BMSCs 來源成骨細胞和內皮細胞復合殼聚糖- 羥基磷灰石多孔支架構建血管化組織工程骨研究_《中國修復重建外科雜志》

作者：

郝增濤 ¹ , 馮衛 ² , 郝廷 ² , 余斌 ¹

1 南方醫科大學南方醫院創傷骨科（廣州，510515）;;
2 內蒙古醫學院第二附屬醫院骨科;

關鍵詞：

BMSCs 組織工程骨血管化骨缺損大鼠

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目的探討大鼠BMSCs 來源的成骨細胞和內皮細胞復合殼聚糖- 羥基磷灰石多孔支架植入大鼠橈骨缺損處的成骨作用和成血管作用。方法取分離培養至第3 代的SD 大鼠BMSCs 行成骨和成內皮細胞誘導并鑒定。分別將內皮細胞（A 組）、成骨細胞（B 組）、混合細胞（成骨細胞和內皮細胞比例為1 ∶ 1，C 組）均勻滴加于殼聚糖- 羥基磷灰石多孔支架上制備3 組細胞- 支架復合物，MTT 檢測支架內細胞增殖活性。取2 月齡雄性SD 大鼠30 只，制作大鼠橈骨5 mm 長缺損模型并分別植入3 組細胞- 支架復合物（n=10）。術后4、8、12 周分別取移植物行HE 染色觀察，CD34免疫組織化學染色計數微血管密度，RT-PCR 法檢測骨橋蛋白（osteopontin，OPN）和骨保護素（osteoprotegrin，OPG）mRNA 表達。結果 BMSCs 成骨誘導7 d 后ALP 染色可見細胞質內藍染顆粒，細胞核呈紅染；內皮細胞誘導14 d 后，CD34 免疫細胞化學染色可見細胞內棕色顆粒。MTT 檢測示3 組細胞活性隨時間延長逐漸升高。HE 染色示，術后12 周A 組未見明顯類骨質形成，而有較密集的微血管結構及較多纖維組織形成；B、C 組可見均質的類骨質，呈條索狀和島狀分布，可見大量成骨樣細胞存在。術后各時間點A、C 組微血管密度均顯著高于B 組（P lt; 0.05）；A 組術后12 周微血管密度高于C 組（P lt; 0.05），其余2 個時間點A、C 組間差異無統計學意義（P gt; 0.05）。A 組3 個時間點OPN 和OPG mRNA 表達水平均較低，與B、C 組比較差異有統計學意義（P lt; 0.05）；B、C 組分別于術后8、12 周OPN mRNA 表達達峰值，4 周時OPG mRNA 表達達峰值。結論 BMSCs 來源的成骨細胞和內皮細胞按1 ∶ 1 比例共培養于殼聚糖- 羥基磷灰石多孔支架作為組織工程骨移植物，可以促進大鼠橈骨缺損部位骨的形成和血管化，促進骨缺損愈合。

引用本文： 郝增濤 ,馮衛,郝廷,余斌. BMSCs 來源成骨細胞和內皮細胞復合殼聚糖- 羥基磷灰石多孔支架構建血管化組織工程骨研究. 中國修復重建外科雜志, 2012, 26(4): 489-494. doi: 復制

1.	Samee M, Kasugai S, Kondo H, et al. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) transfection to human periosteal cells enhances osteoblast differentiation and bone formation. J Pharmacol Sci, 2008, 108(1): 18-31.
2.	Mott DA, Mailhot J, Cuenin MF, et al. Enhancement of osteoblast proliferation in vitro by selective enrichment of demineralized freeze-dried bone allograft with specific growth factors. J Oral Implantol, 2002, 28(2): 57-66.
3.	Ekenbäck SB, Linder LE, Lönnies H. Effect of four dental varnishes on the colonization of cariogenic bacteria on exposed sound root surfaces. Caries Res, 2000, 34(1): 70-74.
4.	Bodde EW, Spauwen PH, Mikos AG, et al. Closing capacity of segmental radius defects in rabbits. J Biomed Mater Res A, 2008, 85(1): 206-217.
5.	Zhao Q, Qian J, Zhou H, et al. In vitro osteoblast-like and endothelial cells’ response to calcium silicate/calcium phosphate cement. Biomed Mater, 2010, 5(3): 35004.
6.	Kagami H, Agata H, Tojo A. Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation. Int J Biochem Cell Biol, 2011, 43(3): 286-289.
7.	Lee WD, Hurtig MB, Kandel RA, et al. Membrane culture of bone marrow stromal cells yields better tissue than pellet culture for engineering cartilage-bone substitute biphasic constructs in a two-step process. Tissue Eng Part C Methods, 2011, 17(9): 939-948.
8.	Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 1968, 6(2): 230-247.
9.	Unger RE, Ghanaati S, Orth C, et al. The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature. Biomaterials, 2010, 31(27): 6959-6967.
10.	陳超, 李琪佳, 孫瑞軍, 等. BMSCs來源的成骨細胞與血管內皮細胞復合于異體凍干顆粒骨的黏附性研究. 中國修復重建外科雜志, 2009, 23(9): 1129-1133.
11.	Zhang Y, Andrukhov O, Berner S, et al. Osteogenic properties of hydrophilic and hydrophobic titanium surfaces evaluated with osteoblast-like cells (MG63) in coculture with human umbilical vein endothelial cells (HUVEC). Dent Mater, 2010, 26(11): 1043-1051.
12.	Deckers MM, Karperien M, van der Bent C, et al. Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endocrinology, 2000, 141(5): 1667-1674.
13.	Kleinheinz J, Stratmann U, Joos U, et al. VEGF-activated angiogenesis during bone regeneration. Oral Maxillofac Surg, 2005, 63(9): 1310-1316.
14.	葛巍立, 謝志堅, 何劍鋒. 兔下頜骨牽張成骨組織中c-fos和OPG及OPGL的表達. 浙江大學學報: 醫學版, 2006, 35(5): 496-500.
15.	Shoji S, Tabuchi M, Miyazawa K, et al. Bisphosphonate inhibits bone turnover in OPG (-/-) mice via a depressive effect on both osteoclasts and osteoblasts. Calcif Tissue Int, 2010, 87(2): 181-192.
16.	Yan MZ, Xu Y, Gong YX, et al. Raloxifene inhibits bone loss and improves bone strength through an Opg-independent mechanism. Endocrine, 2010, 37(1): 55-61.
17.	Kon T, Cho TJ, Aiaawa T, et al. Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res, 2001, 16(6): 1004-1014.
18.	張建新, 徐展望, 常峰. 組織工程化人工骨修復骨缺損的實驗研究. 中國矯形外科雜志, 2009, 17(16): 1258-1261.
19.	Cai L, Wang Q, Gu C, et al. Vascular and micro-environmental influences on MSC-coral hydroxyapatite construct-based bone tissue engineering. Biomaterials, 2011, 32(33): 8497-8505.
20.	Santos MI, Reis RL. Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges. Macromol Biosci, 2010, 10(1): 12-27.

1. Samee M, Kasugai S, Kondo H, et al. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) transfection to human periosteal cells enhances osteoblast differentiation and bone formation. J Pharmacol Sci, 2008, 108(1): 18-31.
2. Mott DA, Mailhot J, Cuenin MF, et al. Enhancement of osteoblast proliferation in vitro by selective enrichment of demineralized freeze-dried bone allograft with specific growth factors. J Oral Implantol, 2002, 28(2): 57-66.
3. Ekenbäck SB, Linder LE, Lönnies H. Effect of four dental varnishes on the colonization of cariogenic bacteria on exposed sound root surfaces. Caries Res, 2000, 34(1): 70-74.
4. Bodde EW, Spauwen PH, Mikos AG, et al. Closing capacity of segmental radius defects in rabbits. J Biomed Mater Res A, 2008, 85(1): 206-217.
5. Zhao Q, Qian J, Zhou H, et al. In vitro osteoblast-like and endothelial cells’ response to calcium silicate/calcium phosphate cement. Biomed Mater, 2010, 5(3): 35004.
6. Kagami H, Agata H, Tojo A. Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation. Int J Biochem Cell Biol, 2011, 43(3): 286-289.
7. Lee WD, Hurtig MB, Kandel RA, et al. Membrane culture of bone marrow stromal cells yields better tissue than pellet culture for engineering cartilage-bone substitute biphasic constructs in a two-step process. Tissue Eng Part C Methods, 2011, 17(9): 939-948.
8. Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 1968, 6(2): 230-247.
9. Unger RE, Ghanaati S, Orth C, et al. The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature. Biomaterials, 2010, 31(27): 6959-6967.
10. 陳超, 李琪佳, 孫瑞軍, 等. BMSCs來源的成骨細胞與血管內皮細胞復合于異體凍干顆粒骨的黏附性研究. 中國修復重建外科雜志, 2009, 23(9): 1129-1133.
11. Zhang Y, Andrukhov O, Berner S, et al. Osteogenic properties of hydrophilic and hydrophobic titanium surfaces evaluated with osteoblast-like cells (MG63) in coculture with human umbilical vein endothelial cells (HUVEC). Dent Mater, 2010, 26(11): 1043-1051.
12. Deckers MM, Karperien M, van der Bent C, et al. Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endocrinology, 2000, 141(5): 1667-1674.
13. Kleinheinz J, Stratmann U, Joos U, et al. VEGF-activated angiogenesis during bone regeneration. Oral Maxillofac Surg, 2005, 63(9): 1310-1316.
14. 葛巍立, 謝志堅, 何劍鋒. 兔下頜骨牽張成骨組織中c-fos和OPG及OPGL的表達. 浙江大學學報: 醫學版, 2006, 35(5): 496-500.
15. Shoji S, Tabuchi M, Miyazawa K, et al. Bisphosphonate inhibits bone turnover in OPG (-/-) mice via a depressive effect on both osteoclasts and osteoblasts. Calcif Tissue Int, 2010, 87(2): 181-192.
16. Yan MZ, Xu Y, Gong YX, et al. Raloxifene inhibits bone loss and improves bone strength through an Opg-independent mechanism. Endocrine, 2010, 37(1): 55-61.
17. Kon T, Cho TJ, Aiaawa T, et al. Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res, 2001, 16(6): 1004-1014.
18. 張建新, 徐展望, 常峰. 組織工程化人工骨修復骨缺損的實驗研究. 中國矯形外科雜志, 2009, 17(16): 1258-1261.
19. Cai L, Wang Q, Gu C, et al. Vascular and micro-environmental influences on MSC-coral hydroxyapatite construct-based bone tissue engineering. Biomaterials, 2011, 32(33): 8497-8505.
20. Santos MI, Reis RL. Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges. Macromol Biosci, 2010, 10(1): 12-27.

《中國修復重建外科雜志》

BMSCs 來源成骨細胞和內皮細胞復合殼聚糖- 羥基磷灰石多孔支架構建血管化組織工程骨研究

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《中國修復重建外科雜志》

BMSCs 來源成骨細胞和內皮細胞復合殼聚糖- 羥基磷灰石多孔支架構建血管化組織工程骨研究

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