The Host Protein CAD Regulates the Replication of FMDV through the Function of Pyrimidines' De Novo Synthesis.

Foot-and-mouth disease virus (FMDV) is a single-stranded picornavirus that causes economically devastating disease in even-hooved animals. There has been little research on the function of host cells during FMDV infection. We aimed to shed light on key host factors associated with FMDV replication during acute infection. We found that HDAC1 overexpression in host cells induced upregulation of FMDV RNA and protein levels. Activation of the AKT-mammalian target of rapamycin (mTOR) signaling pathway using bpV(HOpic) or SC79 also promoted FMDV replication. Furthermore, short hairpin RNA (shRNA)-induced suppression of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), a transcription factor downstream of the AKT-mTOR signaling pathway, resulted in downregulation of FMDV RNA and protein levels. Coimmunoprecipitation assays showed that the ACTase domain of CAD could interact with the FMDV 2C protein, suggesting that the ACTase domain of CAD may be critical in FMDV replication. CAD proteins participate in de novo pyrimidine synthesis. Inhibition of FMDV replication by deletion of the ACTase domain of CAD in host cells could be reversed by supplementation with uracil. These results revealed that the contribution of the CAD ACTase domain to FMDV replication is dependent on de novo pyrimidine synthesis. Our research shows that HDAC1 promotes FMDV replication by regulating de novo pyrimidine synthesis from CAD via the AKT-mTOR signaling pathway. IMPORTANCE Foot-and-mouth disease virus is an animal virus of the Picornaviridae family that seriously harms the development of animal husbandry and foreign trade of related products, and there is still a lack of effective means to control its harm. Replication complexes would generate during FMDV replication to ensure efficient replication cycles. 2C is a common viral protein in the replication complex of Picornaviridae virus, which is thought to be an essential component of membrane rearrangement and viral replication complex formation. The host protein CAD is a key protein in the pyrimidines de novo synthesis. In our research, the interaction of CAD and FMDV 2C was demonstrated in FMDV-infected BHK-21 cells, and it colocalized with 2C in the replication complex. The inhibition of the expression of FMDV 3D protein through interference with CAD and supplementation with exogenous pyrimidines reversed this inhibition, suggesting that FMDV might recruit CAD through the 2C protein to ensure pyrimidine supply during replication. In addition, we also found that FMDV infection decreased the expression of the host protein HDAC1 and ultimately inhibited CAD activity through the AKT-mTOR signaling pathway. These results revealed a unique means of counteracting the virus in BHK-21 cells lacking the interferon (IFN) signaling pathway. In conclusion, our study provides some potential targets for the development of drugs against FMDV.

Long-term three-dimensional condylar remodeling during presurgical orthodontics and after orthognathic surgery of mandibular retrognathia with high mandibular plane angle.

OBJECTIVES: Patients with high mandibular plane facial morphology are the most dominant facial type who experience TMJ abnormalities with resultant condylar resorption, affecting the orthodontic and orthognathic treatment outcomes. The study aimed to quantitatively assess the three-dimensional condylar remodeling during the presurgical orthodontics and after orthognathic surgery of the retrognathic mandible with a high mandibular plane angle. The study also investigated the correlation between the resultant remodeling based on the hypothesis that condylar resorption following orthognathic surgery is a part of a progressive presurgical resorption process. MATERIALS AND METHODS: The study included adults with mandibular retrognathism and high mandibular plane angle who have computed tomography scans (CT) obtained before any treatment (T0), after completion of presurgical treatment before surgery (T1), and at long-term follow-up after surgery (T2). DICOM of CT scan was gathered and processed using ITK-SNAP and 3D Slicer software. The interval between T0 and T1 was represented as a presurgical phase, while between T1 and T2 was defined as a postsurgical phase (T1-T2). RESULTS: Twenty-five patients (50 condyles) were included with a mean age of 23 ± 3.2 years. The mean of the follow-up during the presurgical phase was 19.8 ± 7.1 months and 15.5 ± 5.5 months during the postsurgical phase. The condylar volume during the presurgical phase (T0-T1) was relatively stable (- 3.3 ± 37.2mm3). However, during the postsurgical phase (T1-T2), the volume was significantly reduced - 113.8 ± 98.3mm3 (P < 0.001). Localized condylar surface resorption during the postsurgical phase was significantly higher than during the presurgical phase (P < 0.05). No correlation was found between the localized condylar surface remodeling during the presurgical and postsurgical phases. However, a negative statistically significant correlation existed between the overall condylar volume changes during the presurgical and postsurgical phases (r = 0.502, P < 0.001). CONCLUSION: Significant condylar resorption following orthognathic surgery of the retrognathic mandible with a high mandibular plane angle might occur regardless of the presurgical status of the condyle. CLINICAL RELEVANCE: The study provided an evidence to be discussed with the patients and considered throughout the treatment of mandibular retrognathia with high mandibular plane angle.

Self-healing hydrogels based on biological macromolecules in wound healing: A review.

The complete healing of skin wounds has been a challenge in clinical treatment. Self-healing hydrogels are special hydrogels formed by distinctive physicochemically reversible bonds, and they are considered promising biomaterials in the biomedical field owing to their inherently good drug-carrying capacity as well as self-healing and repair abilities. Moreover, natural polymeric materials have received considerable attention in skin tissue engineering owing to their low cytotoxicity, low immunogenicity, and excellent biodegradation rates. In this paper, we review recent advances in the design of self-healing hydrogels based on natural polymers for skin-wound healing applications. First, we outline a variety of natural polymers that can be used to construct self-healing hydrogel systems and highlight the advantages and disadvantages of different natural polymers. We then describe the principle of self-healing hydrogels in terms of two different crosslinking mechanisms-physical and chemical-and dissect their performance characteristics based on the practical needs of skin-trauma applications. Next, we outline the biological mechanisms involved in the healing of skin wounds and describe the current application strategies for self-healing hydrogels based on these mechanisms. Finally, we analyze and summarize the challenges and prospects of natural-material-based self-healing hydrogels for skin applications.

Single-Cell Sequencing Yields Insights in the Evolution of Foot-and-Mouth Disease Virus Persistent Infection.

Foot-and-mouth disease virus (FMDV) could cause acute infection in host cells, or they could coexist with host cells to generate persistent infection. In persistent infection, the virus could survive for a long time in the host and could be transmitted between different host cells. In the case of FMDV-persistent infection cell line, there is a remarkable significant cellular heterogeneity in the FMDV-persistent infection cell line due to differences of viral load in the individual cells within the cell line. However, the mechanisms of FMDV-persistent infection are not well understood. It is now generally accepted that multiple factors contribute to the coevolution of viruses and cells during the course of persistent infection. The outcome would influence the development of persistent FMDV infection conjointly, reaching a state of equilibrium ultimately. Therefore, in order to elucidate the mechanism of cellular heterogeneity in FMDV-persistent infection cell line, single-cell sequencing was performed on BHK-Op, and pseudotime trajectory plot was draw through cell cluster. Based on the cell clusters, we predicted the development and progression of the FMDV-persistent infection. It could be well explained by the fact that, in BHK-Op cells, there are a fraction of infected cells and a fraction of virus-exposed but uninfected bystander cells. By further comparing the transcripts in cell clusters, we found that these genes were involved in changes in ribosome biogenesis, cell cycle, and intracellular signaling including the interferon signaling pathway and mitogen-activated protein kinase (MAPK) signaling pathway. Through comprehensive cross-tabulation analysis of differential expressed genes in various cluster of cells, we identified a high association of Fos, a downstream transcription factor of the MAPK/extracellular signal-regulated kinase (ERK) signaling pathway, with viral replication during the formation of FMDV-persistent infection. Through the further study of Fos, we found that downregulation of Fos facilitates viral clearance during FMDV-persistent infection. Upregulation of c-Raf, which is the upstream of the MAPK/ERK signaling pathway, could promote FMDV replication through downregulation of Fos. Our research is the first to provide insight into the mechanism of the formation FMDV-persistent infection through single-cell sequencing using persistent infection cell line. Pseudotime trajectory analysis was the first time to apply for FMDV-persistent infection cell line. Our work highlights the detailed overview of the evolution of FMDV-persistent infection. We also analyzed the differential expressed genes in the replication or elimination of FMDV within the host. We found that the MAPK/ERK signaling pathway and its downstream transcription factor Fos play an important role in FMDV-persistent infection.

Next-generation sequencing sheds light on the interaction between virus and cell during foot-and-mouth disease virus persistent infection.

