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Abstract

Chemotherapy-induced oral mucositis (CIOM) is a debilitating complication with limited therapeutic options. Mesenchymal stem cells (MSCs) promote tissue repair through paracrine signaling, and stem cell-derived nanovesicles (SC-NVs) have emerged as a scalable, cell-free therapeutic alternative. However, the regenerative potential of SC-NVs has not been investigated in the context of CIOM. This study evaluated the regenerative effects of SC-NVs derived from human adipose-derived MSCs (AD MSCs) in vitro assays and a rat CIOM model. Transcriptomic profiling showed enrichment of wound healing and angiogenesis-related genes in AD MSCs. SC-NVs were produced by serial extrusion and characterized by nanoscale size and reproducible protein content. Following intralesional injection, SC-NVs localized to the ulcer bed and remained detectable for up to 5 days. SC-NV treatment enhanced epithelial regeneration, promoted angiogenesis, and reduced inflammatory markers in vitro and in vivo. These findings support SC-NVs as a scalable, cell-free therapeutic platform for CIOM.

Graphical Abstract

This study presents a novel therapy using nanovesicles derived from human adipose mesenchymal stem cells to treat oral mucositis after chemotherapy. These nanovesicles, with high scalability and key regenerative bioactivity, enhance tissue regeneration by promoting cell proliferation, angiogenesis, and reducing inflammation. Therapeutic efficacy was demonstrated in both human cell–based assays and a chemotherapy-induced oral mucositis rat model. This graphical abstract was generated using Google Gemini (Imagen 3) based on prompts designed by the authors.

Keywords

mesenchymal stem cells extracellular vesicles antineoplastic agents oral mucositis tissue regeneration

Introduction

Cytotoxic anticancer drugs are essential in cancer therapy, used in both curative and palliative settings.¹ However, their nonspecific toxicity toward rapidly proliferating normal cells often results in serious adverse effects.² One of the most common and debilitating side effects is cytotoxic anticancer drug-induced oral mucositis (CIOM), which affects 20%–40% of patients undergoing standard-dose chemotherapy.^3,4 The incidence and severity of CIOM varies depending on the chemotherapeutic agent used and the patient’s biological susceptibility.⁵ Among commonly used agents, 5-fluorouracil (5-FU), doxorubicin, and methotrexate are particularly well-documented inducers of CIOM.⁶ CIOM typically manifests 5–10 days after chemotherapy and persists for 7–14 days, causing significant oral pain and poor oral intake, leading to increased morbidity and reduced quality of life.^3,4 Despite its clinical importance, no universally effective strategies exist for preventing or treating CIOM. Current management options, such as oral cryotherapy, keratinocyte growth factors (e.g. palifermin), anti-inflammatory agents (e.g. benzydamine), and oral hygiene regimens,⁷ often exhibit inconsistent efficacy, high costs, or limited applicability to specific chemotherapy protocols.⁸ Inadequate management may require dose reduction or interruption of cancer treatment, further worsening patient outcomes.^3,8

Stem cell-based therapies have attracted increasing attention for their regenerative potential in diseases with limited treatment options.⁹ Among various types, mesenchymal stem cells (MSCs)—derived from sources such as adipose tissue, bone marrow, and umbilical cord—have emerged as particularly promising due to their multipotency, immunomodulatory functions, and accessibility.^10,11 MSCs promote tissue regeneration by secreting bioactive molecules, including cytokines, growth factors, and extracellular vesicles (EVs), which collectively facilitate inflammation resolution, angiogenesis, and cell proliferation.^12–14 However, despite their therapeutic promise, the clinical translation of MSC-based therapies faces practical challenges, including variability in cell survival, delivery efficiency, and safety-related considerations, highlighting the need for improved therapeutic strategies.^15,16

To address these issues, cell-free therapeutic strategies have been developed as alternatives to MSC-based therapies, aiming to reproduce their regenerative effects without the use of viable cells.¹⁷ These strategies employ MSC-derived products, such as EVs, secretomes, and intracellular contents. Among these, EVs (e.g. exosomes, microvesicles) have been widely studied for their regenerative effects, particularly in wound healing, due to their ability to deliver paracrine factors while avoiding the limitations of cell-based therapies.^18–21 EVs deliver various nucleic acids and proteins to recipient cells, modulating intracellular signaling and gene expression.²² However, EV biogenesis yields low quantities, requiring substantial time and cost for therapeutic-scale production, which presents significant obstacles to their clinical application.^23,24

Recently, stem cell-derived nanovesicles (SC-NVs)—produced by mechanical disruption of MSCs—have emerged as a scalable and reproducible alternative to naturally secreted EVs. SC-NVs are membrane-bound vesicle-like structures that encapsulate intracellular components and preserve many of the regenerative functions of EVs, while offering advantages in manufacturing yield and batch-to-batch consistency.^25,26 Their therapeutic efficacy has been demonstrated in cardiovascular diseases,²⁷ allergies,^28,29 and other pathological conditions.³⁰ However, the regenerative potential of SC-NVs has not yet been investigated in the context of CIOM. Therefore, this study aims to evaluate the therapeutic efficacy of SC-NVs derived from human adipose-derived MSCs (AD MSCs) in promoting mucosal regeneration in a rat model of CIOM.

Materials and methods

Isolation of AD MSCs from Human Adipose Tissue

All experiments involving the isolation of AD MSCs from human adipose tissue were conducted in the Republic of Korea and approved by the Institutional Review Boards (IRBs) of Dongguk University Hospital (IRB No. DUIH2020-03-012-002) and Dongguk University (IRB No. DUIRB-202210-18). AD MSCs were isolated as previously reported.^31,32 Human adipose tissue was obtained from the infrapatellar fat pad of anonymized adult donors (approximately 30–60 years of age), of whom approximately 80% were female. Adipose tissue was washed with Dulbecco‘s Phosphate-Buffered Saline (DPBS; Hyclone, USA) containing 2% penicillin/streptomycin (P/S; Hyclone), then digested with 0.5 mg mL⁻¹ collagenase (Sigma-Aldrich, USA) in low-glucose Dulbecco‘s Modified Eagle Medium (DMEM-LG; Hyclone) supplemented with 1% P/S at 37°C for 45 min with gentle agitation. The digested mixture was centrifuged at 300g for 10 min at 24°C, and the mature adipocyte-containing fat layer was removed. The remaining cell pellet was filtered through a 40 μm strainer and washed with DMEM-LG, followed by another centrifugation at 1000g for 10 min at 24°C, consistent with previously reported stromal vascular fraction (SVF) isolation protocols.³² The isolated cells were cultured in DMEM-LG supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% P/S at 37°C in a humidified 5% CO₂ incubator, and non-adherent cells were removed after 24 h. The adherent, spindle-shaped fibroblast-like cells were expanded and subsequently referred to as AD MSCs. The isolated AD MSCs were cultured in DMEM-LG supplemented with 10% FBS and 1% P/S at 37°C in a humidified 5% CO₂ incubator. Cells were subcultured at a seeding density of 7 × 10³ cells cm⁻² with medium changes every 2 days until reacting approximately 80% confluency. All AD MSCs used in this study were derived from a single batch obtained from one donated adipose tissue sample, and AD MSCs at passage 3 were used for all experiments.

RNA gene sequencing of AD MSCs

To assess gene expression profiles related to wound healing and angiogenesis, total RNA was extracted from AD MSCs using TRIzol™ reagent (Thermo Fisher Scientific, USA). RNA integrity was assessed using an Agilent 4200 TapeStation system (Agilent Technologies, USA), and RNA concentration was determined with a Qubit fluorometer (Thermo Fisher Scientific). After mRNA isolation using a Poly(A) RNA Selection Kit (Lexogen, Austria), libraries were prepared using the CORALL RNA-Seq V2 Library Prep Kit (Lexogen) according to the manufacturer’s instructions. Isolated mRNA was subjected to cDNA synthesis and fragmentation, followed by indexing using the Lexogen 12-nt Unique Dual Indexing V2. Library enrichment was performed by PCR. Libraries were then assessed using a TapeStation 4200 system to evaluate fragment size distribution. Library quantification was carried out by qPCR using the KAPA Library Quantification Kit (Kapa Biosystems, MA, USA). High-throughput sequencing was performed on a NovaSeq 6000 platform (Illumina, USA) using 100-bp paired-end reads.

Raw sequencing data were quality-checked using FastQC (v0.12.1). Adapter sequences and low-quality reads were removed using Fastp (v0.23.1). Trimmed reads were aligned to the reference genome using STAR (v2.7.10.b), and transcript abundance was quantified using Salmon (v1.10.0). Read counts were normalized using a Trimmed Mean of M-values (TMM) combined with Counts Per Million (CPM) normalization method implemented in the Python conorm package (v1.2.0). Data mining and graphical visualization were performed using ExDEGA v5.3.0 (Ebiogen Inc., Republic of Korea). Genes exhibiting normalized log₂ expression values greater than 1 were considered robustly expressed and selected for subsequent analysis. Genes associated with wound healing and angiogenesis were identified based on expression levels and functional annotation using QuickGO (https://www.ebi.ac.uk/QuickGO/). Statistical analysis of gene expression was conducted using two-tailed Student’s t-tests implemented in the Python SciPy library. Multiple testing correction was performed using the Benjamini–Hochberg false discovery rate (FDR) method via the statsmodels package (fdr_bh). All sequencing and analysis were conducted at Ebiogen.

Preparation of stem cell-derived nanovesicles (SC-NVs)

AD MSCs were seeded at a density of 7 × 10³ cells cm⁻² and cultured for 5 days until reaching approximately 80% confluency. Cells were then harvested by trypsinization and collected for subsequent experiments. The SC-NV preparation protocol was adapted and modified from previously reported methods.^26,33,34 About 1 × 10⁶ of AD MSCs were resuspended in 1 mL of PBS and subjected to five freeze-thaw cycles, consisting of immersion in liquid nitrogen for 10 min followed by complete thawing at room temperature. Following freeze-thawing, the cell suspension was passed 10 times through nitrocellulose membranes with pore sizes of 1.0, 0.45, and 0.2 μm using a mini-extruder (Avanti Polar Lipids, USA). Following membrane extrusion, the nanovesicle suspension was concentrated by centrifugal filtration using a 10 kDa molecular weight cutoff centrifugal filter (Amicon Ultra, Sigma-Aldrich) by centrifugation at 18,000g for 20 min at 4°C. For PKH26 labeling, 1 × 10⁶ AD MSCs were resuspended in 1 mL of DPBS, and 4 μL of PKH26 fluorescent dye (Sigma-Aldrich) was added. The cell suspension was incubated for 15 min a room temperature, followed by centrifugation at 300g for 3 min at 4°C to remove excess free dye. PKH26-labeled SC-NVs were then generated using the same extrusion protocol described above. Fluorescence intensity was confirmed using a Cytation3 reader (BioTek, USA) with excitation at 551 nm and emission detected at 567 nm. Prepared SC-NVs were used immediately for downstream experiments.

Nanoparticle tracking analysis (NTA)

SC-NV size distribution and concentration were measured using an LM10 NTA system (Malvern Panalytical, UK). Prepared SC-NVs were diluted 1:100 in PBS to obtain particle concentrations within the optimal measurement range for NTA analysis. Measurements were conducted under consistent settings: camera level 7, shutter speed 32.5 ms, and gain 250. Data were analyzed with the manufacturer’s software.

Cryo-transmission electron microscopy (Cryo-TEM)

SC-NVs were imaged using a Cryo-TEM system. 3 μL of SC-NVs (1 × 10⁹ particles mL⁻¹) were applied to a 400-mesh copper grid coated with carbon-formvar film (Electron Microscopy Sciences, USA) and imaged using a Tecnai G2-F20 Cryo-TEM (FEI, USA) operating at 200 kV and −80°C. Images were acquired at a nominal magnification of 25,000×, and at least three randomly selected fields from independent grid regions were imaged to confirm SC-NV morphology.

Western blot analysis

Western blot analysis was performed to assess extracellular vesicle-associated and intracellular marker expression in SC-NVs and parent AD MSCs. Samples were lysed in RIPA buffer (Sigma Aldrich) supplemented with protease inhibitor (Merck, USA) and phosphatase inhibitor (Sigma Aldrich), followed by centrifugation at 18,000g at 4°C for 30 min to remove insoluble debris. The resulting supernatants were collected, and total protein concentrations were determined using a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific). Equal amounts of protein were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently transferred onto nitrocellulose membranes using a Tris–glycine–SDS–methanol transfer buffer (Bio-Rad, USA). Membranes were blocked with 5% (w/v) skim milk (BD Difco™, USA) prepared in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) for 1 h at room temperature. The blocked membranes were then incubated with primary antibodies against CD63, HSP70, and calnexin (Santa Cruz, USA), diluted in TBS-T containing 5% (w/v) bovine serum albumin at 4°C overnight. After washing with TBS-T, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Cell signaling, USA) diluted in TBS-T containing 5% (w/v) skim milk. Protein bands were detected using an enhanced chemiluminescence reagent (Amersham™, UK) and visualized using a chemiluminescence imaging system (Bio-Rad).

Bicinchoninic acid (BCA) assay

SC-NV protein concentration was determined using the Pierce™ BCA Protein Assay Kit to quantify and normalize the total protein content of SC-NVs, as well as to assess batch-to-batch reproducibility of SC-NV production. SC-NVs (10 μL) were mixed with 190 μL of BCA reagent (50:1, Reagent A:B) in a 96-well plate (SPL, Korea), incubated at 37 °C for 30 min, and absorbance measured at 562 nm using a Cytation3 reader.

In vivo retention of PKH26-labeled SC-NVs

For in vivo retention analysis, PKH26-labeled SC-NVs were prepared at a concentration of 1 × 10⁷ particles mL⁻¹, as confirmed by nanoparticle tracking analysis following the labeling process. A volume of 0.1 mL, corresponding to 1 × 10⁶ SC-NVs, was locally injected into the ulcer bed of 24 anesthetized rats using a 26-gage needle, targeting the exposed underlying muscle tissue. Ulcer tissues were harvested at 0, 1, 2, and 5 days post-injection (designated as D2, D3, D4, and D7, respectively). Collected samples were frozen, embedded in optimal cutting temperature (OCT) compound (Sakura Finetek), and cryosectioned. Fluorescence images were acquired using an Olympus BX53F fluorescence microscope (Olympus, Tokyo, Japan) equipped with cellSens software, at 400× magnification. For each sample, images were obtained from six randomly selected microscopic fields. Fluorescence signal intensity and localization were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

In vitro evaluation of the regenerative potential of SC-NVs

To assess the regenerative effects of SC-NVs, human keratinocytes (HaCaT cells) and human umbilical vein endothelial cells (HUVECs) were cultured and treated with varying concentrations of SC-NVs. HaCaT cells were obtained from ABM (Canada), and HUVECs were obtained from ATCC (USA). Both cell types were cultured at 37°C in a humidified atmosphere with 5% CO₂. HaCaT cells were maintained in high-glucose DMEM (Hyclone) supplemented with 10% FBS and 1% P/S, while HUVECs were cultured in Endothelial Cell Growth Medium-2 BulletKit (ATCC) on 0.1% gelatin-coated plates. Cells were seeded at a density of 1 × 10⁴ cells cm⁻², and treated with 0 (untreated negative control), 1 × 10⁷, or 1 × 10⁸ SC-NV particles mL⁻¹. Unless otherwise specified, analyses were performed at cell-type-specific time points, with HUVECs analyzed on day 1 and HaCaT cells analyzed on day 2 after SC-NV treatment. To comprehensively evaluate regenerative responses, cellular proliferation, migration, angiogenic activity, and regeneration-related gene and protein expression were assessed using the complementary in vitro assays described below.

Cell proliferation assay

To evaluate the proliferation effect of SC-NVs, HaCaT cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan). Proliferation was evaluated on day 2 after SC-NV treatment. Two days after treatment, 150 μL of a 1:10 CCK-8/medium mix was added per well in a 96-well plate, incubated at 37°C for 1 h, and absorbance was measured at 450 nm using a Cytation3 reader.

Wound-healing assay

To assess the effect of SC-NVs on keratinocyte migration and wound closure, a scratch assay was performed using HaCaT cells. Confluent HaCaT cells in 12-well plates were scratched with a sterile 200 μL pipette tip, followed by application of SC-NV-containing medium. Images were taken at 0, 6, and 24 h using an optical microscope (Olympus, Japan). Wound closure was quantified using ImageJ with the Wound Healing Size Tool.

Gene expression analysis

To investigate molecular changes associated with SC-NV-mediated regenerative responses, quantitative real-time polymerase chain reaction (qRT-PCR) was performed in SC-NV-treated HaCaT and HUVEC cells. HaCaT cells were harvested on day 2, while HUVECs were harvested on day 1 after SC-NV treatment. Total RNA was extracted from SC-NV-treated cells using TRIzol™, purified with chloroform and isopropanol, washed with 75% ethanol, and resuspended in RNase-free water. cDNA was synthesized from 1 μg of RNA using the PrimeScript™ RT Kit (Takara Bio Inc., Japan). qRT-PCR was performed using Power SYBR^® Green PCR Master Mix (Applied Biosystems, UK) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). Cycling conditions were: 95°C for 20 s, then 40 cycles of 95°C for 3 s and 60°C for 30 s, followed by melting curve analysis. Gene expression was normalized using the ΔΔCt method. Primer sequences are listed in Table S1.

Protein expression analysis using immunofluorescence (IF) staining

To further validate SC-NV-induced regenerative signaling at the protein level, immunofluorescence staining was performed in HaCaT and HUVEC cells. HaCaT cells were analyzed on day 2, and HUVECs were analyzed on day 1 after SC-NV treatment. Cells were fixed with 4% paraformaldehyde (Biosesang) for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 30 min, and blocked with 1% BSA for 30 min. Cells were incubated with primary antibodies (1:200) overnight at 4°C, washed, and incubated with fluorophore-conjugated secondary antibodies (1:500) for 1 h at room temperature in the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were obtained using an AXIO Imager.Z2 with Apotome.2 (ZEISS, Germany) and analyzed using ImageJ. Antibodies are listed in Table S2.

Tube formation assay

To evaluate the pro-angiogenic effect of SC-NVs, a tube formation assay was conducted using HUVECs on day 1 after SC-NV treatment. 1 × 10⁴ cells cm⁻² of HUVECs were seeded in 48-well plates pre-coated with growth factor-reduced Matrigel (Corning, USA) and treated with 0, 1 × 10⁷, or 1 × 10⁸ SC-NVs mL⁻¹. After 24 h, cells were stained with 6 μM Calcein-AM (Thermo Fisher Scientific) for 30 min, and imaged using Cytation3. Tube length was quantified using ImageJ with the Angiogenesis Analyzer.

In vivo therapeutic effect of SC-NVs

The CIOM model was approved by the Institutional Animal Care and Use Committee (IACUC) of Dongguk University (Approval No. 2022-12246). Six-week-old male Sprague-Dawley rats (Orient Bio, Republic of Korea) were housed in a pathogen-free facility under controlled conditions (12-h light/dark cycle, 21°C–23°C, 40%–60% humidity) with ad libitum food and water. On day 0 (D0), rats received a single intraperitoneal (IP) injection of 200 mg kg⁻¹ 5-fluorouracil (5-FU; Alfa Aesar, MA, USA) dissolved in PBS after anesthesia with isoflurane (Hana Pharm Co, Republic of Korea) On D2, a 5-mm-diameter of buccal ulcer was created using a biopsy punch (Paramount Surgimed, India) exposing the underlying muscle. White Blood Cell (WBC) counts and body weights were recorded on D0, D2, D4, and D7. In accordance with IACUC guidelines, animals were closely monitored throughout the experimental period, and humane euthanasia were applied in cases of excessive body weight loss (>20%) or severe deterioration in general condition. Sixty CIOM rats were divided into three groups: C+/U− (no ulcer), C+/U+/S− (ulcer only, negative control), and C+/U+/S+ (ulcer + SC-NVs). On D2, 0.1 mL of PBS containing 1 × 10⁶ SC-NVs was injected into the buccal ulcer bed under anesthesia. Ten rats per group were sacrificed on D4 and D7. Ulcer diameters were measured with a precision ruler (Monami, Korea).

Histological and immunohistochemical (IHC) analysis

Harvested buccal tissues were fixed in 4% paraformaldehyde for 24 h at room temperature, paraffin-embedded, and sectioned at a thickness of 4 μm. Mayer’s Hematoxylin (Abcam, UK) and eosin Y (Abcam, UK) staining was performed according to standard histological protocols. For immunohistochemistry, paraffin sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0). After blocking, sections were incubated with primary antibodies against interleukin-6 (IL6), cluster of differentiation 31 (CD31), and Ki-67 (Table S3) at 4°C for 24 h. Immunoreactivity was detected using an HRP-conjugated secondary antibody system, followed by visualization with 3,3′-diaminobenzidine (DAB; Cat# K3468, Dako, Carpinteria, CA, USA). Sections were counterstained with hematoxylin. Images were acquired using an epifluorescence optical microscope (BX53F; Olympus, Tokyo, Japan) equipped with cellSens software at 200× and 400× magnification. For each sample, at least five randomly selected fields from independent tissue regions were analyzed. Quantification of IL6 and CD31 staining intensity, as well as Ki-67-positive cell counts, was performed using ImageJ software.

Statistical analysis

In all figures, exact n values represent independent experiments performed for each condition. Statistical analyses were conducted using GraphPad Prism ver. 10.1.2 (GraphPad Software, CA, USA). All data are presented as means ± standard error of the mean (SEM). Two-tailed Student’s t-tests were used for comparisons between the two groups. One-way ANOVA with Tukey’s multiple comparison post-test was used for multiple group comparisons. Two-way ANOVA with Bonferroni post hoc test was used for comparisons involving two variables. The statistical significance values were set at n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

Overview of SC-NV generation, molecular rationale, and regenerative outcomes

Figure 1 provides an integrated overview of the generation, molecular rationale, and regenerative effects of AD MSC-derived SC-NVs. AD MSCs were isolated from human adipose tissue and first characterized at the transcriptomic level, revealing enrichment of gene signatures associated with wound healing and angiogenesis, supporting their selection as a biologically relevant source for regenerative nanovesicle production (Figure 1(a)). SC-NVs were subsequently generated from AD MSCs through freeze–thaw cycling and serial membrane extrusion and were characterized as nanoscale, vesicle-like structures with reproducible protein content. The regenerative activity of SC-NVs was next evaluated in vitro. SC-NV treatment enhanced proliferation and accelerated wound closure in human keratinocyte (HaCaT) cells, while promoting angiogenic formation in human umbilical vein endothelial cells (HUVECs), indicating coordinated effects on epithelial repair and vascular regeneration (Figure 1(b)). In vivo, SC-NVs exhibited sustained retention within the ulcer bed following intralesional injection in a chemotherapy-induced oral mucositis (CIOM) rat model. Therapeutic evaluation demonstrated reduced inflammatory signaling, increased epithelial proliferation, and enhanced angiogenesis, as evidenced by modulation of IL-6, Ki-67, and CD31 expression, respectively (Figure 1(c)). Collectively, these results establish SC-NVs as a biologically active, cell-free therapeutic modality capable of promoting mucosal regeneration through anti-inflammatory, pro-proliferative, and pro-angiogenic mechanisms.

Figure 1.

Schematic representation of the generation and regenerative effects of AD MSC-derived SC-NVs. (a) Schematic overview of the generation of stem cell-derived nanovesicles (SC-NVs) from adipose-derived mesenchymal stem cells (AD MSCs). Transcriptomic profiling revealed enrichment of wound healing- and angiogenesis-related genes, supporting AD MSCs as a biologically relevant source for SC-NV production. (b) In vitro regenerative responses induced by SC-NVs, including enhanced keratinocyte proliferation and wound closure in HaCaT cells and increased angiogenic formation in HUVECs. (c) In vivo retention and therapeutic effects of SC-NVs in a chemotherapy-induced oral mucositis (CIOM) model, showing localization to the ulcer bed, reduced inflammation, and enhanced epithelial regeneration and angiogenesis.

Gene expression profile of MSCs in pro-regenerative potential

To assess the intrinsic regenerative potential of AD MSCs isolated from human adipose tissue, transcriptomic profiling was performed using RNA sequencing. Gene ontology analysis revealed enrichment of gene sets associated with positive regulation of wound healing and angiogenesis. Heatmap analysis demonstrated prominent expression of genes involved in extracellular matrix organization, cellular migration, and angiogenic signaling pathways (Figure 2(a)). Among genes functionally annotated to wound healing and angiogenesis, a substantial proportion showed pronounced expression (normalized log₂ expression > 1) within the AD MSC transcriptome, including genes related to wound healing (41 of 61 genes) and angiogenesis (115 of 160 genes). Radar chart visualization further illustrated strong expression of representative genes such as ANXA1, ACTG1, ITGB1, MYLK, and DDR2 associated with wound healing, as well as HIF1A, CHI3L1, ITGA5, and GREM1 associated with angiogenesis (Figure 2(b)). Collectively, these data indicate that the AD MSCs used in this study exhibit a robust intrinsic gene expression profile enriched for pathways relevant to tissue regeneration and neovascularization, supporting their suitability as a source cell population for regenerative nanovesicle production.

Figure 2.

Characterization of Mesenchymal Stem Cell-Derived Nanovesicles (SC-NVs) and in vivo retention assay of PKH26-labeled SC-NVs. (a) RNA gene profile of AD MSCs in angiogenesis and wound-healing gene ontology (GO) analysis. (b) Radar chart showing significantly expressed genes (log2-transformed, normalized data) involved in the positive regulation of angiogenesis and wound healing. (c) Size distribution and concentration of SC-NVs, as measured by Nanoparticle Tracking Analysis (NTA). (d) Cryo-Transmission Electron Microscopy (Cryo-TEM) image of SC-NVs showing spherical morphology (<200 nm, white arrowheads; scale bar = 200 nm). (e) Western blot analysis of SC-NVs and AD MSCs showing expression of CD63 and HSP70 (positive markers) and calnexin (negative marker) for extracellular vesicles, respectively. (f) Quantification of protein concentrations in SC-NVs using the Bicinchoninic Acid (BCA) assay. Data are expressed as mean ± SEM (n = 3). (g) Fluorescence intensity of PKH26-labeled SC-NVs. Data are expressed as mean ± SEM (n = 3) and analyzed using an unpaired t-test (****p < 0.0001). (h) Fluorescence microscopy images of PKH26-labeled SC-NVs in the muscle layer of the buccal ulcer bed at days 0 (D2), 1 (D3), 2 (D4), and 5 (D7) post-injection (scale bar = 40 μm). (i) Quantitative analysis of the fluorescence intensity of PKH26-labeled SCM-NVs over time (n = 6). Data represent mean ± SEM and are analyzed using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

Characteristics and in vivo retention of SC-NVs

SC-NVs were generated from human AD MSCs and characterized to assess their physical properties and in vivo retention. NTA revealed a uniform size distribution of SC-NVs, with an average diameter of 176.2 ± 3.9 nm and a particle concentration of 2.75 × 10⁹ ± 6.76 × 10⁷ particles mL⁻¹ (Figure 2(c)). Cryo-TEM imaging confirmed the spherical morphology of SC-NVs with diameters below 200 nm (Figure 2(d)). Western blot analysis demonstrated strong expression of the extracellular vesicle-associated marker CD63 and HSP70 in SC-NVs, while the intracellular negative marker calnexin was minimally detected or absent, supporting the vesicle-like characterization of SC-NVs (Figure 2(e)). Protein content measured using a Bicinchoninic Acid (BCA) assay showed a mean concentration of 0.571 ± 0.011 mg mL⁻¹ per 1 × 10⁶ AD MSCs (Figure 2(f)).

To assess SC-NV delivery efficiency, SC-NVs were labeled with PKH26 dye. Fluorescence analysis demonstrated a 9.99-fold increase in signal intensity in labeled SC-NVs compared to controls (p < 0.0001, Figure 2(g)), confirming successful labeling. Following injection into the buccal ulcer bed of CIOM rats, PKH26-labeled SC-NVs were visualized in the muscle layer of the ulcer site by fluorescence microscopy on the day of injection (D2; Figure 2(h)). Although the signal decreased over time, fluorescence remained detectable up to D7. Quantitative analysis indicated a significant reduction in fluorescence-positive area from D2 to D3 (p = 0.0142) and D4 (p = 0.0012), with continued detectability at D7 (p = 0.0002, Figure 2(i)), supporting sustained retention of SC-NVs at the injection site.

In vitro regenerative effects of SC-NVs

To assess the regenerative efficacy of SC-NVs, in vitro experiments were conducted using HaCaT cells and HUVECs (Supplemental Figures 1 and 2). Treatment with SC-NVs at concentrations starting from 1 × 10⁷ particles mL⁻¹ significantly enhanced HaCaT proliferation, with a maximum increase of 144% as determined by colorimetric assay (p = 0.0066, Figure 3(a)). In scratch assays from HaCaT cells, SC-NVs accelerated wound closure at 6 and 24 h post-treatment at concentrations of 1 × 10⁷ and 1 × 10⁸ particles mL⁻¹ (Figure 3(b)). Quantitative analysis demonstrated wound closure rates of 24.7% (p = 0.1609) and 67.6% (p = 0.0016) in SC-NV-treated cells at 6 and 24 h, respectively, compared to 16.2% and 50.4% in untreated controls (negative control, Figure 3(c)). At a molecular level, SC-NV treatment to HaCaT cells significantly upregulated the expression of the proliferation marker, proliferating cell nuclear antigen (PCNA, p = 0.0146, Figure 3(d)), while also downregulating interleukin 6 (IL6) expression (p = 0.0390, Figure 3(e)). Immunofluorescence staining of marker of proliferation Ki-67 (Ki67) further confirmed enhanced proliferation, with fluorescence intensity increased by 2.71-fold (p = 0.0007) and 3.86-fold (p < 0.0001) at 1 × 10⁷ and 1 × 10⁸ particles mL⁻¹, respectively (Figure 3(f)).

Figure 3.

Wound healing- and angiogenesis-related effects of SC-NVs in human cells. (a) Cell proliferation of human keratinocytes, HaCaT cells (D2) after SC-NVs treatment (n = 3). (b) Representative images from wound-healing assays of SC-NVs on HaCaT cell showing initial defect sites (red lines) and closed regions (yellow lines; scale bar = 300 μm). (c) Quantification of wound area closure over 24 h (n = 5). mRNA expressions of (d) PCNA and (e) IL6 markers treated with SC-NVs on HaCaT cells (n = 3). (f) Representative immunofluorescence images of KI67 (green) from HaCaT cells, co-stained with DAPI (blue) for nuclear indication; fluorescence intensities of KI67 proteins are quantified (scale bar = 100 μm, n = 3). (g) Representative tube formation images of HUVECs after SC-NVs treatment, stained with Calcein AM (green; scale bar = 500 μm). (h) Quantitative analysis of total tube length from tube formation (n = 3). mRNA expression of (i) ANG1 and (j) CD31 markers treated with SC-NVs on HUVECs (n = 3). (k) Representative immunofluorescence images of CD31 (red) from HUVECs, co-stained with DAPI (blue) for nuclear indication; fluorescence intensities of CD31 proteins are quantified (scale bar = 100 μm, n = 3). All data are expressed as mean ± SEM and analyzed using one-way ANOVA (n.s.: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

In HUVECs, SC-NVs stimulated angiogenic activity, evidenced by increased tube formation on Matrigel (Figure 3(g)). Quantitative measurement revealed significantly longer tube lengths in SC-NV-treated HUVECs (3337 μm, p = 0.0148 and 4045 μm, p = 0.0014) compared to controls (2190.33 μm; Figure 3(h)). Additionally, qRT-PCR demonstrated significant upregulation of angiopoietin 1 (ANG1, p = 0.0043) and platelet and endothelial cell adhesion molecule 1 (CD31, p = 0.0079) expression following SC-NV treatment (Figure 3(i) and (j)). Immunofluorescence analysis further showed a 1.95-fold increase in CD31 protein expression (p = 0.0328, Figure 3(k)). These results indicate that SC-NVs enhance keratinocyte proliferation and wound healing, and angiogenic function in endothelial cells, supporting their potential for tissue regeneration.

In vivo evaluation of buccal ulcer healing in the CIOM Rat Model

To evaluate the in vivo therapeutic effect of SC-NVs, a CIOM rat model was established via administration of 5-FU, which effectively induced immunosuppression, as demonstrated by a sustained decrease in WBC counts from D2 to D7 (Figure 4(a)). Compared to the normal control (NC) group, WBC levels in the 5-FU-treated groups were reduced by more than 50% (p < 0.0001). Additionally, persistent weight loss was observed in the 5-FU groups, with average body weight decreasing to 189.177 g by D7, in contrast to the NC group, which gained weight to an average of 214.844 g (p < 0.0001, (Figure 4(b)). These findings confirm the successful establishment of the CIOM model.

Figure 4.

Healing efficacy of SC-NV on the cytotoxic anticancer drug-induced oral mucositis (CIOM) rat model. Evaluation of CIOM in rats induced by 5-Fluorouracil (5-FU) through (a) leukopenia levels and (b) weight reduction from D2 to D7 (n ⩾ 5). (c) Macroscopic images of buccal ulcers formed using a biopsy punch at D2, with ulcer size measurements at selected time points. (d) Quantification of ulcer size showing a reduction in maximum ulcer diameter in the SC-NVs injected group (C+/U+/S+) compared to the non-injected group (C+/U+/S−; n = 5). (e) Hematoxylin and eosin (H&E) staining of buccal ulcers in the CIOM rat model. In the C+/U+/S+ group, proliferation of the basal cell layer at D4 (yellow arrow) and healed epithelial layer at D7 (yellow arrowhead) are indicated (scale bar = 20 μm). All data are expressed as mean ± SEM and analyzed using two-way ANOVA (**p < 0.01, ****p < 0.0001).

Macroscopic evaluation of buccal ulcers showed progressive healing in all groups, with granulation tissue formation by D4 and partial resolution by D7 (Figure 4(c)). Notably, the SC-NV-treated group (C+/U+/S+) exhibited visibly smaller ulcers by D7 compared to the untreated ulcer group (C+/U+/S−, negative control). Quantitative analysis revealed that the SC-NV-injected group had a significantly smaller maximal ulcer diameter (3.118 mm) than the non-injected group (4.26 mm, p < 0.0001, Figure 4(d)). Histological analysis using H&E staining demonstrated epithelial exfoliation and inflammatory cell infiltration in all ulcerated tissues (Figure 4(e)). On D4, high levels of basal cell proliferation were observed in the SC-NV-injected group (yellow arrow). By D7, this group showed enhanced epithelial regeneration, including re-epithelialization, thickened basal layers, and increased numbers of nucleated cells in both basal and suprabasal layers. In several SC-NV-treated animals, the epithelium was largely restored (yellow arrowhead), although full keratinization of the squamous layer had not yet occurred. These observations suggest that SC-NVs promote epithelial repair and mucosal recovery in CIOM lesions.

Immunohistochemical (IHC) evaluation of inflammation, angiogenesis, and proliferation by SC-NVs in buccal ulcers

To investigate the mechanism underlying SC-NV-mediated regeneration, IHC staining was conducted for IL6 (inflammation), CD31 (angiogenesis), and Ki67 (proliferation) in buccal ulcer tissues at D4 and D7. IL6 expression was observed in basal and suprabasal epithelial layers, excluding the keratinized surface epithelium (Figure 5(a)). At D4, IL6 intensity increased significantly in both ulcerated groups (C+/U+/S− and C+/U+/S+) compared to the ulcer-free control (C+/U−) (p < 0.001 and p = 0.003, respectively; Figure 5(b)). By D7, IL6 levels in the SC-NV-treated group were significantly lower than in the non-treated group (p = 0.037), with no significant difference compared to the ulcer-free group (Figure 5(c)). These findings indicate that SC-NVs effectively attenuate inflammation in CIOM.

Figure 5.

Regenerating Potential of SC-NVs in the Epithelium. Immunohistochemical (IHC) staining of buccal ulcer tissues was analyzed. Representative IHC images of (a) IL6, (d) CD31, and (g) Ki67 in the buccal ulcer tissue at D4 and D7. (Scale bar = 20 μm) Quantification of IL6 expression at (b) D4 and (c) D7. Positively expressing CD31 cells are counted at (e) D4 and (f) D7. Ki67-positive cells are counted at (h) D4 and (i) D7. All data are expressed as mean ± SEM (n > 8) and analyzed using one-way ANOVA (n.s.: not significant, *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001).

CD31-positive endothelial cells were primarily localized in the submucosal region beneath the regenerating epithelium (Figure 5(d)). On D4, no statistically significant differences in CD31 expression were observed among groups (Figure 5(e)). However, by D7, CD31 expression in the non-treated ulcer group was significantly lower than in the ulcer-free group (p = 0.0004), while the SC-NV-treated group exhibited significantly increased CD31 expression compared to the non-treated group (p = 0.0009; Figure 5(f)), indicating enhanced angiogenesis.

Ki67 expression was confined to the basal and lower epithelial layers (Figure 5(g)). On D4, the number of Ki67-positive cells was significantly lower in the non-treated ulcer group compared to both the SC-NV-treated group (p = 0.001) and the ulcer-free group (p = 0.0007; Figure 5(h)). By D7, the SC-NV-treated group had the highest Ki67 expression, significantly exceeding both the non-treated group (p < 0.0001) and the ulcer-free control (p = 0.0069; Figure 5(i)). These IHC findings suggest that SC-NVs promote mucosal healing in CIOM by reducing inflammation, enhancing neovascularization, and stimulating epithelial cell proliferation.

Discussion

This study demonstrates the regenerative potential of SC-NVs derived from human AD MSCs as a cell-free therapeutic approach in a rat model of CIOM. CIOM is a debilitating complication of chemotherapy, characterized by impaired cell proliferation, delayed wound healing, reduced angiogenesis, and prolonged inflammation.³ Despite extensive clinical efforts, effective treatment options for CIOM remain limited. According to the MASCC/ISOO Clinical Practice Guidelines for the Management of Mucositis Secondary to Cancer Therapy, multiple pharmacological interventions targeting inflammation, epithelial injury, and microbial dysbiosis have been evaluated³⁵; however, none have demonstrated consistently robust therapeutic efficacy across diverse clinical settings. Even keratinocyte growth factor-1 (KGF-1, palifermin), the only FDA-approved agent for mucositis, has shown benefit primarily in patients with hematologic malignancies, with limited efficacy in other cancer populations.³⁶ As a result, many patients with CIOM continue to rely largely on supportive care, including opioid analgesics and nutritional support.

To support the selection of AD MSCs as a source cell population, we performed transcriptomic profiling of human AD MSCs. Gene ontology analysis revealed elevated expression of key regenerative genes involved in angiogenesis (e.g. HIF1A, CHI3L1, ITGA5, GREM1) and wound healing (e.g. ANXA1, ACTG1, ITGB1, DDR2, MYLK). These genes are associated with extracellular matrix remodeling,³⁷ cytoskeletal reorganization,^38,39 integrin signaling,⁴⁰ and hypoxia-induced responses⁴¹—all essential for tissue repair.^42,43 Consistent with these findings, previous studies have demonstrated that AD MSCs harbor abundant regenerative factors that contribute to angiogenesis, wound healing, and tissue remodeling.⁴⁴ Together, these transcriptomic features provide a molecular rationale for the use of AD MSC-derived nanovesicles as a biologically relevant cell-free therapeutic platform for mucosal regeneration.

SC-NVs retain the regenerative bioactivity of their parental AD MSCs and exhibit physical properties similar to naturally secreted EVs, particularly exosomes.⁴⁵ Produced via mechanical extrusion, these nanovesicles encapsulate molecular constituents from the host cells and function through paracrine mechanisms to regulate target cell behavior. A significant advantage of SC-NVs is their higher production yield and protein content, which addresses scalability challenges commonly encountered in EV-based therapies.^46,47 Quantitative protein analysis demonstrated highly reproducible SC-NV production, with a mean protein concentration of 0.571 ± 0.011 mg mL⁻¹ per 1 × 10⁶ AD MSCs, corresponding to approximately 1.9% variation across batches. This minimal variability further supports the robustness of the extrusion-based fabrication process and its suitability for translational applications.

In vivo, SC-NVs exhibited sustained retention within ulcer tissues up to 5 days following intralesional injection (D7) and localized effectively to the muscle layer of the buccal ulcer bed. Their outer membrane, which retains native lipids, proteins, and carbohydrates derived from AD MSCs,⁴⁸ likely contributes to tissue interaction and retention properties inherent to AD MSC membranes.^49,50 Importantly, SC-NVs were evaluated in this study as an independent, cell-free therapeutic modality with demonstrated stability, reproducibility, and translational potential.

SC-NVs also modulated immune responses during CIOM healing. Chemotherapeutic agents like 5-FU prolong inflammation by inducing sustained cytokine production, including IL6, which delays mucosal recovery.^51,52 In this study, SC-NV treatment significantly reduced IL6 expression in ulcer tissues by D5, suggesting a timely shift from the pro-inflammatory to anti-inflammatory phase—critical for efficient wound resolution.⁵³ Angiogenesis, another key regenerative process impaired by chemotherapy,⁵⁴ was restored following SC-NV treatment. Impaired angiogenesis is a hallmark of CIOM, as it is crucial for wound healing.^55,56 SC-NV treatment increased CD31 and ANG1 expression, coinciding with the transcriptomic upregulation of pro-angiogenic genes such as CHI3L1, ITGA5, and HIF1A. These genes are involved in VEGF and PI3K/Akt signaling pathways,⁵⁷ which regulate endothelial proliferation, vessel permeability, and vascular remodeling.^37,40 Notably, HIF1A is a hypoxia-responsive transcription factor essential for new vessel formation,⁴¹ suggesting that SC-NVs may promote both initiation and stabilization of angiogenesis. The anti-inflammatory effects of SC-NVs likely further supported angiogenic responses by relieving cytokine-mediated endothelial inhibition.

In addition to modulating inflammation and promoting angiogenesis, SC-NVs directly enhanced epithelial regeneration. 5-FU damages basal epithelial cells, impairing mucosal renewal.⁸ SC-NV treatment restored cell proliferation both in vitro and in vivo, evidenced by increased HaCaT migration and PCNA expression, as well as elevated Ki67 levels in basal layers of ulcer tissue. These effects were linked to high expression of genes like ITGB1, ACTG1, and MYLK in AD MSCs—genes involved in integrin-mediated adhesion, cytoskeletal dynamics, and contractility.⁵⁸ These molecular components activate downstream MAPK/ERK and mTOR signaling pathways, central to epithelial proliferation and tissue remodeling.²⁹ Thus, SC-NVs likely orchestrate epithelial repair through coordinated regulation of cell cycle entry, migration, and matrix interaction, collectively accelerating mucosal closure.

To model the CIOM condition, we utilized 5-FU, a chemotherapeutic agent known to induce CIOM through multiple cytotoxic mechanisms. 5-FU exerts its cytotoxic effects by inhibiting thymidylate synthase, integrating into RNA and DNA, generating reactive oxygen species, and initiating inflammation in the oral epithelium.⁵⁹ These actions lead to basal epithelial cell apoptosis and mucosal adhesion disruption, both hallmarks of CIOM pathophysiology.^60,61 Although 5-FU effectively induces CIOM-like conditions, mechanical injury was required to initiate ulceration, introducing an external variable not directly caused by chemotherapy. Additionally, measuring ulcer dimensions in the curved and elastic oral mucosa of rats presents technical challenges, limiting measurement accuracy.

Despite these limitations, our data demonstrate that SC-NVs significantly promote epithelial healing, inflammation resolution, and angiogenesis in the CIOM model. These findings are consistent with emerging evidence that MSC-derived extracellular vesicles exert therapeutic effects in refractory diseases, including CIOM and metabolic disorders, by modulating inflammation, angiogenesis, and tissue regeneration.^62,63 Transcriptomic profiling in this study was used to characterize the intrinsic pro-regenerative gene signature of AD MSCs and to provide a molecular rationale for their selection as a nanovesicle source. Because this study was not designed to directly compare MSCs, MSC-derived nanovesicles, extracellular vesicles from other cellular sources, or standard therapeutic interventions, such comparative analyses were beyond its scope. Future studies incorporating these comparisons as positive controls would provide deeper mechanistic insight and further clarify the relative therapeutic potential of SC-NVs.

In addition, identifying the specific molecular cargo responsible for these regenerative effects, such as miRNAs, proteins, or lipids, and further optimize SC-NV engineering through surface modification or co-delivery strategies remain important directions for future research. Moreover, SC-NVs can be co-formulated with bioactive cargo and incorporated into bioprintable hydrogel bioinks to fabricate anatomically relevant, volumetric tissue-engineered constructs, enabling localized and sustained delivery at the defect site.^64,65 Overall, SC-NVs represent a potent, reproducible, and clinically adaptable cell-free therapy for CIOM and potentially other mucosal injuries.

Conclusion

This study demonstrates that SC-NVs derived from human AD MSCs promote mucosal healing in a CIOM model by modulating inflammation, enhancing angiogenesis, and stimulating epithelial proliferation. Despite partial clearance from the ulcer site, SC-NVs were detectable in surrounding tissues for up to 5 days, during which they exerted therapeutic effects. With their high yield, reproducibility, and exosome-comparable efficacy, SC-NVs offer a promising and scalable cell-free alternative for CIOM treatment.

Supplemental Material

sj-docx-1-tej-10.1177_20417314261430133 – Supplemental material for Adipose mesenchymal stem cell-derived nanovesicles as a therapeutic strategy for oral mucosal regeneration after chemotherapy in a rat model

Supplemental material, sj-docx-1-tej-10.1177_20417314261430133 for Adipose mesenchymal stem cell-derived nanovesicles as a therapeutic strategy for oral mucosal regeneration after chemotherapy in a rat model by Hyeop Oh, Deogil Kim, Sun Jun Lee, Jung Eun Choi, Soo-Hong Lee and Bo Hae Kim in Journal of Tissue Engineering

Footnotes

ORCID iDs

Hyeop Oh

Deogil Kim

Soo-Hong Lee

Bo Hae Kim

Ethical considerations

All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Dongguk University (Approval No. 2022-12246) and were conducted in accordance with institutional and national guidelines for animal research and welfare. For human-derived materials, the isolation of mesenchymal stem cells (MSCs) from adipose tissue was approved by the Institutional Review Board (IRB) of Dongguk University Hospital (IRB No. DUIH2020-03-012-002). The subsequent research use of isolated MSCs was approved by the IRB of Dongguk University (IRB No. DUIRB-202210-18). All donors provided written informed consent for the use of their tissue in research, in compliance with the Declaration of Helsinki and applicable ethical regulations.

Consent to participate

Written informed consent was obtained from all human donors for the collection and research use of adipose tissue-derived mesenchymal stem cells (MSCs), in accordance with protocols approved by the Institutional Review Boards of Dongguk University Hospital and Dongguk University.

Consent for publication

Not applicable.

Author contributions

Hyeop Oh: Data curation, Formal analysis, Investigation, Visualization, Writing – original draft. Deogil Kim: Data curation, Formal analysis, Funding acquisition, Investigation, Validation, Visualization, Writing – original draft. Sun Jun Lee: Data curation, Formal analysis, Methodology. Jung Eun Choi: Data curation, Formal analysis. Soo-Hong Lee: Conceptualization, Project administration, Supervision, Validation, Writing – review & editing. Bo Hae Kim: Conceptualization, Funding acquisition, Methodology, Supervision, Writing – review & editing.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Dongguk University Research Fund of 2024, the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00344950, RS-2023-00214410) and the Korea Environment Industry & Technology Institute (KEITI) through the Technology Development Project for Safety Management of Household Chemical Products, funded by the Korea Ministry of Environment (MOE) (RS-2025-02223058).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

The data presented in this study are available upon request from the corresponding author.

Supplemental material

Supplemental material for this article is available online.

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