Download Method for autologous single skin cell isolation for regenerative cell

DOI: 10.5301/IJAO.2011.6508
Int J Artif Organs 2011 ; 34 ( 3): 271- 279
ORIGINAL ARTICLE
Method for autologous single skin cell isolation for
regenerative cell spray transplantation with
non-cultured cells
Jörg C. Gerlach1, Christa Johnen2, Christian Ottoman3, Kirsten Bräutigam2, Jörn Plettig4, Claudia
Belfekroun3, Sandra Münch3, Bernd Hartmann3
Departments of Surgery and Bioengineering, McGowan Institute for Regenerative Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania - USA
2
Charité, Campus Virchow, AG Experimental Surgery, University Medicine Berlin, Berlin - Germany
3
Burn Center, Department for Plastic Surgery, Unfallkrankenhaus Trauma Hospital Berlin, Berlin - Germany
4
StemCell Systems GmbH, Berlin - Germany
1
ABSTRACT
Background: There is a therapeutic gap for patients with deep partial-thickness wounds (Grade IIb) of
moderate size that were initially not treated with split- or mesh grafting to avoid overgrafting, but developed delayed wound healing around two weeks after injury - at which time grafting is typically not
indicated anymore. Delayed wound healing is often associated with esthetically unsatisfactory results
and sometimes functional problems. An innovative cell isolation method for cell spray transplantation
at the point of care, which eliminates cell culture prior to treatment, was implemented for this population of burn patients in our center.
Methods: Autologous skin cell spray transplantation was initiated by taking healthy skin. The dermal/epidermal layers were separated using enzymatic digestion with 40 min dispase application, followed by 15
min trypsin application for basal kerationcyte isolation, 7 min cell washing by centrifugation, followed by
transferring the cells for spraying into Ringer lactate solution. The procedure was performed on site in a
single session immediately following the biopsy. After sharp wound debridement, cells were immediately
transplanted by deposition with a cell sprayer for even distribution of the cell suspension.
Results and conclusions: Eight patients were treated (mean age 30.3 years, mean burn total body
surface area 14%, mean Abbreviated Burn Severity Index (5 points). The mean time to complete
re-epithelialization was 12.6 days. All patients exhibited wound healing with improved esthetic and
functional quality. Our initial experience for the use of non-cultured cells using a two-enzyme approach
with cell washing suggests shortened time for wound closure, suggesting that the method may potentially avoid longer-term complications.
KEY WORDS: Burns, Keratinocytes, Isolation, Spraying, Transplantation
Accepted: March 15, 2011
INTRODUCTION
Skin cell isolation techniques are of interest for regenerative therapy development for burns. While partial-thickness
(Grade IIa; mid dermal) burns are treated conservatively,
and split skin or mesh-grafting is a routine indication in
third-degree burns, the indication to grafting in deep second-degree (Grade IIb) wound defects in some patients
exhibiting a borderline wound severity can be controversial. These patients are sometimes not treated with grafting
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Autologous skin cell isolation
to avoid overgrafting, for example, on the face and hands.
After deciding not to treat this specific patient group with
grafting, most of the patients start sufficient spontaneous
healing within the first week under observation and intense
wound care, but some patients in this population develop
delayed wound healing over several months with complications, including infections. In these specific cases the
long-term outcome, often with scar formation, skin discoloration and skin hypertrophy, and sometimes contractions
is unsatisfactory (1). While such a development can typically be predicted 10 to 14 days after non mesh-grafting of
the burn injury if the wound is not dry and reepithelialized,
performing split skin grafting at this point is problematic.
Single skin cell spray grafting of cultured cells was recently
introduced (2), and we already implemented the method
by using a processor controlled pneumatic cell sprayer for
better controlled cell deposition for cultured autologous
keratinocytes (3). Avoiding in vitro cell expansion in culture,
by which the basal keratinocytes, considered to be important for wound regeneration, differentiate and lose their
progenitor character, appears to be promising (4-7). Autologous skin cell transplantation in an intraoperative setting is based on removing healthy skin in the dermal layer
from a non-prominent healthy skin area using a dermatom,
followed by cell isolation at the point of care and immediate cell spray grafting to the burn site during the same
operating session. Gravante et al used the Avita ReCell
kit (Nedlands, Australia) and compared cell spray transplantation with mesh grafting in partial-thickness wounds
and concluded that both methods produce similar results,
while cell grafting was described as a well tolerated, safe
and reliable technique (8).
Considering these results, we did not depart from the usual
mesh grafting treatment, but considered this method only
for patients in the aforementioned situation. We believe that
the method described, involving trypsin digestion only and
without cell washing by centrifugation, can be improved
by: a) adding cell washing with a centrifuge to better remove remaining enzymes; and b) an initial step of dispase
application to first separate the dermis from the epidermis prior to the trypsin application, so that the trypsin can
easier reach the cells of the basal keratinocyte layer in the
epidermal interface of the epidermis.
We describe the use of this modified two-enzymatical step
isolation technique involving dispase, trypsin, and cell
washing by centrifugation and we report our clinical implementation for the initial eight patients.
272
MATERIALS AND METHODS
Patients
Autologous skin cell transplantation was performed in
our center on eight patients between 2007 and 2008.
After preparing the logistics for the therapy, all patients
were treated who met the inclusion or exclusion criteria
described below and presented with the following clinical indications: partial-thickness wounds (Grade IIb) with
remaining dermal structures of moderate size that were
initially not treated with split mesh grafting to avoid overgrafting, but experienced delayed wound healing with no
significant re-epithelialization within two weeks after injury. The interval between initial trauma and cell application
was in all cases at least 14 days. The interventions were
performed one time only in all cases. Some treatments
were performed in areas exhibiting critical wound healing
and often unsatisfying cosmetic results: two patients with
burns on the face, and two patients with burns on the
hands. Eligibility criteria for cell spray grafting included
partial-thickness burn injuries that required initial surgical debridement under anesthesia and follow-up wound
treatment with enzymatic debridement. Exclusion criteria were pre-existing local and systemic infections that
required antibiotic treatment for more than 2 days prior
to grafting; hypersensitivity to trypsin or other enzymatic
wound treatments; a high anesthesiology risk. Patients
received routine pre-operative investigations and a second final revision of the surgical indications/decisions.
The patients included 6 males and 2 females; the mean age
was 30.3 years (9 months-58 years). The patients initially
exhibited a mean burn total body surface area (TBSA) of
14% (3-40%). The mean Abbreviated Burn Severity Index
(ABSI) (9) was 5 points (3-8 points). Details on the patients
and wound locations are summarized in Table I.
Informed consent was obtained in all cases, and the
individual’s own decision after providing detailed information was respected in any case. We explained to
the patients that their decision would have no impact
on potential alternative conservative treatment. No patient to whom we suggested the procedure declined the
proposal; see below for Institutional Review Board (IRB)
approval.
All procedures were performed in a burn center operating
room and under general anesthesia.
© 2011 Wichtig Editore - ISSN 0391-3988
Gerlach et al
TABLE I - PATIENT, TRAUMA AND WOUND CHARACTERISTICS OF CELL SPRAY TRANSPLANTATION PATIENTS
Patient
Sex
Age
Cause of
trauma
Initial burn regions
Initial
TBSA burn
(%)
Initial
ABSI
(points)
Treated burn
regions
Treated BSA
(%)
1
F
35 years
Blaze
Both hands, arms, thighs, backside
40
8
Right thigh, right
knee
2
0.5
2
M
18 years
Scald
Penis, scrotum, both thighs, belly
15
4
Penis
3
F
4 years
Scald
Left leg
5
4
Left leg
5
4
M
42 years
Explosion
Right hand, both hips, legs; penis
5
5
Penis
0.5
5
M
39 years
Blaze
Face, forehead, right shoulder,
right hand
10
5
Face, forehead
4
6
M
58 years
Explosion
Face, both hands
15
7
Face, left hand
5
7
M
9 month
Scald
Both arms and dorsal thigh
20
3
Arm circular left
6
8
M
46 years Electro burn
Both hands, head, neck
3
5
Hand left
1
TBSA = total body surface area; ABSI = Abbreviated Burn Severity Index; BSA = body surface area; “Initial” refers to the diagnosis at the day of the initial trauma.
