TISSUE ENGINEERING

Volume 12, Number 9, 2006

# Mary Ann Liebert, Inc.

Update on Tissue-Engineered Biological Dressings

M. EHRENREICH, M.D., and Z. RUSZCZAK, M.D., Ph.D.

ABSTRACT

Tissue-engineered biological dressings offer promise in the treatment of burns, chronic ulcers, donor site
and other surgical wounds, and a variety of blistering and desquamating dermatologic conditions. For
example, the prevalence of diabetic foot ulcers ranges from 4.4% to 10.5% of diabetics, resulting in 82,000
lower extremity amputations annually; venous leg ulcers affect 0.18% to 1.35% of the population; and
pressure ulcers are found in 5.0% to 8.8% of institutionalized patients and 14.8% of patients in acute care
facilities. Despite the large number of potential beneficiaries, cellular tissue-engineered products have
suffered setbacks in recent years and have garnered considerably lower market share than commercial
promoters anticipated. The mechanism of action of these products is not universally agreed upon, but
delivery of growth factors and extracellular matrix components to the wound is thought to be important;
graft ‘‘take’’ is not usually considered to occur. These ‘‘engineered’’ products do not specifically match a
treatment modality to an underlying pathology. Clinical effect is often modest, and sometimes not justi-
fiable from a cost–benefit perspective. Nevertheless, clinical reports in the literature of uses of tissue-
engineered biological dressings continue to mount, indicating that these products are finding niche
applications where clinical utility is high and the cost can be defended. Despite commercial setbacks, the
first-approved products, DermagraftÒ, ApligrafÒ, and Cultured Epidermal Autograft (EpicelÒ) are still
being marketed, and new ones, such as OrCelÒ, continue to be developed. The major indications for these
products are summarized and a brief review of the available clinical literature is offered.

INTRODUCTION graftÒ and ApligrafÒ have, for various reasons, suffered

setbacks in recent years with considerably lower market
1

T HE TERM TISSUE ENGINEERING HAS NUMEROUS DEFINITIONS,

but a broad one that we prefer is ‘‘methods that either
promote biologic repair or regeneration of tissues or organs
by providing signaling, structural or cellular elements (other
than autografting of minimally manipulated tissue), or re-
place organ or tissue function with systems that contain
living tissue or cells.’’ The arsenal of tissue engineering can
include biomaterials, cells, growth factors, other signaling
molecules, and engineering components such as pumps,
tubes, bioreactors, and oxygenators.
Tissue-engineered biological dressings offer promise in
the treatment of burns, chronic ulcers (Table 1), donor site
and other surgical wounds, and a variety of blistering and
desquamating dermatologic conditions. Despite this po-
tential, cellular tissue-engineered products such a Derma-
share than the commercial promoters anticipated. Never-
theless, these devices continue to find application and im-
provements may facilitate broader use in the future.
The challenges in bringing the first living products
through U.S. Food and Drug Administration (FDA) ap-
proval and to commercialization should not be understated.
The time between initiating clinical trials in venous leg
ulcers (VLUs) and product launch for Apligraf was ap-
proximately 68 months (Table 2). Gaining an additional
indication in diabetic foot ulcers required an additional
51 months from the commencement of pivotal studies
(Table 3). This timeline does not include the bench work
and preclinical research requisite before research in humans
could begin. It is hoped that despite the relatively modest
impact of these products in clinical practice they will pave

Department of Dermatology, New Jersey Medical School, Newark, New Jersey.

2407
2408 EHRENREICH AND RUSZCZAK

TABLE 1. SELECTED CHRONIC WOUND DATA TABLE 3. APLIGRAF DEVELOPMENT MILESTONES FOR DIABETIC
FOOT ULCERS (DFU)
Other burden
Wound type Prevalence of disease Time between
Milestone Date milestones
Diabetic foot Prevalence of Results in 82,000
ulcers 4.4–10.5% of lower extremity Commence Apligraf March 1996 —
diabetic population amputations Pivotal Trial for DFU
annually; cost Complete enrollment November 1998 32 months
Medicare $1.5 Complete follow-up May 1999 6 months
billion in 1995 Submit PMA December 1999 7 months
Venous Prevalence of Reduce patient FDA approval for DFU June 2000 6 months
leg ulcers 0.18–1.35%; quality of life, but Total 51 months
approximately does not usually
600,000 wounds in lead to amputation;
the United States average cost per
The skin’s continuity and integrity can be compromised
month of venous
leg ulcer care
as a result of trauma, including surgery, or it can become
estimated at $2400 damaged secondary to an underlying pathology, such as
Pressure Prevalence 5.0–8.8% The majority heal reduced venous or arterial circulation. Wounds can afflict
ulcers of institutionalized readily, but a only the epidermis or can extend through the thicker dermal
population, and complex layer. Generally, for a given wound size, the deeper the
14.8% of patients full-thickness wound, the greater its clinical significance.
in acute care ulcer can cost up Skin consists of 2 distinct layers: the epidermis and
facilities to $70,000 to heal. the dermis. The thin epidermis comprises rapidly dividing
keratinocytes, which produce keratin, a strong structural
protein. The topmost layer of the epidermis is the stratum
corneum, a thin water-tight layer of dead, flattened cells.
the way for next-generation tissue-engineering devices that
The thicker dermis, situated below the epidermis, is a
can ameliorate critical donor organ shortages.
complex association of fibroblasts and extracellular matrix
(ECM), accounting for skin’s mechanical integrity and elas-
ticity. The skin’s blood vessels, nerve fibers, and lympha-
NEED FOR TISSUE-ENGINEERED
tics all reside within this layer. The epidermis is nourished
BIOLOGICAL DRESSINGS by diffusion of small molecules from the dermis. The skin’s
appendages, namely hair follicles and sweat glands, traverse
The skin is the largest organ of the human body. It is the
both levels. Beneath the dermis is the hypodermis, a loose
first line of defense against infection, prevents dehydration,
connective layer comprising primarily adipose tissue.
and helps to regulate body temperature through changes in
Wounds of different etiology, depth, and surface area
blood flow and sweat production. It is also a major sensory
must be treated differently and have dissimilar clinical
organ.
prognoses. Normal wound healing originates from pro-
liferating epidermal cells in the appendages of the deep
TABLE 2. APLIGRAF DEVELOPMENT MILESTONES FOR VENOUS dermal layer, such as hair follicles. In-growth from the
LEG ULCERS wound edges also occurs but is ineffective for wounds larger
Time between than a few centimeters. Wound healing without a dermal
Milestone Date milestones layer results in contracture. It can be concluded that large or
deep wounds and wounds that obliterate the skin’s appen-
Commence Late 1992 — dages are of particular clinical challenge. A distinction
Apligraf should be made between wound healing, which results in
Pivotal Trial
scarring, and regeneration, which implies the recapitula-
for VLU
tion of structure and function that is identical to uninjured
Complete October 1994 Approximately
enrollment 24 months tissue.
Announce data May 1995 7 months The ideal synthetic wound dressing or biologic skin
Submit PMA October 1995 5 months substitute should have the following characteristics, en-
FDA Panel January 1998 27 months umerated for the most part by Pruitt and Levine more than
FDA approval May 1998 4 months 20 years ago2 and added to by ourselves and others.3
Product launch June 1998 1 month

Absence of antigenicity
Total Approximately
Tissue compatible
68 months
Absence of local or systemic toxicity
UPDATE ON TISSUE-ENGINEERED BIOLOGICAL DRESSINGS

Impermeable to exogenous microorganisms

Water vapor transmission similar to normal skin

Rapid and sustained adherence to wound surface

Conformal to surface irregularities

Elastic to permit motion of underlying tissue

Resistant to linear and shear stresses

Tensile strength to resist fragmentation

Inhibition of wound surface flora and bacteria

Long shelf life, minimal storage requirements

Biodegradable (for permanent membranes)

