1. Infrapatellar fat pad aggravates degeneration of acute traumatized cartilage: a possible role for interleukin-6 
J. He, Y. Jiang, P.G. Alexander, V. Ulici, Y. Zhu, S. Wu, R.S. Tuan 
Osteoarthritis and Cartilage 
Volume 25, Issue 1, Pages (January 2017)
DOI: /j.joca
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Early stage responses of articular cartilage after traumatic impact. (A) Cell viability at the surface of cartilage after 7 days of culture (live cells, green with calcein-AM; dead cells, red with ethidium homodimer-1). Decreased chondrocyte viability, and some spindle-shaped cells were detected in 36 MPa group. N = 3; Bar, 200 μm. (B) SEM view of cartilage surface revealed fissuring of the tissue matrix and destruction of the fibrous network after impact. N = 3; Bar, 1 μm. (C) Safranin-O staining (red, sulfated proteoglycans; green, loss of proteoglycan stain, suggesting tissue degeneration after impact). A reduction in matrix sulfated GAG in areas below the superficial zone of the articular cartilage was observed after impact. N = 6; Bar, 40 μm. (D) PGE2 levels in the medium indicate the release of PGE2 after impact (day 1, 2, 4, 7), with the highest release seen on day 2 (P = 0.0011). (E) GAG levels measured in the medium at different time points indicate the release of GAG after impact (day 1, 2, 4, 7) with the highest level on day 2 (P < 0.0001). For (E) and (D), three cartilage plugs were pooled as one sample, three samples were tested in each group, and the experiment was repeated three times. N = 9; biological donors = 27. Two-way ANOVA was used. The measurement is normalized to wet weight of cartilage. 0 MPa, non-impacted group; 36 MPa, impacted group. SEM, scanning electron microscopy; PGE2, prostaglandin E2; GAG, glycosaminoglycan.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Exposure to FP-CM enhances degeneration of traumatized cartilage. (A) Sagittal sections of central impacted region of the articular cartilage were stained with Safranin O (red, presence of sulfated GAG; green, loss of GAG and cartilaginous matrix degeneration). GAG loss in the 4 groups: IC+FP-CM >> (C+FP-CM = IC+BM) > C+BM, suggesting that a 2-day treatment of FP-CM aggravated degeneration of the traumatized cartilage, but had little effect on healthy cartilage. Arrows indicate cartilage surface. Bar, 200 μm. N = 3. (B) Assay of GAG in culture medium. GAG levels in the 4 groups: IC+FP-CM > IC+BM > (C+BM = C+FP-CM). Three cartilage plugs were pooled as one sample, three samples were tested in each group, and the experiment was repeated three times. N = 9; biological donors = 27. One-way ANOVA was used. The measurement is normalized to wet weight of cartilage. 0 MPa, non-impacted group; 36 MPa, impacted group. C+BM: non-impacted cartilage in basal medium; IC+BM: impacted cartilage in basal medium; C+FP-CM: non-impacted cartilage in IPFP-conditioned medium; IC+FP-CM: impacted cartilage in IPFP-conditioned medium.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
Effects of FP-CM on degeneration of traumatized cartilage, and comparison with the effects of IL-6. (A–C) q-PCR analysis of gene expression in bovine cartilage explants treated with BM or FP-CM after 2 days of culture. The results were calculated using the ΔΔCt method and normalized to 18S rRNA as the housekeeping gene. (D) ELISA analysis of IL-6 released in the medium by IPFP, and in cartilage explants cultures treated with BM or FP-CM. (E, F) Comparison between the effects of FP-CM and IL-6 on gene expression of (E) MMP13 and (F) COX2. three cartilage plugs were pooled as one sample, three samples were tested in each group, and all experiments were repeated three times with samples from different donors. N = 9; biological donor = 27. One-way ANOVA was used. C+BM, non-impacted cartilage in basal medium; IC+BM, impacted cartilage in basal medium; C+FP-CM, non-impacted cartilage in IPFP-conditioned medium; IC+FP-CM, impacted cartilage in IPFP-conditioned medium. COL2, type II collagen; AGG, aggrecan; ATS4, ADAMTS4; ATS5, ADAMTS5; COX2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; TIMP, tissue inhibitor of metalloproteinases; MMP, matrix metalloproteinases; IL, interleukin.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Effects of traumatized cartilage conditioned medium (TC-CM) on IPFP-derived adipocytes and stromal cells. (A) Experimental scheme of ceiling culture to isolate DFAT and ADSC. For details see Materials and methods. (B) Morphology of ADSC and DFAT at second passage examined by phase contrast microscopy. Bar: 10 μm. (C) Colony formation units (CFU) test of ADSC and DFAT at passage 2. Left: colonies were stained with Crystal Violet, a 3 mm diameter red dot shown at bottom right. Right: Significantly different CFU characteristics of bovine ADSC and DFAT, P =  (pooled cells from 5 IPFPs as one sample, three technical repeats were performed for each test, and the experiment was repeated five times). N = 5; biological donors = 25. Student's t test was used. (D) qRT-PCR analysis of gene expression in DFAT and ADSC cultures treated with TC-CM after 24 h. The results were calculated using the ΔΔCt method, with 18S rRNA as the housekeeping gene. Pooled cells from 5 IPFPs were used as one sample, three technical repeats were performed for each test, and the experiment was repeated three times. N = 3; biological donors = 15. One-way ANOVA was used. 0A, ADSC cultured with TC-CM0; 36A, ADSC cultured with TC-CM36; 0D, DFAT cultured with TC-CM0; 36D, DFAT cultured with TC-CM36. ADSC, adipose-derived stromal cells; DFAT, dedifferentiated fat cells; TC-CM0, 0 MPa traumatized (non-impacted) cartilage-conditioned medium; TC-CM36, 36 MPa traumatized cartilage-conditioned medium. IL, interleukin; FGF, fibroblast growth factor; LPL, lipoprotein lipase; PPAR, peroxisome proliferator-activated receptor.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

6. Supplementary Fig. 1
Histological observations of cartilage matrix degeneration between treatment with FP-CM and IL-6. Articular cartilage sections stained with Safranin O (red, presence of sulfated GAG; green, loss of GAG and cartilaginous matrix degeneration). GAG loss tendency in the 6 groups: 36 MPa IPFP-CM >> 36 MPa IL-6 > (0 MPa IL-6; 36 MPa Basal Medium; 0 MPa IPFP-CM) > 0 MPa Basal Medium, suggesting that IL-6 might not the only factor to aggravate degeneration of the traumatized cartilage, but it still play an important and might a negative role in cartilage degeneration. Arrows indicate cartilage surface. Bar, 200 μm. 0 MPa: C+BM: non-impacted cartilage; 36 MPa: impacted cartilage. IPFP-CM: IPFP-conditioned medium; IL-6: Interleukin-6 in basal medium, 200 pg/mL.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions