ABSTRACT
Nerve tissue engineering (NTE) is one of the most promising methods to restore central nerve systems in human health care. Three-dimensional (3D) distribution and growth of cells within the porous scaffold composed of nanofibers are of clinical significance for NTE. In this study, an attempt was made to develop and characterize the use of fibrin gel and human endometrial stem cells (hEnSCs)-derived neuron-like cells simultaneously to support cell behavior especially neuron outgrowth. The structural and mechanical characteristics of fibrin gel scaffold were examined with SEM and rheometer. Also, hEnSCs-derived neuron-like cells were cultured in fibrin gel and were subsequently analyzed with immunofluorescent staining against neuronal markers. In parallel, the survival and growth rates of the cells were determined by MTT assay and neurite extension. At the end, cell-matrix interactions were investigated with SEM and TEM micrographs. Mechanical properties of fabricated scaffold were studied and results indicated appropriate choice of material, SEM and TEM showed excellent integration of cells with nanofibers regarding the relation between cells and fibrin gel. Immunofluorescent staining of fibrin gel after 6 days of cell seeding and culture demonstrated well expanded and incorporated network of neurons. In addition, viability, proliferation, and neuronal growth of seeded cells were analyzed at days 1, 3, and 6. Comparing those results with 2D culture of seeded cells showed positive effect of 3D culture. Taken together, the results suggest that fibrin can provide a suitable, three-dimensional scaffold for neuronal survival and outgrowth for regeneration of the central nervous system.
Subject(s)
Endometrium/cytology , Fibrin/pharmacology , Nerve Tissue/physiology , Neurogenesis/drug effects , Neurons/cytology , Stem Cells/cytology , Tissue Engineering/methods , Cell Separation , Cell Shape/drug effects , Cell Survival/drug effects , Cells, Cultured , Cells, Immobilized/cytology , Cells, Immobilized/drug effects , Cells, Immobilized/metabolism , Female , Fibrin/ultrastructure , Gels/pharmacology , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Nerve Tissue/drug effects , Neurites/drug effects , Neurites/metabolism , Neurons/drug effects , Neurons/ultrastructure , Rheology/drug effectsABSTRACT
Neural tissue engineering is one of the most promising strategies for treatment of nerve tissue injuries. Three-dimensional (3D) environment mimics in vivo conditions for cells. 3D distribution and growth of the cells within the scaffold are both important for neural tissue engineering. In this study, endometrial stromal cell-derived oligodendrocyte progenitor cells (EnSC-derived OPCs) were cultured in fibrin gel and cell differentiation and viability were evaluated after 8 days of post-culture. The structural and mechanical characteristics of fibrin gel-like scaffold were examined with rheological analysis. EnSCs were isolated from donor tissue and were induced to OPCs with growth factors (FGF2/EGF/PDGF-AA) for 12 days, then were cultured in fibrin gel with Triiodothyronine (T3) medium for another 8 days. The viability of cells was analyzed using MTT assay for a period of 8 days culturing in a fibrin matrix. Structure of fibrin matrix and cell morphology was analyzed with SEM. TEM, immunostaining and quantitative RT-PCR was performed for OPCs markers after cell culturing in fibrin matrix. Cell viability is enhanced in fibrin matrix after 8 days. SEM and TEM show that cells are in good integration with nano-fibers. Moreover, immunohistochemistry and quantitative RT-PCR of OPCs differentiation markers showed that Olig2, Sox10, PDGFRa, CNP, and A2B5 are expressed after 8 days culturing within fibrin matrix. Fibrin can provide a suitable 3-D scaffold for EnSCs differentiated cells for the regeneration of CNS.
