Heterotopic osteogenesis study of tissue engineered bone by co-culture of vascular endothelial cells and adipose-derived stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Fuke ¹ ,  ZHANG Hong ² , LI Yanlin ¹ , LIU Liu ³ , HE Chuan ¹ , CAI Guofeng ¹ , SONG En ¹

1. Department of Sports Medicine, the First Affiliated Hospital of Kunming Medical University, Kunming Yunnan, 650031, P.R.China;
2. Department of Psychiatry, the First Affiliated Hospital of Kunming Medical University, Kunming Yunnan, 650031, P.R.China;
3. Department of Plastic Surgery, the First Affiliated Hospital of Kunming Medical University, Kunming Yunnan, 650031, P.R.China;

Corresponding author：

ZHANG Hong, Email: 526028544@qq.com

Keywords：

Adipose-derived stem cells; vascular endothelial cells; co-culture; tissue engineered bone; heterotopic osteogenesis

DOI：

10.7507/1002-1892.201808111

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Objective To investigate the heterotopic osteogenesis of tissue engineered bone using the co-culture system of vascular endothelial cells (VECs) and adipose-derived stem cells (ADSCs) as seed cells.Methods The partially deproteinized biological bone (PDPBB) was prepared by fibronectin combined with partially deproteinized bone (PDPB). The ADSCs of 18-week-old Sprague Dawley (SD) rats and VECs of cord blood of full-term pregnant SD rats were isolated and cultured. Three kinds of tissue engineered bone were constructed in vitro: PDPBB+VECs (group A), PDPBB+ADSCs (group B), PDPBB+co-cultured cells (VECs∶ADSCs was 1∶1, group C), and PDPBB was used as control group (group D). Scanning electron microscopy was performed at 10 days after cell transplantation to observe cell adhesion on scaffolds. Forty-eight 18-week-old SD rats were randomly divided into groups A, B, C, and D, with 12 rats in each group. Four kinds of scaffolds, A, B, C, and D, were implanted into the femoral muscle bags of rats in corresponding groups. The animals were killed at 2, 4, 8, and 12 weeks after operation for gross observation, HE staining and Masson staining histological observation, and the amount of bone collagen was measured quantitatively by Masson staining section.Results Scanning electron microscopy showed that the pores were interconnected in PDPB materials, and a large number of lamellar protein crystals on the surface of PDPBB modified by fibronection were loosely attached to the surface of the scaffold. After 10 days of co-culture PDPBB and cells, a large number of cells attached to PDPBB and piled up with each other to form cell clusters in group C. Polygonal cells and spindle cells were mixed and distributed, and some cells grew along bone trabeculae to form cell layers. Gross observation showed that the granulation tissue began to grow into the material pore at 2 weeks after operation. In group C, a large number of white cartilage-like substances were gradually produced on the surface of the material after 4 weeks, and the surface of the material was uneven. At 12 weeks, the amount of blood vessels on the surface of group A increased, and the material showed consolidation; there was a little white cartilage-like material on the surface of group B, but the pore size of the material did not decrease significantly; in group D, the pore size of the material did not decrease significantly. Histological observation showed that there was no significant difference in the amount of bone collagen between groups at 2 weeks after operation (F=2.551, P=0.088); at 4, 8, and 12 weeks after operation, the amount of bone collagen in group C was significantly higher than that in other 3 groups, and that in group B was higher than that in group D (P<0.05); there was no significant difference between group A and groups B, D (P>0.05).Conclusion The ability of heterotopic osteogenesis of tissue engineered bone constructed by co-culture VECs and ADSCs was the strongest.

Citation： WANG Fuke, ZHANG Hong, LI Yanlin, LIU Liu, HE Chuan, CAI Guofeng, SONG En. Heterotopic osteogenesis study of tissue engineered bone by co-culture of vascular endothelial cells and adipose-derived stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(10): 1310-1319. doi: 10.7507/1002-1892.201808111 Copy

1.	Florczyk SJ, Leung M, Li Z, et al. Evaluation of three-dimensional porous chitosan-alginate scaffolds in rat calvarial defects for bone regeneration applications. J Biomed Mater Res A, 2013, 101(10): 2974-2983.
2.	詹健峰, 刘徐妹, 于博. 载 BMP-2 多肽 P24 的巯基化壳聚糖水凝胶诱导异位成骨的实验研究. 中国修复重建外科杂志, 2018, 32(9): 1144-1149.
3.	王福科, 刘流, 赵德萍, 等. 体外血管内皮细胞和脂肪干细胞联合培养对脂肪干细胞成骨分化的影响. 中国修复重建外科杂志, 2010, 24(4): 399-405.
4.	王福科, 刘流, 何晓光, 等. 血管内皮细胞和脂肪干细胞体外联合培养细胞的增殖. 中国组织工程研究与临床康复, 2010, 14(32): 6001-6005.
5.	李彦林, 杨志明, 解慧琪, 等. 生物衍生骨支架材料的体外细胞相容性实验研究. 中国修复重建外科杂志, 2001, 15(4): 227-231.
6.	杨浩, 李彦林, 韩睿, 等. 异种骨衍生材料修复节段性骨缺损的实验研究. 昆明医学院学报, 2004, 25(1): 14-18.
7.	李彦林, 杨志明, 解慧琪, 等. 生物衍生组织工程骨支架材料的制备及理化特性. 生物医学工程学杂志, 2002, 19(1): 10-12.
8.	王福科, 刘流, 代晓明, 等. 纤维粘连蛋白对部分脱蛋白骨细胞相容性影响研究. 昆明医学院学报, 2010, 31(1): 9-14.
9.	裴雪涛. 干细胞实验指南. 北京: 科学出版社, 2006: 83-100.
10.	王福科, 刘流, 赵德萍, 等. 大鼠脐血来源单个核细胞诱导为内皮祖细胞的免疫荧光检测. 中国生物美容, 2009, 1(4): 10-15.
11.	王福科, 赵德萍, 代晓明, 等. 不同类型血清对大鼠脂肪干细胞分离培养的影响. 昆明医学院学报, 2010, 31(3): 4-10.
12.	Han X, Liu L, Wang F, et al. Reconstruction of tissue-engineered bone with bone marrow mesenchymal stem cells and partially deproteinized bone in vitro. Cell Biol Int, 2012, 36(11): 1049-1053.
13.	刘流, 王福科, 赵德萍, 等. 联合培养 VECs 与 ADSCs 与部分脱蛋白生物骨体外构建组织工程骨的实验研究. 中国生物美容, 2010, 1(1): 1-9.
14.	宁钰, 秦文, 任亚辉, 等. 载淫羊藿苷/凹凸棒石/Ⅰ型胶原/聚己内酯复合支架修复兔胫骨缺损的实验研究. 中国修复重建外科杂志, 2019, 33(9): 1-13.
15.	冯立, 曹晓建, 任永信. 纤维粘连蛋白与碱性成纤维细胞生长因子对成骨细胞黏附的协同刺激作用. 中国修复重建外科杂志, 2007, 21(4): 390-395.
16.	García AJ, Reyes CD. Bio-adhesive surfaces to promote osteoblast differentiation and bone formation. J Dent Res, 2005, 84(5): 407-413.
17.	Williams JT, Southerland SS, Souza J, et al. Cells isolated from adult human skeletal muscle capable of differentiating into multiple mesodermal phenotypes. Am Surg, 1999, 65(1): 22-26.
