(131e) Granular Hydrogel Scaffolds Control MSC Derived Extracellular Vesicle Delivery to Modulate Inflammation in Rat Calvarial Defect

Repair of critical-sized craniofacial bone defects remains a major challenge in regenerative medicine, and underlying conditions, such as post-menopausal osteoporosis, can further complicate bone healing. To promote bone regeneration, many clinical trials deliver bone marrow derived mesenchymal stem/stromal cells (MSCs) and rely on their secreted factors (e.g., growth factors, chemokines, extracellular vesicles (EVs)) to improve healing. Despite promising pre-clinical results, the direct delivery of MSCs in vivo leads to low survival and rapid clearance due to acute inflammation, (i.e., <5% of MSCs survive 24 hours post-transplantation), which has limited successful translation to new clinical therapies. Thus, in this study we present an alternative to direct delivery of MSCs, focusing on an innovative strategy of MSC-secreted EV delivery, targeted towards resolving inflammation and facilitating bone regeneration. Initially, we use our versatile microgel scaffolds to in vitro modulate the MSC microenvironment and shift EV protein and mi-RNA content towards an anti-inflammatory profile. Subsequently, using our Strain-Promoted Azide-Alkyne Click Chemistry reaction (SPAAC), we engineer a granular biodegradable scaffold for the controlled release of azide-modified anti-inflammatory EVs in rat critical-sized calvarial defect.

Specifically, in the first part of this study, we fabricate PEG-DBCO-Azide-PEG granular hydrogel scaffolds for bone marrow MSC ex vivo cell culture. Microgels (~200µm) are created using bulk emulsification of a large number of microgels, while maintaining a relatively low microgel diameter polydispersity (Fig. 1a). The microgels are functionalized with biomimetic peptides (hexapeptides-WKYMVm) to shift the MSC secretome towards an anti-inflammatory profile. Harvested EVs (Fig. 1b) show increased protein levels of anti-inflammatory cytokines (IL-4/10) as well as an array of miRNAs (mi-R146a, 125a, 223), key regulators of M2a macrophage polarization. Hierarchical clustering analysis of proteomics data using pairwise comparison from harvested EVs without or with biomimetic peptides depicts upregulation of differentially expressed anti-inflammatory cytokines in the latter culture condition. Co-culture of anti-inflammatory EVs with M0 macrophages from osteoporotic rats (OVX) indicate significant upregulation of anti-inflammatory marker CD206, in comparison to chemically induced differentiation through IL-4 and IL-10. Taken together this data indicates that our functionalized granular microgel scaffold is a powerful tool for modulating MSC microenvironment to influence MSC secretion towards immunomodulation or potentially other profiles (i.e. osteogenesis).

In the second part of this study, we engineer a granular microgel scaffold for the controlled in vivo delivery of the previously harvested anti-inflammatory EVs in a rat critical size calvarial defect model. Firstly, we metabolically glycoengineer MSCs and their secreted EVs through a ManNAz analog of ManNAc that replaces sialic acid membrane sugars with Azide groups, thus allowing to click EVs on DBCO-excess granular microgels. After transplantation in a critical size calvarial defect of rats, we observed that macrophages infiltrating the EV laden microgel scaffolds polarized towards an M2a phenotype over 3 days (Fig. 1c), ultimately resolving inflammation over 7 days, significantly faster than the scaffolds without EVs. Ultimately, the granular microgels bio-degraded over 4-6 weeks, thanks to the hydrolysis of their ester groups allowing for more efficient osteoanabolic activity. Overall, we have utilized granular hydrogels to modulate MSC microenvironment and engineered an innovative acellular system for the controlled delivery of anti-inflammatory EVs for in vivo bone regeneration.