Rapid Bioorthogonal Chemistry Enables in Situ Modulation of the Stem Cell Behavior in 3D without External Triggers

Chemical modification of engineered microenvironments surrounding living cells represents a means for directing cellular behaviors through cell-matrix interactions. Presented here is a temporally controlled method for modulating the properties of biomimetic, synthetic extracellular matrices (ECM) during live cell culture employing the rapid, bioorthogonal tetrazine ligation with trans-cyclooctene (TCO) dienophiles. This approach is diffusion-controlled, cytocompatible, and does not rely on light, catalysts, or other external triggers. Human bone-marrow-derived mesenchymal stem cells (hMSCs) were initially entrapped in a hydrogel prepared using hyaluronic acid carrying sulfhydryl groups (HA-SH) and a hydrophilic polymer bearing both acrylate and tetrazine groups (POM-AT). Inclusion of a matrix metalloprotease (MMP)-degradable peptidic cross-linker enabled hMSC-mediated remodeling of the synthetic environment. The resultant network displayed dangling tetrazine groups for subsequent conjugation with TCO derivatives. Two days later, the stiffness of the matrix was increased by adding chemically modified HA carrying multiple copies of TCO (HA-TCO) to the hMSC growth media surrounding the cell-laden gel construct. In response, cells developed small processes radially around the cell body without a significant alteration of the overall shape. By contrast, modification of the 3D matrix with a TCO-tagged cell-adhesive motif caused the resident cells to undergo significant actin polymerization, changing from a rounded shape to spindle morphology with long cellular processes. After additional 7 days of culture in the growth media, quantitative analysis showed that, at the mRNA level, RGD tagging upregulated cellular expression of MMP1, but downregulated the expression of collagen I/III and tenascin C. RGD tagging, however, was not sufficient to induce the classic osteoblastic, chondrogenic, adipogenic, or fibroblastic/myofibroblastic differentiation. The modular approach allows facile manipulation of synthetic ECM to modulate cell behavior, thus potentially applicable to the engineering of functional tissues or tissue models.