I. Core Research Summary

• Seven-Parameter Quantitative Regulation System

All process parameters are adjusted by changing Deposition rate, solvent evaporation rate, and spray beam focusing performance Three core mechanisms govern the morphology of the formed structure; by identifying the optimal ranges for each parameter, the optimized gelatin surface texture is stabilized to a narrow width of 20 μm, exhibiting uniformity with no protruding droplets.

• Bio-friendly low-temperature molding solution

Conventional high temperatures can denature proteins; by jointly optimizing process parameters, high‑precision grid patterns can be fabricated at physiological temperature (37°C), while fully preserving the matrix’s cell‑adhesion functionality.

• Defect Controllability and Suppression Strategy

The root causes of three types of defects—coffee rings, droplet coalescence, and edge overflow—are elucidated, and corresponding parameter‑tuning strategies are proposed. A high focus ratio, moderate substrate temperature, and reduced printing distance can significantly mitigate edge uplift.

• Validation of the Versatility of Multi-Bioink Systems

The optimized parameter system can be directly applied to poly‑L‑lysine (PLL) bioink, enabling the stable fabrication of 20‑μm micro‑wires. NG10815 neuronal cells exhibit specific adhesion to the printed patterns, facilitating the formation of oriented neuronal networks.

• Cross-Material Standardization Optimization Framework

A universal step-by-step parameter‑tuning workflow has been established, enabling rapid optimization of the optimal process for novel biohydrogels, proteins, and peptide inks, thereby significantly shortening the R&D iteration cycle for bioprinting.

II. Core Working Principle and Unique Process Advantages of Aerosol-Jet Bioprinting

(1) Complete Bio-Micro-Pattern Printing Process

• Prepare a gelatin/poly-L-lysine–based aqueous bioink and maintain it at a constant temperature to keep the ink in a sol state.
• The ultrasonic module atomizes the bio‑solution into microdroplets and delivers them via a saturated nitrogen carrier gas, thereby preventing rapid solvent evaporation during the printing process.
• A coaxial sheath gas forms a focused jet, and the CNC nozzle deposits biomaterials at designated points along the CAD‑defined path.
• The temperature-controlled platform precisely regulates the solvent evaporation rate, enabling in-situ crosslinking to form micron-scale biological patterns.
• Cells are seeded after UV sterilization, enabling a spatially controlled microenvironment for cell culture.

(II) Five Core Process Advantages (Compared with Microcontact Printing and Inkjet Bioprinting)

• Ultra-high fabrication resolution, far surpassing conventional bioprinting.

Stably achieves 20 μm‑uniform bio‑lines, with inkjet deposition as low as 50 μm; enables the fabrication of high‑precision grids and cross‑patterned biomimetic extracellular matrix structures, suitable for single‑cell‑level in vitro studies.

• Ultra-wide viscosity compatibility range for bioinks

It can print hydrogels and protein solutions with viscosities ranging from 1 to 10,000 mPa·s; low-concentration peptides and highly crosslinked matrices can all be stably atomized, whereas inkjet printing is suitable only for low-viscosity, dilute solutions.

• Moldless digital direct writing, rapid iteration

No lithography or PDMS molds are required; CAD‑based pattern modifications can be printed instantly, and a single experimental batch can complete multiple gradient‑parameter comparisons in just a few hours, eliminating the lead time associated with mold fabrication.

• Low-temperature gentle processing fully preserves bioactivity.

Printing can be performed at physiological temperature (37°C), without high‑temperature baking that would degrade the protein’s RGD adhesion motifs, thereby preserving the functionality of bio‑based matrices such as gelatin and laminin.

• Targeted, zoned, precise deposition—no full‑area coverage.

The nozzle enables site-specific printing of localized bioadhesive regions, while the remaining substrate surface remains inert, allowing precise control over cell‑seeding areas and supporting compartmentalized co‑culture platforms.

III. Five Core Technological Innovations of This Study

Innovation 1: The world’s first systematic quantitative parameter model for bio‑ink aerosolization systems.

Innovation: 2–37°C Physiological Low-Temperature High-Precision Printing with Coordinated Parameter Strategies

Innovation: Three Major Forming Mechanisms and Their Categorized Regulatory Logic

Innovative Comprehensive Solution for Fully Mitigating Bioprinting Defects

Innovation: A Universal Optimization Framework for Five Types of Biomaterials

IV. Existing Process Challenges and Corresponding Solutions for Overcoming Them

Challenge 1: Bioinks are sensitive to high temperatures; conventional high-temperature processing can easily lead to enzyme deactivation and broadened feature widths.

Challenge 2: A single parameter change can simultaneously trigger multiple variations in linewidth and thickness, leaving no standardized approach to hyperparameter tuning.

Challenge 3: When printing with low-concentration bioinks, coffee-ring effects and uneven cell distribution are prone to occur.

