Dkk-1-TNF-α crosstalk regulates MC3T3E1 pre-osteoblast proliferation and differentiation under mechanical stress through the ERK signaling pathway.

The study aims to explore the role of the ERK signaling pathway in the crosstalk between Dkk-1 and TNF-α in MC3T3E1 pre-osteoblasts under cyclic tensile/compressive stress. A forced four-point bending system was used to apply cyclic uniaxial tensile/compressive strain (2000 µ, 0.5 Hz) to MC3T3E1 cells. Dkk-1 and TNF-α expression were upregulated in MC3T3E1 cells under compressive strain. Cell proliferation, the cell cycle, osteogenesis-related gene (Wnt5a, Runx2, Osterix) expression, ß-catenin expression, and the p-ERK/ERK ratio were significantly enhanced, whereas apoptosis, the RANKL/OPG ratio, and TNF-α expression were significantly attenuated, by Dkk-1 silencing. Dkk-1 expression increased and the effects of Dkk-1 silencing were reversed when exogenous TNF-α was added. Mechanically, TNF-α crosstalked with Dkk-1 through ERK signaling in MC3T3E1 cells. ERK signaling blockade impaired Dkk-1-induced TNF-α expression and TNF-α-mediated Dkk-1 expression. Dkk-1 and TNF-α crosstalked, partially through ERK signaling, in MC3T3E1 cells under compressive/tensile strain, synergistically modulating various biological behaviors of the cells. These findings not only provide mechanical insight into the cellular events and molecular regulation of orthodontic tooth movement (OTM), but also aid the development of novel strategies to accelerate OTM.

Miniscrews for orthodontic anchorage: analysis of risk factors correlated with the progressive susceptibility to failure.

INTRODUCTION: The phenomenon of orthodontic anchorage miniscrews loosening after being implanted several times happens in daily clinical practice, and the reasons need to be traced. This study aimed to investigate the underlying risk factors influencing the progressive susceptibility of orthodontic miniscrews to failure. METHODS: Overall, 889 miniscrews were successively inserted into 347 patients because some loosened or fell off once, twice, or more before achieving their purposes. The number of miniscrew failures (ie, once, twice, or more) was defined as progressive susceptibility to failure. The clinical indicators were assessed via univariate analysis, multicollinearity diagnosis, and Poisson log-linear regression model with stepwise calculation to screen out. RESULTS: The progressive susceptibility of miniscrews to failure was proved to be affected by the age of patients, the onset of force application, site of placement, and appliance type. Age and onset of force application presented a negative relationship with susceptibility. Miniscrews inserted in the palatal region appeared to be more stable than the forepart of the arch. In contrast, the retromaxillary and retromandibular areas obtained the lowest stability. The patients with fixed appliances were more unlikely to suffer progressive failure than removable appliances. In addition, the larger number of screws inserted in each patient, the greater probability of failure. CONCLUSIONS: Younger people with removable appliances that miniscrews inserted in the retromaxillary or retromandibular regions and earlier onsets of loading had a higher progressive susceptibility to loosening. Meanwhile, the failure rate was elevated with the increasing number of screws per patient received.

A Janus, robust, biodegradable bacterial cellulose/Ti3C2Tx MXene bilayer membranes for guided bone regeneration.

Guided bone regeneration (GBR) stands as an essential modality for craniomaxillofacial bone defect repair, yet challenges like mechanical weakness, inappropriate degradability, limited bioactivity, and intricate manufacturing of GBR membranes hindered the clinical efficacy. Herein, we developed a Janus bacterial cellulose(BC)/MXene membrane through a facile vacuum filtration and etching strategy. This Janus membrane displayed an asymmetric bilayer structure with interfacial compatibility, where the dense layer impeded cell invasion and the porous layer maintained stable space for osteogenesis. Incorporating BC with Ti3C2Tx MXene significantly enhanced the mechanical robustness and flexibility of the material, enabling clinical operability and lasting GBR membrane supports. It also contributed to a suitable biodegradation rate, which aligned with the long-term bone repair period. After demonstrating the desirable biocompatibility, barrier role, and osteogenic capability in vitro, the membrane's regenerative potential was also confirmed in a rat cranial defect model. The excellent bone repair performance could be attributed to the osteogenic capability of MXene nanosheets, the morphological cues of the porous layer, as well as the long-lasting, stable regeneration space provided by the GBR membrane. Thus, our work presented a facile, robust, long-lasting, and biodegradable BC/MXene GBR membrane, offering a practical solution to craniomaxillofacial bone defect repair.

Circ_0036490 and DKK1 competitively bind miR-29a to promote lipopolysaccharides-induced human gingival fibroblasts injury.

MicroRNA (miRNA) plays a regulatory role in periodontitis. This study aimed to explore whether miR-29a could affect lipopolysaccharides (LPSs)-induced injury in human gingival fibroblasts (HGFs) through the competitive endogenous RNAs (ceRNA) mechanism. Periodontal ligament (PDL) tissues and HGFs were derived from patients with periodontitis and healthy volunteers. Periodontitis cell model was established by treating HGFs with LPS. Expression levels of circ_0036490, miR-29a, and DKK1 were evaluated by the reverse transcription quantitative real-time PCR (RT-qPCR) method. Western blotting assay was performed to assess protein expression levels of pyroptosis-related proteins and Wnt signalling related proteins. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. Concentration of lactate dehydrogenase (LDH), interleukin (IL)-1ß, and IL-18 were determined by Enzyme-linked immunosorbent assay (ELISA). Pyroptosis rate were determined by flow cytometry assay to evaluate pyroptosis. The interaction between miR-29a and circ_0036490 or DKK1 was verified by dual-luciferase reporter and RNA pull-down assays. MiR-29a expression was lower in PDL tissues of patients with periodontitis than that in healthy group; likewise, miR-29a was also downregulated in LPS-treated HGFs. Overexpression of miR-29a increased cell viability and decreased pyroptosis of HGFs induced by LPS while inhibition of miR-29a exerted the opposite role. MiR-29a binds to circ_0036490 and elevation of circ_0036490 contributed to dysfuntion of LPS-treated HGFs and reversed the protection function of elevated miR-29a. In addition, miR-29a targets DKK1. Overexpression of DKK1 abrogated the effects of overexpressed miR-29a on cell vaibility, pyroptosis, and protein levels of Wnt signalling pathway of LPS-treated HGFs. Circ_0036490 and DKK1 competitively bind miR-29a to promote LPS-induced HGF injury in vitro. Wnt pathway inactivated by LPS was activated by miR-29a. Thence, miR-29a may be a promising target for periodontitis.

What operative or anatomic factors affect dental arch development in the cleft patient?

PURPOSE: There has been dispute about the exact factors influencing dental arch development in adult cleft palate patients, so we designed this study to investigate the effects of operative and anatomic factors on the development of dental arch morphology. PATIENTS AND METHODS: A retrospective cohort study was conducted among 3 groups of patients (operated, unoperated, and normal) from West China College of Stomatology, Sichuan University, Chengdu, China. The differences in dental arch morphology, including length and width of the upper and lower dental arches, inclination of the palatal shelf, and palatal height, were analyzed by variance analysis with SPSS software, version 13.0 (IBM, Armonk, NY). RESULTS: We enrolled 90 individuals: 30 unoperated adults with bilateral cleft lip and palate, 30 adults with operated bilateral cleft lip and palate, and 30 normal adults. The widths of all upper and posterior lower arches, lengths of the anterior upper arch, palatal height, and palatal shelf inclination in the operated group were smaller than those in the unoperated group; the lengths and widths of the anterior upper arch were smaller whereas the widths of the posterior upper and lower arches, palatal height, and palatal shelf inclination were greater in the unoperated group compared with the normal group. CONCLUSIONS: Operated cleft patients show the most severe deformation of the maxillary arch, especially in the anterior part. There is an intrinsic palatal tissue deficiency in cleft patients, whereas the maxillary arch deformation in unoperated cleft patients is limited to the anterior region only.

