The authors prefer to 3D print collagen in its natural form, but this comes with a challenge. Unmodified collagen transforms into a gel by forming links between individual structures, and this process is difficult to control at physiological pH values of 7.4.

To resolve this issue, the authors prepared collagen bioinks by dissolving collagen in an acidic solution. By dispensing this bioink in a buffered support gel, the pH of the collagen ink is quickly neutralized to 7.4 which promotes the linking process.

This allowed the authors to 3D print unmodified collagen, with higher density and strength, in complex shapes and structures.

FRESH-printed collagen disks had more cells and much deeper cell penetration in comparison to cast collagen disks. These results shows that FRESH-printed implants provide a better platform for host cells to move into and grow.

The authors were able to control the porosity of the 3D printed filaments by controlling the salt concentration and pH of the support bath.

Authors reached the following conclusions with their FRESH-printed functional tri-leaflet heart valves:

First, the valve had well-separated leaflets, and was structurally robust enough.

Second, the leaflets (flaps) functioned properly, repeatedly opening and closing when mounted to a pump system which generates a pulsating flow, mimicking the heart pumping blood.

Third, less than 15% regurgitation was observed, meaning less than 15% of the fluid went backwards through the valve as the leaflets closed. Regurgitation is an indication of a leaky heart valve.

Fourth, maximum fluid pressures measured across the printed valve were within the physiological range observed in human heart.

Finally, authors were able to culture cells on the leaflets which demonstrates their biocompatibility and potential for developing artificial implants.

Previous experiments have only demonstrated small-scale applications such as printing a ventricle and a valve. So, in their last experiment, the authors printed an infant-sized heart to show the larger-scale printing capability of FRESH v2.0 towards printing a fully functional artificial organ.

This result confirmed that the authors were able to print open vessels at small scales (100 microns). This is significant because engineering small-scale vasculature has historically been a major challenge in bioprinting.

MRI uses magnetic field and radio-frequency waves to create detailed 3-dimensional pictures of organs within the body. The authors developed a 3D model of the adult human heart was developed by using magnetic resonance imaging (MRI) images.

Due to low resolution of MRI imaging, their 3D heart model included valves and large vessels, but was missing the smaller-scale vessels. The authors later added the small vessels to their model by using a special computer program. The authors then used this 3D model to FRESH-print human heart.

The authors observed the printed ventricle as it contracted to see how the walls thickened during the contractions. This was done because wall thickening is a typical behavior of ventricle contraction.

The pH of support bath was set to 7.4 using a buffer agent (50 mM HEPES), which produced the best results for 3D printing.

Additionally, this 3D printing mechanism is compatible with other 3D printing methods based on alternative gelation methods. FRESH v2.0 enables orthogonal printing, which means you can use it in combination with other 3D printing methods to create hybrid structures on the same scaffold.

Maybe annotate this as an overall conclusion of the paper

The authors aren't quite ready to develop full organs yet, but the work done in this paper has demonstrated several leaps towards this goal: a model of a live coronary artery, collagen disks that promoted vascularization, printed ventricles and heart valves that showed typical physiological function, and a precise recreation of a full-scale heart structure.

The collagen 3D printing method was able to completely and accurately reproduce all the components and features of the heart from their computer model.

A heart valve was printed with the collagen gel and was strengthened using standardized methods.

Heart valves are structures with flaps of tissue (leaflets) that open and close. This allows blood to flow out to the rest of the body, and prevents it from flowing back into the heart (regurgitation). The valve printed here was 28 mm which is within human range, and was tri-leaflet meaning it contained 3 flaps.

This is an important experiment because heart valves experience extreme forces in the body. If the overall goal for the authors is to 3D print full organs, they need to verify that their collagen can withstand these forces.

During peak systole, the highest pressure generated as the ventricle contracts, the inner void of the ventricle decreased in area by about 5%. This means the walls of the printed ventricle thickened while the ventricle contracted at 60 bpm.

On its own, the ventricle could beat about once per 2 seconds (30 bpm). By using additional stimulation, the authors could get the ventricle to beat from 60-120 bpm.

Electrical activity causes contraction of the cardiomyocytes, and the calcium imaging shows presence of this electrical activity. Additionally, the calcium was observed to propagate (move) through the ventricle at a velocity of 2 centimeters per second

The test print verified that 96% of the cardiomyocytes printed using the bio-ink were able to to survive in the printed collagen.

The authors 3D printed a left ventricle, the bottom left chamber of the heart that pumps blood out to the body. They printed collagen gel for structure, and also used a cellular ink containing human embryonic stem cell-derived cardiomyocytes (hESC-CMs) which are the contracting cells in the heart.

The ventricle was designed as an ellipsoidal shell with an outer and inner wall printed from collagen gel. The hESC-CMs were printed in the space between the two walls along with 2% cardiac fibroblasts, cells that produce connective tissue.

The authors FRESH printed a heart ventricle (two large chambers at the bottom of the heart that collect and expel blood) using collagen support material and cardiomyocytes (functional cells that generate contractile force in the heart). The printed ventricle mimicked several key features of a typical human heart: electrical activity, contractions, and wall thickening during the peak systole (maximum pressure reached while the ventricle contracts to pump blood out to the body).

These results are of great significance in the field of tissue engineering. 3D printed tissues and organs can greatly improve treatment methods such as organ transplants. The authors’ ability to print a ventricle that mimics the human heart is a giant leap towards this goal.

Two types of collagen disks were created: i) solid disks from a mold, and ii) porous disks printed by FRESH v2.0.

These disks were implanted under the skin of live rats to observe cell movement into the porous matrix as a first step towards formation of a network of blood vessels.

After 3 and 7 days, the disks were removed and tested for this result.

After leaving the two disks underneath the rodent skin for 10 days, the authors observed that the collagen disks containing VEGF showed much more vessel formation than the control disks.

Vascularization shows that the body has "accepted" the collagen disks, which is a significant result.

Cells filled the printed, porous collagen disks within 3 days which restructured the collagen, indicating healthy tissue formation. Very few cells filled the solid disks which indicates tissue formation was unsuccessful.

Around the static tube (no perfusion), only one layer of C2Cl2 cells remained alive after five days. In contrast, in the tube which the cells were fed (perfusion), almost all of the cells were alive after 5 days. The cells formed a tight tissue surrounding the tube that leads to compaction of the collagen.

This result proved that the authors were able to 3D print an artificial tube with living cells mimicking a live coronary artery.

In the second version of FRESH, the authors wanted to improve the gelatin microparticles used in the support bath. To do this, they used coacervation which is a chemical method of producing polymer droplets.

The particles produced with this method were smaller, more spherical, and had less size variation. Additionally, the method allowed the elasticity and strength of the particles to be varied.

As stated previously, the FRESH method uses a supporting gel that contains tiny particles to support the bio-inks (such as collagen-containing gel) as they are printed. The supporting gel is thermoreversible which means its properties can be changed with heat.

FRESH printed collagen disks (containing fibrinogen and VEGF) promoted movement of C2C12 cells (cell infiltration) into the porous structure of the collagen. They also promoted formation of blood vessel-like structures (vascularization). Overall these findings indicate that this 3D printing technique produces tissue-like structures closely mimicking physiological blood vessel formation.

This is the key result from the authors’ work. Their modified 3D printing method (FRESH) allowed them to reliably print collagen, an important protein in the extracellular matrix. This breakthrough introduces the possibility of constructing various tissues and organ components with high resolution.

In short, the printing method dispenses a gel within another gel. Both gels contain tiny particles. The gel that is printed contains collagen which forms the scaffold for the artificial tissues and organs. The second gel mainly serves as a support structure and is melted away after printing.

By injecting a fluorescent protein (lectin) into the rodent's vein, the authors were able to demonstrate the formation of different sized blood vessels into a network in the collagen disks. This proves the success of using VEGF

The authors 3D printed a model of a small coronary artery with type 1 collagen (1.4 mm inner diameter, ~300-micron thick wall). C2Cl2 cells in a collagen gel were then printed around the tube to test its ability to support tissue.

Over 5 days, one tube was left static (no perfusion) and the other underwent active perfusion in which the cells were fed.

Here, the authors showed that the formed vessels also contained red blood cells. To do this, they used multiphoton microscopy. This method uses photon absorption and infrared light to suppress image background and reduce light scattering. These two effects allow for a deeper tissue penetration.

FRESH v2.0 allowed authors to print collagen filaments 10 times finer than v1.0, resulting in ability to print features as small as 20 microns.

The authors were successful in designing and fabricating human heart components with 3D printed collagen.