Diamond & Related Materials 19 (2010) 1300–1306

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Diamond & Related Materials

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In vivo preliminary evaluation of bone-microcrystalline and bone-nanocrystallinediamond interfaces

Ana Amélia Rodrigues a,⁎, Vitor Baranauskas b, Helder José Ceragioli b,Alfredo Carlos Peterlevitz b, William Dias Belangero a

a Orthopedic Biomaterials Laboratory, School of Medical Sciences, University of Campinas, SP, Brazilb Department of Semiconductors, Instruments and Photonics, School of Electrical and Computer Engineering, University of Campinas, SP, Brazil

⁎ Corresponding author. Laboratório de BiomateriaisCiências Médicas, Universidade Estadual de Campinas, Rn° 126, Cidade Universitária “Zeferino Vaz”, 13083-887+55 19 3521 7498.

E-mail address: labimo@fcm.unicamp.br (A.A. Rodri

0925-9635/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.diamond.2010.06.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 March 2009Received in revised form 1 June 2010Accepted 11 June 2010Available online 22 June 2010

Keywords:Chemical vapor deposited diamondBiomaterialsImplantsBone prostheses

Chemical vapor deposited diamond is a new potential biomedical material which has the advantage ofchemical inertness, extreme hardness and low coefficient of friction, among others. In orthopedics andmaxillofacial surgery, these properties could improve implant performance, reducing metallic corrosion,particle wear, inflammatory reactions and bone loss. In the present study, two types of chemical vapordeposition (CVD) diamonds have been analyzed: microcrystalline diamonds (MD) and nanocrystallinediamonds (ND), both produced by hot-filament chemical vapor deposition. The diamond tubes werepreviously characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) andRaman scattering spectroscopy (RSS). The aim of this study was to verify the interface between bone and MDand ND, surgically implanted in the femoral diaphysis of Wistar rats, after 4 and 8 weeks. The outcome wasevaluated by scanning electron and optical microscopy using a semi quantitative method. The results suggestthat nanocrystalline diamonds (ND) elicits a richer biological response than microcrystalline diamonds (MD)when in interaction with bone.

em Ortopedia, Faculdade deua Tessália Vieira de Camargo,, Campinas, SP, Brazil. Tel./fax:

gues).

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Stainless steel ASTM F138 and titanium alloys like titanium–

aluminum–vanadium (TiAlV), titanium–aluminum–niobium(TiAlNb) and titanium–molybdenum (TiMo) have been the mainmaterials used to manufacture orthopedic and dental implants [1–4]. However, these materials can undergo corrosion with conse-quent deposition of metal ions and bone resorption around theimplant [5–13]. The effect on tissues and cells has been widelystudied in vivo and in vitro [14–23]. It has been found that ions oftitanium (Ti) and cobalt (Co) could inhibit specific cellularfunctions such as alkaline phosphatase (ALP) activity andextracellular calcification [20, 24–31]. Epidemiological studiesfound that tumor incidence increased in patients with metallicimplants [32]. Experimental studies in vivo have shown thatparticles of chromium (Cr), cobalt (Co), aluminum (Al), iron (Fe),manganese (Mn), molybdenum (Mo), nickel (Ni), silicon (Si),titanium (Ti), vanadium (V) and zirconium (Zr) can induce theformation of tumors in different tissues [32, 33]. For this reason, it

is essential to seek new materials completely inert in contact withbody tissues and fluids that should have neither their physico-chemical properties nor their morphology modified over time.

Following this line of thought, the diamond obtained by thechemical vapor deposition (CVD) method possesses a uniquecombination of properties—mechanical stability, high corrosionresistance, extreme hardness, high thermal conductivity, lowcoefficient of friction, chemical inertia and biocompatibility [34–40]—that makes it highly suited for applications in medicine,especially in the manufacturing or coating of implants.

Diamond with different surface roughness and morphology canbe obtained by CVD and these surface properties play importantroles on tissue reaction. Previous studies have suggested thatadhesion, spreading and cell proliferation on the surface ofmaterials can be affected by roughness [41–43]. It has beenshown that cell adhesion on polymethylmethacrylate (PMMA)increases significantly with increasing roughness. According toLampin et al. [42], PMMA promoted cell adhesion because of anenhanced roughness that favored the adsorption of adhesiveproteins.

Other researchers evaluated cell adhesion on alloys withdifferent roughness, pointing out that adhesion is favored onrougher surfaces and suggesting that fibroblasts and human bonemarrow cells can detect changes in roughness and initiate cellresponse [44–46]. They concluded that osteointegration is corre-lated with an increased surface roughness of the implants [45, 46].

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Anselme et al. [47] evaluated the adhesion and proliferation ofhuman primary osteoblasts on titanium surfaceswithmicro andmacro-roughness and found that cultured human osteoblasts prefer surfaceswith relatively high roughness (10–100 μm). However, Curtis et al. [48]demonstrated that surface roughness in the range of 130 nm could beenough to induce cellular adhesion and proliferation.

Recently, Kalbacova et al. [49] presented a correlation betweensurface roughness of nanocrystalline diamond films and osteoblastbehavior. Their results showed that films with roughness of 20 and270 nm stimulated adhesion and increased differentiation of oste-oblast cells when compared with smooth polystyrene surface.However, the same was not observed for diamond films with aroughness of 500 nm, which exhibited results similar to that ofsmooth polystyrene. Other researchers confirmed these results [50–52].

Based on the potential application of diamond as implantmaterial and considering the influence of surface properties on thetissue response, it was decided to investigate preliminarily thebone-microcrystalline diamond (MD) and bone-nanocrystallinediamond (ND) interfaces in vivo. For that purpose, self-sustainablecylindrical diamond tubes previously characterized by scanningelectron microscopy (SEM), atomic force microscopy (AFM) andRaman scattering spectroscopy (RSS) have been implanted in thefemur of Wistar rats.

2. Materials and methods

2.1. Preparation of CVD diamonds

Diamond tubes were produced by hot-filament chemical vapordeposition using tungsten wires as cylindrical substrates. Wires of0.8 mm diameter have been used. After coating, the wires wereremoved by chemical attack with heated hydrogen peroxide,resulting in self-sustained diamond tubes of 0.8 mm innerdiameter and 1.2 mm outer diameter. Prior to implantation, thetubes were laser cut in 10 mm long pieces. Ethanol highly dilutedin pure hydrogen or in hydrogen with 65% argon was used as thecarbon precursor [53, 54]. Two types of diamond structures wereproduced: microcrystalline diamond—MD (in pure hydrogen) ornanocrystalline diamond—ND (in hydrogen and 65% argon).

Raman scattering spectroscopy (RSS), atomic force microscopy(AFM) and scanning electron microscopy (SEM) have been used toevaluate the samples of microcrystalline (MD) and nanocrystalline(ND) diamonds.

2.2. Animal subjects

Twenty male Wistar rats, 20 weeks old and weighing approxi-mately 300 gwere divided into 4 groups:MD tubes were implanted intwo groups and ND tubes in the other two groups; follow-up was at 4and 8 weeks for all groups.

This study has been carried out in accordance with the ethicalprinciples approved by the Ethics Committee in Animal Experimen-tation of the University of Campinas, São Paulo, Brazil.

2.3. Surface characterization of the diamond samples

2.3.1. Scanning electron microscopy (SEM)The MD and ND samples were dehydrated with ethanol, critical

point dried (Blazers CDT 030) and gold-coated in a sputter coater(Balzers CDT 050). The MD and ND surfaces were observed andphotographed with SEM (JEOL 5800).

2.3.2. Raman scattering spectroscopy analysis (RSS)Raman spectra were recorded at ambient temperature using a

Renishaw Invia Raman Microscope system, which employed argonlaser for excitation (λ=514.5 nm) at a laser power of about6 mW.

2.3.3. Atomic force microscopy (AFM)AFM imaging and roughness measurements of the MD and ND

surface morphologies were conducted at an AFM AutoProbe CP(Park Scientific Instruments, Sunnyvale, USA) operating in non-contact mode, using silicon pyramidal tips and a resonancefrequency of 157 kHz. Representative surface scans (20×20 μm2

each) at three different locations were obtained for MD and NDsamples.

2.4. Surgical procedure

The animals were anesthetized by intravenous flow with 0.2 ml ofketamine and xylazine. An incision of approximately 2 cm was madeon the side of the thigh exposing the femoral diaphysis (Fig. 1A). A 45ºoblique drilling was performed parallel to the long axis of thediaphysis with a 1.2 mm diameter drill until reaching the medullarcavity (Fig. 1B). The site was irrigated with saline solution for removalof bone fragments before the insertion of MD and ND tubes, whichwere introduced nearly half their length, remaining the rest outsidethe bone (Fig. 1C).

After 4 and 8 weeks, the animals were sacrificed by deepeningof anesthesia. After disarticulation of the knee and hip, the softtissue was removed, except at the region where the tubesprotruded.

2.5. Macroscopic analysis (MA)

After hip and knee disarticulation, the femur with the insertedtube was maintained at low temperature with ice and later examinedwith a stereomicroscopy lens (Zeiss STM-11)—40× attached to adigital camera (Canon G5 Power) to identify the growing tissue on thetube tips.

2.6. Optical microscopy (OM)

After that, the femurs were kept in formaldehyde solution 10% for24 h and decalcified in nitric acid at 7% for 10 days. The bone wascarefully sectioned to remove the tube. The piece of bone containingthe empty hole left by the tube was subjected to 5 μm thick semi-serial cuts and stained with Hematoxylin Eosin (HE) and MassonTrichrome (MT). The slides were observed under an opticalmicroscope (Leica DMLB) with a program designed to capture andanalyze images (Leica QWIN). The purpose of OM was to study theimplant–bone interface as well as the presence of monocytes,macrophages and giant cells.

2.7. Scanning electronic microscopy (SEM)

Prior to observation under Scanning Electronic Microscopy(JEOL 5800), the tubes were immediately critical point dried(Balzers CTD-030) and metallized with gold in a sputter coater(Balzers SCD-050). The purpose of SEM was to observe the surfaceof the MD and ND tubes. The apparent area of the surface coveredby some kind of tissue was measured using a millimeter grid. Thepercentage of tissue coating the tubes was calculated as the ratiobetween the apparent area covered by tissue and the totalapparent area.

Fig. 2. Scanning electronic microscopy (SEM) of CVD diamond: (A) SEM ofmicrocrystalline diamond (MD); (B) SEM of nanocrystalline diamond (ND). Scalebar=50 μm.

Fig. 1. Surgical procedure: (A) exposure of the femoral diaphysis side with the aid ofretractors; (B) oblique drilling at approximately 45° performed with helical drill; (C)tube of diamond CVD implanted into the bone defect.

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3. Results

3.1. Scanning electron microscopy (SEM) of the diamond tubes

It was observed that MD and ND had different surfacemorphologies. The MD surface was formed by triangular shapedgrains (Fig. 2A), and the ND surface had a cauliflower-like aspect(Fig. 2B).

3.2. Raman scattering spectroscopy analysis (RSS) of the diamond tubes

Fig. 3A–B shows the typical Raman spectra of MD and ND samples.The main Raman active mode of cubic diamond was observed as an

intense peak around 1333 cm−1 in all spectra. The ND spectrum(Fig. 3B) exhibits peaks at 1140 cm−1, 1333 cm−1, 1350 cm−1,1470 cm−1 and 1570 cm−1.

3.3. Atomic force microscopy (AFM) of the diamond tubes

It was not possible to obtain images and roughness measurementsfrom theMD sample, once it presented peak-valley height beyond theresolution of the equipment used (∼8 μm).

Fig. 4 shows a typical 3-D AFM image of the ND surface. Theseveral aligned triangular features that can be seen, are in factimage artifacts due to the pyramidal shape of the probe tip. Theaverage roughness of the ND sample, based on three 20×20 μm2

scanned areas, was calculated as 455 nm (Fig. 4).

3.4. Macroscopic analysis (MA) of the bone with implant

The ND tubes after 4 and 8-week follow-up had virtually the entiretip covered by a conjunctive tissue. However, the ends of the MDtubes at the same follow-up time were not covered in all observations(Fig. 5A–D).

3.5. Optical microscopy (OM) of the implant–bone interface

After 4 weeks of follow-up, the presence of dense connectivetissue of variable thickness (Fig. 6A) between the ND tubes and

Fig. 3. Raman spectra of MD (A) and ND (B) samples.

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bone was noticed. After 8 weeks, there was an increase in thethickness of this membrane without inflammatory cells (Fig. 6B).

Between the MD tubes and bone, connective fibrous tissue wasformed after 4 weeks (Fig. 6C). After 8 weeks, this membrane wasbetter organized and thinner in comparison to the ND group at thesame time (Fig. 6D).

Table 1 shows an overview of the distribution of connective fibroustissue found around the tubes.

Fig. 4. Surface morphology of the ND sample (3-D imaging with tip artifacts).

3.6. Scanning electronic microscopy (SEM) of the implants

It was identified on the surface of ND tubes by SEM (Fig. 7B andD) a connective tissue like a fibrous tissue. However, less connectivetissue was found at 4 and 8-week follow-up on MD tubes (Fig. 7Aand C).

The average percentage of ND tubes covered by tissue after4 weeks was 98.2%, while for MD tubes it was 1.6% for the sameperiod. After 8 weeks, it was 94.6% and 1.3%, respectively (Table 1).

4. Discussion

The surface morphologies seen on the SEM-images of the MD(formed by triangular shaped grains) and ND (cauliflower-like surfaceaspect) samples used in this work (Fig. 2A and B) are in agreementwith the typical morphologies exhibited by this kind of diamonds [34,35, 53, 54].

The carbon character of the samples was determined by RSS.The spectrum of the MD samples exhibited a peak at 1333 cm−1

(Fig. 3A). Compared with natural diamond (peak at 1332 cm−1),the samples are in a state of minor compressive stress, what maybe attributed to differences in thermal expansion coefficientsbetween the diamond and the W wire substrate. The MD spectrum(Fig. 3A) indicates that the MD tubes are of high quality diamond.The ND spectrum (Fig. 3B) shows peaks at 1140 cm−1, 1333 cm−1,1350 cm−1, 1470 cm−1 and 1570 cm−1, which are expectedfeatures for nanocrystalline diamond. The peaks that appear at1350 cm−1 and 1570 cm−1 correspond to the D and G modes ofdisordered carbon. There has been some controversy regarding thenature of the peak at 1140 cm−1 [55, 56], since some authorsbelieve that it originates from confined phonon modes innanocrystalline diamonds and others claim that it is associatedwith C–C sp2 vibrations at hydrogenated grain boundaries, wherepolyacetylene may be present [56]. The peak at 1470 cm−1 mayalso be assigned to polyacetylene [56].

The surface roughness of the samples was estimated by AFM.The average value obtained for the ND samples (455 nm)approaches that of the roughest samples used by Kalbacova [49].Once it was impossible to measure the roughness of MD samples,as their peak-valley height was beyond the resolution of theequipment, it can only be affirmed that their roughness was above8 μm.

Macroscopic analysis and SEM-imaging indicated a more pro-nounced tissue growth on the surface of the ND tubes than on the MDones, despite the follow-up period (Figs. 5A–D and 7A–D and Table 1).This result was confirmed by OM, where it observed a more organizedtissue at the bone–ND interface than at the bone–MD one (Fig. 6).Although the tissue adhered on the ND diamond tubes has not beenidentified, its histological aspect was characteristic of dense connec-tive tissue, what suggests that ND elicits a richer biological responsethan MD.

