Clinical and laboratory studies of mesenchymal stem cells in long bone fracture nonunion

Treatment of atrophic long bone fracture nonunion is challenging with current therapeutic interventions including bone morphogenetic proteins (BM Ps) or autologous mesenchymal stem cells (MSCs). In this work it was hypothesised that, total MSC numbers and their responsiveness to BMPs in the nonunion setting were compromised, leading to poor healing. Additionally, the rationale for systemic injection of MSCs to repair bone is based on a contentious concept of their widespread circulation. To address the number and functional competence of iliac crest bone marrow MSCs in nonunion, this study employed colony forming unit fibroblast and osteoblast assays (CFU-F, CFU-O) and flow cytometry enumeration of the CD45lowCD271+ cell population, to compare nonunions (n=11) with united long bone fracture patients (n=11). Unexpectedly, total number of MSCs, was higher in nonunion; however their proliferative capacity, was lower. No response to BMP-7, assessed by CFU-F, CFU-O and calcium deposition assays, was found in both nonunion and union study groups. Possible mechanical translocation of MSCs into the venous circulation was investigated using matched antecubital venous blood from the upper limb (UL) and lower limb femoral venous blood (LL) samples from nonunion patients undergoing reaming with reamer irrigation aspiration (RIA) (n=12) and other non-reaming procedures (NR, n=12) with control groups including UL from early rheumatoid arthritis (RA, n=11) and healthy controls (n=12). CFU-F assay results revealed the presence of MSC colonies in LL at higher frequencies than UL samples in RIA and NR, (8LL, 2PB) and (SLL, 1 PB) respectively. None were detected in the UL of RA and controls. Altogether, these results indicate a functional defect in proliferative capacity of MSCs in nonunion. MSCs however are unlikely to circulate and contribute to reduced healing at fracture sites. This points towards a generalized systemic effect of the nonunion state on MSC dynamics, which should be further explored in future.