Synthesis N,N,N-trimethyl chitosan(TMC)

Trimethyl chitosan (TMC) chloride was synthesized and characterized following a procedure similar to that published by Abueva et al. [28]. Low molecular weight chitosan (molecular weight of 50–190 KDa from the crustacean, Sigma-Aldrich, USA) was used to make TMC with N-methyl-2-pyrrolidone (NMP, Sigma-Aldrich, USA), sodium iodide (Merck Millipore, USA) and iodomethane (Sigma-Aldrich, USA). TMC was dialyzed for purification and then lyophilized to collect the product.

Formulation and degradation test of thermosensitive hydrogel

A 4% (w/v) stock solution of synthesized TMC was prepared by dissolving it in Dulbecco’s phosphate-buffered saline (DPBS). Additionally, a series of β-glycerophosphate solutions (β-gp) were prepared by creating a 20% (w/v) stock solution. The chitosan solution was prepared in an ice bath on top of the stirrer and continuously mixed while adding β-gp solution dropwise. The different formulations based on TMC and β-gp composition are listed in Fig. 1A. Upon complete mixing Each solution was pipetted (400 µl) into separate wells in a 24-well plate then the plate was placed in a 37 °C incubator for 1 min. After incubation, the plate was inverted to evaluate the gelation of each of the solution. This simple test validates that the formulations that formed stable gels were able to resist flow while non-gelled solutions showed low viscosity upon inversion. Formulations that successfully solidify into a gel resisted flow upon inversion of the plate.

Fig. 1

Hydrogel fabrication optimization and degradation measurement. (A) The images of gelled hydrogels according to the concentrations of TMC and β-gp. Gelation results were observed at 37 ℃ by mixing 1% and 2% TMC with different concentrations of β-gp. (B) Images of gelled samples during the degradation test observed every other day for 7 days. The arrow indicates the hydrogel fragments

A degradation test was conducted by placing 500 µL of the hydrogel onto a confocal dish and allowing it to solidify. After the hydrogel solidified, 2 mL of saline was added, and the dish was placed in a 37℃ incubator. The hydrogel was visually observed daily for a period of 7 days to monitor any signs of fragmentation or dissolution.

Isolation and characterization of human tonsil-derived mesenchymal stem cells (hTMSCs)hTMSCs isolation and culture

Human tonsil-derived mesenchymal stem cell isolation and culture using discarded tonsils were obtained from children who underwent a tonsillectomy at the Dankook University Hospital (DKUH, Cheonan, Korea). We obtained written informed consent from the parents or guardians of the children who participated in the study (DKUH 2022-04-012).

The tonsil tissue was extensively washed with phosphate-buffered saline (PBS), containing 1% penicillin − streptomycin (Corning, USA), followed by digestion with collagenase type I (Sigma, St. Louis, MO, USA) for 30 min at 37 °C. The pellet was filtered through a 100 μm and 40 μm nylon mesh. Suspended cells were incubated overnight in Dulbecco’s Modified Eagle Medium (DMEM) (Corning, USA) containing 10% fetal bovine serum (FBS) (Corning, NY, USA) and 1% penicillin − streptomycin at 37 °C with 5% CO2. Non-adherent cells were removed by extensive washing with PBS, and adherent cells were maintained and subcultured. The cells were cultured in DMEM supplemented with 10% FBS and the media was changed every 2 days. When the TMSCs reached 80–90% confluence, they were treated with 0.25% trypsin-EDTA (Corning, USA) for 5 min. The detached cells were collected by centrifugation at 1200 rpm for 5 min.

Flow cytometric analysis

Flow cytometric analysis was used to characterize the phenotypes of isolated hTMSCs. At least 5 × 104 cells dispersed in 100µL of PBS containing 0.5% bovine serum albumin (BSA) (37,525, Gibco) were incubated with conjugated monoclonal antibodies (PE/CD90, APC/CD105, PE/CD34, BD) for hTMSCs. Labeled cells were analyzed by flow cytometry using flow cytometry (Beckman Coulter).

Preparation of combined treatment sample

In order to conduct a combined treatment, a composite sample containing both hydrogel and stem cells was prepared. Following the preparation of the hydrogel, the cultured stem cells were harvested and diluted in DPBS and then mixed using a stirrer. The quantity of stem cells mixed with the diluent was adjusted to be within 5% of the total volume of the hydrogel to be produced. The resulting composite treatment sample was obtained by dispersing 2 × 106 cells in 100µL of the hydrogel.

Cytotoxicity test of hTMSCs in TMC hydrogel

According to the manufacturer’s directions, the cytotoxicity in the hydrogel with hTMSCs was determined by the LIVE/DEAD™ Cell Imaging Kit (Invitrogen, USA). Briefly, cells were washed once with DPBS and then incubated with live green and dead red solutions (Live/Dead Cell Imaging Kit, Invitrogen, USA) for 15 min at room temperature in media without FBS. The cells were then imaged using EVOS with phase contrast, red fluorescence, and green fluorescence channels. image analysis was performed on EVOS M7000 (Invitrogen, USA) fluorescence micrographs (n = 10) using ImageJ software (1.53a version; National Institutes of Health).

In vitro co-culture assay

The RAW 264.7 macrophage cells (1 × 106 cells) were plated in 24 mm polyester membrane transwell inserts (0.4 μm pore size, Corning, USA) and allowed to attach for 24 h prior to treatment with lipopolysaccharide (LPS) Sigma-Aldrich, USA) for M1 phenotype induction. Macrophage cells were treated with 1 µg/mL LPS in DMEM medium supplemented with 10% FBS and 1% P/S for 24 h. The transwell inserts’ lower compartments were seeded with hTMSCs (1 × 106 cells), and placed with 2 mL hydrogel or 2 mL hydrogel with hTMSCs of the same cell number the following day. Co-culture with macrophage cells was kept for 24 h in DMEM medium supplemented with 10% FBS and 1% P/S. All cultures were kept at 37oC and 5% CO2. Macrophage cells were collected the next day and prepared for RNA extraction.

In vivo oral ulcer induction and treatment

Thirty-two male SD rats aged 7 weeks were used for this study. Rats were randomly separated into four different groups, including control (n = 8), stem cell (n = 8), hydrogel (n = 8), and combined therapy (n = 8). In each group, four animals were used per experiment for tissue analysis, and the remaining 4 animals were used for molecular biological analyses. The animals were tested after a week of adaptation, and 4 rats were housed per cage. All animals received food and water and were maintained on a 12-hour light/dark cycle in a climate-controlled animal room within the university research facility. All aspect of animal research was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Dankook University (DKU-23-037).

Animal modeling followed a similar procedure as previously published by Ryu et al. [31]. This study induced oral ulceration in rats by injecting 15 µL of 60% acetic acid into the right oral mucosa after anesthesia. After 3 days, the presence or absence of oral ulcers was assessed in each rat, followed by a submucosal injection of 100 µL of each respective treatment into the ulcer area. Animals in the positive control group were injected with only PBS and were designated as sham. Treatments containing stem cells (hTMSCs group and Hydrogel + hTMSCs) were prepared with 1 × 106 cells. The size of the ulcer was measured using digital photos taken on Day 0, Day 1, Day 3, and Day 7.

Histological analysis

Rats were euthanized using CO2 inhalation on Day 3 and Day 7 following the treatment, and their oral mucosa was surgically removed for subsequent histological examinations. The wound area was excised and fixed from each mucosa in a solution of 4% Paraformaldehyde. The fixed tissues were then processed, embedded in paraffin, and sliced into sections of 4 μm thicknesses. Following deparaffinization and rehydration, the prepared specimens were mounted on slides and subjected to staining with hematoxylin and eosin (H&E). The H&E staining was employed to assess the thickness of the mucosal layer and count blood vessels to evaluate wound healing. Additionally, Masson’s trichrome (MT) staining was performed to examine the collagen content and the pattern of collagen arrangement in the wound area. Using FIJI software (ImageJ with built-in plugins), the fibrous tissue composition of the regenerated tissue was quantified by performing blue color thresholding and measuring the area occupied by the segmented blue-stained collagenous tissue within the tissue area of each image [32, 33].

Immunofluorescence (IF) was performed to visualize implanted stem cell detection and immunohistochemical (IHC) staining was also performed to visualize neutrophil expression and inflammation cytokine detection. In brief, de-paraffined and rehydrated tissue sections were processed for antigen retrieval by immersion in citrate buffer (10 mM, pH 6.0) using the microwave. Tissue sections were then blocked with 3% BSA and incubated with primary antibodies, either Neutrophil elastase (1: 100; Abcam, UK), Arginase-1 (Arg-1) (1:200; Invitrogen, USA) or Inter Luekin (IL)- 1 beta (1:200; Abcam, UK) at 4 °C overnight. IF was treated Ku80(1:200; Cell signaling) and Thy-1(BD biosciences, USA) with fluorescent conjugated secondary antibody (Alexa Fluor 488, PE; Abcam, UK) and DAPI stain (nuclear stain). IHC was treated with horseradish peroxidase-conjugated secondary antibody and color-developing reagent from the EnVision Detection System Peroxidase kit (Dako Co. Denmark). Slides were then counterstained with hematoxylin to provide contrast for parts stained using the antibody. The quantitative analysis of the IHC staining was conducted using FIJI software -ImageJ with built-in plugins. High-magnification images were loaded as virtual stacks and processed with HDab color deconvolution to separate out brown-positive staining from all other colors. Thresholding was then applied, followed by particle analysis processing. The summarized values of each image were used to determine the area coverage of the brown-positive stain.

Reverse-transcription polymerase chain reaction (RT-PCR)

Oral mucosa samples were processed by homogenizing each sample in a tube containing 1 mL of TRIzol reagent (Invitrogen, USA). The RNA extraction procedure was carried out using RNA extraction kits (Hybrid-R, Geneall, Biotechnology, Seoul, Korea), and the purity and concentration were assessed using Nanodrop 2000 (Thermo Fisher Science, USA). To generate cDNA, the purified RNA underwent synthesis using HyperScript™ RT Master Mix (Geneall Biotechnology, South Korea). Five genes were selected for analysis, and detailed primer information is described in Table 1. The quantification and analysis of mRNA expression were performed using the RT-PCR method employing the 7500 Real-Time PCR system (Thermo Fisher Science, USA).

The primer sequences, including the forward and reverse primers (Table 1), were designed by consulting primer-BLAST in the NCBI database and were procured from Macrogen (Macrogen, South Korea). The GAPDH gene was utilized as an internal control for normalization purposes to ensure standardized sample amounts.

Table 1 Primer sequences of the RT-PCRStatistical analysis

The statistical analysis and data presentation were conducted using the GraphPad Prism 8.0 software (GraphPad Software Inc., USA). The data from independent experiments were expressed as mean ± SD and statistical significance was determined using unpaired t-tests, two-way ANOVA, one-way ANOVA, and Tukey’s or Sidak’s multiple comparison post-tests.