To define how bone formation is controlled, we need to identify transitions from stem/progenitor cells to osteoblasts. Osteoblast precursors first commit to the lineage by expressing Sp7 (osterix) and when differentiated into osteoblasts, express high levels of Col1a1.
To define stages of osteoblast differentiation, we studied 12 week old (young adult) mice with fluorescence targeted to the osteoblast lineage (OsxCherry) or to mature osteoblasts (Col1a1GFP). Additionally, we generated an osteoblast lineage dual-reporter (DR) mouse that distinguishes osteoblasts from osteoblast precursors by these fluorescent markers, and assessed distribution of these cells on bone surfaces by cryohistology.
In Col1a1GFP mice, Col1a1GFP+ cells covered ~30% of all bone surfaces, which were almost entirely active bone forming surfaces (identified by Alizarin Red labelling). In contrast, in OsxCherry mice, >90% of periosteal and endocortical surfaces, and 52% of trabecular bone surfaces had OsxCherry+ cells. Within OsxCherry+ cells, 78% and 92% were on active bone forming surfaces (calcein-labelled) on endocortical and trabecular bone, respectively. However, on the periosteal surface only 29% of OsxCherry+ cells resided on actively forming bone. By flow cytometry, periosteal OsxCherry+ cells had a 3-5x greater proportion of cells expressing progenitor markers (Sca1+CD51+) than bone marrow OsxCherry+ cells.
By crossing OsxCherry and Col1a1GFP mice to generate DR mice, we could distinguish osteoblast precursor cells (OsxCherry+Col1a1GFP-) from osteoblasts (OsxCherry+Col1a1GFPhi). This confirmed the distinct distributions of cell populations on bone surfaces: the periosteum was lined predominantly by OsxCherry+Col1a1GFP- cells whilst other surfaces were covered by OsxCherry+Col1a1GFPhi cells. Furthermore, periosteal and bone marrow stromal cells from DR mice expressed changes in fluorescence as they differentiated in vitro.
We have developed a model that distinguishes between osteoblast precursors and osteoblasts in vivo and in vitro. We also identified osteoblast precursors are the predominant cell type residing on periosteal, but not trabecular and endocortical, surfaces under physiological conditions.