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Titanium mesh (Ti-mesh) for guided bone regeneration (GBR) approaches has been extensively considered to offer space maintenance in reconstructing the alveolar ridge within bone defects due to its superb mechanical properties and biocompatibility. However, soft tissue invasion across the pores of the Ti-mesh and intrinsically limited bioactivity of the titanium substrates often hinder satisfactory clinical outcomes in GBR treatments. Here, a cell recognitive osteogenic barrier coating was proposed using a bioengineered mussel adhesive protein (MAP) fused with Alg-Gly-Asp (RGD) peptide to achieve highly accelerated bone regeneration. The fusion bioadhesive MAP-RGD exhibited outstanding performance as a bioactive physical barrier that enabled effective cell occlusion and a prolonged, localized delivery of bone morphogenetic protein-2 (BMP-2). The MAP-RGD@BMP-2 coating promoted in vitro cellular behaviors and osteogenic commitments of mesenchymal stem cells (MSCs) via the synergistic crosstalk effects of the RGD peptide and BMP-2 in a surface-bound manner. The facile gluing of MAP-RGD@BMP-2 onto the Ti-mesh led to a distinguishable acceleration of the in vivo formation of new bone in terms of quantity and maturity in a rat calvarial defect. Hence, our protein-based cell recognitive osteogenic barrier coating can be an excellent therapeutic platform to improve the clinical predictability of GBR treatment.
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Limited regenerative capacity of the nervous system makes treating traumatic nerve injuries with conventional polymer-based nerve grafting a challenging task. Consequently, utilizing natural polymers and biomimetic topologies became obvious strategies for nerve conduit designs. As a bioinspired natural polymer from a marine organism, mussel adhesive proteins (MAPs) fused with biofunctional peptides from extracellular matrix (ECM) were engineered for accelerated nerve regeneration by enhancing cell adhesion, proliferation, neural differentiation, and neurite formation. To physically promote contact guidance of neural and Schwann cells and to achieve guided nerve regeneration, MAP was fabricated into an electrospun aligned nanofiber conduit by introducing synthetic polymer poly(lactic-co-glycolic acid) (PLGA) to control solubility and mechanical property. In vitro and in vivo experiments demonstrated that the multi-dimensional tactics of combining adhesiveness from MAP, integrin-mediated interaction from ECM peptides (in particular, IKVAV derived from laminin α1 chain), and contact guidance from aligned nanofibers synergistically accelerated functional nerve regeneration. Thus, MAP-based multi-dimensional approach provides new opportunities for neural regenerative applications including nerve grafting. STATEMENT OF SIGNIFICANCE: Findings in neural regeneration indicate that a bioinspired polymer-based nerve conduit design should harmoniously constitute various factors, such as biocompatibility, neurotrophic molecule, biodegradability, and contact guidance. Here, we engineered three fusion proteins of mussel-derived adhesive protein with ECM-derived biofunctional peptides to simultaneously provide biocompatibility and integrin-based interactions. In addition, a fabrication of robust aligned nanofiber conduits containing the fusion proteins realized suitable biodegradability and contact guidance. Thus, our multi-dimensional strategy on conduit design provided outstanding biocompatibility, biodegradability, integrin-interaction, and contact guidance to achieve an accelerated functional nerve regeneration. We believe that our bioengineered mussel adhesive protein-based multi-dimensional strategy would offer new insights into the design of nerve tissue engineering biomaterials.
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Regeneración Tisular Dirigida , Nanofibras , Regeneración Nerviosa , Proteínas , Nervio Ciático , Animales , Nanofibras/química , Nanofibras/uso terapéutico , Células PC12 , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/farmacología , Proteínas/química , Proteínas/farmacología , Ratas , Células de Schwann/metabolismo , Células de Schwann/patología , Nervio Ciático/lesiones , Nervio Ciático/patología , Nervio Ciático/fisiología , Ingeniería de TejidosRESUMEN
Stem cell therapies are emerging regenerative treatments for ischemic and chronic diseases. Although high cell retention and prompt angiogenesis are prerequisites to improving efficacy, advancements have not yet been developed. Here, we proposed long-term surviving and angiogenesis-inducing stem cell with high cell retention thanks to fluid immiscible liquid micro-droplets bio-inspired by a glue modality 'complex coacervate' found in the sandcastle worm. Formed by the Coulombic force between polycationic MAP and polyanionic hyaluronic acid, the exploited coacervate micro-droplets enabled the encapsulation of stem cells. The underwater adhesiveness facilitated integrating the encapsulated stem cells onto various surfaces with impressive cell retention after facile injection. Stem cells encapsulated in the coacervate platform formed cell clusters capable of pre-adjusting to hypoxia by expressing hypoxia-inducible factor 1α (HIF-1α), increasing viability and reducing apoptosis under hypoxia and ischemia as well as normoxia. Interestingly, multipotent and angiogenic factors were significantly enhanced by HIF-1α expression. In the in vivo evaluation, the coacervate platform showed impressive angiogenesis with biocompatibility and long-term cell retention capacity with sustainable release as protein factories. Therefore, the proposed MAP-based water-immiscible, injectable, sticky, and bioactive 3D coacervate micro-droplets offers a promising tool for chronic diseases in body fluid-rich environments. STATEMENT OF SIGNIFICANCE: High cell retention, long-term survival, and rapid angiogenesis are prerequisites of successful stem cell therapy. However, no previous advancements have simultaneously satisfied all of these requirements. In this work, we clearly developed a novel, revolutionary stem cell carrier platform with underwater adhesiveness from a mussel-derived glue protein and water immiscibility from a sandcastle-worm-inspired glue modality via 'complex coacervation'. To the best of our knowledge, no report has emerged employing coacervate as a stem cell therapeutic platform. This fluid-immiscible, injectable, sticky, and bioactive 3-dimensional stem cell micro-droplets demonstrated the excellent stem cell retention and viability under hypoxia environments and enhanced multipotent and angiogenic effects with minimal immune response.
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Organismos Acuáticos/química , Materiales Biomiméticos/farmacología , Células Inmovilizadas/citología , Células Madre Multipotentes/citología , Neovascularización Fisiológica , Proteínas/farmacología , Animales , Hipoxia de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Inmovilizadas/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Hialurónico/farmacología , Inyecciones , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Ratas , AguaRESUMEN
Silica nanoparticles (SiNPs) have been utilized to construct bioactive nanostructures comprising surface topographic features and bioactivity that enhances the activity of bone cells onto titanium-based implants. However, there have been no previous attempts to create microrough surfaces based on SiNP nanostructures even though microroughness is established as a characteristic that provides beneficial effects in improving the biomechanical interlocking of titanium implants. Herein, a protein-based SiNP coating is proposed as an osteopromotive surface functionalization approach to create microroughness on titanium implant surfaces. A bioengineered recombinant mussel adhesive protein fused with a silica-precipitating R5 peptide (R5-MAP) enables direct control of the microroughness of the surface through the multilayer assembly of SiNP nanostructures under mild conditions. The assembled SiNP nanostructure significantly enhances the in vitro osteogenic cellular behaviors of preosteoblasts in a roughness-dependent manner and promotes the in vivo bone tissue formation on a titanium implant within a calvarial defect site. Thus, the R5-MAP-based SiNP nanostructure assembly could be practically applied to accelerate bone-tissue growth to improve the stability and prolong the lifetime of medical implantable devices.
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Nanoestructuras , Adhesivos , Animales , Bivalvos , Materiales Biocompatibles Revestidos , Diatomeas , Dióxido de Silicio , Propiedades de Superficie , TitanioRESUMEN
Skin scarring after deep dermal injuries is a major clinical problem due to the current therapies limited to established scars with poor understanding of healing mechanisms. From investigation of aberrations within the extracellular matrix involved in pathophysiologic scarring, it was revealed that one of the main factors responsible for impaired healing is abnormal collagen reorganization. Here, inspired by the fundamental roles of decorin, a collagen-targeting proteoglycan, in collagen remodeling, we created a scar-preventive collagen-targeting glue consisting of a newly designed collagen-binding mussel adhesive protein and a specific glycosaminoglycan. The collagen-targeting glue specifically bound to type I collagen in a dose-dependent manner and regulated the rate and the degree of fibrillogenesis. In a rat skin excisional model, the collagen-targeting glue successfully accelerated initial wound regeneration as defined by effective reepithelialization, neovascularization, and rapid collagen synthesis. Moreover, the improved dermal collagen architecture was demonstrated by uniform size of collagen fibrils, their regular packing, and a restoration of healthy tissue component. Collectively, our natural healing-inspired collagen-targeting glue may be a promising therapeutic option for improving the healing rate with high-quality and effective scar inhibition.
