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The Longevity of a Bilayered Skin Substitute After Application to Venous Ulcers
Tania J. Phillips, MD;
Jasmin Manzoor, MD;
Adriana Rojas, MD;
Cary Isaacs, MS;
Polly Carson, CWS;
Michael Sabolinski, MD;
Jan Young, PhD;
Vincent Falanga, MD
Arch Dermatol. 2002;138:1079-1081.
ABSTRACT
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Background A bilayered skin substitute composed of allogeneic keratinocytes and
fibroblasts in a collagen gel has been approved by the US Food and Drug Administration
for the treatment of venous and diabetic ulcers. Its mechanism of action has
not been fully determined.
Objective To determine the longevity of allogeneic fibroblasts and keratinocytes
in a bilayered skin substitute in patients with venous leg ulcers.
Methods Ten patients with venous leg ulcers were treated with a bilayered skin
substitute on day 0, days 3 to 5, and weeks 1 through 3. Biopsy specimens
of the grafted wound were taken. We used polymerase chain reaction analysis
to determine whether allogeneic DNA was present in the biopsy specimens.
Results We detected allogeneic DNA in 2 of 8 specimens at 1 month after initial
grafting. Neither of the 2 patients showed persistence of allogeneic DNA at
2 months after initial grafting.
Conclusions Allogeneic cells from a bilayered skin substitute do not appear to survive
permanently after grafting for treatment of venous leg ulcers. Other mechanisms
of action might include cytokine release, structural support, or provision
of a moist wound environment.
INTRODUCTION
A BILAYERED skin substitute (BSS) (Apligraf; Novartis, Inc, Canton,
Mass) is a viable skin replacement that has been approved by the US Food and
Drug Administration for the treatment of venous and diabetic ulcers. The epidermal
layer is formed by serial passage of human keratinocytes that are seeded onto
a contracted dermal matrix in culture medium. A well-differentiated stratum
corneum develops after exposure of the keratinocytes to an air-liquid interface.
The dermal layer is composed of human fibroblasts in a bovine type I collagen
lattice. These fibroblasts divide, multiply, and produce new collagen. Although
matrix proteins and cytokines found in human skin are present in the BSS,
it does not contain Langerhans cells, melanocytes, macrophages, lymphocytes,
blood vessels, or hair follicles.
At present, the BSS has not been shown to be immunogenic in treated
patients. In vitro studies demonstrated that the BSS does not contain cells
that constitutively express class II major histocompatibility complex antigens
such as macrophages, lymphocytes, and Langerhans cells. These immunogenic
cells are lost during serial culture and expansion of human keratinocytes
and fibroblasts.1-2 When tested
in a mixed lymphocyte reaction assay, cultured human keratinocytes and fibroblasts
did not trigger a proliferative immune response, even when class II antigen
expression was induced by interferon- .2
Therefore, it is possible that the BSS might promote healing by surviving
permanently in the wound bed. However, experience in other models suggests
that the BSS may be eventually replaced by host tissues.3-5
The purpose of the present study was to determine whether allogeneic
DNA from the BSS could be detected at different times after BSS grafting of
venous ulcers.
PATIENTS AND METHODS
Patients with venous ulcers of greater than 1 month's duration (confirmed
by means of duplex ultrasonographic findings) and an ankle-brachial pressure
index of greater than 0.65 were enrolled into the study.
Patients enrolled in other investigational studies within the past 3
months, patients previously treated with the BSS, and patients with HLA class
I antigens common to the patient and the BSS were excluded from participating.
All patients received up to 5 applications of the BSS during the 3-week
treatment. At the screening visit (days -7 to -5), blood was collected
as a DNA control sample. The BSS was applied to a clean, debrided wound after
thorough irrigation with isotonic sodium chloride solution. Oozing or bleeding
caused by debridement was stopped by means of gentle pressure. The graft was
covered with a nonadherent dressing, premeasured gauze, and a self-adherent
elastic wrap from the metatarsals to the tibial plateau. At 4, 8, and 12 weeks
after the first BSS application, 3-mm punch biopsy specimens were taken from
the area judged by the investigator (T.J.P. or V.F.) to be most likely to
contain BSS. If the BSS was not visible on the ulcer, specimens were taken
from the center of the wound. Biopsy sites were rotated so that the specimen
was not taken from the same location at each visit. When DNA of the BSS could
not be detected in the specimen, no further samples were taken.
To test for the persistence of the BSS on patients, we used the expression
of specific HLA genes by BSS cells. The HLA phenotypes of the BSS have been
determined previously (data not shown). We used this information to create
sequence-specific primers directed toward the DNA of the BSS. In the present
study, HLA-DQB1*0201–specific primers were used (Figure 1). Keratinocytes and fibroblasts
from the BSS carry this gene. Primers were amplified using polymerase chain
reaction (PCR) analysis.6
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Figure 1. Titration of the bilayered skin
substitute (BSS) DNA that is positive for HLA-DQB1*0201 and mixed
with specific amounts of DNA that is negative for HLA-DQB1*0201.
The polymerase chain reaction (PCR) analysis requires 1 ng of DNA and results
in a 205–base pair (bp) product. The product of the first lane (after
the DNA marker) was amplified from 1000 ng of HLA-DQB1*0201–positive
DNA. The product of the second lane was amplified from 250 ng of HLA-DQB1*0201–positive DNA mixed with 750 ng of HLA-DQB1*0201–negative
DNA. The third lane has 100 ng of HLA-DQB1*0201–positive
DNA mixed with 900 ng of HLA-DQB1*0201–negative DNA. The
fourth lane has 50 ng of HLA-DQB1*0201–positive DNA mixed
with 950 ng of HLA-DQB1*0201–negative DNA. The fifth lane
has 25 ng of HLA-DQB1*0201–positive DNA mixed with 975
ng of HLA-DQB1*0201–negative DNA. The sixth lane contains
1000 ng of HLA-DQB1*0201–negative DNA. We used this test
to establish the sensitivity of the assay, in this case 25 ng of specific
target sequence. In other words, we can detect the specific sequence in a
biopsy specimen that contains as little as 2.5% DNA from the BSS. The titration
was used to monitor functioning of the PCR analysis within our specifications
and was run every time patient samples underwent testing. It was not used
as a standard curve for quantification.
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RESULTS
Fourteen patients were enrolled into the study. Ten patients demonstrated
results of tissue typing in which HLA antigens were not common with those
of the BSS. These patients underwent evaluation for persistence of BSS cells.
Eight of these 10 patients underwent testing for persistence at 4 weeks. Two
ulcers healed completely during the study. The DNA from the BSS could not
be detected in biopsy specimens from 6 patients (Figure 2). Results of PCR testing showed that 2 of these wounds
demonstrated the BSS DNA at 4 weeks (Figure
3). No correlation between the clinical appearance of the wound
and the BSS persistence was found. Neither wound showed persistence of DNA
from the BSS at 8 weeks. One of these ulcers healed; the other did not.
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Figure 2. Lanes PM-01-06 and PM-01-07 are
samples from study patients 4 weeks after application of the bilayered skin
substitute (BSS). No target DNA can be detected in these lanes. A titration
of DNA in the following lanes was similar to that in Figure 1, which shows
that the polymerase chain reaction (PCR) analysis is working within the stated
sensitivity, and that DNA from the BSS could not be detected in the biopsy
specimens from these patients. bp indicates base pair.
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Figure 3. Samples from study patients PM-02-05
and PM-02-06 show persistence of DNA from bilayered skin substitute (BSS)
at 4 weeks. We detected HLA-DQB4*0201 DNA in biopsy specimens
4 weeks after initial treatment. Lane 1 shows the positive control, with DNA
isolated from BSS keratinocytes. Lanes A and B show DNA from the BSS in specimens
from both patients collected 4 weeks after the initial treatment.
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COMMENT
The BSS has been approved by the US Food and Drug Administration for
the treatment of venous and diabetic ulcers. The healing process has not been
entirely elucidated.
