**6. Effects of modulating inflammatory response therapy in patients with osteoporosis and chronic periodontitis**

Mechanical removal of plaque and calculus from dental surfaces is considered the standard treatment for chronic periodontitis. Contrary to the spectacular evolution in the field of etiopathogenesis of periodontal disease, its treatment has changed very little, in principle. Scaling and root planing remain the "gold standard" of periodontitis treatment.

The importance of the host inflammatory response in periodontal pathogenesis presents the opportunity to explore new therapeutic strategies by means of modulating this response. Modulation therapy can be combined with conventional therapeutic methods to reduce the bacterial load.

To date, the only systemic therapy approved as a modulator of the response in periodontal disease is with sub-antimicrobial doses of doxycycline, which inhibits the activity of matrix metalloproteinases (MMPs) (trade name: Periostat). Doxycycline in sub-antimicrobial doses inhibits MMP activity by synergistic mechanisms, independent of antibiotic properties. Primary studies have shown that the use of tetracycline predominantly inhibits MMPs in excess of periodontitis compared to constitutional MMPs. In vitro studies have shown that MMP-13 is more sensitive to tetracycline inhibitory concentrations than MMP-8 and MMP-1 (fibroblastic collagenase) is the least sensitive.

Doxycycline has been shown to be more effective than other tetracyclines in reducing collagenase activity in crevicular fluid in patients with chronic periodontitis [39]. Doxycycline has a lower inhibitory concentration than minocycline (IC50 = 15 IM compared to IC50 = 190IM) or tetracycline (IC50 = 350IM); thus, a lower dose of doxycycline than minocycline or tetracycline is required to reduce a certain level of collagenase by 50% [40]. Moreover, doxycycline is more effective in blocking the activity of neutrophil collagenase (MMP-8) than the activity of MMP-1 (fibroblastic collagenase), demonstrating that its use is a safe way to reduce pathological collagenase levels without affecting healthy tissues.

We conducted a study in a group of 26 subjects, with the purpose to analyse changes in periodontal clinical parameters that modulation therapy of the host response with sub-antimicrobial doses of doxycycline can exert in patients with periodontal disease and osteoporosis. Patients were randomly divided into two groups: the study group (n = 17), which underwent classical debridement therapy (scaling and root planing) plus sub-antimicrobial doses of doxycycline (20 mg twice daily) for 3 months, and control group (n = 18), which followed only classical debridement therapy [41].

We analysed the following periodontal parameters: probing depth, level of clinical attachment, PBI and PI index at baseline (pre-therapeutic), on the last day of medication, and 3 months after medication completion (6 months from baseline). The sites were grouped according to the probing depth in: group 1 - superficial (0-3 mm); group 2 - moderate (4-6 mm) and group 3 - deep (≥7 mm) [41].

In the present study, 30 patients were initially enrolled, but 4 of them failed to complete doxycycline therapy. Therefore, the study resulted in the use of two groups: the study group (13 subjects) and the control group (13 subjects). There was no statistically significant difference between groups at baseline in terms of probing depth. No significant differences were observed in the sites with an initial depth of 0–3 mm (p > 0.05). Significant reductions in probing depth were observed at sites with an initial depth of 4-6 mm and ≥ 7 mm (p < 0.025) [41].

Although the mean value of pocket reduction for sites with an initial depth of 4-6 mm and ≥ 7 mm was higher for the study group than for the control group

(1.80 mm versus 1.46 mm for moderate pockets and 3.38 mm versus of 2.57 mm for deep pockets), the difference did not reach the significance threshold (p > 0.05). Analysis of sites with an initial depth ≥ 7 mm showed that an increased percentage of sites was reduced by at least 3 mm following doxycycline administration (66.4%), compared to the group without modulation therapy (55.1%) at 3 months, without a statistically significant difference between groups (p > 0.05) [41].

However, at 6 months the percentage of sites with an improvement in depth ≥ 3 mm was significantly higher (p = 0.011) for the group with modulation therapy (73.4%) compared to the group that followed only classical therapy (49.7%) [41]. At baseline, there were no statistically significant differences in the level of clinical attachment in the sub-grouped sites by probing depth between the main study groups (p > 0.05). Sites with moderate depths and deep sites showed significant improvements in attachment level at 3 and 6 months, compared to baseline (p < 0.025). Sites with an initial depth of 0–3 mm did not show significant changes in attachment during the study period (p > 0.05) [41].

Sites with an initial depth of 0–3 mm in the control group (without doxycycline therapy) showed a slight decrease in attachment (−0.04 mm at 3 months, −0.03 mm at 6 months). On the other hand, the sites with the initial depth of 0-3 mm in the study group showed a slight gain of attachment (0.11 mm at 3 months, 0.14 mm at 6 months), but without significant differences between groups (p > 0.05).

Although the average attachment gains for sites with an initial depth of 4-6 mm and ≥ 7 mm was higher for the study group than for the control group (1.12 mm compared to 0.78 mm for sites with moderate depths; 2.15 mm compared to of 1.76 mm compared to the deep sites), the statistical analysis did not show a level of significance (p > 0.05) [41].

GDP and PI values showed significant improvements between baseline and reevaluations at 3 and 6 months (p < 0.025). The reduction in GDP and IP was similar for both groups (p > 0.05).

Periodontal treatment, over time, has focused on reducing the bacterial load and disorganizing the biofilm by mechanical methods. However, recent research has led to a paradigm shift in the evolution of periodontal disease. Thus, it is known today that the lesions that appear at the level of superficial and deep periodontal tissues are a result of the activation of the host's immune-inflammatory defence mechanisms [42].

In addition to the classic periodontal therapy, scaling and root planing, which aims to disorganize the bacterial biofilm and reduce the inflammatory load, new adjunct methods have been postulated, with etiological therapeutic effect in the periodontopathic patient. Among them, the modulation therapy of the host's inflammatory response with pharmacological agents has acquired important dimensions, precisely because of its effectiveness. The success of such therapy is all the more important as it affects a systemically affected area.

Doxycycline has the ability to inhibit the activity of matrix metalloproteinases (MMPs), a capacity confirmed in numerous studies. Minocycline, doxycycline, and tetracycline inhibit collagenolytic activity, while non-tetracycline antibiotics have no effect on collagenase [40]. It was recognized in the mid-1980s that inhibition of collagenolysis by tetracyclines is a new therapeutic method in the management of periodontal disease.

The effects of doxycycline are, in addition to direct inhibition of active matrix metalloproteinases by cationic chelation and inhibition of oxidative activation of latent MMPs, and inhibition of the expression of inflammatory cytokines (IL-1, IL-6, TNFα) and PG-E2; seeks and inhibits the formation of oxygen-reactive species produced by neutrophils; protects the α1-proteinase inhibitor, thus indirectly

#### *The Role of Osteoporosis as a Systemic Risk Factor for Periodontal Disease DOI: http://dx.doi.org/10.5772/intechopen.96800*

reducing tissue proteinase activity; reduces osteoclastic activity and bone resorption; inhibits osteoclastic MMPs.

Doxycycline contributes to decreased conjunctival lysis by inhibiting proinflammatory mediators and cytokines (including IL-1 and TNFα) [43], as well as by increasing collagen production, osteoblast activity and bone formation [41]; this last aspect is of major importance especially for patients with osteoporosis, whose bone capital is affected.

A major concern with long-term administration of doxycycline has been associated with the development of antibiotic resistance. Indeed, when antimicrobial doses of tetracycline were used (250 mg daily, 2–7 years), up to 77% of patients' flora showed resistance to tetracycline [44]. Given this problem, sub-antimicrobial doses (20 mg doxycycline versus 50 or 100 mg) were prepared [41]. One of the preliminary experiments with this new formula clearly demonstrated that such doses (20 mg twice daily), administered 2 weeks, inhibited collagenase activity by 60–80% in gingival tissue in patients with chronic periodontitis [41]. Collagenase activity was significantly reduced in the crevicular fluid collected from these patients. Subsequent studies have indicated that this drug regimen can prevent the progression of periodontitis without the patient developing microorganisms resistant to doxycycline or other types of side effects [42].

The 3-month doxycycline regimen was well tolerated and no adverse reactions (gastrointestinal disorders, etc.) were reported. This may suggest that doxycycline modulation therapy is a safe approach in the long-term treatment of chronic periodontitis.

In the present study we observed improvements in clinical parameters (probing depth, level of clinical attachment, bleeding index, plaque index) both for the study group (with adjunctive modulation therapy) and for the control group (which followed only classical scaling-root planing therapy), improvements that were maintained throughout the study.

Caton et al. [45] established that reductions in probing depth of at least 3 mm represent relevant, clinically significant improvements. In the present study, the percentage of sites of great depth (≥7 mm) that showed reductions of at least 3 mm was significantly higher at 6 months for the group with modulation therapy. This result is of special importance, given that sites with such depth are candidates for surgical procedures. Therefore, it can be hypothesized that adjunctive doxycycline therapy may reduce the likelihood of surgical procedures as well as the discomfort caused by them [41].

We also demonstrated that the sites with relatively small depths (0-3 mm) in the study group showed a slight gain of attachment, while these sites in the control group showed a slight loss of attachment. This supports the efficacy of host response modulation therapy by administering sub-antimicrobial doses of doxycycline.

Studies are needed to evaluate the efficacy of very long-term sub-antimicrobial doses of doxycycline in periodontal therapy and in the prevention of loss of dentalperiodontal units. The financial benefit derived from adjunctive therapy must also be evaluated (can this minimize costs by avoiding the need for periodontal surgery?).

It is suggested that doxycycline-based products be developed to support plasma concentrations for 24 hours by administering a single dose daily.

Given the increased variability of pathogenic pathways with a role in periodontal destruction (e.g., the cytokine group IL-1 is much more complex today), a more diverse range of host response modulators is also needed [46]. Moreover, most biological responses involve a variety of mechanisms; thus, blocking a single inflammatory pathway may not result in the desired result because receptor-mediated

responses can be activated by alternative pathways. Therefore, a poly-pharmaceutical approach is needed to modify a number of different pathways associated with inflammation and tissue destruction.

Lipoxins are another group of compounds that can alter the inflammatory response in periodontal tissue. These mediators are released during the inflammatory response and have the effect of decreasing inflammation and modulating its disappearance. Lipoxins block the secretion of IL-1β from neutrophils and block the migration of neutrophils following exposure to *Porphyromonas gingivalis* [47].

Osteotrophic factors such as hormonal or endocrine-related (vitamin D3, parathyroid hormone), cytokines (IL-1, IL-6, IL-11 and IL-17), growth factors (TNFα, morphogenetic protein-2) and others molecules (PG-E2, LyT activator CD40 ligand and glucocorticoids) increase the expression of the RANKL gene in osteoblastic/stromal cells.

Sequentially, RANKL mediates the signal for ostoclastogenesis through RANK or preosteoclastic cells. Thus, the RANKL/RANK interaction is responsible for the differentiation and maturation of osteoclast precursor cells with osteoclast formation. Osteoprotegrin acts by binding to RANKL, inhibiting osteoclastic development.

In periodontal disease the first to investigate the role of RANKL in bone resorption was Teng [48]. It inoculated *Aggregatibacter actinomycetemcomitans* in mice lacking endogenous LyT and LyB and receiving human CD4 cells; Thus, CD4 + activation, RANKL stimulation and bone resorption were initiated, concluding that RANKL expression plays a significant role in bone destruction in periodontitis. Crotti et al. [49] observed a hyper-expression of RANKL in inflamed periodontal tissues, as well as an increased RANKL/osteoprotegrin ratio to healthy subjects.

There are numerous animal studies as well as preliminary human studies demonstrating inhibition of RANKL function by osteoprotegrin treatment, reducing the number of osteoclastic cells and, implicitly, bone resorption from periodontal disease. Of course, more in-depth studies are needed to certify the most effective therapeutic approach to this molecular interaction.

Fatty acids have been proposed to reduce chronic inflammation in arthritis patients by decreasing the release of LTB4 from neutrophils and IL-1 from monocytes. Local application of Omega-3 polyunsaturated fatty acids has been successful in patients with inflammatory diseases such as psoriasis, as well as in models with experimental periodontitis in animals. The mechanism of action is based on decreased leukocyte chemotaxis, expression of molecular adhesion and production of inflammatory cytokines. Offenbacher demonstrated the inhibition of PG-E2 production by eicosapentanoic or docosahexanoic acid administration, with effects similar to ibuprofen in patients with periodontal disease [50].

Vardar evaluated the use of omega-3 fatty acids in order to block arachidonic acid cascade in mice with experimentally induced periodontal disease [51]. This would inhibit the production of cyclooxygenase-derived prostanoids and lipooxygenase-derived leukotrienes. The authors relied on two aspects: leukotriene B4 (mediator of arachidonic acid) plays an important role in bone resorption and inhibition of COX with NSAIDs would cause the accumulation of arachidonic acid that is metabolized by lipo-oxygenase, causing continuous bone loss. The authors also administered a combination of omega-3 fatty acids with celecoxib, seeking a synergistic anti-inflammatory effect. Combination therapy resulted in significant decreases in prostaglandin, leukotriene B4 and PAF levels; no effect on bone lysis was observed (this may be due to the short evaluation period).

Elkhouli published the results of a study of 40 patients with at least one grade II furcation defect; patients were divided into two groups: the first group performed allografting, which was associated with therapy with omega-3 polyunsaturated fatty acids and low-dose aspirin; in the second group (control) only allografting

#### *The Role of Osteoporosis as a Systemic Risk Factor for Periodontal Disease DOI: http://dx.doi.org/10.5772/intechopen.96800*

was performed, following a placebo adjunctive therapy. At 3 and 6 months, the clinical parameters (plaque index, gingival index, bleeding index, probing depth, level of clinical attachment) were evaluated, as well as biochemical markers in the crevicular fluid (IL-1b and IL-10) [52]. The results were very good for the test group compared to the control group (reduction of probing depth, gain of clinical attachment, significant modulating effect for IL-1b and IL-10 levels).

There are proven clinical results regarding monounsaturated fatty acid substitution; they influence blood pressure, clotting, endothelial activation, inflammation and thermogenic capacity in cardiac patients, they prevent obesity and other metabolic diseases.

Hasturk demonstrated in a study in rabbits with experimentally induced periodontitis with *Porphyromonas gingivalis* that topical application of the tetradecanol-1 complex (1-TDC: mixture of esterified monounsaturated fatty acids) causes an inhibitory effect on the inflammatory cascade of the host response [53].

Further studies are needed to evaluate the impact of this modulation therapy in patients with periodontal disease and osteoporosis at the molecular level (by examination of the crevicular fluid), on pro-inflammatory cytokines, and at the systemic level, by assessing bone mineral density (correlation with pre- and post-therapeutic T score).
