The only currently approved anabolic therapy that increases bone mass and reduces fracture risk in patients with osteoporosis is parathyroid hormone (PTH). To investigate how PTH increases bone mass and improves bone strength, I have investigated the function of two gene products regulated by PTH in osteoblasts on bone structure and strength (gp130 and ephrinB2).

Gp130 deletion in late osteoblasts/osteocytes resulted in greater femoral dimensions indicating a role for osteocytic gp130 in maintaining bone width. Although the bending load required to fracture these bones was not altered, they showed a significant reduction in material strength which was associated with a greater proportion of disorganised woven bone suggesting that the increased bone width may have been a compensatory mechanism for poor bone quality. This indicated that gp130 in late osteoblasts/osteocytes maintains the material strength of the cortical bone matrix collagen production and the deposition of organised lamellar bone.

The role of another PTH downstream target, ephrinB2, in controlling bone strength and quality was investigated in both osteoblasts and osteocytes. Work conducted prior to my PhD showed that specific deletion of ephrinB2 within the entire osteoblast lineage in 12- week old female mice caused osteoblast apoptosis and delayed initiation of bone mineralisation, indicating a role for ephrinB2 in osteoblasts that promotes mineralisation by preventing apoptosis. During my PhD, I found that mice lacking ephrinB2 in the osteoblast lineage had slender bones which were more compliant when assessed by mechanical testing. Despite their greater bending ability, their bones were more fragile but they exhibited no change in bone composition measured by synchrotron-based Fourier transform infrared microscopy (sFTIRM). This suggested that the previously observed delay in the initiation of mineralisation caused by ephrinB2-deficient osteoblasts may have resulted in the fragile bone phenotype.

Since ephrinB2 is expressed throughout the entire osteoblast differentiation pathway, I next sought to determine the role of ephrinB2 in late osteoblasts/osteocytes in 12-week old female mice. Mice lacking ephrinB2 in osteocytes showed an opposing strength phenotype compared to the earlier deletion of ephrinB2 mentioned above. Their bones were more brittle due to greater mineral deposition and parallel stretching/compression of the collagen fibres detected by sFTIRM, highlighting stage-specific roles of ephrinB2 during osteoblast differentiation that regulate bone size and material composition. Correlation analysis revealed that the relationships between collagen fibre alignment and bending strength and between mineral composition and material toughness that normally exist in control mice were lost in bones from mice lacking ephrinB2 in late osteoblasts/osteocytes. This demonstrated that ephrinB2 within late osteoblasts/osteocytes restrains mineral deposition to maintain bone strength.

I next investigated the trabecular bone structure in these mice and found that deletion of ephrinB2 in late osteoblasts/osteocytes resulted in greater trabecular bone containing enlarged osteoclast size observed in vivo suggesting impaired osteoclast function. This may also relate to greater carbonate content within the bone mineral which requires further investigation.

Finally, I assessed whether osteocytic ephrinB2 is required for the anabolic action of PTH. In contrast to impaired anabolic effect of PTH in mice lacking ephrinB2 within the entire osteoblast lineage, PTH treatment in mice lacking ephrinB2 in late osteoblasts/osteocytes did not significantly impair the anabolic action of PTH on trabecular and cortical bone formation. This led me to use the sFTIRM technique to analyse bone composition in vehicle and PTH-treated bones from control mice. This showed that bone deposited during PTH treatment undergoes a normal process of collagen maturation and mineral accrual.

In conclusion, downstream targets of PTH, gp130 and ephrinB2 play distinct roles in regulating bone size, strength and quality. This highlights that osteoblasts utilise these proteins during the formation of bone matrix and the initiation of its mineralisation. In contrast, osteocytes maintain the quality and composition of bone matrix and mineral by regulating mineral accrual and collagen arrangement. Understanding how specific genes expressed in these cells can control bone strength, coupled with the sFTIRM technique developed during my PhD, can provide a new way for investigating different causes of bone fragility. Current diagnostic tests cannot fully predict risk of fracture and treatment options are not patient specific and are limited in their use. In the long term, this study may allow specific targeting of cell types to increase bone mass and improve bone quality which could lead to the development of different personalised treatments for bone fragility.