Granular hydrogels for the controlled delivery of immunomodulatory and angiogenic extracellular vesicles to enhance bone tissue regeneration

PROJECT SUMMARY The healing of large bone defects resulting from traumatic injuries, fracture nonunion, and tumor resection remains a significant clinical challenge. Approximately 500,000 patients undergo bone transplants each year in the US alone and bone diseases and their complications account for half of chronic disease among individuals greater than 50 years old. Common surgical interventions such as autologous, allogenic, or xenogeneic bone grafts can suffer from serious limitations (e.g., availability of grafts, donor site morbidity and pain, incompatibility, immunogenic reactions, and infectious disease). Therefore, researchers have explored tissue engineering approaches to develop suitable bone replacements or regeneration strategies; however, these approaches can be complicated by a heavy, persistent immune response and inadequate vascularization throughout large constructs in vivo. We aim to use a multifactorial approach to regenerating bone tissue by harnessing the regenerative potential of the MSC secretome, specifically extracellular vesicles (EVs), in combination with controlled released from porous biomaterial scaffolds. We hypothesize that controlled release of anti- inflammatory and pro-angiogenic EVs from granular hydrogels will create a pro-healing microenvironment and promote scaffold vascularization, improving overall bone formation by endogenous cells in critical-sized defects. First, we will systematically identify granular hydrogel properties (e.g., porosity, stiffness, bioactive molecule presentation) that promote MSC secretion of EVs enriched with anti-inflammatory and pro-angiogenic factors (Aim 1). Next, we will use glycoengineering approaches to produce modified EVs that can be conjugated to our granular scaffolds via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistries before encapsulating cells within EV-laden scaffolds to investigate the influence anti-inflammatory and pro-angiogenic EVs on macrophage polarization and vascularization, respectively, in vitro (Aim 2). We will then fabricate granular hydrogels using heterogeneous populations of microgels to vary in vivo degradation and enable temporal control over EV release profiles. We will conduct a short-term (7 days) rat subcutaneous implant study to test the capacity of EV-laden granular scaffolds to modulate early inflammation and vessel invasion, providing iterative feedback on scaffold design. Finally, we will interrogate the ability of these complex granular scaffolds to modulate inflammation, vascularization, and osteogenesis to promote bone regeneration by endogenous cells in a rat critical-sized calvarial defect model (Aim 3). This proposal will utilize sophisticated experimental techniques to explore the complexities of cell-cell, cell-matrix, and cell-EV interactions. The contributions of this proposal would significantly impact the wide array of regenerative medicine strategies focused on granular hydrogels, extracellular vesicle therapeutics, and bone tissue engineering.