Correlation between tooth decay and insulin resistance in normal weight males prompts a role for myo-inositol as a regenerative factor in dentistry and oral surgery: a feasibility study

Abstract

Background: In an era of precision and stratified medicine, homogeneity in population-based cohorts, stringent causative entry, and pattern analysis of datasets are key elements to investigate medical treatments. Adhering to these principles, we collected in vivo and in vitro data pointing to an insulin-sensitizing/insulin-mimetic effect of myo-inositol (MYO) relevant to cell regeneration in dentistry and oral surgery. Confirmation of this possibility was obtained by in silico analysis of the relation between in vivo and in vitro results (the so-called bed-to-benchside reverse translational approach).

Results: Fourteen subjects over the 266 screened were young adult, normal weight, euglycemic, sedentary males having normal appetite, free diet, with a regular three-times-a-day eating schedule, standard dental hygiene, and negligible malocclusion/enamel defects. Occlusal caries were detected by fluorescence videoscanning, whereas body composition and energy balance were estimated with plicometry, predictive equations, and handgrip. Statistically significant correlations (Pearson r coefficient) were found between the number of occlusal caries and anthropometric indexes predicting insulin resistance (IR) in relation to the abdominal/visceral fat mass, fat-free mass, muscular strength, and energy expenditure adjusted to the fat and muscle stores. This indicated a role for IR in affecting dentin reparative processes. Consistently, in vitro administration of MYO to HUVEC and Swiss NIH3T3 cells in concentrations corresponding to those administered in vivo to reduce IR resulted in statistically significant cell replication (ANOVA/Turkey tests), suggesting that MYO has the potential to counteract inhibitory effects of IR on dental vascular and stromal cells turnover. Finally, in in silico experiments, quantitative evaluation (WOE and information value) of a bioinformatic Clinical Outcome Pathway confirmed that in vitro trophic effects of MYO could be transferred in vivo with high predictability, providing robust credence of its efficacy for oral health.

Conclusion: Our reverse bed-to-benchside data indicate that MYO might antagonize the detrimental effects of IR on tooth decay. This provides feasibility for clinical studies on MYO as a regenerative factor in dentistry and oral surgery, including dysmetabolic/aging conditions, bone reconstruction in oral destructive/necrotic disorders, dental implants, and for empowering the efficacy of a number of tissue engineering methodologies in dentistry and oral surgery.

Keywords: body composition; caries; endothelial cell; energy balance; insulin resistance; mesenchymal stromal cell; myo-inositol; tooth decay.

FIGURE 1

Correlation between the number of occlusal caries and anthropometric indexes of IR in the total sample of young males studied. Note a direct, statistically significant correlation involving >60% of indexes related to visceral fat (BMI, WC, WHR, WtHR, and BRI), suggesting that caries are related to a likely MUHNW phenotype. ABSI, a body shape index; BMI, body mass index; BRI, body roundness index; CI, conicity index; FM, fat mass percentage after arcsin transformation; WC, waist circumference; WHR, waist-to-hip ratio; WHtR, waist-to-height ratio; NS, not statistically significant (data courtesy of Noemi Coppola, pre-doctoral fellow 2022–2023, Course of Human Nutrition Sciences, UNIPR, Parma, Italy).

FIGURE 2

Correlation between the number of occlusal caries and energy expenditure adjusted to the anthropometric indexes of IR related to visceral fat in the total sample of young males studied. Note constancy of an inverse, statistically significant correlation supporting the association of caries with a likely MUHNW phenotype. BMI, body mass index; BRI, body roundness index; REE, resting energy expenditure; TEE, total energy expenditure; WC, waist circumference (data courtesy of Noemi Coppola, pre-doctoral fellow 2022–2023, Course of Human Nutrition Sciences, UNIPR, Parma, Italy).

FIGURE 3

Correlation between the number of occlusal caries and anthropometric indexes of IR related to fat-free mass and skeletal muscle in the total sample of young males studied. Note a direct, statistically significant correlation suggesting contribution of the skeletal muscle mass to the likely MUHNW phenotype expected to lead to the recorded caries. FFMI, fat-free mass index; NGS, normalized grip strength (data courtesy of Noemi Coppola, pre-doctoral fellow 2022–2023, Course of Human Nutrition Sciences, UNIPR, Parma, Italy).

FIGURE 4

Correlation between the number of occlusal caries and energy expenditure adjusted to the anthropometric indexes of IR related to the skeletal muscle mass in the total sample of young males studied. Note constancy of an inverse, statistically significant correlation supporting the association of caries with a likely MUHNW phenotype. FFMI, fat-free mass index; REE, resting energy expenditure; TEE, total energy expenditure (data courtesy of Noemi Coppola, pre-doctoral fellow 2022–2023, Course of Human Nutrition Sciences, UNIPR, Parma, Italy).

FIGURE 5

Correlation between the number of occlusal caries and composite anthropometry-dependent IR score in the total sample of young males studied. Note a direct, statistically significant correlation supporting a role of IR in the development of caries, as expected in MUHNW subjects. ADIRs, anthropometry-dependent IR score (data courtesy of Noemi Coppola, pre-doctoral fellow 2022–2023, Course of Human Nutrition Sciences, UNIPR, Parma, Italy).

