Lineage tracking of mesenchymal and endothelial progenitors in BMP-induced bone formation - PubMed (original) (raw)

Lineage tracking of mesenchymal and endothelial progenitors in BMP-induced bone formation

Mille Kolind et al. Bone. 2015 Dec.

Abstract

To better understand the relative contributions of mesenchymal and endothelial progenitor cells to rhBMP-2 induced bone formation, we examined the distribution of lineage-labeled cells in Tie2-Cre:Ai9 and αSMA-creERT2:Col2.3-GFP:Ai9 reporter mice. Established orthopedic models of ectopic bone formation in the hind limb and spine fusion were employed. Tie2-lineage cells were found extensively in the ectopic bone and spine fusion masses, but co-staining was only seen with tartrate-resistant acid phosphatase (TRAP) activity (osteoclasts) and CD31 immunohistochemistry (vascular endothelial cells), and not alkaline phosphatase (AP) activity (osteoblasts). To further confirm the lack of a functional contribution of Tie2-lineage cells to BMP-induced bone, we developed conditional knockout mice where Tie2-lineage cells are rendered null for key bone transcription factor osterix (Tie2-cre:Osx(fx/fx) mice). Conditional knockout mice showed no difference in BMP-induced bone formation compared to littermate controls. Pulse labeling of mesenchymal cells with Tamoxifen in mice undergoing spine fusion revealed that αSMA-lineage cells contributed to the osteoblastic lineage (Col2.3-GFP), but not to endothelial cells or osteoclast populations. These data indicate that the αSMA+ and Tie2+ progenitor lineages make distinct cellular contributions to bone formation, angiogenesis, and resorption/remodeling.

Keywords: BMPs; Bone morphogenetic proteins; Endothelial cells; Lineage tracking; Spine fusion; Tie2; Transgenic reporter mice.

Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Confocal fluorescent images of Tie2-lineage cells (tdTomato, red) in rhBMP-2 induced muscle, native bone, bone marrow, and rhBMP-2 induced ectopic bone. Images are shown at the muscle/ectopic bone interface (A) within an ectopic bone nodule (B), and within the native bone of the femur (C). Text labels, m = muscle, eb = ectopic bone, bm = bone marrow, cb = cortical bone. Scale bar = 50 μm.

Figure 2

Figure 2

Fluorescent images of ectopic bone showing _Tie2_-lineage cells (tdTomato) overlaid with vascular marker CD31 (A) or chondrocyte marker SOX9 (B). _Tie2_-lineage cells were found to demark the new blood vessels in the ectopic bone but not cartilage islands. Scale bar = 50 μm

Figure 3

Figure 3

Fluorescent images of ectopic bone showing _Tie2_-lineage cells (tdTomato) overlaid with tartrate-resistant acid phosphatase/TRAP activity stain (ELF97, green) to label osteoclasts (A, zoom in B). No overlap was seen between _Tie2_-lineage cells with an alkaline phosphatase/AP activity stain (ELF97) to label osteoblasts (C, zoom in D). Scale bar = 50 μm.

Figure 4

Figure 4

rhBMP-2 induced ectopic bone formation in Tie2-cre-Osxfx/fx mice and littermate controls. Representative XRs (A, B) and microCT reconstructions (C, D) are shown for the specimens corresponding to the median bone volume for each group. Littermate controls (A, C) were compared to Tie2-cre-Osxfx/fx conditional knockout (cKO) mice (B, D). The bone nodules showed a cortical shell with some trabecular-like elements visualized using Picro Sirius Red/Alcian Blue staining (E). MicroCT quantification revealed no significant difference in bone volume of ectopic rhBMP-2 induced bone with deletion of the Osx gene in _Tie2_-lineage cells (F).

Figure 5

Figure 5

X-ray images showing the progression of rhBMP-2 induced spine fusion. After implantation of collagen sponges loaded with rhBMP-2, no mineralized bone was seen at D2, but a fusion mass was apparent in all specimens by D10 and D17. Anterior-posterior and lateral views are shown for all time points.

Figure 6

Figure 6

Fluorescent images of spine fusion bone masses with co-labeling for α_SMA_-lineage cells expressing tdTomato (red), _Col2.3_-GFP (green), and a TRAP activity stain (blue). At D2, no incorporation of α_SMA_-lineage progenitors was seen within the native spinous processes (green) or the implanted collagen sponge. At D10 and D17, _Col2.3_-GFP+ cells in the fusion mass were shown to originate from α_SMA_-lineage progenitors. No tdTomato+ signal was seen with TRAP+ cells at any time point. Scale bar = 50 μm.

References

    1. Wan M, Cao X. BMP signaling in skeletal development. Biochem Biophys Res Commun. 2005;328(3):651–7. - PubMed
    1. Axelrad TW, Einhorn TA. Bone morphogenetic proteins in orthopaedic surgery. Cytokine Growth Factor Rev. 2009;20(5–6):481–8. - PubMed
    1. Lounev VY, Ramachandran R, Wosczyna MN, Yamamoto M, Maidment AD, Shore EM, Glaser DL, Goldhamer DJ, Kaplan FS. Identification of progenitor cells that contribute to heterotopic skeletogenesis. J Bone Joint Surg Am. 2009;91(3):652–63. - PMC - PubMed
    1. Schindeler A, Liu R, Little DG. The contribution of different cell lineages to bone repair: exploring a role for muscle stem cells. Differentiation. 2009;77(1):12–8. - PubMed
    1. Liu R, Schindeler A, Little DG. The potential role of muscle in bone repair. J Musculoskelet Neuronal Interact. 2010;10(1):71–6. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources