Effects of macrophage with Wnt5a over-expression on inflammation and chondrogenic differentiation in co-culture system

Abstract

To construct and compare macrophage by gene modification of Wnt5a which co-cultured by human bone marrow-derived mesenchymal stem cells (MSCs) with two dimension (2D) and bone marrow aspirates in vitr transwell, in order to investigate the effect of Wnt5a signaling on cartilage homeostasis through regulation of macrophage pro-inflammatory responses. Macrophages, MSCs and bone marrow aspirates specimens were extracted from 6 patients with severe knee deformities undergoing total knee arthroplasty(2 males, 4 females, aged from 58 to 71 years old) from September 2015 to December 2018. The synovial tissues of knee joints were exposed to type II collagenase and obtained single cell suspensions, and the purity of macrophages was determined by Ficoll gradient centrifugation and anti-CD14 antibody flow cytometry. The macrophages were transduced with IFN-γ combined with TNF-α for 48 h, and rAAV-lacZ or Wnt5a transfected for 24 h, then co-cultured model by human bone marrow-derived mesenchymal stem cells (MSCs) with two dimension (2D) and bone marrow aspirates in vitro transwell. HE staining, toluidine blue staining, X-gal staining and anti-Wnt5a, anti-II, X collagen immunohistochemical staining and enzyme-linked immunosorbent assay were applied to detect cell morphology and proliferation (cellularity), viral transfection efficiency respectively. Results of anti-CD68 immunohistochemistry showed macrophage of patients with osteoarthritis increased obviously. Anti-CD 14 flow cytometry confirmed that percentage of isolated synovial macrophages was 90.31%. The level of monocyte chemotactic protein-1 in supernatants was significantly increased after stimulation with IFN-γ and TNF-α, indicating that the macrophages were activated and at proinflammatory condition. After transduction with rAAV-lacZ for 3 days, X-gal staining indicated that lacZ gene transfer could efficiently transduce the activated macrophages with the efficiency over 97.50% for at least 21 days. After transfection macrophages by rAAV-Wnt5a stimulated expression of Wnt5a, and enhanced expression of Wnt5a between two models and inhibited the secretion of MCP-1. The expression of MCP-1 in Wnt5a group was 14.76 and 61.51 pg/ml respectively. In addition, rAAV-Wnt5a gene transfer could promote cell proliferation, chondrogenic differentiation and cartilage matrix synthesis. Under the condition of macrophage and MSCs 2D monolayer or aspirates transwe ll co-culture, rAAV-mediated over-expression of Wnt5a could promote maintenance of cartilage homeostasis and chondrogenic differentiation of MSCs via macrophage inflammatory response, macrophages may affect cartilage homeostasis and MSCs chondrogenesis through Wnt5a signaling pathway.