One-Step Alignment of Cell-Embedded Collagen Promotes Spontaneous Osteogenesis of Mesenchymal Stem Cells for Bone Regeneration

Recent advancements in biomaterials for tissue engineering have focused on developing biomimetic materials engineered to induce precise cellular responses and facilitate tissue regeneration through biomolecular recognition. These biomimetic materials replicate numerous functions of the extracellular matrix (ECM) such as delivering biological signals for cell-matrix interactions to enhance tissue regeneration.<br/>One such application within tissue engineering is bone regeneration, where strategies have increasingly emphasized the use of stem cells embedded in hydrogels. Hydrogels are regarded as excellent candidates for addressing bone defects due to their similarity to natural ECM and their capacity for functionalization, enabling them to mimic the physiological environment and regulate cell fates and tissue regeneration.<br/>In this study, we developed the three-dimensional (3D) aligned collagen embedding human mesenchymal stem cells (hMSCs) for bone regeneration, aiming to elucidate the impact of collagen alignment on the osteogenic differentiation of hMSCs. Our research focused on mimicking the structure of the natural bone matrix by applying one-step mechanical strain to hMSCs-embedded collagen. Notably, hMSCs in aligned 3D collagen spontaneously differentiated into osteoblasts without the need for any inducing agents. We demonstrated that the BMP2-Smad1/5 signaling pathway, which strongly promotes osteogenic differentiation of hMSCs, is activated by aligned 3D collagen, as evidenced by analyzing the distribution and orientation of focal adhesions (FAs), measured by integrin α2 (ITGA2) and collagen immunofluorescence.<br/>We confirmed the efficacy of this approach in promoting new bone formation through<i> in vivo</i> studies using a critical-sized rat calvarial defect model. The bone volume percentage within the defect was significantly higher in the 3D aligned hMSCs embedded collagen group compared to the randomly oriented hMSCs embedded collagen and the bare collagen group in 8 weeks post-surgery. Histological and morphometric analyses revealed substantial bone remodeling and enhanced structural integrity. Our comprehensive findings from both <i>in vitro</i> and <i>in vivo</i> studies suggest that these engineered 3D collagen scaffolds embedding stem cells have considerable promise for clinical application for rapid and effective bone regeneration.