Modulating Inflammation During Tendon-To-Bone Healing Via IKKβ Inhibition

• Format: Other/Unknown Material
• Language: English
• Published: Morressier 2017
• Subjects: SCAR
• Online Access: https://openresearchlibrary.org/viewer/b205b01d-272d-44c3-9c1a-3d508e14d913; https://openresearchlibrary.org/ext/api/media/b205b01d-272d-44c3-9c1a-3d508e14d913/assets/external_content.pdf; https://doi.org/10.26226/morressier.5c0a5c97f0ad58000b08a25b

INTRODUCTION: More than 250,000 rotator cuff repairs are performed annually, yet successful healing of tendon-to-bone attachment is rarely achieved [1]. Although low levels of inflammatory cytokines are important in initiating the repair process, excessive inflammation, commonly seen in adult tendinopathy, impairs healing outcomes [2]. The nuclear factor u03baB (NF-u03baB) signaling pathway has been implicated in the early phases of tendon degeneration, and dramatic up regulation of this inflammatory pathway is seen during tendon healing [3,4]. Specifically inhibiting the inflammatory IKKu03b2-dependent mechanisms of NF-u03baB signaling, rather than complete inhibition of the pathway, following repair may improve clinical outcomes. Therefore, the objective of this study was to evaluate the therapeutic potential of IKKu03b2 inhibition for managing the excessive inflammation that persists after rotator cuff repair and thereby enhance tendon-to-bone healing.METHODS: Patient biopsy cell culture: Human study protocol were approved by the Ethics Committee under ACA No.99/101. Full informed consent was obtained from all patients. Primary tendon cells were isolated from hamstring tendons (n=3, ages:22-44y) taken from patients undergoing anterior cruciate ligament autograft surgery. Cells were isolated, passed to P2, plated, expanded for 48h, and treated with 1 ng Interleukin-1u03b2, 50M IKKu03b2 Inhibitor, or both for 4 hours, and compared to vehicle-treated controls. The supernatant was collected to evaluate inflammatory cytokine concentration. One-way ANOVA with Dunnetu2019s post-hoc analysis was performed to compare cytokine production. Rotator cuff animal model: A total of 52 adult Sprague-Dawley rats were used, as approved by the Columbia University Institutional Animal Care and Use Committee. Bilateral surgical supraspinatus tendon injury-and-repair was performed, as previously described [5]. 42 rats were injured and repaired and 10 rats were used as healthy normal controls (Normal group). Surgical animals were either left untreated (CTRL group) or administered IKKu03b2 small molecule inhibitor (ACHP Hydrochloride) dissolved in 0.5 % sodium carboxymethyl cellulose (Sigma). The IKKu03b2 inhibitor was delivered orally (5 mg/kg) once a day for 7 days after surgery (ACHP group). Following acute injury and repair, all animals were allowed free cage activity and sacrificed after 3 days, 14 days, or 28 days of healing. Gene expression outcomes for the tendon-to-bone attachment site were evaluated at all time points. A subset of samples from each time point were processed for histology to evaluate healing at the repair site. Two-way ANOVAs (for group and time) were performed to compare gene expression levels. p < 0.05 was considered significant. All statistical analyses were performed using GraphPad Prism 7.RESULTS: Treating human tendon cells with IL-1u03b2 significantly increased expression of NF-u03baB target genes IL6 and CCL2. Pharmacological inhibition of IKKu03b2 abrogated these effects in vitro, with reduced gene expression and cytokine production (Fig. 1). In vivo, ACHP treatment dramatically reduced NF-u03baB target genes CCL2, IL6, and COX2 expression over 80% compared to the non-treated group at day 3. These genes were also suppressed at day 14 and day 28 in the ACHP group. Accordingly, MMP3 and MMP13 expression were also dramatically reduced in the presence of ACHP at day 3. The tenogenesis gene SCX was significantly increased at day 14 with ACHP treatment (Fig.2 n=5/group). Consistently, pentachrome staining revealed an increase in collagen content (yellow) at the repair site following ACHP treatment at days 3 and 14 compared to the non-treated group (Fig. 3, n=5/group). DISCUSSION: Inhibition of IKKu03b2 successfully attenuated cytokine and chemokine production in purified tendon fibroblasts subjected to pro-inflammatory stimulus in vitro. The result demonstrated the potential for this inhibitor to reduce inflammation during tendon-to-bone healing in vivo. Similar to published tendon injury and healing models [2,6], the expression of pro-inflammatory genes rose dramatically in rats three days post injury and repair. Oral treatment with IKKu03b2 inhibitor led to significant reduction in the expression of inflammation-related NF-u03baB pathway target genes compared to untreated control. Furthermore, the ACHP-treated group showed enhanced tenogenesis and extracellular matrix (i.e., collagen) production. High levels of pro-inflammatory cytokines during the early inflammatory stages of healing lead to scar formation and impaired healing [2]. Pharmacological inhibition of IKKu03b2 prevented the excessive inflammation that occurs at the early crucial stages of healing and prompted remodeling. In summary, this study showed that suppression of inflammatory NF-u03baB pathway though molecular targeting of IKKu03b2 may enhance outcomes after rotator cuff repair. Future work will examine mechanical and bone morphometric outcomes with inhibitor treatment at longer timepoints. SIGNIFICANCE: The study demonstrated the potential of an IKKu03b2 inhibitor treatment to modulate excessive inflammation and enhance tendon-to-bone attachment healing after repair.