Feature: deceptive appearances – engineering cartilage
The tiny area of uncertainty that is inevitably left by circumstantial evidence – however compelling – poses a dilemma for scientists, as well as juries. At Guy’s Hospital in London, Dr Eileen Gentleman believes the gap between what has been proven as certain and what has to be inferred from contextual clues has been hampering progress in the field of tissue engineering.
For the past 15 years, researchers have been growing bone and cartilage in the laboratory. Biological texts show that these lab-grown tissues have the appearance, texture, and protein and mineral components of bone and cartilage. But once they are tested in an animal, these tissues simply don’t behave quite like the natural tissues they are supposed to replicate.
“When I got into this field, I saw paper after paper in which people described putting cells in a material like a polymer, creating a tissue that resembles cartilage, and testing it in an animal,” says Dr Gentleman. “The results are OK, but they’ve never been good enough to take forward to clinical trials with people.”
She believes the missing part of the equation is an engineering perspective. “Joints are remarkable feats of engineering, but efforts to grow them in the lab have focused mostly on their biology.”
Cartilage and osteoarthritis
That means that so far, aside from joint replacement, there is still limited help for the 10m or so people in the UK estimated to suffer from osteoarthritis. This painful, crippling disease is an extremely complex one involving many different processes and is still poorly understood, although there’s a general consensus that it seems to start with some sort of damage or tear to the cartilage.
Cartilage is the rubbery, slippery tissue that cushions the ends of bones in our joints, absorbing shock and allowing them to slide comfortably against each other. It consists of layers of tissue with distinctly different compositions and structures, which become harder and more calcified towards the surface.
The different layers merge seamlessly with each other and eventually transition equally seamlessly into the bone at the surface, forming a fully integrated ‘osteochondrial interface’ of bone and cartilage. However, unlike bone (which is full of blood vessels and hence very good at self-repair), cartilage contains almost no blood vessels – and no blood cells to help it heal.
Instead, once cartilage is torn, it starts to degrade over many years. That process is compounded by inflammation and aggravation to the surrounding tissue, eventually leading to the extremely painful degeneration of the whole joint – including the underlying bone.
Current surgical interventions can’t stop that degenerative process, although they bring patients some short-term relief from pain. One involves scraping pieces of torn cartilage from the joint and poking the bone underneath to break it slightly (a technique called microfracture) so blood can come up through the bone and attempt to heal the area.
The tissue that then forms isn’t normal cartilage, however. “You get something called fibrocartilage, which doesn’t function like natural cartilage. It’s not the nice, soft, slick stuff that you should have cushioning your joint.”
The alternative technique, mosaicplasty, involves punching a little plug of cartilage and bone from a part of the joint that’s not load-bearing or obviously diseased and using it to ‘fill in’ the lesion in the load-bearing part.
However, says Dr Gentleman, “When you have osteoarthritis in your knee, it’s not just in that one part, it’s in the whole knee. So the plug might look healthy or less worn down, but the cells in it are probably being exposed to all this inflammation, so they might well be diseased too.”
Hence the goal of tissue engineers: to create plugs of healthy bone and cartilage in the laboratory that will function like the ‘real’ cartilage in human joints. This would mean they could prevent the degenerative process before it develops into osteoarthritis, and repair and restore the joint.
It looks like a duck
Dr Gentleman – who is also a Senior Research Fellow at King’s College London – has been awarded a Wellcome Trust Research Career Development Fellowship to take up the gauntlet. Her background is in biomedical engineering, and she now plans to add the methods and perspectives of engineering to those of biology.
Biologists attempting to create cartilage and bone over the past 15 years have typically tested the mechanical properties of their laboratory-grown tissue – for example, whether it is rubbery and resilient enough when pressure is applied. “Obviously the cartilage-bone has to support load and body. That’s its function. So you have to make sure that it’s hard enough to do that.”
