From the H. N. Smith Research Unit of the Department of Physiology, Royal College of Surgeons, London.

Transcription

1 HEALING OF SUTURED SKIN INCISIONS: A MICROSCOPIC STUDY 1 By J. A. BUNTINE, F.R.A.C.S. 2 From the H. N. Smith Research Unit of the Department of Physiology, Royal College of Surgeons, London. THIS paper deals with the process of scab formation and its influence on healing of epidermis in exposed sutured incisions. The findings reported in humans and pigs are similar tothose previously described in rats. Additional findings in rats are also presented. The previous report of healing of exposed sutured incisions in rats (Buntine, I967) described the formation of a line of demarcation between viable and non-viable tissue. This line, consisting of a layer of leucocytes, extended through the upper portions of the skin edges and through intervening coagulum. Ingrowth of epidermis healing across the incision could be accounted for by growth along this previously formed line of demarcation. Hair follicles crossed by the line contributed to the healing sheet of epidermis. All structures superficial to the line of demarcation lost their identity and partially fused to form a scab (consisting of dead dermis, epidermis, keratin, blood clot, leucocytes and exudate). The scab was eventually freed by desquamation of healed epidermis. A similar process occurred at suture punctures. Accurate surface apposition of the skin edges was associated with less tissue loss as scab and faster healing of epidermis. MATERIALS AND METHODS Rats.--The microscopic preparations described in detail in the previous report (Buntine, I967) were re-examined. Also, a further five sutured midline dorsal incisions in Wistar rats were excised after one month and microscopic slides prepared by the same method. The complete set of slides studied began with specimens taken immediately after suture, then at four hours, and at increasing intervals up to one month. Pigs.--Sutured skin incisions at one and two weeks were obtained from each of two female "large white" pigs of three months and about 25 kg., killed following an experiment involving multiple skin incisions on the flanks. The skin had been sutured with only moderate care and then exposed without protection from rubbing. Only incisions which appeared to heal rapidly without complications were examined. After fixation in IO per cent. formol-saline, sutures were removed and sections prepared from paraffin blocks. Some slides were stained with Ehrlich's h~ematoxylin and eosin ; others with Weigert's iron h~ematoxylin and van Gieson's picrofuchsin (specific for collagen). Humans.--Compared with the ease of collecting old scars, recently sutured incisions were difficult to obtain. The earliest specimen was from autopsy, specimens at one week were from delaying procedures, and one at one month from a staged operation. The sutured portion of a tracheostomy incision was removed from a patient who died I4 hours after operation. Specimens of five wounds, one week old, were obtained from tissue discarded following delaying procedures (four abdominal tube pedicles and one buttock flap). All incisions had been carefully though not meticulously sutured, covered 1 Paper delivered at a meeting of the British Association of Plastic Surgeons at Bristol, July Present address : 4o4 Albert Street, East Melbourne 3oo2, Victoria, Australia. 126

2 HEALING OF SUTURED SKIN INCISIONS lightly with dry gauze until oozing ceased, and then exposed. Healing appeared to be uncomplicated, but perhaps slightly slower than union of incisions with a vigorous blood supply on each side. The specimens were fixed in Zenker's fluid. Long runs of serial sections were cut from paraffin blocks, and most slides were stained with Ehrlich's hrematoxylin and eosin. Use of Polarised Light.--All slides were examined with normal illumination and with polarised light. The best results were obtained by resting one polarising disc directly on the coverslip (possible only with low-power objectives) and the other on the substage condenser. When the upper disc was placed in the tube of the microscope (essential with high-power objectives) the relative brightness of birefringent structures was less, even with objectives specially designed for polarised light. As dust is highly birefringent, it was necessary'to use clean materials in preparing the slides and to wipe all glass surfaces between the polarising discs. To compare isotropic and anisotropic structures, one disc was frequently rotated through a small angle. Especially with higher magnifications a bright " Zenon" lamp assisted recognition of slightly birefringent structures. I27 OBSERVATIONS Rats.--Birefringence of collagen depended on the relationship of the axis of the fibre to that of the light. Even slight rippling of sections resulted in an appearance FIG. I Exposed, sutured, skin incision in a rat at two days. Weigert's iron hrematoxylin and van Gieson's picrofuchsin. x 65. Healing of epidermis beneath the scab is complete except for a small interval on the right. The dark layer immediately above healed epidermis consists of densely packed leucocytes. This is the zone of demarcation formed between viable and non-viable tissue. (Compare with Figs. 2 and 3.) of broad striations across longitudinally arranged fibres. Collagen fibres divided transversely usually appeared dark. Birefringence of keratin was more variable but less affected by orientation of the squames. Polarised light greatly assisted recognition of collagen in scabs (compare Fig. I and Fig. 2). In Figure I collagen and coagulum in the scab are indistinguishable. In marked contrast to the bright red colour of normal collagen stained by picrofuchsin, that in the scab is yellow (non-specific staining).i

