Monitoring the first stages of the regeneration of bone defects

The different strategies of tissue engineering for functional reconstruction of critical-size bone defects require a thorough knowledge of physiological mechanisms of bone repair. Bone healing is a complex process affected by various mediators. Several investigations have studied the gene expression 1 to 3 days after an acute or experimental fracture. Little is known about the humoral and cellular in vivo reaction in the early stages of bone healing. In contrast to other methods of molecule sampling and detection, which usually lead to the inhibition of the biological activity following complex sample preparation and quantification, microdialysis is a real-time monitoring technique which can be applied in living tissues providing a strong link between analytical methodology and biochemistry. In this study, the optimal conditions for microdialysis in a critical size rat long bone defect model for both in vivo and in vitro analyses were developed. Mediators and components of the extracellular matrix occurring in the first 24 to 48 hours of bone healing locally and systemically were monitored via microdialysis and blood sampling, respectively. Furthermore, novel proteins and their modulation were explored during this time frame. In vitro microdialysis was used to optimize the condition for protein recovery. Addition of bovine serum albumin (BSA) resulted in an enhanced recovery of interleukin (IL)-6. The maximal relative recovery (RR) was from 15.0% without BSA and 23.6% with BSA, while the maximal RR of transforming growth factor (TGF)-β1 was 11.2% with BSA and the concentration of TGF-β1 was below the detection limit of enzyme-linked immunosorbent assay (ELISA) without BSA. Using in vivo microdialysis, total protein concentrations varied between 0.20±0.12 mg/mL and 0.44±0.18 mg/mL. Among the mediators produced in the fracture hematoma within 24 h after the injury, IL-6 was secreted with the highest concentration of 309.1 pg/mL between 12 and 15 h after creation of the critical size bone defect. Meanwhile, the detectable concentrations of TGF-β1 in microdialysates ranged from 3.6 to 44.0 pg/mL and in blood plasma TGF-β1 was constantly producted ranging from 656.3 to 8398.2 pg/mL for 24 h after bone defct. Moreover, another constant producted growth factor in blood plasma was PDGF-BB and the concentration ranged from 222.1 to 589.4 pg/mL for 8 h after bone defect. Using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), 36 proteins were identified in the microdialysates over 8 h, and 884 proteins were identified on probes which were implanted into the bone defect over 24 h. Among the proteins identified in the hematoma, only a minority originated from the extracellular space. Protein analysis indicated five pathways associated with bone healing that were overrepresented after creating soft tissue and bone defects, of which FGF signaling was specific for bone defects. Furthermore, C-X-C motif ligands CXCL-1, CXCL-2, CXCL-3, CXCL-4, CXCL-5, CXCL-7, rodent bone protein (RoBo-1), insulin-like growth factor (IGF)-I, and chitinase-3-like protein 1 were detected in the fracture hematoma. These proteins are potentially associated to early bone healing. As seen by histological analysis, polymorphonuclear leukocytes (PMNs) and lymphocytes penetrated into the fracture hematoma immediately after surgery and peaked at 24 h. This study for the first time presents data from both the local and systemic acute response to bone and soft tissue injury in a small animal model. The results of mcrodialysis sampling may serve as a baseline for future investigations on different models and time frames. Several proteins and pathways have been identifeid as potentially important for early bone regeneration warranting in depth analysis in further studies.:I. Table of content II. List of abbreviations 1 Summary 2 Introduction 2.1 The process of bone healing 2.1.1 Stages of fracture healing 2.1.2 Early stage of inflammation 2.2 Clinical challenges 2.3 Microdialysis 2.3.1 The principle of Microdialysis 2.3.2 Parameters influencing the recovery 2.4 Aim of this study 3 Materials 3.1 Materials, devices and animals 3.2 Chemicals 3.3 Buffers and solutions 4 Methods 4.1 Background 4.2 In vitro microdialysis 4.2.1 Preparation of the protein solution 4.2.2 Microdialysis sampling procedure 4.3 In vivo microdialysis 4.3.1 Surgical procedure 4.3.2 Sample collection 4.4 Plasma samples 4.5 Determination of the fluid recovery 4.6 Determination of the relative recovery 4.7 Total protein measurement 4.8 Cytokine and growth factor analysis 4.8.1 IL-1β, IL-6, TNF-α and PDGF-BB ELISA 4.8.2 VEGF ELISA 4.8.3 TGF-β1 ELISA 4.8.4 BMP-2 ELISA 4.8.5 Proteome profilerTM array 4.9 Proteomic analysis 4.10 Histological analysis 4.11 Statistical analysis 5 Results 5.1 Protein selection 5.2 Determination of fluid recovery in vitro and in vivo 5.3 Determination of relative recovery (RR) in vitro 5.4 Determination of total protein concentration in vivo 5.5 Determination of cytokine and growth factor concentration in the microdialysate in vivo 5.5.1 IL-6 concentration 5.5.2 TGF-β1 concentration 5.5.3 IL-1β concentration 5.5.4 TNF-α concentration 5.5.5 PDGF-BB, BMP-2 and VEGF concentration 5.6 Determination of further cytokines and chemokines in the microdialysate in vivo 5.7 Protein determination using HPLC-MS/MS analysis 5.7.1 Proteins in the microdialysate 5.7.2 Proteins on the surface of the probe 5.8 Protein annotation 5.9 Determination of cytokines and growth factors in the blood plasma 5.9.1 Determination of IL-6 in the blood plasma 5.9.2 Determination of TGF-β1 in the blood plasma 5.9.3 Determination of PDGF-BB in the blood plasma 5.10 Histological analysis of the hematoma 6 Discussion 6.1 Fluid recovery 6.2 Influence of the crystalloid perfusate on relative recovery 6.3 Relative recovery of cytokines and growth factors in vitro 6.4 In vivo microdialysis 6.4.1 Total protein concentration 6.4.2 Annotation of proteins in hematoma identified by HPLC-MS/MS 6.4.3 Identification of cytokines and bone related proteins 6.5 The humoral inflammatory response 6.6 Cellular response 7 Conclusions 8 References 9 Appendix 9.1 Figure index 9.2 Table index III. Eidesstattliche Erklärung IV. Selbständigkeitserklärung V. AcknowledgementsZur Entwicklung neuer Strategien der Geweberegenerierung in kritischen Knochendefekten, die sich durch Selbstheilungsprozesse nicht schließen, ist das Verständnis der beteiligten physiologischen Prozesse essentiell. Der Wiederaufbau von Gewebe, wie etwa während Knochenheilungsprozesse ist komplex reguliert und erfordert das koordinierte Zusammenspiel einer Vielzahl von Zellen und Mediatoren. Obwohl bereits in zahlreichen Studien die Veränderungen in der Genexpression in den ersten 3 Tagen nach einer akuten oder experimentell induzierten Fraktur untersucht wurden, ist noch immer wenig über die zellulären und humoralen Vorgänge in den frühen Phasen der Knochenheilung in vivo bekannt. Gebräuchliche Analysemethoden erfordern komplexe Verfahren zur Probenentnahme und Nachweisreaktionen währenddessen die biologische Aktivität der untersuchten Mediatoren häufig graduell verloren geht. Die Mikrodialyse hingegen kann in Echtzeit am lebenden Objekt und am Ort der Verletzung durchgeführt werden und bildet somit eine erfolgsversprechende Plattform um die Probengewinnung noch enger mit der anschließenden biochemischen Nachweistechnik zu verbinden. Im Rahmen dieser Arbeit wurden die optimalen Konditionen zur Mikrodialyse erstmals an einem kritischen Defektmodell eines Ratten-Röhrenknochens zur in vivo und in vitro Applikation ermittelt. Dazu wurde das Vorkommen verschiedener Komponenten der extrazellulären Matrix und ausgewählter Mediatoren während der ersten 24 bis 48 Stunden der Knochenheilung überwacht. Neben der durch Mikrodialyse gewonnenen Proben wurden auch Blutproben verarbeitet um sowohl die lokale, als auch systemische Konzentration der untersuchten Proteine zu erfassen. Durch eine Proteomanalyse konnten zudem bislang in diesem Prozess unbekannte Moleküle identifiziert und verfolgt werden. Zur Optimierung der Mikrodialyse wurden zunächst die Bedingungen hinsichtlich der Proteinrückgewinnung verbessert. Durch den Zusatz von Rinderserumalbumin (BSA) konnte die Rückgewinnung von Interleukin (IL)-6 erhöht werden. Die maximale relative Rückgewinnung (RR) konnte von 15.0% ohne BSA auf 23.6% mit BSA gesteigert werden. Noch dramatischer war dieser Effekt für den transforming growth factor (TGF)-β1 von dessen eingesetzter Menge in vitro 11.2% detektiert werden konnte, während in der BSA-freien Dialyselösung kein TGF-β1 nachgewiesen wurde. Die RR blieb stets unter der Detektionsgrenze des verwendeten enzyme-linked immunosorbent assay (ELISA). In vivo-Dialysate enthielten totale Proteinkonzentrationen zwischen 