Preparation and Properties of Chitosan-Based Thermosensitive Hydrogel and Its Positive Influence on the Stability in Dental Implant System as Sealant and Lubricant

242
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/21/4304-0242/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
OBJECTIVE: In order to reduce complications accom
panied with dental implant restoration, this study strives
to prepare a novel sealant and lubricant that can be
used in dental implant systems as well as to evaluate its
characteristics.
STUDY DESIGN: Chitosan (CS), β-glycerophosphate
pentahydrate (β-GP), and nano silver (nAg) were used
to prepare thermosensitive hydrogel. According to the
different volume ratios of CS to β-GP, 3 experimental
groups were established, namely 16/4, 13/7, and 10/10
groups. Their morphology, composition, and chemical
properties were analyzed via SEM, EDS, and FTIR.
In addition, the effect of the hydrogel on the stability of
dental implant-abutment connection was investigated
by removal torque test combined with dynamic cyclic
loading experiment. The maximum fracture load was
measured under different lubricating conditions by elec
tronic universal testing machine. The cytotoxicity and in
vitro antibacterial effect of the hydrogel were examined
respectively by CCK-8 test and the spread plate method.
RESULTS: The CS/β-GP/nAg thermosensitive hydro-
gel was successfully prepared in this study, which was
found to be a porous structure through SEM. The re-
moval torque test and the dynamic cyclic loading ex-
periment showed that the removal torque of the experi
mental group was greater than that of the control group.
Preparation and Properties of Chitosan-Based
Thermosensitive Hydrogel and Its Positive
Influence on the Stability in Dental Implant
System as Sealant and Lubricant
Chunqing Ye, M.M., Xinjie Cai, M.D., Fushi Wang, M.D., Yi Zhou, M.D.,
Tao Jiang, M.D., and Yining Wang, M.D.
From The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral Biomedicine
Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province; and the Department of Pros
thodontics, Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, China.
Chunqing Ye is Student, State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral
Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University.
Xinjie Cai is Physician, State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral
Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, and Department of Prosthodontics, Hos-
pital of Stomatology, Wuhan University.
Fushi Wang is Physician, State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of
Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, and Department of Pros
thodontics,
Hospital of Stomatology, Wuhan University.
Yi Zhou is Associate Professor, State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory
of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, and Department of Prosthodontics,
Hospital of Stomatology, Wuhan University.
Tao Jiang is Professor, Department of Prosthodontics, Hospital of Stomatology, Wuhan University.
Yining Wang is Professor, Department of Prosthodontics, Hospital of Stomatology, Wuhan University.
Chunqing Ye and Xinjie Cai contributed equally.
Address correspondence to: Tao Jiang, M.D., and Yining Wang, M.D., Department of Prosthodontics, Hospital of Stomatology, Wuhan
University, Luoyu Road 237, Hongshan District, Wuhan 430079, Hubei Province, China (jiangtao2006@whu.edu.cn and wang.yn@
whu.edu.cn).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

Volume 43, Number 4/August 2021 243
Chitosan-Based Thermosensitive Hydrogel
Furthermore, the single load-to-fracture test indicated
that the 16/4 group had the greatest maximum bear
ing load. The in vitro cytotoxicity test using rat bone
marrow stromal cells (rBMSCs) and human gingival
fibroblast cells (hGFCs) showed no cytotoxicity in all 3
groups. The 3 experimental groups had obvious antibac-
terial effects against E. coli, S. aureus, and P. gingivalis.
CONCLUSION: A nontoxic antibacterial CS/β-GP/nAg
thermosensitive hydrogel for lubricating purpose was
successfully fabricated. When the volume ratio of CS
to β-GP was 16/4, this thermosensitive hydrogel dem
onstrated better sealing and lubricating abilities and
had a positive influence on the reliability of dental
implant-abutment connection. (Anal Quant Cytopa
thol Histpathol 2021;43:242–254)
Keywords: abutment, dental implant, dental im-
plant restoration, dental sealant, lubrication, ther-
mosensitive hydrogel.
Based on the “osseointegration” theory, dental im-
plant has already become one of the routine ther-
apy options for teeth rehabilitation after 50 years’
development. More importantly, dental implant
has been widely used in clinical practice because
of its advantages, namely strong stability, high
masticatory efficiency, and excellent biocompati-
bility.1-3 However, some mechanical or biological
complications are inevitable in clinical practice
which can even lead to implant failure. The mech
anical complications mainly include screw loos-
ening or fracture occurring in the implant and
abutment4; biological complications mainly include
mucositis, peri-implantitis, and marginal bone re-
sorption.5,6 Screw loosening is one of the most
common mechanical complications in clinical prac-
tice.7-9 The main causes include improper appli-
cation of force, improper occlusal force, improper
screw diameter, stress deformation, and screw cor-
rosion due to microleakage of salivary bacteria, all
of which result in insufficient pre-tightening force
between the implant and abutment.10 In this re-
gard, on the one hand, liquid or solid lubricants
can be used to reduce friction between the implant
and the abutment to improve the pre-tightening
force and stability of the abutment.11,12 On the
other hand, low friction is not conducive to the
stability of the abutment. Therefore, it is particu-
larly important to select materials with variable
friction in implant. In addition, there is a micro-
gap between the implant and the abutment, into
which bacteria can easily penetrate for multipli-
cation, forming biofilms that are difficult to re-
move.13-15 At present, the commonly used block-
ing materials in clinical and experimental practice
mainly include Gapseal blocking gel (Hager &
Werken GmbH & Co. KG, Duisburg, Germany),
O-ring sealing ring, and Chlorhexidine gel.16 How-
ever, since these materials have poor antibacterial
ability and persistence, they cannot completely pre-
vent bacteria from entering the micro-gap. There-
fore, it is urgent to find a blocking material with
long duration and strong antibacterial ability to
solve this problem.
With favorable biocompatibility and phase tran
sition characteristics, the materials of thermosen-
sitive hydrogel present a liquid state at low tem-
peratures and can transform into a gel at 37°C.17,18
Furthermore, the characteristic of thermosensitiv-
ity can also lead to changes in its physical prop-
erties, such as variation in the friction coefficient
that accompanies the liquid-gel phase transition.19
Therefore, thermosensitive hydrogel can be ap-
plied in the implant system to improve abutment
stability. With excellent biodegradability and bio-
compatibility, chitosan (CS) is widely used in bio-
medical and cosmetic fields.20 β-glycerophosphate
pentahydrate (β-GP) is able to catalyze the phase
transition of CS solution at physiological tempera-
ture.21 In addition, thermosensitive hydrogel can
be loaded with antibacterial agents. The common-
ly used antibacterial substance povidone-iodine
(PVP-I) can be efficiently disinfected and sterilized
with low toxicity.22 However, PVP-I offers a too-
short efficacy on the wound mucosa to maintain
durable antimicrobial property. As a physical ster-
ilization, nano silver (nAg) antibacterial agent has
broad-spectrum antibacterial ability to antagonize
gram-negative bacteria, fungi, and even viruses,
etc. Meanwhile, nAg has shown the advantages—
low cytotoxicity, strong stability, low effective con-
centration, less possibility to cause drug resistance,
and so on. Additionally, it also possess
es a certain
repair capacity for the injured surface. In recent
years, nAg particles have been widely applied in
the biomedical field, for example, nAg dressings,
nAg gels, nAg toothpastes, and implant surfaces
like catheters and artificial blood vessels.23 There-
fore, we prepared a CS/β-GP/nAg thermosen-
sitive hydrogel in this study and evaluated its
feasibility as a sealant and lubricant between the
implant and the abutment.
In the present study, a CS/β-GP/nAg thermo-
sensitive hydrogel was prepared to investigate its

244 Analytical and Quantitative Cytopathology and Histopathology®
Ye et al
degradation performance, antibacterial properties,
and cytotoxicity. Afterwards, dynamic cyclic load-
ing experiment was performed to simulate masti-
catory forces between the implant and abutment.
Moreover, a single load-to-fracture test was added
to provide a chewing environment that is more
consistent to the real situation, thereby to record
the peak flexural strength between the implant and
abutment.
Materials and Methods
Materials
Experimental materials such as CS (molecular
weight=179.17 kDa; deacetylation degree ≥95%)
and β-GP (molecular weight=306.11 gmol−1) were
purchased from Shanghai Aladdin Co, Ltd. As
for the implants and abutments in the current
study (Figure 1), they were customized with
grade-2 commercially pure titanium (BAOTi;
Shanxi, China). In particular, the loading crowns
were in accordance with the experimental stan-
dard ISO/FDIS14801: 2014(E), with a hemisphere-
shaped part on the top to absorb load. Moreover,
other chemical reagents with analytical grade were
utilized.
Preparation of CS/β-GP/nAg Thermosensitive
Hydrogel
First of all, chitosan (CS) powder was dissolved
in acetic acid (0.18 M), while β-GP powder was
soluble in tri-distilled water. According to the Lee-
Meisel method,24 nAg was then obtained by reduc-
ing silver nitrate (1.7 mL of 1% AgNO3) with tri
sodium citrate (2 mL of 1% Na3C6H5O7) in 100 mL
water under heating at about 90°C. The obtained
nAg colloid was identified by ultraviolet-visible
absorption spectroscopy (an absorption peak at
about 400 nm). Then, nAg colloid was mixed with
CS solution under stirring to obtain a homogene-
ous CS/nAg solution. After that, the β-GP solution
and the CS/nAg solution were cooled in an ice
bath for 15 minutes. The β-GP solution was then
dropped into the CS/nAg solution and stirred for
15 minutes in an ice bath. Finally, they were kept
still on ice to eliminate bubbles.
Experimental Groups
According to the different volume ratios of CS
solution to β-GP solution, this experimental study
was divided into three groups: 16/4 group, 13/7
group, and 10/10 group (Table I), and bare im-
plants without any interference served as a control
group.
Thermosensitive Property
A simple tube inversion method25,26 was utilized
to investigate the thermosensitive property. Gener-
ally speaking, the CS/β-GP/nAg solution was
added to an Eppendorf tube which was placed in
a 37°C water bath. After that, the tube was taken
out every 10 seconds to observe the fluidity of
the solution. The gelation time was defined as no
change in gel state when the tube was inverted for
30 seconds.
Figure 1 The component parts of implant assembly used in this study. (A) The implant. (B) The abutment. (C) The single loading crown.
(D) The assembled implant abutment. (E) Each specimen was restored with an identical loading crown using standard dental cement for
the abutment.

In Vitro Degradation Test
The hydrogel (0.5 mL gel) was immersed in ster-
ilized PBS containing lysozyme (1.5 μg/mL) at
37°C. The PBS-lysozyme solution was changed
daily to maintain the activity of the enzyme. After
immersion for 1, 4, 7, 14, 21, and 28 days, these
samples were taken out from the solution, rinsed
with deionized water, and then dried under vac-
uum.27 The degradation degree was calculated as
follows:
D= (W0–Wt)/W0,
where W0 is the weight of dried gel before im-
mersion, and Wt is the weight of dried gel at the
specific time of sample collection.
Surface Morphology and Chemical Properties of
CS/β-GP/nAg Thermosensitive Hydrogel
After being converted to the gel state, 10 mL
hydrogel was frozen overnight at −80°C. Then, it
was lyophilized at −6°C for 48 hours in a lyophil-
izer (OHRIST BETA 1-15, Germany). The surface
morphology of the CS/β-GP/nAg hydrogel was
then observed under SEM (FEI, Eindhoven, The
Netherlands). Subsequently, qualitative elemental
analysis was performed with a Quanta-200 EDS
(FEI, Hillsboro, Oregon, USA). In addition, the
dried samples were ground and then mixed with
KBr for ATR-FTIR spectrometry (Thermo Nicolet
5700; Thermo Fisher Scientific, Waltham, Massa-
chusetts, USA).
Implant-Abutment Connection Stability
With bare implants as the control group, three
experimental groups of hydrogels were added to
the corresponding implants (10 pairs of implants
and abutments in each group). The abutment was
then tightened to 32 Ncm using a calibrated digi-
tal torque meter. Afterwards, both the assembled
implants and abutments were immersed into ice
in order to maintain the liquid gel. The abutment
was again tightened to 32 Ncm after 5 minutes
due to stress relaxation and standard record.28
Subsequently, all samples were placed at 37°C for
5 minutes to obtain a liquid-to-gel transformation.
In addition, the maximum removal torque was
recorded with a calibrated digital torque meter.
Finally, Gapseal gel and Vaseline were used in
the micro-gap between implant and abutment to
compare with this thermosensitive hydrogel in this
removal torque test.
Since the materials applied in the micro-gap be-
tween implant and abutment might be affected by
the gingival crevicular fluid which existed in the
micro-gap, the assembled implants and abutments
were immersed in PBS solution at 37°C for 14 days
in order to record the removal torque of abutments.
Eighteen new implant specimens embedded in
a customized metal fixture were randomly divid-
ed into 6 groups: 16/4 group, 13/7 group, 10/10
group, control group, Gapseal group, and Vaseline
group according to different lubricating condi-
tions (n=3). Firstly, abutments from each group
were tightened into implants to 32 Ncm with a
calibrated digital torque meter. Subsequently, the
assembled implants and abutments from the 16/4
group, 13/7 group, and 10/10 group were placed
in ice to keep the hydrogel in a liquid state. Next,
the abutment was again tightened to 32 Ncm
after 5 minutes because of preload loss. Then, the
three experimental groups were placed at 37°C for
5 minutes to ensure that the hydrogel was con
verted into gel. To simulate clinical bone loss, all
implants from the six groups were vertically em-
bedded in fixture at a distance of 3.0 mm±0.5 mm
apically from the top platform. Beyond that, each
specimen was restored by an identical loading
Table I Experimental Groups
Vcs: Vcs Vβ-GP VnAg Vsum
Group Vβ-GP (mL) (mL) (mL) (mL)
16/4 16:4 0.72 0.18 0.1 1
13/7 13:7 0.585 0.315 0.1 1
10/10 10:10 0.45 0.45 0.1 1
Control — 0 0 0 0
Figure 2 In vitro degradation results of hydrogel solutions from
different groups.

Ye et al
crown with standard dental cement for the abut-
ment. The implant specimen embedded in fixture
was obliquely fixed in the specimen holder. The
loading device would set load on the loading
member. The dynamic cyclic loading experiment
was conducted according to the guidelines of
ISO/FDIS 14801:2014(E), followed by a 30° off-
axis loading in a fatigue testing machine (Equip-
ment number: SL-EP-0064, model specification:
UGH6000. 29.4 N~294 N of 30° off-axis fatigue
force was applied at 15 Hz over the loading
crown. In this test 600,000 cycles for every speci-
men was performed to simulate the implant func-
tion within 6 months.29 At the end of the dynam-
ic cyclic loading experiment, the peak removal
torque was determined as previously described.
Single Load-to-Fracture (SLF) Test
Hydrogel from the 16/4 group and Gapseal gel
from Gapseal group were added to the corre-
sponding implants, while bare implants were
serving as control group (4 pairs of implants and
abutments per group). The abutment was then
tightened to 32 Ncm using a calibrated digital
Figure 3 Energy spectrum diagrams of hydrogel from the 16/4 group (A), the 13/7 group (B), and the 10/10 group (C).
Figure 4
SEM images of hydrogel from
groups 16/4 (A and D), 13/7 (B
and E), and 10/10 (C and F).

torque meter. Afterwards, the assembled implants
and abutments of the 16/4 group were immersed
into ice in order to maintain the liquid gel. The
abutment was again tightened to 32 Ncm after 5
minutes due to stress relaxation and standard rec-
ord. Subsequently, samples of the 16/4 group were
placed at 37°C for 5 minutes to obtain a liquid-to-
gel transformation. Finally, the maximum remov-
al torque was recorded with a calibrated digital
torque meter.
All the implants of three groups were vertically
embedded in fixture at a distance of 3.0±0.5 mm
apically from the top platform of the fixture sur-
face. The SLF test was conducted according to
the guidelines of ISO/FDIS 14801:2014(E), fol-
lowed by a 30° off-axis loading in an electronic
universal testing machine (Equipment number:
SL-EP-0021, model specification: UTM4304). Dur-
ing the experiment, a flat tungsten carbide indent
er set the load on the loading crown at a cross-
head speed of 1 mm/min. The loading value kept
increasing until the specimen fractured. Conse-
quently, the maximum loading value was defined
as the maximum fracture load.30
Antimicrobial Properties of CS/β-GP/nAg
Thermosensitive Hydrogel
E. coli (ATCC 25922), S. aureus (ATCC 25923),
and P. gingivalis (ATCC 33277) were selected to
assess the antibacterial ability of CS/β-GP/nAg
thermosensitive hydrogel. E. coli was cultured
with Luria-Bertani (LB) medium. S. aureus was
cultured with Tryptic Soy Broth (TSB) medium.
After incubation for 12 hours at 37°C, the bacterial
concentration was adjusted to 104–105 CFU mL−1.
After that, 1 mL of hydrogel with different volume
ratios was then immersed in the bacterial suspen-
sions (1 mL) and cultured for 4 hours at 37°C with
constant shaking at 200 rpm/min in a constant
shaking incubator. Subsequently, the bacterial sus-
pensions were diluted and spread on plates, and
the CFU were counted.
In addition, P. gingivalis was cultured anaerobi-
cally with TSB medium containing hemin, vitamin
K, and yeast. After being cultured for 36 hours at
37°C, the bacterial concentration was adjusted to
104–105 CFU mL−1. Then the bacterial suspension
was added to Eppendorf tubes with 1 mL of new
ly prepared gel from different groups and jointly
cultured for 24 hours at 37°C under an anaerobic
environment. Lastly, the bacterial suspension was
diluted and spread on the plate to count CFU.
Cytotoxicity of CS/β-GP/nAg Thermosensitive
Hydrogel
The experiment was approved by the Animal Care
and Experiment Committee of Wuhan University.
After alleviating the pain of 6-week-old rats, rat
bone marrow stromal cells (rBMSCs) were ex-
tracted and flushed. The cells were then cultured
in alpha minimum essential medium (α-MEM;
SH30265; HyClone, Logan, Utah, USA) contain-
ing fetal bovine serum (SH30068; HyClone) and
penicillin-streptomycin (SV30010; HyClone). Spe-
cifically, α-MEM was put in a humidified incu-
bator with 5% CO2 at 37°C. When the confluence
reached about 80%, cells were subcultured at a
ratio of 1:3 and cells at passages 3–5 were used
for experiment. But beyond that, human gingival
fibroblast cells (hGFCs) were collected from the
residual gingiva of wisdom teeth which came from
clinical patients. All patients voluntarily signed
the informed consent before receiving the wisdom
tooth extraction surgery. The gingival tissue was
cut into 1 mm diameter pieces which were joint-
ly cultured with fetal bovine serum and penicillin-
streptomycin in a low glucose Dulbecco’s-modified
Eagle’s medium (SH30021; HyClone). Similarly,
this medium was placed in a humidified incubator
with 5% CO2 at 37°C. Then, the cells were sub
cultured at a ratio of 1:2 when their confluence
reached approximately 80%. Cells at passages 3–5
were used for the experiment.
The cytotoxicity of the CS/β-GP/nAg thermo-
Figure 5 FTIR images of CS, β-GP, and nAg in the 16/4 group,
13/7 group, and 10/10 group.

Ye et al
sensitive hydrogel for rBMSCs and hGFCs was
evaluated using an in vitro CCK-8 assay (Dojindo,
Japan). First, the newly prepared gel (10 μL) was
immersed into cell culture medium to prepare
sample extracts. After cell culture medium (5×
104/mL) was seeded in a 24-well plate for one
day, the medium was replaced by the extract.
Subsequently, being cultured in the extracts for 1,
4, and 7 days, the medium was again replaced by
300 μL cell culture medium containing 10% CCK-
8. After one-hour incubation at 37°C, the superna-
tant was transferred to a 96-well plate and mea-
sured at 450 nm using an ELX808 ultra microplate
reader (BioTek Instruments, Inc., Winooski, Ver-
mont, USA).
Statistical Analysis
Experimental data were expressed as mean±stan-
dard deviation (SD). The results were analyzed by
one-way ANOVA with SPSS 16.0 together with
Tukey’s test. Results were considered significant at
p<0.05.
Results
Physical Properties of CS/β-GP/nAg Thermosensitive
Hydrogel
The newly prepared CS/β-GP/nAg solution was
a viscous, pale yellow liquid that transformed into
a gel state at 37°C. The results suggested that the
16/4 group had the longest gelation time (260±
10 s), while the 10/10 group had the lowest gela-
tion time (110±5 s). The gelation time of the 13/7
group was 135±10 s.
The in vitro degradation results of hydrogel
solutions with different volume ratios (Figure 2)
showed that the 16/4 group had the slowest de-
gradation rate, followed by the 13/7 group, while
the 10/10 group had the fastest degradation rate.
In addition, the degradation degrees of samples
from groups 16/4, 13/7, and 10/10 within 28 days
were 7.581%, 12.550%, and 15.950%, respectively.
The composition and surface morphology of
hydrogel from different groups were further ob
served by EDS and SEM (Figure 3). With the vol-
ume ratio of CS to β-GP increasing, the propor-
tion of elements C provided by CS gradually
decreased. On the contrary, the proportion of ele-
ments P provided by β-GP relatively increased. Ad-
ditionally, the mass ratio of Ag atoms did not
change evidently. Based on these results, it could
be concluded that although the volume ratio of
CS to β-GP was different, the composition of the
hydrogel was unchanged. However, owing to dif-
ferent elemental mass ratio, hydrogel from experi-
mental groups demonstrated various surface mor-
phology.
In order to investigate the morphological differ-
ences caused by different ratios, the structure of
hydrogel was observed via SEM. The images (Fig-
ure 4) showed that the hydrogel formed a porous
structure due to the cross-linking of CS chains.
When the volume ratio of CS to β-GP decreased,
the pore sizes of the macropores in all three groups
became larger, which were 32.22±15.25 µm (16/4
group), 55.11±21.62 µm (13/7), and 66.67±27.44 µm
(10/10 group), respectively.
Figure 6 Removal torque test of abutment, results of dynamic cyclic loading experiment, and results of single load-to-fracture test.
(A) Removal torque test of abutment (without immersion versus after immersion). (B) Results of dynamic cyclic loading experiment.
(C) Results of single load-to-fracture test. *p<0.05, **p<0.01, ***p<0.001.

Chemical Properties of CS/β-GP/nAg
The chemical properties of hydrogel solutions
with different ratios were investigated by infrared
spectrometer. The results (Figure 5) showed that
the spectrum of single CS demonstrated the N-H
bond bending resonance peak of II-NH2 at 1598.7
cm−1 together with the C-N bond stretching vibra-
tion peak at 1382.7 cm−1. The spectral peaks of sin
gle β-GP at 1060 cm−1 and 974 cm−1 revealed the
stretching vibration of the phosphate group. Since
the metal had no absorption band in the infra-
red spectrum, the infrared transmittance of single
nAg was relatively high.
These characteristic peaks of CS, β-GP, and nAg
could be found when they were mixed together to
prepare CS/β-GP/nAg hydrogel. The N-H bond
bending resonance shift of II-NH2 in CS was 1566.3
cm−1 and the C-N stretching vibration shift was
1431.2 cm−1, which indicated that OH− and PO42−
in β-GP interacted with NH3+ in CS as well as Ag
in nano silver.
Implant-Abutment Connection Stability
The removal torque test could indirectly reflect the
abutment stability. The abutment was more stable
with greater removal torque.31 According to the
test result (Figure 6A), regardless of whether the
test was performed immediately or after immer-
sion for 14 days, the removal torque of abutments
in the 16/4 group (28.06±1.26 Ncm without im-
mersion and 28.74±2.89 Ncm after immersion)
and the 13/7 group (28.23±2.61 Ncm without im-
mersion and 28.18±2.60 Ncm after immersion)
were significantly greater than those in the con
trol group (24.9±2.38 Ncm without immersion
and 24.78±2.51 Ncm after immersion), the Gapseal
group (24.66±2.21 Ncm without immersion and
Figure 7 Antimicrobial test results for CS/β-GP/nAg hydrogel. (A) Representative bacterial culture plates. (B) Colony-forming number of
E. coli. (C) Colony-forming number of S. aureus. (D) Colony-forming number of P. gingivalis. ***p<0.001.

Ye et al
carried out via spread plate method. The results
(Figure 7) showed that, as compared with the con-
trol group, the number of viable bacteria in the three
experimental groups was significantly reduced.
Cytotoxicity Assay Analysis. In order to test the
toxicity of hydrogels to cells, rBMSCs and hGFCs
were cultured with hydrogel extracts. The results
(Figure 8) indicated that rBMSC (Figure 8A) pro-
liferation ability of the 16/4 and 10/10 groups was
significantly enhanced. The rBMSC proliferation
ability in the 13/7 group had no obvious difference
over time as compared with the control group. As
for hGFCs (Figure 8B), no significant difference
could be found in cell proliferation among the three
groups on the first day. However, at day 4 and day
7, the proliferation ability of the cells in all three
groups was significantly increased, and the 16/4
group had the strongest proliferation ability. These
results indicated that CS/β-GP/nAg hydrogel
could significantly promote the growth of rBMSCs
and hGFCs, and the hydrogel from the 16/4 group
had the strongest ability to promote cell growth.
Discussion
In this study, a CS/β-GP/nAg thermosensitive
hydrogel was successfully fabricated which was
applied in a dental implant system to address the
biological and mechanical complications in-clinic.
The degradation degrees of the three experi
mental groups within 28 days were 7.581% in the
16/4 group, 12.550% in the 13/7group, and 15.950%
24.47±1.65 Ncm after immersion), and the Vaseline
group (24.95±2.56 Ncm without immersion and
24.52±2.74 Ncm after immersion), indicating that
the stability of the abutment in the 16/4 group and
the 13/7 group was better than that in the control
group, Gapseal group, and Vaseline group.
After 600,000 fatigue cycles, the results (Figure
6B) suggested that there was 27.53±0.93 N remov-
al torque in the 16/4 group, 26.1±0.6557 N in the
13/7 group, 25.67±1.274 N in the 10/10 group,
14.93±2.48 N in the Gapseal group, 13.23±3.05 N
in the Vaseline group, while the removal torque of
the control group was 18.3±1.75 N. Thus, it could
be said that the 16/4 group had the highest remov
al torque as well as the best stability, both of which
were more in line with clinical needs.
Single Load-to-Fracture Test (SLF)
As shown in Figure 6C, the 16/4 group had the
greatest maximum bearing load (1262±40.28 N),
which was significantly higher than that of the
control group (1076±45.31 N). On the contrary, the
Gapseal group had the smallest maximum bearing
load (1173±31.1 N), indicating that thermosensitive
hydrogel could increase the mechanical strength
between implant and abutment, which was more
suitable for clinical application.
Study on Biological Characteristics of CS/β-GP/nAg
Antimicrobial Properties. The quantitative determi-
nation on E. coli, S. aureus, and P. gingivalis was
Figure 8 Cytotoxicity test of CS/β-GP/nAg hydrogel on rBMSCs and hGFCs. (A) Rat bone marrow stromal cells. (B) Human gingival
fibroblast cells. *p<0.05, **p<0.01, and ***p<0.001.

the corresponding antibacterial ability would be
slowly weakened. In conclusion, the 16/4 group
had the best antibacterial effect with the lowest
β-GP content, while the 10/10 group had the worst
antibacterial effect with the highest β-GP content.
The implant-abutment connection stability
is closely related to the friction force and pre-
tightening force. Currently, most studies aim to
increase pre-tightening force as well as to reduce
the friction between the abutment and the im-
plant by using a liquid lubricant or a solid lubricant
coating.47-49 However, the influence of abutment
stability on the implant system is ignored. In this
study, removal torque test was utilized to indi-
rectly reflect the stability of the abutment. It was
found that whether the hydrogel was tested im-
mediately or after immersion for 14 days, the
removal torque of the three experimental groups
were significantly greater than those of the control
group, which indicated that the abutments of the
three experimental groups were more stable than
those of the control group. Moreover, the remov-
al torques of the Gapseal and Vaseline groups
were not significantly improved as compared with
the control group, suggesting that the removal
torques of the Gapseal and Vaseline groups were
less than those of the three experimental groups.
These results indicated that CS/β-GP/nAg ther-
mosensitive hydrogel could significantly improve
the stability of the abutment, rather than Gapseal
and Vaseline. This hydrogel was also used as a
lubricant due to its phase transition characteris-
tics. Accompanied by phase transition, friction co-
efficient would be low in a liquid state and high in
a gel state. Therefore, the hydrogel in the present
experiment remained liquid when added to the
implant and converted into gel before tighten-
ing the abutment. Under this condition, the fric
tion coefficient would be relatively low, and the
pre-tightening force would be enhanced. However,
when it was converted to the gel state, the friction
coefficient increased correspondingly. As a result,
the variation of friction coefficient provided a
large pre-tightening force and less friction. In this
experiment, the 16/4 group and the 13/7 group
had stronger removal torque than did the control
group. In other words, the friction force between
implant and abutment would not be changed;
thus, their removal torque was not significantly
different from that of the control group. More-
over, whether the hydrogel was tested immedi-
ately or after immersion for 14 days, there was
in the 10/10 group, which were lower than those
in the previous study.32 As CS concentration be
came larger, cross-linking degree of the hydrogel
might be increased. Consequently, the degrada-
tion rate was relatively reduced.33 Therefore, the
16/4 group had the highest CS concentration and
cross-linking degree, so its degradation degree
was the lowest. The CS/β-GP/nAg thermosensi-
tive hydrogel presented a porous structure under
SEM that contributed to the release of nAg from
the hydrogel. Moreover, the released nAg could
have a local antibacterial effect.34 Because silver
is more toxic to prokaryotic cells than eukaryotic
cells, silver has excellent inhibition of microbial
growth and proliferation and low cytotoxicity to
organism cells. The nano-scale silver particles have
high specific surface area, high contact probabil-
ity with bacteria, and strong chemical activity.
The atoms on their surface are easy to bond with
other chemical groups, and the antibacterial effect
is much stronger than that of micron silver parti-
cles. Therefore, nAg particles are widely used in
biomedical fields, such as nAg dressing, nAg gel,
nAg toothpaste, and internal plant surfaces such
as catheters, artificial blood vessels, and so on.35-38
Stevens et al39 showed that nAg particle coating on
medical catheters has a good bacteriostatic effect
and can accelerate blood coagulation. Tiecheng et
al40 showed that ceramic glazed tiles containing
silver antibacterial agents have a good antibacterial
effect and antibacterial persistence. A new type of
coating was prepared by loading a silver antimi-
crobial agent into calcium phosphate,41 which has
excellent antibacterial properties. The antibacterial
ability of polyphthalamide-silver composites was
studied by Kumar et al.42 The experimental results
showed that polyphthalamide-silver composites
had good bactericidal effect. The Ag-SiO2 films pre-
pared by Baheiraei et al43 by sol-gel method have
good antibacterial properties against Escherichia
coli and Staphylococcus aureus.
The further antibacterial experiments showed
that all three experimental groups had signifi-
cant antibacterial effects against E. coli, S. aureus,
and P. gingivalis, which coincided with previous
reports.44-46 Meanwhile, it could be found that
with the increase of β-GP content, the antibacte-
rial ability of thermosensitive hydrogel was grad-
ually weakened. The possible explanation was that
alkaline β-GP reacted with nAg and eventually gen-
erated silver hydroxide, which would be further
decomposed into silver oxide and water. Therefore,

Ye et al
16/4, the degradation degree of the thermosensi-
tive hydrogel was the lowest, which could greatly
improve the stability of dental implant-abutment
connection and masticatory function load. There-
fore, this thermosensitive hydrogel can play the
role of lubricant and sealant, which can not only
effectively reduce the occurrence of complications
during dental implants, but also improve the clini-
cal success rate of implants.
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1, 4, and 7, and the 13/7 group demonstrated the
fastest cell growth rate. However, a previous study
conducted by Cao et al22 found that CS/β-GP/
PVP-I thermosensitive hydrogel using povidone-
iodine (PVP-I) as an antibacterial agent may inhibit
the growth of human rBMSCs in the 16/4 group
on days 4 and 7. So we considered that the ther-
mosensitive hydrogel using nAg as an antibacterial
agent displayed a weaker cytotoxicity, better anti-
bacterial effect, as well as better biocompatibility.
In this study, a nontoxic and antibacterial CS/
β-GP/nAg thermosensitive hydrogel with excellent
lubricity was successfully prepared. It was found
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Preparation and Properties of Chitosan-Based Thermosensitive Hydrogel and Its Positive Influence on the Stability in Dental Implant System as Sealant and Lubricant

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Preparation and Properties of Chitosan-Based Thermosensitive Hydrogel and Its Positive Influence on the Stability in Dental Implant System as Sealant and Lubricant