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MicroRNAs (miRNAs) regulate gene expression by post-transcriptional inhibition of target genes. Proangiogenic small extracellular vesicles (sEVs; popularly identified with the name "exosomes") with a composite cargo of miRNAs are secreted by cultured stem cells and present in human biological fluids. Lipid nanoparticles (LNPs) represent an advanced platform for clinically approved delivery of RNA therapeutics. In this study, we aimed to (1) identify the miRNAs responsible for sEV-induced angiogenesis; (2) develop the prototype of bioinspired "artificial exosomes" (AEs) combining LNPs with a proangiogenic miRNA, and (3) validate the angiogenic potential of the bioinspired AEs. We previously reported that human sEVs from bone marrow (BM)-CD34+ cells and pericardial fluid (PF) are proangiogenic. Here, we have shown that sEVs secreted from saphenous vein pericytes and BM mesenchymal stem cells also promote angiogenesis. Analysis of miRNA datasets available in-house or datamined from GEO identified the let-7 family as common miRNA signature of the proangiogenic sEVs. LNPs with either hsa-let-7b-5p or cyanine 5 (Cy5)-conjugated Caenorhabditis elegans miR-39 (Cy5-cel-miR-39; control miRNA) were prepared using microfluidic micromixing. let-7b-5p-AEs did not cause toxicity and transferred functionally active let-7b-5p to recipient endothelial cells (ECs). let-7b-AEs also improved EC survival under hypoxia and angiogenesis in vitro and in vivo. Bioinspired proangiogenic AEs could be further developed into innovative nanomedicine products targeting ischemic diseases.
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Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Liposomas/química , MicroARNs/metabolismo , Nanopartículas/química , Neovascularización Fisiológica , Líquido Pericárdico/fisiología , Animales , Exosomas/genética , Vesículas Extracelulares/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Ratones , MicroARNs/genéticaRESUMEN
Therapies based on circulating proangiogenic cells (PACs) have shown promise in ischemic disease models but require further optimization to reach the bedside. Ischemia-associated hypoxia robustly increases microRNA-210 (miR-210) expression in several cell types, including endothelial cells (ECs). In ECs, miR-210 represses EphrinA3 (EFNA3), inducing proangiogenic responses. This study provides new mechanistic evidences for a role of miR-210 in PACs. PACs were obtained from either adult peripheral blood or cord blood. miR-210 expression was modulated with either an inhibitory complementary oligonucleotide (anti-miR-210) or a miRNA mimic (pre-miR-210). Scramble and absence of transfection served as controls. As expected, hypoxia increased miR-210 in PACs. In vivo, migration toward and adhesion to the ischemic endothelium facilitate the proangiogenic actions of transplanted PACs. In vitro, PAC migration toward SDF-1α/CXCL12 was impaired by anti-miR-210 and enhanced by pre-miR-210. Moreover, pre-miR-210 increased PAC adhesion to ECs and supported angiogenic responses in co-cultured ECs. These responses were not associated with changes in extracellular miR-210 and were abrogated by lentivirus-mediated EFNA3 overexpression. Finally, ex-vivo pre-miR-210 transfection predisposed PACs to induce post-ischemic therapeutic neovascularization and blood flow recovery in an immunodeficient mouse limb ischemia model. In conclusion, miR-210 modulates PAC functions and improves their therapeutic potential in limb ischemia.
