The silk scaffold has seamless
junction between the cartilage and bone portions
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.
