Experimental study on tissue engineered cartilage constructed by three-dimensional bioprinted human adipose-derived stem cells combined with gelatin methacryloyl_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

MU Lin , ZENG Jinshi , HUANG Yuanliang , LIN Yanxian , JIANG Haiyue ,  TENG Li

Department of Craniomaxillofacial Surgery, Plastic Surgery Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, 100144, P.R.China;

Corresponding?author：

TENG Li, Email: tenglidr@sina.com

Keywords：

Tissue engineered cartilage; three-dimensional bioprinting; adipose-derived stem cells; biomaterials

DOI：

10.7507/1002-1892.202101049

Video：

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Objective To explore the feasibility of three-dimensional (3D) bioprinted adipose-derived stem cells (ADSCs) combined with gelatin methacryloyl (GelMA) to construct tissue engineered cartilage.Methods Adipose tissue voluntarily donated by liposuction patients was collected to isolate and culture human ADSCs (hADSCs). The third generation cells were mixed with GelMA hydrogel and photoinitiator to make biological ink. The hADSCs-GelMA composite scaffold was prepared by 3D bioprinting technology, and it was observed in general, and observed by scanning electron microscope after cultured for 1 day and chondrogenic induction culture for 14 days. After cultured for 1, 4, and 7 days, the composite scaffolds were taken for live/dead cell staining to observe cell survival rate; and cell counting kit 8 (CCK-8) method was used to detect cell proliferation. The composite scaffold samples cultured in cartilage induction for 14 days were taken as the experimental group, and the composite scaffolds cultured in complete medium for 14 days were used as the control group. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect cartilage formation. The relative expression levels of the mRNA of cartilage matrix gene [(aggrecan, ACAN)], chondrogenic regulatory factor (SOX9), cartilage-specific gene [collagen type Ⅱ A1 (COLⅡA1)], and cartilage hypertrophy marker gene [collagen type ⅩA1 (COLⅩA1)] were detected. The 3D bioprinted hADSCs-GelMA composite scaffold (experimental group) and the blank GelMA hydrogel scaffold without cells (control group) cultured for 14 days of chondrogenesis were implanted into the subcutaneous pockets of the back of nude mice respectively, and the materials were taken after 4 weeks, and gross observation, Safranin O staining, Alcian blue staining, and collagen type Ⅱ immunohistochemical staining were performed to observe the cartilage formation in the composite scaffold.Results Macroscope and scanning electron microscope observations showed that the hADSCs-GelMA composite scaffolds had a stable and regular structure. The cell viability could be maintained at 80%-90% at 1, 4, and 7 days after printing, and the differences between different time points were significant (P<0.05). The results of CCK-8 experiment showed that the cells in the scaffold showed continuous proliferation after printing. After 14 days of chondrogenic induction and culture on the composite scaffold, the expressions of ACAN, SOX9, and COLⅡA1 were significantly up-regulated (P<0.05), the expression of COLⅩA1 was significantly down-regulated (P<0.05). The scaffold was taken out at 4 weeks after implantation. The structure of the scaffold was complete and clear. Histological and immunohistochemical results showed that cartilage matrix and collagen type Ⅱ were deposited, and there was cartilage lacuna formation, which confirmed the formation of cartilage tissue.Conclusion The 3D bioprinted hADSCs-GelMA composite scaffold has a stable 3D structure and high cell viability, and can be induced differentiation into cartilage tissue, which can be used to construct tissue engineered cartilage in vivo and in vitro.

Citation： MU Lin, ZENG Jinshi, HUANG Yuanliang, LIN Yanxian, JIANG Haiyue, TENG Li. Experimental study on tissue engineered cartilage constructed by three-dimensional bioprinted human adipose-derived stem cells combined with gelatin methacryloyl. Chinese Journal of Reparative and Reconstructive Surgery, 2021, 35(7): 896-903. doi: 10.7507/1002-1892.202101049 Copy

