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Progression of nanoscale material testing techniques has produced new perspectives for studying the effect of musculoskeletal diseases on the weight-bearing tissues. Growing understanding of small scale mechanical behavior enhances prevention and treatment of osteoporosis and osteoarthrosis, as well as engineering of artificial prosthesis. Nanoscale indentation testing may be conducted with an atomic force microscope (AFM). Several different calibration and data analysis methods have been introduced for the AFM-nanoindentation. The main goal of this paper is to review the most used calibration methods, especially for analyzing the cantilever spring constant. A particular interest on the testing of bone microstructure is taken into account. Also, this paper describes the initial method of analyzing bone structures using nanoindentation with the Atomic Force Microscope, developed by the research team and relates the issues and problems encountered in the validation process.