Xinhua Sun

N -AC- L -Leu-PEI-mediated miR-34a delivery improves osteogenic differentiation under orthodontic force N-AC-L-Leu-PEI-mediated miR-34a delivery improves osteogenic differentiation under orthodontic force

An oligodeoxynucleotide (ODN), with its design based on human mitochondrial DNA (MT01), has been demonstrated to promote the proliferation and activation of osteoblasts. However, critical issues such as poor oligonucleotide stability in the presence of nucleases currently limit its broad application. This study evaluated whether N-isopropylacrylamide-modified polyethylenimine (PEN) nanoparticles could effectively deliver ODN MT01 locally with low or no toxicity and promote osteoblast differentiation in vitro. Our data demonstrate that at a w/w ratio of 6 : 1, PEN/MT01 nanoparticles were effectively transfected into MG63 cells without cytotoxicity, whereby they promoted cell proliferation in vitro. Quantitative PCR and western blotting analyses showed that the PEN/MT01 complexes enhanced mRNA 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.

This study aims to synthesize MTO1 (a kind of oligodeoxynucleotides) and N-isopropylacrylamide-modified polyethylenimines (PEN) complexes (MT01/PEN) as well as to investigate the effect of the complexes on the expression of osteoprotegerin (OPG) and the receptor activator of nuclear factor κB ligand (RANKL) in the human osteoblast-like cell line MG63. MG63 cells were transfected by MT01/PEN complexes formed with three different mass ratios (1:2, 1:4, 1:6) of MT01 to PEN. MT01 and MT01-s were used as positive control. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction were performed to estimate the amount of OPG and RANKL released into the culture media and in MG63 at 24, 48, 72 h. MG63 responded to the MT01/PEN complexes by significantly upregulating the OPG on the protein and mRNA levels (P < 0.05). The protein and mRNA levels of RANKL were lower in most of the groups with complexes, and the OPG/RANKL ratio were higher (P < 0.05). MG63 were affected by the MT01/PEN complexes with different mass ratios, particularly when the ratio was 1:6. MT01 can enhance the promotion of ossification by establishing the delivery system with PEN.

To observe the expression of MMP-2 in the periodontal tissues of molars in Beagle dogs after different times of intrusion by mini-screw with cyclic intrusive force. Three mature Beagle dogs were used for the experiment. On the buccal and palatal sites of the left maxillary second and third premolars, a minis-crew was placed at the inter-radicular septa separately, intruding the tooth with 150 g initial force, which would be reinforced every 4 weeks. The teeth of left side as the experimental group were divided into 3 subgroups, as being intruded 12, 24 and 36 weeks, and the right side as blank control. Then the dogs were sacrificed, and the second and the third premolars with the surrounding periodontal tissue were cut down, fixed, decalcified, wrapped and cut into slices. Immunohistochemical staining with MMP-2 was performed. The average optical density (OD) of MMP-2 was calculated with IPP software, which represented the expression of MMP-2. SPSS 17.0 software package was used for statistical analysis. Immunohistochemical staining revealed that the expression of MMP-2 in control group was low. After tooth intrusion, MMP-2 expression significantly increased in the periodontal tissues of molars, and reached the maximum in the group of 24 weeks. Then MMP-2 expression decreased in the 36-week group but still significantly higher than the control group. There was no significant difference among the 3 subgroups for different intrusion times(P>0.05). MMP-2 participates in remodeling of the periodontal tissue during tooth intrusion. The expression of MMP-2 is not significantly increased with the extension of the intrusion time with cyclic intrusive force, which suggests that with the use of mini-screw to intrude the molars with cyclic intrusive force, the periodontal tissues of the intruded tooth maintain dynamic balance of bone remodeling.

To investigate the cytotoxicity of laser-welded nickel titanium (NiTi) and stainless steel composite archwire. The NiTi and stainless steel composite archwire (CoAW) laser-welded with pure copper inrerplayer was studied with methyl thiazolyl tetrazolium (MTT) test in vitro. The cytotoxicity of CoAW was compared with stainless steel archwire and NiTi archwire. Two tests were carried out. Test 1: the immersed solution of CoAW was diluted to five grades (50%, 40%, 30%, 20%, 10%). The cytotoxicity in vitro of these agents was assayed on murine fibroblast cell L929 line with MTT test at 24 and 48 hours. Test 2: the immeresed solution of CoAW, NiTi archwires and stainless steel archwires was diluted to four grads (100%, 75%, 50%, 25%). The cytotoxity of three kinds of material was compared at 48 hours. The results of all samples revealed level 0-1 cytotoxicity. In test 1, the same grade solution optical density (except 20%) at 24 hours was statistically lower than at 48 hours. In test 2, the optical density of CoAW solution (1.964 ± 0.122, 2.084 ± 0.056, 2.056 ± 0.071, 2.096 ± 0.050) was statistically lower than the same grade solution of stainless steel archwire (2.168 ± 0.091, 2.227 ± 0.160, 2.302 ± 0.052, 2.301 ± 0.060) and NiTi archwire (2.138 ± 0.105, 2.262 ± 0.050, 2.271 ± 0.082, 2.294 ± 0.056) (P < 0.05). The MTT test of CoAW in vitro showed that cytotoxicity was related to concentration and time. The cytotoxicity of the CoAW was more serious than that of stainless steel and NiTi archwires. However, CoAW belonged to secure rang of material toxicity reaction.

