Crown: Journal of Dentistry and Health Research

CRISPRi-Mediated Repression of gtfB Attenuates Streptococcus mutans Virulence and Promotes Ecological Homeostasis in a Preclinical Cariogenic Biofilm Model

Khairiel Anwar — 2025-10-13

Introduction: Streptococcus mutans is a primary etiological agent of dental caries, largely due to its capacity to form robust, acidogenic biofilms. This virulence is critically dependent on glucosyltransferases, particularly GtfB, which synthesizes the adhesive extracellular glucan matrix. Conventional antimicrobial strategies often lack specificity, leading to oral dysbiosis. This study aimed to develop and evaluate a highly targeted CRISPR interference (CRISPRi) system to silence the gtfB gene in S. mutans, thereby inhibiting its cariogenic potential without adversely affecting the viability of key oral commensal species.

Methods: A CRISPRi system, comprising a nuclease-deactivated Cas9 (dCas9) and a single guide RNA (sgRNA) targeting the gtfB promoter, was engineered into S. mutans UA159. The efficacy of gtfB silencing was quantified via qRT-PCR. The consequential effects on bacterial growth kinetics, insoluble glucan synthesis, and single-species biofilm formation were assessed using spectrophotometry, anthrone assays, crystal violet staining, and confocal laser scanning microscopy (CLSM). The ecological impact was investigated in a multi-species biofilm model containing S. mutans and the commensal bacteria Streptococcus gordonii, Streptococcus oralis, and Actinomyces naeslundii, with microbial composition analyzed by species-specific qPCR. All research activities were conducted in Indonesia.

Results: The CRISPRi system induced a profound and specific downregulation of gtfB mRNA expression by over 98% (p<0.001) in the engineered S. mutans strain compared to the wild-type. This silencing did not impair bacterial planktonic growth. However, it led to a significant reduction in insoluble glucan production by 85% (p<0.001) and a corresponding 79% decrease in total biofilm biomass (p<0.001). CLSM imaging confirmed the formation of structurally deficient biofilms with minimal extracellular matrix. In the multi-species model, repression of S. mutans virulence significantly altered the biofilm ecology, resulting in a 65% reduction in the proportional abundance of S. mutans and a concomitant increase in the representation of commensal species, thereby fostering a community structure more aligned with oral health.

Conclusion: Targeted repression of the gtfB gene using a CRISPRi-based approach effectively 'disarms' S. mutans, neutralizing its primary cariogenic mechanism without being bactericidal. This strategy not only attenuates its virulence but also shifts the ecological balance in favor of beneficial commensal bacteria. These findings underscore the therapeutic potential of gene-targeted virulence modulation as a precise, ecologically-sound strategy for the prevention and treatment of dental caries.

Clinical and Inflammatory Efficacy of a Novel Bioactive Borate Glass Air-Abrasion Powder for Peri-implant Mucositis Treatment: A Split-Mouth Randomized Controlled Clinical Trial

Nur Diana — 2025-10-14

Introduction: Peri-implant mucositis (PIM) is a prevalent inflammatory condition requiring effective biofilm management. This study aimed to evaluate the clinical and inflammatory efficacy of a novel bioactive borate glass (BBG) air-abrasion powder compared to a standard glycine-based powder for treating PIM.

Methods: This was a split-mouth randomized controlled trial conducted at the Palembang, Indonesia. Forty-two patients with two implants each, both diagnosed with PIM (Bleeding on Probing [BOP] positive, Probing Pocket Depth [PPD] 4 mm), were enrolled. In each patient, one implant site was randomly assigned to receive sub- and supragingival air-abrasion with the BBG powder (Test Group), while the contralateral implant received treatment with glycine powder (Control Group). Clinical parameters, including Modified Plaque Index (mPI), Modified Gingival Index (mGI), PPD, and BOP, were recorded at baseline (T0), 4 weeks (T1), and 12 weeks (T2). Peri-implant sulcular fluid (PISF) was collected to quantify levels of Interleukin-1 Beta (IL-1β) and Tumor Necrosis Factor-Alpha (TNF-α). Patient-reported discomfort was assessed using a Visual Analog Scale (VAS).

Results: Both groups showed significant improvements in all clinical parameters from T0 to T2 (p < 0.001). At the 12-week follow-up (T2), the Test group demonstrated a statistically significant greater reduction in mean PPD (Test: mm vs. Control: mm; p < 0.001) and a higher percentage of BOP resolution (Test: 88.1% vs. Control: 66.7%; p = 0.012). Furthermore, the reduction in IL-1β and TNF-α concentrations from T0 to T2 was significantly greater in the BBG group (p < 0.01 for both). Both treatments were well-tolerated with low VAS scores.

Conclusion: Within the limitations of this study, non-surgical treatment of peri-implant mucositis using the novel bioactive borate glass air-abrasion powder resulted in superior clinical and inflammatory outcomes compared to standard glycine powder. This bioactive approach presents a promising advancement in peri-implant maintenance therapy.

In Vitro and In Vivo Efficacy of a Novel Strontium-Doped Bioactive Glass Hydrogel for Dentin-Pulp Complex Regeneration

Rinna Azrida — 2025-10-14

Introduction: The regeneration of the dentin-pulp complex is a major challenge in vital pulp therapy. This study aimed to develop and evaluate a novel injectable hydrogel composed of strontium-doped bioactive glass (Sr-BG) in a methacrylated gelatin (GelMA) matrix to promote dentin-pulp complex regeneration. Strontium was added for its dual therapeutic effects of enhancing odontogenic differentiation and inhibiting bacterial activity.

Methods: We synthesized Sr-BG nanoparticles using a sol-gel method and characterized them with X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The nanoparticles were then incorporated into a GelMA hydrogel. We assessed the material's physical properties, including its swelling ratio, degradation rate, and ion release profiles (Si, Ca, P, Sr). We also evaluated its in vitro biocompatibility and odontogenic potential using human dental pulp stem cells (hDPSCs), assessing cell viability (MTT assay), alkaline phosphatase (ALP) activity, and the expression of odontogenic markers (DSPP, DMP-1, RUNX2) via RT-qPCR. We tested its antibacterial properties against Streptococcus mutans. For the in vivo evaluation, the hydrogel was used as a pulp capping agent in the mechanically exposed molars of Wistar rats. After 4 and 8 weeks, we assessed tissue regeneration using histological analysis (H&E and Masson's trichrome staining) and micro-computed tomography (micro-CT).

Results: The synthesized Sr-BG nanoparticles were amorphous with a particle size of about 80-120 nm. The Sr-BG/GelMA hydrogel exhibited controlled swelling and degradation, along with a sustained release of therapeutic ions. In vitro, the hydrogel demonstrated excellent biocompatibility and significantly upregulated ALP activity and the expression of DSPP, DMP-1, and RUNX2 in hDPSCs compared to the control group (p < 0.05). The material also showed significant antibacterial activity against S. mutans. In vivo, histological analysis revealed the formation of a thick, continuous, and well-organized tertiary dentin bridge with minimal inflammation in the Sr-BG/GelMA group at 8 weeks. Micro-CT analysis confirmed a significantly greater volume and density of newly formed mineralized tissue compared to control groups treated with calcium hydroxide.

Conclusion: The novel strontium-doped bioactive glass hydrogel showed significant potential for dentin-pulp complex regeneration. Its combined osteoinductive, angiogenic, and antibacterial properties make it a promising biomaterial for advanced vital pulp therapy, offering a superior alternative to traditional pulp capping agents.

Metatranscriptomic Profiling of the Subgingival Microbiome in Peri-implantitis versus Healthy Implants: Identifying Key Dysbiotic Pathways

Rheina Weisch Fedre — 2025-10-14

Introduction: Peri-implantitis is a primary cause of dental implant failure, characterized by inflammatory destruction of supporting tissues. While microbial dysbiosis is implicated, the functional activities of the subgingival microbiome that drive disease pathogenesis remain poorly understood. This study aimed to elucidate the key functional and metabolic shifts in the subgingival microbiome associated with peri-implantitis using metatranscriptomic analysis.

Methods: This cross-sectional study involved twenty patients, ten with healthy implants (HI) and ten diagnosed with peri-implantitis (PI). Subgingival biofilm samples were collected from the deepest peri-implant sulcus of each subject. Total RNA was extracted, followed by library preparation and sequencing on an Illumina NovaSeq platform. Bioinformatic analysis included quality control, taxonomic profiling using Kraken2, and functional annotation against the KEGG and Gene Ontology databases. Differential gene expression analysis was performed using DESeq2 to identify microbial transcriptional signatures distinguishing the PI and HI groups.

Results: The metatranscriptome of the PI group exhibited significantly higher microbial diversity and a distinct taxonomic composition, with a notable enrichment of transcripts from species such as Porphyrononas gingivalis, Tannerella forsythia, and Fusobacterium nucleatum. In contrast, the HI group was dominated by transcripts from commensal streptococci. Functional analysis revealed a significant upregulation of pathways related to bacterial virulence, including lipopolysaccharide (LPS) biosynthesis, bacterial secretion systems (Type IV), and iron acquisition in the PI group. Furthermore, pathways associated with amino acid metabolism, particularly arginine and tryptophan degradation, were highly active, suggesting a proteolytic environment. Conversely, the HI metatranscriptome showed enrichment in carbohydrate metabolism and fermentation pathways.

Conclusions: The subgingival microbiome in peri-implantitis is not only taxonomically distinct but also functionally primed for pathogenicity. The active transcription of genes related to virulence, inflammation induction, and proteolytic metabolism highlights the key dysbiotic pathways that likely contribute to tissue destruction. These findings provide a deeper understanding of the functional gene expression profile in peri-implantitis and suggest potential targets for future diagnostic and therapeutic strategies aimed at modulating microbial activity rather than merely eliminating specific taxa.

Three-Year Clinical Performance of Silver Diamine Fluoride (SDF) versus Glass Ionomer Cement in Arresting Carious Lesions in Primary Molars: A Community-Based Cohort Study

Winata Putri — 2025-10-15

Introduction: Early Childhood Caries (ECC) is a significant global health problem. Minimally invasive treatments like Silver Diamine Fluoride (SDF) and Glass Ionomer Cement (GIC) are crucial, but long-term comparative effectiveness data from real-world community settings are scarce. This study aimed to compare the three-year clinical performance of 38% SDF versus high-viscosity GIC in arresting active carious lesions in the primary molars of preschool children.

Methods: This study was designed as a three-year, prospective, non-randomized, community-based cohort study in an underserved urban population in South Sumatra, Indonesia. A total of 450 children aged 3-5 years with at least one active cavitated carious lesion (ICDAS 5/6) in a primary molar were enrolled. Following parental consent and choice, lesions were treated with either a single application of 38% SDF or a high-viscosity GIC restoration using the Atraumatic Restorative Treatment (ART) technique. Calibrated examiners assessed the lesions for caries arrest at 6, 12, 24, and 36 months using standardized visual-tactile criteria. The primary outcome was the proportion of arrested lesions. Survival analysis was performed using Kaplan-Meier curves and a Cox proportional hazards model.

Results: A total of 620 lesions (309 SDF, 311 GIC) were treated and followed. At the 36-month follow-up, the caries arrest rate in the SDF group was 81.2%, which was significantly higher than the 64.8% arrest rate observed in the GIC group (χ² = 24.5, p < 0.001). The Kaplan-Meier survival analysis demonstrated a significantly higher probability of lesions remaining in an arrested state in the SDF group over the three-year period (log-rank test, p < 0.001). The Cox regression model identified the treatment modality as the primary predictor of failure, with GIC having a hazard ratio of 2.15 (95% CI: 1.55-2.98) compared to SDF.

Conclusion: Within the parameters of this community-based cohort study, a single application of 38% SDF was significantly more effective in arresting active carious lesions in primary molars over a three-year period than high-viscosity GIC applied via the ART technique. These findings support the prioritization of SDF in public health programs for managing ECC.