Assessment of engineered constructs in vitro
SF scaffold exhibited a pore size of approximately 100–150 um in diameter by SEM examination (Fig. 2A). In the EC group, the cells adhered to the scaffold with filopodia formation. After four weeks of culturing, the chondrocyte-scaffold constructs (EC) exhibited an ivory-like appearance. H&E staining showed that cartilage lacuna formed and the scaffold partly degraded during 4w in vitro culture. Safranin-O staining showed that cartilage-specific ECM such as glycosaminoglycan (GAG) was expressed (Fig. 2B).
qRT-PCR (Fig. 2C) showed that the expression of COL 2, COL 1 and Aggrecan was increased in engineered cartilage during in vitro culturing, and showed no significant difference with the native auricular cartilage at 4w (all P > 0.05).
In vivo histological and biochemical evaluation of the engineered cartilage
The constructs were autologously implanted in the dorsal back of the donor rabbit subcutaneously after 4w of culture. At 2w, 4w and 8w post-implantation, constructs were harvested and submitted for histological examination. In the AC group, the cartilage grafts had no significant changes macroscopically for 8w, while H&E staining showed a partial absorption of cartilage tissue at 8w in vivo, showing as the cartilage tissue layer became thinner (Fig. 3). In the EC group, the accumulations of GAG as well as collagen was observed at 8w in vivo. In the SF group, the scaffold was wrapped with connective tissue and gradually degenerated, together with leukocytes and fibroblasts infiltration over time (Fig. 3).
Immunohistochemical staining of ECM-featured proteins in the engineered cartilage after in vivo transplantation (Fig. 4A-O) showed positive for COL 2, ELN and COL 1, and their expression increased over time. Picrosirius red staining was performed to observe the collagen organization within the engineered cartilage (Fig. 4P-Y). Using polarized light microscopy, the type I collagen, which appear as brilliant (strongly birefringent) red-yellow fibers and the greenish collagen III fibers (weakly birefringent) were clearly visualized in the perichondrium of the rabbit ear cartilage. In the EC group, the brilliant red-yellow fibers and green fibers increased over time, suggesting the maturation and deposition of collagen fibers.
Cartilage specific protein quantification test further confirmed the histological results. The autologous auricular cartilage grafts gradually degraded after implantation, showing that the content of GAG, HYP, and COL 1 decreased over time. Besides, the GAG, HYP, COL 1, and COL 2 contents of the AC grafts were significantly higher than those of the EC constructs at 2w in vivo, while there was no significant difference between the two groups at 4w and 8w. In addition, the ELN content was relatively stable in both EC and AC groups after implantation (Fig. 4Z).
In vivo MRI evaluation of the engineered cartilage
The PROSET, PDW VISTA SPAIR, 3D T2 VISTA, 2D MIXED T2 Multislice, and SAG TE multiecho sequences were performed to detect the grafts 1w after implantation. We found that the 2D MIXED T2 Multislice sequence provided higher quality images within a short time and was more sensitive to cartilaginous tissue compared with the other sequence (Fig. 5). Thus, we used 2D MIXED T2 Multislice to generate T2 mapping images in this study (Fig. 6A). The quantitative results (Fig. 6B) showed that the average T2 value of grafts in the AC group was significantly lower than that in the EC and SF groups from 1w to 4w after implantation (P < 0.001). At 8w in vivo, there was no significant difference in T2 values between AC and EC groups (P = 0.085). The average T2 value of the grafts in the SF group also decreased (144.1 ± 2.83 ms and 100.4 ± 13.13 ms in 1w and 8w in vivo, respectively), but it was still significantly higher than that in the EC and AC groups after 8w.
Correlation analysis between the T2 value and biochemical indicators value
To further determine the engineered cartilage maturity, we quantified the ECM-featured proteins at 1w, 2w, 4w and 8w after implantation. The results showed that the contents of HYP, ELN, GAG, COL 2, and COL 1 gradually increased over a period of eight weeks in vivo (Fig. 7A). HYP content significantly increased from the second week in vivo, while GAG and Col 2 increased significantly from the fourth week, and ELN protein content increased significantly until the eighth week in vivo. Notably, T2 values were more sensitive to the in vivo maturation, showing significant differences at all four-time points.
To clarify the feasibility of using T2 values to indicate the biochemical index of the engineered cartilage, the Pearson correlation analysis between the T2 value and biochemical indicators value was done (Fig. 7B). We found that T2 significantly correlated with total collagen, ELN and GAG contents (collagen: r = -0.946, P < 0.001; ELN: r= -0.939, P < 0.001; GAG: r = -0.933, P < 0.001). Moderate correlations were found between T2 and specific COL 1 or COL 2 content (COL 1: r = -0.837, P = 0.005; COL 2: r = -0.797, P = 0.01).