0,20±0,12 mg/mL und 0,44±0,18 mg/mL. Von den innerhalb von 24 h nach Verletzung im Frakturhämatom produzierten Mediatoren wurde IL-6 am stärksten exprimiert. Die höchsten Konzentrationen (309,1pg/mL) konnten hierfür nach 12 bis 15 Stunden nach Einführung des Defekts gemessen werden. Die Konzentrationslevel von TGF-β1 hingegegen betrug nur 3,6 bis 44,0 pg/mL.Mittels high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), konnten 36 Proteine in den über 8 Stunden gewonnenen Mikrodialysaten, und 884 Proteine von Explantaten, die 24 h im Knochendefekt integriert waren, identifiziert werden. Von den im Frakturhämatom identifizierten Proteinen war nur eine Minderheit extrazellulären Ursprungs. Durch die Proteomanalyse konnten fünf Signalwegskaskaden identifiziert werden. Von diesen trat „FGF (fibroblast growth factor) signaling“ ausschließlich in Knochendefekten, nicht jedoch in den zur Kontrolle mitgeführten reinen Weichgewebedefekten auf. Im Frakturhämatom konnten die, C-X-C motif-Liganden CXCL-1, CXCL-2,CXCL-3, CXCL-4, CXCL-5, CXCL-7, rodent bone protein (RoBo-1), insulin-like growth factor (IGF)-I, und das chitinase-3-like protein 1 nachgewiesen werden. Die identifizierten Proteine könnten von Bedeutung für die Steuerung früher Knochenheilungsprozesse sein. Histologische Untersuchungen zeigten, dass polymorphkernige Leukozyten (PMNs) und Lymphozyten sofort nach der Operation in das Frakturhämatom einwandern und ihre Anzahl nach etwa 24 h ihr Maximum erreicht. Diese Studie präsentiert erstmals Daten der lokal und systemisch ablaufenden zellulären und humoralen Vorgänge als Antwort auf einen Weichgewebs-bzw. Knochendefekt in einem Nagetier-Kleintiermodell. Die Mikrodialyse-Resultate stellen eine vielversprechende Grundlage für zukünftige Untersuchungen in anderen Modellen dar. Außerdem bilden die hier identifizierten Proteine und Signalwege eine Gruppe potenter Kandidaten für weiterführende Untersuchungen zur Knochenregeration.:I. Table of content II. List of abbreviations 1 Summary 2 Introduction 2.1 The process of bone healing 2.1.1 Stages of fracture healing 2.1.2 Early stage of inflammation 2.2 Clinical challenges 2.3 Microdialysis 2.3.1 The principle of Microdialysis 2.3.2 Parameters influencing the recovery 2.4 Aim of this study 3 Materials 3.1 Materials, devices and animals 3.2 Chemicals 3.3 Buffers and solutions 4 Methods 4.1 Background 4.2 In vitro microdialysis 4.2.1 Preparation of the protein solution 4.2.2 Microdialysis sampling procedure 4.3 In vivo microdialysis 4.3.1 Surgical procedure 4.3.2 Sample collection 4.4 Plasma samples 4.5 Determination of the fluid recovery 4.6 Determination of the relative recovery 4.7 Total protein measurement 4.8 Cytokine and growth factor analysis 4.8.1 IL-1β, IL-6, TNF-α and PDGF-BB ELISA 4.8.2 VEGF ELISA 4.8.3 TGF-β1 ELISA 4.8.4 BMP-2 ELISA 4.8.5 Proteome profilerTM array 4.9 Proteomic analysis 4.10 Histological analysis 4.11 Statistical analysis 5 Results 5.1 Protein selection 5.2 Determination of fluid recovery in vitro and in vivo 5.3 Determination of relative recovery (RR) in vitro 5.4 Determination of total protein concentration in vivo 5.5 Determination of cytokine and growth factor concentration in the microdialysate in vivo 5.5.1 IL-6 concentration 5.5.2 TGF-β1 concentration 5.5.3 IL-1β concentration 5.5.4 TNF-α concentration 5.5.5 PDGF-BB, BMP-2 and VEGF concentration 5.6 Determination of further cytokines and chemokines in the microdialysate in vivo 5.7 Protein determination using HPLC-MS/MS analysis 5.7.1 Proteins in the microdialysate 5.7.2 Proteins on the surface of the probe 5.8 Protein annotation 5.9 Determination of cytokines and growth factors in the blood plasma 5.9.1 Determination of IL-6 in the blood plasma 5.9.2 Determination of TGF-β1 in the blood plasma 5.9.3 Determination of PDGF-BB in the blood plasma 5.10 Histological analysis of the hematoma 6 Discussion 6.1 Fluid recovery 6.2 Influence of the crystalloid perfusate on relative recovery 6.3 Relative recovery of cytokines and growth factors in vitro 6.4 In vivo microdialysis 6.4.1 Total protein concentration 6.4.2 Annotation of proteins in hematoma identified by HPLC-MS/MS 6.4.3 Identification of cytokines and bone related proteins 6.5 The humoral inflammatory response 6.6 Cellular response 7 Conclusions 8 References 9 Appendix 9.1 Figure index 9.2 Table index III. Eidesstattliche Erklärung IV. Selbständigkeitserklärung V. Acknowledgement

