In Vitro and In Vivo Biocompatibility Testing of Ti-6Al-7Nb Alloywith and without Plasma-Sprayed Hydroxyapatite Coating

I. C. Lavos-Valereto,1 S. Wolynec, M. C. Z. Deboni, B. Konig, Jr.3

1 Department of Metallurgical and Materials Engineering, Polytechnic School, University of Sao Paulo, Brazil

2 Faculty of Dentistry, University of Sao Paulo, Brazil

3 Faculty of Biomedical Science, University of Sao Paulo, Brazil

Received 9 August 2000; revised 18 April 2001; accepted 29 June 2001Published online 00 Month 2001; DOI 10.1002/jbm.0000

Abstract: TheTi-6Al-7Nb alloy hasbeen recently developed for biomedical use, particularlyfor orthopedics and dental applications. Osteosynthesis has been used to analyze biocompat-ibilit y and osseoconduction properties. The interaction of the implant with its biologicalenvironment, the formation of the implant material/tissue interface, and the long-term successor failur e of integration in the human body is strongly connected with the surface propertiesof the implant device. This study was undertaken to evaluate the processes involved inbiological responses of the Ti-6Al-7Nb alloy with and without hydroxyapatite coatings withboth in vitro and in vivo tests. The results were analyzed by scanning electron microscopy(SEM) and energy-dispersive x-ray (EDX) microanalysis. The morphology of the in vitro andin vivo testing results with hydroxyapatite coating was similar to those obtained on theuncoated samples. A mineralized extracellular matri x was formed on all materials. Observa-tion of the interface between the cell layer and substrata showed the presence of calcium andphosphorous-rich globular depositsassociated with collagen fiberson all materials in vitro andin vivo. A higher density of these globular deposits was observed in all samples. © 2001 JohnWiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 58: 727–733, 2001

Keywords: osteoblastic-like cells; cell growth; Ti-6Al-7Nb dental implants; dog test; hy-droxyapatite

INTRODUCTION

Increasing attention has been given to the development ofnew biomaterials promoting wound healing in humans. Clin-ical applications in both medical and dental practice, haveincreased during thepast two decades. However, this increas-ing useof biomaterialshascaused agreater need for effectiveand reproducible test systems for these materials.1

The biological performance of a biomaterial has to take intoaccount thematerial’smechanical, physical, and chemical prop-erties, as well as the host response. Biological evaluation playsa crucial role when these materials are intended for human use.The selection of materials for medical applications is usuallybased on considerations of biocompatibility. Testing the bio-compatibility of a specific material ensuresthat thematerial willnot have any toxic effect on cells. The biocompatibility of amaterial canbeevaluatedby invitroand invivo tests, but invitro

testing does not exclude in vivo testing. The in vitro methodsprovide necessary and useful results to be added to those foundin the in vivo testing of potential biomaterials. In vitro testing isusually accepted as afirst-choice method for testing toxicity ofa material. There is a great variety of in vitro parameters oftoxicity, such as cell death, reduced cell adhesion, altered cellmorphology, reduced cell proliferation, and reduced biosyn-thetic activity. These parameters may give warnings regardingmaterial toxicity.2–4

Among many in vivo evaluation tests, the osteointegrationmethods are used to measure the biocompatibility of in-traosseous implants. The inflammatory response to the im-plant is also examined.5,6 Animal experimentation has beenused to obtain information on biological responses to im-plants. Many in vivo implants studies have attempted toprovide an understanding of the implant–bone interface bymeasuring the mechanical performance of this interface invarious ways. Others have attempted to describe the struc-tural nature of the bone–implant interface, of various bioma-terials, by meansof qualitativeand/or quantitativeestimates.7

Sometimes an unwanted leakage of toxic metal ions fromthe implant causesan inflammatory response in thesurround-

Correspondence to: I. C. Lavos-Valareto, Rua Pelotas, 541 Apto. 1004, 04012-0002 Sao Paulo, Brazil (e-mail: iclvaler@usp.br)

Contract grant sponsor: Fundacao de Amparo a Pesquisa do Estado de Sao Paulo(FAPESP); contract grant number 99/08554-6

© 2001 John Wiley & Sons, Inc.

727

ing tissue, resulting in the formation of a fibrous encapsula-tion of the implant. However, an inflammatory response isnormal after the insertion of implants, as was reported byDonath and his collaborators.8–11 In recent years, titaniumand its alloys have been the favorite materials in most surgi-cal implant operations in the dental area, showing an excel-lent biocompatibility. Metallic implants, to fulfill their func-tions, must first be accepted by the body. This acceptance orbiocompatibility is improved by coating the surface in contactwith living tissues with Ca-phosphates, especially hydroxy-apatite. Metals are by far the oldest materials used in surgicalprocedures. In medical applications the titanium and titaniumalloys are relatively new materials compared to the stainlesssteels and the cobalt-based alloys. The main drawback oftitanium and its alloys is their poor wear resistance.9–16

Calcium phosphate ceramics have a strong resemblance tohydroxyapatite, the most important mineral in living bone.The hydroxyapatite is the inorganic component of bones andteeth. It has been identified as a bioceramic with bioactiveproperties suitable for bone substitution and interfacing layersin surgical implants.7,15–19

The purposes of this work were first, to compare thegrowth viability of osteoblastic-like cells from Ti-6Al-7Nballoy samples with and without plasma-sprayed hydroxyap-atite coating; and second to evaluate in dogs the osseointe-gration behavior of implants with and without hydroxyapatitecoating. Scanning electron microscopy (SEM) was used toobserve the material surfaces and the immediately adjacenttissue. Energy-dispersive x-ray (EDX) microanalysis wasused to verify if biomaterials with and without hydroxyapatitecould induce different rates of osteoblast differentiation andmineralization.

MATERIALS AND METHODS

Hydroxyapatite Plasma-Sprayed (HA-Coated) Samples

The Ti-6Al-7Nb alloy samples were coated with hydroxyap-atite with the use of SG-100 (Miller Thermal Inc.®) plasma-spraying equipment. The plasma torch was preheated withargon gas (Ar), and hydrogen (H2) and nitrogen (N2) gaseswere also used, respectively, as primary fuel and carriergas.20,21 The Ti-6Al-7Nb alloy samples were coated withhydroxyapatite (HA) (F. J. Brodmann, 2015M-1) powderswith grain sizes ranging from 44 to 111mm.20

In Vitro Samples

Twenty-four 2.0-mm-thick disk-shaped Ti-6Al-7Nb alloysamples were cut from a 12-mm diameter rod supplied byIMI Titanium Limited [England/IMI-367 (SN056512]. The12 uncoated discs were first ground with 220–1200-grit SiCpapers and then polished with a 6-mm alumina slurry, fol-lowed by a 1-mm alumina-20% chrome-oxide suspension,and then a 1-mm alumina slurry. The other 12 discs were HAcoated.22

In Vivo Samples

For the in vivo tests 12 Ti-6Al-7Nb alloy implants weremachined from the above rod as cylinder-shaped screws.They were about 10 mm long and 4.0 mm in diameter. Six ofthese implants remained uncoated; the other six were HA-coated.21

Sterilization of Samples

All samples, both the disk-shaped ones used for thein vitrotests and the cylinder-shaped screw implants usedin vivo,were ultrasonically degreased in acetone plus ethyl alcoholsolution for 10 min each. They were then air dried andsterilized by gamma radiation, the source of which was cobalt60 (Co). The total dose of radiation per sample was 25 kGy.20

Cell Culture

Osteoblast-like cells (OSTEO-1)23,24 were used forin vitrotests. These cells were obtained from the digestion process ofnewborn rat calvaria. Before the test, with the use of thehistochemic (alkaline phosphatase positive cells) and immu-nohystochemic methods (positively for osteonectin, type Icollagen and bone siaoloprotein), they were characterized asosteoblastic.25,26These cells were subcultivated over the ma-terial samples in order to evaluate their behavior. The osteo-blast-like cells were carefully seeded into 35-mm culturedishes at a concentration rate of 104 cells/ml over the samplesand were left 10 minutes at 37 °C for initial cellular adhesionto the material surface before more 5 ml of Dulbecco’smodified Eagle’s culture medium, supplemented with 10%fetal bovine serum and antibiotics, was been added to thesedishes. This medium was changed every 48 h and the cultureswere maintained at 37 °C in a humidified CO2 atmo-sphere.23,24

Surgical Technique

Both sides of mongrel dogs’ mandible were drilled transcor-tically. The animals weighed 22–24 kg. The surgical proce-dure was carried out by first sedating the animals with the useof Rompun intramuscular injection, and the administering ageneral intravenous anesthesia injection of sodium thiopentalas needed. Routine infiltration anesthesia of Lindocaine 2%was administered at the surgical sites. All inferior premolarswere removed bilaterally. After 12 weeks the animals wereanesthetized again and a full-thickness flap was obtained inthe alveolar ridge. Three insertion beds were created on eachside of the bone for implantation under saline irrigation,according to a routinely used surgical protocol. The implantswere inserted in the first, second, and third premolar teethalveolar sites.20,21

Preparation of the Implant/Bone Block

After a healing period of 16 weeks the animals were sacri-ficed and the implant containing premolar alveolar partsprepared for analysis. The tissue blocks containing the im-

728 LAVOS-VALERETO ET AL.

plants were immediately fixed in 10% buffered formalinsolution, rinsed in distilled water, and dehydrated in an alco-hol series from 70 to 99% ethanol solution for 24 h. Aftertreatment with xylol for 24 and 48 h, the specimens wereembedded in a light-curing metacrylate resin. Each implantblock was sectioned longitudinally to the long axis of theimplant into 80-mm slices. Afterwards they were ground withthe use of 600–1200-grit SiC papers and polished with a6–1-mm alumina slurry.20,21

SEM and EDX

The surface morphology of thein vitro and in vivo sampleswas examined by scanning electron microscopy (SEM)(BALZERS, SCD 030 model). A thin gold film was depositedover the specimens by vacuum evaporation through a heat-ing-under-vacuum method. The SEM samples were exam-ined at accelerating voltages of 15 kV. The Ca/P ratio of allsamples was determined by energy-dispersive x-ray (EDX)(CAMBRIDGE, Stereoscan 240 model). The data were col-lected in a multichannel detector coupled to a microcomputer.The spectra were observed with Kontron Elektronik GmbH(KS 3000) program with respect to available stopping pow-ers. The specimens were gold sputter-coated, and analyzed byEDX at accelerating voltages of 20 kV.

RESULTS

In Vitro

During the in vitro testing, the osteoblast attached to thehydroxyapatite-coated Ti-6Al-7Nb surface produced a largeamount of extracellular matrix, as can be observed in Figure1 and in more detail in Figure 2. These figures, obtainedduring SEM, show the morphology of the hydroxyapatite-coated Ti-6Al-7Nb samples seeded with osteoblast-like cells(OSTEO-1)20,22–24after 15 days ofin vitro testing. It can benoticed that the cell layer covers most of the surface of thesample. Moreover, bundles of collagen fibrils are seen in

intimate contact with the coating of the metal, which iscovered with globular deposits, and the calcified globules.The extracellular matrix is characterized by a bone-like tissuethat covers the template and permeates its entire thickness.The EDX microanalysis of the surface of these samplesdetected the following elements: calcium (Ca), phosphorous(P), oxygen (O), and carbon (C). Ca and P emission intensi-ties due to the presence of the hydroxyapatite surface layerdominated the spectrum. This analysis also indicated that allglobules are rich in calcium and phosphorous.

The SEM observation of the uncoated Ti-6Al-7Nb sam-ples showed that the osteoblast-like cell (OSTEO-1)22 cul-tures reproduce on these metals in a similar morphology toeach other, and also similar to that observed for the culture onhydroxyapatite-coated samples. This morphology is shown inFigure 3 and in more detail in Figure 4. It can be observedthat a compact cell layer was formed on top of the material.Moreover, this upper layer of the cell culture shows thepresence of polygonal and round cells with extensive cyto-

Figure 1. Scanning electron micrograph of osteoblasts cultured onhydroxyapatite-coated Ti-6Al-7-Nb alloy after 15 days in vitro. Thesurface of the cell layer is complete, and osteoblasts (arrowhead) andcollagen fibers can be identified: 17003.

Figure 2. Scanning electron micrograph of osteoblasts cultured onhydroxyapatite-coated Ti-6Al-7-Nb alloy after 15 days in vitro. This isa higher-magnification micrograph showing the interface between thecell layer and the substratum, where globular deposits (arrow) can beidentified: 60003.

Figure 3. Scanning electron micrograph of osteoblasts cultured onuncoated Ti-6Al-7-Nb alloy after 15 days in vitro. This is a higher-magnification micrograph of extracellular matrix showing the exis-tence of globular deposits (arrows), where bundles of collagen fibersare incorporated (arrowheads). Original magnification: 17003.

729TESTING OF TI-6AL-7NB ALLOY

plasm processes. The extracellular matrix seems to be com-posed mainly of collagen fibers, but the presence of globulesin a complex net of collagen was also detected (Figure 4).The EDX microanalysis indicated that these globules are richin calcium and phosphorus.

In Vivo

Both hydroxyapatite-coated and uncoated Ti-6Al-7Nb in-traosseous screw-shaped implants were inserted into mandib-ular bone of mongrel dogs and then removed, after a healingperiod of 16 weeks, for SEM examination and EDX micro-analysis.

The results of SEM examination of intraosseous uncoatedimplants are presented in Figures 5–7. In these figures boththe bone tissue surface and the medulla cell layer are ob-served, as well as the implant/bone interface. There wasalways a fine gap at the interface and, as seen in Figures 5 and6 in the region opposite to the implant/bone interface, thebone layer communicates with a partially calcified collagen

(osteoid), followed by the medullary tissue. It was also ob-served that the bone tissue near the implant surface is of ahigh quality, resembling the compact bone, which helps an-chor the implanted screw.

The results of SEM examination of intraosseous coatedimplants are given in Figures 8–10. It can be noticed that thegap at the implant/bone interface is much narrower and seemsto disappear in some areas. On the other hand, it can also beseen that new bone, in the form of clusters, is formed near thehydroxyapatite. This bone is much thicker than the one situ-ated alongside the uncoated surface. Looking at all the dataobtained, it seems likely that the greatest amount of bone isformed near the head and the base of implants and that theimplant itself is not very far from the compact bone. Figures9 and 10 show the newly formed bone around the head andthe neck of the implant screw, which are smooth and un-coated. A general view can be appreciated in Figure 8. Figure10 shows a very peculiar ossification zone.

Figure 4. Scanning electron micrograph of osteoblasts cultured onuncoated Ti-6Al-7-Nb alloy after 15 days in vitro. Polygonal cells areobserved with the presence of the extracellular matrix composedmainly of collagen fibers and globular deposits. Original magnifica-tion: 60003.

Figure 5. Scanning electron micrograph of an intraosseous uncoatedTi-6Al-7-Nb alloy implant after a healing period of 16 weeks in themandibular bone of a mongrel dog. The bone–implant interface has anarrow space and the bone is well developed along the implantsurface (arrow). Original magnification: 503.

Figure 6. Scanning electron micrograph of an intraosseous uncoatedTi-6Al-7-Nb alloy implant after a healing period of 16 weeks in themandibular bone of a mongrel dog. The bone structure is well defined(w), and partially calcified collagenous areas (osteoid) (arrow) can beseen near the base of the implant (*). The medullary cavity is indicated(arrow head). Original magnification: 433.

Figure 7. Scanning electron micrograph of an intraosseous uncoatedTi-6Al-7-Nb alloy implant after a healing period of 16 weeks in themandibular bone of a mongrel dog. The ossification follows thethread’s contour of the implant. Original magnification: 5143.

730 LAVOS-VALERETO ET AL.

Sixteen weeks after implantation, there were significantdifferences between the implants (uncoated and hydroxyap-atite-coated Ti-6Al-7Nb alloy) at the bone–implant interfacezone. The gap present at the uncoated implant/bone interfacedisappears when coated samples are used. By comparing theresults it may be concluded that the highest activity of newbone formation occurred at the coated implants. A timeincreasing activity was detected in the osteons area, as well asat the interface.

