Scanning electron microscopic analysis of adherent bacterial biofilms associated with peri-implantitis.

OBJECTIVES: Peri-implantitis (PI) is caused by bacteria in the peri-implant space but the consensus on microbial profile is still lacking. Current microbial sampling of PI lesions has largely focused on analyzing bacterial species that have been shed from the implant surface and captured in the pocket fluid. The purpose of the present study was to investigate the morphotypes of bacteria in biofilm covering the implant threads and explore whether certain morphotypes were associated with PI. METHODS: Fourteen failed implants were removed and instantly processed for scanning electron microscope analysis. The implants were imaged at three equally divided sub-crestal levels of the exposed area. Bacterial morphotypes were identified and quantified by three examiners. Mobility and years in function were correlated to the presence of different morphotypes. RESULTS: The implants demonstrated the presence of variable bacterial morphotypes that did not correlate to disease progression in our study. Some implants were dominated by filaments and others showed the presence of combinations of cocci/rods or spirilles/spirochetes. In general, all implants showed variable morphologic biofilm composition. However, individual implants tended to have similar composition throughout the entire implant. Rods and filaments were dominant morphotypes throughout the surfaces and cocci showed increased presence toward the apical third. There were some differences in the biofilm morphology with mobility and time in function. CONCLUSIONS: The profiles of bacterial biofilm morphotypes in failing implants with similar clinical presentations were highly variable. While there were significant differences between implants, similar morphotypes in individual implants were often found throughout the entire surface.

Effect of Air Polishing on the Treatment of Peri-Implant Diseases: A Systematic Review and Meta-Analysis.

Peri-implant diseases have become one of the notable biological complications of postrehabilitation with implant-supported restorations. Effective modalities for decontamination of biofilm deposits around implant surfaces are critical for resolution of the inflammation. Air polishing is one of the recommended clinical methods for treating peri-implant diseases. This systematic review assessed clinical evidence on efficacy of using air polishing technology for the management of peri-implant diseases, including peri-implant mucositis and peri-implantitis. Four electronic databases from January 1990 to December 2022 were searched to identify the relative human randomized clinical trials that applied air polishing for nonsurgical and surgical treatment of peri-implant mucositis and peri-implantitis. Twelve articles were selected. For treating peri-implant mucositis, air polishing showed a comparable effect to ultrasonic scaling in the reduction of bleeding on probing (BOP) and probing pocket depth (PPD). The nonsurgical approach of air polishing in treating peri-implantitis varied in the reduction of BOP, PPD, and clinical attachment level (CAL) in evaluated studies. Air polishing in the surgical treatment of peri-implantitis was comparable to mechanical cleaning, implantoplasty, and the use of Ti-brush, in regards to the significant reduction of BOP, PPD, and CAL, as well as the improvement of the bone level between baseline and follow-ups. The standardized mean difference with a 95% confidence interval of the studied parameters was estimated using the random effect model; however, statistical differences were not detected between air polishing and comparative modalities in the treatment of peri-implantitis. Within the limitations of this review, the application of air polishing did not result in more favorable outcomes in the treatment of peri-implant diseases compared to other modalities.

Gingival epithelial cell-derived microvesicles activate mineralization in gingival fibroblasts.

Soft tissue calcification occurs in many parts of the body, including the gingival tissue. Epithelial cell-derived MVs can control many functions in fibroblasts but their role in regulating mineralization has not been explored. We hypothesized that microvesicles (MVs) derived from gingival epithelial cells could regulate calcification of gingival fibroblast cultures in osteogenic environment. Human gingival fibroblasts (HGFs) were cultured in osteogenic differentiation medium with or without human gingival epithelial cell-derived MV stimulation. Mineralization of the cultures, localization of the MVs and mineral deposits in the HGF cultures were assessed. Gene expression changes associated with MV exposure were analyzed using gene expression profiling and real-time qPCR. Within a week of exposure, epithelial MVs stimulated robust mineralization of HGF cultures that was further enhanced by four weeks. The MVs taken up by the HGF's did not calcify themselves but induced intracellular accumulation of minerals. HGF gene expression profiling after short exposure to MVs demonstrated relative dominance of inflammation-related genes that showed increases in gene expression. In later cultures, OSX, BSP and MMPs were significantly upregulated by the MVs. These results suggest for the first time that epithelial cells maybe associated with the ectopic mineralization process often observed in the soft tissues.

