The Intra-Articular Hematoma Immediately after Ankle Fracture Causes Cartilage Damage That is Partially Attenuated by Anti-Inflammatory Agents

Abstract

Category:Ankle Arthritis; Basic Sciences/BiologicsIntroduction/Purpose:Post-traumatic osteoarthritis (PTOA) is a frequent cause of disability. The most common predisposing factor for ankle PTOA is intra-articular ankle fracture. It has been hypothesized that an early inflammatory response after intra- articular injury could lead to cartilage damage. Our group recently demonstrated significantly elevated inflammatory cytokines in the synovial fluid fracture hematoma (SFFH) immediately after intra-articular ankle fracture. The negative effect that this inflammatory environment has on cartilage is unknown but may be the reason for PTOA development. The purpose of this study was to determine if cartilage occurred when exposed to intra-articular SFFH and if anti-inflammatory agents could attenuate this damage.Methods:SFFH was obtained from 52 intra-articular ankle fractures at the time of surgery. Samples were divided into three groups based on time from fracture: acute (0-2 days), intermediate (3-9 days), late (>=10 days). A biopsy punch was used to create 5mm cartilage discs from the medial and lateral shoulders of fresh human talus cartilage. The cartilage discs were weighed and cultured in standard chondrocyte media (SCM) for 3 days. Next, the discs were randomly assigned to one of 4 groups (n=10 each) and cultured as follows for 6 days: A=control, SCM only; Group B=acute SFFH+SCM; Group C=intermediate SFFH+SCM; Group D=late SFFH+SCM. After 6 days of culture, the discs were rinsed and transferred to new wells and cultured for 3 more days in SCM only, to determine inflammatory cytokine and MMP release from the cartilage discs (cartilage damage). The post SFFH spent culture media was collected. The initial and post SFFH culture spent media were analyzed for inflammatory cytokines, MMPs, CTXII (cartilage breakdown marker), and sGAG. Safranin-O staining was then performed on the discs. The initial and post SFFH media from groups A, B, C, and D were compared to determine if there was a difference in damage caused by SFFH based on time from fracture. Based on the results, Group B (early) was felt to cause the most damage. Therefore, the following treatment groups were created and cultured exactly as before: Group E=same as Group B + IL-1Ra treatment; Group F=same as Group B + doxycycline treatment. Groups B, E, and F were compared to determine if the addition of IL-1Ra or doxycycline (known MMP inhibitor) attenuated the damage.Results:Early, intermediate, and late SFFH all caused some degree of cartilage damage in the form of significantly elevated inflammatory cytokine and MMP production from the cartilage discs (Figure 1). The early SFFH (Group B) was noted to cause significantly elevated production of IL-6, IL-8, CTXII, and decreased safranin-O staining. Therefore, it was chosen to compare with the therapeutic anti-inflammatory agents. The addition of IL-1Ra or doxycycline significantly reduced the production of IL-6, IL-8, CTXII, and MMPs 3, 9, and 10 (Figure 2).Conclusion:This is the first study to show that intra-articular hematoma immediately after ankle fracture causes cartilage damage that can be partially attenuated by current clinically available therapeutic agents. Additionally, further research should be performed regarding the safety and efficacy of anti-inflammatory agents to prevent ankle PTOA.