3D Printing Bone: A Novel Way to Study Bone Diseases In Vitro

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The development of new drugs and therapeutic treatments is a highly complex and elongated process beginning with cell culture studies which progress to animal models before the clinical trials are reached. On average, the development of a new type of medicine takes at least ten years with a likelihood of approval at 9.6%. These numbers can be improved by ensuring that the foundation for the drug is based on a clinically relevant system 1 . In this article, we will focus on how 3D printing bone can be a novel way to improve the drug discovery process for bone diseases by providing a way to evaluate them in vitro.

2D Cell Culture System For Bone Disease

Up until today, the preferred method for studying cellular responses, and molecular biology in general, has been the culturing of cell strands in plastic well plates (hereafter referred to as 2D cell culture systems). The reasoning for this is primarily that it is a highly reproducible and simple method, where the homogenous concentrations of growth factors and nutrients across the cell monolayer leads to a controlled proliferation. However, native cells are not usually found in monolayers and their morphology is therefore altered in response to 2D culturing and the lack of extracellular matrix orientation, heterogeneity and 3D tissue orientation 2–5. This leads to polarization of the cells and ultimately altered responses to stimuli as compared to in vivo conditions.  

3D Cell Culture System 2D Cell Culture System For Bone Disease

To overcome these challenges, we need a new cell culture system that allows the cells to keep their natural morphology as well as their natural orientation and interactions within an extracellular matrix 6–9. Such systems have been known since 1906 where the first 3D cell culture system was established and is continuously being improved and optimized to achieve a low-cost method for analyzing cells in their native state 5

 Despite being studied for more than a century, the development of reliable 3D cell culture systems is still in the early stages and facing difficulties with tissue-mimicking structures and lack of vasculature 10 , amongst others. Several 3D cell culture systems have been developed and used already with some of them being spheroid cultures, hydrogels, and different types of polymer-containing scaffolds 2,11.

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Spheroid cultures cover most of the 3D techniques in which mechanical support is not integrated and the cell orientation instead relies on a non-adherent surface to promote cell-to-cell attachment and clotting 2,12. Some of the advantages in this system is the improvement of proliferation and viability, secretion of cytokines, chemokines and angiogenic factors as well as expression of early stem cell transcription factors 12,13. Unfortunately, the spheroid system is limited in size as nutrient access to the core and the diffusion gradient is reduced in response to an increase in size 12

Hydrogels, as opposed to the spheroid cultures, can be adjusted in regard to biochemical and mechanical properties thereby providing a tissue-like structure using biopolymers. Here, however, problems occur as to control the matrix degradation in coordination with the cellular tissue formation. This could, perhaps, be corrected by using different types of scaffolds – either prefabricated or building block-based scaffolds 14,15. The prefabricated scaffolds provide an elastic matrix with micro-architectures and integrated cells, but is, sadly, quite rough on the cells and should therefore be improved in that regard 16–18

The development of our P3D Scaffold introduced the first-ever polymer-free, structural and porous 3D printed 3D cell culture system19. Our scaffolds allow cells to engage in the bone-like environment by maintaining their cell-to-cell and cell-to-extracellular matrix interactions ultimately enabling a tissue-like structure, cell proliferation, and extracellular matrix production. 

Can we teach old dogs new tricks? 

Despite the general consensus that the future within in vitro studies requires a shift from 2D cell culture systems towards 3D cell culture systems, the transition has proven to be difficult. One of the primary difficulties regarding the shift from 2D to 3D is that all laboratory analyses have been optimized towards usage in 2D cell culture systems. Taking on a new cell culture method, such as the 3D cell culture systems, therefore requires labor-intensive laboratory work in order to further optimize existing protocols or even developing new protocols in order to perform the same studies in 3D as in 2D.  

Another obstacle for the usage of 3D cell culture systems is that most of the reference values/levels for cells grown in vitro is based on the 2D systems. As several studies have shown that cells better retain their normal morphology and gene expression in 3D cell culture systems, we are forced to acknowledge that the current reference values might not be useful in relation to cells cultured in 3D. 

Clinical Studies Should Begin with Thorough, Reliable, and Reproducible In Vitro Studies 

Although researchers are reluctant to embrace the new 3D cell culture systems, we highly believe that a shift from 2D to 3D is needed in order to obtain more lifelike results and even more to reduce the gap between in vitro studies and animal trials, thereby reducing the usage of laboratory animals in the future.

 To achieve this, we have developed our P3D scaffolds which mimic in vivo bone conditions. The bio-ceramic scaffolds are made of beta-tricalcium phosphate which is the main mineral in human bones. The scaffolds are furthermore porous and thereby allow vascularization of the system as well as mimics a natural habitat for bacteria and cancer cells. As the scaffolds are 3D printed, we are able to adjust the scaffolds and tailor them towards specific research needs, such as osteoporosis disease models or biofilm formation studies. We are even able to utilize the 3D bioprinting method to mimic the different types of bones such as trabecular and cortical bone. Additionally, the scaffolds are suitable for subcutaneous implantation in experimental animals making them a good candidate for a more humane tumor model, as our scaffolds can be seeded with cancerous cells prior to implantation, thereby obtaining a tumor quicker, ultimately resulting in less distress for the animal. 

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Scanning Electron Microscopy images of the P3D Scaffold at different magnifications ranging from 40 X to 15,000 X magnification. Images acquired by Luiz Eduardo Carneiro Campos and his research group at Fluminense Federal University, Brazil.

We are currently conducting preliminary validation studies of the P3D scaffolds, which have shown a significant tendency of the scaffolds inducing a collagen-producing response in osteoblasts, quicker than the traditional 2D cell culture method. We found that the differentiation of mesenchymal stem cells to osteoblasts is as good, if not better, as the differentiation occurring in traditional 2D cell culture systems. 

In the future, Particle3D aims to further utilize our proprietary 3D bioprinting technology to develop customized models for different areas of research such as osteoporosis and biofilm formation around implants. We are currently developing a hybrid P3D scaffold with the incorporation of a collagen matrix. By combining a collagen matrix with our scaffold and utilizing our proprietary 3D bioprinting technique, we are able to mimic the structure of healthy and osteoporotic bone as well as the nuances in between. We are, furthermore, currently designing scaffolds optimized for biofilm studies to ameliorate the understanding of biofilm formation around implants. – Knowledge that is beneficial for the clinical system in general and, more specifically, the knowledge that will allow us to further enhance our P3D Bone solutions and improve the post-surgery period for patients. 

References:

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8. Mirabelli P, Coppola L, Salvatore M. Cancer Cell Lines Are Useful Model Systems for Medical Research. Cancers. 2019;11(8). doi:10.3390/CANCERS11081098

9. van Staveren WCG, Solís DYW, Hébrant A, Detours V, Dumont JE, Maenhaut C. Human cancer cell lines: Experimental models for cancer cells in situ? For cancer stem cells? Biochimica et biophysica acta. 2009;1795(2):92-103. doi:10.1016/J.BBCAN.2008.12.004

10. Bleijs M, Wetering M, Clevers H, Drost J. Xenograft and organoid model systems in cancer research. The EMBO journal. 2019;38(15). doi:10.15252/EMBJ.2019101654

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13. Zhang Q, Nguyen AL, Shi S, et al. Three-dimensional spheroid culture of human gingiva-derived mesenchymal stem cells enhances mitigation of chemotherapy-induced oral mucositis. Stem cells and development. 2012;21(6):937-947. doi:10.1089/SCD.2011.0252

14. Han LH, Tong X, Yang F. Photo-crosslinkable PEG-based microribbons for forming 3D macroporous scaffolds with decoupled niche properties. Advanced materials (Deerfield Beach, Fla). 2014;26(11):1757-1762. doi:10.1002/ADMA.201304805

15. Han LH, Conrad B, Chung MT, et al. Winner of the Young Investigator Award of the Society for Biomaterials at the 10th World Biomaterials Congress, May 17-22, 2016, Montreal QC, Canada: Microribbon-based hydrogels accelerate stem cell-based bone regeneration in a mouse critical-size cranial defect model. Journal of biomedical materials research Part A. 2016;104(6):1321-1331. doi:10.1002/JBM.A.35715

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19. Slots C, Jensen MB, Ditzel N, et al. Simple additive manufacturing of an osteoconductive ceramic using suspension melt extrusion. Dental Materials. 2017;33(2):198-208. doi:10.1016/J.DENTAL.2016.11.012

About the Author:

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Sif Sofie Dahl is the Business Developer at Particle3D. She holds a master’s degree in Biomedicine and has several years of experience within setup and usage of 3D cell culture systems for in vitro studies. Sif Sofie Dahl is interested in using 3D bioprinting techniques to develop and optimize in vitro studies to decrease the need for animal experiments and improve the clinical development of new pharmaceutical drugs.

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