Dermatology Online Journal

Case report: A case of pyoderma gangrenosum with intractable leg ulcers treated by allogeneic cultured dermal substitutes
Seiko Toyozawa¹, Yuki Yamamoto¹,Takeshi Nishide¹, Akiko Kishioka¹, Nobuo Kanazawa¹, Yasuhiro Matsumoto², Yoshimitsu Kuroyanagi², Fukumi Furukawa¹
Dermatology Online Journal 14 (11): 17

1. Department of Dermatology, Wakayama Medical University, Wakayama, Japan. seikotoy@wakayama-med.ac.jp
2. R&D Center for Artificial Skin, School of Allied Health Sciences, Kitasato University, Kanagawa, Japan

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Abstract

Pyoderma gangrenosum (PG) is an idiopathic, inflammatory, ulcerative disease of undetermined cause. Both topical and systemic treatments of corticosteroids and cyclosporine are commonly used for the ulcers of PG, but these ulcers are often intractable despite treatment. We employed allogeneic cultured dermal substitutes (CDS) in a patient with intractable ulcers due to PG. The CDS was prepared by cultured human fibroblasts on two-layered sponges of extracellular matrix such as hyaluronic acid and atelo-collagen. In the present case, re-epithelization and healthy granulation were induced by the CDS without increasing the dosage of systemic prednisolone. Then the subsequent autologous skin graft was successfully performed. This indicates that CDS is one of the useful tools for the treatment of intractable ulcers in patients with PG.

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Introduction

Recently, a variety of skin substitutes have been developed for the treatment of skin ulcers [1, 2, 3, 4, 5]. In Japan, Kuroyanagi and his group developed novel allogeneic cultured dermal substitutes (CDS) and clinical trials of this CDS have been carried out in 31 medical centers across Japan with the support of the Millennium Project of the Japanese Ministry of Health, Labor and Welfare [6]. These CDS were prepared by culturing fibroblasts on a two-layered, spongy matrix of hyaluronic acid and atelo-collagen. Allogeneic CDS fail to survive permanently on the wound surface, but can release several growth factors and extracellular matrix components such as fibronectin, which are necessary for wound healing. Another characteristic of the CDS is that the spongy matrix itself, consisting of hyaluronic acid and atelo-collagen, promote the healing of severe skin defects. The results of a multi-center clinical study suggested that this allogeneic CDS could provide an excellent wound bed [7, 8].

The pathogenesis of pyoderma gangrenosum (PG) is poorly understood and various diseases such as inflammatory bowel disease, arthritis, paraproteinemias, and hematologic malignancies are associated with PG [9]. The ulcers that occur in patients with PG are often intractable. Generally, the systemic administration of corticosteroids is the mainstay of treatment for PG. Pulse therapies of corticosteroids or cyclophosphamide, plus cyclosporine are administered in severe cases. Recently, we applied these allogeneic CDS to a patient with intractable ulcer due to PG. In our case, conventional topical therapies had not been effective and the daily dosage of corticosteroids could not be increased due to side effects. Herein we describe the clinical details of a case of PG with intractable leg ulcer.

Case report

Figure 1
Figure 1. Leg ulcer (10 x 5 cm) on admission of a 69-year-old female

A 69-year-old female noticed an ulcer (2 x 2 cm) on her left lower leg in June, 2005. The patient had a past history of rheumatoid arthritis (RA) for 25 years. She had been treated with various topical agents, but the size of the ulcer increased. She visited our department on August 24, 2005. We observed an ulcer (10 x 5 cm) on her left lower leg, the bottom of which contained a yellow necrotic substance (Fig. 1). Laboratory investigation showed a slight inflammatory reaction and anemia, but rheumatoid factor was negative. A skin biopsy specimen obtained from a lesion of the left lower leg showed a neutrophilic infiltration in both superficial and deep dermis (Figs. 2 & 3). No vasculitis was seen. Bacterial culture obtained from the ulcer at the first visit was positive for Pseudomonas aeruginosa, however it became negative after 2 weeks of saline washes without using antibiotics. She was taking prednisolone at a dose of 10mg per day.

Figure 2	Figure 3

Figure 4
Figure 4. Debrided wound surface

Physical examinations and blood tests to evaluate the patient for other associated causes of PG were negative and thus we diagnosed her as PG complicated by RA. Although systemic prednisolone was administered up to 30 mg/day for PG on admission, the ulcer showed no wound healing. On September 16, we started to apply the CDS to the ulcer after debridement (Fig. 4). The wound size decreased and healthy granulation tissue was formed after 6 weeks of CDS treatment (Fig 5.), but a tendon remained exposed on the surface of the wound. After 2 more weeks of treatment with the CDS, an autologous skin graft was performed on November 25, 2005. Two months later, the grafted skin showed complete re-epithelization (Fig 6.). The clinical course was followed up for 2 years (continuing prednisolone at 10mg/day); no recurrence of the ulcer occurred.

Figure 5	Figure 6
Figure 5. Wound appearance after 6 weeks of treatment with allogeneic CDS Figure 6. Clinical appearance at 2 months after autologous skin grafting

Preparation of cultured dermal substitutes(CDS)

The spongy matrix was prepared by the method described in our previous articles [10, 11, 12, 13]. The piece of skin used in these CDS was derived from a young patient during surgical excision. All procedures were in compliance with the ethical guidelines of Osaka Medical College. The method by which the fibroblasts were obtained are described in our previous our articles [6, 11, 12], and were cultivated in culture medium to establish cell banking in liquid nitrogen. The cells were checked for viruses such as HBV, HCV, HIV, HTLV-1, and parvovirus.

Fibroblasts obtained from successive cultivation of the cryo-preserved cells were seeded onto the two-layered sponge. The number of fibroblasts on the two-layered sponge was adjusted to 1x10⁵ cells/cm². Then, the CDS were treated in the manner described in our previous articles [11, 12]. The CDS were frozen in the culture dish, then cryopreserved in a freezer at -152°C [6, 15], and then shipped to the Wakayama Medical University Hospital where it was preserved at -85°C. Before clinical application, the CDS were thawed by the method described in our previous articles [6, 15].

Prior to using allogeneic CDS, all wounds with necrotic tissue were debrided and rinsed sufficiently with saline solution. The CDS was placed cell-seeded side down onto the wound surface and conventional ointment-gauze dressing and sterile dry gauze dressings were applied to protect the CDS. The CDS were applied repeatedly at intervals of 3-5 days. Without any signs of infection on the wound surface, the interval between dressing changes was increased to 5-7 days. The study protocol was approved by the Ethics Committee of the Wakayama Medical University Hospital and each patient gave their written informed consent.

Discussion

Pyoderma gangrenosum (PG) was first described by Brunsting, Goeckerman, and O'Leary in 1930 [15]. PG is an idiopathic inflammatory disease of unknown etiology, frequently associated with an underlying systemic condition such as inflammatory bowel disease, hematological malignancy, rheumatoid arthritis, and paraproteinemias [16]. No single or effective treatment exists and few controlled trials for treatment have been done. Most clinicians use a stepwise approach with both topical and systemic treatments. Immunosuppression is the mainstay of treatment and the most commonly used drugs are corticosteroids and cyclosporine [17]. Topical treatments such as highly potent topical corticosteroids (occasionally underneath occlusive dressings), and injections of triamcinolone are often used adjunctively [16].

This patient was being treated with prednisolone for rheumatoid arthritis. The dosage of the prednisolone was increased after admission but that was not effective. In addition, various topical treatments were not effective. It was difficult to increase the dosage of the prednisolone further because of side effects and we used the allogeneic CDS as a non-invasive treatment at the sites where the mild debridement was performed.

A few reports have supported the usefulness of split-thickness skin grafting during concurrent systemic corticosteroid therapy [18, 19, 20]. Cultured keratinocyte autografts and allografts have also been reported to be useful in some cases [21, 22]. Our present case is the first successful one in which allogeneic cultured fibroblasts were applied to leg ulcers of PG.

Although allogeneic CDS fail to survive permanently on the patient's skin defect, these CDS produce a variety of biologically-active substances [23]. The CDS is characterized by a scaffold for cell implantation that promotes wound healing by itself. This case report suggests that these allogeneic CDS are useful for the treatment of intractable skin defects in patients with PG.

References

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