Researchers at the University of Pittsburgh’s Center for Cellular and Molecular Engineering have developed a small bioreactor that grows constructs of bone and cartilage in a single chamber.
Researchers at the University of Pittsburgh’s Center for Cellular and Molecular Engineering have developed a small bioreactor that grows constructs of bone and cartilage in a single chamber. The bioreactors produce constructs that resemble natural joints and are small enough to fit into 96-well arrays. This opens the door to medium-throughput and perhaps even high-throughput screening study of bone-cartilage interactions, osteoarthritis, and other joint diseases, and medications to stop their progression.
Osteoarthritis is a degenerative joint disease that affects about one-third of all adults 65 and older. It is painful and frequently debilitating enough to place hip and knee replacement among the most common of all major elective surgeries. Researchers hope to replace surgery with medication or even bone and cartilage implants.
To study how joint diseases evolve and whether a drug holds promise, they must test the combined bone and cartilage of the joint. This is because natural bone and cartilage interact with one another, exchanging nutrients, cytokines, and hormones that affect one another’s behavior.
The only way to know what drugs are effective and safe is to test them together. Unfortunately, no one has succeeded in developing a bioreactor to grow those joint constructs in large enough quantities for mass testing. This is because while bone and cartilage consist of cells embedded in extracellular matrix, they differ greatly from each other.
While bone is stiff and highly vascularized, cartilage has no blood vessels and is more flexible. More importantly, they require very different growth environments, including media, growth factors, and oxygen levels. Yet they cannot be grown and tested alone, because of their intimate interactions.
So, while researchers have grown bone and cartilage in bioreactors, they have not been able to grow both in the same reactor at the same time. Instead, researchers who want to study joint interactions on three-dimensional models in the lab usually harvest tissue from animals or human cadavers.
At Pittsburgh’s Center for Cellular and Molecular Engineering, research assistant professor Riccardo Gottardi has developed a two-chamber bioreactor that connects the two joint components but delivers separate signals to each by controlling the fluid flow through the chambers.
Gottardi did not originally set out to build a bioreactor.
“I originally planned to study bone-cartilage crosstalk, so I started looking around for a bioreactor to grow samples,” he says. “I was sure somebody must have done this before, but I found nothing but complex systems that could not be scaled up for high or even medium throughput.”
He decided to build a bioreactor that was simple, small, robust, and easy to place in the well of a 96-well plate used to culture tissues.
Gottardi’s first iteration consists of two chambers, one for bone and one for cartilage. He starts with an extracellular matrix (ECM), and seeds both sides with the stem cells needed to grow bone and cartilage.
The ECM is more porous on the surface and denser in the middle. This reduces but does not stop the two-way movement of biochemicals through the ECM from the growing bone and cartilage on either side. “We want the tissues in there to see their own environment, but also interact with one another,” Gottardi says.
The bioreactor has two sets of inlets and outlets, one set on the bottom of the reactor to provide fluids for bone growth and other on the top to serve the cartilage.
“As long as we maintain the same flow rate, there is little mixing of the two fluid streams,” Gottardi says. “As the bone-cartilage grows thicker, we can vary the pressure of the two streams more without mixing. We adjust the pressure, so the nutrients, oxygen, and growth factors continue to move through the ECM and then onto the next bioreactor.”
The bioreactor fits into one of 96 wells on the assay plate. Its inlets and outlets align with pipes that move fluids through all the bioreactors on the plate.
“The first bioreactor has high throughput and can cultivate a lot of tissues,” Gottardi continues. “The resulting tissues are 7-8 millimeters thick, about 3 millimeters of cartilage and 3-4 mm of bone underneath, which is what we have in our joints. They are uniform, so they are good for screening drugs at multiple concentrations.”
The problem is that the samples are too large to review with a conventional optical microscope, which can penetrate only 0.5-1.0 mm. To look at them through a microscope, researchers must remove and slice each sample.
Gottardi’s second bioreactor array was developed to automate testing using microscopes.
“Researchers may have a gene reporter in their samples that produces a fluorescent protein when some specific event happens, such as the cells making collagen,” he explained. “You could screen the array automatically using a microscope to monitor which ones turn on or off, something that can happen very quickly. That might show us what signals turn a certain set of genes on or off. But to do that, you have to have smaller bioreactors that microscopes can see through.”
While Gottardi retains the diameter of the old bioreactor so it fits in a conventional 96-plate assay, he reduced its height to 1 mm. He also changed the orientation of the bioreactor, so that it grows vertically, bone on the left and cartilage on the right, so a microscope could see both sides of the construct at once.
The reactor consists of an inner chamber, which holds the tissue, surrounded by a ring that distributes the growth fluids to the inner chamber and removes them as they exit.
Microfluidics poses a critical challenge to the new design.
“The very small fluid pathways are dominated by surface interactions, and air bubbles are more of an issue,” Gottardi said. “The pathways are also sensitive to changes in pressure on two sides. That’s the challenge we’re working on now.”
One way to control bubbles and pressure variations is through the design of the fluid’s pathway. Gottardi opts for 3D printing those channels. While he can build a more tortuous path using conventional technologies, 3D printing enables him to optimize the path and microfluidics using all three dimensions.
The downside is that he is pushing 3D printing to the limit of its resolution. He is also test-printing different materials to find the best balance of transparency and the flexibility needed to load cells so they grow properly.
“We’re optimizing the design and standard operating conditions,” Gottardi said. “When we have those, the array will be fairly reproducible.”
Gottardi’s first goal is to develop an assay system that would enable researchers to study osteoarthritis and other bone-cartilage diseases on human tissue in a faster, more methodical way. One day, similar bioreactors could help regenerate damaged tissues.
The larger bioreactor is ready for commercialization, and the second getting closer. Gottardi is looking for investors interested in creating a startup or licensing the technology for commercial development.
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