Genotype-phenotype analysis of three Chinese families with Jervell and Lange-Nielsen syndrome

Long QT syndrome (LQTS) is characterized by QT prolongation, syncope and sudden death. This study aims to explore the causes, clinical manifestations and therapeutic outcomes of Jervell and Lange-Nielsen syndrome (JLNS), a rare form of LQTS with congenital sensorineural deafness, in Chinese individuals.Three JLNS kindreds from the Chinese National LQTS Registry were investigated. Mutational screening of KCNQ1 and KCNE1 genes was performed by polymerase chain reaction and direct DNA sequence analysis. LQTS phenotype and therapeutic outcomes were evaluated for all probands and family members.We identified 7 KCNQ1 mutations. c.1032_1117dup (p.Ser373TrpfsX10) and c.1319delT (p.Val440AlafsX26) were novel, causing JLNS in a 16-year-old boy with a QTc (QT interval corrected for heart rate) of 620 ms and recurrent syncope. c.605-2A>G and c.815G>A (p.Gly272Asp) caused JLNS in a 12-year-old girl and her 5-year-old brother, showing QTc of 590 to 600 ms and recurrent syncope. The fourth JLNS case, a 46-year-old man carrying c.1032G>A (p.Ala344Alasp) and c.569G>A (p.Arg190Gln) and with QTc of 460 ms, has been syncope-free since age 30. His 16-year-old daughter carries novel missense mutation c.574C>T (p.Arg192Cys) and c.1032G>A(p.Ala344Alasp) and displayed a severe phenotype of Romano-Ward syndrome (RWS) characterized by a QTc of 530 ms and recurrent syncope with normal hearing. Both the father and daughter also carried c.253G>A (p.Asp85Asn; rs1805128), a rare single nucleotide polymorphism (SNP) on KCNE1. Bizarre T waves were seen in 3/4 JLNS patients. Symptoms were improved and T wave abnormalities became less abnormal after appropriate treatment.This study broadens the mutation and phenotype spectrums of JLNS. Compound heterozygous KCNQ1 mutations can result in both JLNS and severe forms of RWS in Chinese individuals.SCI(E)CPCI-S(ISTP)PubMed0MEETING ABSTRACT267-75

My traces learn what you did in the dark: recovering secret signals without key guesses

