REJUVENATION RESEARCHVolume 11, Number 4, 2008© Mary Ann Liebert, Inc.DOI: 10.1089/rej.2008.0781

Dissertations

A Survey of Selected Theses Relevant to Combating Aging

Aubrey D.N.J. de Grey

IN THIS SECTION, I continue the series, begun in issue 10(1), of surveys highlighting a small selection of recently completeddoctoral theses with particular relevance to the fields covered by Rejuvenation Research.1–4 While it has become common

for thesis work to appear in the general academic literature, it remains valuable to scan the thesis databases for importantadvances that one might otherwise have missed.

Unraveling Tissue Regeneration Using Chemical Genetics

Lijoy Mathew, Ph.D.Oregon State University, 2008

The emerging field of regenerative medicine is mainly approached by two different aspects. First is the use of stem cell-based models to generate a suite of differentiated cells for therapeutic applications and the alternative approach is to uti-lize the nonmammalian models that have the inherent capacity to regenerate their body parts. Zebrafish caudal fin regen-eration is a well-established research system to understand the basic principles of tissue regeneration. We combined atoxicological, a chemical genetic, and a candidate gene approach to define the molecular signaling pathways important forregeneration. TCDD, an aryl hydrocarbon receptor (AHR) ligand, was used as a chemical probe to impair regeneration, andwe identified that AHR2 and ARNT1 are the in vivo molecular partners for TCDD-mediated inhibition of regeneration. Wefurther performed a global genomic analysis in the regenerating fin tissue after TCDD exposure to identify the downstreamtarget genes modulated by AHR activation. Functional grouping of the differentially expressed genes by TCDD revealedmis-expression of Wnt signaling genes as well as Wnt target genes, suggestive of a cross talk between AHR and Wnt sig-naling pathways. We hypothesized that, mis-expression of R-Spondin1, a TCDD-induced gene as well as a novel ligand forWnt co-receptor LRP6, was responsible for the differential expression of the Wnt target genes. Partial antisense repressionof R-Spondin1 or LRP6 prevented the inhibition of regeneration by TCDD, indicating that mis-induction of R-Spondin1,which mediates through LRP6, is absolutely required for TCDD-mediated inhibitory effect on fin regeneration. Under-standing the advantages of chemicals to probe tissue regeneration, we developed a rapid throughput regeneration assay toidentify additional small molecules that modulated regeneration. Glucocorticoids were identified as inhibitors of regener-ation, and we demonstrated that glucocorticoid receptor activation is absolutely required for mediating the inhibition of re-generation. We further illustrated that signaling from exogenous glucocorticoids impairs blastema formation and limits re-generative capability in vertebrates through an acute inflammation-independent mechanism and also report that neutrophilsand macrophages are not required for fin regeneration. Finally, we performed a comparative global genomic analysis be-tween different zebrafish regeneration models and identified raldh2, a rate-limiting enzyme for retinoic acid (RA) synthe-sis as a candidate gene across the distinct regeneration models. We demonstrated that, in addition to the well-establishedrole of RA signaling during the later phase of regenerative outgrowth, this signaling pathway is also critical for the initia-tion of regeneration, suggesting a dual phase of RA signaling during fin regeneration. Collectively, our results obtainedthrough different experimental approaches suggest that epimorphic regeneration is completed by a well-orchestrated pro-cess of multiple molecular signaling events.

Comment: The study of natural regeneration of complex structures (such as limbs) in vertebrates has fascinated biologists for centuriesand is of immense interest today for what it can offer regenerative medicine. However, the species with the most spectacular and best-known regenerative capacities are unfortunately not those blessed with a substantial body of more general biological (and especially ge-netic) knowledge.5 The serendipitous discovery of a highly regeneration-competent strain of mice is one exception to that rule, but ithas proven highly refractory to genetic analysis.6 The zebrafish, with its great advantages in terms of both genetics and physiology, hasconsiderable potential to spearhead our increasing understanding of vertebrate regeneration,7,8 as this thesis illustrates.

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Methods to Produce Silk Fibroin Film Biomaterials for Applications in Corneal Tissue Regeneration

Brian Lawrence, Ph.D.Tufts University, 2008

Silk fibroin derived from the Bombyx mori silk worm cocoon offers a unique choice in biomaterial selection for uses in tis-sue engineering and regenerative medicine. This is primarily due to fibroin’s nonimmunogenic response upon in vivo im-plantation; controllable material degradation rates; tunable mechanical properties; and ambient processing conditions. Silkfibroin films can be generated to produce simplified geometries with highly controlled material properties that offer sub-strates capable of successfully interfacing with biological systems. In addition, a variety of silk film processing methods ex-ist to tailor these biomaterial properties to a given application. Current biomaterials for use in corneal tissue regenerationdo not possess the range of material properties and processing combinations as offered by silk fibroin. Therefore, studieswere carried out to assess silk fibroin film processing methods and in vitro corneal cell response to better characterize thesebiomaterials for use in cornea-related applications. Processing methods were developed to generate silk film biomaterialswith high-resolution surface patterns, controllable film thickness and porosity, and ensured sterility. Silk films generatedfrom these methods were found to support corneal fibroblast growth, guide cell and extracellular matrix (ECM) alignment,and could be assembled to form 3D corneal tissue constructs. The results from these studies demonstrate that silk films of-fer a new option in biomaterial choice for the development of clinically relevant corneal devices to aid in tissue regenera-tion.

Comment: Whatever advances are made in stimulating spontaneous regeneration of complex structures, it is almost certain that suchtherapies will always need to be complemented by more invasive surgical therapies to rebuild certain tissues9,10—and the cornea, beingrelatively accessible, is a prime example. The qualities of silk fibroin noted and exploited in this thesis are among the major challengesto the development of foreign material that can support the growth and maturation of human tissues in situ. Further work in this areashould be eagerly awaited.

Identification of Factors Affecting Bovine Somatic Cell Nuclear Transfer Efficiency andCharacterization of Transcriptional Profiles of Nuclear Transfer Embryos and Cotyledons

Kenneth Aston, Ph.D.Utah State University, 2008

Since the production of the first sheep by somatic cell nuclear transfer, a great deal of effort has been made to improveefficiency and to understand nuclear reprogramming mechanisms. Unfortunately efficiency remains low, and nuclearreprogramming mechanisms remain uncharacterized. The objectives of this research were to identify factors associatedwith somatic cell nuclear transfer efficiency and to analyze the transcriptome of blastocyst-stage clone and control em-bryos and cotyledonary tissue in an effort to elucidate mechanisms responsible for the low developmental efficiencyand high post-implantation losses. The experiments reported here identify factors, including oocyte source and timingof activation following nuclear transfer, that yield improved efficiencies. It was determined that the use of cow oocytesfor somatic cell nuclear transfer results in improved in vitro development and increased pregnancy rates. These datafurther indicate prolonged exposure of the donor nucleus to preactivated oocyte cytoplasm results in increased nuclearfragmentation and reduced developmental efficiency in vitro. Several aberrantly expressed genes were identified in nu-clear transfer blastocysts and cotyledons that could impact cloning efficiency. Major histocompatibility complex I anddownregulator of transcription 1 were overexpressed in nuclear transfer blastocysts, and retinol binding protein 1 wasoverexpressed in nuclear transfer cotyledons. The functions of these genes in immune response, transcriptional regu-lation, and retinol binding and transport make them attractive candidates for further nuclear transfer research. Ex-pression levels of six developmentally important genes were analyzed in various stages of preimplantation nucleartransfer embryos by real-time polymerase chain reaction to determine the timing of nuclear reprogramming followingnuclear transfer. Five of the six genes were aberrantly expressed at multiple developmental stages, however by the blas-tocyst stage only one gene was aberrantly expressed. These data indicate that reprogramming is delayed in nucleartransfer embryos resulting in over- or underexpression of developmentally important genes during early embryogen-esis. These experiments report factors associated with improved nuclear transfer efficiency; provide insight into po-tential mechanisms for low developmental rates, abnormal placentation, and fetal loss of clones; and characterize thetiming of nuclear reprogramming following somatic cell nuclear transfer.

Comment: Nuclear transfer,11–13 as readers of this journal will be aware, ostensibly seems in danger of being entirely supplanted bythe dedifferentiation technique recently pioneered by Yamanaka.14 However, as noted by Jaenisch in the interview in this issue, thereare likely to be considerable differences in the gene regulation processes involved in the two methods, with inevitable consequences forthe imperfections of epigenetic regulation that result.15 As such, studies such as this thesis are still necessary, both for NT cells and for

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induced pluripotent stem (iPS cells) to minimize those imperfections and to identify which of the two techniques is preferable for a par-ticular application.

