Copper-free clickable alginate microcapsules promote early osteogenic differentiation by in situ conjugation of RGD-DWIVA branched peptides.

Extracellular environments regulate cell proliferation, differentiation, and function through dynamic interactions between cells and the extracellular matrix (ECM), as well as through soluble biochemical cues such as growth factors, all of which are governed by coupled biochemical reactions and mass transport processes. To engineer such environments in a controllable manner, we developed azide-cyclooctyne clickable alginate microcapsules that allow bioactive peptides to be introduced by in situ conjugation via strain-promoted azide-alkyne cycloaddition (SPAAC). Osteoblastic cells were encapsulated in cyclooctyne-modified alginate microcapsules, and branched bone morphogenetic protein 2 (BMP-2) mimetic peptides bearing azide groups were added to the culture medium. The peptides diffused into the microcapsules and selectively reacted with cyclooctyne groups without nonspecific reactions, resulting in spatially confined accumulation controlled by diffusion and click reaction kinetics. This localized enrichment of osteogenic signals promoted early osteogenic differentiation of the encapsulated cells, as indicated by activation of the Col1a1-GFP reporter. From a bioreaction engineering perspective, the present system demonstrates an in situ conjugation strategy for spatiotemporal regulation of bioactive molecules within microstructured materials, enabling modulation of early osteogenic responses without direct chemical modification of cells. This bioinspired in situ conjugation approach based on the click chemistry provides a versatile platform for biomedical, bioindustrial, and biochemical applications.