Ebook Textbook of aging skin: Part 1

(BQ) Part 1 book Textbook of aging skin presents the following contents: Histology, physiology, specialized skin - Genital, rheology, metabolism, molecular biology, endocrinology, stratum corneum, endogenous and exogenous factors in skin aging.

Textbook of Aging Skin Miranda A Farage, Kenneth W Miller and Howard I Maibach Editors Textbook of Aging Skin With 366 Figures and 156 Tables Editors: Miranda A Farage, Ph.D Principal Scientist The Procter & Gamble Company 6110 Center Hill Avenue Cincinnati, OH 45224 USA Kenneth W Miller, Ph.D Associate Director The Procter & Gamble Company 6110 Center Hill Avenue Cincinnati, OH 45224 USA Howard I Maibach, M.D Professor Department of Dermatology University of California, School of Medicine San Francisco, CA 94122 USA Library of Congress Control Number: 2009938632 ISBN 978-3-540-89655-5 This publication is available also as: Electronic publication under ISBN 978-3-540-89656-2 Print and electronic bundle under ISBN 978-3-540-89935-8 This work is subject to copyright All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer Violations are liable to prosecution under the German Copyright Law ß Springer-Verlag Berlin Heidelberg 2010 The use of general descriptive names, registered names, trademarks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book In every individual case the user must check such information by consulting the relevant literature Springer is part of Springer Science+Business Media www.springer.com Publishing Editor: Tobias Kemme MRW Editor: Sandra Fabiani Printed on acid‐free paper Dedicated to those on a forgotten and sometimes lonely and scary aging journey—much more dignity and respect are deserved for all of you – MAF, KWM and HIM When things go wrong as they sometimes will, When the road you’re trudging seems all uphill, When the funds are low and the debts are high, And you want to smile but you have to sigh, When care is pressing you down a bit Rest if you must, but don’t you quit Success is failure turned inside out, The silver tint on the clouds of doubt, And you can never tell how close you are, It may be near when it seems afar So, stick to the fight when you’re hardest hit It’s when things go wrong that you mustn’t quit —Unknown Acknowledgments Deep appreciation and grateful thank-yous are extended to the significant efforts of many people who contributed both knowingly and indirectly to this book, by dedicating their valuable time in preparing their chapters This book represents the fruits of a jointly conceived and executed venture, and has also benefited from global and diverse partners A special thank-you to Dr Mark Dato and Mr Ron Visscher for generously offering their time and expertise in peerreviewing the relevant chapters and extending their immense support to this book No praise is excessive for their efforts, and our heartfelt gratitude goes to them We would like to single out Mr Anil Joseph Chandy (Springer Reference Editorial Office) for a special recognition of his great effort, time, discipline, and dedication in moving this book forward in a timely and organized manner We extend our appreciation to Ms Marion Philipp and Ms Ellen Blasig (Springer Heidelberg) too for the same Last but not least, we acknowledge the assistance provided by Dr Deborah A Hutchins, Ms Zeinab Schwen, Ms Wendy Wippel, Ms Gayle Entrup, Ms Jan Tremaine, Ms Peggy Firth, and Dr T L Nusair for this book Their collective recommendations and input have vastly improved the texts assembled here Above all, we extend our everlasting gratitude and love to our parents, who inspired us and to our families and children, who supported and encouraged us all the way with their incredible patience Only their continuous care, unconditional love, and incomparable sacrifice made all this possible, and easy to achieve Miranda A Farage Kenneth W Miller Howard I Maibach Cincinnati and San Francisco October 2009 Foreword The population is aging rapidly Centenarians are no longer a rarity The fastest growing segment of the population in the United States is people over 80 In the next 25 years, half of the population in the United States will be aged over 50 These shifts will have a tremendous impact on the delivery of healthcare to the elderly and will require a new awareness of how cutaneous disorders affect the quality of life, comprising a heavy burden on health and wellbeing Physicians and healthcare workers are woefully ignorant of the distress, discomfort, and anxieties of people afflicted by disorders of the skin There exists a widespread misconception that skin disorders are simply cosmetic nuisances that can be self-treated by a great assortment of anti-aging creams and lotions available at the local drug store Most of these include high-sounding ingredients such as antioxidants, vitamins, nutrients, botanicals, and ancient folkloristic remedies, the efficacy and safety of which have never been tested