Effect of Physiological Stimuli on the Differentiation of Embryonic Stem Cell-DerivedCardiomyocytes for Cardiac Tissue Engineering

Valerie Shimko, Ph.D.Tulane University School of Science and Engineering, 2008

Cardiovascular disease is the leading cause of morbidity and mortality in developed countries and is the number one causeof death in the United States. Current treatments include drug therapy, gene therapy, surgical interventions, heart trans-plants, and tissue engineering. The goal of this dissertation was to combine the sciences of genetic and tissue engineeringto create a three dimensional (3D) engineered construct with cardiomyocytes derived from genetically selected embryonicstem (ES) cells that could be used as an in vitro model to study the effects of mechanical and electrical stimulation on thedifferentiation of ES cells. To investigate this, two custom devices were built to deliver precise, reproducible, and long-termmechanical and electrical stimulation regiments to the constructs. To optimize construct constituents and test the efficacyof these devices, 3D scaffolds containing HL-1 cells were studied. These experiments showed that an optimal ratio of cellsand ECM proteins (0.2 mg collagen, 0.01 mg fibronectin, and 6 � 106 cells) was necessary to create constructs with the struc-tural integrity necessary for use in the devices. Also, the mechanical loading device was shown to be efficacious by en-couraging cellular alignment with distinct sarcomeric structures, development of dense cell boundary layers, and homo-geneous cell distributions throughout the construct’s central regions. Parallel experiments using constructs containing EScells revealed that 0.4 mg collagen, 0.01 mg fibronectin and 6 � 106 cells were optimal. These experiments led to the dis-covery of a synergistic relationship between mechanical and electrical stimulation. A sequential regimen of electrical stim-ulation, followed by mechanical loading, resulted in the most significant switch from the expression of fetal genes (a-skele-tal actin and b-MHC) to adult cardiac genes (a-cardiac actin and a-MHC). Also, these constructs contained myofiber likestructures, organized sarcomeric structures, and distinct gap junctions similar to those found in the native neonatal heart.This dissertation takes steps toward the development of a 3D model of the developing heart using a renewable cell sourceembedded in a defined ECM scaffold and exposed to mechanical and electrical stimuli, and provides the experimental foun-dation for those developing alternative treatments for those suffering from heart disease.

Comment: One of the primary challenges in tissue engineering has always been the provision of the right chemical environment forappropriate differentiation of cells seeding a scaffold16,17; this is the problem that led to the idea of growing tissues in the abdominalcavity, expressed so succinctly by Atala with the aphorism “the body is the best bioreactor.” But in addition, and especially for thoseorgans whose function is largely mechanical, the development of correct structure typically requires appropriate loading of the nascentorgan, which is not nearly so easily achieved. In this work, substantial progress is reported in possibly the greatest such challenge ofall, the construction of the most complex and highly stressed mechanical organ of all.18–20

Derivation of Cardiomyocytes from Embryonic Stem Cells and Development ofTechniques to Study Cardiac Lineage

Achuta Guddati, Ph.D.Northwestern University, 2008

Embryonic stem (ES) cells have the potential to differentiate into ectodermal, mesodermal, and endodermal derivatives.This property makes them a valuable source of tissue-specific progenitor and differentiated cells that can be used for cellreplacement therapy. The first goal of this thesis is to test if cardiogenesis from ES cells can be influenced by the overex-pression of different proteins. We find that cardiogenesis is significantly enhanced by the overexpression of the c isoformof the transcription factor Paired-Like Homeodomain Transcription Factor (Pitx2c). This effect was verified morphologi-cally by differentiating ES cells with Pitx2c and immunostaining for cardiomyocyte-specific proteins. Similarly, changes inRNA transcripts specific to cardiomyocytes during the process of differentiation were also recorded by means of quantita-tive RT-PCR (QRT PCR). Finally, the contractile properties of these cells were measured by means of calcium imaging andtheir action potential characteristics were measured. We transplanted these cells into a mouse model of myocardial infarc-tion and found significant improvement of cardiac function. We subsequently subjected Pitx2-ES cells to hypoxic condi-tions and observed that the supernatant also caused significant functional recovery when injected into infarcted mice. Thesecond goal of this thesis is to study the development of cardiomyocytes from ES cells by utilizing a series of stage-specificpromoters to drive different fluorophores. We designed a system where three different constructs carrying the promotersof cardiac specific genes would express proteins that would heterodimerize and drive a response element expressing anti-biotic resistance. This interdependence ensures the presence of the constructs in the nonsilenced loci of the genome. Weused this system to insert the promoters of NK2 transcription factor related, locus 5 (Nkx2.5), myocyte-specific enhancer

