Enhancing The Osteogenic Capacity Of MG63 Cells Through N-isopropylacrylamide-modified Polyethylenimine-mediated Oligodeoxynucleotide MT01 Delivery

Bone loss caused by periodontal disease,trauma,congenital defects or tumor is a major worldwide health problem.Research shows that,ODN MT01 can promote osteoblast of rat maturation and activation;reduce rat alveolar bone absorption that caused by periodontitis;regulate expression levels of osteogenic related factors like intracellular Runx-2,Collagen-I and OPG,promote bone marrow mesenchymal stem cells differentiating into osteoblasts.The above research suggests,ODN MT01 may have regulatory effects on bone remodeling.What is most important in the clinical application of ODN is protecting the ODN from DNase and delivering the ODN to the TLR9 of target cells.Although the MT01 is characterized by good water solubility,low cytotoxicity and specific combination of the binding target site,it difficult used for clinical application owe to short half-time,low molecular weights and poor stability.Chemical modification of ODN is an effective technique to protect against degradation by DNase.However,several severe side effects caused by the modification of DNA backbone have been reported.In recent years,research has been conducted on delivery systems for ODNs using nanoparticles.The development of clinically applicable ODN MT01 is therefore expected to grow in the future.It is important to develop a safe delivery systems for MT01 in NPs.Gene therapy requires the transfer of therapeutic genes,or more usually their complementary DNAs(c DNAs),into suitable cells.Viruses serve as the basis formany vectorstransfer due to their own geneticmaterial very efficiently to the cells they infect.However,because of residual safety concerns when using viral vectors,as well as their cost and complexity,there is sustained interest in using non-viral vectors to deliver therapeutic DNA.Non-viral vectors have been widely used as gene delivery systems due to their low immunogenicity,high gene-loading capacity,flexible designability,potential safety and low production cost.Among these carriers,branched polyethylenimine(PEI)has exhibited superior genetransfection efficiency both in vitro and in vivo,because its highpositive charge and proton-buffering capacity.However,high positive charge density can lead to membrane-disruptive properties and thus cause severe problems,such as high cytotoxicity and hemolysis,and serum instability of thenanoparticles.To overcome these disadvantages,chemical modification of PEI to improve its transfection efficiency and/or to decrease its cytotoxicity has been actively investigated,especially grafting of hydrophobic groups.In the present research,a PEI derivative(namely PEN)has been constructed through modification of PEI25 K with N-isopropylacrylamide via Michael addition.The derivative PEN was employed as a carrier to achieve MT01 delivery using the human osteoblast-like cell line(MG63)and rat cranial defectsas models.Then,we investigated its ability to induce osteogenesis in vitro and in vivo.The main contents are as follows: Experiment 1 The preparation structure and properties of PEN/MT01 complexThe derivative PEN was constructed through modification of PEI25 K with N-isopropylacrylamide via Michael addition.The PEN was blended with MT01 by different mass ration.Using the Malvin particle potential to test the particle size and potential of PEN/MT01 complex and by agarose gel electrophoresis to determinate the ability of PEN with MT01.The transfection efficiency and proliferation rate in MG63 cells with different mass ration were detected by fluorescence imaging technique,flow cytometry and MTT,which was to determine the optiomal transfection mass ratio.The results showed that the particle size of PEI/MT01 were in the 34.8-283.9 nm range and the surface of complex is positively charged.With the mass ratio of greater than or equal to 0.5:1,MT01 was fully loaded by PEN to form a stable complex.What is more,when the mass ratio was 6:1,PEN/MT01 nanoparticles were effectively transfected into MG63 cells without cytotoxicity,whereby they promoted cell proliferation in vitro.Experiment 2 The effects of PEN/MT01 on bone formation in vitroPEN/MT01 complexes were transfected into MG63 cell line in vitro,and the capability of inducing osteogenic differentiation was evaluated by ALP stain,realtime-PCR and wesernblot.Real-time PCR and western blotting analyses showed that the PEN/MT01 complexes enhanced m RNA and protein expression of osteoprotegerin,Sp7,and runt-related transcription factor 2,and decreased levels of receptor activator of nuclear factor kappa-B ligand,which are markers indicative of MG63 differentiation into preosteoblasts or mature osteoblasts.This approach demonstrates the potential of using PEN as a biocompatible gene therapy carrier to address regeneration of bone defects.Experiment 3 The effects of PEN/MT01 on bone formation in vivoTo evaluate bone healing ability,we conducted animal experiments with each material.A circular 5mm diameter defects were prepared on the side of rat calvarial bone.After 10 weeks post-operation,all specimens were assessed using HE,immunohistochemistry and Micro-CT to evaluate the bone regenration.HE staining showed that there are many bone cores in the PEN/MT01 group.Micro-CT anlysis indicated that more new bone formation was observed in PEN/MT01 group than in tother groups.Our study demonstrated that the delivery of MT01 into MG63 cells was successfully achieved using the PEN derivative.Following PEN-mediated MT01 transfection,osteoblastic differentiation of MG63 cells was efficiently promoted.Using non chemical modification of ODN with PEN represents a simple and effective alternative approach,and PEN-mediated MT01 delivery has great potential for alveolar bone regeneration.