Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons facilitate movement by transmitting forces from muscle to bone. Although tendon injury is common, these are quite difficult to treat, and the outcome for patients is often poor. Currently, all treatments of tendon injury involve some kind of physiotherapy, and this reflects the fact that mechanical forces play such a central role in tendon biology.

Good experimental models for studying tendon damage and repair don't really exist, so my lab is actively developing new models that can better capture important features of tendon physiology and pathophysiology. In previous studies, we could show that the tendon core, which represents the load-bearing part of the tendon, has by itself a very limited repair capacity. Combined with other research in the field, we hypothesized that a injured core would recruit cells from the extrinsic tendon compartment to help it heal.

Tissue-engineered tendon model system may provide a loadable 3D environment, but don't match the intricacies of an in vivo extracellular matrix. Explant model systems do, but are often difficult to keep alive and mechanically load over longer periods of time, or lack the extrinsic compartment that is central for the repair processes. Our unique model system combines the advantages of marine tail tendon-derived core explants with those of 3D hydrogel based systems.

It provides a loadable, in vivo-like core matrix, alongside an artificial extrinsic compartment. Its composition can be tuned to the research hypothesis and the biomimetic cross-compartmental barrier in between the two. Our hybrid hydrogel explant assembloids are in a prime position to study tendon core biology, matrix structure function interactions, and cross-compartmental interactions between specific cell populations in a fine-tunable micro environment.

Discoveries from the studies conducted with the system will guide in vivo research and treatment development. Begin by sterilizing the mouse skin using 80%ethanol and transferring the mouse to a sterile biosafety hood. To isolate the tail tendon core explants, use a scalpel to cut the tail at its base.

Then grip the tip of the tail with tweezers and wiggle it to break the skin. Gently pull the tweezers away to expose the tendon core explants. Place the tendon core explants in a standard culture medium and separate them from the tail using a fresh scalpel blade.

Next, to isolate the tendon fibroblasts, make a transversal incision in the middle of the mouse foot using a scalpel. From each end of this incision, make a perpendicular cut along the sides of the hind legs, up to the hips. Then use tweezers to fixate the skin flap at the foot and peel away the skin covering the calf muscles.

Separate the Achilles tendon from the calcaneus bone with a fresh scalpel blade. Fixate the loose end with tweezers and separate the other end from the gastrocnemius muscle. Transfer the tendon to PBS and remove the remaining muscle tissue.

Then, transfer the tendon to the digestion media. Next, use scissors to cut at the hip joint and separate the hind legs from the body. Wash the hind legs in cold PBS.

Remove the muscles with the scalpel and place them into a Petri dish on ice. While the Petri dish is on ice, mince the muscle tissue into pieces smaller than one millimeter cube. Transfer the minced muscle tissue to digestion media.

After removing the leftover muscle tissues, wash the leftover bones in cold PBS. Place the bones in fresh cold PBS and gradually remove the epiphyses to expose the bone marrow. Equip a syringe with a 0.4 by 25 millimeter injection needle and fill it with 10 milliliters of macrophage culture medium.

Hold each bone over a 50 milliliter plastic tube and insert the needle about one millimeter deep into the bone marrow from the top. Flush out the bone marrow by emptying the syringe. To begin, prepare the clamp holders and metal clamps.

Place matching clamp holders with one metal clamp each into the mounting station. Place wet autoclaved paper pieces on top of the metal clamps. Cut the paper along the inner borders of the clamps with the scalpel.

Cut two additional smaller paper pieces and keep them wet. Using pointed tweezers, place eight core explants on the paper between the metal clamps with their end points on the clamps. Cover the end points of the core explants with smaller paper pieces and put metal clamps on top.

Use a screwdriver and small screws to fixate the core explants between the metal clamps and the clamp holder. Carefully transfer the clamped core implants into silicone culture chambers. And fill these chambers with two milliliters of standard cell culture medium.

Then, fixate the assembloid with additional screws. To prepare the collagen hydrogel, first, remove target cells from tissue culture dishes using a cell detachment solution. Then centrifuge at 400g for five minutes at room temperature and resuspend in one milliliter of standard culture medium.

Next, prepare a cross-linking solution by mixing 10 microliters of PBS, 1.28 microliters of one molar NaOH, and 8.72 microliters of double-distilled water per assembloid. Add the cell suspension of up to 12 assembloids and place the tube on ice. Adjust the cell suspension such that the following final concentrations are achieved.

Separately, prepare a collagen 1 solution by adding 80 microliters of collagen 1 per assembloid and place the tube on ice. Once the solutions are ready on ice, aspirate the cell culture medium from the chambers containing the clamped core explants. Add collagen 1 solution to the crosslinking solution and mix by pipetting quickly without creating bubbles.

Add 200 microliters of the mixed solution into the groves in the silicone chambers to cover individual tendon core explants. Let the hydrogels polymerize for 50 minutes at 37 degrees Celsius. Then, carefully fill the silicone culture chambers with 1.5 milliliters of the respective co-culture medium by pipetting it into the corners of the chambers.

Place the chambers in a large Petri dish or a sterile box before putting them into an incubator. Culture the assembloids in appropriate conditions, changing the culture medium twice a week. Remove the assembloids from the clamps with scissors.

If required, use tweezers to separate the core explants from the external hydrogel compartment. Upon culturing the assembloids in lesion-like culture conditions for over 21 days, it was observed that the core explant remained mechanically stretchable, unchanged in appearance, visually distinguishable, and physically separatable from the surrounding hydrogel. Further, the surrounding hydrogel compacted over time, depending on the seeded cell population, with Achilles tendon-derived fibroblasts contracting their surrounding hydrogel the fastest.

Confocal microscopy assessment for viability, morphology and cell spreading in the 3D collagen hydrogel revealed that the viability was retained during assembloid culture until at least day seven. Further, the gene expression of Vegfa and Mmps increased strongly in core explants as seen by transcriptome analysis. Similarly, secretome analysis detected the presence of cytokines, such as vascular endothelial growth factor in the core explants.