J. Soc. Cosmet. Chem., 32, 15-26 (January/February 1981)
`
`Use of nonintrusive tests to monitor age-associated
`changes in human skin
`
`GARY L. GROVE, PH.D., ROBERT M. LAVKER, PH.D., ERHARD HOELZLE,
`M.D., and ALBERT M. KLIGMAN, M.D., PH.D., Simon Greenberg Foundation, Skin Study
`Center, 3901 Market Street, Philadelphia, PA 19104.
`
`Received July 24, 1980. Presented at the Society of Cosmetic Chemists' Annual Scientific Seminar,
`San Francisco, CA, May 15-16, 1980.
`
`Synopsis
`Nonintrusive tests can be used to objectively characterize and quantitatively evaluate in vivo those changes
`in the physiological properties of skin due to aging. Such a testing procedure presents no untowards risks
`and has proven to be quite palatable to normal healthy volunteers. A number of age-associated changes in
`skin structure and function can be monitored in this manner. These include changes in SKIN SURFACE
`ANATOMY such as loss of DERMATOGLYPHICS and altered patterns of CORNEOCYTE
`DESQUAMATION. Physiological decrements such as diminished eccrine sweating, epidermal CELL
`RENEWAL and HEALING of superficial skin wounds can also be evaluated. Moreover, by using a
`variety of excitants which when placed on the skin induce specific reactions, viz. erythema, wheals,
`stinging, etc., an age associated loss to express these reactions has been demonstrated.
`
`INTRODUCTION
`
`It seems likely that changes which occur in human skin with advancing age may be of
`some value in monitoring the senescent process not only for that organ but perhaps
`for the entire individual as well (1). Such an ability to measure aging biologically on the
`basis of structural and functional alterations in skin rather than just chronologically on
`the basis of birth certificate information would be of great benefit to the investigative
`gerontologist. Unfortunately, most testing procedures which are used to study skin
`structure and function require biopsies or some other surgical manipulation to be
`performed. There is no doubt that most human subjects, especially those with no
`dermatological problems, find these invasive testing procedures objectionable. What is
`really needed is a testing strategy that is palatable to human volunteers. Such
`procedures should be conveniently administered, cause little or no discomfort, present
`no untoward risks and leave no permanent scars or pigmentary changes.
`The object of this paper is to summarize some preliminary results of a pilot study of
`aging human skin designed primarily to test the feasibility of this nonintrusive
`
`approach. This is a cross-sectional investigation and involves a comparison of two
`
`15
`
`L'OREAL USA, INC. EX. 1021
`
`

`

`16
`
`.JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
`
`age-cohorts: young adults 20-35 years of age and older adults 60-75 years of age. Both
`
`
`
`groups are comprised of normal healthy white volunteers (6 males and 6 females in
`
`
`
`
`
`each) who had given informed consent. To avoid differences due to changes in
`
`
`
`
`
`environmental conditions or protocols, both cohorts were tested concurrently and all
`
`
`
`assessments were made independently by three graders.
`
`REPLICAS AND SKIN SURFACE BIOPSIES
`The skin surface is organized into complex patterns of ridges and furrows which
`
`
`
`
`
`
`
`
`
`
`presumably enable the stratum corneum to undergo deformation in a variety of
`
`
`
`
`
`directions without subsequent loss of integrity (2). With the exception of palmar
`
`
`
`
`
`
`surfaces ("finger prints") information on the dermatoglyphics of the remaining body
`
`
`
`areas is minimal, especially regarding age changes.
`
`
`
`
`With this in mind a special technique has been developed which for the
`
`
`
`
`
`studying dermatoglyphics, is superior to the usual replicas using si
`
`purpose of
`impressions.
`(cid:127)icone or dental wax
`
`
`
`This entails applying a thin layer of polyvinylidine chloride emulsion
`
`
`
`(Duran, Merck) which is actually a liquid form of Saran Warp©. When dry, a thin sheet
`
`
`
`
`can be peeled off which is an exact negative replica of the underlying skin surface. This
`
`
`
`
`
`translucent specimen serves as a permanent record of the dermatoglyphics of that
`
`
`
`
`
`subject. Recent studies indicate that the dermatoglyphics of older adults are highly
`
`
`
`
`
`
`
`irregular and lack the orderly arrangement of geometric patterns typical of the young
`
`
`
`
`
`
`(3). Exposed areas seem to exhibit the greatest alteration in dermatoglyphics with
`
`
`
`
`patterns in some areas of the dorsal hand being almost totally obliterated (Figure 1).
`
`(cid:127)
`
`,.--, (cid:127)
`
`(cid:127)
`
`-,(cid:127)
`
`g
`
`.
`
`(cid:127)
`
`..(cid:127)'..' (cid:127):" .
`
`Figure 1. a) Dermatoglyphic pattern of the dotsurn of the hand of the young. Major (ML) and secondary
`
`
`
`
`
`
`
`
`
`
`
`
`(SL) l(cid:127)es traverse to form a highly ordered pattern of triangles. WithM the triangles the corneocytes are
`
`
`
`
`
`
`
`arranged in a honeycomb fashion (arrow) (x 15). b) Dermatoglyphic pattern of the dotsurn of the hand of
`
`
`
`
`
`
`
`the old. Note the lack of an ordered geometric pa(cid:127)ern due to absence of secondary IMes. Remnants of the
`
`
`
`
`
`honeycombed corneocytes are occasionally obse(cid:127)ed (arrow) (x 15).
`
`

