Efficient osteogenic differentiation of mesenchymal stromal cells (MSCs) is crucial to accelerate bone formation. In this context, the use of extracellular matrix (ECM) as natural 3D framework mimicking in vivo tissue architecture is of interest. The aim of this study was to generate a devitalized human osteogenic MSC-derived ECM and to investigate its impact on MSC osteogenic differentiation to improve MSC properties in bone regeneration. The devitalized ECM significantly enhanced MSC adhesion and proliferation. Osteogenic differentiation and mineralization of MSCs on the ECM were quicker than in standard conditions. The presence of ECM promoted in vivo bone formation by MSCs in a mouse model of ectopic calcification. We analyzed the ECM composition by mass spectrometry, detecting 846 proteins. Of these, 473 proteins were shared with the human bone proteome we previously described, demonstrating high homology to an in vivo microenvironment. Bioinformatic analysis of the 846 proteins showed involvement in adhesion and osteogenic differentiation, confirming the ECM composition as key modulator of MSC behavior. In addition to known ECM components, proteomic analysis revealed novel ECM functions, which could improve culture conditions. In summary, this study provides a simplified method to obtain an in vitro MSC-derived ECM that enhances osteogenic differentiation and could be applied as natural biomaterial to accelerate bone regeneration.

The accuracy of measurements in Amplitude Modulation Atomic Force Microscopy (AFM) is directly related to the geometry of the tip. The AFM tip is characterized by its radius of curvature, which could suffer from alterations due to repetitive mechanical contact with the surface. An estimation of the tip change would allow the user to assess the quality during imaging. In this work, we introduce a method for tip radius evaluation based on the nonlinear dynamic response of the AFM cantilever. A nonlinear fitting procedure is used to match several curves with softening nonlinearity in the noncontact regime. By performing measurements in this regime, we are able to maximize the influence of the tip radius on the AFM probe response, and this can be exploited to estimate with good accuracy the AFM tip radius.