Effect of total sonicated Aggregatibacter actinomycetemcomitans fragments on gingival stem/progenitor cells

Abstract

Background: Aggregatibacter-actinomycetemcomitans (A.actinomycetemcomitans) are strongly associated with localized-aggressive-periodontitis (LAgP). The study's aim was to test for the first time the effect of total sonicated A.actinomycetemcomitans-bacterial-fragments on gingival mesenchymal stem/progenitor cells' (G-MSCs) proliferation and regenerative gene expression in-vitro.

Material and methods: G-MSCs were isolated, characterized, expanded and stimulated by total sonicated A.actinomycetemcomitans-bacterial-fragments (0 (negative-control), 15, 60, 120 and 240µg/ml; serovar-b; n=6/group). Cellular proliferation and NF-κβ (NFKB1), Alkaline Phosphatase (ALPL), Collagen-I (COL1A1), Collagen-III (COL3A1), Osteonectin (SPARC) and Osteopontin (SPP1) m-RNA expression were assessed via reverse-transcription-polymerase-chain-reaction (RT-PCR) at 24, 48 and 72 hours and CFUs-ability evaluated at twelve days.

Results: G-MSCs demonstrated stem/progenitor cells' characteristics. A.actinomycetemcomitans-bacterial-fragments (up to 72 hours) resulted in marked G-MSCs' proliferation over-time (p<0.001) and elevated NFKB1 (p=0.017), COL1A1 (p=0.025), SPARC (p=0.025), decreased ALPL (p=0.017), with no significant differences for COL3A1 and SPP1 expression or stimulation times (p>0.05; Friedman-test). Longer-term stimulation for twelve days reduced G-MSCs' CFUs.

Conclusions: Sonicated A.actinomycetemcomitans-bacterial-fragments' exert beneficial short-term effects on G-MSCs' proliferative and non-mineralized tissue forming aptitude. Results shed new light on the importance of periodontal treatment for LAgP patients, using power driven sonic/ultrasonic devices, which, in addition to reducing the subgingival microbial load, produces cell-stimulatory A.actinomycetemcomitans-bacterial-fragments, with positive attributes on tissue reparative/regenerative responses of tissue resident stem/progenitor cells in their niche.

Figure 1

Isolation and characterization of G-MSCs. (A) Microscopic appearance of outgrowing cells from free gingival margin connective tissue. (B) Microscopic appearance of CFUs of G-MSCs stained with crystal violet. (C) Gram-staining of Aggregatibacter actinomycetemcomitans (A.actinomycetemcomitans) colonies. (D) Alizarin Red staining of G-MSCs after osteogenic induction. (E) Oil Red O staining of G-MSCs after adipogenic stimulation. (F) Alcian Blue and nuclear-fast-red counter staining of G-MSCS after chondrogenic stimulation. (G) Alizarin Red staining of G-MSCs cultured in basic medium. (H) Oil Red O staining of G-MSCs cultured in basic medium. (I) Alcian Blue and nuclear-fast-red counter staining of G-MSCs cultured in basic medium.

Figure 2

Cell proliferation and CFUs assay of G-MSCs with different concentration of total sonicated A.actinomycetemcomitans-bacterial fragments. (A) MTT results (box plots with medians and quartiles) of G-MSCs proliferation at 24, 48 and 72 hours and concentrations 0 (negative-control), 15, 60, 120 and 240 µg/ml A.actinomycetemcomitans-bacterial fragments (n=6/group). Significant differences between time-points with asterisks (***, p<0.001; Wilcoxon-signed-rank-test). (B) Crystal violet staining of G-MSCs CFUs for twelve days in concentrations 0 (negative-control), 15, 60, 120 and 240 µg/ml A.actinomycetemcomitans-bacterial fragments.

Figure 3

m-RNA expression (box plots with medians and quartiles) of NF-κβ (NFKB1) and the regenerative transcription factors: alkaline phosphatase (ALPL), Collagen-I (COL1A1), Collagen-III (COL3A1), Osteonectin (SPARC) and Osteopontin (SPP1) in G-MSCs at 24, 48 and 72 hours and concentrations 0 (negative-control), 15, 60, 120 and 240 µg/ml of total sonicated A.actinomycetemcomitans-bacterial fragments. Significant differences are marked with asterisks (*; p<0.05, **; p<0.01, Wilcoxon-signed-rank-test).