`

`NONINTRUSIVE
`
`TESTING
`
`OF SKIN
`
`17
`
`Such a loss of dermatoglyphics may be correlated with a decrease in skin elasticity and
`an increased tendency to crack and fissure. The underlying events for this change are
`not completely understood but may be related to atrophic changes in the dermis (4).
`By changing the nature of the replica material one can obtain information regarding
`other structural and functional changes in aged skin. For example, the silicone
`technique as originally devised by Sarkany and Gaylarde (5) and later improved by
`Harris, Polk and Willis (6) can be used to evaluate sweating. To take an imprint a
`mixture of silicone base and catalyst (Syringe Elasticon, Kerr) is applied as a thin film
`immediately after drying the skin surface. Because sweat is immiscible with silicone,
`each sweat droplet forms a globular hole in the silicone layer. This rubbery sheet can
`then be peeled off and forms a permanent record for which the density and output of
`active sweat glands can be estimated.
`Preliminary results using this procedure suggest that sweating capacity is diminished in
`older adults (Figure 2). Previous studies by Silver, et al. (7) have indicated that the
`number of digtal sweat glands visualized by a starch-iodine film technique decreases
`with advancing age. By using these nonintrusive approaches in conjunction with
`pharmacological agents known to promote (Pilocarpine) or block (Scopalomine)
`sweating, additional insight on the nature of the apparent age-related differences in
`eccrine gland function should be obtained.
`The skin surface biopsy method of Marks and Dawber (8) results in the removal of a
`sheet of horny cells from the superficial stratum corneum, usually five to six cell layers.
`It should be emphasized that this is not a replica technique but actually removes the
`outermost portion of the horny layer and thus provides biological material which can
`be subsequently analyzed in a variety of ways. To obtain this sample, one drop of
`
`-
`
`Figure 2. a) Sweat gland imprint from the volar forearm of the young. Light circles represent
`perforations in the film of silicone material formed by sweat droplets from functioning sweat glands
`(x 15). b) Sweat gland imprint from the volar forearm of the old. Note diminished amount of functioning
`sweat glands (x 15).
`
`

`

`18
`
`JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
`
`Figure 5. Scanning electron micrograph of a skin surface biopsy from the calf of the young. In addition
`to the easily recognized ridges which constitute the dermatoglyphic pattern, the desquamation of
`
`corneocytes in single and small groups can also be resolved (arrows) (x 250).
`
`cyanoacrylate adhesive (Aron-alpha, Vigor Co.) is placed on a 3 x ! glass slide which
`is then pressed against the area to be sampled. After approximately 30 s, the glass slide
`is removed with its adherent horny layer. This specimen can then be used to study the
`dermatoglyphic patterns as well as the configuration and patterns of desquamation
`with the scanning electron microscope (Figure 3).
`
`INDIRECT
`
`ASSESSMENT
`
`OF EPIDERMAL
`
`PROLIFERATIVE
`
`ACTIVITY
`
`Horny cells are continually being lost into the environment due to exfoliation.
`Normally, the renewal system of the epidermis operates under steady-state kinetics;
`thus for every cell lost, a new cell must be produced in the basal layer. Since all the
`intervening layers are of a simple transit type, cytokinetic analysis of the stratum
`corneum provides indirect assessment of epidermal cell proliferation (9,!0).
`One parameter which can be measured in such a renewal system is transit time, i.e., the
`time required for a cell to travel through a compartment. Since horny cells are tightly
`
`

