Injectable MMP1-sensitive microspheres with spatiotemporally controlled exosome release promote neovascularized bone healing

Bone marrow mesenchymal stromal cell-derived exosomes (BMSC-Exos) can recruit stem cells for bone repair, with neovessels serving as the main migratory channel for stem cells to the injury site. However, existing exosome (Exo) delivery strategies cannot reach the angiogenesis phase following bone injury. To that end, an enzyme-sensitive Exo delivery material that responds to neovessel formation during the angiogenesis phase was designed in the present study to achieve spatiotemporally controlled Exo release. Herein, matrix metalloproteinase-1 (MMP1) was found to be highly expressed in neovascularized bone; as a result, we proposed an injectable MMP1-sensitive hydrogel microspheres (KGE) made using a microfluidic chip prepared by mixing self-assembling peptide (KLDL-MMP1), GelMA, and BMSC-Exos. The results revealed that KGE microspheres had a uniform diameter of 50-70 µm, ideal for minimally invasive injection and could release exosomes in response to MMP1 expression. In vitro experiments demonstrated that KGE had less cytotoxicity and could promote the migration and osteodifferentiation of BMSCs. Furthermore, in vivo experiments confirmed that KGE could promote bone repair during angiogenesis by recruiting CD90+ stem cells via neovessels. Collectively, our results suggest that injectable enzyme-responsive KGE microspheres could be a promising Exo-secreting material for accelerating neovascularized bone healing. STATEMENT OF SIGNIFICANCE: Exosomes can spread through blood vessels and activate stem cells to participate in bone repair, but under normal circumstances, exosomes lacking sustained-release delivery materials cannot be maintained until the angiogenesis phase. In this study, we found that MMP1 was highly expressed in neovascularized bone, then we proposed an MMP1-sensitive injectable microsphere that carries exosomes and responds temporally and spatially to neovascularization, which maximizes the ability of exosomes to recruit stem cells. Different from previous strategies that focus on promoting angiogenesis to accelerate bone healing, this is a brand new delivery strategy that is stimuli-responsive to neovessel formation. In addition, the preparation of self-assembled peptide microspheres by a microfluidic chip is also proposed for the first time.