ARTICLE
Auteur(s) : Seiko
Toyozawa1, Yuki Yamamoto1, Akiko
Kishioka1, Nozomi Yonei1, Nobuo
Kanazawa1, Yasuhiro Matsumoto2, Yoshimitsu
Kuroyanagi2, Fukumi Furukawa1
1Department of Dermatology, School
of Medicine, Wakayama Medical University, 811-1, Kimiidera,
Wakayama, Japan 641-0012
2R&D Center for Artificial Skin, School
of Allied Health Sciences, Kitasato University, Kanagawa,
Japan
accepté le 22 Juin 2009
During the last ten years, a variety of skin substitutes have
been developed for treating skin ulcers [1-5]. Kuroyanagi and his
group developed novel allogeneic cultured dermal substitutes (CDSs)
with the support of the Regenerating Medical Millennium Project of
the Japanese Ministry of Health, Labor and Welfare [6]. CDSs were
prepared by culturing fibroblasts on two-layered, spongy matrices
of hyaluronic acid (HA) and atelo-collagen (Col). Allogeneic CDSs
did not permanently survive on wound surfaces, but released many
biologically active substances and extracellular matrix components,
which are necessary for wound healing. CDSs aid spongy matrices by
promoting the healing of severe skin defects due to burn wounds,
ulcers, traumatic skin injures and cutaneous vasculitis [7, 8].
Systemic lupus erythematosus (SLE) is a disease of unknown
etiology in which tissues and cells are damaged by autoimmune
inflammation. Skin ulcerations and gangrene may occur as a result
of active vasculitis, an association with an antiphospholipid
syndrome or both conditions. Vasculitic leg ulcers are present in
about 5% of SLE patients [9]. Ulcers in patients with SLE are
intractable or non-healing due to various factors including
vasculitis and the use of immunosuppressant therapies.
In this report, we describe the effects of CDSs on ulcers from 3
SLE subjects who lacked antiphospholipid antibodies and were
resistant to various conventional therapies, including basic
fibroblast growth factor (bFGF), and prostagrandinE1
ointment.
Materials and methods
Preparation of cultured dermal substitutes (CDSs)
Human cultured fibroblasts were obtained as previously described
[6, 11, 12], and preserved in liquid nitrogen (LN2).
Cells were checked for viruses such as HBV, HCV, HIV, HTLV-1 and
parvovirus. Spongy matrices were prepared by a method described in
previous studies [10-13].
Cultured fibroblasts obtained from successively cultivating
cryopreserved cells were seeded onto two-layered sponges (1 ×
105 cells/cm2). Afterwards, CDSs were
cultured for 1week in culture medium supplemented with 10% serum
[11, 12]. CDSs were frozen in culture dishes, and cryopreserved in
a freezer at – 152 °C [6, 14]. Before clinical
application, CDSs were thawed as previously described [6, 14].
Briefly, cryopreserved CDSs were thawed by placing a polystyrene
dish containing cryopreserved CDSs in a foam polystyrene box at
room temperature for 30 min, and then submerged in a water
bath at 37 °C, followed by rinsing with lactated
Ringer’s solution 3 times to remove extra substances [6, 14].
Application of allogeneic CDSs
Prior to using allogeneic CDSs, necrotic tissues within wounds were
surgically removed and rinsed with saline solution. CDSs were
placed cell-seeded side down onto wound surfaces at intervals of
3-7 days. Conventional ointment-gauze dressings and sterile
dry gauze dressings were applied to protect the CDSs. The study
protocol was approved by the ethics committee of the Wakayama
Medical University Hospital, and each study subject provided
written informed consent.
Case reports
Three cases fulfilled the SLE criteria outlined by the American
College of Rheumatology, but had no antiphospholipid antibodies or
antineutrophil cytoplasmic antibodies (ANCA).
Case 1: A 26 year-old female injured her left lower
leg in September 2002. She had a 10-year history of SLE, and lupus
nephritis had supervened at the beginning of the disease. As a
course of treatment, she was administered prednisolone, and/or
cyclosporine. The maximum dose of prednisolone was 60 mg/day.
She had an abrasion as a result of an accident on her left lower
leg, and was administered prednisolone (12.5 mg/day) without
SLE. The ulcer enlarged (6 × 4 cm), but various conventional
topical therapies including bFGF were ineffective. A wound
debridement was performed (figure 1A), and CDS
applications were started on April 18, 2003 (figure 1B). The wound size
decreased, and healthy granulation tissue formed after 6 weeks of
CDS treatment (figure
1C). On May 30, an autologous split-thickness skin graft
was performed. One month later, the autologous grafted skin showed
complete re-epithelization (figure 1D), and still
remains in good condition.
Case 2: A 68 year-old female was diagnosed with SLE
and secondary Sjogren syndrome in 2002, and was administered
prednisolone (20 mg/day). A painful edematous erythema
with central whitish skin necrosis and scattered purpura appeared
on her left lower leg in July, 2007. Laboratory investigations
revealed the following: white blood cell count,
12,600/mm3, C reactive protein (CRP), 17.18 mg/dL
[normal range: up to 0.4 mg/dL]. Staphylococcus aureus was
detected in the ulcer, therefore we considered that the ulcer was
triggered by the bacterial infection, and then we started to treat
with antibiotics. The dosage of prednisolone was reduced to
15 mg/day and the wound surface was washed with saline. The
bacterial culture became negative after 2 weeks of treatment.
However, the skin defect remained (figure 2A). Since several
topical conservative treatments had proved ineffective (such as
iodine ointment and sulfadiazine silver), we applied the CDS after
wound debridement on July 23, 2007 (figure 2B). Healthy
granulation tissues formed after 4 weeks of CDS treatment (figure 2C), and an
autologous split-thickness skin graft was performed on August 24,
2007. One month later, the autologous grafted skin showed almost
complete re-epithelization (figure 2D), and still
remains in good condition.
Case 3: A 38-year-old female had an injury on her
left lower leg as a result of a traffic accident in July, 2007. She
had suffered from SLE for 9 years, and had had several pulse
glucocorticoid therapies. When she had the accident, she was
administered prednisolone (10 mg/day), cyclosporine
(90 mg/day) and mizolibin (150 mg/day). The wound did not
show any sign of re-epithelization after several conservative
treatments including bFGF, and PGE1 ointment. Laboratory
investigations revealed no SLE activity. A debridement of the
wound was performed, and a CDS was applied on October 9, 2007. The
wound size decreased, and healthy granulation tissues formed after
5 weeks of CDS treatment. On November 13, an autologous
split-thickness skin graft was performed. Two months later, the
autologous grafted skin showed complete re-epithelization, and
still remains in good condition.
Discussion
Although allogeneic CDSs fail to survive permanently on skin
defects, CDSs produce a variety of biologically active substances
including growth factors and angiogenic factors including vascular
endothelial growth factor (VEGF), bFGF, keratinocyte growth factor
(KGF), platelet-derived growth factor (PDGF), transforming growth
factor β (TGF-β), interleukin-6 (IL-6), IL-8 [6, 15], and CDSs are
characterized by scaffolds for cell implantation, which by
themselves promote wound healing. The two-layered spongy matrix of
HA and Col was found to have higher potency for promoting wound
healing, compared with a collagen spongy matrix, in a preliminary
animal study [10]. When a CDS is applied to a wound surface in
clinical use, the spongy structure degrades within about 1 week
[12]. Both HA and Col molecules seem to be involved in the wound
healing process.
Allogeneic CDSs have been used to provide effective therapies
for patients with severe wounds, including burns, chronic ulcers,
traumatic skin defects and excise wounds from the removal of giant
pigmented nevi [7, 8, 16]. However, to our knowledge, our present
cases are the first reported in the literature, in which allogeneic
cultured fibroblasts were applied to leg ulcers involving SLE.
The primary treatment for SLE is the administration of
immunosuppressants including corticosteroids, cyclophosphamides,
azathioprines and cyclosporines [17]. Therefore, above all, skin
ulcers in SLE patients are highly intractable because of the
effects of various factors in the underlying vasculitis and the
effects of drug treatments. Severe skin ulcers may also be
associated with other systemic vascular, infective and
immunological diseases, such as antiphospholipid syndrome.
In the present 3 cases, each subject was medicated by
immunosuppressants for a long time, and various topical treatments
were ineffective. After applications of allogeneic CDSs, all 3
cases showed rapid granulation formation acceptable for secondary
skin grafts.
These case reports suggest that allogeneic CDSs are useful for
treating non-healing or intractable skin ulcers in patients with
SLE.
Acknowledgement
Financial support: none. Conflict of interest: none.
References
1 Adam J, Richard AF. Cutaneous Wound Healing. New
England J Med 1999; 341: 738-46.
2 Green H, Kehindez O, Thomas J. Growth of
cultured human epidermal cells into multiple epithelia suitable for
grafting. Proc Natl Acad Sci USA 1979; 76: 5665-8.
3 Bell E, Ehrlich HP, Sher S, et al.
Development and use of a living skin equivalent. Plast Reconstr
Surg 1981; 67: 386-92.
4 Cooper ML, Hansbrough JF, Spielvogel RL,
et al. In vivo optimization of a living dermal substitute
employing cultured human fibroblasts on a biodegradable
polyglycolic acid or polygractin mesh. Biomaterials 1991; 12:
243-8.
5 Hansbrough JF, Morgan J, Greenleaf G.
Development of a temporary living skin replacement composed of
human neonatal fibroblasts cultured in Biobrane, a synthetic
dressing material. Surgery 1994; 115: 633-44.
6 Kuroyanagi Y, Kubo K, Matsui H, et al.
Establishment of banking system for allogeneic cultured dermal
substitute. Artif Organs 2004; 28: 13-21.
7 Harima N, Asai S, Wako M, et al. Clinical
trials using allogeneic cultured dermal substitutes for skin
ulcers. Jpn J Dermatol 2003; 113: 253-64; (abstract in
English).
8 Oka H, Fujitsu M, Suenobu K, et al.
Clinical trials with allogeneic cultured dermal substitute(CDS) for
the treatment of burns and skin ulcers. Jpn J Burn Injuries 2002;
28: 333-42; (abstract in English).
9 Daniel J, Bevra H. Clinical aspect of vasculitis and
selected cutaneovascular manifestations of systemic lupus
erythematosus. In: Dubois’ Lupus Erythematosus. Lippincott Williams
& Wilkins, 2007: 702-3.
10 Kubo K, Kuroyanagi Y. Spongy matrix of hyaluronic
acid and collagen for cultured dermal substitute; evaluation in
animal test. J Artif Organ 2003; 6: 64-70.
11 Kubo K, Kuroyanagi Y. Characterization of cultured
dermal substitute composed of spongy matrix of hyaluronic acid and
collagen combined with fibroblasts. J Artif Organ 2003; 6:
138-44.
12 Kubo K, Kuroyanagi Y. Development cultured dermal
substitute composed of hyaluronic acid and collagen combined with
fibroblasts; Fundamental evaluation. J Biomater Sci Polymer Edn
2003; 14: 625-41.
13 Ohtani T, Okamoto K, Kaminaka C, et al.
Digital gangrene associated with idiopathic hypereosinophilia:
treatment with allogeneic cultured dermal substitute (CDS). Eur J
Dermatol 2004; 14: 168-71.
14 Kubo K, Kuroyanagi Y. Development of cultured
dermal substitute composed of spongy matrix of hyaluronic acid and
atelo-collagen combined with fibroblasts; cryopreservation. Artif
Organs 2004; 28: 182-8.
15 Kubo K, Kuroyanagi YA. Study of cytokines released
from fibroblasts in cultured dermal substitute. J Artif Organs
2005; 29: 845-9.
16 Fujimori Y, Ueda K, Omiya Y, et al.
Clinical trials with allogeneic cultured dermal substitutes(CDS)
for full-thickness skin defects. J Jpn PRS 2003; 23: 475-84.
17 Goldblatt F, Isenberg DA. New therapies for
systemic lupus erythematosus. Clin Exp Immunol 2005; 140:
205-12.
|
Printable version
Version PDF
