3D Bioprinting Vascular Tissue (CVD)

A literature review of 3D Bioprinting Vascular Tissue as a Therapy for Cardiovascular Disease-related Injury.

Cardiovascular Disease 

Cardiovascular disease (CVD) is an overarching term used to describe diseases related to the heart and blood vessels (Duan, 2016). Physiologically, CVD presents itself as a mechanical, electrical or functional change that constricts normal blood flow throughout the body. Example of these changes include build-up of plaque in a blood vessel (atherosclerosis), loss of electrical integrity (bradyarrhymia) or changes to systolic and diastolic blood pressure (isolated hypertension) (Monteiro et al., 2017; Lee et al., 2020). CVD is responsible for approximately 1/3 of all deaths, with the figure rising in both developing and developed countries (Deaton et al., 2011) When age-adjusting prevalence rates in the Australian population, CVD is generally decreasing amongst middle and older-aged individuals (Mathers et al., 2001). However, as the whole population grows the crude number of CVDrelated cases increases. This adds tremendous pressure onto the healthcare system to deal with the burden of disease, and negatively affects an individual’s quality of life (Pandya et al., 2013). Current treatment options depend on a person’s specific diagnosis. In general, blood thinning medication, bypass surgery and mechanical interventions such as stents are used. In many cases, these options can address the symptomatic and localised damage, however, these treatments are not perfect or sufficient enough as a long-term solution. Issues related to medication non-adherence and drug-drug interactions present efficacy and toxicity concerns (Stephenson, 2009; Addison et al., 2011). Problems with endothelial injury and organ tissue shortage presents surgical complications while thrombotic occlusion caused by newly employed stents is a long-term durability concern (Verrier & Boyle, 1996; Honda & Fitzgerald, 2003). These problems call for more personalised treatment options that limits the disease’s ability to harm, while respecting the body’s native, biological environment. As such, novel treatment options are anticipated to revolutionise how our society thinks about and handles CVD-related issues.

Tissue Engineering 

Regenerative medicine is an emerging field that recapitulates the physiochemical and biomechanical features of a native system so damaged tissue, vessels and organs are artificially grown for replacement surgery (Cui et al., 2018).This field distinguishes itself from medication treatments as it directly addresses the cause of symptomatic distress without causing whole body effects. This field also distinguishes itself from current surgical interventions as the engineering products are physiologically viable, as opposed to mechanical devices that are biomimetic. Three-dimensional (3D) bioprinting is a subdivision of this field that precisely manufactures a functional component for clinical or research purposes. Looking at Figure 1 and using tissue generation as an example, healthy cells are first harvested and then added to a media matrix that provides the required conditions for cell growth and structurally supports cell proliferation (Jana & Lerman, 2015; Zhang et al., 2016). This media is typically a hydrogel made from polyethylene glycol and sodium alginate as this mix is a printable material with the ability to withstand the mechanical strain tissues ordinarily face (Taylor et al., 2012). Using computer-aided design and a 3D bioprinter, the cell-hydrogel mix is laid down in a bottom-up, tiered manner so architectural fidelity between the printed tissue and native forms is preserved. The printed construct is then chemically cross-linked to ensure stability, which in the case of alginate solutions, occurs in the presence of Ca2+ ions (Dababneh & Ozbolat, 2014). The printed tissue is then analysed under static and dynamic conditions to ensure suitability for surgical implementation.

CVD & 3D Bioprinting 

In the case of CVD, coronary vessels, cardiac valves and myocardium tissue are clinically significant as most damage occurs to these regions (Wang et al., 2018). 3D bioprinting for these distinct parts is hugely beneficial as cell type specificity, spatial distribution and composition complexity can be replicated in accordance to the damaged region. Notable CVD injuries, and how they can be benefited by 3D bioprinting, include myocardial infarctions that have impacted the heart’s wall (Wang et al., 2018). Bioprinting allows for a controlled re-creation of the inner endocardium, middle myocardium or outer pericardium so functionality is restored to where it is lost. Contrasted to current treatments this approach can positively impact a patient’s quality of life as underlying scarring and cell lost is mitigated while injured tissue is directly replaced (Lovell & Mathur, 2004). Figure 2 presents the findings of a study that contrasted the differences between an engineered and native tissue. Optically, there is a difference in what the tissues look like but histologically they are very similar. Furthermore, this study found that extracellular components such as collagen and glycosaminoglycans (GAGs) were cultivated at similar percentages to that of native tissues. This is an important finding as it shows tissue-specific markers of cellular health can be attained through 3D printing mechanisms. Ultimately, findings like this contribute to the notion that bioprinting is a viable treatment option for CVD.

3D Bioprinting Limitations & Future Experiments 

1.1 Mechanical and Chemical Considerations 

When assessing the efficacy of any dispensing technology the effects of shear stress must be considered. Shear stress is the frictional force made by a moving substance against its surrounding environment (Malek, 1999). In the case of printing technologies, shear stress is influenced by nozzle diameter, printing pressure and viscosity of the cell/media mix (Blaeser et al., 2015). When bioprinting cardiovascular tissue, shear stress is an important mechanical problem to overcome. Firstly, vascular tissue generation is dependent on a high-density of multiple cell types that have differing shear thresholds for their unique cellular functions (Zhang et al., 2016). For example, shear stress on cardiac myocytes is important in cell alignment and vascular remodelling, whereas on endothelial cells it has anti-atherosclerotic effects (Shyu, 2009). Functionally, differences in shear sensitivity is related to where cells are proximal to blood flow. In healthy, in vivo conditions, any negative effects caused by shear are mitigated as cells are only exposed to a level they have evolved to tolerate. However, in bioprinting, the printed material is a conglomerate of all cell types needed by the construct which makes specifying shear stress thresholds difficult. This is a hard concern to overcome as higher viscosity and smaller nozzle diameter are useful parameters that provide greater printing precision but inadvertently increase the propensity for issues. These issues include cell signalling and protein expression defects (Blaeser et al., 2015).

Additionally, assessing the extent of shear stress damage is difficult as only short-term issues are detectable with long-term effects not immediately noticeable (Zhang et al., 2016). This raises questions surrounding the efficacy of 3D bioprinting as a therapy for CVD. This is as the engineered tissue may be impacted by fabrication methods and might not be suitable for individual’s whose native vasculature is also exhibiting high shear stress; like those with CVD caused by inflammation. In this case, systemic dysregulation reduces arterial stretch which will exacerbate any shear-induced problems to the engineered construct unless disease treatment also accounts for pharmacological interventions (Lu & Kassab, 2011; Cecchi et al., 2011). Superficially, this mechanical issue is solved by incorporating drug-interventions alongside implementation of these components. However, success in the development of hydrogels for 3D bioprinting has come at the cost of using toxic reactions for cross-linking and evidence of structures developing a necrotic core (Hong et al., 2015; Bejleri et al., 2018). This is a chemical consideration to drug-intervention for printed constructs as local shear stress might be addressed but unforeseeable drug interactions with the cross-linked hydrogels may also occur. Currently, there is no consensus on the issue as some studies demonstrate an effect between cross- linking and drug interactions, whereas others refute this claim (Lan & Starly, 2011; Paradee et al., 2012). This is a directive for future experiments that should evaluate common CVD drugs and their impact on bioprinted materials. It is imperative to get a better idea of whether an interaction exists, and if so, how it may affect treatment outcomes. 

1.2 Differences Across Bioprinters 

Differences across bioprinters results in variations in tissue generation. These differences exist in the form of manufacturing styles that uniquely layer the cell/gel mix (Cui et al., 2018). Figure 3 demonstrates three common 3D printers that can be used in cardiovascular tissue engineering and visually depicts the differences in their approaches. These differences may be beneficial as they allow for a nuanced approach to tissue printing and can be made specific to a patient’s condition. However, this also means lack of consistency across printers and requires careful consideration of which one is most suitable to use. Cui et al. (2018) presented an overview of these bioprinters describing the inkjet printer as low-cost but prone to clogging and requiring chemical cross-linking as ordinary mechanical cues are not present. These limitations are partially addressed by an extrusion printer that can print majority of biomaterials, limiting the need for chemical cross-linking. Conversely, this printer demonstrates low tissue viability due to use of high-shear stress and pressure. Lastly, stereolithography (laser-assisted bioprinting) is a photochemical process wherein cells are made highly cross-linkable by photopolymerization but are also prone to harmful downstream toxicity. These differences are clinically relevant to CVD as each printer-type needs to be evaluated against the benefit it brings to a patient versus the inherent constraints in its design. This is an issue as not every clinical setting will have access to all bioprinter types but if the construct is not suitable to the patient’s needs, CVD may be exacerbated or contribute to other disease-states. Future experiments should evaluate how these differences affect outcomes and model bioprinted tissue in various CVD-related injuries to determine which printer is most suitable to which conditions.

1.3 Risk Factors 

In many ways, CVD is exacerbated by lifestyle choices such as high salt/fat diets, physical inactivity and smoking (Scarborough et al., 2011). These behaviours increase an individual’s likelihood for disease due to risk factors like adiposity occurring (Mozaffarian et al., 2008). When assessing the interventive benefits of 3D printing it becomes obvious that bioprinted constructs are only a symptomatic treatment to damage in a localised area. Analogously, 3D bioprinting is like a band-aid to the factors perpetuating CVD which, if left untreated, may cause greater complications. Other risk factors such as oxidative stress and inflammation pose greater concerns as they are not directly related to pleiotropic lifestyle effects and require specific intervention (Oesterle et al., 2017). In this context, current research is progressively accumulating data on whether engineered tissues will survive in a pathological environment, however, recreating this in vitro is a considerable challenge (Cui et al., 2018). This is as there is a broad range and extent of factors that augment CVD. To understand these effects, current experiments are evaluating bioprinted tissue in the context of relevant bioactive agents such von Willebrand factor (Cui et al., 2016). However, these studies are rare and usually in relation to other bodily systems. 

Furthermore, little to no research has been conducted on the propensity of 3D printed tissues to later contribute to disease, such as form atherosclerotic plaques. This lapse in research might be explained by the newness of this technology and that majority of experiments are still validating proof-of-concepts. This is a directive for future experiments that could use a longitudinal survival analysis in mouse models to contrast Kaplan-Meir survival rates of common CVD treatments to 3D bioprinted grafts. Here, it is hypothesised that 3D bioprinted constructs will result in better outcomes only when combined with pharmacological interventions as it is necessary to control the local vasculature. Similarly, to assess how a perpetually pathological environment affects a 3D construct, a cross-sectional analysis where a gene like Apolipoprotein E (ApoE) is removed in mouse models and a printed tissue is implanted to assess how it reacts in that environment. After a certain time, the construct can be cross-sectioned to look for any markers of apoptosis, necrosis or inflammation. Here, it is hypothesised that the 3D construct will not be influenced by the diseased environment so long as it was grown from cells that had all necessary genes for survival. 

Conclusion 

Overall, 3D bioprinting presents a new way to address tissue and organ damage caused by CVD. This technology capitalises on engineering principles that allow for greater precision in construct manufacturing whilst replicating native tissue. This means that surgical intervention uses physiologically viable constructs as opposed to relying on drug-therapies or biomimetic metal stents. Before 3D bioprinting can be fully accepted, issues surrounding mechanical/chemical limitations, differences across printers and how the construct will react to CVD risk factors must be addressed. As our understanding of this technology gets better and there are solutions to these restraints, it is anticipated that 3D bioprinting will become a part of common practise as it has a lot of potential in remedying CVD-induced injury that current technologies fail to address. 
 

