Qian Zhang,Xingyuan Qu,Chen Liang,Hongyan Li,Siyu Du,Chang Wang,Yuandong Xie,Yi Zheng,Lei Wang
Objective: This study aimed to investigate the regulatory effect of N-isopropylacrylamide-modified polyethyleneimine (PEN)-delivered oligodeoxynucleotide (ODN) MT01 on bone regeneration in vitro and in vivo . Methods: A polyethylenimine (PEI) derivative, PEN, was constructed through Michael addition and employed as a carrier for ODN MT01 transfection. PEN/MT01 nanocomposites were characterized using agarose gel retardation assay, size distribution, zeta potential and transmission electron microscopy. The Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of PEN on cell viability. Alkaline phosphatase (ALP) staining was used to detect the osteogenic differentiation ability of PEN/MT01 nanocomposite. Real-time quantitative PCR (q RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the regulatory effects of PEN/MT01 nanocomposite on osteogenic differentiation gene expression. Rat model was observed using the skull defect method and verified using micro-computed tomography (CT), serum biochemical indices, hematoxylin and eosin (H&E) staining and Immunohistochemistry (IHC). Results: PEN had good biological properties and could deliver MT01 well to achieve efficient transmission of MT01. PEN/MT01 nanocomposites were effectively transfected into MC3T3-E1 cells at a ratio of 6.0. CCK-8 assay displayed that PEN had no cytotoxicity to MC3T3-E1 cells. Additionally, PEN/MT01 nanocomposites could promote the expression of osteogenic genes. In vivo results revealed that PEN/MT01 nanocomposites could promote bone regeneration more effectively than the other groups. Conclusion: PEN has good biocompatibility and low toxicity, which is a good carrier for ODN MT01. PEN-delivered MT01 can be potentially employed as a useful approach to achieving bone regeneration.
Bone regeneration involves a cascade of sophisticated, multiple-staged cellular and molecular events, where early-phase stem cell recruitment mediated by chemokines and late-phase osteo-differentiation induced by pro-osteogenic factors play the crucial roles. Herein, enlightened by a bone physiological and regenerative mechanism, the multilayer nanofibrous membranes (PLLA@SDF-1 α @MT01) consisting of PLLA/MT01 micro-sol electrospun nanofibers as intima and PLLA/PEG/SDF-1 α electrospun nanofibers as adventitia are fabricated through micro-sol electrospinning and manual multi-layer stacking technologies. In vitro releasing profiles show that PLLA@SDF-1 α @MT01 represents the rapid release of stromal cell-derived SDF-1 α (SDF-1 α ) in the outer layers, while with long-term sustained release of MT01 in the inner layer. Owing to interconnected porosity like the natural bone extracellular matrix and improved hydrophilia, PLLA@SDF-1 α @MT01 manifests good biocompatibility both in vitro and in vivo. Furthermore, PLLA@SDF-1 α @MT01 can promote bone marrow mesenchymal stem cells (BMSCs) migration by amplifying the SDF-1 α /CXCR4 axis and stimulating BMSCs osteo-differentiation via activating the MAPK pathway in vitro. PLLA@SDF-1 α @MT01, with a programmed dual-delivery system, exhibits the synergetic promotion of bone regeneration and vascularization by emulating key characteristics of the staged bone repair in vivo. Overall, PLLA@SDF-1 α @MT01 that mimics the endogenous cascades of bone regeneration can enrich the physiology-mimetic staged regenerative strategy and represent a promising tissue-engineered scaffold for the bone defect.
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