2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside–stimulated dental pulp stem cells-derived exosomes for wound healing and bone regeneration
Alveolar bone repair post-trauma, surgery, or periodontal breakdown is intricate, especially when surgical complications or chronic diseases are present, making gingival wound healing challenging. Clinicians need to manage inflammation, curb bacterial infection, and minimize invasive procedures to successfully repair oral and maxillofacial injuries and prevent issues like secondary tissue damage. Tissue engineering, using cell therapy for immune modulation and tissue repair, is increasingly applied in regenerative medicine for reconstruction or functional restoration. , The limitations of cell therapy have led researchers to increasingly focus on the paracrine effects of mesenchymal stem cells (MSCs). , Research on conditioned medium has shown that MSC-derived extracellular vesicles (EVs) are crucial in cell therapy, mediating immune responses, disease progression, and drug delivery. Dental stem cells are a valuable resource for regenerative therapies because they are readily accessible through minimally invasive collection methods. , In particular, dental pulp stem cells (DPSCs) exhibit remarkable proliferation and multilineage differentiation abilities, rendering them suitable for immune modulation, neural repair, and wound healing purposes. Relative to MSC-EVs from other sources, DPSC-EVs reportedly exhibit superior therapeutic efficacy in dental diseases, neurological disorders, and wound healing. In vivo experiments have also demonstrated that MSC-EVs play a crucial role in bone defect repair. However, the regenerative ability of DPSC-EVs remains to be explored. Notably, 2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG) is a water-soluble polyphenolic compound that can scavenge free radicals and has antilipidemic properties. Studies show that THSG boosts osteogenic differentiation in hDPSCs and accelerates bone formation in a rat bone defect model. , It may regulate osteogenic differentiation by inhibiting the expression of KDM5A. Cell therapy success depends on stem cell-secreted substances, and EVs from hDPSCs may promote bone healing. Research shows THSG-stimulated hDPSCs culture medium enhances wound healing in human skin and gingival fibroblasts (HGFs). Therefore, we investigated whether THSG-stimulated hDPSCs exosomes could improve oral wound healing and enhance osteogenic differentiation in alveolar bone defects. 

