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In this study, we employed a combination of electrospinning and electrospray techniques to fabricate wound dressings with a particle-fiber structure, providing dual characteristics of oxygen-releasing and intrinsic antioxidant properties, simultaneously. The electrospun part of the dressing was prepared from a blend of polycaprolactone/gallic acid-grafted-gelatin (GA-g-GE), enabling intrinsic ROS scavenging. To the best of our knowledge, this is the first time that PCL/GA-g-GE was fabricated by electrospinning. Furthermore, polyvinyl pyrrolidone (PVP) microparticles, containing calcium peroxide nanoparticles (CNPs), were considered as the oxygen production agent through the electrospray part. The CNP content was 1% and 3% w/w of PVP while biopolymer:PCL was 10% w/w. The fabricated structures were characterized in terms of fiber/particle morphology, elemental analysis, oxygen release behavior, ROS inhibition capacity, and water contact angle assessments. The covalent bonding of gallic acid to gelatin was confirmed by 1H-NMR, UV spectroscopy, and FTIR. According to the SEM results, the morphology of the prepared PCL/biopolymer fibers was bead-free and with a uniform average diameter. The analysis of released oxygen showed that by increasing the weight percentage of CNPs from 1 to 3 wt%, the amount of released oxygen increased from 120 mmHg to 195 mmHg in 24 h, which remained almost constant until 72 h. The obtained DPPH assay results revealed that the introduction of GA-g-GE into the fibrous structure could significantly improve the antioxidant properties of wound dressing compared to the control group without CNPs and modified gelatine. In vitro, the fabricated wound dressings were evaluated in terms of biocompatibility and the potential of the dressing to protect human dermal fibroblasts under oxidative stress and hypoxia conditions by an MTT assay. The presence of GA-g-GE led to remarkable protection of the cells against oxidative stress and hypoxia conditions. In vivo studies revealed that the incorporation of intrinsic ROS inhibition and oxygen-releasing properties could significantly accelerate the wound closure rate during the experimental period (7, 14, and 21 days). Additionally, histopathological investigations in terms of H&E and Masson's trichrome staining showed that the incorporation of the two mentioned capabilities remarkably facilitated the wound-healing process.
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Background: The cartilage tissue lacks blood vessels, which is composed of chondrocytes and ECM. Due to this vessel-less structure, it is difficult to repair cartilage tissue damages. One of the new methods to repair cartilage damage is to use tissue engineering. In the present study, it was attempted to simulate a three-dimensional environment similar to the natural ECM of cartilage tissue by using hydrogels made of natural materials, including Chitosan and different ratios of Alginate. Material and methods: Chitosan, alginate and Chitosan/Alginate hydrogels were fabricated. Fourier Transform Infrared, XRD, swelling ratio, porosity measurement and degradation tests were applied to scaffolds characterization. After that, human adipose derived-mesenchymal stem cells (hADMSCs) were cultured on the hydrogels and then their viability and chondrogenic differentiation capacity were studied. Safranin O and Alcian blue staining, immunofluorescence staining and real time RT-PCR were used as analytical methods for chondrogenic differentiation potential evaluation of hADMSCs when cultured on the hydrogels. Results: The highest degradation rate was detected in Chitosan/Alginate (1:0.5) group The scaffold biocompatibility results revealed that the viability of the cells cultured on the hydrogels groups was not significantly different with the cells cultured in the control group. Safranin O staining, Alcian blue staining, immunofluorescence staining and real time PCR results revealed that the chondrogenic differentiation potential of the hADMSCs when grown on the Chitosan/Alginate hydrogel (1:0.5) was significantly higher than those cell grown on the other groups. Conclusion: Taken together, these results suggest that Chitosan/Alginate hydrogel (1:0.5) could be a promising candidate for cartilage tissue engineering applications.
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OBJECTIVE: Mesenchymal stem cells (MSCs) are widely recognized as a promising cell type for therapeutic applications due to their ability to secrete and regenerate bioactive molecules. For effective bone healing, it is crucial to select a scaffold that can support, induce, and restore biological function. Evaluating the scaffold should involve assessing MSC survival, proliferation, and differentiation. The principal aim of this investigation was to formulate composite nanofibrous scaffolds apt for applications in bone tissue engineering. MATERIALS AND METHODS: In this experimental study, nanofibrous scaffolds were fabricated using Poly-L-lactic acid (PLLA) polymer. The PLLA fibers' surface was modified by integrating collagen and hydroxyapatite (HA) nanoparticles. RESULTS: The findings demonstrated that the collagen- and nanohydroxyapatite-modified electrospun PLLA scaffold positively influenced the attachment, growth, and osteogenic differentiation of MSCs. CONCLUSION: Coating the nanofiber scaffold with collagen and nanoparticle HA significantly enhanced the osteogenic differentiation of MSCs on electrospun PLLA scaffolds.
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Vaccine potency is typically evaluated using an assay that acts as a surrogate for biological activity. Although in vivo vaccines better represent human immunological responses, in vitro assays are preferred due to lower variability, higher throughput, easier validation and ethical considerations. In in vitro determination of Human Papillomavirus (HPV), Virus-like particle (VLP) vaccine potency currently depends on monoclonal antibody assays. However, these reagents are hard to obtain and currently are not available commercially. In this work, a polyclonal antiserum-based immunoassay was developed to evaluate the relative potency of Alhydrogel formulated HPV 16 VLPs. The repeatability and specificity were evaluated, and found that the assay was sensitive to small amounts of non-VLP HPV 16 L1 proteins. Finally, the assay was tested in comparison to the mouse effective dose 50 (ED50) assay on a limited number of batches. The agreement between these results suggests this test as a suitable surrogate for the in vivo test.
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Infecciones por Papillomavirus , Vacunas contra Papillomavirus , Animales , Ratones , Humanos , Papillomavirus Humano 16 , Anticuerpos Antivirales , Inmunoensayo/métodos , Proteínas de la CápsideRESUMEN
Nanofibrous scaffolds have attracted much attention in bladder reconstruction approaches due to their excellent mechanical properties. In addition, their biological properties can be improved by combination with biological materials. Taking into account the advantages of nanofibrous scaffolds and decellularized extracellular matrix (dECM) in tissue engineering, scaffolds of poly-L-lactic acid (PLLA) coated with decellularized human amnion membrane (hAM) or sheep bladder (SB)-derived ECM proteins are developed (amECM-coated PLLA and sbECM-coated PLLA, respectively). The bladder regenerative potential of modified electrospun PLLA scaffolds is investigated in rabbits. The presence of ECM proteins is confirmed on the nanofibers' surface. Coating the surface of the PLLA nanofibers improves cell adhesion and proliferation. Histological and immunohistochemical evaluations show that rabbits subjected to cystoplasty with a multilayered PLLA scaffold show de novo formation and maturation of the multilayered urothelial layer. However, smooth muscle bundles (myosin heavy chain [MHC] and α-smooth muscle actin [α-SMA] positive) are detected only in ECM-coated PLLA groups. All groups show no evidence of a diverticulumor fistula in the urinary bladder. These results suggest that the biofunctionalization of electrospun PLLA nanofibers with ECM proteins can be a promising option for bladder tissue engineering. Furthermore, hAM can also replace animal-sourced ECM proteins in bladder tissue regeneration approaches.
