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Transplantation of islets in type 1 diabetes (T1D) is limited by poor islet engraftment into the liver, with two to three donor pancreases required per recipient. We aimed to condition the liver to enhance islet engraftment to improve long-term graft function. Diabetic mice received a non-curative islet transplant (n = 400 islets) via the hepatic portal vein (HPV) with fibroblast growth factor 7-loaded galactosylated poly(DL-lactide-co-glycolic acid) (FGF7-GAL-PLGA) particles; 26-µm diameter particles specifically targeted the liver, promoting hepatocyte proliferation in short-term experiments: in mice receiving 0.1-mg FGF7-GAL-PLGA particles (60-ng FGF7) vs vehicle, cell proliferation was induced specifically in the liver with greater efficacy and specificity than subcutaneous FGF7 (1.25 mg/kg ×2 doses; ~75-µg FGF7). Numbers of engrafted islets and vascularization were greater in liver sections of mice receiving islets and FGF7-GAL-PLGA particles vs mice receiving islets alone, 72 h posttransplant. More mice (six of eight) that received islets and FGF7-GAL-PLGA particles normalized blood glucose concentrations by 30-days posttransplant, versus zero of eight mice receiving islets alone with no evidence of increased proliferation of cells within the liver at this stage and normal liver function tests. This work shows that liver-targeted FGF7-GAL-PLGA particles achieve selective FGF7 delivery to the liver-promoting islet engraftment to help normalize blood glucose levels with a good safety profile.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Animales , Glucemia , Factor 7 de Crecimiento de Fibroblastos , Supervivencia de Injerto , RatonesRESUMEN
Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application.
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Péptidos de Penetración Celular/metabolismo , Sistemas de Liberación de Medicamentos , Glicosaminoglicanos/metabolismo , Secuencias de Aminoácidos , Animales , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Detergentes/farmacología , Endocitosis/efectos de los fármacos , Genoma , Proteínas de Homeodominio/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Integrasas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Desarrollo de Músculos/efectos de los fármacos , Proteína MioD/metabolismo , Células 3T3 NIH , Proteína Homeótica Nanog , Nanopartículas , Ácidos Nucleicos/metabolismo , Estructura Terciaria de Proteína , Solubilidad , Tripsina/metabolismoRESUMEN
The ability of materials to define the architecture and microenvironment experienced by cells provides new opportunities to direct the fate of human pluripotent stem cells (HPSCs) [Robinton DA, Daley GQ (2012) Nature 481(7381):295-305]. However, the conditions required for self-renewal vs. differentiation of HPSCs are different, and a single system that efficiently achieves both outcomes is not available [Giobbe GG, et al. (2012) Biotechnol Bioeng 109(12):3119-3132]. We have addressed this dual need by developing a hydrogel-based material that uses ionic de-cross-linking to remove a self-renewal permissive hydrogel (alginate) and switch to a differentiation-permissive microenvironment (collagen). Adjusting the timing of this switch can preferentially steer the HPSC differentiation to mimic lineage commitment during gastrulation to ectoderm (early switch) or mesoderm/endoderm (late switch). As an exemplar differentiated cell type, we showed that directing early lineage specification using this single system can promote cardiogenesis with increased gene expression in high-density cell populations. This work will facilitate regenerative medicine by allowing in situ HPSC expansion to be coupled with early lineage specification within defined tissue geometries.
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Diferenciación Celular/efectos de los fármacos , Hidrogeles/farmacología , Células Madre Pluripotentes/fisiología , Nicho de Células Madre/efectos de los fármacos , Alginatos , Carbocianinas , Colágeno , Ácido Glucurónico , Ácidos Hexurónicos , Humanos , Células Madre Pluripotentes/efectos de los fármacos , Medicina Regenerativa/métodos , Análisis EspectralRESUMEN
Spinal fusion is a prevalent surgical intervention for degenerative spinal diseases, with increasing demand driven by ageing populations. The coexistence of multiple chronic conditions, termed multimorbidity, often complicates surgical outcomes, making advanced bone grafts crucial for successful fusions. This paper reviews the development, clinical application, and controversies surrounding the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in spinal fusion surgeries. A comprehensive narrative review was conducted, focusing on literature from January 1980 to January 2024, sourced from PubMed and Google Scholar. Studies included those examining rhBMP-2 specifically in spinal fusion contexts, excluding other bone morphogenetic proteins (BMPs) and non-spinal applications. This review presents an overarching synopsis of rhBMP-2, its development history and clinical efficacy, the emergence of side effects, and evolving patterns of clinical use. As discussed in this review, clinical practice has adjusted usage and dosages to mitigate adverse effects, yet the need for safer delivery mechanisms persists. rhBMP-2 remains a potent osteoinductive agent with comparable fusion success, as measured by radiographic fusion and good clinical outcomes, to autologous grafts but poses unique risks. This review sets out how further research is essential to optimise the delivery of rhBMP-2 to reduce side effects. Enhanced understanding and innovation of spatio-temporal presentation relative to endogenous BMP could significantly improve patient outcomes in spinal fusion surgeries. The review contributes to the growing body of literature on the use of rhBMP-2 in spine surgery and discusses changing patterns of clinical use over time.
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We demonstrate a new, scalable, simple, and generally applicable two-step method to prepare hollow colloidosomes. First, a high volume fraction oil-in-water emulsion was prepared. The oil phase consisted of CH2Cl2 containing a hydrophobic structural polymer, such as polycaprolactone (PCL) or polystyrene (PS), which was fed into the water phase. The water phase contained poly(vinylalcohol), poly(N-isopropylacrylamide), or a range of cationic graft copolymer surfactants. The emulsion was rotary evaporated to rapidly remove CH2Cl2. This caused precipitation of PCL or PS particles which became kinetically trapped at the periphery of the droplets and formed the shell of the hollow colloidosomes. Interestingly, the PCL colloidosomes were birefringent. The colloidosome yield increased and the polydispersity decreased when the preparation scale was increased. One example colloidosome system consisted of hollow PCL colloidosomes stabilized by PVA. This system should have potential biomaterial applications due to the known biocompatibility of PCL and PVA.
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Polímeros/química , Solventes/química , Coloides , Interacciones Hidrofóbicas e Hidrofílicas , Aceites/química , Tensoactivos/química , Factores de Tiempo , Volatilización , Agua/químicaRESUMEN
Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches.
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Membranas/química , Polímeros , Ingeniería de Tejidos/métodos , Alilamina/química , Animales , Línea Celular , Dimetilpolisiloxanos/química , Elastómeros , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Células Madre Pluripotentes , Propiedades de SuperficieRESUMEN
The limited ability of the heart to regenerate has prompted development of new systems to produce cardiomyocytes for therapeutics. While differentiation of human embryonic stem cells (hESCs) into cardiomyocytes has been well documented, the process remains inefficient and/or expensive, and progress would be facilitated by better understanding the early genetic events that cause cardiac specification. By maintaining a transgenic cardiac-specific MYH6-monomeric red fluorescent protein (mRFP) reporter hESC line in conditions that promote pluripotency, we tested the ability of combinations of 15 genes to induce cardiac specification. Screening identified GATA4 plus TBX5 as the minimum requirement to activate the cardiac gene regulatory network and produce mRFP(+) cells, while a combination of GATA4, TBX5, NKX2.5, and BAF60c (GTNB) was necessary to generate beating cardiomyocytes positive for cTnI and α-actinin. Including the chemotherapeutic agent, Ara-C, from day 10 of induced differentiation enriched for cTnI/α-actinin double positive cells to 45%. Transient expression of GTNB for 5-7 days was necessary to activate the cardiogenesis through progenitor intermediates in a manner consistent with normal heart development. This system provides a route to test the effect of different factors on human cardiac differentiation and will be useful in understanding the network failures that underlie disease phenotypes.
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Diferenciación Celular , Células Madre Embrionarias/citología , Redes Reguladoras de Genes , Actinina/metabolismo , Línea Celular , Proteínas Cromosómicas no Histona , Clonación Molecular , Citarabina/farmacología , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Corazón/embriología , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Lentivirus/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Miocitos Cardíacos/citología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Fluorescente RojaRESUMEN
The field of tissue engineering is generating new scaffolds, bioreactors and methods for stimulating cells within complex cultures, with the aim of recreating the conditions under which cells form functional tissues. Hitherto, the primary focus of this field has been on clinical applications. However, there are many methods of in vitro tissue engineering that represent new opportunities in 3D cell culture and could be the basis for new replacement methods that either replace the use of a tissue isolated from an animal or the use of a living animal. This chapter presents an overview of tissue engineering and provides tissue-specific examples of recent advances.
