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This case‑series study was performed to determine wound for application. Keratinocytes are cultivated through
[8]
reduction and healing following use of a new cultured Rheinwald and Green’s described method. Cells are
allogeneic keratinocyte sheet in the management of incubated with a specific medium at 37°C for 20–30 min
chronic ulcers in diabetic patients. Cultured allogeneic to inactivate Trypsin‑EDTA, and then centrifugated and
keratinocytes on a hyaluronic acid scaffold have recently suspended in a medium without epidermal growth
been demonstrated to be effective in the treatment factor (EGF). The cellular pellet is located on a feeder
of chronic ulcers, but specific studies on a diabetic layer of mouse‑derived fibroblasts (3t3) inactivated by
[6]
group have not yet been described. We reviewed 16 irradiation, and incubated at 37°C with 5% CO . The
2
chronic ulcers in diabetic patients treated with these feeder layer secretes proteins of extracellular matrix and
novel epidermal substitutes, and discussed the potential growth factors, promoting adhesion, and proliferation
benefits, scientific evidence, and safety in the management of keratinocytes. Irradiation of 3t3 serves blocks the
of this complication. The review board of University of Milan replication of these cells. EGF is added to the culture
approved this study. medium 72 h following seeding on the feeder layers. The
medium is changed every 2 days until semi‑confluence is
METHODS achieved. The sheets can be used fresh, within 21 days of
From donor cadavers (brain‑dead), without infectious production, or can be cryopreserved in dimethyl sulfoxide
microorganisms (hepatitis B virus, hepatitis C virus, and stored at −80°C, thus guaranteeing viability for
human immunodeficiency virus, human T‑lymphotropic 2 years.
virus, cytomegalovirus, and negative to the treponema Patient anamnestic data and outcomes were reviewed
pallidum hemagglutination test), autoimmune, genetic, through a case series study of 11 patients with
or infective skin pathologies, a 2 cm × 2 cm biopsy is well‑controlled diabetes type 2, with 16 legs and ankle
performed from a sample of glabrous skin. The transfer of chronic ulcers, unresponsive to previous conventional
the biopsy is performed with a temperature of 4°C, in a therapies (i.e. repeated use of advanced modern dressing
sterile container with gentamicin and amphotericin B, to for many cycles with a mean duration of 18 months),
Niguarda Ca’ Granda Hospital, the Regional Tissue Bank treated with the new skin substitutes from 2011.
of Lombardia, Italy. The Skin Bank qualifies, collects, All patients followed the same surgical protocol: chronic
processes, validates, cryopreserves, and distributes wounds incurable with other reconstructive options,
skin taken from donor subjects. The biopsy is then such as wound dressings, acellular skin substitutes, and
sterilized and placed in Dulbecco’s modified Eagle’s split‑thickness autografts, underwent the application of
medium (DMEM) containing Dispase II (bacterial enzyme) the novel allogeneic skin substitutes in the operating
for 18 h at 4°C or 37°C for about 4 h, in order to split room by the same surgical team. Before the application
the epidermis from the dermis. The biopsy is then treated of sheets, all the wounds were debrided surgically to
with Trypsin‑Ethylenediaminetetraacetic acid (EDTA) in achieve wound bed preparation, and accurate hemostasis
DMEM to isolate keratinocytes. The isolated cells can be was performed. The skin substitutes were applied once
cultivated in a specific culture medium. directly to the wound bed without sutures.

HYAFF11 (Fidia Advanced Biopolymer S.r.l., Abano Patients were observed weekly for a follow‑up period
Terme, Italy) is the biomaterial, composed of hyaluronic of at least 40–70 days. The follow‑up was performed
acid totally esterified with benzyl ester. Due to its chemical by the physician team at the outpatient wound healing
properties, HYAFF11 releases benzyl alcohol and hyaluronic clinic of the Wound Care Unit (Monza, Italy). The wound
acid to the wound microenvironment because of hydrolysis dressing was the same for the postoperative period
of the ester bonds caused by the water in the wound and for every control visits: a multicomponent bandage
exudate. The scaffold is a porous structure, composed of with nonadherent gauze, and polyurethane foam
macropores and micropores, constituting the geometrical with silver (Biatain Ag, Coloplast) to prevent bacterial
mesh of the matrix. Macropores (diameter of 0.5 mm) superinfection.
allow cellular distribution on the scaffold and the drainage
of exudate when the sheet is put on the wound bed. At the entry of the study, anamnestic data collected
Micropores (diameter of 40 µm, 6250 poers/cm ) allow included age, sex, smoking status, and the presence of
2
neovascularization of the construct after the application, hypertension, end‑stage renal disease, vascular diseases,
and cellular migration from the superior to the inferior autoimmune disorders, neurologic or cardiologic
face of the sheet, that is, the face in contact with a wound problems, or burns. Every chronic wound was classified
bed. HYAFF11 forms a two‑dimensional matrix, 20‑mm at the entry of the study for dimension, location, the
[7]
presence of local infection (absent, mild, moderate,
thick, for epidermal substitutes, and a three‑dimensional severe), and wound characteristics such as wound bed,
scaffold for dermal constructs.
perilesional skin disorders, borders, and exudate, to
Skin substitute preparation is divided into two phases: the calculate the wound bed score (WBS), as defined by
first phase consists of the primary culture of allogeneic Falanga et al. Diagnosis of infection was based on
[9]
cells in a specific culture medium, and the second consists culture obtained with a sterile rongeur. The results of this
of the seeding of cells on a hyaluronic acid scaffold for culture guided the appropriate use of systemic antibiotics.
cell expansion with adhesion. The sheets are then ready Multiple ulcers were considered different chronic wounds.

Plast Aesthet Res || Vol 1 || Issue 2 || Sep 2014 75