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, 劉徐妹, 于博. 載 BMP-2 多肽 P24 的巰基化殼聚糖水凝膠誘導異位成骨的實驗研究. 中國修復重建外科雜志, 2018, 32(9): 1144-1149.
3.	王福科, 劉流, 趙德萍, 等. 體外血管內皮細胞和脂肪干細胞聯合培養對脂肪干細胞成骨分化的影響. 中國修復重建外科雜志, 2010, 24(4): 399-405.
4.	王福科, 劉流, 何曉光, 等. 血管內皮細胞和脂肪干細胞體外聯合培養細胞的增殖. 中國組織工程研究與臨床康復, 2010, 14(32): 6001-6005.
5.	李彥林, 楊志明, 解慧琪, 等. 生物衍生骨支架材料的體外細胞相容性實驗研究. 中國修復重建外科雜志, 2001, 15(4): 227-231.
6.	楊浩, 李彥林, 韓睿, 等. 異種骨衍生材料修復節段性骨缺損的實驗研究. 昆明醫學院學報, 2004, 25(1): 14-18.
7.	李彥林, 楊志明, 解慧琪, 等. 生物衍生組織工程骨支架材料的制備及理化特性. 生物醫學工程學雜志, 2002, 19(1): 10-12.
8.	王福科, 劉流, 代曉明, 等. 纖維粘連蛋白對部分脫蛋白骨細胞相容性影響研究. 昆明醫學院學報, 2010, 31(1): 9-14.
9.	裴雪濤. 干細胞實驗指南. 北京: 科學出版社, 2006: 83-100.
10.	王福科, 劉流, 趙德萍, 等. 大鼠臍血來源單個核細胞誘導為內皮祖細胞的免疫熒光檢測. 中國生物美容, 2009, 1(4): 10-15.
11.	王福科, 趙德萍, 代曉明, 等. 不同類型血清對大鼠脂肪干細胞分離培養的影響. 昆明醫學院學報, 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.	劉流, 王福科, 趙德萍, 等. 聯合培養 VECs 與 ADSCs 與部分脫蛋白生物骨體外構建組織工程骨的實驗研究. 中國生物美容, 2010, 1(1): 1-9.
14.	寧鈺, 秦文, 任亞輝, 等. 載淫羊藿苷/凹凸棒石/Ⅰ型膠原/聚己內酯復合支架修復兔脛骨缺損的實驗研究. 中國修復重建外科雜志, 2019, 33(9): 1-13.
15.	馮立, 曹曉建, 任永信. 纖維粘連蛋白與堿性成纖維細胞生長因子對成骨細胞黏附的協同刺激作用. 中國修復重建外科雜志, 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.	孫源, 林紅, 吳子征, 等. 聯合培養時誘導的內皮細胞對自體骨髓基質干細胞成骨作用的影響. 中華創傷骨科雜志, 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, 楊瑞年, 王福科, 等. BMP2 的表達對聯合培養體系中骨髓間充質干細胞增殖和成骨分化的影響. 中華顯微外科雜志, 2013, 36(4): 352-355.
28.	楊瑞年, 劉流, 王福科, 等. 聯合培養體系中對骨髓間充質干細胞 Bmi-1 表達的影響. 中國組織工程研究, 2012, 16(14): 2524-2529.
29.	楊瑞年, 劉流, 王福科, 等. 血管內皮細胞對聯合培養體系中骨髓間充質干細胞侏儒癥相關轉錄因子表達及成骨分化的影響. 中國組織工程研究, 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, 劉徐妹, 于博. 載 BMP-2 多肽 P24 的巰基化殼聚糖水凝膠誘導異位成骨的實驗研究. 中國修復重建外科雜志, 2018, 32(9): 1144-1149.
3. 王福科, 劉流, 趙德萍, 等. 體外血管內皮細胞和脂肪干細胞聯合培養對脂肪干細胞成骨分化的影響. 中國修復重建外科雜志, 2010, 24(4): 399-405.
4. 王福科, 劉流, 何曉光, 等. 血管內皮細胞和脂肪干細胞體外聯合培養細胞的增殖. 中國組織工程研究與臨床康復, 2010, 14(32): 6001-6005.
5. 李彥林, 楊志明, 解慧琪, 等. 生物衍生骨支架材料的體外細胞相容性實驗研究. 中國修復重建外科雜志, 2001, 15(4): 227-231.
6. 楊浩, 李彥林, 韓睿, 等. 異種骨衍生材料修復節段性骨缺損的實驗研究. 昆明醫學院學報, 2004, 25(1): 14-18.
7. 李彥林, 楊志明, 解慧琪, 等. 生物衍生組織工程骨支架材料的制備及理化特性. 生物醫學工程學雜志, 2002, 19(1): 10-12.
8. 王福科, 劉流, 代曉明, 等. 纖維粘連蛋白對部分脫蛋白骨細胞相容性影響研究. 昆明醫學院學報, 2010, 31(1): 9-14.
9. 裴雪濤. 干細胞實驗指南. 北京: 科學出版社, 2006: 83-100.
10. 王福科, 劉流, 趙德萍, 等. 大鼠臍血來源單個核細胞誘導為內皮祖細胞的免疫熒光檢測. 中國生物美容, 2009, 1(4): 10-15.
11. 王福科, 趙德萍, 代曉明, 等. 不同類型血清對大鼠脂肪干細胞分離培養的影響. 昆明醫學院學報, 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. 劉流, 王福科, 趙德萍, 等. 聯合培養 VECs 與 ADSCs 與部分脫蛋白生物骨體外構建組織工程骨的實驗研究. 中國生物美容, 2010, 1(1): 1-9.
14. 寧鈺, 秦文, 任亞輝, 等. 載淫羊藿苷/凹凸棒石/Ⅰ型膠原/聚己內酯復合支架修復兔脛骨缺損的實驗研究. 中國修復重建外科雜志, 2019, 33(9): 1-13.
15. 馮立, 曹曉建, 任永信. 纖維粘連蛋白與堿性成纖維細胞生長因子對成骨細胞黏附的協同刺激作用. 中國修復重建外科雜志, 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. 孫源, 林紅, 吳子征, 等. 聯合培養時誘導的內皮細胞對自體骨髓基質干細胞成骨作用的影響. 中華創傷骨科雜志, 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, 楊瑞年, 王福科, 等. BMP2 的表達對聯合培養體系中骨髓間充質干細胞增殖和成骨分化的影響. 中華顯微外科雜志, 2013, 36(4): 352-355.
28. 楊瑞年, 劉流, 王福科, 等. 聯合培養體系中對骨髓間充質干細胞 Bmi-1 表達的影響. 中國組織工程研究, 2012, 16(14): 2524-2529.
29. 楊瑞年, 劉流, 王福科, 等. 血管內皮細胞對聯合培養體系中骨髓間充質干細胞侏儒癥相關轉錄因子表達及成骨分化的影響. 中國組織工程研究, 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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