Foot-and-mouth disease virus (FMDV) infection can be either persistent or acute in susceptible animals. The mechanisms involved in FMDV replication and clearance during persistent infection remain unclear. To identify host factors that are critical for FMDV replication during persistent infection, we used RNA-seq to compare the transcriptomes of infected (BHK-Op) cells and bystander (BHK-VEC) cells, which are exposed to FMDV but not infected. In total, 1917 genes were differentially expressed between BHK-Op cells and BHK-VEC cells, which were involved in ribosome biogenesis, cell cycle, and dilated cardiomyopathy. We further identified host genes potentially involved in viral clearance during persistent FMDV infection by comprehensive crossover analysis of differentially expressed genes in ancestral host cells, evolved infected host cells, and evolved bystander cells, which are resistant to infection by wild-type FMDV and FMDV-Op that co-evolved with host cells during persistent infection. Among the identified genes were Cav1 and Ccnd1. Subsequent experiments showed that knockdown of Cav1 and Ccnd1 in host cells significantly promoted and inhibited FMDV replication, respectively, confirming that the overexpression of Cav1 and the downregulation of Ccnd1 contribute to virus clearance during persistent FMDV infection. In addition, we found that BHK-Op cells contained mixtures of multiple genotypes of FMDV viruses, shedding light on the diversity of FMDV genotypes during persistent infection. Our findings provide a detailed overview of the responses of infected cells and bystander cells to persistent FMDV infection.

Recognizing adsorption of Cd(â ¡) by a novel core-shell mesoporous ion-imprinted polymer: Characterization, binding mechanism and practical application.

A novel monodispersed Cd(II) ion-imprinted polymer (IIP) was synthesized inside core-shell mesoporous silica (C-SMS) particles to improve the diffusion kinetics of the polymer. The synthesized IIP@C-SMS was characterized and subsequently used in solid-phase extraction (SPE) for the selective adsorption of Cd(II) in aquatic samples. The results indicated that IIP had been successfully assembled inside the C-SMS particles with a high specific surface area (546.3 m2 g-1) and uniform mesoporous size (2.07 nm). The obtained IIP@C-SMS takes only 15 min to reach the adsorption equilibrium due to the highly developed mesoporous structure. IIP@C-SMS also presented a maximal adsorption capacity (201.9 µmol g-1) for Cd(II), which was much higher than that of NIP@C-SMS (80.3 µmol g-1). The relative selectivity coefficient of IIP@C-SMS for Cd(II)/M(II) (M = Cu(II), Pb(II), Cr(II), and Ni(II)) were 7.15, 8.70, 7.18, and 7.36, respectively, further confirming the satisfactory selectivity of IIP@C-SMS. The adsorption isotherms of IIP@C-SMS toward Cd(II) could be described by Langmuir model; whereas the adsorption kinetics could be fitted by the pseudo-second-order model, indicating chemisorption was the rate-limiting step. The FT-IR, ITC and XPS analysis further confirmed that the Cd(II)-induced cavities during the ion-imprinting process and the coordination between Cd(II) and -SH groups were the main adsorption mechanism. Furthermore, in real samples, IIP@C-SMS-SPE adsorbed approximately 93-104% of Cd(II). This work provides new insights for the design of novel macroporous sorbents for Cd(II).

Pullout strength of miniscrews placed in anterior mandibles of adult and adolescent dogs: a microcomputed tomographic analysis.

INTRODUCTION: Miniscrews often loosen during orthodontic treatment, especially in teenage patients. The purposes of this study were to explore the differences of the pullout strengths of miniscrews placed in the anterior mandibles of adolescent and adult dogs and the structural parameters of peri-miniscrew bone, and to analyze the correlation between the pullout strengths and the variables of the peri-miniscrew bone structure. METHODS: Eight adult beagles and 8 young beagles with early permanent dentitions were used as experimental subjects. Two miniscrews were symmetrically placed in the anterior mandible of each dog several minutes before death. The bone density, relative bone volume, and cortical bone thickness were evaluated by micro-computed tomography, and the pullout strength of the miniscrew was tested with a testing machine. Regression analyses were used to study the relationship between pullout strength and bone density, relative bone volume, and cortical bone thickness. RESULTS: The values of bone density, relative bone volume, cortical bone thickness, and pullout strength were 781.94 + or - 21.46 mg of hydroxyapatite per cubic centimeter, 0.62 + or - 0.33, 1.14 + or - 0.11 mm, and 218.40 + or - 24.50 N for the adult dogs; and 713.61 + or - 13.08 mg of hydroxyapatite per cubic centimeter, 0.57 + or - 0.20, 1.07 + or - 0.86 mm, and 130.82 + or - 2.20 N for the young dogs, respectively. All pairs of pullout force and bone structural parameters had significant correlation coefficients. The pullout force showed the strongest correlation with bone density and the weakest with cortical bone thickness. CONCLUSIONS: The values of bone density, relative bone volume, cortical bone thickness, and the pullout strength of the adult group were higher than those of the young group. Furthermore, bone density is more sensitive in terms of showing pullout force compared with relative bone volume and cortical bone thickness.

