还是Maratea，我又来啦，每隔2年来一次，这是第三次到意大利拿破勒斯附近的山区来开会，这次酒店又爆满~(≧▽≦)/~啦啦啦，学生甚至都要4个人住一间，very hot。

按惯例，第一天是教育辅导类的讲座，共4个报告。

第一个报告

Metals: Elemental Physiology and Pathogenesis

Aaron Barchowsky University of Pittsburgh

只是讲了镁和锌，和一张幻灯关于La

All things arepoisonous, only the dose makes it non-poisonous

Paracelsus 1564

Mg Essential for VitaminD metabolism that regulates Ca uptake and bone deposition

Excess of Mgcontaining drugs (antiacid)

Occupational (MgO):metal fume fever

Mg toxicity can bereversed by Ca

Mg promotes bonedevelopment and repair

Of the total Mg inthe body, half is in the bone as an essential structural element in the HA bonematrix and as cell associated pool for homestasis.

Mg promoteosteoblast differentitation(dose dependent) and inhibits osteoclasts

MgSO4 NaSO4 (ActaBiomater 10: 2834-42, 2014)

Brandao ImmunolRes (2013) 55: 261-269

Ion selectivity

pH sensitivity

Mg2+/Mg-ATPinhibition

TRPM6/7 channelsare regulated by receptors

Krapivinsky Cell157:1061-72 2014

Wrighton Nat RevMol Cell Biol 15: 417 2014

Mg promotesendothelial cell migration

Keedler and Ham Invitro Cell Dev Biol 23: 481-491, 1987

Unfortunately manytumors are have a growth advantage due to Mg

Lapidos et alAngiogenesis 4: 21-28, 2001

Zn: the elementalcoordinator

Zn coordinatedproteins and enzymes include DNA/RNA binding proteins, proteases, kinases,channels,…

Zn is the onlyelement to appear in all enzyme classes

Zn excess impairedCu and Fe adsorption

Zn channels andtransporters: Mammary Gland Biol Neoplasia 19: 59-71, 2014

Free Zn is nothealthy Zn

The intracellularconcentration of Zn is 200microM, but the amount of free Zn is PM to low nM.

Zn promotes boneformation by promoting osteoblast differentiation and activity suppressingosteoclastogenesis and resorption.

Regulate ALPactivity and stability

Zn in signalingmolecules is essential for VEGF.

La

Generally viewedas non-toxic in the amounts used in absorbable alloys

Used in mMconcentrations as MRI contrast dyes and phosphate binder in renal disease

第二个报告

Challenges for the investigation of biometal degradationin vitro and in vivo by analytical methods

Carla Vogt

Overviews:scientific tasks

Optimizedsynthesis, mechanical stability

Multielementanalysis for main, minor and trace components

Investigation ofgradients due to degradation processes(2D,3D,4D)

Interaction withcells and biomolecules

Selected analytes:Mg, Zn, Al, Li, Y,La, Ce, …Ca, P, Fe

Coatings:degradable polymers, HA and CaP fluorides, proteins

Sample forms

Matrix:Liquids-human fresh plasma, real body fluid

, solids-bodycomponents(organ bone), solid corrosion products

Gases-evloved during corrosion, gas introducedinto

Analytical methods

Selection criteria

Deliveredinformation/selectivity

Limits ofdetection/sensitivity

Multielementcapabilities

2D or 3D capabilities

Resolution inx-y(and z)

Availability

Money

Sample amount,sample demage

Risk ofcontamination

Access toreference materials(matrices liver, kidney, muscle, brain, bone, serum,plasma…)

Available Methods

(1)SEM-EDX

Topographic informationby SEM

Elementalcomposition by BSE and EDX

Solid degradationproducts

Phase composition

adherence of cells

Problem:normalized brightness in mapping images-not proportional to conc.

Technicalrealization:

Cryo-TEM/SEM

AtmosphericSEM(ASEM)

Fluid cell TEM

NatureNanotechnology 6 (2011) 695

J. Struct. Biol.183(2013) 270

(2) X-raydiffraction

Solid degradationproducts

Phase composition

crystallinity

problem: in databases often insufficient information about REE

WE43 in DMEM:Mg(OH)2+MGCO3, no MgCl2

In HBSS:Mg(OH)2+MgCL2+unknown, no MgCO3

Formation ofproducts determined by the medium, only minor influence by alloy composition

Internationalregulation/standardization of experimental setup is missing

(3) XRF x-rayfluorescence spectroscopy

Elemental distributionacross sample

Enrichment effects

Solid degradationproducts

Problem: noelements with Z<11; LOD vary by 2 orders and depend on technique

Acta Biomater 9(2013) 861

XANES-x-rayabsorption near edge spectroscopy

EXAFS-extendedx-ray absorption fine structure

Oxidation states

Bonding partners

Crystal structure

Magnesium 2009,Wiley-VCH, p.1163-1174, Vogt et al.

(4) PIXE particleinduced x-ray spectrometry

Elementaldistribution across sample

Enrichmentseffects

Solid degradationproducts

Very expensive, noelements with Z<11, better resolution than XRF

NIM B 181(2001)511, 516, Reinert, Reibetanz, Butz

Magnesium 2009,Wiley-VCH, p.1163-1174, Vogt et al. (micro-PIXE)

(5) RBS Rutherfordbackscattering

Elementaldistribution within top 1-2 micrometer of a sample

Layerthickness(nm)

Character andposition of lattice defects

Problems: lowsensitivity for light elements

Acta Biomater7(2011) 2751 Necula et al.

(6) SIMS secondaryion Mass Spectrometry

Distribution ofelements and molecules within a sample

Surface layercomposition(sub-nm)

Character andposition of lattice defects

Alloy LEA442 inbone after several weeks of degradations

Polymer coating onstent

(7) ICP-MSinductively coupled plasma MS

Elementconcentrations from mg/g to ag/g

Sampledigestion-method applicable to most media(blood, urine, alloys, bone, liver)

Difficulties inhandling high salt lads or higher amounts of organics

Let assume to put1g Mg impant in a 1Kg rat

Let assume thealloy contains 10% of aln alloy element 0.1g/Kg(100ppm)

Much morerealistic 1mg of Mg implant in a 1 kg rat 0.1mg/kg(100ppb)

LA-ICP-MS

Elementconcentration for trace elements

Elementaldistribution in x-y-z

JAAS 24(2009)181-186 Gruhl, Witte, Vogt

98Y  90Zr

(8) XPSphotoelectron spectroscopy

Electronicstructure of elements

Bonding forms

Adsorbed layers onsurfaces

Surfacesensitive(0.1-1nm)

Sold degradationproducts after 14 days treatment of alloy E11 in HBSS

Correlation withresults from IR and XRD

(9) IR Infraredspectroscopy

Structure oforganics and inorganics

Mapping ofdistribution

MW of polymers

FTIR spectra ofcorrosion products for Mg alloys

Magnesium 2009,Wiley-VCH, p.1163-1174, Vogt et al.

IR spectrum for a5X5 micrometer with protein

(10) Raman

Structure oforganics and inorganics

Mapping ofdistribution

Phase transitions

PDLLA coating onMg alloy stent

Raman Spectrosc.Vano Misiun, Vogt (in press)

Embedding materialPMMA

implant-PDLLA-PMMA

测试的影响因素

tips for pipette 会带来Al的污染，use colorless tips

polishingpowders—artefacts in solid state spectroscopy

Cutting tools,enhanced values for trace elements (ceramic knife)

Certifiedreference materials

Doped biomaterials,normalized to 13C or other isotopes pf the sample

Matrix matchedreference materials with internal standards

Agarose gels

Polymer films

Synthetic compositematerials

第三个报告

Evaluation of Biocompatibility UsingIn Vitro Methods: Interpretation and Limitations Arie Bruinink, EPMA

Adv BiochemEngin/Biotechnol (2012) 126: 117-152

BiocompatibilityCytotoxicity (sub) toxicity

ISO 10993-5

Colonization

Multiple celltypes contact with the implant

Co-culture

The populationsize affected by other cell types.

Surface chemistryaffect cell-population dynamics

Events afterimplantation of a biomaterials in soft tissue

A material can perdefinition by itself NOT be biocompatible, but only in the context of a definedapplication and defined surface design.

Biodegradablematerials based on Mg, Zn, Fe

用MgCl2测，Mg2+ is cytotoxic> 30mM用Mg2+ in NaCl测则无毒。

Leidi et al, 2011Magneisum Research

Lin et al, 2010.Br. J. Anaest 104, 44-51 MgSO4 on Anti-inflammatory

Effect ofparticulated Mg Colloids & Surface B interface 88(2011)471-486

第四个报告

IRON METABOLISM IN HUMANS

Jeanette Maier

Universita di Milano

Iron distributionMale 2-6 g, Female 2 g

Function: Otransportation&storage, electron transport& energy metabolism,…

Fe is essentialfor cell proliferation

Fe is absolutelynecessary for respiration

Fe homeostasis

Fe adsorption

Fe uptake

Iron-regulatingprotein are Fe sensors and coordinate the regulation of the synthesis offerritin and transferrin

Ferritin ---Majoriron storage protein, can contain up to 4000 Fe atoms, liver contain 60%Ferritin in our body.

Transferrin

HFE-beta2microglobulin interacts with transferrin

Fe is released andtransported in the cytosol by DMT

Ferroportion, theonly cellular Fe exporter

Which cellsrelease iron in the blood?

Who is playing thecentral role in iron metabolism?

HEPCIDIN

Hepcidin bindsferroportin, induces its internalization and degradation reduced release of Feby enterocytes and macrophages.

Iron deficiency

Iron overload