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J. Anat. (1991), 177, 195-207 With 8 figures Printed in Great Britain
Variability of fibroblast morphology in vivo: a silver impregnation study on human digital dermis and subcutis G. E. K. NOVOTNY AND C. GNOTH Department of Neuroanatomy, University of Dusseldorf, Moorenstrasse 5,
4000 Dusseldorf 1, Germany
(Accepted 5 April 1991) INTRODUCTION
Connective tissue research is a vast field with an enormous volume of literature. However, attention has primarily been focused either on collagen and the extracellular matrix, or on fibroblasts in tissue culture. Few publications deal with fibroblasts in situ, and then mostly in very restricted regions and situations (Bulger & Nagle, 1973; Komuro, Desaki & Fujiwara, 1982; Squier & Magnes, 1983; Desaki, Fujiwara & Komuro, 1984; Pieraggi et al. 1985; Eyden, Watson, Harris & Howell, 1986; Sugita, Ishibashi, Shiotani & Yoshioka, 1988). Although it is clear that fibroblasts produce collagen and the matrix, their role in the organisation of these elements is mainly considered to be passive (Tomasek, Hay & Fujiwara, 1982), and considerable energy has been expended upon the study of the arrangement of collagen in various connective tissue regions, especially the dermis (Montagna & Carlisle, 1979; Pfaller, Schuler, Schmidt & Dworschak, 1979; Holbrook, 1982; Holbrook, Byers & Pinnell, 1982; Lavker, Zheng & Dong, 1987; Othani, Ushiki, Taguchi & Kikuta, 1988), as well as on explanations of a particular arrangement in terms of stress and strain forces (Bellows, Melcher & Aubin, 1981; McGaw, 1986). Differences in size between fibroblasts have been noted in the past (Maximow, 1906), though without any subsequent attempt at systematisation or correlation with the organisational form of the connective tissue. In cell culture, differences in proliferation potential between fibroblasts derived from specific regions have been described (Harper & Grove, 1979; Schafer, Pandy, Ferguson & Davis, 1985), but the morphological diversity has been attributed to degenerative processes (Bayreuther et al. 1988), or substratum effects (Tomasek et al. 1982; Yoshizato, Taira & Shioya, 1984; Olmo et al. 1988). Using a new silver impregnation technique (Novotny & Gommert-Novotny, 1990), it is possible to visualise fibroblasts in their total extent in paraffin serial sections. From initial observations it would seem that fibroblasts have a much more systematic relationship to the extracellular structures and are by no means passively confined to convenient interstices, but take an active part in the development of the extracellular organisation. Here we wish to demonstrate that, even within the very confined area of the adult human digital dermis and subcutis, there may be considerable differences in size and morphology of fibroblasts. These may be related to the 3-dimensional collagen architecture of the connective tissue, which is in turn related to the stratification of the digital dermis and subcutis. Correspondence to: Professor G. E. K. Novotny, Abteilung fur Neuroanatomie, Zentrum fur Anatomie und Hirnforschung, Medizinische Einrichtungen der Universitat Diisseldorf, Moorenstrasse 5, 4000 Dusseldorf 1, Germany.
196
G. E. K. NOVOTNY AND C. GNOTH MATERIAL AND METHODS
Digital dermis and subcutis was available from 8 individuals of both sexes aged from 14 to 81 years, with at least 1 representative per decennium. The material was obtained at routine autopsy and fixed by immersion in 10 % formaldehyde. Dehydration and embedding in paraffin (Paraplast Plus) was carried out according to conventional procedures. Sections ranging from 10 to 40 ,um in thickness were cut, mounted, and silver impregnated according to published methods (Novotny & Gommert-Novotny, 1990). Sections were evaluated in a Leitz Orthoplan or Dialux microscope, using transmitted light, interference contrast, or circularly polarised light. For purposes of comparison, frozen sections from biopsy material obtained during a facial operation were reacted with monoclonal antibodies against vimentin (B6hringer, clone Va). The impression obtained from the qualitative observations was further substantiated by measurement. Papillary dermis fibroblasts were compared with subcutis fibroblasts using material obtained from a 54-year-old male. The parameters measured included the greater and lesser diameter of the cell body, the length of the longest cell process, and the number of cell processes. Material from 14-, 37- and 81-year-old females, and from 23- and 54-year-old males was used for measurements of subcutis fibroblasts. In these instances, only the length of the longest cell process was determined. With the exception of the cell body size, where only 50 cells were measured, all measurements were performed on 100 cells per sample and on 40 ,um sections. The selection of cells for measurement posed problems. Since fibroblasts form a continuous network with their cell processes at the light microscopical level, it is impossible to determine the true limits of a cell in the majority of cases. In order to obtain reasonably comparable data, cells for measurement were selected according to the following criteria. (1) The cell body is located within the central part of the section thickness. (2) The cell processes are distinct from those of other fibroblasts in their total course. (3) The terminations of the cell processes are clearly distinguishable, totally within the section thickness, and not terminating at the surface of the section. Cells satisfying these criteria were rare. They were located by scanning the section in the classical meander pattern. Measurements were performed on the first 100 cells found. RESULTS
As illustrated in Figure 1, the adult digital integument can be clearly divided into a papillary dermis, reticular dermis and subcutis. Each of these regions contains
characteristic fibroblast populations, differing markedly from those of the other layers. They are described sequentially in the following section. Papillary dermis The fibroblasts of the papillary dermis are characteristically small cells, of 3dimensional stellate form, with thin processes extending in all directions (Fig. 2a), which frequently seem to merge with those of neighbouring cells. Within the uppermost layer of the papillary dermis, the subepidermal band (Holbrook, 1982), fibroblasts are frequently oriented horizontally, with processes extending parallel to the epidermis, their terminals curving upwards towards the latter (Fig. 2b). A little below these 'horizontal' cells, a 'second line' of 'basket '-shaped fibroblasts may be found (Fig. 2 c). All fibroblasts of the papillary dermis extend their processes to several
Fibroblast morphology
.
P
197
f~ ~~~A
47~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ;iS I,~~~~~~~~~~
Fig. 1. Low power view ( x 50) of a section of human digital dermis and subcutis, demonstrating the layers from which the further illustrations have been taken at higher magnification. P, papillary dermis (Figs 2, 8); R, reticular dermis (Fig. 3); S, subcutis (Figs 4-7). F, fat lobule (Fig. 6); L, tangential sections of concentric collagenous lamellae around a fat lobule (Fig. 5); Pc, pacinian corpuscle; Re, retinaculum cutis (Fig. 7).
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G. E. K. NOVOTNY AND C. GNOTH
Fig. 2. Fibroblasts of the papillary dermis. (a) Typical stellate fibroblast (arrow), with processes in all directions. (b) Fibroblast of the subepidermal band, with horizontal processes curving up to the epidermis (arrow). (c) Basket shaped fibroblast All magnifications x 1200.
of the fine collagenous bundles interwoven within this layer a single cell is thus not confined to 1 collagen bundle. Towards the reticular dermis the size of the cells gradually increases. Reticular dermis Within this layer the fibroblasts are larger than those of the papillary dermis. The collagenous bundles are coarser. In the upper part of this region the fibroblasts are stellate and resemble those of the papillary dermis. In the deeper part of this layer the fibroblasts are predominantly associated with a single very coarse collagen bundle, whereby their processes extend along its length (Fig. 3 a-c). These fibroblasts therefore take on a unipolar or bipolar aspect. The processes either branch in a 'Y' form along the extent of a bundle, or short stubby processes in a transverse orientation give rise to a long longitudinal process in a 'T' junction (Fig. 3a, b). The fibroblasts are predominantly located on the surface of the bundles, though some are inside them (Fig. 3 c). The distribution of fibroblasts is uneven - some collagenous bundles appear nearly black because of the high number of processes associated with them - while others are totally devoid of processes (Fig. 3 c). In instances with a dense population of fibroblasts it is almost always impossible to define the extent of a single cell, as the processes are so closely applied to each other that a boundary cannot be resolved by light microscopy. Between the interwoven collagenous bundles loose collagen can also be found that fills the interstices. This loose collagen is associated with fibroblasts that are oriented 3-dimensionally, with processes extending in all directions, having only occasional contacts with collagenous bundles (Fig. 3d). These fibroblasts may be termed 'interstitial'. Subcutis The architecture of the subcutis is determined by fat lobules, sweat glands, and groups of pacinian corpuscles. Around these 'voluminous' structures concentrically arranged laminae of loose collagen are found (Fig. 4). Each lamina is approximately 10 ,am thick and the fibroblasts are located between adjacent laminae. They are very large cells, with processes of considerable length - the longest found measured 180 ,um. They predominantly extend along the surface of the laminae and only rarely penetrate from one to another. The cells thus appear 2-dimensional, having a stellate aspect from 1 surface and appearing bipolar when viewed at right angles to
Fibroblast morphology
199
All-ma: .......
MR."I'M
....
......
....
Fig. 3. Fibroblasts of the reticular dermis. (a) Cell bodies of fibroblasts arrayed along a coarser collagen bundle with T-shaped branching of a process (arrow). (b) A fibroblast (Cb, cell body) on the surface of a coarse collagenous bundle with longitudinally oriented processes exhibiting Y-shaped branching (arrows). (c) Cross-sections of collagenous bundles demonstrating the location of cell bodies (large arrows) and processes (arrowheads) on the surface and within the bundles. Small arrows indicate processes in longitudinally sectioned bundles. (d) 'Interstitial' fibroblasts (arrows) with processes extending in all directions. All magnifications x 1200.
200
G. E. K. NOVOTNY AND C. GNOTH
Fig. 4. Medium power view of concentrically arrayed collagenous lamellae (L) around fat lobules in the subcutis. The open arrow indicates cross-sectioned lamellae; the large arrow points to the tangentially sectioned portion of the lamellae. The small arrow indicates fibroblasts applied to a fat cell. F, fat lobule. Magnification x 250.
the surface of the laminae (Fig. 5 a, b). The processes of neighbouring cells are closely apposed to each other, so that it is frequently impossible to determine their true extent. The cells thus form a dense interconnected network (Fig. Sc). Fibroblasts with shorter processes are found directly applied to individual adipocytes (Figs 4, 6), pacinian corpuscles, or coils of sweat glands. Smaller fibroblasts with fewer processes are also found around blood vessels, which have a separate system of very fine concentric collagenous lamellae. Within the subcutis bundles of denser collagen may also be observed, originating in the reticular dermis and penetrating to the periost (retinacula cutis). These exhibit the same fibroblast population as is associated with large collagenous bundles in the reticular dermis (Fig. 7). Their processes are oriented predominantly along the long axis of the bundles, whereas some again are associated with loose collagen between the bundles as interstitial fibroblasts. Measurements The results of the measurements performed on individual cells are summarised in Tables 1 and 2. They support the impression obtained from the qualitative observations. DISCUSSION
Silver impregnation is not a specific stain for fibroblasts. All cells are visualised to some extent with this method, if only through staining of the nucleus. However, the cytoplasm of fibroblasts is particularly intensely stained, especially the processes - the
Fibroblast morphology
201
Fig. 5. Subcutis fibroblasts. (a) Fibroblasts (arrows) as seen in cross-sectioned collagenous lamellae. (b) A fibroblast from a tangentially sectioned lamella, showing the considerable extent of the processes. (c) Fibroblasts in a tangential section from between 2 collagenous lamellae, demonstrating their stellate shape and contiguity of their processes. All magnifications x 1200.
more dendrite-like the process, the denser the impregnation. Extracellular material is lightly impregnated. Collagen bundles may be differentiated from the homogenous matrix, elastin lamellae remain unstained and, with few exceptions, elastin fibres are not visible. This generalised staining enables a precise localisation of individual cells,
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G. E. K. NOVOTNY AND C. GNOTH
Fig. 6. A fibroblast applied to a single fat cell. Note plump cell body, stellate shape and short processes. Magnification x 1200.
Fig. 7. Fibroblasts within retinacula cutis in the subcutis. (a) Cross-section of a retinaculum cutis, showing cell bodies (large arrows) and processes (small arrows) within the collagen bundles. The majority of processes are cross-sectioned. (b) A retinaculum cutis in longitudinal section, demonstrating the longitudinally arrayed cell processes. Both magnifications x 1200.
203
Fibroblast morphology
Table 1. Measurements of.fibroblasts in digital skin of a 54-year-old male* Cell body size (,um)
Papillary dermis
Greater diameter
Lesser diameter
7 30 (1-8)
3 7 (1 0)
Length of longest cell process (,um)
Number of cell processes
22 15 (5 9) (n = 100) 75 9 (21-8) (n = 100)
5 27 (1-7) (n = 100) 6 3 (2 5) (n = 100)
(n = 46)
Subcutis
15 6 (4 5) *
8-2 (2 8) (n = 50) Means with SD in parentheses.
Table 2. Average number of processes per cell and average length of the longest process of subcutis fibroblasts in relation to age of subjects Age (years)* ... Average number of cell processes (SD) Average length of longest cell process in um (SD)
14
23
37
54
81
4-3 (25)
61 (28)
65 (27)
6-3 (25)
7-7 (22)
48-1 (15-1)
59-5 (18-0)
71 3 (20 5)
75-9(21 8)
83-0 (19 0)
*
n = 100 for each subject.
as a further help to their identification. Thus endothelium or Schwann cells are clearly defined by their relation to vessels or nerves respectively, in addition to their individual cell morphology. Particularly in the papillary dermis, various leukocytes are frequently concentrated around vessels, or within lymph vessels and may easily be recognised by their size and nuclear structure (Fig. 8 a). Mast cells are very evident through their granules, which are stained (Fig. 8a). According to Wood et al. (1985), tissue macrophages are nondendritic and thus cannot be confused with fibroblasts. In fact, a few cells can be found with a very pale cytoplasm, containing various inclusions, which might be lysosomes (Fig. 8 b). These cells frequently possess an irregular nucleus and outline with short, unbranched protrusions, quite unlike those of fibroblasts. We presume these to be macrophages. Their numbers are greatly increased in scar tissue, where intermediate forms ranging to large vacuolated cells are also present. These cells are currently being subjected to further scrutiny. The overwhelming majority of cells are of the type described as fibroblasts in this study. They are extensively interconnected via their processes and nonfibroblastic cells are not part of this network. Facial skin reacted with antibody against vimentin may be seen to contain very similar cells, with vimentin immunoreactivity in processes that are nearly as long as those demonstrated by silver impregnation, but which frequently fail to form a continuous network (Fig. 8 c). At the same time the reaction reveals dendritic cells within the epidermis, which are presumably Langerhans cells. Silver impregnation does not visualise these cells within the epidermis. It is important to note that, as with all cell identifications relying upon morphological criteria, not every cell within a section can be classified unequivocally. Even so, the number of cells that cannot be clearly identified as fibroblast or nonfibroblast is such a small proportion of the total that they are of little consequence. In our opinion, it is highly unlikely that any cells other than fibroblasts were included in our measurements.
204
G. E. K. NOVOTNY AND C. GNOTH
ee"I . .8. :..s. t~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . . Fig. 8. Nonfibroblast cells and vimentin immunoreactivity in the papillary dermis. (a) A group of leukocytes (small arrows) and a mast cell (larger arrow) in the vicinity of a blood vessel (sectioned on the left). Note smaller size of the leukocytes and their irregular nuclei, as well as the absence of cell processes. For comparison, there are fibroblasts in the upper part of the illustration. (b) A presumed macrophage. Note unbranched protoplasmic protrusions containing inclusions, and the irregular nucleus. (c) Facial skin. Immunocytochemical localisation of vimentin in a dendritic cell (arrow) below the epidermis (top). All magnifications x 1200.
The selection of cells for measurement poses a problem. According to standard morphometric methodology, it is essential that the selection of entities for measurement is random. This is clearly impossible in a situation where the limits of cells are obscured by numerous interconnections. Therefore the cells actually measured are exceptional and form a very small subpopulation. However, as far as could be judged, the selected cells did not differ appreciably from the other fibroblasts within the region, except that all processes were distinct. Possibly the selected cells were in a stage before mitosis, where the processes are beginning to withdraw from their interconnections. Any errors introduced by the procedure of selection apply equally to all measurements. Although differences in the size of fibroblasts were noted in the early observations of Maximow (1906), who considered those in the reticular dermis to be larger than others, there have been no subsequent investigations on this subject. The present study confirms that considerable differences in size exist, but we have found the fibroblasts of the subcutis to be of equal size or even larger. More important than the differences in size are the variations in total morphology. Differences in fibroblast morphology are mentioned in textbooks of histology; however, the range is restricted between spindle-shaped and stellate, with the
Fibroblast morphology
205
implication that these forms are present indiscriminately within any given region of connective tissue. Our findings demonstrate that the precise morphology, which may even permit a more detailed subdivision than the one we have presented, is strictly related to the organisation of the collagen within the region. Table 1 shows that there are considerable differences in size between papillary and subcutis fibroblasts. The figures obtained from this one adult case fully support the obvious differences that may be noted qualitatively in all other adult cases. It was not possible to perform comparable measurements on the reticular dermis, because the large number and close apposition of the processes to wavy and crinkled collagen bundles prevents their clear individual delimitation. An interesting question pertains to the reason for such morphological diversity. At birth and in the first years of life, the fibroblast population is still quite homogeneous. The adult status is achieved during a stepwise process of differentiation (Gnoth and Novotny, unpublished observations). Since the different cell types form a mixed population in the border zones between regions, and the subcutis shows heterogeneous types between the retinacula and the perilobular regions, we consider that at least three distinct fibroblast subpopulations are present: papillary, reticular and subcutaneous. Functional differences in growth kinetics, packing density and age effects between papillary and reticular fibroblasts have already been observed under cell-culture conditions (Harper & Grove, 1979; Schafer, Pandy, Ferguson & Davis, 1985). In these experiments, papillary fibroblasts were shown to possess a greater 'in vitro growth potential' and a longer replication life span than reticular fibroblasts, especially in those subpopulations obtained from older individuals. This may account for the obvious difference in the number of fibroblasts in the dermal layers and subcutis in adults. Tajima & Pinnell (1981) have shown that there are no differences in relative collagen synthesis by fibroblast populations at confluent densities. Taking this finding as a basis, it may be postulated that fibroblasts are capable of producing and maintaining a specific volume of collagen per unit time. A higher rate of collagen turnover would therefore require a higher cell density, as is seen in the papillary dermis. A lower collagen turnover will enable the fibroblasts to maintain a larger volume and so the processes increase in length in the deeper layers of the dermis and in the subcutis. Within all regions the fibroblasts basically form an interconnected network with their processes. The different shapes may be considered to be a consequence of growth. The development of fat lobules in the subcutis displaces the collagen concentrically - the 3-dimensional network is thus compressed in 1 axis, forming concentric layers with the enclosed fibroblasts reduced in the radial dimension. In compensation, the tangential extent can increase. Growing retinacula and large bundles will lead to extension of the fibroblasts in the longitudinal axis of the bundle, leading to the bipolar aspect found in these regions. A further point of interest is the question of age changes in fibroblasts. Numerous studies have shown that the number of fibroblasts decreases with age (Andrew, Behnke & Sato, 1965; Holbrook, 1982; Lindner, 1983; Gilchrest, 1984; Pieraggi et al. 1985; Fenske & Lober, 1986; Schmiegelow, Niissgen, Grasedyck & Lindner, 1986). Our material supports these findings: the differences within the reticular dermis and the subcutis between the young and old material are so pronounced that morphometry is superfluous, and in any case the number of individuals available for this study is too small for a morphometric analysis. However, even in the oldest case (81 years), all fibroblasts are still interconnected via their processes. Since the cells are less closely packed, it would be expected that the processes must be longer in the older individuals.
206
G. E. K. NOVOTNY AND C. GNOTH Table 2 shows this to be the case. We have not been able to obtain sufficient material to perform a statistically based morphometric analysis, but the measurements from the individual cases offer no indication whatever that the length or number of processes decrease with age. This finding is in direct contradiction to the report by Pieraggi et al. (1985), who claimed that older fibroblasts lose their processes. We are also unable to confirm these authors in their contention that the fibroblasts become dissociated from the collagenous bundles. However, our figures are based on subcutis fibroblasts, whereas Pieraggi et al. (1985) studied the papillary dermis. Due to the much greater density of the fibroblast population in the papillary dermis, comparable measurements are much more difficult in this region. We further attribute the discrepancy between the observations to the use of electron microscopy by these authors. The longer processes of older fibroblasts are thinner than the more protoplasmic processes of younger cells. Thinner processes will be more difficult to find sectioned longitudinally at the EM level, and so the impression is derived that the cells have fewer and shorter processes. The dissociation between collagen and cells in older individuals may be due either to differential shrinkage during embedding, or be related to sampling errors. As stated in the Results section, fibroblasts are most unevenly distributed. Such uneven distributions are very prone to errors due to insufficient sample size. A testable consequence of our hypothesis is that the collagenous lamellae of the subcutis should decrease in thickness with age. We are currently engaged in trying to gather sufficient material for a statistical basis broad enough to allow a definite conclusion. REFERENCES
ANDREW, W., BEHNKE, R. H. & SATO, T. (1965). Changes with advancing age in the cell population of human dermis. Gerontologia 10, 1-19. BAYREUTHER, K., RODEMANN, H. P., HOMMEL, R., DiTTMANN, K., ALBIEZ, M. & FRANCZ, P. I. (1988). Human
skin fibroblasts in vitro differentiate along a terminal cell lineage. Proceedings of the National Academy of Science 85, 5112-5116. BELLOWS, C. G., MELCHER, A. H. & AUBIN, J. E. (1981). Contraction and organisation of collagen gels by cells cultured from periodontal ligament, gingiva and bone suggests functional differences between cell types. Journal of Cell Science 50, 299-314. BULGER, R. E. & NAGLE, R. B. (1973). Ultrastructure of the interstitium in the rabbit kidney. American Journal of Anatomy 136, 183-204. DESAKI, J., FUJIWARA, T. & KOMURO, T. (1984). A cellular reticulum of fibroblast-like cells in the rat intestine: scanning and transmission electron microscopy. Archivum histologicumjaponicum 47, 179-186.
EYDEN, B. P., WATSON, R. J., HARRIS, M. & HOWELL, A. (1986). Intralobular stromal fibroblasts in the resting
mammary gland: ultrastructural properties and intercellular relationships. Journal of Submicroscopical Cytology 18, 397-408. FENSKE, N. A. & LOBER, C. W. (1986). Structural and functional changes of normal aging skin. Journal of the American Academy of Dermatology 15, 571-585. GILCHREST, B. A. (1984). Age-associated changes in normal skin. In Skin and Aging Processes (ed. B. A. Gilchrest), pp. 17-35. Boca Raton, Fl: CRC Press Inc. HARPER, R. A. & GROVE, G. (1979). Human skin fibroblasts derived from papillary and reticular dermis: differences in growth potential in vitro. Science 204, 526-527. HOLBROOK, K. A. (1982). A histological comparison of infant and adult skin. In Neonatal Skin - Structure and Function (ed. H. I. Maibach & K. E. Boisets), pp. 1-31. New York: Marcel Dekker. HOLBROOK, K. A., BYERS, P. H. & PINNELL, S. R. (1982). The structure and function of dermal connective tissue in normal individuals and patients with inherited connective tissue disorders. Scanning Electron Microscopy 4, 1731-1744. KOMURO, T., DESAKI, J. & FUJIWARA, T. (1982). Reevaluation of the fibroblasts and/or fibroblast-like cells. Acta anatomica nipponica 57, 332-333. LAVKER, R. M., ZHENG, P. & DONG, G. (1987). Aged skin: a study by light, transmission electron, and scanning electron microscopy. Journal of Investigative Dermatology 83(Suppl. 3), 44s-51s. LINDNER, J. (1983). Haut-Alterung (Ubersicht): Morphologie und Biochemie. Verhandlungen der Deutschen Dermatologischen Gesellschqft 75, 93-99. human
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MAXIMoW, A. (1906). Uber die Zellformen des lockeren Bindegewebes. Archivfuer mikroskopische Anatomie 67, 680-757. MCGAW, T. W. (1986). The effect of tension on collagen remodelling by fibroblasts: a stereological ultrastructural study. Cell and Tissue Research 14, 229 235. MONTAGNA, W. & CARLISLE, K. (1979). Structural changes in aging human skin. Journal of Investigative Dermatology 73, 47-53. NOVOTNY, G. E. K. & GOMMERT-NOVOTNY, E. (1990). A simple procedure for demonstrating the overall morphology of fibroblasts in routine histological preparations of adult tissues, using silver impregnation. Journal of Microscopy 159, 99-107. OLMO, N., LIZARBE, M. A., TURNEY, J., MOLLER, K. P. & GAVILANES, J. G. (1988). Cell morphology, proliferation and collagen synthesis of human fibroblasts cultured on sepolite-collagen complexes. Journal of Biomedical Material Research 22, 257-270. OTHANI, O., USHIKI, T., TAGUCHI, T. & KIKUTA, A. (1988). Collagen fibrillar networks as skeletal frameworks: a demonstration by cell-maceration/scanning electron microscope method. Archivum Histologicum et Cvtolologicum 51, 249-261. PFALLER, K., SCHULER, G., SCHMIDT, W. & DWORSCHAK, K. (1979). A critical reevaluation of fibre arrangement in the mid-zone (stratum reticulare) of human corium. Anatomischer Anzeiger 145, 404-412. PIERAGGI, M., BOUISSoU, H., ANGELIER, C., UHART, D., MAGNOL, J. P. & KOKOLO, J. (1985). Le fibroblaste. Annales de Pathologie (Paris) 5, 65-76. SCHAFER, 1. A., PANDY, M., FERGUSON, R. & DAVIS, B. R. (1985). Comparative observation of fibroblasts derived from the papillary and reticular dermis of infants and adults: growth kinetics, packing density at confluence and surface morphology. Mechanisms of Ageing and Development 31, 275-293. SCHMIEGELOW, P., NUSSGEN, A., GRASEDYCK, K. & LINDNER, J. (1986). Hautveranderungen im hohen Lebensalter - korrespondieren biochemische Befunde zur Morphologie? Zeitschrift fur Gerontologie 19, 179-189. SQUIER, C. A. & MAGNES, C. (1983). Spatial relationships between fibroblasts during the growth of rat-tail tendon. Cell and Tissue Research 234, 17-29. SQUIER, C. A. & BAUSCH, W. H. (1984). Three-dimensional organization of fibroblasts and collagen fibrils in rat tail tendon. Cell and Tissue Research 238, 319-327. SUGITA, A., ISHIBASHI, R., SHIOTANI, N. & YOSHIOKA, H. (1988). Morphological features of iris fibroblasts in dilator muscle region. Japanese Journal of Ophthalmology 32, 151-158. TAJIMA, S. & PINNELL, S. R. (1981). Collagen synthesis by human skin fibroblasts in culture: studies of fibroblasts explanted from papillary and reticular dermis. Journal of Investigative Dermatology 77, 410-412. TOMASEK, J. J., HAY, E. D. & FUJIWARA, K. (1982). Collagen modulates cell shape and cytoskeleton of embryonic corneal fibroma fibroblasts: distribution of actin, x-actin, and myosin. Developmental Biology 92, 107-122. WOOD, G. S., TURNER, R. R., SHIUBRA, R.-A., ENG, L. & WARNKE, R. A. (1985). Human dendritic cells and macrophages. In situ immunophenotypic definition of subsets that exhibit specific morphologic and microenvironmental characteristics. Anmerican Journal of Pathology 119, 73-82. YOSHIZATO, K., TAIRA, T. & SHIOYA, N. (1984). Collagen-dependent growth suppression and changes in the shape of human dermal fibroblasts. Annals of Plastic Surgery 13, 9-14.
J. Anat. (1991), 177, 195-207 With 8 figures Printed in Great Britain
Variability of fibroblast morphology in vivo: a silver impregnation study on human digital dermis and subcutis G. E. K. NOVOTNY AND C. GNOTH Department of Neuroanatomy, University of Dusseldorf, Moorenstrasse 5,
4000 Dusseldorf 1, Germany
(Accepted 5 April 1991) INTRODUCTION
Connective tissue research is a vast field with an enormous volume of literature. However, attention has primarily been focused either on collagen and the extracellular matrix, or on fibroblasts in tissue culture. Few publications deal with fibroblasts in situ, and then mostly in very restricted regions and situations (Bulger & Nagle, 1973; Komuro, Desaki & Fujiwara, 1982; Squier & Magnes, 1983; Desaki, Fujiwara & Komuro, 1984; Pieraggi et al. 1985; Eyden, Watson, Harris & Howell, 1986; Sugita, Ishibashi, Shiotani & Yoshioka, 1988). Although it is clear that fibroblasts produce collagen and the matrix, their role in the organisation of these elements is mainly considered to be passive (Tomasek, Hay & Fujiwara, 1982), and considerable energy has been expended upon the study of the arrangement of collagen in various connective tissue regions, especially the dermis (Montagna & Carlisle, 1979; Pfaller, Schuler, Schmidt & Dworschak, 1979; Holbrook, 1982; Holbrook, Byers & Pinnell, 1982; Lavker, Zheng & Dong, 1987; Othani, Ushiki, Taguchi & Kikuta, 1988), as well as on explanations of a particular arrangement in terms of stress and strain forces (Bellows, Melcher & Aubin, 1981; McGaw, 1986). Differences in size between fibroblasts have been noted in the past (Maximow, 1906), though without any subsequent attempt at systematisation or correlation with the organisational form of the connective tissue. In cell culture, differences in proliferation potential between fibroblasts derived from specific regions have been described (Harper & Grove, 1979; Schafer, Pandy, Ferguson & Davis, 1985), but the morphological diversity has been attributed to degenerative processes (Bayreuther et al. 1988), or substratum effects (Tomasek et al. 1982; Yoshizato, Taira & Shioya, 1984; Olmo et al. 1988). Using a new silver impregnation technique (Novotny & Gommert-Novotny, 1990), it is possible to visualise fibroblasts in their total extent in paraffin serial sections. From initial observations it would seem that fibroblasts have a much more systematic relationship to the extracellular structures and are by no means passively confined to convenient interstices, but take an active part in the development of the extracellular organisation. Here we wish to demonstrate that, even within the very confined area of the adult human digital dermis and subcutis, there may be considerable differences in size and morphology of fibroblasts. These may be related to the 3-dimensional collagen architecture of the connective tissue, which is in turn related to the stratification of the digital dermis and subcutis. Correspondence to: Professor G. E. K. Novotny, Abteilung fur Neuroanatomie, Zentrum fur Anatomie und Hirnforschung, Medizinische Einrichtungen der Universitat Diisseldorf, Moorenstrasse 5, 4000 Dusseldorf 1, Germany.
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G. E. K. NOVOTNY AND C. GNOTH MATERIAL AND METHODS
Digital dermis and subcutis was available from 8 individuals of both sexes aged from 14 to 81 years, with at least 1 representative per decennium. The material was obtained at routine autopsy and fixed by immersion in 10 % formaldehyde. Dehydration and embedding in paraffin (Paraplast Plus) was carried out according to conventional procedures. Sections ranging from 10 to 40 ,um in thickness were cut, mounted, and silver impregnated according to published methods (Novotny & Gommert-Novotny, 1990). Sections were evaluated in a Leitz Orthoplan or Dialux microscope, using transmitted light, interference contrast, or circularly polarised light. For purposes of comparison, frozen sections from biopsy material obtained during a facial operation were reacted with monoclonal antibodies against vimentin (B6hringer, clone Va). The impression obtained from the qualitative observations was further substantiated by measurement. Papillary dermis fibroblasts were compared with subcutis fibroblasts using material obtained from a 54-year-old male. The parameters measured included the greater and lesser diameter of the cell body, the length of the longest cell process, and the number of cell processes. Material from 14-, 37- and 81-year-old females, and from 23- and 54-year-old males was used for measurements of subcutis fibroblasts. In these instances, only the length of the longest cell process was determined. With the exception of the cell body size, where only 50 cells were measured, all measurements were performed on 100 cells per sample and on 40 ,um sections. The selection of cells for measurement posed problems. Since fibroblasts form a continuous network with their cell processes at the light microscopical level, it is impossible to determine the true limits of a cell in the majority of cases. In order to obtain reasonably comparable data, cells for measurement were selected according to the following criteria. (1) The cell body is located within the central part of the section thickness. (2) The cell processes are distinct from those of other fibroblasts in their total course. (3) The terminations of the cell processes are clearly distinguishable, totally within the section thickness, and not terminating at the surface of the section. Cells satisfying these criteria were rare. They were located by scanning the section in the classical meander pattern. Measurements were performed on the first 100 cells found. RESULTS
As illustrated in Figure 1, the adult digital integument can be clearly divided into a papillary dermis, reticular dermis and subcutis. Each of these regions contains
characteristic fibroblast populations, differing markedly from those of the other layers. They are described sequentially in the following section. Papillary dermis The fibroblasts of the papillary dermis are characteristically small cells, of 3dimensional stellate form, with thin processes extending in all directions (Fig. 2a), which frequently seem to merge with those of neighbouring cells. Within the uppermost layer of the papillary dermis, the subepidermal band (Holbrook, 1982), fibroblasts are frequently oriented horizontally, with processes extending parallel to the epidermis, their terminals curving upwards towards the latter (Fig. 2b). A little below these 'horizontal' cells, a 'second line' of 'basket '-shaped fibroblasts may be found (Fig. 2 c). All fibroblasts of the papillary dermis extend their processes to several
Fibroblast morphology
.
P
197
f~ ~~~A
47~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ;iS I,~~~~~~~~~~
Fig. 1. Low power view ( x 50) of a section of human digital dermis and subcutis, demonstrating the layers from which the further illustrations have been taken at higher magnification. P, papillary dermis (Figs 2, 8); R, reticular dermis (Fig. 3); S, subcutis (Figs 4-7). F, fat lobule (Fig. 6); L, tangential sections of concentric collagenous lamellae around a fat lobule (Fig. 5); Pc, pacinian corpuscle; Re, retinaculum cutis (Fig. 7).
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Fig. 2. Fibroblasts of the papillary dermis. (a) Typical stellate fibroblast (arrow), with processes in all directions. (b) Fibroblast of the subepidermal band, with horizontal processes curving up to the epidermis (arrow). (c) Basket shaped fibroblast All magnifications x 1200.
of the fine collagenous bundles interwoven within this layer a single cell is thus not confined to 1 collagen bundle. Towards the reticular dermis the size of the cells gradually increases. Reticular dermis Within this layer the fibroblasts are larger than those of the papillary dermis. The collagenous bundles are coarser. In the upper part of this region the fibroblasts are stellate and resemble those of the papillary dermis. In the deeper part of this layer the fibroblasts are predominantly associated with a single very coarse collagen bundle, whereby their processes extend along its length (Fig. 3 a-c). These fibroblasts therefore take on a unipolar or bipolar aspect. The processes either branch in a 'Y' form along the extent of a bundle, or short stubby processes in a transverse orientation give rise to a long longitudinal process in a 'T' junction (Fig. 3a, b). The fibroblasts are predominantly located on the surface of the bundles, though some are inside them (Fig. 3 c). The distribution of fibroblasts is uneven - some collagenous bundles appear nearly black because of the high number of processes associated with them - while others are totally devoid of processes (Fig. 3 c). In instances with a dense population of fibroblasts it is almost always impossible to define the extent of a single cell, as the processes are so closely applied to each other that a boundary cannot be resolved by light microscopy. Between the interwoven collagenous bundles loose collagen can also be found that fills the interstices. This loose collagen is associated with fibroblasts that are oriented 3-dimensionally, with processes extending in all directions, having only occasional contacts with collagenous bundles (Fig. 3d). These fibroblasts may be termed 'interstitial'. Subcutis The architecture of the subcutis is determined by fat lobules, sweat glands, and groups of pacinian corpuscles. Around these 'voluminous' structures concentrically arranged laminae of loose collagen are found (Fig. 4). Each lamina is approximately 10 ,am thick and the fibroblasts are located between adjacent laminae. They are very large cells, with processes of considerable length - the longest found measured 180 ,um. They predominantly extend along the surface of the laminae and only rarely penetrate from one to another. The cells thus appear 2-dimensional, having a stellate aspect from 1 surface and appearing bipolar when viewed at right angles to
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199
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MR."I'M
....
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Fig. 3. Fibroblasts of the reticular dermis. (a) Cell bodies of fibroblasts arrayed along a coarser collagen bundle with T-shaped branching of a process (arrow). (b) A fibroblast (Cb, cell body) on the surface of a coarse collagenous bundle with longitudinally oriented processes exhibiting Y-shaped branching (arrows). (c) Cross-sections of collagenous bundles demonstrating the location of cell bodies (large arrows) and processes (arrowheads) on the surface and within the bundles. Small arrows indicate processes in longitudinally sectioned bundles. (d) 'Interstitial' fibroblasts (arrows) with processes extending in all directions. All magnifications x 1200.
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G. E. K. NOVOTNY AND C. GNOTH
Fig. 4. Medium power view of concentrically arrayed collagenous lamellae (L) around fat lobules in the subcutis. The open arrow indicates cross-sectioned lamellae; the large arrow points to the tangentially sectioned portion of the lamellae. The small arrow indicates fibroblasts applied to a fat cell. F, fat lobule. Magnification x 250.
the surface of the laminae (Fig. 5 a, b). The processes of neighbouring cells are closely apposed to each other, so that it is frequently impossible to determine their true extent. The cells thus form a dense interconnected network (Fig. Sc). Fibroblasts with shorter processes are found directly applied to individual adipocytes (Figs 4, 6), pacinian corpuscles, or coils of sweat glands. Smaller fibroblasts with fewer processes are also found around blood vessels, which have a separate system of very fine concentric collagenous lamellae. Within the subcutis bundles of denser collagen may also be observed, originating in the reticular dermis and penetrating to the periost (retinacula cutis). These exhibit the same fibroblast population as is associated with large collagenous bundles in the reticular dermis (Fig. 7). Their processes are oriented predominantly along the long axis of the bundles, whereas some again are associated with loose collagen between the bundles as interstitial fibroblasts. Measurements The results of the measurements performed on individual cells are summarised in Tables 1 and 2. They support the impression obtained from the qualitative observations. DISCUSSION
Silver impregnation is not a specific stain for fibroblasts. All cells are visualised to some extent with this method, if only through staining of the nucleus. However, the cytoplasm of fibroblasts is particularly intensely stained, especially the processes - the
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Fig. 5. Subcutis fibroblasts. (a) Fibroblasts (arrows) as seen in cross-sectioned collagenous lamellae. (b) A fibroblast from a tangentially sectioned lamella, showing the considerable extent of the processes. (c) Fibroblasts in a tangential section from between 2 collagenous lamellae, demonstrating their stellate shape and contiguity of their processes. All magnifications x 1200.
more dendrite-like the process, the denser the impregnation. Extracellular material is lightly impregnated. Collagen bundles may be differentiated from the homogenous matrix, elastin lamellae remain unstained and, with few exceptions, elastin fibres are not visible. This generalised staining enables a precise localisation of individual cells,
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G. E. K. NOVOTNY AND C. GNOTH
Fig. 6. A fibroblast applied to a single fat cell. Note plump cell body, stellate shape and short processes. Magnification x 1200.
Fig. 7. Fibroblasts within retinacula cutis in the subcutis. (a) Cross-section of a retinaculum cutis, showing cell bodies (large arrows) and processes (small arrows) within the collagen bundles. The majority of processes are cross-sectioned. (b) A retinaculum cutis in longitudinal section, demonstrating the longitudinally arrayed cell processes. Both magnifications x 1200.
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Fibroblast morphology
Table 1. Measurements of.fibroblasts in digital skin of a 54-year-old male* Cell body size (,um)
Papillary dermis
Greater diameter
Lesser diameter
7 30 (1-8)
3 7 (1 0)
Length of longest cell process (,um)
Number of cell processes
22 15 (5 9) (n = 100) 75 9 (21-8) (n = 100)
5 27 (1-7) (n = 100) 6 3 (2 5) (n = 100)
(n = 46)
Subcutis
15 6 (4 5) *
8-2 (2 8) (n = 50) Means with SD in parentheses.
Table 2. Average number of processes per cell and average length of the longest process of subcutis fibroblasts in relation to age of subjects Age (years)* ... Average number of cell processes (SD) Average length of longest cell process in um (SD)
14
23
37
54
81
4-3 (25)
61 (28)
65 (27)
6-3 (25)
7-7 (22)
48-1 (15-1)
59-5 (18-0)
71 3 (20 5)
75-9(21 8)
83-0 (19 0)
*
n = 100 for each subject.
as a further help to their identification. Thus endothelium or Schwann cells are clearly defined by their relation to vessels or nerves respectively, in addition to their individual cell morphology. Particularly in the papillary dermis, various leukocytes are frequently concentrated around vessels, or within lymph vessels and may easily be recognised by their size and nuclear structure (Fig. 8 a). Mast cells are very evident through their granules, which are stained (Fig. 8a). According to Wood et al. (1985), tissue macrophages are nondendritic and thus cannot be confused with fibroblasts. In fact, a few cells can be found with a very pale cytoplasm, containing various inclusions, which might be lysosomes (Fig. 8 b). These cells frequently possess an irregular nucleus and outline with short, unbranched protrusions, quite unlike those of fibroblasts. We presume these to be macrophages. Their numbers are greatly increased in scar tissue, where intermediate forms ranging to large vacuolated cells are also present. These cells are currently being subjected to further scrutiny. The overwhelming majority of cells are of the type described as fibroblasts in this study. They are extensively interconnected via their processes and nonfibroblastic cells are not part of this network. Facial skin reacted with antibody against vimentin may be seen to contain very similar cells, with vimentin immunoreactivity in processes that are nearly as long as those demonstrated by silver impregnation, but which frequently fail to form a continuous network (Fig. 8 c). At the same time the reaction reveals dendritic cells within the epidermis, which are presumably Langerhans cells. Silver impregnation does not visualise these cells within the epidermis. It is important to note that, as with all cell identifications relying upon morphological criteria, not every cell within a section can be classified unequivocally. Even so, the number of cells that cannot be clearly identified as fibroblast or nonfibroblast is such a small proportion of the total that they are of little consequence. In our opinion, it is highly unlikely that any cells other than fibroblasts were included in our measurements.
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G. E. K. NOVOTNY AND C. GNOTH
ee"I . .8. :..s. t~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . . Fig. 8. Nonfibroblast cells and vimentin immunoreactivity in the papillary dermis. (a) A group of leukocytes (small arrows) and a mast cell (larger arrow) in the vicinity of a blood vessel (sectioned on the left). Note smaller size of the leukocytes and their irregular nuclei, as well as the absence of cell processes. For comparison, there are fibroblasts in the upper part of the illustration. (b) A presumed macrophage. Note unbranched protoplasmic protrusions containing inclusions, and the irregular nucleus. (c) Facial skin. Immunocytochemical localisation of vimentin in a dendritic cell (arrow) below the epidermis (top). All magnifications x 1200.
The selection of cells for measurement poses a problem. According to standard morphometric methodology, it is essential that the selection of entities for measurement is random. This is clearly impossible in a situation where the limits of cells are obscured by numerous interconnections. Therefore the cells actually measured are exceptional and form a very small subpopulation. However, as far as could be judged, the selected cells did not differ appreciably from the other fibroblasts within the region, except that all processes were distinct. Possibly the selected cells were in a stage before mitosis, where the processes are beginning to withdraw from their interconnections. Any errors introduced by the procedure of selection apply equally to all measurements. Although differences in the size of fibroblasts were noted in the early observations of Maximow (1906), who considered those in the reticular dermis to be larger than others, there have been no subsequent investigations on this subject. The present study confirms that considerable differences in size exist, but we have found the fibroblasts of the subcutis to be of equal size or even larger. More important than the differences in size are the variations in total morphology. Differences in fibroblast morphology are mentioned in textbooks of histology; however, the range is restricted between spindle-shaped and stellate, with the
Fibroblast morphology
205
implication that these forms are present indiscriminately within any given region of connective tissue. Our findings demonstrate that the precise morphology, which may even permit a more detailed subdivision than the one we have presented, is strictly related to the organisation of the collagen within the region. Table 1 shows that there are considerable differences in size between papillary and subcutis fibroblasts. The figures obtained from this one adult case fully support the obvious differences that may be noted qualitatively in all other adult cases. It was not possible to perform comparable measurements on the reticular dermis, because the large number and close apposition of the processes to wavy and crinkled collagen bundles prevents their clear individual delimitation. An interesting question pertains to the reason for such morphological diversity. At birth and in the first years of life, the fibroblast population is still quite homogeneous. The adult status is achieved during a stepwise process of differentiation (Gnoth and Novotny, unpublished observations). Since the different cell types form a mixed population in the border zones between regions, and the subcutis shows heterogeneous types between the retinacula and the perilobular regions, we consider that at least three distinct fibroblast subpopulations are present: papillary, reticular and subcutaneous. Functional differences in growth kinetics, packing density and age effects between papillary and reticular fibroblasts have already been observed under cell-culture conditions (Harper & Grove, 1979; Schafer, Pandy, Ferguson & Davis, 1985). In these experiments, papillary fibroblasts were shown to possess a greater 'in vitro growth potential' and a longer replication life span than reticular fibroblasts, especially in those subpopulations obtained from older individuals. This may account for the obvious difference in the number of fibroblasts in the dermal layers and subcutis in adults. Tajima & Pinnell (1981) have shown that there are no differences in relative collagen synthesis by fibroblast populations at confluent densities. Taking this finding as a basis, it may be postulated that fibroblasts are capable of producing and maintaining a specific volume of collagen per unit time. A higher rate of collagen turnover would therefore require a higher cell density, as is seen in the papillary dermis. A lower collagen turnover will enable the fibroblasts to maintain a larger volume and so the processes increase in length in the deeper layers of the dermis and in the subcutis. Within all regions the fibroblasts basically form an interconnected network with their processes. The different shapes may be considered to be a consequence of growth. The development of fat lobules in the subcutis displaces the collagen concentrically - the 3-dimensional network is thus compressed in 1 axis, forming concentric layers with the enclosed fibroblasts reduced in the radial dimension. In compensation, the tangential extent can increase. Growing retinacula and large bundles will lead to extension of the fibroblasts in the longitudinal axis of the bundle, leading to the bipolar aspect found in these regions. A further point of interest is the question of age changes in fibroblasts. Numerous studies have shown that the number of fibroblasts decreases with age (Andrew, Behnke & Sato, 1965; Holbrook, 1982; Lindner, 1983; Gilchrest, 1984; Pieraggi et al. 1985; Fenske & Lober, 1986; Schmiegelow, Niissgen, Grasedyck & Lindner, 1986). Our material supports these findings: the differences within the reticular dermis and the subcutis between the young and old material are so pronounced that morphometry is superfluous, and in any case the number of individuals available for this study is too small for a morphometric analysis. However, even in the oldest case (81 years), all fibroblasts are still interconnected via their processes. Since the cells are less closely packed, it would be expected that the processes must be longer in the older individuals.
206
G. E. K. NOVOTNY AND C. GNOTH Table 2 shows this to be the case. We have not been able to obtain sufficient material to perform a statistically based morphometric analysis, but the measurements from the individual cases offer no indication whatever that the length or number of processes decrease with age. This finding is in direct contradiction to the report by Pieraggi et al. (1985), who claimed that older fibroblasts lose their processes. We are also unable to confirm these authors in their contention that the fibroblasts become dissociated from the collagenous bundles. However, our figures are based on subcutis fibroblasts, whereas Pieraggi et al. (1985) studied the papillary dermis. Due to the much greater density of the fibroblast population in the papillary dermis, comparable measurements are much more difficult in this region. We further attribute the discrepancy between the observations to the use of electron microscopy by these authors. The longer processes of older fibroblasts are thinner than the more protoplasmic processes of younger cells. Thinner processes will be more difficult to find sectioned longitudinally at the EM level, and so the impression is derived that the cells have fewer and shorter processes. The dissociation between collagen and cells in older individuals may be due either to differential shrinkage during embedding, or be related to sampling errors. As stated in the Results section, fibroblasts are most unevenly distributed. Such uneven distributions are very prone to errors due to insufficient sample size. A testable consequence of our hypothesis is that the collagenous lamellae of the subcutis should decrease in thickness with age. We are currently engaged in trying to gather sufficient material for a statistical basis broad enough to allow a definite conclusion. REFERENCES
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