They offer little more than hope in a bottle The fact is that common skin diseases may not often be lethal but can ruin enjoyment of life Chronic itchy rashes can be maddening, lowering one’s self-esteem, embarrassing, interfering with sleep, and often accompanied by depression, social isolation, and deterioration of appearance; they can also be uncomfortable, and, not least, costly to treat The elderly commonly take 15–20 oral supplements daily to fight the ailments of old age These are generally useless and may be harmful, often interacting adversely with prescription drugs The elderly often resort to alternative medicines instead of seeing their doctor to obtain FDA-approved drugs, and also often skip their daily doses to save money Noncompliance is common Misdiagnosis and mistreatment of the elderly by health-care workers are common National surveys show that skin diseases increase steadily throughout our lifespan Old people may have as many as 5–10 coexistent cutaneous problems that are worthy of medical attention Moreover, the clinical manifestations of skin diseases in the aged often have different appearances than in the young, confounding diagnosis Importantly, healing of chronic lesions, especially ulcers, is impaired in the elderly Immunity is weakened, increasing susceptibility to infections Response to treatment is slower, leading to noncompliance Adverse drug reactions are common and too commonly not suspected Management of chronic conditions is difficult and frustrating The above litany of problems makes this textbook edited by Farage, Miller, and Maibach a welcome addition to the literature It is invaluable as a reference resource covering exhaustively an enormous number of clinical conditions No topic is neglected including cosmetic treatments The numerous contributions are by highly qualified experts who have a published record of expertise This comprehensive volume is also practical and relevant to the everyday world of clinical practice The information will be useful to physicians, manufacturers of drugs and skincare products, educators, investigators, nursing home personnel, estheticians, and federal regulators This first edition is up-to-date, including much new material that belongs to the shelves of every library, which deals with geriatric problems Dermatologists especially will be remiss if they not put this volume within easy reach for consultation as they encounter a swelling clientele of aging patients Albert M Kligman M.D., Ph.D Professor Emeritus University of Pennsylvania Philadelphia, PA USA Preface The skin is a portal of knowledge on aging From its softness and smoothness in infancy, through its suppleness in youth, to its wrinkled texture in elders, the skin displays the most visible and accessible manifestations of aging Due to falling birth rates and rising life expectancies in industrialized countries, the average age of the population is increasing Research interest in the process of aging has grown and people are becoming obsessed with looking and “staying” young Although excellent compendia exist on the subject of aging skin, the body of knowledge is burgeoning Consequently, this handbook compiles information into one comprehensive reference It covers a range of topics, from the basics of skin structure and function, to the cellular and molecular mechanisms of aging, to the latest bioengineering instruments used to assess age-related changes in the skin The Nobel Prize in Physiology and Medicine awarded in 2009 to Drs E H Blackburn, C W Greider and J W Szostak will stimulate research that will ameliorate the effects of aging on the organ systems of both humans and animals This textbook will simplify approaches when the skin may be an efficient approach to aging based on Dr Blackburn’s team research The skin approachability and the opportunities to work on humans will provide us in the near future with rapid therapeutic and preventive applications Contributors are internationally recognized experts from multiple disciplines germane to this topic We gratefully acknowledge all contributors for sharing their time and expertise We expect this handbook to be valuable to researchers and students with an interest in aging skin Because research progress in this area is so rapid, we hope to update this compendium periodically as advances in the field dictate The editors welcome suggestions for the second edition Miranda A Farage, Kenneth W Miller, and Howard I Maibach October 2009 462 48 The Use of Reconstructed Skin to Create New In Vitro Models Table 48.1 Aging: theories and mechanisms Classical theories ● Oxidative stress ● Alteration of the genome /mutations ● ‘‘Error catastrophe’’ (L.E Orgel 1963) ● Programmed aging-genetic clock: Limited replicative protential (Hayflick 1966) ● Auto immune responses, deterioration of the immune system ● Accumulation of toxic metabolites ● Formation of cross links Proposed interpretation of ‘‘old theories’’ ● Oxidative stress: DNA, proteins, lipids, mitochondria ● DNA – Repair (mutations) – Function (expression) – Replication (telomere length) ● Proteins: structure and function – posttranslational modifications – Glycation (AGE, cross-links) – Transglutaminase (cross-links) – Farnesylation – Methylation, etc Figure 48.1 Histology of reconstructed skin grown at the air–liquid interphase Complete differentiation was observed when keratinocytes were classically grown on the surface of the dermal equivalent in order to form the epidermis (a), as well as in a simplified system in which keratinocytes were embedded like fibroblasts in the collagen lattice to grow and form spheres (b) Bar, 25 mm effect of vitamin C [8], has been extensively investigated in this system [7] This 3D system has the advantage of allowing for a very precise description of the effect of such compounds both on the epidermis and the dermis, as well as the dermal–epidermal junction Moreover, the possibility of using samples in which dermal fibroblasts have been eliminated, allows for studies of the relative roles of these two tissues Therefore, it seemed that working at making new reconstructed skin models would be a valuable approach for making in vitro models of skin aging Strategies for Skin Aging Studies Using Reconstructed Skin As previously mentioned aging is a complex phenomenon Despite much progress in the field of reconstructed skin The Use of Reconstructed Skin to Create New In Vitro Models during the two previous decades, producing models of skin aging using reconstructed skin remains a difficult challenge In fact, there were two main ways to approach this question First reconstructed skin can be made ‘‘as usual’’ and then submitted to a mechanism presumably leading to skin aging or involved in skin aging The advantage of this strategy is to provide information about early events of aging The disadvantage is that it is unlikely that aging actually occurs because aging is a long-term phenomenon due to chronic exposure to adverse effects, while in vitro cultures are strongly limited in time However, a successful follow-up of this strategy has been made in the context of UV light studies aiming at approaching photoaging Secondly and alternatively, it is also possible to fully reproduce a given mechanism of skin aging in order to mimic aging in reconstructed skin The advantage of this approach is that the effect of a given mechanism of skin aging will be actually reproduced and its consequences evaluated in vitro, while the disadvantage of this approach is that a single mechanism of aging will be used although aging is a complex multifactorial phenomenon Thus, it seems restricting This strategy has been followed in order to mimic chronological aging in vitro using the glycation reaction and interesting results were obtained These two well-defined strategies and their respective advantages are summarized in > Table 48.2 However, there is a different way to look at aging, which was recently developed consisting of considering cellular populations and their changes as a function of aging in vivo in order to reproduce these changes in the reconstructed skin system A study on fibroblasts subpopulations was done, which is discussed later in this chapter 48 Current Models of Skin Aging UV Exposure as an Approach of Photoaging Photoaging is due to solar exposure The effects of photoaging have been well described [9] The UV domains seem to be the key domains of solar light involved in skin aging especially UVB and UVA, which correspond to the two wavelength domains that reach human beings on the earth It is also well known that UVB radiations are more energetic, but less penetrating than UVA radiations Therefore, it is likely that in real life UVB will affect preferentially the surface of the skin while UVA will affect the skin in its depth To approach these questions, reconstructed skin was made and submitted to both UVB and UVA single exposures performing dose–response and kinetic experiments It was found that UVB preferentially affects the epidermal compartment producing complex alterations involving both DNA lesions and modifications of the epidermal differentiation pathway [10] The most obvious effect of UVB exposure was the production of apoptotic keratinocytes easily histologically recognizable, also called sun burn cells, which constitute obvious markers of the effect of UVB and are also representative of their effect in real life It was also found that UVA preferentially affects the dermal compartment by inducing apoptosis of the most superficial fibroblasts at doses at which keratinocytes seem to be unaffected suggesting differential resistance of these two cell types to UVA radiations [11] Table 48.2 Strategies used in the reconstruction of skin to create in vitro models of skin aging Method Initiation Method Reproduction Photoaging: UV exposure Description of the effects of UV light on the Collagen is pre-exposed