In this study, we attempted to define the low survival of BMSCs overexpressing NGF in response to fracture inflammation and to improve it to promote regeneration of bone defect repair. Initially, we used lentiviral vectors loaded with overexpressed NGF genes to transfect BMSCs to determine their relationship with P75NTR and pyroptosis. Nerve Growth Factor induces osteogenic differentiation of various cells to promote fracture healing. For example, Yang et al. investigated the mechanism of action of NGF in the mouse embryonic osteogenic precursor cell line MC3T3-E, and the results showed that NGF promoted the proliferation and osteogenic differentiation of MC3T3-E while increasing the expression of BMP-2 [35]. Additionally, NGF promotes angiogenesis and trophic regeneration of skeletal sensory nerves. And vascular and nerve regeneration can significantly promote the process of bone repair. Recent studies have demonstrated that NGF stimulates the migration of skeletal cells during early bone repair and exerts various cell-specific functions [36]. Indeed, Our previous experiments indicated that cell survival was relatively low after NGF overexpression transfection of BMSCs, and P75NTR silencing combined with NGF overexpression double gene co-transfection of BMSCs demonstrated a good osteogenic capacity [37].
P75NTR is involved in various cell responses, such as apoptosis, survival, migration, and axonal growth. P75NTR has been reported to activate NF-κB signaling, up-regulate MMP-9 and VEGF expression, induce inflammation, and disrupt the blood-brain barrier in astrocytes [38]. Inhibition of P75NTR expression is known to improve the survival of neuro differentiated BMSCs [39] and enhance osteogenic differentiation of rat BMSCs [20]. Here, we found that overexpression of NGF can lead to the increase of P75NTR, and the change of P75NTR expression can regulate the expression of pyrogen related proteins in BMSCs with positive correlation. Consequently, these results support our suspect that the reduced survival of BMSCs overexpressing NGF in the fracture inflammatory response may be associated with P75NTR-induced pyroptosis. Combined with our previous studies, here our focus is to improve the pyroptosis response during the transplantation of BMSCs overexpressing NGF during the healing process of bone defects, in order to increase the survival rate of BMSCs for accelerated repair of bone defects.
In order to clarify the low survival of BMSCs overexpressing NGF during the healing process of bone defects, we selected NLRP3, IL-1β and GSDMD, which are key factors in the process of pyroptosis. Activation of NLRP3 inflammatory vesicles is the key to pyroptosis. This was reported by Wang et al., they found that NLRP3 assembles into NLRP3 inflammatory vesicles upon sensing certain pathogens, and then cuts GSDMD, promotes the secretion of IL-1β and IL-18, and causes inflammatory cell death [40]. Moreover, GSDMD has been reported to be closely related to pyroptosis and inflammation, and GSDMD is cleaved to produce N-GSDMD fragments, forming pores that increase membrane permeability, leading to pyroptosis and IL-1β release [41]. Both classical and non-classical pathways of pyroptosis require activation of GSDMD to lead to the generation of pyroptosis. Therefore, in this study, NLRP3 and GSDMD were selected as entry points for the verification of pyroptosis. This provides a target selection scheme for inhibiting pyroptosis and solving the problem of low cell survival rate in bone defect healing.
In addition, we also found that the pyroptosis of BMSCs overexpressing NGF during fracture inflammation can be blocked by NSA. During pyroptosis, NSA prevents pyroptosis by inhibiting the aggregation of long chains of GSDMD on the cell membrane. Furthermore, Zhang et al. showed that NSA could affect the mRNA and protein expression of pyroptosis related genes, inhibit the secretion of IL-6, TNF-α and IL-1β by osteoblasts, and they also found that NSA promoted the proliferation and differentiation of osteoblasts by inhibiting of NLRP3/caspase-1/GSDMD pyroptosis pathway [42]. Therefore, we selected NSA as a pyroptosis inhibitor. NSA can inhibit the pyroptosis reaction of BMSCs overexpressing NGF during the healing of bone defects in order to improve the survival rate of BMSCs. Our findings highlight the potential of NSA in inhibiting the pyroptosis of NGF+/BMSCs, making it a therapy to improve the low survival of BMSCs overexpressing NGF. Notably, the therapeutic NSA concentrations used in this study did not exhibit cytotoxic effects on cells.
In vitro having demonstrated that NSA can inhibit the pyroptosis of NGF+/BMSCs in fracture inflammation, we proceeded to the final and most important aim of this study which focused on determining the ability of NGF+/BMSCs-NSA-Sca to promote bone regeneration and defect repair in vivo, especially in a rat model of femoral condylar defect. In this study, allograft bone was used as a scaffold to implant BMSCs loaded with overexpressed NGF. Allograft bone as a graft has strong mechanical strength, good biocompatibility, and bone conductivity, and making it a better choice for repairing bone defects [43]. Consistent with previous studies [44], allogeneic bone scaffolds could effectively support the BMSCs attachment and expansion in our experiments. We constructed novel grafts to repair rats bone defects by combining NGF-overexpressed BMSCs with allograft bone and adding pyroptosis inhibitors. Additionally, previous studies have used a rat femoral condylar defect model to assess bone regeneration and defect repair in vivo [45]. Another study revealed that the critical defect size that could not spontaneously be repaired was 3.5/5.0 mm [46]. Therefore, we evaluated the osteogenic capacity of NGF+/BMSCs-NSA combined with allograft bone scaffolds in a surgical model of bone defects in rats femoral condyles with a diameter of 4.5 mm and a depth of 5.0 mm. Micro-CT analysis and histological evaluation revealed that NGF+/BMSCS-NSA scaffolds significantly enhanced new bone formation compared to scaffolds with BMSCs alone. Furthermore, WB and PCR results showed that NGF+/BMSCs-NSA-Sca group had relatively low pyroptosis genes and relatively high osteogenic genes. This demonstrated that NGF+/BMSCs combined with NSA can increase the transplantation survival rate of BMSCs by inhibiting pyroptosis, and thus participate in bone regeneration to accelerate the repair of bone defects. Overall, the results suggest that the potential of the NGF+/BMSCs-NSA-Sca system as a novel therapy for bone defects.
Through the above studies, we believe that NGF+/BMSCs-NSA-Sca is helpful to develop a new theoretical basis for the treatment of bone defects. However, the specific molecular mechanisms and pathway of pyroptosis in BMSCs overexpressing NGF are not yet clear, and whether it is related to the P75NTR-mediated NF-κB pathway. Therefore, specifically, our future work will focus on elucidate the relationship between pyroptosis in BMSCs overexpressing NGF and the NF-κB pathway in the inflammatory environment of fractures.