0022-202Xj82j7903-09:ls$02.00jO THE .JOURNAL OF INVESTIGATIVE DERMATOLOGY. 79:93s-1048, 1982 Copyright © 1982 by The Williams & Wilkins Co.

Vol. 79, Supplement I

Printed in U.S.A.

Structure of the Dermal Matrix during Development and in the Adult

LYNNE T. SMITH, KAREN A. HOLBROOK, AND PETER H. BYERS

Departments of Biological Structure, Departments of Biological Structure and Medicine (Dermatology) and Departments of Pathology and

Medicine (Medical Genetics) University of Washington School of Medicine Seattle, Washington, U.S.A.

To describe a normal adult dermis is a seemingly sim­ple task considering the diverse microscopic methods available for examination of the tissue, staining proce­dures to delineate the fibrous and cellular components, and immunolabeling techniques to identify precisely the various fibrous elements. Yet it is not simple because the range of normal in any of the dermal components has never been surveyed. There are well recognized age­related changes in the dermis; the tissue can be modified by environmental insults (e.g., actinic damage) and al­terations can occur in tissue of individuals with inherited disorders of connective tissue metabolism, other meta­bolic diseases (e.g., diabetes) and in those receiving top­ically applied or systemic medication. From our own experience there is also marked individual variability (at any age) in the connective tissue architecture and its fibrous components. Thus, we are describing the struc­ture of a normal dermis without demonstrating the range of normal in any one of its elements. Reference will occasionally be made to abnormal conditions of the ma­tix since through these deviations it is possible to under­stand more about the normal.

Structural and biochemical properties of the dermal connective tissue in human embryos and fetuses have been described in a number of studies, but ill only a few instances was the goal of the research focused on this problem; instead, fetal tissue was used for comparative purposes in aging studies, or a certain characteristic of the fetal dermis was pointed out along with the descrip­tion of another structure (e.g., hair follicle). In the few instances where a sequential study was carried out on one matrix component during development, an animal (pig, chick) was selected for the work.

The data from these studies have been summarized with our own histologic and ultrastructural observations to provide an overview of human dermal embryogenesis (Table I). An inevi­table consequence of this kind of review is the finding of large gaps in the data; more generalizations exist about changes during dermal differentiation than hard data, hence there is as yet relatively little understanding of matrix interactions during dermal development.

MATERIALS AND METHODS

Embryonic and fetal human skin was obtained through the Central Laboratory for Human Embryology at the University of Washington. Samples from these specimens and from the extensor surface of the upper arm of normal adults were immersed in 1/2 strength Karnovsky's

This work was supported by grants from the NIH (AM-21557, AM-21897), a clinical research grant from the March of Dimes-Birth Defects Foundation (6-298) and a supply grant from the Poncin Fund. Dr. Byers is an established investigator of the American Heart Association.

Reprint requests to: Lynne T. Smith, Department of Biological Structure, University of Washington, School of Medicine, Seattle, Washington 98195.

Abbreviations: CH4-S, CH6-S: chondroit 4- and 6-sulfate DS: derma tan sulfate GAG: glycosaminoglycan HA: hyaluronic acid

93s

fixative [1] buffered in 0.1 M cacodylate buffer and fixed for 2-4 hr in cold. Samples were washed in buffer, postfixed in 1% OS04 in distilled water for an additional hour, soaked in 1% aqueous uranyl acetate for 1 hr to stain en bloc, and dehydrated through a graded series of alcohols. Samples for light microscopy and transmission electron mi­croscopy were further dehydrated into propylene oxide and embedded in Epon by conventional methods [2]. For scanning electron microscopy samples were dehydrated through an alcohol-freon series to 100% freon 113 (Genesolv) and critical point dried in the pressure chamber of a BOMAR critical point bomb using freon 13 as the transitional fluid [2]. Dried specimens were mounted on aluminum stubs, sputter coated with gold palladium alloy and viewed in an ETEC scanning electron microscope at 20 kv accelerating voltage and in the secondary electron mode. Embedded specimens were prepared in 1 /lm sections for light microscopy and 80 nm sections for TEM. Thick sections were stained by the method of Richardson, Jarrett, and Finke [4], thin sections were stained with 1% phosphotungstic acid in water, saturated uranyl ace­tate, and with lead citrate [5] and viewed in a Philips 201 transmission electron microscope.

