Evaluation of Trans-Resveratrol as a Treatment for Periodontitis

Periodontitis is a globally prevalent inflammation-mediated disease that can result in varying degrees of destruction to the tissues supporting the teeth. The microbial pathogenic dysbiosis, oxidative stress, and deregulated inflammation, found in patients with periodontitis, make it a multifaceted condition that is dif-ficult to fully resolve. Further to this, periodontitis has been associated with other systemic inflammatory conditions. Trans-resveratrol (3,5,4 ′ -trihydroxy-trans-stil-bene) is a plant-derived molecule present in many foods, which have been shown to exhibit antimicrobial, antioxidant, anti-inflammatory, and regenerative properties. However, trans-resveratrol has been reported to have physicochemical shortcom-ings, which make its clinical translation a challenge. This review outlines a critical analysis of identified samples from the scientific literature that was conducted to assess the potential of RES as a viable therapeutic for periodontitis. The potential for the improvement of the limiting pharmacological profile of trans-resveratrol via nanoformulation is also explored.


Introduction
Periodontal disease (PD) is a chronic condition accompanied by a progressive pathogenic biofilm that continuously triggers inflammation, potentially resulting in the loss of both soft and bony periodontal tissues. Ultimately, in severe cases, edentulism may result (Figure 1) [1].
Although aspects such as age, genetics, or sex can affect the chance of developing PD, there are also modifiable risk factors that have been identified. That is, smoking, nutrition (e.g., low vitamin D and calcium), and poorly managed diseases (e.g., diabetes, rheumatoid arthritis, and obesity) as well as stress, have also been found to play a significant role in susceptibility [2][3][4].
According to several epidemiological reports, the prevalence of PD is increasing over time. In fact, current publications indicate that approximately 10% of the global population presents with severe periodontitis, while almost half of the remaining 90% of all adults present with a less severe form of the disease. By and large, the most conservative estimate places the prevalence of PD at approximately 50% of the adult population worldwide [1,[5][6][7].
Since people suffering from PD may experience chronic pain and tissue destruction, which can lead to anxiety and depression, the overall loss of quality

Other inflammation-mediated conditions associated with PD
The conflicting reports, regarding the extent and severity of PD in the epidemiological literature, do not change the legitimate growing concern around the prevalence of the disease, especially when one considers the many inflammationmediated systemic diseases with which it has been associated. For example, several reports indicate that PD can potentially increase the chance of developing heart disease [14][15][16][17][18][19][20], neurodegenerative disease [21][22][23], and autoimmune disease [24,25] (Figure 2).

Healthy periodontium and the pathogenesis of periodontitis
The periodontium consists of the tooth's surrounding anatomical structures, which include, from superficial to deep, the gingiva, gingival ligament, root cementum, and alveolar bone (Figure 3).
In a healthy periodontium, the supportive anatomical structures adhere to the tooth by way of connective and epithelial tissue types [35]. The epithelia exist as different subtypes around the erupted tooth and have been described as the first line of defence, protecting the underlying tissues of the periodontium from microbial infiltration from the oral cavity (Figure 4) [35]. The pathogenesis of PD first involves a shift in the oral milieu which optimizes the formation of a dysbiotic  microbial biofilm, resulting gingival inflammation, which then progresses to the subgingival region (Figure 4) [36].
Clinically, people suffering from PD present with bleeding gingiva upon probing and varying degrees of detachment (i.e., clinical attachment loss [CAL]) of the gingiva from the tooth as measured with a periodontal probe (ada.org) ( Figure 5).

Porphyromonas gingivalis and its central role in the pathogenesis of PD
Understanding further details of the disease process of PD from the perspective of the oral microbiome can assist in the creation of novel preventative and treatment applications.
Overall, bacteria, fungi, viruses, and protozoa are among the estimated 1000 microbial species that make up the oral microbiome. However, more than 700 microbes are bacterial, giving investigators a rationale for focusing on the bacterial taxa of the oral cavity, when examining health status [36][37][38].
Keystone pathogen, P. gingivalis (Pg), is a Gram-negative, anaerobic, non-motile, a-saccharolytic bacteria, which is a part of the normal flora of the subgingival region of the oral cavity, becomes an opportunistic pathogen when the microenvironmental factors permit it to thrive [39].  Evaluation of Trans-Resveratrol as a Treatment for Periodontitis DOI: http://dx.doi.org /10.5772/intechopen.101477 Also of note, is lipopolysaccharide (LPS), a feature found in the cell walls of Gram-negative bacteria, which triggers an inflammatory response as a pathogenassociated molecular pattern (PAMP). inflammation [40].
Detection by the host complement system is avoided due to the capsule, which is seen in most strains of Pg [41]. Also, the bacterial virulence factors, FimA and Mfa, which are proteins that make up the bacterial appendages, fimbriae, and pili, allow Pg to adhere to the periodontal cells whilst encouraging agglutination between bacteria, thus promoting the formation of a pathogenic biofilm [39,[42][43][44] (Figure 6).
Following adherence to the gingival epithelia, Pg can enter into its host cell with ease, due to the secretion of the serine phosphatase, SerB, which enters the host cell and triggers the de-polymerisation of cytoskeletal actin microfilaments [45] (Figure 6).
For increased success in gaining an intracellular foothold, Pg also employs a sophisticated secretory system (e.g., Type IX Secretory System [T9SS]), which spans the periplasmic space and allows for the passage of its secretory products from the cytoplasm into the extracellular environment [46,47].
Further to this, gingipains are involved in the manipulation of the host immune system, making them key players in tissue destruction through chronic inflammation. For example, gingipains have been found to degrade many cytokines as well as the CD4 and CD8 integral membrane proteins of T lymphocytes, creating interference within the host's adaptive immune system [46,[48][49][50].
Other significant virulence factors of Pg include outer membrane vesicles (OMVs), which are released from most Gram-negative bacteria and can infiltrate places that the bacteria cannot. However, those derived from Pg, are armed with an outer membrane layer, consisting of a capsule, LPS, and gingipains, enclosing an internal compartment loaded with effector proteins and other macromolecules such as nucleic acids. Indeed, OMVs are pro-inflammatory agents of cytotoxic destruction aiding in biofilm formation, as well as the manipulation and evasion of the host immune response [46,53] (Figure 6).

Pg links to PD-related diseases
Common and frequent activities like mastication and oral care, have been found to release oral pathogens and their components into the lymphatic and cardiovascular systems of PD patients. Therefore, periodontal Pg infections likely act as pathogenic reservoirs, possibly promoting certain systemic diseases [52,54].

Neurodegenerative disease
A 2021 study by Franciotii et al. hypothesised that there is a "bidirectional oral-brain" highway through which neurodegenerative processes are stimulated by pro-inflammatory oral processes and vice versa [55].
Most importantly, initiatives towards the innovation of preventative measures for PD have been recommended, especially since the global population is ageing [55].

Head and neck cancer
The reports regarding Pg infection as a risk factor for oral squamous cell and oesophageal carcinoma, align with the emerging perspective in the clinical arena linking chronic systemic inflammation to serious disease states [23, 56-59].

Cardiovascular disease
Regarding PD as it relates to cardiovascular disease, decades of literature reflect a close association [15,19,60]. DNA (i.e., 16S rDNA) from Pg has been identified in atheroma isolated from patients with coronary heart disease through PCR analysis [61]. Interestingly, Pg may also encourage atherosclerosis by switching HDL properties from antiatherogenic to proatherogenic via the manipulation of monocytes [62].
Further to this, Pg has been shown to invade and multiply within coronary endothelia in vitro, whilst damaging the smooth muscle cells and possibly distorting the vasodilatory mechanism of the central arterial system [63,64].
Overall, the literature encourages appreciation of the clinical significance of the assault on the coronary endothelia demonstrated by Pg, especially since the vasculature acts as a vital line of defence for the cardiovascular system [63, 65].