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Colágeno/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Adhesivos Tisulares/química , Adhesivos Tisulares/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Animales , Colágeno Tipo I/química , Colágeno Tipo I/uso terapéutico , Decorina/química , Decorina/uso terapéutico , Electroforesis en Gel de Poliacrilamida , Femenino , Glicosaminoglicanos , Humanos , Ratones , Microscopía Electrónica de Transmisión , Células 3T3 NIH , Proteínas/química , Proteínas/uso terapéutico , Proteoglicanos/química , Proteoglicanos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
Nanofibrous scaffolds have been assessed as one of many promising tissue engineering scaffolds to be utilized for wound-healing applications. Previously, we reported multi-functionalized electrospun nanofibrous scaffolds blended with mussel adhesive protein (MAP) and polycaprolactone (PCL), which provide durable mechanical strength, cell-friendly environments, and a substantial ability to capture diverse bioactive molecules without any surface modifications. In the present work, we applied the blended nanofibrous mats of MAP and PCL for in vivo skin wound healing. The nanofibrous mats showed accelerated regeneration in a rat skin wound-healing model, which might be attributed to a highly compatible environment for keratinocyte cell growth, an ability to capture inherent growth factors, and an efficient exudate absorption capacity. Thus, this work would suggest that adhesive property of scaffold could be a factor of successful application for wound healing. The MAP-blended nanofibers could also be potentially exploited for diverse tissue regeneration applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 218-225, 2017.
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Nanofibras , Poliésteres , Proteínas , Piel , Cicatrización de Heridas/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Nanofibras/química , Nanofibras/uso terapéutico , Poliésteres/química , Poliésteres/farmacología , Proteínas/química , Proteínas/farmacología , Ratas , Ratas Sprague-Dawley , Piel/lesiones , Piel/metabolismo , Piel/patologíaRESUMEN
Xenogenic bone substitutes are commonly used during orthopedic reconstructive procedures to assist bone regeneration. However, huge amounts of blood accompanied with massive bone loss usually increase the difficulty of placing the xenograft into the bony defect. Additionally, the lack of an organic matrix leads to a decrease in the mechanical strength of the bone-grafted site. For effective bone grafting, this study aims at developing a mussel adhesion-employed bone graft binder with great blood-resistance and enhanced mechanical properties. The distinguishing water (or blood) resistance of the binder originates from sandcastle worm-inspired complex coacervation using negatively charged hyaluronic acid (HA) and a positively charged recombinant mussel adhesive protein (rMAP) containing tyrosine residues. The rMAP/HA coacervate stabilizes the agglomerated bone graft in the presence of blood. Moreover, the rMAP/HA composite binder enhances the mechanical and hemostatic properties of the bone graft agglomerate. These outstanding features improve the osteoconductivity of the agglomerate and subsequently promote in vivo bone regeneration. Thus, the blood-resistant coacervated mussel protein glue is a promising binding material for effective bone grafting and can be successfully expanded to general bone tissue engineering.
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Bivalvos/metabolismo , Regeneración Ósea/efectos de los fármacos , Sustitutos de Huesos/farmacología , Osteogénesis/efectos de los fármacos , Proteínas/farmacología , Animales , Huesos/efectos de los fármacos , Ácido Hialurónico/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Tirosina/farmacologíaRESUMEN
The injector for the main driver linear accelerator of the Rare Isotope Science Project in Korea, has been developed to allow heavy ions up to uranium to be delivered to the inflight fragmentation system. The critical components of the injector are the superconducting electron cyclotron resonance (ECR) ion sources, the radio frequency quadrupole (RFQ), and matching systems for low and medium energy beams. We have built superconducting magnets for the ECR ion source, and a prototype with one segment of the RFQ structure, with the aim of developing a design that can satisfy our specifications, demonstrate stable operation, and prove results to compare the design simulation.
