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Aging in Skin of Color: Disruption to Elastic Fiber Organization Is Detrimental to Skin’s Biomechanical Function

  • Abigail Kate Langton
    Affiliations
    Centre for Dermatology Research, The University of Manchester and Salford Royal NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK

    National Institute for Health Research, Manchester Biomedical Research Centre, Manchester University National Health Service Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK
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  • Sabrina Alessi
    Affiliations
    Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Mark Hann
    Affiliations
    Centre for Biostatistics, The University of Manchester, Manchester Academic Health Science Centre, Manchester, UK
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  • Anna Lien-Lun Chien
    Affiliations
    Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Sewon Kang
    Affiliations
    Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Christopher Ernest Maitland Griffiths
    Affiliations
    Centre for Dermatology Research, The University of Manchester and Salford Royal NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK

    National Institute for Health Research, Manchester Biomedical Research Centre, Manchester University National Health Service Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK
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  • Rachel Elizabeth Beatrice Watson
    Correspondence
    Correspondence: Rachel Elizabeth Beatrice Watson, Centre for Dermatology Research, The University of Manchester and Salford Royal National Health Service Foundation Trust, Manchester Academic Health Science Centre, Manchester, 1.721 Stopford Building, Oxford Road, Manchester, M13 9PT, UK.
    Affiliations
    Centre for Dermatology Research, The University of Manchester and Salford Royal NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK

    National Institute for Health Research, Manchester Biomedical Research Centre, Manchester University National Health Service Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK
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Open AccessPublished:November 04, 2018DOI:https://doi.org/10.1016/j.jid.2018.10.026
      Skin aging is a complex process involving the additive effects of time-dependent intrinsic aging and changes elicited via skin’s interaction with the environment. Maintaining optimal skin function is essential for healthy aging across global populations; yet most research focuses on lightly pigmented skin (Fitzpatrick phototypes I–III), with little emphasis on skin of color (Fitzpatrick phototypes V–VI). Here, we explore the biomechanical and histologic consequences of aging in black African-American volunteers. We found that healthy young buttock and dorsal forearm skin was biomechanically resilient, highly elastic, and characterized histologically by strong interdigitation of rete ridges, abundant organized fibrillar collagen, and plentiful arrays of elastic fibers. In contrast, intrinsically aged buttock skin was significantly less resilient, less elastic, and was accompanied by effacement of rete ridges with reduced deposition of both elastic fibers and fibrillar collagens. In chronically photoexposed dorsal forearm, significant impairment of all biomechanical functions was identified, with complete flattening of rete ridges and marked depletion of elastic fibers and fibrillar collagens. We conclude that in skin of color, both intrinsic aging and photoaging significantly impact skin function and composition, despite the additional photoprotective properties of increased melanin. Improved public health advice regarding the consequences of chronic photoexposure and the importance of multimodal photoprotection use for all is of global significance.

      Abbreviations:

      DEJ (dermal–epidermal junction), ECM (extracellular matrix), FRM (fibrillin-rich microfibrils)

      Introduction

      Skin aging is a complex process and involves the convergence of two distinct mechanisms: the subtle time-dependent effects of intrinsic aging, and the changes brought to bear on our skin by its constant interaction with the environment, predominantly chronic sun exposure. Historically, the majority of skin aging research has focused on defining the properties of lightly pigmented skin; intrinsic aging causes subtle changes to tissue structure (
      • Montagna W.
      • Carlisle K.
      Structural changes in aging human skin.
      ) and function (
      • Escoffier C.
      • de Rigal J.
      • Rochefort A.
      • Vasselet R.
      • Leveque J.L.
      • Agache P.G.
      Age-related mechanical properties of human skin: an in vivo study.
      ); consequently, it is relatively smooth, finely wrinkled, and unblemished, even in extreme old age. In contrast, at photoexposed anatomic sites, photoaging affects both the epidermis and dermis; epidermal changes include atrophy and flattening of the dermal–epidermal junction (DEJ) (
      • Allan A.K.
      The area of the dermo-epidermal junction in human skin.
      ), while in the dermis the accumulation of abnormally deposited amorphous elastin—termed solar elastosis (
      • Kligman A.M.
      Early destructive effect of sunlight on human skin.
      )—and disintegration of the well-organized elastic fiber network containing microfibrils rich in fibrillin (
      • Watson R.E.B.
      • Griffiths C.E.M.
      • Craven N.M.
      • Shuttleworth C.A.
      • Kielty C.M.
      Fibrillin-rich microfibrils are reduced in photoaged skin. Distribution at the dermal-epidermal junction.
      ) and fibulin-5 (
      • Kadoya K.
      • Sasaki T.
      • Kostka G.
      • Timpl R.
      • Matsuzaki K.
      • Kumagai N.
      • et al.
      Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis.
      ) prevail. The loss of elastic fiber integrity and DEJ effacement lead to a marked decline in skin elasticity (
      • Langton A.K.
      • Graham H.K.
      • McConnell J.C.
      • Sherratt M.J.
      • Griffiths C.E.M.
      • Watson R.E.B.
      Organization of the dermal matrix impacts the biomechanical properties of skin.
      ) and manifest clinically as deep coarse wrinkles and skin laxity (
      • Griffiths C.E.
      • Wang T.S.
      • Hamilton T.A.
      • Voorhees J.J.
      • Ellis C.N.
      A photonumeric scale for the assessment of cutaneous photodamage.
      ). Optimal skin health is desirable for all individuals; hence, a subspecialty of dermatology research has emerged that involves understanding skin of color (Fitzpatrick skin types IV–VI) (
      • Dadzie O.E.
      Skin of colour: an emerging subspeciality of dermatology.
      ). In highly pigmented skin, aging at photoexposed sites appears to manifest at a significantly slower rate and with less coarse wrinkling and laxity than is apparent in lightly pigmented skin (
      • Chien A.L.
      • Qi J.
      • Grandhi R.
      • Kim N.
      • Cesar S.S.A.
      • Harris-Tryon T.
      • et al.
      Effect of age, gender, and sun exposure on ethnic skin photoaging: evidence gathered using a new photonumeric scale.
      ). However, skin changes that manifest at photoprotected sites, such as buttock and upper inner arm, appear to be similar between individuals regardless of skin pigmentation (
      • Chien A.L.
      • Qi J.
      • Grandhi R.
      • Harris-Tryon T.
      • Kim N.
      • Jang M.S.
      • et al.
      Chronological aging in African-American skin: a reliable photonumeric scale demonstrates age and body mass index as contributing factors.
      ).
      Comparative studies of young black and white individuals have attempted to understand at the histologic level what is considered to be the defining characteristics of healthy skin. Such studies have identified differences in epidermal thickness and morphology, DEJ interdigitation and dermal extracellular matrix (ECM) composition (
      • Girardeau S.
      • Mine S.
      • Pageon H.
      • Asselineau D.
      The Caucasian and African skin types differ morphologically and functionally in their dermal component.
      ,
      • Girardeau-Hubert S.
      • Pageon H.
      • Asselineau D.
      In vivo and in vitro approaches in understanding the differences between Caucasian and African skin types: specific involvement of the papillary dermis.
      ,
      • Langton A.K.
      • Sherratt M.J.
      • Sellers W.I.
      • Griffiths C.E.
      • Watson R.E.
      Geographical ancestry is a key determinant of epidermal morphology and dermal composition.
      ). Skin biomechanical function has also been assessed in diverse cohorts and identified that skin architecture—both epidermal morphology and elastic fiber arrangement—are essential for providing skin’s optimal biomechanical properties (
      • Langton A.K.
      • Graham H.K.
      • McConnell J.C.
      • Sherratt M.J.
      • Griffiths C.E.M.
      • Watson R.E.B.
      Organization of the dermal matrix impacts the biomechanical properties of skin.
      ).
      An appreciation of skin biomechanics is important for our understanding of skin health and, despite numerous comparative studies characterizing these properties in health (
      • Dobrev H.P.
      A study of human skin mechanical properties by means of Cutometer.
      ,
      • Malm M.
      • Samman M.
      • Serup J.
      In vivo skin elasticity of 22 anatomical sites: the vertical gradient of skin extensibility and implications in gravitational aging.
      ), disease (
      • Dobrev H.P.
      In vivo study of skin mechanical properties in patients with systemic sclerosis.
      ,
      • Dobrev H.
      In vivo study of skin mechanical properties in psoriasis vulgaris.
      ,
      • Dobrev H.
      In vivo study of skin mechanical properties in Raynaud's phenomenon.
      ), and aging (
      • Dobrev H.
      Application of Cutometer area parameters for the study of human skin fatigue.
      ,
      • Krueger N.
      • Luebberding S.
      • Oltmer M.
      • Streker M.
      • Kerscher M.
      Age-related changes in skin mechanical properties: a quantitative evaluation of 120 female subjects.
      ) to date, no such studies have been performed examining the effects of aging on individuals with skin of color. With this in mind, the aims of the current study were: (i) to characterize the biomechanical properties of young and aged, photoprotected and photoexposed skin from cohorts of black African-American individuals and; (ii) relate these biomechanical properties to the underlying architecture of the epidermis and organization of the dermal ECM.

      Results

      The biomechanical properties of skin of color were determined using Cutometer (Courage + Khazaka Electronic, Köln, Germany) and ballistometer (Diastron, Andover, UK) devices. The Cutometer uses a probe to apply a negative pressure to the surface of the skin for a defined time period. The degree of skin deformation into the probe is recorded before the skin is allowed to recover on removal of the suction. The pressure–release cycle is repeated and a time–strain curve is generated (Figure 1a ), from which a number of biomechanical properties are calculated (Table 1) (
      • Dobrev H.
      Cutometer.
      ). The ballistometer is an additional dynamic testing device for skin elasticity that records and analyzes the rebound pattern of a small hammer striking the skin’s surface (Figure 1b and Table 1).
      Figure thumbnail gr1
      Figure 1Biomechanical testing reveals significantly reduced skin function in aged and photoaged skin of color. Graphic representation of the biomechanical properties obtained from application of the Cutometer (a) and ballistometer (b) to the skin. The biomechanical properties of black African-American buttock and forearm skin were determined using the Cutometer in mode 1 with a 4-mm aperture probe (c, e) and the ballistometer (d, f). Curves generated using these devices indicated that the biomechanical properties of young and intrinsically aged buttock skin were similar (c, d); however, intrinsically aged skin was significantly less resilient, had reduced elasticity and fatigue was apparent. In contrast, the Cutometer and ballistometer curves from young and chronically photoexposed forearm (e, f) reveal significant differences in all biomechanical properties between cohorts.
      Table 1Cutometer and Ballistometer Parameters
      ParametersDescription
      Cutometer parameters
       F3Surface area that envelopes the curves (the larger, the better)
       R0 (Uf)Height of the first maximal skin deformation
       R1 (Uf – Ua)Residual deformation, i.e., whether skin can return to its original position (the smaller, the better)
       R2 (Ua/Uf)Gross elasticity (closer to 1 = perfectly elastic)
       R4Skin fatigue (difference between last minimum value and first minimum value)
       R5 (Ur/Ue)Net elasticity (closer to 1 = more elastic)
       R6 (Uv/Ue)Viscoelastic to elastic ratio
       R7 Ur/UfElastic recovery (closer to 1 = more elastic)
       R9Hysteresis (difference between the last and the first maximal skin deformation)
       Ua–UrDelayed retraction
      Ballistometer parameters
       IndentationPeak penetration depth of the probe tip beneath the skin surface level (in mm)
       AlphaRate of energy damping (large values indicate non-elastic damping materials)
       Coefficient of restitutionBounce height relative to start height (large values indicate high elasticity of the sample)
       AreaSum of the area under the curve described by the probe vs. resting level of the surface of the skin

