burns 37 (2011) e19e23

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Case report

Autologous skin cell spray-transplantation for a deep dermal

burn patient in an ambulant treatment room setting
Jorg C. Gerlach a,*, Christa Johnen d, Eric McCoy c, Kirsten Brautigam b,
Jorn Plettig d, Alain Corcos c
a

Departments of Surgery and Bioengineering, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
Charite, Campus Virchow, AG Experimental Surgery, University Medicine Berlin, Berlin, Germany
c
University of Pittsburgh Medical Center, UPMC Mercy Hospital Burn Center, Pittsburgh, PA, USA
d
Stem Cell Systems, Berlin, Germany
b

article info
Article history:
Accepted 24 January 2011

1.

Introduction

While superficial partial-thickness burn wounds are treated

conservatively and split-thickness skin grafting (STSG),
with or without mesh expansion, is unequivocally
indicated for full-thickness burns, the indication for STSG
to a deep partial-thickness burn wound remains obscure,
if not controversial. Delayed healing with its associated
complications (i.e. hypertrophic scarring, contracture, infection, and unsatisfactory psycho-social adjustment)
[1] must be balanced against overgrafting, particularly
when the hands and face are involved [2]. Delays in deepdermal wound healing can typically be appreciated by
week 2 of conservative management. Unfortunately, this
is a particularly problematic stage for traditional skin
grafting.
Autologous single-cell skin grafting as an alternative
treatment in this situation has been described previously by
Wood and co-workers [35]. Much like a traditional STSG,
autologous skin-cell transplantation is based on removing
healthy skin, at a superficial dermal depth, using a derma-

tome, from a non-prominent, unburned area of the body in an

operative setting. Skin cell isolation is performed immediately
after harvest and spray-grafting to the burn site is applied
during the same operative session. In comparing skin cell
spray-grafting with STSG in the deep dermal wound, Gravante
et al. found skin cell grafting to be well tolerated and similar in
results to STSG [6]. The advantage of a reduced donor skin
area for spray-grafting, however, is associated with a nondesirable prolonged general anesthesia time required for the
cell isolation.
Cell spray-grafting that could be enabled in an outpatient
treatment room setting without general anesthesia would
present advantages. We have modified this technique for
patients in whom a late stage STSG may be problematic but
for whom conservative treatments have been unsatisfactory. In contrast to Wood et al., we employ a modified twoenzyme isolation technique involving dispase and trypsin,
together with cell washing by centrifugation. While cell
washing removes remaining enzymes prior to grafting, the
application of dispase enables to separate dermis from
epidermis and 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 the conventional
technique. We report our modified method and our clinical
implementation in an outpatient setting for a patient
that exhibited delayed wound healing after conservative
therapy of a moderate deep partial thickness burn
wound.

* Corresponding author at: McGowan Institute for Regenerative Medicine, University of Pittsburgh, 3025 East Carson Street, Pittsburgh, PA
15203, USA. Tel.: +1 412 383 7640; fax: +1 412 383 9460.
E-mail address: jgerlach@pitt.edu (J.C. Gerlach).
0305-4179/$36.00 # 2011 Elsevier Ltd and ISBI. All rights reserved.
doi:10.1016/j.burns.2011.01.022

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burns 37 (2011) e19e23

2.

Methods

2.1.

Patient

Autologous skin-cell spray transplantation was performed at our

center on a 43 years old male patient in 2009. The Institutional
Review Board, through its Technology and Innovative Practice
Assessment Committee, approved the procedure. The patient
suffered a gasoline flame burn to his face, right shoulder, arm
and elbow. The total body surface area (TBSA) was 7%.

maintained under the direct supervision of the surgeon who

performed the procedure. All devices and solutions employed
were routine to clinical practice available at any burn center
and were disposable. During donor skin processing, and prior
to transplantation, the burn wound was prepared using
Hibiclens1 solution and gentle mechanical debridement until
bleeding spots occurred. To reduce pain, 400 mcg of fentanyl
(Actiq1, Cephalon, Inc., Frazer, PA) was administration orally.
Hemostasis was performed with regular gauze.

2.4.
2.2.

The patient presented to our burn center 5 days after the

injury. He was initially treated in an outside facility with
antibiotic ointment to the face and silver sulfadiazine cream
on the arm and shoulder, which he applied daily at home. He
was referred to our facility by his primary care physician for
concerns of delayed wound healing of the arm and shoulder.
On our initial evaluation he was diagnosed to have a deep
partial-thickness burn to the shoulder and arm with an
inflammatory eschar. While the wounds on face and neck
were relatively superficial, the area of concern on the
shoulder, elbow and arm were moderate deep partialthickness and measured 5% TBSA. No tangential wound
excision or escharotomy was performed; he did, however,
undergo a mild mechanical debridement and began daily local
wound care using collagenase (Santyl1, Healthpoint, Ltd., San
Antonio, TX) ointment and polysporin powder (Johnson and
Johnson CCI, Skillman, NJ) along with daily inspection in our
hydrotherapy unit. His eschar began to lift; however, he still
did not have significant reepithelialization of the wound and
was thus considered a candidate for single-cell spray
application as an alternative to STSG. The patient is physically
active in his profession and we were concerned that without
some epithelial grafting, a hypertrophic scar or constrictive
contraction could develop across the elbow joint. Informed
consent was obtained and alternative treatments explained.
We explained to the patient that the decision would have no
impact on potential alternative treatment. Pre-existing local
and systemic infections, hypersensitivity to trypsin or other
enzymatic wound treatments and the risks associated with
anesthesia were excluded.

