Biocompatibility studies are carried out either in two dimensional monolayer culture or in animal studies. Bone organ cultures are therefore required in order to reduce the number of animal studies performed, while at the same time ensuring a more natural environment than that provided by monolayer culture of isolated cells. Due to the three dimensional nature of bone explants, assays that determine the distribution of viable cells are required, however dense mineralised bone is not easily penetrated by soluble factors. We sought to compare a number of non-radioactive viability methods in order to assess their suitability for use with cancellous bone. Fluorescent live/dead staining, MTT activity and lactate dehydrogenase detection were all investigated on either whole bone explants (9.5 mm in diameter, 5 mm high) or on sections of explants. All these assays are routinely used in 2 dimensional cell culture systems, yet each required modifications to be suitable for use with cancellous bone. Factors such as penetration of reagent, incubation time, assay temperature and ease of determining viable cells were all compared. It was demonstrated that penetration of the reagents into whole cores was a major problem which easily led to artefacts that could be overcome by preparing 250 µm unfixed sections. Fluorescent live/dead staining had extra complications caused by the autofluorescence of the bone generating a high signal to noise ratio, making assessment of osteocyte viability impossible. MTT staining was difficult to interpret due to the punctate nature of the stain. We found that lactate dehydrogenase staining of 250 µm thick unfixed sections led to excellent viability determination of osteocytes within the mineralised matrix. It also maintained marrow structure. Decreasing the viscosity of the LDH assay solution used in published methods led to a greatly improved penetration into the calcified matrix. Quantification of thick sections is aided by using the autofluorescence of the bone to highlight the darkly stained osteocytes against the fluorescing bone.

The goal of this study was to assess the effect of the addition of TGFb 3, alone or in combination with loading, on the survival of osteocytes in 3D human explant cancellous bone during long-term culture in an ex vivo loading bioreactor. Human cancellous bone explants were cultured for up to 14 days with or without TGFb 3 (15 ng ml À1) and with or without loading (300 cycles, at 1 Hz, producing 4000 microstrain). Bone core response was visualized using undecalcified histology with morphological methods after embedding with Technovit 9100 New 1 resin. Histological examination revealed normal gross level bone structure with or without the application of load or the addition of TGFb 3 . The viability of the osteocytes within the bone was assessed by lactate dehydrogenase (LDH) activity. We demonstrate that this ex vivo loading bioreactor is able to maintain a high percentage (over 50%) of viable osteocytes throughout the bone explants after 14 days in ex vivo culture. Further to this, the combination of daily loading and TGFb 3 administration produced superior osteocyte survival at the core centres when compared to loading or TGFb alone.

BackgroundBone graft studies lack standardized controls. We aim to present a quick and reliable method for the intra-operative generation of acellular bone explants.MethodsTherefore, ovine cancellous bone explants from the iliac crest were prepared and used to test several methods for the induction of cell death. Over night heat inactivation was used as positive treatment control, methods to be investigated included UV light, or X- ray exposure, incubation in a hypotonic solution (salt-free water) and a short cycle of repeated freezing and thawing.ResultsViability of treated and 2 days cultured bone explants was investigated by lactate dehydrogenase assay. Non-treated cultured control explants maintained around 50% osteocyte viability, while osteocyte survival after the positive treatment control was abolished. The most dramatic loss in cell viability, together with a low standard deviation, was a repeated cycle of freezing and thawing.ConclusionsTo summarize, we present a freeze-thaw method for the creation of acellular bone explants, which is easy to perform, not time-consuming and provides consistent results.

Severe side effects of cocaine consumption are vasoocclusive events such as myocardial infarction and stroke. We have hypothesized that cocaine could affect red blood cells (RBCs) and alter the rheological behaviour of blood. Heparinized blood from healthy volunteers was incubated with a final hematocrit of 45% with increasing cocaine concentrations: 0, 10, 100, 1000, and 10'000 mol/L plasma. Time dependence of the shape change was tested in phosphate buffered saline containing cocaine. RBCs were fixed in 1% glutaraldehyde for morphological analysis. Blood viscosity was measured with a Couette Viscometer (Contraves LS 30) at 37 • C and a shear rate of 69.5 s -1 . RBC aggregation was assessed with a Myrenne aggregometer. Cocaine induced a dose-dependent stomatocytic shape transformation of RBCs, which was more pronounced in buffer than in plasma (plasma protein binding of the drug). Stomatocytosis occurs when a drug intercalates preferentially in the inner half of the membrane lipid bilayer. It was a time-dependent process with two components, an almost instant shape change occurring within 1 s, followed by a gradual further shape change during 10 min. Stomatocytosis was reversible by resuspension of the RBCs in cocaine-free buffer. This stomatocytic shape change increased whole blood viscosity at high shear rate from 5.69 ± 0.31 mPa.s to 6.39 ± 0.34 mPa.s for control and 10'000 mol/L cocaine, respectively (p < 0.01). RBC aggregation was not affected by the shape change. These effects occurred at a cocaine concentration, which is several-fold above those measured in vivo. Therefore, it is unlikely that hemorheological factors are involved in vascular events after cocaine consumption.

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