The effect of mesenchymal stem cell-derived exosomes and 3D hyaluronan-based scaffold on tendon graft healing in the bone tunnel: An experimental study

Objective: This study aimed to evaluate the effect of a biocompatible, degradable, high-mechanical property three-dimensional hyaluronan-based scaffold that allows mesenchymal stem cell adhesion and proliferation, and exosomes derived from mesenchymal stem cells, on the healing of tendon grafts within the bone tunnel. Another objective was to create an enthesis unit with physiological layers. Materials and Methods: This prospective study was conducted at the Selçuk University Experimental Medical Application and Research Center (SUDAM) Animal Experiments Ethics Committee with approval dated 26.07.2024 and numbered 2024-53. Animal experiments were performed at the Selçuk University Experimental Medical Application and Research Center. A total of 4 groups were formed. Group 1 underwent surgery only, Group 2 received mesenchymal stem cell (MSC)-derived exosomes in addition to surgery, Group 3 received a 3D hyaluronan-based polycaprolactone (PCL) scaffold in addition to surgery, and Group 4 received a scaffold implanted with exosomes. Half of the samples were harvested at the 4th week and the other half at the 8th week. Histological, immunohistochemical, and biomechanical evaluations were performed. Results: A total of 64 experimental animals were used in the study. The animals were divided into four equal groups (16 rats per group, n=32): Group 1 (Control), Group 2 (Exosome), Group 3 (Scaffold), and Group 4 (Exosome and Scaffold). When the effect of the applications on vascularity was examined in the 4th-week samples, the Exosome and Scaffold group showed a significant superiority compared to the Control group (p<0.05), while in the 8th-week samples, the Exosome group and the Exosome and Scaffold group were superior to the Control group (p<0.05). When the effect of the applications on cellular activity was examined in the 4th and 8th-week samples, a significant increase was found in Group 2 and Group 4 compared to Group 1 (p<0.05). Similarly, when the results of both periods regarding the effect on collagen fibril continuity, parallelism, and density were examined, a statistically significant superiority was found in the Exosome group and the Exosome and Scaffold group compared to the Control group that underwent surgery only. In immunohistochemical examinations, the superiority of Group 2 and Group 4 over the Control group (Group 1) was found to be significant in terms of FGF-2 (fibroblast growth factor 2) and BMP-2 (bone morphogenetic protein 2) staining in the 4th and 8th-week samples (p<0.05). In addition, the Exosome and Scaffold group showed a significantly higher level of FGF-2 and BMP-2 staining in both periods compared to the Scaffold group, which received only the scaffold in addition to surgery. In the 8th-week samples, a significant increase in BMP-2 staining was detected in the Scaffold group compared to the Control group. According to the ultimate tensile strength results at the 4th and 8th weeks in terms of biomechanical evaluation, the Group 2, Group 3, and Group 4 samples, which received additional applications to surgery, showed a significant superiority over the Control group that underwent surgery only (p<0.05). In the evaluation among the superior groups, the Exosome and Scaffold group showed a significant increase compared to Group 2 (exosome only) and Group 3 (scaffold only). Conclusion: This study demonstrates that mesenchymal stem cell-derived exosomes implanted on 3D hyaluronic acid-based PCL and TCP scaffolds significantly improve tendon graft healing in a rat tendon-bone tunnel model. The findings suggest that this treatment combination contributes to enthesis region healing and potentially offers an effective strategy in tendon-bone healing. Keywords: Tendon-bone junction, exosomes, hyaluronan-based scaffold