In side channel attack (SCA) studies, it is widely believed that unprotected implementations leak information about the intermediate states of the internal cryptographic process. However, directly recovering the intermediate states is not common practice in today\u27s SCA study. Instead, most SCAs exploit the leakages in a guess-and-determine way, where they take a partial key guess, compute the corresponding intermediate states, then try to identify which one fits the observed leakages better. In this paper, we ask whether it is possible to take the other way around---directly learning the intermediate states from the side channel leakages. Under certain circumstances, we find that the intermediate states can be efficiently recovered with the well-studied Independent Component Analysis (ICA). Specifically, we propose several methods to convert the side channel leakages into effective ICA observations. For more robust recovery, we also present a specialized ICA algorithm which exploits the specific features of circuit signals. Experiments confirm the validity of our analysis in various circumstances, where most intermediate states can be correctly recovered with only a few hundred traces. To our knowledge, this is the first attempt to directly recover the intermediate states in a completely non-profiled setting. Our approach brings new possibilities to the current SCA study, including building an alternative SCA distinguisher, directly attacking the middle encryption rounds and reverse engineering with fewer restrictions. Considering its potential in more advanced applications, we believe our ICA-based SCA deserves more research attention in the future study

Evaluation of the Agronomic Impacts on Yield-Scaled N2O Emission from Wheat and Maize Fields in China

Contemporary crop production faces dual challenges of increasing crop yield while simultaneously reducing greenhouse gas emission. An integrated evaluation of the mitigation potential of yield-scaled nitrous oxide (N2O) emission by adjusting cropping practices can benefit the innovation of climate smart cropping. This study conducted a meta-analysis to assess the impact of cropping systems and soil management practices on area- and yield-scaled N2O emissions during wheat and maize growing seasons in China. Results showed that the yield-scaled N2O emissions of winter wheat-upland crops rotation and single spring maize systems were respectively 64.6% and 40.2% lower than that of winter wheat-rice and summer maize-upland crops rotation systems. Compared to conventional N fertilizer, application of nitrification inhibitors and controlled-release fertilizers significantly decreased yield-scaled N2O emission by 41.7% and 22.0%, respectively. Crop straw returning showed no significant impacts on area- and yield-scaled N2O emissions. The effect of manure on yield-scaled N2O emission highly depended on its application mode. No tillage significantly increased the yield-scaled N2O emission as compared to conventional tillage. The above findings demonstrate that there is great potential to increase wheat and maize yields with lower N2O emissions through innovative cropping technique in China

Mitochondrial DNA Mutations Induced by Carbon Ions Radiation: A Preliminary Study

Heavy-ion irradiation-induced nuclear DNA damage and mutations have been studied comprehensively. However, there is no information about the deleterious effect of heavy-ion irradiation on mitochondrial DNA (mtDNA). In this study, 2 typical mtDNA mutations were examined, including 4977 deletions and D310 point mutations. The 4977 deletions were quantified by real-time polymerase chain reaction, and D310 point mutations were analyzed by direct sequencing and a specific enzyme digestion genotyping method. Results showed that carbon ions radiation can induce temporal fluctuation of mtDNA 4977 deletions in 72 hours after irradiation, while survived clones were free from this deletion. Carbon ions induced more D310 mutations than X-rays, and the single-cell heteroplasmy was eliminated. This is the first study investigating mtDNA mutations induced by carbon ions irradiation in vitro. These findings would provide fundamental information for further investigation of radiation-induced mitochondrial biogenesis

Sustained-Release Solid Dispersion of High-Melting-Point and Insoluble Resveratrol Prepared through Hot Melt Extrusion to Improve Its Solubility and Bioavailability

This study aimed to prepare a sustained-release solid dispersion of poorly water-soluble resveratrol (RES) with high melting point in a single hot melt extrusion step. A hydrophobic–hydrophilic polymeric blend (Eudragit RS and PEG6000) was used to control the release of RES. With the dispersive mixing and high shear forces of hot melt extrusion, the thermodynamic properties and dispersion of RES were changed to improve its solubility. The effects of the formulation were investigated through univariate analysis to optimize the preparation of the sustained-release solid dispersion. In vitro and in vivo studies were performed to evaluate the prepared RES/RS/PEG6000 sustained-release solid dispersion. The physical state of the solid dispersion was characterized using differential scanning calorimetry and X-ray diffraction. Surface properties of the dispersion were visualized using scanning electron microscopy, and the chemical interaction between RES and excipients was detected through Fourier-transform infrared spectroscopy. Results suggested that the optimized sustained-release solid dispersion was obtained when the mass ratio of RES-polymeric blend was 1:5, the ratio of PEG6000 was 35%, the barrel temperature was 170 °C, and the screw speed was 80 rpm. In vitro studies demonstrated that the solid dispersion showed a good sustained release effect. The cumulative release of RES reached 82.42% until 12 h and was fit by the Weibull model. In addition, the saturated solubility was 2.28 times higher than that of the bulk RES. In vitro studies demonstrated that the half-life increased from 3.78 to 7.09 h, and the bioavailability improved to 140.38%. The crystalline RES was transformed into the amorphous one, and RES was highly dispersed in the polymeric blend matrix