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factor 2c (Mef2c), alpha myosin heavy chain (a-MHC), and myosin light chain 2-ventricular isoform (MLC2v) in murine EScells and such that the cardiac differentiation of these cells could be visualized by the various fluorophores driven by thesepromoters. This system will be a powerful tool to study the effects of various morphogens on cardiac differentiation of EScells and will also be useful for cell-transplantation studies.

Comment: If environment were all, we would be able to introduce pluripotent cells into a damaged tissue and leave them to their owndevices in the repair of that tissue. Unfortunately, the differences between development and repair are large enough that such cells areinsufficiently instructed by that environment, and the risk of teratomas results. Thus, a dominant theme within stem cell therapy is theidentification of factors that can nudge initially over-versatile cells some way along a desired lineage, leaving them sufficiently undif-ferentiated to be able to heal the damaged tissue but not to do anything else. Since we still know so little about the detailed composi-tion of the extracellular and paracrine factors that influence this process,21–23 the identification of specific genes (especially transcrip-tion factors) controlling it in a given context gives a potential way to sidestep our ignorance and induce the appropriate differentiationof such cells even in a sub-optimal environment.24

The Role of Cyclooxygenase-2 and Parathyroid Hormone in Mesenchymal ProgenitorCells during Senescent Fracture Repair

Amish Naik, Ph.D.University of Rochester, 2008

For over thirty years it has been well established in the clinical setting that the rate and quality of fracture healing declineswith age. This results in prolonged pain, increased risk of mortality and morbidity, and many times permanent disability.The literature suggests that aging results in discrete morphological and molecular changes, which include delays in chon-drogenesis and endochondral ossification, two vital processes in bone repair. Little progress, however, has been made tocomprehensively define mechanisms underlying these differences and to develop new therapeutic strategies to improvefracture repair in humans and animal models. The current thesis confirms that aged mouse fractures have a decreased rateof healing associated with delayed chondrogenesis and chondrocyte maturation, decreased fracture callus mineralization,and vascularization, and dysregulation of genes anabolic to fracture repair such as cyclooxygenase-2 (COX-2) and bonemorphogenetic protein-2 (BMP-2). COX-2 is the rate-limiting enzyme involved in prostaglandin E2 (PGE2) synthesis andis a critically important enzyme for bone repair. Senescent regulation of these critical genes leads to perturbation in tem-porally distal events such as chondrogenesis, bone formation, and remodeling. Adjuvant therapy with an EP4-selective ag-onist rescues inefficiencies in chondrogenesis and reduced bone formation observed in aging. Furthermore, interventionwith parathyroid hormone (PTH) increased chondrogenesis and bone formation in fractured aged mice to levels exceedingthose observed in young controls. PTH therapy likely induces Indian Hedgehog (Ihh) release from prehypertrophic chon-drocytes enhancing chondrocyte proliferation and osteoblast development through modulation of Runx2. Overall this the-sis examines the role of periosteal progenitor cells and their function in bone repair in the context of aging. The systemicand microenvironment of the aged animal provides less than optimal inflammatory and hormonal stimuli to promote theproliferation and differentiation of precursors into the cellular components of fracture repair. When given sufficient stim-ulus, aged mice demonstrate healing parameters similar to young animals. This thesis, essentially, provides the frameworkfor future investigation into mesenchymal stem cells and their role in senescent fracture healing.

