息

Kim Seon Ae, Park Ho Youn, Shin Yong-Woon, Go Eun Jeong, Kim Young Ju, Kim Yoo Chang, Shetty Asode Ananthram, Kim Seok Jung

机构信息

Department of Orthopedic Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.

Department of Orthopaedic Surgery, College of Medicine, The Inje University of Korea, Seoul, Republic of Korea.

出版信息

Ann Transl Med. 2020 Nov;8(21):1406. doi: 10.21037/atm-20-2215.

DOI:10.21037/atm-20-2215

PMID:33313151

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7723525/

Abstract

BACKGROUND

With increasing life expectancy, stem cell therapy is receiving increasing attention. However, its application is restricted by ethical concerns. Hence a need exists for design of safe procedures for stem cell procurement. Here, we investigated whether hemovac blood (HVB) is an appropriate stem cell source.

METHODS

HVB concentrates (HVBCs) from 20 total knee arthroplasty (TKA) patients and bone marrow aspirate (BMA) concentrates (BMACs) from 15 patients who underwent knee cartilage repair were comparatively evaluated. A bone marrow aspiration needle was inserted into the anterior superior iliac spine. Aspiration was performed using a 50-mL syringe, including 4 mL of anticoagulant, followed by centrifugation to obtain BMACs. To obtain HVBCs, blood was aspirated from the hemovac immediately after TKA surgery. Different cell types were enumerated. Isolation of BMA and HVB mononuclear cells was performed using density gradient centrifugation. Non-hematopoietic fibroblast colonies were quantified by colony forming unit-fibroblast assay surface marker analysis of HVB, HVBC, BMA, and BMAC was performed via flow cytometry. Mesenchymal stem cells (MSCs) isolated from HVBCs and BMACs were examined for osteogenic, adipogenic, and chondrogenic differentiation potential. Gene expression analysis was performed by quantitative real-time polymerase chain reaction (qRT-PCR).

RESULTS

The number of cells from HVB and HVBC was significantly lower than from BMA and BMAC; however, the number of colonies in HVBC and BMAC did not differ significantly (P>0.05). Isolated cells from both sources had a fibroblast-like appearance, adhered to culture flasks, and formed colonies. Under different culture conditions, MSC-specific surface markers (CD29, CD44, CD90, CD105), osteogenic markers [RUNX2, osteopontin, osteocalcin, and alkaline phosphatase (ALP)] and adipogenic markers (PPARγ and C/EBPα) were expressed. Moreover, SOX9, type II collagen, and aggrecan were significantly upregulated upon chondrogenic differentiation.

CONCLUSIONS

HVB from TKA patients is a useful source of stem cells for research.

摘要

背景

随着预期寿命的增加，干细胞疗法受到越来越多的关注。然而，其应用受到伦理问题的限制。因此，需要设计安全的干细胞采集程序。在此，我们研究了负压引流血液（HVB）是否是合适的干细胞来源。

方法

对20例全膝关节置换术（TKA）患者的HVB浓缩物（HVBCs）和15例接受膝关节软骨修复患者的骨髓抽吸物（BMA）浓缩物（BMACs）进行了比较评估。将骨髓穿刺针插入髂前上棘。使用50 mL注射器进行抽吸，其中包括4 mL抗凝剂，随后进行离心以获得BMACs。为了获得HVBCs，在TKA手术后立即从负压引流装置中抽吸血液。对不同细胞类型进行计数。使用密度梯度离心法分离BMA和HVB单核细胞。通过集落形成单位-成纤维细胞测定法对非造血成纤维细胞集落进行定量，通过流式细胞术对HVB、HVBC、BMA和BMAC进行表面标志物分析。检测从HVBCs和BMACs中分离的间充质干细胞（MSCs）的成骨、成脂和成软骨分化潜能。通过定量实时聚合酶链反应（qRT-PCR）进行基因表达分析。

结果

HVB和HVBC中的细胞数量显著低于BMA和BMAC中的细胞数量；然而，HVBC和BMAC中的集落数量没有显著差异（P>0.05）。从这两种来源分离的细胞具有成纤维细胞样外观，附着于培养瓶并形成集落。在不同培养条件下，表达了MSC特异性表面标志物（CD29、CD44、CD90、CD105）、成骨标志物[RUNX2、骨桥蛋白、骨钙素和碱性磷酸酶（ALP）]和成脂标志物（PPARγ和C/EBPα）。此外，在软骨分化时，SOX9、II型胶原蛋白和聚集蛋白聚糖显著上调。