18.	Nakagawa T, Tagawa T. Ultrastructural study of direct bone formation induced by BMPs-collagen complex implanted into an ectopic site. Oral Dis, 2000, 6(3): 172-179.
19.	Li H, Daculsi R, Grellier M, et al. The role of vascular actors in two dimensional dialogue of human bone marrow stromal cell and endothelial cell for inducing self-assembled network. PLoS One, 2011, 6(2): e16767.
20.	Wang DS, Miura M, Demura H, et al. Anabolic effects of 1,25-dihydroxyvitamin D3 on osteoblasts are enhanced by vascular endothelial growth factor produced by osteoblasts and by growth factors produced by endothelial cells. Endocrinology, 1997, 138(7): 2953-2962.
21.	Inglis S, Christensen D, Wilson DI, et al. Human endothelial and foetal femur-derived stem cell co-cultures modulate osteogenesis and angiogenesis. Stem Cell Res Ther, 2016, 7: 13.
22.	Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature, 2014, 507(7492): 323-328.
23.	Liang Y, Wen L, Shang F, et al. Endothelial progenitors enhanced the osteogenic capacities of mesenchymal stem cells in vitro and in a rat alveolar bone defect model. Arch Oral Biol, 2016, 68: 123-130.
24.	Verseijden F, Posthumus-van Sluijs SJ, Pavljasevic P, et al. Adult human bone marrow- and adipose tissue-derived stromal cells support the formation of prevascular-like structures from endothelial cells in vitro. Tissue Eng Part A, 2010, 16(1): 101-114.
25.	孙源, 林红, 吴子征, 等. 联合培养时诱导的内皮细胞对自体骨髓基质干细胞成骨作用的影响. 中华创伤骨科杂志, 2006, 8(10): 919-923.
26.	Zhao X, Liu L, Wang FK, et al. Coculture of vascular endothelial cells and adipose-derived stem cells as a source for bone engineering. Ann Plast Surg, 2012, 69(1): 91-98.
27.	董苑, 杨瑞年, 王福科, 等. BMP2 的表达对联合培养体系中骨髓间充质干细胞增殖和成骨分化的影响. 中华显微外科杂志, 2013, 36(4): 352-355.
28.	杨瑞年, 刘流, 王福科, 等. 联合培养体系中对骨髓间充质干细胞 Bmi-1 表达的影响. 中国组织工程研究, 2012, 16(14): 2524-2529.
29.	杨瑞年, 刘流, 王福科, 等. 血管内皮细胞对联合培养体系中骨髓间充质干细胞侏儒症相关转录因子表达及成骨分化的影响. 中国组织工程研究, 2012, 16(19): 3447-3449.

