Fabricating spatially functionalized 3D-printed scaffolds for osteochondral tissue engineering
Three-dimensional (3D) printing of biodegradable polymers has rapidly become a popular approach to create scaffolds for tissue engineering. This technique enables fabrication of complex architectures and layer-by-layer spatial control of multiple components with high resolution. The resulting scaffolds can also present distinct chemical groups or bioactive cues on the surface to guide cell behavior. However, surface functionalization often includes one or more post-fabrication processing steps, which typically produce biomaterials with homogeneously distributed chemistries that fail to mimic the biochemical organization found in native tissues. As an alternative, our laboratory developed a novel method that combines solvent-cast 3D printing with peptide-polymer conjugates to spatially present multiple biochemical cues in a single scaffold without requiring post-fabrication modification. Here, we describe a detailed, stepwise protocol to fabricate peptide-functionalized scaffolds and characterize their physical architecture and biochemical spatial organization. We used these 3D-printed scaffolds to direct human mesenchymal stem cell differentiation and osteochondral tissue formation by controlling the spatial presentation of cartilage-promoting and bone-promoting peptides. This protocol also describes how to seed scaffolds and evaluate matrix deposition driven by peptide organization.
1. Ansari S, Khorshidi S, Karkhaneh A (2019) Engineering of gradient osteochondral tissue: From nature to lab. Acta Biomater 87: 41–54.
2. Di Luca A, Van Blitterswijk C, Moroni L (2015) The osteochondral interface as a gradient tissue: From development to the fabrication of gradient scaffolds for regenerative medicine. Birth Defects Res Part C Embryo Today Rev 105: 34–52.
3. O’Brien FJ (2011) Biomaterials & scaffolds for tissue engineering. Mater Today 14: 88–95.
4. Rashidi H, Yang J, Shakesheff KM (2014) Surface engineering of synthetic polymer materials for tissue engineering and regenerative medicine applications. Biomater Sci 2: 1318–1331.
5. Stevens MM (2005) Exploring and Engineering the Cell Surface Interface. Science 310: 1135–1138.
6. Holmes B, Bulusu K, Plesniak M, Zhang LG (2016) A synergistic approach to the design, fabrication and evaluation of 3D printed micro and nano featured scaffolds for vascularized bone tissue repair. Nanotechnology 27: 064001.
7. Zhao Y, Tan K, Zhou Y, Ye Z, Tan WS (2016) A combinatorial variation in surface chemistry and pore size of three-dimensional porous poly(ε-caprolactone) scaffolds modulates the behaviors of mesenchymal stem cells. Mater Sci Eng C 59: 193–202.
8. Cai Y, Li J, Poh CK, Tan HC, San Thian E, Hsi Fuh JY, et al (2013) Collagen grafted 3D polycaprolactone scaffolds for enhanced cartilage regeneration. J Mater Chem B 1: 5971.
9. Sun X-Y, Shankar R, Börner HG, Ghosh TK, Spontak RJ (2007) Field-Driven Biofunctionalization of Polymer Fiber Surfaces during Electrospinning. Adv Mater 19: 87–91.
10. Gentsch R, Pippig F, Schmidt S, Cernoch P, Polleux J, Börner HG (2011) Single-Step Electrospinning to Bioactive Polymer Nanofibers. Macromolecules 44: 453–461.
11. Li C, Ouyang L, Armstrong JPK, Stevens MM (2021) Advances in the Fabrication of Biomaterials for Gradient Tissue Engineering. Trends Biotechnol 39:150-164.
12. Calejo I, Costa-Almeida R, Reis RL, Gomes ME (2020) A Physiology-Inspired Multifactorial Toolbox in Soft-to-Hard Musculoskeletal Interface Tissue Engineering. Trends Biotechnol 38: 83–98.
13. Khorshidi S, Karkhaneh A (2018) A review on gradient hydrogel/fiber scaffolds for osteochondral regeneration. J Tissue Eng Regen Med 2: e1974–e1990.
14. Camacho P, Busari H, Seims KB, Schwarzenberg P, Dailey HL, Chow LW (2019) 3D printing with peptide–polymer conjugates for single-step fabrication of spatially functionalized scaffolds. Biomater Sci 7: 4237–4247.
15. Chow LW (2018) Electrospinning Functionalized Polymers for Use as Tissue Engineering Scaffolds. In: Chawla K, editor. Biomaterials for Tissue Engineering Methods in Molecular Biology, vol 1758. New York, NY: Humana Press. pp. 27–39.
16. Harrison RH, Steele JAM, Chapman R, Gormley AJ, Chow LW, Mahat MM, Muzamir M, Podhorska L, Palgrave RG, Payne DJ, Hettiaratchy SP, Dunlop IE, Stevens MM (2015) Modular and Versatile Spatial Functionalization of Tissue Engineering Scaffolds through Fiber-Initiated Controlled Radical Polymerization. Adv Funct Mater 25: 5748–5757.
17. Chow LW, Armgarth A, St-Pierre J-P, Bertazzo S, Gentilini C, Aurisicchio C, McCullen SD, Steele JAM, Stevens MM (2014) Peptide-Directed Spatial Organization of Biomolecules in Dynamic Gradient Scaffolds. Adv Healthc Mater 3: 1381–1386.
18. Guo S-Z, Gosselin F, Guerin N, Lanouette A-M, Heuzey M-C, Therriault D (2013) Solvent-Cast Three-Dimensional Printing of Multifunctional Microsystems. Small 9: 4118–4122.
19. Parmar PA, St-Pierre J-P, Chow LW, Puetzer JL, Stoichevska V, Peng YY, Werkmeister JA, Ramshaw JAM, Stevens MM (2016) Harnessing the Versatility of Bacterial Collagen to Improve the Chondrogenic Potential of Porous Collagen Scaffolds. Adv Healthc Mater 5: 1656–1666.
20. Karaman O, Kumar A, Moeinzadeh S, He X, Cui T, Jabbari E (2016) Effect of surface modification of nanofibres with glutamic acid peptide on calcium phosphate nucleation and osteogenic differentiation of marrow stromal cells. J Tissue Eng Regen Med 10: E132–E146.