Functionally engineered extracellular vesicles improve bone regeneration.

Lineage specific differentiation of host mesenchymal stem cells (MSCs) is a necessary step for bone repair/regeneration. Clinically, growth factors such as bone morphogenetic protein 2 (BMP2) are used to enhance/hasten this process to heal critical sized defects. However, the clinical application of such growth factors is fraught with dosage challenges as well as immunological and ectopic complications. The identification of extracellular vesicles (EVs) as active components of the MSC secretome suggest alternative approaches to enhancing bone regeneration. Based on our earlier studies on the properties of EVs from lineage specified MSCs, this study sought to engineer EVs to enhance osteogenic differentiation. To generate MSC EVs with enhanced osteoinductive abilities, genetically modified human bone marrow derived MSCs (HMSCs) were generated by constitutively expressing BMP2. We hypothesized that these cells would generate functionally engineered EVs (FEEs) with enhanced osteoinductive properties. Our results show that these FEEs maintained the general physical and biochemical characteristics of naïve HMSC EVs in the form of size distribution, EV marker expression and endocytic properties but show increased bone regenerative potential compared to MSC EVs in a rat calvarial defect model in vivo. Mechanistic studies revealed that although BMP2 was constitutively expressed in the parental cells, the corresponding EVs (FEEs) do not contain BMP2 protein as an EV constituent. Further investigations revealed that the FEEs potentiate the BMP2 signaling cascade possibly due to an altered miRNA composition. Collectively, these studies indicate that EVs' functionality may be engineered by genetic modification of the parental MSCs to induce osteoinduction and bone regeneration. SIGNIFICANCE STATEMENT: With mounting evidence for the potential of MSC EVs in treatment of diseases and regeneration of tissues, it is imperative to evaluate if they can be modified for application specificity. The results presented here indicate the possibility for generating Functionally Engineered EVs (FEEs) from MSC sources. As a proof of concept approach, we have shown that EVs derived from genetically modified MSCs (BMP2 overexpression) can be effective as biomimetic substitutes for growth factors for enhanced tissue-specific regeneration (bone regeneration) in vivo. Mechanistic studies highlight the role of EV miRNAs in inducing pathway-specific changes. We believe that this study will be useful to researchers evaluating EVs for regenerative medicine applications.