Organs on Demand
3-D printing has made inroads in the clinic, but
constructing functional complex organs still faces major hurdles.
September 1, 2013
RECONSTRUCTION: Wake Forest postdoctoral fellow Hyun-Wook Kang operates
a 3-D printer that is making a kidney prototype with cells and
biomaterials.IMAGE COURTESY OF WAKE FOREST INSTITUTE FOR REGENERATIVE
On a stage in front of an audience of thousands, a futuristic-looking
machine squirted gel from a nozzle. Layer by layer, it built up the
material, shaping it into a curved, pink, kidney-shape structure based
on a medical CT scan of a real organ.
It was 2011, and Anthony Atala, director of the Wake Forest Institute
for Regenerative Medicine, was demonstrating his progress in using
three-dimensional (3-D) printing to make a kidney during his TED Talk.
Like a TV chef pulling a previously baked casserole from the oven, Atala
soon held a bean-shape object in his gloved hands. “Here it is,” he
said. “You can actually see the kidney as it was printed earlier today.”
The audience erupted into cheers.
But Atala had not made a functional human kidney, as he at times
seemed to imply and as the Agence France-Presse reported in a widely
disseminated article. “A surgeon specializing in regenerative medicine .
. . ‘printed’ a real kidney using a machine that eliminates the need for
donors when it comes to organ transplants,” it read.
BODY PARTS: Anthony Atala has printed a kidney prototype (left) and
biomaterial scaffolds for growing new ears (top right) and finger bones
(bottom).IMAGE COURTESY OF WAKE FOREST INSTITUTE FOR REGENERATIVE
MEDICINE Wake Forest quickly tried to stem the misleading coverage.
“Reports in the media that Dr. Anthony Atala printed a real kidney at
the TED conference in Long Beach, Calif., are completely inaccurate,”
stated a press release issued by the university following the media
coverage. Rather, Atala had printed only a kidney-shape “mold” made of
biocompatible materials combined with cells. The prototype, as Atala
calls it, lacked the kidney’s intricate inner structures, such as the
fine networks of vessels called glomeruli that allow the organ to filter
waste materials from the blood. The prototype could not have functioned
as a real organ and thus was not ready for transplantation prime time;
that may only be possible “many years from now,” cautioned the press
Atala’s kidney prototype represents both the promise of 3-D printing
in a medical context and the hurdles that tissue engineers have yet to
clear. With recent technological advances, using 3-D printing to shape
gels embedded with living cells into the general form of organs has
become a relatively achievable task. Printing a liver or a kidney that
functions in the same way and with the same efficiency as a real organ,
however, is a different story. The most formidable obstacle standing in
the way of functioning 3-D–printed organs is the difficulty of
replicating the branching networks of veins, arteries, and capillaries
that nourish the body’s tissues and filter out waste. In most organs,
cells must be within 150 to 200 microns—the width of a few human
hairs—of the nearest capillary to survive.
As researchers modify and build devices that print with ever greater
precision, and invent new biomaterials to serve as ink for these
machines, they have been able to make substantial progress on printing
ears, spinal discs, heart valves, and bone, which are moving towards the
clinic. (See illustration below.) Similarly, simple engineered tissues,
such as tracheas and bladders made from cells seeded onto biocompatible
scaffolds and created without the use of 3-D printing, have already been
inserted into patients. But these tissues have thrived only because they
are thin enough not to require extensive infiltration by blood vessels.
“[Vascularization has] been something that’s been worked on for 20
years,” says Jennifer Lewis, a materials engineer at Harvard University
who is designing printers she hopes will produce vascularized tissues.
“It’s plagued a number of advances.”
Achieving vascularization may be the biggest challenge that faces
researchers attempting to 3-D print organs, but 3-D printing could also
be the very technology to solve this problem. Researchers are harnessing
3-D printers to build tiny, hierarchical networks of blood vessels to
supply increasingly complex 3-D–printed organs with blood.
“For me the holy grail of tissue engineering is to fabricate tissues
with their own vascular network,” says Jason Spector, an associate
professor of plastic surgery at Weill Cornell Medical College, who is
working on printing ears and other tissues. “Once you can make that,
everything else is cake.”
From playthings to medical devices
TIP: This printer, customized by Harvard’s Jennifer Lewis, can be used
to print structures ranging from scaffolding for cell and tissue culture
(as shown) to microvascular templates for blood vessel growth.COURTESY
OF JENNIFER LEWISSUPPORT
STRUCTURE: A 3-D–printed tracheal splint made of bioresorbable
polycaprolactone is designed to fit around an infant’s collapsing
airway.COURTESY OF JENNIFER LEWISThese days, 3-D printing, which has
been around since the 1980s, calls to mind baubles such as iPhone cases,
high-fashion shoes, personalized sex toys, and even working guns.
There’s a growing market for personal printers—relatively inexpensive
machines that print at fairly low resolution, often using proprietary
polymers—for producing such items at home.
But playthings, accessories, and weapons aside, 3-D printing has also
made incursions into the medical device business. The 3-D–printing
industry brought in $2.204 billion in 2012, $361 million of which was
revenue from 3-D printing for medical and dental uses, according to the
2013 Wohlers Report. And researchers are now testing the feasibility of
using printers to create patient-specific tissues and organs that may
one day be used to supplement scarce donor body parts.
Medical 3-D printing takes advantage of two major printer types. One
type, used by Atala to print kidney prototypes, extrudes a pliable
material, often a melted polymer or a gel, through a nozzle, building up
the desired shape layer by layer according to a computerized blueprint.
The second type of printer operates by shooting a laser or a binding
material at a bed of powder and solidifying it in a highly specific
pattern. As the laser or binding agent moves through the powder, layer
by layer, it builds a solid structure embedded in powder, which is
dusted off when the job is done. The powder can be a polymer, or it can
be metal particles, useful for creating implants such as hip joints.
OF THE LABORATORY OF JONATHAN BUTCHER, CORNELL UNIVERSITYFAB
MACHINE: University of Nottingham tissue engineer Kevin Shakesheff uses
a Fab@Home printer (top) to engineer bone. Others have used the
open-source printer for printing heart valves (bottom), ears, and
more.COURTESY OF THE LABORATORY OF JONATHAN BUTCHER, CORNELL
UNIVERSITYPhysicians already rely on 3-D–printed hearing aids, cups for
hip implants, dental crowns and bridges, and now cranial implants,
modeled on scans of patients’ bodies. Researchers even printed a
customized titanium lower-jaw bone last year for a patient in the
Netherlands. There is also a booming business in 3-D–printed surgical
guides—implements somewhat like draftsman’s rulers and compasses—which
doctors place over surgical sites to guide their drills and knives as
they bore and cut into flesh and bone.
Physicians also order 3-D–printed plastic replicas based on scans of
patients’ actual body parts—a hip joint that needs replacing, for
example, or a patient’s abdominal circulatory system—to practice
upcoming surgeries using realistic models. The Mayo Clinic orthopedics
department in Arizona found custom surgical models so helpful, doctors
there decided to purchase their very own 3-D printer last summer.
And with the success of 3-D–printed implants, it did not take long
for tissue engineers to decide they could adapt 3-D printers to extrude
biologically compatible scaffolds and cells to construct whole organs.
In printing his kidney-shape structure at the TED conference, Atala was
demonstrating the technology’s ability to print a complex structure that
was a hybrid between an implant and an organ: a scaffold made from a
biocompatible and bioresorbable gel mixed with living cells that could
conceivably, if placed in the proper environment, grow into living
“The human body has tissues that are very highly structured,” says
Kevin Shakesheff, a tissue engineer at the University of Nottingham who
is working on printing bone. “Their actual architecture is essential to
how the tissue works. The level of control that the human body has is
something we can now replicate with 3-D printing.”
Indeed, researchers are hoping to introduce even more 3-D–printed
tissues into the clinic and into patient’s bodies in the coming decade.
For instance, 3-D–printed vertebral discs and small pieces of bone are
being tested in animals, while ears, heart valves, and more are being
printed in the lab. But one physiological fact continues to stand in the
way of a true 3-D printing revolution that could potentially save
thousands of lives by stocking operating rooms with a steady supply of
replacement body parts: the complexity of the vascular system that
supplies organs with blood.
“You could print things up that look exactly like a tissue or an organ
if you have a CT scan or MRI-derived data, but unless you can hook it to
[the body’s] blood supply . . . it will die,” says Spector.
REPRAP.ORG AND LICENSED UNDER GPLDESIGNING
VASCULATURE (clockwise from top): The RepRap Prusa Mendel, an
open-source printer that Rice University’s Jordan Miller modified to
print vascular templates for engineered tissues; a 3-D–printed
carbohydrate template for vascular channels, which will be dissolved
after it is encapsulated in cells and biomaterials, leaving vascular
channels in living tissue; a diagram shows vascular channels
encapsulated in a cell-filled biocompatible gel.COURTESY OF
REPRAP.ORG AND LICENSED UNDER GPLThough supplying blood to
3-D–printed organs is the major stumbling block preventing such implants
from becoming a reality, capillaries can, to some extent, branch out
from already-existing blood vessels and into transplanted tissues on
their own. Researchers have seen signs of spontaneous vascularization in
small areas of engineered tissue, such as in healed rat bone defects
around 3 millimeters in diameter.7
But can undirected capillary growth provide the hierarchical,
all-penetrating networks that complex tissues and organs depend on?
Spector says it’s not likely. He deals with microvascular networks in
his medical practice, hooking tissue grafts into the intricate vascular
plumbing of their new hosts, and he has come to realize how difficult it
is to achieve the level of vascularization needed for a transplanted
tissue to thrive. “I have yet to see anything close to [an engineered
solid organ] that will survive in a real clinical situation,” he says.
Rather than letting blood vessels spontaneously branch off and expand
into engineered tissues, some researchers are crafting templates for
more orderly vascular growth. The idea is to create hierarchical
microvascular networks that will guide the endothelial cells that line
blood vessels to form tubes along predetermined courses. Some see 3-D
printing as the best way to accomplish that goal.
Printing such tiny negative spaces, however, is easier said than
done. Capillaries can be as small as a few microns in diameter. Even
with high-resolution printers, such tiny vascular structures would
likely collapse, especially when printed into a soft, biocompatible gel.
Harvard’s Lewis, who serves as the university’s Hansjörg Wyss
Professor of Biologically Inspired Engineering, is interrogating this
problem using a customized, high-resolution 3-D printer that can form
microchannels in biocompatible gels. “We can print hydrogel materials
down at the micron-length scale, smaller than other groups can print
anything,” Lewis says. The smallest microvascular channels her group has
been able to print are around 10 microns in diameter.
VESSELS: Endothelial cells (red) line the walls of Miller’s 3-D–printed
vascular structures.COURTESY OF JORDAN MILLERTo solve the problem of
collapsing channels, she prints them in “fugitive ink”—a substance
designed to melt away after forming the channel’s pattern. For her
fugitive ink, Lewis settled on Pluronic F127, a gel often used in
eyeglass lens cleaner and cosmetics. Pluronic F127 is made up of three
parts—the two poles of the molecule are hydrophilic while the middle
segment is hydrophobic. It also has an unusual property. “Most
materials, when you cool them down, they solidify,” says Lewis. “This
material liquefies when you cool it down.”
Lewis also used Pluronic F127 as the matrix into which she prints the
channels, but she modified the matrix molecules so that they polymerize,
and thus solidify, in the presence of UV light. This allows her to firm
up the matrix before cooling the gel so that the fugitive ink melts
away. Taking advantage of her printer’s fine-tipped nozzle, she printed
a capillary network of fluorescently labeled fugitive ink into the Jello-like
matrix.8 “We were able to show for the first time a way to pattern
hydrogels with these vascular channels,” she says.
The next step, Lewis says, is to take advantage of the
self-organizing quality of endothelial cells in her own 3-D–printed
constructs, seeding her printed vascular structures with these blood
vessel–lining cells. (See “Crowd Control,” The Scientist, July 2013, for
a more in-depth look at how endothelial cells coordinate such behavior.)
She will rely on the tendency of the finest capillaries to grow
spontaneously out of larger microvascular structures. “It’s not trivial,
but biology will work,” she says. “Once you give [them] a reasonable
environment, the cells are happy.”
Pour some sugar
Inspired by some of Lewis’s early work with “fugitive inks,” Jordan
Miller, formerly a postdoc in Christopher Chen’s lab at the University
of Pennsylvania and now an assistant professor of bioengineering at Rice
University, created his own technique for 3-D printing of
vasculature-mimicking channels. Using a simple open-source 3-D printer,
he constructed a carbohydrate lattice made from a combination of simple
and complex sugars.
“A lot of Jennifer Lewis’s work is very inspiring, but the machines
she is using are very high-end,” he says. “They have incredible
precision, but they are not duplicable by anyone else.”
Miller saw potential in the Frostruder, a printer originally used to
extrude sugar frosting for printing fancy designs onto edible treats.
With help from members of the RepRap community—a group founded in 2005
with the purpose of designing self-replicating, open-source
printers—Miller adapted a RepRap printer to incorporate elements of the
food printer’s design and was soon able to print dissolvable lattices of
Miller decided to use a process called “3-D sacrificial molding” that
is akin to the lost-wax method used by sculptors. His printer deposits
filaments of carbohydrate on top of each other in sequence so they are
self-supporting. Miller then covers the entire lattice structure in a
protective layer of a biodegradable polymer. After pouring and
crosslinking a cell-filled gel over the carbohydrate lattice, he
dissolves away the lattice with an aqueous solution.9 (See photographs
Miller’s channels are not as small as Lewis’s—his channels range from
150 microns to around a millimeter in diameter. However, when he and
colleagues seeded his channels with endothelial cells, they lined the
interiors of the channels and even began to penetrate the surrounding
cell-gel mixture. Miller says he hopes that by guiding blood cells into
the larger channels, he can set the stage for endothelial cells to
spontaneously form their own capillary networks. “We may not have to
print an entire capillary bed,” Miller says.
Miller is also working on building more expensive, high-resolution
printers in case the cells aren’t capable of forming capillaries on
their own. But, he says, it’s possible that endothelial cells, if seeded
into a predefined set of capillary channels, might not follow the
planned architecture anyway. “If we had put them in a capillary bed
initially, they would probably remodel it [based] on local needs.”
Miller has successfully pumped human blood through his constructs in
vitro, and he plans to cooperate with a surgeon to connect one of his
printed tissues to the vascular system of a rat to see how long he can
get blood to flow through his channels.
In addition to being relatively cheap, Miller’s method is fast. “The
big challenge in that field is [that] a lot of the interesting cell
types we would want to build into large-scale tissues—things like liver
cells—[are] not going to survive the several hours in the extruder
nozzle, long enough to build something the size of the human liver,” he
says. Quickly pouring the cells and gels over the 3-D–printed lattice is
easier on fragile cells than the arduous process of printing.
The disadvantage is, of course, that the researchers can’t control
the exact placement of the cells. Someone planning to print using
multiple cell types, for instance, might not want to pour them out over
a lattice willy-nilly—although Miller says that cells are surprisingly
good at organizing themselves even when poured into a gel.
“I wouldn’t say this is the end-all be-all solution to tissue
engineering,” says Miller, but “it’s allowed us to take the next step.”
He has already shown, in versions of his constructs printed with rat
liver cells or with human embryonic kidney cells, that the cells near
the channels survive longer than the cells deeper in the gel, suggesting
that the faux vasculature is doing its job. He says that even if his
tissues are nowhere near ready for implantation into humans, at least he
can now keep cells alive for longer in order to do in vitro experiments
to understand better what they need to thrive over the long term.
Numerous other groups are also trying their hand at 3-D printing of
vasculature, and should any of them prove successful, it would pave the
way for tissue engineering on a grander scale than ever before. Growing
and implanting larger swaths of bone or skin may become feasible, and
producing more-complex organs like hearts or kidneys might become more
realistic. “It’s a pretty competitive landscape right now,” says Lewis,
who is continuing work with her Pluronic F127 fugitive ink, as well as
experimenting with growing living cells in various kinds of
extracellular matrix material. “In the next 2, 3, 4 years, you’ll see
lots of groups publishing on this idea—that they can create
deterministic patterns of vascular growth,” agrees Cornell biomedical
engineer Lawrence Bonassar, who is working on his own version of
“It’s just a really fun technology,” says Miller. “We’ve made
thousands of these structures and every time they print it’s just
PDFLUCY READING-IKKANDA3-D printing allows tissue engineers
to fabricate more-complex shapes and to more precisely mix
materials than does the process of growing organs by seeding
cells onto handmade scaffolds. Anthony Atala—whose artificial
bladders, made by growing a patient’s own urothelial and muscle
cells on collagen and polyglycolic acid (PGA) scaffolds, are now
in clinical trials—says he is working on bioprinting a multitude
of tissues and organs, including muscles, bones, tracheas, ears,
noses, and kidneys. His goal is to design bioprinters that can
print usable engineered tissues at all levels of complexity.
Many other researchers are also forging ahead with 3-D printing
projects that may reach patients in the near future. Of the
projects below, only the tracheal splint has thus far made it
into a human patient, but some engineered tissues are now being
tested in animals.