Protective and therapeutic effects of nanoliposomal quercetin on acute liver injury in rats.

BACKGROUND: Quercetin, a pigment (flavonoid) found in many plants and foods, has good effects on protecting liver function but poor solubility and bioavailability in vivo. A drug delivery system can improve the accumulation and bioavailability of quercetin in liver. In this study, we used liposomal nanoparticles to entrap quercetin and evaluated its protective and therapeutic effects on drug-induced liver injury in rats. METHODS: The nanoliposomal quercetin was prepared by a thin film evaporation-high pressure homogenization method and characterized by morphology, particle size and drug content. Acute liver injury was induced in rats by composite factors, including carbon tetrachloride injection, high-fat corn powder intake and ethanol drinking. After pure quercetin or nanoliposomal quercetin treatment, liver function was evaluated by detecting serum levels of glutamic-pyruvic transaminase (GPT), glutamic-oxal acetic transaminase (GOT) and direct bilirubin (DBIL). Histology of injured liver tissues was evaluated by hematoxylin and eosin staining. RESULTS: On histology, liposomal nanoparticles loading quercetin were evenly distributed spherical particles. The nanoliposomal quercetin showed high bioactivity and bioavailability in rat liver and markedly attenuated the liver index and pathologic changes in injured liver tissue. With nanoliposomal quercetin treatment, the serum levels of GPT, GOT and DBIL were significantly better than treated with pure quercetin. Using liposomal nanoparticles to entrap quercetin might be an effective strategy to reduce hepatic injury and protect hepatocytes against damage. CONCLUSION: Liposomal nanoparticles may improve the solubility and bioavailability of quercetin in liver. Furthermore, nanoliposomal quercetin could effectively protect rats against acute liver injury and may be a new hepatoprotective and therapeutic agent for patients with liver diseases.

Establishment of a three-dimensional culture and mechanical loading system for skeletal myoblasts.

Establishment of a three-dimensional (3-D) culture and mechanical loading system which simulates the in vivo environment is critical in cytomechanical studies. The present article attempts to do this by integrating porous PLGA scaffolds with a four-point bending strain unit. Three types of PLGA scaffolds with three average pore sizes were synthesized, i.e., type I (60-88 microm), type II (88-100 microm) and type III (100-125 microm). To establish the 3-D mechanical loading system, PLGA membrane was integrated with conventional force-loading plates and the third passage skeletal myoblasts from neonatal Sprague-Dawley (SD) rats were seeded. Small PLGA membranes were put in 24-well plates followed by cell implantation and MTT assay was performed on days 1, 2, 4, 6 and 8 to compare biocompatibility of the three types of scaffolds. After 3 days' culture, many more cells had grown in type II than in type I or type III under fluorescence microscopy. In the MTT assay, OD of type II was significantly higher (P < 0.05) than the other two, especially at the early stage. As type II proved to be the best among the three, it was used as the scaffold in the preliminary mechanical loading study and 4000 mu-strain cyclic uniaxial strain was imposed. The system worked well and it was found that short to median time of stretching enhances while prolonged time of stretching inhibits cell proliferative activity of the 3-D cultured skeletal myoblasts(P < 0.05). It is concluded that the combination of PLGA scaffolds with a four-point bending strain unit provides a satisfactory 3-D mechanical loading system.

Static magnetic field combined with functional appliances: a new approach to enhance mandibular growth in Class II malocclusion.

