4-2.1 Scanning electron microscopy
An SEM photograph of gelatin/C6S/C4S/HA modified tricopolymer scaffold is showed in Figure 58. It showed a highly porous structure which had an average 40um pore size ranging from 15um to 60um, and all of the pores in the tricopolymer scaffolds were interconnected.
Figure 58 An SEM photograph of gelatin/C6S/C4S/HA modified tricopolymer scaffold (400×).
SEM photographs of cell-scaffold hybrids which were taken immediately after cell seeding are showed in Figure 59. It showed that the seeded cells deeply penetrated and evenly distributed in the scaffold. Also, SEM photographs of the control and experimental cell-scaffold hybrids group were taken at the end of 1, 2, and 4-week culture, and the results are showed in Figure 60, 61, and 62, respectively.
Figure 59 SEM photographs of cell-scaffold hybrids which were taken immediately after cell seeding. (a) 400× (b) 1500×
Figure 60 SEM photographs of cell-scaffold hybrids which were taken at the end of 1-week culture. (a) Control group (3000×) (b) Betulin-treated experimental group (1000×).
75 um 20 um
30 um 10 um
Figure 61 SEM photographs of cell-scaffold hybrids which were taken at the end of 2-week culture. (a) Control group (2000×) (b) Betulin-treated experimental group (1200×).
Figure 62 SEM photographs of cell-scaffold hybrids which were taken at the end of 4-week culture. (a) Control group (3000×) (b) Betulin-treated experimental group.
The chondrocytes were just in the phase of cell division (1500×).
15 um 25 um
20 um 10 um
4-2.2 WST-1 Assay for Cell Proliferation
The OD450 of cell-scaffold hybrids control group and cell-scaffold hybrids experimental group which were treated with 0.32ug/ml betulin were obtained after 1-week, 2-week, and 4-week culture, and the scaffolds without cells seeded were used as the negative control group. The results were summarized in Figure 63, which showed that all of cell-scaffold hybrids treated with betulin had statistically significant and higher OD values than cell-scaffold hybrids control group at the end of each tested week. At the end of 1-week culture, the OD values of the experimental group and the control group are 1.129 ± 0.025 and 1.080 ± 0.019 respectively, and the p value was 0.006 lower than 0.05. At the end of 2-week culture, the OD values
between the experimental group and the control group are 1.428 ± 0.145 and 1.280 ± 0.083 where the p value was 0.03 and lower than 0.05. Besides this, at the end of 4-week culture, the OD values of the experimental group and the control group are 1.936 ± 0.169 and 1.648 ± 0.033 (p=0.009<0.05).
The results also showed that the longer the cells were cultured in 3D environment, the higher the OD values obtained. For instances, the OD450 of 4-week experimental group was significantly higher than the 2-week one (p = 1.79×10-6), and the OD450 of 2-week experimental group was also higher than the 1-week one (p = 0.0003).
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2
1 Week 2 Weeks 4 Weeks
O.D. 450nm
Scaffold Only
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold Hybrids
*
*
*
Figure 63 WST-1 assay of three-dimensional scaffold culture. In WST-1 assay, all of cell-scaffold hybrids experimental groups had statistically significant and higher OD values than cell-scaffold hybrids control groups.
4-2.3 Total DNA for Cell Proliferation Quantification
The total DNA amount of cell-scaffold hybrids control group and experimental group which were treated with 0.32ug/ml betulin were obtained after 1-week, 2-week, and 4-week culture. The results were summarized in Figure 64 and showed that all of cell-scaffold hybrids treated with betulin had higher DNA amount than cell-scaffold hybrids control group at the end of each tested week. At the end of 1-week culture, the DNA amount of the experimental group and the control group are 6.428 ± 0.428 and 5.082 ± 0.590 respectively, and the p value was 0.15. At the end of 2-week culture, the DNA amount between the experimental group and the control group are 6.718 ± 0.626 and 5.873 ± 0.025 where the p value was 0.15. Besides this, at the end of 4-week culture, the DNA amount of the experimental group and the control group are 12.153 ± 0.544 and 10.775 ± 0.110 (p=0.13).
The results also showed that the longer the cells were cultured in 3D environment, the higher the DNA amount obtained. For instances, the DNA amount of 4-week experimental group was significantly higher than the 2-week one (p = 0.02<0.05).
0 2 4 6 8 10 12 14
1 Week 2 Weeks 4 Weeks
DNA conc. (ug/ml)
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold Hybrids
*
Figure 64 Total DNA assay of three-dimensional scaffold culture. In total DNA assay, all of cell-scaffold hybrids experimental groups had higher DNA amount than cell-scaffold hybrids control groups.
4-2.4 DMMB Assay for Sulfated Glycosaminoglycans Content
When conducting DMMB assay, a linear standard curve should be obtained from GAG standards: 0–100 mg/mL of chondroitin-6-sulfate (C-4384, Sigma), which were then used to estimate the GAG content in each experimental sample. The OD570
obtained from C6S standards are summarized in Figure 65. The linear regression trend line of C6S standards was: y = -198.2x + 198.83.
y = -198.2x + 198.83 R2 = 0.9853
0 10 20 30 40 50 60 70 80 90 100
0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
OD 595nm value
C6S concentration ug/ml
Figure 65 Linear standard curve of glycosaminoglycans standards. The absorbance at 570 nm was measured. The linear regression trend line of glycosaminoglycans standards was: y = -198.2x + 198.83.