      Intrinsic aging compromises the biomechanical properties of black African-American skin

      The biomechanical properties of young buttock and dorsal forearm skin were first assessed using the Cutometer. Time–strain curves generated using this regime (Figure 1c–1e) indicated that the biomechanical properties of young buttock and forearm skin were similar; young skin is resilient (capable of returning to its original position following deformation; R1), exhibits minimal fatigue (R4), and is highly elastic (R2, R5, and R7). Comparisons between young and intrinsically aged buttock (Figure 1c and Table 2) revealed that overall time–strain curve shape (F3 envelope), total deformation (R0), and immediate deformation (Ue) were not significantly different. However, there was a decline in the ability of intrinsically aged skin to return to its original position after deformation (R1; P < 0.01) and increased fatigue was noted (R4; P < 0.01). Three parameters that measure different aspects of skin elasticity were all significantly impaired in intrinsically aged skin (R2: P < 0.01, R5: P < 0.001, and R7: P < 0.001). There were, however, no differences noted in viscoelastic properties (Uv, R6, and Ua-Ur) or hysteresis (R9) as a function of increasing age. Skin biomechanical testing of the buttock using the ballistometer (Figure 1d) identified no significant differences in indentation, alpha, coefficient of restitution, or area between cohorts (Table 2).
      Table 2Biomechanical Properties of Buttock and Forearm Skin
      ParameterButtock SkinForearm Skin
      Young, mean ± SEMOld, mean ± SEMP-ValueYoung, mean ± SEMOld, mean ± SEMP-Value
      Cutometry, 4 mm
       F3, mm253.4 ± 2.250.7 ± 1.8NS45.6 ± 1.836.0 ± 2.0<0.001
       Ue, mm0.72 ± 0.030.71 ± 0.03NS0.66 ± 0.020.58 ± 0.020.05
       Uv, mm0.22 ± 0.010.24 ± 0.01NS0.17 ± 0.010.21 ± 0.01<0.01
       Ua-Ur, mm0.32 ± 0.020.36 ± 0.02NS0.25 ± 0.010.33 ± 0.01<0.001
       R0, mm0.94 ± 0.040.95 ± 0.03NS0.83 ± 0.030.78 ± 0.03NS
       R1, mm0.08 ± 0.010.12 ± 0.01<0.010.08 ± 0.010.17 ± 0.01<0.001
       R2, AU0.92 ± 0.010.88 ± 0.01<0.010.90 ± 0.010.78 ± 0.01<0.001
       R4, mm0.15 ± 0.010.21 ± 0.02<0.010.16 ± 0.010.30 ± 0.02<0.001
       R5, AU0.77 ± 0.020.66 ± 0.02<0.0010.75 ± 0.020.49 ± 0.02<0.001
       R6, AU0.32 ± 0.010.34 ± 0.01NS0.26 ± 0.010.36 ± 0.01<0.001
       R7, AU0.58 ± 0.010.50 ± 0.01<0.0010.59 ± 0.010.36 ± 0.01<0.001
       R9, mm0.10 ± 0.010.10 ± 0.01NS0.08 ± 0.010.10 ± 0.01<0.01
      Ballistometry
       Indentation, mm0.71 ± 0.020.71 ± 0.02NS0.68 ± 0.020.63 ± 0.02NS
       Alpha, AU0.02 ± 0.00070.02 ± 0.0008NS0.02 ± 0.0010.03 ± 0.001<0.001
       CoR, AU0.80 ± 0.0060.79 ± 0.007NS0.81 ± 0.0070.75 ± 0.009<0.001
       Area, mm2103.1 ± 4.9103.8 ± 6.2NS107.6 ± 5.182.0 ± 5.8<0.01
      AU, arbitrary units; CoR, coefficient of restitution; NS, not significant; SEM, standard error of mean.

      Chronic photoexposure exacerbates the deterioration of skin’s biomechanical properties over that observed in intrinsically aged skin

      Skin biomechanical properties were further determined for extensor forearm, an anatomic site often chronically photoexposed. Cutometer time–strain curves indicated that marked differences in biomechanical properties were apparent at this site (Figure 1e and Table 2); overall curve shape was significantly different between cohorts (F3 envelope; P < 0.001) and, although aged forearm could be deformed to the same extent as that of young volunteers (R0; P = 0.304), there was a significant decline in immediate deformation (Ue; P < 0.05) of aged skin that was compensated for by an increase in the viscoelastic part of skin deformation (Uv: P < 0.01; R6: P < 0.001; Ua-Ur: P < 0.001). For all other biomechanical properties, aged forearm skin showed significant decline; it was less resilient (R1; P < 0.01), displayed both fatigue (R4; P < 0.001) and hysteresis (R9; P < 0.01), and all elasticity parameters were significantly impaired (R2, R5 and R7; all P < 0.001) compared to the same anatomic site of young volunteers.
      In agreement with Cutometer findings, data from the ballistometer further confirmed marked differences in skin’s biomechanical properties between young and aged photoexposed forearm. While indentation was not significantly different between cohorts, a significant decrease in skin elasticity (coefficient of restitution; P < 0.001) and area (P < 0.01), combined with a significant increase in alpha (P < 0.001), were indicative of aged forearm skin being more energy dampening and less elastic than that of forearm skin of young volunteers (Figure 1f and Table 2).
      Modeling of the curves generated by the Cutometer device was performed to understand the relative impact of deformation and relaxation of the tissue; in the pressure (deformation) cycles, coefficients for the rate of change were very large, indicating a very rapid initial change followed by an almost constant level of deformation for the remainder of the cycle. This identified that deformation rates were greater in older skin (Supplementary Table 1 online). In the relaxation cycles, the coefficients denoting rate of deformation and power (applied to time) are likely to be correlated. The power coefficients—across cycle, location and age—were very similar and so rates of deformation (here, returning to normal or baseline values) appeared to be greater in younger skin. This rate of change further appeared to be greater in tissue chronically photoexposed (forearm) between the young and aged volunteers when compared to the response observed in buttock skin (Supplementary Table 1).
      Hence, in intrinsically aged skin of color subtle variations in biomechanical properties existed when compared to young skin and were largely due to differences in skin elasticity (but not viscoelasticity), while parameters relating to deformation were comparable. However, in aged forearm skin, virtually all biomechanical properties were markedly different from those exhibited in young forearm. Therefore, if one defines optimal biomechanical skin function as the ability to deform and return to its original position without the onset of fatigue, then these data shows that intrinsically aged, and to a greater extent, chronically photoexposed, skin does not behave mechanically in an optimal manner.

      Intrinsic aging in aged skin of color impacts epidermal thickness alone while photoexposure results in additional flattening of the DEJ

      In addition to noninvasive biomechanical measurements, all volunteers were assessed at both anatomic sites for their pigmentary phenotype using a Chroma meter (Konica Minolta, Warrington, UK) and classified according to their individual typology angle (
      • Del Bino S.
      • Bernerd F.
      Variations in skin colour and the biological consequences of ultraviolet radiation exposure.
      ). Skin biopsies were also obtained from all volunteers at both test sites and processed for histologic investigation using the Warthin-Starry method for detection of melanin (
      • Joly-Tonetti N.
      • Wibawa J.I.
      • Bell M.
      • Tobin D.
      Melanin fate in the human epidermis: a reassessment of how best to detect and analyse histologically.
      ). Our study cohort demonstrated the diversity of pigmentation levels in black African-American individuals, with skin types ranging from lightest to darkest (Figure 2a ). A Pearson product-moment correlation coefficient was computed to assess the relationship between an individual’s individual typology angle and their epidermal melanin content (Figure 2b); a negative correlation exists between the two variables (r = –0.598, n = 78, P < 0.001) indicating that those individuals with a higher melanin content (as detected histologically) also have a lower individual typology angle value—indicative of a darker skin color phenotype. Furthermore, younger individuals tend to have darker skin than the aged cohort (Figure 2c).
      Figure thumbnail gr2
      Figure 2Epidermal thickness and DEJ convolution are significantly reduced in both intrinsically aged and chronically photoexposed skin of color. Our study cohort demonstrated the diversity of pigmentation levels in black African-American individuals, with skin types ranging from lightest to darkest (a). A Pearson product-moment correlation coefficient was computed to assess the relationship between an individual’s individual typology angle and their epidermal melanin content (b); a negative correlation exists between the two variables (r = –0.598, n = 78, P < 0.001). Furthermore, younger individuals tend to have darker skin than the aged cohort (c). Morphometric measurements of epidermal thickness and DEJ convolution index were analyzed using cryosections from young and aged African-American skin at buttock and forearm anatomic sites (d). Young skin had a significantly thicker epidermis and a more convoluted DEJ compared to intrinsically aged and chronically photoexposed skin (e). Scale bar = 100 μm. P < 0.05, ∗∗∗P < 0.001. DEJ, dermal–epidermal junction.
      Next, cryosections were stained with hematoxylin and eosin (Figure 2d) and epidermal morphometrics (Figure 2e) assessed for all volunteers. Young buttock and forearm were largely indistinguishable from one another with regard to epidermal thickness (mean ± standard error of mean; buttock: 52.44 ± 0.78 μm; forearm: 49.50 ± 0.93 μm), with strong interdigitation of rete ridges at the DEJ apparent at both anatomic sites (convolution index; buttock: 2.05 ± 0.06 arbitrary units; forearm: 1.51 ± 0.04 arbitrary units). In aged skin, epidermal thickness was significantly reduced at both buttock and forearm sites (buttock: 39.04 ± 1.73 μm; forearm: 40.12 ± 1.42 μm; both P < 0.001). In intrinsically aged buttock, although significantly reduced, interdigitation of rete ridges persisted (1.84 ± 0.08 arbitrary units; P < 0.05), whereas in chronically photoexposed forearm, near complete effacement of rete ridges and flattening of the DEJ were apparent (1.21 ± 0.03 arbitrary units; P < 0.001). Thus, it appears that epidermal thinning is characteristic of intrinsic aging in skin of color; whereas effacement of rete ridges is severely exacerbated by chronic photoexposure.

      Aging in skin of color is characterized by dermal matrix reorganization

      The biomechanical property of elasticity was significantly impaired in both intrinsically aged and chronically photoexposed skin of color; hence, we next performed immunohistochemical analyses of the major dermal elastic fiber components elastin (Figure 3a ), fibrillin-rich microfibrils (FRMs; Figure 3b) and fibulin-5 (Figure 3c) on biopsy samples of buttock and forearm. In young individuals at photoprotected buttock and photoexposed forearm sites, elastic fibers were arranged in distinctive candelabra-like arrays, connecting oxytalan fibers of the DEJ to elaunin fibers of the superficial papillary dermis. Immunohistochemical staining of intrinsically aged buttock skin identified a depletion of these structures at the DEJ; loss of elastic fiber architecture was accompanied by significant reductions in abundance for both FRMs (P < 0.001; Figure 3b) and fibulin-5 (P < 0.001; Figure 3c). Loss of elastic fiber architecture and abundance was further exacerbated in chronically photoexposed forearm; elastin and FRMs were severely truncated at the DEJ and their abundance was significantly reduced compared to young skin (elastin: P < 0.001; Figure 3a; FRM: P < 0.001; Figure 3b). Similarly, there was almost complete depletion of fibulin-5 at the DEJ and throughout the papillary dermis of the forearm which had been chronically photoexposed (P < 0.001; Figure 3c).
      Figure thumbnail gr3
      Figure 3Aging impacts the organization and abundance of elastic fibers and collagen I. Elastin (a), fibrillin-rich microfibrils (b), and fibulin-5 (c) were arranged in distinctive candelabra-like arrays in the superficial papillary dermis of young buttock and forearm. In contrast, intrinsically aged buttock and chronically photoexposed forearm showed significant depletion and remodeling of elastic fibers proximal to the dermal–epidermal junction. Picrosirius Red staining viewed under polarized light identified abundant organized fibrillar collagens in young buttock and forearm (d). In aged skin of color, the abundance of organized fibrillar collagen was significantly reduced in both intrinsically aged buttock and chronically photoexposed forearm. Immunofluorescent detection of mature collagen I (e) further confirmed that the overall intensity was significantly reduced in the papillary dermis of aged buttock and forearm. Scale bar = 50 μm. ∗∗P < 0.01, ∗∗∗P < 0.001.
      Fibrillar collagens within the dermal ECM provide skin with the biomechanical property of tensile strength; therefore, we next examined the abundance of organized fibrillar collagens within skin of color. Organized fibrillar collagens, when visualized by Picrosirius Red staining and polarized light microscopy, were not altered between body sites of young individuals (Figure 3d). However, both intrinsically aged buttock and chronically photoexposed forearm displayed significant loss of organized fibrillar collagens as compared to young subjects (P < 0.01 respectively; Figure 3d). Immunofluorescent detection of mature collagen I (Figure 3e) further confirmed that the overall intensity of collagen I was significantly reduced in the papillary dermis of aged buttock and forearm (P < 0.01 respectively; Figure 3e).

      Impaired biomechanical function is associated with altered epidermal morphology and reduced elastic fiber organization

      Using an X-Y-Z plot, the relationship between epidermal morphology (as measured by DEJ convolution) and FRM organization (as measured by the abundance of microfibril bundles at the DEJ) and biomechanical function (F3 Cutometer parameter) was explored (Figure 4). Using this visualization method, young buttock, young forearm, and intrinsically aged buttock all share similar biomechanical properties and exhibit the architectural features of strong rete ridge interdigitation and arborizing FRMs at the DEJ. However, chronically photoexposed forearm does not share these properties with the other groups; rather, these individuals cluster as a cohort where effacement of rete ridges, combined with significant truncation of FRMs at the DEJ, is strongly associated with a marked decline in in vivo biomechanical function.
      Figure thumbnail gr4
      Figure 4Association between biomechanical properties and skin architecture. An X-Y-Z plot (a) revealed that young African-American buttock (solid black circles) and intrinsically aged buttock (solid red circles) share similar biomechanical properties (F3 parameter) and architectural features as measured by DEJ convolution and elastic fiber abundance. Similarly, young African-American forearm (open black circles) shares these properties. However, chronically photoexposed African-American forearm (open red circles) did not share these properties with the other groups and clustered on the X-Y-Z plot at a position of reduced DEJ convolution, low elastic fiber abundance, and impaired biomechanical function. DEJ, dermal–epidermal junction.

      Discussion

      In this study, we established in aged skin of color that loss of DEJ convolution, disruption to elastic fiber arrangement, and reduced collagen organization appear to be detrimental to skin’s biomechanical behavior. Cutometry and ballistometry are useful methods that describe related, but not identical, aspects of skin biomechanics (
      • Woo M.S.
      • Moon K.J.
      • Jung H.Y.
      • Park S.R.
      • Moon T.K.
      • Kim N.S.
      • et al.
      Comparison of skin elasticity test results from the Ballistometer® and Cutometer®.
      ). The differences in measuring principle suggest that cutometry predominantly measures skin elasticity, while ballistometry predominantly measures stiffness (
      • Jemec G.B.
      • Selvaag E.
      • Agren M.
      • Wulf H.C.
      Measurement of the mechanical properties of skin with ballistometer and suction cup.
      ). Our findings suggest that ballistometry is a less-sensitive method than cutometry, as this device failed to identify any discernible differences between young and intrinsically aged buttock. That said, the cutometry time–strain curves for photoprotected buttock demonstrate how well the biomechanical function of intrinsically aged skin is preserved compared to young buttock skin (F3 parameter)—implying that this anatomic site largely functions at close to its optimal level for the life course of the individual. However, for chronically photoexposed forearm both devices were concordant in their findings; in the aged cohort all biomechanical properties were significantly impaired except for indentation (ballistometry) and total deformation (R0; cutometry)—essentially the same biomechanical measure (
      • Boyer G.
      • Laquieze L.
      • Le Bot A.
      • Laquieze S.
      • Zahouani H.
      Dynamic indentation on human skin in vivo: ageing effects.
      ). Previous studies of skin aging using individuals of Fitzpatrick phototypes I–III have identified several biomechanical parameters that change as a result of chronic photoexposure, including a decrease in immediate deformation (Ue) and deterioration of skin elasticity (R2, R5, and R7) (
      • Takema Y.
      • Yorimoto Y.
      • Kawai M.
      • Imokawa G.
      Age-related changes in the elastic properties and thickness of human facial skin.
      ), an increase in the prevalence of viscoelastic over elastic skin deformation (R6), and onset of skin fatigue (R4 and R9) (
      • Dobrev H.
      Application of Cutometer area parameters for the study of human skin fatigue.
      ). Similarly, in the current study, we identify that for skin of color after multiple deformations, chronically photoexposed forearm suffers a progressive loss of resilience, increased fatigue, with each subsequent curve having a lower amplitude and elastic retraction than observed for young skin. Furthermore, our results are consistent with those of others in that chronic photoexposure significantly increased the viscoelastic element of skin deformation (Uv, Ua-Ur, and R6), leading to hysteresis (the “creep” phenomenon) at the expense of elasticity (R2, R5, and R7) (
      • Boyer G.
      • Laquieze L.
      • Le Bot A.
      • Laquieze S.
      • Zahouani H.
      Dynamic indentation on human skin in vivo: ageing effects.
      ,
      • Cua A.B.
      • Wilhelm K.P.
      • Maibach H.I.
      Elastic properties of human skin: relation to age, sex, and anatomical region.
      ,
      • Dobrev H.
      Use of Cutometer to assess epidermal hydration.
      ,
      • Escoffier C.
      • de Rigal J.
      • Rochefort A.
      • Vasselet R.
      • Leveque J.L.
      • Agache P.G.
      Age-related mechanical properties of human skin: an in vivo study.
      ). Hence, skin of color at chronically photoexposed sites becomes less elastic and more viscous with age.
      Although it is difficult to assign skin biomechanical parameters to any single structural element of skin, the strength of this current study was in the obtainment of skin biopsies from all volunteers at both anatomic sites, allowing interrogation of the relationship between the tissues’ biomechanical properties and its underlying structure and the composition of its dermal ECM. In skin of color we show that reduced DEJ convolution and remodeling of the dermal elastic fiber network and, to a lesser extent, the fibrillar collagen matrix, result in modest impairment to biomechanical function in intrinsically aged skin. When superimposed onto this background of intrinsic aging, the effects of chronic photoexposure result in almost complete flattening of the DEJ and a profound loss of abundance and remodeling of dermal elastic fiber architecture leading to detrimental biomechanical properties. The interpretation of such results leads us to propose several hypotheses. The decrease in skin elasticity that occurs after 70 years of age might be, in part, the consequence of DEJ flattening resulting in a more fragile epidermal–dermal interface and an epidermis that is less resistant to shearing forces (
      • Lavker R.M.
      • Zheng P.S.
      • Dong G.
      Aged skin: a study by light, transmission electron, and scanning electron microscopy.
      ). Similarly, decreased skin elasticity and resilience could be related to the clear organizational changes occurring in the dermal elastic fiber network (
      • Daly C.H.
      • Odland G.F.
      Age-related changes in the mechanical properties of human skin.
      ,
      • Kadoya K.
      • Sasaki T.
      • Kostka G.
      • Timpl R.
      • Matsuzaki K.
      • Kumagai N.
      • et al.
      Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis.
      ,
      • Watson R.E.B.
      • Griffiths C.E.M.
      • Craven N.M.
      • Shuttleworth C.A.
      • Kielty C.M.
      Fibrillin-rich microfibrils are reduced in photoaged skin. Distribution at the dermal-epidermal junction.
      ). Compaction of the skin and widespread epidermal and dermal atrophy, in conjunction with loss of organized fibrillar collagen and increased collagen cross-linking, could lead to reduced tensile strength and stiffer skin (
      • Boyer G.
      • Laquieze L.
      • Le Bot A.
      • Laquieze S.
      • Zahouani H.
      Dynamic indentation on human skin in vivo: ageing effects.
      ,
      • Lovell C.R.
      • Smolenski K.A.
      • Duance V.C.
      • Light N.D.
      • Young S.
      • Dyson M.
      Type I and III collagen content and fibre distribution in normal human skin during ageing.
      ).
      In our aged skin of color cohort, we did not detect the characteristic accumulation of dystrophic elastin within the dermis, termed solar elastosis (
      • Han A.
      • Chien A.L.
      • Kang S.
      Photoaging.
      ,
      • Kligman A.M.
      Early destructive effect of sunlight on human skin.
      ). Solar elastosis has been implicated as a significant driver of altered biomechanical function in lightly pigmented skin (
      • Langton A.K.
      • Graham H.K.
      • McConnell J.C.
      • Sherratt M.J.
      • Griffiths C.E.M.
      • Watson R.E.B.
      Organization of the dermal matrix impacts the biomechanical properties of skin.
      ); however, its absence in chronically photoexposed skin of color suggests that it is not only the deposition of dystrophic elastin that causes loss of biomechanical function, but also, the remodeling of FRM in the superficial papillary dermis that impacts skin’s elastic function. The absence of solar elastosis in highly pigmented skin suggests that the mechanisms underlying skin aging, at least at photoexposed sites, may differ from those observed in lightly pigmented skin. It would be interesting to further extend these biomechanical studies to include additional anatomical sites, such as the photoprotected volar forearm and photoexposed temporal region (
      • Dobrev H.
