Influence of intradiscal medication on nucleus pulposus cells.

BACKGROUND CONTEXT: Eugene Carragee was the first to prove that provocative discography may contribute to intervertebral disc degeneration. Disc degeneration can be induced either by mechanical trauma caused by the puncturing needle or as a pharmacological effect of the drugs instilled into the disc. PURPOSE: The aim of this study was to test the influence of cortisone, lidocaine, and iopamidol on nucleus pulposus cells under an in vitro setting. STUDY DESIGN: Controlled in vitro study is the design type. METHODS: The nucleus pulposus was excised from 12 bovine tail intervertebral discs and monolayer cell cultures were generated. The cultures were divided into four sample groups and incubated in either standard cell culture medium (control group) or medium supplemented with the test substances. The dose rate was adapted based on a total dose of 3 mL iopamidol, 1 mL lidocaine, and 10 mg cortisone per nucleus pulposus. Cell count, viability, proliferation, and differentiation features were analyzed. The study was supported by DePuy. No conflicts of interest arise from this support. RESULTS: After 24 hours, a significant decrease in cell counts was observed in all three test groups. Population doubling time was 16 hours in the control group cultured in standard medium and increased to 21 hours (cortisone), 25 hours (iopamidol), and 38 hours (lidocaine) after incubation in discography medication (p<.001). Cell viability was slightly, but not significantly decreased in all medication groups. Cells incubated in Lidocaine were significantly smaller (p<.01) and showed clearly reduced pseudopodia formation. Incubation in lidocaine and iopamidol also significantly reduced glycosaminoglycan synthesis. CONCLUSION: Although only a small decrease in cell viability was observed in all three substances tested, cell count and proliferation decreased significantly. Incubation in lidocaine inhibited pseudopodia formation and might therefore interfere with intercellular signalling and cell migration. Glycosaminoglycan synthesis was significantly decreased after contact with lidocaine as well as Iopamidol. These observations suggest that all three medications tested might interfere with biological repair mechanisms of the intervertebral disc and therefore contribute to a further degeneration.

Introducing the concept of the 3Rs into tissue engineering research.

Tissue engineering, defined as using a combination of cultured cells and biodegradable scaffolds to repair tissue damaged by injury or disease, represents a booming sector of biomedical research. Animal experimentation is routinely performed prior to clinical trials. The presented study tries to translate the aspect of the 3Rs to tissue engineering research: Cell culture protocols were adapted to antibiotic free and serum free conditions. Biomaterials (Bio-Gide and a collagen sponge prototype) were pre-tested using the HET-CAM assay. CAM-testing suggested a protocol change for application of the Bio-Gide scaffold and demonstrated unsuitable material properties of the collagen sponge. Application of 3R compliant protocols for tissue engineering research led to increased cell proliferation, higher synthesis of extracellular matrix molecules, reduced dedifferentiation and more information about the biomaterials at an early experimental stage. Tissue engineering research can therefore profit from the increased efforts to validate in vitro alternatives and supplements to animal testing.

The mandatory CAM testing of cells and scaffolds for tissue engineering: benefits for the three Rs of cooperation with the vaccine industry.

In cooperation with BAXTER Vaccine AG, which supplies incubated special pathogen-free chicken eggs (including a full veterinary record), a permanent hen's egg chorio-allantoic membrane test (HET-CAM) unit has been established, where angiogenesis testing, cell culture, and digital and histological analyses are performed. At the Core Unit for Biomedical Research, the location of the animal testing facility of the Medical University Vienna, cell-scaffold constructs must be evaluated in vitro and in ovo prior to eventual in vivo tissue engineering experiments. The animal testing advisory committee requires that new test proposals are first evaluated by using cell culture and HET-CAM models. Approvals for in vivo experiments are postponed and not issued prior to in vitro/in ovo evaluation. Examples are presented of protocols planned for in vivo studies on cell seeded scaffolds, which were refined after in vitro/in ovo evaluations.

[Good cell culture practice--implementation of a relational cell culture database]. / Good Cell Culture Practice - Entwicklung einer relationalen Datenbank für die Zellkultur.

The claim for cell culture to provide validable in vitro models for biomedical research postulates evasion of possible fatal record keeping errors. A prototype of a relational computer database for IBM-compatible personal computers using Microsoft(r) Windows 95/98/2000 and NT for administration of cell culture data has been developed using Microsoft(r) Access 98 (Microsoft Corporation, Redmond, USA), -Access Basic, -Visual Basic and Structured Query Language (SQL) (IBM Corporation, Armonk, USA), and was tested successfully. The modular software application manages the many aspects of cell culture laboratory record keeping like detailed information on tissue donor, primary cell isolation/cell line origin, immunohistochemical/molecular biological characterisation, cell countings at passaging/subcultivation/cell aliquotation and cryopreservation. One main feature is a collection of all methods performed at our cell culture laboratory, where linked tables and files store specific informations. Entries into the database are checked via validation rules for correctness to avoid mistakes. The developed prototype has been demonstrated to be an adaptable, reliable tool for improving quality of information storage according to Good Scientific Practice (GSP), Good Cell Culture Practice (GCCP) and general ISO certification trends.

[The "3R" concept in the Internet I]

More and more members of the scientific community interested in 3R related topics of animal experiments are using Internet-Homepages as a low-budget forum to present either their scientific results, or opinions on specific subjects or to announce publications. Supra/inter/national authorities/universities/laboratories are also offering information, documents, access to databases and much more to the interested public. Therefore the Internet is an advantageous new medium for 3R related subjects.