Objective:To investigate the effect of cytoskeleton-associated protein 4 (CKAP4) gene knockout on maxillary expansion osteogenesis and its regulatory mechanism on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSC).Methods:Nineteen wild type (WT) and nineteen CKAP4 gene knockout (Ckap4 -/-) mice aged 6-8 weeks were selected to establish a mouse model of rapid maxillary expansion. Samples were taken on the 7th and 14th day after the operation. Micro-CT and HE staining were used to evaluate bone regeneration. Tissue proteins in the modeled area were collected, and Western blotting analysis (WB) was used to detect the protein expression levels of alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). BMSC were isolated from WT and Ckap4 -/- mice. The expression of surface markers CD29, Sca-1, CD44, CD45, CD34, and CD11b was detected by flow cytometry, and cell proliferation ability was detected by 5-ethynyl-2'-deoxyuridine (EdU). After 7 days of osteogenic induction, real-time fluorescence quantitative PCR (RT-qPCR) and WB were used to detect the expression levels of RUXN2, ALP, OCN, protein kinase B (AKT), and phosphorylated protein kinase B (p-AKT). After 21 days, alizarin red staining and cetyl pyridine chloride quantification were used to detect the differences in mineralized nodule formation in each group. In CKAP4 gene knockout BMSC, the small-molecule AKT agonist sc79 (4 μg/ml) was added as the intervention group (Ckap4 -/- +sc79), and dimethyl sulfoxide (DMSO) treatment was used as the control group (Ckap4 -/- +DMSO). After osteogenic induction, RT-qPCR, WB, and alizarin red staining were used to compare the osteogenic differentiation differences between the two groups of cells. Results:The micro-CT results showed that at 7 days and 14 days after surgery, the new bone volume in the Ckap4 -/- group [(0.070±0.010) and (0.146±0.019) mm 3] was significantly lower than that in the WT group [(0.094±0.006) and (0.196±0.013) mm 3] (both P<0.01). HE-stained histological sections showed that the area of new bone tissue in the Ckap4 -/- group at 7 days and 14 days after surgery [(0.101±0.008) and (0.158±0.010) mm 2] was also significantly lower than that in the WT group [(0.116±0.005) and (0.183±0.008) mm 2] (both P<0.05). WB was used to detect the tissue proteins in the maxillary modeling area of mice in the two groups 7 days after surgery. The results showed that the expression levels of ALP, RUNX2 and OCN in the Ckap4 -/- group were significantly lower than those in the WT group. BMSC from wild-type mice and CKAP4 knockout mice were both positively expressed for CD29, CD44, and Sca-1, and basically not expressed for CD45, CD34, and CD11b. EdU assay showed that there was no significant difference in the proliferation ability of cells in the two groups. After 21 days of osteogenic induction of BMSC, alizarin red staining results showed that the number of mineralized nodules in the Ckap4 -/- group was significantly less than that in the WT group. After adding sc79, the number of mineralized nodules increased significantly, which was consistent with the results of cetyl pyridine chloride quantification. After 7 days of osteogenic induction, It was found that the expression levels of ALP, RUNX2, and OCN in the CKAP4 -/-group (0.751±0.066, 0.484±0.040, 0.679±0.063) were significantly lower than those in the WT group (1.000±0.113, 1.000±0.081, 1.000±0.113) (all P<0.001). The results of WB were consistent with those of RT-qPCR. At the same time, the WB results showed that the level of p-AKT protein in the CKAP4 -/-group (0.518±0.114) was significantly lower than that in the WT group (1.000±0.234) ( P<0.05). After treatment with sc79 for 7 days of osteogenic induction, RT-qPCR was used to detect the gene expression levels of ALP, RUNX2, and OCN. The results showed that the expression levels in the CKAP4 -/-+sc79 group (2.755±0.353, 4.800±0.990, 2.524±0.137) were significantly higher than those in the CKAP4 -/-+DMSO group (1.000±0.078, 1.000±0.247, 1.000±0.175) (all P<0.001). Conclusions:CKAP4 knockout inhibits the osteogenic differentiation of BMSC by reducing the activity of the PI3K/AKT signaling pathway, thereby suppressing osteogenesis in maxillary expansion.

