Summary

Mesenchymal stromal cell exosomes with fibrin sealant biologically enhance ACL primary repair, possibly by augmenting ACL cell functions.

Abstract

Background

The unacceptably high failure rates of primary repair in anterior cruciate ligament (ACL) tears have led to its widespread abandonment and the transition towards reconstruction. However, ACL reconstruction also has drawbacks and suboptimal outcomes continue to persist despite advancements in surgical techniques.

With the resurgence of interest in primary ACL repair, several approaches including the use of mesenchymal stem/stromal cells (MSCs) have been proposed. Although use of MSCs for tissue repair was first predicated on their differentiation potential, it is now accepted that these cells mediate tissue repair through secretion of trophic factors, particularly extracellular vesicles including exosomes.

We hypothesize that MSC exosomes delivered using a fibrin sealant can enhance primary ACL repair, and aim to investigate the efficacy of MSC exosomes for primary ACL repair in a rabbit model.

Method

ACL tears were surgically created in the unilateral knee joints of 12 rabbits through arthrotomies performed with a medial parapatellar approach. These rabbits were randomly allocated to 2 treatment groups (n=6 per group): (1) MSC exosomes and fibrin sealant (Exosome+FS), and (2) FS alone. 400µl containing either 200µg exosomes in 200µl phosphate-buffered saline (PBS) mixed with 200µl FS, or 200µl PBS mixed with 200µl FS were applied intraoperatively after ACL suture repair and allowed to solidify before closure.

Magnetic resonance imaging (MRI) was performed at 6 and 12 weeks post-operatively. At 12 weeks, animals were euthanized and samples were harvested for histology and immunohistochemistry. To assess the quality of ACL repair, immunohistochemistry for types I and III collagen was performed in addition to assessment of general morphology and degree of ligamentous integrity restoration.

Cell cultures utilizing rabbit ACL cells were performed to investigate the cellular processes mediated by MSC exosomes during ACL repair. Cell metabolic activity, total DNA content, cell migration in response to treatment, and amount of collagen deposition were measured at 4, 24, 48 and 72h post-treatment. Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) was performed to examine the genes associated with proliferation, migration, and matrix synthesis.

Results

5 out of 6 rabbits in the Exosome+FS group showed sustained ACL healing on MRI from 6 to 12 weeks, compared to only 1 of the 6 rabbits in the FS group at both timepoints. This finding in the Exosome+FS group correlated well with complete morphological restoration of ligamentous integrity and rich deposition of predominantly type I collagen, similar to the native ACL. In contrast, the FS group had mainly type III collagen with ligamentous integrity restored to some degree.

Using ACL cell cultures, we demonstrated that MSC exosomes dose-dependently enhanced cell metabolic activity (p=0.002), proliferation (p<0.001), and migration (p<0.001). Exosome treatment also suppressed ACL matrix degradation and enhanced collagen synthesis at 48h post-treatment (p=0.015). These findings were supported by increased gene expression associated with proliferation (PCNA, FGF-2; p<0.001), migration (FGF-2; p<0.001), and matrix synthesis (COL3A1, COL5A1, TGF-ß1; p<0.001, Tenascin C, Decorin; p<0.01) with exosome treatment compared to control.

Conclusion

MSC exosomes with fibrin sealant biologically enhance ACL primary repair, possibly by augmenting ACL cell functions.