RESUMO
Transplantation of mesenchymal stromal cells (MSC) presents a promising approach not only for the replacement of lost or degenerated cells in diseased organs but also for local drug delivery. It can potentially be used to enhance the safety and efficacy of inner ear surgeries such as cochlear implantation. Options for enhancing the effects of MSC therapy include modulating cell behaviour with customized bio-matrixes or modulating their behaviour by ex vivo transfection of the cells with a variety of genes. In this study, we demonstrate that MSC delivered to the inner ear of guinea pigs or to decellularized cochleae preferentially bind to areas of high heparin concentration. This presents an opportunity for modulating cell behaviour ex vivo. We evaluated the effect of carboxymethylglucose sulfate (Cacicol®), a heparan sulfate analogue on spiral ganglion cells and MSC and demonstrated support of neuronal survival and support of stem cell proliferation.
Assuntos
Orelha Interna/cirurgia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Gânglio Espiral da Cóclea/cirurgia , Nicho de Células-Tronco , Animais , Adesão Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Orelha Interna/efeitos dos fármacos , Orelha Interna/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/farmacologia , Cobaias , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Ratos Sprague-Dawley , Gânglio Espiral da Cóclea/efeitos dos fármacos , Gânglio Espiral da Cóclea/metabolismo , Técnicas de Cultura de TecidosRESUMO
The treatment of choice for profound sensorineural hearing loss (SNHL) is direct electrical stimulation of spiral ganglion cells (SGC) via a cochlear implant (CI). The number and excitability of SGC seem to be critical for the success that can be achieved via CI treatment. However, SNHL is associated with degeneration of SGC. Long-term drug delivery to the inner ear for improving SGC survival may be achieved by functionalisation of CI electrodes with cells providing growth factors. Therefore, the capacity of brain-derived neurotrophic factor (BDNF)-secreting NIH3T3 cells grown on cylindrically shaped silicone elastomers (SE) to exert local and sustained neuroprotective effects was assessed in vitro and in vivo. An in vitro model to investigate adhesion and cell growth of lentivirally modified NIH3T3 cells synthesising BDNF on SE was established. The bioactivity of BDNF was characterised by co-cultivation of SGC with cell-coated SE. In addition, cell-coated SE were implanted into deafened guinea pigs. The recombinant NIH3T3 cells proliferated on silicone surfaces during 14 days of cultivation and expressed significantly increasing BDNF levels. Enhanced survival rates and neurite outgrowth of SGC demonstrated the bioactivity of BDNF in vitro. Implantation of SE with adhering BDNF-secreting NIH3T3 cells into the cochleae of systemically deafened guinea pigs induced a significant increase in SGC survival in comparison to SE without cell coating. Our data demonstrate a novel approach of cell-based long-term drug delivery to support SGC survival in vitro and in vivo. This therapeutic strategy--once transferred to cells suitable for clinical application--may improve CI performance.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Surdez/cirurgia , Fibroblastos/transplante , Neurônios/metabolismo , Comunicação Parácrina , Elastômeros de Silicone/química , Gânglio Espiral da Cóclea/cirurgia , Animais , Animais Recém-Nascidos , Fator Neurotrófico Derivado do Encéfalo/genética , Adesão Celular , Técnicas de Cultura de Células , Proliferação de Células , Sobrevivência Celular , Técnicas de Cocultura , Surdez/induzido quimicamente , Surdez/metabolismo , Surdez/patologia , Modelos Animais de Doenças , Ácido Etacrínico , Feminino , Fibroblastos/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Cobaias , Humanos , Canamicina , Lentivirus/genética , Masculino , Camundongos , Células NIH 3T3 , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Gânglio Espiral da Cóclea/metabolismo , Gânglio Espiral da Cóclea/patologia , Fatores de Tempo , TransfecçãoRESUMO
HYPOTHESIS: Recombinantly modified cells deliver neurotrophic factors with the capacity to induce differentiation and the outgrowth of neurites of rat pheochromocytoma cells 12 (PC12) serving as a neuronal model. BACKGROUND: The benefit of cochlea implant (CI) is depending, among other factors, on the number of surviving spiral ganglion neurons (SGN). Studies have shown that the external application of neurotrophic factors in combination with electrical stimulation increases the survival rate of SGN after ototrauma. Therefore, functionalization of electrodes with recombinantly modified cells providing neurotrophic factors to the SGN for inducing survival mechanisms may be an approach to realize drug delivery to the cochlea. METHODS: Murine NIH3T3 cells were recombinantly modified with an infectious lentiviral monocistronic and bicistronic system to synthesize glial cell line-derived neurotrophic factor and the green fluorescent protein. Free glial cell line-derived neurotrophic factor from the supernatant of the modified NIH3T3 cells was added to rat PC12, and the neuronal-like outgrowth was determined for 10 days. RESULTS: A significant neuronal-like outgrowth appeared as early as Day 3 after the application of the supernatant. CONCLUSION: The results indicate that the established in vitro model represents a powerful basic model for determining signal pathways between neuronal-like processing PC12 cells and cellular drug delivery systems.
