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Dysfunction of the central nervous system (CNS) following traumatic brain injuries (TBI), spinal cord injuries (SCI), or strokes remains challenging to address using existing medications and cell-based therapies. Although therapeutic cell administration, such as stem cells and neuronal progenitor cells (NPCs), have shown promise in regenerative properties, they have failed to provide substantial benefits. However, the development of living cortical tissue engineered grafts, created by encapsulating these cells within an extracellular matrix (ECM) mimetic hydrogel scaffold, presents a promising functional replacement for damaged cortex in cases of stroke, SCI, and TBI. These grafts facilitate neural network repair and regeneration following CNS injuries. Given that natural glycosaminoglycans (GAGs) are a major constituent of the CNS, GAG-based hydrogels hold potential for the next generation of CNS healing therapies and in vitro modeling of CNS diseases. Brain-specific GAGs not only offer structural and biochemical signaling support to encapsulated neural cells but also modulate the inflammatory response in lesioned brain tissue, facilitating host integration and regeneration. This review briefly discusses different roles of GAGs and their related proteoglycan counterparts in healthy and diseases brain and explores current trends and advancements in GAG-based biomaterials for treating CNS injuries and modeling diseases. Additionally, it examines injectable, 3D bioprintable, and conductive GAG-based scaffolds, highlighting their clinical potential for in vitro modeling of patient-specific neural dysfunction and their ability to enhance CNS regeneration and repair following CNS injury in vivo.
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Materiales Biocompatibles , Enfermedades del Sistema Nervioso Central , Glicosaminoglicanos , Glicosaminoglicanos/metabolismo , Humanos , Animales , Materiales Biocompatibles/química , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Hidrogeles/químicaRESUMEN
Visualizing cells, tissues, and their components specifically without interference with cellular functions, such as biochemical reactions, and cellular viability remains important for biomedical researchers worldwide. For an improved understanding of disease progression, tissue formation during development, and tissue regeneration, labeling extracellular matrix (ECM) components secreted by cells persists is required. Bioorthogonal chemistry approaches offer solutions to visualizing and labeling ECM constituents without interfering with other chemical or biological events. Although biorthogonal chemistry has been studied extensively for several applications, this review summarizes the recent advancements in using biorthogonal chemistry specifically for metabolic labeling and visualization of ECM proteins and glycosaminoglycans that are secreted by cells and living tissues. Challenges, limitations, and future directions surrounding biorthogonal chemistry involved in the labeling of ECM components are discussed. Finally, potential solutions for improvements to biorthogonal chemical approaches are suggested. This would provide theoretical guidance for labeling and visualization of de novo proteins and polysaccharides present in ECM that are cell-secreted for example during tissue remodeling or in vitro differentiation of stem cells.
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Mesenchymal stem cells (MSCs) therapy is a promising approach for treating inflammatory diseases due to their immunosuppressive and tissue repair characteristics. However, allogenic transplantation of MSCs induces thrombotic complications in some patients which limits its potential for clinical translation. To address this challenge, we have exploited the bioactivity of heparin, a well-known anticoagulant and immunosuppressive polysaccharide that is widely used in clinics. We have developed a smart layer-by-layer (LbL) coating strategy using gelatin and heparin polymers exploiting their overall positive and negative charges that enabled efficient complexation with the MSCs' glycocalyx. The stable coating of MSCs suppressed complement attack and mitigated thrombotic activation as demonstrated in human whole blood. Gratifyingly, the MSC coating retained its immunosuppressive properties and differentiation potential when exposed to inflammatory conditions and differentiation factors. We believe the simple coating procedure of MSCs will increase allogenic tolerance and circumvent the major challenge of MSCs transplantation.
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Biomimética , Células Madre Mesenquimatosas , Humanos , Polielectrolitos , Heparina , Diferenciación Celular , InmunosupresoresRESUMEN
Hyaluronic acid (HA), one of the main components of the extracellular matrix (ECM), is extensively used in the design of hydrogels and nanoparticles for different biomedical applications due to its critical role in vivo, degradability by endogenous enzymes, and absence of immunogenicity. HA-based hydrogels and nanoparticles have been developed by utilizing different crosslinking chemistries. The development of such crosslinking chemistries indicates that even subtle differences in the structure of reactive groups or the procedure of crosslinking may have a profound impact on the intended mechanical, physical and biological outcomes. There are widespread examples of modified HA polymers that can form either covalently or physically crosslinked biomaterials. More recently, studies have been focused on dynamic covalent crosslinked HA-based biomaterials since these types of crosslinking allow the preparation of dynamic structures with the ability to form in situ, be injectable, and have self-healing properties. In this review, HA-based hydrogels and nanomaterials that are crosslinked by dynamic-covalent coupling (DCC) chemistry have been critically assessed.
