AccScience Publishing / IJB / Volume 10 / Issue 6 / DOI: 10.36922/ijb.4566
RESEARCH ARTICLE

Collagen bioinks redefined: Optimizing ionic strength and growth factor delivery for cartilage tissue engineering

Murad Redzheb1* Yordan Sbirkov2,3 Atanas Valev1 Vasil Dzharov3 Hristi Petrova1 Tatyana Damyanova2 Ani Georgieva1,4 Victoria Sarafian2,3
Show Less
1 MatriChem Ltd., Sofia, Bulgaria
2 Department of Medical Biology, Faculty of Medicine, Medical University of Plovdiv, Plovdiv, Bulgaria
3 Department of Molecular and Regenerative Medicine, Research Institute at Medical University of Plovdiv (RIMU-Plovdiv), Plovdiv, Bulgaria
4 Section Pathology, Institute of Experimental Morphology, Pathology and Anthropology with Museumn Academy of Sciences, Sofia, Bulgaria
IJB 2024, 10(6), 4566 https://doi.org/10.36922/ijb.4566
Submitted: 18 August 2024 | Accepted: 29 September 2024 | Published: 30 September 2024
© 2024 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract

Tissue engineering of hyaline cartilage for regenerative medicine and the treatment of osteoarthritis has advanced significantly over the past decade, driven by developments in 3D bioprinting and biomaterials science. Despite these advances, standardized biofabrication protocols approved for clinical applications remain elusive, underscoring the need for research into widely accessible, non-immunogenic, and biocompatible bioinks that support chondrogenesis. This study proposes a strategy to improve the gelation kinetics of collagen bioinks by fine-tuning their ionic strength and reports a highly efficient sequestration of TGF-β1 within them, alongside their compatibility with bioprinting live chondrocytes and adipose-derived stem cells for cartilage tissue engineering. By adjusting sodium chloride and phosphate-buffered saline (PBS) concentrations, we demonstrate that reduced ionic strengths accelerate gelation, facilitating high-fidelity bioprinting while supporting high cell viability and proliferation. Furthermore, at 1% collagen concentration, the hydrogel effectively immobilized TGF-β1, with less than 0.5% released over two weeks, indicating potent sequestration capability. Using adipose-derived mesenchymal stromal cells, histomorphological and transcriptomic analyses reveal that the presence of TGF-β1 significantly enhances chondrogenesis. These results underscore the neglected role of ionic strength in optimizing collagen ink properties for advanced bioprinting applications and highlight the potential of collagen hydrogels as effective carriers for sustained growth factor delivery, paving the way for successful cartilage tissue engineering strategies.

