Preliminary study on the preparation of antler powder/chitosan/ß-glycerophosphate sodium/polyvinyl alcohol porous hydrogel scaffolds and their osteogenic effects.

Introduction: The production of bone-like structural scaffolds through bone tissue engineering technology is a promising method for bone regeneration to repair bone defects. Deer antler, an easily harvested and abundantly sourced initial bone tissue structure, resembles the composition and structure of human cancellous bone and can serve as a new material for allogeneic bone transplantation. Methods: This study involved the preparation and characterization of antler powder/chitosan/ß-glycerophosphate sodium/polyvinyl alcohol (AP/CS/ß-GP/PVA) porous hydrogel scaffolds to verify their material properties and osteogenic mechanisms. The microstructure, hydrophilicity, and mechanical properties of the scaffolds were studied using Scanning Electron Microscopy (SEM), contact angle measurement, and a universal material testing machine. The interactions between the various components were investigated using Fourier-Transform Infrared Spectroscopy (FTIR). Biocompatibility, osteogenic properties, and expression of osteogenesis-related proteins of the scaffolds were evaluated through Cell Counting Kit-8 (CCK-8) assays, alkaline phosphatase staining, Alizarin Red staining, live/dead cell staining, and Western blot analysis. Results: The results showed that as the content of deer antler powder increased, both the hydrophilicity and mechanical properties of the scaffold materials improved, while the porosity slightly decreased with an increase in deer antler powder content. Cell culture experiments demonstrated that scaffolds with a higher proportion of deer antler powder were beneficial for the proliferation and differentiation of mouse pre-osteoblast (MC3T3-E1) cells, with the scaffolds containing 10% and 8% deer antler powder showing the best effects. The upregulation of RUNX2, OCN, OSX, and OPN protein expression may promote differentiation. Discussion: Therefore, the AP/CS/ß-GP/PVA hydrogel scaffolds have the potential to become a promising biomaterial for bone tissue engineering.

Interaction-Based Perspective for Designing Polymer Biomaterial: A Strategic Approach to the Chitosan-Glycerophosphate System.

The conventional approach for developing any polymeric biomaterial is to follow protocols available in the literature and/or perform trial-and-error runs without a scientific basis. Here, we propose an analysis of a complex overlay of molecular interactions between drugs and polymers that provides a strategic pathway for biomaterial development. First, this work provides an innovative interaction-based method for developing an ocular formulation involving in situ gelling chitosan, gelatin, and glycerophosphate systems. A systematic interaction study is conducted based on the measurement of hydrodynamic radius, zeta potential, and viscosity with the sequential addition of formulation components. The increase in the hydrodynamic radius of the polymer with the addition of drugs can be interpreted as better diffusion of the drug inside the charged polymer chains and vice versa. Based on the knowledge of these interactions, a formulation has been designed that shows better drug release results with extended and sustained release compared to literature protocols, hence accentuating the importance of this study. An in-depth analysis of interactions can lead to a better understanding of the system. Second, we demonstrate the development of two dual-drug biomaterial systems, i.e., an in situ gelling and a liquid formulation at ocular surface temperature from the same polymers, which can be used as an ocular antiglaucoma formulation. Prior knowledge of the interactions between the drug polymers can be used to design a better formulation. The demonstrated application of this interaction-based protocol development can be extended universally to any biomaterial. This would provide a comprehensive idea about the properties and interactions of polymers and drugs, which can also serve as a base/starting point for a new formulation/biomaterial development.

Topical ocular administration using thermosensitive chitosan-glycerophosphate-PRP hydrogels for improved ocular bioavailability.