Some authors postulated that the presence of a dermal-like substitute
in the BSS may create conditions sufficiently different from cultured epidermal
allografts to allow long-term engraftment.7
In patients with acute wounds due to epidermolysis bullosa, extensive erosions
were treated with the BSS, and the treated areas remained blister free, with
clinical evidence of graft take and no signs or symptoms of acute graft rejection.8-9 Molecular genetic testing using a specific
marker for the tissue-engineered cells confirmed persistence of the skin substitute
in 4 (33%) of 12 patients with these acute wounds at 4 weeks.10
Delayed rejection of allograft may occur in these patients because of immune
tolerance to allogeneic tissue in early life8, 11
or because of abnormalities in immune function in patients with epidermolysis
bullosa.12-15
Alternately, the mechanism of action of cultured allogeneic tissue may be
different in acute wounds compared with chronic wounds. In other acute wounds
(eg, split-thickness skin-graft donor sites), persistence of allogeneic DNA
from the BSS occurred in 3 (27%) of 11 patients at 4 weeks.7, 10
Additional data are needed to elucidate whether the BSS persists for longer
than 4 weeks in acute wounds.
In chronic wounds, clinical investigators have observed clinical graft
take and temporary persistence of the BSS in approximately 41% of patients
in whom the wounds healed. Clinical remodeling of the graft and probable replacement
with the patient's own skin appeared to occur in at least 63% of these patients.16 The authors commented that the BSS could benefit
wounds as a temporary skin replacement or as a stimulus for wound healing.
In the small group of patients with chronic venous ulcers undergoing
testing in this study, allogeneic DNA from a BSS could not be detected at
2 months after grafting. A weakness in this study is that during the period
of observation, only 2 of the patients experienced complete healing. Persistence
is probably related to successful initial graft take and wound closure, and
it is difficult to determine persistence in an unhealed wound. If a wound
heals completely and rapidly after application of the BSS, persistence of
allogeneic cells would be more likely, as occurred in patient PM-02-05. However,
allogeneic DNA was also detected in patient PM-02-06, who did not experience
complete wound healing. Our data did not support survival of allogeneic BSS
cells at 8 weeks after grafting for treatment of venous leg ulcers. However,
given that the initial graft take of the BSS depends on biological and immunological
factors, the results of this study do not allow us to clearly determine the
reason for the lack of long-term persistence of the BSS cells. Venous ulcers
are highly inflammatory, another possible mechanism for no persistence in
this condition.
If the BSS does not survive permanently in chronic wounds, several possible
mechanisms of action exist. The BSS acts to provide immediate wound coverage
and as a barrier to protect the wound from injury, infection, and desiccation.
It also provides a moist environment that would be favorable to wound healing.
Fibroblasts and keratinocytes, which are found in the BSS, are known to release
large numbers of growth factors and cytokines that could stimulate rapid wound
healing from the wound margins and from adnexal elements from within the wound
bed. These include interleukins 1, 3, 6, and 8; transforming growth factors 
and  ; granulocyte-macrophage colony-stimulating factor; basic fibroblast
growth factor; platelet-derived growth factor; tumor necrosis factor ;
and others.17-18 The combination
of human keratinocytes and human dermal fibroblasts in the skin substitute
may act in a synergistic manner. For example, insulin-like growth factor-I
is not found in cultures of pure human keratinocytes or of pure human dermal
fibroblasts, but is present in the BSS (J.Y., unpublished data, September
1997).
The matrix components of the graft may act as a substrate to allow recruitment
of cells into the wound. The dermal fibroblasts in the construct may contribute
to the formation of new dermal tissue through matrix biosynthesis.19-20 The mechanisms of action of this
skin substitute are of great interest and should be investigated further.
However, whatever the mechanism, the BSS offers a new treatment modality for
patients with venous and diabetic ulcers, and potentially for patients with
other types of wounds.
AUTHOR INFORMATION
Accepted for publication August 6, 2001.
This study was supported by a grant from Organogenesis, Inc.
Corresponding author and reprints: Tania J. Phillips, MD, Department
of Dermatology, Boston University School of Medicine, 609 Albany St, Boston,
MA 02118.
From the Department of Dermatology, Boston University School of Medicine,
Boston, Mass (Drs Phillips, Manzoor, Rojas, and Falanga and Ms Carson), and
Organogenesis, Inc, Canton, Mass (Mr Isaacs and Drs Sabolinski and Young).
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