2.3.

Wound treatment and follow-up

Initial wound treatment after trauma

Cell spray-transplantation

The entire procedure was performed in an outpatient

treatment room. On post-burn day #10 the patient underwent
single-cell transplantation. His right hip supplied the donor
skin. A 2 cm  2 cm graft was taken to a depth of 0.008 in using
a Weck1 blade (Teleflex Medical, Research Triangle Park, NC)
after chlorhexidine preparation (Hibiclens1, Molnlycke Health
Care US, LLC, Norcross, GA) and sub-cutaneous 2% lidocaine
with epinephrine (Hospira, Inc., Lake Forest, IL).
The skin cell isolation method is described below. The
tissue was procured into a single cell suspension for cell spraytransplantation. The cell isolation procedure was performed
on site in the same room immediately prior to cell transplantation. Following donor skin harvest, the tissue was handled
under surgically sterile conditions until application and was

Following cell spray-grafting the wound was dressed in

Adaptic1 (Johnson and Johnson Medical Limited, Gargrave,
UK) and wrapped in dry gauze (Kerlix1, Tyco Healthcare Group
LP, Mansfield, MA). For clinical follow-up evaluation to assess
burn scars we used the Vancouver Scar Scale [8].

2.5.

Skin cell isolation

The biopsy treatment includes a modification of a previously

described [7] two-enzyme approach of tissue digestion using
dispase and trypsin, and cell washing using conventional
clinical cell centrifugation. The following steps for autologous
skin-cell spray transplantation were performed; this procedure required approximately 70 min:
A) The donor area 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 Biosciences, Bedford, USA) and is cut into
3 mm  4 mm pieces by the surgeon with a surgical
disposable scalpel. We took care that the specimen would
not dry out.
B) The cell separation, performed by a specialized laboratory
technician, involves
1) initial separation of dermis and epidermis for 2540 min
in a 2.5 unit/ml Dispase II (#4942078, Roche Diagnostics,
Mannheim, Germany) containing Ringers lactate solution (Lactated Ringers Injection USP 1000 ml bag, Baxter,
Deerfield, IL) at 37 8C in Petri-dishes (BD);
2) mechanical separation of dermis and epidermis of each
skin piece using forceps and scalpel, performed in a dry
Petri-dish (the specimen must not dry out), the dermis
parts are not used further;
3) transferring the epidermal pieces in a 15 ml Falcon tube
(BD) with Ringers lactate solution, to be washed from
the enzyme;
4) placing the epidermal pieces into a single cell suspension for 1015 min in a 5 ml 0.05% trypsin/0.02% EDTA
containing solution, at 37 8C in 15 ml Falcon tubes to
allow isolation from epidermis including from the
interface layer of epidermis and dermis that contains
regenerative basal keratinocytes; followed by stopping
with 7.5 ml clinical grade Ringers lactate solution
containing 10% of previously taken patient-own serum,
for 30 s;
5) sieving the cell suspension through a 70 micrometer
50 ml Falcon tube sieve (all BD);
6) centrifugation for 7 min at 100  g for cell washing to
remove the enzymes; and

burns 37 (2011) e19e23

7) placing the cells into clinical grade Ringers lactate

solution.At this stage, cell counting in a Neubauer
chamber showed a yield between 0.5 and 4  106 cells/
cm2 skin, with a trypan-blue viability of the cells
between 97 and 99%. Using a sterile disposable pipette
(BD), the cells are transferred into sterile disposable
syringes (BD) for cell deposition by spraying. We
resuspended into a 1  106 cell/ml suspension, which
we divided into three 2 ml syringes (BD) for cell spraying;
C) One syringe at a time, the cell spraying was performed
using fine needle spraying out of a syringe (BD) with a 30G
needle (BD) through which the suspended cells are
immediately spray-transplanted onto to the burn site of
the patient by spray deposition.
Some cells from the remaining suspension in the syringe
were taken into culture, for control. The n = 3 culture dishes
(25 cm2, BD) seeded showed regular seeding and attachment
behavior and regular in vitro follow up culture.

3.

Results

Using skin-cell spray transplantation grafting, the expansion ratio of skin donor site to treatment surface area was
approximately 1:20, indicating that we could cover a larger
area
than with a traditional graft. The wound dressing was
[()TD$FIG]

e21

removed on post skin cell spray-transplantation grafting day

4. The patient was noted to have complete reepithelialization
of the dry wound. Wound dressings were not required
anymore and the clinical picture improved with subsequent
visits. No infections, inflammation or any adverse effects or
complications were seen. The aesthetic and functional quality
of reepithelialization at the three-, six-, and twelve-month
follow-up was considered excellent. A discoloration was
observed, which decreased between months three and six
but had almost disappeared at month twelve. Fig. 1 shows the
patients wounds before and after skin cell spray-transplantation. For clinical result evaluation, we used the Vancouver
Scar Scale to assess the burn scar, which was 3 points after
three months (2M and 1V) and 2 points (1M) after six months
and 1 point after twelve months (1M).

4.

Discussion

The use of enzymatically isolated [2] and in vitro expanded

cells cultured to skin cell sheets [9,10] has been described to
reduce mortality [11] and pain [12]. However, the take rates
appear not reliable due to limitations of the method, e.g. blister
formation and the loss of some skin cell populations during in
vitro expansion and the use of keratinocytes differentiated to
sheets, often yields cosmetically unsatisfactory regeneration
[13]. To address blister formation and subsequent sheet

Fig. 1 Male 43 years old patient presenting deep dermal burns after kerosene explosion trauma. Upper panel from left to
right: Skin cell-spray grafting 10 days past trauma (treatment day 0) on arm and shoulder prior to wound refreshment; cell
isolation setting in outpatient room; cell spraying in outpatient room. Lower panel from left to right: Wound at follow-up after
four days post cell-spray transplantation; after three months; after six months; and after twelve months.

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burns 37 (2011) e19e23

detachment, an application of single cells after expansion was

introduced [14,15] which we also employed previously [16].
The concept of non-cultured cell spray-grafting enabled by
immediate skin-cell preparation and application avoiding in
vitro culture is based on the application of single cells sprayed
onto the wound that can proliferate in the regenerating skin.
Here, the cells grow to close the wounds from multiple starting
points, and thus a larger ratio of donor to wound size is
possible compared to conventional grafting. While providing
smaller cell numbers, techniques that do not feature in vitro
cell expansion appear promising, as basal keratinocytes can
differentiate and lose these regenerative properties during
culture [2026]. Some authors describe a reduction of time to
wound closure, which is generally known to contribute to a
positive outcome [17,18]. Gravante et al., however, pointed out
that mesh grafting and skin-cell spray-grafting offer comparable results and reepithelialization times and report that
melanocyte grafting may not be sufficient [6]. The use of noncultured cells was discussed controversially [19], since the cell
numbers to be offered for wound regeneration are lower than
with cell sheets.
Modifications of the isolation technique, as described here,
can focus on the provision of cells from the basal keratinocyte
layer. We employed a modified two-enzyme isolation technique that separates the dermis from the epidermis first and
thus allows the subsequently used trypsin to specifically reach
the regenerative keratinocytes in the epidermal part of the
interface between epidermis and dermis. We also introduced
an improved enzyme removal. The reproducible content and
typical amount of basal keratinocytes, however, must be
properly assessed in future studies.
We introduced skin-cell spray transplantation in our center
to provide a therapy to a relatively rare subset of burn patients;
specifically, when delayed wound healing occurs after a
decision to avoid STSG. We chose an innovative practice
approach since planning clinical studies in such a patient
group and our limited patient population appeared challenging. Our first case on the method reported indicates that the
use of sprayed autologous skin-cells in suspension can result
in rapid reepithelialization and an acceptable cosmetic
outcome (Fig. 1). We can confirm that a significant discolorization [5,19] occurred using single cell isolation, which should
indicate that the wound was severely injured; but we saw that
this reversed to almost normal cosmetic appearance between
six and twelve month post grafting. Our results, however,
summarize a clinical evaluation with subjective assessment of
the burn depth only, a comparison to the normal healing time
without the cell spray transplantation cannot be given. To give
statements on whether we accelerated healing and improved
cosmetic and functional outcomes, we cannot present objective markers for the assessment of the initial state of the
injury. The perspective for this report was thought to be on a
modified cell isolation method, easiness of the process and
patient tolerance. While we have not used methods for
determining more objectively if the wound healed faster than
without this treatment, such as laser Doppler sonography, it
was the impression of the burn surgeon that the wound would
not have healed to dry, uniform epithelium by day 4 with
conventional wound care only. Employing such methods,
however, should be considered for clinical studies.

To conclude, we describe a novel treatment method option

for second-degree burns of moderate size that do not heal in a
timely manner following conservative management. The
procedure could be performed in an outpatient setting, and
our initial clinical data show interesting results while
complications were not seen. In addition, we suspect that
such a treatment may be of special interest for areas
exhibiting critical wound healing such as the face or hands.
Larger future clinical studies could be of interest and we are
available to provide training on the method to interested
centers.

Conflict of interest
None identified.

Acknowledgement
The work was sponsored by a grant from the University of
Pittsburgh Medical Center, UPMC, USA.
Author contributions: A.C. designed, coordinated and performed the clinical work, analyzed data and wrote the
manuscript. J.G. designed and coordinated the cell isolation
work, developed methods for cell isolation and spraying,
analyzed data and wrote the manuscript. C.J., K.B., E.M. and J.P.
developed methods for cell spraying, developed methods forand performed the cell isolation and characterization,
performed laboratory analysis, analyzed data and provided
discussions.

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