Building Cartilage Scaffolds the Microfluidic Way

Cartilage

Our bodies are pretty much amazing. We can get hurt, and our bodies will heal our cuts and bones (with the right support). But not everything heals so easily, like cartilage. The cartilage in our joints is called hyaline cartilage and can be damaged from trauma or diseases like osteoarthritis. The other cartilages like elastic cartilage (found in our ears and nose) and fibrocartilage (found on tendons and ligaments) are a bit of a different story. The hyaline cartilage found on the articular surfaces of our bones can't heal like other parts of our body because it doesn't contain any blood vessels. The blood vessels would normally provide the cells and proteins to the damaged tissue. So, without blood, the damaged tissue pretty much does, nothing. Enter tissue engineering.

Cartilage Engineering

When faced with something that won't fix itself, our initial impulse is to replace it. That was our first reaction too, but we can't replace cartilage with just anything. It is a very complex and dynamic tissue. Ideally we would replace it with fresh cartilage, but it's not so easy to grow. The engineered cartilage must have a functional shape, achieve specific mechanical properties and not cause an immunogenic response when implanted in the body. In order to encourage cartilage cells (called chondrocytes) to form tissue in three dimensions instead of the two-dimensional bottom of a dish, tissue engineers have been developing scaffolds. Scaffolds have four desired traits:

  • Highly porous with interconnected network for cell growth and transport of nutrients and metabolic waste
  • Biocompatible and bioresorbable so that it can be replaced by the tissue
  • Ideal surface for cell attachment and proliferation
  • Mimic cartilage mechanical properties

 

Alginate ( source )  

Alginate (source) 

Choosing the right material is pretty important, but devising a way to create a porous 3D network is also vital. Cells can't be cut off from transport of nutrients and waste, even though it's crazy to believe that chondrocytes only make up 1% of the volume in cartilage. Researchers at National Taiwan University have developed a new method to build cartilage scaffolds using a polymer called alginate, which is a gum extracted from seaweed. It has been used previously in other scaffolds, but the main advance made by the researchers is how it is manipulated.

 

Microfluidic-generated Scaffold

Scaffold_Microfluidic_Setup.jpg
Honeycomb Alginate Scaffold

Honeycomb Alginate Scaffold

The work by Feng-Huei Lin et al. is entitled "A highly organized three-dimensional alginate scaffold for cartilage tissue engineering prepared by microfluidic technology" and appears in the October issue of Biomaterials. The authors have developed a novel microfluidic method for creating the alginate-based scaffold. As depicted in their figure, alginate droplets are formed around nitrogen gas. Their formation is highly controlled resulting in monodisperse droplets, which means that they're all (statistically) the same in size and shape. These droplets fall from the device into a solution containing calcium ions. The calcium ions (Ca2+) cause the alginate to form a gel. But before that happens, the droplets form a pretty honeycomb pattern. While this looks nice, it has a very important function. Remember when I said that scaffolds need to have interconnected networks? Well the monodisperse droplets are able to align so that they fit together perfectly, creating hexagonal patterns around each droplet. Once the droplets have gelated, a vacuum is applied which removes the air bubbles and connects the network.

This technique has seen some promising results when looking at how the cells attach, proliferate and survive. But some forms of alginate have been known to cause immunogenic responses which would be unattractive. Any resulting engineered tissue would need to be mechanically tested, which was not performed in this study.

Overall, this research was pretty interesting. It's obviously relevant to us humans, but it also excites me because it is a form of therapeutic microfluidics. As you can see from the rest of my posts, a lot of microfluidic technology is used in diagnostics. Both are equally important, but occur in different frequencies, so you can understand why this would have a place in my heart.

Note: For more information on tissue engineering, check out Nova's great episode on it, Replacing Body Parts.

 

References:

Hutmacher, D. (2000). Scaffolds in tissue engineering bone and cartilage Biomaterials, 21 (24), 2529-2543 DOI: 10.1016/S0142-9612(00)00121-6

Temenoff, J., & Mikos, A. (2000). Review: tissue engineering for regeneration of articular cartilage Biomaterials, 21 (5), 431-440 DOI: 10.1016/S0142-9612(99)00213-6

Wang, C., Yang, K., Lin, K., Liu, H., & Lin, F. (2011). A highly organized three-dimensional alginate scaffold for cartilage tissue engineering prepared by microfluidic technology Biomaterials, 32 (29), 7118-7126 DOI: 10.1016/j.biomaterials.2011.06.018

Creating Droplets in Microfluidic Devices with Ultraviolet Light

Digital Microfluidics Background

With the widespread use of electronics, we often use the word ‘digital,’ but we might not always think about what it actually means. For those of you who have never taken a class in electrical engineering, or never learned Latin (from the word digitus), the word describes anything that is discrete as opposed to continuous. Digital has also been applied to a type of microfluidics. With the definition in hand, you might guess that digital microfluidics does not describe continuous fluid flow through channels at the micro- scale, but instead is made of droplets. Discrete droplets can be implemented in a variety of assays or devices, as they would allow for complete control of the fluid, instead of a continuous stream of fluid that may not be thoroughly mixed. Regardless of the intended use of the droplets, they must first be created.

