Chapter I
A. The structure and physical property of cell-derived ECM scaffold
The cell-derived ECM scaffold was made using porcine chondrocytes as described above. According to the gross examination, the disc-shaped scaffold was approximately 6 mm in diameter and had a white macrostructure with uniform pores (Fig. 4). By SEM images, a highly uniform porous microstructure was observed with the peripheral region of the scaffold densely coated (Fig. 5, white arrowheads). The dense peripheral coat was trimmed off using a 6 mm biopsy punch, resulting in a final cell-derived ECM scaffold with a standardized dimension of 6 mm in diameter and 3 mm in thickness.
The constructs had approximately 504±108㎛ of pore diameter, 90±10.4% porosity and
905±204 m2/g of surface area. The tensile strength was measured as 0.34±0.09 MPa (Table 2).
Fig. 4. The schematic illustration shows the process of making cell-derived ECM scaffold. Experimental details are described in Materials and Methods. A white
cylinder-shape of porous scaffold was finally produced with 6 mm in diameter and 2~3 mm in thickness. The scale bar is in the millimeter (mm) unit.
Table. 2. Physical properties of the cell-derived ECM scaffold.
Fig. 5. The SEM image of cell-derived ECM scaffold (x30). A cross section of the cell-derived ECM scaffold was observed by scanning electron microscope (SEM). (A) The exterior of the scaffold was densely coated with micro-pores (white arrows). (B) A highly uniform porous microstructure was observed in the central region of the scaffold.
B. Distribution and morphology of seeded chondrocytes on scaffold
In the SEM images taken after 1 day of cell seeding, many chondrocytes were evenly distributed on the cell-derived ECM scaffold in a round shape, whereas a few cells were attached to the PGA scaffold in a spindle shape (Figs. 6A and 6C). After 7 days of
culture, the newly synthesized ECM was found in both scaffolds, but, more densely populated cells were observed in the cell-derived ECM scaffold (Figs. 6B and 6D).
Fig. 6. The destribution and morphology of chondrocytes (arrows) seeded on the PGA (A, B) and the cell-derived ECM scaffolds (C, D), respectively. The images were taken by SEM at 1 (A, C) and 7 days (B, D) after culture in vitro (x1000).
C. Gross morphology of neocartilage tissue
When the chondrocytes in the PGA and ECM scaffolds were cultured until 4 weeks, they appeared to mature over time to form a neocartilage-like tissue. The neocartilage constructs of the ECM scaffold was a silvery white cartilage-like morphology after 1 week of culture, and showed a smooth glossy surface gradually with time (Figs. 7E-H).
In contrast, the constructs of PGA scaffolds was white in color and showed many surface nodes at 4 weeks (Figs. 7A-D).
Fig. 7. The gross morphology of neocartilage. The gross morphology of the chondrocytes-seeded PGA (A-D) and ECM (E-H) scaffolds was observed. The chondrocytes/ECM scaffold developed smooth and glossy surface along with culture time (E-H), while the chondrocytes/PGA scaffold showed a cartilage-like tissue of white color with many surface nodes at 4weeks (D). The scale bar is in the millimeter (mm) unit.
D. The changes in the volume and compressive strength of neocartilage tissue No reduction in the volume of the constructs was observed during the culture in both groups of PGA and ECM scaffolds. However, it was rather increased at 4 weeks in the ECM scaffold group with the statically significance (Fig. 8 A). The compressive strength of the neocartilage tissue in the ECM scaffold group was increased gradually and significantly with time as measured with 7.5±0.8, 13.7±3.5, 15.7±2.1 and 21.5±2.2
KPa at 2 days, 1, 2, and 4 weeks, respectively (Fig. 8 B). However, it was gradually and significantly decreased with time in the PGA scaffold group, which showed 26.5,
±
compressive strength of the ECM scaffold group was much lower than that of the PGA scaffold group at 2 days but became higher by more than 2 folds at 4 weeks.
Fig. 8. The volume and compressive strength of the neocartilage tissue. The volume (A) and compressive strength (B) of the chondrocytes-seeded PGA (solid bars) or ECM (hatch bards) scaffold was measured at 2 days, 1 week, 2 weeks and 4 weekds after cutlrue as described in Materials and Methods. Data were presented by mean values ± standar deviations (SD). The statistical significance was calculated between the ECM scaffold samples at 4 weeks versus those at the other time points or versus the PGA scaffold samples at 4 weeks by Student-Newman-Keuls Multiple comparisons test (n=4; *p<0.05, **p<0.01, ***p<0.001).
