RESUMEN
The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.
Asunto(s)
Bombyx/virología , Catepsinas/metabolismo , Quitinasas/metabolismo , Nucleopoliedrovirus/enzimología , Animales , Bombyx/metabolismo , Catepsinas/genética , Quitinasas/genética , Glándulas Exocrinas/metabolismo , Glándulas Exocrinas/virología , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/crecimiento & desarrollo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Growth factors play a crucial role in wound healing in general and are promising tools for the treatment of chronic wounds as they can restore the physiological wound healing process. In growth factor-loaded wound dressings, human epidermal growth factor (EGF) is released in a burst and washed out quickly. The developed matrix consists of recombinant EGF produced in transgenic silkworms as a fusion protein with the fibroin light chain. The covalent linkage prevents EGF from draining into the surrounding tissue while presenting the growth factor on the surface. EGF-functionalized silk membranes and nonwovens lead to a 2.5-fold increase in the cell number of fibroblasts, while retaining full bioactivity even after e-beam sterilization. EGF is long-term presented without burst release and significantly reduces the wound area by 15% in an in vitro wound model. Hence, the cost-effective production of a biomaterial using transgenic silkworm larvae in combination with a growth factor paves the way for a promising new multifactorial wound cover for chronic wound healing. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2643-2652, 2018.
Asunto(s)
Bombyx/química , Factor de Crecimiento Epidérmico/farmacología , Seda/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Partículas beta , Materiales Biocompatibles/farmacología , Línea Celular , Fibroínas/farmacología , Humanos , Larva/efectos de los fármacos , Masculino , Ratones , Modelos Biológicos , Proteínas Recombinantes/farmacología , Propiedades de SuperficieRESUMEN
AIM: The purpose of the present study was to find inexpensive and non-toxic additives enhancing and accelerating the osteogenesis of mesenchymal stem cells in vitro, which can be used for tissue engineering of bone material. MATERIALS AND METHODS: Osteogenic differentiation of rat mesenchymal stem cells was carried-out using classic differentiation medium containing or lacking purmorphamine, statins or oxysterols, respectively. Cell proliferation, alkaline phosphatase activity, calcium sedimentation and expression of bone matrix protein genes were measured to monitor differentiation. RESULTS: Purmorphamine substantially suppressed proliferation, enhanced and accelerated alkaline phosphatase activity and calcium sedimentation and increased the expression of osteopontin and osteocalcin in rat mesenchymal stem cells in vitro. A similar osteogenesis-promoting effect was observed for oxysterols but not for the two statins. CONCLUSION: Purmorphamine and oxysterols promote and accelerate osteogenesis of mesenchymal stem cells in vitro suggesting their potential application for tissue engineering of bone material.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Morfolinas/farmacología , Osteogénesis/efectos de los fármacos , Purinas/farmacología , Esteroides/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Madre Mesenquimatosas/metabolismo , Ratas , Factores de TiempoRESUMEN
AIM: The present study aimed to find bone substitutes to enhance osteogenic differentiation of mesenchymal stem cells in three-dimensional scaffolds in the absence of dexamethasone. MATERIALS AND METHODS: Seven commercial bone substitutes were added to a three-dimensional fibrin-matrix containing rat mesenchymal stem cells in a biocompatible poly-L-lactic-acid mesh. Cell viability, cytotoxicity and alkaline phosphatase activity were followed for three weeks. Expression of bone markers was examined by qualitative evaluation of corresponding transcripts. RESULTS: Six out of the seven bone derivatives exhibited an osteogenic-enhancing effect. CONCLUSION: The osteogenic-enhancing effect of the evaluated bone substitutes suggests their potential clinical application for preparation of autologous bone replacement material in three-dimensional carriers.