Physical activity is a key determinant of bone mass and health, however during adulthood and ageing there appears to be a decrease in the ability to respond positively to exercise which is variable between individuals. While exercise is known to protect against the osteopo-rotic process with modest increases in BMD the exact cellular and molecular responses are poorly understood.

We have studied the effect of mechanical stimulation on bone histomorphometric parameters, osteocyte viability and gene expression in human trabecular bone maintained in a 3D bioreactor.

Trabecular bone cores were prepared from femoral head tissue removed from patients undergoing total hip arthroplasty and maintained in the bioreactor system for 3 (n= 4 patients), 7 (n=5 patients) or 28 days (n=1 patient). Cores (n=3 per patient) were either frozen directly on preparation (T0), placed in the bioreactor system and subjected to Mechanical stimulation (3000 μstrain in jumping exercise waveform repeated at 1Hz for 5 minutes daily) or maintained in the bioreactor system with no mechanical stimulation as control. After the experimental period total cell numbers, cell viability and apoptosis were determined in un-decalcified cryosections at specific distances throughout the bone cores by nuclear staining (DAPI), lactate dehydrogenase activity (LDH) and Nick Translation Assay respectively. Consecutive sections were collected and RNA extracted for gene expression analysis.

Mechanical stimulation was shown to increase Bone Formation Rate (BFR) as determined by Calcein label/ distance to bone surface in the 28 day experiment (BFR mcm/day Control 0.01 ± 0.0035 vs Load 0.055 ± 0.0036 p=0.0022). Expression of bone formation markers such as Alkaline Phosphatase and Collagen Type I was shown to increase in all patients however there was an individual variation in the response of Osteopontin to mechanical stimulation as determined by quantitative real time PCR expression analysis. Numbers of viable osteocytes at T0 varied between individual patients however viability was significantly increased and apoptosis decreased in association with mechanical stimulation compared to control in all patient samples examined (p to 0.021). Our data tend to support animal model findings relating to the osteocyte saving effects of exercise and provide an insight into the molecular detail of the exercise response in human bone.

Introduction: Evidence exists concerning the anti-oxidant properties of oestrogen in protecting neuronal cells from oxidative stress. The withdrawal of oestrogen after menopause is the major factor determining age related bone loss and apoptotic death of osteocytes. While oestrogen replacement demonstrates clear oestrogen receptor mediated benefits to bone cells little is known regarding oestrogens’ anti-oxidant effects in bone.

Methods: Here we have used MLO-Y4 osteocyte-like cell line to determine whether oestrogen saving effects on osteocytes involves its activities as an anti-oxidant.

MLO-Y4 cells were treated with physiological doses (10⁻⁸)M of either 17-beta E₂ or the oestrogen receptor inactive stereoisomer 17-alpha E₂ with or without the specific oestrogen receptor antagonist ICI 182,780 prior to the addition of 0.4milliM 30% (v/v) H₂O₂. Cellular apoptosis was determined using morphological and biochemical criteria.

Results: H₂O₂ induced an increase in apoptosis of MLO-Y4 (14.3 ± 3 SD vs control 1.4 ± 0.9). Pre-treatment of the cells with 17-beta E₂ significantly reduced H₂O₂ induced apoptosis (2.4 ± 0.96). Pre-treatment of cells with 17-alpha E₂ or ICI 182,780 also reduced oxidant induced apoptosis to 3.4 ± 1.5 SD and 7.0 ± 2.3 respectively.

The cellular production of reactive oxygen species was determined using the free radical indicator 2′7′- dichlorodihydrofluorescein diacetate. H₂O₂ induced increases in the number of ROS positive cells (34.6 ± 9.07 SD vs control 0.22 ± 0.39 SD). In contrast pre-treatment with both 17-beta E₂ and 17-alpha E₂ reduced the number of ROS positive cells associated with H₂O₂ treatment (Fig 1).

Conclusion: These data suggest that oestrogens ability to save osteocytes from oxidant induced death is independent of the oestrogen receptor and may be related to oestrogens known activity as an anti-oxidant. This raises the possibility that loss of osteocytes during oestrogen insufficiency may occur through a failure to suppress the activity of naturally occurring or disease associated production of oxidant molecules.

Clinical use of glucocorticoids engenders deleterious changes in bone fragility and initiates apoptosis in osteoblasts and osteocytes. The pathways leading to corticosteroid-induced death in bone remain unclear. Similarly little is known about the effects of ‘bone sparing’ bisphosphonates on osteocytes in vivo. We investigated the effects of bisphosphonates (BPs) on dexamethasone (Dex)-induced apoptosis in the murine osteocyte cell line, MLO-Y4 and studied the putative pathways involved by intervention with inhibitors of signalling molecules, such as p42/44 MAPK and protein kinase A (PKA). Cells were preincubated with N- & non N-containing BPs and/or inhibitors before insult with Dex or H₂O₂ for 5 hrs. Apoptotic morphology was revealed by acridine orange staining. Activation of p42/44 was identified using Western blotting and in situ immunocytochemistry in the presence or absence of serum.

Both N- & non N-containing BPs were shown to protect against cell death. The addition of inhibitors of p42/44 and PKA blocked the action of Dex. H₂O₂-induced apoptosis was not blocked by BPs or by any of the inhibitors. Dex appeared to activate p42/44 only in serum supplemented cultures. These data suggest that glucocorticoid but not oxidant-induced osteocyte apoptosis involves activation of p42/44 and that bisphosphonate engendered cell rescue is brought about by inhibition of these MAPK’s. Studies using truncated BPs that lack anti-resorptive activity, and therefore do not interrupt bone remodelling showed that these BPs were also able to protect osteocytes from glucocorticoid-induced death. The ability of bisphosphonates to influence MAPK activation and cell death in the osteocyte opens up exciting possibilities for pharmaceutical intervention during age and steroid hormone related osteocyte loss.