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Nanofibras , Andamios del Tejido , Humanos , Conejos , Animales , Ovinos , Andamios del Tejido/química , Nanofibras/química , Vejiga Urinaria , Amnios , Ingeniería de Tejidos/métodos , Poliésteres/farmacología , Poliésteres/química , Proteínas de la Matriz Extracelular , Músculo LisoRESUMEN
Self-healing and autologous bone graft of calvaraial defects can be challenging. Therefore, the fabrication of scaffolds for its rapid and effective repair is a promising field of research. This paper provided a comparative study on the ability of Three-dimensional (3D) printed polycaprolactone (PCL) scaffolds and PCL-modified with the hydroxyapatite (HA) and bioglasses (BG) bioceramics scaffolds in newly bone formed in calvaria defect area. The studied 3D-printed PCL scaffolds were fabricated by fused deposition layer-by-layer modeling. After the evaluation of cell adhesion on the surface of the scaffolds, they were implanted into a rat calvarial defect model. The rats were divided into four groups with scaffold graft including PCL, PCL/HA, PCL/BG, and PCL/HA/BG and a non-explant control group. The capacity of the 3D-printed scaffolds in calvarial bone regeneration was investigated using micro computed tomography scan, histological and immunohistochemistry analyses. Lastly, the expression levels of several bone related genes as well as the expression of miR-20a and miR-17-5p as positive regulators and miR-125a as a negative regulator in osteogenesis pathways were also investigated. The results of this comparative study have showed that PCL scaffolds with HA and BG bioceramics have a great range of potential applications in the field of calvaria defect treatment.
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MicroARNs , Andamios del Tejido , Ratas , Animales , Microtomografía por Rayos X , Osteogénesis , Regeneración Ósea , Durapatita/farmacología , Cráneo/diagnóstico por imagen , Impresión Tridimensional , MicroARNs/farmacología , Poliésteres/farmacología , Ingeniería de Tejidos/métodosRESUMEN
The developing fetus is wrapped by a human amniotic membrane or amnion. Amnion is a promising human tissue allograft in clinical application because of its chemical composition, collagen-based, and mechanical properties of the extracellular matrix. In addition, amnion contains cells and growth factors; therefore, meets the essential parameters of tissue engineering. No donor morbidity, easy processing and storage, fewer ethical issue, anti-inflammatory, antioxidant, antibacterial, and non-immunogenic properties are other advantages of amnion usage. For these reasons, amnion can resolve some bottlenecks in the regenerative medicine issues such as tissue engineering and cell therapy. Over the last decades, biomedical applications of amnion have evolved from a simple sheet for skin or cornea repair to high-technology applications such as amnion nanocomposite, powder, or hydrogel for the regeneration of cartilage, muscle, tendon, and heart. Furthermore, amnion has anticancer as well as drug/cell delivery capacity. This review highlights various ancient and new applications of amnion in research and clinical applications, from regenerative medicine to cancer therapy, focusing on articles published during the last decade that also revealed information regarding amnion-based products. Challenges and future perspectives of the amnion in regenerative medicine are also discussed.
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Amnios , Medicina Regenerativa , Humanos , Amnios/química , Ingeniería de Tejidos , PielRESUMEN
Background: A combination of bioceramics and polymeric materials has attracted the research community's interest in bone tissue engineering. These composites are essential to support cell attachment, proliferation, and osteogenesis differentiation, which are vital as a classic strategy in bone tissue engineering. In this study, NiFe2O4/ZnO-coated poly L-Lactide (PLLA) was employed as a scaffold to evaluate the osteogenic differentiation capability of human adipose tissue derived mesenchymal stem cells (hAMSCs). Material and methods: The electrospun PLLA nanofibers were fabricated, coated with nanocomposite (NiFe2O4/ZnO), and evaluated by the water contact angle (WCA), tensile test, attenuated total reflectance fourier-transform infrared (ATR-FTIR) and scanning electron microscopy (SEM). Then, the osteogenic differentiation potential of hAMSCs was assessed using NiFe2O4/ZnO-coated PLLA compared to tissue culture plastic (TCP) and a simple scaffold (PLLA) in vitro conditions. Results: The adhesion, proliferation, and differentiation of hAMSCs were supported by the mechanical and biological properties of the NiFe2O4/ZnO-coated PLLA scaffold, according to SEM and 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining patterns. During bone differentiation, Alkaline phosphatase (ALP) enzyme activity, biomineralization, calcium content, and osteogenic gene expression (ALP, Osteonectin, Osteocalcin, Collagen type I, and Runx2) were higher on NiFe2O4/ZnO-coated PLLA scaffold than on PLLA scaffold and TCP. Conclusion: Based on our results, the osteogenic differentiation of hAMSCs on the improved biological scaffold (PLLA coated with NiFe2O4/ZnO) could accelerate due to the stimulating effect of this nanocomposite.
