/. Embryol. exp. Morph. Vol. 32, 2, pp. 297-323, 1974 297 Printed in Great Britain

Dedifferentiation, redifferentiation and bundle formation of smooth muscle cells in

tissue culture : the influence of cell number and nerve fibres

ByJULTE H. CHAMLEY,1 G O R D O N R.CAMPBELL 1

AND G E O F F R E Y BURNSTOCK 1

From the Department of Zoology, University of Melbourne

S U M M A R Y

Smooth muscle from newborn guinea-pig vas deferens was enzymically dispersed into single cells or small clumps and grown in culture in the presence or absence of sympathetic ganglion expiants.

Most single smooth muscle cells gradually lost their typical ultrastructural features and contractile properties during the first few days in culture. At 7 days of culture these de­differentiated smooth muscle cells underwent extensive proliferation. If sufficient cells were present in the culture inoculate, a continuous monolayer formed at about 9 days of culture and redifferentiation of smooth muscle began. At 11-12 days of culture the cells reaggregated into clumps, began to contract spontaneously, and formed into well-organized muscle bundles in two layers at right angles, resembling the muscle layer organization of the in vivo vas deferens. In cultures where a continuous monolayer was not formed at 9 days, isolated cells did not redifferentiate. The process of dedifferentiation and proliferation was delayed in those smooth muscle cells which had sympathetic nerve fibres in close association.

Clumps of vas deferens tissue which were not fully dispersed by the enzyme treatment did not dedifferentiate with time in culture but muscle bundles were disrupted and asyn­chronous contractions resulted. After 8-12 days of culture the muscle bundles reformed and foci of synchronous contractions developed. Nerve fibres appeared to accelerate bundle and nexus formation in this situation, with synchronous contractions resuming at 3-5 days.

The relation of these findings to the process of wound healing in smooth muscle tissues in vivo is discussed.

INTRODUCTION

Little is known about the factors involved in the regeneration of damaged smooth muscle tissues. Some reports suggest that regeneration occurs through proliferation of dedifferentiated smooth muscle cells (Berry, 1920; Murray, Schrodt & Berg, 1966; Poole, Cromwell & Benditt, 1971), while others consider that migration of fully differentiated muscle cells is the major factor (Ross, Thompson, Bynum & Thompson, 1966; Ross et al. 1969). Other reports

1 Authors' address: Department of Zoology, University of Melbourne, Parkville 3052, Victoria, Australia.

298 J. H. C H A M L E Y A N D O T H E R S

suggest that smooth muscle cells can be derived from connective tissue cells (Bird & Willis, 1965) or from 'little-differentiated spare elements' (Nad, 1966).

Recent studies, where fragments of smooth muscle tissues have been trans­planted into the anterior eye chamber of the adult rat and guinea-pig, have produced further evidence for dedifferentiation of smooth muscle cells before proliferation and ^differentiation (Malmfors, Furness, Campbell & Burnstock, 1970; Burnstock, Gannon, Malmfors & Rogers, 1971; Campbell, Uehara, Malmfors & Burnstock, 1971a; Rogers, 1972). Similar changes have been shown in studies of expiants of pig aorta in tissue culture (Jarmolych et al. 1968; Fritz, Jarmolych & Daoud, 1970).

Studies of the re-innervation process of the anterior eye chamber transplants suggest that the presence of nerve fibres may play some role in muscle bundle formation (Malmfors et al. 1970; Campbell et al. 1971 a). However, Rogers (1972) suggests that contact interactions between smooth muscle cells is the major factor in this process.

The present report describes the changes which occur when both single cells and clumps of smooth muscle tissue from the guinea-pig vas deferens are grown in culture, and discusses the results in relation to the mechanism of smooth muscle regeneration in wound healing. Tissue culture of single cells has a great advantage in studying this phenomenon, as phase-contrast obser­vations of the same cells can be made through successive stages with time. Ultrastructural studies of these stages provide further information. The direct influence of nerve fibres and cellular contacts on smooth muscle regeneration can also be examined.

MATERIALS AND METHODS

Vasa deferentia from newborn guinea-pigs were stripped of serosa, cut into 1 mm3 fragments and dispersed by the action of 0-5 % collagenase and 0-125 % trypsin. The cells were gently centrifuged, resuspended in nutrient medium and adjusted to 5 x 105 cells/ml ('densely seeded') or 103 cells/ml ('sparsely seeded'). Two millilitres of the cell suspensions were then injected into modified Rose chambers, making a total of 106 cells in densely seeded cultures and 2 x 103 cells in sparsely seeded cultures. Some of the chambers already contained expiants of newborn guinea-pig sympathetic ganglia under strips of dialysing cellophane, | in. wide, on carbon- and collagen-coated glass coverslips. The ganglia had been in culture for 7 days, by which time the nerve fibres had grown to the edges of the cellophane, where they could interact with the cells from the vas deferens. After 24 h the entire culture area was covered with a strip of dialysing cellophane 1 in. wide. Details of the procedures are given in a previous report (Mark, Chamley & Burnstock, 1973).

In cultures of both vas deferens alone and vas deferens grown with sympa­thetic ganglia, the nutrient medium consisted of medium 199 to which 20%

Smooth muscle in tissue culture 299 foetal calf serum, 5 mg/ml glucose, 0-05 i.u./ml insulin and 100 i.u./ml penicillin were added. In addition, nerve growth factor (NGF) (Burroughs-Wellcome, England) was added to the medium of half of the cultures with sympathetic ganglia to a final concentration of 1 i.u./ml. The medium was changed twice weekly and the cultures incubated at 37 °C for up to 3^ months. They were examined and photographed with phase-contrast microscopy (Zeiss Standard RA microscope, Zeiss Ikon camera) and time-lapse microcinematography (Wild microscope equipped with a Zeiss heating stage, Bolex camera and Packard-Wild time-lapse equipment). Film records were analysed with the aid of a Photo-optical Data Analyzer (Model Z24-A, L.W. Photo Inc., Van Nuys, California). A total of 200 cultures were studied (50 densely seeded cultures with and without ganglion expiants, 50 sparsely seeded cultures with and without ganglion expiants).

For electron microscopy, cultures were fixed in phosphate-buffered 5% glutaraldehyde (pH 7-4) for 30 min, washed in buffer for 1 h, then postfixed in buffered 1 % osmium tetroxide and 2 % uranyl acetate for 1 h each. After rapid dehydration through a graded series of alcohols, the cultures were embedded in Araldite. Following polymerization, cultures were split off their coverslips and areas containing cells previously selected by phase-contrast microscopy mounted and prepared for sectioning (La Vail, 1968). Thin sections were usually cut parallel to the plane of the coverslip on Huxley-Cambridge or LKB ultramicrotomes, stained with lead citrate and examined in an Hitachi HU IIB electron microscope. The newborn guinea-pig vas deferens was fixed in situ by dropping buffered 5 % glutaraldehyde on it for 5 min. The tissue was then removed and fixed for another hour. The same fixation procedure was then carried out as for the cultures.

RESULTS

Smooth muscle cells from the in situ newborn guinea-pig vas deferens

Muscle cells from both the longitudinal and circular layers of the vas deferens lay in tightly packed parallel bundles and were separated from adjacent muscle cells by a gap of between 30 nm and 1 fim (Fig. 1). Thick (12-14 nm in diameter) and thin (4-8 nm in diameter) myofilaments with associated dark bodies occupied most of the cytoplasm. Organelles such as mitochondria, Golgi apparatus and ribosomes were usually confined to the nuclear region. Plasma-lemmal vesicles were associated with the plasma membrane and a basal lamina was present.

Single smooth muscle cells in culture

After attachment and flattening (1-2 days), approximately 70% of the cells in culture had a distinct smooth muscle morphology under phase-contrast

300 J. H. CHAMLEY AND OTHERS

Fig. 1. Smooth muscle cells from the newborn guinea-pig vas deferens. The cells are tightly packed, the gap between them seldom exceeding 50 nm. Myofilaments fill most of the cell, and few other organelles are present, n, Nerves.

microscopy (see Chamley, Campbell & Burnstock, 1973) being ribbon-shaped (100-150 x 10-15 /im) with phase-dense cytoplasm, few visible organelles and an oval nucleus containing several small pale nucleoli (Figs. 2A, 5 A). They contracted spontaneously at rates of 2-7 times per min, and a small number (less than 5 %) underwent mitosis while in this differentiated state (see Chamley & Campbell, 1974). Fibroblasts comprised the remaining 30 % of cells in the cultures. They were flat, of variable size and shape (50-200 x 20-50 jum), and contained a large clear nucleus with 1-5 dark nucleoli (Fig. 9 A). Their cytoplasm was granular in the region of the nucleus and clear at the periphery. They did not contract, but migrated actively and underwent frequent mitoses.

Smooth muscle in tissue culture 301

Ultrastructurally, cells in culture were classified as smooth muscle using the same criteria as Ross (1971); that is, the presence of a basal lamina, plasma­lemmal vesicles and bundles of myofilaments with associated dark bodies. The criteria used to define their state of differentiation were the type and density of myofilaments ; it is well documented that in smooth muscle, thin filaments are formed first followed by thick filaments, with the number of both increasing during differentiation (Yamauchi & Burnstock, 1969; Bennett & Cobb, 1969; Imaizumi & Kuwabura, 1971). The characteristics of smooth muscle cells from the newborn guinea-pig vas deferens will be taken as those of fully differentiated smooth muscle.

After 2 days in culture the majority of smooth muscle cells had an ultra-structural appearance similar to the cells of the newborn guinea-pig vas deferens. Both thick and thin filaments and dark bodies were present, but few dense areas along the plasma membrane could be distinguished. There was a small increase in the number of organelles and these extended throughout the cyto­plasm. Little basal lamina surrounded the muscle cells, probably due to the protein-digesting action of trypsin. Most cells had finger-like projections, 40-100 nm in diameter, protruding from their surface for distances of up to 3-4 /.im.

Fibroblasts could also be distinguished with electron microscopy. Most of their cytoplasm was filled with free ribosomes. Subjacent to the plasma mem­brane were microfilaments 6-8 nm diameter, occasionally with associated focal regions of dense staining. Another filament type 10 nm in diameter (see Uehara, Campbell & Burnstock, 1971) was found in loose fascicles in the endoplasmic region of the cell. Microtubules were also present. No basal lamina surrounded these cells, and plasmalemmal vesicles were seldom encountered.

After 5 days in culture the smooth muscle cells underwent little gross mor­phological change (Fig. 2B). However, ultrastructurally they showed a further increase in the number of organelles, particularly granular endoplasmic reticulum and free ribosomes (Fig. 3). Filament bundles tended to be found towards the periphery of the cell, and usually contained dark bodies. Some cells contained both thick and thin filaments, while others contained only thin filaments. Plasmalemmal vesicles were numerous and a basal lamina surrounded each cell, but again, very few dense areas were seen along the plasma membrane. '10 nm filaments' were often seen among the organelles. A few cells contained large clusters of these filaments orientated in a random, swirling configuration as previously observed in cultured smooth muscle cells of the chicken gizzard (Campbell, Uehara, Mark & Burnstock, 1971 b; Campbell, Chamley and Burnstock, 1974).

By 7 days of culture, contractions in all cells had ceased (Table 1). At this stage more than 95 % of the smooth muscle cells began to widen, resemble fibroblasts as seen with phase-contrast microscopy (Fig. 2C) and undergo intense proliferation, such that the number of cells increased by a factor of

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Smooth muscle in tissue culture 303 10-20 by day 9 (see Figs. 5 A, B). A small number of cells (less than 5%) still maintained their smooth muscle morphology although they ceased spontaneous contraction. The dedifferentiated cells contained filament bundles usually confined to the periphery of the cell. A large number of organelles were present. Ribosomes were particularly abundant, both free and in the form of rosettes (Fig. 4). Mitochondria were usually long and narrow and often branched. Few thick filaments were found in the bundles, which ran throughout the cells. The size of the thin filament bundles varied from cell to cell and from area to area within the same cell, as did the number of dark bodies usually found within them. Areas of filament bundles without dark bodies would have made it difficult to distinguish these cells from fibroblasts (Movat & Fernando, 1962; McNutt, Culp & Black, 1973), but the presence of basal lamina and plasma-lemmal vesicles allowed this distinction to be made. Again, a number of cells contained large clusters of tightly intermingled ' 10 nm filaments'.

In densely seeded cultures sufficient mitoses had occurred for confluence (i.e. a continuous monolayer) to result after 9 days of culture (Fig. 5B). However, in sparsely seeded cultures the cells were generally still isolated at this time. Consequently, after 9 days in culture the muscle cells from densely and sparsely seeded cultures tended to behave differently and will therefore be described separately.

Densely seeded cultures

Muscle cells after 9 days in culture were both longer and narrower than those in 7-day cultures (Fig. 5B). None was observed to contract spontaneously (Table 1). Most of the cells lay parallel to each other, separated by a gap of 30-50 nm, and contained many organelles, particularly dilated granular endo­plasmic reticulum. Lipid droplets were also present. At this time they had begun to redifferentiate, reaching a stage equivalent to cells between 5 and 7 days in culture. Where cells were separated from each other by a gap of 2-3 fim, finger-like processes, similar to those described previously, extended from one cell to another. Few thick filaments were noted in the filament bundles, and no dense areas were seen along the plasma membrane. Plasma-lemmal vesicles were, however, quite plentiful and a basal lamina was present. A few cells containing small clusters of ' 10 nm filaments' were seen.

FIGURE 2

The same ribbon-shaped smooth muscle cell from the newborn guinea-pig vas deferens, shown with increasing time in culture.

(A) After 3 days in vitro. (B) After 5 days in vitro. (C) After 7 days in vitro. (D) After 9 days in vitro. Note that the muscle cell has dedifferentiated and

undergone mitosis (arrows). 20 E M B 32

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Fig. 3. Smooth muscle cell from the newborn guinea-pig vas deferens after 5 days in vitro. The myofilament bundle contains both thick and thin filaments; dark bodies (db) are amongst these myofilaments. Plasmalemmal vesicles (p) are present along the plasma membrane. There is an increase in free ribosomes and granular endoplasmic reticulum. Note the basal lamina and the finger-like projection (arrow) protruding from the surface of the cell.

T

Smooth muscle in tissue culture 305

Table 1.

Rates of spontaneous contraction of smooth muscle cells with time in culture. Eight separate experiments for each of the six muscle and nerve-muscle arrangements were carried out; within each arrangement the same qualitative results were obtained. As the initial rate of spontaneous contraction varied within each arrangement, representative examples with the same initial rate are shown here, so that direct comparisons can be made.

Contractions/m in

Days in culture ... 2 3 4 5 6 7 9 10 11 12

Single cell 2 2 1-5 2 1-5 0 0 0 0 0 Single cell with nerve 2 2 2 2 1-5 1 0 0 0 0 Partially dispersed clump 6 6 7 6 5 6 6 5 6 6* of tissue

Partially dispersed clump 6 6 7* 7 6 6 7 7 6 7 of tissue with nerve

Monolayer of cells — — — — 0 0 0 0 0 0 Reaggregated clump 7*

* Day when foci of synchronous contractions first appear.

On about day 11 or 12 an unusual phenomenon occurred; cells spontaneously began to draw up into clumps (Fig. 5C). The clumps were loosely packed at first, but by the following day had become very dense (Fig. 5D). This phenomenon did not occur in small, isolated areas, but over the entire culture surface. The clumps that formed had definite structures, being long and narrow (0-5 x 0-1 mm to 3-0 x 0-5 mm) and rod- or ring-shaped, always with two layers of cells at right angles to each other (Fig. 6). As soon as the clumps formed, most or all of the constituent cells resumed a narrow, phase-dense appearance and began to contract spontaneously at a rate of about 7 times per min (Table 1). Contractions were usually only observed in the cells of the long axis of the clump (Fig. 6B), but on two occasions contractions were seen in the cells of the short axis as well (Fig. 6C). Foci of synchronous contraction appeared immediately the reaggregated clumps formed, and corresponded to areas where cells were aligned in parallel. The rate of contraction of both foci and asynchronously contracting cells of the clump was maintained to about day 17, after which time the health of the clumps deteriorated, due to acidity problems in the medium of the excessively thick cultures.

Ultrastructurally, the cells in the reaggregated clumps appeared well dif­ferentiated, containing an abundance of both thick (12-14 nm) and thin (4-8 nm) filaments in bundles. Dark bodies were present among the bundles, and dense areas were often present along the plasma membrane (Fig. 7). Organelles were few in number and consisted mainly of free ribosomes and mitochondria. Plasmalemmal vesicles were plentiful and the majority of cells were surrounded by a basal lamina. No clusters of '10 nm filaments' were seen. Nexuses were occasionally seen between cells (Fig. 7B).

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Fig. 4. Smooth muscle cell from the newborn guinea-pig vas deferens after 7 days in vitro. The myofilament bundle contains only thin filaments; dark bodies (db) are amongst these myofilaments. The granular endoplasmic reticulum (er) is dilated and contains electron-dense flocculent material. The cisternae appear to be connected to the nuclear envelope (arrow), r, Free ribosomes.

Smooth muscle in tissue culture 307 In those areas of the culture where reaggregation did not occur, the muscle

cells were not as differentiated as those in reaggregated clumps, nor did they contract spontaneously.

Sparsely seeded cultures

After 9 days in culture, all but a few cells were markedly dedifferentiated and contained an abundance of free ribosomes and other cellular organelles (Figs. 2D, 8). Filament bundles were present; these usually contained only thin myofilaments confined to the periphery of the cell, and were not as plentiful as those in cells which had been 7 days in culture (Fig. 8). Dark bodies were present in the filament bundles. Again, clusters of ' 10 nm filaments' were noted in a number of cells. A basal lamina was present around the cells and plasmalemmal vesicles were found.

In sparsely seeded cultures (Fig. 9 A) a monolayer did not form for about 3 weeks (Fig. 9B). However, in contrast to densely seeded cultures which began to redifferentiate immediately, the cells did not resume an elongated, narrow, phase-dense appearance (characteristic of differentiated smooth muscle cells in culture) until 1 week later (Fig. 9C). These characteristics were main­tained for several months, during which time strap-like formations developed, often with two layers of cells at right angles to each other (Fig. 9D). At no time after the initial dedifferentiation were the cells seen to contract spon­taneously. Filament bundles, usually containing only thin filaments, were con­fined to the periphery. The cells contained an abundance of mitochondria and large networks of smooth tubules forming tubular and oblate profiles, similar to those described in cultured chicken gizzard smooth muscle cells (Campbell et al 1971 b). Some cells appeared to contain little else but clusters of ' 10 nm filaments' running in random directions.

On 11 occasions, cells in sparsely seeded cultures spontaneously reaggregated into clumps after 6-8 weeks. They contained a slight increase in the number of filament bundles compared with those which had not reaggregated. Many bundles contained only thin filaments, but some contained a few thick filaments. A large amount of smooth endoplasmic reticulum was noted in these cells, and a few clusters of ' lOnm filaments' were also present. Nexuses were occasionally seen. No spontaneous contractions were seen in the cells of the reaggregated clumps.

Clumps of partially dispersed smooth muscle tissue in culture

Clumps of vas deferens tissue which had not fully dispersed in the enzyme treatment were sometimes placed in the cultures. They varied from small (20-30 cells) to quite large (several hundred cells). The outline of cells within the clumps could generally be distinguished, by far the great majority of cells being ribbon-shaped and orientated randomly to each other.

During the first few days in culture the contractions of cells within the

308 J. H. CHAMLEY AND OTHERS

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Smooth muscle in tissue culture 309

clumps were asynchronous, each cell contracting independently. Muscle cells contained both thick and thin filaments, although a predominance of thick filaments existed. The cells were often separated by quite large distances (2-3 (im) although protrusions from some cells made contact with others. Few nexuses were seen. Little basal lamina was present around these cells (Fig. 9B), again probably as the result of trypsinization.

The rate of spontaneous contraction of the muscle cells in the clumps did not diminish with time in culture, and after 8-12 days up to six foci of syn­chronous contraction appeared within each clump (17 observations). These foci each consisted of about a dozen cells aligned parallel to each other. Often the contraction of one focus was seen to pull another, which then contracted. On a few occasions, massive contractions of the whole clump were seen. The muscle cells contained both thick and thin filaments. They were often irregular in shape, but a number of cells were long and strap-like and were close together, forming bundles. Nexuses were occasionally seen. Dense areas along the plasma membrane were usually present as well as a basal lamina. Many cells contained large amounts of glycogen.

Some smooth muscle cells migrated from the clumps during the first 5 or 6 days in culture. Once free of the clumps, they behaved as single cells and underwent the same reactions as described above for single cells in culture. The degree of differentiation of the migrated cells appeared to be inversely proportional to the distance from the clump. This, apparently, was in spite of the close relationship often existing between the cells (Fig. 10).

Influence of sympathetic nerve fibres on smooth muscle differentiation in culture

In sparsely seeded cultures the morphology of 37 out of 58 single smooth muscle cells with nerve fibres in close association was retained to a high degree at days 9-11, when more than 95 % of those cells in the same culture with no nerve fibres in close association had dedifferentiated (Fig. I IA; also compare with Fig. 2D). They contained an abundance of filaments, both thick and thin, and few organelles were present (Fig. 11B). Their contraction rate, how­ever, was about the same as for cells with no nerve fibres in association, and

FIGURE 5

The same field of a densely seeded culture of cells from newborn guinea-pig vas deferens, shown with increasing time in culture.

(A) After 2 days in vitro, m, Muscle cells;/, fibroblasts. (B) After 9 days in vitro. A monolayer consisting mainly of dedifferentiated

ribbon-shaped smooth muscle cells and fibroblasts has formed. (C) After 11 days in vitro. The monolayer has begun to spontaneously draw up

into a clump. At this stage, contractions begin. (D) After 12 days in vitro. The reaggregated clump has become very dense.

