Identification of circulating murine CD34+OCN+ cells

Abstract

Background aims: Previous studies identified a circulating human osteoblastic population that expressed osteocalcin (OCN), increased following fracture and pubertal growth, and formed mineralized colonies in vitro and bone in vivo. A subpopulation expressed CD34, a hematopoietic/endothelial marker. These findings led to our hypothesis that hematopoietic-derived CD34⁺OCN⁺ cells exist in the circulation of mice and are modulated after fracture.

Methods: Flow cytometry was used to identify CD34⁺OCN⁺ cells in male B6.SJL-Ptprc^aPepc^b/BoyJ and Vav-Cre/mTmG (VavR) mice. Non-stabilized tibial fractures were created by three-point bend. Fractures were longitudinally imaged by micro-computed tomography, and immunofluorescent staining was used to evaluate CD34⁺OCN⁺ cells within fracture callus. AMD3100 (10 mg/kg) was injected subcutaneously for 3 days and the CD34⁺OCN⁺ population was evaluated by flow cytometry.

Results: Circulating CD34⁺OCN⁺ cells were identified in mice and confirmed to be of hematopoietic origin (CD45⁺; Vav1⁺) using two mouse models. Both circulating and bone marrow-derived CD34⁺OCN⁺ cells peaked three weeks post-non-stabilized tibial fracture, suggesting association with cartilage callus transition to bone and early mineralization. Co-expression of CD34 and OCN in the fracture callus at two weeks post-fracture was observed. By three weeks, there was 2.1-fold increase in number of CD34⁺OCN⁺ cells, and these were observed throughout the fracture callus. AMD3100 altered CD34⁺OCN⁺ cell levels in peripheral blood and bone marrow.

Discussion: Together, these data demonstrate a murine CD34⁺OCN⁺ circulating population that may be directly involved in fracture repair. Future studies will molecularly characterize CD34⁺OCN⁺ cells, determine mechanisms regulating their contribution, and examine if their number correlates with improved fracture healing outcomes.

Keywords: circulating osteoprogenitor; fracture; hematopoietic stem cell; mesenchymal stromal cell; mobilization.