CDC42-mediated Wnt signaling facilitates odontogenic differentiation of DPCs during tooth root elongation.

BACKGROUND: CDC42 is a member of Rho GTPase family, acting as a molecular switch to regulate cytoskeleton organization and junction maturation of epithelium in organ development. Tooth root pattern is a highly complicated and dynamic process that dependens on interaction of epithelium and mesenchyme. However, there is a lack of understanding of the role of CDC42 during tooth root elongation. METHODS: The dynamic expression of CDC42 was traced during tooth development through immunofluorescence staining. Then we constructed a model of lentivirus or inhibitor mediated Cdc42 knockdown in Herwig's epithelial root sheath (HERS) cells and dental papilla cells (DPCs), respectively. Long-term influence of CDC42 abnormality was assessed via renal capsule transplantation and in situ injection of alveolar socket. RESULTS: CDC42 displayed a dynamic spatiotemporal pattern, with abundant expression in HERS cells and apical DPCs in developing root. Lentivirus-mediated Cdc42 knockdown in HERS cells didn't disrupt cell junctions as well as epithelium-mesenchyme transition. However, inhibition of CDC42 in DPCs undermined cell proliferation, migration and odontogenic differentiation. Wnt/ß-catenin signaling as the downstream target of CDC42 modulated DPCs' odontogenic differentiation. The transplantation and in situ injection experiments verified that loss of CDC42 impeded root extension via inhibiting the proliferation and differentiation of DPCs. CONCLUSIONS: We innovatively revealed that CDC42 was responsible for guiding root elongation in a mesenchyme-specific manner. Furthermore, CDC42-mediated canonical Wnt signaling regulated odontogenic differentiation of DPCs during root formation.

PPAR-γ activation promotes xenogenic bioroot regeneration by attenuating the xenograft induced-oxidative stress.

Xenogenic organ transplantation has been considered the most promising strategy in providing possible substitutes with the physiological function of the failing organs as well as solving the problem of insufficient donor sources. However, the xenograft, suffered from immune rejection and ischemia-reperfusion injury (IRI), causes massive reactive oxygen species (ROS) expression and the subsequent cell apoptosis, leading to the xenograft failure. Our previous study found a positive role of PPAR-γ in anti-inflammation through its immunomodulation effects, which inspires us to apply PPAR-γ agonist rosiglitazone (RSG) to address survival issue of xenograft with the potential to eliminate the excessive ROS. In this study, xenogenic bioroot was constructed by wrapping the dental follicle cells (DFC) with porcine extracellular matrix (pECM). The hydrogen peroxide (H2O2)-induced DFC was pretreated with RSG to observe its protection on the damaged biological function. Immunoflourescence staining and transmission electron microscope were used to detect the intracellular ROS level. SD rat orthotopic transplantation model and superoxide dismutase 1 (SOD1) knockout mice subcutaneous transplantation model were applied to explore the regenerative outcome of the xenograft. It showed that RSG pretreatment significantly reduced the adverse effects of H2O2 on DFC with decreased intracellular ROS expression and alleviated mitochondrial damage. In vivo results confirmed RSG administration substantially enhanced the host's antioxidant capacity with reduced osteoclasts formation and increased periodontal ligament-like tissue regeneration efficiency, maximumly maintaining the xenograft function. We considered that RSG preconditioning could preserve the biological properties of the transplanted stem cells under oxidative stress (OS) microenvironment and promote organ regeneration by attenuating the inflammatory reaction and OS injury.

Combined orthopedic-orthodontic treatments of adolescent skeletal open-bite with severe molar-incisor hypomineralization: a case report and literature review.

The treatment of adolescent skeletal open-bite malocclusion with severe molar-incisor hypomineralization (MIH) remains challenging. Though conducive to open-bite treatment and endodontic management, early molar extraction may trigger a series of negative impacts on occlusion and stomatognathic development. In addition, molars' crown restoration was shown to worsen open-bite malocclusion considering the intrinsic vertical increment of hyperdivergent growth. This case report describes the successful multidisciplinary therapy combined with orthopedic and orthodontic treatment of a 10.2-year-old girl with mixed dentition, a protruding profile and skeletal open-bite malocclusion with severe MIH and crowding. During the mixed and early permanent dentition, function regulator-4 (FR-4), resin-bonding transpalatal arch (TPA) and modified spring-loaded bite blocks were implemented to correct abnormal swallowing and control the facial vertical growth. Radiographic results, including the counterclockwise rotation of the occlusion plane, decreasing mandibular angle and increasing posterior-anterior face height ratio accompanied by obvious mandibular vertical growth, indicated that the performed orthopedic treatments efficiently controlled hyperdivergent open-bite growth during puberty. After the maxillary and mandibular second molars were occluded, all first permanent molars were extracted, and fixed appliances combined with implant anchorage were used to correct malocclusion and convex profile. Ultimately, a stable Class I functional occlusion and satisfying facial improvement were achieved and maintained following a 2-year follow-up.

Generating bioactive and antiseptic interfaces with nano-silver hydroxyapatite-based coatings by pulsed electrochemical deposition for long-term efficient cervical soft tissue sealing.

Infections related to osseointegrated implants have sparked the interest in studying titanium modification for long-term effective soft tissue sealing. Constructing a silver (Ag)-hydroxyapatite (HA) coating is regarded as an effective strategy for integrating antibiosis with osteanagenesis; however, the outcome for long-term cervical soft tissue sealing in vivo is compromised. It is challenging to construct an Ag-HA coating for long-term efficient soft tissue integration that instills a maximum antibacterial effect while retaining favorable bioactivity to normal gingival mesenchymal cells in vivo. In this study, we employed gradient concentrations of Ag/CaP by pulsed electrochemical deposition to fabricate optimal Ag-HA nanocoatings. By physicochemical analyses, these uniform coatings were mainly formed with spherical metallic and hydroxyapatite nanoparticles, which facilitated good hydrophilicity, moderate rough surfaces and corrosion protection. Furthermore, the nanocoating of the 1.5Ag/CaP group exhibited superior performances in dental follicle cells' proliferation, osteogenic differentiation and antibacterial properties mainly through direct contact inhibition and partially through sustained silver ion release, which resulted in functional cervical soft tissue sealing in beagles lasting for one year. Our investigations provide a feasible strategy to balance the long-term antibacterial demand and bioactive induction around osseointegrated implants for long-term efficient cervical soft tissue sealing.

Smurf1 regulates ameloblast polarization by ubiquitination-mediated degradation of RhoA.

Cell polarity is essential for ameloblast differentiation and enamel formation. Smurf1 can mediate cell polarization through ubiquitination degradation of specific substrates. But it remains unclear whether Smurf1 could regulate ameloblast polarity and the underlying mechanism. Here, immuno-fluorescence staining and RT-qPCR were applied to detect the expression of Smurf1 and F-actin. A mouse lower incisor defect model was constructed. Scanning electron microscope, rat lower incisor culture, western blot, wound healing assay and trans-well migration assay were performed to detect the influence of Smurf1 knockdown on ameloblast. IF double staining, western blot and co-immunoprecipitation were conducted to detect the interaction between Smurf1 and RhoA. The in vivo experiment was also performed. We found that Smurf1 was mainly expressed in the membrane and cell cortex of ameloblast, similar to F-actin. Smurf1 expression increased along ameloblast polarization and differentiation. After knocking down Smurf1, the cytoskeleton and cell morphology changed and the cell polarity was damaged. Smurf1 regulated ameloblast polarity through ubiquitination degradation of activated RhoA in vitro. Local knockdown of Smurf1 in rat lower incisor ameloblast resulted in ameloblast polarity loss, enamel matrix secretion disorder and chalky enamel, but RhoA inhibitor Y-27632 could reverse this effect. Collectively, Smurf1 could regulate the polarization of ameloblast through ubiquitination degradation of activated RhoA, which contributed to the knowledge of tooth development and provided new research ideas for cell polarity.

ROS-Mediated Enamel Formation Disturbance Characterized by Alternative Cervical Loop Cell Proliferation and Downregulation of RhoA/ROCK in Ameloblasts.

Reactive oxygen stress (ROS) is generally accepted as a signal transducer for coordinating the growth and differentiation of tissues and organs in the oral and maxillofacial region. Although ROS has been confirmed to affect the development of enamel, it is not yet known that the specific mechanism of ROS accumulation induced enamel defects. Given the lack of knowledge of the role of ROS in enamel, the aim of the study is to determine how oxidative stress affects cervical cells and ameloblast cells. Using SOD1 knockout mice, we identified a relationship between ROS fluctuations and abnormal enamel structure with HE staining, micro-CT, and scanning electron microscope. Increased ROS induced by H2O2, certified by the DCFH probe, has resulted in a dual effect on the proliferation and differentiation of cervical cells, indicating a higher tendency to proliferate at low ROS concentrations. Ameloblasts transfected with SOD1 siRNA showed a significant reduction of RhoA and ROCK. This study investigates for the first time that SOD1-mediated ROS accumulation disrupted normal enamel structure through alternative cervical loop cell proliferation and downregulation of RhoA and ROCK in ameloblasts, demonstrating the convoluted role of ROS in monitoring the progress of enamel defects.

Analysis of complex chromosomal rearrangements using a combination of current molecular cytogenetic techniques.

BACKGROUND: Using combined fluorescence in situ hybridization (FISH) and high-throughput whole-genome sequencing (WGS) molecular cytogenetic technology, we aim to analyze the junction breakpoints of complex chromosome rearrangements (CCR) that were difficult to identify by conventional karyotyping analysis and further characterize the genetic causes of recurrent spontaneous abortion. RESULTS: By leveraging a combination of current molecular techniques, including chromosome karyotype analysis, FISH, and WGS, we comprehensively characterized the extremely complex chromosomal abnormalities in this patient with recurrent spontaneous abortions. Here, we demonstrated that combining these current established molecular techniques is an effective and efficient workflow to identify the structural abnormalities of complex chromosomes and locate the rearrangement of DNA fragments. CONCLUSIONS: In conclusion, leveraging results from multiple molecular and cytogenetic techniques can provide the most comprehensive genetic analysis for genetic etiology research, diagnosis, and genetic counseling for patients with recurrent spontaneous abortion and embryonic abortion.

Xenoextracellular matrix-rosiglitazone complex-mediated immune evasion promotes xenogenic bioengineered root regeneration by altering M1/M2 macrophage polarization.

Xenogenic extracellular matrix (xECM)-based organ transplantation will be a promising approach to address the problem of donor shortage for allotransplantation, which has achieved great success in organ regeneration. However, current approaches to utilize xECM-based organ have limited capacity to yield the host a biofriendly microenvironment for long-term immunity homeostasis, compromising the application of these xenografts for repairing and replacing damaged tissues. As the key innate immune cells, macrophages directly determine the prognosis of xenografts in long term. However, it has not been fully elucidated that how to modulate their biological behavior for microenvironment homeostasis in tissue reconstruction. In this study, we report a robust strategy to impart an immunosuppressive surface to naturally sponge-like porous xECM scaffolds by loading rosiglitazone (RSG) to activate peroxisome proliferators receptors-γ (PPAR-γ). The resultant xECM-RSG complex, enabling RSG to be delivered sequentially and continuously to cells without obvious systemic side effects, is recognized as "self" to escape immune monitoring in local immunoregulation by downregulating the expression of proinflammatory NOS2+ M1 macrophages and oxygen species (ROS) through suppressing NF-κB expression, greatly facilitating the regeneration of enthesis anchoring between the transplanted xenograft and host in both heterotopic and orthotopic models. The newly formed bio-root is morphologically and biomechanically equivalent to native tooth root with a significant expression of odontogenic differentiation-related critical proteins. Therefore, the PPAR-γ-NF-κB axis activated by the xECM-RSG complex enables the xenografts to converse towards M2 macrophages with a modest immunosuppressive capacity for facilitating in xECM-based tissue or organ regeneration.

Improvement of ECM-based bioroot regeneration via N-acetylcysteine-induced antioxidative effects.

BACKGROUND: The low survival rate or dysfunction of extracellular matrix (ECM)-based engineered organs caused by the adverse effects of unfavourable local microenvironments on seed cell viability and stemness, especially the effects of excessive reactive oxygen species (ROS), prompted us to examine the importance of controlling oxidative damage for tissue transplantation and regeneration. We sought to improve the tolerance of seed cells to the transplant microenvironment via antioxidant pathways, thus promoting transplant efficiency and achieving better tissue regeneration. METHODS: We improved the antioxidative properties of ECM-based bioroots with higher glutathione contents in dental follicle stem cells (DFCs) by pretreating cells or loading scaffolds with the antioxidant NAC. Additionally, we developed an in situ rat alveolar fossa implantation model to evaluate the long-term therapeutic effects of NAC in bioroot transplantation. RESULTS: The results showed that NAC decreased H2O2-induced cellular damage and maintained the differentiation potential of DFCs. The transplantation experiments further verified that NAC protected the biological properties of DFCs by repressing replacement resorption or ankylosis, thus facilitating bioroot regeneration. CONCLUSIONS: The following findings suggest that NAC could significantly protect stem cell viability and stemness during oxidative stress and exert better and prolonged effects in bioroot intragrafts.

The Dual Effects of Reactive Oxygen Species on the Mandibular Alveolar Bone Formation in SOD1 Knockout Mice: Promotion or Inhibition.

The status of reactive oxygen species (ROS) correlates closely with the normal development of the oral and maxillofacial tissues. Oxidative stress caused by ROS accumulation not only affects the development of enamel and dentin but also causes pathological changes in periodontal tissues (periodontal ligament and alveolar bone) that surround the root of the tooth. Although previous studies have shown that ROS accumulation plays a pathologic role in some oral and maxillofacial tissues, the effects of ROS on alveolar bone development remain unclear. In this study, we focused on mandibular alveolar bone development of mice deficient in superoxide dismutase1 (SOD1). Analyses were performed using microcomputerized tomography (micro-CT), TRAP staining, immunohistochemical (IHC) staining, and enzyme-linked immunosorbent assay (ELISA). We found for the first time that slightly higher ROS in mandibular alveolar bone of SOD1(-/-) mice at early ages (2-4 months) caused a distinct enlargement in bone size and increased bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), and osteopontin (OPN). With ROS accumulation to oxidative stress level, increased trabecular bone separation (Tb.Sp) and decreased expression of ALP, Runx2, and OPN were found in SOD1(-/-) mice at 6 months. Additionally, dosing with N-acetylcysteine (NAC) effectively mitigated bone loss and normalized expression of ALP, Runx2, and OPN. These results indicate that redox imbalance caused by SOD1 deficiency has dual effects (promotion or inhibition) on mandibular alveolar bone development, which is closely related to the concentration of ROS and the stage of growth. We present a valuable model here for investigating the effects of ROS on mandibular alveolar bone formation and highlight important roles of ROS in regulating tissue development and pathological states, illustrating the complexity of the redox signal.

Xenogeneic native decellularized matrix carrying PPARγ activator RSG regulating macrophage polarization to promote ligament-to-bone regeneration.

Host immune response to tissue engineering tissues or organs directly determines the graft survival and the integration with host. Our and other previous studies have successfully regenerated the organs/tissues based on allogeneic native decellularized matrix (aNDM). But the very limited aNDM clinically hinders the artificial organs/tissues application to resolve the native organs/tissues loss with high incidence. However, the xenogeneic NDM will induce host immune rejection leading to the transplantation failure. This study constructed the xenogeneic (porcine) NDM (xNDM) which carried the immunoregulator Rosiglitazone (xNDM-RSG), a synthetic highly selective agonist of peroxisome proliferator-activated receptor-γ (PPARγ), evaluated xNDM's physical and chemical characterization, immunomodulatory properties, and its effect on the tissue regeneration. Results showed that the xNDM-RSG did not affect the proliferation and differentiation of odontogenic stem cells. In addition, the xNDM-RSG could also effectively decrease the expression of IL-1 and TNFα, and increase the expression of IL-10 and TGFß to enable a favorable immunomodulation and promote the ligament-to-bone regeneration by PPARγ to induce the alternative activated macrophages (M2 macrophages) antagonizing classically activated macrophages (M1 macrophages). Meanwhile the xNDM-RSG obviously reduces the implants absorption and promotes the regenerated ligament-to-bone expressing the key proteins (ALP, OPN, DSP) which are relative to the native dental and bone. This study demonstrated that protein adsorption could aggravate the immune inflammatory reaction, whereas, xNDM-RSG could effectively control the host immune response to accelerate tissue reparation and regeneration by facilitating the macrophage polarization, which highlighted a new strategy for improving the transplantation survival of the artificial organ or tissue based on the xenogeneic decellularized biomaterials.

Blood cell parameters for screening and diagnosis of hereditary spherocytosis.

BACKGROUND: There is currently no single index for the diagnostic screening of hereditary spherocytosis (HS). However, hematology analyzers are widely used in hospital laboratories because of their highly automated performance and quality control procedure, and detection of some blood cell parameters may be useful for the early screening of HS. METHODS: We investigated the values of blood cell parameters for the screening and differential diagnosis of HS. We performed a descriptive study of 482 samples (67 cases of HS, 59 cases of G6PD deficiency, 57 cases of AIHA, 199 cases of thalassemia, and 100 cases of healthy controls) that were run on Beckman Coulter LH780 Hematology Analyzer. RESULTS: HS was characterized by increased MCHC, decreased MRV, MSCV-MCV < 0, and increased Ret with no concomitant increase in IRF. The areas under the ROC curves were MSCV-MCV (0.97; 95% CI 0.95-1.0) > MRV (0.94; 95% CI 0.91-0.97) > MCHC (0.92; 95% CI 0.88-0.97) > Ret/IRF (0.77; 95% CI 0.7-0.84). MSCV-MCV ≤ 0.6 fl was valuable parameter for the diagnostic screening of HS, with a sensitivity of 95.5% and specificity of 94.9%. CONCLUSION: These indices have high reference values for differentiating HS from thalassemia, AIHA, and G6PD deficiency.

Molecular Genetic Mechanisms of Hereditary Spherocytosis: Current Perspectives.

With the widespread use of genetic diagnostic technologies, many novel mutations have been identified in hereditary spherocytosis (HS)-related genes, including SPTA1, SPTB, ANK1, SLC4A1, and EPB42. However, mutations in HS-related genes are dispersed and nonspecific in the diagnosis of some HS patients, indicating significant heterogeneity in the molecular deficiency of HS. It is necessary to provide the molecular and genetic characteristics of these 5 genes for clinicians to examine HS. Here, we reviewed the recent proposed molecular genetic mechanisms of HS.

Mean reticulocyte volume: a specific parameter to screen for hereditary spherocytosis.

This study assessed the value of mean reticulocyte volume (MRV) for differential diagnosis of hereditary spherocytosis (HS) so as to develop conventional and new specific screen indexes. Subjects in this study were divided into three groups: 53 cases in HS group, 217 cases in hemolytic anemia control group (109 cases of thalassemia (THAL), 56 cases of glucose-6-phosphate dehydrogenase G6PD deficiency anemia, and 52 cases of autoimmune hemolytic anemia (AIHA)), and 100 cases in healthy control group. We analyzed erythrocyte and reticulocyte parameters including MRV, mean sphered corpuscular volume, mean corpuscular hemoglobin concentration, and immature reticulocyte fraction. Results demonstrated that MRV was significantly lower in the HS group but significantly higher in the AIHA and G6PD deficiency anemia groups than that in the healthy control group (P = 0.000). MRV was not significantly different between the AIHA and G6PD deficiency anemia groups (P = 0.977) and between the healthy control and THAL groups (P = 0.168). The area under the ROC curve of MRV for diagnosis of HS was 0.942, with a standard error of 0.019, 95% confidence interval of 0.905-0.979, and optimal critical diagnosis point of 95.77 fL. When the MRV was ≤95.77 fL, the sensitivity and specificity for diagnosis of HS were 86.80% and 91.20%, respectively. Therefore, MRV is a general and specific new index for screening HS and important for differential diagnosis of different types of hemolytic anemia.

[Imported falciparum malaria: one case report and literature review].

This paper reports the hematology screening and parasite morphological features of one case of imported falciparum malaria, and reviews the relevant literature.