Damage to joint cartilage from injury or osteoarthritis is challenging to repair because cartilage can’t repair itself. Joint cartilage doesn’t have blood vessels, so it isn’t regularly rejuvenated like other tissues, and there is no way for the body to transport new cells to an injury. Current treatments include microfracture, which breaks the bone under the injury to cause bleeding and presumably introduce new cells to start the healing process, and implanting autologous chondrocytes,1 also gathered by causing a secondary injury. Both heal an injury by making another injury. Researchers continue to look for better methods.
A Possible Solution
Researchers at University Paris Diderot recently produced compact, functional cartilage-like tissues by compacting mesenchymal stem cells with magnets. However, to heal an injury, new tissue needs to be protected from frictional shear stress so a scaffold seems like it would be necessary to provide mechanical support.
The challenge in putting cells on a scaffold, then, is to reach sufficient cell density that cartilage cells, or chondrocytes, can grow and form cartilage tissue. The University Paris Diderot researchers extended their magnetic method to gather cells into discrete and compact areas of a polymer scaffold.
Pullulan and dextran polysaccharides were used to create the scaffold. Natural polymers like these have advantages when used as a cell therapy scaffold because they are biocompatible, and they facilitate cell adhesion and differentiation.
“Here we demonstrate that pullulan/dextran scaffolds, previously shown to confine stem cells, not only retain MSC [mesenchymal stem cells] but also allow them to differentiate into chondrocytes. Using magnetic forces to attract and retain the cells within the scaffold, we enhanced MSC seeding density and condensation. When we combined this magnetic condensation technology with dynamic differentiation in a bioreactor, MSC differentiation into chondrocytes within the scaffold constructs was markedly improved.” – Nathalie Luciani, et al.2
The Experiment
The researchers made ferrous magnetic nanoparticles about 8 to 9 nanometers in diameter that were stabilized in water. The stem cells were incubated in the nanoparticle solution for 30 minutes, then rinsed before incubating overnight in a normal growth medium. During that 30 minutes, the nanoparticles were absorbed or diffused into the cells, after which the cells could be directed by magnetic fields. It had previously been shown that the 5 picograms of iron that ended up in each cell did not have a noticeable effect on normal cell behavior.
Natural polymers pullulan and dextran were crosslinked together and made into a foam before being freeze-dried and rehydrated, a process that creates a regular pore structure that cells can move through. The scaffolds were made 7 mm thick to simulate the thickness of cartilage in the patella.
A 3 by 3 grid of magnets was placed under a scaffold and stem cells were spread on top. This group was compared to a scaffold with no magnets. The cells were maintained for 4 days in this initial stage using chondrogenic medium that encourages stem cells to become chondrocytes, the cells that form cartilage. After the four days, cells were clearly concentrated in a grid pattern above the magnets.
Experimental groups were then divided again. Half of the cells were grown for 21 days in static culture and half were grown in a bioreactor that continuously spun on two axes with a continuous flow of growth medium. Cell aggregates that formed above the magnets were still visible after all 25 days of culture even though the magnets were removed after the first 4 days.
Further testing showed that both magnetic seeding and dynamic culture in the bioreactor improved maturation of the stem cells into chondrocytes. More extracellular matrix was formed and the high ratio of collagen type II to type I found in normal articular cartilage was seen in the scaffolds that were placed over the magnets and then into the bioreactor. The collagen was even seen to form fibrils in a similar fashion seen in native cartilage.
The Discovery
Cell compaction and nutrient diffusion are critical steps for chondrogenesis. These requirements were met with magnetic seeding (compaction) into a porous scaffold made from natural polymers that was placed into bioreactor (nutrient diffusion).
This was the first successful differentiation of mesenchymal stem cells into chondrocytes within the core of a scaffold.
Because the scaffolds are a relevant size for replacing damaged cartilage, this proposed solution could be a promising strategy for healing injuries to cartilage without causing a second orthopedic injury in the process.
- Autologous means it comes from the same individual, and chondrocytes are cells that build cartilage; autologous chondrocytes are then cartilage cells from a different site in the same individual.
- Nathalie Luciani, Vicard Du, Florence Gazeau, Alain Richert, Didier Letourneur, Catherine Le Visage, Clair Wilhelm. “Successful chondrogenesis within scaffolds, using magnetic stem cell confinement and bioreactor maturation.” Acta Biomaterialia 37, 101-110, 2016.