Characterization and assessment of new fibrillar collagen inks and bioinks for 3D printing and bioprinting
Collagen is a cornerstone protein for tissue engineering and 3D bioprinting due to its outstanding biocompatibility, low immunogenicity, and natural abundance in human tissues. Nonetheless, it still poses some important challenges, such as complicated and limited extraction processes, usually accompanied by batchto-batch reproducibility and influence of factors, such as temperature, pH, and ionic strength. In this work, we evaluated the suitability and performance of new, fibrillar type I collagen as standardized and reproducible collagen source for 3D printing and bioprinting. The acidic, native fibrous collagen formulation (5% w/w) performed remarkably during 3D printing, which was possible to print constructs of up to 27 layers without collapsing. On the other hand, the fibrous collagen mass has been modified to provide a fast, reliable, and easily neutralizable process. The neutralization with TRIS-HCl enabled the inclusion of cells without hindering printability. The cell-laden constructs were printed under mild conditions (50–80 kPa, pneumatic 3D printing), providing remarkable cellular viability (>90%) as well as a stable platform for cell growth and proliferation in vitro. Therefore, the native, type I collagen masses characterized in this work offer a reproducible and reliable source of collagen for 3D printing and bioprinting purposes.
1. Souza AG, Silva IBB, Campos-Fernandez E, et al., 2018, Comparative assay of 2D and 3D cell culture models: Proliferation, gene expression and anticancer drug response. Curr Pharm Des, 24(15):1689–1694. https://doi.org/10.2174/1381612824666180404152304
2. Zhang B, Gao L, Ma L, et al., 2019, 3D bioprinting: A novel avenue for manufacturing tissues and organs. Engineering, 5(4):777–794. https://doi.org/10.1016/J.ENG.2019.03.009
3. Ng WL, Chua CK, Shen YF, 2019, Print me an organ! Why we are not there yet. Prog Polym Sci, 97(101145):1–45. https://doi.org/10.1016/J.PROGPOLYMSCI.2019.101145
4. 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):1–23. https://doi.org/10.1016/J.MTBIO.2019.100008
5. ASTM International, 2021, ISO/ASTM 52900:2021(E). Additive manufacturing—General Principles—Fundamentals and Vocabulary. ASTM International, Geneva, 1–5.
6. Zhuang P, Ng WL, An J, et al., 2019, Layer-by-layer ultraviolet assisted extrusion-based (UAE) bioprinting of hydrogel constructs with high aspect ratio for soft tissue engineering applications. PLoS One, 14(6):e0216776. https://doi.org/10.1371/JOURNAL.PONE.0216776
7. Jiang T, Munguia-Lopez JG, Flores-Torres S, et al., 2019, Extrusion bioprinting of soft materials: An emerging technique for biological model fabrication. Appl Phys Rev, 6(1):011310. https://doi.org/10.1063/1.5059393
8. Li X, Liu B, Pei B, et al., 2020, Inkjet bioprinting of biomaterials. Chem Rev, 120(19):10793–10833. https://doi.org/10.1021/ACS.CHEMREV.0C00008/ASSET/ IMAGES/LARGE/CR0C00008_0028.JPEG
9. Ng WL, Huang X, Shkolnikov V, et al., 2022, Controlling droplet impact velocity and droplet volume: Key factors to achieving high cell viability in sub-nanoliter droplet-based bioprinting. Int J Bioprinting, 8(1):1–17. https://doi.org/10.18063/IJB.V8I1.424
10. Ng WL, Lee JM, Zhou M, et al., 2020, Vat polymerization-based bioprinting—Process, materials, applications and regulatory challenges. Biofabrication, 12(2):022001. https://doi.org/10.1088/1758-5090/AB6034
11. Li W, Mille LS, Robledo JA, et al., 2020, Recent advances in formulating and processing biomaterial inks for vat polymerization-based 3D printing. Adv Healthc Mater, 9(15):2000156. https://doi.org/10.1002/ADHM.202000156
12. Garcia-Villen F, Ruiz-Alonso S, Lafuente-Merchan M, et al., 2021, Clay minerals as bioink ingredients for 3D printing and 3D bioprinting: Application in tissue engineering and regenerative medicine. Pharmaceutics, 13(1806):1–46 [Online]. Available: https://pubmed.ncbi.nlm.nih.gov/34834221/
13. Heid S, Boccaccini AR, 2020, Advancing bioinks for 3D bioprinting using reactive fillers: A review. Acta Biomater, 113:1–22. https://doi.org/10.1016/j.actbio.2020.06.040
14. Schwab A, Levato R, D’Este M, et al., 2020, Printability and shape fidelity of bioinks in 3D bioprinting. Chem Rev, 120(19):11028–11055. https://doi.org/10.1021/acs.chemrev.0c00084
15. Groll J, Burdick JA, Cho DW, et al., 2019, A definition of bioinks and their distinction from biomaterial inks. Biofabrication, 11(1):013001. https://doi.org/10.1088/1758-5090/aaec52
16. Postlethwaite AE, Seyer JM, Kang AH, 1978, Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen-derived peptides. Proc Natl Acad Sci U S A, 75(2):871. https://doi.org/10.1073/PNAS.75.2.871
17. Min Lee J, Kang Qiang Suen S, Long Ng W, et al., 2021, Bioprinting of collagen: Considerations, potentials, and applications. Macromol Biosci, 21(1):2000280. https://doi.org/10.1002/MABI.202000280
18. Marques CF, Diogo GS, Pina S, et al., 2019, Collagen-based bioinks for hard tissue engineering applications: A comprehensive review. J Mater Sci Mater Med, 30(3):1–12. https://doi.org/10.1007/S10856-019-6234-X/TABLES/1
19. Damodaran S, Parkin KL, 2017, Philadelphia, Pennsylvania, United States. Fennema’s Food Chemistry, 5th edn, CRC Press, Philadelphia, Pennsylvania, United States.
20. Schrieber R, Gareis H, 2007, Redhill, SURRE, United Kingdom. Gelatine Handbook: Theory and Industrial Practice, Wiley-VCH, Redhill, SURRE, United Kingdom.
21. Schmidt MM, Dornelles RCP, Mello RO, et al., 2016, Collagen extraction process. Int Food Res J, 23(3):913–922. Accessed: July 14, 2022. [Online]. Available: https://www.cabdirect.org/cabdirect/abstract/20163160817
22. Suurs P, Barbut S, 2020, Collagen use for co-extruded sausage casings—A review. Trends Food Sci Technol, 102:91–101. https://doi.org/10.1016/J.TIFS.2020.06.011
23. Stepanovska J, Otahal M, Hanzalek K, et al., 2021, pH modification of high-concentrated collagen bioinks as a factor affecting cell viability, mechanical properties, and printability. Gels, 7(4):252. https://doi.org/10.3390/GELS7040252
24. Osidak EO, Kozhukhov VI, Osidak MS, et al., 2020, Collagen as bioink for bioprinting: A comprehensive review. Int J Bioprinting, 6(3):1–10. https://doi.org/10.18063/IJB.V6I3.270
25. Stuart K, Panitch A, 2008, Influence of chondroitin sulfate on collagen gel structure and mechanical properties at physiologically relevant levels. Biopolymers, 89(10):841–851. https://doi.org/10.1002/BIP.21024
26. Morozova S, Muthukumar M, 2018, Electrostatic effects in collagen fibril formation. J Chem Phys, 149(16):163333. https://doi.org/10.1063/1.5036526
27. Li Y, Qiao C, Shi L, et al., 2014, Viscosity of collagen solutions: Influence of concentration, temperature, adsorption, and role of intermolecular interactions. J Macromollecular Sci B, 53(5):893–901. https://doi.org/10.1080/00222348.2013.852059
28. Osidak EO, Karalkin PA, Osidak MS, et al., 2019, Viscoll collagen solution as a novel bioink for direct 3D bioprinting. J Mater Sci Mater Med, 30(3):1–12. https://doi.org/10.1007/S10856-019-6233-Y/FIGURES/6
29. Duan L, Li J, Li C, et al., 2013, Effects of NaCl on the rheological behavior of collagen solution. Korea-Australia Rheol J, 25(3):137–144. https://doi.org/10.1007/S13367-013-0014-9
30. Newman S, Cloıp M, Allain C, et al., 1997, Viscosity and elasticity during relevance to matrix-driven translocation. Biopolymers, 41:337–347. https://doi.org/10.1002/(SICI)1097-0282(199703)41:3
31. Shin YJ, Shafranek RT, Tsui JH, et al., 2021, 3D bioprinting of mechanically tuned bioinks derived from cardiac decellularized extracellular matrix. Acta Biomater, 119:75–88. https://doi.org/10.1016/j.actbio.2020.11.006
32. Gao T, Gillispie GJ, Copus JS, et al., 2018, Optimization of gelatin-alginate composite bioink printability using rheological parameters: A systematic approach. Biofabrication, 10(3):1–9. https://doi.org/10.1088/1758-5090/AACDC7
33. Lewis JL, Johnson SL, Oegema TR, 2004, Interfibrillar collagen bonding exists in matrix produced by chondrocytes in culture: Evidence by electron microscopy. Tissue Engineering. https://home.liebertpub.com/ten, 8(6):989–995. https://doi.org/10.1089/107632702320934083
34. Oechsle AM, Häupler M, Gibis M, et al., 2015, Modulation of the rheological properties and microstructure of collagen by addition of co-gelling proteins. Food Hydrocoll, 49:118– 126 [Online]. Available: https://www.sciencedirect.com/science/article/pii/ S0268005X15001265
35. Boularaoui S, Al Hussein G, Khan KA, et al., 2020, An overview of extrusion-based bioprinting with a focus on induced shear stress and its effect on cell viability. Bioprinting, 20:e00093. https://doi.org/10.1016/J.BPRINT.2020.E00093
36. Malekpour A, Chen X, 2022, Printability and cell viability in extrusion-based bioprinting from experimental, computational, and machine learning views. J Funct Biomater, 13(2):40. https://doi.org/10.3390/JFB13020040
37. Cunha JP, 2022, Tham (tromethamine injection). https://www.rxlist.com/tham-drug.htm (Accessed July 14, 2022).
38. Rhee S, Puetzer JL, Mason BN, et al., 2016, 3D bioprinting of spatially heterogeneous collagen constructs for cartilage tissue engineering. ACS Biomater Sci Eng, 2(10):1800–1805. https://doi.org/10.1021/ACSBIOMATERIALS.6B00288/ ASSET/IMAGES/LARGE/AB-2016-00288V_0007.JPEG
39. Diegelmann RF, Cohen IK, McCoy BJ, 1979, Growth kinetics and collagen synthesis of normal skin, normal scar and keloid fibroblasts in vitro. J Cell Physiol, 98(2):341–346. https://doi.org/10.1002/JCP.1040980210
40. Ueno H, Nakamura F, Murakami M, et al., 2001, Evaluation effects of chitosan for the extracellular matrix production by fibroblasts and the growth factors production by macrophages. Biomaterials, 22(15):2125–2130. https://doi.org/10.1016/S0142-9612(00)00401-4
41. Freundlich B, Bomalaski JS, Neilson E, et al., 1986, Regulation of fibroblast proliferation and collagen synthesis by cytokines. Immunol Today, 7(10):303–307. https://doi.org/10.1016/0167-5699(86)90067-8
42. Goldberg B, Green H, 1964, An analysis of collagen secretion by established mouse fibroblast lines. J Cell Biol, 22:227–258. Accessed: May 19, 2022. [Online]. Available: http://rupress.org/jcb/article-pdf/22/1/227/1401992/227.pdf
43. Kosir MA, Quinn CCV, Wang W, et al., 2000, Matrix glycosaminoglycans in the growth phase of fibroblasts: More of the story in wound healing. J Surg Res, 92(1):45–52. https://doi.org/10.1006/JSRE.2000.5840
44. Gold KA, Saha B, Rajeeva Pandian, NL, et al., 2021, 3D bioprinted multicellular vascular models. Adv Healthc Mater, 2101141:1–14. https://doi.org/10.1002/adhm.202101141
45. Tian X, Chen X, 2014, Effects of cell density on mechanical properties of alginate hydrogel tissue scaffolds. J Biomimetics Biomater Tissue Eng, 19:77–85. https://doi.org/10.4028/WWW.SCIENTIFIC.NET/ JBBTE.19.77
46. Buckley CT, Thorpe SD, O’Brien FJ, et al., 2009, The effect of concentration, thermal history and cell seeding density on the initial mechanical properties of agarose hydrogels. J Mech Behav Biomed Mater, 2(5):512–521. https://doi.org/10.1016/J.JMBBM.2008.12.007
47. Singh G, Chanda A, 2021, Mechanical properties of whole-body soft human tissues: A review. Biomed Mater, 16(6):062004. https://doi.org/10.1088/1748-605X/AC2B7A
48. Tang-Schomer MD, White JD, Tien LW, et al., 2014, Bioengineered functional brain-like cortical tissue. Proc Natl Acad Sci U S A, 111(38):13811–13816. ht tp s : / / d oi . o rg/10.1073/PNAS.1324214111/-/ DCSUPPLEMENTAL/PNAS.201324214SI.PDF
49. Engler AJ, Sen S, Sweeney HL, et al., 2006, Matrix elasticity directs stem cell lineage specification. Cell, 126(4):677–689. https://doi.org/10.1016/j.cell.2006.06.044
50. Schmitt T, Kajave N, Cai HH, et al., 2021, In vitro characterization of xeno-free clinically relevant human collagen and its applicability in cell-laden 3D bioprinting. J Biomater Appl, 35(8):912–923. https://doi.org/10.1177/0885328220959162
51. Stepanovska J, Supova M, Hanzalek K, et al., 2021, Collagen bioinks for bioprinting: A systematic review of hydrogel properties, bioprinting parameters, protocols, and bioprinted structure characteristics. Biomedicines, 9(9):1–30. https://doi.org/10.3390/BIOMEDICINES9091137
52. Chen C, Zhao ML, Zhang RK, et al., 2017, Collagen/heparin sulfate scaffolds fabricated by a 3D bioprinter improved mechanical properties and neurological function after spinal cord injury in rats. J Biomed Mater Res A, 105(5):1324–1332. https://doi.org/10.1002/JBM.A.36011
53. Antoine EE, Vlachos PP, Rylander MN, 2015, Tunable collagen I hydrogels for engineered physiological tissue micro-environments. PLoS One, 10(3):e0122500. https://doi.org/10.1371/JOURNAL.PONE.0122500