`

`NONINTRUSIVE
`
`TESTING
`
`OF SKIN
`
`19
`
`Non-Intrusive Assessments of Epidermal Proliferative Activity (cid:127)
`
`I
`Table
`
`Transit Time
`Number of
`Turnover Rate
`(days)
`Cell Layers
`(h/layer)
`
`19.8 + 1.39
`28.1 _+ 2.66
`
`17.7 + 2.02
`25.5 + 2.63
`
`17.0 + 0.83
`16.8 + 0.66
`
`14.3 + 0.61
`13.9 -+ 0.81
`
`28.3 + 1.2
`$0.8 + 3.8
`
`30.0 + 2.6
`46.6 + 6.5
`
`Site
`
`Volar Forearm
`<35
`>60
`
`tipper Inner Arm
`<35
`>60
`
`(cid:127)Results are means _+ S.E.
`
`bound, they move in unison through the stratum corneum. Thus, in this special case,
`transit time is equivalent to turnover time, i.e., the time required for a compartment to
`renew or replace itself. This value can be measured nonintrusively by determining with
`the aid of a Wood's lamp, the time required for a fluorescent marker (Dansyl Chloride)
`to disappear from fully stained horny layer (10). This dye binds avidly to the horny
`
`layer only and its rate of disappearance is not influenced by washing or protecting the
`surface. Our preliminary results with Dansyl Chloride Disappearing Method (Table I)
`indicate that for both the volar forearm and upper inner arm, stratum corneum transit
`times of young adult subjects are approximately 18 to 20 days. In older subjects, this
`was lengthened about eight to nine days at both sites indicating that epidermal
`proliferation decreases with age.
`It should be emphasized that transit time values can be misleading unless they are
`related to the number of cell layers (11). If cell proliferation is the same, the time for a
`cell to reach the skin surface will be longer if there are more cell layers. Thus, transit
`times should be corrected for differences in number of cell layers and converted to
`turnover rates. Estimating cell layers entails raising up small blisters with ammonium
`hydroxide, cryostat sectioning the blister roof and treating it with alkali (12). The
`horny cells swell up and the number of cell layers can easily be counted. Our
`preliminary results (Table I) reveal a site difference but no age differences with regard
`to the number of cell layers in the stratum corneum. Thus, the increased transit time
`values observed in older subjects is truly a reflection of diminished proliferative
`activity. In fact, the calculated turnover values indicate that on the average it takes
`about 30 hours to replace each horny cell layer in younger subjects and about 45 hours
`for older subjects.
`In addition to providing a stratum corneum sample for cell layer counts, the blister
`roofs can be subjected to additional types of analyses, such as moisture avidity or
`physiochemical properties (13). It is apparent that much will be learned about the
`structural and functional properties of the stratum corneum from such a comprehen-
`sive examination of blister roof samples.
`
`WOUND
`
`HEALING
`
`The unroofed blister site represents a reasonable standard superficial wound at which
`the dermatoglyphics have been completely obliterated. Thus, by observing the
`restoration of the original markings we can objectively evaluate the rate of wound
`
`

`

`20
`
`JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
`
`Week
`
`I.
`
`Week 2
`
`Week 5
`
`.
`
`ß
`
`..
`
`[::;;:' t __
`
`:'"- (cid:127)
`
`......
`E-]No Dermatoglyphics (cid:127)Minor
`Lines Filling In
`:::(cid:127)Mojor Lines Returning (cid:127)Complete Restoration
`
`Figure 4. Wound healing following unroofing of NH4OH blisters on volar forearms of human subjects.
`
`healing. Figure 4 shows some preliminary results with regard to the volar forearm site.
`Note that by the end of the first week, all young subjects had begun to reestablish
`major lines, while half of the older ones still show a complete absence of lines. By the
`end of the second week, most of the young and many of the old had reestablished all
`major lines and were beginning to fill in the minor ones. The differences in rate of
`wound healing become even more pronounced in subsequent weeks. By the fourth
`week, all young subjects had completely restored their dermatoglyphic pattern. In
`contrast, only 25% of the older group had achieved this stage, and in fact, one
`individual had not even completed reestablishment of the major lines. By the end of
`the sixth week, all but one of the older subjects had completely restored their
`dermatoglyphics. A similar progression of events was observed in the upper inner arm
`site.
`
`BLISTER
`
`FORMATION
`
`The ability to raise up blisters has prov&d useful in a variety of ways in this
`investigation. Frosch and Kligman (14) originally described the induction of blisters by
`ammonium hydroxide (NH4OH). In this procedure, a 1:1 aqueous solution of NH4OH
`is placed in a 14-mm well drilled in an acrylic plastic block which is held snugly to the
`skin surface. During the exposure, the site is examined under good lighting at 30-s
`intervals. The time required for the appearance of tiny vesicles around the follicles is
`termed minimal blistering time (MBT). The exposure is continued and the additional
`time required for a fully tense blister to form, blister filling time (BFT), is also
`measured.
`
`Table II compares blister formation of the two age groups on the volar forearm and
`upper inner arm. At both sites, the young achieved an MBT response after 13 to 14 min
`of exposure, while in striking contrast, the older subjects as a group took only half as
`long. At the present time, it is not clear what accounts for these differences.
`
`

`

`NONINTRUSIVE
`
`TESTING-
`
`OF SKIN
`
`21
`
`II
`Table
`Blister Formation Induced by NH4-OH Exposure
`M.B.T. x
`
`12.5 + 1.68
`6.6 + 0.87
`
`14.0 + 2.11
`6.1 _+ 0.87
`
`Site
`
`Volar Forearm
`<35
`>60
`
`Upper Inner Arm
`<35
`>60
`
`B.F.T. 2
`
`11.7 + 1.96
`24.8 + 5.66
`
`17.3 + 3.00
`31.5 -+ 6.79
`
`(cid:127)Minimal Blistering Time, mean _+ S.E. in min.
`2Blister filling Time, mean _+ S.E. in min.
`
`Table II also shows that in younger subjects, within 10 to 15 min after achieving an
`MBT, a tense blister appears. By contrast, Blister Filling Time is over 25 min for older
`subjects. Indeed, in two cases, the experiment was terminated at 90 minutes without
`obtaining a blister even though a follicular (MBT) pattern was evident. This suggests
`that integumental reactivity may be impaired in older subjects.
`To gain additional insight into this problem, a brief survey of integumental reactivity
`was conducted using a variety of excitants which when placed on the skin induce
`specific reactions, viz. erythema, wheals, vasodilation, stinging, etc. Preliminary
`findings reveal that in general older subjects as a group are less reactive (Figure 5).
`Such an age-related loss in the capacity to express certain inflammatory reactions has
`serious implications. For one thing, it means that certain clinical signs may be muted
`or missing entirely, making diagnosis of even common dermatological problems
`extremely difficult in older patients. Moreover, in various studies, e.g., allergic contact
`sensitization, the results may be meaningless unless it is reasonably certain that older
`panel members possess an adequate effector system for expressing the response.
`
`EXFOLIATIVE
`
`CYTOLOGY
`
`Since cells are continually being shed from the stratum corneum it becomes a simple
`matter to collect them for subsequent analysis (15). One method of collecting these
`
`corneocytes is by the sticky slide technique (16). In this procedure, a glass 3 x 1 slide
`coated with either tacky adhesive or double-sided scotch tape is pressed firmly on the
`skin surface to be examined. Upon removal, the specimen which retains the
`topographical relationships of the horny cells (corneocytes) can be stained for
`visualization. This method seems to be especially useful in studying the pattern of
`desquamation. Figure 6 shows sticky tape slides taken from the legs of a normal
`subject and one with dry skin. Note the appearance of large clumps in the dry skin
`preparation indicating that the corneocytes are shed as large aggregates rather than
`small clusters of individuals as is normal.
`
`Another method for examining the cytology of exfoliated cells is the detergent scrub
`technique (17). In this procedure, a glass well covering 3.8 cm 2 in area is held firmly to
`the skin. One ml of buffered 0.1% Triton X-100 © is added and the skin surface gently
`rubbed with a teflon scrubber for one minute. The resulting wash fluid which contains
`individual horny cells can then be processed in a variety of ways.
`
`

