Lentivirus-mediated silencing of P75 neurotrophin receptor combined with nerve growth factor overexpression and transfection of bone marrow mesenchymal stem cells combined with demineralized bone matrix for heterotopic osteogenesis_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHEN Junyi , WANG Ning , ZHANG Heng , ZHANG Xianping , ZHAO Limin , ZHU Lunjing , LI Zhijun ,  BEI Chaoyong

Department of Orthopedics for Limb Trauma, Affiliated Hospital of Guilin Medical University, Guilin Guangxi, 541000, P.R.China;

Corresponding author：

BEI Chaoyong, Email: beichy2008@126.com

Keywords：

P75 neurotrophic protein receptor; nerve growth factor; bone marrow mesenchymal stem cells; cell activity; decalcified bone matrix; ectopic osteogenesis; rat

DOI：

10.7507/1002-1892.202003166

Video：

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Objective To investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis.Methods BMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model (n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR.Results At 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B (P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days (P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A (P<0.05).Conclusion Silencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.

Citation： CHEN Junyi, WANG Ning, ZHANG Heng, ZHANG Xianping, ZHAO Limin, ZHU Lunjing, LI Zhijun, BEI Chaoyong. Lentivirus-mediated silencing of P75 neurotrophin receptor combined with nerve growth factor overexpression and transfection of bone marrow mesenchymal stem cells combined with demineralized bone matrix for heterotopic osteogenesis. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(11): 1438-1445. doi: 10.7507/1002-1892.202003166 Copy

1.	Gaston MS, Simpson AH. Inhibition of fracture healing. J Bone Joint Surg (Br), 2007, 89(12): 1553-1560.
2.	Calori GM, Albisetti W, Agus A, et al. Risk factors contributing to fracture non-unions. Injury, 2007, 38 Suppl 2: S11-18.
3.	Chu W, Gan Y, Zhuang Y, et al. Mesenchymal stem cells and porous β-tricalcium phosphate composites prepared through stem cell screen-enrich-combine (-biomaterials) circulating system for the repair of critical size bone defects in goat tibia. Stem Cell Res Ther, 2018, 9(1): 157.
4.	Song SH, Kim SG, Kim SE, et al. Is DBM beneficial for the enhancement of bony consolidation in distraction osteogenesis? A randomized controlled trial. Biomed Res Int, 2015, 2015: 281738.
5.	Moss SD, Joganic E, Manwaring KH, et al. Transplanted demineralized bone graft in cranial reconstructive surgery. Pediatr Neurosurg, 1995, 23(4): 199-204.
6.	García R, Aguiar J, Alberti E, et al. Bone marrow stromal cells produce nerve growth factor and glial cell line-derived neurotrophic factors. Biochem Biophys Res Commun, 2004, 316(3): 753-754.
7.	Saglam A, Kim S, Ahn K, et al. BMP2 shows neurotrophic effects including neuroprotection against neurodegeneration. Neuroreport, 2014, 25(8): 549-555.
8.	乔海兵, 邢开宇. 改良碱性磷酸酶染色法. 实用医技杂志, 2006, 13(6): 1011-1012.
9.	陈旭芳. 血清碱性磷酸酶检测恶性肿瘤骨转移诊断价值的探讨. 中华肿瘤防治杂志, 2009, 16(12): 953-954.
10.	Rackwitz L, Reichert JC, Haversath M, et al. Cell-based and future therapeutic strategies for femoral head necrosis. Orthopade, 2018, 47(9): 770-776.
11.	Caporali A, Meloni M, Miller AM, et al. Soluble ST2 is regulated by p75 neurotrophin receptor and predicts mortality in diabetic patients with critical limb ischemia. Arterioscler Thromb Vasc Biol, 2012, 32(12): e149-160.
12.	Meuchel LW, Thompson MA, Cassivi SD, et al. Neurotrophins induce nitric oxide generation in human pulmonary artery endothelial cells. Cardiovasc Res, 2011, 91(4): 668-676.
13.	Zhang J, Zhao J, Bai Y, et al. Effects of p75 neurotrophin receptor on regulating hypoxia-induced angiogenic factors in retinal pigment epithelial cells. Mol Cell Biochem, 2015, 398(1-2): 123-134.
14.	Koga T, Lee SY, Niikura T, et al. Effect of low-intensity pulsed ultrasound on bone morphogenetic protein 7-induced osteogenic differentiation of human nonunion tissue-derived cells in vitro. J Ultrasound Med, 2013, 32(6): 915-922.
15.	Karnes JM, Daffner SD, Watkins CM. Multiple roles of tumor necrosis factor-alpha in fracture healing. Bone, 2015, 78: 87-93.
16.	李家勇, 王铭, 彭称飞, 等. P75NTR 在兔骨折不愈合局部组织中的表达及意义. 中国骨质疏松杂志, 2017, 23(4): 437-440.
17.	朱伦井, 段江涛, 黄义杰, 等. 过表达 P75 神经生长因子受体负向调控骨髓间充质干细胞的成骨分化. 中国组织工程研究, 2020, 24(1): 20-26.
18.	Maes C, Kobayashi T, Selig MK, et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev Cell, 2010, 19(2): 329-344.
19.	Caporali A, Meloni M, Nailor A, et al. p75(NTR)-dependent activation of NF-κB regulates microRNA-503 transcription and pericyte-endothelial crosstalk in diabetes after limb ischaemia. Nat Commun, 2015, 6: 8024.
20.	Shanab AY, Mysona BA, Matragoon S, et al. Silencing p75(NTR) prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina. Mol Ther Methods Clin Dev, 2015, 2: 15013.
21.	Barcelona PF, Sitaras N, Galan A, et al. p75NTR and its ligand ProNGF activate paracrine mechanisms etiological to the vascular, inflammatory, and neurodegenerative pathologies of diabetic retinopathy. J Neurosci, 2016, 36(34): 8826-8841.
22.	Schwabe P, Simon P, Kronbach Z, et al. A pilot study investigating the histology and growth factor content of human non-union tissue. Int Orthop, 2014, 38(12): 2623-2629.
23.	Assimakopoulou M, Kondyli M, Gatzounis G, et al. Neurotrophin receptors expression and JNK pathway activation in human astrocytomas. BMC Cancer, 2007, 7: 202.
24.	Suhl KH, Park JB, Park EY, et al. Effect of nerve growth factor and its transforming tyrosine kinase protein and low-affinity nerve growth factor receptors on apoptosis of notochordal cells. Int Orthop, 2012, 36(8): 1747-1753.