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As biodegradable scaffolds, protein hydrogels have considerable potential, particularly for bioartificial organs and three-dimensional space-filling materials. However, their low strength and stiffness have been considered to be limitations for enduring physiological stimuli. Therefore, protein hydrogels have been commonly utilized as delivery vehicles rather than as supporting materials. In this work, sea anemone tentacle-derived recombinant silk-like protein (aneroin) was evaluated as a potential material for a mechanically durable protein hydrogel. Inspired by the natural hardening mechanism, photoinitiated dityrosine cross-linking was employed to fabricate an aneroin hydrogel. It was determined that the fabricated aneroin hydrogel was approximately 10-fold stiffer than mammalian cardiac or skeletal muscle. The aneroin hydrogel provided not only structural support but also an adequate environment for cells. It exhibited an adequate swelling ability and microstructure, which are beneficial for facilitating mass transport and cell proliferation. Based on its mechanical and biological properties, this aneroin hydrogel could be used in various biomedical applications, such as cell-containing patches, biomolecule carriers, and artificial extracellular matrices.
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Materiales Biocompatibles/química , Reactivos de Enlaces Cruzados/química , Ácido Hialurónico/química , Hidrogeles/farmacología , Proteínas Recombinantes/farmacología , Tirosina/análogos & derivados , Animales , Materiales Biocompatibles/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Dureza , Pruebas de Dureza , Hidrogeles/química , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Luz , Ratones , Células 3T3 NIH , Procesos Fotoquímicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Anémonas de Mar/química , Seda/química , Ingeniería de Tejidos/métodos , Andamios del Tejido , Tirosina/químicaRESUMEN
Urinary fistulas, abnormal openings of a urinary tract organ, are serious complications and conventional management strategies are not satisfactory. For more effective and non-invasive fistula repair, fluid tissue adhesives or sealants have been suggested. However, conventional products do not provide a suitable solution due to safety problems and poor underwater adhesion under physiological conditions. Herein, we proposed a unique water-immiscible mussel protein-based bioadhesive (WIMBA) exhibiting strong underwater adhesion which was employed by two adhesion strategies of marine organisms; 3,4-dihydroxy-l-phenylalanine (DOPA)-mediated strong adhesion and water-immiscible coacervation. The developed biocompatible WIMBA successfully sealed ex vivo urinary fistulas and provided good durability and high compliance. Thus, WIMBA could be used as a promising sealant for urinary fistula management with further expansion to diverse internal body applications.
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Proteínas/uso terapéutico , Fístula Urinaria/tratamiento farmacológico , Agua/química , Animales , Bivalvos , Dihidroxifenilalanina/farmacología , Masculino , Presión , Proteínas/farmacología , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Adherencias Tisulares/patologíaRESUMEN
Currently approved surgical tissue glues do not satisfy the requirements for ideal bioadhesives due to limited adhesion in wet conditions and severe cytotoxicity. Herein, we report a new light-activated, mussel protein-based bioadhesive (LAMBA) inspired by mussel adhesion and insect dityrosine crosslinking chemistry. LAMBA exhibited substantially stronger bulk wet tissue adhesion than commercially available fibrin glue and good biocompatibility in both in vitro and in vivo studies. Besides, the easily tunable, light-activated crosslinking enabled an effective on-demand wound closure and facilitated wound healing. Based on these outstanding properties, LAMBA holds great potential as an ideal surgical tissue glue for diverse medical applications, including sutureless wound closures of skin and internal organs.