to UV radiations for the reconstructed skin system at equilibrium (= epidermal induction of dermal modifications potentially differentiation completed) relevant to photoaging CHOSEN NOT CHOSEN Chronological aging: glycation Induction of glycation by culturing the dermal equivalent in the presence of sugar (in excess) TESTED BUT NOT CHOSEN (toxic effects) Benefit obtained This strategy allows: in the selected ● A description of precocious effects of UV strategy radiations but not established photoaging ● The complete reconstructed skin is UV exposed Application for anti-aging studies Collagen is pre-incubated with sugar (ribose or glucose) prior use for preparing reconstructed skin CHOSEN This strategy allows: An approach of established chronological aging but only one mechanism is involved In vitro photoprotection studies: solar filter studies by Anti-glycation molecules can be tested as an topical application on reconstructed skin approach to select new anti-aging agents 463 464 48 The Use of Reconstructed Skin to Create New In Vitro Models Interestingly, the kinetic experiments showed that these effects were reversible since epidermal differentiation was rapidly normalized, while the dermal compartment also recovered its fibroblast population after a few days However, as opposed to cellular recovery a slight irreversible reduction of the dermal thickness was noticed after UVA exposure due to collagen degradation by collagenase production This observation may constitute the basis of an approach of the mechanisms of photoaging using the reconstructed skin system These findings prompted the definition of markers of the specific effects of both UVB and UVA, that is, sunburn cells or fibroblast disappearance, which became very useful for studies in vitro, topically applied solar filters, which led to the conclusion that relevant in vitro photoprotection studies were possible [12] Glycation of the Collagen as an Approach of Chronological Aging Glycation is a nonenzymatically driven reaction between free amine groups like those of amino acids like lysine and arginine and circulating reducing sugars like glucose This reaction, also known as the Maillard reaction, leads to socalled advanced glycation end products (AGEs), which are eventually involved in the formation of cross-links between macromolecules This reaction, which is a chemical reaction preferentially affects tissues characterized by poor renewal and is therefore thought to play an important role in aging [13] In skin, glycation is thought to affect dermal macromolecules, especially those known for having a very slow turnover like collagen and elastin and because the formation of cross-links may play a role in the alteration of Figure 48.2 Scanning electron microscopy (a, b), bar mm and transmission electron microscopy (c, d), bar 1.14 mm showing the collagen fibers in the dermal equivalents of reconstructed skin when collagen was not pre-glycated (a, c) or glycated (b, d) Note that the collagen fibers seem to be more densely packed when collagen was pre-glycated The Use of Reconstructed Skin to Create New In Vitro Models mechanical properties of skin like stiffness appearing as a function of age Therefore, an investigation was done on the effect of glycation in the reconstructed skin model Pre-glycation of the collagen was used to avoid possible toxic effects on cultured skin cells due to prolonged exposure to high concentrations of sugar The morphology of reconstructed skin was found to be altered (> Fig 48.2) In addition, several skin markers of interest were found to be modified in a way recalling aging in vivo For instance, Matrix Metallo Proteinase or MMP1, as well as the distribution of certain integrins like the b1 integrin were found to be modified in epidermis in a way similar to aged skin in vivo (> Fig 48.3) At first sight it was surprising to find modifications in epidermis, but it was demonstrated that diffusible factors were involved suggesting that dermal–epidermal interactions were modified when fibroblasts were in contact with glycated collagen This also suggests that communication between the two tissues 48 can be affected during aging The results in detail [14] and the findings are schematically summarized in > Fig 48.4 The main conclusions were that glycation of collagen is sufficient to reproduce some manifestations of skin aging, which shows that glycation is probably an important mechanism involved in skin aging, and that the modified reconstructed skin obtained by glycation represents a model of skin aging It is also of interest to note that this system allows studying the anti-glycation effect of various molecules or extracts, which therefore represent anti-aging candidates [15] Specific Role of Papillary Fibroblasts in Aging The dermal part of skin is histologically heterogeneous The superficial or papillary dermis, which is close to Figure 48.3 Immunolabeling of carboxy methyl lysine or CML (a–c) and