When implanted in mice, the silk construct was not rejected even at the end of four weeks, say Biman Mandal (right) and Bibhas Bhunia.
Researchers at IIT Guwahati have fabricated a silk-based spinal disc construct that mimics human intervertebral disc both in form and function. The construct allows annulus fibrosus cells or AF-like differentiated cells to grow and proliferate and deposit normal extracellular matrix. The construct was found to be biocompatible when implanted in mice.
Using mulberry silk, researchers from Indian Institute of Technology (IIT) Guwahati have fabricated a spinal biodisc construct that mimics human intervertebral disc both in form and function. When approved for use in humans, the biodisc can be used for spinal disc replacement therapy.
Degeneration of the intervertebral disc causing low back pain is a globally common health problem and a major cause of disability, according to the World Health Organisation. Spinal injury is also quite common and outcomes of surgical intervention are “disappointing”. Constructs fabricated by other research teams have their own limitations.
The biodisc fabricated by a team led by Prof. Biman Mandal from the Department of Biosciences and Bioengineering at IIT Guwahati closely resembles the natural spinal disc both in anatomical form and load-bearing property. The biodisc allowed the growth and proliferation of disc-like cells and extracellular matrix in studies carried out in the lab. It was found to be biocompatible when implanted in mice. The results were published in the journal Proceedings of the National Academy of Sciences (PNAS).
For animal trials, the team intends to implant the silk construct in animal models that have a degraded disk. It will first be implanted in rats to test the ability of the construct to integrate with the vertebrate. The functionality will then be tested in larger animals such as sheep and goats. “It will take at least a year before we can start animal trials,” Prof. Mandal says.
Preparing the construct
“The most challenging part was replicating the anatomical form of the disk (annulus fibrosus which is the the tough circular exterior of the intervertebral disc that surrounds the soft inner core). We used a novel approach to fabricate a cross-aligned multilamellar scaffold,” says Prof. Mandal. Human disc is marked by 20-25 cross-aligned layers where successive layers are at 30 degrees angle to its vertical axis but in alternate directions; this structure gives the disc the superior compressive strength.
To fabricate the lamellar scaffold, the researchers used a silicon polymer mould that has two hollow chambers divided by a copper metal plate. The silk solution was poured in one chamber and liquid nitrogen in the other. The low temperature (-196 degree C) of liquid nitrogen freezes the silk solution in one direction. “The water crystals grow like needles or channels in one direction. This gives directionality,” he says. When the structure is freeze dried, the frozen water evaporates leaving behind linear pores in the scaffold.
The scaffold was cut into tiny strips at 30 degree angle and wrapped around a mould in such a way that successive layers were cross-aligned at 30 degrees.
The construct had a load-bearing capacity (compressive strength) comparable with the human disk. Constructs seeded with cells taken from the disk of a pig or human mesenchymal stem cells (a type of stem cells) that became AF-like cells were able to grow and multiply within the structures. The cells were able to deposit normal extracellular matrix. “The ability of cells to grow, proliferate and deposit extracellular matrix is a validation of the construct’s ability to support cell growth,” says Bibhas Bhunia from IIT Kolkata and first author of the paper.
Seeding the construct
“We can either use patient’s own annulus fibrosus (AF) cells or use mesenchymal stem cells that have differentiated into AF-like cells to seed the construct before implantation,” says Bhunia.
The lamellar pores in the construct behave like a template for the cells to get aligned in a particular direction. “As a result, the cells will be forced to follow and retain the 30 degree criss-cross architecture similar to native human disc,” Prof. Mandal says.
When implanted in mice, the silk construct was not rejected even at the end of four weeks. “Macrophages were seen around the implant for a week after implantation but subsequently they clear out. This is proof that the construct is biocompatible,” he says.
The silk protein was tuned with secondary structure to slow down the rate of silk degradation. “It will take a few years for the silk-based construct to degrade. The construct will last till the native cells create their own intervertebral structure. If the construct degrades quickly then the structure may fail in its load-bearing capability,” he says.
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