The transplantation of tissue-engineered scaffolds with stem cells is a promising therapeutic approach for bone defect repair. To improve the therapeutic efficacy of this approach, in this study, a novel biofunctional live tissue-engineered bone-like graft was designed and constructed using a fibrin scaffold loaded with TG2 gene-modified ectomesenchymal stem cells (TG2-EMSCs) derived from nasal respiratory mucosa for bone defect repair. Autocalcification of the cell-free fibrin gel in osteogenic medium with additional alkaline phosphatase (ALP) and the osteogenic differentiation of TG2-EMSCs on the fibrin scaffold were assessed in vitro. The results indicated that the cell-free fibrin gel could autocalcify in the osteogenic medium with ALP and that the overexpression of TG2 by TG2-EMSCs could promote the osteogenic differentiation of these stem cells in the fibrin scaffold. Moreover, TG2 could enhance the deposition of extracellular matrix proteins in the fibrin scaffold, followed by calcification of the bone matrix in vitro. After transplantation into critical-sized cranial defects in rats, the functional tissue-engineered bone-like grafts improved bone regeneration. These results indicate that this tissue-engineered bone-like graft could improve the process of bone defect repair.

Ectomesenchymal stem cells (EMSCs) represent a type of adult stem cells derived from the cranial neural crest. These cells are capable of self-renewal and have the potential for multidirectional differentiation. Tissue transglutaminase type 2 (TG2) is a ubiquitously expressed member of the transglutaminase family of Ca 2+ -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 TG2-overexpressing EMSC cell line (TG2-EMSCs) 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 TG2-EMSCs 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 TG2-EMSCs. Notably, TG2-EMSC transplantation in an animal model of spinal cord injury (SCI), TG2-EMSCs differentiated into neuron-like 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 EMSC-based transplantation therapy in patients with SCI.

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