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RESEARCH ARTICLE
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Low Surface Accessible Area NanoCoral TiO2 for the

Reduction of Foreign Body Reaction During Implantation
Fanyu Zhang, Haoning Qi, Wenting Mo, Yueqi Ni, Qin Zhao, Yulan Wang, Shuting Jiang,
Qinchao Tang, Yihong Cheng, Xiangheng Xiao, and Yufeng Zhang*

1. Introduction
The entry of implants triggers the secretion of damage associated molecular
patterns (DAMPs) that recruit dendritic cells (DCs) and results in subsequent Implants play an important role in many as-
foreign body reaction (FBR). Though several studies have illustrated that the pects of clinical treatments. Immune sys-
tem participates in the subsequence of tis-
surface accessible area (SAA) of implants plays a key role in the process of
sue regeneration after implantation.[1] After
DAMPs release and absorption, the effect of SAA on the immune reaction still implantation, a large number of damage as-
remains unknown. Here, a series of TiO2 plates with different SAA is sociated molecular patterns (DAMPs) will
fabricated to investigate the relationship between SAA and FBR. Compared be released into the body, which trigger the
with larger SAA surface, the aggregation of DC is significantly inhibited by foreign body reaction (FBR).[2] Followed by
fibrous encapsulation, FBR always affects
lower SAA surface. Total internal reflection microscopy (TIRFM) and molecular
implants’ function negatively.[3] Therefore,
dynamic (MD) simulation show that although high mobility group box 1 how to reduce the FBR after implantation
(HMGB1) is adsorbed more on plates with lower SAA, the exposure ratio of is decisive to the quality of implants.
cysteine (CYS) residue in HMGB1 is significantly decreased in lower SAA DAMPs stimulate the immune system
group. The lower exposure of CYS reduces the activation of Toll-like receptors through pattern recognition receptors
4 (TLR4), which down-regulates the expression of myeloid differentiation (PRRs), such as toll like receptor, thus
affecting subsequent tissue regeneration.[4]
factor (Myd88)-TNF receptor associated factor 6 (TRAF6) to inhibit nuclear
Besides, previous studies have proved
factor kappa B (NF-𝜿B) signaling. Generally, this study reveals the mechanism that biomaterials-induced DAMPs could
of how SAA, a nanoscale property, affects FBR from perspective of DAMPs, have impact on immune cell function.[5]
and provides a new direction for designing better biocompatible implants. Dendritic cell (DC) is considered to be
the strongest antigen presenting cells
(APCs) and can be aggregated and activated
under stimulus of DAMPs.[6] After binding
DAMPs with specific PRRs, DC will express high levels of cos-
timulatory or cosuppressive molecules that determine immune
activation or suppression.[7] Therefore, it is of great scientific sig-
nificance to study how to reduce the aggregation and activation
F. Zhang, H. Qi, W. Mo, Y. Ni, Q. Zhao, Y. Wang, S. Jiang, Q. Tang,
of DC triggered by DAMPs after implantation.
Y. Cheng, Y. Zhang Recent studies have shown that different surface modifications
The State Key Laboratory Breeding Base of Basic Science of Stomatology of implants will lead to different immune reactions.[8] The sur-
(Hubei MOST) & Key Laboratory of Oral Biomedicine Ministry of face modifications of implants are mostly by chemical or physi-
Education cal methods, such as acid etching, sandblasting, anodizing, sput-
School & Hospital of Stomatology
Wuhan University ter grinding, etc.[9] Existing studies focus on macroscopical prop-
Wuhan 430079, P. R. China erties such as roughness, hydrophilicity, and hydrophobicity on
E-mail: zyf@whu.edu.cn immune cells.[10] For submicrometer and nanomodification, sur-
X. Xiao face accessible area (SAA) plays an important role in protein ad-
School of Physics and Technology sorption and function.[11] Surface-to-volume ratio leads to an in-
Wuhan University
Wuhan 430072, P. R. China
crease of surface energy and renders nanoparticle more biologi-
Y. Zhang
cally active.[12] Additionally, researches have illustrated that SAA
Medical Research Institute could be potential for the release and absorption of DAMPs.[13]
School of Medicine However, there is a lack of effective and unified evaluation stan-
Wuhan University dards to evaluate this property on implants, and it is difficult to
Wuhan 430071, P. R. China exclude the role of surface roughness in this process. In this re-
gard, it is suggested that implants with different SAA may regu-
The ORCID identification number(s) for the author(s) of this article late FBR to implants.
can be found under https://doi.org/10.1002/adhm.202200382 In this study, we prepared TiO2 plates with different SAA and
DOI: 10.1002/adhm.202200382 same roughness to investigate the relationship between SAA

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Figure 1. Modification and characterization of plates with different SAA. a) Diagram of the synthesis of plates with different SAA. b) Morphology of
plates with different SAA detected by FE-SEM (scale bar = 1 μm, top; 200 nm, bottom). c) AFM images of plates with different SAA (scale bar = 2 μm).
d) XPS analysis of element content of plates with different SAA. The black arrow pointed to the characteristic peak of Ti2p. e) Surface accessible area
for each group collected from AFM images (vertical area = 6.25 μm2 , n = 5). f) Roughness for each group collected from AFM images (vertical area =
6.25 μm2 , n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ns means no significance.

and immune responses. The higher SAA group was named
NanoFerns (NF) because the morphology of these plates was sim-
ilar to ferns. For lower SAA group, plates were labeled as NanoCo-
ral (NC). The results showed that compared with the unstruc-
tured TiO2 Plane (Plane) and NF group, NC group had the least
activation of DC. Multiple DAMPs on the surface were analyzed
by enzyme linked immunosorbent assay (ELISA). It had been
found that HMGB1, a crucial DAMP inducing Toll-like recep-
tors after implantation, had significantly different amount of ad-
sorption on different SAA surface.[14] According to the results of
molecular dynamics (MD) simulations, NC group had a worse ex-
posure of CYS of HMGB1, which inhibited HMGB1 protein ac-
tivation of the dendritic cell through Toll-like receptors 4 (TLR4)-
myeloid differentiation factor 88 (Myd88) pathway. By inhibiting
TLR4, NC group reduced the recruitment of DC and activation of
nuclear factor kappa B (NF-𝜅B). Our findings proposed a novel
perspective to focus on the different SAA of implants. Subse-
quently, this property could lead to different immune reactions
through HMGB1-TLR4-Myd88 pathway. This work would pro-
vide a new idea to manufacture more biocompatible implants on
nanoscale.
2. Results and Discussion
2.1. Preparation and Characterization of NC and NF
To prepare plates with lower SAA, we coated silicon wafers with
chromium. Chromium could react with Au+ , which hinder the
reaction between Au+ and silicon, and form loose porous surface
on silicon wafers.[15] This porous surface helped the Au in twigs
deposit thicker on the surface. The NF group was formed with-
out chromium surface (Figure S1, Supporting Information). NC
and NF plates were synthesized by magnetron sputtering (Figure
1a). Morphology of the plates was examined by field emission
scanning electron microscopes (FE-SEM) and atomic force mi-
croscope (AFM) (Figure 1b,c). Images of FE-SEM indicated that
the NF had a more ferns-like surface, while the NC group had
a coral-like surface. Meanwhile, it was showed that the plates in
the NC group had more dispersed pores on their surfaces, which
made their SAA lower. In order to guarantee the biocompati-
bility, all surfaces were then covered a thin film of titanium by
magnetron sputtering. The results of X-ray photoelectron spec-
troscopy (XPS) analysis showed that Ti2p peak appears on the

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surface of all three materials, which proved the validity of mag-
netron sputtering titanium layer. The consistency of the surface
element types was ensured, while the difference of the surface
morphology was maintained (Figure 1d). In order to accurately
define the morphology, we used AFM to analyze the SAA of two
groups. The SAA of NF group was 19.73 ± 2.35 μm2 , much higher
than that of the NC group (14.29 ± 3.31 μm2 ) (Figure 1e). It came
out that both groups had same roughness in micrometer-level,
which fitted our former suppose (Figure 1f). To exclude the im-
pact of other characters, X-rays diffraction (XRD) and water con-
tact angle (WCA) were examined (Figures S2 and S3, Supporting
Information). There was no significant difference in crystal form
and hydrophilicity between the two groups. Generally, these re-
sults showed that the prepared materials had significant differ-
ence only in the physical property of SAA.
2.2. NC Reduced the Aggregation and Activation of DC
In order to investigate the effects of SAA on the aggregation
and activation of immune system, we constructed an in vivo ex-
periment. Plates with different SAA were implanted in 8 weeks
C57 mice. After 3 days, tissue around plates was harvested and
analyzed by flow cytometry. The early phase of FBR to the im-
plant is dominated by antigen-presenting function of APCs.[16]
We detected the aggregation of leukocyte (CD45+) and DC
(CD11c+/MHC2+) around the plates.[17] The results showed that
NC group could inhibit the aggregation of DC compared with
NF (Figure 2a,b), while leukocyte showed no difference among
all groups (Figure S4, Supporting Information). Interestingly, the
plane group also showed highly aggregation of DC, which might
attribute to the low roughness of plane surface.[10a] TLR4 is one
of the receptors of DAMP produced during implantation and is a
classic indicator of DC activation.[18] So we measured the ratio of
TLR4+DC and the proportion of TLR4+DC triggered by NF was
significantly higher than that by NC (Figure 2c,d). Images of im-
munohistochemistry also showed that proportion of TLR4+ DC
in NC group was the least (Figure 2e,f). These reminded us that
NC would inhibit the aggregation and activation of DC.
DAMPs are generally considered as vital stimulators to toll-
like receptor during implantation.[19] Hence, the quantity of sev-
eral classical DAMPs during implantation absorbed on plates
collected after in vivo experiment was detected by ELISA (Fig-
ure 2g).[20] Comparing to other DAMPs, the total amount of
HMGB1 was inversely related to SAA (Figure 2f; and Figure S5,
Supporting Information). HMGB1 is a crucial signal protein in
TLR4 activated immune pathway, and would be highly released
during implantation.[14] To ensure the function of HMGB1 in the
process of TLR4 activation, in vitro experiments were performed.
20 μg mL−1 HMGB1 was preincubated 24 h for each plate. We
examined the expression of TLR4 in DC using the western blot-
ting analysis. The results showed that NC group down-regulated
TLR4 expression in the DC2.4 cells compared to other groups
(Figure S6, Supporting Information). Images of immunofluores-
cence showed that compared to NF, the activation of TLR4 de-
creased in the NC group (Figure 2h). However, the activation of
TLR4 remained the same under treatment of phosphate buffered
saline (PBS). This indicated that HMGB1 played a key role in in-
hibiting TLR4 activation of DC on low SAA plates.
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To further explore the relationship between SAA and the acti-
vation of DC, we then synthesized a series of SAA-level plates
(Figure S7, Supporting Information). Through the 3D optical
profilometer, we ensured that all the surfaces had the same arith-
metic mean height (AMH), which meant the surfaces roughness
remained the same (Figure S8, Supporting Information). After
incubated HMGB1 for 24 h, data of flow cytometry was collected.
Results showed that the proportion of TLR4+DC and CD86+DC
dropped with the decrease of SAA (Figure S9, Supporting Infor-
mation). Therefore, we believed that materials with lower SAA
could effectively reduce the FBR of DC through HMGB1.
2.3. HMGB1 had a More Compact Adsorption Effect on NC
Surface
Encouraged by these in vitro and in vivo results, we then in-
vestigated the adsorption of HMGB1 on different SAA surfaces.
We first examined the movement of the protein on the surface.
As described in our previous work, TIRFM is a reliable tool for
studying the interaction between interfaces and proteins.[21] It il-
luminates only about 100 nm above the substrate, avoiding un-
wanted fluorescent interference, and visualizing and tracking the
movement of individual molecules. HMGB1 adsorption images
on different plates taken by TIRFM were collected by using a
customized single-molecule tracking algorithm. Specifically, the
position information of every protein was extracted from each
frame and successive coordinate changes were then generated
into trajectories (Figure 3a). According to the intermittent molec-
ular hopping model, the power-law relation MSD = 4D(∆t) 𝛼 was
used to fit the ensemble mean square displacement (MSD). The 𝛼
value could distinguish between different motion patterns. The
subdiffusion of HMGB1 on NC surface (𝛼 = 0.5199) was more
obvious than that on NF (𝛼 = 0.6502), which meant that some
proteins might be trapped by the NC surface due to the strong
interaction (Figure 3b). Displacement distributions curve G (Δx,
t) was plotted to represent the probability that a protein molecule
moved a distance (Δx) during 50 ms (t). Apparently, the longer
the Δx was, the smaller the probability of HMGB1 appearing on
NC became, which showed that proteins on NC were unlikely
to move a long way during one jump (Figure 3c). The results
of protein movements revealed that the HMGB1 adsorbed bet-
ter on NC surface, thus moving slower and having more limited
travel distance. When comparing the proportion of trajectories
length, HMGB1 on NC surface had shorter travel distance than
that on NF surface, which indicated that the migration distance of
HMGB1 on NF plates was significantly smaller than that on NC
plates (Figure 3d). To further explain the adsorption of HMGB1
on the surface, the adhesion force between HMGB1 and differ-
ent SAA surfaces was detected by AFM special probe. The re-
sults showed that mean adhesion force of HMGB1 on NF (88.11
pN) was weaker than that on NC (116.4 pN) (Figure 3e). To con-
firm the results, we supplemented FE-SEM images of HMGB1
adsorption on plates with different SAA to intuitively detect the
adsorption of HMGB1 (Figure 3f). Meanwhile, for detection of
HMGB1 adsorption capacity, absorbed HMGB1 was collected to
detect micro-BCA (Figure 3g). The results showed that the ad-
sorption of HMGB1 on NC group was significantly higher than
that on NF group. These results indicated that the ability of NC
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Figure 2. NC group specifically inhibited the aggregation and activation of DC. a) Flow cytometry analysis of proportion of DC (CD11c+/MHC2+) on
different surfaces. CON represents the control group. b) Statistical analysis of flow cytometry results in a (n = 4). c) Flow cytometry analysis of proportion
of TLR4+DC on different surfaces. CON represents the control group. d) Statistical analysis of flow cytometry results in c) (n = 5). e) Statistical analysis
of Immunohistochemistry results of f) (n = 3). f) Immunohistochemistry images of TLR4 staining after implantation (scale bar = 200 μm). The muscle
tissues (dashed line) and TLR4-activated DC (black arrow) were indicated. g) The quantity of HMGB1 absorption on different surfaces detected by ELISA
(n = 15). h) Immunofluorescence staining and quantification of TLR4 in DC cells under different treatments (scale bar = 20 μm, n = 5). *p < 0.05, **p
< 0.01, ***p < 0.001, ****p < 0.0001 and ns means no significance.

surface to imprison HMGB1 protein was significantly higher
than that of NF group in terms of total amount, adhesion force,
and motion range.
2.4. NC Surface Inhibited the Exposure of CYS in HMGB1
Before the formal simulation, The HMGB1 protein (constructed
by SwissModel and CHARMMGUI) was simulated separately in
aqueous solution, and the results showed that the protein had a
good equilibrium in aqueous solution. After equilibrium in so-
lution, HMGB1 and two different TiO2 surface models with dif-
ferent SAA were placed in a unified virtual box. The final system
we used to simulate the absorption of NC and NF on HMGB1
under experimental conditions was shown as Figure 4a (Videos
S1 and S2, Supporting Information). The whole process was sim-
ulated by 5 ns, and root-mean-square deviation (RMSD) results
showed that the interaction between protein and two plates both
reached equilibrium state at 5 ns (Figure S10, Supporting In-
formation). The analysis of the Coulomb force and LJ potential
energy showed that the HMGB1 adsorption energy of NC was
higher than that of NF, which was consistent with the forego-
ing (Figure 4b,c). After reaching equilibrium, the CYS associated
with TLR4 activation in HMGB1 was closer to the substrate in

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Figure 3. HMGB1 had a more compact adsorption effect on NC surface. a) Diagram of protein movement in different groups. White points in red
circles meant the same protein in a 50 ms movement trail. b) Mean squared displacement versus time interval. The experiment data were fitted using
MSD = 4D(∆t) 𝛼 and 𝛼 value was shown along the curve. c) Step-size distribution of HMGB1 on two groups at ∆t = 50 ms. d) Trajectories distance
distribution histogram of HMGB1 on two groups. e) Adhesion force of HMGB1 on two groups was measured by AFM. The peak value meant the final
effective adhesion force. f) The adhesion of HMGB1 on the plates was observed by SEM after incubation for 1 day. The areas blue dotted line around
represented the protein. g) BCA assay of HMGB1 adsorption quantity on different materials by incubation concentration of 20 μg mL−1 . The statistic
taken into account was the concentration of the remaining protein not adsorbed (n = 9). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns
means no significance.

NC, while in the NF group it was closer to the solution envi-
ronment (Figure 4d). For the HMGB1 protein, the CYS region
is an important target site for activating TLR4 and subsequent
pathways and previous study has managed to affect the signal-
ing function of HMGB1 by blocking this binding.[2b] Therefore,
it is assumed that the exposure of this site on different surfaces
with SAA determines the activation of HMGB1 and its down-
stream pathway. We then calculated the total exposure area of
the two groups and the proportion of CYS in the total exposure
(Figure 4e,f). Despite the whole exposure area of HMGB1 was
increased in NC, the proportion of CYS exposure in NC Group
was smaller than that in NF group. This indicated that the CYS
group of HMGB1 was blocked in NC Group. We further explored
the cause of this particular state. By analyzing the deformation
state of the two groups, it was found that the 185–215 amino
acids of HMGB1 were in close contact with the surface of TiO2 .
Base on this finding, next we performed a statistical analysis of
root-mean-square fluctuation (RMSF) for each amino acid of the
protein (Figure 4g). Results showed that this part of amino acid
sequence was relatively unstable, and greatly affected the overall
protein structure. The motion amplitude of the unstable region
(185–215 residues) at the end of HMGB1 was larger in NC Group.

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Figure 4. NC surface blocked CYS of HMGB1, which activated TLR4. a) The state of HMGB1 on different surfaces by GROMACS (Top: 0 ns; Bottom:
5 ns). b) A time-dependent curve of coulomb force of HMGB1 on different surfaces. c) A time-dependent curve of LJ force of HMGB1 on different
surfaces. d) Schematic diagram of secondary structures of HMGB1 on different surfaces by VMD, CYS (red parts). e) A time-dependent curve of SASA
of HMGB1 on different surfaces. f) The proportion of CYS exposure in HMGB1 on different surfaces over time. g) A time-dependent curve of RMSF of
every residue on different surfaces. 185th residue was marked as dashed line.

It was prompted that the end tail unstable region of HMGB1 was
self-stabilized in the absence of substrate (Video S3, Supporting
Information). On the surface of lower SAA, the region would be
unstable after contact with TiO2 . This would destroy its equilib-
rium and lead to great changes in the structure of the protein,
thus hindering the exposure of CYS.
2.5. Validation of NC for Inhibiting HMGB1-TLR4-Myd88
Pathway
The activation of TLR4 would trigger series of intercellular
reaction.[22] Protein–protein interaction analysis showed that
TLR4 was strongly associated with downstream inflammatory
markers, such as Myd88, inducible nitric oxide synthase (iNOS),
TRAF6, NF-𝜅B, etc. (Figure 5a).[23] Myd88 is often regarded as
the classical downstream of TLR4, so we detected the expression
of Myd88 and related RNA.[24] In order to further verify the role of
HMGB1-TLR4, real-time quantitative polymerase chain reaction
(qPCR) was performed. Results showed that there was no signif-
icant difference in the inflammatory markers of Myd88, TRAF6,
NF-𝜅B, and iNOS in the downstream of TLR4 without HMGB1
(Figure S11, Supporting Information). However, the inflamma-
tory markers of the downstream of TLR4 were significantly inhib-
ited in NC group after HMGB1 was involved (Figure 5b). The ac-
tivation of DC often showed an increase in iNOS.[25] Immunoflu-
orescence images showed that the fluorescence intensity of
iNOS remained to be the least on NC (Figure 5c,d). The results

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Figure 5. Validation of NC for inhibiting HMGB1-TLR4-Myd88 classical pathway. a) Coexpression of inflammatory markers in DC-associated TLR4-
Myd88-NF-𝜅B pathway in mice with GENEMANIA database. b) qPCR results of relative iNOS, Myd88, TRAF6, and NF-𝜅B mRNA expressions in cells on
different surfaces after 1-day HMGB1 incubation (n = 3). c) Immunofluorescence staining of iNOS in DC seeded on different surfaces after incubation
of HMGB1 (scale bar = 20 μm). d) Quantitative analysis of immunofluorescence images in (c) (n = 11). *p < 0.05, **p < 0.01, ***p < 0.001, ****p
< 0.0001 and ns means no significance.

of Western blotting showed that NC group down-regulated iNOS 3. Conclusions

expression in the DC2.4 cells compared to other groups (Fig-
ure S11, Supporting Information). In conclusion, these results In this study, we clarified the relationship between plates with
indicated that lower SAA might down-regulated HMGB1-TLR4- different SAA and DAMPs. By this way we proposed a new
Myd88 pathway and led to reduction of FBR (Figure 6). improvement direction of implants modification. This method

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Figure 6. Schematic diagram of NC for reducing the immune recognition of Implants. First, a large amount of HMGB1 was produced during implanta-
tion. Next, the unstable end of HMGB1 had better adsorption on NC, resulting in the reduction of CYS residues exposure. Last, binding of CYS to TLR4
caused DC aggregation and activation of TLR4-Myd88-NF-𝜅B pathway. Finally, morphology of NC helped reducing the immune recognition of DC.
relied on the redox of HAuCl4 with monocrystalline silicon, and
could be adjusted by increasing the reaction time or changing the
metal coating on the monocrystalline silicon surface. Through
in vivo and in vitro experiments, plates with lower SAA were
proved to obtain lower FBR by reduction of DC aggregation and
activation. By detecting DAMPs adsorption on different plates,
we located HMGB1. The movement limitation and adhesion
force of HMGB1 in NC group were higher than those in NF
group, and HMGB1 also had higher total adsorption capacity.
Using molecular dynamics to simulate the interaction between
surface and protein, it was found that NC group significantly
inhibited CYS of HMGB1. The effect of SAA on the subsequent
immune recognition of the implants was studied without chang-
ing other physical parameters. Our study not only elucidated the
effect of SAA on the reduction of FBR, but also provided a new
idea for surface modification of implants.
4. Experimental Section
Materials: Chromium thin films were deposited on N-type monocrys-
talline silicon wafers by direct current magnetron sputtering (JSD-400, An-
hui Jiashuo Vacuum, China) using a chromium target (99.99%, 5.1 cm in
diameter and 0.5 mm in thickness, ELETM, China) in argon atmosphere.
The distance between the target and the base plate was fixed at 100 mm.
During the sputtering process, the power density was kept at 100 W and
the temperature was kept at 300 °C. The operating pressure was 0.5 Pa; the
argon flow rate was kept at 5 sccm. And the sputtering time was 20 min.
The treated substrate was immersed in a mixture of 10 × 10−3 m HAuCl4
and a buffer solution containing 11.3% NH4 F and 2.3% HF and left to
react at room temperature. For NC-1 and NC-2, the reaction time was 10
min in solvent system for NC-1, while for NC-2 it was 15 min. For NF-1
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and NF-2, the concentration of HAuCl4 was halved in NF-1 (10 × 10−3 m)
compared with that in NF-2 (20 × 10−3 m). The surface structures of NC
and NF structures were obtained after deionized water cleaning, spray dry-
ing with high purity nitrogen, and heating at 120 °C for 15 min. The thin
titanium coating was prepared by DC magnetron sputtering (Titanium tar-
get, 99.999%, 5.1 cm in diameter and 0.5 mm in thickness, ELETM, China).
Other conditions were the same as mentioned above. Plates were kept in
vacuum during the whole sample preparation process. Before and after
the cell culture, the plate was sterilized with ultraviolet light for 1 h.
Morphology: The morphology of the plates was observed by FE-SEM
(Zeiss, UK) and atomic force microscope (SPM9700, Japan). As for the
measurement of surface accessible area and roughness, data were ob-
tained randomly by sampling the same size of the AFM images. The aver-
age surface accessible area was calculated by 5 images and 3 images for
average roughness.
Cell Culture: DC2.4 (Cobioer Biotechnology Co., Ltd, China) cells
were cultured in dulbecco’s modified eagle medium (DMEM) (HyClone,
Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS)
in a mild 5% CO2 atmosphere. For the HMGB1 incubated group, the cells
were cultured in DMEM with 500 ng mL−1 HMGB1 at 37 °C overnight.
In Vivo Study: All in vivo experiments were performed on C57 mice.
The mice were all female and 8 weeks old. The animal research program
was approved by the Animal Research Committee of School of Stomatol-
ogy, Wuhan University, China (A31/2020). After anesthesia, the femur was
cut through the skin and muscles by 1.5 cm. A 1 mm diameter anterior and
posterior cortical channel perpendicular to the axis was then constructed.
All plates were cut into 0.3 × 0.3 cm2 . After high-temperature and high-
pressure disinfection, plates were placed gently between the femur and the
musculature (one implant per femur). As for CON group, mice were oper-
ated on but not implanted. After 3 days, mice were over-anaesthetized and
soft tissue around the materials was removed. In flow cytometry analysis,
the tissue was sliced and treated with collagenase (Sigma-Aldrich, USA)
to produce a single cell. The antibodies for Flow cytometry were: CD11c
(1:200), CD45 (1:200), MHC2 (1:200), and TLR4 (1:200) (All purchased
© 2022 Wiley-VCH GmbH

www.advancedsciencenews.com
from Biolegend, USA). Immunohistochemical techniques including im-
mobilization, dehydration, paraffin embedding, section, and staining of
TLR4 (1:200, Abclonal, China) were used.
Flow Cytometry: Cells were seeded in equal quantities on plates of the
same size and collected from the plates after 24 h. After resuscitation by
PBS (Hyclone, USA), cells were stained with CD86 and TLR4 antibody
(CD86, 1:200; TLR4,1:200, Biolegend, USA) at 4 °C for 10 min. Flow cy-
tometry was performed by BD LSRFortessaX-20 (BD, USA) and analyzed
by FlowJo.
Immunofluorescence Staining: Cells were seeded on the plates for 24 h.
After immobilized with 4% paraformaldehyde, cells were then incubated
with CD86 and TLR4 antibody (CD86, 1:200; TLR4,1:200, iNOS,1:200 Ab-
clonal, China) at 4 °C for 12 h. After washing with PBS three times, anti-
rabbit IgG was stained for 1 h (DyLight 594, 1:200, Abclonal, China). Label-
ing the nucleus with DAPI (Beyotime, China). Photos were taken by Zeiss
Axio Imager A2 (Zeiss, Germany).
Western Blot: DC2.4 cells were cultured on plates with different SAA
for 24 h. After 24 h cells were collected and lysed with RIPA buffer (Be-
yotime, China). After centrifugation, supernatant were collected and de-
natured at 96 °C for 5 min with 5× loading buffer (Beyotime, China).
Then, proteins were loaded in 10% SDS-PAGE and transferred onto a
polyvinylidene difluoride (PVDF) membrane. Rabbit anti-toll-like receptors
4 (1:1000, Servicebio, China), rabbit anti-inducible nitric oxide synthase
(1:1000, Abclonal, China), rabbit anti-𝛼-tubulin (1:2000, Abclonal, China)
were used as primary antibodies. Then, anti-rabbit IgG (1:10 000, Protein-
Tech Group, Inc., USA) were used as secondary antibodies. The bands
were illuminated by enhanced chemiluminescence (ECL) system (Amer-
sham Pharmacia Biotech, USA).
Qpcr: After cultured on plates for 24 h, total RNA was extracted by
RNAiso Plus reagent (TaKaRa, Japan) and quantified by Nanodrop2000
(Thermo Fisher, USA). By adding ddH20, the RNA of each group was
maintained at 1 μg. Template was reverse-transcribed into cDNA (Vazyme
Biotech Co., Ltd, China). RT-PCR was performed by a CFX 384 Real-Time
system (Bio-Rad, USA), and was normalized to GAPDH by ΔΔCt and cal-
ibrated to plane. The basic sequence is listed in Table S1 (Supporting In-
formation).
Protein Adsorption: Plates with different SAA were placed in 12-well
plates. After washing with PBS, 20 μg mL−1 HMGB1 was incubated for
each plate at 37 °C. After 2 h, the total amount of HMGB1 unabsorbed
on plates with different SAA was collected and determined by Micro BCA
protein assay kit (Boster, China).
ELISA: The materials were obtained from in vivo study. After blocked
with 2% BSA at 37 °C for 1 h, HMGB1, S100, HSP90 antibody (1:1000,
Abclonal, China) was cultured for each plate overnight at 4 °C overnight.
After washing three times with PBS, plates were incubated with the
Goat anti-mouse IgG (1:5000, noncloned, USA) at 37 °C for 1 h. After
washing three times with PBS, plates were then incubated in 3,3′, 5,5′-
tetramethylbenzidine (TMB, Solarbio, China) for 20 min. The reaction su-
pernatant was terminated by 1 m HCl of equal volume and determined at
450 nm by Spectramax i3x (Molecular Devices, USA).
Single-Molecule Tracking: The substrates were cleaned ultrasonically
with acetone, ethanol, and ultrapure water thoroughly and then dried by
nitrogen. Rhodamine B labeled HMGB1 were diluted in PBS to ≈10−9 –
10−11 m. The substrate was set on the sample stage of the inverted Nikon
Eclipse Ti2-E (Nikon, Japan) equipped with a Nikon CFI Apo TIRF 100×
1.49 NA oil immersion objective. About 50 μL protein solution was added
onto the substrate surface and a green He–Ne laser (𝜆exc = 532 nm) was
then adjusted to illuminate the solid–liquid interface. For each experiment,
5 min movie was taken, and the protein movement on the substrate was
recorded with 50 ms per frame (40 × 40 μm2 ). The protein dots on each
frame of the movies were localized and the continuous coordinate changes
was obtained by a custom-designed tracking algorithm. The linking thresh-
old which was used to recognize the same protein molecule on sequential
two frames was 1.12 μm.[26] All of the calculation were based on trajecto-
ries that lasted between 10 and 600 steps.
Adhesion Force Measurements: The protein functionalization of the
AFM tip has been well established before.[27] The gold-coated AFM sili-
con nitride tips (radius = 30 nm, NPG-10, Bruker, USA) were immersed in
Adv. Healthcare Mater. 2022, 11, 2200382 2200382 (9 of 10)
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1 × 10−3 m HS(CH2 )16 COOH solution (50% aqueous ethanol solution)
at 37 °C for 12 h. The tips were rinsed by ethanol three times and then
dried by nitrogen. 9.58 mL N,N-Dimethylformamide (DMF), 0.28 mL tri-
ethylamine and 0.14 mL trifluoroacetic anhydride were mixed and used
to immerse the tips for 20 min. After being rinsed by dichloromethane
three times and dried by nitrogen, the tips were incubated with 5 mg mL−1
HMGB1 for 12 h. Then the tips were washed by PBS gently and dried by ni-
trogen. The adhesion force measurement was conducted on AFM (Bruker
Multimode 8, USA) under the contact mode in PBS. The substrates were
set on the sample stage respectively for measurement. The effective spring
constant of the functionalized cantilevers was calibrated before measure-
ment, which was between 0.1208 N m−1 . The loading rate was 200 nm
s−1 and the loading force was 30 nN.[22] About 50 force–distance curves
at the randomly chosen spots were recorded and the maximal adhesion
force upon retraction was extracted.
Arithmetic Mean Height: Arithmetic mean heights of plates with dif-
ferent SAA were measured by 3D optical profilometer (ST400, NANOVEA,
USA). Guidelines have been given for the measurement and specification
in ISO 25178.
Molecular Dynamics: Simulations were performed by the Gromacs
2016.4 package[28] and the Charmm36 force field and CHARMMGUI and
SwissModel.[29] The full-scale model of HMGB1 was derived from the re-
sults of previous studies,[30] and the protein movement in aqueous so-
lution was simulated using CHARMMGUI and SwissModel. The plates
of TiO2 were made through Material Studio 8.0. On the basis of pre-
vious studies, all parameters required in the simulations were obtained
from previous study.[31] The HMGB1 and plates were initially placed in a
12.8624 × 11.9437 × 35 nm3 rectangle virtual box with a vacuum layer of
20 nm by z direction inside. The minimum distance between the protein
and the substrate surface was 10 nm. Water molecules were dissolved in
simple point charge model (SPC) in simulation box. Potassium ion (POT)
and chloride ion (CLA) were involved as neutralism system. SPC names
the representative files for water molecules, POT and CLA are the names
of the ions potassium and chloride added for the electro-neutral equilib-
rium. To minimize the system energy, the gradient descent method was
included. A125 ps V-type thermostats was engaged in all MD simulations
and collected as NVT. After equilibrium, 5 ns simulation was obtained for
each model. The long-range electrostatic interactions as the particle grid
Ewald (PME) method were described. The control temperature of the V-
type thermostat was 298 K. These equations of motion were integrated in
2 fs time step. Three repeat simulation work for each plate was done. To
investigate the protein-surface interaction, the Coulomb force and LJ po-
tential energy of the protein was collected. Solvent accessible surface area
(SASA) was involved to illustrate the exposure of the total and local regions
of the protein. Define SASA percentages to illustrate CYS exposures
SASA% = SASACYS ∕SASAHMGB1 × 100% (1)
SASACYS and SASAHMGB1 represent the solvent accessible surface area
of CYS and bulk proteins, respectively. The trajectory was visualized and
analyzed by VMD software.
Data Analysis: All the experiments in this paper have been repeated at
least three times. GraphPad Prism 8 was used for statistical analysis. Stu-
dent’s t-test was performed between the two different groups. ANOVA was
performed to compare samples from more than two groups. The Nonlin-
ear regression method (y = A * XB) was used to fit the MSD curve. The
p-values lower than 0.05 were considered statistically significant.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgements
F.Z. and H.Q. contributed equally to this work. This work was supported
by the National Natural Science Foundation of China (Nos. 82025011 and
© 2022 Wiley-VCH GmbH

www.advancedsciencenews.com
82100975); the China National Postdoctoral Program for Innovative Tal-
ents (No. BX2021227). The Ethics Committee approved all animal exper-
iments of Wuhan University School and Hospital of Stomatology (ethical
Approval No. A31/2020). The animal experiment process complied with all
relevant ethics. After initial online publication, Figure 5b was removed on
July 6, 2022, as this is already present in Figure S11, Supporting Informa-
tion. The rest of the panels in Figure 5, and in-text citations thereof, were
re-labeled accordingly. This correction does not affect the overall results
and conclusions of this work.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available from the cor-
responding author upon reasonable request.
Keywords
DAMP, foreign body reaction, HMGB1, nanomodification, surface acces-
sible area
Received: February 17, 2022
Revised: April 11, 2022
Published online: May 18, 2022
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