We refunctioned a standard single-extruder 3D printer made to print plastic, the Creality Ender 3 Neo, into the first DIY dual-channel extrusion-based bioprinter. For the creation of this printer, we had to complete 3 major hardware modifications:

1. Build a syringe pump
2. Create a new extrusion head
3. Add a second extruder

Building a Syringe Pump

A syringe pump is a device that pushes the syringe plunger to squeeze fluid out of a syringe. While these exist on the market, they are not fully compatible with our printer. Using a market syringe pump would require using an external circuit board to run the syringe pump and communicate with the printer about when to start or stop extruding, and how fast to extrude. This communication needs to take place to sync the printer and extrusion so that the printed product is as accurate as possible to the loaded computer-aided design (CAD).

So, we created a novel syringe pump. Making our own cut costs by repurposing a motor from the plastic extruder. Additionally, we made a custom syringe pump driven by the motherboard of the printer. Our syringe pump was created using CAD and was 3D printed. We chose to make the syringe pump to drive two separate syringes for two different bioinks to be extruded in the same print, allowing us to print our bacteria and yeast hydrogels in parallel. The other benefit of this design is that it can easily be modified if more extruders were needed. This syringe pump sits alongside the printer.

1. Stepper motor
2. Motor holder
3. Motor to lead screw clamp
4. Lead screw
5. Linear shaft
6. Plunger holder
7. Linear ball bearing
8. Syringe holder
9. Syringe
10. Tubing
11. Female Luer lock to barbed adapter
12. Shaft holder
13. Syringe plunger
14. Lead nut (not visible)

Creating a New Extrusion Head

The next major design change was the extrusion head. Extrusion heads typically contain a hotend, which melts the plastic filament for printing. The typical hotend used by plastic printers was incompatible with the syringe pump and our bioinks as they contain live bacteria and yeast, which are temperature sensitive.

We designed a new extrusion head to address this. We decided to use needles to give us precise control over extrusion width, so these needed to be attached to the x-axis trolley. Since the syringe pump was alongside the printer, the syringe needed to be connected to the needles through tubing. Our extruder head design allowed for the needle to be changed without detaching the hosing, using clamps that slide on and off the main structure to allow needle access. Additionally, there is a top slider to allow the tubing to be taken off the trolley if needed.

Adding a Second Extruder

Our decision to engineer a dual-channel syringe pump required us to make additional hardware modifications to make the pump functional. Single extruder printers are not made for running two extruders; they lack ports on the motherboard for the additional motor and motor driver we wanted to add, so the motherboard needed to be replaced with one that accommodates multiple extruders. Our bioprinter uses two extrusion nozzles a fixed distance apart, and operates two extruder motors off of the main printer motherboard instead of with an external circuit, which has been used in other designs.

Additionally, a thermistor, which is a resistor that acts as a temperature sensor, needed to be added for the new extruder so the code could run correctly. A thermistor was added to a port on the motherboard for the second extruder’s thermistor using jumper cables and a 100k resistor. This allows the printer to sense the temperature of the second extruder, which is necessary due to the safety code put in place on the printer to prevent overheating of an unmodified printer that heats up the hotends. The new motherboard needed to be flashed with code compiled by our team from open source examples specific to the number of extruders and the specifications of the attached components — including motors, endstops, coordinate system, which was done in Marlin, the standard 3D printer firmware.

Syringe Pump

The stepper motor (1) is connected to the lead screw (4) by a clamp (3), and rotates to move the plunger holder (6) that holds the end of the plunger (13). The plunger holder has a lead screw nut (14) pressed into it to make it move along the lead screw, and the plunger holder runs along the linear shaft (5), which gives a little more travel length than the length of the syringe. The motor is secured using the motor holder (2) and the linear shaft is held by the shaft holders (12). The syringe sits in the syringe holder, and all of the parts except for the plunger holder are secured to the base plate with screws. The tubing (10) is connected to the syringe using Luer lock adapters (11), and so the bioink can be extruded by the syringe through the tubing to the extrusion head (not pictured). Adjustments made from V3 include changing the hole pattern for the base plate to better fit the shaft holder, motor holder and syringe holder. Once the hole pattern was changed, the syringe pump ran smoothly, and it runs efficiently for our application with our design timeline constraints. Descriptions of all parts are in the downloadable printer components guide.

Extrusion Head

The extrusion head was constructed in three slidable parts to allow the needle height and sizes to be customized for print optimization tests. In the CAD model, Part 1 (light blue piece) has a mounting hole pattern of two slots, so that the height could be changed by loosening the mount screws, moving the extrusion head, and tightening the screws to secure the new height. Next, Part 2 (dark blue piece) assists with needle stabilization to keep the needles from moving during extrusion. Part 3 (gray) is the sliding top holder, which keeps the Luer lock adapters locked in at a specific height. Both sliding parts 2 and 3 can be used for easy needle or tubing switches, and there are grips on both sliding parts for easy removal.

For the needle size, we decided on using a needle with a 0.5 mm inner diameter after consulting with Ram Surya Gona from the Meyer lab. After testing this needle, it was determined to print well, and the stepper motor still moved well with the force of pushing the bioink through this size needle.

Petri Dish Rig

We conducted printing tests with alginate, which needs to be printed on solid media containing calcium chloride ions. The Petri dish containing solid media needs to be held in place, so the Petri dish rig was designed. A two-part track lies diagonally across the print bed, and two clamp parts slide along this track. One of the diagonal track pieces has an end stop to center it, and both have small overhangs that surround the corners of the print bed to align the track. The clamp pieces are put on the track, and then screwed together to make a press fit clamp for Petri dishes. The diagonal tracks are connected with electrical tape on the top and bottom, and are secured on the edges.

New Motherboard

The motherboard original to the printer had only one port for an extrusion motor. Because of this, a new motherboard was needed to accommodate the second channel of the syringe pump. We decided we needed a new motherboard, and chose the MKS Gen L V2.1 motherboard since it is made for dual-extruder motors, and corresponding hotend, fan, and thermistor. This board also uses the firmware language Marlin, and so it is easy to adapt to our printer since Marlin is a printer standard. Creality, the company who made the plastic printer we are converting, is open source, so we looked at the source code examples to start understanding Marlin, but the firmware for configuring the new board was manually written. All specs for the board are on GitHub, making it a good choice for this project.

New SD Card Reader

The one downside of the new board was determined after testing. There is no SD card reader, so getting data to the printer requires application downloads to a connected computer, which initially made it less user-friendly. Because of this, a new SD card reader was added using the pin schematics found on GitHub. After testing the SD card reader, it was a good addition to the printer because the SD card reader is easier for even less-experienced users to operate. If the SD card reader had not been added, an open source software called Pronterface would have needed to be used to load Gcode, or 3D printer software, to the motherboard — which isn’t especially intuitive.

New Thermistor

A thermistor is essentially a resistor. When heat changes, the resistance will lessen, so the change in resistance gives an accurate reading of the temperature wherever the resistor is, which in this case is the hotend. After trying to operate the printer without a thermistor for the second extruder, the printer threw error messages because it could not sense temperature, which the code reads as dangerous. Because of this, we determined that a second thermistor needed to be added, and so we plugged a 100K resistor into the thermistor spot for extruder 2. We determined the resistance value for the thermistor by looking at the specs of thermistors. When this resistor was added, normal temperature readings were sent to the printer, and it could be operated. The test using the homemade thermistor also told us that the printer is unaware of the presence or absence of a hotend because it could operate extruder 2 with just a thermistor. We were able to remove the hotend from extruder 1, which makes it safer because there is no exposed heating element that could accidentally be touched. Additionally, the temperature readout was the same for the stock thermistor and the resistor used as a thermistor, meaning that the homemade resistor was fairly accurate and can be used in the place of a stock thermistor.

Other

The extrusion motor that came with the printer was removed from its mount so that it could be used in the syringe pump. Repurposing parts that came with the printer to build our own syringe pump makes our bioprinter more cost-effective and ensures the printer firmware is compatible with the motor.

The extrusion head case was removed, along with the hotend, and the thermistor was detached from the hotend. This was done because the extrusion head case obstructed where the new needle mount needed to be. The hotend also needed to be removed for safety to prevent burns. The thermistor was detached from the hotend so it could be used on the printer. Thermal regulation via the thermistors is required by the firmware for safety, so the thermistor from the printer was still needed.

The old motherboard was removed, along with the fan and metal plate that enclosed it. Everything was plugged into the new motherboard according to the GitHub wiring specs, and new motor drivers were installed. This was a functional improvement because it allowed us to build the syringe pump with two channels and operate two extruders.

We conducted two print tests to assess the quality and consistency of our prints. Before fully assembling our bioprinter, we printed by hand.

