Einfluß verschiedener Desinfektions- und Sterilisationsverfahren für allogene Knochentransplantate auf die Osteoblastenfunktion in-vitro

One important reason for different clinical outcome after bone allografting might be the alteration of cell adherence, viability, and differentiation by different processing methods. In order to assess the influence of eight different sterilisation and disinfection methods for bone allografts on adhesion, proliferation, and differentiation of bovine periosteal osteoblasts, cells were grown in culture and then plated onto pieces of human bone allografts. We tested autoclaved bone (AUT), demineralised and low-temperature-plasma sterilised bone (D-LTP), ethylene oxide sterilised bone (EtO), 80°C-thermodisinfected bone (80°C), and chemical solvent disinfected bone (CSD). The seeding efficiency was determined during the first hour of incubation to detect the number of attached cells but before mitosis starts. The cell viability was tested after a culture period of 7 and 21 days using the MTT-assay and determination of the total DNA content. Tests to confirm the osteoblastic function and differentiation of the cells included measuring of the total protein amount, histochemical alkaline phosphatase staining and quantitative determination of AP-activity, ELISA for osteocalcin and SDS-PAGE for collagens. Results showed that heat processing of bone matrix clearly influenced the adhesion, proliferation and differentiation of osteoblasts. Further, serum protein mediated cell adhesion upon bone allograft surfaces was altered by different processing methods. Most cells adhered to D-LTP-bone. Highest proliferation rates of periosteal osteoblasts as measured via MTT-activity and total DNA-content were also found within the D-LTP-group after one and three weeks. Highest expression levels of solved and ECM-proteins were found in CLD- and D-LTP groups, followed by the 80°C-group. Extracellular matrix proteins (e.g. Collagen Type I) are necessary for the attachment, migration, proliferation, and differentiation of osteoblasts. The present study clealy shows that different processing methods directly influence the gene expression of collagens Type I and III. The highest expression levels of this ECM-marker was found in the D-LTP-Group, followed by 80°C, AUT, CLD, and EtO-groups. Corresponding to this findings, expression of specific differentiation markers of osteoblastic phenotype AP and osteocalcin were significantly increased in D-LTP group. Growth factor activity (TGF?s, BMP?s) of the allograft matrix may be responsible for these effects. The study reports an in-vitro model to examine the influence of different processing methods for allogenic bone grafts on single cell types. For the first time a direct change of osteoblast growth and funtion caused by processed bone grafts was shown in the present work.