Chemical Modification Of Three-dimensional Porous Scaffolds And Their Applications In In Vitro Culture Of Mesenchymal Stem Cells And Chondrocytes

Tissue engineering aims at fabricating tissue replacements for repairing/regenerating defected tissues/organs by relying on a combination of cells, biomaterials and soluble factors. Both mesenchymal stem cells (MSCs) and adult cells such as chondrocytes are important cell sources. On the other hand, it has been recognized that both pore size and chemical functional groups in three-dimensional porous scaffolds can have significant influences on cellular behaviors. However, in literature, most studies pay attention to the effects of individual properties of biomaterials, and how cells respond in a combination of physicochemical factors has been less understood.To this end, in our study, firstly, we prepared polycaprolactone (PCL) membranes with relatively smooth surface and PCL three-dimensional porous scaffolds with different pore sizes (100-200 μm,200-300 μm and 300-450 μm) and optimized conditions of hydrolysis and aminolysis on both membranes and porous scaffolds. Then, we investigated the influence of different chemically modified PCL membranes on human MSCs (hMSCs) attachment and growth. In addition, we focused on the synergistic roles of pore size and surface chemical modification on the responses of hMSCs in three-dimensional PCL porous scaffolds, including cell adhesion, migration, proliferation and differentiation. Finally, we also seeded rACs at different densities on three-dimensional PCL porous scaffolds to investigate the effects of seeding density and pore size on chondrocyte phenotype.The following findings were obtained. (I) By selecting appropriate conditions for chemical modifications, surface topography of PCL membranes remained smooth and water contact angle decreased upon chemical modification. (II) Hydrolyzed PCL membranes promoted the adhesion, spreading and proliferation of hMSCs. However, the influence of ammonolyzed PCL membranes on cell behavior is strongly associated with the degree of modification. (Ⅲ) The water contact angle of chemically modified PCL porous scaffolds also decreased, with porous structures well maintained. It was noticed that hMSCs could either grow along or across the wall of pores. In general, chemically modified scaffolds were more favorable to cell attachment and proliferation. Concerning pore size, the scaffolds with pore size of 200-300 μm were more advantageous in cell attachment and proliferation. (Ⅳ) It was found that all chemically modified scaffolds were more conducive to osteogenic differentiation of hMSCs. Instead, hydrolyzed scaffolds and scaffolds with pore size of 200-450 μm were superior for chondrogenic differentiation of hMSCs. (V) rACs demonstrated varying degrees of dedifferentiation in the scaffolds with different pore size. While rACs proliferated faster at the seeding density of 5x104 cells/scaffold than 30×104 cells/scaffold, a seeding density of 30×104 cells/scaffold was the best to maintain the phenotype of rACs. (VI) Growth of rACs and glycosaminoglycans (GAG) production were slightly higher in scaffolds with pore size of 300-450 μm. In addition, rACs could migrate deeper inside in these scaffolds.In conclusion, both surface chemical modification and pore size of porous scaffolds have a significant impact on adhesion, proliferation and differentiation of hMSCs, as well as maintaining the phenotype of rACs, and physical and chemical properties of biomaterials could have acted synergistically on hMSCs. These findings will potentially provide solid foundation to understand cell-biomaterials interactions for tissue engineering.