We have developed a novel technique to analyse bone, using imaging mass cytometry (IMC) without the constraints of using immunofluorescent histochemistry. IMC can measure the expression of over 40 proteins simultaneously, without autofluorescence. We analysed mitochondrial respiratory chain (RC) protein deficiencies in human bone which are thought to contribute to osteoporosis with increasing age.

Osteoporosis is characterised by reduced bone mineral density (BMD) and fragility fractures. Humans accumulate mitochondrial mutations and RC deficiency with age and this has been linked to the changing phenotype in advancing age and age-related disease. Mitochondrial mutations are detectable from the age of 30 onwards, coincidently the age BMD begins to decline. Mitochondria contain their own genome which accumulates somatic variants at around 10 times the rate of nuclear DNA. Once these mutations exceed a threshold, RC deficiency and cellular dysfunction occur. The PolgD257A/D257A mouse model expresses a proof-reading deficient version of PolgA, a mtDNA polymerase. These mice accumulate mutations 3-5 times higher than wild-type mice showing enhanced levels of age-related osteoporosis and RC deficiency in osteoblasts.

Bone samples were analysed from young and old patients, developing a protocol and analysis framework for IMC in bone tissue sections to analyse osteoblasts in-situ for RC deficiency.

Samples from the femoral neck of 10 older healthy volunteers aged 40 – 85 were compared with samples from young patients aged 1-19. We have identified RC complex I defect in osteoblasts from 6 of the older volunteers, complex II defects in 2 of the older volunteers, complex IV defect in just 1 older volunteer, and complex V defect in 4 of the older volunteers.

These observations are consistent with the PolgD257A/D257A mouse-model and suggest that RC deficiency, due to age-related pathogenic mitochondrial DNA mutations, may play a significant role in the pathogenesis of human age-related osteoporosis.

The pathogenesis of falling bone mineral density (BMD) as a universal feature of advancing age is poorly understood¹. Frequently culminating in the development of osteoporosis, the process is attributable to more than 500,000 fragility fractures occurring every year in the UK Such injuries are associated with great levels of morbidity, mortality and a £3.5 billion cost to the healthcare economy².

With age, humans are known to accumulate somatic mitochondrial DNA (mtDNA) mutations in mitotic and post mitotic tissue, and stem cell precursors³. Compelling evidence in recent years, particularly that provided by animal models suggests that these mutations are intrinsic to the ageing process^4–6. We provide evidence for the first time that mitochondrial dysfunction contributes significantly to the failure of bone homeostasis and falling BMD.

We have utilised a mouse model that accumulates mtDNA mutations at 3–5 times the rate of normal mice, consequently ageing and developing osteoporosis prematurely⁷, to clearly demonstrate that osteoblasts are vulnerable to mtDNA mutations. We have developed a new quadruple immunofluorescent assay to show that mitochondrial respiratory chain dysfunction occurs in osteoblasts as a consequence (p < 0.0001). We show that this mitochondrial dysfunction is associated with reduced BMD in female and male mice by 7 (p = 0.003) and 11 (p = 0.0003) months of age respectively. Using osteoblasts derived from mesenchymal stem cells extracted from male and female mice with mitochondrial dysfunction aged 4, 7 and 11 months, we demonstrate a vastly reduced capacity to produce new mineralised bone in vitro when compared to wild type cell lines (p < 0.0001). Exercise was found to have no beneficial effect on osteoblast and whole bone phenotype in this mouse model. It is likely that mtDNA mutations accumulating over a longer time period in human ageing have significantly detrimental effects on bone biology and diminishing BMD.