Fermented lingonberry juice's effects on active MMP‐8 (aMMP‐8), bleeding on probing (BOP), and visible plaque index (VPI) in dental implants—A clinical pilot mouthwash study

Abstract Objectives We aimed to study the effects of fermented lingonberry juice (FLJ) as a mouthwash on the levels of active matrix metalloproteinase‐8 (aMMP‐8) in peri‐implant sulcular fluid (PISF), bleeding on probing (BOP), and visible plaque index (VPI). We hypothesized that FLJ rinsing could reduce inflammation (aMMP‐8 and BOP) and microbial load (VPI) in the oral cavity, especially around dental implants. Materials and Methods A clinical pilot study was performed using FLJ as a mouthwash. The inclusion criteria were at least one dental implant in the anterior or posterior areas with a screw‐retained crown. Ten participants used 10 ml of mouthwash twice a day for 15 days, and 10 participants served as the control group. Point‐of‐care tests (POCTs) were used to measure aMMP‐8 levels in the PISF, and BOP and VPI were recorded at the beginning of the trial and after 15 and 30 days. Results The FLJ mouthwash had a reductive effect on aMMP‐8, VPI, and BOP in the mouthwash group; however, there was no significant difference compared to the control group. The difference in VPI and BOP levels between the groups diminished after the lingonberry regimen ended. The decrease in aMMP‐8 levels appeared to continue even after discontinuation of the mouthwash regimen. Conclusion The reduction in the amount of plaque, aMMP‐8, and BOP by FLJ was promising and continuous considering the relatively short study period and sample size. FLJ is a natural and safe supplement for oral and dental implant home care. Further studies are required to verify these promising results.

Previous studies have shown that polyphenols in the high molecular size fractions of crowberry, blackcurrant, bilberry, and lingonberry juices exert an antiaggregation effect on plaque colonizers in vitro (Riihinen et al., 2011). Biofilm formation on dental implant surfaces may be reduced by several treatment modalities (Garaicoa et al., 2020). Chlorhexidine (CHX) mouthwash and gels used in oral home care have been evaluated by several studies (Bescos et al., 2020;Fiorillo, 2019;James et al., 2017); however, a range of negative side effects has been reported, including discoloration of teeth, taste disturbance/alteration, and oral mucosa symptoms such as soreness.
Compared to CHX mouthwash, FLJ mouthwash has not been reported to exert these side effects.
There are no previous studies on the effects of lingonberries on dental implants, and because of the versatility and safety of lingonberry polyphenols in reducing oral inflammation and microbial load, FLJ appears to be a promising treatment candidate.
1.2 | Dental implants, peri-implant mucositis, and peri-implantitis Dental implants are a common treatment for replacing missing teeth.
Dental implants require careful home care as infection and inflammation progress faster in dental implants than in natural teeth, and the treatment of dental mucositis is regarded to be more challenging than that of periodontitis (Golub et al., 2020). There is a variety of bacteria and yeasts in the oral cavity that form biofilms on all surfaces in a fluid system, as well as on hard, nonshedding surfaces such as oral implants (Bescos et al., 2020;Cortelli et al., 2013). Due to the high profusion of bacterial exposure in the mouth, the host tissue responds with a protective mechanism that initially leads to the destruction of soft tissues. This initial inflammation on dental implants is termed peri-implant mucositis; if it is not diagnosed and treated, it may lead to irreversible inflammation that also extends to the hard tissue and develops into peri-implantitis (Belibasakis et al., 2015;Derks & Tomasi, 2015;L. Heitz-Mayfield & Salvi, 2018;L. J. Heitz-Mayfield, 2008;Mikolai et al., 2020;F. Schwarz et al., 2018). Patient home care combined with early clinical diagnosis is a very important element for implant success and maintenance.
Mucositis and initial peri-implantitis can be effectively treated conservatively.

| aMMP-8 and point-of-care testing
Clinical observations are required for the diagnosis of dental implant tissue health. Traditionally, clinical evaluation is performed by measuring the BOP, implant pocket depth, and by performing X-ray evaluations (Berglundh et al., 2018). However, there is an alternative method with which to assess the condition of the tissues around implants. Biomarker-based aMMP-8 point-of-care testing (POCT) has been extensively studied and is an accurate and validated method Deng et al., 2021;Golub et al., 2020;Lähteenmäki et al., 2020;Räisänen et al., 2018Räisänen et al., , 2019Sorsa, Alassiri, et al., 2020;Ziebolz et al., 2017). The aMMP-8 test can be used to detect active and early collagenolysis in dental implant tissues.
aMMP-8 is a lateral flow peri-implant chair-side/POCT technology validated for periodontal/peri-implant diagnosis and screening.
Compared to the standard evaluation, it strengthens the diagnostic procedure by accounting for disease activity (Golub et al., 2020;Lähteenmäki et al., 2020;Sorsa, Alassiri, et al., 2020;Sorsa, Bacigalupo, et al., 2020). The outcome of the test can be read visually as one (test negative) or two (test positive) lines that emerge in a reading window, similar to the classical point-of-care pregnancy test ( Figure 1). The results can also be read quantitatively with a device/reader that provides the quantitative and numerical value of the test in 5-7 min.
This study aimed to evaluate the effects of FLJ, used as a mouthwash, on mucositis inflammation in dental implants. The effects were observed and evaluated by recording VPI, BOP, and aMMP-8 levels.  Procter & Gamble, Cincinnati, OH, USA) and their normal home care tools for 30 days before, during, and after the lingonberry oral rinse period.
FLJ was used as a mouthwash. Its concentrated, naturally occurring sugars were reduced, and there were no additives. Ten participants (Group 1, FLJ group) used 10 ml of FLJ twice daily (30 s) for 15 days, after which they discontinued the mouthwash for 15 days (washout period). Ten participants (Group 2, control group) did not use mouthwash. According to our previous clinical oral fermented lingonberry mouthwash study, a 15-day trial length was suitable for this pilot study. The washout period is sufficient as long as the mouthwash period enables observation of the clearance of the treatment effect (Pärnänen et al., 2019).  Table 2. Peri-implant health was defined as the absence of ≥4-mm probing depths, BOP, erythema, swelling, and suppuration around the implant without radiographic bone loss. Peri-implant mucositis was defined as the presence of BOP with or without erythema, swelling, and/or suppuration around the implant and without radiographic bone loss. Peri-implantitis F I G U R E 1 Sample strip placements in implant sulci. Photographs of an active matrix metalloproteinase-8 point-of-care sample collection container, peri-implant sulcular fluid collection strip, and dipstick test with a reading window. Note: Peri-implant health was defined as the absence of ≥4-mm probing depths, BOP, erythema, and swelling and suppuration around the implant, all without radiographic bone loss. Peri-implant mucositis was defined as the presence of BOP with or without erythema, swelling, and/or suppuration around the implant and without radiographic bone loss. Periimplantitis was defined as the presence of ≥4-mm probing depths and BOP with or without erythema, swelling, and/or suppuration around the implant, together with radiographic bone loss ≥2 mm (Berglundh et al., 2018).
was defined as the presence of ≥4-mm probing depths and BOP with or without erythema, swelling, and/or suppuration around the implant, together with radiographic bone loss ≥2 mm (Berglundh et al., 2018).
At the same time points as above, an additional sulcus fluid rinse was collected for chair-side aMMP-8 POC tests (Implant-Safe ® /ORALyzer ® ; Dentognostics, Jena, Germany), as described in the study by Lähteenmäki et al. (2020). The general patient characteristics are summarized in Table 1.

Differences in categorical and continuous variables between the FLJ
and control groups were analyzed using Fisher's exact test and a t test, respectively. The effects of the FLJ mouthwash on aMMP-8 levels, VPI, and BOP were measured in the FLJ and control groups (at baseline, 15 days, and 30 days) ( Table 3). The differences in the mean levels of aMMP-8, VPI, and BOP were investigated using a mixeddesign repeated-measures ANOVA analysis (Figure 2

| RESULTS
The patient characteristics are presented in Table 1. Fifty percent of the participants in the FLJ group were men, and 30% were men in the control group (p = .650). The mean ages of the patients were 75.1 and 68.3 years in the FLJ and control groups, respectively (p = .068).
There were no significant differences in the prevalence of smoking or comorbidities between the FLJ and control groups.

| DISCUSSION
This study was the first of its kind to examine the effects of FLJ on parameters that reflect the course of peri-implant health and disease using indices and to additionally assess disease activity using the aMMP-8 POCT. In the current study, 15 days of using the FLJ mouthwash seemed to have similar decreasing effects on PISF aMMP-8, visible plaque, and BOP levels around dental implants. In the control group, a similar, although smaller, decrease was seen in visible plaque levels, but not in aMMP-8 or BOP levels, after the first 15 days. Statistically significant differences in the mean aMMP-8, VPI, and BOP levels between the FLJ and control groups were not observed in this study. A previous clinical study (Pärnänen et al., 2019) demonstrated that FLJ mouthwash decreased whole-mouth aMMP-8, BOP, and VPI measured by traditional periodontal techniques in patients with natural teeth. This suggests that the FLJ mouthwash period of only 15 days may have been too short a period of time to achieve site-specific effects instead of whole-mouth effects. This is supported by the findings of this study where the differences in aMMP-8, VPI, and BOP between the groups diminished after the lingonberry regimen ended. Furthermore, exclusion of the effect of outliers revealed that aMMP-8 levels continued to decrease in the FLJ group, even after discontinuation of the mouthwash regimen.
This suggests that the effect of the FLJ mouthwash could be seen earlier in the visible plaque levels, while the antiproteolytic effects lasted longer or were delayed for aMMP-8 levels that are known to T A B L E 3 Results of intra-and intergroup comparisons of aMMP-8, VPI, and BOP at baseline, after 15 days of mouthwash use, and after a 15-day washout period  indicate and reflect the risk of ongoing collagenolytic peri-implant tissue destruction (Lähteenmäki et al., 2020;Pärnänen et al., 2020). F I G U R E 2 Boxplots of active matrix metalloprotease-8 (aMMP-8, ng/ml), visible plaque index (VPI), and bleeding on probing (BOP) data among patients in the fermented lingonberry juice (FLJ) mouthwash and control groups as well as for both groups together (with and without a patient with outlier values) at three time points (at baseline, 15 days, and 30 days). The line represents the group mean with a 95% confidence interval.
The treatment of peri-implant mucositis, especially peri-implantitis, is challenging. The current study was a clinical pilot study to evaluate the effects of a natural substance as an alternative adjunctive therapy for these conditions. Further studies with larger patient cohorts and significantly longer study periods are required to verify and extend the results of the present study. FLJ appears to be a promising and safe aid in oral home care for reducing plaque levels, gingival bleeding, and inflammation around dental implants and throughout the mouth.
Lingonberries are known to have anti-inflammatory, antioxidant (radical oxygen scavenging), antiproteolytic, and antimicrobial properties . FLJ does not inhibit Lactobacilli growth, and according to Cereda et al. (2018), probiotic Lactobacilli have also been proposed to have beneficial effects on chemoradiotherapy-related oral mucositis and peri-implant mucositis. Currently, CHX is regarded as the gold standard for the treatment of oral and implant-related infections.
However, there are indications that repeated exposure to the most commonly used antiseptics, such as CHX, may induce resistance to oral bacteria in deep biofilms, which has never been demonstrated for lingonberries (S. R. Schwarz et al., 2021).

| CONCLUSION
This study was the first of its kind to examine the effects of FLJ on the parameters that reflect dental implant health and disease using indices and to additionally assess the collagenase activity in PISF

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.