1. Bone morphogenetic protein 2/SMAD signalling in human ligamentocytes of degenerated and aged anterior cruciate ligaments 
K. Ruschke, C. Meier, M. Ullah, A.-C. Krebs, K. Silberreis, B. Kohl, P. Knaus, M. Jagielski, S. Arens, G. Schulze-Tanzil 
Osteoarthritis and Cartilage 
Volume 24, Issue 10, Pages (October 2016)
DOI: /j.joca
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Representative histological images of a nearly unchanged (A1–D1) and heavily degenerated (A2, A3, C2, D2 and D3) samples and surrounding synovial sheaths. Samples were stained with HE (A1–B3), AB (C1,3) or van Kossa (D1,3). Altered cell morphology and distribution, disintegration of the extracellular matrix, inflammation of the synovial layer and increased sGAGs (blue) and calcium deposition (black) were detectable. Arrows indicate calcium deposits. Scale bars: 200 μm.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Correlations between histopathological score systems. (A) Synovitis and degeneration scores revealed a significant correlation indicating that a high grade of degeneration increases the development of a synovitis in the knee. (B) Calcium deposition measured by the van Kossa score and ACL degeneration did not show a significant correlation. Spearman's non parametric correlation coefficient is shown. Degeneration and van Kossa score: n = 45. Synovitis score: n = 20.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
The BMP2 responsive BMP receptors ALK3, ALK6 and BMPRII are expressed in human ACL cells. Serum starved and unstimulated ACL cells were analyzed for the gene expression of ALK3 (A), ALK6 (B) and BMPRII (C) by qPCR. Mean values of the MNE ± 95% confidence intervals (n = 20) are shown. Samples were normalized to the reference gene HPRT.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Western blot and densitometric evaluation of ACL cells of two representative donors indicating either BMP2 responsiveness or BMP2 non-responsiveness with respect to SMAD1/5/8 phosphorylation. (A) Quantification of western blot analysis depicting phosphoprotein levels normalized to β-actin. Serum starved ACL fibroblasts were stimulated for 5, 10, 15, 30 and 60 min with 5 nM BMP2. The non-responsive donor (black bars) did not respond to BMP2 stimulations but showed higher basal P-SMAD1/5/8 level. The responsive donor (gray bars) showed high activation of SMAD1/5/8 compared to unstimulated controls. (B) Corresponding western blots for P-SMAD1/5/8 and β-actin of one non-responder (non-resp.) and one responder (resp.), which were used for quantifications are depicted.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
Differential ID1 gene expression in human ACL cells of patients with OA or high basal P-SMAD1/5/8 level. Serum starved ACL fibroblasts were stimulated for 1, 2 and 4 h with 5 nM BMP2 under serum-starved conditions. BMP2 stimulation of ACL fibroblasts resulted in a significant increase in the early target gene ID1 at each time point during the stimulation (indicated with a and b for significant BMP2 induced expression increase in all groups compared to unstimulated controls). (A) The OA group (n = 16) showed significantly less basal ID1 expression in the samples which remained unstimulated for 1 and 4 h compared to the cells derived from non-OA samples (n = 4) (B). In ACL cells showing high basal activation levels of SMAD1/5/8 by phosphorylation (n = 3), the ID1 gene expression was significantly increased in the cells which remained unstimulated for 1 and 2 h. Mean values of the MNE ± 95% confidence intervals (n = 20) are shown. Samples were normalized to the reference gene HPRT. a: P ≤ 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
Relative gene expression of BMP6 and BMP4 in human ACL fibroblasts in response to BMP2 treatment. Gene expression of BMP6 in serum starved ACL cells was not altered by stimulation with 5 nM BMP2 over the whole time kinetics of 1, 2 or 4 h. (A) Gene expression of BMP6 was significantly higher (except for the samples that remained unstimulated for 4 h) in ACLs of older patients (n = 10) compared to younger individuals (n = 10). (B) Gene expression of BMP6 was increased in unstimulated and stimulated ACLs of non-responders (n = 4) compared to responders (n = 16) on the level of phosphorylation of SMAD1/5/8. (C) Gene expression of BMP4 was slightly decreased by BMP2 stimulation in all patients (n = 20). Mean values of the MNE ± 95% confidence intervals (n = 20) are shown. Samples were normalized to the reference gene HPRT. a: P ≤ 0.05; b: P ≤ 0.01.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

8. Fig. 7
Relative gene expression of inhibitory factors of the BMP pathway. Serum starved ACL fibroblasts were stimulated for 1, 2 and 4 h with 5 nM BMP2 under serum-free conditions. (A) BMP2 stimulation seemed to increase the expression of the inhibitory SMAD protein SMAD6 in OA-derived fibroblasts and P-SMAD1/5/8 responders. The expression of SMAD6 was reduced in the OA group (n = 16) with significant difference in the 4 h BMP2 stimulation time point. (B) In ACL samples with high basal SMAD1/5/8 phosphorylation status (n = 3) the gene expression levels of SMAD6 were significantly increased in the 1 and 2 h stimulation controls. (C) Gene expression of the extracellularly acting BMP2 antagonist noggin was clearly and early induced by BMP2 stimulations but showed no differential expression in different groups (n = 20). Mean values of the MNE ± 95% confidence intervals are shown. Samples were normalized to the reference gene HPRT. a: P ≤ 0.05; b: P ≤ 0.01.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

9. Fig. 8
Gene expression of tenogenic, but not chondrogenic markers are upregulated by BMP2 stimulation. Serum starved ACL fibroblasts were stimulated for 1, 2 and 4 h with 5 nM BMP2 under serum-free conditions. (A) BMP2 stimulation upregulated the expression of SCX, a marker for tendons and ligaments in younger (n = 10) and older (n = 10) patients and showed a general higher gene expression in older patients. (B) The chondrogenic transcription factor SOX9 is not regulated by BMP2 stimulations but shows higher expression in the non-responder group (n = 4) compared to responders (n = 16). (C) Since SOX9 has essential roles also in the endochondral bone formation the expression of SOX9 in our unstimulated 1 h samples correlated significantly with the appearance of calcium deposits in the ACL tissues quantified by scoring of van Kossa stainings. Mean values of the MNE ± 95% confidence intervals are shown. Samples were normalized to the reference gene HPRT. a: P ≤ 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions