溶胶凝胶法载银抗菌研究

Antimicrobial Effects and Human Gingival Biocompatibility of Hydroxyapatite Sol–Gel Coatings

Ren-Jei Chung,1Ming-Fa Hsieh,2,*Chine-Wen Huang,1Li-Hsiang Perng,3Hsiao-Wei Wen,4

Tsung-Shune Chin1

1Department of Materials Science and Engineering,National Tsing Hua University,101,Sec2,Kuang-Fu Road,Hsinchu 300,Taiwan,Republic of China

2Biomedical Engineering Center,Industrial Technology Research Institute,195Chung Hsing Road,Sec4Chu Tung, Hsinchu310,Taiwan,Republic of China

3Department of Chemical Engineering,Cheng Shiu University,840,Chengcing Road,Niaosong Township,Kaohsiung83305, Taiwan,Republic of China

4Nuclear Science and Technology Development Center,National Tsing Hua University,101,Sec2,Kuang-Fu Road,Hsinchu 300,Taiwan,Republic of China

Received7December2004;revised27March2005;accepted6April2005

Published online3August2005in Wiley InterScience(https://www.360docs.net/doc/0818527079.html,).DOI:10.1002/jbm.b.30365

Abstract:The sol–gel method was employed to synthesize hydroxyapatite(HAp)coatings

modi?ed with Ag or Zn ions onto Ti-6Al-4V substrate.A bacterial strain Streptococcus mutans

(S.mutans)and a human gingival?broblast(HGF-1)cell line were used to investigate the

antimicrobial effect and biocompatibility,respectively.HAp coatings containing100ppm Ag?

ions suppressed the growth of S.mutans.An apparent inhibition zone around the HAp coating

was further observed at Ag?concentration up to10,000ppm.However,for coatings contain-

ing Zn2?ions,a clear inhibition zone was observed at Zn2?concentration of10,000ppm.

Nevertheless,the results of HGF-1cultivation demonstrated that the Zn2?-modi?ed HAp

coatings exhibited better attachment and spread of HGF-1than did the Ag?-modi?ed coat-

ings.Zn2?modi?ed HAp coatings also increased the plating ef?ciency of HGF-1cells.The

cytotoxicity associated with the addition of Ag and the cell-conductive capacity associated with

the addition of Zn are proportional to the added concentration,from100to10,000ppm.The

dosages of both Ag?and Zn2?ions that should be added to HAp coatings were considered to

prevent infection and improve biocompatibility.The results of this study ensure that HAp

coatings modi?ed with a moderate amount of Ag/Zn ef?ciently resist microorganisms and

improve biocompatibility.?2005Wiley Periodicals,Inc.J Biomed Mater Res Part B:Appl Biomater76B:

169–178,2006

Keywords:hydroxyapatite;biocompatibility/hard tissue;Streptococcus mutans;human gin-

gival?broblast;antimicrobial;coatings

INTRODUCTION

Because they are corrosion-resistant,mechanically strong, tough,and reusable,titanium alloys have been applied in surgical implantations since the1940s.1,2Ti-6A1-4V is most commonly used in bony and dental restoration,especially in implant tooth roots.

Clinical studies have revealed that periimplant in?amma-tory diseases around dental implants may cause periimplan-titis and the loss of supporting bone.3,4Besides mechanical factors such as implant geometry and surface properties, bacterial contamination are also the major issue,leading to failure of dental implants.Pathogenesis of these diseases can be attributed to bacteria that normally reside in oral cavity. Some cariogenic bacterial strains,for example Streptococcus mutans(S.mutans),nourish dental implants in the presence of sucrose.5,6They produce extracellular glucan bio?lms on the surface of dental implants,resulting in accumulation of organic acid synthesized by the bacteria.Low pH in local environment persists over a long period will eventually lead to the erosion of hard tissues emerged as a result.It will

*Present address:Food Science and Technology Department,Cornell University,

Geneva,NY14456

Correspondence to:M.-F.Hsieh(e-mail:mfhsieh@https://www.360docs.net/doc/0818527079.html,.tw)

Contract grant sponsor:National Science Council of the Republic of China;contract

grant number:NSC91-2216-E-230-001

?2005Wiley Periodicals,Inc.

169

furthermore result in serious in?ammation and infection.Nevertheless,intra-oral translocation of other microorgan-isms makes healing process of periimplantitis much more complicated.Therefore,efforts on eliminating colonization of pathogenic species have been involved in the therapy of periimplant in?ammatory diseases and precautionary oral hygiene,3,4and for products preservation as well.

Hydroxyapatite [Ca 10(PO)6(OH)2,HAp]being the main inorganic component of hard tissues of vertebrates 7–9has been synthesized for various biomedical applications.10,11It has been coated on orthopedic and dental implants.In the previous studies,implants with HAp coating have many advantages:direct bony growth and early mineralization at the interface,providing greater ?xation strength in the early stage arisen from the ingrowth of bone tissue;restoration of injury through bony-induction ability of HAp;preventing acute foreign-body-effect caused by metal implant.There are various processes to coat HAp on titanium alloys,10such as plasma spraying,ion sputtering,chemical vapor deposition,electrochemical deposition,sol–gel route,and thermal de-composition,and so on.Among them,the sol–gel route is of relatively low temperature,the composition can be con-trolled,and it is suitable for large or irregular surface coat-ing.11

The ion exchange of HAp with metal ions is promising to create versatile functions of HAp in various applica-tions.12–14The present study is to synthesize metal-ion–added HAp coating and investigate its antimicrobial ef-fects against S.mutans .In the past decades,several metal ions have been used as antimicrobial elements,such as Ag ?,Zn 2?and Cu 2?,and so on 15,16in clinics.Among these,two metal ions were studied in this work:Ag ?and Zn 2?.The Ag-added or Zn-added HAp coatings were prepared via sol–gel method and then were investigated by X-ray diffraction for its crystalline phases.Moreover,it is required to further perceive whether this material still persists in good biocompatibility with peripheral gingival cells.In this regard,the cellular behavior of human gingi-val ?broblast on HAp coatings (pure and Ag/Zn added ones)on Ti-6A1-4V substrates was evaluated in vitro

.

Figure 1.The XRD patterns of HAp coatings on Ti-6Al-4V with dif-ferent concentrations of (A)AgNO 3and (B)Zn(NO 3)2added to the precursors.

TABLE I.The Chemical Composition of Sol–Gel Precursors for Ag ?or Zn 2?Ion-Added HAp Coatings

Sample AgNO 3(mol)Zn(NO 3)2?6H 2O (mol)Ca(NO 3)2?4H 2O (mol)

TEP (mol)Pure HAp 0.010.006Ag 100ppm 1.862E-40.010.006Ag 1,000ppm 1.862E-50.010.006Ag 10,000ppm 1.862E-6

0.010.006Zn 100ppm 3.073E-40.010.006Zn 1,000ppm 3.073E-50.010.006Zn 10,000ppm

3.073E-6

0.01

0.006

TABLE II.The Actual Ca/P Molar Ratio and Concentration of Additive Metal Ions of HAp Precursor and Powders Calcined at 600°C

Sample

Ca/P Molar Ratio [Ag ?]or [Zn 2?]

Precursor without added metal 1.67Pure HAp powder 1.70HAp/Ag 100ppm 1.71210ppm HAp/Ag 1,000ppm 1.73931ppm HAp/Ag 10,000ppm 1.7610,235ppm HAp/Zn 100ppm 1.74150ppm HAp/Zn 1000ppm 1.681,148ppm HAp/Zn 10,000ppm

1.71

9,871ppm

170CHUNG ET AL.

MATERIALS AND METHODS

Preparation of Sol–Gel Precursors and HAp Coatings

The metal-ion–added HAp coatings,with precursor compo-sitions listed in Table I,were prepared by sol–gel technique,followed by a rapid thermal process.17Precursors with cal-cium to phosphorus molar ratio of 1.67were prepared by mixing Ca(NO 3)2-4H 2O,triethyl phosphate [(C 2H 5O)3PO,TEP)and AgNO 3(or/and Zn(NO 3)2]in 2-methoxy ethanol.The precursors were aged in closed containers at 80–90°C for 16h followed by drying in opened containers at the same temperature until its viscosity reached 100?10cP.The pH values were recorded throughout the aging and drying steps.The HAp gels were spin-coated on Ti-6Al-4V alloy sub-strates that had been surface blasted by 500–850-?m Al 2O 3beads,and then predried at 150°C,followed by a rapid heating to 400°C (at a ramp 300°C/min)in ambient environ-ment and holding for 3min.The coatings were repeatedly deposited four more times,and the ?nal thick coatings were subjected to a rapid heating to 600°C (at a ramp 300°C/min)and holding for 1min.

Chemical and Physical Characterizations of the HAp

Powders and Coatings

Due to insuf?cient quantity of the coatings for detailed chem-ical characterization,powders with the same stoichiometry as the coatings were also prepared.The preparation procedure was to calcine the said gels following the same heating process.The concentrations of silver or zinc and Ca/P molar ratio in the calcined HAp powders were analyzed by an inductively coupled plasma mass spectrometer (Perkin Elmer,SCIEX ELAN 5000).The precursors receiving aging and drying steps (mentioned in the last paragraph)were calcined at 500°C in alumina crucibles.Twenty-?ve micro-grams of as-calcined powders were digested by 3mL HNO 3(70%)and 0.5mL H 2O 2in microwave oven (Milestone Co.,mls 1200)for 2min.The resultants were then analyzed.The HAp coatings were examined by an X-ray diffractom-eter (Geiger ?ex D/MAX II B,Rigaku Co.)with the use of Cu K ?radiation.The JCPDS (Joint Committee on Powder Dif-fraction Standards)for HAp (Card No.09-0432)and CaO (Card No.37-1497)were used to identify phase composi-tions.18The surface morphology of HAp coatings was

ob-

Figure 2.FE-SEM morphology of HAp coatings added with different concentrations of (A)pure HAp,(B)100ppm Ag,(C)1,000ppm Ag,(D)10,000ppm Ag,(E)1,000ppm Zn,and (F)10,000ppm Zn.

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HYDROXYAPATITE SOL–GEL COATINGS

served by a ?eld emission scanning electron microscope (FE-SEM,model JSM-6500F,JEOL,Japan).

In Vitro Antimicrobial Tests and the Cell Responses to

the HAp Coatings

To avoid biological contamination during in vitro studies,all samples were sterilized by gamma-ray irradiation with a total dosage of 20kGy before experiments.S.mutans (ATCC 25175)was cultured at 37°C in the brain heart infusion medium (BHI,Di?co.Co.)and brain heart infusion agar (BHIA,Di?co Co.)for cell propagation and antimicrobial tests,respectively.The antimicrobial tests of HAp coatings were conducted in BHIA solid media with the spread-plate method.0.1mL of S.mutans cell suspension (1.5?104CFU/mL)was spread on a 12-cm BHIA plate,and a sample of the HAp coating side was put on it for a time period of 48h.

Human gingival ?broblast (HGF-1,ATCC CRL-2014)from passages numbered 6–20was cultured in Dulbecco’s modi?ed eagle medium (DMEM)supplemented with heat-inactivated fetal bovine serum (10%v/v)in an incubator at

37°C,with 5%CO 2and a saturated humidi?ed atmosphere.0.1mL of HGF-1cell suspension (105cells/mL)was seeded directly onto the HAp surface of the Ti-6Al-4V substrates in a 24-well dish,and then incubated for 65h.Triplicates were used under each condition.

A stereo optical microscope (SMZ-U,Nikon),supple-mented with re?ective light with a ?ber-optic light source (Nikon)and FE-SEM were used to observe the morphologies of the HAp/Ti-6Al-4V samples.The HGF-1cells were then stained with crystal violet and observed under a stereo optical microscope.The sample for FE-SEM observation was pre-pared as a separate HAp/Ti-6Al-4V sample seeded with HGF-1cells treated with 3mL solution of 2.5%glutaralde-hyde for 1h at room temperature.Then the samples were gently washed twice with 3mL of phosphate buffered saline (PBS)to remove any suspended cells,and dehydrated through a graded series of alcohol solutions (40–100%),each for 15min.The samples underwent critical point drying (CPD)and were coated with platinum for FE-SEM examina-tion.

