Application of the cultured epidermal autograft "JACE(®") for treatment of severe burns: Results of a 6-year multicenter surveillance in Japan.

BACKGROUND: In the 1970s, Green et al. developed a method that involved culturing keratinocyte sheets and used for treatment of burns. Since then, the take rate of cultured epidermal autograft (CEA) onto fascia, granulation tissue, or allografts has been extensively reported, while that on an artificial dermis in a large case series is not. Moreover, the contribution of CEA to patient survival has not been analyzed in a multicenter study. METHODS: We conducted a 6-year multicenter surveillance on the application of the CEA "JACE(®") for treatment of burns >30% total body surface area (TBSA) across 118 Japanese hospitals. This surveillance included 216 patients and 718 graft sites for efficacy analysis. The CEA take rate at 4 weeks after grafting was evaluated, and safety was monitored until 52 weeks. In addition, the survival curve obtained in this study and the data obtained from the Tokyo Burn Unit Association (TBUA) were compared. RESULTS: The mean CEA take rates at week 4 were 66% (sites) and 68% (patients), and the rate on the artificial dermis was 65% for 226 sites. CEA application combined with wide split-thickness auto or patch autograft increased the CEA take rate. On comparison with the data obtained from the TBUA, which included data on individuals with burns of the same severity, CEA application was found to contribute to patient survival until 7 weeks after burn. CONCLUSIONS: We reported the take rate of CEA based on a 6-year multicenter surveillance. From our results, we found that the application of CEA is a useful treatment for the patients with extensive burns.

Changes in the expression of epidermal differentiation markers at sites where cultured epithelial autografts were transplanted onto wounds from burn scar excision.

This study investigated the recovery process during which grafted cultured epithelium formed normal epidermis. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) after transplantation. These samples were stained for monoclonal antibodies against filaggrin, transglutaminase (TG), cytokeratin 6 and involucrin. Their expressions were examined in the epidermis. The expression patterns were classified using a six-grade scale. The grades of filaggrin and TG were significantly higher at stage 3 and 4 compared with stage 1. There was a marginally significant increase in the grade of cytokeratin 6 at stage 3 and it was significantly higher at stage 4 compared with stage 1. These results showed that wound healing continued at a molecular level until the end of stage 3. The recovery of involucrin was delayed compared with that of other markers. TG and involucrin are thought to be regulated independently at the grafted sites.

Changes in fibrillin-1 expression, elastin expression and skin surface texture at sites of cultured epithelial autograft transplantation onto wounds from burn scar excision.

This study investigated the recovery process during which grafted cultured epithelium generated skin elasticity and skin surface microarchitecture. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) of transplantation. These samples were evaluated by taking replicas of skin surface, and histological changes of fibrillin-1 and elastin. The expression patterns were classified using a grading scale. The grade of skin surface texture was significantly higher at stage 3 and marginally significantly higher at stage 4 compared with stage 1. The grade of fibrillin-1 was marginally significantly higher at stage 3 and significantly higher at stage 4 compared with stage 1. The grade of elastin was marginally significantly higher at stage 4 compared with stage 1. These results showed that it is important for patients to have skin care and avoid external forces for at least 18 months after transplantation.

Treatment of vitiligo with autologous cultured keratinocytes in 27 cases.

BACKGROUND: Vitiligo is an acquired depigmentation of the skin characterized by white spots with well-defined margins, causing psychological stress in patients due to cosmetic concerns. We examined 27 patients who underwent vitiligo treatment using autologous cultured keratinocytes. METHODS: The study comprised 20 patients with segmental vitiligo and seven patients with generalized vitiligo, and they were followed up for at least 1 year postoperatively. In all 27 cases, topical steroid or ultraviolet therapy had been previously performed by dermatologists, but this treatment had been ineffective. The patients' vitiligo had stabilized. The patients were treated using keratinocytes obtained from primary culture using Green's techniques or from first passage. Dispase treatment was used to detach the stratified cultured epithelial sheets from their culture dishes. The detached sheets shrank to approximately one half to two thirds of their original size on the culture dish. After the recipient site was completely epithelialized, the skin was exposed to sunlight. RESULTS: For patients with segmental vitiligo, 12 had a good therapeutic outcome (90 % or more repigmentation) after the first surgery. This number increased to 14 when patients with multiple surgeries were included. There were six patients with fair outcomes (50-90 % repigmentation), and no patients with poor outcomes (50 % or less repigmentation). For patients with generalized vitiligo, no patients had a good outcome despite multiple surgeries. There were three patients with fair outcomes, and four patients with no change outcomes. CONCLUSIONS: Cultured keratinocyte grafting was a more effective treatment for segmental vitiligo than for generalized vitiligo. Level of Evidence: Level IV, therapeutic study.

Improvement of hind-limb paralysis following traumatic spinal cord injury in rats by grafting normal human keratinocytes: new cell-therapy strategy for nerve regeneration.

Somatic (adult) stem cells are thought to have pluripotency, just as do embryotic stem (ES) cells. We investigated the possibility that grafted epithelial keratinocytes could induce spinal cord regeneration in an animal model of spinal cord injury (SCI). Normal human keratinocytes were cultured by the routine technique, and normal human dermal fibroblasts were cultured by a similar method as a control group. SCI model was prepared by dropping a 10-g weight onto the exposed spinal cord of rats from a height of 25 mm, and 8 days later, the cultured cells were grafted into the injury site. Motor function was significantly improved in the cultured-keratinocyte-grafted group compared with that in the fibroblast-grafted group. After functional observation, human nestin- and nuclei-positive cells were found at the grafted spinal cord. Grafted cultured keratinocytes induced in vitro morphological changes in the neural induction medium. These results indicated one possibility that some of the grafted cultured keratinocytes survived and could have contributed to neural regeneration. On the other hand, it should be noted that the grafted cultured keratinocytes secreted a large amount of enzymes and/or growth factors. Therefore, another possibility is that the grafted-keratinocyte-derived factors could induce survived cell growth and endogenous neural differentiation of spinal-nerve-derived stem cells surrounding the injured spinal cord, leading to functional recovery. Epithelial stem cell therapy may be applied clinically in the near future to treat SCI.

Activation of heparanase by ultraviolet B irradiation leads to functional loss of basement membrane at the dermal-epidermal junction in human skin.

Recently, we reported that heparanase plays important roles in barrier-disrupted skin, leading to increased interaction of growth factors between epidermis and dermis and facilitating various cutaneous changes, including epidermal hyperplasia and wrinkle formation. However, the role of heparanase in sun-exposed skin remains unknown. Here, we show that heparanase in human keratinocytes is activated by ultraviolet B (UVB) exposure and that heparan sulfate of perlecan is markedly degraded in UVB-irradiated human skin. The degradation of heparan sulfate resulted in a marked reduction of binding activity of the basement membrane for vascular endothelial growth factor, fibroblast growth factor-2 and -7 at the dermal-epidermal junction. Degradation of heparan sulfate was observed not only in acutely UVB-irradiated skin, but also in skin chronically exposed to sun. Interestingly, heparan sulfate was found to be degraded in sun-exposed skin, but not in sun-protected skin. These findings suggest that chronic UVB exposure activates heparanase, leading to degradation of heparan sulfate in the basement membrane and increased growth factor interaction between epidermis and dermis. These changes may facilitate photo-aging.

Application for regenerative medicine of epithelial cell culture-vistas of cultured epithelium.

This review describes culture techniques for the epithelial system as well as trends in the clinical application of cultured keratinocytes in our department and the possibility of applying the techniques to other organs. Cultured epithelium and cultured dermis in particular have considerably preceded regeneration of other organs in the field of regenerative medicine. Since 1988 we have grafted cultured keratinocytes by the Rheinwald-Green modified method in at least 500 patients with large skin defects. As a result of the establishment of a culture technique for individual patients, it is now possible to prepare enough regenerated epithelium to cover the body surface area of as many as 10 adult patients in approximately three weeks after collecting 1 cm(2) of skin, and then remaining cultured keratinocytes can be cryo-preserved for two-stage dermatoplasty at another site. This procedure makes it possible to avoid frequent skin collection from the same patient and thereby improves patients' quality of life and activities of daily living. On the other hand, to solve the problem of regenerated epithelium shrinking and problems with graft efficiency on dermis defect lesion, we have developed a proteinase-resistant regenerated dermis by mixing a certain protein with a fibrin scaffold. Recently we also took the initiative in grafting hybrid-type regenerated trachea in an animal experiment by using the epithelial and dermal cell culture technique, and some results of the graft were obtained.

[Treatment of burns by grafting of cultured epithelium].

The most serious problem in the treatment of extensive burns is a lack of sufficient healthy skin for coverage of the affected area. Several methods have been used for the coverage of extensive burn wounds. The grafting of cultured epithelium is a potentially effective method for a permanent covering of the wound, particularly in patients with extensive burns. The condition of the recipient site is the most important factor in the success of cultured epithelium grafting and the preservation of the dermis or dermal components in the burned area will enhance the grafting process. We recommend that prior to the grafting of cultured epithelial cells, the burn wounds should be excised and covered with an allograft or artificial dermis during the first 2 weeks after admission. An allograft of cultured epithelium is also useful. This method accelerates the epithelialization of both the burn and donor sites. It is expected that cultured epithelial cell grafts will prove to be an effective treatment not only for extensive burns but also to epithelialize small area burn wounds and resurfaced burn scars.

Re-epithelialisation and the possible involvement of the transcription factor, basonuclin.

This article briefly summarises the basic mechanism of re-epithelialisation and discusses the possible role of the cell-type-specific transcription factor, basonuclin. Re-epithelialisation is initiated by a signal resulting from the absence of neighbouring cells at the wound edge. Basal cells at the wound edge become flattened and lose their intercellular desmosomes and substratum attachment. The amount of cytoplasmic actinomyosin filaments that insert into the new adhesion complexes is increased, and contraction of those filaments produces cell movement. The epithelial cells at the wound edge migrate on a provisional matrix using the newly expressed integrin receptors. Once re-epithelialisation is complete, the epithelial cells revert to the normal phenotype of basal epidermal cells, firmly attach to the newly developed basement membrane zone through hemidesmosomes and resume standard differentiation. Protein synthesis increases in the epidermal cells at the wound edge during re-epithelialisation. Active protein synthesis requires accelerated transcription of ribosomal RNA genes. The transcription factor basonuclin binds to the ribosomal RNA gene promoter and increases the transcription of the genes. Therefore, it is speculated that basonuclin in epithelial cells is required in the process of re-epithelialisation.

Determination of nitrotyrosine and related compounds in biological specimens by competitive enzyme immunoassay.

A gas mediator, nitric oxide is converted to peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent oxidant, which injures various tissues and organs by nitration of the tyrosine residues of proteins, and it enhances the late response of inflammation. The determination of nitrated tyrosine, nitrotyrosine, which is a stable final metabolite of peroxynitrite, provides an important indicator of tissue disorders caused by peroxynitrite. This paper reports a competitive solid-phase immunoassay for measuring nitrotyrosine in various biological specimens. In this study, peroxidase-conjugated nitrotyrosine was prepared by reaction of nitrotyrosine with 1,4-benzoquinone treatment, and then it was allowed to compete with nitrotyrosine on an anti-nitrotyrosine antibody-coated 96-well multiplate. No amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at the concentrations higher than 1000 times the concentration of nitrotyrosine. The limit of detection of free nitrotyrosine was approximately 500 pg/mL (2 nM) at a competition ratio (B/B(o)%) of 80%. The newly developed enzyme immunoassay (EIA) method was used for assay of nitrotyrosine in biological specimens, with the following results: (i) Lipopolysaccharide (LPS) activation of RAW264.7 cells induced a significant increase in nitrotyrosine production compared to that with nonactivated cells. N(omega)-nitro-L-arginine methyl ester decreased nitrotyrosine production with either LPS-activated or nonactivated RAW cells. There is a relationship between nitrotyrosine production and nitrite ion. (ii) The nitrotyrosine level detected in the plasma specimens from healthy volunteers was 35.21 +/- 4.87 ng/mL (135.4 +/- 18.7 nM). (iii) The concentration of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients was 41.40 +/- 20.96 ng/mL (159.02 +/- 80.6 nM). Thus, the EIA method combines sensitivity and specificity with the ability to process a large number of specimens to quantify nitrotyrosine produced with in vivo and in vitro sources.

Gigantomastia induced by bucillamine.

Drug-induced mammary hyperplasias have been reported as rare complications of D-penicillamine and Neothetazone. The authors report the first case of bucillamine-induced giant mammary hyperplasia. Bucillamine is used as an antirheumatic drug that is structurally analogous to D-penicillamine. A 25-year-old woman with rheumatoid arthritis for the past 5 years started to develop gradual enlargement of her breasts 15 months before presentation. She had been on a combined treatment of steroid and lobenzarit disodium for the first 3 years, and then continued with a combined treatment of steroid and bucillamine for the following years until she was found to have pulmonary tuberculosis, at which time the steroid was suspended 10 months before she visited the authors' clinic. An almost total breast reduction was performed; 5 kg of right breast tissue and 7 kg of left breast tissue were excised. Retrospectively, bucillamine was believed to be the cause of the giant hypertrophy because of its structural similarity to D-penicillamine, which was the subject of an abundance of reports of mammary hyperplasia.

Permanent restoration of human skin treated with cultured epithelium grafting--wound healing by stem cell based tissue engineering--.

The technique of epidermal cell culture developed by Green and colleagues made a breakthrough in the treatment of massive wounds in vivo with grown cells in vitro. In the past two decades, progress of culture methods and clinical practice have been made and now it is possible to treat extensive skin defect with large amounts of cultured epithelium. Since 1985, we have been successfully used cultured epidermis as autografts for the permanent coverage of full-thickness burn wounds or excised burn scars, giant nevi, tattoos and so on. Furthermore, cultured epidermis has been available as allografts to promote the healing of chronic skin ulcers or deep dermal burn. In this paper we describe our clinical experience of cultured epithelium grafting for the treatment of wounds and predict new trial of wound management and regeneration based on tissue engineering concept.