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Hidrogeles , Nanoestructuras , Hidrogeles/química , Ácido Hialurónico/química , Materiales Biocompatibles/química , Matriz ExtracelularRESUMEN
Innovative scaffold designs that modulate the local inflammatory microenvironment through favorable macrophage polarization and suppressing oxidative stress are needed for successful clinical translation of regenerative cell therapies and graft integration. We herein report derivation of a hydrazone-crosslinked gallol functionalized hyaluronic acid (HA-GA)-based hydrogel that displayed outstanding viscoelastic properties and immunomodulatory characteristics. Grafting of 6% gallol (GA) to a HA-backbone formed an interpenetrative network by promoting an additional crosslink between the gallol groups in addition to hydrazone crosslinking. This significantly enhanced the mechanical stability and displayed shear-thinning/self-healing characteristics, facilitated tissue adhesive properties to porcine tissue and also displayed radical scavenging properties, protecting encapsulated fibroblasts from peroxide challenge. The THP-1 human macrophage cell line or primary bone-marrow-derived murine macrophages cultured within HA-GA gels displayed selective polarization to a predominantly anti-inflammatory phenotype by upregulating IL4ra, IL-10, TGF-ß, and TGF-ßR1 expression when compared with HA-HA gels. Conversely, culturing of pro-inflammatory activated primary murine macrophages in HA-GA gels resulted in a significant reduction of pro-inflammatory TNF-α, IL-1ß, SOCS3 and IL-6 marker expression, and upregulated expression of anti-inflammatory cytokines including TGF-ß. Finally, when the gels were implanted subcutaneously into healthy mice, we observed infiltration of pro-inflammatory myeloid cells in HA-HA gels, while immunosuppressive phenotypes were observed within the HA-GA gels. Taken together these data suggest that HA-GA gels are an ideal injectable scaffold for viable immunotherapeutic interventions. STATEMENT OF SIGNIFICANCE: Host immune response against the implanted scaffolds that are designed to deliver stem cells or therapeutic proteins in vivo significantly limits the functional outcome. For this reason, we have designed immunomodulatory injectable scaffolds that can favorably polarize the recruited macrophages and impart antioxidant properties to suppress oxidative stress. Specifically, we have tailored a hyaluronic acid-based extracellular matrix mimetic injectable scaffold that is grafted with immunomodulatory gallol moiety. Gallol functionalization of hydrogel not only enhanced the mechanical properties of the scaffold by forming an interpenetrating network but also induced antioxidant properties, tissue adhesive properties, and polarized primary murine macrophages to immunosuppressive phenotype. We believe such immunoresponsive implants will pave the way for developing the next-generation of biomaterials for regenerative medicine applications.
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Hidrogeles , Adhesivos Tisulares , Animales , Antioxidantes , Ácido Hialurónico/farmacología , Hidrazonas , Hidrogeles/farmacología , Macrófagos , Ratones , Fenotipo , Porcinos , Factor de Crecimiento Transformador betaRESUMEN
Human pluripotent stem cells (hPSC) derived neurons are emerging as a powerful tool for studying neurobiology, disease pathology, and modeling. Due to the lack of platforms available for housing and growing hPSC-derived neurons, a pressing need exists to tailor a brain-mimetic 3D scaffold that recapitulates tissue composition and favourably regulates neuronal network formation. Despite the progress in engineering biomimetic scaffolds, an ideal brain-mimetic scaffold is still elusive. We bioengineered a physiologically relevant 3D scaffold by integrating brain-like extracellular matrix (ECM) components and chemical cues. Culturing hPSCs-neurons in hyaluronic acid (HA) gels and HA-chondroitin sulfate (HA-CS) composite gels showed that the CS component prevails as the predominant factor for the growth of neuronal cells, albeit to modest efficacy. Covalent grafting of dopamine (DA) moieties to the HA-CS gel (HADA-CS) enhanced the scaffold stability and stimulated the gel's remodeling properties by entrapping cell-secreted laminin, and binding brain-derived neurotrophic factor (BDNF). Neurons cultured in the scaffold expressed Col1, Col11, and ITGB4; important for cell adhesion and cell-ECM signaling. Thus, the HA-CS scaffold with integrated chemical cues (DA) supported neuronal growth and network formation. This scaffold offers a valuable tool for tissue engineering and disease modeling and helps in bridging the gap between animal models and human diseases by providing biomimetic neurophysiology. STATEMENT OF SIGNIFICANCE: Developing a brain mimetic 3D scaffold that supports neuronal growth could potentially be useful to study neurobiology, disease pathology, and disease modeling. However, culturing human induced pluripotent stem cells (hiPSC) and human embryonic stem cells (ESCs) derived neurons in a 3D matrix is extremely challenging as neurons are very sensitive cells and require tailored composition, viscoelasticity, and chemical cues. This article identified the key chemical cues necessary for designing neuronal matrix that trap the cell-produced ECM and neurotrophic factors and remodel the matrix and supports neurite outgrowth. The tailored injectable scaffold possesses self-healing/shear-thinning property which is useful to design injectable gels for regenerative medicine and disease modeling that provides biomimetic neurophysiology.
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Biomimética , Células Madre Pluripotentes Inducidas , Animales , Encéfalo , Matriz Extracelular/metabolismo , Humanos , Neuronas , Andamios del Tejido/químicaRESUMEN
Histone Deacetylase (HDAC) enzymes are upregulated in cancer leading to the development of HDAC inhibiting compounds, several of which are currently in clinical trials. Side effects associated with toxicity and non-specific targeting indicate the need for efficient drug delivery approaches and tumor specific targeting to enhance HDAC efficacy in solid tumor cancers. SAHA encapsulation within F127 micelles functionalized with a surface hyaluronic acid moiety, was developed to target endometrial cancer cells expressing elevated levels of CD44. In vitro viability and morphology analyses was conducted in both 2D and 3D models to assess the translational potential of this approach. Encapsulation enhanced SAHA delivery and activity, demonstrating increased cytotoxic efficacy in 2D and 3D endometrial cancer models. High-content imaging showed improved nanoparticle internalization in 2D and CD44 enhanced penetration in 3D models. In addition, the nano-delivery system enhanced spheroid penetration resulting in cell growth suppression, p21 associated cell cycle arrest, as well as overcoming the formation of an EMT associated phenotype observed in free drug treated type II endometrial cancer cells. This study demonstrates that targeted nanoparticle delivery of SAHA could provide the basis for improving its efficacy in endometrial cancer. Using 3D models for endometrial cancer allows the elucidation of nanoparticle performance and CD44 targeting, likely through penetration and retention within the tumor model.
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High-density lipoproteins (HDLs) are a group of different subpopulations of sialylated particles that have an essential role in the reverse cholesterol transport (RCT) pathway. Importantly, changes in the protein and lipid composition of HDLs may lead to the formation of particles with reduced atheroprotective properties. Here, we show that Streptococcus pneumoniae pneumolysin (PLY) and neuraminidase A (NanA) impair HDL function by causing chemical and structural modifications of HDLs. The proteomic, lipidomic, cellular, and biochemical analysis revealed that PLY and NanA induce significant changes in sialic acid, protein, and lipid compositions of HDL. The modified HDL particles have reduced cholesterol acceptor potential from activated macrophages, elevated levels of malondialdehyde adducts, and show significantly increased complement activating capacity. These results suggest that accumulation of these modified HDL particles in the arterial intima may present a trigger for complement activation, inflammatory response, and thereby promote atherogenic disease progression.
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There is an unmet need to develop strategies that allow site-specific delivery of short interfering RNA (siRNA) without any associated toxicity. To address this challenge, we have developed a novel siRNA delivery platform using chemically modified pluronic F108 as an amphiphilic polymer with a releasable bioactive disulfide functionality. The micelles exhibited thermoresponsive properties and showed a hydrodynamic size of â¼291 nm in DLS and â¼200-250 nm in SEM at 37 °C. The grafting of free disulfide pyridyl groups enhanced the transfection efficiency and was successfully demonstrated in human colon carcinoma (HCT116; 88%) and glioma cell lines (U87; 90%), non-cancerous human dermal fibroblast (HDF; 90%) cells as well as in mouse embryonic stem (mES; 54%) cells. To demonstrate the versatility of our modular nanocarrier design, we conjugated the MDGI receptor targeting COOP peptide on the particle surface that allowed the targeted delivery of the cargo molecules to human patent-derived primary BT-13 gliospheres. Transfection experiments with this design resulted in â¼65% silencing of STAT3 mRNA in BT-13 gliospheres, while only â¼20% of gene silencing was observed in the absence of the peptide. We believe that our delivery method solves current problems related to the targeted delivery of RNAi drugs for potential in vivo applications.
