Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Summary

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Abstract

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

Introduction

The goal of regenerative medicine is to enhance tissue regeneration and healing through the application of engineered therapeutics. One approach involves the fabrication of biomaterial constructs that closely emulate the composition and structure of native tissues. When designing these constructs, a critical consideration is the native underlying extracellular matrix (ECM), which provides instructional cues for cell proliferation, migration, and differentiation, ultimately driving tissue regeneration^1,2. The native ECM is predominantly comprised of fibrillar collagen^3,4. In highly organized tissues such as skeletal muscle and tendon, these ECMs exhibit an aligned patterning, which is key to supporting force transmission across the tissue as well as motor function^5,6.

The utility of collagen as a biomaterial for tissue engineering applications extends beyond its extensive prevalence in native tissues, as it also displays good biocompatibility, biodegradability, and cell affinity^7,8,9. Recent advancements in the development of collagen-based biomaterials have focused on the refinement of biophysical properties to better guide the mechanical and biochemical cues that are essential for specific tissue regeneration. Creating collagen biomaterials with a fibrillar structure in vitro is relatively simple, as acid-soluble collagen molecules are capable of self-assembling into nanofibrils when exposed to warm temperatures and neutral pH^10,11. However, without guided manipulation, these fibrils will form into random networks, rendering them unsuitable for anisotropic tissue engineering applications. Techniques capable of producing aligned fibrils include electrospinning and wet-spinning, along with other solution extrusion techniques^12,13, freeze drying¹⁴, electrochemical or magnetic fabrication^15,16, shear flow deposition¹⁷, flow-induced crystallization¹⁸, gel-extrusion¹⁹, strain-induced alignment²⁰, and force-guided alignment through the manipulation of fluid flow^21,22. However, many of these methods are either not highly compatible with collagen, produce alignment on the micro-scale but not on a nanoscale, necessitate additional post-processing steps to induce alignment and stability or require highly specialized equipment and reagents, making their widespread adoption challenging.

The current study presents a facile shear-based method for the fabrication of nanofibrillar collagen, with aligned or randomly oriented fibril patterning, without the need for specialized equipment such as syringe pumps or electrospinners. This technique capitalizes on the pH dependency of collagen fibrillogenesis. Application of shear force to acidic monomeric collagen within a neutralizing buffer promotes aligned fibrillogenesis along the direction of shear. Similarly, collagen can be induced to undergo fibrillogenesis in the absence of shear, which produces fibrils with random patterns. The resulting 3D collagen strips, comprised of either aligned or randomly patterned nanofibrillar collagen, can serve as tools to study the effects of nanoscale patterning on cell behavior. Additionally, they offer diverse possibilities for both anisotropic and isotropic tissue engineering applications^{23,24,25,26,27}. The patterning may be customized to match the structure of the native tissue of interest. The collagen strips may also be combined into various-sized bundles to serve as a transplanted engineered therapeutic in diverse sizes and shapes of injuries.

Protocol

1. Preparation of dialyzed collagen ( Figure 1)

1. Cut dialysis tubing to a length of approximately 3 inches. Use tubing with the following specifications: 32 mm flat width, 20 mm inflated diameter, and 4.8 nm pore size.
2. Rehydrate dialysis tubing in ultra-pure water.
3. Clip one end of the tubing with a dialysis tubing clip.
4. Transfer 5-6 mL of rat tail collagen-type I (concentration: 9-10 mg/mL in 0.02 N acetic acid) into the tubing using a 10 mL syringe equipped with an 18 G needle. Clip the other end of the tubing to close.
5. Place a 0.5-1 cm thick layer of polyethylene glycol (PEG) flakes on the bottom of a glass dish, and then place the collagen-filled dialysis tubing on top of the PEG layer.
   NOTE: A 13 inch x 9 inch x 2 inch glass dish can typically accommodate a row of 4-5 collagen filled tubes. Dish size can be adjusted based on the volume of collagen needed.
6. Add more PEG flakes to completely cover the tubing and place the tray into a 4 °C fridge.
7. Every 10-15 min, remove the wet PEG flakes from the surface of the dialysis tubing and re-cover the tubing with a fresh layer of dry PEG. Return the dish to the 4 °C fridge.
8. Repeat step 1.7 two more times, and after 30-35 min, rinse off the wet PEG flakes using tap water.
9. Rinse the tubing once more using ultra-pure water and pat the tubing dry with a tissue.
10. Unclip one end of the tubing and transfer the dialyzed collagen (final concentration: ~30 mg/mL) into microcentrifuge tubes. Briefly spin down air bubbles in the collagen using a mini-centrifuge (2,000 x g, ≤30 s, room temperature [RT]) and store at 4° C for later use.
11. One day prior to use, load the collagen into syringes.
    1. Using a 16-gauge needle, draw up ~1 mL of dialyzed collagen into a 1 mL syringe.
       NOTE: Collagen is a viscous substance. The needle head may need to be pre-loaded with collagen prior to drawing up more collagen. Remove the needle head and expel large air pockets as necessary
    2. Remove the needle and wrap the syringe head with parafilm.
    3. Store the syringe filled with collagen upright at 4° C overnight to allow small air bubbles to pop.

2. Preparation of glass chips ( Figure 1)

CAUTION: Utilize eye protection for this portion of the protocol.

NOTE: Specially coated hydrophobic glass slides are used for cell culture experiments. The slides must be handled by the edges to prevent the removal of the surface coating. Perform the glass-cutting procedure on a suitable cutting surface.

1. Measure and mark out the desired dimensions of the glass chip using a permanent marker
   1. If creating 8-strip scaffolds, mark out approximately 1 cm increments along the long edge of the slide. Adjust dimensions as necessary based on the desired scaffold size.
2. Using a straight edge (i.e., a 6 well plate edge), glide the glass cutter down the length of the first measurement to score the glass.
3. The slide will either break along the scored line on its own or flip the slide over onto a tissue (hydrophobic side down) and manually apply pressure to the scored region.
4. Mark the coated side of the hydrophobic glass slide using an ethanol marker pen.
5. Rinse the glass chip in ultra-pure water to remove residual glass dust.
6. Dry the glass chips either using a benchtop nozzle for pressurized air or by stacking them at an angle in the fume hood.
7. Repeat as needed for the required number of chips. Store the chips at RT. For best cell culture results, use prepared chips within 7 days.

3. Preparation of 10x phosphate buffered saline (PBS)

1. Fill a glass bottle with 500 mL of ultra-pure water.
2. Add 10 PBS tablets to the bottle.
   NOTE: This protocol assumes that the tablet ratio for 1x PBS is 1 tablet per 500 mL. Adjust the number of tablets as needed if using a different tablet size or brand.
3. Add a stir bar to the bottle and place on a stir plate until the tablets are dissolved.
4. On the day of hydrogel fabrication, transfer the needed volume of 10x PBS to 50 mL conical tubes.
5. Place the PBS-filled tubes in a 37 °C water bath for at least 15 min prior to collagen fabrication.

4. Fabrication of aligned collagen nanofibrils (Figure 2)

1. Remove a syringe of collagen from the fridge and attach a blunt 22 G needle.
2. Place a glass slide in the lid of a 4-well plate.
3. Cover the slide with enough warmed 37 °C 10x PBS to submerge it (30-50 mL).
4. Holding the syringe at approximately a 30-45° angle, manually extrude thin strips of collagen onto the glass chip using a velocity of ~340 mm/s and a flow rate of ~3.2 mL/min.
   NOTE: Complete the collagen extrusion within 30-60 s of adding the warmed PBS to ensure the PBS does not cool excessively.
5. Wait for 1-2 min to allow the collagen to complete fibrillogenesis or until the collagen turns an opaque white color.
6. Use forceps to cut the collagen strips to length. Ensure the strips are long enough so 3-5 mm can be folded under either end of the chip.
7. One at a time, gently drape the strips of collagen lengthwise (parallel to scored region) over a prepared glass chip. Tuck the edges under the chip.
8. Continue placing collagen strips until the desired dimensions are achieved.
9. Position the newly formed collagen hydrogel across two wells of a 12-well plate. This enables airflow during the drying process and prevents the tail ends of the collagen from adhering to undesired surfaces.
10. Leave hydrogels on the benchtop to dry for 1-3 h or until PBS salt crystals cover 50%-90% of the hydrogel surface.
    NOTE: Drying time will vary depending on hydrogel size and the presence of excess liquid.
11. Submerge each hydrogel in 1x PBS for about 30-60 s or until salt crystals are dissolved.
12. Gently dab off excess PBS with a tissue.
13. Place hydrogels back on the well plate and leave to dry in a fume hood overnight.
14. Store the dried hydrogels at RT for about 1 week.

