Ultrastructural Characterization of Human Gingival Fibroblasts in 3D Culture.

Cell spheroids are applied in various fields of research, such as the fabrication of three-dimensional artificial tissues in vitro, disease modeling, stem cell research, regenerative therapy, and biotechnology. A preclinical 3D culture model of primary human gingival fibroblasts free of external factors and/or chemical inducers is presented herein. The ultrastructure of the spheroids was characterized to establish a cellular model for the study of periodontal tissue regeneration. The liquid overlay technique was used with agarose to generate spheroids. Fibroblasts in 2D culture and cell spheroids were characterized by immunofluorescence, and cell spheroids were characterized by optical and scanning electron microscopy, energy-dispersive X-ray spectroscopy, backscattered electrons, and Fourier transform infrared spectroscopy. Ostegenic related genes were analyzed by RT-qPCR. Gingival fibroblasts formed spheroids spontaneously and showed amorphous calcium phosphate nanoparticle deposits on their surface. The results suggest that human gingival fibroblasts have an intrinsic potential to generate a mineralized niche in 3D culture.

Acidogenic and aciduric properties of Streptococcus mutans serotype c according to its genomic variability.

Streptococcus mutans (S. mutans) has a wide genetic diversity that contributes to its phenotypic heterogeneity, and may be related to attributes associated with acidogenicity and aciduricity. The aim of this study was to evaluate the acidogenic and aciduric properties of S. mutans serotype c isolates from saliva of schoolchildren according to the genomic variability. S. mutans isolates were identified by polymerase chain reaction. Fifty S. mutans serotype c isolates were genotyped by pulsed field gel electrophoresis and tested for their ability to produce and resist acid. Three specific genotypes were identified in the caries-active group and only one in the caries-free group. Although isolates were similarly acidogenic, an exclusive caries-active genotype had the greatest glycolytic activity. In contrast, isolates exhibited variable aciduricity, and three caries-active genotypes were the least aciduric. We concluded that there is genetic variability within serotype c. Acid production was similar regardless of the caries status but correlated with the number of genotypes. In addition, resistance to acid could be an important characteristic for the establishment and colonisation of specific genotypes in children with caries. However, it is important to evaluate children's intrinsic characteristics and other phenotypic properties to explain the physiopathological behaviour of the different genotypes.

Composición y función salival en niños con labio con/sin paladar hendido no sindrómico / Salivary composition and function in children with non-syndromic cl w/wo p / Composição e função salivar em crianças com fissuras labiais e/ou palatinas não sindrômicas

Resumen Introducción y Objetivo: La saliva cumple una importante función en la homeostasis oral, aunque no es evaluada rutinariamente en pacientes con labio con/sin paladar hendido (LH±PH). Este estudio pretendió comparar las características salivares de niños con LH±PH no sindrómico vs. niños sin esta diferencia craneofacial. Materiales y métodos: Se incluyeron 17 niños con LH±PH no sindrómico y 25 niños sin LH±PH, de los cuales se colectaron muestras de saliva estimulada (SE) y no estimulada (SNE). Se realizaron pruebas fisicoquímicas, cuantificación de iones (Ca, Mg y P), proteína total y recuento de streptococcus mutans en ambos grupos para ambas muestras de saliva. Para establecer comparaciones entre grupos se realizó la prueba no paramétrica de Mann-Whitney. Resultados: Hubo diferencias estadísticamente significativas en el pH de SE y SNE, así como en proteína total y capacidad amortiguadora de SNE. El pH (p=0,002) y capacidad amortiguadora (p=0,023) de SNE de los niños con LH±PH fueron menores, mientras que la concentración de proteína total (p=0,003) fue mayor con respecto al grupo control. Se encontró mayor proporción de estreptococo mutans en SNE de niños con LH±PH (6,39x104 UFC mL-1) respecto al grupo control (2,21x104 UFC mL-1), aunque sin detectar diferencias estadísticamente significativas (p=0,243). Conclusiones: Estos hallazgos sugieren una alteración en la función y composición de la SNE en pacientes con LH±PH no sindrómico, que correspondería a una alteración en la formación y función de las glándulas salivales en estos individuos que requiere ser investigada.

Abstract Introduction and objective: Saliva plays an important role in oral homeostasis, although it is not routinely evaluated in patients with CL±P. This study aimed to compare these characteristics in children with non-syndromic CL±P vs. children without this craniofacial difference. Materials and methods: This descriptive study included 17 children with non-syndromic CL±P from Fundación Clínica Noel and 25 children without CL±P. Stimulated (SS) and non-stimulated saliva (NSS) from both groups were collected for physicochemical tests, quantification of ions (Ca, Mg and P), total protein and streptococcus mutans count. Results: There were statistically significant differences in pH of SS and NSS, as well as in total protein concentration and buffer capacity in NSS. The pH (p = 0.002) and buffering capacity (p = 0.023) of NSS of CL±P children decreased, while total protein concentration (p = 0.003) increased compared to the control group. A higher streptococcus mutans count was found in NSS of CL±P children (6.39x104 CFU UFC mL-1) compared to the control group (2.21x104 UFC mL-1), although without detecting statistically significant differences (p = 0.243). Conclusions: These findings suggest an alteration in the function and composition of NSS in patients with non-syndromic CL±P; which could possibly be explained from the genetic/biological point of view by an alteration in the salivary glands formation and function in these individuals that requires further investigation.

Resumo Introdução e objetivo: A saliva desempenha um papel fundamental na homeostase oral, não é avaliada rotineiramente em pacientes com FL/P. Portanto, este estudo objetivou indagar essas características em crianças com FL/P não sindrômico. Materiais e métodos: 17 crianças com FL/P não sindrômico da Fundación Clínica Noel, e 25 crianças sem FL/P foram incluídas neste estudo. Amostras de saliva estimulada (SE) e saliva não estimulada (SNE) foram coletadas para realização de testes físico-químicos, quantificação de íons (Ca, Mg e P), proteína total e contagem de streptococcus mutans. Resultados: Houve diferenças estatisticamente significativas no pH de SE e SNE, bem como na concentração de proteína total e capacidade tampão de SNE. O pH (p=0,002) e a capacidade tampão (p=0,023) de SNE de crianças com FL/P foram menores, enquanto a concentração de proteína total (p=0,003) foi maior, quando comparado ao grupo controle. Um contagem maior de streptococcus mutans foi encontrada na saliva não estimulada de crianças com FL/P (6,39x104 UFC mL-1) em comparação ao grupo controle (2,21x104 UFC mL-1), embora sem detectar diferenças estatisticamente significativas (p=0,243). Conclusão: Esses achados sugerem uma alteração na função e composição da SNE em pacientes com FL/P não sindrômico; isso possivelmente poderia ser explicado do ponto de vista genético/biológico por uma alteração na formação e função das glândulas salivares nesses indivíduos que requer investigação.

Optimized liquid-based cytology for the cellular and molecular analysis of oral keratinocytes: A promising diagnostic tool.

BACKGROUND: Liquid-based cytology (LBC) has improved exfoliative cytology by facilitating the extraction of more precise information from epithelial cells. The aim of this study was to optimize a protocol using a conventional cytobrush to perform LBC, obtaining oral keratinocytes for their further cellular and molecular analysis. METHODS: LBC was performed in 30 healthy donors from buccal mucosa. We evaluated the use of diethyl pyrocarbonate (DEPC)-treated Dulbecco's Modified Eagle Medium (DMEM) medium right after the collection of the cells. Cell morphology and viability were determined by Orcein staining and flow cytometry, respectively. RNA was extracted by the trizol method, and evaluated with spectrometry and electrophoresis. Finally, RNA was copied into cDNA and GAPDH and TLR2 genes were amplified by reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) using specific primers. RESULTS: Only DEPC-treated DMEM preserved the viability of intact intraepithelial keratinocytes. RNA quantity and quality improves in samples treated with DEPC. RNA integrity is comparable with a cell line control. GAPDH gene was successfully amplified by RT-PCR and RT-qPCR. CONCLUSIONS: Therefore, LBC performed under these conditions becomes a reproducible technique for the retrieval of intraepithelial oral keratinocytes with good cell viability for cytomorphometric analysis, and extraction of good RNA quality suitable for molecular analyses such as PCR. We propose this LBC protocol as a complementary method to the cellular and molecular study of oral mucosa pathologies; however, it requires further study.

