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51 Insights Into Bmp-7 Signalling Of Rotator Cuff Tenocytes
  1. Franka Klatte-Schulz1,2,
  2. Gerry Giese1,2,
  3. Karen Ruschke3,
  4. Regina Puts1,2,
  5. Petra Knaus2,3,
  6. Britt Wildemann1,2
  1. 1Julius Wolff Institut, Charité-Universitaetsmedizin Berlin
  2. 2Berlin-Brandenburger Center for Regenerative Therapies, Charité-Universitaetsmedizin Berlin
  3. 3Institute for Chemistry and Biochemistry, Free University Berlin, Germany

Abstract

Introduction We reported previously that important cellular characteristics such as the cell activity and the expression and synthesis of collagen I can be strongly enhanced by stimulation of tenocytes with BMP-7 [Klatte-Schulz et al. 2014]. Since tendon healing after surgical reconstructions still represents a challenging topic, due to complications such as retears or non-healing of the tendon tissue, the augmentation of the healing using growth factors like BMP-7 may be a possible treatment option. What remains unknown is how BMP-7 signals in tenocytes, which was analysed in the present study.

Methods Tenocytes of 6 male donors (63–69 years) isolated from biopsies of supraspinatus tendon tears were seeded in cell culture dishes, cultured for 3–5 days and stimulated with 200 or 1000 ng/ml rhBMP-7. Protein lysate samples were collected after 15, 30, 60 and 120 min and the phosphorylation of Smad1/5/8 was analysed using Western Blotting technique. RNA was isolated after 1, 2, 4 and 8 h from the cells. The gene expression of BMP-Receptors (BMPR-Ia, BMPR-Ib, BMPRII), Activin receptors (ActR-I, ActR-IIa, ActR-IIb), Smad 1, 4, 5, 8 and the BMP target gene ID1, as well as scleraxis, osteocalcin and collagen II was analysed by Real-Time PCR. Statistics: Mann-Whitney-U Test, p ≤ 0.05, Bonferroni Holm Correction

Results The relative phosphorylation level of Smad1/5/8 was significantly increased 30 and 60 min after stimulation with BMP-7 compared to unstimulated control cells (Figure 1).

Abstract 51 Figure 1
Abstract 51 Figure 1

Smad phosphorylation relative to total Smad and GAPDH and normalized to unstimulated control

The expression of BMPR-Ib was increased the most compared to both other type I receptors 4 and 8 h after stimulation. In the type II receptor group, BMPR-II showed the highest increase after 2 to 8 h, while ActR-IIb was down-regulated after 2 h and 4 h. The expression of Smad1 and 4 was slightly increased and Smad5 showed no increase of expression. Smad8 expression was strongly up-regulated 4 h and 8 h after stimulation (Figure 2). The expression of ID1 was increased already at 1 h and 2 h and strongly enhanced 8 h after stimulation. Scleraxis expression showed a strong increase at 1, 2 and 4 h and a massive increase at 8 h after stimulation. No change in the osteocalcin and collagen II expression was found after BMP-7 stimulation.

Abstract 51 Figure 2
Abstract 51 Figure 2

Smad8 expression relative to 18 s and normalised to control.

Discussion The study showed that the type I receptor BMPR-Ib and the type II receptor BMPR-II seem to be the most regulated receptors on their mRNA level after tenocyte stimulation with BMP-7. Additionally, the receptor-regulated Smad8 seems to represent the essential signalling molecule in tenocytes after BMP-7 stimulation. An important role of Smad8 in tenogenic differentiation was reported previously. [Hoffmann et al., 2006] The strong increase of the tendon related gene scleraxis in the cells may underline the positive effect of BMP-7 treatment for rotator cuff repair.

References Klatte-Schulz, et al. J Orthop Res. 2014;32(1):129–37

Hoffmann, et al. J Clin Invest. 2006;116(4):940–52

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