The Modic Vertebral Endplate and Marrow Changes

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芳基重氮化修饰玻碳电极和金电极

The modiﬁcation of glassy carbon and gold electrodes with aryl diazonium salt:The impact of the electrode materialson the rate of heterogeneous electron transferGuozhen Liu,Jingquan Liu,Till Bo ¨cking,Paul K.Eggers,J.Justin Gooding*School of Chemistry,The University of New South Wales,Sydney,NSW 2052,AustraliaReceived 1December 2004;accepted 23March 2005Available online 23May 2005AbstractThe heterogeneous electron-transfer properties of ferrocenemethylamine coupled to a series of mixed 4-carboxyphenyl/phenyl monolayers on glassy carbon (GC)and gold electrodes were investigated,by cyclic voltammetry,in aqueous buﬀer solutions.The electrodes were derivatized in a step-wise process.Electrochemical reduction of mixtures of 4-carboxyphenyl and phenyl dia-zonium salts on the electrode surfaces yielded stable monolayers.The introduction of carboxylic acid moieties onto the surfaces was veriﬁed by X-ray photoelectron spectroscopy.Subsequently the 4-carboxyphenyl moieties were activated using water-soluble carbo-diimide and N -hydroxysuccinimide and reacted with ferrocenemethylamine.The rate constants of electron transfer through the monolayer systems were determined from cyclic voltammograms using the Marcus theory for electron transfer and were found to be an order of magnitude higher for the ferrocene-modiﬁed monolayer systems on gold than those on GC electrodes.The results suggest the electrode material has an important inﬂuence on the rate of electron transfer.Ó2005Elsevier B.V.All rights reserved.Keywords:Self-assembled monolayers;Electron transfer;Carbon;Gold;Diazonium salts1.IntroductionThe modiﬁcation of conducting surfaces with mono-layers has received extensive research interest of late be-cause of their utility as model systems for understanding electron transfer [1,2],molecular electronics [3,4],bio-electronics [5,6]and sensors [7]amongst other applica-tions.The most popular chemistry for forming monolayers on electrode surfaces is alkanethiol self-assembly onto coinage metals,in particular gold [8],although other systems have also attracted interest such as silanes on metal oxide electrodes [9]and alkenes on highly doped silicon [10].The attractiveness of gold–thiol chemistry is that well ordered monolayers can be formed relatively easily,with a reasonably strong bond formed between the organic molecule and the electrode,and that a diverse range of molecules can be synthesized with which to modify an electrode.The advantages of gold–thiol chemistry are somewhat oﬀset by a number of disadvantages,including alkanethiols being oxida-tively or reductively desorbed at potentials typically out-side the window deﬁned by À800to +800mV versus Ag/AgCl.Other disadvantages include:alkanethiols being desorbed at temperatures over 100°C,gold being a highly mobile surface which results in the monolayers moving across the electrode surface,the gold–thiolate bond being prone to oxidation and the gold/thiol junc-tion creating a rather large tunneling barrier ($2eV)[11].The last point regarding a large tunneling barrier0301-0104/$-see front matter Ó2005Elsevier B.V.All rights reserved.doi:10.1016/j.chemphys.2005.03.033*Corresponding author.E-mail address:justin.gooding@.au (J.J.Gooding)./locate/chemphysChemical Physics 319(2005)136–146has implications for the rate of electron transfer from the organic monolayer to the electrode which is impor-tant for all molecular scale devices where communica-tion with the macroscopic world is achieved through electron transport.We are interested in alternative monolayer systems to gold–thiol chemistry which overcome some of the disad-vantages but do not severely compromise the advanta-ges of gold/thiol chemistry.The electrochemical reduction of aryl diazonium salts is one possible alterna-tive which has most frequently been used as a method for the covalent derivatization of glassy carbon(GC) surfaces[12–14].The reduction reaction results in the loss of the N2and the formation of a carbon–carbon covalent bond which is strong,stable over both time and temperature,non-polar and conjugated[11].Thus, the conjugated carbon network in the GC electrode can be thought of as continuing into the monolayer sys-tem rather than the abrupt change from electrons being in a metallic environment to an organic environment. The continuity of the electrode material into the mono-layer has resulted in the suggestion that GC electrodes modiﬁed by aryl diazonium salts have the potential to reduce the barrier towards electron transfer from the carbon electrode into the monolayer[11].McCreery and coworkers[15–18]have extensively studied the elec-tron transfer kinetics of GC surfaces in diﬀerent redox probe solutions.However,to the best of our knowledge heterogeneous electron transfer between redox active molecules and GC electrodes through aryl diazonium salt derived monolayers has yet to be investigated.Nor has the notion that the C–C bond will allow eﬃcient electron transfer.The attractiveness of aryl diazonium salts are en-hanced further by recent studies showing they can also be grafted to a variety of metal[19,20]and semicon-ductor[21]surfaces as well as carbon nanotubes[22]. This feature raises the exciting possibility of one monolayer forming system being suitable for a large range of electrode materials for a diverse range of applications.This possibility is helped by a rich array of diﬀerent diazonium salts which have now been pre-pared including molecular wires[23]and polyethylene glycol terminated molecules designed to resist protein adsorption[24,25].The purpose of this study is to modify GC and gold substrates using mixtures of aryl diazonium salt molecules(introducing phenyl and4-carboxyphenyl groups onto the surface)and to com-pare the kinetics of electron transfer to GC and gold surfaces from the same ferrocene-based monolayer sys-tem.A similar ferrocene-based system prepared by a mixed self-assembled monolayers(SAMs)of4-merca-ptobenzoic acid(MBA)and1-propanethiol(PT)has also been prepared on gold surfaces and the rates of electron transfer have been studied for further comparison.2.Experimental2.1.Reagents and materialsTetrabutylammonium tetraﬂuoroborate(NBu4BF4), sodium tetraﬂuoroborate(NaBF4),p-aminobenzoic acid,aniline,4-mercaptobenzoic acid(MBA),1-propa-nethiol(PT),ferricyanide(K4Fe(CN)6),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC),N-hydroxysuccinimide(NHS),N-[2-hydroxy-ethyl]piperazine-N0-[2-ethanesulfonic acid](HEPES), ferrocenecarboxaldehyde,sodium cyanoborohydride and acetonitrile(CH3CN,HPLC grade)were obtained from Sigma(Sydney,Australia).Benzoic acid diazo-nium tetraﬂuoroborate and benzene diazonium tetra-ﬂuoroborate were synthesized according to the method by Saby et al.[26].Ferrocenemethylamine was synthe-sized using the procedure from Kraatz[27].Reagent grade dipotassium orthophosphate,potassium dihydro-gen orthophosphate,potassium chloride,sodium hydroxide,sodium chloride,sodium nitrite,ammonium acetate,sulphuric acid,hydrochloric acid,methanol and diethyl ether were purchased from Ajax Chemicals Pty. Ltd.(Sydney,Australia).All reagents were used as re-ceived,and aqueous solutions were prepared with puri-ﬁed water(18M X cmÀ1,Millipore,Sydney,Australia). Phosphate buﬀer solutions used in this work contained 0.05M KCl and0.05M K2HPO4/KH2PO4and were ad-justed with NaOH or HCl solution.2.2.Modiﬁcation of electrodesThe GC and gold electrodes were modiﬁed with dia-zonium salts followed by attachment of ferrocenemeth-ylamine as depicted in Scheme1.The GC electrodes were purchased commercially(Bioanalytical System Inc.,USA)as3-mm-diameter rods.The electrodes were polished successively in1.0,0.3,and0.05l m alumina slurries made from dry Buehler alumina and Milli-Q water on microcloth pads(Buehler,Lake Bluﬀ,IL, USA).The electrodes were thoroughly rinsed with Milli-Q water and sonicated in Milli-Q water for 5min between polishing steps.Before derivatization, the electrodes were dried with an argon gas stream. The bare GC electrodes had an electrochemical rough-ness factor(the ratio of the electrochemical area to geo-metric area)of 1.43.Surface derivatization of GC electrodes was performed in a solution of1mM aryl diazonium salt and0.1M NaBu4BF4in acetonitrile using cyclic voltammetry(CV)with a scan rate of 100mV sÀ1for two cycles between+1.0andÀ1.0V. The diazonium salt solution was deaerated with argon for at least15min prior to derivatization.The elec-trodes were rinsed with copious amounts of acetonitrile and then water and dried under a stream of argon prior to the next step.G.Liu et al./Chemical Physics319(2005)136–146137Poly-crystalline gold electrodes,prepared as de-scribed previously[28],were polished to a mirror-like ﬁnish with 1.0l m alumina,followed by0.3and 0.05l m alumina slurry on microcloth pad.After re-moval of trace alumina from the surface,by rinsing with water and brief cleaning in an ultrasonic bath with eth-anol and then water,electrochemical cleaning in0.05M H2SO4by cycling the electrodes betweenÀ0.3and1.5V was carried out until a reproducible CV was obtained. Before derivatization,the cleaned electrodes were rinsed with water and dried under a stream of argon.The derivatization of gold electrodes with a mixture of dia-zonium salts was conducted in exactly the same manner as described for the carbon electrodes.The alkanethiol modiﬁed gold electrodes were prepared by immersing the gold electrodes in a1mM mixed thiol solution(mer-captobenzoic acid and propanethiol with diﬀerent dilu-tion ratios)in ethanol overnight(see Scheme2).The electrode was rinsed with copious amounts of ethanol, then water andﬁnally dried under a stream of argon prior to the next step.Covalent attachment of ferrocenemethylamine to car-boxylic acid terminated monolayers followed the proce-dures described by Liu et al.[29].The modiﬁed surfaces were incubated in an aqueous solution of10mM N-hydroxysuccinimide(NHS)and40mM1-ethyl-3-(3-di-methyl aminopropyl)carbodiimide hydrochloride (EDC)for1h.After the activation,the electrodes were rinsed with water and incubated in a5mM ferrocenem-ethylamine solution in HEPES buﬀer pH7.3for24h.2.3.Electrochemical measurementsAll electrochemical measurements were performed with a BAS-100B electrochemical analyser(Bioanalyti-cal System fayette,IL)and a conventional three-electrode system,comprising a GC or a gold work-ing electrode,a platinum foil as the auxiliary electrode, and a Ag/AgCl3.0M NaCl electrode(from BAS)as ref-erence.All potentials were reported versus the Ag/AgCl reference electrode at room temperature.All CV mea-surements were conducted in pH7.0phosphate buﬀer. The area under the Faradaic peaks in the CVs of the fer-rocene modiﬁed electrodes were used to determine the surface coverage of ferrocene.The rate constants for electron transfer were calculated from the variation in peak potential over a wide range of scan rates.For elec-trodes with prominent redox peaks the rate constants were determined byﬁtting the variation in peak poten-tial with scan rate using the Marcus theory for electron transfer as described previously[30–32]whilst at low surface coverages of ferrocene the rates of electronScheme2.Schematic of ferrocenemethylamine immobilized covalently on mixed monolayers of MBA and PT on gold surfaces. 138G.Liu et al./Chemical Physics319(2005)136–146transfer were determined using the Laviron[33]formal-ism.This was because peak shape and position was very sensitive background subtraction at with small redox peaks and thereforeﬁtting the entire background sub-tracted CV peak as required for our Marcus simulation became unreliable.When both methods were employed on the same data very similar rate constants for electron transfer were obtained.2.4.XPS measurementsXP spectra were obtained using an EscaLab220-IXL spectrometer with a monochromated Al K a source (1486.6eV),hemispherical analyzer and multichannel detector.The spectra were accumulated at a take-oﬀan-gle of90°with a0.79mm2spot size at a pressure of less than10À8mbar.3.Results3.1.Aryl diazonium salt modiﬁed glassy carbon electrodesGlassy carbon electrodes were modiﬁed with diazo-nium salts via electrochemical reduction of an aryl dia-zonium salt(1mM in acetonitrile)with0.1M tetrabutylammonium tetraﬂuoroborate as background electrolyte.Theﬁrst sweep showed the characteristic reduction peak atÀ0.16V versus Ag/AgCl with no associated oxidation peak indicative of the loss of N2 and the formation of a4-carboxyphenyl radical fol-lowed by covalent binding to the carbon surface[34]. Subsequent scans showed no electrochemistry indicative of a passivated electrode.The passivation of the GC sur-faces after the modiﬁcation with aryl diazonium salts was conﬁrmed using potassium ferricyanide as a redox probe.Fig.1shows a cyclic voltammogram before and after modiﬁcation with(4-carboxyphenyl)diazonium tetraﬂuoroborate in1mM ferricyanide in a0.05M phosphate buﬀer(0.05M KCl,pH7.0)at a scan rate of100mV sÀ1.After the modiﬁcation of the surface with the aryl diazonium salts,the redox peaks of ferricy-anide observed with bare GC electrodes were almost completely suppressed.This gave strong evidence that a uniform monolayer which blocked access of ferricya-nide to the electrode had formed on the GC surfaces. Based on the area of the reduction peak during the mod-iﬁcation of the GC electrode surface with the aryl diazo-nium salt,the coverage of the4-carboxyphenyl moieties was calculated to be7.4·10À10mol cmÀ2.The reported surface coverage on GC substrates varies in the range of 4–30·10À10mol cmÀ2[35]with the theoretical maxi-mum surface coverage[29]for a monolayer on GC sur-faces being12·10À10mol cmÀ2.The surface coverage of7.4·10À10mol cmÀ2indicates the GC electrode was modiﬁed with a monolayer rather than multilayers of aryl groups as has been reported by some workers[36–38].The modiﬁcation of the GC electrode by electro-chemical reduction of(4-carboxyphenyl)diazonium tet-raﬂuoroborate was conﬁrmed by X-ray photoelectron spectroscopy(XPS).Survey spectra showed the expected carbon1s and oxygen1s peaks at$284and$532eV, respectively,and also a small nitrogen1s peak at $400eV(Fig.2).The level of oxygen was increased in comparison to the bare GC surface as expected for the introduction of4-carboxyphenyl groups onto the sur-face.The presence of the small nitrogen1s peak in the survey spectra was partially due to nitrogen containing species already detectable on the unmodiﬁed GC elec-trodes,which has been observed previously[13,34].Fur-thermore,nitrogen species with a binding energy of $400eV can be introduced onto the surface during the modiﬁcation reaction.It has been proposed that these are due to a hydrazine generated by reaction of the dia-zonium salt with phenol groups on the GC surface[26].Fig.2.XP survey spectrum and carbon1s narrow scan(inset)of a GCelectrode modiﬁed by electrochemical reduction of(4-carboxyphenyl)diazonium tetraﬂuoroborate.G.Liu et al./Chemical Physics319(2005)136–146139Nitrogen1s narrow scans(not shown)were consistent with the formation of low levels of the hydrazine,which exhibited a slightly diﬀerent binding energy to that of the nitrogen species already present on the bare GC electrode.The carbon1s narrow scan(Fig.2,inset)showed a peak centred at288.8eV,which was typical of the car-bon of the carboxylic acid group[13].This peak was ab-sent from the carbon1s narrow scan of unmodiﬁed GC surfaces.The carbon1s peak at284.4eV was slightly broadened compared to the graphitic peak of an unmodiﬁed GC surface and was assigned to the gra-phitic carbon of the underlying GC electrode and the aromatic carbons of the monolayer.The pronounced asymmetry of this peak with a broad shoulder on the high binding energy side($286.2eV)was attributed to an oxidized species present on the GC surface and or-ganic contaminants adsorbed on the monolayer.After modiﬁcation of the GC electrode surface with the aryl diazonium salt the next step in the fabrication of the modiﬁed electrodes was the attachment of ferro-cene.CVs measured in an aqueous solution of0.05M phosphate buﬀer(0.05M KCl,pH7.0)at a scan rate of100mV sÀ1before and after the immobilization of ferrocene on the4-carboxyphenyl modiﬁed GC elec-trode are shown in Fig.3.The strong redox peaks after the attachment of ferrocene showed linear variation in peak current with scan rate indicating that the ferrocene was surface bound.In the absence of EDC and NHS such that no covalent coupling of the ferrocene could occur,only very weak redox peaks due to physisorption were observed.The CVs of the ferrocene coupled to the 4-carboxyphenyl monolayers show non-ideal behaviour[1]with regards to peak separation at slow scan rates(D E p=79mV rather than the ideal D E p=0mV)and the full width half maximum(greater than200mV rather than the ideal E FWHM=90.6mV/n where in this case n=1).With regards to both peak separation and the E FWHM the non-ideal behaviour has been attributed to the ferrocene molecules being located in a range of environments with a range of formal electrode potentials (E0)[39,40].We[29,41]and others[42]have noted pre-viously that fabricating redox active SAMs by assem-bling the SAM and then attaching the redox molecule, results in broader FWHM than observed with electrodes where a redox active alkanethiol was attached directly to the electrode.The reason for modifying electrodes in this step-wise manner,where the monolayer is formed and then the redox active molecule attached,rather than synthesizing a pure redox active self-assembling mole-cule followed by assembly on the electrode,is because in applications of our interest,bioelectronics,the step-wise strategy is the only viable approach.With a monolayer containing only4-carboxyphenyl moieties the number of redox active molecules attached to the surface,as determined from the charge passed under the Faradaic peaks in the ferrocene modiﬁed electrode,is approximately(0.073±0.012)·10À10 mol cmÀ2with a close to unity ratio of anodic to cathodic peak areas(see Table1).Comparing the surface coverage of4-carboxyphenyl groups of7.4·10À10mol cmÀ2,to that of the number of redox centres attached indicates that only approximately10%of the 4-carboxyphenyl monolayers had a ferrocene attached. At this surface coverage the average area per ferrocene molecule,assuming homogeneous distribution,is 2.2nm2which suggests there is a high possibility of interaction between redox active centres[29,42].Interaction between redox active centres has been re-ported to decrease the reorganization energy and in-crease the electron transmission eﬃciency[4,43,44], hence providing an anomalously high measure of the rate constant for electron transfer.As a consequence, the number of coupling points within the monolayer that the ferrocene could couple was reduced by forming mixed monolayers composed of the4-carboxyphenyl diazonium salt and the phenyl diazonium salt(Table 1).Table1shows that the surface coverage initially in-creased with the spacing of the coupling points followed by the more expected decrease as the number of cou-pling points decreased.The reason for the initial in-crease in surface coverage of ferrocene as the solution composition from which the monolayer forms changes from entirely4-carboxyphenyl diazonium salt to a1:1 ratio of4-carboxyphenyl to phenyl diazonium salt is un-clear.The percentage of carboxyl groups to be activated using EDC/NHS,as used here,in a SAM composed of entirely carboxylic acids has been shown to be approxi-mately50%[45]which is equivalent to all the4-carboxy-phenyl groups being activated in a1:1monolayer. Furthermore,the relative surface coverages of the4-carboxyphenyl to ferrocene is10:1in the entirely4-carb-oxyphenyl monolayer so there should be excess coupling points for the ferrocene to attach.Therefore,it is sug-gested that the introduction of a second component into the monolayer(the phenyl diluent)in eﬀect introduces a hydrophobic component into the monolayer.As ferro-cene has been shown previously to adsorb onto the sur-face of hydrophobic self-assembled monolayers[41,46]. Therefore,it is proposed that more ferrocene is attached when the phenyl component is introduced into the monolayer because the surface is more energetically favourable location for the ferrocene compared with an entirely carboxyphenyl monolayer.The rate constant for electron transfer was deter-mined from the variation in peak position between the anodic and cathodic scans as a function of scan rate. In this study,the variation in peak potential over a wide range of scan rates wasﬁtted using the Marcus theory for electron transfer as described previously[30–32] rather than the Laviron[33]formalism which relies on simple Butler–Volmer kinetics and gives rate constants for electron transfer which are sensitive to the choice of sweep rates investigated.Table1shows that across the spectrum of dilution ratios investigated the rate con-stant for electron transfer(k app)is approximately15–20sÀ1.3.2.Aryl diazonium salt modiﬁed gold electrodesGold electrodes were modiﬁed with aryl diazonium salts via electrochemical reduction in exactly the same manner to the GC electrodes.The reduction peak for the attachment of the aryl diazonium salt onto the gold electrode,observed in theﬁrst sweep,was shifted anod-ically230mV relative to carbon being at+70mV versus Ag/AgCl.Subsequent sweeps showed no electrochemis-try indicating a monolayer coverage of4-carboxyphenyl moieties on the electrode surface.The4-carboxyphenyl monolayer blocked access of potassium ferricyanide to the electrode in a similar manner to that depicted in Fig.1for the carbon electrode but to a lesser extent. The coverage of the4-carboxyphenyl moieties on the electrode surface was6.4·10À10mol cmÀ2which was lower than the7.4·10À10mol cmÀ2observed on GC electrodes and hence lower than the theoretical maxi-mum surface coverage[34]for a monolayer of 12·10À10mol cmÀ2.The lower surface coverage could be a reﬂection of the aryl diazonium salt not being nor-mal to the surface of the gold,as suggested by infra-red spectroscopy[20].Again,the presence of a monolayer or submonolayer of aryl diazonium salt on the gold elec-trode is important due to the possibilities of obtaining multilayers with aryl diazonium salts as shown for both carbon[36–38]and metal surfaces[20].An XP survey spectrum of gold modiﬁed with(4-carboxyphenyl)diazonium tetraﬂuoroborate showed the expected1s peaks of carbon and oxygen at$285 and$532eV,respectively,but no signiﬁcant evidence of a nitrogen1s peak(Fig.4).The carbon1s envelope (Fig.4,inset)wasﬁtted with four peaks at288.7, 286.2,284.6and283.9eV assigned to the carboxylic acid moieties,C–O species,the aromatic carbons of the monolayer and the metal-bonded carbon,respectively. The binding energy observed for the carboxylic acid group on gold was consistent with that observed on the GC surface.The inclusion of the metal carbide peak is exceedingly tentative as a goodﬁt to the spectra could be obtained without the presence of this peak.The assignment of the metal carbide peak is based on theTable1Some parameters of ferrocenemethylamine immobilized on GC electrodes modiﬁed with mixed monolayers of4-carboxyphenyl and phenyl moieties.D E p is recorded at a scan rate of100mV sÀ1[Benzyl]/[benzoic acid]E0(mV)D E(mV)E FWHM(mV)C(pmol cmÀ2)C a/C c k app(sÀ1) 0264±1579±10241±1072.8±11.60.89±0.0717±10 1279±1378±14213±19100.3±10.4 1.03±0.0428±10 5292±989±25227±2567.4±10.70.94±0.0515±5 10298±793±10262±3848.1±6.90.88±0.1116±2 20304±17101±21220±2929.4±3.40.71±0.1615±10 40317±19107±10289±1413.3±2.00.77±0.1310±2Fig.4.XP survey spectrum and carbon1s narrow scan(inset)of agold electrode modiﬁed by electrochemical reduction of(4-carboxy-phenyl)diazonium tetraﬂuoroborate.G.Liu et al./Chemical Physics319(2005)136–146141precedence of Pinson and co-workers[19,20,47]who have previously proposed the existence of such a peak for the electroreduction of diazonium salts onto metal surfaces.On iron surfaces the case for a metal–carbide peak is compelling with a very pronounced shoulder when a high resolution instrument is used[47]with the intensity of this shoulder sensitive to take-oﬀangle indi-cating it is a surface bound species.However,on copper electrodes[20]and other examples on iron[19]the shoulder on the carbon1s spectra is less pronounced similar to the observations on gold here.The electrochemical parameters after the attachment of ferrocene to the4-carboxyphenyl modiﬁed gold elec-trodes are shown in Table2.The trends were very sim-ilar to the GC modiﬁed electrodes with broader than ideal E FWHM and non-ideal D E p at slow scan rates. The surface coverage of ferrocene with diﬀerent ratios of diluent to4-carboxyphenyl were slightly lower than those on GC in common with the lower coverage in gen-eral of the aryl diazonium salts on gold compared with GC.Most importantly,the rates of electron transfer measured on the gold modiﬁed surface were signiﬁcantly greater than that observed on carbon.Typically rates of more than a100sÀ1were observed,which was approx-imately one order of magnitude higher than for the same monolayer system on GC electrodes.The values of the rate constants at the low surface coverage of ferrocene (last three entries in the table)were particularly diﬃcult to determine because with small redox peaks back-ground subtraction can have a large impact on the peak positions.As a consequence the rate constants quoted represent the lower limits and therefore we expect the true rate constant is closer to that observed at the1:5 monolayer.3.3.Aryl thiol modiﬁed gold electrodesFor comparison with the monolayers formed by elec-trochemical reduction of aryl diazonium salts we also prepared mixed monolayers of aryl thiol self-assembled monolayers on gold electrodes with attached ferrocene moieties as shown in Scheme2.The rate constants deter-mined for this equivalent aryl thiol system were in the order of103sÀ1(at the limits of what can be measured electrochemically)which was approximately5–10times the values observed for the aryl diazonium salt–gold sys-tem but two orders of magnitude higher than those ob-served for the aryl diazonium salt–GC system.These observations indicate that the metal surface has a signif-icant eﬀect on the rate of electron transfer.4.DiscussionThe rate constants for electron transfer are remark-ably slower for the carbon electrodes relative to the gold electrodes.This is contrary to the suggestion that with diazonium salt modiﬁed carbon electrodes the continu-ity of conjugated carbon network from the electrode into the monolayer will result in a lower barrier for elec-tron transfer than with organic monolayers on metallic electrodes[11].The question that arises is why there is a diﬀerence in rate constants of around one order of magnitude for the same redox active molecule connected to electrodes by the same bridge molecule?The Marcus–Hush expression for electron transfer between a donor and acceptor through an organic bridge in solution includes terms for electronic coupling between the donor and acceptor,the Gibbs free energy for electron transfer(the driving force,D G ET)and the nuclear reorganization energy(k)of the redox molecule as a consequence of its change in oxidation state[48]. For a given donor and acceptor pair the rate of electron transfer decays exponentially with distance according to a proportionality constant,the b value,sometimes called a damping factor.When the organic bridge is anchored to an electrode such that it can act as the donor and/or acceptor the situation is complicated somewhat as the electronic properties of the electrode can also play a role in the rate of electron transfer[2].Equations describing the rate constant for electron transfer now incorporate terms related to the Fermi levels of the electrode and the eﬀective density of electronic states near the Fermi level.In this study,the only changes between the mono-layer systems studied relate to the electrode material and the bond to the electrode.Hence,the reorganization en-ergy and the driving force will remain unchanged.The electronic coupling may be inﬂuenced by the electrodeTable2Some parameters of ferrocenemethylamine immobilized on gold electrodes modiﬁed with mixed monolayers of4-carboxyphenyl and phenyl moieties.D E p is recorded at a scan rate of100mV sÀ1[Benzyl]/[benzoic acid]E0(mV)D E(mV)E FWHM(mV)C(pmol cmÀ2)C a/C c k app(sÀ1) 0268±1281±14209±1149.3±7.60.87±0.13257±41 1277±2585±9191±1780.6±5.90.86±0.09530±42 5280±1875±10227±853.7±5.40.72±0.14211±23 10282±1492±12260±1525.8±4.00.92±0.0783±50 20292±1989±8272±1413.3±2.40.76±0.0369±50 40317±2181±16308±107.1±1.00.93±0.0268±50 142G.Liu et al./Chemical Physics319(2005)136–146。

Lamellar spacing in cuboid hydroxyapatite scaffolds regulates bone formation by human bone marrow st

Lamellar Spacing in Cuboid HydroxyapatiteScaffolds Regulates Bone Formation by HumanBone Marrow Stromal CellsMahesh H.Mankani,M.D.,1Shahrzad Afghani,B.S.,1Jaime Franco,Ph.D.,2Max Launey,Ph.D.,2Sally Marshall,Ph.D.,3Grayson W.Marshall,D.D.S.,M.P.H.,Ph.D.,3Robert Nissenson,Ph.D.,4Janice Lee,D.D.S.,M.D.,M.S.,5Antoni P.Tomsia,Ph.D.,2and Eduardo Saiz,Ph.D.2,6Background:A major goal in bone engineering is the creation of large volume constructs (scaffolds and stem cells)that bear load.The scaffolds must satisfy two competing requirements—they need be sufﬁciently porous to allow nutrient ﬂow to maintain cell viability,yet sufﬁciently dense to bear load.We studied the effect of scaffold macroporosity on bone formation and scaffold strength,for bone formed by human bone marrow stromal cells.Methods:Rigid cubical hydroxyapatite/tricalcium phosphate scaffolds were produced by robo-casting.The ceramic line thickness was held constant,but the distance between adjacent lines was either 50,100,200,500,or 1000m m.Cultured human bone marrow stromal cells were combined with the scaffolds in vitro ;transplants were placed into the subcutis of immunodeﬁcient mice.Transplants were harvested 9,18,23,38,or 50weeks later.Bone formation and scaffold strength were analyzed using histology and compression testing.Results:Sixty transplants were evaluated.Cortical bone increased with transplant age,and was greatest among 500m m transplants.In contrast,maximum transplant strength was greatest among 200m m transplants.Conclusions:Lamellar spacing within scaffolds regulates the extent of bone formation;500m m yields the most new bone,whereas 200m m yields the strongest transplants.IntroductionT he ultimate goal in the engineering of new bone is theformation of large volume,weight-bearing constructs.Thus far,large volume transplants made with particulatescaffolds have successfully closed defects in large animalmodels.1The next threshold in this process is the creation ofscaffolds that bear weight while simultaneously supportingtransplanted skeletal stem cells.A major limitation to successfulcell transplantation in this setting is the difﬁculty in providingnutritional support to the cells in the interior of the scaffold.These cells are typically supported by diffusion of nutrientsfrom the periphery during the short term,and by vasculariza-tion over the long term.The scaffold must therefore satisfy twocompeting primary design requirements—it must be porousenough to allow nutrient ﬂow to maintain cell viability,and yetit must be dense enough to bear loads.Additional but lesscritical design parameters include (1)the capability to undergo programmed resorption,and (2)the ability to bind and even-tually release angiogenic growth factors.Although these competing requirements are well recog-nized,a detailed examination of the effect of scaffold mac-roporosity has not been completed.For our purposes,porosity may be sub-divided according to its scale.Micro-porosity refers to pores on the micrometer and sub-micrometer scale,while macroporosity reﬂects pore sizes ranging from 20m m to 1mm.Microporosity,in association with surface roughness,inﬂuences osteoblast adhesion and differentiation.In contrast,macroporosity inﬂuences the density of adherent cells and the ability of capillaries and larger caliber vessels to grow into the scaffold.The purpose of this study was to evaluate bone formation and scaffold strength as a function of macroporosity.Novel hydroxyapatite/tricalcium phosphate (HA/TCP)lattice-work 1Department of Surgery,University of California–San Francisco,San Francisco,California.2Materials Sciences Division,Lawrence Berkeley National Laboratory,Berkeley,California.3Preventive and Restorative Dental Sciences,University of California–San Francisco,San Francisco,California.4Endocrine Unit,VA Medical Center (111N),Departments of Medicine and Physiology,University of California–San Francisco,San Francisco,California.5Department of Oral and Maxillofacial Surgery,University of California–San Francisco,San Francisco,California.6Department of Materials,Center for Advanced Structural Ceramics,UK Center for Structural Ceramics,Imperial College,Royal School of Mines,London,United Kingdom.TISSUE ENGINEERING:Part AVolume 17,Numbers 11and 12,2011ªMary Ann Liebert,Inc.DOI:10.1089/ten.tea.2010.0573Bone as percentage of tissue in the transplant, exclusive of scaffold0.0010.0020.0030.0040.0050.0060.0070.00Lamellar spacing (micrometers)%Bone as percentage of total transplantD E 0.005.0010.0015.0020.0025.0030.0035.0040.00100050020010050Lamellar spacing (micrometers)%1 = p<0.05 relative to 500 micrometer group2 = p<0.01 relative to 500 micrometer group3 = p<0.005 relative to500 micrometer group4 = p<0.001 relative to500 micrometer group5 = p<0.0005 relative to500 micrometer group1 = p<0.05 relative to 500 micrometer group2 = p<0.01 relative to 500 micrometer group3 = p<0.005 relative to 500 micrometer group4 = p<0.001 relative to 500 micrometer group5 = p<0.0005 relative to500 micrometer group Bone at center of scaffold, as percentage of tissue in the transplant, exclusive of scaffoldLamellar spacing (micrometers)%Bone at center of scaffold, as percentage of total transplantLamellar spacing (micrometers)%* p<0.05 relative to 500micrometer group FGscaffolds were constructed using robocasting such that themacroporosity ranged from 50to 1000m m in a con-trolled fashion.Scaffolds were seeded with preset numbers ofculture-expanded human bone marrow stromal cells(hBMSCs),placed into the subcutis of Bg-Nu-XID mice,har-vested at time points ranging from 9to 50weeks,and ana-lyzed via histomorphometry and strength testing.Materials and MethodsScaffold preparationCeramic-based inks for robocasting were prepared bydispersing mixtures of HA powder (Trans-Tech)and TCPpowder (Keramat)(65/35wt%)with an average particle sizeof 2.4m m in Pluronic ÒF-127water-based solutions withPluronic contents ranging between 10and 30wt%.The dis-persion of the ceramic powders was performed inside anice/water bath to reverse the gellation process and reducethe viscosity.2Approximately 2.5wt%of 1-Octanol (Sigma-Aldrich)in water was added to the mixture to minimize thepresence of bubbles.The suspensions were homogenizedusing an 800W stirrer with variable speed (Dewalt DW236,0–800rpm).After dispersing the powders,4wt%of com-mercial corn syrup in water was added to the ink to enhancethe adhesion between printed layers.The ink was mixedthoroughly for 10min and sieved afterward through a100m m nylon mesh to achieve optimum homogenization andminimize the presence of particle aggregates.The inks wereloaded into a 10mL syringe (BD)that was gently tapped toeliminate air bubbles.The inks were used to print standard ceramic grids with arobotic deposition device (Robocad 3.0,3-D Inks).The diam-eter of the printing nozzle was 250m m.(EFD-precision tips,RI).The grids were printed inside a reservoir of nonwettingoil (Lamplight Ò)on an aluminum oxide plate of 1mm thick-ness.To avoid deformations due to uneven shrinkage duringdrying and sintering,a layer of permanent marker (Sharpie;Sandford)was applied on the surface of the alumina sheetfollowed by a second layer of commercial corn syrup depos-ited by spin coating (WS-400B-6NPP-LITE;Laurell Technolo-gies Corporation).After printing,the plates were tilted *45°to remove the excess oil and dried for 24h at room tempera-ture.The pieces were ﬁred at 600°C (1°C.min -1,heating rate)for 2h to evaporate the organics,followed by sintering for 2hat 1275°C with a heating and cooling rate of 5°C.min -1.Thecomposition and microstructure of the sintered grids werecharacterized by X-ray diffraction and scanning electron mi-croscopy.After sintering,the grids consisted of printed linesof *200m m diameter with pore sizes varying systematicallybetween 50and 1000m m.The printed lines had *10vol%ofmicroporosity with micro-pore sizes in the micrometer range.Scaffold structures measuring 8·8·5mm were producedusing these techniques (Fig.1A).Transplant preparation,placement,and recovery Surgical specimens were obtained containing fragments of normal unaffected bone with bone marrow from a patient undergoing reconstructive surgery.The male patient,aged 19years,underwent iliac crest bone harvest for an orthopedic reconstructive procedure.Tissue procurement proceeded in accordance with NIH regulations governing the use of human subjects (Protocols 94-D-0188).Multi-colony-derived strains of BMSCs were derived from the bone marrow in a manner previously described.3Brieﬂy,a single-cell suspension of bone marrow cells was cultured in growth medium consisting of a MEM,2mM L-glutamine,100U/mL penicillin,100m g/mL streptomycin sulfate (Invitrogen),10-8M dexamethasone (Sigma),10-4M L-ascorbic acid phosphate magnesium salt n-hydrate (Wako),and 20%fetal bovine serum of a pre-selected lot (Equitech-Bio,Inc.).In our hands,20%fetal bovine serum promotes faster cell proliferation without inducing cellular senescence.After 24h,nonadherent cells were re-moved by extensive washing.The cells were then incubated at 37°C in an atmosphere of 100%humidity and 5%CO 2.The medium was changed weekly following initial plating,when the cell density in the tissue culture ﬂasks was of such low density that the media remained at a physiologic pH during the entire week.For subsequent passaging,when cell density was substantially higher in the ﬂasks,cells underwent either passaging or media change approximately every 3days.Cells at passage 3were cryo-preserved in liquid nitrogen.For this set of studies,a single vial containing 2.0million cells was thawed,plated,and further passaged until sufﬁcient cells were available.Upon approaching conﬂuence at passage 5,BMSCs were trypsin-released and transferred to Falcon six-well plates,each well receiving 1million BMSCs and a set of 5scaffolds representing the different lamellar spacing.BMSCs were permitted to adhere and proliferate on the scaffolds.Cells were found to adhere and proliferate on the scaffolds within a day of plating,forming multilayer networks within 3days (Fig.1B).After 1week,the scaffolds were recovered from the plates and transplanted.Eight-month-old immunocompromised Bg-Nu/Nu-Xid female mice (Harlan-Sprague Dawley)served as transplant recipients.All animals were cared for according to the poli-cies and principles established by the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals.Operations were performed in accordance to speciﬁcations of an approved UCSF animal research protocol (AN077250).Mice were anesthetized with inhalational iso-ﬂurane and oxygen.Transplants were placed in the subcu-taneous space along the back,with each mouse receiving all ﬁve types of scaffolds.Incisions were closed with suture.Twelve mice were given a total of 60transplants.The mice were sacriﬁced at 9,18,23,38,or 50weeks postoperatively with inhaled CO 2and their transplants harvested.FIG.1.(A)Scanning electron microscopic image of 200m m scaffold before culture.(B)Transmission light microscopic image of a scaffold 3days after seeding with human BMSCs.(s,scaffold;c,BMSCs).(C)Demonstration of center 1/9th of transplant,depicted by the area enclosed by the center black rectangle.(D)Percentage of bone within the entire transplant,including both transplant periphery and center.(E)Percentage of bone within the tissue (nonscaffold)portions of the transplant.Transplant periphery and center are both included.(F)Percentage of bone within the center of the transplant.(G)Percentage of bone within the tissue (nonscaffold)portions of the transplant,taken only from the transplant center.BMSC,bone marrow stromal cells.Color images available online at /tea‰LAMELLAR SPACING REGULATES BONE FORMATION1617Histomorphometry of bone sectionsAll of the transplants wereﬁxed in4%phosphate-buffered formalin freshly prepared from paraformaldehyde.Follow-ing an overnightﬁxation at4°C,the transplants were sus-pended in phosphate-buffered saline.The transplants were demineralized in buffered10%EDTA,dehydrated,embed-ded in parafﬁn,and sectioned.Sections were deparafﬁnized, hydrated,and stained with hematoxylin and eosin.A single section from the interior of each transplant was obtained,stained with hematoxylin and eosin,and examined to quantify the areas that had either formed or not formed bone. Areas of bone formation(B)were easily distinguished from the scaffold(S),ﬁbro-vascular tissue(FV),and hematopoietic tissue (H).B,S,FV,H,and the total transplant area(T)were then measured for each section.As expected,B+S+FV+H=T. All histologic images were magniﬁed using a Zeiss Axioplan microscope(Zeiss),and they were captured using the micro-scope-imaging program Spot Advanced(Diagnostic Instru-ments,Inc.).Area measurements were obtained with ImageJ version1.36(National Institutes of Health).The B,S,FV,H,and T values of the sections for each transplant were averaged to compute an overall set of values for each transplant.The transplants harvested at23weeks were further analyzed to quantify bone formation within the interior-most portion of the transplant.Each transplant was divided into nine equal portions using a3·3grid,and the center-most portion was assessed for the amount of bone formation(Fig.1C). Statistical comparisons of average outcome levels between groups characterized by scaffold size and sacriﬁce time were based on two-way analysis of variance regression models, including sacriﬁce time by scaffold size interaction terms to allow investigation of time effects.These models were used to obtain group-speciﬁc means and95%conﬁdence intervals as well as pairwise t-test comparisons of means between groups. All analyses were conducted using SAS(version9.12). Mechanical testing of transplantsThe compressive strength of the scaffolds was determined by performing uniaxial tests on the retrieved scaffolds.To avoid damage,the intact specimens were placed on to the test stage in a direction parallel to the printed planer.Pre-vious studies indicate that the compressive response of the scaffolds is very isotropic.4The tests were carried out in air on a universal testing machine at a constant crosshead speed of1mm/min.The load–displacement curve was registered during the tests.The compressive strength of the structure was calculated as the maximum applied load divided by the measured square section of the sample.Identiﬁcation of donor cellsThe human-speciﬁc repetitive alu sequence,which com-prises about5%of the total human genome,can be applied for identiﬁcation of human cells.5We used in situ hybrid-ization for the alu sequence to study the origin of tissues formed in the transplants.The digoxigenin-labeled probe speciﬁc for the alu sequence was prepared by PCR,including 1·PCR buffer(Perkin Elmer),0.1mM dATP,0.1mM dCTP, 0.1mM dGTP,0.065mM dTTP,0.035mM digoxigenin-11-dUTP(Boehringer Mannheim Corp.),10pmol of speciﬁc primers,and100ng of human genomic DNA.The following primers were created on the basis of previously reported sequences6:sense,5¢-GTGGCTCACGCCTGTAATCC-3¢,and antisense,5¢-TTTTTTGAGACGGAGTCTCGC-3¢.The meth-od for in situ hybridization of HA containing transplants has been previously described.3Sections deparafﬁnized with xylene and ethanol were immersed in0.2N HCl at room temperature for7min and then incubated in1mg/mL pep-sin in0.01N HCl at37°C for10min.After washing in phosphate-buffered saline,the sections were treated with 0.25%acetic acid containing0.1M triethanolamine(pH8.0) for10min and prehybridized with50%deionized formam-ide containing4·SSC at37°C for15min.The sections were then hybridized with1ng/m L digoxigenin-labeled probe in hybridization buffer(1·Denhardt’s solution,5%dextran sulfate,0.2mg/mL salmon sperm DNA,4·SSC,50%deio-nized formamide)at42°C for3h after the denaturation step at95°C for3min.After washing with2·SSC and0.1·SSC, digoxigenin-labeled DNA was detected by immunohisto-chemistry using anti-digoxigenin alkaline phosphatase-conjugated Fab fragments(Boehringer Mannheim Corp.). Transplants harvested at18weeks were analyzed.ResultsA total of60transplants were evaluated,including20 harvested at9weeks,10at18weeks,10at23weeks,10at38 weeks,and10at50weeks.Histomorphometry demonstrated that a lamellar spacing of500m m favored bone formation more than a lamellar spacing of50,100,200,or1000m m,for both bone as a frac-tion of the entire transplant(Fig.1D)and for bone as a fraction of only the tissue(nonscaffold)component of the transplant(Fig.1E).Bone formation within the transplant interior was separately assessed at23weeks,the study midpoint,by an examination of the central one-ninth of the transplants;again,the500m m scaffolds had signiﬁcantly greater amounts of bone than the other groups(Fig.1F,G), whereas the1000and200m m scaffolds had negligible amounts of bone within the center of the transplants. Transplant histologyEspecially among the later transplants,bone formation was extensive,and in many areas,bone associated with individual portions of the scaffold appeared to coalesce,forming exten-sive regions of lamellar bone in close proximity to the scaffold (Fig.2A).Older transplants contained abundant hematopoi-etic tissue and occasional adipocytes,all of which were spa-tially associated with the new bone(Fig.2B).A modicum of ﬁbrovascular tissue was found among transplants from23 weeks onward.Older transplants exhibited near complete ﬁlling of the scaffold pores with a combination of bone and hematopoiesis(Fig.2C).All transplants and peri-transplant tissues were characterized by the absence of an inﬂammatory reaction.In summary,relative to young(9week old)trans-plants,the older(50week)transplants exhibited reduced amounts of scaffold,increased amounts of bone and hema-topoiesis,and loss ofﬁbrous tissue.Identiﬁcation of donor cellsThe human alu gene sequence was used to follow the fate of the transplanted cells.Alu served as a marker for donor1618MANKANI ET AL.cell activity because it is not present in the mouse recipientcells.Unstained tissue sections from transplants were eval-uated with a digoxigenin-labeled probe speciﬁc for the alusequence.Alu was detected in osteoblasts and osteocytessurrounding the lattice-work of the scaffolds,and wasprevalent within and on the newly formed bone in thescaffolds.These ﬁndings conﬁrmed that the osteogenic cellswere of donor origin rather than originating from the localmicroenvironment (Fig.3A–D).Alu was restricted to thenew bone and was absent from the peri-transplant tissues.Concurrent positive and negative controls conﬁrmed locali-zation to human cells.These ﬁndings were consistent withour earlier studies.7–9Mechanical testing of transplantsIn compression testing of fresh scaffolds that were devoidof cells,strength increased 35-fold as pore size decreasedfrom 1000to 50m m (Fig.4A).After 23weeks in the mice,strength had increased in all scaffolds,consistent with new bone formation.As transplants were harvested at later time points,the relationship between pore size,bone formation,and strength became more complicated.In large pore scaf-folds (1000and 500m m),strength steadily increased up to the ﬁnal harvest time point of 50weeks,in roughly parallel fashion to the amount of newly formed bone.In contrast,strength of small pore scaffolds (100and 50m m)steadily decreased after 23weeks,eventually reaching parity with their pretransplant strength,despite the formation of sub-stantial amounts of bone.This decrease in strength was not associated with any change in the cross-sectional area of the scaffolds (Fig.4B).The stress–strain relationships of the fresh scaffold,before cell inﬁltration,were consistent with a ceramic,showing brittle behavior (Fig.4C),whereas those relationships among mature transplants,which were extensively inﬁltrated with ﬁbrovascular tissue and marrow,were more consistent with aplastic.FIG.2.(A)Transplant harvested at 23weeks,with 100m m lamellar spacing.Bone is lamellar rather than woven,and is intimately associated with scaffold.Magniﬁcation:20·.(B)Transplant harvested at 23weeks,with 500m m lamellar spacing.Bone is lamellar.Territories of bone and scaffold encompass regions of hematopoiesis.Magniﬁcation:20·.(C)Transplant harvested at 38weeks,with 500m m lamellar spacing.Bone ﬁlls the majority of nonscaffold spaces in this section.Magniﬁ-cation:5·(b,bone;fv,ﬁbrovascular tissue;s,scaffold;h,hematopoietic tissue)Stain:hematoxylin and eosin;parafﬁn embedding following demineralization.Color images available online at /teaLAMELLAR SPACING REGULATES BONE FORMATION 1619DiscussionParticulate and rigid ceramic scaffolds have a role assupports for osteogenic stem cells in bone tissue engineer-ing.Particulate scaffolds,with which our group has greaterin vivo experience,can be combined ex vivo with culture-expanded BMSCs and placed into animal recipients,wherethey will successfully form bone within 8weeks.7,9–13Innude mice,bone formation can occur with rodent BMSCs orhBMSCs,in either orthotopic or heterotopic locations.7,13Such transplants have successfully closed critical-sized cal-varial defects in mice and dogs.1,7The loose cohesion be-tween particles appears to facilitate nourishment andeventual vascularization of cells within the entire crosssection of these transplants,even those as thick as 8mm.1Despite these attractive features,particulate scaffolds’maindrawback is their inability to bear mechanical loads duringbone formation,making them poor candidates for imme-diate load-bearing reconstruction of long-bone defects or fractures.Rigid ceramic scaffolds,in contrast,can be formulated to bear substantial loads from the time of placement.However,earlier rigid scaffolds have not been successful at promoting bone formation by BMSCs because they were unable to balance the competing demands of porosity and strength.Strong scaffolds,for instance,have been too dense to support cell survival;conversely,porous scaffolds maintain cell via-bility but cannot support loads.Our group is now attempt-ing to develop novel strong scaffolds that simultaneously support cell survival and bone formation.2,4,14–16Despite our initial success,actually understanding how to optimize scaffold geometry,structure,and composition re-mains a formidable challenge,given the myriad number of parameters that can potentially be optimized.This effort has been somewhat simpliﬁed by earlier investigators who fo-cused on improving the osteoconductive properties ofrigidFIG.3.(A)Transplant harvested at 18weeks,with 1000m m lamellar spacing.Scaffold lamellae are widely spaced.Bone remains associated with scaffold.Magniﬁcation:5·.(B)Histologic section at same location as in (A).Conﬁrmation of the donor origin of the newly formed bone.In situ hybridization to ALU is localized to osteocytes in the new bone,and is absent in the peri-transplant tissues.Magniﬁcation:5·.(C)Higher powered image of histologic section seen in (A).Magniﬁcation:20·.(D)Histologic section at same location as in (C).Conﬁrmation of the donor origin of the newly formed bone.In situ hybridization to ALU is localized to osteocytes in the new bone,and in BMSCs persisting from the time of transplantation.Magniﬁcation:20·(b,bone;s,scaffold;arrows,alu -positive cells)Stain (A,C):hematoxylin and eosin;parafﬁn embedding following demineralization.Color images available online at /tea1620MANKANI ET AL.scaffolds.17Those studies typically involved placing thescaffold adjacent to normal bone and then measuring the rateand amount of bone ingrowth into the scaffold.Manipula-tion of HA and TCP ratios,micro-porosity,macro-porosity,and surface texture have optimized the ability of thesescaffolds to support new bone ingrowth.Unfortunately,these studies have typically examined bone formation at thesurface of the scaffold rather than at its interior,because theyoccur over short time-frames,so they could not address questions involving penetration of bone into the scaffold interior.Additionally,none of these studies have included co-transplantation of human osteoprogenitor cells,as our study has.18–21This study was designed to offer several novel sets of observations.First,observations were made over an entire year,rather than the typical 8to 16weeks found in most other papers in this ﬁeld,demonstrating that bone forma-tion continues to increase until at least 38weeks.Second,FIG. 4.(A)Compressive strength of the entire trans-plant,at 0,23,38,and 50weeks.(B)Percentage of scaffold within the entire transplant.Transplant periphery and center are both included.(C)Stress–strain relationships for 1000m m scaffold before introduction of cells (blue lower left curve)and following 50weeks of transplantation (red upper curve).Color images available online at www /teaLAMELLAR SPACING REGULATES BONE FORMATION 1621histologic and mechanical observations were made syn-chronously,demonstrating that they are optimized at dif-ferent pore sizes,and therefore need to both be measured to better describe the effect of pore size on scaffold perfor-mance.This study was also designed to evaluate the interplay between scaffold surface area and the density of hBMSCs within the scaffold on bone formation.New bone formation ﬁrst occurs on the scaffold surface,so that scaffolds with substantial surface areas would be expected to promote substantial bone.Yet,linear increases in cell density lead to exponential increases in bone formation,perhaps due to paracrine signaling among the cells,so that scaffolds with substantial voids which wereﬁlled with cells would be ex-pected to also promote bone formation.Our study attempted to identify which of these conditions(substantial surface area vs.high cell density)might best promote bone formation. The experiments were thus established with an expectation that cell density in the scaffolds would increase as lamellar spacing increased.In this study,we created via robo-casting a set of HA scaffolds differing only in the distance between adjacent la-mellae.The thickness of the lamellae,their texture,and their microporosity were kept consistent from scaffold to scaffold. We cultured hBMSCs onto the scaffolds ex vivo and then transplanted the constructs into nude mice.Scaffolds were observed at time points ranging from9to50weeks.By histology,bone formation within each scaffold was quanti-ﬁed across the entire cross section and speciﬁcally at its in-terior.In situ hybridization was used to conﬁrm a donor source for the bone and osteoblasts.Scaffold strength was measured in the scaffolds before cell seeding and then at23, 38,and50weeks.We observed bone formation at theﬁrst time-point(9 weeks)at the scaffold periphery.With increasing time, overall bone formation increased steadily,proceeding radi-ally mellar spacing of500m m was most support-ive of bone formation at the latest time-points of38and50 weeks in the overall transplant,and especially supported bone formation within the scaffold interior at the interme-diate time-point of23weeks.The scaffolds also supported the formation of a marrow organ,consistent with our ex-pectations of a bone-supporting scaffold.The osteocytes within the newly formed bone and the osteoblasts lining the bone were conﬁrmed to be of human origin using in situ hybridization for Alu,and were therefore the transplanted hBMSCs or their progeny.No hBMSCs independent of newly formed were identiﬁed,and no cartilage was seen in any section.No tumors were observed in the transplants or elsewhere in the animals,consistent with our14-year expe-rience transplanting hBMSCs into nude mice.Transplant compressive strength offered additional in-sights into scaffold performance.Among fresh scaffolds, compressive strength predictably increased as lamellar spacing decreased.Following transplantation and then har-vest at the23-week time-point,bone had developed in all transplants and was associated with increases in compres-sive strength in all groups.At later time-points,however,the association between greater bone formation and greater compressive strength was lost,in that transplants with nar-row lamellar spacing had diminished strength despite an increase bone formation and no change in scaffold volume.The loss of scaffold strength has two possible explanations—it could be due to a weakening of the inherent scaffold structure over time,most likely due to resorption of the more soluble b-TCP from the scaffold,or it might be due to propagation of micro-fractures within the scaffold.22Distin-guishing between these two explanations would require additional testing of the transplants in a fresh experiment, perhaps using X-ray microtomography and SEM to quantify the degree of in vivo resorption.In our current study,the compressive strength of our scaffolds reﬂected an interplay between bone formation and scaffold resorption.It is perhaps not surprising,then,that scaffold histology and mechanical strength do not necessar-ily coincide—the transplants with the greatest bone forma-tion(500m m spacing)were not those with the greatest strength(200m m spacing).These results highlight the im-portance of using multiple measures of scaffold success (histologic appearance and mechanical performance)to help predict scaffold performance.An ideal experimental system would permit us to examine the impact of bone formation on scaffold strength separately from the impact of scaffold resorption.Unfortunately,that is difﬁcult to achieve in our model,in which transplanted BMSCs support both bone formation and the formation of a marrow microenvironment;this new marrow in turn may accelerate the scaffold resorption process by introducing osteoclasts to the scaffold.Thus,bone formation supports hematopoiesis,which in turn supports scaffold resorption. The most obvious method for assessing scaffold resorption in the absence of bone formation might have been inclusion of a cell-free transplant in the mouse experiment.While this transplant would have failed to support new bone formation, it would also have failed to form a marrow microenviron-ment,so it would not undergo as substantial a degree of resorption as a BMSC-imbued transplant.Ourﬁndings support the hypothesis that lamellar spacing helps determine the amount of new bone that forms on HA scaffolds as well as the compressive strength of these scaf-folds after1year.We speculate that spacing that is too wide may not provide a sufﬁciently high density of BMSCs to stimulate bone formation,23whereas spacing that is too narrow may impede short-term nourishment of the cells and long-term vascular ingrowth.Theseﬁndings are roughly consistent with those in our prior study using particulate scaffolds,in which particles smaller than100m m and larger than1000m m promoted markedly less bone formation.10The investigation of the speciﬁc impact of lamellar spacing on vascular invasion,and the impact of vascular invasion on bone formation,might shed light on these speculations. However,we have observed that successful development of bone cannot occur without adequate vascularization.BMSCs do attract vascularization,so it can be reasonably assumed that in each particular transplant,the degree of bone for-mation reﬂects and is supported by the corresponding de-gree of vascularization.Ourﬁndings also suggest that transplant strength represents the result of a complex inter-play between new bone formation and scaffold resorption. Our results suggest that optimization of scaffold geome-try,by varying lamellar spacing as we did here,or by varying other parameters such as HA/TCP ratios or surface roughness,may prove sufﬁcient by itself to promote bone formation in the interior of moderately sized rigid scaffolds.1622MANKANI ET AL.。

研究生学术英语高原第七单元课文翻译

为什么材料的历史是真正的文化历史？1.每样东西都是由某种东西构成的。

如果把混凝土、玻璃、纺织品、金属和其他材料从我们的生活中拿走，我们就只能赤身裸体，在泥泞的田野里瑟瑟发抖。

我们生活的复杂性在很大程度上是由物质财富赋予的，如果没有我们的文明，我们将很快恢复到动物行为:使我们成为人类的是我们的衣服、我们的家、我们的城市、我们的东西，我们通过我们的习俗和语言赋予这些东西生命。

如果你去过灾区，这一点就会变得非常明显。

然而，物质世界不仅仅是我们技术和文化的展示，它是我们的一部分，我们发明它，我们创造它，它造就了我们。

2.材料的根本重要性从各个文明时代的命名——石器时代、铁器时代和青铜时代——就可以清楚地看出，每个新时代都由一种新材料带来。

钢铁是维多利亚时代的主要材料，工程师们可以充分发挥他们的梦想，建造悬索桥、铁路、蒸汽机和客轮。

Isambard Kingdom Brunel 将其作为改造世界的宣言，并播下现代主义的种子。

20世纪常被誉为硅的时代，在材料科学取得突破后，迎来了硅芯片和信息革命。

然而,其他新材料的万花筒也彻底改变了现代生活。

建筑师将大量生产的平板玻璃与结构钢结合在一起，建造摩天大楼，从而发明了一种新型的城市生活。

塑料改变了我们的家庭和衣着。

聚合物被用来制造电影胶片，并引入了一种新的视觉文化——电影。

铝合金和镍高温合金的发展使我们能够廉价飞行，并加速了文化的碰撞。

医疗陶瓷和牙科陶瓷让我们得以重建自我，重新定义残疾和衰老——正如“整形手术”一词所暗示的那样，材料往往是修复我们的功能(髋关节置换)或增强我们的特征(隆胸硅胶植入物)的新疗法的关键。

3.我对材料的痴迷始于青少年时期。

我对他们的默默无闻感到困惑，尽管他们就在我们身边。

有多少人能看出铝和钢的区别?木头之间明显不同，但有多少人能说出原因?塑料是混杂的;谁知道聚乙烯和聚丙烯的区别?最终，我进入牛津大学(Oxford University)材料科学系攻读学位，接着攻读喷气发动机合金博士学位，现在是伦敦大学学院(University College London)材料与社会教授和制造研究所(Institute of Making)主任。

人体结构学 Human Structure学习通章节答案期末考试题库2023年

人体结构学 Human Structure学习通超星课后章节答案期末考试题库2023年1.Which bones belong to the shoulder girdle?答案:Scapula###Clavicle2.The paired cerebral bones are答案:parietal bone###temporal bone3.Shoulder joint is formed by答案:head of humerus###glenoid cavity of scapula4.Please deseribe the location and openings of the paranasal sinuses答案:答5.Please describe the formation, main structures and communications of the middle Cranial fossa.答案:答6.Please describe the joints of the vertebral bodies.答案:答7.Please describe the joints of the vertebral arches.答案:答8.Please describe the composition, characteristics and movements of theshoulder joint.答案:答9.Which bone belongs to the long bone?答案:Femur10.Which bones belong to the irregular bone?答案:Vertebra###Sphenoid bone11.The blood- testis barrier does NOT include the答案:gap junction between adjacent spermatogomia12.Which of the following description is true about the primordial follicles答案:The primordial follicle consists of a primary oocyte and a layer of flat follicle cells.13.(英文答题,第一空填1个单词,第二空3个单词)The axial bone contains fromup downwards _____ and_____.答案:skull###bonesoftrunk14.About the component of nephron, the correct option is答案:renal corpuscle, proximal tubules, distal tubules and thin segment15.About the features of proximal tubule, the WRONG option is答案:The cytoplasm of epithelial cell is weakly basophilic.16. A patient presents in your office after having a positive result on a homepregnancy test. Her menstrual cycle has always been the classic 28-day cycle discussed in textbooks, with ovulation occurring on the 14th day following the start of menstruation. Her menstrual period began on August 19.2019.You estimate her EDD to be答案:on May 26, 202017.Which of the following is NOT considered one of the fetal membranes答案:buccopharyngeal membrane18.Which bone does not form the anterior cranial fossa?答案:Temporal bone19.Which bone forms both the middle and posterior cranial fossa?答案:Temporal bone20.(英文答题,第一空填1个单词,第二空1个单词,第三空2个单词)Thesternum consists from up downwards of_____ , _______ and ______ .答案:manubrium###body###xiphoidprocess21.Of the following statements about epididymis, the WRONG option is答案:The ductus epididymis is lined with a simple columnar epithelium22.Of the following statements about trachea, the WRONG option is答案:The adventitia is constructed of the elastic cartilage rings.23.The interalveolar septum does NOT contain答案:ciliated cell24.All the following cells are included in the spermatogenic epithelium, EXCEPTthe答案:Leydig cells25.Please describe the general features of the vertebrae.答案:答26.Which bone forms the posteroinferior part of the bony nasal septum?答案:Vomer27.Drawing pictures of Thoracic vertebra from anterior and lateral view.答案:答28.Which bone does not form the thoracic Cage?答案:Sacrum29.About the scapula, which of the statements is not true?答案:It has three borders, three angles and three surfaces.30.About the component of the renal corpuscle, the WRONG option is答案:At the vascular pole, the efferent arteriole enters the glomerulus.31.Of the following statements about podocyte, the correct option is答案:They form the visceral layer of the Bowman's capsule.32.An infant is born with a sacrococcygeal teratoma. Biopsy(组织活检) andhistologic analysis reveal that it contains intestinal epithelia, cardiac muscle, cartilage, and integument tissue. You counsel the mother that the tumor is benign（良性的）and recommend surgical removal. This tumor was caused by which developmental anomaly?答案:Failure of primitive streak regression33.Which of the following structure is NOT included in the secondary follicle?答案:secondary oocyte34.All the following are from mesoderm EXCEPT the答案:spinal cord35.Which of the following descriptions is NOT true about the corpus luteum?答案:The corpus luteum continues to produce estrogen and progesterone during the whole process of pregnancy.36.Of the following statements about the alveolus of lung, the WRONG option is答案:It opens on the wall of terminal bronchioles.37.Which of the following descriptions is NOT true about the secretory phase ofa menstrual cycle?答案:The basal layer of endometrium becomes thicker .38.Of the following statements about Leydig cells, the correct option is答案:It secretes testosterone.39.Of the following statements about macula densa, the WRONG option is答案:It is derived from smooth muscle fibers of afferent arteriole.40.Of the following statements concerning terminal bronchioles, the WRONGoption is答案:They have some mixed gland.41.Of the following options, the blood-air barrier does NOT contain答案:typeⅡalveolar cells42.All the following cells are included in the spermatogenic cells, EXCEPT答案:Sertoli cells43.Which of the following does not belong to the joints of the vertebral arches?答案:Anterior longitudinal ligament44.Human chorionic gonadotropin is produced by the答案:syncytiotrophoblast45.Which of the followings is not enclosed in the articular capsule of shoulderjoint ?答案:Tendon of the short head of the biceps46.The pathway connecting the infratemporal fossa with the orbit is答案:inferior orbital fissure47.When does ovulation occur in a menstrual cycle?答案:the 14th day。

翻译

石墨为基础的单一细菌分辨率装置和DNA晶体管：石墨衍生物纳米微型生物成份的界面连接技术摘要建立“大接触面积”与微生物和哺乳动物细胞中的敏感纳米界面将导致生物诊断和生物医学方面有价值的工具和设备的发展。

化学改性石墨（CMG）的纳米结构与他们的微型区域、敏感的电气性能与修改的化学功能是生物细胞和生物分子尺度方面的生物装置的极好的选择。

在此,我们报告一种新型CMG-型的制作和运作：（I）单细菌生物装置(ii)无标签的DNA传感器，及（iii）细菌的DNA/蛋白质和电解质的化学晶体管。

在p型CMG，细菌生物装置与单细菌附件产生的约1400载流子是非常敏感。

同样的,拴在石墨上的单链DNA与它的互补DNA 混合，以便可逆增加空穴密度到5.61 ×1012 cm-2.。

我们进一步证明（a）通过操控表面组织,对装置灵敏的控制（b）通过改变表面极性使极性特异性的转换（c）在厚的CMG的表面和尖的CMG 皱纹上的DNA的优先连接物在过去十年中，已经出现了对制造与电，光，热活跃的纳米材料有关纳米/生物界面的大量研究，这使得生物医学、生物驱使设备、生物检测和诊断学领域极大地前进。

当前一代用零维（0D）纳米粒子、一维（1D）纳米线和二维网络建造的电活性纳米生物装置显示了对分子的（DNA，三磷酸腺苷，蛋白质等）和纳米（病毒等）生物成份的优良检测和接口技术能力。

然而,在区域尺寸不相容在使用个别的零维和一维纳米结构时具有挑战性，这种纳米结构是为与大尺寸的微生物建造强的界面或保留他们在网络上。

化学改性的石墨有二维纳米结构和可调表面化学，它能与生物系统界面在无几何限制和在不损害微生物附着的完整性强烈接触。

最近，化学的和几何学的石墨处理显现出极大的潜力来控制半金属和半导体之间的带间隙。

此外，其低电气噪声（低电荷散射）和弹道输运，石墨纳米结构已合成各种电子和光电子应用产品，如气体传感器、晶体管、太阳能电池和液晶元素。

然而，还没有石墨烯在生物装置上应用的报告。

细胞生物学期末复习 Cell Biology 英文

1.I n te rmedia te Filaments (~10 nm)1.Intro1.Main function: enable cells to withstand the mechanical stress that occurs when cells are stretched.2.toughest and most durable of the three types of cytoskeletal filaments.3.Can be found in cytoplasm as well as the nucleus. A mesh of intermediate filaments the nuclear lamina,underlies and strengthens the nuclear envelope in all eucaryotic cells.2.Intermediate ﬁlaments are strong and ropelike1.Elongated fibrous protein, each composed of an N-terminal globular head, a C-terminal globular tail, and acentral elongated rod domain, is the subunit of intermediate filaments. The rod domain consists of anextended α-helical region that enables pairs of intermediate filament proteins to form stable dimers bywrapping around each other in a coiled-coil configuration. Two of these coiled-coiled dimers then associateby noncovalent bonding to form a tetramer. The tetramers then bind to one another end to end and side byside, and also by noncovalent bonding, to generate the final ropelike intermediate filament. Eight tetramersare twisted into a ropelike filament.2.the globular head and tail regions, which are exposed on the surface of the filament, allow it to interact withother components of their cytoplasm. The globular domains vary greatly in both size and amino acidsequence from one intermediate filament protein to another.3.Intermediate ﬁlaments strengthen cells against mechanical stress1.Present in large numbers: along the length of nerve cell axons, providing essential internal reinforcement tocell extensions; in muscle cells and in epithelial cells.2.In all these cells, intermediate filaments, by stretching and distributing the effect of locally applied forces,keep cells and their membranes from breaking in response to mechanical shear.3.Intermediate filaments can be grouped into four classes: (1)keratin filaments in epithelial cells, hair andnails; (2)vimentin and vimentin-related filaments in connective-tissue cells, muscle cells, and supportingcells of the nervous system (glial cells); (3)neurofilaments in nerve cells; and (4)nuclear lamins, whichstrengthen the nuclear membrane of all animal cells4.Keratin filaments typically span the interiors of epithelial cells from one side of the cell to the other, andfilaments in adjacent epithelial cells are indirectly connected through cell-cell junctions called desmosomes.This cabling of high tensile strength, formed by the filaments through the epithelial sheet, distributes thestress that occurs when the skin is stretched.5.Many intermediate filaments are further stabilized and reinforced by accessory proteins, such as plectin, thatcross-link the filament bundles into strong arrays. In addition to holding together bundles of intermediatefilaments (particularly vimentin), these proteins link intermediate filaments to microtubules, to actinfilaments, and to adhesive structures in the desmosomes.4.The nuclear envelope is supported by a meshwork of intermediate ﬁlaments1.Intermediate filaments lining and strengthening the inside surface of the inner nuclear membrane areorganized as a two-dimensional mesh. The intermediate filaments within this tough nuclear lamina areconstructed from a class of intermediate filament proteins called lamins.2.Intermediate filaments of the nuclear lamina disassemble and re-form at each cell division, when the nuclearenvelope breaks down during mitosis and then re-forms in each daughter cell.3.Disassembly and reassembly of the nuclear lamina are controlled by the phosphorylation anddephosphorylation of the lamins by protein kinases. When the lamins are phosphorylated, the consequentconformational change weakens the binding between the tetramers and causes the filament to fall apart.Dephosphorylation at the end of mitosis causes the lamins to reassemble.4.Defects in a particular nuclear lamin are associated with certain types of progeria - rare disorders that causeaffected individuals to appear to age prematurely.2.M icrotubule s1.Intro1.Microtubules are long and stiff hollow tubes of protein that can rapidly disassemble in one location andreassemble in another.2.In a typical animal cell, microtubules grow out from a small structure near the center of the cell calledcentrosome.3.Extending out toward the cell periphery, they create a system of tracks within the cell, along which vesicles,organelles, and other cell components are moved. These and other systems of cytoplasmic microtubules arethe part of the cytoskeleton mainly responsible for anchoring membrane-enclosed organelles within the celland for guiding intracellular transport.4.When a cell enters mitosis, the cytoplasmic microtubules disassemble and then reassemble into an intricatestructure called the mitotic spindle. The mitotic spindle provides the machinery that will segregate thechromosomes equally into the two daughter cells just before a cell divides.5.Microtubules can also form permanent structures called cilia and flagella as a means of propulsion or tosweep fluid over the cell surface. The core of a eucaryotic cilium or flagellum consists of a highly organized and stable bundle of microtubules.2.Microtubules are hollow tubes with structurally distinct ends1.Microtubules are built from subunits-molecules of tubulin-each of which is itself a dimer composed of twovery similar globular proteins called α-tubulin and β-tubulin, bound tightly together by noncovalentbonding.2.The tubulin dimers stack together, again by noncovalent bonding, to form the wall of the hollow cylindricalmicrotubule. This tubelike structure is made of 13 parallel protofilaments, each a linear chain of tubulindimers with α- and β-tubulin alternating along its length. Each protofilament has a structural polarity, with α-tubulin exposed at one end and β-tubulin at the other, and this polarity is the same for all theprotofilaments giving a structural polarity to the microtubule as a whole. β-tubulin end = plus end; α-tubulin end = minus end.3.In vitro, tubulin dimers will add to either end of a growing microtubule, although more rapidly to the plusend than the minus end. This polarity is crucial both of the assembly of microtubules and for their role once they are formed. If they had no polarity, they could not serve their function in defining a direction forintracellular transport, for example.3.The Centrosome is the major microtubule-organizing center in animal cells1.Microtubules are formed by outgrowth from centrosome, which is typically close to the cell nucleus whenthe cell is not in mitosis. It organized the array of microtubules that radiates outward from it through thecytoplasm. Centrosomes contain hundreds of ring-shaped structures formed from another type of tubulin, γ-tubulin ring serves as the starting point, or nucleation site, for the growth of one microtubule. The αβ-tubulin dimers add to the γ-tubulin ring in a specific orientation, with the result that the minus end of each microtubule is embedded in the centrosome and growth occurs only at the plus end.2.The centrosome in most animal cells also contains a pair of centrioles, each made of a cylindrical array ofshort microtubules.3.It is much harder to start a new microtubule from scratch, by first assembling a ring of αβ-tubulin dimers,than to add such dimers to a preexisting microtubule structure. By providing organizing centers containing nucleation sites, and keeping the concentration of free αβ-tubulin dimers low, cells can thus control where microtubules form.4.Growing microtubules show dynamic instability1.Dynamic instability stems from the intrinsic capacity of tubulin molecules to hydrolyze GTP. Each freetubulin dimer contains one tightly bound GTP molecule that is hydrolyzed to GDP (still tightly bound)shortly after the subunit is added to a growing microtubule.2.When polymerization is proceeding rapidly, tubulin molecules add to the end of the microtubule faster thanthe GTP they carry is hydrolyzed. The end of a growing microtubule is therefore composed entirely of GTP-tubulin subunits, forming what is know as a GTP cap. In this situation, the growing microtubule willcontinue to grow. Because of the randomness of chemical processes, however, it will occasionally happen that tubulin at the free end of the microtubule hydrolyzes its GTP before the next tubulin has been added, so that the free ends of protofilaments are now composed of GDP-tubulin subunits. This change tips thebalance in favor of disassembly. Because the rest of the microtubule is composed of GDP-tubulin, oncedepolymerization has started, it will tend to continue, often at a catastrophic rate; the microtubule starts to shrink rapidly and continuously, and may even disappear.5.Microtubules are maintained by a balance of assembly and disassembly1. A microtubule growing out from the centrosome can be prevented from disassembling if its plus end issomehow permanently stabilized by attachment to another molecule or cell structure so as to prevent tubulin depolymerization. If stabilized by attachment to a structure in a more distant region of the cell, themicrotubule will establish a relatively stable link between that structure and the centrosome.2.If a cell in mitosis is exposed to the drug colchicine, which binds tightly to free tubulin and prevents itspolymerization into microtubules, the mitotic spindle rapidly disappears and the cell stalls in the middle of mitosis, unable to partition its chromosomes into two groups.3.The drug taxol has the opposite action at the molecular level. It binds tightly to microtubules and preventsthem from losing subunits. Because new subunits can still be added, the microtubules can grow but notshrink.6.Microtubules organize the interior of the cell1.As cells enter mitosis, microtubules become more dynamic to enable them to disassemble rapidly and thenreassemble into the mitotic spindle. When a cell has differentiated into a specialized cell type and taken on a definite fixed structure, the dynamic instability is often suppressed. Stabilized microtubules serve tomaintain the organization of the cell.2.In the nerve cell, all the microtubules in the axon point in the same direction, with their plus ends toward theaxon terminal.7.Motor proteins drive intracellular transport1.Mitochondria and the smaller membrane-enclosed organelles and vesicles move in small, jerky steps. Thissaltatory movement is more sustained and directional than Brownian movement.2.Both microtubules and actin filaments are involved in saltatory movement. In both cases, the movements aregenerated by motor proteins, which use the energy derived from repeated cycles of ATP hydrolysis to travelsteadily along the actin filament or the microtubule in a single direction. At the same time, these motorproteins also attach to other cell components and thus transport this cargo along the filaments.3.The kinesins move toward the plus end of a microtubule (away from the centrosome), while the dyneinsmove toward the minus end (toward the centrosome). These kinesins and dyneins are both dimers with twoglobular ATP-binding heads and a single tail. The heads interact with microtubules in a stereospecificmanner, so that the motor protein will attach to a microtubule in only one direction. The tail of a motorprotein generally binds stably to some cell component, such as a vesicle or an organelle, and therebydetermines the type of cargo that the motor protein transports.anelles move along microtubules1.As the cell develops and the endoplasmic reticulum grows, kinesins attached to the outside of theendoplasmic reticulum membrane (via receptor proteins) pull it outward along microtubules, stretching itlike a net. Dyneins, similarly attached to the Golgi membranes, pull the Golgi apparatus the other way along microtubules inward toward the cell center.9.Cilia and ﬂagella contain stable microtubules moved dynein1. A single cilium contains a core of stable microtubules, arranged in a bundle, that grow from a basal body inthe cytoplasm; the basal body serves as the organizing center for the cilium.2.Cilia move fluid over the surface of a cell or propel single cells through a fluid.3.The flagella are much like cilia in structure but are usually much longer.4. A cross section through a cilium shows nine doublet microtubules arrange in a ring around a pair of singlemicrotubules.5.The movement of a cilium of a flagellum is produced by the bending of its core as the microtubules slideagainst each other. Ciliary dynein generates the bending motion of the core.3.A c ti n ﬁlaments (~7 nm)1.Actin ﬁlaments are thin and ﬂexible1.Each filament is a twisted chain of identical globular actin molecules, all of which “point” in the samedirection along the axis of the chain.2.Actin filament has a structural polarity, with a plus end and a minus end.2.Actin and tubulin polymerize by similar mechanisms1.Actin filaments can grow by the addition of actin monomers at either end, but the rate of growth is faster atthe plus end than at the minus end. A naked actin filament is unstable, and it can disassemble from bothends. Each free actin monomer carries ATP which is hydrolyzed to ADP soon after the incorporation of theactin monomer into the filament. Hydrolysis of ATP to ADP in an actin filament reduces the strength ofbinding between monomers and decreases the stability of the polymer. Nucleotide hydrolysis promotesdepolymerization, helping the cell to disassemble filaments after they have formed.2.Toxins such as cytochalasins prevent actin polymerization; toxins such as phalloidin stabilize actinfilaments against depolymerization.3.Many proteins bind to actin and modify its properties1.Cells contain small proteins, such as thymosin and profilin, that bind to actin monomers in the cytosol,preventing them from adding to the ends of actin filaments. These proteins play a crucial role in regulatingactin polymerization.2.When actin filaments are needed, proteins called formins and actin-related proteins (ARPs) both controlactin assembly at the advancing front of a migrating cell.4.An actin-rich cortex underlies the plasma membrane of most eucaryotic cells1.Actin is highly concentrated in cell cortex, a layer just beneath the plasma membrane. In this region, actinfilaments are linked by actin-binding proteins into a meshwork that supports the outer surface of the cell and gives it mechanical strength.5.Cell crawling depends on actin1.Cell crawling includes three essential processes: (1) the cell pushes out protrusions at its “front,” or leadingedge; (2) these protrusions adhere to the surface over which the cell is crawling; and (3) the rest of the celldrags itself forward by traction on these anchorage points.2.In the first step, the leading edge of a crawling fibroblast in culture regularly extends thin, sheetlikelamellipodia, which contain a dense meshwork of actin filaments, oriented so that most of the filamentshave their plus ends close to the plasma membrane. Many cells also extend thin, stiff protrusions calledfilopodia, both at the leading edge and elsewhere on their surface. Both lamellipodia and filopodia areexploratory, motile structures that form and retract with great speed. Both are thought to be generated by the rapid local growth of actin filaments, which assemble close to the plasma membrane and elongate by theaddition of actin monomers at their plus ends. In this way the filaments push out the membrane withouttearing it.3.Actin-related proteins (ARPS) promotes the formation of a web of branched actin filaments in lamellipodia.Formins attach to the growing ends of actin filament and promote the addition of new monomers to formstraight unbranched filaments.4.When the lamellipodia and filopodia touch down on a favorable patch of surface, they stick. Integrinsadhere to molecules in the extracellular matrix that surrounds cells or on the surface of a neighboring cellover which the moving cell is crawling. On the intracellular face of the crawling cell’s membrane, integrinscapture actin filaments, thereby creating a robust anchorage for the system of actin filaments inside thecrawling cell.6.Actin associates with myosin to form contractile structures1.Myosin bind to and hydrolyze ATP, which provides the energy for their movement along actin filamentsfrom the minus end of the filament toward the plus end.2.Myosin-I and myosin-II are most abundant. Myosin-I molecules have only one head domain and a tail.1.Overview of th e ce ll cycl e1.The eucaryotic cell cycle is divided into four phases1.2 most dramatic events: mitosis and cytokinesis. These two processes together constitute the M phase of thecell cycle.2.The period between one M phase and the next is called interphase. It consists of three phases: S phase (thecell replicates its nuclear DNA) and G1 and G2 phase (between S and M). During the gap phases, the cellmonitors the internal and external environments to ensure that conditions are suitable and its preparations are complete before it commits itself to S or M.3.During all of interphase, a cell generally continues to transcribe genes, synthesize proteins, and grow in mass.2.A cell-cycle control system triggers the major processes of the cell cycle1.The cell-cycle control system guarantees that the events of the cell cycle occur in a set sequence and that eachprocess has been complete before the next one begins. The cell-cycle control system achieves this bymolecular brakes that can stop the cycle at various checkpoints.2.3 checkpoints: one checkpoint operates in G1 and allows the cell to confirm that the environment is favorablefor cell proliferation before committing to S phase. If extracellular conditions are unfavorable, cells can delay progress through G1 and may even enter a specialized resting state know as G0. Another checkpoint operates in G2 and ensures that cells do not enter mitosis until damaged DNA has been repaired and DNA replication is complete. A third checkpoint operates during mitosis and ensures that the replicated chromosomes areproperly attached to a cytoskeletal machine, called the mitotic spindle, before the spindle pull thechromosomes apart and distributes them into the two daughter cells.2.Th e ce ll-cycle con tr ol sys tem1.The cell-cycle control system depends on cyclically activated protein kinases called Cdks1.The protein kinases at the core of cell-cycle control systems are present in proliferating cells throughout thecell cycle. The activity of each of these kinases rises and falls in a cyclical fashion.2.Switching the kinases on and off at the appropriate times is partly the responsibility of another set of proteinsin the control system-the cyclins. Cyclins have to bind to the cell-cycle kinases before the kinases canbecome enzymatically active. Kinases of cell-cycle are therefore called cyclin-dependent protein kinases (or Cdks).2.The activity of Cdks is also regulated by phosphorylation and dephosphorylation1.Cyclin concentrations increase gradually, but the activity of associated cyclin-Cdk complexes tends to switchon abruptly at the appropriate time in the cell cycle.2.For a cyclin-Cdk to be maximally active, the Cdk has to be phosphorylated at one site by a specific proteinkinase and dephosphorylated at other sites by a specific protein phosphatase.3.Different cyclin-cdk complexes trigger different steps in the cell cycle1.The cyclin that acts in G2 to trigger entry into M phase is called M cyclin, and the active complex it formswith its Cdk is called M-Cdk. Distinct cyclins, called S cyclins and G1/S cyclins, bind to a distinct Cdkprotein late in G1 to form S-Cdk and G1/S-Cdk, respectively, and trigger S phase. Other cyclins, called G1cyclins, act earlier in G1 and bind to other Cdk proteins to form G1-Cdks, which help drive the cell throughG1 toward S phase.4.The cell-cycle control system also depends on cyclical proteolysis1.The concentration of each type of cyclin rises gradually but falls sharply. This abrupt fall results from thetargeted degradation of the cyclin protein. Specific enzyme complexes add ubiquitin chains to the appropriate cyclin, which is then directed to the proteasome for destruction.5.Proteins that inhibit Cdks can arrest the cell cycle at speciﬁc checkpoints1.Some of the molecular brakes rely on Cdk inhibitor proteins that block the assembly or activity of one ormore cyclin-Cdk complexes.2.As a general rule, mammalian cells will multiply only if they are stimulated to do so by extracellular signalscalled mitogens produced by other cells. If deprived of such signals, the cell cycle arrests at a G1 check-point; and, if the cell is deprived for long enough, it will withdraw from the cell cycle and enter the non-proliferating state G0.3.The G1 checkpoint is sometimes called Start.3.S phas e1.S-Cdk initiates DNA replication and helps block re-replication1.DNA replication begins at origins of replication, nucleotide sequences that are scattered along eachchromosome. These sequences recruit specific proteins that control the initiation and completion of DNAreplication. One multiprotein complex, the origin recognition complex (ORC) remains bound to origins ofreplication throughout the cell cycle, where it serves as a sort of landing pad for additional regulatory proteins that bind before the start of S phase.2.One of the regulatory proteins, called Cdc6, is present at low levels during most of the cell cycle, but itsconcentration increases transiently in early G1. When Cdc6 binds to ORCs in G1, it promotes the binding of additional proteins to form a pre-replicative complex. Once the pre-replicative complex has been assembled, the replication origin is ready to “fire”. The activation of S-Cdk in late G1 then “pulls the trigger”, initiating DNA replication.3.S-Cdk also helps prevent re-replication of the DNA. Activated S-Cdk helps phosphorylate Cdc6, causing itand other proteins in the pre-replicative complex to dissociate from the ORC after an origin has fired. Thisdisassembly prevents replication from occurring again at the same origin. In addition to promotingdissociation, phosphorylation of Cdc6 by S-Cdk marks it for degradation, ensuring that DNA replication isnot reinitiated later in the same cell cycle.2.Cohesins help hold the Sister Chromatids of each replicated chromosome together1.The sister chromatids are held together by protein complexes called cohesins. Cohesins form protein ringsthat surround the two sister chromatids, keeping them united.3.DNA damage checkpoints help prevent the replication of damaged DNA1.DNA damage checkpoint in G1 is especially well understood. DNA damage causes an increase in both theconcentration and activity of a protein called p53, which is a transcription regulator that activates thetranscription of a gene encoding a Cdk inhibitor protein called p21. The p21 protein binds to G1/S-Cdk andS-Cdk, preventing them from driving the cell into S phase. The arrest of the cell cycle in G1 gives the celltime to repair the damaged DNA before replicating it. If the DNA damage is too severe to be repaired, p53can induce the cell to kill itself by undergoing apoptosis.2.If p53 is defective, the unrestrained replication of damaged DNA leads to a high rate of mutation and theproduction of cells that tend to become cancerous.3.The activity of cyclin-Cdk complexes is inhibited by phosphorylation at particular sites. For the cell toprogress into mitosis, M-Cdk has to be activated by the removal of these inhibitory phosphates by a specific protein phosphatase. When DNA is damaged, this activating protein phosphatase is itself inhibited, so theinhibitory phosphates are not removed from M-Cdk. As a result, M-Cdk remains inactive and M phase cannot be initiated until DNA replication is complete and any DNA damage is repaired.4.M phas e1.M-Cdk drives entry into M phase and mitosis1.M-Cdk triggers the condensation of the replicated chromosome into compact, rod-like structures, readingthem for segregation, and ti also induces the assembly of the mitotic spindle that will separate the condensed chromosomes and segregate them into the two daughter cells.2.M-Cdk activation begins with the accumulation of M cyclin. Synthesis of M cyclin starts immediately after Sphase. M-Cdk complexes, when they first form, are inactive. The sudden activation of a protein phosphatase (Cdc25) that removes the inhibitory phosphates holding M-Cdk activity in check.3.Once activated, each M-Cdk complex can indirectly activate more M-Cdk, by phosphorylating and activatingmore Cdc25. In addition, activated M-Cdk also inhibits the inhibitory kinase Wee1, further promoting theactivation of M-Cdk. The overall consequence is that, once the activation of M0Cdk begins, there is anexplosive increase in M-Cdk activity that drives the cell abruptly from G2 into M phase.2.Condensins help conﬁgure duplicated chromosome for separation1.Protein complexes, called condensins, help carry out this chromosome condensation. The M-Cdk thatinitiates entry into M phase triggers the assembly of condensin complexes onto DNA by phosphorylatingsome of the condensin subunits.2.Both cohesins and condensins form ring structures, and, together, the two types of protein rings help toconfigure the replicated chromosomes for mitosis.3.The cytoskeleton carries out both mitosis and cytokinesis1.The mitotic spindle is composed of microtubules and the various proteins that interact with them, includingmicrotubule-associated motor proteins.2.Contractile ring consists mainly of actin filaments and myosin filaments arranged in a ring around the equatorof the cell. It starts to assemble just beneath the plasma membrane toward the end of mitosis.3.Both structures rapidly disassemble after they have performed their tasks.4.M phase is conventionally divided into six stages1.The first five stages of M phase (prophase, prometaphase, metaphase, anaphase, and telophase) constitutemitosis. Cytokinesis constitutes the sixth stage, and it overlaps in time with the end of mitosis.2.Prophase=replicated chromosomes condense and mitotic spindle begins to assemble outside the nucleus.Prometaphase=the nuclear envelope breaks down, allowing the spindle microtubules to bind to thechromosomes. Metaphase=the mitotic spindle gathers all of the chromosomes to the center (equator) of thespindle. Anaphase=the two sister chromatids in each replicated chromosome synchronously split apart, andthe spindle draws them to opposite poles of the cell. Telophase=a nuclear envelope reassembles around each of the two sets of separated chromosome to form two nuclei. Cytokinesis begins in anaphase and continuesthrough telophase. It is when nucleus and cytoplasm of each of the daughter cells return into interphase,signaling the end of M phase.1.E x tr ace ll ular ma tr ix and connec ti ve ti ssue s1.Intro1.The strength of plant tissue comes from the cell walls, formed like boxes, that enclose, protect, and constrainthe shape of each of its cells.2.The cell wall is a type of extracellular matrix that the plant cell secretes around itself.2.Plant cells have tough external walls1.Osmotic swelling of the cell, limited by the resistance of the cell wall, can keep the chamber distended, and amass of such swollen chambers cemented together forms a semirigid tissue.2.Most newly formed cells in a multicellular plant initially make thin primary cell walls that can slowly expandto accommodate cell growth, the driving force for growth is the swelling pressure, called the turgor pressure, that develops as the result of an osmotic imbalance between the interior of the cell and its surroundings. Once growth stops and the wall no longer needs to expand, a more rigid secondary cell wall is often produced,either by thickening of the primary wall or by deposition of new layers with a different compositionunderneath the old ones.3.Cellulose microﬁbrils give the plant cell wall its tensile strength1.Plant cells derive their tensile strength from long fibers oriented along the lines of stress. In higher plants, thelong fibers are generally made from the polysaccharide cellulose, the most abundant organic macromolecule on Earth. These cellulose microfibrils are interwoven with other polysaccharides and some structuralproteins, to resists compression as well as tension. In woody tissue, a highly cross-linked network of ligninassociated with cellulose is deposited to make the matrix more rigid and waterproof.2.Because the cellulose microfibrils resist stretching, their orientation governs the direction in which thegrowing cell enlarges.3.Cellulose synthesis is mediated by cellulose synthase complexes. Cellulose is synthesized outside the cell, onthe outer surface of the cell by enzyme complexes embedded in the plasma membrane. These complexestransport sugar monomers across the membrane and incorporate them into a set of growing polymer chains at their points of membrane attachment. Each set of chains forms a cellulose microfibril.4.Just beneath the plasma membrane, microtubules are aligned exactly with the cellulose microfibrils outsidethe cell. These microtubules are thought to serve as tracks to guide the movement of the enzyme complexes.In this way, the cytoskeleton controls the shape of the plant cell and the modeling of plant tissues.4.Animal connective tissues consist largely of extracellular matrix1.4 major types of tissues in animals: connective, epithelial, nervous, and muscular.2.In connective tissues, extracellular matrix is plentiful and carries the mechanical load. In other tissues,extracellular matrix is scanty, and the cells are directly joined to one another and carry the mechanical loadthemselves.3.Animal connective tissues are enormously varied in strength and location. They can be tough and flexible liketendons and ligaments or dermis of the skin, hard and dense like bone, resilient and shock-absorbing likecartilage, or soft and transparent like the jelly that fills the interior of the eye.4.The tensile strength is chiefly provided by collagen.5.Collagen provides tensile strength in animal connective tissues1.Collagen molecule has a long, stiff, triple-stranded helical structure, in which three collagen polypeptidechains are wound around one another in a ropelike superhelix. These molecules in turn assemble into ordered polymers called collagen fibrils. These can pack together into still thicker collagen fibers.2.Connective-tissue cells that manufacture and inhabit the matrix go by various names: in skin, tendon, andmany other connective tissues they are called fibroblasts; int bone they are called osteoblasts. They makeboth the collagen and other organic components of the matrix. Almost all of these molecules are synthesized intracellularly and then secreted in the standard way, by exocytosis.3.Collagen avoids premature assembly before secreting by secreting procollagen, with additional peptides ateach end that obstruct assembly into collagen fibrils. Extracellular enzymes called procollagen proteinases。

火山的地质：地球的构造英语作文

The Geology of Volcanoes:Earth's StructureVolcanoes,majestic and powerful geological features,provide a window into the inner workings of our planet.This essay explores the significance of volcanoes in understanding Earth's structure and the geological processes that shape our world.Formation of VolcanoesVolcanoes are formed when molten rock,known as magma,rises to the surface through cracks in the Earth's crust.This magma is generated deep within the Earth,in regions of intense heat and pressure.As the magma reaches the surface,it erupts,releasing gases,ash,and lava, shaping the landscape around it.Plate Tectonics and VolcanismThe formation of volcanoes is closely linked to plate tectonics,the theory that describes the movement and interaction of Earth's lithospheric plates.Volcanoes are commonly found along plate boundaries,where plates converge,diverge,or slide past each other. These interactions create zones of intense geological activity,leading to volcanic eruptions.Subduction Zones and Volcanic ArcsOne of the most common types of volcanoes is found in subduction zones,where one tectonic plate is forced beneath another.As the subducting plate sinks into the Earth's mantle,it heats up and releases water and other volatile substances.These substances rise into the overlying mantle,melting the rock and generating magma.This magma then rises to the surface,forming a volcanic arc,such as the Pacific Ring of Fire.Hotspots and Mantle PlumesIn addition to plate boundaries,volcanoes can also form over hotspots, areas of intense heat within the Earth's mantle.Hotspots are thought to be caused by mantle plumes,columns of hot,upwelling material from deep within the Earth.As the tectonic plate moves over the hotspot,achain of volcanoes is formed,with the youngest volcano located above the hotspot.Volcanic LandformsVolcanic eruptions create a variety of landforms,each with its unique characteristics.Shield volcanoes,such as those found in Hawaii,are broad and gently sloping,formed by the accumulation of fluid lava flows. Stratovolcanoes,like Mount Fuji in Japan,are tall and steep,composed of alternating layers of lava and ash.Calderas are large,basin-shaped depressions that form when the summit of a volcano collapses following a massive eruption.Volcanic Hazards and BenefitsVolcanic eruptions can pose significant hazards to human populations and the environment.These hazards include pyroclastic flows,ashfall, lava flows,and volcanic gases.However,volcanoes also bring benefits. Volcanic soils are highly fertile,supporting agriculture and providing nutrients for plant growth.Geothermal energy,harnessed from volcanic activity,is a renewable and clean source of power.Understanding Earth's StructureStudying volcanoes provides valuable insights into the inner workings of our planet.By analyzing volcanic rocks,scientists can determine the composition and temperature of Earth's interior.Monitoring volcanic activity helps us understand the processes that drive plate tectonics and the potential for future eruptions.Volcanoes are a key piece of the puzzle in unraveling the mysteries of Earth's structure.ConclusionVolcanoes offer a glimpse into the complex and dynamic nature of our planet.They are a manifestation of the forces that shape Earth's surface and provide valuable information about its internal structure. Understanding the geology of volcanoes is crucial for predicting and mitigating volcanic hazards and for unraveling the mysteries of our planet's past and future.Let us continue to study and appreciate theseawe-inspiring geological features,recognizing their significance in expanding our knowledge of Earth's construction.。

超声增强的输送的物料进入并通过皮肤翻译

超声增强的输送的物料进入并通过皮肤翻译Ultrasound-enhanced delivery of materials into and through the skinA method for enhancing the permeability of the skin or other biological membrane to a material such as a drug is disclosed. In the method, the drug is delivered in conjunction with ultrasound having a frequency of above about 10 MHz. The method may also be used in conjunction with chemical permeation enhancers and/or with iontophoresis.图片(11)权利要求(21)We claim:1. A method for enhancing the rate of permeation of a drug medium into a selected intact area of an individual's body surface, which method comprises:(a) applying ultrasound having a frequency of above 10 MHz to said selected area, at an intensity and for a period of timeeffective to enhance the permeability of said selected area;(b) contacting the selected area with the drug medium; and(c) effecting passage of said drug medium into and through said selected area by means of iontophoresis.2. The method of claim 1, wherein said ultrasound frequency is in the range of about 15 MHz to 50 MHz.3. The method of claim 2, wherein said ultrasound frequency is in the range of about 15 to 25 MHz.4. The method of claim 1, wherein said period of time is in the range of about 5 to 45 minutes.5. The method of claim 4, wherein said period of time is in the range of about 5 to 30 minutes.6. The method of claim 1, wherein said period of time is less than about 10 minutes.7. The method of claim 1, wherein said intensity of said ultrasound is less than about 5.0W/cm.sup.2.8. The method of claim 7, wherein said intensity of said ultrasound is in the range of about 0.01 to 5.0 W/cm.sup.2.9. The method of claim 8, wherein said intensity of said ultrasound is in the range of about 0.05 to 3.0 W/cm.sup.2.10. The method of claim 1, wherein said area of the stratum corneum is in the range of about 1 to 100 cm.sup.2.11. The method of claim 10, wherein said area of the stratum corneum is in the range of about 5 to 100 cm.sup.2.12. The method of claim 11, wherein said area of the stratum corneum is in the range of about 10 to 50 cm.sup.2.13. The method of claim 1 wherein said drug medium comprises a drug and a coupling agent effective to transfer said ultrasound to the body from an ultrasound source.14. The method of claim 13 wherein said coupling agent is a polymer or a gel.15. The method of claim 13 wherein said coupling agent is selected from the group consisting of glycerin, water, and propylene glycol.16. The method of claim 1 wherein said drug medium further comprises a chemical permeation enhancer.17. The method of claim 1, wherein steps (a) and (b) are carried out approximately simultaneously.18. The method of claim 1, wherein step (b) is carried out before step (a).19. The method of claim 1, wherein step (a) is carried out before step (b).20. The method of claim 1, wherein the ultrasound is applied continuously.21. The method of claim 1, wherein the ultrasound is pulsed.说明This application is a division of application Ser. No. 07/844,732 filed Mar. 2, 1992, now U.S. Pat. No. 5,231,975 which is a divisional of application Ser. No. 07/484,560, now U.S. Pat. No. 5,115,805, filed Feb. 23, 1990.TECHNICAL FIELDThis invention relates generally to the field of drug delivery. More particularly, the invention relates to a method of enhancing the rate of permeation of topically, transmucosally or transdermally applied materials using high frequency ultrasound.BACKGROUNDThe delivery of drugs through the skin ("transdermal drug delivery" or "TDD") provides many advantages; primarily, such a means of delivery is a comfortable, convenient and non-invasiveway of administering drugs. The variable rates of absorption and metabolism encountered in oral treatment are avoided, and other inherent inconveniences--e.g., gastrointestinal irritation and the like--are eliminated as well. Transdermal drug delivery also makes possible a high degree of control over blood concentrations of any particular drug.Skin is a structurally complex, relatively impermeable membrane. Molecules moving from the environment into and through intact skin must first penetrate the stratum corneum and any material on its surface. They must then penetrate the viable epidermis, the papillary dermis, and the capillary walls into the blood stream or lymph channels. To be so absorbed, molecules must overcome a different resistance to penetration in each type of tissue. Transport across the skin membrane is thus a complex phenomenon. However, it is the stratum corneum, a layer approximately 5-15 micrometers thick over most of the body, which presents the primary barrier to absorption of topical compositions or transdermally administered drugs. It is believed to be the high degree of keratinization within its cells as well as their dense packing and cementation by ordered, semicrystalline lipids which create in many cases a substantially impermeable barrier to drug penetration. Applicability of transdermal drug delivery is thus presently limited, because the skin is such an excellent barrier to the ingress of topically applied materials. For example, many of the new peptides and proteins now produced as a result of the biotechnology revolution cannot be delivered across the skin in sufficient quantities due to their naturally low rates of skin permeability.Various methods have been used to increase skin permeability, and in particular to increase the permeability of thestratum corneum (i.e., so as to achieve enhanced penetration, through the skin, of the drug to be administered transdermally). The primary focus has been on the use of chemical enhancers, i.e., wherein drug is coadministered with a penetration enhancing agent (or "permeation enhancer"). While such compounds are effective in increasing the rate at which drug is delivered through the skin, there are drawbacks with many permeation enhancers which limit their use. For example, many permeation enhancers are associated with deleterious effects on the skin (e.g., irritation). In addition, control of drug delivery with chemical enhancement can be quite difficult.Iontophoresis has also been used to increase the permeability of skin to drugs, and involves (1) the application of an external electric field, and (2) topical delivery of an ionized form of drug (or of a neutral drug carried with the water flux associated with ion transport, i.e., via "electroosmosis"). While permeation enhancement via iontophoresis has, as with chemical enhancers, been effective, there are problems with control of drug delivery and the degree of irreversible skin damage induced by the transmembrane passage of current.The presently disclosed and claimed method involves the use of ultrasound to decrease the barrier function of the stratum corneum and thus increase the rate at which a drug may be delivered through the skin. "Ultrasound" is defined as mechanical pressure waves with frequencies above 20,000 Hz (see, e.g., H. Lutz et al., Manual of Ultrasound: 1. Basic Physical and Technical Principles (Berlin: Springer-Verlag, 1984)).As discussed by P. Tyle et al. in Pharmaceutical Research 6(5):355-361 (1989), drug penetration achieved via "sonophoresis" (the movement of drugs through skin under theinfluence of an ultrasonic perturbation; see D. M. Skauen and G. M. Zentner, Int. J. Pharmaceutics 20:235-245 (1984)), is believed to result from thermal, mechanical and chemical alteration of biological tissues by the applied ultrasonic waves. Unlike iontophoresis, the risk of skin damage appears to be low.Applications of ultrasound to drug delivery have been discussed in the literature. See, for example: P. Tyle et al., supra (which provides an overview of sonophoresis); S. Miyazaki et al., J. Pharm. Pharmacol. 40:716-717 (1988) (controlled release of insulin from a polymer implant using ultrasound); J. Kost et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater.16(141):294-295 (1989) (overview of the effect of ultrasound on the permeability of human skin and synthetic membranes); H. Benson et al., Physical Therapy 69(2):113-118 (1989) (effect of ultrasound on the percutaneous absorption of benzydamine); E. Novak, Arch. Phys. Medicine & Rehab. 45:231-232 (1964) (enhanced penetration of lidocaine through intact skin using ultrasound); J. E. Griffin et al., Amer. J. Phys. Medicine 44(1):20-25 (1965) (ultrasonic penetration of cortisol into pig tissue); J. E. Griffin et al., J. Amer. Phys. Therapy Assoc.46:18-26 (1966) (overview of the use of ultrasonic energy in drug therapy); J. E. Griffin et al., Phys. Therapy 47(7):594-601 (1967) (ultrasonic penetration of hydrocortisone); J. E. Griffin et al., Phys. Therapy 48(12):1336-1344 (1968) (ultrasonic penetration of cortisol into pig tissue); J. E. Griffin et al., Amer. J. Phys. Medicine 51(2):62-72 (1972) (same); J. C. McElnay, Int. J. Pharmaceutics 40:105-110 (1987) (the effect of ultrasound on the percutaneous absorption of fluocinolone acetonide); and C. Escoffier et al., Bioeng. Skin 2:87-94 (1986) (in vitro study of the velocity of ultrasound in skin).In addition to the aforementioned art, U.S. Pat. Nos. 4,767,402 and 4,780,212 to Kost et al. relate specifically to the use of specific frequencies of ultrasound to enhance the rate of permeation of a drug through human skin or through a synthetic membrane.While the application of ultrasound in conjunction with drug delivery is thus known, results have for the most part been disappointing, i.e., enhancement of skin permeability has been relatively low.SUMMARY OF THE INVENTIONThe present invention provides a novel method for enhancing the rate of permeation of a given material through a selected intact area of an individual's body surface. The method comprises contacting the selected intact area with the material and applying ultrasound to the contacted area. The ultrasound preferably has a frequency of above about 10 MHz, and is continued at an intensity and for a period of time sufficient to enhance the rate of permeation of the material into and through the body surface. The ultrasound can also be used to pretreat the selected area of the body surface in preparation for drug delivery, or for diagnostic purposes, i.e., to enable non-invasive sampling of physiologic material beneath the skin or body surface.In addition to enhancing the rate of permeation of a material, the present invention involves increasing the permeability of a biological membrane such as the stratum corneum by applying ultrasound having a frequency of above about 10 MHz to the membrane at an intensity and for a period of time sufficient to give rise to increased permeability of the membrane. Once the permeability of the membrane has been increased, it is possible to apply a material thereto and obtain an increased rate of flowof the material through the membrane.It is accordingly a primary object of the invention to address the aforementioned deficiencies of the prior art by providing a method of enhancing the permeability of biological membranes and thus allow for an increased rate of delivery of material therethrough.It is another object of the invention to provide such a method which is effective with or without chemical permeation enhancers.It is still another object of the invention to minimize lag time in such a method and provide a relatively short total treatment time.It is yet another object of the invention to provide such a method in which drug delivery is effected using ultrasound.It is a further object of the invention to enable sampling of tissue beneath the skin or other body surface by application of high frequency (>10 MHz) ultrasound thereto.A further feature of the invention is that it preferably involves ultrasound of a frequency greater than about 10 MHz.Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.BRIEF DESCRIPTION OF THE DRAWINGSFIGS. 1A, 1B and 1C are theoretical plots of energy dissipation within the skin barrier versus frequency of applied ultrasound.FIGS. 2, 3 and 4 are graphic representations of the amount of salicylic acid recovered from the stratum corneum after ultrasound treatment at different frequencies.FIGS. 5 and 6 represent the results of experiments similar to those summarized in FIGS. 2, 3 and 4, but with a shorter treatment time.FIGS. 7, 8, 9 and 10 are plots of enhancement versus "tape-strip number," as described in the Example.FIG. 11 illustrates the effect of ultrasound on the systemic availability of salicylic acid following topical application.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTSBefore the present method of enhancing the rate of permeation of a material through a biological membrane and enhancing the permeability of membranes using ultrasound are disclosed and described, it is to be understood that this invention is not limited to the particular process steps and materials disclosed herein as such process steps and materials may, of course, vary. It is alto to be understood that the terminology used herein is used for purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims.It must be noted that as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a drug" includes mixtures of drugs and their pharmaceutically acceptable salts, reference to "an ultrasound device" includes one or more ultrasound devices of the type necessary for carrying out the present invention, and reference to "the method of administration" includes one or more different methods of administration known to those skilled in the art or which will become known to those skilled in the art upon reading this disclosure.In one aspect of the invention a method is provided forenhancing the permeation of a given material such as a drug, pharmacologically active agent, or diagnostic agent into and/or through a biological membrane on an individual's body surface, which method comprises: (a) contacting the membrane with the chosen material in a pharmacologically acceptable carrier medium; and (b) applying ultrasound of an intensity and for a treatment time effective to produce delivery of the material through the membrane. The material is preferably a drug and it is preferable to obtain a desired blood level of the drug in the individual. The ultrasound is of a frequency and intensity effective to increase the permeability of the selected area to the applied drug over that which would be obtained without ultrasound. The ultrasound preferably has a frequency of more than 10 MHz, and may be applied either continuously or pulsed, preferably continuously. The ultrasound may be applied to the skin either before or after application of the drug medium so long as administration of the ultrasound and the drug medium is relatively simultaneous, i.e., the ultrasound is applied within about 6, more preferably within about 4, most preferably within about 2 minutes of drug application.The invention is useful for achieving transdermal permeation of pharmacologically active agents which otherwise would be quite difficult to deliver through the skin or other body surface. For example, proteinaceous drugs and other high molecular weight pharmacologically active agents are ideal candidates for transdermal, transmucosal or topical delivery using the presently disclosed method. In an alternative embodiment, agents useful for diagnostic purposes may also be delivered into and/or through the body surface using the present method.The invention is also useful as a non-invasive diagnostictechnique, i.e., in enabling the sampling of physiologic material from beneath the skin or other body surface and into a collection (and/or evaluation) chamber.The present invention will employ, unless otherwise indicated, conventional pharmaceutical methodology and more specifically conventional methodology used in connection with transdermal delivery of pharmaceutically active compounds and enhancers.In describing the present invention, the following terminology will be used in accordance with the definitions set out below.A "biological membrane" is intended to mean a membrane material present within a living organism which separates one area of the organism from another and, more specifically, which separates the organism from its outer environment. Skin and mucous membranes are thus included."Penetration enhancement" or "permeation enhancement" as used herein relates to an increase in the permeability of skin to a material such as a pharmacologically active agent, i.e., so as to increase the rate at which the material permeates into and through the skin. The present invention involves enhancement of permeation through the use of ultrasound, and, in particular, through the use of ultrasound having a frequency of greater than 10 MHz."Transdermal" (or "percutaneous") shall mean passage of a material into and through the skin to achieve effective therapeutic blood levels or deep tissue therapeutic levels. While the invention is described herein primarily in terms of "transdermal" administration, it will be appreciated by those skilled in the art that the presently disclosed and claimed methodalso encompasses the "transmucosal" and "topical" administration of drugs using ultrasound. "Transmucosal" is intended to mean passage of any given material through a mucosal membrane of a living organism and more specifically shall refer to the passage of a materialfrom the outside environment of the organism, through a mucous membrane and into the organism. "Transmucosal" administration thus includes delivery of drugs through either nasal or buccal tissue. By "topical" administration is meant local administration of a topical pharmacologically active agent to the skin as in, for example, the treatment of various skin disorders or the administration of a local anaesthetic. "Topical" delivery can involve penetration of a drug into the skin but not through it, i.e., topical administration does not involve actual passage of a drug into the bloodstream."Carriers" or "vehicles" as used herein refer to carrier materials without pharmacological activity which are suitable for administration with other pharmaceutically active materials, and include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is nontoxic and which does not interact with the drug to be administered in a deleterious manner. Examples of suitable carriers for use herein include water, mineral oil, silicone, inorganic gels, aqueous emulsions, liquid sugars, waxes, petroleum jelly, and a variety of other oils and polymeric materials.By the term "pharmacologically active agent" or "drug" as used herein is meant any chemical material or compound suitable for transdermal or transmucosal administration which can either (1) have a prophylactic effect on the organism and prevent an undesired biological effect such as preventing aninfection, (2) alleviates a condition caused by a disease such as alleviating pain caused as a result of a disease, or (3) either alleviates or completely eliminates the disease from the organism. The effect of the agent may be local, such as providing for a local anaesthetic effect or it may be systemic. Such substances include the broad classes of compounds normally delivered through body surfaces and membranes, including skin. In general, this includes: anti-infectives such as antibiotics and antiviral agents; analgesics and analgesic combinations; anorexics; antihelminthics; antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants; antidiabetic agents; antidiarrheals; antihistamines; antiinflammatory agents; antimigraine preparations; antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics; antipyretics; antispasmodics; anticholinergics; sympathomimetics; xanthine derivatives; cardiovascular preparations including potassium and calcium channel blockers, beta-blockers, and antiarrhythmics; antihypertensives; diuretics; vasodilators including general coronary, peripheral and cerebral; central nervous system stimulants; cough and cold preparations, including decongestants; hormones such as estradiol and other steroids, including corticosteroids; hypnotics; immunosuppressives; muscle relaxants; parasympatholytics; psychostimulants; sedatives; and tranquilizers. By the method of the present invention, both ionized and nonionzed drugs may be delivered, as can drugs of either high or low molecular weight.Proteinaceous and polypeptide drugs represent a preferred class of drugs for use in conjunction with the presently disclosed and claimed invention. Such drugs cannot generally be administered orally in that they Are often destroyed in the G.I.tract or metabolized in the liver. Further, due to the high molecular weight of most polypeptide drugs, conventional transdermal delivery systems are not generally effective. It is also desirable to use the methodof the invention in conjunction with drugs to which the permeability of the skin is relatively low, or which give rise to a long lag-time (application of ultrasound as described herein has been found to significantly reduce the lag-time involved with the transdermal administration of most drugs).By a "therapeutically effective" amount of a pharmacologically active agent is meant a nontoxic but sufficient amount of a compound to provide the desired therapeutic effect. The desired therapeutic effect may be a prophylactic effect, in preventing a disease, an effect which alleviates a system of the disease, or a curative effect which either eliminates or aids in the elimination of the disease.As noted above, the present invention is a method for enhancing the rate of permeation of a drug through an intact area of an individual's body surface, preferably the human skin. The method involves transdermal administration of a selected drug in conjunction with ultrasound. Ultrasound causes thermal, mechanical and chemical alterations of biological tissue, thereby enhancing the rate of permeation of a given material therethrough.While not wishing to be bound by theory, applicants propose that the use of higher frequency ultrasound as disclosed herein specifically enhances the permeation of the drug through the outer layer of skin, i.e., the stratum corneum, by causing momentary and reversible perturbations within (and thus short-term, reversible reduction in the barrier function of) the layer ofthe stratum corneum. It will be appreciated by those skilled in the art of transdermal drug delivery that a number of factors related to the present method will vary with the drug to be administered, the disease or injury to be treated, the age of the selected individual, the location of the skin to which the drug is applied, and the like.As noted above, "ultrasound" is ultrasonic radiation of a frequency above 20,000 Hz. As may be deduced from the literature cited above, ultrasound used for most medical purposes typically employs frequencies ranging from 1.6 to about 10 MHz. The present invention, by contrast, employs ultrasound frequencies of greater than about 10 MHz, preferably in the range of about 15 to 50 MHz, most preferably in the range of about 15 to 25 MHz. It should be emphasized that these ranges are intended to be merely illustrative of the preferred embodiment; in some cases higher or lower frequencies may be used.The ultrasound may be pulsed or continuous, but is preferably continuous when lower frequencies are used. At very high frequencies, pulsed application will generally be preferred so as to enable dissipation of generated heat.The preferred intensity of the applied ultrasound is less than about 5.0 W/cm.sup.2, more preferably is in the range of about 0.01 to 5.0 W/cm.sup.2, and most preferably is in the range of 0.05 to 3.0 W/cm.sup.2. The total treatment time, i.e., the period over which drug and ultrasound are administered, will vary depending on the drug administered, the disease or injury treated, etc., but will generally be on the order of about 30 seconds to 60 minutes, preferably 5 to 45 minutes, more preferably 5 to 30 minutes, and most preferably 5 to 10minutes. It should be noted that the aforementioned ranges represent suggested, or preferred, treatment times, but are not in any way intended to be limiting. Longer or shorter times may be possible and in some cases desirable. Virtually any type of device may be used to administer the ultrasound, providing that the device is callable of producing the higher frequency ultrasonic waves required by the present method. A device will typically have a power source such as a small battery, a transducer, a reservoir in which the drug medium is housed (and which may or may not be refillable), and a means to attach the system to the desired skin site.As ultrasound does not transmit well in air, a liquid medium is generally needed to efficiently and rapidly transmit ultrasound between the ultrasound applicator and the skin. As explained by P. Tyle et al., cited above, the selected drug medium should contain a "coupling" or "contacting" agent typically used in conjunction with ultrasound. The coupling agent should have an absorption coefficient similar to that of water, and furthermore be nonstaining, nonirritating to the skin, and slow drying. It is clearly preferred that the coupling agent retain a paste or gel consistency during the time period of ultrasound administration so that contact is maintained between the ultrasound source and the skin. Examples of preferred coupling agents are mixtures of mineral oil and glycerine and propylene glycol, oil/water emulsions, and a water-based gel. A solid-state, non-crystalline polymeric film having the above-mentioned characteristics may also be used. The drug medium may also contain a carrier or vehicle, as defined alone.A transdermal patch as well known in the art may be used in conjunction with the present invention, i.e., to deliver the drugmedium to the skin. The "patch", however, must have the properties of the coupling agent as described in the preceding paragraph so as to enable transmission of the ultrasound from the applicator, through the patch, to the skin.As noted earlier in this section, virtually any chemical material or compound suitable for transdermal, transmucosal or topical administration may be administered using the present method. Again, the present invention is particularly useful to enhance delivery of proteinaceous and other high molecular weight drugs.The method of the invention is preferably carried out as follows. The drug medium, i.e., containing the selected drug or drugs in conjunction with the coupling agent and optionally a carrier or vehicle material, is applied to an area of intact body surface. Ultrasound preferably having a frequency greater than about 10 MHz may be applied before or after application of the drug medium, but is preferably applied immediately before application of the drug so as to "pretreat" the skin prior to drug administration.It should also be pointed out that the present method may be used in conjunction with a chemical permeation enhancer as known in the art, wherein the ultrasound enables the use of much lower concentrations of permeation enhancer--thus minimizing skin irritation and other problems frequently associated with such compounds--than would be possible in the absence of ultrasound. The permeation enhancer may be incorporated into the drug medium or it maybe applied in a conventional transdermal patch after pretreatment of the body surface with ultrasound.The present invention may also be used in conjunction with。

法布里珀罗基模共振英文

法布里珀罗基模共振英文The Fabryperot ResonanceOptics, the study of light and its properties, has been a subject of fascination for scientists and researchers for centuries. One of the fundamental phenomena in optics is the Fabry-Perot resonance, named after the French physicists Charles Fabry and Alfred Perot, who first described it in the late 19th century. This resonance effect has numerous applications in various fields, ranging from telecommunications to quantum physics, and its understanding is crucial in the development of advanced optical technologies.The Fabry-Perot resonance occurs when light is reflected multiple times between two parallel, partially reflective surfaces, known as mirrors. This creates a standing wave pattern within the cavity formed by the mirrors, where the light waves interfere constructively and destructively to produce a series of sharp peaks and valleys in the transmitted and reflected light intensity. The specific wavelengths at which the constructive interference occurs are known as the resonant wavelengths of the Fabry-Perot cavity.The resonant wavelengths of a Fabry-Perot cavity are determined bythe distance between the mirrors, the refractive index of the material within the cavity, and the wavelength of the incident light. When the optical path length, which is the product of the refractive index and the physical distance between the mirrors, is an integer multiple of the wavelength of the incident light, the light waves interfere constructively, resulting in a high-intensity transmission through the cavity. Conversely, when the optical path length is not an integer multiple of the wavelength, the light waves interfere destructively, leading to a low-intensity transmission.The sharpness of the resonant peaks in a Fabry-Perot cavity is determined by the reflectivity of the mirrors. Highly reflective mirrors result in a higher finesse, which is a measure of the ratio of the spacing between the resonant peaks to their width. This high finesse allows for the creation of narrow-linewidth, high-resolution optical filters and laser cavities, which are essential components in various optical systems.One of the key applications of the Fabry-Perot resonance is in the field of optical telecommunications. Fiber-optic communication systems often utilize Fabry-Perot filters to select specific wavelength channels for data transmission, enabling the efficient use of the available bandwidth in fiber-optic networks. These filters can be tuned by adjusting the mirror separation or the refractive index of the cavity, allowing for dynamic wavelength selection andreconfiguration of the communication system.Another important application of the Fabry-Perot resonance is in the field of laser technology. Fabry-Perot cavities are commonly used as the optical resonator in various types of lasers, providing the necessary feedback to sustain the lasing process. The high finesse of the Fabry-Perot cavity allows for the generation of highly monochromatic and coherent light, which is crucial for applications such as spectroscopy, interferometry, and precision metrology.In the realm of quantum physics, the Fabry-Perot resonance plays a crucial role in the study of cavity quantum electrodynamics (cQED). In cQED, atoms or other quantum systems are placed inside a Fabry-Perot cavity, where the strong interaction between the atoms and the confined electromagnetic field can lead to the observation of fascinating quantum phenomena, such as the Purcell effect, vacuum Rabi oscillations, and the generation of nonclassical states of light.Furthermore, the Fabry-Perot resonance has found applications in the field of optical sensing, where it is used to detect small changes in physical parameters, such as displacement, pressure, or temperature. The high sensitivity and stability of Fabry-Perot interferometers make them valuable tools in various sensing and measurement applications, ranging from seismic monitoring to the detection of gravitational waves.The Fabry-Perot resonance is a fundamental concept in optics that has enabled the development of numerous advanced optical technologies. Its versatility and importance in various fields of science and engineering have made it a subject of continuous research and innovation. As the field of optics continues to advance, the Fabry-Perot resonance will undoubtedly play an increasingly crucial role in shaping the future of optical systems and applications.。

The Role of Mould Fluxes in Continuous Casting-So Simple Yet So Comples

Fig.1.Schematic drawing of the various slag layers formed in the mould.©2003ISIJof the ﬂuxes have property values which are consistent with those derived from empirical rules. Thus there is simplicity in the way that mould ﬂuxes perform the required func-The complexity arises from the huge range of “in mould conditions” that the ﬂux has to deal with. The large number of variables in the continuous casting process and their ef-fect on both the surface quality of the product and on process control are shown in Fig. 2. The mould ﬂux is ex-pected to compensate for many of the variations in casting conditions by being “ﬂexible” and “forgiving”. Given the large differences in the casting conditions at different plants, it is not surprising that ﬂuxes known to work suc-cessfully on one plant, frequently do not perform so well on another plant.The principal factors affecting ﬂux performance are:Casting conditions (casting speed, V c , oscillation charac-teristics).Steel grade and mould dimensions.Mould level control (which can lead to depressions etc .).Metal ﬂow since turbulent ﬂow can lead to several prob-lems eg gas and slag entrapment.In this study we will examine the effects of mould ﬂux on lubrication and heat transfer and how ﬂux performance is affected by both casting conditions and steel grade being cast. Then we will look at how the mould ﬂux is expected to deal with problems caused by turbulent metal ﬂow in the mould and how recent developments affecting metal ﬂow control may help to simplify the tasks carried out by mould Mould Flux LubricationThe liquid mould ﬂux lubricates the steel strand. It is im-portant that there is liquid lubrication throughout the strand since problems (such as star cracking) can occur if the ﬂux crystallises completely in the lower half of the mould and liquid lubrication is lost.4)The liquid friction (F l ) is given by Eq. (1) where V m is the velocity of the mould. It can be seen that the friction decreases as the viscosity (h ) decreas-es and the liquid ﬂux ﬁlm thickness (d l ) increases.F l ϭh (V m ϪV m )/d l (1)Powder consumption provides a measure of the lubrication supplied and it is very dependent upon mould size since the friction increases as the distance from the corner increases.Thus frictional forces are much larger in slabs Ͼblooms billets. Powder consumption (Q t ) is usually measured as kg ﬂux (tonne steel)Ϫ1. However, Q t can be converted to with units of kg ﬂux m Ϫ2(of mould) using Eq. (2),Q s ϭf *.Q t .7.6/R ϭd l r (2)where f * is the fraction of powder producing slag, density of the liquid slag and R is the surface area to vol-ume of the mould and is given by 2(w ϩt )/wt where are the thickness of the mould. The effect of mould dimen-sions on powder consumption, Q s , can be clearly seen in Fi g. 3since R has values of Ͻ10 for slabs, 10–15 for blooms, Ͼ20 for billets and Ͼ30 for thin slabs.2003ISIJ1480Fig.2.Schematic diagram showing the cause of various defects and operational problems.Fig.3.Powder consumption, Q s as a function of the parameter,R ϭ(surface area/volume).ϭ2/(RϪ5) (3)Empirical rules for the selection of ﬂuxes for “optimum casting” in terms of casting speed and ﬂux viscosity haveWolf subsequently converted these into empirical rules to derive the required powder consump-However, powder consumption is also dependent upon other casting parameters such as the oscillation char-acteristics, solidiﬁcation or break temperature etc. The vari-reported for powder consumptionTable 1. Itoyama17)reported a model contained contributions from (i) ﬂow emanating from the molten pool, (ii) ﬂow between parallel plates (mould and strand), (iii) the oscillation of the mould and(iv) slag trapped in oscillation marks (Qom ).In order to test the validity of these various relationships we have collected plant data from steel-plants all round the world when casting slabs, blooms, billets and thin slabs. Database 1 was collected from plant data from single tri-als for billet-bloom-slab and thin slab-casting in Ͼ30 steel-works. The following data were included: powder con-, mould dimensions, casting speed, steel composition and ﬂux chemical composition, viscosity and. The variation in powder consump-tion values for runs carried out under similar casting condi-%.19)Database 2 contained all the information listed for Database 1 plus the oscillation characteristics, frequency). Mean powder consumption values were derived in trials with identical casting conditions but% variation in the casting speed. The varia-tion of averaged powder consumption values is less than The performances of the various relationships werechecked by comparing the calculated Qs against the mea-; the results are given in Fig. 4.It can be seen from Fig. 4 and Table 1 thatthe Tsutsumi, Maeda and Kwon relations provide thesfollowed by modiﬁed Wolf andthe required viscosity (at 1573K) of the ﬂux can now be calculated for the speciﬁc casting conditions and mould dimensions. (The Tsutsumi relation16)has been adopted when the oscillation characteristics areavailable and the modiﬁed Wolf relationthese data are not available.)(iii)there are deviations from these relations for billet-casting where frequently high-viscosity ﬂuxes areused to minimise problems related to turbulent metalﬂow (e.g.slag entrapment) because the lubrication re-quirements for billets are low.It should also be noted that low powder consumption can occur when casting steels containing Ti, due to the copious formation of TiN which prevents the inﬁltration of liquid slag into the mould/strand gap.It is our belief that these high viscosity ﬂuxes operate ona different principal to conventional ﬂuxes. Most conven-tional ﬂuxes show a marked increase in viscosity when crystalline solids are formed at the break temperature (on cooling (Fig. 5(a)). However, high-viscosity slags form super-cooled liquids on cooling (Fig. 5(b)) which persist down to their glass transition temperatures,(dPa s)ϭ1013.4). No break temperature would be recorded with this type of slag. Furthermore, a super-cooled liquid will move with the strand despite having a high viscosity value.There are also a small number of ﬂuxes (about 10which work well enough in practice but do not ﬁt the re-quirements derived from the empirical rules.Thus, in summary, the only ﬂux properties which are im-portant for lubrication are the viscosity and the break tem-perature.3.Mould Flux and Heat TransferHeat transfer in the mould can be conveniently classiﬁed into vertical and horizontal heat transfer. Decreased vertical heat transfer has been reported20)of pinholes and (ii) the depth of oscillation marks by reduc-ing the length of steel meniscus. However, it is the horizon-tal heat transfer between steel shell and mould which is the more important since it has such a signiﬁcant effect on the surface quality of the steel.Horizontal heat transfer is complex involving two mech-anisms, namely, lattice or phonon conductivity (diation conductivity (kR). Radiation conductivity involves absorption and re-emission of radiated energy and can be the dominant mechanism in glassy materials at high tem-1481peratures.21–23)However, k R can be signiﬁcantly decreased by the presence in the slag ﬁlm of (i) crystallites which scatter the radiation and (ii) transition metal oxides (e.g.FeO) which absorb the radiation. It has been estimated that ϭ10–30% k c 21–23)for heat transfer across slag ﬁlms formed during slab casting. However, it may be much more signiﬁcant in glassy slag ﬁlms formed using high-viscosity ﬂuxes for billet casting. The overall resistance to thermal transfer (R *total ) between shell and mould can be regarded as a series of resistances as shown in Fig. 6and Eq. (4).R *total ϭR *Cu/sl ϩ(d /k )l ϩ(d /k )gl ϩ(d /k )cry (4)where R *Cu/sl is the interfacial resistance and d and k are thethickness and thermal conductivity of the layers in the slag ﬁlm and subscripts l , gl and cry denote the liquid, glass and crystalline layers, respectively.Y amauchi 23)introduced the contribution from radiation conduction as a parallel resistance. The most signiﬁcant terms affecting (R total ) are (i) R Cu/sl and (ii) the thickness of the solid slag ﬁlm i.e.d solid ϭd gl ϩd cry . The interfacial resis-tance R Cu/sl was found to increase with (i) increasing solid slag thickness, d solid and (ii) increasing crystallinity. This is best understood as an increase in R Cu/sl results from an in-crease in the thickness of an air gap, formed as glass trans-forms into the more-dense, crystalline phase (r cry Ͼr gl Thus the two key parameters are (i) the thickness of the solid slag ﬁlm (d solid ) and (ii) the % crystalline phase devel-oped in the slag ﬁlm.Longitudinal cracking in medium carbon (MC) steels re-sults from the 4% mismatch in the thermal shrinkage coef-2003ISIJ1482Fig.5.Arrhenius plots showing log 10viscosity (dPa s) versus reciprocal temperature (K Ϫ1) for (a) conventional ﬂux and (b) high-viscosity ﬂux for billet-casting.Fig.6.Schematic diagram showing the thermal resistances be-tween shell and mould.Fig.4.Measured versus calculated powder consumption values using (a) Wolf equation, (b) modiﬁed Wolf equation, (c)Ogibayashi equation, (d) Jenkins equation, (e) Modiﬁed Jenkins equation, (f) Tsutsumi equation, (g) Maeda equa-tion and (h) Kwon equation.Fig.7.Heat ﬂux as function of liquidus and solidus temperatures of ﬂux.23)Fig.8.Break temperature as a function of ﬂux viscosity.Fig.9.Flow diagram for model to calculate required powder consumption, viscosity and break temperature.19) Problems and Defects4.1.Control of Metal FlowThe complexity in mould ﬂux performance arises when we try to use the ﬂux to combat problems other than those related to lubrication and heat transfer. One example is using the mould ﬂux to deal with problems arising from turbulent metal ﬂow (e.g.slag and gas entrapment and SEN erosion). One way of reducing these problems is to use a high-viscosity ﬂux but this has the disadvantage that it re-duces powder consumption.©2003ISIJ10.Schematic drawing showing double roll ﬂow.ϭ1393K–%MO: b T solϭ1515K–x(MO) where xϭmole3.The effect of different ﬂux components on relevant properties. (a Tbrfraction and MOϭoxide and c refers to %F:)ISIJ1484There is a basic simplicity in the way ﬂuxes work since there are only three properties determining the opti-mum lubrication and heat transfer for the given casting con-ditions, mould dimensions and steel grade, namely, the vis-cosity, break temperature and % crystallinity in the slag When the mould ﬂux is used to combat other prob-slag entrapment due to turbulent metal ﬂow) this frequently leads to the use of ﬂuxes which do not provide optimal lubrication and heat transfer.Several new devices could help to reduce turbu-lence in the metal ﬂow and if these were successfully im-plemented they would allow the mould ﬂux to concentrate on providing optimum lubrication and horizontal heat trans-fer.AcknowledegementsThe authors would like to thank Dr. Adrian Normanton (Corus Teesside Technology Centre), Mr. Tim Mallaband (Metallurgica UK), Ms. Carolina Bezerra (Carboox, Brazil) and Dr. Shuji Takeuchi (Kawasaki Steel) for valuable dis-cussions and the provision of information.Nomenclaturedl:Thickness (m)f:Frequency of oscillation (Hz)f*:Fraction of powder containing slagQs:Powder consumption (kg1485。

骨髓间充质干细胞的主要表面标志

骨髓间充质干细胞的主要表面标志1 骨髓间充质干细胞的发现和来源骨髓组织中有多种细胞成分，除基质细胞等已经分化的细胞外，还含有两类多潜能干细胞：造血干细胞和间充质干细胞。

1987 年Friedenstein 等发现在塑料培养皿中培养的贴壁的骨髓单个细胞在一定条件下可分化为多种类型的细胞，而且经过20－30个培养周期仍能保持其多向分化潜能。

由于骨髓中的这种多能细胞能够分化为多种中胚层来源的间质细胞, 故称之为间充质干细胞(Mesenchymal stem cells，MSCs),或间质祖细胞(MPCs)，是成人多能干细胞的一类。

早期分离培养时，发现其形状呈成纤维细胞样而称其为成纤维细胞集落形成单位(Colony-forming unit-fibroblast，CFU-F)，或骨髓基质成纤维细胞(Marrow stromal fibroblast，MSF)。

Friedenstein AJ , Chailakhyan RK, Gerasimov UV. Bone marrow o steogenic stem cells: in vit ro cult ivat ion and t ransp lantat ion in diffusion chambers. Cell T issue Kinet, 1987, 20 (3) : 263-267]2 鉴于其强大的增殖能力及多向分化潜能，可在体外长期培养和遗传背景较稳定，而且用自体干细胞诱导构建的组织不涉及伦理问题，也不存在MHC限制，所以骨髓间充质干细胞日益受到重视。

但是与造血干细胞等其他细胞相比，骨髓中MSCs的数量非常少，约占整个骨髓有核细胞的十万分之一，并随年龄的增加，细胞数量逐渐减少。

因此，如何简便有效地从骨髓中获取高纯度的MSCs显得尤为重要，寻找高度特异性的MSCs的表面抗原也就成为MSCs研究中的一项重要任务和目标。

不仅如此，一种同样来源于骨髓、贴壁生长、被认为更原始（可以分化为MSCs）也具有更强增殖能力的干细胞也被鉴定，它就是多能成体祖细胞（multipotent adult progenitor cell (MAPC) or mesodermal progenitor cell(MPC)）[Reyes, M., Lund, T., Leuvik, T., Aguiar, D., Koodie, L., Verfaillie,C.M. (2001) Purification and in vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98, 2615-2625]，因能和MSCs一起被纯化而统称BM stromal stem cell。

3D打印个性化髋臼接骨板设计生物力学研究与分析

生物骨科材料与临床研究O rthopaedic B iomechanics M aterials A nd C linical S tudy2020年 12月第17卷第6期.41.doi: 10.3969/j.issn. 1672-5972.2020.06.010文章编号：swgk201 头12-002773D 打印个性化髓臼接骨板设计生物力学研究与分析王晖】王猛2［摘要］目的应用三维有限元方法研究接骨板内固定疗法治疗體臼后壁骨折，探讨接骨板与骨面不同间隙对體臼应力分布的影响。

方法基于真实人体體关节CT 扫描数据，设计出适用于體臼后壁骨折内固定的个性化接骨板，利用Mimics 、Geomagic 、Abaqus 等软件构建接骨板内固定競关节三维有限元模型，选择成人缓慢行走时单脚承重的受力状态对模型加载，分析接骨板与骨面不同间隙(0mm> 0.25 mm. 0.5 mm. 0.75 mm)对競臼生物力学的影响。

结果随着接骨板与骨界面间隙的增大，接骨板与體臼后壁碎片上所受最大应力随着间隙的增大而略微减小，但应力分布情况并没有太大的变化。

结论将本文有限元仿真结果结合临床骨治疗生物固定理念，可以发现个性化接骨板与骨界面间隙并不是越小越好，在允许范围内，应将接骨板与骨界面之间留有一定间隙。

［关键词］生物力学；个性化接骨板；接触间隙［中图分类号］R683.3 ［文献标识码］BBiomechanical research and analysis of 3D printed personalized acetabular bone plate designWangHui 1, WangMeng 2. 1 Department of O rthopedics, Handan Hospital, General H ospital ofJizbong E nergy F engfeng Group, Handan Hebei, 056001; 2 Shanxi A erospace Qinghua Equipment Co., Ltd., Changzhi S hanxi, 046011, China [Abstract] Objective To study the treatment of acetabular posterior wall fractures with bone plate internal fixation by using three-dimensional finite element method, and explore the effect of different gaps between bone plate and bone sur face on acetabular stress distribution. Methods Based on CT scan data of real human hip joints, Mimics, Geomagic, Abaqus, and other software were used to construct a three-dimensional finite element model of the hip plate with fixedhip joints. The loading state of t he single foot was selected when the adult was walking slowly to load the model and ana lyze the bone plate and bone of d ifferent surface gaps (0 mm, 0.25 mm, 0.5 mm, 0.75 mm) on acetabular biomechanics.Results With the increase of t he gap between the bone plate and the bone interface, the maximum stress on the fragmentsof the bone plate and the posterior wall of the acetabulum decreases slightly with the increase of the gap, but the stress distribution has not changed much. Conclusion Combining the finite element simulation results of this article with the clinical bone treatment bio-fixation concept, we can find that the gap between the plate and the bone interface is not assmall as possible. Within the allowable range, a certain gap should be left between the plate and the bone interface.[Key words] Biomechanics; Personalized bone plate; Contact gap髓关节的主要作用是将人体上半身的重量传递到下肢, 属于人体承重的大关节，正常人体的髓臼由坐骨、耻骨和骼骨组成，与股骨头组合在一起形成髓关节冋。

去金属伪影技术联合第四代迭代重建技术在腰椎内固定术后的应用价值

DOI：10.3969/j.issn.1672-9463.2021.03.009去金属伪影技术联合第四代迭代重建技术在腰椎内固定术后的应用价值朱维聪曾慧芸范林［摘要］目的探讨飞利浦Brilliance iCT256去金属伪影技术(O-MAR)联合第四代迭代重建技术(iDose4)在腰椎内固定术后的应用价值。

方法选取我院腰椎内固定术后拟行腰椎GT检查的患者共32例，均行飞利浦BriUiance iCT 256容积扫描并使用iDose4技术重建获得图像，在扫描参数中勾选O-MAR选项，单次扫描同时得到O-MAR技术处理前后的图像数据。

由2名高年资放射医师评价去伪影前后图像螺钉周围软组织、金属与骨质界面软组织、椎间盘、椎弓根及椎体的显示情况，分别测量并比较图像中低密度金属伪影最严重区域软组织、同层面右侧腰大肌、螺钉长轴中心处及同层面受伪影影响最小区域软组织的CT值和SD值，并计算伪影指数(AI)值。

结果去伪影后R0U的CT值大于去伪影前(P<0.05);去伪影后ROI2和ROI3的CT值小于去伪影前(P<0.05);ROI4的CT值在去伪影前后差异无统计学意义(P>0.05)o去伪影后ROI1和R0I3的SD值和AI值均小于去伪影前(PO.05);去伪影后ROI2的SD值和AI 值大于去伪影前(PO.05)；ROI4的SD值在去伪影前后差异无统计学意义(P>0.05)o在螺钉周围软组织、金属与骨质界面软组织及椎弓根显示方面，去伪影后的图像评分明显高于去伪影前(PO.05)；而在椎体及椎间盘显示方面，去伪影后的图像评分低于去伪影前(P<0.05)o结论应用0-MAR技术联合iDose4技术可以减轻金属伪影，有助于准确观察椎弓根螺钉的位置及周围的组织结构，在腰椎内固定术后情况的评估中具有较大的应用价值。

［关键词］去金属伪影技术金属伪影X线计算机体层成像The application value of O-MAR combined with iDose4after lumbar internal fixation Zhu Weicong,Zeng Huiyun,Fan Lin. Dongguan Kanghua Hospital,Dongguan523000［Abstract］Objective To explore the application value of Philips Brilliance iCT256Orthopedic metal artifact reduction(O-MAR)combined with the fourth generation iterative reconstruction technology(iDose4)after lumbar internal fixation.Methods Selected32patients with lumbar CT examination in our hospital after lumbar spinal internal fixation surgery,all of them accepted Philips Brilliance iCT256volume scanning,combined with iDose4 reconstruction image,checked the O-MAR option in the scanning parameters,at the same time in a single scan to get0—MAR technology image data before and after treatment.Two senior radiologists evaluated the display situation of soft tissues around screws and soft tissue at the interface between metal and bone interface,intervertebral disc, vertebral pedicle and cone before and after metal artifact removal,The CT and SD values of soft tissue in the most serious area of low density metal artifacts,the right psoas major muscle in the same plane,the center of long axis of screw,the least affected area in the same plane and artifact index were measured and counted.Results The CT value of the ROI1after artifact removal was higher than that before artifact removal(P<0.05).CT value of ROI 2and ROI3after artifact removal was lower than that before artifact removal(P<0.05).There was no significant difference in CT value of ROI4before and after artifact removal(P>0.05).AI and SD values of ROI1and ROI 3after artifact removal were lower than those before artifact removal(P<0.05).AI and SD values of ROI2after artifact removal was greater than that before artifact removal(P<0.05).There was no significant difference in SD value of ROI4before and after artifact removal.In the soft tissue and metal—bone interface around screws and vertebral pedicle,the image score after artifact removal was significantly higher than before artifact removal(P<0.05). In terms of the display situation of intervertebral disc vertebral pedicle,the image score after artifact removal was lower than before artifact removal(P<0.05).Conclusion Application of O-MAR combined with iDose4technique作者单位:东莞康华医院放射科，广东东莞523000can reduce the metal artifacts and help to accurately observe the pedicle screw position and the surrounding tissue structure,It has great application value in the valuation of the situation after lumbar internal fixation.[Key words]Orthopedic metal artifact reduction Metal artifact X—ray computer tomography腰椎内固定术后,影像学检查成为准确定位植入物位置、判断有无并发症,以及后期随访观察疗效的主要方法。

MAGMAIRON帮助文件（翻译）资料

MAGMAIRON帮助文件（翻译）资料目录1 Introduction2 Theory2.1 热物理数据2.2 合金成分2.3 灰铸铁仿真2.3.1共晶核2.3.2石墨形态-层状石墨2.3.3灰铁和白口铁的凝固2.3.4固态转变2.3.5硬度和材料特性2.3.6弹性模量-杨氏模量2.4球墨铸铁仿真2.4.1石墨形核2.4.2球墨铸铁固态转变（共析转变）2.4.3球墨铸铁的珠光体分解2.4.4机械性能2.4.5弹性模量-杨氏模量2.5蠕墨铸铁仿真2.6铸铁收缩和疏松的形成2.6.1凝固收缩2.6.2砂型/芯子的变形2.6.3疏松形成和压力特性2.6.4石墨聚集因子2.6.5疏松级别模拟的说明3 How to Use MAGMAiron3.1概述3.2MAGMA数据库3.2.1铸铁数据集3.2.2一般参数3.2.3铸铁成分3.2.4铸铁类型/石墨种类3.2.5型砂成分3.2.6金相照片数据/单位面积形核数/铁素体、珠光体分布形核数3.3仿真3.3.1概述3.3.2窗口——铸铁3.3.3铸铁模拟菜单3.4结果演示/后处理3.4.1结果-概述3.4.2金相照片等——球墨铸铁的微观结构3.4.3单位系统4小结- 怎么办4.1铸铁的具体数据4.2项目定义4.3几何建模4.4仿真设置4.5结果显示4.6其他信息1 Introduction介绍MAGMAiron是微观建模软件，可以模拟凝固、固相转变及在铸铁中凝固中相关的物理现象。

MAGMAiron是一个附加的模块，可以模拟灰铸铁、球墨铸铁和蠕墨铸铁的凝固过程和固态相变过程。

冶金质量、工艺条件对铸铁合金的性能有很大的影响。

微观组织和铸件的力学性能不仅取决于铸造的流动过程，而且还由以下下参数决定：1）合金成分、2）金属处理、3）微量元素和杂质、4）熔炼炉、钢包金属液的处理（除氧、镁处理）5）孕育材料的类型和数量6）孕育法析出相的晶粒长大动力学和冷却条件决定了实际的微观组织的形成，因此必须考虑凝固、疏松和固态相变过程，它们共同影响铸铁的机械性能。