E. Chemical composition of neocartilage tissue
The total contents of DNA, GAG and collagens were examined in the neocartilage-like tissues from the PGA and ECM scaffold groups during the culture (Fig. 9). The DNA content of the neocartilage-like tissue was gradually and significantly increased with time in both groups. The DNA content of ECM scaffold group increased more than 3 folds at 4 weeks, when compared with that of the ECM scaffold itself (Fig. 9A). The GAG content also increased with time in all groups. The amount of GAG was 276.5±20.6 µg in the ECM scaffold itself and 378.5±65.6, 396.7±128.9, 1302.8±65.4 and 1450±30 µg in the neocartilage tissue with the ECM scaffold at 2 days, 1, 2, and 4
weeks, respectively. The GAG content of the ECM scaffold group was significantly higher that that of the PGA scaffold group until 2 weeks, but they were quite similar at 4 weeks (Fig. 9B). Although the collagen content also increased gradually with time in both groups, the ECM scaffold group showed much higher values than the PGA group at all time points (Fig. 9 C). The collagen content in the ECM scaffold group was more than 4 folds of the initial amount at 4 weeks. The amount of newly synthesized GAG and collagen from the seeded cells was calculated to be about 1173.5 and 1474.2 µg, respectively, in the ECM scaffold group at 4 weeks.
Fig. 9. Chemical analysis for the neocartilage tissues. The neocartilage constructs from the chondrocytes-seeded PGA (solid bars) or ECM (hatch bards) scaffold were measured for the contents of DNA (A), GAG (B) and collagen (C), respectively as described in Materials and Methods. The statistical significance was analyzed between the ECM scaffold samples at 4 weeks versus those at the other time points or versus the PGA scaffold samples at 4 weeks by Student-Newman-Keuls Multiple comparisons test (n=3; ***p<0.001).
F. Histological assay of the neocartilage tissue
The expression of sulfated proteoglycan and type II collagen was then examined by histological observation in the neocartilage-like tissue. The Safranin-O staining of samples confirmed the accumulation of sulfated proteoglycans, which filled gradually with time the porous space and uniformly distributed within the cell-derived ECM
group decreased slowly with time probably indicating biodegradation of the scaffold (Fig. 10M-P, black arrows). In the PGA scaffold group, the sulfated proteoglycan was accumulated only after 2 weeks and distributed at peripheral area only (Fig. 10A-H).
Immunohistochemical analysis also showed that the synthesis of type II collagen was uniformly observed in the pericellular and pore regions in the ECM scaffold group (Fig.
11G-11L). Just like the sulfated proteoglycans, the expression was collagen type II was gradually increased with time but they were accumulated only in the peripheral area starting after 2 weeks of culture in the PGA scaffold group (Fig. 11A-11F).
Fig. 10. Expression of sulfated proteoglycans was examined in the neocartilage-like tissues with Safranin O staining. The proteoglycans was gradually accumulated in both of the PGA and ECM scaffolds with culture time, however the pattern of their distribution was significantly differanted between two groups. The magnification was
Fig. 11. Expression of type II collagen in the neocartilage-like tissues. The neocartilage tissues from the PGA and ECM scaffolds were subjected to immunostaining for type II collagen along with culture time. As in the distribution of proteoglycan, the pattern of collagen II distribution was also significantly differanted between two groups. The magnification was x20 (A-C and G-I) and x200 (D-F and J-L), respectively.
Chapter II
A. Morphology, volume and histology of neocartilage tissue
When the chondrocyte-seeded ECM scaffold was allowed to grow for 1, 2, and 3 weeks in the nude mice, we observed a smooth, white-colored surface and a relatively hard cartilage-like tissue based on the pinch test. There was also no apparent change in tissue size in the experimental group 1 week after implantation (Fig. 12). In fact, the volumes of the neocartilage were 45.3 ± 3.6 mm3, 48.1 ± 6.3 mm3, and 52.2 ± 2.1 mm3
at the 1st, 2nd, and 3rd weeks, respectively, in the experimental group (Fig. 13). The average volume gradually increased, although there were no significant statistical differences among the specimens of the experimental group. Comparatively, it decreased significantly in the control group by the 3rd week of implantation.
Histological staining with Safranin O (Fig. 14A-F) and Alcian blue (Fig. 14G-L) exhibited a sustained accumulation of the sulfated proteoglycan and, thus, gradually filled the pore spaces in the scaffold in the experimental group. Cartilaginous ECM appeared as early as the 1st week of implantation and it was more prominent at the peripheral region than at the core at the 3rd week of implantation. However, the positive staining was not observed in the pore space of the ECM scaffold, and fibrous tissue filled in the control group.
Fig. 12. The gross morphology of neocartilage tissues. The neocartilage tissue had a white color and smooth surface, and was a relatively hard, cartilage-like tissue on the pinch test. It was notable that there was no apparent change in tissue size in the experimental group after 1 week of implantation.
Fig. 13. The volume of neocartilage tissues. The volume of neocartilage tissue increased gradually and was about 72% of the initial volume at the 3rd week in the experimental group. Comparatively, it decreased continuously by time and significantly
Fig. 14. Histological stainings of neocartilage tissue. Histological stainings with Safranin O (A-F) and Alcian blue (G-L) exhibited a sustained accumulation of the sulfated proteoglycan, and the pore space in the scaffold was thus gradually filled in the experimental group. However, the positive staining was not observed in the control group.
B. Analyzing phenotypes of chondrocytes: RT-PCR analysis
The biomaterial induced a comparable constant gene expression of types I and II collagen over a period 3 weeks in both groups. Type II collagen gene was expressed constantly in the experimental group throughout the experimental period; however, neither types I nor II collagen gene was expressed in the control group. And type I
collagen gene was also expressed in the experimental group, but it decreased gradually with time in the culture (Fig. 15).
Fig. 15. RT-PCR analysis. Type II collagen was expressed constantly in the experimental group during the entire period, while the expression of type I collagen decreased gradually by time (lanes d-f). However, types I and II collagen genes were not expressed in the control group (lanes a-c). a and d, 1 week; b and e, 2 weeks; c and f, 3 weeks.
C. Chemical assay for neocartilage tissues
The water content was 89.4 ± 1.3%, 91.1 ± 0.5%, and 90.5 ± 0.8% in the control group and 89.1 ± 0.5%, 87.8 ± 1.1%, and 87.0 ± 0.4% in the experimental group at the 1st, 2nd, and 3rd weeks, respectively (n=4). Statistically, the water content was not significantly different in the control group; whereas, it decreased gradually in the experimental group during the study period (Fig. 16A). Measured from the papain-digested samples, the total GAG content was 62.6 ± 13.1 µg/mg, 56.3 ± 17.8 µg/mg, and 22.3 ± 7.4 µg/mg in the control group (dry weight) and 75.6 ± 19.8 µg/mg, 171.4 ± 15.3 µg/mg, and 201.3 ± 13.4 µg/mg in the experimental group at the 1st, 2nd, and 3rd
weeks, respectively (n=4). The GAG content decreased gradually in the control group;
whereas, it increased steadily with time in the experimental group, reaching 66% of the native cartilage at the 3rd week (Fig. 16B). Compared with the GAG content, the total DNA content decreased significantly in both groups after the 2nd week of the study (Fig.
16C). The DNA-normalized GAG content increased significantly in the experimental group; whereas, it decreased continually in the control group over time (Fig. 16D).
Fig. 16. Chemical assay. The water content decreased gradually by time in the experimental group; however, it was not significantly changed in the control group (A).
The GAG content gradually decreased in the control group, whereas it increased gradually in the experimental group over time and reached about 66% of the native cartilage at the 3rd week (B). However, the total DNA content decreased significantly in both groups at the 2nd week post-implantation (C). The GAG content normalized by the DNA content increased significantly from 2 weeks in the experimental group; whereas, it decreased continually in the control group over time (D). *p<0.05, **p<0.01, and
D. Collagen analysis
Immunohistochemistry identified type II collagen synthesized in the neocartilage tissues. With the protein deposited in the pericellular and pore regions, it was apparent that more type II collagen accumulated over time in the experimental group (Fig. 17D-F). However, type II collagen was not detected in the control group (Fig. 17A-C).
Western blot analysis revealed that while type II collagen was clearly detected throughout the study period, it was especially prominent at the 3rd week in the experimental group; however, it was not expressed at any time in the control group (Fig.
18). On the contrary, type I collagen was not expressed at all in either group (data not show).
Fig. 17. Immunohistochemistry analysis. Immunohistochemistry identified type II collagen synthesized in the neocartilage tissues in the experimental group (D-F).
However, it was not detected in the control group (A-C).
Fig. 18. Western blot analysis. Western blot analysis revealed that type II collagen was clearly detected throughout the experimental period, particularly prominent at 3 weeks, in the experimental group (lanes d-f). However, it was not expressed at any time points in the control group (lanes a-c). a and d, 1 week; b and e, 2 weeks; c and f, 3 weeks.
F. Compressive strength of neocartilage tissue
For the mechanical properties of the neocartilage, the specimens were tested for ultimate compressive strength at a point of 10% strain. The result was 0.45 ± 0.06 MPa, 0.8 ± 0.14 MPa, 1.18 ± 0.17 MPa (n=4) in the experimental group at the 1st, 2nd, and 3rd weeks, respectively (Fig. 19). This result demonstrated that the compressive strength was increased significantly along with the time points in the experimental group.
However, it could not be measured in the control group because of its fragility.
Fig. 19. The compressive strength of the neocartilage tissue. The compressive strength of the neocartilage tissue increased significantly by time in the experimental group. However, it could not be measured in the control group because of its fragility.
**p<0.01 and ***p<0.001.
Chapter III
A. Gross finding of cartilage defects
The gross appearance of cartilage defects right after implantation of the engineered cartilages was shown in Fig. 20. The implants were stably inserted on the defect sites (Fig. 20A). At 1 month after surgery, the repaired defects in groups 3 and 4 showed a smooth and glistening appearance and continuity with the surrounding host cartilage tissue. In contrast, the defect was not repaired well in group 1 (control) and filled partially with fibrous tissues in group 2. At 3 months, the white and glistening appearance of repaired tissues was shown on defects in all groups. However, the smooth and hard surface of repaired tissue was observed in groups 2, 3 and 4 by forceps test, while the slightly rough surface with many fissures was shown in group 1 (Fig.
20B).
A Initial
B 1 month
3 months
Fig. 20. Gross findings of the cartilage defect with implants. (A) Gross image of the engineered cartilages right after implantation in the defect site. (B) Gross image of the repaired cartilages at 1 and 3 months after implantation. The macroscopically smooth and glistening tissues were appearance with time.
B. Histological evaluation
In the Soffranin-O staining, the defect was partially filled with fibrous tissues in groups 1 and 2 at 1 month, which was not integrated with surrounding host cartilage and bone (Fig. 21A, B, E, F). In contrast, it was repaired to hyaline cartilage-like tissues at 1 month partially in group 3 and completely in group 4, respectively (Fig. 21C, D, G, H). The repaired tissues were integrated successfully to the host cartilage but the subchondral bone was not remodeled in all groups. At 3 months, the fibrous/hyaline cartilage was regenerated and partially integrated to the surrounding host cartilage in groups 1, 2 and 3 (Fig. 21I, J, K, M, N, O). Among them, the rough surface of repaired tissue was found in group 1 and the subchondral bone was partially remodeled in groups 1, 2 and 3. In contrast, the hyaline cartilage tissue with a mature matrix and a columnar organization of chondrocytes was observed in group 4. Moreover, the subchondral bone was well remodeled in the group (Fig. 21L, P).
The total ICRS histological score increased significantly along with time in all groups (Fig. 22). At 1 month, the ICRS score was higher in groups 3 and 4 than in groups 1 and 2, while it was higher in group 4 at 3 months.
1 month
3 months
Group 1 Group 2 Group 3 Group 4
Fig. 21. Histological assay of repaired cartilage on defects. Safranin-O staining of repaired cartilages from each group at 1 and 3 months post-implantation. A, C, E, G, I, K, M and O: x40; B, D, F, H, J, L, N and P: x100.
Fig. 22. The ICRS histological score. The ICRS score was determined from the results of the Safranin-O staining according to the rating system shown in Table 1.
C. Expression of type II collagens
The immunohistochemistry identified the expression of type II collagen was gradually increasing along with time at the pericellular region in the repaired tissues of groups 2, 3 and 4 (Fig. 23). It was not much detected in group 1 both at 1 and 3 month. The most significant expression of type II collagen with dark brown color was observed in zonal-structure in group 4 at 3 months (Fig. 4H).
1 month
3 months
Group 1 Group 2 Group 3 Group 4 Fig.23. Immunohistochemical staining for repaired cartilage. Immunohistochemical staining of type II collagen synthesized in the repaired tissues at 1 and 3 months after implantation (x200).