*3.2. Clinical and Radiographic Peri-Implant Parameters*

The peri-implant mean PD (*p* < 0.05) and mesial (*p* < 0.05) and distal (*p* < 0.05) CBL were significantly higher among group-1 than group-2 and control group. However, the peri-implant mean BOP (*p* < 0.05) and PI (*p* < 0.05) were significantly higher in group-2 when compared with group-1 and the control group. Overall, the peri-implant mean PI (*p* < 0.05), BOP (*p* < 0.05), PD (*p* < 0.05), and CBL (*p* < 0.05) were significantly higher among test groups than the control group. The mean SPP score was highest in group-2 (3.4 ± 1) when compared with group-1 (1.3 ± 1) and the control group (0 ± 0), respectively (Table 2).

**Table 2.** Peri-implant clinical parameters of the research groups.


Dissimilar lowercase letters indicate a statistically significant difference (*p* < 0.05) between groups along rows. Abbreviations: BOP = bleeding on probing; CBL = crestal bone loss; PD = probing depth; PI = plaque index; SPP = self-perceived pain.

#### *3.3. Peri-Implant Crevicular Fluid Cytokine Levels and Self-Perceived Pain*

PICF levels of TNF-α, MMP-1, and IL-8 were raised significantly among group-1 and group-2 than the control group (Table 3).



Dissimilar lowercase letters indicate a statistically significant difference (*p* < 0.05) between groups along rows.

Statistically, a significant association was observed between SPP and TNF-α, MMP-1, and IL-8 expression in the PICF of test groups and control group as per bivariate multiple logistic regression analysis (Table 4).


**Table 4.** Statistical comparisons of the SPP score with PICF cytokines levels.

Abbreviations: CI = confidence interval; IL-8 = interleukin-8; MMP-1 = matrix metalloproteinase-1; OR = odds ratio; PICF = peri-implant crevicular fluid; SPP = self-perceived pain; TNF-α = tumor necrosis factor-alpha.

## **4. Discussion**

It is postulated that SPP, peri-implant clinical, and radiographic parameters are worse among cigarette smokers with peri-implantitis than never smokers without peri-implantitis. In this study, the clinic-radiographic peri-implant parameters were poorer among cigarette smokers with peri-implantitis than never smokers without peri-implantitis; no statistically significant correlation was observed between SPP and expression of TNF-α, MMP-1, and IL-8. However, several variables might have altered the findings, therefore, these results should be interpreted with extreme caution.

A unique ecological niche is formed by the gingival sulcus for bacterial colonization. The bacterial biofilm in the gingival sulcus promotes inflammation in the surrounding connective tissues [32]. An inflammatory process is triggered by the imbalance between host response and microbial challenge at the implant–soft tissue interface [33]. Cytokines, including IL-6, IL-1B, TNF-a, are released from the cells of neutrophils, macrophages, connective tissue fibroblasts, dendritic cells, and gingival epithelium. Moreover, a number of enzymes, including aspartate aminotransferase, alkaline phosphatase, and MMPs, are formed by osteoclasts, fibroblasts, and neutrophils, resulting in the degradation of connective tissue collagen and alveolar bone [34,35]. Over 90 different molecular components in gingival crevicular fluid have been assessed for potential periodontal disease diagnosis linked with the natural dentition [36]. However, considerably fewer PICF components have been investigated around dental implants.

In the current study, certain disparities were also observed. For example, clinical peri-implant examination revealed that PI and BOP were significantly raised in never smokers with peri-implantitis than smokers with peri-implantitis and the control group. Moreover, in cigarette smokers, BOP is masked as nicotine causes the vasoconstriction of gingival blood vessels [37]. From a clinical perspective, the findings of this study conform

to the proposed hypothesis as scores of peri-implant PD are significantly elevated among cigarette smokers than never smokers. One explanation for this finding is that cell death and formation of matrix metalloproteinases are induced and enhanced by cigarette smoke extracts, respectively, leading to the deterioration of the extracellular matrix proteins such as collagen [38]. Furthermore, the expression for RAGE is increased four times by nicotine in human gingival cells [39]; an increased AGE/RAGE interaction aggravates periodontal tissue inflammation [39].

In the current study, smokers and never smokers with peri-implantitis did not show a significant difference in CBL. Among all three groups, the subjects had a mean age between 38 and 42 years. It is well-established that advancing or increasing age is a predisposing factor for the increased loss of alveolar bone around dental implants and natural dentition [14]. In one study, Javed et al. [25] reported that alveolar bone loss around teeth was significantly less among younger non-smokers (≤45 years of age) than old non-diabetic smokers (≥65 years of age). Furthermore, subjects in group-1 (smokers with peri-implantitis) had a cigarette smoking history of one cigarette per day over the past two years. It is possible that participants with such a brief history of tobacco smoking may exhibit better radiographic bone levels when compared with subjects with a prolonged history of cigarette smoking, (i.e., >15 packs per year) [14]. However, further research is required for testing these hypotheses.

The current study reports that TNF-α, MMP-1, and IL-8 levels were significantly raised among smokers and never smokers with peri-implantitis than never smokers without periimplantitis. TNF-α, MMP-1, and IL-8 appear to have a crucial role in the destruction of the peri-implant tissue. TNF-α is a proinflammatory mediator and its level reflects the bacterial amount and the levels of inflammation [40]. MMPs are considered to be involved in wound repair, normal tissue turnover, and periodontal destruction; MMP-1 may initiate the deterioration of the extracellular matrix. Overproduction of MMP-1 may cause accelerated degradation of the matrix in pathologic conditions including peri-implantitis [41]. IL-8 increase causes the activation of polymorphonuclear cells and influences their selective migration from gingival blood vessels. This leads to an increased amount of cells in a limited time and produces inflammatory conditions. Hence, an excessive quantity of IL-8 might indicate the incipient stage of peri-implantitis [42]. Several studies have reported that the secretion of TNF-α, MMP-1, and IL-8 is upregulated by nicotine in tobacco; which appears to play a vital role in the destruction of alveolar bone around dental implants and natural dentition [17]. Elevated levels of these proinflammatory cytokines have been observed in the PICF of patients with peri-implantitis [43]. Moreover, a meta-analysis reported that cigarette smoking jeopardizes bone-to-implant contact by impairing new bone formation around the dental implant [44]. Another explanation in this regard is that cigarette smoking escalates the production and accumulation of AGEs in periodontal tissues [39]. Strong interfaces between AGEs and RAGEs have been linked with the production of ROS that promotes oxidative burst within gingival tissues, functional changes of phagocytosis and chemotaxis of polymorphonuclear cells (PMNs), alleviated formation of antibodies, increased attachment of bacterial adhesion, and raised local and systemic burden by escalating the expression of cytokines in the crevicular fluid and serum [45]. These mechanisms have been associated with the inflammation of oral connective tissues and destruction of alveolar bone around dental implants and natural teeth in cigarette smokers [16].

In literature, a dearth of reports exist that assessed SPP and its association with the inflammatory cytokines' levels discharged saliva, serum, or PICF. In this aspect, the authors hypothesized that PICF levels of TNF-α, MMP-1, IL-8 are raised in smokers with peri-implantitis with high SPP scores than non-smokers with and without periimplantitis. The findings of the present study are in opposition with this hypothesis as no statistically significant association between SPP and PICF levels of TNF-α, MMP-1, IL-8. One justification in this regard might be that an array of factors such as coping strategies, cognition, expectations, beliefs, and interpretation influence pain evaluation.

Furthermore, from these findings, it is not possible to calculate the minimum amount of proinflammatory cytokines required to evoke SPP in individuals with peri-implantitis. Despite a well-documented association between pain conditions and cytokines [46,47], the association between pain perception and cytokine expression in oral fluids remains unclear and poorly understood. Hence, further research is required in this regard.

Although questionnaires are not particularly reliable regarding clinical peri-implant parameters, including PI, PD, and BOP [48], they are valid and reliable tools for evaluation of SPP and other oral symptoms [48]. Moreover, validated and well-designed questionnaires could too yield valuable data in survey-based epidemiological large sample-size reports [26]. In the current study, NPRS was utilized to assess the SPP following the recommendations by Downie and co-workers [5] almost forty years ago, who reported that the NPRS (0–10) was a better method to assess SPP than other scales including the four-point descriptive and the visual analogue scales. Because NPRS was the only utilized scale for assessing the SPP, it is difficult to either contradict or support the Downie study [5]. Based on the present study's findings, the NPRS appears to be an efficient scale for pain evaluation in periodontology and implant dentistry. However, future studies utilizing several pain evaluation scales are required to identify the most efficient and suitable pain scale that can be employed on individuals with peri-implant diseases. It is important to recognize the limitations of this study. First, the cross-sectional study and self-reported results that depend on the subject's recall capacities might have influenced the outcomes of our findings. The assessment of pain biomarker(s) would have helped to evaluate the pain parameter on the molecular level. Second, strict criteria were adopted regarding patient selection. For example, patients with endocrine disorders and tobacco-product consumers were excluded. As habitually consuming tobacco products is a predisposing factor of peri-implant diseases [49], it was speculated that the levels of PICF proinflammatory cytokines and the severity of SPP are less in never smokers than smokers with peri-implantitis. The primary strength of this study is the assessment of local proinflammatory cytokines. In terms of accuracy, the use of bitewing radiographs in this study provided better-standardized measurements. Future research studies using several pain evaluation scales and biomarkers related to pain and dental implant therapy are required to identify the most efficient and suitable diagnostic criterion that could be employed on individuals with peri-implant diseases. In addition, longitudinal reports might help to evaluate the PICF biomarkers' evolution over time for better understanding in terms of peri-implant infections among smokers and non-smokers.

#### **5. Conclusions**

Within the limitations of the present study, proinflammatory biomarkers were higher in smokers with peri-implantitis than never smokers with and without peri-implantitis, with a significant association between the proinflammatory cytokines and self-perceived pain.

**Author Contributions:** Conceptualization, F.A.A., F.K.A. and M.N.A.; methodology, F.A.A., F.K.A., E.M.A., T.A. and M.N.A. software.; validation, A.M.A., S.A.-S., F.V., M.N.A. and T.A. formal analysis, M.N.A., E.M.A., S.A.-S. and F.K.A.; investigation, F.A.A., F.K.A., A.M.A., S.A.-S. and F.V.; resources, S.A.-S., E.M.A.; data curation, M.N.A., A.M.A. and F.A.A.; writing—original draft preparation, F.V., S.A.-S. and P.A.; writing—review and editing, F.A.A., A.M.A. and F.K.A.; supervision, F.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors are grateful to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs, Research Chair for Biological Research in Dental Health.

**Institutional Review Board Statement:** This study was submitted, reviewed, and approved by King Saud University, Riyadh, Saudi Arabia (UDRC/019-12). The ethical standards of the 1964 Helsinki declaration and national and/or institutional research committee were strictly followed while performing all the procedures. Informed written consent was obtained from each subject before conducting any procedures. Additional information on the study was provided verbally by the study investigator or in a written format.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data is available on contact from the corresponding author.

**Acknowledgments:** The authors are grateful to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs, Research Chair for Biological Research in Dental Health.

**Conflicts of Interest:** The authors declare no conflict of interest.
