Spatial Orientation of Microtubules in Contractile Fibroblasts In Vivo ROSS RUDOLPH AND MARILYN WOODWARD Division of Plastic Surgery and the Department of Surgery, University of California, San Diego, and the Veterans Administration Hospital, La Jolla, California 921 61 ABSTRACT Contracting fibrous tissues from skin wounds in pigs, and scars around silicone implants in humans, contained fibroblasts t h a t had multiple bundles of 60-80 A microfilaments with electron-dense bodies, features typical of contractile fibroblasts both in vivo (myofibroblasts) and in vitro. These in vivo fibroblasts contained many 220 A diameter microtubules, which paralleled plasma membranes in the long axis of the cells. The spatial orientation of the microtubules in these myofibroblasts strongly suggests a bracing or scaffolding function. Extensive study of cultured fibroblasts t h a t a r e both mobile and contractile has demonstrated microtubules and microfilaments t h a t appear intimately related to cell movement and mitosis (Porter, ’76; Goldman e t al., ’73). Morphologically similar fibroblasts occur in contracting wounds and scars in living animals and humans. Gabbiani and associates (’72, ’76) described contractile fibroblasts, which they named “myofibroblasts,” t h a t share electron microscopic characteristics of smooth muscle cells and fibroblasts. Gabbiani e t al. (’72, ’76) described these cells as containing bundles of 60-80 A microfilaments with electron dense bodies, convoluted nuclei, desmosomes, and basal lamina. Immunologically (Gabbiani e t al., ’73) and pharmacologically (Gabbiani e t al., ’72; Majno et al., ’711, myofibroblasts s h a r e many characteristics of smooth muscle cells and are thought to be responsible for wound and scar contraction in vivo. Gabbiani e t al.’s descriptions of in vivo contractile fibroblasts did not stress t h e presence of microtubules, but recent studies using colchicine t o control wound contraction in vivo (Ehrlich e t al., ’77) indicate t h e need to determine if microtubules in myofibroblasts have a n orientation t h a t might explain this drug effect. MATERIALS AND METHODS Tissue harvesting On the backs of ten Pitman-Moore 30-40 kg pigs, two 5 x 5 cm full thickness skin wounds were created. These wounds were protected ANAT. REC. (1978) 191: 169-182. with a leather coat for two weeks, and healed by slow contraction and epithelialization. From each wound, multiple small biopsies measuring 1 x 1 x 3 mm were taken weekly for eight weeks, and then every four weeks for a total of 20 weeks of observation. A total of 35 biopsies was taken from t h e wounds, with care being taken to avoid biopsying the same area. Weekly area measurements of t h e granulating wounds were done via tracings of clear plastic followed by planimetry. Human specimens were obtained from contracted tissue around twenty silicone breast implants, at the time of surgical revision. Tissue preparation Tissue for light microscopy was embedded in paraffin blocks and stained with hematoxylin and eosin after formaldehyde fixation. The specimens for electron microscopy were placed immediately in room temperature (21-22OC) solution of 4% paraformaldehyde and 5% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.2-7.4.After four hours of fixation, tissues were rinsed three times with cacodylate buffer a n d postfixed w i t h 2% cacodylate buffered osmium tetroxide at 4°C. Tissue blocks were progressively dehydrated with ethanol, followed by propylene oxide and then propylene oxide-Epon mixtures, proceeding to Epon. Epon blocks were placed in Received Oct. 4, ‘77. Accepted Jan. 13, ‘78. ‘ This investigation was supported in part by the Medical Research Service of the Veterans Administration, and by NIH Grant G M 24648-01. 169 170 ROSS RUDOLPH AND MARILYN WOODWARD embedding molds with fresh Epon, and cured overnight a t 60°C. Thick sections were stained with methylene blue. Thin sections were stained with uranyl acetate and lead citrate, and were examined at 16,000 to 30,000 x using a Zeiss 10 electron microscope. Myofibroblast population was estimated by seeking microfilament bundles in cells in thin sections; from each tissue block ( 5 5 in all), thick sections were cut and used for orientation of thin sections. Thick section face blocks usually contained 60-80 cells, which were examined for myofibroblast characteristics. RESULTS Pig wounds. Wounds within the first week of surgery became thickly crusted with a non-purulent exudate, and began to contract. Rapid contraction of the wounds occurred, with decrease to 42.1 % Standard Error of the Mean 5.2% surface area at four weeks. By eight weeks, wound area was stable at 25.0 2 S.E.M. 7.0% of surface area. Weekly biopsies under light microscopy showed a typical appearance of granulation tissue containing new fibroblasts, new collagen bundles, and multiple small capillaries. During the eight weeks t h a t i t took wounds to heal, the granulation tissue matured with formation of larger collagen bundles and growth of epithelium across the wound. Human tissue. Gross scar around 20 silicone breast implants showed contraction leading to hardness and rounding up of the silicone implant, typical of the contraction process t h a t can occur around such implants (Domanskis and Owsley, ’76; Wilflingseder e t al., ’74). Light microscopy showed dense connective tissue, with collagen bundles parallel to the surface of the scar around the implant. Electron microscopy In the pig wounds, within the first three weeks many fibroblasts were seen containing abundant Golgi apparatus and rough endoplasmic reticulum with small surface area, indicating active protein synthesis (fig. 1). These fibroblasts were surrounded by collagen bundles. A large number of the fibroblasts, estimated to be as high as 80% by two weeks, contained features characteristic of both fibroblasts and smooth muscle cells. Fibroblast cytoplasm contained large bundles of parallel 60-80 A filaments with electron-dense bodies (figs. 1-5).Paralleling the occurrence of these microfilament bundles in fibroblasts, micro- tubules were seen with specific orientation. In fibroblasts in 2-week-old healing pig wounds (fig. l), microtubules of considerable length were seen t h a t paralleled the large bundles of microfilaments. Figure 2 shows another fibroblast in a two week old contracting wound, with microtubules t h a t appear to run directly into bundles of fine parallel filaments a t the edge of the cell membrane. Well defined desmosomes were also seen (fig. 3) in these fibroblasts. Figure 4 shows a fibroblast from a wound at five weeks, containing long parallel microtubules. In addition, microtubules appear to crisscross t h e cell. Microtubules remained plentiful during t h e two-to-eight week period when the large bundles of microfilaments, typical of in vivo “myofibroblasts” were also plentiful. After 8 and 12 weeks of observation, wound myofibroblasts became less plentiful and at 16 to 20 weeks, no cells were seen containing the large bundles of parallel microfilaments. Microtubules were rarely seen in fibroblasts in these wounds, and instead 100-120 A intermediate filaments were especially prominent. When microtubules were present, they were seen only a s short lengths and of random orientation, without the specific spatial orientation found in the fibroblasts seen earlier. In human contracted scar (fig. 5 ) , fibroblasts were also seen containing large bundles of microfilaments with electron-dense bodies. Spatially oriented microtubules paralleled the microfilament bundles, and joined smaller bundles at the edge of t h e cells. DISCUSSION Wound contraction is a basic process of animal wound healing, and this process has recently been identified as a function of specialized fibroblasts (Gabbiani e t al., ’72, ’76; Majno et al., ’71). These contractile fibroblasts, in addition to having rough endoplasmic reticulum and being surrounded by collagen, have large bundles of fine parallel microfilaments with electron-dense bodies. The microfilament bundles are similar to those seen in smooth muscle cells, and are theorized (Gabbiani e t al., ’72, ’76) as being the active component of the in vivo contractile fibroblast. These fibroblasts also contain convoluted nuclei, desmosomes, and basal lamina, characteristics again of smooth muscle cells. Immunologic staining with fluorescein a n tibody to human smooth muscle cells heavily stains t h e myofibroblasts (Gabbiani e t al., MICROTUBULES IN CONTRACTILE FIBROBLASTS ’73). Pharmacologic treatment of human and animal granulation tissue strips with agents t h a t would normally s t i m u l a t e o r relax smooth muscle (Gabbiani et al., ’72; Majno et al., ’72; Madden e t al., ’74; Ryan e t al., ’74) similarly stimulates or relaxes t h e granulation tissue, again suggesting t h a t i t contains cells t h a t react like smooth muscle cells. Fibroblasts with smooth muscle-like features have also been found in normal organs such as adrenal gland (Bressler, ’731, rat testicle (Gorgas and Bock, ’741, duodenal villi (Giildner e t al., ’721, and lung (Kapanci e t al., ’74). Pathologically contracted tissues such as burn scars, cirrhosis and Dupuytren’s contracture also contain myofibroblasts (Ryan e t al., ‘74; Gabbiani e t al., ’72). Many studies of contractile fibroblasts in tissue culture have shown structural characteristics much like those seen in vivo. Mobile fibroblasts in tissue culture contain bundles of microfilaments (Wessells e t al., ’71; Vasiliev and Gelfand, ’761, along with larger (100120 A) intermediate filaments, and microtubules. Probably t h e myofibroblasts of Gabbiani e t al., and the contractile fibroblasts seen in tissue culture, are the same cells. In teleological terms, probably fibroblasts in tissue culture have contractile properties because in t h e living animal they contract and close wounds. In tissue culture studies, substances such as colchicine and cytochalasin B have specific effects on contractile fibroblasts. Cytochalasin B causes disruption of the bundles of microfilaments (Wessells e t al., ’711, while colchicine prevents assembly of microtubules (Vasiliev and Gelfand, ’76). Recent studies by Ehrlich e t al. (’77) utilized these agents not in tissue culture but in live animals to see if drug effects observed in vitro had parallels in living tissue. Contracting wounds were treated topically with these agents, and i t was found t h a t colchicine but not cytochalasin B inhibited the contraction of wounds. Thus, Ehrlich e t al. (’77) stated t h a t microtubules might play a significant role in t h e contraction of myofibroblasts in vivo. Microtubules have not been previously included as a prominent feature of myofibroblasts. In studies from Gabbiani’s group (Ryan et al., ’74), description was made of “scattered microtubules” of 220 A diameter but no specific spatial orientation was noted. Our study shows t h a t in contractile fibroblasts in vivo, microtubules are quite prominent but only 171 during a relatively limited time span when t h e classic myofibroblasts are plentiful (Rudolph e t al., ’77). The microtubules occur parallel to the long bundles of 60-80 A microfilaments. In addition, both in humans and in pigs, they occur parallel to and joining smaller bundles of microfilaments at the cell borders. The especially close relationship of the microtubules and microfilaments is particularly evident in figures 4 and 5 , and suggests t h a t microtubules may be important in facilitating effective microfilament contraction. The spatial orientation of the microtubules - parallel to t h e long axis of the fibroblasts and occasionally crossing the cell a t long angles - suggests a bracing or scaffolding function. Microtubules have been considered as important structural elements of cellular cytoskeleton (Porter, ’761, probably without significant inherent contractile ability. Thus, t h e inhibitory effect of colchicine on wound contraction may be via reduction of structural bracing within myofibroblasts, preventing effective cellular Contraction. LITERATURE CITED Bressler, R. S. 1973 Myoid cells in the capsule of the adrenal gland and in monolayers derived from cultured adrenal capsules. Anat. Rec., 177: 525-531. Domanskis, E. J., and J. Q. Owsley 1976 Histological investigation of the etiology of capsule contracture following augmentation mammaplasty. Plast. Reconstruc. Surg., 58: 689-693. Ehrlich, H. P., G. Grislis and T. K. Hunt 1977 Evidence for the involvement of microtubules in wound contraction. Am. J. Surgery, 133: 706-709. Gabbiani, G., B. J. Hirschel, G. B. Ryan, P. R. Statkov and G. Majno 1972 Granulation tissue as a contractile organ: A study of structure and function. J. Exp. Med., 135: 719-734. Gabbiani, G., M. LeLouis, A. J. Bailey, S. Bazin and A. Delaunay 1976 Collagen and myofibroblasts of granulation tissue. Virchow’s Arch. B. Cell. Path., 21: 133-145. Gabbiani, G., G. B. Ryan, J. P. Lamelin, P. Vassalli, G. Majno, C. A. Bouvier, A. Cruchaud and E. F. Luscher 1973 Human smooth muscle auto-antibody: Its identification as antiactin