Article

Development of Dispersion Process to Improve Quality of

Hyaluronic Acid Filler Crosslinked with 1,4-Butanediol
Diglycidyl Ether
Sunglim Choi * , Jin Cheol Cho, Seunghwa Lee and Seong Jin Lee

CHA Meditech Co., Ltd., 119 Techno 2-ro (#206, Migeun Techno World, Yongsan-Dong), Yuseong-gu,
Daejeon 34116, Republic of Korea; cho928@chamc.co.kr (J.C.C.); lshlsh0106@chamc.co.kr (S.L.);
sjlee@chamc.co.kr (S.J.L.)
* Correspondence: slchoi85@chamc.co.kr; Tel.: +82-10-9489-4489

Abstract: This study proposes a new and simple process that improves the quality of a hyaluronic
acid (HA) filler crosslinked with 1,4-butanediol diglycidyl ether (BDDE) using solution dispersion at
a low temperature. This process involves the solvent being dispersed among the solute naturally after
the mixing process. The process used in this study involved two reactions. First, the solution was
dispersed among HA molecules (Mw = ~0.7 MDa) creating a well-homogenized mixture. Second,
the decomposition and synthesis of HA occurred naturally in an aqueous alkaline solution (>pH 11),
the weight average molar mass (Mw) was adjusted (Mw = ~143,000), and the crosslinking surface
area was expanded, allowing for a high degree of crosslinking. Therefore, the viscoelasticity and
cohesion of the filler increased with the new method compared to the previous process both at the lab
scale (previous process:new process, viscosity (cP) = 24M:43M, storage modulus (Pa) = 306:538, loss
modulus (Pa) = 33:61, and tack (N) = 0.24:0.43) and at the factory scale (previous process:new process,
complex viscosity (cP) = 19M:26M, storage modulus (Pa) = 229:314, loss modulus (Pa) = 71:107, and
tack (N) = 0.35:0.43).

Keywords: hyaluronic acid; BDDE crosslinker; dermal filler; dispersion process

Citation: Choi, S.; Cho, J.C.; Lee, S.;

Lee, S.J. Development of Dispersion
Process to Improve Quality of
1. Introduction
Hyaluronic Acid Filler Crosslinked
with 1,4-Butanediol Diglycidyl Ether. Dermal fillers are generally used to remove wrinkles, restore volume, and provide
Polymers 2024, 16, 3323. https:// moisture. They are injected directly into skin layers; therefore, they must be made of
doi.org/10.3390/polym16233323 materials that are either similar to skin tissue or do not cause adverse reactions [1–9].
In the early stages of dermal filler development, materials such as collagen [2], poly
Academic Editor: Alberto Romero
methyl methacrylate (PMMA) [3], and dextran [4] were used to manufacture the fillers.
García
One advantage of using these materials to produce fillers is the fact that they are semi-
Received: 25 October 2024 permanent; however, they are difficult to remove from the different layers of the skin.
Revised: 21 November 2024 If these materials cause adverse reactions, such as allergies, surgical treatment may be
Accepted: 22 November 2024 required to remove them from the skin [5], which is disadvantageous in terms of both cost
Published: 27 November 2024 and health.
At present, most dermal fillers are made using hyaluronic acid (HA). This choice of
material is based on the fact that HA is decomposed by enzymes such as hyaluronidase, and
Copyright: © 2024 by the authors.
the substances that are produced in the process do not cause harm to the body. However,
Licensee MDPI, Basel, Switzerland.
the advantages and disadvantages of using HA are identical; HA is decomposed in the
This article is an open access article
body, and, as a result, the filler material is not maintained in the skin layer [6–10]. To
distributed under the terms and address the above disadvantage, various crosslinkers have been reacted with HA, with this
conditions of the Creative Commons process increasing the duration of the filler remaining in the skin layer [10–13].
Attribution (CC BY) license (https:// Among them, 1,4-butanediol diglycidyl ether (BDDE) is commonly used as a crosslinker
creativecommons.org/licenses/by/ because the crosslinking method and required conditions are simple; the ring-opening
4.0/).

Polymers 2024, 16, 3323. https://doi.org/10.3390/polym16233323 https://www.mdpi.com/journal/polymers

Polymers 2024, 16, 3323 2 of 13

reaction of the epoxy group in BDDE occurs at a relatively low temperature. In addition,
the reaction requires only a basic (pH > 11) solution [8,13,14].
However, the manufacturing of HA fillers crosslinked with BDDE (HA-BDDE crosslinked
fillers) faces three difficulties.
First, when poly HA is dissolved in an aqueous solution at a high concentration,
common methods such as stirring, are limited to produce a homogeneous solution [15–18].
Therefore, the compound must be mixed manually using a Teflon bar; however, this
method is prone to human error. At present, the use of planetary mixers means that this
problem has, for the most part, been solved [19]. However, it is difficult to produce a
homogeneous solution.
Second, the above problem means that a relatively large amount of unreacted BDDE
with HA remains in the mixture or is hydrolyzed. This, then, results in the filler having low
yield, elasticity, and viscosity [20,21].
Third, the remaining BDDE and oxidized BDDE in the filler are toxic and can cause
allergic reactions. Additionally, pendant BDDE, in which one side of the epoxy group of
BDDE reacts with the primary alcohol group within a single HA unit, may also lead to
other similar side effects [21,22]. For this reason, ensuring the correct crosslinking ratio
between HA and BDDE is especially important. Furthermore, as the crosslinking ratio
increases, the elasticity and viscosity of the filler increase, which improves the quality of
the filler [9].
For the above reasons, by mixing HA and a NaOH solution, an attempt was made to
produce a more homogeneous mixture compared to those made using other processes. [23].
HA is a hydrophilic compound, and the aqueous solution can disperse among the HA
chains. Therefore, the ‘dispersion’ process was hereby introduced into filler production;
this provided enough time for the NaOH solution to disperse among the poly HA chains at
a suitable temperature. This process is expected to produce a homogeneous material.
Chemically, HA chains are connected and cleaved simultaneously in pH > 11 due
to hydrolysis and dehydration [24]. If the reaction rate of hydrolysis and dehydration
of HA can be controlled, HA with appropriately low number-average molar mass (Mn)
and weight-average molar mass (Mw) can be produced. This process will enable the
production of poly HA chains of an appropriate length, ensuring a sufficient surface area
for the reaction with BDDE. However, if the HA chains become too short, the elasticity,
viscosity, and cohesion of the fillers will decrease [25]. Therefore, by controlling the temper-
ature and duration of the ‘dispersion’ process, the optimal conditions for this process can
be determined.
Due to the efficiency of the dispersion process, the time of the dispersion process is
limited to one day, and the temperature of this process is lower than room temperature (rt).
In this study, the optimal time and temperature of the dispersion process at a lab
scale were determined and introduced into the production process of the filler. Thereafter,
the elasticity, viscosity, and cohesion of the filler materials were measured to determine
the improvement in filler quality [26,27]. In addition, the degree of modification (MoD)
was measured to determine the improvement in the crosslinking ratio between HA and
BDDE [28].

2. Methods of Synthesis and Analysis

2.1. Synthesis of the HA-BDDE-Crosslinked Filler
2.1.1. Batch A
First, 15 g of HA ([η] = 1.4, η = intrinsic viscosity, Hyundai Bioland Co., Ltd., Cheongju,
Republic of Korea, Mw = ~0.7 MDa) was placed into a planetary centrifugal mixer case,
and 0.25 N of NaOH solution was added to produce 17 wt% of HA solution based on the
molecular weight of sodium hyaluronate. Next, the mixture was blended using a planetary
centrifugal mixer (ARE-500, THINKY, Chiyoda, Japan) for 10 min at 1000 rpm. This process
was repeated twice. Thereafter, the mixture was blended for 10 min at 1000 rpm and
deformed for 5 min at 2000 rpm. At this point, the mixer case was maintained at a low
 using a planetary centrifugal mixer (ARE-500, THINKY, Chiyoda, Japan) for 1
Polymers 2024, 16, 3323
1000 rpm. This process was repeated twice. Thereafter, the mixture 3was of 13 blende

min at 1000 rpm and deformed for 5 min at 2000 rpm. At this point, the mixer c
maintained at a low temperature using a cooling adapter. The reference mixture w
temperature using a cooling adapter. The reference mixture was synthesized following the
thesized
previous following
process [22], asthe previous
shown process
in Figure 1. [22], as shown in Figure 1.

Previous process New process

HA + 0.25 M HA + 0.25 M
NaOH solution NaOH solution

BDDE 3.5 mol%

Dispersion process
Mix

Crosslinking BDDE 3.5 mol%

25 °C, 24 h Mix

Crosslinking
25 °C, 24 h

Figure
Figure 1. 1. Chart
Chart of difference
of the the difference
betweenbetween the process
the previous previousandprocess and the
the new process new process
(dispersion (d
process at a low temperature).
process at a low temperature).