3 I28 BRITISH JOURNAL OF PLASTIC SURGERY FIG. 2 Photomicrograph with crossed polarised filters of a section almost identical to that in Figure I. This adjacent serial section was stained by the same method. Bright structures are birefringent: hairs, collagen fibres, and to a lesser extent keratin squames or partially keratinised epidermal cells. Note that collagen fibres in the scab have retained normal birefringence. (See Fig. 3.) 00t~ '. : I f.r a oo o FIG. 3 A diagrammatic tracing of Figures I and 2. Coagulum of the scab is stippled~ Surface epidermis (viable and non-viable), epidermis of hair follicles and epidermis growing beneath the scab is shaded black. The original skin edges are indicated by interrupted lines. Arrows indicate sites of downgrowth of epidermis. K, Loose keratin. E, Dead epidermis of the scab continuous with keratin overlying adjacent skin. S, Portions of the scab consisting of sloughed dermis. H, Dead hair follicle in the scab. G, Sebaceous gland in the line of epidermal growth (this and the hair follicle to its left have contributed to healing of surface epidermis). X, Continuity of collagen fibres in viable dermis and slough (about to be nipped off by growth of epidermis from each side).

4 HEALING OF SUTURED SKIN INCISIONS 129 However, Figure 2 demonstrates that scab collagen has retained normal birefringence. Birefringence of keratin is also visible but not of great help in this particular slide. (Figure 3 is a labelled tracing of Figures I and 2.) Small prolongations of the advancing sheet of epidermis often extended into recesses between collagen fibres of a scab. These intrusions subsequently became keratinised, or partially keratinised, and remained adherent to the lower surface of the scab after it had separated. The upper surface of the scab was also partly covered by keratin (from original surface epidermis). Detached scabs thus consisted of a core of dermis and coagulum virtually encapsulated by keratin. Even the core contained some keratin (complete hair follicles in sloughed dermis ; hairs and keratin debris in coagulum). Epidermis growing along the line of demarcation between viable and non-viable dermis was at first directly supported by dermis, but within a few days it was lifted from the dermis by fibroplastic tissue. At one week the defect resulting from loss of dermis was partially filled by thickened epidermis and fibroplastic tissue. By one month it was more completely filled by scar (derived from fibroplastic tissue), the epidermis being thinner than normal at this stage. Because of superficial loss of dermis as scab, the scar was usually wider at the surface than at,a deeper level. Pigs.--Without comparison with the full sequence in rats the structure of scabs, first examined at one week, would have been difficult to interpret. Even with polarised filters collagen was not easy to identify at this stage ; however, keratin remained unchanged. Dead skin margins in scabs could be identified by birefringence of keratin of overlying epidermis and contained hair follicles. The lower surfaces of scabs freed by desquamation could also be identified by adherent keratin squames. Sometimes small blood vessels, containing eosinophilic plugs, were visible in necrotic dermis. Dermal scabs were present in all incisions examined at one week. Healing of epidermis over the incision was usually complete and the defect resulting from loss of dermis was partially filled by fibroplastic tissue. When the divided skin edges overlapped part of the everted edge was lost as scab, but there was little or no loss from the inverted margin. The line of demarcation through the overhanging edge was vertical to the surface, but the scab was elevated and rotated by a wedge of fibroplastic tissue (Figs. 4 and 5). The largest scabs occurred where surface apposition had been least accurate. Tongues of keratinised epidermis often extended for considerable distances into dermal components of scabs. Sometimes a layer of dried leucocytes represented a line of demarcation, along which epidermal growth occurred before necrosis extended to a deeper level. Irregular growth of epidermis from transected hair follicles into fibroplastic tissue was occasionally observed. Large dermal scabs were also present at suture punctures (Figs. 4 and 5). By two weeks all scabs had separated, and the resulting defects were largely filled by immature scar. Scars were often wider at the surface, consistent with loss of superficial dermis as scab. (However, in Figures 4 and 5 the scar is even wider at a deeper level.) Humans.--Sloughing of superficial dermis was observed in the incision inflicted 14 hours before death. Collagen fibres in dry necrotic dermis had clumped together producing a false appearance of swelling of individual fibres (an appearance of hyaline change). Necrotic dermis was infiltrated by leucocytes but the line of demarcation from viable dermis was poorly defined. In three of the five incisions at seven days, slightly inaccurate surface apposition was associated with superficial marginal necrosis and incomplete healing of epidermis. The partially separated scabs consisted of necrotic dermis, epidermis, keratin, blood clot, leucocytes and exudate. When the skin edges overlapped, loss occurred only from the everted margin (Figs. 6 and 7). These Figures demonstrate necrosis of overlapping dermis and epidermis, growth of epidermis from the inverted margin through the substance of the overhanging edge,

