Progress in Tissue Engineering - Suitable Cells

In most situations, building an implantable tissue from a patient’s own cells would be ideal because they are compatible with that person’s immune system. Realistically, such implants might also face fewer regulatory hurdles because the material is derived from the patient’s own body. The ability of normal cells to multiply in culture is limited, however, making it difficult to generate sufficient tissue for an implant. Socalled adult stem cells from the patient’s body or from a donor are somewhat more prolific, and they can be isolated from many sources, including blood, bone, muscle, blood vessels, skin, hair follicles, intestine, brain and liver.

Adult stem cells—which occur in adult tissues and are able to give rise to a variety of cell types characteristic of their native tissue—are difficult to identify, however, because they do not look very different from regular cells. Scientists therefore must look for distinctive surface proteins that serve as molecular markers to flag stem cells. The identification of additional markers would make it considerably easier to work with adult stem cells in tissue-engineering applications. Fortunately, over the past few years a number of major advances have been made, including development of novel methods of isolating the cells and inducing them to proliferate and to differentiate into various tissue types in culture.

Notably, Christopher Chen and Dennis Discher, both at the University of Pennsylvania, have demonstrated that mesenchymal stem cells, which are typically derived from muscle, bone or fat, will respond to mechanical cues from their surroundings. They have been shown to endifferentiate into the tissue that most closely resembles the stiffness of the substrate material on which they are growing. Other researchers have also shown that chemical signals from the substrate and surrounding environment are important for directing the differentiation of adult stem cells into one tissue type or another. Scientists disagree, though, about whether adult stem cells are able to give rise to cells outside their own tissue family—for instance, whether a mesenchymal stem cell could generate liver cells.

In contrast to adult stem cells, embryonic stem (ES) cells are easy to expand in culture and can differentiate into all the cell types of the human body. Langer, along with Shulamit Levenberg of the Technion-Israel Institute of Technology in Haifa and her colleagues, has demonstrated that ES cells can even be made to differentiate into a desired tissue type right on tissue-engineering scaffolds. This capability suggests the potential to make 3-D tissues on scaffolds directly from differentiating ES cells. These cells do present various challenges, however.

Directing the uniform differentiation of ES cells into the desired cell types is still quite difficult. In attempts to mimic the complex natural microenvironment of ES cells and to optimize their differentiation, investigators are testing many conditions simultaneously to find the right combination of cues from different materials and matrix chemicals. They are also screening various small molecules as well as signaling proteins to identify factors that control “stemness”—the cells’ ability to give rise to differentiated progeny while remaining undifferentiated themselves, ready to produce more new cells as needed.

Those insights could also be applied to producing cells with the capabilities of embryonic cells but fewer of the drawbacks. Beyond the difficulties just outlined, scientists are still unable to predict the behavior of transplanted stem cells in patients. Undifferentiated ES cells can form tumors, for instance, creating a risk of cancer if the cells are not all successfully differentiated before transplantation. In addition, researchers have been making efforts to address the ethical issues associated with deriving ES cells from human embryos by exploring approaches to producing ES-like cells from nonembryonic sources.

In the past couple of years remarkable progress has been made in producing ES-like cells from regular adult body tissue, such as skin cells. These altered cells, known as induced pluripotent stem (iPS) cells, are emerging as an exciting alternative to ES cells as a renewable resource for tissue engineering. In 2007 Shiro Yamanaka, then at Kyoto University, and James A. Thomson of the University of Wisconsin–Madison first showed that cells of adult tissue can be transformed to a primitive iPS state by reactivating a number of genetic pathways that are believed to underlie stemness. Reintroducing as few as four master regulatory genes into adult skin cells, for instance, caused the cells to revert to a primitive embryonic cell type. The early experiments used a virus to insert those genes into the cells, a technique that would be too dangerous to use in tissues destined for patients. More recent research has shown that a safer nonviral technique can be adapted to activate the same repertoire of stemness genes and even that activation of just a single regulatory gene may be sufficient. The rapid progress in this area has tissue engineers hopeful that soon a patient’s own cells, endowed with ES cell capabilities, could become the ideal material for building tissue constructs. And even as we experiment with these different cell types, tissue engineers are also refining our building methods.

Source of Information : Scientific American(2009-05)

Progress in Tissue Engineering - Delivering Life’s Blood

One reason that tissues such as skin and cartilage were among the first to be ready for human testing is that they do not require extensive internal vasculature. But most tissues do, and the difficulty of providing a blood supply has always limited the size of engineered tissues. Consequently, many scientists are focusing on designing blood vessels and incorporating them in engineered tissues. Any tissue that is more than a few 100 microns thick needs a vascular system because every cell in a tissue needs to be close enough to capillaries to absorb the oxygen and nutrients that diffuse constantly out of those tiny vessels. When deprived of these fuels, cells quickly become irreparably damaged. In the past few years a number of new approaches to building blood vessels—both outside tissues and within them—have been devised. Many techniques rely on an improved understanding of the environmental needs of endothelial cells (which form capillaries and line larger vessels), as well as an advanced ability to sculpt materials at extremely small scales. For example, when endothelial cells are laid on a bed of
scaffolding material whose surface is patterned with nanoscale grooves—1,000th the diameter of a human hair—they are encouraged to form a network of capillarylike tubes. The grooves mimic the texture of body tissues that endothelial cells rest against while forming natural blood vessels, thus providing an important environmental signal.

Microfabrication, the set of techniques used to etch microelectronics chips for computers and mobile phones, has also been employed to make capillary networks. Vacanti, with Jeffrey T. Borenstein of the Draper Laboratory in Cambridge, Mass., has generated arrays of microchannels to mimic tissue capillary networks directly within degradable polymer scaffolds, for instance. Inside these channels, endothelial cells can be cultured to form blood vessels while also acting as a natural barrier that minimizes the fouling effect of blood on the scaffold materials. An alternative is to use a membrane filter to separate the blood-carrying channels from the functional cells in a tissue construct. Another method for keeping cells and blood separate but close enough to exchange a variety of molecules is to suspend them within hydrogels, which are gelatinlike materials made from hydrated networks of polymers. Hydrogels chemically resemble the natural matrix that surrounds all cells within tissues. The functional cells can be encapsulated inside the material, and channels running through the gel can be lined with endothelial cells to engineer tissue like structures with a protovasculature.

Research from the laboratories of Laura Niklason of Yale University and Langer has shown that larger blood vessels can be generated by exposing scaffolds seeded with smooth muscle cells and endothelial cells to pulsating conditions inside a bioreactor. Arteries made in this environment, which is designed to simulate the flow of blood through vessels in the body, are mechanically robust and remain functional after being transplanted into animals. In addition to enabling tissue engineers to incorporate such vessels into larger constructs, the engineered tubes by themselves may provide grafts for bypass surgery in patients with atherosclerosis.

Although the ability to engineer capillarylike structures and larger blood vessels outside the body is a significant breakthrough, a working engineered tissue implant will have to connect quickly with the recipient’s own blood supply if the construct is to survive. Coaxing the body to form new vasculature is therefore an equally important aspect of this work. David Mooney of Harvard University, for example, has demonstrated that the controlled release of chemical growth factors from polymeric beads or from scaffold material itself can promote the formation of blood vessels that penetrate implanted tissue constructs.

Pervasis Therapeutics, with which Langer and Vacanti are affiliated, is conducting advanced clinical trials in which a variation of this principle is applied to healing a vascular injury. A three-dimensional scaffold containing smooth muscle and endothelial cells is transplanted adjacent to the site of the injury to provide growthstimulating signals and to promote natural rebuilding of the damaged blood vessel.

Despite these advances, a number of challenges still remain in making large vascularized tissues and vascular grafts, and scientists have not yet completely solved this problem. New blood vessels grow and penetrate an implanted tissue construct slowly, causing many of the construct’s cells to die for lack of a blood supply immediately after implantation. For this reason, tissue-engineering approaches that include a vascular system prefabricated within the tissue construct are very likely to be necessary for large transplants. Such prefabricated vessels may also be combined with controlled release of blood vessel–recruiting growth factors to induce further growth of the construct’s vessels. Because integrating the engineered vasculatures and those of the host is also critical, researchers need a better understanding of the cross talk between the host tissue cells and implanted cells to foster their connection. This need to decipher more of the signals that cells exchange with one another and with their environments also extends to other aspects of building a successful tissue implant, such as selecting the best biological raw materials.

Source of Information : Scientific American(2009-05)