Challenge 4: Excessively high atomization flow rates can easily lead to droplet agglomeration and localized protrusion defects.

Challenge 5: Different bioinks exhibit significant variations in viscosity and solvent composition, making it difficult to establish universal parameter‑tuning guidelines.

V. Future Diversified Industrial Application Scenarios

1. In vitro cellular models in neuroscience

2. Stem Cell Differentiation Regulation Platform

3. Functional Modification of Microfluidic Organ-on-a-Chip Devices

4. High-Throughput Cellular Biosensing Chip

5. Surface Modification of Biomimetic Scaffolds in Tissue Engineering

Figure 1: (a) The aerosol jet printing process can be divided into two main steps: 1) Bio‑material ink droplets are generated via ultrasonic atomization and carried by a saturated nitrogen carrier gas, which is produced by passing nitrogen through a deionized water bubbler; 2) Prior to deposition of the aerosol jet onto the substrate surface mounted on a moving heated stage, the nozzle is focused using a sheath gas. (b) Pneumatic focusing is achieved by introducing a sheath flow within the print head. The flow rates of the atomizer and the sheath gas (in sccm) determine the focusing ratio (FR).

Figure 2: (a) The parameters that influence the overall line quality in aerosol jet printing can be categorized into four groups, each associated with the aerosol jetting process, ink properties, environmental conditions, and device settings. Adapted from Salary [25]. (b) Colored triangles denote the three core parameter groups that determine the line quality in aerosol jet printing: material deposition rate, evaporation rate, and aerosol jet focusing. As shown, critical printing parameters simultaneously affect one or more of these processes, giving rise to complex interdependencies.

Table 1: Maximum and minimum test values for each parameter, along with their reference values. Parameters marked with an asterisk were tested at a stage temperature of 55°C, rather than the reference temperature of 75°C.

Figure 4: (a) After applying a 50-point moving average along the longitudinal axis of the 3D optical profilometry image, the average line height and width were measured from a 1.98 µm cross-sectional slice. (b) Both the average line height and the average line width were determined between edge points located at 10% of the maximum line height above the substrate. Under 3D optical profilometry, gelatin typically forms (b) a single unimodal line or (c) a double‑ridged line with blurred edges due to splashing effects. Note: The height scale has been exaggerated to emphasize the line morphology; in reality, the actual line profiles are much smoother than those depicted in the schematic.

Figure 5: (a) Comparison of average line width and height at different printing speeds (mm/s); (b–d) Representative results from typical cross-sections, illustrating the effect of increasing printing speed (1–8 mm/s) on line morphology.

Figure 6: (a) Comparison of average line width and height at different nebulizer flow rates (sccm); (b–d) Representative results from typical cross-sections, illustrating the effect of increasing the nebulizer flow rate (25–50 sccm) on line morphology.

Figure 7: (a) Comparison of average line width and height at different focus ratios; (b–d) Representative results from typical cross-sections, illustrating the effect of increasing the focus ratio (1–5) on line morphology.

Figure 8: (a) Comparison of average line width and height as a function of working distance (mm); (b–d) Representative results from typical cross-sections, illustrating the effect of increasing working distance (1–6 mm) on line morphology.

Figure 9: (a) Comparison of the changes in average linewidth and line height as the reaction-stage temperature (°C) increases; (b–d) Representative results from typical cross-sections, illustrating the effect of increasing reaction-stage temperature (35–115°C) on spectral line morphology.

Figure 10: (a) Comparison of average line width and height as the nozzle diameter (µm) increases; (b–d) Representative results from typical cross-sections, illustrating the effect of increasing nozzle diameter (150–300 µm) on line morphology.

Figure 11: (a) Comparison of average line width and height as a function of concentration (weight percent); (b–d) Representative results from typical cross-sections, illustrating the effect of increasing concentration (1–5 wt%) on line morphology.

Table 2: Summary of Parameter Thresholds Affecting Spatter Volume Based on the Control Parameters Used

Table 3: Summary of the Effects of Adjusting Key Printing Parameters on Line Width, Height, and Variability

Figure 12: (a) Gelatin printed using a grid pattern at 37°C, achieved by moving along the pre-existing lines at a 90° angle, and demonstrating the potential for designing more complex patterns. Scale bar = 100 µm. (b) A three-dimensional cross-sectional view of the finished line morphology, revealing the coffee-ring effect, yet without droplet formation and with minimal spattering. Note: The height scale has been exaggerated to emphasize the line morphology; in reality, the lines are much smoother than depicted in the cross-section.

Figure 13: (a) Parallel lines of poly‑L‑lysine, approximately 20 µm wide, were patterned onto a glass slide. (b) NG‑108‑15 cells were seeded onto the poly‑L‑lysine‑patterned surface and cultured for 48 hours, followed by a three‑day serum‑deprivation treatment prior to imaging. Scale bar = 50 µm.