Altered composition of the oral microbiome in integrin beta 6-deficient mouse.

In periodontal disease (PD), bacterial biofilms suppress ß6 integrin expression transforming growth factor-ß1 signaling, resulting in gingival inflammation and bone loss. ß6 integrin-null (Itgb6-/- ) mice develop spontaneous PD. The aim of this study was to unravel potential differences in oral microbiome in wild-type (WT) and Itgb6-/- FVB mice. Mouse oral microbiome was analyzed from 3- and 6-month-old WT and Itgb6-/- . The periodontal inflammation and spontaneous bone loss were present in 3-month-old and advanced in 6-month-old Itgb6-/- mice. The observed amplicon sequence variants (ASVs) of alpha diversity showed close similarity in 3-month-old and 6-month-old Itgb6-/- mice. Chao1 and ACE methods revealed that the microbiome in Itgb6-/- mice showed less diversity compared to the WT. UniFrac Principal Coordinate analyses (PCoA) showed a clear spatial segregation and clustering between Itgb6-/- and WT mice in general, and between 3-month- and 6-month-old WT mice. Weighted PCoA showed the tight clustering and distinct separation of individual mouse samples within Itgb6-/- and WT. The most abundant microbial classes varied between the sample groups. However, the genus Aggregatibacter significantly increased in the 6-month-old Itgb6-/- mice. ß6 integrin-deficient mice develop periodontal inflammation that may relate to dysbiosis in the microbiome that further promotes the disease process.

Decontamination of multispecies oral biofilm from rough implant surface by airflow with glycine.

OBJECTIVES: Decontamination of biofilm-colonized rough implant surfaces remains challenging. We investigated the effect of airflow with glycine powder (AFG) on decontamination of mature oral multispecies biofilm from a sandblasted and acid etched (SLA) titanium surface. MATERIALS AND METHODS: Subgingival dental plaque was cultured on SLA disks anaerobically for 21 days. AFG with various settings and distances was applied directly on the disks with or without previous rinse of 0.9% NaCl. The specimens were then analyzed through scanning electron microscope and remaining bacteria on the implant surface were quantified and statistically compared. RESULTS: Mature oral biofilm with cocci and rods as major morphotypes, as well as spiral- and filamentous-shaped organisms, was formed on the untreated disks. Saline rinsing removed the thick biofilm layer but left numerous of coccoid bacteria in rough surface pits. AFG effectively removed most of the bacteria from the pits. Both 25% and 50% power settings were equally effective at 3-mm distance. With 50% power, AFG successfully removed bacteria at both 3- and 6-mm distance. When AFG was applied on native biofilm without prior rinsing with saline, it effectively removed the biofilm including bacteria in the pits. CONCLUSION: Application of AFG appears effective in removing bacteria from rough implant surfaces.

Diagnostic and prognostic value of Beclin 1 expression in melanoma: a meta-analysis.

Autophagy plays a complicated role in the occurrence and development of cancer. Beclin 1 is a significant autophagy-related protein that plays an essential role in tumorigenesis, but its expression is controversial in melanoma. In this meta-analysis, we searched seven studies involving 638 melanoma patients. PubMed, Web of Science, Google Scholar, Elsevier, and Chinese National Knowledge Infrastructure were used for literature retrieval. The I2 index was used to assess heterogeneity. The expression of Beclin 1 in the primary melanoma group was significantly lower than the non-tumor group tissues (P < 0.01), while higher than the metastatic melanoma group (P < 0.01). Beclin 1 expression status could not distinguish between patients with melanoma by sex (male vs. female), lymph node metastasis (metastasis vs. non-metastasis), melanin deposition (present vs. absent), ulcer formation (present vs. absent), tumor necrosis status (present vs. absent), and Breslow thickness (<1.5 mm vs. ≥1.5 mm) for the subgroups (all P values > 0.05). Different expression intensities of Beclin 1 did not affect the overall survival and disease-free survival of melanoma patients. This study showed a trend of low expression of Beclin 1 in melanoma; patients with low expression of Beclin 1 were prone to the possibility of distant metastasis. The inconsistent profile of Beclin 1 expression in the prognosis of melanoma patients warrants further clinical investigation.

Mechanism of bone marrow mesenchymal stem cells secreting miR-26a exosomes affecting high glucose-induced skin fibroblasts function by regulating TLR4/NF-κB signaling.

OBJECTIVE: The aim of this study was to investigate the molecular mechanism of human bone marrow mesenchymal stem cells (hMSCs) secreting miR-26a exosomes on the function of skin fibroblasts. METHODS: Exosomes from hMSCs were extracted and identified by transmission electron microscopy, particle size was analyzed and protein markers were detected. Then, the exosomes were co-cultured with human skin fibroblasts (BJ). CCK-8, Annexin V/P and Transwell assays were used to detect the proliferation, apoptosis, and migration of BJ cells. In addition, the expressions of miR-26a, related proteins, and related inflammatory factors were detected by qRT-PCR, western blotting, and ELISA. RESULTS: Compared with the high glucose group, the proliferation rate, migration rate, and the expression of α-SMA, bcl-2, TLR4, NF-κB, TNF-α, IL-6, IL- and IL-1 were significantly decreased in the high glucose + MSC-Exo-miR-26a mimics group, while the apoptosis rate and the expression of miR-26a, cleaved-caspase 3, cleaved-caspase 9 and Bax were significantly increased. The results of the high glucose + MSC-Exo-miR-26a inhibitor group were the opposite. CONCLUSION: These results suggest that hMSCs cells secreting miR-26a exosomes inhibited the proliferation, migration, and transdifferentiation of high glucose-induced BJ cells, and promoted cell apoptosis, which may be related to the TLR4/NF-κB signaling pathway.

Decontamination of rough implant surfaces colonized by multispecies oral biofilm by application of leukocyte- and platelet-rich fibrin.

BACKGROUND: Decontamination of biofilm-infected rough implant surfaces is challenging. Platelet rich blood products have been shown to have anti-microbial properties against periodontal pathogens. Our aim was to investigate the effect of a potential biological implant surface disinfectant, leukocyte- and platelet-rich fibrin (L-PRF), on a mature oral multispecies biofilm on a rough titanium surface. METHODS: Sandblasted, large grit, acid-etched (SLA) titanium disks were inoculated with subgingival dental plaque and cultured anaerobically for 21 days. The L-PRF membranes were collected from 12 donors in three trials (four donors in each trial). The disks were rinsed with 0.9% NaCl and exposed to the cell-rich portion of the L-PRF membranes for 48 hours followed by scanning electron microscope (SEM) analysis immediately or after rinsing with 0.9% NaCl prior to fixation. The presence of platelet factor-4 in the rinse samples was analyzed by Western blotting. Remaining bacteria were quantified from SEM images of the implant surfaces and their numbers statistically compared. RESULTS: The L-PRF-treated samples without rinsing displayed numerous cells with multiple pseudopodia in immediate contact with bacteria that appeared perforated and increased in size. The cells were identified as platelets based on morphological criteria and by positive reaction for platelet factor-4 by Western blotting. After post-treatment rinsing, the L-PRF-treated disks displayed a significant reduction in bacterial counts (in average 92% reduction). CONCLUSION: Application of L-PRF significantly reduced bacterial counts on contaminated SLA titanium surface, most likely through anti-microbial action by platelets.

Leucocyte- and platelet-rich fibrin regulates expression of genes related to early wound healing in human gingival fibroblasts.

BACKGROUND: Leucocyte- and platelet-rich fibrin (L-PRF) is a blood-derived biomaterial rich in leucocytes and platelets embedded in a high-density fibrin network that can be compressed into a membrane and used in surgical applications to stimulate tissue regeneration and wound healing, especially in oral cavity. This study aimed to determine the combined effects of the growth factors and cells present in L-PRF on fibroblasts that directly face the L-PRF membranes placed during surgical procedures. METHODS: The effect of L-PRF from six donors on the expression of 84 key wound healing genes in normal human gingival fibroblasts was tested by RT-qPCR. RESULTS: L-PRF significantly regulated the expression of 33 fibroblast genes (39%), including interleukins, myofibroblast-, extracellular matrix- and angiogenesis-associated genes, and matrix metalloproteinase-1 and -3. L-PRF regulated fibroblast gene expression both time- and dose-dependently, and the effects were mediated by mitogen-activated protein kinases ERK1/2, JNK and p38. L-PRF also stimulated fibroblast wound closure and promoted the ability of fibroblasts to induce endothelial tube formation. L-PRF-induced gene expression changes in fibroblast were similar to those observed in early human and pig wounds. CONCLUSIONS: This study provides new insights into the biological mechanism by which L-PRF regulates key gingival fibroblast functions important in wound healing.

Epidermal growth factor receptor signaling suppresses αvß6 integrin and promotes periodontal inflammation and bone loss.

In periodontal disease (PD), bacterial biofilms cause gingival inflammation, leading to bone loss. In healthy individuals, αvß6 integrin in junctional epithelium maintains anti-inflammatory transforming growth factor-ß1 (TGF-ß1) signaling, whereas its expression is lost in individuals with PD. Bacterial biofilms suppress ß6 integrin expression in cultured gingival epithelial cells (GECs) by attenuating TGF-ß1 signaling, leading to an enhanced pro-inflammatory response. In the present study, we show that GEC exposure to biofilms induced activation of mitogen-activated protein kinases and epidermal growth factor receptor (EGFR). Inhibition of EGFR and ERK stunted both the biofilm-induced ITGB6 suppression and IL1B stimulation. Furthermore, biofilm induced the expression of endogenous EGFR ligands that suppressed ITGB6 and stimulated IL1B expression, indicating that the effects of the biofilm were mediated by autocrine EGFR signaling. Biofilm and EGFR ligands induced inhibitory phosphorylation of the TGF-ß1 signaling mediator Smad3 at S208. Overexpression of a phosphorylation-defective mutant of Smad3 (S208A) reduced the ß6 integrin suppression. Furthermore, inhibition of EGFR signaling significantly reduced bone loss and inflammation in an experimental PD model. Thus, EGFR inhibition may provide a target for clinical therapies to prevent inflammation and bone loss in PD.

Human keratinocyte-derived microvesicle miRNA-21 promotes skin wound healing in diabetic rats through facilitating fibroblast function and angiogenesis.

Skin wound healing is a complex physiological process that maintains the integrity of the skin tissues, involving a variety of distinct cell types and signaling molecules. The specific signaling pathways or extracellular cues that govern the healing processes remain elusive. Microvesicles (MVs) have recently emerged as critical mediators of cell communication by delivery of genetic materials to target cells. In this study, we found the direct delivery of HEKa-MVs expressing miR-21 mimics significantly promoted the healing of skin wound in diabetic rats. In-depth studies showed that MV miR-21 promoted fibroblast migration, differentiation, and contraction, induced a pro-angiogenic process of endothelial cells and mediated a pro-inflammatory response. Mechanically, MV miR-21 might target specific essential effector mRNA in fibroblasts such as MMP-1, MMP-3, TIMP3, and TIMP4 to increase MMPs expression and enzymatic activities. Moreover, MV miR-21 regulated ɑ-SMA and N-cadherin to induce fibroblast-myofibroblast differentiation. MV miR-21 up-regulated the IL-6 and IL-8 expressions and their secretion to amplify the immune response. Furthermore, MV miR-21 down-regulated PTEN and RECK in protein level, and activate MAPK/ERK signaling cascade, thereby promoting fibroblast functions. Thus, our study has provided for the first time the basis for the potential application of HEKa-MVs, and MV miR-21 in particular for wound healing.

Inflammasome and cytokine expression profiling in experimental periodontitis in the integrin ß6 null mouse.

Epithelial αvß6 integrin participates in immune surveillance in many organs, including the gastrointestinal track. Expression of αvß6 integrin is reduced in the junctional epithelium of the gingiva in periodontal diseases, and mutations in the ITGB6 gene are associated with these diseases in humans and mice. The aim of this study was to unravel potential differences in the inflammatory responses in the periodontal tissues of FVB wild-type (WT) and ß6 integrin-null (Itgb6-/-) mice, using a ligature-induced periodontitis model and assessing inflammation, bone loss and expression profiles of 34 genes associated with periodontal disease. Using micro-CT and histology, we demonstrated more advanced inflammation and bone loss in the control and ligatured Itgb6-/- mice compared to the WT animals. Neutrophil and macrophage marker genes were significantly upregulated by ligation in both WT and Itgb6-/- mice while the expression of T-cell and B-cell markers was downregulated, suggesting acute-type of inflammation. Expression of inflammasome NLRP3-related genes Nlpr3 and Il1b was also significantly increased in both groups. However, the expression of Il18 was significantly lower in non-ligatured Itgb6-/- mice than in the WT mice and was further downregulated in both groups by the ligatures. IL-18 mediates many effects of the AIM2 inflammasome, including regulation of the microbiome. Interestingly, expression of Aim2 was significantly lower in both control and ligatured Itgb6-/- mice than in WT animals. Overall, ligature-induced periodontitis was associated with increased expression of pro-inflammatory cytokines, chemokines and osteoclastogenic regulatory molecules. Another significant difference between the Itgb6-/- and WT mice was that mRNA expression of the anti-inflammatory cytokine IL-10 was increased in ligatured WT mice but reduced in the Itgb6-/- mice. In conclusion, αvß6 integrin in junctional epithelium of the gingiva appears to positively regulate the expression of the AIM2 inflammasome and anti-inflammatory IL-10, thus providing protection against periodontal inflammation.

Integrin αvß6: Structure, function and role in health and disease.

Integrins are cell surface receptors that traditionally mediate cell-to-extracellular matrix and cell-to-cell adhesion. They can, however, also bind a large repertoire of other molecules. Integrin αvß6 is exclusively expressed in epithelial cells where it can, for example, serve as a fibronectin receptor. However, its hallmark function is to activate transforming growth factor-ß1 (TGF-ß1) to modulate innate immune surveillance in lungs and skin and along the gastrointestinal tract, and to maintain epithelial stem cell quiescence. The loss of αvß6 integrin function in mice and humans leads to an altered immune response in lungs and skin, amelogenesis imperfecta, periodontal disease and, in some cases, alopecia. Elevated αvß6 integrin expression and aberrant TGF-ß1 activation and function are associated with organ fibrosis and cancer. Therefore, αvß6 integrin serves as an attractive target for cancer imaging and for fibrosis and cancer therapy.

Suppression of αvß6 Integrin Expression by Polymicrobial Oral Biofilms in Gingival Epithelial Cells.

Periodontal diseases manifest by the formation of deep pockets between the gingiva and teeth where multispecies bacterial biofilms flourish, causing inflammation and bone loss. Epithelial cell receptor αvß6 integrin that regulates inflammation by activating the anti-inflammatory cytokine transforming growth factor-ß1, is highly expressed in healthy junctional epithelium that connects the gingiva to the tooth enamel. However, its expression is attenuated in human periodontal disease. Moreover, Itgb6 -/- mice display increased periodontal inflammation compared to wild-type mice. We hypothesized that bacterial biofilms present in the periodontal pockets suppress αvß6 integrin levels in periodontal disease and that this change aggravates inflammation. To this end, we generated three-week-old multi-species oral biofilms in vitro and treated cultured gingival epithelial cells (GECs) with their extracts. The biofilm extracts caused suppression of ß6 integrin expression and upregulation of pro-inflammatory cytokines, including interleukin-1ß and -6. Furthermore, GECs with ß6 integrin siRNA knockdown showed increased interleukin-1ß expression, indicating that αvß6 integrin-deficiency is associated with pro-inflammatory cytokine responsiveness. FSL-1, a synthetic bacterial lipopeptide, also suppressed ß6 integrin expression in GECs. Therefore, biofilm components, including lipopeptides, may downregulate αvß6 integrin expression in the pocket epithelium and thus promote epithelial cell-driven pro-inflammatory response in periodontal disease.

Chemotherapeutic decontamination of dental implants colonized by mature multispecies oral biofilm.

AIM: No studies have tested disinfectants on mature multispecies oral biofilms on titanium substrata. The aim of this study was to investigate the efficacy of commonly used antimicrobial agents in decontamination of multispecies mature oral biofilm on sandblasted, large-grit, acid-etched (SLA) titanium implants. METHODS: SLA titanium disks were inoculated with dental plaque and cultured anaerobically for 21 days. The disks were rinsed with 0.9% NaCl, exposed for 2 min. to tetracycline paste, 1% Chlorhexidine gel (CHX), 35% phosphoric acid gel (Etch) or a novel chemical formula (0.3% cetrimide, 0.1% CHX and 0.5% EDTA) and then rinsed again with 0.9% NaCl. Bacteria were quantified from scanning electron micrographs of the implant surfaces. Living bacteria were quantified with confocal laser scanning microscopy (CLSM). RESULTS AND CONCLUSIONS: Rinsing the surfaces with 0.9% NaCl removed the majority of the biofilm. However, bacteria persisted in all specimens and none of the disinfectants was superior to the double saline rinse group. CLSM analysis showed that CHX and Etch groups had a statistically significant reduction of viable bacteria, although small. Overall the results show that many disinfection agents used in the clinic are ineffective in biofilm removal and leave live bacteria on the surface.