Low cost

Minimize nursing care of wound

Minimize patient discomfort

Translucent properties to allow direct observation of
healing

Reduce heal-time

Not increase rate of infection

Patient acceptance
CHRONIC SKIN ULCERS
Several products have been introduced in recent years
that contain living cells. The initial FDA approvals for 2
products, Dermagraft and Apligraf, were in diabetic foot
ulcers and VLUs, respectively, but numerous studies ad-
dress the use of these products in burns, surgical wounds,
and desquamating dermatologic disorders such as epi-
dermolysis bullosa (EB).
It is important to note that chronic skin ulcers are not
frequently autografted, as the graft take is often poor. Al-
though today it is thought by many that Apligraf 4 and
Dermagraft exert their wound healing effects by delivering
growth factors and ECM components to the wound, they
were originally thought of as ‘‘skin substitutes.’’ The use of
these devices on chronic wounds demanded a paradigm
shift qualitatively different from their use in severe burns,
where the advantages of autografting were already well
established.
But the mechanism of action of these products is still
unresolved. For example, in chronic wounds, graft ‘‘take’’
has been noted with Apligraf in approximately 41% of
patients.5 Histological analysis of 14 wounds treated with
Apligraf indicated an increase in mucin production,6
prompting the author to propose that Apligraf may stimu-
late a fetal pattern of wound healing.
In general, these expensive wound care modalities are
second-line therapies. According to an article by Singer and
Clark in the New England Journal of Medicine, ‘‘con-
servative and time-honored methods of wound care should
be attempted first.’’7 This was echoed by Phillips in Ar-
chives of Dermatology, stating, ‘‘Biological skin substitutes
do not appear appropriate as first-line therapy for venous
ulcers.’’8 It has been pointed out that the utility of the living
cells has never been conclusively demonstrated in a trial
2409
comparing living product with a device rendered inviable
with gamma radiation.9,10 A number of animal studies
support the idea that living cells are more beneficial than
matrix or carrier alone,11,12 but others have not found this
to be the case.13,14
Diabetic foot ulcers
Peripheral neuropathy and vascular disease, when cou-
pled with minor injury and infection, often result in foot
ulceration in diabetics. These ulcers can be entirely neu-
ropathic, entirely ischemic, or result from a combination of
causes. Pathological changes in wound healing in the dia-
betic often render these ulcers slow to heal. The impaired
healing may result from numerous factors including chan-
ges in growth factor milieu, impaired fibroblast function,
defects in ECM environment, and decreased immunologic
function. These ulcers can have deleterious effects on
quality of life.15
Estimates of the prevalence of diabetic foot ulcers vary
depending on the study.16 The highest reported rate, 10.5%,
was in a population of 1780 non-insulin-dependent diabetics
in southern Wisconsin, while the lowest rate, 4.4%, was in
617 patients (unstated type of diabetes) in Stockholm
County, Sweden. Fifteen percent of diabetic individuals are
affected within their lifetime,16 resulting in approximately
82,000 lower extremity amputations annually.17 In 1995,
lower extremity ulcers cost Medicare $1.5 billion.18
The most commonly used grading system for diabetic foot
ulcer is the Wagner Grading System,19 which defines Grade
1 as superficial; Grade 2 as ulcer extension to ligament,
tendon, joint capsule, or fascia, but without osteomyelitis;
Grade 3 as a deep ulcer with infection or osteomyelitis; and
Grades 4 and 5 as including localized or extensive gangrene,
respectively.
The majority of ulcers heal if identified and properly
treated with dressings, topical medications, weigh off-
loading, and possibly sharp débridement.20 Many, however,
are resistant to treatment and ultimately require vascular
surgery or sometimes a skin graft. But many ulcers present
as Grade 4 or 5 and do not have sufficient granulation tissue
to be suitable for a graft of any type. This group was ex-
cluded from the Dermagraft pivotal trial.21 Also, by the time
a diabetic foot is at risk of amputation the cause is almost
always gangrene or infection,22,23 making it unlikely that
tissue-engineered biological dressings will be able to sal-
vage the foot. Infection is, not surprisingly, an exclusion
criterion for most clinical trials involving diabetic foot ul-
cers. One investigator has indicated that all diabetic neuro-
pathic foot ulcers should be assessed using quantitative skin
biopsies, as bacterial load has been show to correlate with
rate of wound healing.24
Many patients at high risk for amputation and suitable for
costly intervention have peripheral vascular disease. This
population was specifically excluded from the Dermagraft
and Apligraf trials. Vascular status is a strong predictor of
2410 EHRENREICH AND RUSZCZAK

both the development25,26 and healing27 of wounds, in-
cluding diabetic foot ulcers. One can anticipate that the
vascular disorder that impairs natural wound healing and the
take of autologous skin graft28 will, in many cases, impair
the efficacy of tissue-engineered biological dressings. It is
thought that the benefit of these products is often from de-
livery of growth factors and ECM components even without
graft take.29 Supporting this concept, increased blood flow to
the diabetic foot following Dermagraft application has been
demonstrated.30,31
The hard-to-heal fraction of Grade 2 and Grade 3 ulcers is
the most likely population to benefit from tissue-engineered
biological dressings. In an effort to stave off amputation,
some physicians will try the product even on Grade 4 ul-
cers, despite low odds of success and no supporting data
from pivotal clinical trials. These devices may also be used
in ischemic patients after they have undergone vascular
surgery.32 Finally, determination of ischemia using non-
invasive methods can be challenging, resulting in product
use in borderline cases.
Recurrence of diabetic foot ulcers is typical.23 This is not
necessarily a failure of treatment but a reflection of the fact
that current treatments address the wound and not under-
lying pathology. Compliance with lifestyle changes, such
as weight offloading, special shoes, and frequent foot in-
spections, is of far greater importance in preventing re-
currence than the mode of ulcer treatment. Aggressive
therapy and good patient compliance alone can reduce
amputation rates by 50%.33 It is unlikely that tissue-
engineered or other wound healing products can reduce the
recurrence rate in a meaningful way other than by sec-
ondary bias towards increased compliance. This is borne
out by clinical studies.20,34,35
Wound healing trials are challenging to conduct and
cannot be compared directly to one another. Nevertheless,
some important diabetic foot ulcer trials are summarized in
Table 4.
Venous leg ulcers (VLUs)
The prevalence of VLUs has been reported as 0.18–
1.35%, depending on inclusion criteria.38 There are be-
lieved to be approximately 600,000 such wounds in the
United States.39,40 Incompetent leg vein valves result in
retrograde blood flow and high macrocirculatory venous
pressure. This leads to elevated microcirculatory pressure
and an increased flux into the perivascular space of large
molecules, including fibrinogen. Fibrinogen polymerizes to
fibrin, creating a cuff and impairing nutrient and waste
exchange, oxygen diffusion, and availability of blood-
borne growth factors. Other factors, such as presence of
leukocytes, are likely to contribute as well.41 VLUs can be

