RESUMEN
To better understand the interactions of cells derived from meningeal tissues with the surfaces of devices used for the treatment of central nervous system disorders, the behavior of primary postnatal day 1 rat meningeal cultures was evaluated on biomaterials of differing surface chemistry. Meningeal cultures in serum containing media were analyzed for attachment, spread cell area, proliferation, the production of extracellular matrix (ECM), and neuronal outgrowth. In general, both cell attachment as well as cell spread area decreased with increasing substrate hydrophobicity, whereas cell division as indicated by BrdU incorporation and time to confluence, was lower on the most hydrophobic materials. We suggest that such differences immediately after cell seeding were most likely mediated by differences in surface adsorption of proteins. In longer-term experiments, most of the materials were colonized by meningeal cultures irrespective of surface chemistry, and all cultures were equally inhibitory to neuronal outgrowth suggesting that over time, cells can modify the substrate perhaps by secretion of extracellular matrix molecule proteins. Our data suggests that cell type-specific differences in response to different biomaterials may play an important role in determining the ultimate nature and composition of the CNS at the host-biomaterial interface.
Asunto(s)
Materiales Biocompatibles , Meninges/citología , Animales , Células Cultivadas , Técnicas de Cocultivo , Matriz Extracelular , Neuronas/citología , RatasRESUMEN
A PEO-containing surface coating was investigated as a means to control neurite outgrowth in the presence of serum. Various ratios of end-group-activated tri-block copolymer Pluronic F108 were used to immobilize the extracellular matrix protein fibronectin (FN). Primary cultures of dorsal root ganglion neurons were cultured on F108-immobilized FN or, as a control, on FN adsorbed from solution directly to polystyrene. Although FN surface concentration could be controlled in a dose-dependent manner by either technique, dose-dependent control of neuronal behaviors was best achieved on F108-immobilized FN. This effect was similar regardless of the presence of serum in the culture medium. F108-immobilized FN supported twofold greater maximal neurite outgrowth than did directly adsorbed FN. Furthermore, at similar surface concentrations, F108-FN was significantly more active in promoting neurite outgrowth. Polypropylene filament bundles treated with F108-immobilized FN supported robust outgrowth from explants of dorsal root ganglia, demonstrating the utility of the surface coating on clinically relevant materials with more complex shapes. The ability to control neuronal behaviors in a serum-resistant manner, coupled with enhanced biologic activity, demonstrates the potential for surfactant-based immobilization as a method for generating biointeractive materials for tissue engineering.
Asunto(s)
Fibronectinas/fisiología , Neuritas/fisiología , Neuronas Aferentes/fisiología , Poloxámero , Tensoactivos , Adsorción , Animales , Animales Recién Nacidos , Bovinos , Adhesión Celular , Células Cultivadas , Medios de Cultivo , Medio de Cultivo Libre de Suero , Fibronectinas/farmacología , Ganglios Espinales/citología , Ganglios Espinales/fisiología , Neuritas/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Poliestirenos , RatasRESUMEN
Soluble factors normally produced by cells of the human body are of increasing importance as potential therapeutic agents. Although considerable progress has been made in understanding the etiology and pathogenesis of disease, in developing animal models and newer experimental therapeutics, few discoveries have been translated into clinically effective ways of delivering the multiple therapeutic agents obtained from living mammalian cells. This review examines the use of transplanted cells as alternatives to conventional delivery systems to deliver a variety of protein based therapeutic agents. The chapter begins with a set of questions to establish the complexity and challenges of this form of drug delivery. The following section focuses the discussion on our understanding of genetic engineering, tissue engineering, and some areas of developmental biology as they relate to the development of this nascent field. Much of the discussion has a neuro/endocrine emphasis. The chapter ends by listing the basic ingredients needed to push the use of transplanted cells toward medical practice and some general comments about future developments.
Asunto(s)
Trasplante de Células , Sistemas de Liberación de Medicamentos , Animales , Movimiento Celular , Regulación de la Expresión Génica , Ingeniería Genética , Vectores Genéticos , HumanosRESUMEN
The behavior of cortical astrocytes was evaluated on a number of medically relevant materials of differing physicochemical properties. This study describes cell attachment, DNA synthesis, production of extracellular matrix (ECM) proteins, and neuronal interactions of perinatal rat astrocytes in vitro. The number of attached astrocytes initially differed among the materials, decreasing with increasing material hydrophobicity. In contrast, the rate of DNA synthesis increased with increasing material hydrophobicity. With the exception of only one material, astrocytes reached confluence by 12 days in culture on all the materials tested. Furthermore, the expression of characteristic ECM proteins and the fundamental ability of astrocytes to support neuronal attachment and growth was qualitatively identical between populations of astrocytes on different materials. The ability of astrocytes to colonize different surfaces initially was mediated via adsorbed serum proteins, as reducing the capacity of a model surface to adsorb proteins inhibited astrocyte colonization for up to 2 weeks in culture. We propose that astrocytes are relatively insensitive to differences in surface chemistries so long as the proteins necessary for cellular attachment are capable of adsorbing to the material to some extent. It seems likely that the ability of astrocytes to produce and remodel a matrix creates a surface environment that eventually becomes similar regardless of the surface chemistry of the underlying material.
Asunto(s)
Astrocitos/fisiología , Corteza Cerebral/citología , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Corteza Cerebral/metabolismo , Medios de Cultivo , ADN/biosíntesis , Matriz Extracelular/metabolismo , Inmunohistoquímica , Neuronas/fisiología , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/farmacología , Biosíntesis de Proteínas , Ratas , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Tensoactivos/farmacologíaRESUMEN
The newly discovered aer locus of Escherichia coli encodes a 506-residue protein with an N terminus that resembles the NifL aerosensor and a C terminus that resembles the flagellar signaling domain of methyl-accepting chemoreceptors. Deletion mutants lacking a functional Aer protein failed to congregate around air bubbles or follow oxygen gradients in soft agar plates. Membranes with overexpressed Aer protein also contained high levels of noncovalently associated flavin adenine dinucleotide (FAD). We propose that Aer is a flavoprotein that mediates positive aerotactic responses in E. coli. Aer may use its FAD prosthetic group as a cellular redox sensor to monitor environmental oxygen levels.
Asunto(s)
Proteínas Bacterianas/fisiología , Escherichia coli/fisiología , Flavoproteínas/fisiología , Transducción de Señal , Oxidación-Reducción , OxígenoRESUMEN
Five proteins (MotA, MotB, FliG, FliM and FliN) may be involved in energizing flagellar rotation in Escherichia coli. To study interactions between the Mot proteins, and between them and the three Fli proteins of the switch-motor complex, we have isolated extragenic suppressors of dominant and partially dominant motB missense mutations. Four of the 13 motB mutations yielded partially allele-specific suppressors. Of the suppressing mutations, 57 are in the motA gene, eight are in fliG, and one is in fliM; no suppressor was identified in fliN. The prevalence of suppressors in fliG suggests that FliG interacts rather directly with the Mot proteins. The behaviour of cells in tethering and swarm assays indicates that the motA suppressors are more efficient than the fliG or fliM suppressors. Some of the suppressing mutations themselves confer distinctive phenotypes in motB+ cells. We propose a model in which mutations affecting residues in or near the putative peptidoglucan-binding region of MotB misalign the stator relative to the rotor. We suggest that most of the suppressors restore motility by introducing compensatory realignments in MotA or FliG.