Kartogenin Combined Platelet-Rich Plasma (PRP) Promoted Tendon-Bone Healing for Anterior Cruciate Ligament (ACL) Reconstruction by Suppressing Inflammatory Response Via targeting AKT/PI3K/NF-κB

Abstract

Anterior cruciate ligament (ACL) rupture is the most common sports injuries and PRP has the potential to be a kartogenin (KGN) carrier to promote collagen fibril organization and cartilage regenerative in the tendon-bone interface. This paper aimed to investigate co-injection of KGN-PRP into the bone tunnels of ACL reconstructions which could enhance tendon-bone healing graft osteointegration effectively. HPLC was used to measured release rate of KGN from KGN-PRP gel. Then, an ACL injury reconstruction model in rabbits was established and the rabbits received saline, PRP, and KGN-PRP injection onto the tendon-bone interface after reconstruction. The tissue was harvested from the tendon-bone interface at 4 weeks and 8 weeks post-surgery, and the sections were stained with Safranin O/fast green to detected tendon-bone healing. Immunochemistry staining was used to analyze VEGF, collagen I, and HIF-1α expression, and ELISA assay was used for detecting IL-6, TNF-α, and COX-2 concentrations. The expression levels of AKT/PI3K/NF-κB-related protein and mRNA were presented by Western blot and qPCR. The release rate of KGN was high within 4 h of KGN-PRP gel and followed by a slow release until 7 days. The Safranin O/fast green staining results indicated that tendon-bone interface in sham and mock group existed gap and tissue disorganization. The KGN + PRP group showed the positive color of the healing interface was more obvious and cartilage tissue began to be generated in large amounts at this interface. The maximum tensile force of KGN-PRP injection tendon-bone healing site was significantly higher than that of PRP group, and KGN-PRP effectively promoted fibro chondrogenesis and tendon-bone healing. The expression of collagen I, VEGF, and HIF-1α in regenerated tissues at the healing interface was significantly increased by KGN-PRP treatment compared with the mock and sham groups. The expressions of IL-6, TNF-α, and COX-2 after KGN-PRP treatment were significantly decreased in tendon-bone interface compared to the mock group. WB and qPCR results showed KGN-PRP treatment effectively inhibits AKT/PI3K/NF-κB activation of inflammatory pathways, thereby reducing the level of inflammation to promote wound healing. PRP is an effective carrier for KGN with the sustained release of KGN. After ACL reconstruction, injection of KGN-PRP gel significantly reduced the inflammatory response and inhibited AKT/PI3K/NF-κB activation in cartilage tissue, which promoted tendon-bone healing.

Keywords: AKT/PI3K/NF-κB; Anterior cruciate ligament reconstruction; Inflammatory response; Kartogenin combined PRP; Tendon-bone healing.