Dense connective tissue healing, such as tendon, is protracted leading to highly variable and unsatisfactory patient outcomes. Biomarkers prognostic of long‐term clinical outcomes is, however, unknown. The present study was designed to investigate the proteomic profile of healing, identify potential biomarkers, and assess their association with the patient's long‐term outcomes after ATR. Quantitative mass spectrometry analysis demonstrated 1423 proteins in healing and contralateral healthy Achilles tendons of 28 ATR patients. Comparing healing at 2 weeks and healthy protein profiles, we identified 821 overlapping, 390 upregulated, and 17 downregulated proteins. Upregulated proteins are related mainly to extracellular matrix organization and metabolism, while downregulated pathways were associated with exocytosis in immune modulation and thrombosis formation. Further proteomic profiling in relation to validated patient outcomes revealed the downregulated pro‐inflammatory complement factor D (CFD) as the most reliable predictive biomarker of successful tendon healing. Our finding showed a comprehensive proteomic landscape and bioinformatics on human connective tissue, indicating subtype‐specific and shared biological processes and proteins in healing and healthy Achilles tendons, as well as in tendons related to good and poor patient outcomes. Inflammatory protein CFD and serpin family B member 1 were finally identified as potential predictive biomarkers of effective healing outcomes when combined the proteomic profiles with a validated clinical database. Following the future elucidation of the mechanisms associated with the identified biomarkers as predictors of good outcomes, our findings could lead to improved prognostic accuracy and development of targeted treatments, thus improving the long‐term healing outcomes for all patients.

Outcomes following human dense connective tissue (DCT) repair are often variable and suboptimal, resulting in compromised function and development of chronic painful degenerative diseases. Moreover, biomarkers and mechanisms that guide good clinical outcomes after DCT injuries are mostly unknown. Here, we characterize the proteomic landscape of DCT repair following human Achilles tendon rupture and its association with long-term patient-reported outcomes. Moreover, the potential regulatory mechanisms of relevant biomarkers were assessed partly by gene silencing experiments. A mass-spectrometry based proteomic approach quantified a large number (769) of proteins, including 51 differentially expressed proteins among 20 good versus 20 poor outcome patients. A novel biomarker, elongation factor-2 (eEF2) was identified as being strongly prognostic of the 1-year clinical outcome. Further bioinformatic and experimental investigation revealed that eEF2 positively regulated autophagy, cell proliferation and migration, as well as reduced cell death and apoptosis, leading to improved DCT repair and outcomes. Findings of eEF2 as novel prognostic biomarker could pave the way for new targeted treatments to improve healing outcomes after DCT injuries.Trial registration: NCT02318472 registered 17 December 2014 and NCT01317160 registered 17 March 2011, with URL http://clinicaltrials.gov/ct2/show/NCT02318472 and http://clinicaltrials.gov/ct2/show/study/NCT01317160.

The suboptimal or protracted regeneration of injured connective tissues often results in significant dysfunction, pain, and functional disability. Despite the prevalence of the condition, few studies have been conducted which focused on biomarkers or key molecules involved in processes governing healing outcomes. To gain insight into injured connective tissue repair, and using the Achilles tendon as a model system, we utilized quantitative proteomic and weighted co-expression network analysis of tissues acquired from Achilles tendon rupture (ATR) patients with different outcomes at 1-year postoperatively. Two modules were detected to be associated with prognosis. The initial analysis identified inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) as a biomarker or hub protein positively associated with better healing outcomes. Additional analysis identified the beneficial role of ITIH4 in inflammation, cell viability, apoptosis, proliferation, wound healing, and for the synthesis of type I collagen in cultured fibroblasts. Functionally, the effects of ITIH4 were found to be mediated by peroxisome proliferator-activated receptor gamma (PPARγ) signaling pathways. Taken together, these findings suggest that ITIH4 plays an important role in processes of connective tissue repair and advocate for the potential of ITIH4 as a therapeutic target for injured connective tissue repair.Trial registrationhttp://clinicaltrials.gov, identifiers NCT02318472, NCT01317160.

Outcomes following human dense connective tissue (DCT) repair are often variable and suboptimal, resulting in compromised function and development of chronic painful degenerative diseases. Moreover, biomarkers and mechanisms that guide good clinical outcomes after DCT injuries are mostly unknown. Here, we characterize the proteomic landscape of DCT repair following human tendon rupture and its association with long-term patient-reported outcome. Moreover, the regulatory mechanisms of relevant biomarkers were assessed partly by gene silencing experiments. A Mass-Spectrometry based proteomic approach quantified a large number (769) of proteins, including 51 differentially expressed proteins among 20 good versus 20 poor outcome patients. A novel biomarker, elongation factor-2 (eEF2) was identified as being strongly prognostic of the 1-year clinical outcome. Further bioinformatic and experimental investigation revealed that eEF2 positively regulated autophagy, cell proliferation and migration, as well as reduced cell death and apoptosis, leading to improved DCT repair and outcomes. Findings of eEF2 as novel prognostic biomarker could pave the way for new targeted treatments to improve healing outcomes after DCT injuries.