Symposium. ESEM 4th International Symposium on Microdamage
ESEM 4th International Symposium on Microdamage 5955 Tu, 14:00-14:15 (P23) Noise-enhanced response o f bone cells to fluid shear stress R.G. Bacabac 1, J.J.W.A. Van Loon 1,2, T.H. Smit 3, J. Klein-Nulend 1. 1Dept
Oral Cell Biology, ACTA-Vrije Universiteit, Amsterdam, The Netherlands, 2Dutch Experiment Support Center, Vrije Universiteit, Amsterdam, The Netherlands, 3 Dept Physics and Medical Technology, VU University Medical Center, Amsterdam, The Netherlands Stochastic resonance is exhibited by non-linear systems, where the response to a small signal is enhanced by noise at an optimum level. It is possible that bone cell mechanosensitivity is enhanced by noise as a mechanism for an amplified response to small stresses. Daily normal loading on bone is expected to occur at strains as low as 10 ~t~. Furthermore, microdamage might impair normal fluid shear stress levels for stimulating bone cells. Since bone formation correlates with mechanical loading, understanding how bone cells might perceive low stresses under normal or special conditions of loading is imperative. Previously, we found that bone cells required an initial stress-kick to respond to fluid shear stress in a rate-dependent manner. This provides a basis for stochastic resonance to occur in bone as a non-linear biological system. Thus, we studied whether noise of varying intensities (0.03-1.4 Pa) enhanced the mechanosensitivity of MLO-Y4 osteocytes in comparison with MC3T3E1 osteoblasts. Nitric oxide (NO) and prostaglandin E 2 (PGE2) production were measured as parameters for bone cell activation. We found that the NO response of MLO-Y4 osteocytes to a small periodic fluid shear stress was acutely enhanced by noise at intensity 0.25 Pa. The NO response of MC3T3E1 osteoblasts to noisy stress was optimum at noise intensity 0.7 Pa. MLO-Y4 osteocytes showed an increase in PGE2 release at noise intensity 0.7 Pa, and MC3T3-E1 osteoblasts showed a peak response at noise intensity 0.42Pa. Our in vitro results also implied differences in stress-thresholds for NO and PGE2 production for MLO-Y4 and MC3T3-E1 bone cells. Since NO and PGE2 regulate bone formation as well as resorption, our results explain how bone cells might cooperate in vivo in driving the mechanical adaptation of bone. The noise-amplified response by bone cells provides a novel paradigm for understanding the osteogenic benefits of low amplitude dynamic loading, and an innovative therapeutic option for preventing bone loss.
$457  M.B. Schaffler, et al. Bone 1995; 6: 521-525.  D. Taylor et al. J. Theoretical. Biology 2003; 225: 65-75. 4332 Tu, 14:30-14:45 (P23) Development o f a novel three-dimensional culture system to study the role o f mechanically damaged osteocytes in the initiation o f targeted bone remodeling T.J. Heino 1, K. Kurata 2, H. Higaki 3, H.K. V ~ n ~ n e n 1. 1Department of Anatomy, Institute of Biomedicine, University of Turku, Finland, 2Department of Biorobotics, Faculty of Engineering, Kyushu Sangyo University, Japan, 3 Department of Mechanical Engineering, Faculty of Engineering, Kyushu Sangyo University, Japan Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Osteocytes are suggested to have a crucial role in the initial resorptive phase of bone turnover after microdamage. To study the role of osteocytes in targeted remodeling, we developed a novel in vitro culture system, in which osteocytes can be locally damaged and their interactions with bone marrow cells studied. Osteocyte-like MLO-Y4 cells were threedimensionally cultured in collagen gel. To apply local mechanical damage, an injection needle was inserted vertically to the wall of the culture dish. A stainless steel wire was installed in the needle and the end inside the gel was bent so that it could scratch the gel. By moving the bent hook backward and forward, local mechanical scratching was applied to the gel-embedded cells, which were then assayed for cell viability. Fibroblastic NIH3T3-3 cells were used as a control. Bone marrow cells were cultured on the top of the mechanically damaged MLO-Y4 cells and the formation of osteoclastic cells was assayed. The M-CSF and RANKL secretion in the conditioned medium was assayed by ELISA. Scratching induced the death of MLO-Y4 cells. When bone marrow cells were cultured over the gel-embedded MLOY4 cells, the application of mechanical scratching induced osteoclastic cell differentiation on gel surface. Osteoclastic cells could be observed in the very restricted region along the scratching path. Additionally, mechanically damaged osteocytes secreted M-CSF and RANKL and the conditioned medium showed the potential to induce osteoclastic cells in bone marrow culture. These findings indicate that soluble factors secreted from damaged osteocytes can locally induce and activate the initial phase of osteoclast formation. This study directly demonstrates the association between the damaged osteocytes and the initiation of resorptive stage in bone remodeling, which would strongly support the existence of the targeted remodeling. 7932 Microdamage in o s t e o p o r o s i s
