*3.4. Arthroscopy and Histological Analysis*

The regeneration of cartilage defect was grossly observed by knee arthroscopy, and a specimen of 2 mm in diameter was taken 1 year after surgery. Nine out of 10 patients in the Kartigen® group and 4 out of 5 patients in the control group consented to second-look arthroscopy. The gross appearance of regenerated cartilage tissues was examined under arthroscope and compared with their corresponding arthroscopic images taken before treatment (Figure 3). Eight out of 9 of the Kartigen® group exhibited smooth and elastic surfaces at the recipient sites (Figure 3A), but none of the control group showed normal or near-normal appearance. Only fibrillated surface was noted (Figure 3B). The ICRS scale is shown in Table 2 with the classification of grade I (normal tissue), grade II (nearly normal tissue), grade III (abnormal), grade IV (severely abnormal).

**Figure 1.** Assessment of knee functions of daily activities by IKDC scores. The knee functions of the patients were significantly improved 16 weeks after the Kartigen® implantation and 28 weeks after microfracture, respectively. The paired *t*-test was conducted to determine the difference between the means of the IKDC scores before and after the treatments at different time points among the same groups. Two years after operation, the IKDC score of the Kartigen® group was higher than the control group. The error bars stand for standard deviations. Asterisks represent the statistical significance: \* *p* < 0.05, \*\*\* *p* < 0.005.

**Figure 2.** VAS assessment. The pain relief was effective and significant in the Kartigen®-treated group, as VAS scores decreased since the 10th week after operation, but the VAS scores flatulated in the control group. The error bars stand for standard deviations. Asterisks represent the statistical significance: \* *p* < 0.05, \*\*\* *p* < 0.005.

**Figure 3.** Arthroscopic images before and 1 year after operation. Images of Kartigen® group (**A**) and microfracture group (**B**).

**Table 2.** ICRS arthroscopic assessment.


The Kartigen® group demonstrated better results in both arthroscopic observations and histological analysis (Figure 4). In the control group, the regenerated cartilage did

not exhibit columnar chondrocyte distribution but only fibrocartilage (Figure 4A). GAG and collagen type II were observed in the biopsy specimens, and a total of 12 specimens (Kartigen®, *n* = 8; control, *n* = 4) are presented in Figure 4B,C. New chondrocytes at defect had the same appearance as the original ones (Figure 4B). However, the chondrocytes in the repaired defect in the Kartigen® group were smaller and denser than those in the original cartilage.

**Figure 4.** Immunohistochemical analyses of biopsy specimens. (**A**) Whole-slide histological images of regenerated cartilage under 4× objective lens. Alcian blue staining and immunochemical study show expression levels of GAG/Collagen type II, respectively, in Kartigen group (**B**) and microfracture group (**C**).
