Six-axis robotic extrusion of hybrid hydrogels for biomimetic airway model fabrication

Abstract

Respiratory diseases remain a major global health burden, motivating the need for improved experimental lung models that capture both anatomical geometry and mechanical compliance. Traditional three-axis 3D printers face limitations in replicating the lung’s curving, branching structures, often resulting in pore collapse or loss of fidelity. In this study, we demonstrate the use of a six-axis robotic extrusion bioprinter to fabricate anatomically inspired airway structures using hybrid hydrogels composed of Alginate (A) and CarboxyMethyl Cellulose (CMC). By systematically tuning hydrogel formulations, we identified a blend (5% Alginate-7% CMC, i.e., A₅C₇) that provides a balance of viscosity, shear-thinning, and diffusion resistance, resulting in enhanced print fidelity and structural stability compared to single-polymer inks. Using this formulation, the robotic platform successfully printed tubular and bifurcating airway constructs that retained lumen geometry, withstanding axial and diametral compression within ranges relevant to lung tissue mechanics. Printability (Pr ≈ 0.92–1.08) analysis confirmed consistent pore fidelity, while mechanical testing demonstrated elastic recovery under loading. A preliminary aerosol deposition test highlighted the feasibility of coupling these constructs with drug delivery studies, though more sensitive measurement methods will be required. Collectively, this work establishes a proof-of-concept fabrication platform for anatomically accurate and mechanically compliant airway models, which can be adapted in future studies to represent both healthy and pathological respiratory states through targeted modifications in geometry, material composition, and cellular integration.

Keywords

3D bioprinting nanofiber robot lung cellulose

Introduction

Breathing is a highly coordinated biomechanical process, relying on the movement of air through a branching network of trachea, bronchi, bronchioles, and alveoli.¹ Capturing this complexity in the laboratory is critical for understanding how inhaled therapeutics reach their targets and how airborne hazards affect respiratory health. Yet, the very features that make the lung effective, the continuously curving airway branches and their dynamic motion during expansion and contraction, make it exceptionally difficult to replicate in vitro.^2,3

Conventional 3D printers, which operate with three fixed linear axes, can reproduce simplified airway geometries. However, these systems are limited when tasked with replicating the smooth bifurcations and continuous curvatures of the bronchial tree.⁴ Moreover, the rigid thermoplastics commonly used in such printers cannot emulate the lung’s elastic, compliant nature, which is central to its function during the breathing cycle. To overcome these limitations, new bioprinting technologies are emerging. A six-axis robotic bioprinter provides additional degrees of freedom, enabling the print head to tilt and rotate. This allows bioink to be deposited on complex curved surfaces without gravity-induced sagging or the stepped artifacts often observed in layer-by-layer approaches.⁵ Such capability is especially relevant for reproducing airway bifurcations, where smooth, seamless deposition is required.⁶

Advances in biomaterials further complement this technological shift. Instead of relying solely on stiff polymers, researchers are now designing soft hydrogel-based bioinks that incorporate bioactive components, supporting both structural fidelity and cellular compatibility. By integrating robotic precision with these cell-friendly hydrogels, it is now possible to build in vitro lung constructs that are anatomically faithful and physiologically responsive. The objective of this study is to demonstrate this integrated approach by using a six-axis robotic arm bioprinter to fabricate lung-inspired structures from next-generation bioinks. In doing so, we aim to bridge a key gap in lung model development: the lack of platforms that can simultaneously achieve realistic geometry, mechanical compliance, and the potential for biological integration.

Advancements in bioprinting technology

Additive manufacturing for tissue engineering has advanced significantly beyond traditional planar, layer-by-layer methods.^7,8 In conventional 3-axis 3D printing, each layer is deposited on a fixed horizontal plane, which can introduce structural weaknesses in geometrically complex features such as branching tubes.⁹ Overhanging regions often require support material and are prone to stair-step artifacts and suboptimal interlayer adhesion.¹⁰ To address these limitations, multi-axis printing techniques have emerged. Notably, Morita et al. demonstrated a multiaxis extrusion approach capable of joining layers along curved paths, significantly reducing surface irregularities in printed hollow hydrogels.⁹ This innovation enables the fabrication of smooth, continuous tubular structures with geometrical fidelity approaching native anatomical forms. Expanding on this concept, robotic arm-based bioprinters now offer dynamic nozzle reorientation during fabrication, unlocking new possibilities for printing complex 3D tissue scaffolds.^11,12

Another notable route to printing curvature-rich and hollow structures without stair-step artifacts or loss of interlayer adhesion is embedded printing in support baths, such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH)¹³ and Suspended Layer Additive Manufacturing (SLAM),¹⁴ which enable extrusion of soft, low-viscosity bioinks in three dimensions with high geometric fidelity and smooth surfaces. While these methods clearly show that our approach is not the only option for fabricating complex hollow geometries, with both FRESH and SLAM successfully producing intricate tubular and vascularized architectures, the present work specifically focuses on exploiting multi-axis robotic printing to achieve continuous curvature and anatomically relevant airway structures.

Biofabrication materials for lung models

Alongside improvements in printing hardware, major progress has been made in the development of bioinks, the foundational materials that enable the fabrication of functional tissue analogues.^15,16,17 Among these, hydrogels remain central due to their high-water content, tunable properties, and inherent biocompatibility. Polymers such as Alginate and gelatin are widely used, as they can be extruded into three-dimensional structures and then crosslinked whether ionically, enzymatically, or thermally, to maintain their architecture.^18,19 While these simple hydrogels offer a supportive matrix for cells, they often fall short in reproducing the mechanical strength and biological signaling characteristic of native tissues.

To overcome these limitations, researchers have increasingly focused on composite bioinks that combine hydrogels with extracellular matrix (ECM) elements or nanomaterials. For example, incorporating collagen or decellularized tissue matrix into Alginate has been shown to promote cell adhesion and encourage tissue-specific behaviors.^20,21 Similarly, nanocomposite hydrogels, reinforced with materials such as nanofibrillated cellulose or silica nanoparticles, enhance printability and mechanical stiffness while preserving a cell-friendly environment.^22,23,24 One study demonstrated that the addition of cellulose nanofibers to a gellan gum hydrogel markedly increased its storage modulus and yield stress, enabling the precise deposition of filaments during printing.²⁵ Notably, these mechanical improvements did not compromise cell viability, underscoring the promise of nanomaterial-reinforced hydrogels as robust scaffolds for engineered tissues, including lung models.

Such formulations can be tailored to replicate the viscoelastic properties of lung tissue, which is essential for capturing the deformation and compliance of airways during respiration. Recent advances have even enabled the bioprinting of small-scale airways containing multiple cell layers using hybrid ECM-based bioinks,²⁶ reflecting a growing sophistication in bioink design aimed at replicating both the structural fidelity and physiological function of native lung tissue.