Line Width Consistency Test

The first print made by the printer was a line test. This test was performed to see the consistency of line width over the course of 40 trials. The experiment was set up with 10 agar-CaCl₂ plates with different molarities of CaCl₂. The plates ranged from 0.1M to 1.0M and were all 0.1M apart.

Each plate was printed with 3 cm lines, and the line widths were measured at four points along the line. The length of the lines were also measured to test consistency. The line test summary data are in the table below; all of the data can be found here.

Syringe Pump Mass Consistency Test

Once we determined that the printer settings were satisfactory for producing a consistent line, we decided to check if the syringe pump could consistently make prints with the same mass of bioink. For this test, the extruder motor was run for 50mm at F750 speed and retracted at 37mm at F2400 speed, with the bioink extruded into a weigh boat. The masses of the trials were then each measured, and we found that the average mass of ink pumped was 0.51g per extrusion distance. The data also showed that the consistency was fairly decent, but changed slightly when the syringe was closer to empty, which is likely because air is occasionally drawn in during the retraction process at the end of the print.

Waffle Consistency Test and Proof of Concept

One of the shapes preferred for diffusion is a series of lines crossing each other, which is a pattern we call the waffle. The waffle shape was first developed with one nozzle to test the path taken by the extrusion head. The waffle is a more complex shape because of how the nozzles’ paths trace over the agar, and it being successfully printed shows competent printing abilities. Once the one needle waffle was made, a two nozzle waffle pattern was made keeping in mind the offset between the extruders. This print was also made successfully over a few trials.

Round 1:

The team had Bruno Coelho, a senior mechanical engineering major and Chief Engineer of University of Rochester Baja SAE who has been interested in printer design for many years, come into the lab for user testing.

During testing, Bruno remarked that the Petri dish rig was a very effective addition to the print bed. After printing line Gcode, Bruno provided recommendations for future changes. Originally, the Petri dishes were at varying heights, and were often made with a low agar height. The part of the printer that the extrusion head is mounted to, which is called the x-carriage, was low enough to hit the walls of the Petri dish, causing the prints to be misshapen. Through this testing, he recommended changing either the height of the needle, or the height of the agar in the Petri dish. Since the CR Touch needs to touch the bed for Z-homing, the needle has to be further from the bed than CR Touch, so the best option for fixing this issue is using petri dishes that have higher agar levels. The final volume of CaCl₂-LB-agar was 35mL per plate, which resulted in a consistent 6.7mm height.

Another recommendation from Bruno was to take a look at how the CR Touch probe restricts the printing area. With the probe, only half of the Petri dish can be printed on due to the CR Touch hitting the Petri dish wall. Bruno recommended moving the CR Touch sensor when not in use, so we removed one of the mounting screws, allowing it to be flipped up and out of the way after Z-homing. When tested, the probe no longer hit the wall, maximizing the printable space on the Petri dish.

Round 2:

For our second round of user testing on the final design of the printer, the team had Showmick Paul, the Wiki Manager from the iGEM Rochester 2022 team Saptasense, which had been a Best Hardware Nominee, come provide the user testing and feedback. Showmick had demonstrated interest in our project and wanted to come see the hardware and software updates for himself.

After a brief description of the project and the role of our bioprinter, Showmick learned how to load the SD card, pull up the desired Gcode, and completed two rounds of printing the 4% alginate bioink with our engineered Creality Ender 3 Neo, along with a comparison to hand extrusions with the glass syringe and 0.5mm needle attached.

He created a pros and cons list of hand extrusion and our bioprinting method:

Showmick’s overall comments on the 3D bioprinter were that he liked the creative design and customizability of the syringe pump system and “inkjet” (extrusion head) that was CADed to fit the needs of the team. With the different sliding parts of the extrusion head, the tubing and the needle tips could be adjusted and taken apart as required. He appreciated how the syringe holders allowed great accessibility to the syringes themselves when needing to refill them with the according bioink. Showmick was very impressed with the specificity at which these custom plastic parts were CADed, and how they were able to function with ease. The team really appreciated his comments and fresh perspetives, and plan to implement some of these changes to improve our hardware.

User testing provided us valuable feedback that we incorporated directly by modifying our 3D bioprinter and adjusting associated bioink-related set-up to print at consistent heights. We also learned of key features users expect and appreciate when using a 3D printer — especially for users who are well-versed in using this technology.

