Author(s): PAULUSSEN, K., GRIFFIN, T., BAAR, K. , Institution: UNIVERSITY OF CALIFORNIA, DAVIS, Country: UNITED STATES, Abstract-ID: 2533

INTRODUCTION:
Exercise induces systemic changes that impact tissue repair and adaptation. One potential mechanism is through the production and release of exosomes (EXO). This study aimed to develop an in vitro screen for testing human serum-derived exosomes on human engineered ligaments (EHL).
METHODS:
EHLs were cultured in vitro, with constructs receiving media containing either complete FBS (CON, n=8) or exosomes isolated from FBS (fbEXO) for the first two experiments to establish an isolation and feeding protocol. EHLs were engineered from human ACL cells and treated with normal growth media until day 8, after which fbEXO were added to reflect 10, 20, or 40% (0.2, 0.4, or 0.8 ml FBS/ml media). Once the optimal percent EXO was established, EHLs were treated with fbEXO for 1, 2, or 3 feeds. All constructs were collected and subjected to tensile testing on day 14. A hydroxyproline assay was run to determine collagen content. Once an optimized protocol was determined for fbEXO, the process was repeated with humEXO. Differences were analyzed using a 1-way ANOVA or student’s unpaired t-test. Significance was set at P<0.05 and all data are presented as mean±SD.
RESULTS:
Increasing the amount of fbEXO within the media resulted in a dose-dependent increase in EHL mechanics (10%: 0.280±0.178 N, 20%: 0.328±198 N, 40%: 0.483±0.365 N; r2=0.858, P=0.077) and collagen content (10%: 100.3±58.17 µg, 20%: 147.9±50.79 µg, 40%: 160.7±32.58 µg; r2:0.211). Incubating EHLs with fbEXO decreased mechanics (1 feed: 0.714±285 N, 2 feeds: 0.809±0.224 N, 3 feeds: 0.318±0.180 N) and collagen (1 feed: 394.30±26.38 µg, 2 feeds: 301.00±69.14 µg, 3 feeds: 150.50±28.25 µg) in a time dependent manner, but at 4 days (2 feeds) there was not a statistical difference between serum and fbEXO (P=0.450). Treating EHLs with 40% humEXO for 4 days (2 feeds) did not significantly decrease maximum tensile load (0.683±0.284 vs. 0.785±0.314 N, P=0.523), failure stress (1.366±0.433 vs. 1.658±0.431 MPa, P=0.214), Young’s Modulus (16.460±3.687 vs. 20.340±4.098 MPa, P=0.077), CSA (0.4914±0.073 vs. 0.466±0.111 mm2, P=0.617) or collagen content (568.7±56.5 vs. 628.8±195.8 µg, P=0.450) compared with CON fed EHLs.
CONCLUSION:
Human serum-derived exosomes support EHL development to the same extent as FBS. These data indicate that serum-derived exosomes modulate tissue development independent of other serum factors. The model also opens new avenues for determining the role of exosomes in muscle-tendon crosstalk in long-term health and training adaptations.