神经生长因子可促进骨折愈合过程中血管内皮生长因子的表达

doi:10.3969/j.issn.2095-4344.2014.24.016

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (24): 3863-3869.doi: 10.3969/j.issn.2095-4344.2014.24.016

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇下一篇

神经生长因子可促进骨折愈合过程中血管内皮生长因子的表达

刘宇鹏^{1, 2}，赵德伟²，王卫明²，张耀²，李放²，刘振刚²

1大连理工大学生物工程学院，辽宁省大连市 116024
2大连大学附属中山医院骨外科，辽宁省大连市 116001

修回日期:2014-05-19 出版日期:2014-06-11 发布日期:2014-06-11
通讯作者: 刘宇鹏，大连理工大学生物工程学院，辽宁省大连市 116024；大连大学附属中山医院骨外科，辽宁省大连市 116001
作者简介:刘宇鹏，男，1972年生，黑龙江省佳木斯市人，汉族，2000年大连医科大学毕业，硕士，主任医师，主要从事骨显微修复及运动医学，骨显微修复及运动医学方面的研究。

Nerve growth factor promotes the expression of vascular endothelial growth factor in the fracture healing process

Liu Yu-peng^{1, 2}, Zhao De-wei², Wang Wei-ming², Zhang Yao², Li Fang², Liu Zhen-gang²

1 Department of Biomedical Engineering, Dalian University of Technology, Dalian 116024, Liaoning Province, China
2 Department of Orthopaedic Surgery, Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, Liaoning Province, China

Revised:2014-05-19 Online:2014-06-11 Published:2014-06-11
Contact: Liu Yu-peng, Master, Chief physician, Department of Biomedical Engineering, Dalian University of Technology, Dalian 116024, Liaoning Province, China; Department of Orthopaedic Surgery, Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, Liaoning Province, China
About author:Liu Yu-peng, Master, Chief physician, Department of Biomedical Engineering, Dalian University of Technology, Dalian 116024, Liaoning Province, China; Department of Orthopaedic Surgery, Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, Liaoning Province, China

摘要/Abstract

摘要：

背景：骨折愈合过程十分复杂，需要多种细胞因子的参与。目前研究较多的细胞因子有骨形态发生蛋白、成纤维细胞生长因子、转化生长因子β、血管内皮细胞生长因子和胰岛素样生长因子，但神经生长因子在骨折愈合过程中对血管内皮细胞生长因子的作用尚不明确。
目的：观察神经生长因子对兔骨折愈合中血管内皮生长因子表达的影响。
方法：实验建立标准兔桡骨骨折模型，分别用神经生长因子、神经生长因子单克隆抗体和生理盐水进行干预，即应用神经生长因子组、拮抗神经生长因子组和对照组。
结果与结论：损伤后24，48 h和损伤后1，3，6，8周Western blot检测骨折端组织血管内皮生长因子蛋白的表达分析结果显示，3组各时间点血管内皮生长因子表达的关系为：应用神经生长因子组>对照组>拮抗神经生长因子组(P < 0.05)。结果证实，在骨折愈合过程当中，应用神经生长因子可以促进血管内皮生长因子表达。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

关键词: 组织构建, 骨组织工程, 血管内皮生长因子, 组织构建与生物活性因子, 神经生长因子, 神经生长因子单克隆抗体, 骨折愈合

Abstract:

BACKGROUND: The fracture healing process is very complex, involving a variety of cytokines. At present, the existing studies focus on bone morphogenetic protein, fibroblast growth factor, transforming growth factor β, vascular endothelial cell growth factor and insulin-like growth factor. The role of nerve growth factor on vascular endothelial cell growth factor during the fracture healing process remains unclear.
OBJECTIVE: To investigate the effect of nerve growth factor on vascular endothelial growth factor expression during the process of fracture healing in rabbits.
METHODS: Radial fracture model established in white rabbits were divided into three groups, they were treated with nerve growth factor, nerve growth factor monoclonal antibody, and saline respectively, serving as nerve growth factor group, antagonizing nerve growth factor group, and control group.
RESULTS AND CONCLUSION: The vascular endothelial growth factor expression was detected with western blot analysis at 24 hours, 48 hours, 1 week, 3 weeks, 6 weeks, 8 weeks post-injury. The results showed that the expression level was the highest in nerve growth factor group, then in control group, and the lowest in antagonizing nerve growth factor group (P < 0.05). Experimental findings suggest that, nerve growth factor can promote the expression of vascular endothelial growth factor in the fracture healing process.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: nerve growth factor, vascular endothelial growth factor, fracture healing, rabbits, proteins

中图分类号:

R318

刘宇鹏，赵德伟，王卫明，张耀，李放，刘振刚. 神经生长因子可促进骨折愈合过程中血管内皮生长因子的表达[J]. 中国组织工程研究, 2014, 18(24): 3863-3869.

Liu Yu-peng, Zhao De-wei, Wang Wei-ming, Zhang Yao, Li Fang, Liu Zhen-gang. Nerve growth factor promotes the expression of vascular endothelial growth factor in the fracture healing process[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(24): 3863-3869.

图/表（结果） 2

Quantitative analysis of experimental animals

All experimental rabbits were involved in the results analysis, without any loss.

General observation

All rabbits in the three groups survived with no deaths and ate properly. The surgical incisions exhibited no signs of infection or purulence and healed at approximately 1 postoperative week. At 24 hours postoperatively, the samples in the three groups all exhibited subcutaneous swelling at the incision sites, with no hematomas formed at the fracture sites. At

48 hours postoperatively, initial hematomas at the fracture sites were formed in all three groups. At 1 week postoperatively, hematoma organization was formed at the fracture sites (NGF group > control group > NGF antagonist group), with discontinuous fractures. At

3 weeks postoperatively, continuous fractures were observed, with primary callus formation, and strong bony connections were formed at the fracture sites (NGF group > control group > NGF antagonist group). At

6 weeks postoperatively, the callus sizes had shrunk compared with the primary calluses (NGF group < control group < NGF antagonist group). At 8 weeks postoperatively, the NGF group and the control group showed no obvious fracture lines, while the NGF antagonist group still showed a small amount of callus.

Western blot analysis and statistical analysis results

Images of immunoblots showing VEGF expression with an internal reference in the bone tissues at different time points in the three groups are shown in Figure 2. The absorbance ratios and the means with standard deviations of VEGF target protein expression versus the internal reference in the bone tissues at different time points in the three groups are shown in Table 1, and their changing trend over time is displayed in Figure 2. Finally, a statistical analysis of variance was conducted for the absorbance ratios of VEGF expression versus the internal reference in the bone tissues at different time points in the three groups (Table 1). In the fracture-healing process, VEGF expression in the callus continuously increased, reaching a peak at postoperative 3 weeks, and then gradually decreased (Figure 2). VEGF expression in the callus was higher in the NGF group than in the other two groups, while VEGF expression in the callus was lower in the NGF antagonist group than in the other two groups (Table 1). The one-way analysis of variance for the absorbance ratios of the target protein VEGF versus the internal reference protein in each group revealed that, compared with the NGF antagonist group and the control group, the results of the NGF group were higher at all time points, with statistically significant differences (P < 0.05). These results indicate that NGF had a positive role in promoting VEGF expression at the fracture site. The differences between the NGF antagonist group and the control group at the 24- and 48-hour and the 8-week time points were not statistically significant (P > 0.05), which may indicate that the NGF antibody concentrations had not yet reached effective levels at the early stages of fracture healing and that the anti-NGF antibody concentration was completely attenuated at the late stage of fracture healing (Table 1).

参考文献

[1] Street J, Bao M, Leo de Guzman, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci U S A. 2002;99(15):9656-9661.
[2] Tong G, Lei J, Zhong C, Cai W, Lin X. The curative effect and safety of collagen sponge with different pH and content of protein implanted into orthopedic bone defect. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2012;29(6): 1125-1130.
[3] Lyons FG, Gleeson JP, Partap S, et al. Novel microhydroxyapatite particles in a collagen scaffold: a bioactive bone void filler? Clin Orthop Relat Res. 2014; 472(4):1318-1328.
[4] Hou J, Wang J, Cao L, et al. Segmental bone regeneration using rhBMP-2-loaded collagen/chitosan microspheres composite scaffold in a rabbit model. Biomed Mater. 2012; 7(3):035002.
[5] Fan BS, Lou JY. Enhancement of angiogenic effect of co-transfection human NGF and VEGF genes in rat bone marrow mesenchymal stem cells. Gene. 2011;485(2): 167-171.
[6] Zhang YG, Lu SB, Wang JF, et al. Experimental study of expression of BMP, TGF-β and bFGF in fracture healing. Zhonghua Chuangshang Zazhi. 2000;3(16):170-172.
[7] Einhorn TA. The science of fracture healing. J Orthop Trauma. 2005;19(10S):S4-S6.
[8] Liu YP, Zhao DW, Lu DC, et al. Expression of nerve growth factors in the fractured bone of rat. Zhongguo Linchuang Kangfu. 2006;0(17):107-109.
[9] Sisask G, Bjurholm A, Ahmed M, et al. Ontogeny of sensory nerves in the developing skeleton. Anat Rec. 1995;243: 234-240.
[10] Sisask G, Silfverswärd CJ, Bjurholm A, et al. Ontogeny of sensory and autonomic nerves in the developing mouse skeleton. Auton Neurosci. 2013;177(2):237-243.
[11] Yasui M, Shiraishi Y, Ozaki N, et al. Nerve growth factor and associated nerve sprouting contribute to local mechanical hyperalgesia in a rat model of bone injury. Eur J Pain. 2012; 16(7):953-965.
[12] Mo Y, Yang Z, Zhao J, et al. Preliminary study on appropriate concentration gradient of nerve growth factor in promoting fracture healing. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2011;25(5):581.
[13] Eppley BL, Snyders RV, Windelmann T, et al. Effects of nerve growth factor on craniofacial onlay bone graft survival: preliminary findings. J Craniofac Surg. 1992;2(4):174-180.
[14] Dobnig H, Sipos A, Jiang Y, et al. Early changes in biochemical markers of bone formation correlate with improvements in bone structure during teriparatide therapy. J Clin Endocrinol Metab. 2005;90(7):3970-3977.
[15] Obrant KJ, Ivaska KK, Gerdhem P, et al. Biochemical markers of bone turnover are influenced by recently sustained fracture. J Bone. 2005;36(5):786-792.
[16] Nakanishi T, Takahashi K, Aoki C, et al. Expression of nerve growth factor family neurotrophins in a mouse osteoblastic cell line. Biochem Biophys Res Commun. 1994; 198(3): 891-897.
[17] Chim SM, Tickner J, Chow ST, et al. Angiogenic factors in bone local environment. Cytokine Growth Factor Rev. 2013;24(3):297-310.
[18] Yasko AW, Lane JM, Fellinger EJ, et al. The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rh-BMP). J Bone Joint Surg (Am). 1992;74:659.
[19] Hayami T, Funaki H, Yaoeda K, et al. Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis. J Rheumatol. 2003;30(10):2207-2217.
[20] Brown AP, Courtney CL, King LM, et al. Cartilage dysplasia and tissue mineralization in the rat following administration of a FGF receptor tyrosine kinase inhibitor. Toxicol Pathol. 2005;33(4):449-455.
[21] Petersen W, Pufe T, Zantop T, et al. Expression of VEGFR-1 and VEGFR-2 in degenerative Achilles tendons. J Orthop Res. 2004;420(3):286-291.
[22] Street JT, Wang JH, Wu QD, et al. The angiogenic response to skeletal injury is preserved in the elderly. J Orthop Res. 2001;19(6):1057-1066.
[23] Jimenez-Andrade JM, Ghilardi JR, Castañeda-Corral G, et al. Preventive or late administration of anti-NGF therapy attenuates tumor-induced nerve sprouting, neuroma formation, and cancer pain.Pain. 2011;152(11):2564-2574.
[24] Mogi M, Kondo A, Kinparak, et al. Anti-apoptotic action of nerve growth factor in mouse osteoblastic cell line. Life Sci. 2000;67(10):1197-1206.
[25] Zhuang YF, Li J. Serum EGF and NGF levels of patients with brain injury and limb fracture. Asian Pac J Trop Med. 2013;6(5):383-386.
[26] Iannone F, De Bari C, Dell'Accio F, et al. Increased expression of nerve growth factor (NGF) and high affinity NGF receptor (p140 TrkA) in human osteoarthritic chondrocytes. Rheumatology (Oxford). 2002;41(12): 1413-1418.
[27] Grills BL, Schuijers JA, Ward AR. Topical application of nerve growth factor improves fracture healing in rats. J Orthop Res. 1997;15(2):235-242.
[28] Jehan F, Naveilhan P, Neveu I, et al. Regulation of NGF, BDNF and LNGFR gene expression in ROS 17/2.8 cells. Molcell Endocrinol. 1996;116(2):149-156.
[29] Lazarovici P, Gazit A, Staniszewska I, et al. Nerve growth factor (NGF) promotes angiogenesis in the quail chorioallantoic membrane. Endothelium. 2006;13(1):51-59.
[30] Furfaro F1, Ang ES, Lareu RR, et al. A histological and micro-CT investigation in to the effect of NGF and EGF on the periodontal, alveolar bone, root and pulpal healing of replanted molars in a rat model-a pilot study. Prog Orthod. 2014;15:2
[31] Garcia R, Aguiar J, Alberti E, et al. Bone marrow stromal cells produce nerve growth factor and glial cell line-derived neurotrophic factors. Biocjem Biophys Res Commun. 2004; 316(3):753-754.
[32] Cao J, Wang L, Lei DL, et al. Local injection of nerve growth factor via a hydrogel enhances bone formation during mandibular distraction osteogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol. 2012;113(1):48-53.
[33] Cai WX, Zheng LW, Li CL, et al. Effect of different rhbmp-2 and tg-vegf ratios on the formation of heterotopic bone and neovessels. Biomed Res Int. 2014;2014:571510.

引言

Fracture is a kind of common disease that seriously affects people’s life and work, it occurs frequently in all kinds of traffic accidents and various natural disasters, the incidence rate of fracture increases year by year. Researchers are paying more and more attention to promote healing of fracture^[1-5].

Fracture healing refers to the regeneration and repair reactions of local bone tissue at a fracture site and is very complex process involving a variety of cells and factors. Fracture healing includes three phases: hematoma inflammation organization, primary callus formation, and bone plate formation and remodeling. Osteoblasts, osteoclasts, and a large number of cytokines participate and function in various phases of the fracture-healing process. These cytokines mainly regulate fracture healing through the processes of cell proliferation and differentiation, matrix synthesis, and calcification^[6-7]. The different healing phases involve different cytokines. Currently, the most studied cytokines involved in fracture healing are bone morphogenetic protein and vascular endothelial growth factor (VEGF).

Recent studies have confirmed that nerve growth factor (NGF) not only exists in normal bone tissue, but also participates in and plays a very important role in the developing skeleton and the fracture-healing process^[8-10]. NGF is a neurotrophic factor and functions to promote the growth, development, and differentiation of central and peripheral neurons, and to maintain their normal survival. NGF can also play repairing and nourishing roles after damage occurs in the nervous system^[11-12]. Research on the role of NGF in promoting fracture healing was initiated by Eppley et al ^[13]. Through the application of NGF in mandibular defect repairs in experimental rabbits, we not only confirmed that NGF could promote axonal regeneration, but also found that newborn osteoid was generated around the regenerated axons. Numerous studies detected little or no ingrowth of nerve fibers in bone tissues with nonunion and heterotopic ossifications^[14-17]. A study by Liu et al found that, NGF and its receptor were expressed in both normal tissue and bone fracture callus. The in vitro expression levels of NGF and its receptor have been studied and detected in cultured bone cells.

Angiogenesis plays an important role in physiological bone growth and remodeling, as well as in pathological bone disorders such as fracture repair, osteonecrosis, and tumor metastasis to bone. Vascularization is required for bone remodeling along the endosteal surface of trabecular bone or Haversian canals within the cortical bone, as well as the homeostasis of the cartilage-subchondral bone interface. Angiogenic factors, produced by cells from a basic multicellular unit within the bone remodeling compartment, regulate local endothelial cells and pericytes. VEGF is a powerful regulating factor of angiogenesis, with strong effects in promoting angiogenesis and the proliferation of endothelial cells. VEGF can promote the regeneration of blood vessels by specifically binding with a receptor on vascular endothelial cells^[18]. Other cytokines fully or partially stimulate angiogenesis via VEGF. The premise of fracture healing is the formation of blood vessels and the recovery of the blood supply at the fracture location. Hayami et al indicated that VEGF induced the ingrowth of vessels in cartilage, leading to endochondral bone augmentation, which is very favorable for fracture healing^[19]. VEGF not only can promote endochondral bone formation, but also significantly promote subperiosteal bone formation^[20]. In addition, VEGF has a direct impact on osteoblasts. One study detected VEGF1 and VEGF2 receptor expression in osteoblasts at fracture sites^[21]. Street et al also found that VEGF played a key regulatory role in fracture healing. The inhibition of endogenous VEGF expression slowed down bone regeneration, and the application of exogenous VEGF could accelerate fracture healing^[22].

How can NGF promote fracture healing? What is the mechanism of fracture healing promoted by NGF? These issues are not very clear.

This study aims to investigate the regulating effect of NGF on the fracture healing-related factor VEGF, and further elucidate the possible mechanism of fracture healing.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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材料方法

Design

A randomized controlled trial.

Time and setting

Experiments were finished at Orthopaedic Laboratory of Affiliated Zhongshan Hospital of Dalian University, China from June 2012 to March 2013.

Materials

A total of 108 healthy New Zealand white rabbits, weighing 2.4-3.2 kg, male or female, were provided by the Medical Experimental Animal Center of Dalian University, China (License No. SCXK (Liao) 2013-0003). These rabbits were randomly divided into three groups: control group, NGF group, and NGF antagonist group, with 36 rabbits in each group.

Methods

Establishing fracture models

Ketamine was administered by slow ear vein injection for anesthesia. The rabbit radius fracture model was established with external fixation using a fiberglass cast. In the first 3 consecutive postoperative days, each rabbit in each group was injected with gentamicin 10 000 U twice a day to prevent infection.

Drug injection

The rabbits in the NGF group received an intramuscular injection of NGF (mouse nerve factor injection, biological activities ≥ 150 000 AU), 200 ng/kg, three times a day for 3 weeks; the rabbits in the NGF antagonist group received an intraperitoneal injection of NGF antibody (Santa Cruz Biotechnology, Inc.) 400 ng/kg, three times a day for 3 weeks; and the rabbits in the control group received an intraperitoneal injection of saline, 1 mL, three times a day for 3 weeks.

Sampling

The rabbits were sacrificed at the 24- and 48-hour and the 1-, 3-, 6-, and 8-week postoperative time points, the tissues were collected and decalcified, and the proteins were extracted. The protein content was measured to generate a standard curve: (y = 0.013 5x + 0.243 3) according to the instructions of Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, China), so that the protein contents could be calculated (Figure 1). VEGF protein expression was detected with western blot analysis.

Western blot analysis

(1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): about 2 hours, at stacking gel voltage of 80 V and separating gel voltage of 100 V.

(2) Transmembrane: The sample was transferred to the PVDF membrane 2 hours at 250 mA current.

(3) Blocking: At 37 ℃, the PVDF membrane was placed in 5% skim milk to block protein solution for 2 hours.

(4) Hybridization: The film was cut according to Mark, and divided into two films: internal reference and VEGF, then added to the rabbit-derived VEGF first antibody, horseradish peroxidase-labeled goat anti-rabbit secondary antibody VEGF, placed in a shaker for 2 hours.

(5) Chemiluminescence.

(6) Exposure, development and fixing.

(7) Western blot analysis.

Main outcome measures

VEGF expression in each group was determined.

Statistical analysis

The results of protein content detection are presented as mean ± SD. The analysis of variance and the least significant difference methods were applied for statistical analysis using SPSS 16.0 statistical software (International Business Machines Corporation, IBM). A P < 0.05 value indicates statistical significance.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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讨论

Fracture healing is a very complex process of tissue repair that mainly includes the two paths, namely endochondral bone formation and intramembranous bone formation. In histocytology, fracture healing is usually divided into three phases: hematoma inflammation organization, primary callus formation, and bone plate formation and remodeling. Osteoblasts, osteoclasts, and a large number of cytokines participate and function in various phases of the fracture-healing process. These cytokines mainly regulate fracture healing through the processes of cell proliferation and differentiation and matrix synthesis and calcification. NGF and VEGF are two very important factors in fracture healing.

NGF is a neurotrophic factor and functions to promote the growth, development, and differentiation of central and peripheral neurons and to maintain their normal survival. NGF can also play repairing and nourishing roles after damage occurs in the nervous system^{[7, 9, 14]}. A study reported anti-NGF therapy can prevent or late administration of nerve sprouting, neuroma formation^[23]. Research on the role of NGF in promoting fracture healing was initiated by Eppley et al. Through the application of NGF in mandibular defect repairs in experimental rabbits, we not only confirmed that NGF could promote axonal regeneration, but also found that newborn osteoid was generated around the regenerated axons. Numerous studies detected little or no ingrowth of nerve fibers in bone tissues with nonunion and heterotopic ossifications^[24]. Zhuang et al found that the serum levels of NGF and EGF were significantly increased when limb fracture combined with brain injury, so EGF and NGF may be involved in the process of fracture healing^[25]. A study by Liu et al found that NGF and its receptor were expressed in both normal tissue and bone fracture callus. The in vitro expression levels of NGF and its receptor have been studied and detected in cultured bone cells. NGF mRNA and protein expression has also been detected in a variety of in vitro osteoblast cell lines^[26-31]. Cao et al ^[32]reported that the application of the Col/nHA/Alg hydrogel as an NGF delivery during the consolidation phase of distraction osteogenesis increased regeneration and new bone formation. The expression of NGF and its receptors in normal bone tissue and calluses in fracture sites suggests that, in both normal physiological metabolism and the fracture-healing process in bone tissue, NGF not only can support and nourish the nerves and induce their ingrowth in bone callus, but also directly or indirectly involved in the healing process through various possible pathways, possibly regulating other factors that promote fracture healing at the fracture site, such as VEGF. Currently, the mechanism by which NGF promotes fracture healing can be summarized as: 1) inducing nerve ingrowth in bone callus; 2) promoting angiogenesis in the callus; 3) promoting the release of neuropeptides; 4) regulating type I and type II collagen expression; 5) regulating 1,25-dihydroxy vitamin D3 intake; 6) promoting osteoblast proliferation and differentiation, and 7) inhibiting osteoclast function. It has been more than 20 years since the accidental discovery of the effect of NGF on fracture healing, but the role of NGF in regulating bone metabolism and promoting fracture healing has not yet been well studied, and the mechanism for promoting fracture healing is still unclear. In this study, the rabbits in the NGF group were injected with NGF and exhibited a shortened postoperative fracture healing time and significantly increased VEGF expression in the callus, indicating that NGF was able to promote fracture healing.

VEGF is a powerful regulating factor of angiogenesis, with strong effects in promoting angiogenesis and the proliferation of endothelial cells. VEGF can promote the regeneration of blood vessels by specifically binding with a receptor on vascular endothelial cells^[17]. Angiogenesis plays an important role in physiological bone growth and remodeling, as well as in pathological bone disorders such as fracture repair, osteonecrosis, and tumor metastasis to bone. Vascularization is required for bone remodeling along the endosteal surface of trabecular bone or Haversian canals within the cortical bone, as well as the homeostasis of the cartilage-subchondral bone interface. Angiogenic factors, produced by cells from a basic multicellular unit within the bone remodeling compartment regulate local endothelial cells and pericytes. Cai et al ^[33] found different rhBMP-2 and TG-VEGF ratios increased angiogenesis. Other cytokines fully or partially stimulate angiogenesis via VEGF. The premise of fracture healing is the formation of blood vessels and the recovery of the blood supply at the fracture location. Hayami et al indicated that VEGF induced the ingrowth of vessels in cartilage, leading to endochondral bone augmentation, which is very favorable for fracture healing. VEGF not only can promote endochondral bone formation, but also significantly promote subperiosteal bone formation. In addition, VEGF has a direct impact on osteoblasts. One study detected VEGF1 and VEGF2 receptor expression in osteoblasts at fracture sites. Street et al also found that VEGF played a key regulatory role in fracture healing. The inhibition of endogenous VEGF expression slowed down bone regeneration, and the application of exogenous VEGF could accelerate fracture healing.

The experimental results in our study demonstrated that VEGF expression levels in calluses in the NGF group were significantly higher than the control group and the NGF antagonist group at various time points. Additionally, VEGF expression occurred earlier and declined slowly. These results demonstrated that NGF can directly promote fracture healing and can also synergistically promote fracture healing, possibly with VEGF. Moreover, VEGF expression levels in calluses in the NGF antagonist group were lower than that in the control group. These differences were not statistically significant at the early and late stages of fracture healing, possibly because the anti-NGF concentrations did not reach effective levels in the early and late stages of fracture healing.

To demonstrate that NGF and VEGF can directly promote angiogenesis similarly to basic fibroblast growth factor, Lazarovici et al compared different doses of NGF with recombinant human VEGF and recombinant human bFGF. The authors found that the pro-angiogenic effect of NGF was similar to the effects of both recombinant human VEGF and bFGF, and that this effect could be blocked by an NGF receptor inhibitor, while a VEGF receptor inhibitor did not show this effect. This study demonstrated that NGF can directly promote local angiogenesis and, thus, can promote fracture healing. The experimental results of our study showed that, in addition to directly promoting angiogenesis, NGF might also promote the secretion of the cytokine VEGF via one or multiple pathways to regulate the fracture-healing process. A limitation of this study is that the promotion of fracture healing by NGF might be achieved by regulating a variety of bone growth factors instead of VEGF only. In the future, we will experimentally investigate the regulating effects of NGF on other cytokines and the interacting mechanisms among the various factors.

The question of how NGF promotes VEGF secretion, thereby promoting angiogenesis at fracture sites and fracture healing, remains to be further investigated in the related fields of molecular biology and genetics. NGF plays a positive regulatory role in VEGF expression in the fracture-healing process. The lack of NGF seriously affects the normal process of fracture healing. The application of NGF can either directly or indirectly promote VEGF expression and can thus contribute to fracture healing.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

神经生长因子可促进骨折愈合过程中血管内皮生长因子的表达

Nerve growth factor promotes the expression of vascular endothelial growth factor in the fracture healing process

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