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.

Bone substitutes have emerged as a promising alternative in surgeries requiring bone grafting, with a large array of materials available for today’s surgeon. Unfortunately, there is currently no definitive method for comparing the potential bone-healing potential of these different materials. We have developed a novel technique for assessing the osteogenic capacity of different bone substitutes in a mechanically-stimulating perfusion bioreactor.

The Zetos(TM) bioreactor system consists of individual flow chambers connected to a low-flow perfusion pump, which recirculates media through samples. The Zetos can be programmed to apply a controlled stress or a controlled strain to each individual sample inside the flow chamber. Since bone formation has been shown to be optimal with short doses of high amplitude strains, test samples were subjected to daily loading corresponding to physiological strain experienced during a jumping exercise (maximum 3000 microStrain).

Three substitute materials representing the range of materials available clinically were tested in the Zetos system; these included collagen, calcium phosphate, and a synthetic polymer. Primary human osteoblasts were seeded onto the substitutes, which were then placed inside the Zetos system and maintained under load or non-load conditions for 14 days. No supplementary osteogenic factors were provided to the cells. The degree of bone formation in the samples was assessed using Von Kossa staining and quantified in terms of the area of new mineral relative to the surface area of the substitute.

No mineralisation was detected in the non-loaded samples. However, in the loaded samples, mineralisa-tion was detected in some of the substitutes. The degree of mineralisation depended on the material: in collagen, an average of 0.22 mm2/mm2 was mineralised; in calcium phosphate, mineralisation averaged 0.0013 mm2/ mm2; but in the loaded polymer samples, no mineralisation was detected.

This indicates that mechanical loading is a sufficient stimulus for bone formation in some materials, even in the absence of other known osteogenic factors. Further, commercial substitutes differ in their ability to support bone formation under conditions of physiological loading. Further development of this technique could allow it to be used as a screening tool for predicting the efficacy of commercial products.

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.

Introduction: Oxidative stress occurs when reactive oxygen species (ROS) are produced faster than they can be removed by cellular defence mechanisms contributing to ageing, many chronic diseases, such as atherosclerosis, RA, Parkinson and Alzheimer’s disease and skeletal pathologies. Here we address the impact of ROS on the viability of early osteogenic precursors in the bone marrow and study the influence of estrogen on this interaction. Cells have a number of mechanisms to protect themselves from ROS, which are constantly being formed in the cell through normal metabolic pathways, such as Vitamin E, C and estrogen. Estrogen has been shown to prevent intracellular accumulation of peroxide and to attenuate oxidant-induced death of neuronal and endothelial cells. In addition, it contributes significantly to bone turnover and relieves postmenopausal symptoms. This study has focused on the potential anti-oxidant properties of estrogen against oxidative on bone marrow stromal cells. stress induced by H₂O₂

Methods: Primary bone marrow stromal cells were pre-treated with several different doses between 10⁻⁶M – 10⁻⁸M of estrogen prior to H₂O₂ administration at 0.08–0.4 mM 30% (v/v) for 2–24h. The cellular production of ROS was determined by using the free radical indicator DCFH-DA. Apoptosis was determined by morphological criteria.

Results: H₂O₂ induced an increase in apoptosis of osteoprogenitor cells (p< 0.05). Determination of apoptosis and cell number by nuclear staining, indicated that pre-treatment of bone marrow stromal cells with 17-beta estradiol reduced the apoptotic response induced by H₂O₂ (p< 0.05) and restored cell number to control levels. In order to test the anti-oxidant activity of estrogen, the dye DCFH-DA was introduced in a cell free system in the presence or absence of 17-beta estradiol and H₂O₂. The same experiment was repeated in the presence of bone marrow stromal cells. H₂O₂ increased both intracellularly and extracellularly oxidant activity and estradiol has the capacity of modifying this activity both inside and outside the cell.

Discussion: These data demonstrate the ability of estrogen, used at physiological doses, to block oxidant-induced apoptosis of osteoprogenitor cells. Estrogen appears to reduce the generation of ROS in these cells. These data could have important implications on the maintenance of osteogenic stem cells during fractures, ageing and disease.

Introduction: The concept of cell senescence has been described as the mechanism responsible for the ageing of tissues, that is a finite ability to replicate and produce new tissue. The senescent cell population is separate and distinct from the cells which are undergoing programmed cell death (apoptosis), and those which are necrosing acutely. Cells reaching the senescent state have an increase in β-galactosidase activity, which is detectable using an established technique for soft tissues including fibroblasts and epithelial tissues. Senescence has not previously been investigated in bone. We have investigated this and hypothesise that new bone formed by distraction osteogenesis will have fewer senescent cells than the adult cortical “old” bone.

Methods: Eight New Zealand white rabbits underwent application of a M100 Orthofix external fixator to the tibia and creation of a mid-diaphyseal osteotomy, using a hand saw. After a seven day latency period, distraction was commenced (0.5mm twice daily) to twenty percent lengthening. After 3 weeks consolidation, the tibae were harvested for histological analysis.Senescent Staining:The sections were stained using a technique described by Faragher, using an X-gal based stain. Sections were incubated for 16 hours at 37 degrees centigrade before counter staining with DAPI. Sections underwent histological analysis and total cell and senescent cell counts performed.

Results: Surprisingly, large numbers of cells within the bone regenerate stained for cell senescence. A mixture of multinucleate and mononucleate cells were present. The location and appearance of the multinucleate cells prompted the use of TRAP staining. This provided support for these cells being osteoclasts.

Discussion: Previously, a high percentage of apoptotic cells and a high rate of cell division has been reported in bone regenerate. The surprisingly high numbers of cells within the newly formed bone staining positively for senescence suggest that there may also be a high senescent cell population. Alternatively, the positive TRAP staining may indicate that the stain is less specific than reported and may be staining osteoclasts and mature macrophages within the bone regenerate.