Fine Tuning the Printer

By Omair Tariq, Systems and Test Engineer

Scope

There are two issues to ensure correct flow of the mixture: Volume and Temperature Control of extrusion apparatus. The extrusion apparatus consists of a syringe and a needle attached to the base of the syringe. Temperature control was addressed by attaching a heater to the body of the syringe. However, the gel was still solidifying at the base of the syringe and at the needle.  This issue was addressed by attaching high-wattage resistors to the base and the needle. The details concerning the correct temperature control are addressed in a separate blog post. The purpose of this test is to determine the ideal needle diameter and Slic3r settings for the 3-D BioPrinter once the problem of temperature control has been addressed.

Equipment Needed

Equipment	Quantity
3-D BioPrinter	1
Vernier Caliper	1
Agarose Solutions of varying percentages	50 ml
Needles with different gauge diameter	1
Laptop with Slic3r and Pronterface Installed	1
USB Cables	2

Table 1. Equipment needed for accuracy testing

Procedure

For the following test prints, a solid 20mm x 20mm x 5 mm cube was printed

1. It was decided to start with a 1% agarose solution and a 18 gauge syringe needle^[1]. This led to an overflow of the liquid from the syringe as illustrated in Figure 1. Due to this overflow, the liquid did not have enough time to solidify. Therefore, the print was stopped even before the first layer was completely printed. The Slic3r settings shown in Table 2 were used for this test print.  

 

Figure 1 Overflowing liquid

 

Perimeter	Value
Nozzle diameter (18 gauge needle inner diameter)	0.8mm
Filament Diameter	40 mm
Travel Speed	50 mm/s
First Layer Speed	20 mm/s
Fill Density	1
Fill Pattern	Concentric
Top/Bottom fill pattern	Concentric

Table 2. Slic3r settings used for 1^st test print

1. It was then decided to switch to a needle with a smaller inner diameter: a 20 gauge needle was chosen.  Subsequently, the nozzle diameter and the filament diameter had to be changed in Slic3r. The new Slic3r settings are shown in Table 3. This yielded a much better result as can be seen in Figure 2. The flow was slow enough to facilitate the gelling of the liquid. Even after gelling, the gel structure was not as strong as expected in that it was not able to hold its weight which was a level 1 requirement. Therefore, It was decided to use 2% agarose gel which would be stronger than 1 % agarose gel after gelling. 2% agarose gel has the same gelling temperature as 1 % agarose gel. Therefore, there was no need to adjust the temperature of the syringe.  It was also decided to use 4% starch in the solution to increase the viscosity of the fluid. This slowed down the flow of the liquid through the needle without the need to change temperature. Starch and Agarose are both carbohydrates, therefore adding starch did not alter the viability or biological nature of the solution.

Table 3. Slic3r settings used for 2^nd test print (Changes highlighted)

Figure 2. Overflowing gel

1. A test print was then carried out using a 2 % agarose, 4 % starch solution (See Table 4 for exact composition of solution).

The resulting solution had just the right amount of viscosity, which justified the use of starch. The final structure was also strong enough to hold its own weight, which justified increasing the concentration of agarose gel. This structure also had the correct dimensions. But there was one problem; instead of being a solid cube like ‘American Cheese’, the block turned out like ‘Swiss Cheese’ with air bubbles in the middle.

Chemical	Quantity
Water	50 ml
Agarose	1 g
Starch	2 g

Table 4. Chemical Composition of 2% Agarose 4 % Starch Solution

  

Figure 3. Test Print using 2% agarose and 4% Starch

1. During the third test print, it was observed that the travel speed of the extruder increased after printing the first layer. This was problematic since it was not possible to adjust the flow of the liquid for two different travel speeds. Even though the first layer turned out fine, the extruding liquid broke off contact with the structure at certain points in the 2^nd layer due to a faster speed. This break in contact in the second layer meant that there was also break in contact in the third and subsequent layers leading to a domino effect. .  We then switched to advance mode in slic3r that gave us a better control of the various speeds of the printer. All these were set to a smaller and equal value of 20 mm/ s. The settings are shown in Table 5. The structure printed using these settings are shown in Figure 4.

Perimeter	Value
Nozzle diameter (20 gauge needle inner diameter)	0.6mm
Filament Diameter	29mm
Travel Speed	20 mm/s
First Layer Speed	20 mm/s
Fill Density	1
Fill Pattern	Concentric
Top/Bottom fill pattern	Concentric
Perimeter Speed	20 mm/s
Small perimeters	20 mm/s
External Perimeters	20 mm/s
Infill	20 mm/s
Solid Infill	20 mm/s
Top solid infill	20 mm/s
Support Material	20 mm/s
Bridges	20 mm/s
Gap Fill	20 mm/s
All Acceleration Controls	0 mm/s2

Table 5. Slic3r settings used for 4^th test print  (Changes from previous step highlighted)

Figure 4 Final Printed Structure

The actual dimensions of this structure were also very close to the expected dimensions.

   

Figure 5. Measurements of Final Printed Structure

Conclusion

A 2% Agarose and 4% starch solution is the ideal solution to be used in the bioprinter. The settings used in Step 4 of this test plan are the ideal settings for the bioprinter when it is being used to print 2% Agarose and 4% Starch solution.

[1] – For needle diameters, check: http://en.wikipedia.org/wiki/Needle_gauge_comparison_chart