`

`22
`
`JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
`
`Age n t s
`
`1 +
`
`2+
`
`3+
`
`Mean Grading Scores
`
`DMSO
`
`HISTAMINE
`
`48/80
`
`ETHYL-N
`
`I COTI NATI
`
`CHLOROFORM-METHANOL
`
`ß
`
`I
`
`LACTIC ACI
`
`Figure 5. Integumental reactivity tests. Values represent mean grading from three independent
`observers.
`
`One extremely important parameter is the corneocyte count, the number of desquama-
`tion cells per square centimeter of skin surface. As such, it provides an indirect
`measure of epidermal proliferative activity since it is these cells which eventually
`
`migrate to the skin surface and are desquamated. In a previous study (18), age-related
`differences were determined in two groups: a) individuals with no history or signs of
`scalp disease and b) individuals with dandruff, a clinically noninflammatory hyperpro-
`liferative scaling disorder of the scalp. The results indicated that dandruff subjects had
`significantly higher corneocyte counts from the scalp than their corresponding
`age-matched controls. In fact, this elevated corneocyte count can be a very reliable
`way to assess the efficacy of various anti-dandruff medications (19). However, what is
`important to the investigative gerontologist is that in both groups, the older subjects
`had significantly lower corneocyte counts than the corresponding younger cohort.
`These results support the conclusion that with advancing age, the proliferative activity
`of the human epidermis is significantly reduced not only in normal individuals but also
`in those with a hyperproliferative disease such as dandruff (18).
`The detergent scrub technique also provides a simple method for the visualization of
`
`

`

`NONINTRUSIVE
`
`TESTING OF SKIN
`
`23
`
`Figure 6. a) Sticky slide from shin area of young. b) Sticky slide from shin area of older subject with a
`dry skin problem.
`
`the corneocytes with the light microscope. The appearance of corneocytes from a
`healthy young subject is shown in Figure 7. Note that these cells lack nuclei, one of
`the results of the terminal differentiation of epidermal cells (20). These cells also tend
`to be polygonal in shape with relatively smooth borders. They show lines at the
`periphery corresponding to regions of overlap with adjacent cells indicating a highly
`structured epidermis. With advancing age, increased frequency of atypical cells (Figure
`8) which lack these general features can be observed. Although clinical significance of
`these unusual cell types is still uncertain, there can be no doubt that these
`cytomorphological alterations have diagnostic importance. Indeed, these specimens
`are akin to those used for Pap Smear cytodiagnosis and exfoliative examination of the
`skin should be equally as rewarding.
`Our studies also indicate that changes in corneocyte size may permit a sensitive
`evaluation of altered skin physiology, especially epidermopoiesis. By using the
`
`projected area feature of the Vickers M-85 microcpectrophotometer, changes in
`corneocyte size can be rapidly and precisely measured (21). Table III gives the results
`obtained for the volar forearm and upper inner arm sites. Note that for both age
`cohorts, the volar forearm cells tend to be smaller than those of the upper inner arm. In
`fact, a size gradient of increasing cell size exists as one goes from the wrist to the axilla.
`Thus, extreme caution should be exercised to sample specific sites if corneocyte size is
`
`to be a meaningful measurement. In this manner an age-related increase in cell size at
`
`these two sites has been demonstrated (15). Previous studies by Piewig, et al. (22) have
`also shown similar age-associated changes in corneocyte size; however, we have been
`unable to confirm any sex differences in this parameter.
`
`

`

`24
`
`JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
`
`Figure 7. Light micrograph showing the morphology of a normal exfoliated corneocyte.
`
`It should be noted that with advancing age, there is an increased variance of cell size
`measurements among the subjects. Figure 9 shows projected area measurements of
`volar forearm cells from another panel of subjects of various ages. Note that the values
`
`are fairly tightly clustered in the early years and exhibit a much wider range later. What
`
`
`
`Figure 8. Light micrograph showing the morphology of some atypical exfoliated corneocytes.
`
`