Class II malocclusion is one of the most common orthodontic problems. The main aetiology of Class II malocclusion is mandibular retrognathia. A variety of functional appliances have been used to stimulate mandibular growth in adolescence, however, the effects remain unsatisfactory. Therefore, new approaches are in need to strengthen the effects of functional appliances. Static magnetic field (SMF), created by permanent magnets, has long been proven to be clinically safe and is well accepted as a practical and non-invasive therapy. Numerous experimental and clinical data suggest that exogenous SMF can make profound effects on a large variety of biological systems. There has been increasing interest in curing bone injuries with SMF. More recently, literatures shed light on the chondrogenic and osteogenic effects of SMF. SMF and functional appliances may well have a synergistic effect in mandibular growth promotion. Based on experimental results and theoretical analysis, it is hypothesized that SMF combined with functional appliances can enhance mandibular growth in Class II malocclusion. A practical clinical design is also put forward.

[Histological study of miniscrew implanted in lower mandibles of juvenile and adult Beagle dogs].

OBJECTIVE: To examine the histological discrepancies between juvenile and adult Beagle dogs at different concrescence times after miniscrew implants. METHODS: Miniscrew implants were performed in six juvenile Beagle dogs and six adult Beagle dogs. The space between the fourth premolar root and first molar root, and the spaces in distal and mesial of M1 root were picked up for the implants of the 48 miniscrews. The lower jaw specimens including the miniscrews were harvested 3 and 12 weeks after the implants for the histological examinations and bone implant contact (BIC) calculations. RESULTS: There was no miniscrew falling off or becoming loose. The miniscrews had favorable biological consistencies with the tissues around them. The osteoblasts and osteoclasts showed active functions in the peri-bones of the minisrews. The BIC became higher when the healing time was prolonged. The juvenile Beagle dogs had lower BIC than the adults 3 weeks after the implants. But the BIC of the juvenile dogs surpassed the adults 12 weeks after the implants. CONCLUSION: The osseointegration of the miniscrews is hindered by the poorer bone quality of the juvenile Beagle dogs. But the early osseointegration deficiency can be made up by the rapid development and growth of the bones of the juvenile dogs.

[Research progress on the relationship between centric relation and orthodontic treatment].

Centric relation (CR) is one of the core research contents in orthodontics, prosthodontics, and gnathology, acting as an important physiological factor in reconstructing the occlusion and adjusting the occlusal relationship. For over a century, CR is still a controversial subject in dentistry. CR has been redefined for several times, and recently, its application has been widened in orthodontics, including orthodontic diagnosis, clinical examination and analysis, and treatment goals. The purpose of this article is to review the definition of CR, its relationship with malocclusion, and the application of this relationship in orthodontic treatment.

Orthodontic Treatment Planning based on Artificial Neural Networks.

In this study, multilayer perceptron artificial neural networks are used to predict orthodontic treatment plans, including the determination of extraction-nonextraction, extraction patterns, and anchorage patterns. The neural network can output the feasibilities of several applicable treatment plans, offering orthodontists flexibility in making decisions. The neural network models show an accuracy of 94.0% for extraction-nonextraction prediction, with an area under the curve (AUC) of 0.982, a sensitivity of 94.6%, and a specificity of 93.8%. The accuracies of the extraction patterns and anchorage patterns are 84.2% and 92.8%, respectively. The most important features for prediction of the neural networks are "crowding, upper arch" "ANB" and "curve of Spee". For handling discrete input features with missing data, the average value method has a better complement performance than the k-nearest neighbors (k-NN) method; for handling continuous features with missing data, k-NN performs better than the other methods most of the time. These results indicate that the proposed method based on artificial neural networks can provide good guidance for orthodontic treatment planning for less-experienced orthodontists.

A novel MSX1 intronic mutation associated with autosomal dominant non-syndromic oligodontia in a large Chinese family pedigree.

BACKGROUND: Tooth agenesis is a common developmental dental anomaly. The aim of the study was to identify the causal genetic mutation in a four-generation Chinese family affected with non-syndromic autosomal dominant tooth agenesis. METHODS: Genome-wide scanning was performed using the Illumina Linkage-12 array. Genotyping of short tandem repeat markers was used to finely map the causative locus. Haplotype analysis and Sanger sequencing was performed to precisely locate the position and nature of the gene defect. RESULTS: Clinical examination of the available 23 family members showed variable tooth agenesis in 10 subjects, ranging from oligodontia to mild hypodontia. Genome-wide scanning and haplotype analyses identified the 4p16.1-p16.3 region with a maximum multi-point LOD score of 3.50, which overlapped with the MSX1 gene. A single heterozygous point mutation IVS1-5 G>A in the MSX1 gene was exclusively detected in the 10 family members affected with tooth agenesis. Sequencing of MSX1 cDNA revealed that the intronic mutation did not affect the normal splicing pattern of the pre-mRNA. However, real-time qPCR analysis of lymphocyte RNA showed that the level of MSX1 mRNA was significantly decreased in individuals heterozygous for the mutation. CONCLUSIONS: We identified and characterized a novel intronic mutation in the MSX1 gene in a large Chinese pedigree, adding to the small repertoire of MSX1 mutations associated with autosomal dominant tooth agenesis. We hypothesize that the variable degree of tooth agenesis observed in each affected individual may be due to sub-optimal levels of MSX1 expression during critical stages tooth development.

