RESUMEN
Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.
Asunto(s)
Fibrinógeno , Mannheimia haemolytica , Serina Proteasas , Animales , Mannheimia haemolytica/enzimología , Ovinos , Bovinos , Fibrinógeno/metabolismo , Concentración de Iones de Hidrógeno , Serina Proteasas/metabolismo , Serina Proteasas/aislamiento & purificación , Temperatura , Proteolisis , Peso Molecular , Gelatina/metabolismo , Estabilidad de Enzimas , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Espectrometría de Masas , Cromatografía por Intercambio Iónico , Porcinos , Factores de Virulencia/metabolismo , Factores de Virulencia/aislamiento & purificaciónRESUMEN
Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.
Asunto(s)
Basigina , Neoplasias de la Mama , Matriz Extracelular , Proteína 1 de la Membrana Asociada a los Lisosomas , Metaloproteinasa 14 de la Matriz , Transportadores de Ácidos Monocarboxílicos , Invasividad Neoplásica , Podosomas , Femenino , Humanos , Basigina/metabolismo , Basigina/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Matriz Extracelular/metabolismo , Gelatina/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 14 de la Matriz/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Invasividad Neoplásica/genética , Podosomas/metabolismoRESUMEN
Radical prostatectomy (RP) can cause neurogenic erectile dysfunction (ED), which negatively affects the quality of life of patients with prostate cancer. Currently, there is a dearth of effective therapeutic strategies. Although stem cell therapy is promising, direct cell transplantation to injured cavernous nerves is constrained by poor cell colonization. In this study, poly-L-lactic acid (PLLA)/gelatin electrospun membranes (PGEM) were fabricated to load bone marrow-derived mesenchymal stem cells (BM-MSCs) as a patch to be placed on injured nerves to alleviate ED. This study aimed to establish a promising and innovative approach to mitigate neurogenic ED post-RP and lay the foundation for modifying surgical procedures. Electrospinning and molecular biotechnology were performed in vitro and in vivo, respectively. It was observed that PGEM enhanced the performance of BM-MSCs and Schwann cells due to their excellent mechanical properties and biocompatibility. The transplanted PGEM and loaded BM-MSCs synergistically improved bilateral cavernous nerve injury-related ED and the corresponding histopathological changes. Nevertheless, transplantation of BM-MSCs alone has been verified to be ineffective. Overall, PGEM can serve as an ideal carrier to supply a more suitable survival environment for BM-MSCs and Schwann cells, thereby promoting the recovery of injured cavernous nerves and erectile function.
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Disfunción Eréctil , Células Madre Mesenquimatosas , Poliésteres , Masculino , Ratas , Animales , Humanos , Disfunción Eréctil/etiología , Disfunción Eréctil/terapia , Gelatina/metabolismo , Pene/inervación , Pene/patología , Médula Ósea/patología , Calidad de Vida , Ratas Sprague-Dawley , Modelos Animales de Enfermedad , Células Madre Mesenquimatosas/metabolismoRESUMEN
One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the so-called metastatic spread. This is allowed thanks to the production of matrix metalloproteinases (MMPs), enzymes capable of degrading a type of collagen abundant in the basal membrane separating the epithelial tissue from the connective one. In this work, we employ a synergistic experimental and mathematical modelling approach to explore the invasion process of tumor cells. A mathematical model composed of reaction-diffusion equations describing the evolution of the tumor cells density on a gelatin substrate, MMPs enzymes concentration and the degradation of the gelatin is proposed. This is completed with a calibration strategy. We perform a sensitivity analysis and explore a parameter estimation technique both on synthetic and experimental data in order to find the optimal parameters that describe the in vitro experiments. A comparison between numerical and experimental solutions ends the work.
Asunto(s)
Podosomas , Humanos , Podosomas/metabolismo , Podosomas/patología , Gelatina/metabolismo , Matriz Extracelular/patología , Modelos Biológicos , Conceptos Matemáticos , Metaloproteinasas de la Matriz/metabolismo , Invasividad Neoplásica/patologíaRESUMEN
BACKGROUND: Currently, society and industry generate huge amounts of plastics worldwide. The ubiquity of microplastics is obvious, but its impact on the animal and human organism remains not fully understood. The digestive tract is one of the first barriers between pathogens and xenobiotics and a living organism. Its proper functioning is extremely important in order to maintain homeostasis. The aim of this study was to determine the effect of microplastic on enteric nervous system and histological structure of swine duodenum. The experiment was carried out on 15 sexually immature gilts, approximately 8 weeks old. The animals were randomly divided into 3 study groups (n = 5/group). The control group received empty gelatin capsules once a day for 28 days, the first research group received daily gelatin capsules with polyethylene terephthalate (PET) particles as a mixture of particles of various sizes (maximum particle size 300 µm) at a dose of 0.1 g/animal/day. The second study group received a dose ten times higher-1 g/animal/day. RESULTS: A dose of 1 g/day/animal causes more changes in the enteric nervous system and in the histological structure of duodenum. Statistically significant differences in the expression of cocaine and amphetamine regulated transcript, galanin, neuronal nitric oxide synthase, substance P, vesicular acetylcholine transporter and vasoactive intestinal peptide between control and high dose group was noted. The histopathological changes were more frequently observed in the pigs receiving higher dose of PET. CONCLUSION: Based on this study it may be assumed, that oral intake of microplastic might have potential negative influence on digestive tract, but it is dose-dependent.
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Microplásticos , Plásticos , Humanos , Porcinos , Animales , Femenino , Tereftalatos Polietilenos/metabolismo , Tereftalatos Polietilenos/farmacología , Gelatina/metabolismo , Gelatina/farmacología , Duodeno/metabolismo , NeuronasRESUMEN
Hair follicle (HF) tissue engineering is promising for hair loss treatment especially for androgenetic alopecia. Physiologically, the initiation of HF morphogenesis relies on the interactions between hair germ mesenchymal and epithelial layers. To simulate this intricate process, in this study, a co-flowing microfluidic-assisted technology was developed to produce dual aqueous microdroplets capturing growth factors and double-layer cells for subsequent use in hair regeneration. Microspheres, called G/HAD, were generated using glycosaminoglycan-based photo-crosslinkable biological macromolecule (HAD) shells and gelatin methacrylate (GelMA) cores to enclose mesenchymal cells (MSCs) and mouse epidermal cells (EPCs). The findings indicated that the glycosaminoglycan-based HAD shells display thermodynamic incompatibility with GelMA cores, resulting in the aqueous phase separation of G/HAD cell spheres. These G/HAD microspheres exhibited favorable characteristics, including sustained growth factor release and wet adhesion properties. After transplantation into the dorsal skin of BALB/c nude mice, G/HAD cell microspheres efficiently induced the regeneration of HFs. This approach enables the mass production of approximately 250 dual-layer microspheres per minute. Thus, this dual-layer microsphere fabrication method holds great potential in improving current hair regeneration techniques and can also be combined with other tissue engineering techniques for various regenerative purposes.
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Gelatina , Glicosaminoglicanos , Ratones , Animales , Gelatina/metabolismo , Microesferas , Glicosaminoglicanos/metabolismo , Metacrilatos , Ratones Desnudos , Biomimética , Cabello , Folículo Piloso , TermodinámicaRESUMEN
Oncolytic Newcastle disease virus is a new type of cancer immunotherapy drug. This paper proposes a scheme for delivering oncolytic viruses using hydrogel microneedles. Gelatin methacryloyl (GelMA) was synthesized by chemical grafting, and GelMA microneedles encapsulating oncolytic Newcastle disease virus (NDV) were prepared by micro-molding and photocrosslinking. The release and expression of NDV were tested by immunofluorescence and hemagglutination experiments. The experiments proved that GelMA was successfully synthesized and had hydrogel characteristics. NDV was evenly dispersed in the allantoic fluid without agglomeration, showing a characteristic virus morphology. NDV particle size was 257.4 ± 1.4 nm, zeta potential was -13.8 ± 0.5 mV, virus titer TCID50 was 107.5/mL, and PFU was 2 × 107/mL, which had a selective killing effect on human liver cancer cells in a dose and time-dependent manner. The NDV@GelMA microneedles were arranged in an orderly cone array, with uniform height and complete needle shape. The distribution of virus-like particles was observed on the surface. GelMA microneedles could successfully penetrate 5% agarose gel and nude mouse skin. Optimal preparation conditions were freeze-drying. We successfully prepared GelMA hydrogel microneedles containing NDV, which could effectively encapsulate NDV but did not detect the release of NDV.
Asunto(s)
Metacrilatos , Viroterapia Oncolítica , Virus Oncolíticos , Animales , Ratones , Humanos , Virus Oncolíticos/genética , Virus Oncolíticos/metabolismo , Virus de la Enfermedad de Newcastle , Gelatina/metabolismo , Hidrogeles/metabolismoRESUMEN
Nifedipine has exhibited to be the oldest primary drug having promising therapeutic potential for hypertension, angina pectoris, and pre-eclampsia treatment which are the most emergency serious complications worldwide. Moreover, for long-term treatment transdermal route of delivery using polymeric dissolving microneedles (DMNs) patches has shown greater advantages, thus enhancing treatment compliance, painless, reducing the daily number of doses, prolonged release in a controlled manner, and variable bioavailability making this an ideal candidate for the transdermal therapeutic system. Here, we fabricated DMN patches made of gelatin and PVP using PDMS molds loaded with nifedipine drugs for a controlled painless delivery for a longer stable duration. The prepared gelatin-PVP (gel-PVP) DMN patches loaded with nifedipine were fabricated by centrifugation casting method. The characterization results displayed excellent mechanical strength of the needles to penetrate the skin. SEM and confocal microscopy showed penetration of the needles up to 567-600 µm using rhodamine B applied to the hairless punctured skin site. FTIR study exhibited no degradation of the drug was observed while fabricating the DMNs patch at different pH 7.4 and 4. Skin resealing test proved that there was immediate resealing of the skin observed within 10-15 min. Further in-vitro drug release profile study was carried out by dissolution method at different pH 7.4 and 4 showed sustained release of the drug up to 96 ± 2% till 48-72 h avoiding polymer or drug loss which was quantified by UV vis spectrophotometer at 235 nm absorbance showed stable release of the drug upto 48-72 h. A stability study carried out by the HPLC method showed the DMN patches loaded with the drug were found to be stable for up to 30 days at 25 °C. This novel preliminary data are the first study to our knowledge introducing these fabricated nifedipine gel-PVP DMN patches were found to be very efficient and showed prolonged controlled release up to 48-72 h thereby treating hypertension in a convenient, painless manner. This DMN patch-formulated design might act as a potential approach leading to a controllable, self-administrative, and rapid transdermal delivery system.
Asunto(s)
Gelatina , Hipertensión , Humanos , Gelatina/metabolismo , Nifedipino , Sistemas de Liberación de Medicamentos/métodos , Piel , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismoRESUMEN
Mandibular continuity defects stem from conditions such as malignancies, trauma, cysts, osteomyelitis and osteoradionecrosis, presenting significant challenges. If mandibular reconstruction fails, it can result in facial collapse, causing significant aesthetic and functional concerns for the patient. In the present study we developed a bio-adhesive Bone Cement (BC) enriched with lyophilised PRF and gelatin to enhance bone repair and induce regeneration. The developed BC consisted of a mixture of Tetracalcium Phosphate (TTCP) and O-Phospho-l-serine (OPLS) in addition to lyophilised Platelet Rich Fibrin (PRF) for sustained growth factor release and gelatin (GE) for improved cement resorption. It is primarily designed for in-situ application, conforming to the shape and size of the defect for effective bone repair and regeneration. The study evaluated four groups: (i) BC (control), (ii) BC-GE (control), (iii) BC-PRF, and (iv) BC-GE-PRF. All the four groups were characterised using FTIR, SEM and XRD. The mechanical studies of the prepared beads exhibited a significant increase in the compressive strength of the PRF loaded bone cement composites. In vitro degradation study of the beads over a 60-day period revealed a significantly higher percentage of bone cement resorption in the gelatin-incorporated groups, BC-GE (44 ± 0.5 %), and BC-GE-PRF (45 ± 2 %). The assessment of growth factor release (TGF-ß and VEGF) using ELISA revealed a prolonged and sustained release of both growth factors over a 28-day period. In vitro studies were performed on human Dental Follicle Stem Cells (DFSCs) to assess cell attachment, proliferation, mineralisation and osteogenic differentiation. These studies clearly depicted that BC-PRF and BC-GE-PRF showed significantly greater proliferation of DFSCs. Furthermore, BC-PRF and BC-GE-PRF samples exhibited notably elevated expression of Runx2 and OPN (osteogenic markers), as well as a higher intensity of alizarin red stain (mineralisation). Therefore, it was concluded that PRF incorporated bioadhesive bone cement composites greatly enhance the cell attachment, proliferation, mineralisation and osteogenic differentiation of the DFSCs. Thus, the PRF and gelatin incorporated bone cement composites is expected to facilitate effective and faster bone regeneration and healing in a wide range of dental and maxillofacial defects.
Asunto(s)
Fibrina Rica en Plaquetas , Humanos , Fibrina Rica en Plaquetas/metabolismo , Osteogénesis , Gelatina/metabolismo , Cementos para Huesos , Péptidos y Proteínas de Señalización Intercelular/metabolismoRESUMEN
We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 µm s-1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 µm s-1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.
Asunto(s)
Lubina , Dorada , Preservación de Semen , Masculino , Animales , Gelatina/farmacología , Gelatina/metabolismo , Motilidad Espermática , Semen , Criopreservación/veterinaria , Espermatozoides/metabolismo , Crioprotectores/farmacología , Crioprotectores/metabolismo , Acuicultura , Nitrógeno , Preservación de Semen/veterinariaRESUMEN
BACKGROUND AIMS: Cell failure and angiogenesis are the key to bladder wall regeneration. Three-dimensional (3D) culture using porous gelatin microspheres (GMs) as a vehicle promotes stem cell proliferation and improves the paracrine capacity of cells. This study aimed to evaluate the therapeutic potential of GMs constructed from adipose-derived mesenchymal stromal cells (ADSCs) (ADSC-GMs) combined with bladder acellular matrix (BAM) in tissue-engineered bladders. METHODS: Isolation of ADSCs, flow cytometry, scanning electron microscopy and cell counting kit-8, ß-galactosidase and enzyme-linked immunosorbent assays were performed in vitro to compare two-dimensional (2D) and 3D cultures. In the in vivo study, male Sprague-Dawley rats were randomly divided into three groups: the BAM replacement alone (BAM) group, ADSCs grown on BAM in replacement (ADSC) group and ADSC-GMs combined with BAM followed by replacement (ADSC-GM) group. Bladder function assessed by urodynamics after 12 weeks of bladder replacement, and the rats were sacrificed at 4 and 12 weeks for further experiments. RESULTS: The in vitro results showed that GM culture promoted ADSC proliferation, inhibited apoptosis and delayed senescence compared with those in the 2D culture. In addition, ADSC-GMs increased the secretion of the angiogenic factors vascular endothelial growth factor, platelet-derived growth factor-BB, and basal fibroblast growth factor. In vivo experiments revealed that ADSC-GMs adhered to the BAM for longer than ADSCs. Moreover, ADSC-GMs significantly promoted the regeneration of bladder vessels and smooth muscle, thereby facilitating the recovery of bladder function. The expression of phosphorylated protein kinase B (AKT) and phosphorylated endothelial nitric oxide synthase (eNOS) was significantly greater in the ADSC-GMs group compared with the BAM and ADSCs groups. CONCLUSIONS: ADSC-GMs increased retention of ADSCs on the BAM, thereby promoting the regeneration and functional recovery of the bladder tissue. ADSC-GMs promoted angiogenesis by activating the AKT/eNOS pathway.
Asunto(s)
Células Madre Mesenquimatosas , Vejiga Urinaria , Ratas , Masculino , Animales , Vejiga Urinaria/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Gelatina/metabolismo , Tejido Adiposo , Ratas Sprague-Dawley , Microesferas , Óxido Nítrico Sintasa de Tipo III/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Porosidad , Transducción de SeñalRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disease marked by mitochondrial dysfunction, amyloid-ß (Aß) aggregation, and neuronal cell loss. G-protein-coupled receptor 55 (GPR55) has been used as a promising target for insulin receptors in diabetes therapy, but GPR55's role in AD is still unidentified. Gelatin (GE) and polyethylene glycol (PEG) polymeric hydrogels are commonly used in the drug delivery system. Therefore, the aim of the present study was the preparation of magnesium hydroxide nanocomposite using Clitoria ternatea (CT) flower extract, GE, and PEG (GE/PEG/Mg(OH)2NCs) by the green precipitation method. The synthesized GE/PEG/Mg(OH)2NCs were used to determine the effect of GPR55 activation of intracerebroventricular administration on streptozotocin (ICV-STC)-induced cholinergic dysfunction, oxidative stress, neuroinflammation, and cognitive deficits. The GE/PEG/Mg(OH)2NCs were administered following bilateral ICV-STC administration (3 mg/kg) in experimental rats. Neurobehavioral assessments were performed using a Morris water maze (MWM) and a passive avoidance test (PA). Cholinergic and antioxidant activity, oxidative stress, and mitochondrial complex activity were estimated in the cortex and hippocampus through biochemical analysis. Inflammatory markers (TNF-α, IL-6, and IL-1ß) were determined using the ELISA method. Our study results demonstrated that the GE/PEG/Mg(OH)2NCs treatment significantly improved spatial and non-spatial memory functions in behavioral studies. Moreover, the treatment with GE/PEG/Mg(OH)2NCs group significantly attenuated cholinergic dysfunction, oxidative stress, and inflammatory markers, and also highly improved anti-oxidant activity (GSH, SOD, CAT, and GPx) in the cortex and hippocampus regions. The western blot results suggest the activation of the GPR55 protein expression through GE/PEG/Mg(OH)2NCs. The histopathological studies showed clear cytoplasm and healthy neurons, effectively promoting neuronal activity. Furthermore, the molecular docking results demonstrated the binding affinity and potential interactions of the compounds with the AChE enzyme. In conclusion, the GE/PEG/Mg(OH)2NCs treated groups showed reduced neurotoxicity and have the potential as a therapeutic agent to effectively target AD.