Formation of extracellular material, such as collagen fibersand bone cells, was detected. An important observation is thatthere was new bone invading the implant helicoidal areacorresponding to the bone medulla that is commonly filledsolely by adipose and hematopoietic tissue. This is a proof ofthe material osteoconductive properties.

The following elements were detected at the sample’ssurface by EDX microanalysis (Figure 11): calcium (Ca),phosphorous (P), oxygen (O), and carbon (C). Ca and Pemission intensities due to the naturally formed apatite sur-

face layer dominated the spectrum. This microanalysismethod also demonstrated that there was new deposition ofcalcified material.

DISCUSSION

In an earlier study20 with the same conditions and parameters,a biocompatibility testing evaluation of the implant/boneinterface was carried out. The titanium implants and hydroxy-apatite-coated titanium implants did show a high osseointe-gration in other reports5,25–27. In the present research, the

Figure 8. Scanning electron micrograph of an intraosseous hydroxy-apatite-coated Ti-6Al-7-Nb alloy implant after a healing period of 16weeks in the mandibular bone of a mongrel dog. The bone is origi-nated in the compact and grows in the implant direction (arrow).Original magnification: 173.

Figure 9. Scanning electron micrograph of an intraosseous hydroxy-apatite-coated Ti-6Al-7-Nb alloy implant after a healing period of 16weeks in the mandibular bone of a mongrel dog. The bone growth ismore intense near the compact layer of the mandible. Original mag-nification: 433.

Figure 10. Scanning electron micrograph of an intraosseous hy-droxyapatite-coated Ti-6Al-7-Nb alloy implant after a healing periodof 16 weeks in the mandibular bone of a mongrel dog. The exhibitedregion is near the neck of the implant, where the bone developed inthe form of a ring. Original magnification: 2053.

Figure 11. EDX spectra of the in vitro and in vivo samples. There wasa complete presence of a Ca and P peak in the spectrum. All EDXspectra were similar and showed the same Ca, P, O, and C elementsand peaks. The Ca concentration is defined to be larger than thatof P.

731TESTING OF TI-6AL-7NB ALLOY

Ti-6Al-7Nb alloy with and without plasma-sprayed hydroxy-apatite coating implants also showed a high osseointegration.The biosafety of a new Ti-6Al-7Nb alloy with and withoutplasma-sprayed hydroxyapatite coating samples was evalu-atedin vitro in cultures of osteoblast-like cells (OSTEO-1),22

andin vivo by intraosseous implantation studies in dog man-dibles. It must be borne in mind that in other studies not allthe coatings were made in the same way as they werehere.3,12,28,29

In the uncoated sample, as noticed in Figures 5–7, a gapwas present in the bone/implant interface. This gap did notappear in the samples coated with hydroxyapatite. The bonetissue, however, was of a very good compacted quality,which is also referred in a previous article.5 The dense tissuesreferred to as osteoid and bone tissue are based in the resultsof previous articles.20,21

In this article the hydroxyapatite coating was done byplasma-spraying onto the surface of a new dental implantalloy. The coating, generated by plasma deposition of thebiologically derived hydroxyapatite powder, with the use ofstandard spray parameters, has been extensively studied. In-vestigations into the thermal spraying of hydroxyapatite coat-ings have shown that variation in operating parameters cancause significant changes to the microstructure and the me-chanical properties of the coating.30 The major advantages ofthese coatings, both tested and used, are their good biologicalcompatibility and relative chemical inertness.31 Plasma-sprayed coatings usually contain a certain percentage ofamorphous phase.32 It seems that this phase is more resistantto dissolution, which is consistent with the fact that samplesimplanted in dogs (Figures 8–10) did not show materialdegradation. In our results it was observed that there was agrowing of the bone tissue from the cortical layer of the bonein direction of the implant surface. As is known, the metal hasan osteoconductive property.20,21

Scanning electron microscopy showed that the surfacesprayed with plasma was much rougher than the plain Ti-6Al-7Nb surface. It was confirmed in these samples that theosteoblast had anchored and spread on the extracellular ma-trix. As in other investigations,3,33,34it resembles the polyg-onal and cubical shapes of normal osteoblast with dorsalruffles and long cellular processes, sometimes resembling themorphology of osteocytes. The isolated osteoblast-like cellpopulations, like the culture of calvarial cells, were found toretain some capacity for osteogenesis. Isolated at cell cul-tures, they express their phenotype, producing an intricate netof collagen fibers in the extracellular matrix that could beobserved covering the material samples. SEM confirmed thetwo types of implants (uncoated and coated) to be nontoxic,as there was no spontaneous detachment of the cells.