5. Fabrication of random collagen nanofibrils (Figure 3)

1. Remove a syringe of collagen from the fridge and attach a blunt 22 G needle.
2. Extrude collagen at ~3.2 mL/min onto a dry glass chip. Extrude enough collagen to obtain similar dimensions to the aligned collagen hydrogels. Prevent air bubble accumulation as necessary.
3. Extrude additional collagen to wrap around the edges of the glass chip. This minimizes the risk of accidental detachment.
4. Use forceps to submerge the extruded collagen into a 50 mL tube of warmed 10x PBS.
5. Wait for 45-60 s for the collagen to complete fibrillogenesis or until the collagen turns an opaque white color.
6. Gently dab off excess PBS with a tissue.
7. Complete steps 4.9-4.14 to rinse and dry the hydrogels

6. Sterilization and rehydration

NOTE: Complete all the following steps in a biosafety cabinet

1. Sterilize the collagen hydrogels and surrounding well plate in 70% ethanol for 15 min.
   1. Briefly elevate the hydrogels halfway through sterilization using forceps to ensure that both the underside of the glass chip and the hydrogel are in contact with ethanol.
2. Remove ethanol and allow hydrogels to air-dry for 10 min.
3. Wash hydrogels 3 times in sterile 1x PBS, for 10 min each, to remove residual ethanol and to rehydrate the hydrogels. Hydrogels are now ready for use (cell culture, etc.). Hydrogels may be briefly left in PBS after the final wash step if not immediately ready for use.

Results

This protocol describes a straightforward shear-based extrusion technique for the fabrication of collagen hydrogels composed of either aligned (Figure 2) or randomly (Figure 3) oriented nanofibrils. Nanofibril patterning relies on the precise control of shear forces, which is achieved through the combined modulation of syringe speed and collagen extrusion rate. Optimal values for inducing fibril alignment have been previously determined to be a syringe velocity ...

Discussion

The critical steps of the protocol can be distilled into three main parts: 1) collagen fibrillogenesis, 2) hydrogel mounting, and 3) washing. The process of collagen fibrillogenesis occurs spontaneously under neutralizing conditions and relies on the self-assembly of individual collagen molecules into larger stabilized fibrillar structures^10,11. This is achieved through the application of a neutralizing buffered medium like 10x PBS that is warmed to 37 °C an...

Materials

Name	Company	Catalog Number	Comments
Collagen I, High Concentration, Rat Tail	Corning	CB354249
Eclipse TE-2000-U microscope	Nikon	TE-2000-U	Inverted microscope
Extra Thick Microscope Slides	Fisher Scientific	22-267-005	Works well for collagen extrusion surface
FEI Helios G3 NanoLab DualBeam	Thermo Fisher Scientific	N/A	Scanning electron microscope
Glass Cutter Tool Set 2mm-20mm Pencil Style Oil Feed Carbide Tip	Amazon/MOARMOR	B07Y1D243H	Option for a glass cutter
Hamilton Kel-F Hub Blunt Point Needles (Luer Lock, 22 G)	Fisher Scientific	14-815-574	Needles for collagen extrusion
Nexterion Slide H 3-D	Schott	NC0782819	Hydrophobic slides
PBS Tablets	Thermo Fisher Scientific	18912014
Polyethylene Glycol 8000	Fisher BioReagents	BP233-1
Seamless Cellulose Dialysis Tubing	Fisher Scientific	S25645G
SnakeSkin Dialysis Clips	Thermo Fisher Scientific	PI68011
Synthware Glass Cutter	Synthware	31-501-927	Option for a glass cutter