Reconstruction of bone due to surgical removal or disease-related bony defects is a clinical challenge. It is known that the immune system exerts positive immunomodulatory effects on tissue repair and regeneration. In this study, we evaluated the in vivo efficacy of autologous neutrophils on bone regeneration using a rabbit calvarial defect model. Methods: Twelve rabbits, each with two surgically created calvarial bone defects (10 mm diameter), were randomly divided into two groups; (i) single application of neutrophils (SA-NP) vs. SA-NP control, and (ii) repetitive application of neutrophils (RA-NP) vs. RA-NP control. The animals were euthanized at 4 and 8 weeks post-operatively and the treatment outcomes were evaluated by micro-computed tomography, histology, and histomorphometric analyses. Results: The micro-CT analysis showed a significantly higher bone volume fraction (bone volume/total volume) in the neutrophil-treated groups, i.e., median interquartile range (IQR) SA-NP (18) and RA-NP (24), compared with the untreated controls, i.e., SA-NP (7) and RA-NP (14) at 4 weeks (p < 0.05). Similarly, new bone area fraction (bone area/total area) was significantly higher in neutrophil-treated groups at 4 weeks (p < 0.05). Both SA-NP and RA-NP had a considerably higher bone volume and bone area at 8 weeks, although the difference was not statistically significant. In addition, immunohistochemical analysis at 8 weeks revealed a higher expression of osteocalcin in both SA-NP and RA-NP groups. Conclusions: The present study provides first hand evidence that autologous neutrophils may have a positive effect on promoting new bone formation. Future studies should be performed with a larger sample size in non-human primate models. If proven feasible, this new promising strategy could bring clinical benefits for bone defects to the field of oral and maxillofacial surgery.
Keywords: bone regeneration; neutrophils; osteogenesis; rabbit calvarial defect model.