P046: Engineering of a 3D cartilage model: study of healthy cartilage exposition during hadrontherapy
Résumé
Objective: Hadronbiology studies have most focused on signaling and repair of DNA damage pathways induced by ionizing radiation in immortalized cells under conditions dramatically different of human homeostasis. Thus, this research area needs to be broadened as the range of normal tissue responses to ionizing radiation are still not fully understood even though it is recognized that these responses are the major limiting factor in the rupture of tissue homeostasis after therapeutic exposure. By using relevant hypoxic tissue models as cartilage, we aim at unveiling the impact of acute radiations on cells fate and function. This work is performed in the context of the ARCHADE project (Advanced Resource Centre for HADrontherapy in Europe), a resource center of research and development for hadrontherapy with carbon ions and a cancer treatment facility by proton therapy, effective in Caen within few years. Methods: A 3D model of reconstructed cartilage is achieved in vitro using a matrix of collagen type 1 and human primary chondrocytes from healthy donors or an immortalized chondrocyte cell line. These models are irradiated using the GANIL facilities (Caen, France) with heavy ions (carbon, neon and oxygen) of various LET (from 30 to 100 kev/μm) or X-rays (as control) with a range of doses 0 - 6 Gy. Effects of irradiation on the differentiation/proliferation balance, viability, genetic stability, oxidative stress and inflammation are analyzed with classical 2D cultures of chondrocytes and a 3D model of reconstructed cartilage.
Results: Colony size distributions show significant differences between normoxia (21% O2) and hypoxia (2% O2) culture conditions of primary chondrocytes. Clonogenicity assays reveal a D10% two times inferior in case of carbon ion irradiation (~35 kev/μm) as compared with X-rays (~1 kev/μm). Survival assays display no necrosis on the 3D model of chondrocytes. Several proteins specifically targeted to investigate ionizing
radiation are studied by western blotting and present characteristic profiles as a unction of radiations, doses and culture conditions.
Conclusion: The presented results show the capacity of our 3D model of reconstructed cartilage to explore the effects of heavy ions on cartilage cells in vitro. This biomaterial is able to reproduce the natural microenvironment of chondrocytes and thus it allows the analysis of cellular responses in conditions close to the biological reality, with no requirement of animal models.
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