A possible issue of articular cartilage degeneration in acute phase after anterior cruciate ligament reconstruction

OBJECTIVES: Intra-articular hematoma are caused by articular injury such as ligament rupture or intra-articular fracture and following knee surgery. It has been reported that intra-articular hematoma leads to cartilage degeneration. Exposure of articular cartilage to low concentrations of blood for a period as short as 2 days has been confirmed to induce the irreversible cartilage damage. Anterior cruciate ligament (ACL) injury is increased risk for developing posttraumatic osteoarthritis (OA). Despite improvement in ACL reconstruction (ACLR), the incidence of posttraumatic OA has remained relatively unchanged over the several decades later. One of cause in OA may be thought to intra-articular hematoma after ACLR. A primary feature in OA is cartilage degeneration, it involves the loss of extracellular matrix components including aggrecan and Type Ⅱ collagen. A distintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4, -5, -9 and matrix metalloprotease (MMP)-2, -9 are reported primary proteases responsible for aggrecan cleavage in OA. The present study was designed to the hypothesis described as above by evaluating the influence of intra-articular hematoma on ADAMTS-4, -5, -9, MMP-2 and -9 activities in acute phase after ACLR. METHODS: Patient sample Intra-articular hematoma was collected from patients, who were underwent primary ACLR with consent to participate in this study, on day1, 4 and 7 after surgery. Day1 samples were collected from drainage tube for approximately 10 ml. Day4 and day7 samples were collected by inserted needle and aspiration under sterile condition. For control sample, it was selected and adopted SF without intra-articular hematoma. To obtain enough amount of SF for control sample on the ipsilateral knee, 20ml of 0.9% saline was injected into the joint cavity prior to arthroscopic surgery. In the laboratory, intra-articular hematoma and SF were centrifuged for 15 min at 3000g and the supernatant were stored at -80℃ until assay. From March 2017 to March 2020, 98 patients who were undergone knee arthroscopic surgery took part in this study. The study protocol was reviewed and approved (accession no.: O-0149) by the local ethics committees at our institutions. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Written informed consent was obtained from the patients for publication of this report. The patients who did not want to take part were not enrolled in this study. Inclusion criteria include patients for primary ACLR in our institute. Exclusion criteria include osteoarthritis with Kellgren-Lawrence classification gradeⅡ or higher on X-ray, cartilage damage with International Cartilage Repair Society (ICRS) cartilage injury classification gradeⅡ or higher based on arthroscopic findings and multi-ligament reconstruction. Enzyme-linked Immunosorbent assay (ELISA) Five proteins (ADAMTS-4, -5, -9 and MMP-2, -9) were analyzed and were measured in intra-articular hematoma and SF using a human ELISA kit (Cloud-Clone Corp, Katy, TX, USA according to manufacturer’s instructions. ADAMTS and MMPs (catalog number): (ADAMTS4, SEK204Hu; ADAMTS5, SEK205Hu; ADAMTS9, SEK209Hu; MMP2, SEA100Hu; MMP9, SEA553Hu) levels of intra-articular hematoma and SF were read off from a standard curve according to the manufacturer’s instruction. RESULTS: Expression levels of each proteins in intra-articular hematoma using ELISA The expression levels of ADAMTS-4 in day4 and day7 samples were significantly higher than in control and day1 samples.(P(c-4)<0.001, P(c-7)<0.001, P(1-4)<0.001, P(1-7)<0.001). The expression levels of ADAMTS-4 in day7 samples also were significantly higher than in day4 samples (P(4-7)<0.05). Those of ADAMTS-5 in day1, day4 and day7 samples were significantly higher than in control samples.(P(c-1)<0.001, P(c-4)<0.001, P(1-7)<0.001). The expression levels of ADAMTS-9 were no significantly differences between each samples. MMP-2 expression levels of day1 and day4 samples were significantly increased compared with the control samples (P (c-1)<0.05, P (c-4)<0.05). The expression levels of MMP-9 were increased only day1 samples, significantly higher than in control, day4 and day7 samples(P(c-1)<0.001, P(1-4)<0.001, P(1-7)<0.001). ELISA analysis of control and post-operative samples : Time-course study In day1 after surgery, expression levels of ADAMTS-5, MMP-2 and MMP-9 were increased significantly compared with those of control samples (ADAMTS-5: P (c-1)<0.001, MMP-2: P (c-1)<0.05, MMP-9: P (c-1)<0.001). In day4, expression of ADAMTS-5 and MMP-2 were increased significantly higher than control samples (respectively, P (c-4)<0.001). MMP-9 levels of day4 were decreased significantly compared with day1 samples (P (1-4)<0.001). In day7, expression of ADAMTS-5 were increased significantly higher than control samples (P (c-7)<0.001). Expression levels of MMP-2 and MMP-9 in day7 were no significantly differences compared with control samples. Those of MMP-9 were decreased significantly lower than day1 samples (P (1-7)<0.001). On the other hand, ADAMTS-4 expression levels revealed significantly increased expression at day4 (P<0.001) and day7 (P<0.001). ADAMTS-4 expression levels were shown to increase in time-course dependency. CONCLUSIONS: Because the expression of ADAMTS-4, -5 and MMP-2, -9 were elevated in acute phase after ACLR, that suggested that intra-articular hematoma after ACLR may contribute to cartilage degeneration. The results suggest that immediate treatment for intra-articular hematoma, such as injection and aspiration, may be needed for prevention of OA. Moreover, these proteins may provide potential for therapeutic targets.