Construction And Poperties Of Three-Dimensional Cell Culture Substrate By Compositing Bacterial Cellulose Into Highly Porous Scaffolds

Ideal three-dimensional(3D) cell culture substrate should simulate the structure and function of the extracellular matrix(ECM) in vivo, with a suitable pore size and porosity, good biocompatibility and mechanical properties. The key to build a 3D cell culture matrix similar to the ECM is to select the appropriate materials, which exhibits biomimetic multi-scale structures composed of microporous structure with nanofibers for more biomimetic cellular environment.Bacterial cellulose(BC) has 3D nanofibrils network architecture which successfully mimics the native ECM. Meanwhile, it possesses desirable physical and mechanical properties, such as high mechanical strength, mouldability and good biocompatibility. As a result, BC is considered as an excellent nanofibrous scaffolds candidate for three-dimensional cell culture matrix. However, the pore size of BC network structure is generally 0.05~5 μm, which can not meet the actual application requirements of 3D cell culture scaffolds. Thus, it is necessary to construct microporous structure based on the BC 3D nanofibrils network.The biological thermoplastic microporous material of polyurethane is used to be a temple to fabricate 3D microporous nanofibrous polyurethane/BC composite scaffolds(PU/BC) by stationary cultivation G. xylinus. The surface decorated on the micropore wall by BC nanofibers(42 ± 13 nm) was fabricated, which is remarkably similar in structure to the native ECM. The pore size and porosity of the PU scaffold and PU/BC have no significant difference. PU/BC scaffold has better water absorption and fluid retention rate.Protein adsorption was evaluated by 3D laser scanning confocal microscopy(3D-LSCM). The result shows that the BC nanofibrous coated on the surface network improve the protein adsorption of scaffolds.The breaking strength of PU/BC scaffold in wet state is 152.8 ± 15.1 KPa, 2.4 times of PU scaffold(64.5 ± 4.4 KPa); elastic modulus of PU/BC scaffold is 1.923 ± 0.152 MPa, 5.5 times of PU scaffold. Cell viability and morphology were evaluated by seeding ADSCs on the scaffolds, using MTT assay and FE-SEM. These results indicate that the 3D microporous nanofibrous scaffolds exhibit good biocompatibility, promote cellular attachment and proliferation. It is anticipated that this 3D microporous nanofibrous scaffold can be applied in the fields of cell supports, which can be used as instructive 3D environments for cell culture.The PGS scaffold with pore size of 81 ± 35μm, porosity of 85.9 ± 6.5% is successfully prepared by salting leaching method. The biological thermosetting microporous material of poly(glycerol sebacate(PGS) is used to be a temple to fabricate 3D microporous nanofibrous PGS/BC composite scaffolds by stationary cultivation G. xylinus. The PGS/BC scaffolds with highly interconnected micropore(76±23μm) and surface decorated on the micropore wall by BC nanofibers(50±17nm) were fabricated. And the pore size and porosity of the PGS scaffold and PGS/BC scaffold have no significant difference. Protein adsorption was evaluated by 3D laser scanning confocal microscopy(3D-LSCM). The result shows that the BC nanofibrous coated on the surface network improve the protein adsorption of scaffolds. The breaking strength of PGS/BC scaffold in wet state is 12.5±2.1 KPa, 4.0 times of PGS scaffold(3.1±0.5 KPa); elastic modulus of PGS/BC scaffold is 28.3±3.1 KPa, which is 8.8 times of PGS scaffold(3.21±0.3 KPa). Cell viability and morphology were evaluated by seeding ADSCs on the scaffolds, using MTT assay and FE-SEM. These results indicate that the 3D microporous nanofibrous scaffolds exhibit good biocompatibility, promote cellular attachment and proliferation. 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.