Murine Supraspinatus Tendon Detachment and Repair Model Augmented With Tendon-Derived, Activated Endothelial Cells: A New Concept in Biologic Enhancement of Tendon-to-Bone Healing

Abstract

Objectives:Biologic interventions are being increasingly used to improve outcome of rotator cuff repair. Individual growth factors and stem cell-based augmentations are among the most popular approaches. In this study we investigated a novel approach in which tendon-derived, activated endothelial cells (tAECs) are used as a source of tissue-specific growth factors to stimulate tendon intrinsic stem cell niche in our established murine model of microsurgical supraspinatus tendon (SST) detachment and repair.Methods:Study protocol was approved by the IACUC. In stage 1, 20 C57BL/6 mice underwent tendon harvest from fore- and hind limbs and endothelial cells isolation and transfection with adenoviral E4 ORF1 and red fluorescent protein labelling using a proprietary protocol. In stage 2, 158 C57BL/6 mice underwent the procedure with implantation of a 10μm fibrin glue bead (FGB) at the repair site and were randomly allocated to study group (SG) and control group (CG). Study mice received 100,000 tAECs suspended in the FGB. Control mice received the FGB only. 3 mice were sacrificed on post-operative day (POD) #3 and #7 for cell viability study. 96 mice were sacrificed at 1, 2, and 4 weeks, with 10 and 6 mice in each group and time point for biomechanical and histologic evaluations, respectively. 56 mice were sacrificed on POD 5, 10, 14, and 28 for gene expression analysis using qPCR, with 7 mice in each group and time point. Biomechanical evaluation consisted of determination of failure force and site. Histologic parameters were cell count (H&E), number of vessels (factor VIII-specific antibody), number of chondrocytes and proteoglycan content (Alcian Blue), and collagen organization (picrosirius red and polarized light microscopy). 12 evaluated genes were aggrecan (ACAN), collagen1α1 (COL1), collagen3α1 (COL3), MKX, MMP-3, 13, and 14, Runx2, scleraxis (Scx), SOX9, tenomodulin (TNM) and VEGFa.Results:Fluorescent microscopy revealed viable tAECs at the repair site on POD #3 with No fluorescent activity on POD #7. Failure force in the SG was 50% higher at 2 weeks (2.50 ± 0.55 N vs 1.86 ± 0.82 N, p = 0.012) with 65% of all failures occurring at SST mid-substance, whereas 90% of failures in the CG occurred at the repair site. There was no significant difference in failure force at 4 weeks. Cell count, number of vessels, and proteoglycan content (Figure 1) were significantly higher in the SG at all time points. The number of chondrocytes was higher in the SG at 1 week (Figure 1). Quantitative analysis showed significantly superior collagen organization in the SG (Figure 2). SCX, MKX, TNM and COL3 expression were up-regulated on POD #10 and decreased on day #28 in both groups. Expression of TNM and COL3 in the SG showed significance difference at 4 weeks. ACAN, SOX9, COL1, MMP14 expression exhibited an increase starting from POD 5, followed by a significant decline towards baseline by POD 28. In the SG, SOX9 peaked at day 14. Expression of VEGFa showed an uptrend at earlier time points (Figure 3).Conclusion:tAECs increased repair strength of SST. Histologic findings were suggestive of a more vigorous cellular and vascular response, higher proteoglycan content, and ultimately, superior collagen organization. A commensurate pattern was observed in expression of relevant genes. This approach opens a new avenue for cell-based biologic augmentation of SST repair with the goal of stimulating the intrinsic stem cell niche in tendon, as opposed to incorporating exogenous cell sources.