2015 ISAKOS Biennial Congress ePoster #317
Influence of Advanced Glycation End Products on Rotator Cuff Derived Cells
Yutaka Mifune, MD, PhD, Kobe JAPAN
Takeshi Kokubu, MD, PhD, Kobe JAPAN
Atsuyuki Inui, MD, PhD, Kobe JAPAN
Tomoyuki Muto, MD, PhD, Kobe JAPAN
Yoshifumi Harada, MD, Kobe JAPAN
Fumiaki Takase, MD, Kobe JAPAN
Yasuhiro Ueda, MD, Kobe JAPAN
Masahiro Kurosaka, MD, Kobe, Hyogo JAPAN
Kobe University Graduate School of Medicine, Kobe, JAPAN
FDA Status Not Applicable
Summary: Advanced glycation end products, which is known as one of the notable factors for senescence, had detrimental effects on rotator cuff derived cells and tissues in the in vitro and ex vivo situation.
Rotator cuff tears could occur with acute injury, but most are the result of age-related degenerative changes. However, there is no report to prove detailed mechanisms of age-related degenerative changes. Recently, advanced glycation end-products (AGEs) have been regarded as one of the notable factors for senescence. Therefore, we hypothesized that AGEs have detrimental effects on rotator cuff derived cells. In the present study, we investigated the influence of AGEs on rotator cuff derived cells in the in vitro and ex vivo situation.
Materials And Methods
Rotator cuff derived cells were obtained from torn edges of human supraspinatus tendons. The cells were cultured in each treated medium; 1) regular medium supplemented with 500 µg/ml AGEs (High AGEs group), 2) regular medium supplemented with 100 µg/ml AGEs (Low AGEs group), and 3) regular medium alone (control group). Cell viability was evaluated by WST assay 3 days after cultivation. We also measured the amount of VEGF that was secreted into the supernatant by ELISA. To detect HIF-1a protein expression, immunofluorescence stain of HIF-1a was performed 24 hours after cultivation. Reactive oxygen species (ROS) are also known to be critical for senescence, therefore the expression of ROS was assessed by immunofluorescence stain 3 days after cultivation. To evaluate the effect of AGEs on cell apoptosis, immunofluorescence staining was performed 3 days after cultivation using the APO-DIRECT Kit. An ex vivo tissue culture was performed to confirm the effect of AGEs on rotator cuff tissue. The scapula-humerus complexes were harvested from SD rats, and then cultured in each treated medium for 7 days; 1) regular medium supplemented with 500 µg/ml AGEs (AGEs group), and 2) regular medium alone (control group). For biomechanical test, the prepared scapula-humerus complex was mounted in a conventional tensile tester. Comparisons among multiple groups were made using analysis of variance (ANOVA) test followed by the Turkey's test. A value of p<0.05 was considered to be statistically significant.
Cell viability in High AGEs group was significantly suppressed when compared with the control group, while there was no significant difference between Low AGEs group and the control group. The estimated concentration of VEGF in the supernatant from cultured cells was significantly greater in the High and Low AGEs groups compared with that measured in the control group. Immunofluorescence staining demonstrated enhancement of HIF-1a and ROS expressions in High and Low AGEs groups when compared to the control group. Immunofluorescence staining for apoptotic cell displayed induction of cell apoptosis in High and Low AGEs groups when compared to the control group. In the ex vivo mechanical testing, the control group showed significant higher tensile strength than the AGEs groups
This study demonstrated that AGEs had detrimental effects on rotator cuff derived cells and tissues. These results indicated that AGEs could cause age-related degenerative changes of rotator cuff, and the reduction of AGEs might prevent rotator cuff from degenerating by senescence.