Integrating robotics with biofabrication

The convergence of multi-axis robotic printing and advanced bioinks is opening new frontiers in lung model engineering. By harnessing robotic arms to precisely deposit cell-laden bioinks in three dimensions, researchers can now fabricate anatomically relevant airway structures that were previously beyond reach. For example, hollow tubes with patient-specific curvature can be printed without sacrificial supports, as the robotic system orients the print head along the surface normal throughout deposition.^7,10 This approach maintains uniform wall thickness and reduces sagging in soft hydrogel constructs. In addition, robotic platforms allow seamless switching between different bioinks or nozzles within a single print, enabling the creation of multi-material constructs such as a supportive scaffold paired with a cell-laden gel.

Previous studies have shown that bioprinted or cast airway-mimetic hydrogels can sustain differentiated epithelial layers and airway-like functions when appropriately seeded and cultured, which motivates the use of anatomically accurate bronchial geometries as a platform rather than a fully validated functional tissue at this stage.^27,28,29 This work is the first to report a multi-axis bioprinting of a bronchial tree segment in soft hydrogel without support materials. The resulting constructs are anatomically accurate and provide a geometrically relevant platform with the potential to support microenvironments resembling ciliated epithelium or respiratory bronchioles in future cell-laden studies. Importantly, the adaptable fabrication approach allows these models to be extended to represent both healthy and pathological respiratory states through targeted modifications in geometry, material composition, and mechanical properties.

Materials and methods

Hybrid hydrogel preparation

Bioinks were formulated using Alginate (Alginic Acid Sodium Salt derived from brown algae) and medium-viscosity CarboxyMethyl Cellulose (CMC), both obtained from Sigma-Aldrich (St Louis, MO, USA) and prepared at a pH of 6.80. Alginate is a widely used, naturally derived biopolymer composed of (1→4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. This negatively charged, linear copolymer dissolves readily in water, supports cell viability, and is known for its excellent biocompatibility. Gelation is primarily mediated through G-blocks, which form ionic crosslinks in the presence of divalent cations, while M and GM blocks contribute to the flexibility of the gel network.

CMC is another anionic, water-soluble biopolymer obtained through the chemical modification of cellulose. Its structure comprises β-D-glucose and β-D-glucopyranose-2-O-(carboxymethyl)-mono-sodium salt units, joined via β-1,4-glycosidic linkages.¹⁵ CMC is commonly employed as a rheological modifier due to its non-toxic, non-allergenic nature and its ability to enhance viscosity and structural stability. Substitution of hydroxyl groups with carboxymethyl moieties improves solubility and thickening capacity.³⁰

Recent work has shown that autoclaving Alginate can reduce molecular weight and modulate viscoelasticity, which in turn can alter cell spreading when cells are encapsulated in the resulting hydrogels. Similarly, studies comparing sterilization methods report that autoclaving may significantly change Alginate’s rheological and printability characteristics, whereas alternatives such as sterile filtration better preserve its physicochemical properties. Given these findings, the current results should be interpreted as representing the properties of non-autoclaved formulations, and future cell-laden studies will explicitly control and report sterilization conditions as an experimental factor.^31,32,33

To investigate the influence of polymer ratios on rheological and printability characteristics, hybrid hydrogel formulations were prepared with varying weight percentages of Alginate and CMC. These formulations were designated as A₅C₅, A₅C₇, and A₅C₈where subscripts represent the percentage of materials. Food-grade colorants were incorporated into each composition to facilitate visual differentiation. Figure 1(a) illustrates the experimental workflow starting with overnight stirring of 5% Alginate and varying concentrations (5%, 7%, 8%) of CMC in deionized water to prepare hybrid hydrogels, and 1(b) shows the resulting formulations are visually assessed for homogeneity. Figure 1(c) highlights the rheological measurements using a 25 mm parallel-plate rheometer under controlled shear rates (0.1–100 s⁻¹) at room temperature. Finally, Figure 1(d) indicates how the printability of the hydrogels is evaluated using a customized bioprinting setup, where nozzle diameter and print height are optimized for consistent filament deposition.

Rheological properties analysis

Rheological measurements (Figure 1(c)) were performed using a rotational rheometer (MCR 102, Anton Paar, Graz, Austria) equipped with a parallel plate geometry (25.0 mm flat plate) and a fixed gap of 1.0 mm. All tests were conducted at room temperature (25°C) to support rapid filament solidification during extrusion. To assess flow characteristics, a steady shear sweep test was carried out across shear rates ranging from 0.1 to 100 s⁻¹. Viscosity data as a function of weight percentage and shear rate were analyzed and plotted using OriginPro 2023b (OriginLab, Northampton, MA, USA).

3D model and printing

Constructs were fabricated using a robot-assisted, extrusion-based 3D bioprinter (BAB 400, Advanced Solutions, Louisville, KY). The print settings included a pressure of 42 psi, an acceleration of 800 mm/s², and a print speed of 5 mm/s. Prepared bioinks were loaded into sterile, a barrel disposable reservoirs (EFD, Nordson, Westlake, OH, USA) and pneumatically dispensed through a dosing nozzle with a 410 µm inner diameter, onto a stationary print bed.

To verify extrusion consistency, various printability characterization tests were conducted. A three-line test was conducted by printing three parallel filaments, each measuring 41.6 mm in length, with a line width of 0.43 mm and a height of 0.2 mm. This test ensures that the extruded filament is uniform and exhibits minimal deviation between lines, confirming stable extrusion performance prior to more complex print geometries. For subsequent printing, a bilayer construct measuring 41.6 mm × 41.6 mm was designed, with a filament spacing of 3 mm. In this context, a bilayer construct refers to a two-layer scaffold in which a second filament layer was printed directly on top of the first layer using the same toolpath geometry. Each layer consisted of a single, planar filament network composed of either pure Alginate, pure CMC, or Alginate–CMC composite formulations (2–14 wt%), and the stacked configuration was used to assess printability by evaluating filament stacking fidelity, pore squareness, and layer-to-layer alignment. Following deposition, printed filaments and scaffolds were sprayed with 4% (w/v) CaCl₂^22,34 solution for physical crosslinking of Alginate. Following crosslinking, no noticeable macroscopic swelling of the printed constructs was observed under the experimental conditions employed in this study. The overall filament dimensions and pore geometries remained stable after crosslinking, suggesting that the selected polymer concentrations and crosslinking parameters effectively limited post-crosslinking volumetric expansion typically associated with alginate-based systems.

Diffusion rate refers to the extent of filament spreading after extrusion and was quantified by measuring the deviation of the printed filament width from the nozzle diameter. Larger deviations indicate greater material diffusion and reduced shape fidelity following deposition. Diffusion rate measurements in this study were conducted using pure Alginate and pure CMC formulations to establish baseline transport behavior as a function of polymer concentration. Diffusion behavior for higher-order hybrid formulations, such as A₅C₇, was not directly measured and therefore should not be assumed to follow a linear combination of the individual polymer responses. We acknowledge that hybrid Alginate–CMC systems may exhibit altered diffusion characteristics due to changes in network density, polymer–polymer interactions, and crosslinking heterogeneity, which can significantly influence mass transport.³⁵ As such, diffusion trends inferred for hybrid formulations were discussed quantitatively through direct experimental quantification in a section ‘Selecting Alginate and CMC based on diffusion rate'.

The model shown in Figure 2 replicates the upper tracheobronchial (TB) region, extending from the mouth to the second bronchial generation. The anatomical geometry was derived from the Human Respiratory Tract (HURT) model.³⁶ The printing process parameters, filament, and 3D scaffolds features are shown in Table 1. These constructs can reasonably be described as primarily architectural at present, but the material system is compatible with future migration-supportive designs. Existing work with Alginate–CMC hydrogels and related Alginate–cellulose systems demonstrates that such matrices can support cell remodeling, spreading, and migration, rather than acting only as inert scaffolds.^35,37,38,39

Table 1.

The printing process parameters, filament, and 3D scaffolds features.

Printing process parameters
Nozzle dia (mm)	Print pressure (psi)	Acceleration (mm/s²)	Print speed (mm/s)
0.41	42	800	5
Filament and scaffold features
Filament (mm)	Filament width (mm)	Layer height (mm)	Line spacing (mm)
41.6	0.43	0.2	3

Mechanical properties analysis

For the bioprinted hydrogel tubular constructs envisioned as lung airway models, it was essential to employ both axial and diametral compression analyses to comprehensively characterize mechanical behavior as shown in Figure 3. This figure illustrates the two mechanical testing configurations used to characterize a hollow bioprinted hydrogel tube for lung model applications. Figure 3(a) illustrates the axial compression, simulating longitudinal loading along the tube’s length, while Figure 3(c) demonstrates the diametral compression, representing radial or circumferential loading. Figure 3(b) links these tests to their relevance in assessing airway mechanics and overall lung compliance. Axial compression assesses longitudinal stiffness and collapse resistance under forces analogous to airway loading or handling stresses, key for structural integrity along the tube’s length. Meanwhile, diametral (or diametral‐tensile) compression, commonly used to infer tensile strength from compressive loading, provides critical insights into the tube wall’s circumferential resistance and failure under radial compression (akin to localized flattening or airway constriction).¹ Hydrogels, with their pronounced viscoelastic and anisotropic behavior, may respond differently depending on loading orientation. Combining both tests offers a full mechanical profile—revealing anisotropy, failure modes, and structural resilience vital for accurate modeling and optimization of lung-mimicking tubular scaffolds.^2,4

Axial compression analysis

Axial compression testing was conducted to evaluate the longitudinal mechanical behavior of the bioprinted tubular constructs when loaded along their vertical axis. This loading mode approximates the type of deformation experienced by airway segments during breathing, where tissues undergo repetitive shortening and elongation. Axial strain $(ε_{axial})$ was calculated from the relative reduction in sample height during loading:

ε_{axial} = \frac{Δ L}{L_{0}} = \frac{L_{0} - L}{L_{0}}

(1)

where

L_{0}

is the original cylinder length and

L

is the compressed length under the applied load. Because hydrogels behave as nearly incompressible materials,^40,41 the original volume of the cylinder was assumed to be constant during compression. The initial volume was calculated as:

V_{0} = A_{0} L_{0}

(2)

where the original cross-sectional area for a hollow cylinder is:

A_{0} = \frac{π}{4} (D_{o}^{2} - D_{i}^{2})

(3)

with

D_{o}

and

D_{i}

representing the outer and inner diameters, respectively. As the construct shortens, its cross-sectional area increases in order to maintain constant volume. The instantaneous area during loading was therefore determined by:

A (L) = \frac{V_{0}}{L}

(4)

This correction captures the lateral bulging behavior commonly observed in hydrated gels under axial compression. True axial stress was computed using the updated area:

σ_{axial} = \frac{F}{A (L)}

(5)

where

F

is the applied compressive force. This formulation ensures accurate estimation of stress even at large strains, where cross-sectional changes become significant.

Young’s modulus (E) was extracted from the linear region of the stress–strain curve:

σ_{axial} = E ε_{axial}

(6)

Only the initial, approximately linear portion of the curve was used to avoid contributions from nonlinear stiffening at higher strains. This analysis provided a quantitative assessment of the cylinder’s compliance, allowing comparison with physiological lung tissue stiffness.

Diametral compression analysis

Diametral compression testing was performed to characterize the circumferential or “ring-like” mechanical response of the bioprinted tubes. This loading mode simulates external forces that flatten the airway, similar to pressures experienced during coughing, airway constriction, or inhalation-driven expansion.^42,43,44 Unlike axial loading, radial compression transforms the initially circular lumen into an ellipse, requiring a more complex geometric analysis. During diametral compression, the internal circular lumen deforms into an ellipse with: (a) Semi-minor axis: aligned with the direction of compression (vertical), (b) Semi-major axis: oriented perpendicular to compression (horizontal).^45,46 The internal perimeter of the lumen was assumed to remain approximately constant during deformation. Using Ramanujan’s first approximation, the elliptical perimeter was estimated as:

P \approx π [3 (a + b) - \sqrt{(3 a + b) (a + 3 b)}]

(7)

and set equal to the original internal circumference:

P_{0} = π D_{i}

(8)

To fully describe the ellipse representing the tube wall, the external semi-axes $(a_{o}, b_{o})$ were derived by enforcing constant wall thickness:

t = a_{o} - a_{i} = b_{o} - b_{i}

(9)

This constraint ensures the wall thickness remains uniform as the lumen reshapes. The original cross-sectional area of the hollow tube was:

A_{original} = \frac{π}{4} (D_{o}^{2} - D_{i}^{2})

(10)

In the deformed state, the new area was computed from the area of the outer and inner ellipses:

A_{elliptical} = π (a_{o} b_{o} - a_{i} b_{i})

(11)

Equality of these two areas ensured incompressibility was respected:

A_{elliptical} = A_{original}

(12)

The radial stress was calculated from the applied load while accounting for the elliptical deformation:

σ_{radial} = \frac{4 F}{π L (a_{o} + a_{i})}

(13)

where

L

is the tube length and

a_{o} + a_{i}

incorporates the expanded geometry across the lateral axis. This formulation provides a more accurate representation of stress distribution in the wall than classical Hertzian or Brazilian disk assumptions, which do not account for hollow tubular geometries.

Cell viability assessment

BxPC3, human pancreatic cancer cells were selected in this study and our published works^22,34 to serve as a robust and well-characterized in vitro model for evaluating the biocompatibility of pure Alginate, pure CMC, and Alginate–CMC composite hydrogels. The primary objective of this cell study was not to replicate tracheobronchial tissue specifically, but rather to assess cytocompatibility, cell survival, and matrix tolerance across different hydrogel compositions. In addition, the use of a cancer cell line enabled preliminary assessment of the potential of Alginate–CMC constructs as drug delivery platforms for cancer-related applications.²² In our recent work, we have also demonstrated the biocompatibility of similar hydrogel systems using human Mesenchymal Stem Cells (hMSCs)⁴⁷ with the intent of directing this material platform toward multiple tissue engineering applications, including lungs, kidney, and bone. Together, these studies highlight the versatility of the Alginate–CMC system and support its adaptability to diverse cell types and biological contexts.

In the present study, cell viability assessments were conducted using pure Alginate and pure CMC formulations. In our previous work, a representative hybrid formulation (4% Alginate–4% CMC, A₄C₄) exhibited improved cell viability compared to pure 4% Alginate, indicating that combining polymers can beneficially modulate the cellular microenvironment relative to single-component hydrogels.²² While higher-order hybrid formulations such as A₅C₇ were not evaluated here, we acknowledge that hybrid compositions may impose distinct cellular responses due to combined effects of matrix stiffness, crosslinking density, and mass transport properties.^48,49 It should also be noted that the cell viability results reported for pure Alginate and pure CMC in this study were obtained from prepared bioinks incubated without extrusion-based printing. In prior work, we demonstrated that increasing applied printing pressure from eight psi to 12 psi reduced Human Embryonic Kidney (HEK) cell viability from approximately 90% to 74%, highlighting the strong influence of processing parameters on post-print cell survival. Accordingly, future studies will focus on incorporating cells into hybrid Alginate–CMC formulations while systematically tuning material composition and printing conditions to enhance post-print viability and recovery.

BxPC3 cell was cultured and maintained in high glucose DMEM, 2 mM Glutamine, and 10% Fetal Bovine Serum (FBS) with 100 µg/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich) in 5% CO₂ at 37°C incubator. The culture medium was changed twice a week. For cell-laden scaffold fabrication, 2 × 10⁶ cells/mL was mixed with Alginate and CMC solutions respectively into disposable barrel reservoir (EFD, Nordson) and dispensed pneumatically through a dosing nozzle (EFD, Nordson, inner diameter 250 µm). The cell viability and cytotoxicity were conducted using LIVE/DEAD assay after four incubation days. Simultaneous fluorescence staining of 1 $μ m o l {mL}^{- 1}$ of calcein-AM and 1 $μ m o l {mL}^{- 1}$ of propidium iodide (PI) are mixed with 2 mL culture medium to stain live and dead cells respectively. After mixing the dye, the scaffold was incubated for 30 min in the dark. Optical and fluorescence images are taken using Lionheart FX automated inverted microscope (BioTek Instruments, Inc) to visualize the live (green) and dead cell (blue) in the scaffold. The z-stack images are captured using 50 µmlayer thickness. The protocol is defined accordingly, and beacon (n = 5) is selected randomly. Laser power and other detector parameters are kept constant throughout the imaging of the different beacons.

Particle deposition measurements

To preliminarily evaluate the feasibility of integrating compliant hydrogel airway constructs with aerosol drug delivery studies, a gravimetry-based aerosol deposition experiment was conducted using a biomimetic hydrogel airway model fabricated via six-axis robotic extrusion bioprinting.

The hydrogel constructs were printed using a hybrid Alginate–CMC formulation (A₅C₇), identified through prior rheological and printability optimization as providing a balance of viscosity, shear-thinning behavior, and structural stability. Printed constructs consisted of cylindrical airway segments representative of proximal airway dimensions and were crosslinked post-printing to preserve lumen geometry.

A Hudson Micro Mist jet nebulizer connected to a Pulmo Aide compressor (Drive DeVilbiss Healthcare, Port Washington, New York, USA) was used to generate aerosolized deionized water as a model carrier fluid. The nebulizer was coupled directly to the hydrogel airway construct via a custom 3D-printed adapter, enabling controlled aerosol exposure of the compliant lumen. Airflow through the system was driven by a downstream vacuum pump to simulate steady inspiratory conditions. Flow rates were maintained at approximately 12–15 L/min and continuously monitored using an inline airflow meter (Alicat Whisper flowmeter, accuracy ±0.8%), corresponding to low-to-moderate adult inspiratory flow during resting tidal breathing. Assuming a typical adult tidal volume of 500 mL and an inspiratory time of 2–3 s, this range reflects physiologically relevant inhalation conditions while enabling steady, repeatable flow through the in vitro airway model.⁵⁰

To capture aerosol particles not retained within the hydrogel airway, the distal airway region was represented by PVC tubing connected to a pass-through filter positioned downstream of the test section. The filter allowed continuous airflow while collecting residual aerosol, enabling confirmation of aerosol delivery through the system. Prior to aerosol exposure, the printed hydrogel construct was weighed using an analytical balance. The nebulizer reservoir was filled with a known volume of deionized water and weighed before and after testing to quantify aerosol delivery. Following a two-minute exposure period, the hydrogel construct was reweighed, and changes in mass were assessedto evaluate potential particle deposition. This gravimetric approach was implemented as an initial, proof-of-concept method to assess whether compliant hydrogel airway models could be integrated into aerosol exposure systems. A schematic and photograph of the experimental setup are shown in Figure 4.

Results and discussion

Selecting Alginate and CMC based on diffusion rate

In developing and selecting bioinks for 3D bioprinting, especially in applications that demand precise geometry and pore fidelity, such as tissue scaffolds or lung models for particle deposition studies, the diffusion rate of the printed material becomes a critical factor.^51,52 In this context, diffusion rate is defined as the percentage change in printed pore area relative to the intended design.²² A high diffusion rate reflects extensive spreading or pore collapse, leading to poor shape fidelity, while a low diffusion rate indicates better geometric stability and preservation of structural detail. As illustrated in Figure 5, Alginate consistently displayed higher diffusion rates across all tested concentrations compared with CMC. At the lowest concentration examined (2% w/v), Alginate exhibited a diffusion rate approaching 100%, corresponding to almost complete pore closure and loss of structural definition. Although diffusion gradually decreased with increasing Alginate concentrations, dropping to ∼78% at 4%, ∼75% at 8%, ∼70% at 12%, and ∼65% at 14%, its tendency toward spreading remained evident. This behavior is likely attributable to Alginate’s low viscosity and moderate gelation characteristics, which limit its ability to maintain complex pore geometry during extrusion.

In contrast to Alginate, CMC exhibited substantially lower diffusion rates at comparable concentrations, reflecting better shape fidelity. At 2% w/v, CMC began with a relatively high diffusion rate (∼95%), but this value dropped sharply to ∼58% at 4% w/v, ∼45% at 8% w/v, ∼25% at 12% w/v, and ∼18% at 14% w/v. This steep inverse relationship between concentration and diffusion rate underscores CMC’s superior capacity to maintain geometric stability at higher concentrations. These trends are evident in Figure 5(a): CMC-printed lattices become progressively sharper and more defined as concentration increases, with crisp pore boundaries and distinct filament separation at 12–14% w/v, features consistent with stable bioink performance during deposition.

By comparison, Alginate constructs show significant spreading and pore collapse across concentrations. At 2% w/v, Alginate grids were nearly indistinguishable from a solid block, confirming near-complete pore closure. Even at 8–12% w/v, the printed grids retained blurred pore edges and fused intersections, illustrating the material’s limitations in maintaining resolution.

Taken together, these findings emphasize the complementary strengths of the two bioinks.

While these results establish clear diffusion-related trends for the individual polymers, they are intended to inform material selection and future hybrid bioink design rather than to claim immediate applicability to fully functional tissue constructs. Alginate offers excellent biocompatibility and efficient ionic crosslinking, while CMC contributes higher viscosity and mechanical stability. To leverage these advantages, hybrid hydrogels combining both polymers were formulated.^22,34 These hybrids were designed to balance print fidelity and post-print stability, particularly for the complex, branching geometries required in lung model fabrication. Among the formulations tested, CMC in the 5–8% w/v range provided the best overall performance, minimizing pore collapse and preserving structural detail. This selection was supported by both quantitative diffusion analysis and qualitative inspection of printed grids (Figure 2).

Cell viability assessment of pure Alginate and CMC

In addition to print fidelity, cell viability was evaluated for pure Alginate and CMC formulations (Figure 5(b)). Across all tested concentrations (2–12% w/v), CMC consistently supported higher viability than Alginate. At 2% w/v, CMC maintained ∼92% live cells, while Alginate supported only ∼34%. At 4% w/v, CMC viability dropped to ∼56% versus Alginate’s ∼29%. By 8% w/v, both materials converged at ∼26–27%, and at 12% w/v, CMC retained ∼17% viability, slightly lower than Alginate’s ∼24%. These data highlight a concentration-dependent trade-off. While increasing concentration improved the print fidelity of both hydrogels, it reduced their ability to support long-term cell survival. The decline was particularly pronounced in CMC, which lost ∼75 percentage points in viability between 2% and 12% w/v, compared to only a ∼10-point reduction for Alginate. This suggests that while CMC excels in achieving high-resolution prints, its dense network at higher concentrations can impede nutrient transport and restrict cell proliferation. In contrast, Alginate, though mechanically weaker, maintains more consistent cell viability across concentrations. These results demonstrate a clear concentration-dependent trade-off between print fidelity and cell viability in pure Alginate and CMC systems, highlighting the need for balanced material design in bioprinting applications. By combining these complementary properties, the A₅C₇ hybrid formulation effectively integrates the print fidelity of CMC with the biocompatibility of Alginate, offering an optimal balance between structural precision and cellular health.

Rheological properties of hybrid hydrogels

As highlighted in the diffusion analysis, CMC and alginate exhibit complementary strengths. At higher concentrations, CMC provides strong shape fidelity due to its high viscosity and lower diffusion rates, whereas alginate offers superior biocompatibility and ionic crosslinking potential. To harness these benefits, we explored hybrid hydrogel formulations for printing geometrically complex constructs such as lung-inspired models.

Although CMC at 12–14% w/v achieved the lowest diffusion and the most stable structures, its high viscosity rendered extrusion impractical. In contrast, pure Alginate was easily extruded but showed poor pore retention due to excessive diffusion. To better understand these differences in flow behavior, we analyzed the viscosity profiles of two representative single-polymer formulations: A₆C₀ (6% Alginate) and A₀C₇ (7% CMC). These formulations were chosen to frame the lower boundary of the printable concentration range near our target design window. A₆C₀, with slightly higher Alginate content than the baseline A₅C₅, demonstrated moderate viscosity and weak shear-thinning behavior. By contrast, A₀C₇ showed a substantial increase in viscosity and stronger shear-thinning, illustrating CMC’s role in stabilizing filament deposition.

Most importantly, combining Alginate and CMC led to a synergistic effect. The hybrid formulation A₅C₇ exhibited both higher viscosity and greater shear-thinning capacity than either single-polymer formulation alone. This behavior is attributed to polymer–polymer interactions and transient entanglement zones, which enhance flow control during extrusion while maintaining stability after deposition.^19,34,53 At equivalent concentrations, neither pure Alginate nor pure CMC could achieve this balance.

High-concentration single-polymer formulations such as A₁₂C₀ and A₀C₁₂ were excluded from comparative rheological analysis because they showed impractical flow behavior, including rapid gelation, erratic filament deposition, and frequent nozzle clogging.⁵⁴ Instead, three hybrid formulations, A₅C₅, A₅C₇, and A₅C₈, were selected for systematic evaluation. These formulations incrementally increased the proportion of CMC, allowing us to study how gradual shifts in rheological properties influence both flow behavior and print fidelity.

The rheological behavior of these bioinks was characterized using steady-shear flow curves and modeled with the power-law equation:

η = K {(\dot{γ})}^{n - 1}

(14)

where

η

is viscosity,

\dot{γ}

is shear rate, K is the consistency index and n, is the flow behavior index.

As shown in Figure 6, all three hybrid formulations exhibited shear-thinning flow behavior, which is characteristic of printable hydrogels. Among them, A₅C₅ demonstrated the lowest viscosity (K ≈ 8.6 × 10⁶ mPa·s) and the highest flow behavior index (n ≈ 0.42), reflecting weaker shear-thinning and therefore reduced shape stability after deposition. In contrast, A₅C₇ displayed intermediate rheological properties (K ≈ 1.01 × 10⁷ mPa·s, n ≈ 0.40), offering a balance between easy extrusion and adequate post-print support. The formulation A₅C₈ achieved the highest viscosity (K ≈ 1.23 × 10⁷ mPa·s) and the strongest shear-thinning behavior (n ≈ 0.35), making it the most resistant to filament spreading and the most structurally stable among the three.

These rheological characteristics are highly desirable for high-resolution bioprinting. Strong shear-thinning facilitates smooth extrusion through the nozzle under applied pressure, while high viscosity following deposition prevents sagging and pore collapse. For lung bioprinting applications, where accurate airway geometry and fine pore resolution are critical to replicating airflow dynamics and particle transport, this balance between flow and stability is essential.

While A₅C₅ is highly extrudable, it may lack sufficient stability to preserve delicate pore structures. A₅C₇ offers a more balanced profile, showing improved reproducibility of printed lattices without excessive extrusion resistance. A₅C₈ exhibits superior shape retention, making it well-suited for replicating intricate structures such as alveolar grids and bronchial branches. However, its high viscosity increases the risk of discontinuous extrusion or rough surface textures during printing. Importantly, the extrusion of A₅C₈ may require pressures exceeding 42 psi, levels that have been associated with reduced cell viability in encapsulated systems. Therefore, A₅C₇ was selected as the optimal formulation, balancing printability, shape fidelity, and biocompatibility. Further print trials will be conducted to refine the formulation and define the best candidate for lung model applications. Overall, the rheological analysis highlights A₅C₇ as a formulation that achieves a practical balance between extrusion stability and post-deposition shape retention within the scope of the current experiments. These results define a preliminary processing window for hybrid Alginate–CMC bioinks; however, additional studies involving cell-laden printing and functional assessment will be necessary to determine their broader applicability to lung tissue modeling.

3D printability test of hydrogels

The comparative pore analysis is presented in Figure 7, which evaluates three parameters—pore area, perimeter, and printability index (Pr), for 16 randomly selected pores (highlighted by green markers in the corresponding image). The printability index (Pr) serves as a dimensionless measure of geometric fidelity, comparing the printed pore shape against its theoretical square design. A value approaching 1.00 represents optimal accuracy.

While the measured pore areas and perimeters show modest variability across the sampled pores, the Pr values remain tightly clustered between ∼0.92 and 1.08. This narrow range indicates that, despite localized differences in extrusion or spreading, the overall deposition process produced pores with consistent geometry and reproducible fidelity. These findings highlight the reliability of the printing parameters in maintaining pore accuracy across multiple sites within a single construct.

Figure 7(a) and 7(b) show single-line filament tests and bilayer grid with 16 randomly selected pores (green dots) for geometric analysis, respectively. Figure 6(c) corresponds to the plot of pore area, perimeter, and printability index (Pr). The hydrogel exhibits consistent pore fidelity, with most Pr values close to unity (ideal square), confirming material stability under extrusion and layered deposition. Printability comparison of bilayer constructs printed with 12% w/v Alginate, 12% w/v CMC, and (f) hybrid A₅C₇ hydrogel (12% w/v total solids) are shown in Figure 7(d), 7(e), and 7(f), respectively. While Alginate shows the highest pore deformation, A₅C₇ maintains sharp, defined pores with minimal spreading. CMC shows moderate pore integrity. These results highlight the enhanced shape fidelity of the hybrid formulation.

Most pores exhibited printability (Pr) values between 0.95 and 1.05, reflecting reliable filament placement and consistent pore geometry throughout the construct. A few deviations were observed: pores 13 and 14 exceeded 1.08 and 1.02, respectively, suggesting local regions of over-deposition or edge thickening. Conversely, pores 3 and 15 fell below 0.94, likely due to under-deposition or filament spreading. Although these deviations are relatively minor, they underscore the importance of spatial uniformity, since small errors can accumulate in multi-layered constructs and ultimately compromise the architecture. Maintaining Pr values close to unity is therefore critical for reproducing anatomically accurate airway structures. Importantly, well-defined pore lattices govern both scaffold porosity and airflow dynamics, making printability a predictive measure for scalable lung tissue models. The unit-cell analysis confirmed that the tested hydrogel could reproducibly generate microstructures with high shape fidelity. This level of reproducibility at the microscale strongly supports the hydrogel’s suitability for fabricating larger, multi-generational bronchial networks, where geometric precision is essential for both biological function and mechanical stability.

Figure 7(d)–(f) further compares pore fidelity in bilayer constructs fabricated with 12% w/v pure Alginate, pure CMC, and the hybrid blend A₅C₇. Among these, A₅C₇ preserved pore geometry most effectively, producing sharp boundaries with minimal diffusion or collapse. CMC displayed moderate deformation, with pores remaining distinguishable but less defined, while Alginate exhibited extensive spreading and structural weakening at the same concentration. These visual observations align with the quantitative diffusion data (Figure 5(a)), where Alginate showed a diffusion rate of ∼65% at 12% w/v, compared with ∼20% for CMC. The superior geometric retention of the A₅C₇ blend highlights the synergistic interaction between Alginate and CMC, balancing viscosity, shear-thinning, and crosslinking behavior to achieve improved performance.

Overall, these results identify A₅C₇ as the most promising bioink for printing high-resolution, structurally stable scaffolds. Its ability to minimize pore collapse and preserve sharp edges at clinically relevant scales makes it particularly well-suited for replicating the intricate geometry of lung tissue. By maintaining both mechanical integrity and compatibility with cell-based applications, A₅C₇ provides a strong foundation for building complex tissue-engineered lung models that are essential for advancing respiratory research. While the present analysis establishes its suitability for producing structurally consistent, high-resolution bilayer scaffolds, further studies incorporating multi-layer constructs and cell-laden systems will be required to fully validate its applicability to functional lung tissue models.

3D printing of a 2^nd generation dual-lobed model

Building on the rheological and printability properties described earlier, the hybrid A₅C₇ hydrogel was employed to fabricate a second-generation dual-lobed construct designed to mimic the upper bronchial generations. This formulation was selected because it combines moderate viscosity (K ≈ 1.01 × 10⁷ mPa·s) with favorable shear-thinning behavior (n ≈ 0.40), providing both smooth extrusion through the nozzle and stable layer-by-layer deposition.

To validate the utility of the six-axis robotic platform prior to fabricating complex bronchial constructs, we performed inclined extrusion experiments with hydrogel cylinders (Figure 8(a)). By tilting the nozzle 30° from the vertical, the robot actively reoriented deposition to follow the construct’s curvature rather than relying solely on planar, layer-by-layer buildup as in gantry-based printers. This approach demonstrated the feasibility of angled deposition without the need for support structures, highlighting a clear distinction from conventional extrusion systems. Under a pneumatic pressure of 42 psi, the theoretical extrusion force at the nozzle tip was calculated to be ∼ 0.038 N. With the nozzle inclined at 30°, the vertical component of this force was reduced to ∼0.033 N, representing a 13% decrease compared to gantry-based vertical deposition. This reduction in vertical compaction minimizes deformation of underlying layers and prevents pore collapse, leading to improved surface fidelity and lumen retention. The visual evidence confirms that angled deposition produced smooth cylindrical geometries with consistent wall thickness, free of sagging or layer misalignment. These observations provide direct qualitative evidence that the robotic arm’s multi-axis control was implemented and beneficial, rather than simply theoretical. The successful demonstration of inclined extrusion established confidence in the platform’s ability to manage delicate soft hydrogel constructs, serving as an essential precursor to the fabrication of bifurcating lung-inspired models without support structures.

As shown in Figure 8(b)–(g), the resulting constructs demonstrated high reproducibility and morphological fidelity, accurately preserving the bifurcation geometry and airway openings characteristic of bronchial anatomy. These features underscore the suitability of A₅C₇ for printing anatomically relevant lung models, where geometric precision is essential for replicating airflow and deposition dynamics.

The printed constructs maintained their intended anatomical design throughout the deposition process, showing minimal deformation and consistent layer fusion. Top and bottom views confirmed uniform material deposition, while isometric perspectives highlighted the clear separation of the dual lobes and the fidelity of their branching junction. Together, these observations validate the hydrogel’s suitability for fabricating geometrically complex soft-tissue models through extrusion-based bioprinting combined with robotic actuation.

Following fabrication, the constructs were stabilized using ionic crosslinking, which enhanced their mechanical integrity. As illustrated in Figure 9(a)–(b), the post-crosslinking models preserved both their overall geometry and airway bifurcations. The isometric and top views further confirmed the fidelity of lumen structures and branching interfaces. Additional handling tests, including vertical suspension and mechanical probing (Figure 9(c)–(d)), demonstrated that the scaffolds could withstand manipulation without collapsing or tearing. This robustness is especially critical for downstream applications, such as airflow simulations and particle deposition studies, where constructs must retain their structural integrity under both physical handling and fluid exposure. The present findings confirm reproducible fabrication and post-crosslinking stability of anatomically accurate bilayer airway constructs, serving as a foundational demonstration of geometric precision. Extending this approach to thicker, cell-laden, and physiologically relevant systems will be essential for translating these constructs toward advanced pulmonary modeling applications.

Mechanical property analysis on bioprinted constructs

Qualitative observations

To assess mechanical resilience and elastic recovery, the crosslinked constructs were subjected to compressive loading. As shown in Figure 9, under a 10 g compressive force (Figure 9(b)), the scaffold retained its geometry with no visible deformation. Under a higher compressive load of 100 g 9(c), the constructs were temporarily flattened, resulting in complete lumen closure. However, minimal manual repositioning restored the original shape, indicating elastic memory and robust structural cohesion. Further tests with lateral compression (Figure 9(d)) revealed the scaffold’s ability to maintain bifurcation integrity even under off-axis loading. These results highlight the hybrid hydrogel’s capacity for elastic recovery and suggest its suitability for modeling deformable airway tissues. This qualitative mechanical assessment is critical for lung research because it demonstrates the scaffold’s ability to undergo large, reversible deformations while preserving airway geometry and bifurcation integrity, key mechanical behaviors required to mimic the cyclic compression, expansion, and elastic recoil experienced by native airways during breathing.^26,29 In next section, these qualitative assessments will be complemented by quantitative strain-recovery analysis to correlate rheological parameters with mechanical behavior. This will support the design of airway scaffolds that replicate physiological actuation under breathing cycles.

Quantitative observations

Axial compression

Under axial compression, where the printed A₅C₇ hydrogel tube was loaded along its longitudinal axis, the stress–strain response was approximately linear over the tested range (Figure 10). The axial stress started at 0.26 kPa at zero strain and increased to 0.65 kPa under a small load at essentially the same strain (ε ≈ 0). As deformation progressed, the stress rose to 1.24 kPa at ε ≈ 0.06, 1.88 kPa at ε ≈ 0.28, 3.09 kPa at ε ≈ 0.53, and reached 3.72 kPa at the maximum measured strain of ε ≈ 0.72. A linear fit to the axial loading data yielded

σ_{a} = 4.77 ε_{a} + 0.58, R^{2} = 0.94,

(15)

indicating a consistent effective axial modulus of ∼4.8 kPa with good correlation.

Diametral compression

When the same tube was subjected to diametral (radial) compression, i.e., flattened across its diameter, the material displayed a higher resistance to deformation over a similar strain range (Figure 10). Radial stress was 0.26 kPa at ε_r ≈ 0.01, increasing to 0.66 kPa at the same small strain. With further loading, stress rose to 1.20 kPa at ε_r ≈ 0.14, 1.98 kPa at ε_r ≈ 0.35, 3.90 kPa at ε_r ≈ 0.66, and reached 6.97 kPa at ε_r ≈ 0.83. The corresponding linear regression,

σ_{r} = 5.15 ε_{r} + 0.41, R^{2} = 0.98,

(16)

reveals a slightly higher effective radial modulus of ∼5.2 kPa and an even stronger linear fit.

At comparable strain levels, the diametral stresses consistently exceeded the axial stresses. For example, near ε ≈ 0.35, the radial stress was approximately 1.98 kPa, while the closest axial data point at ε ≈ 0.28 showed a stress of 1.88 kPa; at the upper end of the range (ε_a ≈ 0.72 vs ε_r ≈ 0.83), the radial stress (6.97 kPa) was nearly 1.9 times the axial stress (3.72 kPa).

This disparity demonstrates that the bioprinted tube is circumferentially stiffer than it is longitudinally, which is biomechanically relevant for airway modeling: enhanced radial rigidity helps maintain lumen patency under external or transmural pressure fluctuations, while a more compliant axial response allows the construct to deform with lung expansion and contraction. This quantitative assessment is critical for lung research because it shows that the bioprinted A₅C₇ tube operates in a lung-relevant stiffness regime (effective axial modulus ≈4.8 kPa, within the reported 1–5 kPa range for lung parenchyma) while simultaneously providing higher circumferential rigidity (effective radial modulus ≈5.2 kPa and stresses up to ∼1.9× the axial value at comparable strains),^26,55 a combination that directly supports lumen patency under pressure fluctuations yet allows compliant axial deformation during breathing cycles. By explicitly resolving stress–strain behavior in both axial and radial loading modes, these measurements go beyond qualitative “soft versus stiff” descriptions and quantitatively benchmark the scaffold against known mechanical requirements of native airways, cyclic expansion, compression, and elastic recoil, thereby establishing a rational basis for using this construct as a mechanically faithful airway segment in lung models. Together, these results demonstrate that the printed tube behaves as a soft, lung-like material in axial loading, while providing enhanced radial support—an advantageous combination for mimicking airway segments that must remain open yet deform with breathing.⁵

Particle deposition

Following fabrication and crosslinking, the hydrogel airway construct maintained structural integrity and lumen patency during coupling with the aerosol delivery system. Prior to testing, the printed hydrogel cylinder weighed 1.273 g and the nebulizer was filled with 5 mL of deionized water, weighing 31.144 g.

After the two-minute aerosol exposure, the nebulizer mass decreased to 30.9 g, corresponding to delivery of approximately 0.91% of the nebulized liquid through the system. Despite confirmed aerosol delivery, the post-test mass of the hydrogel construct decreased to 1.235 g, representing an approximate 3% reduction relative to its initial mass. No measurable mass gain of the hydrogel airway was detected following exposure under the tested conditions.

This preliminary aerosol deposition experiment demonstrates the feasibility of integrating mechanically compliant, hydrogel-based airway constructs fabricated via six-axis robotic bioprinting with aerosol delivery systems. The hydrogel model successfully withstood coupling, airflow, and aerosol exposure without collapse or loss of lumen integrity, supporting its suitability as a platform for future drug delivery investigations.

