Healing by Design

You’ve torn your rotator cuff delivering heat across the plate. What now? You’re in for tendon-repair surgery, significant rehab, and the ever-present risk of re-injury. But tissue engineers want to change all that.

For decades medical researchers have been looking into what makes tissues grow and heal effectively. The factors are as varied as the tissues they study, but one constant remains: Cells heal better with a framework to grow on. Called extra cellular matrices (ECMs), these frameworks occur naturally in healthy tissue. Tissue engineers are investigating new ways to artificially create and deliver the scaffolding that can speed healing.

In damaged tissues, such as torn tendons or bones that fail to knit, a scaffold can be surgically implanted to support the body’s natural construction processes. Natural ECMs provide mechanical support to growing cells, but they also give them places to anchor and contain molecules that trigger the body’s repair mechanisms.

Researchers are evaluating different types of natural ECMs and investigating cells’ ability to assemble themselves without a scaffold. They’re also applying what they’ve learned to develop synthetic scaffolds from biodegradable polymers that can mimic and, perhaps, improve on ECM functions.

Natural Healing
Natural ECMs come from healthy tissues in the patient’s body or from tissues in other mammals. Parts of the skin, the small intestine, the sack around the heart (pericardium), and the tissue between layers of muscle (fascia) are already used surgically as scaffolds after they are treated to remove living or dead cells.

Dr. Kathleen Derwin of the Cleveland Clinic has compared the tensile responses of commercially available natural ECMs to each other and to the tendons damaged in rotator-cuff injuries.

“We can mechanically load the scaffold to represent real-world situations,” Derwin said. The loads and frequencies are similar to what the body would experience during normal motion, 0.5 to 1 Hz for skeletal tendons.

“We have found that fascia has much better mechanical properties, much more similar to tendon, than the others do,” she said. The linear portion of fascia’s stress strain curve lies close to that of a healthy human tendon. Other ECMs had moduli an order of magnitude lower and stretched 5 to 10% more before picking up load.

Taking tension load is the tendon’s main function. Other tissues that are loaded in compression, like cartilage, or have nonloadbearing functions may benefit from the other available ECMs or from scaffolding that has yet to be developed.

Patching process
Picking the right ECM for a particular application is only part of the problem. The next step is actually installing it in the body.

For a rotator-cuff repair, surgeons trim the ECM to about 1 2 in. or less to fit the patient’s injury. They surgically attach it to the upper end of the arm’s humerus bone, where a tendon would have attached, and then spread the other end over the deltoid muscle, securing it with biodegradable sutures.

Culturing the patient’s cells onto an ECM in a lab environment and then transplanting a complete tendon or other body part into the patient, an approach some researchers are investigating, is a tempting prospect. But doctors would have to harvest cells from the patient weeks before surgery in a separate procedure. To spare the patient this double invasion, Derwin is pursuing a different approach.

“If you can add a growth factor that attracts the patient’s cells to the scaffold when you put it in the body, that’s a whole lot easier and cheaper,” she said. “You can put the treated scaffold on a shelf, the surgeon can open it in the operating room, dip it in saline to get it wet, and sew it in.”

Future hurdles include proving that the approach works in humans and securing FDA approval.

Another major stumbling block is the need to create tools to install such a patch arthroscopically. Most rotator-cuff surgeries today are minimally invasive arthroscopic repairs. The few patients who get a commercially available ECM have to submit to higher-risk open surgery. Without the equipment and techniques to install a scaffold arthroscopically, Derwin’s approach, or a similar polymer patch, would be unlikely to catch on among surgeons.

Without a net
Some tissues have demonstrated that they don’t need a scaffold at all. The University of Michigan’s Artificial Heart Lab put rat heart cells in growth media on a protein-rich substrate. They began to organize themselves and spontaneously contract within 48 hr. By the time 10 days had passed, the cells had self-assembled into a cylinder anchored at both ends with preset sutures.

Researchers tested each construct by attaching one end of it to an optical force transducer and exposing the construct to electrical shocks paced at frequencies similar to human heartbeats, 1 to 7 Hz. The constructs responded with regular contractions, like a healthy human heart, especially at the lower frequencies. However, the active force, the strength of the contractions, was around an order of magnitude lower than that of healthy cardiac tissue.

“To bridge that gap you would need mechanical stimulation,” said Dr. Ravi Birla, the lab’s director. “The heart is constantly beating so you need to replicate that within a lab environment.” Birla’s lab has already done work showing a 3.5 increase in active force in response to mechanical stimulation.

The constructs would also need to be supported by microperfusion, continuous fluid flow similar to blood flow in the body. Larger constructs would need vascularization, a structure of vessels to bring nutrients to and remove waste from cells that are not near the construct surface.

Chemical stimulation turns out to be important, too. The constructs have already been shown to increase their active force with the addition of calcium and epinephrine, chemicals that are essential to heart function. Other hormones and growth factors could further improve the constructs’ function.

As the technology develops, Birla hopes to “bring everything together into one big bioreactor system that can give (a heart construct) electrical, mechanical, and chemical stimulation.”

Heart muscle is only one part of the effort in Birla’s lab. Veins, arteries, and valves are all under development. Many of these structures are too complex to be formed by self-assembly.

