Osteoblast interactions with calcium phosphate ceramics modified by coating with type I collagen.

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.

Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation.

Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at -78 degrees C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10% (v/v) dimethyl sulphoxide (ME2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90% (v/v) FCS plus 10% (v/v) ME2SO maintain 79.7+/-6.5% of the monolayer area as viable cells with normal morphology (by image analysis), 112.7+/-14.2% protein concentration, 55.4+/-4.2% carboxyfluorescein diacetate de-acetylation, 27.2+/-7.5% kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3+/-7.3% testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.

Metabolism and distribution of phenanthridine trypanocides in Trypanosoma brucei.

Phenanthridine trypanocides (isometamidium chloride hydrochloride, ISM, and Ethidium bromide, EBr) have been widely used to treat African trypanosomiasis in livestock for more than 40 years. Their main action is to inhibit nucleic acid synthesis in trypanosome parasites, by intercalation between the DNA base pairs. They can also linearise selectively kinetoplast DNA minicircles; a form of mitochondrial DNA unique to this group of parasites. However, the metabolism of these compounds by trypanosomes has not been reported. Indeed, it is not known whether or not their metabolism by the parasite contributes to their activity, selective toxicity for these parasites or to the development of chemoresistance. Therefore, we studied the metabolism of EBr and ISM, and their distribution in Trypanosoma brucei (TREU 927) using high performance liquid chromatography (HPLC), liquid chromatography combined with mass spectrometry (LC-MS) and confocal laser scanning microscopy (CLSM). Incubation of EBr with trypanosomes led to the formation of a small amount (0.606+/-0.191%) of one metabolite (MI). Ion chromatograms extracted from an LC-MS analysis using electrospray ionisation (ESI), showed that the difference in mass between the parent compound and its metabolite was 30. This may correspond to the addition of a hydroxyl and a methyl group. No metabolites could be detected for ISM. The distribution of the two drugs in trypanosomes was investigated by CLSM, using their intrinsic fluorescence. ISM and EBr showed differences in their distribution in trypanosomes. ISM had a greater affinity for the kinetoplast than EBr and it stained other organelles like the flagellum; in contrast the distribution of EBr was more diffuse.

Cryopreservation of rat hepatocyte monolayers: cell viability and cytochrome P450 content in post-thaw cultures.

Cryopreservation of primary hepatocyte monolayers may provide a means of long-term storage of the cells for in vitro studies of xenobiotic metabolism and toxicity. Rat hepatocytes can be stored at -70 degrees C as simple monolayers attached to collagen-coated dishes, and post-thaw cultures can be continued for up to 72 h. Throughout this post-thaw period viability of the cells was demonstrated by retention of intracellular fluorescence after exposure to carboxyfluorescein diacetate (CFDA) and examination by confocal laser scanning microscopy (CLSM). CLSM images revealed an uneven distribution of CFDA-derived fluorescence within hepatocytes post-thaw, particularly in Williams' E medium, indicating generation and retention of carboxyfluorescein within the intracellular organelles. The membranes of the intracellular organelles appear to be less sensitive to freeze/thaw damage than the cell membrane. Viability was not compromised with storage for up to 28 days at -70 degrees C. Cytochrome P450 content was retained in post-thaw culture to a similar extent as in non-frozen cultures. Cryopreserved rat hepatocyte monolayers may provide a useful in vitro model for studying xenobiotic metabolism and toxicity.