There are currently three techniques to generate droplets:

  1. electrowetting
  2. dielectrophoresis
  3. emulsification

Electrowetting essentially uses an electric field to change how fluid interacts with the surface. It can make the surface more or less attracted to water, causing a fluid such as water to 'hug' the substrate or 'ball up' into a droplet. Manipulation of the electric field would provide control of the locations of droplets and how they move. Check out the videos from Dr. Richard Fair's laboratory at Duke that illustrate the formation and transportation of droplets using electrowetting.

Dielectrophoresis occurs when nonuniform electric fields cause polarizable particles to move. The application of dielectrophoresis for microfluidics was proposed by Dr. Thomas B Jones in the Journal of Applied Physics in 2001. Water is attracted to the regions where the electric field is the strongest. This movie from Jones does not demonstrate the formation of droplet formation, but it does illustrate its control over water.

Finally, the process of emulsification describes a system of two fluids in which one fluid is dispersed throughout the other. Think water and oil and the droplets you can create when you shake it around. This first requires at least two fluids to be used (I say at least two, because multiple emulsions can be achieved, as seen here) and an external stimulus. An external stimulus is often needed to cause stable droplet formation. This can occur at junctions, where specific geometry, along with control of flow rates can cause emulsification. You can see a video at the company RainDance's website. They have more information on the subject, and I recommend that you check out their other videos on that page, especially 'Loading droplets.' It's like a gumball machine!

UV Controlled Droplet Formation

While the ability to digitize fluid is valuable, its regulation can be increasingly more prized. Does the digitization have an on/off switch? Do you have to change the flow rates of the system to revert back to continuous flow, or physically move components that are responsible for pinching the droplets? The capability to switch between continuous and digital flow could serve to reduce the footprint of the device. Why make the system larger just to incorporate streams and droplets when it can happen in the same place? This would lend elegance to the design of the device. The sophistication would be improved if this could be accomplished without moving parts. The most reliable instruments and devices have fewer moving parts that could break down. This might be what Damien Baigl et al. from École Normale Supérieure in France had in mind. Their research, which was featured on the cover of the 2011 Issue 16 of Lab on a Chip, proposes a method to emulsify droplets with Ultraviolet (UV) light. Their paper entitled, "Photoreversible fragmentation of a liquid interface for micro-droplet generation by light actuation" describes an emulsification system that is controlled by the use of UV light.

To start, the system has water-in-oil flow. But the water contains a surfactant AzoTAB. When UV light is applied to this compound, a double covalent bond switches (from trans to cis). This causes the surfactant to become more polar and decrease the wettability with the surface of the device. The lowered wettability causes the water with AzoTAB to form droplets. Initially the researchers created a junction that was capable of emulsification depending on the flow rates of the fluid. They were also able to find a combination of flow rates that would not normally create droplets, but digitized with UV light.

While this is a nice feature, it partially relies on the preexisting structure that is capable of emulsification. They next presented a design that does not constrict the fluids. This was also able to cause droplet formation with UV light. It was demonstrated that the presence and absence of UV light resulted in digital and continuous flow. This partially fulfills the desired versatility I discussed earlier. But I think that in addition to being able to generate droplets and streams at whim, it is also advantageous to convert droplets back into a stream. The authors briefly mention this, and it seems that the application of blue light can reverse the switch and produce streams. I think the greatest part of this setup is the ability to change the same unit of fluid between continuous and digital.

But what does this research really get us? Well, nothing at first. This isn't a complete device like Dr. Sam Sia's mchip that can detect HIV. But this will surely be incorporated into a device. It really is a tool that can be applied in different ways along with other tools to create a full device. I'll update you further once this has been incorporated and used further.

Reference:

ResearchBlogging.org

Diguet, A., Li, H., Queyriaux, N., Chen, Y., & Baigl, D. (2011). Photoreversible fragmentation of a liquid interface for micro-droplet generation by light actuation Lab on a Chip, 11 (16), 2666-2669 DOI: 10.1039/C1LC20328B

Videos reproduced by permission of Damien Baigl and The Royal Society of Chemistry from Lab Chip, 2011, 11, 2666-2669, DOI: 10.1039/C1LC20328B.