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Myofibroblasts in human granulation tissue
M Y O F I B R O B L A S T S IN H U M A N GRANULATION TISSUE* Graeme B. Ryan, M.B., B.S., Ph.D.,-~ Walter J. Cliff, M.B., B.Chir., D.Phil.,$ Giulio Gabbiani, M.D., Ph.D.,w Claude Irld, ll
Denys Momandon, M.D.,82 Peter R. Statkov, M.D.,# and Guido Majno, M.D.**
Abstract Fibroblasts o f h u m a n g r a n u l a t i o n t i s s u e s d e v e l o p structural a n d functional features typical o f s m o o t h muscle cells: 1. T h e y contain massive bundles o f intracytoplasmic microfilaments. 2. T h e i r cytoplasm shows i m m u n o f l u o r e s c e n t labeling with h u m a n antis m o o t h muscle s e r u m . 3. T h e nuclei have m u h i p l e indentations a n d d e e p folds, indicative o f contraction. 4. T h e r e are cell-to-cell a n d cell-to-stroma connections w h e r e b y cellular contraction could be t r a n s m i t t e d to the tissue as a whole. 5. Strips o f g r a n u l a t i o n tissue, w h e n tested pharmacologically ill vitro, contract o r relax similarly to s m o o t h muscle. It is p r o p o s e d that these modified fibroblasts, or "myofibroblasts," are responsible for the contraction o f granulation tissue, a process useful in closing w o u n d s t h o u g h potentially h a r m f u l in o t h e r situations.
*Aided by grants from tile Fonds National Suisse de la Recherche Scientifique (3.356.70 and "~.460.70), and from Z)ma S. A., Nyon, Switzerland. tGuest Investigator, Delmrtment of Pathology, University of Geneva, Geneva, Switzerland. Present appointment: Assistant Professor of l'athology, ttarvard Medical School, Boston, Massachusetts. r Fellow, Department of Experimental l'athology, John Curtin School of Medical Research, Australian National University, Canberra, Australia. w
Professor of l'athology, University of Geneva, Geneva, Switzerland.
llMedical Student, University of Geneva, Gcneva, Switzerland. 82
Surgeon, Department of Surgery, University of Geneva, Geneva, Switzerland.
9#Guest Investigator, Department of Pathology, University of Geneva, Geneva, Switzerland. **l'rofcssor of l'atlmlogv University of Geneva. tlead, l)cpartmcnt of Pathology, llfpital Cantonal, Geneva, Switzerland.
55
HUMAN PATHOLOGY--VOI.UME 5, NUMBER 1 Jamtmy 1974 We Imve recently shown, in the rat, that fibroblasts o f c o n t r a c t i n g granulation tissues come to resemble s m o o t h muscle cells? -4 This resemblance is m o r p h o l o g i c : pltarmacologic, 2 chemical, -~ and antigenic? We have called these cells "myofibroblasts" and have p r o p o s e d that it is by their active contraction that granulation tissue s h r i n k s - w h e t h e r to close a w o u n d , constrict a lumen, or d e f o r m a heart valve? This p a p e r describes the presence o f myofibroblasts in h u m a n granulation tissues.
MATERIALS AND METHODS
Clinical Material Biopsy samples o f granulation tissue were obtained f r o m f o u r patients r e f e r r e d to the Plastic S u r g e r y Unit at the Cantonal Hospital o f Geneva: CASE 1. A 53 )'ear old man had a total laryngectomy with a permanent tracheostomy for a carcinonm of the larynx. Ten days later the wound above the tracheostomy broke down, resuhing in a pharyngeal fistula that drained saliva to the outside of the neck. Two months after the initial operation, the fistula was closed using a local skin flap to recreate the pharyngeal mucosa and a dehopectoral flap for the surface skin defect; at this operation we obtained a specimen of granulation tissue from the external opening of the fistula. Four weeks later the base of the deltopectoral flap was restored to its normal position, and we obtained another sample of tissue, this time from the granulating wound on the chest. CASE 2. A 41 )'ear old woman had a bilateral subcutaneous nmstectomy for fibrocystic disease. At the same operation, a Silastic mamnmry prosthesis* was implanted on each side. Two weeks later a henmtoma had developed on top of the right prosthesis; this was aspirated several times over the next few weeks. Two months after the initial procedure there was no more fluid accunmlation, but the prosthesis began to show progressive upward displacement and the patient complained of local tightness and pain. These symptoms persisted and the patient was again submitted to surgery 10 months later. The prosthesis was completely surrounded by a capsule that was 4 to 5 ram. tlfick inferiorly but only I mm. thick superiorly. Tiffs capsule was partially removed 56
*Cronin type, Dow Corning, Midland, Michigan.
(providing us with specimens) and tim implant was set back in the original cavity. CASE 3. A 47 )ear old woman had a bilateral subcutaneous mastectomy for fibrocystic disease. At the same operation a silicone Natural-Y mammary prosthesist was implanted on each side. The postoperative course was complicated by skin necrosis, 3 cm. in diameter, in the left areolar region; tiffs necessitated removal of tile prosthesis (which was not noticeably displaced) after four weeks. We obtained a sample of granulation tissue from around the prosthesis at this stage. CASE 4. A 34 )'ear old woman with a grossly pendulous abdomen following pregnancy lind a subcutaneous lipectomy and a plasty of the rectus abdolninis muscles. One week later she developed a collection of fluid under the wound. This was opened, leaving a round wound 4 cm. in diameter tlmt was allowed to heal by second intention. A biopsy was taken from granulation tissue at the base of the wound four weeks after the initial procedure. NORMAL SKIN'. Biopsy samples were obtained from two patients: one was from the wrist of a 27 )'ear old man undergoing surgery for a carpal tunnel syndrome; the other was taken from the abdomen of a 32 )'ear old alcoholic male during an operation to implant a disulfiram (Antabuse) pellet.
Histologic Examination Tissues for histologic examination were fixed in 10 per cent buffered f o r m a lin, d e h y d r a t e d , and e m b e d d e d in paraffin. Sections were stained with hematoxylineosin, periodic acid-Sclfiff, Masson trichronae, cresyl violet, Mallory's p h o s p h o tungstic acid, and van Gieson stains.
Electron Microscopic Examination Tissues for electron microscopy were cut into cubes o f 1 ram. or less, a n d fixed at r o o m t e m p e r a t u r e by immersion for five h o u r s in 3 per cent g l u t a r a l d e h y d e in cacodylate buffer at pH 7.2 to 7.4. The), were left in buffer at 4 ~ C. overnight, postfixed at 4 ~ C. for two hours in 2 per cent o s m i u m tetroxide in collidine buffer at p H 7.3 to 7.4, d e h y d r a t e d in alcohol, and e m b e d d e d in E p o n 812. Sections approximate'ly lp. tlfick were cut with glass knives on a Reichert u l t r a m i c r o t o m e or LKB tAshley type, Edward .}Veck & Company, Long Island City, N.Y.
MYOFIBROBLASTS IN HUMAN GRANULATION TISSUE-RvAN VT AL. Ultratome and stained with naethylene blue and azur II. Thin sections, were cut with diamond knives, mounted on noncoated grids, stained with uranyl acetate and lead citrate, coated with a tlfin layer of carbon, and examined with a Plfilips 300 electron microscope.
hnm unofluorescence Human antismooth muscle serum ("smooth muscle autoantibody" serum) was obtained from a 16 )'ear old girl with active chronic hepatitis, a Tiffs serum reacted with smooth muscle of rat stonaach at dilutions up to 1/320; tests for antimitochondrial and anti-DNA antibodies and for antinuclear factor were negative. To demonstrate the presence of s|nooth muscle antigens, the double layer technique was used) Unfixed cryostat sections of tissue were treated for 30 minutes with the smooth muscle autoantibody sertun (diluted 1 part in 20 parts of phosplmte buffered saline). The sections were then washed in phosphate buffered saline and stained for 30 minutes with fluoresceinconjugated IgG fraction of goat antiserum to I|uman IgG.* After rewaslfing with phospliate buffered saline and mounting in 90 per cent glycerol in pl|osphate buffered saline, the level of fluorescence was compared with that in control sections treated with normal human serum instead of smooth muscle antoantibody serum. Photographs were taken on a Zeiss UV photomicroscope with a UGI or II excitor filter and a Zeiss No. 50/65 barrier filter, using Anscochrome color slide film (500 daylight). The same sections were then stained with l|enmtoxylin and eosin and rephotographed with visible light.
Contractility in Vitro Snmll strips of tissue (with dimensions of approximately 1 by 1 by 12 mm. and weighing I0 to 15 nag.) were tested plmrmacologically in a specially devised microchamber. In tlfis system the tissue was suspended vertically (by a 6-0 silk suture) from a fixed, rigid horizontal post, and its lower end was attached (also by 6-0 silk) to a point near the freely mobile tip of a very *Code 64-170, Miles-Seravac,Lausanne,Switzerland.
finely drawn, flexible glass filament. The The whole assembly was bathed in 10 nil. of Tyrode's solution that was contint|ally bubbled with 95 per cent oxygen plus 5 per cent carbon dioxide, and kept at a constant 37 ~ C. by a radiant heat source. The tip of the glass filament was observed through a light microscope, and contraction or relaxation of the strip of tissue was detected vist|ally by the movement of the tip of the filament in relation to the graduatious of a measuring ocular. Drugs were added via a hole in the top of the chamber; these included angiotensin (Hypertensin), epinephrine hydrochloride (Adrenalin) and papaverine hydrochloride, norepinephrine (Arterenol), lfistamine dihydrochloride, serotonin creatinine sulfate (5I|ydroxytryptanfine) and dimethyl sulfoxide (DMSO), vasopressin, prostaglandins Ft a and E~, promethazine I|ydrochloride (I'henergan), diphenhydramine hydrochloride (Benadryl), chlorpheniramine maleate (Chlor-Trimeton), a 4:1 mixture of oxytheophylline and tlaeophylline (Cordaline), cytoclmlasin B, and potassium cyanide.
RESULTS
HistoIogic Examination In each case light microscopic stud)' showed the granulation tissue to consist mainly of "fibroblasts," small blood vessels, scattered neutrophilic pol)'morplmnuclear leukocytes, mononuclear cells, and nmcropimges (Fig. 1). The fibroblasts lind pale, oval, or elongated nuclei and abundant cytoplasm with long extensions; some nuclei showed cross banding like that already reported in the myofibroblasts of Du.puytren's contracture3 No other indication as to the contractile nature of these cells was given by histologic study, as expected.
Electron Microscopic Examination Control fibroblasts (from normal humall dermis) showed morphology conforming with standard descriptions: s In typical cells the nuclear contour was smooth and the cytoplasm contained prominent cisternae of rough endoplasmic
57
Jamtmy1974
H U M A N P A T H O L O G Y - - V O L U M E 5, NUMBER 1
Figure 1. Light micrograph of human granulation tissue showing "fibroblasts," small blood vessels, and inllammator)" cells. Case 4. (tlematoxylin and eosin stain, x 160.)
features to distinguish it from a conventional f i b r o b l a s t - i n d e e d , in some ways, it resembled a smooth muscle cell: 1. Massive bundles of long intracytoplasmic microfilaments (Figs9 3 to 6). Most o f t h e microfilaments measured approxi-
reticulum and some mitochondria. Intracytoplasmic microfilaments were few in n u m b e r (Fig. 2). In all the h u m a n granulation tissues examined we f o u n d that the p r e d o m i n a n t connective tissue-cell had three striking
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MYOFIBROBLASTS IN HUMAN GRANULATION TISSUE--RYAXETAL.
Figure 3. Electron micrograph of myofibroblast in Imman granulation tissue showing abundant rough endoplasmic reticuluna and multiple smooth muscle-like cytoplasmic microfilaments arranged in discrete bundles (arrows), one of which lies in a nuclear fold. (Case 1, first biopsy. X 10,500.)
mately 50/~ in diameter; in addition, there were some larger ones, a p p r o x i m a t e l y 100 /~ in diameter, as well as scattered microtubules, a p p r o x i m a t e l y 220/~, in diameter. T h e thin and thick microfilaments were aggregated into bundles tlmt usually ran parallel to the long axis o f the ceil. Many "dense bodies" (or " a t t a c h m e n t sites") were present in the bundles. In most cells the bundles were large and multiple and were distributed like discrete "lnuscles" in different parts o f the cell. Sometimes, however, the bundles were confluent, tlms giving an a p p e a r a n c e very like a classic smooth muscle c e l l - e x c e p t for the presence o f a b u n d a n t cisternae o f r o u g h endoplasmic reticulum (typical o f fibroblasts) elsewhere in the cytoplasm (Figs. 3 to 6). Occasional cells had only one o r two narrow bundles; in such cases the bundles were usually located just beneath the cell m e m b r a n e . T h e only fibroblast seen in mitosis (in case 3) had very few microfilaments.
2. Nuclear deformations. Many o f the nuclei o f these "muscular" fibroblasts had multiple indentations o1" d e e p folds (Fig. 3). 3. Surface differentiation. Intel'cellular connections between such cells were frequent: these were o f the macula a d h e r e n s ("desnaosonle") and nexus ("gap" junction) types (Figs. 7, 9). 9 In additiou, material resetnbling basal lamina was often f o u n d close to the cell surface (Figs. 7, 8, 10). In such cases, there was sometimes a dense zone in the microfilam e n t b u n d l e immediately beneath the plasmalemma, thus r e s u h i n g in a henaidesmosome-like complex (Fig. 10). T h e basal lamina material was not u n c o m m o n l y seen r u n n i n g off into the stroma away fi'om the cell (usually in a direction parallel to the intracytoplasmic' microfilaments) and sometimes c o n n e c t i n g with the basal lamina a r o u n d a n o t h e r cell. Although we have not formally e x c l u d e d the possibility that a g1:azing section n e a r a cell surface (Text continued on page 63.)
59
H U M A N I ' A T H O L O G Y - - V O L U M E 5, NUMBER 1
Janua0'1974
Figure 4. Two large intracellular "muscles" (stars) fanning through cytoplasm of m)ofibroblast and separated b)' endoplasmic reticulum. (Case 1, first biopsy, x 15,200.)
60
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Detail of myofibroblast; ,rote "dense bodies" (arrows) among microfilament bundles. (Case 4.
Figure 6. Detail of two microfilamem bundles separated by endoplasmir reticulum; note microtubule (arrow). (Case 4. • 27,500.)
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Figure 7. Connection between two myofibroblasts: d~isco,uplicated junction is o f the macula a d h e r e n s type (horizontal arrow) in one part and o f nexps type (vertical arrow) in a n o t h e r part. Note basal lamina-like material (star). (Case 4. x 2.5,.500.)
62
Figure 8. Presuulptive connections o f myofibroblasts to stroma via basal lamina-like "microtendons" (arrows). (Case 4. x 25,.500.)
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Figure 9. Intercellular connection of nexus type (arrow); note microtubules in cell at left. (Case 4. x 87,000.)
Figure 10. Basal lalnina material (Sial') applied to surface of myofibroblast; note dense zones (arrows) beneath plasmalemlna, resuhing iq hemidesmosome-like complexes. (Case 1, first biopsy. x 43,000.)
is responsible for this "microtendinous" appearance (Fig. 8), it is most likely that such structures participate in some way in the transmission of cellular pull to other tissue compouents.
and case 4). Tile pooled restdts are as follows. Contraction was induced with prostaglandin Fia 1 x I0 -r to I x 10 -~ gln./ ml., final concentration in bath (Fig. 12 shows the dose-response cnrve), angiotensin 1 x 10 -6 gm./ml., vasopressin 0.01 to 0.1 I.U./ml., promethazine hydrochloride I x 10 -5 gm.hnl., d i p h e n l w d r a m i n e hydrochloride 1 • 10 -3 gm./ml., and cldorpheniramiue maleate 1 x 10 -4 gm./ml. No significant contraction occurred with 5h y d r o x y t r y p t a m i n e 1 x 10 -5 gm./ml. (as was fotmd with rat skiu wounds, a h h o u g h other kinds o f rat granulation tissue gave good responses'-"4), histainine 1 x 10 -~ gin./ml., epinephrine 1 x 10 -7 gm./ml., or norepinephrine 1 • 10 -5 gm./ml. Relaxation was induced with prostaglandin E1 1 x 10 -5 gm./ml. (Fig. 12), Cordaline (a theophylline preparation) 1 • 10 -3 gin./ ml., papaverine 1 x 10 -4 gm./ml., and potassitnn cyanide 1 x 10 -a gm./ml. Cytochalasin B 1 • 10 -5 gm./nfl, also caused relaxation; DMSO alone did not affect strips in the concentration used as a sol~'ent for cytochalasin B (1 per cent D*ISO, final concentration in the bath). Control strips o f normal dermis and subcutaneous tissue neither contracted nor relaxed with any o f the foregoing agents.
ImmunoJhtore$cence All the samples of h u m a n granulation tissue showed striking cellular fluorescence after treatment with h u m a n antismooth muscle serum (Fig. 11). This labeling occurred in the cytoplasm o f elongated cells, presumably corresponding to the smooth muscle-like cells seen by electron microscopic examination. No labeling occurred after treatment with normal htnnan serum (Fig. 1 I). Control sections of normal skin treated with antismooth muscle serum showed labeling of vasctflar smooth muscle but not of fibroblasts.
Contractility in Vitro Strips of granulation tissue were tested pharmacologically (like strips of smooth muscle) when sufficient biopsy material was obtained (case 1 - b o t h biopsies, case 2,
63
HUMAN PATHOLOGY--VOLUME 5, NUMBER 1
Janumy1974
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Figure 1l. Fluorescence microscopy of human granulation tisstte. A, Control section treated with normal human serum followed by antilmlnan lgG: no specific labeling. (x 160.)B, Section treated with Imman antismooth muscle sermn followed b)" antihuman lgG: bright specific fluorescence of myofibroblasts. (• 160.) C, Higher power view of cytoplasmic labeling of inyofibroblasts. (• 500.) (Case 1, first biopsy.)
DISCUSSION
64
Tiffs study shows that in h u m a n granulation tissue fibroblasts develop cytoplasmic features very similar to those o f smooth muscle: by electron microscopic examination, a h h o u g h retaining the areas o f a b u n d a n t endoplasmic reticulum typical o f fibroblasts, it was observed that the)' become e q u i p p e d with nmssive bnndles o f intracytoplasmic microfilaments (with scattered "dense bodies"). F u r t h e r m o r e , as d e m o n s t r a t e d by i m m u n o f l u o r e s c e n c e , they contain antigenic material characteristic o f smooth muscle cells. We have earlier p r o p o s e d that such cells, wltich we call "myofibroblasts," are
responsible for tlte contraction o f granulation tissue in e x p e r i m e n t a l animals. H T h e present work indicates tltat the same may be true for l m m a n granulation tissue: 1. T h e nuclear indentations and folds give indirect evidence o f contraction: tile)" mimic tile nuclear changes in c o n t r a c t e d smooth muscle, 1~myocardium, 11 and venular e n d o t h e l i u m ) 2 9. T h e surface differentiations give a means w h e r e b y cellular contraction could explain an overall shrinkage o f the tissue: myofibroblasts connect with one a n o t h e r (via junctions o f the macula a d h e r e n s and nexus types a) and with tile stroma (via attachments to basal lamina material). 3. W h e n tested pharmacologically,
MYOFIBROBLASTS IN IIUMAN GRANULATION TISSUE-RYAx ETAL.
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Figure 12. Contraction shown in vitro by strip of human granulation tissue in response to increasing doses of prostaglandin Fta (1 x i0 -7 to ! x 10-s gin./ ml.), followed b)" relaxation in response to prostaglandin E 1 (1 X 10-5 gm./ml.), (Case 1, first biopsy.)
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strips o f granulation tissue behave similarly to smooth muscle: they contract with certain smooth muscle stimulants and relax with smooth muscle relaxants. H u m a n granulation tissue contraction can t h e r e f o r e be explained on the following basis: Fibroblasts progressively develop intracytoplasmic "muscles" as well as cell-to-cell and cell-to-stroma connections; these myofibroblasts contract (either spontaneously o r in response to endogenous mediators) at the same time that collagen is laid down (thus providing a "lock-step" system) and so tile whole tissue shrinks. In o t h e r words, granulation tissue becomes a contractile o r g a n ? This, o f course, is o f p r i m a r y i m p o r t a n c e in closing a skin wound, but in o t h e r circumstances it can be disastrous. O u r case 2 (tile patient in whom t h e r e was displacement o f a breast prosthesis) is a relatively m i n o r example. More serious are such problems as postburn disfigurements and postinflammatory luminal strictures. It is also likely that the vah, ular deformities o f chronic r h e u m a t i c heart disease are d u e to a similar process; this is a particularly interesting example because it illustrates how a p p a r e n t l y logical was the old belief/3
(now o u t m o d e d 14) that attributed connective tissue contraction to collagen shrinkage: normal vah'es and c h o r d a e consist o f m a t n r e collagen and little else, and so also do chronically d e f o r m e d vah'es and c h o r d a e - h o w e v e r , pathologists do not often see tile stage in between, i.e., when there is invoh'ement o f these structures by active granulation tissue. Cells uhrastructurally similar to myofibroblasts have been seen in certain o t h e r h u m a n tissues: aortic intimal thickenings, 15 ganglia o f the wrist, 1G cirrhosis o f the liver, t7 and the p a h n a r nodules o f Dupuytren's disease3 T h e significance o f myofibroblasts in t h e s e o t h e r tissues is not clear, apart f r o m D u p u y t r e n ' s disease in which they are ahnost certainly responsible for the traction d e f o r m i t y o f the fingers. We have recently shown that the cells o f D u p u y t r e n ' s nodules are specificall)" labeled with antismooth muscle serum. In this context it should be stressed that we d o not p r o p o s e that D u p u y t r e n ' s nodules are c o m p o s e d o f reactive granulation tissue; D u p u y t r e n ' s disease is one o f the so-called "fibromatoses" for which the causes are u n k n o w n ) s Why have myofibi'oblasts not already
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H U M A N I ' A T H O L O G Y - - V O L U M E 5, NUMBER 1 Jammr'r 1974
66
b e e n d e s c r i b e d in h n n m n g r a n u l a t i o n tissue? Ross a n d O d l a n d t9 e x a m i n e d the h e a l i n g o f skin w o u n d s in I m m a n subjects w i t h o u t o b s e r v i n g myofibroblasts. But these w e r e small linear w o u n d s , a n d so the d i s c r e p a n c y is u n d e r s t a n d a b l e : w h e n c o m p a r e d with o p e n w o u n d s , linear w o u n d s heal with little o r n o c o n t r a c t i o n . T h i s leads us to t h r e e m o r e q u e s t i o n s : W h a t is t h e o r i g i n o f myofibroblasts? W h a t are the stimuli f o r their f o r m a t i o n ? W h a t l m p p e n s to t h e m ? N o n e o f these q u e s t i o n s can be a n s w e r e d yet. It is p r o b a b l e that the m y o fibroblasts arise locally fi'om m o r e c o n ventional fibroblasts o r fl'om primitive m e s e n c h y m a l cells. A d e r i v a t i o n f r o m vascttlar s m o o t h m u s c l e cells is also a possibility, b u t we t h i n k tlfis unlikely b e c a u s e in e x a n f i n i n g t h e earl)" d e v e l o p m e n t o f g r a n u l a t i o n tissue in e x p e r i m e n tal animals, we f o u n d a p r o g r e s s i v e acc u m u l a t i o n o f m i c r o f i l a m e n t s in cells that w e r e fibroblastic in a p p e a r a n c e ( r a t h e r tlmn a loss o f m i c r o f i l a m e n t s f r o m s m o o t h m u s c l e cells: i n d e e d , t r u e s m o o t h ntuscle cells w e r e n o t seen o u t s i d e b l o o d vessel walls)? "a F u r t h e r e v i d e n c e f o r a fibroblastic o r i g i n o f m y o f i b r o b l a s t s is that fibroblasts cultivated in vitro d e v e l o p extensive i n t r a c y t o p l a s m i c fibrillar systems "-'~ a n d intercellular c o n n e c t i o n Q t virtually identical to t h o s e o f g r a n u l a t i o n tissue myofibroblasts. W e have recently c o n f i r m e d that the s a m e u h r a s t r n c t u r h l c h a n g e s o c c u r in cells g r o w i n g o u t fi'om e x p l a n t c u l t u r e s o f a d u l t rat dermis. M o r e over, we have f o u n d cytoplasntic "streaks" ( p r e s u m a b l y c o r r e s p o n d i n g to " m u s c l e " btmdles) w h e n such cells a r e stained with a n t i s m o o t h muscle s e r u m . It seems, t h e n , t h a t w h e n fibroblasts a r e suitably trigg e r e d ( w h e t h e r in a w o u n d o r by b e i n g placed in vitro), t h e y d e v e l o p a m u s c u l a r a p p a r a t u s that enables t h e m to contract. It will be i m p o r t a n t to find o u t h o w tiffs t r i g g e r i n g o c c u r s a n d w h e t h e r it can be m o d i f i e d in s o m e way. Finally, we d o n o t yet k n o w w h a t h a p p e n s to myofibroblasts: p r e s u m a b l y they die a n d d i s i n t e g r a t e wlten c o n t r a c t i o n is cotnpleted. T h e m e c h a n i s n t f o r this is o b s c u r e , b u t it is also i m p o r t a n t to investigate b e c a u s e a b n o r m a l persiste n c e o f myofibroblasts ntay be r e s p o n s i b l e f o r c o n v e r t i n g a beneficial w o u n d c o n t r a c tion to a n u n e s t h e t i c o r c r i p p l i n g c o n tracture.
ACKNO~,VLEDGMENTS
W e t h a n k D o c t o r s A. C r u c h a u d a n d I. N i c o d o f the C a n t o n a l Hospital o f G e n e v a f o r kindly p r o v i d i n g samples o f antisera; Dr. J. E. Pike o f the U p j o h n Co., Kalam a z o o , Michigan, f o r samples o f prostag l a n d i n s Fto~ a n d Et; Miss M. C. Clottu, Miss A. L o m e t t o , a n d Mrs. A. Fiaux f o r excellent technical help; a n d Messrs. J. C. R u m b e l i a n d E. D e n k i n g e r f o r p h o t o g r a p h i c work.
REFERENCES
1. Gabbiani, G., Ryan, G. B., and Majno, G.: Presence of modified fibroblasts in granulation tissue and their possible role in wotlnd contraction. Experientia, 27:549, 1971. 2. Majno, G., Gabbiani, G., ttirschel, 11. J., Ryan, G. B., and Statkov, P. R.: Contraction of granulation tissue in vitro: similarity to smooth muscle. Science, 173:548, 197 I. 3. llirscfiel, B. J., Gabbiani, G., Ryan, G. B., and Majno, G.: Fibroblasts of granulation tissue: immunofluorescent staining with antismooth muscle serum. Proc. Soc. Exp. Biol. Med., 138:'166, 1971. 4. Gabbiani, G., Hirschel, B. J., Ryan, G. B., Statkov, 1'. R., and Majno, G.: Granulation tissue as a conlractile organ: a study of structure and fimction.J. Exp. Med., 135:719, 1972. 5. Nairn, R. C.: Fluorescent Protein Tracing. Ed. 3. Edinburgh, E. & S. Livingstone Ltd., 1969. 6. Cliff, W. J., Statkov, 1'. R., and Majno, G.: A simple technique for pharmacological studies of small strips of contractile tissue: the "inicrobath.'" Unpublished study. 7. Gabblani, G., and Majno, G.: Dul)uytrcn's contracture: fibroblast contraction? An ultrastructural study. Amer. J. l'athol., 66:131, 1972. 8. Ross, R.: The connective tissue fiber forming" cell. In Gould, B. S. (Editor): Treatise on Collagen. '2. Biology of Collagcn (Pt. A). New York, Academic Press, Inc., 1968, 1I. I. 9. Weinstein, R. S., and McNntt, N. S.: Cell junctions. New Eng. J. Med., 286:521, 1972. 10. l.ane, B. P.: Alterations in the cytologic detail of intestinal smooth muscle in various stages of contraction. J. Cell Biol., 27:199, 1965. 11. Bloom, S., and Cancilla, P. A.: Conformational changes in ntyocardial nuclei of rats. Circ. Res., 24:189, 1969. 12. Majno, G., Shea, S. M., and l.eventhal, M.: Endothelial contraction induced by histaminetype mediators: an electron microscopic stud)'. J. Cell Biol., 42:647, 1969. 13. l'ayling Wright, G.: An Introduction to Pathology. Ed. 2. l.ondon, I.ongmans Green & Co., l.td., 1954, p. 219. 14. Abercrombie, M., Flint, M. H., and James, D. W.: Wound contraction in relation to collagen formation in scorbutic guinea pigs. J. Embryol. Exp. Morph., 4:167, 1956.
MYOFIBROBLASTS
IN H U M A N
15. Geer, J. C.: Fine structure of h u n m n aortic intimal thickening and fatty streaks. Lab. Invest., 14:1764, 1965. 16. Ghadially, F. N., and Mehta, I'. N.: Muhifimctional mesenchylnal cells resembling smooth muscle cells in ganglia of the wrist. Ann. Rlleum. Dis., 30:31, 1971. 17. Bhathal, I'. S.: Presence of modified fibroblasts in cirrhotic livers in man. l'athology, 4:i39, 1972. 18. Enzinger, F. N., Lattes, R., and Torloni, l-i.: Types histologiques des Tumeurs des Tissus
GRANULATION
TISSUE-RvA,~
ET AL.