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Enfermedad de Alzheimer , Inhibidores de la Colinesterasa , Nanopartículas , Enfermedades Neurodegenerativas , Animales , Ratas , Acetilcolinesterasa/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Antioxidantes/farmacología , Colinérgicos/metabolismo , Colinérgicos/farmacología , Colinérgicos/uso terapéutico , Modelos Animales de Enfermedad , Gelatina/metabolismo , Gelatina/farmacología , Gelatina/uso terapéutico , Hipocampo/metabolismo , Hidróxido de Magnesio/metabolismo , Hidróxido de Magnesio/farmacología , Hidróxido de Magnesio/uso terapéutico , Simulación del Acoplamiento Molecular , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Estrés Oxidativo , Polietilenglicoles/farmacología , Polietilenglicoles/metabolismo , Polietilenglicoles/uso terapéutico , Receptores de Cannabinoides/metabolismo , Receptores de Cannabinoides/uso terapéutico , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Nanopartículas/química , Nanopartículas/uso terapéuticoRESUMEN
BACKGROUND: In recent years, treated dentin matrix (TDM) has been introduced as a bioactive hydrogel for dentin regeneration in DPC. However, no study has introduced TDM as a photocrosslinkable hydrogel with a natural photoinitiating system. Therefore, the present study aimed to explore the synthesis, characterizations and grafting optimization of injectable gelatin- glycidyl methacrylate (GMA)/TDM hydrogels as a novel photocrosslinkable pulp capping agent for dentin regeneration. METHODS: G-GMA/TDM hydrogel was photocrosslinked using a new two-component photoinitiating system composed of riboflavin as a photoinitiator under visible light and glycine as a first time coinitiator with riboflavin. The grafting reaction conditions of G-GMA/TDM e.g. GMA concentration and reaction time were optimized. The kinetic parameters e.g. grafting efficiency (GE) and grafting percentage (GP%) were calculated to optimize the grafting reaction, while yield (%) was determined to monitor the formation of the hydrogel. Moreover, G-GMA/TDM hydrogels were characterized by swelling ratio, degradation degree, and cytotoxicity. The instrumental characterizations e.g. FTIR, 1H-NMR, SEM and TGA, were investigated for verifying the grafting reaction. Statistical analysis was performed using F test (ANOVA) and Post Hoc Test (P = 0.05). RESULTS: The grafting reaction dramatically increased with an increase of both GMA concentration and reaction time. It was realized that the swelling degree and degradation rate of G-GMA/TDM hydrogels were significantly reduced by increasing the GMA concentration and prolonging the reaction time. When compared to the safe low and moderate GMA content hydrogels (0.048, 0.097 M) and shorter reaction times (6, 12, 24 h), G-GMA/TDM with high GMA contents (0.195, 0.391 M) and a prolonged reaction time (48 h) demonstrated cytotoxic effects against cells using the MTT assay. Also, the morphological surface of G-GMA/TDM freeze-dried gels was found more compacted, smooth and uniform due to the grafting process. Significant thermal stability was noticed due to the grafting reaction of G-GMA/TDM throughout the TGA results. CONCLUSIONS: G-GMA/TDM composite hydrogel formed by the riboflavin/glycine photoinitiating system is a potential bioactive and biocompatible system for in-situ crosslinking the activated-light pulp capping agent for dentin regeneration.
Asunto(s)
Gelatina , Materiales de Recubrimiento Pulpar y Pulpectomía , Humanos , Gelatina/metabolismo , Hidrogeles/química , Hidrogeles/metabolismo , Regeneración , Dentina/metabolismoRESUMEN
OBJECTIVES: We aimed to explore the osteogenic potential of periodontal ligament stem cells (PDLSCs) in bioprinted methacrylate gelatine (GelMA) hydrogels in vitro and in vivo. MATERIALS AND METHODS: PDLSCs in GelMA hydrogels at various concentrations (3%, 5%, and 10%) were bioprinted. The mechanical properties (stiffness, nanostructure, swelling, and degradation properties) of bioprinted constructs and the biological properties (cell viability, proliferation, spreading, osteogenic differentiation, and cell survival in vivo) of PDLSCs in bioprinted constructs were evaluated. Then, the effect of bioprinted constructs on bone regeneration was investigated using a mouse cranial defect model. RESULTS: Ten percent GelMA printed constructs had a higher compression modulus, smaller porosity, lower swelling rate, and lower degradation rate than 3% GelMA. PDLSCs in bioprinted 10% GelMA bioprinted constructs showed lower cell viability, less cell spreading, upregulated osteogenic differentiation in vitro, and lower cell survival in vivo. Moreover, upregulated expression of ephrinB2 and EphB4 protein and their phosphorylated forms were found in PDLSCs in 10% GelMA bioprinted constructs, and inhibition of eprhinB2/EphB4 signalling reversed the enhanced osteogenic differentiation of PDLSCs in 10% GelMA. The in vivo experiment showed that 10% GelMA bioprinted constructs with PDLSCs contributed to more new bone formation than 10% GelMA constructs without PDLSCs and constructs with lower GelMA concentrations. CONCLUSIONS: Bioprinted PDLSCs with high-concentrated GelMA hydrogels exhibited enhanced osteogenic differentiation partially through upregulated ephrinB2/EphB4 signalling in vitro and promoted bone regeneration in vivo, which might be more appropriate for future bone regeneration applications. CLINICAL RELEVANCE: Bone defects are a common clinical oral problem. Our results provide a promising strategy for bone regeneration through bioprinting PDLSCs in GelMA hydrogels.
Asunto(s)
Hidrogeles , Osteogénesis , Hidrogeles/farmacología , Hidrogeles/química , Hidrogeles/metabolismo , Ligamento Periodontal , Gelatina/farmacología , Gelatina/química , Gelatina/metabolismo , Células Madre , Regeneración Ósea , Diferenciación Celular , Células CultivadasRESUMEN
Craniofacial bone defects induced by congenital malformations, trauma, or diseases frequently challenge the orthodontic or restorative treatment. Stem cell-based bone regenerative approaches emerged as a promising method to resolve bone defects. Microenvironment physical cues, such as the matrix elastic modulus or matrix topography, regulate stem cell differentiation via multiple genes. We constructed gelatin methacryloyl (GelMA), a well-known scaffold, to investigate the impact of elastic modulus on osteogenic differentiation in a three-dimensional environment. Confocal microscope was used to observe and assess the condensates fission and fusion. New bone formation was evaluated by micro-computed tomography at 6 weeks in calvarial defect rat. We found that the light curing increased elastic modulus of GelMA, and the pore size of GelMA decreased. The expression of osteogenic markers was inhibited in hBMSCs cultured in the low-elastic-modulus GelMA. In contrast, the expression of YAP, TAZ and TEAD was increased in the hBMSCs in the low-elastic-modulus GelMA. Furthermore, YAP assembled via liquid-liquid phase separation (LLPS) into condensates that were sensitive to 1'6-hexanediol. YAP recruit TAZ and TEAD4, but not RUNX2 into the condensates. In vivo, we also found that hBMSCs in high-elastic-modulus GelMA was more apt to form new bone. This study provides new insight into the mechanism of osteogenic differentiation. Reagents that can regulate the elastic modulus of substrate or LLPS may be applied to promote bone regeneration.
Asunto(s)
Células Madre Mesenquimatosas , Osteogénesis , Ratas , Animales , Hidrogeles/farmacología , Hidrogeles/metabolismo , Módulo de Elasticidad , Microtomografía por Rayos X , Diferenciación Celular , Gelatina/metabolismoRESUMEN
Porous gelatin microspheres (GMSs) were constructed to enhance the neuroprotective effects of fibroblast growth factor 10 (FGF10) against spinal cord injury (SCI). The GMSs were prepared using a waterinoil emulsion, followed by crosslinking, washing and drying. The blank GMSs had a mean particle size of 35 µm, with a coarse and porous surface. FGF10 was encapsulated within bulk GMSs via diffusion. To evaluate the effects of the FGF10GMSs, locomotion tests were performed as a measure of the functional recovery of rats. Hematoxylin and eosin and Nissl staining were used to quantify tissue injury, and Evans blue staining was used to evaluate bloodspinal cord barrier restoration. Western blotting and TUNEL assays were employed to assess apoptotic activity. Immunohistochemical staining of neurofilament antibodies (NF200) was used to evaluate axonal rehabilitation. Compared with the groups intravenously administered FGF10 alone, disruption of the bloodspinal cord barrier and tissue injury were attenuated in the FGF10GMS group; this group also showed less neuronal apoptosis, as well as enhanced neuronal and axonal rehabilitation. Implantable porous GMSs could serve as carriers for FGF10 in the treatment of SCI.
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Gelatina , Traumatismos de la Médula Espinal , Ratas , Animales , Gelatina/metabolismo , Gelatina/farmacología , Ratas Sprague-Dawley , Microesferas , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factor 10 de Crecimiento de Fibroblastos/farmacología , Porosidad , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Recuperación de la FunciónRESUMEN
Metastasis is one of the major causes for cancer mortality. Its early steps comprise of invasion of basement membrane and migration. Thus, it is hypothesized that a platform, that allows quantification and grading of migration capability of cells can potentially be used for predicting metastatic potential. Two-dimensional (2D) models have been rendered inadequate for modelling in-vivo microenvironment due to various reasons. To attenuate homogeneity observed in 2D, three-dimensional (3D) platforms supplemented with bioinspired components have been designed. Unfortunately, till date there are no simple models to capture the migration of cells in 3D along with quantification of the process. In this study, we report an alginate-collagen based 3D model system, which can predict the migratory property of the cells within 72 h. The micron size of the scaffold enabled faster readout and the optimum pore-size provided conducive cellular growth environment. The platform's ability to allow observation of cellular migration was validated by encapsulating cells with transiently upregulated matrix metalloprotease 9 (MMP9), which has been reported to play a significant role in migration of cells during metastasis. The readout for migration was clustering of cells in the microscaffolds detected in a short span of 48 h. The observed clustering in MMP9 upregulated cells was validated by observing changes in the epithelial-mesenchymal transition (EMT) markers. Thus, this simple 3D platform can be used to study migration and predict the metastatic potential of cells.
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Alginatos , Movimiento Celular , Colágeno , Andamios del Tejido , Alginatos/química , Alginatos/metabolismo , Biomarcadores de Tumor/metabolismo , Cadherinas/metabolismo , Colágeno/química , Colágeno/metabolismo , Transición Epitelial-Mesenquimal , Gelatina/metabolismo , Microfluídica , Metástasis de la Neoplasia , Porosidad , Andamios del Tejido/química , Factores de Transcripción Twist/metabolismo , Humanos , Línea Celular TumoralRESUMEN
Myocardial infarction (MI) is a major cause of death worldwide. After the occurrence of MI, the heart frequently undergoes serious pathological remodeling, leading to excessive dilation, electrical disconnection between cardiac cells, and fatal functional damage. Hence, extensive efforts have been made to suppress pathological remodeling and promote the repair of the infarcted heart. In this study, we developed a hydrogel cardiac patch that can provide mechanical support, electrical conduction, and tissue adhesiveness to aid in the recovery of an infarcted heart function. Specifically, we developed a conductive and adhesive hydrogel (CAH) by combining the two-dimensional titanium carbide (Ti3C2Tx) MXene with natural biocompatible polymers [i.e., gelatin and dextran aldehyde (dex-ald)]. The CAH was formed within 250 s of mixing the precursor solution and could be painted. The hydrogel containing 3.0 mg/mL MXene, 10% gelatin, and 5% dex-ald exhibited appropriate material characteristics for cardiac patch applications, including a uniform distribution of MXene, a high electrical conductivity (18.3 mS/cm), cardiac tissue-like elasticity (30.4 kPa), strong tissue adhesion (6.8 kPa), and resistance to various mechanical deformations. The CAH was cytocompatible and induced cardiomyocyte (CM) maturation in vitro, as indicated by the upregulation of connexin 43 expression and a faster beating rate. Furthermore, CAH could be painted onto the heart tissue and remained stably adhered to the beating epicardium. In vivo animal studies revealed that CAH cardiac patch treatment significantly improved cardiac function and alleviated the pathological remodeling of an infarcted heart. Thus, we believe that our MXene-based CAH can potentially serve as a promising platform for the effective repair of various electroactive tissues including the heart, muscle, and nerve tissues.
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Hidrogeles , Infarto del Miocardio , Animales , Hidrogeles/farmacología , Gelatina/metabolismo , Adhesivos/farmacología , Infarto del Miocardio/patología , Miocitos Cardíacos , Polímeros/farmacología , Conductividad EléctricaRESUMEN
Pleurodeles waltl is coming to light as a model animal, especially in regeneration studies, but deep studies on the molecular mechanisms have been limited due to the absence of primary tissue cells for wide usage. Therefore, we aimed to grow primary cells from limb tissue of P. waltl for in vitro experiments. Limb tissues were cut into small pieces and seeded as "explants" on culture dishes coated with fibronectin and gelatin. Compared to the control without coating, both fibronectin and gelatin supported quicker outgrowth of cells from explants and faster cell adhesion, and fibronectin showed significantly better performance than gelatin. Interestingly, the doubling time of cells on fibronectin- and gelatin-coated surfaces was almost the same (42.39 ± 2.79 h vs. 42.91 ± 3.69 h) and was not significantly different from that on non-coated plates (49.64 ± 3.63 h). The cryopreserved cells were successfully recovered and showed a multiplication capacity that was similar to that of fresh cells. Senescent cells were barely detected even after long-term sub-culture (>15 passages). Moreover, enhanced fluorescence of MitoSOX™ Red in cells under H2 O2 exposure confirmed the respondence to chemical stimuli. Collectively, our results show that we are able to grow enough good-quality cells from P. waltl limb tissue for in vitro experiments, and fibronectin coating provides the best biocompatible environment for cell outgrowth and attachment.
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Fibronectinas , Pleurodeles , Animales , Fibronectinas/farmacología , Fibronectinas/metabolismo , Pleurodeles/metabolismo , Gelatina/farmacología , Gelatina/metabolismoRESUMEN
Despite significant advancements in tissue engineering and regenerative medicine during the last two decades, the fabrication of proper scaffolds with appropriate cells can still be considered a critical achievement in this field. Hypoxia is a major stumbling block to chronic wound healing, which restrains tissue engineering plans because a lack of oxygen may cause cell death. This study evaluated the cocultured human keratinocytes and human adipose-derived mesenchymal stem cells (AMSCs) on a multilayer oxygen-releasing electrospun scaffold based on PU/PCL.Sodium percarbonate (SPC)-gelatin/PU. The scaffold was characterized using Fourier transform infrared (FTIR) and scanning electron microscopy (SEM) methods. Flow cytometry confirmed mesenchymal stem cells, and then the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and DAPI staining were used to assess the in vitro biocompatibility of the scaffold. The experimental results showed that the multilayer electrospun scaffold containing 2.5% SPC could efficiently produce oxygen. Furthermore, according to cell viability results, this structure makes a suitable substrate for the coculture of keratinocytes and AMSCs. Gene expression analysis of various markers such as Involucrin, Cytokeratin 10, and Cytokeratin 14 after 14 days confirmed that keratinocytes and AMSCs coculture on PU/PCL.SPC-gelatin/PU electrospun scaffold promotes dermal differentiation and epithelial proliferation compared to keratinocytes single-cell culture. Therefore, our study supports using oxygen-releasing scaffolds as a potential strategy to hasten skin tissue regeneration. Based on the results, this structure is suggested as a promising candidate for cell-based skin tissue engineering. Given that the developed oxygen-generating polymeric electrospun scaffolds could be used as part of a future strategy for skin tissue engineering, the PU/PCL.SPC-gelatin/PU hybrid electrospun multilayer scaffold in combination with keratinocyte/AMSC coculture is proposed as an effective substrate for skin tissue engineering and regenerative medicine platforms.