Magnoflorine With Hyaluronic Acid Gel Regulate Subchondral Bone Regeneration And Attenuate Articular Cartilage Degeneration On The Early Stage Of Spontaneous Osteoarthritis

Abstract

Objective: Aim to address Chinese medicine magnoflorine combined with hyaluronic acid (HA) gel effectively promoting trabecular bone remodeling and attenuate cartilage degeneration on the early stage of spontaneous knee osteoarthritis (OA) through the association between micro-CT (μCT) imaging and histological staining. Methods: The MC3T3 cell (osteoblast) viability of vary concentrations of magnoflorine treatment was detected by XTT. The cell proliferation was reflected by cell cycle assay. Bone formation was detected by Alizarin red staining. The proinflammatory cytokine and chemokines from blood and synovial fluid samples were measured. The 5 month Dunkin-Hartley guinea pig as a spontaneous OA model was made a standardized bone defection with 1mm diameter and 5 mm depth on the tibial medial side close to cartilage. Guinea pigs (n=5/group) were once intra-bone injected 50 ng magnoflorine, HA gel, 50 ng magnoflorine + HA gel, and no treatment, respectively. All collected limbs were performed μCT scan and histological staining 2 month post-surgery. Results: The cell viability of MC3T3 treated with 25 ug/ml magnoflorine, and the S phase of cell cycle were significantly increased. The proinflammatory cytokine and chemokines detected from synovial fluid were significantly decreased in 50 ng magnoflorine + HA gel treated guinea pigs. For in vivo study, with 50 ng magnoflorine + HA gel treated guinea pigs, there had an increased parameters related to trabecular bone remodeling, such as BV/TV, BMD, Tb.N, Tb.Th, Conn.Dn, and decreased parameters like Th.Sp, Th.Pf and DA, which implied trabecular bone regeneration. Furthermore, the decreased modified OARSI scores, lower tidemark thickness, higher ratio of hyaline cartilage (HC)/ calcified cartilage (CC) area and the fractal dimension (FD), which was implied the roughness of SBP, were reflected significant improvement of cartilage degeneration, when compared to no treatment and HA treated group. Finally, there was a significant positive association between FD and the area ratio of HC/CC measured by histological staining and a significant negative association between FD and OARSI scores. Conclusion: Our study elucidated the potential benefit of magnoflorine combined with HA gel to induce trabecular bone regeneration and regulate cartilage degeneration as well as the relationship between SBP and cartilage on the early stage of spontaneous OA. It also demonstrated the possibility to diagnose pathogenesis of early OA using μCT analysis for clinical assessment.