Dual-Crosslinking of Gelatin-Based Hydrogels: Promising Compositions for a 3D Printed Organotypic Bone Model
Abstract
:1. Introduction
2. Methods and Instrumentation
2.1. Materials
2.2. GelMA Synthesis
2.3. Degree of Functionalization
2.4. Hygrogel Ink Preparation
2.5. Rheology
2.6. Cell Culture
2.7. 3D Bioprinting
2.8. Printing Accuracy
2.9. Scaffold Characterization
2.9.1. Surface Morphology
2.9.2. Swelling and Degradation
2.9.3. Compression Testing
2.9.4. Cell Viability and Morphology
2.9.5. Osteogenic Differentiation ALP ELlSA
2.9.6. Histology
2.10. Statistics
3. Result and Discussion
3.1. Ink Formulation and Rheological Characteristics of the Alginate/Gelatin/GelMA Scaffold
3.2. Fabrication and Morphological Characteristics of 3D Bioprinted Scaffolds
3.3. Physical and Mechanical Characterization of Alginate/Gelatin/GelMA Scaffolds
3.4. Cell Viability and Morphology
3.5. MSC Osteogenesis and Formation of Mineralized ECM within 3D Bioprinted Scaffolds
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Zhang, J.; Wehrle, E.; Vetsch, J.R.; Paul, G.R.; Rubert, M.; Müller, R. Hydrogel-based 3D bioprinting: A comprehensive review on cell-laden hydrogels, bioink formulations, and future perspectives. Appl. Mater. Today 2020, 18, 100479. [Google Scholar] [CrossRef]
- Hutchings, G.; Moncrieff, L.; Dompe, C.; Janowicz, K.; Sibiak, R.; Bryja, A.; Jankowski, M.; Mozdziak, P.; Bukowska, D.; Antosik, P.; et al. Bone Regeneration, Reconstruction and Use of Osteogenic Cells; from Basic Knowledge, Animal Models to Clinical Trials. J. Clin. Med. 2020, 9, 139. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bai, X.; Gao, M.; Syed, S.; Zhuang, J.; Xu, X.; Zhang, X.-Q. Bioactive hydrogels for bone regeneration. Bioact. Mater. 2018, 3, 401–417. [Google Scholar] [CrossRef] [PubMed]
- Habraken, W.; Habibovic, P.; Epple, M.; Bohner, M. Calcium phosphates in biomedical applications: Materials for the future? Mater. Today 2015, 19, 69–87. [Google Scholar] [CrossRef]
- Krakow, D. Osteogenesis Imperfecta. In Obstetric Imaging: Fetal Diagnosis and Care; Elsevier: Amsterdam, The Netherlands, 2018; pp. 270–273.e1. [Google Scholar] [CrossRef]
- Hubrecht, R.C.; Carter, E. The 3Rs and Humane Experimental Technique: Implementing Change. Animals 2019, 9, 754. [Google Scholar] [CrossRef] [Green Version]
- Bédard, P.; Gauvin, S.; Ferland, K.; Caneparo, C.; Pellerin, È.; Chabaud, S.; Bolduc, S. Innovative Human Three-Dimensional Tissue-Engineered Models as an Alternative to Animal Testing. Bioengineering 2020, 7, 115. [Google Scholar] [CrossRef]
- Liu, T.; Weng, W.; Zhang, Y.; Sun, X.; Yang, H. Applications of Gelatin Methacryloyl (GelMA) Hydrogels in Microfluidic Technique-Assisted Tissue Engineering. Molecules 2020, 25, 5305. [Google Scholar] [CrossRef]
- Kalaskar, D.M. 3D Printing in Medicine, 2nd ed.; Woodhead Publishing: Sawston, UK, 2022; Available online: https://www.elsevier.com/books/3d-printing-in-medicine/kalaskar/978-0-323-89831-7 (accessed on 10 May 2023).