TABLE 4. SELECTED DIABETIC FOOT ULCER CLINICAL TRIAL RESULTS

% Complete healinga

Standard
N Active of care p Endpointb

Dermagraft-2nd trial (interim 182 30 23 0.176 Healing within 12 weeks

analysis)c
Dermagraft-2nd trial 143 28 14 0.035 Healing within 12 weeks
(interim analysis;
ulcers>6-week duration)
Dermagraft-2nd trial 245 30 18.3 0.023 Healing within 12 weeks
(ulcers>6-week duration)
Apligraf pivotal trial 208 56 38 <0.01 Healing within 12 weeks
Dermagraft-1st pivotal trial 281 38.5 31.7 0.14 Healing within 12 weeks
Dermagraft-1st pivotal trial 226 51.0 31.7 <0.01 Healing within 12 weeks
with supplemental studyd
Regranex (PDGF-BB)36 922 43 33 Unadjusted statistical analysis Healing within 20 weeks;
of 4 studies; some showed hard-to-heal ulcer
significance, and some did not. population
See product label for details of
individual trials.
Regranex (PDGF-BB)—baseline 874 50 36 0.007 Healing within 20 weeks;
ulcer area <10 cm2, hard-to-heal ulcer
meta-analysis37 population
OrCel pilot study 40 35 20 NA Healing within 12 weeks
a
Direct comparisons between studies are not possible because of many variables that change from trial to trial.
b
Time to healing is an important endpoint but has not been used in this table for ease of comparison.
c
The first Dermagraft trial data is calculated on evaluable patients while the second is calculated on an intent-to-treat basis, making comparisons
inappropriate.
d
Additional references for Dermagraft and Apligraf can be found in the text of the article.
UPDATE ON TISSUE-ENGINEERED BIOLOGICAL DRESSINGS
staged as I through IV using the same system described for
pressure ulcers (described below), or are sometimes simply
described as superficial or deep. Several studies use the
similarly styled International Association Enterostomal
Therapy (IAET) classification.
Unlike diabetic foot ulcers, VLUs rarely progress to
osteomyelitis, gangrene, or infection. Nevertheless, these
persistent wounds substantially reduce patient quality of
life.42 The average cost per month of VLU care has been
estimated at $2400.43
The mainstay of therapy addresses the underlying pa-
thology. Compression bandages are applied, along with leg
elevation, to return venous pressure to a healthy range, while
local wound care facilitates healing. A review of 1200 re-
ports on VLU clinical trials (prior to the introduction of
tissue-engineered biological dressings) stated that no ther-
apy has proven superior to compression therapy.44 Fifty to
eighty-six percent of VLUs heal within 2–3 months with an
occlusive dressing and compression therapy.39,45,46 Con-
formity with such a regimen is often not possible. Many
cases of hard-to-heal venous ulcers are due to poor patient
compliance, as is the high incidence of ulcer recurrence. It is
possible that in the context of noncompliance with standard
care, tissue-engineered biological dressings will be less ef-
fective than observed in controlled clinical trials.
It is important to note that before any kind of wound
dressing is applied, the wound should be appropriately
prepared to enhance both the effectiveness of the dressing
and the self-healing ability of the wound. To achieve op-
timal healing, wounds must not be infected, they should
contain as much vascularized wound bed as possible, and
they should be free of exudate.47
Heal rates are significantly lower in ulcers of long dura-
tion, as indicated in the Apligraf trial, in which only 19% of
wounds of greater than 1-year duration healed within 6
months with compression therapy alone (see later).48 Simi-
larly, in a study of chronic VLUs analyzing the effects of
sharp débridement, only 16% of débrided ulcers and 4.3%
of nondébrided ulcers healed completely by 8–20 weeks.49
Ulcer recurrence is high, depending largely on com-
pliance with compression stockings. In one prospective
study, only 44% of patients were free of recurrence at
5 years.50 In the Apligraf pivotal trial, the recurrence rate
was approximately 20% at 12 months for both treatment and
control (18.1% Apligraf vs. 22.2% control, p ¼ ns).34 This is
not surprising because Apligraf treats symptomology and
not pathology. This is consistent with the RegranexÒ
(PDGF-BB) trials which indicated an ulcer recurrence rate
at 3 months of approximately 30% across all groups.20,36,37
Heal rates as low as 20% have been reported in VLUs
following split-thickness skin grafting (STSG),28 though
others report rates as high as 65%.51 This is not surprising
as STSG does not affect the venous circulation. This
treatment should generally be reserved for cases that fail
conventional therapy.41,52 As with diabetic foot ulcers, skin
grafts are not likely to be effective in the presence of
2411
arterial vascular disease. However, tissue-engineered bio-
logical dressings can provide growth factors and responsive
cells for a short while, possibly stimulating wound healing.
As mentioned, these products are thought to act more in the
capacity of an intelligent drug delivery system than a true
graft. It is not entirely clear why the heal rate with tissue-
engineered products (see Apligraf below) is higher than
most reports in the literature for STSG, but the mechanisms
seem to be unique. It is thought that tissue-engineered
biological dressings, such as Apligraf, should be attempted
before skin grafting.41
Given the high cost of Apligraf and Dermagraft, a number
of studies have been conducted analyzing the economic
considerations of using such products in VLUs. In a retro-
spective analysis of 13 patients with 21 ulcers, with a mean
ulcer duration and size of 23.9 months and 13.5 cm2, re-
spectively, the cost per change in unit ulcer size was lower
with Apligraf than with conventional care as observed prior
to Apligraf in the 5 patients for whom data were available.53
Whether cost per change in unit ulcer size is an appropriate
yardstick as opposed to cost per healed ulcer is certainly
debatable. Another economic model concluded that over
3 months Apligraf provided a benefit of 22 ulcer days
avoided at an incremental cost of $304. The product’s cost-
effectiveness increased with longer time horizons and with
patients with ulcers of greater than 1-year duration.54 In an
additional model, annual medical cost of treating patients
with hard-to-heal VLUs was $20,041 with Apligraf as
compared with $27,493 for Unna’s boot.55
Wound healing trials cannot be compared directly to one
another. Nevertheless, for frame of reference, the results of
several VLU trials are summarized in Table 5.
It has been the trend in chronic skin ulcer trials that pilot
data, which may reflect the best centers or other bias, are
superior to that seen for the trial as a whole (Table 6).
Pressure ulcers
Skin breakdown can result from pressure, shear forces,
and friction, typically on the sacrum or heel. These ulcers
occur most often in bed- or chair-bound populations, such
as the elderly, morbid, or paralyzed. They are usually
classified as Stage I through Stage IV according to the
National Pressure Ulcer Advisory Panel For Pressure Ul-
cers.58 Stage I is nonblanchable erythema of intact skin,
which presages ulceration; Stage II is partial-thickness skin
loss; Stage III is a full-thickness lesion that can extend
down to, but not through, fascia; and Stage IV is full-
thickness skin loss with extensive destruction of underlying
structures, such as bone and muscle.
Pressure ulcers occur in both acute and long-term care
facilities. It is reported that 5.0–8.8% of institutionalized
patients have one or more pressure ulcers,59 though certain
populations have a prevalence of over 20%.60 A 1995
survey of 265 acute care hospitals reported pressure ulcer
prevalence of 10.1%.61 In a 1999 survey of 356 acute care
2412 EHRENREICH AND RUSZCZAK

TABLE 5. SELECTED VENOUS LEG ULCER CLINICAL TRIAL RESULTS

% Complete healing

Standard
N Active of care p Endpoint

Apligraf—efficacy cohort 240 55.4 49.1 NS Wound closure by 6 months

Apligraf—ulcers 120 47.2 18.8 <0.05 Wound closure by 6 months
> 1-year duration
Apligraf—efficacy 240 56.8 39.8 0.02 Wound closure by 6 months
cohort adjusted Cox
KGF-256 144a 53–55%b 36% NA 90% wound closure at 12 weeks
OrCel (CCS)—pilot 36c 52.9 26.3 NA Wound closure at 12 weeks
(Planimetric analysis)
OrCel Pivotald 136 59 36 NA Wound closure at 12 weeks
a
In 3 arms.
b
53% in 20-mcg arm and 55% in 60-mcg arm.
c
27 completed the study.
d
FDA has required a confirmatory study.
Additional references for Dermagraft, Apligraf, and OrCel can be found in the body of the article.

facilities the prevalence was 14.8%, with 7.1% being no- The costs of pressure ulcers to the healthcare system are
socomial in origin.62 It is hoped that the prevalence of high but not as a rule due to complications of the ulcer, as
pressure ulcers will decline in the future as more attention indicated by the fact that most pressure ulcers are super-
is paid to prevention. In addition to the high cost of care ficial. Costs in an acute care setting are related to routine
that pressure ulcers necessitate, they are often deemed wound dressing and nursing care. The implication is that
de facto signs of neglect. The threat of litigation is a potent additional expenditures for pressure sore prevention can be
motivator to augment preventive measures. justified but that high-cost treatments to heal pressure ul-
An excessive amount of nursing time is spent on wound cers are in most cases unnecessary. But a complex full-
dressing for pressure ulcers. Standard therapy consists of thickness pressure ulcer, which can cost up to $70,000 to
keeping the wound moist using conventional saline-soaked heal,62 could certainly benefit from treatment modalities
gauze, hydrogel, hydrocolloid, or alginate dressings. Most that can decrease this burden.
pressure ulcers (*80%) are superficial (stages I and II).61,62 In a long-term care setting it was found that 80% of the
The majority of these heal readily after 1–2 weeks of total cost of pressure ulcer treatment was generated by the
treatment. Stage III and stage IV ulcers usually require 4% of patients who required hospitalization.63 If these
débridement and with proper care can heal in 6–12 weeks. high-risk patients can be identified in advance and if tissue-
Deep ulcers that fail to show significant healing within the engineered biological dressings can reduce hospitalization
first few weeks of treatment often require surgical inter- rates then the high cost of these products can be justified in
vention such as a myocutaneous flap graft. Often, sufficient this subpopulation.
granulation tissue is not present to perform a split-thickness Many hard-to-heal and hard-to-prevent pressure ulcers
skin graft. As with other skin ulcers, tissue-engineered occur in patients expected to expire shortly from other
biological dressings in the case of pressure ulcers is more causes. In the 1999 pressure ulcer survey, 61% of patients
likely to act as a growth factor delivery vehicle than a true with pressure ulcers were older than 70. The highest in-
graft. cidence of pressure ulcers (21.5%) occurs in the intensive
care unit.62 These patients may be unlikely to receive
treatment for their ulcers. Given the fact that many persons
TABLE 6. PILOT VS. PIVOTAL TRIAL RESULTS with pressure ulcers are poor or elderly, attainment of
Medicare and Medicaid reimbursement can be critical to
Product Ulcer type Pilot Pivotal using tissue-engineered biological dressings.
Regranex Pressure 23% vs. 0% Failure
Dermagraft Diabetic 50% vs. 8% 39% vs. 32% Apligraf
30% vs. 18%
Apligraf (formerly Graftskin and Living Skin Equiva-
Apligraf57 Venous 70% vs. 29% 57% vs. 40%
lent), developed by Organogenesis Inc. (Canton, MA), is a
Diabetic 75% vs. 41% 56% vs. 39%
bilayer approximating the structure of normal skin. In 1981,
CCS Venous 53% vs. 26% 59% vs. 36%
Dr. Eugene Bell succeeded in growing a living skin-
Diabetic 56% vs. 29% NA
equivalent (LSE) in vitro and, in animal studies, grafting
UPDATE ON TISSUE-ENGINEERED BIOLOGICAL DRESSINGS
the construct onto the donors of the cells.64 The skin-
equivalent comprised a dermal equivalent derived from
fibroblasts in a contracted type I collagen matrix and an
epidermis generated by keratinocytes seeded onto the
dermal equivalent. By December 1986, Organogenesis re-
ported that the LSE had been successfully grafted onto rats
in more than 700 experiments.65 This technology went on
to become Apligraf, the first medical device containing
living allogeneic cells to be approved by the FDA.
The dermal equivalent layer of Apligraf is produced by
neonatal foreskin fibroblasts organizing a bovine collagen
solution into a contracted matrix. The epidermal equivalent
layer comprises neonatal keratinocytes seeded on this layer.
During the manufacturing process, the epidermal layer is
exposed to oxygen, giving rise to the stratum corneum.
Apligraf contains no dermal appendages. As with Derma-
graft (see below), cells do not survive long-term in the
wound. Rather, Apligraf promotes fibrovascular ingrowth
and epithelialization. In most cases, the graft does not
‘‘take’’ nor does it ‘‘close’’ the wound.29 The mechanism of
action of Apligraf has not been clearly elucidated but has
been theorized to work in 3 ways: graft ‘‘take,’’ temporary
wound closure with reepithelialization from the wound
margins, and stimulation of wound healing through growth
factors and ECM components without even temporary
persistence in the wound.66 In a 10-patient VLU study, only
2 of 8 patients evaluated at 4 weeks were positive for
Apligraf-derived DNA. Neither of these patients showed
Apligraf-derived DNA at 8 weeks.34 In clinical trials,
Apligraf did not elicit an antibody response to bovine
collagen or class I HLA antigens on the fibroblasts or
epidermal cells. T-cell response was not observed nor was
there any sign of clinical rejection,67,68 consistent with
previous studies in the hu-PBL-SCID mouse model.69
Histological analysis has indicated a foreign body granulo-
matous response in some patients, though this was not as-
sociated with a poor clinical outcome.6
Apligraf, supplied as a circular disk 75 mm in diameter
(44 cm2) and 0.75 mm thick, has a limited shelf life, originally
5 days but recently increased to 10.70 The product is supplied
in a polyethylene bag containing a 10% CO2/air ratio and
agarose nutrient medium and must be stored at 208C–238C
until use. It is quite expensive71 at £14.20 per cm2.
Wound bed preparation is critical. Débridement of ne-
crotic tissue, control of local infection, and control of edema
to eliminate exudates must be undertaken before treatment.
Apligraf for many applications can be meshed or slit to allow
drainage. Because Apligraf does not ‘‘take’’ as a skin graft,
its appearance on the wound can vary (see below). Usually, 1
or 2 applications of Apligraf occur with minimal wound care
necessary in the interim. Additional application of Apligraf
is usually not covered by Medicare.72
The major adverse event is suspected wound infection at
the study site, reported in 29.2% of patients receiving
Apligraf in the VLU trial versus only 14.0% of control.34
Diagnosis of wound infection can be complicated by the
2413
yellow or white appearance of Apligraf after it becomes
hydrated with wound fluid.6,34 This may have led to an
overestimation of infected ulcers in the Apligraf arm. This
interpretation is supported by the diabetic foot ulcer trial, in
which suspected wound infection at the study site occurred
less frequently in the Apligraf arm (10.7%) versus control
(13.5%),34 suggesting a learning curve with the product.
A clinical trial in VLUs commenced in late 1992, was
completely enrolled by October 1994, and data from it
announced in May 1995. Although the Apligraf Premarket
Approval Application (PMA) was submitted to the FDA in
October 1995, it was not until January 1998 that it was
reviewed by an FDA panel, which voted in favor of ap-
proval without conditions. Apligraf was ultimately approved
in May 1998 for use in VLUs of greater than 1-month
duration. In 2000, the label was expanded to include the
treatment of diabetic foot ulcers of greater than 3-week
duration, without tendon, muscle, capsule, or bone exposure.
Apligraf was initially launched by Novartis (Basel, Swit-
zerland) in June of 1998. However, in early 2000 Novartis
transferred all rights in the product back to Organogenesis.
Currently, Apligraf is available only in the United States and
Canada.
Apligraf for chronic skin ulcers
The approval of Apligraf in VLU was based on a 240-
subject, 15-center, randomized, controlled clinical trial in
which the primary endpoints were frequency of, and time to,
wound closure by 6 months. For the study population as a
whole, use of Apligraf did not result in a statistically sig-
nificant difference versus control on the primary endpoint
of wound closure by 6 months (55.4% vs. 49.1%, p ¼
0.37).34,48,73 Unadjusted data did not show a benefit on
median time to wound closure versus control (140 days vs.
181 days, p ¼ 0.39). However, a statistical analysis that
corrects for covariates (Cox Proportional Hazards Model)
revealed an advantage to Apligraf on both median time
to wound closure (99 days vs. 184 days, p < 0.01) and on
wound closure by 6 months (56.8% vs. 39.8%, p ¼
0.02.)34,48,73 When the data were stratified by ulcer duration,
Apligraf was shown to be equivalent to control in ulcers of
less than 1-year duration (65.5% vs. 72.6%) and superior to
control in ulcers of greater than 1-year duration (47% vs.
19%).48,73,74
Reports on small series outside the context of clinical
trials continue to support use of Apligraf in VLU. In a ret-
rospective analysis of 13 patients with VLUs of nearly 2-
year duration, Apligraf resulted in a 60.5% reduction in ulcer
size versus baseline, but only 1 ulcer achieved 100% closure
within 3 months.53 Although encouraging, this also high-
lights the center-to-center variability and context depen-
dence of successful wound care.
A 1999 consensus statement concluded that Apli-
graf should be considered if patients fail to respond to
compression therapy by 4 weeks.75 There is some evidence
2414
that the initial rate of healing, measured as early as 3 weeks
from the start of compression therapy, can predict whether
a VLU will ultimately prove hard to heal.76 Another author
more conservatively recommends Apligraf or cell therapy
