Arch Dermatol Res (1990) 282:434-441 Archives of
Dynamic ultrastructural changes of the connective tissue sheath of human hair follicles during hair cycle M. Ito and Y. Sato Department of Dermatology, Niigata University School of Medicine, Niigata 951, Japan Received March 17, 1990
Summary. Ultrastructural changes of the connective tissue sheath (CTS), including the hyaline membrane, of human hair follicles during the hair cycle, were studied in normal scalp skin specimens. In early anagen, the CTS was composed of a thin basal lamina and surrounding collagen tissue. The collagen tissue gradually thickened during the development of the hair and hair follicle. In mature anagen hair follicles, the collagen tissue was separated into three layers. The inner collagen layer, just outside the basal lamina, was thin and composed of collagen fibres running longitudinally parallel to the hair axis. The middle collagen layer was very thick with its collagen fibres running transversely against the hair axis and surrounding the inner hair tissue. Many fibroblasts were present among the collagen fibres in the middle layer, whereas the inner layer contained almost none. In the outer collagen layer, collagen fibres ran in various directions parallel to the outer surface of the outer root sheath cells. In late anagen, the basal lamina became very thick. In catagen, the basal lamine and the inner collagen layer became corrugated and showed oedematous change and degeneration. Surrounding fibroblasts showed active production of new collagen fibres, which seemed to fill the spaces left by the retraction of the hair follicle and hyaline membrane. These ultrastructural changes of the CTS show that there may be dynamic metabolic changes of the connective tissue around human hair follicles during the hair cycle. Key words: Hair cycle - H u m a n hair follicle - Connective tissue sheath - Hyaline membrane - Ultrastructure
The hair tissues show cyclic morphological changes from anagen through catagen and telogen, to anagen again. The changes are called the 'hair cycle': hair grows in anagen, stops growing in catagen, falls out at the end of Offprint requests to .' M. Ito.
telogen, and then regenerates in the next anagen. Morphologically [2, 8], the anagen hair follicle is composed of several cell layers, producing a hair by the cell division in the hair matrix in the hair bulb. The lower portion below the bulge of the hair follicle, to which an arrector pili muscle adheres, is called the 'transient portion', because it changes morphologically during the hair cycle . In catagen, the cell division in the hair matrix ceases, the hair bulb becomes atrophic, and the lower end (presumptive club) of the hair-forming cell layers gradually ascends, leaving an epithelial cord . Then, the epithelial cord gradually becomes retracted and finally disappears, resulting in formation of a telogen hair tissue [7, 8]. During the hair cycle, the connective tissue sheath (CTS), which surrounds the hair epithelial tissue, is also known to show histological changes. The hyaline membrane, in particular, shows dramatic changes. This is a membranous structure located just outside the outer root sheath (ORS) and is positively stained with periodic acid Schiff staining . It is relatively thick in the lower portion of the anagen hair follicle, becomes very thick in catagen, and finally disappears in telogen [7, 8]. The structures of the CTS and the changes of the hyaline membrane have been observed under the electron microscope mainly in mouse hair follicles [8, 10, 11, 13, 14]. In human hair follicles, however, ultrastructural details of the CTS, including the hyaline membrane, have not been shown, except for a recent brief report . In the present study, the ultrastructure of the CTS and its changes during the human hair cycle, normal scalp hair follicles in various stages were studied electron microscopically.
Materials and methods Normal scalp skin specimens were obtained from three Japanese (two males and a female), two Caucasians (a male and a female) and a Negroid (female),aged from 16 to 32 years old. Each specimen was cut into small pieces, each of which contained one or a few complete hair follicles. These materials were double-fixed in 5%
M. Ito and Y. Sato: Ultrastructure of connective tissue sheath
Fig. 1 a, b. Light micrographs of a catagen hair follicle, a middle part; b lower part. The hyaline membrane (opposite-pointing arrows) is corrugated and thick around the retracting epithelial cord (E), especially around the lower end of the cord (E). Similar areas to 1 and 2 are shown electron microscopically in Figs. 6a and 7, respectively. Asterisk, club hair root; C, connective tissue sheath; v, blood vessel. Epon-embedded tissue. Toluidine blue stain. • 230
Fig. 2. Ultrastructure of connective tissue sheath of an early anagen hair follicle. The connective tissue sheath (C) is composed of a thin basal lamina (arrowheads), elongated-shaped fibroblasts (f) and collagen bundles running in various directions parallel to the outer surface of the outer root sheath cells (O). Uranyi acetate-lead citrate stain. • 12,300
M. [to and Y. Sato: Ultrastructure of connective tissue sheath
Fig. 3a, b. Ultrastructure of connective tissue sheath of mature anagen hair follicles, a Low-power view. The connective tissue sheath (C) is composed of a linear basal lamina (arrowheads), the inner collagen layer (In), the middle collagen layer (M/), and the outer collagen layer (Ou). In the last two layers, many fibroblasts (f) are embedded, b Enlargement of the outer root sheath side of the connective tissue sheaths. The inner collagen layer (In) consists of
collagen fibres running longitudinally parallel to the hair long axis, whereas the collagen fibres in the middle layer (Mi) run transversely against the hair axis surrounding the inner hair structures. The basal lamina (arrowheads) partly reveals lamination and thickening (small outline arrows). O, Outer root sheath. Uranyl acetate-lead citrate stain, a x 1,800; b x 6,400
glutaraldheyde in 0.1 M cacodylate buffer (pH 7.4) and 1% osmium tetroxide in the same buffer, dehydrated in alcohol solutions of graded concentrations and propylene oxide, and embedded in Epon 812 or Araldite. Longitudinal thick sections of the embedded hair follicles were cut with diamond knives on a Sorvall MT-5000 ultramicrotome, stained with toluidine blue, and observed under a light microscope. Ultrathin sections were then similarly cut, doublestained with 1% or 15% uranyl acetate and Reynolds' lead citrate , and observed in a JEM 100S electron microscope.
U n d e r the electron microscope, the CTS in early anagen is composed of a basal lamina, adjacent to the outer border of the ORS, and surrounding collagen tissue. The basal lamina was a b o u t 0.05 lam thick and ran linearly along the outer surface of the ORS cells (Fig. 2). The whole thickness of the collagen tissues was a b o u t 5 gm. M a n y elongated fibroblasts were sandwiched between several thin lamellar layers of collagen fibres, which ran in various directions parallel to the outer surface of the ORS cells. Some fibroblasts were located very closely to the outer surface of the basal lamina (Fig. 2). In mature anagen hair follicles, the collagen tissue was a b o u t 6 0 - 8 0 g m thick and separated into three distinct layers (Fig. 3 a). The inner collagen layer, just outside the basal lamine, was relatively thin (about 3 lam) and was composed of collagen fibres running longitudinally parallel to the hair long axis (Fig. 3 a, b). The middle layer was a b o u t 30 gm thick, and its collagen fibres ran transversely against the hair axis surrounding the inner hair tissue. In this layer m a n y fibroblasts and a few blood vessels were present a m o n g the collagen fibres, whereas the inner layer contained almost no cells (Fig. 3 a, b). The outer collagen layer consisted of collagen fibres, which ran in various
Results U n d e r the light microscope, a few early anagen hair follicles, which are a short anagen structure with a compact hair dermal papilla in its hair bulb, were found. M a n y mature anagen hair follicles producing hairs were observed in each skin specimen. Although the n u m b e r of hair follicles in catagen or telogen was fewer than that in anagen, hair tissues in various stages in early catagen through late catagen (Fig. l a, b) to telogen were obtained. The present ultrastructural observations concentrated on changes of the CTS around the transient portions of the hair follicles.
M. Ito and Y. Sato: Ultrastructure of connective tissue sheath
Fig. 4a, b. Late anagen hair folIicle, a Low-power view. The basal lamina (opposite-pointing arrowheads) is uniformly thick, b Enlargement of the area indicated by the asterisk in Fig. 4a. The basal lamina consists of amorphous, fine granular or filamentous components and partly lamina-like structures, f, fibroblast; He,
Henl+'s layer; Im, innermost cell layer of the outer root sheath; In, inner collagen layer; Mi, middle collagen layer; O, outer root sheath; t, trichohyaline granule. Uranyl acetate-lead citrate stain, a x 6,300; b x 24,800
M. Ito and Y. Sato: Ultrastructure of connective tissue sheath
Fig. 5. Basal lamina in early catagen. The thick basal lamina (opposite-pointing arrowheads) covers the atrophic hair bulb composed of a remaining melanocyte (M) and a few outer root sheath cells (O). The basal lamina structure (b) facing the hair dermal papilla (P) is thin and irregularly laminated. The inner collagen layer (In) shows an oedematous change, m, melanosomes. Uranyl acetate-lead citrate stain, x 14,100
directions parallel to the outer surface of the ORS cells, and cellular structures such as fibroblasts (Fig: 3 a), blood vessels and some fat cells. Although the basal lamina was fundamentally a linear structure of a b o u t 0.2 gm thickness, it showed partial duplication or further lamination, forming portions a b o u t 1.5 gm thick (Fig. 3 b). In some hair follicles, which could have been in late anagen, the basal lamina became entirely thickened ( 0 . 8 - 2 . 4 g m thick), consisting of amorphous, fine granular or filamentous components and partly lamina-like structures (Fig. 4 a, b). In early catagen hair follicles, whose hair m a t r i x cells had just started cell degeneration, the thick basal lamina became m o r e homogeneous and the surrounding inner collagen layer showed oedematous change (Fig. 5). In relatively late catagen, the upper part of the hair follicle included a club hair root (Fig. I a), while its lower part was a thin epithelial cord showing gradual retraction (Fig. l a, b). The hyaline m e m b r a n e of the CTS around the epithelial cord became corrugated and partly very thickened, especially around the lower end of the epithelial cord (Fig. 1 b). In this area an extremely thick ( 5 0 - 1 0 0 gm) wavy basal lamina was found. Ultrastructurally, this basal lamina was amorphous, had lost its own electron density and fine filamentous components, and contained some collagen fibres (Fig. 6a, b).
The inner collagen layer around the basal lamina zone was also wavy (Fig. 6 a). Surrounding the inner collagen layer, m a n y fibroblasts having b r o a d cytoplasm, which was rich in rough endoplasmic reticula, were present (Fig. 6a, c). In the stroma adjacent to the fibroblasts, there was an abundance of slightly electron dense a m o r p h o u s substances and fine collagen fibres (Fig. 6 a, c). The fibroblasts often contained membrane-limited structures containing a few collagen fibres in the cytoplasm, especially facing the inner collagen layer (Fig. 6 a, b, c). Such intracytoplasmic collagen fibres m a y result f r o m fibre formation of collagen precursors in endoplasmic reticula rather than from phagocytosis of stromal collagen fibres, because there )
Fig. 6 a - e . Connective tissue sheath in catagen, a Ultrastructures ~ of a similar area to i in Fig. 1b. Around the corrugated inner collagen layer (In), many fibroblasts (/) are present and have broad cytoplasm rich in rough endoplasmic reticula (er). b Basal lamina and inner collagen layer. The extremely thick basal lamina (asterisk) is oedematous and contains some fine collagen fibres, e Enlargement of the area indicated by thick arrow in Fig. 6a. Intracytoplasmic collagen fibres (c) included in a limit membrane are seen in the fibroblasts (1). In the stroma surrounding the fibroblasts, abundant slightly electron-dense amorphous substances (stars) and fine collagen fibres can be seen. Uranyl acetate-lead citrate stain, a x 5,000; b x 23,500; c x 11,750
M. Ito and Y. Sato : Ultrastructure of connective tissue sheath
M. Ito and Y. Sato: Ultrastructure of connective tissue sheath
Fig. 7. Connective tissue sheath in catagen. Ultrastructures of a similar area to 2 in Fig. 1a. The basal lamina (opposite-pointing arrowheads) is relativelythick and corrugated.f, fibroblast; In, inner
coIlagen layer; Mi, middle collagen layer; O, outer root sheath. Uranyl acetate-lead citrate stain, x 11,750
were no lysosomal structures in the vicinity of the intracytoplasmic collagen fibres, and the collagen fibres themselves showed no degeneration (Fig. 6 b, c). Only a few lysosomal structures were occasionally found in their cytoplasm. These ultrastructural findings seemed to indicate an active production of new collagen by the CTS fibroblasts in catagen. In an upper part of the same hair follicle, the basal lamina was 0 . 4 - 1 . 3 gm in thickness and showed slight undulation (Fig. 7). At this level, the collagen layers surrounding the basal lamina showed similar structures (Fig. 7) to those of the corresponding portion in anagen (Figs. 3a, b, 4a).
medulla layer, whereas the hair cortex layer of human hairs is well developed and often occupies most of the volume of the hair shaft . From routine histological observations, we have noted that the human anagen hair bulbs include a spherical, well-developed hair dermal papilla, while the animal hair dermal papilla shows a more oval or slender shape, and that a blood capillary penetrates into the hair dermal papilla of humans, but not into that of the animals. Ultrastructurally, the hair cortical cells in humans and guinea-pigs produce small trichohyaline granules before keratinization, but mouse hair cortical cells do not . The keratinized inner root sheath layer gradually becomes thin and atrophic and disappears in the isthmus portion in the animal anagen hair follicles, whereas in humans it abruptly becomes atrophic and disappears [3, 4]. Therefore, investigators need to study the inter-species similarities and differences in biology and morphology of the CTS also around hair follicles. Histologically, the hyaline membrane around the transient portion of the hair follicle, becomes very thick in catagen and gradually absorbed with the retraction of the hair epithelial tissue until telogen. These findings are common to animals [1, 8] and humans . However, ultrastructurally, the basal lamina, just outside the ORS, is thick in some anagen and all catagen hair follicles in humans, as shown in the present study, whereas in mice and guinea pigs the basal lamina is a thin structure even in catagen [8, 10, 11, 13, 14]. Furthermore, in catagen, the basal lamina shows fine pleating around an atrophic epithelial cord in the animals, while it displays gentle waves or corrugations in humans. This difference seems
Discussion The ultrastructure of the hair cycle has been studied in animals such as mice [8, 10, 11, 13, 14], guinea-pigs  and dogs . These animals have been studied because they readily provide hair follicle specimens and show (especially mice) a hair cycle synchronized among all hairs , offering a skin sample containing uniform hair structures in the stage of the hair cycle desired by the investigators. Although hairs and hair follicles of these animals biologically and structurally closely resemble those of humans, there are some differences already known between the species. The hair cycle is synchronous in mice, whereas it is asynchronous in humans and guinea-pigs. The durations of anagen, catagen and telogen are much shorter in mice than in humans . The hair follicles are narrower and shorter in mice and guinea-pigs than in humans. The hairs of these animals have a relatively wide
M. Ito and Y. Sato: Ultrastructure of connective tissue sheath to be caused by the difference in its thickness as described above. The present study has shown that in humans, the basal lamina gradually becomes thickened during the anagen period (compare Figs. 2, 3 b and 4 a) from 0.05 gm in early anagen hair follicles, to 0.2 gm in some mature anagen follicles, and to 0 . 8 - 2.4 ~tm in late anagen. This concept seems to be supported by the finding that the basal lamina in an anagen hair follicle as shown in Fig. 3 b reveals partly a linear, thin structure and partly duplication or lamination, forming partially thickened portions. Observation of the thickened basal lamina at high magnification (see Fig. 4b) shows that it consists of amorphous, fine granular or filamentous components, similar to those in thin basal laminae, and lamina-like structures. Although the whole CTS is much thicker in human than in animal anagen hair follicles, in both it is composed of three collagen layers. The inner collagen layer, just outside the basal lamina, consists of collagen fibres running parallel to the hair long axis, and contains almost no cells . As shown in the present study, in early anagen such a layer is absent and some fibroblasts are located close to the outer surface of the basal lamina. Therefore, the inner collagen layer may be formed gradually during the development of the hair follicle until mature anagen. The middle collagen layer in the mature anagen is very thick and composed o f collagen fibres running transversely against the hair axis; many fibroblasts are embedded among these fibres . This layer seems to be formed by a gradual increase in the number of such fibres also in the inner part of the CTS in early anagen. The outer collagen layer of the CTS in mature anagen structurally resembles the whole collagen tissue of the CTS in early anagen (compare Fig. 3 a with Fig. 2). In catagen, the inner collagen layer becomes wavy with corrugation of the thick basal lamina, resulting in disarrangement of its collagen fibres as shown in Figs. 5 and 6a. Comparing Figs. 1 a, b, 6a and b with the histological findings of human catagen hair follicles illustrated in reference 8 (Fig. 21) and reference 7 (Fig. 5), the complex of the basal lamina and the inner collagen layer of the CTS appears to correspond to the histological thickened hyaline membrane. Many cells are present in the collagenous tissue around the thickened and corrugated hyaline membrane, but no cells are present within the hyaline membrane itself [7, 8]. The present ultrastructural study has shown that fibroblasts are present only outside the wavy inner collagen layer around the thickened basal lamina. The fibroblasts in catagen CTS, especially around the lower end of the retracting epithelial cord, showed very active collagen synthesis, as shown in Fig. 6 a and c. This seems to be contradictory to the concept that many macrophages gather around the catagen hair follicles and play a role in absorption of the hyaline membrane tissue . Indeed, some of the fibroblasts occasionally contained a few lysosomal structures, but the number of such structures was not great. On the other hand, the basal lamina had lost its fine filamentous structures, had become extremely thick and oedematous, and contained a few collagen fibres (see Fig. 6a and b). These fibres
44! seem to derive from the inner part of the inner collagen layer. F r o m these findings, the mechanism of retraction of the hyaline membrane in catagen may be explained as follows. As the catagen epithelial cord retracts, the thick basal lamina and the inner collagen layer around the lower end of the epithelial cord is digested by enzymes, which are probably produced by the epithelial cells. The consequent degeneration and corrugation leads to a shrinkage of their internal volume. The surrounding fibroblasts produce and add new collagen fibres to the inner collagen layer to compensate for the degeneration. Thus, in catagen, the CTS fibroblasts may play a role in filling with new collagen tissue the spaces left by the retraction of the epithelial cord and hyaline membrane. Alternatively, the newly formed collagen tissue may push the retracting tissues upwards. To obtain further information on this mechanism, histochemical and biochemical studies of hair follicles during the hair cycle are required. The dynamic morphological changes of the CTS shown in the present study suggest that there may be active metabolic changes of the connective tissue around human hair follicles during the hair cycle. References
1. A1-Bagdadi FK, Titkemeyer CW, Lovell EJ (1979) Histology of the hair cycle in male Beagle dogs. Am J Vet Res 40:17341741 2. Hashimoto K, Shibazaki S (1967) Ultrastructural study on differentiation and function of hair. In: Kobori T, Montagna W (eds) Biology and disease of the hair. University of Tokyo Press, Tokyo, pp 23- 57 3. Ito M (1986) The innermost cell layer of the outer root sheath in anagen hair follicle: light and electron microscopic study. Arch Dermatol Res 279:112-119 4. Ito M (1988) Electron microscopic study on cell differentiation in anagen hair follicles in mice. J Invest Dermatol 90 : 65 - 72 5. Ito M, Hashimoto K (1982) Trichohyaline granules in hair cortex. J Invest Dermatol 79 : 392- 398 6. Ito M, Hashimoto K, Organisciak DT (1982) Ultrastructural, histochemical, and biochemical studies of the melanin metabolism in eye and skin of pallid mice. J Invest Dermato178:414424 7. Kligman AM (1959) The human hair cycle. J Invest Dermatol 33:307-316 8. Montagna W, Parakkal PF (1974) The pilary apparatus. In: The structure and function of skin. Academic Press, New York, pp 172-275 9. Muto K, Ono T, Arao T (1981) Ultrastructural observations on the glassy membrane. J Clin Electron Microscopy (Tokyo) 14:5-6 10. Parakkal PF (1969) Ultrastructural changes of the basal lamina during the hair growth cycle. J Cell Biol 40 : 561 - 564 11. Parakkal PF (1969) Role ofmacrophages in collagen resorption during hair growth cycle. J Ultrastruct Res 29:210-217 12. Reynolds ES (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Ceil Biol 17:208 --212 13. Rogers GE (1957) Electron microscope observations on the glassy layer of the hair follicle. Exp Cell Res 13:521 - 528 14. Sugiyama S, Takahashi M, Kamimura M (1976) The ultrastructure of the hair follicles in early and late catagen, with special reference to the alteration of the junctional structure between the dermal papilla and epithelial component. J Ultrastruct Res 54:359 - 373