Construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal printing technology_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

 CHEN Mingxue ¹ , WU Jiang ^1,2 , YIN Han ² , SUI Xiang ² , LIU Shuyun ² , GUO Quanyi ²

1. Department of Orthopaedics, Beijing Jishuitan Hospital Affiliated to Capital Medical University, Beijing, 100035, P. R. China;
2. Institute of Orthopedics, the First Medical Center of the Chinese PLA General Hospital, Beijing Key Laboratory of Orthopaedic Regenerative Medicine, Key Laboratory of Orthopaedic War Trauma, Beijing, 100853, P. R. China;

Corresponding author：

CHEN Mingxue, Email: chenmingxue1@126.com

Keywords：

Tissue engineered scaffold; low temperature deposition three-dimensional printing technology; meniscus; decellularized matrix

DOI：

10.7507/1002-1892.202402063

Video：

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Objective To investigate the construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal (3D) printing technology and evaluate its biocompatibility. Methods The fresh pig meniscus was decellularized by improved physicochemical method to obtain decellularized meniscus matrix homogenate. Gross observation, HE staining, and DAPI staining were used to observe the decellularization effect. Toluidine blue staining, safranin O staining, and sirius red staining were used to evaluate the retention of mucopolysaccharide and collagen. Then, the decellularized meniscus matrix bioink was prepared, and the new tissue engineered meniscus scaffold was prepared by low temperature deposition 3D printing technology. Scanning electron microscopy was used to observe the microstructure. After co-culture with adipose-derived stem cells, the cell compatibility of the scaffolds was observed by cell counting kit 8 (CCK-8), and the cell activity and morphology were observed by dead/live cell staining and cytoskeleton staining. The inflammatory cell infiltration and degradation of the scaffolds were evaluated by subcutaneous experiment in rats. Results The decellularized meniscus matrix homogenate appeared as a transparent gel. DAPI and histological staining showed that the immunogenic nucleic acids were effectively removed and the active components of mucopolysaccharide and collagen were remained. The new tissue engineered meniscus scaffolds was constructed by low temperature deposition 3D printing technology and it had macroporous-microporous microstructures under scanning electron microscopy. CCK-8 test showed that the scaffolds had good cell compatibility. Dead/live cell staining showed that the scaffold could effectively maintain cell viability (>90%). Cytoskeleton staining showed that the scaffolds were benefit for cell adhesion and spreading. After 1 week of subcutaneous implantation of the scaffolds in rats, there was a mild inflammatory response, but no significant inflammatory response was observed after 3 weeks, and the scaffolds gradually degraded. Conclusion The novel tissue engineered meniscus scaffold constructed by low temperature deposition 3D printing technology has a graded macroporous-microporous microstructure and good cytocompatibility, which is conducive to cell adhesion and growth, laying the foundation for the in vivo research of tissue engineered meniscus scaffolds in the next step.

Citation： CHEN Mingxue, WU Jiang, YIN Han, SUI Xiang, LIU Shuyun, GUO Quanyi. Construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal printing technology. Chinese Journal of Reparative and Reconstructive Surgery, 2024, 38(6): 748-754. doi: 10.7507/1002-1892.202402063 Copy

