Biomaterials 216 (2019) 119267

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Review

Emerging and innovative approaches for wound healing and skin T

regeneration: Current status and advances
Dimple Chouhana, Namit Deya, Nandana Bhardwajb, Biman B. Mandala,*
a
Biomaterial and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India
b
Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Guwahati, Guwahati, 781125, Assam, India

A R T I C LE I N FO A B S T R A C T

Keywords: Current advances in skin tissue engineering and wound healing augur well for the development of split or full
Wound healing thickness skin substitutes to recapitulating the native functional skin. These engineered skin substitutes have
Skin regeneration fared successfully in recent years with exploration of various emerging technologies. As a result, recent clinical
Vascularization practice has been highly evolved incorporating various engineered skin substitutes as an adjunct to accelerate
Immunomodulation
healing and improvement of quality of life in long-term. This review seeks to bring the researchers through
3D bioprinting
various emerging and innovative approaches being developed and utilized for accelerating wound healing and
Tissue engineering
skin regeneration. In order to attempt this, we reviewed various design considerations for skin repair and impact
of several smart technologies viz., in situ 3D printing, portable bioprinters, electrosprayers and in situ forming
hydrogels that have significantly improved wound healing and skin therapeutics. Furthermore, numerous cel-
lular therapies such as effect of immunomodulation, stromal vascular fraction treatments, micro RNA (miRNA)
and small interfering RNA (siRNA) based skin therapeutics have been thoroughly discussed. Finally, an update of
clinical trials along with critical analysis of properties and benefits of different emerging technologies in healing
certain types of wounds, prime challenges and future prospects in skin tissue engineering are discussed.

1. Introduction to its next phase of proliferation. The proliferation phase comprises of

Skin wounds represent a major healthcare problem owing to an
increasing number of trauma and pathophysiological conditions.
Normal wound healing process includes a very well-orchestrated and
regulated process consisting of series of events such as haemostasis,
inflammation, proliferation and extracellular matrix (ECM) remodelling
[1]. The four healing phases involve interactions between various types
of cells, bioactive factors and a supporting platform, which is usually
the natural ECM secreted by cells [1]. This normal healing process gets
severely dysregulated in case of pathophysiological conditions. Large
trauma wounds due to burns or accidents result in loss of majority of
skin tissue and thus fail to heal [2]. Majority of non-healing wounds are
associated with accidents or disease conditions like diabetes. Such
conditions hamper the normal healing pattern of skin tissue and has
been a major social and financial burden since decades.
The wound healing cascade begins with hemostasis and inflamma-
tion. This stage involves recruitment of blood platelets and immune
cells to control loss of blood and clearing of pathogens [3]. The initially
recruited immune cells play a major role in secreting chemokines and
growth factors, which attract cells and thereby lead the healing process
numerous events like granulation tissue development (formation of
provisional ECM), angiogenesis (formation of blood vessels) and re-
epithelialization (formation of epidermal skin layer), thereby resulting
in wound contraction. This particular phase is regulated through
crosstalk between various cells, mainly macrophages, fibroblasts, en-
dothelial cells and keratinocytes. The final phase constitutes remodel-
ling event, in which the previously formed matrix is slowly morphed
towards forming either a functional skin or a semi/non-functional scar
tissue [3].
In case of non-healing wounds, for example diabetic wounds, the
healing process gets obstructed at the very initial phase and gradually
turns to be chronic due to failure in healing [4]. Depending on the type
of chronic wound (like pressure sores, venous ulcers or diabetic
wounds), the process may get hindered during any of the four stages. In
case of large burn wounds, natural healing is affected due to significant
loss of skin tissue or failure to develop a provisional ECM matrix as a
result of tissue necrosis [2,5]. Therefore, clinical interventions are es-
sential for healing such wounds. The most successful strategy is to
provide an artificial matrix in the form of wound dressing or skin graft,
which serves as a provisional matrix, and thereby aid in the healing

*
Corresponding author. Department of Biosciences & Bioengineering Indian Institute of Technology Guwahati, India.
E-mail addresses: biman.mandal@iitg.ac.in, mandal.biman@gmail.com (B.B. Mandal).

https://doi.org/10.1016/j.biomaterials.2019.119267
Received 26 January 2019; Received in revised form 25 May 2019; Accepted 8 June 2019
Available online 13 June 2019
0142-9612/ © 2019 Elsevier Ltd. All rights reserved.

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D. Chouhan, et al. Biomaterials 216 (2019) 119267

Fig. 1. (I) Schematic representation depicting concept of wound healing process assisted by a dressing material or skin graft. Wound healing process comprises of
three major phases and their key events as described in the diagram. (II) The functions of bioactive scaffolds or constructs are depicted in the illustration describing
how such matrices may trigger any of the key events of wound healing process for skin regeneration applications.

process [6]. Researchers have been developing numerous types of
wound dressing materials or skin substitutes and trying to mimic the
healing-wound or skin microenvironment through cutting edge tech-
nologies over the years [6]. The natural wound milieu consists of ECM
platform having cell adhesive sites, instructive biochemical cues,
growth factors and various types of cells [7]. Mimicking the whole
microenvironment is very challenging and therefore most tissue en-
gineers focus on the structural framework that is of prime importance in
healing.
Significant improvement in the development of bioactive templates
and pro-regenerative matrices has been going on because such matrices
trigger the key events of natural healing cascade and help in the re-
generation of functional skin tissue. Overall, the platform of a wound
dressing or skin graft assists in healing the wounds at a faster rate in
addition to the inherent healing process as illustrated in the schematic
image (Fig. 1). Furthermore, depending on the type of functionalization
of matrix, a specific stage or event of healing process can be stimulated
by certain biomolecules like growth factors, cytokines, chemokines and
cell adhesive peptides [8–10]. The present review will introduce the
readers towards recent and emerging technologies being developed for
the improvement of skin tissue engineering and wound healing. Herein,
we have briefly reviewed scaffold fabrication approaches to fabricate
tissue-engineered skin and bioactive wound dressings. In this review,
we have started with the discussion about different scaffold design
considerations, functionalization, immunomodulation and vasculariza-
tion strategies. Furthermore, various smart technologies such as por-
table skin repair devices and spraying technologies developed in recent
years in this field have been discussed and reviewed.
Artificial construct not only requires ideal surface, material, struc-
tural and mechanical properties but also cell instructive and cell con-
ducive cues to guide the cells for efficient tissue regeneration [11–13].
The bioengineered matrix should be non-immunogenic, biocompatible,
bio-resorbable, and porous in structure with sufficient mechanical
strength [1,11,14]. Numerous natural biopolymers have been explored
in this area within the last 30 years like cellulose, collagen, chitosan,
fibrin, gelatin, silk fibroin, silk sericin, hyaluronic acid and many others
[15–18]. In addition, synthetic polymers like poly(vinyl alcohol) (PVA),
poly(glycolic acid) (PGA), poly(lactic acid) (PLA), polycaprolactone
(PCL), nylon and silicone have also significantly contributed in the
development of next generation wound dressings [19]. Apart from
providing a physical support, the platform of artificial constructs offers
barrier properties, moisture retention properties, and aids in tissue in-
growth at the wound site by acting as an artificial provisional matrix.
Although various types of commercially available skin grafts are well
described in numerous literature, a rational approach towards addres-
sing the recent emerging techniques is of interest in further adopting
efficient wound care therapeutics in future. The present study aims to
highlight the progress of last few years in developing various types of
tissue-engineered constructs with a special focus on vascularized skin
grafts and immunomodulation approach.
Technological advancements in the last 2-3 decades have resulted in
many Food and Drug Administration (FDA) approved bioengineered
scaffolds and wound dressings, which are commercially available for
wound healing applications [20]. This has increased the survival rates
of burn and trauma patients. However, ‘cost to heal factor’ is still a
major concern because most of the advancements in wound care

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D. Chouhan, et al.
therapeutics are highly expensive. Burn injuries and chronic wounds
affect more than 11 million and 6.5 million people respectively every
year; and the estimated healthcare cost may go beyond $14 billion
considering the present situation [21]. Bridging the gap between
number of patients and available wound care regimens is the ultimate
aim of most researchers in order to treat millions of affected patients.
This has motivated researchers to develop different cost-effective
wound care solutions since the last decade [10,22,23]. Here, we criti-
cally appraise the research efforts concerned with the cutting-edge
technologies for wound repair and regeneration. We would also touch
upon the most recent advances in skin tissue engineering like devel-
opment of pre-vascularized grafts, immunomodulatory constructs and
siRNA/miRNA based therapeutics, which represent the next generation
wound care technology. The review also provides an updated overview
on improvisation on the autologous cell delivery with special dedicated
discussions on portable bioprinters, spray devices and in situ hydrogels
for immediate first-aid. Finally, we aim to provide an insight on the
future directions and perspective of the improved wound healing
technology and their possible impact in clinical practice.
2. Recent innovative strategies in wound healing and skin
regeneration
Skin tissue engineering is a complex process involving appropriate
choice of biomaterial, cell selection and designing of suitable platform
in order to mimic structural and functional properties of skin [6,19]. In
last five years, fabrication of a pro-regenerative construct has been a
major focus of research in wound repair and regeneration [24–26]. The
principal aim is to develop scaffolds containing cell instructive cues to
restore the damaged skin in a regenerative manner as depicted in Fig. 1.
Cells not only require a platform to reside and proliferate, but also need
instructive cues in the form of a suitable niche to grow in a regenerative
microenvironment [3,27]. These cues are fine orchestra of signalling
molecules along with physical properties of scaffolds. Functionalization
of scaffolds with bioactive molecules is a commonly explored approach
wherein growth factors, antimicrobial molecules, bioactive nano-
particles or liposomes, cell binding peptides and other specific additives
are used for fostering chemical signalling in the construct [10,28–32].
There are two main types of matrix functionalization approaches – 1)
functionalize the construct that acts as a cargo to slowly deliver the
bioactive compounds at wound site and 2) permanent binding of
bioactive molecule or its functional mimic that stays with the construct
and stimulate cells. Both the approaches help in accelerating the wound
repair process. Slow and sustained release of growth factors or other
molecules from scaffolds has been mostly exploited in maintaining cell-
instructive cues, which in turn helps in wound repair and skin re-
generation processes [9,21,33]. The later approach has also been suc-
cessful in maintaining permanent bioactivity in a wound dressing ma-
terial [10].
Functionalization of wound dressing has been performed targeting
all the four phases of healing using specific bioactive molecules or
materials. For example, addition of antimicrobial peptides or antibiotic
drugs to provide antimicrobial property for prevention and clearance of
bacterial colonization during the very initial phase of wounds.
Incorporation of growth factors like epidermal growth factor (EGF),
basic fibroblast growth factor (bFGF), vascular endothelial growth
factor (VEGF) or platelet derived growth factor (PDGF) to stimulate
growth, proliferation and migration of various cells during the middle
phase of healing. Addition of immunomodulatory molecules like IL-4 in
the construct holds potential to modulate the macrophage functions for
wound repair and regeneration throughout the process of healing.
Finally, tuning the deposition of ECM through cell interacting materials
or molecules in the last phase, i.e. remodelling stage of healing. In this
context, exploration of novel biomaterials has led to new discoveries by
understanding the cell-material interactions. Macrophage reprogram-
ming by modified polymer properties is an intense area of research now
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Biomaterials 216 (2019) 119267
a days [34–36]. ECM remodelling can be tuned by using specific ma-
terials that interact with fibroblasts and thereby regulate ECM deposi-
tion and orientation of deposited collagen fibers. For example, poly-L-
arginine based materials possess potential to regulate synthesis of col-
lagen and their orientation via cell-material interactions [37]. Bioma-
terials like silk fibroin (SF) possess inherent wound healing properties
via NF-κβ signalling [38]. Silk has also been extensively used in nu-
merous drug delivery and tissue engineering applications owing to its
regenerative properties [39–44]. Some varieties of SF (non-mulberry
silk) hold extraordinary cell interacting properties to regulate the de-
position of ECM during wound repair process [24]. Therefore, nu-
merous strategies have been implied to accelerate the healing process
by triggering specific phase or stages of wound healing.
Another approach for skin regeneration therapeutics lies in the
structural engineering of scaffolds or hydrogels [19,45]. Herein, our
efforts have largely been focused on various types of matrices used in
skin tissue engineering and their properties. Skin tissue engineering has
been explored world-wide based on various construct designs like
woven/non-woven mat, nanofibrous matrices, microporous scaffolds,
bilayer or trilayer constructs, hydrogel lattices, injectable gels, micro-
spheres and three-dimensional (3D) bioprinted grafts [45–51]. Fur-
thermore, particular design of a scaffold also depicts its application for
a particular type of wound. For example, electrospun nanofibrous ma-
trices have largely been utilized in the form of wound dressing and not
as dermal replacement due to limitations in cell migration within the
construct [9,52]. Dermal replacements are mostly in the form of hy-
drogel lattice or microporous scaffolds which could be permanently
applied at the wound site either via one-step or two-step grafting sur-
gery [23,45,53]. The microporous network or hydrogel environment
helps in migration of cells within the construct and thereby aid tissue
ingrowth; hence, such constructs could be permanently implanted.
However, it should meet the essential characteristics like its integration
to the wound bed, vascularization or graft take capacity, biodegrad-
ability, immunocompatibility and most importantly its capability to
repair the wound defect while supporting the remodelling events that
would occur throughout a patient's life [15,23,26,45].
2.1. Biofabrication of artificial templates for skin regeneration
Artificial templates for skin regeneration commenced with the de-
velopment of thin cell sheets. The optimized cell culture conditions
have facilitated lab-grown cell sheets of autologous origin to treat
partial thickness skin wounds. In order to attempt improved epithelial
regeneration, thin films and cell sheets have demonstrated remarkable
healing properties [54,55]. Cultured epithelial autografts (CEA) is a cell
sheet of autologous keratinocytes cultured in vitro for superficial
wounds like 1st degree or 2nd degree burns depending on the depth of
defective skin [56]. Being motivated by the efficiency of CEA, other
types of cell sheets were also developed using bone marrow derived
stem cells (BMSCs) and adipose derived mesenchymal stem cells
(ADMSCs) [54,57]. In a recent study, cell sheet made up of ADMSCs
demonstrated scar-free healing owing to their cytokine secretions,
leading to downregulation of transforming growth factor-beta 1 (TGF-
β1) and alpha smooth muscle actin (α-SMA) in fibroblasts [54]. Such
cell sheets like CEA are highly efficient; however, its application on
deep and large burn wounds is a major limitation. To cope with this
limitation, CEA was sandwiched between meshed skin grafts and used
to cover more than 30% of the total body surface area in a clinical study
carried out on burn patients [56]. This minimized donor skin source
and resulted in highly efficient autologous grafts. Such strategies not
only improved the conventional approach of autologous grafts but
could also be utilized to heal larger wounds.
Nevertheless, full thickness wounds like 3rd degree burns require a
platform in the form of artificial skin graft to repair the condition [45].
The application of biomaterials and plethora of fabrication strategies
being utilized in tissue engineering has benefitted the field of wound
D. Chouhan, et al. Biomaterials 216 (2019) 119267

repair and regeneration. The platform on which an artificial skin is
generally constructed is termed as a scaffold and can be of any format
and any biomaterial [11]. Several natural and synthetic biopolymers
have been explored to develop functional skin grafts [6]. A detailed
literature of such biomaterials in this field has been reviewed in our
recent article [6]. In this review, we will specifically focus on the recent
progress in different types of tissue-engineered constructs and the
strategies applied in their design. Scaffold structure promotes vital
characteristics for cell recruitment in case of wound healing. Various
strategies for designing the constructs with favourable biophysical and
biochemical milieu can direct cellular behaviour and function. In a
study, a wound dressing with bilayer membrane was developed having
a pore size of 1.12 ± 0.25 μm in the outer layer and
135.91 ± 30.75 μm in inner layer. The outer layer served the purpose
of protective barrier against bacteria and inner layer guided cellular
growth leading to accelerated wound healing [50]. Similarly, a three-
layer sandwich scaffold with gradient pore size was developed in order
to closely mimic the dermal layer of skin. The sandwich scaffold with
larger pore size (166.9 μm) in both outer layers and lower pore size
(87.7 μm) in the middle layer demonstrated faster wound healing
compared with the homogenous scaffold having uniform pore size [58].
Thus, pore size has been considered as an important factor while de-
signing scaffolds for skin engineering. The guidance thus provided to
cells in the form of tailored cues also facilitates restoration of cellular
functions and induction of directed ingrowth and differentiation of
cells. In a recent study, microfluidic technique was used to generate
microsphere building blocks that created microporous annealed particle
(MAP) scaffolds for cell delivery and wound healing applications
(Fig. 2.I.) [48].
In case of wound healing, scaffold acts as a 3D provisional support
to interact with cells, guiding them spatially and temporally towards
tissue formation and regeneration. The foremost aim while designing a
tissue-engineered graft is that it should mimic the physical properties of
normal skin and thus resemble the native skin architecture [20]. From
the last few years, there has been a substantial paradigm shift in the
scaffold design considerations due to introduction of electrospinning
and 3D bioprinting technology [59,60]. Nanofibrous matrices have
been proven to be a suitable candidate for delivery of bioactive mole-
cules like growth factors, antibiotics and other small molecules [59,61].
The nanofibrous platform containing magnetic particles can be used to
stimulate cells under pulsed magnetic field as shown in our recent study
[62]. Proliferation of human dermal fibroblasts (HDF) cultured on
magnetic nanofibrous platform was found to be increased when sti-
mulated under the stimulation of magnetic actuation in comparison to
non-stimulated conditions [62]. In another study, the nanofibrous
matrix made up of SF biopolymer showed significantly higher potential
for cell recruitment and accelerated wound healing when compared
with flat thin film [61]. This marked difference was attributed to the
nano-range architecture provided by the electrospun mat, thus be-
stowing improved cellular adhesion and migration. These are excellent
cell recruiters because the nanoscale dimensions of nanofibers help in
cell binding and thus aid in their migration towards wound site [24].
However, majority of the electrospun nanofibrous mats have been ap-
plied as wound dressing material, as they lack microporous structures
and therefore cell infiltration within the construct is a major limitation
[10]. Nanofibrous mats are easy to functionalize via adsorption method
due to high surface to volume ratio. In our recent work, SF based ma-
trices were functionalized with recombinant spider silk fusion proteins
via silk-silk interaction by a simple coating technique as shown in
Fig. 2.II. [10]. The spider silk fusion proteins functionalized with cell

Fig. 2. The composite image shows various design considerations, functionalization of constructs and their cellular response for wound healing applications: (I) The
images depicts fabrication of microsphere hydrogel building blocks as injectable scaffolds using microfluidic technology for cell delivery and wound healing ap-
plications (reproduced with permission from Ref. [48]). ©2015 Springer Nature. (II) Illustration of bioactive silk substrates for skin tissue-engineering applications:
silk fibroin bulk constructs (microporous scaffold and nanofibrous mats) are top-coated with 4RepCT spider silk proteins containing RGD motifs of fibronectin (FN), a
growth factor motif (bFGF), and AMP (cationic antimicrobial peptides) (reprinted with permission from Ref. [10]). ©2018 American Chemical Society. (III) (a)
Fabrication of various silk based nanofibrous matrices using electrospinning technique showing morphology of developed mats; PVA = poly(vinyl alcohol); PVABM
= (PVA + Bombyx mori silk fibroin); PVAAA = (PVA + Antheraea assama silk fibroin); PVAPR = (PVA + Philosamia ricini silk fibroin), (b) Images of in vitro scratch
assay on cell monolayers depict effect of different bioactive molecules on the migration of fibroblasts and keratinocytes (reproduced with permission from Ref. [9]).
©
2017 John Wiley and Sons. (IV) Images of cultured matrices demonstrate morphology of HDF and HaCaT cells grown on various nanofibrous mats and live-dead
assay (reproduced with permission from Ref. [24]). ©2016 Acta Materialia Inc. Published by Elsevier Ltd.

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D. Chouhan, et al. Biomaterials 216 (2019) 119267

Fig. 3. Schematic representation of developmental stages of 3D bioprinting technique for designing a complex skin architecture in future: (I) Determining the layered
structure of skin for designing a computer aided design (CAD) program, (II) Development of a suitable bioink for skin bioprinting, (III) Designing a simple bilayer skin
construct using two types of bioinks for fabricating dermal and epidermal structures and (IV) Designing a complex skin structure containing adipocytes, blood
capillaries, dermal, epidermal cells and its assessment strategies. The diagram depicts the flow of advancement done in the field of skin tissue engineering using 3D
bioprinting technology and its future scope.

binding RGD (Arg-Gly-Asp) peptide, growth factor peptide and anti-
microbial peptide bestowed intended functions to the SF based wound
dressings [10]. In our previous work, SF based nanofibrous mats were
directly functionalized with growth factors and antimicrobial peptides
prior to electrospinning the solutions [9,24]. Various varieties of SF
protein were electrospun when blended with PVA to generate smooth
nanofibers as shown in Fig. 2.III. The study revealed cell migration
effects of various growth factors and antibacterial LL37 peptide, as
analysed by scratch assay on the monolayers of HDF and HaCaT cells
[9]. Variation in the morphology of cells could also be observed due
differences in the SF variety owing to differences in the cell-material
interactions as shown in Fig. 2.IV. [24].
The emergence of 3D bioprinting revolutionised the whole tissue
engineering field, as the technique has improved the biomimetic aspect
manifold. Significant progress has been made in the fabrication of ar-
tificial skin by 3D printing technology [49,63]. This facilitated layer by
layer precise positioning of specific cells in a controlled manner [64].
Fig. 3 illustrates the concept and development stages of 3D bioprinting
of skin starting with development of a suitable bioink, formation of
bilayer tissue and finally biofabrication of a complex skin tissue. A bi-
layer 3D bioprinted skin graft was fabricated using fibrin gel containing
human plasma, HDF cells and keratinocytes obtained from human skin
biopsies. The printed skin of 100 cm2 size was developed in less than
35 min and healing efficiency was examined in nude mice [64]. In
comparison to conventional techniques, where laboratory grown skin
grafts take relatively long time (3–4 weeks), 3D printing thus shows
huge potential in treating burn or traumatic wounds in emergency si-
tuations. A recent study demonstrated 3D bioprinting of bilayer skin
structure to produce dermal and epidermal layers with cellular
structuration as a proof of concept [49]. In another study, 3D bio-
printing was applied to fabricate skin equivalent with perfusable vas-
cular channels of human umbilical vein endothelial cells (HUVEC)
within the cultured construct [63]. The customized set up was con-
nected with an external pump and tubes for continuous nutrition supply
to the biofabricated skin-equivalent. Further improvements on skin
generation using this technology involve bioprinting approaches of skin
containing vascular cells, epidermal progenitor stem cells and mela-
nocytes to achieve generation functional skin grafts [60,64,65].
Porous constructs like microporous scaffolds have been considered
suitable for fabricating tissue engineered skin grafts, especially in case
of dermal replacements. The pore size may vary from 20 to 500 μm
depending on the biomaterial used and fabrication methods like freeze-
drying, particle leaching or others [66]. The most significant feature of
porous scaffolds is interconnected pores, which provide a 3D micro-
environment for cellular migration and ingrowth. Matrices with pores
in micro range furnish spatial distribution of cells throughout the
construct and thus coordinate numerous cell fate processes, including a
myriad of signals for angiogenesis and graft take [67,68]. Such complex
array of biophysical and biochemical signals is difficult if cells do not
have interconnectivity. We highlight the role of various types of ma-
trices recently designed to mimic the full thickness skin grafts.
Integra, also referred to as dermis regeneration template (DRT) is a
perfect example of microporous scaffold made up of collagen type I and
chondroitin 6-sulfate [5,20,69]. It has been commercially used as
dermal graft since decades owing to the skin regeneration properties. It
has an optimum pore size ranging from 20 to 125 μm allowing migra-
tion of contractile myofibroblasts cells within the scaffold, thus healing
wounds with minimum scarring [5]. In this context, it is worth

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D. Chouhan, et al. Biomaterials 216 (2019) 119267

Fig. 4. Representation of injectable system using hydrogels and scaffolds: (I)Application of in situ forming silk hydrogel for burn wound healing and (II) in vivo results
depicting comparable healing efficacy of silk fibroin (SF) with that of collagen (Col) as depicted by gross wound images and Masson's Trichrome (MT) histology
(reproduced with permission from Ref. [23]). ©2018 John Wiley and Sons. (III) Schematic diagram shows application of antimicrobial bioadhesives using tannin
inspired gelatin biomaterial for wound healing (reproduced with permission from Ref. [30]). ©2018 Acta Materialia Inc. Published by Elsevier Ltd. (IV) Illustration
shows application of microfluidics technology in the biofabrication of hollow cellulose microspheres used as injectable scaffolds (reproduced with permission from
Ref. [78]). ©2016 John Wiley and Sons.

mentioning that skin regeneration also depends on the graft thickness.
In a comparative study, scaffolds made up of collagen-chitosan blend
with different graft thickness showed better neodermal regeneration by
0.5 mm thick graft than 2 mm thick [70]. In another study, Matriderm
graft of 1 mm thickness exhibited higher cell density post implantation
when compared to Integra having 1.3 mm thickness [53]. This clearly
indicates that a lot of physical properties need to be considered while
designing porous scaffold for skin regeneration applications.
The advent of biomaterials and modern amenities to tailor scaffolds
and hydrogels has yielded attractive strategies to address the wound
healing. The architecture of natural ECM with components like collagen
or fibrin appear as a hydrated gel-like matrix with intricate fibrillar
architecture [17]. From the structural perspective, hydrogels closely
mimic the native ECM structure [71,72]. The skin extracellular mi-
croenvironment, which surrounds cells, is a highly hydrated network,
due to presence of glycosaminoglycan chains. Hydrogels are hydro-
philic lattices of polymeric network or protein fibrils interwoven within
a hydrated network due to chemical or physical crosslinking [26,73].
Collagen gel is a perfect example of such hydrated network, which has
shown great potential in developing functional living skin in vitro
[15,17]. Hydrogel made up of other biomaterials also hold excellent
potential in skin regeneration and wound healing applications
[23,45,74]. Instructive hydrogel that carries signals for scarless wound
healing through macrophage polarization has been a recent develop-
ment in the field of skin tissue engineering [26]. The constructs target
the macrophages to become regenerative type and thus control the
secretion of signalling molecules [26]. Such hydrogel matrices not only
provide a structural support to the cells for their ingrowth but also
carries information to be passed. The flow of information between cells
and the surrounding matrix is highly bidirectional and thus the ultimate
fate of healing can be tuned and designed.
Apart from the structure, mechanical stiffness of matrices also car-
ries information that could modulate cellular events like formation of
blood capillaries within the matrix. In a study, vascularization within
the construct was controlled by tuning the stiffness of fibrin matrix
[75]. Cells have a tight connection between their cytoskeleton and the
surrounding matrix. Cells sense and respond to mechanical properties
provided by the external matrix. The shape of cells and their migration
behaviour differ significantly depending on the mechanical stiffness of
the matrix. Such rational design principles have been put forward to
control cellular behaviour by tuning biomechanical and biomolecular
cues. Such technologies allow researchers to not only explore an array
of modern biomaterials but also enable tuning of its properties like
stiffness, porosity, degradability and cargo release. This encourages us
to develop advanced materials that can be more effective, patient
compliant and cost effective in comparison to the pre-existing treat-
ments.
2.2. In situ forming injectable hydrogels and bioadhesives
In the progress of strategies for wound repair and regeneration, in
situ forming hydrogels and injectable systems have largely been ex-
plored in the last 5 years. For example, thermosensitive gels and pH
responsive gels behave as solution but forms hydrogel at near-physio-
logic conditions like 37 °C and physiological pH [23,51]. This facilitates
both in situ crosslinking within the biopolymer and also with native
wound edge tissue. Another advantage of this strategy is that it is easy
to apply and does not require additional glue or sutures because gels

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D. Chouhan, et al. Biomaterials 216 (2019) 119267

Fig. 5. Composite images show smart devices for wound healing in emergency situations: (I) Handheld skin bioprinter illustrating design of the microfluidic cartridge
to print skin tissue directly on the cutaneous wounds (II) In vivo study represent in situ bioprinting in large animal models and healing efficacy as examined by
trichrome staining (reproduced with permission from Ref. [79]). ©2018 Royal Society of Chemistry via Copyright Clearance Center. (III) Customized spraying device
fabricated by a 3D printer for spraying PRP onto cutaneous wounds; the applicate reservoir tube contains PRP to be sprayed using propellant (aerosol can) directly on
the wounds (reproduced with permission from Ref. [84]). ©2018 Elsevier B.V. (IV) Schematic representation of cell isolation process from donor skin to obtain
autologous skin cells for cell spray applications (reproduced with permission from Ref. [81]). ©2016 Elsevier Ltd. and ISBI. (V) Application of portable electro-
spinning device for covering the wounds with personalized nanofibrous dressings. (reproduced with permission from Ref. [52]). ©2016 Royal Society of Chemistry
via Copyright Clearance Center.

take the exact shape of wound defect. Such injectable systems have also
been used for cell delivery applications [51]. This demonstrates future
possibility of delivering autologous stem cells or autologous tissue
specific cells in the gel, which crosslinks at the wound site and forms a
cellular matrix immediately. These systems have been effectively uti-
lized for treatment of severe wounds like 3rd degree burns and diabetic
wounds [23,51]. In our recent work for treatment of burn wounds, we
developed in situ forming injectable SF hydrogel using a cross-linker
free approach (Fig. 4.I.). SF from two different silk varieties self-as-
sembled to form a hydrogel within 10 min of application at the wound
site in burn rat model [23]. The hydrogel not only provided hydrated
environment to the burn wounds, but also acted as a supporting niche
helping in regenerating the wounded skin (Fig. 4.II.).
In another study, pH and glucose responsive hydrogels containing
insulin and fibroblast cells were developed for treating diabetic wounds
[51]. The injectable hydrogels were fabricated through the cross-
linking of Schiff's base and phenylboronate ester using PVA, phe-
nylboronic-modified chitosan and benzaldehyde-capped poly(ethylene
glycol) [51]. Chemical crosslinking for the fabrication of hydrogels is a
well-known approach and have also been utilized for developing
bioadhesives [30]. Inspired by plant-derived tannins, Guo et al. devel-
oped gelatin based bioadhesives using one-step Michael addition of
tannic acid, which were further crosslinked by silver nitrate (Fig. 4.III.)
[30]. The tannin-inspired gelatin bioadhesives were developed irre-
spective of the gelatin source (fish, bovine, or porcine) and proved to be
cost-effective [30]. Such bioadhesives hold great promise for various
applications in wound healing, tissue sealant, hemostatic material, in-
jectable system and cell/drug delivery. Another example of such
crosslinking chemistry includes fabrication of light-activated, mussel
protein-based bioadhesive (LAMBA) inspired by mussel adhesion and
insect dityrosine crosslinking [76]. Herein, tyrosine-rich mussel ad-
hesive protein was formulated into an adhesive hydrogel by photo-
chemical crosslinking using blue light. The LAMBA system demon-
strated excellent hemostatic activity to be applied as a waterproof
sealant for healing critical wounds [76]. In another study, injectable
nanoengineered hemostats were developed using natural poly-
saccharide kappa-carrageenan functionalized with synthetic nanosili-
cates for enhanced wound healing [77]. The nanocomposite hydrogels
enhanced cell adhesion and increased binding of platelets, thereby re-
ducing blood clotting time [77].
More pertinent for wound healing, microfluidic technology has
shown immense potential in the fabrication of injectable microspheres
[48,78]. Assembly of annealed microgel building blocks formed an in-
jectable MAP scaffold with interconnected pores by microfluidic fab-
rication (Fig. 2.I.) [48]. The study demonstrated ‘plug and play’ nature
of the generated microgel based building blocks, showing in situ tissue
regrowth and regeneration. Another example of application of micro-
fluidic platform includes fabrication of hollow bacterial cellulose mi-
crospheres to develop injectable scaffold for wound healing applica-
tions (Fig. 4.IV.) [78]. The cellulose microspheres were developed on a
platform of microfluidics generated core–shell structured micro-
particles. The core of alginate and shell of agarose material provided
suitable environment to encapsulate the specific bacteria for a long-
term static culture. The bacterial cellulose secreted by bacteria was
inherently entangled within the shell of microparticle to form the de-
sired hollow microspheres. Upon assembly, these hollow microspheres
generated novel injectable scaffold with potential of cell proliferation,
tissue ingrowth and accelerated wound-healing [78]. Utilization of in-
jectable hydrogels with or without crosslinkers can be considered user
friendly even for the patient. Introduction of microfluidics based

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D. Chouhan, et al.
fabrication of annealed microgels and microspheres is an added feature
in the field of injectable systems and can be an attractive avenue of
investment in this domain.
2.3. Smart technologies based innovative approaches for skin therapeutics
2.3.1. Portable 3D skin printers
Technical advancement in recent years has led to the development
of handheld skin printer to be applied for in situ formation of skin tis-
sues at the wound site (Fig. 5.I. and II.). The remarkable work by Ha-
kimi et al. represented development of a portable 3D bioprinter
(weight < 0.8 kg) that enabled bioprinting of skin tissue sheets using a
microfluidic cartridge [79]. The study illustrated in situ formation of
skin sheets in the porcine and murine wound models as a direct treat-
ment using skin specific cells in the bioink. Such portable 3D printers
can be revolutionary in the current healthcare market, as patients do
not have to wait for the laboratory grown cellular skin grafts. This
technology can also be used for emergency cases like burn trauma cases
and can be carried in ambulances for immediate treatment in real time.
2.3.2. Spray based technological approach
Of critical importance for large-sized cutaneous wounds, cell spray
system demands special mention in this review. Such cell spray system
has been developed long back using Badger 100G airbrush and de-
monstrated successful delivery of bovine dermal fibroblast with more
than 50% cell viability [80]. Viability of cells post-spray is an important
concern since direct spraying may induce cellular damage. The problem
was resolved by the aerosol delivery method with encouraging out-
comes. In a recent study, researchers have optimized calculations for
non-cultured autologous skin cell-spray grafting [81]. The calculation
was performed by considering variables like yield of donor site skin
tissue, cell density, distribution area, spray volume and the percentage
of surface coverage. The mathematical approach demonstrated possible
ratios of 1:80 to 1:100 (donor site area: treatment area); thereby re-
vealing significant improvement over the conventional mesh grafting
technique [81]. In a comparative study against fibrin membrane
transfer system, aerosol cell spray was found to be effective with pre-
confluent keratinocytes [82]. Cell suspension experiences various types
of stress conditions like hydrostatic, shear or elongation stress when
sprayed directly onto a wound [82]. As such, aerosol delivery can be
effective for superficial wounds like first degree or second degree
wounds. Cell spray systems using aerosols or airbrush not only requires
an efficient sprayer, but also need tunable air pressure system and
tunable nozzle diameter to obtain high cell viability [80,82].
Although aerosol based cell spray technique has shown huge po-
tential in re-epithelialization of superficial wounds, the technique is
further improved using biopolymers for severe cutaneous injuries.
Recent work on the delivery of human keratinocytes using micro-
carriers and compressed collagen demonstrates the excellent utilization
of spray technique [83]. Support of biomaterial based microcarriers
could further improve cell viability, a major concern in direct cell
spraying due to lack of cell-adhesion in the suspension conditions.
Gelatin microcarriers provided cell adhesion sites and thereby in-
creased the overall distribution of cells in the keratinocyte cell therapy
while maintaining the clinically-relevant time-frame [83]. The success
of cell spray system also encouraged application of natural wound
healing agents like haemoglobin and platelet-rich plasma (PRP)
[84,85]. A clinical study on haemoglobin spray in the treatment of
chronic wounds is a perfect example, which demonstrated its effec-
tiveness in patients with chronic wounds [85]. In a recent study, PRP
spray applicator was developed using 3D printing technology for
treating wounds (Fig. 5.III.) [84]. PRP is known for its wound healing
efficacy; however, its application as a spray system required a custo-
mized spraying device. Duregger et al. developed a lightweight spray
applicator for spraying PRP, which could develop a sprayed film of vital
platelets on the wounds in real time [84]. The PRP spray system also
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Biomaterials 216 (2019) 119267
enhanced the growth factor release due to even distribution and drop
size in micro range (125–145 μm). The novel system depicted treatment
of large-area wounds with a small volume of blood using the handheld
portable PRP spray applicator [84]. In this context, the optimized
methods to mince skin biopsies and isolate skin cells or chopped tissues
as illustrated in Fig. 5.IV. could be applied in combination with the
customized cell spray systems for immediate first-aid. Such examples of
customized cell spray equipment paves way for next generation smart
system for efficient delivery of autologous skin cells for wound healing
applications.
2.3.3. Portable electrospinning apparatus
Electrospinning has attracted substantial interest in the technolo-
gical and healthcare sectors in recent years. This technique enables easy
fabrication of nanofibrous mats in large scale [86]. The paradigm shift
in the fabrication of nanofibrous wound dressing strategy using elec-
trospinning technology has already been discussed before. The latest
technology further developed improvised the electrospinning instru-
ment by miniaturizing its size and led to the development of handheld
electrospinning device [87]. A portable instrument with broad range
polymer specificity is easy to use and reduce wound edema or pain. In a
study, a portable handheld electrohydrodynamic multi-needle spray
gun was developed for practical day-to-day applications using the frame
of Dremel glue gun [88]. The glue gun was modified into the multi-
needle electrospray gun. Different variations of similar handheld elec-
trospinning apparatus were also developed, which were battery oper-
ated by further minimizing the size (Fig. 5.V.) [52,89]. The new device
weighed only 120 g and could electrospun various polymers such as
polyvinylpyrrolidone (PVP), PCL, polystyrene, PLA and poly(vinylidene
fluoride) (PVDF). In situ deposition of a nanofibrous dressing directly on
the cutaneous wounds demonstrate advanced healthcare system. The
approach can also be utilized to fabricate patient-specific dressings by
incorporating particular drugs or molecules in the electrospinning so-
lution [52,89]. Such an equipment if used in first aid or in emergency
cases can generate wound patches at the injury site to stop wound
bleeding and provide instant dressing. Portable electrospinning equip-
ment could be a temporary solution in case of emergencies; however, in
case of large size wound defects, the acellular nanofibrous patch face
drawback in long-term treatment. Similar to portable electrospinning,
the handheld cell spray and bioprinting techniques developed recently
could be an effective solution to deliver living autologous cells over the
wounds.
3. Immunomodulation and vascularization based approach
Principal goals in wound management include vascularization in the
regenerated tissue to achieve rapid wound healing and skin regenera-
tion. Many pioneering approaches have utilized cellular and molecular
biology for the development of pre-vascularized skin grafts over the
past decade [17,29]. During the course of development of advanced
cellular therapies, a greater comprehension of basic biology has been
considered by the researchers in recent years. Targeting immune system
for enhanced angiogenesis has recently been explored leading to sig-
nificant advances in wound healing therapy [46,90]. This approach
stands apart from the orthodox approach of using only endothelial cells
in making vascularized grafts. Clearly, immune system has a major role
in the healing process because inflammation is the very initial phase of
wound healing [90,91]. The early immune response delivers signals to
the subsequent phases through cytokines and chemokines. These strides
have ultimately led to advancements in skin regeneration. Therefore,
tailoring the immune response or modulating the behaviour of immune
cells may impart major breakthroughs in wound repair and skin tissue
engineering in near future.
At the early phase of wound healing, inflammation occurs and re-
cruitment of neutrophils and macrophages takes place [91,92]. The
macrophages release various signals, which can be either pro-
D. Chouhan, et al. Biomaterials 216 (2019) 119267

Fig. 6. (I) Schematic illustration shows model of cell migration using pro-inflammatory macrophages (M1 phenotype); immune cells induce migration of endothelial
cells in the scaffolds through their secretions (reproduced with permission from Ref. [93]). ©2017 Oxford University Press. (II) Representative image of modulating
MSCs cultured on scaffold to prime the cells and utilize the cell communication network toward tissue repair/regeneration. (reproduced with permission from Ref.
[97]). ©2017 The authors, published by Elsevier Ltd. (III) Illustration of study design to stimulate macrophages for switching phenotypes (M1/M2 and M2/M1) using
specific cytokines in the cultured media for tissue engineering applications (reproduced with permission from Ref. [96]). ©2014 Elsevier Ltd. (IV) Schematic diagram
represents the effect of integrin α2β1 inhibition on macrophage polarity using IL-4 functionalized gelatin methacrylate (GelMA) hydrogel and future applications to
obtain tailored immune response by priming human monocytic THP-1 cells. (reproduced with permission from Ref. [98]). ©2017 John Wiley and Sons.

inflammatory or anti-inflammatory and thus fate of the wound is
decided accordingly [91]. In a normal wound, macrophages switch to
become a helping cell and release growth factors, thereby leading the
wound to the next healing phase [91]. In this stage, a new stroma
(called as granulation tissue) begins to develop due to recruitment of
various cells, especially fibroblasts and endothelial cells. Numerous
new blood capillaries sprout in the neostroma and this process is known
as angiogenesis. The crosstalk between immune cells and endothelial
cells plays a major role in the rate of healing process, showing their
interdependence during the tissue repair. Macrophages deliver a con-
tinuing supply of cytokines essential for cell growth and angiogenesis;
and the newly formed blood vessels carry oxygen and nutrients ne-
cessary for cell metabolism [91,92]. The chemokines secreted by
macrophages have also been shown to induce migration of endothelial
under in vitro conditions [93]. The study revealed migration of en-
dothelial cells in a porous scaffold due to secretions of pro-in-
flammatory macrophages (M1 phenotype) seeded on the bottom of
culture well as shown in Fig. 6.I. This interplay between immune
system and vascularization has recently been exploited in various
strategies to increase the wound healing rate and develop pre-vascu-
larized skin grafts. In the subsequent sections, we will briefly discuss
the strategies for immunomodulation and vascularization enhancement
focusing on the recent progress and improvement in this area.
3.1. Tailoring immune system towards skin regeneration
Macrophages are the key players in inflammatory response against
any implant or biomaterial. They are the essential regulators of repair
process, as they can be involved in advancing or suppressing the in-
flammation. Therefore, targeting macrophages may correct the immune
states and direct cells towards tissue repair and regeneration. During
the wound healing process, macrophages can be polarized into either
M1-type pro-inflammatory or M2-type anti-inflammatory macrophage.
Both the types of macrophages are present at wound site during dif-
ferent stages of healing. However, under various pathologic condition
they can remain in either of the polarized type and may result in sig-
nificantly different outcome. Following the plasticity of these cells,
researchers have developed strategies to harness the power of macro-
phages for tissue regeneration. For example, pro-regenerative hydrogels
or scaffolds can be fabricated, which polarize the macrophages to M2
phenotype and thereby restore the wounded tissues both structurally
and functionally. In a study, elastomers were functionalized with he-
parin and interleukin-4 (IL-4) by supramolecular self-assembly to po-
larize the macrophages into regenerative M2 type [8]. They could also
be translated into 3D electrospun matrix for possible wound healing
and tissue engineering application by harnessing the regenerative be-
haviour of host macrophages.
Macrophage phenotype also depends on the 3D microenvironment
provided by various biomaterials. Fibrin, a natural biomaterial present
in blood clot post-injury is the most studied material in wound healing
research. It acts as a provisional matrix and influences macrophage
behaviour by stimulating secretions of anti-inflammatory cytokines.
Moreover, when fibrin was added to collagen matrix, the macrophages
secreted anti-inflammatory cytokine interleukin-10 (IL-10) in response
to the dual material [94]. Although fibrin exerts a protective effect on
macrophages by showing anti-inflammatory activation; fibrinogen

