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Figure8.2 Hyaluronic acid benzyl ester.
degrees of esterification, typically performed with benzyl alcohol, have been experi-
®
mented with. HYAFF
is a registered HA benzyl ester (see Figure8.2), employed in
particular for wound healing[29] and cartilage tissue repair[30].
Cutaneous lesions generated by burns, ulcers, or trauma are usually cured with
skin grafts; however, if the covering process cannot be immediately performed,
biomaterialbased patches are employed to induce the correct recovery and regeneration of the damaged area[31]. HYAFFbased medical devices have been successfully employed for the regeneration of cutaneous lesions, and the different
esterification degrees and formulation methodologies allow for modulation of the
final shape/geometries of the device[32]. HYAFF has also been employed in chondrogenic tissue engineering applications for its ability to induce cartilage formation[33]. In vitro studies employing 3D HYAFFbased matrices showed the ability
of autologous chondrocytes to restore the physiological signatures of hyaline cartilage[34], whereas invivo studies demonstrated that seeding with autologous chondrocytes induces native cartilage tissue formation with optimal integration in the
surrounding tissue[33]. Hyalograft C, a medical device based on cartilage autograft
obtained from patient chondrocytes cultured on HYAFF, was introduced for surgical arthroscopic techniques in 1999 [35, 36]. In 2013, following an European
Medicines Agency (EMA) report on manufacturing practices, Hyalograft C was
withdrawn from the European market[37]. Today, methodologies for treatment of
cartilage include new approaches in which autologous chondrocytes are delivered
into damaged tissues with HA or collagenbased injectable biomaterials[38]. HA is
also employed, in different formulations, in ophthalmic viscoelastic devices (OVDs)
or dermatological therapies[39]. The first use of untreated HA in ophthalmic surgery was reported in cornea transplantation and, today, it is currently employed in
ocular surgeries for corneal protection and vitreous replacement [40]. Food and
Drug Administration (FDA) approved the first viscosurgical device based on HA in
®
1983, under the trademark Healon
[41, 42]. Healaflow®, an FDAapproved HA
crosslinked with 1,4butanediol diglycidyl ether (BDDE), is employed in vitreoretinal surgery[43]. Other ophthalmic applications include the modification of contact
lenses with HA solutions. Recently, HA has been exploited to improve contact lens
performance or obtain a controlled release of HA in the eye[44, 45]. Other FDA
approved products based on crosslinked HA include hydrogels in which the
crosslinking is performed with divinyl sulphone (DVS), such as commercial dermal

8.2 Biomaterials and Medical Devices: Natural and Synthetic Strategies
defect fillers (Hylaform®, CaptiqueTM, Prevelle®, Lift®, and Varioderm®). In the
cosmetic field, the most employed HAcrosslinked hydrogels are based on the men-
®
tioned HADVS and HABDDE (Restylane
, Juvéderm®, Teosyal®, and Hyabell®) as
injectable materials[39, 46–48].
Sulfated polysaccharides, normally linked to proteins as PGs, have impressive biochemical and structural roles in the regulation of tissue morphology and cell development and have consequently been extensively employed in medical devices and
studied for clinical translation. Among them, CS, a sulfated linear polysaccharide
based on repeating units of glucuronic acid (GlcA) and Nacetylgalactosamine
(GalNAc) (Figure 8.1), has been largely employed in the biomaterials field
[2, 49–51]. The sulfation pattern of CS, usually at the positions4 and 6, affects the
physiological role. CS is found in many tissues and organs of the human body, from
central nervous system (CNS) to cartilage, skin, bones, and blood vessels. In particular, the major efforts in the development of CSbased medical devices are focused on
the replacement or treatment of cartilage defects and the regeneration of CNS functions. CS is often employed in combination with other natural polymers to generate
®
hybrid medical devices. Esoxx
is a hybrid medical device employed as mucosal bar-
rier for the treatment of gastroesophageal reflux symptoms based on HA and CS
®
embedded in Lutrol
F 127, a poloxamer with bioadhesive properties [52]. HA/
CSbased medical devices are also employed as injectable hydrogel to treat osteoar-
®
thritic (OA) knee, as reported for Structovial CS
[53]. In this formulation, CS has a
double role: optimizing the HA rheological properties and regulating the cartilage
metabolism and remodeling[53]. CS is also extensively employed in formulations
with glucosamine for oral administration as Symptomatic Slow Acting Drugs for
Osteoarthritis (SYSADOA), even if this use is still debated and several clinical studies are ongoing[54]. Other sulfated polysaccharides exploited in clinical translation
are HS and heparin (Figure8.1). HS is heavily expressed in the ECM and on the cell
surface as HSPGs . The structure and length of the polysaccharide chains are highly
variable due to the different sulfation degrees, the different combinations of disaccharide components, and, like for other ECM polysaccharides, the variable molecular weight [55]. The typical disaccharide components consist of 40–60% of
βglucuronic acid (GlcA) or αiduronic acid (IdoA) linked by (1 → 4) glycosidic
bonds to glucosamine, which can be Nacetylated or Nsulfated[56, 57]. Mimetics
of HS have been developed by derivatizing dextran with carboxymethyl, carboxymethyl benzylamide, and carboxymethyl benzylamide sulfonate groups[58, 59]. An
®
example is a commercially available product named RGTA
the treatment of chronic skin damages (CACIPLIQ20
®
(Cacicol20
). Other applications of RGTA are under study and include osteochon-
, currently employed for
®
) and for corneal regeneration
dral, gastroenterological, cardiac, and nervous system applications[60]. Heparin
contains the same monosaccharides of HS, which can also be, in this case, sulfated
as Ido(2S) and GlcA(2S), and the GlcN unit can be Nsulfated (GlcNS) or N acetylated
(GlcNAc) with differential patterns and degrees[61]. The substitution patterns with
Osulfate and Nacetyl groups result in a wide range of complex structures. Also,
heparin has been largely investigated and employed in the pharmaceutical and
227

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228
material device fields for different applications, even if, considering its main role as
an anticoagulant, the use of heparinbased medical devices is mainly focused on
bloodcontacting materials[61, 62]. Temporary or permanent devices like hemodialysis catheters, coronary or vascular stents and grafts, bypass devices, and extracorporeal circulation devices exploit heparincoated materials[57, 62–64]. The two
general methods employed for the development of heparincoated devices are based
on eluting technologies and noneluting technologies. Eluting technologies include
all the approaches in which heparin is released in a controlled way, taking advantage of noncovalent coatings based on physical or ionic interactions [62, 65, 66].
These methods are employed when heparin delivery is needed to prevent acute and
local devicerelated blood clots. The other approach is based on the covalent functionalization of the medical device surface with heparin[67, 68]. This approach is
desirable when longterm thromboresistance is needed at the material surface.
Different conjugation methodologies exploit the direct coupling of complementary
functional groups already present in heparin and the material surface counterpart,
or alternatively introduce new chemoselective orthogonal functional groups and
properly tailored linkers to better control heparin exposition and consequently the
antithrombotic activity. The chemical nature of the medical device will influence
the choice in light of the functional groups eventually present at the surface or,
alternatively, introducible upon proper treatment. The most employed conjugation
methodologies are based on carbodiimide chemistry, exploiting the carboxyl group
of heparin, or oxidation mediated by periodate, for controlled oxidative degradation
to generate some aldehydes[69]. A coating material based on heparin, polyethylene
oxide, and sulphonate groups covalently conjugated to hydrophilic modified poly-
TM
ethyleneimine (ASTUTE
and licensed to Medtronic as Trillium
dialysis catheters[62, 64]. Another example is Bioline
technology) has been developed by BioInteractions Ltd
®
to cover cardiopulmonary bypass and hemo-
®
, developed by Jostra AG and
now a property of Maquet Medical, a material obtained with heparin linked to
human albumin employed for coating vascular grafts and cardiopulmonary
bypass[63, 70]. The conjugation of heparin to polyamine carriers, performed by
introducing etherofunctional crosslinkers, is currently employed as Corline
Heparine Surface Technology, developed by Corline System AB. Many other coating
methodologies employed for the modification of medical device surfaces to control
the interface between blood components and material surface, and to minimize
blood clot formation and thrombolytic events are reported with other commercial
examples in Table8.1.
®
8.2.1.2 Polysaccharides fromPlants, Algae, Animal, and
Microbial Fermentation
The large availability of polysaccharides derived from plants, algae, and animals
and the possibility to produce them by microbial fermentation make them very
attractive and cheaper starting materials[13]. Several studies are focused on the
biocompatibility of such polysaccharides, and many of them are already in clinical
applications. These polysaccharides present an interesting variety of biochemical
and physical properties that can eventually be advantageously exploited in

8.2 Biomaterials and Medical Devices: Natural and Synthetic Strategies
Table8.1 Examples of commercial heparin-coating technologies.
Polymer Commercial names Application References
229
HA benzyl ester
Sodium hyaluronate
HA–BDDE
HA–DVS and
HA–BDDE
HA–CS embedded in
®
Lutrol
HA–CS
Mimetics of
HS–derivatized
dextran with
sulfonate groups
Heparin, polyethylene
oxide, and sulfonate
groups covalently
conjugated to
polyethyleneimine
Heparin linked to
human albumin
Heparin linked by
reductive amination
to matrices bearing
amino groups
Heparin UV
photocrosslinked
Layer by layer
coatings. The last layer
(4) is coated with
polyethyleneimine
linked to Heparin by
reductive amination
Heparin and
benzalkonium
chloride blended
HYAFF
®
Osteochondral
regeneration, wound
[29, 30,
32–34]
healing
®
Healon
Healaflow
®
HADVS (Hylaform
TM
Captique
Varioderm
(Restylane
Teosyal
Esoxx
, Prevelle®, Lift®,
®
) HABDDE
®
, Juvéderm®,
®
, Hyabell®)
®
®
,
Viscosurgical devices [41, 42]
Vitreoretinal
[43]
surgery[43]
Dermal defect fillers [39, 46–48]
Treatment of gastro
[52]
esophageal reflux
symptoms
Structovial CS
®
Treatment of
osteoarthritic (OA)
[53]
knee
®
RGTA
(CACIPLIQ20® and
®
Cacicol20
)
ASTUTE™/TRILLIUM
coating
BIOLINE
®
Coating
Chronic skin
damages and
corneal regeneration
Hemodialysis
®
catheters, bypass
devices
Vascular grafts,
extracorporeal
[58–60]
[62, 64]
[63, 70]
circulation devices
CARMEDA
®
Surface (CBAS
Surface)
BioActive
®
Heparin
Extracorporeal
circulation devices,
stentgrafts, vascular
grafts, ventricular
[62, 71]
assist devices
PHOTOLINK
®
Various medical
devices
[62]
Hepamed™ Coronary stents [62, 72]
DURAFLO II
®
Extracorporeal
circulation devices
[62]

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230
biomedical applications. Furthermore, they can be properly modified to control the
properties and final morphologies of a large plethora of formulations. The main
nonhuman polysaccharides exploited for biomedical devices are alginates, carrageenan, and fucoidan (derived from algae), dextran, pullulan, and gellan (derived
from microbes), and chitosan (derived from animals) (see Figure8.3).
–
+
COO
O
HO
HO
G
Na+ –OCC
O
HO
R
2
O
OH
O
OH
HO
OH
1
O
HO
O
OH
Alginate
OH
HO
O
O
NH
2
Chitosan
OH
O
O
R
1
Carrageenan
R= –OH, OSO
O
O
OH
OH
O
HO
Pullulan
OH
O
HO
R
OH
M
O
OH
5
R
O
–
3
OH
Na
NH
2
O
O
n
OH
3
O
O
R
4
n
O
OH
O
O
HO
HO
O
HO
OH
O
O
HO
n
HOOC
O
OH
O
HO
O
HO
Gellan gum
HO
HO
OH
HO
HO
OH
O
HO
OH
O
O
OH
O
O
O
OH
HO
Dextran
OH
Fucoidan
O
OH
OH
O
OH
O
O
HO
OH
O
HO
OSO
OH
OH
O
OH
O
HO
O
O
OSO
O
O
–
OSO
3
–
3
HO
O
O
O
O
O
n
n
O
O
–
OSO
3
–
3
Figure8.3 Examples of nonhuman polysaccharides employed for biomedical device
formulations.

8.2 Biomaterials and Medical Devices: Natural and Synthetic Strategies
Alginates are linear polysaccharides obtained from seaweed composed of β
mannuronate (M) and αguluronate (G) residues linked by (1,4) glycosidic
bond[73]. The polysaccharide structures are organized in blocks with consecutive
M (MMMMMM) or G (GGGGGG) residues or alternating M and G residues
(GMGMGM), depending on the algae from which they are extracted. The hydrogel
properties of alginates are related to the M/G ratio and sequence, the Gblock length,
and finally the molecular weight[74]. Alginates are widely investigated for biomedical applications thanks to their biocompatibility and the ability to form hydrogels by
2+
simple addition of divalent cations like Ca
, and several of them have found appli-
cations even in clinics. Alginate dressings have been experimented in wound heal-
TM
ing, and are commercially available as Algicell
, AlgiSite M™, Comfeel Plus™,
Kaltostat™, Sorbsan™, and Tegagen™; they show an improved ability to support
rapid healing through epithelialization[75]. Alginatebased biomaterials are also
employed for dental impression applications[76] and for the treatment of gastro
esophageal reflux [77, 78]. Other interesting biopolymers derived from algae are
carrageenan and fucoidan. Both are sulfated polysaccharides, derived, respectively,
from red and brown algae[79]. Carrageenans are sulfate galactans composed of
galactose (Gal) and 3,6anhydrogalactose with alternating α(1→3) and β(1→4)
glycosidic bonds; depending on the structure and sulfation patterns, they are classified as γ, β, δ, α, μ, κ, λ, ν, ι and θcarrageenans. Carrageenans are currently
employed as food additives and nutraceuticals; however, several studies highlight
their potential use in drugdelivery systems, tissue engineering, and wound healing[80]. Fucoidans are highly sulfated polysaccharides based on repeating units of
α(1→3)fucopyranose, often alternated with α(1→4)fucopyranose units. They
can also contain acetate groups and various glycosyl side branches (i.e. glucuronic
acid, mannose, glucose, galactose, or xylose), depending on the type of brown algae.
Like carrageenan, fucoidan is currently employed in the nutraceutical and food
industry; however, several biomedical applications in the medical devices, drug
delivery, and tissue engineering fields are under investigation.[81].
Polysaccharides derived from microbial sources are extensively studied for cosmetic, pharmaceutical, nutraceutical, and biomedical applications. Dextran is one
of the most employed; it is α1,6polyαglucoside with α1,3 branches produced
by microbial fermentation employing nonpathogenic Leuconostoc mesenteroides,
Saccharomyces cerevisiae, Lactobacillus plantarum, and Lactobacillus sanfrancisco.
Dextran is used in vascular surgery and antiplatelet applications thanks to its
antithrombotic properties; furthermore, it finds application in ophthalmic and diag-
®
nostic applications. Dextran 70
(MMW 70.000 kDa) and Dextran 40® (LMW
40.000 kDa) are employed, respectively, as plasma substitutes and to increase blood
flow in ischemic limbs[82, 83]. Lowmolecularweight dextran sulfate (LMWDS) is
also under study (Phase II clinical trial) to reduce graft rejection for pancreatic islet
transplantation[84]. Dextran is also extensively investigated as coating for diagnostic and therapeutic nanoparticles; in particular, dextrancoated magnetic nanoparticles are employed as diagnostic agents for MRI and as therapeutic nanoparticles for
iron deficiency [85]. Other interesting examples of microbial polysaccharides
include pullulan, employed for oral care and cosmetic applications[86], bacterial
231

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232
cellulose, employed for topical woundhealing applications[87], and gellan gum,
employed in the development of oral, ophthalmic, and spray nasal formulations for
biomedical, cosmetic, and nutraceutical applications[88, 89]. All the cited bacterial
polysaccharides are also under investigation for other biomedical applications,
including wound healing and regenerative medicine[88].
Chitosan is the most commonly used polysaccharide from animal sources; it is
obtained from crustacean shell waste. Many chitosanbased biomaterials are commercially available, and some are in clinical trials for wound healing, pharmaceutical, diagnostic, and cosmetic applications. Formulations and medical devices based
on chitosan are usually obtained by ionic assembling with anionic polymers or by
covalent linkages performed on the amino groups by carbodiimide chemistry.
Another approach is based on the partial oxidation of chitosan with periodate
generating aldehyde groups for subsequent chemoselective reactions (Table8.2).
Table8.2 Examples ofcommercial medical devices and formulations produced
fromvegetal, microbial, or animal-derived polysaccharides.
Polysaccharide Commercial names Origin Application References
Alginate Algicell™, AlgiSite
Sodium alginate Neocolloi
Potassium alginate Blueprint Cremix®,
Sodium alginate Liquid Gaviscon
Carrageenan Carragelose
Dextran MMW Dextran 70
Dextran LMW Dextran 40
Dextran sulfate LMW Ibsolvmir
Dextrancoated iron
nanoparticles
Dextrancoated iron
nanoparticles
M™, Comfeel
Plus™, Kaltostat,
Sorbsan™,
Tegagen™
®
,
Zhermack
Plus
Dentsply DeTrey
Endorem
Combidex
CosmoFer, INFeD,
Ferrisat, DexFerrum,
and DexIron
®
; Palgat
®
, 3M ESPE
®
®
®
®
,
®
®
®
®
®
Algae Wound dressing [75]
Algae Dental impression [76]
Dental impression [76]
Algae Gastroesophageal
reflux
Algae Antiviral eye
drops, nasal spray
Microbial Plasma substitute [82, 83]
Microbial Plasma substitute [82, 83]
Microbial Islet transplant [84]
Microbial Imaging liver
cancer
(endoderm) and
lymph node
metastases
(Combidex)
Microbial Iron deficiency
therapy
[77, 78]
[85]
[85]

8.2 Biomaterials and Medical Devices: Natural and Synthetic Strategies
Table8.2 (Continued)
Polysaccharide Commercial names Origin Application References
233
Pullulan Listerin
Bacterial cellulose Bioprocess
Gellan Gum Grindsted
Gellan Gum Gelrite
Chitosan HemCon
Chitosan and
cellulose
Chitosan–
glycerophosphate
Chitosandextran CD gel Animal
®
and Biofill
®
and
Kelcogel
others
Chitoflex
Chitoseal
BSTCarGel
©
®
®
®
Microbial Oral care [88]
®
, XCell®,
®
®
Gellan Microbial Nutraceutical [91]
many
,
, Celox
®
Microbial Wound healing [87, 90]
Microbial Drug release,
Animal Hemostatic
®
Animal
and
microbial
Animal Cartilage repair [93]
and
microbial
ophthalmic, nasal
formulations,
cosmetic
formulations
bleeding
treatment and
bandages
Bleeding wounds [93]
Surgical
applications,
wound healing
8.2.2 Carbohydrates asSignaling Molecules: Opportunities
inTissue Engineering and Regenerative Medicine
[89, 92]
[93]
[94, 95]
Tissue engineering and regenerative medicine strategies are based on two main pillars: (i) biomaterial scaffolds mimicking the ECM and (ii) stem cells able to regenerate tissues damaged from trauma or pathologies. The biomaterial scaffold can be
implanted or just used to culture the human cells that will be implanted/administered subsequently. The role of the biomaterial scaffold in this case consists in homing the cells and driving their fate to the required regeneration. In this context,
glycans are deeply investigated not only for their structural role to generate cell
hospitable hydrogels but also for their role as signaling molecules capable of modulating the cell’s fate. Recent studies have demonstrated that differential cell fate
modulation is strictly dependent on the glycan epitopes present in the ECM[96, 97].
Considering that both stem cells and ECM are heavily glycosylated, it is easily
understandable that a “glycocode” plays a key role in cellsignaling induction and
transduction[4, 98]. Morphological and functional tissue morphogenesis, related to
healthy and pathological states and even aging, is related to N and Oglycosylation
of ECM proteins and cell–ECM communications[99]. Alteration in glycosylation of
ECM and extracellular components has been characterized in pathological conditions like cancer[100–103], fibrosis[104, 105], and inflammatory diseases[106–108].

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Collagen, laminin, and fibronectin are the most known examples of ECM proteins
in which glycosylation varies between physiological and pathological conditions
and in tissue morphogenesis[23, 109, 110]. ECM–cell crosstalk events are mediated
by carbohydrate–carbohydrate and, more significantly, by carbohydrate–protein
(lectins) interactions[3, 111–113]. The crosstalk will result in different structural
organizations of the ECM responsible for morphogenesis. Therefore, once the correlation between cell fate and ECM morphological and biomolecular properties
(including glycosylation) is clarified, it will be possible to design artificial ECM able
to regenerate the required tissue. Even if glycoproteomic and lectin studies are in
rapid development, we still need a clear and reliable picture of the main glycosignature changes related to morphogenesis and the occurrence of pathologies.
8.3 Carbohydrates inAnimal-Derived Medical
Devices: Friends or Foes?
Among implantable medical devices, animalderived organs or tissues are frequently
the best choice for tissue substitution or surgical applications[114]. They not only
provide the nanometrically correct architecture for the required functionality but
also possess the biochemical composition ideal for cell adhesion and consequent tissue regeneration. As a matter of fact, even in synthetic medical devices, animal
derived polymers are often employed as coatings to optimize the interactions between
tissue and material surfaces or to control the medical device performances[115–117].
Bovine pericardium or bovine/porcine patches are employed for the reinforcement
of shapes and lines in soft tissue surgery applications. Collagen and gelatinderived
meshes and coatings, as well as bovine/porcinebased devices, are employed in
wound dressing and vascular surgery applications. Animalderived dural grafts
[118, 119] and nerve conducts[120, 121] are also employed in neurosurgery.
Animalderived materials, however, suffer from a big inconvenience: the presence
of xenoantigens like αGal, Neu5Gc (N-Glycolylneuraminic acid), and Forssman
antigen (GalNAcα1, 3GalNAcβ1, 3Galα1, 4Galβ1, and 4GlcCer) [122, 123] (see
Figure8.4) that elicit an immune response in humans.
Strategies like genomic editing and chemical immunosuppression are under
investigation to limit xenoantibody production and avoid undesirable early graft
rejection[122, 124, 125]. In December 2020, transgenic pigs with depletion of α
Gal–named GalSafe pigs–were approved by FDA for human food consumption
and possible biomedical uses [126]. Decellularized animal tissues were also
developed with the aim to limit xenoantigens from cellular components; however, the immunological response of these devices is often still in place, probably
due to the glycosylation of ECM components[125]. The most efficient solution is
probably a coating able to mask the xenoantigens responsible for immunoresponse. Glutaraldehyde fixation [127, 128] is an option. Treatment of animal
derived biological tissues with a low concentration of glutaraldehyde enhances
structural performance, maintains sterility, and reduces antigenicity. Although

OH
HO
Forssman antigen
r
235
COOH
O
O
OH
HO
O
OH
O Ce
O
OH
HO
OH
OH
OH
α-Gal
NHAc
OH
HO
O
HO
O
OH
H
N
HO
O
Neu5Gc
O
OH
OH
O
NHAc
OH
OH
O
O
OH
O
HO
O
OH
O
OH
Figure8.4 Xenoantigens responsible for immunoresponse in humans.
the advantages of this kind of coating are wellestablished, the calcification phenomena induced at the surfaces of the devices are still a remarkable limitation[129]. To reduce the risk of calcification, antimineralization treatments have
been developed, such as the use of alphaamino oleic acid (AOA) employed in
®
Mosaic
bioprosthesis (Medtronic), which also improves durability and hemodynamic properties. The AOA reacts with the free aldehyde groups of glutaraldehyde by Schiff base covalent linkages, avoiding calcium nucleation and
precipitation[130]. Glutaraldehyde fixation is able to mask around 50% of αGal
antigens[131], but at the same time, it increases oxidative and enzymatic degradation of the tissue. Recently, a new treatment available for glutaraldehydecoated
®
tissues has been patented (FACTA
), able to totally mask αGal epitopes in
animalderived medical devices, reducing oxidative degradation and thrombotic
risk [132].
8.4 Glycoengineering Application
toRegenerative Medicine
Metabolic glycoengineering (MGE) approaches were developed in the late 1980s
and are revealed today as valuable tools to study the expression of selected glycans
in pathological phenomena and to modulate glycosignature expression [133–135].
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