If we had more time to continue the engineering process on the printer, we would have liked to implement the following suggestions from our users:

1. For the extrusion pump, Bruno suggested three different options for improving the syringe pump pressure. One suggestion was to add gearing, which allows us to still use a weaker, cheaper stepper motor while applying more force. Another option would be to use a larger diameter syringe, which would require less force to generate the necessary pressure for bioink extrusion; comparing two syringes of different diameters, a syringe with a 2-unit diameter will result in four times the pressure from the same applied force as compared to a syringe with a 1-unit diameter because of the area. The final option Bruno helped conceptualize was changing the tubing. We have both low and high pressure tubing, and the low pressure tubing is currently being used on the printer. The alginate bioink we are using to print is relatively viscous, so it requires higher extrusion pressure via needles. Because the tubing being used is for low pressures, the tubing can expand, which creates uneven movement of the extruder and causes an increase in tube volume, which in turn reduces the pressure at the needle. The higher pressure tubing has nylon to help it resist expansion. We had originally decided against the high pressure tubing due to the larger inner diameter, which reduced the amount of bioink that could be extruded due to the larger volume of ink that would stay in the tubing. From Bruno’s feedback, higher pressure tubing would be a good option if there was a smaller inner diameter that could be purchased.


2. Showmick recommended fixing the tubing to the side of the printers so it would not get in the way of the extrusion head and Z-homing. Next, instead of utilizing a hex key and loosening the screw attaching the CR Touch to allow maximized printing on a Petri dish, he recommended more automated technology. Thus, the bulky size of the CR Touch would no longer be a limiting factor to the types of prints possible as it runs into the walls of the Petri dish. This could include a laser to determine height of the build plate via the time it takes for the light to be emitted and received. RoSynth had originally contemplated magnetic technology to treat the Z-homing process similar to a GPS. If we were to have more time to develop this project, the automatic outward swing of the CR Touch would be implemented, and the auto-bed leveling technique of the Creality Ender 3 Neo printer would be modified.


3. An improvement that would benefit the printing of the waffle shape would be changing the needle heights so that the needle not being used could be raised out of the way. A proposed method for going about this would be using a small servo to raise and lower the needles, so while one needle is raised, the other is lowered into printing position. From a software standpoint, this would be difficult and would likely require an external microprocessor board that would take signals from the extruder motors and respond with servo movement.

Selecting a bioink is a highly specific process depending on the application. Bioinks vary across properties including pore size, biocompatibility, and structural integrity, factors that affect the viability of microbes loaded within and the fidelity of the 3D-printed structure to the coded shape.

More importantly, different bioinks have different crosslinking mechanisms, the process during printing through which a bioink undergoes sol-gel transition from a liquid to a gel state. A common crosslinking method is the addition of metal ions on the print surface, so that immediately after extrusion, the physical contact between the bioink and ion crosslinker initiates the transition to a hydrogel state. Other bioinks undergo sol-gel transition on a temperature basis, while others require a photoinitiator [1]. We prioritized bioinks with simple, practical crosslinking mechanisms that would integrate with our 3D bioprinter. To make our 3D bioprinting system as simple and easy-to-use as possible, we preferred bioinks that required either physical crosslinkers or undergo temperature or pH sensitive sol-gel transitions. These crosslinking mechanisms are feasible to accomodate with a home-built printer, through printing on a surface with the crosslinker ion or adjusting the temperature of the print bed.

To optimize our bioinks for our parallel culture system, we first considered five common bioinks used in 3D extrusion-based bioprinting, and not specific to microbial cultures: [2]

1. Alginate: Alginate is extracted from brown seaweed and is used extensively in microbial printing. It is crosslinked with calcium ions, typically with calcium chloride.
2. Chitosan: Chitosan is obtained from chitin and is used in tissue engineering.
3. Collagen: Collagen is used in tissue engineering for its characteristically high cell biocompatibility with mammalian cells, and is temperature and pH sensitive.
4. Gelatin methacrylate (GelMA): GelMA also has high biocompatibility and is used for biomedical applications, and has temperature sensitive crosslinking.
5. Pluronic F-127: Pluronic-based bioinks have good printability and have temperature sensitive crosslinking.

However, after further research, we learned that chitosan has antimicrobial properties and thus likely would not be suitable for our bacteria and yeast co-culture [2]. The other bioinks were established as relatively non-toxic.

We then finalized four bioinks to experimentally test, customize, and characterize for our parallel culture system: alginate, collagen, GelMA, and pluronic F-127.

For these four bioinks, we identified biocompatibility and diffusivity as factors of interest to conduct bioink testing.

To test the predictions of the Multiscale Model for Solute Diffusion (MSDM) model, we conducted diffusion tests for four of our hydrogels: alginate, collagen, GelMA, and pluronic F-127. We picked two dyes, Alizarin and Coomassie Blue, to test the diffusion of different sized molecules through different hydrogels.

Alizarin red was chosen as one of the dyes since it has a similar molecular weight (240.211 Da) as salvianic acid A (198.173 Da) [3]. To compare the diffusion of a smaller dye with a larger dye, we chose a second dye of Coomassie Blue with a larger molecular weight (695.584 Da) than all our intermediates and product [4]. Therefore, the diffusion of coomassie blue would demonstrate that molecules smaller than the dye, such as our product rosmarinic acid, would successfully be able to diffuse in the hydrogels.

Materials

Safety

Alizarin is a mild irritant and Coomassie Blue has no known hazards [4].

Protocol

Assumptions

Analysis

We took images of the gels at six different time points: 1 min, 2 min, 3 min, 1 hr, 2 hr, and 3 hr. We standardized the location, lighting of the pictures, and the phone used to prevent confounding variables in our analysis. Weights of the gels were measured before and after the experiment to observe differences in absorption, swelling, and gel breakdown. Using ImageJ, an imaging processing program, we applied color deconvolution to each image to isolate a single color on the image and measure the mean intensity value. For diffusion tests ran with Alizarin, the isolated color was red, while for Coomassie Blue, the isolated color was blue. To keep measurements consistent across the gels, we selected a region of interest (ROI) of 100 pixels x 100 pixels to measure the intensity values. As we measured mean intensity, a smaller intensity value meant the color was closer to pure blue or red depending on the dye. Values closer to 255, which is the max, represent white. We chose the same area of the gel to analyze across the time points. Some deviations did occur when the gel broke down, leading to the original area being smaller than 100 pixels x 100 pixels.

We used the mean intensity values to find diffusion proportions for each gel and each dye. Using the mean intensity value of the gel before diffusion as the standard, we subtracted the mean intensity value at the different time points from the standard. Next we divided by the standard mean intensity value to get a diffusion proportion, which is a measure of relative diffusivity since it shows the change in dye concentration compared to the starting point. For example, in the Alizarin alginate experiment, the mean intensity value of alginate was 180.42 brightness/pixels² and the mean intensity value of alginate at 1 min was 106.57 brightness/pixels². To find the diffusion proportion we did (180.42 - 106.57)/180.42 to get 0.41.

For the following images, the leftmost bottom and top images are the measurements of the length and width of the hydrogel. For the Petri dish images, each Petri dish has four compartments, the bottom left is 5mL of dye, the top left is distilled water to rinse the hydrogel, and the top right is the hydrogel picture.

Results

When observing the hydrogels during the diffusion experiment, it was clear which hydrogels started to disintegrate. During both diffusion experiments using pluronic 25%, the hydrogel completely dissolved in the dye by the one hour mark, resulting in a large net loss of mass (Figure 5). Conversely, collagen had the largest gain in mass, likely due to absorption of the dye.

When analyzing diffusion over time, alginate, collagen, and GelMA had relatively high diffusion proportions (Figure 6). Mean red intensity and mean blue intensity were measured before the hydrogels were placed in the dye to obtain a standard of zero diffusion. From the MDSM model, the predicted relative diffusivity of salvianic acid A in the hydrogels from highest to lowest were collagen, GelMA, alginate, then pluronic F-127. The diffusion proportions obtained from diffusing Alizarin were similar in trend to those predicted for salvianic acid A. In the experiment, the diffusion proportion of alizarin was highest in alginate, followed by collagen, GelMA, and then pluronic F-127. Pluronic F-127 had consistently the lowest relative diffusivity, and GelMA had a lower relative diffusivity than collagen. Alginate reached a diffusion proportion of around 0.85 at the 2 hour time point, collagen reached 0.72 at 3 hours, GelMA around 0.66 at 3 hours, and pluronic F-127 reached around 0.35 after 1 min (Figure 6). Differences between predicted values and the experimental data could be attributed to the fact that the max diffusion wasn’t reached during the time frame of 3 hours. If the experiment was run for longer, it’s possible that diffusion proportions would be closer to our predicted relative diffusivity because enough time would be given for the hydrogel to take in more dye and reach its max diffusivity. Another factor is our alizarin dye is a bit larger than salvianic acid A, which would result in a lower relative diffusivity than salvianic acid A. Some hydrogels were starting to disintegrate during the experiment, which could result in a lower intensity measure and diffusion proportion than expected.