PURPOSE: One of the difficulties in the pharmacy field is the delivery of drugs for the eyes. Topical therapy is one of the most common methods for treating eye diseases. Due to their unique properties, including biocompatibility and suitable degradation, hydrogels are appropriate for biological purposes. Platelet-rich plasma (PRP), as a designated concentration of platelets, is in a smaller volume than the plasma and is considered a rich source of growth factor that has been used in recent years, including applications in eye diseases including corneal wound healing, improvement of dry eye and post-LASIK syndrome. METHODS: The present study was performed to fabricate Chitosan (CS) and glycerophosphate (GP) based hydrogels that are temperature-sensitive for PRP and investigate their effect on ocular stem cells. RESULTS: CS-GP-based temperature-sensitive hydrogels containing PRP were successfully fabricated using CS and GP. This hydrogel is liquid at ambient temperature and a gel at ocular temperature. Rheology, FTIR, and SEM tests assessed the properties of the hydrogels. The results of the MTT test showed that the hydrogel made with the optimal formulation was not toxic to LSC cell lines. CONCLUSIONS: Given this, CS-GP-based hydrogels can be applied as a biocompatible formulation in ocular medication administration with increased bioavailability at the ocular surface and topical delivery of PRP.

The Remineralization Effect of Calcium Glycerophosphate in Fluoride Mouth Rinse on Demineralized Primary Enamel: An in vitro Study.

Aim: To evaluate the remineralization effect of a fluoride mouth rinse containing calcium glycerophosphate in fluoride mouth rinse based on the surface microhardness of demineralized primary enamel. Materials and Methods: 40 sound primary incisors were placed into self-curing acrylic resin and subjected to a demineralizing solution for 5 days, resulting in the formation of artificial caries. The teeth were categorized into four groups (n = 10): group I artificial saliva, group II sodium fluoride, group III sodium fluoride + sodium monofluorophosphate, and group IV sodium monofluorophosphate + calcium glycerophosphate. The specimens received a pH cycling procedure and were submerged twice in their assigned groups for 7 days. The baseline, after demineralization, and after remineralization surface microhardness values were determined. One-way analysis of variance (ANOVA) was used to analyze the mean surface microhardness between groups and one-way repeated measures ANOVA for the mean surface microhardness within each group and Bonferroni's for multiple comparisons at 95% confidence level. The percentage recovery surface microhardness was determined by calculating the average surface microhardness. Results: After demineralization, the mean surface microhardness in all groups significantly decreased. After remineralization, group I had the lowest surface microhardness values and the percentage recovery surface microhardness (P value < 0.001), and group IV had the highest surface microhardness values and the percentage recovery surface microhardness (P value < 0.001). No significant difference was found between groups II and III (P value = 0.365). Conclusions: Fluoride mouth rinse containing calcium glycerophosphate has a remineralization effect on demineralized primary enamel.

Long-termin vitroculture of 3D brain tissue model based on chitosan thermogel.

Methods for studying brain function and disease heavily rely onin vivoanimal models,ex-vivotissue slices, and 2D cell culture platforms. These methods all have limitations that significantly impact the clinical translatability of results. Consequently, models able to better recapitulate some aspects ofin vivohuman brain are needed as additional preclinical tools. In this context, 3D hydrogel-basedin vitromodels of the brain are considered promising tools. To create a 3D brain-on-a-chip model, a hydrogel capable of sustaining neuronal maturation over extended culture periods is required. Among biopolymeric hydrogels, chitosan-ß-glycerophosphate (CHITO-ß-GP) thermogels have demonstrated their versatility and applicability in the biomedical field over the years. In this study, we investigated the ability of this thermogel to encapsulate neuronal cells and support the functional maturation of a 3D neuronal network in long-term cultures. To the best of our knowledge, we demonstrated for the first time that CHITO-ß-GP thermogel possesses optimal characteristics for promoting neuronal growth and the development of an electrophysiologically functional neuronal network derived from both primary rat neurons and neurons differentiated from human induced pluripotent stem cells (h-iPSCs) co-cultured with astrocytes. Specifically, two different formulations were firstly characterized by rheological, mechanical and injectability tests. Primary nervous cells and neurons differentiated from h-iPSCs were embedded into the two thermogel formulations. The 3D cultures were then deeply characterized by immunocytochemistry, confocal microscopy, and electrophysiological recordings, employing both 2D and 3D micro-electrode arrays. The thermogels supported the long-term culture of neuronal networks for up to 100 d. In conclusion, CHITO-ß-GP thermogels exhibit excellent mechanical properties, stability over time under culture conditions, and bioactivity toward nervous cells. Therefore, they are excellent candidates as artificial extracellular matrices in brain-on-a-chip models, with applications in neurodegenerative disease modeling, drug screening, and neurotoxicity evaluation.

