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Our Energy Future
June 1, 2002 Volume 32 Issue 2

Integration of Molecular and Macroscopic Scales in Tissue Engineering

Saturday, June 1, 2002

Remarks delivered by Linda G. Griffith at the National Academy of Engineering 1999 National Meeting, Arnold and Mabel Beckman Center, Irving CA.

The field of tissue engineering has emerged over the past decade, driven by a diverse range of clinical needs for replacement of diseased or damaged tissue and for delivering genetically engineered cells to patients (Langer and Vacanti, 1993). Although many artificial prosthetic devices are available to replace connective tissues such as joints, heart valves, blood vessels, and breasts, few synthetic devices are able to perform adequately over the lifetime of a patient, and devices vary greatly in their abilities to completely replace all of the functions of native tissue. No long-term replacements are available for some connective tissues, including heart, small-diameter blood vessels, and skin. Clinical needs are even more dire for such organs as livers, which can now be replaced only by organ transplantation.

The goal of tissue engineers is to meet these clinical needs by creating living three-dimensional tissues and organs using cells obtained from readily available sources such as biopsy of a patient's own (autologous) tissue or foreign tissue that would be discarded after surgical procedures such as circumcision. In every case the approach is to break the donor material down to the level of individual cells and then coax the isolated cells into forming a tissue structure of the appropriate size and/or shape by using a physical "scaffold" to organize cells on a macrosopic scale and providing molecular cues to stimulate appropriate cell growth, migration, and differentiation.

The scientific foundation for this new field lies in molecular cell biology, which has enabled the identification of hundreds of molecules involved in controlling cellular behavior from the external environment and development of new methods for assessing cellular responses. The engineering challenges in building on this science to meet clinical needs are at least twofold. The first challenge is to understand quantitatively how cells respond to these molecular signals and integrate multiple inputs to generate a given response - a significant challenge considering that the number of molecules identified so far represents only a fraction of the total that exist in the normal tissue environment. The second challenge is then to arrange cells in an appropriate three-dimensional configuration and present molecular signals in an appropriate spatial and temporal fashion so that the individual cells will grow and form the desired tissue structures - and do so in a way that can be carried out reproducibly, economically, and on a large scale. The specific examples described here are derived from work at the Massachusetts Institute of Technology and represent applications ranging from near term (less than one year) to very long term (more than 10 years).

Near Term - Connective Tissues
Tissues that have received the greatest attention from the commercial side - and thus are presumed to be feasible with current technology for near-term application in the clinic - include skin and cartilage. A common feature of these tissues is their relative avascularity and acellularity over dimensions important for tissue function, the ability to regenerate functional tissue from a single donor cell type, and the relative lack of cell-cell interactions in the normal tissue structure. Further, the cells in cartilage and skin grow readily in culture, allowing the number of cells from a single piece of donor tissue to be increased by several orders of magnitude. These factors make cartilage and skin highly attractive products for the emerging tissue engineering industry.

Among the many functions that cartilage serves in the human body, providing shape to such features as the outer ear and the nose is the least demanding from a tissue engineering perspective. Cartilage cells (chondrocytes) can readily be obtained by enzymatically digesting donor material to free cells from the extracellular matrix (ECM). Isolated chondrocytes placed in culture under appropriate conditions (e.g., in an agarose gel) exhibit a striking intrinsic ability to secrete ECM and form a stiff tissue similar to native cartilage. This property becomes useful for tissue engineering when the appropriate scaffold is used to direct tissue formation.

Our lab and many others have focused on synthetic bioresorbable polyesters in the polylactide/polyglycolide family as materials for scaffold construction in tissue engineering, as these materials have good mechanical properties, a long and favorable clinical record, are processable by solvent or thermal techniques, and break down by hydrolysis in body fluids to yield natural metabolites. In a collaboration initiated by a plastic surgeon at Boston Children's Hospital, we demonstrated that cartilage-like tissue in the shape of a human outer ear could be formed either in culture or by implanting beneath the skin a porous ear-shaped scaffold seeded with chondrocytes (Vacanti et al., 1992). The polyesters were chosen so that the degradation in mechanical strength of the scaffold was commensurate with the gain in mechanical strength of the tissue being formed by chondrocyte-secreted ECM, allowing the implant to retain its shape over the two-month period required for cartilage to form.

This sort of approach is being commercialized, notably for replacement of dermis of skin in diabetic ulcers and burn victims (Cooper et al., 1991). These tissues lead the way in determining what kinds of manufacturing and regulatory procedures will be needed for more complex applications.

Long Term - Vascularized Tissues
With few exceptions, tissues in the body are permeated by vascular networks to supply essential nutrients and regulatory factors. The distance between capillaries generally ranges from 20 to 200 microns, or about one to 10 cell diameters. The need for vascularity at almost the cellular scale is a major impediment to most cell-based approaches to tissue regeneration. Thus, tissues for which the microvascular network contributes strongly to overall tissue function, such as liver, have been more difficult to engineer and are correspondingly farther from clinical application. Highly vascularized tissues also tend to comprise several cell types arranged in a hierarchical structure, further complicating their reproduction by tissue engineering approaches. Cells derived from such tissues, such as hepatocytes from liver, often lose tissue-related functions when placed in culture; presumably, the hierarchy of structure also conveys a hierarchy of molecular control of cell behavior.

A place to start with such tissues is, then, understanding of the molecular signals that govern cell behavior. Virtually every aspect of cell behavior is governed at some level by interactions of transmembrane receptor molecules on the cell surface with ligands in the extracellular environment, and these complicated interactions are now being elucidated with the aid of engineering insights and analysis (Lauffenburger and Linderman, 1993). One example of a molecular control system is epidermal growth factor (EGF), which binds to the epidermal growth factor receptor (EGFR) to stimulate a diverse array of cell behaviors, including cell division. EGF was discovered in the early 1960s but has not yet entered clinical application despite many efforts to develop it and the wide range of effects it exerts under well-controlled in vitro conditions. It is inherently difficult to control local concentration of the peptide in vivo because of diffusive spread, cell uptake, and degradation of EGF. Further, when cells bind EGF, the EGF-EGFR complex is internalized and often degraded, leading to down regulation of receptors and attenuation of cellular responses. EGF is normally present in a soluble form, and some literature suggests that aspects of EGFR signaling can occur inside the cell after internalization. Nonetheless, we have shown that EGF retains biological activity when covalently tethered to the culture substrate where it has mobility but is physically prevented from entering the cell or diffusing away; further, cell response can be tuned by the density of tethered EGF ligand presented (Kuhl and Griffith-Cima, 1996). In addition to applications in tissue engineering, the ability to manipulate ligand presentation by purely physical means is now becoming a powerful tool for fundamental understanding and control of receptor function, complementing tools derived from molecular cell biology. The use of molecularly designed polymers is becoming a new tool for probing cell behaviors.

Moving to the macroscopic level, issues of how groups of cells can self-organize is important. It is becoming more apparent that the molecular signals from ECM may at least partially govern cell behavior by measurable biophysical outcomes such as the relative magnitude of adhesive bonds (Lauffenburger and Horwitz, 1996). For example, the morphology of aggregates of hepatocytes in culture - spread or spheriodal - can be predicted on the basis of the relative magnitudes of cell-substrate adhesion strength and cell contractile forces, and the morphology of more complex structures obtained from mixed hepatocyte/endothelial cultures can also be predicted based on relative cell-cell and cell-substrate adhesion strengths (Powers et al., 1997). We are exploiting this self-organizing ability of cells to generate three-dimensional tissue with a perfused microvascular structure in vitro starting from dispersed cells and a scaffold of precisely defined architecture and surface chemistry. Our initial focus is on trying to re-create the smallest functional unit in a tissue, the capillary bed, which has dimensions of 0.1 to 0.8 mm depending on the tissue and is ~0.8 mm for liver. We have developed and implemented new materials processing techniques to create scaffolds for this purpose, a technique that will allow integration of molecular cues, such as tethered EGF, with macroscopic signals (Griffith et al., 1997).

On the in vivo therapeutic side, our ultimate aim is to create a liver that can be transplanted directly into the portal vein of a patient beginning with a complex hierarchical scaffold seeded with cells. To create scaffolds with this high degree of complexity, we have implemented a solid free-form fabrication (SFF) technique, the 3 Dimensional Printing (3DPTM) process. In SFF techniques, devices are built as a series of thin sequential layers. We have adapted the original 3DPTM process, developed for fabrication of ceramics and metals, to a range of polymeric and composite organic/inorganic materials important in tissue engineering and drug delivery. This technology is currently being commercialized.

In addition to the huge clinical needs for vascularized tissues, in vitro models of vascularized human tissues are desperately needed in the pharmaceuticals industry for determining the effects of drugs on humans and for basic research into disease states and development. Generation of tissue that can be controllably perfused in vitro and maintained in culture for extended periods will enhance virtually every effort to study tissue functions in vitro. We are implementing silicon microfabrication technology to create a miniature "liver on a chip" to allow rapid screening of drugs and other compounds.

Cooper, M. L., J. F. Hansbrough, R. L. Spielvogel, R. Cohen, R. L. Bartel, and G. Naughton. 1991. In vivo optimization of a living dermal substitute employing cultured human fibroblasts on a biodegradable polyglycolic acid or polyglactin mesh. Biomaterials 12(2):243-248.

Griffith, L., B. Wu, M. J. Cima, B. Chaignaud, and J. P. Vacanti. 1997. In vitro organogenesis of liver tissue. Annals of the New York Academy of Sciences 831(December 31):382-397.

Kuhl, P. R., and L. G. Griffith-Cima. 1996. Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase. Nature Medicine 2(9):1022-1027.

Lauffenburger, D. A., and A. F. Horwitz. 1996. Cell migration: A physically integrated molecular process. Cell 84(3):359-369.

Lauffenburger, D. A., and J. L. Linderman. 1993. Receptors: Models for Binding, Trafficking, and Signaling. New York: Oxford University Press.

Langer, R., and J. P. Vacanti. 1993. Tissue engineering. Science 260(5110):920-926.

Powers, M. J., R. E. Rodriguez, and L. G. Griffith. 1997. Cell-substratum adhesion strength as a determinant of hepatocyte aggregate morphology. Biotechnology and Bioengineering 53(4):415-426.

Vacanti, C. A., L. G. Cima, D. Rodkowski, and J. Upton. 1992. Tissue engineered growth of new cartilage in the shape of a human ear using synthetic polymers seeded with chondrocytes. Pp. 323-330 in Tissue-Inducing Biomaterials, Materials Research Society Symposium Proceedings, Vol. 252, L. G. Cima and E. S. Ron, eds. Pittsburgh, Pa.: Materials Research Society.

About the Author
Linda G. Griffith is Associate Professor, Department of Chemical Engineering and Division of Bioengineering and Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts. The author was a participant in the 1998 Frontiers of Engineering Symposium. The Symposium publication Reports on the Leading Edge: Engineering from the 1998 NAE Symposium on Frontiers of Engineering is available online.