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Biomimetic strategies for the
deputization of proteoglycan
functions
Ibrahim F. Rehan
1
,
2
*, Asmaa Elnagar
2
, František Zigo
3
*,
Ahmed Sayed-Ahmed
4
and Shuhei Yamada
2
1
Department of Husbandry and Development of Animal Wealth, Faculty of Veterinary Medicine,
Menoufia University, Shebin Alkom, Egypt,
2
Department of Pathobiochemistry, Faculty of Pharmacy,
Meijo University, Nagoya, Aichi, Japan,
3
Department of Animal Nutrition and Husbandry, University of
Veterinary Medicine and Pharmacy, Košice, Slovakia,
4
Department of Anatomy and Embryology, Faculty
of Veterinary Medicine, Menoufia University, Shebin Alkom, Egypt
Proteoglycans (PGs), which have glycosaminoglycan chains attached to their
protein cores, are essential for maintaining the morphology and function of
healthy body tissues. Extracellular PGs perform various functions, classified into
the following four categories: i) the modulation of tissue mechanical properties;
ii) the regulation and protection of the extracellular matrix; iii) protein
sequestration; and iv) the regulation of cell signaling. The depletion of PGs
may significantly impair tissue function, encompassing compromised
mechanical characteristics and unregulated inflammatory responses. Since
PGs play critical roles in the function of healthy tissues and their synthesis is
complex, the development of PG mimetic molecules that recapitulate PG
functions for tissue engineering and therapeutic applications has attracted the
interest of researchers for more than 20 years. These approaches have ranged
from semisynthetic graft copolymers to recombinant PG domains produced by
cells that have undergone genetic modifications. This review discusses some
essential extracellular PG functions and approaches to mimicking these
functions.
KEYWORDS
proteoglycan, glycosaminoglycan, mimetic molecule, graft copolymer, therapeutic
application
1 Introduction
Core proteins that are modified with a single or multiple glycosaminoglycan (GAG)
chains are identified as a common class of biomolecules referred to as proteoglycans (PGs).
GAGs are divided into two categories: sulfated and non-sulfated GAGs. PGs are the main in
vivo source of sulfated GAGs containing heparin, heparan sulfate (HS), keratan sulfate (KS),
chondroitin sulfate (CS), and dermatan sulfate (DS). Hyaluronan/hyaluronic acid (HA) is a
non-sulfated GAG that is a main component of the extracellular matrix (ECM), but is not
linked to a core protein (Schaefer and Schaefer, 2010).
Even though extracellular molecules make up the majority of PGs, they are also found
inside cells or bound to cell membranes. Extracellular PGs may be further separated into
three groups (Silbert, 2021). The first group consists of small leucine-rich PGs (SLRPs),
which are primarily involved in osteogenesis and bone remodeling (Walimbe and Panitch,
2020). This group contains decorin and biglycan PGs, which consist of a single or multiple
GAGs and a small protein core with a leucine-rich domain. SLRPs are divided into five
OPEN ACCESS
EDITED BY
Achilleas D. Theocharis,
University of Patras, Greece
REVIEWED BY
Pundrik Jaiswal,
National Institutes of Health (NIH), United States
Raymond Alexander Alfred Smith,
The University of Queensland, Australia
Sandra Rother,
Saarland University, Germany
*CORRESPONDENCE
Ibrahim F. Rehan,
ibrahim.rehan@vet.menofia.edu.eg
František Zigo,
frantisek.zigo@uvlf.sk
RECEIVED 26 February 2024
ACCEPTED 15 July 2024
PUBLISHED 06 August 2024
CITATION
Rehan IF, Elnagar A, Zigo F, Sayed-Ahmed A and
Yamada S (2024), Biomimetic strategies for the
deputization of proteoglycan functions.
Front. Cell Dev. Biol. 12:1391769.
doi: 10.3389/fcell.2024.1391769
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Frontiers in Cell and Developmental Biology frontiersin.org01
TYPE Mini Review
PUBLISHED 06 August 2024
DOI 10.3389/fcell.2024.1391769
distinct classes based on their genetic lineage and protein homology:
three canonical SLRP classes, types I–III, and two non-canonical
classes, types IV and V (Iozzo and Schaefer, 2015). While lumican is
a member of SLRP class II and carries 2–4 KS chains, decorin and
biglycan are members of SLRP class I and carry one and two CS/DS
chains, respectively. SLRPs contain leucine-rich repeat units.
Chondroadherin, nyctalopin, tsukushi, podocan, podocan-like 1,
and testican-1, -2, and -3 are non-canonical members that share
structural homology and have common functional characteristics
despite not carrying GAG side chains (Iozzo and Schaefer, 2015;
Gesteria et al., 2023). The second group is modular PGs, such as
perlecan, the N-terminal domain I (amino acids 1–195) of which has
three long 70- to 100-kDa GAG chains (Melrose, 2020). Perlecan is
one of the HS-PGs, but transiently displays native CS chains during
tissue morphogenesis. These CS chains are expressed by progenitor
cell populations during tissue development (Schaefer and Schaefer,
2010;Iozzo and Schaefer, 2015;Sao and Risbud, 2024). Moreover,
agrin is a large PG whose best-characterised role is in developing
the neuromuscular junction during embryogenesis. It is named
based on its involvement in the aggregation of acetylcholine
receptors during synaptogenesis. In humans, this protein is
encoded by the AGRN gene (Groffen et al., 1998;Kröger and
Schröder, 2002;Rupp et al., 2015). There are three potential HS
attachment sites within the primary structure of agrin, but it is
thought that only two of these carry HS chains when the protein is
expressed. Agrin may play an important role in the basement
membrane of the microvasculature and synaptic plasticity. Also,
agrin may be involved in blood-brain formation and/or function
(Donahue et al., 1999;Wolburg et al., 2009)anditinfluences Aβ
homeostasis (Rauch et al., 2011).
The third group is hyalectans, flexible HA-binding PGs that
have four members: aggrecan, versican, neurocan, and brevican.
Three distinct domains make up their core proteins: a HA-
binding domain, a central domain containing GAG
attachment sites, and a lectin-like domain. Depending on
PGs, between three and 100 GAG chains are attached to the
core proteins (Schaefer and Schaefer, 2010). Moreover, modular
non-HA-binding PGs are primarily found in the basement
membrane of tissues and have several forms (Schaefer and
Schaefer, 2010). Extracellular PGs have attracted the most
attention for the replication of physiological conditions
encountered by cells in their natural tissue environment.
Therefore, the main focus of this review will be extracellular
PGs and attempts to mimic their functions.
2 Functions of PGs
The activities of PGs may be classified into four primary
categories: a) the modulation of tissue mechanical properties; b)
the regulation and protection of the ECM structure; c) protein
sequestration; and d) the regulation of cell signaling. The structural
composition of PGs is significantly affected by a number of factors,
such as the size of the core protein and the type and number of
conjugated GAGs. PGs have several physiological roles inside the
human body. This section will concentrate on a few selected
examples that are significant within the discipline of tissue
engineering.
2.1 Modulation of tissue mechanical
properties
GAGs exhibit a higher degree of hydrophilicity than the
majority of other ECM constituents. This enhanced
hydrophilicity is attributed to their sulfate and carboxylate
groups, which provide negative charges that promote the
absorption of water into tissue. Water absorption typically occurs
in cartilage as well as embryonic developmental tissues (Arciniegas
et al., 2004), and brain perineural nets (Ueno et al., 2018),
particularly in regions abundant in the PG aggrecan (Knudson
and Knudson, 2001;Kiani et al., 2002). The core protein of
aggrecan is conjugated with approximately 60 KS and 100 CS
chains. To immobilize HA chains in the ECM of cartilage, the
core protein of the aggrecan molecule connects to HA via a link
protein. This binding process results in the formation of large
aggregates with a strong negative charge, which is important for
enhancing the compressive stiffness of tissue as well as its ability to
absorb water (Kiani et al., 2002).
Degenerative cartilage diseases, such as osteoarthritis (OA), may
be caused by articular surface loss, which occurs as a result of
progressive inflammation as well as excessive catabolic enzyme
production, including aggrecanases, hyaluronidases, and matrix
metalloproteases (MMPs) (Glyn-Jones et al., 2015). Close to the
HA and aggrecan junction, aggrecanases enzymatically break the
aggrecan core protein, releasing it from the network structure and
facilitating its dispersion into synovial fluid (Lohmander et al.,
1993). The first phase of OA is distinguished by the depletion of
aggrecan, which reduced the ability of cartilage to maintain water.
The promotion of cartilage loss is facilitated by an increase in the
exposure of other ECM components to MMPs and hyaluronidases,
and aggrecan loss promotes cartilage loss (Lohmander et al., 1993;
Glyn-Jones et al., 2015). Previous studies extensively examined the
biological and mechanical characteristics of aggrecan in cartilage
(Kiani et al., 2002;Huang and Wu, 2008).
2.2 Regulation and protection of the ECM
The ECM mostly contains fibrillar collagen, which is involved in
regulating the tissue structure such as skin, tendon, and mammary
gland by providing cellular support (Sun, 2021;Melrose, 2024). The
appropriate control of the production and structure of collagen
fibrils is critical for ensuring the normal formation of the ECM and
maintaining the functional properties of tissues. Decorin, biglycan,
fibromodulin, and lumican are examples of SLRPs involved in the
assembly of the ECM (Chen and Birk, 2013). Decorin is a PG that
harbors a single GAG chain primarily composed of DS. In patients
with Ehlers-Danlos syndrome who lack Carbohydrate
Sulfotransferase 14 (Chst14), this DS chain is substituted with CS.
Decorin is a myokine that aids in the development of skeletal muscle
in Chst14-deficient mice and is essential for the assembly of collagen
fibrils (Miyake et al., 2010). Moreover, the binding of the core
protein of SLRPs to collagen markedly affects the regulation of fibril
development during collagen fibril growth (Douglas et al., 2006).
The primary role of the decorin core protein is to facilitate the
connection between PG and collagen. However, GAGs linked to the
core protein also participate in charge-based interactions with
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surrounding collagen fibrils (Henninger et al., 2007). PGs and
collagen fibril networks contribute to the properties and structure
of the ECM, including mechanical stability and hydration (Robinson
et al., 2005;Robinson et al., 2017).
Along with managing the organization as well as synthesis of
collagen, SLRPs prevent its degradation by proteolysis. SLRPs
safeguard collagen against destruction by shielding the external
surface of collagen fibrils, thereby impeding the interaction
between proteases and collagen (Geng et al., 2006). PGs other
than SLRPs have been shown to protect against matrix
proteolytic degradation. Aggrecan was found to impede the
degradation of collagen II by inhibiting the access of MMPs to
its significant bottlebrush structure and excluded volume (Pratta
et al., 2003). In degenerative diseases, the degradation of PGs by
proteases exposes collagen, rendering it susceptible to degradation
(Ni et al., 2014). Further details on the effects of PGs on the matrix
structure are provided in a number of reviews (Chen and Birk, 2013;
Pang et al., 2019).
Elastins and fibronectin play a crucial role in the maturation and
tissue specificity of the ECM. The binding of DS-PGs to plasma
fibronectin (pFN) inhibits its interaction with multiple cell surface
determinants (Laterra et al., 1983a;Laterra et al., 1983b;Woods
et al., 1984). The binding of DS-PGs to pFN can interfere with the
binding of pFN to the 140-kD glycoprotein receptor (Brown and
Juliano, 1985;Cben et al., 1985;Giancotti et al., 1985;Pytela et al.,
1985) or a possible second receptor for fibronectin (Aplin et al.,
1981;Nagata et al., 1985;Urushihara and Yamada, 1986;Waite et al.,
1987). Evidence for a conformational change upon GAG binding
has been recently reported for human pFN (Tooney et al., 1983;
Ostedund et al., 1985).
2.3 Sequestration of proteins
GAGs interact with not only matrix components, but also many
different types of proteins, including morphogens, proteases,
chemokines, and growth factors (GFs), to change their biological
activity. These interactions are accomplished by the cationic domain
of a protein, which consists of clusters of basic residues surrounded
by one or two non-basic residues (Cardin and Weintraub, 1989;
Fromm et al., 1997;Muñoz and Linhardt, 2004). However, the
composition of the cationic domains of GAG-binding proteins
varies, suggesting that specific sequences may not necessarily be
required for interactions with GAGs. Alternatively, proteins may
effectively bind to GAGs using a similar spatial structural motif, in
which basic residues are close to one another in space, but not
necessarily in the amino acid sequence (Zhang et al., 2019). In some
case, the sulfation pattern of GAGs may also affect how proteins
bind to them. For example, the primary binding interaction between
the heparin-binding (HB) protein antithrombin III and heparin is
mediated by a distinct sulfation pattern of pentasaccharides
GlcNAc(6-O-sulfate)-GlcA-GlcN (2-N-sulfate, 3-O-sulfate, 6-
O-sulfate)-IdoA (2-O-sulfate)-GlcN (2-N-sulfate, 3-O-sulfate, 6-
O-sulfate (Yamada, 2019), where GlcNAc, GlcA, GlcN, and IdoA
represent N-acetyl-D-glucosamine, D-glucuronic acid,
D-glucosamine, and L-iduronic acid, respectively, observed on a
heparin molecule subset (Lindahl et al., 1980;Atha et al., 1985;
Merry et al., 2022). Moreover, the sequence required for the binding
to basic fibroblast growth factor (bFGF) was a pentasaccharide
containing N-sulfated GlcN residues and a 2-O-sulfated IdoA
residue (Maccarana et al., 1993;Purushothaman et al., 2012). A
previous study reported that the saccharide sequence of 6-O-sulfated
oligosaccharide required for FGF signaling (Fernig D.G and
Gallagher J.T., 1994;Lindahl et al., 1999;Gallagher, 2001;
Turnbull et al., 2001;Kreuger et al., 2005;Sugaya et al., 2008).
Binding to GAGs immobilizes the protein, thus constraining
and regulating its biological activity. The majority of membrane-
bound PGs can also function as soluble autocrine or paracrine
effectors as their extracellular domains, are enzymatically cleaved
and released from the cell surface. In particular, the ectodomain
shedding of syndecans, a major family of cell surface HS PGs, is an
important posttranslational mechanism that modulates diverse
pathophysiological processes. Syndecan shedding is a tightly
controlled process that regulates the onset, progression, and
resolution of various infectious and noninfectious inflammatory
diseases (Nam and Park, 2012;García et al., 2016). HS-PGs bind to a
number of GFs, such as members of the FGF (Ornitz, 2000), vascular
endothelial growth factor (VEGF) as stated by Robinson et al., 2006,
and platelet-derived growth factor (PDGF) families. HS-PG perlecan
is ubiquitous throughout the body, but is mainly localized in the
basement membrane (Whitelock et al., 2008). Perlecan plays a
crucial role in establishing GF gradients to ensure proper tissue
development by binding to GFs. This was confirmed using perlecan
knockout models, in which the lack of perlecan led to tissue defects,
including impairments in endochondral ossification and
cardiovascular development (Arikawa-Hirasawa et al., 1999;
Costell et al., 1999;Zoeller et al., 2008). The formation of tissues
is disrupted by the lack of perlecan, while embryos experience
premature mortality, thereby highlighting the crucial role of
perlecan in maintaining optimal organ functionality. More details
are provided in a number of reviews (Whitelock et al., 2008;Zoeller
et al., 2008).
2.4 Regulation of cell signaling
PG-rich layers, such as the endothelial glycocalyx present in the
vasculature, may prevent cell-cell interactions because of the anionic
charge and steric hindrance of PGs (Reitsma et al., 2007;Tarbell and
Cancel, 2016;Melrose, 2024). Consequently, no direct interactions
may exist between vascular endothelial cells and circulating
erythrocytes, leukocytes, and platelets in the blood. Upon injury
to the glycocalyx from physical stress or the enzymatic degradation
of GAGs, surface proteins on the endothelium, including selectins,
VCAM, and ICAM, become visible (Lipowsky, 2012). Furthermore,
the glycocalyx is involved in leukocyte recruitment, activation,
arrest, and migration into the surrounding tissues. Effective
artery repair necessitates this process. However, the inadequate
restoration of the glycocalyx results in the unregulated activation
of leukocytes, which induces uncontrolled inflammation at sites of
injury (Reitsma et al., 2007). In addition to being structural proteins,
PGs play a major role in signal transduction with regulatory
functions in various cellular processes. Being mostly extracellular,
they are upstream of many signaling cascades. They are capable of
affecting intracellular phosphorylation events and modulating
distinct pathways, including those driven by bone morphogenetic
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protein/transforming growth factor superfamily members, receptor
tyrosine kinases, the insulin-like growth factor-I receptor, and Toll-
like receptors. Mechanistic insights into the molecular and cellular
functions of PGs have revealed both the sophistication of these
regulatory proteins and the challenges that remain in uncovering the
entirety of their biological functions (Schaefer and Schaefer, 2010).
3 Biomimetic strategies to replace PGs
Due to the significant contributions of PGs to the functions of
healthy tissues, researchers have focused on developing methods
that recapitulate these functions. Various techniques that simulate
PG functions, including the use of recombinant PG domains and the
development of semi-synthetic PG mimics, have been reported (see
Figure 1). Four main goals to mimetic PG functions are
highlighted herein.
3.1 GAG bottlebrush graft copolymers
The use of graft copolymers to replicate the three-dimensional
bottle brush-like structure of hyalectans, such as aggrecan, is one
method for recapitulating the functions of PGs (Figure 1A). The
types of biomimetic molecules are listed in Table 1. The grafting of
CS preparations with a single terminal primary amine group to a
synthetic poly (acryloyl chloride) backbone creates an aggrecan
mimetic (Sarker et al., 2012;Prudnikova et al., 2017;Prudnikova
et al., 2018;Clarke et al., 2024)(Figure 2A). Biomimetic PGs have
been synthesized by combining 174 CS molecules (~22 kDa) that
were grafted onto a linear polymer of poly (acrylic acid) (PAA)
backbone utilizing a reaction between a primary amine at the
terminal end of CS and the acrylic acid groups in the PAA
backbone (Prudnikova et al., 2018). This technique may be used
to create small and large PG mimics with sizes ranging from a 10-
kDa polyacrylate core with 7–8 CS chains attached (Prudnikova
et al., 2017) to a 250-kDa core polymer with 60 CS chains attached
(Prudnikova et al., 2018). The swelling of the molecule was found to
be superior to that of aggrecan and unconjugated CS (Prudnikova
et al., 2017). Moreover, much like SLRPs indicating the polymers
behave like SLRPs with regards to controlling collagen fibril
formation (Moorehead et al., 2019). Since negatively charged
PAA did not affect the morphology of collagen fibrils, the CS
structure rather than core proteins was considered to be
important for controlling fibril formation. CS, either in its free-
floating or PG form, interacts with collagen to regulate the kinetics
of fibril formation and changes in diameters and band spacing
(Prudnikova et al., 2018).
One further method used to simulate aggrecan activity is a graft
copolymer made of heparin or CS chains attached to the HA
backbone through their reducing ends (Figure 2B)(Place et al.,
2014b;Pauly et al., 2017). Sarkar et al. (2012) attempted to create
graft copolymers in distinct compositions by utilizing four different
ratios of either CS or heparin to HA-N-[β-maleimidopropionic acid]
hydrazide trifluoroacetic acid salt (BMPH) (Prudnikova et al., 2018).
Graft copolymers with 1:1, 1:3, 1:10, and 1:30 ratios were produced
by mixing a stoichiometric amount of heparin or CS with HA-
BMPH (one polymer chain per thiol group in the HA-SH
FIGURE 1
PG functions are recapitulated by biomimetic methods. (A) GAG bottlebrush polymers, graft copolymers are used to replicate the bottle brush-like
structure of hyalectans like aggrecan, replicating their functions by grafting CS preparations to a synthetic poly (acryloyl chloride) backbone; (B) polymer/
substrate-binding peptide conjugates, functional mimetics can be created by grafting substrate-binding peptides onto polymer matrices, which bind to
HA or collagen, providing protection or imitating PGs organizational characteristics within the ECM; (C) recombinant PG (perlecan) domains,
recombinant PG domains, such as terminal recombinant perlecan domains (rPlnDs), are used to replicate structural conformation of PGs while allowing
for modification of their characteristics; and (D) GAG-incorporating materials, several research groups have opted for integrating GAGs into biomaterials
instead of developing bespoke compounds, with heparin and CS being commo nly used due to their commercial accessibility. A number of strategies have
been developed to mimic the functions of PGs, ranging from the application of recombinant PG domains to the synthesis of PG mimetics. Blue dotted
lines, red dotted lines, green dotted lines, a mixture of blue dotted and straight lines, a mixture of red dotted and straight lines, black lines, or red stars
represent CS, HA, heparin, CS-bristle, HA-binding peptide, synthetic polymer, and GF, respectively.
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intermediate). The 1:1 ratio showed the highest graft density. Only
1/30 of the stoichiometric amount of CS or heparin was made when
the 1:30 ratio was used. After converting the carboxylic acid groups
of HA to hydrazides, the graft polymer was synthesized by reductive
amination. Smaller molecular weight polysaccharides decreased the
modulus of the hydrogels, while larger molecular weight
polysaccharides and graft copolymers increased the modulus
(Pauly et al., 2017). The modulus of an agarose hydrogel was
enhanced by the addition of the CS graft copolymer, and a
similar effect was observed with the addition of free CS. These
two additives were suggested to affect mechanical properties
through different mechanisms. Free CS increased the modulus by
chain entanglement, while the CS graft copolymer increased the
modulus through osmotic pressure. However, the incorporation of
the CS graft copolymer in agarose gels reduced cell viability,
indicating the need for the further optimization of the mimetic.
Although these mimetic strategies imitate the brush structure of
hyalectan PGs by grafting GAGs onto a core polymer, they do not
encompass the functions of the core protein (Pauly et al., 2017).
Brush PGs are intentionally engineered with a reduced binding
affinity towards HA in order to promote tissue localization and
minimize unintended molecular diffusion (Pratta et al., 2003;Pauly
et al., 2017;Moorehead et al., 2019). Within the realm of therapeutic
interventions, this process may give rise to a gradual increase in the
dosages of hyalectan PGs required to maintain optimal performance
(Maneiro et al., 2004). However, the advantages of increased
resistance to proteolytic breakdown, which may be accomplished
by replacing the protein core with an alternative polymer, may
outweigh the disadvantages that arise from the loss of protein core
functions. Further research is required to obtain a more accurate
assessment of the equilibrium between functional imitation and
effective therapeutics (Pratta et al., 2003;Pauly et al., 2017;
Moorehead et al., 2019;Murphy et al., 2019;Hwang et al., 2020;
Lanzi et al., 2020;Rizzo et al., 2022;Yao et al., 2022;Zhang et al.,
TABLE 1 The types of biomimetic molecules of PGs.
# Type Molecule/
Peptide
Details References
(A) Biomimetic Molecule
1 GAG-PAA BPG250 BPG250 contains of 174 CS molecule (~22 kDa), which were
grafted onto a polymer of PAA backbone utilizing a reaction
between a primary amine at the terminal end of CS and the
acrylic acid groups in the PAA backbone (Figure 2A)
Sharma (2012), Prudnikova et al. (2018), Phillips et al. (2019a)
BPG10 BPG10 consists of a ~10 kDa synthetic PAA core, decorated
with ~5–7 CS-GAG bristles (Figure 2A)
Kahle et al. (2022).
2 GAG-
BMPH-HA
Heparin-
BMPH-HA
Graft copolymers of heparin or CS with HA-BMPH (one
polymer chain per thiol group in the HA-SH intermediate)
(Figure 2B)
Sarkar et al. (2012),Place et al. (2014a), Pauly et al. (2017),
Prudnikova et al. (2018)
CS-BMPH-HA
3 DS-PLL PCNs Prepared by a polymer-polymer pair reaction method and
characterized for physicochemical properties. It is DS with
poly-
L
-lysine (DS-PLL)
Zandi et al. (2020).
4 GAG-poly
glycerol
Star-like PG Grafting high molecular weight GAGs such as heparin or CS to
hyperbranched synthetic cores like polyglycerol using oxime
condensation (Figure 2C)
Novoa-Carballal et al. (2018).
5 CS-collagen CSCL Crosslinking CS onto a collagen-based scaffold. Corradetti et al. (2016).
6 Collagen-
HA-GAG
aCol-aHA-GAG Chemically modified the collagen and HA are co-precipitated
with GAGs. A bio-inspired nano-material recapitulating the
composition, ultra-structure and function of the GAG-rich
ECM is fabricated
Yang et al. (2023).
(B) Peptide-based Mimetic Strategies
1 Peptide-
CS/DS
GAH-oxidized CS The peptide-glycan compounds prepared by this strategy
include peptides that bind to ECM and are conjugated to a
GAG backbone. These peptides, such as HA-binding peptides
(GAH), are grafted to the oxidized CS/DS backbone
(Figure 2D)
Bernhard and Panitch (2012), Sharma et al. (2013a), Zhang,
et al. (2014), Lawrence et al. (2015), Sharma et al. (2016),
Twitchell et al. (2020)
DS-SILY Collagen-binding peptides (SILY) conjugated to a DS backbone Paderi and Panitch (2008), Scott et al. (2013)
Lubricin mimic Attaching type II collagen- and HA-binding peptides to a CS
backbone
Lawrence et al. (2015).
2 Peptide-PEG HA-binding
peptide-PEG
HA-binding peptides are grafted onto PEG Singh et al. (2014), Faust et al. (2018)
aCol-aHA; aminated collagen-aminated hyaluronan/hyaluronic acid-glycosaminoglycan; BMPH, N-[β-maleimidopropionic acid] hydrazide trifluoroacetic acid salt; BPGs, biomimetic
proteoglycans; CS, chondroitin sulfate; CSCL, collagen-based scaffold; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronan/hyaluronic acid; PAA, polymer of poly(acrylic
acid); PCM, pericellular matrix; PCN, polyelectrolyte complex nanoparticles; PEG, poly(ethylene glycol); PLL; poly-
L-
lysine.
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2022;Abdelfadiel et al., 2023;Heide et al., 2023;Kardeby et al., 2023;
Xie et al., 2023;Zhang et al., 2024).
3.2 Peptide-based mimetic strategies
An alternative approach to fabricating functional mimetics
comprises the incorporation of substrate-binding peptides onto
polymer matrices (Figure 1B). These peptides have a propensity
to bind to HA or collagen, resembling the binding domains of
hyalectans or SLRPs. It is possible to offer protection or imitate the
organizational characteristics of PGs within the ECM after grafting
these peptides onto polymers.
The peptide-glycan compounds prepared by this strategy
include peptides that bind to the ECM and are conjugated to a
GAG backbone (Petrovic, 2024). These peptides, such as HA-
binding peptides (GAH), are grafted to the oxidized CS backbone
(Figure 2C). Although PGs often have a single ECM-binding
domain, mimetics were generated by grafting 5–15 peptides onto
the GAG backbone, taking advantage of avidity to enhance target
binding. This methodology utilizes avidity as a means to augment
binding affinity towards the desired target. The use of an aggrecan
mimetic, a CS backbone grafted with HA-binding peptides,
demonstrated that this strategy supported the protection of the
ECM against its proteolytic degradation (Bernhard and Panitch,
2012;Sharma et al., 2013b;Zhang, et al., 2014;Sharma et al., 2016). A
lubricin mimetic was created by combining HA-binding peptides
with a CS backbone (Lawrence et al., 2015;Twitchell et al., 2020),
and it was placed on the surface of articular cartilage, which lowered
the coefficient of friction. Previous studies demonstrated that a
synthetic compound resembling decorin offered a safeguard
against the breakdown of proteins by enzymes and controlled the
formation of collagen fibers. This is achieved by the compound’s
interaction with collagen via specific peptide sequences that are
connected to the decorin DS backbone (Paderi et al., 2009;Stuart
et al., 2011).
This approach effectively restored the glycocalyx after vascular
endothelial denudation by the collagen-binding decorin mimetic
(Scott et al., 2013), called DS-SILY, which markedly inhibited
platelet activation by covering exposed collagen, thereby aiding in
the suppression of vascular intimal hyperplasia. Furthermore, DS
grafted with selectin-binding peptides was utilized to protect an
inflamed endothelium (Dehghani et al., 2020). Both mimics were
located in the damaged vessel to recapitulate the GAG barrier by
preventing vessel interactions with neutrophils and circulating
platelets. Since peptides that bind to HA have been shown to
reduce friction on the surface of articular cartilage (Singh et al.,
2014;Lawrence et al., 2015), a lubricin mimic was developed by
grafting HA-binding peptides onto poly (ethylene glycol) (PEG)
(Singh et al., 2014;Faust et al., 2018;Zhou et al., 2022). The PEG-
based lubricin mimic was administered through an intravenous
injection into an OA animal. The molecule subsequently bound to
HA, causing HA to accumulate in the cartilage and reduce joint
friction (Singh et al., 2014). The application of this specific molecule
FIGURE 2
Representative graft-copolymer PG techniques are shown: (A) CS grafted to a poly (acryloyl chloride) backbone, modified from Prudnikova et al.
(2018);(B) the HA backbone was grafted with heparin or CS, modified from Place et al. (2014a);(C) Grafting high molecular weight GAGs such as heparin
and CS to hyperbranched synthetic cores like polyglycerol using oxime condensation, modified from Novoa-Carballal et al. (2018);and(D) the oxidized
CS/DS backbone was conjugated to the linking compound BMPH and reacted with the synthetic HA-binding peptide GAHWQFNALTVRGGGC
(referred to as GAH) or WYRGRLGC (collagen type II binding peptide, referred to as WYRGRL), modified from Bernhard and Panitch (2012),Sharma et al.
(2016), respectively.
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TABLE 2 Summary of PGs and their therapeutic applications.
PGs Predominant
GAG
Therapeutic application Function References
Glypican 1–6 HS Ischemic wound healing Developmental morphogenesis Monteforte et al. (2016).
Suppressing metastasis in gastric
cancer
Han et al. (2016).
Syndecan 1–4 HS Diabetic wound healing Cell adhesion; binding to FGF
and other growth factors
Das et al. (2016a),Das et al. (2016b), Das et al. (2016c)
Aggrecan CS, KS Osteoarthritis Mechanical support; forms large
aggregates with HA
Bernhard and Panitch (2012), Sarkar et al. (2012),
Sharma et al. (2013a), Place et al. (2014a), Place et al.
(2014b), Sharma et al. (2016), Prudnikova et al.
(2017), Prudnikova et al. (2018), Phillips et al.
(2019b)
Decorin DS Macular degeneration, diabetic
retinopathy, diabetic macular edema
TGF-ß binding and
Fibrillogenesis
Devore et al. (2010).
Corneal wound healing Grisanti et al. (2005), Hill et al. (2018)
Anti-scarring Stuart et al. (2011), Ahmed et al. (2014), Wang et al.
(2016)
Oncosupression Yang et al. (2015), Wang et al. (2016), Oh et al. (2017a),
Liu et al. (2017)
Abdominal aortic aneurysm Shen et al. (2017).
Vascular neointimal hyperplasia Paderi et al. (2011), Scott and Panitch (2014), Scott et al.
(2017)
Lumican KS Corneal wound healing Cell adhesion Chakravarti (2002), Gesteira et al. (2017)
Bacterial lung infections Shao et al. (2012), Shao et al. (2013)
Scarring Yeh et al. (2005), Liu et al. (2013), Yamanaka et al.
(2013), Zhao et al. (2016)
Melanoma Zeltz et al. (2009), Pietraszek et al. (2013)
Biglycan CS Duchenne muscular dystrophy Cell adhesion Amenta et al. (2011), Ito et al. (2017), Fallon and
McNally (2018)
Fibromodulin KS Diabetic wounds and neuropathy Cell adhesion and fibrillogenesis Jian et al. (2013), Zheng et al. (2014), Jazi et al. (2016)
Neointimal hyperplasia Ranjzad et al. (2009).
Bone regeneration Zheng et al. (2012), Li et al. (2016)
Tendon healing Delalande et al. (2015).
Breast cancer metastasis Dawoody et al. (2017).
Lubricin None Osteoarthritis Lubrication Ludwig et al. (2012), Wathier et al. (2013), Lawrence
et al. (2015), Iqbal et al. (2016), Larson et al. (2016),
Lakin et al. (2019)
Ocular applications, dry eye Oh et al. (2017b), Lambiase et al. (2017), Samsom et al.
(2018)
Perlecan HS Cartilage regeneration Stability of basement membranes
and providing filtration barrier
French et al. (2002), Yang et al. (2006), Jha et al. (2009),
Srinivasan et al. (2012)
Ischemic wound healing Aviezer et al. (1994), Zoeller et al. (2009)
Neurovascular dysfunction Clarke et al. (2012), Parham et al. (2016)
Stroke and vascular dementia Lee et al. (2011), Marcelo and Bix (2015)
Neointimal hyperplasia Rnjak-Kovacina et al. (2016).
CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; HS, heparan sulfate; KS, keratan sulfate; PG, proteoglycan.
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in a murine model of OA suppressed disease progression, which
appeared to be attributed to the higher concentration of HA on the
cartilage surface (Faust et al., 2018).
Moreover, as shown in Figure 2C, grafting high molecular
weight GAGs such as heparin and CS to hyperbranched synthetic
cores like polyglycerol using oxime condensation (Novoa-Carballal
et al., 2018).
Although the structures of these molecules differ from those of
the PGs they mimic, they have the functions of the binding domains
on the core proteins of PGs, leading to their local retention in order
to extend the duration of their therapeutic effect. Nevertheless, it is
important to note that the ability of these synthetic analogs to fully
replicate the functions of PGs may be limited due to their smaller
dimensions than PGs and the utilization of their peptides may
render them more vulnerable to destruction by peptidases
(Figure 2D). Moreover, in Table 2, we have summarized the
therapeutic applications of PGs, modified from recent
publications (Walimbe and Panitch, 2020;Rizzo et al., 2022;
Zhang et al., 2022;Yao et al., 2022;Abdelfadiel et al., 2023;
Heide et al., 2023;Kardeby et al., 2023;Xie et al., 2023;Zhang
et al., 2024).
3.3 Recombinant protein domains
Recombinant PG domains represent an alternative approach for
replicating the structural conformation of PGs, while it retains the
flexibility to change their characteristics (Figure 1C). While
pioneering researchers have focused on the roles of GAGs and
PGs for years, in the last two decades the staggering potential of PGs
to modulate tissue environments has been more broadly
appreciated. Their multifunctional biological processes, in
particular, their ability to bind and sequester GFs and interact
with various ECM molecules and influence cellular signaling
events, makes them extremely attractive drug conjugates for
multiple disease indications. Table 2 summarizes common PGs
and their therapeutic applications. Clinical translation of these
molecules, however, remains a challenge. Due to the advent of
recombinant technology, adenoviral and non-viral gene transfers
are attractive alternatives to purify native PGs, a task that is
considered extremely difficult and time intensive. However, while
recombinant technology can synthesize core proteins of PGs fairly
consistently, their post-translational GAG chain modifications
remain a challenge. Some GAG chain structures require enzymes
in the Golgi apparatus only found in multicellular eukaryotes, but
absent in single celled organisms used to synthesize recombinant
PGs. Effectively conveying the mechanism of action of these drugs
also remains a significant challenge, due to the diverse processes
with which these molecules interact.
Terminal recombinant perlecan domains (rPlnDs) with the
native attachment of HS chains are commonly utilized for this
purpose (Whitelock et al., 2008). These rPlnDs may be produced by
transfecting mammalian cells, leading to the production of the core
protein and the HS chains to be connected thereafter (Chiu et al.,
2016;Hubka et al., 2019). To regulate the interaction, exhibition, and
liberation of GFs that have an affinity for heparin, this methodology
was employed to fix rPlnDs onto diverse surfaces (Knox and Merry,
2002;Marneros and Olsen, 2005;Whitelock et al., 2008). The
Farach-Carson group conducted an experiment wherein they
utilized rPlnDI to demonstrate the prolonged liberation of BMP-
2 from a scaffold made of poly (ε-caprolactone) (PCL) through the
process of electrospinning (Chiu et al., 2016). The achievement of
this task involved the covalent attachment of rPlnDI to scaffold
fibers before the addition of BMP-2 (DeCarlo et al., 2012;Chiu et al.,
2016). The immobilization of rPnlDI resulted in the greater loading
of BMP-2 within the scaffold than with a control PCL scaffold.
Additionally, the immobilized scaffold exhibited the prolonged
release of BMP-2 over an extended duration. The research team
also employed a custom-designed microfluidic device fabricated by
3D printing technology to generate varying concentrations of
rPlnDI. These concentration gradients were subsequently utilized
to induce corresponding gradients of FGF-2 within the hydrogel
matrix (Hubka et al., 2019). In contrast to hydrogels with a uniform
distribution of FGF-2, the presence of a gradient of FGF-2 facilitated
enhanced cell migration. Furthermore, rPnlDI was successfully
attached to microparticles to facilitate the regulated release of
GFs, such as BMP-2. The release kinetics of this technique were
superior to the distribution of GF by a free delivery system (Jha
et al., 2009).
Moreover, the anabolic process of chondrocyte aggrecan
recombination is very dynamic during the development of OA.
The domains of aggrecan protein contain several cutting sites
susceptible to MMPs, ADAMTS, and other enzymes (Santamaria
and Yamamoto, 2020). Aggrecan plays an important role in
mediating chondrocyte-chondrocyte and chondrocyte-matrix
interactions through its ability to bind HA (Kiani et al., 2001;
Kiani et al., 2002;Miwa et al., 2006). Given that the biological
function of aggrecan depends on fixing charges in the cartilage ECM,
tethering the PG is vital. The lectican PGs are incorporated into the
ECM through specific interactions with HA and other ECM
components. These interactions are mediated through their
globular domains. Recent findings of missense mutations in the
aggrecan genes in patients with different skeletal disorders
emphasize the importance of the globular domains of aggrecan
(Aspberg, 2012).
In the central and peripheral nervous system, versican is
expressed by glial cells and is implicated in the regulation of
cell adhesion, migration, pattern formation, and regeneration.
Recombinant versican specifically binds HA and does not bind
to heparin or CS. The transfected fibroblasts make a 78-kDa
truncated form of versican that also binds HA but not the
related polysaccharides, showing that the HA-binding activity
resides at the N-terminus of versican. The binding of versican
to HA is substrate-concentration -dependent and time-dependent,
and can be competed HA with unlabeled versican. Versican
interacts with tenascin-R via its C-typelectindomain(Asperg
et al., 1995). This interaction has been shown to contribute to the
formation of perineuronal nets around neuronal cells towards the
end latter stages of brain development, a process that is thought to
inhibit synaptic plasticity (Carulli et al., 2006;Anlar and Gunel-
Ozcan, 2012). Through the same lectin domain versican can also
interact with fibulin-1, fibulin-2 and fibrillin-1 (Asperg et al., 1999;
Olin et al., 2001;Isogai et al., 2002). As these proteins are associated
with elastic microfibrils, it is hypothesized that these complexes
may also play a role in regulating elastogenesis (Wight, 2002;
Bashur and Ramamurthi, 2012).
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Several studies have examined the recombinant domain V of the
Cterminus of perlecan. Endorepellin has been characterized as a
potent anti-angiogenic molecule with a stated form covering the
domain from Glu3687 to Ser4391. However, a large form known as
rPlnDV, which contains the amino acids Leu3626 to Ser4391, has
been shown to promote angiogenesis (Rnjak-Kovacina et al., 2017).
Despite the two reported recombinant domains sharing the integrin
α2β1-binding site, the latter has an HS or CS chain attached, whereas
endorepellin has no GAG chains, indicating the significance of the
GAG chain in the signal transduction of angiogenic GFs (Rnjak-
Kovacina et al., 2017). Lin et al. (2020) examined whether the
mimetic of the large GAG-bound molecule promotes
angiogenesis by potentiating available GFs both in their soluble
and immobilized forms to silk fibroin scaffolds. Following the
removal of GAG chains from rPlnDV, cells did not produce
proangiogenic signals, indicating that GAG chains are necessary
for GF potentiation.
Concerning the potential of an immune response to the protein
core of recombinant PGs. For instance, syndecan, perlecan, biglycan,
decorin, lumican, and especially versican, provide fine control of
innate immunity by binding to a number of recognized
immunoregulatory molecules—chemokines, cytokines, GFs, and
MMPs—mostly mediated by ionic charge interactions, as
extensively discussed elsewhere (Schmidtchen et al., 2001;
Venkatesan et al., 2002;Gill et al., 2010;Kehlet et al., 2017;Kang
et al., 2018). Therefore, innate immune signaling by PGs indicates
the implications and direction to define great progress in this area of
ECM biology. Since recombinant PG domains comprise both GAG
chains and parts of the core protein, the molecules produced are
similar to their respective PGs in terms of both form and function.
This is the most intricate and costly mimetic approach because it
requires genetically modified cells to synthesize molecules.
3.4 GAG-functionalized materials
To recapitulate the GF-binding ability of PGs, many groups have
opted to use unmodified GAGs as biomaterials, rather than
synthesizing specialized molecules. Heparin is commercially
available and which is generally derived from porcine intestine
(Kim et al., 2018;Levinson et al., 2019;Hettiaratchi et al., 2020;
Subbiah et al., 2020;Wang et al., 2020), as well as CS preparations
(Karumbaiah et al., 2015;Betancur et al., 2017;Birdwhistell et al.,
2018), respectively, are commonly employed in this context due to
their widespread commercial accessibility.
A modular system of biohybrid hydrogels based on covalently
cross-linked heparin and star-shaped poly (ethylene glycols) (star-
PEG) in which network characteristics can be gradually varied while
heparin contents remain constant (Freudenberg et al., 2009).
