| Tissue engineering scaffold is designed to biomimetic the cellular microenvironment in vivo. The cells are inherently sensitive to their three-dimensional(3D) environments from the macroscale, microscale, to the nanoscale of chemistry and topography. The 3D microporous and nanofibrous structure of the extracellular matrix(ECM) provides a natural network to guide cell behavior, support cells proliferation and differentiation to hierarchical organization within organs.Therefore, a key challenge of the new generation of tissue engineering scaffolds is to design and fabrication 3D scaffolds that exhibit biomimetic multi-scale structures composed of microporous structure with nanofibers for more biomimetic cellular environment.Although several strategies, including multi-layer sintering, liquid-assisted collection, adding porogen and hybrid nano-/microfiber, have been performed to prepare 3D highly porous nanofibrous scaffolds, none of these methods provided access to obtain ideal scaffolds mimicking the ECM structure with simple processes and tunable architectures. Bacterial cellulose(BC) has recently received extensive attention as an excellent nanofibrous scaffold candidating for tissue regeneration benefit from its refined 3D nanofibrous network architecture which successfully mimics the native ECM. Meanwhile, it possesses desirable physical and mechanical properties, such as high mechanical strength, mouldability, biocompatibility and unique nanostructure. Recently, much interest has been given on the development of medical applications for artificial skins, blood vessels, bone, cartilage, urinary and tympanic membrane. However, the low cell penetration capability and bioactivity due to the relatively dense nanostructure of the nanofibrous network and single cellulose component have limited the application of BC for tissues repairing.Therefore, to fabricate such 3D microporous nanofibrous scaffolds for clinical application, we must control the multi-scale structures with defined macro-, microand nanostructure. We have addressed the challenges and limitations on current technologies to control and improve the surface of nanofibrous structure and microporous architecture of BC.(1) The different nanofiber densities and topography of BC nanofibrous network in surface were obtained by regulating the initial bacterial density. These nanoscale alterations in topography increase cell-material interactions, promote cell attachment, proliferation and prevent apoptosis. With the initial bacterial density is increased, the diameter of BC nanofiber slightly increases and the fiber density increases significantly. When the initial bacterial density is more than 105 cells/mL, the evident dendritic structures of BC nanofibers composed of main fibers, branching fibers and nanofibrils were observed at the first time. Cell viability and morphology were evaluated by seeding adipose-derived stem cells(ADSCs) on the surface of BC membranes, using the laser scanning confocal microscopy(LSCM), LIVE/DEAD? viability/cytotoxicity assay and field emission scanning electron microscopy(FE-SEM). It revealed that cell viability and number increase due to the increased of nanofiber density and presence of dendritic structures. Furthermore, cell morphology with abundant pseudopods tightly adhered to the nanofibers and formed integrated cell-fiber constructs was observed on the surface of BC by LSCM. The results demonstrated that BC membranes with incearsed fiber density and dendritic structure exhibit good mechanical strength and biological properties, promoting cellular attachment, proliferation and maintainable bioactivity.(2) A significant problem limiting the application of BC nanofibrous scaffolds for tissue regeneration is the nanoscale pores that inhibit cell infiltration and vascularization in their 3D structure. In this paper, a facile method was used to fabricate 3D microporous/nanofibrous Gelatin/BC composite scaffolds(Gel/BC) by stationary cultivation G. xylinus using microporous gelatin scaffold as a template. The Gel/BC scaffolds with highly interconnected micro-pore(171 ± 71 ?m) and surface decorated on the micropore walls by BC nanofibers(25.2 ± 7.0 nm) were fabricated, which are remarkably similar in structure to the native extracellular matrix(ECM). Cell distribution, viability and morphology were evaluated by seeding ADSCs on the scaffolds, using the 3D-LSCM, LIVE/DEAD? viability/cytotoxicity assay and FE-SEM. In vivo biocompatibility was evaluated by subcutaneous implantation using a dog model for 2 weeks. These results indicated that the 3D microporous nanofibrous scaffolds exhibit good biocompatibility, promoting cellular attachment, proliferation and maintain cellular phenotype, improving cellular infiltration and vascularization. It is anticipated that this 3D microporous nanofibrous scaffold can be applied in the fields such as medical implants, cell supports, and materials, which can be used as instructive 3D environments for tissue regeneration.(3) Using the method of template synthesis, gelatin pore wall in the 3D highly porous scaffolds were coated by BC nanofibrous network. The different nanofiber densities of network in 3D were obtained by regulating the initial bacterial density(3.8×103, 3.8×105, 3.8×107 cells/mL, respectively), while the micro-pore size, porosity and nanofiber diameter can be kept relatively constant. It provides an excellent model to explore the impact of nanofiber density on cell behavior in 3D highly porous nanofibrous scaffolds. The mechanical property and degradation in vitro and in vivo were evaluated. The rate of mass loss and water uptake increased with the increase of nanofiber density, and the mechanical performance was improved significantly. To investigate the effects of changes in nanofiber density of 3D scaffolds on cell behavior, ADSCs were cultured in the scaffolds. These results indicated that the 3D highly porous scaffolds with incearsed fiber density supported cell vitality, attachment, proliferation and even vascularization.(4) By analyzing the multi-scale structures of urethral tissue, we designed and constructed a bilayer scaffold composed of microporous section of Gel/BC and dense section of BC nanofibrils network to biomimetic urethral acellular matrix. The results showed that the bilayer scaffold(BC-Gel/BC) could support keratincocytes and muscle cells adhesion and growth in vitro for its microporous and nanofibrous property. BC-Gel/BC composites can withstand a higher mechanical load than gelatin alone. It also suggested that BC-Gel/BC bilayer scaffolds with keratincocytes and muscle cells enhance the repair in dog urethral defect models, resulting in patent urethra. Improved organized muscle bundles and epithelial layer were observed in animals treated with BC-Gel/BC scaffold seeded by keratincocytes and muscle cells. This study suggested that the BC-Gel/BC-107 scaffold can realize successful repair of long segment urethral defects(5cm). |
PDF Full Text Request