Close to the bone: investigations into bone tissue mineralisation and mechanobiology of osteoporosis

Abstract

Osteoporosis is a metabolic skeletal disease characterized by low bone mass, depleted micro-architecture and reduced strength. The public health costs of osteoporosis relate almost entirely to the fractures that are the clinical manifestation of the disease and it presents a significant cause of morbidity in today’s ageing population. Oestrogen deficiency during the menopause is the primary causative factor for postmenopausal osteoporosis and although much is known about the pathophysiology of the disease, including dysregulated bone cell function whereby more bone is digested than is formed; the underlying mechanisms involved have not yet been delineated. Recent studies have suggested that although overall strength is decreased following osteoporotic bone loss, the remaining bone tissue is stronger and stiffer, suggesting an alteration in bone tissue composition. Bisphosphonates are among drug treatments administered to tackle bone loss, however the incidence of osteoporotic fractures still remains high. Furthermore, the precise effect of drug treatment on bone tissue mineralisation is unknown.

The global aim of this thesis is to discern the alterations in the quantity and distribution of bone mineral during osteoporosis. Specifically, it is sought to test the hypotheses that bone mineral distribution is altered at a tissue level following oestrogen deficiency and bisphosphonate treatment and that oestrogen depletion alters normal mineralisation and mechano-responsiveness of bone cells. Quantitative backscattered imaging (qBEI) on a scanning electron microscope was used to examine individual bone trabeculae from the proximal femur of ovariectomised sheep (oestrogen deficient state), aged matched control sheep and sheep treated with the bisphosphonate Zoledronic acid. It was found that oestrogen deficiency caused significantly higher mineral heterogeneity within trabeculae (site speficic within the femur) and along a common osteoporotic fracture line. Bone mineralisation was diminished with prolonged oestrogen deficiency and conversely was higher in older healthy sheep compared to younger control sheep. Furthermore, significantly lower mineral heterogeneity was found in OVX sheep treated with Zoledronic acid compared to untreated OVX sheep. These results indicate that changes in bone tissue mineralisation during oestrogen deficiency may be a contributing factor for reduced mechanical strength during osteoporosis, while drug induced increased homogeneity may contribute to the ability of Zoledronic acid to prevent fracture occurrence during oestrogen deficiency.

The next study aimed to delineate the mechanisms responsible for such altered mineral distribution. Osteoblast and osteocyte cells were pre-treated with oestrogen and the effects of oestrogen deficiency were evaluated by subsequently withdrawing oestrogen from cells, or blocking oestrogen receptors using an oestrogen antagonist, fulvestrant. Specifically, alkaline phosphatase expression was investigated using p-nitrophenyl phosphate (pNPP), proliferation by assessing DNA content, calcium production using alizarin red assay and apoptosis by measuring for caspase 3/7 activity. Although mineral production was significantly increased by oestrogen pre-treatment, a further increase in mineral production and apoptosis were observed following oestrogen withdrawal from cells. These observations increase our understanding of the mechanisms controlling bone formation and bone cell death and may aid in the development of enhanced therapeutics for the treatment of osteoporosis.

The final study of this thesis aimed to determine if the mechano-biological response of osteoblasts is impaired during oestrogen deficiency and whether changes in bone mineralisation may be related to altered bone formation in response to mechanical stimulation. Osteoblasts were pre-treated with oestrogen and subsequently oestrogen was withdrawn from cell cultures and their responses under fluid shear stress were evaluated. Firstly, daily loading cycles, using an orbital rotator, were applied to cells and mineralisation and cell viability (using alamar blue assay) were assessed after 7 and 14 days. In a separate experiment, following 2 and 7 days of oestrogen withdrawal, osteoblasts were exposed to 2 hours of shear stress in a custom designed parallel plate bioreactor. PGE2 was quantified in cell culture conditioned media using an immunoassay kit. It was found that orbital fluid flow induced shear stress significantly increased mineral production by bone cells and that under an applied shear stress, mineral production was decreased during oestrogen withdrawal. It was also observed that mechanical loading and oestrogen are required in unison to promote mineral production.

PGE2 release was significantly increased with applied laminar flow, but was decreased by oestrogen withdrawal. Together, these studies provide evidence that bone cells become accustomed to levels of circulating oestrogen and that diminished oestrogen causes osteocyte apoptosis, increased osteoblast mineralisation and altered mechano-sensitivity. These changes might explain the decreased mean concentrations of mineral, together with increased mineral heterogeneity, from our earlier in vivo studies. Therefore, the results of the thesis provide a unique insight into why the tightly coupled mechanisms of matching bone’s structure and composition to the loads it experiences are disrupted when levels of circulating oestrogen are depleted.

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