Blowup of Accidental Images as a Passageway to Discovery: Insights into the Interaction between Hydroxyapatite Nanoparticles and Human Mesenchymal Stem Cells

Abstract

Despite decades of research into the interaction between cells and nanoparticles, there is a lack of consensus regarding how specific physicochemical characteristics of the nanoparticles, including chemical composition, crystallinity, size, morphology, charge, and aspect ratio, among others, govern their internalization and intracellular fate. Methodological novelties offer new perspectives on the same old problematics, and often translate into an improved understanding of the given topic. Inspired by an analogy with the theme of the movie, Lisbon Story, a conceptually unconventional method for gaining insight into the interaction between nanoparticles and cells is proposed here. It involves the random, “Take 1” capture of an atomic force micrograph showing the interaction of human mesenchymal stem cells and clusters of spherical hydroxyapatite nanoparticles with a broad distribution of sizes and shapes, the blowup of its segments, and their detailed qualitative inspection. This method led to the derivation of three illustrative hypotheses, some of which were refuted and some corroborated. Specifically, the presupposition that there is an inverse relationship between the cellular uptake efficiency and the size of nanoparticle clusters was confirmed, both empirically and through a literature meta-analysis, but the idea that the geometry of these clusters affects the uptake was refuted. The definite presence of morphological determinants of the cellular uptake at the level of elementary particles, not clusters thereof, however, was confirmed in an alternative experiment. Likewise, immunofluorescent studies demonstrated that relatively large and irregularly shaped nanoparticle clusters do get internalized and localized to the perinuclear area, where they engage in an intimate interaction with the cell nucleus. The proposed enhancement of the binding between cells and biomaterials by increasing the surface ruffling consequential to the nanoparticle uptake - in analogy with the enhanced cell adhesion achieved by introducing topographic irregularities to smooth biomaterial surfaces - was also confirmed by showing that the uptake improves the stem cell adhesion. The uptake also augmented the stem cell viability and the proliferative capacity of cells reseeded with this internal nanoparticle cargo on a fresh surface, albeit with moderate levels of statistical significance and the caveat of its presumed dependence on the cell type, the nanoparticle chemistry and dose, and the overall stage in the transition of the multipotent cells toward an osteoprogenitor lineage.