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Micelas , Poloxámero , Animales , Línea Celular Tumoral , Ratones , Oxidación-Reducción , ARN Interferente Pequeño/metabolismo , TransfecciónRESUMEN
Mesenchymal stem/stromal cells (MSCs) evoke great excitement for treating different human diseases due to their ability to home inflamed tissues, suppress inflammation, and promote tissue regeneration. Despite great promises, clinical trial results are disappointing as allotransplantation of MSCs trigger thrombotic activity and are damaged by the complement system, compromising their survival and function. To overcome this, a new strategy is presented by the silencing of tissue factor (TF), a transmembrane protein that mediates procoagulant activity. Novel Pluronic-based micelles are designed with the pendant pyridyl disulfide group, which are used to conjugate TF-targeting siRNA by the thiol-exchange reaction. This nanocarrier design effectively delivered the payload to MSCs resulting in â¼72% TF knockdown (KD) without significant cytotoxicity. Hematological evaluation of MSCs and TF-KD MSCs in an ex vivo human whole blood model revealed a significant reduction in an instant-blood-mediated-inflammatory reaction as evidenced by reduced platelet aggregation (93% of free platelets in the TF-KD group, compared to 22% in untreated bone marrow-derived MSCs) and thrombin-antithrombin complex formation. Effective TF silencing induced higher MSC differentiation in osteogenic and adipogenic media and showed stronger paracrine suppression of proinflammatory cytokines in macrophages and higher stimulation in the presence of endotoxins. Thus, TF silencing can produce functional cells with higher fidelity, efficacy, and functions.
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Células Madre Mesenquimatosas , Diferenciación Celular , Células Cultivadas , Humanos , Micelas , Comunicación Paracrina , Poloxámero , Tromboplastina/genéticaRESUMEN
Over the past few years, mesenchymal stem (or stromal) cells (MSCs) have garnered enormous interest due to their therapeutic value especially for their multilineage differentiation potential leading to regenerative medicine applications. MSCs undergo physiological changes upon in vitro expansion resulting in expression of different receptors, thereby inducing high variabilities in therapeutic efficacy. Therefore, understanding the biochemical cues that influence the native local signals on differentiation or proliferation of these cells is very important. There have been several reports that in vitro culture of MSCs in low oxygen gradient (or hypoxic conditions) upregulates the stemness markers and promotes cell proliferation in an undifferentiated state, as hypoxia mimics the conditions the progenitor cells experience within the tissue. However, different studies report different oxygen gradients and culture conditions causing ambiguity in their interpretation of the results. In this progress report, it is aimed to summarize recent studies in the field with specific focus on conflicting results reported during the application of hypoxic conditions for improving the proliferation or differentiation of MSCs. Further, it is tried to decipher the factors that can affect characteristics of MSC under hypoxia and suggest a few techniques that could be combined with hypoxic cell culture to better recapitulate the MSC tissue niche.
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Células Madre Mesenquimatosas , Técnicas de Cultivo de Célula , Diferenciación Celular , Hipoxia de la Célula , Proliferación Celular , Células Cultivadas , OxígenoRESUMEN
Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we develop gut organoid cocultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate that murine and human ILC1 secrete transforming growth factor ß1, driving expansion of CD44v6+ epithelial crypts. ILC1 additionally express MMP9 and drive gene signatures indicative of extracellular matrix remodelling. We therefore encapsulated human epithelial-mesenchymal intestinal organoids in MMP-sensitive, synthetic hydrogels designed to form efficient networks at low polymer concentrations. Harnessing this defined system, we demonstrate that ILC1 drive matrix softening and stiffening, which we suggest occurs through balanced matrix degradation and deposition. Our platform enabled us to elucidate previously undescribed interactions between ILC1 and their microenvironment, which suggest that they may exacerbate fibrosis and tumour growth when enriched in inflamed patient tissues.
