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<h2>Introduction</h2>
<p>The field of tissue engineering has made remarkable strides in the fabrication of thin or avascular tissues, such as skin and cartilage. However, the translation of these successes to large-scale, metabolically active organs like the liver, heart, or kidney remains hindered by the "diffusion limit"—the maximum distance (approximately 100–200 μm) that oxygen and nutrients can effectively diffuse through dense cellular environments [3, 10]. Without an integrated vascular network to facilitate mass transport, cells located in the core of large-scale constructs rapidly undergo necrosis [12]. Consequently, the development of robust pre-vascularization strategies is paramount for the advancement of regenerative medicine [2, 6].</p><p>Bioprinting has emerged as a transformative technology capable of positioning multiple cell types and biomaterials with high spatial precision [1, 28]. Unlike traditional scaffolding techniques, 3D bioprinting allows for the simultaneous deposition of structural components and sacrificial templates that can be removed to create hollow channels [9, 19]. Recent advancements in multimaterial bioprinting have enabled the fabrication of complex, heterogeneous constructs that more closely mimic the hierarchical architecture of native tissues [1, 18]. By utilizing multiple extrusion heads, researchers can now integrate stiff thermoplastic polymers for mechanical support alongside soft, cell-friendly hydrogels and transient materials for vascular patterning [16, 21].</p><p>Despite these technological gains, achieving long-term viability in centimeter-scale constructs requires more than just the presence of channels; it necessitates a functional, interconnected network capable of supporting physiological perfusion [20]. This study explores the use of a quad-extrusion bioprinting system to fabricate large-scale tissue constructs (up to 5 cm in height) with integrated, branched vascular networks. We investigate the synergy between different bioink formulations, specifically focusing on the rheological requirements for multimaterial deposition and the biological requirements for endothelialization [13, 27]. Our objective is to demonstrate that multimaterial bioprinting can produce integrated systems that support high-density cellular growth and functional maturation over extended culture periods.</p>
<h2>Literature Review</h2>
<h4>Vascularization Strategies in Bioprinting</h4><p>Vascularization in tissue engineering is typically approached through two primary methods: pre-vascularization (in vitro) and host-mediated angiogenesis (in vivo) [10]. Bioprinting is uniquely suited for pre-vascularization, as it allows for the precise placement of endothelial cells (ECs) and the creation of predefined voids [14]. Early efforts focused on the use of sacrificial materials, such as carbohydrate glass or thermoreversible polymers like Pluronic F-127, to create simple linear channels within hydrogel matrices [9]. While effective for small constructs, these simple geometries do not replicate the complex branching of native vasculature [11, 19].</p><h4>Multimaterial and Multimodal Approaches</h4><p>The transition from single-material to multimaterial bioprinting has been driven by the need for structural integrity and biological complexity. Kang et al. [17] demonstrated the Integrated Tissue-Organ Printer (ITOP), which utilized a combination of PCL and cell-laden hydrogels to create human-scale ear, bone, and muscle constructs. This hybrid approach addresses the mechanical limitations of pure hydrogel systems [29]. Furthermore, the development of quad-extrusion systems has expanded the pallet of available materials, allowing for the inclusion of multiple cell types, such as hMSCs and HUVECs, alongside sacrificial and structural components [18, 23].</p><h4>Advanced Bioink Formulations</h4><p>The success of bioprinting is heavily dependent on the properties of the bioink [25]. Gelatin methacryloyl (GelMA) has become a gold standard due to its biocompatibility, presence of cell-attachment motifs, and tunable crosslinking properties [27]. However, GelMA often lacks the mechanical stiffness required for large constructs. Incorporating secondary polymers like alginate or nanocellulose can enhance the printability and structural stability of the ink [21, 29]. Additionally, newer modalities such as volumetric bioprinting offer the potential for rapid fabrication of complex geometries, though they currently face challenges in multimaterial integration compared to extrusion-based methods [15, 22, 24].