Print Parameter Optimization Guide

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Whether you have a novel material that you need to characterize or you want to further optimize a more common material, it’s critical to ensure that your print parameters result in a construct that fits your criteria. Before you follow this protocol, a good question to ask yourself is what are you optimizing for? Is it for strength? Speed? Cell viability? This article will walk you through the steps for proper print parameter optimization to ensure you have a successful experiment.

Extrusion Test

For clean and repeatable prints, you need to ensure that you have consistent extrusion of your material. Under-extrusion can lead to poor layer building while over-extrusion can lead to sloppy prints from dragging your needle through the material.

Results: This protocol will help you determine the material’s print pressure.

  1. Fill a syringe with your material and load it into the Allevi. 
  2. In the Allevi software, press and hold the extruder button. Release to stop extruding. 
  3. If there was no extrusion or uneven extrusion, increase the pressure by 5 PSI.
  4. Repeat steps 2 and 3 until the material extrudes evenly. 
  5. Repeat this process to get three pressures the material extrudes at, varying from a slow to fast extrusion.

Notes to consider:

  1. Viscosity considerations
  2. There are a variety of factors that can affect the pressure needed for extrusion. Take note of what needle gauge and shape you are using. To lower the pressure needed for extrusion, try using a lower gauge or a tapered needle. For more information on needles, check out our Guide to Picking Your Needle.
  3. Material volume loaded into the syringe can also have an effect on pressure. Generally, material volume fluxes of ~ 2 ml can affect the pressure needed for extrusion.

Troubleshooting tips:

  1. Each time you increase the pressure, be sure to turn off the pressure then turn it back on again. Otherwise, the pressure settings on the dial may not accurately portray actual pressure output.
  2. Be aware of what temperature you are completing the extrusion test at. Many materials change viscosity when heated or cooled. If you needed to heat or cool the material before loading into the syringe, take note of the amount of time allowed to reach printing temperature for future tests. Allevi 1, 3 and 6 offer a wide range of printing temperatures: from 4 °C to 160 °C. Allevi 2 can heat up extruder up to 160 °C.
  3. If the desired material takes a long time to cool or heat to desired print temperature, try placing the loaded syringe in an ice bath or heated water bath to speed the process.

Volume Test

A volume test allows for the estimation of volume extruded from the printer. By comparing a series of known volumes to yours. This will also be helpful if you plan on printing dots of a certain volume.

Results: This protocol will help you determine the material’s flow rate.

To find the relationship between weight and volume:

  1. Weigh a series of known volumes of the desired material. We recommend using at least 3 volumes.
  2. Plot the weights of these measurements against volume and use a line of best fit to estimate weight increase by volume (see figure 7 and equation 2).
  3. Load a syringe with your material into the Allevi at the temperature and pressure you plan to print.
  4. Extrude the material for equal amounts of time, then measure to obtain a series of weights per time printed (see figure 7 and equation 3). From these weights and the weight by volume estimate from the previous plot, you can estimate the amount of volume extruded over a period of time (see equation 4)

Crosslinking Test

Results: This protocol will help you determine the material’s crosslinking settings

Line Test

Once a range of pressures for extrusion has been determined, feed rate, path height, and print speed must be optimized. This can be completed through a line test in which various lines are extruded at different print speeds (Figure 1). A line test using g-code provided by Allevi should be completed for each pressure and needle to be tested. Once completed, the lines can be analyzed via brightfield microscopy for accuracy and precision.

Results: This protocol will help you determine the material’s print speed, layer height, and line resolution for a given pressure and needle.

g-code line tests for print parameter optimization

Our pre-sliced g-code is written to make editing it easy. The g-code is set up in the section as shown below.

Sample Line test Print File g-code

  1. Determine 3 print speeds and 3 layer heights you want to test. 
    • Note: Needle tip diameter is a good layer height to start with.
  2. Download the Line Test g-code above. Open it in a g-code or text editor.
  3. Edit the g-code to set layer heights.
    • Layer height remains the same across rows (A, B, C). Edit the layer heights for these rows by changing the Z value. You should only need to change 3 values. Lines 20, 60, and 101
  4. Edit the g-code to set speeds.
    • Speeds remain the same across columns (1, 2, 3). Edit the speeds for these columns by changing the F value. You will need to edit the speed for each section (9 total).
    • Note: In g-code, speed is written in the unit of ‘mm/min’ rather than ‘mm/s’. This means that ‘mm/s’ speeds are multiplied by 60.
  5. Save the file.
  6. Load your material into a syringe, attach your desired needle, and set the pressure to your previously determined value.
  7. Print your Line Test.gcode file. 
  8. (Optional) Use brightfield imaging and an image analysis software (such as ImageJ) to determine resolution and variance.

Troubleshooting tips:

  1. Start with a wide range of speeds and path heights, then test a narrower range to optimize.
  2. If you find your line width is larger than desired, a smaller gauge may allow for finer extrusion. However, this might affect extrusion pressure.
  3. Larger line widths than expected may also be caused by incorrect path heights, which can cause the material to smudge. Try increasing path height.

Z-stack Print Calibration

To further check/test/confirm/optimize path height, the print settings determined from the line test should be tested/confirmed through a Z-stack calibration. This print will determine whether your material can repeatably build upon itself layer-by-layer at your print settings. The accuracy of Z-direction can be determined by measuring the final height of the construct and comparing it to the height from the design file.

Results: This test will help you determine Z-resolution as well as check the layer height and crosslinking profile. 

  1. Go the Allevi Bioprint Online. Connect to your printer a.
  2. In the Print File section, click ‘Add file’ and go to Allevi Files and select the 10mm cylinder.
  3. Enter your layer height and print speed in the slicer settings. Leave the infill set to ‘None’.
  4. Set your pressure, add any crosslinking settings.
  5. Click print.

Troubleshooting tips:

  1. If you run into issues with z-stack calibration, try altering path height as well as crosslinking time between layers. Try pausing the print and crosslinking the first few layers for increased amounts of time before increasing height.
  2. If you are still having issues after increasing the crosslinking time of initial layers, you can also try enlarging the width of layers for increased support.
  3. If the needle starts to run into previously printed layers, your path height is likely too small. Try increasing path height.
  4. If the material extrudes only in droplets as layers increase, either the path height is too large or the print speed is too slow. Try either decreasing path height or increasing print speed.

Single Layer Lattice Print Test

Multi-Layer Lattice Print Test

This final calibration tests all optimized parameters from previous calibrations, as well as complexity in 3D dimensions. Print multi-layer lattices of varying sizes with previously determined print parameters. This calibration test is meant as a final step to confirm all previously optimized parameters. If this test is not optimized, you may need to return to previous steps to recalibrate certain print settings.

Results: This calibration step allows for the final testing of optimized print parameters.

X/Y Resolution Print Test (Optional)

Once pressure, gauge, path height, print speed, and feed rate are optimized and finalized, the material spatial resolution in x and y directions can be determined. This can be completed through a resolution test with the STL provided by Allevi.

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