5 130 BRITISH JOURNAL OF PLASTIC SURGERY and growth of epidermis towards the incision from the lateral part of the overlapping margin. The line of demarcation is poorly defined. Two of the incisions were completely healed at one week and no dermal elements could be identified with certainty in the FIG. 4 Exposed, sutured incision in a pig at one week. Weigert's iron hmmatoxylin and van Gieson's picrofuchsin, x 5. The dark scab on the left is above a suture track (the oval space) while the elongated scab on the right overlies the incision. (See Fig. 5.) A f FIG. 5 A diagrammatic tracing of Figure 4- Surface epidermis, epidermis growing beneath scabs and epidermis of hair follicles is solid black. Around the suture scab, epidermis has extended more than half way through the dermis and is still growing, but healing is complete beneath the incision scab. The small amount of coagulum on the scab is stippled. The arrow indicates elevation and rotation of the scab by a wedge of fibroplastie tissue. D, Dermis (crosshatched). A, Adipose tissue. P, Panniculus carnosus (cross-hatched). S, Suture puncture scab on the left and incision scab on the right. Both consist almost entirely of dermis heavily infiltrated by leucocytes. F, Fibroplastic (scar forming) tissue. scabs (but it was known that a scab had been removed from one of the wounds). Tissue loss as scab also occurred at suture punctures and beneath the external portions of sutures (presumably due to pressure necrosis). In three incisions growth of epidermis into fibroplastic tissue was observed at a deeper level between the skin edges. Small solid and tubular epidermal outgrowths appeared to proliferate from transected sweat

6 HEALING OF SUTURED SKIN INCISIONS glands. The one-month-old scar was oblique and noticeably wider near the surface. The contour of both scar and adjacent skin was consistent with replacement by scar of tissue lost from an overlapping margin. I3I FIG. 6 Exposed, sutured, incision in human skin at one week. (H & E) 36. The buttock of a 49-year-old female patient was incised to delay a flap. At the surface the skin margins overlap but lower down dermal apposition is sufficiently close to obscure the line of the incision. (See Fig. 7.) t~ FIG. 7 A diagrammatic tracing of Figure 6. Coagulum is stippled. Surface epidermis (viable and non=viable) and healing epidermis is shaded black. Arrows indicate the direction of epidermal growth. The interrupted line at the lower right corner indicates the line of the incision. Epidermis over the whole of the illustrated portion of the overlapping skin edge has sloughed and is being replaced by epidermis from adjacent undamaged skin (seen as a distinct second layer near the upper right corner of the diagram). K) Keratin. S, Sloughed dermis of the overhanging skin edge. E, Epidermis of the inverted skin edge (inturned at the time of suture), e, Epidermis which has grown from the inverted margin on to the under surface of the overlapping edge. For the greater part this lies on the divided surface of the dermis but growth has commenced through the substance of the dermis beneath the necrotic layer. -

7 I32 BRITISH JOURNAL OF PLASTIC SURGERY DISCUSSION In 1936 Arey, describing healing of apposed incisions, noted growth of epidermis into scabs formed from dried coagulum and necrotic fragments of tissue. A relevant quotation from the detailed review by Arey has already appeared in this Journal (Gillman et al., 1953). However, the nature of scabs formed by exposed unsutured wounds has been fully recognised only recently (Hinman et al., 1963) and scabs of sutured incisions have attracted little attention. As in unsutured wounds, sloughing of dermis and overlying epidermis occurs in exposed sutured incisions where inaccurate apposition leaves portions of the divided surfaces exposed. The poorer the surface apposition, the greater is the loss from the skin edges. Due to loss of dermis and overlying epidermis in the scab, the eventual width of the defect in epidermal cover may be several times that of the initial gap between the divided margins of epidermis. Scar tissue forms beneath epidermis healing across the defect (i.e. dermis lost as scab t~nds to be replaced by scar). None of the incisions was meticulously sutured (taking into account the small size of the rats), therefore the extent to which very carefully sutured incisions form dermal scabs remains unknown. Loss from everted margins (but not from inverted margins) results in some degree of" planing down" of stepped incisions. Hyaline change of the type seen in the I4-hour human incision has been described previously ; drying of exposed dermis causes a false appearance of hyaline change in collagen due to clumping together of the individual fibres (Buntine, 1966). As drying may cause superficial marginal necrosis, dressings might be expected to reduce the extent of necrosis. However, accurate surface apposition is more important. SUMMARY Imperfect apposition of the margins of exposed sutured incisions results in necrosis of dermis due to exposure of portions of the divided surface. Healing epidermis, growing down through the skin edges, follows the most superficial plane capable of supporting its growth (sometimes obvious as a line of demarcation formed by leucocytes). Dry necrotic skin edges and coagulum superficial to the line of epidermal growth partially fuse to form the scab, which is often virtually encapsulated by keratin. The extent of tissue loss as scab is reduced by accurate surface approximation. This is important because dermis lost as scab tends to be replaced by scar. The process of scab formation "planes down" stepped incisions to some extent. Plugs extruded from suture tracks are similar in all respects to incision scabs. These findings apply to humans, pigs and rats. The study was supported by an Edward Lumley Surgical Research Fellowship of the Royal Australasian College of Surgeons. I wish to thank the staff of the Victorian Plastic Surgery Unit, Preston, Australia, for allowing biopsy of healing human incisions, T. J. S. Patterson for generously providing pig specimens, and Professors E. IF. Gault and D. Slome for facilities and technical assistance. REFERENCES AREY, L. B. (1936). Physiol. Rev., I6, 327. BtlNTINE, J. A. (1966). Nature, Lond., 212, 21o. BUNTINE, J. A. (1967). Br. J. Surg., 54, 699. GILLMAN, T., PENN, J., BRONKS, D., and Roux, M. (I953). Br. J. plast. Surg., 6, X53- HINMAN, C. D., MAIBACH, H., and WINTER, G. D. (1963). Nature, Lond., 2oo, 377- Addendum.--Attention is directed to a recent comprehensive account of healing of sutured incisions in pigs by OROMAN, L. J,, and GILLMAN, T. (I966), Archs Surg., Chicago, 93, 857.