基于多色流式细胞术的细胞表型分析可识别骨关节炎软骨下骨髓腔中的骨祖细胞和炎性细胞。

Multicolor flow cytometry-based cellular phenotyping identifies osteoprogenitors and inflammatory cells in the osteoarthritic subchondral bone marrow compartment.

作者信息

Pippenger B E, Duhr R, Muraro M G, Pagenstert G I, Hügle T, Geurts J

机构信息

Osteoarthritis Research Center Basel, Orthopaedic Department, University Hospital Basel, Basel, Switzerland.

Department of Surgery, University Hospital Basel, University of Basel, Basel, Switzerland; Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland.

出版信息

Osteoarthritis Cartilage. 2015 Nov;23(11):1865-9. doi: 10.1016/j.joca.2015.07.021.

DOI:10.1016/j.joca.2015.07.021

PMID:26521732

Abstract

PURPOSE

The cellular component of subchondral bone is thought to be responsible for aberrant bone remodeling in osteoarthritis (OA). Direct phenotypical analysis of the cellular compartment is critical to better understand the OA disease process. This study provides proof-of-principle for flow cytometry-based phenotyping of isolated subchondral trabecular bone (STB) marrow cells without prior use of cell culture techniques.

METHODS

Tibial plateaus were obtained from OA patients undergoing total knee arthroplasty. Subchondral bone chips were digested with collagenase IA and single cell suspensions were directly phenotyped using flow cytometry. Cells were analyzed for the expression of alkaline phosphatase (ALP) as osteoblast/osteoprogenitor marker and monocyte/macrophage markers (CD14, CD68, HLA-DR, CD115).

RESULTS

MTT staining revealed abundant viable cells in the bone marrow compartment of STB prior to digestion, which were efficiently released by collagenase. Within the CD45-negative cell fraction, approximately 20% of the cells were positive for the early osteoblast/osteoprogenitor marker ALP. Within the CD45+ hematopoietic cell fraction, the majority of cells were of monocytic origin (>80%) displaying strong surface expression of CD14. Discreet macrophage populations (CD14+/HLA-DR+/CD68+) and putative osteoclast progenitors (CD45+/HLA-DR-/CD115+) were unequivocally identified. Osteoblast, macrophage and osteoclast progenitor presence in the subchondral bone unit (SBU) was confirmed by (immuno)histochemical staining for osteocalcin, CD68 and tartrate-resistant acid phosphatase, respectively.

CONCLUSIONS

Flow cytometric analysis is a valuable methodology to study the cellular compartment of STB marrow. This method provides a proof of principle that the whole resident cell population can be directly phenotypically characterized without the prior use of cell culture techniques.

摘要

目的

人们认为，软骨下骨的细胞成分是骨关节炎（OA）中异常骨重塑的原因。对细胞区室进行直接表型分析对于更好地理解OA疾病过程至关重要。本研究为基于流式细胞术对分离的软骨下小梁骨（STB）骨髓细胞进行表型分析提供了原理证明，且无需事先使用细胞培养技术。

方法

从接受全膝关节置换术的OA患者获取胫骨平台。用IA型胶原酶消化软骨下骨碎片，并用流式细胞术直接对单细胞悬液进行表型分析。分析细胞中成骨细胞/骨祖细胞标志物碱性磷酸酶（ALP）以及单核细胞/巨噬细胞标志物（CD14、CD68、HLA-DR、CD115）的表达情况。

结果

MTT染色显示，消化前STB骨髓区室中有大量活细胞，胶原酶可有效释放这些细胞。在CD45阴性细胞组分中，约20%的细胞早期成骨细胞/骨祖细胞标志物ALP呈阳性。在CD45+造血细胞组分中，大多数细胞起源于单核细胞（>80%），CD14表面表达强烈。明确鉴定出离散的巨噬细胞群体（CD14+/HLA-DR+/CD68+）和假定的破骨细胞祖细胞（CD45+/HLA-DR-/CD115+）。分别通过骨钙素、CD68和抗酒石酸酸性磷酸酶的（免疫）组织化学染色证实了软骨下骨单位（SBU）中存在成骨细胞、巨噬细胞和破骨细胞祖细胞。