In situ mechanical and molecular investigations of collagen/apatite biomimetic composites combining Raman spectroscopy and stress-strain analysis.

We report the design, fabrication and application of a novel micro-electromechanical device coupled to a confocal Raman microscope that enables in situ molecular investigations of micro-fibers under uniaxial tensile load. This device allows for the mechanical study of micro-fibers with diameters in the range between 10 and 100μm and lengths of several hundred micrometers. By exerting forces in the mN range, the device enables an important force range to be accessed between that of atomic force microscopy and macroscopic stress-strain measurement devices. The load is varied using a stiffness-calibrated glass micro-needle driven by a piezo-translator during simultaneous Raman microscopy imaging. The method enables experiments probing the molecular response of micro-fibers to external stress. This set-up was applied to biomimetic non-mineralized and mineralized collagen micro-fibers revealing that above 30% mineralization the proline-related Raman band shows a pronounced response to stress, which is not observed in non-mineralized collagen. This molecular response coincides with a strong increase in the Young's modulus from 0.5 to 6GPa for 0% and 70% mineralized collagen, respectively. Our results are consistent with a progressive interlocking of the collagen triple-helices by apatite nanocrystals as the degree of mineralization increases. STATEMENT OF SIGNIFICANCE Collagen and apatite are the main constituents regulating the mechanical properties of bone. Hence, an improved understanding of the impact of mineralization on these properties is of large interest for the scientific community. This paper presents systematic studies of synthetic collagen microfibers with increasing apatite content and their response to tensile stress by using a novel self-made electromechanical device combined with a Raman spectrometer for molecular level studies. The impact of apatite on the mechanical and molecular response of collagen is evaluated giving important insights into the interaction between the mineral and organic phases. Therefore our findings expand the fundamental understanding of the mechanics of the apatite/collagen system relevant for the design of bio-composites with similar bio-mimicking properties for e.g. bone regrowth in medical applications.