修复再生受损的牙髓组织,恢复患牙天然的牙髓功能,逐渐成为治疗牙髓疾病的新的重要目标.浓缩生长因子是第3 代经自体全血离心后获得的血小板浓缩制品,富含大量三维网状结构的纤维蛋白和丰富的生长因子,对组织再生具有很强的促进作用,可作为牙髓再生的良好支架材料.本文就浓缩生长因子在牙髓组织再生中的作用机制进行综述,旨在为进一步的研究和临床应用提供理论支持.

Posttranslational glutamylation/deglutamylation balance in tubulins influences dendritic maturation and neuronal survival of cerebellar Purkinje neurons (PNs). PNs and some additional neuronal types degenerate in several spontaneous, independently occurring Purkinje cell degeneration (pcd) mice featuring mutant neuronal nuclear protein induced by axotomy (Nna1), a deglutamylase gene. This defective deglutamylase allows glutamylases to form hyperglutamylated tubulins. In pcd, all PNs die during postnatal “adolescence.” Neurons in some additional brain regions also die, mostly later than PNs. We show in laser capture microdissected single PNs, in cerebellar granule cell neuronal clusters, and in dissected hippocampus and substantia nigra that deglutamase mRNA and protein were virtually absent before pcd PNs degenerated, whereas glutaminase mRNA and protein remained normal. Hyperglutamylated microtubules and dimeric tubulins accumulated in pcd PNs and were involved in pcd PN death by glutamylase/deglutamylase imbalance. Importantly, treatment with a microtubule depolymerizer corrected the glutamylation/deglutamylation ratio, increasing PN survival. Further, before onset of neuronal death, pcd PNs displayed prominent basal polylisosomal masses rich in ER. We propose a “seesaw” metamorphic model summarizing mutant Nna1-induced tubulin hyperglutamylation, the pcd’s PN phenotype, and report that the neuronal disorder involved ER stress, unfolded protein response, and protein synthesis inhibition preceding PN death by apoptosis/necroptosis.

The molecular pathogenesis of bipolar disorder (BPD) is poorly understood. Using human-induced pluripotent stem cells (hiPSCs) to unravel such mechanisms in polygenic diseases is generally challenging. However, hiPSCs from BPD patients responsive to lithium offered unique opportunities to discern lithium's target and hence gain molecular insight into BPD. By profiling the proteomics of BDP-hiPSC-derived neurons, we found that lithium alters the phosphorylation state of collapsin response mediator protein-2 (CRMP2). Active non-phosphorylated CRMP2, which binds cytoskeleton, is present throughout the neuron; inactive phosphorylated CRMP2, which dissociates from cytoskeleton, exits dendritic spines. CRMP2 elimination yields aberrant dendritogenesis with diminished spine density and lost lithium responsiveness (LiR). The "set-point" for the ratio of pCRMP2: CRMP2 is elevated uniquely in hiPSC-derived neurons from LiR BPD patients, but not with other psychiatric (including lithium-nonresponsive BPD) and neurological disorders. Lithium (and other pathway modulators) lowers pCRMP2, increasing spine area and density. Human BPD brains show similarly elevated ratios and diminished spine densities; lithium therapy normalizes the ratios and spines. Consistent with such "spine-opathies," human LiR BPD neurons with abnormal ratios evince abnormally steep slopes for calcium flux; lithium normalizes both. Behaviorally, transgenic mice that reproduce lithium's postulated site-of-action in dephosphorylating CRMP2 emulate LiR in BPD. These data suggest that the " lithium response pathway" in BPD governs CRMP2's phosphorylation, which regulates cytoskeletal organization, particularly in spines, modulating neural networks. Aberrations in the posttranslational regulation of this developmentally critical molecule may underlie LiR BPD pathogenesis. Instructively, examining the proteomic profile in hiPSCs of a functional agent-even one whose mechanism-of-action is unknown-might reveal otherwise inscrutable intracellular pathogenic pathways.

