- Mar 2020
we developed an approach that uses rapid pH change to drive collagen self-assembly within a buffered support material, enabling us to (i) use chemically unmodified collagen as a bio-ink, (ii) enhance mechanical properties by using high collagen concentrations of 12 to 24 mg/ml, and (iii) create complex structural and functional tissue architectures. To accomplish this, we developed a substantially improved second generation of the freeform reversible embedding of suspended hydrogels (FRESH v2.0)
For 3D printing of tissue components, the authors prefer to use collagen in its natural form, but this comes with a challenge. The 3D printing is performed by dispensing a stable bioink composed of collagen through a nozzle. During printing, unmodified collagen transforms into a gel through a self-assembly process (formation of links between the individual collagen structures). This self-assembly process is generally very difficult to control as it is challenging to dissolve collagen and prepare a stable bioink at physiological pH values of 7.4.
To resolve this issue, the authors prepared bioinks by dissolving collagen in an acidic solution. Then, the authors dispensed this bioink into a support gel which rapidly changes the pH of the bioink (neutralizing it to pH=7.4), thereby promoting the self-assembly process. This allowed the authors to 3D print unmodified collagen, with higher density and strength, in complex shapes and structures.
Although the 3D bioprinting of a fully functional organ is yet to be achieved, we now have the ability to build constructs that start to recapitulate the structural, mechanical, and biological properties of native tissues.
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.
We used μCT imaging to confirm reproduction of all the anatomical structures contained within the 3D model of the heart, including the atrial and ventricular chambers, trabeculae, and pulmonary and aortic valves
The collagen 3D printing method was able to completely and accurately reproduce all the components and features of the heart from their computer model.
Finally, to demonstrate organ-scale FRESH v2.0 printing capabilities and the potential to engineer larger scaffolds, we printed a neonatal-scale human heart from collagen
Previous experiments have only demonstrated small-scale applications of their printing method. So, in their last experiment, the authors printed a collagen structure of an infant-sized heart to show the larger-scale abilities of FRESH v2.0.
We confirmed the patency of vessels ~100 μm in diameter by optically clearing and reperfusing the multiscale vasculature
This result confirmed that open vessels in the small scale (100 microns) were able to be printed. This is significant because engineering small-scale vasculature has historically been a major challenge in these 3D printing techniques.
A magnetic resonance imaging (MRI)–derived computer-aided design (CAD) model of an adult human heart was created, complete with internal structures such as valves, trabeculae, large veins, and arteries, but lacking smaller-scale vessels.
A full scale human heart was developed on computer software. The overall structure, valves, and large vessels could be created by MRI and computer aided design, but smaller vessels (~100 microns) had to be created using a special computer program.
A small subsection of this heart was selected and printed from the collagen
Mechanical function was demonstrated by mounting the valve in a flow system with a pulsatile pump to simulate physiologic pressures, and we observed cyclical opening and closing of the valve leaflets
Several functional conclusions are found in the remainder of this paragraph.
First, it was found that the leaflets (flaps) in the printed valve opened and closed as expected when mounted to a pump system.
Second, less than 15% regurgitation was observed, meaning less than 15% of the fluid went backwards through the valve as the leaflets closed. This is within typical physiological range.
Third, the pressures observed in the printed valve were roughly middle-range. They were higher than some typical heart valves but lower than others.
Finally, human umbilical vein endothelial cells (HUVECs) were cultured on the collagen leaflets and formed a confluent monolayer, a single layer covering the entire area of the leaflets. HUVECs are used for various physiological studies.
we printed a tri-leaflet heart valve 28 mm in diameter
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.
The decrease in cross-sectional area of the interior chamber during peak systole showed a maximum of ~5% at 1-Hz pacing (N = 4)
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.
We imaged the ventricles top-down to quantify motion of the inner and outer walls (Fig. 3K).
The authors observed the heart 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 ventricles had a baseline spontaneous beat rate of ~0.5 Hz and could be captured and paced at 1 and 2 Hz by means of field stimulation (Fig. 3J).
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.
Calcium imaging revealed contracting hESC-CMs throughout the entire printed ventricles, with directional wave propagation in the direction of the printed hESC-CMs observed from the side (Fig. 3, D and E) and top (Fig. 3, F and G) during spontaneous contractions in multiple ventricles (N = 3) (movie S6).
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
A test print design (fig. S11A) verified that the collagen pH was neutralized quickly enough to maintain ~96% post-printing viability by LIVE/DEAD staining
The test print verified that 96% of the cardiomyocytes printed using the bio-ink were able to to survive in the printed collagen.
We next FRESH-printed a model of the left ventricle of the heart using human stem cell–derived cardiomyocytes.
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.
Cardiac ventricles printed with human cardiomyocytes showed synchronized contractions, directional action potential propagation, and wall thickening up to 14% during peak systole.
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.
Collagen disks 5 mm thick and 10 mm in diameter were cast in a mold or printed and implanted in an in vivo murine subcutaneous vascularization model
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.
Collagen disks that were FRESH-printed with VEGF and extracted after 10 days in vivo showed enhanced vascularization relative to cast controls
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.
the number of cells in the constructs was significantly greater for the printed collagen at 3 and 7 days compared to cast control
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.
demonstrating viability and active remodeling of the gel through cell-driven compaction.
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.
A pH 7.4 support bath with 50 mM HEPES was the optimal balance between individual strand resolution and strand-to-strand adhesion and was versatile, enabling FRESH printing of multiple bio-inks with orthogonal gelation mechanisms including collagen-based inks, alginate, fibrinogen, and methacrylated hyaluronic acid in the same print by adding CaCl2, thrombin, and UV light exposure
A pH of 7.4 obtained by using a buffer agent (50 mm HEPES) in the support gel produced the best results
Additionally, this 3D printing mechanism is compatible with other 3D printing methods relying on alternative gelation methods based on alginate, fibrinogen and methacrylated hyaluronic acid. Orthogonal printing means you can mix and match 3-D printing methods to create hybrid structures on the same scaffold.
In FRESH v2.0, we developed a coacervation approach to generate gelatin microparticles with (i) uniform spherical morphology (Fig. 1D), (ii) reduced polydispersity (Fig. 1E), (iii) decreased particle diameter of ~25 μm (Fig. 1F), and (iv) tunable storage modulus and yield stress
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.
FRESH works by extruding bio-inks within a thermoreversible support bath
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.
the addition of VEGF to the printed collagen resulted in widespread vascularization,
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.
We present a method to 3D-bioprint collagen using freeform reversible embedding of suspended hydrogels (FRESH) to engineer components of the human heart at various scales, from capillaries to the full organ.
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.
Tail vein injection of fluorescent lectin confirmed an extensive host-derived vascular network with vessels ranging from 8 to 50 μm in diameter throughout the printed collagen disk
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
We first focused on FRESH-printing a simplified model of a small coronary artery–scale linear tube from collagen
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.
Multiphoton microscopy enabled deeper imaging into the printed constructs and showed vessels containing red blood cells at depths of at least 200 μm
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 improves the resolution
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.
- Feb 2020
less than the aortic and mitral valves
The collagen valve allowed less than 15% of the fluid to flow back during opening and closing. This phenomenon is common even in healthy heart valves, and the value found here is consistent with human hearts.
Also, The collagen valve was able to withstand pressures higher than some heart valves, but lower than others.
engineer tissue components of the human heart
The authors were successful in designing and fabricating human heart components with 3D printed collagen.