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NATURE BIOTECHNOLOGY VOLUME 23 NUMBER 10 OCTOBER 2005 1237
rapid changes and customizing its physiology to the ambient conditions. However, this is not to imply that nature will always devise a means of clearing obnoxious chemicals in the envi-ronment, and the toll on human life and health remains immense.
Horizontal gene transfer and gene expansion are the most likely mechanisms for generating diversity of reductive dehalogenases and dechlo-rination potential. Nevertheless, the number of independent integration events observed in these genomes is remarkably high given the recent divergence of these two strains (implied by the level of identity of conserved regions). Similar scenarios have been described in clinical isolates of Gram-negative pathogens with regard to capture and spread of antibiotic-resistancegene cassettes or integrons (mobile DNA ele-ments), allowing these pathogens to rapidly adapt to widespread use of antibiotics9.
Surprisingly, the genome data reveal appar-ently identical shortcomings in the biosynthetic abilities of both Dehalococcoides strains that are not borne out by culture requirements in the lab. Strain 195 requires a complex growth medium containing mixed culture extracts, whereas strain CBDB1 does not. The medium requirements of strain 195 were thought to be explained by this bacterium’s lack of enzymes for methionine, glutamate and cofactor
synthesis. The new finding that these enzymes are also absent in strain CBDB1, which grows in a simpler medium, presents a conundrum.
The study of Kube et al. exemplifies the tremendous value of microbial genomics for unraveling the molecular physiology of organ-isms and provides insights into the substrate specificities of reductive dehalogenases and into the evolution of dehalorespiration. By capitalizing on what we have learned, we may be able to design more effective approaches for removing chlorinated toxic metabolites from soil and water—a proposal of potentially vast economic and ecological importance. In addi-tion, such genomes undoubtedly serve as a model for studying the evolution of catabolic pathways in bacteria.
1. Kube, M. et al. Nat. Biotechnol. 23, 1269–1273 (2005).
2. Seshadri, R. et al. Science 307, 105–108 (2005).3. Maymo-Gatell, X., Chien, Y., Gossett, J.M. & Zinder,
S.H. Science 276, 1568–1571 (1997).4. Fennell, D.E., Carroll, A.B., Gossett, J.M. & Zinder, S.H.
Environ. Sci. Technol. 35, 1830–1839 (2001).5. Major, D.W. et al. Environ. Sci. Technol. 36, 5106–
5116 (2002).6. Fennell, D.E., Nijenhuis, I., Wilson, S.F., Zinder, S.H.
& Haggblom, M.M. Environ. Sci. Technol. 38, 2075–2081 (2004).
7. Bunge, M. et al. Nature 421, 357–360 (2003).8. Adrian, L., Szewzyk, U., Wecke, J. & Gorisch, H. Nature
408, 580–583 (2000).9. Rowe-Magnus, D.A. & Mazel, D. Int. J. Med. Microbiol.
292, 115–125 (2002).
It takes a village to grow a tissueDavid L Kaplan, Randall T Moon & Gordana Vunjak-Novakovic
A recent meeting emphasized the importance of integrating concepts from developmental biology and other disciplines into tissue engineering.
Tissue engineering aims to grow tissue replace-ments by combining cells and matrices under defined laboratory conditions1. The shortage of donor tissues and organs and the increas-
ing demand for tissue repair from an aging population have catalyzed research in the field. A US National Institutes of Health–sponsored workshop, ‘Tissue Engineering: The Next Generation,’ held in Boston on May 2–4, 2005, brought together about 300 participants to explore the future of tissue engineering. A major theme of the meeting was the need to replace the current largely empirical approaches by mechanistic ones and to incorporate approaches from multiple disciplines, in particular, from developmental biology, which offers a wealth of practical and theoretical insights gleaned from hundreds of years of studying embryos.
Current therapeutic approaches with replacement tissues are rather empiri-cal (described by Laura Niklason, Duke University, as “tissue try this”), owing to the
enormous complexity of the cells, scaffolds and stimuli that must be screened and the fact that the clinical problem is not always well articulated at the outset. But the field is embracing more systematic studies in which concepts from engineering, develop-mental biology, genomics, proteomics, cell and molecular biology and systems biologyare considered in concert to rapidly iden-tify the most promising strategies. Previous approaches involving inert, nondegradable, nondecorated or homogenous biomaterials are being replaced with more complex designs that direct cell interactions towards tissue assembly and that degrade at rates commensurate with new tissue formation. At the same time, ter-minally differentiated cells and immortal cell lines are being substituted by progenitor and stem cells to exploit or study cell plasticity.
Most tissue engineering studies have used a single cell type to generate simple tissues in vitro. Stem and progenitor cells, which have the capacity to differentiate into multiple cell lineages and form complex tissues, hold great promise, but challenges include access to specific cell lines, control of differentia-tion and difficulties in handling (according to Mick Bhatia, University of Western Ontario, 40% of these cells die in culture). Workshop participants recognized the need to establish databanks of transfected cells with appropri-ate reporters to track cell differentiation and tissue development. To exploit stem cells for tissue engineering, one must precisely control their local environmental conditions, includ-ing scaffold-derived signals and the levels of nutrients, and molecular and physical regu-latory factors. For example, scaffold design can be manipulated to influence the impact of physical stress (Donald Ingber, Harvard University). The selection of culture param-eters is one area where developmental biology is likely to provide sound principles.
The use of multiple cell types and combi-nations of differentiated and progenitor cells will require the development of readouts or reporters linked to genes involved in selective differentiation pathways that are activated by cell-cell and cell-matrix interactions. Here, too, developmental biology should help in deter-mining the right combinations of Hedgehog, Notch, Wnt and other signaling factors needed to direct cell fate and function3.
Scaffolds are evolving from passive, non-degradable materials to a new generation of designs that actively communicate with sur-rounding cells. Instead of relying on addition of soluble growth factors, these designs aim to deliver multiple factors with orchestration of temporal and spatial control, recapitulating sig-naling cascades to optimize tissue outcomes4.
David L. Kaplan is in the Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, USA; Randall T. Moon is in the Department of Pharmacology, University of Washington School of Medicine and Howard Hughes Medical Institute, Box 357750, Seattle, Washington 98195 USA; and GordanaVunjak-Novakovic is in the Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace, 1210 Amsterdam Avenue, New York, New York 10025, USA.e-mail: [email protected]
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Scaffolds are beginning to accommodate cell adhesion, spreading, replication and theformation of the new extracellular matrix5. Even more advanced designs are required to support gradient structures. Emerging tools include complex polymers, such as hydro-gels containing signaling factors; biopolymer hybrids; block copolymers; genetic engineer-ing of proteins to incorporate cell-binding domains, cytokines and self-assembling or cross-linking domains; and consideration of structural and mechanical design.
In general, monolithic scaffold and tissue structures are being replaced by designs that mimic more complex biological structures. The workshop emphasized in particular tis-sue interfaces (e.g., osteochondral and vascu-lar interfaces) and intra-tissue gradients. For example, scaffolds can be designed to deliver a series of three signaling factors involved in vascularization, including vascular endothe-lial growth factor and platelet-derived growth factor, in a temporally controlled fashion to simulate in vivo blood vessel formation (David Mooney, Harvard University).
As well as advances in engineering the scaf-folds upon which cells are seeded, progress is also being made in bioreactor design to address two main needs: to monitor tissues grown in vitro in real time, and to overcome oxygen and nutrient transport limitations in a way that
enables engineering of compact, clinically sized tissues. Recent improvements include theimplementation of pulsatile interstitial flow, complex mechanical signaling6 and electrical stimulation7. Biomimetic bioreactor designs can also permit the tissue of interest to be subjected to regulatory signals similar to those in the native tissue (Laura Niklason, Duke University; Milica Radisic, Michael Sefton, University of Toronto). Insight from devel-opmental biology will be essential in under-standing the extent to which such signals are required for tissue formation in vitro. Future refinements will include methods for combin-ing nondestructive near-real-time imaging and metabolic and structural assessment of tissues (e.g., using magnetic resonance imag-ing, microcomputer tomography, optical tools and proteomics).
The theme of biomimetics extends all the way to storage and preservation of cells and tissues. For example, the pathways activated in animals undergoing suspended animation to survive freezing conditions can be trans-lated into technologies for preserving tissues (Mehmet Toner, Harvard Medical School).
Functional tissue engineering continues to identify important mechanical and structural design parameters for scaffolds and tissue analogs. Challenges persist, because function means different things to different people: to
a biologist it is often molecular and cellular, whereas to an engineer or clinician it is struc-tural and physical.
Much can be learned about building com-plex structures from building simple ones. It remains to be determined whether the design of native tissues must be reproduced to achieve adequate function, and whether we must reca-pitulate development or merely take advantage of appropriate developmental mechanisms. Considering the intrinsic differences between fetal development and adult tissue regen-eration, it may be more important to control adaptation and remodeling (Frederick Schoen, Harvard Medical School). It is not enough to think about the primary site of injury; one must consider the system being repaired in the context of tissue growth, regeneration and degeneration (Robert Sah, UC San Diego).
Integration of in vitro–generated tissues with the host is an area of intense interest, as it requires not only a metabolic and structural match to the in vivo environment, but also functional vasculature and nerves to sustain function after implantation. At the meeting, David Mooney asked whether we must all become vascular engineers, given the impor-tance of oxygen transport to cells both in vitro and after implantation; engineers require a highly vascularized bed to obtain compact and functional tissues. One possible solution is to stack prefabricated elements of living cells and vasculature (Joseph Vacanti, Harvard Medical School). This technique should be fairly dynamic, allowing a variety of ways to sculpt tissues.
Further understanding of the interacting components in engineered tissue should also enable the time for tissue formation to be compressed (that is, the concept of acceler-ated development; David Butler, University of Cincinnati). The body forms a tissue over many years, and yet tissue engineers are expected to create new tissues for implantation in a matter of weeks. Insights from developmental biologycan help in understanding and mitigating the effects of accelerated development on the structure and function of engineered tissue.
As a means of validating and monitoring the quality of tissue constructs, one approach pro-posed at the meeting was to develop imaging and biomarker technologies in parallel with engineered tissues and consider such tech-nologies in the design process (Martha Gray, Massachusetts Institute of Technology). This approach could not only help guide functional tissue engineering by providing real-time insight into the effects of culture parameters on tissue development, but also accelerate the delivery of engineered constructs to patients by reducing culture time to a minimum and
Figure 1 Electrical stimulation of cardiac myocytes cultured in a three-dimensional collagen scaffold for 8 days triggered the assembly of synchronously beating cardiac tissue with substantial ultrastructural organization (green stain, β-myosin heavy chain; blue stain, cell nuclei; data from ref. 7).
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by improving the match between the in vivo target site and the construct’s structure and function.
As tissue engineering approaches become more sophisticated, attention will turn to applications in drug discovery and biomedi-cal research. With regard to pharmaceuti-cal discovery, screening of drug leads using engineered human tissues is likely to become more commonplace as bioreactors are scaled to array formats and human stem cells are harnessed for in vitro tissue formation. Such approaches should reduce the use of animals while providing more precise control over the properties of engineered grafts and facilitat-ing drug screening. Engineered human tissues
can also serve as controllable models of disease formation and progression.
As yet, however, the challenge of understand-ing basic cell biology limits the extent to which tissue engineering can fully embrace rational design principles. For rational and reproduc-ible tissue engineering, infrastructural changes are needed to stimulate cross-disciplinary research among biologists, materials scien-tists, bioengineers and clinicians. One means of accomplishing this would be through the use of improved electronic communication, such as blogs and shared websites. Indeed, searchable blogs would allow students and postdocs to share unpublishable yet useful data and to com-ment on each others’ work. Several participants
at the NIH meeting noted that a change in NIH grant-application rules to allow more than one principal investigator would facilitate sharing of scientific credit for accomplishments gener-ated by multiple laboratories.
1. Langer, R. & Vacanti, J.P. Science 260, 920–926 (1993).
2. Lanza, R.P., Langer, R. & Vacanti, J. Principles of Tissue Engineering, edn. 2 (Academic Press, San Diego, California, 2000).
3. Moon, R.T., Bowerman, B., Boutros, M. & Perrimon, N. Science 296, 1644–1646 (2002).
4. Shea, L.D., Smiley, E., Bonadio, J. & Mooney, D.J. Nat. Biotechnol. 17, 551–554 (1999).
5. Chen, C.S. et al. Science 276, 1425–1428 (1997).6. Altman, G.H. et al. J. Biomech. Eng. 124, 742–749
(2002).7. Radisic, M. et al. Proc. Natl. Acad. Sci. USA 101,
18129–18134 (2004).
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