biomolecules

Article
Can Hyaluronic Acid Combined with Chondroitin Sulfate in
Viscosupplementation of Knee Osteoarthritis Improve Pain
Symptoms and Mobility?
Augustin Dima 1,† , Magda Dragosloveanu 2 , Andreea Ramona Romila 3 , Alexandru Cristea 1 , Georgiana Marinică 4 ,
Alexandru-Tiberiu Dănilă 4,5,† , Alexandru Mandici 4,5 , Daniel Cojocariu 4,5 , Robert-Alexandru Vlad 6, * ,
Adriana Ciurba 6 and Magdalena Bîrsan 6,7

1 Department of Medical Rehabilitation-Orthopedics and Traumatology, National Institute of Rehabilitation,

Physical Medicine and Balneoclimatology, 2 Sf. Dumitru Street, Sector 3, 030167 Bucharest, Romania;
draugustindima@yahoo.com (A.D.); dr.alexandrucristea@gmail.com (A.C.)
2 Department of Rehabilitation, University of Medicine and Pharmacy “Carol Davila”, 8 Eroii Sanitari Street,
Sector 5, 050474 Bucharest, Romania; magda.dragosloveanu@umfcd.ro
3 Department of Rehabilitation, Physical Medicine and Balneology II, National Institute of Rehabilitation,
Physical Medicine and Balneoclimatology, 2 Sf. Dumitru Street, Sector 3, 030167 Bucharest, Romania;
andreea.romila@gmail.com
4 Medical and Pharmacovigilance Department, Rompharm Company SRL, 1A Eroilor Street,
075100 Otopeni, Romania; alexandru-mandici@email.umfiasi.ro (A.M.);
daniel-cojocariu@email.umfiasi.ro (D.C.)
5 Department Pharmaceutical Sciences II, Faculty of Pharmacy, Grigore T Popa University of Medicine and
Pharmacy Iasi, 16 Universitatii Street, 700115 Iasi, Romania
6 Department of Pharmaceutical Technology and Cosmetology, Faculty of Pharmacy, George Emil Palade
University of Medicine, Pharmacy, Science, and Technology, 38 Gheorghe Marinescu Street,
540142 Targu Mures, Romania; magdalena.birsan@umfiasi.ro (M.B.)
7 Department of Pharmaceutical Industry and Pharmaceutical Biotechnologies, Grigore T Popa University of
Medicine and Pharmacy Iasi, 16 Universitatii Street, 700115 Iasi, Romania
Citation: Dima, A.; Dragosloveanu, * Correspondence: robert.vlad@umfst.ro
† These authors contributed equally to this work.
M.; Romila, A.R.; Cristea, A.;
Marinică, G.; Dănilă, A.-T.; Mandici,
A.; Cojocariu, D.; Vlad, R.-A.; Ciurba, Abstract: The objective of the present study was to assess the effect of intra-articular Hyaluronic acid
A.; et al. Can Hyaluronic Acid (HA) and Chondroitin sulfate (CS) supplementation (Hialurom® Hondro (HH)) on pain symptoms
Combined with Chondroitin Sulfate and joint mobility. In total, 60 mg/mL sodium hyaluronate and 90 mg/mL CS were administered to
in Viscosupplementation of Knee 21 patients (17 females and 4 males) respecting the in-force requirements, excluding patients with
Osteoarthritis Improve Pain some specific comorbidities. In addition to the clinical study (where the pain intensity (severity) and
Symptoms and Mobility? Biomolecules joint mobility were assessed), rheological characterization was conducted evaluating the following
2024, 14, 832. https://doi.org/
parameters: elastic modulus (G′ ), loss modulus (G′′ ) oscillatory frequency (fc) at 0.5 Hz and 2.5 Hz,
10.3390/biom14070832
crossover frequency (fc), relaxation time (λ) where it was noticed that the addition of chondroitin
Academic Editor: Davide Vigetti sulfate (CS) to sodium hyaluronate (SH) significantly enhances and improves the viscoelastic prop-
erties, particularly at higher shear frequencies. A significant decrease in pain intensity felt by the
Received: 3 June 2024
Revised: 3 July 2024
subjects was found, from 7.48 (according to Wong–Baker scale)—pain close to 8 (the patient is unable
Accepted: 9 July 2024 to perform most activities), to more reduced values of 5.86—at 6 weeks after injection, 4.81—at
Published: 11 July 2024 3 months after injection, and 5.24—at 6 months after injection, improvements in symptoms was fast
and durable. Data related to the evolution of joint mobility show that at 6 weeks after injection, the
mobility of joints increased by 17.8% and at 6 months by 35.61%. No serious adverse events were
reported with undesired effects so that they would impose additional measures. Better resistance
Copyright: © 2024 by the authors. to enzymatic degradation and free radicals could be expected from the synergic combination of
Licensee MDPI, Basel, Switzerland.
sodium hyaluronate and chondroitin sodium sulfate, this having a special importance for the patients,
This article is an open access article
granting them the ability to perform more ample movements and reducing dependency on attendants,
distributed under the terms and
thus increasing quality of life.
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Biomolecules 2024, 14, 832. https://doi.org/10.3390/biom14070832 https://www.mdpi.com/journal/biomolecules

Biomolecules 2024, 14, 832 2 of 14

Keywords: hyaluronic acid intra-articular; chondroitin sulfate; viscosupplementation; knee

osteoarthritis; pre-filled syringe; infiltrations

1. Introduction
Osteoarthritis (OA) represents the major cause of disability in older adults, leading to
loss of function, pain, and a decreased quality of life. Estimates suggest that approximately
528 million people worldwide are affected by OA which affects sleep quality, mood, and
participation in everyday life, limiting a person’s ability to self-manage other conditions,
such as diabetes and hypertension [1]. The disease most commonly affects the joints in
the knees, hands, feet, and spine and is relatively common in shoulder and hip joints.
While knee OA is related to aging, it is also associated with a variety of risk factors, such
as obesity, lack of exercise, occupational injury, and trauma [2,3]. Over the course of
OA, articular cartilage gets severely degraded leading to constant or intermittent pain
of different intensities, frequently associated with stiffness, swelling, and joint mobility
loss. Although most changes occur in the cartilage, the entire joint is affected, including
ligaments, synovium, and subchondral bone. Initially, chronic overloading and impaired
biomechanics were thought to be the main causes of OA, which subsequently led to joint
articular destruction and inflammation [4,5]. However, OA seems to be triggered by a
complex process involving metabolic and inflammatory factors, such as active synovitis
and systemic inflammation [6]. Osteoarthritis (OA) is a prevalent condition, affecting a
significant portion of the population. While the incidence increases with age, with the
highest rates between 55 and 64 years old for knee OA, it is important to note that more than
half of individuals with symptomatic knee OA are younger than 65 years old. Furthermore,
women are disproportionately affected by OA, with 78% of adults with OA occurring in
females. An even more disproportionate share of 18% of the population age 65 and older
have OA (43%). This compares to 46% with osteoarthritis in 34% of the population aged
45 to 64 years [7]. This prevalence is likely to rise in the coming years, as the global
population ages rapidly. According to World Health Organization (WHO) in 2020, there
were already 1 billion people aged 60 or over worldwide, and this figure is projected
to reach 1.4 billion by 2030, representing one in six people globally [8]. Therefore, OA
represents a growing public health concern, demanding continued research into prevention,
management, and treatment strategies.
OA management should be focused initially on non-pharmacological interventions
such as weight loss and exercise, followed by pharmacologic interventions such as non-
steroidal anti-inflammatory drug (NSAIDs) (systemic and/or topical) intra-articular (IA)
corticosteroids and/or hyaluronic acid (HA), whereas surgery is deemed as last resort
solution [9–13]. Pharmacological interventions mainly focus on long-term pain alleviation
by using either selective or non-selective NSAIDs. Although efficient in alleviating OA-
related pain, their long-term use results in various adverse events, including gastric ulcers,
bleeding, and renal failure [14,15]. Avoiding systemic adverse events, NSAIDs imply that
alternative therapies such as viscosupplementation should be considered. Although inva-
sive, intraarticular (IA) HA administration is regarded as generally safe and has targeted
results, intending to reinforce synovial fluid viscoelasticity [16,17]. Pain and disability
management on a chronic or relapsing course may ask for polymodal therapies, such as
physical agents, platelet-rich plasma injections, and botulinum toxin administration [18].
Particularly regarding knee OA management by IA-HA viscosupplementation, there
is no consensus between national and international guidelines in recommending IA-HA
administration in OA-affected joints. The American College of Rheumatology recommends
it for patients with no response to conventional treatments and patients with contraindica-
tions to surgery [19].
Biomolecules 2024, 14, 832 3 of 14

HA represents a major component of synovial fluid and cartilage. Due to its viscoelas-
tic and rheological properties, it decreases articular friction and protects soft tissue against
trauma, being responsible for cushioning and lubricating synovial joints. However, its vis-
cosity decreases as shear forces increase, an essential behavior for lubricating joints during
rapid joint movement. Conversely, high viscosity at low shear forces is required for joint sta-
bilization [13,20]. Chondroitin sulfate (CS) has a gel-like structure and plays a major role in
maintaining the structural integrity of tissues by linking to monomers with high molecular
weights, being mainly located around the cartilage of the joints. Furthermore, CS inhibits
extracellular proteases involved in connective tissue metabolism, and cartilage cytokine
production and induces articular chondrocytes apoptosis [21]. More than 40 years have
passed since the first FDA-approved IA injection of HA as sodium hyaluronate (SH) for
the treatment of pain in patients with knee OA in 1997. Its approval was based on positive
outcomes during clinical trial investigation in patients with knee OA and safety regarding
administration [22]. Later it has been noted that SH and CS association in aqueous solution
seems to increase solution’s viscosity. One potential explanation resides in the increased
viscosity when associating CS and SH via hydrogen bonds between N-acetylamino groups,
increasing their molecular size, while also having a crosslinking tendency of long fractions,
further leading to an increase in viscosity [20,21,23]. Based on these observations, CS
could be used to improve HA rheological properties to significantly improve synovial fluid
properties and enhance lubrication. Binding to core proteins through N and O linkages
leads to aggregates of monomers with high molecular weights. The proteoglycan aggregate
has viscoelastic and hydration properties and an ability to interact with the adjacent tissue
through electric charges leading to cartilage tissue protection. Non-animal SH and its
natural crosslinking with CS leads to increased bioavailability, with mechanical and physic-
ochemical properties similar to human synovial fluid. These biopolymers act as a scaffold,
binding other matrix molecules including aggrecan, being involved in several important
biological functions such as cell adhesion and cell motility regulation, cell differentiation
and proliferation, and providing biomechanical properties [21]. Researchers identify HA
as a major supplementation for a wide range of degenerative joint diseases, particularly
hemophilic arthropathy which shares many features with osteoarthritis [23,24].
Considering all the aspects, the objective of the present study was to assess after a
single injection the effect of intra-articular HA-CS supplementation on pain symptoms and
joint mobility, as well as its safety. In the literature, there are very few or incomplete data,
which supports and highlights the importance of this study.

2. Materials and Methods

2.1. Materials
In the present study, a commercially available viscoelastic solution (Hialurom Hondro® ,
Rompharm Company S.R.L., Otopeni, Romania) was investigated. HH is a sterile, isotonic,
viscoelastic solution containing two highly purified and natural crosslinked biopolymers in
phosphate buffer, sodium hyaluronate with an average molecular weight of 3000 kDa and
chondroitin sodium sulfate with an average molecular weight of 25 kDa [25]. Hialurom
Hondro® as biomatrix consists of sodium hyaluronate (SH) 60 mg/3 mL derived from
bacterial fermentation and chondroitin sodium sulfate (CS) 90 mg/3 mL produced from
bovine tracheal cartilage. The viscoelastic solution was administered intra-articular by
an orthopedic specialist to patients with stage II or stage III knee osteoarthritis (KOA)
(Figure 1).
Biomolecules 2024, 14, x FOR PEER REVIEW 4 of 14

