Engineering Organs Heart and Esophagus

Jan 05, 03:42 AM

The potential for tissue engineering to transform the practice of medicine is enormous. Chemical engineers will play important roles in this revolution. The conceptual ideas underlying tissue engineering evolved in the 1970s and were shaped into a more modern form in the late 1980s and early 1990s (1). The literature on this subject is extensive, and includes many general overviews (2-4). Tissue engineering has received good research funding, the attention of researchers excited by the intellectual frontiers, and much media interest. Clinical successes have been achieved (5), and humans are being fitted with lab-grown organs and replacement tissues. New companies are entrepreneurially exploring this frontier.

The opportunities for chemical engineers to contribute to this growing enterprise are numerous. This article focuses on how we might create two muscular tissues - heart and esophagus. It also addresses issues relevant to the tissue engineering field as a whole, as well as to the specific contributions that chemical engineers can make to this emerging frontier.

My own definition of tissue engineering is concise, yet it expresses the essence of the endeavor: "Tissue engineering exploits ideas and methodologies from biology, medicine, materials science and engineering to grow living tissues and organs for repair or replacement of damaged and dis eased tissues and organs."

A National Institutes of Health (NIH) website reports that the U.S. spends $400 billion each year on tissue loss or end-stage organ failure. The synthetic materials that go into medical devices (a $150-billion industry) do save lives and improve the quality of life for millions. However, medical devices rarely work as well as the healthy, natural body part and are associated with numerous complications. To swap out a defective organ or a tissue for a living, functioning replacement is the essence of the tissue engineering revolution that is coming to medicine.

Along with organ and tissue repair, many other exciting outcomes could stem from a mature tissue engineering endeavor, such as:

* tissue and organ repair and replacement

* cosmetic enhancement/alteration

* living devices to treat diseases of the kidney and liver, and Parkinson's disease

* food production (muscle tissue)

* biosensors (living tissues for sensing)

* implantable drug-delivery devices based on living tissue

* production of biological molecules from living tissues

* studies of human disease and pathologies

* drug toxicology testing

* basic studies on tissue formation and biological development.

Addressing key issues

Before a tissue engineering enterprise can translate those possibilities into realities that benefit people and generate economic value, we must come to grips with some profound issues.

Angiogenesis/innervation. Angiogenesis is the formation of new blood vessels to provide oxygen and nutrition to the tissue and remove wastes. Innervation is the ingrowth of nerve tissue to electrically connect the new tissue or organ to the body. Angiogenesis and innervation seem to be biologically connected, and new nerve sprouts follow the growth of new capillary-dimension blood vessels.

From a mass-transport standpoint, a cell must be within approximately 300 um of a capillary in order for the latter to provide sufficient oxygen and nutrients. Some tissues, such as heart, have exceptionally high oxygen needs and require even closer blood vessel proximity. Other tissues, such as cartilage, have very low demand for oxygen.

Numerous approaches to enhancing angiogenesis have appeared in the literature and some of them will be discussed here. Concepts such as synthetic blood-vessel networks to carry oxygen to developing tissue have also been proposed.

Inflammation/healing. Inflammation is defined in medical textbooks as the normal process of injury repair in vascularized tissue. Upon injury (i.e., implantation of a biomaterial), macrophages are recruited to the damage site, where they destroy or engulf foreign material (including bacteria) and release cytokines that brings other cell types to the wound to implement the repair.

The repair process, which is influenced by many factors, can be regenerative or it can lead to scarring (fibrosis). If regenerative healing can be encouraged and fibrotic healing minimized, this will positively contribute to the regrowth and integration of engineered tissues.

Recently, macrophage phenotypes have been identified that may make precision control of healing and regeneration possible. An Ml phenotype is associated with phagocytitic and fibrotic outcomes, while an M2 phenotype should predict angiogenesis and regeneration (6).

Surgical integration. Tissue-engineered constructs are typically started in an in vitro bioreactor. After a period of time for cell growth, and possibly with mechanical conditioning, they must be removed from the bioreactor and surgically placed in vivo. This is a critical period for an engineered tissue or organ, in that blood vessels and nerves must rapidly infiltrate the implant, and the construct must be mechanically anchored and positioned in a meaningful anatomical location.

Biomechanics. Some tissues, such as cartilage, are relatively easy to grow. However, lab-grown cartilage does not have the superb compressive properties of articular cartilage.

The goal should be to create new tissues with mechanical properties that closely match the part they replace. In blood vessel tissue engineering, it has been demonstrated that exposing the developing blood vessel to pulsatile stresses in the bioreactor has vastly improved burst strength - comparable to that of anatomically normal arteries (7). This process of exposing developing tissues to cyclic mechanical loads is often called conditioning.

Cell sources. Potential cell sources include autologous cells (taken from the patient who will receive the engineered tissue or organ), allogeneic cells (derived from cells obtained from one or more humans and used widely in the human population), xenogeneic (derived from animals and used in humans), adult stem cells (either autologous or allogeneic), and human embryonic stem cells (an allogeneic source).

Autologous cells will generally integrate well into the individual who donated those cells. However, "off-theshelf replacement parts are not practical from autologous cells, and the utility of these cells is related to how long the patient can wait for the new tissue or organ grown from his or her own cells. Allogenic and xenogeneic cells can trigger immunologic responses, although some cell lines, such as fibroblasts, seem minimally immunogenic. Adult stem cells have much potential, but at this time they cannot be differentiated to all cell types. Embryonic stem cells can make all tissues and organs (once we learn to harness them), but there are potential immunologic problems, as well as political and ethical concerns.

Manufacturing and bioreactors. A new field of tissue and organ manufacture is evolving at this time. The development of bioreactors for the production of engineered tissues is an endeavor where chemical engineers can make significant contributions. The challenges involve the need for extreme sterile environments, mass transport concerns, complex mechanical designs (where mechanically conditioning the tissue is important), and regulatory issues.

Market realities and business models. When two prominent, pioneering tissue-engineering companies went bankrupt a few years ago, some expressed concern for the future of the field. However, the products developed by both of these companies are back on the market. Furthermore, many new tissue-engineering companies have been launched, and some of them are receiving substantial funding.

Two business models dominate the field:

* manufacture a tissue-engineered product, freeze it, and sell it as an off-the-shelf part

* take a biopsy of a patient's cells, send them to a central facility, grow them into a tissue or organ, and return that tissue- engineered construct to the physician for implantation in the patient.

Only when these considerations are seriously addressed and the challenges met, will tissue engineering be on a fast track to repair people and capture that $400-billion market.

Repairing a damaged heart

Heart failure and cardiovascular diseases are the number one killers in the U.S. and in most of the developed world. In the U.S., almost half a million people die each year from coronary heart disease.

Acute myocardial infarction (AMI) leads to damage to a portion of the heart muscle. The heart has essentially no capacity to heal this damage. The injured site heals as scar tissue, the heart wall thins, the functionality of the heart is seriously impaired, and congestive heart failure often develops - leading to a steady downhill spiral for the patient. If we could replace, repair or regrow the damaged portion of the heart muscle, this steady progression to congestive heart failure might be prevented.

There are four central challenges to replace or repair heart muscle damaged from AMI. Because the predominant muscle cell in the heart, the cardiomyocyte, has essentially no proliferative capacity, we must generate sufficient numbers of these cells to repair hearts. To meet the typically high oxygen demand of cardiomyocytes, we must rapidly bring oxygen to this growing tissue. The heart has an elegant electrical interconnectivity that must be restored for true repair to have occurred. Finally, heart muscle is comprised of mechanically tough, aligned cell units that must be recapitulated in the engineered heart tissue. To address these challenges, the BioEngineered Allogenic Tissue (BEAT) program at the Univ. of Washington, funded by an NTH Bioengineering Research Partnership (BRP) grant, has adopted a systems approach to engineer heart muscle. Coordinated, multidisciplinary teams are working toward the common goal of developing an implantable heart-muscle replacement. Figure 1 suggests how the output of the various teams will coalesce to heart muscle.

Figure 2 illustrates some of the complexity of heart muscle. It features: aligned cardiomyocte cells that are mechanically and electrically interconnected by structures referred to as intercalated disks; a blood vessel within one cell of every cell; and a tough extracellular matrix. This complex structure must be reproduced in the tissue-engineered product.

Another view of heart muscle is presented in the colorized scanning electron micrograph in Figure 3. This shows, on a real specimen of heart muscle, the essential close apposition between blood vessels (orange) and heart muscle cells, and the Purkinje cells (green) that are responsible for the electrical conduction and communication in heart muscle.

Space limitations preclude discussion of all the technologies from the eight BEAT research groups now working to emulate the biological structure in Figures 2 and 3. However, a few key technologies will be highlighted.

A porous, polymeric scaffold made by a templating process is used to orient cardiomyocyte cells into parallel bundles, and also to enhance angiogenesis around the implant seeded with the cells. This angiogenic enhancement is based on observations in the biomaterials field dating back to the 1960s reporting that some implanted porous materials led to enhanced blood vessel formation in the vicinity of the material. A body of published literature that followed up on the early vascularization observations supported the concept that certain pore sizes were pro-angiogenic (8, 9). The problem was that all of the implants in those studies had a broad range of pore sizes. If small and large pores were antiangiogenic, while some intermediate-sized pores were enhancing the blood vessel formation, the influence of the small and large pores might have been working against the desired angiogenesis induced by the correct pore size.

We endeavored to make porous, interconnected structures with all pores of one size, allowing us to systematically explore pore size and its effect on healing (10). These materials were made by: sieving microspheres to get tightly controlled size fractions; packing these spheres into crystallinelike arrays; gently sintering the spheres so they fuse at contact points; surrounding the spheres with monomer and a crosslinking agent; polymerizing the monomer; and solventextracting the spheres from the crosslinked polymer (sphere templating). Figure 4 is an image of one of these materials.

The materials were characterized with permeation measurements and image analysis (11). Implantation studies demonstrated that when the pore size was approximately 35 um dia., profoundly enhanced angiogenesis occurred in the vicinity of the implant and within its pore structure (10).

To create spaces specifically for cardiomyocytes, Derek Mortisen in our laboratory used parallel fiber bundles dispersed within the sphere templates to create a scaffold with long parallel channels for cardiac cells and a porous structure optimized for angiogenesis. Initial subcutaneous implantation experiments in a mouse showed that blood vessels grew into the construct and that seeded cardiomyocyte cells survived well.

Thus, we could potentially address die issues of aligning cardiomyocytes (in the tubular channels) and rapidly inducing blood vessel formation to provide nutrients for the cells.

We still had to tackle the issue of where these cells would come from, since cardiac cells are non-proliferating. Charles Murry, Todd McDevitt and Michael LaRamme, partners in the BRP program, developed methods tiiat differentiate proliferating cardiomyocytes from human embryonic stem cells (the approved line H7) (12). Other BEAT partners are researching: electrical conduction in the heart muscle; furtiier enhancing angiogenesis witii endotiielial cells; surgical implantation; imaging; slowing scar formation and heart muscle damage at the infarction site; biomechanical conditioning; and delivery of cytokines and genes. We aim to have implantable heart muscle in three years.

Engineering a new esophagus

Esophageal injury and cancer can lead to surgical removal of the esophagus. After tiiis procedure, long-term survival rates are poor. An American Cancer Society estimate predicts that 15,560 new esophageal cancer cases will be diagnosed in the U.S. in 2007. Cancer of the esophagus is seen much more frequentiy in otiier countries such as Iran, northern China, India, and soutiiern Africa, where occurrence rates are 10 to 100 times higher than in the U.S.

With funding from the A*Star Agency in Singapore, we launched a project to explore whether we could tissue-engineer esophagus as a surgical replacement for an excised esophagus. The schematic diagram in Figure 5 represents die challenge, and allows us to strategically plan an approach to create an esophagus.

After developing a primary cell line of esophageal epithelium, we explored methods to appropriately differentiate them into a normal multilayer structure with columnar basal cells and increasingly stratified cells in the upper layers. Rat esophageal epithelial cells were seeded onto a variety of porous scaffold materials, including poly(lactic acid), lactic-glycolic copolymers, polycaprolactone, and decellularized skin (Alloderm) (13).

On the decellularized skin, cell stratification was excellent. In later experiments, these cells were seeded onto decellularized rat esophagus, and again stratification was excellent. This suggested that on a substrate sending the correct signals to the cells, appropriate differentiation would occur.

Consistent with this result, Kerm Sin Chian at the Nanyang Technological Univ. developed technology to decellularize pig esophagus. Since pigs are anatomically about the same size as humans, reasonably high on the phylogenetic tree, and already used to supply materials for human medical devices, such technology might ultimately lead to a clinically useful esophagus.

Additional technologies that might be coupled with decellularized pig esophagus include: surface immobilization of specific Notch ligands to further enhance cell differentiation (14); deposition of oriented, electrospun meshes (radially or longitudinally) for muscle cells; bioreactor development; and gene delivery.

Looking ahead

Tissue engineering is here today -living implants are going into humans. However, the science of tissue engineering is just getting off the ground. Comparing cells seeded randomly into a porous scaffold with a real tissue, it is apparent that there is a large gap, intellectual and technological, that must be bridged to truly grow tissues that emulate the originals. In fact, at this time, we depend on some inherent organizational capacity of biology to take our primitive cell-seeded scaffolds all the way to an analog of real tissue. We have little understanding of this process.

Development of a systematic science associated with cell attachment, proliferation and differentiation, especially in three- dimensional scaffolds and with multiple cell types, will bring tissue engineering from an empirical and inventive technology to a refined engineering practice. Studies from developmental biology will help us to understand how tissues develop naturally and how signaling proteins might lead to engineering control of tissue reconstruction. Advances in the understanding of inflammation and angiogenesis will permit a better understanding of integration with the body of an engineered tissue construct. Manufacturing technology will need to be evolved to permit us to make, for example, 100,000- plus esophagi that might be needed in replacements worldwide.

Finally, business models will create a profitable tissueengineering enterprise that will encourage industry investment, leading to further advances and tissue/organ replacement solutions that are not even envisioned today.

Literature Cited

1. Langer R., and J. P. Vacant!, 'Tissue Engineering," Science, 260, pp. 920-926(1993).

2. Palsson, B. O., and S. N. Bhatia, 'Tissue Engineering," Prentice Hall, Upper Saddle River, NJ (2003).

3. Atala, A., et aL, "Principles of Regenerative Medicine," Academic Press, Burlington, MA (2007).

4. Lanza, R., et aL, "Principles of Tissue Engineering," Academic Press, Burlington, MA (2007).

5. Atala, A., et aL, "Tissue-Engineered Autologous Bladders for Patients Needing Cystoplasty," Lancet, 367, pp. 1241-1246 (2006).

6. Mantovani, A., "Macrophage Diversity and Polarization: in vivo Veritas," Blood, 108 (2), pp. 408-409 (2006).

7. Niklason, L. E., et aL, "Functional Arteries Grown in vitro," Science, 284, pp. 489-493 (1999).

8. B ranker, J. H., et aL, "Neovascularization of Synthetic Membranes Directed by Membrane Microarchitecture," J. Biomed. Mater. Res., 29, pp. 1517-1524(1995).

9. Sharkawy, A.A., et aL, "Engineering the Tissue which Encapsulates Subcutaneous Implants. I. Diffusion Properties," J Biomed Mater. Res., 37, pp. 401-412 (1997).

10. Marshall A. J., et aL, "Biomaterials with Tightly Controlled Pore Size that Promote Vascular In-Growth." ACS Polymer Preprints, AS (2). pp. 100-101 (2004).

11. Marshall, A., and B. D. Ratner, "Quantitative Characterization of Sphere-Templated Porous Biomaterials," AlChE Journal, 51 (4), pp. 1221-1232(2005). 12. McDevitt, T.C., et aL, "Proliferation of Cardiomyocytes Derived from Human Embryonic Stem Cells is Mediated via the IGF/PI 3-kinase/Akt Signaling Pathway," Journal of Molecular and Cellular Cardiology. 39, pp. 865-873 (2005).

13. Beckstead, B.L., et aL, "Esophageal Epithelial Cell Interaction with Synthetic and Natural Scaffolds for Tissue Engineering," Biomaterials, 26, pp. 62176228(2005).

14. Beckstead B. L., et aL, "Mimicking Cell-Cell Interactions at the Biomaterial-Cell Interface for Control of Stem Cell Differentiation," J. Biomed Mater. Research A, 79A (1), pp. 94-103 (2006).

BUDDY D. RATNER

UNIV. OF WASHINGTON

ENGINEERED BIOMATERIALS (UWEB)

Acknowledgements

Generous support was received from grants NSF EEC-9529161 (UWEB Engineering Research Center), NIH R24 HL64387 (BEAT BRP), and the A*Star-funded Singapore-Univ. of Washington Alliance (SUWA).

BUDDY D. RATNER is professor of bioengineering and chemical engineering at the Univ. of Washington and, since 1996 has served as director of University of Washington Engineered Biomaterials (UWEB) (Dept. of Bioengineering, Univ. of Washington, Box 355061, William H. Foege Building, Room N330J, Seattle, WA 98195; Phone: (206) 685- 1005; Fax: (206) 616-9763; E-mail: ratner@uweb.engr.washington.edu; Website: www.uweb.engr.washington.edu). From 1985 to 1996, he directed the NIH-funded National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO). His current research interests include biomaterials, tissue engineering, polymers, biocompatibility, surface analysis of organic materials, self- assembly, nanobiotechnology and RF-plasma thin film deposition. He holds a PhD in polymer chemistry from the Polytechnic Institute of Brooklyn.

Copyright American Institute of Chemical Engineers Dec 2007

(c) 2007 Chemical Engineering Progress. Provided by ProQuest Information and Learning. All rights Reserved. Engineering Organs Heart and Esophagus
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