Biocompatibility study of nitrided titanium alloy for bone implantation by laser flow cytometry, laser scanning cytometry and confocal microscopy

Elzbieta Czarnowska 1Agnieszka Sowinska 1Bożena Cukrowska 1Michał Godlewski 2Tadeusz Wierzchoń 3

1. The Children Memorial Health Institute, Department of Pathology, al. Dzieci Polskich 20, Warszawa 04-736, Poland
2. Warsaw Agricultural University, Department of Animal Physiology, Warszawa, Poland
3. Warsaw University of Technology (PW), Warszawa, Poland


One of the parameters that allow assessment of the biocompatibility of innovative materials for implant and prosthesis is cellular adhesion. Therefore the aim of our study was to evaluate the ability of fibroblasts and osteoblasts to adhere to samples with TiN + Ti2N + AlphaTi(N) surface layers produced on titanium alloy by glow discharge nitriding processes in comparison to titanium alloy using laser flow cytometry (FACS), laser scanning cytometry (LSC), and confocal microscopy. Cells were cultured on samples for 48 hours. Data obtained from FACS showed the presence of fibronectin receptors on cells that adhered to both titanium alloy and nitrided surface layers. However, live cells did not express fibronectin, in contrast to dead cells that positively stained for this protein what could suggest intracellular storage of fibronectin. Fibronectin production by adhered cells was confirmed by investigations in LSC and confocal microscopy. Both intra- and extracellular presence of fibronectin in cells adhering to both type of samples was shown. The amount of dead cells harvested from samples was about 30% for both types of biomaterials, as measured by FACS. LSC analysis showed that 5-12% of adhered cells died through mechanisms of apoptosis. These results suggest that strong adhesion to material blocks the mechanisms involved in cell spreading, which can induce cell apoptosis.
In conclusion, results confirms the good biocompatibility of TiN + Ti2N + AlphaTi(N) surface layers and its potential in bone surgery applications. Employment of LSC together with confocal microscopy for analysis of cell behaviour in contact with biomaterials seems to be very useful, but FACS analysis of chemically harvested cells should be carefully taken into account in the study of cell adhesion processes.

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Presentation: oral at E-MRS Fall Meeting 2003, Symposium E, by Agnieszka Sowinska
See On-line Journal of E-MRS Fall Meeting 2003

Submitted: 2003-05-13 12:59
Revised:   2009-06-08 12:55
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