Bioactive Materials 23 (2023) 53–68

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Bioactive Materials
journal homepage: www.keaipublishing.com/en/journals/bioactive-materials

Promoting oral mucosal wound healing using a DCS-RuB2A2 hydrogel

based on a photoreactive antibacterial and sustained release of BMSCs
Wenxin Qi a, b, Naijun Dong a, b, Lingling Wu a, Xueqi Zhang a, He Li c, Hao Wu a, Natalie Ward d,
Jian Yu e, f, He Liu e, Jiao Wang a, *, Xiaoyong Deng g, **, Robert Chunhua Zhao a, h, i, j, ***
a
School of Life Sciences, Shanghai University, Shanghai, China
b
School of Medicine, Shanghai University, Shanghai, China
c
Beijing University of Chemical Technology, Beijing, China
d
Banner Ocotillo Medical Center, 1405 S Alma School Rd, Chandler, AZ, 85286, USA
e
Division of Endodontics, Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, V6T 1Z3, Canada
f
The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory for Oral Biomedicine Ministry of Education, School and
Hospital of Stomatology, Wuhan University, Wuhan, 430079, China
g
School of Environmental and Chemical Engineering, Shanghai University, Shanghai, PR China
h
Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China
i
Centre of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences, Beijing, China
j
Beijing Key Laboratory of New Drug Development and Clinical Trial of Stem Cell Therapy (BZ0381), Beijing, China

A R T I C L E I N F O A B S T R A C T

Keywords: The high occurrence rate and difficulties in symptom control are listed as the major problems of oral mucosal
Oral mucosa disease by medical professionals. Following the development of oral mucosal lesions, the oral microenvironment
Light-responsive changes, immunity declines, and continuous bacterial stimulation causes wound infection. Traditional antibac­
Hydrogel
terial drugs are ineffective for oral mucosal lesions. To overcome this problem, a light-responsive antibacterial
DCS
hydrogel containing sustained-release BMSCs was inspired by the trauma environment in the oral cavity, which
BMSCs
is different from that on the body surface since it mostly remains under dark conditions. In the absence of light,
the hydrogel seals the wound to form a barrier, exerts a natural bacteriostatic effect, and prevents invasion by
foreign bacteria. Simultaneously, mesenchymal stem cells are presented, and the released growth factors and
other substances have excellent anti-inflammatory and angiogenic effects, which result in rapid repair of the
damaged site. Under light conditions, after photo-induced shedding of the hydrogel, RuB2A exerts an antibac­
terial effect accompanied by degradation of the hydrogel. Results in a rat oral mucosal repair model demonstrate
that DCS-RuB2A2-BMSCs could rapidly repair the oral mucosa within 4 days. Sequencing data provide ideas for
further analysis of the intrinsic molecular mechanisms and signaling pathways. Taken together, our results
suggest that this light-responsive antibacterial hydrogel loaded with BMSCs can be used for rapid wound repair
and may advance the development of therapeutic strategies for the treatment of clinical oral mucosal defects.

1. Introduction the shedding of inflammatory necrotic tissue [2], thereby affecting the
oral mucosa and soft tissues including ulcerative lesions of oral mucosa
Oral diseases are some of the most common occurring worldwide represented by recurrent aphthous ulcers [3], which seriously affect the
[1]. Among them, oral mucosal defects are a series of diseases caused by quality of patients’ life. The causes of mouth ulcers are various and

Peer review under responsibility of KeAi Communications Co., Ltd.

* Corresponding author.
** Corresponding author.
*** Corresponding author. School of Life Sciences, Shanghai University, Shanghai, [email protected]
E-mail addresses: [email protected] (W. Qi), [email protected] (N. Dong), [email protected] (L. Wu), [email protected] (X. Zhang),
[email protected] (H. Li), [email protected] (H. Wu), [email protected] (N. Ward), [email protected] (J. Yu), [email protected] (H. Liu),
[email protected] (J. Wang), [email protected] (X. Deng).

https://doi.org/10.1016/j.bioactmat.2022.10.027
Received 31 August 2022; Received in revised form 18 October 2022; Accepted 25 October 2022
Available online 9 November 2022
2452-199X/© 2022 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
W. Qi et al. Bioactive Materials 23 (2023) 53–68

complex, such as an imbalance in the oral microbiota [3], fatigue, stress, shown to migrate to sites of injury and accelerate repair in vivo [31–34].
and complications from diabetes [4], and there exist many types of oral These cells are involved in all three phases of the wound-healing pro­
ulcers, which leads to difficulty in accurate diagnosis. Nevertheless, due cess, and can also enhance healing by immune modulation and pro­
to the dynamic environment of the oral cavity, there are few effective duction of growth factors, which enhance neovascularization and
treatments [5]. re-epithelialization, stimulate angiogenesis, and accelerate repair
The human oral cavity contains complex microbiota, changes in [35–37]. One case study reported that increased wound closure occurs
which are affected by many factors such as exogenous food intake, following administration of MSCs due to accelerated dermal fibroblast
physical diseases, and drug treatment. An imbalance of microbiota in the and keratinocyte migration [38].
oral cavity can trigger various oral mucosal diseases. Candida albicans Chitosan (CS), a linear aminopolysaccharide composed of β (1–4)
can induce dysbiosis of mucosal bacteria, destroy oral immune defense, glycosidic linkages attached to glucosamine and N-acetylglucosamine
and cause oral infectious diseases [6]. Oral mucosal patch disease, units formed by the N-deacetylation of chitin [39], is a low-cost bio­
represented by oral lichen planus, is caused by a disturbance in the level logical material. In recent years, CS has received extensive attention due
of fungi in the oral cavity [7,8]. Recurrent aphthous ulcers may be to its excellent hemostatic efficacy, antibacterial effects, tissue adhesion,
associated with an increase in Escherichia coli and a decrease in the and biocompatibility [40–42]. The hemostatic mechanism of CS is
abundance of Streptococcus [9], and infection of cytotoxic T lymphocytes mainly polycationic mucoadhesion; positively charged CS readily binds
by microbiota causes mucosal epithelial cell damage, leading to ulcer­ to negatively charged sialic acid on mucosa, red blood cells, and plate­
ation [10]. Studies have shown that probiotics promote oral epithelial lets, stimulating red blood cell adhesion, fibrinogen adsorption, platelet
wound healing and reduce proinflammatory cytokine secretion in vitro, activation, and the coagulation cascade [43–45]. Moreover, CS has
which is beneficial for the treatment of oral lichen planus [11]. One excellent antibacterial abilities due to the binding of its positively
study [12] found that most infections in the oral cavity are caused by charged NH+ 3 groups to negatively charged bacterial surfaces [46–48].
endogenous oral bacteria that have overcome the host immune system N-alkylated CS was created by grafting alkyl groups onto the amino
or underlying defects in the innate or specific immune systems. S. aureus groups, which better promotes the activation and aggregation of plate­
is a gram-positive member of the Firmicutes phylum, which is lets and red blood cells to further improve hemostatic efficiency [49,50];
commonly found on cutaneous and mucosal surfaces in healthy however, the antibacterial activity of alkylated CS decreases as the
mammalian hosts as a commensal organism. However, viral infection or substitution degree or carbon chain length increases [51]. In addition to
immunodeficiency can lead to invasion and infection [13]. Another the cationic antibacterial properties of CS, the grafted C-12 alkyl chains
study showed that IL-17 signaling is required for initial barrier defense enhance the bacteriostatic properties by intercalating into the outer
to S. aureus in oral mucosal infections. A further study reported the membrane of E. coli cells [52]. The surface, porosity, and hydrophobicity
occurrence of a case of Lemierre syndrome secondary to an odontogenic of N-alkylated CS nanofibrous membranes (NACS-NM) and the cross­
infection in the context of necrotizing mediastinitis and anterior opti­ linked structure of nanofibers can accelerate blood coagulation and
cobacteria [14]; therefore, dynamic changes in oral microbiota may be platelet activation [53]. Moreover, recent studies have found that
an even more important initiator of oral disease. microneedle patches with a dodecyl CS (DCS) coating can anchor to the
Common products currently on the market for the treatment of oral cell membrane and allow blood cells to coagulate, thereby rapidly
ulcers are composed of hyaluronic acid as a hydrogel or mouthwash promoting hemostasis [54]; therefore, this DCS hydrogel can be used as
combined with other substances possessing healing and soothing prop­ an excellent base material for intraoral wound repair.
erties [15]. Traditional formulations have several limitations: they are Wound repair in the oral cavity is different from the skin surface. The
easily removed by tongue movement, remain in the mouth for only a oral cavity is always in a moist and dark environment with many mi­
short period of time [16], do not display simultaneous antibacterial and croorganisms. Wound repair materials for oral mucosa must have
damage repair properties; and can not firm antibacterial properties and certain adhesion and antibacterial properties [55]. Further, the toxicity
low cytotoxicity; therefore, the development of oral wound healing of the material itself must be considered, in addition to whether it has
materials that promote tissue repair and possess broad-spectrum anti­ good degradability and biocompatibility after entering the gastrointes­
bacterial effects is imperative. Owing to its broad-spectrum antibacterial tinal tract. Functional polymers that can be photochemically manipu­
effect and potential to promote wound healing, photodynamic therapy lated by exogenous light have immense promise in materials science
(PDT) has a favorable effect on local, superficial infections and is suit­ [56]. Photoresponsive units can be attached as pendant groups or
able for the treatment of oral ulcers [17]. Recent studies have shown incorporated into the main chain of photocleavable polymers, which
that PDT treatment mediated by the photosensitizer, indocyanine green, degrade to smaller molecules during a light-driven response. Photo­
can accelerate the healing of traumatic oral ulcers in rats [18]. cleavable polymers also have significant potential in biological appli­
Wound healing undergoes a continuous process of hemostasis, cations, particularly in the spatiotemporal patterning of cellular
inflammation, proliferation, and structural remodeling of damaged tis­ microenvironments in tissue engineering, smart drug-delivery, and
sue [19], involving multiple cell types and various biological processes; diagnostic systems [57,58]. The dark environment of the oral cavity also
therefore, dressings that can quickly stop bleeding and possess anti­ provides natural conditions for light-responsive materials. The inspira­
bacterial and anti-inflammatory properties are important for wound tion for the present study also stems from this fact; a photoactive
healing and the generation of granulation tissue. A variety of hemostatic ruthenium crosslinker enabled the creation of a hydrogel that responds
materials have been developed, such as polyethylene glycol [20], chi­ with unique speed and efficiency to visible light exposure. Silver
tosan [21,22], and nanoclay [23]. In addition, the inflammatory nanoparticles (Ag NPs) have been shown to prevent the growth and
response often accompanies the process of hemostasis [24], and anti­ reproduction of many bacteria, such as Bacillus cereus, Staphylococcus
oxidant and antibacterial effects, immune regulation, and angiogenesis aureus, Escherichia coli, Klebsiella pneumoniae, and Candida albicans,
all play anti-inflammatory roles [25]. Interestingly, hydrogel has been through the binding of Ag to biomolecules present in bacterial walls
shown in multiple studies to display good biocompatibility for hemo­ [59–61]. In addition, it has been proposed that Ag NPs generate reactive
stasis, in addition to successful antibacterial and drug delivery [26–29]. oxygen species and free radicals, which lead to apoptosis and prevent
Moreover, inflammatory responses affect the structural regeneration of their replication. Ruthenium, as a metal cation, may also play a role in
tissue and are regulated by various cytokines and growth factors [30]. killing microorganisms in a similar manner within the same concen­
During wound healing, reducing the increase in pro-inflammatory cy­ tration range as Ag. Various Ru-based DNA photocleavers are known to
tokines (IL-6) and promoting the proliferation of endothelial cells damage DNA through an oxidative mechanism [62]. Different positively
(CD31) reduces pathological pain and accelerates repair. charged Ru(II) polypyridyl complexes have been demonstrated to bind
Bone marrow-derived mesenchymal stem cells (BMSCs) have been to the negatively charged outer membrane of gram-negative bacteria

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and intercalate the dppn ligand into the membrane [63,64]. In the Furthermore, the three typical vibrational peaks of CS, the stretching
present study, CS and the photocrosslinking agent Ru bipyridine were vibration of the –CO bond (approximately 1640 cm− 1), the bending
used to synthesize photoresponsive hydrogels. The picolinaldehyde vibration of the –NH2 group (approximately 1580 cm− 1), and the sym­
group is shed after visible light irradiation, and Ru bipyridine plays a metric vibration of the –CN bond (approximately 1310 cm− 1) can also be
bactericidal role in the humid environment. Moreover, Ru-crosslinked seen. In comparison with CS, DCS displays a clear absorption peak at
polymer systems provide even greater spatiotemporal control over ma­ 2800–2900 cm− 1, with a more pronounced –CH2 antisymmetric
terial properties, such as storage modulus and porosity, as well as the stretching peak (approximately 2920 cm− 1) and a significantly stronger
regulation of drug delivery profiles and cellular function in biomedical –CH3 antisymmetric stretching peak (approximately 2850 cm− 1). DCS
applications. Therefore, this light-responsive hydrogel can be regarded shows no peak at 2810–2720 cm− 1, indicating that the alkyl aldehyde
as a good stem cell presentation system. Under dark conditions, the impurity has been removed. The amino absorption peak (approximately
material has no killing effect and its 3D structure is conducive to the 1580 cm− 1) is weaker, while the –NH bond absorption peak (approxi­
growth and sustained release of BMSCs. This light-responsive antibac­ mately 1560 cm− 1) is significantly enhanced. Moreover, the –CH2 and
terial hydrogel capable of presenting BMSCs has broad application –CH3 variable angle bending vibration peaks (approximately 1460
prospects in the wound repair of oral mucosa. cm− 1) are also present, suggesting linkage of the alkyl group to the
amino group of CS and the synthesis of DCS (Fig. 2E). Previous literature
2. Results and discussion [65] has demonstrated that different aldehyde group substitutions
greatly affect the hemostatic properties of CS; therefore, we speculated
2.1. The preparation and application of the DCS-RuB2A2-BMSCs that other substitutions may show better effects. By changing the molar
hydrogel ratio of dodecanal to CS, DCS with differing degrees of substitution was
generated. Additionally, the strength of these bending, vibrational, and
The base material of the hydrogel was chitosan, and dodecylalde­ tensile peaks was also directly related to the different feeding ratios, and
hyde was used to replace the amino group, obtaining dodecyl chitosan the intensity of the –NH bending vibration peak increased as the molar
(DCS). The molar ratio of the raw materials was set as 0.4:1 to achieve ratio of dodecanal to CS became higher. These data indicate that the
the best hemostatic performance of DCS and simultaneously aid platelet alkyl group is successfully linked to the amino group on CS and that the
aggregation and red blood cell adsorption. Ru(bpy)2Cl2 solution was intensity of the characteristic peak of groups varied with the molar ratio
refluxed at 80 ◦ C for 5 h under argon protection, after which Cl was (Fig. 2F). Fig. 2G is a photograph of the hydrogels of DCS with differing
replaced with 3-pyridinecarboxaldehyde. The material was separated, degrees of substitution.
evaporated to dryness, and purified to obtain a brown-black solid, which To generate an effective, visible light-responsive hydrogel system,
was subsequently dissolved in water to yield Ru(bipyridine)2(3-pyr­ we synthesized a photorelease crosslinker, Ru(bipyridine)2(3-pyr­
idinecarboxaldehyde)2 (RuB2A2: B is 2,2′ -bipyridine and A is 3-pyridine­ idinecarboxaldehyde)2 (RuB2A2: B is 2,2′ -bipyridine and A is-
carboxaldehyde). Under acidic conditions, DCS and RuB2A2 undergo a pyridinecarboxaldehyde, Fig. 1, Fig. 2H). NMR results demonstrated
Schiff base crosslinking reaction to form a dense network-like gel. The successful synthesis of RuB2A2 (Supplementary Fig. 1). RuB2A2 un­
amide bond formed by the crosslinking of DCS and RuB2A2 greatly en­ dergoes substitution of a single pyridine in water to yield RuB2A(H2O)
hances the performance of the gel. Under light irradiation, 3-pyridine­ and free ligand A, a process which occurs in less than 120 s (Fig. 2I, J)
carboxaldehyde falls off, the DCS-RuB2A2 hydrogel decomposes, and and conforms to the Ru(II) polypyridyl complex shedding process
DCS and RuB2A/RuB2 act independently. The dodecyl group of DCS described in the literature [58,66]. Fig. 2I demonstrates that RuB2A2
anchors the bacterial outer membrane, and the released RuB2A/RuB2 substitution occurs in a manner typical for a metal-to-ligand charge
attaches to the cell surface and causes changes in cell permeability, transfer (MLCT) and displays comparable spectra to those of known Ru
resulting in the escape of cellular components and interference with the (II) polypyridyl complexes. The UV–Vis absorption spectra suggest that
role of enzymes in cellular metabolism. At the same time, RuB2A/RuB2 photoreaction cleaves one ligand, resulting in red shift of the absorption
ruptures the membrane and causes damage to DNA, thereby exerting a peak from 446 nm to 469 nm RuB2A2 exhibits strong absorbance to 510
bactericidal effect. Following application of DCS-RuB2A2-BMSCs to oral nm, with a tail extending past 560 nm. The peak wavelength map
mucosal defects, the hydrogel firstly attaches to the wound and DCS (Fig. 2J) can be divided into two stages: the first stage (0–120 s) is
causes rapid hemostasis and platelet aggregation. Under dark condi­ attributed to the photorelease of the first 3-pyridinecarboxaldehyde, and
tions, the hydrogel slowly releases BMSCs and the cytokines in the the second stage corresponds to the stable state of the single chain
BMSCs microenvironment play an anti-inflammatory role, as well as RuB2A.
promoting the formation of new blood vessels. After the slow release of By analyzing changes in the peak wavelength of RuB2A2 with illu­
BMSCs into the wound environment, the hydrogel can be irradiated with mination time, the results show that the peak wavelength of RuB2A2
a light source to exert an antibacterial effect. tends to be stable after 120 s of illumination, suggesting complete
photolysis of only one Ru–pyridine bond (Fig. 2J). HPLC analysis
2.2. Characterization of DCS-RuB2A2 following the photodegradation of the crosslinker further confirmed the
photolysis products as RuB2A(H2O) and free ligand A (Fig. 2K).
To achieve rapid hemostasis and a synergistic antibacterial light- RuB2A2 has a good crosslinking reaction with DCS, which is attrib­
responsive hydrogel system, we first performed dodecyl substitution of uted to the fact that the aldehyde group of ligand A in RuB2A2 can un­
common CS. Previous research [49] has shown that alkyl substitution dergo a Schiff base reaction with the amino group of DCS under acidic or
can enhance platelet activation and blood cell adsorption. Scanning basic conditions to form better mechanical properties. In addition to the
electron microscopy (SEM) analysis was performed to characterize the adhesive hydrogel (Fig. 2H), the hemostatic properties of DCS are
microstructure of the hydrogel (Fig. 2A–D). CS was fragmented, with a retained and there is also a good photoresponse effect. SEM analysis was
flat, smooth surface and a small number of pores, whereas DCS had performed to characterize the microstructure of the DCS-RuB2A2
markedly more pores and the gel was flocculent. This sponge-like hydrogel (Fig. 2L and M). The structure of the crosslinked RuB2A2
structure greatly enhances the adsorption effect. hydrogel is obviously denser, indicating better mechanical properties,
The chemical structures of CS and DCS were characterized by Fourier which are closely related to the plasticity and adhesion properties of the
transform infrared spectroscopy (FTIR) (Fig. 2E). In the original CS gel in Fig. 2H. The FTIR results of RuB2A2 and DCS crosslinked with Ru
sample, the vibrational –OH group peak of the glycopolymer (approxi­ (bipyridine)2(3-pyridinecarboxaldehyde)2 (DCS-RuB2A2) are shown in
mately 3440 cm− 1) and the stretching peak of the methyl and methylene Fig. 2N. EDX analysis demonstrates that the Ru element has been suc­
groups (approximately 2800–2900 cm− 1) can be observed. cessfully crosslinked to the DCS hydrogel (Fig. 2O–Q).

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Fig. 1. Schematic showing the preparation and application of the DCS-RuB2A2-BMSCs hydrogel.

2.3. Coagulation and hemostatic properties of DCS between 0.4 DCS and CS was significant (p < 0.0001).

To determine the hemostatic properties of DCS-RuB2A2, a model of

hemostasis in the mouse tail was employed (Fig. 3A). It can be seen that 2.4. In vitro antibacterial performance
the bleeding volume in the materials group following treatment of the
tail wound is significantly less than that in the gauze group, and the To further explore the antibacterial ability of RuB2A2, the potential
hemostatic effect of DCS is better than that of CS. The hemostasis time antimicrobial activities of RuB2A2 were investigated using Escherichia
and amount of blood loss for each group are shown in Fig. 3C and D. coli and Staphylococcus aureus. Following the addition of RuB2A2 to co­
These results demonstrated that as the substitution degree of aldehyde cultures of E. coli and S. aureus, SEM characterization of the bacterial
groups in DCS increases, the bleeding volume and hemostasis time de­ surface was markedly altered, and objects appeared to be attached to the
creases and subsequently gradually increases, and the hemostatic effect surface, in addition to some of the bacterial contents being extracellular
was best at a substitution degree of 0.4 DCS. Blood clotting time (BCT) (Fig. 4A). This suggests that RuB2A2 attaches to the cell membrane of
was used to measure the blood coagulation properties of the materials. bacteria, changing the permeability of the cell membrane and allowing
Blood flowed freely when extracted from the vessel; however, under the part of their contents to escape, thereby exerting a bactericidal effect.
action of the hemostatic material, the blood clotted and formed a stable Different concentrations of RuB2A2 and bacteria were mixed and spread
clot. Fig. 3B shows the state of blood coagulation after treatment with on a nutrient agar plate (Fig. 4B). After culture at 37 ◦ C for 18 h, the
different materials. It can be seen that the blank group (blood only) is number of E. coli (Fig. 4C) and S. aureus (Fig. 4D) were counted. An
not completely coagulated despite being left to stand for a long time. An obvious antibacterial effect can be observed at a concentration of 0.125
obvious stratification phenomenon is formed between CS, 0.1 DCS, and mg/mL RuB2A2. As the concentration of RuB2A2 increases, the anti­
0.2 DCS, which may be due to the small intermolecular space, which bacterial ability is gradually enhanced, but the inhibitory effect on E. coli
cannot deeply adsorb blood. Encouragingly, 0.3 DCS, 0.4 DCS, and 0.6 is higher than that on S. aureus. In conjunction with the results of the
DCS are all infiltrated by blood, similar to the gel sponge, which can subsequent cytotoxicity test, it can be seen that at a concentration of
absorb all the blood in the pores of the gel molecules, thus exerting the 0.125 mg/mL, RuB2A2 significantly inhibits the growth of two repre­
effect of rapid coagulation. Fig. 3E shows that the time for the CS group sentative bacteria but has little effect on cell viability; therefore, this
to form a blood clot was approximately 400 s, while that for 0.4 DCS was concentration of RuB2A2 was used in the hydrogel for successful
no longer than 100 s. It can be clearly observed that 0.4 DCS had a much biocompatibility and antibacterial properties. Subsequently, the effect
stronger promoting effect on clotting than CS, and the difference of different treatments on the antibacterial properties of the hydrogels
was explored using the zone of inhibition (ZOI) method (Fig. 4E). The

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Fig. 2. Performance characterization of DCS-Ru2BA2 and exploration of the crosslinking mechanism. SEM images of CS (A, B) and DCS (C, D); scale bars 10
μm and 5 μm. (E) FTIR spectra of CS and DCS. (F) FTIR spectra of DCS with differing degrees of substitution. (G) Photographs of CS and DCS. (H) Photographs of DCS-
RuB2A2. (I) UV–Vis spectral evolution of RuB2A2 (100 μmol L− 1) in deionized water with increasing irradiation time (λex = 450 nm, 14 mW cm− 2). (J) The
wavelength corresponding to the UV absorption peak of RuB2A2 varies with illumination time. Pink represents the first stage and blue represents the second stage. (K)
HPLC analysis of the photodegradation of crosslinkers under dark conditions (blue) and after illumination (orange). (L, M) SEM images of DCS-RuB2A2; scale bars 10
μm and 1 μm. (N) FTIR spectra of DCS-RuB2A2. (O-Q) Mapping images of C, O, and Ru elements of DCS-RuB2A2 in (L).

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Fig. 3. Coagulation and hemostatic properties of DCS. (A) Photographs of blood loss after tail docking in mice. (B) In vitro coagulation profiles following
treatment with CS and DCS with differing degrees of substitution. (C) Histogram of blood loss in mice. (D) Histogram of hemostatic time in mice. (E) Statistical
histogram of blood clotting time. (n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

inhibitory effect of RuB2A2-DCS on E. coli (Fig. 4F) and S. aureus approximately 100%. RuB2A2 was crosslinked to our light-responsive
(Fig. 4G) under light conditions was significantly greater than that of the material and shown to have a good bacteriostatic effect (Fig. 4 B–G).
other groups, and the inhibitory effect on S. aureus was stronger. This It was necessary to further explore the optimal concentration at the level
indicates that light is necessary for RuB2A2-DCS to exert a bacteriostatic of epithelial cells. HEK 293T cells were cultured in the presence of
effect. different concentrations of RuB2A for 24 h and 48 h under light condi­
tions. RuB2A at a concentration of no more than 0.5 mg/mL shows good
2.5. Cytocompatibility of DCS-RuB2A2 biocompatibility after 24 h, with a cell viability of higher than 50%.
After 48 h, the cytotoxicity in the group treated with RuB2A at a con­
The biosafety of DCS-RuB2A2 is crucial for the use of hydrogels as centration of less than 0.125 mg/mL is still within an acceptable range
wound hemostasis and healing dressings. HEK 293T, adherent- (Fig. 5C and D). For a more insightful observation of cell survival,
dependent epithelioid cells, were used here as a model to mimic oral Hoechst 33342 and Propidium Iodide (PI) staining was used to differ­
epithelial cells in vitro. Cytotoxicity was assessed by exposing HEK 293T entiate between live and dead cells. Hoechst 33342 can penetrate cell
cells to CS (control group) and differing substitution degrees of DCS in membranes to label live cells with green fluorescence, but PI cannot
leaching solution for 24 h and 48 h. After treatment, cell viability was penetrate or stain normal cells with intact cell membranes. In the case of
measured using a Cell Counting Kit-8 (CCK-8). Fig. 5A and B shows that necrotic cells, the integrity of their cell membranes is lost and they can
both CS and DCS have good biocompatibility. In particular, after 48 h of be stained red. The survival rate and number of dead cells were detected
coculture, the viability of cells treated with CS did not change signifi­ 24 h after treatment with different concentrations of RuB2A. Fig. 5G and
cantly as compared with that at 24 h; however, the viability of cells Supplementary Fig. 2 shows that at concentrations below 0.125 mg/mL,
treated with 0.4 DCS (degree of substitution) was improved to RuB2A has a negligible effect on cell viability. Comprehensively

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Fig. 4. In vitro antibacterial performance. (A) SEM images of E. coli and S. aureus treated with the control and RuB2A2. (B) Growth of E. coli and S. aureus with
different concentrations of RuB2A2 added to the agar plates. Colony numbers of E. coli (C) and S. aureus (D). (E) ZOI method to test the effect of light on the
antibacterial properties of the hydrogel and its components. The areas of the inhibition zone for E. coli (F) and S. aureus (G) in each group were counted.

considering the bacteriostatic effect and cytotoxicity, 0.125 mg/mL is 2.6. Paracrine effects of BMSCs
considered the optimal concentration that produces minimal toxicity to
normal cells in the long-term and also displays a certain bacteriostatic In recent years, stem cells have been shown to benefit angiogenesis
effect. Since the oral environment is mostly under dark conditions, the through paracrine action, and at the same time, they can release cyto­
antibacterial effect of DCS-RuB2A2 under non-light conditions was kines, growth factors, chemokines, and extracellular vesicles to promote
general (Fig. 5E and F). RuB2A formed by picolinaldehyde shedding tissue regeneration and repair. Accordingly, to accelerate the healing of
after illumination exerts an antibacterial effect. HEK 293T cells were oral mucosal defects, BMSCs were loaded onto DCS-RuB2A2, and the
cultured in the presence of 0.4 DCS for 24 h and 48 h to explore the effect loose and hollow properties of the hydrogel provided a 3D structure
of light on cell viability. In comparison with the control and DCS-treated more conducive to their growth and release of cytokines. BMSCs were
groups, the cell viability of the DCS-RuB2A2-treated and DCS-RuB2A co-cultured with HEK 293T cells (Fig. 6A) to explore the effect of the
(light)-treated groups is decreased. The cell viability following DCS- BMSCs microenvironment on the growth, proliferation, and migration of
RuB2A2 treatment is 80–90% at both 24 h and 48 h under dark condi­ epithelial cells. The CCK8 assay was used to evaluate the proliferative
tions, while that following DCS-RuB2A (light) treatment is slightly lower ability of HEK 293T cells by detecting the OD values at 6 h, 12 h, 24 h,
at 75–85%. It can be seen that the light conditions significantly reduce 36 h, and 48 h. The results show that the proliferation ability of HEK
cell viability, indicating that DCS-RuB2A2 has a good light-controlled 293T cells co-cultured with BMSCs is significantly increased, suggesting
effect and the oral environment will not affect the normal epithelium. that the microenvironment of BMSCs is conducive to the growth and
Material have lower cytotoxicity and good biocompatibility, which is proliferation of epithelial cells (Fig. 6B). The cell scratch assay was used
the premise of hydrogels as wound healing dressings in the oral cavity. to test cell migration ability in the presence of BMSCs, and Fig. 6C and D
demonstrates that the size of the wound healing area between cell
scratches was significantly lower than that of the control group,

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Fig. 5. Cytocompatibility of DCS-RuB2A2. The CCK-8 method detected the cytotoxicity of CS and DCS with differing degrees of substitution (A, B) and RuB2A2 at
different concentrations (C, D). (E, F) CCK-8 assays for the biocompatibility of DCS-RuB2A2 under light and dark conditions. (G) (PI/Hoechst) live and dead cell
staining images and statistical analysis of HEK 293T cells cultured for 24 h, scale bars 50 μm (n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

suggesting that the microenvironment of BMSCs significantly enhances factors in a short period of time to promote the regulation of down­
the migration ability of epithelial cells. Lipopolysaccharide (LPS) is an stream signaling pathways, a mechanism that provides the possibility
inflammatory stimulator often used to construct cellular inflammatory for the rapid healing of oral wounds.
models. To explore the BMSCs paracrine effect on epithelial cells, an
inflammatory model of HEK 293T cells was constructed using an 2.7. Application of DCS-RuB2A2-BMSCs to promote mucosal defect
appropriate concentration of LPS, following which BMSCs were cocul­ healing in a rat model
tured. The expression levels of transforming growth factor beta (TGF-β),
platelet-derived growth factor (PDGF), epidermal growth factor (EGF), To determine the in vivo wound closure efficacy of the photoresponse
fibroblast growth factor (FGF), and vascular endothelial growth factor hydrogels containing BMSCs, oral mucosal defects were created in the
(VEGF) were measured. Interestingly, the mRNA expression levels of mouths of rats and an infection was established with E. coli and S. aureus.
TGF-β, PDGF, EGF, FGF, and VEGF were significantly increased Subsequently, 1 × 104 BMSCs were resuspended in 10 μL PBS, injected
following stimulation by the BMSCs microenvironment (Fig. 6E–I), into 3g DCS-RuB2A2 hydrogel, and allowed to incubate in the dark for 2
demonstrating that BMSCs can rapidly elevate the expression of growth min. The prepared samples were applied at the wound sites (Fig. 7A).

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Fig. 6. Paracrine effects of BMSCs. (A) Schematic diagram of the co-culture of BMSCs and HEK 293T cells. (B) CCK-8 assay to detect cell viability after co-culture of
HEK 293T cells and BMSCs. (C) Cell scratch assay to detect the cell migration ability of HEK 293T cells after co-culture with BMSCs. (D) Statistical histogram of the
cell migration ability after BMSCs co-culture. (E–I) Growth factor expression levels in HEK 293T cells after co-culture with BMSCs (n ≥ 3, *p < 0.05, **p < 0.01, ***p
< 0.001, ****p < 0.0001).

Fig. 7B shows images of the oral mucosal defects taken at different time Furthermore, following treatment with different materials, the
intervals after treatment with the control and DCS-RuB2A2 hydrogels in mucus was extracted from the oral wound into the medium and cultured
the presence and absence of BMSCs. There is no sign of inflammation or for 8 h at 37 ◦ C. Although external adverse stimulation is one of the main
infection in the DCS-RuB2A2-BMSCs-treated group and growth of new causes of inflammation, fewer bacteria were detected in the DCS-
oral mucosa can be observed, which resulted in a reduced wound area. RuB2A2-BMSCs- and DCS-RuB2A2-treated groups (Fig. 7D and E).
On day 4, the wounds treated with DCS-RuB2A2-BMSCs and DCS-RuB2A2 To further investigate the biological mechanism of the wound heal­
were almost completely healed and the oral mucosa was closed, whereas ing process, hematoxylin-eosin (H&E) staining and immunohistochem­
the control group still had obvious oral mucosal defects. In particular, ical analysis were performed on mouse wound tissue sections. Wounds
the oral mucosa in the DCS-RuB2A2-BMSC-treated group became very on day 4 of being covered with DCS-RuB2A2-BMSCs and the other
smooth after healing. Interestingly, DCS-RuB2A2-BMSCs and DCS- samples are shown in Fig. 8A. The surface of wounds treated with DCS-
RuB2A2 both had valuable wound closure effects in comparison with the RuB2A2-BMSCs and DCS-RuB2A2 show complete formation of new
DCS and control groups; therefore, both DCS-RuB2A2 and BMSCs can epithelium, while the control group still shows obvious oral mucosal
enhance wound healing in vivo. The results in Fig. 7C show that on day 4, defects as observed by H&E staining (Fig. 8A). As the main component of
the wounds treated with DCS-RuB2A2-BMSCs were significantly reduced the extracellular matrix, collagen binds to cell surface receptors and
as compared with the control. These results may be due to the material participates in signal transduction processes. In addition, collagen plays
properties of DCS-RuB2A2 hydrogels, such as the antibacterial and an important role in cell proliferation, migration, differentiation, and
platelet recruitment effects, which can enhance adhesion at the wound other cellular activities to promote adhesion, movement, growth, and
site, promoting wound contraction and closure during the early stages of deposition of cells on new connective tissue. At the same time, collagen
the wound healing process. Moreover, the introduction of BMSCs pro­ induces chemotaxis of fibroblasts to accelerate wound repair and
moted the release of many cytokines, which rapidly accumulate at the improve the quality of wound healing [67]. After 4 days of treatment,
site of inflammation and promote repair, which is beneficial for reduced retrieved tissues were subjected to Masson’s trichrome staining to
infection in the wound area during the initial stages of healing. identify collagen (Fig. 8A). It can be seen from the intensity and area of

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Fig. 7. Application of DCS-RuB2A2-BMSCs to promote mucosal defect healing in a rat model. (A) Schematic diagram of the construction of the rat oral mucosal
defect model. (B) Photographs of oral mucosal defects in rats. (C) The wound area in (B) was measured. (D) Schematic diagram of the local bacterial culture of the
extracted affected area. (E) OD values for the bacterial cultures at 600 nm after 8 h (n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

blue staining that there are significant differences in collagen deposition (Fig. 8A and D) show that DCS-RuB2A2-BMSCs promotes the production
among the experimental groups. The DCS-RuB2A2-BMSCs group has the of CD31 in wound tissue, and the blood vessel density is significantly
highest collagen deposition, indicating that this material has the best increased as compared with that in the other groups. These results
wound healing effect. Furthermore, DCS-RuB2A2-BMSCs-treated indicate that the risk of infection is reduced during the early stages of
wounds display more densely packed collagen fibers in the extracellular wound healing due to hemostasis initiated by DCS, in addition to the
matrix, which is in contrast to other samples that display loosely packed recruitment of platelets and the antibacterial effect of RuB2A2. The
collagen fibers. incorporation of BMSCs improves the production of cytokines and che­
Cytokines such as IL-6, TNF-α, and platelet endothelial cell adhesion mokines around the wound and simultaneously stimulates angiogenesis,
molecule-1 (CD31) play an important role in the process of wound providing a low-inflammatory environment to enable wound repair to
healing. Immunohistochemistry was used to explore the regulation of rapidly enter the proliferative stage of healing. Moreover, fibroblasts are
cytokines by incorporated BMSCs. As shown in Fig. 8B and C, in com­ derived from mesenchymal stem cells, which can produce a large
parison with the control group, the levels of IL-6 and TNF-α are signif­ amount of collagen, promoting the repair of wounds and aiding rapid
icantly reduced in the DCS-RuB2A2-BMSCs group. At this time, the recovery in approximately 4 days.
wound has entered the mature stage of healing and the inflammatory
factors are reduced. On the contrary, the control group is still in the
inflammatory or proliferative stage, in which the levels of IL-6 and TNF- 2.8. Bioinformatics analysis of oral mucosal defects after treatment
α remain high. Treatment with DCS-RuB2A2-BMSCs greatly accelerates
wound healing. The process of wound repair is accompanied by the To further explore the intrinsic molecular mechanisms of wound
formation of granulation tissue and deposition of collagen, and the repair by DCS-RuB2A2-BMSCs, oral wound tissue samples were collected
formation of new blood vessels is also indispensable. CD31 is an on day 4 for RNA sequencing (RNAseq). The heatmap of differentially
angiogenesis marker, staining and quantitative analysis of which expressed genes is shown in Fig. 9A. After administration of the
hydrogel, 241 significantly differentially expressed genes (DEGs) were

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Fig. 8. Histological evaluation of the oral mucosal trauma healing process. (A) Images of H&E and Masson’s trichrome staining, in addition to immunochemical
staining of the inflammatory factors IL-6 and TNF-α and angiogenesis factors at the wound site in rats treated with different materials for 4 days. (B–D) Quantitation
of IL-6, TNF-α, and angiogenesis factor levels in (A) (n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

identified, 147 of which were upregulated and 94 were downregulated, invasion of epithelial cells, viral myocarditis, neutrophil extracellular
according to the empirical bayes method (fold change ≥2; p value < trap formation and inflammatory mediator regulation of TRP channels
0.05). These include genes closely related to inflammation, such as IL- (Fig. 9D, Supplementary Fig. 3D. These results suggest that the key
17b, IL-Z11. A significant difference was found between the tran­ enriched items of DEGs are the bacterial invasion of epithelial cells,
scriptomics profiles of the control and hydrogel-treated groups, and immune system, metabolism, inflammation improvement concentrated
enrichment analysis was conducted based on the DEGs. Hierarchical in the extracellular matrix, neutrophil action, cellular action on growth
cluster analysis separated and screened the DEGs between wound tissues factors, and downstream signaling pathway regulation. This indicates
from the control and hydrogel-treated rats. Reactome enrichment that DCS-RuB2A2-BMSCs ameliorates bacterial-induced infections, re­
analysis and reactome annotations analysis demonstrates that the DEGs duces inflammation due to the paracrine effects of BMSCs, improves the
are mainly involved in the immune system, neutrophil degranulation, wound microenvironment by regulating cytokines, and promotes wound
hemostasis and extracellular matrix organization (Fig. 9B, Supplemen­ repair.
tary Figs. 3A and B). These results suggest that wound healing is closely
related to these processes, DCS-RuB2A2-BMSCs also promoted wound 3. Conclusions
healing by improving these effects. Gene ontology (GO) enrichment
analysis and GO annotations analysis revealed a focus on response to We designed and constructed a light-responsive antimicrobial
biotic stimulus, response to cytokine, cellular response to cytokine hydrogel that can present BMSCs for the topical treatment of oral
stimulus, response to bacterium and virus (Fig. 9C, Supplementary mucosal wounds. The developed hydrogel is based on dodecyl chitosan,
Fig. 3C). This indicated that the rapid wound healing in the DCS- which is loose and porous and possesses good hemostatic and coagula­
RuB2A2-BMSCs group was attributed to the paracrine effect of BMSCs, tion effects, in addition to being capable of activating platelets to pro­
which greatly promoted the release of growth factors and the killing mote wound healing. A light-responsive antibacterial system was
effect on bacteria and viruses. Kyoto Encyclopedia of Genes and Ge­ constructed using ruthenium bipyridine-crosslinked DCS, which has a
nomes (KEGG) enrichment analysis and KEGG annotations analysis of larger storage modulus and porosity under dark conditions, and is
DEGs found enrichments in regulation of actin cytoskeleton, bacterial suitable for the presentation and release of BMSCs. This moist, soft, 3D

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Fig. 9. Global assessment of the wound microenvironment using RNA-seq after treatment with the photoresponse material. (A) Heat map of the upregulated
and downregulated genes in the wound microenvironment after treatment with the photoresponse material (fold change ≥2 and p < 0.05). (B) Reactome enrichment
analysis of the DEGs. (C)Gene ontology (GO) enrichment analysis of the DEGs. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of
the DEGs.

culture system is conducive to the release of growth factors and cyto­ fragmented elastin and collagen, and loss of water, leading to dry skin
kines at the wound site. Following exposure to light for a period of time, and increased wrinkles [68]. Accumulation of senescent cells in the
the pyridine carboxaldehyde in DCS-RuB2A2 becomes detached, and elderly leads to increased inflammation, decreased skin immune func­
RuB2/RuB2A is removed from the gel network and anchors to the bac­ tion, and higher susceptibility to bacterial infections [69,70]. Wound
terial membrane with help from the long dodecyl chain of DCS, which healing is also affected by age, leading to chronic wounds. Clinical data
plays a local sterilization effect. The system has good biocompatibility demonstrate that oral mucosal wounds in the elderly recover more
and broad-spectrum antibacterial properties. In addition, when com­ slowly than those in younger individuals [71]. Oral infection leads to
bined with the anti-inflammatory and repair-promoting effects of inflammation, which reduces the capacity of oral mucosal wounds to
BMSCs, the material can repair oral mucosal wounds in a short period of recover and may even increase the risk of systemic diseases, such as
time. Our study shows that this multifunctional light-responsive atherosclerosis, coronary heart disease, stroke, chronic obstructive
hydrogel has commercialization potential and further supports the pulmonary disease, diabetes, cancer, and rheumatoid arthritis. There­
development of innovative treatments for oral mucosal diseases. fore, it is meaningful to research and develop materials to promote oral
Aging causes skin to become more vulnerable to mechanical damage, mucosal healing in aged subjects. Current bioactive materials are used to
in addition to causing changes in skin microbiome, increases in promote wound contracture and significantly increase collagen

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deposition at the injured site, thus accelerating the repair of skin wounds nutrient agar plates. After culture at 37 ◦ C for 18 h, the plate was pho­
[72,73]. As an effective bactericidal and early anti-inflammatory ma­ tographed and the number of colonies were recorded. The effect of light
terial, DCS-RuB2A2-BMSCs should be further explored to evaluate on the bacteriostatic properties of the hydrogel and RuB2A2 was eval­
whether it exerts a significant reparation effect in aged subjects and uated using the zone of inhibition (ZOI) method. A 100-μL aliquot of
other severe wound models that are difficult to repair. bacterial suspension (104 CFU/mL) was spread evenly on each nutrient
agar plate, after which the hydrogel and its components were placed on
4. Experimental section top. After culture at 37 ◦ C for 18 h, the RuB2A2 and 0.4 DCS + RuB2A2
groups were treated with light at the same time, and the size of the ZOI
4.1. Materials was observed.

Ru(bpy)2Cl2 and 3-pyridinecarboxaldehyde were purchased from 4.5. Animals

Beijing Huawei Ruike Chemical Co., Ltd. Paraformaldehyde-
glutaraldehyde in PBS was bought from ShanghaiMacklin Biochemical C57BL/6 mice and SD rats (6–8 weeks old) were purchased from
Co., Ltd. Chitosan was obtained from Sigma-Aldrich Co., Ltd. Chloral Vital River Laboratory Animal Technology (Shanghai, China). All ani­
hydrate was procured from Sangon Biotech (Shanghai) Co., Ltd. mals were bred and maintained under specific pathogen-free conditions.
Deionized AMTA, N, N-dimethylformamide (DMF), and sodium cyano­ Animal use and experimental procedures were approved by the Animal
borohydride were purchased from Shanghai Aladdin Biochemical Care and Use Committee of Shanghai University. The HEK 293T cell line
Technology Co., Ltd. Anhydrous ethanol (C2H5OH) was bought from was obtained from the Cell Culture Center of the Chinese Academy of
China Medicine (Group) Shanghai Chemical Reagent Corp.I. All chem­ Medical Sciences.
ical reagents were used as received without further purification. Water
(18.2 MΩ cm) obtained from a Milli-Q system (Millipore, Bedford, MA) 4.6. Isolation and culture of cells
was used for all experiments.
Isolation and culture of BMSCs was performed as previously reported
4.2. Preparation of RuB2A2 [74,75]. The isolated cells and HEK 293T cells were cultured in com­
plete DM/F12 (Thermo Scientific, Gibco) and DMEM (Biological In­
A mass of 200 mg (0.412 mmol) Ru(bpy)2Cl2 was dissolved in 10 mL dustries) supplemented with 10% FBS (Thermo Scientific, Gibco).
water under argon protection and refluxed for 5 h at 80 ◦ C. A solution of
264 mg (2.472 mmol) 3-pyridinecarbaldehyde in 10 mL ethanol was 4.7. Mouse tail hemostasis test
added dropwise to the above reaction, which was subsequently refluxed
overnight. After the reaction, ethanol was removed and the solid was A similar hemostatic procedure was performed in a mouse tail
cooled to room temperature. 1 M NH4PF6 was added and the solution amputation model. Approximately 50% was cut off the length of the tail,
was extracted with dichloromethane (DCM). The lower organic phase and the blood was allowed to drip onto a pre-weighed filter paper.
was taken, dried over magnesium sulfate, and filtered, and the solvent Subsequently, the mouse tails were inserted into tubes containing
was evaporated to dryness to obtain a brown-red solid. A DCM to hydrogels with differing degrees of substitution, and the tail blood was
methanol ratio of 6:1 was used as the eluent for silica gel chromatog­ gently absorbed. The tails were removed every 30 s to absorb the blood
raphy column separation to remove excess 3-pyridinecarboxaldehyde, with filter paper, and images were taken. A group treated with gauze
and the pure orange-red solution was collected. The solvent was evap­ served as a control. Blood loss and time to hemostasis were monitored.
orated to dryness, and methanol was subsequently used as the solvent to
pass through a chloride ion-exchange column. Methanol was evaporated 4.8. Blood clotting time (BCT)
to dryness to yield the final product as a brown-red solid.
An equal amount of CS or DCS was placed in a glass vial, preheated
4.3. Synthesis of dodecyl-modified chitosan (DCS) with the extracted blood at 37 ◦ C for 5 min, and then 200 μL blood was
added to the surface of the material. CaCl2 (25 mM) was pipetted into
The synthesis of DCS was performed based on previous work with the tube and incubated at 37 ◦ C. Timing from this point, the tube was
some modifications. Specifically, 1 g chitosan (CS) was added to 50 mL tilted every 30 s until the blood had completely coagulated; this is the
2% acetic acid aqueous solution at room temperature with stirring to BCT. Each group was measured at least three times.
dissolve. To make the reaction more homogeneous, different amounts of
dodecanal (molar ratio of dodecanal to chitosan: 0.1:1, 0.2:1, 0.3:1, 4.9. Cytotoxicity assessment of the hydrogel and its components
0.4:1, 0.6:1, 0.8:1) was dissolved in 40 mL ethanol, and this solution was
then added to the chitosan solution under constant stirring until it had HEK 293T cells were seeded at 4 × 103 cells per well (in 100 μL) on a
dissolved. Subsequently, excessive sodium borohydride (NaBH4:CS = 96-well plate and allowed to attach for 24 h. Different materials were
3:1) was added to this mixture in small amounts and stirred at room then added to the wells and incubated for a further 24 h. The CCK-8 test
temperature until it dissolved. An appropriate amount of sodium hy­ was used to determine the cell viability in the experimental group and
droxide (NaOH) solution was added to adjust the pH of the mixed so­ expressed as a percentage of viable cells relative to the control.
lution to 7.0, which was then stirred at room temperature overnight. The Hoechst 33342/propidium iodide (PI) double staining assay: For live
following day, the precipitated DCS was washed at least three times with and dead cell visualization, HEK 293T (1 × 105) cells were seeded on a
70–100% ethanol until the pH value was neutral. Finally, the pre­ cover slip in a 24-well plate with different gel treatments under 5% CO2
cipitates were dried to obtain DCS powder. at 37 ◦ C. After 24 h of incubation, the supernatant was removed, and
cells were washed with cold PBS. HEK 293T cells were labeled with
4.4. In vitro antibacterial assay of DCS-RuB2A2 Hoechst 33342 and PI at 37 ◦ C for 20 min, after which the cells were
washed three times with PBS and visualized using a Leica fluorescence
Taking Escherichia coli and Staphylococcus aureus (E. coli and microscope (Germany).
S. aureus) as model bacteria, the antibacterial ability of RuB2A2 at
different concentrations was assessed using the spread plate count 4.10. Oral mucosal trauma model construction
method. After mixing 5 mL bacterial suspension (104 CFU/mL) with
different concentrations of RuB2A2, the mixture was spread evenly on Male SD rats (average weight 200 g) were anesthetized by

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intraperitoneal injection of 7% chloral hydrate (0.1 mL/10 g). Trauma Science Foundation of China (81971324).
was established on the oral mucosa of rats using a dental drill (round,
approximately 5 mm in diameter), and the wound was then infected Appendix A. Supplementary data
with 20 μL 108 CFU/mL E. coli and S. aureus. After 1 h, the experimental
group was treated with hydrogel samples, while the control group was Supplementary data to this article can be found online at https://doi.
treated with saline. On days 0, 1, 2, and 4 after induction of the wound, org/10.1016/j.bioactmat.2022.10.027.
photographs of the different groups were taken using a camera at a fixed
height to record the size and appearance of the wounds. The wound References
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