Reagents and cell culture We maintained hDPSCs in low-glucose Dulbecco's modified Eagle's medium (10-014-CM, Corning, NY, USA) with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (Invitrogen, Grand Island, NY, USA). THSG was extracted as previously described and dissolved in dimethyl sulfoxide (Invitrogen) for cell treatment. , The other cell cultures are presented in the supplementary data. Preparation of conditioned medium The hDPSCs were cultured in growth media with medium containing 0.25 % stripped FBS for cell starvation for 1 day. To enhance the stimulation, 100 μM THSG was further evaluated based on previous study. The cells were either treated or not treated with 10 or 100 μM THSG in 0.25 % stripped FBS medium for 48 h. Other experimental procedures for the purification of conditioned medium are provided in the supplementary data. Isolation and identification of hDPSC-derived exosomes For tangential flow filtration (TFF), we used a KrosFlo Map.03 Cross Flow TFF pump (Spectrum Laboratories, Rancho Dominguez, CA, USA) fitted with a 300-kDa molecular weight cut-off mPES hollow fiber MidiKros filter module. The control-CM and THSG-CM were aspirated from a conical bottle, pumped through the hollow fiber system, and recirculated into the conical bottle. The pressure of the pump system was monitored using KrosFlo digital pressure transducers and a monitor (Spectrum Laboratories). Adjustable clamps were used to maintain a low transmembrane pressure of 5 PSI, which minimized the loss of small exosomes into the permeate. To achieve buffer exchange and remove any remaining phenol red, the concentrated solution was washed with 10 volumes of 1 × PBS. Finally, the exosomes obtained from the control-CM and THSG-CM were sterilized by loading them in 0.22-μm syringe filters. For the identification of exosomes, we used transmission electron microscopy (TEM), particle size analysis, and surface antigen detection. Detailed procedures are provided in the Supplementary data. Cell viability assay HGFs and MC3T3 cells were seeded at cell densities of 1000 and 3000 cells per well, respectively, in 96-well culture plates containing growth medium and then incubated for 24 h at 37 °C in a 5 % CO 2 environment. Subsequently, the medium was replaced with 5 % stripped serum for cell starvation, and then the THSG-Exo (10 μM, 100 μM) were added to the medium with 0.25 % stripped FBS; after 0, 3, and 5 days of continuous culture, the medium was replaced with 10 % CCK-8, and incubation proceeded at 37 °C for 1–4 h; the absorbance was measured hourly with a VersaMax microplate reader (Molecular Device, San Jose, CA, USA) at a wavelength of 450 nm. Wound healing assay For our wound healing assay, we first placed a culture-insert 2 well (ibidi GmbH, Martinsried, Germany) into a 6-cm cell culture dish and then seeded the HGF cells at a density of a 2.5 × 10 6 cells/cm 2 . Photographs of the wound healing process were taken on days 0, 6, and 9 of culture. The size of the wound healing area was analyzed and calculated using Image J software (version 1.52a, NIH, USA). Detailed experimental procedures are presented in the Supplementary data. Alizarin red staining MC3T3 cells were cultured at a density of 1 × 10 5 cells/well in 12-well culture plates in MEM-α with 10 % FBS until 80 % confluence was achieved. Subsequently, the growth medium was replaced with mineralization medium (MEM-α medium containing 50 μM ascorbic acid, 10 nM dexamethasone, 20 mM β-glycerophosphate; Sigma–Aldrich, St. Louis, MO, USA) containing exosomes. Thereafter, the medium was changed every 2 days. After 1, 2, and 3 weeks of cultivation in each treatment, the hDPSCs were washed with PBS and fixed with 4 % paraformaldehyde (Sigma–Aldrich). The cells in the culture plate were stained with 2 % Alizarin Red S (Catalog No.: TMS-008-C, Merck Millipore, St. Louis, MO, USA) for 20 min. Finally, distilled water was used to remove the stained background from each well, and photographs were taken for documentation. Rat alveolar bone defect model In the first procedure, bilateral maxillary first molars were extracted and allowed to heal for 4 weeks ( ). In the second surgery, a 2.0 mm diameter, 0.5 mm width, and 1 mm depth alveolar bone defect was created in the healed bone to quantify and confirm defect consistency. The rats were randomly divided into the following 5 groups: PBS, low-dose DPSC-Exo, high-dose DPSC-Exo, low-dose THSG-Exo, and high-dose THSG-Exo (3 rats/group). In the PBS group, PBS and Matrigel were placed in the alveolar bone defect, whereas in the DPSC-Exo and THSG-Exo groups, approximately 1 × 10 5 or 1 × 10 6 exosomes and Matrigel were placed in the bone defect. The remaining steps for the rat alveolar bone defect model are provided in the supplementary data. Radiographic analysis of hard tissue with micro-CT Detailed experimental procedures to assess bone status are presented in the Supplementary data. Osteoblast and immunohistochemical analysis Three antibodies were used in the immunohistochemistry (IHC) assay: proliferating cell nuclear antigen (PCNA), osteopontin (OPN), and vascular endothelial growth factor (VEGF). The procedures for analyzing osteoblasts are detailed in the supplementary data. Proteomics analysis Complete exosome proteomics involved three steps: gel-assisted digestion, LC-MS analysis, and protein identification, with details in the Supplementary data. Statistical analysis Data were analyzed using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance, followed by the Tukey–Kramer multiple comparison test, was conducted. P < 0.05 indicated statistical significance. 

We maintained hDPSCs in low-glucose Dulbecco's modified Eagle's medium (10-014-CM, Corning, NY, USA) with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (Invitrogen, Grand Island, NY, USA). THSG was extracted as previously described and dissolved in dimethyl sulfoxide (Invitrogen) for cell treatment. , The other cell cultures are presented in the supplementary data. 

The hDPSCs were cultured in growth media with medium containing 0.25 % stripped FBS for cell starvation for 1 day. To enhance the stimulation, 100 μM THSG was further evaluated based on previous study. The cells were either treated or not treated with 10 or 100 μM THSG in 0.25 % stripped FBS medium for 48 h. Other experimental procedures for the purification of conditioned medium are provided in the supplementary data. 

For tangential flow filtration (TFF), we used a KrosFlo Map.03 Cross Flow TFF pump (Spectrum Laboratories, Rancho Dominguez, CA, USA) fitted with a 300-kDa molecular weight cut-off mPES hollow fiber MidiKros filter module. The control-CM and THSG-CM were aspirated from a conical bottle, pumped through the hollow fiber system, and recirculated into the conical bottle. The pressure of the pump system was monitored using KrosFlo digital pressure transducers and a monitor (Spectrum Laboratories). Adjustable clamps were used to maintain a low transmembrane pressure of 5 PSI, which minimized the loss of small exosomes into the permeate. To achieve buffer exchange and remove any remaining phenol red, the concentrated solution was washed with 10 volumes of 1 × PBS. Finally, the exosomes obtained from the control-CM and THSG-CM were sterilized by loading them in 0.22-μm syringe filters. For the identification of exosomes, we used transmission electron microscopy (TEM), particle size analysis, and surface antigen detection. Detailed procedures are provided in the Supplementary data. 

HGFs and MC3T3 cells were seeded at cell densities of 1000 and 3000 cells per well, respectively, in 96-well culture plates containing growth medium and then incubated for 24 h at 37 °C in a 5 % CO 2 environment. Subsequently, the medium was replaced with 5 % stripped serum for cell starvation, and then the THSG-Exo (10 μM, 100 μM) were added to the medium with 0.25 % stripped FBS; after 0, 3, and 5 days of continuous culture, the medium was replaced with 10 % CCK-8, and incubation proceeded at 37 °C for 1–4 h; the absorbance was measured hourly with a VersaMax microplate reader (Molecular Device, San Jose, CA, USA) at a wavelength of 450 nm. 

For our wound healing assay, we first placed a culture-insert 2 well (ibidi GmbH, Martinsried, Germany) into a 6-cm cell culture dish and then seeded the HGF cells at a density of a 2.5 × 10 6 cells/cm 2 . Photographs of the wound healing process were taken on days 0, 6, and 9 of culture. The size of the wound healing area was analyzed and calculated using Image J software (version 1.52a, NIH, USA). Detailed experimental procedures are presented in the Supplementary data. 

MC3T3 cells were cultured at a density of 1 × 10 5 cells/well in 12-well culture plates in MEM-α with 10 % FBS until 80 % confluence was achieved. Subsequently, the growth medium was replaced with mineralization medium (MEM-α medium containing 50 μM ascorbic acid, 10 nM dexamethasone, 20 mM β-glycerophosphate; Sigma–Aldrich, St. Louis, MO, USA) containing exosomes. Thereafter, the medium was changed every 2 days. After 1, 2, and 3 weeks of cultivation in each treatment, the hDPSCs were washed with PBS and fixed with 4 % paraformaldehyde (Sigma–Aldrich). The cells in the culture plate were stained with 2 % Alizarin Red S (Catalog No.: TMS-008-C, Merck Millipore, St. Louis, MO, USA) for 20 min. Finally, distilled water was used to remove the stained background from each well, and photographs were taken for documentation. 

In the first procedure, bilateral maxillary first molars were extracted and allowed to heal for 4 weeks ( ). In the second surgery, a 2.0 mm diameter, 0.5 mm width, and 1 mm depth alveolar bone defect was created in the healed bone to quantify and confirm defect consistency. The rats were randomly divided into the following 5 groups: PBS, low-dose DPSC-Exo, high-dose DPSC-Exo, low-dose THSG-Exo, and high-dose THSG-Exo (3 rats/group). In the PBS group, PBS and Matrigel were placed in the alveolar bone defect, whereas in the DPSC-Exo and THSG-Exo groups, approximately 1 × 10 5 or 1 × 10 6 exosomes and Matrigel were placed in the bone defect. The remaining steps for the rat alveolar bone defect model are provided in the supplementary data. 

Detailed experimental procedures to assess bone status are presented in the Supplementary data. 

Three antibodies were used in the immunohistochemistry (IHC) assay: proliferating cell nuclear antigen (PCNA), osteopontin (OPN), and vascular endothelial growth factor (VEGF). The procedures for analyzing osteoblasts are detailed in the supplementary data. 

Complete exosome proteomics involved three steps: gel-assisted digestion, LC-MS analysis, and protein identification, with details in the Supplementary data. 

Data were analyzed using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance, followed by the Tukey–Kramer multiple comparison test, was conducted. P < 0.05 indicated statistical significance. 