ß-TCP from 3D-printed composite scaffolds acts as an effective phosphate source during osteogenic differentiation of human mesenchymal stromal cells.

Introduction: Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are often combined with calcium phosphate (CaP)-based 3D-printed scaffolds with the goal of creating a bone substitute that can repair segmental bone defects. In vitro, the induction of osteogenic differentiation traditionally requires, among other supplements, the addition of ß-glycerophosphate (BGP), which acts as a phosphate source. The aim of this study is to investigate whether phosphate contained within the 3D-printed scaffolds can effectively be used as a phosphate source during hBM-MSC in vitro osteogenesis. Methods: hBM-MSCs are cultured on 3D-printed discs composed of poly (lactic-co-glycolic acid) (PLGA) and ß-tricalcium phosphate (ß-TCP) for 28 days under osteogenic conditions, with and without the supplementation of BGP. The effects of BGP removal on various cellular parameters, including cell metabolic activity, alkaline phosphatase (ALP) presence and activity, proliferation, osteogenic gene expression, levels of free phosphate in the media and mineralisation, are assessed. Results: The removal of exogenous BGP increases cell metabolic activity, ALP activity, proliferation, and gene expression of matrix-related (COL1A1, IBSP, SPP1), transcriptional (SP7, RUNX2/SOX9, PPARγ) and phosphate-related (ALPL, ENPP1, ANKH, PHOSPHO1) markers in a donor dependent manner. BGP removal leads to decreased free phosphate concentration in the media and maintained of mineral deposition staining. Discussion: Our findings demonstrate the detrimental impact of exogenous BGP on hBM-MSCs cultured on a phosphate-based material and propose ß-TCP embedded within 3D-printed scaffold as a sufficient phosphate source for hBM-MSCs during osteogenesis. The presented study provides novel insights into the interaction of hBM-MSCs with 3D-printed CaP based materials, an essential aspect for the advancement of bone tissue engineering strategies aimed at repairing segmental defects.

Corrigendum: Evidence for the cytoplasmic localization of the L-α-Glycerophosphate oxidase in members of the "Mycoplasma mycoides cluster".

[This corrects the article DOI: 10.3389/fmicb.2019.01344.].

Remineralization Effect of Calcium Glycerophosphate in Fluoride Mouth Rinse on Eroded Human Enamel: An In Vitro Study.

Aims and Objectives: The aim of this in vitro study was to evaluate the remineralization effect of calcium glycerophosphate (CaGP) in fluoride mouth rinse on permanent enamel eroded by a soft drink. Materials and Methods: Forty sound permanent premolars were embedded in self-curing acrylic resin and immersed in Coca-Cola to create erosive lesions. The teeth were divided into four groups (n = 10): Group I artificial saliva; Group II sodium fluoride; Group III sodium fluoride + sodium monofluorophosphate; and Group IV sodium monofluorophosphate + CaGP. The specimens in the assigned groups underwent pH cycling for ten days. The baseline, after erosion, and after remineralization surface microhardness (SMH) values were determined. The data were analyzed by one-way analysis of variance (ANOVA). The mean SMH value between groups and one-way repeated measures ANOVA for the mean SMH value within each group and Bonferroni's for multiple comparisons at a 95% confidence level were determined. The average SMH was used and calculated as the percentage recovery of SMH. Results: After being eroded by the cola soft drink, the mean SMH values in all groups were significantly decreased. After remineralization, Group I had the lowest %SMHR. The %SMHR of Groups II, III, and IV were significantly higher than Group I (P < 0.001). However, there were no significant differences among Groups II, III, and IV (P > 0.05). Conclusions: Fluoride mouth rinse with and without CaGP showed similar efficacies in remineralizing eroded permanent enamel.

Hydrogels and Nanoscaffolds for Long-Term Intraparenchymal Therapeutic Delivery After Stroke.

Stroke remains a leading cause of adult disability with treatments limited to thrombolytic therapies that are severely limited by a narrow therapeutic window. The potential of hundreds of other therapeutic agents cannot be evaluated due to their poor ability to cross the blood-brain barrier. Recently, biopolymer hydrogels have shown promise at overcoming these obstacles via the delivering of therapeutic molecules (pharmacological, mRNA, stem cells, etc.) to injured nervous tissue to afford functional recovery in rodent models of stroke. To date, we have tested different biopolymer hydrogels in mouse models of stroke for their ability to promote post-stroke recovery and for in situ delivery of growth factors, small pharmacological compounds, siRNAs, and stem cells. Here, we describe practical instructions on how to prepare various biopolymer hydrogels in house with further guidance on how to use them for intracerebral administration of therapeutic agents in preclinical stroke models.

Evaluation of different remineralization agents in the treatment of natural caries-affected dentin in permanent teeth.

BACKGROUND: In the preventive treatment protocol, providing remineralization of the tissue in demineralized dentin caries is an important step. OBJECTIVE: In this in vitro study, the effectiveness of remineralization agents in natural caries-affected dentin (NCAD) were investigated. METHODS: Forty caries slices were prepared from permanent molar dentin with International Caries Detection and Assessment System (ICDAS 2) (Code 3). The interventions with 8 days pH cycling were as follows: Deionized water (DW); 5% Sodium Fluoride (5% NaF) Varnish; Casein Phosphopeptide-Amorphous Calcium Fluoride Phosphate (CPP-ACFP); Calcium Glyserophosphate (CaGP) + Magnesium (Mg) + Xylitol. DIAGNOdent (Laser Fluorescence, LF), Surface Microhardness (SMH), and X-ray Fluorescence (XRF) Spectroscopy measurements were calculated before and after pH cycling. RESULTS: LF values decreased between 5% NaF, CCP-ACFP and CaGP. NCAD treated with 5% NaF, CaGP and CCP-ACFP exhibited statistically higher hardness compared to the control group. After 5% NaF application, SMH values were significantly higher than the others. There was no statistically significant difference between baseline and after pH cycling hardness of the control group. After cycling, XRF showed that Ca and P concentrations were increased in all groups. CONCLUSION: The application of agents used in the study could be recommended and promoted as a treatment option of caries dentin for conventional management of caries.

A Thermosensitive, Chitosan-Based Hydrogel as Delivery System for Antibacterial Liposomes to Surgical Site Infections.

Prophylaxis and the treatment of surgical site infections (SSIs) with antibiotics frequently fail due to the antibiotic resistance of bacteria and the ability of bacteria to reside in biofilms (i.e., bacterial clusters in a protective matrix). Therefore, alternative antibacterial treatments are required to combat biofilm infections. The combination of diethyldithiocarbamate (DDC-) and copper ions (Cu2+) exhibited antibiofilm activity against the staphylococci species associated with SSIs; however, the formation of a water-insoluble Cu(DDC)2 complex limits its application to SSIs. Here, we describe the development and antibiofilm activity of an injectable gel containing a liposomal formulation of Cu(DDC)2 and Cu2+ (lipogel). Lyophilized liposomes were incorporated into a mixture of chitosan (CS) and beta-glycerophosphate (ßGP), and the thermosensitive gelling properties of CS-ßGP and the lipogel were determined. The liposomes remained stable after lyophilization over six months at 4-6 °C and -20 °C. The sol-gel transition of the gel and lipogel occurred between 33 and 39 °C, independently of sterilization or storage at -20 °C. CS-ßGP is biocompatible and the liposomes were released over time. The lipogel prevented biofilm formation over 2 days and killed 98.7% of the methicillin-resistant Staphylococcus aureus and 99.9% of the Staphylococcus epidermidis biofilms. Therefore, the lipogel is a promising new prophylaxis and treatment strategy for local application to SSIs.

Reverse and Forward Electron Flow-Induced H2O2 Formation Is Decreased in α-Ketoglutarate Dehydrogenase (α-KGDH) Subunit (E2 or E3) Heterozygote Knock Out Animals.

α-ketoglutarate dehydrogenase complex (KGDHc), or 2-oxoglutarate dehydrogenase complex (OGDHc) is a rate-limiting enzyme in the tricarboxylic acid cycle, that has been identified in neurodegenerative diseases such as in Alzheimer's disease. The aim of the present study was to establish the role of the KGDHc and its subunits in the bioenergetics and reactive oxygen species (ROS) homeostasis of brain mitochondria. To study the bioenergetic profile of KGDHc, genetically modified mouse strains were used having a heterozygous knock out (KO) either in the dihydrolipoyl succinyltransferase (DLST+/-) or in the dihydrolipoyl dehydrogenase (DLD+/-) subunit. Mitochondrial oxygen consumption, hydrogen peroxide (H2O2) production, and expression of antioxidant enzymes were measured in isolated mouse brain mitochondria. Here, we demonstrate that the ADP-stimulated respiration of mitochondria was partially arrested in the transgenic animals when utilizing α-ketoglutarate (α-KG or 2-OG) as a fuel substrate. Succinate and α-glycerophosphate (α-GP), however, did not show this effect. The H2O2 production in mitochondria energized with α-KG was decreased after inhibiting the adenine nucleotide translocase and Complex I (CI) in the transgenic strains compared to the controls. Similarly, the reverse electron transfer (RET)-evoked H2O2 formation supported by succinate or α-GP were inhibited in mitochondria isolated from the transgenic animals. The decrease of RET-evoked ROS production by DLST+/- or DLD+/- KO-s puts the emphasis of the KGDHc in the pathomechanism of ischemia-reperfusion evoked oxidative stress. Supporting this notion, expression of the antioxidant enzyme glutathione peroxidase was also decreased in the KGDHc transgenic animals suggesting the attenuation of ROS-producing characteristics of KGDHc. These findings confirm the contribution of the KGDHc to the mitochondrial ROS production and in the pathomechanism of ischemia-reperfusion injury.

PTP1B Inhibition Improves Mitochondrial Dynamics to Alleviate Calcific Aortic Valve Disease Via Regulating OPA1 Homeostasis.

There are currently no pharmacological therapies for calcific aortic valve disease (CAVD). Here, we evaluated the role of protein tyrosine phosphatase 1B (PTP1B) inhibition in CAVD. Up-regulation of PTP1B was critically involved in calcified human aortic valve, and PTP1B inhibition had beneficial effects in preventing fibrocalcific response in valvular interstitial cells and LDLR-/- mice. In addition, we reported a novel function of PTP1B in regulating mitochondrial homeostasis by interacting with the OPA1 isoform transition in valvular interstitial cell osteogenesis. Thus, these findings have identified PTP1B as a potential target for preventing aortic valve calcification in patients with CAVD.

Androgen receptor functions in pericentral hepatocytes to decrease gluconeogenesis and avoid hyperglycemia and obesity in male mice.

BACKGROUND: Although the impact of hepatic androgen receptor (AR) pathway on liver pathogenesis was documented, its physiological function in normal liver is remained unclear. This study aims to investigate if hepatic AR acts on metabolism, the major liver function, using a hepatic-specific AR-transgenic (H-ARTG) mouse model. METHODS: We established the albumin promoter driven H-ARTG mice and included wild type (WT) and H-ARKO mice for study. The body weight, specific metabolic parameters and results from various tolerance tests were compared in different groups of mice fed a chow diet, from 2 to 18 months of age. Glucose feeding and insulin treatment were used to study the expression and zonal distribution pattern of AR and related genes in liver at different prandial stages. RESULTS: The body weight of H-ARTG mice fed a chow diet was 15 % lower than that of wild-type mice, preceded by lower blood glucose and liver triglyceride levels caused by AR reduced hepatic gluconeogenesis. The opposite phenotypes identified in H-ARKO and castrated H-ARTG mice support the critical role of activated AR in decreasing gluconeogenesis and triglyceride levels in liver. Hepatic AR acting by enhancing the expression of cytosolic glycerol-3-phosphate dehydrogenase (cGPDH), a key of glycerophosphate shuttle, was identified as one mechanism to decrease gluconeogenesis from glycerol. We further found AR normally expressed in zone 3 of hepatic lobules. Its level fluctuates dependent on the demand of glucose, decreased by fasting but increased by glucose uptake or insulin stimulation. CONCLUSION: AR is a newly identified zone 3 hepatic gene with function in reducing blood glucose and body weight in mice. It suggests that stabilization of hepatic AR is a new direction to prevent hyperglycemia, obesity and nonalcoholic fatty liver disease (NAFLD) in males.

Thermosensitive injectable hydrogel based on chitosan-polygalacturonic acid polyelectrolyte complexes for bone tissue engineering.

An extracellular matrix (ECM) mimicking a 3D microenvironment is an essential requirement to achieve desirable repair or regeneration of damaged tissue or organ. In this context, hydrogels may be able to create an appropriate 3D microenvironment. The lack of mechanical stability limits their application. This study prepared and characterized thermosensitive injectable hydrogels based on chitosan and polygalacturonic acid (PgA). A method of producing novel biomimetic polymeric-based injectable hydrogel using hydrothermal assisted hydrolysis is introduced. The synthesized hydrogels showed good compressive stiffness. We have also studied the possible chemistry of the materials in the hydrogel network. The biocompatibility and gelation time of the hydrogel was optimized by adding ß-glycerophosphate (ßGP) and hydroxyapatite. The synthesized liquid formulation can turn into gel at 37 °C. The biocompatibility for MG63 cells within 3D hydrogels was investigated. Scanning electron microscopy revealed that the PEC fibers are uniformly distributed in the hydrogel matrix. MTT assay and confocal imaging were employed to observe cytotoxicity and proliferation of cells cultured in the hydrogels with and without an osteogenic medium. Alkaline phosphatase activity (ALP) and collagen production in cell-cultured hydrogel were also measured to evaluate osteoblast activity. The cellular responses to various types of hydrogels cultured at a 14-day culture appeared to be superior in the hydrogels with gelatin incorporated and hydrothermally treated PEC fibers. These results indicated that hydrothermal treatment and inclusion of gelatin in the chitosan-ßGP hydrogel system enhanced the hydrogel bioactivity and mechanical properties. Overall, improved cellular proliferation, osteogenic differentiation, and stable physical network with uniform distribution of fibrous matrix in-vitro were achieved.

Effect of Remineralization Agents on Molar-Incisor Hypomineralization-Affected Incisors: A Randomized Controlled Clinical Trial.