1.	Holzer LA, Leithner A, Holzer G. Surgery versus physical therapy for meniscal tear and osteoarthritis. N Engl J Med, 2013, 369(7): 677. doi: 10.1056/NEJMc1307177.
2.	Jiang D, Zhang ZZ, Zhao F, et al. The radiated deep-frozen xenogenic meniscal tissue regenerated the total meniscus with chondroprotection. Sci Rep, 2018, 8(1): 9041. doi: 10.1038/s41598-018-27016-w.
3.	Barceló X, Eichholz K, Gonçalves I, et al. Bioprinting of scaled-up meniscal grafts by spatially patterning phenotypically distinct meniscus progenitor cells within melt electrowritten scaffolds. Biofabrication, 2023, 16(1). doi: 10.1088/1758-5090/ad0ab9.
4.	Li M, Yin H, Chen M, et al. STS loaded PCL-MECM based hydrogel hybrid scaffolds promote meniscal regeneration via modulating macrophage phenotype polarization. Biomater Sci, 2023, 11(8): 2759-2774.
5.	Chen M, Feng Z, Guo W, et al. PCL-MECM-based hydrogel hybrid scaffolds and meniscal fibrochondrocytes promote whole meniscus regeneration in a rabbit meniscectomy model. ACS Appl Mater Interfaces, 2019, 11(44): 41626-41639.
6.	Noh S, Jin YJ, Shin DI, et al. Selective extracellular matrix guided mesenchymal stem cell self-aggregate engineering for replication of meniscal zonal tissue gradient in a porcine meniscectomy model. Adv Healthc Mater, 2023, 12(27): e2301180. doi: 10.1002/adhm.202301180.
7.	Zhu M, Li W, Dong X, et al. In vivo engineered extracellular matrix scaffolds with instructive niches for oriented tissue regeneration. Nat Commun, 2019, 10(1): 4620. doi: 10.1038/s41467-019-12545-3.
8.	Kim MK, Jeong W, Lee SM, et al. Decellularized extracellular matrix-based bio-ink with enhanced 3D printability and mechanical properties. Biofabrication, 2020, 12(2): 025003. doi: 10.1088/1758-5090/ab5d80.
9.	Jian Z, Zhuang T, Qinyu T, et al. 3D bioprinting of a biomimetic meniscal scaffold for application in tissue engineering. Bioact Mater, 2020, 6(6): 1711-1726.
10.	Liu L, Xiong Z, Yan Y, et al. Porous morphology, porosity, mechanical properties of poly (alpha-hydroxy acid)-tricalcium phosphate composite scaffolds fabricated by low-temperature deposition. J Biomed Mater Res A, 2007, 82(3): 618-629.
11.	Sun T, Wang J, Huang H, et al. Low-temperature deposition manufacturing technology: a novel 3D printing method for bone scaffolds. Front Bioeng Biotechnol, 2023, 11: 1222102. doi: 10.3389/fbioe.2023.1222102.
12.	Chen M, Li Y, Liu S, et al. Hierarchical macro-microporous WPU-ECM scaffolds combined with microfracture promote in situ articular cartilage regeneration in rabbits. Bioact Mater, 2020, 6(7): 1932-1944.
13.	Sun X, Zhu Y, Yin HY, et al. Differentiation of adipose-derived stem cells into Schwann cell-like cells through intermittent induction: potential advantage of cellular transient memory function. Stem Cell Res Ther, 2018, 9(1): 133. doi: 10.1186/s13287-018-0884-3.
14.	Liu L, Xian Y, Wang W, et al. Meniscus-inspired self-lubricating and friction-responsive hydrogels for protecting articular cartilage and improving exercise. ACS Nano, 2023, 17(23): 24308-24319.
15.	Kwon H, Brown WE, Lee CA, et al. Surgical and tissue engineering strategies for articular cartilage and meniscus repair. Nat Rev Rheumatol, 2019, 15(9): 550-570.
16.	Makris EA, Hadidi P, Athanasiou KA. The knee meniscus: structure-function, pathophysiology, current repair techniques, and prospects for regeneration. Biomaterials, 2011, 32(30): 7411-7431.
17.	Liu W, Wang D, Huang J, et al. Low-temperature deposition manufacturing: A novel and promising rapid prototyping technology for the fabrication of tissue-engineered scaffold. Mater Sci Eng C Mater Biol Appl, 2017, 70(Pt 2): 976-982.
18.	Lian M, Sun B, Han Y, et al. A low-temperature-printed hierarchical porous sponge-like scaffold that promotes cell-material interaction and modulates paracrine activity of MSCs for vascularized bone regeneration. Biomaterials, 2021, 274: 120841. doi: 10.1016/j.biomaterials.2021.120841.
19.	Sun T, Meng C, Ding Q, et al. In situ bone regeneration with sequential delivery of aptamer and BMP2 from an ECM-based scaffold fabricated by cryogenic free-form extrusion. Bioact Mater, 2021, 6(11): 4163-4175.
20.	Li N, Guo R, Zhang ZJ. Bioink formulations for bone tissue regeneration. Front Bioeng Biotechnol, 2021, 9: 630488. doi: 10.3389/fbioe.2021.630488.
21.	Zhao L, Zhou Y, Zhang J, et al. Natural polymer-based hydrogels: from polymer to biomedical applications. Pharmaceutics, 2023, 15(10): 2514. doi: 10.3390/pharmaceutics15102514.
22.	Nam SY, Park SH. ECM based bioink for tissue mimetic 3D bioprinting. Adv Exp Med Biol, 2018, 1064: 335-353.
23.	Jung CS, Kim BK, Lee J, et al. Development of printable natural cartilage matrix bioink for 3D printing of irregular tissue shape. Tissue Eng Regen Med, 2017, 15(2): 155-162.
24.	陈明学, 郭维民, 沈师, 等. 半月板细胞外基质-海藻酸水凝胶的制备及其对半月板细胞的影响. 中国医药生物技术, 2017, 12(6): 505-512.
25.	Dai P, Zou T, Zhao W, et al. Short-term transplantation effect of a tissue-engineered meniscus constructed using drilled allogeneic acellular meniscus and BMSCs. Front Vet Sci, 2023, 10: 1266018. doi: 10.3389/fvets.2023.1266018.
26.	Yuan Z, Liu S, Hao C, et al. AMECM/DCB scaffold prompts successful total meniscus reconstruction in a rabbit total meniscectomy model. Biomaterials, 2016, 111: 13-26.
27.	Zhou Y, Hu J, Li B, et al. Towards the clinical translation of 3D PLGA/β-TCP/Mg composite scaffold for cranial bone regeneration. Materials (Basel), 2024, 17(2): 352. doi: 10.3390/ma17020352.