Graphical abstract
Keywords
Collagen
Bioink
Bioprinting
Cartilage
Chondrocytes
Gelation kinetics
TGF-β1
Funding
This work was supported by the Bulgarian National Innovation Fund (grant number: 13IF-02-9/07.12.2022).
Conflict of interest
The authors declared the following potential conflicts of interest concerning the research, authorship, and/or publication of this article: M.R. is the majority owner and Managing Director of MatriChem, Ltd., which markets the collagen used in this study. A.V., H.P., and A.G. are MatriChem employees. No conflicts of interest concerning the research, authorship, and/or publications exist for all other authors.
References
  1. Dhavalikar P, Lan Z, Kar R, Salhadar K, Gaharwar AK, Cosgriff-Hernandez E. Biomedical applications of additive manufacturing. In: Biomaterials Science. 4th ed. San Diego, USA: Elsevier; 2020:623-639. doi: 10.1016/B978-0-12-816137-1.00040-4
  2. Sagi I, Afratis NA. Collagen. Vol 1944. In: Sagi I, Afratis NA, eds. New York, NY: Springer; 2019. doi: 10.1007/978-1-4939-9095-5
  3. Osidak EO, Kozhukhov VI, Osidak MS, Domogatsky SP. Collagen as bioink for bioprinting: a comprehensive review. Int J Bioprint. 2024;6(3):270. doi: 10.18063/ijb.v6i3.270
  4. Li Z, Ruan C, Niu X. Collagen-based bioinks for regenerative medicine: fabrication, application and prospective. Med Nov Technol Devices. 2023;17(37):100211. doi: 10.1016/j.medntd.2023.100211
  5. Stepanovska J, Supova M, Hanzalek K, Broz A, Matejka R. Collagen bioinks for bioprinting: a systematic review of hydrogel properties, bioprinting parameters, protocols, and bioprinted structure characteristics. Biomedicines. 2021;9(9):1137. doi: 10.3390/biomedicines9091137
  6. Marques CF, Diogo GS, Pina S, Oliveira JM, Silva TH, Reis RL. Collagen-based bioinks for hard tissue engineering applications: a comprehensive review. J Mater Sci Mater Med. 2019;30(3):32. doi: 10.1007/s10856-019-6234-x
  7. Li Y, Asadi A, Monroe MR, Douglas EP. pH effects on collagen fibrillogenesis in vitro: electrostatic interactions and phosphate binding. Mater Sci Eng C. 2009;29(5):1643-1649. doi: 10.1016/j.msec.2009.01.001
  8. Rezabeigi E, Griffanti G, Nazhat SN. Effect of fibrillization ph on gelation viscoelasticity and properties of biofabricated dense collagen matrices via gel aspiration-ejection. Int J Mol Sci. 2023;24(4):3889. doi: 10.3390/ijms24043889
  9. Kalbitzer L, Pompe T. Fibril growth kinetics link buffer conditions and topology of 3D collagen I networks. Acta Biomater. 2018;67:206-214. doi: 10.1016/j.actbio.2017.11.051
  10. Li Y, Douglas EP. Effects of various salts on structural polymorphism of reconstituted type I collagen fibrils. Colloids Surf B Biointerfaces. 2013;112:42-50. doi: 10.1016/j.colsurfb.2013.07.037
  11. Achilli M, Mantovani D. Tailoring mechanical properties of collagen-based scaffolds for vascular tissue engineering: the effects of pH, temperature and ionic strength on gelation. Polymers (Basel). 2010;2(4):664-680. doi: 10.3390/polym2040664
  12. Stamov DR, Stock E, Franz CM, Jähnke T, Haschke H. Imaging collagen type I fibrillogenesis with high spatiotemporal resolution. Ultramicroscopy. 2015;149:86-94. doi: 10.1016/j.ultramic.2014.10.003
  13. Gobeaux F, Mosser G, Anglo A, et al. Fibrillogenesis in dense collagen solutions: a physicochemical study. J Mol Biol. 2008;376(5):1509-1522. doi: 10.1016/j.jmb.2007.12.047
  14. Zhang J, Wei B, He L, et al. Systematic modulation of gelation dynamics of snakehead (Channa argus) skin collagen by environmental parameters. Macromol Res. 2017;25(11):1105-1114. doi: 10.1007/s13233-017-5149-y
  15. Harris JR, Reiber A. Influence of saline and pH on collagen type I fibrillogenesis in vitro: fibril polymorphism and colloidal gold labelling. Micron. 2007;38(5):513-521. doi: 10.1016/j.micron.2006.07.026
  16. Harris JR, Soliakov A, Lewis RJ. In vitro fibrillogenesis of collagen type I in varying ionic and pH conditions. Micron. 2013;49:60-68. doi: 10.1016/j.micron.2013.03.004
  17. Salinas-Fernandez S, Garcia O, Kelly DJ, Buckley CT. The influence of pH and salt concentration on the microstructure and mechanical properties of meniscus extracellular matrix-derived implants. J Biomed Mater Res A. 2024;112(3):359-372. doi: 10.1002/jbm.a.37634