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Células de la Médula Ósea/citología , Médula Ósea/fisiología , Miembro Posterior/citología , Isquemia/genética , Isquemia/terapia , MicroARNs/genética , Neovascularización Fisiológica/fisiología , Adulto , Animales , Línea Celular , Quimiocina CXCL12/genética , Células Endoteliales/citología , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Transfección/métodosRESUMEN
Effective chemotherapy delivery for glioblastoma multiforme (GBM) is limited by drug transport across the blood-brain barrier and poor efficacy of single agents. Polymer-drug conjugates can be used to deliver drug combinations with a ratiometric dosing. However, the behaviors and effectiveness of this system have never been well investigated in GBM models. Here, we report flexible conjugates of hyaluronic acid (HA) with camptothecin (CPT) and doxorubicin (DOX) delivered into the brain using focused ultrasound (FUS). In vitro toxicity assays reveal that DOX-CPT exhibited synergistic action against GBM in a ratio-dependent manner when delivered as HA conjugates. FUS is employed to improve penetration of DOX-HA-CPT conjugates into the brain in vivo in a murine GBM model. Small-angle x-ray scattering characterizations of the conjugates show that the DOX:CPT ratio affects the polymer chain flexibility. Conjugates with the highest flexibility yield the highest efficacy in treating mouse GBM in vivo. Our results demonstrate the association of FUS-enhanced delivery of combination chemotherapy and the drug-ratio-dependent flexibility of the HA conjugates. Drug ratio in the polymer nanocomplex may thus be employed as a key factor to modulate FUS drug delivery efficiency via controlling the polymer flexibility. Our characterizations also highlight the significance of understanding the flexibility of drug carriers in ultrasound-mediated drug delivery systems.
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The blood-brain barrier (BBB), a crucial protection mechanism in the central nervous system (CNS), is a selective barrier comprised of endothelial cells. It hampers the development of therapeutic and diagnostic tools for neurological diseases due to the poor penetration of most of these agents. Rationally engineered nanoparticles (NP) can facilitate the transport of therapeutic and diagnostic agents across the BBB. However, evaluating BBB penetration by NP majorly relies on the use of expensive and time-consuming animal experiments with low throughput. In vitro BBB models composed of brain endothelial cells can be a useful tool to rapidly screen multiple NP formulations to compare their BBB penetration ability and identify optimal formulations for in vivo validation. In this protocol, we present an in vitro model of BBB developed using murine cerebral cortex endothelial cells (bEnd.3). bEnd.3 is a commercially available, easy to manipulate cell line that forms tight junctions with potent paracellular barrier property. The protocol includes culturing of bEnd.3 cells, establishment of the in vitro model, and assessing NP permeability. We believe that, due to its simplicity and consistency, this step-by-step protocol can be easily used by researchers to screen NP-based drug delivery systems for BBB penetration. Graphic abstract.
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Endothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is relatively limited. Here, we screened a library of microRNA (miR) mimics and identified 25 miRs capable of enhancing the survival of ECs exposed to ischemia-mimicking conditions. In vitro, we showed that miR-425-5p, one of the hits, was able to enhance EC survival and migration. In vivo, using a mouse Matrigel plug assay, we showed that ECs transfected with miR-425-5p displayed enhanced survival compared with scramble-transfected ECs. Mechanistically, we showed that miR-425-5p modulated the PTEN/PI3K/AKT pathway and inhibition of miR-425-5p target genes (DACH1, PTEN, RGS5, and VASH1) phenocopied the pro-survival. For the in vivo delivery of miR-425-5p, we modulated small extracellular vesicles (sEVs) with miR-425-5p and showed, in vitro, that miR-425-5p-modulated sEVs were (1) capable of enhancing the survival of ECs exposed to ischemia-mimic conditions, and (2) efficiently internalized by skin cells. Finally, using a streptozotocin-induced diabetic wound healing mouse model, we showed that, compared with miR-scrambled-modulated sEVs, topical administration of miR-425-5p-modulated sEVs significantly enhanced wound healing, a process mediated by enhanced vascularization and skin re-epithelialization.
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Small interfering RNA (siRNA)-based therapeutics can mitigate the long-term sequelae of traumatic brain injury (TBI) but suffer from poor permeability across the blood-brain barrier (BBB). One approach to overcoming this challenge involves treatment administration while BBB is transiently breached after injury. However, it offers a limited window for therapeutic intervention and is applicable to only a subset of injuries with substantially breached BBB. We report a nanoparticle platform for BBB pathophysiology-independent delivery of siRNA in TBI. We achieved this by combined modulation of surface chemistry and coating density on nanoparticles, which maximized their active transport across BBB. Engineered nanoparticles injected within or outside the window of breached BBB in TBI mice showed threefold higher brain accumulation compared to nonengineered PEGylated nanoparticles and 50% gene silencing. Together, our data suggest that this nanoparticle platform is a promising next-generation drug delivery approach for the treatment of TBI.
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Lesiones Traumáticas del Encéfalo , Nanopartículas , Animales , Barrera Hematoencefálica , Encéfalo , Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/terapia , Ratones , ARN Interferente Pequeño/genéticaRESUMEN
Small extracellular vesicles (sEVs) are those nanovesicles 30-150 nm in size with a role in cell signalling and potential as biomarkers of disease. Nanoparticle tracking analysis (NTA) techniques are commonly used to measure sEV concentration in biofluids. However, this quantification technique can be susceptible to sample handing and machine settings. Moreover, some classes of lipoproteins are of similar sizes and could therefore confound sEV quantification, particularly in blood-derived preparations, such serum and plasma. Here we have provided methodological information on NTA measurements and systematically investigated potential factors that could interfere with the reliability and repeatability of results obtained when looking at neat biofluids (i.e., human serum and pericardial fluid) obtained from patients undergoing cardiac surgery and from healthy controls. Data suggest that variables that can affect vesicle quantification include the level of contamination from lipoproteins, number of sample freeze/thaw cycles, sample filtration, using saline-based diluents, video length and keeping the number of particles per frame within defined limits. Those parameters that are of less concern include focus, the "Maximum Jump" setting and the number of videos recorded. However, if these settings are clearly inappropriate the results obtained will be spurious. Similarly, good experimental practice suggests that multiple videos should be recorded. In conclusion, NTA is a perfectible, but still commonly used system for sEVs analyses. Provided users handle their samples with a highly robust and consistent protocol, and accurately report these aspects, they can obtain data that could potentially translate into new clinical biomarkers for diagnosis and monitoring of cardiovascular disease.
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BACKGROUND: Endothelial Progenitor Cells (EPCs) are a circulating stem cell population with in vivo capacity of promoting angiogenesis after ischemic events. Despite the promising preclinical data, their potential integration with reperfusion therapies and hemodynamic evolution of stroke patients is still unknown. Our aim was to determine the association of EPCs with acute, subacute and chronic hemodynamic features. METHODS: In this prospective study, we included consecutive patients with ages between 18 and 80years and non-lacunar ischemic stroke within the territory of a middle cerebral artery. All patients were subject to hemodynamic evaluation by ultrasound at baseline, seven days and three months. We quantified cerebral blood flow (CBF) and assessed early recanalization and collateral flow. Hemorrhagic transformation was graded in Magnetic Resonance imaging performed at seven days. EPCs were isolated from peripheral venous blood collected in the first 24h and seven days, counted and submitted to functional in vitro tests. RESULTS: We included 45 patients with a median age of 70±10years. The angiogenic and migratory capacities of EPCs were associated with increased collateral flow in the acute stage and day seven CBF, without statistically significant associations with recanalization nor haemorrhagic transformation. The number of EPCs was not associated with any hemodynamic variable. CONCLUSIONS: The functional properties of EPCs are associated with acute and subacute stroke hemodynamics, with no effect on haemorrhagic transformation.
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Células Progenitoras Endoteliales/fisiología , Hemodinámica/fisiología , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/fisiopatología , Accidente Cerebrovascular/terapia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Isquemia Encefálica/complicaciones , Arteria Carótida Interna/fisiopatología , Circulación Cerebrovascular/fisiología , Femenino , Hemorragia/diagnóstico por imagen , Hemorragia/etiología , Hemorragia/terapia , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Accidente Cerebrovascular/diagnóstico por imagen , Ultrasonografía Doppler Transcraneal , Adulto JovenRESUMEN
OBJECTIVE: To study the association among endothelial progenitor cells (EPCs), subacute blood-brain barrier (BBB) permeability, and clinical outcome after ischemic stroke, determining the micro RNAs of EPCs responsible for good clinical outcome. METHODS: We included consecutive patients with nonlacunar acute ischemic strokes in the territory of a middle cerebral artery and ages between 18 and 80 years. Clinical outcome was defined as modified Rankin Scale score at 3 months. Neuroimaging was performed at day 0 and 7 by MRI, including assessment of BBB permeability by dynamic contrast enhancement. EPCs were isolated from peripheral venous blood, quantified, and submitted to in vitro functional tests, including migratory and angiogenic assays. Stroke hemodynamics were evaluated serially by ultrasound. Statistical significance was set at p < 0.05. RESULTS: We included 45 patients; mean age was 70.0 ± 10.0 years. The in vitro functional properties of EPCs were associated with BBB permeability, particularly at day 7. The number of each EPC subset at both timepoints was not associated with BBB permeability. Permeability of BBB at day 7 was independently associated with improved clinical outcome (odds ratio 0.897; 95% confidence interval 0.816-0.986; p = 0.025). The EPCs (CD34+ cell subset) of patients with good clinical outcome showed 24 differentially expressed miRNAs, with a common effect on adherens junction pathway. CONCLUSIONS: The functional properties of EPCs are associated with enhanced subacute permeability of BBB and improved clinical outcome after acute ischemic stroke.
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Barrera Hematoencefálica/metabolismo , Isquemia Encefálica/metabolismo , Permeabilidad Capilar/fisiología , Células Progenitoras Endoteliales/metabolismo , Accidente Cerebrovascular/metabolismo , Anciano , Antígenos CD34/metabolismo , Barrera Hematoencefálica/diagnóstico por imagen , Isquemia Encefálica/diagnóstico por imagen , Isquemia Encefálica/patología , Movimiento Celular/fisiología , Células Cultivadas , Células Progenitoras Endoteliales/patología , Femenino , Hemodinámica , Humanos , Estudios Longitudinales , Masculino , MicroARNs/metabolismo , Pronóstico , Estudios Prospectivos , Accidente Cerebrovascular/diagnóstico por imagen , Accidente Cerebrovascular/patologíaRESUMEN
Insulin and/or heparin immobilized/co-immobilized non-woven polyester fabric (NWPF) discs were developed for the cultivation of L929 mouse fibroblasts in low-serum media. At first, NWPF discs were hydrolyzed to obtain a carboxylic acid group-introduced matrix (NWPF-hydrolyzed). Insulin and heparin co-immobilized NWPF (NWPF-insulin-heparin) was prepared by the grafting of PEO onto NWPF-hydrolyzed disc (NWPF-PEO), followed by the reaction first with insulin and then heparin. In the presence of spacer arm, PEO, the amount of immobilized insulin molecules significantly increased from 6.96 to 84.45 microg/cm(2). The amount of heparin bound to the NWPF-PEO (5.93 microg/cm(2)) was higher than that of the insulin immobilized surface (4.59 microg/cm(2)). Insulin and heparin immobilized NWPF discs were observed with fluorescence microscopy by labeling the insulin and heparin with 8-anilino-1-naphthalene sulfonic acid (ANS) or fluorescein isothiocyanate (FITC), respectively. L929 fibroblasts were used to check the cell adhesion and cell growth capabilities of modified NWPF discs in low-serum media (containing 5% fetal bovine serum). Optical photographs showed that after 2nd day of the culture, fibroblastic cells spread along the length of modified fibers, eventually filling the interfiber space. At the end of 6-day growth period, cell yield in the presence of immobilized heparin was a little bit higher than that of the immobilized insulin. Co-immobilized (insulin/heparin) NWPF discs did not accelerate the cell growth as well as insulin or heparin immobilized discs.
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Fibroblastos/citología , Heparina/química , Insulina/química , Poliésteres/química , Animales , Bovinos , Técnicas de Cultivo de Célula , Línea Celular , Medios de Cultivo , Ratones , Factores de TiempoRESUMEN
Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)-VEGF165, have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases.Soluble vascular endothelial growth factor (VEGF) enhances vascular engraftment of transplanted cells but the efficacy is low. Here, the authors show that VEGF-immobilized microparticles prolong survival of endothelial progenitors in vitro and in vivo by downregulating miR17 and upregulating CDKN1A and ZNF652.
