Mesenchymal Stem Cell Exosome and Fibrin Sealant Composite Enhances Rabbit Anterior Cruciate Ligament Repair

Abstract

Background: The anterior cruciate ligament (ACL) fails to heal after rupture, leading to joint instability and an increased risk of osteoarthritis. Mesenchymal stem/stromal cell (MSC) exosomes have reported wide-ranging therapeutic efficacy; however, their potential for augmenting ACL repair remains to be investigated.

Purpose: To evaluate the use of MSC exosomes with fibrin sealant on biological augmentation of ACL healing after suture repair and their effects on ACL fibroblast functions.

Study design: Controlled laboratory study.

Methods: Twelve rabbit knees underwent ACL transection and suture repair. MSC exosome and fibrin composite (Exosome+Fibrin) or fibrin (Fibrin) alone was used to supplement the suture repair in 6 knees. ACL repair was assessed by magnetic resonance imaging at 6 and 12 weeks postoperatively and by histologic and immunohistochemical analyses at 12 weeks. To investigate the mechanisms through which MSC exosomes augment ACL repair, metabolic activity, proliferation, migration, and matrix synthesis assays were performed using the primary ACL fibroblasts. RNA sequencing was also performed to assess global gene expression changes in exosome-treated ACL fibroblasts.

Results: Based on magnetic resonance imaging findings, 5 of 6 Exosome+Fibrin-treated ACLs were completely or partially healed, as opposed to 5 of 6 Fibrin-treated ACLs appearing torn at 6 and 12 weeks postoperatively. Additionally, 4 of 6 Exosome+Fibrin-treated ACLs were isointense, as compared with 5 of 6 Fibrin-treated ACLs that were hyperintense, indicating improved remodeling and maturation of the repaired ACLs with Exosome+Fibrin treatment. Histologically, Exosome+Fibrin-treated ACLs showed more organized collagen fibers and abundant collagen deposition, with a high amount of collagen I and relatively lower amount of collagen III, which are consistent with the matrix structure and composition of the normal ACL. Cell culture studies using ACL fibroblasts showed that MSC exosomes enhanced proliferation, migration, and collagen synthesis and deposition, which are cellular processes relevant to ACL repair. Further gene set enrichment analysis revealed key pathways mediated by MSC exosomes in enhancing proliferation and migration while reducing matrix degradation of ACL fibroblasts.

Conclusion: The combination of MSC exosomes and fibrin sealant (Exosome+Fibrin) applied to a suture repair enhanced the morphologic and histologic properties of the ACL in a rabbit model, and these improvements could be attributed to the augmented functions of ACL fibroblasts with exosome treatment.

Clinical relevance: This work supports the use of MSC exosomes in biological augmentation of ACL healing after suture repair.

Keywords: anterior cruciate ligament; exosomes; extracellular vesicles; mesenchymal stem/stromal cells; repair.

Figure 1.

Surgical procedure and study design. (A) Exposure of the intercondylar notch and transection of the ACL at the midsubstance. Approximation of the transected ACL ends and primary suture repair. (B) The rabbits were randomly allocated into 2 groups: ACL repairs treated with MSC exosomes and fibrin sealant (Exosome+Fibrin; n = 6) and ACL repairs treated with fibrin sealant (Fibrin; n = 6). MRI was performed at 6 and 12 weeks, and histologic and immunohistochemical analyses were performed at 12 weeks postoperatively. The sample size for the number of joints is indicated. ACL, anterior cruciate ligament; MRI, magnetic resonance imaging; MSC, mesenchymal stem/stromal cell.

Figure 2.

Schematic illustration of the surgical procedure and application of the Exosome+Fibrin composite. (A) Intact ACL preincision. (B) Incision made with surgical blade. (C) First suture placement. (D) Second suture placed after the first suture was pulled to minimize the gap. (E) First suture secured with surgeon's knot. (F) Second suture secured with surgeon's knot. (G) Excess suture trimmed for clean closure and (H) MSC exosomes applied with fibrin sealant for enhanced healing. ACL, anterior cruciate ligament; MSC, mesenchymal stem/stromal cell.

Figure 3.

3T MRI analysis of ACL repair. (A, B) 3T MRI of the rabbit knees was performed at 6 and 12 weeks postoperatively. Representative images of the ACLs achieving the best, median, and worst repair (n = 6). Completely healed ACL, gray arrow; partially healed, white arrow; torn, black arrow. ACL, anterior cruciate ligament; MRI, magnetic resonance imaging.

Figure 4.

7T MRI analysis of ACL repair. 7T MRI of the rabbit knees was performed at 12 weeks postoperatively. Representative images of the ACLs presented at the sagittal view show the best, median, and worst repair (n = 6). Isointense ACL, white arrow; hyperintense, black arrow. ACL, anterior cruciate ligament; MRI, magnetic resonance imaging.

Figure 5.

Histologic assessment of ACL repair. (A, B) Histologic analysis by hematoxylin and eosin (HE) and Masson trichrome (MT) staining. Representative images of the ACLs achieving the best, median, and worst ACL repair (n = 6). Completely healed ACL, green arrowhead; partially healed, yellow arrowhead; torn, red arrowhead. Scale bar: 2 mm. ACL, anterior cruciate ligament.

Figure 6.

Ligament-specific matrix expression of ACL repair. (A, B) Immunohistochemical staining for collagen I and collagen III. Representative images of the ACLs achieving the best, median, and worst ACL repair (n = 6). Scale bar: 2 mm. ACL, anterior cruciate ligament.

Figure 7.

Effects of MSC exosomes on functions of ACL fibroblasts. (A) MTS metabolic assay (left) and DNA assay (right) show potent dose- and time-dependent effects of MSC exosomes on metabolic activity and proliferation of ACL fibroblasts. (B) Transwell migration assay demonstrates dose-dependent effect of MSC exosomes on migration of ACL fibroblasts. (C) Quantification of total collagen deposited and soluble collagen released into the culture supernatants by ACL fibroblasts indicates that MSC exosomes significantly enhance collagen synthesis and deposition while reducing the loss of collagen into the culture media. Data are presented as mean ± SD. Compared with the PBS group within the same time point (n = 4): *P < .05. **P < .01. ***P < .001. Compared with 4 hours within the same treatment group (n = 4): ^#P < .05. ^##P < .01. ^###P < .001. Scale bar: 200 µm. ACL, anterior cruciate ligament; HPF, high-power field; MSC, mesenchymal stem/stromal cell; PBS, phosphate-buffered saline.

Figure 8.

Gene set enrichment analysis of primary ACL fibroblasts, comparing MSC exosome and PBS treatment groups. (A) KEGG pathway analysis highlights key pathways for cell proliferation, migration, and matrix degradation, with positive scores for events such as cell cycle and DNA replication and negative scores for pathways including focal adhesion and glycosaminoglycan degradation. (B) Reactome analysis reveals the pivotal roles of cell cycle progression, including the various mitotic phases. (C) Hallmark analysis demonstrates upregulation of signaling pathways such as mTORC1 signaling, which plays an important role in regulating cell growth, proliferation, and extracellular matrix biosynthesis. n = 3 per group. ACL, anterior cruciate ligament; KEGG, Kyoto Encyclopaedia of Genes and Genomes; MSC, mesenchymal stem/stromal cell; mTORC1, mammalian target of rapamycin complex 1; PBS, phosphate-buffered saline.