However, gravimetric analysis did not reveal measurable aerosol mass deposition within the hydrogel construct. Although aerosol delivery was confirmed by the reduction in nebulizer reservoir mass, the absence of detectable mass gain highlights limitations of bulk gravimetric methods when applied to hydrated, compliant materials. The high intrinsic water content of Alginate–CMC hydrogels, combined with their susceptibility to handling-induced deformation and transient moisture loss, likely obscures small deposition signals. The observed reduction in hydrogel mass following testing is likely attributable to experimental handling, compression within the coupling adapter, and short-term evaporation during exposure and weighing. These effects underscore the challenge of quantifying aerosol deposition in soft, water-rich biomaterials using methods traditionally applied to rigid thermoplastic airway models.

Importantly, the goal of this experiment was not to quantify deposition efficiency, but rather to establish proof-of-concept compatibility between bioprinted hydrogel airways and aerosol exposure hardware. In this context, the results highlight the need for more sensitive and spatially resolved measurement techniques—such as fluorescent or radiolabeled tracers, localized imaging, or qualitative visualization—to accurately assess deposition behavior in compliant biomimetic airway models.

Overall, these findings complement the demonstrated print fidelity and mechanical resilience of the Alginate–CMC airway constructs, supporting their potential use in future studies of aerosol transport and drug delivery. With appropriate refinements in deposition measurement strategies, this platform can be extended to investigate both healthy and pathological respiratory states through targeted modifications in geometry, material composition, and functionalization.

Future direction

We plan to advance the platform toward the fabrication of third generation and deeper bronchial branches using six-axis robotic printing to preserve lumen continuity and bifurcation fidelity, thereby enabling investigation of aerosol transport and deposition across increasingly complex lung architectures. Such models will allow quantitative comparison of spatial deposition patterns across defined airway generations systematic evaluation of deposition profiles under a range of conditions, systematic variation of particle aerodynamic diameter, steady versus transient inspiratory flow rates, and controlled inhalation profiles representative of healthy and diseased lungs. including variations in particle size, airflow rate, and inhalation dynamics. To overcome the limitations of gravimetric analysis identified in this study, future deposition experiments will transition from traditional gravimetric methods to qualitative and semi-quantitative approaches, including fluorescent or dye-labeled aerosols, enabling visualization of relative drug distribution and spatial deposition patterns within compliant hydrogel airways. This approach will enable direct comparison between rigid and compliant airway models under identical flow conditions, establishing the impact of tissue compliance on aerosol transport.

Another critical next phase involves the biological integration of living cells within the hydrogel constructs. Previous studies using the A₄C₄ formulation demonstrated excellent shape fidelity in structures up to 1.2 cm in height while maintaining ∼89% cell viability after 15 days of culture.^22,34 Building on these results, the A₅C₇ formulation will be systematically evaluated for biocompatibility, epithelial attachment, and barrier formation and suitability in both cell-laden and cell-seeded constructs, enabling development of a functional airway lining..

This study emphasized process development, hydrogel formulation, and mechanical characterization of printed constructs, while the integration of living cells and spatially resolved aerosol deposition measurements areand quantitative aerosol deposition analysis based on fluorescent imaging are intentionally reserved for future work. To this end, although the current constructs were printed at 42 psi to maximize fabrication speed, future efforts will decouple printing speed from extrusion pressure through optimization of nozzle geometry and extrusion profiles to improve cell survival without sacrificing structural fidelity. Reduce extrusion pressure to improve cell survival. Optimization of print parameters will emphasize the balance between cell viability and structural integrity.

In terms of biological relevance, we will incorporate human bronchial epithelial cells (HBECs), which provide a physiologically relevant model of the upper airway mucosa,⁵⁶ alongside A549 alveolar epithelial cells, widely employed in distal lung toxicity and uptake studies.⁵⁷ These co-culture systems will enable investigation of how aerosolized therapeutics and particulates interact with airway and alveolar epithelium under controlled mechanical and flow conditions.

Ultimately, these studies will extend beyond construct fabrication to mechanistically evaluate aerosol–tissue interactions examine how aerosolized particles interact with embedded cells, focusing on endpoints such as cell viability, oxidative stress, and inflammatory signaling pathways. Collectively, these efforts aim to establish a tunable, compliant in vitro lung platform capable of isolating the roles of geometry, material compliance, and cellular response in respiratory drug delivery and inhalation toxicology.

Conclusions

This study demonstrates the potential of a robot-assisted, six-axis bioprinting platform combined with hybrid Alginate–CMC hydrogels to fabricate anatomically realistic and mechanically compliant lung models and subsequent particle deposition measurements. By systematically tuning hydrogel formulations and rheological properties, we identified A₅C₇ as the optimal bioink, offering a balance of printability, shape fidelity, and biocompatibility. The extrusion tests, pore fidelity analyses, and compression studies confirmed that A₅C₇ enables reproducible fabrication of bifurcating airway constructs that maintain lumen integrity under deformation and recover elastically, capturing key features of respiratory mechanics. These findings validate the feasibility of engineering soft, branching airway models that overcome the structural limitations of conventional 3-axis printing and single-polymer hydrogels. The fabricated constructs establish a geometrically accurate and mechanically stable platform that can be leveraged in future studies to investigate airflow dynamics, aerosol deposition, and toxicological interactions, following dedicated flow characterization and biological validation. Looking forward, integration of human bronchial and alveolar epithelial cells, along with expansion into deeper bronchial generations, will transform this platform into a biologically active and physiologically relevant lung model. This study extends additive manufacturing capabilities by demonstrating that complex organ geometries can be produced in soft hydrogels via robotic multi-axis control, which could be applied to other tissue models or customized scaffold fabrication. Together, this work establishes a path toward next-generation in vitro systems that bridge engineering innovation with respiratory biology, advancing applications in drug delivery, inhalation toxicology, and regenerative medicine.

Footnotes

Acknowledgements

The authors gratefully acknowledge the contributions of students, Tanush Senthil Kumar and Chris Nuzzolo, who assisted in data collection for this study. Their dedication, attention to detail, and persistence were invaluable in ensuring the quality and completeness of the dataset.

ORCID iD

Ahasan Habib

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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Six-axis robotic extrusion of hybrid hydrogels for biomimetic airway model fabrication

Abstract

Keywords

Introduction

Advancements in bioprinting technology

Biofabrication materials for lung models

Integrating robotics with biofabrication

Materials and methods

Hybrid hydrogel preparation

Rheological properties analysis

3D model and printing

Mechanical properties analysis

Axial compression analysis

Diametral compression analysis

Cell viability assessment

Particle deposition measurements

Results and discussion

Selecting Alginate and CMC based on diffusion rate

Cell viability assessment of pure Alginate and CMC

Rheological properties of hybrid hydrogels

3D printability test of hydrogels

3D printing of a 2nd generation dual-lobed model

Mechanical property analysis on bioprinted constructs

Qualitative observations

Quantitative observations

Axial compression

Diametral compression

Particle deposition

Future direction

Conclusions

Footnotes

Acknowledgements

ORCID iD

Funding

Declaration of conflicting interests

Appendix

References

3D printing of a 2^nd generation dual-lobed model