Respiratory disease
Mortality risks from aspiration pneumonia are high in geriatric populations [66]. Of note, in vitro studies have identified Pg as a potent pro-inflammatory agent in isolated respiratory epithelia cells [67]. Additional in vitro studies identified Pg-derived OMVs as significant bacterial virulence factors which connect PD to respiratory disease [68].

Liver disease
It is worth noting that a significant correlation (P < 0.05) between non-alcoholic steatohepatitis (NASH) and oral Pg, has been reported. Furthermore, following treatment for PD, an improvement of liver function, displayed by the normalisation of AST and ALT, has been demonstrated [54,69].

Diabetes mellitus
The relationship between PD and diabetes mellitus (DM) has also been studied with respect to Pg. For example, gingipains carried by OMVs derived from oral Pg, decreased the insulin sensitivity of hepatocytes whilst hepatocytes invaded by Pg were also found to display a decrease in glycogen synthesis in vitro (human) and in vivo (mouse model) [70].

Rheumatoid arthritis
DNA sequences from Pg have been isolated from the synovial fluid and bloodstream of patients with rheumatoid arthritis (RA). Further to this, the consistently reported relationship between an oral Pg infection and RA has encouraged medical clinicians to place more emphasis on the oral health of their patients [71].

Adverse pregnancy outcomes
Pg DNA has also been detected in the amniotic fluid, umbilical cord, and placenta of women who encountered pregnancy complications such as preeclampsia and preterm birth [72]. Additionally, results from animal studies suggest that the mechanism involves the direct invasion and damage of the uterine and placental tissue [65].
The adage that correlation does not mean causation, should be considered, and although Pg cannot be the sole etiological agent of all the systemic diseases with which it is associated, there is accumulating evidence demonstrating its value as a modifiable risk factor for the prevention, management, and treatment of PD and other systemic diseases (Figure 7) [65].

Treatment of PD
Typically, treatment for periodontitis includes physical removal of the biofilm and calculus from under the gingiva by way of scaling and root planning (SRP) followed by comprehensive care (CC) (www.NHS.uk; www.ADA.org) (Figure 8). Whereas, in cases where more severe destruction has occurred, flap surgery is performed, which is often accompanied by expensive reconstructive treatments and/or procedures. In all cases of PD, patients are advised to adhere to lifelong CC to mitigate any further destruction [73][74][75][76].
Adjunct therapies are often combined to optimise results following SRP [76]. For example, one type of host modulation therapy (HMT) consisting of a subantimicrobial dose of doxycycline (SDD), is an internationally approved adjunct treatment for PD. SDD acts through the inhibition of the pathogenic collagenase activity in the host, thus decreasing inflammation and tissue destruction [77].
Interestingly, some naturally occurring phytonutrients also may work through the management of the host inflammatory response. For example, chemically modified curcumin has been shown to be safe and effective for the treatment of PD and other inflammation-mediated diseases in animal models [77][78][79]. Another bioactive phytonutrient of interest is trans-resveratrol, which in combination with curcumin, has been gaining attention as a supplement for the prevention and treatment of PD and other inflammation-mediated conditions [80].

Resveratrol: a bioactive polyphenol with attractive medicinal properties
Trans-resveratrol (trans-3,5,4′-trihydroxystilbene) (RES) is a polyphenol that can be sourced from various edible plants, which has demonstrated antioxidant, anti-inflammatory, antimicrobial, anticancer, and restorative properties [81][82][83][84]. Therefore, RES is positioned in alignment with the treatment principles for PD and the diseases with which it has been associated (Figure 9).
Even though RES is found in a breadth of plant-based foods (e.g., red wine, berries, peanuts, and dark chocolate), the naturally occurring concentrations of RES are not substantial enough (e.g., 0.1-0.7 mg/L in red wine) to reasonably attain the therapeutic values reported in the scientific literature (e.g., an oral dose of approximately 10 mg/kg body) [86][87][88].
Consequently, the purified and optimised extracts of RES are often used in research and some products have been made commercially available as  However, RES is a hydrophobic molecule and therefore, like other promising phytotherapeutics such as curcumin, has poor water solubility (<0.05 mg/mL). RES has also been found to rapidly metabolise in vivo and revert to its less stable isomer when exposed to light, demonstrating its instability and photosensitivity, respectively [85].
Additionally, the low oral bioavailability of RES has been considered a significant obstacle to its clinical translation, resulting in the development of drug carrier models. In fact, there is ample evidence indicating that nano-formulation may be a successful strategy to improve the pharmacological indices of RES under physiological conditions [89][90][91].
Interestingly, the design of functional foods also includes the application of nanotechnology, via the incorporation of liposomal nanocarriers or other nanoencapsulated systems. In this way, the therapeutic potential of customised, effective, and stable fortified foods with specific pharmacokinetic parameters, such as steady time-release, can be investigated [92].
Indeed, both oral and buccal delivery systems, such as those possible via functional food design, have plausible applications regarding PD therapeutics, especially since the primary target area for treatment is in the oral cavity. In fact, many nano-formulations also aim to enhance the delivery and efficacy of targeted therapeutics by engineering combinations of selected bioactive molecules that offer specific properties that promise to optimise the probability of the desired treatment outcome [93].

The attenuation of inflammatory processes by RES in vitro
The modulation of deregulated inflammation, which has been consistently reported for RES in the in vitro reports within the literature, is a central treatment principle for a viable therapeutic for PD. Additionally, in vitro studies allow for a breadth of experiment parameter manipulation not afforded by in vivo studies. So, although such studies cannot probe disease development and treatment, they can support the elucidation of mechanisms of action, thus identifying potential molecular targets for therapeutic applications.
For example, studies that used LPS-stimulated human gingival fibroblasts (HGFs), found through ELISA, and MTT assays, that RES significantly decreased IL-6 and IL-8, but did not increase cell viability. Interestingly, once RES was combined with the polyphenol silymarin (SIL), the viability increased in combination  see left), which is the more stable, and therefore bioactive form compared to its isomer, cis-resveratrol (see right) (Gambini et al. [85]).
with the decrease in IL-6, IL-8 as well as TNF-α, suggesting that RES-± SIL have a more widespread modulatory effect on LPS-induced inflammation [94,95].
Additionally, in 2014, Fordham et al. examined the effect of RES (plus antioxidants, phloretin, silymarin, hesperetin) on LPS-stimulated peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors. ELISA showed that RES decreased the secretion of IL-1β, IL-6, and IFN-ɣ in the LPS-induced PBMCs. Further to this, TNF-α was attenuated at the level of mRNA, as determined by RT-PCR. The researchers concluded that hesperetin and RES significantly inhibited (p < 0.05) the inflammatory response in LPS-stimulated PBMCs [96].

The influence of RES on regenerative processes in periodontal cells
RES has also shown promise regarding the restoration of periodontal tissue, which is a crucial part of the complete treatment of PD. For example, in a complex human in vitro and in situ study, Wang and colleagues reported that RES preserved cell aggregation and osteo-differentiation of normal human periodontal ligamental stem cells (HPLSCs) treated with TNF-α. In this study, histological analysis confirmed that RES treatment (even pre-implantation) improved regeneration in tissue originating from both healthy and pro-inflammatory microenvironments [97].
In accordance, Yuan and colleagues also found through histochemical analysis, RT-PCR, Western blot, and ELISA, that RES attenuated TNF-α -induced osteogenic suppression in HPLSCs in vitro [98].