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2.	Zhang YS, Yue K, Aleman J, et al. 3D bioprinting for tissue and organ fabrication. Ann Biomed Eng, 2017, 45(1): 148-163.
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7.	宋楊, 王曉飛, 王宇光, 等. 人脂肪間充質干細胞與生物材料共混物三維打印體的體內成骨. 北京大學學報 (醫學版), 2016, 48(1): 45-50.
8.	Tasnim N, De la Vega L, Anil Kumar S, et al. 3D bioprinting stem cell derived tissues. Cell Mol Bioeng, 2018, 11(4): 219-240.
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13.	Kilian D, Ahlfeld T, Akkineni AR, et al. 3D Bioprinting of osteochondral tissue substitutes-in vitro-chondrogenesis in multi-layered mineralized constructs. Sci Rep, 2020, 10(1): 8277. doi: 10.1038/s41598-020-65050-9.
14.	Mouser VH, Melchels FP, Visser J, et al. Yield stress determines bioprintability of hydrogels based on gelatin-methacryloyl and gellan gum for cartilage bioprinting. Biofabrication, 2016, 8(3): 035003. doi: 10.1088/1758-5090/8/3/035003.
15.	Loessner D, Meinert C, Kaemmerer E, et al. Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms. Nat Protoc, 2016, 11(4): 727-746.
16.	Daly AC, Critchley SE, Rencsok EM, et al. A comparison of different bioinks for 3D bioprinting of fibrocartilage and hyaline cartilage. Biofabrication, 2016, 8(4): 045002. doi: 10.1088/1758-5090/8/4/045002.
17.	Gu Y, Zhang L, Du X, et al. Reversible physical crosslinking strategy with optimal temperature for 3D bioprinting of human chondrocyte-laden gelatin methacryloyl bioink. J Biomater Appl, 2018, 33(5): 609-618.
18.	De Moor L, Fernandez S, Vercruysse C, et al. Hybrid bioprinting of chondrogenically induced human mesenchymal stem cell spheroids. Front Bioeng Biotechnol, 2020, 8: 484. doi: 10.3389/fbioe.2020.00484.
19.	Zheng CX, Sui BD, Liu N, et al. Adipose mesenchymal stem cells from osteoporotic donors preserve functionality and modulate systemic inflammatory microenvironment in osteoporotic cytotherapy. Sci Rep, 2018, 8(1): 5215. doi: 10.1038/s41598-018-23098-8.
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21.	Billiet T, Gevaert E, De Schryver T, et al. The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. Biomaterials, 2014, 35(1): 49-62.
22.	Luo C, Xie R, Zhang J, et al. Low-temperature three-dimensional printing of tissue cartilage engineered with gelatin methacrylamide. Tissue Eng Part C Methods, 2020, 26(6): 306-316.
23.	Kosik-Kozio? A, Costantini M, Mróz A, et al. 3D bioprinted hydrogel model incorporating β-tricalcium phosphate for calcified cartilage tissue engineering. Biofabrication, 2019, 11(3): 035016. doi: 10.1088/1758-5090/ab15cb.
24.	Nguyen D, H?gg DA, Forsman A, et al. Cartilage tissue engineering by the 3D bioprinting of iPS cells in a nanocellulose/alginate bioink. Sci Rep, 2017, 7(1): 658. doi: 10.1038/s41598-017-00690-y.
25.	Kolan KCR, Semon JA, Bromet B, et al. Bioprinting with human stem cell-laden alginate-gelatin bioink and bioactive glass for tissue engineering. Int J Bioprint, 2019, 5(2.2): 204. doi: 10.18063/ijb.v5i2.2.204.
26.	Olate-Moya F, Arens L, Wilhelm M, et al. Chondroinductive alginate-based hydrogels having graphene oxide for 3D printed scaffold fabrication. ACS Appl Mater Interfaces, 2020, 12(4): 4343-4357.

