Establishment and biological effect evaluation of prevascularized porous β-tricalcium phosphate tissue engineered bone_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HUANG Mengquan , FAN Jian , MA Ziyang ,  LI Jing ,  LU Yajie

Department of Orthopedics, the First Affiliated Hospital of Air Force Medical University of Chinese PLA, Xi’an Shaanxi, 710032, P. R. China;

Corresponding?author：

LI Jing, Email: 13359265058@189.cn; LU Yajie, Email: yajiel_fmmu@foxmail.com

Keywords：

Tissue engineered bone; bone marrow mesenchymal stem cells; endothelial progenitor cells; prevascularized scaffold; β-tricalcium phosphate; rabbit

DOI：

10.7507/1002-1892.202202010

Video：

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Objective To evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. Methods EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group (n=6) and porous β-TCP scaffold as the control group (n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. Results The isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel (P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group (P<0.05), except for area fraction at 4 weeks after implantation (P>0.05). Conclusion The prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.

Citation： HUANG Mengquan, FAN Jian, MA Ziyang, LI Jing, LU Yajie. Establishment and biological effect evaluation of prevascularized porous β-tricalcium phosphate tissue engineered bone. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(5): 625-632. doi: 10.7507/1002-1892.202202010 Copy

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12.	Gurel Pekozer G, Torun Kose G, Hasirci V. Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells. Microvasc Res, 2016, 108: 1-9.
13.	賈智明, 郭海林, 陳方. 組織工程組織血管化的研究進展. 組織工程與重建外科雜志, 2018, 14(1): 39-42.
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15.	Yong KW, Choi JR, Choi JY, et al. Recent advances in mechanically loaded human mesenchymal stem cells for bone tissue engineering. Int J Mol Sci, 2020, 21(16): 5816. doi: 10.3390/ijms21165816.
16.	Kocherova I, Bryja A, Mozdziak P, et al. Human umbilical vein endothelial cells (HUVECs) co-culture with osteogenic cells: From molecular communication to engineering prevascularised bone grafts. J Clin Med, 2019, 8(10): 1602. doi: 10.3390/jcm8101602.
17.	廖欣宇, 王福科, 李彥林, 等. 聯合培養血管內皮細胞和脂肪干細胞與部分脫蛋白生物骨構建組織工程骨修復頜骨缺損. 中國組織工程研究, 2022, 26(13): 2027-2033.
18.	Kong L, Wang Y, Wang H, et al. Conditioned media from endothelial progenitor cells cultured in simulated microgravity promote angiogenesis and bone fracture healing. Stem Cell Res Ther, 2021, 12(1): 47. doi: 10.1186/s13287-020-02074-y.
19.	Bouland C, Philippart P, Dequanter D, et al. Cross-talk between mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs) in bone regeneration. Front Cell Dev Biol, 2021, 9: 674084. doi: 10.3389/fcell.2021.674084.
20.	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.
21.	Li G, Wang X, Cao J, et al. Coculture of peripheral blood CD34+ cell and mesenchymal stem cell sheets increase the formation of bone in calvarial critical-size defects in rabbits. Br J Oral Maxillofac Surg, 2014, 52(2): 134-139.
22.	Kawecki F, Galbraith T, Clafshenkel WP, et al. In vitro prevascularization of self-assembled human bone-like tissues and preclinical assessment using a rat calvarial bone defect model. Materials (Basel), 2021, 14(8): 2023. doi: 10.3390/ma14082023.
23.	Li Z, Yang A, Yin X, et al. Mesenchymal stem cells promote endothelial progenitor cell migration, vascularization, and bone repair in tissue-engineered constructs via activating CXCR2-Src-PKL/Vav2-Rac1. FASEB J, 2018, 32(4): 2197-2211.
24.	Tamari T, Kawar-Jaraisy R, Doppelt O, et al. The paracrine role of endothelial cells in bone formation via CXCR4/SDF-1 pathway. Cells, 2020, 9(6): 1325. doi: 10.3390/cells9061325.

