IIT Guwahati researchers have created a bioartificial liver model grown within a 3D silk scaffold. The scaffold made by mixing mulberry and non-mulberry silk fibroins showed enhanced liver-specific functions. When implanted in animals, the scaffold was found to be biocompatible. The researchers are now validating the scaffold with primary human liver cells and human stem cell-derived liver cells.
After successfully creating an implantable bioartificial pancreas model grown within a 3D silk scaffold, researchers at the Indian Institute of Technology (IIT) Guwahati have taken the first step in creating a bioengineered liver model that might help patients with liver problems.
A team led by Prof. Biman Mandal from the Department of Biosciences and Bioengineering at IIT Guwahati has successfully created a bioartificial liver model grown within a 3D silk scaffold. The silk scaffold is capable of supporting the growth and distribution, and sustaining the functionality of liver cells. While liver cell lines were used for optimising the scaffolds, testing was done using liver cells taken from neonatal rats.
In studies carried out in petri dishes in the lab, the researchers found that the scaffold made from mixing mulberry and non-mulberry silk fibroins showed enhanced liver-specific functions.
Enhanced liver functions
“The blend scaffold showed enhanced liver-specific functions such as increased albumin production and urea synthesis, and enhanced detoxification. There was characteristic bidirectional diffusion of nutrients into liver clusters and secreted bio-products from the clusters,” says Guru Janani from the Department of Biosciences and Bioengineering at IIT Guwahati and first author of a paper published in the journal Acta Biomaterialia. “The extracellular matrix (ECM) production was higher; the higher the ECM production the faster is the liver regeneration.”
Besides being metabolically active, gene expression studies and functionality tests demonstrated that optimal-sized (less than 100 micrometre) liver clusters formed on the scaffold maintained liver-specific functions for 21 days compared with scaffolds made of mulberry and non-mulberry silk fibroins.
Tests carried out for four weeks on animal models showed that the scaffolds were both hemocompatible and immunocompatible. The scaffolds were subcutaneously implanted to test for biocompatibility.
Once successful tested in animal models and humans, the scaffolds can potentially be used for regenerating bioartificial liver for human transplantation. The 3D platforms may also come handy for high-throughput drug testing by pharmaceutical companies. Currently, animals are used for carrying out such tests and non-animal model made of human liver cells will have distinct advantages and also reduce the number of animals sacrificed. They can also be used for creating cirrhosis disease model for drug development.
Blend of two silk varieties
The researchers tested scaffolds made of mulberry and non-mulberry silk fibroins and a blend of the two silk varieties in petri dishes in the lab and in animal models. The blend of the two silk varieties mixed in equal proportions was found to be superior on several counts to the other two varieties used individually. The blend showed better ability to retain stable primary liver clusters that exhibited optimal size, higher proliferation leading to high cell density, prolonged cell survival and better functionality, paving the way for its use in liver tissue engineering at a later stage.
Unlike the mulberry silk, the non-mulberry silk has cell binding sites (RGD) in them that help in better cell attachment and proliferation. Also, the water-loving (hydrophilic) nature of non-mulberry silk allows for better cell attachment. Being a structural protein, the mulberry silk fibroins render strength to the 3D scaffold. The two silk varieties performed equally well on most other parameters. So a blend of the two varieties offered the best combination.
“People were using synthetic RGD to mulberry silk to improve cell attachment and growth. We used the non-mulberry silk instead, which naturally has the cell binding site property, to produce the blend,” Prof. Mandal says.
“Individually, the silk varieties have their own limitations as a liver scaffold. We knew from the morphology of liver that we need a scaffold that offers the best of the two. So blending the two varieties was a planned experiment. We had blended it for a different purpose but found it to be suitable for liver scaffold,” he recalls.
The team is collaborating with Prof. Stephen Badylak, Deputy Director of McGowan Institute for Regenerative Medicine, U.S. to undertake animal trials in liver cirrhosis rodents for studying the potential of silk matrices in aiding the liver regeneration. “We now validating with primary human liver cells and human stem cell-derived liver cells on the blend scaffold to make it clinically more viable,” Prof. Mandal says.