The acid phosphatase (ACP)assay was used to measure the population of HGF-1cells.19The ACP assay is sensitive and suitable for the evaluation of small cell population.The mechanism is used to determine the activity of the cytosolic acid phosphatase with the use of a synthetic substrate of p -nitrophenylphosphate (P NPP).PNPP reacted with ACP followed by formation of p -nitrophenol.The concentration of p -nitrophenol can be measured by UV-VIS spectrometer.So the concentration of ACP in the HGF-1cells can be quanti-tatively determined.After plating and culturing the cells on the coatings for 48h,a 500-?L aliquot of buffer,containing 0.1M sodium acetate (pH 5.5,J.T.Baker Chemical,Phil-lipsburg,NJ),0.1%(v/v)Triton X-100(BDH Chemicals)and 10mM p -nitrophenyl phosphate (Sigma 104phosphatase substrate,Sigma Chemical),was added to each culture well,and the culture wells were kept in the incubator at 37°C for 2h.The reaction was stopped by the addition of 50?L 1M NaOH to each well,and the wells were measured at a wave-length of 405nm with the use of an enzyme-linked immu-nosorbent assay reader.

RESULTS AND DISCUSSION Material Characterization

Prepared Sol–Gel Precursors.Sol–gel derived HAp

ceramic is sensitive to the aging process,especially when organic phosphate-or phosphite-based precursors were used.17,20The sol solutions under aging were transparent in spite of silver or zinc additions,and pH values de-creased from 3.0–4.0to 2.0in a period of 16h.Note that the sol solutions became pale yellow at the end of aging.Theoretically,the chemistry of the aging process is com-plicated because of two concurrent reactions:the hydroly-sis of phosphate and the formation of Ca-P intermedi-ates.20Thus metal ions,Ag ?or Zn 2?dissolved in the precursors may affect Ca-P intermediates because of

sub-

Figure 2.(continued)

172CHUNG ET AL.

stitutions by Ag ?or Zn 2?in the Ca-P intermediates.However,no differences in color and pH values of sol solutions were seen in the aging process,and the sol remained transparent without any precipitate.

The effect of Ag ?or Zn 2?addition appeared signi?cant in the drying and calcination.The phase evolved after precur-sors were spin-coated onto Ti-6Al-4V substrates and rapidly calcinated,and showed HAp main phase along with an im-pure phase CaO.Figure 1displays XRD patterns of HAp coating.The CaO impurity was enhanced with increasing metal addition,which revealed a positive substitution trend of the additive metal ions.21It was inferred that the substitution of silver or zinc ions in Ca-P intermediates of aged sol resulted in more free calcium ions.Thus,upon rapid heating of HAp precursors coated on the substrates,calcium ions reacted with surrounding oxygen,leading to CaO by-prod-ucts.Yet the effect of substrate material was also suspected as one of the reasons.HAp deposited over silicon substrates also contained CaO phase,regardless of silver or zinc addition.Lopatin et https://www.360docs.net/doc/0818527079.html,ed silicon wafers coated with borophosphate silicate as substrates and reported CaO formation as a minor phase.22

Chemical Compositions of HAp Powder.Table II sum-

marizes the Ca/P molar ratio of HAp powders prepared at 600°C.The ratios were in the range of 1.67(precursors)to 1.76(10,000ppm silver added).The higher Ca/P ratios,except precursors,can be anticipated as organic phosphorus compounds are more susceptible to evaporate upon calcining in an open-air oven as compared with calcium nitrate.On the other hand,in previous reports,species of carbonated HAp [for instance,Ca 10(PO 4)6-X (CO 3)3/2X (OH)2]are commonly observed in arti?cial and bioinspired apatites.21The substi-tute carbonate groups would lead to raising of Ca/P ratio in a reasonable manner.Phase purity represented in Figure 1ensures the ef?cacious production of crystalline HAp.

Table II also shows the measured concentration of additive metal ions.The actual silver concentration in sample Ag 100ppm was 210ppm,which is two times more than the desig-nated amount in the precursors (Table I,Ag 100ppm).In the HAp powders using the precursors of Zn 1,000ppm and Zn 10,000ppm,the actual zinc concentrations were 1148ppm and 9871ppm,respectively.The silver and zinc concentra-tions in the HAp powders seem not to match with designated precursors exactly;however,the measured data are within

a

Figure 3.Antimicrobial tests of HAp coatings on BHIA (with a ruler in millimeters):(A)pure HAp coatings;(B)HAp coatings with 100ppm Ag added (arrow indicated the effect due to poor spreading of precursor);(C)HAp coatings with 10,000ppm Ag added;(D)HAp coatings with 1,000ppm Zn added;(E)HAp coatings with 10,000ppm Zn added;(F)PSA coated on blasted Ti-6Al-4V substrate (samples a and c)and pure HAp coatings (samples b and d).Samples c and d were washed by phosphate buffer and distilled water three times.[Color ?gure can be viewed in the online issue,which is available at https://www.360docs.net/doc/0818527079.html,.]

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HYDROXYAPATITE SOL–GEL COATINGS

reasonable error range,and most importantly the increasing trend persists.There is no evidence of phase separation for the additives.Synthesized coatings are homogeneous and with distinguishable characteristics,as in the original design.

HAp Coating.The total thickness of the coating was

around 20?m for ?ve repeated spinning coats.The FE-SEM micrograph revealed that pure HAp had a rough and porous surface with pore sizes ranging from 1to 10?m [Figure 2(A)].The porous structure is a characteristic feature of rapid thermal process.The micrographs also showed that,with increasing added concentration of Ag,the surface of HAp coatings was more compact [Figure 2(B–D)].On the other hand,increasing Zn addition leads to ?ner microstructure with cellulose morphology [Figure 2(E–F)].

Compared to plasma spraying and the biomimetic method,10,23the sol–gel route for HAp coating is an econom-ical and time-saving process.It is also suitable for coating complex surfaces.Another advantage is that the incorpora-tion reagents are able to homogeneously distribute without phase separation.The coating thickness can be adjusted through precursor viscosity and sequential coatings.Regard-ing about the antibiotic carriers,the reagent is also distributed deep in the coating;a steady and long-period releasing effect is anticipated.

In Vitro Antimicrobial Tests of HAp Coatings

S.mutans was used as the tested bacterial strain.The interactions between S.mutans and HAp coating modi?ed with metal ions were observed.S.mutans ,a Gram-positive bacteria,is an etio-logical agent of human caries and plaques,and has been exten-sively investigated in the pathogenesis of oral infection.A mi-croassay conducted for bacterial adherence to HAp revealed that S.mutans attaches onto the HAp surface by glucan-mediated accretion.24Calcium-phosphate coatings on dental implants are usually loaded with antibiotics to prevent postoperative bacterial infectionly.25However,the prevalence of bacterial antibiotic resistance has become a serious issue in medical communities around the world;this study seeks to develop HAp coatings to which silver or zinc has been added,aiming at tackling the issues of antibiotic resistance.

In the antimicrobial tests,the growth of S.mutans around the coating layers of pure HAp were observed,the microbial colonies were uniformly distributed [Figure 3(A)].However,the Ag 100ppm coating suppressed the growth of the S.mutans that surrounded the sample [Figure 3(B)].Some bac-terial colonies remained close to the corners of the coating [indicated by an arrow in Figure 3(B),because of the poor spreading of the precursor during the spin-coating process.Furthermore,the HAp ?lm added with 10,000ppm Ag com-pletely inhibited the growth [Figure 3(C)].Adding 1,000ppm Zn to the HAp coatings [Figure 3(D)]did not clearly suppress the growth,but adding 10,000ppm Zn did [Figure 3(E)].The use of antibiotics as a substitute for metal ions and the incorporation into the porous HAp coatings that serve as carriers were also evaluated.An antibiotic complex of peni-cillin (10,000units/mL),streptomycin (10mg/mL),and am-photericin (0.025mg/mL)(PSA)was absorbed directly onto Ti-6Al-4V substrate [Figure 3(F),samples a and c]or on pure HAp coatings [Figure 3(F),samples b and d].Samples c and d in Figure 3(F)were washed with the phosphate buffer and distilled water three times each.Neither the coated [Figure 3(F),sample d]nor the noncoated [Figure 3(F),sample c]batch exhibited a stronger antimicrobial effect.Figure 3(F),samples a and b show obvious antimicrobial phenomenon.The antibiotic complex immobilized by the substrate surface and the coating effectively inhibited the proliferation of bac-teria.However,after they had been washed,only coated samples exhibited slight antimicrobial ef?cacy [Figure 3(F),sample d];treated naked Ti-6Al-4V substrate exhibited no antimicrobial ability,unlike in Figure 3(F),sample a.It is suggested that the surface of HAp coatings did not strongly trap PSA antibiotics,which were easily removed by ?uid.Stiger,Groot,and Layrolle conducted a similar investigation by absorbing tobramycin into biomimetic HAp coatings or plasma-sprayed HAp coatings.They found that 90%of the antimicrobial agent was released within 3h.25Radin et al.and Campbell et https://www.360docs.net/doc/0818527079.html,ed multilayer coatings as barriers to retard the quick release of antibiotics and thereby cause long-term release.However,the outer coating also inhibited mammal cell adhesion,even though the release period was extended to as long as 72h.

