Conjugation of IL-33 to Microporous Annealed Particle Scaffolds Enhances Type 2-Like Immune Responses In Vitro and In Vivo.

The inflammatory foreign body response (FBR) is the main driver of biomaterial implant failure. Current strategies to mitigate the onset of a FBR include modification of the implant surface, release of anti-inflammatory drugs, and cell-scale implant porosity. The microporous annealed particle (MAP) scaffold platform is an injectable, porous biomaterial composed of individual microgels, which are annealed in situ to provide a structurally stable scaffold with cell-scale microporosity. MAP scaffold does not induce a discernible foreign body response in vivo and, therefore, can be used a "blank canvas" for biomaterial-mediated immunomodulation. Damage associated molecular patterns (DAMPs), such as IL-33, are potent regulators of type 2 immunity that play an important role in tissue repair. In this manuscript, IL-33 is conjugated to the microgel building-blocks of MAP scaffold to generate a bioactive material (IL33-MAP) capable of stimulating macrophages in vitro via a ST-2 receptor dependent pathway and modulating immune cell recruitment to the implant site in vivo, which indicates an upregulation of a type 2-like immune response and downregulation of a type 1-like immune response.

Delivery of Dissociated Islets Cells within Microporous Annealed Particle Scaffold to Treat Type 1 Diabetes.

Type 1 diabetes (T1D) is caused by the autoimmune loss of insulin-producing beta cells in the pancreas. The only clinical approach to patient management of blood glucose that doesn't require exogenous insulin is pancreas or islet transplantation. Unfortunately, donor islets are scarce and there is substantial islet loss immediately after transplantation due, in part, to the local inflammatory response. The delivery of stem cell-derived beta cells (e.g., from induced pluripotent stem cells) and dissociated islet cells hold promise as a treatment for T1D; however, these cells typically require re-aggregation in vitro prior to implantation. Microporous scaffolds have shown high potential to serve as a vehicle for organization, survival, and function of insulin-producing cells. In this study, we investigated the use of microporous annealed particle (MAP) scaffold for delivery of enzymatically dissociated islet cells, a model beta cell source, within the scaffold's interconnected pores. We found that MAP-based cell delivery enables survival and function of dissociated islets cells both in vitro and in an in vivo mouse model of T1D.

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold.

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that can form a structurally stable scaffold with cell-scale porosity in situ following a secondary light-based chemical crosslinking step (i.e., annealing). MAP scaffold has shown success in a variety of regenerative medicine applications, including dermal wound healing, vocal fold augmentation, and stem cell delivery. This paper describes the methods for synthesis and characterization of poly(ethylene glycol) (PEG) microgels as the building blocks to form a MAP scaffold. These methods include the synthesis of a custom annealing macromer (MethMAL), determination of microgel precursor gelation kinetics, microfluidic device fabrication, microfluidic generation of microgels, microgel purification, and basic scaffold characterization, including microgel sizing and scaffold annealing. Specifically, the high-throughput microfluidic methods described herein can produce large volumes of microgels that can be used to generate MAP scaffolds for any desired application, especially in the field of regenerative medicine.

Spatially heterogeneous epidermal growth factor release from microporous annealed particle (MAP) hydrogel for improved wound closure.

Microporous annealed particle (MAP) hydrogel has been a promising scaffold platform technology to promote immediate tissue integration in injured tissue environments. The addition of growth factors has the potential to accelerate tissue integration and enhance scaffold-mediated healing. Growth factor releasing scaffolds face the translational hurdle of limited solubilized protein shelf stability; however, to address this hurdle we present a lyophilized MAP scaffold which can be effectively rehydrated directly prior to use. Our new approach includes a heterogenous MAP scaffold wherein 5% of the microgels contain immobilized heparin loaded with epidermal growth factor (EGF) at 1 µg mL-1. We demonstrate that these scaffolds, which are directly loaded with EGF following lyophilization maintain equivalent properties to scaffolds loaded passively via diffusion into water-swollen microgels, including EGF release profiles and cell migration studies that did not significantly differ. Further, these heterogeneous scaffolds exhibit a significant increase in cellular migration in vitro and quicker re-epithelialization in vivo. This progress on spatially heterogenous growth factor release from MAP scaffolds has great potential to improve complex wound treatment and advance the field of growth factor releasing scaffolds.

Synthesis of sonicated fibrin nanoparticles that modulate fibrin clot polymerization and enhance angiogenic responses.

Chronic wounds can occur when the healing process is disrupted and the wound remains in a prolonged inflammatory stage that leads to severe tissue damage and poor healing outcomes. Clinically used treatments, such as high density, FDA-approved fibrin sealants, do not provide an optimal environment for native cell proliferation and subsequent tissue regeneration. Therefore, new treatments outside the confines of these conventional fibrin bulk gel therapies are required. We have previously developed flowable, low-density fibrin nanoparticles that, when coupled to keratinocyte growth factor, promote cell migration and epithelial wound closure in vivo. Here, we report a new high throughput method for generating the fibrin nanoparticles using probe sonication, which is less time intensive than the previously reported microfluidic method, and investigate the ability of the sonicated fibrin nanoparticles (SFBN) to promote clot formation and cell migration in vitro. The SFBNs can form a fibrin gel when combined with fibrinogen in the absence of exogenous thrombin, and the polymerization rate and fiber density in these fibrin clots is tunable based on SFBN concentration. Furthermore, fibrin gels made with SFBNs support cell migration in an in vitro angiogenic sprouting assay, which is relevant for wound healing. In this report, we show that SFBNs may be a promising wound healing therapy that can be easily produced and delivered in a flowable formulation.

Biomimetic microgels with controllable deformability improve healing outcomes.

Platelets mediate hemostasis by aggregating and binding to fibrin to promote clotting. Over time, platelets contract the fibrin network to induce clot retraction, which contributes to wound healing outcomes by increasing clot stability and improving blood flow to ischemic tissue. In this study, we describe the development of hollow platelet-like particles (PLPs) that mimic the native platelet function of clot retraction in a controlled manner and demonstrate that clot retraction-inducing PLPs promote healing in vivo. PLPs are created by coupling fibrin-binding antibodies to CoreShell (CS) or hollow N-isopropylacrylamide (NIPAm) microgels with varying degrees of shell crosslinking. We demonstrate that hollow microgels with loosely crosslinked shells display a high degree of deformability and mimic activated platelet morphology, while intact CS microgels and hollow microgels with increased crosslinking in the shell do not. When coupled to a fibrin-binding antibody to create PLPs, hollow particles with low degrees of shell crosslinking cause fibrin clot collapse in vitro, recapitulating the clot retraction function of platelets, while other particle types do not. Furthermore, hollow PLPs with low degrees of shell crosslinking improve some wound healing outcomes in vivo.