0041-1337/04/7703-350/0
TRANSPLANTATION
Copyright © 2004 by Lippincott Williams & Wilkins, Inc.
Vol. 77, 350–355, No. 3, February 15, 2004
Printed in U.S.A.
HUMAN PLASMA AS A DERMAL SCAFFOLD FOR THE
GENERATION OF A COMPLETELY AUTOLOGOUS
BIOENGINEERED SKIN
SARA G. LLAMES,1 MARCELA DEL RIO,2 FERNANDO LARCHER,2 EVA GARCÍA,1 MARTA GARCÍA,2
MARÍA JOSÉ ESCAMEZ,2 JOSE L. JORCANO,2 PURIFICACIÓN HOLGUÍN,3 AND ALVARO MEANA1,4
Background. Keratinocyte cultures have been used
for the treatment of severe burn patients. Here, we
describe a new cultured bioengineered skin based on
(1) keratinocytes and fibroblasts obtained from a single skin biopsy and (2) a dermal matrix based on human plasma. A high expansion capacity achieved by
keratinocytes grown on this plasma-based matrix is
reported. In addition, the results of successful preclinical and clinical tests are presented.
Methods. Keratinocytes and fibroblasts were obtained
by a double enzymatic digestion (trypsin and collagenase, respectively). In this setting, human fibroblasts are
embedded in a clotted plasma-based matrix that serves
as a three-dimensional scaffold. Human keratinocytes
are seeded on the plasma-based scaffold to form the epidermal component of the skin construct. Regeneration
performance of the plasma-based bioengineered skin
was tested on immunodeficient mice as a preclinical
approach. Finally, this skin equivalent was grafted on
two severely burned patients.
Results. Keratinocytes seeded on the plasma-based
scaffold grew to confluence, allowing a 1,000-fold cultured-area expansion after 24 to 26 days of culture.
Experimental transplantation of human keratinocytes
expanded on the engineered plasma scaffold yielded
optimum epidermal architecture and phenotype, including the expression of structural intracellular proteins and basement-membrane components. In addition, we report here the successful engraftment and
stable skin regeneration in two severely burned patients at 1 and 2 years follow-up.
Conclusions. Our data demonstrate that this new
dermal equivalent allows for (1) generation of large
bioengineered skin surfaces, (2) restoration of both
the epidermal and dermal skin compartments, and (3)
functional epidermal stem-cell preservation.
great in vitro expansion capacity. However, the lack of a
healthy dermal component caused by damage induced by the
burn represents a serious handicap for graft take and aesthetic and functional outcome of this therapeutic strategy
(2–5). Current approaches aimed at dermal conditioning before epidermal sheet grafting that rely on the use of either
skin allografts (cadaver skin) or artificial dermis made by
tissue engineering procedures have been shown to improve
the regenerative process (2, 3).
One attractive alternative to pure keratinocyte grafts is
the association of keratinocytes with dermal equivalents (6).
Various methodologies of the culture of these skin equivalents and their clinical efficacy have been reported (7, 8).
However, the use of these skin equivalents for the treatment
of severely burned patients is limited by the insufficient
keratinocyte expansion capacity when they are grown on
these artificial dermal matrices (9). We have recently overcome this problem by developing an artificial dermis based
on live human fibroblast-containing fibrin gels (10). Recent
work by our group demonstrated the applicability of that
skin equivalent in preclinical cutaneous tissue engineering
and gene therapy studies (10 –13). In the original system, the
fibroblasts and keratinocytes were obtained from different
donors (allogeneic cells), whereas fibrin was prepared from
cryoprecipitates. In the present study, we describe and characterize an improved and whole autologous bioengineered
skin based on (1) the use of both autologous fibroblasts and
keratinocytes obtained from a single biopsy and (2) the use of
clotted human plasma as a three-dimensional dermal scaffold in which fibroblasts are embedded. In addition, we
present long-term follow-up results of two burned patients
receiving grafts with this new autologous bioengineered skin.
Cultured keratinocyte sheets are an alternative to the
split-thickness skin grafts for permanent coverage of deep
burns (1). Keratinocyte cultures offer the advantage of their
MATERIALS AND METHODS
Obtaining Cells for Culture
For preclinical grafts, primary cells (keratinocytes and fibroblasts)
were isolated from three different skin-donor biopsies. Whole biopThis work was supported by Fis 99/1001, by Ministerio de Ciencia y sies were minced using surgical scissors, with no previous dermalTecnología (grant BMC 2001–1018), and by Fundación Marcelino Botín. epidermal separation. The fragments obtained were enzymatically
Sara Llanes and Marcela Del Rio contributed equally to this work. digested with trypsin (0.05%)/ethylenediaminetetraacetic acid
1
Centro Comunitario de Sangre y Tejidos del Principado de As- (EDTA, 0.02%) (T/E). Every 30 minutes, T/E was changed for a fresh
turias, Emilio Rodriguez Vigil s/n, Oviedo, Spain.
T/E mixture. The removed T/E was inactivated with serum-contain2
Ciemat, Epithelial Damage, Repair and Tissue Engineering, Ma- ing culture medium (Dulbecco’s modified minimal essential medium
drid, Spain.
[DMEM] with 10% fetal calf serum [FCS]) and centrifuged at 1,400
3
Department of Plastic Surgery, Hospital Universitario de Getafe, rpm for 10 minutes. The pellet was resuspended in culture medium,
Madrid, Spain.
and cells were counted. The procedure was repeated until no more
4
Address for correspondence: Alvaro Meana, Centro Comuni- cells were obtained from the sample. The T/E solution was then
tario de Sangre y Tejidos del Principado de Asturias, Emilio completely eliminated, and the remaining skin biopsy was introRodriguez Vigil s/n, 33006 Oviedo, Spain. E-mail: investigacion@ duced in a collagenase solution (Type I, 2 mg/mL) (Sigma, St. Louis,
c-transfusion-asturias.com.
MO) until its complete disaggregation (between 8 –12 hours). The
Received 19 May 2003. Accepted 20 November 2003.
collagenase solution was filtered through a 60 ␮m filter (Falcon,
DOI: 10.1097/01.TP.0000112381.80964.85
350
February 15, 2004
351
LLAMES ET AL.
Beckton-Dikinson, San Jose, CA) and centrifuged at 1,400 rpm for 10
minutes. The pellet was resuspended in culture medium, and cells
were counted.
Primary Keratinocyte Culture
Cells obtained after T/E digestion were seeded in a 75 cm2 culture
flask (2⫻106 cells/flask) in the presence of 6⫻106 lethally irradiated
3T3 cells (European collection of animal cell culture, n° 85022108)
and cultured following the method initially described by Rheinwald
and Green (14). The keratinocyte culture medium was a mixture of
DMEM/HAM-F-12 supplemented with 10% FCS, epidermal growth
factor, insulin, cholera toxin, hydrocortisone, triiodo-thyronine, and
adenine as previously described (10). The medium was changed
every 48 to 72 hours. Subconfluent primary keratinocyte cultures
were treated with T/E and propagated on the engineered plasmabased scaffold (see below).
Fibroblast Culture
Cells obtained from biopsies after collagenase digestion were
seeded (100,000 cells/cm2) in the absence of lethally irradiated 3T3
using DMEM supplemented with 10% FCS as the culture medium
(primary fibroblast culture). The medium was changed every 72
hours. When an increase in the fibroblast-like cells was observed,
cells were trypsinized and seeded at twice the original surface (a 2:1
passage with respect to primary culture area).
The secondary culture was maintained until keratinocyte subconfluence. Fibroblasts were then detached from the flask by T/E treatment, counted, and used as dermal cells in the plasma-based dermal
equivalent.
Preparation of Plasma-Based Dermal Equivalent
A plasma scaffold populated with autologous fibroblasts was used
as the dermal component of the bioengineered skin. Fresh frozen
plasma was obtained from voluntary donors of the local blood bank
(Centro Comunitario de Tejidos del Principado de Asturias) according to the standards of the American Association of Blood Banks (15).
The plasma-based dermal equivalent was prepared as follows:
6 to 7.5⫻104 cultured fibroblasts were resuspended in 10 mL of
plasma containing 10 mg of tranexamic acid (Amchafibrin, FidesEcofarm, Barcelona, Spain), the final volume was adjusted to 23 mL
by adding ClNa 0.9%, and, finally, 2 mL of Cl2Ca 1% were added. The
mixture was placed in a tissue culture flask (75 cm2) and allowed to
solidify at 37°C in a CO2 incubator for 30 minutes.
Keratinocyte Expansion on the Plasma-Based Dermal Equivalent
Keratinocytes were seeded on the plasma-fibroblast dermal equivalent at various densities. In two cases, 8 to 9⫻104 cells/cm2 were
plated (a 1:15 passage expansion ratio from the primary culture). In
the third case, 6 to 7⫻104 cells/cm2 (an expansion ratio of 1/20) were
used. The culture medium was the same as that used for the primary
keratinocyte culture on feeder layer. When a 1:15 expansion rate was
used, keratinocytes growing on plasma gels reached confluence
within 11 to 12 days and were ready for grafting.
Experimental Grafting Protocol
Plasma-based bioengineered skin was fixed to a nonpetroleum
gauze with an inorganic polymer glue (Histoacryl, B/Braun Aesculap, Tuttlingen, Spain) and manually detached from the culture flask
(10). The bioengineered skin was placed orthotopically on the backs
of nu/nu mice. Ten mice received grafts in two series of grafting
experiments. Mice were aseptically cleansed, and full-thickness 3
cm2 wounds were created on the dorsum of mice to match a 3 cm2
skin equivalent piece cut from the original 75 cm2 culture product.
Devitalized mouse skin was used as a biologic bandage to protect and
hold the skin substitute in place during the take process (13). Grafting was performed under sterile conditions using 6-week-old male
mice purchased from the Jackson Laboratory (Bar Harbor, ME) and
housed in pathogen-free conditions for the duration of the experiment at the CIEMAT Laboratory Animals Facility (Spanish registration number 28079 –21). All experimental procedures were carried out according to European and Spanish laws and regulations
(European Convention 123, Spanish R.D223/88 and O.M13–10 – 89 of
the Ministry of Agricultural, Food and Fisheries, animal protection
biosafety and bioethics guidelines).
Patients
Two patients suffering from flame burn injuries were selected
at the Hospital Universitario de Getafe (Madrid, Spain) for autologous plasma-based bioengineered skin transplantation. Patient 1
was a 28-year-old male with 50% burn of total body surface area
(TBSA) and full-thickness burns of 35% TBSA. Patient 2 was a
17-year-old male with 85% burn of TBSA and full-thickness burns
of 60% TBSA. All procedures followed were approved by the ethical committee of the institution. Full-thickness skin biopsies of 2
cm2 (patient 1) and of 2.5 cm2 (patient 2) were taken, placed in
keratinocyte growth medium, and transferred refrigerated to the
laboratory within 24 hours. Family informed consents were requested. Keratinocytes and fibroblasts were cultured as described
above, and autologous plasma-based bioengineered skin was prepared as above.
The patients’ wound bed was prepared with cadaver skin as previously described (2, 16). Debridement was made down to the fascia
and followed immediately by meshed cadaver-skin transplantation.
Depending on autologous skin availability, meshed autologous skin
transplantation was also performed. When cultured autologous plasma-based bioengineered skin was ready, the epidermal layer of cadaver skin was removed with an electric dermatome. The bioengineered skin was then carefully placed on the dermal bed by hand
only and covered with petroleum gauze. The gauze was changed
every 48 hours. Microbiologic samples were taken when bacterial
contamination was suspected, and topic preventive antibiotic treatment was applied to the putatively affected areas.
Histologic Studies
The cultured bioengineered skin, the regenerated human skin
xenografts, and the regenerated autologous skin of patients were
fixed in 10% formalin for paraffin embedding and hematoxylin-eosin
staining. Samples from regenerated human skin on mice were taken
at 4, 8, and 16 weeks postgrafting. In experimental grafting protocols, formalin or ethanol fixed sections were stained using specific
antibodies against human involucrin (cloneSY-5, Sigma), human
vimentin (V9, BioGenex, San Ramon, CA), keratin 5 (polyclonal
AF138, BabCO, Berkeley, CA), keratin 10 (monoclonal AE2, ICN
Biomedicals, Cleveland, OH), and loricrin (polyclonal AF-62,
BabCO). To determine the dermoepidermal junction, ethanol-fixed
sections were immunostained with specific antibodies against basal
membrane laminin (Clone LAM-89, Sigma). The Masson trichrome
(Accustain Trichrome Stains, Sigma) was used as a marker of mature fibers of collagen.
RESULTS
Optimized Recovery of Keratinocytes and Fibroblasts from
the Same Biopsy
Although most standard protocols for isolation of human
skin cells make use of the same trypsin digestion to obtain
either keratinocytes or fibroblasts, the collagenase step
within the double sequential enzymatic digestion procedure
(see Methods) renders an enriched population of fibroblasts.
Although a few epidermal colonies persisted in the fibroblast
culture, they differentiated and were rapidly overgrown by
fibroblasts (Fig. 1A). The large number of fibroblasts obtained in this manner avoids the need to use allogenic dermal
352
TRANSPLANTATION
Vol. 77, No. 3
TABLE 1. Cell yield and expansion rates
Biopsy 1
2
Size of biopsy (cm )
Age of donor
Cell extraction rate (cells
⫻106/cm2 skin
biopsy)
T/E
Collagenase
Fibroblast (⫻106)
obtained in culture
(days)
Seeding density in dermis
(fibroblasts/cm2)
Final cultured area (cm2)
Expansion rate (final
cultured area/initial
biopsy size)
Days
12
18
2.4
1.8
16 (13)
Biopsy 2
8
28
2.5
1.4
8.5 (12)
Biopsy 3
6
24
1.6
1.25
6 (14)
1,000
750
800
16,000
⫻1,330
11,250
⫻1,400
7,500
⫻1,250
25
23
26
T/E, trypsin-ethylenediaminetetraacetic acid.
FIGURE 1. Phase contrast image of cultures (magnification
ⴛ100). (A) Primary fibroblast culture; (arrow) keratinocyte
colony. (B) Fibroblasts in plasma-based scaffold at 24 hours
and at day 5 (C). (D) Keratinocyte colony growing on the
plasma-based dermal equivalent surface (day 5).
cells for the generation of the bioengineered skin (Table 1)
(and see below). The cell yield as well as the density of
fibroblasts that were embedded in the plasma scaffold are
shown in Table 1. It is worth noting that, although a large
number of cells were obtained from the collagenase digestion,
only a fraction of the total population can properly attach and
grow in the primary culture. Thus, the actual cell yield is
higher than it appears.
Generation of the Plasma-Based Bioengineered Human Skin
In a previous study, we demonstrated that live allogenic
human fibroblasts embedded in fibrin gels obtained from
blood cryoprecipitates (10) supported clonal growth of human
keratinocytes without the need of lethally irradiated mouse
3T3 cells. To obtain a less time-consuming and more costeffective clinically suitable product, we tested whether blood
cryoprecipitate preparations could be substituted by whole
plasma. In fact, in the presence of calcium, tranexamic acidtreated plasma containing dermal fibroblasts coagulated
quickly at 37°C, achieving maximum strength in about 30
minutes. Culture flasks containing the clotted plasma gels
could be manipulated without damaging the dermal equivalent. Fibroblast growth behavior was then studied. At the
time of plasma clotting, fibroblasts appeared round, and
spreading could be observed 24 hours later (Fig. 1B). After
acquiring their typical spindle-like shape (after 36 – 48
hours), fibroblasts started to proliferate and, by the end of
the composite culture (days 11–14), reached a very high
density (Fig. 1C).
As in the case of fibrin-fibroblast gels (10), keratinocytes
seeded on the fibroblast-containing plasma scaffold demonstrated excellent growth features. Thus, isolated cells
attached to the dermal equivalent surface 24 hours after
seeding gave rise to expanding epithelial colonies (Fig.
1D), which finally occupied the whole surface of the dermal
equivalent. Epidermal cell confluence was attained be-
tween days 11 and 14 (depending on expansion). Absence
of carry-over 3T3 cells in the transplantable bioengineered
skin was demonstrated by polymerase chain reaction analysis for mouse DNA (data not shown). Fibroblast-containing plasma-based dermal equivalents did not contract during culture period, although a slight decrease in thickness
was observed as from day 10.
Experimental Grafting of Full-Thickness Wounds with the
Plasma-Based Human Bioengineered Skin
The regeneration performance of the plasma-based bioengineered skin was evaluated in an immunodeficient mouse
model. Experimental grafting was performed using a recently described surgical procedure (13).
All transplanted bioengineered grafts were adherent to the
wound bed throughout the study. Within 15 days after grafting, devitalized mouse skin, used as a biologic bandage,
sloughed off, and human epidermis became readily visible
(Fig. 2A). Grafts displayed fine wrinkles and clinical signs of
hyperkeratosis, both typical of native human skin. The regenerated human skin on mice was easy to lift and move on
the dorsal fascia.
Grafting of keratinocytes expanded in vitro on the engineered plasma-scaffold allowed for optimum restoration of
the epidermal phenotype, including the synthesis of intracellular structural proteins (keratins K5 and K10), other differentiation markers (involucrin and loricrin), and basement
components (laminin). Moreover, tissue architecture 4 weeks
after grafting closely resembled human interfollicular epidermis (Fig. 2B). A healthy and mature tissue architecture was
preserved even at 16 weeks, the longest follow-up time, corresponding to approximately four epidermal turn-overs according to previous reports (17, 18) (Fig. 2D and data not
shown) The basal stratum was present as a compartment of
mostly elongated cells clearly distinguishable by labeling
with an antibody recognizing keratin K5 (Fig. 2E). Accordingly, there was a strong suprabasal expression of keratin
K10 and involucrin as well as of the late differentiation
marker loricrin in upper regions corresponding to the granular layer, as evidenced by microscopy observation (Fig. 2, C,
E, and F).
February 15, 2004
LLAMES ET AL.
FIGURE 2. (A) Clinical appearance of regenerated human
skin (dotted square) on back of a nude mouse at 16 weeks
postgrafting. (B) Histologic appearance of the regenerated
human skin 4 weeks postgrafting. (C) Human involucrin immunostaining at the junction of regenerated human skin
(HS) and murine receptor (MS) (4 weeks postgrafting). (D)
Human vimentin immunostaining at the junction of regenerated human skin and murine receptor (16 weeks postgrafting). (E) Keratin K5 immunostaining (green) and keratin K10
immunostaining (red) of the regenerated human skin (4
weeks postgrafting). (F) Loricrin immunostaining of the regenerated human skin (4 weeks postgrafting). (G) Masson’s
trichrome staining of the regenerated human skin. (H) Laminin immunostaining of the dermoepidermal junction.
Immunostaining of histologic sections with human-specific
antibodies directed against epidermal involucrin or dermal
vimentin clearly demonstrated the boundary between mouse
and human tissue (Figs. 2, C and D). In addition, this immunostaining showed the persistence of human epidermis (Fig.
2C) and of human fibroblasts in the dermal compartment of
the regenerated skin 16 weeks after grafting (Fig. 2D). Four
weeks after transplantation, human fibroblasts were embedded in a well-vascularized, mature, collagen-rich matrix as
seen in sections stained with Masson’s trichrome. (Fig. 2G).
Concomitantly, the epidermal-dermal junction of the graft
was clearly outlined by labeling with an antilaminin antibody (Fig. 2H). Long-term preservation of epidermal architecture, only achievable by several epidermal turn-overs, is a
353
FIGURE 3. Patient 1. (A) Cultured plasma-based bioengineered skin prepared from autologous keratinocytes and fibroblasts and immunostained with human pankeratin antibody (magnification ⴛ100). (B) Patient leg covered with the
plasma-based bioengineered skin. (C) Clinical appearance of
the regenerated skin at 2 months follow-up.
definitive sign of in vivo functional epidermal stem-cell
persistence.
Treatment Of Massive Full-Thickness Burned Patients with
the Autologous Plasma-Based Bioengineered Skin
Twenty-four days after the skin biopsy was taken from
patient 1, cultured autologous plasma scaffold-based bioengineered skin (2,500 cm2) was applied onto the wound bed of
the patient’s legs (Fig. 3, A and B). The take (evaluated by
visual inspection 15 days after grafting) was 95%. Epidermal
regeneration analyzed 2 months after grafting was complete
(100% of the grafted area). At 2 years follow-up, epidermal
regeneration was stable, with good cosmetic outcome, as
shown in Figure 3C.
354
TRANSPLANTATION
FIGURE 4. Patient 2. (A) Clinical appearance of the regenerated skin at 21 days follow-up. (B) Clinical appearance of the
regenerated skin at 1 year follow-up. (C) Histologic appearance of the regenerated skin at 3 weeks follow-up. (D) Involucrin immunostaining of the regenerated human skin (3 weeks
postgrafting).
Similar results were obtained in patient 2. Figure 4 shows
clinical and histologic results obtained for patient 2. Twentyfive days after the skin biopsy was taken, the cadaver epidermal layer was removed, and cultured autologous plasmabased bioengineered skin (3,700 cm2) was grafted on the
chest, abdomen, and thighs. The take down 15 days after
grafting was approximately 80%. In addition, histologic analysis from a punch biopsy (3 weeks postgrafting) showed a
well-organized neoepidermis. The regenerated skin appeared
differentiated, with a well-developed stratum granulosum
and corneum (Fig. 4C), and re-expressed involucrin at its
normal suprabasal location (Fig. 4D). Epidermal regeneration at 1 year follow-up (last point evaluated) was again
stable, with satisfactory aesthetic results (Fig. 4B). Histiotypic morphology was also preserved at 1 year follow-up
(data not shown). Even at the longest follow-up times, neither of the patients evaluated exhibited epidermal blistering
or skin retractions.
Permanent take obtained with keratinocytes expanded on
the engineered plasma-based dermal matrix suggests that
this new technology allows for functional epidermal stem
cells, persisting both in vitro during expansion and in vivo
after transplantation (19).
DISCUSSION
We and others have recently demonstrated the advantages
of growing human keratinocytes on fibrin matrices (10 –13,
Vol. 77, No. 3
16, 20). One of the most important properties of fibrin is that
it allows maintenance of the epidermal cell “stemness,” a
requisite for permanent skin regeneration (13, 16, 20). However, DeLuca, Barrandon, and their collaborators use commercial fibrin only as a cell carrier because the technique
they use to grow keratinocytes still relies on the presence of
irradiated mouse 3T3 cells as a feeder layer (16, 20). We went
on to provide evidence that the presence of live allogenic
human fibroblasts embedded in a cryoprecipitate-derived fibrin matrix supports the clonal growth of keratinocytes without the need of feeder cells (10). In the present study, we
show, in addition, that the autologous human fibroblasts
persist after grafting and contribute to dermal repair.
Here, we describe a major improvement to the fibrin-based
human keratinocyte culture technique that involves both the
use of clotted plasma as a dermal scaffold and autologous
fibroblasts as keratinocyte growth support. The use of
plasma instead of cryoprecipitate-derived fibrin represents a
great advantage regarding dermal equivalent preparation.
Thus, whereas plasma clotting requires a one-step calcium
addition, preparation of cryoprecipitate-derived fibrin gels is
a more laborious, time-consuming, and expensive procedure.
Preliminary comparative results also indicate that plasmabased dermal equivalents perform better than cryo-based
equivalents in terms of keratinocyte expansion abilities
(Meana, unpublished data, 2001). Several dermal equivalents of diverse composition have been developed: acellular
dermis from cadaver skin (18, 21), type I collagen associated
or not associated with proteoglycans (22, 23), and poly-Llactide-poly-l-glycolide structures (24). However, none of
them show the advantages of human plasma. The presence of
a whole-blood set of cytokines, attachment factors, and platelet-derived growth factors (25) in the plasma-based dermal
scaffold is likely to offer a highly proliferative environment
for keratinocytes. The elevated cost of raw materials and
poor keratinocyte expansion factor on available substrates
other than fibrin–plasma have precluded their routine use
for seriously burned patients who require large skin surfaces.
In addition to being a robust keratinocyte growth substrate,
the fibroblast-containing plasma scaffold fulfills a major objective, namely, transient dermal repair. This is particularly
true for patients who have suffered important losses of dermal component coupled with the epithelial wound. Clotted
plasma is the naturally occurring temporary wound cover
involved in efficient re-epithelization and connective-tissue
reorganization. In fact, when markers of epidermal differentiation and dermal regeneration were analyzed in the grafted
immunodeficient mice, normal features of human-skin maturation were detected early after grafting.
Whereas autologous keratinocytes, used for the definitive
cover of grafted wounds, are essential to avoid immunologic
rejection, the allogenic fibroblast rejection process has not
been adequately characterized. However, recent reports demonstrate a beneficial role of autologous fibroblasts in wound
repair in terms of functional and aesthetic results (26 –29).
Improved enzymatic cell dissociation of biopsies enabled us
to obtain sufficient primary cells to prepare bioengineered
skin containing live autologous fibroblasts for its dermal
component. Moreover, we have been able to demonstrate
long-term maintenance of these cells in the dermis of grafted
mice.
February 15, 2004
LLAMES ET AL.
The new plasma-based bioengineered skin described here
offers the possibility of a fully autologous cultured skin replacement in which both cells and plasma come from patients
themselves. However, donor plasma would be necessary
when availability of autologous plasma is limited in patients
suffering extensive and acute skin losses. Conversely,
chronic skin defects requiring sequential autologous skin
replacement interventions, such as giant nevus, may profit
from this new therapeutic strategy.
It has been suggested that inappropriate culture conditions could cause an irreversible loss of stemness. A recent
and very pertinent study proposed that any new dermal
equivalent or even any new epidermal graft-carrier, aimed at
permanent skin regeneration, should be properly tested for
its capacity to maintain functional epidermal stem cells in
vitro (16). Different tests correlate well with stem-cell persistence in vitro (16). However, even with stem-cell founders
(holoclones) in hand, it is relatively difficult to predict their in
vivo potential. This is because stemness should be determined, at least in a first attempt, retrospectively by evaluating the in vivo progeny of stem cells and inferring the behavior of their ancestral parent (19). In vivo long-term
persistence of experimental grafts on immunodeficient mice
is perhaps, although laborious, the best way to do this. Using
this in vivo test, we have safely determined that the stem-cell
compartment, the required component of any tissue-engineered self-renewal organ, was successfully preserved using
this new keratinocyte culture technology.[[21]]
Acknowledgments. We wish to thank Almudena Holguin and
Blanca Duarte for excellent technical assistance and Jesus Martinez
for animal care. S.L. is a recipient of a predoctoral fellowship from
Fundación Botı́n. E.G. is a recipient of a predoctoral fellowship
from FICYT (Asturias). M.G. is a recipient of a predoctoral fellowship
from CAM. We are also indebted to Jose L. Jorcano for expert advice.
REFERENCES
1. Barret JP, Wolf SE, Desai MH, et al. Cost-efficacy of cultured epidermal
autografts in massive pediatric burns. Ann Surg 2000; 231: 869.
2. Hickerson WL, Compton C, Fletchall S, et al. Cultured epidermal autografts and allodermis combination for permanent burn wound coverage. Burns 1994; 20: S52.
3. Rennekampff HO, Kiessig V, Griffey S, et al. Acellular human dermis
promotes cultured keratinocyte engraftment. J Burn Care Rehabil
1997; 18: 535.
4. Cuono C, Langdon R, Mc Guire J. Use of cultured epidermal autografts
and dermal allografts as skin replacement after burn injury. Lancet
1986; 1: 1124.
5. Archambault M, Yaar M, Gilcherst BA. Keratinocytes and fibroblasts in a
human skin equivalent model enhance melanocyte survival and melanin synthesis after ultraviolet irradiation. J Invest Dermatol 1995; 104:
859.
6. Sheridan RL, Tomkins RG. Skin substitutes in burns. Burns 1999; 25: 97.
7. Hansbrough JF, Boyce ST, Cooper ML, et al. Burn wound closure with
cultured autologous keratinocytes and fibroblasts attached to a collagen-glycosaminoglycan substrate. JAMA 1989; 262: 2125.
8. Kim BM, Suzuki S, Nishimura Y, et al. Cellular artificial skin substitute
produced by short period simultaneous culture of fibroblasts and keratinocytes. Br J Plast Surg 1999; 52: 573.
355
9. Kremer M, Lang E, Berger AC. Evaluation of dermal-epidermal skin
equivalents (“composite-skin”) of human keratinocytes in a collagenglycosaminoglycan matrix (Integra artificial skin). Br J Plast Surg
2000; 53: 459.
10. Meana A, Iglesias J, Del Rio M, et al. Large surface of cultured human
epithelium obtained on a dermal matrix based on live fibroblast-containing fibrin gels. Burns 1998; 24: 621.
11. Rio MD, Larcher F, Meana A, et al. Nonviral transfer of genes to pig
primary keratinocytes. Induction of angiogenesis by composite grafts of
modified keratinocytes overexpressing VEGF driven by a keratin promoter. Gene Ther 1999; 6(10): 1734.
12. Larcher F, Del Rio M, Serrano F, et al. A cutaneous gene therapy approach
to human leptin deficiencies: correction of the murine ob/ob phenotype
using leptin-targeted keratinocyte grafts. FASEB J 2001; 15(9): 1529.
13. Del Rio M, Larcher F, Serrano F, et al. A preclinical model for the analysis
of genetically modified human skin in vivo. Hum Gene Ther 2002; 13:
959.
14. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal
keratinocytes: the formation of keratinizing colonies from single cells.
Cell 1975; 6: 331.
15. Walker RH. Technical Manual [ed. 10]. Bethesda, MD, American Association of Blood Banks 1990, p 46.
16. Pellegrini G, Ranno R, Stracuzzi G, et al. The control of epidermal stem
cells (holoclones) in the treatment of massive full-thickness burns with
autologous keratinocytes cultured on fibrin. Transplantation 1999; 68:
868.
17. Dover R, Wright NA. Epidermal cell kinetics. In: Fitzpatrick T and Freedberg I, eds. Dermatology in general medicine. London, McGraw-Hill
1999, pp. 160 –171.
18. Potten CS, Booth C. Keratinocyte stem cells: a commentary. J Invest
Dermatol. 2002; 119: 888.
19. Compton CC, Nadire KB, Regauer S, et al. Cultured human sole-derived
keratinocyte grafts re-express site-specific differentiation after transplantation. Differentiation 1998; 64: 45.
20. Ronfard V, Rives JM, Neveux Y, et al. Long-term regeneration of human
epidermis on third degree burns transplanted with autologous cultured
epithelium grown on a fibrin matrix. Transplantation 2000; 70(11):
1588.
21. Chakrabarty KH, Dawson RA, Harris P, et al. Development of autologous
human dermal-epidermal composites based on sterilized human allodermis for clinical use. Br J Dermatol 1999; 141: 811.
22. Bell E, Ehrlich HP, Buttle DJ, et al. Living tissue formed in vitro and
accepted as skin equivalent tissue of full thickness. Science 1981; 211:
1052.
23. Buttler CE, Yannas IV, Compton C, et al. Comparison of cultured keratinocytes seeded into a collagen GAG matrix for skin replacements. Br J
Plast Surg 1999; 52: 127.
24. Beumer GJ, van Blitterswijk CA, Bakker D, et al. Cell-seeding and in vitro
biocompatibility evaluation of polymeric matrices of PEO/PBT copolymers and PLLA. Biomaterials 1993; 14: 598.
25. Marx RE, Carlson ER, Eichstaedt RM, et al. Platelet rich plasma, growth
factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 1998; 85: 638.
26. Svensjo T, Yao F, Pomahac B, et al. Cultured autologous fibroblasts
augment epidermal repair. Transplantation 2002; 73: 1033.
27. Ferguson PC, Boynton EL, Wunder JS, et al. Intradermal injection of
autologous dermal fibroblasts improves wound healing in irradiated
skin. J Surg Res 1999; 85: 331.
28. Caruso DM, Schuh WH, Al-Kasspooles MF, et al. Cultured composite
autografts as coverage for an extensive body surface area burn: case
report and review of the technology. Burns 1999; 25: 771.
29. Lamme EN, Van Leeuwen RT, Brandsma K, et al. Higher numbers of
autologous fibroblasts in an artificial dermal substitute improve tissue
regeneration and modulate scar tissue formation. J Pathol 2000; 190:
595.