RESUMO
Oligo(polyethylene glycol) fumarate (OPF) hydrogel has been employed in musculoskeletal tissue engineering for photoencapsulation of chondrocytes and as a matrix for marrow stromal cells differentiation. In this study, we have studied the application of OPF hydrogel for coencapsulation of DNA and bone cells and examined whether coencapsulation can enhance gene transfer by maintaining the DNA within the cellular microenvironment. Our results showed that plasmid DNA encoding green fluorescence protein (GFP), coencapsulated with bone tumor cells, was capable of transfecting the cells, and the transfected tumor cells continuously expressed GFP protein over the time course of study (21 days). Furthermore, we have examined the coencapsulation of estrogen receptor (ER) encoding plasmid DNA and human fetal osteoblast cells (hFOB) that lack endogenous ER. Our results show that the transfected cells responded to estrogen as alkaline phosphatase (ALP), and estrogen response element (ERE)-directed luciferase enzyme activities increased with estrogen treatment. Taken together, these studies show that OPF hydrogel could be further explored for targeted gene delivery in bone and other tissues encapsulated within the hydrogels.
Assuntos
DNA/metabolismo , Estrogênios/farmacologia , Técnicas de Transferência de Genes , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/metabolismo , Osteoblastos/citologia , Osteoblastos/enzimologia , Receptores de Estrogênio/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , TransfecçãoRESUMO
Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.