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Ingeniería de Tejidos/métodos , Humanos , Especificidad de Órganos , Andamios del TejidoRESUMEN
BACKGROUND & AIMS: Intrahepatic drug delivery from implantable scaffolds is being developed as a strategy to modulate growth and enhance regeneration at the time of liver resection. In this study we examine the effects of scaffolds containing hepatocyte growth factor, epidermal growth factor, fibroblast growth factor 1, fibroblast growth factor 2, and liver-derived extracellular matrix (L-ECM) when implanted into normal and partially hepatectomized rat livers. METHODS: Scaffolds loaded with combinations of growth factors and L-ECM were implanted into normal livers (controls=L-ECM, polymer or sham) and livers following partial hepatectomy (controls=partial hepatectomy or sham). The primary end points were hepatocyte DNA synthesis and liver tissue penetration into scaffolds. Secondary end points included non-parenchymal cell DNA synthesis, liver weight analysis, liver function, and histological characterisation of the peri-implant parenchyma. RESULTS: Four days after implantation in normal livers, there was significantly more hepatocyte proliferation around growth factor scaffolds than controls. Seven days after implantation, there was significantly more tissue penetration into growth factor scaffolds than control scaffolds. ED-1 and desmin positive cells were present in the pores of scaffolds. Two days after partial hepatectomy, there was significantly more hepatocyte proliferation around scaffold implanted livers than after partial hepatectomy alone. CONCLUSIONS: Growth factors and L-ECM accelerated non-parenchymal cell migration into scaffolds and increased hepatocyte and non-parenchymal cell proliferation around them. These results demonstrate the potential for intrahepatic implantation of scaffolds containing growth factors and L-ECM to modulate growth in the normal and regenerating liver.
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Movimiento Celular , Matriz Extracelular/fisiología , Hepatocitos/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Regeneración Hepática , Animales , Aspartato Aminotransferasas/sangre , Proliferación Celular , Factor de Crecimiento Epidérmico/fisiología , Factor 1 de Crecimiento de Fibroblastos/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Hepatectomía , Factor de Crecimiento de Hepatocito/fisiología , Masculino , Ratas , Ratas WistarRESUMEN
Thermoresponsive copolymers continue to attract a great deal of interest in the literature. In particular, those based on ethylene oxide-containing methacrylates have excellent potential for biomaterial applications. Recently, some of us reported a study of thermoresponsive cationic graft copolymers containing poly(N-isopropylacrylamide), PNIPAm, (Liu et al., Langmuir, 24, 7099). Here, we report an improved version of this new family of copolymers. In the present study, we replaced the PNIPAm side chains with poly(2-(2-methyoxyethoxy)ethylmethacrylate), PMeO(2)MA. These new, nonacrylamide containing, cationic graft copolymers were prepared using atom transfer radical polymerization (ATRP) and a macroinitiator. They contained poly(trimethylamonium)-aminoethyl methacrylate and PMeO(2)MA, i.e., PTMA(+)(x)-g-(PMeO(2)MA(n))(y). They were investigated using variable-temperature turbidity, photon correlation spectroscopy (PCS), electrophoretic mobility, and (1)H NMR measurements. For one system, four critical temperatures were measured and used to propose a mechanism for the thermally triggered changes that occur in solution. All of the copolymers existed as unimolecular micelles at 20 °C. They underwent reversible aggregation with heating. The extent of aggregation was controlled by the length of the side chains. TEM showed evidence of micellar aggregates. The thermally responsive behaviors of our new copolymers are compared to those for the cationic PNIPAm graft copolymers reported by Liu et al. Our new cationic copolymers retained their positive charge at all temperatures studied, have high zeta potentials at 37 °C, and are good candidates for conferring thermoresponsiveness to negatively charged biomaterial surfaces.
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Metacrilatos/química , Polímeros/química , Cationes , Espectroscopía de Resonancia Magnética , Micelas , Microscopía Electrónica de Transmisión , TemperaturaRESUMEN
OBJECTIVE: Vasculogenic progenitor cell therapy for ischemic diseases bears great potential but still requires further optimization for justifying its clinical application. Here, we investigated the effects of in vivo tissue engineering by combining vasculogenic progenitors with injectable scaffolds releasing controlled amounts of proangiogenic growth factors. METHODS AND RESULTS: We produced biodegradable, injectable polylactic coglycolic acid-based scaffolds releasing single factors or combinations of vascular endothelial growth factor, hepatocyte growth factor, and angiopoietin-1. Dual and triple combinations of scaffold-released growth factors were superior to single release. In murine hindlimb ischemia models, scaffolds releasing dual (vascular endothelial growth factor and hepatocyte growth factor) or triple combinations improved effects of cord blood-derived vasculogenic progenitors. Increased migration, homing, and incorporation of vasculogenic progenitors into the vasculature augmented capillary density, translating into improved blood perfusion. Most importantly, scaffold-released triple combinations including the vessel stabilizer angiopoietin-1 enhanced the number of perivascular smooth muscle actin(+) vascular smooth muscle cells, indicating more efficient vessel stabilization. CONCLUSIONS: Vasculogenic progenitor cell therapy is significantly enhanced by in vivo tissue engineering providing a proangiogenic and provasculogenic growth factor-enriched microenvironment. Therefore, combined use of scaffold-released growth factors and cell therapy improves neovascularization in ischemic diseases and may translate into more pronounced clinical effects.
