Protocol
For designing and fabricating structured tissue engineering scaffolds with varied porosity architecture we use CAD/CAM software, and 3D Printing.
2-1- Scaffold Design using CAD software
The first step in the fabrication process is the design of the tissue engineering scaffold using CAD software. CAD allows for precise control over the scaffold's geometry, pore size, and interconnectivity. During this stage, the specific requirements of the target tissue or organ are taken into consideration to customize the scaffold accordingly. Solidworks, Materialise Mimics, Materialise 3-matic, and etc. are the common software for designing custom scaffolds.
2-2- CAM software (Prusa Slicer)
Using CAM Software (Prusa Slicer) for 3D Printing Tissue Engineering Scaffold:
1) Launch Prusa Slicer and Import STL Geometry: Click on "Add" or drag-and-drop the STL file of your scaffold design (designed in CAD softwares like Solidworks, MIMICS, 3-matic,…) into Prusa Slicer's interface. The software will load and display the 3D model.
2) Printer and Material Selection: Choose your 3D printer model from the list of supported printers. Select the appropriate material (e.g., biocompatible filament) from the material profiles available, and choose 0.5 mm for nozzle diameter.
3) Temperature Settings: Navigate to the "Filament" settings. Set the nozzle and bed temperatures according to the recommendations for your chosen material. Proper temperatures ensure optimal adhesion and print quality. For PLA material nozzle and bed temperature set to 190 ℃ and 30 ℃ respectively.
4) Layer Thickness and Print Speed: In the "Print Settings" section, adjust the layer thickness (also known as layer height) based on your desired scaffold porosity and print resolution (For our study layer thickness was 0.2 mm). Modify print speeds to balance speed and print quality.
5) Pore Design and Density: Prusa Slicer provides you with the flexibility to finely tailor the pore design and density of your tissue engineering scaffold through precise adjustment of the infill settings. This essential feature empowers you to create scaffolds with optimized structural characteristics. Within Prusa Slicer, choose from a variety of infill patterns to define the spatial arrangement of pores within your scaffold. Options such as grid, honeycomb, gyroid, cubic, and more are available. Select an infill pattern that aligns with your scaffold's intended function and the specific cellular responses you seek to encourage. After choosing infill pattern, finelytune the infill density. Higher infill density translates to a more solid scaffold with fewer voids, suitable for load-bearing applications. Lower infill density promotes higher porosity, facilitating cellular infiltration and nutrient diffusion for tissue regeneration. Depending on your tissue engineering goals, you can adapt the infill settings to create scaffolds with porosity optimized for different tissues. For example, bone scaffolds may benefit from a denser infill to mimic natural bone density, while softer tissues like cartilage could thrive in a scaffold with higher porosity.
6) Visualize Before Printing: Use Prusa Slicer's 3D preview to visualize how the scaffold will be printed layer by layer. This helps you identify potential issues and make adjustments before initiating the printing process (Figure 1).
7) Support Structures (Optional): If your scaffold design requires support structures, enable them in the settings. Prusa Slicer will automatically generate supports to ensure overhangs and complex geometries are properly printed.
8) Generate G-code: Once you're satisfied with the settings, click on "Slice Now." Prusa Slicer will process your model and generate G-code instructions that your 3D printer will follow.
9) Export G-code: After slicing is complete, click on "Export G-code" and choose a location to save the G-code file. This file contains the detailed instructions for the printer, including movement paths, temperatures, and extrusion rates.
Figure 1. Different layers of scaffold formation in the Prusa Slicer (visualize printing path before real printing)
2-3- 3D Printing Process
1) Material Selection and Preparation: Choose a suitable filament material for your tissue engineering scaffold. We used PLA filament (Gando). Ensure the filament is stored in a dry and controlled environment to prevent moisture absorption.
2) Load Filament: Insert the chosen filament into the 3D printer's filament guide or extruder assembly. If material are in granule type, load the granules into the printer's material feeding system such as metal syringe. The granules will be fed into the extruder for layer-by-layer deposition.
3) Printer Setup: Power on the FDM (Fused Deposition Modeling) 3D printer machine (Sizan ECO, Sizan Co., Kashan, Iran) and ensure it is properly calibrated. Make sure the print bed is clean and level, and the nozzle is at the appropriate starting position.
4) Transfer G-code to 3D Printer: Transfer the exported G-code file form Prusa Slicer to your 3D printer using a USB drive, SD card, or other appropriate method.
5) Start Printing: Initiate the 3D printing process through the machine software interface. The printer will start heating the nozzle and bed to the specified temperatures. After heating completed, the printer will follow the instructions in the G-code to create your scaffold layer by layer.
6) Printing Progress: Monitor the printing progress closely, especially during the initial layers. Ensure that the filament adheres properly to the print bed and that the layers are being deposited accurately.
7) Post-Printing Care: Once printing is complete, allow the print to cool before removing it from the print bed. Carefully detach any support structures, if present, using appropriate tools.
8) Finishing Touches: Inspect the printed scaffold for any imperfections, such as stringing or layer inconsistencies. Trim or sand any rough edges if necessary.
9) Quality Control: Perform quality checks on the printed scaffold, ensuring that dimensions and structural integrity meet your specifications.
10) Storage and Handling: Store the printed scaffold in a controlled environment to prevent exposure to moisture or UV light, which can degrade the material over time.
Results and Discussion
Successful 3D printing of diverse scaffold designs has yielded a range of fabricated structures characterized by varying pore shapes and sizes (Figure 2). Notably, the print times for each scaffold exhibit a significant disparity, spanning from mere minutes to several hours. The scaffolds were tailored to cater to the regeneration needs of various tissues, including bone, cartilage, and skin.
Figure 2. Left: Scaffolds on 3d printing bed at the end of the printing process, Right: Different scaffolds with diverse shape and pores.
Our study presents a notable advancement in the field of tissue engineering through the precise customization of scaffold architectures. By meticulously manipulating pore shape and size, we offer a pathway to fabricating tissue-engineered constructs that closely emulate the intricate microenvironments of natural tissues. Fine-tuning of scaffold mechanical properties enhances the potential for generating constructs with tailored strength and structural integrity, thereby catering to diverse tissue applications such as bone and cartilage regeneration. Opting for granule-type materials or hydrogels in 3D printing, as opposed to conventional filaments, could potentially limit the array of biomaterials accessible for the fabrication of the intended scaffold designs. This limitation arises from the possibility that these alternatives might not fully replicate the intricately designed pore structures and shapes as designed. However, the successful design and fabrication of tissue engineering scaffolds with diverse porosity architecture using CAD/CAM, and 3D printing demonstrate the immense potential of this approach in the field of regenerative medicine (10-14). As these fabrication techniques continue to evolve, we can expect significant advancements in tissue engineering and regenerative medicine, ultimately benefiting countless patients in need of effective and personalized therapeutic solutions.