- Hospodiuk, M.; Dey, M.; Sosnoski, D.; Ozbolat, I.T. The bioink: A comprehensive review on bioprintable materials. Biotechnol. Adv. 2017, 35, 217–239. [Google Scholar] [CrossRef] [Green Version]
- Zhu, W.; Ma, X.; Gou, M.; Mei, D.; Zhang, K.; Chen, S. 3D printing of functional biomaterials for tissue engineering. Curr. Opin. Biotechnol. 2016, 40, 103–112. [Google Scholar] [CrossRef] [Green Version]
- Lu, J.; Chen, Y.; Ding, M.; Fan, X.; Hu, J.; Chen, Y.; Li, J.; Li, Z.; Liu, W. A 4arm-PEG macromolecule crosslinked chitosan hydrogels as antibacterial wound dressing. Carbohydr. Polym. 2021, 277, 118871. [Google Scholar] [CrossRef]
- Kim, W.; Kim, G. Collagen/bioceramic-based composite bioink to fabricate a porous 3D hASCs-laden structure for bone tissue regeneration. Biofabrication 2019, 12, 015007. [Google Scholar] [CrossRef] [PubMed]
- Chen, Y.-W.; Shen, Y.-F.; Ho, C.-C.; Yu, J.; Wu, Y.-H.A.; Wang, K.; Shih, C.-T.; Shie, M.-Y. Osteogenic and angiogenic potentials of the cell-laden hydrogel/mussel-inspired calcium silicate complex hierarchical porous scaffold fabricated by 3D bioprinting. Mater. Sci. Eng. C 2018, 91, 679–687. [Google Scholar] [CrossRef] [PubMed]
- Malda, J.; Visser, J.; Melchels, F.P.; Jüngst, T.; Hennink, W.E.; Dhert, W.J.A.; Groll, J.; Hutmacher, D.W. 25th Anniversary Article: Engineering Hydrogels for Biofabrication. Adv. Mater. 2013, 25, 5011–5028. [Google Scholar] [CrossRef] [PubMed]
- Peng, H.; Poovaiah, N.; Forrester, M.; Cochran, E.; Wang, Q. Ex Vivo Culture of Primary Intestinal Stem Cells in Collagen Gels and Foams. ACS Biomater. Sci. Eng. 2014, 1, 37–42. [Google Scholar] [CrossRef]
- Ahmed, T.; Dare, E.V.; Hincke, M. Fibrin: A Versatile Scaffold for Tissue Engineering Applications. Tissue Eng. Part B Rev. 2008, 14, 199–215. [Google Scholar] [CrossRef]
- Jeon, O.; Lee, Y.; Hinton, T.; Feinberg, A.; Alsberg, E. Cryopreserved cell-laden alginate microgel bioink for 3D bioprinting of living tissues. Mater. Today Chem. 2019, 12, 61–70. [Google Scholar] [CrossRef]
- Zhang, J.; Wehrle, E.; Vetsch, J.R.; Paul, G.R.; Rubert, M.; Mueller, R. Alginate dependent changes of physical properties in 3D bioprinted cell-laden porous scaffolds affect cell viability and cell morphology. Biomed. Mater. 2019, 14, 065009. [Google Scholar] [CrossRef]
- Gorski, J.P. Biomineralization of bone: A fresh view of the roles of non-collagenous proteins. Front. Biosci. 2011, 16, 2598–2621. [Google Scholar] [CrossRef] [Green Version]
- Xu, Y.; Zhang, F.; Zhai, W.; Cheng, S.; Li, J.; Wang, Y. Unraveling of Advances in 3D-Printed Polymer-Based Bone Scaffolds. Polymers 2022, 14, 566. [Google Scholar] [CrossRef]
- Lam, T.; Dehne, T.; Krüger, J.P.; Hondke, S.; Endres, M.; Thomas, A.; Lauster, R.; Sittinger, M.; Kloke, L. Photopolymerizable gelatin and hyaluronic acid for stereolithographic 3D bioprinting of tissue-engineered cartilage. J. Biomed. Mater. Res. Part B Appl. Biomater. 2019, 107, 2649–2657. [Google Scholar] [CrossRef] [Green Version]
