Local Transplantation of Granulocyte Colony-Stimulating Factor-Mobilized Human Peripheral Blood Mononuclear Cells for Unhealing Bone Fractures

Abstract

We previously reported the therapeutic potential of human peripheral blood (hPB) CD34 + cells for bone fracture healing via vasculogenesis/angiogenesis and osteogenesis. Transplantation of not only hPB CD34 + cells but also hPB total mononuclear cells (MNCs) has shown their therapeutic efficiency for enhancing ischemic neovascularization. Compared with transplantation of purified hPB CD34 + cells, transplantation of hPB MNCs is more attractive due to its simple method of cell isolation and inexpensive cost performance in the clinical setting. Thus, in this report, we attempted to test a hypothesis that granulocyte colony-stimulating factor-mobilized (GM) hPB MNC transplantation could also contribute to fracture healing via vasculogenesis/angiogenesis and osteogenesis. Nude rats with unhealing fractures received local administration of the following materials with atelocollagen: 1 × 10 7 GM hPB MNCs (Hi group), 1 × 10 6 GM hPB MNCs (Lo group), or PBS (PBS group). Immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated human cell-derived vasculogenesis and osteogenesis in the Hi and Lo groups, but not in the PBS group at week 1. Intrinsic angiogenesis and osteogenesis assessed by rat capillary, osteoblast density, and real-time RT-PCR analysis was significantly enhanced in the Hi group compared to the other groups. Blood flow assessment by laser doppler perfusion imaging showed a significantly higher blood flow ratio at week 1 in the Hi group compared with the other groups. Morphological fracture healing was radiographically and histologically confirmed in about 30% of animals in the Hi group at week 8, whereas all animals in the other groups resulted in nonunion. Local transplantation of GM hPB MNCs contributes to fracture healing via vasculogenesis/angiogenesis and osteogenesis.

Emerging Role of Circulating Calcifying Cells in the Bone-Vascular Axis

“…Therefore, a series of studies have explored the use of PB CD34+ cells or EPCs for bone fracture healing. 83–86 Using a mouse bone fracture model, these studies first confirmed that fractures trigger EPC mobilization and incorporation into fracture sites. 83 The overlapping origin of endothelial and osteogenic markers was confirmed by Matsumoto et al 84 showing that about 20% of human peripheral blood CD34+ cells expressed the mRNA for OC.…”

“…85 Importantly, CD34+ cells were more effective than mononuclear cells (MNCs) in inducing fracture healing, suggesting that stem/progenitor cell properties were involved. 86 Following these series of studies, researchers demonstrated fracture-induced mobilization of EPCs for bone healing in mice, rats, and humans. 87–89 EPCs were also reported to contribute to bone healing in rat segmental bone defect model and critical-sized bone defects in sheep.…”

Local Transplantation of Granulocyte Colony Stimulating Factor-Mobilized CD34+ Cells for Patients With Femoral and Tibial Nonunion: Pilot Clinical Trial

“…Infusion of the crude BM cells might cause similarly unfavorable events in the case of fracture. In addition, we reported the advantages of CD34 + cell transplantation over MNCs for fracture healing [21].…”

“…Next, we attempted local transplantation of CD34 + cells with atelocollagen gel, a bioabsorbable scaffold, in the same animal model and demonstrated the similar effect at the lower dose compared with the systemic administration [20]. In addition, we reported the advantages of CD34 + cell transplantation over mononuclear cells (MNCs) for fracture healing [21].…”

Superior Potential of CD34-Positive Cells Compared to Total Mononuclear Cells for Healing of Nonunion following Bone Fracture

“…In both of our previous studies focusing on the local transplantation of human GM-PB CD34 + cells and human GM-PB MNCs immediately after the fracture, immunohistochemistry and real-time RT-PCR for human-specific markers around the fracture sites indicated that one of the mechanisms underlying the angiogenic and osteogenic effects of these cells may be direct differentiation into endothelial and osteoblastic lineages. Furthermore, paracrine effects of the transplanted cells on resident cells were also proved by quantitative real-time RT-PCR for rat-specific primers for angiogenic and osteogenic growth factors or cytokines (14,48). Regarding the therapeutic potential of GM-PB MNCs and CD34 + cells for fracture healing, the union rate of fractures following human GM-PB MNC local transplantation in our previous study (33% at week 8) (14) was obviously lower compared with the union rate after human GM-PB CD34 + cell transplantation demonstrated in another study (100% at week 8) (48).…”