| Interactions of stem cell with their environment result in the development of new materials-based approaches to control stem cell behavior toward cellular culture and tissue regeneration applications. The materials can provide cues based on chemistry, mechanics, structure, and molecule delivery that control stem cell fate and matrix formation. As a crucial factor in tissue engineering and regenerative medicine, the scaffold should possess appropriate pore size and porosity, good pore interconnectivity, and appropriate mechanical properties to function as a physical template to support cell adhesion, proliferation and differentiation, and provides channels for the exchange of nutrients and wastes. The ability of three-dimensional (3-D) scaffolds to spatially control stem cell spreading and subsequent fate is of great importance in tissue engineering applications due to the heterogeneous nature of tissues. The typical scaffolds include hydrogels, sponges, electrospun fibers, unvoven meshes and bounded microspheres et. al. Among which, the microspheres scaffold has attracted much focus because it has the advantages of extremely large surface area for cell binding and proliferation, and ability for precise control of the cell growth.Many methods have been applied for preparation of the 3-D scaffolds. Among which, the particle-leaching method is frequently adopted because of the controlled pore size and distribution, which is mainly determined by the size of the porogens, and the controlled morphology on the pore walls, which is a key factor to determine the cell and tissue response in vitro and in vivo. Herein, a porogen-leaching method combined with thermally induced phase separation (TIPS) was applied to fabricate polycaprolactone (PCL) scaffolds and microspheres. Following with a routine solution infiltration, freeze-drying and porogen-leaching process, the porous scaffolds were normally prepared at an initial solution temperature of 25℃. However, the PCL anisotropic particles with the smooth and fuzzy surfaces toward the gelatin porogen and the solution, respectively, were unexpectedly obtained when the initial solution temperature was maintained at 37℃. The freezing temperature was a governing factor for formation of the different PCL products too, while the coarsening time and the PCL concentration within 10-20% had no substantial influence. The PCL anisotropic particles are highly crystallized than the PCL raw materials. To clarify the intrinsic mechanisms, the temperature, cloud point, crystalline ability, and particle size in the solution were quantified. It is demonstrated that the sponges are formed by the traditional liquid-liquid demixing for the 25℃ solution, whereas the anisotropic particles are obtained by the solid-liquid demixing for the 37℃ solution and under the assistance of gelatin particles as nucleation sites.Bearing the complicated fabrication process of 3-D scaffold by porogen-leaching method, a microspheres-aggregated scaffold with ultra big pores and fuzzy microspheres is fabricated by incubating PCL/tetrahydrofuran (THF) solution in a -20 ℃ refrigerator, following by freeze-drying. Formation of the scaffold is mainly governed by the crystallization of the PCL polymer at appropriate conditions. All the 10-20% PCL/THF solutions yield the microspheres-aggregated scaffolds when their temperature is higher than 37℃, whereas the 10-15% solutions form dense membranes when the temperature is below 25℃. The size of the microspheres and pores are as large as 70-150 μm and 170-816μm, respectively. The structure parameters of the microspheres-aggregated scaffold such as the microsphere size, pore size and porosity could be controlled by the fabrication parameters too. This type of microspheres-aggregated scaffolds with fuzzy surface on the microspheres could better enhance the spreading and maintain the viability of bone mesenchymal stem cells (BMSCs). They also showed a stronger tendency in inducing the chondrocyte rather than the osteogenic differentiation of BMSCs as confirmed by the assays of glycosaminoglycan (GAGs) secretion and alkaline phosphatase (ALP) activity and expression of type Ⅰ collagen and type II collagen.Besides the physical topography, substrates with varying surface chemistry induce diverse cellular responses. Substrate surface properties play an important role in protein adsorption kinetics and their folded conformation, which in turn influence cellular activities. These surface-engineered biomaterials having active receptor binding domains can be used to modulate the integrin-signaling pathway in a defined manner to control stem cell fate. Herein, the PCL microspheres-aggregated scaffold with defined topography was modified by galatosylated chitosan (GC) to achieve the differentiation of BMSCs toward hepatocytes. The COOH groups on PCL microspheres-aggregated scaffold was much lower than the ordinary porous scaffold after hydrolyzed in 1M/L NaOH solution. By the amidation between the GC and the hydrolyzed PCL microspheres-aggregated scaffold, the GC molecules with a galactosylated substitution degree of 65% were covalently immobilized onto the scaffold surface. The GC content on the microspheres-aggregated scaffold is much lower than that of the ordinary porous scaffold. Most of the incorporated GC could be remained on the hydrolyzed PCL microspheres-aggregated scaffold and porous scaffold after incubation in phosphate buffered saline (PBS) for certain time. BMSCs culture in vitro presented good viability and a higher level of albumin secretion on the PCL microspheres-aggregated scaffold and the GC modified one than those of the ordinary scaffold. This property could be contributed to the topography and surface chemistry of the scaffold.It is well known that natural biomaterials may provide efficient adhesion sites for cell attachment and a wide range of biological signals. Collagen is a significant constituent of the natural extracellular matrix (ECM), and collagenous scaffolds have been used extensively in tissue engineering due to their unique properties:hemostatic, low antigenicity, appropriate mechanical stiffness, promotion of cell attachment and growth. Herein, collagen was used to modify the PCL microspheres-aggregated scaffold after alkaline hydrolyzation. The cell viability was improved considerably. The BMSCs secreted more ALP and less GAGs on the collagen-modified PCL microspheres-aggregated scaffold than on the blank one, indicating a stronger tendency in inducing BMSCs to osteogenic differentiation. |
PDF Full Text Request