The glycosaminoglycans content of cell-scaffold hybrids control groups and cell-scaffold hybrids experimental groups which were treated with 0.32ug/ml betulin was obtained after 1-week, 2-week, and 4-week culture and summarized in Figure 66, and the scaffolds without cells seeded were used as the negative control group.
At the end of 1-week culture, betulin-treated experimental group didn’t show a higher GAG content than control group (79.365 ±2.690 ug/ml vs. 84.518 ±4.092 ug/ml, p = 0.017). However, at the end of 2-week culture, betulin-treated experimental groups showed statistically significant higher GAG content than their relative control groups (94.924 ± 0.862 vs. 80.158 ±8.611, P = 0.0177<0.05). Similarly, at the end of 4-week culture, betulin-treated experimental groups showed higher but not statistically significant GAG content than their relative control groups.
The results also showed that when the cell-scaffold hybrids were treated with betulin, at the end of 2-week culture, the amount of GAGs got maxima. Since former research and the experimental result here have shown that the GAG originally entrapped in the scaffold substrate can not be retained by the scaffold and is lost into the culture medium during the in vitro culture period (data showed in Figure 67), the GAGs content increase of the experimental group means that the GAGs were newly secreted by chondrocytes themselves. Here, we didn’t discover that the longer the cells were cultured in 3D environment, the higher the GAGs content existed.
0
Figure 66 The GAG contents of cell-scaffold hybrids control groups and betulin-treated cell-scaffold hybrids experimental groups after cultured for 1, 2, and 4 weeks were shown.
Figure 67 The GAG contents of the scaffolds without cells seeded negative control
4-2.5 Real-time Reverse-Transcriptase Polymerase Chain Reaction for mRNA Expression Quantification
In real-time PCR, the -△Ct values of each target gene were normalized by the house-keeping gene, GAPDH, and relative expressions of each target gene between the experimental groups and the control group are shown by the - CT and △ summarized as follows.
4-2.5.1 mRNA Expression of Collagens
The expression of type II collagen, type I collagen and type X collagen were shown in Figure 68, Figure 69 and Figure 70, respectively. Type II collagen, one of the major components in the extracellular matrix of articular cartilage, increased with the culture time when cell-scaffold hybrids were treated by betulin (Figure 68). Also, at the end of each tested week, betulin-treated experimental groups showed statistically significant higher type II collagen gene expression than their relative control groups. In contrast, in the control groups, the expression of type II collagen didn’t show constant increase within culture time. For Type I collagen gene, although at the end of the first week betulin-treated experimental groups showed statistically significant higher type I collagen gene expression than the control groups (Figure 69).
However, at the end of 2 and 4 weeks, the type I collagen gene expression of control groups increased dramatically, and betulin-treated experimental groups showed statistically significant lower type I collagen gene expression than their relative control groups. When chondrocytes cultured in the betulin-treated experimental group, type X collagen expression was kept in a very low level, and at the end of 1 and 2 weeks it was significantly lower then the control group (Figure 70).
-8
Relative type II collagen expression (- △Ct)
Cell-Scaffold Hybrids Control collagen gene expression were obtained. In the bar chart, – Ct was shown by mean △ with S.D. (* means p-value<0.01).
Relative type I collagen expression (- △Ct)
Cell-Scaffold Hybrids Control collagen gene expression were obtained. In the bar chart, – Ct was shown by mean △ with S.D. (* means p-value<0.01)
-6 -5 -4 -3 -2 -1 0 1 2
1 Week 2 Weeks 4 Weeks
Relative type X collagen expression (- △Ct)
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold Hybrids
*
*
Figure 70 At the end of 1, 2, and 4-week, the values of –△Ct of relative type X collagen gene expression were obtained. In the bar chart, – Ct was shown by mean △ with S.D. (* means p-value<0.01).
4-2.5.2 mRNA Expression of Proteoglycans
The expression of proteoglycans including aggrecan and decorin were shown in Figure 71 and Figure 72, respectively. In the betulin-treated experimental groups, at the end of each tested week, mRNA expression of aggrecan showed statistically significant higher than their relative control groups. Moreover, at the end of 2 weeks, betulin successfully increased the expression of aggrecan to the maximum. Similarly, mRNA expression of decorin also showed statistically significant higher than their relative control groups at the end of 1 week and 2 weeks.
0
Relative aggrecan expression (- △Ct)
Cell-Scaffold Hybrids Control gene expression were obtained. In the bar chart, – Ct was shown by m△ ean with S.D.
(* means p-value<0.01).