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Smooth muscle in tissue culture 311 most ceased contracting by days 7 or 8 (Table 1). Morphologically, they did not dedifferentiate to the same state as muscle cells with no nerve fibres until after about 17 days and did not undergo mitosis until this time.

Similarly, in densely seeded cultures, the close association of sympathetic nerve fibres with single smooth muscle cells appeared to decrease their rate of dedifferentiation, but did not prevent cessation of spontaneous contraction after days 7 or 8.

Clumps of partially dispersed vas deferens tissue with nerve fibres penetrating them contracted asynchronously at about the same rate and strength as clumps in the same culture with no nerves (Table 1). They also spread out in a similar manner, revealing cells in the same highly differentiated state. This state was maintained for many weeks, provided the cells remained within the clump. Foci of synchronous contraction also developed. In large clumps with relatively few nerve fibres per cell, the foci appeared at about the same time as in clumps with no nerves (days 8-12; 15 observations). However, in small clumps with a relatively large density of nerve fibres, foci (about the same number as in clumps with no nerves) were seen much earlier (3-5 days; 19 observations). Massive contractions of the whole clump were seen more frequently in clumps with nerves (nine observations) than in clumps with no nerves (three obser­vations). No difference in the degree of differentiation or in bundle formation could be detected between these clumps and those without nerve. However, an increase of approximately 50% in the number of nexuses was found.

DISCUSSION

For most smooth muscle cells of the newborn guinea-pig vas deferens in culture, dedifferentiation is a necessary prerequisite for proliferation. When a sufficient number of mitoses of the dedifferentiated cells has resulted in a continuous monolayer being formed, the cells cease proliferation and begin to redifferentiate. Monolayers form within a certain period (usually 9-11 days of culture) then spontaneously reaggregate into clumps. The cells of the clump redifferentiate further, begin to contract spontaneously, and become arranged into two layers at right angles, resembling the longitudinal and circular muscle layers of an intact vas deferens.

Although freshly dissociated cell suspensions have been shown on many occasions to reaggregate spontaneously into masses resembling their original

FIGURE 6

(A) A spontaneously reaggregated clump of vas deferens cells from newborn guinea-pig, 12 days in vitro.

(B, C) Higher magnifications at different foci of the field within the rectangle in (A), showing that the reaggregated clump consists of two layers of cells at right angles. Arrows indicate the orientation of the cell layers.

312 J. H. CHAMLEY AND OTHERS

Smooth muscle in tissue culture 313

tissue within the first 24 h of settling (Moscona, 1965), only a few previous reports of spontaneous reaggregation of cells in monolayer culture have been found. Ebelling & Fischer (1922) and Drew (1923) described 'pulling together' of cells into three-dimensional tubular structures when cultures of fibroblasts and epithelial cells were merged. Spontaneous reaggregation into clumps of dissociated dorsal root and sympathetic ganglia (Cohen, Nicol & Richter, 1964) and of spinal cord and brain neurons (Crain & Bornstein, 1972) has also been reported. Haibert, Bruderer & Lin (1971) found that dissociated cardiac muscle cells, when grown on polystyrene coverslips, pull up into compact, spherical masses approximately 2 mm in diameter, closely resembling miniature hearts. The mechanism by which the spontaneous reaggregation occurs is not under­stood. It is, however, widely believed that cells possess type-specific differences in adhesiveness, and that reaggregation occurs through the progressive building up of old cellular alliances after random collisions (Moscona, 1965). Jones (1966) proposed that cell adhesion involved the participation of an actomyosin-like protein located at the cell surface. This view was supported by the reduction of adhesiveness through adsorption onto the cell surface of antibodies directed specifically against myosin-ATPase (Jones, Kemp & Gröschel-Stewart, 1970; Gröschel-Stewart, Jones & Kemp, 1970; Kemp, Jones & Gröschel-Stewart, 1971, 1973). It was also shown that the antigenic character of the myosin differed between different tissues (Jones et al. 1970).

Partially dispersed and spontaneously reaggregated clumps of newborn guinea-pig vas deferens in culture are more differentiated than monolayers of cells, which in turn are more differentiated than isolated cells. That a mass effect is important for differentiation is well known (Weiss & Amprino, 1940; Grobstein & Zwilling, 1953; Grobstein, 1959, 1965). However, as cells from clumps of vas deferens are more differentiated than those of monolayer cultures, a three-dimensional structure as well as a mass effect appears to be important. Indeed, in experimental reaggregation studies of a variety of tissues (reviewed by Grobstein, 1965; Moscona, 1965) it was found that reaggregated cells from freshly dissociated tissues not only maintain their morphology better than monolayer cultures containing the same number of cells, but their hormone (Hilfer, 1962) and enzyme levels (Seeds, 1971), their response to various agents

FIGURE 7

(A) Smooth muscle cells from a reaggregated clump of newborn guinea-pig vas deferens after 12 days in vitro. Note the close relationship between cells. Thick (12-14 nm) (thick arrows) and thin (4-8 nm) (thin arrows) myofilaments are present with associated dark bodies (db). p, Plasmalemmal vesicles; da, dense area along the plasma membrane.

(B) Nexus between two muscle cells from the same reaggregated clump of newborn guinea-pig vas deferens cells shown in (A), p, Plasmalemmal vesicles, (x 149000.)

314 J. H. CHAMLEY AND OTHERS

FIGURE 8

Cells from a sparsely seeded culture of newborn guinea-pig vas deferens after 9 days in vitro. Note the abundance of free ribosomes, granular endoplasmic reticulum, mitochondria and plasmalemma vesicles (p). Myofilament bundles are confined to the periphery and consist of only thin filaments (arrow), db, Dark body; bl, basal lamina.

(Morris & Moscona, 1970; McDonald, Sachs & DeHaan, 1972) and their bioelectric activity (Crain & Bornstein, 1972) more closely resemble the normal.

There appears to be parallel behaviour between cells in densely and sparsely seeded cultures of smooth muscle and cells in small and large wounds in smooth muscle tissues. In small wounds - for example, those due to a small amount

Smooth muscle in tissue culture 315 of damage in anterior eye chamber transplants of vas deferens (Campbell et al. 1971 a) - the damaged region is rapidly filled with dedifferentiated and proliferating smooth muscle cells. The time course for this process and for the subsequent redifferentiation is similar to that in densely seeded cultures in vitro. In sparsely seeded cultures, and in larger wounds such as experimental incisions in blood vessels in situ (Murray et al. 1966) and extensively damaged anterior eye chamber transplants (Campbell et al. 1971a), the time taken for the culture area or the damaged region to be filled with dedifferentiated and proliferating smooth muscle cells is much longer - presumably due, at least in part, to the many more mitoses each cell must undergo so that the space is filled. It has also been suggested that a cell microexudate from each cell stimulates mitosis, and that at low cell densities longer periods are required for a complete carpet of this microexudate to be formed, resulting in delay or inhibition of mitosis (Yaoi & Kanaseki, 1972). These two factors (i.e. the number of mitoses each cell must undergo and the time the cells remain undifferentiated) are the most likely reasons why, in both large wounds and sparsely seeded cultures, the smooth muscle cells never completely regain their differentiation (see Holtzer, Abbott, Lash & Holtzer, 1960).

Other observations in single cell cultures may also be relevant to wound healing of smooth muscle tissues. For example, in small wounds, as in densely seeded cultures, confluence or cell contact may be the stimulus for redifferen­tiation, reaggregation and the formation of muscle layers and bundles. The reaggregation process may, in fact, be involved in the observed drawing together of wound margins during repair of all tissues. This is usually attributed solely to the contraction of granulation tissue which contains 'myofibroblasts cells' (Gabbiani, Ryan & Majno, 1971 ; Majno et al 1971 ; Gabbiani et al. 1972). These cells are morphologically very similar to cultured smooth muscle cells undergoing reaggregation. Consequently, the drawing together of wound margins may also involve a similar process to the reaggregation of smooth muscle cells in tissue culture.

The observation that small numbers of single smooth muscle cells are capable of mitosis while in a differentiated state (Chamley & Campbell, 1974), and other cells (it is not yet known whether they are the same cells) are capable of retaining their differentiation for a greater length of time than the majority of cells, may also be of importance in wound healing of smooth muscle tissues. A small number of differentiated smooth muscle cells have also been seen throughout all stages in anterior eye chamber transplants of vas deferens (Campbell et al. 1971a). If similar cells do exist in wound healing in situ, then they may help to maintain the integrity of the region when most of the smooth muscle cells have dedifferentiated. They may also play some part in directing the processes of redifferentiation or bundle formation.

