1. Identification of small molecular compounds and fabrication of its aqueous solution by laser-ablation, expanding primordial cartilage 
D. Ikegami, T. Iwai, S. Ryo, N. Gu, T. Sugiyama, I. Oh, H. Yoshikawa, N. Tsumaki 
Osteoarthritis and Cartilage 
Volume 19, Issue 2, Pages (February 2011)
DOI: /j.joca
Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
BPIB and its related molecules enhanced the growth of cartilage in organ culture. The metatarsal primordial cartilage from 14.5 d.p.c. mouse forelimbs was dissected and cultured (day 0). On the following day, the medium was changed to medium with or without compounds (day 1). (A) Searching for compounds that enhance the growth of cartilage explants in organ culture. Cartilage explants were cultured in the presence or absence of compounds for 6 days. Relative mean areas of cartilage explants after culture (day 7). Mean areas of cartilage cultured in the absence of compounds were set as 1. P values were determined by the Student’s t-test. Error bars show means±95% CIs. (B) Cartilage explants were cultured in the presence (40μM) or absence of compounds. Top, explants on day 1 and 7. Bottom, For quantification of cartilage growth, areas of cartilage explants were measured on images, and the area on day 7 was divided by the area on day 1. Mean ratios of explant areas on day 7 to that on day 1 (n=6, except for that SB is n=4). Error bars show means±95% CIs. *P< (Welch’s t-test) as compared with the samples cultured in the absence of compounds. (C) Inhibition of TGF-β signals by compounds. ATDC5 cells were transfected with p3TP-luc and cultured in medium supplemented with TGF-β1 (2ng/ml) and in the presence (10μM) or absence of compounds. (n=4, except for that BPIB is n=3). P value was determined by the Welch’s t-test.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Impairment of BPIB-induced enhancement of cartilage growth by addition of TGF-β1. Left, cartilage explant on day 7. Right, mean ratios of explant areas on day 7 to that on day 1 (n=3). P values were determined by the Student’s t-test.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
BPIB and BMPs synergistically enhance cartilage growth. Cartilage explants were cultured in the presence or absence of BPIB and GDF5 for 6 days (day 1–day 7). (A) BPIB and its related molecules synergistically enhanced cartilage growth induced by GDF5. Left, cartilage explant on day 7. Right, mean ratios of explant areas on day 7 to that on day 1 (n=3). Error bars show means±95% CIs. *P< (Student’s t-test). Scale bars, 1mm. (B) In situ mRNA expression analysis of cartilage explants. Semiserial sections of cartilage explants on day 7 were stained with Safranin O-fast green-iron hematoxylin and hybridized with type II collagen gene (Col2a1) and type X collagen gene (Col10a1) cRNA probes. Red color staining with safranin O suggests the presence of glycosaminoglycans, a component of cartilage matrix. Type II collagen is a major cartilage-specific matrix component. Type X collagen is a maker for hypertrophic cartilage. Scale bar, 300μm.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
BPIB increased glycosaminoglycan content and synergistically enhanced cartilage growth with BMP2. (A) Sulfated glycosaminoglycan content in organ-cultured primordial cartilage in the presence or absence of BPIB and GDF5. P values were determined by the Student’s t-test. (B) BPIB synergistically enhanced the expansion of primordial cartilage induced by BMP2. Left, cartilage explant on day 7. Scale bar, 1mm. Right, mean ratios of explant areas on day 7 to that on day 1 (BMP2 (–), n=3; BMP2 (+), n=4). P values were determined by the Welch’s t-test. Error bars show means±95% CIs.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
Effects of BPIB on cartilage were abolished without perichondrium. (A) Proliferation rates of chondrocytes in cartilage explant cultured in the presence of BMP2 or BPIB for 3 days (days 1–4) increased as compared with those in the absence of BMP2 and BPIB, as indicated by the increased numbers of BrdU-positive cells. Immunohistochemistry of cartilage explants on day 4 with anti-BrdU antibodies. Scale bar, 50μm. Mean ratios (±95% CIs) of BrdU-positive nuclei (n=3) were: control (–), 3.22±1.08%; BMP2 (500ng/ml) 13.56±6.15% (P=0.0021); and BPIB (10μM) 9.28±2.80% (P<0.0001). P value (Student’s t-test) as compared with the control (–). (B) After perichondrium was stripped from cartilage by partial digestion with collagenase, metatarsal explants were cultured in the presence or absence of BMP2 or BPIB for 3 days (days 1–4). Growth of stripped cartilage was enhanced by addition of BMP2, but not by addition of BPIB. Shown are cartilage explants on day 1 and 4. Scale bar, 1mm. Mean ratios (±95% CIs) of explant areas on day 4 (mm2) to that on day 1 (mm2) (n=3, except for that BPIB is n=4) were: control (–), 1.36±0.45; BMP2 (500ng/ml) 3.30±1.05 (P=0.0019); and BPIB (10μM) 1.53±0.40 (P=0.3592). P value (Student’s t-test) as compared with the control (–). (C) Metatarsal explants were cultured in the presence or absence of BPIB or FGF2 for 6 days (days 1–7). BPIB-induced cartilage expansion was reduced by addition of FGF2. Left, cartilage explants on day 7. Scale bar, 1mm. Right, mean ratios of explant areas on day 7 to that on day 1 (n=3). P values were determined by the Student’s t-test.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
BPIB dispersed by laser irradiation was more stable and enhanced growth of cartilage in organ culture more effectively than BPIB colloidal solution prepared by using DMSO. (A) Stabilities of BPIB/laser aqueous and BPIB/DMSO colloidal solution at various concentrations. After preparation of solutions (day 0), solutions were left to stand for 7 or 11 days, and the absorption spectra were measured. Absorptions of 43μM BPIB/laser aqueous and BPIB/DMSO colloidal solution just after preparation by dilution (day 0) are set as 1. (B) Top, Cartilage explants cultured in BPIB/laser aqueous medium and BPIB/DMSO colloidal medium on day 1, day 7 and day 11. Bottom, Growth of cartilage explants for 6 days (left graph) and 10 days (right graph) (n=6). P values were determined by the Welch’s t-test. Error bars show means±95% CIs.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions