Effects of Mouthwashes on the Oral Microbiome and Systemic Health

PROJECT AIMSThe aim of this project is to determine the role of different mouthwashes on the oralmicrobiome and its relationship with cardiovascular health. We will achieve this byconducting a double‐blinded randomised clinical trial (pilot study) in a primary caredental setting, to investigate (i) the composition of the oral microbiome in differentoral niches in periodontal health and disease, and (ii) determine how the oral microbiomeis altered by the use of different mouthwashes during oral health and disease, witheither physiological saline, essential oil (EOs), CPCs, hydrogen peroxide (H2O2); allversus placebo (water).OVERVIEWWhile antimicrobial mouthwashes are proven to be clinically effective for management ofcertain oral microbial diseases, recent studies (Bescos et al 2025, Gallard et al 2025)suggest tha, in addition to targeting bacteria responsible for gum diseases such asgingivitis and periodontitis, they may harm healthy bacteria and disturb the balance andprotective role of the oral microbiome (dysbiosis).Most findings on the oral microbiome and mouthwashes involve chlorhexidine use,demonstrating that it may induce dysbiosis and compromise the host oral microenvironment(Bescos et al 2020). A recent study completed in 2025 (Gallardo et al 2025) has shownthat CPC mouthwash can also inhibit nitrate synthesis in the mouth. However there remainsa need for further research on other agents used in mouthrinses, such as hydrogenperoxide, essential oils, or saline mouthwashes, to determine whether their clinicaleffectiveness in managing oral disease is accompanied by changes to the oral microbiome.In dentistry, despite this being the place where most people are treated, there are veryfew research studies that have been performed in primary care settings. Hence this studywill be designed for delivery in primary care, to produce 'real‐life' data on a patientcohort more typical of general dental practice.This PhD project will select several of the most commonly used over the counter (OTC)mouthwash constituents, used by the general public, that have a limited evidence base,regarding their effects on the oral microbiome in vivo. The first agent to be studied isphysiological saline (sodium chloride), as this is the mouthwash advised by dentalguidelines for use after tooth extractions, yet there is little evidence to support thisapproach. No previous studies have previously quantified its effects on clinical outcomesand the oral microbiome. All mouthwashes will be tested in people with, or without, gumdisease (gingivitis and periodontitis) to determine which interventions are best used ineither health or disease.BACKGROUND 1. Introduction to the Oral MicrobiomeThe oral cavity is one of the most ecologically diverse habitats in the human body,hosting hundreds of microorganisms such as bacteria, archaea, fungi, protozoa, andviruses that coexist within biofilms on both soft and hard oral tissues. The oralmicrobiome is now understood to not be a passive community but is rather a highlydynamic, interactive, immunologically and metabolically active system, not justorally but both in oral and systemic health (Kilian et al. 2016).Historically, the study of oral microorganisms was restricted by culture‐basedtechniques. While these methods were important in identifying cariogenic speciessuch as Streptococcus mutans and periodontal pathogens such as Porphyromonasgingivalis, they dramatically underestimated microbial diversity because only aminority of oral species can be cultured under laboratory conditions. With theintroduction of 16S ribosomal RNA (rRNA) gene sequencing and later shotgunmetagenomics, the true complexity of the oral microbiome was revealed. These methodsidentified more than 700 bacterial taxa, along with a wide variety of fungi (e.g.,Candida spp.), viruses (including bacteriophages), and protozoa. (Wirth et al.2022).The oral microbiome is not the same everywhere in the mouth; it is site‐specific.Different areas, such as saliva, plaque, the tongue and gingival crevicular fluid(GCF), each host their own unique mix of microorganisms. These communities areshaped by factors like the immune system, pH levels, oxygen, and availablenutrients. Because of this, oral health and disease cannot be fully understood bystudying saliva alone; samples from multiple sites are needed to capture the fullpicture. In each of these niches, in oral health these populations are diverse,whereas in oral disease, the oral microbiome becomes less diverse and pathogenicspecies predominate. For example, in supra‐gingival plaque, one NGS study foundhigher abundance of bacteria associated with gingivitis, such as Fusobacterium,Treponema and Campylobacter and lower abundance of bacteria associated with oral andcardiovascular health, such as Neisseria, Actinomyces, and Rothia (Wirth at al.2022). 2. Dysbiosis of oral microbiomeDysbiosis is a disruption of microbial balance leading to a dominance of pathogenicspecies and loss of symbiosis. In the oral cavity, dysbiosis underlies the hostresponse and clinical outcomes for most prevalent dental diseases, including dentalcaries, gingivitis and periodontal diseases.2.1. Dysbiosis of oral microbiome in oral diseaseDental caries (tooth decay) is mainly caused by Streptococcus mutans. This bacteriumfeeds on sugars from food and produces acid, which demineralises the enamel anddentine. It often hides in the pits and grooves of teeth and forms sticky biofilms.When S. mutans combines with fungi like Candida albicans, the biofilms become evenstronger and more harmful (Kim et al 2017). This partnership is especially linked toearly childhood caries. Because tooth decay is still the most common diseaseworldwide, researchers are exploring new treatments, such as probiotics that add"good bacteria" and medicines that can target S. mutans more directly. (Morrison etal 2023)Plaque results in the inflammation of gingival tissue and gingivitis in some formsare estimated to affect up to 90% of the world's population at one time or another.It is reversible, but if left untreated can be a precursor to irreversibleperiodontal diseases. In humans, gingivitis typically arises in response tocommunities of bacteria (dental plaque) attached to the surface of teeth(supra‐gingivally) in people with poor oral hygiene. Clinically, gingivalinflammation is seen as bleeding when the gingival is probed, as well as redness andswelling of the gingival marginal tissues, without any signs of periodontalattachment loss. Being reversible, gingivitis is a condition that can be managedeffectively with therapy and improved oral hygiene. However, if left untreated, itcan progress to periodontitis (Manzoor et al 2024)Gingivitis can be linked to bacteria such as Leptotrichia buccalis and Prevotellaspecies, as these are found to predominate in the plaque of people with gingivitis(Scannapieco et al 2021, Lie et al 2001) These bacteria can trigger the body'simmune system to release inflammatory molecules, causing redness, swelling, andbleeding of the gums. While Leptotrichia is normally harmless, it may play a part ingum inflammation when conditions in the mouth change. Prevotella species are moredirectly involved, as they are often found in higher numbers when gums becomeinflamed.Periodontitis manifests clinically with bleeding on probing (BOP) from deeperperiodontal pockets and eventually tooth mobility and loss due to the irreversibledestruction of alveolar bone, which holds teeth in place. Pockets are first createddue to localised host inflammation and swelling in response to pathogenicGram‐negative bacteria present in plaque below the gumline (sub‐gingival), such asPorphyromonas gingivalis, Fusobacterium, Treponema and Tannerella species (Kilian etal,2016). The inflammatory mediators that are released into the pocket in responseto this infection, also destroy bone, which further deepens the pockets and makesoral hygiene even more challenging.Two of the most important bacteria that predominate in the oral microbiome of peoplewith periodontal disease include Porphyromonas gingivalis and Fusobacteriumnucleatum.P. gingivalis is a Gram‐negative, anaerobic bacterium that grows without oxygen'This bacterium thrives when the mouth's microbial balance, or microbiome, isdisturbed. In these conditions, it triggers inflammation, which contributes to thedevelopment of gum disease. One of the reasons P. gingivalis is so harmful is itsability to invade human cells, resist some antibiotics, and use thin, hair‐likestructures called fimbriae to attach to and enter host cells. Beyond gum disease, itis also linked to oral and digestive cancers. It encourages cells to grow whileavoiding normal cell death, blocks tumour‐suppressor proteins such as p53, andtriggers cell changes (epithelial‐mesenchymal transition, EMT) that allow cancer tospread. Because of these factors, P. gingivalis is considered a dangerous pathogenthat not only causes periodontal disease but also increases the risk of oral cancer.F. nucleatum is another Gram‐negative, anaerobic bacterium that is very common inthe mouth. While it was once thought to be harmless, it is now recognised as animportant disease‐causing organism. It has also been found outside the oral cavityin several conditions, including bowel disease, colorectal cancer, Crohn's disease,arthritis, meningitis, appendicitis, and pregnancy complications. Within the mouth,F. nucleatum plays a key role in dental biofilms, acting as a bridge that connectsdifferent bacterial species. It uses proteins such as FadA and Fap2 to stick tohuman cells, provoke inflammation, and promote tumor growth. The bacteriumstimulates immune signals like IL‐6, IL‐8, IL‐17, and TNF‐α. While these signalssupport healing in healthy conditions, in disease they contribute to DNA damage andcancer. F. nucleatum is also capable of resisting antibiotics and reducing theeffectiveness of certain cancer treatments. It is often found alongside P.gingivalis in oral cancers, and together these bacteria strongly enhance pathwaysthat promote cancer growth. (Morrison et al 2023)2.2. Treatment of oral diseaseAt the dentist, gingivitis is managed with improved oral hygiene instruction; OHI(tooth brushing and interdental cleaning); dentists and dental hygiene therapistspromote at home oral hygiene regimens, as the most important factor for thestabilisation of the disease. In periodontitis, OHI is accompanied by non‐surgicaltherapy performed within the dental practice, usually every 3 months (supra‐ andsub‐gingival scaling with ultrasonic or hand scalers known as PMPR), to reducebacterial load within the periodontium. Indeed, the diversity of the oral microbiomeincreases after PMPR, and PMPR leads to immediate reduction in periodontalinflammation (Johnston et al,2023). This is because bacterial load is reduced, suchthat the structure of the plaque is less favourable for many of the pathogenicanaerobic bacteria associated with oral disease. Despite this, many patients do notmaintain improvements in periodontal health after OHI and PMPR, and new adjunctivemeasures such as mouthwash are often sought, for example in patients with impaireddexterity, and other mental or physical health conditions which can impairengagement.(https://www.nice.org.uk/guidance/ng48/chapter/Recommendations#daily‐mouth‐care ) 3. Links Between Oral Microbiome and Systemic HealthImportantly, dysbiosis is not just the presence of pathogens but an imbalance whereprotective microorganisms decline and pathogenic taxa dominate. In the past decade,the interest in the oral microbiome by researchers has grown, as an important linkin systemic health. There is a growing body of evidence linking periodontal diseaseand systemic diseases, such as diabetes, cardiovascular disease, Alzheimer's (Longet al 2017, Maintre et al 2021 & Preshaw et al 2019). The evidence forcardiovascular disease is some of the most compelling and there are severalsystematic reviews and meta‐analysis that report on the association betweenperiodontal disease and cardiovascular disease, including hypertension. Thesystematic review by Munoz‐Aguilera found a clear link between periodontitis andhypertension. Patients with moderate to severe gum disease were about 20% morelikely to have high blood pressure than those without, and the risk increased withdisease severity. On average, people with periodontitis had higher blood pressurereadings; around 4.5 mmHg systolic and 2 mmHg diastolic. These findings suggest thatperiodontitis may be a modifiable risk factor for hypertension. The review alsoconsidered possible mechanisms. Periodontitis can drive systemic inflammation,releasing molecules like CRP, IL‐6, and TNF‐α that impair blood vessel function.Oral bacteria such as Porphyromonas gingivalis may also directly affect vascularhealth and blood pressure. Overall, the evidence indicates that gum disease andhypertension are closely linked, and periodontal therapy could play a role inreducing cardiovascular risk.There are several different mechanisms that have been proposed for thisrelationship: (1) systemic inflammation due to cytokines released into thebloodstream during bacterial dysbiosis, (2) bacteraemia (pathogenic oral bacteriaentering the bloodstream), (3) the oral bacterial enterosalivary pathway (withsystemic nitric oxide [NO] release), and (4) genetic susceptibility (Alrashdan et al2023). Genetic factors, pre‐existing disease and the host response also play a partin terms of the amount of inflammation and clinical outcomes observed in response tooral microbiome dysbiosis. However, this study will focus on the salivary‐enteronitrate reducing pathway involving nitric oxide, as our previous studies havedemonstrated that this is affected by mouthwashes (Bondonno, Liu et al. 2015, Bescoset al, 2020, Woessner, Smoliga et al. 2016, Mitsui and Harasawa 2017)3.1 Cardiovascular HealthIn health there are bacteria on the dorsum of the tongue (and thus saliva), thatconvert nitrate in food to nitrite, which is swallowed and converted into nitricoxide (NO) in the gut wall to maintain a lower blood pressure. Nitrates and nitriteswidely exist in soil, water, and plants. Humans get their nitrates through food,mainly through green vegetables. These dietary nitrates are stable but when incontact with symbiotic bacteria in the oral cavity and stomach, they get convertedto nitrite and nitric oxide (NO). NO is the metabolic product of dietary nitrate andplays an important role in protecting cardiovascular system and gastric mucosa. (Ma,Hu et al. 2018). Via this mechanism therefore, nitrate‐reducing oral bacteria canmodule blood pressure.A diagram of a diagram of the internal organsDescription automatically generatedFig 1. Nitrate‐nitrite‐NO pathwayDiagram adopted by M. Cana‐Bishop from H. S. Alzahrani et al 2020; The role ofdietary nitrate and the oral microbiome on blood pressure and vascular tone,Cambridge University Press: 07 December 2020In the oral cavity these nitrate‐nitrite reducing bacteria tend to be in the deepcrypts of the posterior part of the tongue (Qu, Wu et al. 2016), but can also beidentified in saliva (Bescos, Ashworth et al. 2020). Those species in the mouth thatare nitrate reducing can be broadly categorised into two groups: strict anaerobes(Veillonella atypica and Veillonella dispar) and facultative anaerobes (Actinomycesodontolyticus and Rothia mucilaginosa). Veillonella species have been identified asthe primary nitrate reducers in the tongue and play a significant role in nitratereduction. Recent research utilising 16S rRNA has revealed a higher prevalence ofPrevotella, Neisseria and Haemophilus on the posterior surface of tongue comparedwith Actinomyces (Qu, Wu et al. 2016). The presence of sufficient nitrate reducingbacteria when exposed to dietary nitrates, therefore, play an important role inprevention of myocardial infarctions, hypertension, and acute stress, due to itsrole as a biological reservoir for NO in hypoxia or acidic conditions. A moderateconsumption of nitrate rich fruits and vegetables such as beetroot, spinach,lettuce, radish and celery, therefore, is a low‐cost way of contribution tocardiovascular health in part via the oral microbiome (Qu, Wu et al. 2016). 4. Mouthwashes as Modifiers of the Oral Microbiome4.1. Clinical effectivenessA huge variety of mouthwashes are available over the counter and can be either chemicalor natural. Some of the most commonly used antimicrobial mouthwashes includechlorhexidine (CHX), hydrogen peroxide (H2O2), cetylpyridinium chloride (CPC), povidoneiodine (PVP‐I), and essential oils (EO) (James et al, 2017; Brookes, McGrath et al.2023). These mouthwashes are used because they are clinically effective in terms ofreducing plaque and bleeding, and in turn gingivitis (McGrath et al, 2023). They alsohave a small degree of clinical effectiveness, particularly chlorhexidine, at reducingpocket depths when used adjunctively alongside OHI and PMPR (da Costa et al, 2017). Thus,they are widely used by patients as an over‐the counter (OTC) treatment to addressproblems such as bleeding gums and bad breath (halitosis). However, they are alsointegrated into the daily oral hygiene routines of healthy individuals, and thus it isimportant to evaluate whether there is oral dysbiosis in this group, as mouthwashesshould ideally be used only when benefits outweigh risks (Brookes et al 2023).4.2. Mechanism of action and clinical effectiveness4.2.1. Chlorhexidine digluconateChlorhexidine has been used in medicine and dentistry since 1950, being found inmouthwashes since1970 (Loe &Schiott,1970). It has been used as an effective antisepticagent for 'killing' both gram positive and negative bacteria, fungi, and certain viruses.A study by Rius‐Salvador et al., 2024 indicated that Chlorhexidine can decrease theinfectivity of both the Influenza A virus and the Respiratory Syncytial virus in vitro.Chlorhexidine is a bacteriostatic at lower concentrations of 0.02%‐0.06%, andbactericidal at the higher concentration of 0.12%. Chlorhexidine is used in dentistry asan antiseptic mouthwash at 0.12%‐0.2% and surface disinfectant at 0.2% for this reason.Chlorhexidine is a positively charged cationic bisbiguanide that can be adsorbed to avariety of negatively charged sites, including mucous membranes, salivary pellicle onteeth, as well as several components of the biofilm on the tooth surfaces, e.g.,bacteria, extracellular polysaccharides, and glycoproteins. In vitro studies have showedthat, at concentrations lower than used clinically, chlorhexidine causes destruction tothe cell membrane and in turn and low molecular weight molecules escape from themicroorganisms. On the other hand, a higher concentration of chlorhexidine can causeprecipitation and coagulation of the proteins in the cytoplasm of the exposed microbes.These properties interfere with biofilm formation and prevent bacterial growth (Rashed,2016).James et al., 2017 undertook a systematic review confirming that the daily chlorhexidinemouthwash (used alongside brushing/flossing) greatly reduces dental plaque over 4‐6weeks, and this effect is maintained up to 6 months. The same study also confirmed ahigh‐ certainty evidence that chlorhexidine reduces gingivitis in people with mild guminflammation, with consistent improvements seen both short‐ and long‐term. In periodontaldisease, studies also prove that it is clinically effectively at reducing periodontalpockets when used adjunctively. Costa et al., 2017 meta‐analysis confirmed thatadjunctive use of chlorhexidine mouth rinse with mechanical scale and root planning (SPR)resulted in slightly greater probing depth reduction than did SRP alone.Chlorhexidine also reduces Streptococcus mutans levels, suggesting possible benefits inpreventing tooth decay, but more long‐term, high‐quality studies are needed to confirmthis. (McGrath et al 2023). In terms of other oral health benefits, a 2019 systematicreview by (Kumbargere Nagraj, Eachempati et al. 2019) found low certainty evidence thatchlorhexidine mouthwash may help reduce bacteria that cause halitosis. A 2022 Cochranereview (Casarin, Matos et al. 2023) found moderate‐certainty evidence that chlorhexidinerinses before and after tooth extraction lower the risk of dry socket.Beyond dentistry, a 2020 Cochrane review (Hasan, Chiu et al. 2023) suggestedchlorhexidine may lower the risk of ventilator‐associated pneumonia in critically illpatients (low‐certainty evidence). During COVID‐19, interest in pre‐proceduralmouthwashes grew, but current evidence is insufficient to confirm benefits for patientoutcomes or healthcare worker safety.4.2.2. Essential oils (EO)It is difficult to find antimicrobial data for essential oils alone, in terms of themechanisms and clinical effectiveness when they are used as a mouthwash, but the base forEO mouthwashes such as in ListerineTM, traditionally includes thymol, methyl salicylateand eucalyptol. Traditionally, the mechanism of action of essential oils is thought to bebased on disruption of cytoplasmic membranes and inhibition of bacterial enzymes,however, despite many studies describing the antimicrobial activities of essential oils,herbal extracts, or their active components, there remains a lack of evidence on theirmechanisms of action (Weber, Bonn et al. 2023). ListerineTM can reduce plaque andbleeding scores and therefore reduce clinical signs of gingivitis (Goutham, Manchanda etal. 2013). It is also recommended adjunctively for periodontal disease however,ListerineTM contains alcohol (plus CPCs in some formulations), which complicatesknowledge and understanding of the true mechanisms on EOs in vivo. Thus, further clinicalstudies are required to elucidate the clinical effects of EOs alone. Alcohol may alsoaffect the oral microenvironment, and whilst unfounded, regarding the risk of alcoholmouthwashes causing cancer (Vlachojannis, Al‐Ahmad et al. 2016) there are still someconcerns about the suitability of daily use of alcohol mouthwashes by pregnant women,those with alcohol dependency and in patients with mucosal injuries. Consequently, thealcohol free EO mouthwashes have been introduced by ListerineTM and others (Spuldaro, etal. 2021).4.2.3 Hydrogen peroxide (H2O2)H2O2 has found its application in dentistry for more than 70 years either in combinationwith salt or in its pure form. H2O2 has been shown to possess a wide spectrum ofantimicrobial activity because it is active against bacteria, yeasts, fungi, viruses, andspores, however these positive outcomes are observed with concentrations greater than 1%(Rashed 2016). H2O2 is a bleaching agent with strong oxidising action that releases freeradicals and disrupts the lipid component of microbial cell wall (Weber et al 2023). Inaddition, H2O2 produces foam when in contact with human tissue, releasing water andoxygen which is believed to contribute to the destruction of anaerobic bacterial species.A 5% concentration of H2O2 causes soft tissue damage, therefore it is used in dentistryas antiseptic mouthwash at concentrations of 1.5%‐3.0% (Brookes, McGrath et al. 2023). Asystematic review done in 2011 (Hossainian, Slot et al. 2011) showed that H2O2mouthwashes do not consistently prevent plaque accumulation when used as a short‐termmono‐therapy. When used as a long‐term adjunct to daily oral hygiene however, one studyindicated that oxygenating mouthwashes reduce gingival redness. This systematic reviewwas limited by what was available in the existing dental literature and found that onlyone study that had an evaluation period of more than 4‐weeks. Therefore, the outcome ofthis review with respect to levels of gingival inflammations was based on a singleexperiment with an estimated low risk of bias. Clearly, this is not sufficient evidenceto draw any conclusions on, regarding the long‐term effects of H2O2 on plaque levels.4.2.4.Cetylpyridinium chloride (CPC)CPCs are found in many commercially available OTC mouthwashes, and are classified asquaternary ammonium compounds (QACs) that are antibacterial because they react withlipids and proteins of cell membrane, causing leakage of low molecule components (VanLeeuwen, Rosema et al. 2015). CPCs can also cause the release cytolytic enzymes, leadingto the lysis of bacterial cells (Brookes, McGrath et al. 2023). It is worth noting thatQACs are also commonly used in various surface spray disinfectants, so have other useswithin the dental surgery (Brookes, McGrath et al. 2023). They are used in mouthwashes atvarying concentrations (0.045%‐0.1%). The results of a clinical trial (Van Leeuwen,Rosema et al. 2015) demonstrated that the use of a 0.07% CPC mouth rinse wassignificantly more effective in reducing plaque scores than the use of the VehicleControl (VC) mouth rinse. However, there were no significant differences between the CPCand VC groups, with respect to bleeding scores observed at 6 months. CPC's may also beeffective in reducing plaque and the levels of anaerobic species of bacteria in plaqueand saliva (Van Leeuwen, Rosema et al. 2015), however, evidence from in vivo studiesremains limited (Brookes et al 2023). Similar to EOs, CPCs are usually used incombination with other effective antibacterial agents, including CHX and alcohol, as wellas fluoride. Thus, further studies are required investigating the antibacterial effectsof CPCs alone, rather than commercially available products whose formulations are verycomplex and change over time.4.2.5 Saline rinsesSaline refers to an isotonic solution containing sodium chloride (NaCl), and water,usually of concentrations of 0.09% when referred to a 'physiological' saline. However, athigher concentrations it may be antibacterial (Osunde, Adebola et al. 2014).Theliterature suggests that 1.4‐1.7% is the appropriate concentration to use in saline mouthrinses (Osunde, Adebola et al. 2014) but there is some uncertainty about this in clinicaldentistry, and hence determining a clinically relevant concentration also forms part ofthis thesis. Although, it is arguably less potent as an antimicrobial agent than some ofthe chemical agents discussed thus far, it has been recognised for its mild antisepticproperties for centuries and is still advised by dental practitioners on a daily basisfor managing oral infections (Sinha, Shil et al. 2024). It is difficult therefore tounderstand, the lack of evidence‐base surrounding the appropriate dose and frequency ofsaline use in dentistry.Based on what evidence does exist, it has been suggested that saltwater or saline mouthrinses can reduce plaque scores and the colony counts in saliva of bacteria in vitro,such as S mutans, L acidophilus, A actinomycetemcomitans, and P gingivalis (Aravinth etal 2017). Saline also reduces the pH within the oral cavity in vivo, and may causebacteria to lose water due to osmosis; thus, it makes sense that saltwater reduces thegrowth of unwanted oral bacteria. As mentioned, dental practitioners often suggestsaltwater rinses for postoperative care after oral surgery, but there is little evidenceto support this recommendation (Duane, Yap et al. 2023), and hence the clinicaleffectiveness and antibacterial mechanisms of saline mouth rises requires furtherinvestigationSaline is also commonly regarded as being beneficial for reducing gingival inflammationand facilitating the healing of oral ulcerative lesions (Montaser et al 2023). An invitro study performed by Huynh et al., in 2016 used human gingival fibroblasts (hGFs)cultured in growth medium already containing a small amount of NaCl (about 0.4%). In thisstudy cells were therefore exposed to slightly higher dosages of NaCl than in salinemouth rinses, showing that 1.8% NaCl was the most effective concentration at stimulatinghGF cell migration, altering the organisation of cytoskeletal molecules (FAK andF‐actin), and enhancing extracellular matrix gene expression (COL1 and Fn). These dataprovided the first scientific evidence to support the application of salt solution asmouthrinse in conjunction with routine oral care to promote oral wound healing. Salinerinses are thus recommended post‐extraction for wound healing as part of nationalguidelines (https://www.nhs.uk/tests‐and‐treatments/wisdom‐tooth‐removal/, Oral healthfoundation, https://www.dentalhealth.org/what‐to‐do‐following‐an‐extraction).They havefurther been shown to alleviate xerostomia, reduce halitosis, and significantly lowerbacterial load within the oral cavity (Kim and Kim 2014), yet evidence on their clinicaleffectiveness due to an antibacterial effect in vivo is virtually absent.4.2.6. Fluoride (mouthwashes)Fluoride is widely used, and it's found in various oral products such as toothpastemouthwash and gels. Mouthwashes are particularly favoured by the public due to their easyuse, and recommendations in current guidelines(https://www.gov.uk/government/publications/delivering‐better‐oral‐health‐an‐evidence‐based‐toolkit‐for‐prevention/chapter‐9‐fluoride )Fluoride mouthwashes have been widely recommended for maintaining oral health, especiallyin the prevention of dental caries. Sodium fluoride is the most common active ingredient.Fluoride concentration in these rinses varies: OTC products usually contain about200‐1000 ppm, while prescription formulations may contain several thousand ppm.Typically, OTC mouthwashes are intended for daily use, whereas prescription‐strengthrinses are used less frequently. Although fluoride is known to have some antiplaqueactivity, there is limited direct evidence on its effectiveness in reducing plaque levelsor in managing gingivitis and periodontal disease when used as a mouthwash (McGrath et al2023). Marinho et al 2016 undertook a systematic review, finding that supervised regularuse of fluoride mouthrinse by children and adolescents was associated with a largereduction in caries compared with control groups, on average by 27%, but there was nosuch similar study in adultsThere is no evidence for effects of mouth rinses with fluoride on dental plaqueaccumulation, gingivitis development or periodontal disease (Giertsen, Emberland et al.1999) in vivo. In vitro, sodium fluoride mouthwash had little effect on P gingivalis, Pintermedia, F nucleatum, and A actinomycetemcomitans; using ex vivo cultures of bacteriafrom the tongue (Brookes et al 2023). Thus, given the widespread use offluoride‐containing mouthwashes, there is much work to be done in terms of quantifyingthe clinical effectiveness of fluoride on periodontal health in vivo and theantimicrobial mechanisms associated with this. Perhaps the known anti‐caries benefits offluoride outweigh the need to know this clinically, but the marketing of thesefluoride‐containing mouthwashes does often pertain to improving gum health.4.2.7. Alternatives: Herbal and Probiotic Rinses.There is growing consumer demand for "natural" products, and within mouthwashes this hasincited interest in alternatives such as:Coconut oil (pulling)SeaweedPropolisProbiotic rinsesCoconut Oil; this is a traditional method of oral hygiene in Ayurvedic medicine that hasrecently become popular in Western populations. It involves swishing around 1 tablespoonof oil in the mouth for 10 to 20 minutes to reduce the populations of bact