International Journal of
Molecular Sciences
Article
New Hyaluronic Acid from Plant Origin to Improve Joint
Protection—An In Vitro Study
Rebecca Galla 1 , Sara Ruga 1 , Silvio Aprile 2 , Sara Ferrari 1 , Arianna Brovero 1 , Giorgio Grosa 2 ,
Claudio Molinari 3 and Francesca Uberti 1, *
Citation: Galla, R.; Ruga, S.; Aprile, S.;
Ferrari, S.; Brovero, A.; Grosa, G.;
Molinari, C.; Uberti, F. New
Hyaluronic Acid from Plant Origin to
Improve Joint Protection—An In
Vitro Study. Int. J. Mol. Sci. 2022, 23,
8114. https://doi.org/10.3390/
ijms23158114
Academic Editor: Elizabeth W.
Bradley
Received: 29 May 2022
Accepted: 18 July 2022
Published: 23 July 2022
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Copyright: © 2022 by the authors.
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This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2022, 23, 8114. https://doi.org/10.3390/ijms23158114
1 Laboratory of Physiology, Department of Translational Medicine, University of Piemonte Orientale,
Via Solaroli 17, 28100 Novara, Italy; rebecca.galla@uniupo.it (R.G.); sara.ruga@uniupo.it (S.R.);
sara.ferrari@uniupo.it (S.F.); arianna.brovero@gmail.com (A.B.)
2 Dipartimento di Scienze del Farmaco, Università del Piemonte Orientale, Largo Donegani 2,
28100 Novara, Italy; silvio.aprile@uniupo.it (S.A.); giorgio.grosa@uniupo.it (G.G.)
3 Dipartimento per lo Sviluppo Sostenibile e la Transizione Ecologica, University of Piemonte Orientale,
13100 Vercelli, Italy; claudio.molinari@uniupo.it
* Correspondence: francesca.uberti@med.uniupo.it
Abstract: Background: In recent decades, hyaluronic acid (HA) has attracted great attention as a new
treatment option for osteoarthritis. Classical therapies are not able to stop the cartilage degeneration
process nor do they favor tissue repair. Nowadays, it is accepted that high molecular weight HA can
reduce inflammation by promoting tissue regeneration; therefore, the aim of this study was to verify
the efficacy of a new high molecular weight HA of plant origin (called GreenIuronic® ) in maintaining
joint homeostasis and preventing the harmful processes of osteoarthritis. Methods: The bioavailability
of GreenIuronic® was investigated in a 3D intestinal barrier model that mimics human oral intake
while excluding damage to the intestinal barrier. Furthermore, the chemical significance and biological
properties of GreenIuronic® were investigated in conditions that simulate osteoarthritis. Results:
Our data demonstrated that GreenIuronic® crosses the intestinal barrier without side effects as it has
a chemical–biological profile, which could be responsible for many specific chondrocyte functions.
Furthermore, in the osteoarthritis model, GreenIuronic® can modulate the molecular mechanism
responsible for preventing and restoring the degradation of cartilage. Conclusion: According to our
results, this new form of HA appears to be well absorbed and distributed to chondrocytes, preserving
their biological activities. Therefore, the oral administration of GreenIuronic® in humans can be
considered a valid strategy to obtain beneficial therapeutic effects during osteoarthritis.
Keywords: osteoarthritis; cartilage inflammation; tissue degradation; high molecular weight hyaluronic
acid; intestinal absorption; chondrocytes
1. Introduction
Osteoarthritis (OA) is a slow progressive joint disorder that causes several disabilities
in the adult population [1]. For a long time, OA was regarded as progressive wear of the
joint cartilage alone. However, recent research has shown that it is an inflammatory disease
of the entire synovial joint, which includes not only the mechanical degeneration of the
articular cartilage, but also the concomitant structural and functional change of the entire
joint, including the synovium, meniscus, periarticular ligaments, and subchondral bone [2].
An important role in infrapatellar fat pad inflammation and fibrosis has also recently
been discovered [3]. Today, the treatment modalities for OA include non-pharmacological
(e.g., physiotherapy), pharmacological (e.g., steroidal and nonsteroidal anti-inflammatory
drugs), or intra-articular (e.g., injection of hyaluronic acid) therapies [4,5]. These classical
therapies can reverse the symptoms only in a small number of cases, but they do not stop
the degeneration process of the cartilage or promote the repair of the tissue. Therefore, the
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022, 23, 8114 2 of 26

development of new therapies is a primary goal, preferably hypothesizing the oral admin-
istration, which remains the preferred route for drug delivery due to its low invasiveness,
high efficiency, and better patient compliance [6]. Obviously, in this case, the bioaccessi-
bility and bioavailability of orally administered compounds need to be investigated in a
preclinical model in order to evaluate the ability to cross the intestinal barrier after oral
administration. During the last decades, hyaluronic acid (HA) has attracted great attention
as a new treatment option for knee OA pain [7–9]. HA is a natural polymer belonging to
the glycosaminoglycan heteropolysaccharides family (GAGs), but unlike these molecules,
it is not sulfated and it is not synthesized by Golgi enzymes [10]. In addition, the native
form appears as a very long polymer, called high-molecular weight HA (HMWHA) [10].
Therefore, the native HA consists of 2000–25,000 disaccharide units, corresponding to
106 –107 Da molecular weight; for that, a long chain contains more than 10,000 units, which
is ~4000 kDa [9,11]. In the biological systems, HMWHA (also called native HA) is degraded
into small fragments named low molecular weight HA (LMWHA) corresponding to dif-
ferent molecular weights; in particular, HMWHA has >1 to 10 MDa; intermediate HA has
>100 to 1000 kDa; and LMWHA has the molecular weight between 1 and 10 kDa [9,12].
Several studies reported that the structural and biological properties of HA within medical,
pharmaceutical, and cosmetic applications analyzing the role of HA in inflammation and
tissue regeneration are related to its specific molecular weight [13,14]. Applications of HA
depend on its biological effects on cell differentiation and proliferation, and on its ability
to lubricate, hydrate, and interact with various receptors present on the cell surface. It is
this interaction that facilitates the exact delivery of drugs, facilitating their internalization
in target sites. The safety, tolerability, and efficacy of HA-based formulations for the treat-
ment of various types of joint diseases have been validated in several studies [10,15]. It
is widely accepted that exogenous hyaluronic acid is incorporated into articular cartilage
where it may have a direct biological effect on chondrocytes to improve joint lubrication
as well described by clinical studies. The concept of viscosupplementation is based upon
the hypothesis that HA administration could improve the rheological properties of joints,
and promote the endogenous synthesis of HMWHA and possibly more functional HA,
thereby improving mobility, and articular function, and decreasing pain. The growing
use of HA in medical practice can be explained by its effectiveness and versatility as well
as its favorable safety profile [16]. Nowadays, sodium HA seems to be the best choice
available on market, since it exerts an analgesic effect by blocking pain receptors in synovial
tissues and holding endogenous pain substances in its molecule [17]. However, it can be
suggested that the characteristic steric configurations of HMWHA are necessary for the
manifestation of the analgesic effect, indicating a possible clinical application of all HA
fragments [17]. Indeed, HA is a Food and Drug Administration (FDA)-approved treatment
for inflammatory conditions, including those affecting the joints, and is also acknowledged
in Europe for its beneficial properties due to the therapeutic potential caused by native
HA, but without toxicity [9]. In particular, in vitro studies demonstrate that only native
HA exerts an inhibitory effect on interleukin (IL)-1βstimulated prostaglandin E2 (PGE2)
production in inflammation-damaged bovine cartilage. This supports the hypothesis that
administration of HMWHA may be an important new strategy to restore proteoglycan
content leading to a new cartilage protective strategy [9,18]. Synovial inflammation and
structural and molecular changes in the joint system should be the target of OA therapies.
For this reason, thanks to its viscoelastic properties, HA therapy has been proposed for
OA [19]. Much research has been conducted on HA combined with anti-inflammatory
drugs, both in clinical trials and in vivo and in vitro studies, and the published data indi-
cate that, with a good level of significance, intra-articular injection of HA combined with
anti-inflammatory drugs can potentially relieve pain in OA knee patients [20]. In the last
years, symptomatic slow-acting drugs for OA have been vastly studied and many studies
have focused the attention on HA, or chondroitin sulfate (CS) combined with nonsteroidal
anti-inflammatory drugs (NSAIDs) to limit the related adverse events in the gastrointestinal
tract, kidney, and cardiovascular system [19]. Finally, to reduce the strong adverse effects
Int. J. Mol. Sci. 2022, 23, 8114 3 of 26

due to drugs, HA may be combined with several agents including lactose-modified chitosan
and cyclodextrin to improve chondroprotection and to stimulate cartilage growth reducing
inflammation [21,22]. Additional studies reported similar beneficial effects of HMWHA in
OA and in other inflammatory conditions [23,24]. The main purpose induced by HMWHA
is to promote chondroprotection and involves several proteins including binding to the
receptor of the differentiation cluster 44 (CD44), which is required to inhibit the expression
of IL-1β, leading to a decline production of matrix metalloproteinases (MMPs) −1, 2, 3, 9,
and 13 [7,25,26]. In addition, HA binds hyaluronan-mediated motility receptors (RHAMM)
to induce chondroprotection as well as CD44 binding [7]. Regarding the mechanism ac-
tivated by the binding of CD44, the most important is the role of the mitogen-activated
protein kinase phosphatase (MKP)-1, which is able to inhibit the production of IL-1β, and
consequently inhibits the MMPs within articular cartilage and finally prevent apoptotic
events in the chondrocyte through the reduction in disintegrin and metalloproteinase ex-
pression with thrombospondin motifs (ADAMTS) [7,27,28]. Another important element is
the production of reactive oxygen species (ROS) and nitric oxide (NO), which are normally
involved in the apoptosis-dependent death of chondrocytes, leading to the degeneration of
cartilage. In this context, the current literature reported that HMWHA after the binding
with CD44 is able to prevent chondrocytes apoptosis by inhibiting PGE2 synthesis and
interleukin activity such as IL-1β, which is responsible for oxidative stress [7,25,29,30].
Regarding the effectiveness of HA on joint tissue, it is important to remember that it can
act as a passive structural molecule or exerts biological effects via a signaling molecule.
Furthermore, since its different mechanism of action depends on the molecular weight,
the link between the molecular weight and its pro and anti-inflammatory activities, the
promotion, and inhibition of the activation of migration, and the blocking or promotion of
the division is also important [10]. Today some details are known about how HA exerts its
different biological functions at different concentrations and molecular weights [31]. For
example, at the level of the intestinal mucosa, the intermediate HA and HMWHA have
antioxidant and antimicrobial properties [32]. Its importance is constantly growing because
this substance regulates tissue homeostasis and its physiological decrease is related to the
aging process that leads to various diseases [32]. Oral administration of exogenous HA has
attracted the attention of researchers as a supplementary therapy to prevent or treat the
aging process of cartilage and related diseases [33]. The purpose of this study was to verify
the efficacy of HMWHA plant-derived HA (called GreenIuronic® ) in maintaining joint
homeostasis in order to prevent all the harmful processes that can trigger the pathology
of OA.