Alteration of maxillary and mandibular growth of adult patients with unoperated isolated cleft palate.

OBJECTIVE: The objective of this study was to investigate the effects of cleft palate itself on the growth of maxilla and mandible. PATIENTS AND METHODS: Fifty-two adult female patients with unoperated isolated cleft palate and 52 adult female individuals with normal occlusion were included in our study. Computer software was used for lateral cephalometry measurement. Manual measurement was performed for dental cast measurements, and sample t test analysis was applied to analyze the differences between the 2 groups using SPSS 17.0. RESULTS: The sella-nasion-subspinale point angle, subspinale-nasion-supramentale point angle, and maxillary arch length of the cleft group were significantly smaller than those of the control group (P < 0.01). Both maxillary and mandibular posterior dental arch widths of the cleft group were significantly larger compared with the control group (P < 0.01), whereas the sella-nasion-supramentale point angle, mandible arch length, palate height, and palate shelf inclination did not differ between the 2 groups. The measurements did not differ between the submucosal cleft and the overt cleft patients. CONCLUSIONS: Cleft palate itself has adverse effects on the maxilla growth with shorter maxillary arch length and wider posterior dental arch width.

Identification of a novel missense mutation in non-syndromic familial multiple supernumerary teeth.

OBJECTIVE: This study intended to evaluate the involvement of genetic factors in the etiology of non-syndromic multiple supernumerary teeth. DESIGN: We filtered the single nucleotide polymorphisms (SNPs) of the proband and his mother with similar phenotypes through whole-genome sequencing (WGS). By integrating multiple databases related to human genome mutations and disease information for mutation annotation, we excluded the SNPs of people without supernumerary teeth. Subsequently, the bioinformatics analysis tools (Sorting Intolerant From Tolerant (SIFT) < 0.05, Polymorphism Phenotyping (PolyPhen) > 0.90) were used to screen out the most correlated SNPs of the disease, besides, Gene Ontology (GO) analysis (P<0.05, FDR<0.05) and Sanger sequencing was applied to further verify the candidate pathogenic mutation point. RESULTS: A novel heterozygous variant in fer-1 like family member 6 (FER1L6) gene likely denoted pathogenicity in non-syndromic familial multiple supernumerary teeth. We identified a cohort of 3499 non-synonymous SNPs (nsSNPs), and only 142 nsSNPs with the score of SIFT < 0.05 and PolyPhen > 0.90 were retained. Then we got 54 nsSNPs from 31 candidate genes through GO analysis. Sanger sequencing revealed a missense variant in exon 31 of the FER1L6 gene, causing a transition from guanine to adenine in position 1447 of protein kinase C conserved region 2. CONCLUSIONS: We identified a novel heterozygous chromosome 8q24.13 mutation of FER1L6, which was a new mutation site identified in non-syndromic familial multiple supernumerary teeth through genetic analysis of a Chinese family.

Three-dimensional, biomimetic electrospun scaffolds reinforced with carbon nanotubes for temporomandibular joint disc regeneration.

Temporomandibular disorder (TMD) remained a huge clinical challenge, with high prevalence but limited, unstable, and only palliative therapeutic methods available. As one of the most vulnerable sites implicated in TMD, the temporomandibular joint disc (TMJD) displayed a complicated microstructure, region-specific fibrocartilaginous distribution, and poor regenerative property, which all further hindered its functional regeneration. To address the problem, with versatile and relatively simple electrospinning (ELS) technique, our study successfully fabricated a biomimetic, three-dimensional poly (ϵ-caprolactone) (PCL)/polylactide (PLA)/carbon nanotubes (CNTs) disc scaffold, whose biconcave gross anatomy and regionally anisotropic microstructure recapitulating those of the native disc. As in vitro results validated the superior mechanical, bioactive, and regenerative properties of the biomimetic scaffolds with optimal CNTs reinforcement, we further performed in vivo experiments. After verifying its biocompatibility and ectopic fibrochondrogenicity in nude mice subcutaneous implantation models, the scaffolds guided disc regeneration and subchondral bone protection were also confirmed orthotopically in rabbits TMJD defected areas, implying the pivotal role of morphological cues in contact-guided tissue regeneration. In conclusion, our work represents a significant advancement in complex, inhomogeneous tissue engineering, providing promising clinical solutions to intractable TMD ailments. STATEMENT OF SIGNIFICANCE: Complex tissue regeneration remains a huge scientific and clinical challenge. Although frequently implicated in temporomandibular joint disorder (TMD), functional regeneration of injured temporomandibular joint disc (TMJD) is extremely hard to achieve, mainly because of the complex anatomy and microstructure with regionally variant, anisotropic fiber alignments in the native disc. In this study, we developed the biomimetic electrospun scaffold with optimal CNTs reinforcement and regionally anisotropic fiber orientations. The excellent mechanical and bioactive properties were confirmed both in vitro and in vivo, effectively promoting defected discs regeneration in rabbits. Besides demonstrating the crucial role of morphological biomimicry in tissue engineering, our work also presents a feasible clinical solution for complex tissue regeneration.

Carbon Nanotube Reinforced Collagen/Hydroxyapatite Scaffolds Improve Bone Tissue Formation In Vitro and In Vivo.

Current bone regeneration strategies faced major challenges in fabricating the bionic scaffolds with nano-structure, constituents and mechanical features of native bone. In this study, we developed a new porous scaffold by adding the multi-walled carbon nanotube (MWCNT) into collagen (Col)/hydroxyapatite (HA) composites. Data showed that 0.5%CNT/Col/HA (0.5%CNT) group was approximately tenfolds stiffer than Col-HA, and it was superior in promoting bone marrow mesenchymal stem proliferation and spreading, mRNA and protein expressions of bone sialoprotein (BSP) and osteocalcin (OCN) than Col-HA group. Moreover, we utilized 0.5%CNT composite to repair the rat calvarial defects (8 mm diameter) in vivo, and observed the new bone formation by 3D reconstruction of micro CT, HE and Masson staining, and BSP, OCN by immunohistochemical analysis. Results showed that newly formed bone in 0.5%CNT group was significantly higher than that in Col-HA group at 12 weeks. These findings highlighted a promising strategy in healing of large area bone defect with MWCNT added into the Col-HA scaffold as they possessed the combined effects of mechanical strength and osteogenicity.

Factors Affecting the Clinical Success Rate of Miniscrew Implants for Orthodontic Treatment.

PURPOSE: The purpose of this study was to evaluate the various factors that influence the success rate of miniscrew implants used as orthodontic anchorage. MATERIALS AND METHODS: Potential confounding variables examined were sex, age, vertical (FMA) and sagittal (ANB) skeletal facial pattern, site of placement (labial and buccal, palatal, and retromandibular triangle), arch of placement (maxilla and mandible), placement soft tissue type, oral hygiene, diameter and length of miniscrew implants, insertion method (predrilled or drill-free), angle of placement, onset and strength of force application, and clinical purpose. The correlations between success rate and overall variables were investigated by logistic regression analysis, and the effect of each variable on the success rate was utilized by variance analysis. RESULTS: One hundred fourteen patients were included with a total of 253 miniscrew implants. The overall success rate was 88.54% with an average loading period of 9.5 months in successful cases. Age, oral hygiene, vertical skeletal facial pattern (FMA), and general placement sites (maxillary and mandibular) presented significant differences in success rates both by logistic regression analysis and variance analysis (P < .05). CONCLUSION: To minimize the failure of miniscrew implants, proper oral hygiene instruction and effective supervision should be given for patients, especially young (< 12 years) high-angle patients with miniscrew implants placed in the mandible.

A comparative cephalometric study for adult operated cleft palate and unoperated cleft palate patients.

OBJECTIVE: To determine the effects of a cleft deformity unilateral cleft lip and palate (UCLP) and the subsequent surgical interventions on maxillary growth. MATERIALS AND METHODS: This retrospective study evaluated the lateral cephalograms of 3 groups of individuals: 40 adult patients with ULCP who underwent surgery for both lip and palate; 40 adult patients with ULCP who underwent surgery for lip only; and 40 age- and gender-matched noncleft controls. Differences in jaw morphology among them were analyzed statistically. RESULTS: Adult UCLP patients in both groups showed maxillary hypoplasia in anteroposterior and vertical directions compared with noncleft control adults. Significant differences (p ≤ 0.01) in the ANB (subspinale-nasion-supramentale angle), NA-FH (the angle formed between Frankfort horizontal plane and the plane from nasion to subspinale), MP-FH (the angle formed between mandibular plane and Frankfort horizontal plane), and GoMe/SN (the ratio between length of mandibular body and length of anterior cranial base) were found between the two UCLP patient groups. Although maxillary growth in the two UCLP groups was less than that in the noncleft control group, the anteroposterior growth in the UCLP patients with palatoplasty was even less than that in the UCLP patients with unoperated palate. CONCLUSIONS: There may be an intrinsic deficiency of maxillary anteroposterior and vertical development in UCLP patients compared with the noncleft controls. Palatoplasty can further limit the anteroposterior growth of maxilla but has no detrimental effect on maxillary vertical development. The mandible is rotated clockwise after palatoplasty in UCLP patients.

Incorporation of aligned PCL-PEG nanofibers into porous chitosan scaffolds improved the orientation of collagen fibers in regenerated periodontium.

The periodontal ligament (PDL) is a group of highly aligned and organized connective tissue fibers that intervenes between the root surface and the alveolar bone. The unique architecture is essential for the specific physiological functionalities of periodontium. The regeneration of periodontium has been extensively studied by researchers, but very few of them pay attention to the alignment of PDL fibers as well as its functionalities. In this study, we fabricated a three-dimensional multilayered scaffold by embedding highly aligned biodegradable poly (ε-caprolactone)-poly(ethylene glycol) (PCE) copolymer electrospun nanofibrous mats into porous chitosan (CHI) to provide topographic cues and guide the oriented regeneration of periodontal tissue. In vitro, compared with random group and porous control, aligned nanofibers embedded scaffold could guide oriented arrangement and elongation of cells with promoted infiltration, viability and increased periodontal ligament-related genes expression. In vivo, aligned nanofibers embedded scaffold showed more organized arrangement of regenerated PDL nearly perpendicular against the root surface with more extensive formation of mature collagen fibers than random group and porous control. Moreover, higher expression level of periostin and more significant formation of tooth-supporting mineralized tissue were presented in the regenerated periodontium of aligned scaffold group. Incorporation of aligned PCE nanofibers into porous CHI proved to be applicable for oriented regeneration of periodontium, which might be further utilized in regeneration of a wide variety of human tissues with a specialized direction. STATEMENT OF SIGNIFICANCE: The regeneration of periodontium has been extensively studied by researchers, but very few of them give attention to the alignment of periodontal ligament (PDL) fibers as well as its functionalities. The key issue is to provide guidance to the orientation of cells with aligned arrangement of collagen fibers perpendicular against the root surface. This study aimed to promote oriented regeneration of periodontium by structural mimicking of scaffolds. The in vitro and in vivo performances of the scaffolds were further evaluated to test the topographic-guiding and periodontium healing potentials. We also think our research may provide ideas in regeneration of a wide variety of human tissues with a specialized direction.

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.

Histomorphometric and biomechanical analyses of the osseointegration of loaded orthodontic microscrews inserted at different cortical bone thickness sites.

OBJECTIVES: The aim of this study was to evaluate the osseointegration of loaded orthodontic microscrews at different cortical bone thickness (CBT) sites. STUDY DESIGN: Forty-eight microscrews were inserted bilaterally in the tibias of 12 beagles, and divided into thick and thin CBT group. After 1, 3, 5, and 7 weeks, the dogs were killed and bone-screw specimens prepared for polyfluorochrome sequential labeling, microscopic computerized tomography (µCT) analysis, and biomechanical pull-out testing. RESULTS: All µCT parameters were increased in the thin CBT group, and decreased in the thick CBT group with time passed. Fmax of microscrews in thick CBT sites reached a peak in week 1, but dropped to lowest levels in week 3. Remodeling, apposition, and measured deposition areas of 3 labels were significantly higher in the thin CBT group than in the thick CBT group (P < .05). CONCLUSIONS: Relatively larger CBT is of benefit for primary stability at early time points, whereas thinner CBT is more appropriate for achieving long-term stability with prolonged time.

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.

Response of immortalized murine cementoblast cells to hypoxia in vitro.

OBJECTIVES: The aim of the study was to investigate the impact of hypoxia on proliferation, apoptosis and mineralization of cementoblast-like cells (OCCM-30) in vitro. METHODS: The effects of different periods of hypoxia (2% O2) on proliferation, apoptosis, cementoblastic potential and root cementum resorption capability of OCCM-30 were evaluated, by using MTT, flow cytometry, alkaline phosphatase (ALP) activity assay, reverse transcription-polymerase chain reaction measurement, enzyme-linked immunosorbent assay and mineralization nodule formation assay. RESULTS: OCCM-30 viability was significantly inhibited by hypoxia while the apoptosis ratio was enhanced in a time-dependent manner; hypoxia inducible factor-1α and vascular endothelial growth factor mRNA were induced by hypoxia in different manners; temporary hypoxia (<24 h) stimulated cementoblastic function of OCCM-30, while long-term hypoxia inhibited it, manifested by decreased mRNA level or release of ALP, osteocalcin, bone sialoprotein, osteopontin and osteoprotegerin. In addition, hypoxia affected mineralized nodule formation of OCCM-30 in a time-dependent fashion; moreover, root cementum resorption function was also induced by hypoxia, manifested by increased receptor activator of nuclear factor kappa B ligand mRNA and protein expression. CONCLUSION: Temporary exposure of OCCM-30 to hypoxia inhibited proliferation, promoted apoptosis and mineralization, while longer duration of hypoxia could inhibit the cementoblast function. The findings may provide theoretical basis for developing novel therapeutics to prevent root resorption during orthodontic treatment.

Effect of placement angle on the stability of loaded titanium microscrews in beagle jaws.

OBJECTIVE: To evaluate the effect of insertion angle on stability of loaded titanium microscrews in beagle jaws. MATERIALS AND METHODS: Forty-eight microscrews were inserted at four different angles (30°, 50°, 70°, and 90°) into the intraradicular zones of the mandibular first molars and third premolars of 12 beagles and immediately loaded with a force of 2 N for 8 weeks. Microcomputed tomography (micro-CT) and biomechanical pull-out tests were used to assess osseointegration of the interface. RESULTS: All micro-CT parameters and maximum pull-out force (FMAX) of the microscrews were affected by insertion angles of microscrews. Higher micro-CT parameters and FMAX were seen for implants inserted at angles between 50° and 70° (P < .05). Excessive oblique and vertical insertion angles resulted in reduced stability (P < .05). CONCLUSION: An insertion angle of 50° to 70° is more favorable than excessive oblique or vertical angles to achieve stability of microscrews.

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.