FIGURE 6

Values of kinetic parameters of human umbilical vein endothelial cells (HUVECs) in monolayer, surrogating endothelial cells of the dental pulp/oral bone. Following administration of different myo-inositol (MYO) concentrations, a statistically significant increase in cell doubling (CD) was observed at each time point (days 4, 6, and 8), suggesting a continuous recruitment of cells entering the cell cycle with all MYO amounts. Similarly, a statistically significant decrease in doubling time (DT) was detected during the first growth period (from days 4–6) with all MYO concentrations. In contrast, no statistically significant changes occurred to DT during the second growth period (from days 6–8), pointing to an initial spurt in cell growth velocity, which is expectedly stabilized by cell crowding and space-dependent constraints in the monolayer. Inverted light microscopy revealed that cells maintained constant spindle/polygonal morphology and integrity of the nuclear shape and structure for all 8 days of culture and with all MYO concentrations, suggesting the stability of the vascular endothelial phenotype. As a representative example of this morphological constancy independent on increased cell crowding in the cultures with MYO, we here show cell morphologies at day 8 (A) = control, (B) = 40 microM MYO, and (C) = 640 microM MYO (insets show enlargements of cell types present in each culture). Microscopy magnification ×5, marker = 100 microns. Each kinetic value and light microscopic image represents the average of four different experiments. Ctrl = control; * = p < 0.05 vs 4 days; ° = p < 0.05 vs 4 days; § = p < 0.05 vs 6 days (data courtesy of Sara Maioli, pre-doctoral fellow 2019–2020, Course of Medical, Veterinary, and Pharmaceutical Biotechnologies, UNIPR, Parma, Italy).

FIGURE 7

Effect of MYO administration to HUVECs in monolayer for 8 days. A statistically significant increase in DNA content of the culture occurred at day 8, with all but one (80 microM) MYO concentrations in respect to untreated controls. * = p < 0.05 treated vs untreated sample; NS = not statistically significant (data courtesy of Sara Maioli, pre-doctoral fellow 2019–2020, Course of Medical, Veterinary, and Pharmaceutical Biotechnologies, UNIPR, Parma, Italy).

FIGURE 8

Values of kinetic parameters of mouse embryonic fibroblasts (Swiss NIH3T3) in monolayer, surrogating fibroblasts and mesenchymal stromal cells (MSCs) of the dental pulp/oral bone. Following administration of different MYO concentrations for 6 days, a statistically significant increase in CD was observed at each time point (days 2, 4, and 6) with all MYO concentrations, suggesting a continuous recruitment of cells entering the cell cycle. Similarly, a statistically significant increase in DT was detected during the entire period of culture (days 2, 4, and 6), pointing to a progressive deceleration in cell growth, an expected constraint from cell crowding in the monolayer. At day 6 of culture, inverted light microscopy revealed that control and MYO-stimulated embryonic fibroblasts gave rise to a very similar dense pavement of adherent elongated cells, suggesting the stability of their fibroblastic/MSCs-like phenotype for the entire period of culture; (A) = control and (B) = 80 microM MYO. Microscopy magnification ×5, marker = 100 microns. Each kinetic value and light microscopic image represents the average of four different experiments. Ctrl = control; * = p < 0.05 vs 4 days; ° = p < 0.05 vs 4 days; § = p < 0.05 vs 6 days (data courtesy of Sara Maioli, pre-doctoral fellow 2019–2020, Course of Medical, Veterinary, and Pharmaceutical Biotechnologies, UNIPR, Parma, Italy).

FIGURE 9

Effect of MYO administration to Swiss NIH3T3 cells in monolayer for 6 days. A statistically significant increase in DNA content of the culture occurred at day 6 with 80 microM MYO concentration in respect to untreated controls. * = p < 0.05 treated vs untreated sample; NS = not statistically significant (data courtesy of Sara Maioli, pre-doctoral fellow 2019–2020, Course of Medical, Veterinary, and Pharmaceutical Biotechnologies, UNIPR, Parma, Italy).

FIGURE 10

Flowchart of a bottom-up theoretical linkage or clinical outcome pathway (COP) for trophic/regenerative actions of MYO, bridging in vitro data to in vivo human evidence. Based on ad hoc selection of the international literature (75 publications), the knowledge chain was constructed using a procedure similar to that for adverse outcome pathways in toxicology (see at Wiki site https://aopwiki.org/). Strength of prediction in the ensuing bioinformatic linkage was provided by the weight of evidence (WOE) and information value (I.V.) expressing a rank of the categorical variable detailed inside each node (the descriptor of the MYO action at that level), and it is reported in the left table below the graph. Note that cumulated I.V. for MYO trophic actions was >0.5, indicating a very high predictive power of the cause–effect relations between the molecular initiation event (MIE) and subsequent measurable responses of MYO labeled as key events (KE) up to the final in vivo real-world evidence or clinical outcome (CO). Current experimental results contributed to the rank values of the categorical variables. A further threshold of credibility of the COP was provided by the spanning of cause–effect links through different levels of biological organization and taxonomic scale up to Homo sapiens, as summarized in the right table below the graph (data courtesy of Sara Maioli, pre-doctoral fellow 2019–2020, Course of Medical, Veterinary, and Pharmaceutical Biotechnologies, UNIPR, Parma, Italy).