Andrea Cavero-Arrivasplata‡
abc,
David Hyram Hernández-Medina‡a,
Irving Isaí Rendón-Morenoad,
Diego Alonso Quevedo-Moreno
g,
Dariush Ebrahimibagha
a,
Sanal Kozhiparambil Chandran
f,
Julio Ernesto Valdivia Silva
b,
Claudia Maribel Luna-Aguirre
acd,
Mario Moisés Alvarez
*acde and
Grissel Trujillo-de Santiago
*acde
aTecnologico de Monterrey, Escuela de Ingeniería y Ciencias, Ave. Eugenio Garza Sada 2501 Sur, Col: Tecnológico, Monterrey, N.L., México 64700. E-mail: grissel@tec.mx; mario.alvarez@tec.mx
bDepartamento de Bioingeniería, Universidad de Ingeniería y Tecnología de Lima, 15063, Lima, Peru
cTecnologico de Monterrey, Expedition-FEMSA, Ave. Eugenio Garza Sada 2501 Sur, Col: Tecnológico, Monterrey, N.L., México, 64700
dTecnologico de Monterrey, Monterrey, Departamento de Mecatrónica, Ave. Eugenio Garza Sada 2501 Sur, Col: Tecnológico, Monterrey, N.L., México, 64700
eForma Foods, Research and Development Unit, Tecnológico de Monterrey, Monterrey, Nuevo León 64849, Mexico
fFacultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, Nuevo León 66455, Mexico
gDepartment of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
First published on 22nd July 2025
Engineering vascularization in hydrogel constructs remains a significant challenge in tissue engineering. Prevascularized hydrogels, engineered with void channels, enhance cell viability but often lack the mechanical stability needed for long-term culture, which is crucial for proper tissue maturation. In this study, we introduce chaotic bioprinting—a chaos-enabled biofabrication strategy—to produce mechanically robust hydrogel prevascularized filaments (with inner void channels) suited for extended culture. Utilizing a Kenics Static Mixer (KSM) printhead with various inlets (4 or 8), we developed fibers with intercalated layers of a myoblast-laden gelatin methacryloyl (GelMA)-alginate bioink, a sacrificial material for channel formation, and a reinforcing alginate scaffold. By optimizing ink ratios, we maximized cell-laden compartments while reinforcing the fiber structure and embedding microchannels for efficient mass and gas transport. Mechanical testing and degradation analysis reveal that optimized fibers achieve sufficient resistance (elastic modulus = 12.8 kPa) to withstand extended periods of cell culture up to 21 days. Additionally, C2C12 myoblasts cultured within these prevascularized and reinforced hydrogel filaments maintained high cell viability (>90%) for more than 21 days and demonstrated superior cell proliferation, spreading, and alignment throughout the filament volume compared to solid fibers (reinforced but without inner void channels). Sacrificial layers created void microchannels, enhancing mass and gas transport, while the reinforcing layers provided structural integrity. Multimaterial chaotic printing enabled the fabrication of mechanically stable, functional constructs with compartmentalized architectures, facilitating extended culture and tissue maturation. Our results demonstrate the potential of this method for engineering thick tissues, including skeletal muscle, and highlight its versatility for various regenerative medicine applications, advancing biofabrication towards thicker and mature tissues.
Recent advances in chaotic bioprinting offer a promising approach to overcoming these limitations. Continuous chaotic bioprinting27–29 is a multi-material extrusion technique that uses chaotic flows to generate multi-compartmentalized hydrogel filaments with alternating layers of different materials.30 This approach relies on using printheads equipped with Kenics Static Mixer (KSM) elements, which induce chaotic flows by iteratively reorienting and splitting the materials as they pass through the printhead.30 In previous work, we have shown applications of chaotic printing in diverse scenarios, and we have demonstrated the versatility of the technique for printing more than two materials31 to produce material constructs with a distinctive microstructure composed of multiple, alternating, and parallel layers of the different materials fed to the printhead. Moreover, using sacrificial inks, we previously demonstrated the ability to print hydrogel filaments with internal microchannels (capillary-size) in a single step, enhancing mass transport and improving metabolic activity and cell differentiation.31,32 Despite these advantages, a significant limitation persists: the hydrogel filaments produced lack mechanical robustness, leading to degradation and erosion within a few days of culture. This instability restricts the construct's potential for extended culture, which is essential for achieving mature, functional tissues.33
In this study, we present an enhanced chaotic bioprinting approach designed to produce mechanically robust hydrogel filaments suitable for prolonged cell culture. Our strategy involves using a multi-port KSM printhead with multiple inlets (8 and 4) and two mixing elements to print hydrogel filaments with intercalated layers of myoblast-laden gelatin methacryloyl (GelMA)-alginate bioink, a sacrificial material to fabricate void channels, and a reinforcing alginate scaffold. This multimaterial design allows us to integrate the presence of cell-laden compartments, reinforce the fiber structure, and incorporate microchannels that facilitate efficient nutrient and gas exchange. By leveraging chaotic flows, we aimed for a construct architecture that balances prevascularization with enhanced mechanical stability, thus advancing biofabrication towards durable, mature tissue models.
The multimaterial printing strategy introduced in this study is versatile and can be adapted to fabricate a wide range of tissues. For the first proof-of-concept demonstration, we chose the fabrication of skeletal muscle fibers due to their relevance across various applications. Skeletal muscle tissue models are critical for fundamental studies on muscle physiology,34,35 aging,36 and muscle-related pathologies,35,37,38 as well as for pharmacological testing.35 Muscle tissue engineering will enable the fabrication of implantable muscle constructs,39,40 and cultured meat production.41 Additionally, skeletal muscle presents unique fabrication challenges, including its highly aligned architecture and the need for extended culture periods to achieve maturation under optimal nutrient and oxygen supply. These requirements align closely with the capabilities of our chaotic bioprinting approach, making skeletal muscle an ideal model to showcase the utility and effectiveness of this multimaterial printing technique.
Here we illustrate the implementation of a multimaterial (bio)printing strategy to develop skeletal-muscle tissue-like fibers with internal prevascularization, made from soft and common hydrogel materials (i.e., alginate and GelMA)45 that can withstand extended incubation periods (up to 28 days).
To develop these tissue constructs, we followed the following logical steps. First, in acellular experiments, we tested different feeding strategies of three distinct inks with complementary functionalities, namely a structural (scaffolding) ink, a sacrificial ink, and a cell-friendly ink. Then, in tensile tests, we evaluated the mechanical properties of hydrogel filaments obtained from these feeding strategies and selected those that exhibited higher young modulus for assessment of erosion resistance under benign agitation and incubation at 37 °C in culture medium. Finally, filament formulations with good performance in these erosion experiments were used in bioprinting experiments in which murine myoblasts were integrated in cell-friendly inks. We evaluated the performance of these constructs through 28 days of culture in terms of viability, metabolic activity, cell density, cell alignment, and muscle tissue maturation.
In chaotic printing, the number and location of these layers can be determined a priori by selecting a suitable geometry of the printhead (i.e., number of mixing elements and number of inlets).29,31,46 In a first set of acellular experiments, we used chaotic printheads equipped with 8 inlets and two KSM elements (Fig. 1A) to coextrude three different inks, namely a structural ink composed of 4% high-viscosity alginate (AlgH), a cell-friendly ink composed of 3% GelMA and 3.5% low-viscosity alginate (AlgL), and a sacrificial ink consisting of 0.6% hydroxyethyl cellulose (HEC) (Fig. 1Bi). This printhead configuration rendered compartmentalized fibers with 16 intercalated layers (Fig. S1†) of the feed materials because the eight feeding streams are reoriented by the first mixing elements and then split by the second mixing element.
Since the multimaterial filament was extruded through a nozzle orifice of 1 mm, each layer exhibits, on average, a thickness of 62.5 μm (1000 μm/16) at the central line of the cross-section. The printing process involved submerging the printhead outlet directly into a calcium chloride (CaCl2) bath to crosslink the alginate molecules, followed by UV light exposure to stabilize the GelMA matrix.
The composition of the sacrificial and cell-loadable inks were determined based on previous reports of our group.32 The scaffolding ink composition was a 4% alginate-based ink.
Our selection of 4% AlgH solution as a scaffolding ink was based on preliminary assays that demonstrate that the use of 6% AlgH results in brittle constructs, while the use of 2% AlgH results in a significantly weak fiber. Therefore 4% AlgH was selected for the subsequent experiments. Indeed, combining alginate with collagen or gelatin to create a mixed bioink is one common approach to improve mechanical strength of scaffolds.47–49 However, here we can micro-compartmentalize our stiffest alginate-based fibers to provide the desired mechanical support, without compromising the compartments loadable with living cells which must be softer and looser matrices. The sacrificial ink, composed of 0.6% (w/v) HEC, leached out during crosslinking to create hollow microchannels to enhance nutrient and gas exchange along the filament's length (Fig. 1Bii).
In chaotic printing, the relative location of the inner layers can be determined a priori by selecting a suitable feeding position for each one of the materials to be used (see also Fig. S1†).31,50 Fig. 1B shows the rationale of the architectural design of our fibers. With the selection of three hydrogel-based materials or inks, we aimed to continuously fabricate long hydrogel filaments containing a compartmentalized architecture composed of multiple repetitions of a sequence of three different material environments, namely solid layers for enhancing mechanical robustness, solid layers suitable for cell proliferation, and sacrificial layers to create prevascularization. Fig. 1Biii shows a brightfield micrograph of a fiber containing inner hollow microchannels and reinforced with AlgH. Light layers correspond to the hollow microchannels produced by the leaching of the sacrificial material, while blue layers correspond to 4% AlgH loaded with colored particles.
We explored a wide variety of formulations and feeding configurations (see Fig. 1C–E; Table 1), their effect on the mechanical properties of the printed fibers and their stability under extended culture conditions.
Type of KSM | Type of fiber | Type of material | Flow rate (mL min−1) | ||
---|---|---|---|---|---|
Support | Matrix | Sacrificial | |||
All concentrations are % (w/v). | |||||
8 inlets, 2 mixing elements | Solid | 2% AlgH | — | — | 1.00 |
Hollow | 2% AlgH | — | 0.6% HEC | 0.50 | |
2% AlgH | 3% GelMA/3.5% AlgL | 0.6% HEC | 0.20 | ||
2% AlgH | 3% GelMA/3.5% AlgL + 3 × 106 cells per mL | 0.6% HEC | 0.15 | ||
2 inlets, 4 mixing elements | Solid | — | 3% GelMA/3.5% AlgL | — | 1.00 |
4% AlgH | 3% GelMA/3.5% AlgL | — | 0.75 | ||
Hollow | — | 3% GelMA/3.5% AlgL | 0.6% HEC | 0.60 |
Of note, achieving multimaterial filaments with precise architecture required careful optimization of flow conditions. Preliminary experiments revealed that, as the number of materials increased, the flow rate needed to decrease to produce fibers with distinct material compartments. The selection of proper flow rates ensured that the inks behaved as Newtonian fluids, resulting in the preservation of the expected multilayered microstructures that are characteristic of chaotic printing and mechanically stable fibers (Table 1). Indeed, using a flow rate of 0.20 mL min−1 produced consistent fibers with clear structural integrity. However, deviations from this rate led to non-Newtonian behavior, nozzle clogging, and irregularities in the printed fibers, compromising their multilayered microstructure or mechanical stability, or both.
To assess the effects of these parameters on mechanical properties and integrity under culture conditions, various formulations and/or configurations were tested, including a solid fiber composed solely of the structural ink as a control (Table 1, Fig. 1D and E). The design strategy also aimed to ensure that cell-laden compartments were adjacent to sacrificial layers, with inter-layer distances not exceeding 200 μm to prevent mass transfer limitations that could impair cell viability. The 62.5 μm layer width achieved by the bioprinting setup, combined with the placement of cell-containing layers near the void channels, effectively addressed these constraints. Originally, we aimed to maximize the number of cell-laden compartments, while striving to distribute structural compartments evenly and minimize their volumetric fraction. Computational simulations guided the rational pre-design of the filament architecture by predicting the placement of materials based on input configurations (see Fig. S1†). Then, we printed solid fibers using color inks (i.e., three distinct inks, each containing solid particles of a distinct color) and demonstrated the similarity between the anticipated and experimental cross-sections (Fig. 1Di and Dii). Image analysis of the area fractions occupied by the different inks further corroborated the consistency between pre-designed and experimental outcomes (Fig. 1Ei and Eii). These findings highlight the robustness of the proposed printing setup for designing complex multimaterial architectures.
The structural ink (4% AlgH) was fed into the chaotic printhead at varying proportions, ranging from 2/8 to 5/8 of the total inlets, with the remaining inlets allocated to sacrificial ink (0.6% HEC) and cell-loadable ink (3% GelMA/3.5% AlgL). A control composed entirely of AlgH (8/8) was also included for comparison. Continuous structured filaments were successfully produced with higher proportions of sacrificial ink (up to 3/8), but these constructs were mechanically weaker, exhibiting tensile strengths below 9 kPa (Fig. 2Aiii and Table 2).
Strain at break | Stress at break (kPa) | Elastic modulus (kPa) | |
---|---|---|---|
Proportion of HEC | |||
2/8 inlets | 0.49 ± 0.14 | 2.15 ± 0.16 | 10.74 ± 0.05 |
4/8 inlets | 0.76 ± 0.16 | 3.10 ± 0.28 | 11.76 ± 1.03 |
6/8 inlets | 0.32 ± 0.08 | 1.32 ± 0.52 | 9.44 ± 0.08 |
Proportion of AlgH | |||
2/8 inlets | 1.05 ± 0.15 | 1.52 ± 0.13 | 4.78 ± 0.72 |
3/8 inlets | 1.07 ± 0.14 | 2.55 ± 0.21 | 8.93 ± 0.81 |
4/8 inlets | 0.76 ± 0.16 | 3.10 ± 0.28 | 11.76 ± 1.03 |
5/8 inlets | 1.52 ± 0.13 | 4.34 ± 0.32 | 12.33 ± 0.36 |
8/8 inlets | 1.29 ± 0.24 | 6.73 ± 1.25 | 17.87 ± 0.21 |
Concentration of AlgH | |||
2% (w/v) | 1.07 ± 0.16 | 2.55 ± 0.28 | 8.93 ± 1.03 |
4% (w/v) | 1.54 ± 0.02 | 6.96 ± 0.45 | 12.80 ± 0.44 |
6% (w/v) | 0.86 ± 0.34 | 7.05 ± 2.84 | 25.83 ± 0.25 |
Stress–strain curves for each configuration (Fig. 2Aiii) and their corresponding elastic modulus values (Fig. 2Aiv) revealed a clear trend: the inclusion of AlgH layers significantly enhanced the mechanical robustness of the hydrogel filaments. Notably, filaments composed entirely of AlgH (8/8) had the highest elastic modulus, reaching 17.87 kPa. This value was statistically different from all other configurations, confirming the critical role of AlgH in providing mechanical reinforcement.
Interestingly, filaments with 5/8 and 4/8 AlgH proportions did not exhibit statistically significant differences in elastic modulus, whereas those with 4/8 and 3/8 AlgH proportions showed significant differences. These results suggest that the mechanical reinforcement provided by the scaffolding ink (4% AlgH) follows an asymptotic trend, with diminishing returns when more than half of the inlets are dedicated to the structural ink.
In the context of practical applications, filaments with 3/8 and 4/8 AlgH were considered suitable for experiments involving living cells, as they offered a good balance between mechanical stability and the availability of cell-loadable compartments (3 and 2 compartments, respectively). Ultimately, we selected the 3/8 AlgH configuration, which demonstrated an elastic modulus of approximately 8.93 kPa, for subsequent experiments. This configuration provided a higher number of GelMA/AlgL layers, maximizing the potential for cell hosting while maintaining sufficient mechanical stability for extended culture.
To evaluate the resistance of the optimized hydrogel filament (3/8 AlgH) to degradation under dynamic conditions, we conducted erosion assays in a rocking cell-culture flask. This system generates a gentle, cyclic movement with low shear stress, simulating benign agitation conditions akin to those encountered in tissue culture bioreactors. The flask completes one cycle of movement, consisting of a half rotation, in one minute (Fig. 2Bi).
The susceptibility of the fibers to erosion was assessed by measuring the percentage of remaining filament mass over time. For comparison, we included three control samples printed using the KSM printhead configurations shown in Fig. 2Bii. These controls comprised (a) a solid fiber reinforced with alginate (3% GelMA/3.5% AlgL + 4% AlgH), (b) a solid fiber without alginate reinforcement (3% GelMA/3.5% AlgL), and (c) a hollow fiber without alginate reinforcement (3% GelMA/3.5% AlgL + 0.6% HEC) (Fig. 2Bii).
The test sample was the optimized multimaterial hollow fiber (3% GelMA/3.5% AlgL + 0.6% HEC + 4% AlgH) that was selected from the mechanical characterization experiments in virtue of demonstrating superior mechanical properties and contained reinforcing AlgH layers, cell-friendly GelMA/AlgL layers, and sacrificial HEC layers to create hollow channels.
Erosion testing results clearly demonstrated the importance of alginate reinforcement in resisting erosion under continuous agitation. Hydrogel filaments with void channels and without alginate reinforcement (GelMA/AlgL + HEC) completely degraded after 24 hours, while solid filaments without reinforcement (GelMA/AlgL) degraded entirely after 72 hours. In contrast, both solid fibers with alginate reinforcement (GelMA/AlgL + AlgH) and hollow fibers with alginate reinforcement (GelMA/AlgL + HEC + AlgH) retained over 75% of their initial mass after 72 hours of continuous agitation (Fig. 2Biii).
Of note, despite the presence of structural reinforcement, prevascularized filaments with alginate layers experienced more mass loss compared to solid reinforced filaments. This should be mainly attributed to the presence of internal channels within the hydrogel filaments which facilitates perfusion and erosion along the inner walls of the construct.
These findings underscore the significance of alginate content in enhancing mechanical stability and resistance to erosion. The higher alginate proportion in reinforced solid fibers (4/8 AlgH) conferred greater mass retention under agitation compared to hollow fibers with a lower alginate proportion (3/8 AlgH). However, the inclusion of channels in the hollow fibers offers critical advantages for mass transfer and prevascularization, justifying their use for long-term tissue culture applications despite their slightly reduced erosion resistance.
As anticipated, incorporating cells into the GelMA/AlgL matrix slightly increased the apparent viscosity (ESI Fig. S2†), likely due to particle–matrix interactions introduced by suspended cells. This increase in viscosity required empirical adjustments in flow rate to maintain high-quality filament formation. Specifically, we reduced the extrusion flow rate by approximately 25%—from 0.20 mL min−1 to 0.15 mL min−1—to maintain consistent filament formation and avoid overpressure at the nozzle, which could otherwise result in irregular deposition or excessive shear stress on the cells. These adjustments also enabled the reproducible production of filaments with a well-defined compartmentalized architecture.
The resulting muscle tissue-like filaments featured internal void channels to enhance mass transport, critical for maintaining cell viability and promoting tissue maturation (Fig. 3Aiii). The cell-laden hydrogel fraction provided a soft matrix conducive to cell anchorage, proliferation, and differentiation—essential for the transformation of myoblasts into multinucleated muscle cells.32,49,51,52 Sacrificial ink layers leached out during crosslinking, creating hollow microchannels along the filament's length to enhance nutrient and gas exchange. Chaotic bioprinting enabled these materials to be arranged in intercalated parallel layers, closely mimicking the highly aligned, organized, and compartmentalized architecture of skeletal muscle (Fig. 3Aiv). We hypothesized that this architecture would favor tissue maturation, resulting in skeletal muscle tissue-like filaments after extended culture.
To evaluate the functionality of the fabricated filaments, we compared cell viability, cell density, cell proliferation, and alignment between reinforced solid and hollow fibers (Fig. 3B and C). While solid reinforced filaments were expected to demonstrate superior mechanical robustness throughout culture, we hypothesized that hollow fibers would outperform solid ones in terms of cell viability and proliferation. The enhanced mass transfer facilitated by the void channels eliminates nutrient and oxygen transport limitations in the hydrogel core, creating a more favorable environment for cell growth and differentiation.
Indeed, reinforced prevascularized filaments exhibited significantly higher cell viability (>80%) compared to solid filaments at all time points (days 1, 7, and 14; Fig. 3B). Cell viability in hollow fibers increased during the first 14 days of culture, reaching values above 98% by day 14. In contrast, solid filaments showed an initial increase in cell viability between days 1 and 7, plateauing at approximately 80% by day 14.
Interestingly, no significant differences in cell density were observed between the two filament types during the first week of culture. However, by day 14, cell density (defined as the number of living cells per unit volume) was significantly higher in hollow filaments compared to solid ones (Fig. 3C).
Notably, in solid fibers, viable cells predominantly resided in the outer sections of the filaments, with more than 95% of the live cells located within 200 μm of the filament surface. Z-Stack microscopy images confirmed remarkable cell proliferation near the surface of solid fibers, with minimal cell presence deeper within the filament core (Fig. 3D). In contrast, hollow filaments supported cell proliferation throughout their entire volume. Z-stack images encompassing the entire thickness of the hollow filaments revealed evenly distributed cells across the cross-section, indicating that the void channels improved mass transfer and supported cell viability and proliferation throughout the construct (Fig. 3E).
These results highlight the advantages of incorporating void microchannels in reinforced hydrogel filaments. The presence of void channels significantly improves cell viability and proliferation in mechanically stable constructs, making them suitable for extended culture and possibly tissue maturation.
In the following set of experiments, we investigated how different printhead configurations and degrees of compartmentalization affected the alignment, proliferation, and maturation of muscle-like fibers. Specifically, we compared hydrogel filaments fabricated using printheads with 4 inlets and 2, 4, or 6 KSM elements. As a control, we also bioprinted filaments using single-inlet printheads where the supportive ink, sacrificial ink, and C2C12 cells in the GelMA/AlgL blend were fully mixed before extrusion.
Cell viability trends mirrored previous observations: cells in hydrogel filaments without void channels (single-inlet printheads) localized primarily near the filament surface, whereas cells in prevascularized filaments with void channels proliferated throughout the entire volume during extended culture (Fig. 4A and B). Reinforced and prevascularized filaments consistently maintained >95% cell viability over 21 days with minimal erosion (Fig. 4Ai and Aii).
Remarkably, filaments with eight or more compartments exhibited significant cell alignment. Confinement within layers narrower than 125 μm appeared to promote multinucleation and alignment, particularly in prevascularized fibers fabricated using 6-KSM-element printheads (Fig. 4B and C). Homogeneous constructs with no compartments (e.g., fibers fabricated using premixed inks extruded through a 1-inlet, 6-KSM-element printhead) showed negligible cell alignment, underscoring the importance of compartmentalization for achieving muscle-like fiber architecture.
These results highlight the dual role of void channels and compartmentalization in facilitating cell alignment (Fig. 4C and D). Void channels improved mass transfer, eliminating nutrient and oxygen limitations, while compartmentalization constrained cells within discrete layers, fostering interactions necessary for alignment and multinucleation. Solid filaments, by contrast, exhibited limited cell spreading and alignment confined to their surface. Prevascularized filaments supported cell alignment and spreading throughout their volume, demonstrating the importance of structural reinforcement with 4% AlgH layers (Fig. 4D). In addition, we performed quantitative PCR (qPCR) to analyze the expression of specific muscle-related biomarkers, MyoG and ACTA1. The expression patterns of these genes offer insights into the differentiation and maturation status of the myoblasts (Fig. 4E). MyoG and ACTA1 are well-established indicators of myogenic differentiation,53 signifying that the cells are progressing towards myotube formation.
In sum, the integration of void channels and compartmentalized architecture, enabled by chaotic bioprinting, proved critical for long-term culture and the maturation of muscle-like fibers. These findings reinforce the practical relevance of using reinforced, prevascularized hydrogel filaments for tissue engineering applications.
In acellular experiments, we optimized fiber composition for mechanical robustness under agitated culture. Optimized fibers composed of 3/8 AlgH (4%), 3/8 GelMA/AlgL, and 2/8 HEC exhibited an elastic modulus of 12.8 kPa, retained more than 65% of their mass after 72 hours of continuous agitation, and supported long-term culture.
To illustrate the usefulness and effectiveness of this fabrication strategy to bioprint muscle-like fibers, we chaotically bioprinted C2C12 cell-laden prevascularized hydrogel filaments mechanically reinforced by only 3/8 4% AlgH content.
The compartmentalized architecture provided by chaotic bioprinting played a pivotal role in sustaining cell viability above 90% at day 14 and facilitating cell proliferation and alignment throughout the fiber volume. Void channels enhanced mass and gas transport, alleviating nutrient and oxygen diffusion limitations within the construct and promoting cell viability and differentiation to muscle-like fibers after a culture period of 21 days.
Reinforced and prevascularized hydrogel filaments demonstrated superior cellular outcomes compared to solid filaments, which exhibited limited mass transport and concentrated cell proliferation within 200 μm near the surface.
The mechanical stability and functionality of the fabricated fibers were heavily influenced by their rationally designed compartmentalized architecture. Sacrificial layers enabled efficient mass transport, while reinforcing layers ensured the structural integrity required for extended culture periods. These features, prevascularization and mechanical reinforcement, collectively supported the alignment and multinucleation of myoblasts, crucial for muscle tissue maturation.
Remarkably, cell elongation, spreading, and a high degree of cell alignment were observed not only at the surface but also within the inner layers of prevascularized and reinforced fibers. In comparison, solid reinforced fibers lacked the mass transport capabilities provided by void channels, resulting in less uniform cell proliferation and alignment. However, the ability to tune the proportions and arrangements of inks in the chaotic bioprinting setup enabled the fabrication of hydrogel fibers with optimized mechanical and functional properties, meeting the requirements for tissue engineering applications.
These findings highlight the versatility of multimaterial chaotic bioprinting for creating complex and functional hydrogel constructs. The technique opens avenues for fabricating thick (>1 mm) tissues with robust mechanical properties, extended culture times, and high cell viability. Beyond skeletal muscle, the potential applications span various tissue engineering and regenerative medicine domains, including organ models and other thick, vascularized tissues.
In summary, chaotic bioprinting allows for the rational design and fabrication of robust, prevascularized hydrogel filaments with tunable compositions and architectures. This versatile biofabrication technique provides an effective platform for developing mature, functional constructs, paving the way for innovations in tissue engineering and regenerative medicine.
GelMA was synthesized according to established protocols.54 A solution containing 6% (w/v) GelMA and 0.2% (w/v) Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; Allevi) was prepared in DPBS and heated at 70 °C for 15 minutes. This GelMA solution was subsequently mixed in a 1:
1 ratio with the 7% (w/v) AlgL solution, yielding a hybrid GelMA-based ink with a final composition of 3% (w/v) GelMA and 3.5% (w/v) AlgL (GelMA/AlgL). All hydrogel solutions were stored at 4 °C until further use.
For the fabrication of hydrogel filaments with channels, a sacrificial ink composed of 0.6% (w/v) HEC was always used. This sacrificial ink coextruded alongside the support ink (4% (w/v) AlgH) and the cell-hosting ink (3% (w/v) GelMA/3.5% (w/v) AlgL), with each ink introduced through separate feeding ports. The coextrusion process was conducted using the KSM printhead at flow rates of 0.15–0.6 mL min−1, adjusted according to the specific material composition.
After printing, hydrogel filaments were crosslinked in a 2% (w/v) calcium chloride bath. For filaments containing 3% (w/v) GelMA/3.5% (w/v) AlgL, an additional photocrosslinking step was performed under UV light (365 nm) for 45 seconds. A detailed summary of the printing conditions for hydrogel filaments with varying material compositions is provided in Table 1.
The Navier–Stokes equations were solved at each node within the 3D mesh under laminar flow conditions, assuming a transient state and no-slip boundary conditions. These simulations yielded detailed velocity field solutions for various feeding strategies, encompassing different flow rates and inlet configurations.
We took these velocity field results to conduct particle tracking to analyze individual and collective particle trajectories. A total of 100000 massless particles were introduced per inlet, with distinct color coding applied to particles originating from different inlets. The trajectories of these particles were tracked, and their crossing patterns at the printhead outlet were recorded to determine the degree of compartmentalization and the resulting microstructure at the printhead outlet.
Cross-section images were captured by cutting a small section of the printed filaments with a blade, wetting the sample with deionized water, and visualizing the cross-sections under an Axio Observer.Z1 microscope (Zeiss, Germany) equipped with Colibri.2 LED illumination, Apotome, and a 10× objective lens.
The proportion of each ink in the cross-section was quantified using ImageJ software. The light blue, black, and orange areas were measured, and the area fraction for each ink was calculated using eqn (1):
Area fraction i = (Ai/Atotal) | (1) |
Type of KSM | Type of fiber | Nomenclature | Proportion of AlgH | Proportion of GelMA/AlgL | Proportion of HEC |
---|---|---|---|---|---|
Analysis of microstructure & tensile tests | |||||
8 inlets, 2 elements | Solid fiber | AlgH | 8/8 inlets | — | — |
Hollow fiber | GelMA/AlgL-HEC-AlgH(2) | 2/8 inlets | 4/8 inlets | 2/8 inlets | |
Hollow fiber | GelMA/AlgL-HEC-AlgH(3) | 3/8 inlets | 3/8 inlets | 2/8 inlets | |
Hollow fiber | GelMA/AlgL-HEC-AlgH(4) | 4/8 inlets | 2/8 inlets | 2/8 inlets | |
Hollow fiber | GelMA/AlgL-HEC-AlgH(5) | 5/8 inlets | 1/8 inlets | 2/8 inlets | |
Degradation analysis | |||||
8 inlets, 2 elements | Hollow fiber | GelMA/AlgL-HEC-AlgH(3) | 3/8 inlets | 3/8 inlets | 2/8 inlets |
2 inlets, 4 elements | Hollow fiber | GelMA/AlgL-HEC | 1/2 inlets | 1/2 inlets | |
Solid fiber | GelMA/AlgL-AlgH | 1/2 inlets | 1/2 inlets | — | |
Solid fiber | GelMA/AlgL | — | 2/2 inlets | — |
For each experiment, hydrogel filaments with a diameter of 1 mm and an initial length of 30 mm were used. The Arduino system recorded displacement (mm) and force (N) data, which were subsequently processed to generate stress–strain curves using eqn (2) and (3):
![]() | (2) |
![]() | (3) |
The elastic modulus (kPa) was determined by calculating the slope of the linear region of the stress–strain curve. Curve fitting was performed using Microsoft Excel's curve fitting function for precise determination of the modulus.
The fibers were submerged in PBS (pH 7.4) at a 1:
1 weight-to-volume ratio (5 grams of fiber in 5 mL of PBS) within the dynamic incubation system described above. Wet mass measurements were taken at specified time points: 8, 12, 24, 48, and 72 hours.
The remaining weight percentage was calculated using eqn 4:
![]() | (4) |
The GelMA solution was subsequently mixed with the 7% (w/v) AlgL solution in a 1:
1 ratio to produce a bioink containing 3% (w/v) GelMA and 3.5% (w/v) AlgL. Before bioprinting, all bioinks were equilibrated to 37 °C to ensure compatibility with C2C12 cells. To incorporate cells, C2C12 myoblasts were detached from T75 flasks, pelleted via centrifugation, and resuspended in the GelMA/AlgL bioink at a final concentration of 3 × 106 cells per mL.
For the fabrication of reinforced solid fibers, a KSM printhead with 2 inlets and 4 mixing elements was used. The support ink consisted of 4% AlgH, while the bioink consisted of 3% GelMA, 3.5% AlgL, and 0.5% LAP with 3 × 106 live C2C12 cells per mL. A printing rate of 0.75 mL min−1 was employed. The fibers were crosslinked by immersion in a 2% CaCl2 bath for 30 seconds, followed by a PBS wash to remove excess CaCl2, and UV exposure at 365 nm for 30 seconds to stabilize the GelMA layer. As with hollow fibers, the solid fibers were cut into 1 cm pieces and cultured in ultralow attachment 12-well plates containing DMEM supplemented with 10% (v/v) FBS and 1% (v/v) antibiotic–antimycotic solution.
Analysis was conducted using the Fourier component method, with orientation angles binned into 13 intervals ranging from −90° to +90°. Importantly, the longitudinal axis of the hydrogel filament was defined as the 0° reference, allowing us to measure the angular deviation of cells relative to the main fiber direction.
The primer sequences used were as follows: GAPDH (housekeeping gene): Forward: 5′-CATCACTGCCACCCAGAAGACTG-3′ and reverse: 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′; MyoG: Forward: 5′-CAGCTCCCTCAACCAGGA-3′ and reverse: 5′-TGCCCCACTCTGGACTG-3′; and ACTA1: Forward: 5′-CGATCTCACCGACTACCTGA-3′ and reverse: 5′-CAGCTTCTCCTTGATGTCGC-3′.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4bm01674b |
‡ These authors contributed equally to this work. |
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