• 1 南方醫(yī)科大學(xué)南方醫(yī)院創(chuàng)傷骨科（廣州，510515）;;
  • 2 內(nèi)蒙古醫(yī)學(xué)院第二附屬醫(yī)院骨科;

目的 探討大鼠BMSCs 來(lái)源的成骨細(xì)胞和內(nèi)皮細(xì)胞復(fù)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(yǎng)至第3 代的SD 大鼠BMSCs 行成骨和成內(nèi)皮細(xì)胞誘導(dǎo)并鑒定。分別將內(nèi)皮細(xì)胞（A 組）、成骨細(xì)胞（B 組）、混合細(xì)胞（成骨細(xì)胞和內(nèi)皮細(xì)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(xì)胞- 支架復(fù)合物，MTT 檢測(cè)支架內(nèi)細(xì)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(zhǎng)缺損模型并分別植入3 組細(xì)胞- 支架復(fù)合物（n=10）。術(shù)后4、8、12 周分別取移植物行HE 染色觀察，CD34免疫組織化學(xué)染色計(jì)數(shù)微血管密度，RT-PCR 法檢測(cè)骨橋蛋白（osteopontin，OPN）和骨保護(hù)素（osteoprotegrin，OPG）mRNA 表達(dá)。 結(jié)果 BMSCs 成骨誘導(dǎo)7 d 后ALP 染色可見(jiàn)細(xì)胞質(zhì)內(nèi)藍(lán)染顆粒，細(xì)胞核呈紅染；內(nèi)皮細(xì)胞誘導(dǎo)14 d 后，CD34 免疫細(xì)胞化學(xué)染色可見(jiàn)細(xì)胞內(nèi)棕色顆粒。MTT 檢測(cè)示3 組細(xì)胞活性隨時(shí)間延長(zhǎng)逐漸升高。HE 染色示，術(shù)后12 周A 組未見(jiàn)明顯類骨質(zhì)形成，而有較密集的微血管結(jié)構(gòu)及較多纖維組織形成；B、C 組可見(jiàn)均質(zhì)的類骨質(zhì)，呈條索狀和島狀分布，可見(jiàn)大量成骨樣細(xì)胞存在。術(shù)后各時(shí)間點(diǎn)A、C 組微血管密度均顯著高于B 組（P  lt; 0.05）；A 組術(shù)后12 周微血管密度高于C 組（P  lt; 0.05），其余2 個(gè)時(shí)間點(diǎn)A、C 組間差異無(wú)統(tǒng)計(jì)學(xué)意義（P  gt; 0.05）。A 組3 個(gè)時(shí)間點(diǎn)OPN 和OPG mRNA 表達(dá)水平均較低，與B、C 組比較差異有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）；B、C 組分別于術(shù)后8、12 周OPN mRNA 表達(dá)達(dá)峰值，4 周時(shí)OPG mRNA 表達(dá)達(dá)峰值。 結(jié)論 BMSCs 來(lái)源的成骨細(xì)胞和內(nèi)皮細(xì)胞按1 ∶ 1 比例共培養(yǎng)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(jìn)大鼠橈骨缺損部位骨的形成和血管化，促進(jìn)骨缺損愈合。

引用本文： 郝增濤 ,馮衛(wèi),郝廷,余斌. BMSCs 來(lái)源成骨細(xì)胞和內(nèi)皮細(xì)胞復(fù)合殼聚糖- 羥基磷灰石多孔支架構(gòu)建血管化組織工程骨研究. 中國(guó)修復(fù)重建外科雜志, 2012, 26(4): 489-494. doi: 復(fù)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(lái)源的成骨細(xì)胞與血管內(nèi)皮細(xì)胞復(fù)合于異體凍干顆粒骨的黏附性研究. 中國(guó)修復(fù)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(jiān), 何劍鋒. 兔下頜骨牽張成骨組織中c-fos和OPG及OPGL的表達(dá). 浙江大學(xué)學(xué)報(bào): 醫(yī)學(xué)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(fù)骨缺損的實(shí)驗(yàn)研究. 中國(guó)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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 10. 陳超, 李琪佳, 孫瑞軍, 等. BMSCs來(lái)源的成骨細(xì)胞與血管內(nèi)皮細(xì)胞復(fù)合于異體凍干顆粒骨的黏附性研究. 中國(guó)修復(fù)重建外科雜志, 2009, 23(9): 1129-1133.
  11. 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. 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. 13. Kleinheinz J, Stratmann U, Joos U, et al. VEGF-activated angiogenesis during bone regeneration. Oral Maxillofac Surg, 2005, 63(9): 1310-1316.
  14. 14. 葛巍立, 謝志堅(jiān), 何劍鋒. 兔下頜骨牽張成骨組織中c-fos和OPG及OPGL的表達(dá). 浙江大學(xué)學(xué)報(bào): 醫(yī)學(xué)版, 2006, 35(5): 496-500.
  15. 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. 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. 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. 18. 張建新, 徐展望, 常峰. 組織工程化人工骨修復(fù)骨缺損的實(shí)驗(yàn)研究. 中國(guó)矯形外科雜志, 2009, 17(16): 1258-1261.
  19. 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. 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.