When looking at the diffusion of Coomassie Blue in the hydrogels, we observe that the diffusion proportion increases over time (Figure 7). Alginate reached a diffusion proportion of around 0.62 at 2 hours, collagen reached 0.78 at 3 hours, GelMA around 0.86 at 3 hours, and pluronic F-127 was low at less than 0.05 at 1 minute. In the experiment, the diffusion proportion of coomassie blue was highest in GelMA, followed by collagen, alginate, and then pluronic F-127. Compared to Alizarin diffusion, the diffusion proportions of coomassie blue in collagen and GelMA were higher, suggesting that larger molecules diffuse better through GelMA and collagen. The diffusion proportion of coomassie blue for alginate was lower than alizarin, suggesting that alginate diffuses smaller molecules better. Pluronic F-127 had a diffusion proportion close to zero, suggesting large molecules don’t diffuse in pluronic F-127 well. Similar to the Alizarin experiment, low diffusion proportions could be due to short experiment time and the breakdown of hydrogels during the experiment.

Conclusion

From the diffusion testing, we conclude that our predicted model was accurate that GelMA, alginate, and collagen had a higher relative diffusivity compared to pluronic F-127 for both molecule sizes.

Abstract

Tensile testing is an important method for assessing the mechanical properties of materials, enabling the study of how different factors, such as the presence of bacteria, affect the behavior of printed materials for the parallel cultured 3D bioprinting system. In this study, the tensile properties of three sample groups were examined: A control group consisting of bacteria-free 4% alginate, samples from groups containing 4% alginate cultured with green fluorescent protein (GFP)-expressing E. coli strains, and samples from groups containing 4% alginate cultured with red fluorescent protein (RFP)-expressing E. coli strains. The aim of this study was to determine the effect of the presence of E. coli and different fluorescent proteins on the tensile properties of alginate materials. For the testing, a 3D-printed sample in a dog bone shape was made, and the structures were then placed on LB plates containing chloramphenicol (chlora). This study provides valuable insights into the mechanical behavior of alginate-based materials under various bacterial conditions. We completed this testing in the Meredith Silberstein's Mechanics for Materials Design (MMD) Lab, located in Cornell University, Ithaca, NY.

Introduction

Tensile testing is a fundamental method for evaluating the mechanical properties of materials, offering insights into their strength, ductility, and resilience. In this study, we investigated the tensile properties of alginate-based samples under different bacterial conditions, specifically focusing on the effects of E. coli expressing green fluorescent protein (GFP) and red fluorescent protein (RFP). The samples under investigation included a control group with 4% alginate without bacteria, 4% alginate cultured with GFP-expressing E. coli strains, and 4% alginate cultured with RFP-expressing E. coli strains. Utilizing the 3D printing technology, we fabricated dogbone-shaped specimens for accurate tensile testing in the Silberstein Lab in Cornell, placing them on LB agar plates with chloramphenicol to maintain bacterial cultures in the - 4°C fridge. Our research aims to uncover how the presence of specific bacteria and fluorescent proteins influences the tensile properties of alginate-based materials, with potential implications for applications like bioprinting, where material integrity is crucial. We hypothesized that the presence of bacteria, whether GFP- or RFP-expressing E. coli, would not induce any unexpected chemical or structural changes in the alginate material that could alter its tensile properties. This study seeks to make valuable contributions to the fields of materials science and biotechnology by enhancing our understanding of how microorganisms and fluorescent proteins affect material behavior.

Why Tensile Tests?

Tensile testing is a basic tool for materials science and engineering and serves a variety of key purposes. It can assess mechanical properties such as Young's modulus and maximum tensile strength, both of which are critical to understanding how materials respond to the forces exerted. In the context of studies of alginate-based materials with and without bacteria, tensile testing is essential to assess the mechanical integrity of these materials under different bacterial conditions. Young's modulus is a key parameter because it quantifies the stiffness of the material and its ability to return to its original shape after deformation, which is critical for predicting its behavior in response to mechanical stress. On the other hand, the maximum tensile strength represents the ultimate resistance of the material to tensile forces, providing insight into its overall structural integrity. This information is critical to assess the applicability of biological inks like alginate-based materials for bioprinting, where the ability to withstand mechanical forces is critical to decide the optimal material and microbe strain to use.

