Human nasal mucosa ectomesenchymal stem cells derived extracellular vesicles loaded omentum/chitosan composite scaffolds enhance skull defects regeneration.

Engineered bone tissue that can promote osteogenic differentiation is considered an ideal substitute for materials to heal bone defects. Extracellular vesicle (EV)-based cell-free regenerative therapies represent an emerging promising alternative for bone tissue engineering. We hypothesized that EVs derived from human nasal mucosa-derived ectomesenchymal stem cells (hEMSCs) can promote bone tissue regeneration. Herein, hEMSCs were cultured with osteogenic induction medium or normal medium to generate two types of EVs. We first demonstrated that the two EVs exhibited strong potential to promote rat suture mesenchymal stem cell (SMSC) osteogenesis by transferring TG2 to SMSCs and regulating extracellular matrix (ECM) synthesis. Next, we developed a composite hydrogel made of porcine omentum and chitosan into which EVs were adsorbed to enable the effective delivery of EVs with sustained release kinetics. Implantation of the EV-loaded hydrogels in a critical-size rat cranial defect model significantly promoted bone regeneration. Therefore, we suggest that our hEMSC-derived EV-loading system can serve as a new therapeutic paradigm for promoting bone tissue regeneration in the clinic.

Tetracycline-grafted mPEG-PLGA micelles for bone-targeting and osteoporotic improvement.

Aim: We aimed to create a nano drug delivery system with tetracycline (TC)-grafted methoxy poly-(ethylene-glycol)‒poly-(D, L-lactic-co-glycolic acid) (mPEG‒PLGA) micelles (TC‒mPEG‒PLGA) with TC and mPEG‒PLGA for potential bone targeting. Prospectively, TC‒mPEG‒PLGA aims to deliver bioactive compounds, such as astragaloside IV (AS), for osteoporotic therapy. Methods: Preparation and evaluation of TC‒mPEG‒PLGA were accomplished via nano-properties, cytotoxicity, uptake by MC3T3-E1 cells, ability of hydroxyapatite targeting and potential bone targeting in vivo, as well as pharmacodynamics in a rat model. Results: The measured particle size of AS-loaded TC‒mPEG‒PLGA micelles was an average of 52.16 ± 2.44 nm, which exhibited a sustained release effect compared to that by free AS. The TC‒mPEG‒PLGA demonstrated low cytotoxicity and was easily taken by MC3T3-E1 cells. Through assaying of bone targeting in vitro and in vivo, we observed that TC‒mPEG‒PLGA could effectively increase AS accumulation in bone. A pharmacodynamics study in mice suggested potentially increased bone mineral density by AS-loaded TC‒mPEG‒PLGA in ovariectomized rats compared to that by free AS. Conclusion: The nano drug delivery system (TC‒mPEG‒PLGA) could target bone in vitro and in vivo, wherein it may be used as a novel delivery method for the enhancement of therapeutic effects of drugs with osteoporotic activity.

Enhanced neural differentiation of neural stem cells by sustained release of Shh from TG2 gene-modified EMSC co-culture in vitro.

As a promising cell therapy, neural crest-derived ectoderm mesenchymal stem cells (EMSCs) secrete high amounts of extracellular matrix (ECM) and neurotrophic factors, promoting neural stem cell (NSC) differentiation into neuronal lineages and aiding tissue regeneration. Additionally, the forced overexpression of secreted proteins can increase the therapeutic efficacy of the secretome. Tissue transglutaminase (TG2) is a ubiquitously expressed member of the transglutaminase family of calcium-dependent crosslinking enzymes, which can stabilize the ECM, inducing smart or living biomaterial to stimulate differentiation and enhance the neurogenesis of NSCs. In this study, we examined the neuronal differentiation of NSCs induced by TG2 gene-modified EMSCs (TG2-EMSCs) in a co-culture model directly. Two weeks after initiating differentiation, levels of the neuronal markers, tubulin beta 3 class III and growth-associated protein 43, were higher in NSCs in the TG2-EMSC co-culture group and those of the astrocytic marker glial fibrillary acidic protein were lower, compared with the control group. These results were confirmed by immunofluorescence, and laminin, fibronectin and sonic hedgehog (Shh) contributed to this effect. The results of western blot analysis and the enzyme-linked immunoassay showed that after TG2-EMSCs were co-cultured for 2 weeks, they expressed much higher levels of Shh than the control group. Moreover, the sustained release of Shh was observed in the TG2-EMSC co-culture group. Overall, our findings indicate that EMSCs can induce the differentiation of NSCs, of which TG2-EMSCs can promote the differentiation of NSCs compared with EMSCs.

Overexpression of TG2 enhances the differentiation of ectomesenchymal stem cells into neuron­like cells and promotes functional recovery in adult rats following spinal cord injury.

Ectomesenchymal stem cells (EMSCs) represent a type of adult stem cells derived from the cranial neural crest. These cells are capable of self­renewal and have the potential for multidirectional differentiation. Tissue transglutaminase type 2 (TG2) is a ubiquitously expressed member of the transglutaminase family of Ca2+­dependent crosslinking enzymes. However, the effect of TG2 on neural differentiation and proliferation of EMSCs remains unknown. To determine whether TG2 improves EMSC proliferation and neurogenesis, a stable TG2­overexpressing EMSC cell line (TG2­EMSCs) was established by using an adenovirus system. Immunofluorescence staining and western blot analyses demonstrated that TG2 overexpression had beneficial effects on the rate of EMSC neurogenesis, and that the proliferative capacity of TG2­EMSCs was higher than that of controls. Furthermore, the results of western blotting revealed that extracellular matrix (ECM) and neurotrophic factors were upregulated during the differentiation of TG2­EMSCs. Notably, TG2­EMSC transplantation in an animal model of spinal cord injury (SCI), TG2­EMSCs differentiated into neuron­like cells and enhanced the repair of SCI. Taken together, these results demonstrated that TG2 gene transfection may offer a novel strategy to enhance EMSC proliferation and neurogenesis in vivo and in vitro, which may ultimately facilitate EMSC­based transplantation therapy in patients with SCI.