Biomolecules 2024, 14, 832 4 of 14

Sodium
hyaluronate
structure

Chondroitin sodium
sulfate structure

The viscoelastic
solution

Figure 1. The knee injection of HH by a specialist physician to patients with stage II or stage III KOA:
Figure 1. The knee injection of HH by a specialist physician to patients with stage II or stage III
the best approach is the path of least obstruction and maximal access to the synovial cavity, which
KOA: the best approach is the path of least obstruction and maximal access to the synovial cavity,
could be superolateral, superomedial, or anteromedial/anterolateral.
which could be superolateral, superomedial, or anteromedial/anterolateral.
2.2. Patient Enrollment and Study Protocol
2.2. Patient
ThisEnrollment and Study
was a monocentric, Protocol confirmatory, randomized, and non-controlled
open-label,
study,
Thisinvolving patients with diagnosed
was a monocentric, open-label, osteoarthritis
confirmatory, (OA).randomized,
Patients’ enrollment was at the
and non-controlled
National Institute of Rehabilitation, Physical Medicine, and Balneoclimatology
study, involving patients with diagnosed osteoarthritis (OA). Patients enrollment was at (Bucharest,
Romania). The study period was between March 10 and November 7 and for inclusion
the National Institute of Rehabilitation, Physical Medicine, and Balneoclimatology (Bu-
criteria, eligible patients were those between 18 and 75 years old and diagnosed with
charest, Romania). The study period was between March 10 and November 7 and for in-
stage II and III OA, according to the Kellgren–Lawrence scale [25]. Exclusion criteria were
clusion criteria,
patients diagnosed eligible
withpatients were those
septic arthritis, those between
who suffered 18 and
from75traumatic
years old and of
events diagnosed
the
with stage
joint II andtoIII
intended OA,those
treat, according to thedisease,
with Paget’s Kellgren–Lawrence scale [25].
gout, major dysplasia, Exclusion
Wilson’s criteria
disease,
were patients diagnosed with septic arthritis, those who suffered
acromegaly, ochronosis, hemochromatosis, Ehlers–Danlos syndrome, Charcot arthropathy, from traumatic events
hypo-/hyper-parathyroidism,
of the joint intended to treat, those active with
synovitis,
Pagetrheumatoid
s disease,arthritis, dermatological
gout, major dysplasia, condi-
Wilson s
tions at the injection site, and patients who underwent arthroscopy
disease, acromegaly, ochronosis, hemochromatosis, Ehlers–Danlos syndrome, Charcot ar- at least 1 year before
or received
thropathy, IA steroid injection or HA at least
hypo-/hyper-parathyroidism, 6 months
active before
synovitis, investigation.
rheumatoid There were
arthritis, dermato-
no patients from vulnerable groups (paediatrics, pregnant or lactating women, patients
logical conditions at the injection site, and patients who underwent arthroscopy at least 1
with hepatic and/or renal impairment, or populations with specific racial and/or ethnic
year before or received IA steroid injection or HA at least 6 months before investigation.
origins).
There were no patients
The primary from vulnerable
endpoints of the study groups (paediatrics,
were pain scores andpregnant or lactating
joint mobility measured women,
patients with hepatic and/or renal impairment, or populations with
using the following parameters: pain intensity felt by participants was quantified using thespecific racial and/or
ethnic origins).scale for rating pain intensity [26]. Lequesne index for lower limb arthrosis
Wong–Baker
wasTheused for pain
primary severity evaluation
endpoints of the study[27].were
Jointpain
mobility was
scores andassessed by measuring
joint mobility measured
movement amplitude in all directions and is rather an expression of
using the following parameters: pain intensity felt by participants was quantified usingthe mobilization mode
theofWong–Baker
a segment than scalea degree of movement
for rating measurement.
pain intensity [26]. Lequesne For a index
better for
assessment
lower limb of its
arthro-
evolution, it was considered that the sum of joint mobility values before injection was
sis was used for pain severity evaluation [27]. Joint mobility was assessed by measuring
100% and the percentage value was calculated for the sums of corresponding values at
movement amplitude in all directions and is rather an expression of the mobilization
weeks 6, 12, and 24, respectively. Joint mobility was assessed with a transparent goniometer
mode of a As
(20 cm). segment than endpoint,
a secondary a degree quantifying
of movement measurement.
adverse For a better
incident occurrence. assessment
Initial clinical of
its evaluation
evolution,took it was considered that the sum of joint mobility values before
place before performing the injection. Follow-up timepoints for evaluations injection was
100%wereand the percentage
at weeks 6, 12, and 24,value was the
assessing calculated for the parameters
aforementioned sums of corresponding
at each visit. values at
weeks 6, 12, to
Prior and 24, respectively.
HA-CS injection, theJoint mobility
injection was properly
site was assessed disinfected
with a transparent
accordinggoniom-
to
clinical settings procedures. After disinfection, any fluid accumulation
eter (20 cm). As a secondary endpoint, quantifying adverse incident occurrence. Initial in the joints was
removed
clinical by arthrocentesis.
evaluation took place Thebefore
volume of HA and CS
performing thesolution for Follow-up
injection. injection wastimepoints
adjusted for
according to the joint size and IA space of each participant to avoid overfilling. A single
evaluations were at weeks 6, 12, and 24, assessing the aforementioned parameters at each
IA injection (20 mg/mL SH and 30 mg/mL CSNa) was administered to each patient after
visit.
initial evaluation.
Prior to HA-CS injection, the injection site was properly disinfected according to clin-
ical settings procedures. After disinfection, any fluid accumulation in the joints was re-
moved by arthrocentesis. The volume of HA and CS solution for injection was adjusted
according to the joint size and IA space of each participant to avoid overfilling. A single
Biomolecules 2024, 14, 832 5 of 14

This study was designed in accordance with the guidelines of the Declaration of
Helsinki and standard EN ISO 14155, Clinical investigation in human subjects—Good
Clinical Practice [28]. The protocol was approved by the Ethics Committee of the Na-
tional Institute of Rehabilitation, Physical Medicine and Balneology, Bucharest, Romania
(no. 2288). Before enrolling, participants involved in the study were properly informed
by the medical staff involved in the investigation and gave their informed consent. The
medical device was administered intra-articulary by an orthopedic specialist.

2.3. Rheological Parameters of Viscosupplement

Rheological parameters for the viscosupplement were obtained with a Malvern
Kinexus Pro+ Rheometer with cone-plate geometry (40 mm plate diameter and 1◦ cone
angle) at 25 ± 0.01 ◦ C or physiological temperature 37 ± 0.01 ◦ C. The tested samples
were equilibrated for ten minutes at working temperature before use, each test run was
duplicated with a fresh sample. Frequency sweep measurements (G′ and G′′ as a function
of frequency) were performed in the mode oscillation-controlled deformation (i.e., 0.2%
deformation kept constant). The frequency sweep range was 0.01–10.0 Hz.
The parameters collected, e.g., elastic modulus (G′ ) and loss modulus (G′′ ) as a function
of oscillatory frequency (fc), were plotted for identifying viscoelastic profiles and reported
as elastic modulus (G′ ) and loss modulus (G′′ ) corresponding to the transition points 0.5 Hz
(representative for walking) and 2.5Hz (representative for running), crossover frequency
(fc) defined as the frequency at which G′ = G′′ , relaxation time (λ (s) meaning the time
it takes to recover to its original state following deformation estimated as the inverse of
the oscillating frequency at which the elastic modulus (G′ ) equals loss modulus (G′′ ). The
frequency sweeps were performed at strain amplitudes that were determined to be in the
linear viscoelastic range [29].
Additional rheological property related to HA-based formulation was considered
the percentage of elasticity, i.e., the proportion of elasticity in HA-based formulation, as a
function of frequency, calculated by applying 100 × G′ /(G′ + G′′ ). Reported values will be
related to the reference frequencies 0.5 and 2.5 Hz, by comparison, HA-based formulations
with or without chondroitin sulfate. Viscosity measurements depending on shear rate were
performed in the dynamic mode viscosity (rotational)—controlled shear by varying shear
speed within 0.01–10.00 s−1 . A 20 mg/mL SH-only viscosupplement served as the control
for the comparison of rheological parameters at 25 ± 0.01 ◦ C.

2.4. Statistical Analysis

As there was no control group, statistical analysis was mainly descriptive. Measured
parameters were expressed as mean ± SD. The admitted error limit for null hypotheses
tests was r = 1%. Significance level thresholds for the alternative hypothesis regarding pain
intensity, Lequesne index, and joint mobility were α = intense, 0.25, and 0.5, respectively.
The average rate of joint mobility variation and joint mobility dispersion were calculated.
For the accuracy measurement, Pearson’s coefficient of skewness was calculated. For
statistical power, a unilateral t-test for independent samples was calculated. A p < 0.05 was
considered significant.

3. Results
A total of 21 patients met the study protocol criteria. As depicted in Table 1, 4 males
(19.1%) and 17 females (80.9%) were enrolled in the study (age 61.8 ± 8.2 years). The mean
age of OA was 7.9 ± 3.6 years. All enrolled patients completed the study.
Biomolecules 2024,
Biomolecules 14,14,
2024, x FOR
832 PEER REVIEW 6 of6 14
of 14

Table
Table 1. Patient’s
1. Patient demographics.
s demographics.

4 male
4 male (19.1%)
(19.1%)
Gender
Gender 17 female
17 female (80.9%)
(80.9%)
AgeAge 61.861.8 ± 8.2
± 8.2 years
years
Mean ageage
Mean of OA
of OA 7.9 7.9
± 3.67 years
± 3.67 years

3.1.3.1.
Pain Intensity
Pain andand
Intensity Severity
Severity
Prior to to
Prior injection, pain
injection, was
pain wasthethe
predominant
predominant symptom
symptom reported
reported (100%
(100%of of
patients),
patients),
followed
followed byby
crackles (66.66%
crackles (66.66%of of
patients), decreased
patients), decreased mobility (52.38%),
mobility (52.38%),and impairment
and impairment of of
walking (47.64%).
walking (47.64%).
Baseline
Baselinevalues for pain
values intensity
for pain and severity
intensity were pre-injection
and severity measurements
were pre-injection (7.48
measurements
± 0.96
(7.48and 17.48
± 0.96 and± 2.34 for±the
17.48 2.34Wong–Baker scale andscale
for the Wong–Baker Lequesne index, respectively).
and Lequesne Con-
index, respectively).
cerning the primary
Concerning endpoint
the primary of the study,
endpoint a significant
of the study, improvement
a significant improvementin thein
pain
theintensity
pain inten-
score
sityhas been
score hasnoted
been at follow-up
noted visits (pvisits
at follow-up < 0.001)
(p <compared to baseline.
0.001) compared The average
to baseline. pain
The average
intensity decreases
pain intensity at weeks
decreases at12 and 24
weeks 12were
and 244.81 ± 0.73
were 4.81 ± 5.24
and 0.73 ±and
0.53, ± 0.53, respectively
respectively
5.24 (Figure
2a). Although
(Figure 2a). the latter shows
Although painshows
the latter with strong discomfort,
pain with when it is compared
strong discomfort, to base-
when it is compared
to (7.48
line baseline (7.48
± 0.96) ± 0.96)
shows shows an improvement
an improvement in patients inquality
patients’ quality
of life. of life.
Analysis Analysis
showed a
showed areduction
significant significantinreduction in pain
pain severity severity atvisits
at follow-up follow-up visitscompared
(p < 0.001) (p < 0.001)tocompared
baseline to
baseline
(Figure 2b).(Figure 2b).

(a) (b)
Figure 2. Significant
Figure reduction
2. Significant in in
reduction pain intensity
pain (a)(a)
intensity (* p(*<p0.001) and
< 0.001) pain
and severity
pain (b)(b)
severity (* p(*<p0.001) at at
< 0.001)
weeks 6, 12 and 24 vs. baseline.
weeks 6, 12 and 24 vs. baseline.

Although
Although significant improvement
significant improvement in in
severity scores
severity have
scores been
have noted,
been noted,thethe
Lequesne
Lequesne
index at baseline (17.48 ± 2.34) suggests an extremely severe OA, condition preserved
index at baseline (17.48 ± 2.34) suggests an extremely severe OA, condition preserved at at
week
week6 (12.76 ±±
6 (12.76 2.35) and
2.35) andweeks 1212
weeks and
and 2424(10.71
(10.71± ±
1.61 and
1.61 and11.33 ±±
11.33 1.39, respectively)
1.39, respectively)
(Figure 2).
(Figure 2).

3.2.3.2.
Joint Mobility
Joint Mobility
AnAn increase in in
increase joint mobility
joint mobilityhas been
has noted
been notedat at
weeks
weeks6, 6,
12,12,
andand2424
byby17.8 ± 3.51%,
± 3.51%,
17.8
31.5
31.5 ± 5.86%,
± 5.86%, andand
35.6135.61 ± 6.85%,
± 6.85%, respectively,
respectively, compared
compared to baseline
to baseline (Figure(Figure 3b),spe-
3b), with with
special
cial importance
importance regarding
regarding patientpatient movement,
movement, as theyaswere
theyallowed
were allowed to perform
to perform higher higher
am-
amplitude
plitude movements
movements (Figure(Figure
3). 3).
The average rate of variation of joint mobility was calculated for each participant
at weeks 6, 12, and 24 and compared to baseline. Weeks 6 and 12, respectively, showed
positive and towards zero values, meaning an increasing trend in their mobility after HA
and CS injection (Figure 4).
molecules 2024, 14, x FOR2024,
Biomolecules PEER 14,REVIEW
832 7 of 14
7 of 14

(a) (b)
Figure 3. Joint mobility as measured in degrees (a) (mean ± SD) and percent (%) increase in joint
mobility (b) (mean ± SD) at weeks 6, 12, and 24 compared to baseline.

The average rate of variation of joint mobility was calculated for each participant at
(a) 6, 12, and 24 and compared to baseline. Weeks 6(b)
weeks and 12, respectively, showed pos-
Figure 3. Joint mobility as measured in degrees (a) (mean ± SD) and percent in
itive and towards zero values, meaning an increasing trend their
(%) mobility
increase after HA and
in joint
Figure 3. Joint mobility as measured in degrees (a) (mean ± SD) and percent (%) increase in joint
mobility (b) CS injection
(mean ± SD) at(Figure 4).12, and 24 compared to baseline.
weeks 6,
mobility (b) (mean ± SD) at weeks 6, 12, and 24 compared to baseline.

The average rate of variation of joint mobility was calculated for each participant at
weeks 6, 12, and 24 and compared to baseline. Weeks 6 and 12, respectively, showed pos-
itive and towards zero values, meaning an increasing trend in their mobility after HA and
CS injection (Figure 4).

Figure
Figure4.4.The
Theaverage
averagerate
rateof
ofjoint
jointmobility
mobilityvariation (average±±SD).
variation(average SD).

Toassess
To assessdata
datacollection
collectionprecision,
precision,joint
jointmobility
mobilitydispersion
dispersionand
andPearson
Pearson’s coefficient
s coefficient
of skewness were calculated. The calculated Pearson’s coefficient of skewness
of skewness were calculated. The calculated Pearson s coefficient of skewness showed showedaa
left, positive skew in all situations, suggesting appropriate data recording (Table
left, positive skew in all situations, suggesting appropriate data recording (Table 2).2).
Figure 4. The average rate of joint mobility variation (average ± SD).
Table2.
Table Pearson’s
2.Pearson coefficientof
s coefficient ofskewness (average±±SD).
skewness(average SD).
To assess data collection precision, joint mobility dispersion and Pearson s coefficient
Timeframe (Weeks)
Timeframe (Weeks) Pearson’s Coefficient
Pearson’s of Skewness
Coefficient of Skewness
of skewness were calculated. The calculated Pearson s coefficient of skewness showed a
Baseline
Baseline 169.82 ± 35.41
169.82 ± 35.41
left, positive skew in all situations, suggesting appropriate data recording (Table 2).
6 6 200.56200.56 ± 39.98
± 39.98
12 223.55 ± 42.71
Table 2. Pearson s coefficient of12skewness
24 (average ± SD).
223.55 ± 42.71
231.21 ± 44.89
24 231.21 ± 44.89
Timeframe (Weeks) Pearson’s Coefficient of Skewness
Baseline
AAunilateral t-testfor
unilateralt-test forindependent
independentsamples 169.82
samples ± 35.41
was
was usedtotocalculate
used calculatethe
thestatistical
statisticalsig-
sig-
nificance of knee joint mobility measurements.
6 of knee joint mobility measurements.
nificance 200.56Results
± 39.98
Results showed a significant difference
showed a significant difference be-
between
tween 12 knee
knee joint
joint mobility
mobility at baseline
at baseline andand at follow-up
223.55
at follow-up (p <(p0.001).
± 42.71 < 0.001).
24 231.21 ± 44.89
3.3. Rheological Measurements of Viscosupplement
3.3. Rheological Measurements of Viscosupplement
The rheological parameters of HHwas ± 0.01
at 25used ◦ C and 37 ± 0.01 ◦ C at 0.5 Hz frequency
A unilateralThe
t-test for independent
rheological samples
parameters of HH at 25 to °C
± 0.01 calculate
and 37 the statistical
± 0.01 °C at 0.5sig-
Hz frequency
which
nificance ofwhich are mobility
knee joint representative for walking
measurements. and 2.5
Results Hz which
showed a is representative
significant difference for
be-running are
are representative for walking and 2.5 Hz which is representative for running are
shown
tween kneeshown in Table
joint mobility 3. The rheological performance of SH (20 mg/mL) and CS (30 mg/mL) for-
in Tableat3.baseline and at follow-up
The rheological (p < of
performance 0.001).
SH (20 mg/mL) and CS (30 mg/mL) for-
mulation vs. another SH formulation without CS (defined as Control, 20 mg/mL SH only)
mulation vs. another SH formulation without CS (defined as Control, 20 mg/mL SH only)
as viscosupplements
3.3. Rheological presented distinctive viscoelastic properties with non-Newtonian
Measurements of Viscosupplement
behavior, which can enhance and improve viscoelastic properties when shear frequencies
The rheological parameters of HH at 25 ± 0.01 °C and 37 ± 0.01 °C at 0.5 Hz frequency
are increased (e.g., from walking to running).
which are representative for walking and 2.5 Hz which is representative for running are
shown in Table 3. The rheological performance of SH (20 mg/mL) and CS (30 mg/mL) for-
mulation vs. another SH formulation without CS (defined as Control, 20 mg/mL SH only)
 behavior, which can enhance and improve viscoelastic properties when shear frequencies
are increased (e.g., from walking to running).

Table 3. Rheological parameters for HH commercial batch.

Biomolecules 2024, 14, 832 8 of 14
Temperature 25 ± 0.01 °C 37 ± 0.01 °C
Reference frequency 0.5 a Hz 2.5 b Hz 0.5 a Hz 2.5 b Hz
3. Rheological
TableElastic parameters
modulus (G′) (Pa) for HH commercial
271.6 batch. 478.3 174.4 198.8
Viscous modulus (G″) (Pa)
Temperature 25 208.7
± 0.01 ◦ C 394.8 37 149.8
± 0.01 ◦ C 181.7
Cross-over frequency (Hz) 0.5 Hz
Reference frequency a 0.096Hz
b c a
0.5 Hz 0.171
b c
2.5 2.5 Hz
Elastic ′ ) (Pa)
modulus (Gtime 271.6 478.3 174.4 198.8
Relaxation (s) 10.4 5.8
Viscous modulus (G′′ ) (Pa) 208.7 394.8 149.8 181.7
Elasticity
Cross-over frequency(%)(Hz) 60.9 c
0.096 70.6 58.2 c
0.171 68.5
—representative
a Relaxation for
timewalking,
(s) and b—2.5 Hz 10.4
representative for running; c—the
5.8 frequency at which
G′ = G″. Elasticity (%) 60.9 70.6 58.2 68.5
a —representative for walking, and b —2.5 Hz representative for running; c —the frequency at which G′ = G′′ .
Oscillatory frequency provides additional information about the structure–property
Oscillatory
relationship frequency provides
of biopolymer solutionadditional information
(i.e., sodium about the
hyaluronate andstructure–property
chondroitin sulfate) as
it is used to determine the viscous and elastic properties of the sample. Bysulfate)
relationship of biopolymer solution (i.e., sodium hyaluronate and chondroitin adding chon-
as it is sulfate
droitin used toto determine the viscous and
sodium hyaluronate the elastic propertiesresponse
high-frequency of the sample. By adding
is defined by the elastic
chondroitin sulfate to sodium hyaluronate the high-frequency response is defined by the
modulus, and the crossover frequency shifts toward lower frequencies or larger relaxation
elastic modulus, and the crossover frequency shifts toward lower frequencies or larger
times. Furthermore,
relaxation at the entanglement
times. Furthermore, region,region,
at the entanglement the elastic modulus
the elastic reaches
modulus a plateau
reaches a at
which
plateaupoint it ispoint
at which independent of frequency
it is independent and behaves
of frequency more
and behaves likelike
more an an
elastic solid
elastic solid(Figures
5(Figures
and 6). 5 and 6).

25 ±at ◦ ◦
5. Visco-elasticity
Figure 5.
Figure Visco-elasticity profile of HH
profile in a in
of HH commercial batch at
a commercial batch 0.01
25 ±C0.01
(a) and (a)±and
°C 37 0.0137C±(b).
0.01 °C
(b).
HH showed a robust shear thinning response at high shear rates (Figure 7), relaxation
times in the order of seconds 5 ÷ 10 s (Table 4), a flattening of the G′ beyond the crossover
frequency, and G′′ within and a wider range of linear viscoelastic response dominated by
the elastic component.
Biomolecules 2024, 14, x FOR PEER REVIEW 9 of 14
Biomolecules 2024, 14, 832 9 of 14

Figure 6. Visco-elasticity profile of positive control commercial batch of HA-hydrogel polymer so-
lution at 25 °C.

HH showed a robust shear thinning response at high shear rates (Figure 7), relaxation
times in the order of seconds 5 ÷ 10 s (Table 4), a flattening of the G′ beyond the crossover
Figure Visco-elasticity
Figure6.6.Visco-elasticity
frequency, and G″ within profile
andof
profile of positive
a wider range
positive control commercial
ofcommercial
control linear batch
viscoelastic
batch of HA-hydrogel
of response polymer
dominated
HA-hydrogel polymer by
so-
solution
lution at 25at 25
°C. ◦ C.
the elastic component.

HH showed a robust shear thinning response at high shear rates (Figure 7), relaxation
times in the order of seconds 5 ÷ 10 s (Table 4), a flattening of the G′ beyond the crossover
frequency, and G″ within and a wider range of linear viscoelastic response dominated by
the elastic component.

Shear rate (s−1) 0.1 0.5 1.0 2.0 5.0 10.0

Dynamic viscosity (Pa∙s) 278 135 101 75 48 28

Dynamic
Figure7.7.Dynamic
Figure viscosity
viscosity profilefor
profile forHH
HHcommercial
commercialbatch.
batch.

Table 4. Comparative rheological parameters for HH vs. Control at 25 ± 0.01 ◦ C.

Table 4. Comparative
Shear rate (s−1) rheological parameters
0.1 for HH 0.5 vs. Control
1.0 at 25 ±2.0
0.01 °C. 5.0 10.0
Viscosupplement
Dynamic viscosity (Pa∙s) 278 HH
135 101 75 Control
48 28
Viscosupplement HH Control b
Reference frequency 0.5 a Hz 2.5 b Hz 0.5 a Hz 2.5 Hz
Figure
Reference 7. Dynamic
frequencyviscosity profile for HH commercial batch.
0.5 Hz478.32.5 Hz 82.6
a b 0.5 Hz
a 2.5 b Hz
Elastic modulus (G′ ) (Pa) 271.6 208.0
Elastic modulus
Viscous (G′) (G
modulus ′′ ) (Pa)
(Pa) 208.7 271.6 394.8 478.3 91.482.6 208.0
136.4
TableCross-over
4. Comparative rheological
frequency (Hz) parameters for0.096 HH vs. c Control at 25 ± 0.01 °C. 0.660 c
Viscous modulus (G″) (Pa) 208.7 394.8 91.4 136.4
Cross-over Relaxation
frequency time(Hz)
(s) 10.4 0.096 c 1.5 c
0.660
Viscosupplement Elasticity (%) 60.9 HH
70.6 47.5 Control60.4
Relaxation time (s) 10.4 1.5 ′ = G′′ .
a —0.5 Hz representative for walking, and b —2.5 Hz representative
Reference frequency 0.5 a Hz for2.5 b Hz c —frequency
running; 0.5 a Hz at which
2.5 bGHz
Elasticity (%) 60.9 70.6 47.5 60.4
aElastic modulus (G′) (Pa) 271.6 478.3
—0.5 Hz representative for walking, and b—2.5 Hz representative for running; c—frequency at
82.6 208.0
4. Discussion
Viscous modulus (G″) (Pa) 208.7 394.8 91.4 136.4
which G′ = G″.
The purpose
Cross-over frequency of (Hz)
this study was to assess the performance 0.096 c and safety0.660 of viscoelastic
c

solution
Relaxation
4. Discussion combining
time (s) Sodium Hyaluronate (20 mg/mL) 10.4 and Chondroitin Sodium 1.5 Sulfate
(30 mg/mL),
Elasticity (%) administered by IA injection in knee OA
60.9performance patients.
70.6 and safetyAll enrolled patients
47.5 of viscoelastic
60.4
The purpose of this study was to assess the
received
a—0.5 Hz a single injection
representative for at baseline,
walking, and after
b—2.5 initial
Hz clinical evaluation,
representative for and were
running; assessedatat
c—frequency
solution combining Sodium Hyaluronate (20 mg/mL) and Chondroitin Sodium Sulfate (30
threeG′follow-up
which =administered
G″. timepoints (6, 12, and 24 weeks). Although pain with strong discomfort
mg/mL), by IA injection in knee OA patients. All enrolled patients received
was considered at baseline, pain intensity and severity decreased significantly at weeks 6,
a single injection at baseline, after initial clinical evaluation, and were assessed at three
4.12, and 24 vs. baseline (p < 0.001).
Discussion
Thepurpose
The mean joint mobility
of this studyatwas
weeks 6, 12, and
to assess 24 showed anand
the performance increase
safety(by
of 17.8%, 31.5%,
viscoelastic
solution combining Sodium Hyaluronate (20 mg/mL) and Chondroitin Sodium Sulfatemean
and 35.61%, respectively) when compared to mean joint mobility at baseline, while the (30
Pearson’s coefficient of skewness showed a left, positive skew
mg/mL), administered by IA injection in knee OA patients. All enrolled patients receivedfor all situations. Unilateral
t-test showed a significant difference between knee joint mobility at baseline and at follow-
a single injection at baseline, after initial clinical evaluation, and were assessed at three
up (p < 0.001). The results obtained were consistent with previous research conducted by
Biomolecules 2024, 14, 832 10 of 14

other authors [26,27,29–33], although differences were noted in IA-HA posology between
our study protocol and others.
In this study protocol, an HA-CSNa injection was administered once at the begin-
ning of the study, whereas other studies administered one or more injections weekly for
the duration of the study. Similar to us, Henrotin et al. (2012) noted an improvement
in pain intensity and functional impairment, quantified by the Lequesne index. Signifi-
cant decreases in pain intensity were similarly noted, at weeks 6 and 12 (p = 0.0008 and
p = 0.0042, respectively) after injection, although no data were recorded at week 24, as in
our study. Injections were administered once a week (days 0, 7, and 14 of the study) and
followed up at weeks 7 and 14 [30].
Consistent results with which ones obtained through this study were noted in a
randomized, blind observer, parallel trial, where a significant improvement in favor of
the HA-treated group vs. placebo was noted in Lequesne index at week 5 (p = 0.03), with
persistent results at week 8 (p = 0.0431) and at week 16 (p = 0.0528), but with no difference
at week 24. The VAS score for pain during walking improved significantly at week 5
and month 6 (p = 0.0087 and p = 0.0049, respectively), whereas VAS score at rest showed
a difference compared to placebo, but not significant [31]. In an RCT, IA-HA injection
showed a significant reduction in Lequesne index at week 5 vs. placebo (from 13.57 ± 1.88 to
7.94 ± 2.53, p < 0.01), in addition to improving total workload of knee flexion and extension
(p < 0.01). IA-HA posology consisted of a weekly injection for a total of five injections [32].
Significant favorable differences from baseline were noted in the HA-treated group
for the VAS scale and Lequesne index at week 4. However, the HA-treated group received
an IA injection a week for 3 weeks (4 injections) [33]. Although using a different index
to measure pain, stiffness, and disability than the Lequesne index, Petrella et al. (2006)
showed, in a double-blinded RCT, a significant improvement in WOMAC scores for knee
pain in the HA-treated group at week 3 (p < 0.05), with no further difference at weeks
6 and 12. Pain assessment using the VAS scale showed similar improvement between
the placebo and HA-treated group, with no further differences at weeks 6 and 12. An
interesting observation resides in the improvement in the knee joint’s range of motion in
our study, whilst there was no improvement noted in the range of knee joint motion at
weeks 6 and 12 compared to baseline (p = 0.89 and 0.59, respectively) in Petrella et al. (2006)
trial [34].
A similar study found an improvement in WOMAC and VAS score for resting pain
at week 4 (p < 0.05) in the HA and placebo-treated group, NSAIDs, misoprostol, and
HA-treated group, and NSAIDs and placebo-treated group compared to baseline. At
week 12, the aforementioned groups had no significant improvement [35]. In a prospective
study, Weinhart (2008) showed a reduction in pain severity score at different stages (at
night, at rest, starting to walk, and exertion pain) after five injections and 4 weeks after
therapy, simultaneously decreasing Lequesne index summation score when measured at
the same periods (3.45 points decrease after five injections and 5.81 points decrease at
4 weeks post-therapy from baseline measurements) [36].
A systematic review of overlapping meta-analyses that included 20,049 patients from
14 meta-analyses (13,698 receiving IA HA, 255 receiving NSAIDs, 294 receiving IA corti-
costeroids and 5702 receiving IA placebo) found that IA HA improved pain and function,
while no clinically relevant differences regarding efficacy when compared with NSAIDs
were found, whereas clinical benefit of IA HA were greater at 5 to 13 weeks and persisted
up to 26 weeks, concluding that IA HA injections represent a viable alternative in patients
with early knee OA [37]. In a Cochrane review, Bellamy et al. (2006) evaluated 76 trials,
in which follow-up periods varied between the day of the last injection and 18 months. A
total of 40 trials investigated whether the difference between HA and placebo exists. The
pooled analyses of the effects of HA vs. placebo support the efficacy of IA HA administra-
tion, especially at weeks 5 and 13 post-injection, showing a percentage improvement from
baseline of 28–54% for pain parameter and 9–32% for function. When compared to NSAIDs,
efficacy was similar, whereas HA compared to IA corticosteroids favors IA HA, especially
Biomolecules 2024, 14, 832 11 of 14

regarding long-term effects [38]. The results of this confirmatory study are consistent with
the findings of previous studies and support the benefit/risk ratio of IA HA and HA-CS
injections in OA patients.
Viscosity (η) is a parameter that can only be measured for bodies in a fluid (liquid)
state, expressing the ability of the fluid to resist the sliding of two adjacent layers of its
mass, during the movement of the mass of fluid through flow. In the case of products
with intra-articular application, structurally fluid systems at ambient temperature, the fluid
state occurs when the system is sheared with a force that exceeds the stress value called
yield threshold (τ0 ), a parameter dependent on the system and its characteristics. After
this threshold, the flow behavior is determined by the resistance that the fluid exhibits
to the shear force (speed gradient, shear rate) applied to it, a property that is expressed
by the value of the tangential stress (τ). Tangential flow stress (τ) and viscosity (η) are
interdependent parameters that vary directly or inversely proportionally, depending on
the shear flow behavior, and each of these two (measurable) parameters can be used to
evaluate the consistency of a product that is administered intra-articularly and comparison
with the properties of synovial fluid from a healthy person. The rheological behavior of
structurally viscous systems is characterized by two values (parameters): the yield point -
τ0 (N/m2 ) and, respectively, the plastic viscosity -η (mPa·s). The properties of the synovial
fluid are different when it comes to a healthy person, and when it comes to a condition
such as osteoarthritis, not only the amount of hyaluronic acid is changed, but also the
visco-elastic properties [29].
The rheological study demonstrates the distinctive viscoelastic properties of the SH
(20 mg/mL) and CS (30 mg/mL) formulation compared to the control (20 mg/mL SH only).
This finding suggests that the addition of chondroitin sulfate (CS) to sodium hyaluronate
(SH) significantly enhances and improves the viscoelastic properties, particularly at higher
shear frequencies (e.g., running vs. walking). The application of oscillatory frequency
provided valuable insights into the structure–property relationship of the biomatrix solu-
tion. The observed shift in the crossover frequency towards lower frequencies upon CS
addition indicates larger relaxation times, suggesting a more entangled network structure.
This aligns with the observed plateau in the elastic modulus (G′ ) at the entanglement
region, signifying a transition towards more elastic solid-like behavior. Furthermore, the
robust shear thinning response of the SH-CS formulation at high shear rates implies shear-
dependent behavior, potentially beneficial for in vivo applications. The relaxation times in
the range of 5–10 s suggest a balance between viscous and elastic components, while the
flattening of G′ beyond the crossover frequency and dominance of G′′ within a wider range
indicate a viscoelastic response dominated by the elastic component. Rheological analysis
showed that chondroitin sulfate markedly increases the viscosity of HA solutions under
physiological conditions, giving further insights into the physiological role of chondroitin
sulfate and chondroitin sulfate proteoglycans in extracellular matrices and body fluids.
Limitations regarding the present study include a small number of participants (21),
and a lack of a prolonged follow-up period to assess the clinical outcome parameters in the
long term. To overcome these limitations, further studies ought to be conducted on a larger
population and a longer follow-up period to assess performance in the long term.
There are many products on the market in Europe that are administered intra-articularly,
but the vast majority only focus on the different compositions and different molecular
weights of hyaluronic acid. The complex biomatrix that includes chondroitin sulfate
and the achievement of viscoelastic properties similar to the synovial fluid of a healthy
patient proves to be a personalized medication that could increase the patient’s degree of
satisfaction after administration [39].
In future studies, we propose to evaluate through a wide post-market questionnaire
the degree of patient satisfaction with a return to daily activities/work correlated with
the assessment of the attending physician, after one cycle of treatment with an HA-CS
combination. Both the patient’s perception and the specialist’s medical judgment are
important for evaluating the patient’s degree of satisfaction. The extremely promising data
Biomolecules 2024, 14, 832 12 of 14

of this product require further investigation with respect to clinical implications under
current medical practice in knee osteoarthritis patients.

5. Conclusions
IA-HA is an established therapy for OA treatment. The first product of sodium
hyaluronate was approved by the FDA in 1997. Even though hyaluronic acid administered
intra-articularly has been used since 1987 in Europe and Japan, through the proposed
study we wanted to come up with eloquent data about the effectiveness of hyaluronic
acid and chondroitin sulfate administered for knee osteoarthritis. The present study was
designed to support data regarding the positive clinical outcomes of HA and CS viscoelastic
solutions for IA injections in patients with OA. Based on our study results, the one-time
injection proved to significantly alleviate OA symptoms until 24 weeks after injection,
which confirms its suitability as a viscoelastic supplement or a replacement for synovial
fluid in human knee joint osteoarthritis. No adverse events have been noted during the
study period.
By adding chondroitin sodium sulfate at a ratio of 3:2 to natural hyaluronic acid
supported natural and physical crosslinking of sodium hyaluronate providing mechanical
robustness, improved rheological properties, simultaneously with preserving biocompati-
bility and biodegradability of the sodium hyaluronate native polymer. At the same time,
better resistance to enzymatic degradation and free radicals could be expected from the
synergic combination of sodium hyaluronate and chondroitin sodium sulfate.

Author Contributions: Conceptualization, A.D., G.M. and A.C. (Alexandru Cristea); methodology,
A.D., A.R.R., G.M. and M.D.; validation, A.C. (Alexandru Cristea), A.D. and M.D.; formal analysis,
A.D. and A.R.R.; investigation, A.D., A.C. (Alexandru Cristea), A.R.R. and M.D.; data curation, A.R.R.;
visualization, A.M., D.C. and A.-T.D.; writing—original draft preparation, A.M., D.C. and A.-T.D.;
writing—review and editing, A.M., D.C., A.-T.D., A.D., M.D., R.-A.V., M.B. and A.C. (Adriana Ciurba);
supervision, A.C. (Alexandru Cristea) and M.B.; project administration, A.C. (Adriana Ciurba), A.D.
and G.M.; funding acquisition, A.D., R.-A.V. and G.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This manuscript is based upon clinical trial results from a study financial supported by
Rompharm Company which provided the medical devices used in the study. The funder was not
involved in the study design, collection, analysis, interpretation of data, the writing of the article, or
the decision to submit it for publication.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved in 26 February 2014 by the Institutional Ethics Committee of the National
Institute of Rehabilitation, Physical Medicine and Balneoclimatology (Bucharest, Romania) (no. 2288)
for studies involving humans.
Informed Consent Statement: Informed consent was obtained from all subjects that were involved
in this study.
Data Availability Statement: Data are contained within the article.
Acknowledgments: The authors would like to personally thank all the principal investigators and
study coordinators for their help with subject recruitment and data collection and to Rompharm
Company for providing Hialurom Hondro® samples used in this study.
Conflicts of Interest: Authors G.M., A.M., D.C., A.-T.D. are employees of Rompharm Company S.R.L.
Authors A.D., A.C. (Alexandru Cristea) declare that they receive support from commercial sources of
funding by Rompharm Company S.R.L. Other authors declare that the research was conducted in the
absence of any commercial or financial relationship that could be construed as a potential conflict
of interest.
Biomolecules 2024, 14, 832 13 of 14

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