Dec 30, 2024

Public workspaceProtocol for FRESH extrusion 3D printing of Type-1 collagen hydrogels photocrosslinked using Ruthenium

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.x54v92r2ml3e/v1
  • Richard Steiner1,2,
  • Jack Buchen1,2,
  • Evan R. Phillips1,3,
  • Christopher R. Fellin1,2,
  • Xiaoning Yuan2,
  • Shailly H. Jariwala1,2
  • 1The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., 6720A Rockledge Drive, Suite 100, Bethesda, MD 20817, USA;
  • 2The Center for Rehabilitation Sciences Research, Department of Physical Medicine and Rehabilitation, Uniformed Services University of Health Sciences, 4301 Jones Bridge Rd, Bethesda, MD 20814, USA;
  • 3CytoSorbents Medical Inc., 305 College Road East, Princeton, New Jersey, 08540, USA
  • Richard Steiner: R.C.S., J.T.B, E.R.P., C.R.F, and S.H.J conceived the process and outlined the content. R.C.S. and J.T.B. contributed equally to writing the protocol and the accompanying article. X.Y. and S.H.J. provided guidance on the scope and content of the protocol. All authors reviewed, revised, and finalized the protocol.;
  • PLOS ONE Lab Protocols
    Tech. support email: plosone@plos.org
Jack T Buchen
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.x54v92r2ml3e/v1
External link: https://doi.org/10.1371/journal.pone.0317350
Protocol Citation: Richard Steiner, Jack Buchen, Evan R. Phillips, Christopher R. Fellin, Xiaoning Yuan, Shailly H. Jariwala 2024. Protocol for FRESH extrusion 3D printing of Type-1 collagen hydrogels photocrosslinked using Ruthenium. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v92r2ml3e/v1
Manuscript citation:
Steiner RC, Buchen JT, Phillips ER, Fellin CR, Yuan X, et al. (2025) FRESH extrusion 3D printing of type-1 collagen hydrogels photocrosslinked using ruthenium. PLOS ONE 20(1): e0317350. https://doi.org/10.1371/journal.pone.0317350
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 10, 2024
Last Modified: December 30, 2024
Protocol Integer ID: 101546
Keywords: Bioprinting, Collagen Bioprinting, FRESH, FRESH Bioprinting, Extrusion 3D Bioprinting, FRESH extrusion 3D printing of Type-1 collagen hydrogels, FRESH extrusion 3D printing of Type-1 collagen hydrogels photocrosslinked using Ruthenium
Funders Acknowledgements:
This study was funded by the Center for Rehabilitation Sciences Research’s(CRSR) In-House Laboratory Independent Research (ILIR) award, Department of Physical Medicine and Rehabilitation, Uniformed Services University, Bethesda, MD, USA
Grant ID: #HU00012320007
Disclaimer
The opinions and assertions expressed herein are those of the authors and do not necessarily reflect the official policy or position of the Uniformed Services University (USU) or the Department of Defense. The opinions and assertions expressed herein are those of the authors and do not necessarily reflect the official policy or position of the Henry M. Jackson Foundation for the Advancement of Military Medicine (HJF), Inc.
Abstract
The extrusion bioprinting of collagen material has many applications relevant to tissue engineering and regenerative medicine. Freeform Reversible Embedding of Suspended Hydrogels (FRESH) technology is capable of 3D printing collagen material with the specifications and details needed for precise tissue guidance, a crucial requirement for effective tissue repair. While FRESH has shown repeated success and reliability for extrusion printing, the mechanical properties of completed collagen prints can be improved further by post-print crosslinking methodologies. Photoinitiator-based crosslinking methods are simple and have proven effective in strengthening protein-based materials. The ruthenium and sodium persulfate photoinitiator system (Ru(bpy)3/SPS) has been suggested as an effective crosslinking method for collagen materials. Herein, we describe the procedure our group has developed to combine extrusion-based 3D printing of type-1 collagen using FRESH technology with Ru(bpy)3/SPS photoinitiated crosslinking methods to improve the strength and stability of 3D printed collagen structures. 
Materials
●      Corning‱ 50 mL centrifuge tubes
●      Kimwipes‱ disposable wipers
●      Allevi Plastic Syringes (5cc); Catalog No. SKU-PSYR5
●      Eppendorf Centrifuge 5810 R 15amp
●      40 x 25mm glass dish
●      30 gage, 1-inch blunt end, lavender, FisnarⓇ; Catalog No. 8001104
●      Allevi 3 systems, SN. 7c1057c
●      Sundee glue tape 
●      Fisherbrand‱ Premium Microcentrifuge Tubes: 2.0mL; Catalog No 05-408-138
●      Aluminum foil
●      Fisherbrand 10ml serological pipettes (2X)
●      Eppendorf Centrifuge 5810 R 15amp
●      Heidolph Instruments Vortex D-91126 Schwabach REAX top 541-10000-01-1
●      40mm x 25mm glass dish
●      Fisherbrand‱ Semimicro Spatula with One Tapered End, One Rounded End 14-374
●      Sundee glue tape 
●      Heidolph Instruments Vortex D-91126 Schwabach REAX top 541-10000-01-1
●      AmScope 3WX2 LED-6WD LED Spot Light with Two 6500K LED Attachments; Catalog Number YAG20201215
●      100-1000 µl pipette, Fisherbrand Elite NU09348 and pipette tips
●      Drummond Scientific Co. Pipetaid XP 
●      Thermoscientific HERRAcell i150 Incubator

