Acrylamide (AM, >98%), ammonium persulfate (APS, >98%), acetic acid (>99.5%), and ethanol were purchased from Sigma-Aldrich Co., Ltd. (Canada). N,N,N′,N′-tetramethyl- 1,2-ethanediamine (TEMED, >98%), Chitosan (CS, deacetylation value, 75–85%), and sodium hydrate (NaOH) were obtained from TCI (Tokyo Chemical Industry Co., Ltd, Japan). Tryptone and yeast extract were obtained from OXOID Co., Ltd (USA). Agar Powder was purchased from Solarbio Co., Ltd (China). Sodium chloride (NaCl, 99.9%) was obtained from JHD Co., Ltd (China). DMEM/F-12 culture medium, trypsin-EDTA (0.25%), penicillin-streptomycin, cell counting kit-8 (CCK-8), and the hematoxylin and eosin (H&E) staining kit were all obtained from Yeasen Biotech Co., Ltd (China). Fetal bovine serum (FBS, Gibco) was purchased from Thermo Fisher Co., Ltd (USA). Chloral hydrate, D₂O, and DCl was obtained from Macklin Biochemical Co., Ltd (China).

Based on our recently reported work with slight modifications, the chitosan-based polymer was synthesized using an in-situ polymerization method in dilute acetic acid (Zhang C. et al., 2019). Chitosan (0.4 g) was dispersed in a dilute acetic acid aqueous solution (1%, 20 mL) and stirred violently at room temperature for 4 h. The insoluble substances were removed by filtration. Then the AM monomer (AM:CS = 7:2 mass ratio) was added to the reaction solution and continuously stirred under N₂ for ~20 min at room temperature. APS and TEMED catalysts (1:1 molar ratio at 3 mol% of the monomer concentration) were then added to the solution and stirred for 10 min. Subsequently, the mixture was heated to 30°C for 24 h. The solution pH was adjusted to 9.0 with a NaOH solution (1 M) to obtain the precipitant. The addition of a few milliliters of ethanol further facilitated precipitation. This purification process was repeated twice. After that, the system was dialyzed for 3 days at room temperature to eliminate unreacted substances (spectrum dialysis membrane, MWCO 10 kDa). This solution was then frozen and dried to obtain a light-yellow solid. The yield of the CP copolymer was ~85%.

FTIR spectra were recorded using a KBr pellet (4,000–400 cm⁻¹) on a Perkin-Elmer FTIR2000 spectrometer (Perkin-Elmer Co., Ltd, UK). An average of 64 scans were taken at room temperature with a resolution of up to 2 cm⁻¹. The coupling sample was dried in a vacuum at 50°C for 1 day before testing. ¹H NMR spectra of conjugates at room temperature were measured with a JEOL ECA 500 MHz NMR spectrometer (JEOL Co., Ltd, Japan). D₂O, containing 0.1 M DCl, was used as the solvent. The chemical shifts were reported in 1/1,000,000 (ppm). The solvent peak of D₂O is δH 4.70.

The antibacterial effects of the CP copolymer were investigated using an inhibition zone method (Lu et al., 2017). Staphylococcus aureus (1,000,000 CFU/mL) and Escherichia coli (1,000,000 CFU/mL) cultures were evenly coated on a solid LB medium plates. Circular copolymer samples (with a diameter of 6 mm) were then deposited on the surface of the solid medium; the plates were then incubated at 37°C for 24 h. The antibacterial effect of the CP copolymer at different concentrations was evaluated by observing the size of the inhibition zones around the hydrogel samples.

Human gingival fibroblasts (HGF-1, Shanghai Sixin Biotech.Co., Ltd., China) were cultured in a cell incubator with DMEM/F12 media, FBS (10%), and penicillin-streptomycin (100 μg/mL) with 5% CO₂ at 37°C.

The viability of HGF-1 cells incubated with the CP copolymer was investigated using a CCK-8 assay. Cells were cultured in sterile 96-well plates at 4.8 × 10⁵ cells/mL (4.8 × 10⁴ cells per hole) with different concentrations of the CP copolymer for 24 h. Next, 10 μL volume of CCK-8 reagent was added to each well, and sterile 96-well plates were incubated for another 3 h. The absorbance of the samples at a wavelength of 450 nm was measured using an enzyme labeling instrument (Perkin Elmer Co., Ltd, USA).

The cell growth evaluation of HGF-1 cells was determined using the CCK-8. When the number of HGF-1 cells reached 2.88 × 10⁵ cells/mL, the cells were cultured in sterile 96-well plates with different concentrations of the CP copolymer for 24 h. Fresh media was replaced every day. Next, 960 μL of CCK-8 was added to the 96-well plates at 12, 24, and 48 h of culture and sterile 96-well plates were incubated for 4 h. The absorbance wavelength (450 nm) was measured with an enzyme labeling instrument, and cell growth evaluation were calculated for each time point from the average OD values.

Filter paper (3 mm × 3 mm) samples with 20 μL of a 70% acetic acid solution were applied (for 1 min) to the gingival mucosa location of healthy mature Wistar rats (Shanghai SLAC Laboratory Animal Co.,Ltd, China) that were anesthetized with 10% chloral hydrate. Application of 70% acetic acid was repeated for two consecutive days to establish the animal model. Animal treatment procedures and feeding were conducted as per the Xiamen University Animal Care Guidelines.

The blood of Wistar rats was collected by retro-orbital extraction into EP tubes that contained heparin. The blood was mixed with the CP copolymer samples (2:1 volume ratio) and incubated in a water bath at 37°C. Untreated rat blood was used as the control group.

After animal models were established, 20 μL of a 5 wt% CP copolymer solution was applied to the festering site in the oral cavity of anesthetized rats [chloral hydrate (10%)] every day. Each experimental group contained three rats, and pictures were taken every 5 days with a camera to measure and record the length and width of the oral cavity using Vernier calipers.

After 10 days of administration, the rats were sacrificed, and tissues were taken isolated and immersed in 15 or 30% sucrose solutions for 24 h. The tissues were frozen and sliced into sections of 7 μm in thickness, then stained with an H&E stain for microscopic observation. All animal treatment procedures and feeding were conducted following the Xiamen University Animal Care Guidelines.

All experimental data are represented as the mean ± standard deviation. All experimental data and figures were analyzed using GraphPad Prism 5.0 and OriginPro 8. Statistical significance was indicated when the p-value was <0.05.

Our recent work presented a simple, one-pot procedure to prepare chitosan conjugate compounds (Zhang C. et al., 2019; Zhang Z. et al., 2019). The CP copolymer was prepared by in-situ free-radical-induced polymerization using TEMED as an accelerator and APS as an initiator in an acetic acid solution (Figure 1). The sulfate radical produced by thermal decomposition of the APS initiator caused AM polymerization to create a PAM long chain structure, which further polymerized with chitosan in situ (Huang et al., 2013; Echeverria et al., 2015; Wang and Yu, 2015). CP copolymers were synthesized with a mass ratio of 3.41:1 (PAM:CS). The characteristic chemical shift peaks of PAM and chitosan could both be observed in the ¹H NMR spectra of the CP copolymer (Figure S1). The specific characteristic peaks were as follows: 2.38–1.10 m (protons of the main chain, br); 0.97 m [-CH(CH₃)₂, br] belong to the PAM polymer; 4.12–2.77 m (protons, br), 2.28–1.98 m [-CH₂-CH(CONH₂)-, br], 1.93–1.88 m (-NHCO-CH₃, br), and 1.79–1.21 m [-CH₂-CH(CONH₂)-, br] belonging to chitosan. The mass ratio (calculated by signal integration from the ¹H NMR spectra) of PAM and chitosan in the conjugates was close to that of the initial ratio of monomer to chitosan. The monomer conversion rate of PAM was typically higher than 99%, and the conjugation yield was higher than 85%. The above data indicated that the polymer was grafted onto the chitosan backbone.

The FTIR spectra of conjugates and chitosan were collected to determine the extent of PAM grafting onto the chitosan backbone (Figure S2). The characteristic peaks of chitosan were seen at 1,081 cm⁻¹ [s, ν(C-O-C) and ν(C-O)], 1,650 cm⁻¹ [s, ν(C=O)], 2,879 cm⁻¹ [m, ν_s(C-H) and ν_as(C-H)], and 3,440 cm⁻¹ [strong and wide, br, ν(O-H) and ν(N-H)]. The FTIR spectrum of the CP copolymer also showed additional peaks at 1,452 cm⁻¹ [m, ν(C-N)] due to the amide group of PAM, 1,658 cm⁻¹ [s, ν(C=O), 2,930 cm⁻¹ (m, ν_as(C-H)], and 3,000–3,750 cm⁻¹ [br, ν(O-H) and ν(N-H)] due to the amide groups of chitosan and PAM. These FTIR results further indicated that PAM grafting onto the chitosan backbone was achieved by in situ free-radical polymerization.

Oral bacterial infections often increase the burden of ulcer wound healing, necessitating effective antibacterial treatments. While the antibacterial activity of chitosan is well-known (Sonis et al., 2014; Zivanovic et al., 2007), its specific antibacterial mechanism has not been definitively established. Dina et al. suggested that chitosan's antibacterial ability is regulated by a number of diatheses, which are concentration-dependent and may be related to binding of the tea polyacid and potential extraction of lipids (Xiao et al., 2000; Dina et al., 2008). Other research indicates that the pH, temperature, and the ionic strength in the microenvironment strongly influences chitosan's antibacterial ability. At an acidic pH (pH < 6) and high temperature (25–37°C), the increase in ionic strength can significantly improve the sterilizing ability of chitosan, but the metal ions can inhibit the antibacterial effects of chitosan (Guo-Jane and Prot, 1999; Chung et al., 2003). In this study, the antibacterial activity of the CP copolymer system was investigated using E. coli and S. aureus (Figure 2). The CP copolymers were transferred to the E. coli and S. aureus mediums, and the growth status of the colonies near the samples was observed. Bacterial cultures were challenged with three CP copolymer concentration gradients (3.0 to 7.5 wt%) to verify the relationship between sample concentration and antimicrobial activity. It was easy to observe that the CP copolymer showed antibacterial activity as the antibacterial area (cm²) increased with the increasing polymer concentration. The high antibacterial activity of the CP copolymer is likely related to the high specific gravity of chitosan.

The ability of the CP copolymer to mediate human fibroblast proliferation in the oral cavity was investigated with human gingival fibroblasts (HGF-1), which are often used to study mucosa-related diseases (Häkkinen et al., 2014; Vahabi et al., 2015). The CP copolymer (0 to 1,000 μg/mL) demonstrated no cytotoxic effects to the HGF-1 cells during 48 h of culture (Figure 3A). A CCK-8 assay was used to determine the effects of the CP copolymer on HGF-1 cell growth (Figure 3B). Surprisingly, the CP copolymer strongly promoted HGF-1 cell growth. Clearly, the CP copolymer system significantly promotes cell DNA replication, thereby promoting cell proliferation. The specific gravity of chitosan and PAM caused the increase in HGF-1 cell proliferation in this system.

Oral ulcerative lesions are often accompanied by bleeding, which causes patient discomfort and is not conducive to wound healing. Therefore, a hemostatic polymer system is needed to treat oral ulcerative lesion diseases (Lee et al., 2014). Natural, biodegradable chitosan is the only positively charged cationic polysaccharide that can interact with negatively charged red blood cells to form a thrombus (Murugan and Ramakrishna, 2006; Yang et al., 2008). Chitosan can also activate platelets and inhibit the dissolution of fibrin, thereby promoting blood coagulation and hemostasis (Wu et al., 2004; Zigang et al., 2004; Li et al., 2016). To investigate the hemostatic effects of the CP copolymer, blood was collected from Wistar rats (in heparinized EP tubes) by retro-orbital blood sampling. The blood was then mixed with the CP copolymer and incubated in a water bath at 37°C. As can be seen from (Figure 4A), the CP copolymer clotted the blood after 30 s. (Figure 4B) shows that the coagulation time of the CP copolymer was much shorter than that of the control due to the combination of the chitosan skeleton and PAM.

As the in vitro experiments showed that the CP copolymer had a promoting effect on the proliferation of human gingival fibroblasts, the in vivo effects of the CP copolymer on oral ulcers were investigated to determine its potential as a therapeutic agent. An oral ulcerative lesion model in Wistar rats was used to investigate the therapeutic effect of the CP copolymer by monitoring the degree of healing every 5 days over a 10-day period (Figure 5A). The swelling area of rats treated with the CP copolymer after 10 days was much lower than the swelling area of rats in the control group.

(Figure 5B) summarizes the change in the gingival ulcer area over 10 days, which showed that the CP copolymer had a reduction of 46% in the ulcer area within 5 days, compared to a 33% reduction in the area in the control group. It is worth mentioning that after 10 days of treatment, the ulcer area in the CP copolymer group decreased by 85%, compared to a 72% decrease in the control group, indicating that the CP copolymer effectively promoted the healing of oral ulcerative lesions. (Figure 6) shows the change in body weight of the Wistar rats, which was used to further determine the treatment effect on oral ulcerative lesions. It can be seen that the body weight of the five control group rats showed an acute downward trend in the first two days, which may be caused by difficulties in eating due to the induced oral ulcerative lesion. The body weights of rats that recovered after treatment with the CP copolymer were higher than those of rats in the control group, indicating that the CP copolymer treatment resulted in a gradual reduction of the oral ulceration symptoms and the recovery of feeding. This indicates that the CP copolymer effectively improved oral festering and promoted wound healing in Wistar rats. The CP copolymer displayed antibacterial effects and simultaneously promoted gingival fibroblast proliferation, making it an effective gingival ulcer treatment.

To further investigate the therapeutic effects of the CP copolymer on rat oral gingival mucosa ulcers, histological sections of gingival ulcer sites were analyzed (Figure 7). The gingival ulcer tissue sections were stained with H&E stain, and then observed under a light microscope. Purple-blue staining (hematoxylin) mainly shows chromatin and nucleic acids, and purple-red staining (eosin) shows the extracellular matrix and cytoplasm. In the untreated control group, the cells between the basal layer and the keratinized layer of the epidermis were relatively loose with clear inflammatory cell infiltration. Compared with the control group, the number of newly formed fibroblasts in the CP copolymer group was significantly increased, and the tissue damage was decreased.