Initial wound treatment after trauma
Patients presented to our burn center initially received an
escharectomy with tangential excision using a curette,
Guillian knife, or a hydrosurgical system (Versajet®; Smith &
Nephew, London, UK) when the wounds exhibited a thick
fibrin coating. Daily wound inspection included enzymatic debridement using collagenase gel (Iruxol® N; Smith &
Nephew, Hull, UK) in combination with fatty gauze (Jelonet®, Smith & Nephew).
Cell spray grafting method
Intra-operative treatment included wound disinfection with
chlorexidine-cetrimide solution. Immediately before cell application we performed sharp surgical spoon debridement
under general anesthesia. Hemostasis was performed with
regular gauze.
The biopsy for the skin cell transplantation was obtained from
a non-involved region, as known from split thickness graft
harvesting, and the tissue was procured into a single-cell
suspension for cell spray transplantation. This cell isolation
procedure is performed on site in the operating room immediately prior to cell transplantation during the same anesthesia
session. Immediately following the biopsy, the tissue is procured under sterile conditions until application; the tissue is
maintained under the direct supervision of the surgeon who
performs the procedure using routine clinical devices, solutions and disposables available in skin burn centers.
The biopsy treatment includes existing methods of enzymatic tissue digestion with dispase and trypsin, and the
washing away of the connective tissue is performed using conventional patient cell centrifugation methods; all
solutions and containers are sterile commercial products.
If not otherwise indicated, all solutions were supplied by
Biochrom AG, Berlin, Germany; and disposables by BD
Biosciences, Bedford, MA, USA.
Specifically, the following steps for autologous skin cellspray transplantation were performed. According to individual enzyme incubation times, this procedure requires
around 70 to 90 minutes.
A) A 2-3 cm x 3-4 cm split skin biopsy (0.2 mm deep) is taken using a dermatom (Humeca D 42; Asclepios, Gutach,
Germany). The biopsy is performed in a routine way, as
known from split mesh skin grafting. The biopsy is transferred into a sterile disposable 100 mm Petri-dish (BD) and
is cut into 4 x 4 mm pieces by the surgeon with a surgical
disposable scalpel (the specimen must not dry out).
B) The cell separation, performed by a specialized laboratory technician, involves:
1) initial separation of dermis and epidermis over 25 to 40
minutes in a 2.5 unit/mL Dispase II (#4942078; Roche
Diagnostics, Mannheim, Germany) containing phosphate buffered saline (PBS 1x without Ca2+, Mg2+) solution at 37°C in Petri-dishes;
2) The mechanical separation of the dermis and epidermis
of each skin piece using forceps and scalpel, performed
in a dry Petri-dish (the specimen must not dry out), with
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Autologous skin cell isolation
no further use of the dermis parts;
3) The epidermal pieces are transferred into a 15 mL Falcon tube with PBS without Ca2+, Mg2+, to wash away the
enzyme.
4) The epidermal pieces are then transferred into 15 mL
Falcon tubes for 10 to15 minutes at 37°C containing a
single cell suspension and a 5 mL 0.05% Trypsin / 0.02%
EDTA (# L2143; Biochrom AG, Berlin, Germany) solution,
to allow for cell isolation from the epidermis, including
from the interface layer of epidermis and regenerative
basal keratinocyte containing dermis; followed by stopping with 7.5 mL Ringer lactate (B Braun, Melsungen,
Germany) containing 10% of the patient’s own serum,
taken previously, over 30 seconds;
5) The cell suspension is sieved through a 70 micrometer
50 mL Falcon tube sieve (all BD);
6) seven minutes of centrifugation at 100 g for cell washing
to remove the enzymes;
7) The cells are put into clinical grade Ringer’s lactate solution (B. Braun, Melsungen, Germany) which is used as a
carrier for cells during the spraying process.
At this stage, cell counting in a Neubauer chamber showed
a yield between 2 to 10 x 106. Using a sterile disposable pipette (BD Biosciences), the cells are transferred into sterile
disposable syringes (BD Biosciences) for cell deposition
by spraying. We typically resuspend the cells in a 1 x 106
cell/ml suspension, which we divided into one to five 2 mL
syringes (BD Biosciences) for cell spraying; the trypan-blue
viability of the cells was between 96% and 98%.
C) One syringe at a time, the syringes are placed in a
processor-controlled pneumatic cell spray device through
which the suspended cells are immediately spray-transplanted onto the burn site of the patient by spray deposition, as described earlier (3), allowing an even distribution
of the cell suspension across a larger surface. Donor site
area to transplant site “expansion” ratios were 1:20 to 1:60,
with a mean of 1:25.
After aspirating the cells for cell spraying from the centrifugation tubes used for cell washing, few cells remained. To
obtain control cultures of the cells sprayed on each patient,
we rinsed the remaining cells left in the container with standard culture medium (Epilife; Gibco, Carlsbad, CA, USA;
with Penicillin/Streptomycin; Biochrom, Berlin, Germany)
and cultured them under standard conditions (Falcon Primaria 25 cm, 2 flasks; BD, Franklin Lake, NJ, USA ) in 5%
CO2 culture incubators. For all patients treated, the cells
pipetted into culture flasks successfully attached, spread,
274
and remained viable. In all cases, we were able to produce
follow-up cultures with typical passage behavior.
Wound treatment and followup
After cell spray grafting, the wounds were covered with
polylactid sheets (Suprathel®; PolyMedics Innovations,
Denkendorf, Germany) combined with fatty gauze (Jelonet®) cotton and elastic bandage. This dressing was used
for one week. Thereafter we used AdapticTM (Johnson and
Johnson, Eticon, Norderstedt, Germany) sheets under
gauze dressings. After the initial days, the wounds were
treated in the same way as after split mesh skin transplantation. The patient followup is also the same as for split
mesh skin transplantation. To assess burn scars for clinical
evaluation we used the Vancouver Scar Scale (10) (Tab. II).
Innovative practice versus clinical study
The skin cell transplantation treatment was planned solely
to enhance the well-being of this unique patient group as
described above; patients with these injuries appear relatively seldom. The method used is innovative, but given the
cited results of Wood et al and Gravante (2, 8) it is not radically new. Skin cell isolation is well known to provide cells
for medical cell applications, a technique already used in
the past in some larger burn centers. Also, the tissue is
stored under sterile operating room conditions throughout the entire procedure until the final cell transplantation
procedure takes place; the tissue is maintained under the
direct supervision of the surgeon who performs the procedure; and the cells undergo minimal manipulation only
with a well characterized and widely accepted enzymatic
cell isolation not involving in vitro culture. Therefore, the
patients were not exposed to radically new procedures or
considerable additional risks.
Considering the comparably low patient number per year
we did not plan a clinical study. We therefore choose to
consider this treatment as the introduction of an innovative practice. We planned to apply the practice on eight
patients, and report to the responsible local Innovative
Practice Advisory Committee of the IRB for re-evaluation.
The procedure was only suggested to individual patients
that had a reasonable expectation of success. Treatment
was suggested to enable wound closure within 2 to 3 more
weeks and reduce the development of long-term wound
healing complications over several months. All patients re-
© 2011 Wichtig Editore - ISSN 0391-3988
Gerlach et al
quired outpatient followup for observation, and we planned
to immediately report any potential adverse events to the
Institutional Patient Safety Committee.
RESULTS
Although we did not perform a clinical study, we agreed
to primary “endpoints” for the patient follow-up charac-
terization: a) time to complete re-epithelialization of the
treated area; and b) the esthetic and functional quality of
re-epithelialization at six months’ followup. As secondary
“endpoints”, we assessed for infections, inflammation, or
any adverse effects or complications. The mean treatment
area was 3% (0.5-6%) body surface area. Some patients
received additional split skin mesh grafting prior to cell
spray grafting, but only to wound areas unrelated to our
analysis. These additional treatments are listed in Tables I
TABLE II - WOUND CHARACTERISTICS OF CELL SPAY GRAFT PATIENTS AFTER CELL SPRAY TRANSPLANTATION GRAFTING
Patient
Treated
II°b BSA
(%)
Region
treated and
amount of
sprayed
cells
Full
Wound healing results at 6 months
re-epithelialization
of treated regions
after day
Vancouver Scar-Scale
Score at six months post cell
spray grafting.
Range from 0 - 14 points (worst)
1
2
Right thigh,
right knee
9.8x105
14
Patient did not appear for followup, report on phone: Good clinical followup, occasional tickling, dry skin, esthetically good picture, no scars at regions treated
2
0.5
Penis
5.8x105
12
Slight hyper and hypopigmentation at region
treated
3
5
Left leg
3.7 x106
16
Entire area: no functional findings, occasional Central area (1%): 7
tickling, dry skin
Central (1%): hyperthrophic scar formation
Peripheral area (4%): 2
Peripheral (4%): hypopigmentation
4
0.5
Penis
4.0x10 6
21
Hypopigmentation at region treated
5
4
Face, forehead
5.0x106
10
Patient did not appear for followup, report on phone: good wound healing, no
functional or esthetic concerns, no scar
6
5
Face, left
hand
9.8x106
12
All areas: no functional or cosmetic
restrictions, except
hand: dry skin
face: partially reddening
Hand: 1
Face: 1
7
6
Arm circular
left
1.2x106
20
All areas: no functional or cosmetic
restrictions, except
upper arm: reddening, partial slight
hypertrophy
lower arm: reddening, partial hypertrophy
Upper arm: 4
Lower arm: 3
8
1
Hand left
2.4x105
16
Hand distal between 2nd/3rd/4th finger:
Hyper- and hypopigmentation and scar
(fingers can be spread apart about 1cm
in palmar position with distinct sensation
of tension, also itching and dryness)
Hand proximal (0.5%): Pink skin
Hand distal (0.5%): rough skin, hyperthrophic
sanguine scar with partial strand formation
Hand distal (0.5%): 9
1
1
Hand proximal (0.5%): 4
All patients were treated with primary debridement and debridement prior to cell application; the interval between initial trauma and cell application was in all cases
at least 14 days.
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Autologous skin cell isolation
Fig. 2 - 58-year-old male patient (Tab. I, Patient 6) presenting areas
of deep dermal burns after explosion trauma. A) Patient 2 days after
trauma during observation time without mesh grafting or cell grafting; B) five days after cell spray grafting; C) after 12 days, 16 days,
and six months at followup.
Fig. 1 - Male patient, 42 years old (Tab. I, Patient 4) presenting areas
of deep dermal burns after propane gas explosion trauma. A) During
cell spray grafting, B) wound at follow up after four weeks and C)
after six months.
and II, but they were not considered in the evaluation described here. The localization of the cell spray transplantation is summarized in Table II, which also summarizes the
yield of the cell isolation, in other words, the therapeutic
cell amount.
The mean interval between trauma and cell spray transplantation was 16 +4 days. Using single spray transplantation grafting, the ratio of skin biopsy to treatment surface could be enlarged from the typical 1:2 to 1:4 in mesh
grafting to 1:20 to 1:60 with a mean of 1:25. Figures 1 to
3 show examples of patients prior to and post cell spray
transplantation. The mean time to full re-epithelialization
was 12.6 days (10-21 days). For clinical result evaluation
at six months, we used the Vancouver Scar Scale (10) to
assess the scar development. The evaluated parameters
are described in Table II. All patients exhibited a constant
improvement of the clinical picture, with at least 12 months
followup. No patient showed side effects or complications.
276
Fig. 3 - 46-year-old male patient (Tab. I, Patient 8) presenting areas of deep dermal burns after electro-burn injury. A) during cell
spray grafting; B) wound at followup after five days; and C) after six
months. The cell spray device in action is visible in A).
© 2011 Wichtig Editore - ISSN 0391-3988
Gerlach et al
Considering the clinical picture of the cases described prior
to application, we believe that the functional and cosmetic
results were excellent after cell spray transplantation. At
this time, however, our information is limited to the feasibility of the cell isolation method.
DISCUSSION
The use of enzymatically isolated (11) and in vitro expanded cells cultured into skin cell-sheets (12) was introduced
around the turn of the century. This application of in vitro
cultured cells grown to sheet grafts was described to reduce mortality and pain (13). The availability and exclusive
use of differentiated keratinocytes, often result in cosmetically unsatisfactory regeneration (14). The take rates are
not consistent due to limitations of the method, for example, blister formation and the loss of some skin cell populations during in vitro expansion. The application of single
sprayed cells after expansion in culture was therefore introduced (3, 15).
Single skin cell transplantation application follows a historic development of burn treatment methods. In the early
1900s, autologous skin 1:1 transplantation was introduced; in the mid-1900s, split skin and mesh grafting was
established. Mesh grafting results in wound closure after a
small “skin lattice” is placed on the wound; cells grow in
the open spaces, thereby filling in the gaps and closing the
wound. While the split ratio is typically 1:4, larger ratios are
often associated with unsatisfactory results. In the 1960s
the Meek technique was introduced (16), in which the split
skin is cut mechanically into even smaller tissue pieces of
around 1 x 1 mm, enabling split ratios of 1:4 to 1:6. The
concept of non-cultured single cell spray grafting follows
this development path, which is based on the application
of single cells sprayed into the wound, where they continue to grow and regenerate skin in the wound. Here, the
cells grow to close the wound from multiple starting points,
thus allowing a larger split ration of typically more than 1:20
to be used. Some authors describe a reduction of time to
wound closure, which is generally known to contribute to
a positive outcome (17). Gravante et al, however, pointed
out that mesh grafting and cell spray grafting enable comparable results and re-epitelialization times (8) and report
that melanocyte grafting may not be sufficient. On the
other hand, the cosmetic aspects of moderate Grade IIb
burn area treatment results are of special interest for burns
on face and hands, a potentially interesting application for
cell-grafting. The 1:20 ratio with cell-grafting allows comparably small biopsy sizes to be taken from non-prominent
body areas, while the typical mesh pattern of mesh grafting on the burn site can be avoided.
The method we describe here enables skin cell spray
transplantation using a modified cell isolation involving a
two-enzyme approach and cell washing. While cell washing removes remaining enzymes prior to grafting, the application of dispase enables the dermis to be separated from
the epidermis; this allows the subsequently used trypsin
to isolate the cells from the epidermis-dermis interface,
specifically the otherwise enclosed layer of the epidermis
that contains regenerative basal keratinocytes. These skin
progenitors are not selectively reached by conventional
techniques. The assessment of a reproducible content and
typical amount of basal keratinocytes is the subject of ongoing studies. We introduced this method in our center to
provide a therapy to individuals with a rare medical condition. For these individuals the decision not to perform mesh
grafting to avoid overgrafting resulted in delayed wound
healing, which could be addressed by our method. Interestingly, our health care provider, BG Germany, reimburses
the amount for this procedure that is otherwise received for
mesh grafting surgery.
Our experience confirms positive results using a processor-controlled cell spray device, reported previously with in
vitro expanded cells (3). The clinical case reports presented
in this paper show that surgical debridement and consecutive application of sprayed autologous cells in suspension
in this patient group results in re-epithelialization within an
acceptable time and acceptable cosmetic outcome (Figs.
1-3). Since we did not include controls, for example, patients sprayed with cells isolated in a standard way, the
specific benefits of the procedure cannot be assessed.
Other limitations of our study include the fact that our results also summarize clinical evaluation data with subjective assessments of the burn depth only; a comparison to
the normal healing time without the cell spray transplantation cannot be given; and a debridement treatment alone
may have contributed to improved healing.
We present, however, a limited number of cases to provide
information on the feasibility of the cell isolation method.
Larger future clinical studies would be of interest and we
are available to provide training on the method to interested centers.
In conclusion, we describe a novel method for skin cell
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Autologous skin cell isolation
isolation in an intra-operative setting at the point of care
and a treatment option with non-cultured cells for patients
with Grade IIb burns that did not receive grafting to avoid
overgrafting and did not heal in the appropriate time. We
believe that our initial clinical data show interesting results
while complications were not seen. For future clinical studies, we propose to consider multicenter studies on a patient group exhibiting all of the following clinical aspects:
a) acute burns with Grade IIb areas of less than 15% body
surface; b) no previous split skin transplantation on the
wound to avoid overgrafting; c) treated over two weeks
conservatively with intense wound care including enzymatical debridement; d) wound closure only up to 50% of
the initial Grade IIb burn area within these two weeks.
Financial support: This work was sponsored by a grant from the Federal Ministry of Education and Research, Germany (BMBF) and the
City of Berlin, Germany.
REFERENCES
0436.2004.07208005.x.
7. Morasso MI, Tomic-Canic M. Epidermal stem cells: the
cradle of epidermal determination, differentiation and
wound healing. Biol Cell 2005; 97: 173-183. doi:10.1042/
BC20040098.
8. Gravante G, Di Fede MD, Araco A, et al. A randomized
trial comparing ReCell® system of epidermal cells delivery versus classic skin grafts for the treatment of deep
partial thickness burns. Burns 2007; 33 (8): 966-972.
doi:10.1016/j.burns.2007.04.011.
9. Tobiasen J, Hiebert JM, Edlich RF. The abbreviated burn
severity index. Ann Emerg Med 1982; 11 (5): 260-262.
doi:10.1016/S0196-0644(82)80096-6.
10. Baryza MJ, Baryza GA. The Vancouver Scar Scale: an administration tool and its interrater reliability. J Burn Care
Rehabil 1995; 16 (5): 535-538. doi:10.1097/00004630199509000-00013.
11. Johnen C, Hartmann B, Steffen I, et al. Skin cell isolation and expansion for cell transplantation is limited in
patients using tobacco, alcohol, or are exhibiting diabetes mellitus. Burns 2006; 32 (2): 194-200. doi:10.1016/j.
burns 2005.10.001.
12. Gallico GG III, O’Connor NE, Compton CC, Kehinde O, Green H. Permanent coverage of large burn
wounds with autologous cultured human epithelium.
N Engl J Med 1984; 311 (7): 448-451. doi:10.1056/
NEJM198408163110706.
1. Gerlach J, Wolf SE, Johnen C, Hartmann B. Innovative
regenerative medicine approaches to skin cell-based
therapy for patients with burn injuries. In: Atala A, Lanza R, Thomson J, Nerem R. Principles of Regenerative
Medicine. Burlington, MA: Elsivier/Academic Press;
2008: 1298-1321.
2. Navarro FA, Stoner ML, Lee HB, Park CS, Wood FM, Orgill DP. Melanocyte repopulation in full-thicknesswounds
using a cell spray apparatus. J Burn Care Rehabil 2001;
22 (1): 41-46. doi:10.1097/00004630-20010100000009.
3. Hartmann B, Ekkernkamp A, Johnen C, Gerlach JC,
Belfekroun C, Küntscher MV. Sprayed cultured epithelial
autografts for deep dermal burns of the face and neck.
Ann Plast Surg 2007; 58 (1): 70-73. doi:10.1097/01.
sap.0000250647.39784.bb.
4. Bickenbach JR, Chism E. Selection and extended growth
of murine epidermal stem cells in culture. Exp Cell Res
1998; 244: 184-195. doi:10.1006/excr.1998.4163.
5. Fuchs E, Raghavan S. Getting under the skin of epidermal morphogenesis. Nat Rev Genet 2002; 3: 199-209.
doi:10.1038/nrg758.
6. Webb A, Li A, Kaur P. Location and phenotype of human adult keratinocyte stem cells of the skin. Differentiation 2004; 72: 387-395. doi:10.1111/j.1432-
278
Conflict of interest statement: None identified.
Address for correspondence:
Jörg C. Gerlach, MD, PhD
McGowan Institute for Regenerative Medicine
University of Pittsburgh
3025 East Carson Street, Suite 238
Pittsburgh PA, 15203, USA
e-mail: [email protected]
© 2011 Wichtig Editore - ISSN 0391-3988
Gerlach et al
13. Munster AM. Cultured skin for massive burns. A prospective, controlled trial. Ann Surg 1996; 224 (3):
372-375, discussion 375-377. doi:10.1097/00000658199609000-00013.
14. Chester DL, Balderson DS, Papini RP. A review of keratinocyte delivery to the wound bed. J Burn Care
Rehabil. 2004; 25 (3): 266-275. doi:10.1097/01.
BCR.0000124749.85552.CD.
15. Harris PA, Leigh IM, Navsaria HA. Pre-confluent keratinocyte grafting: the future for cultured skin replace-
ments? Burns 1998; 24 (7): 591-593.
16. Meek CP. Extensive severe burn treated with enzymatic
debridement and microdermagrafting: case report. Am
Surg 1963; 29: 61.
17. Yanaga H, Udoh Y, Yamauchi T, et al. Cryopreserved
cultured epidermal allografts achieved early closure of
wounds and reduced scar formation in deep partialthickness burn wounds (DDB) and split-thickness skin
donor sites of pediatric patients. Burns 2001; 27 (7):
689-698. doi:10.1016/S0305-4179(01)00008-0.
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