1. Florczyk SJ, Leung M, Li Z, et al. Evaluation of three-dimensional porous chitosan-alginate scaffolds in rat calvarial defects for bone regeneration applications. J Biomed Mater Res A, 2013, 101(10): 2974-2983.
2. 詹健峰, 刘徐妹, 于博. 载 BMP-2 多肽 P24 的巯基化壳聚糖水凝胶诱导异位成骨的实验研究. 中国修复重建外科杂志, 2018, 32(9): 1144-1149.
3. 王福科, 刘流, 赵德萍, 等. 体外血管内皮细胞和脂肪干细胞联合培养对脂肪干细胞成骨分化的影响. 中国修复重建外科杂志, 2010, 24(4): 399-405.
4. 王福科, 刘流, 何晓光, 等. 血管内皮细胞和脂肪干细胞体外联合培养细胞的增殖. 中国组织工程研究与临床康复, 2010, 14(32): 6001-6005.
5. 李彦林, 杨志明, 解慧琪, 等. 生物衍生骨支架材料的体外细胞相容性实验研究. 中国修复重建外科杂志, 2001, 15(4): 227-231.
6. 杨浩, 李彦林, 韩睿, 等. 异种骨衍生材料修复节段性骨缺损的实验研究. 昆明医学院学报, 2004, 25(1): 14-18.
7. 李彦林, 杨志明, 解慧琪, 等. 生物衍生组织工程骨支架材料的制备及理化特性. 生物医学工程学杂志, 2002, 19(1): 10-12.
8. 王福科, 刘流, 代晓明, 等. 纤维粘连蛋白对部分脱蛋白骨细胞相容性影响研究. 昆明医学院学报, 2010, 31(1): 9-14.
9. 裴雪涛. 干细胞实验指南. 北京: 科学出版社, 2006: 83-100.
10. 王福科, 刘流, 赵德萍, 等. 大鼠脐血来源单个核细胞诱导为内皮祖细胞的免疫荧光检测. 中国生物美容, 2009, 1(4): 10-15.
11. 王福科, 赵德萍, 代晓明, 等. 不同类型血清对大鼠脂肪干细胞分离培养的影响. 昆明医学院学报, 2010, 31(3): 4-10.
12. Han X, Liu L, Wang F, et al. Reconstruction of tissue-engineered bone with bone marrow mesenchymal stem cells and partially deproteinized bone in vitro. Cell Biol Int, 2012, 36(11): 1049-1053.
13. 刘流, 王福科, 赵德萍, 等. 联合培养 VECs 与 ADSCs 与部分脱蛋白生物骨体外构建组织工程骨的实验研究. 中国生物美容, 2010, 1(1): 1-9.
14. 宁钰, 秦文, 任亚辉, 等. 载淫羊藿苷/凹凸棒石/Ⅰ型胶原/聚己内酯复合支架修复兔胫骨缺损的实验研究. 中国修复重建外科杂志, 2019, 33(9): 1-13.
15. 冯立, 曹晓建, 任永信. 纤维粘连蛋白与碱性成纤维细胞生长因子对成骨细胞黏附的协同刺激作用. 中国修复重建外科杂志, 2007, 21(4): 390-395.
16. García AJ, Reyes CD. Bio-adhesive surfaces to promote osteoblast differentiation and bone formation. J Dent Res, 2005, 84(5): 407-413.
17. Williams JT, Southerland SS, Souza J, et al. Cells isolated from adult human skeletal muscle capable of differentiating into multiple mesodermal phenotypes. Am Surg, 1999, 65(1): 22-26.
18. Nakagawa T, Tagawa T. Ultrastructural study of direct bone formation induced by BMPs-collagen complex implanted into an ectopic site. Oral Dis, 2000, 6(3): 172-179.
19. Li H, Daculsi R, Grellier M, et al. The role of vascular actors in two dimensional dialogue of human bone marrow stromal cell and endothelial cell for inducing self-assembled network. PLoS One, 2011, 6(2): e16767.
20. Wang DS, Miura M, Demura H, et al. Anabolic effects of 1,25-dihydroxyvitamin D3 on osteoblasts are enhanced by vascular endothelial growth factor produced by osteoblasts and by growth factors produced by endothelial cells. Endocrinology, 1997, 138(7): 2953-2962.
21. Inglis S, Christensen D, Wilson DI, et al. Human endothelial and foetal femur-derived stem cell co-cultures modulate osteogenesis and angiogenesis. Stem Cell Res Ther, 2016, 7: 13.
22. Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature, 2014, 507(7492): 323-328.
23. Liang Y, Wen L, Shang F, et al. Endothelial progenitors enhanced the osteogenic capacities of mesenchymal stem cells in vitro and in a rat alveolar bone defect model. Arch Oral Biol, 2016, 68: 123-130.
24. Verseijden F, Posthumus-van Sluijs SJ, Pavljasevic P, et al. Adult human bone marrow- and adipose tissue-derived stromal cells support the formation of prevascular-like structures from endothelial cells in vitro. Tissue Eng Part A, 2010, 16(1): 101-114.
25. 孙源, 林红, 吴子征, 等. 联合培养时诱导的内皮细胞对自体骨髓基质干细胞成骨作用的影响. 中华创伤骨科杂志, 2006, 8(10): 919-923.
26. Zhao X, Liu L, Wang FK, et al. Coculture of vascular endothelial cells and adipose-derived stem cells as a source for bone engineering. Ann Plast Surg, 2012, 69(1): 91-98.
27. 董苑, 杨瑞年, 王福科, 等. BMP2 的表达对联合培养体系中骨髓间充质干细胞增殖和成骨分化的影响. 中华显微外科杂志, 2013, 36(4): 352-355.
28. 杨瑞年, 刘流, 王福科, 等. 联合培养体系中对骨髓间充质干细胞 Bmi-1 表达的影响. 中国组织工程研究, 2012, 16(14): 2524-2529.
29. 杨瑞年, 刘流, 王福科, 等. 血管内皮细胞对联合培养体系中骨髓间充质干细胞侏儒症相关转录因子表达及成骨分化的影响. 中国组织工程研究, 2012, 16(19): 3447-3449.

Chinese Journal of Reparative and Reconstructive Surgery

Heterotopic osteogenesis study of tissue engineered bone by co-culture of vascular endothelial cells and adipose-derived stem cells

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