Evaluation of Ilex guayusa and Piper marginatum Extract Cytotoxicity on Human Dental Pulp Mesenchymal Stem Cells.

BACKGROUND: Amelogenesis imperfecta is a hereditary disorder affecting dental enamel. Among its phenotypes, hypocalcified AI is characterized by mineral deficiency, leading to tissue wear and, consequently, dental sensitivity. Excessive fluoride intake (through drinking water, fluoride supplements, toothpaste, or by ingesting products such as pesticides or insecticides) can lead to a condition known as dental fluorosis, which manifests as stains and teeth discoloration affecting their structure. Our recent studies have shown that extracts from Colombian native plants, Ilex guayusa and Piper marginatum, deposit mineral ions such as phosphate and orthophosphate into the dental enamel structure; however, it is unknown whether these extracts produce toxic effects on the dental pulp. OBJECTIVE: To assess cytotoxicity effects on human dental pulp stem cells (hDPSCs) exposed to extracts isolated from I. guayusa and P. marginatum and, hence, their safety for clinical use. METHODS: Raman spectroscopy, fluorescence microscopy, and flow cytometry techniques were employed. For Raman spectroscopy, hDPSCs were seeded onto nanobiochips designed to provide surface-enhanced Raman spectroscopy (SERS effect), which enhances their Raman signal by several orders of magnitude. After eight days in culture, I. guayusa and P. marginatum extracts at different concentrations (10, 50, and 100 ppm) were added. Raman measurements were performed at 0, 12, and 24 h following extract application. Fluorescence microscopy was conducted using an OLIMPUS fv1000 microscope, a live-dead assay was performed using a kit employing a BD FACS Canto TM II flow cytometer, and data analysis was determined using a FlowJo program. RESULTS: The Raman spectroscopy results showed spectra consistent with viable cells. These findings were corroborated using fluorescence microscopy and flow cytometry techniques, confirming high cellular viability. CONCLUSIONS: The analyzed extracts exhibited low cytotoxicity, suggesting that they could be safely applied on enamel for remineralization purposes. The use of nanobiochips for SERS effect improved the cell viability assessment.

In vitro mineral apposition analysis of two Colombian plant extracts on Amelogenesis imperfecta teeth.

OBJECTIVE: To determine if native Colombian Piper marginatum Jacq. and Ilex guayusa Loes plant extracts have a remineralizing effect on teeth with Amelogenesis imperfecta in comparison with the commercial products Clinpro-3M and Recaldent™. MATERIAL AND METHODS: An in vitro study was carried out with 128 human teeth slices (64 healthy and 64 with Amelogenesis imperfecta) on which an initial Raman spectroscopy was performed followed by Raman spectroscopies at 0, 24, 48, and 72 h to determine possible remineralization by observing mineral increase or decrease as a result of P. marginatum Jacq. and I. guayusa Loes extract application in comparison to control substance (Clinpro and Recaldent™) application. Obtained data were analyzed using a bivariate method with a t unidirectional test. Significant differences among groups were determined by an ANOVA with Dunnett post hoc tests. RESULTS: Native I. guayusa Loes and P. marginatum Jacq. Colombian plants extracts exhibited phosphate and orthophosphate mineral apposition, where P. marginatum Jacq. presented better results. CONCLUSIONS: Native Colombian I. guayusa Loes and P. marginatum Jacq plant extract might in the future be useful for dental tissue remineralization, as they induced phosphate and orthophosphate mineral apposition, main components of tooth enamel. These types of natural compounds can become an alternative to fluorine, whose ingestion is harmful to the human body.

Clinical and Molecular Study of the NOG Gene in Families with Mandibular Micrognathism.

OBJECTIVES: Previous studies showed that noggin gene (NOG) sequence alterations, as well as epigenetic factors, could influence mandibular development. The aim of this study was to analyze clinical characteristics, NOG gene sequences, and promoter methylation sites in patients with mandibular micrognathism. MATERIALS AND METHODS: A total of 35 individuals of five Colombian families were subject to clinical and cephalometric analysis for mandibular micrognathism. One nonaffected individual of each family was included as a control. DNA was isolated from whole blood sample from all individuals by salting out method. Nine NOG gene fragments were amplified by polymerase chain reaction (PCR) and sequenced. Identification of CpG islands for methylation analysis at the NOG gene promoter was performed by MSP-PCR kit (Qiagen R). STATISTICAL ANALYSIS: A descriptive statistical analysis was carried out evaluating the presence or absence of genetics variants and the methylation sites in the NOG gene. RESULTS: NOG sequence results of affected individuals with mandibular micrognathism for one of the families studied demonstrated that they were heterozygous for 672 C/A (new mutation). For a second family, individuals were heterozygous for 567 G/C (single nucleotide polymorphism [SNP] RS116716909). For DNA analyzed from all patients studied, no methylations were observed at the NOG gene promoter region. CONCLUSION: Our results suggested that 672 C/A and 567 G/C variants could be involved in the presence of mandibular micrognathism. Moreover, lack of methylation sites at the NOG gene promoter region of all individuals studied suggests possibly other epigenetic factors could modulate mandibular growth. The search of genetic variants related with mandibular micrognathism will allow to predict in an integral way the development patterns of the patients and therefore establish a better clinical treatment.

Analysis of In Vitro Osteoblast Culture on Scaffolds for Future Bone Regeneration Purposes in Dentistry.

One of the main focuses of tissue engineering is to search for tridimensional scaffold materials, complying with nature's properties for tissue regeneration. Determining material biocompatibility is a fundamental step in considering its use. Therefore, the purpose of this study was to analyze osteoblast cell adhesion and viability on different materials to determine which was more compatible for future bone regeneration. Tridimensional structures were fabricated with hydroxyapatite, collagen, and porous silica. The bovine bone was used as material control. Biocompatibility was determined by seeding primary osteoblasts on each tridimensional structure. Cellular morphology was assessed by SEM and viability through confocal microscopy. Osteoblast colonization was observed on all evaluated materials' surface, revealing they did not elicit osteoblast cytotoxicity. Analyses of four different materials studied with diverse compositions and characteristics showed that adhesiveness was best seen for HA and viability for collagen. In general, the results of this investigation suggest these materials can be used in combination, as scaffolds intended for bone regeneration in dental and medical fields.