Vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor mixed microcapsules in promoting angiogenic differentiation of rat bone marrow mesenchymal stem cells <i>in vitro</i>_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHAO Shengli ¹ , YIN Jie ² , YU Qinghe ¹ , ZHANG Guowei ¹ ,  MIN Shaoxiong ¹

1. Department of Spinal Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou Guangdong, 510282, P.R.China;
2. Department of Hand Surgery, Ningbo No.6 Hospital, Ningbo Zhejiang, 315040, P.R.China;

Corresponding author：

MIN Shaoxiong, Email: msxbear24@163.com

Keywords：

Bone marrow mesenchymal stem cells; vascular endothelial growth factor; basic fibroblast growth factor; polylactide-polyethyleneglycol-polylactic acid copolymer; endothelial differentiation

DOI：

10.7507/1002-1892.201808099

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor (VEGF/PELA/bFGF) mixed microcapsules in promoting the angiogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods The BMSCs were isolated by the method of whole bone marrow adherent, and sub-cultured. The passage 3 BMSCs were identified by Wright-Giemsa staining and flow cytometry, and used for subsequent experiments. VEGF/PELA/bFGF (group A), PELA/bFGF (group B), VEGF/PELA (group C), and PELA (group D) microcapsules were prepared. The biodegradable ability and cytotoxicity of PELA microcapsule were determined, and the slow-released ability of VEGF/PELA/bFGF mixed microcapsules was measured. The passage 3 BMSCs were co-cultured with the extracts of groups A, B, C, and D, separately. At 1, 3, 7, 14, and 20 days after being cultured, the morphological changes of induced BMSCs were recorded. At 21 days, the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein (Dil-ac-LDL) and FITC-labeled ulex europaeus agglutinin I (FITC-UEA-I) uptake ability. The tube-forming ability of the induced cells on Matrigel was also verified. The differences of the vascularize indexes in nodes, master junctions, master segments, and tot.master segments length in 4 groups were summarized and analyzed. Results The isolated and cultured cells were identified as BMSCs. The degradation time of PELA was more than 20 days. There was no significant effect on cell viability under co-culture conditions. At 20 days, the cumulative release of VEGF in the mixed microcapsules exceeded 95%, and the quantity of bFGF exceeded 80%. The morphology of cells in groups A, B, and C were changed. The cells in groups A and B showed the typical change of cobble-stone morphology. The numbers of double fluorescent labeled cells observed by fluorescence microscope were the most in group A, and decreases from group B and group C, with the lowest in group D. The cells in groups A and B formed a grid-like structure on Matrigel. Quantitative analysis showed that the differences in the number of nodes, master junctions, master segments, and tot.master segments length between groups A, B and groups C, D were significant (P<0.05). The number of nodes and the tot.master segments length of group A were more than those of group B (P<0.05). There was no significant differences in the number of master junctions and master segments between group A and group B (P>0.05). Conclusion VEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs in vitro.

Citation： ZHAO Shengli, YIN Jie, YU Qinghe, ZHANG Guowei, MIN Shaoxiong. Vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor mixed microcapsules in promoting angiogenic differentiation of rat bone marrow mesenchymal stem cells in vitro. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(2): 243-251. doi: 10.7507/1002-1892.201808099 Copy