- Singh, Y.P.; Bandyopadhyay, A.; Mandal, B.B. 3D Bioprinting Using Cross-Linker-Free Silk–Gelatin Bioink for Cartilage Tissue Engineering. ACS Appl. Mater. Interfaces 2019, 11, 33684–33696. [Google Scholar] [CrossRef] [PubMed]
- Łabowska, M.B.; Cierluk, K.; Jankowska, A.M.; Kulbacka, J.; Detyna, J.; Michalak, I. A Review on the Adaption of Alginate-Gelatin Hydrogels for 3D Cultures and Bioprinting. Materials 2021, 14, 858. [Google Scholar] [CrossRef]
- Wu, Y.; Xiang, Y.; Fang, J.; Li, X.; Lin, Z.; Dai, G.; Yin, J.; Wei, P.; Zhang, D. The influence of the stiffness of GelMA substrate on the outgrowth of PC12 cells. Biosci. Rep. 2019, 39, BSR20181748. [Google Scholar] [CrossRef] [Green Version]
- Lin, R.-Z.; Chen, Y.-C.; Moreno-Luna, R.; Khademhosseini, A.; Melero-Martin, J.M. Transdermal regulation of vascular network bioengineering using a photopolymerizable methacrylated gelatin hydrogel. Biomaterials 2013, 34, 6785–6796. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- LOuyang, L.; Highley, C.B.; Sun, W.; Burdick, J.A. A Generalizable Strategy for the 3D Bioprinting of Hydrogels from Nonviscous Photo-crosslinkable Inks. Adv. Mater. 2016, 29, 1604983. [Google Scholar] [CrossRef]
- Liu, W.; Heinrich, M.A.; Zhou, Y.; Akpek, A.; Hu, N.; Liu, X.; Guan, X.; Zhong, Z.; Jin, X.; Khademhosseini, A.; et al. Extrusion Bioprinting of Shear-Thinning Gelatin Methacryloyl Bioinks. Adv. Healthc. Mater. 2017, 6, 1601451. [Google Scholar] [CrossRef] [PubMed]
- Yin, J.; Yan, M.; Wang, Y.; Fu, J.; Suo, H. 3D Bioprinting of Low-Concentration Cell-Laden Gelatin Methacrylate (GelMA) Bioinks with a Two-Step Cross-linking Strategy. ACS Appl. Mater. Interfaces 2018, 10, 6849–6857. [Google Scholar] [CrossRef]
- Cidonio, G.; Alcala-Orozco, C.R.; Lim, K.S.; Glinka, M.; Mutreja, I.; Kim, Y.-H.; Dawson, J.I.; Woodfield, T.B.F.; Oreffo, R.O.C. Osteogenic and angiogenic tissue formation in high fidelity nanocomposite Laponite-gelatin bioinks. Biofabrication 2019, 11, 035027. [Google Scholar] [CrossRef]
- CLiu, C.; Yang, G.; Zhou, M.; Zhang, X.; Wu, X.; Wu, P.; Gu, X.; Jiang, X. Magnesium Ammonium Phosphate Composite Cell-Laden Hydrogel Promotes Osteogenesis and Angiogenesis In Vitro. ACS Omega 2021, 6, 9449–9459. [Google Scholar] [CrossRef]
- Goto, R.; Nishida, E.; Kobayashi, S.; Aino, M.; Ohno, T.; Iwamura, Y.; Kikuchi, T.; Hayashi, J.-I.; Yamamoto, G.; Asakura, M.; et al. Gelatin Methacryloyl–Riboflavin (GelMA–RF) Hydrogels for Bone Regeneration. Int. J. Mol. Sci. 2021, 22, 1635. [Google Scholar] [CrossRef]
- Tavares, M.T.; Gaspar, V.M.; Monteiro, M.V.; Farinha, J.P.S.; Baleizão, C.; Mano, J.F. GelMA/bioactive silica nanocomposite bioinks for stem cell osteogenic differentiation. Biofabrication 2021, 13, 035012. [Google Scholar] [CrossRef] [PubMed]
