ELLIS E T AL.: HEALING OF PERITONEUM 471

THE HEALING OF PERITONEUM UNDER NORMAL AND PATHOLOGICAL CONDITIONS

BY HAROLD ELLIS PROFESSOR OP SURGERY, WESTMINSTXR HOSPITAL

WALFORD HARRISON SENIOR REGISTRAR I N PATHOLOGY, WESTMINSTER HOSPITAL

AND T. B. HUGH SURGICAL REGISTRAR, WGSTMINSTER HOSPITAL

IT has been widely held that, following the destruc- tion of an area of peritoneal mesothelium, repair is effected by the proliferation of surrounding meso- thelial cells and their inward migration from the margins of the damaged area (Le Gros Clark, 1958). However, this hypothesis was based not on experi- mental observations but on analogy with ‘centripetal healing’, which is readily demonstrable in skin and rectal mucous membrane.

Serious doubt was first cast on this concept by the work of Robbins, Brunschwig, and Foote (1949), who investigated the healing of parietal peritoneum in dogs and suggested that the new serosa formed not only by ingrowth from the undamaged serosa at the peri- phery but also by transformation of fibroblasts which had migrated upwards from the base of the wound. Williams (r955), studying the healing of parietal defects in rabbits, believed that the new mesothelium arose in the main from underlying connective-tissue cells, since healing was rapid and simultaneous all over the area of the wound.

Cameron, Hassan, and De (1957), studying the healing of tangential liver wounds in rats, observed tiny islands of cells on the surface of the wounds within 24 hours of injury and believed that these were mesothelial cells which, after becoming detached from nearby peritoneum in contact with the raw area, acted as grafts. Proliferating cells appeared to spread out from these islands to form most of the new meso- thelium, although some healing also took place from mesothelium at thc periphery of the wound.

Johnson and Whitting (1962) and Bridges and Whitting (1964) investigated the repair of parietal peritoneum in the rabbit and rat respectively. They supported the conclusions of Cameron and others (1957), but also suggested that in some circumstances macrophages might contribute to the healing process by transforming into mesothelial cells.

The present investigation was undertaken in the hope of clarifying a subject which had received relatively little attention in proportion to its impor- tance in surgical practice, and on which widely divergent views are held as to the origin of the new serosal layer. Moreover, in contrast to the extensive studies on healing of skin and subcutaneous tissues, no experimental investigations have been carried out on factors which may affect peritoneal healing, a sub- ject of considerable importance in the healing of laparotomy wounds and peritoneal defects which are left after operations such as excision of the colon.

We here report studies on the effect on this process of age, cortisone in high dosage, diabetes, and scurvy.

METHODS I. Healing Process in Normal Adult Rats.-

Ninety-three male rats were operated upon under open ether. Strict asepsis was not observed but no case became infected. The abdomen was opened by a midline incision and on either side a piece of parietal peritoneum varying from 0.5 to 2 cm. square was excised together with a sliver of underlying muscle. Twenty of these animals were stained intravitally with trypan blue, 6-12 ml. of a I per cent solution of the dye being injected subcutaneously in divided doses during the 5 days before operation.

In another group of 11 animals a thin square sheet of sterile polythene was held in place over the defect on one side by an atraumatic silk suture at each corner, while on the other side the defect was left uncovered. Both defects were covered by polythenc in a further 18 rats, while in 3 animals patches were sewn over intact peritoneum.

The rats were sacrificed at 12 and 24 hours, then daily for 5 days and at gradually longer intervals for a further 3 weeks. After careful inspection for the presence of adhesions, the defects were excised to- gether with the underlying abdominal muscle and a ring of intact peritoneum, The specimens were pinned flat on cork and fixed in a 10 per cent formol saline. They were then bisected through the centre of the defect and the tissue processed and embedded in paraffin. Sections were cut at 5 p, and ten sections at 50 p intervals were stained with haematoxylin and eosin. Adjacent sections were stained with phospho- tungstic acid haematoxylin, van Gieson, 0.1 per cent aqueous toluidine blue, and by the silver impreg- nation method of Gordon and Sweet. In 18 animals the defects were not excised. Instead a small spatula was drawn very lightly across each defect and smears made on glass slides. Smears were also taken from normal peritoneum. The slides were fixed in alcohokther and stained by Papanicolau’s method.

2. Healing Process in Immature Animals.- Nine 4-week-old male Wistar rats of average weight IOO g. were used. Bilateral defects measuring 0.5 cm. square were created on each flank wall after laparo- tomy under ether anaesthesia. The animals were sacrificed and the defects prepared for study as described above.

472 BRIT. J. SURG., 1965, Vol. 52, No. 6, JUNE

3. Healing under Pathological Conditions.- a. Cortisone.-Nine male Wistar rats weighing

200-250 g. were given I mg. of cortisone acetate subcutaneously for 2 days prior to laparotomy and I mg. daily thereafter. One-centimetre-square defects were fashioned. The animals were sacrificed and prepared as described in section I .

b. Diabetes.-Seven male Wistar rats weighing 200-250 g. were used. Diabetes mellitus was induced as described by Rosen and Enquist (1961) using alloxan (150 mg. per kilo) subcutaneously in rats

a glistening but slightly irregular surface. The colour gradually faded, the surface became quite smooth, and by the end of the first week healing appeared to be complete. The operation area remained visible for a further 2 weeks as a somewhat opaque ‘milky’ patch in an otherwise continuous glistening sheet of peri- toneum, but after this time it became difficult to distinguish from its surrounds. Adhesions were con- fined to 15 out of 133 uncovered defects.

In 43 out of the 47 polythene-covered defects the surface of the polythene was partially or completely

FIG. r.-Normal adult rat, defect a t 12 hours. Fibrinous exudate infiltrated mainly by monocytes and histiocytes. Muscle- fibres at base, necrotic muscle-fibre on right. H. and E. ( x 575.)

which had been fasting for 48 hours. Thereafter 1.5 units of protamine zinc insulin was given sub- cutaneously daily to prevent cachexia and dehydra- tion. Blood-sugar levels remained at over 300 mg. per cent (65-135 mg. per cent in normal controls).

Peritoneal defects I cm. square were created z days after administration of alloxan, the animals sacrificed daily, and the defect prepared for investigation as already described.

c. Scurvy.-Since vitamin-C deficiency is virtually impossible to produce in the rat, this factor was studied in the guinea-pig.

Eleven guinea-pigs, weighing 4-450 g. before the experiment, were given a diet of 70 per cent crushed oats, 25 per cent bran, and 5 per cent skim milk with added iron and vitamins A and D. After 16 days on this diet, peritoneal defects were made on each flank wall with full aseptic precautions. At the time of laparotomy all of these animals had clinical signs of scurvy. Animals surviving longer than 5 days after operation were given 0.5 mg. of ascorbic acid by pipette on alternate days to reduce the high mortality.

In 6 control guinea-pigs on a normal diet with added vitamin C similar defects were made.

In each group the animals were sacrificed at 24,48, and 72 hours, and then at gradually longer intervals up to 3 weeks. The defects were prepared in a similar manner to the rat series.

RESULTS I. Normal Adult Rats- Gross Appearances.-During the first 2 days the

uncovered peritoneal wounds were red in colour with

FIG. z.--Normal adult rat, defect at 24 hours. Scanty exudate with monocytes, histiocytes, and pol morphs Some of the larger cells on the surface may be mesotgelid. H. and E. ( x 575.)

obscured by adhesions which appeared to develop initially to the sutures at each comer. The margins of the polythene were applied to the parietal peri- toneum between the sutures except in 6 cases where adhesions were found beneath the polythene. In 2 animals (at 16 and 21 days) the polythene on either side lay free in a cyst formed between heaped-up adhesions and the surface of the wound. The four defects without adhesions were all examined within 3 days of operation.

Histological Appearances of Uncovered Defects.- At 12 and 24 hours the smaller defects were covered by a fibrinous exudate through which red cells and nucleated cells were evenly scattered, being equally numerous in the deeper, the more superficial, and surface layers (Fig. I). However, in some areas there was a thin, acellular layer of fibrin which separated the cells on its free surface from the main cellular reaction in the base of the wound and within the exudate. The base of the wound consisted of de- generate muscle-fibres, and the perimysial reticulin framework with its related fibroblasts was clearly seen in silver-impregnated sections. In the larger defects the overlying exudate tended to be scanty and the wound surface was often formed by necrotic muscle- fibres (Fig. 2). The ‘cellular reaction’ consisted of mononuclear cells, polymorphonuclear leucocytes, and smaller numbers of lymphocytes, mast cells, and eosinophils. The mononuclear cells were of two main types, although others of uncertain identity, possibly primitive mesenchymal cells, were present. Cells of the first type were readily identifiable as histiocytes, which in the vitally stained animals had cytoplasm heavily laden with coarse trypan blue granules. These

ELLIS E T AL.: HEALING OF PERITONEUM 473

cells were plentiful throughout the exudate filling the wound and between muscle-fibres for some distance around the defect. The second type of mononuclear cell was most numerous in the more superficial layers of the exudate. Small trypan blue granules were occasionally seen but phagocytosis of debris was not observed. I t was concluded that these cells were blood monocytes.

About 50 per cent of the surface was covered by cells, leaving intervening areas of bare fibrin. Many of the surface cells closelyresembled monocytes or, where

FIG. 3.-Normal adult rat, defect at 48 hours. Almost con- tinuous surface layer of cells resembling monocytes. H. and E. ( X 57s.)

confident identification was not possible, could be matched with similar mononuclear cells of uncertain type within the exudate. Scattered polymorpho- nuclear leucocytes were also present, together with occasional cells similar to those of mesothelium. Adjacent, intact peritoneum showed no change at this stage and mitoscs wcre rarely seen either here, within the exudate, or on its surface.

At 48 hours much of the surface of the exudate was covered by cells which formed an uninterrupted layer in some areas (Fig. 3). As at 24 hours the majority of these cells resembled monocytes while the larger mesothelial-like cells formed a small proportion of the total. Within the exudate histiocytes and mono- cytes predominated, with smaller numbers of poly- morphonuclears and occasional mast cells also present. At this stage mesothelial cells in the adjacent un- damaged serosa were becoming rounded and plumper and their cytoplasm more basophilic. Young fibro- blasts were by now appearing in the base of the wound. Cells in mitosis were seen on the surface, within the exudate, and in mesothelial cells just beyond the wound margins.

By the third day dramatic changes had occurred (Fig. 4). The wound surface was covered by a con- tinuous layer of cells, which now resembled the active mesothelial cells at the edge of the defect with which they formed a continuous layer. Trypan blue could no longer be identified in any of these surface cells. Numerous fibroblasts were now advancing from the base and sides of the defect and as organization of the exudate progressed during the next few days the fibrin was absorbed and histiocytes and monocytes dwindled in numbers. There were fairly numerous mitotic figures on the wound surface, within the exudate, and in cells of the intact mesothelium at the wound edges.

At 4 days the new surface layer was more uniformly flattened and the cells now closely resembled the underlying fibroblasts which could be identified with some confidence because they were closely surrounded by reticulin fibres. Both types of cell still showed fairly numerous mitoses.

B y rhe fifrh day (Fig. 5) the new mesothelium and the adjacent undamaged mesothelium formed a con- tinuous layer of flattened cells identical in appearance in all cases, so that their junction could not be identi- fied. These cells became increasingly similar to

FIG. 4.-Normal adult rat, defect at 72 hours. Surface layer of flattened cells morphologically resembling both active mesothelial cells and fibroblasts. H. and E. ( x 575.)

FIG. s.--Normal adult rat defect at 5 days. Surface layer of adjacent normal peritoneum. flattened cells closely resemblin

Thick layer of underlying fibrobfasts. H. and E. ( x 290.)

normal, inactive mesothelium and by 7-10 days mitotic figures were no longer seen. The granulation tissue became less vascular and fibroblasts replaced the histiocytes when absorption of the fibrin and of damaged muscle-fibres was complete. Collagen was laid down and after 2 weeks a thin layer of fibrous tissue covered by mesothelium was left at the site of the original defect. Later sections show little further change, although the fibrous tissue appeared to con- tract and to reduce the size of the scar considerably.

Microscopy of Peritoneal Smears.- a . Normal peritoneum: Cells were scanty but were

predominantly of mesothelial type with smaller numbers of polymorphonuclear leucocytes, mono- cytes, lymphocytes, and mast cells. The mesothelial cells were large, with oval or round nuclei, in which the

474 BRIT. 3. SURG., 1965, Vol. 52, No. 6, JUNE

pattern was finely stippled, and one or two nucleoli were sometimes visible. I n sharp contrast, the mono- cytes were much smaller, and their nuclei showed the characteristic rcniform outline, while the chromatin pattern was coarse and the nuclear membrane dense and well defined.

b. Wound surfaces: At 24 and 48 hours the smears were highly cellular (Fig. 6). The cells were made up of monocytes, mcsothelial cells, and cells intermediate between these two types. After the second day the

the site of wounding. At 7 days there remained only a faint slightly opaque area otherwise indistinguish- able from normal peritoneum.

Microscopically in the first 24 hours there was only a scanty exudate containing a few mononuclear cells. At 48 hours the cellular infiltrate had increased and the surface layer of cells was definitely flattened. In addition, some of the cells in the exudate could be recognized as fibroblasts. At 72 hours the defects were filled by quite mature connective tissue with

FIG. 6.-Kormal adult rat. Smear from defect at 48 hours. Large mcsothelial-like cells and smaller monocytes with reniform nuclei. Other cells intermediate in size with oval nuclei. Papanicolau. ( x 575. )

mesothelial cells increased in number and at 5 and 7 days the smears consisted mainly of large sheets of these cells. During the second to fifth days these cells showed numerous mitoses, but after this time they assumed the inactive form of lining cells from normal peritoneum.

Histological Appearances of Defects covered by Poly- thene.-During the first 48 hours the appearances of covered defects were similar to those of uncovered dcfects. 'The fibrinous exudate deep to the polythene contained principally monocytes and polymorpho- nuclear leucocytes and the same types were scattered on the surface. However, there were marked differ- ences after the second day, a new mesothelium failing to form for at least 2 weeks (Fig. 7). During this time little change took place in the surface cells which con- sisted largely of monocytes. These remained isolated from each other, separated by wide bare areas of fibrin or, later, of fibrous tissue. Mesothelial-like cells were not seen at this stage. However, at 3 and 4 weeks the wound surface became covered by a layer of flattened cells which resembled mesothelium.

It was not possible to study the process of peri- tonealization of the exudate superficial to the poly- thene because of the dense adhesions which developed in nearly all cases.

2. Immaturity.-The rapidity with which the defects healed in this group was striking. Macro- scopically the defect had acquired a smooth surface by 48 hours, and at 5 days it was difficult to detect

FIG. normal adult rat, I I days. Rase of pol thene- covered defect showing no sign of formation of ncw mesothlurn. Surface formed by scattered monocytes. 14. and 1:. ( '" 290.)

numerous elongated fibroblasts, and a flattened sur- face layer. A small amount of reticulin could be seen at this stage, especially immediately beneath the surface layer.

From the fifth day onwards the wound showed very little change apart from becoming less cellular, and the appearance at 14 days resembled that at I week. 3. Pathological Conditions.- a. Cortisone.-There was no detectable difference

in the healing of the defects in this group when com- pared with normal controls. All defects were covered by a smooth shining layer in 5-7 days and at 10 days were not distinguishable histologically or macro- scopically from the control defects.

b. Diabetes.-Healing of the defects was compar- able both macroscopically and microscopically to the control animals. All defects were covered with a smooth surface layer by the end of the first week. Two of the animals developed infection in the laparo- tomy wounds. Only one of the defects was covered by adhesions, an incidence comparable with the controls.

c. Scum.y.--In this guinea-pig group there was a great difference in the healing of the defects when compared with normal controls.

