Uncultured Autogenous Adipose-derived Regenerative Cells Promote Bone Formation During Distraction Osteogenesis in Rats

Abstract

Adipose-derived stem cells have recently shown differentiation potential in multiple mesenchymal lineages in vitro and in vivo. These cells can be easily isolated in large amounts from autologous adipose tissue and used without culturing or differentiation induction, which may make them relatively easy to use for clinical purposes; however, their use has not been tested in a distraction osteogenesis model. The question of this animal study in a rodent model of distraction osteogenesis was whether uncultured adipose-derived regenerative cells (ADRCs), which can easily be isolated in large amounts from autologous adipose tissue and contain several types of stem and regenerative cells, promote bone formation in distraction osteogenesis. We evaluated this using several tools: (1) radiographic analysis of bone density; (2) histological analysis of the callus that formed; (3) biomechanical testing; (4) DiI labeling (a method of membrane staining for postimplant celltracing); and (5) real-time polymerase chain reaction. Sixty rats were randomly assigned to three groups. Physiological saline (control group), Type I collagen gel (collagen group), or a mixture of ADRC and Type I collagen gel (ADRC group) was injected into the distracted callus immediately after distraction termination. To a rat femur an external fixator was applied at a rate of 0.8 mm/day for 8 days. The bone density of the distracted callus in the ADRC group increased by 46% (p = 0.003, Cohen's d = 10.2, 95% confidence interval [CI] ± 0.180) compared with the control group at 6 weeks after injection. The fracture strength in the ADRC group increased by 66% (p = 0.006, Cohen's d = 1.32, 95% CI ± 0.180) compared with the control group at 6 weeks after injection. Real-time reverse transcription-polymerase chain reaction of the distracted callus from the ADRC group had higher levels of bone morphogenetic protein-2 (7.4 times higher), vascular endothelial growth factor A (6.8 times higher), and stromal cell-derived factor-1 (4.3 times higher). Cell labeling in the newly formed bone showed the ADRCs differentiated into osseous tissue at 3 weeks after injection. The injection of ADRCs promoted bone formation in the distracted callus and this mechanism involves both osteogenic differentiation and secretion of humoral factors such as bone morphogenetic protein-2 or vascular endothelial growth factor A that promotes osteogenesis or angiogenesis. The availability of an easily accessible cell source may greatly facilitate the development of new cell-based therapies for regenerative medicine applications in the distraction osteogenesis.