Fig 1.
Pure SF and SF/CS scaffold after lyophilization.
(A) pure SF in powder form is difficult to grip and has poor mechanical properties. (B) The SF/CS sample is yellowish white with a rough surface and porous internal structure.
Fig 2.
EM image of pure SF and pure CS.
(A) pure SF after lyophilization showing a lamellar, curled structure with no typical pore structure. (B) pure CS after lyophilization with small pores, poor connectivity, and uneven sizes.
Fig 3.
SEM images of SF/CS scaffold materials.
(A) In the 20% SF-80% CS group, a porous network structure appeared. The thickness of the pore walls was uniform, yet the shape and size of the pores were not very regular, and the connectivity between the pores was poor. The mean pore diameter was 100±20.56 μm. (B) In the 40% SF -60% CS group, the pore size was uniform, and the pore walls were a single layer with a smooth surface and uniform thickness. The connectivity between the pores was good, and the pores themselves were round and relatively uniform with a mean pore size of 150±28.56 μm. (C) In the 60% SF-40% CS group, an irregular laminar curled structure appeared. The connectivity between the pores was relatively good. The mean pore size was 210±23.71 μm. (D) In the 80% SF-20% CS group, the pore size was large and the wall of each pore was not smooth, but composed of multiple irregular laminar structures. The curling was more notable than the 60% SF-40% CS group, and the pore shapes were not very uniform. The mean pore size was 300±23.43 μm.
Table 1.
Porosities of SF/CS composite scaffold with different mass ratios ±s (n = 3).
Fig 4.
Fourier transform infrared spectra of SF/CS scaffold materials with different SF and CS mass ratios.
Fig 5.
XRD spectra of SF/CS scaffold materials with different SF and CS mass ratios.
Fig 6.
EDS spectrum of pure CS and pure SF and 40% SF-60% CS.
The relative mass ratios of elements C, N, O of the 40% SF-60% CS group (C) were between that of pure SF (B) and pure CS (A). This suggests that only simple blending between SF and CS occurs and no new substance or element emerges.
Table 2.
Water absorption and swelling ratios of SF/CS composite scaffold with different mass ratios ±s (n = 3).
Table 3.
The pH values and degradation ratios of the 40% SF-60% CS group ±s (n = 3).
Fig 7.
Change in the pH of the lysate and the degradation rate in the 40% SF-60% CS group.
(A) pH fluctuated in a small range of 7.42~7.62; (B)The scaffold lost only 18.25% of its original weight after 64 days.
Fig 8.
MG-63 cell growth on the scaffold.
At 6 h after inoculation, the cells started to adhere to the walls of the scaffold material; at 24 h the adhesion completed and the cell volumes increased. At day 2, the cells became spindle-shaped or triangular, showing junctions between the growth projections; at 3 d the cells were tightly connected to each other, and the walls and pores of the scaffold material were all covered with cells.
Fig 9.
Fluorescence image showing MG-63 cell adhesion to the scaffold material.
At 12 h after inoculation, the cells uniformly adhered to the pore walls of the scaffold.
Table 4.
Adhesion rates of MG-63 cells at different time points (±s, n = 3).
Fig 10.
Adhesion rates of MG-63 cells at different time points.
The adhesion rates of both experimental groups were significantly higher than that of the blank control side group. At 1 h and 3 h, the adhesion rate of the 40% SF-60% CS group was significantly higher than that of the 100% CS group; at 5 h, the two groups did not differ significantly.
Fig 11.
Fluorescence image showing MG-63 cell growth on the scaffold material.
At 3 h a very small number of cells adhered to the scaffold, but at 6 h and 12 h the number of adhered MG-63 cells increased substantially. At 1 d the cell nuclei began to show dispersed and uniform blue fluorescence. At 2 d the fluorescence intensity increased substantially, and at 3 d a granular fluorescence was observed due to increased cell density.
Table 5.
MG-63 proliferation rates at different time points (±s, n = 6).
Fig 12.
MG-63 cell proliferation revealed by OD values in different scaffold groups and at different time points.
The MG-63 cell proliferation rates of both experimental groups were significantly higher than that of the blank control slide group. On day 3, the proliferation rate of the 40% SF-60% CS group was significantly higher than that of the 100% CS group. On day 1 and 5, the two experimental groups did not differ significantly.
Table 6.
Activities of ALP secreted by MG-63 cells on different scaffolds at different time points (±s, n = 6).
Fig 13.
Secretion of ALP by MG-63 cells in different scaffold groups and at different time points.
ALP secretion by MG-63 cells in the two experimental groups was significantly stronger than that in the blank control group. On day 7, 14, and 21 the 40% SF-60% CS group facilitated osteogenesis significantly more than the 100% CS group.
Fig 14.
The mineralization of MG-63 cells on the scaffold.
(A) Osteoblasts secreted a spherical matrix, which clustered together. (B) Osteoblasts grew in multi-layers and clustered into groups. (C and D) The matrix secreted by the osteoblasts was mineralized, forming calcified nodules.