Comparative study of stem cell recruitment during postnatal bone formation
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INTRODUCTION: Post-natal fractures are among some of the most common orthopedic injuries with up to 10% resulting in delayed or non-unions. Understanding the early mechanism of fracture repair via stem cell recruitment will allow for more specific therapeutics to be developed. Two post-natal bone formation models were used for this study, ectopic bone and fracture repair; both show primarily endochondral ossification. To investigate the role of stem cells during post-natal bone formation, two markers were selected, based on previous results, Prx1 and Pax7. The Prx1 gene is expressed by skeletal progenitor cells within the periosteal tissues while the Pax7 gene is expressed by skeletal muscle precursor cells. OBJECTIVES: The purpose of this study is to follow stem cell lineages that arise from Prx1 and Pax7 expressing cells in both ectopic bone growth and fracture repair models. METHODS: Prx1 CreER-GFP mice (Kawanami et al., 2010) and Pax7 CreER (Jackson Laboratories) mice were individually crossed with the RosaAi14 reporter (dTomato) animal. These animals were then crossed with immunodeficient Rag1 mice allowing for implantation of human demineralized bone matrix. Six different control groups were analyzed with multiple mice used for each. These groups included no injury with Tamoxifen followed by three days (short term), seventeen days (intermediate) or 28 days and onward (long term) harvest, oil injections followed by three days (short term) or 30 days (long term) harvest, no injections, and Cre negative. Two methods of post-natal bone formation were studied, ectopic bone growth and fracture repair. For ectopic bone growth, demineralized bone matrix (DBM) was implanted on either the periosteum surface or and within skeletal muscle of the upper hind-limb. Samples were then harvested on day 16 or day 31 post-surgery. For post-natal fracture repair, mice received a transverse stabilized fracture and samples were harvested on day 5 or day 23. All samples were then fixed, decalcified, and standard frozen histology was performed. Images were collected with a fluorescent microscope to detect the presence of dTomato tagged Prx1 and Pax7 derived cells. Ratios of positive cells were calculated using total number of nuclei within the regions of interest. RESULTS: Prx1 control animals that received no Tamoxifen injections showed about 4-16% positively labeled cells. Cre+ control that received Tamoxifen washout showed the highest percentage of cells, 10-20%. Control animals that receive oil pulse and oil washout showed similar results, about 6-20%. Interestingly, the Cre- control group showed a high number of labeled cells, about 6-12%. Prx1 positive cells were seen throughout the bone (16-30%) and callous (43-70%) in the fracture model and throughout the bone (43%) and implant (30-47%) in the ectopic bone development model. Pax7 control animals that received no tamoxifen injections and animals that received tamoxifen long term injections both showed the highest percentage of cells, 2% in the muscle. No response was seen in the bone or marrow for either of the control groups. No Pax7 derived cells were seen in the bone or callous in the fracture model or the bone and implant in the ectopic bone model. CONCLUSIONS: Previous research has shown Prx1 cells to be localized to the periosteum. This study confirmed those results and showed an increase in Prx1 derived cells in the callous in the fracture model. An increase in Prx1 was also seen in the implant in the ectopic bone growth model. However, at a later time point, the number of Prx1 cells decreased in both the inner callous and implant suggesting either the presence of bone remodeling or a different population of stem cells contributing at later in time. With regards to Pax7, previous research has suggested a role in postnatal bone formation. However, this study showed that no Pax7 derived cells were seen in bone or the fracture callous in the fracture model nor in the implant in the ectopic bone growth model. Therefore, it does not appear Pax7 has any role in postnatal bone formation.