Xenogeneic Endothelial Progenitor Cell Therapy in Variably Immune-Deficient Small Animal Models of Nonunion

Non-union and bone defects remain a major challenge in orthopedic trauma surgery. Though the current standard treatment is autologous bone grafting, this approach has many shortcomings. A promising experimental alternative to this treatment is endothelial progenitor cell (EPC) therapy. While EPCs have been shown to be highly effective in preclinical models of non-union, this therapy has not been evaluated in clinical trials. A major barrier to translation is the lack of evidence outlining the effectiveness of human EPCs for bone healing, with rodent EPCs used in preclinical work potentially differing from humans EPCs in several facets. Thus, the effectiveness of human EPC therapy for the treatment of non-union remains unknown and must be tested prior to proceeding to clinical trials. This requires translational studies evaluating human EPCs in an appropriate model of non-union, necessitating the xenografting of EPCs, and a corresponding animal model capable of accepting or tolerating such cells. Accordingly, this study sought to evaluate potential models of non-union for the study of xenogeneic EPC therapy. Porcine bone marrow derived EPCs (pBM-EPC) were used to provide a xenogeneic source of EPCs in lieu of human EPCs for this work. EPCs were isolated from bone marrow samples collected from Yorkshire pigs by adapting established methods of EPC isolation and culture. Following culture, cells were seeded onto collagen gel foam scaffolds at 2×10 6 cells per scaffold. These cells were then delivered to animals from one of the three following groups of immunodeficient rats: 1) Rowett Nude (RNU) rats, which are congenitally T cell deficient (n = 6), 2) Immunosuppressed (IS) Fischer 344 rats, rendered functionally T and partially B cell deficient by a 1.33 mg/kg/d dose of FK506 beginning the day of treatment (n = 5), and 3) Sprague Dawley Recombination activator 2 Interleukin-2 receptor-ɣ (SRG) rats, which are T-, B- and NK- cell deficient due to a double gene knockout (n = 5). All animals had a five to six mm critical sized defect generated in their right femur to model non-union. Defects were stabilized with a mini-plate and screws and all animals received pBM-EPC seeded gel foam scaffolds to the defect site. Post-operatively, animals were kept for 10 weeks with serial x-rays collected throughout. At 10 weeks, femora were harvested for assessment of bone healing by x-ray scoring, microCT analysis, and torsional biomechanical testing. SRG animals demonstrated an 80% rate of union, compared to 0% in the IS and RNU rats, with correspondingly greater radiographic scores (p < 0.05). Similarly, the SRG animals demonstrated greater total bone volume and bone volume fraction on microCT analyses compared to RNU and IS rats (p < 0.05). Finally, SRG animals demonstrated greater ultimate torque, yield point and maximum stiffness compared to RNU and IS animals (p < 0.05 for all outcomes). Overall, these results suggest that the SRG rat is a viable model to study xenogeneic EPC therapy for the treatment of non-union, with the degree of bone healing correlateing to the immunodeficency of the recipient.