Implanting cartilage alone or injecting cells found in healthy cartilage (chondrocytes) at the site of injury to heal the damaged cartilage in patients with osteoarthritis does not produce favourable results. Similarly, implanting two different scaffolds joined together to simultaneously regenerate the cartilage and reconstruct the bone too has many limitations.
The problem arises because the interface between the cartilage and bone scaffolds, which are made of different materials, is not connected but has a distinct boundary. As a result, the interface tends to delaminate and degrade. Now, researchers from Indian Institute of Technology (IIT) Guwahati have addressed this shortcoming by fabricating a silk scaffold where the junction between the cartilage and bone scaffold is continuous and seamless and hence less prone to damage under load-bearing environment of the joint.
A team led by Biman B. Mandal from the Department of Biosciences and Bioengineering has fabricated the biphasic scaffold where the top portion is highly porous and spongy thus mimicking the cartilage, while the bottom portion is reinforced with silk fibre thus imparting more stiffness and less porous to mimic the bone. Since the entire scaffold is made of silk, the interface merges with one another and is seamless despite having different porosities and stiffness. The results of the study were published in the Journal of Materials Chemistry B.
The researchers made scaffolds using both wild silkworm (Antheraea assamensis) and mulberry silk (Bombyx mori) and found scaffolds made of non-mulberry silk were superior to the one made of mulberry silk in all respects.
“To make the biphasic scaffold we prepared silk solution by completely dissolving the silk. We then added chopped silk fibres to the solution so the bottom half portion of the scaffold becomes fibre-reinforced silk composite while the rest of the top portion had only the silk solution,” says Prof. Mandal. The solution is processed by reducing the temperature to –20 degree C and then vacuum dried to remove water. The top portion of the scaffold is highly porous and soft like a sponge whereas the bottom portion is less porous and strong. The biphasic scaffold was treated with alcohol to make it water-insoluble.
The porosity is intended for neighbouring cells to migrate, infiltrate and regenerate in the scaffold, and support better nutrient exchange. The less porous silk-reinforced scaffold portion allows bone cells to optimally grow. Owing to the RGD sequence in the non-mulberry silk, more cells tend to migrate to the scaffold and proliferate.
Seeding stem cells
“It is also possible to take a patient’s bone marrow stem cells and seed them on the scaffold. The stem cells will differentiate to become mature cartilage-like and bone-like cells. Our scaffold is amenable to stem cell seeding and differentiation protocols,” Prof. Mandal says.
Validation of the scaffold that was seeded with cartilage and bone cells was first done through in vitro studies. “We saw elevated levels of cell proliferation, extra-cellular matrix deposition and higher tissue-specific gene expression within the construct. These proved that the construct was compatible and good,” says Yogendra Pratap Singh from IIT Guwahati and first author of the paper. “The cartilage cells prefer a softer matrix compared to bone cells and our construct was suitable for both types of cells to proliferate.”
The compatibility and ability of the scaffold to regenerate cartilage and bone was then tested in rabbits. The scaffolds were studied eight weeks after implantation. “The fibre-reinforced scaffold allowed more bone formation, while regeneration and complete repair of the cartilage was seen,” says Singh, who currently is a Newton-Bhabha Fellow at the University of Sheffield. “We found the non-mulberry silk scaffold outperformed the mulberry silk one.”
The researchers found the non-mulberry silk scaffold had 1.5 and 0.5 times more bone and cartilage cells respectively attached than in the mulberry silk scaffold. Gene expression was nearly double in the non-mulberry silk scaffold than in the mulberry silk scaffold. Also, the extra-cellular matrix in both the cartilage and bone portion of the scaffold increased six-fold in 14 days.
The fibre-reinforced scaffold mimicking the bone was 10 times stronger than the sponge-like portion. Greater compressive and tensile strength of fibre-reinforced scaffold are desirable.
The researchers are planning to test the scaffold on larger animals, preferably pigs.