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Light at wavelength 405 nm is an effective bactericide. Previous studies showed that exposing mammalian cells to 405 nm light at 36 J/cm(2) (a bactericidal dose) had no significant effect on normal cell function, although at higher doses (54 J/cm(2)), mammalian cell death became evident. This research demonstrates that mammalian and bacterial cell toxicity induced by 405 nm light exposure is accompanied by reactive oxygen species production, as detected by generation of fluorescence from 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate. As indicators of the resulting oxidative stress in mammalian cells, a decrease in intracellular reduced glutathione content and a corresponding increase in the efflux of oxidised glutathione were observed from 405 nm light treated cells. The mammalian cells were significantly protected from dying at 54 J/cm(2) in the presence of catalase, which detoxifies H2O2. Bacterial cells were significantly protected by sodium pyruvate (H2O2 scavenger) and by a combination of free radical scavengers (sodium pyruvate, dimethyl thiourea (OH scavenger) and catalase) at 162 and 324 J/cm(2). Results therefore suggested that the cytotoxic mechanism of 405 nm light in mammalian cells and bacteria could be oxidative stress involving predominantly H2O2 generation, with other ROS contributing to the damage.

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Complications associated with the use of autogenous bone in the repair or replacement of tissue lost through injury or disease have driven the search for alternative sources of graft material. Bioceramics containing hydroxyapatite (HA), tricalcium phosphate (TCP), or composites that combine the best properties of both of these materials are among the principal candidates. In this study, we have investigated the in vitro proliferation, morphology, and viability of an immortalized rat osteoblast cell line cultured on HA, TCP, and composites of the two in the ratios 75:25 (H75), 50:50 (H50), and 25:75 (H25) for 28 days. The biocompatibility of each material was examined in the presence and absence of a collagen coating. With the exception of H50, cell proliferation, quantified by carboxyfluorescein fluorescence, was enhanced by collagen coating of all materials for the first 14 days, although at later time points cell numbers were unaffected. It is notable that the collagen coating was least stable on H50, the only material not to show enhancement of cell growth on coating. Confocal laser scanning microscopy confirmed that cell growth was more extensive on coated materials over the first 7-14 days in culture, and the development of cell extensions and bridges across the pores in the materials was observed. Results indicate that collagen coating of calcium phosphate ceramics may also increase their compatibility and osseointegration in vivo.

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Artificial skin substitutes based on autologous keratinocytes are being developed for grafting onto burns patients. In order to be used successfully in the clinic, these skin substitutes need to have sufficient strength to allow ease of handling. This may be achieved by crosslinking the collagen substratum on which the cells are cultured. The influence of potential crosslinking agents on the tensile properties of acellular collagen gels has been investigated, including the glycosaminoglycan, chondroitin-6-sulphate (Ch6SO4), the water-soluble carbodiimide crosslinking agents 1-ethyl-3-(3-diaminopropyl) carbodiimide (EDAC), and 1,1-carbonyldiimidazole (CDI), and the polyamines, putrescine and diaminohexane. Values for Young's modulus, maximum load, stress, displacement and percentage strain at maximum load were generated by subjecting the samples to a tear propagation test. Incorporation of 20% Ch6SO4 into collagen gels caused a significant increase in the Young's modulus, maximum load and stress at maximum load. Crosslinking treatment with EDAC, CDI or polyamines had little further benefit, and in many cases resulted in a decrease in particular parameters. In terms of mechanical strength, the best crosslinking combination proved to be the combination of CDI and diaminohexane, with results either improved or maintained when compared with the control no treatment variants. However, previous experience suggests that the use of CDI as a crosslinking reagent may inhibit infiltration and proliferation of fibroblasts in the substratum and it may be necessary to reach a compromise to obtain the best combination of biological and mechanical properties for artificial skin substitutes.

Reconstituted collagen hydrogels are often used for in vitro studies of cell-matrix interaction and as scaffolds for tissue engineering. Understanding the mechanical and transport behaviours of collagen hydrogels is therefore extremely important, albeit difficult due to their very high water content (typically >99.5%). In the present study the mechanical behaviour of collagen hydrogels in confined compression was investigated using biphasic theory (J Biomechemical Engineering 102 (1980) 73), to ascertain whether the technique is sufficiently sensitive to determine differences in the characteristics of hydrogels of between 0.2% and 0.4% collagen. Peak stress, equilibrium stress, aggregate modulus and hydraulic permeability of the hydrogels exhibited sensitivity to collagen content, demonstrating that the technique is clearly able to discriminate between hydrogels with small differences in collagen content and may also be sensitive to factors that affect matrix remodelling. The results also offer additional insight into the deformation-dependent permeability of collagen hydrogels. This study suggests that confined compression, together with biphasic theory, is a suitable technique for assessing the mechanical properties of collagen hydrogels.