Development of 3D Printed Scaffolds Containing Decellularized Plants and Investigation of Their Basic Cell Interactions

Year 2024, Volume: 52 Issue: 6, 303 - 318, 12.12.2024

Sedat Odabaş , Melek İpek Ertuğrul , Fulya Özdemir , Zeliha Esra Çakmak , Süveydanas Çakıcı , Şükrü Kaan Konaklı , Melissa Kılıç

https://doi.org/10.15671/hjbc.1561887

Abstract

The decellularization process fundamentally removes the cellular content of the tissue (nuclear material and other nucleic acid components) without disrupting the structural integrity of the tissue. It is an effective approach, especially for obtaining three-dimensional (3D) biomaterials composed of the extracellular matrix (ECM), which provides tissue biomechanical support. In the literature, studies have shown that after the decellularization process, animal-derived decellularized tissues have been combined with various biopolymers to prepare composite scaffolds using different techniques. In recent years, due to their structural features, decellularization studies of plant-derived tissues have also gained prominence alongside animal tissues. In this study, succulent plants were chosen as the plant tissue, and the purpose was to prepare hybrid scaffolds by combining decellularized succulent tissues with alginate structures. The study aimed to investigate the fundamental cell-material interactions and cartilage-specific differentiation parameters using mesenchymal stem cells. Succulent plant leaves were decellularized using a solution containing Triton X-100 and SDS. The water-retaining parts were separated from other tissues, lyophilized, and turned into a powder. This approach was employed to preserve biomolecules with water-retaining capacity in powdered form. To determine the efficiency of the decellularization process, the quantities of DNA and proteins were assessed and compared. Due to their high water-absorbing capacity, the succulent plants' water-retaining structures were combined with alginate biopolymer at various viscosity levels to prepare an ink suitable for 3D printing. After printing, the resulting scaffolds' degradation and swelling behavior, chemical composition, structural characterization, and thermal properties were examined. In the final phase, a fundamental investigation was carried out on cell-material interactions using L929 mouse fibroblast cells and human mesenchymal stem cells on 3D printed scaffolds. The interactions within the prepared hybrid scaffolds were analyzed through basic cytotoxicity tests.

Keywords

Succulent plant, plant decellularization, biomaterials

Ethical Statement

The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript

Supporting Institution

TUBITAK

Project Number

120M986

Thanks

This article was dedicated in memory of Prof. Dr. Kadriye Tuzlakoğlu, who passed away in 2022.

Hücresizleştirilmiş Bitki İçeren 3B Baskılı Doku İskelelerinin Hazırlanması ve Temel Hücre Etkileşimlerinin İncelenmesi

Abstract

Hücresizleştirme işlemi, dokularda biyomekanik destek sağlayan ve hücre dışı matristen (ECM) oluşan 3B biyomalzemelerin elde edilmesinde kullanılmak üzere, dokudaki hücresel içeriğin dokunun yapısal bütünlüğünü bozmadan uzaklaştırılması işlemidir. Literatürde yapılan çalışmalar, hücresizleştirilmiş hayvan kaynaklı dokuların, çeşitli biyopolimerlerle farklı teknikler kullanılarak birleştirilerek kompozit doku iskeleleri hazırlandığını ortaya koymuştur. Son yıllarda yapısal özellikleri nedeniyle hayvan dokularının yanı sıra bitki kaynaklı dokuların da hücresizleştirme çalışmaları ön plana çıkmıştır. Bu çalışmada, hücresizleştirilmiş sukulent dokuları aljinat ile birleştirilerek hibrit doku iskeleleri hazırlanması amaçlanmıştır. Çalışmanın amacı mezenkimal kök hücreleri kullanılarak temel hücre-malzeme etkileşimlerini ve kıkırdak farklılaşma parametrelerini araştırmaktır. Kullanılan metoda göre, etli bitki yaprakları hücresizleştirilip su tutan kısımlar diğer dokulardan ayrıldı, liyofilize edildi ve toz haline getirildi. Bu yaklaşım, su tutma kapasitesine sahip biyomolekülleri toz halinde korumak için kullanıldı. Hücresizleştirme sürecinin verimliliğini belirlemek için DNA ve protein miktarları değerlendirilip karşılaştırıldı. Yüksek su tutma kapasiteleri nedeniyle etli bitkilerin su tutan yapıları, 3B baskıya uygun bir mürekkep hazırlamak için çeşitli konsantrasyonlarda aljinat biyopolimeri ile birleştirildi. Baskıdan sonra, elde edilen iskelelerin bozunma ve şişme davranışı, kimyasal bileşimi, yapısal karakterizasyonu ve termal özellikleri incelendi. Son aşamada, 3B yazdırılmış iskeleler üzerinde L929 fare fibroblast hücreleri ve insan mezenkimal kök hücreleri kullanılarak hücre-malzeme etkileşimi üzerine temel bir çalışma yürütüldü. Hazırlanan hibrit iskelelerinde hücre-malzeme etkileşimi temel sitotoksisite testleri kullanılarak incelenmiştir.

Keywords

Sukulent bitkisi, bitki hücresizleştirme işlemi, biyomalzemeler

Project Number

120M986

References

• 1. T. J. Keane, S. F. Badylak, Biomaterials for tissue engineering applications. In Seminars in Pediatric Surgery. WB Saunders, 23 (2014) 112- 118.
• 2. C. Migliaresi, A. Motta, Scaffolds for tissue engineering: Biological design, materials, and fabrication. CRC Press, 2014.
• 3. S. F. Badylak, B. N. Brown, T. W. Gilbert, Tissue engineering with decellularized tissues. In the book: Biomaterials Science: An Introduction to Materials. Elsevier, 2013.
• 4. S. F. Badylak, D. Taylor, K. Uygun, Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds. J Biomed Mater Res Part A 103(4) (2015) 1498–1508.
• 5. T. W. Gilbert, T. L. Sellaro, S. F. Badylak, Decellularization of tissues and organs. Biomaterials, 27(19) (2006) 3675-3683.
• 6. T. W. Gilbert, Strategies for tissue and organ decellularization. Journal of cellular biochemistry, 113(7) (2012) 2217-2222.
• 7. P. M. Crapo, T. W. Gilbert, S. F. Badylak, An overview of tissue and whole organ decellularization processes. Biomaterials, 32(12) (2011) 3233-3243.
• 8. J. R. Gershlak, S. Hernandez, G. Fontana, L. R. Perreault, K. J. Hansen, S. A. Larson, M. W. Rolle, Crossing kingdoms: using decellularized plants as perfusable tissue engineering scaffolds. Biomaterials, 125 (2017) 13-22.
• 9. G. Fontana, J. Gershlak, M. Adamski, J. S. Lee, S. Matsumoto, H. D. Le, W. L. Murphy, Biofunctionalized plants as diverse biomaterials for human cell culture. Advanced Healthcare Materials, 6(8) (2017) 1601225.
• 10. D. J. Modulevsky, C. Lefebvre, K. Haase, Z. Al-Rekabi, A. E. Pelling, Apple derived cellulose scaffolds for 3D mammalian cell culture. PloS one, 9(5) (2014) e97835.
• 11. D. J. Modulevsky, C. M. Cuerrier, A. E. Pelling, Biocompatibility of subcutaneously implanted plant-derived cellulose biomaterials. PloS one, 11(6) (2016) e0157894.
• 12. M. Toker-Bayraktar, M. İ. Ertugrul, S. Odabas, B. Garipcan, A typical method for decellularization of plants as biomaterials. MethodsX, 11, (2023) 102385.
• 13. M. Toker-Bayraktar, B. Erenay, B. Altun, S. Odabaş, B. Garipcan, Plant-derived biomaterials and scaffolds. Cellulose, 30(5) (2023) 2731-2751.
• 14. M. Thippan, M. Dhoolappa, K. T. Lakshmishree, P. Sheela, R. V. Prasad, Morphology of medicinal plant leaves for their functional vascularity: A novel approach for tissue engineering applications. IJCS, 7(3) (2019) 55-58.
• 15. H. Griffiths, J. Males, Succulent plants, Current biology: CB, 27 (2017) R890–R896.
• 16. L. I. Ahl, J. Mravec, B. Jørgensen, P. J. Rudall, N. Rønsted, O. M. Grace, Dynamics of intracellular mannan and cell wall folding in the drought responses of succulent Aloe species, Plant, cell & environment, 42 (2019) 2458-2471.
• 17. J. Jia, D. J. Richards, S. Pollard, Y. Tan, J. Rodriguez, R. P. Visconti, Y. Mei, Engineering alginate as bioink for bioprinting. Acta biomaterialia, 10(10) (2014) 4323-4331.
• 18. S. Naghieh, M. D. Sarker, E. Abelseth, X. Chen, Indirect 3D bioprinting and characterization of alginate scaffolds for potential nerve tissue engineering applications. Journal of the mechanical behavior of biomedical materials, 93 (2019)183-193.