  18. Zhu S, Yuan Q, Yin T, et al. Self-assembly of collagen-based biomaterials: preparation, characterizations and biomedical applications. J Mater Chem B. 2018;6(18):2650-2676. doi: 10.1039/c7tb02999c
  19. Cooke ME, Rosenzweig DH. The rheology of direct and suspended extrusion bioprinting. APL Bioeng. 2021;5(1):011502. doi: 10.1063/5.0031475
  20. Xing JY, Yang L, Li YL. Effect of anions on type I collagen fibrillogenesis in aqueous solution. ISWREP 2011 – Proc 2011 Int Symp Water Resour Environ Prot. 2011;4: 2975-2978. doi: 10.1109/ISWREP.2011.5893502
  21. Zhu J, Kaufman LJ. Collagen I self-assembly: revealing the developing structures that generate turbidity. Biophys J. 2014;106(8):1822-1831. doi: 10.1016/j.bpj.2014.03.011
  22. Diamantides N, Wang L, Pruiksma T, et al. Correlating rheological properties and printability of collagen bioinks: the effects of riboflavin photocrosslinking and pH. Biofabrication. 2017;9(3):034102. doi: 10.1088/1758-5090/aa780f
  23. Yang YL, Motte S, Kaufman LJ. Pore size variable type I collagen gels and their interaction with glioma cells. Biomaterials. 2010;31(21):5678-5688. doi: 10.1016/j.biomaterials.2010.03.039
  24. Lai G, Li Y, Li G. Effect of concentration and temperature on the rheological behavior of collagen solution. Int J Biol Macromol. 2008;42(3):285-291. doi: 10.1016/j.ijbiomac.2007.12.010
  25. Xie J, Bao M, Bruekers SMC, Huck WTS. Collagen gels with different fibrillar microarchitectures elicit different cellular responses. ACS Appl Mater Interfaces. 2017;9(23):19630-19637. doi: 10.1021/acsami.7b03883
  26. Tran-Ba KH, Lee DJ, Zhu J, Paeng K, Kaufman LJ. Confocal rheology probes the structure and mechanics of collagen through the sol-gel transition. Biophys J. 2017;113(8): 1882-1892. doi: 10.1016/j.bpj.2017.08.025
  27. Sarrigiannidis SO, Rey JM, Dobre O, González-García C, Dalby MJ, Salmeron-Sanchez M. A tough act to follow: collagen hydrogel modifications to improve mechanical and growth factor loading capabilities. Mater Today Bio. 2021;10(January):100098. doi: 10.1016/j.mtbio.2021.100098
  28. Goebel EJ, Hart KN, McCoy JC, Thompson TB. Structural biology of the TGFβ family. Exp Biol Med. 2019;244(17): 1530-1546. doi: 10.1177/1535370219880894
  29. Shah SS, Mithoefer K. Current applications of growth factors for knee cartilage repair and osteoarthritis treatment. Curr Rev Musculoskelet Med. 2020;13(6):641-650. doi: 10.1007/s12178-020-09664-6
  30. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res. 1998;238(1):265-272. doi: 10.1006/excr.1997.3858
  31. Kundu J, Shim JH, Jang J, Kim SW, Cho DW. An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering. J Tissue Eng Regen Med. 2015;9(11):1286-1297. doi: 10.1002/term.1682
  32. Zhu W, Cui H, Boualam B, et al. 3D bioprinting mesenchymal stem cell-laden construct with core–shell nanospheres for cartilage tissue engineering. Nanotechnology. 2018;29(18):185101. doi: 10.1088/1361-6528/aaafa1
  33. Hauptstein J, Forster L, Nadernezhad A, Groll J, Teßmar J, Blunk T. Tethered TGF-β1 in a hyaluronic acid-based bioink for bioprinting cartilaginous tissues. Int J Mol Sci. 2022;23(2):924. doi: 10.3390/ijms23020924
  34. Komsa-Penkova R, Stavreva G, Belemezova K, Kyurkchiev S, Todinova S, Altankov G. Mesenchymal stem-cell remodeling of adsorbed type-I collagen – the effect of collagen oxidation. Int J Mol Sci. 2022;23(6):3058. doi: 10.3390/ijms23063058
  35. Ge SX, Jung D, Jung D, Yao R. ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics. 2020;36(8):2628-2629. doi: 10.1093/bioinformatics/btz931
  36. Szklarczyk D, Kirsch R, Koutrouli M, et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023;51(1D):D638-D646. doi: 10.1093/nar/gkac1000
  37. Deyl Z, Mikšík I, Eckhardt A. Preparative procedures and purity assessment of collagen proteins. J Chromatogr B Anal Technol Biomed Life Sci. 2003;790(1-2):245-275. doi: 10.1016/S1570-0232(03)00158-2
  38. Drzewiecki KE, Grisham DR, Parmar AS, Nanda V, Shreiber DI. Circular dichroism spectroscopy of collagen fibrillogenesis: a new use for an old technique. Biophys J. 2016;111(11):2377-2386. doi: 10.1016/j.bpj.2016.10.023
  39. Mertz EL, Leikin S. Interactions of inorganic phosphate and sulfate anions with collagen. Biochemistry. 2004;43(47):14901-14912. doi: 10.1021/bi048788b
  40. Hayashi T, Nagai Y. Factors affecting the interactions of collagen molecules as observed by in vitro fibril formation: III. non-helical regions of the collagen molecules. J Biochem. 1974;76(1):177-186. doi: 10.1093/oxfordjournals.jbchem.a130543
  41. Weinstock A, King PC, Wuthier RE. The ion-binding characteristics of reconstituted collagen. Biochem J. 1967;104(3):705_b1-705_b1. doi: 10.1042/bj1040705_b1a
  42. Freudenberg U, Behrens SH, Welzel PB, et al. Electrostatic interactions modulate the conformation of collagen I. Biophys J. 2007;92(6):2108-2119. doi: 10.1529/biophysj.106.094284
  43. Oh S, Nguyen QD, Chung KH, Lee H. Bundling of collagen fibrils using sodium sulfate for biomimetic cell culturing. ACS Omega. 2020;5(7):3444-3452. doi: 10.1021/acsomega.9b03704
  44. Lien YH, Stern R, Fu JCC, Siegel RC. Inhibition of collagen fibril formation in vitro and subsequent cross-linking by glucose. Science. 1984;225(4669):1489-1491. doi: 10.1126/science.6147899
  45. Sbirkov Y, Molander D, Milet C, et al. A colorectal cancer 3D bioprinting workflow as a platform for disease modeling and chemotherapeuticsScreening. Front Bioeng Biotechnol. 2021;9(November):1-12. doi: 10.3389/fbioe.2021.755563
  46. Chaudhuri O, Cooper-White J, Janmey PA, Mooney DJ, Shenoy VB. Effects of extracellular matrix viscoelasticity on cellular behaviour. Nature. 2020;584(7822):535-546. doi: 10.1038/s41586-020-2612-2
  47. Schinagl RM, Gurskis D, Chen AC, Sah RL. Depth-dependent confined compression modulus of full-thickness bovine articular cartilage. J Orthop Res. 1997;15(4):499-506. doi: 10.1002/jor.1100150404
  48. Beketov EE, Isaeva EV, Yakovleva ND, et al. Bioprinting of cartilage with bioink based on high-concentration collagen and chondrocytes. Int J Mol Sci. 2021;22(21):11351. doi: 10.3390/ijms222111351
  49. Isaeva EV, Beketov EE, Demyashkin GA, et al. Cartilage formation in vivo using high concentration collagen-based bioink with MSC and decellularized ECM granules. Int J Mol Sci. 2022;23(5):2703. doi: 10.3390/ijms23052703
  50. Gospodinova A, Nankov V, Tomov S, Redzheb M, Petrov PD. Extrusion bioprinting of hydroxyethylcellulose-based bioink for cervical tumor model. Carbohydr Polym. 2021;260(February):117793. doi: 10.1016/j.carbpol.2021.117793
  51. Sbirkov Y, Redzheb M, Forraz N, McGuckin C, Sarafian V. High hopes for the biofabrication of articular cartilage – what lies beyond the horizon of tissue engineering and 3D bioprinting? Biomedicines. 2024;12(3):665. doi: 10.3390/biomedicines12030665
  52. Cui X, Gao G, Yonezawa T, Dai G. Human cartilage tissue fabrication using three-dimensional inkjet printing technology. J Vis Exp. 2014;(88):1-5. doi: 10.3791/51294
  53. Bourin P, Bunnell BA, Casteilla L, et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International So. Cytotherapy. 2013;15(6):641-648. doi: 10.1016/j.jcyt.2013.02.006
  54. Sang S, Mao X, Cao Y, et al. 3D bioprinting using synovium-derived MSC-laden photo-cross-linked ECM bioink for cartilage regeneration. ACS Appl Mater Interfaces. 2023;15(7):8895-8913. doi: 10.1021/acsami.2c19058
  55. Grafe I, Alexander S, Peterson JR, et al. TGF-β family signaling in mesenchymal differentiation. Cold Spring Harb Perspect Biol. 2018;10(5):1-50. doi: 10.1101/cshperspect.a022202
  56. Baba AB, Rah B, Bhat GR, et al. Transforming growth factor-beta (TGF-β) signaling in cancer-A betrayal within. Front Pharmacol. 2022;13(February):1-16. doi: 10.3389/fphar.2022.791272
  57. Stromps JP, Paul NE, Rath B, Nourbakhsh M, Bernhagen J, Pallua N. Chondrogenic differentiation of human adipose-derived stem cells: a new path in articular cartilage defect management? Biomed Res Int. 2014;2014:740926. doi: 10.1155/2014/740926

 

 

 



Share
Back to top
International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing