CL283

Wound closure with human keratinocytes
cultured on a polyurethane dressing
overlaid on a cultured human dermal
replacement
Hans O. Rennekampff, MD,John F. Hansbrough, MD, VereDa Kiessig, BS,
SalwanAbiezzi, and Vlrgll Woods Jr., MD, San Diego,Calif
Background. Bum excisionlollowed by immediate lvound coveragehas becomethe clinical standard lor
managing extensivebum injuries in much 01 the world. VVhensufficient autogralt skin to achieve
permanent lvound closureis unavailable, cell culture technologyhas made the use 01 cultured human
keratinocyte(HK) sheetsclinically leasible. VVhereas
previous techniqueshavelocused on devetopment01
multilayered, differentiated HK sheets,OUTattention has beendrawn to using HK in a highly
proliferative, lessdifferentiated state. Time requirementslor preparation 01 multistratijied cultured HK
are high, and preparatory steps may destroyimportant integrin adhesion molecules.
Methods. We describethe use01 HK cultured to single layer confluente on a polyurethane
membrane(HD), lvith serumjree medium. HK-HD gralts lvere transplanted tolull-thickness wounds on
athymic mice (n = 31). A secondgroup DImite (DG-HK-HD, n = 28) receiveda living human dermal
replacementcontaining cultured fibroblasts beforeplacement 01 HK-HD. Control mice receivedHD
alone (n = 4). Basementmembraneproteins on healed wounds and surfaceintegrins on cultured HK
were identijied by means01 immunostaining and direct microscopicvisualization.
Results. HK cultured just to the confluent state on polyurethane membranewerepositive lor integrins
(X5and (X6,major integrins on proliferating HK Histologic analysis showedepithelialized wounds in
all groups alter 21 days. Using an anti-human involucrin antibody we demonstratedthe presente01
HK in 64.5 % 01 the HK-HD group, 61 % 01 the DG-HK-HD group, and 0% in the HD group. Mice
that receivedthe living human dennal replacementcontaining cultured fibroblasts in combination with
HK-HD gralts devetopeda thick, lvell-vascularized neodermis.Strong laminin and collagen N
staining lvas observedin wound aTeascoveredwith HK.
Conclusions. Thesedata show that lull-thickness wounds can be closedby application 01 a single layer
01proliferating HK cultured on a biocompatiblepolyurethane membrane.1'his technique is an
alternative to the use01 multilayered, differentiated HK sheets.Preparation timeslor HK-HD gralts
should be signijicantly shorter than requiredlor multilayered HK sheets,technical efforts should be less,
and more extensivel/Jound aTeascould be covered. (Surgery 1996,'120:16-22.)~
From theDepartmentso/ Surgery,Medicine,and Orthopedics,
Universityo/ California,Sanniego
Medical Genter,SanDiego,Calif
WHENTHERi\W.INJURIES
to the skin are extensive, the
availability of donar sites for autografting is limited. Although temporary skin substitutes have been useful for
short-term wound coverage,eventuallypermanentgrafting with autologous skin grafts is necessary. An ap,.H. O. RennekampITissupported byagrdntfrom Deutsche Forschungsgemeinschaft.
Accepted for publication Oct. 11, 1995.
Reprint requests: Oliver RennekampIT, MD, Department of Surgery
8896, Universii). ofCalifomia, San Diego Medical Cerner, 200 W. Arbor Dr., San Diego, CA 92103.
Copyright @ 1996 by Mosby-Year Book, Inc.
003%060/96/S5.00
16
SURGERY
+ O 11/56/71954
proach to accelerate fue availability of graftable tissue is
fue expansion of autologous skin by culturing human
keratinocytes (HK) in fue laboratory. Advances in serial
subculture techniques of human epiderrnal celIs result
in high proliferation rates ofHK and fue ability to generate large amounts of epithelium suitable for grafting.l
The time period required for fue culture of suitable epithelium is at least 3 to 4 weeks because fue celI sheets
must be physicalIy removed from fue culture surface
and transferred to the wound; fue sheetsmust be at least
four to six layers in thickness to withstand fue trauma.