A Pt (IV) complex which combined the bioactivities of both oxaliplatin and demethylcantharidin (DMC) is synthesized and delivered by a polymer–drug conjugate for combination chemotherapy. Oxaliplatin is released from the polymer–drug conjugate within cancer cell by reduction to attack nuclear DNA, while a dose of DMC is also hydrolyzed subsequently to block DNA damage‐induced defense mechanisms by serine/threonine phosphatase 2A (PP2A) inhibition. In vitro evaluation shows that the polymer–drug conjugate with dual modes of action upon cancer cells displays higher cytotoxicity against SKOV‐3 cells than that of free drugs. This enhanced cytotoxicity is attributed to the synergistic effect between oxaliplatin and DMC, as well as the effective intracellular internalization of the micelles observed by confocal laser scanning microscopy (CLSM) imaging.

To evaluate casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and several fluoride products in inhibiting enamel demineralization. Seventy healthy premolars extracted before orthodontic treatment were divided into seven groups (A: CPP-ACP; B: CPP-ACP + fluoride mouth rinses; C: fluoride mouth rinses; D: fluoride glass ionomer protection; E: fluoride resin binder; F: fluoride varnish; G: control group). There were ten samples in each group. The teeth were dipped into an artificial caries solution ten minutes at a time, then applied separately with CPP-ACP, CPP-ACP + fluoride mouth rinses, fluoride mouth rinses, fluoride glass ionomer protection, fluoride resin binder and fluoride varnish on labial enamel. The samples were dipped into an artificial saliva solution (37°C). Then all samples were examined with atomic force microscope (AFM) at the end of first, second and third monthes. At the end of the first month, surface roughness in group D [(114 ± 1) nm] was significantly lower than that in group G[(172 ± 9) nm]. At the end of the second month and the third month, significant difference was found in surface roughness between group G and the rest of groups (P < 0.05); between group B and the rest of groups; and between group C and groups (D, E and F). CPP-ACP and fluoride could both inhibit enamel demineralization in vitro. Solid fluoride (groups D, E and F) had better results than liquid fluoride (group C).

To investigate the effect of oligodeoxynucleotides (ODNs) on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts, in order to find a candidate ODN with potential for the treatment of periodontitis, a series of ODNs were designed and selected to test their effect on the promotion of the differentiation of BMSCs to osteoblasts in vitro and on the repair of periodontal tissue in rats with periodontitis. It was found that MT01, one of the ODNs with the sequences of human mitochondrial DNA, stimulated the proliferation of BMSCs, the differentiation of BMSCs to osteoblasts and mRNA expression of bone-associated factors including Runx2, Osterix, OPG, RANKL and collagen I in vitro. In vivo study showed that MT01 prevented the loss of alveolar bone in the rats with periodontitis and induced the production of proteins of OPG and Osterix in the bone tissue. These results indicated that MT01 could induce differentiation of BMSCs to osteoblasts and inhibit the alveolar bone absorption in rats with periodontitis.

The paper explored the regulatory role of oligodeoxynucleotides (ODNs) with specific sequences in the proliferation and activation of osteoblast, using human osteoblast-like cell line MG 63 as the model. Through the administration of ODNs to MG 63 cells at a concentration of 1.0 µg/mL, ODN MT01 with positive effects on proliferation and activation of osteoblast was selected from 11 different ODNs by methyl thiazolyl tetrazolium (MTT) assay and alkaline phosphatase (ALP) activity measurement. To get a deeper insight into the molecular mechanism, effects of ODN MT01 treatment on the expression level of Sp7, runx-2, collagen-I, osteoprotegerin (OPG) and RANK ligand (RANKL) were determined using quantitative real time PCR and Western blotting. Remarkably, the mRNA and protein expression levels of Sp7, runx-2, collagen-I and OPG were improved after ODN MT01 treatment. Meanwhile, the protein expression level of RANKL was dramatically decreased. These results suggested that ODN MT01 had a significant impact in facilitating osteogenic proliferation and activation, and provided a direct evidence for the notion that single strand ODN could regulate the balance of bone formation and resorption, and thus was of great potential in the rebuilding of alveolar bone.

The aim of this study was to quantify the changes in post-orthodontic demineralized enamel lesion surface areas after using casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). Ten orthodontic patients (mean age, 17.7 years) were involved in this study who had multiple decalcified enamel lesion after fixed orthodontic therapy. Ten percent of CPP-ACP paste (GC Tooth Mousse, Recaldent, Europe) was taught to spread on tooth surface two times per day immediately after tooth brush. Standardized intraoral images were taken before and after using it. Image analysis software Image-Pro Plus was used to quantify the visible areas of the demineralized lesions which were expressed as a percentage of total labial surface areas of each tooth. The measurement data before and after using CPP-ACP for 1 month and 2 months were analyzed by paired t test. The measurement of image was repeated again 1 month later to determine the repeatability of the method by calculating the intraclass correlation coefficient between the two results of analysis with SPSS13.0 software package. CPP-ACP significantly reduced the visible enamel demineralization (P<0.01, paired t test). The mean reduction in lesion size after treatment was 4.89%(SD:2.05%; 95% CI: 1.93%-8.77%) for 1 month, and 2 months was 8.359%(SD: 3.472%; 95% CI: 3.76%-15.01%). The intraclass correlation coefficient between the two measurements was 0.968. CPP-ACP can effectively improve the long-standing postorthodontic demineralized enamel white lesion, and the intraoral image analysis is an reliable and repeatable method to measure white spot lesions. Supported by Research Fund from Bureau of Science and Technology of Changchun City (Grant No. 2008108).