دانلود رایگان مقاله عدم همکاری سلول خونساز به تشکیل استخوان

ABstract

Despite years of extensive investigation, the cellular origin of heterotopic ossification (HO) has not been fully elucidated. We have previously shown that circulating bone marrow-derived osteoblast progenitor cells, characterized by the immunophenotype CD45−/CD44+/CXCR4+, contributed to the formation of heterotopic bone induced by bone morphogenetic protein (BMP)-2. In contrast, other reports have demonstrated the contribution of CD45+ hematopoietic derived cells to HO. Therefore, in this study, we developed a novel triple transgenic mouse strain that allows us to visualize CD45+ cells with red fluorescence and mature osteoblasts with green fluorescence. These mice were generated by crossing CD45-Cre mice with Z/RED mice that express DsRed, a variant of red fluorescent protein, after Cre-mediated recombination, and then crossing with Col2.3GFP mice that express green fluorescent protein (GFP) in mature osteoblasts. Utilizing this model, we were able to investigate if hematopoietic derived cells have the potential to give rise to mature osteoblasts. Analyses of this triple transgenic mouse model demonstrated that DsRed and GFP did not co-localize in either normal skeletogenesis, bone regeneration after fracture, or HO. This indicates that in these conditions hematopoietic derived cells do not differentiate into mature osteoblasts. Interestingly, we observed the presence of previously unidentified DsRed positive bone lining cells (red BLCs) which are derived from hematopoietic cells but lack CD45 expression. These red BLCs fail to produce GFP even under in vitro osteogenic conditions. These findings indicate that, even though both osteoblasts and hematopoietic cells are developmentally derived from mesoderm, hematopoietic derived cells do not contribute to osteogenesis in fracture healing or HO.

4. Discussion

Although many efforts have been made, the cellular origin of osteoblasts responsible for HO is a long standing question which has yet to be solved [20]. Various candidate cells, both hematopoietic and non-hematopoietic, derived from different germ layers have been shown to potentially contribute to HO [1,3–7,12,21–24]. We have previously demonstrated that circulating osteoblast progenitor cells originating from bone marrow contributed to the development of heterotopic bone induced by BMP-2 [3,4]. These circulating osteoblast progenitor cells are negative for CD45, suggesting a non-hematopoietic origin of HO osteoblasts [4]. Contrary to our findings, others have reported that CD45 positive circulating cells formed HO [5,8,25,26]. Therefore, in this study, we traced hematopoietic cells using CD45-Cre mice and investigated whether hematopoietic derived cells contributed to skeletogenesis, HO and fracture healing. The advantage of using the Cre/loxP system is the ability to trace descendant cells derived from Cre-expressing cells, even if they discontinue expressing Cre. In our mouse model, cells that express CD45 at any developmental stage become DsRed positive and remain positive for DsRed even if CD45 expression turns off. In other words, all DsRed positive cells in our experimental animals originated from CD45 positive hematopoietic cells. Our results showed that over 90% of CD45 positive cells expressed DsRed, validating the efficiency of Cre-mediated recombination in our animal model. Additionally, we combined this Cre/loxP murine system with Col2.3GFP mice which are known to label mature osteoblasts with GFP [17], allowing us to evaluate if hematopoietic derived cells can give rise to mature osteoblasts. Our analyses of these triple transgenic mice demonstrated that hematopoietic derived cells did not differentiate into mature osteoblasts either in developmental skeletogenesis, in HO, or in bone regeneration after fracture. These results were supported by our findings that hematopoietic derived cells did not have the potential to become mature osteoblasts even under in vitro osteogenic conditions. Taken together, the results led us to conclude that hematopoietic derived cells are not responsible for heterotopic bone formation as osteoblasts in murine models. Possible explanations for the discovered inconsistencies with the previous findings by Suda et al. showing the contribution of CD45 positive circulating osteogenic precursor (COP) cells to heterotopic bone formation in patients with FOP [5] could be: 1) species differences between humans and mice; 2) our HO model induced by BMP-2 might not exactly reflect the pathogenesis and pathophysiology of patients with FOP; and 3) imperfection of Cre-mediated recombination, despite over 90% efficiency, might fail to label the CD45 positive COP cells, resulting in the failure to detect the contribution of these cells to HO.