      Application of Cutometer area parameters for the study of human skin fatigue.
      ). Similarly, Asian individuals are also classified as having skin of color and, to date, just a few studies have examined the effects of skin aging on this diverse population. Skin elasticity is compromised in photoaged Asian facial skin (
      • Ahn S.
      • Kim S.
      • Lee H.
      • et al.
      Correlation between a Cutometer and quantitative evaluation using Moire topography in age-related skin elasticity.
      ,
      • Nam G.W.
      • Kim E.J.
      • Jung Y.C.
      • Jeong C.B.
      • Shin K.H.
      • Lee H.K.
      Differences in skin properties of Korean Women at the initial aging phase.
      ,
      • Park S.Y.
      • Shin Y.K.
      • Kim H.T.
      • et al.
      A single-center, randomized, double-blind, placebo-controlled study on the efficacy and safety of “enzyme-treated red ginseng powder complex (BG11001)” for antiwrinkle and proelasticity in individuals with healthy skin.
      ); however, it remains unknown to what extent other biomechanical properties are affected. Performing a thorough assessment of both biomechanical and histologic analyses of young and aged Asian skin is therefore warranted.
      Irrespective of level of constitutive pigmentation, commonalities exist between histologic composition and skin biomechanical function. Deterioration of biomechanical properties, alterations to epidermal morphology and remodeling of dermal ECM appear to materialize in photoexposed forearm in individuals with skin of color by the 8th decade. In contrast, these properties are evident in lightly pigmented forearm by the 3rd decade (
      • Langton A.K.
      • Alessi S.
      • Chien A.L.
      • Kang S.
      • Griffiths C.E.M.
      • Watson R.E.B.
      The impact of ageing and chronic sun exposure on the biomechanical function and histological composition of black African-American skin.
      ). High pigmentation in black skin is thought to provide a 20–70× level of protection from UVR (
      • Gloster H.M.
      • Neal K.
      Skin cancer in skin of color.
      ,
      • Norval M.
      • Kellett P.
      • Wright C.Y.
      The incidence and body site of skin cancers in the population groups of South Africa.
      ) and appears to preserve skin architecture and biomechanical function by an additional 40–50 years compared to white skin. The eventual deterioration of skin function and architecture might, however, coincide with the reduced pigmentation and reddening of skin that has been identified in individuals with skin of color aged 65 years or older versus those aged 18–30 years (
      • Chien A.L.
      • Suh J.
      • Cesar S.S.A.
      • Fischer A.H.
      • Cheng N.
      • Poon F.
      • et al.
      Pigmentation in African American skin decreases with skin aging.
      ).
      The long-lived nature of dermal ECM components (
      • Shapiro S.D.
      • Endicott S.K.
      • Province M.A.
      • Pierce J.A.
      • Campbell E.J.
      Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weapons-related radiocarbon.
      ) makes them particularly susceptible to gradual accumulation of damage. During photoaging, one important driver of ECM remodeling in lightly pigmented skin is the ultraviolet-induction of matrix metalloproteinases (
      • Fisher G.J.
      • Wang Z.Q.
      • Datta S.C.
      • Varani J.
      • Kang S.
      • Voorhees J.J.
      Pathophysiology of premature skin aging induced by ultraviolet light.
      ). However, it has been demonstrated that the UV induction of metalloproteinases is attenuated in skin of color (
      • Fisher G.J.
      • Kang S.
      • Varani J.
      • Bata-Csorgo Z.
      • Wan Y.
      • Datta S.
      • et al.
      Mechanisms of photoaging and chronological skin aging.
      ,
      • Wang F.
      • Garza L.A.
      • Cho S.
      • Kafi R.
      • Hammerberg C.
      • Quan T.
      • et al.
      Effect of increased pigmentation on the antifibrotic response of human skin to UV-A1 phototherapy.
      ). An additional mechanism shown to play a role in mediating damage to ECM components is oxidative stress induced by reactive oxygen species (see review by
      • Naidoo K.
      • Hanna R.
      • Birch-Machin M.A.
      What is the role of mitochondrial dysfunction in skin photoaging?.
      ). Human skin is exposed to reactive oxygen species generated from both environmental sources, such as photoexposure, but also by airborne pollutants and particulate matter, diet, and endogenous oxidative metabolism (
      • Vierkotter A.
      • Krutmann J.
      Environmental influences on skin aging and ethnic-specific manifestations.
      ). In addition to UVR, exposure to infrared A—which comprises one-third of total solar energy—is capable of directly affecting cells located in the epidermis, dermis, and subcutis (reviewed in
      • Schieke S.M.
      • Schroeder P.
      • Krutmann J.
      Cutaneous effects of infrared radiation: from clinical observations to molecular response mechanisms.
      ). infrared A induces skin damage akin to that caused by UVR (
      • Schroeder P.
      • Calles C.
      • Benesova T.
      • Macaluso F.
      • Krutmann J.
      Photoprotection beyond ultraviolet radiation--effective sun protection has to include protection against infrared A radiation-induced skin damage.
      ), and can trigger both the induction of metalloproteinases (
      • Schroeder P.
      • Lademann J.
      • Darvin M.E.
      • Stege H.
      • Marks C.
      • Bruhnke S.
      • et al.
      Infrared radiation-induced matrix metalloproteinase in human skin: implications for protection.
      ) and an increase in intra-mitochondrial production of reactive oxygen species (
      • Darvin M.E.
      • Haag S.
      • Meinke M.
      • Zastrow L.
      • Sterry W.
      • Lademann J.
      Radical production by infrared A irradiation in human tissue.
      ,
      • Schroeder P.
      • Pohl C.
      • Calles C.
      • Marks C.
      • Wild S.
      • Krutmann J.
      Cellular response to infrared radiation involves retrograde mitochondrial signaling.
      ). Thus, although high constitutive pigmentation may protect skin from the deleterious dermal remodeling events that are triggered by UVR exposure, skin of color may accumulate time-related damage via exposure to infrared, visible light, pollution, and particulate matter—all of which generate reactive oxygen species.
      The results presented here provide a detailed insight into the interplay between skin architecture and its effect on biomechanical function; an appreciation of these properties is important for our understanding of skin health and the impact of disease and aging. Our data examine the consequences of cutaneous aging in skin of color and identify that despite the photoprotective properties of melanin, chronic photoexposure exacerbates features of aging skin at both the histologic and functional level. This further promotes the need for improved public health advice regarding the consequences of chronic sun exposure and the importance of multimodal photoprotection use for all, regardless of ethnicity.
      A better understanding of skin health is of global importance, as highlighted by the recent World Health Organization report on aging (
      • Blume-Peytavi U.
      • Kottner J.
      • Sterry W.
      • Hodin M.W.
      • Griffiths T.W.
      • Watson R.E.
      • et al.
      Age-associated skin conditions and diseases: current perspectives and future options.
      ); only when we understand how to promote and maintain skin health throughout the life course—in all of its diversity—will we be able to make significant improvements to the clinical management of skin disease (be that age-related or not) and relieve the socioeconomic burden related to an aging global population.

      Materials and Methods

      Participants

      Healthy, black African-American (Fitzpatrick skin phototype V–VI; mean age ± standard deviation; young: 23.9 ± 3.6 years; men: n = 4; women: n = 17; old: 70.8 ± 5.9 years; men: n = 7; women: n = 11) volunteers were recruited to the study. Local ethical approval was obtained from The Johns Hopkins Institutional Review Board. Written informed consent was obtained from the participants and the study adhered to Declaration of Helsinki principles. Basic demographic information was collected and participants were asked to self-declare their ethnicity.

      Measurement of skin biomechanical properties and colorimetry

      Test sites were selected on the buttock (at the midpoint between the intergluteal cleft and the lateral border) and forearm (at the midpoint between the dorsal, proximal wrist crease and olecranon process). The Cutometer MPA580 (Courage + Khazaka Electronic) with a 4-mm aperture probe (mode 1; 3-second suction followed by 3-second relaxation period, for a total of 10 cycles using a negative pressure of 450 mbar) and the ballistometer were applied to three adjacent but nonoverlapping areas at each anatomical test site. A Chroma Meter (CR-400; Konica Minolta) was used to measure the L* and b* parameters of the standard CIE L*a*b* color space at both anatomic sites. Further details are provided in Supplementary Materials and Methods online.

      Biomechanical data modeling

      To further interrogate the biomechanical behavior, a standard exponential regression model of deformation on time was fitted to data collected in the deformation cycle, while a Weibull-type regression model was fitted to data collected in the relaxation cycle. In the latter case, time (T) was better modeled as a power function (i.e., Tp) rather than linearly (i.e., T) as indicated by a higher model-adjusted R2 and smaller residuals. The full functional form of both models is presented in Supplementary Materials and Methods. Regression coefficients for the second, sixth, and tenth (of 10) deformation-relaxation cycles were analyzed, that is, an early, mid, and late cycle, to assess their consistency of biomechanical response.

      Biopsy procurement and sample preparation

      Once all noninvasive measurements had been completed, 6-mm diameter punch biopsies were obtained from all volunteers at the two anatomic sites. Each skin biopsy was obtained under 1% lidocaine local anesthesia. At the time of procurement, biopsies were snap-frozen in liquid nitrogen and stored at –80°C. Biopsies were cryosectioned at 7 μm in a single run, using the same blade and the same cryostat settings.

      Histologic staining and immunohistochemistry

      Epidermal melanin was assessed using the modified Warthin-Starry procedure (
      • Joly-Tonetti N.
      • Wibawa J.I.
      • Bell M.
      • Tobin D.
      Melanin fate in the human epidermis: a reassessment of how best to detect and analyse histologically.
      ), epidermal morphology was assessed using hematoxylin and eosin staining and Picrosirius Red staining for fibrillar collagens. Immunohistochemistry was performed using mouse monoclonal antibodies to detect elastin (clone BA4, dilution 1:500; Sigma-Aldrich, Watford, UK) and FRMs (clone 11C1.3, dilution 1:1000; Neomarkers, Runcorn, UK). Rabbit polyclonal antibody was used to detect fibulin-5 (catalog #HPA000848; dilution 1:500; Sigma-Aldrich, St Louis, MO). See Supplementary Materials and Methods for detailed protocols.

      Microscopy, image analysis, and statistical testing

      Brightfield and cross-polarized images were captured using a BX53 microscope (Olympus Industrial, Southend-on-Sea, UK) and image analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD) (
      • Abramoff M.D.
      • Magelhaes P.J.
      • Ram S.J.
      Image processing with ImageJ.
      ). DEJ convolution index was measured using the method described previously (
      • Langton A.K.
      • Sherratt M.J.
      • Sellers W.I.
      • Griffiths C.E.
      • Watson R.E.
      Geographical ancestry is a key determinant of epidermal morphology and dermal composition.
      ). Statistical analysis was performed using GraphPad Prism, version 7.01 (GraphPad Software, La Jolla, CA). Results were considered significant if P < 0.05 (95% confidence level).

      Conflict of Interest

      The Centre for Dermatology Research is in receipt of research grants from Walgreens Boots Alliance and Unilever UK Limited. Chris E.M. Griffiths is Director of CGSkin Limited. The remaining authors state no conflict of interest.

      Acknowledgments

      Christopher E.M. Griffiths is a National Institute for Health Research Senior Investigator. Christopher Griffiths and Rachel E. B. Watson are supported in part by the National Institute for Health Research Manchester Biomedical Research Centre . This study was funded by a program grant from Walgreens Boots Alliance .

      Supplementary Material

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