RESULTS

Dermal Thickness and Regional Organization

The boundaries of the dermis are the dermal-epidermal junc­tion distally and the subcutaneous fat proximally. Between these 2 limits the thickness of the dermis can vary from 1 -4 mm with the thickest regions on the back and thigh and thinnest found on the face. Two (sometimes 3) regions are described on the basis of size and arrangement of the collage­nous fibers [7]. The uppermost of these is the papillary dermis; it is proximal to the epidermis and usually forms no more than 1/10 full dermal thickness. Fibrous elements of the papillary dermis follow the basal lamina into the reticular dermis where they form sheathes about follicles, sebaceous glands, sweat glands, and ducts. The sheathes are referred to collectively as the periadnexal dermis [8]. Papillary and periadnexal dermis have the same fibrous organization and biochemical composi­tion, thus have sometimes been grouped together as a distinct anatomic unit, the adventitial dermis [9] .

The boundary between the papillary and reticular dermis is often marked by an abrupt transition in size of collagen bundles and/or horizontal vessels of the subpapillary plexus (Fig 1a,b). The reticular dermis forms the bulk of the dermis (Fig. 2a-c). It blends with the subcutaneous fat at its deepest most extent and receives distributing vessels, nerves, and lymphatics through that layer. There are many structural and functional differences between the papillary and reticular dermis: collagen types, organization of collagen fibers, elastic fiber structure, cellular populations, vessel and nerve patterns, and relation­ships with appendages. These are summarized on Table II.

Two additional zones of the dermis can also be identified in normal tissue but are often seen more prominently in individ­uals with inherited connective tissue diseases. A fine, but densely organized network of collagen fibrils is often apparent proximal to the dermal-epidermal junction, and, at the light microscopic level gives the impression that the basal lamina is exceptionally thick (Fig 3). This zone in normal adult dermis is stained consistently and exclusively with antibodies to type I pro collagen [8]. Other studies suggest that fibronectin is also present in this region [ 10, 11]. This zone may be equivalent to the proximal collagen matrix which appears early during devel­opment (see below).

A second region which may merit independent designation is

948 SMITH, HOLBROOK, & BYERS Vol. 79, Supplement 1

TAHLE I. Chrono[o{<ic chan{<cs in embl�vonic/fet(ll dermal nmnective tissue durin{< development -�- -------

Months --- ------�-

Structural Organizat ion First Trimester Second Trimester Third Trimester I{eference

---- --------Dermal-subcutaneous boundary Proximal collagen matrix Horizontal layers of fibrous ma-

tl'ix

2

+

+

:l 5

+ +

+ + +

+ + +

-- � -- � -----�-

Ij R !)

+ + + +

+ + + + 53 + + + + 49"

Pattern interrupted on downgrowth of appendages

Papillary and reticular regions + + + + + + 49 Dermal papillae + + + + +

Panniculus adiposus + + + + + + 49" Cleavage lines + + + + + + 4R Collagenous Fibrous Matrix Collagen present by ultrastruc- .) + + + + + + + + 52-54

tural observation Collagen present by biochemical

analysis: Type I 'J + + + + + + + + 57-5H(I

Type III .) + + + + + + + + 57-59 Type V (ages unspecified) fi1

Hatio ,,1 (l):,,(lIl) .) .) ') 0.R:1 0.fi:1 ? .) 2.7:1 2.7:1 57-59 Uncbanging ratio throughout

development (Y, collagen nitrogen of total skin .) .) .) .) 20(" .) .) ') fi3(1, 55

nitrogen Collagen solubility .) .) .) ? .) ? ? ? 24(1, li5 Enzymes associated with post-

translational modification of collagen

relative activity = (adult Prolyl hydroxylase .) ? .) JI)()(i;. ') .) ') .) 1:i('! "?'() fi1,0:1

4()X more active 8x more active Lysyl hydroxylase .) .) .) than adult .) .) than adult li2

fiX more active G lucosylatransferase .) ? t han adult ? .) .) ? ? ') 56

4X more active Galactosyltransferase .) .) than adult .) .) 'J ') .) .) 5(j

Collagenease .) ? + + + + + + + Bauer (unpub-lished)

Elastic Fibrous Matrix Microfibrils 'J + + + + + + + + [)4 Elastin matrix + + + + + + (io,67 Elastic fibrous networks + + + + IiIi.li7 Mature elastic fibers Water content .) .) ') (J2(,¥ 9<Y! ? ? Rae! 55 Hyaluronic acid higb ? ? ? ? ? ') ? IiR,69 Cbondroiten 4-,Ii-sulfate .) ') .) 'J .) .) 'J ') .) li8 Dennatan sulfate .) ') 'J Increasing .) ? ') 68 G Iycoprot eins .) .) .) .) ? ? ') .) .)

--��- ------ -----�- � -�----

., New data of S mitb and Holbrook,

the upper portion of the reticular dermis (the third zone of the dennis defined by others, [ 12, 13] (Fig la,b, 2b). This region could be called an intermediate dermis because the size of collagen bundles and the diameters of collagen fibrils are inter­mediate between the papillary and reticular dermis. Elastic fibers are partially "mature" in structure and morphologic alterations of the matrix in the skin of patients with inherited connective tissue disorders may be expressed differently in this area than in the remainder of the dermis. Craik and McNeil [ 12] and Brown [ 13] identify the "midzone" as a region of more densely compacted fibrous connective tissue when compared with the deep reticular dermis. Collagen fiber organization in that zone has been described as precise, and nonrandom, with the orientation dictated by the relative mechanical stresses exerted on the skin [14] . The ability to preserve this precise pattern, however, requires in situ fixation of the whole body!

Collagenous Fibrous Matrix

Figure 4 demonstrates a dermal fibroblast and the major elements of the fibrous matrix: collagen, elastic, and proteogly-

can-glycosaminoglycans. Collagen Types I, III, IV, and V, Type II trimer and Type I pro collagen collagen are present in the dermis in varying amounts and in specific locations dependent upon the stages in development (see below). Type I collagen forms the majority of the reticular dennis. Type I fibrils have been shown by immunocytolabeling studies to be "thick" col­lagen fibrils (60-100 nm) , while the small diameter "thin" fibrils (2()-40 nm) have been identified as Type III [15]. It is not yet resolved whether Types I and III collagen co-exist within a single fibril.

Type III collagen accounts for only 10-15% of dermal collagen yet this small amount is undoubtedly significant, as individuals affected with Type IV Ehlers-Danlos syndrome, a disorder in which Type III collagen is reduced substantially in skin and other tissues [16,17], have thin and transparent skin with com­promised mechanical properties. Type III collagen is localized in the papillary dermis in fine filamentous structures (Fig 1a,b), in the reticular dermis surrounding vessels, in the sheaths of pilosebaceous structures and apocrine and eccrine glands and encasing fat cells of the hypodermis [8].

July 1982 STRUCTURE OF ADULT AND FETAL DERMAL MATRIX 958

FIG 1. SEM (a) and LM (b) images of adult papillary and intermediate dermis. The broken line shows the approximate separation between the 2 zones. Note the smaller fiber bundle size in the papillary dermis (all X 400).

Type IV collagen is present wherever there is basal lamina: at the dermal-epidermal junction, surrounding vessels and epi­dermally derived appendages, and smooth muscle cells of the arrector pili muscle. Additional collagens are also present in the dermis in minor amounts; type I trimer has been identified in normal and pathologic skin [18] and type V collagen has been found as a component of the basement membrane. Other com­ponents of the basement membrane (iaminin, bullous pemphi­goid antigen, fibronectin and glycoprotein (GP)-2) are other topics of this symposium and have been reviewed elsewhere [19]. Fibronectin is a collagen-associated glycoprotein found on fibroblastic cells and co-distributed with collagen fibrils in both the papillary and reticular demis. It is dispersed in a linear organization in the papillary dermis, but forms a reticular pattern in the reticular dermis. Fibronectin shows enhanced immunofluorescent staining in the papillary dermis, around vessels, and in the sheaths of nerves, pilosebaceous units and eccrine sweat glands [ 10] .

ELASTIC FIBROUS MATRIX

Elastic fibers comprise only 4% of the adult dermal proteins [20) yet they form a surprisingly extensive network in the skin [21) (Fig 5). Mature elastic fibers have 2 components, a micro­fibrillar scaffolding and a matrix of elastin "cement." Of these, the elastin constitutes as much as 90% of the fiber [22]. The microfibrils are 10-12 nm tubular-appearing structures com­posed of 2 glycoproteins [23]. These are biochemically distinct

from elastin within which they are embedded, exposed only at the surface or as darkly staining linear streaks within the unstained matrix. Alone, the microfibrils have no mechanical properties [24]. An individual fibroblast can secrete more than one type of collagen, and elastin, simultaneously [25,26], al­though during development collagen and elastin are synthesized as independent events; collagen synthesis is presumed to occur ahead of synthesis of the elastin matrix [27].

Schemes of elastic fiber fibrillogenesis have been proposed on the basis of biochemical studies [28) and of ultrastructural observations of elastic fibers in the skin [29-31]' The most immature forms are found in the papillary dermis; these are composed of little more than bundles of microfibrils which extend from the basal lamina into the papillary dermis, often at right angles to the epidermis (Fig 6a). Such fibers have been named oxytalan [29] and are believed to serve a role in anchor­ing the basal lamina and to form a template upon which elastin is secreted at deeper levels of the dermis. Oxytalan fibers branch and form a horizontal plexus in the upper reticular dermis (intermediate dermis). Small amounts of stainable elastic ma­trix (presumably not cross linked) are secreted onto the micro­fibrillar bundles in this region (Fig 6b). These fibers are called elaunin [29]. Large mature band-like or sheet-like elastic fibers are restricted to the reticular dermis. The elastin resists staining with the usual cationic dyes as a consequence of the highly cross-linked elastin matrix (Fig 5). Large elastic fibers border and interlace between the collagen fiber bundles (Fig 2b,c),

968 SMITH, HOLBROOK, & BYERS Vol. 79, Supplement 1

FIG 2. SEM (a) and LM (b, c) images of collagen fiber bundles in the adult dermis. Compare the sizes of fiber bundles (C) in the intermediate (b) and deep reticular (ct, c) regions. Elastic fibers (arrows) border the collagen fiber bundles (all x 1,100).

Charactt-'ri:-;t ic.-;

Matrix oq;anizat ion

Collagc'n types Fibronectin

Collagenase activity Elast ic fibers

Cellularity Fibroblast morphology Fibroblast proliferation Vascular or�anization

TABLE [I. Comparison of the structure and composition of the papillwy and reticular dermis

Papillar.\' dermis

Loose, fine collagen fibers

I, Ill, Type I proco llagen Present in a linear pattern and lllay concen­

trate along dermal-epidermal junet ion, greater intensity of immunofluorescent staining

Virtually all of the activity Immature oxytalan fibers (microfibrillar

bundles)

Notable cellular density Active by morphologic criteria; basophilic Greater proliferative capacity Microcirculation; capillary loops

Hetil'ular dprmis

Dense; intermediat e and coarse collagen fi­bers

I, III Present in reticular pattern; low intensity

witb immunofluoreocent otaining except around vessels and in sheat hes of epider­mal appendages

Little if any activity Intermediat e elaunin and fully mat ure fibers

(microfibrilo plus varv ing degrees of elas­tin)

Sparsely cellular Less active by morphologic criteria Less proliferative capacity Distributing vesoels; microcirculation asso­

ciated with appendages

I) 10,11

July 1982

FIG 3. LM image of adult epidermis and papillary dermis showing the narrow region of diffuse collagen subjacent to the basal lamina (arrows) (x 400).

FIG 4. TEM image of fibroblastic cell (FB) from adult reticular dermis showing normal collagen (C) and elastic (E) matrix components Ix il,6(0).

appear to increase within the dermis with progressive age and show a progressive tendency toward spontaneous degenerative changes [32] . Elastic fibers are also found in the walls of cutaneous vessels and contribute to the sheathes of epidermal appendages. An elastic network intermixed with collagen forms around the permanent portion of the hair follicle (above the

STRUCTURE OF ADULT AND FETAL DERMAL MATRIX 975

bulge) . Collagen alone forms the sheath below the bulge [33]. An additional accumulation of elastic connective tissue is as· sociated with the terminus of the follicle as a "fluffy body" [34]. It may persist with regression of the hair (telogen) but seems to form anew with each anagen phase [33].

The sheath of the sweat gland also contains elastic fibers but these are primarily bundles of microfibrils which intermingle with the collagen. Collagen fibrils assemble individually (not in bundles) around the gland and duct into 2 circumferential layers separated by fibroblastic cells. The overall organization of the connective tissue in the sheath of both the gland and duct, and the immature state of the elastic fibers, may provide for greater flexibility of this coiled structure within the dermis

[35].

