Pereira Et Al. (2013) - Advanced Biofabrication Strategies For Skin Regeneration and Repair

603
REVIEW
ISSN 1743-5889 10.2217/NNM.13.50 2013 Future Medicine Ltd Nanomedicine (2013) 8(4), 603621
Advanced biofabrication strategies for skin
regeneration and repair
Skin damage & wound healing
Skin is the largest organ of the body, serving
primarily as a protective barrier against the
environment [13]. It also helps to prevent body
dehydration and constitutes a physical barrier,
limiting the penetration of potentially harmful
agents to internal organs. The skin has a three-
layer structure composed of epidermis, dermis
and hypodermis (FIGURE 1) [46]. The epidermis,
the superfcial layer, is mainly composed of kera-
tinocytes but also contains other cell types, such
as Langerhans cells and melanocytes [5,7], provid-
ing a barrier against infection and moisture loss.
The dermal layer, situated below the epidermis,
is responsible for the elasticity and mechanical
integrity of the skin. It contains vascularized
extracellular matrix (ECM) rich in collagen, elas-
tin and glycosaminoglycans [3,5,6]. The cellular
components of the dermal layer include fbro-
blasts, endothelial cells, smooth muscle cells and
mast cells [3,5]. The hypodermis, located below
the dermis, is mainly composed of adipose tis-
sue and collagen, and mainly acts as an energy
source [4,5,7].
Skin damage is mainly caused by burn injuries,
chronic wounds (venous, pressure and leg ulcers),
excision of skin, tumors and other dermato logical
conditions [2,8]. According to WHO, 300,000
deaths are annually attributed to burn injuries,
while 6 million patients worldwide suffer from
burns every year [4]. Additionally, more than
6 million individuals suffer from chronic skin
ulcers [4]. In the USA alone more than 3 million
patients suffer from chronic wounds [9].
Skin has a natural healing ability that gener-
ates, at the moment of injury, a complex cascade
of highly integrated and overlapping events of
hemostasis, infammation, migration, prolif-
eration and maturation, as illustrated in FIGURE 2
[1012]. This complex, dynamic and continuous
process relies on the interaction between cel-
lular components, growth factors and cyto-
kines, acting in concert to repair the damaged
tissue [13,14].
Treatment of skin lesions
The treatment of skin lesions is a critical issue in
healthcare, requiring the need to consider several
parameters affecting the healing process, such
as the wound type (e.g., burn, ulcer, acute and
chronic wound), the wound depth (e.g., epi-
dermal, superf icial partial-thickness, deep
partial-thickness and full-thickness wounds),
the patients health (e.g., diabetes and persistent
infections) and the level of the exudate [14]. Cur-
rently, a great variety of wound-care products are
available for the treatment of different types of
skin lesions (TABLE 1).
Solutions, creams & ointments
Liquid and semi-solid formulations, such as
solutions, creams and ointments, have been
Skin is the largest organ of human body, acting as a barrier with protective, immunologic and sensorial
functions. Its permanent exposure to the external environment can result in different kinds of damage
with loss of variable volumes of extracellular matrix. For the treatment of skin lesions, several strategies
are currently available, such as the application of autografts, allografts, wound dressings and tissue-
engineered substitutes. Although proven clinically effective, these strategies are still characterized by key
limitations such as patient morbidity, inadequate vascularization, low adherence to the wound bed, the
inability to reproduce skin appendages and high manufacturing costs. Advanced strategies based on both
bottom-up and top-down approaches offer an effective, permanent and viable alternative to solve the
abovementioned drawbacks by combining biomaterials, cells, growth factors and advanced
biomanufacturing techniques. This review details recent advances in skin regeneration and repair
strategies, and describes their major advantages and limitations. Future prospects for skin regeneration
are also outlined.
KEYWORDS: bottom-up approach in situ biofabrication nanoscale structure
skin regeneration tissue engineering top-down approach wound healing
Rben F Pereira
1,2,3
,
Cristna C Barrias
2
,
Pedro L Granja
2,3,4