They have also used gene-expression analysis and histological stains to look for important constituents of the material, such as calcium in bone and type II collagen in cartilage. “You put a slice of the tissue on a slide under a microscope and add a dye that binds to calcium or one that binds to type II collagen.
“If the stains show that the right proteins, minerals and other components are present – and the material has the right mechanical properties – that’s how you demonstrate that you’ve done tissue engineering.”
Herein lies the tissue engineering equivalent of the smoking gun scenario. “Just because biological tests indicate a tissue looks like bone and feels like bone, doesn’t actually mean it is bone,” says Dr Gentleman. “Lots of forms of mineral contain calcium, but they’re not all bone.”
This is where an engineering perspective becomes important. To look at how close a match these laboratory-generated tissues really are to native bone and cartilage, Dr Gentleman and her colleagues supplemented the biological analyses with engineering tests, such as bio-Raman microspectroscopy.
“You shine a laser on the material, and the way the light scatters gives you an idea of the bonds between its components. Different mineral types form different bonds, so you get a much more precise picture of what is actually present.”
The engineering tests revealed differences between the composition of the engineered tissues and that of normal bone that were not previously visible.
Is it a duck?
If a lab-grown tissue seems from some tests to be the real thing but isn’t really, then it won’t behave like it once it has been implanted in a human body.
Bone cells in the body carry out many biological and structural functions. They degrade and resorb, maintain tissue metabolism, exchange nutrients and waste, build the extracellular matrix, mineralize and contain, synthesize, transport, and secrete.
Engineered bone and cartilage able to regenerate the joints of living people must generate cells that will carry out these myriad, highly specialized functions in precisely the same way they do in native tissue. They must also merge seamlessly with each other to create the integrated ‘osteochondrial interface’ of bone and cartilage that is so crucial to pain-free, fully functioning joints.
That’s a larger-scale engineering challenge that is made harder by the fact that cartilage doesn’t repair itself: “Even if you do create something in the lab that has exactly the same components as cartilage, how do you then get it into the lesion in the joint and get it to heal in there and integrate with the normal cartilage and bone?”
To get past that hurdle, many studies have attempted to make layered constructs, such as cell-seeded scaffolds. “People have tended to take some cells, stick them to a biocompatible material or scaffold, then put that structure in the joint and hope some magic will happen.
“But as any engineer will tell you, if you just glue two pieces together, as soon as you put a shearing force on it they peel apart. The only thing holding them together is whatever glue you used. So with all these sliding forces in the joint, it is probably not what you want to do.”
A whole interface
With her Wellcome Trust Research Career Development Fellowship, therefore, Dr Gentleman aims to be a lot more thorough in the laboratory than she believes people have been to date, before pushing forward with animal studies.
Her aim is to use an engineering approach to create a whole osteochondral interface in which bone and cartilage transition seamlessly into each other like they do in the body. “That’s the only way it will effectively transmit loads to the underlying bone. And because bone will heal, it will heal the construct into the joint.”
At the same time, she plans to take inspiration from biology to enhance the engineering techniques by mimicking what the body does in a developing embryo. “We’ll use the human stem cells that form the osteochondrial interface in the embryo and continue to live in your bone marrow when you are an adult.
“Stem cells take something that is essentially a whole lot of nothing and create the extracellular matrix – the part of your tissues outside the cells that gives them structural support – out of collagen, sugars, carbohydrates and minerals. The aim is to apply a stimulus to convince them that they’re in the part of the body where they would make either cartilage or bone. Hopefully, that will encourage them to do so.”
Dr Gentleman hopes this will produce constructs that are closer to the native tissue in their biology and their engineering than previous attempts have been: “It’s not just a matter of getting the engineering right, as well as the biology. It’s a matter of getting them both correct at the same time.”
If she’s successful, there is a better chance these constructs will function like the real thing in people’s bodies, offering hope to the estimated 10m people in the UK who are affected by osteoarthritis.