antibody and a study of its binding to “non-muscular” cells. Am. J. Path., 72: 473-484. Goldman, R. D., G. Berg, A. Bushnell, C. Chang, L. Dickerman, N. Hopkins, M. Miller, R. Pollack and E. Wang 1973 Fibrillar systems in cell motility. In: Locomotion of Tissue Cells. Elsevier, Amsterdam, pp. 83-107. Gorgas, K., and P. Bdck 1974 Myofibroblasts in the rat testicular capsule. Cell Tissue Res., 154: 533-541. Guldner, F. H., J. R. Wolff and D. Grafkeyserlingk 1972 Fibroblasts as a part of the contractile system in duodenal villi of rat. Z. Zellforsch. Mikrosk. Anat., 135: 349-360. Kapanci, Y., A. Assimacopoulos, C. Irle, A. Zwahlen and G. Gabbiani 1974 “Contractile interstitial cells” in pulmonary alveolar septa; A possible regulator of ventilationiperfusion ratio? J. Cell. Biol., 60: 375-392. Madden, J . W., D. Morton, J r . and E. E. Peacock, 172 ROSS RUDOLPH AND MARILYN WOODWARD Jr. 1974 Contraction of experimental wounds. I. I n hibiting wound contraction by using a topical smooth muscle antagonist. Surgery, 76: 8-15. Majno, G., G. Gabbiani. B. J. Hirschel. G . B. Ryan a n d P. R. Statkov 1971 Contraction of granulation tissue in uitro: Similarity to smooth muscle. Science, 173: 548-550. Porter, K. R. 1976 Motility in cells. I n : Cell Motility. Cold Spring Harbor Conference on Cell Proliferation. Vol. 111. Cold S p r i n g Harbor Laboratory, New York. pp. 1-28. Rudolph, R.. S. Guber, M. Suzuki a n d M. Woodward 1977 T h e life cycle of t h e myofibroblast. Surg.. Gyn.. & Obstet., 145: 389-394. Ryan, G. B.. W. J. Cliff, G. Gabbiani, C. Irle, D. Montandon. P. Statkov a n d G. Majno 1974 Myofibroblasts in h u m a n granulation tissue. H u m a n P a t h . , 5: 55-67. Vasiliev, J. M., a n d I. M. Gelfand 1976 Effects of colcemid on morphogenic processes a n d locomotion of fibroblasts. In: Cell Motility, Cold Spring Harbor Conference on Cell Proliferation. Vol. 111. Cold Spring Harbor Laboratory, New York, pp. 279-304. Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0.Bradley, M. A. Luduena, E. L. Taylor, J. T. Wrenn a n d K. M. Yamada 1971 Microfilaments i n cellular a n d developmental processes. Science, 171: 135.143. Wilflingseder, P., A. Propst a n d G. Mikuz 1974 Constructive fibrosis following silicone implants in mammary a u g mentation. Chir. Plastica 2: 215-229. PLATE 1 EXPLANATION OF FIGURE 1 Fibroblast from a 2-week-old contracting wound in a pig. Rough endoplasmic reticulum is active with many ribosomes. Long microtubules (arrows1 of 220 A diameter a r e prominent, r u n n i n g parallel to t h e cell membrane a n d t o each other. A bundle of 60 8, parallel microfilaments with electron-dense bodies (asterisk) is typical of in vivo contractile fibroblast (“myofibroblast”). X 40,000. MICROTUBULES I N CONTRACTILE FIBROBLASTS Ross Rudolph and Marilyn Woodward PLATE 1 173 PLATE 2 EXPLANAlION OF F I G U R E 2 Terminal process of a fibroblast from a 2-week-old wound in a pig. Microtubules a r e frequent and appear to r u n parallel and into short bundles of 60 A microfilaments a t cell membrane, some of which extend into t h e extracellular space (arrow). Pinocytic vesicles and rough endoplasmic reticulum a r e prominent. X 45,000. 174 MICKOTUBULES IiX CONTHACTILE FIBROBLASTS Ross Rudolph and Marilyn Woodward PLATE 2 175 PLATE 3 EXPLANATION OF FIGURE 3 Myofibroblasts in a 2-week-old wound in a pig. Desmosome and gap junction (asterisk a t upper left) are present. Microtubules (arrows) parallel the cell membranes. X 40,000. 176 MICROTUBULES IN CONTRACTILE FIBROB1,ASTS Ross Rudolph a n d Marilyn Woodward 177 PLATE 4 EXPLANATION OF FIGURE 4 Fibroblast in a 5-week-old contracting wound in a pig. Prominent long microtubules . microfilaments with electron-dense bodies parallel thick bundles of 60-80 h (asterisk).Microtubules also traverse the cell a t an oblique angle, suggesting a bracing function (arrows). X 40,000. 178 MICROTUBU1,ES IN ('ONTRACTILE FIBROBLASTS Ross Rudolph a n d M a r i l y n Woodward PI.A'TE 4 179 PLATE 5 EXPLANATION OF FIGURE 5 Fibroblast in a human contracting fibrous scar around a silicone rubber implant, four weeks after onset of contraction. Prominent microtubules (arrows) appear to join large bundles of microfilarnents. x 40,000. 180 MICROTUBULES I N CON'IKA('TILE FIBKOBLASTS Ross Kudolph a n d Marilyn Wondward PI.ATE 5 181
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