Moreover, Levinson et al. (2019) established a hydrogel
composed of HA and heparin, which was a carrier for TGF-
β1 and chondrocytes (Figure 1D). This study described the
utilization of two hydrogels, one of which incorporated HA
crosslinked with heparin, while the other hydrogel consisted of
heparin without any cross-linking. Both gels were formulated with
TGF-β. The gel with cross-linked heparin showed a sustained release
profile, whereas the gel with uncross-linked heparin exhibited a
bigger initial burst release of GF. The production of collagen II and
GAG was greater by encapsulated cells than by those cultured in
medium supplemented with TGF-β(Levinson et al., 2019). In an
investigation of the treatment of ischemic wounds, Kim et al. (2018)
successfully trapped VEGF using a gelatin cryogel functionalized
with heparin. Since the level of heparin integrated was elevated, a
larger amount of VEGF was preserved over time. In a rat ischemia
hind limb model, the construct was implanted with NIH-3T3
fibroblasts and VEGF-laden cryogels. The induction of
angiogenesis was weaker by gels loaded with only VEGF or cells
alone than by a gelatin cryogel functionalized with heparin,
suggesting that sequestered GFs by sulfated GAGs have an
impact on tissue engineering. Other research groups reported
similar findings, namely, the enhancement of tissue strength was
achieved through the sequestration of GFs by their interaction with
sulfated GAGs (Yu et al., 2000;David et al., 2014;Diana et al., 2017;
Daniel et al., 2019;Bryce et al., 2023). The spatial patterning of GFs
may be achieved by incorporating GAGs using techniques such as
controlled deposition (Oh et al., 2018) and 3D printing (Wang et al.,
2020). Moreover, an injectable clinical biomaterial must meet
marketing, regulatory, and financial constraints to provide
affordable products that can be approved, deployed to the clinic,
and used by physicians. Many HA-derived hydrogels can deliver
cells and therapeutic agents for tissue repair and regeneration
(Burdick and Prestwich, 2011).
Sulfated HA, especially high-sulfated HA (hs-HA), blocks
Heparanase (Hpse)-mediated enzymatic actions and cellular
functions, that is, invasion into the surrounding ECM and Hpse-
mediated upregulation of the chemokine CCL2 released from colon-
26 carcinoma cells. Therefore, sulfated HA is potentially considered
as anti-metastatic and anti-inflammatory agent via inhibition of
Hpse functions (Shi et al., 2022). The sulfation involves the C-6 and
C-4 positions of glucosamine and the C-2 and C-3 positions of
glucuronic acid. Moreover, sulfated HA has also been employed in
the sequestration of GFs. The advantage of this strategy is that the
degree of sulfation may be controlled, thereby lowering the chance of
heparin intrinsic antithrombotic activity (Feng et al., 2017;Thönes
et al., 2019).
The sequestration and controlled release or presentation of GFs,
such as FGF-2 (Karumbaiah et al., 2015), VEGF (Kim et al., 2018;
Subbiah et al., 2020), transforming growth factor-beta (TGF-β)
(Birdwhistell et al., 2018;Levinson et al., 2019), BMP-2
(Hettiaratchi et al., 2020;Subbiah et al., 2020), and nerve growth
factor (Oh et al., 2018), have been achieved by integrating these
proteins into materials.
4 Conclusion and prospective
Efforts to mimic PGs in the cellular environment have mainly
focused on ECM PGs and membrane-anchored PGs. PG mimetic
strategies have varied, with most incorporating GAGs. However, due
to the complexity of PG structures, no mimic fully replicates their
functions. Bottlebrush copolymers, which lack HA-binding sites,
may regulate swelling behavior, but do not fully replicate their
structural characteristics. Recombinant PG domains can
accurately replicate native PGs; nevertheless, PG biosynthesis and
genetic engineering pose challenges to the scaling up of recombinant
technology. Due to their greater degree of control over synthesis and
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Rehan et al. 10.3389/fcell.2024.1391769
optimization, cost effectiveness, and ability to impart bioactivity
comparable to that of their native equivalents, PG mimetics are
becoming more common. However, there are difficult challenges
with synthetic methods when it comes to producing quantities that
are beneficial to the market. The next-generation of PG therapeutics
to target a wide range of disorders is being developed by scientists for
the synthetic GAG and sequencing, as well as knowledge of the
kinetics of PG binding interactions with growth factors. To improve
binding interactions, important characteristics of GAG length and
sulfation along with interactions with core proteins are being
changed. Interest in concentrating on the precise and regulated
release of these PGs is also growing. For improved treatments,
engineering PGs to sequester and regulate GF release is being
investigated. To develop targeted and effective treatments,
strategies for targeting particular tissues are being investigated.
One such strategy involves taking use of core proteins”capacity
to attach to collagen or HA. In order to manage and adjust particular
sulfation patterns, binding potential, and specificity of mimetics to
build novel ways of influencing disease states, synthetic approaches
are being used to get around the variety of native PGs. Ultimately,
researchers are developing new mimetics that imitate not just the
structure but also the functionality of PGs. The links between PG
structure and function are still mostly unknown. However, early
preclinical research has demonstrated the potential of PG therapies
to lead the way in novel therapeutic approaches and advancements
across a range of disease indications, including diabetes, cancer,
osteoarthritis, wound healing, and hypertrophic scarring. Overall,
the discovery of PG- and GAG-based therapeutics is bringing the
importance of the extracellular matrix (ECM) for tissue health and
cell function back into focus and creating new opportunities for the
creation of bioinspired and targeted medication classes.
Author contributions
IR: Conceptualization, Data curation, Formal Analysis,
Investigation, Project administration, Resources, Software,
Supervision, Validation, Visualization, Writing–original draft,
Writing–review and editing. AE: Conceptualization, Data curation,
Formal Analysis, Investigation, Project administration, Resources,
Validation, Visualization, Writing–original draft, Writing–review and
editing. FZ: Conceptualization, Data curation, Formal Analysis, Funding
acquisition, Investigation, Project administration, Resources, Software,
Supervision, Validation, Visualization, Writing–original draft,
Writing–review and editing. AS-A: Conceptualization, Funding
acquisition, Project administration, Supervision, Validation,
Writing–review and editing. SY: Conceptualization, Funding
acquisition, Project administration, Supervision, Validation,
Writing–review and editing.
Funding
The author(s) declare that financial support was received for the
research, authorship, and/or publication of this article. This work
was supported by Grants-in-Aid for Scientific Research (C) from the
Japan Society for the Promotion of Science (21K06552 and
24K09805 to SY) as well as a Grant-in-Aid for Research Center
for Pathogenesis of Intractable Diseases from the Research Institute
of Meijo University (SY). Also, this work was supported by the
Slovak grants VEGA no. 1/0162/23 and KEGA no.
011UVLF-4/2024.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher.
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