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Researchers improve living tissues with 3-D printed vascular networks made from sugar
Researchers improve living tissues with 3-D printed vascular networks made from sugar
Monday, July 2, 2012Researchers are hopeful that new advances in tissue engineering and regenerative medicine could one day make a replacement liver from a patient's own cells, or animal muscle tissue that could be cut into steaks without ever being inside a cow. Bioengineers can already make 2D structures out of many kinds of tissue, but one of the major roadblocks to making the jump to 3D is keeping the cells within large structures from suffocating; organs have complicated 3D blood vessel networks that are still impossible to recreate in the laboratory.
Now, University of Pennsylvania researchers have developed an innovative solution to this perfusion problem: they've shown that 3D printed templates of filament networks can be used to rapidly create vasculature and improve the function of engineered living tissues.
The research was conducted by a team led by postdoctoral fellow Jordan S. Miller and Christopher S. Chen, the Skirkanich Professor of Innovation in the Department of Bioengineering at Penn, along with Sangeeta N. Bhatia, Wilson Professor at the Massachusetts Institute of Technology, and postdoctoral fellow Kelly R. Stevens in Bhatia's laboratory.
Their work was published in the journal Nature Materials.
Without a vascular system ? a highway for delivering nutrients and removing waste products ? living cells on the inside of a 3D tissue structure quickly die. Thin tissues grown from a few layers of cells don't have this problem, as all of the cells have direct access to nutrients and oxygen. Bioengineers have therefore explored 3D printing as a way to prototype tissues containing large volumes of living cells.
The most commonly explored techniques are layer-by-layer fabrication, or bioprinting, where single layers or droplets of cells and gel are created and then assembled together one drop at a time, somewhat like building a stack of LEGOs.
Such "additive manufacturing" methods can make complex shapes out of a variety of materials, but vasculature remains a major challenge when printing with cells. Hollow channels made in this way have structural seams running between the layers, and the pressure of fluid pumping through them can push the seams apart. More important, many potentially useful cell types, like liver cells, cannot readily survive the rigors of direct 3D bioprinting.
To get around this problem, Penn researchers turned the printing process inside out.
Rather than trying to print a large volume of tissue and leave hollow channels for vasculature in a layer-by-layer approach, Chen and colleagues focused on the vasculature first and designed free-standing 3D filament networks in the shape of a vascular system that sat inside a mold. As in lost-wax casting, a technique that has been used to make sculptures for thousands of years, the team's approach allowed for the mold and vascular template to be removed once the cells were added and formed a solid tissue enveloping the filaments.
"Sometimes the simplest solutions come from going back to basics," Miller said. "I got the first hint at this solution when I visited a Body Worlds exhibit, where you can see plastic casts of free-standing, whole organ vasculature."
This rapid casting technique hinged on the researchers developing a material that is rigid enough to exist as a 3D network of cylindrical filaments but which can also easily dissolve in water without toxic effects on cells. They also needed to make the material compatible with a 3D printer so they could make reproducible vascular networks orders of magnitude faster, and at larger scale and higher complexity, than possible in a layer-by-layer bioprinting approach.
After much testing, the team found the perfect mix of material properties in a humble material: sugar. Sugars are mechanically strong and make up the majority of organic biomass on the planet in the form of cellulose, but their building blocks are also typically added and dissolved into nutrient media that help cells grow.
"We tested many different sugar formulations until we were able to optimize all of these characteristics together," Miller said. "Since there's no single type of gel that's going to be optimal for every kind of engineered tissue, we also wanted to develop a sugar formula that would be broadly compatible with any cell type or water-based gel."
The formula they settled on ? a combination of sucrose and glucose along with dextran for structural reinforcement ? is printed with a RepRap, an open-source 3D printer with a custom-designed extruder and controlling software. An important step in stabilizing the sugar after printing, templates are coated in a thin layer of a degradable polymer derived from corn. This coating allows the sugar template to be dissolved and to flow out of the gel through the channels they create without inhibiting the solidification of the gel or damaging the growing cells nearby. Once the sugar is removed, the researchers start flowing fluid through the vascular architecture and cells begin to receive nutrients and oxygen similar to the exchange that naturally happens in the body.
The whole process is quick and inexpensive, allowing the researchers to switch with ease between computer simulations and physical models of multiple vascular configurations.
"This new platform technology, from the cell's perspective, makes tissue formation a gentle and quick journey," Chen said, "because cells are only exposed to a few minutes of manual pipetting and a single step of being poured into the molds before getting nourished by our vascular network."
The researchers showed that human blood vessel cells injected throughout the vascular networks spontaneously generated new capillary sprouts to increase the network's reach, much in the way blood vessels in the body naturally grow. The team then created gels containing primary liver cells to test whether their technique could improve their function.
When the researchers pumped nutrient-rich media through the gel's template-fashioned vascular system, the entrapped liver cells boosted their production of albumin and urea, natural components of blood and urine, respectively, which are important measures of liver-cell function and health. There was also clear evidence of increased cell survival around the perfused vascular channels.
And theoretical modeling of nutrient transport in these perfused gels showed a striking resemblance to observed cell-survival patterns, opening up the possibility of using live-cell data to refine computer models to better design vascular architectures.
Though these engineered tissues were not equivalent to a fully functioning liver, the researchers used cell densities that approached clinical relevance, suggesting that their printed vascular system could eventually be used to further research in lab-grown organs and organoids.
"The therapeutic window for human-liver therapy is estimated at one to 10 billion functional liver cells," Bhatia said. "With this work, we've brought engineered liver tissues orders of magnitude closer to that goal, but at tens of millions of liver cells per gel we've still got a ways to go.
"More work will be needed to learn how to directly connect these types of vascular networks to natural blood vessels while at the same time investigating fundamental interactions between the liver cells and the patterned vasculature. It's an exciting future ahead."
With promising indications that their vascular networks will be compatible with all types of cells and gels, the team believes their 3D printing method will be a scalable solution for a wide variety of cell- and tissue-based applications because all organ vasculature follows similar architectural patterns.
"Cell biologists like the idea of 3D printing to make vascularized tissues in principle, but they would need to have an expert in house and highly specialized equipment to even attempt it," Miller said. "That's no longer the case; we've made these sugar-based vascular templates stable enough to ship to labs around the world."
Beyond integrating well with the world of tissue engineering, the researchers' work epitomizes the philosophy that drives much of the open source 3D printing community.
"We launched this project from innovations rooted in RepRap and MakerBot technology and their supporting worldwide communities," Miller said. "A RepRap 3D printer is a tiny fraction of the cost of commercial 3D printers, and, more important, its open-source nature means you can freely modify it. Many of our additions to the project are already in the wild."
Several of the custom parts of the RepRap printer the researchers used to make the vascular templates were printed in plastic on another RepRap. Miller will teach a class on building and using these types of printers at a workshop this summer and will continue tinkering with his own designs.
"We want to redesign the printer from scratch and focus it entirely on cell biology, tissue engineering and regenerative medicine applications," Miller said.
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University of Pennsylvania: http://www.upenn.edu/pennnews
Thanks to University of Pennsylvania for this article.
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Sharapova loses at Wimbledon, will drop from No. 1
Sabine Lisicki of Germany reacts winning against Maria Sharapova of Russia during a fourth round single match at the All England Lawn Tennis Championships at Wimbledon, England, Monday, July 2, 2012. (AP Photo/Sang Tan)
Sabine Lisicki of Germany reacts winning against Maria Sharapova of Russia during a fourth round single match at the All England Lawn Tennis Championships at Wimbledon, England, Monday, July 2, 2012. (AP Photo/Sang Tan)
Maria Sharapova of Russia, right, congratulates Sabine Lisicki of Germany after she won a fourth round singles match at the All England Lawn Tennis Championships at Wimbledon, England, Monday, July 2, 2012. (AP Photo/Sang Tan)
Maria Sharapova of Russia reacts during a fourth round singles match against Sabine Lisicki of Germany at the All England Lawn Tennis Championships at Wimbledon, England, Monday, July 2, 2012. (AP Photo/Sang Tan)
Serena Williams of the United States reacts after defeating Yaroslava Shvedova of Kazakhstan during a fourth round singles match at the All England Lawn Tennis Championships at Wimbledon, England, Monday, July 2, 2012. (AP Photo/Kirsty Wigglesworth)
Serena Williams of the United States prepares for a fourth round women's singles match against Yaroslava Shvedova of Kazakhstan at the All England Lawn Tennis Championships at Wimbledon, England, Monday, July 2, 2012. (AP Photo/Kirsty Wigglesworth)
WIMBLEDON, England (AP) ? All at once, there was a frenzy of activity at a wet and windy All England Club early Monday afternoon.
