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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).
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.
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