Background:

Facilitating the healing process of injured anterior cruciate ligament (ACL) tissue is crucial for patients to safely return to sports. Stem cell derived exosomes have shown positive effects on enhancing the regeneration of injured tendons/ligaments. However, clinical application of exosomes in terms of storage and pre-assembly is challenging. We hypothesized that lyophilized exosomes derived from human umbilical cord stem cells (hUSC-EXs) could enhance the cell activity of chronically injured ACL cells.

Materials and Methods:

We harvested the 8 weeks injured ACL cells from rabbit under IACUC (No.110232) approval. The studied exosomes were purified from the culture medium of human umbilical cord stem cells (IRB approval No. A202205014), lyophilized to store, and hydrated for use. We compared exosome treated cells with non-exosome treated cells (control group) from the same rabbits. We examined the cell viability, proliferation, migration capability and gene expression of type I and III collagen, TGFβ , VEGF , and tenogenesis in the 8 weeks injured ACL cells after hUSC-EX treatment.

Results:

After hydration, the average size of hUSC-EXs was 85.2 nm, and the cells tested positive for the Alix, TSG101, CD9, CD63, and CD81 proteins but negative for the α-Tubulin protein. Compared with no exosome treatment, hUSC-EX treatment significantly improved the cell viability, proliferation and migration capability of 8 weeks injured ACL cells. In addition, the expression of collagen synthesis, TGFβ , VEGF , and tenogenesis gene were all significantly increased in the 8 weeks injured ACL cells after hUSC-EX delivery.

Discussion:

Lyophilized exosomes are easily stored and readily usable after hydration, thereby preserving their characteristic properties. Treatment with lyophilized hUSC-EXs improved the activity and gene expression of 8 weeks injured ACL cells.

Conclusion:

Lyophilized hUSC-EXs preserve the characteristics of exosomes and can improve chronically injured(8 weeks) ACL cells. Lyophilized hUSC-EXs could serve as effective and safe biomaterials that are ready to use at room temperature to enhance cell activity in patients with partial ACL tears and after remnant preservation ACL reconstruction.