T-420 SEN-I GAKKAISHI (XO (46)

(Received March 31, 1982)

STUDY ON THE LOCATION OF GRAFTED POLYMER IN

CORTEX, CUTICLE, AND CELL MEMBRANE COMPLEX:

BONDING FORCES BETWEEN FIBER COMPONENTS AND AGGREGATION STATE OF THEIR STRUCTURAL CONSTITUENTS

By Kozo Arai

(Department of Chemistry, Faculty of Technology, Gunma University, Kiryu, Gunma 376, Japan)

Abstract

Wool fibers were grafted with methyl methacrylate by using a lithium bromide and potassium

persulfate system. The location of grafted polymer in wool fiber was determined by transmission electron microscopy. It was found that the amount of polymer deposition was decreased in the

order: endocuticle ?? cytoplasmic remnant ?? intermacrofibrillar material intercellular membrane in

cortex>orthocortex>paracortex>exocuticle>a-layer. This order was inverse to that of sulfur

content. The rigidity of cellular components was discussed in terms of the amount of polymer

deposition. In this context, the rigidity of the endocuticle is considered to be almost the same as

that of the 8-layer in the cell membrane complex between cuticle cells, and that of cytoplasmic

components such as cytoplasmic nuclear remnant and intermacrofibrillar materials. On the basis of

these finding, mechanical properties of wool in terms of the fiber components are briefly discussed.

INTRODUCTION

Wool fibers contain two types of cells, viz.,

cuticle and cortical cells. The cuticle cells consist

of external epicuticle, exocuticle, and endocuticle.

The cortical cells are divided into two different

types of cells termed as orthocortical and para

cortical cells which occupy about 90% of the wool

fibers. They are separated from one another by a

cell membrane complex with three layer structure.

The cortex consists of d-helical microfibril em

bedded in a matrix. Wool fiber is thus a composite

material with a variety of function on mechanical,

chemical, and physical properties.

In order to understand the relation between the

structure and property of wool fiber, it is very

important to elucidate the aggregation state of

various wool fiber components, i.e., various types

of cells in cortex, cuticle and cell membrane com

plex, and the bonding forces between the fiber components. However, no detailed study has so far been reported, especially on the bonding forces

between the fiber components. During the course of studies on graft copolymerization onto wool,

we developed a new staining method to differentiate the grafted poly (methyl methacrylate) from the wool fiber components in a thin cross

section1,2 The observation of longitudinal or transverse cross-sections of grafted wool fibers by high-resolution electron micrography may give valuable informations about the aggregation state

of the cells as well as the bonding forces between the cells. The aim of this report is to demonstrate the dimensional changes of the wool fiber com

ponents by grafting at ultrastructural level.

EXPERIMENTAL

Materials.

Purification of Merino and Lincoln wool fibers was carried out by a Soxhlet extraction with

acetone for 24 h, washing with petroleum ether for 4 h, successive washing with cold water after

washing with ethanol for 20 h at room tempera

*1 Grafting onto Wool. XI. Part X of this series: Quantitative Analysis of the High-Angle X-Ray Diffraction Intensity from Grafted and Ungrafted Wool Keratin appeared in Int. J. Biol. Macromol., 2, 361-367 (1980).

(47) Vol. 38, No.10 (1982) T-421

ture, and then air-drying. Reduced Merino wool fibers were prepared by the treatment with 0.227

N sodium thioglycolate (TGA) solution (pH 5.4) at 30°C for 24 h. The thiol and disulfide contents

determined by a polarographic method3 were 526.amoles and 188.umoles per gram wool, respectively.

Graft copolymerization.The purified wool and TGA-reduced wool fibers

were grafted with methyl methacrylate (MMA) by varying the time of reaction, using the LiBr-K2 S2 08 initiating system (1 g wool, 27.5 g LiBr, 0.2 g K2S2O8, 22.5 g butyl carbitol, 44.8 g H2O and 5 g MMA) at 30°C, unless otherwise specified.

The grafted fibers were washed with water only and then air-dried, since no homopolymerization occurs under the reaction conditions employed here". The extent of grafting was defined as a

percentage of weight increase to the original weight of the wool fibers. The extent of grafting of the

graft copolymers which had been prepared from Merino wool by the reaction for 45 min and 4 h was 54.0% and 114%, respectively. The reduced

Merino wool was treated for 2 h and the extent of

grafting was 93.8%. A copolymer from Lincoln wool was also prepared by grafting of MMA at 40°C for 4 h.