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Supervivencia Celular , Células Progenitoras Endoteliales/metabolismo , MicroARNs/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Tratamiento Basado en Trasplante de Células y Tejidos , Micropartículas Derivadas de Células , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Unión al ADN/genética , Células Progenitoras Endoteliales/citología , Sangre Fetal/citología , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Isquemia/metabolismo , Isquemia/terapia , Neovascularización Fisiológica , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Chronic skin wounds affect ≈3% of persons aged >60years (Davies et al., 2007) [1]. These wounds are typically difficult to heal by conventional therapies and in many cases they get infected making even harder the regeneration process. The antimicrobial peptide (AMP) LL37 combines antimicrobial with pro-regenerative properties and thus represents a promising topical therapy to address both problems. Here, we investigated the wound healing potential of soluble and immobilized LL37 (LL37-conjugated gold nanoparticles, LL37-Au NPs), both in vitro (migration of keratinocytes) and in vivo (skin wound healing). Our results show that LL37-Au NPs, but not LL37 peptide, have the capacity to prolong the phosphorylation of EGFR and ERK1/2 and enhance the migratory properties of keratinocytes in a large in vitro wound model. We further report that both LL37 and LL37-Au NPs promote keratinocyte migration by the transactivation of EGFR, a process that seems to be initiated at the P2X7 receptor, as confirmed by chemical and genetic inhibition studies. Finally, we show in vivo that LL37-Au NPs have higher wound healing activity than LL37 peptide in a splinted mouse full thickness excisional model. Animal wounds treated by LL37-Au NPs have higher expression of collagen, IL6 and VEGF than the ones treated with LL37 peptide or NPs without LL37. Altogether, the conjugation of AMPs to NPs offers a promising platform to enhance their pro-regenerative properties.
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Catelicidinas/administración & dosificación , Oro/administración & dosificación , Nanopartículas del Metal/administración & dosificación , Regeneración/efectos de los fármacos , Fenómenos Fisiológicos de la Piel , Cicatrización de Heridas/efectos de los fármacos , Animales , Péptidos Catiónicos Antimicrobianos , Catelicidinas/química , Catelicidinas/uso terapéutico , Línea Celular , Femenino , Oro/química , Oro/uso terapéutico , Humanos , Nanopartículas del Metal/química , Nanopartículas del Metal/uso terapéutico , RatonesRESUMEN
Several clinical trials are exploring therapeutic effect of human CD34(+) cells in ischemic diseases, including myocardial infarction. Unfortunately, most of the cells die few days after delivery. Herein we show that lysophosphatidic acid (LPA)-treated human umbilical cord blood-derived CD34(+) cells cultured under hypoxic and serum-deprived conditions present 2.2-fold and 1.3-fold higher survival relatively to non-treated cells and prostaglandin E2-treated cells, respectively. The pro-survival effect of LPA is concentration- and time-dependent and it is mediated by the activation of peroxisome proliferator-activator receptor γ (PPARγ) and downstream, by the activation of pro-survival ERK and Akt signaling pathways and the inhibition of mitochondrial apoptotic pathway. In hypoxia and serum-deprived culture conditions, LPA induces CD34(+) cell proliferation without maintaining the their undifferentiating state, and enhances IL-8, IL-6 and G-CSF secretion during the first 12 h compared to non-treated cells. LPA-treated CD34(+) cells delivered in fibrin gels have enhanced survival and improved cardiac fractional shortening at 2 weeks on rat infarcted hearts as compared to hearts treated with placebo. We have developed a new platform to enhance the survival of CD34(+) cells using a natural and cost-effective ligand and demonstrated its utility in the preservation of the functionality of the heart after infarction.
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Antígenos CD34/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Isquemia/metabolismo , Lisofosfolípidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Caspasa 9/metabolismo , Diferenciación Celular/efectos de los fármacos , Hipoxia de la Célula , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Trasplante de Células Madre de Sangre del Cordón Umbilical , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Humanos , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/mortalidad , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/terapia , PPAR gamma/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Resultado del Tratamiento , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/ß-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.
