Mechanical force is an osteoinductive factor that plays an important role in bone growth and repair in vivo (Carter et al. 1988). Many in vitro studies have shown that osteoblasts and osteocytes respond to mechanical loads such as stretch and fluid-flow induced shear stresses, with initiation of signalling pathways (Reilly et al 2003). The underlying mechanisms by which bone cells respond to mechanical signals are difficult to investigate in a 3-D environment, because of reduced nutrient delivery to cells and difficulties in analysis. We are developing a model to analyse the effects of mechanical compression on matrix forming osteoblasts in a 3-D system. Our model uses polyurethane (PU) open cell foam scaffolds, MLO-A5 osteoblast-like cells (Kato et al 2001) and a sterile fluid filled biodynamic loading chamber (Bose). We have shown using a cell proliferation assay (Promega) that cells survive well and proliferate in the PU scaffolds. Cell number after 15 days of culture was four times that after 5 days of culture. To examine the effect of mechanical stimulation on osteoblastic cells we seeded MLO-A5, kindly donated by Dr. L. Bonewald, at densities of 125,000 cells per scaffold in PU foam cylinders, 10 mm thick and 25 mm diameter. The cell seeded PU scaffolds were dynamically loaded in compression at 1Hz, 5% strain in a sterile fluid-filled chamber. Loading was applied for 2 hours per day at days 5, 7 and 9 of culture. In between loading cycles, scaffolds were cultured in an incubator in standard conditions. Preliminary data indicates that the cells survived loading but final cell number was reduced compared to unloaded controls by 30%. However, the scaffold stiffness (Young’s modulus) increased in loaded samples over time (days) which may be an indication of increased matrix production. Fluorescence microscopy indicated that loaded cells were distributed in dense clusters whereas unloaded cells were distributed evenly throughout the scaffold. In conclusion, this model has the potential to answer questions about cell survival, distribution and matrix production in 3-D, in response to mechanical signals.
The purpose of this study was to examine the effects of cement-free implant fixation on microperfusion in the vicinity of the bone-implant interface and to elucidate the effects of mechanical loading on interstitial fluid flow. Experiments were conducted on both forelimbs of sheep (n=8, age: 4–7 years) using an ex vivo model. Immediately after euthanasia, forelimbs were amputated and a system of perfusion with Procion red (0,08 %) as flow indicator was established. In one group (4 animals), an prosthesis was inserted into the reamed intramedullary cavity of the metacarpus. In a second group (4 animals) no implant was inserted. For each pair, one limb (chosen randomly) was subjected to cyclic loading. Loading was applied at a rate of 1 Hz for 5 minutes. Infusion lasted 5 minutes in all limbs. After the experiment histological cross sections were taken and analysed for the amount of tracer present. Twelve regions were marked on the slide prior to examination and acquired under fluorescence mode. The average pixel intensity of each field of view, was measured using ‘Scion Image’ software. The mean (± standard deviation) of the 12 readings (pixel intensities) for each group were as follows: Non-implanted group, loaded: 83.31 (± 13.56); Non-implanted group, unloaded 80.80 (± 9.22); Implanted group, loaded: 71.86 (± 19.28); Implanted group, unloaded 66.79 (± 15.52). Anova analysis showed the effect of loading not to be significant statistically (p = 0.082) but the effect of implant to be highly significant (p0.0001). Implant fixation and mechanical loading affect both microperfusion and interstitial fluid flow modulated mass transport in bony tissue surrounding implants. It appears that the presence of an implant per se reduces perfusion as well as fluid flow in the vicinity of the bone-implant interface. Within subchondral bone loading does not have a significant effect on transport of small molecular weight tracers.
