Discussion

The aim of this study was to evaluate oral mucosal lesions in terms of clinical presence, visual grading, and histopathological characteristics following replacement of tobacco-based snus with either dry or moist nicotine pouch formulations over a four-week period. A secondary aim was to compare salivary protein expression after substitution with these products over the same duration. The findings indicate that replacing snus with non-tobacco-based nicotine pouches led to gradual improvement of pre-existing oral mucosal lesions in healthy snus users during the study period. However, despite these clinical improvements, the histological characteristics of these lesions, including epithelial thickness and inflammatory cell presence, remained unchanged. Furthermore, no statistically significant differences were observed in total protein levels or in MCP-1, IL-1β, and TNF-α expression in saliva. However, IL-8 levels were significantly increased in the dry formulation group.

The use of NPs has increased markedly in recent years [14, 15]. Because NPs are neither combusted nor derived from tobacco leaf, they could offer a lower-risk alternative to traditional nicotine products [16]. However, as they are relatively new to the market, scientific evidence regarding their effects on the oral environment remains limited. In particular, further research is needed to clarify their impact on oral immune responses, saliva composition, the histological health of oral soft tissues.

Oral mucosal lesions associated with smokeless tobacco (snus) have previously been documented at the pouch placement site [1, 13]. These lesions are generally reversible within weeks after cessation and do not appear to be premalignant [17]. Although the underlying mechanisms are not fully understood, local chemical and mechanical irritation caused by snus constituents has been proposed to contribute to these mucosal changes [18, 19]. The oral mucosa is susceptible to chemical and mechanical irritation because of its permeability and vascularization [20, 21]. Such irritation may contribute to local inflammatory responses in the oral mucosa. A previous study by Axéll et al. demonstrated histological inflammation at lesion sites in snus users [13]. Inflammation involves various white blood cells that can release mediators such as cytokines, which contribute to local immune and inflammatory processes.

In the present study, replacement of snus with non-tobacco-based nicotine pouches resulted in a clear reduction in lesion severity in both groups. Lesion severity at Visit 5 was significantly reduced in both the dry and moist formulation groups compared with Visit 3. These findings are consistent with those of a previous study reporting improvement of pre-existing mucosal lesions following substitution of regular snus with non-tobacco-based nicotine pouches [8].

A correlation has previously been suggested between the clinical grading of snus-induced lesions and corresponding histologic changes, including histological inflammation in clinically severe lesions [22]. Building on these findings, we examined the presence of inflammatory cells in biopsies collected from lesion sites at Visit 3, when participants were still using tobacco-based snus, and at Visit 5, after four weeks of non-tobacco-based NP use. We also assessed epithelial thickness, as changes in thickness may reflect tissue responses to chronic irritation or inflammation [23]. The results showed no significant differences in the percentage of inflammatory cells or in epithelial thickness between Visit 3 and Visit 5 in either the dry formulation or the moist formulation groups. However, although not statistically significant, a slight reduction in inflammatory cells was observed in the moist formulation group at Visit 5.

Given the diverse range of cell types present in the oral cavity, including those in the pulp, gingival crevicular fluid, and epithelium [24, 25], many of these cells may come into contact with substances released from snus, potentially triggering inflammation, as previously suggested by Axéll et al. [13]. Cytokines play an important role in inflammatory regulation and salivary cytokine levels have previously been associated with inflammatory oral conditions, including gingivitis, periodontitis, and oral lesions [26–29]. Elevated levels of IL-1, IL-6, IL-8, and TNF-α have also been reported in the saliva of snus users compared with non-tobacco-based controls [30]. In the present study, no significant differences were observed in total protein levels or in the pro-inflammatory cytokines IL-1β, TNF-α, or MCP-1 between Visit 3 and Visit 5. However, IL-8 levels were significantly increased in the dry formulation group. This finding was unexpected, as inflammatory cytokine levels were hypothesized to decrease following the observed clinical improvement in lesion severity. IL-8 has previously been implicated in inflammatory and tissue repair processes [31]. However, the biological significance of the observed increase remains unclear and should therefore be interpreted cautiously. In addition, a potential contribution of nicotine itself to the observed IL-8 response cannot be excluded.

Within the limitations of the present study, substitution of regular snus with non-tobacco-based nicotine pouches was associated with improvement of pre-existing snus-induced oral lesions. However, it cannot be excluded that the observed improvements may partly reflect reduced exposure to tobacco constituents or spontaneous lesion regression over time. Although the study was designed as a longitudinal within-subject comparison, the absence of a continued snus-use control group or a nicotine-abstinent control group limits the ability to draw definitive conclusions regarding causality. In addition, participants were allowed to select among available flavours and could change flavour during the intervention period. Although this approach reflected real-world product use, it cannot be excluded that flavouring agents may have influenced oral mucosal responses independently of the nicotine pouch formulation itself. The present study was not designed to evaluate flavour-specific effects. Compliance with exclusive nicotine pouch use was monitored through electronic product-use diaries completed by the participants. However, no objective measures were used to verify compliance, and therefore occasional use of tobacco-based snus or other nicotine products during the intervention period cannot be completely excluded. Furthermore, the effects observed in this study relate only to pre-existing lesions and may not be generalizable to individuals without prior tobacco or nicotine exposure. Histological analysis showed no significant differences in inflammatory cell presence or epithelial thickness between Visit 3 and Visit 5. These findings should, however, be interpreted with caution. Several biopsy samples were excluded from the analyses due to technical and alignment-related issues, reducing the number of evaluable specimens and thereby potentially limiting the ability to detect subtle tissue-level changes. Furthermore, inflammatory cell assessment was based on morphology-based quantification of total inflammatory cells in H&E-stained sections rather than immunohistochemical characterization of specific inflammatory cell populations. Consequently, more subtle changes in inflammatory cell composition or phenotype may have remained undetected despite the observed clinical improvement in lesion severity.

Analysis of saliva revealed that participants randomized to the dry formulation exhibited significantly increased IL-8 levels at Visit 5 compared with Visit 3. One limitation of the present study is that the exact cause of this increase remains unclear. The observed increase was not accompanied by significant changes in other major pro-inflammatory cytokines, suggesting that it may not reflect a broader inflammatory response. Another limitation is the relatively short duration of the study. While four weeks may not fully capture the long-term effects of non-tobacco-based nicotine pouch use, previous studies on snus-induced lesions have demonstrated that mucosal changes caused by smokeless tobacco are generally reversible within a relatively short period, often within 2–6 weeks after cessation or substitution of use [17, 32]. Therefore, a 4-week intervention period was considered sufficient to evaluate early clinical changes in lesion severity following product substitution.

Future studies including more detailed characterization of immune cell populations and local tissue expression of IL-8 are warranted to further clarify the biological mechanisms underlying lesion improvement and the observed cytokine changes.