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Insectos/química , Luz , Proteínas/farmacología , Adhesivos Tisulares/farmacología , Animales , Bivalvos/química , Muerte Celular/efectos de los fármacos , Línea Celular , Cianoacrilatos/farmacología , Adhesivo de Tejido de Fibrina/farmacología , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Piel/efectos de los fármacos , Piel/patología , Sus scrofa , Suturas , Tirosina/análogos & derivados , Tirosina/química , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Xenograft bone substitutes, such as deproteinized bovine bone mineral (DBBM), have been widely employed as osteoconductive structural materials for bone tissue engineering. However, the loss of xenograft bone substitute particles in defects has been a major limitation, along with a lack of osteoinductive function. Mussel adhesive protein (MAP), a remarkable and powerful adhesive biomaterial in nature, can attach to various substrates, even in wet environments. Its adhesive and water-resistant abilities are considered to be mainly derived from the reduced catechol form, 3,4-dihydroxyphenylalanine (DOPA), of its tyrosine residues. Here, we evaluated the use of DOPA-containing MAP as a functional binder biomaterial to effectively retain DBBM particles at the defect site during in vivo bone regeneration. We observed that DOPA-containing MAP was able to bind DBBM particles easily to make an aggregate, and grafted DBBM particles were not lost in a defect in the rat calvaria during the healing period. Importantly, grafting of a DOPA-containing MAP-bound DBBM aggregate resulted in remarkably accelerated in vivo bone regeneration and even bone remodeling. Interestingly, we found that the DOPA residues in the modified MAP had an osteoinductive ability based on clear observation of the in vivo maturation of new bones with a similar bone density to the normal bone and of the in vitro osteogenic differentiation of osteoblast cells. Collectively, DOPA-containing MAP is a promising functional binder biomaterial for xenograft bone substitute-assisted bone regeneration with enhanced osteoconductivity and acquired osteoinductivity. This mussel glue could also be successfully utilized as a potential biomaterial for general bone tissue engineering.
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Successful titanium implantation strongly depends on early fixation through an osseointegration between the titanium fixture and adjacent bone tissue. From a clinical perspective, rapid recruitment of functional biomolecules from the blood and osteogenic cell binding is critical for osseointegration immediately after implant insertion. Thus, surface modifications aiming to improve the interactions between the blood and implant and to enhance the binding of osteogenic cells onto the implant surface can contribute to successful osseointegration. Mussel adhesive proteins (MAPs) derived from marine mussels have been considered as promising bioadhesives that have strong adhesion and coating abilities onto organic and inorganic surfaces, even in wet environments. Here, we investigated the in vitro and in vivo osteostimulating ability of the bioengineered mussel glue MAP-RGD, which is a recombinant MAP fused with an Arg-Gly-Asp (RGD) peptide, an effective cell recognition motif for activating intracellular signaling pathways, using a titanium mesh (Ti-mesh) as a model titanium implant. We found that the in vitro cell behaviors of pre-osteoblast cells, such as attachment, proliferation, spreading, and osteogenic differentiation, increased significantly on the MAP-RGD-coated Ti-mesh surface. In vitro blood responses including blood wetting, blood clotting, and platelet adhesion were also highly enhanced on the MAP-RGD-coated surface. Importantly, implantation of the MAP-RGD-coated Ti-mesh resulted in a remarkable acceleration of in vivo bone regeneration and maturation of a new bone in a rat calvarial defect. Consequently, the bioengineered mussel glue can be successfully utilized as an osteostimulating bone bioadhesive for titanium implant applications with further expansion to general bone tissue engineering.
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During implant surgeries, antibacterial agents are needed to prevent bacterial infections, which can cause the formation of biofilms between implanted materials and tissue. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on various surfaces even in wet environment. Here, we proposed a novel surface-independent antibacterial coating strategy based on the fusion of MAP to a silver-binding peptide, which can synthesize silver nanoparticles having broad antibacterial activity. This sticky recombinant fusion protein enabled the efficient coating on target surface and the easy generation of silver nanoparticles on the coated-surface under mild condition. The biosynthesized silver nanoparticles showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria and also revealed good cytocompatibility with mammalian cells. In this coating strategy, MAP-silver binding peptide fusion proteins provide hybrid environment incorporating inorganic silver nanoparticle and simultaneously mediate the interaction of silver nanoparticle with surroundings. Moreover, the silver nanoparticles were fully synthesized on various surfaces including metal, plastic, and glass by a simple, surface-independent coating manner, and they were also successfully synthesized on a nanofiber surface fabricated by electrospinning of the fusion protein. Thus, this facile surface-independent silver nanoparticle-generating antibacterial coating has great potential to be used for the prevention of bacterial infection in diverse biomedical fields.