• 19. R. Russo, M. Malinconico, G. Santagata, Effect of cross-linking with calcium ions on the physical properties of alginate films. Biomacromolecules, 8(10) (2007) 3193-3197.
• 20. S. Odabas, Functional Polysaccharides Blended Collagen Cryogels, Hacettepe J. Biol. & Chem., 46 1(2018) 113–120.
• 21. M. A. Godshall, Interference of plant polysaccharides and tannin in the Coomassie Blue G250 test for protein. Journal of Food Science, 48(4) (1983) 1346-1347.
• 22. S. P. Banik, S. Pal, S. Ghorai, S. Chowdhury, S. Khowala, Interference of sugars in the Coomassie Blue G dye-binding assay of proteins. Analytical biochemistry, 386(1) (2009) 113- 115.
• 23. C. C. Piras, D. K. Smith, Multicomponent polysaccharide alginate-based bioinks. Journal of Materials Chemistry B. 8 (2020) 8171-8188.
• 24. S. A. Wilson, L. M. Cross, C. W. Peak, A. K. Gaharwar, Shear-Thinning and Thermo- Reversible Nanoengineered Inks for 3D Bioprinting. ACS Appl Mater Interfaces. 20;9(50) (2017) 43449-43458.
• 25. P. Thara, Y. Beeran, V. Kokol, Effect of Nozzle Diameter and Cross-Linking on the Micro-Structure, Compressive, and Biodegradation Properties of 3D-Printed Gelatin/Collagen/Hydroxyapatite Hydrogel. Bioprinting, 31 (2023) e00266:9.
• 26. B. Abderrahim, E. Abderrahman, A. Mohamed, T. Fatima, T. Abdesselam, O. Krim, Kinetic thermal degradation of cellulose, polybutylene succinate, and a green composite: a comparative study. World J. Environ. Eng, 3(4) (2015) 95-110.
• 27. M. Aprilliza, Characterization, and properties of sodium alginate from brown algae used as an eco-friendly superabsorbent. In IOP conference series: materials science and engineering 188 (1) (2017) 012019.
• 28. ISO 10993-5 Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity, (2009).
• 29. E. Y. Abdul-Hafeez, M. A. Orabi, O. H. Ibrahim, O. Ilinskaya, N. S. Karamova, In vitro cytotoxic activity of certain succulent plants against human colon, breast and liver cancer cell lines. South African Journal of Botany, 131 (2020) 295-301.
• 30. L. H. Du Plessis, J. H. Hamman, In vitro evaluation of the cytotoxic and apoptogenic properties of aloe whole leaf and gel materials. Drug and Chemical Toxicology, 37(2) (2014) 169-177.
• 31. X. Guo, N. Mei, Aloe vera: A review of toxicity and adverse clinical effects. Journal of Environmental Science and Health, Part C, 34(2) (2016) 77-96.
• 32. M. Ayran, A. Y. Dirican, E. Saatcioglu, S. Ulag, A. Sahin, B. Aksu, A. Ficai, 3D-Printed PCL Scaffolds Combined with Juglone for Skin Tissue Engineering. Bioengineering, 9(9) (2022) 427.
• 33. S. Rahman, P. Carter, N. Bhattarai, Aloe Vera for Tissue Engineering Applications. J. Funct. Biomater, 8(1):6 (2017).
• 34. P. Jithendra, J. M. M. Mohamed, D. Annamalai, R. H. Al-Serwi, A. M. Ibrahim, M. El-Sherbiny, A. M. Rajam, M. Eldesoqui, N. Mansour, Biopolymer collagen-chitosan scaffold containing Aloe vera for chondrogenic efficacy on cartilage tissue engineering. Int J Biol Macromol, 248 (2023).