| Although so many kinds of scaffolds could be used in bone tissue engineering, each of them has some disadvantages so as to they are difficultly satisfied with the large requirements for bone repair, bone substitutes and bone remodeling. It becomes a key point to fabricate a novel or a modified bone tissue engineering scaffold which could mimic extracellular matrices (ECM) and have a suitable mechanical property with a good effect of osteogenesis. In this paper, we developed a novel micro-nano alternating multilayer scaffold for bone tissue engineering to investigate the osteogenetic differentiation by encapsulated rat bone marrow mesenchymal stem cells (rBMSCs) and bone morphogenetic protein-2(BMP-2).Firstly, we prepared-poly (ε-caprolactone)(PCL) nanofibers by electrospinning. The surface morphologies and the average diameter of the PCL nanofibers were measured by a scanning electron microscope (SEM). The PCL fibers were smooth, uniform and randomly orientated in appearance with an average diameter of282nm. We also have designed and fabricated a microfluidic device, which was used to form calcium alginate microbeads. The characterization of the microbeads was measured by a stereoscopic microscope and a rheometer. The microbeads which had an average diameter of318μm, are very uniform and have round shapes with a high monodispersity. The rheo logical characterization has already yielded the quantitative information on the viscoelastic properties and guaranteed the three-dimensional cross-linked networks of the calcium alginate hydrogel. The novel micro-nano alternating multilayer scaffold was formed by incorporation of the electrospun nanofibers and the calcium alginate microbeads generated from a microfluidic device.Secondly, the primary rat bone marrow mesenchymal stem cells (rBMSCs) were obtained from10day-old newborn Sprague-Dawley rats. The survival and proliferation of rBMSCs which was encapsulated into calcium alginate microbeads were investigated by live/dead stained and Alamar blue assay. It was found that the encapsulated rBMSCs were alive in a large number with a high viability (>85%). Encapsulated rBMSCs had a slow continuous proliferation compared with that seeded on the tissue culture plate polystyrene (TCP). rBMSCs and BMP-2were both encapsulated into calcium alginate microbeads or micro-nano alternating multilayer scaffold to evaluate alkaline phosphatase (ALP) activity. It was shown that the highest ALP activity was found in rBMSCs which were encapsulated into the micro-nano alternating multilayer scaffold containing BMP-2(MN+BMP+B) both on day14and day21. The mineral deposition was qualitatively and quantitatively detected by Alizarin Red S staining in vitro on day21. The highest mineral deposition of rBMSCs was also presented in MN+BMP+B group, which could be comparable to the result of ALP activity on day21.Finally, in vivo animal model was established by subcutaneously implanting micro-nano alternating multilayer scaffold into the back of rats to measure the ectopic bone formation. Histological and immunohistochemical assessments were detected on4and8weeks after implantation. According to hematoxylin and eosin (H&E) staining, the new tissue had already grown into all the scaffolds after8weeks. Moreover, except for the pure micro-nano scaffold, the microbeads and the micro-nano scaffold encapsulating rBMSCs with/without BMP-2pruduced the new bone-liked structure. Furthermore, immunohistochemical staining of osteocalcin showed the positive immunoreaction in the microbeads and the micro-nano scaffold encapsulating rBMSCs with/without BMP-2, which pronounced the new bone formation was undergoing. In conclusion, the nano-micro alternating multilayer scaffold could be a novel biomaterial to be preferable potential applied in bone tissue engineering. |
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