Mechanobiologically induced bone-like nodules: Matrix characterization from micro to nanoscale

In bone tissue engineering, stem cells are known to form inhomogeneous bone-like nodules on a micrometric scale. Herein, micro- and nano-infrared (IR) micro-spectroscopies were used to decipher the chemical composition of the bone-like nodule. Histological and immunohistochemical analyses revealed a cohesive tissue with bone-markers positive cells surrounded by dense mineralized type-I collagen. Micro-IR gathered complementary information indicating a non-mature collagen at the top and periphery and a mature collagen within the nodule. Atomic force microscopy combined to IR (AFM-IR) analyses showed distinct spectra of “cell” and “collagen” rich areas. In contrast to the “cell” area, spectra of “collagen” area revealed the presence of carbohydrate moieties of collagen and/or the presence of glycoproteins. However, it was not possible to determine the collagen maturity, due to strong bands overlapping and/or possible protein orientation effects. Such findings could help developing protocols to allow a reliable characterization of in vitro generated complex bone tissues. The present work highlights the use of micro- and nano-infrared spectroscopies, two spatially resolved technics from a few tens of μm (μ-IR) to a few tens of nm (AFM-IR), to characterize the collagen maturation and mineral crystallinity and to evidence the spatial distribution of protein conformations in an in vitro formed bone-like organoid, providing thus a qualitative and semi-quantitative information on the matrix compounds and maturation. The gathered data support the potential use of μ-IR and AFM-IR as reliable tools to decipher the chemical content of in vitro engineered bone organoid tissue. [Display omitted] •Differentiation of stem cell and formation of bone-like nodules•Micro- and nano-infrared spectroscopies to characterize a bone-like nodule•Spatially resolved technics from a few tens of microns to a few tens of nanometers•Bone-like nodule collagen maturity and spatial distribution of proteins conformations•Micro- and nano-infrared spectroscopies as reliable tools to decipher bone-like nodules chemical content