Sterilization of Ceramic Sharpening Stones.

Traditionally, periodontal hand instruments are honed or sharpened during patient care as they dull easily during contact with enamel, calculus and cementum. This approach is taught in dental and hygiene schools around the world and remains the standard of care. Recently, some professional organizations have questioned whether this practice should be abandoned because of safety issues. Questions have been raised whether sharpening stones can be properly sterilized and whether the sharpening of contaminated instruments poses a health hazard for the provider. Using bacteria culture techniques and scanning electron microscopy, we tested whether contaminated ceramic sharpening stones can be sterilized. Our results demonstrate that the stones were sterile after being subjected to the manufacturer's sterilization protocol. In addition, over the last year, no incidents related to periodontal instrument sharpening have been reported among nearly 400 students at the faculty of dentistry, University of British Columbia, where chair-side sharpening is taught. Therefore, we conclude that ceramic sharpening stones can be sterilized using normal office protocols and that chair-side sharpening adds little risk beyond routine handling of operatory or periodontal instruments during patient care when proper protocols are followed.

Keratinocyte Microvesicles Regulate the Expression of Multiple Genes in Dermal Fibroblasts.

Extracellular vesicles released from cells regulate many normal and pathological conditions. Little is known about the role of epidermal keratinocyte microvesicles (KC-MVs) in epithelial-stromal interaction that is essential for wound healing. We investigated, therefore, whether MV-like structures are present in human wounds and whether they affect wound healing-associated gene expression in dermal fibroblasts. In human wounds, MV-like vesicles were observed during active epithelial migration and early granulation tissue formation. When KC-MVs derived from keratinocyte-like cells (HaCaT) were added to fibroblast cultures, expression of 21 genes was significantly regulated (P<0.05) out of 80 genes investigated, including matrix metalloproteinase-1 and -3, interleukin-6 and -8, and genes associated with transforming growth factor-ß signaling. Similar changes were observed at the protein level. MVs from normal epidermal keratinocytes showed similar response to HaCaT cells. KC-MVs activated ERK1/2, JNK, Smad, and p38 signaling pathways in fibroblasts with ERK1/2 signaling having the most prominent role in the MV-induced gene expression changes. KC-MVs stimulated fibroblast migration and induced fibroblast-mediated endothelial tube formation but did not affect collagen gel contraction by fibroblasts. The results demonstrate that keratinocyte microvesicles have a strong and a specific regulatory effect on fibroblasts that may modulate several aspects of wound healing.

A quantitative diagnostic method for oral mucous precancerosis by Rose Bengal fluorescence spectroscopy.

A novel in vivo fluorescence spectroscopic diagnostic method has been developed in an animal model to make a quantified precancer diagnosis. In the study, 40 golden hamsters were randomly divided into four groups (groups A, B, C, and D), with group A being the control group and the other three groups being inducted at different precancer stages. A 1% Rose Bengal (RB) solution was used for the fluorescence spectroscopic diagnosis. A parameter K defined as K = I(RB)/I(auto) was introduced to reflect the amount of RB in the tissue, where I(RB) and I(auto) represent the fluorescence peak intensity of the RB in the tissue and the autofluorescence intensity of tissue at 580 nm, respectively. The average K values of the four groups were calculated and statistically analyzed by analysis of variance (ANOVA), which revealed statistically significant differences within each group as well as between groups (p < 0.001). After analysis by Clementine 11.1 C&R Tree modeling (CART), the following diagnostic criteria were set: normal, K ≤ 8.91; simple hyperplasia, 8.91 < K ≤ 41.92; mild dysplasia, 41.92 < K ≤ 70.79; moderate and severe dysplasia, K >70.79. The sensitivity and specificity to detect precancerous lesions compared with scalpel biopsy were calculated. The results of this study showed that the spectrofluorometric method mediated by RB could accurately discriminate different precancer stages.