Engineered Small Extra-Cellular Vesicles for Endogenous Mesenchymal Stem Cells Recruitment and in situ Periodontal Tissue Regeneration

OBJECTIVES: Exo released by immune-regulatory dendritic cells (regDCs) have been used as an effective nanodelivery system for the reprogramming of immune response at inflammatory bone defects in mice. This study aims to determine the efficacy of engineered SDF-1-loaded immune-regulatory DC-derived exosomes (SDF-1exo) in MSC recruitment and bone regeneration. METHODS: Exo were purified from human and murine regDCs. Exo were loaded with SDF-1 using ultrasonication. In vitro studies of exo cargo including resistance of SDF-1 to proteolysis, MSC recruitment, and osteogenic differentiation were carried out. The therapeutic effect of SDF-1exo in hyaluronic acid-based hydrogel carrier was then tested in mandibular defects in a murine model. Five groups (n=5) were tested: group 1 (negative control), group 2 (carrier alone), group 3 (carrier with unloaded exo), group 4 (carrier with free SDF-1) and group 5 (carrier with SDF-1-exo). Biodistribution of DiI-prelabeled SDF-1-exo was performed using in-vivo imaging. Specimens were collected at 2- and 4-weeks post-operative. 3D volumetric micro-CT, histologic, qPCR, and IHC analyses were performed. RESULTS: SDF-1exo protected its cargo against lysine gingipains and DPP4 proteolytic cleavage and promoted MSC migration, and osteogenesis. TEM revealed SDF-1 localization in the exo lumen and in the transmembrane domain. Blocking experiments revealed that sustainable SDF-1 signaling required interaction between SDF-1exo and CXCR4 receptor. In vivo, SDF-1exo showed a high affinity at the defect sites and sustained SDF-1 expression during the first 2 weeks of healing, promoting MSC migration. Significantly greater bone maturation in the SDF-1exo group was observed at 4 weeks. CONCLUSIONS: This study demonstrated the efficacy of SDF-1exo delivery in promoting bone regeneration. IMPLICATIONS: This study provides the basis for a novel natural nano-therapeutic strategy for periodontal tissue regeneration in humans.