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D. Chouhan, et al.
(soluble precursor of fibrin) demonstrated opposite effect by stimu-
lating pro-inflammatory activation [94]. Therefore, fibrin and its pre-
cursor fibrinogen act as real players in the natural wound healing
process and can be used as a valuable immunomodulatory material for
tissue repair and regeneration applications. In another study, allogeneic
PRP has also been shown to induce differentiation of monocyte to
dendritic cells and promote wound healing by stimulating an anti-in-
flammatory microenvironment [95]. Being motivated by this, re-
searchers have applied the same strategy to develop im-
munomodulatory wound dressings using synthetic polymer poly
(hydroxybutyrate) (PHB). The electrospun PHB dressing showed
greater angiogenic and M2 macrophage polarization properties under in
vivo conditions [46].
In a recent study, researchers have also utilized immunomodulatory
properties of mesenchymal stem cells (MSCs) by developing surgical
meshes coated with MSCs [28]. The cryopreserved MSCs coated mesh
was like an ‘off the shelf’ product and demonstrated local im-
munomodulation of macrophages towards an anti-inflammatory M2
phenotype [28]. Therefore, 3D microenvironment can be tuned to steer
the host's immune system using various biomaterials, cells or bioactive
molecules for achieving a natural healing response [96,97]. The path-
ways to tune macrophage behaviour through MSCs and monocytes for
wound healing and tissue engineering applications has been depicted in
Fig. 6.II. and III. respectively. These findings provide new insights into
designing immunomodulatory wound dressings for constructive re-
modelling during skin repair and thus manipulate wound healing in
regenerative manner. The immunomodulatory and multi-functionalized
constructs could mimic plethora of signals by interacting with the cells
and guide them towards proper tissue regeneration. Such bioactive
systems hold great potential for the development of smart and cell re-
sponsive skin grafts or dressing materials.
3.2. Pre-vascularized skin grafts and crosstalk with immune system
Formation of blood vessels in the wounded tissue during healing
process is a common phenomenon and is known as angiogenesis [27].
In this process, new vessels sprout from existing vessels to establish the
supply of oxygen and nutrients to the newly formed tissue at the injured
site. Vascularization is a complex process requiring interaction between
various cells, the ECM and numerous growth factors [27]. However, in
certain pathophysiological conditions like diabetic wounds or large
burn wounds, this natural angiogenesis process is severely obstructed.
Artificially developed implants on such wounds often fail to develop
vascularized network and clinical outcomes remain poor. To cope with
this problem, fabrication of pre-vascularized skin grafts has been a re-
cent area of research, where the bioengineered tissue is already well
developed with a vascular network. Several strategies have been ex-
plored with the aim of developing pre-vascularized constructs using
cellular therapy, growth factor delivery, and use of bioactive materials.
Co-culture system has been the most explored and highly efficient
strategy for developing pre-vascularized grafts so far. Endothelial cells,
which make wall of the blood capillaries, have cellular crosstalk with
fibroblast cells. Co-culture of both the cells has been very successful in
bioengineering the vascularized dermo-epidermal skin grafts [17].
Human dermal microvascular endothelial cells (HDMECs) when co-
cultured with HDF in 3D hydrogels developed vasculature networks
within 3 weeks [17]. It was also shown that HDMECs when cultured
alone in the hydrogels failed to develop capillary like structures proving
the significance of co-culture system. Marino et al. went one step ahead
by developing skin grafts with lymph vessels along with blood vessel
network [17]. The tissue engineered skin grafts containing blood and
lymph vessels when transplanted on wounds of nude rats demonstrated
connections with existing vessels and formed functional skin layers. The
bioengineered grafts also drained the wound fluid away through pre-
formed vessels thereby helping in accelerated wound healing [17]. In
another study, researchers used growth factor therapy by encapsulating
10
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Biomaterials 216 (2019) 119267
a cocktail of growth factors along with endothelial colony forming cells
to develop vascularized grafts [74]. The growth factor cocktail con-
sisted of recombinant human VEGF165, bFGF, angiopoietin-1, tumor
necrosis factor alpha (TNF-α) and stromal cell-derived factor-1 (SDF-1)
and the constructs demonstrated accelerated diabetic wound healing
[74]. Among all the mentioned growth factors, VEGF is the most es-
sential growth factor for vascularization and it is also a chemoattractant
secreted by macrophages during the wound healing process.
Not only this, natural biomaterials and their composition strongly
affects the M1–M2 macrophage polarization. Integrin mediated inter-
actions between macrophages and biomaterial have also been found to
be responsible for cell polarization [98]. A matrix lacking cell attach-
ment motifs showed proinflammatory M1 phenotype, on the other hand
the matrix containing cell attachment sites demonstrated anti-in-
flammatory M2 phenotype (Fig. 6.IV.) [98]. This suggests that cell
binding motifs if present in biomaterials may play crucial roles in im-
munomodulation. Macrophages also play crucial roles in angiogenesis,
as they secrete a lot of factors that affect both endothelial cells and
smooth muscle cells (SMCs; another cell component of blood vessels)
[99]. Crosstalk between macrophages and SMCs demonstrated in-
creased VEGF secretion by the former and sprouting of new blood
vessels by their co-cultured conditioned media in matrigel lattice [99].
Macrophages, both M1 (pro-inflammatory) and M2 (anti-in-
flammatory), are known to direct the behaviour of other cells [90,100].
In a recent work, researchers engineered the macrophages to attain
control on their behaviour for activation of endothelial cells [90]. Using
a chemical inducer of dimerization (CID) technology, tunable pro-in-
flammatory macrophages were designed to activate receptor-specific
pathways. Macrophage polarization could be easily controlled by sup-
plementing and withdrawing the CID drug when needed. The study
demonstrated the ability to control the inflammatory response and
possible manipulation of the host response, depicting a classic example
of bioengineering for wound healing therapeutics [90]. Overall, im-
munomodulation of grafts is a vital area of research, as it contributes
majorly towards underlying aspects of graft acceptance in patients.
Tailoring macrophage infiltration, switching its phenotype and estab-
lishing angiogenesis are essential to the cause of graft acceptance, thus
encouraging further investigation in this branch.
3.3. Stromal vascular fraction (SVF) in skin engineering and wound healing
SVF or stromal vascular fraction is a cocktail of cells that can be
obtained from adipose tissue through enzymatic digestion or mechan-
ical dissociation [15,101]. The source of adipose tissue can be animal
fat or lipoaspirates obtained from human patients. Although SVF has
been found to be effective in treatment of multiple conditions; its role in
wound healing is appealing. The phenomenon of wound healing is an
interesting crossroad of multiple factors including fibroblast migration
and maturation, ECM deposition, loss of inflammation and cytokine
generation. However, the major challenge is to create efficient vascu-
larization in grafts that can provide factors promoting the aforesaid in a
regulated non-diffusion dependent manner. The popularity of SVF in
wound healing is gaining momentum as it seems to contain all com-
ponents that not only take care of the underpinned properties of ac-
celerated scarless wound healing, but can also be valuable in terms of
providing vascularization. This can in turn enormously contribute to-
wards better graft acceptance for full thickness skin grafts and wound
patch.
Attempts to characterize the cellular and ECM components of SVF
are under scrutiny. The presence of endothelial, mesenchymal/stromal
cell -associated markers, stem cell markers and hematopoietic markers
were observed in the SVF cellular fraction (Fig. 7.I.) [15,71]. Fabrica-
tion of SVF based pre-vascularized grafts and their integration with host
skin demonstrate the benefits of SVF application in healing critical
wounds (Fig. 7.II.) [71]. Besides this, the use of SVF alone and SVF
-ECM gel have been found to be beneficial in deep acute wounds and in
D. Chouhan, et al.
excisional mice wound models [102,103]. It has been found to be ef-
fective in hyperglycemic wound conditions, stem cell therapy for
wound regeneration and also in hair follicle growth promotion for
adipose tissue mediated baldness treatment [104–106]. The technique
of nanofat grafting; SVF-ECM gel transplantation and pre-micro-
vascularized graft placement are other application modes of SVF in
different forms in wound healing [107–109]. Integra template seeded
with GFP tagged adipose tissue-derived microvascular fragments
(GFP+-ad-MVF) demonstrated success of such pre-vascularized artifi-
cial skin grafts in skin engineering applications as shown in Fig. 7.III.)
[107].
The application of SVF in regenerative medicine is becoming con-
spicuous with increasing characterization of its components. SVF, par-
ticularly in wound healing can prove to be immensely beneficial as it
provides a repertoire of cells, which can be differentiated to adipogenic,
osteogenic and smooth muscle cell lineages [110]. Fat transplant has
been a long lasting practice in treating scars and aesthetic physical
augmentation. The use of SVF is a further improvement towards the
same, as it has been implicated in scarless healing and better graft ac-
ceptance [111]. The presence of adipose derived stem cells in the
fraction along with its microenvironment (the ECM) provided by the
SVF can enable better potential and endurance to the stem cell based
therapies. Overall, it is seldom in nature that the most desired com-
ponents for therapy are found in a well-balanced cluster. SVF seems to
be such a component, which if harnessed properly can be an ingenious
approach towards regeneration therapy.
11
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Biomaterials 216 (2019) 119267
Fig. 7. Various strategies of developing pre-vascu-
larized skin grafts: (I and II) Isolation and phenotypic
characterization of white adipose tissue derived en-
dothelial cells (watECs) and adipose stem cells
(watASCs) from SVF and in vivo analysis of bilayer
skin substitutes (seeded with SVF) showing human
CD31+ capillaries (red) and rat CD31+ capillaries
(green) after the transplantation (reprinted with
permission from Ref. [71]). ©2015 Springer Nature.
(III) Image shows seeding of Integra skin graft with
GFP+-ad-MVF and in vivo analysis of the implanted
pre-vascularized Integra in the dorsal skinfold
chamber observation window of full-thickness skin
defect. (reproduced with permission from Ref.
[107]). ©2016 The authors, published by Elsevier,
Inc. on behalf of the society for Investigative Der-
matology.
4. SiRNA based skin therapeutics
Among the nucleic acid based wound healing strategies, utilization
of siRNA and miRNA based wound healing has gained momentum over
the past few years. Small interfering RNA is a small, synthetic RNA of
21–25 nucleotides length that can specifically carry out silencing of
intended genes through knockdown of messenger RNA (mRNA) specific
to the gene of interest [112]. Both the siRNA and miRNA are short
stretches of nucleotide RNA duplex and holds potential to inhibit mRNA
translation through various ways as illustrated in Fig. 8.I. Reduction in
gene expression has major implications in multiple disease manage-
ment strategies. Among these wound healing treatments, siRNA owes a
special mention due to its complicated interplay of myriad of cellular
pathways that prevail among the four stages of the process. Silencing
gene targets like p53, matrix metalloproteinases (MMPs), transforming
growth factor beta receptor 1 (TGFBR1) and others have shown fa-
vourable response towards management of difficult to heal wounds
[113–115]. Expression of connective tissue growth factor (CTGF) was
targeted through siRNA loaded customized skin sutures for scarless
wound healing applications in a recent study. Development of the su-
tures using layer-by-layer (LbL) assembly facilitated controlled delivery
of siRNA through ultrathin polymer coating of sutures (Fig. 8.II.) [113].
The study revealed scarless healing outcomes in a 3rd degree burn in-
duced scar animal model, establishing the promising approach of RNA
interference (RNAi) in skin therapeutics. This has encouraged re-
searchers to look into more such targets, which if silenced could in turn
affect a large number of downstream target genes that can concordantly
act to hasten up the process.
The application of siRNA in other disease conditions although has
D. Chouhan, et al. Biomaterials 216 (2019) 119267

Fig. 8. (I) Schematic representation of (a) siRNA and miRNA complementarity towards target recognition site of mRNA and (b) mechanisms of gene silencing by
miRNA and siRNA in the cytoplasm of cells via formation of RNA-induced silencing complex (RISC). Both miRNA and siRNA share common endonucleolytic cleavage
mechanism of mRNA to inhibit the target mRNA; however, miRNA holds additional gene silencing methods like mRNA degradation and translation repression. (II)
Schematic cartoon of Layer-by-layer (LbL) assembly architecture to develop sutures with local delivery of siRNA (reproduced with permission from Ref. [113]).
©
2016 Elsevier Ltd. (III) Development of siRNA loaded wound dressing fabricated using LbL coating comprising siMMP-9 for accelerated wound healing rate; the
gross wound images demonstrate healing effects of siRNA loaded bandage owing to the sustained release of siMMP-9 (reproduced and modified with permission from
Ref. [47]). ©2015 John Wiley and Sons.

been prevalent for some time now; its utility in wound healing became
relevant with the elucidation of gene expression profile during wound
healing [116]. The practice of RNA interference is plausible when
compared to direct gene delivery as it can modulate prominent gene
expression at the site of action that can lead to a cascade of downstream
effects. However, the major drawback in RNAi therapy is its decreased
bioavailability, rapid degradation and transient expression mechanism.
Hence, multiple efforts have been undertaken by researchers to address
such issues. The major approach is using a delivery medium to carry the
RNA to its site of action that can enhance the permeability, retention
and half-life of the treatment so that a prolonged expression efficiency
can be achieved. The application of siRNA in wound healing seems
feasible since it can be applied locally, thus obliterating the drawbacks
of systemic administration. With the appropriate choice of delivery
vehicles, RNAi is and will continue to be a prominent approach for
accelerated wound healing.
Wound healing is a multistep process characterized by changing cell
phenotype, variations in protein expression, and the production of ac-
tive biomolecules [117]. It also accompanies upregulation or down-
regulation of multiple genes that can either hasten or hinder the pro-
cess. In this context, siRNAs have been targeted against PHD2, MMP9,
CTGF, TGF-β type 1 receptor and p53 in both chronic and diabetic
wound healing [47,113–115,118]. Such targets pertain to different
stages of wound healing like chronic inflammation (MMP9); migration
and vascularization observed in the subsequent proliferation and mi-
gration stages respectively (PHD2, p53) followed by the ultimate re-
modelling stage (HOX B13) [47,112,118]. siRNA targeting connective
tissue growth factor (CTGF) and TGF-β type 1 receptor has been found
to be beneficial for treatment of scars [113,115]. In this context, mul-
tiple delivery vehicles encapsulating siRNAs like scaffolds, agarose
matrix, polymeric star shaped cationic carriers and cationic lipid na-
noparticles have been utilized with encouraging results [119–123].
Apart from this, layer by layer deposition of self-assembled dressings
containing siRNAs, MMP-2 siRNA incorporated LPEI-immobilized na-
nofibrous meshes and GM3S thiolated siRNA tagged gold nanoparticles
(NPs) deserves special mention in terms of innovation, stability and
efficacy in difficult to heal diabetic wound conditions [124,125].
Taking advantage of the LbL assembly, commercially available nylon
bandage was functionalized with siMMP-9 to knockdown the target
gene for diabetic wound healing applications [47]. Coatings in nan-
ometer scale were generated to incorporate and deliver siRNA at a
sustained rate (for 2 weeks) in the wound bed for rapid wound closure.
Application of siRNA loaded wound healing in mouse model revealed
accelerated healing rate with rapid re-epithelialization and early
granulation tissue formation (Fig. 8.III.) [47].
The success of siRNA treatment using appropriate delivery vehicles
is crucial, as it will ultimately make way for alternative nucleic acid
based approaches like miRNA, or short hairpin (shRNA) in wound
healing. ShRNA mediated silencing of PHD2 in diabetic wounds and
ischemic wound is a shift towards this direction [126]. The safety,
specificity and the effectiveness of this strategy holds promise in the
field of wound healing treatment and thus will pave way to more other
such nucleotide based treatments. These newer candidates along with
the advent of CRISPR-Cas-9 technology will provide a plethora of
choices for researchers to explore various modalities of genetic ma-
nipulation towards wound healing thus promoting safe, effective and

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D. Chouhan, et al. Biomaterials 216 (2019) 119267

personalized next generation medication. therapeutics. Apart from the traditional viral vectors, non-viral carriers
are equally gaining momentum in this respect. For example, miR-29b
loaded functionalized collagen scaffolds efficiently delivered the anti-
5. MicroRNA based skin therapeutics
fibrotic exogenous miRNA at the wound site to modulate ECM re-
modelling [132]. The implanted scaffolds demonstrated improved col-
The discussion on nucleotide-based treatments relevant in wound
lagen type III/I ratios showing reduced wound contraction in vivo and
healing is incomplete without incorporating the context of miRNAs.
ideal skin regeneration [132]. In another study, miRNA-132 was re-
Although both miRNA and siRNA share similar structures (~25 nu-
plenished in the wounds by developing liposome-formulated synthetic
cleotide RNA duplex with 2 nucleotides 3'overhang), their mode of
double-stranded miR-132 mimics, which led to improved healing in
action is different [127]. Unlike siRNAs that are designed prioritizing
diabetic mice [135]. Comparative study between healthy wounds and
specificity for binding, miRNAs are comparatively imprecise in terms of
diabetic cutaneous ulcers revealed differences in their appearance and
complementarity to their target and hence can target multiple genes as
miR-132 expression, as analysed by tissue histology and in situ hy-
depicted in Fig. 8.Ia [127]. siRNA regulate the gene expression by en-
bridization (Fig. 9.II.) [135]. The liposome based miR-132 delivery via
donucleolytic cleavage of mRNA; whereas miRNA do translational re-
pluronic F-127 gel on human ex vivo skin wounds also demonstrated
pression of the target mRNA and also degrade the target mRNA as il-
enhanced re-epithelialization [135].
lustrated in Fig. 8.Ib [127]. Upon reaching the cytoplasm, miRNA acts
Recent advancements in nanotechnology have unlocked greater
on the target mRNA by forming RISC containing miRNA, Dicer and
avenues in the field of drug delivery applications that have also been
Argonaute (Ago) protein, where the miRNA pairs with the 3′ UTR of
utilized for DNA/RNA delivery. The development of smart light-acti-
target mRNA and either inhibit the translation of mRNA or degrade/
vatable miRNA-loaded nanocarriers using plasmonic gold nanocarriers
cleave the same [127]. The two major approaches of miRNA-based
is a perfect example (Fig. 10.I.). The plasmonic gold nanocarrier de-
therapy include - 1) restoration of miRNA expression by delivering
monstrated sequential release of two miRNAs (miR-302a and miR-155)
synthetic miRNA via scaffold or viral vector-based carriers and 2) in-
in endothelial cells and depicted varied response working as an optical
hibiting or silencing a pathology associated miRNA by chemically
switch for on-demand delivery. The study also demonstrated how order
modified anti-miRNA oligonucleotides or antagonistic sequence (an-
of release of miRNA can modulate wound healing outcomes (Fig. 10.II.)
tagomir).
by targeting different cellular pathways involved in the process and in
The association of miRNA to wound healing is well established now.
turn regulating gene expression in a spatiotemporal manner [136]. In
Information on gene profiling in case of burn or diabetic wounds
another study, polymeric nanoparticles were functionalized to achieve
through microarray techniques has opened various therapeutic ap-
sustained delivery of proangiogenic miRNA-132 via poly lactic (glycolic
proaches involving miRNA targeting or delivery [128,129]. Liu et al.
acid) (PLGA) nanoparticles for improving vascularization [29]. Such
identified 18 miRNAs being upregulated and 65 as downregulated in
advanced systems of miRNA delivery hold great potential if combined
their study, investigating the differential expression profiling of
with the conventional systems of developing skin grafts. For example,
miRNAs in cutaneous wounds of diabetic rat model. Similar study has
the proangiogenic-miRNA loaded nanocarriers can be incorporated in
been carried out in burn inflicted dermis, revealing 34 downregulated
suitable hydrogel (such as collagen gel) to fabricate pre-vascularized
and 32 upregulated miRNAs [128]. Apart from this, various miRNAs
skin grafts in vitro. Similarly, different cells can be easily targeted to
have been shown to regulate angiogenesis, fibroblast and keratinocyte
develop an artificial bilayer or trilayer skin graft using miRNA therapy.
proliferation, bacterial clearance, ECM remodelling and scar manage-
Silencing mRNA may lead to systemic side effects owing to its
ment through its involvement in major wound healing pathways in a
multiple targets in various signalling pathways. However, sophisticated
variety of wound bed environment [130–134]. Fig. 9.I. illustrates nu-
gene expression analysis of wound beds has been able to eradicate such
merous miRNAs and their roles in governing the behaviour of a variety
risks to a greater extent through recognizing exclusive targets involved
of skin cells and wound healing pathways.
in the pathophysiology of the process. This has led to the development
RNA delivery techniques have been very advanced in the last five
of a plethora of anti-miRNA-based strategies parallel to that of the
years, leading to the development of functionalized wound dressings or
functional miRNA transfection approach. Light-inducible synthetic
scaffolds carrying stable miRNA or anti-miRNA molecules for skin re-
antimiR-92a and Locked Nucleic Acid (LNA)-antimiR- 26a exhibit
generation applications. Delivery of both functional miRNA and anti-
progress made in this respect [137,138]. While the former led to im-
miRNA is laced with equal difficulties as in case of siRNA. Hence, in-
proved cell proliferation and angiogenesis in a diabetic wound healing
vestigation of delivery strategies is forefront towards miRNA-based

Fig. 9. (I) Schematic image shows various miRNAs involved during the wound healing process (reproduced with permission from Ref. [131]). ©2015 Elsevier B. V.
(II) Comparative images of healthy wound vs. diabetic cutaneous ulcer; images represent miR-132 expression analysis in the wound samples of healthy donors and
those with diabetic foot ulcers (DFUs) via in situ hybridization (reproduced with permission from Ref. [135]). ©2017 The authors, published by Elsevier, Inc. on
behalf of the society for Investigative Dermatology.

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D. Chouhan, et al.
model, the later promoted angiogenesis and wound closure through
induction of the SMAD1 signalling pathway. Another study involving
synthetic microRNA-92a inhibitor (MRG-110) reported accelerated
angiogenesis and wound healing in both diabetic mice and normal pig
models [139]. The LNA-modified anti-miRNA oligonucleotide (MRG-
110) targets the antiangiogenic miRNA miR-92a-3p present in wound
beds, hence demonstrating its efficacy in both acute and chronic wound
conditions. The pre-clinical study revealed better results in comparison
to positive controls like rhVEGF-165 and rhPDGF-BB, underpinning
miRNA-based strategy as an improvement on the conventional growth
factor delivery-based approach in wound healing.
The discussion on miRNA based wound healing treatment can be
concluded mentioning specific cell derived exosomal miRNAs.
Exosomal miRNAs derived from umbilical cord mesenchymal stem
cells, human amniotic epithelial cells and human umbilical cord blood
plasma have proven to be pragmatic in wound healing studies
[130,140,141]. Exosomes, a typical membrane vesicle has been iden-
tified as a kind of paracrine factor released by a majority of cells, in-
cluding stem cells that can promote wound healing [142,143]. Having
identified miRNA as key component of exosomes that promote healing,
exosomes are set to be key mediators in the subsequent generation of
wound healing treatment strategy. In a recent study, functionalized
wound dressings made up of chitosan biomaterial were developed in-
corporating the exosomes derived from synovium mesenchymal stem
cells, which overexpressed microRNA-126 [32]. The dressing offered
sustained release of exosomes and thereby aided in diabetic wound
healing promotion. Having said that, exosomes although has been itself
defined as a sort of delivery vehicle, additional delivery strategies in
wound dressings can prove to be a further drive towards enhancement
of its utility and efficacy in wound healing studies.
6. An update on the clinical trials
The clinical implementation of technology is necessary to validate
proof of concept and pre-clinical studies performed in animal models.
Clinical study not only reveals the treatment outcomes, but also unfolds
the basic information like cost of treatment, patient safety and risks
14
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Biomaterials 216 (2019) 119267
Fig. 10. (I) Schematic representation of fabrication
of light activated miR-dsDNA-AuNR conjugates using
miR-155 and miR302a for wound healing applica-
tion. The nanoconjugates are temperature sensitive
that can be regulated through near infrared irradia-
tion, thereby leading to DNA dehybridization, which
ultimately impacts the release of two miRNAs with
different kinetics (II) In vivo analysis of the nano-
conjugates in a mouse model of wound healing
showing healing outcomes of wounds treated with i,
only OECs and ii/iii, OECs incubated with two dif-
ferent types of miR-dsDNA-AuNR conjugates (re-
produced with permission from Ref. [136]). ©2018
American Chemical Society.
involved during the particular treatment. Efficacy of wound dressings
and skin grafts have been constantly under evaluation through the
regulated clinical trials on patients in multiple clinical set-ups over a
period of 5–10 years [20]. The aim of clinical study is to transfer the
technology from bench to bedside for harnessing the emerging wound
management strategies and realistic advancements over the conven-
tional methods. Clinicians and scientists are nowadays applying math-
ematical calculations, statistical methods and resource expenditure as-
sessment to demonstrate the results in a more realistic and accurate
manner. Herein, we try to summarize the outcomes of some recent trial
reports on the bioengineered skin grafts, cryopreserved decellularized
or cadaveric based skin constructs and emerging usage of SVF on pa-
tients.
Clinical practices have evolved in the past few years by taking trial
deigns, patient population, accurate wound measurements and specia-
lized tools into consideration. Integra, the well-explored acellular
bioengineered skin graft has been constantly under frequent clinical
trials since last 3 decades. It is a porous scaffold made up of collagen
and glycosaminoglycan (GAG) covered with silicone layer. It is also
considered as a well-established DRT and is the best example of tissue
engineering technology for wound healing applications. In a recent
clinical trial on severely wounded military soldiers, Integra was found
to be highly successful in the management of complex and devastating
wounds with a success rate of 78–86% [2]. Integra is also commonly
used as a positive control in clinical trials of novel constructs like Cel-
lonexTM (a viscose cellulose sponge), which revealed comparable
healing efficacy of the cellulose based construct in a recent study [69].
In a comparative study with Integra, Hyalomatrix demonstrated better
cell regulation, ECM production and stimulation activity; whereas, In-
tegra showed dermal regeneration with more elastic, physical and
mechanical properties [144].
Another well-studied commercially available dermal substitute is
Dermagraft, which is made up of polyglactin mesh seeded with HDF
[145]. It serves as a cryopreserved cellular dermal substitute for chronic
wounds and has been found to be very effective in treating DFU and
venous leg ulcers [20,145]. Application of SVF in combination with
other treatments is an emerging therapeutic approach for treating burns
D. Chouhan, et al.
and scars, as revealed by a recent clinical study [111]. Successful
clinical trials on various well-established artificially developed living
cellular grafts like Apligraf, CEA and Orcel reveal their safety and ef-
ficacy [20,56]. In the past five years, huge exploration and clinical
study on the utilization of decellularized human amniotic membrane
and umblical cord (AM/UC) in treating cutaneous wounds has opened
up their potential benefits. The amniotic membrane, although an allo-
graft, is safe to use because the expression of human leukocyte antigen
(HLA) (HLA-A, -B, -DR antigens) is absent and thus such membranes do
not cause immunological rejection [20]. Grafts made up of such de-
cellularized tissues include Epifix (dehydrated human amnion/chorion
membrane), Grafix (placental allograft) and Neox (cryopreserved
human AM/UC tissue dressing) [146–148]. Their clinical trial on ex-
treme diabetic foot ulcers not only revealed their safety but also their
cost effectivity and efficient treatment. Recent report on the clinical
trial of a fibrin patch containing concentrated leukocytes and blood
platelets revealed excellent outcomes in treating DFU and also its fea-
sibility in clinical practice [149]. Application of cadaveric dermal al-
lografts on trauma wounds has been a common practice since centuries.
Although allogenic in nature, skin allografts are still considered as a
good option for treating a variety of wounds ranging from chronic
wounds to burn and trauma injuries. In a recent comparative study,
Theraskin (cryopreserved human skin allograft) proved to be cost ef-
fective (42.2% decrease in cost) in comparison to the bioengineered
skin Apligraf with no significant differences in the healing outcomes
[150]. TheraSkin cohort was $2495.33/subject in comparison to Apli-
graf cohort $4316.67/subject [150].
Although many novel types of constructs and dressing materials
have been developed in the past 2-3 decades, only a handful of products
have undergone clinical trials. Prospective and retrospective analysis of
clinical trials of the commercially available acellular and cellular skin
grafts provide a clear picture of their utility. Such studies have side by
side improved our understanding of the pathophysiology of hard to heal
wounds like chronic wounds and burn injuries. The clinical trials done
so far thus represent encouraging results in the field of wound healing
and could motivate the experts to conduct such studies on emerging
techniques and novel bioengineered grafts. We also stress upon the
future possibility of long term, multicenter follow up studies and timely
validation of the outcomes for further improvement. Overall, more re-
search is indeed needed to determine the clinical efficacy of various
novel therapeutic options to translate the technology from bench to
bedside.
7. Conclusion and future scope
Wound chronicity and poor healing associated with diabetic and
trauma wounds are serious clinical problems, affecting millions of
people around the world. Expensive treatments and long-term hospital-
stays further worsen the situation, thus creating a major economic
burden on the healthcare system. Tissue engineering concept together
with recent technological advancements has led to obtaining efficient
fabrication processes, which ultimately aim towards affordability when
produced in large scale. Technological advancement in the field of
electronics has benefitted various aspects of healthcare system. Devices
like electrospinning unit, 3D printers, spray systems are vivid examples
in this field. Further improvement in the systems has led to the devel-
opment of their portable versions in recent years. Handheld devices like
portable 3D printers and electrosprayers represent a powerful tool in
the next generation first aid systems, which may also be established in
ambulances or emergency wards. Of critical importance for autologous
cellular therapy, injectable hydrogels and cell spray systems could be
the solution for immediate wound repair surgeries in hospitals in near
future. The interdisciplinary research in the field of biomedical en-
gineering and tissue regeneration therapeutics might lead to develop-
ment of such new innovative treatment strategies.
Development of a variety of constructs in the form of wound
15
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Biomaterials 216 (2019) 119267
dressing and skin grafts provide a clear picture of the advanced ther-
apeutic strategies over the conventional gauze treatment. Ongoing re-
search in the exploration of bioactive molecules and functionalization
of constructs with those compounds has led to the fabrication of smart
wound dressings containing functional biomolecules. Improvement in
the bioprocess engineering may provide more solutions in producing
high yield of recombinant proteins like growth factors or cytokines of
human origin at an affordable cost. Recombinantly produced bioma-
terials have also emerged owing to the advanced genetic engineering
techniques and purification strategies. Production of materials con-
taining cell binding or cell stimulating peptides using recombinant DNA
technology at a large scale may be fruitful in developing bioactive
materials. Matrices made up of such materials like spider silk fusion
proteins represent one such example of multifunctional bioactive
wound dressing [10]. Exploration of novel biomaterials is also an up-
coming area of intense research in skin engineering applications.
Within the last decade, substantial emphasis has been directed to-
ward cellular therapies including pre-vascularized skin grafts or living
skin equivalents and immunomodulatory scaffolds, which recapitulate
multiple features of the native skin or normal wound milieu. The in-
corporation of various cells and bioactive factors into scaffolds has re-
sulted in achieving cell-cell and cell-matrix interactions leading to full
tissue regeneration. Researchers working on treating chronic wounds
also aim to normalize the wound conditions to achieve a regulated
wound healing cascade. The understanding of biochemical pathways
involved in the wound healing process has promoted the development
of bioactive constructs and making of engineered cells to attain regu-
lated tissue regeneration. Understanding of major signalling pathways
during wound healing process might be useful in targeting the key
regulators using siRNA or miRNA therapy. Significant improvement in
the development of wound dressings has been going on with additives
like antimicrobial molecules, immunomodulatory cytokines, growth
factors, miRNA, siRNA and exosomes. The gradual improvement in skin
bioengineering with the help of cutting-edge technology and knowledge
of molecular signalling pathways would further motivate the re-
searchers to develop efficient wound care therapeutics in future.
Notwithstanding the fact that conventional treatments were highly
successful in promoting the wound contraction and repair to some ex-
tent. In this review, we specially focussed on the recent developments in
the field of wound healing that have emerged as an improvement over
the existing strategies. The recent emerging techniques are unfolding
novel pathways and strategies to develop wound specific treatment
approach. The combined effort of various gene editing tools, material
science engineering and interdisciplinary science holds immense po-
tential in revolutionizing the current scenario. With the novel techno-
logical developments in biomedical sciences, continuous improvement
in the skin regeneration and repair is going on at a faster rate. Overall,
we support the idea that the innovative techniques in combination with
the conventional methods hold great potential in future. The future
trend of regenerative medicine and clinical studies on the same may
further open gates for newer treatments that may lead to painless, faster
and scarless healing of wounds.
Acknowledgment
BBM thankfully acknowledges funding support from Department of
Biotechnology (DBT) & Department of Science and Technology (DST),
Government of India.
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Abbreviations
3D: 3 dimensional
RNA: ribonucleic acid
miRNA: micro RNA
siRNA: small interfering RNA
ECM: extracellular matrix
PVA: poly(vinyl alcohol)
PGA: poly(glycolic acid)
PLA: poly(lactic acid)
PCL: polycaprolactone
FDA: food and drug administration
CEA: cultured epithelial autografts
BMSCs: bone marrow derived stem cells
ADMSCs: adipose derived mesenchymal stem cells
TGF-β1: transforming growth factor-beta 1
α-SMA: alpha smooth muscle actin
HDF: human dermal fibroblast
NHDF: normal human dermal fibroblasts
HUVEC: human umbilical vein endothelial cells
DRT: dermis regeneration template
FN: fibronectin
RGD: Arg-Gly-Asp
bFGF: basic fibroblast growth factor
AMP: antimicrobial peptide
SF: silk fibroin
D. Chouhan, et al. Biomaterials 216 (2019) 119267

LAMBA: light-activated, mussel protein-based bioadhesive GFP: green fluorescent protein

COL: collagen SMA: smooth muscle actin
PRP: platelet rich plasma MMPs: matrix metalloproteinases
PVP: polyvinylpyrrolidone TGFBR1: transforming growth factor beta receptor 1
PVDF: poly(vinylidene fluoride) RNAi: RNA interference
IL-4: interleukin-4 mRNA: messenger RNA
IL-10: interleukin-10 CTGF: connective tissue growth factor
PHB: poly(hydroxybutyrate) LbL: layer by layer
MSCs: mesenchymal stem cells NPs: nanoparticles
GelMA: gelatin methacrylate shRNA: short hairpin RNA
HDMECs: Human dermal microvascular endothelial cells CRISPR: clustered regularly interspaced short palindromic repeats
VEGF: vascular endothelial growth factor RISC: RNA-induced silencing complex
EGF: epidermal growth factor PLGA: poly lactic (glycolic acid)
PDGF: platelet derived growth factor LNA: locked nucleic acid
TNF-α: tumor necrosis factor alpha rhPDGF-BB: recombinant human platelet derived growth factor
SDF-1: stromal cell-derived factor-1 DFUs: diabetic foot ulcers
SMCs: smooth muscle cells GAG: glycosaminoglycan
CID: chemical inducer of dimerization HLA: human leukocyte antigen
SVF: stromal vascular fraction AM: amniotic membrane
watECs: white adipose tissue derived endothelial cells UC: umbilical cord
watASCs: white adipose tissue derived adipose stem cells OECs: outgrowth endothelial cells
ad-MVF: adipose tissue-derived microvascular fragments

19

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