Materials

Sample prepared using the bioink protocol:

Load the 3D printer to print the dogbone shape and ensure the thickness of the gauge with enough printing materials, and place the structure on the LB plates with chlora.

Safety

There are several potential hazards associated with the use of green fluorescent protein-expressing E. coli and RFP-expressing E. coli in laboratory settings. These hazards primarily relate to biocontainment and biosecurity measures, including the risk of accidental release into the environment or contamination of personnel and equipment. Additionally, there is a risk of accidental exposure to live bacteria, particularly during handling of cultures or during disposal, which could lead to infection or allergic reactions in laboratory personnel. To mitigate these hazards, biosafety protocols including the use of appropriate personal protective equipment were strictly followed when working with GFP and RFP-expressing E. coli strains in research processes and every equipment was sanitized with the ethanol spray after operations.

Protocol

Sample Measurement: Measure the gage length (both wide and narrow section), gage width, and thickness of the material accordingly.

Completing Tensile Testing

Assumptions

First, each set of samples was assumed to be representative of the entire population and to exhibit consistent material properties, disregarding any potential sample-to-sample variability. Second, it was assumed that the tensile behavior of the alginate-based material remained linearly elastic within the strain range chosen for Young's modulus determination. Furthermore, the assumption was that the loading conditions and testing equipment remained consistent for all samples to ensure the reliability of the results. Additionally, tensile tests were performed at a constant temperature and humidity, with the assumption that changes in environmental conditions did not significantly affect the properties of the materials under examination. We hypothesized that the introduction of bacteria, whether in the form of GFP- or RFP-expressing E. coli, would not lead to unforeseen alterations in the chemical or structural composition of the alginate material that might impact its tensile characteristics.

Analysis Methods

In this study, the tensile testing method was utilized to generate force-displacement curves for various sample groups, including the control, green fluorescent protein-expressing E. coli, and RFP-expressing E. coli groups. To achieve this, dog bone-shaped specimens created using 3D printing technology were fixed to the tensile tester. Tensile tests were conducted by incrementally applying tensile loads while recording the corresponding displacement values. The resulting data, which represented the applied force and associated displacement, was collected in real-time and subsequently exported for further analysis using MATLAB. Young's modulus and the ultimate tensile strength for each material were then computed based on the force-displacement curves, providing a dependable quantitative analysis of the mechanical properties of the alginate-based materials in each sample group [5].

Using the data and points from the elastic region and the point at the end of the proportional limit, the stiffness (K), Young’s Modulus (E), the Maximum Force Applied, the Original Cross-sectional Area, and the Ultimate Tensile Strength can be calculated using the following equations:

    Stiffness (K) = Change in Load Applied/ Change in Displacement                                 
    Material's elastic Modulus (E) = Stress / Strain
    Stress = Force / Area 
    Strain = Deformation / Original Strength 
    Ultimate Tensile Stress (UTS) = Maximum Load Applied / Original Cross Sectional Area
  

Conclusion

From the tensile testing, we concluded that the presence of bacteria, whether GFP- or RFP-expressing E. coli, did not significantly induce any unexpected chemical or structural changes in the alginate material that could alter its tensile properties with the similar results from the three different sample groups.

Future Plan

Our focus was to explore the influence of bacterial presence and fluorescent proteins (GFP and RFP) on alginate-based materials, and by using E. coli consistently, we ensured a controlled and reproducible environment for our investigations. By conducting tensile testing with bacteria, the future plan is to conduct similar testing with yeast to see if hydrogels laden with yeast experience similar stress and strain curves as bacteria.

Acknowledgments

Special Acknowledgments to the assistance provided by the Silberstein Lab, Department of Mechanical and Aerospace Engineering, Cornell University, and the Meyer Lab, Department of Biology, University of Rochester.

We utilized fluorescent cell testing to assess the cell viability as well as cell growth behavior in bacteria expressing red fluorescent protein (RFP) and yeast expressing yellow fluorescent protein (YFP). Fluorescent intensity emitted from cells is a good indicator of cell growth. We performed many tests to verify cell viability in hydrogels and cell growth behavior in our hydrogels. The results and their interpretations can be found in the cell viability portion of the Wet lab tab.