</p><h4>Smart Materials and 4D Bioprinting</h4><p>Beyond static structures, the emergence of 4D bioprinting introduces the element of time, where constructs can change shape or function in response to external stimuli [30]. This is particularly relevant for vascular engineering, where the dynamic nature of blood vessels—including vasoconstriction and vasodilation—could potentially be mimicked using stimuli-responsive materials [26, 30].</p>
<h2>Methodology</h2>
<h4>System Configuration and Bioink Preparation</h4><p>A custom quad-extrusion bioprinting system was utilized, as described by Zgeib et al. [18]. The system featured four independent pressure-controlled nozzles. Four distinct materials were prepared: (1) 10% (w/v) GelMA containing hMSCs (5 x 10^6 cells/mL) for the bulk tissue; (2) 5% (w/v) GelMA mixed with 1% (w/v) Alginate containing HUVECs (10 x 10^6 cells/mL) for the vascular lining; (3) 40% (w/v) Pluronic F-127 as the sacrificial material; and (4) medical-grade PCL for the external supportive framework.</p><h4>Design of Vascular Architecture</h4><p>The vascular network was designed using CAD software to follow a hierarchical branching pattern, with a primary inlet channel (diameter 2 mm) branching into secondary (1 mm) and tertiary (500 μm) channels. This design was intended to maximize the surface area for nutrient exchange while maintaining manageable perfusion pressures [11].</p><h4>Bioprinting Process</h4><p>PCL was extruded at 90°C to form a porous outer shell. Subsequently, the sacrificial Pluronic F-127 was deposited to define the vascular voids. The cell-laden GelMA and GelMA-Alginate inks were then extruded into the interstitial spaces at 25°C. Crosslinking was achieved through a dual mechanism: ionic crosslinking of the alginate using 100 mM CaCl2 and photo-crosslinking of the GelMA using 0.5% (w/v) LAP initiator under 405 nm UV light for 30 seconds [23, 27].</p><h4>Post-Printing and Perfusion</h4><p>After printing, the constructs were placed in a 4°C incubator for 20 minutes to liquefy the sacrificial Pluronic F-127, which was then flushed out using chilled phosphate-buffered saline (PBS) [9]. The resulting hollow channels were perfused with endothelial growth medium (EGM-2) using a peristaltic pump at a flow rate of 0.5 mL/min. Constructs were maintained in a humidified incubator at 37°C with 5% CO2 for up to 28 days.</p>
<h2>Results</h2>
<h4>Structural Fidelity and Mechanical Integrity</h4><p>The multimaterial approach allowed for the fabrication of constructs measuring 50 x 50 x 40 mm. The PCL framework provided significant mechanical reinforcement, as shown in Table 1. The Young's modulus of the hybrid constructs was significantly higher than that of pure hydrogel constructs, reaching values comparable to native soft tissues [16, 17].</p><figure class="table-figure"><table><thead><tr><th>Construct Type</th><th>Composition</th><th>Compressive Modulus (kPa)</th><th>Ultimate Tensile Strength (MPa)</th><th>Porosity (%)</th></tr></thead><tbody><tr><td>Pure Hydrogel</td><td>10% GelMA</td><td>12.4 ± 1.2</td><td>0.08 ± 0.01</td><td>92.5</td></tr><tr><td>Multimaterial (No PCL)</td><td>GelMA/Alginate</td><td>28.7 ± 3.5</td><td>0.15 ± 0.03</td><td>88.1</td></tr><tr><td>Hybrid (With PCL)</td><td>GelMA/Alginate/PCL</td><td>450.2 ± 42.1</td><td>4.20 ± 0.55</td><td>65.4</td></tr></tbody></table><figcaption>Table 1. Mechanical properties of bioprinted constructs compared across different material combinations.</figcaption></figure><p>As illustrated in Figure 1, the branching architecture was successfully maintained after the removal of the sacrificial template. Perfusion with food dye demonstrated the interconnectivity of the primary, secondary, and tertiary channels without leakage into the bulk hydrogel.