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

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Multicolor flow cytometry-based cellular phenotyping identifies osteoprogenitors and inflammatory cells in the osteoarthritic subchondral bone marrow compartment B.E. Pippenger, R. Duhr, M.G. Muraro, G.I. Pagenstert, T. Hügle, J. Geurts Osteoarthritis and Cartilage Volume 23, Issue 11, Pages 1865-1869 (November 2015) DOI: 10.1016/j.joca.2015.07.021 Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Marrow cell extraction from the SBU of tibial plateaus. (A) Histological section of the SBU from an osteoarthritic knee tibial plateau stained with hematoxylin and eosin, showing a thickening of the subchondral cortical plate (SCP, dotted line) and increased marrow fibrosis in STB subjacent to regions of pronounced cartilage damage. (B) MTT staining (purple) of trimmed bone pieces for viable cells present before (left) or after (right) 4 h digestion with collagenase. Scale bars 500 μm (A). (C) Flow cytometry analysis starting with the identification of nucleated cells in the bone digest by gating only events positive for DAPI. Subsequent selection of single cell events, using a combination of FSC-H and FSC-A. Staining of single cells against CD45 showing the existence of subpopulations of hematopoietic and non-hematopoietic origin in the bone digest. Osteoarthritis and Cartilage 2015 23, 1865-1869DOI: (10.1016/j.joca.2015.07.021) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Cellular phenotyping by multi-color flow cytometry and immunohistochemistry. (A) Identification of CD14+ monocytes (upper left quadrant) and CD14+/CD68+ macrophages (upper right quadrant) among cells from the CD45+ fraction, isolated by magnetic bead separation. Complementary immunohistochemical staining showing mononuclear CD68-positive cells in the subchondral bone marrow. (B) Staining against CD115 and HLA-DR of CD45+ cells, revealing a subpopulation of osteoclast progenitors (lower right quadrant: CD45+/HLA-DR−/CD115+). Histological staining for TRAP+ multinucleated osteoclasts and mononuclear osteoclasts precursors (arrowheads). (C) Expression of ALP in the CD45-subpopulation, indicating the presence of osteoprogenitors/early osteoblasts. Immunohistochemical staining against osteocalcin showing positivity in bone-lining cells. Original magnifications 200× (A, C) and 100× (B). Osteoarthritis and Cartilage 2015 23, 1865-1869DOI: (10.1016/j.joca.2015.07.021) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions