Exosome-loaded human amniotic membrane hydrogel modulates regenerative responses of human dental pulp stem cells

Abstract

Context:

Human dental pulp stem cell (hDPSC)-derived exosomes may support dentin–pulp regeneration, but free exosomes have limited local retention. Human amniotic membrane hydrogel may serve as a scaffold for exosome delivery.

Aim:

This study evaluated the effect of exosome-loaded human amniotic membrane (hAM-Exo) hydrogel on hDPSC responses compared with free exosomes under basal and lipopolysaccharide (LPS)-stimulated conditions.

Materials and Methods:

hDPSCs were treated with free exosomes or hAM-Exo at 0.5%, 1%, and 5%, with or without LPS. Viability, cytocompatibility, migration, vascular endothelial growth factor-A (VEGF-A) secretion, and dentin sialophosphoprotein (DSPP) expression were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, live/dead, scratch, and enzyme-linked immunosorbent assay (ELISA) assays. Data were analyzed using two-way analysis of variance and post hoc tests.

Results:

hAM-Exo was cytocompatible in all groups and showed a more stable metabolic profile than free exosomes at 96 h. hAM-Exo 1% significantly increased VEGF-A secretion under basal conditions at 48 h. Under LPS stimulation, hAM-Exo better preserved VEGF-A secretion than free exosomes, while hAM-Exo at all concentrations significantly enhanced DSPP expression on days 7 and 14.

Conclusions:

hAM-Exo modulated regenerative-related hDPSC responses under basal and inflammatory conditions compared with free exosomes, supporting scaffold-assisted exosome delivery for cell-free regenerative endodontics.

INTRODUCTION

Regenerative endodontics aims to restore the dentin–pulp complex function through cell survival, migration, angiogenesis, and odontoblast-like differentiation.[1] Human dental pulp stem cells (hDPSCs) support this process through proliferative, migratory, angiogenic, and dentinogenic potential.[2,3] Dental stem cell-derived exosomes are promising cell-free mediators that regulate dentinogenic, angiogenic, and reparative responses.[4,5,6] However, free exosomes may disperse rapidly, especially in an inflammatory microenvironment. Human amniotic membrane (hAM) hydrogel may support local exosome delivery and tissue repair.[7,8] Therefore, this study evaluated exosome-loaded hAM (hAM-Exo) effects on hDPSCs’ regenerative responses compared with free exosomes under basal and lipopolysaccharide (LPS)-stimulated conditions.

MATERIALS AND METHODS

This in vitro experimental study evaluated the biological response of recipient hDPSCs after treatment with free exosomes and hAM-Exo. This study was approved by the Ethical Committee of the Faculty of Dentistry, Universitas Indonesia (No. 91/Ethical approval/FKGUI/X/2024; Protocol No. 070940924). hDPSCs were cultured in α-modified Eagle’s medium supplemented with fetal bovine serum and antibiotics under standard conditions at 37°C and 5% CO₂ (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The cells were characterized using mesenchymal stem cell-associated markers and were used when positive for CD73 and CD105 antibodies and negative for CD45 antibody (Elabscience, Houston, TX, USA).

Exosomes were isolated from three-dimensional (3D) spheroid-cultured hDPSCs, as this culture system has been shown to better support cell–cell interactions and paracrine activity, and prepared based on a previously established protocol from the authors’ unpublished observations.[9,10] Conditioned medium was collected and processed using the minimate EVO Tangential Flow Filtration System (Pall Corporation, Port Washington, NY, USA). The exosome-enriched samples were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA; ViewSizer 3000, Horiba, Kyoto, Japan), and flow cytometry for CD9, CD63, and CD81 (Elabscience, Houston, TX, USA). These complementary methods were applied in accordance with extracellular vesicle reporting guidelines for morphology, particle characterization, and protein marker assessment.[11]

A sterile preprepared hAM hydrogel from the Indonesian Medical Education and Research Institute was used as the exosome scaffold.[12] The 3D hDPSC-derived exosomes were loaded into the hAM hydrogel using an elution-based mixing method to obtain final concentrations of 0.5%, 1%, and 5% (v/v). The mixture was gently homogenized under sterile conditions before application to recipient hDPSCs. The treatment workflow is summarized in Supplementary Figure 1 ^{(636.9KB, tif)} .

The basal condition consisted of untreated control, free exosome groups, and hAM-Exo groups at 0.5%, 1%, and 5%. The inflammatory condition was induced using 100 ng/mL LPS from Escherichia coli O26:B6 (L2654, Sigma-Aldrich, St. Louis, MO, USA). Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 72 and 96 h and live/dead assay at 24 h. Migration was evaluated using a scratch assay at 3, 6, and 24 h. Vascular endothelial growth factor-A (VEGF-A) secretion was measured by ELISA at 48 and 72 h, while dentin sialophosphoprotein (DSPP) expression was measured on days 7, 14, and 21. Data were analyzed using two-way analysis of variance followed by post hoc tests, with significance set at P < 0.05.

RESULTS

Exosomes derived from 3D cultured hDPSCs were characterized by TEM, NTA, and flow cytometry. TEM revealed the typical spherical morphology of exosomes, NTA confirmed particle size distribution within the expected range, and flow cytometry demonstrated the expression of CD63, CD9, and CD81 [Figure 1a-d].

Figure 1.

Characterization of three-dimensional human dental pulp stem cell-derived exosomes. (a) Transmission electron microscopy. (b) Nanoparticle tracking analysis. (c) Fluorescence microscopy image of labeled exosomes. (d) Flow cytometry analysis of exosomal markers CD63, CD9, and CD81. PE-A: Phycoerythrin-area, FITC-A: Fluorescein isothiocyanate-area, APC-A: Allophycocyanin-area, Exo-hDPSC: Exosomes derived from human dental pulp stem cells

The effects of free exosomes and hAM-Exo on hDPSC viability, cytocompatibility, and migration were evaluated using live/dead assays, MTT, and scratch [Figure 2a-c]. Live/dead staining confirmed good cytocompatibility in all groups at 24 h [Figure 2a]. MTT analysis showed that metabolic activity was influenced by treatment type, concentration, observation time, and LPS stimulation [Figure 2b]. Under basal conditions, free exosomes induced a stronger metabolic response at 72 h, particularly at lower concentrations, whereas hAM-Exo exerted a more moderate effect. At 96 h, free exosomes reduced metabolic activity with a significant decrease at 1%, while hAM-Exo did not significantly affect cell metabolism. Under LPS stimulation, neither treatment significantly altered metabolic activity compared with the LPS control.

Figure 2.

Functional responses of human dental pulp stem cells after free exosome and exosome-loaded human amniotic membrane treatment. (a) Live/dead assay. (b) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (c) Scratch assay. (d) Vascular endothelial growth factor-A secretion. (e) Dentin sialophosphoprotein expression. Data: mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. LPS: Lipopolysaccharide, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, VEGF-A: Vascular endothelial growth factor-A, ELISA: Enzyme-linked immunosorbent assay, hAM-Exo: Exosome-loaded human amniotic membrane

Scratch assay demonstrated progressive wound closure over time in all groups [Figure 2c]. Under basal conditions, 5% hAM-Exo significantly reduced wound closure, whereas 1% free exosomes significantly enhanced wound closure under LPS-stimulated conditions at 24 h. Representative scratch assay images are provided in Supplementary Figure 2 ^{(747.1KB, tif)} .

VEGF-A secretion was modulated by treatment type and inflammatory condition. At 48 h under basal conditions, VEGF-A levels were generally comparable among groups; however, hAM-Exo 1% significantly increased VEGF-A secretion compared with the control. At 72 h under basal conditions, free exosomes 0.5% significantly decreased VEGF-A secretion compared with the control. Under LPS-stimulated conditions at both 48 and 72 h, free exosomes at all tested concentrations significantly reduced VEGF-A secretion compared with the LPS control, whereas among the hAM-Exo groups only hAM-Exo 0.5% significantly reduced VEGF-A secretion relative to the LPS control [Figure 2d].

DSPP expression showed a time-dependent and condition-dependent response. Under basal conditions, free exosomes increased DSPP expression at day 7, with a significant effect observed at the 5% concentration relative to the control [Figure 2e]. By day 14, all concentrations of free exosomes, as well as hAM-Exo at 0.5% and 1%, significantly suppressed DSPP expression compared with the control. On day 21, no significant differences in DSPP expression were detected among the treated groups compared with the control [Figure 2e]. Under LPS-stimulated conditions, free exosomes at 0.5% and 1%, and hAM-Exo at all tested concentrations significantly upregulated DSPP expression compared with the LPS-treated group at day 7. At day 14, hAM-Exo at all concentrations continued to significantly enhance DSPP expression relative to the LPS-treated group. Notably, by day 21, all treatment conditions markedly reduced DSPP expression compared with the LPS-treated group [Figure 2e].

DISCUSSION

The present study examined the effects of free hDPSC-derived exosomes and hAM-Exo on hDPSC responses under basal and LPS-stimulated conditions. The findings demonstrated that neither free exosomes nor hAM-Exo affected hDPSC viability. Changes in cell migration were observed at a later time point, with reduced migration in the hAM-Exo group, whereas free exosomes promoted wound closure under basal and LPS-treated conditions, respectively. In addition, VEGF-A secretion varied with treatment modality and inflammatory status, whereas DSPP expression exhibited time- and condition-dependent patterns. These findings are consistent with the biological properties of exosomes as complex extracellular vesicles containing proteins, lipids, and regulatory RNAs, whose effects may vary depending on the condition of recipient cells and the surrounding microenvironment.[4,5,13]

The present study demonstrated that the influences of both free exosomes and hAM-Exo on hDPSCs viability and cellular metabolic activity were modest. In this regard, the viability findings showed that free exosomes elicited strong metabolic responses at 72 h, which later subsided at 96 h. The metabolic response was not dose-dependent, suggesting that increasing exosome concentration does not yield greater biological activity. Correspondingly, the exosome isolated from osteogenic induction of DPSCs did not markedly change cellular metabolic activity in bone marrow-derived stem cells at 72 h.[14] The hAM-Exo groups showed a more moderate profile in both basal and LPS-stimulated conditions, as cellular metabolic activities were not significantly different from the control or LPS control conditions. This may indicate that the hydrogel matrix modifies how exosomes interact with recipient cells rather than acting as an immediate stimulant. Live/dead assay confirmed that both free exosomes and hAM-Exo were cytocompatible, which is essential for any scaffold or delivery system intended for dentin–pulp regeneration.[15,16,17]

Exosomes’ influence on cell migration in an in vitro scratch assay depended on the exposure format and duration. Free exosomes enhanced wound closure under LPS-stimulated conditions, whereas hAM-Exo attenuated wound closure under basal conditions. This finding may be related to the hydrogel matrix, which can influence how exosomes are presented to the cell surface and how rapidly vesicles interact with recipient cells. Hydrogel-based systems are increasingly considered useful in dentin–pulp regeneration because they can provide a 3D matrix and support local delivery of bioactive molecules.[13,14] The lower migration in selected exosome groups under basal conditions may reflect context-dependent regulation rather than direct inhibition. In stable physiological conditions, exosome signals may be directed more toward cellular regulation or differentiation than active wound closure. Under LPS stimulation, inflammatory stress may activate repair-related signaling, allowing exosomes to support cellular adaptation and migration.[4,5,6,12,15,17]

VEGF-A was evaluated because angiogenesis is essential for dentin–pulp regeneration.[18] Free exosomes showed variable VEGF-A responses, whereas selected hAM-Exo groups maintained VEGF-A under LPS stimulation. The reduction of VEGF-A at 72 h in free hDPSC-derived exosome groups may reflect immunomodulatory regulation rather than impaired angiogenic potential. Under LPS stimulation, VEGF-A may increase as part of inflammatory stress. Therefore, decreased VEGF-A after exosome treatment may indicate a reduction of excessive inflammatory-related angiogenic signaling. This differs from platelet-derived exosomes, which can enhance hDPSC migration and VEGF-A expression.[19]

DSPP was used as a dentinogenic-related marker because it is associated with odontoblast-like differentiation and dentin mineralization.[20,21] This is consistent with previous evidence showing that platelet-rich plasma-derived exosomes increased odontogenic-related markers, including transforming growth factor-beta 1 and DSPP expression, in hDPSCs.[22] The DSPP response was more complex and varied across time points, which is biologically reasonable because dentinogenic activity is slower than early viability or migration. Under basal conditions, hAM-Exo did not consistently produce the highest DSPP expression across all groups; however, it preserved DSPP expression under selected conditions, whereas free exosomes enhanced expression only at the early time point and were subsequently associated with reduced DSPP levels relative to the control. This finding may suggest that the controlled release of exosomes from hAM contributes to the sustained maintenance of DSPP expression. Therefore, under basal conditions, the effect of hAM hydrogel should be interpreted as a modulation of dentinogenic-related activity rather than a simple enhancement.

Inflammation is an inevitable consequence of microbial exposure, and low-grade inflammation has been shown to promote dentin–pulp healing and regeneration.[17] However, excessive or persistent inflammation impairs cell survival and odontogenic differentiation.[20,21] The ability of selected hAM-Exo groups to maintain VEGF-A expression could support its potential influence on angiogenesis, the process crucially involved in regeneration and healing. In addition, hAM-Exo groups enhanced DSPP expression in response to LPS stimulation, suggesting that scaffold-assisted exosome delivery supports regenerative signaling under less favorable biological conditions.

The novelty of this study lies in shifting the focus from exosome production to scaffold-assisted exosome delivery. Previous evidence supports the regenerative role of dental stem cell-derived exosomes and the potential of hAM hydrogel as a bioactive scaffold.[4,5,6,7,8] The present study directly compares free hDPSC-derived exosomes and hAM-Exo under basal and inflammatory conditions, providing information on how hAM hydrogel assists hDPSC exosome delivery and affects hDPSC responses. Recent evidence also indicates that exosome-loaded scaffolds enhance dentin–pulp regeneration, underscoring the importance of delivery systems in acellular regenerative endodontics.[23,24] In this study, hAM hydrogel did not merely act as a passive carrier but appeared to influence the pattern of exosome-mediated responses, as distinct cell responses were observed compared with those exposed to free exosomes. This distinction is important because regenerative endodontic strategies require not only potent biological signals but also an appropriate delivery system that supports local and sustained interaction with target cells.

Notwithstanding these noteworthy findings, the present study had several limitations. Exosome release kinetics from hAM hydrogel were not evaluated, so the release pattern remains interpretative. Exosomal cargo, including microRNAs and specific proteins, was not profiled; thus, the active ingredient influencing cell function remains unknown. Hydrogel-only groups were not investigated, so the independent effect of the hAM hydrogel itself cannot be fully disentangled from the combined effect of the hydrogel and exosomes. Future studies are indeed necessary and should include hydrogel-only controls, release kinetics analysis, molecular pathway evaluation, and in vivo validation.

CONCLUSIONS

hAM-Exo was cytocompatible with hDPSCs and modulated the biological responses of hDPSCs in a distinct manner compared with free exosomes under both basal and LPS-stimulated conditions. Free exosomes produced strong responses in selected parameters, while hAM-Exo showed a more scaffold-assisted modulatory profile, especially in VEGF-A and selected DSPP responses under inflammatory stimulation. Both systems were cytocompatible with hDPSCs. These findings support hAM hydrogel as a bioactive scaffold for exosome delivery in cell-free regenerative endodontic strategies.

Institutional review board statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Dental Research Ethics Committee (KEPKG), Faculty of Dentistry, Universitas Indonesia (No. 91/Ethical approval/FKGUI/X/2024; Protocol No. 070940924).

Declaration of generative AI and AI-assisted technologies in the writing process

During the writing process of this manuscript, the authors used ChatGPT (OpenAI) for language refinement and grammar assistance. The authors reviewed and edited the manuscript and take full responsibility for the final content.

Conflicts of interest

There are no conflicts of interest.

Supplementary Figure 1

Experimental workflow of free exosome and exosome-loaded human amniotic membrane hydrogel treatment in human dental pulp stem cells. 3D: Three-dimensional, hDPSC: Human dental pulp stem cell, LPS: Lipopolysaccharide, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, VEGF-A: Vascular endothelial growth factor-A, ELISA: Enzyme-linked immunosorbent assay, DSPP: Dentin sialophosphoprotein, hAM-Exo: Exosome-loaded human amniotic membrane

Supplementary Figure 2

Representative scratch assay images under basal and lipopolysaccharide-stimulated conditions. LPS: Lipopolysaccharide, hAM-Exo: Exosome-loaded human amniotic membrane

Funding Statement

This research was funded by the Indonesian Education Scholarship (BPI), Center for Higher Education Funding and Assessment (PPAPT), Indonesia Endowment Fund for Education (LPDP), and Universitas Indonesia Research Grant No. NKB-331/UN2.RST/HKP.05.00/2024. The funding sources supported different components of the study.