RESUMEN
Two silklike proteins, [TGRGDSPAGG(GAGAGS)3AS]5 (FS5) and [TGRGDSPA-(GVPGV)2GG(GAGAGS)3AS]8 (FES8) were designed to demonstrate the superior performance as biomaterials of silklike proteins. The former protein consists of the crystalline domain sequence, (GAGAGS)n from Bombyx mori silk fibroin and cell-adhesive sequence TGRGDSPA coming from fibronectin-containing RGD triplet. The additional sequence (GVPGV)n from elastin was included in the latter protein. The considerably higher cell-adhesion activities of these proteins for NHDF and VERO cells were observed by comparing with those of silklike materials without RGD sequences and also the crystalline fraction of B. mori silk fibroin. This tendency was independent of the treatments, 4.5M LiClO4 or formic acid (FA), on silklike proteins. Their activities are also higher than those of commercial Fibronectin F for NHDF cell. Their structural characterization was studied using 13C solid-state NMR. Although the overlapped peaks in usual 13C CP/MAS NMR spectra make the detailed structural analysis difficult, the methyl resonance regions observed using dipolar dephasing NMR were very useful for the analysis. The presence of both random coil and beta-sheet structures was observed in these proteins clearly. The content of beta-sheet structure in both proteins increases after FA treatment when compared with the lyophilized samples. The production of electrospun nanofibers from their hexafluoroacetone solution was also tried. The silklike protein FES8 could prepare nonwoven silk fibers although FS5 could not.
Asunto(s)
Bombyx/química , Elastina/química , Elastina/genética , Fibroínas/química , Fibroínas/genética , Fibronectinas/química , Fibronectinas/genética , Seda/química , Seda/genética , Acetona/análogos & derivados , Acetona/química , Animales , Secuencia de Bases , Materiales Biocompatibles/química , Adhesión Celular/efectos de los fármacos , Chlorocebus aethiops , Escherichia coli/metabolismo , Fluorocarburos/química , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Nanotubos , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Andamios del Tejido , Células VeroRESUMEN
A novel, pure, synthetic material is presented that promotes the repair of full-thickness skin wounds. The active component is tropoelastin and leverages its ability to promote new blood vessel formation and its cell recruiting properties to accelerate wound repair. Key to the technology is the use of a novel heat-based, stabilized form of human tropoelastin which allows for tunable resorption. This implantable material contributes a tailored insert that can be shaped to the wound bed, where it hydrates to form a conformable protein hydrogel. Significant benefits in the extent of wound healing, dermal repair, and regeneration of mature epithelium in healthy pigs are demonstrated. The implant is compatible with initial co-treatment with full- and split-thickness skin grafts. The implant's superiority to sterile bandaging, commercial hydrogel and dermal regeneration template products is shown. On this basis, a new concept for a prefabricated tissue repair material for point-of-care treatment of open wounds is provided.
Asunto(s)
Implantes Absorbibles , Dermis , Hidrogel de Polietilenoglicol-Dimetacrilato , Andamios del Tejido , Tropoelastina , Cicatrización de Heridas/efectos de los fármacos , Animales , Autoinjertos/trasplante , Vasos Sanguíneos/metabolismo , Dermis/lesiones , Dermis/metabolismo , Dermis/patología , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Ratones , Porcinos , Tropoelastina/química , Tropoelastina/farmacologíaRESUMEN
The purpose of this study is to create a new silk fibroin scaffold with sufficient three-dimensional morphology and porous structure for cartilage formation. We have applied sucrose particles sized around 300 to 500 microm as porogens compared to equal-sized salt particles. After the porogen was leached out with water, scaffolds were prepared with fibroin derived from sucrose/hexafluoroisopropanol (Su/H) or salt/water (Sa/W) based composites. A compression test indicated that the Sa/W fibroin was much harder than the Su/H fibroin, but a protease enzyme digested the Sa/W fibroin more quickly than Su/H fibroin. Rabbit ear chondrocytes were seeded onto the scaffolds for 4-8 week in vitro culture and histological analyses were performed. The distribution of cartilage formation in Safranin O staining was more homogenous in Su/H fibroin than that of Sa/W fibroin. The overall amount of cartilage was significantly better in the Su/H fibroin than that in the Sa/W fibroin. However, the inner structure of pore wall in the Sa/W fibroin was rough and microporous with cartilage matrix deposition, while that in the Su/H fibroin was thin and homogenous. Since mature cartilage gradually regenerates to fill the porous space, slowly degradable Su/H fibroin should be a better candidate for cartilage formation.