DIFFUSE EXTRACELLULAR MATRIX

The third class of connective tissue components in the skin includes the proteoglycan and glycosaminoglycan (GAG) rna· trix or "ground substance." In the adult this matrix is composed of sulfated GAGs derma tan sulfate (DS), chondroitin 4· and 6· sulfate (CH4·S, CH6·S) and the nonsulfated GAG hyaluronic

acid (HA) [36,37]. It has been shown that the various GAGs interact with collagen to influence both the rate and structure of collagen fibril formation [37,38] and elastic fiber structure [40]. The presence of DS in skin has been correlated with the formation of coarse collagen fibrils; it is absent in tissues with thin and presumably "immature" collagen fibrils [41].

FIG 5. SEM image of elastic fibrous network in adult dermis. The collagen has been removed by autoclaving. Micrograph courtesy of T. Tsuji, R. Lavker and A. Kligman [21J (x 3(0).

988 SMITH, HOLBROOK, & BYERS Vol. 79, Supplement 1

FIG 6. TEM images of papillary (a) and intermediate (b) regions of the adult dermis showing immature stages of elastic fibers (all X 12,000).

Proteoglycans and GAGs are removed from tissue prepared for electron microscopy unless cationic dyes are added to the fixative to render them insoluble. Several dyes may serve this purpose (e.g., alcian blue [42] or thorium [43]), although ruthe­nium red is usually the dye of choice because it reacts with GAGs, anionic proteoglycans and lipids [44,45] and is reduced by OS04 which then stains a fine network connected by 100-200 A diameter particles. Ruthenium red positive material is asso­ciated with the surface of dermal mesenchymal cells, collagen fibrils at their major period bands, basement membranes, elastic fibers and forms a meshwork in the seemingly empty space between bundles and fibers of the structured connective tissue matrix.

Smooth Muscle Cells and Matrix Synthesis

Smooth muscle cells of the dermis are also capable of syn­thesizing and secreting collagen (predominantly type III) and elastic fibers [46]. In the normal dermis. smooth muscle cells are found in the vessel wall and the arrector pili muscle of the follicle. This population of cells probably contributes little if any to the overall matrix of the dermis. However, hyperplastic development and unusual localization of individual smooth

muscle cells between bundles of collagen fiber are characteristic of abnormal dermis of patients with inherited disorders of connective tissue metabolism. In particular, individuals with Ehlers-Danlos syndrome Type IV have large bundles of smooth muscle in the papillary and reticular dermis which are too large, too isolated and observed with too great a frequency to be accounted for by sections through arrector pili muscle. The observation is curious for the reason that smooth muscle cells produce a high proportion of Type III collagen, yet the tissue of these patients is deficient in this collagen.

DEVELOPMENT OF THE DERMIS IN THE HUMAN EMBRYO AND FETUS

The cellular dermis of the early embryo is gradually changed into a fibrous dermis, a transition which has been described as a "ripening of the dermis" by Lynch [47]. In this process changes occur in the density and size of collagen bundles, the diameter of collagen fibrils, the types of collagen present, its solubility and degree of post translational modification, the com­position and amount of GAGs, the ratio of GAGs to collagen, the maturity of the elastic fiber and the overall water content of the dermis.

July 1982

! ,

. . .

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STRUCTURE OF ADULT AND FETAL DERMAL MATRIX

. .' --"':' .

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FIG 7. LM images of fetal dermis at progressive ages of development. Note the increasing amount of connective tissue matrix and organization of fiber bundles in the intercellular space (x 95).

20WK

" ,

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FIG 8. LM images showing the development of the subcutaneous panniculus adiposus in 15- [IJ, 18- ( b) and 20-week ( c) fetuses. Adipose cells

(A) assemble and accumulate within arcades (arroll's) of subcutaneous fibrous connective tissue (x 70).

100s SMITH, HOLBROOK, & BYERS

ORGANIZATION OF THE EMBRYONIC/FETAL DERMIS

Three stages of dermal development can be identified arbi­trarily on the basis of tissue organization at the light micro­scopic level. Stages 1 and 2 are embryonic; stage iI, the longest, begins in the third month and is a continuum of progressive changes in matrix development (Fig 7a-f). The first stage characterizes the dermis of the 1-2-mo embryo. All of the subepidermal tissue is organized into an open cellular network which appears to be devoid of fibrous marix (Fig 7a). Studies by transmission and scanning electron microscopy show, how­ever, that fibers are present in the extracellular space associated primarily with the basal lamina and the mesenchymal/fibro­blast cell surface (see below). There is no apparent inferior boundary separating the dermis from the hypodermis (Fig. 7a).

The second stage of dermal organization begins at the end of the second month. It is defined in relation to vascular devel­opment in that large subcutaneous vessels form a distinct border which demarcates the dermis from subcutaneous tissue (Fig 7b) . The assignment of these vessels as the inferior bound­ary of the dermis is possible only retrospectively after a com­plete series of sections of skin from embryos and fetuses of progressive age is examined. This stage is further characterized by the alignment of cells and deposition of matrix at the dermal­epidermal junction.

By the end of the first trimester the dermis already has finely organized, uniformly dense fiber bundles throughout (Fig 7c). Matrix accumulates progressively during the second and third trimesters (stage 3 dermal development) in variable patterns depending upon the region and the internal forces of develop­ment and rate of growth [48]. Thus, patterns of dermal orga­nization change throughout development and after birth until adulthood. The fibrous dermis of the regions we routinely sample is organized in layers of alternating direction, planar to the epidermal surface, and is interrupted only where epidermal appendages extend into the dermis. This pattern has been recognized by others [49].

Differentiation of papillary and reticular regions on the basis of differing fibrous organization occurs in the fifth month, although at an earlier stage, distinction of the 2 regions is suggested by a greater concentration of cells proximal to the epidermis, particularly in association with the basal lamina. A vascular boundary between papillary and reticular demis as is seen in the adult is not evident until the third trimester (Fig 7f).

Organization of the subcutaneous tissue begins in the fourth month. Arcade-like assemblies of connective tissue form a framework for the association of differentiating adipocytes (Fig Sa-c). At first, individual fat cells are sparse, but within the fifth month a sizable accumulation of fat, visible by light mi­croscopy, forms the pannicular adiposus [49]. Fujita et al [50] have described the cellular differentiation of this tissue at the ultrastructural level.

At birth, the dermis is transitional between fetal and adult in the pattern of organization, thickness and size of component fiber bundles. A significant portion of the fibrous connective tissue is synthesized postnatally [51].

DEVELOPMENT OF THE COLLAGENOUS FIBROUS MATRIX

Stage 1 dermal development was described as a cellular phase on the basis of an obvious cellular network and no apparent indication of a fibrous matrix. By ultrastructural studies, how­ever, it can be seen that the earliest embryos (36-45 days) investigated already have a significant quantity of matrix (Fig 9) in which there are 2 distinct populations of extracellular fibers. One type of fiber is straight or curved. banded and argyrophilic. It is approximately 25 nm in diameter and is morphologically equivalent to collagen [52,53] (Fig 9a). The second type of fiber appears tubular, is 8-10 nm in diameter

Vol. 79, Supplement 1

FIG 9. TEM (a) and SEM (b) images of dermis from a 7-week-old fetus. Fibroblasts are abundant with few fine diameter collagen fibrils ( C) associated at the cell surfaces (Inset) and at the dermal-epidermal junction (b). The extent of the fibrous matrix is best seen by SEM (b). (x 5,400, x 22,600, X 5,150).

July 1982

and has the morphologic characteristics of the microfibrillar component of the elastic fiber [54]. Both types of fibers are closely associated with the cell surface, the banded fibrils in smaU

' (5-20 fibrils) loosely associated bundles and the smaller

fibrils as random and tangled-appearing threads. A second major site of matrix accumulation at this early stage is in a proximal collagen network associated with the basal lamina. This zone is a 600-1100 A feltwork composed of interlacing banded fibrils [5:3] (Fig 9b).

By stage two, collagen bundles are clearly evident throughout the dermis. The proximal collagen matrix can be seen at the light level, fiber bundles have increased in size and the matrix has begun to lose its cellular network appearance.

Matrix secretion appears to accelerate in the second trimester (during stage three), although even at 14-16 weeks collagenous nitgrogen is only 20% of total skin nitrogen [55]. Fiber bundles increase in size and density (Fig 7d-f, lOa-c), papillary and reticular regions are defined and individual collagen fibrils increase in diameter depending upon the zone of the dermis (see below).

With one exception [56], all of the published biochemical studies of fetal collagen synthesis and posttranslational modi­fication have been performed on tissue obtained from fetuses 4 mo and older. Our own biochemical and ultrastructural data however, have demonstrated the presence of types I, III, and V during the first trimester (Smith, unpublished).

Studies in 2 laboratories have provided data showing that human fetal collagen is largely type III. While both Epstein [57] and Sykes et al [58,59] have shown that the percent of type III collagen in fetal skin (16-45% versus 1O-15'?{, adult) is ele­vated, Type III collagen clearly coexists with another collagen species, but in what distribution and how each type influences the fibril diameter and the overall properties of the fetal dermis, is completely unknown. The relative proportion of type III collagen was shown to decline in the third trimester, although during this period the collagen nitrogen as percent total nitro­gen increases from 20% to 6.3% [55]. The importance of abun­dant type III collagen during the fetal period has been suggested to play a role in growth and remodelling of the dermis [57] to form a template necessary for deposition of type I collagen in a normal pattern of organization and to interact specifically with, and perhaps to influence, the developing epithelium [57,60]. In general, type III collagen is present in tissues requir­ing greater compliance; this is consistent with the needs of a developing integument.

New data of Kirsch et al [61] show different relative analysis of fetal collagens. Constant ratios of types I, III, and V collagens were measured at all stages of development, although they did find the extent of hydroxylation of types I and III collagen greatest in the youngest fetal skin, declining with age. They interpreted these data to suggest that the changes in hydrox­ylation (and thus with glycosylation) could regulate changes in fibril diameters during development.

Our measurements of fibril diameters during development (Fig 11) have shown that collagen fibrils in the first trimester embryo/fetus already depend upon the zone of the dermis within which they are found. They measure 9-17 nm in the proximal collagen matrix but 25 nm where they are associated with cells. Fibril diameters increase in the second trimester in both regions and the outlines of the individual fibrils become "crisp," evidently associated with less diffuse matrix. In the fourth month, fibrils in the presumptive papillary dermis are 25-.35 nm in diameter, while those in the deeper (reticular) region are .35-45 nm. In the fifth through seventh months, fibrils of the papillary dermis, like those in the papillary dermis of many adults, show a range of diameters from 25-60 nm. Fibrils in the reticular dermis have a more constant diameter between 45 and 60 nm. Fibrils in fetuses of all ages are widely spaced within fibers and fiber bundles, separated by a signifi­cant amount of diffuse and finely filamentous material.

The studies of Kirsch et al [61] confirm an earlier report that

STRUCTURE OF ADULT AND FETAL DERMAL MATRIX lOIs

FIG 10. LM (a), TEM (b) and SEM (e) images of the dermis from a 15-week-old fetus. Collagen fiber bundles (C) are cell associated and occupy tbe intercellular space. Several fihroblastic cells (FB) and a nerve (N) are evident (x :300, x 4,500, x 4,1(0).

lysyl hydroxylase in the 4-mo fetus is about 40x greater than in the adult. The activity of the enzyme declines sharply in the third trimester to a level approximately 8 x greater in the newborn than in the adult [62]. A similar pattern of high fetal activity and decline toward adulthood was also demonstrated for the enzyme proline hydroxylase [6.3]. The change in the activity has been shown further to correlate with a change of the enzyme from an active to an inactive form [64 J.

Glycosylation of collagen is related to hydroxylation as glu­cose and galactose are added through transferase enzymes to the hydroxylysine residues. Studies with fetal skin have also shown a higher activity of galactosyl transferase (4 x increase)

1028 SMITH, HOLBROOK, & BYERS Vol. 79, Supplement 1

® FIG 1 1 . TEM images of collagen fibrils showing increasing diameters in each region with progressive age. Pd = papjjjary dermis, ID =

intermediate dermis, and RD = reticular dermis (A X 37,(00) .

and glycosyltransferase (7 x increase) enzymes prenatally [56] . Hydroxylation and glycosylation, together, then may have a bearing on regulation of collagen fibril formation and most certainly influence collagen solubility. Even at birth, 24% of dermal collagen is soluble compared with 1% in the adult [65].

The circumstances of degradation and turnover of collagen in fetal dermis are another unknown of collagen metabolism during development, although collagen fibrils within phagocytic vacuoles are a common finding in the dermis of a second trimester fetus and preliminary data of Bauer (unpublished) suggest that collagenase activity can be detected in the dermis early in development.

The "transitional" quality of the dermis described for its overall organization at birth reflects to a large extent the transitional nature of the collagen. Collagen fibrils of both regions are smaller in diameter and fiber bundles of the reticular dermis are smaller than in the adult [51] . Collagenous protein accounts for 63% of total skin protein at birth, 71% at 6 mo compared with 90% in the adult [55] .

DEVELOPMENT OF THE ELASTIC FIBROUS MATRIX

Microfibril-like structures were identified in even the very youngest embryonic tissue [54] . At first these are disoriented and tangled appearing, but by the third month are organized in parallel alignment in small bundles. In the fourth month, small amounts of elastin matrix are deposited onto the microfibrillar assemblies. These structures can only be seen ultrastructurally. In the sixth month, fine granular elastic fibers, limited in distribution to the reticular dermis, can be seen by light mi­croscopy in tissue stained with elastic stains [66] . The elastic matrix increases sufficiently by the eighth month to define a reticular elastic network in both the papillary and reticular dermis. Elastic fibers in the reticular dermis at this stage have been described as "robust" [67] , although by electron micros­copy it is apparent that the elastic fibers in all regions are immature (elaunin-like) in matrix density and staining charac­teristics (Fig 1 2 ) . The framework of elastic fibrous connective tissue is present at birth in an adult pattern of distribution but complete elaboration of the elastic matrix is not evident before the first or second year postnatally [51] giving the impression histologically that this component in infant skin is sparse com­pared with that of the adult [60]. From the morphologic studies of elastic fibers, it would appear that their role in the mechanical properties of fetal skin would be insignificant.

DIFFUSE EXTRACELLULAR MATRIX

The glycoproteins and glycosaminoglycans of the diffuse extracellular matrix and the changes that they undergo in composition during development are one of the most poorly documented aspects of human dermal development. Most of the work in this field appears limited to animal tissues other than human. Embryonic dermis is described as a watery, PAS t gel which has a high hyaluronic acid content and glycogen [49,68]. With progressive development a decline in HA and an increase in dermatan sulfate occurs. The water content in fetal skin at 14 and 16 weeks is approximately 90%; most of this is

FIG 12. TEM images of dermal matrix from a 26-week-old fetus showing the state of elastic fiber development. The arrows in ( a ) indicate the distribution of the immature elastic fibers at the periphery of collagen bundles ( C ) . The relative amounts of elastin and microfi­brillar components are evident in ( b ) (arrows ) (x 2,500, x 15 , 100) .

probably associated with the GAGs and non fibrous proteins. Changes in the GAG content of embryonic skin have been documented for the pig dermis [69]; correlative histologic and biochemical data from this study suggested that the maturity of collagen bundles and appearance of large diameter fibers might be correlated with the presence of DS in the matrix [69]. The depression of HA synthesis and a corresponding elevation of CS have been identified with cessation of cell migration and establishment of a tissue [70] , presumably a change in dermal development from phases one and two to three. Quantitative relationships between GAGs and collagen fibrillogenesis also have been demonstrated in embryonic chick skin [71] , but such studies in human fetal dermis do not appear to have been accomplished.

COMMENT

Developing systems and abnormal adult systems (either ex­perimentally manipulated or naturally occurring genetic de­fects) are ideal for the study of interrelationships among matrix components. In human dermis, there has been almost no effort in that direction with fetal dermis, most likely because of tissue limitations and also because individual laboratories focus on

one particular aspect of the connective tissue matrix. Yet, here is a model system with which we might be able to answer questions such as what type III collagen means to the structure of the fibril, the overall structure, organization and function of the dermis, whether or how it is involved in epithelial mainte­nance, how various GAGs influence (regulate ?) the develop­ment of fibril and fiber organization, and how collagens are independently or co-distributed at early stages of architectural "planning" of the dermis. The apparent simplicity of this tissue,

July 1982

particularly in the early embryo, presumably free of environ­ment assaults, should provide an ideal opportunity to investi­gate the interaction of matrix molecules in tissue.

The authors gratefully acknowledge the technical assistance of Mrs. Mary Hoff, Ms. Carolyn Foster and Mr. Gary Hanson. The manuscript was expertly prepared by Ms. Doris Ringer.

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