Dual Delivery Of MiRNA-2861 And FK506 By PLGA/Gelatin Nanofibrous Scaffold To Promote Bone Regeneration

Background: Critical size bone defects or bone loss arising from trauma, tumor and inflammation not only influence mastication, pronunciation and beautiful appearance, but also brings heavy burden to patients on health and life. Therefore, many scholars of domestic and overseas have been studying the hot spots that finding the optimal treatment of bone defect and obtain the better result of bone regeneration. In recent years, with the quick development of bone tissue engineering, various novel biomedical scaffolds have been widely used for meeting the different requirements of clinical. However, the microenvironment of bone injury site is very complicated due to a variety of factors, such as inflammation, hypoxia, high lactate and etc. The traditional bioengineering scaffold typically rely on the delivery of single gene or drug to bone injury, this may explain the limited clinical treatment effect of many strategies. The diameter of nanofiber build by electrospinning method was nanoscale which give the nanofiber large surface area, the porous three-dimensional structure similar to the extracellular matrix, which will be beneficial to adhesion, stretching and growth of cells, making it as one of the best choice for tissue engineering scaffolds. According to the micro-environment and healing process of bone injury, delivering multi targeted bioactive molecule to the local bone defect may shorten the period of bone repair.Objective: To fabricate PLGA/Gelatin electrospinning nanofibrous scaffold for releasing the mi RNA – 2861 which can promote the bone regeneration, and using FK506 to inhibit the inflammation reaction, so that they can promote the bone healing synergistically.Material and method: PEI(MW=1800) as a gene carrier, it can transfer miRN A into BMSC. The osteogenesis-relative gene expression and mi RNA-2861 expression were separately detected with RT-PCR technique, and both of them were stained by Alizarin red. MTT colorimetry tests were performed to exam cell growth in various dose of FK506. Measure the ALP activity of cell after stimulated directly by LPS. Fabricating the P LGA/Gelatin complex scaffold by electrospinning and characterized by SEM to measure the surface appearance parameters. Blending the FK506 into spinning liquid to fabricate the drug- loaded scaffold, the PEI/mi RN A complex are adsorbed by interaction of static electricity in the space of nanofibers. Finally, the mi RN A and FK506 scaffold were obtained by freeze-drying. The cumulative in vitro release profile of mi RN A and FK506 were detected separately. Fabricate the FK506/mi R-2861 loaded electrospun nanofibrous scaffold and evaluate the ability of osteogenesis in vitro and in vivo.Result: According to SEM results, the PLGA/Gelatin electrospinning nanofibers had cross- linked by N HS/EDC with smooth aesthetic surface, and the porosity of nanofibers after crosslinked still maintained at a high level. The release profile of mi RN A were measured by Ribo Green Kit, the result suggested that mi RN A had a burst release from the scaffold, about 31% in 3 hours; And the cumulative release was about 61% in 7D. Until 14 days mi R-2861 still released continuously. Owing to the FK506 was encapsulated in the nanofibers, so the drug release was late and slow with the degradation of scaffold. The results of RT-PCR and Alizarin red showed, PEI/mi RNA complex could enhanced the level of bone relate gene expression, and promoted the formation of calcium nodules. The results of MTT assay indicated that the different concentrations of FK506 were nontoxic to the growth of cells. But the 100 n M FK506 exhibits slight promotion of cell proliferation. The results of RT-PCR and Alizarin red suggested that FK506 could induce BMSCs differentiation at an mid and late stage under inflammation conditions. Meantime, the immune-suppressing effect of FK506 could enhance the ALP activity and promote the gene expression during the period of 3 days to 14 days. No matter in vitro or in vivo, the FK506/mi RN A-2861 loaded PLGA/Gelatin nanofibrous scaffold is the most potential group to promote the osteogenesis by their synergistic effect.Conclusion: The method is effective for preparing the PLGA/Gelatin nanofibrous scaffold to deliver mi RNA and FK506. And it is hopeful to promote the local bone regeneration in critical defect by the synergistic effect of mi RNA and FK506 which lead to the osteogenic differentiation and ease of inflammation.