The Contribution Of Tie2-Lineage Cells To rhbmp-2 Induced Bone Formation

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1 The Contribution Of Tie2-Lineage Cells To rhbmp-2 Induced Bone Formation Mille P. Kolind, Ph.D 1, Alastair Aiken 1, Kathy Mikulec 1, Lauren Peacock 1, David Little 1,2, Aaron Schindeler, PhD 1,2. 1 Orthopaedic Research and Biotechnology, The Children's Hospital at Westmead, Sydney, Australia, 2 University of Sydney, Sydney, Australia. Disclosures: M.P. Kolind: None. A. Aiken: None. K. Mikulec: None. L. Peacock: None. D. Little: 5; Amgen, Novartis AG, Celgene, N8 Medical. A. Schindeler: 5; N8 Medical. Introduction: Recombinant human bone morphogenetic proteins (rhbmps) can promote osteogenic differentiation and their implantation can produce ectopic bone and cartilage. The exact cellular origin and location of the cells that contribute to ectopic bone formation is still in a matter of controversy. Multiple progenitor lineages including myogenic cells, vascular endothelial cells, pericytes, haematopoietic cells, and circulating osteoprogenitors have been hypothesized to be involved (1). Tie2 has been used as a specific vascular endothelial cell marker in lineage tracing studies and using a Tie2-cre Rosa26R reporter mouse it was reported that this lineage significantly contributes to heterotopic ossification (2). Further studies have shown that vascular endothelial cells are multipotential in the context of ALK2 mutations, which underlie the genetic disease Fibrodysplasia Ossificans Progressiva (FOP) (3). However a more recent study reports that only a sub-population of Tie2 lineage cells that lack expression of other vascular endothelial markers such as CD31 can give rise to cartilage and bone cells (4). Tie2 is also expressed in haematopoietic cells and cells from this lineage are also labelled in reporter mice, although there is little support for the concept that this lineage can contribute to osteoblastic bone formation. In the present study we have used genetic mouse models to fluorescently track Tie2-lineage cells and their descendants in ectopic bone formation induced by rhbmp-2. We further addressed the functional contribution of Tie2-lineage cells to bone formation using conditional knock-out mice, where Tie2-lineage cells were unable to differentiate into osteoblasts. Methods: The vascular endothelial cell population was permanently labelled by crossing the Tie2 (B6.Cg-Tg (Tek-cre) 12Flv) lineage-specific Cre mice with the Cre-dependent reporter mice Ai9(B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato). The conditional knockout mice was created by crossing the Tie2-cre mice with the Osxflox/flox mice generated by Prof Benoit de Crombrugghe (5), to generate Tie2-Cre; Osxflox/flox conditional knockout mice. Control littermates consisted of mice negative for Cre. Ectopic bone was induced by bilateral intramuscular implantation of a collagen-ceramic scaffold doped with 10 µg rhbmp-2 (Medtronic). At 7 and 14 days after surgery contribution of vascular endothelial cells to bone formation were studied by immunofluorescence (n=3 mice per time point). 21 days post implantation the ectopic bone volume were quantified by micro-ct. Group sizes were n=12. Mice were housed at the Children's Hospital at Westmead (CHW) Transgenic Facility and at the Westmead Hospital Department of Animal Care. Animal experimentation was approved by the CHW/CMRI Animal Ethics Committee. Results: In rhbmp-2 induced ectopic bone there was a strong representation of Tie2 lineage (Tie2+) cells as expected (Fig. 1a & 1c). In the marrow space small vessels and small marrow-like cells were labelled, indicating that the Tie2-reporter labels haematopoietic cells in addition to vascular endothelial cells (Fig. 1b). Immunofluorescent staining using the endothelial marker (PECAM-1, CD31) confirmed that most Tie2+ cells in the early condensation/cartilage phase were also CD31+ (Fig 2a). CD31- /Tie2+ cells were also identified and these cells appeared as single cells close to areas of vascularisation (Fig 2a, arrows). The majority of Sox9+ chondrocytes were not Tie2+ (Fig 2b), but a very small number of Sox9+/Tie2+ double positive cells were detected (Fig 2b, asterisks). During the ossification stage TRAP+/Tie2+ double positive cells were detected in large numbers (Fig. 2c). However, alkaline phosphatase (AP) staining did not show any Tie2+/AP+ cells (Fig. 2d). Bone volume from of the 3 wk samples from Tie2-Cre; Osxflox/flox mice as compared to control littermates. (n=24) were not significant different. Discussion: In conclusion, our results show that the Tie2+ progenitor cells are abundantly present in the BMP-2 induced bone formed. The majority of these cells express the endothelial marker CD31, and we believe that they have a role in vascularisation and not make a significant direct contribution to the osteoblast population. Data from the functional study did not identify a significant difference in the bone volume in Tie2-Cre; Osxflox/flox mice, further supporting the concept that the majority of the anabolic contribution to ectopic bone does not come from Tie2-lineage cells. The finding that Tie2 lineage cells contribute to TRAP+ cells lining the ectopic bone surfaces may explain the findings of previous reporter studies that lacked double labelling (2). Instead we hypothesize the vascular endothelial cells are critical for attracting or facilitating the migration of bone progenitor cells to the ossification site, rather than undergoing osteoblastic transdifferentiation. Significance: This study brings new information regarding the contribution from vascular endothelial and haematopoietic cell to bone formation in ectopic bone studies that has direct translational relevance to stem cell approaches for improving bone repair. These data may also be relevant to strategies aimed at reducing pathological bone formation, including in FOP and myositis ossificans. Acknowledgments: This work was supported by grant funding from the National Health & Medical Research Council of Australia. The KRI Microscope Facility provided the Leica TCS SP5 confocal laser scanning microscope. References: References (1)Liu R et al. The potential role of muscle in bone repair. J Musculoskelet Neuronal Interact. 2010;10:71-6.

2 (2)Lounev VY et al. Identification of progenitor cells that contribute to heterotopic skeletogenesis. J Bone Joint Surg Am. 2009;91: (3)Medici D et al. Conversion of vascular endothelial cells into multipotent stem-like cells. Nat Med. 2010;16: (4)Wosczyna MN et al. Multipotent progenitors resident in skeletal muscle interstitium exhibit robust BMP-dependent osteogenic activity and mediate heterotopic ossification. J Bone Miner Res. 2012;27: (5)Akiyama H et al. Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors. Proc Natl Acad Sci USA. 2005;102:14665-

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