3D Bioprinting of Biomimetic Bilayered Scaffold Consisting of Decellularized Extracellular Matrix and Silk Fibroin for Osteochondral Repair
Recently, three-dimensional (3D) bioprinting technology is becoming an appealing approach for osteochondral repair. However, it is challenging to develop a bilayered scaffold with anisotropic structural properties to mimic a native osteochondral tissue. Herein, we developed a bioink consisting of decellularized extracellular matrix and silk fibroin to print the bilayered scaffold. The bilayered scaffold mimics the natural osteochondral tissue by controlling the composition, mechanical properties, and growth factor release in each layer of the scaffold. The in vitro results show that each layer of scaffolds had a suitable mechanical strength and degradation rate. Furthermore, the scaffolds encapsulating transforming growth factor-beta (TGF-β) and bone morphogenetic protein-2 (BMP-2) can act as a controlled release system and promote directed differentiation of bone marrow-derived mesenchymal stem cells. Furthermore, the in vivo experiments suggested that the scaffolds loaded with growth factors promoted osteochondral regeneration in the rabbit knee joint model. Consequently, the biomimetic bilayered scaffold loaded with TGF-β and BMP-2 would be a promising strategy for osteochondral repair.
1. Lewis PB, McCarty LP 3rd, Kang RW, et al., 2006, Basic Science and Treatment Options for Articular Cartilage Injuries. J Orthop Sports Phys Ther, 36:717–27.
2. Kilian D, Ahlfeld T, Akkineni AR, et al., 2020, 3D Bioprinting of Osteochondral Tissue Substitutes in Vitro-Chondrogenesis in Multi-layered Mineralized Constructs. Sci Rep, 10:8277.
3. Hu J, Zou WZ, Li L, et al., 2020, Overexpressing Runx2 of BMSCs Improve the Repairment of Knee Cartilage Defects. Curr Gene Ther, 20:395–404.
4. Stefani RM, Barbosa S, Tan AR, et al., 2002, Pulsed Electromagnetic Fields Promote Repair of Focal Articular Cartilage Defects with Engineered Osteochondral Constructs. Biotechnol Bioeng, 117:1584–96.
5. Levingstone TJ, Ramesh A, Brady RY, et al., 2016, Cell-free Multi-layered Collagen-based Scaffolds Demonstrate Layer Specific Regeneration of Functional Osteochondral Tissue in Caprine Joints. Biomaterials, 87:69–81.
6. Khanarian NT, Haney NM, Burga RA, et al., 2012, A Functional Agarose-hydroxyapatite Scaffold for Osteochondral Interface Regeneration. Biomaterials, 33:5247–58.
7. Abdollahiyan P, Oroojalian F, Mokhtarzadeh A, et al., 2002, Hydrogel-Based 3D Bioprinting for Bone and Cartilage Tissue Engineering. Biotechnol J, 2020:e2000095.
8. Abasalizadeh F, Moghaddam SV, Alizadeh E, et al., 2020, Alginate-based Hydrogels as Drug Delivery Vehicles in Cancer Treatment and their Applications in Wound Dressing and 3D Bioprinting. J Biol Eng, 14:8.
9. Zuo Q, Cui W, Liu F, et al., 2016, Utilizing Tissue-engineered Cartilage or BMNC-PLGA Composites to Fill Empty Spaces during Autologous Osteochondral Mosaicplasty in Porcine Knees. J Tissue Eng Regen Med, 10:916–26.
10. Cui W, Wang Q, Chen G, et al., 2011, Repair of Articular Cartilage Defects with Tissue-engineered Osteochondral Composites in Pigs. J Biosci Bioeng, 111:493–500.
11. Qiao Z, Lian M, Han Y, et al., 2021, Bioinspired Stratified Electro written Fiber-reinforced Hydrogel Constructs with Layer-specific Induction Capacity for Functional Osteochondral Regeneration. Biomaterials, 266:120385.
12. Cao R, Zhan A, Ci Z, et al., 2021, A Biomimetic Biphasic Scaffold Consisting of Decellularized Cartilage and Decalcified Bone Matrixes for Osteochondral Defect Repair. Front Cell Dev Biol, 9:639006.
13. Ashammakhi N, Ahadian S, Xu C, et al., 2019, Bioinks and Bioprinting Technologies to Make Heterogeneous and Biomimetic Tissue Constructs. Mater Today Bio, 1:100008.
14. Antich C, de Vicente J, Jimenez G, et al., 2002, Bio-inspired Hydrogel Composed of Hyaluronic Acid and Alginate as a Potential Bioink for 3D Bioprinting of Articular Cartilage Engineering Constructs. Acta Biomater, 106:114–23.
15. Zhang W, Ling C, Zhang A, et al., 2020, An All-silk-derived Functional Nanosphere Matrix for Sequential Biomolecule Delivery and In Situ Osteochondral Regeneration. Bioact Mater, vol. 5, no. 4, pp. 832-843, 2020.