Top-seeded and 2004 Wimbledon champion Maria Sharapova, a big hitter in her own right, was overpowered in a 6-4, 6-3 loss to No. 15 Sabine Lisicki. Four-time title winner Serena Williams was locked in a three-set tussle against a wild-card entry from Kazakhstan who is ranked 65th but is responsible for the only perfect set in women's professional tennis. Defending champion Petra Kvitova was trying to come back after dropping her opening set.
Oh, and over on Centre Court, there was the not-so-insignificant matter of 16-time Grand Slam champion Roger Federer's medical timeout to get treatment for his aching back.
The start of Week 2 at Wimbledon has been dubbed "Manic Monday," because it's the only major tournament that schedules all 16 fourth-round singles matches on one day.
Sure lived up to that moniker this year, even if rain prevented five of the eight men's matches from finishing.
The most newsworthy result was the abrupt end of Sharapova's bid to become the first woman since Williams in 2002 to win the French Open and Wimbledon in the same year. Less than a month after completing a career Grand Slam in Paris to return to No. 1, Sharapova bowed out against someone she had beaten the three other times they met. She will be replaced atop the rankings next week.
"Nothing is easy. Certainly not a Wimbledon title," Sharapova said. "So I don't know if it's easier or tougher now than it was years ago, but I don't think it's ever easier."
Federer, seeking a seventh trophy at the grass-court Grand Slam, beat Xavier Malisse 7-6 (1), 6-1, 4-6, 6-3 to reach a 33rd consecutive major quarterfinal, adding to his record. After the seventh game, Federer got help from a trainer for his back. When he returned, his play didn't appear to suffer all that much, other than slower-than-usual serves. On the other hand, Federer capped the match with a 122 mph ace.
"Honestly, I'm not too worried. I've had bad backs over the years. I've been around. They go as quick as they came," he said. "But of course I have to keep an eye on it now."
Federer now faces No. 26 Mikhail Youzhny, a 6-3, 5-7, 6-4, 6-7 (5), 7-5 winner over Denis Istomin. Federer is 13-0 against Youzhny, who chose to look on the bright side, saying: "I have one more chance."
The only other man assured a spot in Wednesday's quarterfinals is No. 1 Novak Djokovic. The defending champion improved to 12-1 against Viktor Troicki, his doubles partner for Serbia at the upcoming London Olympics, by winning 6-3, 6-1, 6-3 under the Centre Court roof.
"Weather is always an obstacle here," Djokovic said.
Two men's matches never started, and three were suspended: No. 4 Andy Murray leads No. 16 Marin Cilic by a set and a break; No. 10 Mardy Fish took the first set against No. 5 Jo-Wilfried Tsonga and they're tied 1-all in the second; No. 31 Florian Mayer leads No. 18 Richard Gasquet 6-3, 2-1.
The women's quarterfinals are set for Tuesday: No. 6 Williams vs. No. 4 Kvitova, who came back to beat No. 24 Francesca Schiavone of Italy 4-6, 7-5, 6-1; Lisicki vs. No. 8 Angelique Kerber, who ended the soon-to-retire Kim Clijsters' last Wimbledon 6-1, 6-1; No. 2 Victoria Azarenka vs. Tamira Paszek; and No. 3 Agnieszka Radwanska vs. No. 17 Maria Kirilenko.
Azarenka, the Australian Open champion, has lost only 14 games so far. The most interesting aspect of her 6-1, 6-0 win over 2008 French Open champion Ana Ivanovic? The pigeon feathers that slowly floated down to the grass after a bird collided with the roof.