2. Results
2.1. Characterization of GreenIuronic®
High-Performance Liquid Chromatography analysis of GreenIuronic® (Figure 1) re-
vealed the presence of HA, which was detected as the corresponding disaccharide ∆Di-HA
generated by chondroitinase AC enzymatic hydrolysis of the Tremella extract. The identity
of this disaccharide was established by comparison with the ∆Di-HA reference standard
and by the protonated and sodiate positive ions detected in its mass spectrum. Moreover,
the same analysis also revealed the absence of chondroitin 4 and 6 mono-sulfates, which
eluted at 6.25 and 5.35 min, respectively. However, because of the absence of sulfate group
in the disaccharide chondroitin 0 sulfate (∆D-0S), arising from chondroitine hydrolysis,
the HPLC method, based on ion-pair retention, did not allow the separation between the
disaccharides ∆Di-HA and ∆D-0S: indeed, they eluted at 2.25 min.
Moreover, since HPLC-UV analysis revealed a possible high concentration of HA
in GreenIuronic® samples, additional experiments were carried out to quantify the con-
tent of glucuronic acid in GreenIuronic® and in sodium hyaluronate samples. As re-
ported in Table 1, the content of glucuronic acid in GreenIuronic® is about 90%, which
is higher than that of sodium hyaluronate (about 62%). These data support what was
nt. J. Mol. Sci. 2022, 23, 8114 4 of 26
Int. J. Mol. Sci. 2022, 23, 8114 4 of 26

previous experiments in HPLC (reported above) about the purity of the GreenIuronic®
observed in previous experiments in HPLC (reported above) about the purity of the
material. ®
GreenIuronic material.
RT: 1.97 min, NL: 9.25 ×106
mAU T: FTMS + p ESI Full ms [80.0000-600.0000]
A 175 240nm,4nm (1.00)
B 100
402.10022

150 ∆Di-HA 90
(2.25 min)
80
125

70
100

Relative Abundance
60

75 50

40
50

30
25
20
380.11841 403.10379
0
10
379.07837 404.10480
min 381.12198
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
0
380 385 390 395 400 405
m/z
mAU mAU
240nm,4nm (1.00)
C 130
240nm,4nm (1.00)
D 80

120 ∆Di-HA
∆Di-6S (5.35 min) 70 (2.25 min)
110
∆Di-0S ∆Di-4S (6.25 min)
100 (2.25 min) 60
90
50
80

70
40
60

50 30

40
20
30

20 10

10
0
0

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min

Figure 1. HPLC-UV
Figure 1. HPLC-UV andandhigh-resolution massspectrometry
high-resolution mass spectrometry (HRMS)
(HRMS) analysis
analysis of GreenIuronic
of GreenIuronic ® after ® after

enzymatic hydrolysis
enzymatic with
hydrolysis withchondroitinase AC.InIn(A,B)
chondroitinase AC. (A,B) HPLC-UV
HPLC-UV chromatogram
chromatogram of GreenIuronic
of GreenIuronic ® ®

sample and and

sample its positive HRMS
its positive HRMSspectrum. In(C,D)
spectrum. In (C,D)HPLC-UV
HPLC-UV chromatograms
chromatograms of a mixture
of a mixture of chon-of chon-
droitin disaccharides
droitin standard
disaccharides ∆Di-0S, ∆Di-4S,
standard ΔDi-0S, ΔDi-4S,and ∆Di-6S
and ΔDi-6S
and aand a solution
solution of the disaccharide
of the disaccharide standard stand-
∆Di-HA of
ard ΔDi-HA ofHA.
HA.
Table 1. Quantification of HA. The % w/w of all HA forms normalized on standard curves generated
Tableusing
1. Quantification of HA. The % w/w of all HA forms normalized on standard curves generated
glucuronic acid standard (ranging from 0 to 2 mg/mL) analyzed at 340 nm by spectrophotome-
usingtryglucuronic acid standard (ranging from 0 to 2 mg/mL) analyzed at 340 nm by spectrophotom-
(Infinite 200 Pro MPlex, Tecan). Data are expressed as means ± standard deviation (SD) (%) of
etry (Infinite 200 Pro MPlex, Tecan).
five independent experiments Datainare
performed expressed as means ± standard deviation (SD) (%) of
triplicates.
five independent experiments performed in triplicates.
Raw Material Mean (%w/w) ± SD
Raw Material
Sodium Hyaluronate
Mean (%w/w) ± SD
62.5 ± 2.121
Sodium Hyaluronate
GreenIuronic® 90.5 62.5 ± 2.121
± 6.364
GreenIuronic® 90.5 ± 6.364
Finally, for the analysis of GreenIuronic® size distribution, agarose gel electrophore-
sis was used
Finally, to define
for the a range
analysis of the molecular
of GreenIuronic weight.
® size Agarose agarose
distribution, gel retards
gelthe elec-
electrophore-
trophoretic mobility
sis was used to define of HA molecules in a molecular weight-dependent manner indicating
a range of the molecular weight. Agarose gel retards the electro-
that GreenIuronic® may be considered the HMWHA (>1650 kDa), as can be seen in Figure 2.
phoretic mobility of HA molecules in a molecular weight-dependent manner indicating
On the contrary, sodium hyaluronate was confirmed to have the lower molecular weight
that GreenIuronic ® may be considered the HMWHA (>1650 kDa), as can be seen in
HA (LMWHA) (between 300 and 500 kDa). These results indicate that GreenIuronic® Figure
2. On the contrary, sodium
molecular weight is higher than hyaluronate was
that of sodium confirmed
hyaluronate andto haveexperiments
further the lowerwere molecular
weight HA (LMWHA)
performed (between
in order to confirm 300 and
the hypothesis that 500
HMWHAkDa).exerts
These
more results
beneficialindicate
effects that
compared to those of LMWHA.
GreenIuronic molecular weight is higher than that of sodium hyaluronate and further
®

experiments were performed in order to confirm the hypothesis that HMWHA exerts
more beneficial effects compared to those of LMWHA.
Int.
Int. J. Mol. Sci. 2022, 23, 8114 55 of
of 26
26

Figure 2.
Figure 2. In
In the
the figure
figure an
an example
example ofof HA
HA molecular
molecular weight
weight determination
determination on
on 1%
1% Agarose
Agarose gel.
gel. The
The
sample loads are described as follows by the abbreviations: M W = standard molecular weight Mega
sample loads are described as follows by the abbreviations: MW = standard molecular weight
+ HiLadder specific for HA detection; L1 = lane empty loaded with 10 µL TAE buffer; L2 = lane
Mega + HiLadder specific for HA detection; L1 = lane empty loaded with 10 µL TAE buffer;
loaded with 100 µg/10 µL Sodium Hyaluronate; L3 = lane empty loaded with 10 µL TAE buffer; L4
L2 = lane loaded with 100 µg/10 µL Sodium® Hyaluronate; L3 = lane empty loaded with 10 µL
= lane loaded with 100 µg/10 µL GreenIuronic ; L5 = lane empty loaded with 10 µL TAE buffer.
TAE buffer; L4 = lane loaded with 100 µg/10 µL GreenIuronic® ; L5 = lane empty loaded with 10 µL
TAE buffer.
2.2. Dose–Response and Time-Course Study of GreenIuronic® on CaCo-2 Cells
Before studying
2.2. Dose–Response andthe permeability
Time-Course and
Study of transport
GreenIuronic ® on CaCo-2 Cells
of GreenIuronic ®, the human immor-

talized
Before studying the permeability and transport of GreenIuronic , thea human
colorectal adenocarcinoma (CaCo-2) cell line was used to perform
® dose–response
immor-
study to
talized exclude adenocarcinoma
colorectal any cytotoxic effects. The analysis
(CaCo-2) cell line waswas used
performed comparing
to perform the effects
a dose–response
of GreenIuronic
study to exclude to
® sodium
any cytotoxichyaluronate,
effects. Thetesting them was
analysis at theperformed
same concentration
comparing (ranging
the ef-
from 0.125 to 1 µg/µL) ® on cell viability and ROS production
fects of GreenIuronic to sodium hyaluronate, testing them at the same concentration in CaCo-2 cells in a time-
course study
(ranging from(from
0.125 2toto 6 h). Theoncell
1 µg/µL) cellviability
viabilityofand theROSCaCo-2 cells, measured
production in CaCo-2 bycells
3-(4,5-
in
a time-course study (from 2 to 6 h). The cell viability of the CaCo-2 cells, measuredand
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, showed time by
concentration-dependent effects of both substances (Figure
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 3A), and the
(MTT) beneficial
assay, showed effects
time
compared
and to control (p < 0.05) were
concentration-dependent effectsmaintained during all(Figure
of both substances periods3A),of stimulation exclud-
and the beneficial
ing any
effects cytotoxicto effect
compared controlat(p all dosage
< 0.05) weretested.
maintained In particular,
during allthe cellsoftreated
periods with
stimulation
GreenIuronic ® 1 µg/µL showed high variability compared to control (p < 0.05) and com-
excluding any cytotoxic effect at all dosage tested. In particular, the cells treated with
pared to other
GreenIuronic ®1 concentrations
µg/µL showed tested
high(p < 0.05) suggesting
variability comparedthat GreenIuronic
to control ® 1 µg/µL is
(p < 0.05) and com-
non-toxic
pared to intestinal
to other epithelial
concentrations cells(p
tested exhibiting the best profile
< 0.05) suggesting that GreenIuronic ®
also compared1toµg/µL sodium is
hyaluronate
non-toxic at the same
to intestinal concentration
epithelial and timethe
cells exhibiting (p best
< 0.05). Additional
profile experiments
also compared were
to sodium
carried out inatorder
hyaluronate the sameto confirm the safety
concentration andoftime
GreenIuronic on intestinal
(p < 0.05).®Additional epitheliumwere
experiments ana-
lyzing ifout
carried theinsubstances tested could
order to confirm induce
the safety oxidative stress.
of GreenIuronic ® onFor this reason,
intestinal ROS produc-
epithelium analyz-
ing
tionifwas
the evaluated
substanceson tested
CaCo-2could induce
cells fromoxidative
2 to 6 h ofstress. For thiswith
stimulations reason,
bothROS production®
GreenIuronic
was evaluated on CaCo-2As cells fromin2 Figure
to 6 h of ®
and sodium hyaluronate. shown 3B,stimulations with both GreenIuronic
none of the concentrations tested was
and
able sodium
to increasehyaluronate. As shownmaintaining
the ROS production in Figure 3B, themnone at of the concentrations
normal tested was
physiological conditions.
able to increase
GreenIuronic ® 1the ROSmaintains
µg/µL production maintaining
a low ROS levelthem during at normal physiological
all periods conditions.
analyzed better than
GreenIuronic ® 1 µg/µL maintains a low ROS level during all periods analyzed better than
the other concentrations tested and all sodium hyaluronate concentrations, and it was
the other concentrations
maintained for all furthertested and all sodium hyaluronate concentrations, and it was
experiments.
maintained for all further experiments.
Int. J. Mol. Sci. 2022, 23, 8114 6 of 26
Int. J. Mol. Sci. 2022, 23, 8114 6 of 26

Figure
Figure 3. Cell
3. Cell viabilityand
viability andROS
ROS production
production on onCaCo-2
CaCo-2 cells. In In
cells. panel (A,B)
panel dose–response
(A,B) studystudy
dose–response on on
cell viability measured by MTT test of both GreenIuronic ® and Sodium Hyaluronate from 2 to 6 h. In
cell viability measured by MTT test of both GreenIuronic and Sodium Hyaluronate from 2 to 6 h.
®
panel (C,D) ® and Sodium Hyaluronate measured by reduction
In panel (C,D)ROS ROSproduction
production of both GreenIuronic
of both GreenIuronic ® and Sodium Hyaluronate measured by re-
in cytochrome
duction C from C
in cytochrome 2 to 6 h. Data
from 2 to 6are Data ±are
h.mean SDmean
of five±independent experiments performed
SD of five independent in per-
experiments
triplicates vs. control values (0% line).
formed in triplicates vs. control values (0% line).
2.3. Permeability Analysis of GreenIuronic® Using an In Vitro Model of Intestinal Barrier
2.3. Permeability Analysis of GreenIuronic® Using an In Vitro Model of Intestinal Barrier
To assess permeability, and to obtain additional information about the GreenIuronic®
To assess
intestinal permeability,
absorption, furtherand to obtainwere
experiments additional information
carried out performingabout the
a 3D in GreenIuronic
vitro model ®

in orderabsorption,
intestinal to mimic thefurther
in vivoexperiments
complexity ofwerethe intestinal
carried outbarrier. In this context,
performing a 3D in1 vitro
µg/µLmodel
GreenIuronic ® and 1 µg/µL sodium hyaluronate were tested from 2 to 6 h in order to
in order to mimic the in vivo complexity of the intestinal barrier. In this context, 1 µg/µL
measure transepithelial
GreenIuronic ® and 1 µg/µL electrical
sodium resistance (TEER)were
hyaluronate values, the apparent
tested from 2 topermeability
6 h in ordercoef-
to meas-
ficient (Papp) values, and the HA concentration to predict their bioavailability. The data
ure transepithelial electrical resistance (TEER) values, the apparent permeability coefficient
obtained show that intestinal adsorption has a physiological trend as can be observed from
(Papp) values, and the HA concentration to predict their bioavailability. The data obtained
the analysis of TEER and tight junction (TJ). In particular, the passage through the intestinal
show that intestinal
epithelium adsorption
demonstrates has asodium
that both physiological
hyaluronatetrend andas GreenIuronic
can be observed from
® were ablethe
to anal-
ysismaintain
of TEERthe and tight junction (TJ). In particular, the passage through the
epithelial integrity increasing the ionic flux of the paracellular exchanges intestinal epithe-
lium demonstrates
across the intestinalthatepithelial
both sodium hyaluronate
compared to control and(p <GreenIuronic
0.0001). Indeed, ® were ®
able to maintain
GreenIuronic
thedemonstrates
epithelial integrity
a betterincreasing the ionic
effect compared to flux
sodiumof the paracellular
hyaluronate exchanges
during all timesacross
of thethe in-
stimulation (p < 0.0001), as reported in Figure 4A. Afterwards, also
testinal epithelial compared to control (p < 0.0001). Indeed, GreenIuronic demonstrates the evaluation
® of TJ a
confirmed these results; indeed, GreenIuronic ® exerted the greatest effects on occludin
better effect compared to sodium hyaluronate during all times of the stimulation (p < 0.0001),
as (p = 0.0286, about 31%, Figure 4B), claudin-1 (p = 0.0299, about 37%, Figure 4C), and zonula
reported in Figure 4A. Afterwards, also the evaluation of TJ confirmed these results; in-
occludens-1 (ZO-1) (p = 0.0299, about 50%, Figure 4D) compared to sodium hyaluronate
deed, GreenIuronic® exerted the greatest effects on occludin (p = 0.0286, about 31%, Figure
and compared to control value (reported as 0 line, p < 0.05). From these encouraging results,
4B),which
claudin-1 (p = 0.0299,
confirmed about
the correct 37%, Figure
functioning 4C),
of the and zonula
intestinal occludens-1
epithelium, further(ZO-1) (p = 0.0299,
experiments
about
were50%, Figure
carried 4D) compared
out measuring to sodium rate,
the permeability hyaluronate
analyzing andthecompared to control value
flux of non-electrolyte
(reported as 0 line, pas<permeability
tracers (expressed 0.05). From coefficient
these encouraging
as reported) results,
and how which
much confirmed the correct
HA has crossed
functioning of the intestinal epithelium, further experiments were carried out measuring
the intestinal barrier to reach the target site. Data obtained from the analysis of the ba- the
solateral environment
permeability rate, analyzing(Figurethe4E) confirmed
flux our previous
of non-electrolyte findings
tracers since the as
(expressed amount of
permeability
coefficient as reported) and how much HA has crossed the intestinal barrier to reach the
target site. Data obtained from the analysis of the basolateral environment (Figure 4E) con-
firmed our previous findings since the amount of GreenIuronic® was higher compared to
sodium hyaluronate (p < 0.05) with a maximum effect at 4 h compared to sodium hyalu-
 Int. J. Mol. Sci. 2022, 23, 8114 7 of 26

Int. J. Mol. Sci. 2022, 23, 8114 7 of 26

GreenIuronic® was higher compared to sodium hyaluronate (p < 0.05) with a maximum
effect at 4 h compared to sodium hyaluronate (about 20%, p < 0.013). In addition, the
data obtained
basolateral levelfrom the quantification
(Figure 4F) supported of
thethe basolateral
hypothesis levelthe
about (Figure 4F) supported
importance the
of predicting
hypothesis about the importance of predicting human absorption; GreenIuronic ® has a
human absorption; GreenIuronic has a higher amount of HA that crosses the barrier and
®
higher amount
reaches the plasmaof HA
levelthat crosses the
compared barrier(p
to control and reachesand
< 0.0001) the compared
plasma level compared
to sodium to
hyalu-
control (p < 0.0001) and compared to sodium hyaluronate (about 30%,
ronate (about 30%, p < 0.0001) with the greatest effects between 4 and 5 h. p < 0.0001) with the
greatest effects between 4 and 5 h.

Figure4.4.
Figure Permeability
Permeability studystudy on CaCo-2
on CaCo-2 cells. Incells.
(A) TEER In Value
(A) TEER usingValue
EVOM3; using
fromEVOM3;
(B–D) thefrom
anal-
(B–D)
ysis of TJthe analysis by
measured of Enzyme-Linked
TJ measured byImmunosorbent
Enzyme-LinkedAssay Immunosorbent
(ELISA) testAssay (ELISA)
(Occludin, test (Oc-
Claudin1, and
cludin, Claudin1, and ZO-1, −6 cm/s
× 10absorption
ZO-1, respectively); in (E) the respectively);
Papp values in inwhich
(E) thedata
Papp values
<0.2 incm/s
× 10−6 which datavery
mean < 0.2poor
with
mean a bioavailability <1%, data
very poor absorption withbetween 0.2 × 10−6 <
a bioavailability and
1%,2 data
× 10−6between
cm/s with × 10−6 and 2 ×
0.2bioavailability 10−6 cm/s
between 1 and
90%, −6 cm/s mean very good absorption−with 6
with bioavailability between 1 and 90%, and data > 2 × 10 cm/s mean very good absorption withHA
and data >2 × 10 a bioavailability over 90%. In (F) a
quantification
bioavailabilitymeasured
over 90%.by InELISA
(F) HAkit. Data are mean
quantification ± SD ofby
measured five independent
ELISA kit. Dataexperiments
are mean ± SD performed
of five
inindependent
triplicates. From (B–D) means±
experiments performedSD in
aretriplicates.
expressedFrom comparing data to
(B–D) means ±control value (0% comparing
SD are expressed line) and * p
<data
0.05 to
vs.control
control;
value (0% line) and * p < 0.05 vs. control; # p < 0.05 vs. Sodium Hyaluronate the
# p < 0.05 vs. Sodium Hyaluronate 1 µg/µL. On the contrary, in (A,E,F) control
1 µg/µL.
samples are specifically reported and both GreenIuronic ® and sodium hyaluronate are p < 0.0001 vs.
On the contrary, in (A,E,F) the control samples are specifically reported and both GreenIuronic® and
control; # p < 0.05 vs. Sodium Hyaluronate 1 µg/µL.
sodium hyaluronate are p < 0.0001 vs. control; # p < 0.05 vs. Sodium Hyaluronate 1 µg/µL.

2.4. Effects of GreenIuronic® Crossed Intestinal Barrier on Chondrocytes.

Since the exogenous hyaluronic acid administered into articular cartilage has a direct
biological effect on chondrocytes, several experiments were carried out to explore the ef-
fect of GreenIuronic®, compared to sodium hyaluronate, on chondrocytes after intestinal
 Int. J. Mol. Sci. 2022, 23, 8114 8 of 26

Int. J. Mol. Sci. 2022, 23, 8114 8 of 26

2.4. Effects of GreenIuronic® Crossed Intestinal Barrier on Chondrocytes
Since the exogenous hyaluronic acid administered into articular cartilage has a direct
biological effect on chondrocytes, several experiments were carried out to explore the
absorption
effect in terms of
of GreenIuronic ® , mitochondrial
compared to sodium metabolism and cell
hyaluronate, on proliferation. As expected
chondrocytes after intestinal(Fig-
ure 5A), both
absorption 1 µg/µL
in terms of GreenIuronic ® and sodiumand
mitochondrial metabolism hyaluronate were ableAs
cell proliferation. to improve
expected cell
viability
(Figure compared
5A), to control
both 1 µg/µL (p < 0.05);® in
GreenIuronic particular,
and GreenIuronic
sodium hyaluronate ® induces the main ef-
were able to improve
® induces the main
fectviability
cell on cell viability
compared (about 50%,(p
to control p << 0.05)
0.05);compared to sodium
in particular, hyaluronate
GreenIuronic reducing ROS
effect on cell viability (about 50%, p
production (about 38% p < 0.05), as reported in Figure 5B. Furthermore, asreducing
< 0.05) compared to sodium hyaluronate reported in
ROS production
Figure (about 38%
5C, GreenIuronic p < 0.05),an
® induces asimprovement
reported in Figure 5B.proliferation
in cell Furthermore,compared
as reportedtoincon-
Figure ® induces an improvement in cell proliferation compared to control
trol (p5C, GreenIuronic
< 0.05), and compared to sodium hyaluronate, by about 60%, indicating that
(p ®
GreenIuronic is able to stimulatehyaluronate,
< 0.05), and compared
® to sodium by about
the proliferative 60%, indicating
activity that GreenIuronic
of chondrocytes. Since the im-
is able to stimulate
portance the proliferative
of the activity activity ofincludes
on cell proliferation chondrocytes. Sincetothe
the ability importance
modulate jointofproduc-
the
activity
tion of HA in cells, the HA quantification in chondrocytes (Figure 5D) revealed that ain
on cell proliferation includes the ability to modulate joint production of HA large
cells, the HA quantification in chondrocytes (Figure 5D) revealed that a large amount of
amount of HA present in GreenIuronic and sodium hyaluronate was captured by chon-
®
HA present in GreenIuronic® and sodium hyaluronate was captured by chondrocytes
drocytes after intestinal passage compared to the control (p < 0.05). In particular, approx-
after intestinal passage compared to the control (p ®< 0.05). In particular, approximately
imately 75% of HA was induced by GreenIuronic compared to sodium hyaluronate (p <
75% of HA was induced by GreenIuronic® compared to sodium hyaluronate (p < 0.05) in
0.05) in chondrocytes,
chondrocytes, confirming confirming
that HMWHA that HMWHA is better
is better utilized byutilized by chondrocytes.
chondrocytes.

®
Figure5.5.Analysis
Figure AnalysisofofGreenIuronic
GreenIuronicand Sodium
® and Sodium Hyaluronate
Hyaluronate on human
on human chondrocyte (T/C-28a2)
chondrocyte (T/C-28a2)
cells
cellsfunctions.
functions.InIn(A)
(A)the
themitochondrial
mitochondrial metabolism
metabolism tested byby
tested MTTMTTtest; in (B)
test; the the
in (B) ROSROS
production;
production;
in
in(C)
(C)the
theproliferation
proliferationanalysis
analysis by by
crystal violet
crystal assay;
violet and in
assay; (D)inthe
and (D)HA thequantification by ELISA
HA quantification by kit.
ELISA
Data are expressed
kit. Data as mean
are expressed as mean ± SD± compared
SD compared to control (0%(0%
to control line)line)
of five independent
of five independentexperiments
experiments
performed triplicates.* *p p< <0.05
performedinintriplicates. 0.05vs.
vs.control;
control;# p# <p 0.05 vs.vs.
< 0.05 Sodium
Sodium Hyaluronate
Hyaluronate 1 µg/µL.
1 µg/µL.

2.5. Effects of HA Crossed Intestinal Barrier on Chondrocytes under OA Condition

From the data obtained under physiological conditions, it can be assumed that
GreenIuronic® is also effective after oral administration and is an important starting point
for determining the success of therapy in joint damage, such as OA. Oxidative stress and
inflammation are known to be involved in cartilage degeneration of OA and it is similarly
Int. J. Mol. Sci. 2022, 23, 8114 9 of 26

2.5. Effects of HA Crossed Intestinal Barrier on Chondrocytes under OA Condition

From the data obtained under physiological conditions, it can be assumed that
GreenIuronic® is also effective after oral administration and is an important starting point
for determining the success of therapy in joint damage, such as OA. Oxidative stress
and inflammation are known to be involved in cartilage degeneration of OA and it is
similarly approved that the degree of anti-inflammatory, immunomodulatory, analgesic,
and anti-OA effects of HA is determined by MW and route of administration. Based on
these results, in the last phase, the in vitro study was conducted by analyzing the effects
of both 1 µg/µL of GreenIuronic® and sodium hyaluronate on T/C-28a2 cells pretreated
with 10 µg/mL of lipopolysaccharide (LPS) for 24 h in order to simulate the condition of
OA. The effects of chondrocyte metabolism were shown in Figure 6 where the beneficial
effects of GreenIuronic® can be observed. Specifically, chondrocytes treated only with
10 µg/mL of LPS significantly reduced cell viability (panel 6A, about 10%) and improved
ROS production (panel 6B about 23%) compared to control (p < 0.05) but this effect was
significantly reduced by the presence of both agents. In particular, GreenIuronic® was
able to counteract these negative effects caused by LPS alone (p < 0.05) better than sodium
hyaluronate (about 47% on cell viability and two times on ROS production, respectively,
p < 0.05). These data were also confirmed by nuclear factor kappa B (NFkB) analysis
(Figure 6C) in which the beneficial potential of GreenIuronic® against inflammation, a key
point in the mechanisms involved during OA processes, was observed. Indeed, the cells
treated with only 10 µg/mL of LPS increased inflammatory processes compared to control
(about 18%, p < 0.05) assuming the beginning of chronic processes leading to cell death,
and this situation was reversed following stimulation with both HA agents. In particular,
1 µg/µL GreenIuronic® was able to reduce the negative effect produced by LPS (about
1.25 times more) better than sodium hyaluronate (about 50%, p < 0.05). This recovery
mechanism was confirmed also by proliferation assay (panel 6D) in which T/C-28a2 cells
lost their proliferative properties when treated only with 10 µg/mL of LPS (p < 0.05 com-
pared to control). On the contrary, both 1 µg/µL GreenIuronic® and sodium hyaluronate
counteract this negative effect compared to control (p < 0.05), but GreenIuronic® was able
to restore the damage by about 62% compared to sodium hyaluronate (p < 0.05), supporting
its use during OA injuries. Finally, this recovery mechanism was also confirmed by the
analysis of HA, which showed that under OA conditions, GreenIuronic® is able to improve
a much higher amount of HA released in stressed chondrocytes than sodium hyaluronate
at the same concentration, approximately by about 21% (p < 0.05).
In order to demonstrate that LPS is able to reproduce the OA condition in vitro leading
to chondrocyte death, additional experiments were carried out to explore the involvement
of the apoptosis process. As reported in Figure 7, several markers related to apoptotic pro-
cesses were evaluated in response to 10 µg/mL of LPS and to both 1 µg/mL GreenIuronic®
and sodium hyaluronate. In particular, the stimulation with 10 µg/mL of LPS treatment on
T/C-28a2 cells enhanced BAX and Caspase 9 activities (Figure 7A,B), about 22% and 18%
compared to control (p < 0.05), indicating a dramatic improvement of the apoptosis process
supporting the chondrocyte death during OA. Contemporary, the stimulation with both
1 µg/µL GreenIuronic® and sodium hyaluronate added after 10 µg/mL of LPS caused
a statistically significant reduction in both these markers. In particular, GreenIuronic®
exerted the main effects compared to sodium hyaluronate on Bax (about 2 times less,
p < 0.05) and Caspase-9 activities (about 1.5 times less, p < 0.05), suggesting that GreenIuronic®
contributes to cell protection. These data were also confirmed by the activation of risk
pathways such as mitogen-activated protein kinases/extracellular signal-regulated kinase
(ERK/MAPK) activity (Figure 7C), which demonstrated that GreenIuronic® reverts the
10 µg/mL LPS damage, activating the survival pathways and restoring the chondrocyte to
normal conditions (about 25% compared to sodium hyaluronate, p < 0.05).
Int.
Int.J.J.Mol.
Mol.Sci.
Sci.2022,
2022,23,
23,8114
8114 10ofof2626
10

®
Figure 6.
Figure 6. GreenIuronic
GreenIuronic® andand Sodium
Sodium Hyaluronate
Hyaluronateeffects
effectsononT/C-28a2
T/C-28a2cellscellsduring
duringOA OAconditions.
conditions.
In (A) mitochondrial metabolism tested by MTT test; in (B) ROS production; in (C) NFkBanalysis
In (A) mitochondrial metabolism tested by MTT test; in (B) ROS production; in (C) NFkB analysis
by ELISA
by ELISA test;
test; in
in (D)
(D) proliferation
proliferation analysis
analysisby bycrystal
crystalviolet;
violet;and
andinin(E)
(E)HA
HAquantification
quantificationbybyELISA
ELISA
kit. Data are mean ±
kit. Data SD of five independent
± SD independent experiments
experiments performed
performedinintriplicates
triplicatesexpressed
expressedasasa a
percentage compared to to control
control (0%
(0% line).
line). **pp<<0.05
0.05vs.vs.control; y py <p 0.05
control; vs.vs.
< 0.05 10 µg/mL of LPS;
10 µg/mL #p<
of LPS;
0.05
# p <vs. Sodium
0.05 Hyaluronate
vs. Sodium 1 µg/µL.
Hyaluronate 1 µg/µL.
Int.Int.
J. Mol. Sci.
J. Mol. Sci.2022,
2022,23,
23,8114
8114 11 of 2611 of 26

Figure7.7.Analysis
Figure Analysisofofthe
themain
main intracellular
intracellularpathways
pathways activated in T/C-28a2
activated cellscells
in T/C-28a2 during AO con-
during AO condi-
ditions. The results demonstrated a reduction in apoptotic pathways
tions. The results demonstrated a reduction in apoptotic pathways and an improvement and an improvement of the
of the sur-
survival pathways
vival pathways supportingthe
supporting theability
ability of
of HA
HA totorestore
restorethe theOA
OA damage.
damage. In In
(A)(A)
BAXBAX activity; in in (B)
activity;
(B) Caspase
Caspase 9 activity;
9 activity; in in
(C)(C) ERK/MAPK
ERK/MAPK activity;
activity; allallthese
theseresults
resultsare
areobtained
obtained from
from specifically
specifically ELISA
ELISA Data are mean ±
kit. Data are mean ± SD of five independent experiments performed in triplicates compared
kit. SD of five independent experiments performed in triplicates compared to the
to the control
control valuevalue (0% line).
(0% line). p < 0.05
* p <*0.05 vs. control; y p y< p0.05
vs. control; < 0.05
vs.vs.
10 10 µg/mL
µg/mL of of
LPS; # p# <p <
LPS; 0.05vs.
0.05 vs.Sodium
Sodium Hyaluronate
Hyaluronate 1 µg/µL. 1 µg/µL.

Finally, to explore the possible effector molecules responsible for the maintenance
Finally, to explore the possible effector molecules responsible for the maintenance of
of chondrocyte wellbeing, the activity of cyclin D1, osteoprotegerin (OPG), and CD44
chondrocyte wellbeing, the activity of cyclin D1, osteoprotegerin (OPG), and CD44 were
were investigated. As reported in Figure 8A–D, 10 µg/mL of LPS confirmed its negative
investigated.
effect on T/C-28a2As reported in Figure
cells compared 8A–D,
to control 100.05)
(p < µg/mL of LPS confirmed
downregulating its negative
OPG activity, CD44 effect
on T/C-28a2
and cyclin D1cells compared
expressions to control
(about 18%, 8%,(p <and0.05)
12%downregulating OPG activity,
compared to control, CD44 and
respectively)
modifying negatively chondrocytes activity. Conversely, both 1 µg/µL GreenIuronic and modi-
cyclin D1 expressions (about 18%, 8%, and 12% compared to control, respectively)
®

fying negatively
sodium hyaluronatechondrocytes activity.
were able to reduce Conversely,
the damage induced both
by 110µg/µL
µg/mLGreenIuronic ® and so-
of LPS (p < 0.05),
confirming the positive
dium hyaluronate wererole
ableof to
HA contained
reduce in two agents
the damage induced in stimulating
by 10 µg/mL chondrocyte
of LPS (p < 0.05),
metabolism. In particular, 1 µg/mL GreenIuronic ® appears to be able to induce main
confirming the positive role of HA contained in two agents in stimulating chondrocyte
effects compared
metabolism. to sodium hyaluronate
In particular, (p < 0.05) to® counteract
1 µg/mL GreenIuronic appears tothe be negative effects of
able to induce main ef-
OA induction. Indeed, 1 µg/mL GreenIuronic® is able to restore the damage induced by
fects compared to sodium hyaluronate (p < 0.05) to counteract the negative effects of OA
10 µg/mL of LPS in all parameters tested (about 60% for OPG, one time more for CD44,
induction. Indeed, 1 µg/mL GreenIuronic® is able to restore the damage induced by 10
and 57% for cyclin D1 expression, p < 0.05), suggesting that it could ameliorate chondrocyte
µg/mL of LPS
pathological in all parameters
conditions by activating tested
them(about
through60% thefor OPG, one
markers time more
responsible for CD44, and
for articular
57% for cyclin
joint homeostasis. D1 expression, p < 0.05), suggesting that it could ameliorate chondrocyte
pathological conditions by activating them through the markers responsible for articular
joint homeostasis.
Int.
Int.J.J.Mol.
Mol.Sci.
Sci.2022,
2022,23,
23,8114
8114 1212
of of
26 26

Figure 8.
Figure Western-blot and
8. Western-blot and densitometric
densitometric analysis
analysis of of the
the main
main intracellular
intracellular pathways
pathways activated
activated in
T/C-28a2 cells during AO conditions. In (A) OPG activity measured by ELISA test,test,
in T/C-28a2 cells during AO conditions. In (A) OPG activity measured by ELISA in the
in (B) (B) CD44,
the
CD44, and in (C) cyclin D1 densitometric analysis of the specific Western blot,
and in (C) cyclin D1 densitometric analysis of the specific Western blot, which is reported as which is reported as an
example
an examplein (D). Data
in (D). areare
Data meanmean ± SD
± SD of five independent
of five independent experiments
experiments performed in triplicates
performed com-
in triplicates
pared to control
compared value
to control (0%(0%
value line). * p*<p0.05
line). < 0.05vs.vs.
control;
control;y yp p< <0.05
0.05vs.
vs.1010µg/mL
µg/mLofofLPS;
LPS;##pp<<0.05
0.05vs.
Sodium
vs. Hyaluronate
Sodium Hyaluronate 1 µg/µL.
1 µg/µL.

3. Discussion
3. Discussion
Current
Current guidelines
guidelinesfor forthe
thetreatment
treatmentofofOA OAsuggest
suggest many
many conventional
conventional approaches
approaches
to improve this chronic condition. For example, pharmacological
to improve this chronic condition. For example, pharmacological treatment, which treatment, which is
is char-
characterized by NSAIDs [34], opioids, and cyclooxygenase (COX)-2-specific drugs, is an
acterized by NSAIDs [34], opioids, and cyclooxygenase (COX)-2-specific drugs, is an ac-
accepted method considered only as a “palliative” method since it reduces the symptoms
cepted method considered only as a “palliative” method since it reduces the symptoms
but does not address the essential problem of cartilage degeneration [35]. In addition,
but does not address the essential problem of cartilage degeneration [35]. In addition, con-
conventional therapies can cause possible side effects, especially for long periods of use,
ventional therapies can cause possible side effects, especially for long periods of use,
which can reduce the compliance at the onset of gastrointestinal, cardiovascular, and other
which
adverse can reduce
effects theFurthermore,
[36]. compliance atthe theconventional
onset of gastrointestinal,
therapies often cardiovascular,
use HA injections and other
to
adverse
treat knee OA and to improve the functions of the knee joint, these methods are called to
effects [36]. Furthermore, the conventional therapies often use HA injections
treat knee OA and to[37].
viscosupplementation improve
It wasthe functions
reported that of the knee joint,
intra-articular these methods
HA improves synovialarefluid
called
viscosupplementation
elasticity and viscosity by [37]. It was reported
decreasing the releasethat intra-articular
of pain mediators and HAproinflammatories
improves synovial
fluid elasticity and viscosity by decreasing the release of pain
from synovial cells [37]. Otherwise, a recent systematic review, based on the analysis mediators and proinflam-
of pain
matories from synovial cells [37]. Otherwise, a recent systematic
relief and functional improvement, concluded that the routine use of HA injections review, based doeson the
analysis
not produce of pain
so manyreliefbenefits
and functional improvement,
for the patient concluded
with no clinical that the
relevance, routine
because usepain
of the of HA
injections
caused [38].does not produce
According so many
to the current benefits
protocol, HA forshould
the patient with no clinical
be administered relevance,
repeatedly into
the joint cavity,
because of the but
painmultiple
causedinjections could cause
[38]. According to the much
currentdiscomfort
protocol,in patients
HA should and increase
be admin-
the risk of complications [39]. Notwithstanding, HA is a useful tool
istered repeatedly into the joint cavity, but multiple injections could cause much discom- in the management of
patients with OA, since clinical data indicate its ability to reduce pain
fort in patients and increase the risk of complications [39]. Notwithstanding, HA is a use- and improve joint
function,
ful tool inwith the potential ability
the management to modify
of patients chondrocytes
with OA, since clinicalactivity
data[40]. Also,its
indicate it should
ability to
be taken
reduce intoand
pain account
improvethatjoint
the administration
function, with the of HA by intraarticular
potential injection
ability to modify can also
chondrocytes
cause adverse
activity effectsit such
[40]. Also, should as infectious
be taken arthritis and cartilage
into account that the damage [41]. Therefore,
administration of HA by thein-
possibility of administering HA orally represents a considerable advantage
traarticular injection can also cause adverse effects such as infectious arthritis and carti- [13]. From this
point of view, several studies have explored new approaches for consistent and pain-free
lage damage [41]. Therefore, the possibility of administering HA orally represents a con-
siderable advantage [13]. From this point of view, several studies have explored new ap-
proaches for consistent and pain-free administration of HA, reporting positive effects after
Int. J. Mol. Sci. 2022, 23, 8114 13 of 26

administration of HA, reporting positive effects after oral administration and suggesting
that it may have beneficial therapeutic effects on patients with OA [39,42,43]. Thus, the
possibility of using HA in a dietary supplement to be taken orally sparked interest in
designing a new nutraceutical (based on plant-derived HA, called GreenIuronic® ) able to
counteract the harmful consequences of OA. With these premises, our chemical analysis
revealed that GreenIuronic® contains a large amount of HA with a chemical profile useful
to be a new nutraceutical.
In addition, the presence of a high molecular weight ingredient related to HA sup-
ports its use to counteract the adverse effects of OA, since high molecular weight HA is
nowadays the best treatment option for knee OA by intra-articular injection [44]. However,
as reported in the literature, high levels of HA in serum after its oral administration in vivo
model are also reported. Indeed, therapeutic efficacy of HA against lameness was found to
be greater with oral than intra-articular administration because this way of administration
dissipates out of the joint the main amount of HA within 14–18 h; HA diffuses out of
tissues via the bloodstream, circulating throughout the body, and is rapidly eliminated [17].
In addition, several human studies have revealed that patients with OA must undergo
clinical visits repeatedly and must undergo the discomfort associated with injections while
also experiencing an increase in complications associated with repeated injections [39].
Considering these obvious and potential drawbacks, it is much more desirable to use HA by
oral administration to improve the condition of OA. Indeed, some studies have suggested
that knee OA symptoms may actually be alleviated by taking HA, and other studies also
report positive effects of orally administered HA on improving joint function in mild to
moderate osteoarthritis of the knee [39,45–47]. In addition, the international evidence-
based guidelines agree that knee OA management requires both non-pharmacological and
pharmacological approaches and suggest initiating a background therapy with chronic
symptomatic slow-acting drugs for OA such as HA by oral administration [48]. Conse-
quently, the effects of GreenIuronic® were analyzed mimicking the human oral ministration
in vitro, since orally administered HA should be absorbed and distributed to the knee
joints where it exerts its biological activities. The results obtained from the 3D model
that mimics intestinal absorption clearly demonstrated that oral administration is possible.
Bioavailability experiments indicated that orally administered HA is effectively absorbed
and biodistributed to the chondrocytes and exerts its biological functions in those tissues.
Indeed, GreenIuronic® has a high amount of HA that reaches the plasma level compared
to control (p < 0.05) and compared to sodium hyaluronate within 4 h and 5 h, confirming
the hypothesis that GreenIuronic® improves the absorption during the physiological time
of intestinal digestion and improving its bioavailability. In addition, GreenIuronic® treat-
ment indicated that a substantial part of HA is absorbed without damaging the intestinal
epithelium; this is a crucial point since HA has a role in decreasing the permeability by
enhancing tight junction proteins. In epithelial cells, the formation of tight junctions plays
an important role in the intestinal barrier, and this is mediated by proteins such as claudins,
occludin, and ZO-1 that are necessary for epithelial barrier activity [49].
These proteins are critical in maintaining homeostatic intestinal permeability in multi-
ple intestinal inflammatory diseases, supporting a gut–joint axis in OA pathogenesis and
progression [50]. In particular, dysbiosis-related gut permeability determined lower mRNA
levels of TJ, ZO-1, and occludin, and higher LPS plasma levels in the in vivo model, which
has a positive association of synovial LPS with inflammation and disease severity in articu-
lar chondrocytes in OA patients [51,52]. For this reason, several studies using nutraceuticals
evaluate the intestinal integrity as the first step to ameliorate pain and disease progression
of OA [50] during chronic nutritional intervention [36], using also HA as a multifunctional
agent [9]; in particular, Kotla et al. demonstrated that the treatment with HA upregulated
the expression of the tight junction proteins claudin and occludin [53]. These three proteins
are pivotal because ZO-1 connects claudin and occludins to the cytoskeleton so they are
indicators of good gut barrier functions [54]. Furthermore, it has been demonstrated that
GreenIuronic® is able to maintain epithelial integrity and the ionic exchanges across the
Int. J. Mol. Sci. 2022, 23, 8114 14 of 26

intestinal barrier, suggesting that this proteoglycan is able to pass the cell monolayer with-
out negatively altering the epithelium. Subsequently, the second important purpose of this
work was to test the ability of GreenIuronic® to stimulate chondrocyte biological activity
under physiological and pathological conditions. As expected, GreenIuronic® was able
to stimulate cell viability and induce chondrocyte proliferation without causing adverse
effects, also compared to conventional HA supplementation Indeed, thanks to the presence
of HMWHA, the beneficial effects of GreenIuronic® on the activity of chondrocytes support
the hypothesis of its use in inflammatory joint conditions. Since OA is a disease of the
whole joint and a multifactorial entity, there are various therapeutic strategies that involve
numerous fields of medicine: rheumatology, orthopedics, geriatrics, psychiatry, general
practitioners, and physiotherapists. The goal of OA therapy is to reduce pain and increase
patients’ quality of life. For this purpose, HA has shown not only beneficial effects on
articular cartilage trophism, but also antinociceptive effects with a significant reduction in
pain [55]. In particular, the beneficial effects of GreenIuronic® have also been confirmed by
the quantity of HA, contained in this new formulation, which reached the target site and
was absorbed into the joint without damaging it. Consequently, the therapeutic effects of
GreenIuronic® on OA conditions may necessarily require the improved absorption of HA.
Moreover, we pre-treated T/C-28a2 cells with 10 µg/mL of LPS to mimic the osteoarthritic
phenotype as reported by Zhang et al. [56]. Our data support the literature [57,58] showing
that HMWHA may bind to CD44 on chondrocytes to exert its biological activities, demon-
strating that the association of HA with CD44 increased the HA absorption/production
suppressing proinflammatory processes. The binding of HA, present in GreenIuronic® ,
to CD44 also suppresses the expression of the apoptosis process, which again contributes
to the damage of chondrocytes and improves its activity, regulating cartilage production,
improving OPG activity, and proliferation process, modulating cyclin D1 expression. Taken
together, these results suggest that GreenIuronic® is the best choice to maintain chondrocyte
behavior during the inflammatory condition related to OA, and therefore, the application
of this innovative HA form could be an excellent strategy to restore OA damage.

4. Materials and Methods

4.1. Agents Preparation
GreenIuronic® was obtained from White Tremella (Silver Ear), which is a traditional
foodstuff with medicinal applications in China [59]. The production process involves
several steps necessary to obtain a final extract and includes a new technology based on
patent N◦ WO2021/250566 from Vivatis Pharma GBHE, Grüner Deich 1–3, 20,097 Hamburg,
Germany. Briefly, the process involves steps of extraction, purification, and refining by
alcohol solution, sieving, and crushing. The resulting powder is then packed and tested for
metals and stored [60,61]. In addition, sodium hyaluronate (Merck Life Science, Rome, Italy)
was tested to verify the mechanism of action of GreenIuronic® . All these substances are
prepared directly in water for HA determination or directly in Dulbecco’s Modified Eagle’s
Medium (DMEM, Merck Life Science, Rome, Italy) without phenol red and supplemented
with 0.5% fetal bovine serum (FBS, Merck Life Science, Rome, Italy), 2 mM L-glutamine
(Merck Life Science, Rome, Italy), and 1% penicillin–streptomycin (Merck Life Science,
Rome, Italy) for biological analysis.

4.2. HPLC Analysis

The determination of the HA was also confirmed by HPLC (Shimadzu, Kyoto, Japan)
analysis according to the method reported in the literature [62], as described in Appendix A
(Appendices A.1 and A.2). Briefly, 20 µL of TRIS buffer (3.0 g TRIZMA base, 4.0 g sodium
acetate trihydrate, 1.46 g sodium chloride, and 50 mg crystalline bovine serum albumin
dissolved in 100 mL of 0.12 M HCl, pH 7.3 with 6 M HCl. All chemicals are purchased from
Merck Life Science, Rome, Italy), 30 µL of chondroitinase AC solution (Merck Life Science,
Rome, Italy) (diluted to 10 U/mL with water), and 20 µL of GreenIuronic® test solution
(200 mg dissolved in 100 mL of water) were pipetted into a conical 1.5 mL vial. The vial
Int. J. Mol. Sci. 2022, 23, 8114 15 of 26

was placed in a warm water bath at 37 ◦ C for 3 h. After cooling at room temperature, the
sample was diluted to 1 mL by adding 930 µL of mobile phase A (reagent purchased from
Merck Life Science, Rome, Italy, and column from Phenomenex Srl, Bologna, Italy) (see
Appendices A.1 and A.2 in Appendix A) and the mixture was analyzed by HPLC-UV and
HPLC-HRMS systems. A control solution was prepared by replacing the enzyme aliquot
with TRIS buffer.

4.3. Colorimetric Determination of Hyaluronic Acid

The assay performed to quantify the concentration of HA on material samples was the
same reported in the literature [63]. Briefly, 1 mg of raw sample was dissolved in 1 mL of
deionized water, and 200 µL of resuspended samples were displaced in new Eppendorf,
diluted with 1.2 mL of sulfuric acid (Merck Life Science, Rome, Italy) with 0.0125 M
tetraborate (Merck Life Science, Rome, Italy), shaken for 20 s and then boiled at 100 ◦ C for
5 min. Once the samples were allowed to cool on ice, 20 µL of 0.15% hydroxydiphenyl
(Merck Life Science, Rome, Italy) (dissolved in 0.5% NaOH, Merck Life Science, Rome,
Italy) was added and stirred; 100 µL of each sample was placed in a 96 multi-well plate
and the absorbance was measured at 340 nm by a spectrophotometer (Infinite 200 Pro
MPlex, Tecan, Männedorf, Switzerland). The data obtained were compared to a calibration
curve generated using glucuronic acid (0, 0.25, 0.5, 1, 1.5, 2 mg/mL Merck Life Science,
Rome, Italy) [64] and the results were expressed as mean (%w/w) ± SD compared to control
(0 line).

4.4. Molecular Weight Determination

The determination of the molecular weight of HA before exploring its biological effects
was carried out using 1% agarose gel, following the method reported in the literature [65].
Briefly, 0.3 g agarose (Merck Life Science, Rome, Italy) was dissolved in 30 mL of Tris-
acetate-EDTA (TAE) buffer (48.5 g tris base, 11.4 mL acetic acid, and 0.5 M EDTA pH 8, all
substances were purchased from Merck Life Science, Rome, Italy) and the solution was
heated for 30 s in a microwave at high power. The gel was poured into the holder and
allowed to solidify before performing a pre-run at 100 V for 45 min, using the Mini-Sub
Cell GT System (Bio-Rad, Hercules, CA, USA). In the meantime, samples were prepared by
dissolving 200 µg of raw samples in 16 µL of TAE buffer 1×. Before running the gel, 4 µL
of loading buffer (0.2% Bromophenol Blue, 1 mL of TAE 1×, and 8.5 mL of glycerol, which
were purchased from Merck Life Science, Rome, Italy) was added to each sample and to
the molecular weights (mixture of 5 µL of Select-HA HiLadder and 5 µL Select-HA Mega
Ladder, Echelon Biosciences, Tebu-Bio Srl, Magenta, Italy). The samples were run at 100 V
until the samples reached 1 cm from the end of the gel. Then, the gel was hydrated in H2 O
for 24 h at room temperature in agitation and then the gel was placed in 30% ethanol with
0.015% Stains All dye (Merck Life Science, Rome, Italy) for 24 h in the dark. The gel was
decolored for 30 min in H2 O in the dark before proceeding with image acquisition using
ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, USA). The image obtained
was analyzed by Image Lab 3.0 software (Bio-Rad Hercules, CA, USA).

4.5. Cell Culture

The human epithelial intestinal cells, CaCo-2, purchased from the American Type Cul-
ture Collection (ATCC), were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient
F-12 Ham (DMEM-F12, Merck Life Science, Rome, Italy) containing 10% FBS (Merck Life
Science, Rome, Italy), 2 mM L-glutamine and 1% penicillin–streptomycin (Merck Life
Science, Rome, Italy) maintaining in an incubator at 37 ◦ C and 5% CO2 [66]. The cells
used for the experiments were at passage numbers between 26 and 32 in order to preserve
the integrative paracellular permeability and transport properties [67] maintaining the
similarity to the intestinal absorption mechanism following oral intake in humans. The cells
were plated in a different manner to perform several experiments including 1 × 104 cells in
96 well plates to study cell viability by MTT-based In Vitro Toxicology Assay Kit (Merck Life
Int. J. Mol. Sci. 2022, 23, 8114 16 of 26

Science, Rome, Italy) and ROS production using cytochrome C (Merck Life Science, Rome,
Italy) in a complete medium. Eight hours before the stimulation the cells were incubated
with DMEM without red phenol and supplemented with 0.5% FBS (Merck Life Science,
Rome, Italy), 2 mM L-glutamine, and 1% penicillin–streptomycin (both from Merck Life
Science, Rome, Italy) at 37 ◦ C to synchronize them. In addition, 2 × 104 cells were plated
on 6.5 mm Transwell® (Corning® Costar® , Merck Life Science, Rome, Italy) with a 0.4 µm
pore polycarbonate membrane insert (Corning® Costar® , Merck Life Science, Rome, Italy)
in a 24 well plate to perform the absorption analyses [68]. Cells plated on the Transwell®
insert were maintained in a complete medium, which was changed every other day on the
basolateral and apical sides for 21 days before the simulations [69]. Before the stimulation,
on the apical side, the medium was brought to pH 6.5 as the pH in the lumen of the small
intestine, while the pH 7.4 on the basolateral side represented blood [70,71]. This in vitro
model is widely used [68,72] and accepted by European Medicines Agency (EMA) and
FDA to predict the absorption, metabolism, and bioavailability of several substances after
oral intake in humans [73,74].
The immortalized human juvenile costal chondrocyte cell line T/C-28a2 (purchased
from Merck Life Science, Rome, Italy) was cultured in DMEM-F12 medium supplemented
with 10% FBS (Merck Life Science, Rome, Italy), 2 mM L-glutamine (Merck Life Science,
Rome, Italy), and antibiotics (50 UI/mL penicillin and 50 µg/mL streptomycin, Merck Life
Science, Rome, Italy)) and maintained in an incubator at 5% CO2 and 95% humidity [75].
This cell line is representative and the most commonly used cells for mimicking joints [76]
and they were used between passages 3 and 10 [77]. For the experiments 1 × 104 cells were
seeded in 96 well plates to study cell viability by MTT-based In Vitro Toxicology Assay
Kit (Merck Life Science, Rome, Italy), ROS production using cytochrome C (Merck Life
Science, Rome, Italy), and Crystal Violet (Merck Life Science, Rome, Italy) in a complete
medium; additionally, 1 × 106 cells were plated on a 6-well to determine HA concentration,
using quantification kit, and to analyze molecular pathways by Western-blot analysis or
ELISA kit.

4.6. Experimental Protocol

In order to analyze the beneficial effects of hyaluronic acid on articular joints in hu-
mans after oral intake, the experiments were divided into two steps; the aim of the first
one was to verify the ability of HA to cross the intestinal barrier in vitro model excluding
negative effects, and of the second one was to check the direct effects on chondrocytes
analyzing several parameters and mechanism of actions. For this reason, in intestinal
CaCo-2 cells, a dose–response study ranging from 0.125 to 1 µg/µL [78] was performed to
assess the concentration able to exert beneficial effects on cell viability and ROS production.
Subsequently, the best concentration of GreenIuronic® and hyaluronic acid salt were tested
on an intestinal in vitro barrier model to verify intestinal integrity through TEER measure-
ment, tight junction analysis by ELISA kit, and permeability assay by Papp measurement,
also analyzing the total amount of hyaluronic acid that had crossed the intestinal barrier.
For all these experiments, cells were treated in a time-dependent manner from 2 to 6 h, as
reported in the literature [66]. In addition, after each stimulation, the basolateral medium
was collected to be used on chondrocytes cells. T/C-28a2, a chondrocyte cell line widely
used to study articular joints, was treated for 3 days [79] and, at the end of stimulation,
the mitochondrial metabolism, cell proliferation, ROS production, and hyaluronic acid
quantification were tested. Finally, in order to mimic OA conditions, further experiments
were performed pre-treating T/C-28a2 with 10 µg/mL of LPS (Merck Life Science, Rome,
Italy) for 24 h [80], and then stimulating with GreenIuronic® and sodium hyaluronate for
3 days to evaluate if they are able to restore the damage. In these conditions, the survival
mechanisms and articular recovery were investigated.
 Int. J. Mol. Sci. 2022, 23, 8114 17 of 26

Int. J. Mol. Sci.

Int. 2022,
J. Mol.23,
Int.
Sci.
8114
J.2022,
Mol. Sci.
23, 8114
2022, 23, 8114 17 of 26 17 of 26 17 o

4.7. Cell Viability

The analysis of cell viability was performed using a classical technique based on the
the manufacturer’s
the manufacturer’s
MTT-based theVitro
In instructions.
manufacturer’sinstructions.
Toxicology Indeed,
instructions.
Assay atIndeed,
the (Merck
Kit end Indeed,
atofthe
stimulation,
end
LifeatScience,
the
of stimulation,
end theofcells
Rome,stimulation,
were
the cells
Italy) incubated
the
[81],werecells
incubated
followingwere incuba
withthe1%manufacturer’s
MTT
with 1%dyewith
MTT
for1% 2 h
dye
instructions. Indeed, at the end of stimulation, the cells were incubatedthen
MTT
in for
an dye
2 h
incubator
in
for an2 hincubator
at
in37
an °C,
incubator
at
5% 37CO°C,2at
, 5%
and
37 CO
°C,
95% 25%
, and
humidity,
CO 95%
2, andhumidity,
and
95% then
humidity,
and and t
the purple theformazan
purple
the formazan
purple
crystals formazan
were
crystalsdissolved
crystals
were dissolved
in
were
an ◦equal
dissolved
in an
volume
with 1% MTT dye for 2 h in an incubator at 37 C, 5% CO2 , and 95% humidity, and then equal
in anof
volume
equal
MTT volume
Solubilization
of MTT of
Solubilization
MTT Solubilizat
Solution. Solution.
The absorbance
the purple Solution.
The absorbance
formazan The
was absorbance
crystalsanalyzed
was analyzed
were bywasspectrophotometer
dissolved analyzed
by
in anspectrophotometer
by spectrophotometer
equal (Infinite
volume of 200
(Infinite
MTT Pro (Infinite
MPlex,
200 Pro200
Solubilization MPlex,
Pro MP
Tecan, Männedorf,
Tecan,The
Solution. Männedorf,
Tecan,
Switzerland)
Männedorf,
absorbance Switzerland)
wasat 570 nm
Switzerland)
analyzed atwith
570 nm correction
at 570 nm
with correction
by spectrophotometer at
with690 nm,
correction
at and
690 nm,
(Infinite results
at200690 nm,
and were
Pro results
and were
MPlex, results w
expressed expressed
Tecan, comparedexpressed
Männedorf, compared
to the compared
control
to the(0%
Switzerland) control
to
at line),
the
570 control
(0%
nmwhichline),
with (0%
represented
which
line),represented
correction whichuntreated
at 690represented
nm,untreated
cells. The
untreated
and results cells.
re-were
Thecells.
re- The
sultsexpressed
reported
sults reported
an sults
increase
reported
an in
increase
the
an percentage
increase
in the percentage
in of
theviable
percentage
of
cells
viable
compared
of cells
viable
compared to the control (0% line), which represented untreated cells. The results compared
to
cells
the compared
control
to the and
control
to the control
and a
indicated indicated
a higher indicated
number
a higher aof
number
higher
viable number
of
cells
viable
plus
of cells
the
viablecontrol.
plus cells
the plus
This
control.
the
strategy
reported an increase in the percentage of viable cells compared to the control and indicated control.
This can
strategy
This
lead strategy
to
cana lead
high can
to alead
highto a h
levelaofhigher
safety
levelnumber
of level
safety
the stimulation
of
ofofsafety
the stimulation
viable of and,
theplus
cells stimulation
consequently,
and,control.
the consequently,
and, toThis
consequently,
a correct to aanalysis
strategy correct
to alead
can of
analysis
correct
thetoresults.
aanalysis
of thelevel
high results.
of the
of results
safety of the stimulation and, consequently, to a correct analysis of the results.
4.8. In Vitro
4.8.Intestinal
In Vitro
4.8. InIntestinal
Barrier
Vitro Model
Intestinal
Barrier Model
Barrier Model
4.8. In Vitro
An intestinal Intestinal
An intestinal
barrier Barrier
An intestinal
model, Model
barrierusingmodel,
barrier CaCo-2
model,
usingcells,
CaCo-2
usingwas CaCo-2
cells,
performed
wascells,
performed
to
was
analyze
performed
to the
analyze
pas-
to analyze
the pas-the p
sage throughAn intestinal
sage the
through
sage barrier
intestinal
through model,
the intestinal
barrier using
the intestinal
ofbarrier CaCo-2
GreenIuronic
of
barrier cells,
GreenIuronic
®ofand was ® performed
GreenIuronic
sodium andhyaluronate,
sodium
® and tosodium
analyze
hyaluronate, the passage
having,
hyaluronate,
ashaving,having
as
through thedestination, ®
a final destination,
a final aintestinal
final barrier
the chondrocyte
destination, ofthe
GreenIuronic
the chondrocyte
where chondrocyte
theywhere
could and
they
where sodium
exertcould
their
theyexerthyaluronate,
could
beneficial
their
exert having,
beneficial
effects.
their as
beneficial
Foreffects.
this a final
For
effects.
thisFor
destination,
reason, the
reason, the
TEERreason,
the chondrocyte
values
TEER the
were
values
TEER where
determined
were
values they could
determined
were
with exert
determined
EVOM3,
with their
EVOM3,beneficial
coupled
with EVOM3,
with effects.
coupled STX2 For
coupled
with this
chopstick
STX2
withreason,
chopstick
STX2 chops
the
electrodesTEER values
electrodes
(World were
electrodes
Precision determined
(World (World
Precision
Instruments, with
Precision EVOM3,
Instruments,
Sarasota,
Instruments,coupled
Sarasota,
FL, USA); with
Sarasota,
FL,
this USA);STX2
assay
FL,this chopstick
USA);
was assay
carried
thiswas electrodes
assay
out
carried
was carried
out
every(World
2 days
everyPrecision
for2 every
21
days Instruments,
daysfor
2 days
until
21 days
for 21Sarasota,
reaching
until
days FL,reaching
reaching
a until
TEER USA);
value
a TEER ≥this
400 assay
avalue
TEERΩcm was
≥ value
400 carried
2 before
Ωcm ≥ 400
the out
2 before
Ωcm every
stimulation
2 the
before2 days
stimulation
the stimulat
for the
[66,82], 21 days
timeuntil
[66,82], required
[66,82],
the timereaching
the
fortimea TEER
required
the cell
requiredvalue
formonolayer
the cell≥ the
for 400 Ωcm
monolayer
formation,
cell
2 before the stimulation [66,82], the time
monolayer
formation,
for cell formation,
differentiation,
for cell differentiation,
for celland differentiation,
for and for and
required
the exposition for
the expositionthe
ofthe cellofmonolayer
theexposition
intestinal
the intestinal
villi.
of theOn formation,
intestinal
day
villi.21, for
Onvilli.
the
day cell
21,differentiation,
medium
On day
the medium
at
21,thetheapicalatand
medium the
and for
apicalthe exposition
atbasolateral
theandapicalbasolateral
and basolate
of the
environments intestinal
environments
was villi.
environments
changed was On todaywas21,
changed
create the
changed
to medium
different
create at the
todifferent
pH
create
conditions:apical
different
pH and
conditions:
pHpH basolateral
around
conditions:
pH6.5 around
atpH environments
the around
6.5
apical
at the 6.5apical
at the ap
was changed
level (acidic
levelpH to
(acidic create
mimicking
levelpH (acidic different
mimicking
lumen
pH mimickingpH
oflumenconditions:
small intestine)
of
lumen
smallofpH around
intestine)
and
small 6.5
pHintestine)
around at
and pH7.4 the
and apical
around
atpH level
thearound
7.4 (acidic
basolateral
at the pH
7.4basolateral
at the basolat
levelmimicking
(neutral
level pH lumen
(neutral
level
mimickingof small
(neutral
pH mimicking
human
pH intestine)
mimicking
blood)
humanand pHThe
[69].
human
blood)around
[69].
blood) 7.4
cells The
were atcells
[69]. the
kept
The basolateral
were
for
cells
15
kept
were
min level
for at
kept
1537 (neutral
min
for
°C at
15 37min °Cat 37
pH CO
and 5% mimicking
and 2, 5%
afterand
CO human
that,
25%
, after
the blood)
COthat,
TEER
2, afterthe [69].
values
that,
TEER The
the
were cells
values
TEER were
measured
were
values kept
measured
again
were formeasured
15again
beforeminthe at 37 ◦ Cof
before
again
start and
the
before
the 5%
start
ex-
theCO
of 2 , ex-
start
the of the
afterperiment
periment that, theperiment
to verify TEER
tothe values
verify were
stabilization
to theverify measuredthe again
stabilization
the
of stabilization
values.
of the before
The
of the
values.
the start
cellsvalues.
The ofcells
were theTheexperiment
stimulated
were
cells to verify
stimulated
were
with stimulated
with w
the stabilization of® the ®
GreenIuronic
GreenIuronic
® andGreenIuronic
sodium andvalues.
® andThe
hyaluronate
sodium cells
sodiumfor 2were
hyaluronate stimulated
hyaluronate
h tofor6 h2 beforefor62with
h to h
the
hbeforeGreenIuronic
tosuccessive
6 h the
before theand
successive
analysis, sodium
successive
analysis,
in- analysis,
in-
hyaluronate for 2 h to 6 h before the successive analysis, including the permeability assay
cluding thecluding
permeability
cluding
the permeability
the
assaypermeability
measured
assay measured
assay
by Papp measured
by
analysis
Pappby [66].
analysis
Papp Briefly,
analysis
[66].the Briefly,
[66].
PappBriefly,
the
(cm/s)
Papp the(cm/s)
Papp (cm
measured by Papp analysis [66]. Briefly, the Papp (cm/s) was calculated with the following
was calculated
was calculated
with
wasthe calculated
following
with thewith following
formula
the following
[66,69]:
formulaformula[66,69]: [66,69]:
formula [66,69]:
Papp==dQ/dt
Papp Papp⇥
dQ/dt 1/m0⇥
= dQ/dt
Papp
1/m0 =⇥dQ/dt
1/A⇥⇥
1/A
1/m0 ⇥
V 1/m0
VDonor
1/A ⇥⇥V1/A
Donor ⇥ V Donor
Donor
dQ: amount dQ:of
dQ: amount amount
substance
dQ: amount
of substance
transported
of substance oftransported
substance
transported
(nmol transported
or (nmol
(nmol μg); or(nmol
or µg); μg); or μg);
dt: incubation
dt: incubation
time
dt: incubationdt:time
(sec);
incubation
time
(sec);(sec);
time (sec);
m0: m0:
m0: amount amount
of of substrate
amount
substrate
m0: amount applied
of substrate
applied
of to donor
substrate
to
applied
donor applied
to compartment
compartment
donor tocompartment
donor (nmol(nmol or µg);
compartment
or (nmol
μg); or(nmol
μg); or μg);
A: surface area of Transwell membrane 2 ); 2
A: surface A:area
surface
ofA:Transwell
surface
area of Transwell
area
membrane
of (cm2(cm
Transwell
membrane );membrane
(cm ); (cm2);
VDonor:
VDonor: VDonor:
volume volume theofdonor
VDonor:
of
volume the
ofdonor
volume
the ofcompartment
compartment
donor thecompartment
donor (cm (cm3(cm
). 3). (cm3).
compartment
3).

Negative
Negative controls
Negative
controlsNegativewithout
without
controls cellswithout
controls
cells
without
were were
cells
tested tested
wereto
cells to exclude
exclude
tested
were totested Transwell
Transwell
exclude
to excludemembrane
Transwell
membrane
Transwell
membraneinfluence.
influence.
membrane
influence.
influen

4.9. Occludin
4.9. Occludin
4.9. OccludinQuantification
Quantification
4.9. Occludin
Quantification
Assay Assay
Quantification
Assay Assay
The
The Human Human
TheOccludin
Human Occludin
The Human
Occludin ELISA
ELISA Occludin
kitELISA
(OCLNkitkit(OCLN
ELISA
kit,
(OCLN kitkit,
kit,MyBiosource,
MyBiosource,
(OCLN MyBiosource,
kit,
San San
MyBiosource,
Diego, San CA,Diego,
Diego,
USA) CA,
SanCA,Diego,
ana-USA)
USA) CA,ana-
USA) a
analyzed
lyzed thelyzed the
occludin
the occludin
lyzed
occludin
presence presence
the occludin
presence
in CaCo-2 in
presenceCaCo-2
incell
CaCo-2
lysates, cell
in CaCo-2 lysates,
cellaccording
lysates, according
cell lysates,
according to
to the manufacturer’s
according the manufacturer’s
to the manufacturer’s
to theinstruc-
manufacturer’s
instruc- instr
instruction
tion [66]. tion
Briefly, [66].
[66].tion
CaCo-2Briefly,
Briefly,
[66].cellsCaCo-2
Briefly,
CaCo-2
wereCaCo-2cells
cells
lysed werewere
with
cellslysedlysed
cold
were withwith cold Phosphate-Buffered
Phosphate-Buffered
lysed
coldwith
Phosphate-Buffered
cold Phosphate-Buffered
Saline (PBS, Saline
Saline
Merck (PBS,
(PBS,
Saline Merck
(PBS, Me
Merck Life Science, Rome, Italy) 1 × , centrifuged at 1500 × g for 10 min at 4 ◦ C, and 100 µL
Life Science,
LifeRome,
Science,
LifeItaly)
Science,
Rome,1×, Italy)
centrifuged
Rome, 1×,Italy)
centrifuged
at1×,
1500×
centrifuged
gatfor
1500×
10 at
min
g 1500×
forat10
4 g°C,
min
forand
at
104min100
°C,μL at
and 4of°C,
100
each
and
μL
◦ C for 90 min.
of
100each
μL of e
of each
sample was sample
sample waswas
transferred
sample transferred
transferred
to
was
thetransferred
strip to the
to well
the to strip
strip
before
thewell well
strip
the beforebefore
incubation
well the theatincubation
before
incubation
the
37 °Cincubation
for
at 37
90 at°C37atfor
min. 37The
90°Cmin.
su-
for 90The min.
su- The
The supernatants were removed, and the strips were incubated with 100 µL of Detection
pernatants pernatants
were removed,
pernatants
were removed,
and
were theremoved,
strips
and the wereand
strips
incubated
thewere
stripsincubated
with
were100 incubated
with
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4.10. Claudin
4.10.1Claudin
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4.10. Claudin
1 Detection
1 Detection
Int. J. Mol. Sci. 2022, 23, 8114 18 of 26

4.10. Claudin 1 Detection

The Human Claudin1 was measured in CaCo-2 lysates by ELISA kit (Cusabio Tech-
nology LLC, Huston, TX, USA), following the manufacturer’s instructions [66]. Briefly,
the cells were lysed with cold PBS 1× (Merck Life Science, Rome, Italy) and centrifuged
at 1500× g for 10 min at 4 ◦ C. Then, 100 µL of each sample was added to the ELISA plate
and incubated at 37 ◦ C for 2 h; after which, the plate was washed and 100 µL of Biotin-
antibody was added to the wells and incubated for 1 h at 37 ◦ C. After this time, the wells
were washed and 100 µL of HRP-avidin were added in each well, and the samples were
incubated for 1 h at 37 ◦ C. Then, 90 µL of TMB Substrate was also added to the samples and
the plate was incubated for 20 min at 37 ◦ C protected from light. At the end, 50 µL of Stop
Solution was used to stop the reaction and the plate was analyzed by a spectrophotometer
(Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) at 450 nm. The concentration was
expressed as pg/mL, comparing data to the standard curve (range from 0 to 1000 pg/mL),
and the results were expressed as percentage (%) versus control (0 line).

4.11. ZO-1 Detection

The Human Tight Junction Protein 1 ELISA kit (MyBiosource, San Diego, CA, USA)
was measured in CaCo-2, following the manufacturer’s instructions [66]. Briefly, the cells
were rinsed with ice-cold PBS 1× (Merck Life Science, Rome, Italy) and processed with
two freeze-thaw cycles; then, cell lysates were centrifuged for 5 min at 5000× g at 4 ◦ C.
After which, 100 µL of each sample were collected and incubated on the ELISA plate at
37 ◦ C for 90 min; after washing, 100 µL of Detection Solution A was added to each well
and incubated for 45 min at 37 ◦ C. The wells were washed and 100 µL Detection Solution B
was added to the samples. After an incubation of 45 min, the wells were washed again and
90 µL of Substrate Solution was added to each well, and then the samples were incubated
for 20 min at 37 ◦ C in the dark. Finally, 50 µL of Stop Solution was added and then the plates
were read by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland)
at 450 nm. The concentration was expressed as pg/mL, comparing data to standard curve
(range from 0 to 1000 pg/mL), and the results were expressed as percentage (%) versus
control (0 line).

4.12. Crystal Violet Staining

At the end of stimulation time, the cells were fixed with 1% glutaraldehyde (Merck
Life Science, Rome, Italy) for 15 min at room temperature, washed, and stained with
100 µL 0.1% aqueous crystal violet (Merck Life Science, Rome, Italy) for 20 min at room
temperature and solubilized with 100 µL 10% acetic acid before reading the absorbance at
595 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland).
The estimated number was determined by comparing data to the control cells normalized
to T0 (measurement at the beginning of the stimulation) [83]. The results were expressed as
percentage (%) versus control (0 line).

4.13. ROS Production

The quantification of superoxide anion release was obtained following a standard
protocol based on the reduction in cytochrome C [83], and the absorbance in culture
supernatants was measured at 550 nm using the spectrophotometer (Infinite 200 Pro MPlex,
Tecan, Männedorf, Switzerland). The O2 rate was expressed as the mean ± SD (%) of
nanomoles per reduced cytochrome C per microgram of protein compared to the control
(0 line) [83].

4.14. Quantification of Hyaluronic Acid in Cell Culture

At the end of stimulations, both cell types were lysed with 100 µL of cold PBS1×
to measure the total HA following the instructions of the Hyaluronic Acid ELISA Kit
(ClueClone). Briefly, 50 µL of sample and reagent A were added to each well and after
gently shaking the plate was incubated for 1 h at 37 ◦ C. At the end, the wells were washed
Int. J. Mol. Sci. 2022, 23, 8114 19 of 26

three times and 100 µL of reagent B was added before incubating the plate for 30 min at
37 ◦ C, then 90 µL of substrate solution was added before incubating the plate for 20 min at
37 ◦ C. At the end, 50 µL of stop solution was added immediately before reading at 450 nm
by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) [84,85].
The results were expressed as means ± SD (%) versus control (0 line).

4.15. ERK/MAPK Activity

The analysis of ERK/MAPK activity was performed using the InstantOneTM ELISA
(Thermo Fisher, Milan, Italy) on chondrocytes lysates [86]. Briefly, 50 µL of lysate samples
prepared in Lysis Buffer were tested in ELISA microplate strips after the incubation for 1 h
at room temperature in a microplate shaker pre-coated with the antibody cocktail. After
that, the strips were incubated with the detection reagent for 20 min before stopping the
reaction with a stop solution. The absorbance was measured by a spectrophotometer at
450 nm (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) and the results were
expressed as means ± SD (%) versus control (0 line).

4.16. OPG Activity

The OPG/TNFRSF11B Duo Set (R&D Systems, Minneapolis, MN, USA) was applied
according to the manufacturer’s instructions to verify the OPG involvement [87]. Briefly,
100 µL of samples or standards were added to the well and incubated for 2 h at room
temperature protected from light and, after washing, 100 µL of the Detection Antibody
was added to each well and incubated as previously described. After 2 h, 100 µL of the
working dilution of Streptavidin-HRP A was added to each well and incubated for 20 min
at room temperature. At the end of the time, 100 µL of Substrate Solution was added to
each well, incubated for 20 min at room temperature, and then 50 µL of Stop Solution was
used to stop the enzymatic reaction. The absorbance of each well was measured at 450 nm
by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) and the
results were interpolated with the standard curve (6.25 to 625 pg/mL) and the results were
expressed as means ± SD (%) compared to control (0 line).

4.17. NFKB Analysis

The NF-kB (p65) Transcriptional factor Assay kit was carried out to analyze the NF-
κB DNA binding activity, following the manufacturer’s instruction (Cayman Chemical
Company, Ann Arbor, MI, USA) [88]. The concentration was calculated by comparing
results to the standard curve (generated by NF-kB (p65) Transcriptional factor positive
control (ranging from 0 to 10 µL/well according to differently scaled dilutions) and reported
as means ± SD (%) compared to control (0 line).

4.18. BAX Assay

BAX activity was determined in chondrocyte lysates using an ELISA kit (Human Bax
ELISA Kit, MyBiosource, San Diego, CA, USA) according to the manufacturer’s instruc-
tions [89]. The absorbance of the samples was measured at 450 nm by a spectrophotometer
(Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) and the results were compared
to the standard curve (range from 0 to 2000 pg/mL) and expressed as means ± SD (%)
normalized to control value (0 line).

4.19. Caspase 9 Assay

The Caspase 9 activity was investigated in chondrocytes lysates by ELISA kit (Caspase
9 Human ELISA Kit, Thermoscientific, Waltham, MA, USA), according to the manufac-
turer’s instructions, reading the sample’s absorbance at 450 nm with a spectrometer (Infinite
200 Pro MPlex, Tecan, Männedorf, Switzerland). The data were obtained by comparison
to a standard curve (ranging from 1.6 to 100 ng/mL), and the results were expressed as
means ± SD (%) compared to control value (0 line) [90].
Int. J. Mol. Sci. 2022, 23, 8114 20 of 26

4.20. Western-Blot Analysis

At the end of each stimulation, chondrocytes were washed with ice-cold PBS 1×
(Merck Life Science, Rome, Italy), and lysed using Complete Tablet Buffer (Roche, Basel,
Switzerland) supplemented with 2 mM sodium orthovanadate (Na3 VO4 ), 1 mM phenyl-
methanesulfonyl fluoride (PMSF) (Merck Life Science, Rome, Italy), 1:50 mix Phosphatase
Inhibitor Cocktail (Merck Life Science, Rome, Italy), and 1:200 mix Protease Inhibitor Cock-
tail (Merck Life Science, Rome, Italy) to obtain a total protein extract that was centrifuged
at 14,000× g for 20 min at 4 ◦ C. Then, 35 µg of proteins for each extract was resolved on 8%
and 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane,
which was incubated overnight with the specific primary antibodies such as Cyclin D1
(1:500, Santa Cruz, CA, USA) and CD44 (1:500, Santa Cruz, CA, USA). All protein expres-
sions were normalized and verified through β-actin detection (1:5000, Merck Life Science,
Rome, Italy), and expressed as mean ± SD (%) compared to control value (0 line).

4.21. Statistical Analysis

Data obtained from each experimental protocol and assay were collected and analyzed
using GraphPad Prism 7 statistical software through mixed variance analysis. In particular, for
all growth curves, bar graphs, and line graphs, five independent experiments were performed
in triplicates and included in the statistical analysis. All time points in growth curves were
presented as the mean of the three biological replicates with mean errors < 5%. The two-tailed
Student’s t-test was followed by Welch’s t test to analyze two groups. Multiple comparisons
between groups were analyzed by two-way ANOVA followed by a two-tailed Dunnett
post hoc test. Error bars in the bar charts and line charts represent the standard deviation.
For TEER analyses, one-way ANOVA followed by Bonferroni post hoc tests was performed
to see if the means were significantly different between groups. All results were expressed
as mean ± SD of at least 5 independent experiments performed in triplicates. Differences
with a p value < 0.05 were considered statistically significant. Data normality was assessed
with the Kolmogorov–Smirnov test.

5. Conclusions
As demonstrated by these findings, the results of our study show that this new form
of plant HA is likely absorbed and distributed to the chondrocytes, while preserving its
biological activities. Although the in vitro data are very clear and promising, in vivo or
even human studies would be needed to confirm these observations, before assuming an
absolute efficacy of this HA extracted from plants. Thus, despite the fact that our data
derived from an in vitro study and, therefore, need further validation, the results of the
present study about the effectiveness in improving chondrocyte function in conditions
that mimic OA, may support the hypothesis that the oral administration of GreenIuronic®
in humans can be considered a valid therapeutic strategy to obtain beneficial therapeutic
effects during OA. In particular, it can be hypothesized that these promising beneficial
effects are relevant not only to the joint, but also to any OA-induced damage.

Author Contributions: Conceptualization, F.U.; methodology, R.G., S.R. and S.A.; software, S.F. and
A.B.; validation, R.G. and S.A.; formal analysis, R.G., S.A. and A.B.; resources, F.U.; writing—original
draft preparation, R.G., S.R., S.A., S.F., A.B., G.G., C.M. and F.U.; visualization, G.G. and C.M.;
supervision, G.G., C.M. and F.U.; project administration, F.U.; funding acquisition, F.U. and C.M. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Int. J. Mol. Sci. 2022, 23, 8114 21 of 26

Data Availability Statement: Raw data are preferably deposited at the Laboratory of Physiology
(C. Molinari), ensuring appropriate measures so that raw data are retained in full forever under
a secure system. The data presented in this study are available on reasonable request from the
corresponding author.
Acknowledgments: The authors thank Vivatis Pharma GBHE, which donated the substances. This
study has been (partially) funded by the Italian Ministry of Education, University and Research
(MIUR) program “Departments of Excellence 2018–2022”, AGING Project–Department of Transla-
tional Medicine, Università del Piemonte Orientale.
Conflicts of Interest: The authors declare no conflict of interest. C.M. and F.U. F.U. and C.M. are
co-founders of noivita Srls.

Abbreviations

ADAMTS disintegrin and metalloproteinase with thrombospondin motifs

ANOVA one-way analysis of variance
CaCo-2 the human immortalized colorectal adenocarcinoma cell line
CD44 differentiation cluster 44
COX-2 cyclooxygenase 2
DMEM/F12 Dulbecco’s modified Eagle’s medium/Nutrient F-12 Ham
EFSA European Food Safety Authority
ELISA Enzyme-Linked Immunosorbent Assay
EMA European Medicines Agency
ERK extracellular signal-regulated kinases
ERK/MAPK mitogen-activated protein kinases/extracellular signal-regulated kinase
FBS fetal bovine serum
FBS fetal bovine serum
FDA US Food and Drug Administration
GAGs glycosaminoglycan heteropolysaccharides family
HA hyaluronic acid
HMWHA high-molecular weight HA
HPLC High-Performance Liquid Chromatography
HRMS high-resolution mass spectrometry
IBD inflammatory bowel disease
IL-1β interleukin (IL)-1β
LMWHA low molecular weight HA
LPS lipopolysaccharide
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MMPs matrix metalloproteinases
MPK-1 mitogen-activated protein kinase phosphatase-1
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide
Na3VO4 sodium orthovanadate
NFkB nuclear factor kappa B
NSAIDs non-steroidal anti-inflammatory drugs
NO nitric oxide (NO)
OA Osteoarthritis
OPG osteoprotegerin
Papp apparent permeability coefficient
PBS phosphate-buffered saline
PGE2 prostaglandin E2
PMSF phenylmethanesulfonyl fluoride
PVDF polyvinylidene difluoride
RHAMM hyaluronan-mediated motility receptors
ROS reactive oxygen species
TEER transepithelial electrical resistance
T/C-28a2 human chondrocyte cells
TJ tight junction
ZO-1 zonula occludens-1
Int. J. Mol. Sci. 2022, 23, 8114 22 of 26

Appendix A
Appendix A.1. HPLC-UV Method
• Column: Phenomenex Synergi Polar 4 µm 150 × 4.6 mm preceded by a Security guard
Polar and kept at room temperature
• Mobile phase A: 340 mg of tetrabutylammonium bisulfate dissolved in 1000 mL of
water HPLC grade.
• Mobile phase B: 340 mg of tetrabutylammonium bisulfate dissolved in 330 mL of
water HPLC grade, then after the solution is at room temperature, brought to 1000 mL
with acetonitrile.
• Wavelength: 240 nm
• Volume of injection: 30 µL
• Flow rate: 1.1 mL/min
• Gradient elution program:

Time (min) Mobile Phase B%

0.00 20
7.00 65
12.00 65
12.50 20
22.50 20

Appendix A.2. HPLC-HRMS Method

• Thermo Scientific Q-Exactive plus
• Column: Phenomenex Synergi Polar 4 µm 150 × 2.0 mm preceded by a Security guard
Polar and kept at room temperature
• Mobile phase A: 0.1% formic acid in water
• Mobile phase B: 0.1% formic acid in acetonitrile
• Volume of injection: 5 µL
• Flow rate: 0.200 mL/min
• Gradient elution program:

Time (min) Mobile Phase B%

0.00 15
4.00 50
9.50 50
10.00 15
15 15

• Positive full scan.

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