ANZ J. Surg.

2002;

72

: 893–895

ORIGINAL ARTICLE

Original Article

INITIAL EXPERIENCE WITH CULTURED EPITHELIAL AUTOGRAFTS IN MASSIVELY BURNT PATIENTS

M

ICHAEL

E

LLIOTT

AND

J

OHN

V

ANDERVORD

Department of Plastic Surgery, Royal North Shore Hospital, Sydney, New South Wales, Australia

Background

:

Prognosis in patients with severe burns is improved by early debridement and coverage with skin. The limitingfactor in these patients is the lack of unburnt skin for harvesting. Cultured epithelial autograft (CEA) sheets provide an alternativemeans of covering debrided skin. This is a report on an initial experience with using CEA sheets.

Methods

:

The two patients in this study had their medical records examined retrospectively.

Results

:

The effective take of the CEA sheets was zero in both patients. One of the patients died approximately 6 weeks after admission.

Conclusions

:

Because of the poor graft take seen in the patients in the present study, the use of CEA sheets only as a form oftemporary biological dressing is recommended.

Key words: burns, cultured skin, epithelial autografts.

Abbreviations

: CEA, cultured epithelial autograft; SSG, split skin grafts; TBSA, total body surface area.

INTRODUCTION

Prognosis in the massively burnt patient is improved by earlyexcision of burnt skin and wound closure with skin grafts. Themajor limiting factor for permanent skin coverage is the lack ofdonor site that can be harvested for split skin grafts (SSG). Burnsup to 40% total body surface area (TBSA) can be adequatelytreated with autologous meshed SSG. With greater percentageburns, cultured epithelial autograft (CEA) sheets provide an addi-tional means to meshed SSG for wound coverage. A 4-cm

2

sample of unburnt skin is taken usually at the same time as initialdebridement and is sent for laboratory manufacture of CEAsheets. This takes 3–4 weeks to occur. In the interim, the debridedburnt skin is temporarily covered by a variety of means such asallograft, xenograft or skin substitutes. Areas grafted with CEAsheets must be handled with care as the grafts are fragile andprone to easy disruption. Graft take is initially assessed at 1 week.Take is extremely variable, with a range of 0–100% reported.

We report on our centre’s first experience in the use of CEAsheets in two patients with massive burns to 75% and 85%TBSA, respectively.

METHODS

Two patients were treated for massive burns from the same bush-fire at Royal North Shore Hospital in 2000. This was the firsttime CEA sheets were used in our centre. Their medical recordswere examined to review our experience in the use of CEA sheetsfor massively burnt patients.

CASE REPORTS

Case A

Patient A is a 32-year-old man who sustained 65% TBSA (95%full thickness) flame burns from a bushfire. Early intubation was

performed due to the presence of inhalation burns. Circum-ferential burns to his legs, arms, chest and abdomen requiredemergency escharotomies. A sample of unburnt skin from theposterior neck was sent for CEA sheet manufacture.

Debridement of burnt tissue down to a healthy subcutaneousfat or muscle fascia was undertaken during the first week and thewounds were dressed with Biobrane (Winthrop Pharmaceuticals,Wound Care Division, Fountain Valley, California, USA).

After a period of 4 weeks, the CEA sheets were available forapplication. They were placed circumferentially around the legsfrom the groin to the ankle after removal of the Biobrane andredebridement of questionable areas to healthy subcutaneous fator fascia and securing of haemostasis. The CEA sheets weredressed with paraffin gauze, surgical gauze, Velband, Exudry andcrepe. The legs were not splinted as the patient remained intu-bated and sedated. During the following week, the patient becameseptic and haemodynamically unstable. The outer dressings onthe legs were replaced due to local infection and broad-spectrumparenteral antibiotics were used.

The legs were formally inspected after the first week and therewas effectively no take of the CEA sheets. The dressings aroundthe groin had shifted downwards and the CEA sheets werebunched up under the dressings. Due to infection of the legs andresulting fat necrosis, the legs were redebrided. The legs wereeventually covered with autologous SSG.

Case B

Patient B was a 34-year-old man who sustained 85% flame burns(95% full thickness) from the same bushfire as Patient A. Earlyintubation was performed due to the presence of inhalation burns.He required emergency escharotomies to his legs, arms, chest andabdomen.

Debridement of burnt skin was undertaken in the first weekand the wounds were covered with Biobrane. A sample ofunburnt skin was taken from the posterior neck and sent for CEAsheet manufacture.

After 3 weeks, the CEA sheets were available for placement.The Biobrane was removed and superficial debridement of areasof questionable viability was undertaken and haemostasis was

M. Elliott

MB BS;

J. Vandervord

FRACS.

Correspondence: Dr M. Elliott, 236 Eastern Valley Way, Willoughby,NSW 2068, Australia.Email: mselliott@optusnet.com.au

Accepted for publication 26 August 2002.

894 ELLIOTT AND VANDERVORD

secured. The CEA sheets were placed circumferentially aroundthe legs from the groin to the ankle and dressed in a similarmanner as Patient A. The legs were not splinted as the patientremained intubated and sedated.

The outer dressings were changed due to contamination andparenteral broad-spectrum antibiotics were given. Swabs takenfrom the groin grew

Actinetobacter baumaronuii

. Graft take wasassessed at 1 week. The effective take of the CEA sheets was zeroand the same problems with shifted dressings and bunching up ofthe grafts were seen. The legs were subsequently redebrided anddressed. Patient B died shortly afterwards.

DISCUSSION

The ability to grow keratinocytes

in vitro

was developed byRheinwald and Green in 1975.

1

The first clinical application wasreported by O’Connor

et al

. in 1981.

2

The CEA sheets are 8–10 cells thick and 75 cm

2

in size.

3

Theyare placed onto the graft site, basal surface down, stapled intoposition and dressed with minimal disruption to the grafts. In ourcase, the patients were hospitalized at Royal North Shore Hos-pital in Sydney, but the grafts were manufactured in Melbourne.The grafts in their flasks were transported to Sydney by flight onthe day of grafting and the final stage of preparation was under-taken at Royal North Shore Hospital.

One of the major factors affecting prognosis of the massivelyburnt patient is early excision of burnt skin and obtaining ade-quate wound coverage to reduce the chance of sepsis, fluid lossand to limit inflammation.

4

The traditional gold standard treat-ment for permanent wound coverage is autologous SSG. Evenwith widely meshed autologous SSG, inadequate donor sitenecessitates the use of additional means of wound coverage.Herein lies the benefit of CEA sheets. Essentially, from a 4-cm

2

sample of unburnt tissue, an endless supply of cultured tissuegrafts can be manufactured to provide the means for permanentskin coverage.

Debridement of burnt skin should be undertaken as early aspossible. This has been shown to decrease morbidity, mortalityand hospitalization time, and is associated with better grafttake.

5,6

A reduction in graft survival and an increased incidence ofwound infection is seen if debridement is delayed beyond10 days. It is debatable as to whether it is better to debride tohealthy subcutaneous tissue or to excise to muscle fascia from theoutset. We decided to debride to healthy bleeding subcutaneousfat or fascia and obtain haemostasis at that stage. Most areasrequired subsequent debridement to muscle fascia due to infec-tion and necrosis of subcutaneous fat.

After debridement, temporary wound coverage is achievedusing a variety of means, including cadaver skin (allograft),porcine skin (xenograft) or temporary skin substitute (such asBiobrane, a biosynthetic temporary bilaminated dressing, Trans-Cyte (Advanced Tissue Sciences, La Jolla, CA, USA) and Integra(Integra Life Sciences Corp, Plainsboro, NJ, USA)).

7,8

As far assubsequent graft take is concerned, allograft is the best temporarydressing until donor site becomes available for harvesting or CEAsheets become available for placement.

5

Allograft stimulates vas-cular growth and the development of an allodermis to whichgrafts will adhere to more readily.

6,9–11

There is, however, theadded risk of transmission of viruses and bacteria. Partial debri-dement of allograft is undertaken prior to the application of CEAsheets, leaving the layer of allodermis as a graft bed. Allograftwas not an option at our centre and we elected to use Biobrane as

a temporary wound dressing. Superficial debridement of ques-tionable areas was undertaken after the Biobrane was removedand haemostasis was secured prior to graft application.

Graft take is initially assessed at 7–10 days and is indicated byareas with a ground glass appearance.

5

Accurate assessment atthis time can be difficult as the grafts are thin and difficulties withreporting graft take at this time have been reported.

Limitations to the use of CEA sheets are the time taken to man-ufacture the grafts, the fragility of the grafts, shrinkage of thegrafts, durability of the grafts and the variable take on burnt areas(Table 1).

5,6

A 4-cm

2

sample of unburnt tissue is taken usually at the sametime as initial debridement. It is then transported to the laboratoryunder specific conditions. The sample is produced into CEAsheets, a process that takes 3–4 weeks, before they are ready to begrafted onto the patient. The debrided skin must be coveredduring that time frame via a variety of means to reduce the chanceof sepsis.

Being only 8–10 cells thick, the CEA sheets are very fragile.They must be handled with care during the production and graft-ing processes. Care must also be taken in dressing the grafts onceapplied, subsequent mobilization of the patient and dressingchanges. We noticed that graft loss around the groin was attribut-able to bunching up of the dressings and disruption of the under-lying grafts, which most likely occurred during movement of thepatient. As both patients were intubated and sedated, we did notuse splints to immobilize the legs as elected by other authors. Theuse of splints may have offered more support to the legs and mayhave reduced the graft movement and loss that we observed.

During the final stage of preparation, the CEA sheets are proneto shrinkage of the order of 7–14%.

3

They are then backed with adressing (e.g. paraffin gauze) and placed onto the patient. Thevariable shrinkage results in small areas of non-confluencebetween grafts. Lateral spread of the epidermal cells is requiredto fill these areas.

Cultured epithelial autograft sheets lack a dermal componentthat provides strength and resistance to disruptive forces.

3,4,7,9,12,13

Dermis also provides a foundation for fibrin and collagen deposi-tion. As a result, CEA sheets are prone to easy disruption withminimal shearing forces. Grafting onto a non-dermal bed requiresthe

de novo

synthesis of a basal lamina, hemidesmosomes andanchoring fibrils. It takes 3–4 weeks before the basal lamina isconfluent, hemidesmosomes are mature and anchoring fibrilsbecome thick. Until this occurs, the CEA sheets are at risk of easydislodgement. Over the long-term, histological analysis revealsthat the CEA sheets resemble normal dermis with demonstrationof a complete dermal/epidermal junction at 3–4 weeks, rete ridge

Table 2.

Benefits of cultured epithelial autograft sheets

Reproducibility of graftsMinimizing fluid and electrolyte lossCosmetic resultsReduction in mortality

Table 1.

Limitations of cultured epithelial autograft sheets

Time taken to manufactureFragilityShrinkageDurabilityVariable take

CEA IN MASSIVELY BURNT PATIENTS 895

formation and full maturation of anchoring fibrils at 1 year.

5

At 1 month, the durability of CEA sheets is similar to SSG at1 week.

4

However, the durability of CEA sheets has beenreported to be comparable with split thin thickness one or moreyears after grafting.

5

Take of CEA sheets is widely variable (0–100%).

11

Factorsinvolved in graft take include graft site preparation, the site to begrafted, graft site infection, and mechanical factors such as shear-ing and pressure.

6

Factors not related to graft survival includeage, sex, race and size of the burn.

5

As mentioned, graft take is better when early debridementoccurs and formation of an allodermis has occurred.

The take on partial thickness burns averages 73%, and on fullthickness burns is lower, averaging 53%.

3

The best take is seenon non-dependant areas of the body such as the anterior chest,abdomen and lower legs.

4,12,14,

Upper extremities, skin over jointsand the face should be grafted with autologous SSG if possible.Graft take is generally poorer on the posterior aspects of the bodyand dependant areas due to pressure factors on the friable grafts.In our patients, the problem areas were mainly around the groin,with bunching up of the dressings and disturbance of the under-lying grafts.

Areas of graft loss must be regrafted and this adds to the totaloperations that are required per patient to achieve permanentwound coverage. Patients treated with CEA sheets usuallyrequire more operations in total compared with patients treatedwith SSG alone.

13

It is well appreciated that the biggest problem in graft sur-vival is wound infection. Infection causes graft site necrosis andsubsequent poor take of CEA sheets.

15

It is indicated by puru-lent exudate, odour, fever and tissue necrosis.

5

Cultured epi-thelial autograft sheets are more sensitive to the effects ofinfection than SSG.

5

The mechanisms include increased vulner-ability to bacterial proteases and cytotoxins during attachmentand growth of the grafts. Both our patients had infected grafts,evident from the contamination of the dressings, and it wasclear that the graft site had extensive areas of fat necrosis,which required redebridement.

The benefits of CEA sheets predominately relate to the repro-ducibility of grafts without the need for waiting for re-epitheliali-zation of donor sites, minimization of fluid and electrolyte lossand improved cosmetic results reported by some (Table 2).

The keratinocytes from the unburnt skin sample are bankedand further CEA sheets can be produced as required, providingessentially an unlimited amount of tissue for grafting. Culturedepithelial autograft sheets provide a barrier to infection, and limitfluid, electrolyte and temperature loss. In comparison with SSG,hypertrophic scarring has been reported to be less, pigmentationgreater and no difference in respect to erythema, blistering andsuppleness has been reported with the use of CEA sheets.

