Discussion
ATR, inflammation, connective tissue injuries, acute ATR
PROLIFERATION, INFLAMMATION, PROLIFERATIVE
In this study, the proteomic composition of good versus poor ATR repair was quantitatively characterized during both the inflammatory and proliferative phases of healing in patients with acute ATR. By combining data from tissue biopsies representing the inflammatory healing stage from the first week after injury and microdialysate samples representing the proliferative healing phase obtained at 2 weeks after repair surgery, our previous and present findings identified CFD as a novel biomarker with strong predictive association with long-term clinical outcomes and potential role in improving tendon repair. Further bioinformatic analysis and mechanistic exploration demonstrated that CFD has a potential role in enhancing tissue healing by regulating Col1a1 synthesis to enhance fibroblast migration during DCT repair, using ATR repair as an example, is a complex process that exhibits partly overlapping healing stages, including inflammation, proliferation and remodeling (Thus, one of the main findings presented is that higher CFD levels during the inflammatory phase induce increased Col1a1 expression, which leads to an enhanced fibroblast migration, and thereby promotes long-term healing quality. Previously, it has been shown that FGF also can serve as a prognostic biomarker during the inflammatory phase (Although the exact pathways by which CFD regulates healing are not known, it has been reported that CFD, a zymogen, exists in two isoforms with potentially different functions (Whether the increased Col1a1 production, due to the regulation by CFD is the sole regulator of long-term clinical outcome in the patients is still unclear. The proteomic profile of the good- and poor outcome patients indicated a significant difference in the level of CFD during inflammation that would impact long-term outcome. The findings regarding the clinical impact of CFD and Col1a1 during inflammation mainly enhancing fibroblast migration creates a feedback loop for later healing stages. This observation is also strengthened by findings from earlier studies demonstrating that fibroblast production of collagen type I synthesis is mainly initiated during the proliferative- and remodeling healing phases (Thus, the current identification of time-based opposing effects of CFD in the regulation of Col1a1 production during Achilles tendon repair may provide a possible explanation regarding the long-term effects of CFD on connective tissue repair. Unfortunately, the exact timeline of these opposing effects of CFD on Col1a1 production during the healing process cannot be determined from the current experimental construct. However, the results from the present studies do demonstrate that opposing effects are initiated during the transitional phase between inflammation, at 1 week post-injury, and proliferation at 2 weeks post-injury.Fibroblast migration plays a vital role during tissue healing from the late inflammation stage by acting as a trigger of inflammation-proliferation transition (The earlier observations at 2 weeks post-injury during the proliferative phase of healing indicated that lower CFD expression was associated with improved long-term clinical patient outcome (Migration of fibroblasts to the site of injury plays an important role during wound healing. Based on the experimental observations, we speculate that the increased Col1a1 expression associated with CFD levels during inflammation and proliferation leads to improved fibroblast migration to promote ATR repair, subsequently leading to an enhanced healing outcome.A compromised healing process may lead to less strength of the repair tissue and limitations with regard to daily activities, which are important assessments during outcome evaluation in the clinic. An impaired fibroblast migration during the later proliferative healing phase may lead to less provisional matrix formation with Col1a1, which presumably leads to more limitations in daily life with less loading of the injured tissue, all causative factors for ATR patients having a prolonged healing time and an unsatisfactory patient-reported outcome (There are several limitations for the study design, one of the main study limitation is that the proteomic profile from the inflammatory healing phase consisted of tendon tissue biopsies (i.e. were tissue-associated), while the proteomic profile from the proliferative healing phase comprised of microdialysates (i.e. were soluble molecules) from the paratenon. This may limit the comparability of proteomic profiles from the two different healing stages. However, earlier studies have demonstrated that microdialysis of the paratenon, which is a biologically active part of the tendon, can capture the proteins of the tendon tissue that control cell proliferation during tendon healing (Taken together, the current findings further establish the potential of CFD to function as a prognostic biomarker of good human Achilles tendon healing. While currently specific to tendon healing, future studies may confirm CFD to be a generic biomarker prognostic of human dense connective tissue repair in general. The bioinformatic analysis and experimental investigation demonstrated that CFD likely promotes tissue repair by enhancing Col1a1 synthesis via in part, to increased fibroblast migration during both inflammation and proliferation. Among the 13 overlapping patients with both tissue biopsies and microdialysates, CFD is significantly higher in tendon tissues in good outcome patients while CFD microdialysate levels were significantly lower in good outcome patients. These findings follow the same pattern as observed and reported in the manuscript. Additionally, we checked the correlation between patient outcome and CFD expression among these 13 patients. We observed that CFD expression is positively associated with better patient outcome in tissues, but negatively associated with better clinical outcome in microdialysates. All these new findings matched our results which were already presented in the manuscript text, so we believe that these new findings strengthened our conclusions. But we also concur that more studies are needed. These findings may also be a highly relevant to the development of both individualized treatment protocols as well as new targeted treatments, which could enhance the long-term healing outcomes for patients suffering with dense connective tissue injuries that currently yield a poor clinical outcome.