Engineering the liver

By: Hadas Goshen and Prof. Yaacov Baruch, Director of the Liver Unit, Rambam Health Care Campus, and Dr. Dror Seliktar of the Biomedical Engineering Department, the Technion

The field of tissue engineering evolved from the ever-increasing need to supply tissues and organs for transplants. In order to regenerate damaged tissue that doesn't regenerate itself independently, either autologous stem cells or donor tissue cells are injected. These cells will integrate into the damaged organ and initiate a healing process. In order to increase the number of implanted cells, they are first "seeded" on a three-dimensional biological scaffold built of polymer. Like a skeleton, the scaffold provides a biomechanical support until the cells develop into functional tissue. Development and construction of these scaffolds is one of the research fields in which biomedical engineering scientists are involved, attempting to base the scaffolds on natural and cell-friendly materials.

The liver seems to be a promising candidate for treatment by tissue implantation. In contrast to some of the organs and tissues in our bodies, which are incapable of regeneration following damage, the liver has regenerative capacity – the capacity of renewed growth. For example, following resection of 70% of the liver volume, a rat liver will regenerate itself within one week, and a human liver will regenerate itself within two months. One of the advantages of implanting cells into the liver, instead of a whole liver transplant, stems from the possibility of promoting recovery of the diseased liver by reinforcement with healthy cells.

"When we attempt to treat hepatic insufficiency, the major problem is donation of a liver for transplantation," says Prof. Yaacov Baruch, Director of the Liver Unit at Rambam Healthcare Campus. "Although we can use a live donor for transplantation, because one hepatic lobe is sufficient and there is no need for the whole liver, the surgery is complicated, and is associated with complications for both the donor and the recipient."

The search for cells for implantation
One of the serious problems associated with transplants is rejection of the transplant, which is recognized as a foreign invader by the recipient's immune system, which attacks the transplant by means of the recipient's white blood cells. "The same problem exists in cell implantation, but to a lesser extent, due to the relatively small number of implanted cells," emphasizes Prof. Baruch. "In the worst case scenario, the immune system will succeed in its mission and destroy the cells, but without causing the damage associated with rejection of the whole organ."

"Implanted cells can correct genetic defects, which cause deficiency or formation of defective enzymes (proteins that participate in metabolic processes). For example, deficiency of an enzyme involved in the metabolism of bilirubin (the degradation product of hemoglobin, the substance responsible for the red color of the blood) leads to excessive accumulation of bilirubin in the blood, which may be harmful in neonates. The implanted cells, which are capable of remaining in the body for a year, are expected to correct this defect."

How can survival be improved?
Even after the appropriate cells have been chosen for implantation – the problems are not yet over; most of the cells (90%-95%) suffer from difficulties that prevent successful implantation, and do not survive. Some of them migrate out of the target organ, some of them either die by programmed cell death (apoptosis), or are recognized as a foreign tissue and destroyed by the immune system.

The final goal of the study is to achieve maximum cell survival, thus enabling them to proliferate and to create functional tissue.
"We must improve the conditions for implantation to increase efficiency," emphasizes Prof. Baruch. "We act to improve the survival percentage in order to achieve functioning of the cells immediately following implantation."

One of the challenges in tissue engineering is supply of nutrients and oxygen to the cells implanted in the tissue. This supply must reach the tissue immediately following implantation. Until blood vessel growth (angiogenesis) begins, there are only a few blood vessels in the implant environment, and the implant is separated from the nearest blood capillaries by several cellular layers.
 Therefore, it is important to provide growth factors promoting rapid blood vessel growth in the implant area during the implantation.

Building the perfect scaffold
In collaboration with Prof. Smadar Cohen of Ben Gurion University of the Negev, Beer Sheva, construction of a three-dimensional "alginate" (a substance extracted from brown seaweed) scaffold was examined. Tiny capsules (3 microns – 3 thousands of a millimeter – in diameter), releasing the growth factor Basic Fibroblast Growth Factor (bFGF) in a continuous and controlled manner, were inserted into the scaffold. It turned out that the continuous release of the growth factor resulted in the formation of mature blood capillaries in the tissue scaffold itself, thus promoting the viability and functioning of the implanted tissue.

The field of tissue engineering gave rise to the need for biological materials capable of forming a three-dimensional structure designed to provide support and life to the cells grown for implantation outside the body, both as a physical support and by signal transduction. Therefore, the ideal three-dimensional scaffold must combine structural elements with biological function. Most of the biomaterials currently used for tissue engineering provide a successful physical support, but fail to provide the environment necessary for maintaining cellular life processes.

Finding the correct balance between structure and function is still a challenge for the scientists. The ability to regulate the interaction between the cells and their environment, using a scaffold with growth-promoting nano-materials inserted in it, may become an advantage in tissue engineering.

Factors endangering the cells
Together with his colleagues, Prof. Baruch is currently involved in several research tracks simultaneously: "One track being examined is injection of hepatic cells into the spleen, enabling them to reach the liver via the bloodstream. The major risk for the cells transferred by the bloodstream is exposure to shear stress, which tears them into pieces. The polymers create a cage-like structure, which protects the cells from shear stress, and from attack by cells of the immune system. Another advantage of the polymers is related to the fact that they are composed of biological material, which is degraded upon completion of its mission. "We can control the rate of polymer degradation by manipulating its composition," emphasizes Prof. Baruch.

The research studies of Dr. Dror Seliktar of the Biomedical Engineering Department, the Technion, involve tissue engineering and regenerative medicine, with emphasis on biomaterials and biomechanics. Dr. Seliktar has developed a hydrogel-based three dimensional scaffold. This is a liquid gel that integrates the two properties essential for a biological scaffold – structure and function, by combining the two components into one piece. The material has been successfully examined in smooth muscle cells, cartilage tissue and cardiomyocytes culture. The implant cells are inserted into the liquid gel, and the timing of polymerization (conversion of the liquid substance into a viscous polymer) is controllable. The liquid is injected into a certain cavity inside the liver, followed by local polymerization in the liver using UV light, similar to the formation of a rigid dental filling.

"My job is to create a scaffold composed of a material that provides an environment enabling development of the implanted cells, and to engineer the properties essential for recovery inside the scaffold," says Dr. Seliktar. His scaffold is composed of fibrinogen – a protein that participates in blood coagulation. In contrast to natural fibrinogen, which is degraded within several weeks, Dr. Seliktar creates a scaffold that is more resistant to degradation, with a controllable degradation rate.

How can the degradation rate be controlled? The protein is cross-linked with a synthetic molecule – a biomedical polymer called polyethylene glycol (PEG). "A cell-friendly sponge-like scaffold is formed, with structural properties enabling attachment of the implant cells to the matrix, followed by proliferation and alignment into a structure typical of the organ. On the other hand, the scaffold is still exposed to the proteases (protein-degrading enzymes) plasmin and collagenase, degrading it at a rate controlled by the ratio between the synthetic and biological components.

The initial substance is a liquid, into which the hepatic cells are inserted. A soft flexible substance resembling gum candy is obtained. This substance is injected into the body, and solidified by UV light that is emitted by a "gun."

Towards an orthopedic application
Dr. Seliktar also emphasizes that liver research is still in its early stage. However, an advanced pre-clinical study is currently being carried out in collaboration with Dr. Eli Peled of Orthopedics Department B at Rambam. A company has been established for application of this same technology in the recovery of damaged cartilage: the natural fibrinogen cross-linked with a synthetic component is injected directly into the site of the injury. "Our technology is most suitable for the treatment of cartilage injuries incurred by sportsmen or soldiers, or due to accidents," emphasizes Dr. Seliktar. "But we don't have any good news for degenerative diseases, such as arthritis, because this problem cannot be solved by tissue replacement."

One of the reasons for the rapid progress towards an orthopedic application is insertion of the network backbone only, without cells, thus avoiding the need to grow blood vessels required to provide nutrients to the cells, and the problems of rejection. Additional feasibility studies are being carried out to examine the same biological scaffold with other cell types – stem cells, cardiomyocytes, smooth muscle cells and neurons, while searching for the compatibility unique to each tissue. "The mechanical properties required for replacing cartilage tissue are entirely different from those required for replacing cardiac tissue damaged by hypoxia due to myocardial infarction, and from those required for replacing hepatic tissue damaged by liver cirrhosis," emphasizes Dr. Seliktar.

How do we detect that an implant is functioning efficiently? In hepatic cells, we detect alignment and metabolic activity typical of liver tissue; in cardiomyocytes, we detect spontaneous contraction, and in neurons, we detect axons protruding through the gel, following creation of the desired path by laser cauterization.