Objective:To investigate the effect of cytoskeleton-associated protein 4 (CKAP4) gene knockout on maxillary expansion osteogenesis and its regulatory mechanism on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSC).Methods:Nineteen wild type (WT) and nineteen CKAP4 gene knockout (Ckap4 -/-) mice aged 6-8 weeks were selected to establish a mouse model of rapid maxillary expansion. Samples were taken on the 7th and 14th day after the operation. Micro-CT and HE staining were used to evaluate bone regeneration. Tissue proteins in the modeled area were collected, and Western blotting analysis (WB) was used to detect the protein expression levels of alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). BMSC were isolated from WT and Ckap4 -/- mice. The expression of surface markers CD29, Sca-1, CD44, CD45, CD34, and CD11b was detected by flow cytometry, and cell proliferation ability was detected by 5-ethynyl-2'-deoxyuridine (EdU). After 7 days of osteogenic induction, real-time fluorescence quantitative PCR (RT-qPCR) and WB were used to detect the expression levels of RUXN2, ALP, OCN, protein kinase B (AKT), and phosphorylated protein kinase B (p-AKT). After 21 days, alizarin red staining and cetyl pyridine chloride quantification were used to detect the differences in mineralized nodule formation in each group. In CKAP4 gene knockout BMSC, the small-molecule AKT agonist sc79 (4 μg/ml) was added as the intervention group (Ckap4 -/- +sc79), and dimethyl sulfoxide (DMSO) treatment was used as the control group (Ckap4 -/- +DMSO). After osteogenic induction, RT-qPCR, WB, and alizarin red staining were used to compare the osteogenic differentiation differences between the two groups of cells. Results:The micro-CT results showed that at 7 days and 14 days after surgery, the new bone volume in the Ckap4 -/- group [(0.070±0.010) and (0.146±0.019) mm 3] was significantly lower than that in the WT group [(0.094±0.006) and (0.196±0.013) mm 3] (both P<0.01). HE-stained histological sections showed that the area of new bone tissue in the Ckap4 -/- group at 7 days and 14 days after surgery [(0.101±0.008) and (0.158±0.010) mm 2] was also significantly lower than that in the WT group [(0.116±0.005) and (0.183±0.008) mm 2] (both P<0.05). WB was used to detect the tissue proteins in the maxillary modeling area of mice in the two groups 7 days after surgery. The results showed that the expression levels of ALP, RUNX2 and OCN in the Ckap4 -/- group were significantly lower than those in the WT group. BMSC from wild-type mice and CKAP4 knockout mice were both positively expressed for CD29, CD44, and Sca-1, and basically not expressed for CD45, CD34, and CD11b. EdU assay showed that there was no significant difference in the proliferation ability of cells in the two groups. After 21 days of osteogenic induction of BMSC, alizarin red staining results showed that the number of mineralized nodules in the Ckap4 -/- group was significantly less than that in the WT group. After adding sc79, the number of mineralized nodules increased significantly, which was consistent with the results of cetyl pyridine chloride quantification. After 7 days of osteogenic induction, It was found that the expression levels of ALP, RUNX2, and OCN in the CKAP4 -/-group (0.751±0.066, 0.484±0.040, 0.679±0.063) were significantly lower than those in the WT group (1.000±0.113, 1.000±0.081, 1.000±0.113) (all P<0.001). The results of WB were consistent with those of RT-qPCR. At the same time, the WB results showed that the level of p-AKT protein in the CKAP4 -/-group (0.518±0.114) was significantly lower than that in the WT group (1.000±0.234) ( P<0.05). After treatment with sc79 for 7 days of osteogenic induction, RT-qPCR was used to detect the gene expression levels of ALP, RUNX2, and OCN. The results showed that the expression levels in the CKAP4 -/-+sc79 group (2.755±0.353, 4.800±0.990, 2.524±0.137) were significantly higher than those in the CKAP4 -/-+DMSO group (1.000±0.078, 1.000±0.247, 1.000±0.175) (all P<0.001). Conclusions:CKAP4 knockout inhibits the osteogenic differentiation of BMSC by reducing the activity of the PI3K/AKT signaling pathway, thereby suppressing osteogenesis in maxillary expansion.