The phenotype of gingival fibroblasts and their potential use in advanced therapies.

Advanced therapies in medicine use stem cells, gene editing, and tissues to treat a wide range of conditions. One of their goals is to stimulate endogenous repair of tissues and organs by manipulating stem cells and their niche, as well as to optimize the intrinsic characteristics and plasticity of differentiated cells in adult tissues. In this context, fibroblasts emerge as an alternative source to stem cells because they share phenotypic and regenerative characteristics. Specifically, fibroblasts of the oral mucosae have been shown to have improved regenerative capacity compared to other fibroblast populations. Additionally, their easy access by means of minimally invasive procedures without generating aesthetic problems, with easy and rapid in vitro expansion and with great capacity to respond to extrinsic factors, make oral fibroblasts an attractive and interesting resource for regenerative medicine. This review summarizes current concepts regarding the phenotypic and functional aspects of human Gingival Fibroblasts and their niche, differentiating them from other fibroblast populations of oral-lining mucosa and skin fibroblasts. Furthermore, some applications are presented in regenerative medicine, emphasizing on the biological potential of human Gingival Fibroblasts.

Oral lichen planus: A chronic inflammatory model to study the regulation of the Toll-like receptor signaling in oral keratinocytes.

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory disease with a strong immune mechanism involved. Although no causal factor is identified in OLP, a cellular hypersensitivity has been associated with its pathophysiology. Furthermore, the chronicity of the disease could cause its malignant transformation. HIGHLIGHT: Herein, we present a literature review focusing on the interrelationship of Toll-like receptors (TLRs) and OLP, mainly on the molecular behavior of oral keratinocytes once TLR signals are activated. A family of transcription factors, the Interferon Regulatory Factor (IRF) family, could be having a novel role in the prognosis of OLP. Specifically, Interferon Regulatory Factor 6 (IRF6) as a key component of the TLR signaling could impart specificity to downstream responses in oral keratinocytes. CONCLUSION: We propose a hypothetical model after TLR2 activation in which a plausible TLR2-IRF6 regulatory mechanism could be a key factor to be evaluated in OLP as a convenient chronic inflammatory model. Further molecular studies are required to fully understand the role of oral keratinocytes in the initiation of OLP.

Genomic and phenotypic diversity of Streptococcus mutans.

BACKGROUND: Streptococcus mutans (S. mutans) is a commensal microorganism found in the human oral cavity. However, due to environmental changes, selective pressures, and the presence of a variable genome, it adapts and may acquire new physiological and metabolic properties that alter dental biofilm homeostasis, promoting the development of dental caries. Although the plasticity and heterogeneity of S. mutans is widely recognized, very little is known about the mechanisms for the expression of pathogenic properties in specific genotypes. HIGHLIGHT: The implementation of molecular biology techniques in the study of S. mutans has provided information on the genomic diversity of this species. This variability is generated by genome rearrangements, natural genetic transformation, and horizontal gene transfer, and continues to grow due to an open pan-genome. The main virulence factors associated with the cariogenic potential of S. mutans include adhesion, acid production (acidogenicity), and acid tolerance (aciduricity), and also show variability. These factors coordinate the modification of the physicochemical properties of the biofilm, which results in the accumulation of S. mutans and other acidogenic and aciduric species in the oral cavity. CONCLUSION: We review the current literature on the main processes that generate S. mutans genomic diversity, as well as the phenotypic variability of its main virulence factors. S. mutans achieves its pathogenesis by sensing the intra- and extracellular environments and regulating gene transcription according to perceived environmental modifications. Consequently, this regulation gives rise to differential synthesis of proteins, allowing this species to potentially express virulence factors.

Genetic diversity of Streptococcus mutans serotype c isolated from white spot and cavitated caries lesions from schoolchildren.

OBJECTIVE: To determine the genetic diversity of Streptococcus mutans (S. mutans) serotype c isolated from white spot and cavitated caries lesions of schoolchildren. METHODS: S. mutans isolates were obtained and identify by Polymerase Chain Reaction (PCR) from 28 schoolchildren. A total of 92 S. mutans isolates, identified as serotype c by PCR, were analyzed by pulsed field gel electrophoresis after digestion of genomic DNA with SmaI enzyme. 62 isolates were obtained from white spot and cavitated caries lesions of schoolchildren that presented both lesions simultaneously and 30 isolates were from saliva and biofilm samples of schoolchildren without dental caries. Cluster analyses were performed using the Dice coefficient of the BioNumerics software version 6.0. RESULTS: It was possible to determine the serotype in 190 isolates out of 255 isolates identified as S. mutans. Serotype c was the most frequent (n = 139), followed by serotype f (n = 31) and serotype e (n = 20). After analyzing the dendograms of the 92 serotype c isolates, this study identified three strains present in both types of lesions, two strains specific to the type of lesion: one strain from the white spot lesion and one strain from the cavitated caries lesion, and five strains specific to children with caries versus four strains for children without caries. CONCLUSION: S. mutans serotype c genetic variability is similar in terms of the number of strains present according to the caries status and type of lesion.

Segundos molares mandibulares permanentes impactados en pacientes de 8 a 15 años del programa de presupuesto participativo de Medellín, Colombia / Impactation of Permanent Mandibular Second Molars in 8 to 15 Year-Old Patients from the Participative Budget Program in Medellín, Colombia

Antecedentes: La impactación de segundos molares mandibulares permanentes (2MM) se presenta con variaciones considerables según el grupo poblacional. Además de los problemas asociados a esta alteración, podrían requerir tratamientos múltiples y complejos. Por lo tanto, el diagnóstico temprano de una alteración en el proceso de erupción del 2MM es imperativo para reducir el riesgo de impactación, la dificultad de tratamiento y la aparición de problemas asociados en las poblaciones. Propósito: Describir las características de impactación del 2MM en pacientes de 8 a 15 años en una muestra de Medellin, Colombia. Métodos: En este estudio descriptivo retrospectivo se evaluaron 1756 radiografias panorámicas de las cuales 95 presentaron impactación de 2MM. Estas se analizaron según sexo, localización en la mandíbula y tipo de inclinación, y se realizaron 13 mediciones lineales y angulares para identificar parámetros dentoesqueléticos característicos del lado de la impactación. Para el análisis de los datos se utilizó estadística descriptiva y la prueba T (p < 0,05). Resultados: La prevalencia de impactación fue del 5,4 %. La impactación bilateral con inclinación mesial fue la más frecuente. El lado izquierdo predominó en impactaciones unilaterales. El único parámetro dentoesquelético característico de impactación fue el ángulo de inclinación del 2MM con respecto al plano oclusal, con mayor severidad en el lado izquierdo. Conclusiones: Un ángulo igual o mayor a 112,94° podría indicar riesgo de impactación contra el primer molar mandibular permanente. Por lo tanto, este parámetro debería incluirse en el análisis radiográfico para el diagnóstico temprano de impactación del 2MM.

Background: Impactation of permanent mandibular second molars (2MM) occurs with significant variations according to the population group. In addition to the problems associated with it, they could require multiple and complex treatments. Therefore, the early diagnosis of an alteration in the 2MM eruption process is a must to reduce the impactation risk, the treatment difficulty and the occurrence of related problems in the populations. Purpose: To describe the 2MM impactation characteristics in an 8 to 15 year-old patient sample in Medellin, Colombia. Methods: This retrospective descriptive study evaluated 1 756 panoramic radiographs and 95 of them showed 2MM impactation. The latter were analyzed based on the sex, location in the mandible and type of inclination; 13 linear and angle measurings were then carried out in order to identify dental-skeletal parameters characteristic in the impactation side. The data analysis used descriptive statistics and a T test (p < 0.05). Results: The impactation prevalence was 5.4% and the bilateral impactation with mesial inclination was the most frequently found. Unilateral impactations prevailed in the left side of the mandible/maxilla. The only dental-skeletal parameter of impactation was the 2MM inclination angle regarding the occlusal plane, with a greater severity on the left side. Conclusions: An angle of 112.94° or greater could indicate an impactation risk on the permanent mandibular first molar. Therefore, this parameter should be included in the radiographic analysis when diagnosing the early 2MM impactation.