Hirotomo Ishida

Clinical application of autologous cultured epithelia for the treatment of burn wounds and burn scars.

This report presents our experience with autologous cultured human epithelia grafting on burn wounds, burn scars, and skin-graft donor sites in seven patients. Dispersed epidermal cells were cultured with 3T3 cells treated with mitomycin C. After 2 to 3 weeks, cultured epithelia (total 350 to 2250 cm2) were grafted to the wound. The results showed that cultured epithelia grafts did not take so completely compared to the meshed skin grafts used for the coverage of burn wounds. However, cultured grafts placed on aseptic wounds adhered well and showed good appearance. In the histologic findings, normal differentiation of epidermal cells was found. Cultured grafts were bordered from subepidermal granulative tissue with basement membrane. A rete ridge and the adnexal structures were absent in the specimens that adhered to the burn wounds. However, in the specimens that took on abraded wounds, a gently sloping rete ridge and elastic fibers were seen. The histologic findings showed structures resembling normal skin.

Suppressive effects of tranilast on the expression of inducible cyclooxygenase (COX2) in interleukin-1β-stimulated fibroblasts

We investigated the effects of tranilast on inducible cyclooxygenase (COX2)-mediated prostaglandin E2 (PGE2) production and enzyme induction in interleukin-lbeta (IL-1beta)-stimulated cultured dermal fibroblasts. IL-1beta enhanced PGE2 production in cultured fibroblasts. Tranilast did not affect constitutive cyclooxygenase (COX1) or COX2 activity in non-stimulated or IL-lbeta-stimulated fibroblasts. However, the COX2 expression induced by IL-1beta was inhibited by tranilast. This result, that IL-1beta-induced COX2 expression was suppressed by tranilast, was confirmed by immunohistochemical analysis. Thus, it is possible for tranilast to regulate PGE2 production by inhibiting COX2 induction.

Physiological roles of endothelium-derived nitric oxide in the epigastric island flaps of rabbits.

Nitric oxide (NO), identified as the mediator of endothelium-dependent relaxation of vascular smooth muscle, is known to cause a number of inflammatory conditions, especially in ischemla/reperfusion injury. This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia followed by reperfusion-influenced serum NO and c-GMP concentrations in the flap. In addition, we also investigated the premedicated effects of the NO synthase inhibitor and heparin on serum NO and c-GMP concentrations in skin flap ischemia/reperfusion. Serum NO concentration after 15,30,45, and 60 minutes of ischemia followed by reperfusion significantly increased compared with that in nonischemic control and elevated flaps. On the contrary, serum NO concentration was suppressed in L-NAME or aminoguanidine pretreated animals with ischemic group. Administration of heparin increased the serum NO concentration in elevated flaps, but suppressed it in ischemic flaps followed by reperfusion. The changes in serum c-GMP and NO concentrations were related in all of the experimental groups. These results suggest that NO may be derived from vascular endothellal cells and dilate peripheral vessels in compensation for ischemia.

Eye socket reconstruction with free flaps in patients who have had postoperative radiotherapy

It is difficult to fit an ocular prosthesis and correct periorbital depression deformity in a patient who has undergone radiotherapy after enucleation of the eye for retinoblastoma during infancy. Simultaneously, developmental disturbances of the eyelids and severe periorbital depression deformities develop. However, due to recent advances in microvascular surgery, free flap transplantation is increasingly used for eye socket reconstruction because of its good circulatory effects with enough volume to augment large periorbital tissue defects. In the present study, we report on our success in using a free groin flap, scapular flaps and parascapular flaps simultaneously for reconstruction of eye sockets and correction of depressed deformities.

Grafting of autologous-cultured epithelium after excision of tattoos

Nine patients with tattoos were treated with cultured epidermal autografts. The tattoos were excised down to the deep dermal layer, and the wounds were covered with cultured epidermal autografts. The areas grafted with cultured epidermal autografts were an average of 907 cm2 in size. The average percentage “take” of the cultured epidermal autografts was 86%. The average time for complete wound epithelialization was 20 days. The pain at the grafted sites was minimal. There was minimal scarring and no tendency for scar contracture. The appearance and elasticity of the grafted sites resembled those of adjacent normal skin 2 1/2 years after grafting. These findings demonstrate that cultured epithelium autografting is a suitable method for coverage of the tangentially excised tattoo wound.

Treatment of giant congenital nevi with cryopreserved allogeneic skin and fresh autologous cultured epithelium

Giant congenital nevi, confluent over 10% to 52% of the body surface area, were treated in 5 pediatric patients with allogeneic skin and autologous cultured epithelium (ACE). The lesions were excised to the adipose tissue and the wound was covered with cryopreserved or fresh allografted skin. Later, the skin surface was abraded and ACE was applied. The size of the cultured graft ranged from 360 to 900 cm2 (average, 630 cm2). In 3 patients the wounds healed well. Allograft cryopreservation averaged 50 days. The average interval to ACE application after skin grafting was 10 days. Histological rejection was minimal. In 2 patients the cultured grafts did not take. The wounds were covered with split-thickness skin grafts. In one such patient, a fresh allograft was used. In the other, ACE was applied 27 days after skin grafting. Vigorous rejection reaction was observed. Cryopreservation of allogeneic skin may be important in the treatment of human leukocyte antigen-mismatch patients with giant nevi, and ACE should be applied within 10 days of skin grafting.

Treatment of nevus of Ota with autologous-cultured epithelium grafting combined with dermabrasion.

Nevus of Ota is an anomaly of the dermal melanocytes, which lies primarily in the deep dermis. It causes discoloration of the face and induces a remarkable disfigurement to the patients. This report demonstrates the favorable results of 3 male patients treated with autologous-cultured epithelium grafting combined with dermabrasion. The lesions on the cheek, forehead, and temple were dermabraded to the deep dermal layer. The cultured epithelium obtained from preauricular skin or medial upper arm were grafted on the wounds. The grafts took well, and most of the grafted site healed after 1 week. Facial appearances improved remarkably. There was minimal scar formation and minimal recurrence of nevus cell. After long-term follow-up, the grafted sites resembled the adjacent normal face skin in terms of appearance and texture.

The effects of endothelin-1 on flap necrosis.

In reconstructive surgery, flap necrosis is a major obstacle. The vasospasm in microvascular circulation can be one of the causes of flap necrosis. The vascular endothelium has an important role in the regulation of vascular tone by producing vasodilating and vasoconstricting factors. Endothelin-1, a 21-amino acid polypeptide isolated from the vascular endothelium, is reported to be one of the most potent vasoconstrictors. The purpose of this study was to determine the significance of endothelin-1 on flap necrosis using a rat skin flap. First, we introduced into the experiment endothelin-1 and ETA receptor antagonists, FR139317, then we tried to determine the time course changes of endothelin-1 levels in the flap. Endothelin-1 antagonist had no effect on survival length, although endothelin-1 injection clearly reduced survival length. The combined treatment of antagonist and injection suppressed the effect of endothelin-1 in reducing survival length. Endothelin-1 levels in the flap base significantly increased from 6 hours, and its levels in the flap periphery presented no remarkable changes. These results may suggest that endothelin-1, especially that produced in the flap base, exerts a major effect on flap microcirculation.

Changes in endothelin-1, 6-Keto-PG-F1α, and TX-B2 in random pattern flaps

In this study we investigated kinetic changes in the ratio of endothelin-1 (ET-1) and eicosanoids (6-keto-PG-F 1α / TX-B 2 ) in 20 x 60 mm random pattern flaps from rats. The ET-1 content of regions A (20 mm from the peripheral end) and B (2040 mm) 6 h after surgery tended to decrease slightly compared to the ET-1 content immediately after surgery. The ET-1 content of region C (20 mm from the flap base) 6 h postoperatively increased significantly compared to that immediately after surgery. The ET-1 content of region C 6 h after surgery was significantly higher compared to that of regions A and B, which were obtained simultaneously. The ratios of eicosanoids in the three regions 6 h after surgery were significantly lower than those immediately after operation. However. the ratio in region A was higher than that in region C, showing that there was a difference in distribution in the flap between ET-1 and eicosanoids. The administration of an ET A receptor antagonist, FR-139317, extended the survival length of the flap. These results suggest that ET-1 can regulate the microcirculation in a flap directly and/or indirectly.

Favorable donor site for epidermal cultivation for the treatment of burn scars with autologous cultured epithelium

It is very important to determine from where we select the donor skin for epidermal cultivation in the treatment of burn scar disfigurement. To prove this point, we compared the appearance and histology of grafted sites according to the different donor sites. Thirty-eight patients with skin color difference and irregular contours of matured burn scars were superficially abraded and underwent autologous cultured epithelial grafting. These patients were followed more than 2 years. The donor skin for epidermal cultivations was taken from the buttock (group 1, 8 patients), sole (group 2, 6 patients), and adjacent to the site of the scar (group 3, 24 patients). In group 3, skin elasticity was also measured after 2 years. Hypo- and hyperpigmentation were well treated with autologous cultured epithelial grafting. The most favorable results in terms of color match were obtained in group 3, where the skin color resembled surrounding normal skin. The skin tension returned to almost normal. In group 2, the histology of the grafted site resembled the sole epidermis. It was concluded that the general principle of conventional skin grafting (i.e., “closer is best”) was also correct in cultured epithelial grafting. Furthermore, it was revealed that cultured epithelium has a site specificity even after grafting.