Characterization of exosomes obtained through TFF The transmission electron microscope (TEM) analysis revealed that the membranes of the exosomes, obtained through TFF, were spherical and remained intact; these characteristics are similar to those of exosomes obtained using the gold standard ultracentrifugation method ( A). After tunable resistive pulse sensing (TRPS) analysis, the sizes of the fresh and lyophilized samples were 100–250 nm and 60–150 mm ( B), respectively, indicating a marked size difference. Flow cytometry revealed that the expression levels of exosome marker CD9 were higher in the THSG-Exo than in the DPSC-Exo ( C). Stimulation of hDPSCs with THSG can prompt an increased production of exosomes. THSG-Exo enhanced cell proliferation of HGFs and MC3T3 cells HGFs cultured with 10 μM THSG-Exo showed a significant increase in proliferation on day 5, while 100 μM THSG-Exo did not ( A). By contrast, MC3T3 cells cultured with THSG-Exo (100 μM) exhibited a significant increase in cell proliferation on day 3; THSG-Exo (10 μM) did not have this effect ( B). The limited proliferation effect of 10 μM THSG-Exo in MC3T3 cells may be due to high osteogenic differentiation rather than proliferation. THSG-Exo accelerated wound healing rate of HGFs Because 10 μM THSG-Exo stimulated cell proliferation, we used HGFs to evaluate whether exosomes can accelerate wound healing. By day 6, wounds in HGFs treated with THSG-Exo were smaller than those treated with DPSC-Exo, with slight intercellular connections observed ( C). The wound area in the THSG-Exo group was significantly smaller than that in the DPSC-Exo group on day 9. The wound area calculation results are presented in D. The wound area of HGFs cultured with THSG-Exo gradually decreased from days 6–9. On day 9, greater wound closure was observed in the THSG-Exo group than in the DPSC-Exo group. The wound area in the THSG-Exo group shrank to 40 % of its original size ( D). These results indicate that THSG-induced exosomes significantly promote cell migration and wound healing in HGFs. THSG-Exo enhanced osteogenic differentiation of MC3T3 cells The MC3T3 cells were cultured in mineralization medium for 1, 2, and 3 weeks for osteoblast differentiation. In the group receiving THSG-Exo (10 μM), calcium nodules were observed on day 7, and in the second and third weeks, an increased number of larger dark red nodules was observed ( E). Micro-CT results of animal bone defect specimens In our analysis, the degree of wound healing in the THSG-Exo group was superior to that in the DPSC-Exo group ( A). We used software to analyze the micro-CT images of the large periodontal defect area and measured 4 values: the relative bone volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular spacing (Tb.Sp) ( B). The THSG-Exo group had a higher BV/TV ratio than the PBS and DPSC-Exo groups, with even low-dose DPSC-Exo showing a higher ratio than PBS, indicating bone regeneration effects of exosomes ( C). Similar trends were observed in terms of Tb. Th and Tb. N, with the THSG-Exo group having higher Tb. Th and Tb. N values than the PBS and DPSC-Exo groups did. Finally, we observed a significant difference in the Tb. Sp values between the 2 low-dose exosome groups; in addition, the Tb. Sp value in the high-dose THSG-Exo group was the lowest of those of all the groups, indicating that the degree of bone osteoporosis in the THSG-Exo group was lower than that in the DPSC-Exo group within the alveolar bony defect model ( C). Micro-CT analysis showed the THSG-Exo group had greater bone regeneration and less bone rarefaction compared to the DPSC-Exo and PBS groups. Histological results of animal bone defect specimens Our results indicated a greater increase in the number of osteoblasts in the 2 exosome groups than in the PBS group ( A). In high doses, THSG-Exo led to the generation of more osteoblasts than did DPSC-Exo, confirming that the addition of THSG-Exo had a more favorable effect on new bone formation within the large bone defect. Moreover, the high dose of THSG-Exo outperformed the low dose. The IHC results indicated that both the low and high doses of THSG-Exo resulted in greater regeneration or osteogenic differentiation of cellular tissues than that which occurred in the other groups ( B). The low-dose DPSC-Exo and THSG-Exo groups showed significantly higher OPN levels than the PBS group, suggesting these exosomes increased OPN expression ( C, left). The THSG-Exo and the DPSC-Exo groups also differed significantly in terms of OPN expression levels, with dose-dependent characteristics. Similar trends were also observed for PCNA ( C, middle). Thus, THSG-Exo increased PCNA and OPN levels, boosting cell proliferation and osteogenic functionality in alveolar bone defect tissues. The expression levels of VEGF were higher in high-dose than in low-dose THSG-Exo ( C, right). However, the VEGF expression levels following both low-dose and high-dose DPSC-Exo treatment did not differ significantly from that in the PBS group. Although VEGF levels didn't significantly increase after DPSC-Exo treatment, it was still detectable in IHC images. Longer treatment may reveal differences in VEGF expression. Proteome comparison between DPSC-Exo and THSG-Exo The proteome results revealed that DPSC-Exo and THSG-Exo contained 180 and 280 proteins, respectively ( A and ). The 2 exosome groups shared 163 proteins, with 66 and 18 proteins being upregulated and downregulated, respectively, and 79 proteins exhibiting no significant differences. In these 2 sets of exosomes, we collectively identified 297 unique proteins, with THSG-Exo representing the majority. A Venn diagram revealed that DPSC-Exo had 17 unique proteins, constituting 5.74 % of the total. Following THSG stimulation, the THSG-Exo produced by hDPSCs had 117 unique proteins, accounting for 39.5 % of the total ( A). The top 10 results for the 117 unique proteins in THSG-Exo across three categories in the GO database (cellular component, molecular function, and biological process) are shown in B: the first 3 results were related to the cellular component category, highlighting that these unique proteins are primarily associated with the composition of exosomes ( ); in the molecular function category, the most prominent pathways were extracellular matrix (ECM) structural constituent, calcium ion binding, and integrin binding, all of which are related to tissue regeneration and repair; and in the biological process category, we observed pathways involving the positive regulation of cell adhesion, cell migration, muscle cell differentiation, and angiogenesis, among others. Subsequently, we searched the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for the 117 unique proteins, and found that they were associated with 26 biologically relevant pathways ( ). Five key pathways for regeneration and repair are ECM receptor interaction, focal adhesion, PI3K–Akt signaling, protein processing in the endoplasmic reticulum, and regulation of the actin cytoskeleton (related to integrin binding pathway) ( B). Finally, we used the Cytoscape program to visualize the links between the KEGG and GO enrichment analyses ( C). Visualization showed that THSG-Exo's unique proteins influence osteogenesis, wound healing, and tissue repair ( ). 