in cases with less than a 10% decrease in wound area over 2
months.41 A severity scale has been developed, using data
from the clinical trial, to evaluate the need for Apligraf on a
VLU.77 Wound duration and ulcer area were found to have
the greatest impact on ulcer healing.
The diabetic foot ulcer PMA supplement was based on a
208-patient trial comparing Apligraf (up to 5 applications)
plus standard of care with standard of care alone. Patients in
the Apligraf group healed 56% of ulcers within 12 weeks, as
compared with 38% for standard of care alone, and had
reduced time to healing (median 65 days vs. 90 days.)67
Apligraf use was associated with a reduction in the ampu-
tation rate versus control (16% vs. 6%). Approximately
9% of patients received 1 application of Apligraf, 10% re-
ceived 2; 13%, 3; 15%, 4; and 53%, 5. The response rate
decreased rapidly with the number of applications. In other
words, if the wound did not respond quickly, it was de-
creasingly likely to heal and was more likely to become
infected.
In a series of 10 diabetic patients with various ulcera-
tions, all wounds were healed in 7 (17 of 20 total wounds
healed in an average of 42 days) when treated with Apli-
graf. In all but one case, a single Apligraf treatment was
sufficient. In 2 of the patients with failures, Apligraf was
placed on necrotic gangrenous toes. The other failure
was in a patient with renal failure, a contralateral below-
knee amputation, and a purulent wound on a failed muscle
flap.78
A 31-patient study compared Apligraf to moist dressing
changes in patients with lower extremity foot ulcers or
wounds, who had undergone revascularization surgery. The
use of Apligraf was associated with a significantly greater
percentage of wounds healed than moist dressing changes
(62% vs. 0% at 8 weeks; 86% vs. 40% at 12 weeks, p < 0.01)
and a decreased median time to complete wound closure
(7 vs. 15 weeks, p < 0.01).32
In pressure ulcers, 13 patients with a total of 21 wounds
were treated with Apligraf; in 7 of these patients all wounds
were healed. Thirteen of twenty-one of the wounds healed
within 29 days after 1 Apligraf application. The patients
also received alternating air therapy.78
Apligraf in burns
The first intended indication for Apligraf was full-
thickness burns. A 15-patient pilot study in this setting
commenced in 1989. Based on encouraging results, a 6-
center, open-label, within-patient controlled trial was un-
dertaken in 1992. Owing to the limited number of sites and
an FDA requirement for 2-year follow-up data, the study
was not fully enrolled until early 1995 and follow-up was not
completed until early 1997.
EHRENREICH AND RUSZCZAK
In the study, physicians had 2 possible protocols to
choose from: either Apligraf could be applied directly to
the wound or meshed Apligraf could be placed over me-
shed autograft. Most patients in the study—40 out of 56—
were treated with meshed Apligraf over autograft. Apligraf
did not influence the rate of underlying autograft take. In
these patients, the use of meshed Apligraf in conjunc-
tion with meshed autograft resulted in better scores on the
Vancouver Scar Assessment Scale than the use of meshed
autograft alone. In most patients, Apligraf-treated burns
were judged to have healed better than those treated with
meshed autograft alone. Investigators rated Apligraf-treated
sites superior to control in 58% of patients, equivalent in
16%, and worse in 16%.79
Despite these results, a labeling for burns was never
sought. Current reports of Apligraf use in burns are scarce,
with one case study in the last 5 years describing its suc-
cessful use on a full-thickness burn to the dorsum of the
foot.80
Apligraf in acute wounds
Meshed Apligraf, in a 20-subject, within-patient con-
trolled study in donor sites, was comparable to meshed
autograft and superior to polyurethane film dressing in both
time to complete healing and reduction in pain.81,82 The
difference in healing time versus polyurethane film (7.3
days vs. 9.5 days), though statistically significant, is not
clinically relevant in most cases. The common denominator
in reducing pain is the use of a biological dressing. Apligraf
does not seem likely to offer an advantage versus dressings
such as BiobraneÒ83 in most instances.
In an uncontrolled pilot study in skin cancer excision, 12
of 15 patients receiving Apligraf had clinical graft take at
7 days.84 They would otherwise have required a skin graft or
been allowed to heal by second intention. One patient tore
off his Apligraf on day 2 and 1 patient became infected. Of
13 evaluable patients, healing was judged to be Good or
Excellent in 11, Fair in 1, and Poor in 1. Anecdotally, pa-
tients in the study who had received Mohs surgery to other
body parts, or in the past, reported that Apligraf produced a
better quality of life, resulting in less pain and fewer dressing
changes. The authors reported that the ‘‘degree of con-
traction was greater than would have occurred with full-
thickness grafts’’ but that the ‘‘the artificial look often given
by full-thickness grafts would offset the advantage of less or
no contraction.’’ It is important to note that a donor wound
was avoided in those that otherwise would have required a
skin graft. Since 8 out of 15 patients were older than 60 years
and subject to delayed donor site healing, this is an
advantage.
In a series of 107 patients with acute wounds, mostly
following skin cancer excision, Apligraf persistence after
one application was reported as ‘‘good’’ to ‘‘excellent’’ in
73.3% of patients at 1 week, 56.6% at 2 weeks, and 53.6% at
1 month.68
UPDATE ON TISSUE-ENGINEERED BIOLOGICAL DRESSINGS
In another series, Apligraf following Mohs or excisional
surgery for skin cancer was compared to healing by sec-
ondary intention in 14 patients (12 evaluable). The 2 groups
were equivalent in terms of healing at 6 months (100% in
both groups), healing rate, and comorbid factors such as
pain and infection. Apligraf use resulted in a more pliable
and less vascular wound as defined by the Vancouver Scar
Assessment Scale, and greater patient satisfaction with the
cosmetic outcome.85 Another report on 2 patients treated
with Apligraf following Mohs surgery indicates complete
healing within 9 weeks with no complications. The authors
considered it ‘‘an excellent alternative for difficult surgical
wounds.’’86 Apligraf has also been used following laser
ablation of a hypertrophic scar.87
The product has been used in 10 patients, retrospectively
identified, with traumatic avulsion wounds and dermal
atrophy related to aging or steroid use. Primary closure in
these cases was not possible because of tissue loss or an-
ticipated necrosis. A skin graft or closure by secondary
intention would otherwise have been necessary. All patients
had 100% wound healing at an average time of 9.2 weeks,
but 2 required reapplication due to ‘‘graft loss.’’88
Apligraf has been used to treat challenging wounds in
other circumstances. Leg and sternal wound complications
are common following coronary artery bypass grafting,
reported at 2.9% and 0.9%, respectively, in one series.89 A
retrospective study of 45 leg and 15 sternal wounds in-
dicated that Apligraf reduced time to healing in both groups
(mean 46 days vs. 84 days in leg wounds; 39 days vs. 62
days in sternal wounds) as compared with standard care.89
A case study reports on a chronic ulcer of 28-year duration
on a right foot amputation stump that healed by 7 weeks
after 1 Apligraf application.90
Apligraf in epidermolysis bullosa
Apligraf has been used to treat EB, a rare inherited dis-
order in which minor trauma results in severe skin blister-
ing.91 There are 3 types of EB, categorized by level of blister
formation. In the most severe form, dystrophic EB, the
blister occurs below the basement membrane in the dermis,
resulting in the equivalent of a full-thickness burn. The af-
flicted newborn’s skin sloughs and blisters at the slightest
touch, resulting in painful ulcerations and permanent scar-
ring, including the so-called ‘‘mitten’’ deformities of the
hands and feet. The cause of the disorder is a defect in an
integrin responsible for basement membrane attachment.
The standard treatment is nonadherent dressings for wound
coverage and surgical approaches such as autologous skin
flaps or split-thickness skin graft. Impaired wound healing,
scarring, and infection can occur at the donor site following
autologous STSG.
Following a case report of the successful use of Apligraf
in an infant with Dowling-Merea variant EB,92 a larger
study was undertaken. In 15 patients with EB (9 dystrophic,
1 junctional, and 5 simplex), with a total of 69 acute
2415
wounds treated with Apligraf, 79% (63/69) were healed at
day 7, 82% (51/62) at 6 weeks, 75% (27/36) at 12 weeks,
and 79% (11/14) at 18 weeks. Quality of life was reported
to improve after treatment for most patients.93
The formation of mitten deformities is a difficult to
manage complication. In 5 patients, Apligraf was used to
dress the fingers after digit release. This resulted in im-
proved range of motion in all cases, but repeated blistering
occurred in several patients at variable intervals.94 The
same author reports on 9 children with EB treated with
Apligraf and indicates 90–100% healing at 5 to 7 days at a
total of 96 sites treated.95 With follow-up periods of 6–36
months, most treated sites remained free of blisters. A case
report of Apligraf in a patient with EB following digit re-
lease for pseudosyndactyly of the fingers describes very
good ‘‘take.’’ At 1-year follow-up, functional gains in the
hands persisted.96
In a 12-year-old boy with EB and previously failed skin
grafting attempts, Apligraf was used to cover the defect
resulting from axillary contracture release. Apligraf and
full-thickness autologous skin grafting was also used to
correct ectropion in this patient. At 1-year follow-up, the
author reports ‘‘Excellent adaptation of the grafts … greatly
improved upper extremity movements.’’97
Apligraf has been used in 2 patients with Herlitz junc-
tional EB, a fatal form of the disease caused by the absence
of laminin-5 in the basement membrane. In 1 infant, 10 of 13
acute or chronic wounds were healed after 3–6 weeks of
treatment. Nine of ten wounds successfully treated after 1
round of treatment were still closed at 18-week follow-up. In
the second patient, however, only 2 of 18 chronic wounds
were healed at 3 weeks.98
In view of the fact that EB is a genetic disease that results
from defects in various ECM components and their recep-
tors, it is doubtful that tissue-engineered products can have
any long-term impact. However, if the wound can be closed
for 3–6 months this would probably be considered clinically
significant.
Apligraf in other skin disorders
The use of Apligraf to repair a cicatricial ectropion in an
infant with harlequin ichthyosis, a congenital scaling con-
dition of the skin, has been reported. Recurrent ectropion
developed, which was retreated with Apligraf at 61 days.
The repair persisted until the child’s expiration from re-
spiratory failure at 6 months. It was suggested that the
persistence may have related to general improvement in the
dermatopathy and not the product.99
Apligraf has been used to treat aplasia cutis congenita, a
condition in which skin is absent from certain areas at birth.
In this case, Apligraf was applied to an 8 cm8 cm full-
thickness defect in the thoracolumbar area. The author re-
ports 80% graft ‘‘take’’ at day 5.100 However, others have
indicated that AlloDermÒ and cultured epidermal autograft
are preferred for the coverage of aplasia cutis congenital.101
2416
A case report describes use of Apligraf to treat chronic
ulcers secondary to bullous morphea, a localized form
of scleroderma. Complete wound healing occurred after
4 months following 1 application.102
Case studies report the use of Apligraf to treat an excised
lesion of actinic purpura in an elderly patient with good
clinical ‘‘take’’ and lack of new skin tears, erosions, or
ulcers103; to heal refractory ulcers in a patient with poly-
arteritis nodosa104; to close steroid-resistant ulcers second-
ary to cutaneous sarcoidosis105; to heal within 6 weeks a
7.6 cm6.9 cm pyoderma gangrenosum lesion, in con-
junction with cyclosporine106; to successfully treat a hy-
droxyurea induced ulcer in an elderly woman with chronic
myelocytic leukemia107; and to heal nonhealing amputation
stumps secondary to purpura fulminans.108
In summary, clinical data support the use of Apligraf in
certain diabetic foot ulcers, VLUs, and a variety of other
applications, such as acute wounds and various ulcerative
and dermatologic conditions. The product’s major defi-
ciencies are the logistics of delivery, being limited by a 10-
day shelf life, high cost, and a variable outcome in the
wound environment ranging from apparent ‘‘take’’ to de-
gradation to a yellow mass that can be confused with
purulence.
DERMAGRAFT
Dermagraft, originally developed by Advance Tissue
Sciences, Inc., and now a product of Smith & Nephew
(London, UK), is composed of neonatal fibroblasts, ori-
ginally derived from human foreskin, cultured onto a
bioresorbable glycolic acid scaffold (polyglactin 910). The
product does not have an epidermal equivalent layer. The
mechanism of action is not entirely elucidated. The cells
deposit into the mesh ECM components such as collagens,
vitronectin, glycosaminoglycans, and growth factors. Der-
magraft stimulates healing, the ingrowth of fibrovascular
tissue, and epithelialization but does not close the wound.
The cells in Dermagraft remain metabolically active but
eventually die off. This is in contrast to TransCyte, an
earlier developed product intended as a temporary dressing
in burns, in which the cells are not viable. It is composed of
human neonatal fibroblasts cultured under aseptic condi-
tions onto the nylon mesh component of Biobrane.109
Dermagraft does not appear to stimulate rejection.21,34,110
As is the case with Apligraf, preparation of the wound
bed is important. Sharp débridement should be used to
create a wound bed suitable for skin grafting.34 Although
clinical trials used up to 8 pieces of Dermagraft over a 12-
week period, in most cases application of more than 1 sheet
is not considered ‘‘reasonable and necessary’’ and is not
reimbursed.111 Dermagraft is supplied frozen as a
5.0 cm7.5 cm sheet and must be stored at –758C, com-
plicating the logistics of its use and delivery. At £7.14
per cm2 it is moderately expensive.71
EHRENREICH AND RUSZCZAK
Dermagraft for chronic skin ulcers
Dermagraft was approved by the FDA in September
2001 for use in treatment of full-thickness diabetic foot
ulcers of greater than 6-week duration that extend through
dermis but without exposed tendon, muscle, joint capsule,
or bone exposure.34
The pivotal study was a 314-patient, controlled, rando-
mized clinical trial comparing Dermagraft plus conven-
tional therapy to conventional therapy alone, consisting of
sharp débridement, saline-moistened gauze, and pressure-
reducing footwear, in plantar diabetic foot ulcers.34 The
efficacy evaluation only included the 245 patients with
ulcers of greater than 6-week duration at enrollment. The
trial allowed for up to 8 Dermagraft applications over
12 weeks. The endpoint was complete wound closure, de-
fined as reepithelialization without drainage, by week 12.
The statistical analysis used in this trial was quite com-
plex, utilizing Bayesian analysis that allowed for informa-
tion from the first part of the trial to be utilized prospectively
in the latter part. This analysis indicated that 30% (39 of 130)
of Dermagraft patients healed compared with 18.3% (21 of
115) of control patients, a statistically significant difference,
in those patients with ulcers of greater than 6-week duration,
a subgroup defined first at an interim analysis.21 At an in-
terim analysis that evaluated 182 patients Dermagraft failed
to show a statistically significant advantage in complete
healing at 12 weeks (30% vs. 23%; p ¼ 0.176).112 If the
healing rate revealed on the interim analyses had been
equivalent to the results seen in an earlier pivotal trial (see
below) then enrollment would have been terminated and a
PMA prepared for filing. In the subset of patients that had
ulcers of greater than 6-week duration, which was 79% of
patients, Dermagraft did show a statistically significant ad-
vantage versus control ( p ¼ 0.035). Twenty-eight percent of
Dermagraft patients healed in this group versus 14% of
control. The statistical plan for the balance of the trial was
therefore modified as noted above. The incidence of ulcer-
related adverse events was lower in the Dermagraft group as
compared with control (19% vs. 32%; p ¼ 0.007).21
The road to approval was a long one. Prior clinical trials
had failed to support an FDA labeling of Dermagraft in
diabetic foot ulcers. In 1996, a 281-subject, single-blinded,
randomized, multicenter clinical trial was completed as-
sessing the use of Dermagraft in the treatment of diabetic
ulcers.113 In the treatment group, Dermagraft was applied
once per week for up to 8 weeks, while the control group
received standard care. The study’s primary endpoint was
week 12 complete healing. The results of this trial were
complicated by the fact that 44% of the treatment group
received Dermagraft that was deemed to be not metaboli-
cally active within a defined range in over half of their
doses. (Protein synthesis by cells in Dermagraft was in-
hibited by 70–98% by cryopreservation but recovered to
45–85% of the precryopreservation value within 48 h if the
cells were in the therapeutic range as defined by the MTT
UPDATE ON TISSUE-ENGINEERED BIOLOGICAL DRESSINGS
reduction assay. Dermagraft outside this metabolic range
showed variable low recovery.114)
This therapeutic range was identified retrospectively fol-
lowing interim analyses. The use of Dermagraft when
evaluated in the prospectively defined treatment group did
not result in a statistically significant change in week 12
complete healing versus control (38.5% vs. 31.7%, p ¼
0.14). An evaluation of the retrospectively defined subgroup
who received Dermagraft within the therapeutic range
showed that Dermagraft in the therapeutic range was sig-
nificantly better than control in achieving week 12 complete
healing (50.8% vs. 31.7%, p ¼ 0.006). At 32-week follow-
up, both the prospectively defined Dermagraft treatment
group and the retrospectively defined therapeutic range
group performed better than control on 32-week complete
healing (Dermagraft 57.5%, Dermagraft therapeutic range
57.7%, control 42.4%, p < 0.05 vs. control). However,
32-week complete healing was not the study’s primary
endpoint.
To confirm the effectiveness of Dermagraft with meta-
bolic activity in the therapeutic range, a 50-subject sup-
plemental study was undertaken in which all subjects
received Dermagraft metabolically active in the therapeutic
range. By week 12, 51.3% of evaluable patients had
achieved complete healing, consistent with the pivotal trial
results. When the supplemental study was combined with
the pivotal study, a significant difference was demonstrated
between Dermagraft and control (51.0% vs. 31.7% p ¼
0.002).115 Needless to say, the combination of an open-
label, noncontrolled supplementary study with a single-
blinded, controlled study is far from ideal. On January 29,
1998, an FDA advisory panel recommended approval of
Dermagraft for diabetic foot ulcers, with the condition that
the product’s sponsor conduct another controlled clinical
trial following approval. However, the FDA in a relatively
rare departure did not follow this panel recommendation
and required the new clinical trial, described above, prior to
approval, resulting in a nearly 4-year delay for the product.
In November 1993, Dermagraft completed enrollment of
a 210-subject pivotal clinical trial evaluating Dermagraft
for use in venous ulcers. An interim analysis in April 1994
indicated that there was no significant difference in week
24 complete healing between the control group and those
treated with a single application of Dermagraft. Six-month
follow-up data did, however, show a statistically significant
reduction in recurrence rate with the use of Dermagraft
versus control (6% vs. 20%).116 This was not considered
compelling enough to warrant further investigation at the
time. A later pilot study of 18 patients with VLUs of more
than 12-week duration compared 4 applications of Der-
magraft plus compression dressings with compression
dressings alone.31 On the study’s primary endpoint of
complete healing by 12 weeks, Dermagraft outperformed
conventional therapy alone (50% vs. 12.5%) but the study
was not sufficiently powered to demonstrate significance
( p ¼ 0.15). However, on a secondary endpoint of reduction
2417
in ulcer area (84% reduction vs. 16%), the results achieved
statistical significance ( p ¼ 0.002), as did the rate of linear
and area healing per week. However, control ulcers were
12.3 cm2 at baseline versus 9.5 cm2 for the Dermagraft-
treated group, complicating analysis.
During 1994, Dermagraft underwent a 50-subject pilot
clinical study in pressure ulcers, evaluating 3 dosing regi-
mens. Week 12 complete healing was the study’s primary
endpoint. The treatment group that received 2 pieces of
Dermagraft every 2 weeks, for a total of 8 pieces, achieved
46% complete wound healing by week 12 versus 25% for
the control group.116 Despite the encouraging pilot data, the
product’s sponsor did not at that time initiate pivotal trials
in pressure ulcers, preferring to focus its resources on trials
in diabetic ulcers.
Dermagraft in other skin disorders
Dermagraft, like Apligraf, may also be of utility in various
ulcerative or blistering skin disorders. For example, there
have been 2 case reports of Dermagraft having been used
successfully in ulcerated necrobiosis lipoidica, a rare skin
condition strongly associated with diabetes mellitus.117,118
Dermagraft in burns
Early in its development, Dermagraft was evaluated in
17 patients as a dermal replacement beneath meshed STSG
in full-thickness burn wounds. The Dermagraft-treated sites
were compared with paired controls sites on each patient
receiving only STSG. The results indicated that take of the
STSG on control sites was actually better than that on the
Dermagraft-treated sites, but not to a statistically significant
degree.110
Dermagraft in acute wounds
Although there are numerous reports of IntegraÒ Artifi-
cial Skin (Integra Lifesciences, Plainsboro, NJ), Alloderm
(LifeCell, Branchburg, NJ), Biobrane (Mylan Laboratories
Canonsburg, PA), and Apligraf being used in plastic and
reconstructive applications, reports describing the use of
Dermagraft are scarcer.
Six patients received Dermagraft to line a buccal fat pad
flap (BFPF).119 This flap is easily raised and can be used to
cover defects in the buccal plane after excision of squa-
mous cell carcinomas. The limitation of this approach is
that it is not epithelialized, resulting in fibrous scar tissue
which in this location can impede speech or swallowing. At
2-year follow-up all patients exhibited no change in texture
at the defect site, ‘‘giving the impression that the defect was
not only reconstructed but regenerated,’’ and exhibited no
functional deficits.
An interesting use of Dermagraft is to promote epithelial
regeneration for secondary closure of complex surgical
wounds. One case study reports its use in a pediatric patient
with a complex surgical history that complicated abdominal
2418
wall closure.120 Five applications of Dermagraft over
8 months is believed by the authors to have contributed to
successful wound closure.
In summary, Dermagraft is easier to use than Apligraf, as
it can be stored frozen whereas Apligraf must be used fresh
within 10 days, but it is more demanding to store than pro-
ducts such as Integra Artificial Skin or AlloDerm. Its clinical
trial history is not as clear cut as one might have hoped, but
the preponderance of evidence is that it does promote wound
healing in diabetic foot ulcers and possibly other chronic
wounds, though the niche for its use is relatively narrow,
requiring uninfected, well-vascularized wounds without
exposed bone or tendon, a perhaps not too common con-
fluence of conditions among hard-to-heal diabetic foot ul-
cers. Its use in other application, such as acute wounds, has
not been as widely reported on as with Apligraf.
ORCEL BILAYER CELLULAR MATRIX
OrCelÒ, previously termed Composite Cultured Skin
(CCS), was invented by Dr. Mark Eisenberg of Australia121
as a treatment for dystrophic EB, a rare genetic disorder in
which the skin is prone to repeated blistering, and further
developed by Ortec International (New York, NY). The
product is not currently available commercially.
Like Apligraf, OrCel is a bilayer dressing resembling
normal skin. The product is composed of type I bovine
collagen in which epidermal keratinocytes and dermal fi-
broblasts, derived from neonatal foreskin tissue, are cul-
tured in 2 layers. Fibroblasts are placed within the porous
sponge while keratinocytes are seeded on the nonporous
side of the matrix. Like Apligraf and Dermagraft, the
product does not contain dermal appendages.122
The major differences between OrCel and Apligraf are that
in OrCel fibroblasts are cultured onto a preformed porous
sponge, while in Apligraf they are cultured in a collagen so-
lution, which then forms a gel matrix; in OrCel the epidermal
layer is not exposed to an air interface, so it does not differ-
entiate into a stratum corneum during manufacture; culture
time for OrCel is 10–14 days as compared with 21 days for
Apligraf122; and OrCel is now produced in a cryopreserved
format whereas Apligraf must be delivered fresh.
The fresh form of OrCel has been previously approved
by FDA (see below) but is not being marketed at this time.
The cryopreserved form was used in a recently completed
clinical trial in VLUs (see below) and will be used in an
upcoming pivotal trial for diabetic foot ulcers.
According to its developers,123 OrCel was developed as a
tissue-engineered biological dressing that delivers ECM
components and growth factors and creates an environment
conducive to wound healing and was never intended to be
viewed as an artificial skin for grafting. One placed in
a wound bed, the device is resorbed in approximately
2 weeks. The duration of OrCel cells in wounds is not
EHRENREICH AND RUSZCZAK
precisely known but, like Dermagraft and Apligraf, is
thought to be limited to about 4 weeks. The fate of these
cells remained an open question at an FDA panel meeting
reviewing OrCel for donor site wounds.124
OrCel in epidermolysis bullosa
Between 1977 and 1998 a series125,126 of 7 patients with
recessive dystrophic epidermolysis bullosa (RDEB) un-
derwent treatment for correction of mitten deformities
using a combination of OrCel, skin flap, and autograft.
Flaps and autograft were applied to web spaces and joints,
while OrCel was applied to donor sites and any remaining
areas on the dorsum of the hand and fingers. OrCel was also
applied as an adjunct to autograft during dressing changes
when areas of deepithelialized tissue developed. All told,
OrCel was used in 15 surgeries. Five of seven patients had
prior surgeries using split-thickness skin autograft and
served as control. When OrCel was used, the need for donor
sites was naturally reduced. In 13 surgeries, the OrCel-
treated donor sites healed in 2 weeks and in 1 surgery in
3 weeks. Long-term follow-up of 3 patients has shown that
donor sites remained free of blisters for 6–10 years. OrCel-
treated donor sites healed without complication. In 2 cases,
OrCel-treated sites were reharvested 2 and 4 years later,
respectively, and produced autografts deemed equivalent to
autografts from nontreated patients.
A further randomized within-patient controlled trial in the
United States enrolled 12 junctional and dystrophic EB pa-
tients with chronic nonhealing wounds. In this study, 3
wounds on each patient were treated with either OrCel, the
collagen sponge component of OrCel, or standard care. At
the discretion of the investigator, OrCel could be reapplied
within the first 4 weeks of the study. No statistically sig-
nificant difference in the incidence of, or time to wound
healing was observed between the 3 treatments.
On November 22, 1999, the product’s sponsor applied
with the Center for Devices and Radiological Health of the
FDA for a Humanitarian Device Exemption (HDE) for the
noncryopreserved version of OrCel for the treatment of re-
cessive dystrophic EB. [The product had previously been
designated as a Humanitarian Use Device (HUD) on April
24, 1998, a status reserved for products intended for diseases
that affects fewer than 4000 individuals annually in the
United States. An HDE application is similar in form and
content to a PMA but is exempt from the effectiveness re-
quirements of a PMA. It can be requested for products
previously designated as an HUD.] On February 21, 2001,
OrCel was awarded an HDE for use in recessive dystro-
phic EB patients with mitten deformity as an adjunct
to autograft. OrCel, by reducing the number and size of do-
nor sites required and the potential for complications was
deemed of ‘‘probable benefit’’ in reconstructive hand sur-
gery in RDEB patients, according to the product’s HDE
label.
UPDATE ON TISSUE-ENGINEERED BIOLOGICAL DRESSINGS 2419

OrCel in burns and donor wounds patients achieved complete wound closure at 12 weeks in
comparison to 39.1% with standard of care.132 For the com-
Ortec, in May 1999, commenced a controlled clinical
pleted trial, 59% of the OrCel-treated group achieved
trial comparing OrCel to Biobrane-L in split-thickness
complete wound healing at 12 weeks versus 36% for stan-
donor sites in burn patients. The multicenter 82-patient
dard of care.133
study had a matched pair design in which each patient had
In April 2005 the FDA requested a confirmatory trial in
2 designated donor sites of equivalent surface area and
VLUs, which will include 60 patients.134
depth127 randomized to receive a single application of ei-
OrCel entered a 40-patient pilot study in diabetic foot
ther OrCel or Biobrane-L. The primary efficacy outcome
ulcers in November 1999 using the fresh version of the
was time to 100% wound closure, defined as complete
product. According to Ortec, a preliminary analysis of 16
reepithelialization, as assessed by blinded photographic
patients indicated that at 12 weeks 56% of the patients
analysis, planimetric measurement for wound size, and
treated with OrCel achieved complete wound closure com-
clinical evaluation. The sites were also assessed for pain,
pared to 29% of the patients treated with standard of care in
itching, signs of breakdown, readiness for recropping, and
that trial.135 For the completed pilot study, 35% of OrCel-
scarring severity during the follow-up period.
treated ulcers healed versus 20% with standard of care.136 In
Ortec filed a PMA with the FDA in March 2001 seeking
ulcers less than 6 cm2, 47% of OrCel-treated ulcers healed
approval in donor site wounds. At a panel meeting on July
versus 23% with standard of care. In February 2005, OrCel
17, 2001, the FDA voted in favor of an approval for OrCel for
received FDA approval to commence pivotal clinical trials in
treatment of donor site wounds, and a PMA was issued on
diabetic foot ulcers, for which it will use the cryopreserved
August 31, 2001.128 According to Ortec,129 the trial showed
product.
a clinically and statistically shorter time to 100% wound
OrCel is not expected to be on the market for at least
closure when OrCel was used compared to current standard
1 year, allowing time for the confirmatory trial in VLUs
of care. The differential in median healing times was 7 days
and FDA review. The diabetic foot ulcer trial is not likely
(15 vs. 22) using photographic analyses, 5 days using pla-
to be initiated before that time.123 As has been the case with
nimetric analyses, and 4 days by investigator assessment. All
Apligraf and Dermagraft before it, the clinical data are
of these differences were statistically significant but not
often not as clear cut as might be desired, as evidenced by
thought to be of great clinical relevance by some panel
the need for a confirmatory trial in VLUs, but is certainly
members. The scarring results, while also showing a statis-
suggestive of efficacy. It is possible that OrCel, being the
tical advantage to OrCel, were deemed not clinically mean-
third product in the class for which approval is sought, is
ingful by the FDA panel. Only 4 of 82 patients required
held to greater scrutiny than earlier products. Although the
recropping procedures and no conclusions about the role of
fresh form of the product is approved under a PMA for
OrCel in facilitating these procedures were able to be made.
donor wounds and under an HDE for RDEB, the challenges
There were no differences in adverse events, including pain,
of distributing a fresh product and the lesson learned from
itching, and infection, between OrCel and Biobrane-L.
Apligraf has no doubt deterred Ortec from pursuing com-
mercialization with this format.
OrCel in chronic skin ulcers
OrCel is also being studied as a dressing for VLUs.
There are no results published in peer-reviewed journals CULTURED EPIDERMAL AUTOGRAFT
and the following discussion is gleaned from Ortec com-
pany filings and press releases. A single case report de- For the sake of completeness, it should be noted that
scribing the use of meshed OrCel in a 9 cm5 cm VLU was cultured epidermal autografts were introduced by Rhein-
first reported in 2002, with complete wound closure at 4 wald and Green in the 1980s137 and should be considered
months.121 the predecessor to the products described above. From a
An interim analysis130 of a feasibility study of 36 patients 1-cm sample, enough skin can be grown in about 3 weeks
showed that at 12 weeks 52.9% of the OrCel-treated patients to cover most of the body. Only epidermis can be cultured
had achieved complete wound closure versus 26.3% of those under these circumstances and a true bilayer skin is not
treated with standard of care. At 24 weeks, 71% of the obtained, limiting potential application. Genzyme Tissue
OrCel-treated patients achieved 100% closure versus 37% for Repair (Cambridge, MA), under the brand name EpicelÒ,
the standard of care.131 The interim analysis data were eval- performs such cell culturing and expansion as a commercial
uated based on planimetric analysis, which tends to show a service. In Europe this service is provided by many hos-
greater difference between product and control than does pital-associated laboratories. Several investigators have
clinical assessment.124 OrCel was found to be safe, with no found cultured epidermal autograft (CEA) to be useful in
adverse events caused directly by the product. the management of extensive burns,138 but in most cases
In a pivotal trial of 136 patients, it was reported that the results obtained are not deemed satisfactory. In a review
in preliminary data for 48 patients 64% of OrCel-treated of 5 years of clinical experience with 28 patients, it was
2420
concluded that ‘‘CEA engraftment continues to be un-
predictable and inconsistent, and hence, it should be used as
only a biologic dressing and experimental adjunct to con-
ventional burn wound coverage with split thickness auto-
graft.’’139 CEA has also been used with some success in the
treatment of leg ulcers.140,141 CEA requires several weeks
to produce, has a low rate of graft take, is very fragile, is
susceptible to infection, and is not suitable for use without a
dermal layer. The take of CEA is also reported to be in-
versely related to the TBSA of burn involvement,142 lim-
iting its utility in exactly those instances when it is most
needed.
To address the shortcomings of CEA, namely fragility,
various improvements have been studied. For exam-
ple, CEA has been grown on fibroblast-seeded LaserskinÒ,
creating a construct termed Composite Biocompatible Skin
Graft (CBSG). Laserskin (Fidia Advanced Biopolymers,
Turin, Italy) is a thin hyaluronic acid membrane that has
been laser-perforated with microholes to facilitate ingrowth
and proliferation of cells. CBSG is reported to require a
lower seeding density than conventional CEA, have a re-
duced culture time as the cells do not need to be grown to
confluence, can be more easily transferred from the culture
dish since it is grown on a transferable membrane, ob-
viating the need for enzymatic digestion, and does not
contract in culture.143
SUMMARY AND CONCLUSION
The development, FDA approval, and commercial launch
of the first living tissue-engineered products were heralded
by much excitement in the medical, scientific, and financial
communities, but time has tempered the enthusiasm for
these products owing to high cost and narrow clinical utility.
It can certainly be said that these early products were tech-
nologies seeking an application as opposed to products truly
‘‘engineered’’ to a particular task. In the future, it is hoped
that less expensive and more clinically effective tissue-
engineered skin products can be developed. Yet, we should
not lose sight of the fact that skin pathology is a multi-
variable process that cannot necessarily be solved with a
construct that histologically resembles skin. For complex
wounds, products must be matched in some fashion to the
underlying pathophysiology.
The principle of using an appropriate biologically active
matrix is now well established for accelerating wound healing
and achieving skin reconstruction. Cellular components mi-
grate to the wound from preexisting cell populations in
adjacent tissue. Increasing evidence suggests that both circu-
lating marrow-derived stem cells and preexisting organ-
specific stem cells can contribute to tissue regeneration.
Transfer studies demonstrate that mesenchymal pre-
cursors can populate various tissues, and that circulating
endothelial progenitor cells can give rise to vasculogenesis.
The availability of such cell types may be the rate-limiting
EHRENREICH AND RUSZCZAK
step for dermal wound healing. Stem cells are important
targets for growth factors applied locally and for gene
therapy. They participate in the natural healing processes
during wound organization and remodeling. The applica-
tion of autologous or allogenic stem cells, either alone or
together with specific growth factors in an appropriate
3-dimensional matrix, may help to initiate and regulate the
de novo reconstruction of missing skin.
Because of technical, ethical, and legislative difficulties
connected with the use of stem cells, interest remains fo-
cused on in vitro–generated skin, which can be transplanted
to the wound bed to replace missing tissue permanently.
Neither the commercially available products nor the pro-
ducts currently described in experimental studies are able to
fully substitute for natural skin. However, much success
has been achieved in the substitution of connective tissue
matrix, creating human dermis. Once dermis is reconstructed,
the closure of the wound surface with in vitro–expanded
epidermis or autologous split-thickness skin grafts is sig-
nificantly easier and more likely to succeed.
Products containing living cells typically require only a
few simple preparative steps, mostly washing or thawing.
Apligraf is essentially ready to use as delivered. The pro-
ducts can be applied to many different types of skin wounds,
despite the limited FDA approval. An advantage of col-
lagen matrix containing products, such as Apligraf, is the
possible acceleration of granulation tissue formation, re-
duction of scarring, and more rapid reepithelization.
Although important issues concerning wound pretreat-
ment, choice of matrix support for cell growth, and the
use of allogenic cells remain to be fully resolved, tissue-
engineered approaches to wound repair still offer sig-
nificant therapeutic possibilities.
Such benefits may include

Reduced donor site morbidity in burn wounds

Increased potential for healing of recalcitrant lesions

Reduced rates of lesion recurrence (as a result of im-
proved dermal quality)

Reduced wound contracture and scarring

More rapid closure (epithelialization) of large acute
excisional wounds

Delivery of exogenous growth factors (autologous,
allogenic, or genetically engineered)

Reduced overall treatment costs and hospital stay
The importance of available tissue-engineered products
should not be discounted on account of modest clinical and
commercial success with first-generation constructs. Al-
though these products were not the success hoped for, im-
portant proof-of-principle was demonstrated in regulatory,
technical, manufacturing, logistical, and commercialization
elements. As the first medical device containing living cells
to be approved by the FDA, Apligraf has potentially paved
the way for all others. In an optimistic vision of the future,
skin is only the beginning of a plethora of tissue-engineered
products that can address the loss of tissue and organ
UPDATE ON TISSUE-ENGINEERED BIOLOGICAL DRESSINGS
function is areas such as bone, heart, liver, kidney, and
neural repair, where clinical cost–benefit may better favor
product adoption. However, the challenges and setbacks in
skin should alert us to the even greater challenges in
achieving success with other tissue.
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