One of the approaches being pursued in hopes of alleviating the imbalance between organ donors and recipients in need of a transplant is the engineering of artificial organs, but artificial tissue also could be used to repair damaged organs so that a transplant might be avoided altogether.
Researchers from Harvard Medical School (Boston) and the Massachusetts Institute of Technology (MIT; Cambridge, Massachusetts) reported in the Dec. 28, 2004, issue of the Proceedings of the National Academy of Sciences (PNAS) on tissue engineering to create functional heart tissue.
The core feature of heart muscle is its ability to beat synchronously in response to electrical stimulation. Heart tissue is thus characterized by cells connected to enable the free flow of electrical impulses from cell to cell (the so-called "gap junctions") and a contractile apparatus. To date, there has been limited success in inducing cultured heart cells to differentiate into mature, functional tissue that shows that ability. Physical therapy for cells, in the form of mechanical stretching, has been shown to improve matters to some degree. But the resulting tissue, while it shows improved ability to contract, still lacks several structural hallmarks of mature heart tissue.
Given that electrical activity is a critical feature of heart muscle function, the authors of the PNAS study decided to apply electrical stimulation to cells isolated from neonatal rat hearts during the culturing process. "We expected some improvement," said Gordana Vunjak-Novakovic, principal research scientist at MIT and senior author of the study. "What was unexpected was the extent of the improvement, and how critical electrical signaling really is." Functionally, she described the tissue as "a patch for a broken heart."
The scientists isolated neonatal rat heart cells, transferred them onto scaffolds, allowed them varying amounts of time to recover from the harvesting procedure and attach, and then stimulated them electrically for five days. Electrical stimulation led to improvements in the molecular, structural and functional properties of the stimulated cells. On the molecular level, there was an increase in proteins necessary for development of gap junctions and a contractile apparatus. Structurally, the cells resembled mature heart cells in several respects, including their alignment with the electrical field, elongated shape and the presence of structural features of muscle cells that enable their contraction. Functionally, the cells showed evidence of electrical coupling (i.e., working gap junctions), and after eight days of stimulation, their ability to contract was about seven times that of non-stimulated controls.
The researchers also treated cultures with drugs designed to interfere with spreading of the electrical stimulation, contraction of the cells in response to the stimulation or the coupling between the two. When contraction was blocked, the cells developed gap junctions, but the development of the contractile apparatus was impaired, reducing but not eliminating contractions even after removal of the drug. Blocking either the spread of electrical stimulation through gap junctions or the downstream effects of electrical stimulation and contraction had more serious effects. Cells remained morphologically immature, and electrical stimulation did not lead to organized contractions even after removal of the drugs.
Vunjak-Novakovic and her colleagues also found that electrical stimulation needs to be timed carefully to coincide with the right level of cellular maturation. During the harvesting of neonatal heart cells, the cells are separated from each other. Before they can benefit from electrical stimulation, cells need to reconnect by laying down new gap junctions, as well as a contractile apparatus, in a period of protein synthesis. "If you stimulate during protein synthesis, you just confuse the cells," she said. The "confused" cells neither differentiate properly nor develop the ability to contract as much as cells that are first allowed a few days in preculture to assemble the necessary proteins. Conversely, waiting too long leads to declining amounts of those proteins, also reducing the effectiveness of electrical stimulation. Vunjak-Novakovic's group also is working with other cell types, and while the exact timing is different, the basic existence of a critical period is the same in the types they have looked at so far.
Asked where her work fits into the spectrum of research applicability, Vunjak-Novakovic cautioned that it was only "the beginning" of the road to the clinic. For any clinical benefit to be realized, at least two things need to happen. First, benefits of the approach need to be demonstrated in vivo: "We need to patch hearts [with the tissue], and show that it integrates and leads to recovery of function," she said.
The researchers also need to show that their approach works with human cells. Experiments with a variety of cell types, including human embryonic stem cells, are ongoing.
Vunjak-Novakovic described a constant interplay of basic scientific and clinical findings and advances. In the biomimetic approach, "we try to mimic normal conditions, but really, very little is known about those conditions. So we build tissue and try to mimic conditions that will allow it to grow. And along the road, we learn things that will allow us to build a little bit better tissue next time."