A Preliminary Study Of In Vitro Cartilage Engineering Combining Magnesium Alloy Scaffold And BMSCs

OBJECTIVE Because of its excellent mechanical properties and biodegradability, magnesium alloy is a promising candidate for bioscaffold material in orthopaedic applications. In this study, we evaluate the histocompatibility of calcium-phosphate-coated porous magnesium alloy and human BMSCs, its potential as cartilage engineering scaffold and the effect of magnesium alloy on chondrogenic differentiation.MATERIALS AND METHODS 1. Primary human BMSCs were isolate, cultured and directed to differentiate into osteoblasts, chondroblasts and lipoblasts to access the multipotential differentiation ability of BMSCs. 2. The porous magnesium alloy scaffolds were prepared by laser drilling. The scaffolds were coated with calcium phosphate coating to enhance its corrosion resistance. Cell adhesion of human BMSCs to the calcium phosphate coating surface was observed through SEM and CLSM. MTT analysis was used to access the effect of leach liquor from both coated and uncoated magnesium alloy scaffolds on cellular activity. 3. Human BMSCs were seeded into calcium-phosphate-coated or uncoated porous magnesium alloy scaffolds. ?-TCP scaffolds were used in control group. Experimental groups and control group were in vitro induced for chondrogenesis for 28 days. Toluidine blue staining, alcian blue staining and type II collagen immunohistochemical staining were used to characterize the formed cartilage-like tissue, in order to identify the formation of cartilage matrix. Real-time PCR were used to assess effect of magnesium alloy on chondrogenic differentiation related genes. 4. Bone morphogenetic proteins(BMPs) in human bone marrow mesenchymal stem cells into osteoblasts play a crucial role. Sclerostin able proteins(BMPs) in combination inhibit osteogenesis BMPs and bone morphogenetic; this part of the study aims to affect the test Sclerostin of BMP-2-induced human cell activity mesenchymal stem cells(BMSCs) in bone marrow and osteogenic differentiation.RESULTS 1. We have successfully isolated and cultured human BMSCs. Three-lineage differentiation experiments showed that it had multipotential differentiation ability. 2.Through SEM and CLSM, we observed that human BMSCs had good adhesion to calcium-phosphate-coated porous magnesium alloy scaffolds and spread well. MTT analysis of alloy leach liquor showed that leach liquor from calcium-phosphate-coated magnesium alloy could improve cellular activity, while leach liquor from uncoated magnesium alloy inhibited cellular activity. 3. Human BMSCs combining with calcium-phosphate-coated porous magnesium alloy scaffolds could be induced to form cartilage-like tissue in vitro. Toluidine blue staining, Alcian blue staining and type II collagen immunohistochemical staining confirmed the production of cartilage matrix. On the other hand, uncoated magnesium alloy failed to produce cartilage-like tissue in induction in vitro, possibly because the hydrogen from its degradation inhibited cell adhesion to the material. Real-time PCR result showed that magnesium alloy promoted the m RNA transcription of collagen II, but had no significant effect on m RNA transcription of Aggreacan and SOX9. 4. Sclerostin can inhibit the expression of BMP-2-induced promotion of human BMSCs osteogenic differentiationCONCLUSIONS Human BMSCs combining with calcium-phosphate-coated magnesium alloy scaffolds can be induced to form cartilage tissue in vitro. Also, the magnesium alloy can promote cell proliferation and up-regulated the expression of cartilage-related genes Aggreacan. But uncoated porous magnesium alloy scaffold degrades too fast, which makes it not suitable for direct cell seeding. The calcium-phosphate-coated porous magnesium alloy scaffold has the potential to be used as cartilage tissue engineering scaffold.