Orthodontic mini-implant stability under continuous or intermittent loading: a histomorphometric and biomechanical analysis.

BACKGROUND: Both continuous and intermittent loadings are commonly applied in orthodontics. Clinical experiences and some studies believed that longer duration of force produce more effect (tooth movement, suture expansion, bone remodeling) than transient forces applied with the same magnitude. Alternatively, others indicated that interruption or recovery periods of various periods between loadings cause more bone remodeling and less root resorption. Therefore, which force is more favorable for osseointegration and stability of orthodontic mini-implant remains to be elucidated. PURPOSE: To evaluate the influence of continuous or intermittent loading on stability of titanium mini-implants. MATERIALS AND METHODS: One hundred ninety-two mini-implants were implanted bilaterally in intraradicular zones of mandibular M1 and P2 in 48 beagles. Loadings were delivered consecutively in continuous group, pauses were given for the last 3 or 7 days of each 2-week reactivation period for intermittent group A and B, respectively. The group unloaded was set as control. After 2, 4, 6 and 8 weeks, the animals were sacrificed and microscopic computerized tomography (µCT), histomorphological observation and pull-out test were applied. RESULTS: The µCT parameters of mini-implants in four groups were gradually increased with loading time prolonged, while the value of peak load at extraction (F(max)) increased and reached summit at week 6, but dropped slightly at week 8. In continuous group, all measurements were lower than those in intermittent groups at all time points (p < .05), and all values in intermittent group B were higher than those in intermittent group A. Histomorphology observation revealed different degrees of bone remodeling with new bone formation in the peri-implants region in different groups. CONCLUSIONS: Intermittent loading regimen is more favorable for obtaining stability than continuous force.

Novel de novo nonsense mutation of the PHEX gene (p.Lys50Ter) in a Chinese patient with hypophosphatemic rickets.

X-linked hypophosphatemic rickets (XLHR), the most common form of inherited rickets, is a dominant disorder characterized by hypophosphatemia, abnormal bone mineralization, and short stature. Mutations in the PHEX gene are major causes of XLHR. Herein, we clinically characterized four unrelated families with hypophosphatemia, bone abnormalities, short stature, and dentin malformation. Mutational analysis of the PHEX gene using Sanger sequencing revealed three recurrent mutations (c.2197T>C, c.1646G>C, and c.2198G>A) and a de novo nonsense mutation (c.148A>T). The novel mutation was not found in any of the unaffected family members or in the 100 healthy controls and was predicted to produce a truncated protein (p.K50X), a truncated form of the PHEX protein caused by nonsense mutations has been frequently detected in XLHR individuals. Thus, our work indicated that the c.148A>T (p.K50X) mutation was the likely pathogenic mutation in individual III-2 in family 2, and that PHEX gene mutations were responsible for XLHR in these Chinese families. These findings expand the mutation spectrum of PHEX and may help us to understand the molecular basis of XLHR in order to facilitate genetic counseling.

Microcomputed tomographic and biomechanical analysis of orthodontic microscrew stability under continuous or intermittent force.

PURPOSE: To evaluate and compare the influence of continuous and intermittent forces on stability of titanium microscrews. MATERIALS AND METHODS: One hundred forty-four microscrews were inserted bilaterally in the intraradicular zones of the maxillary first molar and second premolar in 36 beagles. Loads were delivered consecutively in the continuous group (n = 12), in cycles of 12 hours on/paused for 12 hours in intermittent group A (n = 12), and in cycles of 24 hours on/paused for 24 hours in intermittent group B (n = 12). The on/off cycles were repeated for 1, 3, 5, or 7 weeks, after which the animals were sacrificed, and microcomputed tomography (micro-CT) and pull-out testing were performed. RESULTS: The micro-CT parameters of the microscrews in all three groups increased gradually with loading time. The value of peak load at extraction (Fmax) increased and reached a peak at week 5 but dropped slightly at week 7. In the continuous group, all measurements were lower than those in the intermittent groups at all times examined. All values in intermittent group A were higher than those in intermittent group B. CONCLUSION: An intermittent loading regimen appears to be more favorable for obtaining stability than continuous loading, and a 12-hour/12-hour on/off loading cycle is superior to a 24-hour/24-hour on/off protocol in promoting bone-implant contact.