OBJECTIVE: The objective of this study was to evaluate the remineralization effect of two different mineral containing agents on white/creamy and yellow/brown demarcated opacities in incisors in children with molar-incisor hypomineralization (MIH) by using laser fluoresence (LF). STUDY DESIGN: Fifty-three children (n=401 lesions) with MIH were randomly divided into three groups: (1)calcium glycerophosphate (CaGP), (2)casein phosphopeptide amorphous calcium fluoride phosphate (CPP-ACFP) and, (3)control (1450 ppm fluoride toothpaste). Remineralization was evaluated by means of LF, at baseline, after one and threemonths. Anova Test for Repeated Measurements in intra-group comparisons in evaluating the effectiveness of remineralization agents. One-way Variance Analysis (ANOVA) and Tukey-Kramer Multiple Comparison test were used in the comparisons between groups and, Student Newman Keuls Multpile Comparison Test was used to determine the differences between the measurement averages in case of p<0.05. RESULTS: There was a significant improvement in MIH-lesions over time in all groups (p<0.001), with no differences between groups. The highest percentage of change was observed in CPP-ACFP in lesions LF≤20 scores and the mean percentage of change LF>20 scores, the highest percentage changes in CaGP. There was no significant difference between the groups over the time for all the used outcome measures (p>0.05). CONCLUSION: The additional use of both mineral containing agents in MIH-affected teeth improved these hypomineralized lesions with mineral deposition. Even if both agents could be used in the hypomineralized teeth with demarcated opacities, future studies are recommended the long-term effect of these mineral containing agents with longer observation and a larger sample size.

Focal adhesion kinase inhibitors prevent osteoblast mineralization in part due to suppression of Akt-mediated stabilization of osterix.

Focal Adhesion Kinase (FAK) is an important regulator of tumor cell proliferation, survival and metastasis. As such it has become a therapeutic target of interest in cancer. Previous studies suggested that use of FAK tyrosine kinase inhibitors (TKIs) blocks osteolysis in in vivo models of bone metastasis. However, from these studies it was not clear whether FAK TKIs blocked bone degradation by osteoclasts or also promoted bone formation by osteoblasts. In this study we evaluated whether use of the FAK TKI PF-562,271 affected the differentiation of pre-osteoblasts, or activity of mature differentiated osteoblasts. MC3T3-E1 pre-osteoblastic cells were treated with various doses of PF-562,271 following 3 or 10 days of differentiation which led to the inhibition of alkaline phosphatase (ALP) expression and reduced viable cell numbers in a dose-dependent manner. MC3T3-E1 cells which had been differentiated for 21 days prior to treatment with PF-562,271 showed a dose dependent decrease in mineralization as assessed by Alizarin Red staining, with concomitant decreased expression of ALP which is known to facilitate the bone mineralization activity of osteoblasts, however mRNA levels of the transcription factors RUNX2 and osterix which are important for osteoblast maturation and mineralization appeared unaffected at this time point. We speculated that this may be due to altered function of RUNX2 protein due to inhibitory phosphorylation by GSK3ß. We found treatment with PF-562,271 resulted in increased GSK3ß activity as measured by reduced levels of phospho-Ser9-GSK3ß which would result in phosphorylation and inhibition of RUNX2. Treatment of 21 day differentiated MC3T3-E1 cells with PF-562,271 in combination with GSK3ß inhibitors partially restored mineralization however this was not statistically significant. As we observed that FAK TKI also resulted in suppression of Akt, which is known to alter osterix protein stability downstream of RUNX2, we examined protein levels by western blot and found a dose-dependent decrease in osterix in FAK TKI treated differentiated MC3T3-E1 cells which is likely responsible for the reduced mineralization observed. Taken together our results suggest that use of FAK TKIs as therapeutics in the bone metastatic setting may block new bone formation as an off-target effect and thereby exacerbate the defective bone regulation that is characteristic of the bone metastatic environment.

Rho kinase inhibitor induced human dental pulp stem cells to differentiate into neurons.

AIMS: Neurological diseases due to neuron loss have become major public health problems. Current treatment reduces symptoms; however, there is no cure for neurological diseases. Therefore, stem cells may be an alternative therapy. Human dental pulp stem cells (hDPSCs) are an attractive source for cell-based approaches due to their high regenerative potential. The Rho kinase (ROCK) inhibitor Y-27632 promoted the neuronal differentiation of several stem cell types. However, its neuronal-inductive effect on hDPSCs has not been reported. Thus, the aim of our study was to investigate whether Y-27632 can induce the neuronal differentiation of hDPSCs. MAIN METHODS: hDPSCs were isolated from human third molars using an enzymatic method and were subsequently characterized. Cytotoxicity was evaluated using an MTT assay. The optimal concentration to induce neural differentiation was assessed using 1-50 µM Y-27632 as evaluated by Cresyl violet and immunofluorescence staining of neurofilaments and ßIII-tubulin, respectively. Ten µM Y-27632 was used for neuronal induction for 72 h, and differentiation was confirmed based on the expression of neurogenic markers (MAP2, Brn3a, and ChAT) and intracellular calcium activity. KEY FINDINGS: Our findings indicate that Y-27632 was not cytotoxic to hDPSCs and 10 µM Y-27632 was the lowest concentration that induced the morphological changes of hDPSCs into neuronal cells with Cresyl violet-positive staining and significantly enhanced the fluorescence intensity of neurofilament and ßIII-tubulin. The neuronal genes' expression and intracellular calcium activity were upregulated after induction with Y-27632. SIGNIFICANCE: At the optimal concentration and time, Rho kinase inhibitor induces hDPSC differentiation into neuronal cells.

Exploring Thermal Sensitivities and Adaptations of Oxidative Phosphorylation Pathways.

Temperature shifts are a major challenge to animals; they drive adaptations in organisms and species, and affect all physiological functions in ectothermic organisms. Understanding the origin and mechanisms of these adaptations is critical for determining whether ectothermic organisms will be able to survive when faced with global climate change. Mitochondrial oxidative phosphorylation is thought to be an important metabolic player in this regard, since the capacity of the mitochondria to produce energy greatly varies according to temperature. However, organism survival and fitness depend not only on how much energy is produced, but, more precisely, on how oxidative phosphorylation is affected and which step of the process dictates thermal sensitivity. These questions need to be addressed from a new perspective involving a complex view of mitochondrial oxidative phosphorylation and its related pathways. In this review, we examine the effect of temperature on the commonly measured pathways, but mainly focus on the potential impact of lesser-studied pathways and related steps, including the electron-transferring flavoprotein pathway, glycerophosphate dehydrogenase, dihydroorotate dehydrogenase, choline dehydrogenase, proline dehydrogenase, and sulfide:quinone oxidoreductase. Our objective is to reveal new avenues of research that can address the impact of temperature on oxidative phosphorylation in all its complexity to better portray the limitations and the potential adaptations of aerobic metabolism.

Effect of casein phosphopeptide amorphous calcium fluoride phosphate and calcium glycerophosphate on incisors with molar-incisor hypomineralization: A cross-over, randomized clinical trial.

BACKGROUND: Within the scope of minimally invasive dentistry, the use of different biocompatible remineralization agents on incisors affected by molar-incisor hypomineralization (MIH) gains importance. OBJECTIVE: To evaluate the effect of casein phosphopeptide amorphous calcium fluoride phosphate (CPP-ACFP) and calcium glycerophosphate (CaGP) in mineral density (MD) of white/creamy and yellow/brown demarcated opacities on incisors affected by MIH by means laser fluorescence (LF). METHODS: As a cross-over, randomized trial, twenty-two children with 167 incisors affected by MIH were recruited and randomly assigned to one of the two different agents and crossed over to other agents with two weeks washout in between. Incisors were examined by using LF at all before and after three months periods. RESULTS: The results of the paired t-tests for determining the period effect between the baseline findings showed significant difference in white/creamy and yellow/brown demarcated opacities of LF values for both groups (p < 0.05). The difference between both groups according to after categorization of 20% increasing in MD in the percent of change before and after application on LF values; was not found statistically significant in white/creamy (p = 0.970) and yellow/brown (p = 0.948) opacities. CONCLUSIONS: The primary outcome was CPP-ACFP and CaGP had a positive effect in decreasing hypomineralization on MIH-affected enamel for three months period.