METTL3 promotes osteogenic differentiation of human umbilical cord mesenchymal stem cells by up-regulating m6A modification of circCTTN.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUCMSCs) are promising seed cells in bone tissue engineering. circRNA and N6-methyladenosine (m6A) RNA methylation play important roles in osteogenic differentiation. Here, we investigated the potential relevance of a critical circRNA, hsa_circ_0003376 (circCTTN), and methyltransferase-like 3 (METTL3) in osteogenic differentiation of hUCMSCs. METHODS: Expression of circCTTN after hUCMSC osteogenic induction was detected by qRT-PCR. Three databases (RMBase v2.0, BERMP, and SRAMP) were used to predict m6A sites of circCTTN. RNA was enriched by methylated RNA immunoprecipitation (MeRIP), followed by quantitative real-time polymerase chain reaction to detect m6A level of circCTTN after METTL3 overexpression and osteogenic induction. RNA pull-down, Western blotting, and protein mass spectrometry were performed to investigate the potential mechanisms by which METTL3 promoted m6A modification of circCTTN. Bioinformatic analyses based on database (STRING) search and co-immunoprecipitation were used to analyze the proteins that interacted with METTL3. RESULTS: Overexpression of METTL3 promoted osteogenic differentiation of hUCMSCs and increased m6A level of circCTTN. Two potential m6A modification sites of circCTTN were predicted. No direct interaction between METTL3 and circCTTN was observed. Thirty-one proteins were pulled down by probes specific for circCTTN, including NOP2, and two m6A reading proteins, EIF3A and SND1. Bioinformatics analysis and co-immunoprecipitation showed that METTL3 interacted with EIF3A indirectly through NOP2. CONCLUSIONS: METTL3 promotes the osteogenic differentiation of hUCMSCs by increasing the m6A level of circCTTN. However, METTL3 does not bind directly to circCTTN. METTL3 interacts with circCTTN indirectly through NOP2 and EIF3A.

Clinical performance of two onlay designs for molars after root canal treatment.

PURPOSE: To evaluate the clinical performance of two proposed onlay designs. METHODS: Molars with occlusal and/or mesial/distal defects after root canal treatment were classified by design into three groups. Onlays without shoulders were the control group (Group C, n = 50). The designed onlays were Group O (n = 50) and the designed mesio-occlusal/disto-occlusal onlays were Group MO/DO (n = 80). All onlays had an occlusal thickness of approximately 1.5-2.0 mm, and the designed onlays had a shoulder depth and width of approximately 1 mm. In Groups C and O, the box-shaped retention was 1.5 mm in depth. The proximal box was connected with a dovetail retention in Group MO/DO. Patients were examined every 6 months and followed for 36 months. Restorations were evaluated by using the modified United States Public Health Service Criteria. Statistical analysis was performed by using Kaplan-Meier analysis, the chi-square test, and Fisher's exact test. RESULTS: No tooth fracture, debonding, secondary caries, or gingivitis was observed in any group. Groups O and MO/DO had satisfactory survival and success rates, and there was no significant difference in performance characteristics among the three groups (P > 0.05). CONCLUSION: The two proposed onlay designs were effective in protecting molars.

A dual role of HIF1α in regulating osteogenesis-angiogenesis coupling.