Synthetic scaffolds
Researchers at Oxford, the Massachusetts Institute of Technology, and the University of Michigan, among others are trying to build tissues like these from the ground up. They are making synthetic scaffolding from nontoxic, biodegradable polymers like polylactic acid (PLA or PLLA) and polyglycolic acid (PGA) or from biomolecules like fibrin, an important factor in blood clotting.

One advantage of using a polymeric scaffold is the ability to control the physical features of the structure. These can be determined with great detail through computed tomography (CT) scanning and magnetic resonance imaging (MRI) and translated into CAD models.

At the Michigan Artificial Heart Lab, negative wax molds have been created from CT-derived heart-valve geometries. Researchers create a heart valve by casting biodegradable polymer into the wax mold and then dissolving the mold. The result is nearly identical to the natural valve.

Another approach is to create the structure directly from the CAD model with solid free-form fabrication (SFF) techniques. There are several widely used versions of SFF, but they all boil down to selectively solidifying areas of a medium that represent a cross section of the design. Then the part is indexed in the Z direction and the next cross section is solidified.

For some tissues, woven polymer fibers can provide strength to support the growing cells. Interstitial spaces between the fibers can provide footholds for the cells and allow nutrients to reach them. Derwin’s lab is investigating these weaves’ utility for tendon applications.

“Polymers have some real advantages,” she said. “You can make reams of polymer. You can sterilize it very easily. You can make it any shape and size you want. With polymer you’ve got the advantage of making millions of them and making them all the same way. The disadvantage is that, although these polymers have been shown to be biodegradable and not toxic, you could have some focal toxicity if the body is not able to dispose of the by-products of the degradation.”

Another advantage of using a polymeric scaffold is the ability to tailor the strength of the scaffold to what’s needed to support the tissue that will grow on it. Isotropic properties can be built in as required.

Home Sweet Scaffold
Creating the structure is relatively easy. Engineers have been using investment casting, rapidprototyping techniques, and woven materials for years. Next comes the hard part: making polymer structure a favorable environment for cell growth. For some structures, that means having pores that will let nutrient molecules reach cells at every point in the structure. Seeding the polymer with biomolecules that cells will recognize might help, too.

Once the structure is ready, cells can be plated onto it. Another strategy is to allow the cells to self-assemble into a sheet in a Petri dish before wrapping the sheet over the structure.

These techniques are further complicated when a biological structure contains two or more cell types. Veins, for instance, are lined with endothelial cells that create a smooth boundary layer. The vein OD is made of skeletal muscle cells and protein building blocks of a future ECM.

A cell-populated scaffold that is installed in the body has one more hurdle to overcome: its own destruction. As the cells on the scaffold create their own natural ECM, the polymeric scaffold should be reabsorbed in the body. The trick is to time the degradation of the synthetic scaffold so its support functions are taken over by the natural ECM as it forms.

Fibrin hydrogel is particularly promising in this respect. Birla’s lab has experimented with it to support the assembly of heart cells. The hydrogel’s molecular structure is similar to what is found in the body. The real advantage of fibrin, however, is the ability to control the way it degrades.

“We can cross-link the fibers. There are various cross-linking agents available; we use genipin. If you stabilize the gel it is simply a stronger gel and by controlling how much you stabilize it you can delay the rate of degradation,” Birla said. Depending on the amount of cross-linking, the fibrin can degrade in a week or take months to be reabsorbed. Heart-muscle constructs assembled on fibrin had the highest active force when compared to self-assembling construct and those using other polymeric scaffolds.

Today’s Technology?
So about that torn rotator cuff: can you benefit from tissue engineering today? Although there are natural ECMs that are approved to augment surgery, there is no conclusive data to say whether they improve your prognosis.

“There is not yet a polymer patch, but I think that’s going to happen in the next year or two,” Derwin told MD. “As for taking any type of scaffold, whether it’s ECM or polymer, and adding a growth factor or using it as a delivery vehicle (for cells), that’s being actively researched. The research might prove that it works in the next year or two, but going through the FDA is going to take longer yet. I would certainly imagine that within the next five years those kinds of things are going to be available.”

At the Artificial Heart Lab, Birla says that a complete artificial heart with living tissue is still science fiction.

“If you look at how long it will take before we can put this back into a patient, nobody can provide a time frame right now because a lot of critical technologies that need to go into it have just not been developed,” he said. “Even the application of patches (to replace diseased cardiac muscle) is a few years away before we can take it to the clinic.”

Birla, like Derwin, will face clinical trials and regulatory approval before any part of his lab’s artificial heart becomes part of the surgeon’s tool kit.

Simplifying Screening
But there are other uses for the advances that have been made to this point. The heart constructs can be a valuable research tool for many other developments in medicine.

“You can use these (heart-muscle) constructs for drug screening for toxic effects,” Birla said. “Something like that can be done pretty much right away.”

Other researchers are tissue engineering organ models especially for the purpose of drug screening. The goals are streamlining the process of evaluating proposed drugs and making drug trials safer for human patients.

In 2004, Machine Design reported that MIT had developed a “micro-liver” using micropatterning technology such as that used to place copper wires on microchips. Precise placement of liver cells, support tissue, and culture media in an array created this artificial liver that behaves, at least chemically, the way a healthy human liver does.

Using human cells means scientists don’t have to guess whether it’s appropriate to extrapolate results from other animals such as rats. The long life of the cells in this device also lets researchers determine what happens when the liver is exposed to these chemicals for weeks or months at a time, something that was not previously possible.