- Rastin, H.; Ormsby, R.T.; Atkins, G.J.; Losic, D. 3D Bioprinting of Methylcellulose/Gelatin-Methacryloyl (MC/GelMA) Bioink with High Shape Integrity. ACS Appl. Bio Mater. 2020, 3, 1815–1826. [Google Scholar] [CrossRef]
- Naficy, S.; Kawakami, S.; Sadegholvaad, S.; Wakisaka, M.; Spinks, G.M. Mechanical properties of interpenetrating polymer network hydrogels based on hybrid ionically and covalently crosslinked networks. J. Appl. Polym. Sci. 2013, 130, 2504–2513. [Google Scholar] [CrossRef]
- Anand, R.; Nimi, N.; Sivadas, V.P.; Lal, L.P.M.R.; Nair, P.D. Dual crosslinked pullulan–gelatin cryogel scaffold for chondrocyte-mediated cartilage repair: Synthesis, characterization and in vitro evaluation. Biomed. Mater. 2022, 17, 015001. [Google Scholar] [CrossRef] [PubMed]
- Hernández-González, A.C.; Téllez-Jurado, L.; Rodríguez-Lorenzo, L.M. Alginate hydrogels for bone tissue engineering, from injectables to bioprinting: A review. Carbohydr. Polym. 2019, 229, 115514. [Google Scholar] [CrossRef]
- Tabriz, A.G.; Hermida, M.A.; Leslie, N.R.; Shu, W. Three-dimensional bioprinting of complex cell laden alginate hydrogel structures. Biofabrication 2015, 7, 045012. [Google Scholar] [CrossRef]
- Pan, T.; Song, W.; Cao, X.; Wang, Y. 3D Bioplotting of Gelatin/Alginate Scaffolds for Tissue Engineering: Influence of Crosslinking Degree and Pore Architecture on Physicochemical Properties. J. Mater. Sci. Technol. 2016, 32, 889–900. [Google Scholar] [CrossRef]
- Claaßen, C.; Claaßen, M.H.; Truffault, V.; Sewald, L.; Tovar, G.E.M.; Borchers, K.; Southan, A. Quantification of Substitution of Gelatin Methacryloyl: Best Practice and Current Pitfalls. Biomacromolecules 2017, 19, 42–52. [Google Scholar] [CrossRef]
- Moazzam, M.; Shehzad, A.; Sultanova, D.; Mukasheva, F.; Trifonov, A.; Berillo, D.; Akilbekova, D. Macroporous 3D printed structures for regenerative medicine applications. Bioprinting 2022, 28, e00254. [Google Scholar] [CrossRef]
- Ma, Z.; He, H.; Deng, C.; Ren, Y.; Lu, D.; Li, W.; Sun, X.; Wang, W.; Zhang, Y.; Xu, Y.; et al. 3D bioprinting of proangiogenic constructs with induced immunomodulatory microenvironments through a dual cross-linking procedure using laponite incorporated bioink. Compos. Part B Eng. 2021, 229, 109399. [Google Scholar] [CrossRef]
- Gungor-Ozkerim, P.S.; Inci, I.; Zhang, Y.S.; Khademhosseini, A.; Dokmeci, M.R. Bioinks for 3D bioprinting: An overview. Biomater. Sci. 2018, 6, 915–946. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sun, X.; Ma, Z.; Zhao, X.; Jin, W.; Zhang, C.; Ma, J.; Qiang, L.; Wang, W.; Deng, Q.; Yang, H.; et al. Three-dimensional bioprinting of multicell-laden scaffolds containing bone morphogenic protein-4 for promoting M2 macrophage polarization and accelerating bone defect repair in diabetes mellitus. Bioact. Mater. 2020, 6, 757–769. [Google Scholar] [CrossRef] [PubMed]
- Yao, B.; Hu, T.; Cui, X.; Song, W.; Fu, X.; Huang, S. Enzymatically degradable alginate/gelatin bioink promotes cellular behavior and degradation in vitro and in vivo. Biofabrication 2019, 11, 045020. [Google Scholar] [CrossRef] [PubMed]
- West, E.R.; Xu, M.; Woodruff, T.; Shea, L.D. Physical properties of alginate hydrogels and their effects on in vitro follicle development. Biomaterials 2007, 28, 4439–4448. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chatterjee, K.; Lin-Gibson, S.; Wallace, W.E.; Parekh, S.H.; Lee, Y.J.; Cicerone, M.T.; Young, M.F.; Simon, C.G., Jr. The effect of 3D hydrogel scaffold modulus on osteoblast differentiation and mineralization revealed by combinatorial screening. Biomaterials 2010, 31, 5051–5062. [Google Scholar] [CrossRef] [Green Version]
- Jain, T.; Baker, H.B.; Gipsov, A.; Fisher, J.P.; Joy, A.; Kaplan, D.S.; Isayeva, I. Impact of cell density on the bioprinting of gelatin methacrylate (GelMA) bioinks. Bioprinting 2021, 22, e00131. [Google Scholar] [CrossRef]
- Khatiwala, C.B.; Peyton, S.; Putnam, A. Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells. Am. J. Physiol. Physiol. 2006, 290, C1640–C1650. [Google Scholar] [CrossRef]
Bioink Formulation | Alginate (mg/mL) | Gelatin (mg/mL) | GelMA (mg/mL) | PBS (mL) |
---|---|---|---|---|
I (1% Alg/4% Gel/5% GelMA) | 100 | 400 | 500 | 8 |
II (1% Alg/8% Gel/2.5% GelMA) | 100 | 800 | 250 | 8 |
III (1% Alg/2% Gel/10% GelMA) | 100 | 200 | 1000 | 8 |
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Shehzad, A.; Mukasheva, F.; Moazzam, M.; Sultanova, D.; Abdikhan, B.; Trifonov, A.; Akilbekova, D. Dual-Crosslinking of Gelatin-Based Hydrogels: Promising Compositions for a 3D Printed Organotypic Bone Model. Bioengineering 2023, 10, 704. https://doi.org/10.3390/bioengineering10060704
Shehzad A, Mukasheva F, Moazzam M, Sultanova D, Abdikhan B, Trifonov A, Akilbekova D. Dual-Crosslinking of Gelatin-Based Hydrogels: Promising Compositions for a 3D Printed Organotypic Bone Model. Bioengineering. 2023; 10(6):704. https://doi.org/10.3390/bioengineering10060704
Chicago/Turabian StyleShehzad, Ahmer, Fariza Mukasheva, Muhammad Moazzam, Dana Sultanova, Birzhan Abdikhan, Alexander Trifonov, and Dana Akilbekova. 2023. "Dual-Crosslinking of Gelatin-Based Hydrogels: Promising Compositions for a 3D Printed Organotypic Bone Model" Bioengineering 10, no. 6: 704. https://doi.org/10.3390/bioengineering10060704
APA StyleShehzad, A., Mukasheva, F., Moazzam, M., Sultanova, D., Abdikhan, B., Trifonov, A., & Akilbekova, D. (2023). Dual-Crosslinking of Gelatin-Based Hydrogels: Promising Compositions for a 3D Printed Organotypic Bone Model. Bioengineering, 10(6), 704. https://doi.org/10.3390/bioengineering10060704