海藻酸钠固定细菌

Whole cell-and protein-based biosensors for the detection ofbioavailable heavy metals in environmental samplesPhilippe Corbisier a,*,Daniel van der Lelie a ,Brigitte Borremans a ,Ann Provoost a ,Victor de Lorenzo b ,Nigel L.Brown c ,Jonathan R.Lloyd c ,Jonathan L.Hobman c ,Elisabeth CsoÈregi d ,Gillis Johansson d ,Bo Mattiasson d aVlaamse Instelling voor Technologisch Onderzoek (VITO),Boeretang 200,B-2400Mol,BelgiumbCentro Nacional de BiotecnologõÂa,Campus de Cantoblanco,Universidad Auto Ânoma,Spain cThe University of Birmingham,School of Biological Sciences,Edgbaston,Birmingham B152TT,UKdDepartment of Biotechnology,Centre for Chemistry and Chemical Engineering,Lund University,PO Box 124,S-22100Lund,SwedenReceived 8July 1998;received in revised form 9October 1998;accepted 11October 1998AbstractThe principal goal of this work was to establish the feasibility of two biosensor technologies with enhanced speci®city and selectivity for the detection of several bioavailable heavy metals in environmental samples.Two parallel strategies have been followed.The ®rst approach was to construct whole cell bacterial biosensors that emit a bioluminescent or ¯uorescent signal in the presence of a biologically available heavy metal.The molecular basis of s -54promoters as sensing elements of environmental pollutants has been determined and a number of metal-induced promoter regions have been identi®ed,sequenced and cloned as promoter cassettes.The speci®city of the promoter cassettes has been determined using lux CDABE reporter systems.Whole cell-biosensors containing metal-induced lux reporter systems have been incorporated into different matrices for their later immobilisation on optic ®bres and characterised in terms of their sensitivity and storage capacity.The second type of sensors was based on the direct interaction between metal-binding proteins and heavy metal ions.In this case,the capacitance changes of the proteins,such as synechoccocal metallothionein (as a GST-SmtA fusion protein)and the mercury regulatory protein,MerR,were detected in the presence of femtomolar to millimolar metal ion concentrations.#1999Elsevier Science B.V .All rights reserved.Keywords:Heavy metals;Bioavailability;Biosensors;Af®nity sensors;Capacitance measurements1.IntroductionMost metal ions can be detected in environmental samples using classical analytical methods such as inductively coupled plasma atomic electron spectro-metry (ICP/AES)or mass spectrometry (ICP/MS),¯ow injection atomic absorption (FIAAS)or electro-chemical methods that include ion selective electro-des,polarography and other voltammetric electrodes.The environmental samples need to be digested under high temperature,pressure and acidic conditions to free the metal ions in solution as a prerequisite for all those methods.However,the total amount ofmetalAnalytica Chimica Acta 387(1999)235±244*Corresponding author.Tel.:+32-14-335-112;fax:+32-14-580-523;e-mail:corbisip@vito.be 0003-2670/99/$±see front matter #1999Elsevier Science B.V .All rights reserved.P I I :S 0003-2670(98)00725-9detected after such an extraction may not always be related to the toxicity of the samples because the original biological availability of metal ions is not taken into account.To quantify the biologically avail-able fraction of metal in environmental samples, different approaches have been followed.The®rst approach is based on the use of soil bacteria that are genetically engineered so that a quanti®able signal is produced when the bacteria are in contact with bioavailable metal ions.A number of fundamental aspects of the regulation of sensing elements have been studied,and new metal-induced regulators have been discovered and characterised.Soil bacteria able to produce a bioluminescent signal in the presence of speci®c metal ions have been constructed by genetic manipulation and immobilised in solid matrices for use with optic®bres.The second approach followed in this work was to follow directly the interaction between metal ions and speci®c puri®ed metal-binding proteins by recording the capacitance changes on metal binding.Both tech-niques are described below.2.Experimental2.1.Construction of heavy metal-induced promotercassettesTwo independent clones containing the copSRA promoter region of the Alcaligenes eutrophus CH34 cop operon and the tetracycline marker(as an Eco RI fragment from miniTn5-luxAB[1])were constructed in plasmid pUC18/S®I.An s®I cassette containing the mercury responsive elements from Tn501[2]was constructed as follows:the E.coli merR gene and mer promoter(P mer TPAD)of Tn501was cloned as a 0.7kb Xba-Eco RI fragment in pUC18/S®I and the tetracycline resistance marker was cloned into the Hin dIII site.An s®I cassette containing the chromate responsive element from the A.eutrophus pMOL28 plasmid[3]was constructed by cloning the chr pro-moter/operator region and the chrB gene of plasmid pMOL28as a1.2kb XbaI-PstI fragment in pUC18/ S®I together with the Eco RI fragment containing the tetracycline resistance gene.The A.eutrophus pbr R promoter/operator region and the partially deleted pbr A gene were cloned as a3.8kb Eco RI fragment in PUC18/S®I,and the tetracycline marker was cloned into the Hin dIII site.2.2.Immobilisation of the whole cell copperbiosensorTo immobilise the strain AE1239in sodium algi-nate(BDH Supplies Poole,Dorset,UK),50ml of fresh bacterial culture was harvested by centrifugation at6000rpm for10min in a JA-20Beckman rotor.The cell pellet was suspended in20ml0.9%(w/w)NaCl, and20ml4%(w/w)sodium alginate was added to the bacterial suspension.Next,a syringe with a needle(ID 1.2mm)was used to create droplets added to200ml 0.2M CaCl2to form the alginate beads.The beads were washed twice with0.9%NaCl and kept in0.9% NaCl at48C until use.To immobilise the whole cell copper biosensor in seaplaque agarose,the same procedure was followed but the cell pellet was sus-pended in9ml0.9%NaCl.Next,2.22g agarose was dissolved in100ml0.9%NaCl with heating,cooled to 308C in a water bath,and5ml bacterial suspension was added.A2mm thick biogel was produced on setting,and discs of4mm diameter were cut from the gel.The biogel was kept at48C in0.9%NaCl until use.2.3.Bioluminescence measurementsFor the copper whole cell-sensor AE1239,the chromate whole cell-sensor AE2440,or the lead whole cell-sensor AE2448,an overnight25ml LB bacterial culture was centrifuged for10min at 10000rpm in a JA-20rotor and resuspended in cool cryoprotectants(1ml/0.1g wet wt).A volume of 150m l cell suspension was dispensed in sterile lyo-philisation glass vials and cooled in ice water before being frozen atÀ408C and lyophilised in a vacuum chamber.The vacuum was released under nitrogen gas and the vials were sealed.About107±108viable cells were obtained after reconstitution in a reconstitution medium(RM).This medium is similar to the Tris medium described previously[4]but Tris buffer was replaced by MOPS buffer and sodium phosphate by sodium- -glycerophosphate to minimise metal chela-tion and precipitation.The RM medium contained 4.68g of NaCl,1.49g of KCl,1.07g of NH4Cl, 430mg of Na2SO4,200mg of MgCl2Á6H20,30mg236P.Corbisier et al./Analytica Chimica Acta387(1999)235±244of CaCl2Á2H20,294mg of Na- -glycerophosphate, 2g of Na-acetate or0.1g of gluconate,20mM of MOPS,pH7.0and one1ml of trace element solution SL7of Biebl and Pfennig[4]in1l of distilled water. The metal salt solutions(20m l)were added to180m l cell suspension and the bioluminescence was recorded over5h at238C in a Lucy1microtitre plate lumino-meter.2.4.Overexpression and purification of heavy-metalbinding proteinsThe mercuric ion-binding regulatory protein,MerR from transposon Tn501,has been puri®ed in large amounts.The protein was overexpressed in E.coli and an extraction protocol using sonication,salt-extrac-tion,and liquid chromatography(LC),modi®ed from [5],was used to purify10mg of MerR.The cloned merR gene from Bacillus[6]was expressed from the bacteriophage T7promoter on plasmid pBS ,via temperature induction(at428C)of T7RNA polymer-ase from plasmid pGP1-2,co-transformed into the E.coli host.After3h induction,the cells were sonicated and the MerR protein puri®ed by LC af®nity chromatography followed by size exclusion chroma-tography.Yields were low(less than100m g l per culture)but0.6mg puri®ed protein was produced for immobilisation.The fusion protein GST-SmtA,containing glu-tathione-S-transferase linked to the synechococcal metallothionein protein,was overexpressed in E.coli from an expression vector pGEX3X(Pharmacia)con-taining smtA,and puri®ed using glutathione sepharose 4B by published methods[7].2.5.Protein immobilisation and capacitancemeasurementsThe fusion proteins GST-SmtA and MerR were produced as described above and dissolved in phos-phate buffered saline(70mM NaCl,1.3mM KCl, 5mM Na2HPO4,0.9mM KH2PO4,pH7.3)contain-ing50%glycerol to a®nal concentration of1mg/ml protein.Thioctic acid and glutaraldehyde(GA)were purchased from Sigma and1-(3-dimethylaminopro-pyl)-3ethyl-carbodiimide hydrochloride(EDC)was obtained from Fluka.1-Dodecanethiol and the gold rods used for the electrodes came from Aldrich.Heavy metal salts CuCl2Á2H20,ZnCl2,HgCl2and Cd(NO3)2Á4H20were from Merck(Darmstadt,Ger-many).Polyethyleneglycol diglycidyl ether (PEGDGE)was obtained from Polysciences(USA). All other reagents were of analytical grade.The biosensors were prepared by immobilising fusion proteins on the gold surface by EDC-mediated coupling,PEGDGE entrapment or GA cooling.In all cases20m l of the dissolved fusion proteins were diluted with480m l100mM borate buffer,pH8.75 and the solution was®ltered through a micro-®lter (Amicon,USA)with a molecular cut-off of3000D. After ultra®ltration,the fusion protein concentration was adjusted to0.04mg/ml in borate buffer.Gold electrodes were cleaned and pretreated with thioctic acid,as described elsewhere[8].The biosensor was arranged as the working elec-trode in a three-electrode system connected to a fast potentiostat.It was placed in a¯ow cell with a dead volume of10m l which was built in-house[8].A platinum foil served as the auxiliary and a platinum wire as the reference electrode.An extra reference electrode(Ag/AgCl)was placed in the outlet stream, as the platinum does not have a de®ned potential.The buffer solution was pumped by a peristaltic pump with a¯ow rate of0.5ml/min through the¯ow cell.Sam-ples were injected into the¯ow via a250m l sample loop.The buffer was10mM borate pH8.75,®ltered through a0.22m m millipore®lter and degassed before use.3.Results and discussion3.1.Development of new genetic toolsA number of genetic tools and the background knowledge necessary for the successful production of metal-responsive strains have been developed.Sub-stantial progress in understanding the molecular basis of s-54promoters as sensors of environmental pollu-tants has been described earlier[9,10].Understanding the mechanism of activation of this regulator was important in designing the gene-based sensors,which are based on the activation of metal-induced promoter/ operator regions.The mechanisms of metalloregula-tion of two promoters of E.coli based on the Fur protein has also been dissected[11].Finally,theP.Corbisier et al./Analytica Chimica Acta387(1999)235±244237exploitation of the outer membrane protein LamB as an anchor of heterologous metal-binding peptides such as the yeast (CUP1)and mammalian (HMT-1A)metallothioneins on the surface of Gram-negative bacteria has been explored [12].A.eutrophus was chosen as a reference organism due to its ability to survive in harsh environments [13].Four different metal-induced promoter cassettes were constructed (Fig.1).Promoters responsive to Cu 2 ,Hg 2 ,Cr 6 /3 and Pb 2 ions were chosen to demon-strate the feasibility of this approach.The pMOL30copper resistance operon from A.eutrophus was located,sequenced and analysed [14].At least eight open reading frames were identi-®ed and designated copSRABCDGF .The copSR genes encode for a two-component regulatory system that was needed as the copper responsive element [15].The copABCD genes encode the structural copper resistance genes similar to the pcoABCD and copABCD genes of the E.coli and Pseudomonas syringae ,respectively [16,17].In contrast to the E.coli and P .syringae copper resistance operons,theregulatory genes copSR are transcribed in the opposite orientation of the structural genes copABCD .The copF gene,transcribed in the opposite direction to copABCD ,encodes a Cu-ef¯ux ATPase similar to the PacS protein of Synechococcus [18].The copSRA promoter region of the A.eutrophus CH34cop operon and the mer regulatory region (merR and mer promoter,P mer TPAD )of Tn 501were cloned in S®I cassettes for the detection of copper and mercury ions,respectively.Once cloned in A.eutrophus to express the LamB [19]or the GFP [20]reporter systems,concentrations of 1m M Cu 2 or 0.01m M HgCl 2should be easily detected.When cloned in front of a Vibrio ®scheri luxCDABE promoterless expression vector such as pMOL877[21],the chrA promoter was mainly induced by Cr 6 compared to Cr 3 ions as shown in Fig.2.Ni,Zn,Co,Al,Cd,Mn,AsO 4,MoO 4,WoO 4,SeO 3and SeO 4in their respective ionic forms did not induce any bioluminescence and were not toxic up to 100m M (data not shown).The use of this fusion to assess the bioavailability and toxicity of chromium in soilsam-Fig. 1.Schematic organisation of the heavy metal-induced promoter Sfi I cassettes.(A)Copper promoter region from plasmid pMOL30from A.eutrophus CH34in both orientations;(B)mercury promoter region from transposon Tn 501;(C)chromate promoter region from A.eutrophus CH34plasmid pMOL28;(D)lead promoter region from plasmid pMOL30.Not all restriction sites areshown.Fig. 2.Bioluminescence induction of A.eutrophus AE2440containing the chr B Áchr A::lux CDABE fusion in the presence of increasing concentration of (*)K 2CrO 4and (~)CrCl 3;( )other tested ions:Ni,Zn,Co,Al,Cd,Mn,AsO 4,MoO 4,WoO 4,SeO 3and SeO 4.The bioluminescence,expressed as a signal to noise ratio,was measured 3h after induction in the reconstitution medium (RM)with 0.1%gluconate as C-source medium.238P .Corbisier et al./Analytica Chimica Acta 387(1999)235±244ples has large potentials since it can easily differenti-ate the trivalent from hexavalent chromium.The lead resistance operon of A.eutrophus pMOL30plasmid has also been cloned and sequenced.It contains two genes,pbr RA,required for regulation and lead resistance,respectively.The pbr R regulator was very similar to the MerR protein (regulator of the mercury resistance operon).The PbrA protein has all characteristics of an ef¯ux ATPase.When cloned in into pMOL877,the pbr R promoter appeared to be speci®cally induced by lead ions as shown in Fig.3.Those four different metal-induced promoter cassettes can now be further assembled in the LamB or GFP reporter systems. The metal-induced bacterial biosensors[22]that were already available have been further immobilised in different solid matrices.3.2.Immobilisation of the bacterial sensorsThe bacterial Cu-sensor AE1239[22]has been immobilised in alginate and agarose gels and chal-lenged to increasing concentrations of Cu2 ions in the LB medium.In both immobilisation matrices,the relative bioluminescent signals were perfectly linear between0and200m M Cu2 and®tted the linear equations were y 1.88x 9.51(R2 0.99)and y 9.55x 221(R2 0.99)for agarose and alginate beads,respectively.As seen from Table1,both immo-bilisation matrices showed similar characteristics, except that the relative signal was higher in agarose gels.The lower sensitivity of the Cu-biosensor in alginate was probably due to an overgrown bacterial culture,since the optical density of the culture was above the optimal value of0.4±0.5.The detection limit was similar to that obtained when cells were in solu-tion,but remained too high for practical applications. Therefore the reaction medium(RM)has been opti-mised to provide suf®cient nutrient supply to the bacterial cells and to avoid heavy-metal chelation with the culture medium[4].When the immobilised cells were tested in this RM medium,the detection limit could be reduced to1m M Cu and the signal was kept linear up to200m M Cu according to the equation y 14.93x 32(R2 0.99)(see also Table1).Fig.3.Specificity of the A.eutrophus CH34containing the pbr RÁpbr A::lux CDABE fusion in the presence of increasing concentration of metal ions.The bioluminescence,expressed as a signal to noise ratio was measured3h after induction in the reconstitution medium(RM)with0.1%gluconate as C-source medium.Table1Characteristics of the whole cell Cu-biosensor AE1239in solution and immobilised in alginateAE1239Absorbanceat! 600nm Relativesignal(S0.5/S0)Detectionlimit(m M)Linear range(m M)Inductiontime(h)RSD0.5mM Cuafter4h(%)Solution0.3416.120up to1000 1.519.5LB/alginate 1.6a 6.9620up to250 1.518.4LB/agarose0.7a13.140up to250 1.5 6.48RM/solution0.10a 6.31up to25 1.50.2RM/alginate0.49a11.61up to200 1.57.5 Measurements were performed in Luria-broth medium(LB)and in a mineral salts medium(0.1%acetate,20mM MOPS,pH7)(RM). RSD:relative standard deviation,the detection limit is calculated as twice the signal to noise ratio.a Calculated figures.P.Corbisier et al./Analytica Chimica Acta387(1999)235±244239The storage stability of immobilised cells was assessed by measuring the emitted bioluminescence for six days (see Fig.4(A)and (B)).Agarose discs lost 84%of their activity within six days,while alginate beads were found to be stable.As seen from these ®gures the alginate immobilised cells displayed much better stability than the cells immobilised in agarose.3.3.Metal ion-specific capacity affinity sensors Protein engineering has opened up the possibility of designing and producing new proteins with,for exam-ple,a higher selectivity than natural ones [23].Also,recent progress in the construction of thin self-assembled monolayer (SAM)-forming molecules [8]has allowed us to construct very sensitive and speci®cmetal ion capacity af®nity sensors.The reactive bio-logical elements have been chosen from proteins known to bind metal ions in a reversible manner.The ®rst step for this technology was to overproduce and purify proteins.We have made the constructs for overexpression of the broader-spectrum MerR from Bacillus [6].Unlike the Tn 501protein,which responds only to mercuric ions,this protein also responds in vivo to the presence of organomercurials and it is know that changes in the very C-terminal amino acid sequence are responsible for this speci®city [24].This broader-spectrum MerR protein from Bacillus ,which responds to both mer-curic ions and organomercurials,was overexpressed and puri®ed by af®nity chromatography.The fusion protein synechococcal GST-SmtA metallothione protein has been produced by protein engineering.The ®nal yield was 4mg protein per l.The Zn-binding characteristics of the fusion protein were con®rmed using atomic adsorption spectroscopy.The fusion protein also binds Cd,Cu and Hg,with pH half dissociation values similar to those reported for commercially available equine metallothionein [23].Future studies can be done with other metal-binding proteins from bacterial resistance determinants,such as MerP and PbrR,which we have recently puri®ed (unpublished data).In order to develop a sensitive af®nity sensor based on capacitance measurement,the immobilisation layer has to be as thin as possible and well-ordered.First,experiments were focused on ®nding the optimal immobilisation procedure (EDC-coupling,PEGDGE-entrapment and GA-crosslinking).So far,EDC coupling has resulted in the highest sensitivities,although experiments are planned with <10%PEGDGE as well.The effect of blocking the electrode surface with insulators was studied.The importance of good insu-lation of the electrode surface has been demonstrated earlier [8].The degree of insulation can be studied by having a small permeable redox couple in solution.Fig.5shows how the blocking increases for each additional layer.For a clean gold surface the redox couple K 3[Fe(CN)6]was oxidised and reduced at the metal surface.A surface covered with self-assembled thiotic acid reduced access to the surface.Immobilisa-tion of the GST-SmtA protein further insulated the surface,but it was not until the treatment with1-Fig.4.(A)Storage stability of immobilised AE1239in (!)alginate or (*)agarose matrices measured after 4h incubation with 0.5mM Cu 2 ;(B)relative storage stability expressed in percentage related to the initial bioluminescence value.240P .Corbisier et al./Analytica Chimica Acta 387(1999)235±244dodecanthiol that the oxidation/reduction peaks com-pletely disappeared.The activity of the optimised GST-SmtA electrodes was studied with copper,cad-mium,mercury and zinc ions and gave a selectivity in the named order (data not shown).These results are consistent with the literature,which states that the GST-SmtA protein has a broad selectivity towards heavy metals[7].Fig.5.Cyclic voltammetry responses recorded in an Fe CN 3À6solution when the measuring electrode was (a)unmodified gold,(b)gold modified with thioctic acid,(c)as in (b)with additional modification with immobilised fusion protein GST-SmtA and (d)as in (c)with additional modification with1-dodecanethiol.Fig.6.Storage stability of the GST-SmtA electrodes.A calibration curve from 10À15to 10À10M Cu 2 was recorded each day and the total capacitance change vs.time was plotted.Between measurements the sensor was stored at 48C in 10À1M borate buffer 8.75.P .Corbisier et al./Analytica Chimica Acta 387(1999)235±244241The GST-SmtA electrodes could be regenerated by injection of 1mM EDTA.It was found that if EDTA was injected just before storage,the biosensor lost activity on overnight storage.However,if the elec-trode was stored in the presence of heavy metal ions and regenerated immediately before taking measure-ments on the following day,no activity loss was observed.This was probably due to the protein being protected from denaturation (possibly oxidation)by binding of the heavy metal to cysteine sulphydryl groups.The stability of these electrodes over 16days is shown in Fig.6.The activity of the GST-SmtA electrodes was stu-died for an extended concentration range (see Fig.7).As shown three distinct parts can be identi®ed,the ®rst (up to 10À5M)being attributed to the capacitance change upon relative non-speci®c binding of the heavy metal to sulphydryl groups,the second (up to 10À2M)Fig.7.Capacitance change of the GST-SmtA electrode vs.Cu 2 concentration in the range 10À15±10À1M.The measurements were performed in 10À1M borate buffer,pH 8.75and a flow rate of 0.5ml/min.Samples with a volume of 250m l wereinjected.Fig.8.Capacitance change vs.heavy metal ion concentration for MerR immobilised electrode:(a)in the presence of Cd 2 ,(b)in the presence of Cu 2 and (c)in the presence of Hg 2 .The measurements were performed in 100mM borate,pH 8.75and a flow rate of 0.5ml/min.Samples with a volume of 250m l were injected.242P .Corbisier et al./Analytica Chimica Acta 387(1999)235±244probably due to the formation of a closed metallothio-nein`cage'containing the metal ions,and the third due to saturation(levelling off).Preliminary experiments were also carried out with an Mer R electrode,immo-bilised by the same method found to be optimal for GST-SmtA protein.This protein is known to be highly speci®c towards Hg2 ions in vivo and in vitro[23]. The protein was immobilised on the sensor surface by the EDC method.The sensitivity was studied for the three different heavy metals;mercury,copper and cadmium.It was found,as expected,that the sensi-tivity was the highest for mercury,and that this electrode showed higher selectivity than the GST-SmtA electrode at low metal ion concentrations (Fig.8).4.ConclusionsThis work has shown the feasibility of technologies for the detection of heavy metal ions based on whole cell-biosensors and on protein-based sensors.A num-ber of metal-induced promoter regions have been identi®ed and arranged in cassettes that can be easily used to activated reporter system such as the lux or GFP reporter genes or the expression of outer mem-brane epitopes that can be easily detected by immu-nochemistry.The high speci®city of such an induced gene expression has been shown for the lead and chromate ions.Preliminary results for immobilised whole cell-sensors have been obtained and demon-strated the applicability of this technology.Af®nity sensors based on proteins were also demon-strated as suitable for monitoring heavy metal ions at trace levels.The metal ion-speci®c capacitance sen-sors have an exceptional sensitivity and a wide oper-ating range.They are also versatile systems because different kinds of recognition elements can be immo-bilised directly in a self-assembling monolayer com-pletely covering the surface of the measuring noble metal electrode.The electrode then becomes selective to those metal ions in the solution that show af®nity to the recognition element on the pared to previously described electrochemical sensors,the pro-tein-based sensor shows many orders of magnitude better sensitivity[25].The whole cell-sensor and the protein-based sensor now need to be tested on real environmental samples.The main advantage of the whole cell-sensors will remain their ability to react only to the bioavailable fraction of metal ions,whereas the protein-based sensor's potential application remains in its high sensitivity towards metals ions.AcknowledgementsWe thank Professor Nigel Robinson(Newcastle)for supplying the pGEX3X-smtA fusion plasmid.NB thanks Kenneth J.Jakeman for technical assistance. PC thanks S.Leth,S.Maltoni and A.Bossus for technical assistance and L.Diels and M.Mergeay for the constructive discussions.This work was sup-ported by the European Commission as part of the contract ENV4-CT95-0141.References[1]M.Herrero,V.de Lorenzo,K.N.Timmis,J.Bacteriol.172(1990)6557.[2]P.A.Lund,N.L.Brown,J.Mol.Biol.205(1989)343.[3]A.Nies,D.H.Nies,S.Silver,J.Biol.Chem.265(1990)5648.[4]P.Corbisier, E.Thiry, A.Masolijn,L.Diels,in: A.K.Campbell,L.J.Kricka,P.E.Stanley(Eds.),Bioluminescence and Chemiluminescence:Fundamentals and Applied Aspects, Wiley,New York,1994,pp.151±155.[5]J.Parkhill,A.Z.Ansari,J.G.Wright,N.L.Brown,T.V.O'Halloran,EMBO J.12(1993)413.[6]J.D.Helmann,Y.Wang,I.Mahler,C.T.Walsh,J.Bacteriol.171(1989)222.[7]A.M.Tommey,J.Shi,W.P.Lindsay,P.E.Urwin,N.J.Robinson,FEBS Lett.292(1991)48.[8]C.Berggren,G.Johansson,Anal.Chem.69(1997)3651.[9]J.PeÂrez-MartõÂn,V.de Lorenzo,J.Mol.Biol.258(1996)562.[10]J.PeÂrez-MartõÂn,V.de Lorenzo,J.Mol.Biol.258(1996)575.[11]L.Escolar,V.de Lorenzo,J.PeÂrez-MartõÂn,Mol.Microbiol.26(1997)799.[12]C.Sousa,A.Cebolla,V.de Lorenzo,Nature Biotechnol.14(1996)1017.[13]M.Mergeay,D.Nies,H.G.Schlegel,J.Gerits,P.Charles,F.J.VanGijsegem,Bacteriology162(1985)328.[14]B.Borremans,A.Provoost,P.Corbisier,M.Mergeay,J.L.Hobman,N.L.Brown,D.van der Lelie,Proceedings of the VI International Congress on Pseudomonas:Molecular Biology and Biotechnology,1997,p.IX.[15]D.A.Rouch,N.L.Brown,Microbiology143(1997)1191.[16]N.L.Brown,S.R.Barrett,J.Camakaris,B.T.O.Lee,D.A.Rouch,Mol.Microbiol.17(1995)1153.[17]S.D.Molls,C.-K.Lim,D.A.Cooksey,Mol.Gen.Genet.244(1994)341.P.Corbisier et al./Analytica Chimica Acta387(1999)235±244243。

Vertebrate Evolutionary Biology Specimens

Vertebrate Evolutionary BiologySpecimensVertebrate evolutionary biology specimens are crucial for understanding the history and development of vertebrate animals. These specimens provide tangible evidence of the changes and adaptations that have occurred over millions of years. By studying these specimens, scientists can piece together the evolutionaryhistory of various vertebrate groups, from fish to mammals. One of the most important aspects of vertebrate evolutionary biology specimens is their diversity. Vertebrates encompass a wide range of animals, from the jawless fish that first appeared over 500 million years ago to the complex mammals that dominate the Earth today. By studying the specimens of different vertebrate groups, scientists can see how these animals have evolved and diversified over time. In addition to diversity, vertebrate evolutionary biology specimens also provide insights into the anatomical changes that have occurred over time. For example, the transition from fish to tetrapods (four-limbed vertebrates) is a key event in vertebrate evolution. By studying the specimens of early tetrapods, scientists can see how their limbs evolved from fins and how their skulls adapted to support their new way of life on land. Furthermore, vertebrate evolutionary biology specimens can help scientists understand the relationships between different vertebrate groups. By comparing the anatomical features of different specimens, scientists can determine which groups are more closely related and which are more distantly related. This information is crucial for constructing accurate evolutionary trees and understanding the overall history of vertebrate animals. Moreover, vertebrate evolutionary biology specimens can also shed light on the ecological roles that different vertebrate groups have played throughout history. For example, studying the specimens of extinct predators like dinosaurs can help scientists understand how these animals interacted with their environments and prey. By studying the specimens of both predators and prey, scientists can reconstruct ancient ecosystems and understand how vertebrates have shaped the world around them. Overall, vertebrate evolutionary biology specimens are invaluable tools for understanding the history and development of vertebrate animals. Through the studyof these specimens, scientists can uncover the diversity, anatomical changes, relationships, and ecological roles of different vertebrate groups throughout millions of years of evolution. By piecing together this information, scientists can gain a deeper understanding of the evolutionary processes that have shaped the vertebrate animals we see today.。