In addition, the culture and preparation processesmar
reduce adhesion properties of fue cultured celIs.
We report the transplantation of single layers of ke-
Surgery
Volume120, ¡Vumber1
RennekamPJJel al.
Skin Sample
HK Suspension
17
HK Culture
Epldennls
Dennis
Panniculus
camosus
,
,
,
,
;V~~
~-.~1!!fI
,..Jj
~
I~~~~~~~~~~
~-H K
Hydroderm@
,,
If'Jj
,.jJ
.-=7
Fig. l. Sequenceofsteps involved in preparation ofHydroderm-HKgrafts and their application to excised
wounds.
ratinocytes, cultured on a synthetic polyurethane membrane (Hydroderm; Wilshire Medical Inc., DalIas, Texas), anta fulI-thickness excised wounds in athymic mice.
In some of the animals we placed the grafts overlaid on
a dermal replacement composed of human fibroblasts
cultured on a biodegradable mesh. We algo examined
the expression of specific integrins during the culture
process and structural characteristics of the healed skin
in this animal model of wound closure.
MATERIAL AND METHODS
Cell culture. Fig. 1 shows fue overall scheme for setting up primary cultures from human skin, establishing
secondary cultures on fue polyurethane membrane,
and then transfer of grafts to excised wounds on
animals. Human keratinocytes (HK) were isolated from
cadaveric donor skin obtained from fue University of
California SanDiego Regional Tissue Bank. Donor skin
was placed overnight in 0.25% trypsin/0.02% ethylenediamine tetraacetic acid (Gibco BRL Life Technologies,
Grand Island, N.Y.) at 40C. Mter isolation into a single
cell suspension, epidermal cells were resuspended and
expanded in number in 75 cm2 polystyrene tissue culture flasksby using Keratinocyte-SFM medium (Gibco).
HK were released from the plastic surface with 0.25%
trypsin/0.02% ethylenediamine tetraacetic acid.
Preparation of fue polyw:ethane-HK grafts. HK were
seeded onto a sypthetic hydrophilic dressing, Hydro-
derm. Hydroderm has high but variable water vapor
permeability and has a biocompatible adhesive coating
applied in a diamond-shaped pattem to one sirle of the
membrane. Hydroderm has the unique characteristic of
markedly but reversibly increasing its moisture-vapor
transmission characteristics (MVfR) when it becomes
moist, facilitating the local control of wound exudates
and wound evaporation.
A frame was designed to keep the membrane flat
while it was submerged in culture medium. A 7 x 8 cm
sterile piece ofHydroderm wasstretched flat in a sterile
polycarbonate frame that was constructed in our biomedical engineering shop. HK were seeded at a density
of 5 x 104 cells/40 cm2 of Hydroderm and red every 3
days with Keratinocyte-SFM medium. Mter 3 to 5 days
when HK had just begun to attain confluent growth
(Fig. 2), the membranewas removed from fue culture
medium and cut into 2 x 2 cm sections, and sections
were transplanted to athymic mice.
Application of Hydroderm/HK
grafts to animal
wounds. Animal experiments were performed with arproval oftheUniversityofCalifomia,
San DiegoAnimal
ResearchCommittee and in accordance with guidelines
prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal
Resources,National ResearchCouncil. Athymic mice, 4
to 8 weeks old (Balb/c-nu/nu; Simonsen Laboratories,
Gilroy, Calif.) ,were kept in isolation rooms. Surgery and
18 Rennekampff
el al.
Surgery
fuiy 1996
Fig. 2. Photomicrograph of cultured HK attached to Hydroderm 4 days after seeding of cell anta membrane. HK havejust reached the stageof confluent growth. Staining with hematoxylin-eosin;original magnification x400.
dressing changes were perfonned in laminar-flow hoods
under anesthesia with 10 mg intraperitoneal Avertin
(Aldrich Chemical Inc., Milwaukee, Wis.) in 0.4 mI normal saline salution. The dorsolateral surface of the
mouse was washed with povidone-iodine and 70%
isopropanol. Full-thickness skin defects (2 x 2 cm) were
created to a depth sparing the panniculus camosis;
grafts were sutured ayer fue defect with 6-0 silk. In fue
first group (n = 4) the defect wascovered with Hydrodenn without keratinocytes, which was kept in Keratinocyte-SFM medium for at least 24 hours before fue
operation. The second group (n= 11) received Hydrodenn seeded with HK (Hydrodenn-HK). In the third
group (n = 14) a 2 x 2 cm piece ofDennagraft, a living
dennal replacement composed of human neonatal
fibroblasts cultured on polyglactin mesh (Advanced
Tissue Sciences Inc., Lajolla, Calif.), was sutured into
the defect and covered with a 2 x 2 cm piece ofHydrodenn-HK Grafts were covered with a bulky gauze dressing (Band-aid; johnson & johnson, New Brunswick,
NJ.), and animals were wrapped circurnferentially.
Mter operation animals were given daily injections of
3 mg ceftazidime (Glaxo Inc., ResearchTriangle Park,
N.C.) intraperitoneally for 7 daysafter operation. Sulfamethoxazole and trimethoprim (Biocraft Laboratories,
Elmwood Park, N J.) were added to autoclaved drinking
water. Animals were examined for the integrity of dressings each dar and were killed on dar 21. Wound areas
were excised through the dorsal muscle bed for histologic evaluation.
Histologic analysis of ceIls and tissues. For light microscopy, tissue samples were flXed in 10% fonnalin,
embedded in paraffin, sectioned at 5 pm thickness,
mounted anta slides, and stained with hematoxylineosin. HK-seeded Hydrodenn (1 x 1 cm) wasmounted
on slides and stained with hematoxylin-eosin.
Immunohistochemical staining was used to identify
specific proteins.ldentification ofhuman involucrin in
healed wounds on fue athymic mice wasobtained after
following fue instructions described in fue Histostain-SP
Kit (Zymed Laboratories Inc., San Francisco, Calif.).
Frozen sections were fixed, rehydrated, and treated with
10% nonimmune goat serum to block nonspecific
binding. Slides were incubated with primary antibody
(rabbit anti-human involucrin; Biomedical Technologies Inc., Stoughton, Mass.) for 60 minutes, rinsed, and
incubated for 10 minutes with biotinylated secondary
goat anti-rabbit antibody. Mter rinsing, enzyme conjugate was applied for 10 minutes and rinsed, and slides
were incubated 10 minutes in Substrate-Chromogen
Mixture (3-amino-9-ethylcarbazole,AEC). Positive staining revealed a red precipitate in fue superficial epidermal layer.
Staining for human collagen type IV and laminin was
performed on 7 11mthick, frozen sections, which were
treated as above. Specimens were incubated with fue
collagen IV primary antibody (rabbit anti-human collagen IV; Chemicon, Temecula, Calif.) and laminin primary antibody, respectively (rabbit anti-human laminin
1;Chemicon). Mter washing,fue biotinylated secondary
antibody \vas applied, followed by an additional rinsing
and then application afilie enzyme conjugate and SuDstrate-Chromogen Mixture (AEC).
Staining for fue integrin Q5and integrin Q6was performed on fue HK~eeded Hydroderm according to fue
instructions of fue Vectastain ABC kit (Vector Laboratories Inc., Burlingame, Calif.). Specimenswere blocked
with horse serum, followed by human AB serum. Sections
were incubated ovemight with fue primary antibody to
fue integrins Q5 (1VF4,2.5 l1g/ml) and Qij (S3-41, 2.5
l1g/ml) , respectively. Mterwashing, a biotinylated horse
anti-mouse secondary antibody wasapplied. Additional
washing was followed by 60 minutes of incubation with
biotinylated alkaline phosphate H and by 25 minutes of
incubation with fue substrate alkaline phosphatase suDstrate kit 1 (Vector red; Vector Laboratories Inc.).
RennekamPff
el al. 19
Surgery
Volume 120, Number 1
/
Fig. 3. A. Photomicrograph ofhealed wound 21 daysafter application ofHydroderm-HK to excisedwound.
Stratified layer of epidermal cells restson thin well-vascularizedlayer of connective tissuecovering skeletal
muscle. Hematoxylin-eosin stain; original magnification x200. B, Healed wound 21 daysafter application
of Hydroderm-HK overlaid on Dermagraft on excisedwound. In contrast to A a very thick layerof well-vascularlzed connective tissue can be seen,containing large numbers of fibroblasts. Hematoxylin-eosinstain;
original magnification x200.
RESULTS
Keratinocyte culture on Hydrodenn membranes.
Initial attempts to culture cells on Hydrodenn were perfonned by immersing fue membrane in plastic tissue
culture dishes. This resulted in wrinkIing of fue Hydroderrn and floatingofthe membrane to fue surface ofthe
culture medium. To avoid wrinkIing of fue Hydroderrn
membrane during the culture process an adjustable
frame wasdeveloped, asdescribed above. In this wayfue
celJscould be uniforrnly seeded onto fue stretched and
flat Hydroderrn, and wrinkIing of fue membrane was
prevented. The HK became adherent to fue Hydrodenn by 24 hou:rs after inoculation of cells to fue membrane surface.
Under light microscopy a sample ofthe cultured graft
(Hydroderrn-HK) 4 days after inoculation just before
transplantation is shown in Fig. 2. Multiple colonies of
HK were seen, resulting in almost confluent cell coverage ofthe membrane. No multilayered epiderrnal afeas
were seen at this time point. Cells seemed to attach and
proliferate on fue polyurethane membrane and on fue
bioadhesive that coated a portion of fue membrane
surface in a crisscross pattem.
During operation fue Hydroderrn-HK was convenient to handle and could be moved easilyfrom fue culture apparatus to the wounds. No shrinkage or tearing
occurred \'(hile culturing celJSon fue membrane or
while transferring or suturing fue Hydroderrn to fue
wound. However, fue markedly convex con tour of fue
dorsum of fue animals, coupled with their constant activity, created partial wrink1ing ofthe Hydrodenn membrane within severa! days of its application to fue
wounds. Wrink1ing mar have resulted in some loss of
adherence of cultured HK to fue wound surface after
transplantation.
Gross appearance of healed wounds. Seeded and
nonseeded Hydroderm were adherent to fue wound
from postoperative dar 2. Peeling of fue Hydrodenn
occurred from dar 17 after grafting. Wounds were
closed in all groups after17 to 21 days,and a silvery pink
appearance of fue woundsurface was observed, which
preceded spontaneous separation of fue Hydrodenn
from fue healing epithelium.
Histologic examination of healed wounds. Mice that
received Hydroderm coverage without HK exhibited
wound closure by epithelialization and marked wound
contraction after 21 days,with onlya small reepithelialized area. Typical photomicrographs of healed wounds
21 days after application of Hydrodenn-HK to an'
excised wound are shown in Fig. 3. Mter application of
Hydrodenn-HK a stratified layer of epidennal cells can
be seen, resting on a thin layer of connective tissue covering skeletal muscle (Fig. 3, A). In Fig. 3, B, a healed
wound is shown 21 days after application of Hydrodenn-HK overlaid on Dermagraft on fue excised wound.
A thick layer of well-vascularized neodennal tissue can
20
RennekamPff
et al.
Surgery
fUI] 1996
Fig. 4. junction of healed,grafted wound and normal mouse skin is shown. Red staining of suprabasalepitheliallayers on right sideof section (belowcornified layer) with anti-human involucrin antibody identifies
human epidermal cells,proving persistenceof transplantedHK. Nonstaining epidermis on leftis murine origin. Hematoxylin-eosinstain; original magnification x200.
be seen, containing large numbers of fibroblasts and
many blood vessels.
Immunohistochemical analysis was used to prove
persistence of HK on fue murine wounds. Animals that
were treated with Keratinocyte-seeded Hydroderm (HDHK) exhibited a "take" of HK in 20 (64.5%) of 31 animals in this group, determined by positive staining for
human involucrin (Fig. 4). Up to 90% of fue length of
fue wound section stained with involucrin. The remainder of fue wound showed reepithelialization with murine keratinocytes, which had migrated from the wound
margins. In wounds that were closed with Hydroderm
atolle, no human skin wasidentified by means of staining for involucrin.
Analysis of fue 28 animals that received Dermagraft
before HK-HD revealed that 17 (61 %) of fue animals
showed positive take of HK, as proved by staining for
human involucrin. The areas of human skin were characterized by a well-defined multilayered epidermis at
dar 21 and a thick, well-vascularized "neodermis" as described above.
Human collagen type IV and laminin were identified
at fue dermal-epidermaljunction by means ofimmunohistochemical staining. No collagen type IV and laminin
were seen in wounds covered with non-HK-seeded Hydroderm. Collagen type IV and laminin expression was
seen in fue animals closed with Hydroderm-HK and
with Hydroderm-HK overlaid on Dermagraft, fue living
dermal replacement. It was our impression thatstronger, more continual expression ofbasementmembrane
antigens was seen in animals that received Dermagraft
folIowed by Hydroderm-HK However, no definitive
conclusions could be drawn because fue staining tech:'
nology does not yield quantifiable data.
Analysis of keratinocyte expression of integrins. Integrin expression on HK wasexamined by means of direct staining of cells attached to fue Hydroderm mem-
brane. HK that were cultured on Hydrodenn exhibited
anti-«6 integrin irnrnunoreactivity at fue cell borders, as
exhibited by others in cells cultured in plastic flasks.2
Staining wasalso seen on areas of fue Hydrodenn where
cells had detached during specirnen preparation. Distinct Q5 integrin expression by attached cells was also
noticed.
DISCUSSION
Numerous reports on fue use of cultured HK sheets
have appeared with variable results. Long-term take is
frequently less than 50%,1.-' and late graft loss and friability of healed skin are reported.4 Early take of
cultured epithelium is affected by storage and handling
of fue cultured sheet grafts.5 Optimal take of cultured
HK sheets mar relate to their adhesion to fue wound.
Keratinocyte adhesion to the wound bed is mediated by
integrins, a family of cell surface receptors.6.7The use
of the enzyme dispase,a neutral protease derived from
BaciUuspolymyxa,to separate epithelial sheets from fue
surface of fue culture flask was an advance in fue development of cultured epithelium. Dispase, a fibronectifiase and type IV collagenase, avoids traumatizing fue
thin, fragile epithelial sheets as they separate from fue
culture flask surface.8 However, dispase-detached HK
sheets mar lose adhesion properties.9 Furthermore,
cultured HK, when forming a multilayered epithelium,
change their characteristics from a proliferating state to
.' a more differentiated epithelium with modified expression of adhesion molecules,10 including loss of fue
a5~1 integrinll that promotes cell adherence to fue
woundl2. 13and basement membrane.
A specialized polyurethane membrane, Hydroderm,
wasused as a support for culturing HK in fuese experiments. Hydroderm has unique characteristics that
make it a satisfactory dressing for moist wounds, and it
also supports fue growth of human censoThe MVfR of
Surgery
Volume 120, Number 1
Hydroderm is a function of fue degree of wetnessof the
membrane. Normal skin has a range of MVTR of 200 to
2000gm-2. day-l , whereas damaged or burned skin mar
have water losses as high as 3000 to 5000 gm-2.day-l.14
Conventional hydrophobic polyether urethane films
with equilibrium water contents of <1 % have MVTRs of
1500 to 2000 gm-2.day-l. However, when coated with a
conventional medical adhesive, fue MVfRfalls to 500 to
1000 gm-2.day-l because fue adhesive blocks water vapor transmission. Conventional films with working
MVTRs of <1000 gm-2.day-l are unsatisfactory for use
on moist wounds because exudates rapidly accumulate.
The high MVTR of Hydroderm results in avoidance of
exudate collections.
Hydroderm, maintained taut in a frame and submerged in culture medium, promoted attachment and
proliferation of HK. Hydroderm with attached HK was
easily removed from fue culture chamber and transferred to fue wound without further manipulation. Ease
of achieving cell transfer to the wound at an early stage
in fue culture process is important. Instead ofwaiting 3
to 4 weeks until multilayered HK sheetsare achieved,
cultured cells can be transferred much earlier to the
wound bed. In addition, enzymatic release of cultured
epithelium from fue culture flask surface and tedious
transfer of fragile cell sheets to a support dressing are
unnecessary. Finally, cultured HK sheetscontract markedly after their enzymatic release, thereby limiting the
wound area that can be closed. Use ofHydroderm as a
support for cultured HK not only permits application of
grafts to fue wound surface far earlier after injury, but
shrinkage of fue cultured graft is avoided during the
transplantation process. Calculations suggestthat at fue
seeding density we used, a smafi section of skin could
yield 500 cm2 ofHydroderm-HKin
17 to 18 days on average, and 14 days later 1 m2could be furnished for fue
patient.
The development and use of effective dermal replacements appear necessary to achieve functional skin
replacements. 1he dermis is now known to play an important role in development and stability of the epidermis. Reconstitution of the dermal-epidermal junction
and in particular fue basement membrane is mandatory
for optimal skin strength and durability .15Full-thickness
wounds that are closed by cultured epithelium frequently exhibit subsequent fragility and breakdown.l, 4
Ultrastructural studies show that after application of
cultured HK sheets healed wounds lack critical anatomic structures such as anchoring fibrils,16 perhaps
contributing to clinical instability of the 'I'ounds. We
have demonstrated that composite grafts, which contain
both demial and epidermal elements, achieve accelerated formation of basement membrane stn¡ctures.l
For thest; reasons we used a living dermal replacement, Dermagraft, in some afilie experimental groups
RennekamPJJ
el al. 21
to achieve both epidermal and dermal replacement in
fue full-thickness wounds. Dermagraít is a living human
dermal replacement tissue that consists of neonatal
fibroblasts that are cultured on a biodegradable polyglacrin mesh. During fue culture process fue cells secrete multiple matrix proteins and multiple growth factors.17,18 Dermagraft supports fue adherence and
growth of overlaid meshed skin grafts on both animals
and human beings and supports growth of cultured keratinocytes in vitro. Because fibroblasts arrear to be
nonantigenic in fue allogeneic situation, Dermagraít
can be clinically used as a permanent dermal replacemento
OUT experiments show that grafting of proliferating
keratinocytes attached to Hydroderm, in conjunction
with their placement on the living dermal replacement
Dermagraft, results in wound closure with a multilayered, differentiated epidermis growing on a well-vascularized tissue thatresembles dermis. Strong and continual staining for laminin and typeIV collagen, important
structural proteins at fue dermal-epidermal junction,
suggeststhat these graíts replace important anatomic
structures in the full-thickness wound. Closure of fue
wounds, or take of the cultured grafts, was equally successful in experimental groups with and without fue inclusion of Dermagraít. Subjectively, staining for specific
basement membrane proteins appeared more intense
and continual in fue wounds that received Dermagraít
before application ofHydroderm-HD, although no firm
conclusions could be drawn in this regard.
Hydroderm is an effective dressing on wounds, controlling exudates because of its high moisture vapor
transmission. As HK proliferation progresses on fue
wounds and fue cells stratify and differentiate, fue Hydroderm separates from fue epithelialized wound. If
this technique can be used in full-thickness wounds on
bumed human beings, it mar be useful for replacing
both dermal and epidermal components by using a
simpler approach than fue use of delicate cultured HK
sheets. In addition, coverage of extensive bum wounds
with a layer of viable autologous keratinocytes, in conjunction with a living humandermal substrate,could be
achieved 1 to 2 weeks earlier compared with cultured
epithelial autograít sheets.
The loss of integrins during enzymatic detachment of
HK sheets mar relate to suboptimal early adherence of
cultured cells to fue wound bed. These results mar relate to the relatively low and inconsistent clinical success'
in grafting dispase-<letached epithelial cell sheets to
wounds. Becauseculture of HK on Hydroderm and direct transfer of fue membrane containing attached cells
to fue wound do not require fue use of enzytnatic treatment to detach fue cells before transplantation, thus
preserving integrin expression, this potencial obstacle to
cultured skin transplantation success might be over-
~
Surgery
22 Rennekampffetal.
july 1996
come by this new process. Clinical trials to test this
methodology in bumed human beings are planned.
9. Poumay Y, Leclercq-Smekens M, Grailly S, et al. Specific internalization ofbasal membrane domains containing the integrin
«6~4 in Dispase-detached cultured human keratinocytes. Eur J
These studies were perfonned with fue assistance of fue
University of California, San Diego, Institute for Biomedical
Engineering Adhesion Receptor Core Facility.
Cell BioI1993;60:12-20.
10. Hotchin NA, Watt FM. Transcriptional and post-translational
regulation of ~1 integrin expression during keratinocyte termi-
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2. Marchisio PC. BondanzaS, CremaDa O. Cancedda R, DeLuca M.
Polarized expression ofintegrin receptors (a6r.4,a2r.¡, a3r.¡, and
avr.5) and their relationship with the cytoskeleton and basement
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1991;112:761-73.
3. Rue LW lII, Cioffi WG, McManus WF, Pruitt BAJr. Wound closure and outcome in extensively bumed patients treated with
cultured autologous keratinocytes. J Trauma 1993;34:662-8.
4. Desai MH, Mlakar JM, McCauley RL, et al. Lack oflong-term durability of cultured keratinocyte bum-wound coverage: a case report.J Bum Care Rehabill991;12:540-5.
5. Poumay Y, Boucher F, Degen A. Leloup R lnhibition ofbasal cell
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6. Hynes RO. lntegrins: a family of cell surface receptors. Cell
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7. Marchisio PC. Bondanza S, Cremona O, et al. Polarized expression of integrin receptors and their relationship with the cytoskeleton and basement membrane matrix in cultured human
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Invest DermatoI1968;51:454-65.
16. Woodley DT, Peterson HD, Herzog SR, et al. Burn wounds
resurfaced by cultured epidermal autograrts show abnormal reconstitution of anchoring fibrils. JAMA 1988;259:2566-71.
17. HansbroughJF, MorganJL, GreenleafGE, Bartel R Composite
grarts of human keratinocytes grown on a polyglactin mesh-<ultured fibroblast dermal substitute function as a bilayer skin
replacement in full-thickness wounds on athymic mice. J Bum
Care RehabiI1993;I4:485-94.
18. HansbroughJF, Doré C, Hansbrough WB. Clinical trials ora living dermal tissue replacement placed beneath meshed, splitthickness skin grarts on excised burn wounds. J Burn Care RehabiI1992;13:519-29.
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