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BACKGROUND: Glioblastoma multiforme (GBM) is an aggressive and lethal brain cancer, which is incurable with standard cancer treatments. miRNAs have great potential to be used for gene therapy due to their ability to modulate several target genes simultaneously. We found miR-429 is downregulated in GBM and has several predicted target genes from the ERBB signaling pathway using bioinformatics tools. ERBB is the most over-activated genetic pathway in GBM patients, which is responsible for augmented cell proliferation and migration in GBM. METHODS AND RESULTS: Here, miR-429 was overexpressed using lentiviral vectors in U-251 and U-87 GBM cells and it was observed that the expression level of several oncogenes of the ERBB pathway, EGFR, PIK3CA, PIK3CB, KRAS, and MYC significantly decreased, as shown by real-time PCR and western blotting. Using the luciferase assay, we showed that miR-429 directly targets MYC, BCL2, and EGFR. In comparison to scrambled control, miR-429 had a significant inhibitory effect on cell proliferation and migration as deduced from MTT and scratch wound assays and induced cell-cycle arrest and apoptosis in flow cytometry. CONCLUSIONS: Altogether, miR-429 seems to be an efficient suppressor of the ERBB genetic signaling pathway and a potential therapeutic for GBM.
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Neoplasias Encefálicas , Glioblastoma , MicroARNs , Humanos , Glioblastoma/genética , Glioblastoma/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Línea Celular Tumoral , Neoplasias Encefálicas/metabolismo , Puntos de Control del Ciclo Celular/genética , Apoptosis/genética , MicroARNs/genética , Proliferación Celular/genética , Transducción de Señal/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Movimiento Celular/genéticaRESUMEN
Bone tissue engineering uses various methods and materials to find suitable scaffolds that regenerate lost bone due to disease or injury. Poly(ε-caprolactone) (PCL) can be used in 3D printing for producing biodegradable scaffolds by fused deposition modeling (FDM). However, the hydrophobic surfaces of PCL and its non-osteogenic nature reduces adhesion and cell bioactivity at the time of implantation. This work aims to enhance bone formation, osteogenic differentiation, and in vitro biocompatibility via PCL scaffolds modification with Hydroxyapatite (HA) and Collagen type I (COL). This study evaluated the osteosupportive capacity, biological behavior, and physicochemical properties of 3D-printed PCL, PCL/HA, PCL/COL, and PCL/HA/COL scaffolds. Biocompatibility and cells proliferation were investigated by seeding human adipose tissue-derived mesenchymal stem cells (hADSCs) onto the scaffolds, which were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and 6-diamidino-2-phenylindole (DAPI) staining. In addition, the bone differentiation potential of the hADSCs was assessed using calcium deposition, alkaline phosphatase (ALP) activity, and bone-related protein and genes. Although all constructed scaffolds support hADSCs proliferation and differentiation, the results showed that scaffold coating with HA and COL can boost these capacities in a synergistic manner. According to the findings, the tricomponent 3D-printed scaffold can be considered as a promising choice for bone tissue regeneration and rebuilding.
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Durapatita , Osteogénesis , Colágeno/química , Durapatita/química , Durapatita/farmacología , Humanos , Poliésteres/química , Impresión Tridimensional , Células Madre , Andamios del Tejido/químicaRESUMEN
Background: Freeze dried bone allograft nanoparticles on a nanofiber membrane may serve as an ideal scaffold for bone regeneration. This study aimed to assess the biological behavior of human mesenchymal stem cells (MSCs) in terms of proliferation and adhesion to nanoparticulate and microparticulate freeze dried bone allograft (FDBA) scaffolds on poly-L-lactic acid (PLLA) nanofiber membrane. Methods: In this experimental study, PLLA nanofiber scaffolds were synthesized by the electrospinning method. The FDBA nanoparticles were synthesized mechanically. The FDBA nanoparticles and microparticles were loaded on the surface of PLLA nanofiber membrane. A total of 64 scaffold samples in four groups of n-FDBA/PLLA, FDBA/PLLA, PLLA and control were placed in 24-well polystyrene tissue culture plates; 16 wells were allocated to each group. Data were analyzed using one-way ANOVA and Bonferroni test. Results: The proliferation rate of MSCs was significantly higher in the nanoparticulate group compared to the microparticulate group at five days (p = 0.034). Assessment of cell morphology at 24 hours revealed spindle-shaped cells with a higher number of appendages in the nanoparticulate group compared to other groups. Conclusion: MSCs on n-FDBA/PLLA scaffold were morphologically more active and flatter with a higher number of cellular appendages, as compared to FDBA/PLLA. It seems that the nanoparticulate scaffold is superior to the microparticulate scaffold in terms of proliferation, attachment, and morphology of MSCs in vitro.
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Células Madre Mesenquimatosas , Nanofibras , Nanopartículas , Proliferación Celular , Humanos , Osteogénesis , Poliésteres/farmacología , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Three dimensional (3D) substrates based on natural and synthetic polymers enhance the osteogenic and mechanical properties of the bone tissue engineering scaffolds. Here, a novel bioactive composite scaffolds from polycaprolactone /kappa-carrageenan were developed for bone regeneration applications. 3D PCL scaffolds were fabricated by 3D printing method followed by coating with carboxymethyl kappa-carrageenan. This organic film was used to create calcium and strontium phosphate layers via a modified alternate soaking process in CaCl 2 /SrCl 2 and Na2HPO4 solutions in which calcium ions were replaced by strontium, with different amounts of strontium in the solutions. Various characterization techniques were executed to analyze the effects of strontium ion on the scaffold properties. The morphological results demonstrated the highly porous with interconnected pores and uniform pore sizes scaffolds. It was indicated that the highest crystallinity and compressive strength were obtained when 100% CaCl2 was replaced by SrCl2 in the solution (P-C-Sr). Incorporation of Sr onto the structure increased the degradation rate of the scaffolds. Mesenchymal stem cells (MSCs) culture on the scaffolds showed that Sr effectively improved attachment and viability of the MSCs and accelerated osteogenic differentiation as revealed by Alkaline phosphatase activity, calcium content and Real Time-Reverse transcription polymerase chain reaction assays.
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Calcio , Osteogénesis , Adsorción , Regeneración Ósea , Calcio/farmacología , Fosfatos de Calcio/farmacología , Carragenina/farmacología , Fosfatos , Poliésteres , Impresión Tridimensional , Estroncio/química , Ingeniería de Tejidos , Andamios del Tejido/químicaRESUMEN
Background: Inflammatory bone resorption in periodontitis can lead to tooth loss. Systemic administration of bisphosphonates such as risedronate for preventing bone resorption can cause adverse effects. Alginate hydrogel (ALG) and poly (lactic acid-co-glycolic acid) (PLGA) microparticles have been studied as drug delivery systems for sustained release of drugs. Therefore, the release pattern of risedronate from PLGA microparticles embedded with ALG was studied as a drug delivery system for sustained release of the drug, which can be used in local administrations. Methods: Risedronate-containing PLGA microparticles were fabricated using double emulsion solvent evaporation technique. Ionic cross-linking method was used to fabricate risedronate-loaded ALG. Risedronate-containing PLGA microparticles were then coated with ALG. The calibration curve of risedronate was traced to measure encapsulation efficiency (EE) and study the release pattern. Scanning electron microscope (SEM) imaging was carried out, and cell toxicity was examined using MTT assay. Statistical analysis of data was carried out using SPSS ver. 20 software, via one-way ANOVA and Tukey's tests. Results: SEM imaging showed open porosities on ALGs. The mean EE of PLGA microparticles for risedronate was 57.14 ± 3.70%. Risedronate released completely after 72 h from ALG, and the cumulative release was significantly higher (p = 0.000) compared to PLGA microspheres coated with ALG, which demonstrated sustained released of risedronate until day 28. Risedronate-loaded ALG showed a significant decrease in gingival fibroblasts cell viability (p < 0.05). Conclusion: Alginate-coated PLGA microspheres could release risedronate in a sustained and controlled way and also did not show cell toxicity. Therefore, they seem to be an appropriate system for risedronate delivery in local applications.
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Alginatos/química , Enfermedades Óseas/tratamiento farmacológico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Ácido Risedrónico/metabolismo , Línea Celular , Preparaciones de Acción Retardada/metabolismo , Hidrogeles/química , MicroesferasRESUMEN
INTRODUCTION: Antimicrobial photodynamic therapy (aPDT) is an adjunctive non-invasive procedure for the management of periodontal tissue infection and deep periodontal pocket decontamination. However, the effects of this procedure on periodontal cells like osteoblasts that play a role in periodontal tissue repair and regeneration is not yet clear.
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Fotoquimioterapia , Proyectos de Investigación , Humanos , Osteoblastos , PeriodoncioRESUMEN
BACKGROUND: Biocompatibility and induction of mineralized tissue formation are the properties expected from a material used in vital pulp therapy and repair of perforations. Cold ceramic (SJM, Iran; CC) is a newly introduced calcium silicate-based cement for above mentioned therapeutic applications. This in-vitro study aimed to compare the effect of CC and White MTA-Angelus (MTA) on cell viability, attachment, odontogenic differentiation, and calcification potential of human dental pulp stem cells (DPSCs) and periodontal ligament fibroblasts (PDLFs). METHODS: Cell viability of DPSCs and PDLFs was assessed using MTT on days 1, 3, 7, and 14 (n = 9) in contact with freshly mixed and set states of CC and MTA. Field emission scanning electron micrographs (FESEM) were taken to evaluate cell-bioceramic interaction (n = 6). Gene expression levels of osteo/odontogenic markers (Dentin sialophosphoprotein, Dentin matrix protein 1, Collagen type I alpha 1, and Alkaline phosphatase (DSPP, DMP1, COL 1A1, and ALP, respectively) (n = 8) were assessed using qrt-PCR. ALP enzymatic activity was evaluated to assess the mineralization potential. A two-way ANOVA test was applied, and p < 0.05 was considered to be statistically significant. RESULTS: The effect of freshly mixed and set MTA and CC on the survival of DPSCs and PDLFs in all study groups was statistically similar and comparable to the positive control group (p > 0.05); the only exception was for the viability of PDLFs in contact with freshly mixed cements on day 1, showing a more significant cytotoxic effect compared to the control and the set state of materials (p < 0.05). PDLFs attached well on CC and MTA. The spread and pseudopodium formation of the cells increased on both samples from day 1 to day 14. Contact of MTA and CC with DPSCs similarly increased expression of all dentinogenesis markers studied on days 7 and 14 compared to the control group (p < 0.001), except for DSPP expression on day 7 (p = 0.46 and p = 0.99 for MTA and CC, respectively). CONCLUSIONS: Within the limitation of this in-vitro study, cold ceramic and MTA-Angelus showed high biocompatibility and induced increased expression of osteo/dentinogenic markers. Therefore, cold ceramic can be a suitable material for vital pulp therapy and the repair of root perforations.
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Compuestos de Calcio , Pulpa Dental , Compuestos de Aluminio/farmacología , Bismuto , Compuestos de Calcio/farmacología , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Cerámica , Combinación de Medicamentos , Fibroblastos , Humanos , Óxidos/farmacología , Ligamento Periodontal , Silicatos/farmacología , Células MadreRESUMEN
Introduction: This study aimed to compare the effects of root biomodification by citric acid and antimicrobial photodynamic therapy (aPDT) with LED and laser on the proliferation of human gingival fibroblasts (HGFs). Methods: This in vitro experimental study evaluated 60 single-rooted teeth extracted due to periodontal disease. The teeth underwent scaling and root planing (SRP), and then 5 × 5 mm blocks were prepared from the cervical area of the teeth 1 mm apical to the cementoenamel junction. The blocks were divided into 4 groups (n=15 blocks): SRP alone (control), SRP + citric acid, SRP + toluidine blue (TBO) + LED light, and SRP + TBO + laser. HGFs were seeded on the surface of the samples, and the methyl thiazolyl tetrazolium (MTT) assay was performed after 24, 48 and 72 hours. Group comparisons were performed using repeated measures ANOVA, while pairwise comparisons of the time points were performed by an LSD test. Results: Cell proliferation was higher in all experimental groups at 48 and 72 hours, compared with 24 hours (P < 0.05). Cell proliferation was significantly different in the citric acid group at 24 hours (P = 0.016) and 48 hours (P = 0.015), compared with other groups. However, cell proliferation was not significantly different in the aPDT group with LED Photosan and a diode laser at 24 and 48 hours (P > 0.05). Conclusion: aPDT and citric acid can enhance the proliferation of HGFs on dentin blocks. Further studies can pave the way for their future use in the clinical setting.
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Steroid-associated osteonecrosis (SAON) is a chronic disease that leads to the destruction and collapse of bone near the joint that is subjected to weight bearing, ultimately resulting in a loss of hip and knee function. Zn2+ ions, as an essential trace element, have functional roles in improving the immunophysiological cellular environment, accelerating bone regeneration, and inhibiting biofilm formation. In this study, we reconstruct SAON lesions with a three-dimensional (3D)-a printed composite made of poly (epsilon-caprolactone) (PCL) and nanoparticulate Willemite (npW). Rabbit bone marrow stem cells were used to evaluate the cytocompatibility and osteogenic differentiation capability of the PCL/npW composite scaffolds. The 2-month bone regeneration was assessed by a Micro-computed tomography (micro-CT) scan and the expression of bone regeneration proteins by Western blot. Compared with the neat PCL group, PCL/npW scaffolds exhibited significantly increased cytocompatibility and osteogenic activity. This finding reveals a new concept for the design of a 3D-printed PCL/npW composite-based bone substitute for the early treatment of osteonecrosis defects.
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Regeneración Ósea/efectos de los fármacos , Fémur/efectos de los fármacos , Nanopartículas/administración & dosificación , Osteogénesis/efectos de los fármacos , Poliésteres/farmacología , Andamios del Tejido/química , Animales , Caproatos/farmacología , Diferenciación Celular/efectos de los fármacos , Lactonas/farmacología , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Osteonecrosis/tratamiento farmacológico , Impresión Tridimensional , Conejos , Silicatos/farmacología , Ingeniería de Tejidos/métodos , Microtomografía por Rayos X/métodos , Compuestos de Zinc/farmacologíaRESUMEN
Glioblastoma is a lethal and incurable cancer. Tumor suppressor miRNAs are promising gene therapy tools for cancer treatment. In silico, we predicted miR-424 as a tumor suppressor. It had several target genes from the epidermal growth factor receptor (ERBB) signaling pathway that are overactive in most glioblastoma cases. We overexpressed miR-424 by lentiviral transduction of U-251 and U-87 glioblastoma cells confirmed with fluorescent microscopy and real-time quantitative PCR (qRT-PCR). Then the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation assay and scratch wound migration assay were performed to investigate the miR-424 tumor suppressor effect in glioblastoma. miR-424's effect on glioblastoma apoptosis and cell-cycle arrest was verified using Annexin V- phosphatidylethanolamine (PE) and 7-minoactinomycin D (7-AAD) apoptosis assay and cell-cycle assay. miR-424 predicted target genes mRNA and protein level were measured after miR-424 overexpression in comparison to the control group by qRT-PCR and western blotting, respectively. We confirmed miR-424 direct target genes by dual-luciferase reporter assay. miR-424 overexpression significantly suppressed cell proliferation and migration rate in glioblastoma cells based on the MTT and scratch assays. Flow cytometry results confirmed that miR-424 promotes apoptosis and cell-cycle arrest in glioblastoma cells. Predicted target genes of miR-424 from the ERBB pathway were downregulated by miR-424 overexpression. qRT-PCR and western blotting showed that KRAS, RAF1, MAP2K1, EGFR, PDGFRA, AKT1, and mTOR mRNA expression levels and KRAS, RAF1, MAP2K1, EGFR, and AKT1 protein level, respectively, had significantly decreased as a result of miR-424 overexpression in comparison to the control group. Dual-luciferase reporter assay confirmed that miR-424 directly targets RAF1 and AKT1 oncogenes. Overall, miR-424 acts as tumor suppressor miRNA in glioblastoma cells.