& Paulo J Bartolo*
1
1
Centre for Rapid & Sustainable
Product Development, Polytechnic
Insttute of Leiria, Portugal
2
Insttuto de Engenharia Biomdica,
Universidade do Porto, Porto, Portugal
3
Insttuto de Cincias Biomdicas Abel
Salazar, Universidade do Porto, Porto,
Portugal
4
Faculdade de Engenharia da
Universidade do Porto, Departamento
de Engenharia Metalrgica &
Materiais, Porto, Portugal
*Author for correspondence:
paulo.bartolo@ipleiria.pt
SPECIAL FOCUS y Advanced nanobiomaterials
for tissue engineering and regenerative medicine
part of
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REVIEW Pereira, Barrias, Granja & Bartolo
widely used for wound healing, disinfection,
cleaning and debridement [1521]. However, the
physical form of these products limits their per-
manency in the wound for acceptable clinical
periods [13]. Consequently, these agents have
been replaced by advanced products that include
wound dressings and skin substitutes.
Wound dressings
Skin lesions involving the loss of high amounts
of skin require the immediate use of a dress-
ing primarily to protect the wound [22]. Wound
dressings have been widely used due to their
relative low cost, ease of use, and effectiveness
to clean, cover and protect the wound from
the external environment. An ideal wound
dressing should provide a moist environment
to the wound bed, remove the excess of exu-
date, avoid maceration, minimize scar forma-
tion, protect the wound from infection and
maintain an adequate exchange of gases [2325].
Wound dressings must be fexible, permeable
to water vapor, ft the lesion region, and exhibit
good adhesion to the wound bed and adequate
mechanical properties [6,2327]. Some dressings
also act as drug delivery systems, incorporating
and releasing therapeutic agents into the wound
bed [24,2833]. Wound dressings can be classifed
according to different parameters, including the
type of material, function in the wound, physi-
cal form or ability to provide a moist environ-
ment to the wound. They can also be classifed
as traditional and modern dressings [13].
Traditional dressings, such as bandages,
gauzes or cotton wool, represent the most
conventional products used for the treatment
of skin lesions, providing a protective barrier
against bacteria and microbes [26]. When applied
to the wound, they absorb large amounts of
exudate, drying the wound bed and avoiding
a moist wound environment, which is widely
recognized to promote the healing process [6,31].
Thus, the use of these materials may inhibit
the healing process [6,32,33]. Owing to the high
rate of fuid absorption, these dressings may also
adhere to the wound bed, making its removal
diffcult and consequently causing pain to the
patient [13].
Modern dressings were developed to address
the limitations of traditional ones. They are
D
e
r
m
i
s
E
p
i
d
e
r
m
i
s
Hair shaft
Sweat gland pore
Sweat gland
Keratinocytes
Melanocyte
Elastin
Fibroblast
Artery
Adipose tissue
Vein
Collagen
H
y
p
o
d
e
r
m
is
Figure 1. Skin structure showing the three layers, the skin appendages and the main
cellular constituents.
www.futuremedicine.com 605 future science group
Advanced biofabrication strategies for skin regeneration & repair REVIEW
able to create and maintain a warm, moist
environment, providing optimal conditions
for an improved healing process [6,13], contrary
to traditional dressings. Modern dressings can
be obtained from either natural [23,24,3437] or
synthetic polymers [28,3841], or a combination
of both [30,4244]. They are available as thin
flms, foams or gels, and can be classifed as
hydrocolloid dressings, alginate dressings and
nonalginate dressings (TABLE 2) [13].
Autografts & allografts
In the case of patients with skin lesions extended
to the dermis or hypodermis regions, a complex
treatment procedure is required. In these cases,
the clinical gold standard procedure relies
on the use of split-thickness skin autografts or
allografts, which contain all of the epidermis
and marginal parts of the dermis [7,45]. However,
autografts are naturally limited in their extent,
inducing scarring at donor sites and lengthy hos-
pital stays, while allografts present ethical and
safety issues related to disease transmission, and
may lead to immune rejection [2,3,5,46,47]. There-
fore, alternatives providing permanent solutions
are required.
Tissue-engineered skin substitutes
Tissue engineering has emerged as a new and
promising feld for the treatment of skin lesions,
combining scaffolds, cells and biomolecular
signals, such as growth factors. It is a multi-
disciplinary feld requiring the combined effort
of clinicians, cell biologists, engineers, material
scientists and geneticists toward the development
of biological substitutes to restore, maintain or
improve tissue function [48].
Despite recent developments in manufac-
turing processes and biomaterials, both auto-
grafts/allografts and wound dressings have sig-
nifcant limitations for skin regeneration, which
were previously mentioned. The main draw-
backs of wound dressings are the low adhesion
to the lesion, impossibility of reproducing skin
appendages and the inability to replace the lost
tissue, particularly the dermis, after severe burns
[2,13]. In order to address these limitations and
solve the problem of the donor graft shortage,
both cellular (e.g., Apligraf; Organogenesis,
MA, USA) and acellular (e.g., Alloderm;
Biohorizons, AL, USA) tissue-engineered skin
substitutes were developed. Skin regeneration
is, in fact, among the few felds where tissue
Fibrin clot Platelet Macrophage
Inflammation Hemostasis
Migration and proliferation Maturation
Macrophage Neutrophil Lymphocyte Mast cell
Late granulation tissue Keratinocyte Epithelial cell Fibroblast
Granulation tissue
Figure 2. Phases of the wound healing process. Hemostasis involves the formation of a brin clot occurring simultaneously with the
inammatory phase, in which inammatory cells are responsible for wound cleaning. During the migration and proliferation phases,
epithelial cells and broblasts migrate to the wound site in order to synthesize the constituents of the extracellular matrix, leading to the
formation of a granulation tissue. In the maturation phase, the composition and properties of the granulation tissue are continuously
remodeled in order to achieve values proximal to the healthy skin.
Adapted with permission from [4].
engineering commercial products are already
available and under clinical utilization. Acellular
constructs are made of natural or synthetic
biomaterials, and can be used in combination
with autografts [2]. Cellular constructs contain
biomaterials and cells, obtained from different
origins including autologous, allogenic or xeno-
geneic [7]. Clinically available skin substitutes
can be broadly divided into epidermal, dermal
and dermoepidermal constructs, as shown in
TABLE 3 [4]. However, available skin substitutes
often suffer from a range of problems including
poor integration due to inadequate vasculariza-
tion, ineffcient adhesion to the wound bed,
scarring at graft margins or the lack of skin
appendages [5,49].
Epidermal substitutes
Epidermal substitutes, used for the treatment
of lesions affecting the epidermis, contain auto-
logous keratinocytes that are generally cultured
in the presence of murine fbroblasts [7]. To
produce epidermal substitutes, the frst step is
a skin biopsy with an extension in the range of
25 cm
2
. The epidermis is then separated from
the dermis, and the keratinocytes isolated and
cultured using an appropriate medium. As this
procedure takes approximately 3 weeks, the
wounds are prim arily treated with temporary
dressings that protect the lesion and stimulate
the healing [2,7]. Major limitations of epidermal
constructs are the fabrication time, high pro-
duction costs, variable engraftment rates and
diffcult handling, as a result of the thin and
fragile nature of the constructs [7]. To overcome
the long fabrication times associated with these
epidermal substitutes, cell populations obtained
from a biopsy can alternatively be directly
sprayed into the lesion (ReCell
; Avita Medical,
Perth, Australia; and Spray
XP; Graco, MN,

USA), allowing the local delivery of epidermal
cells [7,50,51]. This strategy allows a faster epithe-
lialization and epidermal maturation. However,
it is not suitable for the treatment of third-degree
burn wounds [7]. Zhu et al. investigated the clini-
cal effcacy of a surface-functionalized medical-
grade polymer disc that enables the delivery of
cultured autologous keratinocytes into burn and
chronic wounds [52]. Skin lesions treated with
this strategy showed improved healing rates and
Table 1. Strategies for the treatment of skin lesions: main advantages and limitations.
Treatment of skin
lesions
Advantages Limitations
Autografts Golden standard in skin regeneration; good adhesion to
the wound bed; provides pain relief; reduced rejection
Limited availability of donor sites; induce scar
formation; patient morbidity; lengthy hospital stays
Allografts Temporary prevention of wound dehydration and
contamination; incorporate into deep wounds
Limited availability; may lead to immune rejection;
transmission of diseases
Creams, solutions and
ointments
Ease of use; provide disinfection, cleaning and
debridement; not expensive in general
Limited skin regeneration; short residence time on the
wound (require frequent administrations)
Traditional dressings Not expensive; provide a protective barrier against the
penetration of exogenous microbes
High absorption capacity; do not provide a moist
environment; adhere to the wound bed; may inhibit
the healing process
Modern dressings Create and maintain a moist wound environment; can be
made from a wide range of materials with different
properties; ability to hydrate the wound and remove
excess exudate
More expensive; low adhesion to the wound bed;
inability to promote the regeneration of lost skin, in
particular the dermal layer
Tissue-engineered
skin substitutes
Promote the regeneration of dermis and epidermis;
prevent uid loss and provide protection from
contamination; may deliver extracellular matrix
components, cytokines, growth factors and drugs to the
wound bed, enhancing the healing process; can be used
in combination with autografts
High manufacturing costs; difcult handling; poor
adhesion to the wound bed; possibility of immune
rejection and transmission of diseases (allogeneic skin
cells); inability to promote the regeneration of
full-thickness wounds; poor vascularization;
impossibility of reproducing skin appendages
In situ biofabrication
of skin substitutes
Provide immediate and effective relief to the patient;
enable the direct fabrication of skin substitutes tting to
the anatomical shape of the defect; allow the controlled
deposition of cells and biomaterials; may eliminate the
use of bioreactors for growing and maturing the tissue
ex vivo; may solve the need for vascularization through
the controlled deposition of endothelial cells
Biofabrication techniques need to be adapted for
in situ biofabrication; may require integration with
imaging techniques to print the skin substitute with
appropriate anatomical shape; requires the use of
printable materials exhibiting adequate biological and
mechanical properties
Data taken from [2,13].
a signifcant reduction in ulcer size. Recently,
Hu et al. developed a skin substitute composed
of human EGF gene-modified HaCaT cells
(an immortal keratinocyte cell line) [53]. The
authors investigated the ability of the construct
to heal burn wounds created in a rat model.
Results indicated that the substitute signifcantly
improved epidermal morphogenesis along with
the formation of thicker and compact complete
epidermis. Other clinical trials were performed
to evaluate the effectiveness of epidermal sub-
stitutes for the regeneration of different skin
lesions such as chronic wounds [54,55] and ulcers
[56,57]. However, further clinical investigation is
necessary to evaluate its effectiveness and safety.
Dermal substitutes
The treatment of full-thickness lesions, affect-
ing both epidermis and dermis, requires the use
of an allograft skin or, alternatively, the use of
dermal constructs [58]. Dermal substitutes can
be produced by using either natural or synthetic
materials [59]. These substitutes prevent wound
contraction, provide good mechanical stability,
and are available in different thicknesses and
compositions [2,58]. Dermal substitutes must be
covered by a permanent epidermal substitute,
usually an autologous split-thickness skin graft
[58]. The substitutes undergo colonization and
vascularization by the underlying cells, leading
to the formation of an autologous neodermis [7].
Dermal substitutes are used for the treatment
of skin ulcers [60,61] and burns [6264]; although
more controlled clinical trials are required,
involving a signifcant number of patients, to
confrm its clinical effectiveness. Recently, Guo
et al. developed a bilayer dermal substitute com-
posed of a covalently crosslinked collagenchi-
tosan sponge and a silicone membrane, which
acts as a temporary protection layer [65]. The
dermal substitute was loaded with plasmid DNA
encoding VEGF-165/N,N,N-trimethyl chitosan
chloride complexes, and tested for the regen-
eration of full-thickness burn wounds in a pig
model. In vitro results showed that the human
umbilical vein endothelial cells remained viable
after culture into the dermal substitute, express-
ing high levels of VEGF. Full-thickness burn
wounds treated with the N,N,N-trimethyl chito-
san chloride/pDNAVEGF dermal substitute
showed the fastest regeneration of the dermis
and vascularization rate. Furthermore, results
Table 2. Commercially available wound dressings.
Wound
dressing type
Properties and application Product Manufacturer
Hydrocolloid
dressings
Promote debridement, angiogenesis and a moist environment;
do not adhere to the wound bed; useful for low-to-moderate
exuding wounds
Granuex
; Aquacel
Conva Tec (NJ, USA)

Tegasorb

3M Healthcare (MN, USA)
Comfeel
Coloplast (Humlebk,
Denmark)
Alginate
dressings
Hydrophilic gel that maintains a moist environment and
protects the wound; absorbent; partial dissolution; indicated
for moderate-to-heavy exuding and bleeding wounds
Sorbsan
Maersk (Copenhagen,
Denmark)
Kaltostat
Conva Tec
Tegagen 3M Healthcare
Nonalginate
dressings
Ability to hydrate the wound bed; low absorption capacity;
promote autolytic debridement; indicated for dry, necrotic and
thick sloughy wounds
Nu-Gel
Johnson & Johnson (NJ, USA)

Purilon
Coloplast
Intrasite
Smith & Nephew Healthcare

(London, UK)
Semi-permeable
lms
Good adhesion with the healthy skin; can be applied as a
secondary dressing; high transparency and exibility; promote
a moist environment; indicated for low-exuding wounds
Opsite

Cutilm

Bioclusive
Johnson & Johnson

Tegaderm 3M Healthcare
Foam dressings High absorption; provide thermal insulation; appropriate for
wounds with moderate-to-high levels of exudate
Lyofoam Conva Tec
Allevyn

Antimicrobial
dressings
Useful for infected wounds; can be applied in both dry and
exuding wounds
Acticoat Smith & Nephew Healthcare
Inadine
Johnson & Johnson

Data taken from [3,13,26,31].
also indicated the positive effect of the con-
struct on angiogenesis, leading to signifcantly
higher numbers of newly formed and mature
blood vessels. Similarly, Philandrianos et al.
compared the effcacy of fve acellular dermal
skin substitutes (Integra
, Integra Life Sciences,