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Antibacterianos/química , Materiales Biocompatibles Revestidos/química , Nanopartículas del Metal/química , Proteínas/química , Nitrato de Plata/química , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Bivalvos/metabolismo , Línea Celular , Materiales Biocompatibles Revestidos/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Ratones , Nanofibras/química , Nanofibras/ultraestructura , Péptidos/genética , Péptidos/metabolismo , Poliestirenos/química , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Propiedades de SuperficieRESUMEN
BACKGROUND: Unique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult. RESULTS: In the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151. CONCLUSIONS: Through this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications.
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Proteínas/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Endotoxinas/análisis , Endotoxinas/toxicidad , Escherichia coli/metabolismo , Cuerpos de Inclusión/metabolismo , Interleucina-6/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Proteínas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Silk has recently been exploited in various fields due to its superior mechanical properties. However, this material's lack of biological functions and relatively poor biodegradation have hindered its wide use in applications related to cells and tissues. Here, we improved the overall characteristics of silkworm silk fibroin (SF) by introduction of RGD peptide-fused recombinant mussel adhesive protein (MAP-RGD). Simple blending of MAP-RGD provided not only bulk-scale adhesive ability but also microscale adhesiveness to cells and various biomolecules. MAP-RGD-blended SF fibers supported enhanced adhesion, proliferation, and spreading of mammalian cells as well as the efficient attachment of biomolecules, including carbohydrate and protein. In addition, the hydrophilicity, swelling, and biodegradability of the MAP-RGD-blended SF material were improved without notable hampering of the original mechanical properties of SF. Therefore, the adhesive silk fibrous scaffold could be successfully used in diverse biomedical engineering applications.
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Materiales Biocompatibles , Fibroínas/química , Oligopéptidos/química , Proteínas/química , Proteínas Recombinantes de Fusión/química , Adhesivos/química , Adhesión Celular , Línea Celular , Proliferación Celular , Humanos , Queratinocitos/citología , Osteoblastos/citología , Andamios del TejidoRESUMEN
A whole cell array biosensor for the efficient detection of neurotoxic organophosphate compounds (OPs) was developed through the immobilization of recombinant Escherichia coli cells containing periplasmic-expressing organophosphorus hydrolase (OPH) onto the surface of a 96-well microplate using mussel adhesive protein (MAP) as a microbial cell-immobilizing linker. Both the paraoxon-hydrolyzing activity and fluorescence microscopy analyses demonstrated that the use of MAP in a whole cell biosensor increased the cell-immobilizing efficiency and enhanced the stability of immobilized cells compared to a simple physical adsorption-based whole cell system. Scanning electron microscopic analyses also showed that the E. coli cells were effectively immobilized on the MAP-coated surface without any pretreatment steps. The whole cell array biosensor system, prepared using optimal MAP coating (50 µg/cm(2)) and cell loading (4 OD(600)), detected paraoxon levels as low as 5 µM with high reproducibility, and its quantitative detection range was ~5-320 µM. The MAP-based whole cell array biosensor showed a good long-term stability for 28 day with 80% retained activity and a reusability of up to 20 times. In addition, paraoxon in tap water was also successfully detected without a reduction in sensitivity. Our results indicate that the proposed MAP-based whole cell array system could be used as a potential platform for a stable and reusable whole cell biosensor.
