(247d) Granular Hydrogel Scaffolds Modulate MSC Microenvironment and Extracellular Vesicle Secretion for Calvarial Defect Bone Regeneration

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 (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. Initially, we use our versatile microgel scaffolds to in vitro modulate the MSC microenvironment and shift EV protein and mi-RNA content towards an osteogenic profile. Subsequently, using our versatile thiol-ene bio-click chemistry we engineer a granular biodegradable scaffold for the controlled release of thiol-modified osteogenic EVs in rat critical-sized calvarial defect over 12 weeks.

Specifically, in the first part of this study, we fabricate granular hydrogel scaffolds for bone marrow MSC ex vivo cell culture with amide-, ester-, or thioester-linked PEG-norbornene (8 arm, 20 kDa) photo-clicked with PEG-thiol (4 arm, 10ka) and 1 mM RGD. Microgels (~200µm) are created using submerged electrospray droplet generation which allows for rapid processing of a large number of microgels, while maintaining a relatively low microgel diameter polydispersity (Fig. 1A). The microgels are functionalized with biomimetic peptides HAVDI and GFOGER (N-Cadherin, Collagen respectively) to shift the MSC secretome towards an osteogenic profile. Harvested EVs (Fig. 1B) show increased protein levels of osteoanabolic factors (PDGF-AA, BMP2, TGF-β1, VEGF) as well as mi-R203 a key regulator of pre-osteoblast marker RUNX2. Hierarchical clustering analysis of proteomics data using pairwise comparison from harvested EVs without or with biomimetic peptides depicts upregulation of differentially expressed osteoanabolic proteins in the latter culture condition. Co-culture of osteogenic EVs with MSCs from osteoporotic rats (OVX) indicate significant upregulation of osteogenic markers RUNX2 and ALP over two weeks (Fig. 1C). Taken together this data indicates that our functionalized granular microgel scaffold is a powerful tool for modulating MSC microenvironment to influence MSC secretion towards osteogenesis or potentially other profiles (i.e. immunomodulation).

In the second part of this study, we engineer a granular microgel scaffold for the controlled in vivo delivery of the previously harvested osteogenic EVs in a rat critical size calvarial defect model. Firstly, we metabolically glycoengineer MSCs and their secreted EVs through a ManNAc analog of Neu5AC that replaces sialic acid membrane sugars with thiols, thus allowing to click EVs on thiol-ene granular microgels. Crosslinking the granular hydrogel scaffold with MMP cleavable peptides allowed for degradation times of 4-6 weeks resulting in a slow and controlled in vitro release of clicked EVs. After transplantation in a critical size calvarial defect of rats, we observed that osteoprogenitor cells infiltrating the EV laden microgel scaffolds expressed mature osteogenic markers (RUNX2, OPN) faster (4-5 weeks) compared to control (6-8 weeks). Overall, we have utilized granular hydrogels to modulate MSC microenvironment and engineered an innovative acellular system for the controlled delivery of pro-osteogenic EVs for in vivo bone regeneration.