In situ recruitment of human bone marrow-derived mesenchymal stem cells using chemokines for articular cartilage regeneration

Abstract

Articular cartilage has a limited capacity for self-regeneration after injury. Bone marrow-derived mesenchymal stem cells (BMSCs) are good sources of repair since they can migrate directly to the injury site and differentiate to articular chondrocytes. To repair articular cartilage injury, various surgical techniques such as subchondral drilling and autologous chondrocytes transplantation are widely used. However, these techniques have severe problems including the fibro-cartilage formation, two-step surgery, limited donor sites for osteochondral autograft and loss-of-chondrogenic potency of ex vivo expanded chondrocytes. Chondral defects do not completely heal due to lack of chondrocytes at the defect site and the insufficient migration of surrounding chondrocytes and BMSCs to the injury site. Therefore, enhancing migration of a large number of BMSCs, chondrogenic progenitor cells, to the injury site is considered to be an effective strategy for cartilage regeneration. The primary purpose of this study is to screen the chemokine receptors and their ligands inducing the chemotaxis of BMSCs and the second is to investigate the chemotaxis of BMSCs in vivo toward selected chemokines which may give rise to a complete regeneration of articular cartilage.BMSCs isolated from human bone marrow were grown in either presence or absence of pro-inflammatory cytokines, such as IL-1β and TNF-α. RT-PCR was performed to evaluate the expression of nineteen chemokine receptors. To evaluate changes in the expression of chemokine receptors by pro-inflammatory cytokines, a reverse dot-blot assay was performed. It was shown that CCR2, CCR4, CCR6, CXCR1, and CXCR2 were expressed in normal BMSCs and increased significantly upon treatment with pro-inflammatory cytokines. To evaluate the effect of chemokines on BMSCs, the ligands of screened chemokine receptors were selected using the “Protein-knowledge database” and other reports. After screening the meaningful chemokine receptors and ligands, MTT assay for cell proliferation and cytotoxicity was performed for MCP-1, MIP-3α, IL-8, and SDF-1α.To determine the effect of chemokine on cell movement, two-dimensional migration assay was performed using wound healing and live cell tracking techniques. The wound healing capacity was increased by MIP-3α and IL-8 more than that by MCP-1 or SDF-1α. These four chemokines significantly increased the migrating mean velocity and total migrated distance of BMSCs. Especially, MIP-3α was most potent in healing capacity among chemokines. To investigate the chemotactic effect of chemokines on BMSCs, in vitro chemotaxis assay was performed. As a result, IL-8 and MIP-3α significantly enhanced the chemotaxis of BMSCs compared to MCP-1, SDF-1α and control. Collectively, IL-8 and MIP-3α were finally selected as candidates for the recruitment of BMSCs. To examine whether chemokine candidates enhance osteogenesis and chondrogenesis of BMSCs, cell differentiation assay was performed. These candidates did not induce ALP expression and calcium deposition in osteogenic differentiated BMSCs. The expression of chondrogenic markers, such as collagen fibers, aggrecan and type II collagen, were also not affected by chondrogenic differentiation with IL-8 and MIP-3α. To evaluate chemotactic effect of either IL-8 or MIP-3α, in vitro/in vivo chemotaxis assay using human BMSCs, chemokines-containing PLGA scaffolds, and nude mice was performed with live in vivo imaging system. In nude mice, the in situ recruitment of human BMSCs toward transplanted either IL-8 or MIP-3α with scaffolds was significantly induced. According to the results of histological analysis, fibroblastic cells recruited into chemokines-containing scaffolds were identified as human BMSCs which were injected into tail vein of mice. In addition, the subcutaneous tissue formation was enhanced by recruited human BMSCs without inflammatory reaction.Conclusively, IL-8 and MIP-3α were significantly enhanced tissue formation by chemotaxis of BMSCs without inflammation. Therefore, this study suggests that either IL-8 or MIP-3α can be useful candidates for regeneration of damaged articular cartilage.