RESUMO
Bioactive recombinant human bone morphogenetic protein-2 (rhBMP-2) was obtained using Escherichia coli pET-25b expression system: 55 mg purified rhBMP-2 were achieved per g cell dry wt, with up to 95% purity. In murine C2C12 cell line, rhBMP-2 induced an increase in the transcription of Smads and of osteogenic markers Runx2/Cbfa1 and Osterix, measured by semi-quantitative RT-PCR. Bioassays performed in human fat-derived stem cells showed an increased activity of the early osteogenic marker, alkaline phosphatase, and the absence of cytotoxicity.
Assuntos
Adipócitos/citologia , Adipócitos/fisiologia , Proteínas Morfogenéticas Ósseas/administração & dosagem , Osteoblastos/citologia , Osteoblastos/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Fator de Crescimento Transformador beta/administração & dosagem , Adipócitos/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Camundongos , Mioblastos/efeitos dos fármacos , Mioblastos/fisiologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Proteínas Recombinantes/administração & dosagem , Células-Tronco/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Biocompatibility, stability, and efficiency remain among the major goals in the development of new drug delivery systems. Herein we describe a facile method for encapsulation of Bone Morphogenetic Protein-2 (BMP-2) at high concentrations and in mild conditions by exploiting the thermo-responsiveness of an elastin-like polymer. Chemically synthesized poly(VPAVG) demonstrates excellent biocompatibility, stability at room temperature, and allows the use of mild conditions and harmless solvents, such as water, for its production. Here, we describe how to use a recombinantly produced poly(VPAVG) polymer to entrap BMP-2 with a high encapsulation efficiency and the methods for in vitro bioactivity assays.
Assuntos
Proteína Morfogenética Óssea 2/administração & dosagem , Elastina/química , Nanopartículas/química , Polímeros/química , Animais , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Sistemas de Liberação de Medicamentos , Camundongos , Nanopartículas/ultraestruturaRESUMO
Elastin-like polymers are a new type of protein-based polymers that display interesting properties in the biomaterial field. Bone morphogenetic proteins (BMPs) are cytokines with a strong ability to promote new bone formation. In this work, we explored the use of elastin-like nanoparticles (average size 237.5+/-3.0 nm), created by thermoresponsive self-assembly, for the combined release of bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-14 (BMP-14). These BMPs could be encapsulated at high efficiency into the elastin-like particles and delivered in a sustained way for 14 days. The activity of the growth factors was retained, as shown by the induction of ALP activity and osteogenic mineralization in C2C12 cells. Increased bioactivity was observed with a combined release of BMP-2 and BMP-14. This approach shows a significant potential for future tissue engineering applications in bone.