1. Hao Z, Li H, Wang Y, et al. Supramolecular peptide nanofiber hydrogels for bone tissue engineering: From multihierarchical fabrications to comprehensive applications. Adv Sci (Weinh), 2022: e2103820. doi: 10.1002/advs.202103820.
2. 秦宇星, 任前貴, 沈佩鋒, 等. 組織工程技術治療骨缺損: 應用于臨床還有多遠? 中國組織工程研究, 2021, 25(29): 4703-4708.
3. Wang W, Yeung KWK. Bone grafts and biomaterials substitutes for bone defect repair: A review. Bioact Mater, 2017, 2(4): 224-247.
4. Lopes SV, Collins MN, Reis RL, et al. Vascularization approaches in tissue engineering: Recent developments on evaluation tests and modulation. ACS Appl Bio Mater, 2021, 4(4): 2941-2956.
5. Sandhurst ES, Jaswandkar SV, Kundu K, et al. Nanoarchitectonics of a microsphere-based scaffold for modeling neurodevelopment and neurological disease. ACS Appl Bio Mater, 2022, 5(2): 528-544.
6. Peng J, Chen L, Peng K, et al. Bone marrow mesenchymal stem cells and endothelial progenitor cells co-culture enhances large segment bone defect repair. J Biomed Nanotechnol, 2019, 15(4): 742-755.
7. Shafiee S, Shariatzadeh S, Zafari A, et al. Recent advances on cell-based co-culture strategies for prevascularization in tissue engineering. Front Bioeng Biotechnol, 2021, 9: 745314. doi: 10.3389/fbioe.2021.745314.
8. 魯亞杰, 李明輝, 龍作堯, 等. 基于血管灌注及硬組織切片技術的骨微血管研究方法. 解剖學雜志, 2019, 42(1): 85-87.
9. 魯亞杰, 李明輝, 龍作堯, 等. 骨組織內微血管可視化研究模型的建立. 中華實驗外科雜志, 2018, 35(12): 2336-2338.
10. Chen X, He W, Sun M, et al. STING inhibition accelerates the bone healing process while enhancing type H vessel formation. FASEB J, 2021, 35(11): e21964. doi: 10.1096/fj.202100069RR.
11. Qin Y, Zhang C. Endothelial progenitor cell-derived extracellular vesicle-meditated cell-to-cell communication regulates the proliferation and osteoblastic differentiation of bone mesenchymal stromal cells. Mol Med Rep, 2017, 16(5): 7018-7024.
12. Gurel Pekozer G, Torun Kose G, Hasirci V. Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells. Microvasc Res, 2016, 108: 1-9.
13. 賈智明, 郭海林, 陳方. 組織工程組織血管化的研究進展. 組織工程與重建外科雜志, 2018, 14(1): 39-42.
14. Lin Z, Zhang X, Fritch MR, et al. Engineering pre-vascularized bone-like tissue from human mesenchymal stem cells through simulating endochondral ossification. Biomaterials, 2022, 283: 121451. doi: 10.1016/j.biomaterials.2022.121451.
15. Yong KW, Choi JR, Choi JY, et al. Recent advances in mechanically loaded human mesenchymal stem cells for bone tissue engineering. Int J Mol Sci, 2020, 21(16): 5816. doi: 10.3390/ijms21165816.
16. Kocherova I, Bryja A, Mozdziak P, et al. Human umbilical vein endothelial cells (HUVECs) co-culture with osteogenic cells: From molecular communication to engineering prevascularised bone grafts. J Clin Med, 2019, 8(10): 1602. doi: 10.3390/jcm8101602.
17. 廖欣宇, 王福科, 李彥林, 等. 聯合培養血管內皮細胞和脂肪干細胞與部分脫蛋白生物骨構建組織工程骨修復頜骨缺損. 中國組織工程研究, 2022, 26(13): 2027-2033.
18. Kong L, Wang Y, Wang H, et al. Conditioned media from endothelial progenitor cells cultured in simulated microgravity promote angiogenesis and bone fracture healing. Stem Cell Res Ther, 2021, 12(1): 47. doi: 10.1186/s13287-020-02074-y.
19. Bouland C, Philippart P, Dequanter D, et al. Cross-talk between mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs) in bone regeneration. Front Cell Dev Biol, 2021, 9: 674084. doi: 10.3389/fcell.2021.674084.
20. 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.
21. Li G, Wang X, Cao J, et al. Coculture of peripheral blood CD34+ cell and mesenchymal stem cell sheets increase the formation of bone in calvarial critical-size defects in rabbits. Br J Oral Maxillofac Surg, 2014, 52(2): 134-139.
22. Kawecki F, Galbraith T, Clafshenkel WP, et al. In vitro prevascularization of self-assembled human bone-like tissues and preclinical assessment using a rat calvarial bone defect model. Materials (Basel), 2021, 14(8): 2023. doi: 10.3390/ma14082023.
23. Li Z, Yang A, Yin X, et al. Mesenchymal stem cells promote endothelial progenitor cell migration, vascularization, and bone repair in tissue-engineered constructs via activating CXCR2-Src-PKL/Vav2-Rac1. FASEB J, 2018, 32(4): 2197-2211.
24. Tamari T, Kawar-Jaraisy R, Doppelt O, et al. The paracrine role of endothelial cells in bone formation via CXCR4/SDF-1 pathway. Cells, 2020, 9(6): 1325. doi: 10.3390/cells9061325.

Chinese Journal of Reparative and Reconstructive Surgery

Establishment and biological effect evaluation of prevascularized porous β-tricalcium phosphate tissue engineered bone

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