Mous. Geneva, World ttealth Organization, 1970, p. 28. 19. Ross, R., and Odland, G.: t t u m a n wound repair. II. hdlamnlatory ceils, epitllelial-nlesencllymal interrelations, and fibrogenesis. J. Cell I',iol., 39:152, 1968. 2(1. Goldberg, B., and Green, It.: An analysis of collagen secretion by established mouse fibroblast lines. J. Cell Biol., 22:227, 1964. 21. Devis, R., and James, D. W.: Close associations between adult guinea-pig fibroblasts in tissue cnlture, studied with the electron microscope. J. Anat., 98:63, 1964. Department of Pathology University of Geneva 40 Boulevard de la Cluse 1211 Geneva 4, Switzerland (Dr. Majno)
67
Denys Momandon, M.D.,82 Peter R. Statkov, M.D.,# and Guido Majno, M.D.**
Abstract Fibroblasts o f h u m a n g r a n u l a t i o n t i s s u e s d e v e l o p structural a n d functional features typical o f s m o o t h muscle cells: 1. T h e y contain massive bundles o f intracytoplasmic microfilaments. 2. T h e i r cytoplasm shows i m m u n o f l u o r e s c e n t labeling with h u m a n antis m o o t h muscle s e r u m . 3. T h e nuclei have m u h i p l e indentations a n d d e e p folds, indicative o f contraction. 4. T h e r e are cell-to-cell a n d cell-to-stroma connections w h e r e b y cellular contraction could be t r a n s m i t t e d to the tissue as a whole. 5. Strips o f g r a n u l a t i o n tissue, w h e n tested pharmacologically ill vitro, contract o r relax similarly to s m o o t h muscle. It is p r o p o s e d that these modified fibroblasts, or "myofibroblasts," are responsible for the contraction o f granulation tissue, a process useful in closing w o u n d s t h o u g h potentially h a r m f u l in o t h e r situations.
*Aided by grants from tile Fonds National Suisse de la Recherche Scientifique (3.356.70 and "~.460.70), and from Z)ma S. A., Nyon, Switzerland. tGuest Investigator, Delmrtment of Pathology, University of Geneva, Geneva, Switzerland. Present appointment: Assistant Professor of l'athology, ttarvard Medical School, Boston, Massachusetts. r Fellow, Department of Experimental l'athology, John Curtin School of Medical Research, Australian National University, Canberra, Australia. w
Professor of l'athology, University of Geneva, Geneva, Switzerland.
llMedical Student, University of Geneva, Gcneva, Switzerland. 82
Surgeon, Department of Surgery, University of Geneva, Geneva, Switzerland.
9#Guest Investigator, Department of Pathology, University of Geneva, Geneva, Switzerland. **l'rofcssor of l'atlmlogv University of Geneva. tlead, l)cpartmcnt of Pathology, llfpital Cantonal, Geneva, Switzerland.
55
HUMAN PATHOLOGY--VOI.UME 5, NUMBER 1 Jamtmy 1974 We Imve recently shown, in the rat, that fibroblasts o f c o n t r a c t i n g granulation tissues come to resemble s m o o t h muscle cells? -4 This resemblance is m o r p h o l o g i c : pltarmacologic, 2 chemical, -~ and antigenic? We have called these cells "myofibroblasts" and have p r o p o s e d that it is by their active contraction that granulation tissue s h r i n k s - w h e t h e r to close a w o u n d , constrict a lumen, or d e f o r m a heart valve? This p a p e r describes the presence o f myofibroblasts in h u m a n granulation tissues.
MATERIALS AND METHODS
Clinical Material Biopsy samples o f granulation tissue were obtained f r o m f o u r patients r e f e r r e d to the Plastic S u r g e r y Unit at the Cantonal Hospital o f Geneva: CASE 1. A 53 )'ear old man had a total laryngectomy with a permanent tracheostomy for a carcinonm of the larynx. Ten days later the wound above the tracheostomy broke down, resuhing in a pharyngeal fistula that drained saliva to the outside of the neck. Two months after the initial operation, the fistula was closed using a local skin flap to recreate the pharyngeal mucosa and a dehopectoral flap for the surface skin defect; at this operation we obtained a specimen of granulation tissue from the external opening of the fistula. Four weeks later the base of the deltopectoral flap was restored to its normal position, and we obtained another sample of tissue, this time from the granulating wound on the chest. CASE 2. A 41 )'ear old woman had a bilateral subcutaneous nmstectomy for fibrocystic disease. At the same operation, a Silastic mamnmry prosthesis* was implanted on each side. Two weeks later a henmtoma had developed on top of the right prosthesis; this was aspirated several times over the next few weeks. Two months after the initial procedure there was no more fluid accunmlation, but the prosthesis began to show progressive upward displacement and the patient complained of local tightness and pain. These symptoms persisted and the patient was again submitted to surgery 10 months later. The prosthesis was completely surrounded by a capsule that was 4 to 5 ram. tlfick inferiorly but only I mm. thick superiorly. Tiffs capsule was partially removed 56
*Cronin type, Dow Corning, Midland, Michigan.
(providing us with specimens) and tim implant was set back in the original cavity. CASE 3. A 47 )ear old woman had a bilateral subcutaneous mastectomy for fibrocystic disease. At the same operation a silicone Natural-Y mammary prosthesist was implanted on each side. The postoperative course was complicated by skin necrosis, 3 cm. in diameter, in the left areolar region; tiffs necessitated removal of tile prosthesis (which was not noticeably displaced) after four weeks. We obtained a sample of granulation tissue from around the prosthesis at this stage. CASE 4. A 34 )'ear old woman with a grossly pendulous abdomen following pregnancy lind a subcutaneous lipectomy and a plasty of the rectus abdolninis muscles. One week later she developed a collection of fluid under the wound. This was opened, leaving a round wound 4 cm. in diameter tlmt was allowed to heal by second intention. A biopsy was taken from granulation tissue at the base of the wound four weeks after the initial procedure. NORMAL SKIN'. Biopsy samples were obtained from two patients: one was from the wrist of a 27 )'ear old man undergoing surgery for a carpal tunnel syndrome; the other was taken from the abdomen of a 32 )'ear old alcoholic male during an operation to implant a disulfiram (Antabuse) pellet.
Histologic Examination Tissues for histologic examination were fixed in 10 per cent buffered f o r m a lin, d e h y d r a t e d , and e m b e d d e d in paraffin. Sections were stained with hematoxylineosin, periodic acid-Sclfiff, Masson trichronae, cresyl violet, Mallory's p h o s p h o tungstic acid, and van Gieson stains.
Electron Microscopic Examination Tissues for electron microscopy were cut into cubes o f 1 ram. or less, a n d fixed at r o o m t e m p e r a t u r e by immersion for five h o u r s in 3 per cent g l u t a r a l d e h y d e in cacodylate buffer at pH 7.2 to 7.4. The), were left in buffer at 4 ~ C. overnight, postfixed at 4 ~ C. for two hours in 2 per cent o s m i u m tetroxide in collidine buffer at p H 7.3 to 7.4, d e h y d r a t e d in alcohol, and e m b e d d e d in E p o n 812. Sections approximate'ly lp. tlfick were cut with glass knives on a Reichert u l t r a m i c r o t o m e or LKB tAshley type, Edward .}Veck & Company, Long Island City, N.Y.
MYOFIBROBLASTS IN HUMAN GRANULATION TISSUE-RvAN VT AL. Ultratome and stained with naethylene blue and azur II. Thin sections, were cut with diamond knives, mounted on noncoated grids, stained with uranyl acetate and lead citrate, coated with a tlfin layer of carbon, and examined with a Plfilips 300 electron microscope.
hnm unofluorescence Human antismooth muscle serum ("smooth muscle autoantibody" serum) was obtained from a 16 )'ear old girl with active chronic hepatitis, a Tiffs serum reacted with smooth muscle of rat stonaach at dilutions up to 1/320; tests for antimitochondrial and anti-DNA antibodies and for antinuclear factor were negative. To demonstrate the presence of s|nooth muscle antigens, the double layer technique was used) Unfixed cryostat sections of tissue were treated for 30 minutes with the smooth muscle autoantibody sertun (diluted 1 part in 20 parts of phosplmte buffered saline). The sections were then washed in phosphate buffered saline and stained for 30 minutes with fluoresceinconjugated IgG fraction of goat antiserum to I|uman IgG.* After rewaslfing with phospliate buffered saline and mounting in 90 per cent glycerol in pl|osphate buffered saline, the level of fluorescence was compared with that in control sections treated with normal human serum instead of smooth muscle antoantibody serum. Photographs were taken on a Zeiss UV photomicroscope with a UGI or II excitor filter and a Zeiss No. 50/65 barrier filter, using Anscochrome color slide film (500 daylight). The same sections were then stained with l|enmtoxylin and eosin and rephotographed with visible light.
Contractility in Vitro Snmll strips of tissue (with dimensions of approximately 1 by 1 by 12 mm. and weighing I0 to 15 nag.) were tested plmrmacologically in a specially devised microchamber. In tlfis system the tissue was suspended vertically (by a 6-0 silk suture) from a fixed, rigid horizontal post, and its lower end was attached (also by 6-0 silk) to a point near the freely mobile tip of a very *Code 64-170, Miles-Seravac,Lausanne,Switzerland.
finely drawn, flexible glass filament. The The whole assembly was bathed in 10 nil. of Tyrode's solution that was contint|ally bubbled with 95 per cent oxygen plus 5 per cent carbon dioxide, and kept at a constant 37 ~ C. by a radiant heat source. The tip of the glass filament was observed through a light microscope, and contraction or relaxation of the strip of tissue was detected vist|ally by the movement of the tip of the filament in relation to the graduatious of a measuring ocular. Drugs were added via a hole in the top of the chamber; these included angiotensin (Hypertensin), epinephrine hydrochloride (Adrenalin) and papaverine hydrochloride, norepinephrine (Arterenol), lfistamine dihydrochloride, serotonin creatinine sulfate (5I|ydroxytryptanfine) and dimethyl sulfoxide (DMSO), vasopressin, prostaglandins Ft a and E~, promethazine I|ydrochloride (I'henergan), diphenhydramine hydrochloride (Benadryl), chlorpheniramine maleate (Chlor-Trimeton), a 4:1 mixture of oxytheophylline and tlaeophylline (Cordaline), cytoclmlasin B, and potassium cyanide.
RESULTS
HistoIogic Examination In each case light microscopic stud)' showed the granulation tissue to consist mainly of "fibroblasts," small blood vessels, scattered neutrophilic pol)'morplmnuclear leukocytes, mononuclear cells, and nmcropimges (Fig. 1). The fibroblasts lind pale, oval, or elongated nuclei and abundant cytoplasm with long extensions; some nuclei showed cross banding like that already reported in the myofibroblasts of Du.puytren's contracture3 No other indication as to the contractile nature of these cells was given by histologic study, as expected.
Electron Microscopic Examination Control fibroblasts (from normal humall dermis) showed morphology conforming with standard descriptions: s In typical cells the nuclear contour was smooth and the cytoplasm contained prominent cisternae of rough endoplasmic
57
Jamtmy1974
H U M A N P A T H O L O G Y - - V O L U M E 5, NUMBER 1
Figure 1. Light micrograph of human granulation tissue showing "fibroblasts," small blood vessels, and inllammator)" cells. Case 4. (tlematoxylin and eosin stain, x 160.)
features to distinguish it from a conventional f i b r o b l a s t - i n d e e d , in some ways, it resembled a smooth muscle cell: 1. Massive bundles of long intracytoplasmic microfilaments (Figs9 3 to 6). Most o f t h e microfilaments measured approxi-
reticulum and some mitochondria. Intracytoplasmic microfilaments were few in n u m b e r (Fig. 2). In all the h u m a n granulation tissues examined we f o u n d that the p r e d o m i n a n t connective tissue-cell had three striking
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MYOFIBROBLASTS IN HUMAN GRANULATION TISSUE--RYAXETAL.
Figure 3. Electron micrograph of myofibroblast in Imman granulation tissue showing abundant rough endoplasmic reticuluna and multiple smooth muscle-like cytoplasmic microfilaments arranged in discrete bundles (arrows), one of which lies in a nuclear fold. (Case 1, first biopsy. X 10,500.)
mately 50/~ in diameter; in addition, there were some larger ones, a p p r o x i m a t e l y 100 /~ in diameter, as well as scattered microtubules, a p p r o x i m a t e l y 220/~, in diameter. T h e thin and thick microfilaments were aggregated into bundles tlmt usually ran parallel to the long axis o f the ceil. Many "dense bodies" (or " a t t a c h m e n t sites") were present in the bundles. In most cells the bundles were large and multiple and were distributed like discrete "lnuscles" in different parts o f the cell. Sometimes, however, the bundles were confluent, tlms giving an a p p e a r a n c e very like a classic smooth muscle c e l l - e x c e p t for the presence o f a b u n d a n t cisternae o f r o u g h endoplasmic reticulum (typical o f fibroblasts) elsewhere in the cytoplasm (Figs. 3 to 6). Occasional cells had only one o r two narrow bundles; in such cases the bundles were usually located just beneath the cell m e m b r a n e . T h e only fibroblast seen in mitosis (in case 3) had very few microfilaments.
2. Nuclear deformations. Many o f the nuclei o f these "muscular" fibroblasts had multiple indentations o1" d e e p folds (Fig. 3). 3. Surface differentiation. Intel'cellular connections between such cells were frequent: these were o f the macula a d h e r e n s ("desnaosonle") and nexus ("gap" junction) types (Figs. 7, 9). 9 In additiou, material resetnbling basal lamina was often f o u n d close to the cell surface (Figs. 7, 8, 10). In such cases, there was sometimes a dense zone in the microfilam e n t b u n d l e immediately beneath the plasmalemma, thus r e s u h i n g in a henaidesmosome-like complex (Fig. 10). T h e basal lamina material was not u n c o m m o n l y seen r u n n i n g off into the stroma away fi'om the cell (usually in a direction parallel to the intracytoplasmic' microfilaments) and sometimes c o n n e c t i n g with the basal lamina a r o u n d a n o t h e r cell. Although we have not formally e x c l u d e d the possibility that a g1:azing section n e a r a cell surface (Text continued on page 63.)
59
H U M A N I ' A T H O L O G Y - - V O L U M E 5, NUMBER 1
Janua0'1974
Figure 4. Two large intracellular "muscles" (stars) fanning through cytoplasm of m)ofibroblast and separated b)' endoplasmic reticulum. (Case 1, first biopsy, x 15,200.)
60
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Detail of myofibroblast; ,rote "dense bodies" (arrows) among microfilament bundles. (Case 4.
Figure 6. Detail of two microfilamem bundles separated by endoplasmir reticulum; note microtubule (arrow). (Case 4. • 27,500.)
61
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62
Figure 8. Presuulptive connections o f myofibroblasts to stroma via basal lamina-like "microtendons" (arrows). (Case 4. x 25,.500.)