1. Phull AR, Eo SH, Abbas Q, et al. Applications of chondrocyte-based cartilage engineering: An overview. Biomed Res Int, 2016, 2016: 1879837. doi: 10.1155/2016/1879837.
2. Zhang YS, Yue K, Aleman J, et al. 3D bioprinting for tissue and organ fabrication. Ann Biomed Eng, 2017, 45(1): 148-163.
3. Matai I, Kaur G, Seyedsalehi A, et al. Progress in 3D bioprinting technology for tissue/organ regenerative engineering. Biomaterials, 2020, 226: 119536. doi: 10.1016/j.biomaterials.2019.119536.
4. Ozbolat IT, Hospodiuk M. Current advances and future perspectives in extrusion-based bioprinting. Biomaterials, 2016, 76: 321-343.
5. Massa S, Sakr MA, Seo J, et al. Bioprinted 3D vascularized tissue model for drug toxicity analysis. Biomicrofluidics, 2017, 11(4): 044109. doi: 10.1063/1.4994708.
6. Kuss MA, Wu S, Wang Y, et al. Prevascularization of 3D printed bone scaffolds by bioactive hydrogels and cell co-culture. J Biomed Mater Res B Appl Biomater, 2018, 106(5): 1788-1798.
7. 宋楊, 王曉飛, 王宇光, 等. 人脂肪間充質干細胞與生物材料共混物三維打印體的體內成骨. 北京大學學報 (醫學版), 2016, 48(1): 45-50.
8. Tasnim N, De la Vega L, Anil Kumar S, et al. 3D bioprinting stem cell derived tissues. Cell Mol Bioeng, 2018, 11(4): 219-240.
9. Camara DAD, Shibli JA, Müller EA, et al. Adipose tissue-derived stem cells: The biologic basis and future directions for tissue engineering. Materials (Basel), 2020, 13(14): 3210. doi: 10.3390/ma13143210.
10. Yue K, Trujillo-de Santiago G, Alvarez MM, et al. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials, 2015, 73: 254-271.
11. 羅春陽, 劉楊, 張嘯, 等. 應用明膠甲基丙烯酰胺低溫3D打印組織工程軟骨. 南京醫科大學學報 (自然科學版), 2020, 40(4): 533-537.
12. Wilkes GH, Wong J, Guilfoyle R. Microtia reconstruction. Plast Reconstr Surg, 2014, 134(3): 464e-479e.
13. Kilian D, Ahlfeld T, Akkineni AR, et al. 3D Bioprinting of osteochondral tissue substitutes-in vitro-chondrogenesis in multi-layered mineralized constructs. Sci Rep, 2020, 10(1): 8277. doi: 10.1038/s41598-020-65050-9.
14. Mouser VH, Melchels FP, Visser J, et al. Yield stress determines bioprintability of hydrogels based on gelatin-methacryloyl and gellan gum for cartilage bioprinting. Biofabrication, 2016, 8(3): 035003. doi: 10.1088/1758-5090/8/3/035003.
15. Loessner D, Meinert C, Kaemmerer E, et al. Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms. Nat Protoc, 2016, 11(4): 727-746.
16. Daly AC, Critchley SE, Rencsok EM, et al. A comparison of different bioinks for 3D bioprinting of fibrocartilage and hyaline cartilage. Biofabrication, 2016, 8(4): 045002. doi: 10.1088/1758-5090/8/4/045002.
17. Gu Y, Zhang L, Du X, et al. Reversible physical crosslinking strategy with optimal temperature for 3D bioprinting of human chondrocyte-laden gelatin methacryloyl bioink. J Biomater Appl, 2018, 33(5): 609-618.
18. De Moor L, Fernandez S, Vercruysse C, et al. Hybrid bioprinting of chondrogenically induced human mesenchymal stem cell spheroids. Front Bioeng Biotechnol, 2020, 8: 484. doi: 10.3389/fbioe.2020.00484.
19. Zheng CX, Sui BD, Liu N, et al. Adipose mesenchymal stem cells from osteoporotic donors preserve functionality and modulate systemic inflammatory microenvironment in osteoporotic cytotherapy. Sci Rep, 2018, 8(1): 5215. doi: 10.1038/s41598-018-23098-8.
20. Li CY, Wu XY, Tong JB, et al. Comparative analysis of human mesenchymal stem cells from bone marrow and adipose tissue under xeno-free conditions for cell therapy. Stem Cell Res Ther, 2015, 6(1): 55. doi: 10.1186/s13287-015-0066-5.
21. Billiet T, Gevaert E, De Schryver T, et al. The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. Biomaterials, 2014, 35(1): 49-62.
22. Luo C, Xie R, Zhang J, et al. Low-temperature three-dimensional printing of tissue cartilage engineered with gelatin methacrylamide. Tissue Eng Part C Methods, 2020, 26(6): 306-316.
23. Kosik-Kozio? A, Costantini M, Mróz A, et al. 3D bioprinted hydrogel model incorporating β-tricalcium phosphate for calcified cartilage tissue engineering. Biofabrication, 2019, 11(3): 035016. doi: 10.1088/1758-5090/ab15cb.
24. Nguyen D, H?gg DA, Forsman A, et al. Cartilage tissue engineering by the 3D bioprinting of iPS cells in a nanocellulose/alginate bioink. Sci Rep, 2017, 7(1): 658. doi: 10.1038/s41598-017-00690-y.
25. Kolan KCR, Semon JA, Bromet B, et al. Bioprinting with human stem cell-laden alginate-gelatin bioink and bioactive glass for tissue engineering. Int J Bioprint, 2019, 5(2.2): 204. doi: 10.18063/ijb.v5i2.2.204.
26. Olate-Moya F, Arens L, Wilhelm M, et al. Chondroinductive alginate-based hydrogels having graphene oxide for 3D printed scaffold fabrication. ACS Appl Mater Interfaces, 2020, 12(4): 4343-4357.

Chinese Journal of Reparative and Reconstructive Surgery

Experimental study on tissue engineered cartilage constructed by three-dimensional bioprinted human adipose-derived stem cells combined with gelatin methacryloyl

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