Osteochondral repair using scaffolds with gradient pore sizes constructed with silk fibroin, chitosan, and nano-hydroxyapatite

Figures & data

Figure 1 Schematic of the preparation of the cell–scaffold complex.

Figure 2 Morphology of passage 3 bone marrow mesenchymal stem cells (A, 40×; B, 100×; C, 200×).

Table 1 Height, diameter, and weight of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds with gradient pore diameters (mean±SD, n=4)

	Height (mm)	Diameter (mm)	Weight (mg)
Scaffold-1	9.20±0.26	5.86±0.50	6.21±0.27
Scaffold-2	9.55±0.35	6.53±0.07	10.29±0.65a
Scaffold-3	9.53±0.23	6.40±0.32	13.80±0.66a,b

Notes:

a P<0.05, vs Scaffold-1.

b P<0.05, vs Scaffold-2.

Figure 3 Appearance of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds: Scaffold-1 (A), Scaffold-2 (B), and Scaffold-3 (C).

Figure 4 Height (A), diameter (B), and weight (C) of silk fibroin/chitosan/nano-hydroxyapatite scaffolds.

Notes: ^aP<0.05, vs Scaffold-1. ^bP<0.05, vs Scaffold-2.

Table 2 Porosity, hot-water dissolution rate, and water swelling rate of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds with gradient pore diameters (mean±SD, %, n=4)

	Porosity	Hot-water dissolution rate	Water swelling rate
Scaffold-1	91.30±3.35	16.57±3.18	3,218.53±84.37
Scaffold-2	89.50±3.11	15.03±2.17	2,573.83±132.51a
Scaffold-3	82.58±1.47a,b	15.65±0.68	1,969.50±90.01a,b

Notes:

a P<0.05, vs Scaffold-1.

b P<0.05, vs Scaffold-2.

Figure 5 Porosity (A), water swelling rate (B), and hot-water dissolution rate (C) of silk fibroin/chitosan/nano-hydroxyapatite scaffolds.

Notes: ^aP<0.05, vs Scaffold-1. ^bP<0.05, vs Scaffold-2.

Figure 6 Hot-water dissolution rate as a function of dissolution time for silk fibroin/chitosan/nano-hydroxyapatite scaffolds.

Figure 7 Hot-water dissolution rate vs dissolution time of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds: (A) Scaffold-1, (B) Scaffold-2, and (C) Scaffold-3.

Figure 8 Stress–strain curves of silk fibroin/chitosan/nano-hydroxyapatite scaffolds: (A) Scaffold-1, (B) Scaffold-2, and (C) Scaffold-3. The averaged curves for comparing the three scaffolds (D). The linear fit curves for comparing the elastic modulus of scaffolds (E).

Table 3 Water swelling rate of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds with gradient pore diameters before and after compression (mean±SD, %, n=4)

	Scaffold-1	Scaffold-2	Scaffold-3
Before compression	3,218.53±84.37	2,573.8±132.5	1,969.5±90.0
After compression	1,571.60±60.29a	2,393.53±68.85	2,018.85±113.41

Note:

a P<0.05, vs water swelling rate before compression.

Figure 9 The water swelling rate of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds with gradient pore diameters before and after compression.

Note: ^aP<0.05, vs water swelling rate before compression.

Table 4 Pore size of the different layers of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds with gradient pore diameters (mean±SD, μm, n=4)

	Scaffold-1	Scaffold-2	Scaffold-3
L1	141.11±11.85	152.60±22.47	133.35±11.24
L2	119.94±9.05a	117.46±15.49a	118.42±19.10
L3	93.10±14.98a,b	102.19±14.33a	94.97±5.68a,b
L4	79.95±8.65a–c	86.29±12.10a,b	74.66±2.70a–c

Notes:

a P<0.05, vs L1.

b P<0.05, vs L2.

c P<0.05, vs L3.

Abbreviation: L, layer.

Figure 10 Scanning electron micrographs (×210) showing the internal structure of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds.

Abbreviation: L, layer.

Figure 11 Pore size of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds (A and B).

Notes: ^aP<0.05, vs L1. ^bP<0.05, vs L2. ^cP<0.05, vs L3.

Abbreviation: L, layer.

Table 5 Optical density of the cells in different layers of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds (mean±SD, n=4)

	1 day	3 days	5 days	7 days	9 days	11 days
L1	0.135±0.019	0.356±0.021	0.778±0.026	1.153±0.047	1.455±0.049	1.190±0.036
L2	0.140±0.021	0.351±0.017	0.706±0.028a	1.098±0.029	1.369±0.013a	1.147±0.010a
L3	0.130±0.013	0.356±0.013	0.683±0.035a	1.086±0.058	1.347±0.046a	1.101±0.014a,b
L4	0.119±0.014	0.362±0.005	0.668±0.018a	1.034±0.046a	1.284±0.037a–c	1.060±0.035a–c

Notes:

a P<0.05, vs L1.

b P<0.05, vs L2.

c P<0.05, vs L3.

Abbreviation: L, layer.

Figure 12 Cell proliferation curves corresponding to the different layers of the cell-scaffold complex.

Note: *P<0.05.

Abbreviation: L, layer.

Figure 13 DAPI staining of the cell-scaffold complex after 9 days of culture (A, 100×; B, 200×).

Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; L, layer.

Figure 14 Scanning electron microscope observation of cell morphology and distribution of the cell-scaffold complex at 9 days of culture.

Abbreviation: L, layer.