RES as an attenuator of risk factors and conditions associated with PD
It has been well-established that PD is associated, to varying degrees, with a collection of modifiable risk factors as well as a myriad of systemic inflammationmediated diseases [24,99]. Hence, studies examining the effect of RES on PD in combination with purported comorbidity, and/or risk factor, could contribute to the argument regarding the breadth of its benefits.
Studies employing the integration of RA, DM, cigarette smoking, or osteoporosis (OP) into the induced-PD model have demonstrated that RES may assist in the mitigation of the periodontal damage contributed by associated risk factors and concomitant conditions. For example, with cigarette smoking added to the animal model, it was found that RES decreased both alveolar bone loss and oxidative stress [100,101]. Additionally, using a ligature-induced PD model, RES was found to reduce alveolar bone loss and attenuate hyperglycemia in diabetic mice [102,103].
Another study, which employed an induced-PD and RA animal model, determined immunoenzymatically, that both Ibuprofen and RES reduced the tissue levels of anti-cyclic citrullinated peptide antibody (ACCPA) by 99 and 72%, respectively (p < 0.05), and RES alone, was reported to reduce serum rheumatoid factor (RF) (p < 0.05) [101].
Interestingly, the results of a study that used an induced-PD model which concurrently induced osteoporosis (OP) by ovariectomising the rats, suggested that RES may reduce alveolar bone loss in oestrogen-deficient rats via the attenuation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, making NADPH oxidase a potential drug target for RES [104].
Also of note, an extensive in vitro study showed the potential for RES to address Pg-related disease, with a particular focus on the prevention of Alzheimer's disease. Using a human in vitro model for neuroinflammation, Bahar and Singarao demonstrated that RES successfully modulated the ROS and deregulated inflammation. A total of 96 genes were analysed in Pg LPS-induced human neuroblastoma cells via qPCR followed by pathway analysis. In this way, RES was found to diminish NF-κB, neuroinflammatory acute phase pathways [105].

Animal studies: the amelioration of ligature-induced PD by RES
Although microbial dysbiosis is a necessary early occurrence in the pathogenesis of PD, the resulting chronic inflammation is the causal factor regarding its progression and continuous tissue destruction [106,107]. Therefore, an effective therapeutic approach for the mitigation of PD would be to address the pathogenetically deregulated inflammatory pathways, mediators, and markers, encouraging the system to return to balance without deleterious side effects.
In a commonly used animal model, PD is induced by fitting a ligature around the neck of pre-selected molar teeth. Typically, PD that is induced in this way predictably presents with significant alveolar bone loss, accompanied by the increased expression of pro-inflammatory genes such as those for IL-1β, IL-6, and TNF-α. Notably, increased mRNA expression of genes coding for osteoclastogenic proteins and receptor activator of nuclear factor-k B ligand (RANKL) has also been reported when applying this model [108].
Morphometric analysis [27, 100,101,103,[109][110][111] and/or Micro-CT [104,[112][113][114] has been employed to demonstrate that RES reduced the alveolar bone loss from experimentally induced PD. The micro-CT analyses also reported improved bone density, suggesting that at the very least, RES has therapeutic potential as an adjunct to traditional SRP. This of course is caveated by emphasising the dependence of this data on the relevance of the PD animal model, and the need for validation with human studies.

Low bioavailability and stability: an obstacle to the clinical translation of RES
The poor water solubility of RES is well established. However, RES is highly stable in aqueous solutions of acidic pH. Moreover, researchers must consider that RES degrades rapidly in buffers of 7.4 pH or higher [115]. For example, RES incorporated into buffered cell medium was found to degrade to 50% of its original concentration within 24 h of incubation at 37°C [115]. Hence, many of the in vitro studies, which assume that the pre-determined RES concentration is consistent for the study duration, are likely to produce misleading results regarding therapeutic dose.

Overcoming therapeutic limitations of RES by the application of nanotechnology
RES has been reported as having notably poor water solubility as well as high sensitivity to heat and pH [115]. Also, since RES is unstable under physiological pH and temperature, in vivo assays are challenging to design and in vitro assays are likely to have low translatability [92,120,121].
Additionally, oral administration of RES has demonstrated unfavourable pharmacokinetics due to its extensive first pass, resulting in the accumulation of potentially recycled conjugates, RES-glucuronides, and RES-sulphates; although these metabolites have also been found to possess biological activity, it may not match that of the parental compound [85].
Previous reports highlighting the physicochemical limitations of RES indicate that meticulous consideration of aqueous solubility, pH, temperature, and light, during the experimental design phase is crucial for the optimisation of clinical translation [122].
Consequently, the search for effective strategies for the improvement of the limited oral bioavailability and stability, is a complex, yet necessary, undertaking for the successful development of RES as a therapeutic.
Regarding RES, improvement of one or more physicochemical and/or pharmacological parameters has been reported when in a nano form, indicating the potential of nanotechnological formulation as a viable strategy for improving its physicochemical stability and pharmacological profile.
Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) are commonly employed to improve the therapeutic potential of hydrophobic drugs such as RES. Furthermore, findings that assessed the pharmacological potential of RES-loaded SLNs and NLCs, indicated their higher stability and sustained release compared to RES in its bulk form [123][124][125][126][127][128].
Further to this, studies seeking out to fortify and/or functionalise foods with RES, reported that nanoencapsulation substantially increased thermostability and photostability whilst retaining or optimising the desired biological activity. For example, an in vitro investigation examining the nano-encapsulation of RES in starch, conducted at pH 7.4 at 37° C, was reported to demonstrate an almost tenfold increase in drug retention following a food extrusion process, as well as higher anti-diabetic, anti-obesity, and antioxidant effects, compared to bulk RES [129].
Similarly, the sustained release of RES from ZEIN-encapsulated nanoparticles (NPs) under physiological conditions (pH 7.4, 37°C) was reported [130] and caseinencapsulated RES NPs, designed by Penlava et al., were found to be stable through a continuous pH range mimicking those of the gastrointestinal compartments (i.e., pH 1.2 for 2 h and pH 6.8 for 2-24 h). Interestingly, the latter study also demonstrated in vivo (using rats), a ten-fold increase in oral availability of casein-nanoencapsulated RES compared to the bulk form as determined by blood plasma assays over a 24 h period following a single oral dose of 15 mg/kg of RES (in ddH 2 O and PEG) or casein-encapsulated RES NPs [131].
These studies and others bring to light the prospect of the customisation of functional foods, to serve as both local and systemic delivery system for the effective prevention, management, and treatment of PD.

Restoration of tissue damage from PD: potential of current Nano RES formulations
Nano-RES formulations intended specifically for the treatment of PD, are only beginning to emerge. For example, Berta et al., reported a nano-formulated REScyclodextrin mouthwash that was found to reduce plaque and bleeding gums in children [132]. Nonetheless, there are several nano RES formulations, intended to treat other conditions, which could, in theory, be studied as potential formulations for PD, with little divergence from the original formula.
For example, in a 2021 study, Li and colleagues produced nano-hydroxyapatite-RES-chitosan (CS) microspheres for bone generation, which could potentially be used to restore bone loss due to PD [133]. Also, electrospun 3-D nano-scaffolds loaded with RES, consisting of a biodegradable polymer (PLA)-biopolymer-gelatin (GEL) nano-scaffold was found to repair cartilage defects in the rat model [134].
Moreover, the successful application of nano-RES as a potential treatment for AD has been reported by Sun et al., who designed a RES-loaded mesoporous selenium-Fcβ-cyclodextrin-Borneol nanoparticle that crossed a blood-brain barrier model [136].

RES has evolved to be a viable agent for the treatment of PD
RES has been shown to execute biological action that alleviates deregulated inflammation, and restores both soft and bony tissues, in vitro, and in vivo, via modulation on the genetic, protein, and cellular level, thus strengthening the case for RES as a therapeutic for PD. Further to this, improvement of the pharmacokinetic and physicochemical limitations of RES has been demonstrated via nanoformulation. There is now much work to be done in identifying and optimising the ideal nanoformulation and administration route to achieve optimal benefit from the activities RES has demonstrated.
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