1.	Johnson EO, Troupis T, Soucacos PN. Tissue-engineered vascularized bone grafts: basic science and clinical relevance to trauma and reconstructive microsurgery. Microsurgery, 2011, 31(3): 176-182.
2.	赵胜利, 史本超, 殷杰, 等. 实现支架材料在修复大段骨缺损时的早期血管化. 中国组织工程研究, 2018, 22(34): 5534-5539.
3.	Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev, 2011, 63(4-5): 300-311.
4.	Liu Y, Ming L, Luo H, et al. Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects. Biomaterials, 2013, 34(38): 9998-10006.
5.	陈家磊, 刘明, 段鑫, 等. BMP-7/聚乳酸-羟基乙酸共聚物缓释微球对兔BMSCs增殖和成软骨分化的影响. 中国修复重建外科杂志, 2018, 32(4): 428-433.
6.	Karoline P, Sandra H, Heinz R, et al. Vascularization mediated by mesenchymal stem cells from bone marrow and adipose tissue: a comparison. Cell Regen, 2015, 4(1): 8-18.
7.	Chen Y, Wang J, Zhu X, et al. The directional migration and differentiation of mesenchymal stem cells toward vascular endothelial cells stimulated by biphasic calcium phosphate ceramic. Regen Biomater, 2017, 5(3): 129-139.
8.	Semenza GL. Vasculogenesis, angiogenesis, and arteriogenesis: mechanisms of blood vessel formation and remodeling. J Cell Biochem, 2007, 102(4): 840-847.
9.	Spanholtz TA, Theodorou P, Holzbach T, et al. Vascular endothelial growth factor (VEGF165) plus basic fibroblast growth factor (bFGF) producing cells induce a mature and stable vascular network-a future therapy for ischemically challenged tissue. J Surg Res, 2011, 171(1): 329-338.
10.	刘晓彤, 沈若武, 卞明心, 等. 大鼠创伤皮肤组织 VEGF、PDGF 和 bFGF 表达及意义. 青岛大学医学院学报, 2016, 52(2): 209-211.
11.	Li X, Yi W, Jin A, et al. Effects of sequentially released BMP-2 and BMP-7 from PELA microcapsule-based scaffolds on the bone regeneration. Am J Transl Res, 2015, 7(8): 1417-1428.
12.	Li X, Min S, Zhao X, et al. Optimization of entrapping conditions to improve the release of BMP-2 from PELA carriers by response surface methodology. Biomed Mater, 2014, 10(1): 015002.
13.	Muschler GF, Nakamoto C, Griffith LG. Engineering principles of clinical cell-based tissue engineering. J Bone Joint Surg (Am), 2004, 86-A(7): 1541-1558.
14.	Filipowska J, Tomaszewski KA, Niedźwiedzki Ł, et al. The role of vasculature in bone development, regeneration and proper systemic functioning. Angiogenesis, 2017, 20(3): 291-302.
15.	王志红, 黄汉, 张斌, 等. 骨髓间充质干细胞促进皮肤创口愈合及 Wnt 信号通路在愈合过程中的作用. 中国现代医学杂志, 2016, 26(15): 56-59.
16.	Majidinia M, Aghazadeh J, Jahanban-Esfahlani R, et al. The roles of Wnt/β-catenin pathway in tissue development and regenerative medicine. J Cell Physiol, 2018, 233(8): 5598-5612.
17.	Kang Y, Ren L, Yang Y. Engineering vascularized bone grafts by integrating a biomimetic periosteum and β-TCP scaffold. ACS Appl Mater Interfaces, 2014, 6(12): 9622-9633.
18.	Clarkin CE, Emery RJ, Pitsillides AA, et al. Evaluation of VEGF-mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast-mediated crosstalk to endothelial cells. J Cell Physiol, 2008, 214(2): 537-544.
19.	Jiang YN, Zhao J, Chu FT, et al. Tension-loaded bone marrow stromal cells potentiate the paracrine osteogenic signaling of co-cultured vascular endothelial cells. Biol Open, 2018, 7(6): bio032482.
20.	何颖, 邹立津, 高蔓蔓, 等. 体外长期培养的猪BMSCs发生转化过程中基因表达谱和DNA甲基化谱关联分析. 中国修复重建外科杂志, 2018, 32(8): 1066-1073.
21.	Barkholt L, Flory E, Jekerle V, et al. Risk of tumorigenicity in mesenchymal stromal cell-based therapies-bridging scientific observations and regulatory viewpoints. Cytotherapy, 2013, 15(7): 753-759.
22.	Dew L, MacNeil S, Chong CK. Vascularization strategies for tissue engineers. Regen Med, 2015, 10(2): 211-224.
23.	Ahluwalia A, Tarnawski AS. Critical role of hypoxia sensor-HIF-1α in VEGF gene activation. Implications for angiogenesis and tissue injury healing. Curr Med Chem, 2012, 19(1): 90-97.
24.	Mattern J, Koomägi R, Volm M. Coexpression of VEGF and bFGF in human epidermoid lung carcinoma is associated with increased vessel density. Anticancer Res, 1997, 17(3C): 2249-2252.
25.	Kuttappan S, Mathew D, Nair MB. Biomimetic composite scaffolds containing bioceramics with collagen/gelatin for bone tissue engineering-A mini review. Int J Biol Macromol, 2016, 93(Pt B): 1390-1401.
26.	Lasprilla AJ, Martinez GA, Lunelli BH, et al. Poly-lactic acid synthesis for application in biomedical devices-A review. Biotechnol Adv, 2012, 30(1): 321-328.