b1 integrin (d–f) in reconstructed skin in the absence of glycation (a, d) or when collagen was pre-glycated either in the presence of glucose (b, e) or ribose (c, f) Note the presence of CML only when collagen was pre-glycated and the corresponding extension of the b1 labeling in most of the suprabasal layers of the epidermis Bar, 25 mm 465 466 48 The Use of Reconstructed Skin to Create New In Vitro Models Figure 48.4 Schematic representation of reconstructed skin produced either in absence of pre-glycated collagen (left) or when preglycated collagen was used (right) Note that basement membrane components (in brown and dark green) are more abundant when pre-glycated collagen was used together with other extracellular matrix molecules of the dermis (as mentioned) As indicated, also note the increased distribution of both b1 (in green) and a6 (in yellow) integrins in the epidermis epidermis is very thin as opposed to the deep dermis or reticular dermis, which constitutes the vast majority of this tissue The reticular dermis is characterized by the accumulation of thick fibers, which are thought to be responsible for the mechanical properties of the dermis It is now known that the fibroblast populations of these two regions are different, but only a small number of laboratories have investigated their relative properties including growth potential [16] So far their fate during aging remains totally unknown Both populations were isolated and then characterized, and their properties investigated as a function of aging This was made possible by numerous isolations of site-matched pairs from donors of increasing age to raise age-dependant collections of these cells; their growth characteristics, cytokine, and other diffusible factor production were studied by cell sorting and by performing cloning experiments Reconstructed skins with dermal compartments containing one population or the other were made and compared by means of dermal contraction and ability to participate to skin reconstruction in terms of capacity to promote epidermal morphogenesis To summarize, the reticular fibroblast population does not seem to be modified by aging as opposed to the papillary fibroblast population The papillary fibroblast population seems to disappear in conditions that comprise different possibilities like (1) apoptosis of papillary fibroblast, (2) replacement of papillary fibroblasts by upward migration of reticular fibroblasts, or (3) differentiation of papillary fibroblasts into reticular fibroblasts during aging This later hypothesis is a very interesting and provocative one currently under investigation because it means that papillary and reticular The Use of Reconstructed Skin to Create New In Vitro Models 48 Figure 48.5 Schematic representation of both young human skin (a: left) and old human skin (a: right) emphasizing the so far not completely elucidated modification occurring in the papillary dermis and the corresponding schematic representation of both young-like skin equivalent (b: left) or old-like skin equivalent (b: right) These schematic representations were made to emphasize the idea that reconstructed skin lacking the papillary dermal component is an easy first approximation of old skin fibroblasts represent different differentiation states of a single cell type The findings and these possibilities were recently published in detail and schematically represented [17] A simplified way to conclude is to consider that a model of reconstructed skin with a dermal compartment containing only reticular fibroblasts is a reasonable approach of a reconstructed skin model of aged skin (> Fig 48.5) 467 468 48 The Use of Reconstructed Skin to Create New In Vitro Models Flexibility of Reconstructed Skin and the Possibility to Standardize the Production: Towards RealSkin Flexibility is a very attractive property of the reconstructed skin model The shape (> Fig 48.6), the dermal thickness (> Fig 48.7), how to make several – at least two – dermal compartments can be varied in laboratories, and to manipulate the cell population content of this or these dermal compartment(s) including destruction of the fibroblast population by osmotic shock (> Fig 48.8) to provide a negative control if necessary Figure 48.6 Macroscopic view of collagen-contracted lattices or dermal equivalents cast in classical round petri dishes or square tissue culture dishes Note that the final form – round versus square – is the consequence of the shape of the tissue culture vessel used Bar, cm Glycation of the collagen represents an example of the possibility to modify the extracellular matrix The use of different fibroblast populations is an illustration of the possibility to change the cellular content in reconstructed skin Such flexibility was critical to create in vitro aging models In addition, it is also possible to standardize the fabrication of the reconstructed skin system to adapt it to production This was achieved by casting directly the fibroblast-contracted collagen gel or dermal equivalent in inserts placed in multiwell plates (> Fig 48.9) This system, which is a full-thickness reconstructed skin system was recently named RealSkin and is very close to the ‘‘historical’’ reconstructed skin system produced classically in individual petri dishes with the help of stainless steel rings (epidermal cell seeding) and stainless steel grids (air– liquid interphase) with the exception of the fact that tension is generated in the dermis It is however accompanied by the presence of more abundant extracellular matrix (ECM) macromolecules (> Fig 48.10) and increased production of MMP1 (> Fig 48.11) Interestingly, tension seems to be correlated to increased resistance to the effect of UVA light (> Fig 48.12) Future Models of Reconstructed Skin with Special Emphasis on Skin Aging Classical modifications of reconstructed skin include modifications of cell populations by enrichment consisting of incorporating new cell types generally in epidermis Figure 48.7 Histology of different types of reconstructed skin, whose dermal compartment was the result of fibroblast-contracted gels of mL (a), 10 mL (b), 20 mL (c) Note that the final thickness of the dermal equivalents obtained is the product of the initial volume used Bar, 25 mm The Use of Reconstructed Skin to Create New In Vitro Models 48 Figure 48.8 Histology of dermal equivalent containing living fibroblasts (a) and corresponding reconstructed skin (b), and dermal equivalents in which fibroblasts were destroyed by osmotic shock (c) and corresponding reconstructed skin (d) Note the presence of fibroblasts in a and b and absence of fibroblasts in c and d Bar, 25 mm Figure 48.9 Macroscopic view of classical reconstructed skin (a) made in individual petri dishes and reconstructed skin made in multiwell plates (b) Bar, cm 469 Figure 48.10 Classical reconstructed skin (a–g) and reconstructed skin produced in multiwell plates (h–n) Macroscopic view (a, h), Histology (b, i), Involucrin (c, j), Collagen IV (d, k), Vimentin (e, l), Procollagen I (f, m), PG4 proteoglycan epitope (dermatan/chondroitin sulfate epitope) (g, n) immunostainings Note increased presence of extracellular matrix material in reconstructed skin produced in multiwell plates as compared to classical reconstructed skin Also note that vimentin labeling, which shows orientation of fibroblasts in the dermal equivalent shows no tension in the dermis of classical reconstructed skin (random orientation) as opposed to tension visible in reconstructed skin produced in multiwell plates (horizontal orientation) Bar, 25 mm 470 48 The Use of Reconstructed Skin to Create New In Vitro Models The Use of Reconstructed Skin to Create New In Vitro Models Figure 48.11 Matrix Metallo Proteinase production in reconstructed skin Note increased production in classical reconstructed skin (in blue) as compared to reconstructed skin produced in multiwell plates (in red) like melanocytes to add pigmentation of the reconstructed skin It would also be of high interest, especially in the context of creating new models of skin aging to add new dermal cells especially endothelial cells as already done by others [18] Endothelial cells, which are the key components of blood vessels including skin blood vessels may interact preferentially with papillary fibroblasts [19] Moreover, dermal papillae, which are the image of skin vasculature form preferentially in the presence of papillary fibroblasts after grafting onto the nude mouse [20] Thus, it is possible to make dermal constructs with two compartments containing papillary fibroblasts and reticular fibroblasts by making serially two gels containing first reticular fibroblasts and then papillary fibroblasts so that it is quite possible to lay down endothelial cells at the interface on top of the first gel before casting the second gel Another expected modification of reconstructed skin of interest would be to introduce changes in the content of extracellular matrix molecules in dermal equivalents Little has been done in that area with the exception of combining collagen with glycosaminoglycans It would be of interest of course to introduce molecules other than collagen in dermal equivalent like elastin, for instance, it would also be of high interest to introduce various types of collagens specific from the different regions of the dermis like adding collagen III to better reproduce the 48 papillary dermis Other strategies in that area would also consist of replacing collagen classically provided by animal species like bovine collagen by human collagen or by replacing the collagen classically provided as individual molecules in solution by preparations in which the organization of collagen in fibers is better preserved in order to be closer to the actual structure and organization of collagen in skin In the context of creating new models of reconstructed skin for skin aging studies it is very interesting to investigate human genetic diseases by creating in vitro models of these diseases, incorporating skin cells isolated and amplified from small biopsies provided by patients For instance, the successful reproduction of the Xeroderma pigmentosum (XP) phenotype by showing lack of repair of DNA lesions in XP reconstructed skin after UV exposure [21] in a way strikingly recalling the in vivo situation Similarly, in the future, it would be of high interest to create new in vitro models of accelerated aging by incorporating human skin cells corresponding to other genetic diseases like Progeria or Werner An interesting point also is that skin is different as a function of the anatomic site For instance, in epidermis in certain regions of the body specific keratins are expressed, which are not seen elsewhere It is well known that palm of foot sole epidermis contains keratin 9, which is not found elsewhere There are certainly many regional differences, yet to be discovered that could become the subject of specific models representing skin of any region of the body like the face versus trunk or else, which may age differently At least this question could be investigated if corresponding reconstructed skins could be made and compared and whether they age the same way Another striking observation is that skin is obviously different as a function of ethnic origin at least by means of pigmentation Recently, it was illustrated that the Caucasian skin type versus African skin type is different not only in terms of pigmentation, but also morphogically in its depth since the dermal–epidermal junction is more invaginated in African skin relative to Caucasian skin (> Fig 48.13) Moreover, the production of certain cytokines like MCP1 is different in the two types of fibroblasts [22] suggesting that not only epidermis, but also dermis could be different Such results raise the question of whether the two types of skin age the same way or not since aging affects preferentially the dermal component of the skin To address this question reconstructed skins are made containing either Caucasian or African skin types keratinocytes and fibroblasts in order to look for differences This is a very interesting preliminary step prior to actual comparative aging studies, which means 471 Figure 48.12 Dose–response experiment of classical reconstructed skin (a–j) and reconstructed skin produced in multiwell plates (k–t) exposed to UVA light: control (a–p), 25 J/cm2 (b–q), 35 J/cm2 (c–r), 45 J/cm2 (d–s), and 55 J/cm2 (e–t) Histology (a–e and k–o), Vimentin labeling (f–j and p–t) Bar, 25 mm 472 48 The Use of Reconstructed Skin to Create New In Vitro Models The Use of Reconstructed Skin to Create New In Vitro Models 48 Figure 48.13 Histology of respectively Caucasian human skin (a) and African human skin (b) Note pronounced dermal–epidermal invaginations in African skin Bar, 25 mm Figure 48.14 Schematic representation of current ‘‘ideal’’ version of both young and old reconstructed skin models as seen at this point defined as being the closest to skin in vivo These so-called ideal models would comprise respectively the use of cells isolated from young or old donors and the fabrication of both papillary and reticular dermal compartments containing respectively papillary and reticular fibroblasts in the presence of a preglycated collagen characterized by thick fibers for the reticular dermis, while normal unglycated collagen would be used for the papillary dermal compartment 473 474 48 The Use of Reconstructed Skin to Create New In Vitro Models making reconstructed skins both age and ethnic origin dependant Finally, there are of course many other possibilities, which are not mentioned in this brief review It is of interest to note that in the near future not only classical anti-aging studies involving topical application of formulations will take place, but also so-called aesthetic approaches of anti-aging will become more and more fashionable However, there is a considerable lack of reliable scientific information in that area Therefore, experiments are initiated aiming at studying the effect produced by dermal fillers in the context of reconstructed skin in order to determine specific effects (beneficial vs adverse) as a function of the type of filler used and specific of the way the filler is distributed Conclusion Such considerations show that it is possible to make more complex reconstructed skin systems by varying simultaneously several parameters For instance, the knowledge of fibroblast collection has made it possible to consider simultaneously the type of fibroblast (papillary vs reticular) the age of the donor (young vs old), and the glycation status of the collagen used (preglycated vs normal) At this point, it is possible to imagine some kind of ideal normal or aged skin made in vitro in the near future For instance, such constructs could comprise in the case of ‘‘young’’ (normal) skin a