A 250% ethyl acrylate (EA) grafted sample was

prepared from Merino wool by using NH4Br-

(NH4)2S2O8 system (1 g wool, 10 g NH4Br, 40g H2 0, 1 g (NH4)2 S2 08, 37.0 g butyl carbitol and 12 g EA) at 30°C for 24 h.

Electron microscopy.The grafted fibers obtained were treated with a

phosphate buffer solution (pH 7.2) containing 2-3% potassium permanganate for 3 h at room temperature, and then thoroughly washed with

same buffer. These samples were immersed in an aqueous solution containing 10% ethanol by volume, and the solution was substituted succes

sively by a solution containing 20%, 30%, 40%,

50%, 60%, 70%, 80%, and 90% ethanol, and then finally by a pure ethanol. Dehydrated samples thus obtained were embedded in Epon 812.

Ultra-thin sections of grafted samples were

prepared by use of a JUM-7 ultramicrotome (Japan Electron Optics Lab.) equipped with a glass knife.

The ultra-thin sections were stained with a satu-rated solution of uranyl acetate for 10 min at

room temperature and washed with water. In the case of the grafted Lincoln wool fibers, the thin

sections were post-stained with lead citrate after the treatment with uranyl acetate. They were then viewed in a JEM-100B electron microscope (Japan Electron Optics Lab.) at 80 kV accelerating voltage .

RESULTS AND DISCUSSION

As shown in Fig. 1, a marked difference in

texture among the two cortical and the cuticle cells can be clearly observed. Fine-granular ortho-cortical macrofibrils and coarser paracortical cells are distinguishable. Exocuticle, endocuticle and

cell membrane in the cuticle becomes also dis

tinguishable. Cytoplasmic nuclear remnant regions in the paracortex are densely stained.

In a graft copolymerization system employed in the present work, i.e., no diffusion-controlled system of MMA, the deposition of grafted polymer

occurs preferentially in the orthocortex regions rather than in the para-type cortex regions' 2 This may be due to the difference in the content of cross-links between the ortho- and paracortex.

Fig. 2 shows an electron micrograph of a cross-section of the 114%-MMA grafted Merino wool.

Fig. 1. Electron micrograph of a thin cross-section of a fine Merino wool fiber, showing th c uticle, macrofibril (M) in the orthocortex

(O) and densely stained nuclear remnant (nr) regions in the paracortex (P). Exocuticle (Ex), endocuticle (En) and cell

membrane complex (cm) are clearly differentiated. The layer-like contrast is due to

variation in thickness of the section.

T-422 SEN-I GAKKAISHI ()Z) (48)

Fig. 2. Electron micrograph of a thin cross-section of the 114% MMA-grafted fiber (Merino wool), showing the appearance of the boundary region of the orthocortex (0)

and paracortex (P). It can be seen that the

polymer deposition occurs homogeneously within a single cortical cell, but the degree

of deposition differs from cell to cell.

A marked difference in the degree of grafting

between the two cortex regions is observed. A boundary region between the ortho- and para-

cortical cells can be also seen. On the paracortical cells, the electron density of the cell differs from

cell to cell. It is apparent that the grafted polymer was deposited in the lightly stained cells more than in the densely stained ones, since only wool materials may be associated with the heavy metals such as uranyl and permanganate ions. Diffusion

of polymers into cells mirrors the overall inter-action forces in keratin aggregates which contain considerable amounts of chemical or physical cross-links. The difference in the degree of polymer

deposition between cells can be seen in the para-cortical cells, but not in the orthocortical cells.

This suggests that there is a considerable difference in the rigidity of the cross-linked texture of

individual cortical cell in the paracortex.In grafted fibers, the cytoplasmic remnant and

intercellular regions which had been stained dense-

ly in the native state, were stained more lightly than the keratinous region by grafting. The

presence of such a large amount of deposited polymer in these regions indicates that there is a large enough space for deposition of grafted poly-

mer without bringing about any distortion in

geometrical sense on the cortical cells. Previously, present author reported that, by grafting of ethyl acrylate up to the extent of grafting of 250%, the

size of the whorled macrofibrils in the ortho-

cortex was increased about 3 times in lateral

dimension, but no perceptible change was observed

in longitudinal dimension of wools,6

Fig. 3. Electron micrograph of a thin cross-section of the 54.0% MMA-grafted fiber (Merino

wool), showing that a large amount of

grafted polymers are deposited in the endocuticle (En), cytoplasmic nuclear remnant

(nr), and cell membrane complex (cm) between the adjacent paracortical cells, and between the cuticle and cortex. The a-layer and the exocuticle (Ex) are stained densely, because of small amounts of polymer deposition.

The cross-sectional view of the paracortical cells

near the cuticular region is shown in Fig. 3. Assum

ing that the variation of the electron density

reflects the amount of the deposited polymer,

the rigidity of histologically different components

in wool fiber may be estimated by visible observa

tion of Fig. 3. It is obvious that the amount of

the polymer deposition decreases in the order:

endocuticle ?? cytoplasmic remnant intercellular

membrane in cortex > cortex> exocuticle> a-layer.

It should be noted that the order is inverse to that

of sulfur content7-10. It is also interesting to

note that the rigidity of endocuticle is almost the

same as that of cytoplasmic components such as

cytoplasmic nuclear remnant. The exocuticle

contains cystine residue higher than the endo

cuticle which is more susceptible to attack by

enzymes10,11 External layer of the exocuticle,

termed a-layer has much high sulfur content7-9

It is not certain, however, whether the polymer

can be located in this region, because of small

amounts of polymer deposition. A previous elec-

(49) Vol. 38, No. 10 (1982) T-423

tron microscopy study on the 250% EA-grafted

wool showed that the polymer deposited within

the a-layer2.

The cell membrane complex adjoining the endo

cuticle to the cortex involves in part a densely

stained layer which is considered to be a high

sulfur material. Such a layer is not present in the

cell membrane complex between the endocuticle

in outer cuticular cell and the exocuticle in inner

cuticular cell. The ordered structure of the cell

membrane complex between cortical cells tends

to be easily deformed by the grafting. This suggests

that the components consist of low-sulfur proteins,

since the grafting occurs selectively in aggregates

with lower content of cystine residues. Cell mem

brane complex in a keratinized fiber is a plasma

membrane, which has been considered to be

concerned mainly with stabilizing cell-cell contacts

during keratinization steps'2 It can be also

observed that the polymer penetrates somewhat

randomly into the adjoining cortical cells and

expands the boundary regions between the cells.

It is inferred, therefore, that the plasma membrane

adheres the continuous cortical cells, but has less

resistance than the cortex to the external forces

such as a longitudinal extension. On the other

hand, the plasma membrane located between

endocuticle and exocuticle tended to crack along

the exocuticular cell boundary owing to the shear

stress during the thin sectioning (see Fig. 3). This

suggests that the adhesion between the two com

ponents by this plasma membrane is very weak.

Fig. 4 shows a high magnification view of the

cross-section of a macrofibril expanded by the

deposition of polymer. The width of the macro

fibril, which is ca. 0.3ƒÊ in the native state,

increased by polymer deposition. The maximum

width by the grafting was ca. 0.6, 0.8, and 0.99

for 54.0% MMA, 114% MMA, and 250% EA

samples, respectively. Concentric layer lines can

be still observed which is characteristic of the

macrofibril in the orthocortex of the native wool.

The grafted polymer can be differentiated from

the keratin, which is an electron-dense material,

in whorl pattern. The spacing of the layer structure

was around 25 to 30 nm, which is 3 or 4 times

larger than that of native wool13 It should be

emphasized that the increase ratio in the spacing

of the layer structure by grafting is almost equal

to that of the macrofibril. This indicates that

homogeneous polymer deposition occurred not

only in the aggregates of histologically different

components, but also in fine structural compo

nents such as microfibrils in the concentric layer

structure of macrofibril.

Fig. 4. A high-resolution electron micrograph of a thin cross-section of the 114% MMA-

grafted fiber (Merino wool), showing the appearance of the concentric layer structure of an orthocortical macrofibril (Mac).

Polymer can be clearly differentiated from densely stained wool material.

Fig. 5 shows a high-resolution electron micro-

graph of the cross-section of the paracortex region of the 114% MMA-grafted fiber. Regular striation

of lamella structure of the paracortex can be seen

together with the delineation of the grafted poly-

mer. The spacing between the densely stained

keratinous layer or lightly stained polymer layers

Fig. 5. A high-resolution electron micrograph of a thin cross-section of the 114% MMA-

grafted fiber (Merino wool), showing the lamella structure of the paracortex (P).