1. Holzer LA, Leithner A, Holzer G. Surgery versus physical therapy for meniscal tear and osteoarthritis. N Engl J Med, 2013, 369(7): 677. doi: 10.1056/NEJMc1307177.
2. Jiang D, Zhang ZZ, Zhao F, et al. The radiated deep-frozen xenogenic meniscal tissue regenerated the total meniscus with chondroprotection. Sci Rep, 2018, 8(1): 9041. doi: 10.1038/s41598-018-27016-w.
3. Barceló X, Eichholz K, Gonçalves I, et al. Bioprinting of scaled-up meniscal grafts by spatially patterning phenotypically distinct meniscus progenitor cells within melt electrowritten scaffolds. Biofabrication, 2023, 16(1). doi: 10.1088/1758-5090/ad0ab9.
4. Li M, Yin H, Chen M, et al. STS loaded PCL-MECM based hydrogel hybrid scaffolds promote meniscal regeneration via modulating macrophage phenotype polarization. Biomater Sci, 2023, 11(8): 2759-2774.
5. Chen M, Feng Z, Guo W, et al. PCL-MECM-based hydrogel hybrid scaffolds and meniscal fibrochondrocytes promote whole meniscus regeneration in a rabbit meniscectomy model. ACS Appl Mater Interfaces, 2019, 11(44): 41626-41639.
6. Noh S, Jin YJ, Shin DI, et al. Selective extracellular matrix guided mesenchymal stem cell self-aggregate engineering for replication of meniscal zonal tissue gradient in a porcine meniscectomy model. Adv Healthc Mater, 2023, 12(27): e2301180. doi: 10.1002/adhm.202301180.
7. Zhu M, Li W, Dong X, et al. In vivo engineered extracellular matrix scaffolds with instructive niches for oriented tissue regeneration. Nat Commun, 2019, 10(1): 4620. doi: 10.1038/s41467-019-12545-3.
8. Kim MK, Jeong W, Lee SM, et al. Decellularized extracellular matrix-based bio-ink with enhanced 3D printability and mechanical properties. Biofabrication, 2020, 12(2): 025003. doi: 10.1088/1758-5090/ab5d80.
9. Jian Z, Zhuang T, Qinyu T, et al. 3D bioprinting of a biomimetic meniscal scaffold for application in tissue engineering. Bioact Mater, 2020, 6(6): 1711-1726.
10. Liu L, Xiong Z, Yan Y, et al. Porous morphology, porosity, mechanical properties of poly (alpha-hydroxy acid)-tricalcium phosphate composite scaffolds fabricated by low-temperature deposition. J Biomed Mater Res A, 2007, 82(3): 618-629.
11. Sun T, Wang J, Huang H, et al. Low-temperature deposition manufacturing technology: a novel 3D printing method for bone scaffolds. Front Bioeng Biotechnol, 2023, 11: 1222102. doi: 10.3389/fbioe.2023.1222102.
12. Chen M, Li Y, Liu S, et al. Hierarchical macro-microporous WPU-ECM scaffolds combined with microfracture promote in situ articular cartilage regeneration in rabbits. Bioact Mater, 2020, 6(7): 1932-1944.
13. Sun X, Zhu Y, Yin HY, et al. Differentiation of adipose-derived stem cells into Schwann cell-like cells through intermittent induction: potential advantage of cellular transient memory function. Stem Cell Res Ther, 2018, 9(1): 133. doi: 10.1186/s13287-018-0884-3.