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Matriz Extracelular/metabolismo , Mucosa Intestinal/metabolismo , Linfocitos/metabolismo , Organoides/metabolismo , Animales , Femenino , Humanos , Mucosa Intestinal/citología , Linfocitos/citología , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Organoides/citología , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Anti-inflammatory drugs such as dexamethasone (DEX) are commonly administered to cancer patients along with anticancer drugs, however, the effect of DEX on human cancers is poorly understood. In this article, we have tailored self-assembled nanoparticles derived from hyaluronic acid (HA) wherein, anti-inflammatory DEX was used as a hydrophobic moiety for inducing amphiphilicity. The HA-DEX micelles were subsequently loaded with chemotherapeutic agent, doxorubicin (DOX) (HA-DEX-DOX) and was utilized to deliver drug cargo to human cancer cells expressing different levels of CD44 receptors. We found that DEX suppressed the cytotoxicity of DOX in HCT116, while it synergistically enhanced cytotoxicity in MCF-7 cells. When we tested DOX and HA-DEX-DOX in an ex-vivo human whole blood, we found activation of complement and the coagulation cascade in one group of donors. Encapsulation of DOX within the nanoparticle core eliminated such deleterious side-effects. The HA-DEX-DOX also polarized bone-marrow-derived anti-inflammatory M2 macrophages, to pro-inflammatory M1 phenotype with the upregulation of the cytokines TNF-α, iNOS and IL-1ß.
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Antiinflamatorios/administración & dosificación , Antibióticos Antineoplásicos/administración & dosificación , Polaridad Celular/efectos de los fármacos , Dexametasona/administración & dosificación , Doxorrubicina/administración & dosificación , Portadores de Fármacos/química , Ácido Hialurónico/química , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Nanopartículas/química , Animales , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Combinación de Medicamentos , Liberación de Fármacos , Células HCT116 , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Ácido Hialurónico/farmacología , Inflamación/tratamiento farmacológico , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Micelas , Fenotipo , Agregación Plaquetaria/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Statins are currently the most prescribed hypercholesterolemia-lowering drugs worldwide, with estimated usage approaching one-sixth of the population. However, statins are known to cause pleiotropic skeletal myopathies in 1.5% to 10% of patients and the mechanisms by which statins induce this response, are not fully understood. In this study, a 3D collagen-based tissue-engineered skeletal muscle construct is utilised as a screening platform to test the efficacy and toxicity of a new delivery system. A hyaluronic acid derived nanoparticle loaded with simvastatin (HA-SIM-NPs) is designed and the effect of free simvastatin and HA-SIM-NPs on cellular, molecular and tissue response is investigated. Morphological ablation of myotubes and lack of de novo myotube formation (regeneration) was evident at the highest concentrations (333.33 µM), independent of delivery vehicle (SIM or HA-SIM-NP). A dose-dependent disruption of the cytoskeleton, reductions in metabolic activity and tissue engineered (TE) construct tissue relaxation was evident in the free drug condition (SIM, 3.33 µM and 33.33 nM). However, most of these changes were ameliorated when SIM was delivered via HA-SIM-NPs. Significantly, homogeneous expressions of MMP2, MMP9, and myogenin in HA-SIM-NPs outlined enhanced regenerative responses compared to SIM. Together, these results outline statin delivery via HA-SIM-NP as an effective delivery mechanism to inhibit deleterious myotoxic side-effects.
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Ácido Hialurónico/química , Músculo Esquelético/citología , Osteogénesis/efectos de los fármacos , Simvastatina/efectos adversos , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Ratones , Músculo Esquelético/química , Músculo Esquelético/efectos de los fármacos , Miogenina/genética , Miotoxicidad , Nanopartículas , Simvastatina/química , Simvastatina/farmacología , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Regeneration of a severely damaged cornea necessitates the delivery of both epithelium-renewing limbal epithelial stem cells (LESCs) and stroma-repairing cells, such as human adipose-derived stem cells (hASCs). Currently, limited strategies exist for the delivery of these therapeutic cells with tissue-like cellular organization. With the added risks related to suturing of corneal implants, there is a pressing need to develop new tissue adhesive biomaterials for corneal regeneration. To address these issues, we grafted dopamine moieties into hydrazone-crosslinked hyaluronic acid (HA-DOPA) hydrogels to impart tissue adhesive properties and facilitate covalent surface modification of the gels with basement membrane proteins or peptides. We achieved tissue-like cellular compartmentalization in the implants by encapsulating hASCs inside the hydrogels, with subsequent conjugation of thiolated collagen IV or laminin peptides and LESC seeding on the hydrogel surface. The encapsulated hASCs in HA-DOPA gels exhibited good proliferation and cell elongation, while the LESCs expressed typical limbal epithelial progenitor markers. Importantly, the compartmentalized HA-DOPA implants displayed excellent tissue adhesion upon implantation in a porcine corneal organ culture model. These results encourage sutureless implantation of functional stem cells as the next generation of corneal regeneration.