26,27

Figure 3.(continued)[Color ?gure can be viewed in the online issue,which is available at https://www.360docs.net/doc/0818527079.html,.]

174CHUNG ET AL.

The prevention and treatment of postsurgical infection remain challenging tasks in orthopedics and dentistry.Infec-tion results in early failure.Sterilization and antiinfection procedures are necessary not only during implantation sur-gery,but also after changing dressings.Hence,a long-term antimicrobial strategy is crucial.The antimicrobial effect of organic antibiotics carried by HAp coatings is manifest pri-marily through physical absorption;thereafter,rapid dissolu-tion is unavoidable.HAp to which Ag or Zn has been added,consolidated by rapid thermal process,features slow release,and the tissue is directly attachable to the bony inductive materials.Restated,adding a moderate or an appropriate speci?c amount of metallic ions to an HAp coating offers advantages of long-term microbial suppression beyond just short-range

effects.

Figure 4.Morphology of HGF-1cells cultured on a ?ask observed through a stereo optical microscope.[Color ?gure can be viewed in the online issue,which is available at

https://www.360docs.net/doc/0818527079.html,.]

Figure 5.Morphology of HGF-1cells attached on different HAp coatings observed through a stereo optical microscope after crystal violet staining (original magni?cation 200?;arrows indicate the cells;the scale bar is 100?m).(A)Pure HAp coatings;(B)1,000ppm Ag added;(C)10,000ppm Ag added;(D)1,000ppm Zn added;(E)10,000ppm Zn added.[Color ?gure can be viewed in the online issue,which is available at https://www.360docs.net/doc/0818527079.html,.]

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HYDROXYAPATITE SOL–GEL COATINGS

Attachment and Plating Ef?ciency Of Human Gingival

Fibroblast to HAp Coatings

Figure 4shows the HGF-1cell morphology observed under a stereo optical microscope on a culture ?ask.The cells had a spindle shape;the cell bodies were 5?m wide and 60–70?m long,and their pseudopods were extended from two distal ends.In the literature,primary gingival ?broblasts extracted from adult humans and plated on calcium-phosphate surfaces exhibited doubling times of a few days to 1week.28The HGF-1cells cultured in the T75?asks were slowly expended;the estimated doubling time was about 2weeks.In this study,the HGF-1cell culture was used to investigate the short-term attachment of cells over HAp coatings.Figure 5presents the morphology of HGF-1cells cultured on HAp coatings for 48h.On the pure HAp coating [Figure 5(A)],attached cells (indicated by arrows)were observed with extended pseudo-pods,revealing that the pure HAp surface was not cytotoxic to the cells.However,cells seeded on HAp coatings to which Ag had been added [Figure 5(B,C),1,000and 10,000ppm respectively)did not spread very well;those on coatings to which Zn had been added [Figure 5(D,E),1,000and 10,000

ppm,respectively]extended their pseudopods and compactly adhered onto the HAp coating surface.Samples were then examined by FE-SEM following CPD.The surface

morphol-

Figure 6.Morphology of HGF-1cells attached on different HAp coatings observed through FE-SEM (original magni?cation 1000?):(A)pure HAp coatings;(B)1,000ppm Ag added;(C)1,000ppm Zn added;(D)10,000ppm Zn

added.

Figure 7.Number of attached HGF-1cells onto different HAp coat-ings evaluated by ACP assay.[Color ?gure can be viewed in the online issue,which is available at https://www.360docs.net/doc/0818527079.html,.]

176CHUNG ET AL.

ogy of the coating changed because of etching by immersion in the medium and CPD processing,providing clearer details of the adherence(Figure6).The pseudopods of the attached cells were in direct contact with the coatings,and the cells presented extended morphologies.Cells on HAp coatings to which Zn had been added seemed to be active.

Figure7plots the results of ACP assay for various HAp coatings after they were cultured for65h.The optimized plating ef?ciency onto the culture dish was30%.When the concentration of added Ag was increased(100to10,000 ppm),the cytotoxicity of the corresponding coating in-creased.Subsequently,fewer cells adhered and subsisted onto the surface.However,as more Zn was added(100to10,000 ppm),more cells survived.Adding Zn to the HAp coating not only promoted the early attachment of the HGF-1cells,but also increased the plating ef?ciency.In vitro evaluation di-rectly revealed the immediate response of the cells to the coating.The in situ environment of implantation is more complex,and animal tests are being conducted to ensure the developed coatings can be used.

Results of the in vitro HGF-1tests reveal that the cells directly adhere to the HAp coatings with added Ag/Zn.The cytotoxicity of Ag-HAp coatings depends on the Ag concen-tration;increasing the amount of added Zn increases the biocompatibility of Zn-HAp.Table III presents the results of antimicrobial and in vitro biocompatibility tests.A combina-tion of Ag and Zn is preferred,and the optimal mixing concentration is currently under investigation.A HAp coating with added Ag and Zn exhibits effective antimicrobial ability and satisfactory biocompatibility.An effort should be made to design an appropriate surface for antibiotic entrapment and organic–inorganic hybrid surface entrapment with Ag/Zn to reduce the cost of dental implants.

CONCLUSIONS

The sol–gel method is a suitable process for preparing Ag?-or Zn2?-added antimicrobial HAp coating,enabling the infection prevention of implantation.In order to achieve early-stage healing and prevent postsurgical in?ammation,a biocompatible and antimicrobial HAp coating on metal im-plant is needed.In this study,adding100ppm Ag ions to the sol–gel precursor calcined HAp coatings on Ti-6Al-4V sub-strates resulted in growth suppression of the pathogen(Strep-tococcus mutans),and an apparent inhibition zone was found for HAp?lm with Ag ion addition up to10,000ppm.For Zn-added HAp,more than1,000ppm Zn is needed to show antimicrobial effect.Zn addition(100–10,000ppm)is bene-?cial for cell attachment and intends to increase the plating ef?ciency of human gingival?broblast cells.

REFERENCES

1.Bothe TR,Beaton LE,Davenport HA.Reaction of bone to

multiple metallic implants.Surg Gynecol Obstet1940;71:598–602.

2.Meffert RM,Langer B,Fritz ME.Dental implants—A review.

J Periodontol1992;63:859–870.

3.Quirynen M,De Soete M,van Steenberghe D.Infectious risks

for oral implants:A review of the literature.Clin Oral Implants Res2002;13:1–19.

4.Grivet M,Morrier JJ,Benay G,Barsotti O.Effect of hydropho-

bicity on in vitro streptococcal adhesion to dental alloys.J Mater Sci Mater Med2000;11:637–642.

5.Bates DG,Navia JM.Chemotherapeutic effect of zinc on Strep-

tococcus mutans and rat dental-caries.Arch Oral Biol1979;24: 799–805.

6.Liljemark WF,Bloomquist C.Human oral microbial ecology

and dental caries and periodontal diseases.Crit Rev Oral Biol Med1996;7:180–198.

7.Jarcho M.Calcium phosphate ceramics as hard tissue prosthet-

ics.Clin Orthop1981;157:259–278.

8.Hench LL.Bioceramics—from concept to clinic.J Am Ceram

Soc1991;74:1487–1510.

9.de Groot K,Wolke JGC,Jansen JA.Calcium phosphate coat-

ings for medical implants.Proc Inst Mech Eng H1998;212: 137–147.

10.Ong JL,Chan DCN.Hydroxyapatites and their use as coatings

in dental implants:A review.Crit Rev Biomed Eng2000;28: 667A–707A.

11.Gross KA,Chai CS,Kannangara GSK,Ben-Nissan B,Hanley

L.Thin hydroxyapatite coatings via sol–gel synthesis.J Mater Sci Mater Med1998;9:839–843.

12.Root MJ.Inhibition of the amorphous calcium phosphate trans-

formation reaction by polyphosphonates and metal ions.Calcif Tissue Int1990;47:112–116.