3,9–11,16

Cultured epithelial autograft sheets heal with a smoother surfaceand avoid the pebbled surface seen with meshed SSG.

Munster, in a prospective series, reported a reduction in mor-tality in patients grafted with CEA sheets compared with controlsfrom 48% to 14% (

P

< 0.007).

13

Opinion differs as to the usefulness of CEA sheets. Some limitits use to that of a biological dressing, whereas others advocateits definitive use as a form of permanent wound coverage.

4

Wefound that its use was limited to a temporary biological dressing asthe take was so poor. Research continues in the formation of ahybrid graft of cultured keratinocytes and dermal components to

which grafts will adhere to more readily. Encouraging results havebeen obtained in the recent development of cultured keratinocytesthat are sprayed onto partial thickness burns or used in conjunctionwith meshed SSG for full thickness burns.

17

Despite our poorresults, we are encouraged by the success of others. In the futurewe shall refine our practice to hopefully improve outcomes.

ACKNOWLEDGEMENT

Many thanks to Ms J. E. Paddle-Ledinek and her team fromMonash University Medical School for their work in the produc-tion of the CEA sheets and guidance in their use.

REFERENCES

1. Rheinwald JG, Green H. Serial cultivation of strains of humanepidermal keratinocytes: the formation of keratinizing coloniesfrom single cells.

Cell

1975;

6

: 331–44.2. O’Connor NE, Mulliken JB, Banks-Schlegal S, Kehinde O,

Green H. Grafting of burns with cultured epithelium preparedfrom autologous epidermal cells.

Lancet

1981;

1

: 75–8.3. Paddle-Ledinek JE, Cruickshank DG, Masterton JP. Skin

replacement by cutured keratinocyte grafts: an Australian experi-ence.

Burns

1997;

23

: 204–11.4. Williamson JS, Snelling CF, Clugston P, Macdonald IB,

Germann E. Cultured epithelial autograft: five years of clinicalexperience with twenty-eight patients.

J. Trauma

1995;

39

:309–19.

5. Odessey R. Addendum: Multicentre experience with culturedepidermal autograft for treatment of burns.

J. Burn Care Rehabil.

1992;

13

: 174–80.6. Haith LR, Patton ML, Goldman WT. Cultured epidermal

autograft and the treatment of the massive burn injury.

J. BurnCare Rehabil.

1992;

13

: 142–6.7. Pandya AN, Woodward B, Parkhouse N. The use of cultured

autologous keratinocytes with integra in the resurfacing of acuteburns.

Plast. Reconstr. Surg.

1998;

102

: 825–8.8. Noordenbos J, Dore C, Hansbrough JF. Safety and efficacy of

TransCyte for the treatment of partial-thickness burns.

J. BurnCare Rehabil.

1999;

20

: 275–81.9. Hansbrough J. Current status of skin replacements for coverage

of extensive burn wounds.

J. Trauma

1990;

30

: S155–60.10. Rivas-Torres MT, Amato D, Arambula-Alvarez H, Kuri-

Harcuch W. Controlled clinical study of skin donor sites anddeep partial-thickness burns treated with cultured epidermal allo-grafts.

Plast. Reconstr. Surg.

1996;

98

: 279–87.11. Hickerson WL, Compton C, Fletchall S, Smith LR. Cultured epi-

dermal autografts and allodermis combination for permanentburn coverage.

Burns

1994;

20

(Suppl. 1.): S52–5.12. Donati L, Magliacani G, Bormioli M, Signorini M, Preis FW.

Clinical experiences with keratinocyte grafts.

Burns

1992;

18

(Suppl. 1.): S19–26.13. Munster AM. Cultured skin for massive burns.

Ann. Surg.

1996;

224

: 372–7.14. Siwy BK, Compton CC. Cultured epidermis.

J. Burn Care Reha-bil.

1992;

13

: 130–7.15. Blight A, Mountford EM, Chesire IM, Clancy JM, Levick PL.

Treatment of full skin thickness burn injury using cultured epi-thelial grafts.

Burns

1991;

17

: 495–8.16. Boyce ST, Goretsky MJ, Greenhalgh DG, Kagan RJ, Rieman

MT, Warden GD. Comparative assessment of cultured skin sub-stitutes and native skin autograft for treatment of full-thicknessburns.

Ann. Surg.

1995;

222

: 743–52.17. Navarro FA, Stoner ML, Park CS

et al.

Sprayed keratinocytesuspensions accelerate epidermal coverage in a porcine micro-wound model.

J. Burn Care Rehabil.

2000;

21

: 513–18.