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Figure 10. Basal lalnina material (Sial') applied to surface of myofibroblast; note dense zones (arrows) beneath plasmalemlna, resuhing iq hemidesmosome-like complexes. (Case 1, first biopsy. x 43,000.)
is responsible for this "microtendinous" appearance (Fig. 8), it is most likely that such structures participate in some way in the transmission of cellular pull to other tissue compouents.
and case 4). Tile pooled restdts are as follows. Contraction was induced with prostaglandin Fia 1 x I0 -r to I x 10 -~ gln./ ml., final concentration in bath (Fig. 12 shows the dose-response cnrve), angiotensin 1 x 10 -6 gm./ml., vasopressin 0.01 to 0.1 I.U./ml., promethazine hydrochloride I x 10 -5 gm.hnl., d i p h e n l w d r a m i n e hydrochloride 1 • 10 -3 gm./ml., and cldorpheniramiue maleate 1 x 10 -4 gm./ml. No significant contraction occurred with 5h y d r o x y t r y p t a m i n e 1 x 10 -5 gm./ml. (as was fotmd with rat skiu wounds, a h h o u g h other kinds o f rat granulation tissue gave good responses'-"4), histainine 1 x 10 -~ gin./ml., epinephrine 1 x 10 -7 gm./ml., or norepinephrine 1 • 10 -5 gm./ml. Relaxation was induced with prostaglandin E1 1 x 10 -5 gm./ml. (Fig. 12), Cordaline (a theophylline preparation) 1 • 10 -3 gin./ ml., papaverine 1 x 10 -4 gm./ml., and potassitnn cyanide 1 x 10 -a gm./ml. Cytochalasin B 1 • 10 -5 gm./nfl, also caused relaxation; DMSO alone did not affect strips in the concentration used as a sol~'ent for cytochalasin B (1 per cent D*ISO, final concentration in the bath). Control strips o f normal dermis and subcutaneous tissue neither contracted nor relaxed with any o f the foregoing agents.
ImmunoJhtore$cence All the samples of h u m a n granulation tissue showed striking cellular fluorescence after treatment with h u m a n antismooth muscle serum (Fig. 11). This labeling occurred in the cytoplasm o f elongated cells, presumably corresponding to the smooth muscle-like cells seen by electron microscopic examination. No labeling occurred after treatment with normal htnnan serum (Fig. 1 I). Control sections of normal skin treated with antismooth muscle serum showed labeling of vasctflar smooth muscle but not of fibroblasts.
Contractility in Vitro Strips of granulation tissue were tested pharmacologically (like strips of smooth muscle) when sufficient biopsy material was obtained (case 1 - b o t h biopsies, case 2,
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HUMAN PATHOLOGY--VOLUME 5, NUMBER 1
Janumy1974
A
Figure 1l. Fluorescence microscopy of human granulation tisstte. A, Control section treated with normal human serum followed by antilmlnan lgG: no specific labeling. (x 160.)B, Section treated with Imman antismooth muscle sermn followed b)" antihuman lgG: bright specific fluorescence of myofibroblasts. (• 160.) C, Higher power view of cytoplasmic labeling of inyofibroblasts. (• 500.) (Case 1, first biopsy.)
DISCUSSION
64
Tiffs study shows that in h u m a n granulation tissue fibroblasts develop cytoplasmic features very similar to those o f smooth muscle: by electron microscopic examination, a h h o u g h retaining the areas o f a b u n d a n t endoplasmic reticulum typical o f fibroblasts, it was observed that the)' become e q u i p p e d with nmssive bnndles o f intracytoplasmic microfilaments (with scattered "dense bodies"). F u r t h e r m o r e , as d e m o n s t r a t e d by i m m u n o f l u o r e s c e n c e , they contain antigenic material characteristic o f smooth muscle cells. We have earlier p r o p o s e d that such cells, wltich we call "myofibroblasts," are
responsible for tlte contraction o f granulation tissue in e x p e r i m e n t a l animals. H T h e present work indicates tltat the same may be true for l m m a n granulation tissue: 1. T h e nuclear indentations and folds give indirect evidence o f contraction: tile)" mimic tile nuclear changes in c o n t r a c t e d smooth muscle, 1~myocardium, 11 and venular e n d o t h e l i u m ) 2 9. T h e surface differentiations give a means w h e r e b y cellular contraction could explain an overall shrinkage o f the tissue: myofibroblasts connect with one a n o t h e r (via junctions o f the macula a d h e r e n s and nexus types a) and with tile stroma (via attachments to basal lamina material). 3. W h e n tested pharmacologically,
MYOFIBROBLASTS IN IIUMAN GRANULATION TISSUE-RYAx ETAL.
.
HUMAN GRANULATION TISSUE EFFECT OF P R O S T A G L A N D I N S F lo~ AND E 1
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.
Figure 12. Contraction shown in vitro by strip of human granulation tissue in response to increasing doses of prostaglandin Fta (1 x i0 -7 to ! x 10-s gin./ ml.), followed b)" relaxation in response to prostaglandin E 1 (1 X 10-5 gm./ml.), (Case 1, first biopsy.)
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strips o f granulation tissue behave similarly to smooth muscle: they contract with certain smooth muscle stimulants and relax with smooth muscle relaxants. H u m a n granulation tissue contraction can t h e r e f o r e be explained on the following basis: Fibroblasts progressively develop intracytoplasmic "muscles" as well as cell-to-cell and cell-to-stroma connections; these myofibroblasts contract (either spontaneously o r in response to endogenous mediators) at the same time that collagen is laid down (thus providing a "lock-step" system) and so tile whole tissue shrinks. In o t h e r words, granulation tissue becomes a contractile o r g a n ? This, o f course, is o f p r i m a r y i m p o r t a n c e in closing a skin wound, but in o t h e r circumstances it can be disastrous. O u r case 2 (tile patient in whom t h e r e was displacement o f a breast prosthesis) is a relatively m i n o r example. More serious are such problems as postburn disfigurements and postinflammatory luminal strictures. It is also likely that the vah, ular deformities o f chronic r h e u m a t i c heart disease are d u e to a similar process; this is a particularly interesting example because it illustrates how a p p a r e n t l y logical was the old belief/3
(now o u t m o d e d 14) that attributed connective tissue contraction to collagen shrinkage: normal vah'es and c h o r d a e consist o f m a t n r e collagen and little else, and so also do chronically d e f o r m e d vah'es and c h o r d a e - h o w e v e r , pathologists do not often see tile stage in between, i.e., when there is invoh'ement o f these structures by active granulation tissue. Cells uhrastructurally similar to myofibroblasts have been seen in certain o t h e r h u m a n tissues: aortic intimal thickenings, 15 ganglia o f the wrist, 1G cirrhosis o f the liver, t7 and the p a h n a r nodules o f Dupuytren's disease3 T h e significance o f myofibroblasts in t h e s e o t h e r tissues is not clear, apart f r o m D u p u y t r e n ' s disease in which they are ahnost certainly responsible for the traction d e f o r m i t y o f the fingers. We have recently shown that the cells o f D u p u y t r e n ' s nodules are specificall)" labeled with antismooth muscle serum. In this context it should be stressed that we d o not p r o p o s e that D u p u y t r e n ' s nodules are c o m p o s e d o f reactive granulation tissue; D u p u y t r e n ' s disease is one o f the so-called "fibromatoses" for which the causes are u n k n o w n ) s Why have myofibi'oblasts not already