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Sustancia Propia/fisiología , Epitelio Corneal/fisiología , Ácido Hialurónico/farmacología , Hidrogeles/farmacología , Regeneración/efectos de los fármacos , Trasplante de Células Madre , Células Madre/citología , Adhesivos Tisulares/farmacología , Animales , Moléculas de Adhesión Celular/metabolismo , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sustancia Propia/efectos de los fármacos , Dihidroxifenilalanina/química , Dopamina/química , Epitelio Corneal/efectos de los fármacos , Femenino , Humanos , Hidrazonas/química , Proteínas Inmovilizadas/metabolismo , Implantes Experimentales , Cinética , Técnicas de Cultivo de Órganos , Reología , PorcinosRESUMEN
Currently, there are limited approaches to tailor 3D scaffolds cross-linked with a stable covalent C-C bond that does not require any catalysts or initiators. We present here the first hydrogels employing aldol condensation chemistry that exhibit exceptional physicochemical properties. We investigated the aldol-cross-linking chemistry using two types of aldehyde-modified hyaluronic acid (HA) derivatives, namely, an enolizable HA-aldehyde (HA-Eal) and a non-enolizable HA-aldehyde (HA-Nal). Hydrogels formed using HA-Eal demonstrate inferior cross-linking efficiency (due to intramolecular loop formation), when compared with hydrogels formed by mixing HA-Eal and HA-NaI leading to a cross-aldol product. The change in mechanical properties as a result of cross-linking at different pH values is determined using rheological measurements and is interpreted in terms of molecular weight between cross-links (Mc). The novel HA cross-aldol hydrogel demonstrate excellent hydrolytic stability and favorable mechanical properties but allow hyaluronidase-mediated enzymatic degradation. Interestingly, residual aldehyde functionality within the aldol product rendered the tissue-adhesive properties by bonding two bone tissues. The aldehyde functionality also facilitated facile post-synthetic modifications with nucleophilic reagents. Finally, we demonstrate that the novel hydrogel is biocompatible with encapsulated stem cells that show a linear rate of expansion in our 3-6 days of study.
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Células Inmovilizadas/metabolismo , Ácido Hialurónico , Hidrogeles , Ensayo de Materiales , Células Madre Mesenquimatosas/metabolismo , Adhesivos Tisulares , Células Inmovilizadas/citología , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Hidrogeles/química , Hidrogeles/farmacología , Concentración de Iones de Hidrógeno , Células Madre Mesenquimatosas/citología , Adhesivos Tisulares/química , Adhesivos Tisulares/farmacologíaRESUMEN
Cardiovascular diseases represent a major socio-economic burden. In recent years, considerable effort has been invested in optimizing cell delivery strategies to advance cell transplantation therapies to restore heart function for example after an infarct. A particular issue is that the implantation of cells using a non-electroconductive matrix potentially causes arrhythmia. Here, we demonstrate that our hydrazide-functionalized nanotubes-pericardial matrix-derived electroconductive biohybrid hydrogel provides a suitable environment for maturation of human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. hiPSC-derived cardiomyocytes exhibited an improved contraction amplitude (>500%) on conductive hydrogels compared to cells cultured on Matrigel®. This was accompanied by increased cellular alignment, enhanced connexin 43 expression, and improved sarcomere organization suggesting maturation of the hiPSC-derived cardiomyocytes. Sarcomeric length of these cells increased from 1.3 to 1.7 µm. Moreover, 3D cell-laden engineered tissues exhibited enhanced calcium handling as well as positive response to external electrical and pharmaceutical stimulation. Collectively, our data indicate that our biohybrid hydrogels consisting of solubilized nanostructured pericardial matrix and electroconductive positively charged hydrazide-conjugated carbon nanotubes provide a promising material for stem cell-based cardiac tissue engineering.