The osteogenic differentiation of PDLSCs is mediated through MEK/ERK and p38 MAPK signalling under hypoxia.

PURPOSE: During orthodontic treatment and chronic periodontitis, the periodontal vasculature is severely impaired by overloaded mechanical force or chronic inflammation. This leads to the hypoxic milieu of the periodontal stem cell niche and ultimately affects periodontal tissue remodelling. However, the role of hypoxia in the regulation of periodontal ligament stem cell (PDLSC) behaviours still remains to be elucidated. The present study was aimed at investigating the effects of hypoxia on osteogenic differentiation, mineralisation and paracrine release of PDLSCs and further demonstrating the involvement of mitogen-activated protein kinase (MAPK) signalling in the process. METHODS: First, PDLSCs were isolated and characterised. Second, the effects of different periods of hypoxia on PDLSC osteogenic potential, mineralisation and paracrine release were investigated. Third, extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 kinase activities under hypoxia were measured. Finally, specific MAPK inhibitors PD98059 and SB203580 were employed to investigate the involvement of two kinases in PDLSC osteogenesis under hypoxia. RESULTS: Immunocytochemical staining and multilineage differentiation assays verified that the isolated cells were PDLSCs. Cell viability, alkaline phosphatase (ALP) activity, messenger RNA (mRNA) and protein levels of runt-related transcription factor 2 (Runx2) and Sp7, mineralisation and prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) release were significantly increased by hypoxia. ERK1/2 and p38 were activated in different ways under hypoxia. Furthermore, hypoxia-stimulated transcription and expression of the above-mentioned osteogenic regulators were also reversed by PD98059 and SB203580 to different degrees. CONCLUSIONS: Exposure of PDLSCs to hypoxia affected their osteogenic potential, mineralisation and paracrine release, and the process involved mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and p38 MAPK signalling.

Coculture with endothelial cells enhances osteogenic differentiation of periodontal ligament stem cells via cyclooxygenase-2/prostaglandin E2/vascular endothelial growth factor signaling under hypoxia.

BACKGROUND: During periodontitis and orthodontic tooth movement, periodontal vasculature is severely impaired, leading to a hypoxic microenvironment of periodontal cells. However, the impact of hypoxia on periodontal cells is poorly defined. The present study investigates responses of cocultured endothelial cells (ECs) and periodontal ligament stem cells (PDLSCs) to hypoxia. METHODS: Osteogenic differentiation, molecular characterization, and various behaviors of PDLSCs and human umbilical venous ECs under hypoxia were assessed by quantitative real-time reverse-transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Moreover, the effect of ECs on PDLSC osteogenic differentiation was tested using NS398 (cyclooxygenase 2 blocker), SU5416 (vascular endothelial growth factor [VEGF] receptor inhibitor), AH6809, L-798106, and L-161982 (EP1/2/3/4 antagonists). RESULTS: First, hypoxia promoted osteogenic differentiation in PDLSCs and enhanced EC migration, whereas PD98059 (extracellular signal-regulated protein kinase [ERK] inhibitor) blocked, and cocultured ECs further enhanced, hypoxia-induced osteogenic differentiation. Second, NS398 impaired EC migration and prostaglandin E2 (PGE2)/VEGF release, whereas cocultured PDLSCs and exogenous PGE2 partially reversed it. Third, NS398 (pretreated ECs) decreased PGE2/VEGF concentrations. NS398-treated ECs and AH6809/SU5416-treated PDLSCs impaired cocultured EC-induced enhancement of PDLSC osteogenic differentiation. CONCLUSIONS: Hypoxia enhances ERK-mediated osteogenic differentiation in PDLSCs. Coculture with EC further augments PDLSC osteogenic differentiation via cyclooxygenase-2/PGE2/VEGF signaling.