The nodules that could be seen in some areas of the SEMobservations can be considered as calcified globules ThroughEDX microanalysis the following elements were detected atthe sample surface: calcium (Ca), phosphorous (P), oxygen(O), and carbon (C). They reinforce the material’s stabilityand the possibility of synthesis of mineralized matrix, asalready established in previous studies.20,22 The materials

tested allowed the cells to have a normal level of activity. Theextracellular matrix is mainly composed of collagen fibers inwhich small globules could be visible (Figures 1–4).

In a previous work,20 cellular viability percentages ob-tained by the Trypan Blue stain method displayed similarvalues (about 80%) for uncoated samples, hydroxyapatite-coated samples, and the control group. It was recognized thatsome cells could have been dispersed trough the mediumafter the seeding over the material, but many have adheredand could be fixed in place to be processed for SEM study(Figures 3 and 4).

At the in vivo assay, bone tissue was seen directly incontact with the coated surface (Figures 8–10) showing theosseoconductive properties, a different situation from thatseen in the uncoated samples. It was also observed that theosseoconductivity has variations according to the distance ofthe implant in relation to the bone compact. The conductivityis more intense when the implant is near this region of thebone. As the mandible is a bone with a relatively narrowcross section, there would be no difficulties with the proxim-ity of the bone compact, which will not be the case in themaxilla, where regional accurate judgments have to be done.

EDX microanalysis was performed in other investiga-tions35 in order to evaluate the Ti-6Al-4V alloy. Comparedwith the present research it was noticed that the Ti-6Al-7Nballoy seems to be more inert,21 and that there is a formationof calcium phosphate at the interface with the depositedmaterial.

This article has shown that the cell cultures, whether thesamples of the alloy are coated or not, exhibit a morphologytypical of osteoblasts, in a way similar to that shown in otherinvestigations,3,34,36,37that is, a polygonal shape with dorsalruffles and cell processes. EDX microanalysis also showedthat there is a calcium-phosphate layer at the alloy surface.

According to the present results, the Ti-6Al-7Nb alloyprovides a very compatible material for osseointegration,with a promising future for its use as an implant material.This alloy was shown to have good stability, no toxicityeffects, high biocompatibility, and relatively good osseocon-duction properties. The behavior of the surface coating aftera long term cannot to be judged with the results presented inpresent article, but we know by previous experiences that thehydroxyapatite coating may osseointegrate and then detachfrom the alloy surface. The bone would then integrate withthe alloy after a possible reabsorption of the coating. Moreresearch is needed to discuss these points of view.

CONCLUSIONS

The results of present study are extremely encouraging for theuse of Ti-6Al-7Nb alloy, with and without hydroxyapatitecoating, as a biomaterial for dental implants. Both thein vitroand thein vivo investigations, carried out on both uncoatedand coated Ti-6Al-7Nb alloys, showed that none of the sam-ples evoked any cytotoxic or inhibition effects on samples

732 LAVOS-VALERETO ET AL.

during the testing period. Moreover, cellular growth, cellproliferation capability, viability of conservation, cell adhe-sion, and mineralization were also demonstrated to occurduring the extent of this study. A bone-like tissue grew, inculture, on all studied materials. The SEM observation of thecell layer on the surface of all the materials showed that themorphology was always similar, consisting of osteoblast-likecells. The EDX showed that the area was made up mainly ofcollagen, where calcium- and phosphorus-rich globules weretrapped. The implant/bone interface showed the presence ofcalcium and phosphorus-rich globule deposits with a higherdensity than on the cell layer surface.

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