Fabrication of antimiR-138 delivered mesenchymal stem cell sheet and its effects on freeze dried allograft bone osteogenesis

YAN Jun; ZHAO Yan-tao; ZHANG Jing; WU Chen; CHEN Jing-yuan; ZHANG Yu-mei

doi:10.3969/j.issn.2095-252X.2015.11.007

Abstract：Objective To verify the feasibility of transfecting the bone mesenchymal stem cells ( BMSC ) sheets with the non-viral way and to confirm the osteogenesis enhancing effects of antimiR-138 delivered BMSC sheet / freeze dried bone ( FDB ) complex.Methods A continuous vitamin C inducing method was used to culture the BMSC sheets, which were later transfected with different Lipofectamine 2000-miRNA lipoplexes. The transfection efficiencies of different formulations were assessed by the miRNA RT-PCR. H&E staining and SEM observation were performed to evaluate the harvested sheets. The ectopic bone regeneration ability of the BMSC sheet / FDB complexes were assessed in immunocompromised mice: H&E or Masson’s Trichrome staining were used to assess the osteogenesis of the BMSC sheet / FDB complexes in SCID mice 4 and 8 weeks after implantation.Results The BMSC sheets were fabricated by a vitamin C inducing method. They could be successfully transfected with antimiR-138 to achieve a transfection efifciency of nearly 85% by a Lipofactamine2000 based transfection formulation after proper adaption and optimization. The in vivo ectopic bone regeneration results conifrmed the robust enhancing effects of antimiR-138 transfection on the bone regeneration ability of BMSC sheets combined with freeze dried allograft bone. Conclusions ( 1 ) Satisfactory transfection efifciency of nearly 85%, good cytocompatibility and unimpaired cell sheet structure can be obtained by properly optimizing the Lipofactamine 2000 based transfection formulation. ( 2 ) The antimiR-138 transfection signiifcantly enhances the osteogenesis of the BMSC sheet/FDB complexes in vivo, showing great clinical signiifcance for bone repair and regeneration.