Lipopolysaccharide-Preconditioned Dental Follicle Stem Cells Derived Small Extracellular Vesicles Treating Periodontitis via Reactive Oxygen Species/Mitogen-Activated Protein Kinase Signaling-Mediated Antioxidant Effect

Figures & data

Figure 1 Identification of cells and sEV. (A a and B a) DFSCs and PDLSCs morphology under an optical microscope. Scale bar: 200 μm. (A b and B b) Representative pictures after osteogenesis induction for 2 weeks. Scale bar:100 μm. (A c and B c) Representative pictures after adipogenesis induction for 2 weeks. Scale bar:100μm. (C) Flow cytometry detection of the cell markers CD90, CD73, CD31, and CD34 in DFSCs. (D) Flow cytometry detection of the cell markers CD90, CD73, CD31, and CD34 in PDLSCs. (E) TEM analysis was performed and verified that D-sEV and L-D-sEV were double-membrane shapes with diameters between 30 and 150 nm. Scale bar: 200 nm. (F) Size distribution of D-sEV and L-D-sEV as determined by NTA. (G) Western blot for Hsp70, Tsg101, CD63, and GAPDH in DFSCs, D-sEV, and L-D-sEV.

Table 1 List of the Up-Regulated Proteins in L-D-sEV vs D-sEV

Protein Name	Gene Name	Molecular Function	Cellular Component	L-D-sEV/D-sEV	t test p value
Glutathione reductase	GSR	Antioxidant activity; oxidoreductase activity; catalytic activity; binding	Mitochondrial	1.9767	0.0146
Protein disulfide-isomerase A6	PDIA6	Catalytic activity; oxidoreductase activity	Endoplasmic reticulum	1.7533	0.0318
Protein S100-A1	S100A1	Binding	Cytoplasmic	1.690	0.027
Peptidyl-prolyl cis-trans isomerase A	PPIA	Catalytic activity; binding	Cytoplasmic	1.5994	0.0234
Angiopoietin-related protein 3	ANGPTL3	Binding; molecular function regulator	Nuclear	1.5170	0.0183
Syndecan-4	SDC4	Binding; molecular transducer activity	Cytoplasmic;Nuclear	1.5144	0.0498
Actin-related protein 2/3 complex subunit 4	ARPC4	Structural molecule activity; binding	Cytoplasmic;Mitochondrial	1.4568	0.0370
Follistatin	FST	Binding; molecular function regulator	Extracellular	1.3701	0.0092
Decorin	DCN	Structural molecule activity; binding	Extracellular;Nuclear	1.3246	0.0026
Thrombospondin-2	THBS2	Binding	Extracellular	1.2355	0.02102
NADP-dependent malic enzyme	ME1	Catalytic activity; binding; oxidoreductase activity	Cytoplasmic	1.2247	0.0367

Table 2 List of the Down-Regulated Proteins in L-D-sEV vs D-sEV

Protein Name	Gene Name	Molecular Function	Cellular Component	L-D-sEV/D-sEV	t test p value
Neurogenic locus notch homolog protein 1	NOTCH1	Transcription regulator activity; binding; molecular function regulator; molecular transducer activity	Extracellular	0.8134	0.0170
L-xylulose reductase	DCXR	Catalytic activity; binding; oxidoreductase activity	Mitochondrial	0.7964	0.0094
Plexin domain-containing protein 2	PLXDC2	Not retrieved	PlasmaMembrane; Nuclear	0.7497	0.0320
Ubiquitin carboxyl-terminal hydrolase isozyme L1	UCHL1	Catalytic activity; binding	Cytoplasmic	0.7369	0.0482
Laminin subunit alpha-1	LAMA1	Structural molecule activity; binding	Extracellular	0.7293	0.0298
Fumarate hydratase, mitochondrial	FH	Catalytic activity; binding	Mitochondrial	0.7174	0.0350
Lysosomal protective protein	CTSA	Catalytic activity; molecular function regulator	Lysosomal	0.7033	0.0350
Collagen alpha-1(XIV) chain	COL14A1	Structural molecule activity; binding	Cytoplasmic; Nuclear	0.6954	0.0239
Inactive tyrosine-protein kinase 7	PTK7	Catalytic activity; binding; molecular transducer activity	Cytoplasmic; Nuclear	0.6896	0.0137
Lactotransferrin	LTF	Catalytic activity; binding; molecular function regulator	Extracellular	0.6133	0.0307
Dual specificity mitogen-activated protein kinase kinase 1	MAP2K1	Catalytic activity; structural molecule activity; binding; molecular function regulator	Cytoplasmic; Nuclear	0.5590	0.0412
Protein sidekick-1	SDK1	Binding	PlasmaMembrane; Nuclear	0.5110	0.0497

Table 3 List of the Detected or Undetected Proteins in L-D-sEV vs D-sEV

Protein Name	Gene Name	Molecular Function	Cellular Component	L-D-sEV	D-sEV
Actin-related protein 2/3 complex subunit 1B	ARPC1B	Structural molecule activity; binding;	Extracellular	Detected	Undetected
Prolyl 4-hydroxylase subunit alpha-2	P4HA2	Catalytic activity; binding; oxidoreductase activity	Endoplasmic reticulum	Detected	Undetected
Superoxide dismutase [Cu-Zn]	SOD1	Antioxidant activity; catalytic activity; binding; oxidoreductase activity	Cytoplasmic	Detected	Undetected
Hepatocyte growth factor receptor	MET	Catalytic activity; binding; molecular transducer activity	PlasmaMembrane	Detected	Undetected
Creatine kinase B-type	CKB	Catalytic activity; binding	Cytoplasmic	Detected	Undetected
Ribonuclease inhibitor	RNH1	Molecular function regulator	Cytoplasmic	Detected	Undetected
Tenascin-X	TNXB	Structural molecule activity; binding	Nuclear	Detected	Undetected
Heterogeneous nuclear ribonucleoproteins A2/B1	HNRNPA2B1	Binding	Nuclear	Detected	Undetected
40S ribosomal protein S3	RPS3	Catalytic activity; structural molecule activity; binding	Cytoplasmic	Detected	Undetected
Palmitoyl-protein thioesterase 1	PPT1	Catalytic activity; binding	Lysosomal	Detected	Undetected
Ephrin type-A receptor 4	EPHA4	Catalytic activity; binding; molecular transducer activity	PlasmaMembrane;Cytoplasmic	Detected	Undetected
Prostaglandin E synthase 3	PTGES3	Catalytic activity; binding	Nuclear	Detected	Undetected
Neuronal pentraxin-1	NPTX1	Binding	Extracellular; Cytoplasmic; Mitochondrial	Detected	Undetected
Protein phosphatase 1A	PPM1A	Catalytic activity; binding	Cytoplasmic	Undetected	Detected
Eukaryotic translation initiation factor 2 subunit 3B	EIF2S3B	Catalytic activity; binding	Cytoplasmic; Mitochondrial	Undetected	Detected

Figure 2 Quantitative proteomic analysis of D-sEV and L-D-sEV. (A) Gene ontology (GO) analysis of the DEPs in L-D-sEV vs D-sEV. The right coordinate axis indicates the number of proteins for each GO annotation, and the lower coordinate axis indicates the GO annotations. Blue stripes indicate biological process categories of DEPs; red stripes indicate categories of molecular functions; yellow stripes indicate categories of cellular components. (B) PPI analysis of some DEPs related to antioxidant activity. (C) KEGG annotation analysis of DEPs in L-D-sEV vs D-sEV.

Figure 3 L-D-sEV inhibited the RANKL/OPG ratio in PDLSCs via ROS/JNK signaling under inflammatory conditions. (A) PDLSCs were cocultured with (100 μg/mL) D-sEV or L-D-sEV, and cell viability was measured by CCK-8 assay. (B) Representative images of the cell migration assay and quantitative analysis. Scale bar: 200 μm. (C) Intracellular ROS levels were measured by flow cytometry. PDLSCs were pretreated with LPS (1 μg/mL, 24 h) and then cocultured with D-sEV or L-D-sEV (100 μg/mL, 4 h or 8 h). (D) Quantitative analysis of intracellular ROS levels. (E) The JNK, p-JNK, ERK, p-ERK, OPG and RANKL protein levels were measured by Western blot. PDLSCs were cocultured with D-sEV or L-D-sEV (100 µg/mL, 48 h). (F) Quantitative analysis of the protein bands in Figure 3E. (G) The JNK, p-JNK, ERK, p-ERK, OPG and RANKL protein levels were measured by Western blot. PDLSCs were stimulated with LPS (1 µg/mL, 24 h) and then cocultured with D-sEV or L-D-sEV (100 µg/mL, 48 h). (H) Quantitative analysis of the protein bands in Figure 3G. (I) The OPG, RANKL, JNK, and p-JNK protein levels were measured by Western blot and quantitative analysis. PDLSCs were incubated with LPS (1 μg/mL, 24 h) and then stimulated with SP600125 (5 µM, 10 µM) for 24 h. (J) DFSC-sEV inhibited the RANKL/OPG ratio via ROS/JNK signaling. Data are shown as the mean ± SEM for three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4 L-D-sEV promoted macrophage proliferation and M2 polarization. (A) Uptake analysis of D-sEV and L-D-sEV by RAW264.7 cells (red: phalloidin, green: DiO-labeled sEV, blue: DAPI-labeled nuclei). Scale bar: 10 μm. (B) RAW264.7 cells were cocultured with (20, 50, or 100 μg/mL) D-sEV or L-D-sEV, and cell viability was measured by CCK-8 assay. (C) The Arg-1 and CD206 gene levels of RAW264.7 cells were measured by qRT–PCR. (D) The percentage of M2 macrophages was measured by flow cytometry. Double-positive cells (CD206 and F4/80) represented M2 macrophages. Data are shown as the mean ± SEM for three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5 L-D-sEV promoted M2 polarization via ROS/ERK signaling. (A) The percentage of M2 macrophages was measured by flow cytometry. RAW264.7 cells were treated with D-sEV or L-D-sEV (50 μg/mL, 6 h) with or without 25 μM H₂O₂ or 10 mM NAC stimulation. (B) The GSSG/GSH ratio of macrophage. RAW264.7 cells were treated with D-sEV or L-D-sEV (50 µg/mL, 6 h). (C) The SOD1, SOD2, ERK and p-ERK protein levels were measured by Western blot. Raw264.7 Cells were first pretreated with or without u1206 (10 µM, 30 min) and then cocultured with or without D-sEV or L-D-sEV (50 µg/mL, 6 h). (D) Quantitative analysis of the protein bands in Figure 5C. (E) Intracellular ROS levels were measured by flow cytometry and quantitative analysis. RAW264.7 cells were first pretreated with or without u1206 (10 µM, 30 min) and then cocultured with or without D-sEV or L-D-sEV (50 µg/mL, 6 h). (F) Illustration showing that sEV promotes M2 polarization via SOD1/ERK-mediated ROS. Data are shown as the mean ± SEM for three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6 Physical and biological characterization of HA/sEV. (A) The schematic diagram of material construction. (B) The dynamic viscosity of the mixture of sEV (dissolved in PBS) and HA with different volume ratios (0.125, 0.25, 0.5). (C) The frequency scanning analysis. (D) Release experiment of the D-sEV-loaded HA. (E) Release experiment of the L-D-sEV-loaded HA. (F) The effect of (5%, 10%, 20%) HA or 5% HA with D-sEV or L-D-sEV (200 μg/mL) on PDLSCs viability was measured by CCK-8 assay. (G) Representative images of the cell migration assay and quantitative analysis. Scale bar: 200 μm. Data are shown as the mean ± SEM for three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7 HA/sEV promoted periodontal regeneration in vivo. (A) Representative micro-CT images showing new bone formation in the PD, PBS, D-sEV, and L-D-sEV groups at 8 weeks post-injection. Scale bar: 1 mm. The red box represents the bone defect size. (B) The BV/TV and Tb. SP comparison. (C) The representative H&E stained sections. Scale bar: 200 μm. (D) The representative Masson stained sections. The black arrow represents Notch. (E) Quantitative analysis of PDL width. (F) The number of vessels in the PDL. (G) Representative images of Periostin immunofluorescence staining in PDL. Scale bar:100 μm. (H) Quantitative analysis of Periostin expression in PDL. (I) Representative images of CD206 immunofluorescence staining in ALB. Scale bar: 100 μm. (J) Quantitative analysis of CD206 expression in ALB. (K) Representative images of RANKL immunofluorescence staining in PDL. Scale bar: 100 μm. (L) Representative images of OPG immunofluorescence staining in PDL. Scale bar: 100 μm. (M) Quantitative analysis of RANKL/OPG ratio in PDL. Data are shown as the mean ± SEM for three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Abbreviations: ALB, alveolar bone; D, dentin; PDL, periodontal ligament; PD, periodontitis group; HA, 0.2% hyaluronic acid gel; PBS, PBS-loaded HA; D-sEV, D-sEV-loaded HA; L-D-sEV, L-D-sEV-loaded HA.

Table 4 Serum IgA, IgG, IgM, C3, C4, and CRP Levels in Canine

	IgA (g/L)	IgG (g/L)	IgM (g/L)	C3 (g/L)	C4 (g/L)	CRP(μg/L)
N	<0.1	1.92	1.08	<0.1	0.06	290
0 W	<0.1	2.00	1.41	<0.1	0.06	280
4 W	<0.1	1.44*	1.33	<0.1	0.06	240
8 W	<0.1	1.70**	1.13	<0.1	0.06	230

Notes: Serum immunoglobulins (IgA, IgG, IgM), complements (C3, C4), and CRP fluctuated slightly around normal levels in the following month after surgery. N, normal condition; 0 W, post-operation (1 month after experimental periodontitis induction); 4W, postoperative for 4 weeks; 8 W, postoperative for 8 weeks. Data are shown as mean. (4 W vs 0 W) *P < 0.05; (8 W vs 0 W) **P < 0.01.