Expert in oral health: COSSMA

photo: Roman Samborskyi/Shutterstock.com

In synergy, several microorganisms ensure a healthy oral flora. If this is out of balance, inflammation and periodontal diseases can develop, which sometimes lead to systemic diseases. Stefan Fellner reports on the positive effects that the use of an extract from medicinal moor can have.

Gingivitis and periodontitis are common oral infection that affects the tissues that surround and support teeth. The prevalence of periodontal disease was reported to range from 20% to 50% around the world and is considered a major health problem¹.

Periodontitis, a chronic inflammatory disease causes tooth loss and deterioration of gingiva, alveolar bone, and periodontal ligaments. Gingivitis, an inflammatory condition of the gingival tissue, is a prerequisite for the development of periodontal disease.

Porphyromonas gingivalis is strongly implicated in the pathogenesis of periodontitis². This bacterium is a gram negative, rod-shaped, obligate anaerobe belonging to the 500 bacterial species living in the oral cavity³. It infects periodontal tissues as a secondary infection through interactions with commensal streptococci⁴. In addition, P. gingivalis induces impairment of oral epithelial barrier functions which potentiate susceptibility to microbial infections, leading to local tissue inflammation responsible for periodontium (tooth-supporting tissues such as gingiva and alveolar bone) and systemic diseases⁵. Recent studies show that P. gingivalis induces premature senescence, a cellular process defined as irreversible cell cycle arrest which contributes to aging and age² related diseases⁶.

Previous studies demonstrated multiple properties of AHE (Alpin Heilmoor Extract), including anti-inflammatory, antioxidant, skin barrier strengthening effects, for better maintenance of healthy skin. Scientists found that it is equally effective in maintaining oral health. Several in vitro and clinical studies were performed to determine the benefits of AHE in maintaining oral health and hygiene.

figure 1: Immediate and long term effect of 5% AHE on P. gingivalis in-vitro. figures: Premium Organic

Antibacterial activity

Patients with periodontal disease have a greater proportion of Gram-negative, proteolytic, bacteria such as P. gingivalis or Tanneralla forsythia, as well as species of Prevotella, Fusobacterium, and Treponema in their subgingival plaques while healthy adults are composed primarily of Gram-positive species belonging to the genera of actinomyces and Streptococcus, and to the Gram-negative genus Veillonella⁷.

P gingivalis is known to be highly proteolytic, with primary activity from a class of endopeptidases known as gin-gipains that are critical virulence factors⁸. Proteolysis actively changes the environmental conditions; importantly, causing a pH increase, thus creating an environment more hospitable to many Gram-negative anaerobes which mediates periodontitis⁹. These bacteria are also responsible for the bad smell related to halitosis. AHE was investigated by quantifying, in gingiva probing samples, the six most important bacterial species indicative for periodontitis, peri-implantitis and halitosis before and after application.

After 24h of contact time, a base tooth-paste with 5% of the ingredient was shown to be 99% effective against P. gingivalis (figure 1). AHE showed high anti-bacterial activity against P. gingivalis and can be applied to prevent periodontal diseases and halitosis.

Reinforcement of oral epithelial barrier

Epithelial cells are interconnected to each other by several specialised transmembrane molecular complexes, among them cell-cell junctions comprising tight junctions, adherens junctions, gap junctions, and desmosomes. They provide the first line of defence in the oral mucosa.

It is known that P. gingivalis impairs oral epithelial barrier partly through targeting grainyhead-like² (GRHL2), an epithelial-specific transcription factor which regulates the expression of the junction proteins⁵.

E-cadherins are components of adherens junctions. These structures ensure intercellular adhesion amongst epithelial cells. They regulate a diverse range of other cellular processes next to adhesion, such as cell shape, division, growth, apoptosis, wound healing, and barrier function.

AHE was found to activate E-Cad B1 expression by 57% and E-Cad-B2 by 25% in cultured HaCaT cells, hence counteracting the invasive properties of P. gingivalis. Therefore, it enhances the oral mucosa barrier thus preventing injuries and penetration of noxious substances that can impair oral health and responsible for non-oral systemic diseases.

figure 2: Dose-dependent reduction of senescent cells by AHE. figures: Premium Organic

Protection against cell senescence

Recent study shows that P. gingivalis induces premature senescence in dendritic cells by direct cellular invasion leading to disruption of immune homeostasis in periodontitis6. Cellular senescence is an irreversible growth arrest evoked by various stimuli, including oxidative, epigenetic, and genotoxic stresses, telomere damage, and oncogene activation^10.

During the senescence process, senescent cells secrete senescence-associated phenotype (SASP) factors (including cytokines, proteases, growth factors, and matrix metalloproteases) that preclude senescent cells clearance by immune system, a process orchestrated by age-related immune system remodeling¹¹. While accumulation of senescent cells has been demonstrated to play a causal role in driving ageing and chronic diseases, their clearance has been shown to delay and reduce the aging phenotype in several tissues of premature and natural aged models.

Defence against infection, improvement of wound healing and the regenerative capability of tissues are also crucial parameters in preventing premature gingival ageing and bacteria invasion. Gingival fibroblasts are the primary cell type present in periodontal connective tissue and maintain gingival tissue integrity by regulating collagen and proteoglycan meta-bolism. However, in response to P. gingivalis LPS, gingival fibroblasts produce several proinflammatory cytokines such as interleukin IL-6 and IL-8 which mediates periodontitis¹².

AHE may therefore act through various potential mechanisms involving gingival fibroblasts and senescent cells that play important roles in the pathogenesis of periodontitis and gingival ageing. Here, senescence-associated β-galactosidase (SA β-gal) was used, a common biomarker of cellular senescences. AHE 1%, 2% and 5% was able to clear by 8%, 14 and 21% the SA β-gal positive cells, respectively, demonstrating that it exhibits significant dose-dependent senolytic properties. The extract can therefore be used to prevent premature aging and consequently to preserve the wound healing and regenerative properties of gums (figure 2).

figure 3: Realtime wound healing assay of 1%, 2% and 5% AHE.figures: Premium Organic

Wound healing and regeneration

Wound healing and regenerative capacity are key properties for a healthy oral mucosa. After injury to periodontal tissues, a sequentially phased healing response is initiated that enables wound closure and partial restoration of tissue structure and function. Fibroblasts, which synthesise and organise the collagen fibres that link alveolar bone and gingiva to the cementum covering the tooth root, play a critical role during periodontal wound healing¹³. Importantly, regeneration of connective tissues involves different cellular activities driven by fibroblasts populations.

These include the cellular proliferation, migration and the secretion of matrix molecules, the organisation of these matrix components into functionally active fibres that finally restore the periodontium. A real-time wound healing assay with NIH-3T3 fibroblasts which followed the wound healing process by live imaging over 48 hours showed that the extract significantly accelerated wound healing process (figure 3), demonstrating its pro-healing properties and regenerative capacity.

figure 4: LPS-induced inflammatory responses in monocytes by AHE. figures: Premium Organic

Inflammation controlled

Chronic inflammation of the gingiva, supporting connective tissues, and the alveolar bone causes periodontal diseases¹⁴. Thus, preventing inflammation is critical in preventing periodontal diseases. P. gingivalis is recognised as a keystone pathogen of the disease7 provoking periodontal microbiota¹⁵. P. gingivalis contributes to the pathogenesis of aggressive periodontitis by inducing high levels of proinflammatory cytokines¹⁶. P. gingivalis lipopolysaccharide (LPS) plays a key role in this process.

First, AHE 1% and 2% reduced by 13%, and 65%, respectively, the release of prostaglandin E2 (PGE2), a proinflammatory lipid mediator, induced by LPS treatment in Raw cells. In addition, the anti-inflammatory of the extract was confirmed thanks to IL-1β or LPS-induced inflammatory responses in monocytes. AHE 2% reduced the release of several proinflammatory mediators including IL-6, IL-8, Isoprostan (figure 4).

Against oxidative stress and glycation

Oxidative stress is characterised by an accumulation of reactive oxygen species (ROS) and plays a key role in the progression of inflammatory diseases including periodontal diseases¹⁷. LPS from P. gingivalis as well as hypoxia, induces a NOX4-dependent increase in H2O2 release in periodontal ligament fibroblasts which may contribute to the development and progression of periodontal diseases in the absence of antioxidative systems¹⁸. P. gingivalis also induces ROS that activate Foxo transcription factors through JNK signaling¹⁹.

Advanced glycation end products (AGEs) are responsible for inducing low intensity chronic inflammation and thereby, for initiating and/or aggravating chronic diseases. Nowadays, research has demonstrated a significant association between AGEs and dental or periodontal pathology. Thus, the effect of AHE on various oxidative stress and glycation markers was evaluated. It was demonstrated that 1% of the extract neutralises H2O2-induced ROS by 43% and was more effective than Trolox at 50 μg/ml, a potent antioxidant molecule. The extract also inhibited IL-1b-induced RCAN1 expression, LPS-induced NO and 8-Iso-PGF2α production, a key biomarker of oxidative stress.

Importantly, the extract increases AGE-R1 expression, an oxidative stress suppressor and a negative regulator of the inflammatory response to AGE. This makes it a potent antioxidant, a property that prolongs cell survival for healthy gums. Finally, it was shown that AHE 1% strongly inhibited IL-1β-induced Rage expression. This data suggests that the extract can inhibit the proinflammatory and pro-oxidative effects of AGEs and can be considered as an anti-glycation active ingredient.

figure 5. Toothshade reduction of AHE. figures: Premium Organic

Reducing tooth shade

Bright teeth are cosmetically attractive. The extract was investigated for its ability to reduce tooth shade. The most prominent shade of the frontal teeth was determined on every subject before and after the period of product application. The volunteers saw an average 15% reduction in the tooth shade demonstrating the whitening effect of AHE (figure 5). 14 of the 20 subjects, who did not apply the placebo, displayed a decrease of tooth shade to brighter teeth with a mean reduction of 21%. The other six subjects as well as the subjects, who applied placebo, did not exhibit a change in tooth shade. Regarding all 20 subjects, who did not apply placebo, the tooth shade was reduced in mean by 15%.

(Plaque is a complex biofilm, that consists of protein, carbohydrates, phosphates, and microorganisms. Plaque formation predisposes to dental caries and periodontal diseases²⁰. Thus, the effective removal of dental plaque is important for maintaining periodontal and oral health. The role of AHE in improving oral hygiene by reducing plaque intensity was determined.

Plaque can be visualised and assessed using a staining method. The plaque index was determined after corresponding staining and flushing with water. The inner and outer area of every tooth was analysed concerning the intensity of plaque. In the mean, it was found that, the plaque index of the 20 subjects, was reduce by 75%.

The degree of inflammation of the gingiva was assessed by determining the gingiva index. The gingiva index, corresponding to the intensity of bleeding at gingival margin following probing, was determined on the inner as well as outer area of every tooth. In the mean the gingiva index of the 20 subjects, was reduced by 77%.

References

1. M. Sanz, “European workshop in periodontal health and cardiovascular disease”European Heart Journal Supplements, vol. 12, no. Suppl B, p. B2, 2010.

2. Singhrao SK, Harding A, Poole S, Kesavalu L, Crean S. Porphyromonas gingivalis Periodontal Infection and Its Putative Links with Alzheimer’s Disease. Buommino E,ed. Mediators Inflamm. 2015;2015:137357. doi:10.1155/2015/137357

3. Mysak J, Podzimek S, Sommerova P, et al. Porphyromonas gingivalis: Major Periodonto-pathic Pathogen Overview. Riera CM, ed. J Immu-nol Res.2014;2014:476068.

4. Sakanaka A, Takeuchi H, Kuboniwa M, Amano A. Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells. Microb Pathog. 2016;94:42-47.

5. Chen W, Alshaikh A, Kim S, Kim J, Chun C, Mehrazarin S, Lee J, Lux R, Kim RH, Shin KH, Park NH, Walentin K, Schmidt-Ott KM, Kang MK. Porphyromonas gingivalis Impairs Oral Epithelial Barrier through Targeting GRHL2. J Dent Res. 2019 Sep;98(10):1150-1158.

6. Elsayed R, Elashiry M, Liu Y, El-Awady A, Hamrick M, Cutler CW. Porphyromonas gingivalis Provokes Exosome Secretion and Paracrine Immune Senescence in Bystander Dendritic Cells. Front Cell Infect Microbiol. 2021 Jun 1;11:669989.

7. Cugini C, Klepac-Ceraj V, Rackaityte E, Riggs JE, Davey ME. Porphyromonas gingivalis: Keep-ing the pathos out of the biont. J Oral Microbiol. 2013;5(2013).

8. Kuramitsu HK. Proteases of Porphyromonas gingivalis: what don’t they do? Oral Microbiol Immunol. 1998;13(5):263-270.

9. Hasturk H, Kantarci A, Goguet-Surmenian E, et al. Resolvin E1 Regulates Inflammation at the Cellular and Tissue Level and Restores Tissue Homeostasis In Vivo. J Immunol. 2007;179(10):7021 LP - 7029.

10. van Deursen JM. The role of senescent cells in ageing. Nature. 2014;509(7501):439-446.

11. Denkinger MD, Leins H, Schirmbeck R, Florian MC, Geiger H. HSC Aging and Senescent Im-mune Remodeling. Trends Immunol. 2015;36(12):815-824.

12. Domon H, Tabeta K, Nakajima T, Yamazaki K. Age-related alterations in gene 13 expression of gingival fibroblasts stimulated with Porphyromo-nas gingivalis. J Periodontal Res. 2014;49(4):536-543.

13. Smith PC, Martinez C, Martinez J, McCulloch CA. Role of Fibroblast Populations in Periodontal Wound Healing and Tissue Remodeling. Front Physiol. 2019;10:270.

14. Williams RC. Periodontal Disease. N Engl J Med. 1990;322(6):373-382.

15. Lee H-J, Kim J-K, Cho J-Y, Lee J-M, Hong S-H. Quantification of Subgingival Bacterial Pathogens at Different Stages of Periodontal Diseases. Curr Microbiol. 2012;65(1):22-27.

16. Gonzales JR, Groeger S, Johansson A, Meyle J. T helper cells from aggressive periodontitis patients produce higher levels of interleukin-1 beta and interleukin-6 in interaction with Porphy-romonas gingivalis. Clin Oral Investig. 2014;18(7):1835-1843.

17. Nguyen TT, Huynh NN-C, Seubbuk S, Nilmoje T, Wanasuntronwong A, Surarit R. Oxidative stress induced by Porphyromonas gingivalis lysate and nicotine in human periodontal ligament fibro-blasts. Odontology. 2019;107(2):133-141.

18. Golz L, Memmert S, Rath-Deschner B, et al. LPS from P. gingivalis and Hypoxia Increases Oxidative Stress in Periodontal Ligament Fibro-blasts and Contributes to Periodontitis. Buommino E, ed. Mediators Inflamm. 2014;2014:986264.

19. Wang Q, Sztukowska M, Ojo A, Scott DA, Wang H, Lamont RJ. FOXO responses to Porphyromo-nas gingivalis in epithelial cells. Cell Microbiol. 2015;17(11):1605-1617.

20. Rosan B, Lamont RJ. Dental plaque formation. Microbes Infect. 2000;2(13):1599-1607.