Introduction
Tissue-engineered constructs offer valuable platforms for in vitro investigation and serve as potential substitutes for diseased or impaired tissues. As a rapidly advancing technology, three-dimensional (3D) bioprinting facilitates tissue engineering by enabling the spatially controlled deposition of biomaterials and living cells into predefined architectures [1]. Furthermore, 3D bioprinting overcomes the microstructural precision limitations typically associated with conventional manufacturing methods [2]. Among the various 3D bioprinting techniques, extrusion-based printing is the most widely used due to its extensive material versatility, ease of implementation, and adaptability across diverse fabrication environments. Guided by computer-aided design (CAD), this method enables the precise tuning of structural parameters, including pore morphology and distribution [3, 4]. 
Hydrogels are considered promising bioinks for 3D bioprinting due to their biocompatibility, biodegradability, and structural similarity to the extracellular matrix (ECM). Furthermore, their high water content facilitates efficient diffusion of nutrients and gases, which is vital for maintaining cell viability within printed constructs [5]. The printability of hydrogels is influenced by multiple factors, including their chemical composition, viscosity, and key printing parameters such as nozzle diameter, temperature, pressure, and flow rate [6, 7]. To ensure smooth extrusion during bioprinting, bioinks are generally formulated with low viscosity [8]. However, increasing viscosity and using finer nozzle diameters can significantly improve the resolution of printed constructs. Despite this advantage, these conditions expose encapsulated cells to elevated shear forces during deposition, which may adversely affect their viability and biological function [9]. Moreover, extrusion pressure is a critical parameter in bioprinting, as elevated pressure levels can produce thicker filament strands during deposition. Strand size in bioprinted scaffolds has a significant influence on the survival and growth of embedded cells. Thicker strands can impede the transport of essential nutrients and oxygen, resulting in suboptimal cellular function [10, 11]. Therefore, careful optimization of these variables is essential for achieving high-quality printed constructs and promoting favorable cellular outcomes. 
Among naturally sourced hydrogels, alginate (Alg), a polysaccharide derived from brown algae, is recognized as one of the most frequently utilized materials in bioprinting applications [12] due to its favorable biocompatibility, non-toxicity, ECM-like physical structure, rapid gelation modulated by divalent cations such as calcium and barium, high aqueous solubility, and low cost [13-15]. However, its limited mechanical strength, poor print fidelity, and lack of intrinsic bioactive motifs for cell adhesion and differentiation remain significant challenges in tissue engineering [16, 17]. Filler nanomaterials, such as carbon-based nanomaterials, play a pivotal role in modulating both the structural and physicochemical properties of printed hydrogels. In addition to enabling customization of scaffold architecture, these additives significantly influence the rheological behavior of bioinks during the bioprinting process. Consequently, their incorporation can directly impact the fidelity, resolution, and overall quality of the final printed scaffold [18-20]. Graphene oxide (GO), a type of carbon-based nanomaterial, contains functional groups such as carboxyl, hydroxyl, and epoxy. These groups enable strong molecular interactions, which enhance the mechanical properties of GO-incorporated hydrogels compared to those without GO [21]. As a result, incorporating GO into Alg hydrogels can enhance both their mechanical strength and printability [22]. This study aims to optimize key 3D printing parameters to fabricate scaffolds with structural and functional properties suitable for tissue engineering applications.

Materials and Methods
Preparation of bioinks
To prepare the bioinks, 6%, 7%, and 8% (w/v) sodium Alg (W201502, Sigma-Aldrich) were dissolved in deionized water and vortexed for 20 minutes to ensure complete dissolution. The resulting Alg solutions were then combined with varying concentrations of GO dispersions at 0, 0.05, 0.1, and 2.0 mg/mL (Graphene X, Iran). The mixtures were stirred continuously for an additional 20 minutes to promote the uniform distribution of GO within the hydrogel matrix.

