Human Bone Marrow Stromal Cell Exosomes Ameliorate Periodontitis

Abstract

Human bone marrow stromal cell (hBMSC)-derived exosomes are promising therapeutics for inflammatory diseases due to their unique microRNA (miRNA) and protein cargos. Periodontal diseases often present with chronicity and corresponding exuberant inflammation, which leads to loss of tooth support. In this study, we explored whether hBMSC exosomes can affect periodontitis progression. hBMSC exosomes were isolated from cell culture medium through sequential ultracentrifugation. miRNAs and proteins that were enriched in hBMSC exosomes were characterized by RNA sequencing and protein array, respectively. hBMSC exosomes significantly suppressed periodontal keystone pathogen Porphyromonas gingivalis-triggered inflammatory response in macrophages in vitro. Transcriptomic analysis suggested that exosomes exerted their effects through regulating cell metabolism, differentiation, and inflammation resolution. In vivo, weekly exosome injection into the gingival tissues reduced the tissue destruction and immune cell infiltration in rat ligature-induced periodontitis model. Collectively, these findings suggest that hBMSC-derived exosomes can potentially be used as a host modulation agent in the management of periodontitis.

Keywords: cell therapy; exosomes; experimental periodontitis; human bone marrow stromal cells; miRNA; periodontal inflammation.

Figure 1.

Characterization of mesenchymal stem cell exosomes. (A) Transmission electron microscopy image of exosomes. (B) The sizes of exosomes were measured by ZetaView NTA. (C) Particle numbers from 3 different exosome batches were measured and normalized to protein concentration. (D) Protein markers were evaluated by Western blot. (E) Exosomes were labeled by PKH26 and the unbound dye was washed off by ultracentrifugation. Labeled exosomes were added to RAW cells (2 µg/mL) for 18 h, and the cells were imaged by florescent microscopy. 10× and 63× presented the lenses that were used. Blue: DAPI. Red: PKH26.

Figure 2.

The RNA and protein components of human bone marrow stromal cell (hBMSC) exosomes. (A) RNA from hBMSCs and their exosomes were extracted, and distinct patterns between cellular and exosomal RNA were noticed. Small RNAs were highly enriched in exosomes. (B) Top 10 most abundant microRNAs (miRNAs) in hBMSC exosomes identified by RNA sequencing. Their quantities in cells are also listed. (C) Top 5 most enriched miRNAs in exosomes. (D) Cytokines and growth factors in hBMSC exosomes and supernatants. Thirty-one of 120 cytokines and growth factors in the array were detected in exosomes, whereas 48 were found in the medium supernatant.

Figure 3.

Exosomes inhibited cytokine gene expression and protein production. (A) RAW264.7 macrophages were treated with Escherichia coli lipopolysaccharide (0.1 µg/mL) and exosomes (2 µg/mL) for 12 h. The interleukin-6 (IL-6) protein in culture medium was analyzed by enzyme-linked immunosorbent assay (ELISA). (B) Reverse transcription polymerase chain reaction (RT-PCR) showed that Il6 gene expression was suppressed after exosome treatment. (C, D) Heat-killed Porphyromonas gingivalis (Pg) was used to stimulated RAW264.7 cells for 12 h with and without exosomes (2 µg/mL). The IL-6 protein expression in culture medium was measured by ELISA (C) and the cytokine gene expressions of ll6, ll1b, and ll10 were measured by RT-PCR (D). *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001.

Figure 4.

The impact of exosomes on transcriptome. RAW264.7 cells were stimulated by heat-killed Pg with and without exosomes (2 µg/mL). (A) RNA sequencing was performed and the top 10 biological processes in gene ontology (GO) analysis were listed and ranked by false discovery rate (FDR). The raw P values were labeled on the side. (B) Reverse transcription polymerase chain reaction (RT-PCR) was applied to measure the expression of a panel of genes identified from RNA sequencing. NT means no Pg or exosome treatment. Pg represents heat-killed Porphyromonas gingivalis bacteria. Exo represents exosome treatment. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 5.

Exosome treatment ameliorated periodontal bone loss in vivo. (A) Experiment timeline. Ligatures were placed around the left maxillary second molar (M2) for 3 wk. Exosomes or saline were injected to gingival tissues once a week. (B) Representative micro–computed tomography images of the ligated and nonligated maxillae. (C) Normalized bone volume/tissue volume (BV/TV) representing the ratio between treatment (left side) versus healthy control (right side). (D) Normalized linear alveolar bone loss (ABL) representing the ratio of treatment (left side)/healthy control (right side). (E) Hematoxylin and eosin staining. Upper panels, low magnification (the scale bars were 200 µm). Lower panels, high magnification of the areas in the yellow boxes (the scale bars were 50 µm). (F) Quantitative measurement of infiltrated lymphocytes in the interproximal gingival tissues.