FIG. 2.
Effect of RGD peptide density of of microgel (NRGD) on MSC's proliferation and differentiation. The microgel diameter was kept constant at 500 μm. (A) (A-1) The dependency of MSC number (N) on cell culture period was controlled by RGD peptide density (NRGD). The number of MSCs (N) was normalized to the number of cells adhered to the RGD-alginate microgels within first 24 h in culture (No). The NRGD of the microgel surface was varied from 6.22×107 (▲) to 3.11×108 (●) and 6.22×108 RGD/mm2 (■). (A-2) The N0 (●) and the cell proliferation rate (k), (■) were significantly increased with NRGD. The differences between the values were statistically significant. (*p<0.05). (B) The mesenchymal stem cells (MSCs) expanded on alginate microgels maintained multipotency. MSCs were isolated from the alginate microgels and re-seeded on poly(styrene) substrate. (B-1) MSCs cultured in adiopogenic media for 18 days were positively stained with Oil Red O (scale 100 μm). (B-2) MSCs cultured in osteogenic media for 18 days were positively stained for matrix mineralization with von Kossa reagents (scale 200 μm). (B-3) MSCs encapsulated in a collagen gel and cultured in chondrogenic media for 17 days were positively stained with Alcian blue (C, Scale: 150 μm). (C) Effects of NRGD on MSC's osteogenic differentiation during cell culture in the osteogenic differentiation media. The extents of cellular osteocalcin secretion (C-1) and osteopontin secretion (C-2) were significantly higher for cells cultured on microgels with a higher RGD density (6.2×108 RGD/mm2). The closed bar represents protein concentration measured at day 7 and the open bar represents that at day 14. The difference between the values was statistically significant (*p<0.05). (D) Increasing NRGD increased the degree of mineralization on microgel surfaces, marked by positive staining with von Kossa reagents in black. The cells were counterstained in red. (D-1), (D-2) and (D-3) represent the microgel surfaces with NRGD of 6.2×107 to 3.1×108 and 6.2×108 RGD/mm2, respectively (scale bar: 100 μm). (E) Increasing NRGD also elevated the chondrogenic differentiation level of MSCs cultured in the chondrogenic differentiation media. The glycosaminoglcan secreted by cells was positively stained by Alcian blue (Scale: 100 μm). (E-1) and (E-2) represent the microgel surfaces with NRGD of 6.2×107 and 6.2×108 RGD/mm2, respectively. The extent of quantified glycosaminoglycan production from cells on beads after 14 days in culture (E-3) (*p < 0.05). Color images available online at www.liebertonline.com/tea