Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Our group's overarching scientific mission is to develop therapeutic treatments for traumatic musculoskeletal injuries using bioengineering strategies. Specifically, we engineer materials with tunable biophysical features to modulate extracellular environments and drive regenerative cellular phenotypes. Our goal is to utilize these materials to ultimately improve healing and advance quality of life.

By applying shear to collagen during fibrogenesis, the organization or anisotropy of the individual collagen fibrils can be controlled to generate scaffolds with highly aligned or randomly pattern fibrils with nanoscale features. These features can guide cellular interactions and cytoskeletal organization along the direction of the fibrils. This protocol can fabricate biomaterials with nanoscale fibrillary patterning without expensive specialized equipment or reagents.

This makes it more accessible than other fibro fabrication techniques and highly effective for inotropic tissue engineering applications. Our results set the stage for adapting this protocol for other extracellular matrix proteins. We're exploring a bio link made from decellularized muscle to see how mimicking both tissue patterning and protein composition can enhance regeneration.

This work also sets the stage for using our biomaterial in a variety of different tissues. We're currently utilizing this technology to generate engineered tissues that can facilitate complex healing of multiple tissue types. And in parallel, we are looking at how to synergize our engineered materials with mechanical stimuli.

We hope to achieve a deeper understanding of how nanoscale features guide cellular phenotypes and to develop more robust engineered therapeutics in the future. To begin, cut the dialysis tubing to approximately three inches and rehydrate it in ultrapure water. Then clip one end of the tubing with a dialysis tubing clip using a 10 milliliter syringe equipped with an 18 gauge needle, transfer five to six milliliters of rat tail collagen type one into the tubing.

After that, clip the other end of the tubing to close it. Next, place a 0.5 to one centimeter thick layer of polyethylene glycol or peg flakes on the bottom of a glass dish. Then place the collagen filled dialysis tubing on the peg layer.

Add more peg flakes to completely cover the tubing and place the dish into a four degrees Celsius refrigerator. Every 10 to 15 minutes, remove the wet peg flakes from the surface of the dialysis tubing. Recover the tubing with a fresh layer of dry peg and return the dish to the four degrees Celsius refrigerator.

After 30 to 35 minutes, remove the wet peg flakes from the surface of the dialysis tubing. Rinse off the wet peg flakes using tap water and pat the tubing dry with a tissue. Then unclip one end of the tubing and transfer the dialyzed collagen into micro centrifuge tubes.

Briefly spin down any air bubbles in the collagen using a mini centrifuge at 2, 000G for up to 30 seconds at room temperature. Store the collagen at four degrees Celsius for later use. One day prior to use, draw approximately one milliliter of dialyzed collagen into a one milliliter syringe with a 16 gauge needle.

Then remove the needle and wrap the syringe head with parafilm. Store the syringe upright at four degrees Celsius overnight to allow the removal of small air bubbles. To begin, remove a syringe of prepared dialyzed collagen from the fridge and attach a blunt 22 gauge needle.

Place a glass slide in the lid of a four well plate and cover the slide with enough warm 37 degrees Celsius PBS to submerge it. Holding the syringe at approximately a 30 to 45 degree angle, manually extrude thin strips of collagen onto the glass slide. Wait for one to two minutes for the collagen to complete fibrogenesis or until it turns opaque white.

Then use forceps to cut the collagen strips to the desired length. One at a time, gently drape the strips of collagen lengthwise parallel to the scored region over a prepared glass chip. Tuck the edges of the strips under the chip.

Continue placing collagen strips until the desired dimensions are achieved. Now, position the newly formed collagen hydrogel across two wells of a well plate. Leave the hydrogels on the benchtop to dry for one to three hours or until PBS salt crystals cover 50%to 90%of the hydrogel surface.

Then submerge each hydrogel in PBS for about 30 to 60 seconds or until the salt crystals are dissolved. Gently dab off excess PBS from the hydrogels with a tissue. Finally, place the hydrogels back on the well plate and leave them to dry in a fume hood overnight.

To begin, remove a syringe of collagen from the fridge and attach a blunt 22 gauge needle. Extrude collagen at approximately 3.2 milliliters per minute onto a dry glass chip. Ensure that enough collagen is extruded to obtain dimensions like the aligned collagen hyderogels.

Now, use forceps to submerge the extruded collagen in a 50 milliliter tube of warmed 10XPBS. Wait 45 to 60 seconds for the collagen to complete fibrogenesis or until it turns opaque white. Then gently dab off excess PBS from the collagen with a tissue.

After rinsing and drying the hydrogels, sterilize them and the surrounding well plate in 70%ethanol for 15 minutes. Briefly elevate the hydrogels halfway through sterilization using forceps to ensure both the underside of the glass chip and the hydrogel are in contact with ethanol. Then remove the ethanol and allow the hydrogels to air dry for 10 minutes.

Wash the hydrogels three times in sterile PBS for 10 minutes each to remove residual ethanol and to rehydrate the hydrogels. Bright field images of primary mouse skeletal muscle myoblasts showed that anisotropic nanotopographical guidance of collagen fibro promotes cellular alignment.