Composite Graphene for the Dimension- and Pore-Size-Mediated Stem Cell Differentiation to Bone Regenerative Medicine.

As one of the most promising means to repair diseased tissues, stem cell therapy with immense potential to differentiate into mature specialized cells has been rapidly developed. However, the clinical application of stem-cell-dominated regenerative medicine was heavily hindered by the loss of pluripotency during the long-term in vitro expansion. Here, a composite three-dimensional (3D) graphene-based biomaterial, denoted as GO-Por-CMP@CaP, with hierarchical pore structure (micro- to macropore), was developed to guide the directional differentiation of human umbilical cord MSCs (hucMSCs) into osteoblasts. GO-Por-CMP@CaP could act as a high-efficiency living composite material without a "dead space", effectively regulating the cellular response. The 3D topological structure generated via the two-step modification on two-dimensional graphene could effectively mimic the natural 3D microenvironment of cells, enhancing the stem cell attachment, which is not only conducive for the proliferation of stem cells but also beneficial for the osteogenic differentiation. Meanwhile, the wide existence of interconnected macropores was favorable for bone ingrowth, capillary formation, as well as the nutrients transportation. Furthermore, the concurrent existence of micro- and mesopores significantly promoted the extracellular matrix (ECM) adsorption, which ensured cellular attachment, leading to multiscale osteointegration. Both in vitro and in vivo assay demonstrated the above three factors collaborated mutually with nanosized calcium phosphate (CaP, with chemical similarities to the inorganic components of bone), which provided abundant adhesive sites to adequately induce osteogenic differentiation in the absence of any soluble growth factors. Proteomic analysis experiments confirmed that GO-Por-CMP@CaP promoted the differentiation of hucMSCs cells into osteoblasts by affecting the PI3K-Akt signaling pathway through the up-regulation of SPP1 protein. Our study offers a pure material-based stem cell differentiation regulating behavior via engineering the dimension and porosity of material, which provides insights into the design and development of substitutes to bone repair materials.