Macroscopically at 24 hours an exudate, sometimes blood-stained, filled the defect. Over the next 2 days there was very little change, so that at 72 hours the wounds appeared as if they had just recently been made. The surface was rough and a little blood- stained, with no evidence of a shiny surface layer. It was not until the seventh day that some healing was apparent, the surface becoming somewhat smooth and shiny, but still clearly different from the surrounding peritoneum. At 10 days the defect was still easily recognizable, but now covered with a complete smooth surface layer. At 2 weeks healing was well advanced,

ELLIS E T AL.: HEALING OF PERITONEUM 475

but the area of the defect still detectable as a slightly red area with a smooth surface.

In the control animals at this 2-week stage the defect had attained its final smooth white opaque appearance with a surface indistinguishable from the surrounding peritoneum.

Histologically, very little difference from the normal controls was detected in the first 48 hours. A cell- filled exudate appeared and some fibroblasts were

PIC. 8.-Scorbutic guinea-pig, defect at 72 hours. There is no sign of a flattened surfacc layer. H. and B. ( x 290.)

recognizable by 72 hours. In some of the scorbutic animals haemorrhages were noted under the adjacent peritoneum. At 72 hours, in contrast to the controls, there was no sign of a flattened surface layer in the scorbutic guinea-pigs (Fig. 8). At this stage the con- nective tissue filling the defect showed a striking contrast to the controls (Pig. g), there being a very loose irregular stroma with many more cells, some being immature fibroblasts. In parts the stroma was broken up by blood-stained effusions. At 5 days the beginning of a flattened surface layer was seen, but did not assume the completely flat single layer (seen in the controls from 5 tc 7 days) until 10 days. At this stage there was still considerable activity in the under- lying connective tissue, with many new capillaries and immature fibroblasts. No collagen was seen in the healing defects in the scorbutic animals up to 21 days, but could be seen in the control animals from the seventh day onwards.

By the twelfth day the defect was well healed with a single flattened surface layer continuous with the adjacent peritoneurn, but there was still considerable cellular activity in the underlying connective tissue with many irregular fibroblasts.

DISCUSSION The present work has confirmed that parietal peri-

toneal defects in normal adult rats and guinea-pigs heal rapidly, a continuous layer of flattened cells forming simultaneously over the whole surface of the defect within 3-5 days. The new mesothelium develops at the same rate in the centre of large (2 x 2 cm.) defects as in small ones (0.5 x 0.5 cm.). These findings strongly suggest that the old concept of peritoneal regeneration by multiplication and inward migration of lining cells from the margins of excised areas cannot play a major part in the healing process, a conclusion which, as described above, has

signs of activity-cellular enlargement, cytoplasmic basophilia, and mitotic figures-in the adjacent, intact mesothelium do indicate that these cells may con- tribute to the peritonealization at the periphery. Similar observations were made by Johnson and Whitting (1962).

A question remaining to be answered is whether the regenerated serosa arises mainly by metaplasia of underlying connective tissue cells (Robbins and others,

FIG. 9.--h’ormal adult guinea-pig, defect at 72 hours. There is a well-marked flattened surface layer. H. and E. ( x 290.)

1949; Brunschwig and Robbins, 1954; Williams, 1955) or by implantation of mesothelial cells which detach from adjacent structures (Cameron and others, 1957; Johnson and Whitting, 1962). The last-named authors, studying the healing of defects in the parietal peritoneum of the rabbit, described a pro- cess similar to that found by Cameron and his co-workers in the rat during the healing of wounds of Glisson’s capsule. They concluded that the cells which appeared rapidly on the surface of the exudate and completely covered it within 3 days had become detached from normal peritoneum in contact with the wound. When the operation site was covered by polythene healing was greatly delayed, but the surface was eventually covered by a layer of cells indis- tinguishable from normal mesothelium, whereas the exudate which formed superficial to the polythene was rapidly covered by mesothelial cells in a manner identical to that of uncovered defects. These authors suggested that monocytes and tissue histiocytes could form mesothelial cells but probably only did so in sites inaccessible to implantation from pre-existing mesothelium.

However, the delay in the healing of these poly- thenc-covered wounds may well be due to the highly artificial conditions of the experiments rather than to the presence of a physical barrier to mesothelial cell implantation. The wound surface is completely sealed off from the rest of the peritoneal cavity and the environment beneath the polythene may be sufficiently abnormal to interfere with the trans- formation of connective-tissue cells into lining cells.

In the technique universally employed to aate in the investigation of peritoneal healing-the fashioning of defects, the sacrifice of animals at regular intervals, and the study of the microscopic appearances by con- ventional histological methods-there are two serious disadvantages. The first is the attempt to interpret a verv raoid dvnamic Drocess from a series of static been reached in several recent studies. However, .I --,

476 BRIT. J. SURG., 1965, Vol. 52, No. 6, JUNE

appearances. The second is the lack of distinctive morphological differences between the various cell types concerned. In the present study some of the latter difficulties were eased by intravital staining (used to distinguish between tissue histiocytes, which were heavily stained, and blood monocytes, which took up very little of the dye), by reticulin staining (used to demonstrate early fibroblast activity), and by examining surface smears of both normal and healing peritoneum.

The striking feature of the appearances of the healing defects was the rapid change which took place in the period between 48 and 72 hours after wounding. Up to the third day, the defect was filled with an inflammatory exudate principally composed of mono- cytes, histiocytes, and polymorphs in a fibrin exudate. By 72 hours these cells had greatly disappeared, the surface of the defect was covered by a continuous layer of cells in which trypan blue could no longer be seen, and at the same time there was exuberant under- lying fibroblast activity. T o us, these appearances strongly suggested a two-stage process : an initial wave of phagocytic cells derived from the blood and the local tissues, responsible for clearing of traumatic debris, followed by a second wave of fibroblasts responsible for the healing of the defects and the differentiation of a new mesothelium.

Although in the first 48 hours of healing both mono- cytes and mesothelial cells were observed on the surface exudate, we consider that these can play no more than a minor role in the production of a new definitive mesothelium. Apart from our interpre- tation of the histological picture, this view is supported by the following:-

I. In vitamin-C depleted animals, a normal inflam- matory exudate appears, yet healing is greatly delayed. It is well known that vitamin-C deficiency affects fibroblast maturation, but this should not impede any process of implantation of mesothelial cells onto the defects.

2. In immature animals the fibroblast reaction occurs more swiftly than in the adult group and in these animals re-peritonealization is correspondingly more rapid.

3. The surface cells of the peritoneum are con- tinually being shed. Even if mesothelial cells do deposit on the defect in its early stages, the sub- sequent mature mesothelium must be continually re-formed from underlying cells; these are unmis- takably fibroblasts.

4. Embryologically the peritoneal mesothelium is of mesodermal origin. Every other mesodermal derivation in the mammal heals fundamentally by a process of fibroblast proliferation. As a teleological argument it would be at least surprising if the peri- toneal serosa were not only an exception to this rule but also were to heal by a unique process of implanta- tion of shed mature cells from adjacent serosal surfaces.

Of perhaps more immediate practical importance is the investigation of factors which may affect the rate of peritoneal healing. T o date we have studied only maturity, vitamin-C deficiency, experimental diabetes, and heavy doses of cortisone on this process. Peritoneal hcaling is considerably more rapid in the immature than in the adult rat, and considerably more delayed in the scorbutic guinea-pig than in the healthy control. Diabetes and cortisone therapy have no obvious effect. The influence of protein depletion, irradiation, and uraemia on peritoneal healing is at present under investigation in this department.

SUMlMARY I. The process of peritoneal healing has been

studied in normal rats and guinea-pigs. 2. It is concluded that the fibroblast is the impor-

tant source of the rapidly re-forming peritoneal membrane.

3. The healing is more rapid in immature animals, delayed by vitamin-(= deficiency and unaffected by experimentally induced diabetes or heavy cortisone dosage.

We would like to thank Professor G. Hadfield, Dr. Ian Dawson, and Dr. James Earle for helpful advice, and Mr. Jack Haines for the photomicro- graphs.

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LE GROS CLARK (1958), The Tissues of the Body, p. 63.

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