1. Johnson EO, Troupis T, Soucacos PN. Tissue-engineered vascularized bone grafts: basic science and clinical relevance to trauma and reconstructive microsurgery. Microsurgery, 2011, 31(3): 176-182.
2. 赵胜利, 史本超, 殷杰, 等. 实现支架材料在修复大段骨缺损时的早期血管化. 中国组织工程研究, 2018, 22(34): 5534-5539.
3. Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev, 2011, 63(4-5): 300-311.
4. Liu Y, Ming L, Luo H, et al. Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects. Biomaterials, 2013, 34(38): 9998-10006.
5. 陈家磊, 刘明, 段鑫, 等. BMP-7/聚乳酸-羟基乙酸共聚物缓释微球对兔BMSCs增殖和成软骨分化的影响. 中国修复重建外科杂志, 2018, 32(4): 428-433.
6. Karoline P, Sandra H, Heinz R, et al. Vascularization mediated by mesenchymal stem cells from bone marrow and adipose tissue: a comparison. Cell Regen, 2015, 4(1): 8-18.
7. Chen Y, Wang J, Zhu X, et al. The directional migration and differentiation of mesenchymal stem cells toward vascular endothelial cells stimulated by biphasic calcium phosphate ceramic. Regen Biomater, 2017, 5(3): 129-139.
8. Semenza GL. Vasculogenesis, angiogenesis, and arteriogenesis: mechanisms of blood vessel formation and remodeling. J Cell Biochem, 2007, 102(4): 840-847.
9. Spanholtz TA, Theodorou P, Holzbach T, et al. Vascular endothelial growth factor (VEGF165) plus basic fibroblast growth factor (bFGF) producing cells induce a mature and stable vascular network-a future therapy for ischemically challenged tissue. J Surg Res, 2011, 171(1): 329-338.
10. 刘晓彤, 沈若武, 卞明心, 等. 大鼠创伤皮肤组织 VEGF、PDGF 和 bFGF 表达及意义. 青岛大学医学院学报, 2016, 52(2): 209-211.
11. Li X, Yi W, Jin A, et al. Effects of sequentially released BMP-2 and BMP-7 from PELA microcapsule-based scaffolds on the bone regeneration. Am J Transl Res, 2015, 7(8): 1417-1428.
12. Li X, Min S, Zhao X, et al. Optimization of entrapping conditions to improve the release of BMP-2 from PELA carriers by response surface methodology. Biomed Mater, 2014, 10(1): 015002.
13. Muschler GF, Nakamoto C, Griffith LG. Engineering principles of clinical cell-based tissue engineering. J Bone Joint Surg (Am), 2004, 86-A(7): 1541-1558.
14. Filipowska J, Tomaszewski KA, Niedźwiedzki Ł, et al. The role of vasculature in bone development, regeneration and proper systemic functioning. Angiogenesis, 2017, 20(3): 291-302.
15. 王志红, 黄汉, 张斌, 等. 骨髓间充质干细胞促进皮肤创口愈合及 Wnt 信号通路在愈合过程中的作用. 中国现代医学杂志, 2016, 26(15): 56-59.
16. Majidinia M, Aghazadeh J, Jahanban-Esfahlani R, et al. The roles of Wnt/β-catenin pathway in tissue development and regenerative medicine. J Cell Physiol, 2018, 233(8): 5598-5612.
17. Kang Y, Ren L, Yang Y. Engineering vascularized bone grafts by integrating a biomimetic periosteum and β-TCP scaffold. ACS Appl Mater Interfaces, 2014, 6(12): 9622-9633.
18. Clarkin CE, Emery RJ, Pitsillides AA, et al. Evaluation of VEGF-mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast-mediated crosstalk to endothelial cells. J Cell Physiol, 2008, 214(2): 537-544.
19. Jiang YN, Zhao J, Chu FT, et al. Tension-loaded bone marrow stromal cells potentiate the paracrine osteogenic signaling of co-cultured vascular endothelial cells. Biol Open, 2018, 7(6): bio032482.
20. 何颖, 邹立津, 高蔓蔓, 等. 体外长期培养的猪BMSCs发生转化过程中基因表达谱和DNA甲基化谱关联分析. 中国修复重建外科杂志, 2018, 32(8): 1066-1073.
21. Barkholt L, Flory E, Jekerle V, et al. Risk of tumorigenicity in mesenchymal stromal cell-based therapies-bridging scientific observations and regulatory viewpoints. Cytotherapy, 2013, 15(7): 753-759.
22. Dew L, MacNeil S, Chong CK. Vascularization strategies for tissue engineers. Regen Med, 2015, 10(2): 211-224.
23. Ahluwalia A, Tarnawski AS. Critical role of hypoxia sensor-HIF-1α in VEGF gene activation. Implications for angiogenesis and tissue injury healing. Curr Med Chem, 2012, 19(1): 90-97.
24. Mattern J, Koomägi R, Volm M. Coexpression of VEGF and bFGF in human epidermoid lung carcinoma is associated with increased vessel density. Anticancer Res, 1997, 17(3C): 2249-2252.
25. Kuttappan S, Mathew D, Nair MB. Biomimetic composite scaffolds containing bioceramics with collagen/gelatin for bone tissue engineering-A mini review. Int J Biol Macromol, 2016, 93(Pt B): 1390-1401.
26. Lasprilla AJ, Martinez GA, Lunelli BH, et al. Poly-lactic acid synthesis for application in biomedical devices-A review. Biotechnol Adv, 2012, 30(1): 321-328.

Chinese Journal of Reparative and Reconstructive Surgery

Vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor mixed microcapsules in promoting angiogenic differentiation of rat bone marrow mesenchymal stem cells in vitro

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content