Eur J Plast Surg (1994) 17:312-315 European " ~ 1 ,dO Journal of m.~' | 6"~ ~.~r~ 4rb

© Springer-Verlag 1994

Grafting of autologous and allogenic cultured epithelium after excision of tattoos N. K u m a g a i

Department of Plastic and Reconstructive Surgery, St. Marianna University School of Medicine, Kawasaki-Shi, Japan

Summary. Two pa t ien t s wi th large t a t t oos were t r ea ted wi th a u t o l o g o u s (auto) and c ryoprese rved a l logeneic (al- Io) cu l tu red ep i the l ium (CE) grafts. These pa t ien t s were fo l lowed up for 21/2 years. The t a t t o o e d areas were ex- cised, a n d the w o u n d s were covered symmet r i ca l l y wi th au to and c ryop re se rved al lo CE grafts. The w o u n d heal- ing of the g r a t ed sites, a p p e a r a n c e and tex ture were simi- lar. In pa t i en t 1, the graf ted sites wi th bo th CE re sembled ad jacen t n o r m a l skin. In pa t i en t 2, there was m i n i m a l scar f o r m a t i o n in b o t h sites. In conclus ion, the excised t a t t o o w o u n d s cou ld be successfully t r ea ted using b o t h types of grafts.

Key words: Cell cu l ture - E p i d e r m a l cells - T ransp l a n t a - t ion - Ta t too

M a n y surgical techniques for t a t t o o r emova l have been used. These techniques inc lude superf ic ial d e r m a b r a s i o n [1, 7], t angen t i a l excis ion wi th a spl i t th ickness skin graft, a n d laser [5]. However , it is genera l ly difficult to t rea t a large t a t t o o where the p igmen t is depos i t ed in the deep dermis o r in the subcu t aneous tissue. Two t a t t o o e d pa- t ients have been successfully t r ea ted wi th au to CE and c ryop re se rved al lo CE. This r e p o r t descr ibes the results o b t a i n e d f rom the s tudy.

Human epidermal cell culture

A small piece of partial thickness skin was harvested with a der- matome under local anesthesia from a site adjacent to the tattooed area. Human epidermal cells were cultured using Green's method [6]. The cells were inoculated into culture dishes (Falcon: 100 mm dish) containing a lethally irradiated 3T3.J2 feeder layer. After three weeks of cultivation (average - two passage), the epidermal cells became confluent and multilayered. At this stage, the epithelium was detached from the dish using dispase, 300 PU/ml (Godo Shusei Co). The epithelial sheets were washed in PBS solution and mountd onto the carrier, basal site up. Chitin membrane (Yunichika Co.), made from crab shell extract, were usually used as the carrier.

Cryopreservation of cultured epithelium

Cryopreserved CE were used as allografts. These grafts were ob- tained from the normal skin of other patients who were tested negative for viral hepatitis, HIV, adult T cell leukemia and syphilitic infection.

After treatment with dispase, the cultured grafts on the chitin membrane were immersed in the solution containing 10% fetal bovine serum (HyClone), 10% glycerol (Wako-Jyunnyaku) and Dulbeco's Modified Eagle's medium (GIBCO). The grafts in the culture dishes were gradually frozen at 4 ° C, 2 hours, - 8 0 ° C, overnight and stored at - 135°C in the deep freezer (Sanyo Co., Japan).

On the day of grafting, frozen CE were rapidly thawed in a water bath at 37 ° C, the medium was removed and the CE were washed in PBS solution.

Materials and methods

Patients

Two patients were males 58 and 19 years old, respectively. The tattooed area were the arm, shoulder, upper chest (patient 1) and back (patient 2). The patients were treated and followed up for more than two years.

Correspondence to: N. Kumagai, MD, Department of Plastic and Reconstructive Surgery, St. Marianna University School of Medicine, 2-16-1, Sugao, Miyamae-ku, Kawasaki-Shi, 216, Japan

Surgical technique

Pigments deposited in the deep dermal layer were removed by serial shave excision using a free hand dermatome. Linear or spotty pig- ment deposited in the fatty tissue was excised and punched out. The wound surfaces were covered with epinephrine-impregnated gauzes for about five minutes. The bleeding points were coagulated com- pletely. The CE grafts were then applied and the chitin membranes were removed. The grafted areas were dressed with vaseline and dry gauze. The grafted sites on the arm were immobilized with a plaster bandage for one week. The first dressing change was usually per- formed at one week after grafting. The vaseline gauzes were careful- ly removed. The "take" of the auto CE grafts and the adherence of

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Fig. l a-d. Treatment of an excised tattoo with cryopreserved allo CE (Patient 1). a Preoperative (left upper arm). b After excision of tattoo. 360 cm 2 of allo CE were applied. c 23 days after grafting. The wound healed completely, d 7 months after grafting

Fig. 2a-d. Histological findings taken from allo CE grafted site (Patient 1). (H&E: x 200). a 7 days post-grafting, b 21 days post-grafting, c 7 months post-grafting, d 11/2 years post- grafting

Fig. 3a, b. Appearance of the auto and allo CE grafted sites (Patient 1). a Preoperative. b Left upper arm: 21/2 years after allo CE grafting. Right upper arm: 2 years after auto CE grafting

Fig. 4a, b. Histology of auto and allo CE grafted sites 3 years after first operation, a Allo CE grafted site. b Auto CE grafted site

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Fig. 5 a-ft. Treatment of excised tattoo with auto and cryopreserved allo CE (Patient 2). a Preoperative. b Auto CE was applied to the left side of the back, and cryopreserved allo CE was applied to right

side of the back. e 13 days after grafting. Allo CE grafted site shows faster healing than auto CE site. d 21/2 years after grafting

the allo CE grafts were evaluated clinically. The wound was covered again using the above dressing methods. The time to complete healing was evaluated in the auto- and allo grafted areas. After completion of the epithelialization, the grafts were managed as if they were conventional skin grafts.

Histology

Biopsies were taken from the grafted area. Sections were prepared and stained with hematoxylin-eosin (H&E) and periodic acid-Shift (PAS).

Results

Cases

Patient 1: A 58-year-old male, with tat toos on his chest, upper limbs and deltoid areas bilaterally. The tatoo on the left arm was excised down to the deep dermal layer, and in some areas, the pigment was excised from the fatty tissue. The allo grafts which were applied to the wounds had been preserved for the three months in the deep freeze. The viability of this CE was assessed by flow cy- tometry one year after cryopreservation. The viability of the epidermal cells were 80%.

The allo CE grafted site (grafted area - 360 cm 2) was reepithelialized completely 23 days after grafting. There was no clinical evidence of rejection (Fig. 1 a-d). There was minimal scar formation and no tendency toward scar contracture after one year. The grafted site became soft. Biopsies taken from the allo grafted site showed no evi- dence of immune rejection using the H&E staining (Fig. 2).

Six months after the first operation, auto CE grafts (grafted area - 1080 cm 2) were applied to the wounds of the right upper arm and left elbow. The graft "take" was 80%, and the wounds healed 20 days after grafting. It

showed the same type of wound healing clinically as in allo grafted sites.

The cosmetic results of both grafted sites were the same, and they resembled the adjacent normal skin with minimal marginal scar 21/2 years after the first operation (Fig. 3).

Three years after the first operation, biopsy specimens were taken from both upper arms. The epithelium of allo CE grafted site was well differentiated and auto CE graft- ed site was hypoplastic. Rete ridge were not observed at both of the grafted sites (Fig. 4).

Patient 2: A 19-year-old male had a large tat too on the back and buttock. The ta t too on the back was excised completely as in Patient 1. The auto CE (grafted area - 1080 cm 2) was applied to the left, while the cryopreserved allo CE (grafted area - 900 cm 2) was applied to the right side of the back. The allo CE used here had been pre- served for about one month in the deep freeze. The viabil- ity of the epidermal cells was not investigated. This pa- tient was kept in the supine position for 10 days after grafting.