NJ, USA; ProDerm
, UDL Laboratories Inc.,

IL, USA; Renoskin
, Symatese, Ivry-le-Temple,
France; Matriderm
2 mm, Ideal Medical Solu-

tions, Wallington, UK; and Hyalomatrix
PA,
Addmedica, Paris, France) to heal full-thickness
wounds created in a porcine model [58]. The
study was conducted in a two-step procedure
through the implantation of the skin substitutes
in the wound, followed by the reconstruction of
the epidermis using an autologous split-thickness
skin graft or cultured epithelial autograft (after
21 days). Results indicated signifcant differ-
ences between the skin substitutes in terms of
dermis incorporation and early wound contrac-
tion. However, no signifcant differences were
noted in the long-term wound retraction. In
respect to long-term macroscopic and micro-
scopic scar qualities, no signifcant differences
were observed between the wounds treated with
the dermal substitutes and the control group.
Dermoepidermal substitutes
Constructs that mimic epidermal and der-
mal layers of the skin are the most advanced
substitutes currently available for clinical use.
These constructs, consisting of keratinocytes
and fbroblasts incorporated into constructs to
form a temporary cover [2], have the potential to
promote the regeneration of dermal and epider-
mal layers, resembling the skin structure. The
main limitations of the dermoepidermal substi-
tutes are the high production costs and failure
to close the wound permanently, due to rejection
of allogeneic cells [2].
Mazlyzam et al. developed an equivalent
bilayer human skin substitute consisting of
human plasma-derived fbrin, fbroblasts and
keratino cytes [66]. For the fabrication of the
bilayer construct, fbroblast cells were embed-
ded into a human fbrin matrix (dermal layer),
and subsequently a layer of human keratino-
cytes within human fbrin (epidermal layer) was
deposited on the top of the construct. Substi-
tutes were implanted at the dorsal part of athy-
mic mice for 4 weeks to evaluate their ability to
promote skin regeneration. Results showed the
formation of a continuous epidermaldermal
junction between the stratifed epidermal and
dermal layers, mimicking the organization of
native human skin.
Although widely recognized that full-thick-
ness wounds more than 1 cm in diameter need
skin grafting to promote healing and prevent the
formation of excessive scarring, the skin grafts
Table 3. Commercially available skin substitutes.
Substitute Product Graft type Cell source Manufacturer
Epidermal
substitutes
Epicel
Cell based Autologous keratinocytes Genzyme Biosurgery (MA, USA)

CellSpray
Cell based Autologous keratinocytes Avita Medical (Perth, Australia)

Myskin Scaffold containing cells Autologous keratinocytes CellTran Ltd (Shefeld, UK)
Laserskin
Scaffold containing cells Autologous keratinocytes Fidia Advanced Biopolymers

(Abano Terme, Italy)
ReCell
Autologous epidermal cell suspension Autologous keratinocytes Avita Medical

Dermal
substitutes
Integra
Cell free Integra NeuroSciences (NJ, USA)

AlloDerm
Cell free LifeCell Corp. (NJ, USA)

Hyalomatrix PA
Cell free Fidia Advanced Biopolymers

Dermagraft
Scaffold containing cells Neonatal allogeneic

broblasts
Advanced BioHealing (CT, USA)
TransCyte
Scaffold containing cells Neonatal allogeneic

broblasts
Advanced BioHealing
Hyalograft 3D Scaffold containing cells Autologous broblasts Fidia Advanced Biopolymers
Dermoepidermal
substitutes
OrCel
Natural-based scaffold containing cells Allogeneic keratinocytes

and broblasts
Ortec International (GA, USA)
Apligraf
Natural-based scaffold containing cells Allogeneic keratinocytes

and broblasts
Organogenesis Inc. (MA, USA)
PolyActive
Synthetic scaffold containing cells Autologous keratinocytes

and broblasts
HC Implants BV (Leiden,
The Netherlands)
currently available are not capable of pro moting
the regeneration of these lesions [2]. Tissue-
engineered skin substitutes are still characterized
by both poor vascularization and the impossi-
bility of reproducing skin appendages such as
hair follicles, sebaceous glands and sweat glands
[2,3,7]. To induce the formation of skin append-
ages, recent works are exploiting the culture of
specifc cell lines (e.g., Schwann cells, hair fol-
licle cells and melanocytes) within scaffolds.
Sriwiriyanont et al. used an engineered skin
substitute model composed of collagenglycos-
aminoglycan, containing murine dermal papilla
cells expressing green fuorescent protein, human
dermal papilla cells and/or human fbroblasts,
to investigate the hair regeneration [67]. In this
work, chimeric hair follicles were successfully
generated in the engineered skin substitute con-
taining combinations of murine dermal papilla
cells and unmodifed or genetically modifed
human epidermal keratinocytes overexpressing
stabilized b-catenin. However, the formed chi-
meric hair follicles were anatomically defcient.
Several other works demon strate the potential of
tissue-engineered skin substitutes for skin regen-
eration [6668], although further clinical evidence
is necessary to assure its clinical effciency and
market costeffectiveness.
Although the feld of skin engineering has
delivered a limited number of products to the
market, there have been several product fail-
ures. Dermoepidermal substitutes Orcel
(Ortec
International, GA, USA) and PolyActive
(HC
Implants BV, Leiden, The Netherlands) have
both failed, and dermal substitute Transcyte

(Advanced BioHealing Inc., CA, USA) has
failed to penetrate the market. These failures
have resulted from several factors, including
poor performance and a prohibitive cost, result-
ing in failed costeffectiveness for medical
reimbursement.
Advanced skin regeneration
strategies
Advanced skin regeneration strategies offer an
effective, permanent and viable alternative to
solve the major drawbacks of currently avail-
able skin grafts. These strategies combine bio-
materials, cells, growth factors and advanced
biomanu facturing techniques for the fabrication
of constructs that mimic skin anatomy, and pro-
mote the regeneration of healthy and vascular-
ized tissues. Two fundamental strategies can be
considered, based on bottom-up or top-down
approaches [48,6973]. These two approaches are
adaptable to computer control, allowing the
realization of several operations in an automatic
way, such as the deposition of cells aggregates,
scaffold fabrication and cell seeding. Their main
difference consists of the use of a scaffold as a
supporting material.
The bottom-up approach employs different
techniques for creating modular tissues that
are then assembled into engineered tissues with
specifc microarchitectural features [74]. Tissue
modules can be created through self-assembled
aggregation, microfabrication of cell-laden
hydrogels, fabrication of cell sheets or direct
printing. The ability of cell aggregates to fuse is
based on the concept of tissue fuidity, according
to which embryonic tissues can be considered as
liquids [75]. The major drawback of this approach
is that some cell types are unable to produce
sufficient ECM, migrate or form cellcell
junctions [71].
The top-down or scaffold-based approach is
based on the use of a temporary scaffold that
provides a substrate for implanted cells and a
physical support to organize the formation of
the new tissue [48,69,76]. In this approach, trans-
planted cells adhere to the scaffold, proliferate,
secrete their own ECM and stimulate new tissue
formation.
Bottom-up approaches for skin
regeneration & repair
Bottom-up approaches are based on the use of
cells or cell aggregates to produce tissue-engi-
neered constructs without the use of scaffolds
as supporting matrices [48,7779]. Usually, these
approaches comprise three key elements: a bio-
ink containing the cell suspension to be printed,
a biopaper, which provides a temporary support
for the deposited bio-inks, and a bioprinter or
robotic dispenser [79,80].
Bottom-up approaches have the potential to
improve the vascularization of the 3D constructs
through the fabrication of intraorgan vascular
trees to perfuse the constructs and ensure their
viability [81,82]. Additionally, they also allow
the deposition of multiple cell types with 3D
organization [48,83], which is relevant for skin
regeneration due to its inherent multilayered
structure.
Different laser-assisted techniques, such as
laser-guided direct writing [84,85] and modifed
laser-induced forward transfer [86,87], or jet-based
processes, such as inkjet printing systems [88,89]
and microdispensing techniques [90,91], have
been developed to print different biomaterials,
cells and growth factors with high precision and
reproducibility.
Modified laser-induced forward transfer
processes comprise two main techniques: the
matrix-assisted pulsed-laser evaporation direct
write or matrix-assisted pulsed-laser direct write,
and the biological laser printing or absorbing
film-assisted laser-induced forward transfer
[89,92,93]. Both processes use a pulsed focused
beam to print cells from a transfer layer or res-
ervoir onto a substrate with micron-scale resolu-
tion. The cell printing can be performed through
the deposition of cells onto an ECM-like layer
with variable thickness (substrate) or by printing
bio-inks consisting of cells embedded in ECM-
like printable bio materials such as hydrogels [87].
The main difference between them is the num-
ber of layers of the target. Biological laser print-
ing comprises one additional layer (conversion
layer or laser absorption layer) that eliminates the
interaction of the laser with the cells, protecting
them from the direct exposure of incident UV
light [92] and improving process reproducibility
[94]. Recently, Koch et al. used a biological laser
printing process to produce 3D skin grafts, con-
sisting of fbroblasts and keratinocytes embed-
ded in collagen (FIGURE 3) [95]. After 10 days of
culture, cells proliferated well and remained via-
ble without mixing. The formation of skin tissue
was confrmed by the formation of basal lamina
between the fbroblasts and keratinocytes, and
the existence of intercellular adherens junctions
between the cells.
Top-down approaches for skin
regeneration & repair
Top-down approaches are based on the fabrica-
tion of porous, biocompatible and biodegrad-
able scaffolds, in which donor cells with or with-
out growth factors are seeded, followed by the
maturation of this 3D assembly in a bioreactor
[48,78,96]. Scaffolds can be made of natural or syn-
thetic materials, as well a combination of both,
mimicking the function of the natural ECM
present in the human body [48,77,97,98]. They are
critical elements for the regeneration of large
defects, where the isolated use of cell therapies
compromise the healing, due to the high volume
of lost ECM [46]. Ideal scaffolds for skin regen-
eration should actively guide tissue formation,
prevent scarring, and retain and deliver cells and
growth factors (TABLE 4).
Laser absorbing layer
Laser pulse
Gel with cells
Figure 3. Biological laser printing process to produce skin grafts. (A) The laser printing
process. (B) Layer-by-layer printed construct composed of broblasts (green) and keratinocytes (red).
(C & D) Histologic cryosection of seven alternating layers of red and green uorescence-labeled
keratinocytes. Each layer contains four sublayers. The construct has a height of approximately 2 mm
and a square base area of 10 10 mm. Scale bars: 500 m.
Reproduced with permission from [95].
Biomanufacturing represents a group of
unconventional fabrication techniques recently
introduced in the medical feld for the produc-
tion of biological structures for tissue engineer-
ing applications. These techniques use additive
technologies, biodegradable and biocompatible
materials, cells and growth factors, to produce
complex 3D scaffolds in a layer-by-layer pattern
from a computer-aided design model, providing
precise control over the pore size and shape, the
percentage of porosity and the pore interconnec-
tivity [48,69,99]. As some of these processes oper-
ate at room temperature, they have been used to
fabricate carriers containing cells and growth fac-
tors without signifcantly affecting cell viability
[100]. Biomanufacturing comprises a wide range
of processes such as vat photopolymerization pro-
cesses, powder bed fusion processes, extrusion-
based processes, inkjet printing processes and
electrospinning [48,69,77,100,101].
Vat photopolymerization processes produce
3D solid objects in a multilayer procedure,
through the selective photoinitiated curing reac-
tion of a liquid photosensitive material contain-
ing a low-molecular weight prepolymer, additives
and photoinitiators [48,69,102,103]. The curing reac-
tion is induced by a light source that irradiates the
liquid polymer and supplies the necessary energy
to bond a large number of small molecules, form-
ing a highly crosslinked polymeric structure
[104]. Vat photopolymerization processes have
been used to induce the alignment of NIH/3T3
mouse embryonic fbroblasts on 3D hydrogels
patterned with acryl-fbronectin [105], and for the
production of 3D constructs, enabling study of
the interactions between different cell types [106].
As a result of their resolution and precision, vat
photopolymerization processes can potentially be
used to reproduce the microenvironment of skin,
allowing for the study of the interaction between
different types of cells, such as fbroblasts, kerati-
nocytes and endothelial cells, leading towards the
development of vascularized constructs.
Several extrusion-based techniques such as 3D
fber deposition [107], precision extruding depo-
sition [108], the bioextruder [109] and the biocell
printing system [110] were developed for tissue
engineering applications. Generally, extrusion-
based processes comprise multiple reservoirs
containing different materials and cells, a move-
able nozzle with variable diameter (501000 m)
controlled by a computer-aided design/manufac-
turing system, and a platform that collects the
printed material [90,91,111114]. Lee et al. developed
a method for the fabrication of 3D skin cell layers
using a multichannel robotic cell printing system
(FIGURE 4) [91]. In this method, instead of printing
the biomaterialcell suspension into a solution
containing the crosslinking agent, the hydrogel
layers are crosslinked by using a nebulized aque-
ous sodium bicarbonate solution before and after
printing each layer. These authors fabricated 3D
multilayer cellhydrogel constructs through the
deposition of a collagen hydrogel precursor, fbro-
blasts and keratinocytes. The constructs were
printed into a planar tissue culture dish and in
a poly(dimethylsiloxane) mold containing 3D
surface contours as a wound model. Fibroblasts
and keratinocytes printed on planar surfaces pre-
sented good proliferation after 4 days of culture,
Table 4. Growth factors for skin regeneration.
Growth
factor
Function Ref.
EGF Stimulates the migration of keratinocytes, the proliferation and differentiation
of broblasts and the synthesis of granulation tissue; induces the proliferation
and differentiation of broblasts, epithelial cells and mesenchymal cells
[4,53,96,146]
IGF-1 Glycoprotein synthesized by the liver and expressed by broblasts that
stimulates the migration and proliferation of keratinocytes
[4,96]
bFGF Induces angiogenesis and formation of granulation tissue; regulates cellular
proliferation
[98,147]
KGF Mediates the proliferation and differentiation of epithelial cells; induces
keratinocyte proliferation and differentiation
[148]
PDGF Stimulates broblasts to produce extracellular matrix; stimulates proliferation
and migration of endothelial and smooth muscle cells
[4,96]
VEGF Stimulates the migration and proliferation of endothelial cells; promotes
angiogenesis and re-epithelialization during the healing process
[4,5,96]
TGF-b Most potent growth factor involved in wound healing; induces migration and
proliferation of broblasts; stimulates collagen synthesis, neovascularization
and formation of granulation tissue
[4,149,150]
while the hydrogelcell layers printed on non-
planar surfaces presented a nonhomogeneous
distribution, showing good proliferation.
Despite great advances in both the devel-
opment of tissue-engineered skin substi-
tutes and the use of either bottom-up or top-
down approaches to produce macroscale and
microscale constructs for skin regeneration,
these products do not yet replicate the multiscale
organization of skin, failing in the reproduction
of the nanoscale of ECM components. The com-
ponents of natural ECM include structural pro-
teins such as collagen and elastin, specialized
proteins such as fbronectin, fbrillin and lam-
inin, and proteoglycans [115], well organized in
the different skin layers. For instance, the basal
lamina in the epidermis contains an ECM fber
layer composed of type IV collagen, laminin,
fbronectin and heparin sulfate proteoglycan
with a well-defned micro topography and nano-
topography, while the dermis comprises a 3D
fbrillar network with diameters in a nanoscale
range (30130 nm) that provides structural
integrity and mechanical strength to the skin
[116119]. In order to promote the regeneration
of skin with biological and mechanical prop-
erties similar to the healthy skin, constructs
should be able to reproduce the microscale and
nanoscale organization of natural ECM com-
ponents, providing an optimal environment for
cell attachment, proliferation and differentiation
[120]. These constructs should also enable the
10
9
8
7
6
5
4
3
2
1
Collagen
FBs KCs
Two layers of collagen
Embedded KCs
Six layers of collagen
Embedded FBs
Two layers of collagen
Figure 4. Bioprinting system and fabrication procedure of 3D hydrogel constructs for skin
regeneration. (A) The multichannel robotic cell printing system: (1) syringes containing cell
suspensions and hydrogel precursors, (2) array of four channel dispensers, (3) target substrate,
(4) horizontal stage, (5) vertical stage, (6) range nder, (7) vertical stage heater/cooler and
(8) optional independent heating/cooling unit for the dispenser. (B) Fabrication procedure of
multilayer cellhydrogel constructs.
FB: Fibroblast; KC: Keritanocyte.
incorporation/attachment of biological signal-
ing molecules, such as growth factors and cell
adhesive peptides, to stimulate cellular activity
[121,122]. Several strategies have been explored to
produce constructs at the nanoscale, including
self-assembly, electrospinning, template synthe-
sis and phase separation [116,123]. Owing to its
operational simplicity, versatility, wide range
of processable materials and ability to produce
nanofbers resembling the collagen structure of
the ECM, there is a great interest on the use
of electrospinning for fabricating nanoscale
structures for skin regeneration.
Electrospinning is the most relevant electro-
chemical process to produce nanoscale meshes
for tissue engineering [10,69,101,124,125]. Nanofbers
with diameters ranging from a few nanometers
to several micrometers can be produced using
either an electrostatically driven jet of a polymer
solution (solution electrospinning) or a polymer
melt (melt electrospinning) [69,101,126128]. The
basic requirements of an electrospinning appa-
ratus are illustrated in FIGURE 5A, including a capil-
lary tube with a needle or pipette, a high-power
voltage supply, electrical wires connecting the
high-power voltage supply to both the capillary
tube, and the collector or target. The collector
can be moved in the vertical direction and in
the xy plane, enabling electrospinning to be an
additive technology.
Nanopatterned electrospun fiber meshes
are very attractive for skin regeneration due to
their high surface-to-volume ratio, porosity and
structural similarity to the meshwork of ECM
fbers of the dermis [127,129,130]. As a result of the
high surface area ratio, these constructs also pro-
mote better cellular interaction and improved
vascularization, while the porosity allows the
drainage of exudate and oxygen permeation
[10,119,124]. Additionally, the dimension of the
electrospun nanofbers are in the range of the
structural components of natural ECM and the
molecules involved in the wound healing process
(1100 nm) [4,120]. The main limitations are the
poor mechanical properties, nonuniform thick-
ness distribution and poor integrity [124]. A wide
range of electro spun natural materials [125,131133],
synthetic materials [22,127,120,134] and combina-
tions of both material types [1,129,130,135,136] have
been used to produce electrospun nanofbers
meshes for skin regeneration applications.
For the regeneration of dermal wounds,
Chong et al. used the electrospinning technique
to produce poly(caprolactone)/gelatin nano-
fbrous meshes on top of a commercial poly-
urethane wound dressing (Tegaderm; 3M
Healthcare, MN, USA) [136]. Human dermal
fbroblasts cultured in these constructs for 7 days
showed good proliferation rates. Yang et al. pro-
duced poly(ethylene glycol)poly(d,l-lactide)
V
Emulsification
High-voltage generator
Collector
High-voltage
generator
Emulsion Water phase
Emulsifier
Oil phase
Syringe needle
Capillary tube
Needle
Collector
High-voltage
power supply
Nanomedicine Future Science Group (2013)
Figure 5. Electrospinning processes to produce ber meshes for skin regeneration. (A) Solution electrospinning process and
(B) emulsion electrospinning process.
fbers with a coresheath structure containing
bFGF through an emulsion electro spinning
process (FIGURE 5B) [127]. The in vitro deliv-
ery studies showed a gradual release of the
growth factor for 4 weeks. The constructs were
also seeded with mouse embryo fbroblasts,
which showed good adhesion, proliferation
and ECM production. Once implanted into
dorsal wounds created on diabetic rats, the
bFGF/poly(ethylene glycol)poly(d,l-lactide)
meshes promoted a faster healing process with
complete re-epithelialization and regeneration
of skin appendages such as hair and sebum. Jin
et al. produced poly(l-lactic acid)-co-poly(e-
capro lactone) nanofbrous scaffolds, with and
without collagen, to investigate the differen-
tiation of bone marrow-derived mesenchymal
stem cells into the epidermal lineage (keratino-
cytes) [135]. Better results were observed in the
case of poly(l-lactic acid)/poly(caprolactone)
constructs with collagen.
Electrospun nanofber meshes can also be
used as drug delivery systems for the release of
bioactive molecules, including natural or syn-
thetic drugs and growth factors [122,127,137,138].
Shalumon et al . prepared sodium algi-
nate/poly(vinyl alcohol) meshes containing
different concentrations of zinc oxide (ZnO)
nanoparticles as an antibacterial agent [137].
Mouse fbroblasts cultured on meshes contain-
ing 0.5 and 1.0 wt% ZnO nanoparticles showed
good adhesion and spreading. For high concen-
trations of ZnO nanoparticles (2 and 5 wt %),
a decrease in cell spreading was observed as a
result of the toxicity effect of these particles.
The antibacterial activity of the nanofbers was
evaluated through diffusion disc tests, using
Staphylococcus aureus and Escherichia coli. These
results confrmed the antibacterial activity of the
nanofbers, which was improved by increasing
the concentration of ZnO nanoparticles.
Despite the growing interest on the use of
nanoscale structures for skin regeneration, the
medical effectiveness of these materials is not
yet confrmed, and only a few studies focus on
their in vivo evaluation [44,127,139].
In situ biofabrication of skin
substitutes
The development of in situ biomanufactur-
ing strategies, such as in situ skin fabrication,
represents one of the major challenges in tissue
engineering [69,77,140143]. Contrary to the tradi-
tional biofabrication approaches, in which con-
structs are produced based on predesigned mod-
els and then cultured in in vitro conditions for
subsequent implantation into the patient, in situ
biofabrication involves the fabrication of substi-
tutes for tissue repair and regeneration directly
in the lesion of the patient. In this case, bio-
fabrication systems are combined with real-time
imaging techniques and path-planning devices,
enabling the controlled deposition of biomateri-
als with or without encapsulated cells into the
lesion site. In situ biofabrication has great poten-
tial for clinical applications, due to its minimally
invasive nature, the possibility of eliminating
the need for postprocessing operations, ability
to fabricate patient-specifc biological substitutes
and reduced intervention time. Theoretically,
this new concept can be applied for the regen-
eration of different tissues, but recent works are
only focused on osteochondral [141], bone [142]
and skin [140] defects. Owing to its layered struc-
ture and multiple cell composition, the regenera-
tion of healthy and vascularized skin remains a
huge challenge. The current strategies for skin
regeneration still present major pitfalls such as
inadequate vascularization, poor adherence to
the wound bed, ineffcient elasticity, inability to
reproduce hair follicles, sweat, sebaceous glands
and pigmentation, the possibility of rejection
and high manufacturing costs. Some of these
limitations could be addressed by the develop-
ment of an in situ biofabrication device, enabling
the direct deposition of cell-laden constructs in a
layer-by-layer fashion, in this way mimicking the
structural and compositional organization of the
skin tissue. The printed skin substitute covers
the wound to prevent contamination, provides
adequate moisture to avoid dehydration, gives
immediate and effective relief to patients, and
induces skin regeneration, due to its functional
composition and cell combination. Recently,
Sofokleous et al. developed a portable electro-
hydrodynamic multineedle system and proposed
its use for the in situ fabrication of polymeric
meshes in the damaged skin site of a patient
(FIGURE 6) [143]. These meshes can be coated, thus
encapsulating drugs, and therapeutic and clot-
ting agents. Poly(lactic-co-glycolic) acid meshes
with an average diameter of 2.3 0.5 m were
reported. The use of a coaxial needle device
allowed the fabrication of bimaterial fbers (aver-
age diameter of 3.9 0.7 m), with a core struc-
ture made by polymethysilsesquioxane coated
with a thin layer of poly(lactic-co-glycolic) acid.
Binder et al. developed a portable inkjet deliv-
ery system for the in situ printing of skin cells into
the lesion site [140]. The potential of the system
to induce the skin regeneration was evaluated
through the printing of human keratinocytes
and fbroblasts into full-thickness skin lesions
(3 2.5 cm) created on mice. Results showed the
complete closure of the wound after 3 weeks, as
well the formation of skin with similar properties
to the healthy skin. Histological analysis revealed
that the new skin contained organized dermal
collagen and a fully formed epidermis.
Despite the tremendous potential of in situ
biofabrication of skin substitutes, this con-
cept is still in its infancy. To be effective, great
challenges need to be addressed: the integra-
tion between biomanufacturing technologies
and imaging techniques; the development of
dedicated software to control the deposition of
different biomaterials and cells, and planning
the surgical intervention; and the development
of advanced multi functional biomaterials,
capable of entrapping different cells types and
maintaining their viability and functionality.
Currently, several strategies are being exp lored
to promote skin regeneration. The most com-
monly used are autografts and allografts, wound
dressings and tissue-engineered skin substitutes.
Although these strategies result in products with
distinct characteristics, they have been used in
combination to improve the healing process. For
example, wound dressings, either traditional or
modern and tissue-engineered skin can be
applied on top of autografts and allografts, pro-
viding a better adhesion to the defect site and
protecting it from fuid loss and contamination
[144,145]. Another approach involves the fabrica-
tion of electrospun meshes on top of commercial
wound dressings in order to obtain a nanofbrous
scaffold with morphological and architectural
features similar to the natural ECM in the skin
[136]. Despite the great potential of the in situ
biofabrication of skin substitutes, this strategy
may also require the combined use of dressings
or cell-free tissue-engineered skin substitutes
to ensure initial adhesion to wound bed and
prevent biomaterial (hydrogel) dehydration.
Conclusion & future perspective
Despite recent advances in the development of
biomaterials and fabrication strategies for skin
repair and regeneration, their clinical use still
requires several drawbacks to be addressed.
Numerous wound-care products were developed,
such as lotions, dressings and tissue-engineered
skin substitutes, and are currently commer-
cialized. Until now, the gold standard for skin
regeneration still relies on the use of autografts
and allografts, which present signifcant limita-
tions as previously discussed. Advanced strate-
gies, combining additive biomanufacturing
processes, bio materials, cells and growth factors,
have emerged as a group of techniques with
high potential for skin regeneration applica-
tions. These techniques allow printing of skin
substitutes in a precise and automated manner,
enabling the production of skin replacements
with properties that resemble both the structure
and the function of native skin. These techniques
enable the development of bottom-up approaches
to fabricate 3D constructs that mimic the orga-
nization of healthy skin and can be used for
the regeneration of full-thickness wounds, for
which an effcient dermoepidermal substitute is
not available.
In order to achieve effective and clinically rele-
vant skin regeneration, relevant challenges to be
addressed in the future include:
The insuffcient vascularization of the regener-
ated skin. This requires the development of
sophisticated printing systems to produce
multi hydrogel constructs with multiple cell
types (e.g., stem cells and endothelial cells) and
the controlled release of angiogenic growth
factors;
Development of advanced in situ crosslinking
multifunctional hydrogels, mimicking the bio-
signaling and mechanical properties of the
skin, and allowing the incorporation of
multiple cell types;
The inability of currently available skin grafts
to reproduce all skin structures, such as hair and
glands. This requires a deeper understanding of
Figure 6. Electrohydrodynamic multineedle system printing a polymeric
mesh directly into the lesion site on the patient.
Executive summary
Skin damage & wound healing
Skin is a multilayer organ with a natural ability to promote regeneration after injury.
The healing of a wound comprises ve integrated and overlapping phases, namely hemostasis, inammation, migration, proliferation
and maturation.
The healing process, involving the interaction between cells, growth factors and cytokines, is highly dependent on the extension of
the lesion and the number of affected layers.
Treatment of skin lesions
The gold standard for skin regeneration remains the use of autografts and allografts, with important limitations such as patient
morbidity, risk of immunogenic rejection and disease transmission.
Solutions, creams and ointments are widely used due to their ability to provide disinfection, cleaning and debridement.
Wound dressings are used as an alternative to the autografts and allografts. Although proven clinically effective, these products
have low adherence to the wound bed and an inability to reproduce skin appendages and promote regeneration of the lost tissue, in
particular the dermis.
Tissue-engineered skin substitutes
Principles of tissue engineering are used in the development of cellular and acellular epidermal, dermal and dermoepidermal skin
substitutes.
Tissue-engineered skin substitutes are designed by combining natural /synthetic polymers with cells, enabling the regeneration of the
different skin layers.
The main limitations of these products rely on the inability to regenerate full-thickness wounds, poor vascularization and the
difculty of reproducing skin appendages.
Further research is necessary to investigate the clinical efciency and market costeffectiveness of tissue-engineered skin
substitutes.
Advanced skin regeneration strategies
Advanced skin regeneration strategies provide a clinical route for the development of biological substitutes for skin regeneration, by
combining biomaterials, cells, growth factors and biomanufacturing techniques.
Bottom-up and top-down approaches are used to produce well-organized 3D multilayer skin substitutes containing broblasts and
keratinocytes.
Nanoscale structures are very attractive for skin regeneration, due to their high surface/volume ratio, porosity, improved
vascularization and dimensions in the range of the structural components of the natural extracellular matrix.
There is a need for clinical trials to investigate the medical effectiveness of these materials.
In situ biofabrication of skin substitutes
In situ biofabrication is a novel concept involving the fabrication of skin substitutes directly in the patient, using a bioprinting device.
It comprises the direct deposition of cell-laden constructs in a layer-by-layer fashion, mimicking the structural and compositional
organization of the skin tissue.
This strategy aims to regenerate healthy and vascular skin, providing immediate relief to the patient.
skin biology and the use of more complex cell-
ular approaches, including the use of genetically
engineered cells;
Applying nano-, micro- and macro-fabrication
technologies for enhancing effcacy and preci-
sion and to reproduce the natural hierarchy
that characterizes the skin;
Increasing the level of automation and indus-
trialization, as traditional laboratory pro-
cesses are often characterized by a high degree
of manual handling operations;
Enhancing multidisciplinarity, linking clini-
cians, biologists and engineers to facilitate
further developments and the clinical
translation of the products being inves tigated;
Scaling up the fabrication strategies under
development toward clinical application.
Financial & competing interests disclosure
The authors are supported by the Strategic Projects
PEST-OE/EME/UI4044/2011 and PEST-C/SAU/
LA0002/2011, and financed by European Regional
Development Fund thr ough the Programa Operacional
Factores de Competitiv idade COMPETE and by
Portuguese funds through Fundao para a Cincia e a
Tecnologia. The authors have no other relevant afliations
or nancial involvement with any organization or entity
with a nancial interest in or nancial conict with the
subject matter or materials discussed in the manuscript apart
from those disclosed.
No writing assistance was utilized in the production of
this manuscript.
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XP; Graco, MN,

Conva Tec (NJ, USA)

Johnson & Johnson (NJ, USA)

Smith & Nephew Healthcare

Smith & Nephew Healthcare

Smith & Nephew Healthcare

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Smith & Nephew Healthcare

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2 mm, Ideal Medical Solu-

Cell based Autologous keratinocytes Genzyme Biosurgery (MA, USA)

Cell based Autologous keratinocytes Avita Medical (Perth, Australia)

Scaffold containing cells Autologous keratinocytes Fidia Advanced Biopolymers

Autologous epidermal cell suspension Autologous keratinocytes Avita Medical

Cell free Integra NeuroSciences (NJ, USA)

Cell free LifeCell Corp. (NJ, USA)

Cell free Fidia Advanced Biopolymers

Scaffold containing cells Neonatal allogeneic

Scaffold containing cells Neonatal allogeneic

Natural-based scaffold containing cells Allogeneic keratinocytes

Natural-based scaffold containing cells Allogeneic keratinocytes

Synthetic scaffold containing cells Autologous keratinocytes

Burn Study Group.

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