It is known that, soon after the implantation, neighboring cellssend and receive chemical and physical signals from each other,surrounding extracellular matrices and external medium. Theimplant interacts with the living tissue, blood and transmembraneproteins in the site of adhesion [57, 58]. Since the cellularresponse is linked to the implant surface properties that modifyseveral cellular responses like initial cell attachment, migration,differentiation and production of new tissues [57–59], it isexpected that such properties influence the tissue response. Asthe two forms of CVD diamond used in this study had similarchemical composition, the surface finishing, the grain size or thehydrophilicity (not measured here) could be the main factorinfluencing the different biological performance of the tubes onbone. Since very few studies have been carried out by now aboutthe behavior of diamond in animal models, the results presented

Fig. 5. Macroscopic analysis: (A) nanocrystalline diamond at 4-week follow-up; (B) nanocrystalline diamond at 8-week follow-up. The presence of conjunctive soft tissue coveringthe end of the tubes (→) can be noticed; (C) microcrystalline diamond at 4-week follow-up; (D) microcrystalline diamond at 8-week follow-up.

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here will be discussed in the light of the in vitro studies. Inprevious in vitro studies, it has been demonstrated that osteoblastcells preferentially adhere to nanocrystalline diamond films with

Fig. 6. Optical microscopy images of ND (A–D) and MD (E–H). (A–B): ND at 4-week followshowing increase in thickness of this dense connective tissue. (E–F): MD at 4-week follow-showing better organization of connective tissue, compared to the 4-week follow-up group.stain (B, D, F and H) with magnification of ×400.

relatively higher surface roughness and hydrophilic properties (O-termined) [60, 61]. Nanocrystalline diamond with relatively highsurface roughness stimulated stronger adhesion, proliferation and

-up, showing thickening of dense connective tissue. (C–D): ND at 8-week follow-up,up, showing irregular formation of connective tissue. (G–H): MD at 8-week follow-up,Hematoxylin Eosin stain (A, C, E and G) with magnification of ×100. Masson Trichrome

Table 1Optical microscopy (OM) where the presence (+) or absence (−) of neoformedmembrane was verified. Scanning electron microscopy (SEM) where the presence (+),absence (−) and percentage of tissue covering the tube were verified.

Material ND MD

Imaging OM SEM OM SEM

Follow-up

Membrane(interface)

Tissuecovering

% tissuecovering

Membrane(interface)

Tissuecovering

% tissuecovering

4 weeks + + 93.0 + − 0+ + 100.0 + + 6.4+ + 100.0 − − 0.0− + 100.0 − − 0.0Average tissuecovering

98.2 Average tissuecovering

1.6

8 weeks − + 100.0 + − 0.0− + 100.0 + − 0.0+ + 73.0 − − 0.0+ + 100.0 + + 6.4+ + − − 0.0Average tissuecovering

94.6 Average tissuecovering

1.3

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increased differentiation of osteoblast cells [49–52, 60, 61].Regarding the grain size, Yang et al. [51] concluded that NDcould facilitate osteoblast proliferation, whereas MD could inhibitit. In the present study, the smoother ND implants elicited a richerbiological response than the rougher MD implants, what suggests

Fig. 7. Electronic micrographs of the tubes: (A) microcrystalline diamond after 4 weeks; (B) n(D) nanocrystalline diamond after 8 weeks. Scale bar=200 μm (panels A and B). Scale bar

that the grain size could be the most important factor of influence.It is worthy to note that the triangular shaped grains of the MDcould have a surface roughness smaller than that of the ND surfaceas a whole.

Hence, in clinical situations that demand tissue proliferation,such as filling of bone or cartilaginous cavities, ND could be better.Furthermore, situations where this proliferation is undesirable, asin guiding nerve fibers growth, MD could be more appropriate.

5. Conclusion

Nanocrystalline CVD diamond with an average surface rough-ness of 455 nm promoted more intense tissue growth after 4 and8 weeks than microcrystalline CVD diamond with surface rough-ness higher than 8 μm, what suggests that the former could besuitable for applications that demand tissue proliferation, whereasthe latter would better suit to applications where this proliferationis undesirable.

Acknowledgments

This work was supported by CNPq and FAPESP. The authorsthank the Department of Applied Physics, Institute of Physics“Gleb Wataghin”, University of Campinas, for atomic forcemicroscopy imaging.

anocrystalline diamond after 4 weeks; (C) microcrystalline diamond after 8 weeks and=500 μm (panels C and D).

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