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References

Addison, C. C., Jenkins, B. W., Sarpong, D., Wilson, G., Champion, C., Sims, J., & White, M. S. (2011). Relationship between Medication Use and Cardiovascular Disease Health Outcomes in the Jackson Heart Study. International Journal of Environmental Research and Public Health, 8(6), 2505–2515. https://doi.org/10.3390/ijerph8062505 

Blaeser, A., Duarte Campos, D. F., Puster, U., Richtering, W., Stevens, M. M., & Fischer, H. (2015). Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity. Advanced Healthcare Materials, 5(3), 326–333. https://doi.org/10.1002/adhm.201500677 

Cecchi, E., Giglioli, C., Valente, S., Lazzeri, C., Gensini, G. F., Abbate, R., & Mannini, L. (2011). Role of hemodynamic shear stress in cardiovascular disease. Atherosclerosis, 214(2), 249– 256. https://doi.org/10.1016/j.atherosclerosis.2010.09.008 Cui, H., Miao, S., Esworthy, T., Zhou, X., Lee, S., 

Liu, C., Yu, Z., Fisher, J. P., Mohiuddin, M., & Zhang, L. G. (2018). 3D bioprinting for cardiovascular regeneration and pharmacology. Advanced Drug Delivery Reviews, 132, 252–269. https://doi.org/10.1016/j.addr.2018.07.014 Cui, H., Zhu, W., Nowicki, M., Zhou, X., Khademhosseini, 

A., & Zhang, L. G. (2016). Hierarchical Fabrication of Engineered Vascularized Bone Biphasic Constructs via Dual 3D Bioprinting: Integrating Regional Bioactive Factors into Architectural Design. Advanced Healthcare Materials, 5(17), 2174–2181. https://doi.org/10.1002/adhm.201600505 

Dababneh, A. B., & Ozbolat, I. T. (2014). Bioprinting Technology: A Current State-of-the-Art Review. Journal of Manufacturing Science and Engineering, 136(6). https://doi.org/10.1115/1.4028512

Deaton, C., Froelicher, E. Sivaraja., Wu, L. Ha., Ho, C., Shishani, K., & Jaarsma, T. (2011). The Global Burden of Cardiovascular Disease. European Journal of Cardiovascular Nursing, 10(2_suppl), S5–S13. https://doi.org/10.1016/s1474-5151(11)00111-3

Duan, B. (2016). State-of-the-Art Review of 3D Bioprinting for Cardiovascular Tissue Engineering. Annals of Biomedical Engineering, 45(1), 195–209. https://doi.org/10.1007/s10439-016-1607-5 

Frantz, C., Stewart, K. M., & Weaver, V. M. (2010). The extracellular matrix at a glance. Journal of Cell Science, 123(24), 4195–4200. https://doi.org/10.1242/jcs.023820 

Honda, Y., & Fitzgerald, P. J. (2003). Stent Thrombosis. Circulation, 108(1), 2–5. https://doi.org/10.1161/01.cir.0000075929.79964.d8 

Hong, S., Sycks, D., Chan, H. F., Lin, S., Lopez, G. P., Guilak, F., Leong, K. W., & Zhao, X. (2015). 3D Printing of Highly Stretchable and Tough Hydrogels into Complex, Cellularized Structures. Advanced Materials, 27(27), 4035–4040. https://doi.org/10.1002/adma.201501099 

Jana, S., & Lerman, A. (2015). Bioprinting a cardiac valve. Biotechnology Advances, 33(8), 1503– 1521. https://doi.org/10.1016/j.biotechadv.2015.07.006 

Lan, S.-F., & Starly, B. (2011). Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities. Toxicology and Applied Pharmacology, 256(1), 62–72. https://doi.org/10.1016/j.taap.2011.07.013 

Lee, H., Yano, Y., Cho, S. M. J., Park, J. H., Park, S., Lloyd-Jones, D. M., & Kim, H. C. (2020). Cardiovascular Risk of Isolated Systolic or Diastolic Hypertension in Young Adults. Circulation, 141(22), 1778–1786. https://doi.org/10.1161/circulationaha.119.044838 

Lovell, M. J., & Mathur, A. (2004). The role of stem cells for treatment of cardiovascular disease. Cell Proliferation, 37(1), 67–87. https://doi.org/10.1111/j.1365-2184.2004.00301.x 

Lu, D., & Kassab, G. S. (2011). Role of shear stress and stretch in vascular mechanobiology. Journal of The Royal Society Interface, 8(63), 1379–1385. https://doi.org/10.1098/rsif.2011.0177

