Meeson, Richard Lawrence; (2018) Stem cell mobilisation and homing to improve fracture healing. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Stem cell homing and migration is regulated through chemokine SDF-1 and its receptor CXCR4. In vitro studies in mice have demonstrated endogenous mobilisation of mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) by administering growth factors and AMD3100, which is an antagonist of CXCR4. The hypothesis of my study was that antagonism of the CXCR4-SDF1 axis would mobilise stem and progenitor cells into the circulation, and by increasing the available pool of cells in early fracture healing, improve bone formation. Peripheral blood MSCs and EPCs were isolated from rats treated with VEGF and AMD3100. Non-mobilised controls did not yield any viable MSC CFUs, whereas mobilised were significantly higher at 2.9±1.8 CFUs/ml blood (p=0.029). The MSCs were CD29 and CD90 positive and CD34 negative, however unlike bone marrow MSCs, they had a mixed CD45 expression. These cells were only able to differentiate down osteogenic lines in vitro. Mobilised EPCs had the typical ‘cobblestone’ morphology, were CD45 and CD34 negative, with low to variable expression of endothelial markers CD31 and VEGFR2, and MSC marker CD29. These cells had variable in vitro tube forming ability and could differentiate down adipogenic and not osteogenic lines. In order to evaluate the potential of endogenous mobilisation on fracture healing, a rat femoral osteotomy model stabilised with an external skeletal fixator was used. Several modifications were made to improve the reliability of the model and are reported in my thesis. The influence of gap size on interfragmentary strain and subsequent healing using this system was evaluated to identify the optimised situation to measure the effect of endogenous mobilisation. Mechanical analysis of the construct stiffness and the interfragmentary strain showed the gap size did not have an influence on the construct stiffness. However, increased gap size significantly reduced day 0 interfragmentary strain (p=0.013), with 1.0mm gap having significantly higher interfragmentary strain than the 2.0mm gap (p=0.029). To evaluate the effect of interfragmentary strain on healing, a femoral osteotomy in 12-14 week old female Wistar rats was stabilised with an osteotomy gap of 1.0mm, 1.5mm and 2.0mm. After five weeks, the 1.0mm gap had the largest callus (0.069um3) and bone volume per microCT slice (0.035um3). As the gap size increased, the bone volume per slice decreased, however significance was not found (p=0.082). Histomorphometry also showed the bone formation deceased as the gap increased. There was an increase in cartilage formation associated with the decrease in bone in the 1.5mm gap, whereas the 2.0mm gap had an increase in fibrous tissue associated with a decrease in cartilage and bone, indicating that the 2.0mm gap was tending towards fibrous non-union. The 1.5mm gap provided a suitable compromise to test endogenous mobilisation, and although the interfragmentary gap strain reduced as the gap enlarged, there was a reduction in osteotomy healing as the gap size increased. VEGF (V), IGF-1 (I) or GCSF (G) combined with AMD3100, or AMD3100 alone (A) were subsequently investigated using the rodent fracture model with a 1.5mm gap. At 5 weeks, groups V, I and A had increased bone formation compared to control animals, however, group A had a significant increase in bone volume (p=0.01) and group I a significant increase in % bone within the callus (p=0.035). Group G on the other hand, showed a decrease in bone volume. Irrespective of whether there was an increased or decreased bone content, all treated groups had an increase in trabecular thickness, or more accurately, thicker segments of woven bone, compared with the controls. Histomorphometric analysis showed decreased cartilage tissue was associated with increased bone in groups with improved healing, and was associated with increased fibrous tissue in poorly performing groups. Overall therefore, AMD3100 given alone significantly increased fracture healing over and above the control level. In conclusion, disruption of SDF1-CXCR4 axis can mobilise stem and progenitor cells, and can boost impaired fracture healing in a rat femoral osteotomy model.

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