optimization-ligature-induced-periodontitis-mouse-model-for

Optimization of the Ligature-induced Periodontitis Mouse Model for Preclinical Research

The experimental model of ligature-induced periodontitis in mice has played a fundamental role in understanding periodontal disease pathogenesis; yet, it ...

University of North Carolina at Chapel Hill

Chitosan films containing distilled pyroligneous extracts of Eucalyptus grandis (DPEC), characterized and developed by Brazilian Agricultural Research Corporation-Embrapa Temperate Agriculture (EMBRAPA-CPACT), were evaluated for antimicrobial activity against Candida albicans, Streptococcus mutans, and Lactobacillus acidophilus by direct contact test. Further, their capacity for the prevention of teeth enamel demineralization and cytotoxicity in vitro were also determined. The natural polymers were tested at different concentrations (1500-7500 µg mL) and the formulation of an experimental fluoride varnish with antimicrobial activity was evaluated by direct contact test, whereas cytotoxicity was analyzed through the colorimetric MTT assay. Preliminary data showed no statistically significant differences in cytotoxicity to NIH/3T3 cell line when DPEC is compared to the control group. On the other hand, the antimicrobial capacity and demineralization effects were found between the test groups at the different concentrations tested. Chitosan films containing distilled pyroligneous extracts of E. grandis may be an effective control strategy to prevent biofilm formation related to dental caries when applied as a protective varnish. They may inhibit the colonization of oral microorganisms and possibly control dental caries through a decrease in pH and impairment of enamel demineralization.

Antimicrobial and cytotoxic capacity of pyroligneous extracts films of Eucalyptus grandis and chitosan for oral applications.

Periodontal destruction can be the result of different known and yet-to-be-discovered biological pathways. Recent human genetic association studies have implicated interferon-gamma inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) with high periodontal interleukin (IL)-1β levels and more destructive disease, but mechanistic evidence is lacking. Here, we sought to experimentally validate these observational associations and better understand IFI16 and AIM2's roles in periodontitis.

Interferon activated gene 204 protects against bone loss in experimental periodontitis.

Inflammatory bowel disease (IBD) is a significant global health problem that involves chronic intestinal inflammation and can involve severe comorbidities, including intestinal fibrosis and inflammation-associated colorectal cancer (CRC). Disease-associated alterations to the intestinal microbiota often include fecal enrichment of , which are strongly implicated in IBD development. This dysbiosis of intestinal flora accompanies changes in microbial metabolites, shaping host:microbe interactions and disease risk. While there have been numerous studies linking specific bacterial taxa with IBD development, our understanding of microbial function in the context of IBD is limited. Several classes of microbial metabolites have been directly implicated in IBD disease progression, including bacterial siderophores and genotoxins. Yet, our microbiota still harbors thousands of uncharacterized microbial products. In-depth discovery and characterization of disease-associated microbial metabolites is necessary to target these products in IBD treatment strategies. Towards improving our understanding of microbiota metabolites in IBD, it is important to recognize how host relevant factors influence microbiota function. For example, changes in host inflammation status, metal availability, interbacterial community structure, and xenobiotics all play an important role in shaping gut microbial ecology. In this minireview, we outline how each of these factors influences gut microbial function, with a specific focus on IBD-associated  metabolites. Importantly, we discuss how altering the intestinal microenvironment could improve the treatment of intestinal inflammation and associated disorders, like intestinal fibrosis and CRC.

Microenvironmental Factors that Shape Bacterial Metabolites in Inflammatory Bowel Disease.

Human periodontal ligament stem cells (PDLSCs) have been studied as a promising strategy in regenerative approaches. The protease-activated receptor 1 (PAR) plays a key role in osteogenesis and has been shown to induce osteogenesis and increase bone formation in PDLSCs. However, little is known about its effects when activated in PDLSCs as a cell sheet construct and how it would impact bone formation as a graft in vivo. Here, PDLSCs were obtained from 3 patients. Groups were divided into control, osteogenic medium and osteogenic medium + PAR activation by TFLLR-NH2 peptide. Cell phenotype was determined by flow cytometry and immunofluorescence. Calcium deposition was quantified by Alizarin Red Staining. Cell sheet microstructure was analyzed through light, scanning electron microscopy and histology and transplanted to Balb/c nude mice. Immunohistochemistry for bone sialoprotein (BSP), integrin β1 and collagen type 1 and histological stains (H&E, Van Giesson, Masson's Trichrome and Von Kossa) were performed on the ex-vivo mineralized tissue after 60 days of implantation in vivo. Ectopic bone formation was evaluated through micro-CT. PAR activation increased calcium deposition in vitro as well as BSP, collagen type 1 and integrin β1 protein expression and higher ectopic bone formation (micro-CT) in vivo.

Osteogenesis in human periodontal ligament stem cell sheets is enhanced by the protease-activated receptor 1 (PAR) in vivo.

We have previously characterized the main osteoimmunological events that occur during ligature periodontitis. This study aims to determine the polymicrobial community shifts that occur during disease development.

Oral biofilm dysbiosis during experimental periodontitis.

Encephalitis is a syndrome characterized by brain damage secondary to an inflammatory process that is manifested by cognitive impairment and altered cerebral spinal fluid analysis; it may evolve with seizures and coma. Despite viral infections representing the main cause of encephalitis in children, respiratory syncytial virus (RSV) and parainfluenza virus are mostly associated with respiratory presentations. Uncommonly, the inflammatory phenomena from encephalitis secondary to viral agents may present with an exacerbated host response, the so-called cytokine storm. The link between these infectious agents and neurologic syndromes resulting in a cytokine storm is rare, and the underlying pathophysiology is still poorly understood.

Encephalitis and cytokine storm secondary to respiratory viruses in children: Two case reports.

Gingipains are important virulence factors present in Porphyromonas gingivalis. Arginine-specific gingipains (RgpA and RgpB) are critically associated with increased proteolytic activity and immune system dysfunction, including neutrophilic activity. In this study, we assessed the impact of gingipains (RgpA and RgpB) on neutrophil function.

Arginine-specific gingipains (RgpA/RgpB) knockdown modulates neutrophil machinery.

Periodontal regeneration is a challenge, and tissue engineering based on periodontal ligament stem cells (PDLSCs) has been shown to be a promising alternative to this process. However, the need for scaffolds has limited the therapeutic use of PDLSCs. In this context, scaffold-free tissue engineering using the cell sheet (CS) technique has been developed as an alternative approach to improve tissue regeneration. Previously, we showed that Protease-activated receptor-1 (PAR1) can regulate PDLSCs. Herein, we evaluate whether PAR1 influences osteogenesis in CSs produced from PDLSCs, without the use of scaffolds. PDLSCs were isolated and immunophenotyped. Then, CSs were obtained by supplementing the culture medium with ascorbic acid (50 µg/mL), and PAR1 was activated through its agonist peptide (100 nM). Scaffold-free 3D CSs were successfully produced from PDLSCs, and they showed higher proliferation potential than isolated PDLSCs. Also, PAR1 activation decreased senescence and improved osteogenic differentiation of CSs by increasing mineralized nodule deposition and alkaline phosphatase concentration; PAR1 also modulated osteogenic markers at the gene and protein levels. We further demonstrated that this effect was regulated by Wnt, TGF-βI, MEK, p38 MAPK, and FGF/VEGF signaling pathways in PDLSCs (p < 0.05%). Overall, PAR1 activation increased osteogenic activity in CSs, emerging as a promising scaffold-free therapeutic approach for periodontal regeneration.

Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation.

Inflammasome initiates inflammation via the maturation of interleukin-1 beta (IL-1β). Periodontitis is a prevalent, male-biased disease characterized by inflammation-driven bone loss, yet the mechanism(s) of this sex bias is unknown. This study explored whether enhanced inflammasome represents a causal mechanism for this bias. Analyses of three separate human studies (>6,200 samples) show that males have significantly higher IL-1β in the gingival crevicular fluid than females during health and periodontitis. This pattern is experimentally reproduced with different versions of the ligature-induced periodontitis mouse model where males show greater IL-1β secretion than females. The inflammasome drives bone resorption in males but not females as revealed by analyses of inflammasome gene-deletion mice. Pharmacologic treatment with a caspase-1/4 inhibitor reduces inflammatory cell infiltration, dampens osteoclastogenesis signaling (via the receptor activator of nuclear factor-kappa B pathway), and prevents bone resorption in males but not females during experimental periodontitis. While ovariectomized females show no change in their nonresponsiveness to caspase-1/4 inhibition, orchiectomized males no longer respond to the inhibition, suggesting the importance of an intact male reproductive system in the mediation of this inhibition. Thus, our study identifies inflammasome activation as causal for male-biased experimental periodontitis and supports sex-stratified studies to foster future advancement of inflammasome therapeutics in periodontics.

Article	Year
Antimicrobial and cytotoxic capacity of pyroligneous extracts films of Eucalyptus grandis and chitosan for oral applications. Scientific reports\| PubMed ID: 34728647	2021
Interferon activated gene 204 protects against bone loss in experimental periodontitis. Journal of periodontology\| PubMed ID: 35404474	2022
Microenvironmental Factors that Shape Bacterial Metabolites in Inflammatory Bowel Disease. Frontiers in cellular and infection microbiology\| PubMed ID: 35959366	2022
Osteogenesis in human periodontal ligament stem cell sheets is enhanced by the protease-activated receptor 1 (PAR) in vivo. Scientific reports\| PubMed ID: 36117187	2022
Oral biofilm dysbiosis during experimental periodontitis. Molecular oral microbiology\| PubMed ID: 36189827	2022
Encephalitis and cytokine storm secondary to respiratory viruses in children: Two case reports. Frontiers in pediatrics\| PubMed ID: 36741098	2022
Arginine-specific gingipains (RgpA/RgpB) knockdown modulates neutrophil machinery. Journal of oral microbiology\| PubMed ID: 38988325	2024
Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation. Brazilian oral research\| PubMed ID: 39258632	2024
Inflammasome targeting for periodontitis prevention is sex dependent. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 41144672	2025

Tomaz Alves

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