SDF-1α/CXCR4信号通路在轴向应力刺激促进骨再生中的作用研究
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摘要
目的通过轴向应力刺激促进骨再生,观察基质细胞衍生因子1α/趋化因子CXC亚族受体4(stromal cell-derived factor 1α/cysteine X cysteine receptor 4,SDF-1α/CXCR4)信号通路变化,探讨轴向应力刺激促进骨再生的机制。方法取72只雄性新西兰大白兔,于右后肢胫骨近端内侧制备直径8 mm圆形皮质骨缺损并脱蛋白松质骨支架修复模型,随机分为3组(n=24)。A组腹腔注射PBS,B组术肢给予应力刺激治疗+腹腔注射PBS,C组术肢给予应力刺激治疗+腹腔注射CXCR4拮抗剂(AMD3100)。术后2、4、8、12周,摄X线片并采用Lane-Sandhu X线评分标准评价骨愈合情况,取标本行HE染色观察新生骨组织及支架降解,免疫组织化学染色观察VEGF、CXCR4表达水平;4、8周取标本Western blot检测SDF-1α及CXCR4蛋白表达水平;12周行Micro-CT检查,计算新生骨体积及新生骨密度。结果 X线片检查示,除术后2周各组骨缺损区及支架无明显变化外,4、8及12周时B组骨愈合评分均高于A、C组(P<0.05)。12周时Micro-CT扫描可见B组骨缺损修复、髓腔再通,新生骨体积及骨密度均高于A、C组(P<0.05)。HE染色显示,术后4周开始B组骨再生及支架降解均明显快于A、C组。免疫组织化学染色示,各组VEGF及CXCR4阳性表达均在4周达峰值;各时间点B组VEGF及CXCR4表达量均显著高于A、C组(P<0.05)。Western blot检测显示,4、8周时B组SDF-1α与CXCR4表达量均显著高于A、C组(P<0.05)。结论轴向应力刺激促进骨再生可能与其促进骨缺损区组织高表达SDF-1α,激活与其下游调控BMSCs募集的CXCR4信号有关。
Objective To observe the change of stromal cell-derived factor 1α/cysteine X cysteine receptor 4(SDF-1α/CXCR4) signaling pathway during the process of axial stress stimulation promoting bone regeneration, and to further explore its mechanism. Methods A total of 72 male New Zealand white rabbits were selected to prepare the single cortical bone defect in diameter of 8 mm at the proximal end of the right tibia that repaired with deproteinized cancellous bone. All models were randomly divided into 3 groups(n=24). Group A was treated with intraperitoneally injection of PBS; Group B was treated with stress stimulation and intraperitoneally injection of PBS; Group C was treated with stress stimulation and intraperitoneally injection of AMD3100 solution. The X-ray films were taken and Lane-Sandhu scores of bone healing were scored at 2, 4, 8, and 12 weeks after operation, while specimens were harvested for HE staining,immunohistochemical staining of vascular endothelial growth factor(VEGF) and CXCR4, and Western blot(SDF-1α and CXCR4). The bone healing area was scanned by Micro-CT at 12 weeks after operation, and the volume and density of new bone were calculated. Results X-ray film showed that the Lane-Sandhu scores of bone healing in group B were significantly higher than those in groups A and C at 4, 8, and 12 weeks after operation(P<0.05). Micro-CT scan showed that the bone defect was repaired in group B and the pulp cavity was re-passed at 12 weeks after operation. The volume and density of new bone were higher in group B than in groups A and C(P<0.05). HE staining showed that the new bone growth in bone defect area and the degradation of scaffolds were faster in group B than in groups A and C after 4 weeks.The immunohistochemical staining showed that the expressions of VEGF and CXCR4 in 3 groups reached the peak at4 weeks, and group B was higher than groups A and C(P<0.05). Western blot analysis showed that the expressions of SDF-1α and CXCR4 in group B were significantly higher than those in groups A and C at 4 and 8 weeks after operation(P<0.05). Conclusion Axial stress stimulation can promote the expression of SDF-1α in bone defect tissue, activate and regulate the CXCR4 signal collected by marrow mesenchymal stem cells, and accelerate bone regeneration in bone defect area.
Objective To observe the change of stromal cell-derived factor 1α/cysteine X cysteine receptor 4(SDF-1α/CXCR4) signaling pathway during the process of axial stress stimulation promoting bone regeneration, and to further explore its mechanism. Methods A total of 72 male New Zealand white rabbits were selected to prepare the single cortical bone defect in diameter of 8 mm at the proximal end of the right tibia that repaired with deproteinized cancellous bone. All models were randomly divided into 3 groups(n=24). Group A was treated with intraperitoneally injection of PBS; Group B was treated with stress stimulation and intraperitoneally injection of PBS; Group C was treated with stress stimulation and intraperitoneally injection of AMD3100 solution. The X-ray films were taken and Lane-Sandhu scores of bone healing were scored at 2, 4, 8, and 12 weeks after operation, while specimens were harvested for HE staining,immunohistochemical staining of vascular endothelial growth factor(VEGF) and CXCR4, and Western blot(SDF-1α and CXCR4). The bone healing area was scanned by Micro-CT at 12 weeks after operation, and the volume and density of new bone were calculated. Results X-ray film showed that the Lane-Sandhu scores of bone healing in group B were significantly higher than those in groups A and C at 4, 8, and 12 weeks after operation(P<0.05). Micro-CT scan showed that the bone defect was repaired in group B and the pulp cavity was re-passed at 12 weeks after operation. The volume and density of new bone were higher in group B than in groups A and C(P<0.05). HE staining showed that the new bone growth in bone defect area and the degradation of scaffolds were faster in group B than in groups A and C after 4 weeks.The immunohistochemical staining showed that the expressions of VEGF and CXCR4 in 3 groups reached the peak at4 weeks, and group B was higher than groups A and C(P<0.05). Western blot analysis showed that the expressions of SDF-1α and CXCR4 in group B were significantly higher than those in groups A and C at 4 and 8 weeks after operation(P<0.05). Conclusion Axial stress stimulation can promote the expression of SDF-1α in bone defect tissue, activate and regulate the CXCR4 signal collected by marrow mesenchymal stem cells, and accelerate bone regeneration in bone defect area.
引文
1 Hwang SJ, Lublinsky S, Seo YK, et al. Extremely small-magnitude accelerations enhance bone regeneration:a preliminary study. Clin Orthop Relat Res, 2009, 467(4):1083-1091.
2 Zhang F, Tsai S, Kato K, et al. Transforming growth factor-beta promotes recruitment of bone marrow cells and bone marrowderived mesenchymal stem cells through stimulation of MCP-1production in vascular smooth muscle cells. J Biol Chem, 2009,284(26):17564-17574.
3 Rollin R, Alvarezlafuente R, Marco F, et al. Abnormal transforming growth factor-beta expression in mesenchymal stem cells from patients with osteoarthritis. J Rheumatol, 2008, 35(5):904-906.
4 Mendelson A, Cheung Yk, Paluch K, et al. Competitive stem cell recruitment by multiple cytotactic cues. Lab Chip, 2013, 13(6):1156-1164.
5张峰,王江泽,丁真奇,等.叩击式骨应力刺激仪促进山羊胫骨骨折愈合的实验研究.中华创伤骨科杂志,2016,18(1):66-73.
6陈德旺,丁真奇,翟文亮,等.骨应力刺激仪叩击治疗促进小腿骨干骨折术后骨愈合的前瞻性研究.中华创伤骨科杂志,2012,14(4):304-308.
7卢庆威,万春友,张弢,等.轴向载荷力学测试在胫腓骨骨折术后外固定器拆除中的临床应用.中国修复重建外科杂志,2016,30(9):1085-1088.
8葛启航,万春友,刘亚北,等.胫腓骨开放骨折Taylor空间支架外固定术后轴向应力刺激对骨折愈合的影响研究.中国修复重建外科杂志,2017, 31(8):931-935.
9刘国浚,黄国锋,高建廷,等.骨应力刺激仪叩击治疗促进兔胫骨缺损内成骨及支架降解的实验研究.中华创伤骨科杂志,2016,18(10):895-901.
10丁真奇.四种移植材料修复兔颅骨缺损的比较研究.重庆:重庆医科大学,1993.
11 Andersson L. Dentin xenografts to experimental bone defects in rabbit tibia are ankylosed and undergo osseous replacement.Dental Traumatol, 2010, 26(5):398-402.
12 Lane JM, Sandhu HS. Current approaches to experimental bone grafting. Orthop Clin North Am, 1987, 18(2):213-225.
13 Brighton CT, Fisher JR Jr, Levine SE, et al. The biochemical pathway mediating the proliferative response of bone cells to a mechanical stimulus. J Bone Joint Surg(Am), 1996, 78(9):1337-1347.
14 Athanasiou KA, Zhu C, Lanctot DR, et al. Fundamentals of biomechanics in tissue engineering of bone. Tissue Eng,2000, 6(4):361-381.
15喻鑫罡,张先龙,曾炳芳.低频可控性微动影响长骨骨折愈合的实验研究.中华创伤骨科杂志,2005, 7(8):744-748.
16 Wallace AL, Draper ER, Strachan RK, et al. The vascular response to fracture micromovement. Clin Orthop Relat Res, 1994,(301):281-290.
17 Ito H. Chemokines in mesenchymal stem cell therapy for bone repair:a novel concept of recruiting mesenchymal stem cells and the possible cell sources. Modern Rheumatol, 2011, 21(2):113-121.
18 Ladage D, Brixius K, Steingen C, et al. Mesenchymal stem cells induce endothelial activation via paracine mechanisms.Endothelium, 2007, 14(2):53-63.
19 Sacchetti B, Funari A, Michienzi S, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell, 2007, 131(2):324-336.
20 Simmons CA, Matlis S, Thornton AJ, et al. Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase(ERK1/2)signaling pathway. J Biomech, 2003, 36(8):1087-1096.
21 Grunewald M, Avraham I, Dor Y, et al. VEGF-induced adult neovascularization:recruitment, retention, and role of accessory cells. Cell, 2006,124(1):175-189.
22 Kitaori T, Ito H, Schwarz EM, et al. Stromal cell-derived factor1/CXCR4 signaling is critical for the recruitment of mesenchymal stem cells to the fracture site during skeletal repair in a mouse model. Arthritis Rheum, 2009, 60(3):813-823.
23 Qian D, Gong J, He Z, et al. Bone marrow-derived mesenchymal stem cells repair necrotic pancreatic tissue and promote angiogenesis by secreting cellular growth factors involved in the SDF-la/CXCR4 axis in rats. Stem Cells Int, 2015,(2015):306836.
24 Granero-Molto F, Weis JA, Miga MI, et al. Regenerative effects of transplanted mesenchymal stem cells in fracture healing. Stem Cells, 2009, 27(8):1887-1898.
25 Ceradini DJ, Kulkarni AR, Callaghan MJ, et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1induction of SDF-1. Nature Medicine, 2004, 10(8):858-864.
26 Uchida D, Begum NM, Almofti A, et al. Possible role of stromalcell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma. Exp Cell Res, 2003, 290(2):289-302.
27 Burns JM, Summers BC, Yu W, et al. A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med, 2006, 203(9):2201-2213.
28 Wang YL, HE XM, Tang W, et al. Effect of stromal cell derived factor-1α/CXCR4/CXCR7 axis on migration of the bone marrow mesenchymal stem cells. Acta Anatomica Sinica,2014,(2014):78-86.
2 Zhang F, Tsai S, Kato K, et al. Transforming growth factor-beta promotes recruitment of bone marrow cells and bone marrowderived mesenchymal stem cells through stimulation of MCP-1production in vascular smooth muscle cells. J Biol Chem, 2009,284(26):17564-17574.
3 Rollin R, Alvarezlafuente R, Marco F, et al. Abnormal transforming growth factor-beta expression in mesenchymal stem cells from patients with osteoarthritis. J Rheumatol, 2008, 35(5):904-906.
4 Mendelson A, Cheung Yk, Paluch K, et al. Competitive stem cell recruitment by multiple cytotactic cues. Lab Chip, 2013, 13(6):1156-1164.
5张峰,王江泽,丁真奇,等.叩击式骨应力刺激仪促进山羊胫骨骨折愈合的实验研究.中华创伤骨科杂志,2016,18(1):66-73.
6陈德旺,丁真奇,翟文亮,等.骨应力刺激仪叩击治疗促进小腿骨干骨折术后骨愈合的前瞻性研究.中华创伤骨科杂志,2012,14(4):304-308.
7卢庆威,万春友,张弢,等.轴向载荷力学测试在胫腓骨骨折术后外固定器拆除中的临床应用.中国修复重建外科杂志,2016,30(9):1085-1088.
8葛启航,万春友,刘亚北,等.胫腓骨开放骨折Taylor空间支架外固定术后轴向应力刺激对骨折愈合的影响研究.中国修复重建外科杂志,2017, 31(8):931-935.
9刘国浚,黄国锋,高建廷,等.骨应力刺激仪叩击治疗促进兔胫骨缺损内成骨及支架降解的实验研究.中华创伤骨科杂志,2016,18(10):895-901.
10丁真奇.四种移植材料修复兔颅骨缺损的比较研究.重庆:重庆医科大学,1993.
11 Andersson L. Dentin xenografts to experimental bone defects in rabbit tibia are ankylosed and undergo osseous replacement.Dental Traumatol, 2010, 26(5):398-402.
12 Lane JM, Sandhu HS. Current approaches to experimental bone grafting. Orthop Clin North Am, 1987, 18(2):213-225.
13 Brighton CT, Fisher JR Jr, Levine SE, et al. The biochemical pathway mediating the proliferative response of bone cells to a mechanical stimulus. J Bone Joint Surg(Am), 1996, 78(9):1337-1347.
14 Athanasiou KA, Zhu C, Lanctot DR, et al. Fundamentals of biomechanics in tissue engineering of bone. Tissue Eng,2000, 6(4):361-381.
15喻鑫罡,张先龙,曾炳芳.低频可控性微动影响长骨骨折愈合的实验研究.中华创伤骨科杂志,2005, 7(8):744-748.
16 Wallace AL, Draper ER, Strachan RK, et al. The vascular response to fracture micromovement. Clin Orthop Relat Res, 1994,(301):281-290.
17 Ito H. Chemokines in mesenchymal stem cell therapy for bone repair:a novel concept of recruiting mesenchymal stem cells and the possible cell sources. Modern Rheumatol, 2011, 21(2):113-121.
18 Ladage D, Brixius K, Steingen C, et al. Mesenchymal stem cells induce endothelial activation via paracine mechanisms.Endothelium, 2007, 14(2):53-63.
19 Sacchetti B, Funari A, Michienzi S, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell, 2007, 131(2):324-336.
20 Simmons CA, Matlis S, Thornton AJ, et al. Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase(ERK1/2)signaling pathway. J Biomech, 2003, 36(8):1087-1096.
21 Grunewald M, Avraham I, Dor Y, et al. VEGF-induced adult neovascularization:recruitment, retention, and role of accessory cells. Cell, 2006,124(1):175-189.
22 Kitaori T, Ito H, Schwarz EM, et al. Stromal cell-derived factor1/CXCR4 signaling is critical for the recruitment of mesenchymal stem cells to the fracture site during skeletal repair in a mouse model. Arthritis Rheum, 2009, 60(3):813-823.
23 Qian D, Gong J, He Z, et al. Bone marrow-derived mesenchymal stem cells repair necrotic pancreatic tissue and promote angiogenesis by secreting cellular growth factors involved in the SDF-la/CXCR4 axis in rats. Stem Cells Int, 2015,(2015):306836.
24 Granero-Molto F, Weis JA, Miga MI, et al. Regenerative effects of transplanted mesenchymal stem cells in fracture healing. Stem Cells, 2009, 27(8):1887-1898.
25 Ceradini DJ, Kulkarni AR, Callaghan MJ, et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1induction of SDF-1. Nature Medicine, 2004, 10(8):858-864.
26 Uchida D, Begum NM, Almofti A, et al. Possible role of stromalcell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma. Exp Cell Res, 2003, 290(2):289-302.
27 Burns JM, Summers BC, Yu W, et al. A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med, 2006, 203(9):2201-2213.
28 Wang YL, HE XM, Tang W, et al. Effect of stromal cell derived factor-1α/CXCR4/CXCR7 axis on migration of the bone marrow mesenchymal stem cells. Acta Anatomica Sinica,2014,(2014):78-86.