"Sometimes it can be annoying when somebody is chewing chips right when you're serving. Doesn't really matter; you just have to stay focused on your game. Whatever is going on around is going on around. It's out of your hands," Azarenka said. "But the feathers? It was fun."
Lisicki certainly had a grand ol' time against Sharapova, smiling all the while.
She used flat, powerful groundstrokes to neutralize Sharapova from the baseline. She also served bigger than Sharapova, reaching 118 mph and collecting six aces. A second-serve winner at 117 mph earned Lisicki's third match point, which she converted with a second-serve ace at 108 mph, then dropped to her knees and shook her fists while Dallas Mavericks star Dirk Nowitzki cheered from her Court 1 guest box. (Michael Jordan's Chicago Bulls running-mate Scottie Pippen was at Williams' match on Court 2.)
"That's my game, to serve well and be aggressive. That's what I did. I think it worked well," Lisicki said. "As soon as I got the break in the second set, I knew, 'I'm going to take it home.'"
Lisicki missed seven months in 2010 because of a left ankle injury ? she's described what she went through as having "to learn how to walk again" ? and dropped out of the top 200. After twisting that ankle in April, Lisicki withdrew from two tournaments and then lost her opening matches at four consecutive events, including the French Open.
But she clearly has taken a liking to the All England Club, having reached the semifinals last year, when she lost to Sharapova.
Despite their history, Sharapova referred to Lisicki as "the girl I played today," rather than by name. Sort of the way Williams' father talked about Yaroslava Shvedova, who gave the 13-time major champion all she could handle over the last two sets before losing 6-1, 2-6, 7-5.
"Whatever her name is, her feet were moving very well," Richard Williams said. "Serena's feet weren't moving."
"Looked like Serena's just not playing. She's not moving forward. Standing still. Getting caught on her back heels too much," he said. "Looked like if the girl took the ball early, she won the point."
In the third round, Shvedova won every single point ? 24 of 24 ? in the first set against French Open runner-up Sara Errani, the first "golden set" by a woman in the 44-year Open era.
When Williams began Monday's match by sailing her first groundstroke, a backhand, long to trail love-15, did that perfect set by Shvedova cross her mind?
"I was worried about it," Williams joked. "I just said, 'Serena, just get a point in this set and try to figure it out.' I definitely thought about it."
Quickly, the question became not whether Williams would win a point ? OK, everyone knew that answer beforehand ? but whether Shvedova would win a game. Call it a "Serena Set": She won 16 of 19 points in one stretch and went ahead 5-0.
But from 2-all in the second, Shvedova began hitting backhand winners at will, serving better and returning well, too, reeling off five games in a row. After the second set ended on a forehand into the net by Williams, she earned a warning from the chair umpire for racket abuse.
Williams already was pushed to a 9-7 third set in the third round, then trailed Shvedova 5-4 in the third. But with her father yelling encouragement from the stands, Williams took the final three games.
They played through drizzles that left Shvedova's prescription glasses tough to see through, so she removed them. And at 5-5, she double-faulted twice in a row to set up break point, then missed a backhand wide. But Shvedova insisted her mistakes had nothing to do with her vision.
"It's just I was a bit nervous," she said.
Plus, of course, that was Williams out there.
"In the right moments," Shvedova explained, "she did the right things."
That included a running, stretching cross-court backhand lob that Shvedova let drop behind her for a winner.
"I was surprised it went in. Maybe it was wind or something," Shvedova said. "Very weird."
Richard Williams' take?
"Actually," he said, "it was luck, to be honest with you."
His daughter acknowledged she "had no intention of hitting that shot. ... I thought I was going for a backhand down the line, and somehow it ended up being a cross court lob. That was not in the plans whatsoever."
She'll play Kvitova in a quarterfinal between the only past Wimbledon champions left in the women's draw, now that Sharapova is gone. Williams is 2-0 against Kvitova, both straight-set victories in 2010, at the Australian Open and Wimbledon.
"She's obviously a great grass-court player, as well as I am," Williams said. "I'll be ready."
___
Follow Howard Fendrich on Twitter at http://twitter.com/HowardFendrich
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