Acceleration of bone repairation by BMSCs overexpressing NGF combined with NSA and allograft bone scaffolds

Abstract

Background: Repairation of bone defects remains a major clinical problem. Constructing bone tissue engineering containing growth factors, stem cells, and material scaffolds to repair bone defects has recently become a hot research topic. Nerve growth factor (NGF) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but the low survival rate of the BMSCs during transplantation remains an unresolved issue. In this study, we investigated the therapeutic effect of BMSCs overexpression of NGF on bone defect by inhibiting pyroptosis.

Methods: The relationship between the low survival rate and pyroptosis of BMSCs overexpressing NGF in localized inflammation of fractures was explored by detecting pyroptosis protein levels. Then, the NGF⁺/BMSCs-NSA-Sca bone tissue engineering was constructed by seeding BMSCs overexpressing NGF on the allograft bone scaffold and adding the pyroptosis inhibitor necrosulfonamide(NSA). The femoral condylar defect model in the Sprague-Dawley (SD) rat was studied by micro-CT, histological, WB and PCR analyses in vitro and in vivo to evaluate the regenerative effect of bone repair.

Results: The pyroptosis that occurs in BMSCs overexpressing NGF is associated with the nerve growth factor receptor (P75NTR) during osteogenic differentiation. Furthermore, NSA can block pyroptosis in BMSCs overexpression NGF. Notably, the analyses using the critical-size femoral condylar defect model indicated that the NGF⁺/BMSCs-NSA-Sca group inhibited pyroptosis significantly and had higher osteogenesis in defects.

Conclusion: NGF⁺/BMSCs-NSA had strong osteogenic properties in repairing bone defects. Moreover, NGF⁺/BMSCs-NSA-Sca mixture developed in this study opens new horizons for developing novel tissue engineering constructs.

Keywords: BMSCs; Bone regeneration; Bone tissue engineering; NGF; P75NTR; Pyroptosis.

Fig. 1

Cell morphology and identification of the BMSCs. A Inverted microscope image revealing the isolated BMSCs (Sale bar: 200/150 μm). B Flow cytometry analysis of BMSCs, and the results showed the following: CD90 ( +), CD29 ( +), CD44 ( +), and CD34 ( −)

Fig. 2

Effect of overexpressing NGF and P75NTR on BMSCs of mineralization and pyroptosis. A Fluorescent microscope image of each group (Sale bar: 50 μm). B Representative image of Alizarin red staining of BMSCs induced in osteogenic medium for 2 and 4 wk (Sale bar: 50 μm). C-E Protein levels of the pyroptosis-related proteins GSDMD and NLRP3, as measured by Western blotting. Corresponding uncropped full‑length gels and blots are presented in Additional file 2. F Fluorescent microscope image of each group (Sale bar: 50 μm). G-J Protein levels of P75NTR, NLRP3 and GSDMD as measured by Western blotting

Fig. 3

Effect of NSA and overexpressing NGF on BMSCs of pyroptosis. A The cell viability of BMSCs after they were treated with different concentrations of NSA for 24 h. B Fluorescent microscope image of each group (Sale bar: 50 μm). C-E Protein levels of the pyroptosis-related proteins GSDMD and IL-1β, as measured by Western blotting.  Corresponding uncropped full‑length gels and blots are presented in Additional file 2

Fig. 4

Characteristics of the scaffolds. A,B SEM observation of the allogenic bone scaffolds (Sale bar: 500/10 μm). C,D SEM observation of the BMSCs cultured on the allogenic bone scaffolds (Sale bar: 10 μm). E FTIR spectra of the allogenic bone scaffolds. F 1H NMR spectra of the allogenic bone scaffolds

Fig. 5

Micro-CT image analysis of femoral condylar bone regeneration. A Reconstructed three-dimensional micro-CT images at 4 wk after surgery. B Reconstructed three-dimensional micro-CT images at 8 wk after surgery. C Comparison of BV/TV in the bone defect area at post-operative 4 and 8 wk. D Comparison of Tb.N in the bone defect area at post-operative 4 and 8 wk. E Comparison of Tb.Th in the bone defect area at post-operative 4 and 8 wk. F Comparison of Tb.Sp in the bone defect area at post-operative 4 and 8 wk

Fig. 6

Histology and immunohistochemistry analysis. A new bone and osteocytes analysis by HE staining for each treatment group at 4 and 8 wk after after surgery. B Masson's trichrome staining at post-operative 4 and 8 wk. C Immunohistochemical assessment of OCN at post-operative 4 and 8 wk. Black arrow represented the positive cells. D The area of new bone was quantified by masson trichrome staining and is presented in the histogram. E The area of positive anti-OCN staining was quantified. (NB: new bone, CT: connective tissue, S: scaffold, Sale bar: 50 μm)

Fig. 7

Immunofluorescence analysis. A Bone tissue from the SD rat femoral condylar defect at 8 wk after after surgery were stained with anti-GSDMD (green) for immunostaining analysis. B Bone tissue were stained with anti-NLRP3 (green) antibody for immunostaining analysis. (Sale bar: 50 μm). C The immunofluorescence intensity of GSDMD in bone sections was quantified and is presented in the histogram. D The immunofluorescence intensity of NLRP3 in bone sections was quantified

Fig. 8

NSA and Over-expression of NGF regulated pyroptosis and Osteogenic related genes at 4w after surgery. A, B mRNA levels of the pyroptosis-related genes GSDMD and IL-1β at 4 weeks after surgery, as determined by real-time PCR. C-G Protein levels of the proteins NGF, GSDMD, NLRP3, IL-1β and OCN at 4 weeks after surgery, as measured by Western blotting. Corresponding uncropped full‑length gels and blots are presented in Additional file 2

Fig. 9

NSA and Over-expression of NGF regulated pyroptosis and Osteogenic related genes at 8w after surgery. A, B mRNA levels of the pyroptosis-related genes GSDMD and IL-1β at 8 weeks after surgery, as determined by real-time PCR. C-G Protein levels of the proteins NGF, GSDMD, NLRP3, IL-1β and OCN at 8 weeks after surgery, as measured by Western blotting. Corresponding uncropped full‑length gels and blots are presented in Additional file 2