The graft "take" of auto CE was 70%. The auto CE grafted site healed 29 days after grafting, while the allo CE grafted site healed in 20 days. Figure 5 shows the clinical course of this patient. The course of wound heal- ing and the appearance were similar in both grafted sites 21/2 years after grafting. There was minimal scar forma- tion.

Discussion

There are many methods of ta t too removal, such as su- perficial dermabrasion [1, 7], tangential excision, and the application of a split thickness skin graft or meshed graft and laser [5]. In our cases, the pigments were deposited in the deep dermal layer and in the subcutaneous tissue.

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The tat tooed areas were large. Therefore, it is difficult to treat them with superficial dermabrasion or laser, be- cause these procedure do not achieve complete removal of the tattoo and the wounds undergo hypertrophic scar formation and contracture. A sure method of removing large tattoos is tangential excision with sheet or meshed grafts. However, treatment is difficult, because of the large amount of skin grafts required and the donor site wound.

Human epidermal cells obtained from small skin sam- ples can be cultured to produce coherent epithelial sheets sufficient to cover the entire body surface [2]. This proce- dure enabled us to treat large tattoos. Nanchahal et al. first reported tat too cases treated with cultured com- posite skin grafts [4]. They applied these grafts to small full thickness skin wounds less than 30 cm 2. The grafted area had a fully differentiated epidermis and a mature dermis withoug adnexae; our cases, however, had large tattoos. Until now, there have been no reports on the results of large area of tattoo removal. Furthermore, there have been no comparative studies of wound healing and cosmetic appearance areas treated with both auto CE or allo CE. This study of tattoo patients was designed to show the results of both type of grafting.

Most tattoo pigments were deposited in the deep der- mal layer. The dermal component and the epidermal ap- pendages were partially preserved during the excision of the tattoo. This is important, as the residual dermal com- ponent plays an important role in take of the grafts and the reconstitution of the skin. Auto CE took almost com- pletely to the excised wounds. The basement membrane was formed within one week of grafting by interaction of auto CE and preserved dermal components. As a result, the regenerated epidermis became nonfragile within one month after grafting. Furthermore, this procedure re- duced the pain, because of early closure of the excised wounds.

Allo CE is acknowledged as a temporary biological dressing material [3]. However, the effects on the wound healing were remarkable. The cryopreserved allo CE

placed on the excised tat too adhered well and exhibited a favorable healing as shown in this study. The number of days required for healing was almost identical compared to auto CE grafting. Furthermore, allo CE showed no evidence of classical rejection either macroscopically, in the form of erythema or blistering, or microscopically, such as leucocyte invasion and epithelial vacuolation.

The graft appearance and texture in both types of grafting resemble each other. This is probably because the donor skin was taken from the adjacent tattooed area and the epidermis reepithelialized in the allo CE grafting was reconstituted by the epidermal cells in the epidermal appendages of the recipient bed. The findings also show that cryopreserved allo CE with good cell viability is as suitable a cover for wounds with preserved epidermal appendages as auto CE grafts.

Acknowledgements. The author wishes to thank Professor Howard Green for kindly donating 3T3.J2 cell line.

References

1. Boo-Chai K (1963) The decorative tattoo: its removal by der- mabrasion. Plast Reconstr Surg 32:559

2. Green H, Kehinde O, Thomas J (1979) Growth of cultured hu- man epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 76:5665

3. Merrwe AE, Mattheyse F J, Bedford M, Heldem PD, Rossouw DJ (1990) Allografted keratinocytes used to accelerate the treatment of burn wounds are replaced by recipient cells. Burns 16:193

4. Nanchahal J, Otto W, Dover R, Dhital SK (1989) Cultured com- posite skin grafts: biological skin equivalents permitting massive expansion. Lancet 22:191

5. Reid WH, Miller ID, Murphy MJ, Paul JP, Evans JH (1990) Q-wiched ruby laser treatment of tattoos : a 9-year experience. Br J Plast Surg 43:663

6. Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331

7. Wheeler ES, Miller TA ,(1976) Tattoo removal by split thickness tangential excision. West J Med 124:272