Cultured epithelium as a skin substitute

Burns (1987) 13, (3). 173-180

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Cultured epithelium

as a skin substitute

A. Eldad,” A. Burt and J. A. Clarke Burns Unit, Queen Mary’s Hospital, Roehampton, Plastic and Maxillofacial Surgery, Shaare-Zedek

London Medical

and the *Department of Center, Jerusalem, israel

B. Gusterson Ludwig Sutton,

institute for Cancer Research Surrey, UK

(London

Summary Twenty-five burn patients with full or partial thickness skin loss received cultured epithelium (CE), allografts or autografts as part of their treatment. Overall, a 30 per cent graft ‘take’ was achieved irrespective of whether the CE was autograft or allograft, fresh or frozen. In the case of deep dermal burns this figure improved to 50 per cent. The surviving grafts merged with split thickness skin grafts ISSG) and adv&cing wound edges. When full rhickness skin loss was grafted, only a patchy take could be achieved and the surviving islands of CE tended not to spread across the wound. No rejection of CE allograft was recorded either clinically or histologically up to 6 months. The technical problems and clinical implications are discussed.

INTRODUCTION IN the treatment of extensive burns the plastic surgeon must overcome the limitations of available donor sites, supply the patient with appropriate temporary coverage to reduce bacterial infection and prevent sepsis, until further harvesting of the same donor sites is possible. Large donor sites are always associated with considerable blood loss, morbidity and the risk of being infected and turning into full thickness skin loss. These problems have been helped by expanded mesh grafts (Tanner et al., 1964) and the use of combined autografts with strips of cadaver allografts (Jackson, 1954; Colson and Prunieras, 1960). These methods attempt to obtain rapid closure of the burn wound, however, the benefit may often be temporary or insufficient. In ad-

Branch), Royal Marsden

Hospital,

dition, cadaveric homografts may be difficult to obtain because of medicolegal, social or religious problems. Recently, the Chinese have reported a successful outcome in major burns from the use of large sheets of allograft interspersed with small pieces of autograft (Yang et al., 1982). The results are. impressive, although this method is extremely time- and labour-consuming. The implications of the prolonged survival of dermal elements following rejection of epidermis by the immune system were realized many years ago and recently put into perspective by Gibson (1986). In 1975 Rheinwald and Green cultured keratinocytes from the human foreskin using a combination of 3T3 feeder layer, cholera toxin and epidermal growth factor (Rheinwald and Green, 1977). The growth of large numbers of cells from an initial small biopsy was used successfully for human skin grafting in 1981 (O’Connor et al., 1981). The possibility of covering the whole body surface from an initial biopsy of 1 cm2 of skin was described some time later (Gallico et al., 1984). The implication for cultured epithelium seemed enormous. A different skin culture technique was used by Bell to produce “Living Skin Equivalent” (Bell et al., 1981). This method attempted to produce dermis and epidermis. A third technique was used by Hefton et al. (1983), who grafted CE allografts to partial thickness bums without apparent rejection. These techniques have not, as yet, gained general acceptance due to the high

174

Burns(1987)Vol. 13/No.3

degree of tissue-culture expertise required. We present our findings using CE obtained by Green’s method, in the treatment of the burned patient.

MATERIALS AND METHODS For autograft purposes a small SSG biopsy (2 x 2cm) was obtained soon after admission. For allograft purposes skin was obtained from SSG of other burned patients or from elective reduction mammaplasties. We found the growth of keratinocytes from donors over 40 years of age disappointing. However, usable cultures from these donors have been produced using a higher primary inoculum. The method used was essentially that described by Green et al. (1979). After mincing and trypsinization of the skin, an inoculum of 2 x IO6 viable cells in suspension was added to each 75ml flask containing irradiated 3T3 mouse fibroblasts. At 12-14 days each primary confluent culture was subcultured into many secondary cultures of the same size. These reached confluence in 10-14

days and the cultures continued to grow to produce a keratinocyte layer, four to eight cells thick, for grafting. Two days before grafting the cholera toxin was eliminated from the medium and bacteriological cultures obtained. On the grafting day the flasks were cut open. CE was detached from the flasks by 0.2 per cent Dispase (Protease) solution incubated for 10min at 37°C + bench for half an hour and mounted on 6 x 8cm petroleum jelly gauze or 6 x 8cm Surfasoft (Mediprof, Holland) with ligaclips. Mounted CE was then transferred from the tissue culture laboratory to the burns unit. It was either grafted immediately following excision of the burn (18 patients) or as a delayed grafting 3-5 days after excision (five patients) or on clean granulating wounds (eight patients). In some patients CE was used both on freshly excised and granulating wounds at different stages. Between August 1985 and March 1986, 25 patients were grafted using CE. The ages of the patients and nature of the skin loss are shown in Table I. These patients received CE either as autografts or allografts. No attempt was made to

Table I. Patients’age and percentage of burns

Patient no.

Age W

Total area of burn (% BSA)

Deep dermal burn (% BSA)

FIJI/ thickness burn I% BSA)

18 5 0 2 1

20 7 3 8 1 2

1

14

2 3 4 5 6

49 84 68 72 74

7 0

76 ii 45

9 40 12 7

: 10 4 0

3: 8 7

18 43 80 61 36 5 x3

5 :

8 1 0 5

25 2 10 20 8 25 3 3 1 4 10 2 6

20 4

z: 11

38 12 3

10 2 10

10 11 12 13 14 15 16 17 18 19 20 21 22 9s

1 75 61 26 30 75

30 3 12 20 10 30 3 5 10 4 10 3 3:

25 Mean

23 47

55 15

0 2 S 2 9

775

Eldad et al.: Cultured epithclwm as a skin substitute

Table II. Cultured epithelium thickness burns

autografts

No. of patients CE autograft (cm*) CE allograft (cm?

and allografts on deep dermal and full

Deep dermal burns (no.)

Full thickness burns In0.l

% CE grafts of total grafts

0 585

800 2545

18 8.5

2 23

tissue-type donor or recipient. Table ff describes CE as autografts and allografts on lhe burns.

Quantitative bacteriological counts were obtained from excised burn wounds in addition to surface swabs. The wound beds were biopsied to ascertain if any dermal or epidermal elements remained. When CE was grafted onto limbs the procedure was carried out without suturing and a standard Jelonet and dry gauze dressing was applied to the area. In more difficult areas of the trunk, shoulder and abdominal wall CE was secured with sutures or staples. In two patients CE was secured using fibrin-thrombin glue. The first dressing was carried out at 5 days. When the carrier (culture mount) was adherent to the wound no attempt was made to dislodge it for a further few days. The grafted area was then dressed with Paratulle and changed regularly. Estimation of graft ‘take’ was initially made at the first dressing change. It was difficult. almost always. to differentiate at this stage between a raw surface and a thin layer of transparent epithelium. By 14 days post-grafting, the CE was generally sufficiently opaque to estimate ‘take’ more accurately. In addition to clinical inspection, the area grafted was biopsied at 7-10 days and at 30 days and in some cases 60 and 180 days post-grafting. Nine patients had CE grafted on 6 : 1 or 3 : 1 mesh autograft in a trial to obtain accelerated healing of these grafts. Toxic effects of various antiseptics

vitro trials with confluent-ready-to-graft CE were conducted in order to find how various local antiseptics influence CE. Discs of povidone iodine, silver sulphadiazine. silver nitrate and furacin were applied to the CE surface in the flasks. The effects were observed by microscopical examination. In

Frozen CE

Flasks of CE were cut open when they reached confluence and were ready for grafting. The medium was replaced with one containing IS per

cent glycerol and 10 per cent calf serum and cooled to 4°C for 1-2 h. The bottom of the flask with CE still attached was sealed in a poiythene bag and frozen in liquid nitrogen for 1-S weeks. Forty-eight hours before the required grafting the bag was removed and the culture thawed rapidly by addition of culture medium at 37°C. Glycerol was washed off and fresh culture medium was added without cholera toxin. CE was then incubated for 4g h at 37°C. Further preparations for grafting were as for fresh CE.

RESULTS In general the results were disappointing. The best CE ‘take’ was achieved when grafted on partial thickness burns. Adherence and ‘take’ were less satisfactory on freshly excised full thickness burns and bad on old granulating bum wounds (Table Iif). Fifteen patients with full thickness burns had CE grafts, six of them (40 per cent) showed complete destruction at the first dressing. About a third of all CE grafted on deep dermal burns or on 6: 1 expanded mesh in full thickness burns showed similar destruction. The overall ‘take’ for deep dermal burns was 50 per cent (Fig. 1). The average ‘take’ for CE grafted on 6 : 1 mesh was 28 per cent (Fig. 2) and only 1X.5 per cent ‘take’ for CE grafted on full thickness burns (Fig. 3, Table IV). However, at the present time, it is impossible to estimate visually the

Table IN. Feilure and ‘take‘ of cultured epithelium on freshly excised or granulating wounds

Wound

bed

Freshly excised Partial thickness burns Full thickness burns Granulating Partial thickness burns Full thickness burns

Failure

Average ‘take’

NO.

I%)

(%I

8 16

25 31

45 27

2 9

33

70 14

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Burns (18871Vol. 13/No. 3

Fig. 1. Patient no. 1. Wound healing at I4 days post-grafting. Notice the difference between the strip of SSG and the CE grafted areas.

Kg. 2. CE allograft on 6 : 1 meshed autograft 7 days post-grafting, Approximatciy 90 per cent wound coverage.

contribution from CE and that from the meshed SSG. CE ‘take’ on full thickness burns was patchy and the surviving islands tended not to spread on the wound when further granulation occurred. In two patients we used autograft CE, one was a failure and only a 10 per cent ‘take’ was achieved in the second patient. In that patient a better ‘take’ was demonstrated where CE allograft was used.

Reasons for poor results In this group of patients the numbers are too small to be significant, but they may suggest that age is not a contraindication for treatment with CE (Table I’). Many mounting materials for CE were tried (bovine collagen. glutaraldehyde-preserved porcine skin, amniotic membranes) but were grossly inferior to petroleum jelly gauze. Only Surfasoft gave similar results to petroleum jelly gauze and

177

Eldad et al.: Cultured epithelium as a skin substitu!e

p;
CE 6 months post-grafting

Patches of unpigmcnted

Tab/e IV. Failure and ‘take’ of cultured epitheiium of varied amounts of dermal elements

CE on deep dermal burns (10 patients) CE on 6 : 1 mesh in a full thickness

burn (9 patients) CE on raw surface of full thickness burn

in the presence

Failure l%l

Average ‘take’ (%l

30

50

33

28

40

18-6

(15 patients)

Tab/e V. ‘lake’ of cultured epithelium Age-group (yrl O-20 21-40 41-60 61-90

in various age-groups

No. of patients

Mean % of burn

Mean CE ‘take’ 1%)

4 7 3 11

27 22 14 9

27.5 17 43 26

was much easier for mounting. Unfortunately. as CE take was only patchy at times. an ingrowth of granulation tissue into the pores of the Surfasoft caused bleeding and pain on removal.

in-vitro-viability testing. The average take of the frozen CE grafts was similar to that of the ‘fresh grafts’.

Investigations

Serial punch biopsies were taken from the CE grafted areas. Only one patient demonstrated histological signs consistent with rejection at 35 days (Fig. 4). At this time there was no clinical

Rejection of ‘tissue banking’

Examining banking of CE allografts: we applied 10 pieces of frozen CE. Viability of the keratinocytes was demonstrated by parallel subculture

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Burns (1987) Vol. 13/No. 3

. *,^.

““.

*

.,*-

of CE allografts to a full thickness skin loss. There is basal cell vacuolation and degeneration in association with a mononuclear cell infiltrate which is centred on this region of the graft (X 1032).

*”

5

da

_ s

:.

-~*$;“‘-

I

I

Fig. 4. Thirty-five days .post-grafting

*

x5

”

^ h.

ant;” - = __

Fig. 6. Patient no. 2. Biopsy from CE grafted area at six months. Note hyperkeratosis, tlat epidermis and absence of melanocytes, as in a secondary healed wound (x 1032). Table V/. Failure: ‘take’ related to quantiative terial counts

No bacterial growth Patient ‘take’ (%I Patient failure (%I

10 15

bac-

No. of ~;3c%‘/ri/g tiss;e >lO 15 15

15 30

Bacteriology

Fig. 5. Same area as in 9-56 days post-grafting. There is no histological evidence of rejection (X 1652).

evidence of rejection. A biopsy taken after 56 days did not demonstrate any rejection characteristics (Fig. 5). All other allografted patients in this group followed up to 6 months did not show any histological signs of rejection (Fig. 6). The method of securing the grafts (suturing, stapling or merely dressing) had no effect on the outcome. In two cases fibrin-thrombin glue was applied in a trial to improve poor adherence of grafts to the wound bed. Adherence was improved dramatically but there was no subsequent take of CE. In a trial to inhibit the destructive proteolytic activity on the wound surface we used local irrigation with Trasylol (Aprotinin, Proteinase inhibitor, Bayer). No ‘take’ was subsequently demonstrated in these wounds.

There was no obvious correlation between the bacteriological quantitative counts and the final results. In some wounds where no bacterial growth was obtained from the wound-bed biopsy, complete failure of graft still occurred. On the other hand, some successful graft ‘takes’ were obtained in the presence of l@organisms/g tissue (Tuhfe VI). Similarly, the percentage ‘take’ in the successfully grafted wounds was not related to the bacterial status of the wound (ruble Vll). Local antiseptics

Iodine-containing dressing (including povidone iodine) were destructive to the growing CE. Onecentitietre discs of povidine iodine-impregnated gauze were applied to ready-to-graft flasks of CE and complete destruction of culture was observed within 48 h. Silver containing local antiseptics (silver sulphadiazine. silver nitrate) caused moderate inhibition of growth, and the least harmful of all tested local antiseptics was Furacin gauze. Petroleum jelly gauze (Jelonet, Paratulle) did not cause any visible disturbance to CE.

179

Eldad et al.: Cultured epithelium as a skin substitute

Table WI. ‘Take’/failure in presence of major bacteriological grown from the burn wounds

Organism Staphylococcus aureus Pseudomonas aeruginosa Faecal-type flora Proteus sp. Strep. P-haemolytic Group G Strep. P-haemolytic Group A

CASE REPORTS Patient 5 A 72-year-old male suffered a partial and full thickness circumferential burn to his left calf after his trousers caught fire. The wound was treated with Paratulle and later with Furacin gauze. At I6 days post-burn the wound was excised down to fat with patches of deep dermal elements in some areas. Three days after excision it was grafted with four pieces of CE (2OOcm’) and one strip of 2 x 5 cm SSG. Seventy per cent epithelial coverage was apparent by the time of the first dressing (Fig. 1). A second CE grafting was tried to cover the raw surface at the posterior aspect of the calf, but failed. This area healed secondarily within 3 weeks.

Patients /%1

‘Take l%l

52 40 36 28 20 12

69 70 66 57 40 33

‘

groups

Failure f%) 31 30 33 43 60 66

4 weeks. The legs needed further SSG 7 weeks postburn. Follow-up did not reveal any signs of rejection in the CE grafted areas.

DISCUSSION Cultured epithelium

as a skin substitute?

while having many theoretical advantages over most current skin substitutes, lacks many characteristics of normal skin. At best it could serve as an outer barrier to prevent the loss of body fluids and heat while at the same time diminishing bacterial invasion. Most importantly in addition, it should reduce or eliminate the demands for extensive donor sites Patient 2 (Gallico and O’Connor, 1985). Furthermore, the A 49-year-old black male suffered 12 per cent mixed successful use of allogenic CE could revoburns to his face, neck, hands and legs from burning lutionize the early excision and grafting of deep paint. His bums were treated by exposure and hand dermal burns without causing further morbidity bags. At I4 days post-burn his wounds were excised. from donor sites. The length of time necessary to He had SSG to his neck, hands and thigh. Six pieces grow culture autografts (3-4 weeks) prevents the of CE alongside strips of SSG autografts covered use of this material for early excision and grafting the burn wounds of his legs. Only a patchy take (about of deep dermal burns. If, however, an equivalent SOper cent) was obtained in the left calf. No take could ratio of ‘take’ and long-term survival can be be demonstrated in the right calf which was heavily achieved for allografts as for autografts, this may contaminated with bacteria. The successful CE allograft in the left calf remained unpigmented (Fig. 3) and lead the way to centralized CE banking, with biopsies up to 6 months did not show any signs of grafts being distributed to several burn units. rejection (Fig. 6). Frozen CE is even more suitable for such a purpose. This possibility can only become a rePatient 25 ality if the technical problems of getting CE to A 23-year-old male suffered 59 per cent partial and full take reliably can be overcome. thickness burns when his jeep caught fire while crossing We could not demonstrate the successful rethe Sahara Desert. The patient was rescued and sults reported by Gallico et al. (Gallico et al., reached the burns unit within 24 h of the accident. He 1984; Gallico and O’Connor, 1985), and only was resuscitated and the burns treated with silver sulpoor ‘take’ could be achieved treating full skin phadiazine. Seventeen days post-burn eschars from hands and legs were excised and grafted with 3 : 1 and thickness bums. These disappointing results have 6 : 1 mesh autografts. Eight pieces of CE allografts were encouraged us to further investigation and trials, grafted on his legs. Abdominal burns were excised and in order to improve the technique. temporarily covered with SSG allografts. Four days Success or failure of CE is established in the later only a patchy ‘take’ of the CE grafts could be first few days after grafting. In those that failed demonstrated, and all these areas needed further graftcomplete disintegration of CE took place within ing or healed secondarily. SSG allografts from the abdomen were removed and replaced by seven pieces 3-5 days; these patients had not been exposed to allograft before, and early biopsies did not reveal of CE allografts and some 3 : I meshed autografts. any signs of hyperacute rejection. CE is a pure These grafts were more successful and the abdominal wall was completely covered with epithelium within keratinocyte culture, with the Langerhans cells, Cultured

epithelium,

Burns(1987)

180

the only cells in the skin to present the HLA-DR complex (Stingle et al., 1978; Braathen and Thorby, 1980), being selected out in the keratinocyte culture process. CE presents, therefore, only much weaker antigenic groups and we did not expect these grafts to be rejected. Six months of clinical and histological follow-up without rejection supports these expectations. Similar results were obtained by Hefton et al. (1983) with CE on deep dermal burns. When the SSG has not taken, we still see the non-adherent, dead skin on the wound surface. CE is much thinner and more sensitive to proteolytic activity. Zamecnic et al. (1945) described the high levels of proteases in the burn patient and the burn wound. These proteases are produced by microorganisms as well as by leucocytes. In some cases they completely digested CE and although we tried to block this process by local irrigation with proteinase inhibitor, we failed to increase percentage take. Further research and trials in this area are needed. The importance of dermal elements to the survival and proliferation of CE was demonstrated by the improved results when CE was grafted on deep dermal burns or widely expanded mesh grafts. Lack of collagen and elastin in the CE can explain its poor adherence to the wound bed (Burlson and Eiseman, 1972; Thornton et al., 1977) and it suggests the need for incorporation of collagen or other dermal elements in the grafts. Finally, and importantly, at times it is almost impossible to estimate how much of a wound coverage is a CE contribution and how much is secondary healing (from wound edges, deep dermal elements or other grafts). There are no differences between the histological appearances of a CE graft and a scar, and unless we are able to identify positively CE on the healed wound we will not be able to evaluate different modalities of improving the take of cultured epithelium grafts. Acknowledgements Mrs A. Burt is a visiting worker in Dr Gusterson’s laboratory at the Ludwig Institute for Cancer Research, Sutton, and is in receipt of an award from South West Thames Regional Health Authority, Research Committee. We are indebted to the photographic department at Roehampton for the illustrations and the secretarial help at the Ludwig Institute. Epidermal growth factor for cell culture was generously supplied by the Chiron Corporation (4560 Horton Street, Emeryville, CA94608, USA).

Vol. 13/No. 3

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Gibson T. (19%) The ‘Second Set’ phenomenon as first shown in skin allografts. An historical case which shows also the behaviour of cell free collagen. Br. J. Plast.

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Green H.. Kehinde 0. and Thomas J. (1979) Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc. Nat/. Acud. Sci. USA 76, I I. Hefton J. M., Madden M. R., Finkelstein J. L. et al. (1983) Grafting of burn patients with allografts of cultured epidermal cells. Lancer i, 428. Jackson D. M. (IYS4) Clinical study of the use of skin homografts for burns. Br. J. Plasm. Surg. 7, 26. O’Connor N. E., Mulliken J. B., Banks-Schlegal S. et al. (1981) Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Luncn I. 75. Rheinwald J. G. and Green H. (1975) Serial cultivation of stains of human epidermal keratinocytes. the formation of keratinisinr! colonies from sinele cells. Cell 6. 331. Rheinwald J. G. and Green H. (1977) Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265, 421. Stingle G., Katz S. I., Shevach E. M. et al. (1978) Analogous functions of macrophages and Langerhans cells in the initiation of the immune response. J. Invest. Dermarol. 71, 59. Tanner J. C., Vandeput J. and Olley J. F. (1964) The mesh skin eraft. Plasr. Reconsrr. Surp. 34. 2X7. Thornton J. W., Tavis M. J., Harney J.“H. et al (1977) Graft adherence to wound surface: collagen-fibrin interactions. Burns 4, 23. Yang C. C., Shin T. S. and Xu W. S. (1982) A Chinese concept of treatment of extensive burns. P/W. Reconslr.

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Zamecnic P. C., Stephenson M. L. and Cope 0. (1945) Peptidase activity of lymph and serum after burns. J. Biol. Chem. 158. 13.5.

Paper accepted I2 December 19%

Correspondewe should be addressed IO: Mr J. A. Clarke, Regional Burns Unit. Queen Mary’s Hospital. Roehampton. London SW15 SPN. UK.