Cerebellar MR imaging has several challenging aspects, due to the fine, repetitive layered structure of cortical folia with underlying axonal pathways. In this MR study, we imaged with high-angular resolution diffusion imaging (HARDI) abnormal cerebellar cortical structure (gray matter) and myelinated axonal pathways (white matter) of a mouse spontaneous mutation, Purkinje cell degeneration (pcd), in which almost all Purkinje neurons degenerate, mainly between postnatal days 20 and 35. Mouse brains at postnatal day 20 (P20) and at 8 months were scanned, and known or expected abnormalities, such as reduction of the white matter volume, disorganized pathways likely linked to parallel fibers, mossy fibers, and other fibers running from/to the cerebellar cortex were observed in mutant mice. Such abnormalities were detected at both an early and a fully advanced degeneration stage. These results suggest that our diffusion MR tractography is useful for early detection and tracking of neuropathology in the cerebellum.

Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens.To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ∼95% of the reduced-representation phosphopeptide probes to be detected in ∼200 samples.The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.

Blood vessel growth from preexisting vessels (angiogenesis) underlies many severe diseases including major blinding retinal diseases such as retinopathy of prematurity (ROP) and aged macular degeneration (AMD). This observation has driven development of antibody inhibitors that block a central factor in AMD, vascular endothelial growth factor (VEGF), from binding to its receptors VEGFR-1 and mainly VEGFR-2. However, some patients are insensitive to current anti-VEGF drugs or develop resistance, and the required repeated intravitreal injection of these large molecules is costly and clinically problematic. We have evaluated a small cyclic retro-inverted peptidomimetic, D(Cys-Leu-Pro-Arg-Cys) [D(CLPRC)], and hereafter named Vasotide, that inhibits retinal angiogenesis by binding selectively to the VEGF receptors VEGFR-1 and neuropilin-1 (NRP-1). Delivery of Vasotide via either eye drops or intraperitoneal injection in a laser-induced monkey model of human wet AMD, a mouse genetic knockout model of the AMD subtype called retinal angiomatous proliferation (RAP), and a mouse oxygen-induced model of ROP decreased retinal angiogenesis in all three animal models. This prototype drug candidate is a promising new dual receptor inhibitor of the VEGF ligand with potential for translation into safer, less-invasive applications to combat pathological angiogenesis in retinal disorders.

Significance Cerebellar Purkinje neurons (PNs) strongly affect motor coordination and learning. PN study has contributed significantly to fundamental concepts of modern neuroscience. The present investigation defines distinctive molecular signaling pathways through which tissue plasminogen activator/plasmin-based proteolysis regulates postnatal PN dendrite development, synapse formation, mitochondrial morphology and function, and PN survival. These pathways involve differentially acting downstream constituents, including protein kinase Cγ, brain-derived neurotrophic factor, and a voltage-dependent anion channel. The metabolic mechanisms established here may apply to the development and degeneration of PNs and additional types of neurons in many animal and human brain diseases in which PNs are notably vulnerable.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by the unremitting degeneration of motor neurons. Multiple processes involving motor neurons and other cell types have been implicated in its pathogenesis. Neural stem cells (NSCs) perform multiple actions within the nervous system to fulfill their functions of organogenesis and homeostasis. We test the hypothesis that transplanted, undifferentiated multipotent migratory NSCs may help to ameliorate an array of pathological mechanisms in the SOD1(G93A) transgenic mouse model of ALS. On the basis of a meta-analysis of 11 independent studies performed by a consortium of ALS investigators, we propose that transplanted NSCs (both mouse and human) can slow both the onset and the progression of clinical signs and prolong survival in ALS mice, particularly if regions sustaining vital functions such as respiration are rendered chimeric. The beneficial effects of transplanted NSCs seem to be mediated by a number of actions including their ability to produce trophic factors, preserve neuromuscular function, and reduce astrogliosis and inflammation. We conclude that the widespread, pleiotropic, modulatory actions exerted by transplanted NSCs may represent an accessible therapeutic application of stem cells for treating ALS and other untreatable degenerative diseases.

Titanium (Ti) and its alloys are used extensively in implants due to their excellent biocompatibility and mechanical properties. However, Ti-based implant materials have specific complications associated with their applications, such as the loosening of implanted host interface owing to unsatisfactory cell adhesion and the susceptibility of the implants to bacterial infections. Hence, a surface that displays selective biointeractivity, i.e., enhancing beneficial host cell responses but inhibiting pathogenic microbial adhesion, would be highly desirable. This study aims to confer long-lasting antibacterial properties and good biocompatibility on Ti via the microarc oxidation technique. The biocompatibility of the Ti surface was evaluated by cytotoxicity test, and the bacteriostasis rate was evaluated by antibacterial efficacy. The results showed that the implant surface might be nontoxic to cell and its long-lasting antibacterial properties could be significantly improved. These results indicate that such microarc oxidation coatings are expected to have good potential in transcutaneous implant applications.

We here propose an updated concept of stem cells (SCs), with an emphasis on neural stem cells (NSCs).The conventional view, which has touched principally on the essential property of lineage multipotency (e.g., the ability of NSCs to differentiate into all neural cells), should be broadened to include the emerging recognition of biofunctional multipotency of SCs to mediate systemic homeostasis, evidenced in NSCs in particular by the secretion of neurotrophic factors.Under this new conceptual context and taking the NSC as a leading example, one may begin to appreciate and seek the "logic" behind the wide range of molecular tactics the NSC appears to serve at successive developmental stages as it integrates into and prepares, modifies, and guides the surrounding CNS micro-and macro-environment towards the formation and self-maintenance of a functioning adult nervous system.We suggest that embracing this view of the "multipotency" of the SCs is pivotal for correctly, efficiently, and optimally exploiting stem cell biology for therapeutic applications, including reconstitution of a dysfunctional CNS.

In the field of induced potency and fate reprogramming, it remains unclear what the best starting cell might be and to what extent a cell need be transported back to a more primitive state for translational purposes. Reprogramming a committed cell back to pluripotence to then instruct it toward a particular specialized cell type is demanding and may increase risks of neoplasia and undesired cell types. Precursor/progenitor cells from the organ of therapeutic concern typically lack only one critical attribute--the capacity for sustained self-renewal. We speculated that this could be induced in a regulatable manner such that cells proliferate only in vitro and differentiate in vivo without the need for promoting pluripotence or specifying lineage identity. As proof-of-concept, we generated and tested the efficiency, safety, engraftability, and therapeutic utility of "induced conditional self-renewing progenitor (ICSP) cells" derived from the human central nervous system (CNS); we conditionally induced self-renewal efficiently within neural progenitors solely by introducing v-myc tightly regulated by a tetracycline (Tet)-on gene expression system. Tet in the culture medium activated myc transcription and translation, allowing efficient expansion of homogeneous, clonal, karyotypically normal human CNS precursors ex vivo; in vivo, where Tet was absent, myc was not expressed, and self-renewal was entirely inactivated (as was tumorigenic potential). Cell proliferation ceased, and differentiation into electrophysiologically active neurons and other CNS cell types in vivo ensued upon transplantation into rats, both during development and after adult injury--with functional improvement and without neoplasia, overgrowth, deformation, emergence of non-neural cell types, phenotypic or genomic instability, or need for immunosuppression. This strategy of inducing self-renewal might be applied to progenitors from other organs and may prove to be a safe, effective, efficient, and practical method for optimizing insights gained from the ability to reprogram cells.

Due to the pronounced oxidative nature of titanium at high temperatures, an excessively thick oxide layer may form on its surface. This oxide layer could adversely affect titanium-porcelain bonding. The aim of the present study was to investigate the effects of micro-arc oxidation on the titanium-porcelain bond strength which was relevant to the titanium oxide layer Twelve cast titanium specimens were prepared following the protocol ISO 9693. The test group was treated with micro-arc oxidation and the control group was treated with sandblasting. Contact angle and surface roughness were detected. SEM, XRD, and SEM/EDS analyses were performed on the titanium surfaces to ascertain bond failure. The groups were compared for their bond strength. The results showed that: there are differences in the surface morphology and the phase components of two groups. In addition, compared with the control group, the contact angle of the MAO group is smaller, but the surface roughness is similar. In the MAO specimen, the oxide layer was thin, and it was compact bonding with titanium and porcelain. The mean bond strength of the micro-arc oxidation group and the control group were 46.46 +/- 4.35 MPa and 33.28 +/- 2.24 MPa, respectively. While the improvement in bond strength was 39.6% for micro-arc oxidation group, it was statistically significant for the control group. These results suggest that the micro-arc oxidation technique is significantly effective in improving the bond strength of titanium-porcelain. Crown Copyright (c) 2009 Published by Elsevier B.V. All rights reserved.

The molecular pathways controlling cerebellar Purkinje cell dendrite formation and maturation are poorly understood. The Purkinje cell degeneration (pcd) mutant mouse is characterized by mutations in Nna1, a gene discovered in an axonal regenerative context, but whose actual function in development and disease is unknown. We found abnormal development of Purkinje cell dendrites in postnatal pcdSid mice and linked this deficit to a deletion mutation in exon 7 of Nna1. With single cell gene profiling and virus-based gene transfer, we analyzed a molecular pathway downstream to Nna1 underlying abnormal Purkinje cell dendritogenesis in pcdSid mice. We discovered that mutant Nna1 dramatically increases intranuclear localization of lysyl oxidase propeptide, which interferes with NF-κB RelA signaling and microtubule-associated protein regulation of microtubule stability, leading to underdevelopment of Purkinje cell dendrites. These findings provide insight into Nna1's role in neuronal development and why its absence renders Purkinje cells more vulnerable.

An oxide layer was formed on the surface of Ti after micro-arc oxidation (MAO) treatment, which contained F-, Cl- and I-. Then the bacterial adhesion test was carried out. The results show that the titanium after surface modification by MAO has certain antibacterial activity to the common flora around the implant, whether they are aerobic or facultative anaerobic bacteria. At the same time, the F-, Cl-, and I- ions in the oxide layer improve antibacterial properties further. Different crystalline structures result in different antibacterial properties. Under low voltage, the antibacterial properties of Ti are better due to the main product of anatase TiO2.

How grafted neural stem cells (NSCs) and their progeny integrate into recipient brain tissue and functionally interact with host cells is as yet unanswered. We report that, in organotypic slice cultures analyzed by ratiometric time-lapse calcium imaging, current-clamp recordings, and dye-coupling methods, an early and essential way in which grafted murine or human NSCs integrate functionally into host neural circuitry and affect host cells is via gap-junctional coupling, even before electrophysiologically mature neuronal differentiation. The gap junctions, which are established rapidly, permit exogenous NSCs to influence directly host network activity, including synchronized calcium transients with host cells in fluctuating networks. The exogenous NSCs also protect host neurons from death and reduce such signs of secondary injury as reactive astrogliosis. To determine whether gap junctions between NSCs and host cells may also mediate neuroprotection in vivo, we examined NSC transplantation in two murine models characterized by degeneration of the same cell type (Purkinje neurons) from different etiologies, namely, the nervous and SCA1 mutants. In both, gap junctions (containing connexin 43) formed between NSCs and host cells at risk, and were associated with rescue of neurons and behavior (when implantation was performed before overt neuron loss). Both in vitro and in vivo beneficial NSC effects were abrogated when gap junction formation or function was suppressed by pharmacologic and/or RNA-inhibition strategies, supporting the pivotal mediation by gap-junctional coupling of some modulatory, homeostatic, and protective actions on host systems as well as establishing a template for the subsequent development of electrochemical synaptic intercellular communication.

In this review, we propose an updated concept of the neural stem cell (NSC). New data of our own and others suggest that the field's conventional view which has touched principally on the essential multipotency of lineage phenotypes (i.e., the ability of NSC to differentiate into all neural cells), should be broadened to include the emerging recognition of the biofunctional multipotency of the NSC to mediate systemic homeostasis. Under this new conceptual context, one may begin to appreciate and seek the "logic" and teleology behind the wide range of molecular tactics the NSC appear to serve at each developmental stage as they integrate into and prepare, modify, and guide the surrounding CNS micro- and macro-environment towards the formation and self-maintenance of a physiologically functioning adult nervous system. We believe that embracing this view of the NSC's "multipotency" is pivotal for correctly, efficiently, and optimally exploiting stem cell biology for therapeutic applications, including reconstituting the dysfunctional CNS.

This study is intended to evaluate the effects of microarc oxidation (MAO) on the biocompatibility of near beta titanium alloy Ti-5Zr-3Sn-5Mo-15Nb (TLM) in vitro. Two porous bioactive Surfaces with different surface characteristics were grown on TLM substrates via MAO process at two different final voltages. Both calcium and phosphorus were incorporated into the oxidized Surfaces, and their content was dependent on the voltage applied. Surface roughness was enhanced on the MAO surfaces, which was higher when a higher voltage was applied. After MAO treatment, water contact angles became smaller and surface energies were increased, especially the polar components, which were also related to the MAO final voltage. Cell culture experiments showed an enhanced osteoblasts adhesion, spread, and viability on the microarc oxidized surfaces, and better cell spread and viability were found on the surface formed at 450 V than that at 300 V. No obvious variations in gene expression of integrin PI (Itg beta 1), core binding factor-alpha 1, osteopontin, collagen type I alpha 2-chain, and fibronectin by osteoblasts were observed on different surfaces. The expression of osteocalcin was strikingly increased on MAO surfaces after 72 h, thus indicating enhanced osteoblasts differentiation on MAO surfaces. Interestingly, obvious enhanced bone morphogenetic proteins (BMP)-2 and BMP-4 expression was observed on MAO surfaces, which may be a reason for the enhanced osteoblasts functions on MAO-modified TLM surfaces. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 92A: 432-440, 2010

The objective of this study was to modify the surface of a novel near beta titanium alloy Ti-5Zr-3Sn-5Mo-15Nb (TLM) by micro-arc oxidation (MAO). The surface characteristics of the TLM surfaces were analyzed and their effects on rat osteoblasts adhesion in vitro were evaluated. A porous bioactive oxide layer was grown on TLM substrate by MAO process. Both calcium and phosphorus were incorporated into the oxidized surfaces and their content was dependent on the applied voltage. Surface roughness was enhanced on the micro-arc oxidized surfaces, which was higher when the high voltage was applied. Surface wettability and surface energy of the TLM surface were increased by the MAO treatment, which were also related to the applied voltage. Cell culture experiment demonstrated a significant enhanced osteoblasts adhesion on the micro-arc oxidized surfaces.

Objective: To evaluate the influence of electrolyte`s concentration at micro-arc oxide treatment on the bonding strength of titanium to porcelain.Methods: CP titanium specimens with size of 25 mm×3 mm×0.5 mm were treated with micro-arc oxidation(MAO) in Na2SiO3 solution of three different concentrations.After ultra-low-fusing porcelain was applied,a three-point-flexure-test was used to evaluate the bonding strength of titanium to porcelain.The surface of the specimens was observed by SEM and EDS,as well as the interface between titanium and porcelain.Results: The bonding strength values between titanium treated with MAO and porcelain was significantly higher then the control group's,Group 20 g/L has the highest bonding strength values.SEM/EDS suggested that a porous thin layer of oxide which contains Si element is created by MAO,and higher concentration of Na2SiO3 leads to more Si element in oxide.Conclusion: MAO treating can improve the bonding strength between titanium and ceramic.Electrolyte's concentration has an effect on the bond strength between titanium and porcelain.

Molecular and cellular interactions coordinating the origin and fate of neural stem cells (NSCs) in the adult brain are far from being understood. We present a protein complex that controls proliferation and migration of adult NSCs destined for the mouse olfactory bulb (OB). Combinatorial selection based on phage display technology revealed a previously unrecognized complex between the soluble protein netrin-4 and laminin γ1 subunit that in turn activates an α6β1 integrin-mediated signaling pathway in NSCs. Differentiation of NSCs is accompanied by a decrease in netrin-4 receptors, indicating that netrin-4 participates in the continual propagation of this stem cell population. Notably, the stem cells themselves do not synthesize netrin-4. Further, we show that netrin-4 is produced by selected GFAP-positive astrocytes positioned close to newborn neurons migrating in the anterior part of the rostral migratory stream (RMS) and within the OB. Our findings present a unique molecular mechanism mediating astrocytic/neuronal crosstalk that regulates ongoing neurogenesis in the adult olfactory system.