Software:
●      Slic3r
●      Repitier 
●      Autodesk Fusion, Shapr3D, or TinkerCAD


Protocol materials
Reagent1X Phosphate Buffered Saline SolutionCatalog #BP2438-4
ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267
Reagent1X Phosphate Buffered Saline SolutionCatalog #BP2438-4
ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267
ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267
ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267
ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267
ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267
Reagent1X Phosphate Buffered Saline SolutionCatalog #BP2438-4
ReagentSodium PersulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6172
ReagentTris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #224758
Reagent1X Phosphate Buffered Saline SolutionCatalog #BP2438-4
ReagentLifeSupportAdvanced BiomatrixCatalog #5244
Reagent1X Phosphate Buffered Saline SolutionCatalog #BP2438-4
ReagentTris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #224758
ReagentSodium PersulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6172
Reagent1X Phosphate Buffered Saline SolutionCatalog #BP2438-4
ReagentTris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #224758
ReagentSodium PersulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6172
Reagent1X Phosphate Buffered Saline SolutionCatalog #BP2438-4
ReagentTris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #224758
ReagentSodium PersulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6172
LifeSupport Preparation
LifeSupport Preparation
10m 45s
10m 45s
Prepare the centrifuge by cooling the temperature to Temperature4 °C

Prepare Amount35 mL of Reagent1X Phosphate Buffered Saline SolutionContributed by usersCatalog #BP2438-4 , and refrigerate until temperature reaches Temperature4 °C .

Retrieve the Advance Biomatrix LifeSupport, which is stored at room temperature.
Prepare an emptyAmount50 mL conical tube for mixing LifeSupport. Separate the LifeSupport powder into two Amount1 g aliquots, each in a separate Amount50 mL conical tube.

To each of the 1g aliquots of LifeSupport, add Amount17.5 mL of Temperature4 °C PBS.

For each conical tube, shake vigorously for Duration00:01:00 by

1m
Shaking horizontally along the length of the tube, and
Tapping the end of the tube against the edge of the table. Continue until the gelatin has been uniformly mixed, and there’re no longer large clumps clinging to the walls of the tube.
Place the conical tubes containing LifeSupport into a Temperature4 °C refrigerator for Duration00:45:00 . This step allows the LifeSupport to fully rehydrate, and ensures the mixture is at an adequate temperature.

45m
Vortex each conical tube for Duration00:00:45 .

45s
Centrifuge the conical tubes at Centrifigation2500 x g for Duration00:05:00 .

5m
Remove conical tubes from the centrifuge, open the caps, and pour off the supernatant. Absorb the remaining supernatant with Kimwipes (low-linting) to remove as much liquid as possible.
Recap and shake the conical tubes again. For each conical tube, shake vigorously for Duration00:01:00 by

1m
Shaking horizontally along the length of the tube, and
Tapping the end of the tube against the edge of the table. Continue until the gelatin has been uniformly mixed, and there are no longer large clumps clinging to the walls of the tube.
Vortex each conical tube for Duration00:00:45

45s
Centrifuge the conical tubes again at Centrifigation2500 x g for Duration00:05:00