IGBT模块3

3Materials3.1Insulating Substrate and MetallizationThe insulating substrate serves as the supporting structure for the circuitry of the power module.1 It acts as the surface for depositing conductive, dielec-tric, and resistive materials that form the passive circuit elements. It is also a base for mechanical support for all active and passive chip components. It must be strong enough to withstand different environmental stresses. Electrically, it must be an insulator to isolate various conductive paths of the circuit. It must be able to withstand an RMS AC voltage (50 to 60 Hz) of 2500 V applied between any terminal and the case, including the base plate, for a one-minute duration. It must have sufﬁcient thermal conductivity to remove the heat generated by the components.In addition, a high degree of surface smoothness is required for adhesion of ﬁlms, ﬁne conductor lines, and spacings. Surface ﬂatness is desirable to minimize processing problems during screen-printing, photomasking, etc. Nonﬂat surfaces do not press uniformly against the base plate. This can potentially lead to microcracks and poor localized thermal conduction. 3.1.1Selection CriteriaThe substrate material most suitable for power applications should be determined by the following electrical, thermal, mechanical, and chemical requirements:2,3•Electrical–High-volume (or insulating) resistivity (> 1012W cm)–High dielectric strength (> 200 v/mil)–Low dielectric constant (< 15)•Thermal–High thermal conductivity (> 30 w/mK for effective thermal conduction)–Matching coefﬁcient of thermal expansion with the power semi-conductor chips (2 to 6 ppm/°C for minimizing thermal stress and matching with Si 2.8 ppm/°C)–High thermal stability (> 1000°C for direct bonded copper [DBC] and brazing operations)•Mechanical–High tensile strength (> 200 MPa)–High ﬂexural strength (> 200 MPa)–Dimensional stability — Hardness–Machineable — Can easily lap, polish, cut, and drill–Good surface ﬁnish (< 1 m m)—If not as ﬁred, then should be able to be lapped to this speciﬁcation–Metallizability — C ompatible with popular metallization tech-niques:•Thin ﬁlm•Thick ﬁlm•Plated copper•Direct bonded copper (DBC)•Active brazed copper (ABC)•Regular brazed copper (RBC)•Chemical–High resistance against acidic, alkaline, and other processing solvents–Low moisture absorption rate–Low toxicity–Chemically inert to plasma process•Density/weight–Low density/weight — Minimize mechanical shock •Maturity–Technology–Suppliers•Cost–As close to that of alumina as possible3.1.2List of Insulating SubstratesNumerous insulating substrates of different materials have been used for semiconductor applications.2-4 The following is a list of the popular ones, grouped by type:•Ceramic substrates–Alumina (Al2O3: 96%, 99%)–Aluminum nitride (AlN)–Beryllia (BeO)–Boron nitride (BN, hex)–Cordierite–Fordterite–Mullite–Steatite–Titanate•Glass substrate–Borosilicate glass•Sapphire substrate–Sapphire•Quartz substrate–Quartz•Si-based substrates–SiC–SiO2–Si3N4•Metal-core substrates–Insulated metal substrates (IMS)•Diamond substrate–Diamond (CVD polycrystalline)Table 3.1 through Table 3.5 show the following properties of these insu-lating substrates:1–20•Mechanical•Thermal•Electrical•Chemical•OtherTABLE 3.1Mechanical Properties of Insulating SubstratesMaterialTensileStrength(MPa)FlexuralStrength(MPa)ElasticModulus(GPa)HardnessSurfaceFinish(m m)Density(kg/m3)CeramicAl2O3(96%)127.4317310.32000 K 1.03970Al2O3(99%)206.93453459 MH 1.03970 AlN3103603101200 K 1.03260 BeO230250345100 K153000 BN (hex)1102200 Corderite5510141600 Forsterite55124902700 Mullite125175Steatite55110902500 Titanate2869693500 GlassBorosilicate509 1.02280 SapphireSapphire3994309 MH 1.03990 QuartzQuartz4814072 5 MH 1.02200 Si-basedSiC174404123160 SiO29630692190 Si3N4969323142400 Metal-coreIMS39262700 DiamondDiamond(CVD)100011807500 K< 1.03500 Note: MH = Moh; K = Knoop.TABLE 3.2Thermal Properties of Insulating SubstratesMaterialThermalConductivity(W/m ˚K)CTE(ppm/˚C)HeatCapacity(J/kg-˚C)MaximumUse Temperature(˚C )MeltingPoint(˚C)CeramicAl2O3(96%)24 6.0765********Al2O3(99%)337.276516002323 AlN150–180 4.6745> 10002677 BeO2707.010472725 BN (hex)60 3.8Corderite4 3.07701250Forsterite4111100Mullite6 4.2Steatite 2.510.51100Titanate410700GlassBorosilicate2 3.2SapphireSapphire41.77.3735QuartzQuartz43 5.581611401938 Si-basedSiC120 4.6675> 10003100 SiO2 1.50.6> 800Si3N470 3.0691> 10002173 Metal-coreIMS425DiamondDiamond (CVD)2000 (Z)1400 (X,Y)1509Resistant tooxidationto 600˚CTABLE 3.3Electrical Properties of Insulating SubstratesMaterial Resistivity(W-cm)DielectricStrength(kV/mm)DielectricConstantat 1 MHzCeramicAl2O3(96%)> 1014129.2Al2O3(99%)> 1014129.9 AlN> 1014158.9 BeO> 101412 6.7 BN (hex) 4.1 Corderite106–1014Forsterite1010–10127.9–11.8 6.2 Mullite> 1012Steatite1011–10137.9–15.7 5.5–7.5 Titanate106–1013 2.0–11.815 GlassBorosilicate> 1014 3.7 SapphireSapphire> 10144810.4 QuartzQuartz101016.1 3.8 Si-basedSiC> 101120–42 SiO2> 1014 3.5–4 Si3N4> 1010106–10 Metal-coreIMS> 1013DiamondDiamond(CVD)> 1011> 100 5.7TABLE 3.4Chemical Properties of Insulating SubstratesMaterialMoistureAbsorption(%)NitricAcidSulfuricAcid(Weight Loss atmg/cm2/day)CausticSoda ToxicityCeramicsAl2O3(96%)0NoAl2O3(99%)00.050.220.04No AlN0No BeO0Yes BN (hex)0No Corderite27No Forsterite0No Mullite5–15No Steatite0No Titanate0No GlassBorosilicate0No SapphireSapphire0000No QuartzQuartz0000No Si-basedSiC00.040.010No SiO20No Si3N40 1.000.400.36No Metal-coreIMS0No DiamondDiamond(CVD)0000NoTABLE 3.5Other Properties of Insulating SubstratesMaterial Metallizability a Machineability b Relative CostCeramicsAl2O3(96%)All, except thin ﬁlm Good1¥Al2O3(99%)All Good2¥AlN All, except thickﬁlmGood4¥BeO All Good (only by approved source)5¥BN (hex)None GoodCorderite Thick ﬁlm Good0.6¥Forsterite Thick ﬁlm Good1¥Mullite Thick ﬁlm Good1¥Steatite Thick ﬁlm Good0.8¥Titanate Thick ﬁlm Good2¥GlassBorosilicate Thin ﬁlm Fair1¥SapphireSapphire Thin ﬁlm Fair10¥QuartzQuartz Thin ﬁlm Fair8¥Si-basedSiC None Fair4¥SiO2GoodSi3N4All Good 2.5¥Metal-coreIMS Thin and thickﬁlmsGoodDiamondDiamond (CVD)Thin and thickﬁlmsGood Higha Metallization: Thin ﬁlm, thick ﬁlm, plated copper, direct bonded copper (DBC), active brazed copper (ABC), regular brazed copper (RBC).b Machineability: lapping, polishing, cutting, drilling, shaping.3.1.3Selected Insulating SubstratesAfter carefully examining the preceding tables and comparing them with the selection criteria, it appears that there are four possible candidates for insulating material that are suitable for power semiconductor applications:•Ceramic substrates:–Al2O3 (96%, 99%)–AlN–BeO•Si-based substrate:–Si3N4CVD polycrystalline diamond substrate has promising potential if:•Heavy Cu metallization can be attached•Cost can be reducedIn the following sections, the strengths and weaknesses of each of these substrates are analyzed and compared.3.1.3.1Alumina (96%, 99%)•Strengths:–Most commonly used ceramic for substrate material and there-fore the most mature technology–Low cost — 1" ¥ 1" size as ﬁred, 96% ~ $0.10; 99% ~ $0.20–Average but adequate all-around characteristics: mechanical, thermal, electrical, and chemical–Easily metallized — thin ﬁlm, thick ﬁlm, plated copper, DBC, ABC, RBC–Easily machineable–Nontoxic–Nonpermeable to gas–Zero moisture absorption rate–Camber ~ 0.003"/1"–Mature technology–Dimensionally stable•Weaknesses:–Thermal conductivity -30 w/k-m is adequate for low- to medi-um-power applications but ineffective for high power. Forexample, a 25-mil thick substrate with 10 mils of C u on bothsides and a 0.4" ¥ 0.4" IGBT chip attached to it has a thermalresistance of about 0.30 w/k-m. This means that for an acceptableT j rise of 30°C, the IGBT chip can only operate at 30 to 40A. Thischip is rated at about 75A and so is greatly underutilized.–Thermal expansion mismatch with Si — 6.0 to 7.2 ppm/ºC for Al2O3 vs. 2.8 ppm/°C for Si.–High dielectric constant.–Average chemical resistance ability against acid.•Comments:–More appropriate for low- to medium-power applications.–High-volume and low-cost applications.–Good material for hermetic packages.–For power applications, 99% Al2O3 is a better compromise be-tween cost and performance than the 96%.–If cost is an issue in the high-power application, then a very thin Al2O3 (10 mil) with 8 mils of Cu on both sides may be consideredas an alternative. In this case, the small thickness will compensatefor the low thermal conductivity of Al203. The trade-offs are inthe isolation voltage, V ISO, and in mechanical fragility.3.1.3.2Aluminum Nitride (AlN)•Strengths:–Very good thermal conductivity — Six times that of Al2O3–Very effective for high-power semiconductor applications–Good thermal expansion matching with Si — 4.6 ppm/°C for AlN vs. 2.8 ppm/°C for Si–Mechanical, thermal, and all other electrical characteristics are average but adequate (comparable to those of Al2O3)–Easily machineable–Nontoxic–Nonpermeable to gas–Zero moisture absorption rate–Inert to most chemicals–Camber ~ 0.003"/1"–Dimensionally stable–Relatively new material, so its technology is not as mature as Al2O3 and beryllia.–DBC metallization on AlN has difﬁculties. Thermal fatigue fail-ure is a major concern for DBC AlN substrate.–Thick-ﬁlm metallization process is not as repeatable and reliable as Al2O3.–Four times more expensive than Al2O3.–Poor resistance against alkaline environment — Requires special cleaning agent.–May decompose into hydrated alumina in the presence of high temperature and humidity.•Comments:–One of the best all-around substrate materials for high-power semiconductor applications.–Due to its average mechanical fracture strength, it is preferred to be used in conjunction with a metal base plate. Otherwise, sub-strate thickness must be about 80 mils if it is exposed directly toexternal environment without a metal base plate.–Thermal fatigue capabilities depend very much on the following:•Bonding technique of Cu foil to the AlN substrate•Design of the Cu foil•Substrate thickness•Particular care must be exercised in selecting the right vendors. 3.1.3.3Beryllia (BeO)•Strengths:–Excellent thermal conductivity — Eight times that of Al2O3–Very effective for high-power semiconductor applications–Mature technology–Thermal, electrical, and all other chemical characteristics are av-erage, but adequate (comparable to those of Al2O3)–Easily metallized — Thin ﬁlm, thick ﬁlm, plated copper, DBC, ABC, and RBC–Nonpermeable to gas–Zero moisture absorption rate–Mature technology–Dimensionally stable–Toxic in both powder and vapor form:•Machining, such as cutting, drilling, and shaping, must be handled only by specialty vendors•Environmental issues are a major concern when disposing of the material–Limited suppliers–Thermal expansion mismatch with Si — 7.0 ppm/r C for BeO vs.2.8 ppm/r C for Si–Mechanical characteristics are below average, with mechanical strength only 60% that of Al2O3–Five times more expensive than Al2O3•Comments:–Used by the defense industry–For power semiconductor applications where thermal manage-ment consideration cannot be served in any other way3.1.3.4Silicon Nitride (Si3N4) — Sintered•Strengths:–Excellent thermal expansion match with Si — 3.0 ppm/r C for Si3N4vs. 2.8 ppm/r C for Si–Very strong mechanically:•Mechanical fractural toughness is more than double those of Al2O3and AlN, and triple that of BeO–Good thermal conductivity — Two-and-a-half times that of Al2O3–Effective for high-power semiconductor applications–Thermal and all other electrical characteristics are average but adequate (comparable to those of Al2O3).–Easily metallized — Thin ﬁlm, thick ﬁlm, plated copper, DBC, ABC, RBC–Easily machineable–Nontoxic–Nonpermeable to gas–Zero moisture absorption rate–Creep resistance–Good high-temperature strength–Good thermal shock resistance–Dimensionally stable–New material with relatively immature technology — Use of this material in the power semiconductor ﬁeld is in the infancy stage.–Limited suppliers.–Weak chemical resistance against acidic environment.–Two to two-and-a-half times more expensive than Al 2O 3.•Comments:–Price will come down as usage increases.–Si 3N 4 is probably the best material to be used as a stand-alone substrate * (no metal base plate) for high-power semiconductor applications. It is also conceivable that power semiconductor chips can be attached to the top side of the substrate, with the bottom side having a ﬁnlike structure †formed or machined for the purpose of heat dissipation. In this way, the heat sink can be integrated directly with the substrate without the use of any mechanical force or thermal interface material.21–Good for applications where thermal fatigue capability is highly desired.Table 3.6 presents a list of suppliers for ceramic substrates.* Patent pending.†Patent pending.TABLE 3.6List of Substrate SuppliersSupplier96% Al 2O 399% Al 2O 3AlNBeO Si 3N 4Brush Wellman X X XCarborumdum X X XCeradyne X X XX CeramTec X X XCoors X X XCuramik X X X Denka X X X X Insaco X X KyoceraX X XX Lambertville X X NGK X X XStellar X XWesgo(Morgan Advanced Ceramics)XXXX3.1.4Metallization on Insulating SubstratesFor power semiconductor applications, the metallizations on the insulating substrates should possess the following characteristics:1,2,3,5,12,13•Thermal:–High thermal conductivity (> 200 w/k-m)–Matching thermal expansion with insulating substrate–High thermal fatigue capability — Greater than 1000 cycles from -40 to 100r C with no failures at the interface with the substrate –High thermal stability — Greater than 1000°C in order to be compatible with the direct bonding and brazing operations •Electrical:–Able to conduct high current density–Low electrical resistivity — Typically, ohmic drop across the met-allization should not exceed one-tenth of the IGBT’s V CESAT •Mechanical:–Strong adhesion to the substrate — High peeling strength–Al wire–bondable–Solderable for standard solder (such as 95Pb/5Sn, etc.)–Applicable to the previous four selected insulating substrates —Al2O3, AlN, BeO, and Si3N4–Compatible with standard processing equipment•Chemical:–Photoetchable — Designed pattern can be easily formed (for the case of thick ﬁlm, this is not required)–High chemical resistance against standard processing solvents–Nontoxic–Good corrosion resistance–Chemically inert•Cost:–Low3.1.5 Types of Metallizations3.1.5.1Metallization TechnologiesThe insulating substrate is typically metallized by one of the methods listed in Table 3.7.TABLE 3.7Metallization TechnologiesThick Film Thin Film Copper Metallization Polymer Sputtering Plated copperCermet Evaporation Direct bonded copperGold Gold Active brazed copperSilver Silver Regular brazed copperCopper CopperPalladium silver AluminumPlatinum goldPlatinum silverRefractoryTungstenMolybdenumMoly-manganese3.1.5.2Types of Metallization2,33.1.5.2.1Thick FilmThick-ﬁlm circuits are typically fabricated by screen-printing a specially formulated paste onto a substrate, which is dried and then ﬁred at high temperature. The paste can be in the form of a conductor, resistor, capacitor, inductor, fuse, varister, transient voltage suppressor, or thermistor. Typical ﬁlm thickness is from 0.5 to 2.0 mils.There are three basic categories of thick ﬁlm:• Polymer• Cermet• Refractory3.1.5.2.1.1Polymer — Polymer thick ﬁlm (PTF) is a polymeric resin mixed with conductive, resistive, or insulating particles. It is ﬁred or cured at temperatures ranging from 85 to 300r C, typically from 120 to 165r C. It is low in cost, limited to low-temperature operation, and widely used in plastic or organic substrates.3.1.5.2.1.2Cermet — This is the most popular type and is applicable to both ceramic and Si-based substrates.It consists of a mixture of the following:•Active element — To establish the function of the ﬁlm•Adhesion element — To provide adhesion to the substrate •Organic binder — To provide proper ﬂuid properties for screen printing •Solvent or thinner — To adjust the viscosityThis mixture is ﬁred at a range from 850 to 1000r C.TABLE 3.8Popular Thick Film MaterialsMaterial Resistively, r (m W/ )Gold3Silver1Copper2Palladium silver30Palladium gold50Platinum gold50The adhesion of the thick ﬁlm to the substrate is above average, and the thermal fatigue capability is rated as fair.Popular thick ﬁlm materials are shown in Table 3.8.The maximum current allowed for each type of material can be calculated from the following:•Cross-sectional area of the metallization•Maximum power dissipation allowed for each materialFor example:•Al2O3 substrate — 4 watt/cm2•BeO substrate — 80 watt/cm2•AlN and Si3N4 substrates are somewhere in betweenBecause the typical thick-ﬁlm thickness is 0.5 mil or 12 m m, it can be seen that the maximum current-carrying capability is about a few amperes.3.1.5.2.1.3Refractory — These are special kinds of cermet thick ﬁlm that can withstand high-temperature operations. They are typically ﬁred at a much higher temperature (1500 to 1600r C) in a reducing atmosphere. The adhesion of these ﬁlms to the substrates is strong, and the thermal fatigue capability is rated as very good. Popular refractory materials are tungsten and molybdenum (Table 3.9).The maximum current carrying capability is limited to 1 to 2 amperes for these materials.TABLE 3.9Popular Refractory MaterialsRefractory Material Resistively, r (m W/ )Tungsten22–32Molybdenum153.1.5.2.2 Thin FilmThin-ﬁlm circuits are typically fabricated by ﬁrst sputtering or evaporating the ﬁlm across the entire surface of the substrate. This ﬁlm is then coated, photoprocessed, and etched to form the desired pattern. Thin ﬁlm offers better deﬁnition and narrower lines than thick ﬁlm. It is most suitable for high-density and high-frequency applications. Thin ﬁlm metallization adheres strongly to the substrate and provides excellent thermal fatigue performance. Wire bondability is also superior to that of thick ﬁlm.Due to the labor and specialty equipment involved, thin-ﬁlm circuits are almost ﬁve to six times more expensive than thick ﬁlm. Also, multilayer thin-ﬁlm structure is extremely difﬁcult and expensive to fabricate. Popular thin-ﬁlm materials include the following:•Gold•Silver•Copper•AluminumTypical thickness is about 0.1 mil (2.5 m m) or less. Current-carrying capa-bility is limited to a few amperes.3.1.5.2.3Copper MetallizationBoth thick-ﬁlm and thin-ﬁlm technologies are limited in their thickness, usually to less than 1 mil (25 m m). This limitation affects their ability to conduct large currents.Copper metallization technology provides three features:•Increased metallization thickness•Improved current-carrying capability•Improved thermal spreadingFour basic techniques are available:•Plated copper•Direct bonded copper (DBC)•Active brazed copper (ABC)•Regular brazed copper (RBC)However, as the copper metallization thickness increases, its thermal expansion mismatch with the insulating substrates becomes more and more pronounced, as shown in Table 3.10.This large mismatch will be manifested during thermal fatigue tests, which can potentially lead to excessive thermal stress and eventually to micro-cracks. Proper metallization design and processing techniques must beTABLE 3.10CTE of Insulating Substrates and Copper MetallizationMaterial CTE (ppm/°C)Al2O3(96%) 6.0AL2O3 (99%)7.2AlN 4.6BeO7.0Si3N4 3.0Cu17.0utilized to minimize or eliminate these cracks. This requires careful screening of suppliers.Copper has the additional problems of adhesion and being very chemically reactive. A barrier layer is required to improve adhesion, and a Ni or Au layer is required on the surface for protection.3.1.5.2.3.1Plated Copper — The concept behind this technique is to build up the thickness of copper material by electroplating. A ﬁlm is ﬁrst deposited onto the substrate, either by a thin-ﬁlm method (sputtering or evaporation) or a thick-ﬁlm process (screen-printing). Typical materials used are Moly/ Mn for thin ﬁlm and copper for thick ﬁlm. A layer of electroless copper may be plated over this ﬁlm surface, followed by electrolytic copper to increase the thickness. The plated copper ﬁlm is then ﬁred at an elevated temperature in a nitrogen atmosphere to improve adhesion. The thermal fatigue strength of plated copper is rated as good. One supplier has successfully performed 1000 cycles from -55 to 150r C without failure for 4-mil thick plated Cu. Maximum copper thickness achievable after electroplating is about 5 to 8 mils, with density of about 70% of regular copper material. Fine patterns can be generated by photolithographic etchings. For thicker ﬁlm, this may result in undercutting and loss of resolution. Current-carrying capability has been reported up to 50 A.3.1.5.2.3.2Direct Bonded Copper3,17,18 — This technique uses a high-tem-perature process to achieve an intimate bond between the copper and the ceramic. There is no solder or any other catalyst used in the interface between the copper and the ceramic surface. Here, the combination of copper and ceramic is heated to a temperature of about 1070r C, slightly below copper’s melting point, in a nitrogen atmosphere. At this temperature, the copper oxide forms a eutectic melt that wets and, when cool, produces a strong bond between copper and ceramic.Copper thickness is typically 8 to 20 mils, and patterns can be formed by photolithographic etching. Fine resolution is difﬁcult due to undercutting. Minimum line width and separation are 20 mils. Copper is usually bonded to both surfaces of the substrate in order to balance thermal expansion.Because a layer of copper oxide is required for the direct bonding process, AlN and Si3N4 ceramics must be treated ﬁrst in order to apply this technique. This involves an additional oxidation process at about 125°C. However, this added oxygen diffuses along grain boundaries and may downgrade the thermal conductivity of AlN and Si3N4.Direct bonded copper offers the following advantages for power packag-ing:•The copper and ceramic tend to act, often bonding, like an integral unit with a single coefﬁcient of thermal expansion. This coefﬁcientis much lower than that of pure copper and more closely matchedto that of the ceramic. It is therefore possible to solder even largechips directly onto the copper layer without risking stress damages.•The copper used is of the high-purity, oxygen-free high-conductivity (OFHC) type. With proper line width and thickness, the metalliza-tion can be of very low electrical resistance and can handle currentwell in excess of 100 A.•The thick layer of copper provides efﬁcient heat spreading from the power chips.3.1.5.2.3.3Active Brazed Copper — The active brazed copper process uti-lizes brazing alloy to form a bonding layer between copper and ceramics.3,16 An example is the alloy of silver, copper, titanium, and zirconium in the ratio 72Ag/28Cu/3TiH2/3Zr.The braze is usually applied in the form of a paste. Most suppliers use the screen-printing technique. The resulting titanium braze layer provides supe-rior adhesion and thermal cycle performance.Active brazed copper has been applied extensively to AlN and lately to Si3N4 substrates, where DBC techniques have experienced difﬁculties. Dur-ing direct bonding operation on these ceramics, oxygen gas must be fed into the critical interface region. This added oxygen will diffuse along grains and reduce the thermal conductivity of the ceramics. Furthermore, the release of gases during the direct bonding process may require a perforated C u foil design. However, this results in a loss of conduction area for current. The advantages of using the ABC process over DBC for these two substrates are as follows:•Better adhesion•Better current-carrying capability•Better thermal-fatigue capability3.1.5.2.3.4Regular Brazed Copper — The technique is similar to that of the active brazed copper, except that it is performed in a vacuum. In this case, typically a thin silver ﬁlm is ﬁrst sputtered onto the substrate. Ag-braze alloy is then applied to the ﬁlm in the form of a paste, and the copper foil is placedon top. The combination is then heated to the melting point of the braze alloy. After brazing, the top foil may be photoetched to form the desired pattern.One supplier noted that the titanium-based brazing alloy is overly active and can potentially lead to nonuniform brazing. Regular Ag-based braze is more controllable and therefore results in a more uniform and stronger adhesion.Table 3.11 and Table 3.12 compare different metallizations available for the insulating substrates.For a conductor of 40 mil width and 12 mil thickness on an Al 2O 3 substrate,the temperature rise for a 100 A operating current is about 17°C. From Table 3.11, we can conclude that the metallizations for high-power semiconductor applications are:•Direct bonded copper (DBC)•Active brazed copper (ABC)•Regular brazed copper (RBC) •Plated copper (possibly)An estimate of the total cost of the insulating substrate per square inch with metallizations at thickness of 25 mils is as shown in Table 3.13.3.1.6Analysis of Copper Metallization SuppliersIt should be noted that the preceding description of the technology is very general. Every supplier has its own unique processing features and design techniques that can affect performance, especially thermal fatigue and cost.Table 3.14 through Table 3.17 provide lists of suppliers and their capabilities.This information is gathered from publications, conferences, or private con-versations.TABLE 3.11Metallization for Different Insulating SubstratesTechnology Al 2O 3AlN BeO Si 3N 4Thin ﬁlm Good Good Good Good Thick ﬁlm Good FairGood FairPlated copper GoodGoodDB C Good GoodABC Good Good RBCGood GoodTABLE 3.12Comparison of Metallization Technologies4,5Technology Adhesion Geometry Process Electrical ThermalFatigueProcessCostThin ﬁlm Good0.002 line< 0.005 thick Good wirebondability;fair solderability1–2 A Good HighThick ﬁlm Fair0.010 line> 0.005 thick Good wirebondability;good solderability2–5 A Fair LowPlated copper Fair0.004 line0.005 thick Good wirebondability;good solderability50 A Fair MediumDBC Good0.020 linetyp 0.020 thick Good wirebondability;good solderability> 200 A a Good forAl2O3andBeOLowABC, RBC Good0.020 linetyp 0.020 thick Good wirebondability;good solderability> 200 A Good for AlNand Si3N4Lowa For a conductor of 40 mil width and 12 mil thickness on a A12O3 substrate, the temperature rise for 100 Amp. operating current isabout 17°C.© 2005 by CRC Press LLC。

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Modic改变的病理学意义和Modic改变与下腰痛和腰椎节段不稳的关系
主要内容
1 2 3 4 5 6 7 概述流行病学鉴别诊断
病理及发病机理
Modic改变与下腰痛 Modic改变与节段不稳自然病史
一、SUMMARY 终板和椎体的Modic改变自从首次被描述已经 20多年了，然而Modic改变的意义还是一个有争议的问题。它和腰椎的正常退变紧密相连，其发生率也随着年龄的增加而增加。Modic改变的发病机制和它与下腰痛和腰部脊柱部分不稳的关系至今尚不完全清楚。本篇文章回顾相关文献和讨论当下可用的相关证据来探讨一下 Modic改变的发病机制和它与下腰痛和腰部脊柱部分不稳的关系这个问题。
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1型改变代表较强烈的炎症反应过程，而2型和3型改变则代表相对静态的过程
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五、Modic改变和下腰痛
在椎间盘退变过程中，与下腰痛联系起来的临床检查中， Modic改变是很重要的组成部分：
1型改变与下腰痛联系最紧密
2型改变与下腰痛联系程度相对较小 1型改变转变为2型改变，临床症状改善 1型改变的范围变大，临床症状加重
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三、鉴别诊断 1型Modic改变vs椎间隙的感染
影像鉴别
• T2显示正常或者低信号 vs 高信号 • 终板完整vsLeabharlann 板破坏 • 脊柱旁和硬脊膜有无炎症改变
临床表现实验室检查
• 红细胞沉降率 • C反应蛋白（100%）
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四、病理及发病机制
1型改变与终板的破裂和县围绕肉芽组织的形成有关，代表着腰椎间盘退行性疾病的炎症期，提示退变过程正在进行 2型改变与红骨髓的脂肪退变有关，代表着腰椎间盘退行性疾病的脂肪期（fatty stage），它与比较稳定的和慢性的病理过程相关
腰椎融合的一些回顾性研究得出以下结论：
• 腰椎融合术后若存在1型改变，那么说明有假关节形成
• 对1型改变的患者行融合术预后较好
• 融合术可以加速1型改变的进程，这可能由于融合术纠正脊柱生物力学的不稳
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七、自然病程 Modic改变的不同型的改变可以相互转换，这些类型的改变代表着同一病理过程的不同时期，而混合型的改变代表转换为真正的一类型的之前的变化
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二、流行病学
腰椎间盘退行性疾病的患者中，Modic改变的发生率在 19%～59%，其中1型改变和2型改变最常见，3型和混合型相对少见，对于1型改变和2型那个更常见这个问题上，意见不一； Modic改变最常见于L4-L5和L5-S1，和年龄相关；
没有椎间盘退行性疾病的患者中，Modic改变比较少见（9.6% 3～10%）；
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五、Modic改变和下腰痛
对患有慢性下腰痛的病人行椎间盘造影时，Modic改变对疼痛再现有非常高的预测价值（88%-91%）和特异性（96%-96.8%），其他的MR检查则不行：
中等的严重的Modic改变预测价值和特异性可以提高到 100% 由于他们研究样本量的限制，当下对Modic改变和下腰痛，还不能得出任何结论
3型改变代表腰椎间盘退行性疾病的硬化期
– 这些改变可能代表椎骨对涉及椎间盘退变过程的反应
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四、病理及发病机制
不断的椎间盘损伤导致髓核产生炎症介质，这些毒性炎症介质可以渗透终板导致局部炎症反应，而这会导致下腰部疼痛的出现受损的终板的血管密度和感觉神经纤维数量的增加，可以加强椎间盘的营养，也可以导致下腰痛症状促炎症介质如白介素-6、白介素-8、PG-E-2可能是导致下腰痛的主要因素
1型改变是不稳定的，可以转换为0型或者2型 2型改变随着时间的推移相对稳定
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七、自然病程
2型改变的稳定性受到质疑：
• 2型改变的稳定性隙要比我们先前认为的要低，猜测在2 型改变的患者中，持续性急性炎症作用导致黄骨髓替代红骨髓 • 腰椎间盘突出是诱发Modic改变的强危险因素 • 1型和2型改变交换着引发腰痛症状，诱发能力相近
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六、Modic改变与节段不稳
Segmental hypermobility（节段运动过度）
• 屈伸胶片上，失状面上平移大于或等于3mm
1型Modic改变提示关节不稳
关节不稳会导致1型Modic改变有1型改变的患者更易有节段不稳
需要做关节融合
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六、Modic改变与节段不稳
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八、结论
1型Modic改变可能是炎症诱发，与下腰部症状和腰椎节段不稳紧密相关，反应腰椎退变和腰椎生物力学上的不稳定，这改变提示腰椎融合预后非常好 2型Modic改变和下腰痛的相关性较小，表示生物力学上的稳定状态 3型Modic改变的确切本质和发病机理的重要性我们还知之甚少。
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Classification
Classification of Modic changes on MR:
T1WI Type 0 Type 1 T2WI association 正常解剖表现水肿、炎症 F1 F2 F3
= ↓
= ↑
Type 2
Type 3
↑
↓
=/↑
↓
黄骨髓代替红骨髓
椎骨骨硬化