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Sustancias de Crecimiento/administración & dosificación , Isquemia/terapia , Angiopoyetina 1/administración & dosificación , Animales , Embrión de Pollo , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Factor de Crecimiento de Hepatocito/administración & dosificación , Miembro Posterior/irrigación sanguínea , Humanos , Isquemia/tratamiento farmacológico , Isquemia/patología , Ácido Láctico , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Trasplante de Células Madre , Ingeniería de Tejidos , Andamios del Tejido , Factor A de Crecimiento Endotelial Vascular/administración & dosificaciónRESUMEN
Mesenchymal stem cells are the focus of intense research in bone development and regeneration. The potential of microparticles as modulating moieties of osteogenic response by utilizing their architectural features is demonstrated herein. Topographically textured microparticles of varying microscale features are produced by exploiting phase-separation of a readily soluble sacrificial component from polylactic acid. The influence of varying topographical features on primary human mesenchymal stem cell attachment, proliferation and markers of osteogenesis is investigated. In the absence of osteoinductive supplements, cells cultured on textured microparticles exhibit notably increased expression of osteogenic markers relative to conventional smooth microparticles. They also exhibit varying morphological, attachment and proliferation responses. Significantly altered gene expression and metabolic profiles are observed, with varying histological characteristics in vivo. This study highlights how tailoring topographical design offers cell-instructive 3D microenvironments which allow manipulation of stem cell fate by eliciting the desired downstream response without use of exogenous osteoinductive factors.
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Células Madre Mesenquimatosas , Osteogénesis , Diferenciación Celular , Células Cultivadas , Humanos , Células Madre , Ingeniería de TejidosRESUMEN
Confocal Raman micro-spectroscopy (CRMS) was used to measure spectral images of immunological synapse formation between dendritic and T cells without using molecular labels or other invasive procedures. The purpose-built inverted CRMS instrument integrated an environmental enclosure and a near-infrared laser to allow measurements on live cells maintained under physiological conditions. The integration of the wide-field fluorescence also enabled viability assays and direct comparison between Raman spectral images and gold-standard immuno-fluorescence images for specific molecules. Raman spectral images of nucleus and proteins were built by fuzzy c-mean clustering method. The Raman images were found to be in good correspondence with the immuno-fluorescence images of DNA and actin. These results indicate that actin is a main contributor to the Raman spectrum of the cytoplasm of dendritic and T cells. While for control cells the Raman spectral images of proteins indicated a more homogeneous distribution of proteins in the cytoplasm of dendritic cells, they indicated a higher accumulation of proteins at the immunological synapses when dendritic cells were pre-treated with laminin. These conclusions were also supported by confocal immuno-fluorescence imaging after cell fixation and labelling. This study demonstrates the potential of CRMS for label-free non-invasive imaging of junctions between live cells. Therefore, this technique may become a useful tool for studying cellular processes in live cells and where non-invasive molecular specific imaging is desirable, such as cell-cell interactions.
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Células Dendríticas/química , Sinapsis Inmunológicas/química , Microscopía Confocal/métodos , Espectrometría Raman/métodos , Linfocitos T/química , Actinas/química , Células Cultivadas , Análisis por Conglomerados , Técnicas de Cocultivo , Células Dendríticas/ultraestructura , Humanos , Sinapsis Inmunológicas/ultraestructura , Laminina/química , Linfocitos T/ultraestructuraRESUMEN
Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses. Graphical abstract.