The transmission electron microscope (TEM) analysis revealed that the membranes of the exosomes, obtained through TFF, were spherical and remained intact; these characteristics are similar to those of exosomes obtained using the gold standard ultracentrifugation method ( A). After tunable resistive pulse sensing (TRPS) analysis, the sizes of the fresh and lyophilized samples were 100–250 nm and 60–150 mm ( B), respectively, indicating a marked size difference. Flow cytometry revealed that the expression levels of exosome marker CD9 were higher in the THSG-Exo than in the DPSC-Exo ( C). Stimulation of hDPSCs with THSG can prompt an increased production of exosomes. 

HGFs cultured with 10 μM THSG-Exo showed a significant increase in proliferation on day 5, while 100 μM THSG-Exo did not ( A). By contrast, MC3T3 cells cultured with THSG-Exo (100 μM) exhibited a significant increase in cell proliferation on day 3; THSG-Exo (10 μM) did not have this effect ( B). The limited proliferation effect of 10 μM THSG-Exo in MC3T3 cells may be due to high osteogenic differentiation rather than proliferation. 

Because 10 μM THSG-Exo stimulated cell proliferation, we used HGFs to evaluate whether exosomes can accelerate wound healing. By day 6, wounds in HGFs treated with THSG-Exo were smaller than those treated with DPSC-Exo, with slight intercellular connections observed ( C). The wound area in the THSG-Exo group was significantly smaller than that in the DPSC-Exo group on day 9. The wound area calculation results are presented in D. The wound area of HGFs cultured with THSG-Exo gradually decreased from days 6–9. On day 9, greater wound closure was observed in the THSG-Exo group than in the DPSC-Exo group. The wound area in the THSG-Exo group shrank to 40 % of its original size ( D). These results indicate that THSG-induced exosomes significantly promote cell migration and wound healing in HGFs. 

The MC3T3 cells were cultured in mineralization medium for 1, 2, and 3 weeks for osteoblast differentiation. In the group receiving THSG-Exo (10 μM), calcium nodules were observed on day 7, and in the second and third weeks, an increased number of larger dark red nodules was observed ( E). 

In our analysis, the degree of wound healing in the THSG-Exo group was superior to that in the DPSC-Exo group ( A). We used software to analyze the micro-CT images of the large periodontal defect area and measured 4 values: the relative bone volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular spacing (Tb.Sp) ( B). The THSG-Exo group had a higher BV/TV ratio than the PBS and DPSC-Exo groups, with even low-dose DPSC-Exo showing a higher ratio than PBS, indicating bone regeneration effects of exosomes ( C). Similar trends were observed in terms of Tb. Th and Tb. N, with the THSG-Exo group having higher Tb. Th and Tb. N values than the PBS and DPSC-Exo groups did. Finally, we observed a significant difference in the Tb. Sp values between the 2 low-dose exosome groups; in addition, the Tb. Sp value in the high-dose THSG-Exo group was the lowest of those of all the groups, indicating that the degree of bone osteoporosis in the THSG-Exo group was lower than that in the DPSC-Exo group within the alveolar bony defect model ( C). Micro-CT analysis showed the THSG-Exo group had greater bone regeneration and less bone rarefaction compared to the DPSC-Exo and PBS groups. 

Our results indicated a greater increase in the number of osteoblasts in the 2 exosome groups than in the PBS group ( A). In high doses, THSG-Exo led to the generation of more osteoblasts than did DPSC-Exo, confirming that the addition of THSG-Exo had a more favorable effect on new bone formation within the large bone defect. Moreover, the high dose of THSG-Exo outperformed the low dose. The IHC results indicated that both the low and high doses of THSG-Exo resulted in greater regeneration or osteogenic differentiation of cellular tissues than that which occurred in the other groups ( B). The low-dose DPSC-Exo and THSG-Exo groups showed significantly higher OPN levels than the PBS group, suggesting these exosomes increased OPN expression ( C, left). The THSG-Exo and the DPSC-Exo groups also differed significantly in terms of OPN expression levels, with dose-dependent characteristics. Similar trends were also observed for PCNA ( C, middle). Thus, THSG-Exo increased PCNA and OPN levels, boosting cell proliferation and osteogenic functionality in alveolar bone defect tissues. The expression levels of VEGF were higher in high-dose than in low-dose THSG-Exo ( C, right). However, the VEGF expression levels following both low-dose and high-dose DPSC-Exo treatment did not differ significantly from that in the PBS group. Although VEGF levels didn't significantly increase after DPSC-Exo treatment, it was still detectable in IHC images. Longer treatment may reveal differences in VEGF expression. 

The proteome results revealed that DPSC-Exo and THSG-Exo contained 180 and 280 proteins, respectively ( A and ). The 2 exosome groups shared 163 proteins, with 66 and 18 proteins being upregulated and downregulated, respectively, and 79 proteins exhibiting no significant differences. In these 2 sets of exosomes, we collectively identified 297 unique proteins, with THSG-Exo representing the majority. A Venn diagram revealed that DPSC-Exo had 17 unique proteins, constituting 5.74 % of the total. Following THSG stimulation, the THSG-Exo produced by hDPSCs had 117 unique proteins, accounting for 39.5 % of the total ( A). The top 10 results for the 117 unique proteins in THSG-Exo across three categories in the GO database (cellular component, molecular function, and biological process) are shown in B: the first 3 results were related to the cellular component category, highlighting that these unique proteins are primarily associated with the composition of exosomes ( ); in the molecular function category, the most prominent pathways were extracellular matrix (ECM) structural constituent, calcium ion binding, and integrin binding, all of which are related to tissue regeneration and repair; and in the biological process category, we observed pathways involving the positive regulation of cell adhesion, cell migration, muscle cell differentiation, and angiogenesis, among others. Subsequently, we searched the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for the 117 unique proteins, and found that they were associated with 26 biologically relevant pathways ( ). Five key pathways for regeneration and repair are ECM receptor interaction, focal adhesion, PI3K–Akt signaling, protein processing in the endoplasmic reticulum, and regulation of the actin cytoskeleton (related to integrin binding pathway) ( B). Finally, we used the Cytoscape program to visualize the links between the KEGG and GO enrichment analyses ( C). Visualization showed that THSG-Exo's unique proteins influence osteogenesis, wound healing, and tissue repair ( ). 

In this study, we elucidated the effects of THSG-induced exosomes derived from hDPSCs in vitro and in vivo . THSG-Exo (10 μM) significantly increased cell proliferation in HGF, but this effect was not observed in MC3T3 cells at the same dosage. The different effects of THSG-Exo concentrations on cell proliferation in HGFs and MC3T3 cells may be attributed to cell type specificity. Zhang et al. observed that bone marrow mesenchymal stem cell exosomes (BMMSC-Exo) induced cell proliferation in MC3T3 cells. However, Shimizu et al. discovered that exosomes derived from DPSCs and gingival mesenchymal stem cells (GMSCs) did not have a proliferative effect in MC3T3 cells. BMMSC-Exo, DPSC-Exo, and GMSC-Exo exhibit cell-specific responses in MC3T3 cells because of the distinct origins of the stem cells. In vitro experiments indicated that 10 μM THSG-Exo does not induce MC3T3 cell proliferation, likely due to involvement in other functions like osteogenic differentiation. Previous studies showed that stimulating human skin fibroblasts with ADSC-EV promoted cell proliferation and migration, increased MMP-1 expression, and phosphorylated AKT; , thus, adipose stem cell exosomes may accelerate wound healing in rat skin wounds through the PI3K–Akt pathway. Wang et al. also reported a significant increase in the expressions of MMP-1 and MMP-3 under the influence of exosomes. In a mouse skin wound model, they achieved a reduction in scar formation and improved wound repair by increasing the ratio of MMP-3 to tissue inhibitor of metalloproteinase, thereby facilitating ECM remodeling. Another team used the secretome of HGFs to accelerate the healing of large skin wounds through the secretome's anti-inflammatory effects, promotion of angiogenesis, and induction of collagen regeneration. However, few studies have considered the promotion of wound healing by DPSC-Exo. Our proteomic findings highlight the PI3K–Akt pathway and MMP proteins in promoting wound closure in HGFs and rat gingival healing via THSG-Exo. In summary, THSG-Exo includes various unique proteins related to wound healing, consistent with our findings that THSG-induced hDPSCs exosomes demonstrate enhanced wound healing abilities. Bone morphogenetic protein-2 (BMP-2) is an FDA-approved (US Food and Drug Administration) osteogenic factor. , Some research shows that BMP-2 is osteoinductive only at optimal doses, while high doses cause inflammation and abnormal bone formation. In this study, we found that THSG-Exo exhibit a greater bone-promoting effect than BMP-2 does. Lee et al. found that DPSC-Exo stimulated jawbone MSCs with osteogenic differentiation efficacy comparable to BMP-2. We propose THSG-Exo as a potential alternative to BMP-2 for bone induction, with BMP-1 as a unique protein in THSG-Exo. Related studies have confirmed that enhancing its expression can promote osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). In addition to BMP-1, THSG-Exo include anti-inflammatory proteins such as tumor necrosis factor (TNF)-α-stimulated gene 6 (TSG-6 or TNFAIP6). It can compete with the receptor activator of nuclear factor kappa-B ligand on the surface of osteoclasts to inhibit bone resorption activity. In vitro experiments have highlighted the ability of TNFAIP6 to inhibit the activation of osteoclasts and promote osteoclast apoptosis. , Moreover, THY-1 (CD90) positively regulates osteogenic differentiation in bone marrow stromal cells and inhibits adipocyte differentiation in obese mice. Our data show that THSG-Exo contains BMP-1 to activate osteoprogenitor cells and osteoprotegerin to inhibit bone-resorbing cells. Most unique proteins in THSG-Exo possess diverse functional properties, such as neuropilin 2 (NRP2). It binds with VEGF, thereby participating in the growth of neuronal axons, vascular neogenesis, and other processes. Angiopoietin-1, an ECM glycoprotein, aids in regeneration and repair by inhibiting apoptosis, promoting migration, maintaining vascular integrity, and inducing angiogenesis and tissue remodeling. Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase family member 2, can promote cell migration and proliferation through lysophosphatidic acid receptor signaling. Additionally, it participates in angiogenesis and tissue repair induced by VEGF. THSG-Exo surpassed DPSC-Exo in in vitro and in vivo experiments for wound healing, osteogenic differentiation, and tissue repair. Both types of exosomes are water-soluble and stable, and can be used clinically with dressings for direct application on soft and hard tissues. THSG-Exo is particularly effective when combined with bone graft materials to address potential gingival atrophy after tooth extraction. 

The authors have no conflicts of interest relevant to this article. 