`

`NONINTRUSIVE
`
`TESTING
`
`OF SKIN
`
`25
`
`III
`Table
`Projected Areas of Corneocyte½
`
`Cohort
`
`<35
`>60
`
`Volar Forearm
`
`838.7 _+ 17.4
`936.4 + 46.1
`
`Upper Inner Arm
`
`984.4 _+ 29.6
`1,076.3 + 36.3
`
`(cid:127)Values are means _+ S.E. in/a 2.
`
`VOLAR
`
`FOREARM
`
`o ß
`
`ß
`
`ß
`ßß
`
`ß
`
`ß
`
`o
`o
`
`ß
`
`o
`
`o
`
`I]00
`
`-
`
`1200
`
`-
`
`I100
`
`-
`
`I000
`
`900
`
`-
`
`-
`
`o
`
`ß
`
`o
`
`800
`
`-
`
`CI(cid:127)
`
`0
`
`Oß
`ß
`
`ß
`ß
`
`700
`
`-
`
`600 --/- I 20
`
`I
`I
`I
`I
`I
`I
`80
`70
`60
`50
`40
`30
`SUBJECT CHRONOLOGICAL AGE
`
`I
`90
`
`Figure 9. Scatter plot showing the projected areas of exfoliated corneocytes from the volar forearm of
`human subjects of different ages.
`
`accounts for this extraordinary spread of values among aged individuals is perhaps one
`of the most important questions we can ask. By correlating ethnic origins, occupa-
`tional histories, life styles, etc. with age-associated differences revealed by our
`nonintrusive testing procedure, we hope to estimate the relative importance of
`genetics, environment and other factors which clearly contribute to "aging." Thus we
`anticipate that the knowledge gained will serve as the foundation for the development
`of effective therapeutic and prophylactic treatment for easing dermatological prob-
`lems associated with "aging."
`
`REFERENCES
`
`(1) A.M. Kligman,J. Invest. Dermatol., 73, 39 (1979).
`(2) F. A. Schellander andJ. T. Headington, Brit. J. Dermatol., 91,507-515 (1974).
`
`

`

`26
`
`JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
`
`(3) R. M. Lavker, F. Kwong and A.M. Kligman,J. Gerontol., 35,348-353 (1980).
`(4) R. A. Lavker,J. Invest. Dermatol., 73, 59-66 (1979).
`(5) I. Sarkany and P. Gaylarde, Brit. J. Dermatol., 80, 601-605 (1968).
`(6) D. R. Harris, B. F. Polk and I. Willis,J. Invest. Dermatol., 58, 78-84 (1972).
`(7) A. F. Silver, W. Montagna and I. Karacan, in W. Montagna (Ed.), Advances in the Biology of Skin, Vol.
`6 (Pergamon Press, New York, 1965), pp 129-150.
`(8) R. Marks and R. P. R. Dawber, Brit. J. Dermatol., 84, 117-123 (1971).
`(9) H. Baker and A.M. Kligman, Arch. Dermatol., 95,408-411 (1967).
`(10) L. H. Jansen, M. T. Hojyo-Tomoka, and A.M. K/igman, Brit. J. Dermatol., 87, 9-12 (1974).
`(11) H. Baker and C. P. Blair, Brit. J. of Dermatol., 80, 367-372 (1968).
`(12) E. Christophers and A.M. Kligman,J. Invest. Dermatol., 42,407-409 (1964).
`(13) D. L. Miller and R. H. Wi/dnauer,J. Invest. Dermatol., 69, 287-289 (1977).
`(14) P.J. Frosch and A.M. Kligman, Brit. J. of Dermatol., 96, 461-473 (1977).
`(15) G. L. Grove,J. Invest. Dermatol., 73, 67-69 (1979).
`(16) H. Goldschmidt and A.M. Kligman, Methods of Cell Removal and Microscopic Techniques, 96, 572-576
`(1967).
`(17) K. McGinley, R. R. Marples and G. Plewig,J. Invest. Dermatol., 53, 107-111 (1969).
`(18) J. j. Leyden, K.J. McGinley and G. L. Grove, in J. Roberts, R. D. Adelman and V.J. Cristofolo
`
`
`
`(Eds.), Pharmacological Intervention in the Aging Process, (Plenum, New York, 1978), pp 297-298.
`(19) J.J. Leyden, K.J. McGinley and A.M. Kligman,J. Soc. Cosm. Chem., 26, 573-580 (1975).
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