Glass ionomer cement (GIC) is widely used as a common filling material in dentistry but still exhibits problems with secondary caries and fractures. Thus, the antibacterial and anti-caries performance of GIC needs to be further improved. In recent years, natural antimicrobial components have become more desirable due to their good biological properties and low drug resistance. In this review, the natural antimicrobial ingredients in GIC modification are classified, reviewed and summarized according to the different sources of antimicrobial ingredients. In terms of animal origin, chitosan and casein phosphopeptide-amorphous calcium phosphate exhibit antimicrobial properties without affecting the mechanical properties of materials; propolis and bioactive enzymes have good biocompatibility; in terms of plant origin, polyphenols help improve the antimicrobial and mechanical properties of the material; arginine has a good remineralization effect; and plant essential oils have a certain ion release effect. In terms of microbial origin, antibiotics greatly improve the antibacterial properties of materials; in addition, the combined application of natural antimicrobial ingredients also exhibited excellent performance. Despite these advantages, the optimal addition concentration and biocompatibility in vivo are questions that need to be further explored before clinical applications can be achieved.

Objective:To investigate the effect of heat shock protein B8 (HspB8) downregulation on retinal ganglion cell (RGC) and retinal function in the mice model of optic nerve injury (ONC).Methods:Adeno-Associated Virus (AAV) 2 AAV2-shHspB8-GFP was constructed to knockdown HspB8. 66 adult male C57/BL6 mice were randomly divided into the control group, the ONC group, the AAV2-shHspB8 group, the ONC+AAV2-shHspB8 group, and the ONC+AAV2-GFP group. There were 10, 20, 16, 10 and 10 mice respectively, and both eyes were used as experimental eyes. Western blot was used to evaluate the expression of HspB8 on day 3 and 7 after ONC. By GFP immunofluorescence staining, the efficacy of AAV2-shHspB8-GFP transfer was accessed. Moreover, it was possible to identify functional and RGC survival differences between groups by optomotor response (OMR), dark adapted full-field flash electroretinogram (ff-ERG), oscillatory potentials (OPs), photopic negative response (PhNR) and retinal flat-mount RGC counting 5 days after ONC. Comparisons between two groups were made using Mann-Whitney U test, unpaired t-test, unpaired t-test with Welch’s correction, one-way ANOVA, and Bonferroni t test. Results:Compared with the control group, the expression of HSPB8 protein in the retina of mice in ONC3 group was significantly increased, and the difference was statistically significant ( F=43.63, P<0.01). Compared with the control group, the ONC group showed obviously lower visual acuity ( P<0.01), lower a-wave, b-wave, OPs, PhNR amplitude, longer b-wave latency ( P<0.05), and the survival rates of RGC in ONC3 group, ONC5 group and ONC7 group decreased in a time-dependent manner( F=384.90, P<0.01). Transfection of AAV2 efficiency was highest on 4 weeks after IVT. Besides, there was no significant differences between the control group and the AAV2-shHspB8 group on visual acuity, ff-ERG, OPs, PhNR and RGC survival ( P>0.05). In comparison of the control group, we found that RGC survival of the ONC5+AAV2-shHspB8 group was significantly elevated ( F=10.62, P<0.01). Conclusions:Expression of HspB8 on the retina can be induced by ONC. The investigation of RGC counting, visual acuity, and ff-ERG revealed that optic nerve injury destructed functionality of mice retina and resulted to RGC death ultimately. The Most crucial finding of this research is that HspB8 knockdown had a neuroprotective effect in RGC after ONC.