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Barrera Hematoencefálica/citología , Células Madre Hematopoyéticas/fisiología , Biomarcadores/metabolismo , Permeabilidad Capilar , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Modelos Biológicos , Pericitos/fisiología , Reproducibilidad de los Resultados , Vía de Señalización WntRESUMEN
The aim of this study is to investigate the effects of heparin-functionalized chitosan scaffolds on the activity of preosteoblasts. The chitosan scaffolds having the pore size of â¼100 µm were prepared by a freeze-drying method. Two different methods for immobilization of heparin to chitosan scaffolds were successfully performed. In the first method, functionalization of the scaffolds was achieved by means of electrostatic interactions between negatively charged heparin and positively charged chitosan. The covalent immobilization of heparin to chitosan scaffolds by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC) and N-hydroxysuccinimide (NHS) was used as a second immobilization method. Morphology, proliferation, and differentiation of MC3T3-E1 preosteoblasts on heparin-functionalized chitosan scaffolds were investigated in vitro. The results indicate that covalently bound heparin containing chitosan scaffolds (CHC) stimulate osteoblast proliferation compared to other scaffolds, that is, unmodified chitosan scaffolds (CH), electrostatically bound heparin containing chitosan scaffolds (EHC), and CH+free heparin (CHF). SEM images also proved the stimulative effect of covalently bound heparin on the proliferation of preosteoblasts. Alkaline phosphatase (ALP) and osteocalcin (OCN) levels of cells proliferated on CHC and EHC were also higher than those for CH and CHF. In vitro studies have demonstrated that chitosan scaffolds increase viability and differentiation of MC3T3-E1 cells especially in the presence of immobilized heparin.
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Materiales Biocompatibles/química , Quitosano/química , Heparina/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Materiales Biocompatibles/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Ratones , Osteogénesis/efectos de los fármacos , Electricidad EstáticaRESUMEN
In this study, low pressure water/O2 plasma treatment was performed in order to obtain COOH functionalities on the surface of poly-ε-caprolactone (PCL) membranes as well as non-woven polyester fabric (NWPF) discs. The plasma treatments were performed in a cylindrical, capacitively coupled RF-plasma-reactor and then following steps were performed: in situ (oxalyl chloride vapors) gas/solid reaction to convert -OH functionalities into -COCl groups; and hydrolysis under open laboratory conditions using air moisture for final-COOH functionalities. COOH and OH functionalities on modified surfaces were detected quantitatively by using fluorescent labeling technique and an UVX 300G sensor. Electron spectroscopy for chemical analysis (ESCA) was used to evaluate the relative surface atomic compositions and the carbon and oxygen linkages located in non-equivalent atomic positions of untreated and modified surfaces. Atomic force microscope (AFM) analysis showed that nanoscale features of the PCL surfaces are dramatically changed during the surface treatments. Scanning electron microscopy (SEM) results indicated the changes in the relatively smooth appearance of the untreated NWPF discs after the plasma treatment. Periodontal ligament (PDL) fibroblasts were used in cell culture studies. Cell culture results showed that plasma treated PCL membranes and NWPF discs were favorable for the PDL cell spreading, growth and viability due to the presence of functional groups and/or nanotopographies on their surfaces.
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Materiales Biocompatibles/química , Fibroblastos/citología , Oxígeno/química , Oxígeno/farmacología , Poliésteres/química , Polietilenglicoles/química , Agua/química , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Gases/química , Humanos , Hidróxidos/química , Membranas Artificiales , Microscopía de Fuerza Atómica/métodos , Microscopía Electrónica de Rastreo/métodos , Tereftalatos Polietilenos , Propiedades de SuperficieRESUMEN
The manipulation of endogenous stem cell populations from the subventricular zone (SVZ), a neurogenic niche, creates an opportunity to induce neurogenesis and influence brain regenerative capacities in the adult brain. Herein, we demonstrate the ability of polyelectrolyte nanoparticles to induce neurogenesis exclusively after being internalized by SVZ stem cells. The nanoparticles are not cytotoxic for concentrations equal or below 10 µg/mL. The internalization process is rapid, and nanoparticles escape endosomal fate in a few hours. Retinoic acid-loaded nanoparticles increase the number of neuronal nuclear protein (NeuN)-positive neurons and functional neurons responding to depolarization with KCl and expressing NMDA receptor subunit type 1 (NR1). These nanoparticles offer an opportunity for in vivo delivery of proneurogenic factors and neurodegenerative disease treatment.