RESUMEN
Several congenital and acquired diseases of the human genito-urinary tract may need, due to lack or destruction of functional tissues, mechanically stable biomaterials as cell carriers for the engineering of these tissues. When using collagen scaffolds, both their capacity to induce tissue regeneration and their biocompatibility are advantageous characteristics to render them apt for tissue engineering. The attachment of extracellular matrix or serum proteins to their surfaces does further improve these characteristics, mimicking a close to natural cell environment. In this study, equine collagen scaffolds (TissueFleece) were modified by coating fetal bovine serum proteins, before human bladder smooth muscle cells were seeded. Cell growth was evaluated by WST-1 proliferation assay and improved when using modified collagen scaffolds. However, cell penetration assessed by histology showed similar results on modified and native scaffolds. These cell-scaffold constructs were further implanted in the dorsal subcutaneous space of athymic mice. In vivo studies showed the presence of the fluorescent-labeled transplanted smooth muscle cells until day 3 and thereafter angiogenesis was induced and infiltration of mouse fibroblasts and polymorphonuclear cells were observed. The latter had completely disappeared after 3 weeks.
Asunto(s)
Proteínas Sanguíneas/química , Colágeno/química , Regeneración Tisular Dirigida/métodos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/trasplante , Ingeniería de Tejidos/métodos , Vejiga Urinaria/citología , Animales , Bovinos , Células Cultivadas , Niño , Materiales Biocompatibles Revestidos/química , Caballos , Humanos , Ensayo de Materiales , Ratones , Ratones Desnudos , Vejiga Urinaria/crecimiento & desarrolloRESUMEN
Reconstruction of the genitourinary tract, using engineered urological tissues, requires a mechanically stable biodegradable and biocompatible scaffold and cultured cells. Such engineered autologous tissue would have many clinical implications. In this study a highly porous biodegradable polyesterurethane-foam, DegraPol was evaluated with tissue engineered human primary bladder smooth muscle cells. The cell-polymer constructs were characterized by histology, scanning electron microscopy, immunohistochemistry and proliferation assays. Smooth muscle cells grown on DegraPol showed the same morphology as when grown on control polystyrene surface. Positive immunostaining with alpha smooth muscle actin indicated the preservation of the specific cell phenotype. Micrographs from scanning electron microscopy showed that the cells grew on the foam surface as well as inside the pores. In addition they grew as cell aggregates within the foam. The smooth muscle cells proliferated on the Degrapol; however, proliferation rate decreased due to apoptosis with time in culture. This study showed that Degrapol has the potential to be used as a scaffold.
Asunto(s)
Materiales Biocompatibles , Miocitos del Músculo Liso , Poliésteres , Poliuretanos , Ingeniería de Tejidos , Apoptosis/fisiología , Adhesión Celular/fisiología , Proliferación Celular , Células Cultivadas , Niño , Humanos , Inmunohistoquímica , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Miocitos del Músculo Liso/ultraestructuraRESUMEN
This study investigates in vitro growth of human urinary tract smooth muscle cells under static conditions and mechanical stimulation. The cells were cultured on collagen type I- and laminin-coated silicon membranes. Using a Flexcell device for mechanical stimulation, a cyclic strain of 0-20% was applied in a strain-stress-time model (stretch, 104 min relaxation, 15 s), imitating physiological bladder filling and voiding. Cell proliferation and alpha-actin, calponin, and caldesmon phenotype marker expression were analyzed. Nonstretched cells showed significant better growth on laminin during the first 8 days, thereafter becoming comparable to cells grown on collagen type I. Cyclic strain significantly reduced cell growth on both surfaces; however, better growth was observed on laminin. Neither the type of surface nor mechanical stimulation influenced the expression pattern of phenotype markers; alpha-actin was predominantly expressed. Coating with the extracellular matrix protein laminin improved in vitro growth of human urinary tract smooth muscle cells.
Asunto(s)
Colágeno Tipo I/farmacología , Laminina/farmacología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Sistema Urinario/citología , Biomarcadores/análisis , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Lactante , Masculino , Miocitos del Músculo Liso/metabolismo , Estrés Mecánico , Vejiga Urinaria/citologíaRESUMEN
In tissue engineering, degradable or non-degradable polymer matrices can act as cell-carrier-scaffolds. Cell adhesion and growth on these scaffolds can be promoted by immobilizing extracellular matrix proteins. Therefore, in this study, polymer poly(ethylene terephthalate) (PET) films were surface modified by graft polymerization of acrylic acid, to subsequently allow collagen (types I and III) immobilization and human smooth muscle cell expansion. The surfaces of PET were activated by plasma, followed by acrylic acid graft polymerization, resulting in covalently bound brushes, containing an average of either 0.22+/-0.1 or 5.93+/-0.87 microg/cm2 of poly(acrylic acid) (PAA). Subsequent electrostatic adsorption of collagen gave a surface concentration of 4.96 and 17.2 microg/cm2, respectively, as determined using radiolabelled 125I collagen. Both PET films grafted with 0.22 microg/cm2 of PAA with or without adsorbed collagen were apt for smooth muscle cell adhesion and proliferation. However, films grafted with 5.93 microg/cm2 were not. PAA-grafted PET films, onto which serum proteins of the culture medium adsorbed spontaneously, proved to be better matrices than films on which collagen has been immobilized. It, therefore, can be speculated that other serum proteins are more important than collagen for the human smooth muscle cell adhesion and growth on surface-modified polymer matrices.