Tissues in the body commonly include a fibrous matrix with exquisite fiber alignment along different directions, which guides cell alignment and spreading [1], [2], [3], [4], [5], [6], [7], [8], [9]. The most abundant fibrous protein of the native extracellular matrix (ECM) in the human body is collagen, and varying, complex patterns of collagen fiber alignment are observed across different tissues, such as the cornea, cartilage, and arteries. [5], [8], [10], [11], [12], [13]. For example, in the cornea, the ECM contains orthogonal sheets of collagen fibrils that are able to transmit light while being mechanically resilient [5], [14]. In articular cartilage, collagen fibril architecture exhibits depth-dependent organization: fibrils are aligned parallel to the surface in the superficial zone, display a more isotropic distribution in the transitional zone, and orient perpendicular to the surface in the deep zone, where they anchor into the calcified cartilage [12]. This zonal arrangement restricts tissue swelling, facilitates osmotic pressurization for load-bearing, and confers both compressive compliance and shear resistance. Arterial collagen fibers are predominantly arranged in a helical pattern, enabling them to reinforce the artery in both the circumferential and axial directions [13], [15].

Collagen fiber alignment can direct many physiological cellular processes, such as migration [16], [17], [18], differentiation [6], [19], and proliferation [20]. For example, cellular alignment has been widely reported to impact cell proliferation [21], matrix secretion [22], [23], and phenotype maintenance [24], [25]. Thus, a common goal in tissue engineering is to control the orientation of collagen fibers to guide cell behavior [6], [7], [16], [26]. To date, several fabrication techniques have been used to create substrates with aligned collagen fibers, including electro-spinning, magnetic flow alignment, microfluidics, mechanical strain devices, extrusion through a tapered nozzle, and gravity-based fluidic alignment [6], [16], [26], [27], [28], [29]. While these aligned collagen constructs have been helpful tools to study cell–matrix interactions such as mechanosensing, cell migration speed, and cell persistence, these techniques are typically limited to unidirectional fiber alignment [16], [30], [31].

Although existing techniques like electrospinning [20], [27], [32], [33] enable nanoscale fiber alignment with high precision, it is typically limited to 2D surfaces or thin scaffolds, lacking structural complexity and spatial control in 3D constructs. Magnetic field–based methods [28], [29], [33] can orient fibers in 3D hydrogels, but they require incorporation of magnetic nanoparticles or specialized materials, and are constrained by the need for strong, spatially uniform magnetic fields. Shear flow–based alignment, such as induced by microfluidic [29] or controlled extrusion [7], [26], [34], [35], can achieve unidirectional alignment but often lacks the ability to spatially modulate alignment direction within a single filament or print path. Mechanical strain–based methods [9], [36], like stretching gels post-printing or during curing, offer bulk alignment but are unsuitable for patterning complex, heterogeneous orientations and often compromise spatial resolution. In addition, many of these conventional approaches rely on discrete, layer-by-layer deposition strategies to alternate fiber alignment direction, which can introduce inter-filamentary gaps and discontinuities while printing complex geometries. Frequent lifting and repositioning of the printhead further interrupts material flow, reducing control over fiber orientation and potentially compromising print fidelity. Creative approaches to overcome these limitations and fabricate radial patterns of collagen fibers have employed Marangoni flow in evaporating, aqueous sessile droplets [37] and microextrusion of discrete filaments in different macroscopic orientations [26]. Yet, it remains challenging to achieve larger constructs with controlled fiber alignment in multiple directions.

3D printing of biological materials has emerged as a reproducible, scalable, and versatile method to fabricate constructs with clinically relevant dimensions and increased complexity [38], [39], [40], [41], [42], [43], [44], [45]. In particular, extrusion-based 3D printing offers ease of use and the potential to produce oriented constructs. Alignment in the extrusion direction has been achieved through different methods for a variety of polymers and fibrous materials. For example, alignment can be induced inside the nozzle by subjecting the material to controlled flow profiles during extrusion; alternatively, alignment can be induced outside of the nozzle by subjecting the printed filament to extensional deformation [7], [34], [35], [46], [47], [48], [49], [50], [51]. For fibrous collagen inks, shear stress-induced fiber alignment has been achieved through extrusion-based 3D printing, resulting in constructs with uniaxially aligned fibers in the direction of printing [7], [26].

Here, we sought to develop an easy to implement and reproducible 3D printing strategy to create constructs with dual-directionally aligned collagen fibers. Specifically, we demonstrate the ability to pattern collagen fibers both parallel to and perpendicular to the printing direction, enabling the fabrication of constructs with more complex patterns of fiber alignment. The primary innovation of this technique lies in its ability to modulate collagen fiber orientation within a single printed filament, enabling transitions between parallel and perpendicular orientations in situ. This facilitates the generation of complex, spatially patterned fiber alignments simply by adjusting the printing parameters during continuous extrusion, without the need for changing the printhead trajectory.

To achieve this, we hypothesized that two different printing regimes could be achieved within a single printing setup. The first regime relies primarily on extensional deformation of the collagen ink within a tapered print nozzle to achieve fiber alignment that is parallel to the print direction. In the second regime, we rationalized that controlling the lateral spreading of the printed filament post-printing can be used to induce collagen fiber alignment perpendicular to the printing direction. To explore this idea, we first theoretically predicted how different printing parameters might impact filament lateral spreading. We then performed a systematic evaluation of the printing parameters for our extrusion-based 3D printer and quantified the collagen fiber alignment patterns for each condition. These experimental observations validated our predictions and allowed us to define the printing parameters for each regime of fiber alignment. We next demonstrated the ability of these parallel and perpendicularly aligned collagen substrates to guide the spreading morphology of cells. Using this new printing strategy, we fabricated specimens with dual-directional collagen fiber alignment in a single print. Taken together, this work introduces a predictable, reproducible, and scalable approach to print biomimetic constructs with complex patterns of collagen fiber directionality to guide cellular alignment, while eliminating the need for external fields, complex device modifications, or material additives. The simplicity and compatibility of this method with standard extrusion-based 3D printing make it broadly applicable for engineering tissue-mimetic collagen architectures with high spatial precision.