16. Ng WL, Chua CK, Shen YF, 2019, Print Me An Organ! Why We Are Not There Yet. Prog Polym Sci, 97:101145.
17. Liu N, Ye X, Yao B, et al., 2021, Advances in 3D Bioprinting Technology for Cardiac Tissue Engineering and Regeneration. Bioact Mater, 6:1388–401.
18. Osidak EO, Kozhukhov VI, Osidak MS, et al., 2020, Collagen as Bioink for Bioprinting: A Comprehensive Review. Int J Bioprint, 6:270.
19. Lee JM, Sing SL, Yeong WY, 2002, Bioprinting of Multimaterials with Computer-aided Design/Computer-aided Manufacturing. Int J Bioprint, 6:245.
20. Gantumur E, Nakahata M, Kojima M, et al., 2002, Extrusion-Based Bioprinting through Glucose-Mediated Enzymatic Hydrogelation. Int J Bioprint, 6:250.
21. Askari M, Naniz MA, Kouhi M, et al., 2020, Recent Progress in Extrusion 3D Bioprinting of Hydrogel Biomaterials for Tissue Regeneration: A Comprehensive Review with Focus on Advanced Fabrication Techniques. Biomater Sci, 9:535–573.
22. Li X, Liu B, Pei B, et al., 2020, Inkjet Bioprinting of Biomaterials. Chem Rev, 120:10793–833.
23. Ng WL, Lee JM, Zhou M, et al., 2002, Vat Polymerization based Bioprinting-process, Materials, Applications and Regulatory Challenges. Biofabrication, 12:022001.
24. Vurat MT, Ergun C, Elcin AE, et al., 2002, 3D Bioprinting of Tissue Models with Customized Bioinks. Adv Exp Med Biol, 1249:67–84.
25. Ning L, Mehta R, Cao C, et al., 2020, Embedded 3D Bioprinting of Gelatin Methacryloyl-Based Constructs with Highly Tunable Structural Fidelity. ACS Appl Mater Interfaces, 12:44563–577.
26. Bicho D, Ajami S, Liu C, et al., 2019, Peptide biofunctionalization of Biomaterials for Osteochondral Tissue Regeneration in Early Stage Osteoarthritis: Challenges and Opportunities. J Mater Chem B, 7:1027–44.
27. Gan D, Xu T, Xing W, et al., 2019, Mussel-inspired Dopamine Oligomer Intercalated tough and Resilient Gelatin Methacryloyl (GelMA) Hydrogels for Cartilage Regeneration. J Mater Chem B, 7:1716–25.
28. Mishbak HH, Cooper G, Bartolo PJ, 2019, Development and Characterization of a Photocurable Alginate Bioink for Three-dimensional Bioprinting. Int J Bioprint, 5:189.
29. Chang R, Nam J, Sun W, 2008, Effects of Dispensing Pressure and Nozzle Diameter on Cell Survival from Solid Freeform Fabrication-based Direct Cell Writing. Tissue Eng Part A, 14:41–8.
30. Pati F, Jang J, Ha DH, et al., 2014, Printing Three-dimensional Tissue Analogues with Decellularized Extracellular Matrix Bioink. Nat Commun, 5:3935.
31. Fedorovich NE, De Wijn JR, Verbout AJ, et al., 2008, Three-dimensional Fiber Deposition of Cell-laden, Viable, Patterned Constructs for Bone Tissue Printing. Tissue Eng Part A, 14:127–33.
32. Abaci A, Guvendiren M, 202, Designing Decellularized Extracellular Matrix-Based Bioinks for 3D Bioprinting. Adv Healthc Mater, 2020:e2000734.
33. Shin YJ, Shafranek RT, Tsui JH, et al., 3D Bioprinting of Mechanically Tuned Bioinks Derived from Cardiac Decellularized Extracellular Matrix. Acta Biomater, 1;119:75–88.
34. Lee H, Yang GH, Kim N, et al., 2018, Fabrication of Micro/Nanoporous Collagen/dECM/Silk-fibroin Biocomposite Scaffolds Using a Low Temperature 3D Printing Process for Bone Tissue Regeneration. Mater Sci Eng C Mater Biol Appl, 84:140–7.
35. Yang Q, Peng J, Guo Q, et al., 2008, A Cartilage ECM-derived 3-D Porous Acellular Matrix Scaffold for In Vivo Cartilage Tissue Engineering with PKH26-labeled Chondrogenic Bone Marrow-derived Mesenchymal Stem Cells. Biomaterials, 29:2378–87.
36. Jang J, Kim TG, Kim BS, et al., 2016, Tailoring Mechanical Properties of Decellularized Extracellular Matrix Bioink by Vitamin B2-Induced Photo-Crosslinking. Acta Biomaterialia, 33:88–95.
37. Zhang X, Liu Y, Luo C, et al., 2002, Crosslinker-free Silk/Decellularized Extracellular Matrix Porous Bioink for 3D Bioprinting-based Cartilage Tissue Engineering. Mater Eng C, 2020:111388.
38. Li Z, Zhang X, Yuan T, et al., 2020, Addition of Platelet-Rich Plasma to Silk Fibroin Hydrogel Bioprinting for Cartilage Regeneration. Tissue Eng Part A, 26:886–95.
39. Gupta S, Alrabaiah H, Christophe M, et al., 2020, Evaluation of Silk-based Bioink during Pre and Post 3D Bioprinting: A Review. J Biomed Mater Res B Appl Biomater, 109:279–93.
40. Almeida HV, Liu Y, Cunniffe GM, et al., 2014, Controlled Release of Transforming Growth Factor-beta3 from Cartilage-extra-cellular-matrix-derived Scaffolds to Promote Chondrogenesis of Human-joint-tissue-derived Stem Cells. Acta Biomater, 10:4400–9.
41. Almeida HV, Cunniffe GM, Vinardell T, et al., 2015, Coupling Freshly Isolated CD44(+) Infrapatellar Fat Pad-Derived Stromal Cells with a TGF-beta3 Eluting Cartilage ECM Derived Scaffold as a Single-Stage Strategy for Promoting Chondrogenesis. Adv Healthc Mater, 4:1043–53.
42. Dickman CT, Russo V, Thain K, et al., 2002, Functional Characterization of 3D Contractile Smooth Muscle Tissues Generated Using a Unique Microfluidic 3D Bioprinting Technology. FASEB J, 34:1652–64.
43. Stanco D, Boffito M, Bogni A, et al., 2002, 3D Bioprinting of Human Adipose-Derived Stem Cells and Their Tenogenic Differentiation in Clinical-Grade Medium. Int J Mol Sci, 21:8694.
44. Sawkins MJ, Bowen W, Dhadda P, et al., 2013, Hydrogels Derived from Demineralized and Decellularized Bone Extracellular Matrix. Acta Biomater, 9:7865–73.
45. Zhang X, Zhai C, Fei H, et al., 2018, Composite Silk-Extracellular Matrix Scaffolds for Enhanced Chondrogenesis of Mesenchymal Stem Cells. Tissue Eng Part C Methods, 24:645–58.
46. Chenjun Z, Qiang Z, Kai S, et al., 2002, Utilizing an Integrated Tri-layered Scaffold with Titanium-Mesh-Cage Base to Repair Cartilage Defects of Knee in Goat Model. Mater Des, 193:108766.
47. Zhai C, Fei H, Hu J, et al., 2018, Repair of Articular Osteochondral Defects Using an Integrated and Biomimetic Trilayered Scaffold. Tissue Eng Part A, 24:1680–92.
48. Crapo PM, Gilbert TW, Badylak SF, 2011, An Overview of Tissue and Whole Organ Decellularization Processes. Biomaterials, 32:3233–43.
49. Schacht K, Jungst T, Schweinlin M, et al., 2015, Biofabrication of Cell-loaded 3D Spider Silk Constructs. Angew Chem Int Ed Engl, 54:2816–20.
50. Ni T, Liu M, Zhang Y, et al., 2002, 3D Bioprinting of Bone Marrow Mesenchymal Stem Cell-Laden Silk Fibroin Double Network Scaffolds for Cartilage Tissue Repair. Bioconjug Chem, 31:1938–47.
51. Chawla S, Midha S, Sharma A, et al., 2018, Silk-Based Bioinks for 3D Bioprinting. Adv Healthc Mater, 7:e1701204.
52. Ding C, Qiao Z, Jiang W, et al., 2013, Regeneration of a Goat Femoral Head Using a Tissue-specific, Biphasic Scaffold Fabricated with CAD/CAM Technology. Biomaterials, 34:6706–16.
53. Cals FL, Hellingman CA, Koevoet W, et al., 2012, Effects of Transforming Growth Factor-beta Subtypes on In Vitro Cartilage Production and Mineralization of Human Bone Marrow Stromal-derived Mesenchymal Stem Cells. J Tissue Eng Regen Med, 6:68–76.
54. Freeman FE, Pitacco P, van Dommelen LH, et al., 2002, 3D Bioprinting Spatiotemporally Defined Patterns of Growth Factors to Tightly Control Tissue Regeneration. Sci Adv, 6:eabb5093.
55. Datta S, Rameshbabu AP, Bankoti K, et al., 2021, Decellularized Bone Matrix/Oleoyl Chitosan Derived Supramolecular Injectable Hydrogel Promotes Efficient Bone Integration. Mater Sci Eng C Mater Biol Appl, 119:111604.
56. Das S, Pati F, Chameettachal S, et al., 2013, Enhanced Redifferentiation of Chondrocytes on Microperiodic Silk/Gelatin Scaffolds: Toward Tailor-made Tissue Engineering. Biomacromolecules, 14:311–21.
57. Huh JE, Koh PS, Seo BK, et al., 2014, Mangiferin Reduces the Inhibition of Chondrogenic Differentiation by IL-1beta in Mesenchymal Stem Cells from Subchondral Bone and Targets Multiple Aspects of the Smad and SOX9 Pathways. Int J Mol Sci, 15:16025–42.