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Materiales Biocompatibles/química , Hidrogeles/química , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Nanotubos de Carbono/química , Pericardio/química , Andamios del Tejido/química , Biomarcadores/metabolismo , Calcio/metabolismo , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Colágeno/química , Conexina 43/metabolismo , Combinación de Medicamentos , Conductividad Eléctrica , Humanos , Laminina/química , Células Madre Mesenquimatosas/citología , Tamaño de la Partícula , Proteoglicanos/químicaRESUMEN
We developed a novel miRNA design that significantly improves strand selection within the RISC complex by engineering the 3' end by adding extra nucleotides. Addition of seven nucleotides at the 3' ends of the miR or miR* strand resulted in a thermodynamic asymmetry at either of the two ends, which resulted in selective RISC recruitment, as demonstrated by a stem-loop PCR experiment. Such selective recruitment was also corroborated at the protein level by western blot analysis. To investigate the functional effect because of selective recruitment, we performed apoptosis and metastasis studies using human colon carcinoma cells (HCT116) and human osteosarcoma cells (MG63). These experiments indicated that recruitment of the miR strand is responsible for inducing apoptosis and inhibiting the invasiveness of cancer cells. Recruitment of the miR* strand, on the other hand, had the opposite effect. To the best of our knowledge, our strand engineering strategy is the first report of improved strand selection of a desired miRNA strand by RISC without using any chemical modifications or mismatches. We believe that such structural modifications of miR34a could mitigate some of the off-target effects of miRNA therapy and would also allow a better understanding of sequence-specific gene regulation. Such a design could also be adapted to other miRNAs to enhance their therapeutic potential.
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The disulfide bond plays a crucial role in protein biology and has been exploited by scientists to develop antibody-drug conjugates, sensors, and for the immobilization other biomolecules to materials surfaces. In spite of its versatile use, the disulfide chemistry suffers from some inevitable limitations such as the need for basic conditions (pH > 8.5), strong oxidants, and long reaction times. We demonstrate here that thiol-substrates containing electron-withdrawing groups at the ß-position influence the deprotonation of the thiol group, which is the key reaction intermediate in the formation of disulfide bonds. Evaluation of reaction kinetics using small molecule substrate such as l-cysteine indicated disulfide formation at a 2.8-fold higher ( k1 = 5.04 × 10-4 min-1) reaction rate as compared to the conventional thiol substrate, namely 3-mercaptopropionic acid ( k1 = 1.80 × 10-4 min-1) at physiological pH (pH 7.4). Interestingly, the same effect could not be observed when N-acetyl-l-cysteine substrate ( k1 = 0.51 × 10-4 min-1) was used. We further grafted such thiol-containing molecules (cysteine, N-acetyl-cysteine, and 3-mercaptopropionic acid) to a biopolymer namely hyaluronic acid (HA) and determined the p Ka value of different thiol groups by spectrophotometric analysis. The electron-withdrawing group at the ß-position reduced the p Ka of the thiol group to 7.0 for HA-cysteine (HA-Cys); 7.4 for N-acetyl cysteine (HA-ActCys); and 8.1 for HA-thiol (HA-SH) derivatives, respectively. These experiments further confirmed that the concentration of thiolate (R-S-) ions could be increased with the presence of electron-withdrawing groups, which could facilitate disulfide cross-linked hydrogel formation at physiological pH. Indeed, HA grafted with cysteine or N-acetyl groups formed hydrogels within 3.5 min or 10 h, respectively, at pH 7.4. After completion of cross-linking reaction, both gels demonstrated a storage modulus G' ≈ 3300-3500 Pa, which indicated comparable levels of cross-linking. The HA-SH gel, on the other hand, did not form any gel at pH 7.4 even after 24 h. Finally, we demonstrated that the newly prepared hydrogels exhibited excellent hydrolytic stability but can be degraded by cell-directed processes (enzymatic and reductive degradation). We believe our study provides a valuable insight on the factors governing the disulfide formation and our results are useful to develop strategies that would facilitate generation of stable thiol functionalized biomolecules or promote fast thiol oxidation according to the biomedical needs.