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Bioensayo/instrumentación , Técnicas Biosensibles/instrumentación , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Análisis de Inyección de Flujo/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Compuestos Organofosforados/análisis , Compuestos Organofosforados/farmacología , Proteínas/metabolismo , Adhesión Celular/fisiología , Diseño de Equipo , Análisis de Falla de Equipo , Compuestos Organofosforados/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The use of mussel adhesive proteins (MAPs) as a surface coating for cell adhesion has been suggested due to their unique properties of biocompatibility and effective adhesion on diverse inorganic and organic surfaces. The surface functionalization of scaffolds or implants using extracellular matrix (ECM) molecules is important for the enhancement of target cell behaviors such as proliferation and differentiation. In the present work, we suggest a new, simple surface functionalization platform based on the charge interactions between the positively charged MAP linker and negatively charged ECM molecules, such as glycosaminoglycans (GAGs). MAP was efficiently coated onto a titanium model surface using its adhesion ability. Then, several GAG molecules, including hyaluronic acid (HA), heparin sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS), were effectively immobilized on the MAP-coated surfaces by charge interactions. Using HA as a model GAG molecule, we found that the proliferation, spreading, and differentiation behaviors of mouse preosteoblast cells were all significantly improved on MAP/HA-layered titanium. In addition, we successfully constructed a multilayer film on a titanium surface with oppositely charged layer-by-layer coatings of MAP and HA. Collectively, our simple MAP-based surface functionalization strategy can be successfully used for the efficient surface immobilization of negatively charged ECM molecules in various tissue engineering and medical implantation applications.
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Glicosaminoglicanos/química , Proteínas/química , Células 3T3 , Animales , Adhesión Celular , Diferenciación Celular , Línea Celular , Proliferación Celular , Sulfatos de Condroitina/química , Dermatán Sulfato/química , Heparitina Sulfato/química , Ácido Hialurónico/química , Ratones , Osteoblastos/citología , Propiedades de Superficie , Ingeniería de Tejidos/métodos , Titanio/químicaRESUMEN
Adhesion of cells to surfaces is a basic and important requirement in cell culture and tissue engineering. Here, we designed artificial extracellular matrix (ECM) mimics for efficient cellular attachment, based on mussel adhesive protein (MAP) fusion with biofunctional peptides originating from ECM materials, including fibronectin, laminin, and collagen. Cellular behaviors, including attachment, proliferation, spreading, viability, and differentiation, were investigated with the artificial ECM material-coated surfaces, using three mammalian cell lines (pre-osteoblast, chondrocyte, and pre-adipocyte). All cell lines examined displayed superior attachment, proliferation, spreading, and survival properties on the MAP-based ECM mimics, compared to other commercially available cell adhesion materials, such as poly-L-lysine and the naturally extracted MAP mixture. Additionally, the degree of differentiation of pre-osteoblast cells on MAP-based ECM mimics was increased. These results collectively demonstrate that the artificial ECM mimics developed in the present work are effective cell adhesion materials. Moreover, we expect that the MAP peptide fusion approach can be extended to other functional tissue-specific motifs.
Asunto(s)
Materiales Biomiméticos/farmacología , Matriz Extracelular/química , Péptidos/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Péptidos/aislamiento & purificación , Propiedades de Superficie/efectos de los fármacosRESUMEN
Adhesion of cells to a surface is a basic and important requirement in the fields of cell culture and tissue engineering. Previously, we constructed the cell adhesive, fp-151-RGD, by fusion of the hybrid mussel adhesive protein, fp-151, and GRGDSP peptide, one of the major cell adhesion recognition motifs; fp-151-RGD efficiently immobilized cells on coated culture surfaces with no protein and surface modifications, and apparently enhanced cell adhesion, proliferation, and spreading abilities. In the present study, we investigated the potential use of fp-151-RGD as a biomimetic extracellular matrix material at the molecular level by elucidating its substantial effects on integrin-mediated adhesion and signaling. Apoptosis derived from serum deprivation was significantly suppressed on the fp-151-RGD-coated surface, indicating that RGD-induced activation of integrin-mediated signaling triggers the pathway for cell survival. Analysis of the phosphorylation of focal adhesion kinase clearly demonstrated activation of focal adhesion kinase, a well-established indicator of integrin-mediated signaling, on the fp-151-RGD-coated surface, leading to significantly enhanced cell behaviors, including proliferation, spreading and survival, and consequently, more efficient cell culture.