Comment: Though nonbiologists often (and naturally) assume that bone is a nonregenerating tissue like teeth, it is of course not:Rather, it is continuously remodeled by osteoclasts and osteoblasts.25 As such, the decline in fracture repair during aging must be un-derstood as a change not in the bone itself but in the function of the cells that maintain it—changes that, as this thesis reports, may bereversible by quite simple interventions. Such interventions must always be evaluated cautiously because of the risk of side-effects, es-pecially the risk of promoting unwanted proliferation; however, those side-effects should likewise be evaluated carefully because it maybe easier to address them than to develop a primary intervention that does not cause them in the first place.

Tendon and Ligament Repair: Regeneration and Maturation

Jing Zhao, M.S.Clemson University, 2008

This thesis constitutes the studies about the two aspects of tendon and ligament tissue engineering: regeneration and mat-uration. Injuries to tendon and ligament are among the most common injuries to the body, particularly in the young andphysically active population. Associated with the problems of incomplete healing and recurrent injury, these injuries arenot only responsible for large health-care cost, but also result in lost work time and individual morbidity. Tissue engineeringholds promise in treating these conditions by replacing the injured tissue with engineered tissue exhibited similar me-chanical and functional characteristics. Collagen plays a central role in tendon and ligament regeneration, as collagen type

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I is responsible for more than 60% and 80% of the dry weight of tendon and ligament structures, respectively. The hierar-chical organization of collagen type I in bundles confers most of the mechanical properties of tendons and ligaments. Con-sequently, tendon or ligament tissue engineering studies are mainly focused on seeding cells into collagen gels. However,up to now, no cell–collagen constructs have been able to achieve sufficient mechanical properties and the complex archi-tecture of the tendon and ligament is never fully reproduced. A major cause for low mechanical property of regeneratingtendon or ligament is the slow maturation. The maturation of the engineered tissue is dominated by the maturation degreeof extracellular matrix, such as collagen crosslink density. To overcome the slow growth and maturation of tissue engi-neering grafts, one goal of this project is to accelerate the tissue maturation using gene therapeutics approach. Therefore,in the first part of this thesis, two different genes, lysyl oxidase (LOX) and decorin, were transfected into fibroblasts byretrovirus infection. LOX initiates the covalent cross-linking of collagen and elastin in the extracellular space by oxidizingspecific lysine residues in these proteins to peptidyl a-aminoadipic-d-semialdehyde (AAS). These aldehyde residues canspontaneously condense with vicinal peptidyl aldehydes or with [varepsilon]-amino groups of peptidyl lysine to generatethe covalent crosslinkages, which stabilize and insolubilize polymeric collagen or elastin fibers in the extracellular matrix.Decorin is considered a key regulator of matrix assembly because it limits collagen fibril formation and thus directs tendonand ligament remodeling due to tensile forces. In this study, we found that the mechanical property of the tissue-engi-neered grafts was significantly increased by over-expressing LOX or decorin gene. And decorin over-expression made themean diameter of the collagen fibers uniform. In the second part of this thesis, the extracellular matrix (ECM)-based hy-drogel system was used to control the delivery of chemotaxic growth factors, such as hepatocyte growth factor (HGF), forrecruiting endogenous stem cells. This approach was used to attract endogenous stem cells to the lesion site for tendon orligament regeneration. In this study, we found that stem cells could be recruited effectively to the local site where HGFwere released by chemically modified hyaluronic acid- (HA) and gelatin- (Gtn) based hydrogels both in vitro and in vivo.

Comment: Unlike bone (see previous abstract), elastic tissues, including human tendons and especially ligaments, are very poorly re-paired by natural processes.26–28 Much progress has been made in purely surgical approaches to this problem, including transplantingmaterial from elsewhere to the site of an injury and also the “weaving” of new connective tissue. However, the highly invasive natureof such therapies will always be a problem, so any progress (even at an early stage of development, such as the work reported here) inimproving the innate regenerative capacity of such tissues is highly welcome.

References

1. de Grey ADNJ. Dissertations. Rejuvenation Res 2007;10:117–122.2. de Grey ADNJ. Dissertations: a survey of selected recent theses relevant to combating aging. Rejuvenation Res 2007;10:245–251.3. de Grey ADNJ. Dissertations: a survey of selected recent theses relevant to combating aging. Rejuvenation Res 2007;10:339–343.4. de Grey ADNJ. Dissertations: a survey of selected recent theses relevant to combating aging. Rejuvenation Res 2007;10:641–646.5. Gardiner DM, Satoh A, Ferris D, Graham G, Bryant SV, Rugg E. Engineering a blastema: steps toward regenerating a limb.

Rejuvenation Res 2007;10:S31.6. Heber-Katz E, Leferovich J, Bedelbaeva K, Gourevitch D, Clark L. Conjecture: Can continuous regeneration lead to immortal-

ity? Studies in the MRL mouse. Rejuvenation Res 2006;9:3–9.7. Patel AN, Kuzman M, Spaddaccio C, Genovese J, Toma C. Scar: is rejuvenation possible? Rejuvenation Res 2007;10:S44.8. Kaushik S, Cuervo AM, Guerin JC. Proteolytic systems in long-lived rockfish. Rejuvenation Res 2007;10:S32.9. Mason C. Regenerative medicine 2.0. Rejuvenation Res 2007;10:S40–S41.

10. Fathi F, A. Kermani AJ, Pirmoradi L, Mowla SJ, Asahara T. Angiogenesis in vitro: vascular tube formation from the differen-tiation of murine embryonic stem cells. Rejuvenation Res 2007;10:S29.

11. Stojkovic M. Timeless human embryonic stem cells. Rejuvenation Res 2007;10:S49.12. Lee CK, Yun JI, Lee ST, Lee EJ, Lee SG, Kim HB, Lim JM, Kim DY, Han JY. Interspecies somatic cell nuclear transfer for es-

tablishing embryonic stem cells with desired genotype. Rejuvenation Res 2007;10:S38.13. Fu W. A novel possible approach to the creation of genetically personalized human embryonic stem-like cell lines. Rejuvena-

tion Res 2007;10:19–25.14. Wernig M, Jaenisch R. Direct reprogramming of somatic cells into pluripotent stem cells. Rejuvenation Res 2007;10:S52.15. Stolzing A, Hescheler J, Sethe S. Fusion and regenerative therapies: is immortality really recessive? Rejuvenation Res

2007;10:571–586.16. Minger SL. Therapeutic applications of human stem cells—prospects and challenges. Rejuvenation Res 2007;10:S42.17. Abramovich A, Fraifeld V. Have we reached the point for in vivo rejuvenation? Rejuvenation Res 2007;10:S19.18. Charniot JC, Vignat N, Rousselot DB, Bogdanova V, Zerhouni K, Monsuez JJ, Artigou JY. Oxidative stress implication in acute

heart failure. Rejuvenation Res 2007;10:S23.19. Larrick JW, Huang M, Lum L, Lee R. Specific targeting of therapeutic stem cells for cardiovascular disease. Rejuvenation Res

2007;10:S37.20. Conboy I, Carlson M, O’Connor M, Conboy MJ. Will stem cell-based tissue replacement work in the old? Rejuvenation Res

2007;10:S24.21. Donnini A, Re F, Orlando F, Provinciali M. Intrinsic and microenvironmental defects are involved in the age-related changes

of Lin- c-kit� hematopoietic progenitor cells. Rejuvenation Res 2007;10:459–472.22. Ksiazek K, Korybalska K. Effect of human peritoneal mesothelial cell senescence on the production of proangiogenic media-

tors and endothelial cell growth. Rejuvenation Res 2007;10:S36.

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23. Shytle RD, Ehrhart J, Tan J, Vila J, Cole M, Sanberg CD, Sanberg PR, Bickford PC. Oxidative stress of neural, hematopoietic,and stem cells: protection by natural compounds. Rejuvenation Res 2007;10:173–178.

24. Goldspink G. Loss of muscle strength during aging studied at the gene level. Rejuvenation Res 2007;10:397–405.25. El-Bialy T. Therapeutic ultrasound applications in craniofacial growth, healing and tissue engineering. Rejuvenation Res

2007;10:367–371.26. Laurencin CT. Musculoskeletal regenerative engineering: taking on the grand challenges. Rejuvenation Res 2007;10:S37.27. Moreau P. Can we influence the “normal” aging of arteries? Rejuvenation Res 2007;10:S43.28. Ellis-Behnke RG, Liang YX, Tay DKC, Schneider GE, So KF. Using nanotechnology to repair the body. Rejuvenation Res

2007;10:S28.—Aubrey D.N.J. de Grey

Editor-in-ChiefMethuselah Foundation

Cambridge, U.K.

Email: aubrey@sens.org

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