Mathers, C. D., Vos, E. T., Stevenson, C. E., & Begg, S. J. (2001). The burden of disease and injury in Australia. Bulletin of the World Health Organization, 79, 1076–1084. https://doi.org/10.1590/S0042-96862001001100013 

Malek, A. M. (1999). Hemodynamic Shear Stress and Its Role in Atherosclerosis. JAMA, 282(21), 2035. https://doi.org/10.1001/jama.282.21.2035 Monteiro, L. M., Vasques-Nóvoa, F., Ferreira, L., Pinto-do-Ó, P., & Nascimento, D. S. (2017). Restoring heart function and electrical integrity: closing the circuit. Npj Regenerative Medicine, 2(1). https://doi.org/10.1038/s41536-017-0015-2 

Mozaffarian, D., Wilson, P. W. F., & Kannel, W. B. (2008). Beyond Established and Novel Risk Factors. Circulation, 117(23), 3031–3038. https://doi.org/10.1161/circulationaha.107.738732 

Oesterle, A., Laufs, U., & Liao, J. K. (2017). Pleiotropic Effects of Statins on the Cardiovascular System. Circulation Research, 120(1), 229–243. https://doi.org/10.1161/circresaha.116.308537 

Pandya, A., Gaziano, T. A., Weinstein, M. C., & Cutler, D. (2013). More Americans Living Longer With Cardiovascular Disease Will Increase Costs While Lowering Quality Of Life. Health Affairs, 32(10), 1706–1714. https://doi.org/10.1377/hlthaff.2013.0449

Paradee, N., Sirivat, A., Niamlang, S., & Prissanaroon-Ouajai, W. (2012). Effects of crosslinking ratio, model drugs, and electric field strength on electrically controlled release for alginate- 8 based hydrogel. Journal of Materials Science: Materials in Medicine, 23(4), 999–1010. https://doi.org/10.1007/s10856-012-4571-0

Pati, F., Jang, J., Ha, D.-H., Won Kim, S., Rhie, J.-W., Shim, J.-H., Kim, D.-H., & Cho, D.-W. (2014). Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink. Nature Communications, 5(1). https://doi.org/10.1038/ncomms4935 

Scarborough, P., Bhatnagar, P., Wickramasinghe, K. K., Allender, S., Foster, C., & Rayner, M. (2011). The economic burden of ill health due to diet, physical inactivity, smoking, alcohol and obesity in the UK: an update to 2006-07 NHS costs. Journal of Public Health, 33(4), 527–535. https://doi.org/10.1093/pubmed/fdr033 

Shyu, K.-G. (2009). Cellular and molecular effects of mechanical stretch on vascular cells and cardiac myocytes. Clinical Science, 116(5), 377–389. https://doi.org/10.1042/cs20080163 

Stephenson, P. H. (2009). Medications and the aging body: an overview of adverse drug reactions. Dspace.Library.Uvic.Ca. http://hdl.handle.net/1828/2148 

Taylor, D., O’Mara, N., Ryan, E., Takaza, M., & Simms, C. (2012). The fracture toughness of soft tissues. Journal of the Mechanical Behavior of Biomedical Materials, 6, 139–147. https://doi.org/10.1016/j.jmbbm.2011.09.018 

Verrier, E. D., & Boyle, E. M. (1996). Endothelial Cell Injury in Cardiovascular Surgery. The Annals of Thoracic Surgery, 62(3), 915–922. https://doi.org/10.1016/s0003-4975(96)00528-0 

Wang, Z., Lee, S. J., Cheng, H.-J., Yoo, J. J., & Atala, A. (2018). 3D bioprinted functional and contractile cardiac tissue constructs. Acta Biomaterialia, 70, 48–56. https://doi.org/10.1016/j.actbio.2018.02.007 

Zhang, Y. S., Yue, K., Aleman, J., Mollazadeh-Moghaddam, K., Bakht, S. M., Yang, J., Jia, W., Dell’Erba, V., Assawes, P., Shin, S. R., Dokmeci, M. R., Oklu, R., & Khademhosseini, A. (2016). 3D Bioprinting for Tissue and Organ Fabrication. Annals of Biomedical Engineering, 45(1), 148–163. https://doi.org/10.1007/s10439-016-1612-8