Summary Injectable hydrogels can provide a scaffold for in situ tissue regrowth and regeneration, however these injected materials require gel degradation prior to tissue reformation limiting their ability to provide physical support. We have created a new class of injectable biomaterial that circumvents this challenge by providing an interconnected microporous network for simultaneous tissue reformation and material degradation. We assemble monodisperse micro-gel building blocks into an interconnected microporous annealed particle (MAP) scaffold. Through microfluidic formation, we tailor the chemical and physical properties of the building blocks, providing downstream control of the physical and chemical properties of the assembled MAP scaffold. In vitro, cells incorporated during MAP scaffold formation proliferated and formed extensive 3D networks within 48 hours. In vivo, the injectable MAP scaffold facilitated cell migration resulting in rapid cutaneous tissue regeneration and tissue structure formation within 5 days. The combination of microporosity and injectability achieved with MAP scaffolds will enable novel routes to tissue regeneration in vivo and tissue creation de novo.

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Hydrogel scaffolds are commonly used as 3D carriers for cells because their properties can be tailored to match natural extra-cellular matrix. Hydrogels may be used in tissue engineering and regenerative medicine to deliver therapeutic cells to injured or diseased tissue through controlled degradation. Hydrolysis and enzymolysis are the two most common mechanisms employed for hydrogel degradation, but neither allows sequential or staged release of cells. In contrast, photodegradation allows external real-time spatial and temporal control over hydrogel degradation, and allows for staged and sequential release of cells. We synthesized and characterized a series of macromers incorporating photodegradbale ortho-nitrobenzyl (o-NB) groups in the macromer backbone. We formed hydrogels from these macromers via redox polymerization and quantified the apparent rate constants of degradation (kapp) of each via photorheology at 370 nm, 10 mW/cm2. Decreasing the number of aryl ethers on the o-NB group increases kapp, and changing the functionality from primary to seconday at the benzylic site dramatically increases kapp. Human mesenchymal stem cells (hMSCs) survive encapsulation in the hydrogels (90% viability post-encapsulation). By exploiting the differences in reactivity of two different o-NB linkers, we quantitatively demonstrate the biased release of one stem cell population (green-fluroescent protein expressing hMSCs) over another (red-fluorescent protein expressing hMSCs).

Microporous annealed particle (MAP) scaffolds are flowable, in situ crosslinked, microporous scaffolds composed of microgel building blocks and were previously shown to accelerate wound healing. To promote more extensive tissue ingrowth before scaffold degradation, we aimed to slow MAP degradation by switching the chirality of the crosslinking peptides from L- to D-amino acids. Unexpectedly, despite showing the predicted slower enzymatic degradation in vitro , D-peptide crosslinked MAP hydrogel (D-MAP) hastened material degradation in vivo and imparted significant tissue regeneration to healed cutaneous wounds, including increased tensile strength and hair neogenesis. MAP scaffolds recruit IL-33 type 2 myeloid cells, which is amplified in the presence of D-peptides. Remarkably, D-MAP elicited significant antigen-specific immunity against the D-chiral peptides, and an intact adaptive immune system was required for the hydrogel-induced skin regeneration. These findings demonstrate that the generation of an adaptive immune response from a biomaterial is sufficient to induce cutaneous regenerative healing despite faster scaffold degradation.