First record of Thalassiosira curviseriata Takano (Bacillariophyta) and its bloom in the East China Sea

A bloom caused by a diatom, Thalassiosira curviseriata Takano (Bacillariophyta), is recorded in the East China Sea for the first time in China during a red fide investigation cruise (MC2005-2) from 27 March to 12 April 2005. This bloom was developed with the competition of Chaetoceros debilis and Skeletonema spp. The highest cell density of T. curviseriata, which has reached 1.27 x 10(6) cells/dm(3), was found in the surface and middle water layers of Stas ZD, ZB in the East China Sea (27.22 degrees similar to 29.48 degrees N, 121.53 degrees similar to 122.98 degrees E) in early spring in 2005. During the blooming period of T. curviseriata, the population with high cell density was found in the water area with temperature of 10 similar to 15 degrees C and salinity of 29.0 similar to 33.5. The percentage of the predominant species, T. curviseriata, has reached 95.8% of total diatom cells at one time in the middle water layer. The morphological characteristics of T. curviseriata, were observed with light microscope (LM) and transmission electronic microscope (TEM). The cells are 5.0 similar to 12.6 mu m in diameter, connecting each other by mucilaginous thread to form spiral and curved chains. Description and LM and TEM images of T. curviseriata are presented. T. curviseriata is ecologically characterized by eurythermy and euryhalinity, and its population variation is affected mainly by silicate, the ratios of phosphorus to silicon and nitrogen to silicon.Major State Basic Research Development Program of China [2001CB409701, 2005CB422305]; Special Prophase Project of Fujian Science and Technology Major Program of China [2005YZ1024]; National Natural Science Foundation of China [40627001, 40476055

Preparation of Solid Dispersion of Polygonum Cuspidatum Extract by Hot Melt Extrusion to Enhance Oral Bioavailability of Resveratrol

The aim of this study was to improve the solubility, bioavailability, and stability of resveratrol (RES-SD) Solid Dispersion in Polygonum cuspidatum extract (PCE) by hot melt extrusion (HME). In addition, the role of the auxiliary substances in PCE was also studied. The solid dispersion of Polygonum cuspidatum extract was prepared by hot-melt extrusion. The optimum formula was selected by single factor design and orthogonal test. The optimum formula was barrel temperature 140 °C, screw rotation speed 40 rpm/min, and the ratio of Polygonum cuspidatum extract to HPMCAS was 1:2. The dissolution test showed that PCE-SD increased the dissolution of RES from 46.75 ± 0.47% to 130.06 ± 0.12%. The pharmacokinetics curve of rats showed that PCE-SD increased AUC0-t of RES from 111,471.22 ± 11.4% to 160,458.968 ± 15.7%, indicating an approximately 1.44-fold increase in absorption. In addition, the rotation speed of PCE-SD screw is less than that of RES-SD screw. The bioavailability of PCE-SD was slightly better than that of RES-SD. PCE-SD is more hygroscopic than RES-SD. PCE-SD increased the solubility and oral bioavailability of RES. The auxiliary substances in Polygonum cuspidatum extract have influence on its preparation technology, stability, and bioavailability

Direct Construction of Optimal Rotational-XOR Diffusion Primitives

As a core component of SPN block cipher and hash function, diffusion layer is mainly introduced by matrices built from maximum distance separable (MDS) codes. Up to now, most MDS constructions require to perform an equivalent or even exhaustive search. In this paper, we study the cyclic structure of rotational-XOR diffusion layer, a commonly used diffusion primitive over