OBJECTIVES: The hypoxia-inducible factor 1-α (HIF1α), a key molecule in mediating bone-vessel crosstalk, has been considered a promising target for treating osteoporosis caused by gonadal hormones. However, senile osteoporosis, with accumulated senescent cells in aged bone, has a distinct pathogenesis. The study aimed at revealing the unknown role of HIF1α in aged bone, thus broadening its practical application in senile osteoporosis. MATERIALS AND METHODS: Femurs and tibias were collected from untreated mice of various ages (2 months old, 10 months old, 18 months old) and treated mice (2 months old, 18 months old) underwent 4-w gavage of 2-methoxyestradiol (a kind of HIF1α inhibitor). Bone-vessel phenotypes were observed by microfil infusion, micro-CT and HE staining. Markers of senescence, osteogenesis, angiogenesis, oxidative stress and expression of HIF1α were detected by senescence ß-galactosidase staining, qRT-PCR, western blot and immunostaining, respectively. Furthermore, bone mesenchymal stem cells from young mice (YBMSCs) and aged mice (ABMSCs) were transfected by knockout siRNA and overexpression plasmid of HIF1α. Senescence ß-galactosidase staining, Cell Counting Kit-8, transwell assay, alkaline phosphatase staining, alizarin red-S staining and angiogenesis tests were utilized to assess the biological properties of two cell types. Then, Pifithrin-α and Nutlin-3a were adopted to intervene p53 of the two cells. Finally, H2O2 on YBMSCs and NAC on ABMSCs were exploited to change their status of oxidative stress to do a deeper detection. RESULTS: Senescent phenotypes, impaired osteogenesis-angiogenesis coupling and increased HIF1α were observed in aged bone and ABMSCs. However, 2-methoxyestradiol improved bone-vessel metabolism of aged mice while damaged that of young mice. Mechanically, HIF1α showed opposed effects in regulating the cell migration and osteogenesis-angiogenesis coupling of YBMSCs and ABMSCs, but no remarked effect on the proliferation of either cell type. Pifithrin-α upregulated the osteogenic and angiogenic markers of HIF1α-siRNA-transfected YBMSCs, and Nutlin-3a alleviated those of HIF1α-siRNA-transfected ABMSCs. The HIF1α-p53 relationship was negative in YBMSCs and NAC-treated ABMSCs, but positive in ABMSCs and H2O2-treated YBMSCs. CONCLUSION: The dual role of HIF1α in osteogenesis-angiogenesis coupling may depend on the ROS-mediated HIF1α-p53 relationship. New awareness about HIF1α will be conducive to its future application in senile osteoporosis.

Involvement of PI3K/Akt signaling pathway in promoting osteogenesis on titanium implant surfaces modified with novel non-thermal atmospheric plasma.

Non-thermal atmospheric plasma (NTAP) modification to induce a hydrophilic titanium (Ti) surface with less carbon contamination, has been demonstrated to boost the osteogenic responses. In this study, we investigated the underlying bone formation mechanism of NTAP-Ti, and the involvement of PI3K/Akt signaling pathway in regulating osteogenic activities on NTAP-Ti surfaces. NTAP was employed for Ti activation, and PI3K inhibitor, LY294002, was applied to the suppression of PI3K/Akt pathway. We systematically and quantitatively detected the cell morphology, attachment, proliferation, osteogenic differentiation and mineralization of MC3T3-E1 mouse preosteoblasts, and molecular expressions involved in osteogenesis and PI3K/Akt signaling pathway in vivo and in vitro. A descent in osteoblast proliferation on Ti surfaces in relation to LY294002. Alkaline phosphatase (ALP) activity, as well as matrix mineralization, was mitigated by PI3K inhibitor in NTAP-Ti. Likewise, the expression levels of osteogenesis-related genes [ALP, osteocalcin (Ocn), osteopontin (Opn) and runt-related transcription factor 2 (Runx2)] on NTAP-Ti were notably attenuated by LY294002, as confirmed by the results of osteogenesis-related proteins (ALP, and Runx2) expression analysis. In addition, the expression of PI3K/Akt signal pathway proteins further verified the inhibition of LY294002 on Ti surfaces modified by NTAP. Collectively, the PI3K/Akt signal pathway was involved in the amelioration of osteogenesis induced by NTAP modification. NTAP treatment for Ti activation is promising in augmented osteogenic potential through the activation of PI3K/Akt signal pathway.

[Response of morphological characteristics of Populus euphratica nebkhas to groundwater in Birkum Desert, west of Taklimakan].

By regarding the east of Birkum desert of the west of Taklimakan as research areas, the paper measured the basic morphological characteristics of Populus euphratica nebkhas through real-time kinematics, got the relevant data of groundwater by manual drilling, and analyzed the response of emorphological characteristics of P. euphratica nebkhas to groundwater. The results showed that the change range of morphological parameters of P. euphratica nebkhas was large. Groundwater infil- trated the research areas from west, south and southwest to northwest and the depth of groundwater ranged from 0.15 m to 83.2 m. Except for the maximums of the length and circumference, the other morphological parameters of P. euphratica nebkhas had a negative relation with the depth of groundwater. The volume of P. euphratica nebkhas was mainly in the range of 63.54-91.24 m3.