Tu, 14:45-15:00 (P23)
6234 Tu, 14:15-14:30 (P23) Osteocyte function in microcrack detection J.G. Hazenberg 1, D. Taylor2, T.C. Lee 1,2. 1Department of Anatomy,
T.C. Lee 1,3, N.J. Mahony 2,3, D. Taylor3. 1Department of Anatomy, Roya/
Royal College of Surgeons in Ireland, Dublin, Ireland, 2Trinity Centre for Bioengineering, Trinity College, Dublin, Ireland
Osteoporosis is a skeletal disorder characterised by compromised bone strength leading to an increased risk of fracture . In 2000, there were an estimated 3.79 million osteoporotic fractures at a cost of 32 billion Euro. The incidence of fracture is dependent on the loading applied to the bone, for example by a fall, and bone strength. Strength, in turn, is determined by both bone quantity and bone quality. Quantity is measured clinically as bone mineral density (BMD) using dual energy X ray absorptiometry (DEXA), but it has limitations as a measure of both fracture risk and of response to therapeutic interventions . Bone quality is determined by collagen, mineralisation, trabecular microarchitecture and microdamage. The precise role of microdamage in osteoporosis has yet to be defined. There is growing evidence that microdamage is an activator of bone remodelling  and that osteoporosis is characterised by an initial increase in the rate of remodelling and, subsequently, by microdamage accumulation. Microdamage has been measured histologically, but this requires excision biopsy, which is invasive and exacerbates fracture risk. A non-invasive method of measuring microdamage accumulation has the potential, along with bone quantity measurements, to aid in fracture risk assessment and thus in targeted therapy. To date microCT  and PET  scanning have shown promise, but a clinically applicable technique is awaited.
Accumulation of microdamage in bone  has been linked to remodelling [2, 3] and to adaptation. How does the body detect these cracks? Recently Taylor et al.  have proposed a new mechanism: rupture of cell processes spanning a crack might occur due to relative crack-face displacements in opening and shear. A theory was developed to predict these displacements and thus estimate the number of broken cell-processes. This paper discusses the experimental work carried out to investigate if propagating cracks can rupture osteocyte cell processes. A UV-epifluorescence microscope was attached to a test rig to observe crack growth during loading of 31 pre-notched bovine bone specimens, and to measure relative displacements across the crack faces. Ten randomly chosen specimens were examined using a scanning electron microscope (SEM). The remaining 21 specimens were used for immuno-histochemical staining with Phalloidin and confocal microscopy analysis. Cracks tended to grow parallel to osteons, being inclined to the load axis at 220 on average and therefore experiencing shear as well as tensile strains. Both SEM and confocal microscopy analysis showed unbroken cell processes crossing crack faces near the crack tip, but in regions where more crack opening and shearing occurred, cell processes were broken. This experimental evidence, coupled with our previous theory  suggests that the number of fractured cell processes could act as a measure of the amount of damage in bone. This could be the so-called 'cellular transducer' which provides the information needed by the body to effect repair, remodelling and adaptation. Acknowledgements: This work was financially supported through the EMBARK Postdoctoral Fellowship by the Irish Research Council for Science. References  H.M. Frost. In: Henry Ford Hosp Bull. 1960; pp. 25-35.  V. Bentolila, et al. Bone 1998; 23: 275-281.
College of Surgeons in Ireland, 2Department of Anatomy, 3 Trinity Centre for Bioengineering, Trinity College, Dublin 2, Ireland
References  Anonymous. Osteoporosis prevention, diagnosis and therapy. Journal of the American Medical Association 2001; 285: 785-795.  Bouxsein ML. Determinants of skeletal fragility. Best practice & research in clinical rheumatology 2005; 19:897-911.  Lee TC, Staines A, Taylor D. Bone adaptation to load: Microdamage as a stimulus for bone remodelling. Journal of Anatomy 2002; 201 : 437-446.  Parkesh R, Lee TC, Gunnlaugsson T, Gowin W. Microdamage in bone: Surface analysis and radiological detection. Journal of Biomechanics, in press.  Li J, Miller MA, Hutchins GD, Burr DB. Imaging bone microdamage in vivo with positron emission tomography. Bone 2005; 37: 819424.