L-PRF Secretome from Both Smokers/Nonsmokers Stimulates Angiogenesis and Osteoblast Differentiation In Vitro.

Leukocyte and Platelet-Rich Fibrin (L-PRF) is part of the second generation of platelet-concentrates. L-PRF derived from nonsmokers has been used in surgical procedures, with its beneficial effects in wound healing being proven to stimulate biological activities such as cell proliferation, angiogenesis, and differentiation. Cigarette smoking exerts detrimental effects on tissue healing and is associated with post-surgical complications; however, evidence about the biological effects of L-PRF derived from smokers is limited. This study evaluated the impact of L-PRF secretome (LPRFS) derived from smokers and nonsmokers on angiogenesis and osteoblast differentiation. LPRFS was obtained by submerging L-PRF membranes derived from smokers or nonsmokers in culture media and was used to treat endothelial cells (HUVEC) or SaOs-2 cells. Angiogenesis was evaluated by tubule formation assay, while osteoblast differentiation was observed by alkaline phosphatase and osterix protein levels, as well as in vitro mineralization. LPRFS treatments increased angiogenesis, alkaline phosphatase, and osterix levels. Treatment with 50% of LPRFS derived from smokers and nonsmokers in the presence of osteogenic factors stimulates in vitro mineralization significantly. Nevertheless, differences between LPRFS derived from smokers and nonsmokers were not found. Both LPRFS stimulated angiogenesis and osteoblast differentiation in vitro; however, clinical studies are required to determine the beneficial effect of LPRFS in smokers.

Osteoblastic differentiation and changes in the redox state in pulp stem cells by laser treatment.

The aim of this study was to determine the effect of low-level laser therapy (LLLT) on cell proliferation, mitochondrial membrane potential changes (∆Ψm), reactive oxygen species (ROS), and osteoblast differentiation of human dental pulp stem cells (hDPSCs). These cells were irradiated with 660- and 940-nm lasers for 5 s, 50 s, and 180 s. Cell proliferation was assessed using the resazurin assay, cell differentiation by RUNX2 and BMP2 expression, and the presence of calcification nodules using alizarin-red S staining. ROS was determined by the dichlorofluorescein-diacetate technique and changes in ∆Ψm by the tetramethylrhodamine-ester assay. Data were analyzed by a Student's t-test and Mann-Whitney U test. The 940-nm wavelength for 5 and 50 s increased proliferation at 4 days postirradiation. After 8 days, a significant decrease in proliferation was observed in all groups. Calcification nodules were evident in all groups, with a greater staining intensity in cells treated with a 940-nm laser for 50 s, an effect that correlated with increased RUNX2 and BMP2 expression. ROS production and Δψm increased independently of irradiation time. In conclusion, photobiomodulation (PBM) with LLLT induced morphological changes and reduced cell proliferation rate, which was associated with osteoblastic differentiation and increased ROS and Δψm, independent of wavelength and time.

Relaxin treatment stimulates the differentiation of mesenchymal stem cells into osteoblasts.

Background/purpose: Several studies have determined that relaxin stimulates differentiation and regulates the activity of mature osteoclasts, but little is known about its effect on the differentiation of mesenchymal cells towards the osteogenic lineage. Therefore, this study aimed to determine the effect of relaxin on the proliferation and differentiation of the osteoblastic lineage of mesenchymal cells derived from human dental pulp (hDPSC). Materials and methods: In this in vitro study, hDPSC were characterized and treated with relaxin at different doses (10-80 ng/ml) and times (1-21 days). Morphology was assessed by microscopy, and proliferation was assessed using a resazurin assay. Osteoblastic differentiation was evaluated by Alizarin Red staining, alkaline phosphatase (ALP) labeling, and changes in the expression of the osteoblastic differentiation genes RUNX2 and BMP2. Results: Relaxin treatment did not induce changes in the proliferation or viability of hDPSCs; however, larger cells and increased cytoplasmic prolongation were observed. Relaxin treatment (20 and 80 ng/ml) significantly increased calcified nodule formation on days 14 and 21. The cytochemical signals for ALP, RUNX2, and BMP2 gene expression were significantly (P < 0.05) increased by the relaxin treatment. Conclusion: Relaxin treatment does not induce changes in hDPSC proliferation but induces morphological changes, increases ALP detection, calcified nodule formation, and increases expression of RUNX2 and BMP2, suggesting the induction of osteoblastic differentiation of hDPSC.

Response of osteoblastic cells to low-level laser treatment: a systematic review.

Low-level laser therapy (LLLT)-induced photobiomodulation (PBM) stimulates bone tissue regeneration by inducing osteoblast differentiation and mitochondrial activation. However, the role of reactive oxygen species (ROS) in this process remains controversial. The aim of this systematic review was to collect and analyze the available literature on the cellular and molecular effects of LLLT on osteoblasts and the role of ROS in this process. A search was conducted in PubMed, ScienceDirect, Scopus, and Web of Science. Studies published in English over the past 15 years were selected. Fourteen articles were included with moderate (n = 9) and low risk of bias (n = 5). Thirteen studies reported the use of diode lasers with wavelengths (λ) between 635 and 980 nm. One study used an Nd:YAG laser (λ1064 nm). The most commonly used λ values were 808 and 635 nm. The energy densities ranged from 0.378 to 78.75 J/cm2, and irradiation times from 1.5 to 300 s. Most studies found increases in proliferation, ATP synthesis, mitochondrial activity, and osteoblastic differentiation related to moderate and dose-dependent increases in intracellular ROS levels. Only two studies reported no significant changes. The data presented heterogeneity owing to the variety of LLLT protocols. Although several studies have shown a positive role of ROS in the induction of proliferation, migration, and differentiation of different cell types, further research is required to determine the specific role of ROS in the osteoblastic cell response and the molecular mechanisms involved in triggering previously reported cellular events.

Human recombinant cementum protein 1, dental pulp stem cells, and PLGA/hydroxyapatite scaffold as substitute biomaterial in critical size osseous defect repair in vivo.

OBJECTIVE: To evaluate the healing response of critical defects in rat calvaria with recombinant cementum protein 1 (hrCEMP-1) combined with human dental pulp stem cells (hDPSC) and polylactide-co-glycolide/hydroxyapatite (PLGA/HA) scaffold. METHODS: The effect of hrCEMP-1 on proliferation and differentiation of human dental stem cells (hDPSCs) toward a mineralizing-like phenotype was evaluated in monolayer and PLGA/HA scaffold by qPCR. 5 mm calvarial defects were created in Wistar rats and filled with: 1) PLGA/HA scaffold; 2) hDPSCs-PLGA/HA scaffold; 3) hrCEMP-1-hDPSc-PLGA/HA scaffold; 4) control (without scaffold). Bone formation was evaluated by histological-histomorphometric analysis, scanning electron microscopy (SEM) and radiographic evaluation. Comparisons between groups were made with a one-way analysis of variance ANOVA and Bonferroni post-hoc test. RESULTS: In vitro results showed that the PLGA/HA scaffold loaded with hrCEMP-1 improved the proliferation and differentiation of hDPSCs towards a mineralization phenotype by inducing mRNA expression of ALP, OSX, RUNX2, OP, and COL-I genes. The hrCEMP-1/hDPSCs/-PLGA/HA scaffold resulted only in connective tissue formed after ten weeks of healing, larger central radiolucency, and a low peripheral density. We showed superior bone growth and repair with a PLGA/HA matrix scaffold alone and containing hDPSCs compared to the hrCEMP/cells group. CONCLUSIONS: PLGA/HA scaffold with hrCEMP-1 induces hDPSC commitment to mineralizing phenotype in vitro, but does not promote critical size osseous defect repair in vivo when it is included in a substitute biomaterial with hDPSc-PLGA/HA scaffold.

Retinoic and ascorbic acids induce osteoblast differentiation from human dental pulp mesenchymal stem cells.

Previous studies have suggested an important role of retinoic acid (RA) and ascorbic acid (AA) in the stimulation of osteoblastic differentiation; however, the function of RA and AA in the osteogenic differentiation from human dental pulp (hDPSCs) remains unclear. OBJECTIVE: This in vitro study investigated the effects of RA and AA on the differentiation of osteoblast from hDPSCs. METHODS: hDPSCs were treated with different doses of RA and AA, separately or in combination (RA â€‹+ â€‹AA). Morphology and cell proliferation were assessed. Osteoblast differentiation was evaluated by alizarin red, alkaline phosphatase staining, and RUNX2 gene expression. RESULTS: A significant reduction was observed in the number of cells treated with RA (26%) and RA â€‹+ â€‹AA (30%) after 12 days of treatment. AA treatment alone induced a 12% reduction in the number of cells. Morphologically, the cells treated with RA and RA â€‹+ â€‹AA were larger and more elongated than the control cells. A mesh pattern was observed in cells treated with AA. Numerous calcified nodules were present in cells treated with RA, AA, and RA â€‹+ â€‹AA. This coincided with increased expression of RUNX2 and high alkaline phosphatase staining levels. CONCLUSIONS: hDPSCs treated with RA and RA â€‹+ â€‹AA showed significant reduction in proliferation, detectable morphological changes, and expression of the key differentiation gene RUNX2, consistent with an osteoblast phenotype. AA induced morphological changes and early formation of calcified nodules. RA had a predominant effect when AA and RA were used together.

Comparison of Cell Viability and Chemical Composition of Six Latest Generation Orthodontic Wires.

Orthodontic wires are made of alloys containing different metals, including nickel. It is important to evaluate their biocompatibility prior to use, owing to their long-term use in patients. This in vitro study compared the cytotoxicity and chemical composition of six latest orthodontic wires: Fantasia®, Tanzo®, FLI®, NT3®, DuoForce®, and Gummetal®. The before-use group consisted of wires that were not used in the mouth, and the after-use group consisted of wires that were used in the mouth for two months. The wires were placed in contact with human gingival fibroblasts (HGF) for 72 h, and cytotoxicity was determined using the resazurin test. The chemical composition and surface characterisation were evaluated by spectrometry and scanning electron microscopy. The groups were compared using ANOVA and Kruskal-Wallis test. Only the FLI® wires produced a 36% reduction in HGF viability (p < 0.05) and presented greater irregularities and loss of polymer structure. After-use wires showed a significant reduction in the percentage of nickel and the appearance of new elements (oxygen and carbon). Therefore, it can be concluded that no toxic ion release was noticed in this study. Rhodium-coated wires were more stable than PTFE-coated wires, and only the FLI® wires showed a slight cytotoxic effect.

Funcionalidad familiar de pacientes dependientes con falla cardíaca / Family functioning of dependent patients with heart failure

Resumen Objetivo: Describir la funcionalidad familiar de pacientes dependientes con falla cardiaca y clase funcional II-C. Métodos: Estudio cuantitativo, observacional de corte transversal, en el que participaron 50 familias entre septiembre y octubre de 2016. La medición se realizó a través de la “Escala de evaluación de la funcionalidad familiar”. Resultados: Se encontró una asociación estadísticamente significativa entre el nivel de escolaridad del informante familiar y el nivel de la funcionalidad de la familia (p 0.006). La funcionalidad familiar total mostró un nivel bajo en el 38 % de las familias participantes. Las dimensiones de mantenimiento y cambio exponen niveles altos de funcionamiento en un 88 y 74 % respectivamente, mientras la dimensión de individuación presenta 70 % en nivel bajo. La dimensión de coherencia muestra un 48 % de niveles altos e intermedios de funcionalidad. Por otra parte, las metas de estabilidad y control se comportan con niveles altos en un 82 % y 84 %, mientras que el crecimiento y la espiritualidad estuvieron con niveles intermedios. Los pacientes del estudio presentan una dependencia funcional moderada y leve. Finalmente, no se encontró asociación estadísticamente significativa entre la dependencia de los pacientes con la falla cardiaca y el nivel de funcionamiento de sus familias. Conclusiones: Las familias con integrantes con falla cardíaca con funcionalidad moderada y leve presentan bajo nivel de funcionamiento familiar, lo cual las afecta en el cumplimiento de su rol como red primaria de apoyo del paciente. Es necesario continuar investigaciones que amplíen la información del comportamiento de los factores sociodemográficos asociados al funcionamiento de la familia con integrante con falla cardiaca.

Abstract Objective: To describe the familial functionality of dependent patients with heart failure with functional class II-C. Methods: A quantitative, cross-sectional observational study was conducted with 50 families, between September and October 2016, to obtain a measurement through the “Family Functionality Assessment Scale”. Results: Between the level of schooling of the family informant and the level of family functionality (p = 0.006) was found a statistically significant association. In 38% of the participating families, the total family functionality showed a low level. The System Maintenance and change dimensions expose high levels of operation by 88% and 74% respectively, whereas the dimension of individuation presents 70% at a low level. The coherence dimension reveals 48% of high and intermediate levels of functionality. On the other hand, the goals of stability and control behave with high levels at 82% and 84%, while growth and spirituality were at intermediate levels. The patients concerning the study have a moderate and mild functional dependence. Ultimately, between the dependence of patients with heart failure and the level of functioning of their families, no statistically significant association was found. Conclusions: Families with members with moderate and mild functional heart failure have a low level of family functioning, which affects the family in fulfilling its role as a primary patient support network. It is necessary to continue research that broadens the information on the behaviour of sociodemographic factors associated with the functioning of the family with a member with heart failure.

Effect of Vitamins D and E on the Proliferation, Viability, and Differentiation of Human Dental Pulp Stem Cells: An In Vitro Study.

INTRODUCTION: The aim of the present study was to determine the effects of vitamins D and E on the proliferation, morphology, and differentiation of human dental pulp stem cells (hDPSCs). METHODS: In this in vitro experimental study, hDPSCs were isolated, characterized, and treated with vitamins D and E, individually and in combination, utilizing different doses and treatment periods. Changes in morphology and cell proliferation were evaluated using light microscopy and the resazurin assay, respectively. Osteoblast differentiation was evaluated with alizarin red S staining and expression of RUNX2, Osterix, and Osteocalcin genes using real-time RT-PCR. RESULTS: Compared with untreated cells, the number of cells significantly reduced following treatment with vitamin D (49%), vitamin E (35%), and vitamins D + E (61%) after 144 h. Compared with cell cultures treated with individual vitamins, cells treated with vitamins D + E demonstrated decreased cell confluence, with more extensive and flatter cytoplasm that initiated the formation of a significantly large number of calcified nodules after 7 days of treatment. After 14 days, treatment with vitamins D, E, and D + E increased the transcription of RUNX2, Osterix, and Osteocalcin genes. CONCLUSIONS: Vitamins D and E induced osteoblastic differentiation of hDPSCs, as evidenced by the decrease in cell proliferation, morphological changes, and the formation of calcified nodules, increasing the expression of differentiation genes. Concurrent treatment with vitamins D + E induces a synergistic effect in differentiation toward an osteoblastic lineage.