The experimental mixture was subjected to dispersion processes with different tem-
Theand
peratures experimental
durations. mixture was subjected to dispersion processes with differ
peratures
When BDDEand (Sigma-Aldrich
durations. Co., Ltd., St. Louis, MO, USA) was added to the mixture,
When BDDE
the concentration was(Sigma-Aldrich
3.5 mol%. Next, Co., Ltd., St.
the mixture Louis,
was MI,using
blended USA)the was added to the
planetary
centrifugal
the concentration was 3.5 mol%. Next, the mixture was blended using the
mixer for 5 min at 1000 rpm. This process was repeated twice. Thereafter, the planet
mixture was deformed for 2 min at 2000 rpm. At this point, the mixer case was maintained
trifugal mixer for 5 min at 1000
at a low temperature using a cooling adapter.
rpm. This process was repeated twice. Thereafter,
tureThewas deformed
mixture for 2 into
was poured minthe at metal
2000 frame
rpm. (20
At ×
this
20 ×point,
20 mmthe
bymixer case
a cell, 120 was maint
× 120
×a 20
lowmm temperature
by a frame), andusing a cooling
the frame adapter.
was placed ◦
in an oven at 25 C for 24 h. The mixture
was transformed into an elastic gel form.
The mixture was poured into the metal The gels (20 ×frame
20 × 20(20
mm× by
20a×gel)
20were
mmplaced
by a cell, 12
in dialysis bags. Afterward, the dialysis bags were placed in 0.9 (w/v)% NaCl solution, and
20 mm by a frame), and the frame was placed in an oven at 25 °C for 24 h. The
the solution was slowly stirred for 5 days. The solution was replaced twice a day.
wasLastly,
transformed into(50
the hydrogels an×elastic
50 × 45 gel
mm form. Thewere
by a gel) gelscrushed
(20 × 20
to ×300
20µmmm by the
using a gel) wer
in dialysis
mesh bags. Afterward,
sieve (DH.Si8038, DAIHAN the dialysis
Scientific, bagsRepublic
Incheon, were placed in 0.9
of Korea), and(w/v)% NaCl solut
the crushed
hydrogels were sterilized
the solution was slowly usingstirred
an autoclave
for 5 (HS-3041VD, HANSHIN
days. The solution MEDICAL
was replaced Co.,twice
Ltd., a day
Incheon, Republic ◦
Lastly, theofhydrogels
Korea) at 120(50C×for 5020× min.
45 mm by a gel) were crushed to 300 µm u
mesh
2.1.2. sieveB(DH.Si8038,
Batches and C DAIHAN Scientific, Incheon, Republic of Korea), and the
hydrogels were
In this step, 160 sterilized
g of HA ([η]using
= 1.4, ηan autoclave
= intrinsic (HS-3041VD,
viscosity, HANSHIN
Hyundai Bioland Co., Ltd.,MEDIC
Ltd., Incheon,
Republic of Korea, Republic
Mw = ~0.7of MDa)
Korea) at 120
(Batch B) °C forg20(Batch
or 320 min.C) was placed into the
planetary mixer case, and 0.25 N of NaOH solution was added to produce 17 wt% of HA
solution based on the molecular weight of sodium hyaluronate. Next, the mixture was
2.1.2. Batches B and C
blended twice using a planetary centrifugal mixer (OST-CMT1811, OSTAR TECH Co., Ltd.,
In this
Pyeongtaek, step, 160
Republic g of HA
of Korea), and ([η]
it was= ultimately
1.4, η = intrinsic
deformedviscosity, Hyundai
for 10 min at Bioland C
600 rpm. At
this point, the mixer was maintained at − 6 ◦ C.
Republic of Korea, Mw = ~0.7 MDa) (Batch B) or 320 g (Batch C) was placed into t
Themixer
etary reference mixture
case, was synthesized
and 0.25 N of NaOH following the was
solution previous
addedprocess, as shown17
to produce in wt% o
Figure 1.
lution based on the molecular weight of sodium hyaluronate. Next, the mixt
The experimental mixtures were subjected to the dispersion process at 18 ◦ C for 24 h.
blended
When BDDEtwice using
was added a planetary
to the mixture, thecentrifugal
concentrationmixer
was 3.5(OST-CMT1811,
mol%. To ensure thatOSTAR
the TE
Ltd., was
BDDE Pyeongtaek, Republic
mixed thoroughly, the of Korea),
mixing and
process it carried
was was ultimately deformed
out in the same manner for
as 10 mi
above. The mixture was poured into the metal frame,
rpm. At this point, the mixer was maintained at −6 °C. and the frame was placed in the oven
The reference mixture was synthesized following the previous process, as s
Figure 1.
The experimental mixtures were subjected to the dispersion process at 18 °C
When BDDE was added to the mixture, the concentration was 3.5 mol%. To ens
Polymers 2024, 16, 3323 4 of 13

at 25 ◦ C (Batch B) or 30 ◦ C (Batch C) for 24 h. The mixture was transformed into an elastic

gel form. The gels (20 × 20 × 20 mm by a gel) were placed in 0.9 (w/v)% NaCl solution
(Batch B) or 1× phosphate-buffered saline (PBS) pH 7.4 solution (Batch C) using a swelling
device (PID-101, SEJIN T&E Co., Ltd., Jeonju, Republic of Korea), and the solution was
stirred slowly for 5 days. The solution was replaced twice a day.
Lastly, the hydrogels (50 × 50 × 45 mm by a gel) were crushed to 300 µm using a
plunger mill (OST-PM60-A01, OSTAR TECH Co., Ltd., Republic of Korea), and the crushed
hydrogels were sterilized using an autoclave (BP-AC680G, Bio Plan, Daejeon, Republic of
Korea) at 120 ◦ C for 20 min.

2.2. Decomposition of HA-BDDE Crosslinked Hydrogel

First, 1 g of the crushed hydrogel was place in a vial (>4 mL) and 1 mg of chondroitinase
ABC from Proteus vulgaris (2 unit/mg, Sigma-Aldrich Co., Ltd., USA) was dissolved in
1× PBS solution pH 7.4 (20× PBS solution (LPS Solution, Daejeon, Republic of Korea)):
Distilled water = 19:1) 10 mL. Thereafter, a solution of 1.2 mL was added to the vial
containing the hydrogel. After the mixture was stirred for 30 min, the vial was placed in
a shaking incubator set to 157 rpm for 5 days. Next, the vial was placed in an ultralow-
temperature freezer (MDF-192, SANYO Electronic Co., Ltd., Osaka, Japan) for 1 h. Lastly,
the mixture was dried using a freeze-dryer (FD8508, ilShinBioBase Co., Ltd., Dongducheon,
Republic of Korea) for 3 days [29].

2.3. Measurement of Viscoelasticity and Cohesion

Using a rheometer (Phisica MCR301, Anton Paar, Ostfildern, Germany), complex
viscosity, storage modulus (G′ ), loss modulus (G′′ ), and tack were measured.
Next, 1 mL of filler material was placed on the ground plate of the rheometer at 25 ◦ C,
and, using a rheometer software (Rheoplus V. 3.62, Anton Paar, Germany), the measuring
plate (Φ = 25 mm) was pulled down until the gap with the ground plate was 1 mm (sample
size: Φ25 × 1 mm). Thereafter, complex viscosity, G′ , and G′′ were measured. Lastly, tack
was measured.

2.4. Measurement of Gel Filtration Chromatography (GFC) and Nuclear Magnetic

Resonance (NMR)
GFC (e2695, Waters, Pleasanton, CA, USA) commissioned the Korea Research Institute
of Chemical Technology (KRICT) to conduct the measurements. To produce the GFC
sample, 1 g of the crushed hydrogel was poured into a vial and placed in an ultralow-
temperature freezer for 1 h. Thereafter, the mixture was dried using a freeze-dryer for
3 days. This powdered material was submitted to KRICT for GFC analysis.
NMR (AVANCE III 600, Burker, Billerica, MA, USA) was either conducted upon
request by Chungnam National University or performed directly. To produce the NMR
sample, 20 mg of the decomposed compound was dissolved in 1 mL of D2 O (Cambridge
Isotope Laboratories, Tewksbury, MA, USA) and poured into an NMR tube (508HP, Norell,
Morganton, NC, USA) [29].

3. Results and Discussion

In previous studies, various methods were employed to enhance the viscoelasticity
and cohesion of fillers. These included using HA with a relatively high Mw [30], incorpo-
rating compounds such as mannitol [31], and employing alternative crosslinkers like poly
(ethylene glycol) diglycidyl ether (PEGDE) [32,33], and divinyl sulfone (DVS) [26,34].
However, this experiment was conducted to enhance the viscoelasticity and cohesion
of the filler through simple process modifications, without altering the composition of
specific substances, as mentioned above. Additionally, efforts were made to improve these
properties beyond the results achieved in previous studies [35] conducted at this facility.
Polymers 2024, 16, x FOR PEER REVIEW 5 of 14
Polymers 2024, 16, 3323 5 of 13

3.1. Experimental Results of Products Manufactured in the Lab

3.1. Experimental Results of Products Manufactured in the Lab
3.1.1.Viscoelasticity
3.1.1. Viscoelasticityand andCohesion
Cohesion
Complexviscosity
Complex viscosity represents
represents the the overall
overall viscosity
viscosity and
and elasticity
elasticity of
of aa material.
material. G′ G′re-
flects its elasticity, whereas G″ ′′ indicates its viscosity.
reflects its elasticity, whereas G indicates its viscosity.
InFigure
In Figure2a–c2a–cand
andTables
TablesS1–S9,
S1–S9,complex
complexviscosity,
viscosity,
G′G′, and
, and G′′G″ show
show a tendency
a tendency to to
increaseover
increase overtime,
time,except ◦
exceptatat4 4 C.
°C.The
Thegraph
graphofofcomplex
complexviscosity
viscosity shows
shows that
that thethe value
value forfor
theprevious
the previousexperiment
experimentwas was roughly
roughly 2.42.4 McP,
McP, andandthe the values
values for experiment
for the the experiment includ-
including
ing the dispersion process were about 4.8 McP◦ at 10 °C and 4.3 McP
the dispersion process were about 4.8 McP at 10 C and 4.3 McP at 18 C for 24 h—roughly◦ at 18 °C for 24 h—
roughly
twice the twice the values.
previous previous values.

(a) Complex viscosity (cP) (b) Storage modulus (G′)

6 × 10⁶ 800

5 × 10⁶
600
4 × 10⁶

G′ (Pa)
cP

3 × 10⁶ 400

2 × 10⁶
200
1 × 10⁶

0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time (h) Time (h)
(c) Loss modulus (G″, Pa) (d) Complex modulus
800
80 700

600
60
G* (Pa)
G″ (Pa)

500

40 400

300
20
200

0 100
0 5 10 15 20 25 0 5 10 15 20 25
Time (h) Time (h)
(e) Tack
0.5

4 °C linear graph
0.4
4 °C spline graph
10 °C linear graph
Tack

10 °C spline graph
0.3
18 °C linear graph
18 °C spline graph

0.2

0 5 10 15 20 25
Time (h)

Figure2.2.(a)
Figure (a)Complex
Complexviscosity
viscosity(cP),
(cP),(b)
(b)storage
storagemodulus
modulus (G(G′),
′ ), (c)(c) loss
loss modulus
modulus (G(G″),
′′ ), (d)(d) complex
complex
modulus (G*), and (e) tack over dispersion time at each dispersion temperature (4 C, 10 C, and
modulus (G*), and (e) tack over dispersion time at each dispersion temperature (4 °C,
◦ 10 °C,
◦ and 18
°C,
◦ n = 3).
18 C, n = 3).

TheGG′
The ′ and
andGG” ′′ of
ofthe
theexperiment
experimentinvolving
involvingthethedispersion
dispersionprocess
process were
were also
also roughly
roughly
twice those
twice those of of the previous experiment. When comparing all values, the elasticity
experiment. When comparing all values, the elasticity and and vis-
cosity ofofthe
viscosity thefiller
fillermaterial
materialincreased
increased over time. Additionally,
Additionally,the thecohesion
cohesionofofthethe filler
filler
materials was
materials was known through the tack tack and
and the
thecomplex
complexmodulus
modulus(G*).(G*).InInFigure
Figure2d,e and
2d,e
and Tables
Tables S10–S15,
S10–S15, tacktackandand
G* G*
cancan also
also be be seen
seen increasing
increasing overtime.
over time.InInthe
theexperiment
experimentin-
including thedispersion
cluding the dispersion process,
process, the values
values were
were0.41~0.43
0.41~0.43for
fortack
tackand
and500~600
500~600PaPaforforG*.
G*.
These values were also roughly twice those of the previous
These values were also roughly twice those of the previous experiment.experiment.
As shown in Figure 3a–c, the elasticity and viscosity increased up to 10 °C, after
which the values slightly decreased until 18 °C. However, in Figure 3e, the tack values can
Polymers 2024, 16, 3323 6 of 13

Polymers 2024, 16, x FOR PEER REVIEW 6 of 14

As shown in Figure 3a–c, the elasticity and viscosity increased up to 10 ◦ C, after which

the values slightly decreased until 18 ◦ C. However, in Figure 3e, the tack values can be
seen increasing
be seen withwith
increasing the temperature. ThereThere
the temperature. was no significant
was difference
no significant in elasticity
difference and
in elasticity
viscosity between 10 ◦ C and 18 ◦ C; however, the cohesion value changed, as increasing the
and viscosity between 10 °C and 18 °C; however, the cohesion value changed, as increas-
tack value
ing the was
tack verywas
value difficult.
very difficult.

(a) Complex viscosity (cP) (b) Storage modulus (G′)

6 × 10⁶ 800

5 × 10⁶
600
4 × 10⁶

G′ (Pa)
cP

3 × 10⁶ 400

2 × 10⁶
200
1 × 10⁶

0 0
276 278 280 282 284 286 288 290 292 276 278 280 282 284 286 288 290 292
HA dispersionTemp (K) HA dispersionTemp (K)
(c) Loss modulus (G″) (d) Complex modulus (G*)
800
80
700

60 600
G* (Pa)
G″ (Pa)

500
40
400
20
300

0 200
276 278 280 282 284 286 288 290 292 276 278 280 282 284 286 288 290 292
HA dispersionTemp (K) HA dispersionTemp (K)
(e) Tack
0.5

0.4

1h
Tack

3h
0.3 24h

0.2

276 278 280 282 284 286 288 290 292

Time (h)

Figure3.3.(a)
Figure (a)Complex
Complexviscosity
viscosity(cP),
(cP),(b)
(b)storage
storagemodulus
modulus ′ ), (c)(c)
(G(G′), loss
loss modulus
modulus ′′ ), (d)(d)
(G(G″), complex
complex
modulus(G*),
modulus (G*),and
and(e)
(e)tack
tackover
overdispersion
dispersiontemperature
temperatureatateach
eachdispersion
dispersiontime
time
(1(1
h,h, 3 h,
3 h, andand
2424
h, h,
n = 3).
n = 3).

Whensummarizing
When summarizingthetheabove
aboveresults,
results,thethetime
timeand
andtemperature
temperature
ofof
thethe dispersion
dispersion
processwere
process determinedtotobebe2424h hatat1818◦ C.
weredetermined °C.

3.1.2.
3.1.2.Molar
MolarMass
Mass
As
Asmentioned
mentionedabove,
above,relatively
relativelylowering
loweringthe
theMn
Mnand
andMw
Mw ofof
polymers increases
polymers their
increases their
surface area. In Figure 4 and Table 1, the Mn and Mw of HA subjected to the dispersion
surface area. In Figure 4 and Table 1, the Mn and Mw of HA subjected to the dispersion
process ◦ C for 24 h are reduced to 1/6 compared to those of unprocessed HA.
processatat18
18 °C for 24 h are reduced to 1/6 compared to those of unprocessed HA.
It was assumed that the change in the molecular weight of HA had affected the
crosslinking ratio between HA and BDDE [36]. This is because the lower the Mw, the larger
the reaction surface area of the polymer. Through the changes in physical properties and
Mw, it was believed that the process hereby described yielded a higher crosslinking ratio
compared to the previous process.
Polymers 2024,16,
Polymers2024, 16,3323
x FOR PEER REVIEW 77 of 14
of 13

18.00
(a)
16.00

92,904
14.00
12.00
10.00
8.00
6.00
MV 4.00

537
2.00
0.00
−2.00
−4.00
−6.00
−8.00
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00
Minutes

7.00
(b)
6.00 714,562
5.00
4.00
3.00
2.00
MV

1.00
557

0.00
−1.00
−2.00
−3.00
−4.00
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00
Minutes

Figure 4.
Figure 4. (a)
(a) GFC
GFC graph
graph of
of 17
17 wt%
wt% HA
HAinin0.25
0.25N
NNaOH
NaOHsolution
solutionincluding
includingthe
thedispersion
dispersion process
process
(18◦°C; 24 h); and (b) GFC graph of 17 wt% HA aqueous solution.
(18 C; 24 h); and (b) GFC graph of 17 wt% HA aqueous solution.

Table It1.was assumed thatmolecular

Number-average the change in the(Mn),
weight molecular weight ofmolecular
weight-average HA had affected the cross-
weight (Mw), and
linking ratio between HA and BDDE [36].
polydispersity under (a) and (b) in Figure 4. This is because the lower the Mw, the larger the
reaction surface area of the polymer. Through the changes in physical properties and Mw,
it was believed that the process hereby
Process Mndescribed
(Da) yielded
Mw a(Da)
higher crosslinking ratio com-
Polydispersity
pared to
◦ the previous process.
18 C and 24 h dispersion 60,620 143,034 2.36
No dispersion 336,990 768,281 2.28
Table 1. Number-average molecular weight (Mn), weight-average molecular weight (Mw), and pol-
ydispersity under (a) and (b) in Figure 4.
3.2. Experimental Results of Products Manufactured in the Factory (Batch B)
3.2.1. The Apparent
Process State Mn (Da) Mw (Da) Polydispersity
18Based
°C andon24the
h dispersion 60,620
above results, the dispersion process 143,034◦ 2.36
at 18 C for 24 h was introduced at
No dispersion
the production scale (Batch B). When 336,990
simply blending768,281
HA in the 0.25 N NaOH 2.28 solution
using a planetary mixer, the mixture was cloudy, and there were also areas where HA was
clumped togetherResults
3.2. Experimental in powder form. Manufactured in the Factory (Batch B)
of Products
After the dispersion
3.2.1. The Apparent State process, the mixture became clear; there were no places where
HA accumulated. Therefore, HA that had undergone the dispersion process seemed to
Based on the above results, the dispersion process at 18 °C for 24 h was introduced at
react better with BDDE than HA that had undergone the previous process (Figure 5).
the production scale (Batch B). When simply blending HA in the 0.25 N NaOH solution
It was assumed that the change in the polydispersity of HA affected the crosslinking
usingbetween
ratio a planetary
HAmixer, the mixture
and BDDE. was cloudy,
However, andnothere
there was were also
difference areaspolydispersity;
in their where HA was
clumped together in powder form.
therefore, increasing the polydispersity of HA does not affect the molar mass of HA during
After the
dispersion. dispersion
During process, the
the dispersion mixture
process, thebecame
reactiveclear; thereiswere
surface no places
expected where
to increase
HA accumulated. Therefore,
compared to untreated HA. HA that had undergone the dispersion process seemed to
react better with BDDE than HA that had undergone the previous process (Figure 5).
 18 °C, 24 h + BDDE
dispersion & Mix
Polymers2024,
Polymers 2024,16,
16,3323
x FOR PEER REVIEW 88 of 14
of 13

Figure 5. Visible transparency

18 °C, 24changes
h in HA in 0.25 N NaOH
+ BDDEsolution after the dispersion process
dispersion
(18 °C; 24 h) and then mixed with BDDE using a revolving–rotating
& Mix mixer.

It was assumed that the change in the polydispersity of HA affected the crosslinking
ratio between HA and BDDE. However, there was no difference in their polydispersity;
therefore, increasing the polydispersity of HA does not affect the molar mass of HA dur-
ing dispersion. During the dispersion process, the reactive surface is expected to increase
Figure 5.
compared
Figure 5. Visible
Visible transparency
to untreated changes in
HA.changes
transparency in HA
HA in
in 0.25
0.25 N
N NaOH
NaOH solution
solution after
after the
the dispersion
dispersion process
process
(18 ◦°C;
(18 24 h)
C; 24 h) and
and then
then mixed
mixed with
with BDDE
BDDE using
using aa revolving–rotating
revolving–rotatingmixer.
mixer.
3.2.2. Viscoelasticity and Cohesion
3.2.2.ItViscoelasticity
was assumedand thatCohesion
the change in the polydispersity of HA affected the crosslinking
As shown in Figure 6a–c and Table S16, the complex viscosity and G′′ of products
ratioAsbetween HA and BDDE.
shown in Figure 6a–c and However,
Table there wascomplex
S16, the no difference in their
viscosity and Gpolydispersity;
of products
manufactured in the dispersion process at 18 °C ◦for 24 h were 1.2 times higher than those
therefore, increasing the polydispersity of HA does not affect the molar
manufactured in the dispersion process at 18 C for 24 h were 1.2 times higher mass of HA than
dur-
of products with no dispersion; G″ was′′1.1 times higher. As shown in Figure 6d,e and
ing dispersion.
those of productsDuring thedispersion;
with no dispersion process,
G was 1.1 thetimes
reactive surface
higher. Asisshown
expected to increase
in Figure 6d,e
Table S17, compared to the cohesion of the products, the tack and G* of products manu-
compared
and to untreated
Table S17, comparedHA.to the cohesion of the products, the tack and G* of products
factured during the dispersion process at 18 °C for◦24 h were 1.2 times higher than those
manufactured during the dispersion process at 18 C for 24 h were 1.2 times higher than
of products with no dispersion. It can be concluded that the viscoelasticity and cohesion
3.2.2. Viscoelasticity
those of products withandnoCohesion
dispersion. It can be concluded that the viscoelasticity and
of products manufactured using the dispersion process were better than those of products
cohesion shown in Figure 6a–c and using
of products manufactured Table the
S16,dispersion process were better
and G′than those of
with As
no dispersion. the complex viscosity of products
products with no dispersion.
manufactured in the dispersion process at 18 °C for 24 h were 1.2 times higher than those
of products with no dispersion; G″ was 1.1 times higher. As shown in Figure 6d,e and
Table S17, compared to the cohesion of the products, the tack and G* of products manu-
factured during the dispersion process at 18 °C for 24 h were 1.2 times higher than those
of products with no dispersion. It can be concluded that the viscoelasticity and cohesion
of products manufactured using the dispersion process were better than those of products
with no dispersion.

Figure 6. Comparison between old and new process at the 1/2 factory scale: (a) complex viscosity
(cP), (b) storage modulus (G′ ), (c) loss modulus (G′′ ), (d) complex modulus (G*), and (e) tack (n = 3).

As mentioned above, increasing the elasticity and viscosity indicated an improvement

in the quality of the filler. However, if the values of the elasticity and viscosity are too
high, the injection pressure of the filler will increase. It is therefore crucial to manufacture
products that account for these factors. When measuring the injection pressure with a 27 G
 (cP), (b) storage modulus (G′), (c) loss modulus (G″), (d) complex modulus (G*), and (e) tack (n = 3).

As mentioned above, increasing the elasticity and viscosity indicated an improve-

ment in the quality of the filler. However, if the values of the elasticity and viscosity are
too high, the injection pressure of the filler will increase. It is therefore crucial to manufac-
Polymers 2024, 16, 3323 9 of 13
ture products that account for these factors. When measuring the injection pressure with
a 27 G needle, it was found that the values were all similar and less than 20 N. In other
words, as a result of the dispersion process, the quality of the filler increased.
needle, it was found that the values were all similar and less than 20 N. In other words, as
a result
3.2.3. of the
Degree ofdispersion process,
Modification (MoD)the quality of the filler increased.
The
3.2.3. stability
Degree of the filler material
of Modification (MoD) is as important as its viscoelasticity and cohesion.
The stability can be determined based on
The stability of the filler material the
is as crosslinking
important as itsratio, because the
viscoelasticity higher
and the
cohesion.
crosslinking ratio, the more rigid the structure of the filler material. Therefore,
The stability can be determined based on the crosslinking ratio, because the higher the the filler’s
decomposition rate the
crosslinking ratio, wasmore
slow.rigid
Thesetheresults mean
structure thatfiller
of the the filler may Therefore,
material. remain in the
the filler’s
body
slightly longer. The crosslinking ratio was analyzed based on the degree
decomposition rate was slow. These results mean that the filler may remain in the body of modification
(MoD) and
slightly the ratio
longer. Thebetween the number
crosslinking ratio wasof hydrogens
analyzed based in C7on andtheC6, 6’. This
degree of number can
modification
be checked in 1H NMR. The equation is as follows [29,37]:
(MoD) and the ratio between the number of hydrogens in C7 and C6, 6 . This number can ′

be checked in 1H NMR. The

MoDequation
(%) = (Iisδ1.65~1.70
as follows
/4)/((I[29,37]:
δ2.00~2.10/3)·100 (1)
The MoD (%) of theMoD filler(%) = (Iδ1.65~1.70
materials was /4)/(I
limitedδ2.00~2.10
to 3.5 /3) ·100 the concentration of
because (1)
the added BDDE was 3.5 mol%.
The
As MoDin
shown (%) of the7filler
Figure materials
and Table S18,was limited
the MoD of to
the3.5 because the concentration
manufactured of the
products through
added
the BDDE was
dispersion 3.5 mol%.
process was 3.43, and the product produced with no dispersion process
As shown
was 3.16. in Figure
The reason 7 and
for these TableisS18,
results thethe
factMoD of the
that the manufactured
crosslinking ratio products through
was higher than
in the previous product. These results therefore show that the stability of the fillers was
the dispersion process was 3.43, and the product produced with no dispersion process in-
3.16. The reason for these results is the fact that the crosslinking ratio was higher than in
creased.
the previous product. These results therefore show that the stability of the fillers increased.

Figure 7. (a) The molecular structure of the BDDE-crosslinked HA dimer; (b) 1H NMR of the product
being produced (no dispersion); (c) 1H NMR of the product made through a new process (18 ◦ C and
24 h dispersion); and (d) graph detailing the MoD (%) of (b,c).

By combining the above results with this finding, the material produced through the
dispersion process in this experiment showed improved quality as a filler.

3.3. Experimental Results of Products Manufactured in the Lab (Batch C)

3.3.1. Viscoelasticity and Cohesion
Based on the above results, the dispersion process was introduced to the production
process (batch C). In Figure 8a–c and Table S19, complex viscosity and G′ increased by
 dispersion process in this experiment showed improved quality as a filler.

3.3. Experimental Results of Products Manufactured in the Lab (Batch C)

3.3.1. Viscoelasticity and Cohesion
Polymers 2024, 16, 3323 10 of 13
Based on the above results, the dispersion process was introduced to the production
process (batch C). In Figure 8a–c and Table S19, complex viscosity and G′ increased by 1.4
times and G″
1.4 times and increased by 1.7 times
G′′ increased by 1.7when
timescomparing the products
when comparing manufactured
the products through
manufactured
dispersion and the non-dispersion products. In Figure 8d,e and Table S20, G*
through dispersion and the non-dispersion products. In Figure 8d,e and Table S20, G* increased
by 1.4 timesbyand
increased 1.4tack increased
times and tackby 1.3 times
increased byin1.3
comparison.
times in comparison.

Figure 8. Comparison between the old and new process at the factory scale: (a) complex viscosity
(cP), (b) storage modulus (G′ ), (c) loss modulus (G′′ ), (d) complex modulus (G*), and (e) tack.

Despite the changes in the crosslinking temperature of the BDDE and the purified
solution during the purification process, the increases in the overall viscoelasticity and
cohesion were greater than those observed in the previous process.

3.3.2. Degree of Modification (MoD)

As shown in Figure 9 and Table S21, the MoD of products manufactured using the
new process increased more than in those manufactured using the old process. That is, the
quality of the material produced through new process was better than that of the material
made through the old process.
 3.3.2. Degree of Modification (MoD)
As shown in Figure 9 and Table S21, the MoD of products manufactured using the
new process increased more than in those manufactured using the old process. That is,
Polymers 2024, 16, 3323
the quality of the material produced through new process was better than that of the ma-
11 of 13
terial made through the old process.

Figure 9. (a)
Figure Graph
9. (a) Graph showing
showing thethe
1H1HNMR NMR of products made
of products made (with nono
(with dispersion) at the
dispersion) factory
at the scale;
factory scale;
(b)(b)
1H1HNMRNMR of of
thethe
products
products made
made through
through thethe
new process
new process °C◦and
(18(18 2424
C and h dispersion) at the
h dispersion) factory
at the factory
scale; and
scale; (c)(c)
and graph
graphof of
thethe
MoD
MoD (%) of of
(%) (a,b).
(a,b).

4. 4. Conclusions
Conclusions
InIn the
the presentstudy,
present study,a anew
newandandsimple
simpleprocess
processwas wasdeveloped
developedand and implemented
implemented in in a
a manufacturing process, even reaching the production stage. Using the
even reaching the production stage. Using the dispersion pro- dispersion process,
three
cess, main
three mainachievements
achievements were accomplished:
were accomplished:
First,
First, thethe process
process reduced
reduced the
the burden
burden placed
placed onon workers
workers inin relation
relation totoproduction
production
andand minimized
minimized human
human errors.
errors. AsAs a result,
a result, workers
workers nono longer
longer needed
needed toto mix
mix thethe materials
materials
directly, to eliminate heterogeneous parts during the mixing
directly, to eliminate heterogeneous parts during the mixing process of HA and the process of HA and the base
base
solution. It also helped to prevent human errors that could occur as
solution. It also helped to prevent human errors that could occur as a result of said mixing.a result of said mixing.
Second,
Second, physical
physical properties
properties such
such as as
thethe viscoelasticity
viscoelasticity and and cohesion
cohesion of of
thethe filler
filler im-im-
proved compared to existing products by introducing our simple
proved compared to existing products by introducing our simple process. As mentioned process. As mentioned
inin the
the results
results above,
above, the
the dispersion
dispersion process
process waswas confirmed
confirmed toto increase
increase the
the viscoelasticity
viscoelasticity
andand cohesiveness
cohesiveness ofof
thethe filler
filler materials,
materials, andand this
this improvement
improvement waswas maintained
maintained under
under allall
production conditions.
production conditions.
Lastly,
Lastly, thethe crosslinking
crosslinking ratio
ratio ofof the
the product
product increased.
increased. AsAsthethe reaction
reaction surface
surface area
area ofof
HA increased through the dispersion process, it became easier for collision reactions toto
HA increased through the dispersion process, it became easier for collision reactions
take place, which was confirmed by MoD (%). This finding demonstrated that the overall
quality-related data improved, including enhancements in viscoelasticity and cohesion.
From a product-regulatory perspective, there was a limitation in not being able to
change the product’s HA concentration in NaOH solution and the amount of crosslinking
agent, so a clearer interpretation of the new process was lacking. However, the process
of homogeneously dissolving a high concentration of HA could improve the crosslinking
efficiency and physical properties of the final product.
Going forward, a method or process to improve the quality of the final products will
be developed, and its applicability to other areas will be explored.

5. Patent
The patent application number is 10-2024-0120713 (Republic of Korea).
Polymers 2024, 16, 3323 12 of 13

Supplementary Materials: The following supporting information can be downloaded at https://www.

mdpi.com/article/10.3390/polym16233323/s1, Tables S1–S15: Complex viscosity, storage modulus
(G′ ), loss modulus (G′′ ), complex modulus (G*), and tack of the filler material using the dispersion
process over time at 4 ◦ C, 10 ◦ C, and 18 ◦ C (n = 3) at the lab scale; Tables S16 and S17: Complex
viscosity, storage modulus (G′ ), loss modulus (G′′ ), complex modulus (G*), and tack of previous and
new processes at the 1/2 factory scale (n = 3); Table S18: MoD (%) of previous and new processes at
the 1/2 factory scale; Tables S19 and S20: Complex viscosity, storage modulus (G′ ), loss modulus
(G′′ ), complex modulus (G*), and tack of previous (n = 1) and new (n = 3) processes at the factory
scale; and Table S21: MoD (%) of previous and new processes at the factory scale.
Author Contributions: Conceptualization, S.C. and J.C.C.; compound synthesis and analysis, S.C.;
writing—original draft preparation, S.C.; writing—review and editing, S.C., J.C.C., S.L. and S.J.L.;
and supervision, J.C.C. and S.J.L. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported by the Technology Innovation Program (RS-2024-00438191,
Development of technology to commercialize non-animal derived cell growth enhancing recombinant
protein materials at GMP level for biopharmacetical production) funded By the Ministry of Trade,
Industry & Energy (MOTIE, Republic of Korea).
Institutional Review Board Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article/Supplementary Materials, further inquiries can be directed to the corresponding author.
Conflicts of Interest: Authors Sunglim Choi, Jin Cheol Cho, Seunghwa Lee and Seong Jin Lee were
employed by the company CHA Meditech Co., Ltd. The authors declare no conflict of interest.

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