Articular cartilage repair

Gross findings and histological evaluation of cartilage defects

week after surgery, the defect area in the Defect group partially remained vacant while in the TGF-m group, the tissue looked a little whitish and was distinguished from the surrounding cartilage. Repaired tissues in the Control and TGF-i group partially filled the defect area and looked a little whitish. At week 8, defects in all groups, except for the TGF-m group, were observed to be filled with repaired tissue, but appeared different from the surrounding cartilage because of their white color. In the TGF-m group, the defect area was filled with tissue that was approach from normal cartilage (Fig. 2-1).

Safranin-O staining showed that defects in the Defect group were most filled with fibrous tissue. In contrast, most tissue, except for the surface layer, stained with safranin-O; however, its surface was quite rough and cells generally lacked orientation in the TGF-m group. In the Control and TGF-i group, defects were filled with fibrous tissue minimally occupied with hyaline-like tissues at week 4. At week 8, fibrous tissue was filled in the Defect group, while the TGF-m group, repaired tissue was well organized with intense ECM; further, the cell distribution was composed of columnar and cluster cells with hyaline character, even though its surface and cartilage-bone were irregular. Repaired tissue in the Control group and TGF-i showed an increased area of metachromatic staining compared to that of 4 weeks; however, it was still occupied by much fibrous-like tissue covering the articular surface (Fig. 2-2).

In Sirius red staining, collagen fiber was found only in small amounts in samples of the Defect, Control and TGF-i groups at week 4, and no oriented pattern was observed. Collagen fibers were found in large amounts and integration was observed between repaired cartilage tissue and host cartilage in the TGF-m group (Fig. 2-2. D-c). At week 8 after surgery, the amount of collagen fibers was increased in all groups; however, oriented arrangement of collagen fibers was not observed in the Defect, Control and TGF-i groups. In the TGF-m group, a well-oriented arrangement of collagen fibers similar to normal cartilage were found at 8 weeks; futhermore, integration between repaired tissue and host tissue was also observed (Fig. 2-2)

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The ICRS histological score showed significant improvement with time in all but the Defect group. It was not until 8 weeks that the TGF-m group and TGF-i group and Control group showed a difference in ICRS score (Number of femoral condyles (N) =5; TGF-m and TGF-i was P =0.021, TGF-m and Control P =0.006) (Fig. 2-3).

Fig. 2-1. Gross observation of the defect area of the cartilage at 4 weeks (A-D) and 8 weeks (E-H) after surgery. In the 4-week samples, D showed a smooth and glistening appearance, and continuity with the surrounding host cartilage tissue was also observed. In contrast, the defect was not repaired in picture A and was filled partially with fibrous tissues in picture B and C. At 8 weeks after surgery, a white and glistening appearance of repaired tissues was shown in all groups.

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Fig. 2-2. Histological observation of repaired cartilage in the defect area of the experimental groups at 4 weeks (A-D) and 8 weeks (E-H) after surgery. And Safranin-O staining image of normal cartilage was also provided (I). The defect was fibrous/hyaline cartilage was regenerated in the all groups at week 4. The defect was partially filled with fibrous tissues in the Defect groups at week 8. At week 8, fibrous/hyaline cartilage was regenerated in the Control and TGF-i groups. In contrast, hyaline cartilage tissues with mature matrix and columnar organization of chondrocytes were observed in the TGF-m groups. Magnification was x20 (a), and x100 (b). Sirius red staining image at 4 weeks (A-c – D-c) and 8 weeks (E-c – H-c) after surgery. An image of normal cartilage was also provided (I-c). Magnification was at x200 (A-c – I-c), respectively.

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Fig. 2-3. ICRS scores at 4 weeks and 8 weeks after surgery. The total ICRS histological score increased significantly along with time in all groups, except for the Defect group. No significant difference was observed between the Control, TGF-i and TGF-m group at 4 weeks; however, at 8 weeks, a significant difference was shown in ICRS score. (N =6, Error bars are standard deviation, *P < 0.05).

42 2.3.2 Chemical assay of repaired tissue

At 8 weeks post-surgery, the amount of GAG in the TGF-m was significantly higher than those in the Defect, Control and TGF-i groups, while there was no statistically significant difference in the amount of GAG in the TGF-m group and normal cartilage. This finding suggested that GAG formation of the TGF-m group approached that of normal cartilage (N

=6, P =0.640) (Fig. 2-4A). Lower levels of DNA content were detected in the Buffy coat group compared to the Defect, Control and TGF-i groups. This finding means that repaired cartilage of the TGF-m group was compatible to hyaline cartilage (N =5, P=0.998) (Fig. 2-4B).

Fig. 2-4. (A) Changes and comparison of GAG contents among the experimental groups at 8 weeks after surgery. GAG contents of the TGF-m group were higher than those of the Defect, Control and TGF-i group. Moreover, they were significantly different in statistical analysis (N =6; P=0, P=0.006, P=0.009). However, the TGF-m group showed no difference from this of the normal tissue in statistical analysis (N =6, P=0.640). (B) Changes and comparison of DNA contents among the experimental groups at 8 weeks after surgery. A lower level of DNA content was detected in the TGF-m group compared with the Defect, Control and TGF-i group. Moreover, they were significantly different in statistical analysis (N=6; P=0, P=0.018, P=0.049). However, the TGF-m group showed no difference from this of the normal tissue in statistical analysis. (N=6, P=0.998). (Error bars are standard deviation, *P <

0.05).

43 2.4 DISCUSSION

Hough it is important to lead regeneration of cartilage on osteochondral defect, it is also significant to have regeneration of subchondral bone on primary defect sites. Moreover, regenerated and host tissues shall be integrated well.

In in vivo experiment, without losing the activity of TGF-β3 in membrane, sustained releasing system was working on differentiation of MSCs as effectively as in the in vitro.

The in vitro and in vivo tests confirmed the effect of constantly released TGF on differentiation of MSCs into chondrocytes, suggesting presence of a biologic activity.

The mechanism wherein drug is released from the ECM multilayer membrane system was not clearly revealed. First of all, drug is presumed to diffuse from the edge of the membrane. Since past experiments found based on analysis that ECM would have low permeability (permeability coefficiency), however, some are expected to be released through the transmembrane route.

2.5 CONCLUSION

We speculate that the sustained release of rhTGF-β3 using the ECM membrane after BMS could be a useful clinical protocol for cartilage repair.

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u CHAPTER Ⅲ

Nondestructive assessment of GAG content and distribution in