Implantation of bone marrow-derived buffy coat can supplement bone marrow stimulation for articular cartilage repair L.H. Jin, B.H. Choi, Y.J. Kim, S.R.

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Implantation of bone marrow-derived buffy coat can supplement bone marrow stimulation for articular cartilage repair L.H. Jin, B.H. Choi, Y.J. Kim, S.R. Park, C.Z. Jin, B.-H. Min Osteoarthritis and Cartilage Volume 19, Issue 12, Pages 1440-1448 (December 2011) DOI: 10.1016/j.joca.2011.07.012 Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Procedure for implantation of autologous buffy coat. Bone marrow was aspirated from rabbit iliac crest. Then, 20μl of buffy coat was separated from bone marrow using a Ficoll gradient centrifugation system and injected into the BMS treated area of articular cartilage, followed by ECM membrane covering. Osteoarthritis and Cartilage 2011 19, 1440-1448DOI: (10.1016/j.joca.2011.07.012) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Gross observation of the defect area of the cartilage at 4weeks (A–D) and 8weeks (E–H) after surgery. In the 4-week samples, C and 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. At 8weeks after surgery, a white and glistening appearance of repaired tissues was shown in all groups. Histological observation of repaired cartilage in the defect area of the experimental groups at 4weeks (I–P) and 8weeks (Q–X) after surgery. And Safranin-O staining image of normal cartilage was also provided (Y). The defect was partially filled with fibrous tissues in the Defect and BMS groups at week 4. In contrast, hyaline cartilage-like tissues were observed partially in the Buffy coat group and completely in the AOTS group. At week 8, fibrous/hyaline cartilage was regenerated in the Defect and the BMS groups. In contrast, hyaline cartilage tissues with mature matrix and columnar organization of chondrocytes were observed in the Buffy coat and AOTS groups. Magnification was ×20 (I–L, Q–T, Y), and ×200 (M–P, U–Z). Osteoarthritis and Cartilage 2011 19, 1440-1448DOI: (10.1016/j.joca.2011.07.012) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Sirius red staining image at 4weeks (A–H) and 8weeks (I–P) after surgery. An image of normal cartilage was also provided (Q). Magnification was at ×100 (A–D, I–L) and ×200 (E–H, M–P), respectively. Osteoarthritis and Cartilage 2011 19, 1440-1448DOI: (10.1016/j.joca.2011.07.012) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 ICRS scores at 4weeks and 8weeks after surgery. The total ICRS histological score increased significantly along with time in all groups. No significant difference was observed between the Buffy coat group and the BMS group at 4weeks; however, at 8weeks, a significant difference was shown in ICRS score. N≥5, P=0.018, P=0. (Error bars are standard deviation, ∗P<0.05). Osteoarthritis and Cartilage 2011 19, 1440-1448DOI: (10.1016/j.joca.2011.07.012) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Immunohistochemistry for collagen type II expression. At 4weeks (A–H) and 8weeks (I–P) after surgery. An image of normal cartilage was also provided (Q). (Magnification of ×20: A–D, I–L and magnification of ×200: E–H, M–P) Expression of type II collagen was gradually increased along with time at the pericellular region in repaired tissues of the BMS, Buffy coat, and AOTS groups. It was not significantly detected in the Defect group at both week 4 and 8. The most significant expression of type II collagen with dark brown color was observed in zonal-structure in the Buffy coat and AOTS groups at 8weeks. Osteoarthritis and Cartilage 2011 19, 1440-1448DOI: (10.1016/j.joca.2011.07.012) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 (A) Changes and comparison of GAG contents among the experimental groups at 8weeks after surgery. GAG contents of the Buffy coat group were higher than those of the Defect group and the BMS group. Moreover, they were significantly different in statistical analysis. However, the Buffy coat group showed no difference from those of the AOTS group and normal groups in statistical analysis. N≥5, P=0.015, P=0.047. (B) Changes and comparison of DNA contents among the experimental groups at 8weeks after surgery. A lower level of DNA content was detected in the Buffy coat group compared with the Defect group and the BMS group. Moreover, they were significantly different in statistical analysis. However, the Buffy coat group showed no difference from those of the AOTS group and normal groups in statistical analysis. N≥5, P=0.045, P=0. (Error bars are standard deviation, ∗P<0.05). Osteoarthritis and Cartilage 2011 19, 1440-1448DOI: (10.1016/j.joca.2011.07.012) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions