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：

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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

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5.	陳家磊, 劉明, 段鑫, 等. BMP-7/聚乳酸-羥基乙酸共聚物緩釋微球對兔BMSCs增殖和成軟骨分化的影響. 中國修復重建外科雜志, 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.
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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.
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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.	何穎, 鄒立津, 高蔓蔓, 等. 體外長期培養的豬BMSCs發生轉化過程中基因表達譜和DNA甲基化譜關聯分析. 中國修復重建外科雜志, 2018, 32(8): 1066-1073.
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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, Koom?gi 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. 趙勝利, 史本超, 殷杰, 等. 實現支架材料在修復大段骨缺損時的早期血管化. 中國組織工程研究, 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. 陳家磊, 劉明, 段鑫, 等. BMP-7/聚乳酸-羥基乙酸共聚物緩釋微球對兔BMSCs增殖和成軟骨分化的影響. 中國修復重建外科雜志, 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. 劉曉彤, 沈若武, 卞明心, 等. 大鼠創傷皮膚組織 VEGF、PDGF 和 bFGF 表達及意義. 青島大學醫學院學報, 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, Nied?wiedzki ?, et al. The role of vasculature in bone development, regeneration and proper systemic functioning. Angiogenesis, 2017, 20(3): 291-302.
15. 王志紅, 黃漢, 張斌, 等. 骨髓間充質干細胞促進皮膚創口愈合及 Wnt 信號通路在愈合過程中的作用. 中國現代醫學雜志, 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. 何穎, 鄒立津, 高蔓蔓, 等. 體外長期培養的豬BMSCs發生轉化過程中基因表達譜和DNA甲基化譜關聯分析. 中國修復重建外科雜志, 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, Koom?gi 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

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