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Encapsulated beta cell transplantation offers a potential cure for a subset of diabetic patients. Once transplanted, beta cell grafts can help to restore glycemic control; however, locating and retrieving cells in the event of graft failure may pose a surgical challenge. Here, a dual-function nanoparticle-loaded hydrogel microcapsule is developed that enables graft retrieval under an applied magnetic field. Additionally, this system facilitates graft localization via magnetic resonance imaging (MRI), and graft isolation from the immune system. Iron oxide nanoparticles encapsulated within alginate hydrogel capsules containing viable islets are transplanted and the in vitro and in vivo retrieval of capsules containing nanoparticles functionalized with various ligands are compared. Capsules containing islets co-encapsulated with COOH-coated nanoparticles restore normal glycemia in immunocompetent diabetic mice for at least 6 weeks, can be visualized using MRI, and are retrievable in a magnetic field. Application of a magnetic field for 90 s via a magnetically assisted retrieval device facilitates rapid retrieval of up to 94% (±3.1%) of the transplant volume 24 h after surgical implantation. This strategy aids monitoring of cell-capsule locations in vivo, facilitates graft removal at the end of the transplant lifetime, and may be applicable to many encapsulated cell transplant systems.
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Diabetes Mellitus Experimental/diagnóstico por imagen , Diabetes Mellitus Experimental/patología , Células Secretoras de Insulina/trasplante , Fenómenos Magnéticos , Imagen por Resonancia Magnética , Animales , Cápsulas , Compuestos Férricos/química , Ratones , Nanopartículas/químicaRESUMEN
Nanoparticles (NPs) are increasingly being developed as biomedical platforms for drug/nucleic acid delivery and imaging. However, in biological fluids, NPs interact with a wide range of proteins that form a coating known as protein corona. Coronae can critically influence self-interaction and binding of other molecules, which can affect toxicity, promote cell activation, and inhibit general or specific cellular uptake. Glycosaminoglycan (GAG)-binding enhanced transduction (GET) is developed to efficiently deliver a variety of cargoes intracellularly; employing GAG-binding peptides, which promote cell targeting, and cell penetrating peptides (CPPs) which enhance endocytotic cell internalization. Herein, it is demonstrated that GET peptide coatings can mediate sustained intracellular transduction of magnetic NPs (MNPs), even in the presence of serum or plasma. NP colloidal stability, physicochemical properties, toxicity and cellular uptake are investigated. Using label-free snapshot proteomics, time-resolved profiles of human plasma coronas formed on functionalized GET-MNPs demonstrate that coronae quickly form (<1 min), with their composition relatively stable but evolving. Importantly GET-MNPs present a subtly different corona composition to MNPs alone, consistent with GAG-binding activities. Understanding how NPs interact with biological systems and can retain enhanced intracellular transduction will facilitate novel drug delivery approaches for cell-type specific targeting of new nanomaterials.
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Sistemas de Liberación de Medicamentos/métodos , Nanopartículas de Magnetita/química , Corona de Proteínas/química , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Células Cultivadas , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Corona de Proteínas/metabolismoRESUMEN
Thermoresponsive and photocrosslinkable polymers can be used as injectable scaffolds in tissue engineering to yield gels in situ with enhanced mechanical properties and stability. They allow easy handling and hold their shapes prior to photopolymerization for clinical practice. Here we report a novel copolymer with both thermoresponsive and photocrosslinkable properties via a facile one-step deactivation enhanced atom transfer radical polymerization (ATRP) using poly(ethylene glycol) methyl ether methylacrylate (PEGMEMA, M(n) = 475) and poly(propylene glycol) methacrylate (PPGMA, M(n) = 375) as monofunctional vinyl monomers and up to 30% of ethylene glycol dimethacrylate (EGDMA) as multifunctional vinyl monomer. The resultant PEGMEMA-PPGMA-EGDMA copolymers have been characterized by gel permeation chromatography (GPC) and 1H NMR analysis, which demonstrate their multivinyl functionality and hyperbranched structures. These water-soluble copolymers show lower critical solution temperature (LCST) behavior at 32 degrees C, which is comparable to poly(N-isopropylacrylamide) (PNIPAM). The copolymers can also be cross-linked by photopolymerization through their multivinyl functional groups. Rheological studies clearly demonstrate that the photocrosslinked gels formed at a temperature above the LCST have higher storage moduli than those prepared at a temperature below the LCST. Moreover, the cross-linking density of the gels can be tuned to tailor their porous structures and mechanical properties by adjusting the composition and concentration of the copolymers. Hydrogels with a broad range of storage moduli from 10 to 400 kPa have been produced.