3D printing process for scaffold construction
Following preparation, the bioink was loaded into the syringe reservoir of an extrusion-based 3D bioprinter N2 (3DPL Co. Ltd., Tehran, Iran), equipped with 3-axis motion control (x, y, and z) and a pneumatic extrusion system operating at pressures below 2 bar. Cube-shaped scaffold models (20×20 mm, 5 layers) were designed using AutoCAD software. The resulting geometries were exported in STL format and subsequently converted into G-code using Simplifier software to enable 3D printing. All samples were printed at room temperature with a consistent printing speed of 60 mm/s [23]. Figure 1 demonstrates the bioprinting process.

Optimization of key 3D bioprinting parameters
To enhance the printability, mechanical stability, and geometric precision of the Alg-GO constructs, critical 3D bioprinting parameters, including hydrogel concentration, extrusion pressure, and needle diameter, were optimized. To determine the optimal Alg concentration, 3D bioprinting was conducted using Alg solutions at 6%, 7%, and 8% (w/v), each combined with varying GO concentrations (0, 0.05, 0.1, and 2 mg/mL). The complete set of bioink formulations is summarized in Table 1.

Subsequently, to address the optimal nozzle diameter, the bioprinting process was performed using 22-G and 25-G nozzles exclusively with the optimized concentration of Alg, i.e. the Alg8-GO-0 formulation. Following this, the Alg8-GO-0, Alg8-GO-1, Alg8-GO-2, and Alg8-GO-3 formulations were bioprinted using the optimized nozzle diameter, while varying the extrusion pressure between 0.85 and 1.4 bars to identify the most suitable pressure for each sample.

Statistical analysis
All experiments were done three times, and the results are shown accordingly.

Results
Bioprinted Alg-based scaffolds often suffer from poor shape fidelity due to insufficient viscosity. Increasing Alg concentration enhanced viscosity and improved structural stability during printing. As illustrated in Figure 2, the 6% Alg formulation (Alg6-GO-0) exhibited acceptable printability.

However, upon incorporating GO, the bioinks failed to maintain adequate structural integrity post-extrusion. Subsequently, the Alg concentration was increased to 7%, and the 3D bioprinting process was repeated. As expected, the Alg7-GO-0 formulation demonstrated acceptable printability; however, Alg7-GO-1 exhibited poor structural uniformity and integrity (Figure 2). Consequently, further printing with Alg7-GO-2 and Alg7-GO-3 at the same concentration was not pursued. In the next phase, samples Alg8-GO-0, Alg8-GO-1, Alg8-GO-2, and Alg8-GO-3 were successfully bioprinted, all displaying satisfactory printability and shape fidelity following the extrusion process (Figure 2). Based on these results, a concentration of 8% was identified as the optimal formulation in this study, providing superior printability and structural integrity compared to lower concentrations.
Printing Alg8-GO-0 with a 25-G nozzle required substantially higher pressure (>1 bar) due to its smaller inner diameter, resulting in thinner filaments and larger pores. The extrusion process lacked continuity, and the hydrogel was deposited discontinuously (Figure 3a).

The incorporation of GO particles further exacerbated this issue. These findings indicate that the 25-G nozzle is unsuitable for this application. In contrast, printing with the 22-G nozzle required lower pressure (<1 bar), produced slightly thicker filaments, and yielded smaller pores (Figure 3b). The extrusion process exhibited acceptable continuity, suggesting that the 22-G nozzle offers a more favorable balance between pressure requirements and print quality.
In the next stage, various extrusion pressures were evaluated across four groups to identify the optimal pressure that provides the highest printing resolution. For Alg8-GO-0, an initial pressure of 0.99 bar produced continuous ink flow but resulted in excessive strand thickness, leading to partial pore closure. Reducing the pressure to 0.85 bar improved both filament diameter and pore uniformity, establishing it as the optimal setting for this group (Figure 4).

Alg8-GO-1, initially printed at 0.85 bar, produced thinner strands and larger pores compared to Alg8-GO-0, although continuity was slightly compromised in some regions. To refine pore size, the pressure was increased to 0.99 bar and then to 1.10 bar; however, inadequate ink flow prompted a further adjustment to 1.25 bar, which yielded structurally acceptable scaffolds and was deemed optimal (Figure 4). For Alg8-GO-2, 0.85 and 0.99 bar pressures were insufficient due to low ink flow and nozzle clogging, probably caused by high surface adhesion forces [24]. At 1.25 bar, consistent flow and desirable strand morphology were achieved (Figure 4). Alg8-GO-3 could not be printed at 0.85 bar, and printing at 0.99 bar resulted in undesirable scaffold features. Ultimately, 1.25 bar was selected, with a mid-process increase to approximately 1.4 bar to overcome nozzle blockage and ensure consistent strand deposition (Figure 4). 

Discussion
The results highlight the importance of optimizing bioink composition and printing parameters for cardiac tissue engineering. Increasing Alg concentration improved viscosity and shape fidelity, with 8% Alg providing the most stable scaffolds by increasing the number of G-blocks, thereby enhancing structural stability during printing [6]. Lower concentrations were insufficient, particularly when GO was incorporated. This may be attributed to the reduced viscosity of the Alg hydrogel observed after incorporating low GO concentrations (<0.2 mg ml⁻¹), may be due to the homogeneous and parallel alignment of GO sheets along the Alg polymer chains without contacting each other [25]. 
As mentioned before, nozzle diameter is a critical parameter in the 3D bioprinting process. The filament width and pore size of the printed constructs are directly influenced by the nozzle's inner diameter [26]. In our study, the 25-G nozzle produced discontinuous filaments and was prone to clogging at the nozzle tip, especially with GO sheets, making it unsuitable. The 22-G nozzle offered a better balance between extrusion pressure and print quality. When cells are present in the bioink, a larger nozzle diameter is preferable, as previous studies on cell-laden Alg hydrogel bioprinting have shown that larger nozzle diameters result in higher cell viability [27]. This improvement is likely due to reduced shear stress, which otherwise adversely affects the viability of encapsulated cells when small-diameter nozzles are used [9].
Extrusion pressure optimization is formulation-dependent. Excessive extrusion pressure generally produces thick strands, while insufficient pressure may result in irregular filament deposition [20, 28]. Additionally, the incorporation of GO into Alg hydrogels results in the formation of thinner filaments and larger pores, likely due to enhanced molecular interactions between Alg and GO [20]. This phenomenon becomes particularly significant when bioinks are cell-laden, as the dimensions of printed strands directly influence the viability and proliferation of encapsulated cells. For example, thicker filaments may hinder the diffusion of essential nutrients and oxygen, potentially compromising cell survival within the scaffold [10, 11]. Consequently, identifying the optimal extrusion pressure for each formulation, based on nozzle diameter and bioink viscosity, is crucial for ensuring structural accuracy in 3D bioprinting.

Conclusion
This study demonstrates that fine-tuning Alg concentration, nozzle diameter, and extrusion pressure is essential for achieving structurally robust and biologically viable scaffolds in 3D bioprinting. An 8% Alg formulation combined with GO enhances printability and mechanical integrity. The 22G nozzle was identified as optimal for maintaining filament continuity and pore uniformity, while extrusion pressures between 0.85 and 1.25 bar produced the most consistent scaffold architecture across formulations. These insights contribute to the development of customized biofabrication strategies for cardiac tissue engineering applications.

Ethical Considerations
Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
This study paper was extracted from the master's thesis of Fatemeh Edrisi, approved by the Department of Modern Technologies in the Engineering, Faculty of Interdisciplinary Science and Technology, Tarbiat Modares University, Tehran, Iran.

Authors' contributions
Conceptualization, and supervision: Nafiseh Baheiraei and Ali Zamanian; Methodology: Nafiseh Baheiraei, Ali Zamanian, and Fatemeh Edrisi; Software, validation, visualization, formal analysis, investigation, data curation, and writing the original draft preparation: Fatemeh Edrisi; Review and editing: Nafiseh Baheiraei and Ali Zamanian; Funding acquisition and resources: Nafiseh Baheiraei.

Conflict of interest
The authors declared no conflict of interest.



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