We discussed in an earlier post the application of natural polymers in clinical treatment of osteoarthritis and other injuries to articular cartilage. Those current therapies are all limited to treating small lesions or holes of only a few millimeters across. Material challenges remain when larger, curved areas of cartilage need to be regenerated in joints. Larger scaffold constructs are more difficult to evenly seed with cells, and are also prone to shrinking and changing shape as the cells grow inside the pores.
A Possible Solution
Christopher R. Rowland, et al., at Washington University in St. Louis addressed this challenge using a decelluarized, cartilage-derived extracellular matrix. Extracellular matrix is produced and maintained by living cells, providing structure to the tissue and giving a solid platform for growth and transmission of chemical cues or messages. Because this matrix is the natural platform for cell growth in the body, there is a lot of interest in using it as a tissue engineering scaffold.
Extracellular matrix contains a wide array of active proteins and relevant chemistry to tissue engineering, particularly when the material is taken from the same type of tissue that needs to be grown. However, it also contains the byproducts of dead cells which would cause a harmful immune response. To prevent this immune response, the matrix is decellularized – processed so that all traces of cells and antigens are gone from the material.
To create an anatomically relevant scaffold for cartilage tissue engineering, the research team at Washington University used cartilage-derived extracellular matrix to create disks and hemispheres (bowl shapes) that were seeded with stem cells from bone marrow.
“Therefore in the current study, we sought to minimize the manipulation of the native cartilage extracellular matrix by only using dehydrothermal treatment to minimally crosslink [cartilage-derived matrix] constructs. … [We] developed a method for fabrication of anatomically relevant, hemispherical CDM constructs seeded with human [mesenchymal stem cells].” — Christopher R. Rowland, et al.1
The cartilage-derived matrix came from the femurs of pigs. Cartilage was shaved off the bone, frozen overnight, lyophilized for another 24 hours, then pulverized into a fine powder before it was treated with a decellularization solution for another 24 hours. After decellularization, it was frozen and lyophilized again, then pulverized again before sieving to remove any particles bigger than 100 micrometers.
To create the tissue engineering scaffold, the matrix powder was suspended in water, put into silicone molds, carefully frozen, then lyophilized. Again. Physical crosslinking between the particles was achieved by heating the lyophilized material in dry air at 120 C (248 F) for 24 hours. This process uses a minimum of modification techniques to create the solid scaffold, retaining the beneficial chemistry of natural tissue.
The freezing step is more significant that would seem at first glance. The scaffold was frozen so that one side freezes first, and the ice crystals grow in a single direction across the scaffold. This is contrasted with ice forming randomly throughout and growing in all directions. Controlling the freezing controlled the orientation of pores in the final scaffold.
Using the same paradigm as seen in clinical applications, stem cells were added to the scaffold and grown for about a month. The first six days are for increasing the number of cells present, and the following 28 days are to change the stem cells to chondrocytes, or cartilage producing cells. This timeline is well within the precedent set by current clinical tissue engineering therapies.
This research was able to demonstrate several improvements to the general field. First, the hemisphere shaped scaffolds did not shrink or distort at all, even after a month with cells growing inside. This is promising for a consistent treatment that does not pull away from the area that needs new cartilage. Second, the research demonstrated that a minimum of chemical modifications will still produce a viable scaffold for tissue engineering. By controlling the porosity of the scaffold with careful freezing techniques, even the a densest scaffold supported a relevant number of therapeutic cells. Finally, the scaffolds were able to be evenly seeded with cells throughout the scaffold which is important for engineering uniform repair tissue.
Challenges remain, such as improving mechanical compression characteristics for the entire month of growing cells and regaining the microstructure of the cartilage before it was pulverized to make the scaffold. However, demonstration of a decellularized matrix scaffold that can be made in relevant shapes, and does not shrink, is an interesting step forward in cartilage tissue engineering.