thick reticular-like dermis containing reticular fibroblasts and made with a thick fibered collagen and a thin papillary dermis containing papillary fibroblasts and made with classical collagen on which keratinocytes would be grown while the corresponding ‘‘aged’’ skin would be made the same way except that the reticular dermal compartment would be made with glycated collagen and the papillary dermal compartment would be almost absent This is schematically represented in > Fig 48.14 The range of these possibilities is important and very promising for the future Cross-references > Aging of Skin Cells in Culture Acknowledgments We would like to thank Anne-Marie Minondo for performing the electron microscopy, and Catherine Olivry for the artwork References Yaar M, Eller MS, Gilchrest BA, et al Fifty years of skin aging J Invest Dermatol Symp Proc 2002;7:51–58 Farage MA, Miller KW, Elsner P, et al Intrinsic and extrinsic factors in skin ageing: a review Int J Cosmet Sci 2008;30:87–95 Boyce ST Cultured skin substitutes: a review Tissue Eng 1996;2:255–266 Bell E, Sher S, Hull B, et al The reconstitution of living skin J Invest Dermatol 1983;81:2S–10S Asselineau D, Prunieras M Reconstruction of ‘‘simplified’’ skin: control of fabrication Br J Dermatol 1984;111:219–221 Asselineau D, Bernhard B, Bailly C, et al Epidermal morphogenesis and induction of the 67 kD keratin polypeptide by culture of human keratinocytes at the liquid-air interface Exp Cell Res 1985;159: 536–539 Asselineau D, Bernhard B, Bailly C, et al Retinoic acid improves epidermal morphogenesis Dev Biol 1989;133:322–335 Marionnet C, Vioux-Chagnoleau C, Pierrard C, et al Morphogenesis of dermo-epidermal junction in a model of reconstructed skin: beneficial effects of vitamin C Exp Dermatol 2006;15(8):625–633 Oikarinen A, Karvonen J, Uitto J, et al Connective tissue alterations in skin exposed to natural and therapeutic UV-radiation Photodermatology 1985;2:15–26 10 Bernerd F, Asselineau D Successive alteration and recovery of epidermal differentiation and morphogenesis after specific UVBdamages in skin reconstructed in vitro Dev Biol 1997;183:123–138 11 Bernerd F, Asselineau D UVA exposure of human skin reconstructed in vitro induces apoptosis of dermal fibroblasts: Subsequent connective tissue repair and implications in photoaging Cell Death Differ 1998;5:792–802 12 Bernerd F, Asselineau D An organotypic model of skin to study photodamage and photoprotection in vitro J Am Acad Dermatol 2008;58:S155–S159 13 Frye EB, Degenhardt TP, Thorpe SR, et al Role of the maillard reaction in aging of tissue proteins J Biol Chem 1998;273: 18714–18719 14 Pageon H, Bakala H, Monnier VM, et al Collagen glycation triggers the formation of aged skin in vitro Eur J Dermatol 2007;17:12–20 15 Pageon H, Te´cher MP, Asselineau D, et al Reconstructed skin modified by glycation of the dermal equivalent as a model for skin aging and its potential use to evaluate anti-glycation molecules Exp Gerontol 2008;43:584–588 16 Harper RA, Grove G Human skin fibroblasts derived from papillary and reticular dermis: Differences in growth potential in vitro Science 1979;204:526–527 17 Mine S, Fortunel NO, Pageon H, et al Aging alters functionally human dermal papillary fibroblasts but not reticular fibroblasts: a new view of skin morphogenesis and ageing PLoS One 2008;3: 1–9 18 Black AF, Berthod F, L’heureux N, et al In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent FASEB J 1998;12:1331–1340 19 Sorrell JM, Baber MA, Caplan AI Human dermal fibroblast subpopulations; differential interactions with vascular endothelial cells in coculture: nonsoluble factors in the extracellular matrix influence interactions Wound Repair Regen 2008;16:300–309 20 Asselineau D, Te´cher MP, Caplan AI, et al Complex reconstructed skin equivalents made with papillary and reticular fibroblast The Use of Reconstructed Skin to Create New In Vitro Models populations incorporated in distinct layers: re-expression of papillary and reticular fibroblast characteristics after grafting onto nude mice J Invest Dermatol 2000;114:863 21 Bernerd F, Asselineau D, Vioux C, et al Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient 48 xeroderma pigmentosum skin in vitro Proc Natl Acad Sci USA 2001;93:7817–7822 22 Girardeau S, Mine S, Pageon H, et al The Caucasian and African skin types differ morphologically and functionally in their dermal component Exp Dermatol 2009;118:704–711 475 ... 11 19 10 2 Solutions and Products for Managing Female Urinary Incontinence 11 21 David J Caracci 10 3 Changes in Vulvar 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Chronology of Procedures 11 75 Alexander S Donath Part 10 7 Global Market Place for the Aged 11 85 Marketing and Product Design of Anti -aging. .. 611 0 Center Hill Avenue Cincinnati, OH 45224 USA miller.kw .1@ pg.com Howard I Maibach, M.D Professor Department of Dermatology University of California, School of Medicine San Francisco, CA 9 412 2