Manipulating Stem Cell Fate with Disordered Bioactive Cues on Surfaces: The Role of Bioactive Ligand Selection

The development of 2D or 3D bioactive platforms for rapidly isolating pure populations of cells from adult stem cells holds promise for advancing the understanding of cellular mechanisms, drug testing, and tissue engineering. Over the years, methods have emerged to synthesize bioactive micro- and nanostructured 2D materials capable of directing stem cell fate. We introduce a novel method for randomly micro- or nanopatterning any protein/peptide onto both 2D and 3D scaffolds via spray technology. Our goal is to investigate the impact of arranging bioactive micropatterns (ordered vs disordered) on surfaces to guide human mesenchymal stem cell (hMSC) differentiation. The spray technology efficiently coats materials with controlled, cost-effective bioactive micropatterns in various sizes and shapes. BMP-2 mimetic peptides were covalently grafted, individually or in combination with RGD peptides, onto activated polyethylene terephthalate (PET) surfaces through a spraying process, incorporating nano/microscale parameters like size, shape, and composition. The study explores different peptide distributions on surfaces and various peptide combinations. Four surfaces were homogeneously functionalized with these peptides (M1 to M4 with various densities of peptides), and six surfaces with disordered micro- and nanopatterns of peptides (S0 to S5 with different sizes of peptide patterns) were synthesized. Fluorescence microscopy assessed peptide distribution, followed by hMSC culture for 2 weeks, and evaluated osteogenic differentiation via immunocytochemistry and RT-qPCR for osteoblast and osteocyte markers. Cells on uniformly peptide-functionalized surfaces exhibited cuboidal forms, while those on surfaces with disordered patterns tended toward columnar or cuboidal shapes. Surfaces S4 and S5 showed dendrite-like formations resembling an osteocyte morphology. S5 showed significant overexpression of osteoblast (OPN) and osteocyte markers (E11, DMP1, and SOST) compared to control surfaces and other micropatterned surfaces. Notably, despite sharing an equivalent quantity of peptides with a homogeneous functionalized surface, S5 displayed a distinct distribution of peptides, resulting in enhanced osteogenic differentiation of hMSCs.