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Surface Modification of Hydroxyapatite

Surface Modi?cation of Hydroxyapatite Nanoparticles with Thermal-Responsive

PNIPAM by ATRP

Junchao Wei,Pan He,Aixue Liu,Xuesi Chen,*Xianhong Wang,Xiabin Jing

Introduction

The surface modi?cation of nanomaterials including hydroxyapatite (HA),silicon,silver,and iron oxide has

been carried out to tune the surface properties such as wettability,adhesion,and surface activity.[1–8]Recently,much work has been focused on the construction of intelligent surfaces with novel properties for perspective applications in arti?cial organs,biofunctional materials,drug delivery systems,bio-and chemosensing,bioelec-tronics,regenerative medicine and the microstructure of organic,polymeric,and hybrid materials and surfaces.[9–12]Among many methods,the connection of stimuli-responsive polymers on the surface of nanomaterials has attracted much attention.One of the most widely researched stimuli-responsive polymers,poly(N -isopropylacrylamide)(PNIPAM),is a typical thermal-responsive polymer and is character-ized by a sudden precipitation on heating and switch from hydrophilic to hydrophobic with a phase transition temperature around 328C.[13]

Full

Paper

J.Wei,P.He,A.Liu,X.Chen,X.Wang,X.Jing

State Key Laboratory of Polymer Physics and Chemistry,

Changchun Institute of Applied Chemistry,Chinese Academy of Sciences,Changchun 130022,P.R.China

Graduate School of Chinese Academy of Sciences,Beijing 100039,P.R.China

Fax:t86-431-85262112;E-mail:xschen@https://www.wendangku.net/doc/5813395580.html,

:Supporting information for this article is available at the bottom of the article’s abstract page,which can be accessed from the journal’s homepage at http://www.mbs-journal.de,or from the author.

Hydroxyapatite (HA)nanoparticles grafted by poly(N -isopropylacrylamide)(PNIPAM)brushes (PNIPAM-g -HA)have been synthesized by the surface-initiated atom transfer radical polymerization (ATRP)of N -isopropylacrylamide (NIPAM).The surface grafting amount of PNIPAM ranges from 15.5%to 46.4%.PNIPAM-g -HA has been characterized by FT-IR spec-troscopy,thermal gravimetric analysis,X-ray diffraction,and scanning electron microscopy (SEM).The UV transmittance spectra and the particle size analysis of PNIPAM-g -HA in aqueous solution demonstrates that the PNIPAM-g -HA possess reversible thermal stimuli responsive properties.An in vitro bioactivity assessment indicates that PNIPAM-g -HA can induce the

mineralization of Ca 2tand HPO 2à

4and possesses an excellent bioactivity.The cell culture results show that the cells adhered to the surface of PNIPAM-g -HA grow better than on HA,and the area of the cells on the surface of PNIPAM-g-HA is much greater than for HA,which proves that the PNIPAM-g -HA has a better biocompatibility than

HA.

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In a conventional cell cul-ture process,cells are attached to the surface of substrates,and they prolifer-ate and produce the extracel-lular cell matrix(ECM).When cells are detached from the substrates,trypsin and ethylenediaminetetraacetic acid(EDTA)are used,which always disrupt the ECM and

crucial cell surface proteins.In comparison with conven-tional cell culture substrates,thermal-responsive PNIPAM surfaces for the culture of cells can be covered with con?uent cell sheets.The detachment of cells from these thermo-responsive PNIPAM surfaces can be done simply by lowering the temperature thereby the newly formed ECM can be kept intact and the use of deleterious protease can be avoided.[14–18]Therefore,PNIPAM has been used to construct thermal-responsive surfaces or used as ther-mal-responsive substrates in the tissue engineering?elds to work as a molecular switch to tune cell adhesion(ON)and cell detachment(OFF).[17–20]

HA is an important material used in hard tissue repairs and replacements because of its similarity to bone minerals in composition,crystallinity,and morphology.[21–23]Thus HA has been widely used to prepare arti?cial bone-like ceramic/polymer composites.Up to now,there have been many methods used to tune the surface properties of HA nanoparticles.Many materials,such as silane coupling agents,[24,25]zirconyl salts,[26]polyacid,[27]isocyanates,[28] poly(caprolactam)(PCL),[29]poly(ethylene glycol methacry-late phosphate),[30]chitosan,[31]Arginine-Glycine-Aspartic acid(RGD)containingpeptide,[32]poly(g-benzyl L-glutamate) (PBLG),[33]and poly(L-lactide)(PLLA)[34]have been tethered to the surface of HA nanoparticles to modify the surface properties.However,all these methods mainly focus on tuning the surface wettability to change the surface adhesion ability of the nanoparticles with the polymer matrix.Shin et al.[35]have prepared a hybrid temperature-responsive HA-PNIPAM gel by the interpenetration of PNIPAM into a sintered HA disc through radical-initiated polymerization of NIPAM,and the hybrid gel can work as a sustained drug release system.Chang et al.[36]proposed a method to coat a silici?ed L3-PNIPAM gel over porous HA and prepared a functional scaffold.However,these two studies focused on the bulk HA materials.As for the surface modi?cation of HA nanoparticles,to the best of our knowledge,there have been few reports about thermal-responsive PNIPAM surfaces constructed on the surface of HA nanoparticles.

In this study,thermal-responsive PNIPAM was con-nected to the surface of HA nanoparticles by a surface initiated ATRP method(Scheme1),and a type of new PNIPAM-grafted HA hybrid material(denoted as PNIPAM-g-HA)was synthesized.PNIPAM-g-HA possesses a smart surface and shows thermal responsive properties.In addition,the hybrid materials also have wonderful bioactivity and biocompatibility,and show potential applications for cell culture in tissue engineering. Experimental Part

Materials

Calcium nitrate tetrahydrate[Ca(NO3)2á4H2O],diammonium hydrogenphosphate[(NH4)2HPO4],and25%ammonium hydroxide (NH3áH2O)were purchased from Beijing Chemical Co.Ltd. 3-Aminopropyl-thiethoxysilane(APS)was purchased from Nanjing Shu Guang Chemical Group Co.Ltd.2-Bromoisobutyl bromide was purchased from Shanghai Jingchun reagent Co.Ltd.N-Isopropylacrylamide(NIPAM)was obtained from Sigma–Aldrich, recrystallized in hexane,and dried under vacuum before use. Cuprous bromide and2,20-dipyridyl(bpy)were purchased from Sinopharm Chemical reagent Co.Ltd.

Synthesis of ATRP Initiator on the Surface of HA Synthesis of HA

The synthesis of HA was carried out based on ref.[37]Brie?y, 500mL of a1M Ca(NO3)2á4H2O aqueous solution was adjusted with NH3áH2O to pH10,and the solution was heated to908C.A volume of500mL of0.6M(NH4)2HPO4at pH10(adjusted with NH3áH2O)was then added dropwise under stirring.The precipitate was maintained in the reaction solution for5h at 908C,and then it was centrifuged at10000rpm for10min and repeatedly washed with distilled water.The obtained HA nanoparticles were lyophilized.

Synthesis of Amino-Functionalized HA(HAAPS)

The synthesis of amino-functionalized HA was based on a method previously proposed by us.[33]Brie?y,APS(0.442g)was added into a mixed solution of180mL of ethanol and20mL of water.The system was stirred for half an hour and then HA(2.0g)was added to the system.The mixture was then stirred for another3h.The pH of the system was adjusted to9–10with NH3áH2O,and the reaction was continued for another3h.After?ltration,the powder was ?rst

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Jing

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dried at room temperature and then cured at1308C to strengthen the silane coating by formation of a polysiloxane network structure.

Synthesis of2-Bromobutyrate-Functionalized HA(HABr)

HAAPS(1.0g)was dispersed in20mL of N,N-dimethylformamide (DMF),and a2-bromoisobutyryl bromide solution(1.0g dissolved in10mL of DMF)was then added dropwise slowly at room temperature.The reaction system was stirred at room temperature for5h.The nanoparticles were isolated by centrifugation and washed three times with DMF and then washed with chloroform twice.The product was dried under vacuum at room temperature. The synthesis route is shown in Scheme1.

Surface-Initiated ATRP of NIPAM

HABr(100mg),NIPAM(480mg),and bpy(12.3mg)were added into 20mL of deionized water in a reaction bottle equipped with a magnetic stir bar.The HABr nanoparticles were dispersed by sonication,and then N2was bubbled into the water for half an hour so that all the O2was removed from the water.The catalyst CuBr (5.4mg)was added into the reaction bottle under N2atmosphere and then the reaction bottle was sealed and stirred at room temperature.After a predetermined time interval,the solid product was collected by centrifugation,and the raw product was washed with water for at least5times by a redispersion process.In the end, the product was lyophilized.

Assessment of in vitro Bioactivity

The in vitro bioactivity of PNIPAM-g-HA was carried out in simulated body?uid(SBF)proposed by Kokubo et al.[38]The ionic composition and concentration of SBF were similar to that of human blood plasma.In the experiment,30mg of PNIPAM-g-HA1, PNIPAM-g-HA6,and HA(control sample)were dipped into30mL of the SBF solution in a50mL polyethylene bottle covered with a tight lid.The bottles were then placed in a shaking box at378C without refreshing the SBF solution.After different time intervals,100m L of liquid was removed from the bottle and analyzed by inductively coupled plasma–mass spectrometry(ICP-MS)to determine the concentrations of Ca and P.

Cell Culture and Morphology Analysis

The PNIPAM-g-HA1,PNIPAM-g-HA6,and pure HA(control sample) were dispersed in deionized water with a density of1wt.-%, respectively.The suspension(50m L)was coated onto a clean cover slide(24mm?24mm)and then dried under vacuum.The prepared specimens were sterilized by UV light for30min.

Marrow stromal cells as stem cells were obtained from the tibia of a mature rabbit.The cells were rinsed three times with0.1M phosphate buffered saline(PBS)by centrifugation and then were cultured in the cell culture?asks with a cell culture medium(DMEM/F12?1:1)supplemented with10%FBS(GIBCO), 10?10à3M HEPES(Sigma),60mgáLà1penicillin(Sigma),and 100mgáLà1streptomycin(Sigma).The culture density was 2.0?104mLà1,and the environment was a humidi?ed incubator at378C and5%CO2.The culture medium was changed every2d.After10d of culture,the monolayer cells were removed from the cell culture?asks by trypsin(2.5mgámLà1)and EDTA (0.2mgámLà1)(1:1,v/v)treatment,and the cells were rinsed three times with0.1M PBS by centrifugation at1000rpm for 5min.

The obtained marrow cells were resuspended in the medium and the cell density was adjusted to1.0?105mLà1.The cells were seeded on cover-slides coated with PNIPAM-g-HA1, PNIPAM-g-HA6and HA,respectively,in6-well cell culture plates(Coster)at a density of1.0?105cells per well,and the materials were sterilized by UV light for20min before cell seeding.A volume of3mL of the medium was then added to each well and mixed with the cells homogeneously.The cells were cultured in a humidi?ed incubator containing5%CO2at 378C for24and48h,respectively.

After a predetermined time interval,the cover-slides were washed three times with PBS,and?xed with3%glutaraldehyde in PBS for8min.After thorough washing,the cells were dyed with ?uorescein isothiocyanate(FITC).The cell attachment and the cell morphology were observed under a reverse microscope(TE2000U, NIKON)and nine pictures at different spots of the cover slide were taken by using a Digital Camera DXM1200F(NIKON).The NIH Image J software was used to analyze the pictures,and the area fractions of cells in each cover-slide were obtained.Statistical analysis(n?9)of the area fractions was performed using descriptive statistics with Origin.

Characterization

The amount of surface-grafted PNIPAM on HA was determined by thermal gravimetric analysis(TGA)using an SDT Q500 thermogravimetric analyzer(TA instruments,Delaware,USA). The TGA measurements were carried out from room tempera-ture to8008C at a heating rate of208Cáminà1.FT-IR spectro-scopy was performed on a Bruker Vertex70FTIR spectrometer and the spectra were collected at64scans with a spectroscopic resolution of2cmà1.The wide-angle X-ray diffraction(WAXD) data from108to708of the samples were measured using a Rigaku D/max2500kVPC X-Ray Diffractometer with a Cu tube anode.The morphology of HA and PNIPAM-g-HA was observed by using a scanning electron microscope(XL30ESEM FEG, PHILIPS).The optical transmittance of the hybrid nanoparticles was acquired on a UV/Vis spectrometer(Shimadzu UV2401-PC) equipped with a temperature controller(Shimadzu S-1700)at a heating rate of0.58Cáminà1.The particle size of PNIPAM-g-HA was measured by a Particle Size Analyzer(Brukehaven90plus). The elemental concentration was analyzed by ICP-MS(TJA POEMS-I).

Results and Discussion

Synthesis of ATRP Initiator on the Surface of HA

HA nanoparticles were synthesized according to the reported method,and the amino-functionalized HA nano-particles were obtained by treating HA with APS according to the reported method.[33]The rod like HA nanoparticles

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were20–40nm in width and100–200nm in length. 2-Bromoisobutyrate-functionalized HA(HABr)nanoparti-cles were prepared by the reaction of the amino-functio-nalized HA with2-bromoisobutyryl bromide.As shown in Figure1b,two weak peaks at1388and1369cmà1are the characteristic deformation vibrations of the two methyl groups of2-bromoisobutyrate residues,which can prove that2-bromoisobutyrate-functionalized HA was synthe-sized.Furthermore,the presence of bromide on the surface of HA was quantitatively determined by ICP-MS, and the results show that the surface density of bromide was13.8mgágà1.In addition,as shown in Table1,there was about1.6%weight loss difference between HAAPS and HABr,which also demonstrated the successful connection of2-bromoisobutyrate on to the surface of the HAAPS nanoparticles.

Surface-Initiated ATRP of NIPAM

Although some reports introduced the use of Cu II in the process of ATRP to ensure an exchange between the dormant and active species,[39]there are also some reports that show it is not necessary to add Cu II for the ATRP of NIPAM.[40,41]The surface initiated ATRP of NIPAM on the surface of HA nanoparticles was conducted in water at room temperature with CuBr/bpy as the catalysts,with-out addition of Cu II,however,the reaction proceeded well, and this may be because the high content of2-brosiobutyrate on the surface of HABr was able to generate enough organic radicals and Cu II to make the reaction go well.The product was washed with water several times so that all the physically adsorbed reagents on the surface of the HA nanoparticles were washed away,and only the polymer chains that were covalently connected could exist on the surface of the HA nanopar-ticles(the product is denoted as PNIPAM-g-HA,see Table 1).In order to demonstrate the absence of non-grafted PNIPAM on the surface of PNIPAM-g-HA,the water used to wash the product was collected and its UV transmit-tance spectra were measured.After?ve washes,the results demonstrated that no PNIPAM was present in the water,which shows the absence of non-grafted polymer on the surface of PNIPAM-g-HA(see Supporting Information 1).

J.Wei,P.He,A.Liu,X.Chen,X.Wang,X.

Jing Figure1.IR spectra of HA(a),HABr(b),PNIPAM-g-HA1(c),and

PNIPAM-g-HA6(d).

Table1.TGA results of HA,HAAPS,PNIPAM-g-HA,and their related parameters.(In the parts of IR characterization,TGA curves,SEM images, XRD curves,assessment of in vitro bioactivity and biocompatibility test,the samples PNIPAM-g-HA1and PNIPAM-g-HA6are used as representatives.)

Samples Polymerization

time

Weight

loss(W)

Weight

residue(W r)

Grafting amount

(M)

h%%% HA– 2.2––

HAAPS– 5.792.0 3.8

HABr–7.390.6 5.6

PNIPAM-g-HA0.50.514.679.815.5

PNIPAM-g-HA1117.976.520.5

PNIPAM-g-HA2221.871.427.5

PNIPAM-g-HA3324.668.932.5

PNIPAM-g-HA4427.865.738.9

PNIPAM-g-HA6631.563.146.4 1240

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The FT-IR spectra of HA,HABr,PNIPAM-g-HA1,and PNIPAM-g-HA6are shown in Figure1.When compared with the spectra of bare HA,some important new peaks appear in the spectra of PNIPAM-g-HA1(Figure1c)and PNIPAM-g-HA6(Figure1d).The peaks at1652and 1547cmà1are ascribed to the amide I band(stretching vibration of C?O)and amide II band(stretching vibration of NàH).Both the peaks at1388and1369cmà1with the same intensity are the characteristic deformation vibration peaks of CàH bonds in the methyl groups of isopropyl. The IR results con?rmed the existence of PNIPAM on the surface of the HA nanoparticles.

The surface grafting amount of PNIPAM on the surface of the nanoparticles was measured by TGA.Figure2shows the TGA curves of HA,HAAPS,HABr,PNIPAM-g-HA1,and PNIPAM-g-HA6.There are two steps of weight loss.The?rst stage is before2008C,and the weight loss is due to the evaporation of the adsorbed water on the surface of samples;the second stage is after2008C,and the weight loss is ascribed to the loss of water adsorbed or contained in the nanoparticles.As for the other samples,the weight losses were mainly a result of the decomposition of organic layers on the surface of HA.According to the TGA curve of PNIPAM-g-HA6(Figure2e),there is about4.3%weight loss before2008C ascribed to loss of adsorbed water,and the weight loss of31.4%in the range of200to8008C(W p)is because of the decomposition of polymer chains.The weight retention(W r%)at8008C is63.1%.The surface grafting amount of organic layers(M%)is de?ned by the following equation:

M%?W pàW0

?100%

where W0is the weight loss of HA and,M represents the mass of organic layers connected on100g of HA.The calculation process was based on the literature.[42]With the difference in reaction time,surface-modi?ed HA with different grafting amounts were synthesized and the results are listed in Table1.

Morphology and Crystalline Results

Surface-initiated ATRP is an ef?cient way to realize the covalent connection of polymer chains on the surface of inorganic particles.However,as a surface modi?cation route,the chemical reaction should not change the bulk properties or original crystalline state and intrinsic proper-ties of the HA nanoparticles.Powder X-ray diffraction(XRD) was used to characterize the crystallinity of HA,HABr,and PNIPAM-g-HA.As shown in Figure3,the characteristic diffraction peaks of the(002),(102),(211),(300),(202),(310), (222),(213),and(411)crystal planes for HA almost remain the same in the XRD curves of HABr and PNIPAM-g-HA.This result shows that the ATRP took place on the surface of the nanoparticles,and after the reaction,the inorganic part of the PNIPAM-g-HA still retained its original crystal structure. However,there was line broadening of the(102),(210), (211),and other crystal planes in the XRD patterns of PNIMAM-g-HA1and PNIPAM-g-HA6(Figure3c and3d), which may be a result of the preferential interaction of the polymer chains and the HA crystal faces.[43]There is also a broad peak around208in the XRD patterns of PNIPAM-g-HA6(Figure3d),which is attributed to the diffraction of PNIPAM brush aggregates on the surface of HA.However, the broad peak is absent in the XRD curve of PNIPAM-g-HA1, and this may be because the PNIPAM chains are shorter than those on PNIAM-g-HA6,and the shorter brushes could not aggregate into the crystalline phase.

Surface Modi?cation of Hydroxyapatite Nanoparticles with Thermal-Responsive

...

Figure2.TGA curves of HA(a),HAAPS(b),HABr(c),PNIPAM-g-HA1 (d),and PNIPAM-g-HA6

(e).Figure3.XRD patterns of HA(a),HABr(b),PNIPAM-g-HA1(c),and PNIPAM-g-HA6(d).

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As shown in Figure4,the structure of HABr is almost the same as that of the needle-like HA,and the surface of the HA particles are relatively neat.After ATRP,PNIPAM brushes are connected on the surface of the HA nanoparticles,so the morphology has changed.The needle-like particles of PNIPAM-g-HA1(Figure4c)are very similar to those of HABr,only a little roughness could be found,and this is because the PNIPAM chains on the surface of the nanoparticles are too short.As for the morphology of PNIPAM-g-HA6,the polymer chains are longer and the needle-like particles are encapsulated by a thin?lm (aggregation of polymer brushes);the nanoparticles aggregate together and the presence of the organic layer is more evident.The results also demonstrated that the PNIPAM brushes are connected to the surface of HA. Thermal Responsive Properties

It is well known that PNIPAM passes through a coil-to-globule transition at a lower critical solution temperature and has been used to construct thermal responsive interfaces.Liu and co-workers[39]reported that when PNIPAM was connected to the surface of silica nanoparti-cles,the hybrid particles also possessed thermal responsive properties.When PNIPAM is connected to the surface of HA nanoparticles,the hybrid material PNIPAM-g-HA may possess thermal responsive properties.As shown in Figure5,the transmittance of the PNIPAM-g-HA6suspen-sion system in water(0.5mgámLà1)changed with an increase of temperature.At a lower temperature,the

PNIPAM brushes on the sur-

face of HA adopted a random

coil conformation and

stretched longer into the

solution.Therefore,the PNI-

PAM brushes prevented the

aggregation of the HA parti-

cles.At a higher temperature,

the polymer brushes started

to collapse and compacted on

the surface of the nanoparti-

cles,and thus the polymer

layers become hydrophobic,

reducing their water solubi-

lity.Under such a condition,

the nanoparticles would

have an aggregation ten-

dency,so the transmittance

was lowered quickly.In addi-

tion,the thermal responsive

properties of PNIPAM-g-HA

were also affected by the

surface grafting amount,

the thermal responsive properties of PNIPAM-g-HA with a grafting amount less than15.5%,such as PNIPAM-g-HA0.5,was not evident(see Supporting Information2,S.Figure2).

Figure6shows the size of PNIPAM-g-HA6hybrid particles in dilute aqueous(less than0.03mgámLà1) solution.When the temperature is lower than308C,the particle size is greater than1000nm,but when heated to 308C,the particle size begins to decrease quickly.This phenomenon is attributed to the phase transition of PNIPAM chains on the surface of the nanoparticles.At a lower temperature,the PNIPAM chains are hydrophilic and

J.Wei,P.He,A.Liu,X.Chen,X.Wang,X.

Jing Figure4.SEM images of HA(a),HABr(b),PNIPAM-g-HA1(c),and PNIPAM-g-HA6

(d).

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stretch freely in the water.When the temperature is higher than 308C,the PNIPAM chains become hydrophobic and compact on the surface of the nanoparticles,and thus the particle size begins to decrease quickly.The results (Figure 7)also demonstrate that the thermal responsive properties of the hybrid particles are reversible.When the hybrid solution is at 258C,the particle size is around 1000–1200nm.While the temperature is increased to 408C,the particle size decreases to 300–350nm because of the collapse of the polymer chains.When the solution is cooled to 258C,the particle size returns to 1000–1200nm again.In vitro Bioactivity Assessment

HA-based materials are always used in the ?eld of hard tissue repair or bone tissue engineering,and the bioactivity properties,which are characterized by formation of apatite

layers on the surface of materials,are always assessed by the immersion of materials in SBF.[38]During the process of

immersion,Ca 2tand HPO 2à

will accumulate at the interface between the bioactive materials and the liquid,and an apatite layer will be formed on the surface of the materials,which is accompanied by the change of ion concentration in the liquid.In our experiment,the in vitro bioactivity of two PNIPAM-g -HA samples (PNIPAM-g -HA1and PNIPAM-g -HA6)and HA (control samples)have been assessed.As shown in Figure 8,during the ?rst two and a half hours,the concentration of Ca 2tfor the three samples decreased quickly,and then the tendency to decrease was slower.Most interestingly,there was even a minor increase of Ca 2tconcentration for PNIPAM-g -HA6observed after17h.After immersion for 30h,the concentration of Ca 2tin the SBF for PNIPAM-g -HA6decreased much more than that for PNIPAM-g -HA1and HA,which illustrates that the PNIPAM-g -HA6has better in vitro bioactivity than PNIPAM-g -HA1and HA.Since the polymer grafting amount

Surface Modi?cation of Hydroxyapatite Nanoparticles with Thermal-Responsive

...

Figure 6.The particle sizes of PNIPAM-g -HA6hybrid particles at different

temperatures.

Figure 7.Reversible change of the particle sizes of PNIPAM-g -HA6at 40and 258

Figure 8.Concentrations of Ca (A)and P (B)in SBF for HA,PNIPAM-g -HA1,and PNIPAM-g -HA6at different time intervals.

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of PNIPAM-g-HA6is greater than that of PNIPAM-g-HA1,we can conclude that the PNIPAM-g-HA with more polymer content has a better bioactivity.As for the changes of the HPO2à4concentration,the total tendency was similar to that of Ca2t.

The change of ion concentrations could be explained by Scheme2.At the initial stage1,because of the negative zeta potential of PNIPAM-g-HA and HA,the cationic ions Ca2taccumulate quickly onto the surface of PNIPAM-g-HA,and a Ca2t-rich micro phase is formed on the surface.Because the polymer brushes on the surface of HA make the contact area between PNIPAM-g-HA and SBF larger than that with HA. The decrease of Ca2tin the SBF for PNIPAM-g-HA6was greater more than for PNIPAM-g-HA1and HA.Thi s may be explained by the higher polymer grafting amount have the larger contact area.In addition the polymer brushes may also act as an anchor point for Ca2tmineralization.During the second stage,HPO2à4ions migrate to the Ca2t-rich micro phase to form a HA layer on the surface,so the contents of Ca and P determined by ICP-MS show a tendency to decrease in the solution and is characteristic of bioactivity.During the immersion process,the bulk PNIPAM-g-HA6also released a certain amount of Ca and P to the solution,which was identi?ed by the minor increase of Ca and P concentrations after17h immersion in the SBF.Actually,the speed of Ca and P release from the bulk materials is quite slow.This release process of Ca and P has an important effect for the material to be used as a bioactive material because the released Ca and P could support raw materials for the formation of new tissues.The decrease of the Ca and P concentration in the SBF proves that the PNIPAM-g-HA possess better bioactivity properties than HA. Biocompatibility

Biocompatibility is an important factor that affects the biomedical application of biomaterials.It was reported that the nanometer-scale alternation of materials could also elicit diverse cell behaviors,ranging from changes in cell

adhesion,cell motility,surface antigen

display,cytoskeleton concentration,to

modulating intracellular signaling path-

ways.[44]HA is always used as an arti?cial

substitute material for surgical treatment

of bone repair,so the marrow osteoblasts

have been used in the in vitro cell culture

experiment here.As shown in Figure9,

after24h of cultivation,only a few cells

are attached to the HA samples,and most

of the cells are round and do not grow very

well.On the contrary,more cells are

adhered on the surface of PNIPAM-g-

HA1and PNIPAM-g-HA6,and most of

the cells grow in a shuttle-like shape, which demonstrates that the biocompatibility of PNI-PAMA-g-HA is better than that of HA.After48h,the cells attached to the samples proliferated and the results (Figure9)show that there are many more cells on the surface of PNIPAM-g-HA1and PNIPAM-g-HA6than on the HA surface.The cells on the PNIPAM-g-HA1and PNIPAM-g-HA6also looked fat and proliferated better.Figure10 demonstrates the quantitative analysis of cell areas attached on the samples,and the cell area fraction on the PNIPAM-g-HA6is twice that of HA.All the results con?rm

J.Wei,P.He,A.Liu,X.Chen,X.Wang,X.

Jing Scheme2.Demonstration of ion changes in the SBF during the immersion

process.

Figure9.Morphology of cells attached to HA(a),PNIPAM-g-HA1

(b),and PNIPAM-g-HA6(c)for24h;and HA(d),PNIPAM-g-HA1(e),

and PNIPAM-g-HA6(f)for48h,respectively.

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1237–1246

?2009WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim DOI:10.1002/mabi.200900256

that the PNIPAM-g-HA hybrid material possesses better biocompatibility than HA.In addition,it was evident that there were more cells adhered to and had proliferated better on the surface of PNIPAM-g-HA6than on PNIPAM-g-HA1, which shows that the PNIPAM-g-HA with a higher PNIPAM grafting amount possesses better biocompatibility than that with a lower grafting amount.

Conclusion

Thermal responsive PNIPAM brushes have been grafted on the surface of HA nanoparticles by ATRP under mild conditions.With different reaction times,PNIPAM-g-HA hybrid particles with different grafting amounts(15.5–46.4%)can be prepared.The PNIPAM-g-HA could induce the mineralization of Ca and P in the SBF solution quickly, which shows that the hybrid particles possess excellent in vitro bioactivity.The PNIPAM-g-HA particles also show reversible thermal-responsive properties around328C and have a better biocompatibility than HA,which indicates that a smart interface has been constructed on the surface of the HA.In addition,The PNIPAM-g-HA hybrid shows better biocompatibility than HA.All the results indicate that the PNIPAM-g-HA hybrid materials will have promising applications in tissue engineering.

Acknowledgements:This work was supported by the National Natural Science Foundation of China(Key project50733003),the National Natural Science Foundation of China—A3Foresight Program(20621140369),Key International Science and Technology Cooperation Project of Ministry of Science and Technology of China(No.20071314),and the Science and Technology Develop-ment project of Jilin Science and Technology Bureau(20060701). Received:July13,2009;Revised:August31,2009;Published online:November6,2009;DOI:10.1002/mabi.200900256 Keywords:atom tranfer radical polymerization(ATRP);bioma-terials;hydroxyapatite;nanoparticles;surface modi?cation

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windows下安装苹果教程。最详细，最全面，最值得看的教程！最详细，最适合新手的教程：如何原版安装mac 从windows到mac os（安装黑苹果目前最详细教程）最近网上有不少如何安装苹果系统的教程，个人感觉都不错，但是有些地方还是不够详细，所以我决定写一个比较详细的教程。鉴于网上有不少类似的教程，所以我的这个安装方法中有不少是借鉴和总结他们的，在此不一一提出，但是对这些作者表示感谢和崇高的敬意。本教程较长，不要因此以为苹果的安装比较复杂，其实我只是让每一步叙述的更加详细，提供更多的方法处理安装，还有就是加入了大量的图片。由于本教程过于详细，所以比较适合新手阅读。安装之前要做好失败的心理准备，新手建议先备份重要数据到别的机器，预防数据丢失。要有足够的耐心和毅力，敢于不断尝试，不自己实践过永远不知道自己电脑是不是能够成功安装，安装成功后的成就感只有自己知道，另外完成安装过程也是学习电脑知识的过程，祝各位能安装顺利。以下是详细过程：安装之前的准备： ################################################### ##################### 硬盘安装原版mac（以10.6.3零售版为例）以下工具或软件下载地址在4楼和5楼，4楼提供10.6.3零售版115网盘下载，5楼提供帖子中所涉及的大部分软件的115网盘下载，126楼提供本教程的pdf格式的下载

所要准备的软件等： ①原版苹果镜像，推荐Mac.OS.X.10.6.3.Retail.dmg ②推荐bootthink2.4.6版本，另外也可以用变色龙，个人喜欢用bootthink 引导，感觉方便。 ③winPE或者windows原版安装光盘（设置C盘为活动分区用），推荐winPE，winPE的详细制作过程及制作需要的软件（附下载地址）下面有，另外一般的Ghost安装盘上都会有winPE的。 ④硬盘安装助手 ⑤XP必备DiskGenius：硬盘分区工具（windows7可不用该工具，系统自带磁盘管理） ⑥Mac.OS.X.10.6.3.Retail的替换文件：替换文件是让苹果系统可以安装在MBR分区上，即windows的分区，有想拿出整块硬盘安装mac的可以不用替换，如何替换看以下的安装过程。不替换的缺点：需要另外找硬盘备份自己硬盘上的所有数据资料，因为硬盘由MBR 分区转变为GPT时会损失所有数据。 ⑦HFS Explorer和Java VM（Java JRE虚拟机），，先安装Java VM （Java JRE虚拟机），才能正常使用HFS Explorer。 ⑧Macdrive或是TransMac，安装了Macdrive可以不用TransMac。 ⑨AMD，intel Atom和Pentium单核的CPU需要准备替换对应的内核主板不能开启AHCI的还需要补丁i系列的处理器（i3，i5，i7）还需要替换10.3.1以上内核，因为2010年4月

Windows7_各个版本原版系统下载

最全的微软msdn原版windows系统镜像和office下载地址集锦

最全的微软msdn原版windows系统镜像和office下载地址集锦随着windows的发展，越来越多的人都热衷于微软的原版系统下载了，相比之前的版本比如winxp版本，windows vista/win7/win8/win8.1/后来的版本在安装方面也比较简单了，所以ghost系统正在渐渐的消失市场，即便如此，很多同学还是找不到微软原版的系统和office下载，今天亦是美网络特意归结了一下微软msdn原版系统和原版office各个版本的下载，希望对正在苦苦找寻原版系统的同学有所帮助。说在前面的话： 1、本文提供的windows和office版本均为微软MSDN原版！ 2、挑选系统版本，一般来说专业版（vol）更适合我们！密钥获取：最新win8/8.1、office2013 key 密钥实时更新或请点击此处订阅网站内容，确认订阅后，激活方式将自动发送到您的邮箱当中！！！以下地址建议使用迅雷下载（点击此处下载迅雷极速版） windows 10 技术预览版简体中文： 32位系统下载：

Windows软件正版、原版和盗版的区别

Windows软件正版、原版和盗版的区别我们先了解一下什么是激活模块和激活码激活模块是软件生产商为了保护知识产权对软件产品进行锁定的模块，主要是锁定软件的功能或使用期限等。激活码是验证解锁这一激活模块的钥匙，可实现使用该软件的全部功能、使用权和提供官方的技术支持等。什么是正版？根据微软官方的释义，正版 Windows 是由 Microsoft 授权和发布的 Windows 副本，经过授权，由 Microsoft 或受信任伙伴提供支持，我的解释是可以通过微软正版Windows软件网络验证的，或者微软正式授权的都可以叫正版。但是这个正版的范围比较大，比如你买了一张盗版精简Windows7的光盘或者上网下载了各种修改版的系统，只要使用激活码成功在正版Windows软件中心通过了认证，这也可以叫做正版。正版有什么好处？使用 Windows 的正版副本能帮助你避免数据遭受盗版软件带来的安全威胁，它允许访问 Microsoft 及其合作伙伴提供的支持以及访问更新和下载，从而可以在电脑中获得最大收益。正版主要是可以得到微软的技术支持。什么是原版？原版只的是微软官方发售的零售版光盘复制的副本，并不一定可以通过微软正版Windows软件网络验证。为什么说不一定可通过正版验证呢？因为如果你安装时使用的激活码未能通过微软软件正版授权许可，或者被微软查封，那么这个系统就是盗版。原版软件只包含了软件本身，并不代表此软件成功安装后就可通过验证，它和激活码是两个概念原版有什么好处？原版软件保证了软件本身的性能安全及稳定，并且包含了全部功能及组件，并不存在恶意插件、流氓软件、病毒和木马等。

windows正版系统+正版密钥

正版Windows 系统下载+正版密钥 2010-05-30 11:49 喜欢正版Windows 系统这是我收集N天后的成果，正版的Windows系统真的很好用，支持正版！大家可以用激活工具激活！现将本收集的下载地址发布出来，希望大家多多支持！ Windows 98 第二版（简体中文）安装序列号：Q99JQ-HVJYX-PGYCY-68GM3-WXT68 安装序列号：Q4G74-6RX2W-MWJVB-HPXHX-HBBXJ 安装序列号：QY7TT-VJ7VG-7QPHY-QXHD3-B838Q Windows Millennium Edition (Window ME)（简体中文）安装序列号：HJPFQ-KXW9C-D7BRJ-JCGB7-Q2DRJ 安装序列号：B6BYC-6T7C3-4PXRW-2XKWB-GYV33 安装序列号：K9KDJ-3XPXY-92WFW-9Q26K-MVRK8 Windows 2000 PRO SP4（简体中文） SerialNumber: BCJTW-2M9JH-M8HHT-KWWWM-3444Y MD5：599d35359320db8c58c7f55da11d3458 Windows XP with sp3 VOL 微软原版（简体中文）文件名:zh-hans_windows_xp_professional_with_service_pack_3_x86_cd_vl_x14-74070.iso 大小: 630237184 字节

MD5: E74D72F3D90456003E9E02BA0FB7DA61 SHA1: D142469D0C3953D8E4A6A490A58052EF52837F0F CRC32: FFFFFFFF 邮寄日期(UTC)：5/2/2008 12:05:18 AM 下载地址：ed2k://|file|zh-hans_windows_xp_professional_with_service_pack_3_x86_cd_vl_x14-74070%20(E https://www.wendangku.net/doc/5813395580.html,).iso|630237184|EC51916C9D9B8B931195EE0D6EE9B40E| Windows XP pro with sp3 VOL 微软原版（简体中文）正版密钥： MRX3F-47B9T-2487J-KWKMF-RPWBY(工行版)（强推此号！！！） QC986-27D34-6M3TY-JJXP9-TBGMD(台湾交大学生版) QHYXK-JCJRX-XXY8Y-2KX2X-CCXGD(广州政府版) MFBF7-2CK8B-93MDB-8MR7T-4QRCQ(北京政府版) T72KM-6GWBP-GX7TD-CXFT2-7WT2B(上海政府版) DG8FV-B9TKY-FRT9J-6CRCC-XPQ4G(上海政府版) BCJTW-2M9JH-M8HHT-KWWWM-3444Y(英文版) CD87T-HFP4C-V7X7H-8VY68-W7D7M(英文版) RFYPJ-BKXH2-26FWP-WB6MT-CYH2Y(英文版) 7HPVP-8VHPV-G7CQ3-BTK2R-TDRF3(英文版) DRXKM-94K47-38QVX-F8K7R-2H7CD(日文版) FCKGW-RHQQ2-YXRKT-8TG6W-2B7Q8(韩文版) VMXC2-M9HKH-DRYGC-FHQ7H-BJY33(0408版) CM3HY-26VYW-6JRYC-X66GX-JVY2D（可用） DP7CM-PD6MC-6BKXT-M8JJ6-RPXGJ（可用） F4297-RCWJP-P482C-YY23Y-XH8W3（可用） HCQ9D-TVCWX-X9QRG-J4B2Y-GR2TT（可用） M6TF9-8XQ2M-YQK9F-7TBB2-XGG88（可用） HH7VV-6P3G9-82TWK-QKJJ3-MXR96（可用） TDWGX-DMF97-BJYDQ-X9DJV-CYHWQ（可用） T8FMX-Q4HQJ-3JW77-JGPDC-FY9DG（可用） G6X78-XG4KV-3MXT7-FT8YM-F3YW3（可用） OEM版：华硕：家庭版: KR63J-B34MB-CVP9K-T478G-8Y3XG 联想：家庭版: PWBPT-6PGKF-TP6MY-299P4-CPXQG (XXXXX-119-0001544-XXXXX) 专业版: FCDGH-QW3DJ-VBC6C-9BYTX-4GKQJ (XXXXX-119-0001553-XXXXX) VF4HT-MPWB8-TWV6R-K6QM4-W6JCM

Windows系统原版镜像下载地址

Win8.1下载地址 Windows 8.1 Pro VL (x86) - DVD (Chinese-Simplified) Win8.1简体中文专业版 32位文件名 cn_windows_8_1_pro_vl_x86_dvd_2972620.iso 大小2.84GB 下载地址： ed2k://|file|cn_windows_8_1_pro_vl_x86_dvd_2972620.iso|3049981952|5B396C3A0BA99 617647D9AFE8403AFA5|/ Windows 8.1 Pro VL (x64) - DVD (Chinese-Simplified)Win8.1简体中文专业版 64位文件名cn_windows_8_1_pro_vl_x64_dvd_2971907.iso 大小3.76GB 下载地址 ed2k://|file|cn_windows_8_1_pro_vl_x64_dvd_2971907.iso|4032598016|1FDA520B3E888 0E2FB00B20439E0826E|/ Windows 8.1 Enterprise (x86) - DVD (Chinese-Simplified)Win8.1简体中文企业版 32位文件名cn_windows_8_1_enterprise_x86_dvd_2972257.iso 文件大小2.84GB 下载地址 ed2k://|file|cn_windows_8_1_enterprise_x86_dvd_2972257.iso|3050842112|6B60ABF82 82F943FE92327463920FB67|/ Windows 8.1 Enterprise (x64) - DVD (Chinese-Simplified)Win8.1简体中文企业版 64位文件名cn_windows_8_1_enterprise_x64_dvd_2971863.iso 文件大小3.76GB 下载地址 ed2k://|file|cn_windows_8_1_enterprise_x64_dvd_2971863.iso|4039327744|08BAF1832 0B8FFC58D4C35BCC7A32012|/ 安装密钥核心版 334NH-RXG76-64THK-C7CKG-D3VPT 专业版 XHQ8N-C3MCJ-RQXB6-WCHYG-C9WKB win10下载地址 Win10正式版32位简体中文版（含家庭版、专业版） ed2k://|file|cn_windows_10_multiple_editions_x86_dvd_6846431.iso|3233482752|B5C706594 F5DC697B2A098420C801112|/

安装Windows7原版系统教程

安装Windows7原版系统教程一、需要的硬件：u盘一个（需要格式化）。二、需要的软件： 1、下载windows ISO格式镜像文件。该文件可以看做是Windows系统的安装文件。下载地址： ed2k://|file|cn_windows_7_ultimate_with_sp1_x86_dvd_u_677486.iso|2653276160|7503E 4B9B8738DFCB95872445C72AEFB|/ 2、去自己的电脑品牌官网下载电脑对应的有线和无线网卡驱动做为备用，因为新装的系统是没有网卡驱动的，如果不提前下载下来会导致装完系统没法上网。下载以后放到除了C盘意外的盘符或者u盘中备用。 3、下载winrar解压软件。下载地址： https://www.wendangku.net/doc/5813395580.html,/plus/download.php?open=2&id=8196&uhash=673c01a1f23336e96 033e2b2 下载以后放到除了C盘意外的盘符或者u盘中备用。 4、下载UltraISO并安装。下载地址： https://www.wendangku.net/doc/5813395580.html,/alading/anquan_soft_down_ub/11522 注册码用户名：王涛注册码：7C81-1689-4046-626F 如果注册码失效，自行百度一个可用的。三、装系统的步骤 1、制作启动盘 1）打开ultraISO，选择文件，选择打开，选择之前下载的window7 ISO格式的镜像。如图： 2）把准备的u盘插入电脑，点击ultraISO的启动，点击写入硬盘映像。

弹出对话框里，选择你的u盘，点击写入。（写入过程u盘会全盘格式化，备份好有用的文件）写入完成以后，你的u盘就是一个系统启动盘了。

微软MSDN官方原版windows8简体中文正式版(3264位)下载

微软MSDN官方原版windows8简体中文正式版（32/64位）下载北京时间8月16日凌晨，微软如约向MSDN付费用户提供了windows8 的下载（点击查看）。MSDN版微软操作系统，一直是公认为“微软原版”， Jeff从来都是建议安装微软原版 windows8。windows8时代就此开启！下面就分享微软MSDN官方原版 windows8简体中文正式版下载并提供相关文件信息： Windows 8 （包含专业版和Core版）MSDN官方原版简体中文（建议下载） 32位（x86）：文件名：cn_windows_8_x86_dvd_915414.iso SHA1：0C4A168E37E38EFB59E8844353B2535017CBC587 下载地址： ed2k://|file|cn_windows_8_x86_dvd_915414.iso|2679801856|9AF10141BFD61BC66D9D64597 58D7749|/ 附：安装密匙 Core版：BN3D2-R7TKB-3YPBD-8DRP2-27GG4 FB4WR-32NVD-4RW79-XQFWH-CYQG3 专业版： NG4HW-VH26C-733KW-K6F98-J8CK4 XKY4K-2NRWR-8F6P2-448RF-CRYQH 64位（x64）：文件名：cn_windows_8_x64_dvd_915407.iso SHA1：A87C4AA85D55CD83BAE9160560D1CB3319DD675C 下载地址： ed2k://|file|cn_windows_8_x64_dvd_915407.iso|3652950016|5C7F8C212BD3A1827866563773 A431C2|/ Windows 8 企业版((Eterprise)MSDN官方原版简体中文 32位（x86）：文件名：cn_windows_8_enterprise_x86_dvd_917682.iso SHA1：951565D8579C5912FB4A407B3B9F715FBDB77EFE 下载地址： ed2k://|file|cn_windows_8_enterprise_x86_dvd_917682.iso|2597502976|7B6541942A16EB54 BC81E84558DF09DF|/ 64位（x64）：文件名：cn_windows_8_enterprise_x64_dvd_917570.iso SHA1：1280BC3A38A7001FDE981FA2E465DEB341478667 下载地址： ed2k://|file|cn_windows_8_enterprise_x64_dvd_917570.iso|3560837120|8CAE8064C4B8F9CD 84941B4FF4A34722|/ Windows 8 批量授权版本（Pro VL ）MSDN官方原版简体中文 32位（x86）：文件名：cn_windows_8_pro_vl_x86_dvd_917720.iso SHA1：EEEF3C3F6F05115C7F7C9C1D19D6A6A6418B5059

windows xp官方原版

1）近日，照牛排在野猪尖分享了雨林木风最后4个系统的下载地址，以及深度系统全系列MD5值及下载地址，喜欢雨林木风及深度的朋友，可以选择下载。尽管雨林木风和深度的系统经过优化会有一些优势，但毕竟是修改过的，有这样或那样的问题。我还是喜欢微软原版的系统，稳定。本文将分享微软原版Windows XP Pro With SP3 VOL MSDN简体中文专业版的下载地址、MD5值及序列号。 2）2008年5月2日，微软推出Windows XP Pro With SP3 VOL MSDN简体中文专业版，这是最经典也是我最喜爱的操作系统之一。我在MSDN（微软开发者网络）的网站上查到，Windows XP Pro With SP3 VOL MSDN简体中文版的全名叫Windows XP Professional with Service Pack 3 (x86) - CD VL (Chinese-Simplified)，更详细的信息如下：文件名： zh-hans_windows_xp_professional_with_service_pack_3_x86_cd_vl_x14-740 70.iso 文件大小：601 MB (630,237,184 字节) 发布日期 (UTC)：5/2/2008 12:05:18 AM 上次更新日期 (UTC)：2/19/2010 11:36:41 AM SHA1：D142469D0C3953D8E4A6A490A58052EF52837F0F MD5：E74D72F3D90456003E9E02BA0FB7DA61 3）微软官方原版Windows XP Pro With SP3 VOL MSDN简体中文专业版的迅雷下载地址为： thunder://QUFodHRwOi8vc29mdC51c2Fpa2EuY24vJUU2JTkzJThEJUU0JUJEJTlDJUU 3JUIzJUJCJUU3JUJCJTlGL3dpbmRvd3MvV2luZG93c1hQL1ZMLUltYWdlL01TRE4vemgt aGFuc193aW5kb3dzX3hwX3Byb2Zlc3Npb25hbF93aXRoX3NlcnZpY2VfcGFja18zX3g4N l9jZF92bF94MTQtNzQwNzAuaXNvWlo 电驴下载地址为： ed2k://|file|zh-hans_windows_xp_professional_with_service_pack_3_x86_ cd_vl_x14-74070%20(https://www.wendangku.net/doc/5813395580.html,).iso|630237184|EC51916C9D9B8B931195EE0D 6EE9B40E| 只要复制上面任何一个下载地址，并启动迅雷新建任务即可开始下载。这是一个ISO镜像文件，下载后，请用校验工具（点此下载）验证一下MD5值。你可以把镜像刻录到光盘，也可以解压后从硬盘安装。 4）至于微软原版Windows XP Pro With SP3 VOL MSDN简体中文专业版的序列号（CD-KEY，即密钥，俗称SN），我在用工行版的正版序列号： MRX3F-47B9T-2487J-KWKMF-RPWBY。你也可以尝试以下VOL版XP序列号： QC986-27D34-6M3TY-JJXP9-TBGMD（台湾交大学生版） HCQ9D-TVCWX-X9QRG-J4B2Y-GR2TT（https://www.wendangku.net/doc/5813395580.html,） DP7CM-PD6MC-6BKXT-M8JJ6-RPXGJ CM3HY-26VYW-6JRYC-X66GX-JVY2D