1. Gaston MS, Simpson AH. Inhibition of fracture healing. J Bone Joint Surg (Br), 2007, 89(12): 1553-1560.
2. Calori GM, Albisetti W, Agus A, et al. Risk factors contributing to fracture non-unions. Injury, 2007, 38 Suppl 2: S11-18.
3. Chu W, Gan Y, Zhuang Y, et al. Mesenchymal stem cells and porous β-tricalcium phosphate composites prepared through stem cell screen-enrich-combine (-biomaterials) circulating system for the repair of critical size bone defects in goat tibia. Stem Cell Res Ther, 2018, 9(1): 157.
4. Song SH, Kim SG, Kim SE, et al. Is DBM beneficial for the enhancement of bony consolidation in distraction osteogenesis? A randomized controlled trial. Biomed Res Int, 2015, 2015: 281738.
5. Moss SD, Joganic E, Manwaring KH, et al. Transplanted demineralized bone graft in cranial reconstructive surgery. Pediatr Neurosurg, 1995, 23(4): 199-204.
6. García R, Aguiar J, Alberti E, et al. Bone marrow stromal cells produce nerve growth factor and glial cell line-derived neurotrophic factors. Biochem Biophys Res Commun, 2004, 316(3): 753-754.
7. Saglam A, Kim S, Ahn K, et al. BMP2 shows neurotrophic effects including neuroprotection against neurodegeneration. Neuroreport, 2014, 25(8): 549-555.
8. 乔海兵, 邢开宇. 改良碱性磷酸酶染色法. 实用医技杂志, 2006, 13(6): 1011-1012.
9. 陈旭芳. 血清碱性磷酸酶检测恶性肿瘤骨转移诊断价值的探讨. 中华肿瘤防治杂志, 2009, 16(12): 953-954.
10. Rackwitz L, Reichert JC, Haversath M, et al. Cell-based and future therapeutic strategies for femoral head necrosis. Orthopade, 2018, 47(9): 770-776.
11. Caporali A, Meloni M, Miller AM, et al. Soluble ST2 is regulated by p75 neurotrophin receptor and predicts mortality in diabetic patients with critical limb ischemia. Arterioscler Thromb Vasc Biol, 2012, 32(12): e149-160.
12. Meuchel LW, Thompson MA, Cassivi SD, et al. Neurotrophins induce nitric oxide generation in human pulmonary artery endothelial cells. Cardiovasc Res, 2011, 91(4): 668-676.
13. Zhang J, Zhao J, Bai Y, et al. Effects of p75 neurotrophin receptor on regulating hypoxia-induced angiogenic factors in retinal pigment epithelial cells. Mol Cell Biochem, 2015, 398(1-2): 123-134.
14. Koga T, Lee SY, Niikura T, et al. Effect of low-intensity pulsed ultrasound on bone morphogenetic protein 7-induced osteogenic differentiation of human nonunion tissue-derived cells in vitro. J Ultrasound Med, 2013, 32(6): 915-922.
15. Karnes JM, Daffner SD, Watkins CM. Multiple roles of tumor necrosis factor-alpha in fracture healing. Bone, 2015, 78: 87-93.
16. 李家勇, 王铭, 彭称飞, 等. P75NTR 在兔骨折不愈合局部组织中的表达及意义. 中国骨质疏松杂志, 2017, 23(4): 437-440.
17. 朱伦井, 段江涛, 黄义杰, 等. 过表达 P75 神经生长因子受体负向调控骨髓间充质干细胞的成骨分化. 中国组织工程研究, 2020, 24(1): 20-26.
18. Maes C, Kobayashi T, Selig MK, et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev Cell, 2010, 19(2): 329-344.
19. Caporali A, Meloni M, Nailor A, et al. p75(NTR)-dependent activation of NF-κB regulates microRNA-503 transcription and pericyte-endothelial crosstalk in diabetes after limb ischaemia. Nat Commun, 2015, 6: 8024.
20. Shanab AY, Mysona BA, Matragoon S, et al. Silencing p75(NTR) prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina. Mol Ther Methods Clin Dev, 2015, 2: 15013.
21. Barcelona PF, Sitaras N, Galan A, et al. p75NTR and its ligand ProNGF activate paracrine mechanisms etiological to the vascular, inflammatory, and neurodegenerative pathologies of diabetic retinopathy. J Neurosci, 2016, 36(34): 8826-8841.
22. Schwabe P, Simon P, Kronbach Z, et al. A pilot study investigating the histology and growth factor content of human non-union tissue. Int Orthop, 2014, 38(12): 2623-2629.
23. Assimakopoulou M, Kondyli M, Gatzounis G, et al. Neurotrophin receptors expression and JNK pathway activation in human astrocytomas. BMC Cancer, 2007, 7: 202.
24. Suhl KH, Park JB, Park EY, et al. Effect of nerve growth factor and its transforming tyrosine kinase protein and low-affinity nerve growth factor receptors on apoptosis of notochordal cells. Int Orthop, 2012, 36(8): 1747-1753.

Chinese Journal of Reparative and Reconstructive Surgery

Lentivirus-mediated silencing of P75 neurotrophin receptor combined with nerve growth factor overexpression and transfection of bone marrow mesenchymal stem cells combined with demineralized bone matrix for heterotopic osteogenesis

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