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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.

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.

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.

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.

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.

Background aims. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of brain and spinal cord motor neurons (MN). The intraspinal and systemic grafting of mesenchymal stromal cells (MSC) was used to treat symptomatic transgenic rats overexpressing human superoxide dismutase 1 (SOD1) in order to alleviate the disease course and prolong the animals ' lifespan. Methods. At the age of 16 weeks (disease onset) the rats received two grafts of MSC expressing green fl uorescent protein (GFPMSC) on the same day, intraspinally (10 5 cells) and intrave- nously (2 � 10 6 cells). Sham-treated animals were injected with phosphate-buffered saline (PBS). Motor activity, grip strength and body weight were tested, followed by immunohistochemical analysis. Results. The combined grafting of MSC into symptomatic rats had a signifi cant effect on motor activity and grip strength starting 4 weeks after transplantation. The lifespan of animals in the treated group was 190 � 3.33 days compared with 179 � 3.6 days in the control group of animals. Treated rats had a larger number of MN at the thoracic and lumbar levels; these MN were of larger size, and the intensity of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining in the somas of apoptotic MN at the thoracic level was much lower than in sham-treated animals. Transplanted GFPMSC survived in the spinal cord until the end stage of the disease and migrated both rostrally and caudally from the injection site. Conclusions . Intraspinal and intravenous transplantation of MSC has a benefi cial and possibly synergistic effect on the lifespan of ALS animals.

Chronic spinal cord injury (SCI) is characterized by tissue loss and a stable functional deficit. While several experimental therapies have proven to be partly successful for the treatment of acute SCI, treatment of chronic SCI is still challenging. We studied whether we can bridge a chronic spinal cord lesion by implantation of our newly developed hydrogel based on 2-hydroxypropyl methacrylamide, either alone or seeded with mesenchymal stem cells (MSCs), and whether this treatment leads to functional improvement. A balloon-induced compression lesion was performed in adult 2-month-old male Wistar rats. Five weeks after injury, HPMA-RGD hydrogels [N-(2-hydroxypropyl)-methacrylamide with attached amino acid sequences--Arg-Gly-Asp] were implanted into the lesion, either with or without seeded MSCs. Animals with chronic SCI served as controls. The animals were behaviorally tested using the Basso–Beattie-Breshnahan (BBB) (motor) and plantar (sensory) tests once a week for 6 months. Behavioral analysis showed a statistically significant improvement in rats with combined treatment, hydrogel and MSCs, compared with the control group (P < 0.05). Although a tendency toward improvement was found in rats treated with hydrogel only, this was not significant. Subsequently, the animals were sacrificed 6 months after SCI, and the spinal cord lesions evaluated histologically. The combined therapy (hydrogel with MSCs) prevented tissue atrophy (P < 0.05), and the hydrogels were infiltrated with axons myelinated with Schwann cells. Blood vessels and astrocytes also grew inside the implant. MSCs were present in the hydrogels even 5 months after implantation. We conclude that 5 weeks after injury, HPMA-RGD hydrogels seeded with MSCs can successfully bridge a spinal cord cavity and provide a scaffold for tissue regeneration. This treatment leads to functional improvement even in chronic SCI.

Shear-induced platelet responses are triggered by VWF binding to the platelet GpIb-IX complex, and there is evidence that this ligand-receptor coupling stimulates transmembranous signaling through the cytoplasmic tail of glycoprotein (Gp) Ib alpha. To investigate the mechanism by which signaling is effected, new molecular interactions involving GpIb-IX that develop in response to pathological shearing stress were examined in intact human platelets. Exposure to shear, but not alpha-thrombin, results in the co-immunoprecipitation of the actin cross-linking protein alpha-actinin with the GpIb-IX complex. Blockers of VWF binding to GpIb alpha or actin polymerization inhibit the association of alpha-actinin with the GpIb-IX complex, but the association of alpha-actinin with the GpIb-IX complex is not affected by inhibiting VWF binding to platelet integrin alpha IIb beta 3 (GpIIb-IIIa). alpha-Actinin becomes tyrosine phosphorylated in response to pathological shear stress, and phosphorylated alpha-actinin associates with GpIb-IX. In resting platelets, class IA heterodimeric phosphatidylinositol 3-kinase (PI 3-K) and protein kinase N (PKN) associate with nonphosphorylated alpha-actinin. Shear stress causes PI 3-K to disassociate from alpha-actinin, while it stimulates PKN binding to alpha-actinin. These results demonstrate that shear-induced VWF binding to GpIb alpha causes enhanced binding of cytoskeletal alpha-actinin to GpIb-IX and suggest that alpha-actinin, perhaps through tyrosine phosphorylation, serves as an adapter for a signaling complex that could regulate VWF-induced platelet aggregation.