65
H U M A N I ' A T H O L O G Y - - V O L U M E 5, NUMBER 1 Jammr'r 1974
66
b e e n d e s c r i b e d in h n n m n g r a n u l a t i o n tissue? Ross a n d O d l a n d t9 e x a m i n e d the h e a l i n g o f skin w o u n d s in I m m a n subjects w i t h o u t o b s e r v i n g myofibroblasts. But these w e r e small linear w o u n d s , a n d so the d i s c r e p a n c y is u n d e r s t a n d a b l e : w h e n c o m p a r e d with o p e n w o u n d s , linear w o u n d s heal with little o r n o c o n t r a c t i o n . T h i s leads us to t h r e e m o r e q u e s t i o n s : W h a t is t h e o r i g i n o f myofibroblasts? W h a t are the stimuli f o r their f o r m a t i o n ? W h a t l m p p e n s to t h e m ? N o n e o f these q u e s t i o n s can be a n s w e r e d yet. It is p r o b a b l e that the m y o fibroblasts arise locally fi'om m o r e c o n ventional fibroblasts o r fl'om primitive m e s e n c h y m a l cells. A d e r i v a t i o n f r o m vascttlar s m o o t h m u s c l e cells is also a possibility, b u t we t h i n k tlfis unlikely b e c a u s e in e x a n f i n i n g t h e earl)" d e v e l o p m e n t o f g r a n u l a t i o n tissue in e x p e r i m e n tal animals, we f o u n d a p r o g r e s s i v e acc u m u l a t i o n o f m i c r o f i l a m e n t s in cells that w e r e fibroblastic in a p p e a r a n c e ( r a t h e r tlmn a loss o f m i c r o f i l a m e n t s f r o m s m o o t h m u s c l e cells: i n d e e d , t r u e s m o o t h ntuscle cells w e r e n o t seen o u t s i d e b l o o d vessel walls)? "a F u r t h e r e v i d e n c e f o r a fibroblastic o r i g i n o f m y o f i b r o b l a s t s is that fibroblasts cultivated in vitro d e v e l o p extensive i n t r a c y t o p l a s m i c fibrillar systems "-'~ a n d intercellular c o n n e c t i o n Q t virtually identical to t h o s e o f g r a n u l a t i o n tissue myofibroblasts. W e have recently c o n f i r m e d that the s a m e u h r a s t r n c t u r h l c h a n g e s o c c u r in cells g r o w i n g o u t fi'om e x p l a n t c u l t u r e s o f a d u l t rat dermis. M o r e over, we have f o u n d cytoplasntic "streaks" ( p r e s u m a b l y c o r r e s p o n d i n g to " m u s c l e " btmdles) w h e n such cells a r e stained with a n t i s m o o t h muscle s e r u m . It seems, t h e n , t h a t w h e n fibroblasts a r e suitably trigg e r e d ( w h e t h e r in a w o u n d o r by b e i n g placed in vitro), t h e y d e v e l o p a m u s c u l a r a p p a r a t u s that enables t h e m to contract. It will be i m p o r t a n t to find o u t h o w tiffs t r i g g e r i n g o c c u r s a n d w h e t h e r it can be m o d i f i e d in s o m e way. Finally, we d o n o t yet k n o w w h a t h a p p e n s to myofibroblasts: p r e s u m a b l y they die a n d d i s i n t e g r a t e wlten c o n t r a c t i o n is cotnpleted. T h e m e c h a n i s n t f o r this is o b s c u r e , b u t it is also i m p o r t a n t to investigate b e c a u s e a b n o r m a l persiste n c e o f myofibroblasts ntay be r e s p o n s i b l e f o r c o n v e r t i n g a beneficial w o u n d c o n t r a c tion to a n u n e s t h e t i c o r c r i p p l i n g c o n tracture.
ACKNO~,VLEDGMENTS
W e t h a n k D o c t o r s A. C r u c h a u d a n d I. N i c o d o f the C a n t o n a l Hospital o f G e n e v a f o r kindly p r o v i d i n g samples o f antisera; Dr. J. E. Pike o f the U p j o h n Co., Kalam a z o o , Michigan, f o r samples o f prostag l a n d i n s Fto~ a n d Et; Miss M. C. Clottu, Miss A. L o m e t t o , a n d Mrs. A. Fiaux f o r excellent technical help; a n d Messrs. J. C. R u m b e l i a n d E. D e n k i n g e r f o r p h o t o g r a p h i c work.
REFERENCES
1. Gabbiani, G., Ryan, G. B., and Majno, G.: Presence of modified fibroblasts in granulation tissue and their possible role in wotlnd contraction. Experientia, 27:549, 1971. 2. Majno, G., Gabbiani, G., ttirschel, 11. J., Ryan, G. B., and Statkov, P. R.: Contraction of granulation tissue in vitro: similarity to smooth muscle. Science, 173:548, 197 I. 3. llirscfiel, B. J., Gabbiani, G., Ryan, G. B., and Majno, G.: Fibroblasts of granulation tissue: immunofluorescent staining with antismooth muscle serum. Proc. Soc. Exp. Biol. Med., 138:'166, 1971. 4. Gabbiani, G., Hirschel, B. J., Ryan, G. B., Statkov, 1'. R., and Majno, G.: Granulation tissue as a conlractile organ: a study of structure and fimction.J. Exp. Med., 135:719, 1972. 5. Nairn, R. C.: Fluorescent Protein Tracing. Ed. 3. Edinburgh, E. & S. Livingstone Ltd., 1969. 6. Cliff, W. J., Statkov, 1'. R., and Majno, G.: A simple technique for pharmacological studies of small strips of contractile tissue: the "inicrobath.'" Unpublished study. 7. Gabblani, G., and Majno, G.: Dul)uytrcn's contracture: fibroblast contraction? An ultrastructural study. Amer. J. l'athol., 66:131, 1972. 8. Ross, R.: The connective tissue fiber forming" cell. In Gould, B. S. (Editor): Treatise on Collagen. '2. Biology of Collagcn (Pt. A). New York, Academic Press, Inc., 1968, 1I. I. 9. Weinstein, R. S., and McNntt, N. S.: Cell junctions. New Eng. J. Med., 286:521, 1972. 10. l.ane, B. P.: Alterations in the cytologic detail of intestinal smooth muscle in various stages of contraction. J. Cell Biol., 27:199, 1965. 11. Bloom, S., and Cancilla, P. A.: Conformational changes in ntyocardial nuclei of rats. Circ. Res., 24:189, 1969. 12. Majno, G., Shea, S. M., and l.eventhal, M.: Endothelial contraction induced by histaminetype mediators: an electron microscopic stud)'. J. Cell Biol., 42:647, 1969. 13. l'ayling Wright, G.: An Introduction to Pathology. Ed. 2. l.ondon, I.ongmans Green & Co., l.td., 1954, p. 219. 14. Abercrombie, M., Flint, M. H., and James, D. W.: Wound contraction in relation to collagen formation in scorbutic guinea pigs. J. Embryol. Exp. Morph., 4:167, 1956.
MYOFIBROBLASTS
IN H U M A N
15. Geer, J. C.: Fine structure of h u n m n aortic intimal thickening and fatty streaks. Lab. Invest., 14:1764, 1965. 16. Ghadially, F. N., and Mehta, I'. N.: Muhifimctional mesenchylnal cells resembling smooth muscle cells in ganglia of the wrist. Ann. Rlleum. Dis., 30:31, 1971. 17. Bhathal, I'. S.: Presence of modified fibroblasts in cirrhotic livers in man. l'athology, 4:i39, 1972. 18. Enzinger, F. N., Lattes, R., and Torloni, l-i.: Types histologiques des Tumeurs des Tissus
GRANULATION
TISSUE-RvA,~
ET AL.
Mous. Geneva, World ttealth Organization, 1970, p. 28. 19. Ross, R., and Odland, G.: t t u m a n wound repair. II. hdlamnlatory ceils, epitllelial-nlesencllymal interrelations, and fibrogenesis. J. Cell I',iol., 39:152, 1968. 2(1. Goldberg, B., and Green, It.: An analysis of collagen secretion by established mouse fibroblast lines. J. Cell Biol., 22:227, 1964. 21. Devis, R., and James, D. W.: Close associations between adult guinea-pig fibroblasts in tissue cnlture, studied with the electron microscope. J. Anat., 98:63, 1964. Department of Pathology University of Geneva 40 Boulevard de la Cluse 1211 Geneva 4, Switzerland (Dr. Majno)
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