14. Liu L, Xian Y, Wang W, et al. Meniscus-inspired self-lubricating and friction-responsive hydrogels for protecting articular cartilage and improving exercise. ACS Nano, 2023, 17(23): 24308-24319.
15. Kwon H, Brown WE, Lee CA, et al. Surgical and tissue engineering strategies for articular cartilage and meniscus repair. Nat Rev Rheumatol, 2019, 15(9): 550-570.
16. Makris EA, Hadidi P, Athanasiou KA. The knee meniscus: structure-function, pathophysiology, current repair techniques, and prospects for regeneration. Biomaterials, 2011, 32(30): 7411-7431.
17. Liu W, Wang D, Huang J, et al. Low-temperature deposition manufacturing: A novel and promising rapid prototyping technology for the fabrication of tissue-engineered scaffold. Mater Sci Eng C Mater Biol Appl, 2017, 70(Pt 2): 976-982.
18. Lian M, Sun B, Han Y, et al. A low-temperature-printed hierarchical porous sponge-like scaffold that promotes cell-material interaction and modulates paracrine activity of MSCs for vascularized bone regeneration. Biomaterials, 2021, 274: 120841. doi: 10.1016/j.biomaterials.2021.120841.
19. Sun T, Meng C, Ding Q, et al. In situ bone regeneration with sequential delivery of aptamer and BMP2 from an ECM-based scaffold fabricated by cryogenic free-form extrusion. Bioact Mater, 2021, 6(11): 4163-4175.
20. Li N, Guo R, Zhang ZJ. Bioink formulations for bone tissue regeneration. Front Bioeng Biotechnol, 2021, 9: 630488. doi: 10.3389/fbioe.2021.630488.
21. Zhao L, Zhou Y, Zhang J, et al. Natural polymer-based hydrogels: from polymer to biomedical applications. Pharmaceutics, 2023, 15(10): 2514. doi: 10.3390/pharmaceutics15102514.
22. Nam SY, Park SH. ECM based bioink for tissue mimetic 3D bioprinting. Adv Exp Med Biol, 2018, 1064: 335-353.
23. Jung CS, Kim BK, Lee J, et al. Development of printable natural cartilage matrix bioink for 3D printing of irregular tissue shape. Tissue Eng Regen Med, 2017, 15(2): 155-162.
24. 陈明学, 郭维民, 沈师, 等. 半月板细胞外基质-海藻酸水凝胶的制备及其对半月板细胞的影响. 中国医药生物技术, 2017, 12(6): 505-512.
25. Dai P, Zou T, Zhao W, et al. Short-term transplantation effect of a tissue-engineered meniscus constructed using drilled allogeneic acellular meniscus and BMSCs. Front Vet Sci, 2023, 10: 1266018. doi: 10.3389/fvets.2023.1266018.
26. Yuan Z, Liu S, Hao C, et al. AMECM/DCB scaffold prompts successful total meniscus reconstruction in a rabbit total meniscectomy model. Biomaterials, 2016, 111: 13-26.
27. Zhou Y, Hu J, Li B, et al. Towards the clinical translation of 3D PLGA/β-TCP/Mg composite scaffold for cranial bone regeneration. Materials (Basel), 2024, 17(2): 352. doi: 10.3390/ma17020352.

Chinese Journal of Reparative and Reconstructive Surgery

Construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal printing technology

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