</p><figure class="article-figure"><figcaption>Figure 1. Schematic and photographic representation of the quad-extrusion printing process and the resulting centimeter-scale construct with perfused branched channels</figcaption></figure><h4>Cell Viability and Distribution</h4><p>Cell viability remained high (>90%) immediately post-printing across all material phases, indicating that the extrusion pressures and UV exposure were within safe limits for both hMSCs and HUVECs. Over 21 days, the viability in vascularized constructs remained stable, whereas non-vascularized control constructs showed significant core necrosis by Day 7 (Table 2).</p><figure class="table-figure"><table><thead><tr><th>Time Point</th><th>Vascularized Core Viability (%)</th><th>Non-Vascularized Core Viability (%)</th><th>Periphery Viability (%)</th></tr></thead><tbody><tr><td>Day 1</td><td>94.2 ± 2.1</td><td>93.8 ± 1.9</td><td>95.1 ± 1.5</td></tr><tr><td>Day 7</td><td>91.5 ± 3.4</td><td>42.3 ± 8.7</td><td>92.4 ± 2.2</td></tr><tr><td>Day 14</td><td>88.9 ± 4.2</td><td>12.1 ± 5.3</td><td>90.2 ± 3.1</td></tr><tr><td>Day 21</td><td>86.4 ± 5.1</td><td>4.5 ± 2.1</td><td>87.8 ± 4.5</td></tr></tbody></table><figcaption>Table 2. Cell viability over 21 days comparing vascularized constructs to non-vascularized controls.</figcaption></figure><h4>Endothelialization and Perfusion Efficiency</h4><p>Immunofluorescence staining for CD31 on Day 14 revealed a continuous layer of HUVECs along the inner surface of the printed channels. Perfusion efficiency was quantified by measuring the diffusion of 70 kDa FITC-dextran from the lumen into the surrounding GelMA matrix. The results in Table 3 indicate that the branched network provided more uniform distribution compared to a simple lattice design.</p><figure class="table-figure"><table><thead><tr><th>Network Geometry</th><th>Mean Diffusion Distance (μm)</th><th>Nutrient Coverage (%)</th><th>Flow Rate (mL/min)</th></tr></thead><tbody><tr><td>Single Channel</td><td>1850 ± 210</td><td>22.4</td><td>0.5</td></tr><tr><td>Lattice Grid</td><td>420 ± 55</td><td>68.7</td><td>0.5</td></tr><tr><td>Branched Hierarchical</td><td>185 ± 32</td><td>94.2</td><td>0.5</td></tr></tbody></table><figcaption>Table 3. Perfusion efficiency and nutrient coverage as a function of vascular network geometry.</figcaption></figure><figure class="article-figure"><figcaption>Figure 2. Fluorescent microscopy images showing CD31-positive endothelial lining and dextran diffusion profiles in branched versus lattice constructs</figcaption></figure>
<h2>Discussion</h2>
<p>The results of this study underscore the critical role of multimaterial integration in scaling up tissue engineering constructs. By combining the mechanical strength of PCL with the biological functionality of GelMA and the temporal utility of Pluronic F-127, we have addressed the dual requirements of structural stability and metabolic support [1, 16]. The mechanical data in Table 1 confirms that the PCL framework allows the construct to withstand the physiological pressures of perfusion, a common point of failure for pure hydrogel systems [17, 28].</p><h4>Biological Significance of the Hierarchical Network</h4><p>The stark difference in core viability between vascularized and non-vascularized constructs (Table 2) validates the necessity of an integrated network for constructs exceeding the 200 μm diffusion limit [3, 12]. Our branched hierarchical design achieved a mean nutrient diffusion distance of 185 μm, effectively ensuring that all cells within the bulk tissue were within reach of the perfused media. This is a significant improvement over simple lattice structures, which often leave "dead zones" in the corners of the grid [11, 19].</p><h4>Bioink Synergies and Endothelialization</h4><p>The use of a GelMA-Alginate blend for the vascular lining proved advantageous. Alginate provided immediate structural definition through ionic crosslinking, while GelMA offered the RGD sequences necessary for HUVEC attachment and spreading [21, 27]. The formation of a confluent CD31-positive endothelium is a crucial step toward creating a truly biomimetic vascular system, as the endothelium acts as a selective barrier and prevents thrombosis in vivo [2, 14].