| Background:Maxillary transverse deficiency is a common maxillofacial bony deformity in orthodontic practice.Rapid maxillary expansion is an important tool for the correction of maxillary transverse deficiency in adolescents by mechanically opening the mid-palatal suture.However,extensive relapse after rapid maxillary expansion has been reported.Inadequate bone formation is one of the main causes of relapse.In current studies on promoting new bone formation in the mid-palatal suture after rapid maxillary expansion,there are still some problems with various promotion methods,such as obvious parameter dependence,unclear mechanisms,and inability to avoid systemic side effects.Therefore,there is still a need to find new methods that can avoid systemic side effects and promote local osteogenesis in the mid-palatal suture.Numerous studies have shown that miR-21 is closely related to the osteogenic differentiation of precursor cells under mechanical force.Our group has found that miR-21 was associated with new bone formation in the mid-palatal suture after rapid maxillary expansion and could promote that in mice.This shows that the application of miR-21 is a very promising therapeutic idea to promote new bone formation in the mid-palatal suture after rapid maxillary expansion,and two key issues need to be addressed to achieve this clinical application:protection of the miR-21 molecule from massive enzymatic cleavage and specific regulation of miR-21 activity locally in the mid-palatal suture.Upconversion nanoparticles can achieve localized bioactivity of nucleic acid molecules in deep tissues by near-infrared light control,which is widely used in bioimaging and disease treatment.However,there is no study on the application of upconversion nanoparticles to deliver nucleic acid molecules to specifically promote local bone tissue formation in the mid-palatal suture after rapid maxillary expansion.In this experiment,a novel miR-21-loaded upconversion nanoparticle will be constructed to promote local bone tissue formation in the mid-palatal suture by near-infrared light control.Objectives:Construction of near-infrared photosensitive miR-21 upconversion nanodelivery particles(UCNP@PEI@PC-miR-21)for achieving miR-21 near-infrared photocontrol to promote new bone formation in the mid-palatal suture after rapid maxillary expansion,providing a new approach to promote localized new bone formation in the mid-palatal suture of the rapid maxillary expansion in the clinic.Research methods:1.Design,synthesis and characterization of nanoparticles.Field transmission electron microscopy and hydrated particle size measurements to evaluate the morphology,dispersion and particle size of UCNPs@PEI;Zeta potential and Fourier infrared spectroscopy to qualitatively analyse the nucleic acid loading capacity of UCNPs@PEI,agarose gel electrophoresis experiments to quantitatively determine the optimal incubation time and maximum nucleic acid loading;agarose gel electrophoresis experiments to evaluate nucleic acid protection and release;The nucleic acid and nanoparticle photosensitisation properties were evaluated by agarose gel electrophoresis and fluorescence spectroscopy.2.Evaluation of nanoparticle cytotoxicity,cellular uptake,and photocontrol of osteogenic properties at the cellular and animal levels.CCK-8 cytotoxicity assay to evaluate the cytotoxicity of nanoparticles;confocal micrographs to verify the cellular uptake of nanoparticles;qRT-PCR and Western Blot analysis of MC3T3-E1 cells treated with nanoparticles under the effect of mechanical traction.The expression of key osteogenic factors ALP,RUNX2,and COLIA1 at the mRNA and protein levels was evaluated at the cellular level to assess the performance of nanoparticles in contributing to osteogenic differentiation by light control;a miR-21 knockout gene(miR-21-/-)mice rapid maxillary expansion model was established,and HE staining and immunohistochemical staining were performed to observe the morphological changes of the mid-palatal suture and the histological changes of ALP.TRAP staining to observe the number and location of osteoclasts and to evaluate the light-controlled bone formation properties of nanoparticles in vivo.Research results:1.The results of field emission transmission electron microscopy,hydrated particle size and zeta potential indicate the successful construction of uniformly dispersed and stable nucleic acid-loaded nanoparticles.Agarose gel electrophoresis experiments showed that the maximum loading mass ratio of nucleic acid was about 25:1 and the optimal loading incubation time was about 150 min.The results of agarose gel electrophoresis experiments and fluorescence spectroscopy analysis showed that the particles possessed light-controlled activation miR-21 properties.2.CCK-8 experiments showed no significant cytotoxicity of the particles in the concentration range of 0.5 mg/mL;the laser confocal microscopy results proved that the particles could successfully enter the cytoplasm and be uniformly distributed.The qRT-PCR and WB results showed that the nanoparticles could upregulate the expression of ALP,RUNX2 and COLIA1,key factors of osteogenic differentiation in MC3T3-E1 cells,at the mRNA and protein levels under light control,in other words,the nanoparticles were verified to have light-controlled osteogenic differentiation properties at the cellular level.The results of HE staining showed a significant widening of the mid-palatal suture,a disorganization of the cellular and fibrous layers in the mid-palatal suture,and an active cell proliferation.The results of immunohistochemical staining showed that the nanoparticles upregulated the expression of ALP in the mid-palatal suture under light control,and the number of TRAP-positive cells in the mid-palatal suture was relatively low and the staining was lighter,suggesting that the local osteoclastic effect in the mid-palatal suture was weakened in the drug-lighted group.Conclusion:We successfully constructed near-infrared light-controlled miR-21 upconversion nanoparticles with good dispersion,good biocompatibility and no obvious cytotoxicity.Under the effect of mechanical force,the nanoparticles could photocontrol the expression of genes related to osteogenic differentiation of osteogenic precursor cells and promote osteogenic differentiation.The nanoparticles photocontrollably promote local osteoblast formation in the mid-palatal suture of miR-21-/-mice under the effect of rapid maxillary expansion,while there are relatively few osteoclasts around the mid-palatal suture and the osteoclastic effect is weak.This novel near-infrared light photosensitive miR-21 upconversion nano-delivery particle can promote local bone tissue formation in the mid-palatal suture during rapid maxillary expansion,providing a new therapeutic idea to promote local new bone formation in the mid-palatal suture during rapid maxillary expansion. |
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