Tenascin C Deletion Impairs Tendon Healing and Functional Recovery After Rotator Cuff Repair

The biological factors that affect healing after rotator cuff repair (RCR) are not well 63 understood. Genetic variants in the extracellular matrix protein Tenascin C (TNC) are associated 64 with impaired tendon healing and it is expressed in rotator cuff tendon tissue after injury, 65 suggesting it may have a role in the repair process. The purpose of the current study was to 66 determine the role of TNC on tendon healing after RCR in a murine model. The supraspinatus 67 tendon was transected and repaired on the left shoulder of Wild-Type (WT-RCR) and Tenascin 68 C null (Tnc - -RCR) mice. Controls included the unoperated, contralateral shoulder of WT-RCR 69 and Tnc - -RCR mice and unoperated shoulders from WT and Tnc - mice. We performed 70 histologic, activity testing, RNA-seq, and biomechanical analyses. At 8-weeks post-RCR, Tnc71 mice had severe bone and tendon defects following rotator cuff repair. Tnc- -RCR mice had 72 reduced activity after rotator cuff repair including reduced wheel rotations, wheel duration, and 73 wheel episode average velocity compared with WT-RCR. Loss of Tnc following RCR altered 74 gene expression in the shoulder, including upregulation of sex hormone and WNT pathways and 75 a downregulation of inflammation and cell cycle pathways. Tnc - mice had similar biomechanical 76 properties after repair as WT. Further research is required to evaluate tissue specific alterations 77 of Tnc, the interactions of Tnc and sex hormone and inflammation pathways as well as possible 78 adjuvants to improve enthesis healing in the setting of reduced TNC function. Overall design: RNA sequencing and enrichment analysis 148 WT-RCR (n=3) and Tnc - -RCR (n=4) were sacrificed at 2 weeks postoperative and used 149 for RNA-seq. RCR was performed on seven mice (WT-RCR (n=3) and Tnc - -RCR (n=4)) and Page 7 of 24 John Wiley & Sons, Inc. Journal of Orthopaedic Research For Peer Review 150 mice were euthanized 2 weeks post-RCR. Whole shoulder joints were dissected and total RNA 151 was isolated as previously described.11 RNA sequencing, quality control, and alignment was 152 performed as previously described.16 Fold-changes were calculated by comparing counts in Tnc153 -RCR samples relative to WT-RCR. Genes with an adjusted p < 0.05 were considered 154 differentially expressed. We used Ingenuity Pathway Analysis (Qiagen) software for functional 155 pathway analysis of the differentially expressed genes identified in this study. The log2 fold 156 change and adjusted p values using Bonferroni method from Deseq2 analysis were used to 157 calculate directionality (Z score) and set canonical pathway p-values (