The layer structures are delineated by the

grafted polymers.

T-424 SEN-I GAKKAISHI ( &Z (50)

was ca. 20-23 rim, which was considerably less than that observed for the orthocortical macrofibril. This suggests that the content of disulfide cross-links in the paracortex is higher than that in the orthocortex. Although the layer structure of macrofibril was expanded in an isotropic manner by the grafting, it is not certain whether the difference in the degree of swelling exists between the peipendicular and parallel directions to the layer lines.

Mesocortical cells which are intermediate in morphology between orthocortical and paracortical cells were found on the cross-section of the 114% MMA-grafted fiber. Fig. 6 shows the meso-type cell region in the paracortex. The regular structure of the "micro fibril + matrix" units can be observed. Figs. 7 and 8 show the 'cross-sections in the

paracortex region of partially reduced Merino wool fiber grafted with MMA. In contrast to native Merino wool fiber, the layer-like structure was not observed (see Fig. 5). The grafted polymer was

finely dispersed around the small keratin aggregates with diameter ca. 2 nm. There is an X-ray evidence that the intensity at the 9.8 A equatorial reflection from the reduced and grafted fibers decreased considerably, but the intensity at the 5.1 A meridional reflection little changed. This means that a dissociation of the d-helical components constituting microfibril occurs in part, but the initial molecular conformation of the keratin is almost retained without being affected by the polymer depositions .

Fig. 6. A high-resolution electron micrograph of a thin cross-section of the 114% MMA-

grafted fiber (Merino wool), showing the meso-type cell in the paracortex region (P). Regular arrangements of wool components can not be observed.

Fig. 7. Electron micrograph of a thin crosssection of the 93.8% MMA-grafted fiber (Merino wool) after treatment with 0.277 N thio

glycolate, showing the appearance of paracortex region. The lamella structure disappears owing to the homogeneously dispersed polymer particles.

Fig. 8. A high-resolution electron micrograph simi

lar to that in Fig. 7. The grafted polymer

is located around the small keratin aggre

gates with diameter of ca. 2 nm.

Fig. 9 shows a longitudinal section of the

Lincoln wool fiber grafted with 73.0% MMA. It

can be seen that the polymer deposition occurs in

the aggregates of the nuclear remnant and the cell

membrane complex. At the histological level it is

unlikely that the grafted polymer brings any

additional non-uniformity along the direction of

fiber axis. It is further noted that the polymer

deposition in nuclear remnant regions results in

no distortion on the geometric form.

Fig. 10 shows a high-magnification view of the

longitudinal section of cuticle cells. The cell

membrane complex in the flat overlapping cuticle

cells consists of three layers, i.e., the ƒÀ-layer

(51) Vol.38, No. 10 (1982) T -425

Fig. 9. Electron micrograph of a thin longitudinal section of the 73.0% MMA-grafted fiber

(Lincoln wool), showing that the polymer deposition occurs parallel to the direction of the fiber axis in the paracortex (P) , and cuticle (cu), nuclear remnant (nr), and cell membrane complex (cm).

Fig. 10. A high magnification view of the cuticle similar to that in Fig. 9. The deposition of polymer occurs in the 6- and S-layers of the cell membrane complex between endocuticle (En) and exocuticle (Ex), and between endocuticle and paracortex (P).

adjoining endocuticle, the middle layer termed as S-layer, and the B-layer adjacent to the surface of the inner exocuticle. It can be seen that grafted

polymer is deposited in the latter B-layer rather more than the former one. This seems to be due to the difference in the content of cross-links, probably that of disulfide cross-links between the two

fl-layers. The endocuticle adheres to the S-layer through the disulfide bonds, but no such disulfide bond exists between the S-layer and the exo

Fig. 11. A high-resolution electron micrograph of a thin longitudinal section of the 114%

MMA-grafted fiber (Merino wool) , showing the intermacrofibrillar region (im)

rich in the deposited polymer .

cuticle" It has been reported by Nakamura et al. 14 that the S-layers between the cuticle cells are not identical to those between the cortical

cells. They have also shown that the amino acid composition of the S-layer between the cuticle cells is similar to that of the endocuticle and that the B-layer adjoining the exocuticle tends to be disintegrated by twisting the fiber's Here, it is noted that the amount of polymer deposition of

the S-layer between the cuticle cells is almost the same as that of the endocuticle. As can be seen in Fig. 10, a large amount of grafted polymers are

deposited in the cell membrane complex between the endocuticle and cortex, especially for the regions of cortical cell side. On the other hand , the ,9- and S-layers in the cell membrane complex between the cortical cells can not be differentiated

from each other, as already indicated in Fig. 3. From the ultrastructural appearance of these layers

in the grafted fiber, however, it may be inferred that the intramacrofibrillar bonding force is con

siderably stronger than the intercellular or intermacrofibrillar one (see Fig. 11). A similar result has been reported by Orwin and Thomsonts

ACKNOWLEDGEMENT

This work was partially supported by Scientific

Research Funds provided by the Education Ministry of Japan, No. 440005, 1981.

T-426 SEN-I GAKKAISHI(報 文) (52)

REFERENCES

1) K. Arai and M. Negishi, J. Polym. Sci, A-1, 9, 1865 (1971).

2) K. Arai, in "Block and Graft Copolymerization" Vol. 1 (R. J. Ceresa, Ed.), Wiley-Interscience, London, 1973, Chpt. 8.

3) S. J. Leach, Austral. J. Chem., 13, 547

(1960).4) K. Arai, S. Komine and M. Negishi, J. Polym.

Sci. A-1, 8, 917 (1970).5) K. Arai, M. Negishi, S. Komine and K. Takeda,

Appl. • Polym. Symp. No. 18, Vol. I, 545

(1971).6) K. Arai, M. Negishi, T. Suda and K. Doi,

J. Polym. Sci. A-1, 9, 1879 (1971).7) J. Sikorski and W. S. Simpson, Nature, 182,

1235 (1958).8) G. E. Rogers, Ann. N. Y. Acad. Sci., 83, 408

(1959).

9) J. A. Swift, J. Roy. Microscop. Soc., 88,449

(1968).10) J. H. Brudbury and K. F. Ley, Aust. J. Biol.

Sci., 25, 1232 (1972).11) J. A. Swift and A. W. Holmes, Text. Res. J.,

35, 1014 (1965).12) D. F. G. Orwin and R. W. Thomson, J. Ultra

struct. Res., 45, 41 (1973).13) R. D. B. Fraser, T. P. MacRae, G. R. Millward,

D. A. D. Parry, E. Suzuki and P. A. Tulloch, Appi. Polym. Symp., No. 18, Vol. I, 65

(1971).14) Y. Nakamura, T. Kanoh, T. Kondo and H.

Inagaki, Proc. Int. Wool Text. Res. Conf., Aachen, II, 23 (1975).

15) Y. Nakamura, Sen-i Gakkaishi, 35, P-248

(1979).16) D. F. G. Orwin and R. W. Thomson, Proc. Int.

Wool Text. Res. Conf., Aachen, II, 173

(1975).

皮質,表 皮細胞,お よび細胞膜錯合体中におけるグラフト

ポ リマーの沈着位置に関する研究:繊 維成分間の結合力と

それらを構成する構成成分の凝集状態

群馬大学工学部 新 井 幸 三

羊 毛繊維 への メタ ク リル酸 メ チルの グ ラフ ト共 重合 は

臭化 リチ ウム ー過硫 酸 カ リ系 を用 いて行 な うた。 羊毛繊

維 中の グラ フ トポ リマ ーの沈着 位置 は透過電 子顕 微鏡法

に よ って決定 され た。 ポ リマー沈着 量は次 の順序 で減少

す るに とが 見い出 され た:エ ン ドキ ュチ クル〓 細胞 核残

遺物 とマ ク ロフ ィブ リル間物 質〉皮 質細胞 中の細 胞膜 〉

オル ソ コル テ ックス〉パ ラコル テ ック ス〉エキ ソキ ュチ

クル〉α一層 の順で ある。 この順序 は硫黄 含量の 順序 と

逆である。ポリマー沈着量に基づいて,細 胞成分のかた

さが議論 された。これ に関連 して,エ ン ドキュチクルの

かたさは表皮細胞間の細胞膜錯合体中の δ一層や細胞核

残遺物あるいはマクロフィブ リル間物質のような細胞質

成分のかたさとほとんど同 じであることが見い出された。

にれらの事実に基づいて,繊 維を構成する成分が羊毛の

力学的性質 にどう関わっているかについても若干考察し

た。