13.Hidaka S,Okamoto Y,Abe K.Elutions of metal-ions from

dental casting alloys and their effect on calcium-phosphate precipitation and transformation.J Biomed Mater Res1994;28: 175–180.

14.Bigi A,Foresti E,Gandol?M,Gazzano M,Rovri N.Inhibiting

effect of zinc on hydroxyapatite crystallization.J Inorg Bio-chem1995;58:49–58.

15.Okamoto Y,Hidaka S.Studies on calcium phosphate precipi-

tation:Effects of metal ions used in dental materials.J Biomed Mater Res1994;28:1403–1410.

16.Kim TN,Feng QL,Kim JO,Wu J,Wang H,Chen GC,Cui FZ.

Antimicrobial effects of metal ions(Ag?,Cu2?,Zn2?)in hydroxyapatite.J Mater Sci Mater Med1998;9:129–134. 17.Hsieh MF,Perng LH,Chin TS,Perng HG.Phase purity of

sol–gel-derived hydroxyapatite ceramic.Biomaterials2001;22: 2601–2607.

18.JCPDS.Joint Committee on Powder Diffraction Standards

(JCPDS)database of standards:Powder diffraction(inorganic and

TABLE III.In Vitro Antimicrobial and Biocompatibility Tests of Ag/Zn Substituted HAp Coatings(Ranging from?to???) Sample Antimicrobial Zone a HGF-1Cell Attachment b Pure HAp??

Ag100ppm???

Ag1,000ppm????

Ag10,000ppm????

Zn100ppm??

Zn1,000ppm??

Zn10,000ppm??????

a Distance of the edge of apparent antimicrobial zone to the coatings(?,2mm;??, 2–5mm;???,?5mm).

b Attached cell number evaluated by ACP assays(?,0–1300cells;??,1300–2600 cells;???,2600–4000cells).177

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organic).Swarthmore,PA:International Centre for Diffraction Data,and American Society for Testing and Materials;2003. 19.Roach HI.New aspects endochondral ossi?cation in the chick:

chondrocyte apoptosis,bone formation by former chondrocytes, and acid phosphatase activity in the endochondral bone matrix.

J Bone Miner Res1997;12:795–805.

20.Liu DM,Troczynski T,Tseng WJ.Aging effect on the phase

evolution of water-based sol–gel hydroxyapatite.Biomaterials 2002;23:1227–1236.

21.Chung RJ,Hsieh MF,Huang KC,Perng LH,Chou FI,Chun TS.

Anti-microbial hydroxyapatite particles synthesized by a sol–gel route.J Sol–Gel Sci Technol.Forthcoming.

22.Lopatin CM,Pizziconi V,Alford TL,Laursen T.Hydroxyapa-

tite powders and thin?lms prepared by a sol–gel technique.

Thin Solid Films1998;326:227–232.

23.Habibovic P,Barrere F,van Blitterswijk CA,de Groot K,

Layrolle P.Biomimetic hydroxyapatite coating on metal im-plants.J Am Ceram Soc2002;85:517–522.24.Schilling KM,Carson RG,Bosko CA,Golikeri GD,Bruinooge

A,Hoyberg K,Waller AM,Hughes NP.A microassay for bacterial adherence to hydroxyapatite.Colloid Surf B1994;3: 31–38.

25.Stiger M,Groot KD,Layrolle P.Incorporation of tobramycin

into biomimetic hydroxyapatite coating on titanium.Biomate-rials2002;23:4143–4153.

26.Radin S,Campbell JT,Ducheyne P,Cuckler JM.Calcium

phosphate ceramic coatings as carriers of vancomycin.Bioma-terials1997;18:777–782.

27.Campbell AA,Song L,Li XS,Nelson BJ,Bottoni C,Brooks

DE,Dejong ES.Development,characterization,and anti-micro-bial ef?cacy of hydroxyapatite-chlorhexidine coatings produced by surface-induced mineralization.J Bone Miner Res2000;53: 400–407.

28.Chou L,Marek B,Wagner WR.Effects of hydroxyapatite

coating crystallinity on biosolubility,cell attachment ef?ciency and proliferation in vitro.Biomaterials1999;20:977–985.

178CHUNG ET AL.