5m
Pour off or absorb any remaining supernatant with a Kim wipe.
Prepare a 40 x 25mm glass dish.
Uncap the first conical tube, and invert the tube into the glass dish so that it’s upside down with the open end inside the glass dish. Tap the tube up and down vertically until the gelatin LifeSupport dislodges and fills the glass dish.
Remove the conical tube from the dish. Using a small spatula, scoop the remaining LifeSupport from the walls of the conical tube, being careful not to introduce new air bubbles. Any LifeSupport that is scooped out of the conical tube should be placed on top of and in the center of the LifeSupport that is sitting in the glass dish.
Carefully but forcefully tap the glass dish flat against a table to get the LifeSupport to spread out and fill the base of the dish. If small air bubbles form during this process, use the spatula to carefully scoop them out, and then tap the LifeSupport against the table again to fill the void.
Uncap the second conical tube, and carefully scoop the LifeSupport out, careful not to create air bubbles. Place the LifeSupport from the second tube on top of the LifeSupport from the first tube. Again, tap against the table to disperse the LifeSupport evenly. Use the spatula to remove any large air bubbles that form.
LifeSupport should achieve a vaseline-like consistency, with no flow of material from side-to-side when tilted.

The LifeSupport can be stored at Temperature4 °C for up to Duration01:00:00 but should be used as quickly as possible to obtain the best results

1h
Ink Preparation
Ink Preparation
5m
5m
Commercially available Amount0 g syringes designed for Allevi 3 systems 3D printers were used to hold and disperse LifeInk 240 during 3D printing. Prepare the 5cc syringes by removing the plunger cap from the plunger and placing it at the bottom of the syringe until it is in contact with the syringe neck.

Using a syringe female-female luer lock adapter, transfer Amount4 mL of ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267 to the 5cc syringes.

To eliminate air bubbles and gaps in the ink, cap the syringe, then centrifuge the syringe at Temperature4 °C and Centrifigation2000 x g forDuration00:05:00

5m
To remove any final air bubbles, use an empty syringe with a 30 gauge needle. Insert the needle into the plunger cap and suck out any air pockets. You may also use a spatula to move the plunger cap down into the empty space, while simultaneously letting air escape out the side of the plunger cap.
Store the prepared syringe at Temperature4 °C until printing.

3D Print File Preparation
3D Print File Preparation
3D files can be designed using standard computer-aided-design (CAD) software. Popular options include AutoCAD, Solidworks, Autodesk Fusion, Shapr3D, etc. Any CAD software that can adequately meet your design needs and can export files as .stl or .obj is acceptable. 
3D files were designed in Autodesk Fusion, Shapr3D, or TinkerCAD. Files were exported in .stl file format. 
After 3D files have been created, they need to be “sliced.” Slicing is the process by which the 3D file is translated into a G-code file. G-code is a software programming language used to control 3D machines, such as CNC machines and 3D printers. The Allevi 3 3D Bioprinter used in this process accepts G-code files. Popular slicing software includes UltiMaker Cura, PrusaSlicer, Slic3r, Repetier, etc. Files for this protocol were sliced using Slic3r and Repetier. 
To slice a 3D CAD file, open Repetier. 
Select “Config”, and select the standard printer configuration.
Click “Load,” then upload your .stl 3D file.
Under “Object Placement,” assign extruder 0 to the “Object Group 1.”
Click “Slicer.” From the dropdown menu titled “Slicer:” select “Slic3r.”
If desired, select “Configuration” to choose your specific slicing parameters (see below), then save and return to the Repetier window.
Ensure that “Extruder 1:” selection lists your extruder that the bioink will be extruded from.
Click “Slice with Slic3r.” Wait for the Slic3r window to pop up.
Click “Export G-code…” Save the Gcode. You will upload it to the Allevi 3 Bioprinter in the next section.
Slicing Parameters: 
 
For our purposes in designing a nerve guide conduit, we used the following parameters:
 
50% Infill (This prevents the object from being too dense and overfilled)
Rectilinear Infill (This is the pattern of infill inside your print)
150% Overlap (This ensures that each individual layer and line is properly connected to the last, allowing for cohesion of layers during crosslinking)
No Perimeter (Typically slicing software will allow you to print additional perimeters around your design with a different set of parameters than the rest of the design. We avoided this for two reasons: 1) Additional perimeters were not factored into our design specifications, thus adding perimeters after the fact would increase the size and deform the shape. 2) In our extensive testing, we found that perimeters, with our particular collagen bioink, tended to shear and separate from the rest of the print)
0.10mm Layer Height (Setting the layer height shorter than the inner diameter of the needle allowed us to force additional vertical overlap, thus creating a well-bonded and cohesive object)
0.17mm Extruder Width (The extruder width is traditionally set at or slightly larger than the inner diameter of the extruder nozzle. In this situation, the inner diameter of our needle was 0.15mm, but through experimentation and testing, we identified 0.17mm to be the best extruder width)
6 mm/s print speed (This is the tool speed at which the extruder moves along it’s path. For our purposes, we identified 6mm/s to be the best print speed, both for print quality and to prevent excessive drag interference.) 
Allevi 3 System Preparation
Allevi 3 System Preparation
Allevi 3 systems should be turned on and connected to a WiFi server without interruption during the printing process.
Make sure working room environmental conditions read to between Temperature17-22 °C with minimal surface vibrations for optimal printing.

Using an empty 40 x 25mm glass dish, center the dish on the print stage then mark the edges of the dish onto the print stage with a marker. Using an empty 5cc syringe with a 1 inch, 30 gauge needle attached, position the needle into the printer head and lock in place with the air compression hose.
On the Allevi interface, we use the directional controls (x,y, and z-axis) to position the tip of the needle to the bottom center of the 40 x 25mm glass dish. We position the tip of the needle so that there is only 0.1mm z-spacing between the needle tip and the glass dish surface. If the position of the needle tip relative to the glass dish is correct and acceptable to the print dimensions, then using the Allevi interface move the print stage on the z-axis down from the needle tip by 25 mm. Remove the empty syringe from the print head and the glass dish from the print stage.
Set the printer head parameters on the interface screen to the following: Ink temp Temperature4 °C , air pressure of 50psi.

Collect the prepared 5cc of LifeInk from the Temperature4 °C fridge. Place into the syringe slot on the printer head and securely attach and lock the air compression hose to the top of the syringe.

Calibrate the extrusion properties of the ink by manually extruding the ink through the length of the needle and observing the rate of droplet formation at the needle tip. Manually extrude several times to clear the line of air bubbles. Make sure to wipe the needle tip with Kim wipe after testing to ensure no residual dried ink will interfere with droplet formation and ink flow.
Upload the Gcode file for the desired print to the Allevi server using the Allevi interface. Ensure the Gcode is selected in the interface screen before printing.
Collect the prepared lifesupport dish from the Temperature4 °C fridge. Add double-sided Sundee glue tape to the bottom of the dish, then place the dish on the printer stage so that the edges of the dish align with the marks on the stage. Ensure the dish is adhered tightly to the stage.

Press the print button on the interface to begin printing.
Crosslinking
Crosslinking
For 25/250 mM Ru/SPS Concentration:
  1. Prepare two 2.0ml microcentrifuge tubes by wrapping them in aluminum foil to prevent light leakage.
  2. Label one tube Ru and the other SPS. 
  3. To each, add Amount1 mL ofReagent1X Phosphate Buffered Saline SolutionContributed by usersCatalog #BP2438-4 . 
  4. To the tube labeled Ru, add Amount0.0187 g of ReagentTris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #224758 . Close the tube and ensure it is adequately wrapped in aluminum foil. 
  5. To the tube labeled SPS, add Amount0.0595 g of ReagentSodium PersulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6172 . Close the tube and ensure it is adequately wrapped in aluminum foil. 
For each tube, vortex for one minute to ensure that the mixture is uniformly distributed




For 50/500 mM Ru/SPS Concentration:
  1. Prepare two 2.0ml microcentrifuge tubes by wrapping them in aluminum foil to prevent light leakage.
  2. Label one tube Ru and the other SPS. 
  3. To each, add Amount1 mL of Reagent1X Phosphate Buffered Saline SolutionContributed by usersCatalog #BP2438-4 . 
  4. To the tube labeled Ru, add Amount0.0374 g of ReagentTris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #224758 . Close the tube and ensure it is adequately wrapped in aluminum foil. 
  5. To the tube labeled SPS, addAmount0.1191 g of ReagentSodium PersulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6172 . Close the tube and ensure it is adequately wrapped in aluminum foil. 
  6. For each tube, vortex for one minute to ensure that the mixture is uniformly distributed. 





For 75/750 mM Ru/SPS Concentration:
  1. Prepare two 2.0ml microcentrifuge tubes by wrapping them in aluminum foil to prevent light leakage.
  2. Label one tube Ru and the other SPS. 
  3. To each, add Amount1 mL of Reagent1X Phosphate Buffered Saline SolutionContributed by usersCatalog #BP2438-4 .  
  4. To the tube labeled Ru, add Amount0.0561 g ofReagentTris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #224758 . Close the tube and ensure it is adequately wrapped in aluminum foil. 
  5. To the tube labeled SPS, add Amount0.1786 g ofReagentSodium PersulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6172 . Close the tube and ensure it is adequately wrapped in aluminum foil. 
  6. For each tube, vortex for one minute to ensure that the mixture is uniformly distributed. 




Determining the Volume to add to different dish sizes:
Calculate the volume of the dish you used for printing, and multiply the volume of the dish by 2%. This will give you the volume of each solution you need to pipette into the dish for crosslinking.  For Example: 40 x 25mm glass dish


Visible Light Crosslinking- Wavelength and Power Density:
We used an AmScope 3WX2 LED-6WD LED Spot Light with Two 6500K LED Attachments that was positioned approximately 75mm away from the sample. 
 
The light produced by this system was 425nm, with a power density of .

Crosslinking the collagen Print
Crosslinking the collagen Print
1h 10m
1h 10m
Place a container of Amount500 mL of Reagent1X Phosphate Buffered Saline SolutionContributed by usersCatalog #BP2438-4 in an incubator or water bath at Temperature37 °C until it comes to temperature.

Place the 40 x 25mm glass dish containing the LifeSupport and completed ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267 collagen print in an incubator at Temperature37 °C for Duration00:45:00 .

45m
Prepare two Amount10 mL serological pipettes and a waste container. Set aside for later use.

Remove the dish from the incubator. Verify that the LifeSupport has melted to form a non-viscous liquid and that the ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267 print has turned an opaque white color. If not, place it back into the incubator and monitor it until a change is observed. If so, proceed to the next step.

Pipette Amount10 mL of LifeSupport out of the dish and place in the waste container. Be careful not to disturb the ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267 bioprint with the pipette.

Pipette Amount10 mL of Temperature37 °C Reagent1X Phosphate Buffered Saline SolutionContributed by usersCatalog #BP2438-4 into the dish containing the LifeSupport and ReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267 bioprint. Be careful not to disturb theReagentLifeink 240 Acidified Collagen Bioink Advanced BiomatrixCatalog #5267 bioprint with the pipette.

Repeat steps 49 & 50 approximately 20 times, or until no gelatin remains in the dish or on the print.
If gelatin remains in the dish or on the print, place the dish back into the Temperature37 °C incubator for Duration00:10:00 , and then repeat steps 5 & 6 until no ReagentLifeSupportAdvanced BiomatrixCatalog #5244  remains.

10m
If using the AmScope 3WX2 LED-6WD LED Spot Light with Two 6500K LED Attachments: Place the 40 x 25mm glass dish on a stage with both LED  lights suspended over it such that all the LED light will be concentrated within the dish. This should be approximately 75mm above the dish if using the same dish size. Turn the brightness control slider up to 10 (max). Do not turn on the LED light at this time. If using another light source: adjust the setting of the light source to emit 425nm light at
Pipette Amount628.4 µL of the ReagentSodium PersulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6172 solution into the 40 x 25mm dish. Discard the pipette tip.

Pipette Amount628.4 µL of the ReagentTris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #224758 solution into the 40 x 25mm dish.

Use the pipette to gently draw liquid in and out, thereby mixing the solutions together in the dish. Complete this procedure approximately 20 times to ensure adequate mixing. Take caution not to disturb the collagen print.
Turn on the LED light. Expose the dish to the light for Duration00:15:00 .

15m
Use a spatula to remove the collagen bioprint from the dish. The crosslinking process is now completed.
Alternatively, you may also use a pipette to draw all of the solution out of the dish, which may make removing the print easier.
Store your completed bioprint suspended in Reagent1X Phosphate Buffered Saline SolutionContributed by usersCatalog #BP2438-4 or deionized water at Temperature4 °C until ready for use.