</p><h4>Comparison with Existing Literature</h4><p>Our findings align with the work of Kang et al. [17] regarding the utility of hybrid PCL-hydrogel systems but extend the application to include complex internal fluidics. While volumetric bioprinting offers faster fabrication times [15, 22], the extrusion-based multimaterial approach remains superior for integrating multiple distinct cell-laden inks and structural polymers in a single process. Furthermore, the use of low-cost quad-extrusion systems [18] democratizes the ability to produce these complex constructs, which were previously limited to high-end, proprietary platforms.</p><h4>Limitations and Future Directions</h4><p>Despite the successful endothelialization, the current model lacks a smooth muscle cell layer, which is essential for physiological vasoreactivity. Future work should explore the deposition of concentric layers of different cell types to better replicate the tunica media and tunica adventitia [1, 13]. Additionally, incorporating 4D materials could allow for the maturation of the vascular network under dynamic mechanical stimuli, further enhancing the functionality of the tissue [26, 30].</p>
<h2>Conclusion</h2>
<p>In this study, we demonstrated the successful multimaterial bioprinting of large-scale tissue constructs with integrated, branched vascular networks. By optimizing the interplay between structural PCL, sacrificial Pluronic F-127, and cell-laden GelMA-Alginate bioinks, we fabricated centimeter-scale tissue analogs that maintained high cell viability and metabolic activity over a 28-day period. The hierarchical branching design effectively bypassed the diffusion limit, providing uniform nutrient distribution to the construct core. Furthermore, the formation of a functional endothelial lining suggests the potential for these constructs to be integrated with host vasculature upon transplantation. This research provides a robust framework for the biofabrication of complex, thick tissues and brings us closer to the goal of engineering whole organs for clinical use.</p>
<h2>References</h2>
<ol class="references">
<li>Dikyol, C., Altunbek, M., Bartolo, P., Koc, B.. Multimaterial bioprinting approaches and their implementations for vascular and vascularized tissues. Bioprinting. 2021;24, e00159. https://doi.org/10.1016/j.bprint.2021.e00159</li>
<li>Lee, H., Bae, H.. Forming vascular networks within functional cardiac tissue constructs. Biomedical Engineering Letters. 2013;3(3), 138-143. https://doi.org/10.1007/s13534-013-0106-y</li>
<li>Ozbolat, I. T.. Bioprinting scale-up tissue and organ constructs for transplantation. Trends in Biotechnology. 2015;33(7), 395-400. https://doi.org/10.1016/j.tibtech.2015.04.005</li>
<li>Bleeker, R., Campbell, R., Hernandez, K., Joyce, J., Spector, J. A.. LOP09. Plastic and Reconstructive Surgery. 2013;132, 500. https://doi.org/10.1097/01.prs.0000433432.97206.33</li>
<li>Bleeker, R., Campbell, R., Hernandez, K., Joyce, J., Spector, J. A.. LOP09. Plastic and Reconstructive Surgery. 2013;132(2), 500. https://doi.org/10.1097/01.prs.0000433340.43100.07</li>
<li>He, J., Mao, M., Li, X., Chua, C. K.. Bioprinting of 3D Functional Tissue Constructs. International Journal of Bioprinting. 2021;7(3), 395. https://doi.org/10.18063/ijb.v7i3.395</li>
<li>Tharakan, S., Khondkar, S., Ilyas, A.. Bioprinting of Stem Cells in Multimaterial Scaffolds and Their Applications in Bone Tissue Engineering. Sensors. 2021;21(22), 7477. https://doi.org/10.3390/s21227477</li>
<li>Zimmerling, A., Zhou, Y., Chen, X.. Bioprinted constructs for respiratory tissue engineering. Bioprinting. 2021;24, e00177. https://doi.org/10.1016/j.bprint.2021.e00177</li>
<li>Fitzsimmons, R. E., Aquilino, M. S., Quigley, J., Chebotarev, O., Tarlan, F., Simmons, C. A.. Generating vascular channels within hydrogel constructs using an economical open-source 3D bioprinter and thermoreversible gels. Bioprinting. 2018;9, 7-18. https://doi.org/10.1016/j.bprint.2018.02.001</li>
<li>Liew, A. W. L., Zhang, Y.. In vitro pre-vascularization strategies for tissue engineered constructs–Bioprinting and others. International Journal of Bioprinting. 2017;3(1), 3-17. https://doi.org/10.18063/ijb.2017.01.008</li>
<li>Liu, X., Wang, X., Zhang, L., Sun, L., Wang, H., Zhao, H.. 3D Liver Tissue Model with Branched Vascular Networks by Multimaterial Bioprinting (Adv. Healthcare Mater. 23/2021). Advanced Healthcare Materials. 2021;10(23). https://doi.org/10.1002/adhm.202170114</li>
<li>Ashammakhi, N., Tamimi, F., Caterson, E. J.. Three-Dimensional Bioprinting, Oxygenated Tissue Constructs, and Intravital Tissue Regeneration. Journal of Craniofacial Surgery. 2021;32(7), 2257-2258. https://doi.org/10.1097/scs.0000000000007721</li>