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Neoplasias Encefálicas/genética , Glioblastoma/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-raf/genética , Apoptosis/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Receptores ErbB/metabolismo , Glioblastoma/patología , Células HEK293 , Humanos , Transducción de Señal/genéticaRESUMEN
Due to the osteoconductive role of bioceramics, use of these bioactive nanocomposite scaffolds that can maintain their structural integrity during bone tissue repair is one of the major goals of tissue engineering. Herein, a nanofibrous poly-L-lactic acid (PLLA) scaffold was fabricated by electrospinning and then gelatin and hydroxyapatite nanoparticles (nHA) were coated over the surface of the scaffold. Osteoconductivity of the fabricated nano-composite scaffolds was then studied while grafted on the rat calvarial defects. Our results indicated that the coating of PLLA scaffold with nHA and gelatin increased the adhesion and growth of the human bone marrow derived mesenchymal stem cells (BM-MSCs) and also significantly increased the level of mineralization over a week culture period. The results of radiographic and histological studies showed that the newly created bone tissue at the defect site was significantly higher in animals treated with nanocomposite scaffolds than the empty scaffolds and control groups. This increase in the defect reconstruction was significantly increased after culturing BM-MSCs on the scaffolds, especially nanocomposite scaffolds. It can be concluded that the combination of nanocomposite scaffolds and BM-MSCs could be a very good candidate for treatment of bone lesions and could be considered as a bony bioimplant.
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Células Madre Mesenquimatosas , Nanocompuestos , Animales , Regeneración Ósea , Durapatita , Humanos , Osteogénesis , Ratas , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
With advances in bone tissue engineering, various materials and methods have been explored to find a better scaffold that can help in improving bone growth and regeneration. Three-dimensional (3D) printing by fused deposition modeling can produce customized scaffolds from biodegradable polyesters such as polycaprolactone (PCL). Although the fabricated PCL scaffolds exhibited a lack of bioactivity and poor cell attachment on their surfaces, herein, using a simple postfabrication modification method with hydroxyapatite (HA) and bioglasses (BGs), we obtained better cell proliferation and attachment. Biological behavior and osteosupportive capacity of the 3D-printed scaffolds including PCL, PCL/HA, PCL/BG, and PCL/HA/BG were evaluated in this study, while human adipose tissue-derived mesenchymal stem cells (hADSCs) were cultured on the scaffolds. The cell morphology, attachment, and proliferation were investigated using scanning electron microscopy (SEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and 4',6-diamidino-2-phenylindole (DAPI) staining. In the next step, the ability of stem cells to differentiate into osteoblasts was evaluated by measuring alkaline phosphatase (ALP) activity, calcium deposition, and bone-related gene and protein expression. In the end, the expression levels of miR-20a, miR-125a, and their target genes were also investigated as positive and negative regulators in osteogenesis pathways. The results showed that the coated scaffolds with bioceramics present a more appropriate surface for cell adhesion and proliferation, as well as efficient potential in inducing osteoconduction and osteointegration compared to PCL alone and control. The PCL/HA/BG scaffold exhibited higher in vitro cell viability and bone formation compared to the other groups, which can be due to the synergistic effect of HA and BG. On the whole, this tricomponent 3D-printing scaffold has a promising prospect for bone tissue engineering applications.