Calcium-MicroRNA Complex-Functionalized Nanotubular Implant Surface for Highly Efficient Transfection and Enhanced Osteogenesis of Mesenchymal Stem Cells.

Controlling mesenchymal stem cell (MSC) differentiation by RNA interference (RNAi) is a promising approach for next-generation regenerative medicine. However, efficient delivery of RNAi therapeutics is still a limiting factor. In this study, we have developed a simple, biocompatible, and highly effective delivery method of small RNA therapeutics into human MSCs (hMSCs) from an implant surface by calcium ions. First, we demonstrated that simple Ca/siRNA targeting green fluorescent protein (GFP) nanocomplexes were able to efficiently silence GFP in GFP-expressing hMSCs with adequate Ca2+ concentration (>5 mM). In addition, a single transfection could obtain a long-lasting silencing effect for more than 2 weeks. All three of the main endocytosis pathways (clathrin- and caveolin-mediated endocytosis and macropinocytosis) were involved in the internalization of the Ca/siRNA complexes by MSCs, and macropinocytosis plays the most dominant role. Furthermore, the Ca/siRNA complexes could be efficiently loaded onto the titanium implant surface when pretreated with anodization to create a nanotube (NT) layer. Because of the hydrophilic property of the NT surface, the Ca/siRNA was quickly loaded (less than 4 h) with high efficiency (nearly 100%), forming an even amorphous coating. The Ca/siRNA-coated NT surface showed an initial burst release of 80% of the siRNA complexes over 2 h, which is adequate to achieve robust gene silencing of attached hMSCs. To demonstrate the therapeutic potential of our Ca/siRNA coating technology, Ca/antimiR-138 complexes were loaded on to the NT surface, which strongly enhanced the osteogenic differentiation of hMSCs. In conclusion, our findings suggest that Ca2+ is an effective and biocompatible carrier to deliver small RNA therapeutics into hMSCs, both in solution and from functionalized surfaces, which provides a novel approach to control the MSC differentiation and tissue regeneration.

Engineered three-dimensional nanofibrous multi-lamellar structure for annulus fibrosus repair.

Repairing annulus fibrosus (AF) defects is one of the most challenging topics in intervertebral disc disease treatment research. The highly oriented native structure offers mechanical functionality to the spine, however manufacturing scaffolds with such a structure still presents a challenge for tissue engineering. Here, a three-dimensional (3D) multi-lamellar scaffold with hierarchically aligned nano- and micro-fibers for AF tissue engineering was successfully developed. Aligned polycaprolactone (PCL) nano-fiber sheets, which were fabricated by electrospinning, were inserted into fused-deposit-modeling (FDM) micro-fibers to build a layer-by-layer structure, with the thickness of each layer being 0.7 mm and the angle of fiber alignment in each adjacent layer being 60°. Human mesenchymal stem cells (hMSCs) were used for in vitro compatibility studies. The architecture of the scaffold was characterized by scanning electron microscopy (SEM). Uniaxial tensile testing showed closed mechanical properties of the scaffold to native AF tissue. The XTT cell viability and DNA quantification of the cells on the multi-lamellar scaffold were found to be significantly higher than the FDM scaffolds without nano-fibers. Confocal microscopy demonstrated that the cells spread evenly on the surface of the electrospun sheet and oriented along the nano-fiber direction. This 3D multi-lamellar scaffold has the advantages of stability from the FDM micro-fibers, and unique characteristics from the aligned electrospun nano-fibers, such as mimicking the extracellular matrix (ECM), and an ultrahigh surface area for improved hMSC attachment, proliferation and contact guidance of cell morphology. The newly designed scaffold mimics the native structure of AF and has a great potential as a substrate for the regeneration of AF.