Mesenchymal stromal cell exosomes enhance dental pulp cell functions and promote pulp-dentin regeneration.

Mesenchymal stromal/stem cell (MSC) therapies are currently being explored for dental pulp regeneration. As the therapeutic effects of MSCs in tissue repair are mediated mainly through the release of extracellular vesicles (EVs) including exosomes, we investigated here the cellular processes and molecular mechanisms modulated by MSC exosomes in dental pulp regeneration. Using dental pulp cell (DPC) cultures, we demonstrated that MSC exosomes could increase DPC migration, proliferation, and odontogenic differentiation. The enhancement of these cellular processes was mediated through exosomal CD73-mediated adenosine receptor activation of AKT and ERK signaling. Consistent with these observations, MSC exosomes increased the expression of dentin matrix proteins and promoted the formation of dentin-like tissue and bridge-like structures in a rat pulp defect model. These effects were comparable to that of mineral trioxide aggregate (MTA) treatment. MSC exosomes also yielded recellularized pulp-dentin tissues in the root canal of endodontically-treated human premolars, following subcutaneous implantation in the mouse dorsum. Together, our findings suggest that MSC exosomes could exert a multi-faceted effect on DPC functions including migration, proliferation and odontogenic differentiation to promote dental pulp regeneration. This study provides the basis for development of MSC exosomes as a cell-free MSC therapeutic alternative for pulp-dentin regeneration.

Small extracellular vesicles from mesenchymal stromal cells: the next therapeutic paradigm for musculoskeletal disorders.

Musculoskeletal disorders are one of the biggest contributors to morbidity and place an enormous burden on the health care system in an aging population. Owing to their immunomodulatory and regenerative properties, mesenchymal stromal/stem cells (MSCs) have demonstrated therapeutic efficacy for treatment of a wide variety of conditions, including musculoskeletal disorders. Although MSCs were originally thought to differentiate and replace injured/diseased tissues, it is now accepted that MSCs mediate tissue repair through secretion of trophic factors, particularly extracellular vesicles (EVs). Endowed with a diverse cargo of bioactive lipids, proteins, nucleic acids and metabolites, MSC-EVs have been shown to elicit diverse cellular responses and interact with many cell types needed in tissue repair. The present review aims to summarize the latest advances in the use of native MSC-EVs for musculoskeletal regeneration, examine the cargo molecules and mechanisms underlying their therapeutic effects, and discuss the progress and challenges in their translation to the clinic.

Mesenchymal Stromal Cell Exosomes Mediate M2-like Macrophage Polarization through CD73/Ecto-5'-Nucleotidase Activity.

Mesenchymal stem/stromal cell (MSC) exosomes have been shown to alleviate immune dysfunction and inflammation in preclinical animal models. This therapeutic effect is attributed, in part, to their ability to promote the polarization of anti-inflammatory M2-like macrophages. One polarization mechanism has been shown to involve the activation of the MyD88-mediated toll-like receptor (TLR) signaling pathway by the presence of extra domain A-fibronectin (EDA-FN) within the MSC exosomes. Here, we uncovered an additional mechanism where MSC exosomes mediate M2-like macrophage polarization through exosomal CD73 activity. Specifically, we observed that polarization of M2-like macrophages by MSC exosomes was abolished in the presence of inhibitors of CD73 activity, adenosine receptors A2A and A2B, and AKT/ERK phosphorylation. These findings suggest that MSC exosomes promote M2-like macrophage polarization by catalyzing the production of adenosine, which then binds to adenosine receptors A2A and A2B to activate AKT/ERK-dependent signaling pathways. Thus, CD73 represents an additional critical attribute of MSC exosomes in mediating M2-like macrophage polarization. These findings have implications for predicting the immunomodulatory potency of MSC exosome preparations.

Mesenchymal Stem Cell Exosomes Promote Functional Osteochondral Repair in a Clinically Relevant Porcine Model.

BACKGROUND: Previous studies have reported the efficacy of human mesenchymal stem cell (MSC) exosomes for the repair of osteochondral defects in rats and rabbits. However, the safety and efficacy of MSC exosomes remain to be validated in a clinically relevant large animal model. PURPOSE: To validate the safety and efficacy of human MSC exosomes for osteochondral repair in a clinically relevant micropig model. STUDY DESIGN: Controlled laboratory study. METHODS: Bilateral osteochondral defects (6-mm diameter and 1-mm depth) were surgically created in the medial femoral condyles in knees of 12 micropigs. The pigs then received 2-mL intra-articular injections of MSC exosomes and hyaluronic acid (HA) (Exosome+HA) or HA alone after surgery and thereafter at 8 and 15 days. Osteochondral repair was assessed by magnetic resonance imaging (MRI) at 15 days and at 2 and 4 months after surgery as well as by macroscopic, histological, biomechanical, and micro-computed tomography (micro-CT) analyses at 4 months after surgery. RESULTS: Exosome+HA-treated defects demonstrated significantly better MRI scores than HA-treated defects at 15 days and at 2 and 4 months. Additionally, Exosome+HA-treated defects demonstrated functional cartilage and subchondral bone repair, with significantly better macroscopic and histological scores and biomechanical properties (Young modulus and stiffness) than HA-treated defects at 4 months. Micro-CT further showed significantly higher bone volume and trabecular thickness in the subchondral bone of Exosome+HA-treated defects than that of HA-treated defects. Importantly, no adverse response or major systemic alteration was observed in any of the animals. CONCLUSION: This study shows that the combination of MSC exosomes and HA administered at a clinically acceptable frequency of 3 weekly intra-articular injections can promote functional cartilage and subchondral bone repair, with significantly improved morphological, histological, and biomechanical outcomes in a clinically relevant porcine model. CLINICAL RELEVANCE: Our findings provide a robust scientific rationale to support a phase 1/2 clinical trial to test MSC exosomes in patients with osteochondral lesions.

Mesenchymal stem cell exosomes enhance periodontal ligament cell functions and promote periodontal regeneration.

Mesenchymal stem cells (MSCs) are potential therapeutics for the treatment of periodontal defects. It is increasingly accepted that MSCs mediate tissue repair through secretion of trophic factors, particularly exosomes. Here, we investigated the therapeutic effects of human MSC exosome-loaded collagen sponge for regeneration of surgically created periodontal intrabony defects in an immunocompetent rat model. We observed that relative to control rats, exosome-treated rats repaired the defects more efficiently with regeneration of periodontal tissues including newly-formed bone and periodontal ligament (PDL). We also observed that concomitant with this, there was increased cellular infiltration and proliferation. We therefore postulated that MSC exosomes enhanced regeneration through increased cellular mobilisation and proliferation. Using PDL cell cultures, we demonstrated that MSC exosomes could increase PDL cell migration and proliferation through CD73-mediated adenosine receptor activation of pro-survival AKT and ERK signalling. Inhibition of AKT or ERK phosphorylation suppressed PDL cell migration and proliferation. Our findings demonstrated for the first time that MSC exosomes enhance periodontal regeneration possibly by increasing PDL migration and proliferation. This study suggests that MSC exosome is a viable ready-to-use and cell-free MSC therapeutic for the treatment of periodontal defects. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cell (MSC) therapies have demonstrated regenerative potential for the treatment of periodontal defects. However, translation of cellular therapies is hampered by challenges in maintaining optimal cell vitality and viability from manufacturing and storage to final delivery to patients. Although the use of MSCs for tissue repair was first predicated on their differentiation potential, the therapeutic efficacy of MSCs has increasingly been attributed to its paracrine secretion, particularly exosomes or small extracellular vesicles. In this study, MSC exosome-loaded collagen sponge enhanced periodontal regeneration in an immunocompetent rat periodontal defect model without any obvious adverse effects. These findings provide the basis for future development of MSC exosomes as a cell-free strategy for periodontal regeneration.

MSC exosomes alleviate temporomandibular joint osteoarthritis by attenuating inflammation and restoring matrix homeostasis.

The efficacy of mesenchymal stem cell (MSC) therapies is increasingly attributed to paracrine secretion, particularly exosomes. In this study, we investigated the role of MSC exosomes in the regulation of inflammatory response, nociceptive behaviour, and condylar cartilage and subchondral bone healing in an immunocompetent rat model of temporomandibular joint osteoarthritis (TMJ-OA). We observed that exosome-mediated repair of osteoarthritic TMJs was characterized by early suppression of pain and degeneration with reduced inflammation, followed by sustained proliferation and gradual improvements in matrix expression and subchondral bone architecture, leading to overall joint restoration and regeneration. Using chondrocyte cultures, we could attribute some of the cellular activities during exosome-mediated joint repair to adenosine activation of AKT, ERK and AMPK signalling. Specifically, MSC exosomes enhanced s-GAG synthesis impeded by IL-1ß, and suppressed IL-1ß-induced nitric oxide and MMP13 production. These effects were partially abrogated by inhibitors of adenosine receptor activation, AKT, ERK and AMPK phosphorylation. Together, our observations suggest that MSC exosomes promote TMJ repair and regeneration in OA through a well-orchestrated mechanism of action that involved multiple cellular processes to restore the matrix and overall joint homeostasis. This study demonstrates the translational potential of a cell-free ready-to-use exosome-based therapeutic for treating TMJ pain and degeneration.