At the present time, very little is known of the factors which stimulate smooth muscle bundle formation. Malmfors et al. (1970) found in anterior eye

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Smooth muscle in tissue culture 317

chamber experiments that muscle bundle formation in vas deferens transplants occurred at about the same time as the appearance of nerve fibres within the transplant. However, they could not determine whether this was a direct effect or a coincidence. Rogers (1972), using the same system with taenia coli trans­plants, suggested that close, specialized contact between the muscle cells is the major factor in bundle formation. In tissue culture, muscle cells in partially dispersed clumps of vas deferens retain their differentiation for many weeks, but muscle bundles are disrupted within the first day of culture and the cells do not contract in synchrony. Electron microscopy of the clumps reveals that the cellular contacts are generally not as close as in non-cultured vas deferens, and few nexuses are present. However, after 8-12 days, cells in extensive areas of the clumps come closer together and a number of nexuses and muscle bundles form. In addition, single, isolated cells after 2 days in culture contain a number of finger-like projections, 40-100 nm in diameter, which protrude for up to 3-4 [im. These projections make contact with other cells, and appear to determine their orientation. From these results in culture it would appear that specialized contact between cells, both in partially dispersed clumps and as isolated cells, is a necessary feature for bundle formation. However, as partially dispersed clumps of tissue into which nerve fibres have grown reform muscle bundles after only 3-5 days in culture, it seems that while innervation is not essential for muscle bundle formation, it substantially accelerates the process.

Isolated smooth muscle cells with nerve fibres in close association maintain their differentiation for longer periods of time than most smooth muscle cells with no nerve fibres in association. This may be due to nerve fibres having selected those muscle cells which are capable of maintaining their differentiation longer, or because nerve fibres have a trophic effect on the muscle cells. The first possibility seems unlikely, since sympathetic nerve fibres form lasting associations with every differentiated smooth muscle cell from the vas deferens that they encounter, as long as other nerve fibres in the culture have not previously formed associations with that cell (Chamley et al. 1973). Changes in vas deferens smooth muscle cells have not been seen ultrastructurally (G. R. Campbell, personal observation) or functionally (Birmingham, 1970)

FIGURE 9

The same region of a sparsely seeded culture of newborn guinea-pig vas deferens cells, shown with increasing time in culture.

(A) After 2 days in vitro, m, Muscle cells ; ƒ, fibroblast. (B) After 22 days in vitro. A monolayer, consisting mainly of dedifferentiated

ribbon-shaped smooth muscle cells and fibroblasts, has formed. (C) After 32 days in vitro. Elongated, narrow, phase-dense cells characteristic

of differentiated smooth muscle cells appear (arrows). (D) After 100 days in vitro. Strap-like formations of elongated smooth muscle

cells in two layers at right angles (arrows) can be seen.

318 J. H. CHAMLEY AND OTHERS

"«s^r^

Smooth muscle in tissue culture 319 after denervation in situ. Thus, the absence of sympathetic nerves does not lead to dedifferentiation. However, the present experiments indicate that their presence will retard this process under circumstances where it would normally occur. The delay in dedifferentiation of smooth muscle cells in close association with nerve fibres is probably responsible for the absence of mitosis in these cells, as in most cases it would appear that dedifferentiation is a necessary prerequisite for smooth muscle proliferation (see also Keller, 1965; Imai, Lee, Lee & Lee, 1969).

The decrease in rate of spontaneous contraction of single, isolated smooth muscle cells with nerve fibres in close association was about the same as for less-differentiated cells with no nerve fibres. As cells with the same ultra-structure in clumps maintain their spontaneous contractility for many weeks, it would appear that in culture other factors as well as ultrastructural differen­tiation affect contractility. For vas deferens smooth muscle, these factors appear to be related to the isolation of the cells.

This study was supported by grants from the Australian Research Grants Committee, the National Heart Foundation of Australia and the Australian and New Zealand Life Insurance Medical Research Fund. J. H. Chamley and G. R. Campbell are holders of Post-doctoral Fellowships from the Australian and New Zealand Life Insurance Medical Research Fund and the National Heart Foundation of Australia, respectively.

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FIGURE 10

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F I G U R E 11

(A) Muscle cell (m) with nerve fibres in close association retains its differentiation to a greater extent than do muscle cells with no nerve fibres (arrows). Also, compare with Fig. 2D. Newborn guinea-pig sympathetic chain 16 days in vitro, vas deferens 9 days in vitro. No NGF.

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(Received 12 November 1973, revised 5 February 1974)