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

Introduction: Biologic interventions are being increasingly used in modern orthopedic practice to improve anatomic and clinical outcome of rotator cuff tendon 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 the intrinsic stem cell niche in tendon in our established murine model of supraspinatus tendon (SST) detachment and repair. Method(s): Study protocol was approved by the IACUC. In stage 1, 20 C57BL/6 mice were euthanized and underwent tendon harvest from fore- and hind limbs bilaterally and endothelial cells isolation using a proprietary protocol with subsequent transfection with adenoviral E4 ORF1 and red fluorescent protein (RFP) labelling. In stage 2, 158 C57BL/6 mice underwent microsurgical SST detachment and repair with implantation of a 10mum fibrin glue (FG) bead at the repair site and were randomly allocated to study and control groups. Study mice received 100,000 tAECs suspended in the FG bead. Control mice received the FG bead only. Three mice were sacrificed on post-operative day (POD) #3 and #7 for cell viability study. Ninety six 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. Fifty six 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 of failure. Histologic parameters were cell count (H&E staining), number of vascular structures (factor VIII-specific antibody), number of chondrocytes and proteoglycan content (Alcian Blue staining), and collagen organization (picrosirius red staining and polarized light microscopy). 12 genes of interest were aggrecan (ACAN), collagen1alpha1 (COL1), collagen3alpha1 (COL3), Mohawk (MKX), metalloproteinases (MMP)-3, 13, 14, Runx2, scleraxis (Scx), SOX9, tenomodulin (TNM) and VEGFa (Vascular Endothelial Growth Factor A). Result(s): Fluorescent microscopy revealed viable tAECs at the repair site on POD #3. No fluorescent activity was detected on POD #7. Failure force in the study group was 50% higher than in the control group at the 2-week time point (2.50 +/- 0.55 N vs 1.86 +/- 0.82 N, p = 0.012). At this time point, 65% of all failures in the study group occurred at SST mid-substance, whereas 90% of failures in the control group occurred at the tendon-bone repair site. There was no significant difference between groups in failure force at the 4-week time point. Cell count, number of vascular structures, and proteoglycan content (Figure 1) were significantly higher in the study group at all time points. The number of chondrocytes was higher in the study group at the 1-week time point (Figure 1). Quantitative analysis showed significantly superior collagen organization in the study group (Figure 2). qRT-PCR analysis revealed trends, though statistical significance was not achieved for all comparisons. SCX, MKX, TNM and COL3 expression were up-regulated on POD #10, decreasing at day #28 in both groups. Expression of TNM and COL3 in the study group showed significance difference at 4-week time point. ACAN, SOX9, COL1, MMP14 expression exhibited an increase starting from POD 5, followed by a significant downtrend towards baseline by POD 28. In the study group, SOX9 peaked at day 14. Expression of VEGFa showed an uptrend at earlier time points (Figure 3). Discussion(s): tAECs increased repair strength in our murine model of SST detachment and repair. Histologic findings were suggestive of a more vigorous cellular and vascular response leading to higher proteoglycan content and ultimately superior collagen organization. A commensurate pattern was observed in expression of relevant genes. (Figure Presented).