0 1 2 3 4 5 6 7 8 9
1 Week 2 Weeks 4 Weeks
Relative decorin expression (- △Ct)
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold
*
*
Figure 72 At the end of 1, 2, and 4-week, the values of –△Ct of relative decorin gene expression were obtained. In the bar chart, – Ct was shown by mean with S.D. (* △ means p-value<0.01).
4-2.5.3 mRNA Expression of ECM Regulators
The mRNA expression of three major factors related to extra-cellular matrix degradation, TIMP-1, MMP-2 and MT1-MMP, were measured, and results were shown in Figure 73, Figure 74 and Figure 75, respectively. The mRNA expression of TIMP-1 increased with the culture time when cell-scaffold hybrids were treated by betulin (Figure 73). Although the mRNA expression of TIMP-1 had no statistical significance between the control group and the experimental group, at the end of 1 week and 2 weeks, the expression value of experimental groups were still higher than the control group.
0 1 2 3 4 5 6 7
1 Week 2 Weeks 4 Weeks
Relative TIMP-1 expression (- △Ct)
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold Hybrids
Figure 73 At the end of 1, 2, and 4-week, the values of –△Ct of relative TIMP-1 gene expression were obtained. In the bar chart, – Ct was shown by mean with S.D. △ (* means p-value<0.01).
In MMP-2 mRNA expression, betulin-treated experimental group successfully kept the expression of MMP-2 in a significant low level although the expression amount was increase with the culture time (Figure 74).
-6 -4 -2 0 2 4 6 8 10
1 Week 2 Weeks 4 Weeks
Relative MMP-2 expression (- △Ct)
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold Hybrids
*
*
*
Figure 74 At the end of 1, 2, and 4-week, the values of –△Ct of relative MMP-2 gene expression were obtained. In the bar chart, – Ct was shown by mean with S.D△ . (* means p-value<0.01).
Although at the end of the first week the mRNA expression of membrane-type 1 metalloproteinase (MT1-MMP) of betulin-treated experimental group was significantly higher that the control group. However, at the end of 2 weeks, mRNA expression of MT1-MMP betulin group showed statistically significant lower value their relative control groups (Figure 75).
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
1 Week 2 Weeks 4 Weeks
Relative MT1-MMP expression (- △Ct)
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold Hybrids
*
*
Figure 75 At the end of 1, 2, and 4-week, the values of –△Ct of relative MT1-MMP gene expression were obtained. In the bar chart, – Ct was shown by mean with S.D. △ (* means p-value<0.01).
4-2.5.4 mRNA Expression of Growth and Differentiation Factors
In TGF-β1, there was no significant difference between the control and experimental group after 1-week culture, and at the end of 2 weeks the TGF-β1 mRNA expression of control group increased to a significant higher value than the betulin-treated experimental group. However, after 4-week culture the mRNA expression of betulin-treated experimental group increased to a significant higher value than the control group (Figure 76).
0 0.5 1 1.5 2 2.5 3
1 Week 2 Weeks 4 Weeks
Relative TGF-β1 expression (- △Ct)
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold Hybrids
* *
Figure 76 At the end of 1, 2, and 4-week, the values of –△Ct of relative TGF-β1 gene expression were obtained. In the bar chart, – Ct was shown by mean with S.D. △ (* means p-value<0.01).
The results of BMP-7 mRNA expression showed that the longer the cells were cultured in betulin-treated 3D environment, the higher the mRNA expression amount obtained. At the end of 1 week and 2 weeks, mRNA expression of BMP-7 successfully increased to a significant higher value than their relative control groups.
-6 -5 -4 -3 -2 -1 0
1 Week 2 Weeks 4 Weeks
Relative BMP expression (- △Ct)
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold Hybrids
*
*
Figure 77 At the end of 1, 2, and 4-week, the values of –△Ct of relative BMP-7 gene expression were obtained. In the bar chart, – Ct was shown by mean with S.D. (* △ means p-value<0.01).
Also, in the results of IGF-1 mRNA expression showed that the longer the cells were cultured in betulin-treated 3D environment, the higher the mRNA expression obtained. At the end of 1 week and 2 weeks, mRNA expression of IGF-1 successfully increased to a significant higher value than their relative control groups.
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
1 Week 2 Weeks 4 Weeks
Relative IGF expression (- △Ct)
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold Hybrids
*
*
Figure 78 At the end of 1, 2, and 4-week, the values of –△Ct of relative IGF-1 gene expression were obtained. In the bar chart, – Ct was shown by mean with S.D. (* △ means p-value<0.01).
4-2.5.5 mRNA Expression of Catabolic Cytokines
When chondrocytes cultured in the 3D environment, IL-1β expression was kept in a quite low level, and at the end of 1 and 2 weeks it was significantly lower then the control group (Figure 79).
-14 -12 -10 -8 -6 -4 -2 0
1 Week 2 Weeks 4 Weeks
Relative IL-1β expression (- △Ct)
Cell-Scaffold Hybrids Control Betulin Treated Cell-Scaffold Hybrids
*
Figure 79 At the end of 1, 2, and 4-week, the values of –△Ct of relative IL-1β gene expression were obtained. In the bar chart, – Ct was shown by mean with S.D. △ (* means p-value<0.01).