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Resinas Acrílicas/síntesis química , Resinas Acrílicas/farmacología , Hidrogeles/química , Metacrilatos/química , Éteres Metílicos/química , Polímeros/síntesis química , Polímeros/farmacología , Resinas Acrílicas/química , Reactivos de Enlaces Cruzados/farmacología , Ensayo de Materiales , Fotoquímica , Polímeros/química , Estrés Mecánico , Temperatura , Rayos UltravioletaRESUMEN
Photo-cross-linked hydrogels from thermoresponsive polymers can be used as advanced injectable biomaterials via a combination of physical interaction (in situ thermal gelation) and covalent cross-links (in situ photopolymerization). This can lead to gels with significantly enhanced mechanical properties compared to non-photo-cross-linked thermoresponsive hydrogels. Moreover, the thermally phase-separated gels have attractive advantages over non-thermoresponsive gels because thermal gelation upon injection allows easy handling and holds the shape of the gels prior to photopolymerization. In this study, water-soluble thermoresponsive copolymers containing multiple methacrylate groups were synthesized via one-step deactivation enhanced atom transfer radical polymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, M(n) = 475), poly(propylene glycol) methacrylate (PPGMA, M(n) = 375), and ethylene glycol dimethacrylate (EGDMA) and were used to form covalent cross-linked hydrogels by photopolymerization. The cross-linking density was found to have a significant influence on the mechanical and swelling properties of the photo-cross-linked gels. Release studies using lysozyme as a model protein demonstrated a sustained release profile that varied dependent on the copolymer composition, cross-linking density, and the temperature. Mouse C2C12 myoblast cells were cultured in the presence of the copolymers at concentrations up to 1 mg/mL. It was found that the majority of the cells remained viable, as assessed by Alamar Blue, lactate dehydrogenase (LDH), and Live/Dead cell viability/cytotoxicity assays. These studies demonstrate that thermoresponsive PEGMEMA-PPGMA-EGDMA copolymers offer potential as in situ photopolymerizable materials for tissue engineering and drug delivery applications through a combination of facile synthesis, enhanced mechanical properties, tunable cross-linking density, low cytotoxicity, and accessible functionality for further structure modifications.
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Resinas Acrílicas/química , Hidrogeles/química , Metacrilatos/química , Proteínas/química , Microscopía Electrónica de Rastreo , Fotoquímica , Reología , Espectrofotometría UltravioletaRESUMEN
The effect of topography in three-dimensional (3D) printed polymer scaffolds on stem cell differentiation is a significantly underexplored area. Compared to two-dimensional (2D) biomaterials on which various well-defined topographies have been incorporated and shown to direct a range of cell behaviors including adhesion, cytoskeleton organization, and differentiation, incorporating topographical features to 3D polymer scaffolds is challenging due to the difficulty of accessing the inside of a porous scaffold. Only the roughened strut surface has been introduced to 3D printed porous scaffolds. Here, a rapid, single-step 3D printing method to fabricate polymeric scaffolds consisting of microstruts (ca. 60 µm) with micro-/nanosurface pores (0.2-2.4 µm) has been developed based on direct ink writing of an agitated viscous polymer solution. The density, size, and alignment of these pores can be controlled by changing the degree of agitation or the speed of printing. Three-dimensional printed scaffolds with micro-/nanoporous struts enhanced chondrogenic and osteogenic differentiation of mesenchymal stem cells (MSCs) without soluble differentiation factors. The topography also selectively affected adhesion, morphology, and differentiation of MSC to chondrogenic and osteogenic lineages depending on the composition of the differentiation medium. This fabrication method can potentially be used for a wide range of polymers where desirable architecture and topography are required.
Asunto(s)
Diferenciación Celular , Condrogénesis , Células Madre Mesenquimatosas/citología , Nanoporos , Osteogénesis , Impresión Tridimensional , Andamios del Tejido/química , Adhesión Celular , Proliferación Celular , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/ultraestructura , Nanoporos/ultraestructura , Poliésteres/química , Solventes , Propiedades de Superficie , TermogravimetríaRESUMEN
Tissue engineering response may be tailored via controlled, sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional (3D) ice-templated collagen scaffolds. However, the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored. Here, we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide) microparticles. We probe the effects of subsequent N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride crosslinking on protein release, using microparticles with different internal protein distributions. Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug. The scaffolds display a homogeneous microparticle distribution, and a reduction in pore size and percolation diameter with increased microparticle addition, although these values did not fall below those reported as necessary for cell invasion. The protein distribution within the microparticles, near the surface or more deeply located within the microparticles, was important in determining the release profile and effect of crosslinking, as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold. Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release. Protein located within the bulk of the microparticles, was protected from the crosslinking reaction and no delay in the overall release profile was seen.