<li>Freeman, S., Ramos, R., Alexis Chando, P., Zhou, L., Reeser, K., Jin, S.. A bioink blend for rotary 3D bioprinting tissue engineered small-diameter vascular constructs. Acta Biomaterialia. 2019;95, 152-164. https://doi.org/10.1016/j.actbio.2019.06.052</li>
<li>Khalil, S., Sun, W.. Bioprinting Endothelial Cells With Alginate for 3D Tissue Constructs. Journal of Biomechanical Engineering. 2009;131(11). https://doi.org/10.1115/1.3128729</li>
<li>Bernal, P. N., Delrot, P., Loterie, D., Li, Y., Malda, J., Moser, C.. Biofabrication: Volumetric Bioprinting of Complex Living‐Tissue Constructs within Seconds (Adv. Mater. 42/2019). Advanced Materials. 2019;31(42). https://doi.org/10.1002/adma.201970302</li>
<li>Nowicki, M., Zhu, W., Sarkar, K., Rao, R., Zhang, L. G.. 3D printing multiphasic osteochondral tissue constructs with nano to micro features via PCL based bioink. Bioprinting. 2020;17, e00066. https://doi.org/10.1016/j.bprint.2019.e00066</li>
<li>Kang, H., Lee, S. J., Ko, I. K., Kengla, C., Yoo, J. J., Atala, A.. A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nature Biotechnology. 2016;34(3), 312-319. https://doi.org/10.1038/nbt.3413</li>
<li>Zgeib, R., Wang, X., Zaeri, A., Zhang, F., Cao, K., C. Chang, R.. Development of a low-cost quad-extrusion 3D bioprinting system for multi-material tissue constructs. International Journal of Bioprinting. 2023;10(1), 0159. https://doi.org/10.36922/ijb.0159</li>
<li>Shao, L., Gao, Q., Xie, C., Fu, J., Xiang, M., He, Y.. Directly coaxial 3D bioprinting of large-scale vascularized tissue constructs. Biofabrication. 2020;12(3), 035014. https://doi.org/10.1088/1758-5090/ab7e76</li>
<li>Moldovan, N. I.. Three-Dimensional Bioprinting of Anatomically Realistic Tissue Constructs for Disease Modeling and Drug Testing. Tissue Engineering Part C: Methods. 2021;27(3), 225-231. https://doi.org/10.1089/ten.tec.2020.0293</li>
<li>Muthukrishnan, L.. Imminent antimicrobial bioink deploying cellulose, alginate, EPS and synthetic polymers for 3D bioprinting of tissue constructs. Carbohydrate Polymers. 2021;260, 117774. https://doi.org/10.1016/j.carbpol.2021.117774</li>
<li>Bernal, P. N., Delrot, P., Loterie, D., Li, Y., Malda, J., Moser, C.. Volumetric Bioprinting of Complex Living‐Tissue Constructs within Seconds. Advanced Materials. 2019;31(42), e1904209-e1904209. https://doi.org/10.1002/adma.201904209</li>
<li>Wu, Z., Su, X., Xu, Y., Kong, B., Sun, W., Mi, S.. Bioprinting three-dimensional cell-laden tissue constructs with controllable degradation. Scientific Reports. 2016;6(1), 24474-24474. https://doi.org/10.1038/srep24474</li>
<li>Ge, Q., Li, Z., Wang, Z., Kowsari, K., Zhang, W., He, X.. Projection micro stereolithography based 3D printing and its applications. International Journal of Extreme Manufacturing. 2020;2(2), 022004-022004. https://doi.org/10.1088/2631-7990/ab8d9a</li>
<li>Donderwinkel, I., Hest, J. C. M. v., Cameron, N. R.. Bio-inks for 3D bioprinting: recent advances and future prospects. Polymer Chemistry. 2017;8(31), 4451-4471. https://doi.org/10.1039/c7py00826k</li>
<li>Schwartz, J. J., Boydston, A. J.. Multimaterial actinic spatial control 3D and 4D printing. Nature Communications. 2019;10(1), 791-791. https://doi.org/10.1038/s41467-019-08639-7</li>
<li>Piao, Y., You, H., Xu, T., Bei, H., Piwko, I. Z., Kwan, Y. Y.. Biomedical applications of gelatin methacryloyl hydrogels. Engineered Regeneration. 2021;2, 47-56. https://doi.org/10.1016/j.engreg.2021.03.002</li>
<li>Davoodi, E., Sarikhani, E., Montazerian, H., Ahadian, S., Costantini, M., Święszkowski, W.. Extrusion and Microfluidic‐Based Bioprinting to Fabricate Biomimetic Tissues and Organs. Advanced Materials Technologies. 2020;5(8). https://doi.org/10.1002/admt.201901044</li>
<li>Li, H., Tan, C., Li, L.. Review of 3D printable hydrogels and constructs. Materials & Design. 2018;159, 20-38. https://doi.org/10.1016/j.matdes.2018.08.023</li>
<li>Ryan, K. R., Down, M. P., Banks, C. E.. Future of additive manufacturing: Overview of 4D and 3D printed smart and advanced materials and their applications. Chemical Engineering Journal. 2020;403, 126162-126162. https://doi.org/10.1016/j.cej.2020.126162</li>
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