15018752330
发表时间:2015-12-08 浏览次数:461次
Introduction
The burden of diabetes-related complications is an inevitable
consequence of the rise in the prevalence of diabetes mellitus
worldwide. The lifetime probability of diabetics to develop a diabetic
chronic ulcer is estimated at 10%-25%. [1]
The risk for patients, in particular with type 2 diabetes, of
undergoing a lower extremity amputation is 23-fold higher than that of a
nondiabetic. [2] Diabetic chronic ulcers definitively represent a significant cause of morbidity, hospitalization, and a huge financial cost.
Risk
factors for diabetic chronic ulcers include vascular anomalies,
peripheral neuropathy, imbalanced foot mechanical forces, impaired joint
mobility, high body mass, foot instability, and history of previous
ulceration or amputation. The standard of care for these wounds, as
defined by the International Working Group on the Diabetic Foot,
requires multidisciplinary management including control of systemic
glucose, extremity vascularization, off-loading, debridement of necrotic
tissue, control of local infection, and patient compliance. [3],[4]
In this holistic approach, for lesions where the healing process is
unsatisfactory, and no other underlying cause exists, there is an
increasing need for more effective therapies that will stimulate healing
of diabetic chronic ulcers. Tissue-engineered products, especially skin
substitutes, both cellular and acellular, are emerging as new local
therapy for the treatment of nonhealing diabetic chronic ulcers. [5]
This case-series study was performed to determine wound reduction and
healing following use of a new cultured allogeneic keratinocyte sheet in
the management of chronic ulcers in diabetic patients. Cultured
allogeneic keratinocytes on a hyaluronic acid scaffold have recently
been demonstrated to be effective in the treatment of chronic ulcers,
but specific studies on a diabetic group have not yet been described. [6]
We reviewed 16 chronic ulcers in diabetic patients treated with these
novel epidermal substitutes, and discussed the potential benefits,
scientific evidence, and safety in the management of this complication.
Methods
From donor cadavers (brain-dead), without infectious microorganisms
(hepatitis B virus, hepatitis C virus, human immunodeficiency virus,
human T-lymphotropic virus, cytomegalovirus, and negative to the
treponema pallidum hemagglutination test), autoimmune, genetic, or
infective skin pathologies, a 2 cm × 2 cm biopsy is performed from a
sample of glabrous skin. The transfer of the biopsy is performed with a
temperature of 4°C, in a sterile container with gentamicin and
amphotericin B, to Niguarda Ca' Granda Hospital, the Regional Tissue
Bank of Lombardia, Italy. The Skin Bank qualifies, collects, processes,
validates, cryopreserves, and distributes skin taken from donor
subjects. The biopsy is then sterilized and placed in Dulbecco's
modified Eagle's medium (DMEM) containing Dispase II (bacterial enzyme)
for 18 h at 4°C or 37°C for about 4 h, in order to split the epidermis
from the dermis. The biopsy is then treated with
Trypsin-Ethylenediaminetetraacetic acid (EDTA) in DMEM to isolate
keratinocytes. The isolated cells can be cultivated in a specific
culture medium.
HYAFF11 (Fidia Advanced Biopolymer S.r.l., Abano
Terme, Italy) is the biomaterial, composed of hyaluronic acid totally
esterified with benzyl ester. Due to its chemical properties, HYAFF11
releases benzyl alcohol and hyaluronic acid to the wound
microenvironment because of hydrolysis of the ester bonds caused by the
water in the wound exudate. The scaffold is a porous structure, composed
of macropores and micropores, constituting the geometrical mesh of the
matrix. Macropores (diameter of 0.5 mm) allow cellular distribution on
the scaffold and the drainage of exudate when the sheet is put on the
wound bed. Micropores (diameter of 40 μm, 6250 poers/cm 2 )
allow neovascularization of the construct after the application, and
cellular migration from the superior to the inferior face of the sheet,
that is, the face in contact with a wound bed. [7]
HYAFF11 forms a two-dimensional matrix, 20-mm thick, for epidermal
substitutes, and a three-dimensional scaffold for dermal constructs.
Skin
substitute preparation is divided into two phases: the first phase
consists of the primary culture of allogeneic cells in a specific
culture medium, and the second consists of the seeding of cells on a
hyaluronic acid scaffold for cell expansion with adhesion. The sheets
are then ready for application. Keratinocytes are cultivated through
Rheinwald and Green's described method. [8]
Cells are incubated with a specific medium at 37°C for 20-30 min to
inactivate Trypsin-EDTA, and then centrifugated and suspended in a
medium without epidermal growth factor (EGF). The cellular pellet is
located on a feeder layer of mouse-derived fibroblasts (3t3) inactivated
by irradiation, and incubated at 37°C with 5% CO 2 . The
feeder layer secretes proteins of extracellular matrix and growth
factors, promoting adhesion, and proliferation of keratinocytes.
Irradiation of 3t3 serves blocks the replication of these cells. EGF is
added to the culture medium 72 h following seeding on the feeder layers.
The medium is changed every 2 days until semi-confluence is achieved.
The sheets can be used fresh, within 21 days of production, or can be
cryopreserved in dimethyl sulfoxide and stored at −80°C, thus
guaranteeing viability for 2 years.
Patient anamnestic data and
outcomes were reviewed through a case series study of 11 patients with
well-controlled diabetes type 2, with 16 legs and ankle chronic ulcers,
unresponsive to previous conventional therapies (i.e. repeated use of
advanced modern dressing for many cycles with a mean duration of 18
months), treated with the new skin substitutes from 2011.
All
patients followed the same surgical protocol: chronic wounds incurable
with other reconstructive options, such as wound dressings, acellular
skin substitutes, and split-thickness autografts, underwent the
application of the novel allogeneic skin substitutes in the operating
room by the same surgical team. Before the application of sheets, all
the wounds were debrided surgically to achieve wound bed preparation,
and accurate hemostasis was performed. The skin substitutes were applied
once directly to the wound bed without sutures.
Patients were
observed weekly for a follow-up period of at least 40-70 days. The
follow-up was performed by the physician team at the outpatient wound
healing clinic of the Wound Care Unit (Monza, Italy). The wound dressing
was the same for the postoperative period and for every control visits:
a multicomponent bandage with nonadherent gauze, and polyurethane foam
with silver (Biatain Ag, Coloplast) to prevent bacterial superinfection.
At
the entry of the study, anamnestic data collected included age, sex,
smoking status, and the presence of hypertension, end-stage renal
disease, vascular diseases, autoimmune disorders, neurologic or
cardiologic problems, or burns. Every chronic wound was classified at
the entry of the study for dimension, location, the presence of local
infection (absent, mild, moderate, severe), and wound characteristics
such as wound bed, perilesional skin disorders, borders, and exudate, to
calculate the wound bed score (WBS), as defined by Falanga et al.[9]
Diagnosis of infection was based on culture obtained with a sterile
rongeur. The results of this culture guided the appropriate use of
systemic antibiotics. Multiple ulcers were considered different chronic
wounds. At the end of follow-up dimensional reduction, the WBS and
presence of the adverse reaction were recorded.
The primary
endpoint of the study was to determine the variation in ulcer dimension
versus t0. Secondary endpoints included the evaluation of variation in
the wound bed and exudate to determine the WBS, the percentage of wound
reduction, and the percentage and time to healing.
Abstracted
data were stored using an Excel Office database (Microsoft Corporation,
Washington, USA) containing fields for clinical data entry. The
statistical analysis was performed considering the patient as a unit of
analysis initially (for anamnestic data), and then the single-chronic
wound (for clinical results). The mean reduction of the skin lesion
during follow-up was verified with the Wilcoxon Signed-Rank Test. The
variation of WBS versus t0 was analyzed with the Friedman test. The
level of statistical significance was fixed to 5% (P < 0.05) to reject the null hypothesis.
Results
Between January of 2011 and December of 2013, 11 patients with diabetes type 2 with 16 wounds underwent an application of allogeneic epidermal substitutes on a hyaluronic acid scaffold. [Table 1] describes the demographics of all patients. Four out of 11 (36%) patients were females, and the mean age of all patients was 75 ± 8.2 years. Among comorbidities, hypertension (7, 63%) and cardiopathy (3, 27%) were the most frequent.
As described by [Table 2],
8 out of 16 (50%) chronic ulcers were located on the ankle or foot, and
the other 50% were located on the lower third of the leg. Five (32%)
ulcers showed a mild to moderate local infection at the entry of the
study; only 1 severe infection was present. The mean preoperative wound
size for all diabetic chronic ulcers was 14.37 ± 9.29 cm 2 ,
and the mean preoperative WBS was 9.6 ± 3.5. During the follow-up
period, no wound advanced to require amputation of the foot or lower
limb, and only one ulcer developed a local severe infection under the
application of the sheet, enlarging the wound dimensions. No other
adverse reactions were recorded.
Six out of 16 (38%) chronic ulcers healed in a mean time of 42 ± 16
days. The application of the new skin substitutes reduced the mean
percentage of the wound dimension by 70% (P = 0.0007). The WBS demonstrated an improvement of 52% (P < 0.0001) at the end of the follow-up period from the score recorded at the entry of the study.
Case example: ankle diabetic ulcers in a 66-year-old patient treated with allogeneic keratinocyte [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5].
Discussion
The chronic wound microenvironment is biologically distinct from the
acute wound milieu: venous and diabetic chronic ulcers are hypothesized
to be trapped in the inflammatory and proliferative phases of normal
healing, respectively. Poor wound healing may be a consequence of
abnormal insulin signaling and hyperglycemia, affecting skin
proliferation and differentiation. [10]
Skin biopsies performed in nondiabetic and diabetic subjects from the
edges of chronic wounds have revealed increased expression of
transforming growth factor (TGF) beta 3 and low expression of TGF-beta
1, resulting in nonhealing. [11] Abnormal expression of insulin-like growth factor type 1 in diabetic skin may also contribute to delayed wound healing. [12]
The damaged biological background of diabetic wounds explains the
necessity to modulate therapeutically the unbalanced levels of growth
factors, signaling molecules, and extracellular matrix proteins. [13]
How these novel skin substitutes work is still not completely
understood. Initially, especially with skin substitutes as cultured
epidermis and living bilayered skin construct, some degree of permanent
engraftment was thought to assist in healing. As shown by DNA and Y
chromosome probes, allogeneic constructs are not the same as autografts:
a true take of allogeneic sheets has not been demonstrated. [14]
The allogeneic skin constructs usually do not stay on the wound for
more than a few weeks, and their function is not to replace tissues or
cells, but instead to stimulate tissue repair as pharmacologic agents,
secreting healing factors in chronic wound microenvironment. The key
role of allogeneic constructs seems to be the secretion of growth
factors and extracellular matrix proteins, and to attract differentiated
or stem cells in the wound milieu. [15]
This clinical case-series study based on the utilization of new
cultured allogeneic keratinocyte sheets showed promising results,
including safety and tolerability of the allogeneic product, good wound
healing rate, a great reduction in wound size in a relatively short
period, and preparation of the wound bed for alternative reconstructive
treatments (i.e. split-thickness autograft). The application of
allogeneic keratinocytes on a hyaluronic acid scaffold may allow
improvement of diabetic leg and foot lesions not amenable to other
therapies or surgical indications, thereby allowing the closure of
nonhealing chronic ulcers, and thus reducing morbidity, cost, and length
of hospitalization. Allogeneic skin substitutes do not require
prolonged operating time or skin biopsy, and are easily applied by the
surgeon in contrast to flaps or autograft. In the multidisciplinary
approach to diabetic chronic wounds, allogeneic skin substitutes on a
hyaluronic acid scaffold may represent a valid alternative when other
possibilities have been exhausted. Keratinocyte sheets were also applied
when mild to moderate local infection was present, resulting in
interesting clinical outcomes. Cultured keratinocytes were, in fact,
resistant to bacterial colonization in excised burns and chronic ulcers. [16]
In such settings and considering the cost of this new product,
allogeneic keratinocytes on a hyaluronic acid scaffold could be
considered a second-line treatment in case of prior treatment failure.
Fortunately,
we had no cases of immunologic response to these allogeneic products in
our case series. Had this occurred, the application of a topical
immunologic suppressant like 5-fluorouracil ointment would have been
recommended.
In the literature, other studies have reported
clinical outcomes for cellular skin substitutes on various other
scaffolds in the treatment of chronic leg and foot diabetic ulcers. [17],[18],[19],[20]
The differences among these studies in results, methods, products,
cost-effectiveness ratio, and follow-up period are highlighted in [Table 3].
However, comparison of the effectiveness is difficult to perform given
the extreme variation in protocols (e.g., skin substitutes were used
multiple times on the same ulcer in some studies, or the end of the
study was not fixed until 100% wound healing was achieved). [21],[22],[23]
However, some clinical features have emerged from these studies
regarding the use of cellular skin substitutes in the management of
chronic diabetic ulcers. The role of allogeneic keratinocytes appears to
be central in the cellular therapy of diabetic wounds, although good
results have been reported with the use of autologous cells. [24],[25]
Unlike allogeneic substitutes, autologous sheets are not available for
use immediately, a skin biopsy is required, and longer times are
necessary for cell processing, with the ever-present risk of ischemia or
osteomyelitis. Cellular skin substitutes are formed by two elements:
cells and scaffold. In the "dynamic reciprocity" model, the
extracellular matrix emerges as capable of influencing wound healing,
acting on the others two characters, cells and signal factors. [26]
Thus, the scaffold and cells are both fundamental in the clinical
outcome of skin substitutes. Hyaluronic acid is a central molecule in
human skin, and its functions are diverse. Hyaluronan influences
hydration of the extracellular matrix, due to its hydroscopic
characteristics, and contributes to the physical and mechanical
properties of the dermis. Hyaluronic acid interacts with a number of
receptors, resulting in the activation of signaling cascades that
influence cell migration, proliferation, and gene expression. Further,
fetal-like regenerative wound healing is characterized by a large amount
of hyaluronic acid deposition. From these observations, a membrane
composed of completely esterified hyaluronic acid was developed, and was
shown to support growth of keratinocytes in vitro and biocompatibility in vivo. [27]
Prior studies on cellular therapy for diabetic wounds have emphasized
repeated debridement, control of bacterial growth, careful moisture
balance to prevent maceration, blood pressure control, management of
blood glucose, and perfusion of the extremity. Wound bed preparation
remains central for cellular skin substitutes application and efficacy.
The present case series study on skin substitutes based on hyaluronic acid scaffold for the therapy of chronic diabetic leg and foot ulcers allows investigation of the clinical results, in order to find evidence for treatment perspectives, and stimulate biochemical research in the field of regenerative medicine. Comprehensive studies will be necessary to evaluate the cost-effectiveness of these therapies before they become acceptable for general use. The goal is to achieve the cellular therapy that "suits" the specific chronic wound microenvironment in diabetic wounds in the future. However, large, randomized, and controlled clinical trials are required to confirm and validate the clinical results of these novel skin substitutes.
References
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